• TABLE OF CONTENTS
HIDE
 Monitoring for exotic Spodoptera...
 Corn expressing Cry1Ab or Cry1F...
 Evaluations of Bollgard[R], Bollgard...
 Physiological basis of fall armyworm...
 Review of fall armyworm (Lepidoptera:...
 Efficacy of Cry1F insecticidal...
 Does secondary plant metabolism...
 Efficacy of Vip3A and Cry1AB transgenic...
 Odorants of the flowers of butterfly...
 Greenhouse trials of Aphidius colemani...
 Fall armyworm (Lepidoptera: Noctuidae)...
 Biology of Eurytoma sivinskii,...
 Seismic behaviors of a leafminer,...
 Toxicity of organosilicone adjuvants...
 Comparison of parasitic hymenoptera...
 Offspring in response to parental...
 Variation of Copaeodes minima and...
 Effect of rainfall and soil moisture...
 Modified agar-based diet for small...
 Body size relationship between...
 Pathogenicity of Beauveria bassiana...
 Antennal sensilla of Toxotrypana...
 Mortality of the lobate lac scale,...
 Rearing Cactoblastis cactorum (Lepidoptera:...
 Exposing entire adult holding rooms...
 Synonymy of Phanerota appendiculata...
 Aspects of the field ecology of...
 Geographic range expansion of Oxyops...
 New host, Host plants, and distribution...
 Removal of fungal contaminants...
 First record of Cuterebra fontinella...
 Fruit flies (Diptera: Tephritidae)...
 Races of Heliconius erato (Nymphalidae:...
 Book reviews
 Volume 91 author index
 Volume 91 subject index
 Back Matter














Group Title: Florida Entomologist
Title: The Florida entomologist
ALL VOLUMES CITATION THUMBNAILS PAGE IMAGE ZOOMABLE
Full Citation
STANDARD VIEW MARC VIEW
Permanent Link: http://ufdc.ufl.edu/UF00098813/00358
 Material Information
Title: The Florida entomologist
Uniform Title: Florida entomologist (Online)
Abbreviated Title: Fla. entomol. (Online)
Physical Description: Serial
Language: English
Creator: Florida Entomological Society
Florida Center for Library Automation
Publisher: Florida Entomological Society
Place of Publication: Gainesville Fla
Gainesville, Fla
Publication Date: December 2008
Frequency: quarterly
regular
 Subjects
Subject: Entomology -- Periodicals   ( lcsh )
Insects -- Periodicals -- Florida   ( lcsh )
Genre: review   ( marcgt )
periodical   ( marcgt )
 Notes
Additional Physical Form: Also issued in print.
System Details: Mode of access: World Wide Web.
Language: In English; summaries in Spanish.
Dates or Sequential Designation: Vol. 4, no. 1 (July 1920)-
Issuing Body: Official organ of the Florida Entomological Society; online publication a joint project of the Florida Entomological Society and the Florida Center for Library Automation.
General Note: Title from caption (JSTOR, viewed Sept. 13, 2006).
General Note: Place of publication varies.
General Note: Latest issue consulted: Vol. 87, no. 4 (Dec. 2004) (JSTOR, viewed Sept. 13, 2006).
 Record Information
Bibliographic ID: UF00098813
Volume ID: VID00358
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: isbn - 0015-4040
issn - 1938-5102
oclc - 33223434
lccn - sn 95026670
 Related Items
Preceded by: Florida buggist (Online)

Table of Contents
    Monitoring for exotic Spodoptera species (Lepidoptera: Noctuidae) in Florida
        Page 517
        Page 518
        Page 519
        Page 520
        Page 521
        Page 522
    Corn expressing Cry1Ab or Cry1F endotoxin for fall armyworm and corn earworm (Lepidoptera: Noctuidae) management in field corn for grain production
        Page 523
        Page 524
        Page 525
        Page 526
        Page 527
        Page 528
        Page 529
        Page 530
    Evaluations of Bollgard[R], Bollgard II[R], and WideStrike[R] technologies against beet and fall armyworm larvae (Lepidoptera: Noctuidae)
        Page 531
        Page 532
        Page 533
        Page 534
        Page 535
        Page 536
    Physiological basis of fall armyworm (Lepidoptera: Noctuidae) resistance in seedlings of maize inbred lines with varying levels of silk maysin
        Page 537
        Page 538
        Page 539
        Page 540
        Page 541
        Page 542
        Page 543
        Page 544
        Page 545
    Review of fall armyworm (Lepidoptera: Noctuidae) Genetic complexity and migration
        Page 546
        Page 547
        Page 548
        Page 549
        Page 550
        Page 551
        Page 552
        Page 553
        Page 554
    Efficacy of Cry1F insecticidal protein in maize and cotton for control of fall armyworm (Lepidoptera: Noctuidae)
        Page 555
        Page 556
        Page 557
        Page 558
        Page 559
        Page 560
        Page 561
        Page 562
        Page 563
        Page 564
        Page 565
    Does secondary plant metabolism provide a mechanism for plant defenses in the tropical soda apple Solanum viarum (Solanales: Solanaceae) against Spodoptera exigua and S. eridania (lepidoptera: noctuidae)?
        Page 566
        Page 567
        Page 568
        Page 569
    Efficacy of Vip3A and Cry1AB transgenic traits in cotton against various lepidopteran pests
        Page 570
        Page 571
        Page 572
        Page 573
        Page 574
        Page 575
    Odorants of the flowers of butterfly bush, Buddleja davidii, as possible attractants of pest species of moths
        Page 576
        Page 577
        Page 578
        Page 579
        Page 580
        Page 581
        Page 582
    Greenhouse trials of Aphidius colemani (Hymenoptera: Braconidae) banker plants for control of aphids (hemiptera: aphididae) in greenhouse spring floral crops
        Page 583
        Page 584
        Page 585
        Page 586
        Page 587
        Page 588
        Page 589
        Page 590
        Page 591
    Fall armyworm (Lepidoptera: Noctuidae) resistance in Texas Bluegrass, Kentucky Bluegrass, and their hybrids (POA SPP.)
        Page 592
        Page 593
        Page 594
        Page 595
        Page 596
        Page 597
    Biology of Eurytoma sivinskii, an unusual eurytomid (hymenoptera) parasitoid of fruit fly (diptera: tephritidae) pupae
        Page 598
        Page 599
        Page 600
        Page 601
        Page 602
        Page 603
    Seismic behaviors of a leafminer, Antispila nysaefoliella (lepidoptera: heliozelidae)
        Page 604
        Page 605
        Page 606
        Page 607
        Page 608
        Page 609
    Toxicity of organosilicone adjuvants and selected pesticides to the Asian citrus psyllid (Hemiptera: Psyllidae) and its parasitoid Tamarixia radiata (Hymenoptera: Eulophidae)
        Page 610
        Page 611
        Page 612
        Page 613
        Page 614
        Page 615
        Page 616
        Page 617
        Page 618
        Page 619
        Page 620
    Comparison of parasitic hymenoptera captured in malaise traps baited with two flowering plants, Lobularia maritima (Brassicales: Brassicaceae) and Spermacoce verticillata (Gentianales: Rubiaceae)
        Page 621
        Page 622
        Page 623
        Page 624
        Page 625
        Page 626
        Page 627
    Offspring in response to parental female densities in the fruit fly parasitoid Diachasmimorpha longicaudata (Hymenoptera: Braconidae: opiinae)
        Page 628
        Page 629
        Page 630
        Page 631
        Page 632
        Page 633
        Page 634
        Page 635
    Variation of Copaeodes minima and the status of Copaeodes rayata (Lepidoptera: Hesperiidae: Hesperiinae)
        Page 636
        Page 637
        Page 638
        Page 639
        Page 640
        Page 641
        Page 642
    Effect of rainfall and soil moisture on survival of adults and immature stages of Anastrepha ludens and A. obliqua (Diptera: Tephritidae) under semi-field conditions
        Page 643
        Page 644
        Page 645
        Page 646
        Page 647
        Page 648
        Page 649
        Page 650
    Modified agar-based diet for small scale laboratory rearing of olive fruit fly, Bactrocera oleae (Diptera: Tephritidae)
        Page 651
        Page 652
        Page 653
        Page 654
        Page 655
        Page 656
    Body size relationship between Sphecius speciosus (Hymenoptera: Crabronidae) and their prey: prey size determines wasp size
        Page 657
        Page 658
        Page 659
        Page 660
        Page 661
        Page 662
        Page 663
    Pathogenicity of Beauveria bassiana (Deuteromycotina: Hyphomycetes) against the white grub Laniifera cyclades (Lepidoptera: Pyralidae) under field and greenhouse conditions
        Page 664
        Page 665
        Page 666
        Page 667
        Page 668
    Antennal sensilla of Toxotrypana curvicauda (Diptera: Tephritidae)
        Page 669
        Page 670
        Page 671
        Page 672
        Page 673
    Mortality of the lobate lac scale, Paratachardina Pseudolobata (Hemiptera: Kerriidae), at near or below Freezing temperatures
        Page 674
        Page 675
        Page 676
        Page 677
        Page 678
    Rearing Cactoblastis cactorum (Lepidoptera: Pyralidae) on a factitious meridic diet at different temperatures and larval densities
        Page 679
        Page 680
        Page 681
        Page 682
        Page 683
        Page 684
        Page 685
    Exposing entire adult holding rooms containing sterile male Mediterranean fruit flies to orange oil increases the mating success of those males in field-cage trials
        Page 686
        Page 687
        Page 688
        Page 689
    Synonymy of Phanerota appendiculata and P. insigniventris (Coleoptera: Staphylinidae: Aleocharinae), and first record of P. appendiculata in Korea
        Page 690
        Page 691
        Page 692
    Aspects of the field ecology of Stenoma catenifer (Lepidoptera: Elachistidae) infesting Hass avocados in Guatemala
        Page 693
        Page 694
    Geographic range expansion of Oxyops vitiosa (Coleoptera: Curculionidae) to the Bahamian archipelago
        Page 695
        Page 696
        Page 697
    New host, Host plants, and distribution records for Horismenus (Hymenoptera: Eulophidae) species in a bruchid beetle parasitoid guild attacking wild type Phaseolus coccineus and P. vulgaris in Central Mexico
        Page 698
        Page 699
        Page 700
        Page 701
    Removal of fungal contaminants and their DNA from the surface of Diaphorina citir (Hemiptera: Psyllidae) prior to a molecular survey of endosymbionts
        Page 702
        Page 703
        Page 704
        Page 705
    First record of Cuterebra fontinella (Diptera: Oestridae) larvae infesting a Florida rat (Rodentia: Muridae)
        Page 706
        Page 707
        Page 708
    Fruit flies (Diptera: Tephritidae) associated with umbu (Spondias tuberose) in the Semiarid region of Bahia, Brazil
        Page 709
        Page 710
    Races of Heliconius erato (Nymphalidae: Heliconiinae) found on different sides of the andes show wing size differences
        Page 711
        Page 712
    Book reviews
        Page 713
        Page 714
        Page 715
    Volume 91 author index
        Page 716
        Page 717
        Page 718
    Volume 91 subject index
        Page 719
        Page 720
        Page 721
    Back Matter
        Page 722
        Page 723
Full Text



Meagher et al.: Exotic Spodoptera Species


MONITORING FOR EXOTIC SPODOPTERA SPECIES
(LEPIDOPTERA: NOCTUIDAE) IN FLORIDA

ROBERT L. MEAGHER', JULIETA BRAMBILA2 AND EDWARD HUNG3
'USDA-ARS Center for Medical, Agricultural and Veterinary Entomology, 1700 SW 23rd Drive, Gainesville, FL 32608

2USDA-APHIS-PPQ, P.O. Box 147100, Gainesville, FL 32614-7100

3Sherwood Farms, Inc., P.O. Box 548, 13613 Honeycomb Rd., Groveland, FL 34736

ABSTRACT

Trapping studies were conducted in 2 Florida locations to determine if 3 Old World
Spodoptera Guen6e species were present. Commercially-produced lures for S. exempt
(Walker), S. littoralis (Boisduval), and S. litura (F.), plus a S. litura lure made by the USDA-
APHIS-CPHST laboratory at Otis ANGB in Massachusetts, were used with plastic Unitraps
and placed near 2 orchid nurseries in Lake and Miami-Dade counties. One S. litura male
moth was identified from collections made in Apr 2007; no other exotic species were found
in either location. However, thousands of resident species were collected, including S. albula
(Walker) (= S. sunia Guen6e), S. dolichos (F.), S. eridania (Stoll), S. exigua (Hiibner), (J. E.
Smith), and S. pulchella (Herrich-Schaffer). This study exposed the amount of labor and
level of technical knowledge needed for scientists involved in finding exotic Spodoptera spe-
cies.

Key Words: Spodoptera, pheromones, trapping, orchid nurseries, exotic species

RESUME

Se llevaron a cabo studios de monitoreo en dos localidades en Florida para detectar la pre-
sencia de tres species ex6ticas de Spodoptera del hemisferio Este. Se usaron cebos comer-
ciales para las species de S. exempta (Walker), S. littoralis (Boisduval), y S. litura (F.),
ademas de un cebo de S. litura fabricado por el laboratorio Otis ANGB de USDA-APHIS-
CPHST en Massachussetts. Se utilizaron trampas de plastico < Unitraps >, las cuales se ins-
talaron cerca de dos viveros de orquideas en los condados de Lake y Miami-Dade. Un espe-
cimen macho de S. litura fu6 identificado en un muestra de abril 2007. Ninguna otra especie
ex6tica fu6 encontrada en las dos localidades. Sin embargo, miles de especimenes nativos
fueron atrapados de las species S. albula (Walker) (= S. sunia Guen6e), S. dolichos (F.), S.
eridania (Stoll), S. exigua (Hiibner), S. frugiperda (J. E. Smith), y S. pulchella (Herrich-
Schaffer). Este proyecto expuso la gran cantidad de trabajo requerido y el nivel de conoci-
miento t6cnico necesario de los investigadores para detectar species ex6ticas de
Spodoptera.


The armyworm genus Spodoptera Guenee
(Noctuidae: Amphipyrinae) contains 30 species
that inhabit 6 continents (Pogue 2002). Species in
temperate North America include several agricul-
tural pests such as S. eridania (Stoll) (southern
armyworm, Liburd et al. 2000), S. exigua (Hib-
ner) (beet armyworm, Chen et al. 2008), S. fru-
giperda (J. E. Smith) (fall armyworm, Nagoshi &
Meagher 2004), S. latifascia (Walker) (Vergara &
Pitre 2001), S. ornithogalli (Guenee) (yellow-
striped armyworm, Liburd et al. 2000), and S.
praefica (Grote) (western yellowstriped army-
worm, Summers 1989). These species feed on a
wide range of grain, row, forage, vegetable, and
ornamental crops. Four other species, S. albula
(Walker) (= S. sunia Guenee), S. androgea (Stoll),
S. dolichos (F.), and S. pulchella (Herrich-
Schaffer) are present in the USA and in Neotropi-
cal regions of the Western Hemisphere (Pogue


2002). The beet armyworm is the only cosmopoli-
tan species in the genus (Pogue 2002), and is prob-
ably Palaearctic in origin. The western yellow-
striped armyworm is restricted to the western
United States in distribution (Pogue 2002).
The Eastern Hemisphere contains 3 species
that are serious agricultural pests. Spodoptera ex-
empta (Walker), the nutgrass armyworm or Afri-
can armyworm, is a migratory pest of cereal and
pasture grasses (Cheke & Tucker 1995) that in-
habits southern Europe, the Ethiopian Region,
the Australasian Region, and various South Pa-
cific islands including Hawaii (Pogue 2002).
Spodoptera littoralis (Boisduval), the Egyptian
cotton leafworm, is a pest of cotton (Amin & Ger-
gis 2006) and other agricultural crops. It ranges
from southern Europe and Africa through the
Middle East and western Asia and several islands
in the Indian Ocean. Spodoptera litura (F.) (to-







Florida Entomologist 91(4)


bacco cutworm, cluster caterpillar, and rice cut-
worm, among other common names) attacks a
wide range of crops including cotton, peanuts
groundnutss), rice, soybeans, vegetables, and or-
namental plants (EPPO/CABI 1997). This species
also has a wide geographical range, inhabiting
western Asia eastward through eastern Asia and
southward to the Australasian Region (Pogue
2002). It is also found throughout the Pacific in-
cluding Hawaii.
Florida is second in the USA in the value of
nursery and greenhouse crops, reaching $1.63 bil-
lion in 2004 (Jerardo 2005). Orchids are the sec-
ond leading potted flowering plant produced in
Florida. Much of the production in Florida in-
volves importing small plants, growing them in
shade houses for 6-12 mo. and then shipping them
to markets in the USA and Canada. Many of the
hundreds of thousands of small plants are im-
ported from locations in Asia (Thailand, Taiwan,
and China) where S. litura is known as a pest.
Pheromone-baited traps provide a technique
to monitor relatively large areas for the presence
of moth species. Pheromone components or blends
have been identified for many of the species cur-
rently in the USA, including S. albula (Bestmann
et al. 1988-published as S. sunia Guenee), S.
dolichos (Lalanne-Cassou et al. 1994), S. eridania
(Mitchell & Tumlinson 1994; Teal et al. 1985), S.
exigua (Deng et al. 2004; Jung et al. 2003; Mitch-
ell & Tumlinson 1994), S. frugiperda (Tumlinson
et al. 1986; Batista-Pereira et al. 2006), S. latifas-
cia (Monti et al. 1995) and S. praefica (Landolt et
al. 2003). Pheromone blends have been synthe-


sized and are commercially available for S. erida-
nia, S. exigua, S. frugiperda, and S. praefica.
Pheromone components and blends have been
identified and are commercially-available for 3 of
the agriculturally-important exotic Spodoptera
species, S. exempta (Cork et al. 1989), S. littoralis
(Dunkelblum et al. 1987; Malo et al. 2000), and S.
litura (Sun et al. 2003; Wei et al. 2004). Many of
the components that make up the blends are sim-
ilar across species (Table 1), but are released in
different ratios. Our objective for this research
was to monitor for exotic Spodoptera species with
pheromone-baited traps placed near orchid nurs-
eries. A secondary objective was to determine
which native species would be attracted to these
lures so that identification of moths could be
made.

MATERIALS AND METHODS

Groveland

The field site was located outside of Groveland,
FL (Lake County) and was composed of shade
houses (52,000 m2) surrounded by mixed natural
vegetation and small home sites. The shade
houses contained over 2 million plants, including
Cymbidium, Dendrobium, and Phalenopsis or-
chids. The orchids are imported from Asia as
young plants and grown for 6-12 months. They
are then shipped to retail stores across the U.S.
and Canada.
Pheromone-baited standard Universal Moth
Traps, "Unitraps" (Great Lakes IPM, Vestaburg,


TABLE 1. PHEROMONE COMPONENTS FOR SELECTED SPODOPTERA SPECIES.

Species Components Reference

albula Z9-14:Ac, Z9-14:OH, Z11-16:Ac, Z9,E12-14:Ac Bestmann et al. 1988
dolichos Z9-14:Ac, Z9,E12-14:Ac Lalanne-Cassou et al. 1994
eridania Z9-14:Ac, Z9,E12-14:Ac, Zll-16:A,Z9,E11-14:Ac Teal et al. 1985; Mitchell & Tumlinson 1994
exempta Z9-14:Ac, Z9,E12-14:Ac, Z11-16:Ac Cork et al. 1989
exigua Z9,E12-14:Ac, Z11-16:Ac, Z9-14:OH Mitchell & Tumlinson 1994; Jung et al. 2003;
Z9,E12-14:Ac, Z9-14:OH Deng et al. 2004
Z9,E12-14:Ac, Z9-14:OH
frugiperda Z9-14:Ac, Z7-12:Ac Tumlinson et al. 1986; Batista-Pereira et al.
Z9-14:Ac, Z7-12:Ac, E7-12:Ac 2006
latifascia Z9-14:Ac, Z9,E12-14:Ac Monti et al. 1995
littoralis Z9,Ell-14:Ac Dunkelblum et al. 1987; Malo et al. 2000
Z9,E11-14:Ac, Z9-14:Ac, E 11-14:Ac
14:Ac, Z11-14:Ac
litura Z9,E11-14:Ac, Z9,E12-14:Ac Sun et al. 2003; Wei et al. 2004
Z9,E11-14:Ac, E11-14:Ac, Z9,E12-14:Ac
Z9-14:Ac

14:Ac = tetradecyl acetate; E7-12:Ac = (E)-7-dodecenyl acetate; Z7-12:Ac = (Z)-7-dodecenyl acetate; Z9-14:OH = (Z)-9-tetradecen-
1-ol; Z9-14:Ac = (Z)-9-tetradecenyl acetate; E11-14:Ac = (E)-11-tetradecenyl acetate; Z11-14:Ac = (Z)-11-tetradecenyl acetate; Z1-
16:Ac = (Z)-11-hexadecenyl acetate; Z9,E11-14:Ac = (Z,E)-9,11-tetradecadienyl acetate; Z9,E12-14:Ac = (Z,E)-9,12-tetradecadienyl
acetate.


December 2008







Meagher et al.: Exotic Spodoptera Species


MI) were used to capture attracted male moths.
These traps are constructed of a white bucket, a
yellow cone on top of the bucket, and a dark green
cover above the cone. Scenturion pheromone
lures (Suterra LLC, Bend, OR) for each species (S.
eridania, S. exempta, S. exigua, S. frugiperda, S.
littoralis, S. litura, and S. praefica) were attached
to corks (#11) with a pin, and the cork was placed
in the middle of the green cover. An additional
lure for S. litura was provided by the USDA-
APHIS CPHST laboratory at Otis ANGB, Cape
Cod, MA. A 2.5 x 2.5 cm piece of Vaportape (Her-
con Environmental, Emigsville, PA) releasing the
pesticide dichlorvos was stapled to a string and
hung inside the bucket to kill captured moths.
Two rows of traps were placed outside of the
shade houses on 1.5-m metal poles at least 30 m
apart, and species order was randomized at each
sample date. Traps were placed 25 Jan 2006 and
samples were collected either biweekly or
monthly until 2 Oct 2006. Pheromone lures and
vaportape were replaced when traps were ser-
viced. It was soon noticed that large numbers of
honey bees were collected in the traps due to a
small apiary less than 1 km east of the site.
Therefore, green buckets were used on one row of
traps closest to the apiary.

Homestead

This site was located southwest of Homestead,
FL (Miami-Dade County) in a mixed agricultural
and urban environment, and contained 26,000 m2
of greenhouse space holding Dendrobium, and
Phalenopsis orchids. One row of pheromone-
baited traps for the same species as Groveland
were placed on the south side of the complex on 24
Jan 2007 and removed 31 Oct 2007. Traps were
serviced every 2 weeks and lures and vaportape
were replaced monthly.


traps only attracted one beet armyworm moth,
the S. eridania-baited traps attracted 13 southern
armyworm moths and 1 S. dolichos moth, and the
S. frugiperda-baited traps attracted 6 fall army-
worm moths. The S. praefica-baited traps did not
attract any Spodoptera species but did attract 2
Plusiinae moths, soybean looper Ci., b, ../. i in-
cludens (Walker) (10) and sharp-stigma looper
Ctenoplusia oxygramma (Geyer) (6).
The lures for the exotic species were much
more active in attracting moths. The S. exempta-
baited traps attracted large numbers of S. albula
(183) and S. latifascia (863), and both species
were present throughout the trapping duration
(Fig. 1). The 2 S. litura-baited traps caught much
fewer moths. The Suterra blend only attracted 1
beet armyworm; the Otis ANGB blend only at-
tracted 3 S. dolichos. Surprisingly, the S. littora-
lis-baited traps did not attract any moths for the
entire season.

Homestead

Many more moths were collected at the Home-
stead location. The beet armyworm and fall army-
worm lures attracted almost exclusively the tar-
get species, with 257 and 682 moths captured, re-
spectively (Fig. 2). The southern armyworm lure
attracted the target species (165), but attracted
more nontarget Spodoptera species including S.
dolichos (237) and S. pulchella (12) (Fig. 3). As at
Groveland, the S. praefica lures did not attract
any Spodoptera species but did attract soybean
loopers (194).
Collections of moths on Apr 20 revealed 1 S. lit-
ura moth in the Suterra blend of the S. litura
pheromone. This was the only exotic moth col-
lected for either location. Otherwise, these traps
only attracted 9 S. dolichos and 2 S. pulchella.


Moth Species Identification is
Groveland
S.exempta lure
Spodoptera species moths were identified with Sxetare
the keys and descriptions of Pogue (2002). The ab- 'a
domen of all specimens was softened and cleared
with hot 10% KOH. Genitalia were extracted with a ,,
fine tweezers and various characters were exam-- s \
ined under a dissecting microscope. The primary
characters used for species identification were, \ 6
not in order of importance, (1) number of core-
mata lobes and the shape of (2) clavus, (3) saccu- 3
lus, (4) costal process, (5) basal sclerite of clasper,
and (6) juxta.

RESULTS ai
2006
Groveland Fig. 1. Numbers of Spodoptera albula (Walker) and S.
latifascia (Walker) adult males collected per night in Uni-
Pheromone lures for the 3 native species traps baited with a lure comprised of the sex pheromone
caught relatively few moths. The S. exigua-baited blend of S. exempta (Walker), Groveland, FL, 2006.







Florida Entomologist 91(4)


2007

Fig. 2. Numbers of Spodoptera exigua (Htibner) and
S. frugiperda (J. E. Smith) adult males collected per
night in Unitraps baited with lures comprised of the sex
pheromone blends for each species, respectively, Home-
stead, FL, 2007.


The Otis-ANGB blend attracted 149 S. dolichos
and 5 S. pulchella (Fig. 4). The S. exempta baited-
traps attracted thousands of native Spodoptera
moths, including S. albula (1896), S. latifascia
(1729) and S. pulchella (28). During May and Jun,
over 15 S. albula moths per night were collected,
whereas the final sampling date resulted in 30 S.
latifascia moths per night (Fig. 5). As at Grove-
land, the S. littoralis-baited traps captured no
Spodoptera species.

DISCUSSION

The main objective of this work was to deter-
mine if any of the 3 exotic Spodoptera species


4
Homestead
S. eidania lure


-- S. mini.
2 S f*chm

a SH rile 8
r A.AA\


20107
Fig. 3. Numbers of Spodoptera eridania (Stoll), S.
dolichos (F.), and S. pulchella (Herrich-Schaffer) adult
males collected per night in Unitraps baited with a lure
comprised of the sex pheromone blend of S. eridania,
Homestead, FL, 2007.


.i ktL J 17
2007
Fig. 4. Numbers of Spodoptera dolichos (F.) and S.
pulchella (Herrich-Schaffer) adult males collected per
night in Unitraps baited with a lure comprised of the
sex pheromone blend of S. litura (F.) (Otis ANGB septa),
Homestead, FL, 2007.


were present. Although 1 S. litura male was
found, subsequent intensive surveys with both
pheromone lures have failed to collect moths (J.
B., unpublished data). Tests are currently being
conducted in India to determine the attractive-
ness of both blends in areas of natural S. litura
populations. The 2 blends attracted different
numbers of the native species S. dolichos. This ap-
parently common species has (Z)9,(E)12-14:Ac
and (Z)9-14:Ac as pheromone components (La-
lanne-Cassou et al. 1994) and appears to be at-
tracted to the 2 double-bonded acetate component
that is in the Otis-ANGB blend.
It was somewhat surprising that the S. lit-
toralis lures did not attract any Spodoptera



Homestead
S. exempts lure
24


2007
Fig. 5. Numbers of Spodoptera albula (Walker), S.
latifascia (Walker), and S. pulchella (Herrich-Schaffer)
adult males collected per night in Unitraps baited with
a lure comprised of the sex pheromone blend of S. ex-
empta (Walker), Homestead, FL, 2007.


December 2008







Meagher et al.: Exotic Spodoptera Species


moths, although the major component of the
blend, (Z)9,(E)11-14:Ac (Dunkelblum et al. 1987;
Malo et al. 2000), is not a major component for
any of the native Spodoptera species. Previous
tests in the Madeira Islands, Portugal, showed
that these lures were effective in attracting S.
littoralis moths (R. Pereira and J. B., unpub-
lished data).
On the other hand, the large number of S. al-
bula and S. latifascia moths attracted to the S. ex-
empta lures agrees with the published reports of
the pheromone blends for these species. The ma-
jor component is (Z)9-14:Ac, with lesser amounts
of(Z)9,(E)12-14:Ac and (Z)11-16:Ac (Bestmann et
al. 1988; Cork et al. 1989; Monti et al. 1995). Com-
mercial lures for S. albula and S. latifascia are
not available, but it appears the lure for S. ex-
empta could be used for these species.
Lures for the 3 Florida pest species, S. erida-
nia, S. exigua, and S. frugiperda attracted the tar-
get moths. The S. eridania lures, however, at-
tracted higher numbers of S. dolichos than the
target species. Both species have (Z)9-14:Ac and
(Z)9,(E)12-14:Ac as part of their pheromone
blends (Lalanne-Cassou et al. 1994; Mitchell &
Tumlinson 1994; Teal et al. 1985). The component
(Z)9,(E)12-14:Ac is known to attract S. dolichos
males (Mitchell & Tumlinson 1973). Cross attrac-
tion between S. eridania and S. exigua, which has
been documented in other studies (Mitchell &
Doolittle 1976; Mitchell & Tumlinson 1994), was
rarely found in our study.
Because several native species were attracted
to the blends designed for the 3 exotic species,
moth identification will be critical to determining
whether S. exempta, S. littoralis, or S. litura is in-
tercepted or becomes established in Florida. Un-
less moths are retrieved quickly, noctuids cap-
tured in bucket traps over a period of several
nights can be worn and difficult to identify based
on forewing characters. Therefore, time-consum-
ing identification with characters of the genitalia
will need to be made. It is hoped that more ento-
mologists can learn these characters and tech-
niques to help identify this important group of
pests.


ACKNOWLEDGMENTS

We thank W. Montgomery (USDA-ARS-SHRS, Mi-
ami, FL) for moth collections and trap maintenance at
the Homestead site. We thank M. G. Pogue (USDA-ARS-
SEL, Washington, D.C.) for verification of the S. litura
specimen. We thank John Adamczyk (Weslaco, TX) and
Rod Nagoshi (Gainesville, FL) for critical review of an
early manuscript. The use of trade, firm, or corporation
names in this publication is for the information and con-
venience of the reader. Such use does not constitute an
official endorsement or approval by the United States
Department of Agriculture or the Agricultural Research
Service of any product or service to the exclusion of oth-
ers that may be suitable.


REFERENCES CITED

AMIN, A. A., AND M. F. GERGIS. 2006. Integrated man-
agement strategies for control of cotton key pests in
middle Egypt. Agron. Res. 4: 121-128.
BATISTA-PEREIRA, L. G., K. STEIN, A. F. DE PAULA, J. A.
MOREIRA, I. CRUZ, M. D. L. C. FIGUEIREDO, J. PERRI,
JR., AND A. G. CORREA. 2006. Isolation, identifica-
tion, synthesis, and field evaluation of the sex pher-
omone of the Brazilian population of Spodoptera fru-
giperda. J. Chem. Ecol. 32: 1085-1099.
BESTMANN, H. J., A. B. ATTYGALLE, J. SCHWARZ, O.
VOSTROWSKY AND W. KNAUF. 1988. Identification of
sex pheromone components of Spodoptera sunia
Guen6e (Lepidoptera: Noctuidae). J. Chem. Ecol. 14:
683-690.
CHEKE, R. A., AND M. R. TUCKER. 1995. An evaluation of
potential economic returns from the strategic control
approach to the management of African armyworm
Spodoptera exempta (Lepidoptera, Noctuidae) popu-
lations in eastern Africa. Crop Prot. 14: 91-103.
CHEN, Y. G., J. R. RUBERSON, AND D. M. OLSON. 2008.
Nitrogen fertilization rate affects feeding, larval per-
formance, and oviposition preference of the beet ar-
myworm, Spodoptera exigua, on cotton. Entomol.
Exp. Appl. 126: 244-255.
CORK, A., J. MURLIS, AND T. MEGENASA. 1989. Identifi-
cation and field testing of additional components of
female sex pheromone of African armyworm,
Spodoptera exempta (Lepidoptera: Noctuidae). J.
Chem. Ecol. 15: 1349-1364.
DENG, J. Y., H. Y. WEI, Y. P. HUANG, AND J. W. DU.
2004. Enhancement of attraction to sex pheromones
of Spodoptera exigua by volatile compounds pro-
duced by host plants. J. Chem. Ecol. 30: 2037-2045.
DUNKELBLUM, E., M. KEHAT, M. HAREL, AND D. GOR-
DON. 1987. Sexual behaviour and pheromone titre of
the Spodoptera littoralis female moth. Entomol.
Exp. Appl. 44: 241-247.
EPPO/CABI. 1997. Quarantine Pests for Europe. 2nd
edition. Edited by I. M. Smith, D. G. McNamara, P.
R. Scott, and M. Holderness, CABI International,
Wallingford, UK, 1425 pp.
JERARDO, A. 2005. Floriculture and nursery crops situ-
ation and outlook yearbook. USDA Econ. Res. Serv.
FLO-2005.
JUNG, C. R., Y. J. PARK, AND K. S. Boo. 2003. Optimal
sex pheromone composition for monitoring
Spodoptera exigua (Lepidoptera: Noctuidae) in Ko-
rea. J. Asia-Pacific Entomol. 6: 175-182.
LALANNE-CASSOU, B., J. F. SIVAIN, L. MONTI, AND
C. MALOSSE. 1994. Description d'une nouvelle es-
pece de Spodoptera de Guyane francaise: S. descoin-
si (Lepidoptera: Noctuidae: Amphipyrinae), d6cou-
verte grace a des attractifs sexuels. Am. Soc.
Entomol. Fr. 20: 25-32.
LANDOLT, P. J., C. SMITHHISIER, T. ADAMS, AND R. S.
ZACK. 2003. An improved multi-component sex at-
tractant for trapping male western yellowstriped ar-
myworm, Spodoptera praefica (Grote) (Lepidoptera:
Noctuidae). Agric. Forest Entomol. 5: 333-339.
LIBURD, O. E., J. E. FUNDERBURK, AND S. M. OLSON.
2000. Effect of biological and chemical insecticides
on Spodoptera species (Lep., Noctuidae) and market-
able yields of tomatoes. J. Agric. Entomol. 124: 19-
25.
MALO, E. A., M. RENOU, AND A. GUERRERO. 2000. Ana-
lytical studies of Spodoptera littoralis sex phero-











mone components by electroantennography and gas
chromatography-electroantennographic detection.
Talanta 52: 525-532.
MITCHELL, E. R., AND R. E. DOOLITTLE. 1976. Sex pher-
omones of Spodoptera exigua, S. eridania, and S. fru-
giperda: bioassay for field activity. J. Econ. Entomol.
69: 324-326.
MITCHELL, E. R., AND J. H. TUMLINSON. 1973. An attrac-
tant for males of Spodoptera dolichos (Lepidoptera:
Noctuidae). Ann. Entomol. Soc. America 66: 917-918.
MITCHELL, E. R., AND J. H. TUMLINSON. 1994. Response
of Spodoptera exigua and S. eridania (Lepidoptera:
Noctuidae) males to synthetic pheromone and S. ex-
igua females. Florida Entomol. 77: 237-247.
MONTI, L., B. LALANNE-CASSOU, P. LUCAS, C. MALOSSE,
AND J.-F. SILVAN. 1995. Differences in sex phero-
mone communication systems of closely related spe-
cies: Spodoptera latifascia (Walker) and S. descoinsi
Lalanne-Cassou & Silvain (Lepidoptera: Noctuidae).
J. Chem. Ecol. 21: 641-660.
NAGOSHI, R. N., AND R. L. MEAGHER. 2004. Behavior
and distribution of the two fall armyworm host
strains in Florida. Florida Entomol. 87: 440-449.
POGUE, M. G. 2002. A world revision of the genus
Spodoptera Guen6e (Lepidoptera: Noctuidae). Mem.
American Entomol. Soc. 43: 1-202.
SUMMERS, C. G. 1989. Effect of selected pests and mul-
tiple pest complexes on alfalfa productivity and
stand persistence. J. Econ. Entomol. 82: 1782-1791.


December 2008


SUN, F., J. W. DU, AND T. H. CHEN. 2003. The behavioral
responses of S. litura (F.) males to the female sex
pheromone in wind tunnel and field trapping tests.
Acta Entomol. Sinica 46: 126-130.
TEAL, P. E. A., E. R. MITCHELL, J. H. TUMLINSON, R. R.
HEATH, AND H. SUGIE. 1985. Identification of volatile
sex pheromone components released by the southern
armyworm, Spodoptera eridania (Cramer). J. Chem.
Ecol. 11: 717-725.
TUMLINSON, J. H., E. R. MITCHELL, AND H. S. YU. 1990.
Analysis and field evaluation of volatile blend emit-
ted by calling virgin females of beet armyworm,
Spodoptera exigua (Htibner). J. Chem. Ecol. 16:
3411-3423.
TUMLINSON, J. H., E. R. MITCHELL, P. E. A. TEAL, R. R.
HEATH, AND L. J. MENGELKOCH. 1986. Sex phero-
mone of fall armyworm, Spodoptera frugiperda (J.
E. Smith) identification of components critical to
attraction in the field. J. Chem. Ecol. 12: 1909-
1926.
VERGARA, O. R., AND H. N. PITRE. 2001. Planting date,
weed management, and insecticide application for
control of lepidopterous pests in intercropped sor-
ghum and maize in southern Honduras. Tropical Ag-
ric. 78: 182-189.
WEI, H., Y. HUANG, AND J. DU. 2004. Sex pheromones
and reproduction behavior of Spodoptera litura
(Fabricius) moths reared from larvae treated with
four insecticides. J. Chem. Ecol. 30: 1457-1466.


Florida Entomologist 91(4)







Buntin: Bt Corn for Grain Production


CORN EXPRESSING CRYIAB OR CRY1F ENDOTOXIN FOR FALL
ARMYWORM AND CORN EARWORM (LEPIDOPTERA: NOCTUIDAE)
MANAGEMENT IN FIELD CORN FOR GRAIN PRODUCTION

G. DAVID BUNTIN
Department of Entomology, University of Georgia-Griffin Campus, 1109 Experiment Street, Griffin, GA 30223


ABSTRACT

Fall armyworm, Spodoptera frugiperda (J. E. Smith), and corn earworm, Helicoverpa zea
(Boddie), perennially cause leaf and ear damage to corn in the southeastern United States.
Transgenic hybrids expressing the CrylAb (MON810 event) or Cry 1F (TC1507 event) in-
secticidal endotoxin from Bacillus thuringiensis (Bt) were evaluated for management of fall
armyworm and corn earworm in central Georgia during 2006 and 2007. Corn was planted
at the recommended time in mid-Apr and in late Jun to simulate a double-crop corn plant-
ing. Both Bt events reduced whorl infestation and damage by fall armyworm, but TC1507
provided greater protection from whorl injury than MON 810 under severe fall armyworm
infestations. Hybrids with the MON810 event usually had less ear infestation by corn ear-
worm than susceptible hybrids, whereas the TC1507 event usually did not reduce ear infes-
tations. Nevertheless, both events prevented ear damage, but there was no consistent
difference between the two Bt traits in preventing ear damage. Bt traits did not affect grain
yield in either year during the first planting when fall armyworm infestations were low.
Both events prevented significant yield loss during the second planting in 2006 when whorl
infestation levels exceeded 50% in susceptible hybrids. Because of the greater activity in pre-
venting whorl damage by fall armyworm, the TC1507 event would be useful in mitigating
the risk of severe lepidopteran damage to later plantings of field corn for grain production
in the southeastern U.S.

Key Words: plant resistance, Spodoptera frugiperda, Helicoverpa zea, transgenic crops,
CrylF, CrylAb, Bt traits

RESUME

El gusano cogollero, Spodoptera frugiperda (J. E. Smith) y el gusano de elote de maiz, Heli-
coverpa zea (Boddie), perennemente causan daio a las hojas y mazorcas de maiz en el su-
reste de los Estados Unidos. Los hibridos transg6nicos que expresan las endotoxinas CrylAb
evento MON810) o Cry 1F evento TC1507) de Bacillus thuringiensis (Bt) con propiedades
de insecticides fueron evaluados para el manejo del gusano cogollero y el gusano del elote de
maiz en la region central del estado de Georgia durante los aios 2006 y 2007. El maiz fue
sembrado al tiempo recomendado en el medio de abril y en la ultima parte dejunio para es-
timular la siembra de un double cultivo de maiz. Ambos events de Bt reducieron la infesta-
ci6n y daio hecho por el gusano cogollero en el cogollo, pero el TC1507 provey6 una
protecci6n mejor al cogollo que MON810 bajo durante infestaciones several del gusano co-
gollero. Los elotes de los hibridos con el event MON810 usualmente tenian un nivel menor
de infestaci6n del gusano de elote que en los hibridos susceptibles, mientras que el event de
TC1507 usualmente redujo las infestaciones del elote. No obstante, ambos events previnie-
ron daio a los elotes, pero no hubo suficientes diferencias entire las caracteristicas de Bt en
cuanto de la prevenci6n del daio al elote. Las caracteristicas de Bt no afectaron el rendi-
miento del grano en ninguno de los dos aios durante la primera siembra cuando las infes-
taciones del gusano cogollero fueron bajas. Ambos events previnieron una p6rdida
significativa en el rendimiento durante la segunda siembra en 2006 cuando el nivel de infes-
taci6n en los cogollos fue mas de 50% en los hibridos susceptibles. Por causa de la mayor ac-
tividad en prevenir el daio al cogollo hecho por el gusano cogollero, el event TC1507 seria
mas util en mitigar el riesgo de daio severe hecho por los lepid6pteros a siembras posterio-
res de maiz del campo para la producci6n de granos en el sureste de los Estados Unidos.


Fall armyworm, Spodoptera frugiperda (J. E. ing direct loss of grain, but fall armyworm also
Smith), and corn earworm, Helicoverpa zea (Bod- may infest ears especially during large infesta-
die), are the most important lepidopteran pests of tions. Insecticidal control to prevent ear damage
corn in the southeastern United States. Fall ar- in field corn is difficult and generally not cost ef-
myworm often infests whorl stage plants causing fective. Typically, early planting times are recom-
leaf injury. Corn earworm often infests ears caus- mended in the Southeast partly to avoid damag-







Florida Entomologist 91(4)


ing levels of both insects, which often occur later
in the season (Buntin 2007). Transgenic corn hy-
brids expressing the insecticidal Cry protein from
Bacillus thuringiensis (Bt) has been available in
the Southeast since 1998, and this technology of-
fers the potential for reducing losses by fall army-
worm and corn earworm in field corn (Buntin et
al. 2001, 2004).
Several events of transgenic Bt corn have been
developed with different modes of toxin expres-
sion (Ostlie et al. 1997). The MON810 event
(Monsanto Co., St. Louis, MO) and a similar event
Btll (Syngenta Crop Sciences, RTP, NC) contain
the CrylAb gene. These events are marketed as
YieldGard corn borer (YGCB) corn, and express
endotoxin in vegetative and reproductive struc-
tures throughout the season (Armstrong et al.
1995; Williams et al. 1997). More recently, a new
transformation event TC1507 expressing a Bt-de-
rived insecticidal protein CrylF is being mar-
keted as Herculex I Insect Protection (HX) (Dow
AgroSciences, Indianapolis, IN). Both MON810
and TC1507 events are very effective against the
European corn borer, Ostrinia nubilalis (Hiibner),
and southwestern corn borer, Diatraea grandi-
osella Dyar (Williams et al. 1998; Graeber et al.
1999; Lauer & Wedberg 1999; Archer et al. 2000;
Abel & Pollan 2004; Allen & Pitre 2006; Siebert et
al. 2008). European corn borer occurs throughout
most of Georgia but usually is not an important
pest of field corn. Southwestern corn borer does
not occur in the coastal plain region of the south-
eastern United States but is present in north-
western Georgia and northern Alabama where it
can cause economic damage (Buntin 2007).
Laboratory feeding trials and small controlled
field trials have shown that hybrids containing
CrylAb endotoxin reduced fall armyworm and
corn earworm growth and survival (Williams et
al. 1997, 1998; Bokonon-Ganta et al. 2003; Abel &
Pollan 2004). However, fall armyworm is less sus-
ceptible to CrylAb endotoxins than southwestern
corn borer (Williams et al. 1977; Abel & Pollan
2004). Storer et al. (2001) showed that corn con-
taining CrylAb (Btll) stunted growth of H. zea
larvae, reduced H. zea adult emergence from Bt-
corn fields by about 75% and delayed adult emer-
gence by 6-12 d. Furthermore, kernel damage was
reduced an average of 80% in Bt hybrids. Buntin
et al. (2001, 2004) conducted a series of trials in
1998-2000 in Georgia with corn planted at the
recommended time and one and two months later,
which showed that MON810 and Btll events pre-
vented whorl damage, kernel damage, and yield
loss by lepidopterans, primarily fall armyworm
and corn earworm, in later plantings at all loca-
tions. Bt traits generally did not improve the per-
formance of corn planted at recommended times
(March and April depending on location), because
these plantings mostly escaped severe lepi-
dopteran damage. However, Bt traits prevented


yield loss of 50% or more in some later plantings.
Nevertheless, field observations indicate that hy-
brids containing the MON810/Btll events can
still suffer substantial whorl damage by fall ar-
myworm when serve infestations occur (GDB,
personal observation). More recently, Siebert et
al. (2008) showed that CrylF provided a high
level of protection in corn against fall armyworm
leaf feeding and whorl damage. Adapted field corn
hybrids expressing CrylF have only become
available in the last few years in the southeastern
United States.
The study objective was to evaluate corn ex-
pressing either CrylAb or CrylF endotoxins for
protection against fall armyworm and corn ear-
worm infestations and damage in field corn. The
effect on corn grain yield also was measured.
Comparisons were done during 2 planting times,
a recommended time in Apr and a late-Jun plant-
ing to simulate a double-crop planting of corn.

MATERIALS AND METHODS

Trials were conducted in 2006 and 2007 at the
University of Georgia Bledsoe Research farm lo-
cated near Williamson (Pike Co.), GA. Soil was an
Appling sandy loam, and tillage was conventional
with chisel plowing followed by disk harrowing.
Before disking 440 kg/ha of 3-18-9 (N-P-K) granu-
lar fertilizer was applied and an additional 112 kg
of nitrogen as ammonium nitrate was side-dress
applied about 20 d after planting. Seed was
planted with a Monosem air-planter at the rate
of 66,700 plants per ha with 76-cm row spacing.
Pendimethalin (Prowl 3.3 EC, BASF, Research
Triangle Park, NC) at 0.71 L/ha and atrazine
(Aatrex 4L, Syngenta Crop Protection, Greens-
boro, NC) at 0.57 L/ha were applied to control
weeds. Seed of all hybrids were treated with
clothianidin seed treatment at 0.25 mg ai per ker-
nel (Poncho 250, Bayer CropSciences, Research
Triangle Park, NC). No other pesticides were ap-
plied. Natural rainfall was supplemented by irri-
gating weekly with 6 cm of water as needed.
The experimental design of all trials was a ran-
domized complete block design of hybrids with 4
replications. In 2006, plots were 4 rows and 10 m
long and in 2007 plots were 8 rows and 10 m long.
Early and late planting trials were conducted in
both years. The first planting at the recommend
time in late Apr (24 Apr 2006 and 30 Apr 2007)
and the second planting was in late Jun (23 Jun
2006 and 26 Jun 2007) to simulate a double-crop
corn planting.
Adapted non-Bt hybrids were compared with
near-isogenic hybrids containing either the trans-
formation event MON810, which expresses the
CrylAb toxin, or the transformation event
TC1507, which expresses the CrylF toxin. Hy-
brids pairs in both years were Dekalb DKC 69-72
(non-Bt) and DKC 69-71 (MON810) (Dekalb


December 2008







Buntin: Bt Corn for Grain Production


TABLE 1. EFFECT OF BT EVENTS ON PERCENTAGE OF FALL ARMYWORM INFESTED WHORLS IN THE SECOND CORN
PLANTING IN 2006 AND 2007.

2006 2007

Brand/Hybrid Bt event 6-leaf 10-leaf 6-leaf 10-leaf

DKC 6972 -57.8 + 3.6 a 60.5 3.9 a 13.1 + 2.2 a 18.9 3.6 a
DKC 6971 MON810 15.2 6.6 cd 37.3 4.3 b 0.3 0.3 b 1.5+ 1.1 bc
Pioneer 31G66 -54.0 4.9 a 63.3 3.0 a 16.7 5.7 a 23.5 3.5 a
Pioneer 31G68 MON810 18.8 10.1 c 39.3 7.2 b 2.2 2.2 b 3.2 2.4 b
Pioneer 31G97 -50.5 6.6 ab 66.0 5.0 a 18.1+ 4.1 a 24.2 4.2 a
Pioneer 31G96 TC1507 10.0 6.0 cd 4.8 2.4 c 0.4 0.4 b 0.0 0.0 c
Pioneer 31N27 -44.3 6.0 ab 62.3 3.6 a 16.6 2.7 a 23.0 4.4 a
Pioneer 31N28 MON810 10.0 1.6 cd 35.5 2.4 b 1.2 1.0 b 2.6 1.8 b
Pioneer 34B97 -34.5 4.5 b 63.1 2.8 a -
Pioneer 34B98 MON810 18.0 5.0 c 36.8 3.5 b -
Pioneer 34B99 TC1507 5.3 2.4 d 8.8 2.6 c -
LSD, 005
Combined -48.2 13.5 x 63.0 6.9 x 19.0 2.6 x 26.5 4.0 x
Combined MON810 15.5 12.3 y 37.2 9.9 y 1.2 1.2 y 2.4 3.3 y
Combined TC1507 7.6 8.8 z 6.8 5.1 z 0.4 0.8 y 0.0 0.0 y
Source of variation2
Hybrid 14.22*** 28.86*** 16.62*** 26.37***
Non-Bt v. MON810 72.80*** 57.50*** 77.95*** 145.25***
Non-Btv. TC1507 51.58*** 205.46*** 36.97*** 61.66***
MON810 v. TC1507 5.29* 95.46*** 0.24ns 4.98*

Means within columns followed by the same lower case letter among hybrids or combined are not significantly different (LSD;
P = 0.05).
*,**, *** indicate significant contrast F value at P < 0.05, P < 0.01, and P < 0.001 respectively; ns, not significant.
2006: Hybrid df= 10, 30; contrast df= 1, 15; 2007: Hybrid df= 7, 21; contrast df = 1, 21.


Seeds, Monsanto Comp., St Louis, MO), Pioneer
Brand 31G66 (non-Bt) and 31G68 (MON810), Pi-
oneer Brand 31N27 (non-Bt) and 31N28
(MON810), Pioneer Brand 31G97 (non-Bt) and
31G96 (TC1507) (Pioneer Hi-bred International,
Des Moines, IA). In 2006, Pioneer Brand 34B97
(non-Bt), 34B98 (MON810), and 34B99 (TC1507)
also were included.
In all trials, stand counts of all rows were made
about 21 d after planting. Whorl defoliation was
assessed by rating all plants in 2 rows per plot at
the 6-leaf stage in each planting date. A second
rating was made in the Jun plantings at about the
10-leaf stage. Plants were rated for damage on a
0-9 scale where 0 is no damage and 9 is whorl and
furl almost completely defoliated (Davis et al.
1992). The damage scale is not linear with ratings
of >4 indicating substantially more damage than
ratings of <3. Twenty to 30 larvae were collected
for species identification from infested whorls in
border rows at the edge of plots. Ear damage was
measured on 20 ears per plot about 2 wk after
green-silk stage after nearly all larvae had exited
the ears in the non-Bt hybrids. Ear damage was
rated in both years by the Widstrom (1967) sys-


tem where 1 = silk feeding, 2 = 1 cm of tip feeding,
and each additional cm of tip feeding counts as
another point. In 2007 final ear damage of 20 ears
per plot was assessed at physiological maturity by
measuring the total area (cm2) of kernel damage.
Grain yield was measured from the 2 center
rows of each plot in 2006 and from rows 2 and 3
in 2007. Grain was harvested with a Hege two-
row corn combine in Aug and Sep when respec-
tive plantings reached about 16-18% moisture
content. Grain weight, test weight, and moisture
content were measured. Grain yields and test
weights were adjusted to 15.5% moisture con-
tent.
Results were analyzed by trial with a RCBD.
Before analysis, percentage data were trans-
formed by square-root arcsine transformation.
Results were compared among all hybrids by
ANOVA, and means were separate by Fisher's
Protected LSD (a = 0.05). Single degree-of-free-
dom contrasts were used to compare hybrids
without and with Bt traits and to compare among
Bt traits (PROC GLM, SAS Institute 2003). Fur-
thermore the effects of Bt traits (non-Bt,
MON810, and TC1507) were averaged across all







Florida Entomologist 91(4)


TABLE 2. EFFECT OF BT EVENTS ON DAMAGE RATING (0-9) F FALL ARMYWORM INFESTED WHORLS IN THE SECOND
CORN PLANTING IN 2006 AND 2007.

2006 2007

Brand/Hybrid Bt event 6-leaf 10-leaf 6-leaf 10-leaf

DKC 6972 -2.57 + 0.31 bc 6.57 0.31 a 4.61+ 0.65 a 6.53 0.11 a
DKC 6971 MON810 1.34 0.11 d 5.55 0.09 b 0.25 0.25 b 1.38 0.85 cd
Pioneer 31G66 -3.04 0.22 ab 6.61 0.21 a 3.40 0.67 a 5.87 0.09 a
Pioneer 31G68 MON810 1.26 0.06 d 5.15 + 0.18 b 0.61 0.60 b 2.75 + 1.03 bc
Pioneer 31G97 -2.46 0.30 c 7.00 0.56 a 3.68 0.60 a 6.44 + 0.17 a
Pioneer 31G96 TC1507 1.35 0.20 d 4.08 0.44 c 0.75 0.75 b 0.0 0.0 d
Pioneer 31N27 -2.18 0.08 c 6.52 0.21 a 3.22 0.23 a 5.91 0.27 a
Pioneer 31N28 MON810 1.33 0.12 d 5.16 + 0.14 b 1.15 + 0.68 b 3.80 0.64 b
Pioneer 34B97 -3.52 + 0.25 a 6.68 0.19 a -
Pioneer 34B98 MON810 1.23 0.09 d 4.99 + 0.14 b -
Pioneer 34B99 TC1507 1.08 0.08 d 4.04 0.38 c -
LSD005 0.53 0.88 1.75 1.59
Combined -2.75 0.65 x 6.68 0.61 x 3.65 0.27 x 6.24 0.12 x
Combined MON810 1.29 0.19 y 5.21 0.33 y 0.67 0.29 y 2.64 0.42 y
Combined TC1507 1.22 0.32 y 4.00 0.76 z 0.75 0.38 y 0.00 0.00 z
Source of variation2
Hybrid 21.22*** 12.80*** 8.07*** 21.38***
Non-Bt v. MON810 138.99*** 41.54*** 39.99*** 61.15***
Non-Bt v. TC1507 92.59*** 87.66*** 12.11** 70.71***
MON810 v. TC1507 0.20ns 21.22*** 0.01ns 17.85***

Means within columns followed by the same lower case letter among hybrids or combined are not significantly different (LSD;
P = 0.05).
*, **, *** indicate significant contrast F value at P < 0.05, P < 0.01, and P < 0.001 respectively; ns, not significant.
22006: Hybrid df= 10, 30; contrast df= 1, 15; 2007: Hybrid df= 7, 21; contrast df = 1, 21.


hybrids and analyzed by one-way ANOVA (PROC
GLM, SAS Institute 2003), and means were sepa-
rated by Protected LSD (a = 0.05).

RESULTS

Plant stand was significantly different
among hybrids in all trials, but most stand dif-
ferences were associated with hybrid pedigree
and not Bt traits (data not shown). In 2006,
stands of the Pioneer 34B series hybrids were
about 20% and 30% less than other hybrids in
the first and second planting dates, respec-
tively. These hybrids were planted in 2007 but
not included in further analysis due to poor
stand establishment.
Whorl infestations in all trials consisted al-
most entirely of fall armyworm. Fall armyworm
infestations were substantially greater in 2006
than 2007 and also were greater in the second
than the first plantings in both years. Whorl in-
festations in the first planting in 2006 ranged
from 0 to 4.75%. Significantly fewer whorls were
infested in hybrids with Bt traits than non-Bt hy-
brids in 2006 (F = 45.2, df= 1, 30;P < 0.0001; data
not shown), but infestations were not different be-


tween Bt traits (F = 0.75, df = 1, 30; P = 0.40).
Likewise, damage ratings of infested whorls in
the first planting in 2006 were significantly lower
in hybrids with Bt traits than susceptible hybrids
(F = 112.3; df = 1, 30; P < 0.0001; data not shown)
but were not different between Bt traits (F = 0.34;
df = 1, 30; P = 0.56). In 2007, whorl infestations
ranged from 0 to 1.4% in the first planting and
were not different among hybrids (F = 1.1, df = 7,
21; P = 0.39, data not shown). Whorl damage rat-
ings in the first planting in 2007 also were not dif-
ferent among hybrids (F = 1.5, df = 7, 21; P =
0.22).
In the second plantings hybrids with Bt traits
had significantly fewer infested whorls at the 6-
and 10-leaf stages than non-Bt hybrids in both
years (Tables 1 and 2). Furthermore, in 2006 hy-
brids with the TC1507 event had significantly
fewer infested whorls and less damage of infested
whorls than hybrids with the MON810 event ex-
cept for damage ratings at the 6-leaf stage. In
2007 whorl damage was not significantly differ-
ent between Bt traits at the 6-leaf stage but was
significantly lower at the 10-leaf stage in the hy-
brid with the TC1507 trait than in hybrids with
the MON810 trait.


December 2008







Buntin: Bt Corn for Grain Production


TABLE 3. EFFECT OF BT EVENTS AND PLANTING TIME ON PERCENTAGE OF INFESTED EARS IN 2006 AND 2007.

2006 2007

Brand/Hybrid Bt event PD1 PD2 PD1 PD2

DKC 6972 -100 0 a 100 0 a 100 0 a 100 0 a
DKC 6971 MON810 98.3 1.8 a 86.8 5.5 bc 97.5 2.5 a 81.7 5.0 c
Pioneer 31G66 -100 0 a 100 0 a 100 0 a 100 0 a
Pioneer 31G68 MON810 100 0 a 96.8 3.3 ab 98.8 1.3 a 68.3 6.8 d
Pioneer 31G97 -100 0 a 96.5 + 2.0 ab 100 0 a 100 0 a
Pioneer 31G96 TC1507 100 0 a 93.3 4.7 de 100 0 a 93.3 2.7 b
Pioneer 31N27 100 0 a 100 0 a 100 0 a 100 0 a
Pioneer 31N28 MON810 96.8 3.3 a 70.3 13.7 c 81.3 6.3 b 63.3 4.3 d
Pioneer 34B97 -100 0 a 100 0 a -
Pioneer 34B98 MON810 98.3 1.8 a 100 0 a -
Pioneer 34B99 TC1507 100 0 a 100 0 a -
LSD0o5 NS -
Combined -100 0 x 99.3 0.71 x 100 0 x 100 0 x
Combined MON810 98.4 0.4 x 88.5 0.21 y 92.5 3.4 y 71.1 5.0 z
Combined TC1507 100 + 0 x 96.7 0.71 xy 100 0. x 93.3 5.1 y
Source of variation2
Hybrid 0.83ns 3.96** 11.42*** 40.62***
Non-Bt v. MON810 4.15* 14.99*** 27.73*** 237.67***
Non-Bt v. TC1507 0.01ns 0.09ns 0.00ns 12.77**
MON810 v. TC1507 2.77ns 3.59ns 13.87*** 42.57***

Means within columns followed by the same lower case letter among hybrids or combined are not significantly different (LSD;
P = 0.05).
*, **, *** indicate significant contrast F value at P < 0.05, P < 0.01, and P < 0.001 respectively; ns, not significant.
'PD1 = recommended mid-Apr planting time, PD2 = mid-Jun planting time.
22006: Hybrid df= 10, 30; contrast df= 1, 15; 2007: Hybrid df= 7, 21; contrast df = 1, 21.


Ear damage in the first planting in both years
and in the second planting in 2007 was caused al-
most entirely by corn earworm. In the second
planting in 2006, ears were infested by both corn
earworm and fall armyworm. The percentage of
infested ears was not different among hybrids in
the first planting in 2006 (Table 3). In the other
trials, hybrids with the MON810 event had signif-
icantly fewer infested ears than the non-Bt hy-
brids. Ear infestations in 2007 also were lower in
hybrids with the MON810 event than hybrids
with the TC1507 event in both plantings. Ear
damage ratings were significantly less in hybrids
with Bt traits than the non-Bt hybrids in all trials
(Table 4). Ear damaging ratings were similar
among Bt traits except the first planting in 2007
where ear damage was less in hybrids with the
MON810 event than the TC1507 event.
Grain yields were not significantly different
between Bt and non-Bt hybrids in the first plant-
ing in either year (Table 5). Hybrids with either
the MON810 or the TC1507 events yielded more
than the non-Bt hybrids in the second planting in
2006, but the yield of hybrids with either Bt traits
were similar. In 2007, grain yield in the second


planting was lower in the non-Bt hybrids than hy-
brids with either type ofBt but this difference was
not significant. Grain test weight was signifi-
cantly affected by hybrid pedigree but was not sig-
nificantly affected by Bt trait in any trial (F = 0.01
- 1.72; df = 1, 30 (2006) or 21 (2007); P = 0.20 -
0.96; data not shown).

DISCUSSION

The Pioneer 34B series hybrids in 2006 pro-
vided a direct comparison of the 2 Bt events with
a non-transformed hybrid with the same base ge-
netics. Results were similar to those previous
listed for the comparison of near-isogenic pairs of
hybrids, which indicates that the broader compar-
ison of Bt activity among hybrids with different
pedigrees is appropriate.
Both Bt events were effective in preventing
whorl infestations and damage by fall armyworm.
Infestation levels in the first planting dates in
both years were substantially below the level of
whorl infestation needed to cause economic losses
(Buntin 1986). Both the MON810 and TC1507
events provide similar levels of protection from







Florida Entomologist 91(4)


TABLE 4. EFFECT OF BT EVENTS AND PLANTING TIME'ON DAMAGE RATING OF INFESTED CORN EARS IN 2006 AND 2007.

2006 2007

Brand/Hybrid Bt event PD1 PD2 PD1 PD2

DKC 6972 -5.34 + 0.24 a 4.47 0.12 b 4.36 0.57 a 4.94 0.24 a
DKC 6971 MON810 3.97 0.34 b 2.32 0.18 de 6 0 e 2.27 0.33 b
2.63 + 0.18 e
Pioneer 31G66 -3.47 0.22 bcd 3.67 0.25 b 3.71 + 0.17 bc 4.70 + 0.19 a
Pioneer 31G68 MON810 3.25 0.17 cd 2.27 0.43 e 0 09 d 1.73 + 0.15 b
3.05 + 0.19 d
Pioneer 31G97 -3.93 0.37 bc 3.43 0.22 bc 3.96 0.02 b 5.21 0.39 a
Pioneer 31G96 TC1507 3.24 + 0.15 d 2.57 0.31 de 1 c 2.14 + 0.13 b
3.46 + 0.13 c
Pioneer 31N27 -4.14 0.45 b 3.87 0.05 ab 3.72 + 0.12 bc 4.85 0.39 a
Pioneer 31N28 MON810 2.47 0.21 e 1.30 0.35 f 1.51 + 0.17 b
1.69 + 0.20 f
Pioneer 34B97 -3.63 + 0.22 bcd 3.90 0.18 ab -
Pioneer 34B98 MON810 2.95 0.18 de 2.97 0.20 cd -
Pioneer 34B99 TC1507 3.23 0.27 d 2.75 0.34 cde -
LSD005 0.69 0.68 0.38 0.80
Combined -4.10 0.27 x 3.87 0.71 x 3.94 0.37 x 4.66 0.78 x
Combined MON810 3.16 0.41 y 2.22 0.21 y 2.46 0.69 z 1.84 0.58 y
Combined TC1507 3.24 0.27 y 2.66 0.71 y 3.46 0.26 y 2.14 0.26 y
Source of variation2
Hybrid 9.93*** 15.09*** 42.29*** 35.96***
Non-Bt v. MON810 33.95*** 110.78*** 192.06*** 181.77***
Non-Bt v. TC1507 5.29* 18.03*** 7.32* 63.35***
MON810 v. TC1507 0.13ns 4.77* 44.63*** 0.88ns

Means within columns followed by the same lower case letter among hybrids or combined are not significantly different (LSD;
P = 0.05).
*,**, *** indicate significant contrast F value at P < 0.05, P < 0.01, and P < 0.001 respectively; ns, not significant.
PD1 = recommended mid-Apr planting time, PD2 = mid-Jun planting time.
22006: Hybrid df= 10, 30; contrast df= 1, 15; 2007: Hybrid df= 7, 21; contrast df = 1, 21.


whorl damage by fall armyworm under low to
moderate infestation levels. However, hybrids
with the MON810 event had substantially more
infested whorls and damage per infested whorl
than hybrids with the TC1507 event under large
infestation levels that occurred during the second
planting in 2006.
In previous studies the MON810 event was ef-
fective in preventing whorl damage under large
infestations that occurred during a fall army-
worm outbreak in 1998 (Buntin et al. 2001) and
more moderate infestations in studies in 1999
and 2000 (Buntin et al. 2004). Fall armyworm is
more sensitive to CrylF endotoxin than CrylAc,
CrylBb, and CrylCa endotoxins (Lou et al. 1999).
Siebert et al. (2008) also demonstrated that
CrylF (TC1507 event) provides high level of pre-
vention to leaf feeding in field corn but did not
evaluate other Bt endotoxins.
In the current study, corn earworm infested
virtually 100% of the ears in susceptible hybrids.
Hybrids with the MON810 event had less ear in-
festation than susceptible hybrids except in the


first planting in 2006, although infestations in all
trials exceeded 63% in all hybrids with MON810
and often had greater than 90% infested ears. In
three of four trials, the TC1507 event did not re-
duce ear infestations. Where a reduction did occur
in the second 2007 planting, the difference was
small. Nevertheless, both Bt traits partly reduced
ear damage ratings in all trials. There was no con-
sistent difference between the 2 Bt traits in pre-
venting ear damage. The reduction in ear damage
was similar to reduction in grain damage of about
52% from earworms and armyworms previously
reported (Buntin et al. 2004). Furthermore,
Storer et al. (2001) showed that corn containing
the MON810 or Btll events reduced kernel dam-
age by an average of 80%. Corn containing
CrylAb endotoxin usually reduces the growth of
H. zea larvae, and reduces and delays H. zea adult
emergence from Bt-corn fields (Storer et al. 2001;
Allen & Pitre 2006).
Bt traits did not prevent grain yield loss in the
first planting in either year. Fall armyworm infes-
tations were low with <5% infested whorls in the


December 2008







Buntin: Bt Corn for Grain Production


TABLE 5. EFFECT OF BT EVENTS AND PLANTING TIME' ON CORN GRAIN YIELD (MG/HA) IN 2006 AND 2007.

2006 2007

Brand/Hybrid Bt event PD1 PD2 PD1 PD2

DKC 6972 -12.60 + 0.30 a 5.43 0.84 b-f 13.40 0.48 a 5.19 0.18 ab
DKC 6971 MON810 11.10 + 0.80 a 7.96 0.60 a 13.00 0.68 a 5.90 + 0.13 a
Pioneer 31G66 -12.21 1.21 a 4.90 0.63 def 11.92 0.43 a 3.98 + 0.16 bc
Pioneer 31G68 MON810 10.62 2.07 a 8.04 1.08 a 13.02 0.34 a 4.32 0.05 b
Pioneer 31G97 -11.02 + 0.76 a 4.75 1.00 ef 13.67 0.75 a 2.65 0.79 cd
Pioneer 31G96 TC1507 12.18 + 0.57 a 6.94 1.53 abc 11.36 0.30 a 4.23 0.29 b
Pioneer 31N27 -11.22 1.07 a 4.36 0.65 f 13.49 0.91 a 1.49 0.53 d
Pioneer 31N28 MON810 11.71 + 0.45 a 7.29 0.80 ab 13.16 + 0.41 a 1.94 0.87 d
Pioneer 34B97 -8.47 1.28 a 5.02 0.58 d-f -
Pioneer 34B98 MON810 8.78 0.84 a 6.78 0.49 a-d -
Pioneer 34B99 TC1507 10.06 1.19 a 6.76 0.71 a-e -
LSD005 NS 2.02 NS 1.39
Combined -11.10 2.30 x 4.90 1.39 x 12.40 2.03 a 3.74 1.86 a
Combined MON810 10.55 4.39 x 7.52 1.49 y 13.06 1.44 a 4.05 1.76 a
Combined TC1507 11.12 2.06 x 6.85 2.22 y 11.36 1.71 a 4.23 0.29 a
Source of variation2
Hybrid 1.88 ns 2.88*** 1.32 ns 10.82***
Non-Bt v. MON810 0.74 ns 27.23*** 0.05 ns 1.6 5ns
Non-Bt v. TC1507 2.14 ns 6.30* 5.36* 5.59*
MON810 v. TC1507 0.49 ns 1.52 ns 4.33* 0.11 ns

Means within columns followed by the same lower case letter among hybrids or combined are not significantly different (LSD;
P = 0.05).
*, **, *** indicate significant contrast F value at P < 0.05, P < 0.01, and P < 0.001 respectively; ns, not significant.
PD1 = recommended mid-Apr planting time, PD2 = mid-Jun planting time.
'2006: Hybrid df= 10, 30; contrast df= 1, 15; 2007: Hybrid df= 7, 21; contrast df= 1, 21.


first planting in both years. During the second
planting, Bt traits prevented significant yield loss
in 2006, but the same trend in 2007 was not sig-
nificant. Previous studies also in Georgia have
found that Bt traits for lepidopteran control usu-
ally only prevent yield loss in late plantings (Bun-
tin et al. 2001, 2004).
Comparing the 4 trials in the current study,
the yield responses to Bt traits were associated
with the level of fall armyworm whorl infesta-
tion and damage with Bt preventing significant
yield loss only during the second planting in
2006 when whorl infestation levels exceeded
50% in susceptible hybrids. Differences in ear in-
festation were fairly minimal between hybrids
with and without Bt traits and not generally as-
sociated with observed differences in grain
yields. Abel & Pitre (2004) also did not find a
yield response to reduction in ear damage by
corn earworm between MON810 trait and sus-
ceptible hybrids. Despite large differences in the
prevention of whorl damage between the 2 Bt
traits in the second plantings, this difference in
damage did not significantly affect grain yield.
Nevertheless it is difficult to convince farmers


that the difference in the level of defoliation seen
in late 2006 in the hybrids with the MON810
event or the TC1507 event was not important.
Because of the greater activity in preventing
whorl damage by fall armyworm, the TC1507
event would be useful in preventing fall army-
worm damage to late planted field corn for grain
production in the southeastern U.S.

ACKNOWLEDGMENTS

I thank William R. Slaughter, Jr. for technical assis-
tance and Xinzhi Ni for reviewing an early version of
this manuscript. I thank Jerry Davis for advice on sta-
tistical procedures used in this study.

REFERENCES CITED

ABEL, C. A., AND M. C. POLLAN. 2004. Field resistance of
Bacillus thuringiensis Berliner transformed maize
to fall armyworm (Lepidoptera: Noctuidae) and
southwestern corn borer (Lepidoptera: Crambidae)
leaf feeding. J. Entomol. Sci. 39: 325-336.
ALLEN, K. C., AND H. N. PITRE. 2006. Influence of trans-
genic corn expressing insecticidal protein of Bacillus
thuringiensis Berliner on natural populations of











corn earworm (Lepidoptera: Noctuidae) and south-
western corn borer (Lepidoptera: Crambidae). J. En-
tomol. Sci. 41: 221-231.
ARCHER, T. L., G. SCHUSTER, C. PATRICK, G. CRONHOLM,
E. D. BYNUM, JR, AND W. P. MORRISON. 2000. Whorl
and stalk damage by European and southwestern
corn borers to four events of Bacillus thuringiensis
transgenic maize. Crop Protect. 19: 181-190.
ARMSTRONG, C. L., G. B. PARKER, J. C. PERSHING, S. M.
BROWN, P. R. SANDERS, D. R. DUNCAN, T. STONE, D.
A. DEAN, D. L. DEBOER, J. HART, A. R. HOWE, F. M.
MORRISH, M. E. PAJEAU, W. L. PETERSON, B. J. RE-
ICH, R. RODRIGUEZ, C. G. SANTINO, S. J. SATO, W.
SCHULER, S. R. SIMS, S. STEHLING, L. J. TARO-
CHIONE, AND M. E. FROMM. 1995. Field evaluation of
European corn borer control in progeny of 173 trans-
genic corn events expressing an insecticidal protein
for Bacillus thuringiensis. Crop Sci. 35: 550-557.
BOKONON-GANTA, A. H., J. S. BERNAL, P. V. PIETRANTO-
NIO, AND M. SETAMOU. 2003. Survivorship and de-
velopment of fall armyworm, Spodoptera frugiperda
(J. E. Smith) (Lepidoptera: Noctuidae) on conven-
tional and transgenic maize cultivars expressing Ba-
cillus thuringiensis Cry9C and CrylA(b) endotoxins.
Int. J. Pest Manage. 49: 169-175.
BUNTIN, G. D. 1986. A review of plant response to fall
armyworm, Spodoptera frugiperda (J. E. Smith), in-
jury in selected field and forage crops. Florida Ento-
mol. 69: 549-559.
BUNTIN, G. D. 2007. Insect management, p. 23-31 In
D. Lee [ed.], A Guide to Corn Production in Georgia
2007. Georgia Agric. Extension Misc. Publ. CSS 03-07.
BUNTIN, G. D., J. N. ALL, R. D. LEE, AND D. M. WILSON.
2004. Plant incorporated Bt resistance for control of
fall armyworm and corn earworm (Lepidoptera: Noc-
tuidae) in corn. J. Econ. Entomol. 97: 1603-1611.
BUNTIN, G. D., R. D. LEE, D. L. WILSON, AND R. M.
MCPHERSON. 2001. Evaluation of YieldGard trans-
genic resistance for control of fall armyworm and
corn earworm (Lepidoptera: Noctuidae) on corn.
Florida Entomol. 84: 37-42.
DAVIS, F. M., S. S. NG, AND W. P. WILLIAMS. 1992. Visu-
al Rating Scales for Screening Whorl-stage Corn for


December 2008


Resistance to Fall Armyworm. Miss. Agric. & Forest.
Exp. Stn. Tech. Bull. 186.
GRAEBER, J. V., E. D. NAFZIGER, AND D. W. MIES. 1999.
Evaluation of transgenic, Bt-containing corn hy-
brids. J. Pod. Agric. 12: 659-663.
LAUER, J., AND J. WEDBERG. 1999. Grain yield of initial
Bt corn hybrid introductions to farmers in the north-
ern corn belt. J. Prod. Agric. 12: 373-376.
Lou, K., D. BANKS, AND M. ADANG. 1999. Toxicity, bind-
ing, and permeability analyses of four Bacillus thur-
ingiensis Cryl 6-endotoxins using brush border mem-
brane vesicles of Spodoptera exigua and Spodoptera
frugiperda. Appl. Environ. Microbiol. 65: 457-464.
OSTLIE, K. R., W. D. HUTCHISON, AND R. L. HELLMICH.
1997. Bt corn and European corn borer. NCR Publ.
602. Univ. of Minnesota, St. Paul, MN.
SAS INSTITUTE. 2003. Version 9.1. SAS Institute, Cary,
NC.
SIEBERT, M. W., K. V. TINDALL, B. R. LEONARD, J. W.
VAN DUYN, AND J. M. BABCOOK. 2008. Evaluation of
corn hybrids expressing CrylF (Herculex I Insect
Protection) against fall armyworm (Lepidoptera:
Noctuidae) in the southern United States. J. Ento-
mol. Sci. 43: 41-51.
STORER, N. P., J. W. VAN DUYN, AND G. G. KENNEDY.
2001. Life history traits of Helicoverpa zea (Lepi-
doptera: Noctuidae) on non-Bt and Bt transgenic
corn hybrids in eastern North Carolina. J. Econ. En-
tomol. 94:1268-1279.
WIDSTROM, N. W. 1967. An evaluation of methods for
measuring corn earworm injury. J. Econ. Entomol.
60: 791-794.
WILLIAMS, W. P., P. M. BUCKLEY, J. B. SAGERS, and J. A.
HANTEN. 1998. Evaluation of transgenic corn for re-
sistance to corn earworm (Lepidoptera: Noctuidae),
fall armyworm (Lepidoptera: Noctuidae), and
southwestern corn borer (Lepidoptera: Noctuidae)
in a laboratory bioassay. J. Agric. Entomol. 15: 105-
112.
WILLIAMS, W. P., J. B. SAGERS, J. A. HANTEN, F. M.
DAVIS, AND P. M. BUCKLEY. 1997. Transgenic corn
evaluated for resistance to fall armyworm and
southwestern corn borer. Crop Sci. 37: 957-962.


Florida Entomologist 91(4)







Adamczyk et al.: Efficacy of Bt Cottons Against Armyworms


EVALUATIONS OF BOLLGARD, BOLLGARD II, AND WIDESTRIKE
TECHNOLOGIES AGAINST BEET AND FALL ARMYWORM LARVAE
(LEPIDOPTERA: NOCTUIDAE)

J. J. ADAMCZYK, JR., S. GREENBERG, J. S. ARMSTRONG, W. J. MULLINS1,
L. B. BRAXTON2, R. B. LASSITER2AND M. W. SIEBERT2
USDA, ARS, KSARC, Beneficial Insects Research Unit, 2413 E. Hwy 83, Weslaco, TX 78501

1Monsanto Co., St. Louis, MO

2Dow AgroSciences LLC, Indianapolis, IN


ABSTRACT

Transgenic cottons containing the Bollgard, Bollgard II and WideStrike traits were
grown in 2005 and 2007 to examine the efficacy against beet armyworm Spodoptera exigua
(Htibner) and fall armyworms S. frugiperda (J. E. Smith). Results suggest that both dual-
gene traits are more efficacious against these armyworm species than Bollgard. In these
studies, WideStrike@ appears to be more efficacious against fall armyworms than Bollgard
II, while Bollgard II is more efficacious against beet armyworms than WideStrike. Pos-
sible reasons for these differences in efficacy are discussed.

Key Words: Spodoptera exigua, Spodoptera frugiperda, armyworms, transgenic cotton


RESUME

Clases de algod6n transg6nicos que tienen las caracteristicas Bollgard, Bollgard II y
WideStrike fueron sembrados durante 2005 y 2007 para examiner la eficacia contra el gu-
sano ejercito de la remolacha, Spodoptera exigua (Hiibner) y el gusano cogollero, S. fru-
giperda (J. E. Smith). Los resultados sugerieron que ambas lines de los duos genes son mas
eficaz contra estas species de gusanos que Bollgard. En estos studios, WideStrike apa-
rece ser mas eficaz contra el gusano cogollero que Bollgard II, mientras que Bollgard II
es mas eficaz contra el gusano ejercito de la remolacha que WideStrike. Se discute las ra-
zones que son posibles para estas diferencias en eficacia.


Since the first CrylAc Bacillus thuringiensis
Berliner (Bt) cotton variety was commercialized
in 1996 (Bollgard , Monsanto Ag. Co., St. Louis,
MO), advancements for insect control with trans-
genic technology has occurred that offer improved
efficacy against many lepidopteran pests. Current
varieties can contain CrylAc alone or they can be
stacked with Cry2Ab (Bollgard II, Monsanto Ag.
Co.) or CrylF (WideStrike, Dow Agrosciences,
Indianapolis, IN).
The beet armyworm, Spodoptera exigua
(Hiibner) is a secondary, but serious migratory
pest of various vegetable and certain row crops
in the southern part of the United States of
America. Although larval feeding on cotton is
primarily concentrated on foliage, larvae can
cause devastating losses in yield during out-
breaks (Hardee & Herzog 1997; Adamczyk et al.
1998). The fall armyworm, S. frugiperda (J. E.
Smith) also is a destructive migratory pest of
many crops in the Western Hemisphere, where it
appears to be more common and widespread
(Sparks 1979; Young 1979). Like the beet army-
worm, this pest has the potential to damage both


conventional and Bollgard@ cotton bolls (Adam-
czyk et al. 1998).
Although certain lepidopteran pests of cotton
are controlled by Bollgard@ cotton (e.g., tobacco
budworms, Heliothis virescens (F.) and pink boll-
worms, Pectinophora gossypiella (Saunders)), the
CrylAc 8-endotoxin in Bollgard@ cotton is less ef-
fective for controlling beet and fall armyworms
(MacIntosh et al. 1990; Adamczyk et al. 1998).
Consequently, outbreaks of these pests on Boll-
gard@ often need full application rates of foliar in-
secticide treatments to keep these populations be-
low economic injury levels (Hood 1997; Smith
1997). The addition of other Cry proteins stacked
with CrylAc (i.e., Bollgard II@ and WideStrike@)
has improved the efficacy against these army-
worms (Adamczyk et al. 2001; Stewart et al. 2001;
Adamczyk & Gore 2004). However, differences in
survivorship of beet and fall armyworm larvae
feeding on Bollgard II@ versus WideStrike cot-
tons have been suggested, but never character-
ized or explained. The purpose of the study was to
examine the efficacy of Bollgard II@ and Wide-
Strike@ against beet and fall armyworms.








Florida Entomologist 91(4)


December 2008


MATERIALS AND METHODS

Field Plots

In May 2005 transgenic cotton varieties con-
taining the Bollgard@, Bollgard II and Wide-
Strike@ traits were planted in research plots in
the Mississippi Delta near Stoneville, MS
(Table 1). Plots consisted of 2 rows (1.0 m centers)
x 10.67 m. All plots were arranged in a random-
ized complete block design with each variety rep-
licated 4 times (once in each block). Only insecti-
cides not active on Lepidoptera were applied to all
plots throughout the season as dictated by local
management practices. In Mar 2007 transgenic
cotton varieties containing the Bollgard, Bollgard
II@ and WideStrike@ traits were planted in strip
plots in the lower Rio Grande Valley of Texas near
Weslaco, TX (Table 1).

Insects

All Lepidoptera utilized in these studies were
obtained from laboratory colonies maintained at
the USDA, ARS located in Stoneville, MS or
Weslaco, TX.


Bioassays with Larvae

In 2005 bioassays were conducted with only
fall armyworm larvae. A single neonate was
placed into individual 9-cm diameter petri dishes
(8) that each contained a moistened filter paper
and a single lower leaf obtained from all plots for
a total of 32 larvae per variety. Cotton plants were
at peak bloom. The plates were covered with cor-
responding lids. After 5 d, surviving larvae were
carefully transferred with a camel-hair brush into
new petri dishes containing fresh filter paper and
a new leaf. This procedure continued until pupa-
tion. At 7 and 10 d, larvae were prodded with a
camel-hair brush and considered alive if coordi-
nated movement was observed. Beginning at 15 d,
plates were checked daily for the presence of pu-
pae. Percent survival was analyzed by REML-
ANOVA, and means were separated according to
Fisher's Protected LSD (Littell et al. 1996; PROC
MIXED, SAS Institute 2001).
In 2007 leaves obtained from various sections
of the plant containing various transgenic traits
were assayed for bioactivity against beet and fall
armyworms. Individual leaves were placed into a
50 x 9-mm Tight-Fit Lid sealing Petri dish (BD
Falcon@ #351006, VWR International). Beet ar-
myworms (5) were placed in a dish containing a
terminal (upper canopy) leaf or a mid-canopy leaf
(10 dishes per variety) for a total of 50 larvae per
variety. Fall armyworm bioassays were conducted
identically, except only mid-canopy leaves were
used. Leaves were also collected from various
times during the growing season. At 5 d after ex-


Z
C)







g-
0
C)
0
C



cc UM C) 4 t U 4 4 t 4 4






ab,
00000a c c0 0 0 0





C) ca ca ca C) & C) aca r cwc^


cn^^^a0' 0^ 2


0 ^t ^5^"950 cc0 000 0 0
o~ o 6 o o o o o 6








'C 'S m 'C )C )3 C3 )C C )
0a0 0 0 00000000
ZZ| Z ZZZ|
II Qlll d


+ + +


w ~
mm:
F ^ '--
0; (M ^
tg ^ 10
,fcfc


In c C)
MI~


C o
-




mmmmmcmm

03 M M M M M
eresss><
a a a a a
00000-00 o
> > ZZZ> "
CL CC CC CC CC CC n n
666 cH H
C)CC))CC) aC
w -
00000 C) C


(M] (M] (M
000? E

5
oooZZ1


CqI
Cl ~' 0 Cl ~

g-, P, ,-qI
or i ~'
C)
gpqpq^ us
96^|^i%
11^C~~


c~ c9 9
00000;I;


HHHHHE-'
0000000
0000000
c~c c~c~~ c c


000000000000000
000000000000000







Adamczyk et al.: Efficacy of Bt Cottons Against Armyworms


posure to cotton tissue, larvae were prodded with
a camel-hair brush and considered alive if coordi-
nated movement was observed. Percent mortality
by trait was analyzed by REML-ANOVA, and
means were separated according to Fisher's Pro-
tected LSD (Littell et al. 1996; PROC MIXED,
SAS Institute 2001).

Bioassays with Egg Masses

Inoculations of beet and fall armyworm egg
masses to leaves located in various sections of the
plant were conducted in 2007. In the laboratory,
egg masses were deposited on nylon cloth placed
on the top of adult rearing cages (3.79-L card-
board containers). For each inoculation, an egg
mass of equal size (ca. 100-300 eggs per 2.54 cm2
cloth sample) was pinned to the underside of a
leaf for all traits and covered with a cage that con-
sisted of a condiment cup (118 mL) (Solo Co.,
Highland Park, IL) coupled with a hard plastic lid
(Fig. 1). Five d after inoculations, the infested leaf
and corresponding cages were harvested and
transported to the laboratory. Leaf damage (0-5)
was estimated with a categorical rating scale
where 0% indicated no leaf damage, while 80-
100% of leaf consumption was given a value of 5.
Damage ratings were analyzed by non-paramet-
ric statistics (SAS Institute 2001).

RESULTS AND DISCUSSION

Bioassays with Larvae

Both Bollgard II and WideStrike had signif-
icantly higher efficacy against fall armyworms
than Bollgard (Fig. 2 and Fig. 3); however, in


both 2005 and 2007, WideStrike had typically
higher efficacy compared to Bollgard II. It is in-
teresting to note that larval development to pupa-
tion was observed for fall armyworms when fed
Bollgard or Bollgard II, but not WideStrike.
In WideStrike isolines containing only CrylF or
CrylAc, Adamczyk & Gore (2004) showed that the
CrylF protein provided greater efficacy compared
to CrylAc against this pest. In addition, previous
studies have shown little efficacy of CrylAc
against fall armyworms (Adamczyk et al. 1998).
We believe that the greater efficacy of Wide-
Strike compared to Bollgard II against fall ar-
myworms is primarily due to the action of the
CrylF protein. Although we did not evaluate iso-
lines containing only CrylF or Cry2Ab, our data
suggests greater efficacy of CrylF versus Cry2Ab
against this pest.
The expression of the CrylF protein affects
beet armyworm survival both temporally and
spatially. When using mid-canopy leaves sampled
throughout the season, no significant differences
(P > 0.05) were observed between the survival of
beet armyworms fed Bollgard II and Wide-
Strike (Fig. 4). However late in the season when
beet armyworms were fed terminal leaves located
in the upper part of the plant (i.e., upper-canopy
leaves), survival of larvae on WideStrike was
very high (>%60) (Fig. 5). Siebert et al. (2009) re-
ported that expression of CrylF protein is great-
est in mature leaves as compared to other struc-
tures such as terminal leaves, squares, flowers,
and bolls, and that protein levels in mature leaves
increases with age. This expression profile for
CrylF is very different than what was reported
for Cry2Ab in Bollgard II, where expression in
all tissue appears to remain relatively constant


Fig. 1. Cages used to enclose egg masses.











100
90 a a
80 a
70-
70 Non-Bt
-60 b
-0 Bollgard
40-
* 40 0 Bollgard II
30 WideStrike
30 b
20 b
10
0-
7 d 10 d Pupation
df= 3. di= 3,9 df = 2.9
F =22.71 F=5976 F=4215
P 0 001 P 0.001 P < 0 DO
No survival

Fig. 2. Mortality of fall armyworms fed lower leaves,
Stoneville, MS 2005.


-- Bollgard
-1- Bollgard 1
-- WideStrike
-- Non-Bt




P 4W


o a a a a a

Days After Planting
Fig. 3. Mortality of fall armyworms at 5 d after expo-
sure for bioassays with mid-canopy leaves sampled
throughout the season, Weslaco, TX 2007.




100 -*- Bollgard
90 -- -- Bollgard II
80 -- WideStrike
70 -0- Non-Bt
60
250 ,,
4030
30 -
20
10
0 1
CM Co C 4 OD V I- I ,,O
(0 t0 a S as d o* "
IfT 1.1II UItI4
Days after Planting


Fig. 4. Mortality of beet armyworms at 5 d after ex-
posure for bioassays with mid-canopy leaves sampled
throughout the season, Weslaco, TX 2007.


December 2008


*- Bollgard
-1- Bollgard II
-- WideStrike
-0- Non-Bt


a o r I I--I"I
Days after Planling -u- ls T


Fig. 5. Mortality of beet armyworms at 5 d after ex-
posure for bioassays with upper-canopy leaves sampled
throughout the season, Weslaco, TX 2007.


over time Adamczyk et al. (2001). In addition,
mortality at >109 DAP of beet armyworms on
WideStrike terminal leaves was similar to what
was observed with Bollgard which also contains
a CrylAc-like transgene that provides little effi-
cacy against both beet and fall armyworms (Stew-
art et al. 2001;Adamczyk et al. 1998;Adamczyk &
Gore 2004). We believe that low levels of CrylF
protein reported for new foliage (e.g., young ter-
minal leaves) as compared to mature, fully ex-
panded leaves may partially explain the low mor-
tality observed against beet armyworms. Fur-
thermore, our field studies with leaves collected
from different portions of the plant supports these
temporal and spatial conclusions. More damage
was observed from beet armyworms feeding on
WideStrike leaves compared to Bollgard II
leaves in the upper canopy compared to the mid-
dle canopy (Fig. 6). Even with the presumed
greater efficacy of CrylF compared to Cry2Ab
against the fall armyworm, this same trend was
observed (Fig. 7). Regardless of transgenic trait, it
is interesting to note that more damage was ob-
served for both armyworm species caged on leaves
located in the upper canopy. This may be partially
explained by greater amounts of secondary plant
compounds, as well as a thicker leaf cuticle, for
leaves located lower in the plant canopy. In some
situations, we believe that beet armyworms may
need supplemental foliar insecticides for control-
ling outbreak populations that may be feeding on
young tissues especially late in the season where
CrylF levels do not have the time to build to pro-
vide adequate control.
Various transgenes that are used to produce
cotton with the WideStrike trait may partially
explain the observed protein expression profile of
CrylF. In particular, the crylF trait in Wide-
Strike is regulated by the (4ocs) DeltaMas 2'
Promoter. Previous studies have shown that the
dual promoter of Agrobacterium tumefaciens


Florida Entomologist 91(4)


-t~







Adamczyk et al.: Efficacy of Bt Cottons Against Armyworms


2.0

to 1.22 1.11 1.00
Nn1.0 l o



Non-Bl WideStrike Bollgard II Bollgrd II
(cv. DP 143) (cv. St 437)

Trait

Fig. 6. Damage ratings for beet armyworms caged in
the field on various cotton leaves located in the upper
(A) and middle (B) canopy, Weslaco, TX 2007. Kiskal-
Wallis Test: upper, P = 0.039; middle, P = 0.001. Culti-
vars: DP 143 BGII/RR & St 4357BGII/RRF (Bollgard
II), Phy 485 WR (WideStrike), Phy 425 RR (Non-Bt).



mannopine synthase (mas) genes is regulated by
plant growth hormones, and that the activity of
the mas dual promoters increases basipetally in
developing plants; it has also been reported that
the apical meristem contains a factor that inhib-
its stimulation of mas promoter activity (Lan-
gridge et al. 1989). However, it is important to
note that WideStrike traits contain enhancers
that make the promoter more constitutive,
whereby losing sensitivity to hormones (US
Patent: 5,955,646). Thus, further research is
needed to determine the various plant mecha-
nisms that confer protein expression for the Wide-
Strike traits.

ACKNOWLEDGMENTS

We thank the efforts of Alex Gomezplata, Carlos
Gracia, Jay Alejandro Frank Garza, and Chuy Cabal-
lero for helping with rearing insects and conducting bio-
assays. Mention of a trademark, warranty, proprietary
product or vendor does not constitute a guarantee by
the USDA and does not imply approval or recommenda-
tion of the product to the exclusion of others that may be
suitable.


0.0


1.88


U 0.33 0.14
Non-Bt WideStrike Boligard II
Trait


Fig. 7. Damage ratings for fall armyworms caged in
the field on various cotton leaves located in the upper
(A), middle (B), and lower (C) canopy, Weslaco, TX 2007.
Kiskal-Wallis Test: upper, P < 0.001; middle, P = 0.002;
lower, P = 0.076. Cultivars: DP 143 BGII/RR (Bollgard
II), Phy 485 WR (WideStrike), Phy 425 RR (Non-Bt).




REFERENCES CITED

ADAMCZYK, JR., J. J., AND J. GORE. 2004. Laboratory
and field performance of cotton containing CrylAc,
CrylF, and both CrylAc and CrylF (WideStrike)
against beet armyworm and fall armyworm larvae
(Lepidoptera: Noctuidae). Florida Entomol. 87: 427-
432.
ADAMCZYK, JR., J. J., L. C. ADAMS, AND D. D. HARDEE.
2001. Field efficacy and seasonal expression profiles


A
. 4.22 0= no damage
1m 1-20% damage
2= 20-40% damage
3= 40-60% damage
4-= 6040% damage
6 80-100% damage

1.56

0.50


27S


0.29 0.14
l "











for terminal leases of single and double Bt toxin cot-
ton genotypes. J. Econ. Entomol. 94: 1589-1593.
ADAMCZYK, JR, J. J., V. J. MASCARENHAS, G. E.
CHURCH, B. R. LEONARD, AND J. B. GRAVES. 1998.
Susceptibility of conventional and transgenic cotton
bolls expressing the Bacillus thuringiensis CrylAc
6-endotoxin to fall armyworm (Lepidoptera: Noctu-
idae) and beet armyworm (Lepidoptera: Noctuidae)
injury. J. Agric. Entomol. 15: 163-171.
HARDEE, D. D., AND G. A. HERZOG. 1997. 50m Annual
conference report on cotton insect research and con-
trol, pp. 809-818 In Proc. Beltwide Cotton Conf., Na-
tional Cotton Council, Memphis, TN.
HOOD, E. 1997. The fall armyworm: and I thought I had
it made, pp. 1223-1224 In Proc. Beltwide Cotton
Conf., National Cotton Council, Memphis, TN.
LANGRIDE, W. H. R., K. J. FITZGERALD, C. KONCZ,
J. SCHELL, AND A. A. SZALAY. 1989. Dual promoter of
Agrobacterium tumefaciens mannopine synthase
genes is regulated by plant growth hormones. Proc.
Natl. Acad. Sci. USA 86: 3219-3223.
LITTRELL, R. C., G. A. MILLIKEN, W. W. STROUP, AND
R. D. WOLFINGER 1996. SAS System for Mixed Mod-
els. SAS Institute, Cary, NC.
MACINTOSH, S. C., T. B. STONE, S. R. SIMS, P. L. HUNST,
J. T. GREENPLATE, P. G. MARRONE, F. J. PERLAK,


December 2008


D. F. FISCHHOFF, AND R. L. FUCHS. 1990. Specificity
and efficacy of purified Bacillus thuringiensis pro-
teins against agronomically important insects. J. In-
vertebr. Pathol. 56: 258-266.
SAS INSTITUTE 2001. Proprietary Software Release 8.2,
Cary, NC, USA.
SIEBERT, M. WILLRICH, T. G. PATTERSON, G. J. GILLES,
S. NOLTING, L. B. BRAXTON, B. R. LEONARD, J. W.
VAN DUYN, AND R. B. LASSITER. 2009. Quantification
of CrylAc and CrylF Bacillus thuringiensis insecti-
cidal proteins in various cotton plant tissue types
containing CrylAc:CrylF. J. Econ. Entomol (In
Press).
SMITH, R. H. 1997. An extension entomologist's 1996 ob-
servations ofBollgard (Bt) technology, pp. 856-857 in
Proc. Beltwide Cotton Conf., National Cotton Coun-
cil, Memphis, TN.
SPARKS, A. N. 1979. A review of the biology of the fall ar-
myworm. Florida Entomol. 62: 82-87.
STEWART, S. D., J. J. ADAMCZYK, JR., K. S. KNIGHTEN,
AND F. M. DAVIS. 2001. Impact of Bt cottons express-
ing one or two insecticidal proteins of Bacillus thur-
ingiensis Berliner on growth and survival of noctuid
(Lepidoptera) larvae. J. Econ. Entomol. 94: 752-760.
YOUNG, J. R. 1979. Fall armyworm: control with insec-
ticides. Florida Entomol. 62: 130-133.


Florida Entomologist 91(4)







Ni et al.: Fall Armyworm Resistance in Maize


PHYSIOLOGICAL BASIS OF FALL ARMYWORM
(LEPIDOPTERA: NOCTUIDAE) RESISTANCE IN SEEDLINGS OF MAIZE
INBRED LINES WITH VARYING LEVELS OF SILK MAYSIN

XINZHI NI, KEDONG DA', G. DAVID BUNTIN1 AND STEVE L. BROWN2
USDA-ARS, Crop Genetics and Breeding Research Unit, Tifton, GA 31793-0748

1Department of Entomology, University of Georgia, Griffin, GA 30223-1797

2Department of Entomology, University of Georgia, Tifton, GA 31793-0748

ABSTRACT

To assess both foliage- and ear-feeding insect resistance in the same maize inbred lines, fall
armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) resistance at the
seedling stage was examined in 6 corn inbred lines, including 4 CIMMYT maize inbred lines
(CML333, CML335, CML 336, and CML338) with varying levels of silk maysin that confers
corn earworm, Helicoverpa zea (Boddie), resistance and controls (fall armyworm-resistant
Mp708 and susceptible AB24E). Fall armyworm injury rating and chlorophyll content were
examined under greenhouse and field conditions. Plant height, plant stem circumference,
and photosynthesis-related measurements were recorded on uninfested and infested plants
only under greenhouse conditions. Injury ratings on CML333, CML336, and CML338 (with
a range of low to high levels of silk maysin) were the same as for the resistant control
(Mp708), and were significantly lower than for the susceptible control AB24E and CML335
(without silk maysin). Plant height, plant stem circumference, and chlorophyll content var-
ied among the 6 inbred lines, but were not consistently correlated to resistance at the seed-
ling stage. Photosynthetic rate was negatively affected by injury in AB24E, CML333,
CML335, and CML336, but not affected in CML338 and Mp708. The reduction in photosyn-
thetic rate of fall armyworm-susceptible AB24E, and in resistant CML333 and CML336 in-
dicated that insect resistance in CML333 and CML336 might not be related to
photosynthetic rate. At the same time, the data suggest that CML338 and Mp708 were tol-
erant to herbivory because no difference in either photosynthetic rate or photosynthetic ca-
pacity was detected in either inbred line between uninfested and injured corn seedlings.
Further examination of photosynthetic capacity based on A/Ci and light response curves
supported this resistance mechanism categorization. This experiment indicated that corn
earworm-resistant corn inbred lines with varying levels of silk maysin could confer resis-
tance to foliage-feeding fall armyworm at its seedling stage.

Key Words: CO2 exchange rate, light response curve, A/Ci curve, host plant resistance, tol-
erance

RESUME

Para evaluar la resistencia de insects que se alimentan del follaje y el elote en las mismas
lines endogamicas de maiz, la resistencia de la etapa de la plantula hacia el gusano cogo-
llero, Spodoptera frugiperda (J. E. Smith) (Lepid6ptero: Noctuidae) fue examinada en 6 li-
neas endogamicas de maiz, incluyendo 4 lines endogamicas de maiz CIMMYT (CML333,
CML335, CML 336 y CML338) con various niveles de maysin en la seda que proven resis-
tencia y control contra el gusano del elote de maiz, Helicoverpa zea (Boddie), (Mp708 resis-
tente al gusano cogollero yAB24E susceptible al gusano cogollero). La tasa del dano causado
por el gusano cogollero y el contenido de clorofila fueron examinados bajo condiciones de in-
vernadero y de campo. La altura de la plant, la circunferencia del tallo de la plant y me-
didas relacionadas con la fotosintesis fueron registrados sobre plants no infestadas y en
plants infestadas bajo condiciones en el invernadero. La tasa de dano sobre CML333,
CML336 y CML338 (con bajos a altos niveles de maysin en la seda) fue igual que el control
resistente (Mp708), y fue significativamente mas baja que en los controls susceptibles
AB24E y CML335 (sin maysin en la seda). La altura de la plant, la circunferencia del tallo
y el contenido de la clorofila varian entire las 6 lines endogamicas, pero no fueron correla-
cionados consistentemente con la resistencia en la etapa de la plantula. La tasa fotosintetica
fue afectada negativamente por el daio en AB24E, CML333, CML335 y CML336, pero no
afectada en CML338 y Mp708. La reducci6n en la tasa fotosintetica de AB24E susceptible al
gusano cogollero, y en las lines resistentes, CML333 y CML336, indic6 la posibilidad que la
resistencia de los insects en CML333 y CML336 no es relacionada con la tasa fotosintetica.
A la vez, los datos sugieren que CML338 y Mp708 son mas tolerantes a las herbivoras por







Florida Entomologist 91(4)


que ninguna diferencia en la tasa fotosint6tica y la capacidad fotosint6tica fue detectada en
ninguna de las dos lines endogamicas entire las plantulas de maiz no infestadas versus en
las plants daiadas. Un examen adicional de la capacidad fotosint6tica basada sobre las
curvas de A/Ci y la respuesta hacia la luz apoy6 esta categorizaci6n del mecanismo de resis-
tencia. Este experiment indic6 que las lines endogamicas de maiz resistentes a gusano del
elote con niveles de maysin en la seda variables pueden proveer resistencia a los gusanos co-
golleros que se alimentan del follaje en la etapa de la plantula.


Fall armyworm, Spodoptera frugiperda (J. E.
Smith) (Lepidoptera: Noctuidae) is one of the
most important whorl-feeding insect pests of corn
production in the southeastern U.S. Corn resis-
tance to S. frugiperda has been studied exten-
sively, and a series of corn germplasm conferring
S. frugiperda resistance has been developed at
Mississippi State, MS (Brooks et al. 2007) and
Tifton, GA (Wiseman et al. 1996) for corn produc-
tion in the southern states. Although corn resis-
tance to 2 whorl-feeding lepidopteran insects
(i.e., fall armyworm and southwestern corn borer,
Diatraea grandiosella Dyar (Lepidoptera: Cram-
bidae)) has been examined previously (Abel &
Adamczyk 2004; Brooks et al. 2007), multiple in-
sect resistance to both whorl- and ear-feeding in-
sects is not well understood. Only reports by Ni et
al. (2007, 2008) have recently examined corn ger-
mplasm for multiple ear-feeding insect resis-
tance. High levels of corn silk maysin has been
considered an important phenotypic trait that
confers ear-feeding corn earworm, Helicoverpa
zea (Boddie), resistance in laboratory bioassays,
but with varying levels of resistance under field
conditions (Rector et al. 2002; Ni et al. 2008).
However, it is still not clear whether inbred lines
with varying levels of resistance to ear-feeding
insects would confer whorl-feeding insect resis-
tance.
In addition, physiological and biochemical
mechanisms of whorl-feeding insect resistance
in corn are not well understood. Several recent
reports have examined the biochemical and
physiological bases for insect resistance in both
piercing-sucking and chewing insect pests on
various crop plants. Oxidation and detoxification
enzymes have been examined as biochemical
bases for Russian wheat aphid, Diuraphis noxia
(Mordvilko), resistance in wheat, barley and oat
(Ni et al. 2001a; Ni & Quisenberry 2003), and
chinch bug resistance in turf grasses (Franzen et
al. 2007). Impact of both piercing-sucking and
chewing insect herbivory on photosynthetic rate
and photosynthesis capacity was examined to es-
tablish baseline information on the physiological
basis of crop plants resistance to insect pests
(Haile et al. 1999; Macedo et al. 2003; Peterson
et al. 2004; Franzen et al. 2007; Macedo et al.
2007). In addition, D. noxia-elicited changes in
photosynthetic pigments were also assessed to
unravel the underlying mechanisms of aphid-
elicited leaf chlorosis and photosynthetic pig-


ment losses (Ni et al. 2001b; Ni et al. 2002; Heng-
Moss et al. 2003).
Thus, we examined the possibility of identify-
ing multiple insect resistance/susceptibility over
multiple growth stages of the corn plants. We ex-
amined S. frugiperda resistance at whorl (V6)
stage in 4 corn inbred lines from CIMMYT
(CML333, CML335, CML336, and CML338) with
varying levels of corn silk maysin with S. fru-
giperda-susceptible (AB24E) and resistant
(Mp708) corn inbred lines as controls. Both green-
house and field artificial infestations of the corn
seedlings with S. frugiperda neonates were con-
ducted. The objectives of this study were: (1) to de-
termine S. frugiperda resistance in seedlings of
the four CML inbred lines with varying silk may-
sin levels; and (2) to elucidate the physiological
basis for fall armyworm resistance and/or suscep-
tibility in these 6 corn inbred lines using photo-
synthetic measurement data.

MATERIALS AND METHODS

Plants and Insects

Six maize inbred lines were used, including 4
CIMMYT inbred lines (CML333, CML335,
CML336, and CML338), and Mp708 and AB24E
as resistant and susceptible controls, respectively,
(Brooks et al. 2007; Ni et al. 2008). The silk may-
sin levels in CML333, CML335, CML336 were
0.17, 0, 0.07%, respectively, of its fresh silk weight
(Ni et al. 2008), and the maysin level in CML338
was 0.48% of its fresh silk weight (unpublished
data). All fall armyworm neonates used in this
study were from a laboratory colony maintained
in the Insectary in the Crop Protection and Man-
agement Unit, USDA-ARS, Tifton, Georgia.

Artificial Insect Infestation and Damage Rating

Experimental plants used in the greenhouse
study were infested individually with 0 or 5 S. fru-
giperda neonates for each of the inbred line en-
tries when the plants were at the 6-leaf (V6)
stage. All plants in the field experimental plots
were planted in a single-row plot 3 m in length,
and were infested with 15-20 S. frugiperda neo-
nates/plant, with the protocol by Davis et al.
(1996). The insect injury ratings were conducted 7
and 14 d after the infestation with a scale of 1-9 as
described by Davis et al. (1992) and Smith et al.


December 2008







Ni et al.: Fall Armyworm Resistance in Maize


(1994). Briefly, 1 = no damage or few pinholes; 2 =
few short holes on several leaves; 3 = short holes
on several leaves; 4 = several leaves with short
holes and a few long lesions; 5 = several holes
with long lesions; 6 = several leaves with lesions <
2.5 cm; 7 = long lesions common on one half of the
leaves; 8 = long lesions common on one half to two
thirds of leaves; and 9 = most leaves with long le-
sions. While insect injury was rated by individual
plants in the greenhouse study, injury rating un-
der field conditions was recorded as the mean for
all plants in an experimental plot.

Plant Height, Stem Circumference, and Leaf Chloro-
phyll Content

Height and circumference of corn plants were
measured after the injury rating to assess the
impact of insect injury on plant vegetative
growth in the greenhouse study. Chlorophyll
content of experimental plants in both green-
house and field experiments was measured with
a SPAD-502 chlorophyll meter (Konica Minolta
Sensing, Inc., Osaka, Japan) on the top ex-
panded leaf with leaf collar of the plants. Leaf
chlorophyll content (pmol m'2) was calculated ac-
cording to a standard curve generated for this
chlorophyll meter (i.e., chlorophyll (pmol m-2) =
10 'M^ 02611), where M is the chlorophyll meter read-
ing (Markwell et al. 1995). While only chloro-
phyll content of the infested plants was mea-
sured for the field experiment, chlorophyll con-
tent of all experimental plants (both infested and
uninfested plants) was measured in the green-
house experiments.

Photosynthetic Measurements

Photosynthesis-related parameters were
measured on the plants used in the greenhouse
study. The photosynthesis rate (also known as
CO, exchange rate) of S. frugiperda-infested
and control plants was assessed with a LI-
6400R portable photosynthesis system (LI-COR
Inc., Lincoln, NE). In addition, the photosyn-
thetic capacity of the infested and control
plants was assessed with CO, (or A/Ci) and light
response curves. Because corn is a C4 plant, the
following parameters were used for the light
and CO, response curves. A light response curve
was generated by the gas exchange rates mea-
sured at light intensities at 2000, 1500, 1000,
500, 200, 100, 50, 20, 0 pmol photons m-2 s-1,
with a constant CO, concentration (400 ppm),
whereas the CO, response curve (also known as
assimilation rate plotted against intercellular
CO, concentration, or A/C, curve) was generated
by the gas exchange rates measured at
COconcentrations at 400, 300, 200, 100, 0, 400,
400, 600, 800 ppm, with a constant light inten-
sity of 1500 pmol photons m-2 s-1.


Experimental Design and Data Analysis

The field experiment utilized a randomized
complete block design with 6 corn-inbred lines as
the treatments. The greenhouse experiment was
conducted with the individual plant as an experi-
mental unit. The greenhouse study was a 6 x 2
factorial experiment that utilized a randomized
complete block design with 3 replications (blocks).
Two trials of the experiment were conducted. All
insect damage ratings and plant parameters were
analyzed by the PROC MIXED procedure and the
means were separated by Fisher's protected LSD
test (a = 0.05) (SAS Institute 2003). Both A/Ci and
light response curves were established with a
polynomial regression model in Sigma Plot (ver-
sion 8.02A) software (SYSTAT, Richmond, CA) at
7 and 14 d after infestation.

RESULTS AND DISCUSSION

Injury Ratings and Chlorophyll Content

Field injury ratings were significantly differ-
ent among the 6 maize inbred lines at both 7 d (F
= 6.1, df = 5, 17; P = 0.002) and 14 d (F = 11.6, df
= 5, 17; P = 0.0001) (Figs. 1A, B). The injury rat-
ings of CML333, CML336, and CML338 were the
same as the resistant control, Mp708, and consis-
tently lower than the susceptible control AB24E.
In contrast, the injury rating of CML335 was not
different from the susceptible control. Chloro-
phyll content measurements 14 d after infesta-
tion were significantly different among infested
plants (F = 9.2, df = 5, 201; P = 0.0001). Leaf chlo-
rophyll content in the injured CML338 and
Mp708 leaves was significantly higher than
AB24E and CML333 (Fig. 1C).
In addition to the injury ratings differing be-
tween uninfested and infested plants (F = 1143.6,
df = 1, 199; P = 0.0001) in the greenhouse study,
injury ratings were significantly different among
inbred lines (F = 3.7, df = 5, 199; P = 0.0033). In-
jury ratings differed between the 7 d and 14 d in-
festation durations (F = 7.0, df = 1, 199; P =
0.0089) and the two-way and three-way interac-
tions (P < 0.01). Thus, all injury rating data were
separately presented by the s so much, once
again.se a issue for your recital or not. phone:229-
387-0852.infestation durations (Figs. 2A, B).
When injury ratings were compared at 7 d after
infestation, higher injury occurred on AB24E
than on CML333, CML336, CML338, and Mp708
(Fig. 2A). This result was consistent with the field
screening data. However at 14 d, injury on AB24E
was not different from CML333, CML336, and
CML338 (Fig. 2B). Leaf chlorophyll content was
significantly different among inbred lines (F =
7.8, df= 5, 200;P = 0.0001), but not affected by ei-
ther infestation type or infestation duration, or by
any of the two- or three-way interactions among






Florida Entomologist 91(4)


December 2008


A
8

a
ab
b bc
C
2
2
o 0
SAL54E CLALSS.5 CLMLS CmLS 63~ Lt3 NqIJ(U
10
EB
a.
Ss


AB24E CML333 CML335 CML336 CL338 Mp708


4w ,4b
- bc bc I
3 bc






U
1 II


AB24E CML333 CML335 CML336 CML338 Mp708
Corn inhd line
Fig. 1. Field assessment of fall armyworm resistance
in the 6 selected corn inbred lines. (A) Damage rating 7
d after infestation; (B) Damage rating 14 d after infes-
tation; (C) Leaf chlorophyll content on damaged leaves
14 d after infestation. Means followed by the same letter
are not significantly different, Fisher's protected LSD
test (a = 0.05).




the inbred lines, infestation types, and infestation
durations (P > 0.05). Thus, the chlorophyll data
were combined and compared only among inbred
lines (Fig. 2C). In contrast to the field chlorophyll
data (Fig. 1C), chlorophyll content ofAB24E and
Mp708 was significantly higher than CML333


AB4E CM333 C0335 OCL336 04338 T708


S a a ab
2



inii
bo







AEB24E C333 CL335 CML336 CML338 Nt70B
Corn inbred ine
Fig. 2. Greenhouse evaluation of fall armyworm re-
sistance on 6 selected corn inbred lines.(A) Damage rat-
ing 7 d after infestation; (B) Damage rating 14 d after
infestation; (C) Leaf chlorophyll content means from the
pooled data collected at 7 and 14 d after infestation.
Means followed by the same letter are not significantly
different, Fisher's protected LSD test (a = 0.05).



and CML338. Both field and greenhouse data
showed that CML333, CML336, CML338, and
Mp708 were resistant to fall armyworm injury
compared to AB24E, although the injury ratings
varied between the field and greenhouse evalua-
tion results.







Ni et al.: Fall Armyworm Resistance in Maize


Plant Height, Stem Circumference, and Photosynthesis
Measurements

Plant height was significantly different among
inbred lines (F = 16.0, df = 5, 220; P = 0.0001), be-
tween uninfested and injured plants (F = 75.3, df
= 1, 220; P = 0.0001), and between 7 and 14-d in-
festation durations (F = 62.0, df = 1, 220; P =
0.0001). Plant height was significantly affected by
infestation duration x infestation type interaction
(F = 9.7, df = 1, 220; P = 0.0021). Plant height 7 d
after infestation was significantly different
among uninfested lines, but not among infested
lines (Fig. 3 A). Plant height of uninfested AB24E,
Mp708, and CML338 were significantly greater
than CML333, CML335, and CML336. Larval in-
festation significantly reduced plant height in all
entries except CML335.
Plant height 14 d after infestation was signif-
icantly affected by infestation types (F = 36.0, df =
1, 120; P = 0.0001), inbred lines (F = 19.0, df = 5,
122; P = 0.0008), and inbred line by infestation
type interactions (F = 4.7, df = 5, 120; P = 0.0005).
Furthermore, uninfested AB24E and CML338
plants were significantly taller than Mp708,
CML333, CML335, and CML336 plants (Fig. 3B).
In contrast, infested CML338 plants were the
tallest and CML336 plants were the shortest (Fig.
3B), which suggested that CML338 was tolerant
to the fall armyworm feeding injury.
Stem circumference was significantly different
among inbred lines (F = 13.0, df = 5, 220; P =
0.0001), and between infestation durations (F =
62.0, df = 1, 220; P = 0.0001), but not affected by
infestation types (F = 0.43, df = 1, 220; P =
0.5134). None of the two-way or three-way inter-
actions were significant (P > 0.05). Thus, data of
both infestation types were pooled and compared
between infestation durations (Fig. 3C). Stem cir-
cumference of AB24E, Mp708, and CML335 was
significantly greater than that of CML333 7 d af-
ter infestation, while 14 d after infestation stem
circumference of AB24E and Mp708 was greater
than that of CML333, CML335, and CML338
(Fig. 3C). The height and stem circumference of
the infested plants indicated that in general,
plant height was negatively affected but stem cir-
cumference was not affected by fall armyworm in-
festation.

Photosynthetic Rate Measurements

Survey measurement of photosynthetic rate
was significantly different among inbred lines (F
= 4.8, df = 5, 198; P = 0.0003), and between infes-
tation types (F = 31.0, df= 1, 198;P = 0.0001). The
photosynthetic rate of experimental plants was
affected by 3 two-way interactions (i.e., inbred
line by infestation type (F = 2.3, df = 5, 198; P =
0.0486), inbred line by infestation duration (F =
3.2, df = 5, 198; P = 0.0092), and infestation type


u au ab ab
bb
I 4

2
Z.2



A824E CML333 CM&335 CML336 C(2338 Mp708
Corn inbred line
Fig. 3. Effect of fall armyworm infestation on plant
growth parameters in the greenhouse study. (A) Plant
height (cm) 7 d after infestation; (B) Plant height (cm)
14 d after infestation; (C) Stem circumference (cm) on
uninfested and injured plants. Bars of the same type
with the same letter (a-c for uninfested, or A-D for in-
fested) are not significantly different, Fisher's protected
LSD test (a = 0.05). The or ** between uninfested and
injured plants within a germplasm entry denotes a sig-
nificant difference at a = 0.05 or a = 0.01, respectively.


by infestation duration (F = 13.7, df = 1, 198; P =
0.0003)). The three-way interaction was not sig-
nificant (P > 0.05). In contrast to the plant height
and stem circumference data, photosynthesis rate
was not affected by either infestation duration or
the three-way interaction of inbred line by infes-


AB4. CMaLs33 C0 s35 CMN3s CAU33$ WM70


AB24E CML333 CML335 (CL336 CM0 338 MpT7M







Florida Entomologist 91(4)


station type by infestation duration (P > 0.05).
Thus, the photosynthesis rate data of both 7 and
14 d measurements were combined and presented
in pairs with infestation types (Fig. 4). Photosyn-
thetic rate of uninfested plants was not signifi-
cantly different (F = 1.7, df = 5, 100; P = 0.1414)
among inbred lines, whereas the photosynthetic
rate of injured plants was different (F = 6.3, df =
5, 106; P = 0.0001). Mp708 had the highest photo-
synthetic rate, while CML335 showed the lowest
photosynthetic rate (Fig. 4). Such variation in
photosynthetic rate among the corn seedlings
with less foliar injury suggested that the corn-in-
bred lines might possess different physiological
mechanisms that confer varying levels of resis-
tance. Furthermore, irrespective of resistance, in-
sect injury significantly reduced photosynthetic
rate in AB24E, CML333, CML335, and CML336,
but had no effect on photosynthetic rate in
CML338 and Mp708 (Fig. 4). The results sug-
gested that the last 2 inbred lines were tolerant to
fall armyworm feeding injury.

Photosynthetic Capacity Measurements

Based on the injury rating data collected from
the field and greenhouse experiments, 5 inbred
lines (Mp708 and AB24E controls plus 3 CIM-
MYT lines with low injury ratings, CML333,
CML336, and CML338) were selected to assess
the impact of infestation on the photosynthetic
capacity of the plants. Photosynthetic capacity
was assessed with CO2 (or A/Ci) and light re-
sponse curves. Insect injury significantly reduced
the light-harvesting capacity of AB24E 7 d
(Fig. 5A), but not 14 d after infestation, nor were


40 --
unrilted
nfested
a a A
E 30 a AB


20
o 0




o



AB24E CML33 CML335 CM&336 CML338 k08
Corn inbred line

Fig. 4. Photosynthetic rates of uninfested control
and fall armyworm-injured corn plants from the 6 in-
bred lines. Bars of the same type with the same letter
(a-c for uninfested, or A-D for infested) are not signifi-
cantly different, Fisher's protected LSD test (a = 0.05).
The or ** between uninfested and injured plants
within a germplasm entry denotes a significant differ-
ence at a = 0.05 or a = 0.01, respectively.


a i.


t 0

i 2U-

l0-


A
o control: y = -2.53 + (45.37x)/(489.29+x)
(r= 0.95, F = 139.8, df= 2, 33, P= 0.0001)


..---. -

4 --l


infested: y =-2.64 +(31.62x)/(305 41+x)
(r= 0.97. F = 277.28 df = 2. 33. P= 0.0001)


0 so 1lo000 1500


0 500 1I00 1500 000 2500

C
o control: y = -3 0 + (44.4x)/(393.88+x)
40 (r=O.89,F=64.72. d= 2.33.P=0.0001)

I --- -1-
4---~ a-
20
. .


o infested: y = -2.62 + (35.44xY(351.75+x)
(r= 0.97. F = 276.47 df = 2, 33, P= 0.0001)

O 500 1000 1500 2000 2500
Light intensity (tunol photon mf2seiC')

Fig. 5. Light response curves of uninfested and the
fall armyworm-injured plants 7 d after infestation. (A)
AB24E (susceptible control); (B) CML333 (with moder-
ate level of silk maysin, 0.17% of fresh silk weight); and
(C) CML336 (with low level of silk maysin, 0.07% of
fresh silk weight).



the A/Ci curves of AB24E affected 7 or 14 d after
infestation. Thus, the reduction of photosynthetic
rate in AB24E might be the result of the reduction
of light-harvesting capacity (or the light reaction)
of the photosynthesis process, but not the carbon
assimilation (or the dark reaction) process. In
contrast, CML333 showed an increase in photo-
synthetic rate in injured plants at high light in-
tensity (> 1000 photons m-2 s-') 7d after infestation
(Fig. 5B). There was no difference in A/Ci curves 7


B
o control: y = -1.97 + (34.06x)(378.61+x)
(r= 0.99, F = 565.47, df= 2. 33. P=0.0001)

S_ -----^-- 5
_-
.,- --. --



S* infested: y = -2.91 + (45.48x)/(452.3+x)
(r= 0.98, F = 337.8 df= 2, 33, P= 0.0001)


December 2008







Ni et al.: Fall Armyworm Resistance in Maize


or 14 d after infestation. Although CML336
showed significantly lower injury ratings than
AB24E, the A/Ci and light response curves were
very similar between AB24E and CML336 (Fig.
5C).
The reduction of photosynthetic capacity in the
inbred lines with low injury ratings might indi-
cate that plants reduced their growth and in-
creased their biosynthesis of secondary metabo-
lites to defend against insect herbivory. Thus
CML333 and CML336 might possess antibiotic
resistance to insect feeding. Significant reduction
in photosynthetic rate in injured susceptible
plants with high injury ratings (i.e., AB24E, and
CML335), and CML333, CML 335, and CML336
seedlings was similar to the previous findings in
D. noxia-injured wheat leaves (Haile et al. 1999),
and common smut (Ustilago maydis L.)-infected
maize leaves (Horst et al. 2006). It is intriguing
that a significant photosynthetic rate reduction
occurred in insect-susceptible, and some but not
all insect-resistant crop plants. Thus, the findings
suggested that photosynthesis might not be di-


rectly related to these insect-resistant inbred
lines with reduced photosynthetic rate, as shown
by the susceptible inbred AB24E.
In contrast, A/Ci and light response curves of
CML338 were different from AB24E, CML333,
and CML336. The A/Ci curves 7 and 14 d after in-
festation showed that injured plants increased
photosynthetic rate in response to the change in
CO2 levels compared with control plants (Figs. 6A,
B), which suggested that plants were tolerant to
insect feeding by compensatory growth. In addi-
tion, the light response curves of CML338 showed
no difference 7 or 14 d after infestation (Figs. 6C,
D), which indicated that injury had no effect on
the light-harvesting capacity of CML388 seed-
lings. This was opposite to the findings of
CML333 and CML336 as shown in Figs. 5A and
B. Using the combination of the photosynthetic
survey data (Fig. 4) and the A/Ci and light re-
sponse curve data (Figs. 5 and 6), we conclude
that CML338 seedlings were tolerant to injury,
but the resistance in CML333 and CML336 might
not be directly related to plant photosynthesis.


o control: y= -0.20 + (31.25xy(140.88+x)
(r= 0,92, F = 76.37. df= 2, 29, P= 0.0001)



I /-- ----
/^^ 5^---'"


Infested: y= -0.77 + (38.93xy( 113.25+x)
S (r= 0.85, F= 37.56, df= 2, 29, P= 0.0001)


1000


CO, lewIl (ppm)
50


o control: y = -2.95 +(48.42x)/(45981+x)
(r= 0.91, F= 81.08, df= 2,3, P=OD 0 1)


T ^ ^----i-





infested: y- -3.16+ (40.64x)/(411.74+
(r= 0.98 F = 501.17df= 2. 33. P= 0


x)1)
001)


a 500 IODO 15s0 2000
Light intensity (pmol photon m sec')


CO level (ppm)


o control: y=-1.6 +(27.84x)/(343.15+x)
(r= 0,86, F = 45.26, df = 2, 33, P= 0.0001)








(r= 0.95, F = 165.31 df = 2, 33 P= 0.0001)


1500 20o 2O00


0 W0O 100


Ught intensity (pmol photon m" sect')


Fig. 6. Photosynthetic capacity measurement of the inbred line CML338 (with high level of silk maysin, 0.48%
of fresh silk weight). A/Ci (or CO2 response) curves recorded 7 (A) and 14 d (B) on uninfested (empty circle) and fall
armyworm-injured plants (filled circle), respectively; and light response curves recorded 7 (C) and 14 d (D) on un-
infested and fall armyworm-injured plants, respectively.


200 400 o00 800 1000 0 20 400 600 00
20 4Cs EOD Boo 1ooD AOO bO Om 80


M


-F


I











The correlation between insect resistance and
plant secondary metabolites (e.g., chlorogenic ac-
ids) needs to be further examined. Furthermore,
neither the A/Ci nor light response curves of
Mp708 were different between uninfested and in-
jured plants 7 or 14 d after infestation, which in-
dicated that Mp708 might be tolerant to the in-
jury as well.
In summary, the current study showed that
the inbred lines CML333 (with moderate silk
maysin), CML336 (with low silk maysin) and
CML338 (with high silk maysin) were resistant to
fall armyworm feeding at the seedling stage, and
CML335 (without silk maysin) was susceptible.
The findings indicate that multiple insect resis-
tance across multiple growth stages of corn plants
is promising, and merits further detailed recipro-
cal examinations between plant growth stages.
Fall armyworm resistance in CML333 and
CML336 was not directly related to photosynthe-
sis, because the reduction in photosynthetic rate
is similar to the susceptible control. At the same
time, CML338 and Mp708 were categorized as
tolerant to insect herbivory because uninfested
and injured plants showed no differences in either
survey measurements of photosynthetic rate,
light response curves, or photosynthetic rate in
the A/Ci curves.


ACKNOWLEDGMENTS

Mention of trade names or commercial products in
this article is solely for the purpose of providing specific
information and does not imply recommendation or en-
dorsement by the U. S. Department of Agriculture. We
thank David G. Riley and Yigen Chen (Department of
Entomology, University of Georgia-Tifton) for critical
reviews of the earlier version of the manuscript.


REFERENCES CITED

ABEL, C. A., AND J. J. ADAMCZYK, JR. 2004. Relative con-
centration of CrylA in maize leaves and cotton bolls
with diverse chlorophyll content and corresponding
larval development of fall armyworm (Lepidoptera:
Noctuidae) and southwestern corn borer (Lepi-
doptera: Crambidae) on maize whorl leaf profiles. J.
Econ. Entomol. 97: 1737-1744.
BROOKS, T. D., B. S. BUSHMAN, W. P. WILLIAMS, M. D.
MCMULLEN, AND P. M. BUCKLEY. 2007. Genetic ba-
sis of resistance to fall armyworm (Lepidoptera: Noc-
tuidae) and southwestern corn borer (Lepidoptera:
Crambidae) leaf-feeding damage in maize. J. Econ.
Entomol. 100: 1470-1475.
DAVIS, F. M., S. S. NG, AND W. P. WILLIAMS. 1992. Visu-
al Rating Scales for Screening Whorl-stage Corn for
Resistance to Fall Armyworm. Technical Bulletin
186, Mississippi Agric. Forestry Exp. Sta., 9 pp.
DAVIS, F. M., B. R. WISEMAN, W. P. WILLIAMS, AND N.
W. WIDSTROM. 1996. Insect colony, planting date,
and plant growth stage effects on screening maize
for leaf-feeding resistance to fall armyworm (Lepi-
doptera: Noctuidae). Florida Entomol. 79: 317-328.


December 2008


FRANZEN, L. D., A. R. GUTSCHE, T. M. HENG-MOSS, L. G.
HIGLEY, G. SARATH, AND J. D. BURD. 2007. Physio-
logical and biochemical responses of resistant and
susceptible wheat to injury by Russian wheat aphid.
J. Econ. Entomol. 100: 1692-1703.
HAILE, F. J., L. G. HIGLEY, X. NI, AND S. S. QUISENBER-
RY. 1999. Physiological and growth tolerance in
wheat to Russian wheat aphid (Homoptera: Aphid-
idae) injury. Environ. Entomol. 28: 787-794.
HENG-MOSS, T. M., X. Ni, T. MACEDO, J. P. MARKWELL,
F. P. BAXENDALE, S. S. QUISENBERRY, AND V. TOL-
MAY. 2003. Comparison of chlorophyll and carotenoid
contents in Russian wheat aphid (Homoptera: Aphi-
didae)-infested wheat isolines. J. Econ. Entomol. 96:
475-481.
HORST, R. J., T. ENGELSDORF, U. SONNEWALD, AND L.
M. VOLL. 2008. Infection of maize leaves with Usti-
lago maydis prevents establishment of C4 photosyn-
thesis. J. Plant Physiol. 165: 19-28.
MACEDO, T., L. HIGLEY, X. NI, AND S. S. QUISENBERRY.
2003. Light activation of Russian wheat aphid-elicit-
ed physiological responses in susceptible wheat. J.
Econ. Entomol. 96:194-201.
MACEDO, T. B., R. K. D. PETERSON, C. L. DAUSZ, AND
D. K. WEAVER. 2007. Photosynthetic responses of
wheat, Triticum aestivum L., to defoliation patterns
on individual leaves. Environ. Entomol. 36: 602-608.
MARKWELL, J., J. C. OSTERMAN, AND J. L. MITCHELL.
1995. Calibration of the Minolta SPAD-502 leaf chlo-
rophyll meter. Photos. Res. 46: 467-472.
NI, X., AND S. S. QUISENBERRY. 2003. Possible roles of
esterase, glutathione S-transferase, and superoxide
dismutase activities in understanding aphid-cereal
interactions. Entomol. Exp. Appl. 108: 187-195.
NI, X., M. D. KRAKOWSKY, G. D. BUNTIN, B. G. RECTOR,
B. GUO, AND M. E. SNOOK. 2008. Identification of
multiple ear-colonizing insect and disease resistance
in CIMMYT maize inbred lines with varying levels
of silk maysin. J. Econ. Entomol. 101: 1455-1465.
NI, X., S. S. QUISENBERRY, T. HENG-MOSS, J. J. MARK-
WELL, G. SARATH, R. KLUCAS, AND F. BAXENDALE.
2001a. Oxidative responses of resistant and suscep-
tible cereal leaves to symptomatic and nonsymptom-
atic cereal aphid (Hemiptera: Aphididae) feeding. J.
Econ. Entomol. 94: 743-751.
NI, X., S. S. QUISENBERRY, T. HENG-MOSS, J. MARK-
WELL, L. HIGLEY, F. BAXENDALE, G. SARATH, AND
R. KLUCAS. 2002. Dynamic change in photosynthet-
ic pigments and chlorophyll degradation elicited by
cereal aphid feeding. Entomol. Exp. Appl. 105: 43-
53.
NI, X., S. S. QUISENBERRY, J. MARKWELL, T. HENG-
MOSS, L. HIGLEY, F. BAXENDALE, G. SARATH, AND
R. KLUCAS. 200 b. In vitro enzymatic chlorophyll ca-
tabolism in wheat elicited by cereal aphid feeding.
Entomol. Exp. Appl. 101: 159-166.
NI, X., W. XU, M. D. KRAKOWSKY, G. D. BUNTIN, S. L.
BROWN, R. D. LEE, AND A. E. COY. 2007. Field screen-
ing of experimental corn hybrids and inbred lines for
multiple ear-feeding insect resistance. J. Econ. Ento-
mol. 100: 1704-1713.
PETERSON, R. K. D., C. L. SHANNON, AND A. W. LENS-
SEN. 2004. Photosynthetic responses of legume spe-
cies to leaf-mass consumption injury. Environ. Ento-
mol. 33: 450-456.
RECTOR, B. G., M. E. SNOOK, AND N. W. WIDSTROM.
2002. Effect of husk characteristics on resistance to
corn earworm (Lepidoptera: Noctuidae) in high-


Florida Entomologist 91(4)







Ni et al.: Fall Armyworm Resistance in Maize


maysin maize populations. J. Econ. Entomol. 95:
1303-1307.
SAS INSTITUTE. 2003. SAS System (version 9.1) for
Windows. SAS Institute, Cary, NC.
SMITH, C. M., Z. R. KHAN, AND M. D. PATHAK. 1994.
Evaluation of plants for insect resistance, pp. 17-114
In C. M. Smith, Z. R. Khan, and M. D. Pathak [eds],


Techniques for Evaluating Insect Resistance in Crop
Plants. CRC Press, Boca Raton, FL.
WISEMAN, B. R., F. M. DAVIS, W. P. WILLIAMS, AND N.
W. WIDSTROM. 1996. Resistance of a maize popula-
tion, FAWCC(C5), to fall armyworm larvae (Lepi-
doptera: Noctuidae). Florida Entomol. 79: 329-
336.







Florida Entomologist 91(4)


December 2008


REVIEW OF FALL ARMYWORM (LEPIDOPTERA: NOCTUIDAE)
GENETIC COMPLEXITY AND MIGRATION

RODNEY N. NAGOSHI AND ROBERT L. MEAGHER
Center for Medical, Agricultural and Veterinary Entomology, USDA-ARS, Gainesville, FL 32608


ABSTRACT

The fall armyworm, Spodoptera frugiperda (J. E. Smith) is a significant economic pest in the
western hemisphere, causing substantial losses in corn, sorghum, forage, and turf grasses.
Although fall armyworm does not survive severe winters, it infests most of the central and
eastern United States and Canada because of annual migrations from overwintering sites in
Florida and Texas. A detailed description of these movements is a prerequisite for identify-
ing the factors that determine the timing and direction of migration and for developing mod-
els that can predict the severity of infestations at the migratory destinations. Complicating
this effort is genetic heterogeneity within the species, which increases phenotypic variabil-
ity. Particularly important are 2 "host strains", defined by a preferential association with ei-
ther large grasses (designated corn-strain), such as corn and sorghum, or smaller grasses
(designated rice-strain), such as rice and bermudagrass. This paper reviews recent studies
examining the genetic complexity of fall armyworm populations, including characteristics of
the 2 strains and the possibility of subgroups within strains. The use of this information to
monitor short and long distance movements is discussed.

Key Words: Spodoptera frugiperda, migration, haplotype, cytochrome oxidase I


RESUME

El gusano cogollero, Spodoptera frugiperda (J. E. Smith) es una plaga econ6micamente sig-
nificativa en el hemisferio occidental, que causa perdidas substanciales en maiz, sorgo, fo-
rraje y c6spedes. Aunque el gusano cogollero no sobrevive los inviernos severos, siempre
infesta la mayor parte de las regions central y este de los Estados Unidos y Canada por la
eigraciones anuales de los sitios de invernaci6n en los estados de Florida y Texas. Una des-
cripci6n detallada de estos movimientos es un prerrequisito para identificar los factors que
determinan el tiempo y la direcci6n de la migraci6n y para desarrollar models que pueden
predecir la severidad de las infestaciones en las destinaciones donde emigra. Complicando
este esfuerzo es la heterogeneidad gen6tica que existe en la especie, la cual aumenta la va-
riabilidad fenotipica. Particularmente important son 2 "cepas de hospedero", definidas por
su asociaci6n preferencial con pastos grandes (designada la cepa de maiz), tales como el maiz
y sorgo, o pastos pequeios (designados como la sepa de arroz), tales como el arroz y past
bermuda. En este articulo se revisa los studios recientes que examinan la complejidad ge-
n6tica de poblaciones del gusano cogollero, incluyendo las caracteristicas de 2 cepas y la po-
sibilidad de subgrupos entire las cepas. Se discute el uso de esta informaci6n para el
monitoreo de su movimiento por a distancias cortas y largas.


The Two Host Strains

Molecular markers remain the most accurate
indicators of strain identity (McMichael & Prow-
ell 1999; Prowell et al. 2004) and the most conve-
nient of these are mitochondrial haplotypes that
can be assayed by polymerase chain reaction
(PCR) amplification methods (Levy et al. 2002;
Meagher & Gallo-Meagher 2003; Nagoshi et al.
2006b). Strain-specific polymorphisms within the
mitochondrial Cytochrome Oxidase I (COI) gene
consistently divide fall armyworm populations
into 2 haplotype groups. The demonstration that
these delineate the host strains is based primarily
on the observation that the haplotypes are asym-
metrically distributed among plant hosts in the


field, with the corn-strain haplotype typically rep-
resenting about 80% of the larvae isolated from
corn and the rice-strain haplotype in over 95% of
the larvae collected from pasture or turf grasses
(Pashley 1989; Prowell 1998).Initial descriptions
of fall armyworm assumed a genetically homoge-
neous population capable of large variations in
such behaviors as plant host choice. This was
shown to be overly simplistic by the demonstra-
tion of 2 morphologically identical host strains as
originally indicated by comparisons of electro-
phoretic protein variants from samples collected
from large grasses (designated corn-strain), such
as corn and sorghum, or smaller grasses (desig-
nated rice-strain), such as rice and bermudagrass
(Pashley et al. 1985; Pashley 1986; Pashley et al.







Nagoshi & Meagher: Fall Armyworm Migration


1987a Pashley 1988b). There are several reports
describing differential effects of plant hosts on the
viability and development of the 2 strains (Pash-
ley 1986; Pashley 1988b; Whitford et al. 1992; Pa-
shley 1993; Pashley et al. 1995; Meagher et al.
2004), as well as different levels of sensitivities to
a variety of pesticides (McCord & Yu 1987; Yu
1991; Veenstra et al. 1995; Adamczyk et al. 1997;
Yu 1999). There has been recent interest in exam-
ining Bacillus thuringiensis Berliner (Bt) suscep-
tibility in fall armyworm, although in many cases
the strain identity of the lines being tested was
unclear (Adamczyk et al. 1997; Lynch et al. 1999;
Adamczyk et al. 2001; Dequech et al. 2005; Po-
lanczyk & Alves 2005; Monnerat et al. 2006; Wil-
liams et al. 2006; Chilcutt et al. 2007). Despite
these studies, a behavioral or physiological bioas-
say capable of reliably distinguishing between
strains has yet to be described.
Studies of fall armyworm populations from
Brazil indicate that subpopulations with the
same host preferences as the rice-strain and corn-
strain are present in South America (Busato et al.
2004; Busato et al. 2005; Nagoshi et al. 2007b;
Machado et al. 2008), and that these can be de-
tected by the same strain-specific mitochondrial
markers used to distinguish North American
strains (Nagoshi et al. 2007b). This supports a rel-
atively ancient divergence of the 2 strains (Lew-
ter et al. 2006), and further suggests that the fac-
tors driving divergence is present throughout
most of the western hemisphere.

Evidence of a Hybrid Subpopulation

The persistence of genetic and physiological dif-
ferences between the host strains strongly suggests
limitations to hybridization between strains. Sup-
porting this hypothesis was the observation of direc-
tional interstrain mating biases. In crosses between
corn-strain females to rice-strain males, no sper-
matophores were transferred to the females, while
the reciprocal cross produced normal levels of fertil-
ity (Pashley & Martin 1987). The hybrid females
produced by interstrain crosses could only success-
fully mate with their hybrid brothers. In compari-
son, the hybrid males could mate with females of ei-
ther strain with near normal fertility. Similar direc-
tional mating bias was observed in field studies us-
ing multilocus genetic analysis where 2 or more
genetic markers were used to identify strains, and
discrepancies in identity were assumed to result
from interstrain hybridization. In one set of studies,
combinations of strain-biased markers from es-
terase allozymes, mitochondrial DNA (mtDNA),
and AFLPs were use to identify samples discordant
for at least one marker (Prowell et al. 2004). A dis-
cordance frequency of 16% was observed with a
slight majority (54%) displaying a mitochondrial cy-
totype consistent with a rice-strain female to corn-
strain male mating.


A second study compared mtDNA with a tan-
dem-repeat sequence called FR (for Fall army-
worm Rice strain), which was reported to be local-
ized to the sex-chromosomes and present in large
clusters only in the rice-strain genome of the Mis-
sissippi populations tested (Lu et al. 1994). A
PCR-based method for detecting FR sequences
was developed that allowed rapid analysis of sin-
gle individuals (Nagoshi & Meagher 2003). In a
multiyear sampling of field collected samples
from several regions we confirmed the rice-strain
biased distribution of FR. Over 70% of the mtR
population sampled in Brazil in 2005 were FR'
compared to 14% of the mtc group (Nagoshi et al.
2008), while about 40% mtR specimens from Texas
(2006-7) and Florida (2003-7) were FR compared
to about 10% of the mtc population (Nagoshi &
Meagher 2003; Nagoshi et al. 2008). Such geo-
graphical and temporal consistency strongly sup-
ports the existence of mating or selection biases
that are preventing a more homogeneous distri-
bution of FR between the 2 strains.
There was an unexpectedly high proportion of
mt FRO samples, which in Florida and Texas rep-
resented the majority of the mt population. It is
possible that this "discordant" class results from
interstrain matings between rice-strain females
and corn-strain males, an explanation supported
by the observation that this genotype displayed a
different plant host distribution pattern than ei-
ther parental strain (Nagoshi et al. 2006a). If this
pattern is a result of interstrain mating then it
suggests a strong bias for rice-strain females mat-
ing with corn-strain males relative to the recipro-
cal pairing (Pashley & Martin 1987), as the recip-
rocal "hybrid" configuration, mtc FR is relatively
rare (Nagoshi & Meagher 2003; Nagoshi et al.
2008).
The observation that the putative hybrids may
behave differently from the parents is potentially
important because it identifies an additional
group besides the host strains whose behavior
must be considered for a complete understanding
of fall armyworm population dynamics. Prowell et
al. (2004) also observed that the majority of the
hybrids with the rice-strain mitochondrial cyto-
type (but other corn-strain markers) were found
in a corn habitat, though they interpreted this as
possibly resulting from increased opportunities
for interstrain mating in corn habitats. While we
cannot distinguish between these explanations,
we note that the behavioral and physiological dif-
ferences reported for the 2 strains would suggest
that interstrain hybridization should have signif-
icant phenotypic consequences.
The possibility of genetic subpopulations in ad-
dition to the 2 strains could explain discrepancies
observed between the published reports on fall ar-
myworm behavior. In particular, 2 laboratories
were unable to find directional differences in in-
terstrain mating in controlled crosses (Whitford







Florida Entomologist 91(4)


et al. 1988; Quisenberry 1991). Instead normal
fertility were observed in all crosses between
strains and when using hybrids. One possibility is
that mating behavior is a labile trait, perhaps
sensitive to artificial rearing and testing condi-
tions (Pashley 1993; Prowell et al. 2004). Alterna-
tively, it may be that the fall armyworm popula-
tion is more genetically complex than that de-
scribed by the 2 strains. There may be a range of
genotypes that exhibit different mating behaviors
and strain specificities such that independently
derived colonies of the same strain may not be
equivalent.

Genome-wide Examination of Genetic Heterogeneity

There have been several studies with genome-
wide methods to study genetic variation in fall ar-
myworm populations. These include the RAPD
(Random Amplification of Polymorphic DNA),
RFLP (Restriction Fragment Length Polymor-
phism) and AFLP (Amplified Fragment Length
Polymorphism) techniques (Lu et al. 1992; Mc-
Michael & Prowell 1999; Busato et al. 2004; Prow-
ell et al. 2004; Busato et al. 2005; Martinelli et al.
2006; Clark et al. 2007). Attempts to identify ge-
netic markers that can distinguish between popu-
lations from geographically distant regions have
given mixed results. A survey of fall armyworm
from maize and cotton plants in Brazil with
RAPD identified some clustering of genotypes ac-
cording to geographical origin, with only limited
gene flow evident between sites. However, AFLP
surveys that sampled populations from such dis-
tantly separated locations as those in Argentina,
Brazil, Mexico, Puerto Rico, and Iowa failed to
find evidence of clusters associated with geogra-
phy (Clark et al. 2007; Martinelli et al. 2007). The
overall results suggest that fall armyworm popu-
lations are genetically heterogeneous with inter-
breeding occurring generally throughout the
western hemisphere (Clark et al. 2007). If so, then
identifying genetic markers that can accurately
and consistently identify the geographical origin
of a population will be difficult by these methods.
These molecular studies bring into question
how the host strains maintain their integrity in
the face of substantial within-strain genetic vari-
ation and evidence of significant interstrain hy-
bridization. One proposed explanation was based
on the observations that strain-specific genetic
markers represented a small minority of the de-
tected polymorphisms, with the majority of these
either mapping to the sex-chromosomes or, in the
case of the mitochondrial haplotypes, displaying a
sex-restricted inheritance pattern (Prowell 1998).
If the sex chromosomes contain the strain-speci-
fying genes then these may exhibit limited gene
flow compared to the more profligate mixing of
the rest of the genome. There is evidence for sex-
chromosome biased speciation in Lepidoptera in


general and fall armyworm specifically (Sperling
1994; Prowell 1998). Therefore, techniques that
use autosomal markers to examine population
structure may not be successful in identifying fall
armyworm strains.
Several studies identified phylogenetic group-
ings consistent with strain designations (Lu et al.
1992; McMichael & Prowell 1999; Busato et al.
2004; Prowell et al. 2004), with some indications
that interstrain hybridization could be detected
(McMichael & Prowell 1999). However, an AFLP
survey comparing populations from the U.S., Cen-
tral America, and South America failed to find
clusters associated with plant host even in com-
parisons between samples collected from ber-
mudagrass and maize, where strain differences
would have been expected (Clark et al. 2007). This
could indicate variability in strain host use, as
has occasionally been observed with rice-strain
predominating on corn (Nagoshi & Meagher
2004; Prowell et al. 2004), or the use of AFLP
markers that were not specific to the strains. The
latter is a distinct possibility given the substan-
tial genetic heterogeneity observed in fall army-
worm populations (Clark et al. 2007), even when
sampling inbred laboratory lines (Lu et al. 1992).

Local Population Movements Between Corn and Cotton
Habitats

The most simple and direct method to identify
the strain infesting a plant is to analyze larvae for
strain identity. But this is problematic for plant
hosts that are secondary or sporadic targets of in-
festations where finding larvae is difficult and
any such collections tend to be biased for unusual
"outbreak" events when infestation levels are ab-
normally high. These may not be representative
of the typical behavior of the species. Another is-
sue is that larval surveys may not be predictive of
adult numbers if there are substantial strain dif-
ferences in fitness during pre-adult stages (Fitt
1989), a major problem when trying to estimate
the contribution of a particular plant host to the
overall fall armyworm population.
A useful method for estimating plant host us-
age was developed for the study of Helicoverpa
zea (Boddie), which like fall armyworm is a pest of
corn and cotton (Gould et al. 2002). The stable
carbon isotope ratio (13C/12C) in adult wings, com-
monly designated as 613C, was used to determine
whether the specimen arose from a C3 (i.e., cotton)
or C4 (i.e., corn) plant host. Studies in Lepi-
doptera have found that while adult behavior can
influence the isotope ratio, the primary determi-
nant is the larval diet (Ponsard et al. 2004). Gould
et al. (2002) was able to show that corn can serve
as a refuge for H. zea infesting Louisiana Bt cot-
ton and used the observed seasonal changes in
the proportions of C3 and C4 moths to infer pat-
terns of plant host use and migratory behavior.


December 2008







Nagoshi & Meagher: Fall Armyworm Migration


This strategy was used to identify of the strain
of fall armyworm most likely to infest cotton in
the U.S. Preliminary studies comparing protein
polymorphisms showed that larvae collected from
cotton in Ecuador were more similar to the
corn-strain than the rice-strain (Pashley 1986),
and genetic examination of Brazilian fall army-
worm populations showed substantial gene flow
between larvae isolated from corn and cotton
fields (Martinelli et al. 2006). While both imply
that the corn-strain infests both corn and cotton,
it is not clear whether this observation applies to
U.S. fall armyworm, the contribution of cotton to
the overall population, or if the rice-strain might
also be a significant cotton pest.
To address these issues, fall armyworms were
collected by pheromone trapping in cotton fields
within the Mississippi delta region during 2004
and 2005 and tested both for their carbon isotope
ratios and strain identity (Nagoshi et al. 2007c).
The prediction was that the fall armyworm sub-
population arising from cotton would display a
higher proportion with a C3 signature in a pattern
corresponding to the availability of cotton at sus-
ceptible development stages. Fall armyworm can
feed on squares, blooms, and bolls and has an ap-
proximately 37-d developmental cycle on cotton
(Pitre & Hogg 1983). Assuming that infestation
began in mid-Jun when approximately half the
crop was squaring, the first adults arising from
cotton should be present by late summer, with
numbers increasing into the fall, and declining af-
ter harvesting. The corn-strain best displayed the
expected pattern. After being largely absent dur-
ing the early cotton growing season, the C3 corn-
strain population increased rapidly in the Aug to
Oct period (Fig. 1). During these collections, the
proportion of corn-strain developing from C3
plants was at its highest levels, reaching averages
of 17% in 2004 and 39% in 2005 that were signif-
icantly higher than the preceding and subsequent
periods. These results are all consistent with the
corn-strain, but not the rice-strain, infesting and
successfully developing on cotton (Nagoshi et al.
2007c). The predominance of adults with the C4
signature early in the growing season further in-
dicates that the infesting population originally
developed on a C4 host, most likely corn since it is
a preferred host of the corn-strain and large
plantings were present within a few miles of the
test site.

Long-range Migration Patterns

Historical studies. Various attempts have been
made to describe the annual migrations of fall ar-
myworm in North America. The only regions in
the U.S. in which the insect is consistently know
to survive the winter are southern Florida and
southern Texas (Luginbill 1928). One of the early
descriptions of migration from these overwinter-


S7]

1B 60



Jun2-Aug2 Aug93-at1
80
2W5







MayZ20 Aug2 Am3 -Oct
mlnsqa W.m" r e nf$qiw-g bo


*C3cs
MC3RS
DCRCS
OC4RS


Oa.2-N 1








onr-oa 27
harv"


Fig. 1. Proportion of field-collected fall armyworm of
a given strain and 1C value in the Mississippi delta re-
gion during the 2004 and 2005 growing seasons. (n) rep-
resents the total number of samples tested during each
time period. Data from Nagoshi et al. (2007c).


ing areas came from observations of the timing of
fall armyworm appearance in selected locations
(Luginbill 1928). Populations in southern Texas
appear to move northward into Oklahoma and
northeasterly following the coastal plain and into
the Mississippi river valley. The southern Florida
populations were believed to migrate to northern
Florida typically by early May and into north-cen-
tral Georgia by Jun, continuing east of the Appa-
lachians into South Carolina by Jul. Fall army-
worm first appears in central Tennessee by mid-
Jul, in southeastern Kansas in late Jul, and in the
Ohio Valley and Maryland during Aug and Sep
(Luginbill 1928).
Synoptic meteorological analysis was used to
calculate atmospheric trajectories that seemed to
generally corroborate observations of fall army-
worm at migratory destinations (Rose et al. 1975;
Westbrook & Sparks 1986; Mitchell et al. 1991).
When coordinated with pheromone trap captures,
it was evident that favorable wind currents were
conducive to the spread of fall armyworm into un-
infested areas (Mitchell et al. 1991). These types
of studies also provided evidence for a reverse
(south to north) migration in the fall (Pair et al.
1987; Mitchell et al. 1991). However, the resolu-
tion of the trap data was not sufficient to identify
the source of the migrant populations.
Pheromone trap methods in combination with
larval collections were used in an extensive sur-
vey of southeastern states during 2 "off-years" in
which widespread fall armyworm outbreaks were
not observed in any location (Pair et al. 1986). The
strategy again was to use the sequential appear-
ance of the insect in different locations to esti-
mate migration movement. It was observed that







Florida Entomologist 91(4)


infestations occurred earlier and were more se-
vere in the Florida panhandle and Baldwin Co.,
AL (located just west of Pensacola, FL) than in
southern Georgia. This suggested to the authors a
northwesterly movement from southern Florida,
perhaps infesting the Mississippi Valley states, as
well as a subsequent northeastern movement into
Georgia and South Carolina (Pair et al. 1986).
Population movements could also be inferred
by a comparison of disease or pesticide resistance
of fall armyworm from different locations. Exam-
ination of resistance to carbaryl and methomyl
suggested that in 1977, infestations of the eastern
states could have originated in both Texas and
Florida while infestations farther west were de-
rived primarily from populations in Texas and
Mexico (Young 1979). However, a different study
comparing pesticide resistance suggested that the
Texas population was isolated from fall army-
worm in Georgia and Florida, and that Missis-
sippi and Florida populations were similarly sep-
arated (Pitre 1988). A comparison of resistance to
nuclear polyhedrosis virus suggested that fall ar-
myworm from Brazil was at least partly isolated
from those around the Gulf of Mexico, but that
there was mixing of populations through Texas,
Louisiana, Florida (Fuxa 1987). While not com-
pletely consistent, these studies suggest regional
differences between fall armyworm populations
that may derive from geographical isolation.
However, a possible complicating factor is that
fall armyworm host strains were not considered
in these studies.
Taken together, the data provide a broad esti-
mation of the direction of fall armyworm migra-
tion into the northern U.S., but not one suffi-
ciently detailed to identify the specific destina-
tions of migrants arising from the overwintering
sites in Florida and Texas. A more direct way to
achieve this objective is to identify genetic mark-
ers that can distinguish between migrants from
Florida and Texas.
Molecular monitoring of migration. Molecular
analysis of the mitochondrial COI gene identified
2 nucleotide sites that were polymorphic within
the corn-strain. The polymorphisms generated 4
haplotype subgroups (labeled as CS-hl, CS-h2,
CS-h3, and CS-h4) that together make up the en-
tire corn-strain population examined (Nagoshi et
al. 2007a). Surveys of Florida corn-strain popula-
tions from different geographical locations, sea-
sons, or plant hosts consistently showed the pat-
tern of CS-h4 > CS-h2 > CS-hl, with only a small
number of the CS-h3 haplotype sporadically
found. These results combined with the relative
stability of the haplotype distribution pattern
during the 4-year period from 2002 to 2006
strongly suggest a homogeneous corn-strain pop-
ulation that is in equilibrium in the state of Flor-
ida. This is not surprising given that the mobility
of fall armyworm should facilitate the rapid mix-


ing of populations. A different haplotype pattern
was observed in Brazil, based on surveys in the 2
nonadjacent states of Parana and Mato Grosso
that lie along the southwestern border. The Brazil
populations display a haplotype relationship of
CS-h2 > CS-hl > CS-h4, with no CS-h3 haplotype
collected. The CS-h4 haplotype, the dominant
subgroup in Florida, was found in less than 10%
of the samples from either Brazilian state.
Most relevant to studies on North American
migration was the observation that haplotype
proportions in Texas fall armyworm from both
pheromone trap and larval collections consis-
tently displayed the pattern of CS-h2 > CS-hl =
CS-h4, with the CS-h3 haplotype detected sporad-
ically and at low levels (Nagoshi et al. 2008). This
haplotype profile appears to be in equilibrium
based on captures at three different locations over
a multiyear period, and is similar to that found in
Brazil corn-strain populations. Because the pri-
mary difference between the profiles lies in the
relative proportions of the CS-h2 and CS-h4 hap-
lotypes, a ratio of the 2 haplotype proportions pro-
vides a simple metric to quickly distinguish be-
tween the profiles. Collections in Florida, Brazil,
and Texas gave CS-h4/CS-h2 ratios of 2.4, 0.05,
and 0.15, respectively. Pairwise comparisons of
the data demonstrated a statistically significant
difference between the CS-h4/CS-h2 ratios ob-
served in Florida populations with those from
Texas or Brazil, but no indication of a significant
difference between Texas and Brazil (Nagoshi et
al. 2007b) (Fig. 2).
If the migration behavior of the corn-strain
haplotypes is the same, and there is no reason to
expect otherwise, then it should be possible to de-
termine the origin of migrant populations by com-
paring their haplotype profiles with those in Flor-
ida and Texas. As a proof of concept, we tested
corn-strain populations in Georgia, Alabama,
Louisiana, and Mississippi, states that lie adja-
cent to Texas or Florida and along an east-west


10


116


214


Elc-

Eles-
LIS~


Brazil


Florida Texas


Fig. 2. Proportions of the corn strain haplotypes
present in samples collected from different locations in
Florida. Numbers above bars indicate number of sam-
ples tested. Data from Nagoshi et al. (2007b).


December 2008







Nagoshi & Meagher: Fall Armyworm Migration


line from the Atlantic Ocean to the Gulf of Mexico
(Nagoshi et al. 2008). The results indicated that
the most eastern of the states, Georgia, was in-
fested by corn-strain populations that were indis-
tinguishable in haplotype distribution from those
overwintering in southern Florida. Corn-strain
populations in Louisiana, Mississippi, and Ala-
bama were statistically indistinguishable to pop-
ulations sampled in central and southern Texas.
This distribution pattern suggests a migratory
pattern similar to that described by Luginbill
(1928), in which the fall armyworm overwintering
in Texas migrate north and eastward through
Lousiana, Mississippi, and into Alabama, while
Florida populations move northward into Georgia
(Fig. 3).
It has been suggested that there is a return mi-
gration of fall armyworm in the autumn during
which the populations in the northern states
move southward to repopulate the overwintering
sites (Pair et al. 1987; Mitchell et al. 1991). If
there is substantial mixing of the overwintering
populations among the migrants followed by a
southern return, then we should see a gradual ho-
mogenization of the Florida and Texas popula-
tions with respect to haplotype proportions. Al-
ternatively, the long-term persistence of the
asymmetrical haplotype distribution would argue
that return migrations either do not occur or that
any genetic mixing is limited in scope. The alter-


native seems to be the case as the haplotype pro-
files of Florida from 2003-2006 and Texas from
2004-2007 has remained relatively constant.

SUMMARY

The highly mobile and genetically diverse fall
armyworm presents many technical challenges
for studies on migration and population dynam-
ics. Traditional methods of field observations,
trap captures, and synoptic weather analysis
have generally supported but only marginally
added to the early estimations of fall armyworm
migration (Luginbill 1928). However, relatively
recent advances in molecular techniques make
possible a far more detailed analysis of fall army-
worm movements that begins to take into account
the presence of genetically define subpopulations.
In the near future, we anticipate mapping the an-
nual migration of the corn-strain population
throughout North America from their overwinter-
ing sites in Texas and Florida, and extending this
analysis to compare the historical movements of
the Brazil and Florida subgroups of this strain in
the Caribbean, South America, and Central
America. As more genetic markers are uncovered
it may be possible to perform analogous studies
on the rice-strain and interstrain hybrid popula-
tions to develop a more complete description of
the migratory behaviors of this important agricul-


Fig. 3. Locations of the corn-strain fall armyworm haplotype profiles in selected states in the southeastern U.S.
Circles with diagonal lines display the Florida profile, clear circles indicate the Texas profile. Data from Nagoshi et
al. (2008).











tural pest. We anticipate that the more detailed
understanding of fall armyworm population
movements will facilitate efforts to find more ac-
curate ways to predict the timing and severity of
infestations and to assess the feasibility of miti-
gating infestations at migratory destinations by
population suppression in the overwintering
sites.

ACKNOWLEDGMENTS

We thank John Adamczyk (USDA-ARS) and Mirian
Hay-Roe (USDA-ARS) for helpful comments on the
manuscript. The use of trade, firm, or corporation
names in this publication is for the information and con-
venience of the reader. Such use does not constitute an
official endorsement or approval by the United States
Department of Agriculture or the Agricultural Research
Service of any product or service to the exclusion of oth-
ers that may be suitable.

REFERENCES CITED

ADAMCZYK, JR., J. J., D. D. HARDEE, L. C. ADAMS, AND
D. V. SUMERFORD. 2001. Correlating differences in
larval survival and development of bollworm (Lepi-
doptera: Noctuidae) and fall armyworm (Lepi-
doptera: Noctuidae) to differential expression of
CrylA(c) delta-endotoxin in various plant parts
among commercial cultivars of transgenic Bacillus
thuringiensis cotton. J. Econ. Entomol. 94: 284-90.
ADAMCZKY, JR., J. J., J. W. HOLLOWAY, B. R. LEONARD,
AND J. B. GRAVES. 1997. Susceptibility of fall army-
worm collected from different plant hosts to selected
insecticides and transgenic Bt cotton. J. Cotton Sci.
1: 21-28.
BARFIELD, C. S., J. L. STIMAC, AND M. A. KELLER 1980.
State-of-the-art for predicting damaging infesta-
tions of fall armyworm. Florida Entomol. 63: 364-
375.
BUSATO, G. R., A. D. GRTZMACHER, M. S. GARCIA, F. P.
GIOLO, M. J. ZOTTI, AND G. J. STEFANELLO, JR 2005.
Compared biology of Spodoptera frugiperda (J. E.
Smith) (Lepidoptera: Noctuidae) populations in corn
and rice leaves. Neotropical Entomol. 34: 743-750.
BUSATO, G. R., A. D. GRTZMACHER, A. C. DE OLIVEIRA,
E. A. VIEIRA, P. D. ZIMMER, M. M. KOPP, J. D. M.
BANDEIRA, AND T. R. MAGALHAES. 2004. Analysis of
the molecular structure and diversity of Spodoptera
frugiperda (J. E. Smith) (Lepidoptera: Noctuidae)
populations associated to the corn and rice crops in
Rio Grande do Sul State, Brazil. Neotropical Ento-
mol. 33: 709-716.
CHILCUTT, C. F., G. N. ODVODY, J. C. CORREA, AND
J. REMMERS. 2007. Effects of Bacillus thuringiensis
transgenic corn on corn earworm and fall armyworm
(Lepidoptera: Noctuidae) densities. J. Econ. Ento-
mol. 100: 327-334.
CLARK, P. L., J. MOLINA-OCHOA, S. MARTINELLI, S. R.
SKODA, D. J. ISENHOUR, D. J. LEE, J. T. KRUMM, AND
J. E. FOSTER 2007. Population variation of the fall
armyworm, Spodoptera frugiperda, in the Western
Hemisphere. J. Insect Sci. 7: available online: insect-
science.org/7.05.
DEQUECH, S. T. B., R. F. P. DA SILVA, AND L. M. FIUZA.
2005. Interaction between Spodoptera frugiperda (J.


December 2008


E. Smith) (Lepidoptera: Noctuidae), Campoletis fla-
vicincta (Ashmead) (Hymenoptera: Ichneumonidae)
and Bacillus thuringiensis aizawai, in laboratory.
Neotropical Entomol. 34: 937-944.
FITT, G. P. 1989. The ecology of Heliothis species in re-
lation to agroecosystems. Annu. Rev. Entomol. 39:
543-562.
FUXA, J. R. 1987. Spodoptera frugiperda susceptibility
to nuclear polyhedrosis virus isolates with reference
to insect migration. Environ. Entomol. 16: 218-223.
GOULD, F., N. BLAIR, M. REID, T. L. RENNIE, J. LOPEZ,
AND S. MICINSKI. 2002. Bacillus thuringiensis-toxin
resistance management: stable isotope assessment
of alternate host use by Helicoverpa zea. Proc. Natl.
Acad. Sci. USA. 99: 16581-6.
LEVY, H. C., A. GARCIA-MARUNIAK, AND J. E. MARUNIAk.
2002. Strain identification of Spodoptera frugiperda
(Lepidoptera: Noctuidae) insects and cell line: PCR-
RFLP of cytochrome oxidase subunit I gene. Florida
Entomol. 85: 186-190.
LEWTER, J. A., A. L. SZALANSKI, R. N. NAGOSHI, R. L.
MEAGHER JR., C. B. OWENS, AND R. G. LUTTRELL.
2006. Genetic variation within and between strains
of the fall armyworm, Spodoptera frugiperda (Lepi-
doptera: Noctuidae). Florida Entomol. 89: 63-67.
LU, Y., M. J. ADANG, D. J. EISENHOUR, AND G. D.
KOCHERT. 1992. Restriction fragment length poly-
morphism analysis of genetic variation in North
American populations of the fall armyworm
Spodoptera frugiperda (Lepidoptera: Noctuidae).
Mol. Ecol. 1: 199-208.
LU, Y. J., G. D. KOCHERT, D. J. ISENHOUR, AND M. J.
ADANG. 1994. Molecular characterization of a strain-
specific repeated DNA sequence in the fall army-
worm Spodoptera frugiperda (Lepidoptera: Noctu-
idae). Insect Mol. Biol. 3: 123-30.
LUGINBILL, P. 1928. The fall armyworm. U.S. Dept. AG-
RIC. TECH. BULL. 34: 1-91.
LYNCH, R. E., B. R. WISEMAN, D. PLAISTED, AND
D. WARNICK. 1999. Evaluation of transgenic sweet
corn hybrids expressing CrylA (b) toxin for resis-
tance to corn earworm and fall armyworm (Lepi-
doptera: Noctuidae). J. Econ. Entomol. 92: 246-52.
MACHADO, V., M. UNDER, V. D. BALDISSERA, J. V. OL-
IVEIRA, L. M. FIiZA, AND R. N. NAGOSHI. 2008.
Spodoptera frugiperda (J. E Smith) (Lepidoptera:
Noctuidae): molecular characterization of host
strains in southern Brazil. Ann. Entomol. Soc. Amer-
ica 101: 619-626.
MARTINELLI, S., R. M. BARATA, M. I. ZUCCHI, M. D. C.
SILVA-FILHO, AND C. OMOTO. 2006. Molecular vari-
ability of Spodoptera frugiperda (Lepidoptera: Noc-
tuidae) populations associated to maize and cotton
crops in Brazil. J. Econ. Entomol. 99: 516-526.
MARTINELLI, S., P. L. CLARK, M. I. ZUCCHI, M. C. SILVA,
J. E. FOSTER, AND C. OMOTO. 2007. Genetic struc-
ture and molecular variability of Spodoptera fru-
giperda (Lepidoptera: Noctuidae) collected in maize
and cotton fields in Brazil. Bull. Entomol. Res. 97:
225-231.
MCCORD, E., AND S. J. YU. 1987. The mechanisms of
carbaryl resistance in the fall armyworm,
Spodoptera frugiperda (J. E. Smith). Pestic. Bio-
chem. Physiol. 27: 114-122.
McMICHAEL, M., AND D. P. PROWELL. 1999. Differences
in amplified fragment-length polymorphisms in fall
armyworm (Lepidoptera: Noctuidae) host strains.
Ann. Entomol. Soc. America 92: 175-181.


Florida Entomologist 91(4)







Nagoshi & Meagher: Fall Armyworm Migration


MEAGHER, JR., R. L., AND M. GALLO-MEAGHER 2003.
Identifying host strains of fall armyworm (Lepi-
doptera: Noctuidae) in Florida using mitochondrial
markers. Florida Entomol. 86: 450-455.
MEAGHER, R. L., R. N. NAGOSHI, C. STUHL, AND E. R.
MITCHELL. 2004. Larval development of fall army-
worm (Lepidoptera: Noctuidae) on different cover
crop plants. Florida Entomol. 87: 454-460.
MITCHELL, E. R., J. N. MCNEIL, J. K. WESTBROOK, J. F.
SILVAIN, B. LALANNE-CASSOU, R. B. CHALFANT, S. D.
PAIR, V. H. WADDILL, A. SOTOMAYOR-RIOS, AND F. I.
PROSHOLD. 1991. Seasonal periodicity of fall army-
worm, (Lepidoptera: Noctuidae) in the Caribbean
basin and northward to Canada. J. Entomol. Sci. 26:
39-50.
MONNERAT, R., E. MARTINS, P. QUEIROZ, S. ORDTUZ, G.
JARAMILLO, G. BENINTENDE, J. COZZI, M. D. REAL,
A. MARTINEZ-RAMIREZ, C. RAUSELL, J. CERON, J. E.
IBARRA, M. C. DEL RINCON-CASTRO, A. M. ESPINOZA,
L. MEZA-BASSO, L. CABRERA, J. SANCHEZ, M. SO-
BERON, AND A. BRAVO. 2006. Genetic variability of
Spodoptera frugiperda Smith (Lepidoptera: Noctu-
idae) populations from Latin America is associated
with variations in susceptibility to Bacillus thuringien-
sis Cry toxins. Appl. Environ. Microbiol. 72: 7029-7035.
NAGOSHI, R. N., AND R. MEAGHER 2003. Fall armyworm
FR sequences map to sex chromosomes and their
distribution in the wild indicate limitations in inter-
strain mating. Insect Mol. Biol. 12: 453-458.
NAGOSHI, R. N., AND R. L. MEAGHER 2004. Seasonal
distribution of fall armyworm (Lepidoptera: Noctu-
idae) host strains in agricultural and turf grass hab-
itats. Environ. Entomol. 33: 881-889.
NAGOSHI, R. N., P. SILVIE, AND R. L. MEAGHER, JR.
2007a. Comparison of haplotype frequencies differ-
entiate fall armyworm (Lepidoptera: Noctuidae)
corn-strain populations from Florida and Brazil. J.
Econ. Entomol. 100: 954-961.
NAGOSHI, R. N., R. L. MEAGHER, G. NUESSLY, AND D. G.
HALL. 2006a. Effects of fall armyworm (Lepidoptera:
Noctuidae) interstrain mating in wild populations.
Environ. Entomol. 35: 561-568.
NAGOSHI, R. N., J. J. ADAMCZYK, R. L. MEAGHER, J.
GORE, AND R. JACKSON. 2007c. Using stable isotope
analysis to examine fall armyworm (Lepidoptera:
Noctuidae) host strains in a cotton habitat. J. Econ.
Entomol. 100: 1569-1576.
Nagoshi, R. N., P. Silvie, R. L. Meagher, Jr., J. Lopez,
AND V. Machado. 2007b. Identification and compari-
son of fall armyworm (Lepidoptera: Noctuidae) host
strains in Brazil, Texas, and Florida. Ann. Entomol.
Soc. America 100: 394-402.
NAGOSHI, R. N., R. L. MEAGHER, J. J. ADAMCZYK, S. K.
BRAMAN, R. L. BRANDENBURG, AND G. NUESSLY.
2006b. New restriction fragment length polymor-
phisms in the cytochrome oxidase I gene facilitate
host strain identification of fall armyworm (Lepi-
doptera: Noctuidae) populations in the southeastern
United States. J. Econ. Entomol. 99: 671-677.
NAGOSHI, R. N., R. L. MEAGHER, K. FLANDERS, J. GORE,
R. JACKSON, J. LOPEZ, J. S. ARMSTRONG, G. D. BUN-
TIN, C. SANSONE, AND B. R. LEONARD. 2008. Using
haplotypes to monitor the migration of fall army-
worm (Lepidoptera: Noctuidae) corn-strain popula-
tions from Texas and Florida. J. Econ. Entomol. 101:
742-749.
PAIR, S. D., J. R. RAULSTON, A. N. SPARKS, J. K. WEST-
BROOK, AND G. K. DOUCE. 1986. Fall armyworm dis-


tribution and population dynamics in the southeast-
ern states. Florida Entomol. 69: 468-487.
PAIR, S. D., J. R. RAULSTON, D. R. RUMMEL, J. K. WEST-
BROOK, W. W. WOLF, A. N. SPARKS, AND M. F.
SCHUSTER 1987. Development and production of
corn earworm and fall armyworm in the Texas high
plains: evidence for reverse fall migration. South-
western Entomol 12: 89-99.
PASHLEY, D. P. 1986. Host-associated genetic differenti-
ation in fall armyworm (Lepidoptera: Noctuidae): a
sibling species complex? Ann. Entomol. Soc. America
79: 898-904.
PASHLEY, D. P. 1988a. The current status of fall army-
worm host strains. Florida Entomol. 71: 227-234.
PASHLEY, D. P. 1988b. Quantitative genetics, develop-
ment, and physiological adaptation in host strains of
fall armyworm. Evolution 42: 93-102.
PASHLEY, D. P. 1989. Host-associated differentiation in
armyworms (Lepidoptera: Noctuidae): an allozymic
and mitochondrial DNA perspective, pp. 103-114 In
H. D. Loxdale and J. der Hollander [eds.], Electro-
phoretic Studies on Agricultural Pests. Clarendon
Press, Oxford.
PASHLEY, D. P. 1993. Causes of host-associated varia-
tion in insect herbivores: an example from fall army-
worm, pp. 351-359 In K. C. Kim and B. A. McPheron
[eds.], Evolution of Insect Pests/Patterns of Varia-
tion. Wiley, New York.
PASHLEY, D. P., AND J. A. MARTIN. 1987. Reproductive
incompatibility between host strains of the fall ar-
myworm (Lepidoptera: Noctuidae). Ann. Entomol.
Soc. America 80: 731-733.
PASHLEY, D. P., S. J. JOHNSON, AND A. N. SPARKS. 1985.
Genetic population structure of migratory moths:
the fall armyworm (Lepidoptera: Noctuidae). Ann.
Entomol. Soc. America 78: 756-762.
PASHLEY, D. P., T. N. HARDY, AND A. M. HAMMOND.
1995. Host effects on developmental and reproduc-
tive traits in fall armyworm strains (Lepidoptera:
Noctuidae). Ann. Entomol. Soc. America 88: 748-755.
PASHLEY, D. P., S. S. QUISENBERRY, AND T. JAMJANYA.
1987a. Impact of fall armyworm (Lepidoptera: Noc-
tuidae) host strains on the evaluation of bermuda-
grass resistance. J. Econ. Entomol. 80: 1127-1130.
PASHLEY, D. P., T. C. SPARKS, S. S. QUISENBERRY,
T. JAMJANYA, AND P. F. DOWD. 1987b. Two fall army-
worm strains feed on corn, rice and bermudagrass.
Louisiana Agric. 30: 8-9.
PITRE, H. N. 1988. Relationship of fall armyworm (Lep-
idoptera: Noctuidae) from Florida, Honduras, Ja-
maica, and Mississippi: susceptibility to insecticides
with reference to migration. Florida Entomol. 71: 56-
61.
PITRE, H. N., AND D. B. HOGG. 1983. Development of the
fall armyworm on cotton, soybean and corn. J. Geor-
gia Entomol. Soc. 18: 182-186.
POLANCZYK, R. A., AND S. B. ALVES. 2005. Biological pa-
rameters of Spodoptera frugiperda (J. E. Smith)
(Lepidoptera: Noctuidae) assayed with Bacillus thu-
ringiensis Berliner. Sci Agr 62: 464-468.
PONSARD, S., M.-T. BETHENOD, A. BONTEMPS, L. PELO-
ZUELO, M.-C. SOUQUAL, AND D. BOURGUEt. 2004.
Carbon stable isotopes: a tool for studying the mat-
ing, oviposition, and spatial distribution of races of
European corn borer, Ostrinia nubilalis, among host
plants in the field. Can. J. Zool. 82: 1177-1185.
PROWELL, D. P. 1998. Sex linkage and speciation in Lep-
idoptera, pp. 309-319 In D. Howard and S. Berlocher











[eds.], Endless Forms: Species and Speciation. Ox-
ford Press, New York.
PROWELL, D. P., M. MCMICHAEL, AND J.-F. SILVAIN.
2004. Multilocus genetic analysis of host use, intro-
gression, and speciation in host strains of fall army-
worm (Lepidoptera: Noctuidae). Ann. Entomol. Soc.
America 97: 1034-1044.
QUISENBERRY, S. S. 1991. Fall armyworm (Lepidoptera:
Noctuidae) host strain reproductive compatibility.
Florida Entomol. 72: 194-199.
ROSE, A. H., R. H. SILVERSIDES, AND O. H. LINDQUIST.
1975. Migration flight by an aphid, Rhopalosiphum
maidis (Hemiptera: Aphididae) and a noctuid,
Spodoptera frugiperda (Lep: Noctuidae). Canadian
Entomol. 107: 567-576.
SPARKS, A. N. 1979. A review of the biology of the fall ar-
myworm. Florida Entomol. 62: 82-86.
SPERLING, F. A. H. 1994. Sex-linked genes and species dif-
ferences in Lepidoptera. Can. Entomol. 126: 807-818.
VEENSTRA, K. H., D. P. PASHLEY, AND J. A. OTTEA. 1995.
Host-plant adaptation in fall armyworm host
strains: comparison of food consumption, utilization,
and detoxication enzyme activities. Ann. Entomol.
Soc. America 88: 80-91.
WESTBROOK, J. K., AND A. N. SPARKS. 1986. The role of
atmospheric transport in the economic fall army-


December 2008


worm (Lepidoptera: Noctuidae) infestations in the
southeastern United States in 1977. Florida Ento-
mol. 69: 494-502.
WHITFORD, F., S. S. QUISENBERRY, AND D. J. MOELLEN-
BECK. 1992. Nutritional response by rice and corn
fall armyworm (Lepidoptera: Noctuidae) strains to
dietary component substitution in artificial diets. J.
Econ. Entomol. 85: 1491-1496.
WHITFORD, F., S. S. QUISENBERRY, T. J. RILEY, AND J. W.
LEE. 1988. Oviposition preference, mating compati-
bility, and development of two fall armyworm
strains. Florida Entomol. 71: 234-243.
WILLIAMS, W. P., P. M. BUCKLEY, AND C. A. DAVES.
2006. Identifying resistance in corn to southwestern
corn borer (Lepidoptera: Crambidae), fall armyworm
(Lepidoptera: Noctuidae), and corn earworm (Lepi-
doptera: Noctuidae). J Agr Urban Entomol. 23: 87-
95.
YOUNG, J. R. 1979. Fall armyworm: control with insec-
ticides. Florida Entomol. 62: 130-133.
YU, S. J. 1991. Insecticide resistance in the fall army-
worm, Spodoptera frugiperda (J. E. Smith). Pestic.
Biochem. Physiol. 39: 84-91.
YU, S. J. 1999. Induction of new glutathione S-trans-
ferase isozymes by allelochemicals in the fall army-
worm. Pestic. Biochem. Physiol. 63: 163-171.


Florida Entomologist 91(4)







Siebert et al.: CrylF For Control of Fall Armyworm


EFFICACY OF CRY1F INSECTICIDAL PROTEIN IN MAIZE AND COTTON FOR
CONTROL OF FALL ARMYWORM (LEPIDOPTERA: NOCTUIDAE)

M. WILLRICH SIEBERT'2, J. M. BABOCK1, S. NOLTING', A. C. SANTOS1, J. J. ADAMCZYK, JR.3, P. A. NEESE1, J. E. KING1,
J. N. JENKINS4, J. MCCARTY4, G. M. LORENZ5, D. D. FROMME6 AND R. B. LASSITER1
'Dow AgroSciences LLC, 9330 Zionsville Rd., Indianapolis, IN 46268

2Corresponding author email: mwillrichsiebert@dow.com

3USDA, ARS, 2413 E. Hwy 83, Weslaco, TX 78596

4USDA, ARS, 810 Hwy 12E, Starkville, MS 39762

5University of Arkansas, 2001 Hwy 70E, Lonoke, AR 72086

6Texas A&M University, P.O. Box 1349, Uvalde, TX 78802

ABSTRACT

Efficacy of maize, Zea mays L., hybrids and cotton, Gossypium hirsutum (L.), varieties ex-
pressing CrylF insecticidal crystal protein of Bacillus thuringiensis (Bt) var. aizawai Ber-
liner (transformation event TC 1507 in corn and event DAS-24236-5 in cotton) was evaluated
for control of fall armyworm, Spodoptera frugiperda (J.E. Smith). Control of natural and ar-
tificial fall armyworm infestations of eggs and various larval stages to 3 CrylF and non-Bt
maize isoline pairs at V4-V7 corn growth stage was evaluated at 10 locations across the
United States and Brazil. Varieties producing the CrylF protein provided high levels of con-
trol. Furthermore, control provided by CrylF-maize hybrids was frequently better than
when fall armyworm were managed with 3 applications of foliar insecticides. Efficacy of
transgenic CrylAc:CrylF cotton against fall armyworm was evaluated for 5 varieties during
anthesis in laboratory and natural infestation field studies in the southern United States.
Laboratory colonies of fall armyworm originally collected from corn, bermudagrass, Cyn-
odon dactylon (L.), cotton, and royal paulownia, Paulownia tomentosa (Thunb.) and deter-
mined to be either the rice or corn-associated host strain, all resulted in low levels of survival
when fed matures leaves of CrylAc:CrylF-cotton. In natural infestation studies, levels of
fall armyworm in squares (flower buds), flowers, and bolls (fruit), were significantly lower in
cotton containing CrylF as compared to non-Bt cotton. These results demonstrate that
maize hybrids and cotton varieties containing CrylF can be an important component in an
overall management program for fall armyworm across a broad range of geographies and
crops.

Key Words: Bacillus thuringiensis var. aizawai, Spodoptera frugiperda, cotton, maize, IPM,
CrylF

RESUME

La eficacia de los hibridos de maiz, Zea mays L. y de las variedades de algod6n, Gossypium
hirsutum (L.), en expresar la protein cristalizada insecticide CrylF de Bacillus thuringien-
sis (Bt) var. aizawai Berliner (transformaci6n event TC1507 en maiz y event DAS-24236-
5 en algod6n) fue evaluada para el control del gusano cogollero. El control de infestaciones
naturales y artificiales de huevos y various estadios de larvas del gusano cogollero en 3 pares
de isolineas de CrylF y de maiz sin Bt en maiz en las etapas V4-V7 del desarrollo, fueron
evaluadas en 10 localidades a trav6s de los Estados Unidos y Brasil. Las variedades que pro-
ducen la protein CrylF proveyeron altos niveles de control. Ademas, el control proveido por
los hibridos CrylF de maiz frecuentemente fue mejor que cuando se aplicaron 3 insecticides
foliares para el manejo del gusano cogollero. La eficacia de algod6n transg6nico
CrylAc:CrylF contra el gusano cogollero fue evaluada en 5 variedades durante la antesis en
los studios de laboratorio y en las infestaciones naturales en el campo en el sur de los Es-
tados Unidos. Las colonies del gusano cogollero en el laboratorio que fueron recolectadas ori-
ginalmente sobre maiz; el past bermuda, Cynodon dactylon (L.); el algod6n; y la paulownia
imperial, Paulownia tomentosa (Thunb.) y que fueran determinadas como la sepa asociada
con arroz o maiz, todas resultaron en tener niveles de sobrevivencia mas bajos cuando las
alimentaron de hojas maduras del algod6n con CrylAc:CrylF. En studios de infestaciones
naturales, los niveles de los gusanos cogolleros en las bracteas, flores y bellotas (fruta), fue-
ron significativamente mas bajos en algod6n que tenia CrylF en comparici6n a algod6n sin







Florida Entomologist 91(4)


Bt. Estos resultados demuestran que los hibridos de maiz y variedades de algod6n que con-
tienen CrylF pueden ser un component important en un program total del manejo de gu-
sano cogollero a trav6s de un amplio rango geografico y rango de hospederos.


Transformation of maize, Zea mays L., and
cotton, Gossypium hirsutum (L.), to express Ba-
cillus thuringiensis insecticidal toxins has re-
sulted in numerous benefits to producers and
agroecosystems. Transgenic crops provide the
opportunity to control insecticide-resistant pest
species, maximize crop yields, conserve benefi-
cial arthropods, and reduce the frequency of ap-
plications of synthetic insecticides (Edge et al.
2001; Shelton et al. 2002). Additionally, trans-
genic technologies can offer control of insect
pests generally difficult to manage with syn-
thetic insecticides. One example of these bene-
fits is illustrated in the management of fall ar-
myworm, Spodoptera frugiperda (J. E. Smith),
with transgenic Bt technology. Biological char-
acteristics of fall armyworm, coupled with oper-
ational factors that result in suboptimal control
with insecticides, make transgenic options at-
tractive for management in maize and cotton.
Fall armyworm can be a destructive pest of cot-
ton and maize produced in the U.S., Central
America, and South America (Sparks 1979; Sena
et al. 2003). In maize, fall armyworms are capable
of causing defoliation during the whorl stage as
well as direct injury to the ear (Labatte 1993;
Davis et al. 1998). Although fall armyworm may
injure maize plants in nearly all stages of devel-
opment, infestations in the U.S. concentrate on
plants that are in whorl stages, particularly in
later-planted maize where these vegetative
stages are synchronized with high moth abun-
dance (Quisenberry 1999). Although considered
an occasional pest in the U.S., fall armyworm is a
primary pest in tropical climates. Factors that
contribute to the elevated pest status of fall army-
worm in those locations include continuous pro-
duction of maize, multiple generations of intense
infestations, and widespread insecticide resis-
tance (Cruz 1995). In Brazil, the potential for
yield losses in maize has ranged from 17 to 38.7%
(Fernandes et al. 2003). Historical reports of sig-
nificant yield loss can also be found in Mexico,
Central America, and Argentina (Perdiguero et
al. 1967; Andrews 1980).
Fall armyworms can be a destructive pest in
cotton. As with maize, fall armyworm is consid-
ered an occasional pest of cotton in the U.S. and a
primary pest of cotton in Brazil (Santos et al.
2005). Moth oviposition and subsequent feeding
by first and second instar larvae commonly occur
on leaves within the lower two-thirds of the plant
canopy (Ali et al. 1990). Later instars predomi-
nately feed on fruiting structures including
squares (flower buds), flowers, and bolls (fruit)
(Ali et al. 1990).


The spatial distribution of fall armyworm eggs
and small larvae in maize and cotton make detec-
tion and control with foliar insecticides challeng-
ing. Insecticide spray coverage in maize is diffi-
cult because larvae are located in the wrapped-up
leaves of the whorl within 1 d of egg eclosion until
larval development is complete (Labatte 1993).
Similarly in cotton, insecticide coverage is inade-
quate for larvae located in lower portions of the
plant canopy and for larger larvae concealed in
fruiting structures. Failure to control fall army-
worm as early instar larvae may be problematic
because fall armyworm become tolerant to insec-
ticides as they increase in size (Yu 1983; Mink &
Luttrell 1989). Selection of an effective insecti-
cide, timing and method of application, and rein-
festation are additional considerations that affect
management of fall armyworm in maize and cot-
ton (Cook et al. 2004; Ghidiu & Andaloro 1993).
Transgenic plants that express Bt proteins
throughout various tissue types and for the dura-
tion of plant development are useful for overcom-
ing many of the limitations associated with man-
aging fall armyworm with synthetic foliar insecti-
cides.
Single-gene Bt maize hybrids and cotton vari-
eties became available to producers during 1996
and contained CrylAb (events MON 810, Bt-176,
and Bt-11) and CrylAc (event MON 531), respec-
tively. The rationale for introducing these trans-
genic technologies was to control pests of global
and economic importance including European
corn borer, Ostrinia nubilalis (Hiibner), and
southwestern corn borer, Diatraea grandiosella
Dyar, in maize and heliothines tobacco budworm,
Heliothis virescens (F.) and bollworm, Helicoverpa
zea (Boddie) and pink bollworm, Pectinophora
gossypiella (Saunders), in cotton (Milfin 1996; Gi-
anessi et al. 2002a; Gianessi et al. 2002b). Bt
maize hybrids containing CrylAb protein provide
excellent control of European corn borer and
southwestern corn borer (Archer et al. 2001; Cas-
tro et al. 2004). However, larval establishment
can occur on CrylAb maize hybrids for other spe-
cies that feed on foliage and ears including fall ar-
myworm, black cutworm, Agrotis ipsilon (Hufna-
gel), western bean cutworm, Richia albicosta
(Smith), and corn earworm, Helicoverpa zea (Bod-
die) (Pilcher et al. 1997; Buntin et al. 2001; Cat-
angui & Berg 2006).
Single-gene Bt cotton varieties contain CrylAc
protein and provide absolute control of tobacco
budworm and pink bollworm (MacIntosh et al.
1990; Tabashnik et al. 2000; Jackson et al. 2003).
However, as with maize, it was recognized that a
single Bt protein could not provide broad spec-


December 2008







Siebert et al.: CrylF For Control of Fall Armyworm


trum lepidopteran control. Thus, bollworm and
other secondary lepidopteran pests infesting sin-
gle-gene Bt cotton would need to be managed with
supplemental insecticides to prevent economic
losses (Gore et al. 2001; Stewart et al. 2001).
Therefore, new Bt proteins can broaden the spec-
trum of lepidopteran activity in both Bt maize hy-
brids and Bt cotton varieties. Combinations of in-
sect resistant traits also aid in resistance man-
agement of target Lepidoptera pest species (Mc-
Gaughey & Whalon 1992; Tabashnik 1994; Gould
1998; Stewart et al. 2001).
Maize hybrids that express the CrylF insecti-
cidal crystal protein ofB. thuringiensis var. aiza-
wai were commercialized in the U.S. in 2003
(event TC1507, Herculex I Insect Protection).
CrylF maize hybrids provide control of not only
European corn borer and southwestern corn
borer, but also provide protection against damag-
ing infestations of other lepidopteran pests in-
cluding sugarcane borer, Diatraea saccharalis
(F.), fall armyworm, black cutworm, and western
bean cutworm (U.S. Environmental Protection
Agency 2005; Catangui & Berg 2006; Siebert et al.
2008). The first dual-toxin Bt cotton varieties
were available during the 2003 growing season
and contained CrylAc and Cry2Ab (event MON
15985). Closely thereafter in 2005, varieties con-
taining combined CrylAc (event DAS-21023-5)
and CrylF (event DAS-24236-5) (WideStrikeTM,
Dow AgroSciences LLC, Indianapolis, IN) also be-
came available to producers. The addition of ei-
ther CrylF or Cry2Ab to CrylAc has allowed for
improved control of secondary pests (Stewart et
al. 2001; Willrich et al. 2005; Greenberg & Adam-
czyk 2007).
The objective of the following series of experi-
ments was to compare the efficacy of maize hy-
brids and cotton varieties containing transgenic
CrylF for control of fall armyworm. CrylF effi-
cacy was compared across numerous geographies
and plant tissue types.

MATERIALS AND METHODS

Laboratory and Field Studies on Maize Producing the
CrylF Bt Protein

Studies evaluating fall armyworm injury to
vegetative (whorl) stage CrylF and non-Bt maize
hybrids were conducted in 3 locations (7 studies)
in the U.S. from 2002-2006 and in 3 locations in
Brazil during 2007 (Table 1). At each location,
Mycogen corn hybrids (Mycogen Seeds, LLC, Indi-
anapolis, IN) producing CrylF were compared to
non-Bt near-isolines. Up to 3 CrylF/non-Bt pairs
were evaluated at a single location (Table 1). In
Brazil, an additional treatment of the non-Bt corn
hybrid, managed with synthetic insecticides tar-
geting fall armyworm, was included for compari-
son. In those studies, methomyl (Lannate 216 g/L


SL, DuPont Crop Protection, Alphaville, SP, Bra-
zil), k-cyhalothrin (Karate Zeon 50 g/L CS, Syn-
genta Crop Protection, Inc., Santo Amaro, SP,
Brazil), and lufenuron (Match 50 g/L EC, Syn-
genta Crop Protection, Inc., Santo Amaro, SP,
Brazil) were applied sequentially at a 4-7 d inter-
val between applications beginning at the initia-
tion of natural infestations. Treatments in the
field were planted in a randomized complete block
design with 4 replications at all locations. Plot
size across locations ranged from 2 to 8 rows (76.2
to 101.6-cm row centers) by 4.0 to 12.1 m in
length. All studies were maintained by agronomic
practices for optimal productivity. Seed used for
testing was treated only with a commercial fungi-
cide and no preventive treatments of soil or foliar
applied insecticides were applied across the test
area to plots not designated to receive such treat-
ments.
Natural fall armyworm infestations or artifi-
cial fall armyworm infestations of 3 immature
stages were used to evaluate efficacy of CrylF
maize hybrids. Artificial infestations occurred at
intervals which corresponded to V4-V6, V4-V8,
V5-V6, V6, V6-V7 and V7 stages of corn develop-
ment (Ritchie et al. 1993). All plants within a sin-
gle center row of each plot were infested at a par-
ticular interval and at each location. Plants were
infested 1 or 2 times during each study. Fall army-
worm eggs and larvae used in these studies were
provided by Dow AgroSciences (Indianapolis, IN)
and had originated from a collection in maize. In-
sects were shipped to test locations as egg masses
laid on wax paper sheets or as 2nd instars reared
on a meridic insect diet (Southland Multispecies
Diet, Southland Products, Inc., Lake Village, AR)
in 236-mL cups (1 egg mass per cup). Fall army-
worm received as eggs were hatched and mixed
with corncob grit and infested into plant whorls
by the technique and a plastic dispensing device
(bazooka) described by Davis & Oswalt (1979).
Second instars were individually placed into the
whorls of corn plants with a fine camel hair paint
brush. Egg masses were cut from wax paper ovi-
position sheets and a single mass containing ap-
proximately 50 eggs was placed into an individual
whorl. At 14 to 24 d after the establishment of ei-
ther natural or artificial infestations, all plants
were rated for leaf-feeding injury on a 0-9 scale
(Davis et al. 1992), where 0 is no visible injury
and 9 is whorl and furl leaves >90% destroyed.

Laboratory and Field Studies on Cotton Producing
CrylF Bt Protein

Studies evaluating survival of fall armyworm
on CrylAc:CrylF cotton plants and non-Bt cotton
varieties were conducted during 2004 to 2007
with fresh tissue laboratory bioassays or natural
infestation field studies. At each test site, cultural
practices including fertility, irrigation, and weed

















TABLE 1. LOCATION AND METHODOLOGIES USED FOR ACROSS TRIAL SUMMARIZATIONS OF CRY1F EFFICACY, AS EXPRESSED IN MAIZE HYBRIDS, AGAINST FALL ARMYWORM IN
THE UNITED STATES AND BRAZIL.

CrylF (non-Bt isoline) Corn
Year Location Hybrid Pairs Evaluated' Methodology For Evaluating Efficacy2

2002 Fowler, IN 2G768 (M2784) Artificial infestation: neonate larvae (15/plant); applied at V7 maize growth stage
Greenville, MS 2G768 (M2784) Artificial infestation: eggs (15/plant) applied to V7 maize growth stage
2003 Fowler, IN 2G768 (M2784) Artificial infestation: neonate larvae (30/plant) applied twice at V5-V6 and V6-V7
maize growth stage
2004 Fowler, IN 2G768 (M2784), 2A812 (2A775),
11084BMR (F717BMR) Artificial infestation: neonate larvae (25/plant) applied twice 3-d apart at V5-V6
maize growth stage
2005 Fowler, IN 2G768 (M2784), 2A812 (2A775),
11084BMR (F717BMR) Artificial infestation: eggs (1 mass/plant, 50 eggs/mass) applied twice at V5-V6 and
V6-V7 maize growth stage
Huxley, IA 2G768 (M2784), 2A812 (2A775),
11084BMR (F717BMR) Artificial infestation: Second instar larvae (10/plant) applied at V4-V8 maize
growth stage
2006 Fowler, IN 2P788 (M2784) Artificial infestation: second instar larvae (20/plant) at V6 maize growth stage
2007 Indian6polis, Minas Gerais, Brazil 2B710 HX (2B710) Natural infestation to whorl stage maize
Rio Verde, Goias, Brazil 2B710 HX (2B710) Natural infestation to whorl stage maize
Jardin6polis, Sao Paulo, Brazil 2B710 HX (2B710) Natural infestation to whorl stage maize

1Mycogen CrylF and non-Bt maize hybrids.
'Maize growth stages described by Ritchie et al. (1993).







Siebert et al.: CrylF For Control of Fall Armyworm


management, as recommended by state extension
guidelines, were used to maintain experimental
plots for optimum productivity. The entire test
area was managed for non-lepidopteran insects
pests, including thrips (Thysanoptera: Thripi-
dae), aphids (Homoptera: Aphididae), stink bugs
(Hemiptera: Pentatomidae), and plant bugs
(Hemiptera: Miridae), by using insecticide chem-
istries with limited activity against lepidopteran
insects. Insecticides used included aldicarb
(Temik150 g/kg, Bayer Crop Science, Research
Triangle Park, NC), dicrotophos (Bidrin480 g/L
E, Amvac Chemical Corporation), and thia-
methoxam (Centric400 g/kg WG, Syngenta Crop
Protection, Greensboro, NC). The data reported
from the following studies is reflective of cotton
managed without insecticides active against lepi-
dopteran pests.
Plots were planted in a randomized block ex-
perimental design at each location. Treatments
were replicated 4 times at each location, with ex-
ception of Wharton, TX and Pine Bluff, AR, which
included 3 replications. At each location, single or
multiple cotton varieties containing
CrylAc:CrylF were compared to a single gene or
non-Bt cotton varieties. CrylAc:CrylF cotton va-
rieties included PHY 440 W, PHY 470 WR, PHY
475 WRF, PHY 485 WRF, and PHY 375 WRF (all
from PhytoGen Seed Company, LLC, Indianap-
olis, IN). Non-Bt cotton varieties included PSC
355, PHY 410 R, and PHY 315 RF. Stoneville
46971B, which produces CrylAc, was the single-
gene Bt cotton variety evaluated. PhytoGen cot-
ton varieties within the same series (i.e., 400s or
300s) are derived from the same parent rather
than being genetic isolines. Plots sizes were 12.2
m long by 4 rows (91.4-101.6 cm centers) at all lo-
cations with the exception of Wharton, TX, where
plots were 12 rows by 272.7 m. All studies were
maintained by agronomic practices for optimal
productivity.
In the natural infestation field studies, the
center rows of each plot were sampled on a weekly
basis beginning at the onset of anthesis and ex-
amined for the presence of fall armyworm larvae.
At the Lonoke and Pine Bluff, AR locations during
2005, whole plant samples of 20 and 60 plants per
plot, respectively, were conducted on each date.
All plant structures (reproductive and vegetative
components) were visually inspected for presence
of fall armyworm larvae. At other locations dur-
ing 2006 and 2007, 4 types of plant structures
were sampled and included squares (flower bud),
flowers, and bolls (fruit). Fruiting forms (40
squares, white flowers, and bolls per plot) were
randomly selected and examined for presence of
surviving fall armyworm larvae on each date.
Larval count data for each plot was transformed
to percent larval infestation based on the number
of structures (i.e., 40) or whole cotton plants (i.e.,
20 or 60) sampled as the denominator. For all nat-


ural infestation field trials, data reported are for
the date of peak larval infestations in the non-Bt
cotton variety.
In fresh tissue bioassays, fall armyworm lar-
vae were infested on mature, fully expanded
leaves. At the Stoneville, MS location, leaves were
collected from all varieties when plants across the
test plots had approximately 5 mainstem nodes
above a sympodial branch with a flower on the
first node (5 NAWF). Leaves collected were lo-
cated 5 mainstem nodes below the terminal apex.
First instars (F2 generation) were placed on an in-
dividual leaf inside a 9.2-cm diameter plastic
Petri dish with a 9.0-cm diameter filter paper and
covered to prevent escape (5 larvae per dish and 5
dishes per variety). Fall armyworm colonies used
in each bioassay were derived from collections
from one of 3 different plant hosts: cotton, ber-
mudagrass, Cynodon dactylon (L.), and royal pau-
lownia, Paulownia tomentosa (Thunb.). A subset
of fall armyworm from these colonies were sub-
jected to genetic analysis to determine their host-
associated strain based on methods as described
by Meagher & Gallo-Meagher (2003). At the
Starkville, MS location, leaves were collected
from cotton at the initiation of anthesis and were
located 4 mainstem nodes below the terminal. A
single 2.54-cm diameter disc was excised from
each leaf and infested with a single, 1st instar fall
armyworm (8 leaf discs per replication). Leaf
discs were placed in individual cells (3.81 cm
length x 4.44 cm width x 2.54 cm depth) of molded
rearing trays (BIO-RT-32, C-D International,
Pittman, NJ) and covered with perforated lids
(Bio-CV4, C-D International, Pittman, NJ). An
agar medium was added to each cell to maintain
leaf turgor. Fall armyworm larvae used were es-
tablished from crosses between wild males col-
lected during 2004 and laboratory reared females
that had previously been maintained in culture.
Larval survival was determined at 4 to 5 d after
infestation in each bioassay The criterion for sur-
vival was the ability of a larva to make coordi-
nated movement when prodded with a camel-hair
brush.

Statistical Analysis

The 0-9 scale rating data obtained from the
field studies in maize were analyzed within each
country by multinomial, ordinal logistic regres-
sion techniques (Minitab 1998). Paired treat-
ments were compared and P-values for the coeffi-
cient estimating change in the logit link function,
odds of observing a 0 score for the first treatment
in the pair relative to second treatment (odds ra-
tio), and 95% confidence intervals were calcu-
lated. In addition, individual plant damage rat-
ings within each replication were averaged and
rounded to the nearest whole number. The fre-
quency of occurrence of each of the 0-9 damage







Florida Entomologist 91(4)


rating values was tabulated across the trial sites
and used to compare among the management
strategies for fall armyworm.
For laboratory and field studies in cotton, per-
cent larval survival and percent larval infestation
data were subjected to Markov chain Monte Carlo
(MCMC) simulations and similar to that of Mila
& Michailides (2006). Analysis of variance tech-
niques were not utilized because data sets were
characterized by non-normal distributions, heter-
ogeneity, and small number of observations.
BRugs software (Bayesian inference with Gibbs
sampling) was used to build 95% credible inter-
vals which were used to compare treatments (R
Development Core Team 2005; Thomas et al.
2006). Credible intervals provide the probability
that a mean is contained within the calculated in-
terval (Box-Steffensmeier et al. 2008). Treatment
means were considered significantly different if
95% credible intervals did not overlap (Carlin &
Lewis 2000).

RESULTS AND DISCUSSION

Efficacy of Corn Hybrid Producing CrylF

Four CrylF and 3 non-Bt maize hybrids were
compared in 7 U.S. field trials for leaf-feeding in-
jury by fall armyworm. Mean ( SEM) injury rat-
ings for CrylF-Bt maize hybrids including
11083BMR, 2A812, 2G768, and 2P788 were 1.4 +
0.5, 1.5 + 0.8, 0.9 + 0.7, and 1.7 + 1.7, respec-
tively. Mean ( SEM) injury ratings for non-Bt
maize hybrids including 2A775, F717BMR, and
M2784 were 7.4 + 0.5, 8.1 + 1.0, and 7.9 + 1.1, re-
spectively. There was no significant difference in
damage among non-Bt corn hybrids (treatment
pair, P-value, odds ratio, 95% confidence limits:
M2784 and F717BMR, 0.693, 1.32, 0.33-5.31;
2A775 and F717BMR, 0.151, 3.77, 0.62-23.06;
2A775 and M2784, 0.137, 2.85, 0.72-11.33;
F717BMR and M2784, 0.693, 0.76, 0.19-3.04). In
addition, there was no significant difference in
damage among CrylF-Bt corn hybrids (treat-
ment pair, P-value, odds ratio, 95% confidence
limits: 2G768 and F11084BMR, 0.174, 2.96,
0.62-14.01; 2A812 and F11084BMR, 0.868, 0.86,
0.13-5.42; 2P788 and F11084BMR, 0.937, 1.07,
0.18-6.24; 2A812 and 2G768, 0.120, 0.29, 0.06-
1.38; 2P788 and 2G768, 0.170, 0.36, 0.09-1.54;
F11084BMR and 2G768,0.174, 0.34, 0.07-1.61;
2P788 and 2A812, 0.800, 1.26, 0.22-7.26;
F11084BMR and 2A812, 0.868, 1.17, 0.18-7.41;
2G768 and 2A812, 0.120, 3.46, 0.72-16.56).
Therefore, injury ratings for all non-Bt and all
CrylF-Bt maize hybrids were combined for anal-
ysis.
Mean leaf-feeding injury for CrylF-Bt maize
hybrids was 1.3 as compared to 7.9 for the non-
Bt corn hybrids (Fig. 1). Injury was significantly
less for CrylF-Bt maize hybrids as compared to


o Cryl F-Bt nmize
ENon-Bt maiz


Fig. 1. Leaf-feeding injury (0-9 scale, Davis et al.
1992) by fall armyworm on a Bt and non-Bt maize hy-
brid, 2002-2006, U.S.


non-Bt maize hybrids (P-value, odds ratio, 95%
confidence intervals: <0.001, 0.00, 0.00-0.00).
Therefore, it is highly unlikely that the lower
damage ratings would be observed on non-Bt
maize hybrids relative to the CrylF-Bt maize hy-
brids. These results were consistent with field
trials reported in the southern U.S. by Siebert et
al. (2008) in which CrylF-Bt maize hybrids pro-
vided significant protection and improved plant
height when exposed to high levels of feeding
pressure from fall armyworm. In addition, Waq-
uil et al. (2002) demonstrated that a maize hy-
brid containing CrylF provided better control of
fall armyworm feeding than hybrids producing
either CrylAb or native host plant resistance
factors.
The use of multinomial techniques for the
Brazil data was not possible (model convergence
not obtained) and was likely due to the small
data set. Leaf feeding injury for the CrylF-Bt
and non-Bt maize hybrids was 1.3 0.4 and 4.0
+ 1.9, respectively, similar to the trends observed
in the U.S. data. Level of injury for non-Bt maize
managed with foliar insecticides was 4.1 1.5.
The range of damage values assigned to CrylF-
Bt maize hybrids, a foliar insecticide program,
and non-Bt maize hybrids were 1-2, 2-7, and 2-7,
respectively, (Fig. 2). Based on this qualitative
analysis, managing fall armyworm with foliar
insecticides on non-Bt corn was similar to dam-
age levels observed on non-Bt maize hybrids.
Furthermore, damage values for these non-Bt
strategies were generally greater as compared to
a management strategy with CrylF-Bt maize
hybrids. A multinomial logistic regression anal-
ysis of the combined U.S. and Brazil CrylF-Bt
and non-Bt data produced results similar to that
of the U.S. alone (P-value, odds ratio: <0.001,
0.00). These results reinforce the improbability
of observing a damage rating lower for a non-Bt
maize hybrid relative to a CrylF-Bt maize hy-
brid across geographies.


December 2008







Siebert et al.: CrylF For Control of Fall Armyworm


O CrylF-Bt nmize
2,6
S5 U Foliar Insecticides/Non-Bl umaiz
4-
Non-Bt rmize
3-





11

Leaf-feeding rating

Fig 2. Comparison oftransgenic and non-transgenic management tactics for control of fall armyworm on maize,
2005, Brazil.


Maize hybrids containing CrylF have provided
effective control of fall armyworm in Argentina
field trials. In Los Altos, Catamarca grain yields
were significantly greater (17%, P < 0.05) for a
corn hybrid containing CrylF as compared to a
non-Bt corn hybrid (Dow AgroSciences, LLC, un-
published data). The results reported here dem-
onstrate that CrylF maize hybrids can serve as
an effective management option for fall army-
worm in South America. In addition, manage-
ment of fall armyworm with CrylF may be a bet-
ter management option as compared to using fo-
liar insecticides on non-Bt maize.


Efficacy of Cotton Varieties Producing CrylF

Survival of fall armyworm larvae did not ex-
ceed 4.0% in laboratory infestations on mature
leaves of PHY 440 W, which contains
CrylAc:CrylF (Table 2). Regardless of the source
(plant host) of laboratory-reared fall armyworm,
larval survival on PHY 440 W was significantly
less than on PSC355 cotton (P < 0.05). Survival of
fall armyworm larvae on Stoneville 4691B, which
contains CrylAc, and Stoneville 474 (non-Bt) was
not significantly different than PSC 355 (non-Bt)
cotton (P > 0.05). Additionally, fall armyworm


TABLE 2. SURVIVAL OF FALL ARMYWORM (FIRST INSTAR) FROM VARIOUS LABORATORY COLONIES EXPOSED TO NON-BT
AND BT COTTON TISSUE IN A FRESH TISSUE BIOASSAY OF MATURE LEAVES

Percent Survival SEM (95% Credible Interval)

Stoneville, MS: Source of Laboratory Reared Fall Armyworm1 Starkville, MS2

Variety Cotton3 Bermudagras4 Royal Paulownia4

PSC 355 96.0 8.9 88.0 17.9 96.0 8.9 81.5 16.1
(68.8 95.5) (59.5 97.8) (68.7 99.3) (69.0 99.3)
Stoneville 474 72.0 17.9 72.0 26.8 84.0 21.9
(47.7 92.6) (47.3 94.2) (55.4 -97.1)
Stoneville 4691B5 64.0 26.1 60.0 14.1 84.0 21.9
(41.7 90.5) (43.7 73.6) (56.2 97.2)
PhytoGen 440 W6 4.0 8.9 4.0 8.9 4.0 8.9 0.0 0.0
(0.7 -30.7) (0.7 -31.4) (0.7 -31.1)

Mean survival within columns are significantly different if 95% credible intervals do not overlap (a = 0.05, MCMC simulations).
'Mature leaves located five mainstem nodes below the terminal. Evaluation at 4 d after infestation.
Mature leaves located four mainstem nodes below the terminal. Evaluation at 5 d after infestation.
Corn-associated strain as determined by genetic analysis.
'Rice-associated strain as determined by genetic analysis.
'Cotton containing CrylAc (MON 531) Bt protein.
'Cotton containing CrylAc (DAS-21023-5) and CrylF (DAS-24236-5) Bt proteins.


I







Florida Entomologist 91(4)


survival was significantly less on PHY 440 W as
compared to Stoneville 4691B.
At Stoneville, MS, genetic analysis of fall ar-
myworm collections indicated the larvae originat-
ing from bermudagrass and royal paulownia to be
of the rice-associated strain and the colony origi-
nating from cotton to be of the corn-associated
strain. Pashley (1986) described these fall army-
worm host strains, which exhibit polymorphisms
at 5 allozyme loci. Identification of fall armyworm
host strains is important because differences in
susceptibility to insecticides and CrylAc Bt toxin
have been demonstrated (Pashley et al. 1987; Ad-
amczyk et al. 1997). Adamczyk et al. (1997) dem-
onstrated that larvae collected from bermuda-
grass and browntop millet, Brachiaria ramosa
(L.), were significantly more sensitive to CrylAc
Bt cotton as compared to larvae collected from
maize. In contrast, results from our studies indi-
cate that corn-associated and rice-associated host
strains of fall armyworm are equally sensitive to
cotton producing CrylAc and CrylF combined,
while little control of either strain was evident in
the CrylAc-only cotton variety.
In field studies, varieties containing
CrylAc:CrylF significantly reduced (P<0.05) lar-
val infestations in whole plants as compared to a
non-Bt cotton variety, PHY 410 R, across 2 loca-
tions (Table 3). There were 6.3, 16.0, and 4.5-fold
reductions in larvae infestations for PHY 440 W,
PHY 470 WR, and PHY 475 WRF, respectively, as
compared to PHY 410 R, at Lonoke, AR. Similarly
at Pine Bluff, AR, there was a 10.6-fold reduction
for PHY 470 WR as compared to PHY 410 R. In
addition, at the Lonoke, AR location there was no
significant difference in larval infestations among
varieties containing CrylAc:CrylF.
Larval infestations were significantly (P<0.05)
reduced in samples of squares, flowers, and bolls
for varieties containing CrylAc:CrylF in Whar-
ton, TX, during 2006 as compared to a non-Bt cot-
ton variety (Table 4). Across structures, percent


larval infestation of PHY 425 RF (non-Bt) and
PHY 485 WRF (CrylAc:CrylF) ranged from 1.7 to
13.3% and 0.0 to 0.8%, respectively. Larval infes-
tations were significantly (P<0.05) reduced in
samples of bolls for PHY 485 WRF and PHY 375
WRF in Lonoke, AR during 2007 as compared to
their respective non-Bt cotton varieties (Table 4).
Significant differences were not detected for lar-
val infestations in flowers at the Lonoke, AR loca-
tion for either the PHY 400 or 300 varietal series.
Across structures and varieties, percent larval in-
festation of non-Bt varieties and CrylAc:CrylF
varieties ranged from 2.5 to 4.4% and 0.0 to 0.6%,
respectively.
Previous studies by Adamczyk & Gore (2004b)
have established that CrylF Bt protein, rather
than CrylAc, in a CrylAc:CrylF variety provides
control of fall armyworm and that synergism be-
tween the two proteins was not apparent. In addi-
tion, commercial cotton varieties containing
CrylAc (event MON 531) have not provided com-
mercially acceptable control of fall armyworm
(Stewart et al. 2001). Similar results were ob-
served in our laboratory bioassay in that survival
was similar between Stoneville 4691B (CrylAc)
and PSC 355 (non-Bt) cotton across three colonies
of fall armyworm. Therefore our results support
the conclusions ofAdamczyk & Gore (2004b), that
control provided by varieties containing CrylAc
and CrylF is attributed to the presence of CrylF
Bt protein.
Fall armyworm infestations are initially estab-
lished on mature leaves and these studies have
demonstrated high levels of mortality (96%) with
a variety containing CrylF. Coincidentally, ex-
pression of CrylF protein is greatest in mature
leaves as compared to other structures including
terminal leaves, squares, flowers, and bolls and
protein levels in mature leaves increases with age
(Dow AgroSciences, LLC, unpublished data). As-
suming there is a positive relationship between
protein expression and control of target insects in


TABLE 3. COMPARISON OF COTTON VARIETIES CONTAINING CRY1AC:CRY1F AND A NON-BT VARIETY FOR CONTROL OF
FALL ARMYWORM IN NATURAL INFESTATION FIELD STUDIES, 2005.

Percent Larval Infestation' SEM (95% Credible Interval)

Variety Lonoke, AR Pine Bluff, AR

PHY 440 W2 6.3 4.8 (2.2 12.6) -
PHY 470 WR 2.5 5.0 (0.3 7.0) 5.6 6.7 (0.2 3.2)
PHY 475 WRF 8.8 8.5 (3.7 16.0) -
PHY 410 R 40.0 10.8 (29.7 50.8) 59.4 39.3 (59.0 72.9)

Mean larval infestations within columns are significantly different if 95% credible intervals do not overlap (a = 0.05, MCMC sim-
ulations).
Peak date of fall armyworm infestations based on percent larval infestation in PHY 410 R non-Bt treatment.
'Cotton varieties containing CrylAc (DAS-21023-5) and CrylF (DAS-24236-5)Bt proteins are denoted by W'.


December 2008







Siebert et al.: CrylF For Control of Fall Armyworm


TABLE 4. COMPARISON OF A CRY1AC:CRY1F COTTON VARIETY AND A RELATED NON-BT COTTON FOR CONTROL OF FALL
ARMYWORM IN NATURAL INFESTATION STUDIES.

Percent Larval Infestations SEM (95% Credible Interval)'

Location/Year Structure PHY 485 WRF2 PHY 425 RF

Wharton, TX 2006 Flower 0.8 1.4 (0.03 3.2) 13.3 8.0 (7.9 20.0)
Square 0.0 0.0 3.3 1.4 (1.0 7.3)
Boll 0.0 + 0.0 1.7 + 2.9 (0.2 4.7)
PHY 485 WRF PHY 425 RF
Lonoke, AR 2007 Flower 0.6 1.3 (0.02 2.5) 2.5 3.5 (0.7 5.5)
Boll 0.0 + 0.0 4.4 + 3.1 (1.8 8.2)
PHY 375 WRF PHY 315 RF
Flower 0.6 1.3 (0.02 2.5) 2.5 2.0 (0.7 5.5)
Boll 0.0 + 0.0 3.8 1.4 (1.4 7.3)

Means larval infestations within rows are significantly different if 95% credible intervals do not overlap (a = 0.05, MCMC sim-
ulations).
'Peak date of fall armyworm infestations based on percent larval infestation in PHY 425 RF and PHY 315 RF non-Bt cotton va-
rieties.
'Cotton varieties containing CrylAc (DAS-21023-5) and CrylF (DAS-24236-5) Bt proteins are denoted by W'.


the field, it is plausible that increasing levels of
CrylF protein in leaves as plants mature could
support these findings.
Larvae that survive on mature leaf tissue in
the field will presumably move to cotton squares,
flowers, and bolls. In our field studies, cotton vari-
eties containing CrylAc:CrylF had significantly
reduced fall armyworm larval densities in these
structures as compared to that of non-Bt cotton
lines. In addition, field efficacy of CrylAc:CrylF
cotton was confirmed for multiple varieties (PHY
440 W, PHY 470 WR, PHY 475 WRF, PHY 485
WRF, PHY 375 WRF). It has been documented
that where protein expression for 2 CrylAc cotton
varieties differed the survival of bollworm also
differed (Adamczyk & Gore 2004a). The results
from our studies suggest deviations in efficacy
among commercial varieties containing combined
CrylAc (event DAS-21023-5) and CrylF (event
DAS-24236-5) for fall armyworm control should
not be anticipated based on a field study in
Lonoke, AR which compared PHY 440 W, PHY
470 WR, and PHY 475 WRF.
Results from our field and laboratory studies
evaluating insecticidal CrylF Bt protein, as ex-
pressed in maize hybrids and cotton varieties, in-
dicate economical levels of efficacy against fall ar-
myworm. These results were validated in field tri-
als conducted in numerous geographies ranging
from the midwestern to the southern U.S. and in
Brazil. In addition, CrylF was confirmed to be ef-
ficacious against a broad range of native fall ar-
myworm infestations, including two host-associ-
ated strains. In cotton, efficacy was also demon-
strated for several plant structures commonly in-
jured by fall armyworm. These results collectively
demonstrate that maize and cotton varieties pro-


during CrylF can be an important component of
an overall management program for fall army-
worm across a broad range of geographies.


ACKNOWLEDGMENTS

The authors gratefully acknowledge the efforts of
our many colleagues at Dow AgroSciences, University of
Arkansas, Texas Cooperative Extension Service, and
USDA-ARS research units in Stoneville, MS and
Starkville, MS. In particular, we thank S. Uhart for con-
tributing data from Argentina and N. Storer, G. Thomp-
son, and B. Braxton for reviewing the manuscript.


REFERENCES CITED

ADAMCZYK, JR., J. J., AND J. GORE. 2004a. Development
of bollworms, Helicoverpa zea, on two commercial
Bollgard cultivars that differ in overall CrylAc lev-
els. J. Insect Sci. 4: 32.
ADAMCZYK, JR., J. J., AND J. GORE. 2004b. Laboratory
and field performance of cotton containing CrylAc,
CrylF, and both CrylAc and CrylF (WideStrike)
against beet armyworm and fall armyworm (Lepi-
doptera: Noctuidae). Florida Entomol. 87: 427-432.
ADAMCZYK, JR., J. J., J. W. HOLLOWAY, B. R. LEONARD,
AND J. B GRAVES. 1997. Susceptibility of fall army-
worm collected from different plant hosts to selected
insecticides and transgenic Bt cotton. J. Cotton. Sci.
1:21-28.
ALI, A. A., R. G. LUTTRELL, AND H. N. PITRE. 1990. Feed-
ing sites and distribution of fall armyworm (Lepi-
doptera: Noctuidae) larvae on cotton. Environ. Ento-
mol. 19: 1060-1067.
ANDREWS, K. L. 1980. The whorlworm, Spodoptera fru-
giperda, in Central America and neighboring area.
Florida Entomol. 63: 456-467.
ARCHER, T. L., C. PATRICK, G. SCHUSTER, G. CRON-
HOLM, E. D. BYNUM, JR., AND W. P. MORRISON. 2001.











Ear and shank damage by corn borers and corn ear-
worm to four events of Bacillus thuringiensis trans-
genic maize. Crop Protection 20: 139-144.
BOX-STEFFENSMEIER, J. M., H. E. BRADY, AND D. COLLI-
ER 2008. The Oxford handbook of political methodol-
ogy. Oxford University Press. 800 pp.
BUNTIN, G. D., R. D. LEE, D. M. WILSON, AND R. M.
MCPHERSON. 2001. Evaluation of YieldGard trans-
genic resistance for control of fall armyworm and
corn earworm (Lepidoptera: Noctuidae) on corn.
Florida Entomol. 84: 37-42.
CARLIN, B. P., AND T. A. LEWIS. 2000. Bayes and empir-
ical bayes methods for data analysis. Chapman &
Hall/CRC, New York, NY.
CASTRO, B. A., B. R. LEONARD, AND T. J. RILEY. 2004.
Management of feeding damage and survival of
southwestern corn borer and sugarcane borer (Lepi-
doptera: Crambidae) with Bacillus thuringiensis
transgenic field corn. J. Econ. Entomol. 97: 2106-
2116.
CATANGUI, M. A., AND R. K. BERG. 2006. Western bean
cutworm, Striacosta albicosta (Smith) (Lepidopter-
an: Noctuidae), as a potential pest of transgenic
CrylAb Bacillus thuringiensis_corn hybrids in South
Dakota. Environ. Entomol. 35: 1439-1452.
COOK, D. R., B. R. LEONARD, AND J. GORE. 2004. Field
and laboratory performance of novel insecticides
against armyworms (Lepidoptera: Noctuidae). Flori-
da Entomol. 87: 433-439.
CRUZ, I. 1995. Fall armyworm on maize. Circular Tecni-
ca-Embrapa Centro Nacional de Pesquisa de Milho
e Sorgo. No. 21. 45 pp.
DAVIS, F. M., AND T. G. OSWALT. 1979. Hand inoculator
for dispensing lepidopterous larvae. SEA, USDA.
Advances in Agricultural Technology, AAT-S-9/Octo-
ber 1979.
DAVIS, F. M., S. S. NG, AND W. P. WILLIAMS. 1992. Visu-
al rating scale for screening whorl-stage corn for re-
sistance to fall armyworm. MS Agric. For. Exp. Sta.
Tech. Bull. 186. 9 pp.
DAVIS, F. M., W. P. WILLIAMS, AND P. M. BUCKLEY.
1998. Growth response of southwestern corn borer
(Lepidoptera: Crambidae) and fall armyworm (Lepi-
doptera: Noctuidae) larvae fed combinations of
whorl leaf tissue from a resistant and susceptible
maize hybrid. J. Econ. Entomol. 91: 1213-1218.
EDGE, J. M., J. H. BENEDICT, J. P. CARROLL, AND H. K.
REDING. 2001. Bollgard cotton: an assessment of glo-
bal economic, environmental, and social benefits. J.
Cotton. Sci. 5:121-136.
FERNANDES, O. D., J. R. P. PARRA, A. F. NETO, R. PICO-
LI, A. F. BORGATTO, AND C. G. B. DEMETRIO. 2003.
Effect of the genetically modified corn MON810 on
fall armyworm, Spodoptera frugiperda (J.E. Smith,
1797) (Lepidoptera: Noctuidae). Revista Brasileira
de Milho e Sorgo. 2: 25-35.
GHIDIU, G. M., AND J. T. ANDALORO. 1993. The relation-
ship between fall armyworm (Lepidoptera: Noctu-
idae) instar and susceptibility to insecticides applied
to sweet corn. Florida Entomol. 76: 549-555.
GIANESSI, L. P., C. S. SILVERS, S. SANKULA, AND J. E.
CARPENTER. 2002a. Plant biotechnology: current and
potential impact for improving pest management in
U.S. agriculture, insect resistant field corn (1). Na-
tional Center for Food and Agricultural Policy. Wash-
ington, D.C. 32 pp. (http://www.ncfap.org/whatwedo/
40Case Studies.php).


December 2008


GIANESSI, L. P., C. S. SILVERS, S. SANKULA, AND J. E.
CARPENTER. 2002b. Plant biotechnology: current and
potential impact for improving pest management in
U.S. agriculture, insect resistant cotton corn (1). Na-
tional Center for Food and Agricultural Policy. Wash-
ington, D.C. 34 pp. (http://www.ncfap.org/whatwedo/
40Case Studies.php).
GORE, J., B. R. LEONARD, AND J. J. ADAMCZYK. 2001.
Bollworm (Lepidoptera: Noctuidae) survival on
'Bollgard' and 'Bollgard II' cotton flower bud and
flower components. J. Econ. Entomol. 94: 1445-1451.
GOULD, F. 1998. Sustainability of transgenic insecticid-
al cultivars: integrating pest genetics and ecology.
Annu. Rev. Entomol. 43: 701-726.
GREENBERG, S. M., AND J. J. ADAMCZYK, JR 2007. Noc-
tuid survivorship and damage in Widestrike, Boll-
gard, and Bollgard II cottons in the lower Rio
Grande valley of Texas, pp. 316-320 In P. Dugger and
D. Richter [eds.], Proc. Beltwide Cotton Conf., 9-12
January 2007, New Orleans, LA. National Cotton
Council, Memphis, TN.
JACKSON, R. E., J. R. BRADLEY, AND J. W. VAN DUYN.
2003. Field performance of transgenic cottons ex-
pressing one or two Bacillus thuringiensis endotox-
ins against bollworm, Helicoverpa zea (Boddie). J.
Cotton Sci. 7: 57-64.
LABATTE, J. M. 1993. Within-plant distribution of fall
armyworm (Lepidoptera: Noctuidae) larvae on corn
during whorl-stage infestation. Florida Entomol. 76:
437-447.
MACINTOSH, S. C., T. B. STONE, S. R. SIMS, P. L. HUNST,
J. T. GREENPLATE, P. G. MARRONE, F. J. PERLAK, D.
A. FISCHOFF, AND R. L. FUCHS. 1990. Specificity and
efficacy of purified Bacillus thuringiensis proteins
against agronomically important insects. J. Inverte-
brate Path. 56: 258-266.
MEAGHER, JR., R. L., AND M. GALLO-MEAGHER 2003.
Identifying host strains of fall armyworm (Lepi-
doptera: Noctuidae) in Florida using mitochondrial
markers. Florida Entomol. 86: 450-455.
MCGAUGHEY, W. H., AND M. E. WHALON. 1992. Manag-
ing insect resistance to Bacillus thuringiensis toxins.
Science 258: 1451-1455.
MIFLIN, B. 1996. A view from industry, pp. 367-386 In G.
L. Persley [ed.], Biotechnology and Integrated Pest
Management. CAB International, Wallingford, UK.
MILA, A. L., AND T. J. MICHAILIDES. 2006. Use of Baye-
sian methods to improve prediction of panicle and
shoot blight severity of pistachio in California. Phy-
topathol. 96: 1142-1147.
MINITAB. 1998. MINITAB 12.21. Minitab, State College,
PA.
MINK, J. S., AND R. G. LUTTRELL. 1989. Mortality of fall
armyworm, Spodoptera frugiperda (Lepidoptera:
Noctuidae) eggs, larvae and adults exposed to sever-
al insecticides on cotton. J. Entomol. Sci. 24: 563-
571.
PASHLEY, D. P. 1986. Host-associated genetic differenti-
ation in fall armyworm: a sibling species complex?
Ann. Entomol. Soc. America 79: 898-904.
PASHLEY, D. P., T. C. SPARKS, S. S. QUISENBERRY, T.
JAMJANYA, AND P. F. DOWD. 1987. Two fall army-
worm strains feed on corn, rice, and bermudagrass.
Louisiana Agric. 30: 8-9.
PERDIGUERO, J. S. J. M. BARRAL, AND M. V. DE STACUL.
1967. Biological Aspects of Plagues of Corn of Region
Chaquefia: Evaluation of Damage. INTA, Station


Florida Entomologist 91(4)







Siebert et al.: CrylF For Control of Fall Armyworm


Experimental Agriculture, Presidency Roque Saenz
Peia. Bull. no. 46. 30 pp.
PILCHER, C. D., M. E. RICE, J. J. OBRYCKI, AND L. C.
LEWIS. 1997. Field and laboratory evaluations of
transgenic Bacillus thuringiensis corn on secondary
lepidopteran pest (Lepidoptera: Noctuidae). J. Econ.
Entomol. 90: 669-678.
QUISENBERRY, S. S. 1999. Armyworms, pp. 49-53 In K.
L. Steffey, M. E. Rice, J. All, D. A. Andow, M. E. Gray,
and J. W. Van Duyn [eds.], Handbook of Corn Insects,
Entomological Society of America, Lanham, MD.
R DEVELOPMENT CORE TEAM. 2005. R: a language and
environment for statistical computing. R Founda-
tion for Statistical Computing. Vienna, Austria. ht-
tp://www.R-project.org.
RITCHIE, S. W., J. J. HANAWA, AND G. O. BENSON. 1993.
How A Corn Plant Develops. Iowa State Univ. Coop.
Ext. Serv. Spec. Rep. 48. 21 pp.
SANTOS, K. B., A. M. MENEGUM, AND P. M. O. J. NEVES.
2005. Biology and consumption of Spodoptera erida-
nia (Cramer) (Lepidoptera: Noctuidae) in different
hosts. Neotropical Entomol. 34: 903-910.
SENA, D. G., F. A. C. PINTO, D. M. QUEIROZ, AND P. A.
VIANA. 2003. Fall armyworm damaged maize plant
identification using digital images. Biosystems Eng.
85: 449-454.
SHELTON, A. M., J. Z. ZHAO, AND R. T. ROUSH. 2002.
Economic, ecological, food safety, and social conse-
quences of the deployment of Bt transgenic plants.
Annu. Rev. Entomol. 47: 845-881.
SIEBERT, M. WILLRICH, K. V. TINDALL, B. R. LEONARD,
J. W. VAN DUYN, AND J. M. BABCOCK. 2008. Evalu-
ation of corn hybrids expressing CrylF (Herculex I
Insect Protection) against fall armyworm (Lepi-
doptera: Noctuidae) in the southern United States.
J. Entomol. Sci. 43: 41-51.
SPARKS, A. N. 1979. A review of the biology of the fall
armyworm. Florida Entomol. 62: 82-87.


STEWART, S. D., J. J. ADAMCZYK, JR., K. S. KNIGHTEN,
AND F. M. DAVIS. 2001. Impact of Bt cottons ex-
pressing one or two insecticidal proteins of Bacil-
lus thuringiensis Berliner on growth and survival
on noctuid (Lepidoptera) larvae. J. Econ. Entomol.
94: 752-760.
TABASHNIK, B. E. 1994. Evolution of resistance to Ba-
cillus thuringiensis. Annu. Rev. Entomol. 39: 47-
49.
TABASHNIK, B. E., A. L. PATIN, T. J. DENNEHY, Y. LIU, Y.
CARRIER, M. A. SIMS, AND L. ANTILLA. 2000. Fre-
quency of resistance to Bacillus thuringiensis in field
populations of pink bollworm. Proc. Natl. Acad. Sci.
USA 97: 12980-12984.
THOMAS, A., B. O'HARA, U. LIGGES, AND S. STURTZ.
2006. Making BUGS Open. R News 6: 12-17.
UNITED STATES ENIVRONMENTAL PROTECTION AGENCY.
2005. Biopesticide registration action document,
Bacillus thuringiensis CrylF corn. http://
www.epa.gov/oppbppdl/ biopesticides/ingredients/
tech_docs/brad_006481.pdf.
WAQUIL, J. M., F. M. FERREIRA VILLELA, AND J. E. FOS-
TER 2002. Resistance of Bt transgenic maize (Zea
mays L.) to fall armyworm, Spodoptera frugiperda
(Smith) (Lepidoptera: Noctuidae). Revista Brasilei-
ra de Milho e Sorgo. 1: 1-11.
WILLRICH, M. M., L. B. BRAXTON, J. S. RICHBURG, R. B.
LASSITER, V. B. LANGSTON, R. A. HAYGOOD, J. M. RI-
CHARDSON, F. J. HAILE, R. M. HUCKABA, J. W. PEL-
LOW, G. D. THOMPSON, AND J. P. MUELLER 2005.
Field and laboratory performance of WideStrike in-
sect protection against secondary lepidopteran
pests, pp. 1262-1268 In Proc. 2005 Beltwide Cotton
Conf, New Orleans, LA, January 4-7, 2005.
Yu, S. J. 1983. Age variation in insecticide susceptibil-
ity and detoxification capacity of fall armyworm
(Lepidoptera: Noctuidae) larvae. J. Econ. Entomol.
76: 219-222.







Florida Entomologist 91(4)


December 2008


DOES SECONDARY PLANT METABOLISM PROVIDE A MECHANISM FOR
PLANT DEFENSES IN THE TROPICAL SODA APPLE SOLANUM VIARUM
(SOLANALES: SOLANACEAE) AGAINST SPODOPTERA EXIGUA AND
S. ERIDANIA (LEPIDOPTERA: NOCTUIDAE)?

R. L. HIX, M. T. KAIRO AND S. REITZ1
Center for Biological Control, CESTA, Florida A & M University, Tallahassee, FL 32307

1USDA-ARS-CMAVE, Tallahassee, FL 32307

ABSTRACT

Survival assays were conducted with beet armyworm Spodoptera exigua (Hiibner) and south-
ern annyworm S. eridania (Stoll) with tropical soda apple Solanum uiarum Dunal, a relative
of tomato. In addition, polyphenol oxidase (PPO) enzyme assays were conducted to determine
if plant defense compounds are being produced by tropical soda apple in response to her-
bivory. Both S. exigua and S. eridania induced plant defenses in tropical soda apple. Signifi-
cantly more S. exigua and S. eridania neonate larvae survived to 2nd instar on non-induced
plants and artificial diet when compared with plants with induced defenses. Tropical soda ap-
ple plants fed on by S. exigua and S. eridania had significantly increased PPO activity.

Key Words: induced resistance, secondary plant defenses, night shade, biological control

RESUME

Se condujeron analysis de supervivencia con palometas de Spodoptera exigua y palometas
surenas de S. eridana en manzana de soda tropical Solanun viarum relative del tomate. Mas
aun, analysis con enzimas de oxidasa de polyphenol (PPO) fueron realizaron para determi-
nar si se produce compuestos secundarios como defense de la plant de manzana de soda tro-
pical en respuesta a herviboros. Tanto Spodoptera exigua como S. eridana parecen inducir en
la plant defenses secundarias. Significativamente mas larvas neonatas de S. exigua y S.
eridana sobrevivieron al segundo estadio en plants no inducidas y dietas artificiales com-
paradas con plants con defenses inducidas. Las plants de manzana de soda tropical ali-
mentadas con S. exigua y S. eridana han aumentado significartivamente la actividad de
PPO.


Translation provided by the authors.


The tropical soda apple (TSA) Solanum vi-
arum Dunal is a nightshade classified by the
State of Florida (FLEPPC 2007) and the USDA as
a noxious weed. Tropical soda apple is native to
Argentina, Brazil, Paraguay, and Uruguay (Gan-
dolfo et al. 2007) and was first reported in Florida
in 1988 (Mullahey et al. 1993). It has invaded
thousands of acres of pasture thereby reducing
carrying capacity, and has invaded natural areas
displacing native plant species (Mullahey &
Colvin 1993; Medal & Cuda 1999). Tropical soda
apple serves as a reservoir for several viruses in-
cluding the tomato spotted wilt virus (TSWV) vec-
tored by thrips including Frankliniella occidenta-
lis (Pergande), the western flower thrips.
Insect herbivory can have effects on both the
plant and the herbivore. For example, tomato
plants respond to herbivory by producing pro-
teinase inhibitors that reduce the palatability
and nutritional quality of the plant. These plant
defense compounds are directed by the octade-
canoid pathway (Karban & Baldwin 1997; Tha-


ler 1999a, 1999b). Herbivore feeding has been
found to induce peroxidase, lipoxygenase,
polyphenol oxidase (PPO) and proteinase inhibi-
tor (PI) activities (Stout et al. 1998) but not nec-
essarily all at the same time by a given insect.
This induced resistance (IR) system directed by
the octadecanoid pathway is triggered by jas-
monates such as jasmonic acid by the feeding of
a broad range of insects (Broadway & Duffey
1986; Karban & Baldwin 1997). These induced
defense proteins can reduce insect fitness, host
preference, and nutritional value as well as in-
crease mortality and work independently of con-
stitutive defenses. Plants produce more than
1000 different volatiles by various plant species
which may attract parasitoids and predators
that attack the herbivore. These include the
"green-leaf" 6-carbon aldehydes, alcohols and
derivatives as well as alkanes, alkenes, alcohols,
ketones, aldehydes, ethers, esters, and carboxy-
lic acids just to name a few. Many of them are not
species specific while others are very specific in







Hix et al.: Induced Resistance in Tropical Soda Apple


which species they attract (D'Allessandro &
Turlings 2005).
The beet armyworm Spodoptera exigua (Hiib-
ner) (Lepidoptera: Noctuidae) was chosen because
it is a generalist herbivore known to induce sec-
ondary plant defenses in tomato, a closely related
species in the genus Solanum (Felton et al. 1992;
Alborn et al. 1997; Alborn et al. 2000). The south-
ern armyworm Spodoptera eridania (Stoll) was
chosen for this study due to its range overlap with
TSA in Florida (R.L.H. unpublished data). The
objectives of this study were to determine if feed-
ing by the generalist herbivores S. exigua and S.
eridania (Stoll) induced secondary plant defenses
in TSA.

MATERIALS AND METHODS

Spodoptera exigua colonies were established
from eggs obtained from Bio-Serv (Frenchtown,
NJ) and S. eridania colonies were established
from wild caterpillars collected feeding on tomato.
Colonies were maintained on S. exigua artificial
diet (Bio-Serv, Frenchtown, NJ). Prior to assays,
caterpillars were allowed to feed on tomato for 24
h.
Tropical soda apple plants were grown from
seed collected from wild plants in Leon Co., Flor-
ida. Plants were planted in pots (12.7 cm x 12.5
cm) with Metro Growing Mix 220 (Sierra Grace
Hort Products, Milpita, CA) (Mullahey & Cornell
1994) and fertilized with Peters 20-20-20 (N) so-
lution and maintained in an environmental
chamber with photoperiod of 16:8 (L:D) or an out-
door shade tent.
Five 4thinstars ofS. exigua were allowed to feed
on 5 TSA plants (i.e., 5 larvae per plant) for a pe-
riod of 3 d. These plants were 45 d old. After this
"induction" period, 100 neonate S. exigua larvae
were placed on each of 5 induced plants, 5 control
plants, and an external control consisting ofS. ex-
igua artificial diet (BioServ, Frenchtown, NJ).
Those molting to the second instar were quanti-
fied. Plants were in individual cages to prevent
caterpillars from moving from plant to plant.
This experiment was conducted in the same
way substituting S. eridania in place of S. exigua.
This experiment was repeated twice for S. exigua
and S. eridania. Means and standard errors were
calculated and subjected to ANOVA followed by
Tukey's HSD where appropriate (a = 0.05). Sta-
tistical analyses were done with JMP IN (SAS In-
stitute 1996).

Polyphenol Oxidase Assay

Five 4th instars of S. exigua were placed on each
of eight 45-d-old TSA plants, and five 4th instars of
S. eridania larvae were placed on each of eight 45-
d-old plants. The control consisted of eight 45-d-
old plants that had never been exposed to herbi-


vores. After 2 d of feeding, a leaf from each plant
was excised at the petiole with a razor blade (n =
8). PPO activity was quantified by the methods of
Felton et al. (1989). The rate of reaction was de-
termined with a spectrophotometer (BioTek) to
measure absorbance at 470 nm every 10 s over 3
min. This reaction rate was then divided by the
mass of the leaf cutting to determine the rate for
that cutting (n = 8). The arcsine-square root
transformation was used on the mean rates and
subjected to ANOVA followed by Tukey's HSD (a
= 0.05).

RESULTS

Significantly more neonate S. exigua larvae
survived to 2nd instar on the non-induced tropical
soda apple and artificial diet than on induced TSA
(F = 12.8, df= 2, 14; P = 0.001). The mean number
surviving on the artificial diet was 91.0 ( 7.3
SEM), on non-induced plants 74.6 ( 12.3 SEM),
and on induced plants 27.3 ( 9.75 SEM) (Fig. 1A).
This experiment was repeated with similar re-
sults (F = 12.6, df= 2, 14; P = 0.001) (Fig. 1B). The
mean survival on the artificial diet was 95.4 ( 6.2
SEM), on non-induced plants 69.1 ( 11.9 SEM),
and on induced plants 30.2 ( 8.8 SEM).
Significantly more neonate S. eridania larvae
survived to the 2nd instar on the non-induced
TSA and artificial diet than did on induced plants
(F = 11.0, df= 2, 14;P = 0.002). The mean survival
on the non-induced plants was 67.16 ( 8.1 SEM),
on artificial diet 93.3 ( 5.9 SEM), and on induced
plants 34.0 ( 10.3 SEM) (Fig.2A). This experi-
ment was repeated with similar results (F = 7.4,
df = 2, 14; P = 0.008) (Fig. 2B). The mean survival
on the artificial diet was 89.0 ( 8.1 SEM), on non-
induced plants 80.0 (10.9 SEM) and on induced
plants 29.0 ( 15.5 SEM).

Polyphenol Oxidase Assay

The PPO activity for both S. exigua and S. eri-
dania induced plants was statistically different
than the non-induced plants (P < 0.008 and P <
0.003, respectively), but means were not different
from each other (P < 0.868). ANOVA was per-
formed on transformed data (F = 8.9, df = 2, 23; P
= 0.002). The data in Fig. 3 are the untransformed
data.

DISCUSSION

Before this study, nothing was known about
secondary plant defenses in tropical soda apple.
We hypothesized that feeding by generalist and/
or specialist herbivores will induce secondary
plant defenses in TSA. Survival assays were con-
ducted with beet armyworm and southern army-
worm because these generalist herbivores are
known to induce secondary plant defenses in to-







Florida Entomologist 91(4)


*O 100
Stoo



- 0
0


I
m
C 21
a
(B


non-Induced Induced


Art Diet


4o 100


~50

C
S0oo


to



c 20
45


non-Induced Induced


B.

so a




20
0



0

non-induced Induced Art Diet
Treatment

Fig. 1. A. Mean number (+ SEM) of neonate
Spodoptera exigua surviving to 2"d instar on non-in-
duced TSA were significantly higher than on induced
TSA plants (P < 0.038). Tukey's HSD n = 5, a = 0.05. B.
The mean number of neonate larvae surviving to 2nd in-
star was significantly higher on non-induced plants
than induced plants (P < 0.029). Tukey's HSD n = 5, a =
0.05. The artificial diet means and non-induced plant
means were not statistically different in either experi-
ment (P > 0.106 andP > 0.153 forA and B, respectively).
Means followed by the same letter are not significantly
different.


mato, a relative of TSA. In addition, polyphenol
oxidase (PPO) enzyme assays were conducted to
determine if secondary plant defense compounds
are being produced in response to herbivory. This
oxidative enzyme is produced by solanaceous
plants including tobacco and tomato (Stout et al.
1996; Thaler et al. 1996; Stout et al. 1998; Thaler
1999a). PPO reacts with substrates in the herbi-
vore gut making essential amino acids unavail-
able (Duffey & Felton 1989; Felton et al. 1992; Ha-
litschke et al. 2001). Based on our results, TSA
plants fed on by S. exigua and S. eridania had sig-
nificantly increased PPO activity. Both S. exigua
and S. eridania appears to induce secondary plant
defenses in TSA.


a a




0







non-Induced Induced Art Diet
Treatment

Fig. 2. A. Mean number ( SEM) of neonate
Spodoptera eridania surviving to 2nd instar on non in-
duced TSA were significantly higher than on induced
TSA plants (P < 0.015). Tukey's HSD n = 5, a = 0.05. B.
Mean number of neonate larvae surviving to 2nd instar
was significantly higher on non-induced plants than in-
duced plants (P < 0.026). Tukey's HSD n = 5, a = 0.05.
The artificial diet means and non-induced plant means
were not statistically different in either experiment (P >
0.498 and P > 0.856 for A and B, respectively). Means fol-
lowed by the same letter are not significantly different.


The tortoise beetle Gratiana boliviana Spa-
eth (Coleoptera: Chrysomellidae) was released
in Florida in 2003 as a classical biological con-
trol agent of TSA (Medal et al. 2003). G. bolivi-
ana and other biological control agents in quar-
antine are specialists that only feed on this spe-
cies. These species-specific agents may provide
negative effects on F occidentalis feeding, egg
laying performance or vector competency. Trop-
ical soda apple is known to be a host of tomato
spotted wilt virus (TSWV). Generalist herbi-
vores like F occidentalis may demonstrate
lower feeding preference for plants with in-
duced plant defenses (Karban & Baldwin 1997;
Gouinguene et al. 2003). We are currently


Art Diet


December 2008







Hix et al.: Induced Resistance in Tropical Soda Apple


0.00 *- -
Nonlnduced BAW Induced SAW Induced
Tropical Soda Apple
Solanum Viarum

Fig. 3. The mean PPO level of Spodoptera exigua in-
duced and S. eridania induced tropical soda apple
leaves were significantly higher than the non-induced
plants (P < 0.008 and P < 0.003, respectively). Tukey's
HSD n = 8, a = 0.05.


studying tropical soda apple induced responses
in context of G. boliviana, S. exigua, and F occi-
dentalis.

ACKNOWLEDGMENTS

This research was funded in part by the USDA-ARS
and Florida A & M University. We thank Mr. George
Benn Marshal for technical assistance.

REFERENCES CITED

ALBORN, H. T., T. C. J. TURLINGS, T. H. JONES, G. S.
STENHAGEN, J. H. LOUGHRIN, AND J. H. TUMLINSON.
1997. An elicitor of plant volatiles from beet army-
worm oral secretion. Science 276: 945-949.
ALBORN, H. T., T. H. JONES, G. S. STENHAGEN, AND J. H.
TUMLINSON. 2000. Identification and synthesis ofvo-
licitin and related components from beet armyworm
oral secretions. J. Chem. Ecol. 26: 203-220.
BROADWAY, R. M., AND S. S. DUFFEY. 1986. Plant pro-
teinase inhibitors: mechanism of action and effect on
the growth and digestive physiology of larval Helio-
this zea and Spodoptera exigua. J. Insect Physiol. 32:
827-833.
D'ALESSANDRO, M., AND T. C. J. TURLINGS. 2005. In situ
modification of herbivore-induced plant odors: a nov-
el approach to study the attractiveness of volatile or-
ganic compounds to parasitic wasps. Chem. Senses
30: 739-753.
DUFFEY, S. S. AND G. W. FELTON. 1989. Plant enzymes
in resistance to insects, pp. 289-313 In J. R. Whitak-
er and P. E. Sonnet [eds], Biocatalysis in Agricultur-
al Biotechnology. Am. Chem. Soc.
FELTON, G. W., K. DONATO, R. J. DEL VECCHIO, AND S.
S. DUFFEY. 1989. Activation of plant foliar oxidases
by insect feeding reduces nutritive quality of foliage
for noctuid herbivores. J. Chem. Ecol. 15: 2667-2694.
FELTON, G. W., K. K. DONATO, R. M. BROADWAY, AND S.
S. DUFFEY. 1992. Impact of oxidized plant phenolics
on the nutritional quality of dietary protein to a noc-
tuid herbivore, Spodoptera exigua. J. Insect Physiol.
38: 277-285.


FLEPPC. 2007. Florida Exotic Pest Plant Council's
2005 List of invasive species. www.fleppc.org.
GANDOLFO, D., F. McKAY, J. C. MEDAL, AND J. P. CUDA.
2007. Open-field host specificity test of Gratiana bo-
liviana (Coleoptera: Chrysomelidae), a biological
control agent of tropical soda apple (Solanaceae) in
the United States. Florida Entomol. 90: 223-228.
GOUINGUENE, S., H. ALBORN, AND T. C. J. TURLINGS.
2003. Induction of volatile emissions in maize by dif-
ferent larval instars of Spodoptera littoralis. J.
Chem Ecol 29: 145-162.
HALITSCHKE, R., U. SCHITTKO, G. POHNERT,
W. BOLAND, AND I. T. BALDWIN. 2001. Molecular in-
teractions between the specialist herbivore Mandu-
ca sexta (Lepidoptera, Sphingidae) and its natural
host Nicotiana attenuata. III. Fatty acid-amino acid
conjugates in herbivore oral secretions are necessary
and sufficient for herbivore-specific plant responses.
Plant Physiol. 125: 711-717.
KARBAN, R., AND I. T. BALDWIN. 1997. Induced Respons-
es to Herbivory. The University of Chicago Press,
Chicago.
MEDAL, J. C., AND J. P. CUDA. 1999. Biological control of
invasive weeds in Florida and the Caribbean region,
In Proc. Caribbean Basin Administrative Group
Workshop on Approaches to Mitigating the Effects of
Exotic Pests on Trade and Agriculture in the Carib-
bean Region, 16-18 June 1999, Homestead, FL. Uni-
versity of Florida-Tropical Research Education Cen-
ter, Homestead.
MEDAL, J. C., D. GANDOLFO, AND J. P. CUDA. 2003. Bi-
ology of Gratiana boliviana, the First Biocontrol
Agent Released to Control Tropical Soda Apple in
the USA. University of Florida-IFAS Extension Cir-
cular ENY-826. 3pp.
MULLAHEY, J. J., AND D. L. COLVIN. 1993. Tropical Soda
Apple: A New Noxious Weed in Florida. University of
Florida Cooperative Extension Service Fact Sheet
WRS-7.
MULLAHEY, J. J., AND J. CORNELL. 1994. Biology of trop-
ical soda apple (Solanum viarum) an introduced
weed in Florida. Weed Technol. 8: 465-469.
MULLAHEY, J. J., M. NEE, R. P. WUNDERLIN, AND K. R.
DELANEY. 1993. Tropical soda apple: a new weed
threat in subtropical regions. Weed Technol. 7: 783-
786.
SAS INSTITUTE. 1996. JMP IN version 3. Cary NC.
STOUT, M. J., K. V. WORKMAN, AND S. S. DUFFEY. 1996.
Identity, spatial distribution, and variability of in-
duced chemical responses in tomato plants. Ento-
mol. Exp. Appl. 79: 255-271.
STOUT, M. J., K. V. WORKMAN, R. M. BOSTOCK, AND S. S.
DUFFEY. 1998. Stimulation and attenuation of in-
duced resistance by elicitors and inhibitors of chem-
ical induction in tomato (Lycopersicon esculentum)
foliage. Entomol. Exp. Appl. 86: 267-279.
THALER, J. S. 1999a. Induced resistance in agricultur-
al crops: effect ofjasmonic acid on herbivory and
yield in tomato plants. Environ. Entomol. 28: 30-
37.
THALER, J. S. 1999b. Jasmonate-inducible plant defens-
es cause increased parasitism of herbivores. Nature
399: 686-688.
THALER, J. S., M. J. STOUT, R. KARBAN, AND S. S. DUF-
FEY. 1996. Exogenous jasmonates simulate insect
wounding in tomato plants (Lycopersicon esculentum)
in the laboratory and field. J. Chem Ecol. 22: 1767-
1781.







Florida Entomologist 91(4)


December 2008


EFFICACY OF VIP3A AND CRY1AB TRANSGENIC TRAITS
IN COTTON AGAINST VARIOUS LEPIDOPTERAN PESTS

J. J. ADAMCZYK, JR. AND J. S. MAHAFFEY1
USDA, ARS, KSARC, Beneficial Insects Research Unit, 2413 E. Hwy 83, Weslaco, TX 78501

1Delta and Pine Land Company, Scott, MS 38772

ABSTRACT

From 2004 through 2005, plots of experimental transgenic cotton lines containing the vege-
tative insecticidal protein, Vip3A; 8-endotoxin, CrylAb; and both Vip3A and CrylAb were
evaluated for efficacy against certain lepidopteran pests. Results showed that the cotton line
containing Vip3A was more efficacious against the beet Spodoptera exigua (Hiibner) and fall
armyworm Spodoptera frugiperda (J. E. Smith) compared to the CrylAb cotton line; how-
ever, the CrylAb cotton line was more efficacious against the tobacco budworm Heliothis
virescens F. compared to the cotton line containing Vip3A. Both the Vip3A and CrylAb cot-
ton lines provided similar mortality against the bollworm Helicoverpa zea (Boddie). No syn-
ergism between Vip3A and CrylAb was observed.


RESUME

Desde el aio 2004 hasta el final de 2005, se evaluaron parcelas experimentales con lines
transg6nicas de algod6n que contienen la protein vegetal insecticide, Vip3A; 6-endotoxin,
CrylAb; y se evaluaron ambas Vip3A y CrylAb por su eficacia contra ciertas plagas lepid6p-
teros. Los resultados mostraron que la linea de algod6n que contiene Vip3A fue mas eficaz
contra el gusano de ejercito de remolacha, Spodoptera exigua (Hiibner) y el gusano cogollero,
Spodoptera frugiperda (J. E. Smith) en comparaci6n con la linea CrylAb de algod6n; sin em-
bargo, la linea CrylAb de algod6n fue mas eficaz contra el gusano del brote de tabaco, Helio-
this virescens F. en comparaci6n con la linea de algod6n con Vip3A. Ambas lines de algod6n
(Vip3A y CrylAb) proveyeron una mortalidad similar contra el gusano de la belota, Helicov-
erpa zea (Boddie). Ningun sinergismo fue observado entire la Vip3A y CrylAb.


Advancements for insect control that utilize
transgenic technology will continue to improve ef-
ficacy against many lepidopteran pests. Current
and experimental cotton varieties can contain
CrylAc alone (Bollgard, Monsanto Ag. Co., St.
Louis, MO), or they can be stacked with Cry2Ab
(Bollgard II, Monsanto Ag. Co.) or CrylF (Wide-
Strike, Dow Agrosciences, Indianapolis, IN).
Furthermore, a novel vegetative insecticidal pro-
tein from B. thuringiensis (Vip3A) stacked with
CrylAb is currently in development (VipCotM,
Syngenta Crop Protection, Greensboro, NC).
The beet armyworm, Spodoptera exigua (Hiib-
ner) is an occasional but serious pest of various
vegetable and row crops in the mid-southern
United States of America. Although larval feeding
on cotton is primarily concentrated on foliage, lar-
vae can cause devastating losses in yield (Hardee
& Herzog 1997; Adamczyk et al. 1998). The fall ar-
myworm, Spodoptera frugiperda (J. E. Smith) also
is a destructive migratory pest of many crops in the
Western Hemisphere (Sparks 1979; Young 1979).
Like the beet armyworm, this pest has the poten-
tial to damage both conventional cotton bolls and
Bollgard cotton bolls (Adamczyk et al. 1998).
Although certain lepidopteran pests of cotton
are controlled by Bollgard cotton (e.g., tobacco


budworms, Heliothis virescens F. and pink boll-
worms, Pectinophora gossypiella (Saunders)), the
CrylAc 8-endotoxin in Bollgard cotton is less ef-
fective for controlling beet and fall armyworms,
and bollworms, Helicoverpa zea (Boddie) (Adamc-
zyk et al. 1998; Henneberry et al. 2001). Conse-
quently, outbreaks of these pests on Bollgard of-
ten need full application rates of foliar insecticide
treatments to keep populations below economic in-
jury levels (Hood 1997; Smith 1997). However, the
addition of other Cry proteins stacked with CrylAc
appears to have improved the efficacy (i.e., Boll-
gard II and WideStrikeTM) against beet and fall
armyworms, and bollworms (Adamczyk et al.
2001; Stewart et al. 2001;Adamczyk & Gore 2004).
The purpose of the study was to examine the effi-
cacy of Vip3A, CrylAb, and CrylAb + Vip3A traits
against various Lepidoptera in laboratory bioas-
says and small experimental field plots.

MATERIALS AND METHODS

Field Plots

From 2004-2005, experimental transgenic cot-
ton lines containing Vip3A (2004 and 2005),
CrylAb (2005), or CrylAb stacked with Vip3A







Adamczyk & Mahaffey: Efficacy of Vip3A and CrylAb Cottons Against Lepidoptera 571


(2005) were planted in research plots in the Mis-
sissippi Delta (Table 1). In 2004, three different
transgenic cotton lines containing the Vip3A
event were planted near Stoneville, MS in early
May with plots consisting of 2 rows (1.0 m centers
x 10.67 m). These events (COT102, COT202 and
COT203) contained different promoters driving
Vip3A expression, where COT202 and COT203
contained a more robust promoter than COT102.
In 2005, eight different transgenic cotton lines
containing Vip3A, CrylAb, or CrylAb stacked
with Vip3A events were planted in late Apr near
Scott, MS with plots consisting of 4 rows (1.0 m
centers x 12.20 m). In addition, 3 different
CrylAb lines that had different insertion events
(02A, 67B, and 69D) were evaluated. All plots
were arranged in a randomized complete block
design with each variety replicated 4 times (once
in each block, except COT102 replicated twice in
each block in 2004). Only insecticides not active
on Lepidoptera were applied to all plots through-
out the season as dictated by local management
practices. All Lepidoptera (beet and fall army-
worms, bollworms, and tobacco budworms) uti-
lized in these studies were obtained from labora-
tory colonies maintained at the USDA, ARS,
Southern Insect Management Research Unit, lo-
cated in Stoneville, MS.

Efficacy of Vip3A Events (2004)

Terminal leaves or flower buds (squares, fall
armyworms only) containing 3 Vip3A events were
assayed for bioactivity against beet armyworms,
fall armyworms, bollworms, and tobacco bud-
worm neonates (Table 1). Squares were utilized to
evaluate activity against fall armyworms because


TABLE 1. BIOASSAYS CONDUCTED WITH COTTON LINES IN
2004 AND 2005.

Year Cotton Lines Trait

2004 COT 102 Vip3A
COT 202 Vip3A
COT 203 Vip3A
Coker 312 None

2005 COT 102 Vip3A
COT 203 Vip3A
02A CrylAb
67B CrylAb
69D CrylAb
02A x COT202 CrylAb + Vip3A
67B x COT202 CrylAb + Vip3A
69D x COT202 CrylAb + Vip3A
Coker 312 None
DP 393 None
DP 491 None
DP 493 None


the larval stage primarily feeds on fruit (Adamc-
zyk et al. 1998). Individual leaves or squares col-
lected from plants that had begun to flower were
placed into a Tight-Fit Lid sealing Petri dish (50 x
9 mm, BD Falcon@ #351006, VWR International).
A single larva was placed in a dish containing a
single terminal leaf or square. Ten dishes per plot
were used for bollworms, while 20 dishes per plot
were used for beet armyworms and tobacco bud-
worms. Thirty dishes per plot were used for fall
armyworms. At various days after exposure to
cotton tissue, larvae were prodded with a camel-
hair brush and considered dead if no coordinated
movement was observed. Percent mortality of ne-
onates was analyzed by REML-ANOVA, and
means were separated by LSMEANS (Littell et
al. 1996; PROC MIXED, SAS Institute 2001).
Because of the historic ability of the tobacco
budworm to cause severe economic losses to cot-
ton, a field experiment was conducted to further
evaluate efficacy of the Vip3A events against this
pest. Two 1-d-old larvae were placed on a single
terminal and covered with a nylon cage to prevent
interplant movement (Fig. 1). Plants were at the
pre-bloom stage at the time of infestations. Ten
cages were utilized per plot. After 2 d, 3 cages per
plot were removed and larval mortality was re-
corded. The remaining cages were removed and
assessed at 5 d after larval infestations. All larvae
that were alive were placed in individual 1-oz
souffle cups and transported to the laboratory in a
cooler containing ice. Larvae were weighed within
2 h.

Efficacy of the Vip3A, CrylAb, and CrylAb + Vip3A
Traits (2005)

Terminal leaves from different cotton lines
containing 2 Vip3A events, 3 CrylAb events, and
3 CrylAb stacked with Vip3A events were as-
sayed as described above for bioactivity against


Fig. 1. Cages used to enclose tobacco budworms on
cotton terminals







Florida Entomologist 91(4)


beet armyworms, fall armyworms, bollworms,
and tobacco budworm neonates (Table 1). Three
larvae (tobacco budworms) or a single larva (all
other species) were placed in a dish containing a
single terminal leaf from plants that had begun to
flower. Ten dishes per plot were utilized for all
species, and mortality was assessed at 5 days af-
ter exposure. Percent mortality of neonates was
combined by trait and analyzed by REML-
ANOVA. Means were separated by LSMEANS
(Littell et al. 1996; PROC MIXED, SAS Institute
2001).

RESULTS AND DISCUSSION

Efficacy of Vip3A Events (2004)

All cotton lines containing the Vip3A event
caused significantly higher mortality against all
examined pests compared to conventional cotton
(Figs. 2-4). COT202 and/or COT203 were signifi-
cantly more efficacious than COT102 against the
bollworm, tobacco budworm, and beet army-




A.
df = 3,15
80 P < 0.001
a _F= 14.33
Sa


40
b
20
b


df- 3, 12
P < 0.001
F= 18.68


U
COT 102 COT 2 COT 2 Coke 312

Vip3A Event

Fig. 2. Efficacy at (A) 2 d after exposure and (B) 4 d
after exposure to terminal leaves containing Vip3A
events against bollworm larvae. Coker 312 is a conven-
tional cotton variety. Bars (Mean SE) with a common
letter are not significantly different (a = 0.05) according
to LSMEANS.


0

o -

Q.


40-


A.


If= 2,13
SF=4510


U.'c


df= 3 13
P= 0.027
F= 4.24


COT 102 COT 20 COT 203 Coke 312


Vip3A Event

Fig. 3. Efficacy at 7 d after exposure to terminal
leaves containing Vip3A events against beet armyworm
(A) and tobacco budworm (B) larvae. Coker 312 is a con-
ventional cotton variety. 100% mortality was observed
for beet armyworms fed cotton containing COT203;
therefore, it was excluded in the ANOVA. Bars (Mean
SE) with a common letter are not significantly different
(a = 0.05) according to LSMEANS.



worms (Figs. 2-3), although not apparent for fall
armyworms fed squares (Fig. 4). All cottons con-
taining the Vip3A event significantly reduced the
number of tobacco budworms recovered alive 5 d
after infestation when caged on terminals, al-
though no cotton caused 100% mortality (Fig. 5).
In addition, the weight of larvae recovered alive
at 5 d after infestation from all cotton with the
Vip3A event was significantly less than those lar-
vae recovered alive from conventional cotton.
However, tobacco budworms recovered from
COT202 and COT203 weighed significantly less
than larvae recovered from COT102 (Fig. 6).

Efficacy of the Vip3A, CrylAb, and CrylAb + Vip3A
Traits (2005)

All cotton lines containing the Vip3A or
CrylAb trait caused significantly higher mortal-
ity against tobacco budworm larvae than conven-
tional cotton (Fig. 7A). In addition, cotton lines


December 2008







Adamczyk & Mahaffey: Efficacy ofVip3A and CrylAb Cottons Against Lepidoptera 573


80
P
C



oa
*E
S o
a

20


o


Vip3A Event

Fig. 4. Efficacy at 3 d after exposure to squares con-
taining Vip3A events against fall armyworm larvae.
Coker 312 is a conventional cotton variety. Bars (Mean
+ SE) with a common letter are not significantly differ-
ent (a = 0.05) according to LSMEANS.

containing the stacked CrylAb + Vip3A trait
caused significantly higher mortality than cotton
containing just the Vip3A trait. There were no sig-
nificant differences in larval mortality when to-
bacco budworm larvae were fed with cotton con-
taining CrylAb alone or stacked with Vip3A.
In contrast, lines that contained just the Vip3A
trait were equally efficacious against bollworm
larvae compared to lines containing just the
CrylAb trait or lines containing the stacked
CrylAb + Vip3A trait (Fig. 7B). Similar to the
data for tobacco budworms, all cotton lines con-
taining Vip3A or CrylAb traits caused signifi-
cantly higher mortality against bollworm larvae
compared to conventional cotton.
For both beet and fall armyworms, cotton lines
containing the Vip3A trait caused significantly
higher morality compared to cotton lines contain-
ing just the CrylAb trait (Fig. 8). All cotton lines
containing Vip3A or CrylAb traits caused signifi-
cantly higher mortality against beet and fall ar-
myworm larvae compared to conventional cotton.
There were no significant differences in larval
mortality when beet and fall armyworm larvae
were fed cotton that contained Vip3A alone or
stacked with CrylAb.
The efficacy of Vip3A and CrylAb against Lep-
idoptera is species-specific. Our study shows that
the cotton lines containing Vip3A are more effica-
cious against beet and fall armyworms compared
to the CrylAb cotton lines, while the opposite re-
lationship is true for tobacco budworms. Previous
studies have shown that certain Lepidoptera,
such as tobacco budworms, are more susceptible
to CrylAc compared to bollworms and fall army-
worms, while CrylF provides greater efficacy
against fall armyworms compared to CrylAc (Ma-
cIntosh et al. 1990; Adamczyk et al. 1998; Adam-


B. df= 3. 13 a
p< 0.0D1
F 52.37






b
b 2 b

COT 102 COT 202 COT 203 Coker 312


Vip3A Event

Fig. 5. Tobacco budworms recovered alive at (A) 2 d
and (B) 5 d after being caged on terminal portions of cot-
ton plants containing Vip3A events. Coker 312 is a con-
ventional cotton variety. Bars (Mean SE) with a
common letter are not significantly different (a = 0.05)
according to LSMEANS.


czyk & Gore 2004). In addition, no additive or syn-
ergistic relationship between the Vip3A and
CrylAb traits in these particular cotton lines was
observed. This is similar to what was reported for
the 2 Cry proteins in WideStrike cotton (Adam-
czyk & Gore 2004). Parker & Livingston (2005) re-
ported that private industry is developing a
transgenic cotton trait containing a Vip3A trait
stacked with CrylAb trait (VipCotTM). This would
provide a much better spectrum of Lepidopteran
control than one with the traits expressed indi-
vidually.

ACKNOWLEDGMENTS

We thank the efforts of Don Hubbard and Jennifer
Holcomb. Furthermore, we thank Dr. Scott Arm-
strong and an anonymous reviewer from Syngenta
Co., for their comments on this manuscript. Mention
of a trademark, warranty, proprietary product or ven-
dor does not constitute a guarantee by the USDA and
does not imply approval or recommendation of the
product to the exclusion of others that may be suit-
able.


I f= 3. 13
Pw0001
Fr1739


COT 203 Coke 312


COT 102 COT 202








Florida Entomologist 91(4)


December 2008


E T
-a
E 16-

o
* c-


dU 3. 69.6
P< 0.001
F=33.73


COT 10 COT 2W COT 23 Clkr 312

Vip3A Event

Fig. 6. Weight of tobacco budworms recovered alive 5
d after being caged on terminal portions of cotton plants
containing Vip3A events. Number (n) of larvae weighed
per Vip3A event. Coker 312 is a conventional cotton vari-
ety. Bars (Mean SE) with a common letter are not sig-
nificantly different (a = 0.05) according to LSMEANS.


A.




F= 192.75
a a




40


20 b

SB C



B. a
T


df= 3. 41
P< 0.001
F = 24.04





b

N-Ie


vu3A CyAb CylAb Vip


Trait

Fig. 7. Efficacy at 5 d after exposure to terminal
leaves containing various transgenic traits (see Table 1,
2005) against tobacco budworm (A) and bollworm (B) lar-
vae. Bars (Mean SE) with a common letter are not sig-
nificantly different (a = 0.05) according to LSMEANS.


so-
80-





40-


= 2-

0
0



CL


A.
df= 3,41
P< 0.001
F 73.28


B.

df 3, 41
P< 0.001
F= 139.09


c

ne


Vlp3 CrlAb CrylAb Vip3


Trait

Fig. 8. Efficacy at 5 d after exposure to terminal
leaves containing various transgenic traits (see Table 1,
2005) against beet (A) and fall armyworm (B) larvae.
Bars (Mean SE) with a common letter are not signifi-
cantly different (a = 0.05) according to LSMEANS.





REFERENCES CITED

ADAMCZYK, JR., J. J., AND J. GORE. 2004. Laboratory
and field performance of cotton containing CrylAc,
CrylF, and both CrylAc and CrylF (WideStrike)
against beet armyworm and fall armyworm larvae
(Lepidoptera: Noctuidae). Florida Entomol. 87: 427-
432.
ADAMCZYK, JR., J. J., L. C. ADAMS, AND D. D. HARDEE.
2001. Field efficacy and seasonal expression profiles
for terminal leases of single and double Bt toxin cot-
ton genotypes. J. Econ. Entomol. 94: 1589-1593.
ADAMCZYK, JR., J. J., V. J. MASCARENHAS, G. E.
CHURCH, B. R. LEONARD, AND J. B. GRAVES. 1998.
Susceptibility of conventional and transgenic cotton
bolls expressing the Bacillus thuringiensis CrylAc
6-endotoxin to fall armyworm (Lepidoptera: Noctu-
idae) and beet armyworm (Lepidoptera: Noctuidae)
injury. J. Agric. Entomol. 15: 163-171.
HARDEE, D. D., AND G. A. HERZOG. 1997. 50m Annual
conference report on cotton insect research and con-
trol, pp. 809-818 In Proc. Beltwide Cotton Conf., Na-
tional Cotton Council, Memphis, TN.







Adamczyk & Mahaffey: Efficacy ofVip3A and CrylAb Cottons Against Lepidoptera 575


HENNEBERRY, T. J., L. FORLOW JECH, AND T. DE LA
TORRE. 2001. Effects oftransgenic cotton on cabbage
looper, tobacco budworm, and beet armyworm (Lep-
idoptera: Noctuidae) larval mortality and develop-
ment and foliage consumption in the laboratory.
Southwestern Entomol. 26: 325-338.
HOOD, E. 1997. The fall armyworm: and I thought I had
it made, pp. 1223-1224 In Proc. Beltwide Cotton
Conf., National Cotton Council, Memphis, TN.
LITTELL, R. C., G. A. MILLIKEN, W. W. STROUP, AND
R. D. WOLFINGER 1996. SAS system for mixed mod-
els. SAS Institute, Cary, NC.
MACINTOSH, S. C., T. B. STONE, S. R. SIMS, P. L. HUNST,
J. T. GREENPLATE, P. G. MARRONE, F. J. PERLAK,
D. F. FISCHHOFF, AND R. L. FUCHS. 1990. Specificity
and efficacy of purified Bacillus thuringiensis pro-
teins against agronomically important insects. J. In-
vertebr. Pathol. 56: 258-266.
PARKER, R. D., AND S. D. LIVINGSTON. 2005. Results of
field experiments with transgenic cotton varieties


(VipCot and WideStrike) in the Texas coastal blend,
pp. 1687-1693 In Proc. Beltwide Cotton Conf., Na-
tional Cotton Council, Memphis, TN.
SAS INSTITUTE 2001. Proprietary Software Release 8.2,
Cary, NC, USA.
SMITH, R. H. 1997. An extension entomologist's 1996 ob-
servations of Bollgard (Bt) technology, pp. 856-857
In Proc. Beltwide Cotton Conf, National Cotton
Council, Memphis, TN.
SPARKS, A. N. 1979. A review of the biology of the fall ar-
myworm. Florida Entomol. 62: 82-87.
STEWART, S. D., J. J. ADAMCZYK, JR, K. S. KNIGHTEN,
AND F. M. DAVIS. 2001. Impact of Bt cottons ex-
pressing one or two insecticidal proteins of Bacillus
thuringiensis Berliner on growth and survival of
noctuid (Lepidoptera) larvae. J. Econ. Entomol. 94:
752-760.
YOUNG, J. R. 1979. Fall armyworm: control with insec-
ticides. Florida Entomol. 62: 130-133.







Florida Entomologist 91(4)


December 2008


ODORANTS OF THE FLOWERS OF BUTTERFLY BUSH, BUDDLEJA DAVIDII,
AS POSSIBLE ATTRACTANTS OF PEST SPECIES OF MOTHS

CHRISTELLE GUEDOT*, PETER J. LANDOLT AND CONSTANCE L. SMITHHISLER
U.S. Department of Agriculture, Agricultural Research Service, 5230 Konnowac Pass Road, Wapato, WA 98951


ABSTRACT

Flowers of the butterfly bush, Buddleja davidii Franch., are visited by butterflies as well as
other insects. Night captures revealed also that moths visit butterfly bush flowers. Moths
captured in traps over flowers included 12 species of Noctuidae, 6 species of Pyralidae, 2 spe-
cies of Geometridae, and 1 tortricid species. The majority of moths trapped at these flowers
were cabbage loopers, Trichoplusia ni (Htibner), and alfalfa loopers, Autographa californica
(Speyer). Both males and females were captured at butterfly bush flowers. Additionally, but-
terflies, bees, wasps, flies, and other insects also were captured. Analysis of volatile com-
pounds collected from air over clusters of butterfly bush flowers yielded the consistent
presence of nine chemicals: benzaldehyde, 6-methyl-5-hepten-2-one, hexyl acetate, 4-oxoiso-
phorone, (E,E)-a-farnesene, (Z)-cinnamaldehyde, dihydrooxoisophorone, P-cyclocitral, and
oxoisophorone oxide. Emitted amounts of these floral odorants averaged 57 ng per h per flo-
ret or 21 pg per h per flower cluster racemee). Five of those floral chemicals, benzaldehyde,
4-oxoisophorone, dihydrooxoisophorone, oxoisophorone oxide, and (E,E)-a-farnesene trig-
gered antennal responses in cabbage looper moths, while benzaldehyde, oxoisophorone ox-
ide, and 4-oxoisophorone also stimulated antennal responses in alfalfa looper moths. Some
of these compounds may be attractants or co-attractants for moths and play a key role in lo-
cating flowers as nectar sources.

Key Words: Lepidoptera, kairomone, floral odor, pollination

RESUME

Las flores del arbusto de mariposa, Buddleja davidii Franch., estan frecuentemente visita-
das por mariposas y otros insects. Las captures hechas en la noche revelaron que las poli-
llas tambi6n visitan las flores del arbusto de mariposa. Las polillas capturadas en las
trampas sobre las flores incluyeron 21 species de Geometridae, Noctuidae, Pyralidae y Tor-
tricidae. La mayoria de las polillas capturadas en las flores pertenecen al gusano medidor
del repollo, Trichoplusia ni (Htibner) y al gusano medidor de alfalfa, Autographa californica
(Speyer). Tanto machos como hembras fueron capturados sobre las flores del arbusto de ma-
riposa. Tambi6n se capturaron mariposas, abejas, avispas, moscas y otros insects. El andli-
sis de los volatiles compuestos recolectados del aire sobre los grupos de flores del arbusto de
mariposa siempre mostr6 la presencia de nueve quimicos: benz aldehido, acetato hexil 6-me-
til-5-hepten-2-uno, 4-oxoisoforone, (E,E)-a-faresene, (Z)-cinnamaldehido, dihidrooxoisofo-
rone, p-ciclocitral y oxido oxoisoforone. Las cantidades emitidas de estos odorantes de las
flores fueron un promedio de 57 ng por hora florecita o 21 pg por hora por cada grupo de flo-
res (racimos). Cinco de estos quimicos de flores, benzaldehido, 4-oxoisoforone, dihidrooxoiso-
forone, oxido oxoisoforone y (E,E)-a-farnesene estimularon una respuesta en las antenas de
las polillas del gusano medidor del repollo, mientras que el benzaldehido, oxido oxoisoforone
y el 4-oxoisoforone tambi6n estimularon una respuesta en las antenas del gusano medidor
de la alfalfa. Algunos de estos quimicos compuestos pueden ser atrayentes o co-atrayentes
para las polillas que juegan un papel clave en localizar las flores como fuentes de nectar.


The butterfly bush, Buddleja davidii Franch.
(Loganiaceae), is native to China and Japan, but is
a common ornamental shrub planted throughout
much of North America and Europe. The shrub has
abundant and colorful flowers that occur as 15 to 40
cm long racemic clusters. Flower color varies from
white to pink, red, blue, and purple. The flower is
strongly fragrant. It is attractive to butterflies (Cor-
bett 2000) and is recommended in horticultural
plantings as a source of nectar for butterflies.
Moths were initially observed orienting to but-
terfly bush flowers on the campus of Washington


State University, Pullman, Washington, USA in
late afternoon (P. J. Landolt, unpublished data).
We hypothesize that moth visitation at these
flowers may result from their response to flower
odor. The odor chemistry of these flowers, re-
ported by Andersson et al. (2002), is very complex
and includes 31 compounds. Antennal responses
for several species of butterflies to B. davidii floral
scent were reported, narrowing down the list of
compounds that could be butterfly attractants
(Andersson 2003;Andersson & Dobson 2003). It is
not yet known which chemicals in the butterfly







Gu6dot et al. Odorants of Butterfly Bush Flowers as Moth Attractants


bush flowers might be detected by or attractive to
moths.
Some species of moths often visit flowers for
nectar, and some of these species arrive at flowers
following upwind attraction to floral odors. This
behavior is particularly pronounced and well
studied for pest species of noctuids in the subfam-
ily Plusiinae. The cabbage looper moth Trichoplu-
sia ni (Hiibner), for example, visits or responds to
odors of flowers of bladderflower, Araujia sericof-
era (Brot.) (Cantelo & Jacobson 1979), glossy abe-
lia, Abelia grandiflora (Andre) (Grant 1971),
night blooming jessamine, Cestrum nocturnum
(Heath et al. 1992), and Japanese honeysuckle
Lonicerajaponica (Thunb.) (Pair & Horvat 1997).
The alfalfa looper Autographa californica
(Speyer) responds to flowers of Oregon grape, Ber-
beris aquifolium (Prursh.) (Landolt & Smith-
hisler 2003), and the silver Y moth Autographa
gamma (L.) is attracted to odors of flowers of
creeping thistle, Cirsium arvense (L.), butterfly
orchid, Platanthera bifolia (L.), soapwort, Sa-
ponaria officianalis (L), and others (Plepys 2001;
Plepys et al. 2002b). Isolation and identification of
the principal constituents of the odors of these
flowers have provided a set of attractive chemi-
cals for several major pest species of moths. For
example, phenylacetaldehyde from flowers of
bladderflower (Cantelo & Jacobson 1979) is at-
tractive to a number of species of moths, and phe-
nylacetaldehyde, benzyl alcohol, 2-phenyletha-
nol, and benzaldehyde, isolated and identified
from flowers of A. grandiflora, are attractive to
cabbage looper moths (Haynes et al. 1991). The
use of some of these floral attractants as lures
holds promise for monitoring and surveying
moths (Lopez et al. 2000; Landolt et al. 2006) or
for management of pest species of moths (Camelo
et al. 2007).
We report here studies to determine if moths
are consistently visiting butterfly bush flowers
and, if so, to identify those moths. We also deter-
mined the compounds that consistently make up
the odor of those flowers and report antennal re-
sponses of cabbage looper and alfalfa looper
moths to floral compounds emitted. This work ad-
dresses in part the hypothesis that moths are vis-
iting butterfly bush flowers as a result of attrac-
tion to flower odor, and provides the identification
of a set of chemicals that are putative attractants
for moths that can be evaluated as such in future
studies.

METHODS AND MATERIALS

In May 2002, butterfly bush plants were pur-
chased from Richard Owen Nursery (Blooming-
ton, IL) and were planted on the grounds of the
USDA, ARS Yakima Agricultural Experiment
Station, near Donald, Yakima County, Washing-
ton, USA. These plants received regular irriga-


tion from lawn sprinklers from mid Apr to mid
Oct. Plants were 1 to 2 m tall when studies were
initiated in Jul, 2003. Blooming occurred from
Jun into Oct of 2003 and 2004. Bushes did not re-
ceive fertilizer or pesticide applications.

Moth Sampling

In order to sample night-flying insects visiting
butterfly bush flowers, a large, white, mesh cone
shaped trap (Scentry Heliothis trap, Gempler's,
Madison, WI) was attached to a vertical pole, and
positioned so that the trap opening was above but
overlapped the top of a cluster of flowers (Landolt
& Smithhisler 2003). Moths visiting these flowers
and then departing might then fly up into the trap
and be unable to escape the top chamber. Traps
were positioned over flowers in late afternoon
(1500 to 1600 h PS.T.) and trapped insects were
collected the following morning (0800 to 0900 h
P.S.T.). Insects at flowers of 5 butterfly bushes
were sampled intermittently, providing a total of
32 trap samples between 2 Jul and 22 Aug 2003,
by sampling 2 bushes per night with Itrap per
bush. Insects in traps were captured with plastic
vials, generally when alive, and were then killed
in a freezer where they remained until identified.

Volatile Collections

Chemical odorants emitted by flower clusters
on butterfly bushes growing outside were col-
lected with a portable volatile collection system.
This volatile collection system consisted of an
electric air suction pump (model E2 M1.5, BOC
Edwards, Wilmington, MA), flow meters (Gilmont
#13, 0-13 L/min, Cole-Palmer, Vernon Hills, IL),
Tedlar gas sampling bags (#40046, 10 L, 12 x 18"
Tedlar snap with septum port, Alltech), and Su-
per Q adsorbent traps (Analytical Research Sys-
tems, Gainesville, FL). Airflow into the suction
pump was from 2 latex air lines provided by a
glass T-shaped tube. One air line pulled air
through a gas sampling bag holding a flower sam-
ple at the end of the air line, while the other air
line pulled air through a gas sampling bag hold-
ing foliage and no flowers. The bag was placed
over a branch with a flower raceme or with foliage
only and was closed up with clamps around the
stem of the branch. Air was pulled from the gas
sampling bag through a septum port and then
through the Super Q adsorbent trap and lastly
through the flow meter at a rate of 2 L per min.
The trap of the volatile collection system con-
tained 30 mg of Super Q (400 mesh) adsorbent in
a 0.635 cm x 6.67 cm long (1/4" x 2 5/8") borosili-
cate glass tube. A backup adsorbent trap of the
same construction, but containing 10 mg of Super
Q, was connected between the first trap and the
vacuum pump to check for breakthrough of chem-
icals passing through the primary first adsorbent







Florida Entomologist 91(4)


trap. These collections were made for a period of 1
h, following a 15 min purge period of the system.
Adsorbent traps were removed, sealed with Teflon
tape and stored in a freezer overnight until they
were extracted the following day. For each collec-
tion from a floral raceme, the number of open flo-
rets was counted so that chemical amounts per
floret could be computed. This procedure was fol-
lowed 9 times in 2003 over 4 different plants, pro-
viding the sampling of odorants from 9 butterfly
bush branches with flowers and 9 branches with-
out flowers. Volatile collections were conducted at
sunset, between 18:30 and 20:30 h P.S.T.

Chemical Analysis

Adsorbent traps were extracted with 600 pL of
10% ether in hexane. One pL aliquots of extracts
from these Super Q traps were analyzed by gas
chromatography mass spectrometry (GC-MS; Ag-
ilent 6890 GC) with a 5973 electron impact mass
selective detector, and a 7683 series autosampler.
The GC was equipped with a DB-1 (J & W Scien-
tific, Folsum, CA) fused silica capillary column,
0.25 mm (ID) x 60 m (length) with 0.25 pm film
thickness. The temperature program used was
40C for 1 min, increasing 15C per min to 200C.
All 9 collections were first analyzed as above, and
then 6 of the extracts were analyzed on a DB-Wax
(J & W Scientific) capillary column of the same di-
mensions with a temperature program of 40C for
2 min, increasing at 10C per min to 180C. Mass
spectra of eluting peaks were matched to those in
the NIST 98 library of compounds to obtain pre-
liminary structural assignments. These assign-
ments were confirmed by comparing the retention
times of butterfly bush compounds with retention
times of known standards, in both types of GC col-
umns, and by comparing mass spectra of butterfly
bush compounds with spectra of known stan-
dards. Pentadecane at 20 pg/mL was used as an
external standard for quantification of the identi-
fied chemicals.
Synthetic chemical standards used in estab-
lishing GC retention times and for comparing
spectroscopic data were purchased from Aldrich
Chemical Co. or Sigma-Aldrich Flavors and Fra-
grances (Milwaukee, WI). These were hexyl ace-
tate, 6-methyl-5-hepten-2-one, benzaldehyde, p-
cyclocitral, 4-oxoisophorone, and (Z)-cinnamalde-
hyde. These were 98-99% pure except for P-cycloc-
itral which was 90% pure. (E,E)-a-farnesene was
extracted with pentane from apples and purified
on silica gel columns to attain 98% purity.

Gas Chromatography with Electroantennographic De-
tection Analysis

One pL aliquots of extracts obtained from vol-
atile collections were analyzed by gas chromatog-
raphy coupled to electroantennographic detection


(GC-EAD) with a Hewlett Packard 5890 Series II
gas chromatograph and an IDAC-232 data acqui-
sition interface with a micromanipulator assem-
bly type IRN-5 (Syntech, The Netherlands). The
gas chromatograph was equipped with a DB-1
(Agilent Technologies, Palo Alto, CA) fused silica
capillary column 0.25 mm (ID) x 60 m (length)
with 0.25 pm film thickness and samples were in-
jected manually in splitless mode. The injector
temperature was 250C and helium was the car-
rier gas. The column temperature program
started at 40C for 2 min, increased to 200C at a
rate of 10C/min and held at 200C for 12 min.
The effluent from the column was split by a OSS-
2 splitter (SGE Analytical Science, Austin, TX) be-
tween the flame ionization detector (FID) and the
electroantennographic detector (EAD) with a ra-
tio FID:EAD of 1:2. The make-up gas connected to
the splitter was nitrogen delivered at a flow rate
of 40 mL/min.

Insects

Eggs of cabbage looper moths were provided by
the Entomology Department at the University of
Georgia, Athens, GA, USA. Larvae were reared
on artificial cabbage looper diet (Southland Prod-
ucts Inc., Lake Village, AR). Males and females
were separated at the pupal stage, and kept (as
pupae and emerged adults) in separate rooms on
reversed light cycle; L16:D8, at 26.0 + 0.1 C and
65-70% relative humidity. Cabbage looper moths
used for GC-EAD analyses were 2-6-d-old virgin
females. Male alfalfa looper moths were captured
in a walk-in light trap at the Yakima Agricultural
Research Laboratory, during Jun and Jul 2007.
These were kept for 1 to 2 d in the laboratory on a
L16: D8 light cycle, at 24.8C and 65-70% RH.
GC-EAD analyses were conducted on male alfalfa
looper moths because of light trap captures. One
antenna per insect was excised at the base of the
antennal scape and fixed between 2 silver elec-
trodes with electrically conductive gel (Spectra
360 electrode gel, Parker Laboratories, Fairfield,
NJ). GC-EAD analyses on volatile collections of
butterfly bush were conducted on 5 separate an-
tennae from 5 different moths and for each moth
species. Antennal responses of identified com-
pounds were confirmed through GC-EAD analy-
sis by exposing 5 cabbage looper antennae to 1 pL
aliquots of a mixture of 20 ng/pL solutions of the
corresponding synthetic standards.

RESULTS

Two hundred ninety three moths were cap-
tured in traps over butterfly bush flowers (Table
1). The greatest numbers were cabbage looper
(52%) and alfalfa looper moths (24%). Other pest
moths captured were the true armyworm Pseuda-
letia unipuncta (Haworth), the corn earworm,


December 2008







Gu6dot et al. Odorants of Butterfly Bush Flowers as Moth Attractants


TABLE 1. NUMBERS OF MOTHS CAPTURED IN HELIOTHIS TRAPS PLACED OVER FLOWER RACEMES OF BUTTERFLY BUSHES AT
THE USDA, ARS YAKIMA AGRICULTURAL RESEARCH LABORATORY, WAPATO, WA. JUL AND AUG 2003.

Moth species # Males # Females Total

Geometridae
Digrammia curvata (Grote) 1 0 1
Pero hubneraria (Guenee) 1 0 1
Noctuidae
Catocalinae
Melipotisjanuaris (Guenee) 1 0 1
Caenurgina erectea (Cramer) 0 1 1
Hadeninae
Dargida procinta (Grote) 0 2 2
Discestra trifolii (Hufnagel) 5 0 5
Lacanobia subjuncta (Grote and Robinson) 1 0 1
Leucania farcta (Grote) 1 0 1
Mamestra configurata (Walker) 0 1 1
Pseudaletia unipuncta (Walker) 9 1 10
Heliothinae
Helicoverpa zea (Boddie) 3 2 5
Plusiinae
Autographa californica (Speyer) 41 28 69
Anagrapha falcifera (Kirby) 2 2 4
Trichoplusia ni (Htibner) 83 69 152
Pyralidae
Crambus cypridalus (Hulst) 11 3 14
Loxostege sticticalis (L.) 6 2 8
Nomaphila nearctia (Munroe) 1 0 1
Prorasia simalis (Grote) 1 0 1
Dioryctria sp. 3 0 3
Udea rubigalis (Guenee) 4 6 10
Tortricidae
Choristoneura occidentalis (Freeman) 2 0 2


bertha armyworm, Mamestra configurata
(Walker), Lacanobia subjuncta (Barnes & Mac-
Dunnough), clover looper, Caenurgina erechtea
(Cramer), and the celery leaftier, Udea rubigalis
(Guenee). In total, 12 species of Noctuidae, 6 spe-
cies of Pyralidae, 2 species of Geometridae, and 1
tortricid species were captured in the traps. In ad-
dition, we caught 28 butterflies, 33 honeybees
Apis mellifera L., 22 other bees and wasps, 277
syrphid flies, 133 other Diptera, 17 Hemiptera, 13
lacewings, 5 beetles, 9 earwigs, 3 damsel flies, and
10 spiders.
Nine compounds were present consistently in
all 9 flower odorant samples and not in foliage
odor samples (Table 2). Those compounds are 4-
oxoisophorone (2,6,6-trimethyl-2-cyclohexene-
1,4-dione); benzaldehyde; (E,E)-a-farnesene; 6-
methyl-5-hepten-2-one; hexyl acetate; oxoiso-
phorone oxide (1,3,3-trimethyl-7-oxabicyclo
[4.1.0]-heptan-2,5-dione); dihydrooxoisophorone
(2,6,6-trimethyl-l,4-cyclohexadione), p-cycloci-
tral, and (Z)-cinnamaldehyde (Fig. 1, chemical
structures). These 9 compounds were initially
identified by mass spectral data and the struc-
tures of 6 of these were then confirmed by compar-


ing retention times and spectral data of those
compounds with those of synthetic standards.
Synthetic samples were not available for oxoiso-
phorone oxide and dihydrooxoisophorone and
those structural assignments are tentative. Mean
amount of odorants emitted per floret was 57.4
6.0 ng/h (mean S.E.). Flower clusters or racemes
averaged 371 38.7 florets, and mean amount of
volatiles per raceme was then 21.3 2.2 pg/h
(mean S.E.). Amounts of chemicals collected
were quite variable, indicated by the large stan-
dard errors for mean amounts of individual chem-
icals in Table 2. The limit of detection was esti-
mated to be 0.1 ng per analysis, which was 60 ng
per collection (0.1 ng/pL x 600 uL/sample).
Five of the floral compounds identified trig-
gered consistent antennal responses in female
cabbage looper moths (Table 2). These 5 com-
pounds were benzaldehyde, 4-oxoisophorone, di-
hydrooxoisophorone, oxoisophorone oxide, and
(E,E)-a-farnesene. Antennal responses to benzal-
dehyde, 4-oxoisophorone, and (E,E)-a-farnesene
were confirmed by exposing female cabbage
looper antennae to the corresponding standards.
Three of the floral compounds, benzaldehyde, ox-







Florida Entomologist 91(4)


December 2008


TABLE 2. MEAN ( SEM) AMOUNTS OF ODORANT COMPOUNDS PER FLORET OBTAINED IN VOLATILE COLLECTIONS FROM
FLOWER RACEMES OF BUTTERFLY BUSHES AND ANTENNAL RESPONSE THROUGH GC-EAD ANALYSIS (EAD) OF
CABBAGE LOOPER (CL) AND ALFALFA LOOPER (AL) MOTHS TO THESE ODORANT COMPOUNDS OBTAINED IN
VOLATILE COLLECTIONS (EXTRACT) AND TO THE CORRESPONDING SYNTHETIC CHEMICALS (SYNTHETIC).

Nanograms Percent of CL EAD to CL EAD to AL EAD to
Compound per floret Blend extract synthetic extract

Benzaldehyde 12.8 1.5 22.4 2.7 + + +
6-Methyl-5-hepten-2-one 1.2 0.2 2.0 0.3
Hexyl acetate 0.8 0.1 1.4 0.2 -
Oxoisophorone oxide 1.8 0.4 3.2 0.6 + nt +
4-Oxoisophorone 29.8 3.4 52.0 6.0 + + +
Dihydrooxoisophorone 1.1 0.3 1.8 0.5 + nt
P-Cyclocitral 1.4 0.3 2.5 0.6 -
(Z)-Cinnamaldehyde 1.3 0.4 2.3 0.7 -
(E,E)-a-Faresene 7.1 2.0 12.4 3.5 + +
Total 57.4 6.0 100%

+: EAD response in at least 3 antennae out of 5 antennae exposed to either the extract or the synthetic chemicals;
-: no EAD response; nt: not tested.


oisophorone oxide, and 4-oxoisophorone, elicited
consistent antennal responses with male alfalfa
looper moths (Table 2).


a0


-e10
[C)i


Fig. 1. Structures of compounds identified from vol-
atile collections made over open flowers of Buddleja
bushes. (a) Hexyl acetate CAS 142-92-7 (Acetic acid,
hexyl ester); (b) 4-Oxoisophorone CAS 1125-21-9 (2,6,6-
trimethyl-2-cyclohexene-1,4-dione); (c) Oxoisophorone
oxide (1,3,3-trimethyl-7-oxa-bicyclo[4.1.0]heptane-2,5-
dione); (d) Dihydrooxoisophorone CAS 20547-99-3
(2,2,6-trimethyl 1,4-cyclohexadione); (e) P-Cyclocitral
CAS 432-25-7 (2,2,6- trimethyl 1-cyclohexene-carboxal-
dehyde); (f) (Z)-Cinnamaldehyde CAS 104-55-2 (2-Pro-
penal, 3-phenyl-).


a


DISCUSSION

Although known for "attractiveness" to day-ac-
tive butterflies (Corbet 2000), the flowers of the
butterfly bush also were visited consistently by
night-flying Lepidoptera. The list of moths
trapped indicates that a diversity of species of
moths visits these flowers. However, most of the
moths trapped were 2 pest species of Noctuidae in
the subfamily Plusiinae; the cabbage looper and
the alfalfa looper. Because our sampling was con-
ducted in Jul and Aug and at 1 site, we expect that
various numbers and additional species of moths
would visit butterfly bush flowers at other times
of the season and at other locations. Several spe-
cies of Plusiinae, including the cabbage looper, al-
falfa looper, and silver Y moth have been reported
as frequent visitors at flowers of other plants.
Cabbage looper moths have been observed at
flowers of A. grandiflora (Grant 1971), alfalfa
looper moths were collected at flowers of Oregon
grape (Landolt & Smithhisler 2003), and the sil-
ver Y moth is an abundant visitor at flowers of
Silene latifolia (Brantjes 1976).
Other pestiferous moth species that were
trapped over butterfly bush flowers were the corn
earworm and the true armyworm. The corn ear-
worm moth feeds at flowers (Hendrix et al. 1987;
Lindgren et al. 1993) and responds to odors of
flowers such as Japanese honeysuckle Lonicera
japonica, and Gaura drummondii (Beerwinkle et
al. 1996; Pair & Horvat 1997; Lopez et al. 2000)
and is also attracted to odors of fermented sugar
baits (Ditman & Cory 1933). The true armyworm
has not been reported as a frequent flower visitor,
but was captured in traps baited with acetic acid
and 3-methyl-1-butanol, a feeding attractant de-
rived from fermented molasses solutions (Utrio &
Eriksson 1977; Landolt & Higbee 2002).


,Jl







Gu6dot et al. Odorants of Butterfly Bush Flowers as Moth Attractants


We hypothesize that moths locate flowers of
butterfly bush from some distance by flying up-
wind in response to the flower odor. Initial obser-
vations in late afternoon of moths at butterfly
bush flowers indicated plume-tracking behavior
(zigzagging upwind flights) at distances up to 2 M
downwind of flower racemes, but these moths
were not identified. We found that these flowers
produce a high amount of odorants that might at-
tract moths. Moth attraction to chemicals emitted
by flowers is well documented. For example, many
moths are attracted to bladderflower, and to phe-
nylacetaldehyde, which is produced by bladder-
flower (Cantelo & Jacobson 1979). The cabbage
looper moth is a frequent visitor at flowers of A.
grandiflora (Grant 1971), which produces the cab-
bage looper attractants phenylacetaldehyde, ben-
zaldehyde, 2-phenylethanol, and benzyl alcohol
(Haynes et al. 1991). Alfalfa loopers visit flowers of
Oregongrape, which produces a blend of odorant
chemicals including the alfalfa looper attractants
phenylacetaldehyde and p-myrcene (Landolt et al.
2001, 2006; Landolt & Smithhisler 2003). Other
examples include moth attraction to flowers of L.
japonica which produces phenylacetaldehyde,
linalool, and cis-jasmone (Pair & Horvat 1997), C.
nocturnum which produces benzaldehyde, benzyl
acetate, and phenylacetaldehyde (Heath et al.
1992), S. latifolia which produces phenylacetalde-
hyde and lilac aldehydes (Ddtterl 2004; Ddtterl et
al. 2006), and Platanthera bifolia which produces
lilac aldehydes (Plepys et al. 2002a).
Five of the 9 compounds found in the odor of
butterfly bush flowers elicited antennal responses
in cabbage looper and 3 compounds in alfalfa
looper moths, suggesting differences in antenna
receptor sensitivity between moth species. Previ-
ous studies with butterflies and butterfly bush
scents showed antennal responses to the same
chemicals these moths responded to, although se-
lective antennal responses to those compounds
also were observed between butterfly species
(Andersson 2003;Andersson & Dobson 2003). Dif-
ferences in antennal responses between moth
species could also indicate gender differences,
with female cabbage looper being responsive to
more compounds than male alfalfa looper, as sug-
gested with the butterfly Heliconius melpomene
(Andersson & Dobson 2003). Four-oxoisophorone
and oxoisophorone oxide were the only 2 com-
pounds that were found to consistently elicit an-
tennal responses in both moth species as well as
in the 4 butterfly species when testing butterfly
bush scent samples (Andersson 2003; Andersson
& Dobson 2003). Four-oxoisophorone is not only
eliciting antennal responses in Lepidoptera spe-
cies, but is active for bees (Ddtterl et al. 2005).
Four-oxoisophorone was identified previously
and in the present study as the most abundant
compound emitted by butterfly bush flowers
(Andersson et al. 2002; Andersson 2003). This


compound is found also in floral scents of Primula
farinosa (Gaskett et al. 2005), Lumnitzera race-
mosa (Azuma et al. 2002), Salix atrocinerea (Dat-
terl et al. 2005), and other plants (Knudsen et al.
2006). Two other compounds in the odor of butter-
fly bush, benzaldehyde and (E,E)-a-farnesene,
are odorants for other moth-visited flowers. Ben-
zaldehyde was found in odor of other plant species
including C. nocturnum (Heath et al. 1992), A.
grandiflora (Haynes et al. 1991), S. latifolia (Jiir-
gens et al. 2002), and B. aquifolium (Landolt &
Smithhisler 2003). (E,E)-a-farnesene was found
in the floral odor of Japanese honeysuckle
(Schlotzhauer et al. 1996). An additional butterfly
bush flower odorant, cinnamaldehyde, which did
not stimulate antennal responses in our study, is
found in odor of flowers of S. latifolia (Jiirgens et
al. 2002), and G. drummondi (Teranishi et al.
1991).
Three to 5 of the 9 compounds produced by but-
terfly bush flowers are biologically relevant com-
pounds for alfalfa looper and cabbage looper
moths. Although it is not completely known which
of these compounds may be involved in attracting
night-flying moths, some are already known to be
behaviorally active for the cabbage looper, alfalfa
looper, and other moths. Benzaldehyde was at-
tractive to cabbage looper moths in a flight tunnel
assay (Haynes et al. 1991; Heath et al. 1992), but
was not attractive to alfalfa looper moths when
field-tested by Landolt et al. (2001) in Washing-
ton, or to soybean looper moths, Pseudoplusia in-
cludens (Walker), when field-tested by Meagher
(2002) in Florida. Further research will be needed
to determine which of these chemicals elicit up-
wind attraction responses in moths such as the
cabbage looper and alfalfa looper.

ACKNOWLEDGMENTS

We thank Daryl Green for technical assistance, and
Michael Strand from the Entomology Department at
University of Georgia, Athens, GA for providing cab-
bage looper eggs. Helpful suggestions to improve the
manuscript were provided by Theresa Pitts-Singer,
David Robacker, and Wee Yee.

REFERENCES CITED

ANDERSSON, S. 2003. Antennal responses to floral
scents in the butterflies Inachis io, Aglais urticae
(Nymphalidae), and Gonepteryx rhamni (Pieridae).
Chemoecol. 13: 13-20.
ANDERSSON, S., AND H. E. M. DOBSON. 2003. Antennal
responses to floral scents in the butterfly Heliconius
melpomene. J. Chem. Ecol. 29: 2319-2330.
ANDERSSON, S., L. A. NILSSON, I. GROWTH, AND G. BERG-
STROM. 2002. Floral scents in butterfly-pollinated
plants: possible convergence in chemical composi-
tion. Bot. J. Linn. Soc. 140: 129-153.
AZUMA, H., M. TOYOTA, Y. ASAKAWA, T. TAKASO, AND
H. TOBE. 2002. Floral scent chemistry of mangrove
plants. J. Plant Res. 115: 47-53.











BEERWINKLE, K. R., T. N. SHAVER, P. D. LINDGREN, AND
J. R. RAULSTON. 1996. Free-choice olfactometer bio-
assay system for evaluating the attractiveness of
plant volatiles to adult Helicoverpa zea. Southwest.
Entomol. 21: 395-405.
BRANTJES, N. B. M. 1976. Senses involved in the visit-
ing of flowers by Cucullia umbratica (Noctuidae:
Lepidoptera). Entomol. Exp. Appl. 20: 1-7.
CAMELO, L. D. A., P. J. LANDOLT, AND R. S. ZACK. 2007.
A kairomone based attract and kill system effective
against alfalfa looper (Lepidoptera: Noctuidae).
J. Econ. Entomol. 100: 366-374.
CANTELO, W. W., AND M. JACOBSON. 1979. Phenylace-
taldehyde attracts moths to bladder flower and to
blacklight traps. Environ. Entomol. 8: 444-447.
CORBET, S. A. 2000. Butterfly nectaring flowers: butter-
fly morphology and flower form. Entomol. Exp. Appl.
96: 289-296.
DITMAN, L. P., AND E. N. CORY. 1933. The response of
corn earworm moths to various sugar solutions. J.
Econ. Entomol. 76: 109-115.
DOTTERL, S. 2004. Importance of Floral Scent Com-
pounds for the Interaction between Silene latifolia
(Caryophyllaceae) and the Nursery Pollinator Hade-
na bicruris (Lepidoptera: Noctuidae). PhD disserta-
tion. University of Bayreuth, Bayreuth, Germany.
DOTTERL, S., U. FUSSEL, A. JURGENS, AND G. AAS. 2005.
1,4-Dimethoxybenzene, a floral scent compound in
willows that attracts an oligolectic bee. J. Chem.
Ecol. 31: 2993-2998.
DOTTERL, S., A. JURGENS, K. SEIFERT, T. LAUBE,
B. WEISSBECKER, AND S. SCHUTZ. 2006 Nursery pol-
lination by a moth in Silene latifolia: the role of
odours in eliciting antennal and behavioral re-
sponses. New Phytol. 169: 707-718.
GASKETT, A. C., E. CONTI, AND F. P. SCHIESTL. 2005.
Floral odor variation in two heterostylous species of
Primula. J. Chem. Ecol. 31: 1223-1228.
GRANT, G. G. 1971. Activity of adult cabbage loopers on
flowers with strong olfactory stimuli. J. Econ. Ento-
mol. 64: 315-316.
HAYNES, K. F., J. Z. ZHAO, AND A. LATIF. 1991. Identifi-
cation of floral compounds from Abelia grandiflora
that stimulate upwind flight in cabbage looper
moths. J. Chem. Ecol. 17: 637-646.
HEATH, R. R., P. J. LANDOLT, B. D. DUEBEN, AND B.
LENCZWESKI. 1992. Identification of floral compounds
of night blooming jessamine attractive to cabbage
looper moths. Environ. Entomol. 21:854-859.
HENDRIX, W. H. III., T. F. MUELLER, J. R. PHILIPS, AND
O. K. DAVIS. 1987. Pollen as indicator of long dis-
tance movement of Heliothis zea (Lepidoptera: Noc-
tuidae). Environ. Entomol. 16: 1148-1151.
JURGENS, A., T. WITT, AND G. GOTTSBERGER. 2002.
Flower scent composition in night flowering Silene
species (Caryophyllaceae). Biochem. Syst. Ecol. 30:
383-397.
KNUDSEN, J. T, R. ERIKSSON, J. GERSHENZON, AND
B. STAHL. 2006. Diversity and distribution of floral
scent. Bot. Rev. 72: 1-120.


December 2008


LANDOLT, P. J., AND B. S. HIGBEE. 2002. Both sexes of
the true armyworm (Lepidoptera: Noctuidae)
trapped with the feeding attractant composed of ace-
tic acid and 3-methyl-l-butanol. Fla. Entomol. 85:
182-185.
LANDOLT, P. J., AND C. L. SMITHHISLER. 2003. Charac-
terization of the floral odor of Oregongrape: possible
feeding attractants for moths. Northwest Sci. 77: 81-
86.
LANDOLT, P. J., T. ADAMS, AND R. S. ZACK. 2006. Field
response of alfalfa looper and cabbage looper moths
(Lepidoptera: Noctuidae; Plusiinae) to single and bi-
nary blends of floral odorants. Environ. Entomol. 35:
276-281.
LANDOLT, P. J., T. ADAMS, H. C. REED, AND R. S. ZACK.
2001. Trapping alfalfa looper moths (Lepidoptera:
Noctuidae) with single and double component floral
chemical lures. Environ. Entomol. 30: 667-672.
LINDGREN, P. D., V. M. BRYANT, JR., J. R. RAULSTON,
M. PENDLETON, J. WESTBROOK, AND G. D. JONES.
1993. Adult feeding host range and migratory activ-
ities of corn earworm, cabbage looper, and celery
looper (Lepidoptera: Noctuidae) moths as evidenced
by attached pollen. J. Econ. Entomol. 86: 1429-1439.
LOPEZ, J. D., JR., T. N. SHAVER, K. R. BEERWINKLE, AND
P. D. LINDGREN. 2000. Feeding attractant and stim-
ulant for adult control of noctuid and/or other lepi-
dopteran species. U.S. Patent 6,074,623. Issued
June 2000. United States Patent Office, Washing-
ton D.C.
MEAGHER, R. L., JR 2002. Trapping noctuid moths with
synthetic floral volatile lures. Entomol. Exp. Appl.
103: 219-226.
PAIR, S. D., AND R. J. HORVAT. 1997. Volatiles of Japa-
nese honeysuckle flowers as attractants for adult
Lepidopteran insects. U.S. Patent 5,665,344. Issued
9 September 1997.
PLEPYS, D. 2001. Odour-mediated Nectar Foraging in
the SilverY Moth,Autographa gamma.Ph.D. disser-
tation, Lund University, Lund, Sweden.
PLEPYS D., F. IBARRA, AND C. LOFSTEDT. 2002a. Volatil-
es from flowers ofPlatanthera bifolia (Orchidaceae)
attractive to the silver Y moth, Autographa gamma
(Lepidoptera: Noctuidae). Oikos 99: 69-74.
PLEPYS D., F. IBARRA, W. FRANCKE, AND C. LOFSTEDT.
2002b. Odour-mediated nectar foraging in the silver
Y moth, Autographa gamma (Lepidoptera: Noctu-
idae): behavioral and electrophysiological respons-
es to floral volatiles. Oikos 99: 75-82.
SCHLOTZHAUER, W. S., S. D. PAIR, AND R. J. HORVAT.
1996. Volatile constituents from the flowers of Japa-
nese honeysuckle (Lonicera japonica). J. Agric. Food
Chem. 44: 206-209.
TERANISHI, R., R. G. BUTTERY, S. KINT, G. TAKEOKA, P.
D. LINDGREN, J. R. RAULSTON, AND T. N. SHAVER
1991. Chemical composition of Gaura drummondii
flower volatiles. J. Essent. Oil Res. 3: 287-288.
UTRIO, P., AND K. ERIKSSON. 1977. Volatile fermenta-
tion products as attractants for Macrolepidoptera.
Ann. Zool. Fenn. 14: 98-104.


Florida Entomologist 91(4)







Van Driesche et al.:Aphidius colemani Banker Plants


GREENHOUSE TRIALS OF APHIDIUS COLEMANI (HYMENOPTERA:
BRACONIDAE) BANKER PLANTS FOR CONTROL OF APHIDS (HEMIPTERA:
APHIDIDAE) IN GREENHOUSE SPRING FLORAL CROPS

R. G. VAN DRIESCHE1, S. LYON1, J. P. SANDERSON2, K. C. BENNETT2, E. J. STANEK III3 AND RUITAO ZHANG3
'PSIS/Division of Entomology, University of Massachusetts, Amherst, MA, 01003, USA

2Department of Entomology, Cornell University, Ithaca, NY, 14583, USA

'School of Public Health, University of Massachusetts, Amherst, MA, 01003, USA


ABSTRACT

Banker plants withAphidius colemani Viereck were tested in greenhouses in Massachusetts
and New York for control of cotton aphid Aphis gossypii Glover, and green peach aphid
Myzus persicae (Sulzer) on 2 spring flower crops, pansies (Viola tricolor hortensis) and Mar-
guerite daisies (Argyranthemum hybrid). Banker plants consisted of pots of barley plants in-
fested with the bird cherry-oat aphid Rhopalosiphum padi (L.), inoculated at the start of the
crop with adults ofA. colemani purchased from a commercial insectary. Initial trials were
conducted in University of Massachusetts greenhouses containing flats of the crop plants.
Sentinel plants in flats were infested uniformly with aphids, and particular greenhouses
were subjected to the presence of banker plants or left as controls. Prior to University trials,
a survey was conducted in commercial greenhouses in Massachusetts and New York to de-
termine the frequency and species of aphid infestation in spring flower crops. After Univer-
sity trials, the efficacy of banker plants was tested in commercial greenhouses in both states.
In surveys of commercial greenhouses, M. persicae was the most frequently detected species,
accounting for 53% of all infestations. In University greenhouse trials, in absence of parasit-
ism, A. gossypii increased fastest on daisy, followed by M. persicae on daisy, M. persicae on
pansy, andA. gossypii on pansy. Parasitoid suppression of population increase was strongest
forA. gossypii on daisy and poorest for M. persicae on pansy. The presence of 2 aphid species
in the same greenhouse did not alter the level of biological control in our trial. In commercial
greenhouses, banker plants failed to control M. persicae deployed on infested pansies as sen-
tinel hosts. In the laboratory, a 12-h exposure to dried residues of pyriproxyfen or
pymetrozine, insecticides commonly used to control aphids, reduced survival ofA. colemani
adults, compared to a water control (82% survival), to 71% and 53%, respectively. Adult par-
asitoid emergence from pesticide-treated aphid mummies was reduced from 68% for the con-
trols to 56% for pyriproxyfen and 62% for pymetrozine.

Key Words: Aphidius colemani, Myzus persicae, Aphis gossypii, banker plants, biological
control, greenhouse flower crops


RESUME

Plantas banqueras con Aphidius colemani Viereck fueron probadas en invernaderos en los
estados de Massachusetts y Nueva York para el control del afido del algod6n, Aphis gossypii
Glover, y el afido verde del durazno, Myzus persicae (Sulzer) sobre 2 cultivos de flores de la
primavera, violetas (Viola tricolor hortensis) y margaritas (hibrido deArgyranthemum). Las
plants banqueras consistieron de plants de cebada sembradas en macetas infestadas con
el afido, Rhopalosiphum padi (L.), inoculadas al principio con adults de A. colemani com-
prados en un insectario commercial. Se realizaron las pruebas iniciales en plants del cultivo
puestas en bandejas en los invernaderos de la Universidad de Massachusetts. Las plants
centinelas puestas en las bandejas fueron infestadas de una manera uniform con afidos, y
ciertos invernaderos fueron sujetos a la presencia de plants banqueras o dejados como un
control. Antes de las pruebas en la Universidad, se realizaron sondeos de los invernaderos
comerciales en Massachusetts y Nueva York para determinar la frecuencia y las species de
los afidos infestando los cultivos de flores de primavera. En los sondeos de invernaderos co-
merciales, M. persicae fue la especie mas frecuentemente detectada, representando 53% de
todas las infestaciones. En las pruebas del invernadero de la Universidad, en la ausencia de
parasitismo, la poblaci6n de A. gossypii aumento mas rdpido sobre las margaritas, seguida
por M. persicae sobre las margaritas, M. persicae sobre las violetas yA. gossypii sobre las vio-
letas. La supresi6n de la poblaci6n de afidos debida a los parasitoides fue mas fuerte paraA.
gossypii sobre las violetas y mas d6bil en M. persicae sobre las violetas. La presencia de 2 es-
pecies de afidos en el mismo invernadero no cambio el nivel de control biol6gico en nuestra
prueba. En los invernaderos comerciales, las plants banqueras fallaron en controlar M. per-







Florida Entomologist 91(4)


sicae puestos sobre las violetas infestadas como hospederos centinelas. En el laboratorio, la
exposici6n de 12 horas a residues secos de piriproxifen o pimetrozin, insecticides comun-
mente usados para controlar afidos, reducieron la sobrevivencia de adults de A. colemani,
en comparaci6n al control con solo agua (82% sobrevivencia), a 71% y 53%, respectivamente.
La emergencia de los adults de parasitoides de las momias de afidos tratadas con pesticide
fue reducida 68% en el control a 56% para piriproxifen y 62% para pimetrozin.


Aphids are a common problem on a wide vari-
ety of greenhouse crops. In a survey of Massachu-
setts flower growers in 1996, growers reported ap-
plying an average of 3 pesticide applications per
crop for aphids, second only to thrips (Smith,
1998). Use of pesticides for control of aphids, how-
ever, can disrupt biological control of other pests.
Current use of aphid biological control in flower
crops has an inadequate research base, and has
largely been guided by insectary recommenda-
tions. On a per capital basis, parasitoids have
greater potential for suppressing aphid popula-
tions in greenhouses than predators because of
their higher intrinsic rates of increase. But even
for parasitoids, price can be an obstacle to use.
The cost of the most commonly used aphid parasi-
toid, Aphidius colemani Viereck, is 7 cents per
adult (at US $22.50 per 500 parasitoid pupae, Ko-
ppert Inc., at Koppert.com, allowing for shipping
cost and non-emergence of some adults). Vasquez
et al. (2006) found that direct releases ofA. cole-
mani provided excellent control of Aphis gossypii
Glover in small, within-greenhouse netted enclo-
sures (2.1 x 6.1 m; = ca 108 sq. ft.) when released
at 5 mixed-sex adults/m2 in each of the first 3
weeks of a 5-week trial. However, at this release
rate, biological control was 4.7 times more expen-
sive than the pesticide standard (imidacloprid).
The limitation of noncompetitive price is most im-
portant in smaller greenhouses producing short-
term crops such as flowers because time for para-
sitoid reproduction during the crop is limited.
A potential solution to the high cost of repeated
mass releases of parasitoids for aphid control in
short duration crops is to place breeding colonies
of parasitoids (called "banker plant systems" or
"open rearing systems") in greenhouses at plant-
ing time, before aphids appear. This approach can
be quite effective against some aphid species in
some crops (e.g., Conte 1998). Banker plant sys-
tems begin with of a colony of a monocot-feeding
aphid such as Rhopalosiphum padi (L.), the bird
cherry-oat aphid, reproducing on a mildew-resis-
tant variety of a monocot such as rye, grown in
pots. Parasitized aphids (mummies) or adult par-
asitoids, purchased from commercial insectaries,
are then placed on such pots at the start of the
crop and pots are changed as needed when plants
deteriorate. This system reduces cost because
only enough parasitoids need be purchased to es-
tablish the initial breeding colonies and time is
gained for 1 or more parasitoid generations to oc-
cur on the alternative non-pest aphid before pest


aphids invade the crop. Banker plant systems for
two Aphidius species (A. colemani and Aphidius
ervi Haliday) are available for use in the United
States.
Three potential problems exist with use of the
A. colemani banker plant system in spring flower
crops. First, this parasitoid does not parasitize all
aphid species that might become important in
some crops, such as the potato aphid, Macrosi-
phum euphorbiae (Thomas), and the foxglove
aphid, Aulacorthum solani (Kaltenbach), requir-
ing spot applications of pesticides to plants in-
fested with these species. To conserve the efficacy
of banker plant systems when such species are
among the aphids present, pesticides compatible
with key parasitoids are needed. Second, banker
plants require watering and possibly fertilization,
can die from plant diseases like mildew, or may
cease to produce parasitoids if all the aphids on
the plant are killed by the parasitoid or other nat-
ural enemies. These problems can be managed by
use of mildew-resistant rye and periodic transfer
of aphids to new rye plants. Third, aphid suppres-
sion is poor if greenhouse temperatures exceed
28C because such temperatures are favorable to
aphids and unfavorable toA. colemani (Goh et al.
2001; Kim & Kim 2003).
Our goal was to better understand the poten-
tial for effective use ofA. colemani banker plants
for aphid control in spring flower crops in the
northeastern US. Our specific objectives were, as
follows: (1) to survey aphids in commercial green-
houses in Massachusetts and New York to deter-
mine if the aphids found most frequently in the
region's spring flowers were species susceptible to
A. colemani; (2) to measure aphid control pro-
vided by A. colemani-banker plants in University
greenhouses filled with various combinations of
M. persicae and A. gossypii on pansy and Mar-
guerite daisy; (3) to assess the efficacy ofA. cole-
mani banker plants in commercial greenhouses
in Massachusetts and New York; and (4) to deter-
mine if 2 widely used insecticides might be com-
patible withA. colemani.

MATERIALS AND METHODS

Aphid Surveys in Greenhouse Floral Crops

In 2004, to determine what species of aphids oc-
curred in greenhouses during the spring flower crop
and the relative frequency of their infestations, we
visited 41 greenhouses, 20 in New York (from 26


December 2008







Van Driesche et al.:Aphidius colemani Banker Plants


May to 10 Jun) and 21 in Massachusetts (from 15
Apr to 25 May). At each greenhouse, 30 plants of
each of the 3 most prevalent flower species were ex-
amined for aphids. In greenhouses in which there
were more than 3 plant species in significant num-
bers, the 90 scouted plants were divided equally
among the most common plants. In Massachusetts,
but not New York, if no aphids were found during
initial scouting of the dominant crops, we checked
additional species known to be especially suscepti-
ble to aphid infestations (ivy geranium, petunia,
and fuchsia) or that were reported by the grower to
be infested, examining 30 plants per species. Sam-
ples of aphids detected were preserved for later
identification. Identifications were made by Su-
zanne Lyon (MA) or K. C. Bennett (NY), following
their training by Susan Halbert of the Florida De-
partment of Agriculture. We then calculated the rel-
ative frequency of infestations by aphid species.

Efficacy ofA. colemani-banker Plants in University
Greenhouses

Sources ofAphids and Plants. For this trial, we
infested plants with one or both species ofA. gos-
sypii and Myzus persicae (Sulzer), which were 2 of
the 3 most commonly encountered species in our
survey (the third, Aulacorthum solani (Kalten
bach) is not attacked by A. colemani, and so could
not be considered for inclusion in this test). Colo-
nies of both of the pest aphids were provided by
Dan Gilrein of Cornell Cooperative Extension in
Riverhead, New York. Aphids were reared in
cages at University of Massachusetts on pansies
(Viola tricolor hortensis, Delta Blotch Mix) and
Marguerite daisies (Argyranthemum hybrid),
which were the plant species subsequently used
in our trials. Choice of plant species was coordi-
nated with choice of aphid species and strain so
that both aphids used were able to feed and repro-
duce on both species of plants. Insecticide-free
plants used in experiments were grown on con-
tract for us by a local greenhouse operator (Five
Acre Farms, Northfield, Massachusetts).
Source, Management, and Number of Banker-
Plants. The monocot-feeding bird cherry-oat aphid
(R. padi), a species not able to infest dicot flower
crops, was used as the aphid on the banker plants.
These aphids were obtained from Melanie Filotas
at Cornell University, Ithaca, New York. Banker
plants consisted of mildew-resistant barley (Hor-
deum vulgare, McGregor barley, of Agri-Culver
seeds, Trumansburg, New York) grown in pots (20
cm diam.). Plants were infested with aphids when
15-20 cm tall. Aphids periodically were moved to
new barley plants as old ones declined in vigor due
to aphid feeding. The number of banker plants per
greenhouse and the number of parasitoids re-
leased per banker plant in our trials at the Univer-
sity of Massachusetts were chosen to be low in cost
and therefore potentially acceptable to growers.


For each 4 x 8 m plastic hoop greenhouse (ca 350 sq
ft., ca 50% filled), we used 1 banker plant, onto
which we released 25 mixed-sex parasitoids once
at the start of the trials. Wasps were purchased
from Koppert Biological Inc. at a cost of $22.50/ 500
mummies (parasitoid pupae in host aphids), which
came to 7 cents per emerged wasp when emer-
gence rate (65%) and shipping were considered.
Given this cost and the fill rate of the greenhouse,
the price for this treatment was $0.11/m2(= $10 per
1000 sq ft) of protected crop.
Experimental Design and Description of Sam-
pling. Four trials were run, 2 each in 2005 and
2006. All trials were run in 4 identical plastic
hoop greenhouses (4 x 8 m) at the University of
Massachusetts. The purpose was to measure the
effect of A. colemani-banker plants on suppres-
sion of aphid densities. We examined 2 aphid spe-
cies (M. persicae and A. gossypii) on 2 plant spe-
cies (pansy and Marguerite daisy) because of the
high plant diversity in spring flower crops. We
structured trials to measure if the presence of a
second aphid host species in the same greenhouse
affected the degree of control. Trial dates corre-
sponded to a slightly early and slightly late spring
flower crop period in each year (trial one, 23 Mar-
11 May, 2005; trial two, 15 Jun- 7 Jul, 2005; trial
three, 16 Feb- 3 Apr, 2006; and trial four, 26 Apr-
2 Jun, 2006). Only 2 trials were retained for anal-
ysis because delay caused the 15 Jun- 7 Jul, 2005
trial to occur mostly after the normal spring
flower production period and as a consequence
this trial experienced hot weather, not typical of
the crop and unfavorable to this parasitoid (Za-
mani et al., 2007). We excluded the 26 Apr- 2 Jun,
2006 trial because parasitoids invaded the control
greenhouse and suppressed aphids.
There were 4 greenhouses in each trial. These
were partially filled with plants purchased as
plugs from a commercial grower (grown without
pesticide use), potted in 10 cm dia pots, grouped 8
per flat (25 x 50 cm), and placed on greenhouse
benches. Each greenhouse contained 30 flats of
pansies and 30 of Marguerite daisies. One banker
plant was placed on a bench beside the crop plants
in each of 3 of the 4 greenhouses, and the fourth
was kept as an untreated control where parasi-
toids were not released. The 3 greenhouses con-
taining banker plants were inoculated with either
(1)A. gossypii only, (2) M. persicae only, or (3) both
aphid species. The control greenhouse, without
parasitoids, contained both species of aphids. In
the single-aphid greenhouses, aphids of the indi-
cated species were taken from our laboratory col-
ony and 5 aphids were placed on 1 flagged sentinel
plant in the middle of each flat (30 of each plant
species) at the start of the trial. In the mixed aphid
greenhouses (the control greenhouse and 1 with a
banker plant), there were 15 plants of each of the
4 aphid species x plant species combinations. In all
greenhouses, plants were grouped by species, not







Florida Entomologist 91(4)


interspersed. Data collected consisted of a total
count of all aphids on each sentinel plant, weekly
for 7 weeks. At the end of each trial, 1 additional
sample was taken by counting all aphids on each
of 30 randomly selected plants of each plant spe-
cies (exclusive of the inoculated sentinel plants) in
single-aphid greenhouses or 15 plants for each
aphid x plant combination in greenhouses with 2
aphid species present.

Efficacy of Banker Plants in Commercial Greenhouses

In 2006, concurrent with the second year of the
trials at the University of Massachusetts, we con-
ducted a modified trial of banker plants at 7 com-
mercial greenhouses growing spring flower crops,
3 in Massachusetts and 4 in New York. Based on
data from our 2004 survey of aphid occurrence in
regional greenhouse spring flower crops, which
showed M. persicae to be much more common
thanA. gossypii (Table 1), we focused on control of
M. persicae to evaluate banker plants. Further-
more, because the same survey showed aphids to
be spotty in their occurrence in greenhouses, we
decided to evaluate the efficacy of banker plants
in greenhouses based on population increase of
aphids deliberately added to test plants in green-
houses, rather than waiting for infestations to de-
velop naturally. At each test greenhouse, we intro-
duced 4 flats (25 x 50 cm), each with 10 pots (11.2
cm dia) of pansies. Two flats were placed in a 60 x
60 x 60 cm "Bug Dorm" cage (from BioQuip Inc.,
Rancho Dominguez, CA), while 2 uncaged flats
were placed next to the cage on the same green-
house bench. Five plants in each flat were inocu-
lated with 2 M. persicae individuals from our col-
onies at the start of the experiment. In each
greenhouse, we placed 1 banker plant (as in the
University of MA trial) per 38 m2 (400 square
feet). Each banker plant, previously infested with
bird cherry-oat aphids, was inoculated with 25
mixed-sex parasitoid adults or aphid mummies.

TABLE 1. SPECIES OF APHIDS FOUND IN A SURVEY OF 41
GREENHOUSES WITH SPRING FLOWER CROPS IN
2004 IN MASSACHUSETTS AND NEW YORK.

No. infestations
detected
Aphid species (% of total)

Mysus persicae (Sulzer) 27 (52.9)
Aulacorthum solani (Kaltenbach) 14 (27.5)
Aphis gossypii (Glover) 3 (5.9)
Aphis sp. 2 (3.9)
Macrosiphum euphorbiae (Thomas) 2 (3.9)
Ovatus crataegarius (Walker) 1 (2.0%)
Macrosiphum sp. 1 (2.0%)
Aphis spiraecola Patch 1 (2.0%)
Total 51(100%)


Whole plant counts of aphids were made weekly
on each of the 20 inoculated plants (5 per flat).
First aphid counts were made either on 21 or 27 of
Mar in Massachusetts and continued either until
26 Apr or 2 May, depending on location. Sampling
in New York greenhouses began either on 4 or 7
Apr and continued until 12 or 21 May.

Compatibility ofA. colemani with Insecticides

The goal of this experiment was to determine if
2 common pesticide products used to control
aphids, formulations of pymetrozine and py-
riproxyfen, were compatible with A. colemani
adults (via contact with freshly dried residues) or
mummies (via direct sprays). If compatible, such
materials might be used to control species not
parasitized byA. colemani.
Pesticide Rates. Pymetrozine (Endeavor 50WG,
manufactured by Novartis) was applied at the la-
bel-recommended rate for aphid suppression (2.5
oz per 100 gallons, = 0.177g product/473 mL, =
0.000374 g ai/mL spray). Pyriproxyfen (Distance
IGR, manufactured by Valent) was used at the
high end of the labeled range for aphids (8 fl. oz per
100 gallons, = 0.3 mL product/473 mL spray, =
0.0000653 g ai/mL spray). Pesticide solutions were
applied with small, hand pumped, spray bottles.
Water was applied as a control.
Wasp Source. All A. colemani used in these ex-
periments were purchased from IPM Laboratories
in Locke, New York (sourced originally from Kop-
pert, Inc.). Wasps were shipped as aphid mum-
mies, and typically adult wasps were just begin-
ning to emerge on the day the shipment arrived.
Exposure of Adult A. colemani to Pesticide Resi-
dues. Adult wasps were exposed to freshly dried res-
idues in glass shell vials (3.7 ml, 15 x 45 mm, Fisher
Scientific) in groups of 10. Vials were treated indi-
vidually with 3 pumps from a spray bottle contain-
ing either an insecticide solution or water, until vial
walls were coated to run off. After 1 h, vials were in-
verted to allow excess liquid to drain out. Two h af-
ter application, vial surfaces were dry, and 10 adult
wasps (unsexed) were aspirated into each vial (= 1
replicate). For ventilation, a 10-mm dia hole was cut
in each vial top and fine-meshed polyester screening
then secured over the mouth of the vial by the re-
mainder of the lid. Wasps were collected with aspi-
rators from emergence containers and allowed to
walk from the aspirator into the test vials. Vials
with wasps were held in a growth chamber at 22 C,
75% RH, and constant light. After 2 and 12 h, vials
were examined under a dissecting microscope and
the number of dead wasps counted. Each treatment
was replicated 30 times.
Exposure of A. colemani Pupae to Pesticide
Sprays. Groups of aphid mummies from which
wasps had not yet emerged were placed on blotting
paper on plastic dishes with a fine paintbrush.
Each group was sprayed directly with one of the


December 2008







Van Driesche et al.:Aphidius colemani Banker Plants


test solutions as described above and allowed to
dry. Each replicate consisted of 10 treated mum-
mies, which were held in a clean vial (3.7 mL, 15 x
45 mm, Fisher Scientific) secured with fine mesh
polyester fabric in place of a lid. Mummies were
held in a growth chamber at 22 C, 75% RH, 16:8
L:D photoperiod for 72 h, and then the number of
emerged wasps were counted. Each treatment was
replicated 40 times (total, 400 treated mummies).

Statistical Analyses

For the trial at the University of Massachusetts,
the response (aphid numbers per plant on initially
inoculated plants only) was recorded each week for
7 weeks on each sentinel plant in each greenhouse.
Each greenhouse contained a unique combination of
aphid species, plant types, and banker plants. These
treatment combinations were randomly assigned to
greenhouses in each trial. We accounted for these
treatment combinations as fixed effects in the anal-
ysis, and included plants in each trial, and trials as
nested random effects. Thus, we represented the
study by a randomized block design with repeated
measures made on each sentinel plant nested in
each block (i.e., trial). We considered the blocks and
plants to be random, and accounted for the repeated
measures that were nested on sentinel plants in
each greenhouse using a mixed model for SAS
PROC Mixed. Plots of the number of aphids per
week were constructed for each plant and aphid
species for each treatment combination in each
block, along with average profiles. The plots indi-
cated exponential growth over time for aphids in
non-banker plant blocks. We took the natural log of
the aphid count (after adding 1 to avoid zero counts)
to linearize the response pattern over time, and
summarized the linear trend for each plant by the
linear trend for a first order orthogonal polynomial
(using 7 equally spaced time points) for each plant
(Kirk, 1995). A mixed model with plants nested in
blocks as random blocks was fit to evaluate differ-
ences between conditions (aphid species, plant spe-
cies, and banker plant effects) on the linear trend in
aphid growth. We examined homogeneity of vari-
ance between trials and between plants for different
conditions prior to conducting statistical tests. The
statistical analysis focused on comparisons between
linear trends equivalent to growth slopes for simple
population growth curve models between condi-
tions. Slopes of the resulting regressions have bio-
logical meaning because they reflect the population
increase of the pest aphids over time in greenhouses
either with or without the treatment being tested (=
parasitoids on banker plants). If parasitism re-
strains aphid population growth rate, data from
greenhouses with this treatment will give regres-
sion lines with lower slopes. After calculating aver-
age slopes associated with each treatment, we com-
pared slopes against the null hypothesis that slopes
were zero (no increase over time) and among each


other. Data on aphid density on non-inoculated
plants in the various treatments at the end of the
trial were analyzed by similar models. The response
corresponded to the natural logarithm of the num-
ber of aphids (plus 1) per plant. For analysis of the
data on wasp survival after exposure to pesticides,
we fit a mixed model accounting for the repeated
measure using vials as random effects to compare
response between conditions (water, pyriproxyfen,
and pymetrozine) and response (number of live
wasps) over time. A one-way analysis of variance
model was used to test for differences in wasp emer-
gence rates at 72 h comparing treatments by water,
pyriproxyfen, and pymetrozine.
RESULTS

Aphid Surveys in Greenhouse Floral Crops
In Massachusetts, aphids were detected in 5 of
the 21 commercial greenhouses surveyed during
the initial sampling period. The most frequently
infested plants were fuchsia (Fuchsia hybrids),
million bells cultivars (Calibrachoa hybrids), and
ivy geranium (Pelargonium peltatum). At 4 green-
houses in which aphids were not initially de-
tected, aphids were later found on ivy geranium,
Petunia sp., Gerbera jamesonii, or Helichrysum
hybrids. In New York, aphids were found at 12 of
20 commercial greenhouses, mostly frequently on
fuchsia, petunia, or Impatiens sp. Of 51 detected
aphid infestations (both states), the 3 most com-
mon aphids were Myzus persicae (52.9% of infes-
tations), Aulacorthum solani (27.5%), and Aphis
gossypii (5.9%) (Table 1).

Efficacy ofA. colemani-Banker Plants in University
Greenhouses
Plant Effects. From highest to lowest, average
growth rates were (1) A. gossypii on daisy (2) M.
persicae on daisy (3) M. persicae on pansy, and (4)
A. gossypii on pansy (Fig. 1). However, population




c -*-GPA + Daisy
i -o-MA+ Pansy
100 D-MA isy


10
S to


1
1 2 3 4 5 6 7
Week No.
Fig. 1. Effect of plant species on growth ofA. gossypii
and M. persicae populations in the absence of Aphidius
colemani. (Data are geometric means of exponentiated
values of the log transformed values used in analysis).











growth rates of M. persicae and A. gossypii colo-
nies (estimated as the slope of the regression of
aphid density vs sample date) developing on ei-
ther pansy or Marguerite daisy in University
greenhouses were not different by either aphid (F
= 0.13, df = 1, 4, P < 0.7366) or plant species (F =
1.09, df= 1, 4, P < 0.3559), nor was the interaction
of aphid by plant species significant (F = 0.71, df
= 1, 4, P < 0.4461). Variances between trials and
between plants within a trial were not equal for
the 4 aphid-plant combinations, but this inequal-
ity was accounted for in the analysis.
Parasitoid Suppression of Aphid Population
Growth. Again, variances between trials and be-
tween plants within a trial were not equal for the
4 aphid-plant combinations, but this inequality
was accounted for in the analysis. The presence
of banker plants in greenhouses had a signifi-
cant effect on the growth of aphid populations (F
= 212.62, df = 1, 351, P < 0.0001). There was a
statistically significant interaction between the
effect of banker plants and plant species (F =
10.41, df= 1, 351, P < 0.0014). In contrast, the in-
teraction of aphid species and the effect of
banker plants was not significant (F = 3.29, df =
1, 351, P < 0.0707). The three way interaction of


December 2008


plant species, aphid species and banker plants
was significant, suggesting that parasitoids re-
spond to both aphids and the plant on which they
must forage for aphids (F = 13.05, df= 1, 351, P
< 0.0003).
In all greenhouses where banker plants were
used, no slopes of lines for aphids vs time were
significantly different from zero (that is, no popu-
lations showed a statistically significant increase
in aphid numbers over time). Results of hypothe-
sis tests of zero slope were GPA/daisy (t = 1.28, df
= 8, P < 0.2367); MA/daisy (t = -0.73, df = 8, P <
0.4887); GPA/pansy (t = 0.71, df = 8, P < 0.4983)
and MA/pansy (t = 0.45, df= 8, P < 0.6651). In con-
trast, in greenhouses without banker plants,
slopes did differ significantly from zero, suggest-
ing real increase in aphid numbers for 3 aphid-
plant combinations (GPA/daisy, t = 5.15, df= 8, P
< 0.0009; MA/daisy, t = 4.03, df = 8, P < 0.0038,
and GPA/pansy, t = 22.73, df = 8, P < 0.0001), but
not for A. gossypii on pansy (t = 2.17, df =8, P =
0.0621).
When slopes of aphid numbers vs time from
greenhouses without banker plants (controls)
were directly compared to slopes of populations in
greenhouses with banker plants (Fig. 2), differ-


--Single Aphid Species
Treated Greenhouse
-- Cnwrl Greenhouse

100 Mixed Aphid Species
Treated Greenhouse


Green Peach Aphid Pansy A


---Single Aphid Specie
Trealed Greerouse
---Control Grnhousa


100 -*-Mixed Aphid Species
Treated Greeniouse



10


Melon Aphid + Pansy C


1 2 3 4
Week No.


--- Sangle Aphid Species
Treated Gremnhouse
-a- Contl Greenhouse
-a- Mixed Aphid Speies
Treated Grenhouse


Green Peach Aphid+ Daisy B


Melon Aphid + Daisy


Z 10
1
2 o
4


2 3 4 5 6
Week No.


2 3 4
Week No.


Fig. 2. Effect of the presence of the braconid parasitoid Aphidius colemani on population growth of A. gossypii
or M. persicae on 2 host plants in single-aphid or mixed-aphid greenhouses. (Data are geometric means of exponen-
tiated values of the log transformed values used in analysis).


Florida Entomologist 91(4)


2 3


1000

I
a. 100
I
t
4 i
10
&


Week No.







Van Driesche et al.:Aphidius colemani Banker Plants


ences were found forA. gossypii on daisy (t = 3.78,
df =8, P = 0.0054) and M. persicae on pansy (t =
2.40, df =8, P = 0.0434), but not for A. gossypii on
pansy (t = 1.90, df =8, P = 0.0938) or M. persicae
on daisy (t = 2.17, df =8, P = 0.0616).
Effect of 1 versus 2 Aphid Species. The pres-
ence of a second host species (here, a second aphid
species), which in some systems can enhance bio-
logical control, had no significant effects in this
case. Rates of increase per aphid species were not
significantly different between banker plant
greenhouses with 1 aphid species vs banker plant
greenhouses with both aphid species present (Fig.
2a,b,c,d). No pairwise comparisons between one-
aphid species and two-aphid species greenhouses,
both with banker plants, were significant (GPA-
D, t = 0.29, df=8, P = 0.7760) (MA-D, t = -1.42, df
=8, P = 0.1942) (GPA-P, t = -0.09, df =8, P =
0.9335) (MA-P, t = 0.18, df=8, P = 0.8639).
Final Aphid Densities in the Crop as a Whole.
On non-inoculated plants, the use of banker
plants suppressed aphids from 73 to 90% relative
to the controls, depending on aphid and plant spe-
cies (Table 2). Comparison of aphid densities
among treatments showed a significant effect of
banker plants (F = 6.56, df= 8, P < 0.0336). How-
ever, no individual pairwise comparisons were
significant between final aphid densities on non-


inoculated plants between greenhouses with and
without banker plants, for any aphid x plant com-
bination.

Efficacy of Banker Plants in Commercial Greenhouses

In commercial greenhouses, use of banker
plants at the rate tested did not provide ade-
quate suppression of M. persicae (the only spe-
cies tested). Population growth on sentinel pan-
sies, inoculated at the start of the trial, was sup-
pressed successfully in only 4 of 7 greenhouses.
Moreover, of these 4, control was due at least in
large part in 2 instances to larvae of syrphid flies
that spontaneously invaded the greenhouses
(Table 3). In only 1 of 7 cases did the banker
plants prevent aphids from increasing in num-
ber.

Compatibility ofA. colemani with 2 Insecticides

At 2 h, adult survival ofA. colemani in the wa-
ter control (98%), was different from survival in
vials treated with Distance (pyriproxyfen) or En-
deavor 50WG (pymetrozine) (both, 89%) (F = 3.21,
df = 2, 88, P=0.0452). By the end of the experi-
ment at 12 h, there were larger differences in
wasp survival among treatments in a one-way


TABLE 2. SUPPRESSION LEVEL AND FINAL APHID DENSITY (#/PLANT, MEAN, STANDARD DEVIATION) IN (A) CONTROL
GREENHOUSES, (B) PARASITOID-TREATED GREENHOUSES WITH 1 APHID SPECIES, AND (C) PARASITOID-
TREATED GREENHOUSES WITH 2 APHID SPECIES IN A TRIAL AT THE UNIVERSITY OF MASSACHUSETTS IN 2005
AND 2006.

(A) Final aphid density (#/plant) in control greenhouses
M. persicae A. gossypii
Trial (in mixed aphid greenhouses) (in mixed aphid greenhouses)
Daisy Pansy Daisy Pansy

1 532.9 562.7 115.2 23.1 3214.4 2750.1 145.6 29.4
2 4.9 6.4 271.6 225.9 2.2 1.5 9.2 14.6

(B) Final aphid density (#/plant) in parasitoid-treated greenhouses with one
aphid species (and % reduction compared to control)
Trial M. persicae A. gossypii
Daisy Pansy Daisy Pansy
1 4.4 2.7 (99%) 3.1 3.9 (97%) 0.4 1.3 (100%) 0.3 0.6 (100%)
2 2.5 3.2 (49%) 54.6 56.8(80%) 0.6 1.3 (73%) 1.8 3,3 (80%)
Ave. suppression 74% 89% 87% 90%

(C) Final aphid density (#/plant) in parasitoid-treated greenhouses with two
aphid species (and % reduction compared to control)
Trial M. persicae A. gossypii
Daisy Pansy Daisy Pansy
1 23.5 12.2 (96%) 0.6 1.3 (100%) 16.5 11.4(100%) 1.1 1,6 (99%)
2 2.5 2.9 (49%) 68.9 73.5 (75%) 1.4 2.1(36%) 2.9 3.9 (70%)
Ave. suppression 73% 88% 68% 85%







Florida Entomologist 91(4)


TABLE 3. LEVEL OF INCREASE IN DENSITY (AS RATIO OF FINAL OR PEAK DENSITY/DENSITY ON FIRST SAMPLE DATE) OF
MYZUS PERSICAE (SULZER) DURING SPRING FLOWER CROPS IN 7 COMMERCIAL GREENHOUSES IN THE NORTH-
EASTERN UNITED STATES, COMPARED TO DENSITIES INSIDE EXCLUSION CAGES, IN THE PRESENCE OF BANKER
PLANTS WITH THE PARASITOID APHIDIUS COLEMANI.


Caged controls


Uncaged treatment
(accessible to parasitoids from banker plants)


BC Success/
Failure?
(S, F)


Final #-fold increase
(or peak') density (last/first sample date)


10.6
563.6
157.5



177.6
415.4
236.4
500.0


17.7
234.8
29.2
(93.9)


66.0
33.5
19.3
57.5
(44.1)


Final Density


57.3
6.0
2.7



0.6
134.6
3.3
206.3


#-fold increase
(last/first sample date)


47.8
3.0
2.1
(17.6)


0.5
41.4
1.0
75.0
(29.5)
24.4


'For caged controls, if aphid densities peaked in middle of trial and then collapsed due to effects on plant quality, increase is cal-
culated using the peak value rather than the final value, to correct for loss of plant quality
Outcomes at growers #2 and #6 were mostly due to syrphids that naturally invaded the greenhouse. Numerous syrphid eggs and
larvae were found on the uncaged test plants.
'Caged control plants collapsed after 3 weeks due to high aphid numbers. Observations on uncaged plants, on which aphid
growth was very low, were made for 6 weeks.


ANOVA (F = 18.54, df = 2, 88, P < 0.0001). Sur-
vival on pymetrozine-treated surfaces was 53%
versus 71% for pyriproxyfen and 82% for the wa-
ter control, with both being different from sur-
vival in the controls (pyriproxyfen, t = 2.21, df =
88, P < 0.0296; pymetrozine, t = 6.01, df = 88, P <
0.0001).
The emergence of adult parasitoids from pesti-
cide-treated aphid mummies was affected by ex-
posure to pesticides (one-way Anova, F= 5.2, df =
2, P < 0.0069). In pairwise comparisons, py-
riproxfen's effect (56%) was different from the wa-
ter control (68%) (F = 10.39, df= 1, P < 0.0016) but
pymetrozine's (65%) was not (F = 2.6, df = 1, P <
0.1098).

DISCUSSION

The presence of banker plants in greenhouses
had a significant effect on the growth of aphid
populations. Aphidius colemani banker plants
placed in 4- x 8-m plastic hoop greenhouses at the
University of Massachusetts significantly sup-
pressed aphids in 2 of the 4 aphid x plant combi-
nations tested (M. persicae on pansy and A. gos-
sypii on daisy), while the other 2 combinations
showed levels of suppression that might have
been significant with greater replication. Least


impact occurred onA. gossypii on pansy. The pres-
ence of a second species of aphid in the green-
house did not have any important effect on the
level of suppression byA. colemani versus green-
houses with only 1 aphid species.
Little control was achieved by banker plants
against M. persicae in commercial greenhouses.
This may have been caused by neglect of banker
plants by some growers (failure to adequately wa-
ter plants, which occurred in New York), too few
aphids on banker plants at the start of the crop
(due to late placement in greenhouses), or use of
too few banker plants per unit area in view of crop
density. The banker plant rate (#/m2 of green-
house floor) used in this research was the same as
our University trials, whose greenhouses were
only partially filled with plants. This banker
plant rate may have been insufficient in commer-
cial greenhouses, which were completely filled
with larger, more densely packed plants. Greater
foliage volume would have increased the area for
parasitoids to search, reducing their efficiency.
In addition to using more banker plants per
m2, control might be improved through better
management of the banker plants to ensure
larger, healthier populations of grain aphids, or
use a different monocot-feeding aphid more suit-
able as a host for A. colemani, since R. padi is not


Wks
in
Site trial


MA
1
2
3
(Ave.)


4 3/63
5 7
6 7
7 7
Ave.


December 2008







Van Driesche et al.:Aphidius colemani Banker Plants


a high quality host for this parasitoid (Ode et al.,
2005). However, the commonness of foxglove
aphids in the northeastern US flower crops could
compromise the use of A. colemani because this
aphid's presence would require spot applications
of pesticides (which could harm parasitoids) or
use of some other natural enemy.

ACKNOWLEDGMENTS

We thank Dan Gilrein and Melanie Filotas of Cor-
nell University for initial colonies ofA. gossypii and M.
persicae (DG) and bird cherry-oat aphid (MF). Financial
support for this research was provided by the Northeast
IPM Program, the New England Greenhouse Confer-
ence, and the Massachusetts Agricultural Experiment
Station (project No. MAS00885, publication No. 3427).

REFERENCES CITED

CONTE, L. 1998. New prospects for the biological control
of Aphis gossypii on protected crops. Informatore Fi-
topatologico 48: 25-30.
GOH, H., J. KIM, AND M. HAN. 2001. Application of
Aphidius colemani Viereck for control of the aphid in


the greenhouse. J. Asia-Pacific Entomol. 4(2): 171-
174.
KIM, Y., AND J. KIM. 2003. Biological control of aphids
on cucumber in plastic greenhouses using banker
plants. Korean J. Appl. Entomol. 42: 81-84.
KIRK, R. E. 1995. Experimental Design. Procedures for
the Behavioral Sciences. Brooks/Cole Publishing
Company, New York.
ODE, P. J., K. R. HOPPER, AND M. COLL. 2005. Oviposi-
tion vs. offspring fitness inAphidius colemani para-
sitizing different aphid species. Entomol. Exp. Appl.
115: 303-310.
SMITH, T. 1998. Survey of integrated pest management
for bedding plants: Part II. Floral Notes 10 (5): 4-5
(March-April issue); see also http://www.umass.edu/
umext/programs/agro/ipm/Reports/plantina.htm
VASQUEZ, G. M., D. B. ORR, AND J. R. BAKER 2006. Effi-
cacy assessment of Aphidius colemani (Hymenoptera:
Bracondiae) for suppression of Aphis gossypii (Ho-
moptera: Aphididae) in greenhouse-grown chrysanthe-
mum. J. Econ. Entomol. 99: 1104-1111.
ZAMANI, A. A., A. TALEBI, Y. FATHIPOUR, AND V. BAN-
IAMERI. 2007. Effect of temperature on life history of
Aphidius colemani and Aphidius matricariae (Hy-
menoptera: Braconidae), two parasitoids of Aphis
gossypii and Myzus persicae. Environ. Entomol. 36:
263-271.







Florida Entomologist 91(4)


December 2008


FALL ARMYWORM (LEPIDOPTERA: NOCTUIDAE) RESISTANCE IN TEXAS
BLUEGRASS, KENTUCKY BLUEGRASS, AND THEIR HYBRIDS (POA SPP.)

JAMES A. REINERT1 AND JAMES C. READ'
'Texas AgriLife Res & Ext Urban Solutions Center, 17360 Coit Rd, Dallas, TX 75252-6599 USA
E-mail j-reinert@tamu.edu


ABSTRACT

The fall armyworm Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae), is a de-
structive pest of many species of both C3 and C4 (cool- and warm-season) turfgrass. No-choice
experiments were conducted to evaluate 13 turfgrass genotypes of various Poa spp. for sus-
ceptibility or resistance to the fall armyworm. All 13 genotypes, including 8 Texas bluegrass
(Poa arachnifera Torr.), 2 Kentucky bluegrass (P. pratensis L.) and 3 Kentucky bluegrass x
Texas bluegrass interspecies hybrids, were antibiotic and produced an accumulated >80%
mortality of neonate larvae before they pupated. 'Reveille' (a hybrid) provided 100% antibio-
sis of larvae within 4 d of feeding. When 4-d-old fall armyworm larvae that had first fed on a
susceptible Poa host were confined on the same 13 Poa genotypes as in the neonate test, a
much higher survival rate was recorded. 'Reveille' produced 94.4% mortality after 3 d of feed-
ing and 100% mortality after 8 d of feeding, while TXKY90-13-16 (another hybrid) provided
100% mortality of larvae within 13 d of feeding. A third hybrid, TXKY90-13-8 was one of the
more susceptible genotypes. For the 4-d-old larvae, 'Baron' and 'Delwood Fine' Kentucky
bluegrass provided only 50 and 22.2%, respectively, mortality after 8 d of larval feeding and
did not produce 100% mortality until pupation. Also, mortality of larvae on the 8 Texas blue-
grass genotypes produced <45% maximum accumulated mortality by pupation or adult emer-
gence.'Laser' rough bluegrass (P. trivialis L.) is an excellent host with <5.6% larval mortality.

Key Words: turfgrass pests, host plant resistance, antibiosis, Poa arachnifera, Poa pratensis,
Poa hybrids


RESUME

El gusano cogollero Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae), es una
plaga destructive para muchas species de c6spedes tipo C3 y C4 (climas frios y templados).
Se llevaron a cabo experiments de no-opci6n para evaluar la susceptibilidad o resistencia
de 13 genotipos de varias species Poa spp. al gusano cogollero. Todos los 13 genotipos, in-
cluyendo 8 Texas bluegrass (Poa arachnifera Torr.), 2 Kentucky bluegrass (P. pratensis L.) y
3 hibridos interespecificos de Kentucky bluegrass x Texas bluegrass fueron antibi6ticos y
produjeron mortalidades >80% en larvas neonatas antes de que puparan. 'Reveille' (un hi-
brido) provey6 una antibiosis de 100% en los primeros 4 dias. Cuando larvas de 4-dias fueron
primero alimentadas con hospederos susceptibles de Poa, y despu6s confinadas a en los mis-
mos 13 genotipos de Poa, se observe una mayor tasa de sobrevivencia. 'Reveille' produjo una
mortalidad de 94.4% despu6s de 3 dias de alimentaci6n y una mortalidad de 100% de des-
pu6s de 8 dias, mientras que TXKY90-13-16 (otro hibrido) provey6 una mortalidad de 100%
dentro de 13 dias. Un tercer hibrido, TXKY90-13-8, fue el mas susceptible de los genotipos.
Para larvas de 4 dias de edad, los cultivares de Kentucky bluegrass 'Baron' y 'Delwood Fine'
proveyeron solo un 50 y 22.2% de mortalidad, respectivamente despu'es de 8 dias de alimen-
taci6n, y no produjeron mortalidades de 100% hasta pupaci6n. Por otro lado, la mortalidad
de larvas en los ocho genotipos de Texas bluegrass fue aproximadamente un 45% de la mor-
talidad maxima tanto en pupaci6n o emergencia de los adults. El c6sped'Laser' rough blue-
grass (P. trivialis L.) es un hospedero excelente, mostrando solo una mortalidad de 5.6%.

Translation provided by the authors.


Genetic plant resistance to pests including mary turfgrass pests regardless of their utiliza-
insects, mites, and diseases is an effective and tion from production fields to installed land-
economical control strategy and should be a ma- scapes, golf courses, or recreational fields.
jor component of every Integrated Pest Manage- When pest resistant cultivars are used in the
ment program when resistant cultivars are landscape, they will help to reduce the need for
available. A major need in the turfgrass indus- pesticide input into urban and suburban land-
try is the continued development of improved scapes and indirectly reduce the potential for
cultivars with inherent resistance to the pri- environmental contamination.







Reinert & Read: Fall Armyworm Resistance in Bluegrass


The fall armyworm (FAW) Spodoptera fru-
giperda (J. E. Smith) (Lepidoptera: Noctuidae) is
a destructive pest of over 50 species of plants
(Luginbill 1928) and it is known to feed on many
species of both C3 and C4 (cool- and warm-season)
turfgrass (Reinert et al. 1997, 1999). Leuck et al.
(1968) first identified resistance to fall armyworm
in bermudagrass. Lynch et al. (1983), Quisen-
berry & Wilson (1985), and Jamjanyu & Quisen-
berry (1988) confirmed the high level of antibiosis
and nonpreference in Tifton 292 and several other
bermudagrass genotypes. Wiseman et al. (1982)
and Chang et al. (1985) reported a high level of re-
sistance to fall armyworm in'Common' centipede-
grass Eremochloa ophiuroides (Munro.) Hack.
and Reinert & Engelke (unpublished manuscript)
discovered high levels of antibiosis in 'Cavalier'
(Zoysia matrella L.) and several other genotypes
of zoysiagrass. Cavalier was introduced as a new
cultivar for its resistance to environmental
stresses including its resistance to the FAW, other
chewing insects, and several diseases. Resistance
to FAW has been characterized among 46 Ken-
tucky bluegrass (Poa pratensis L.) cultivars (Rein-
ert et al. 2004b). The potential for use of insects
and mites resistant turfgrass cultivars was sum-
marized by Reinert et al. (2004a), but they also
emphasize the considerable lack of known infor-
mation on host response to insects and mites in
the turfgrass ecosystem.
There is a need across the southern United
Stated for a perennial cool-season grass for use as
turf and for a permanent winter pasture for live-
stock. The existing cool-season perennial grasses
(C3 grasses), primarily tall fescue (Festuca arun-
dinacea Schreb.) that are available require higher
rainfall and more temperate climates than the C4
grasses that are typically utilized throughout the
southern United States. Texas bluegrass (P.
arachnifera Torr.) is a C3 grass and its hybrids
with Kentucky bluegrass (P pratensis L.) have
considerable potential in these drier and hotter
regions (Read 1994, 2001).
The purpose of this research was to evaluate
genotypes of Texas bluegrass, Kentucky blue-
grass, and their interspecies hybrids and to iden-
tify potential resistance to the FAW, which is one
of the primary pests of turfgrasses and forage
grasses across the southern region and much of
the Eastern and Midwestern U.S.

MATERIALS AND METHODS

Poa genotypes and interspecies hybrids (Ta-
bles 1 and 2) were maintained in the greenhouse
and cultured in plastic pots (15.24 cm. top diam.,
12.7 cm bottom diam. by 17.7 cm tall) and fertil-
ized bi-weekly with Peter's 20-20-20 (NPK) at ap-
proximately 170 ppm. Leaf and stem clippings
from these plants were used to bioassay FAW lar-
vae in no-choice laboratory feeding experiments.


Clippings were taken from several plants for each
genotype and mixed together so that a represen-
tative sample of the grass was always used to feed
the FAW larvae. Each experiment was set up in
the laboratory with plastic Petri dishes (9-cm
diam. x 20 mm deep) as feeding chambers for lar-
vae. Each feeding chamber was provided with two
water saturated 7.5-cm filter paper discs. Water
was added to the filter paper as needed through-
out the experiments to keep it saturated and
maintain the grass cuttings. Each dish was pro-
vided with a small amount of fresh leaf tissue (ca.
3 g) of the respective Poa genotype.
Grass was added or replaced daily or every-
other-day throughout the experiment so that tur-
gid fresh grass was always available to the devel-
oping larvae. For these experiments, eggs of the
corn strain of FAW were obtained from the lab col-
ony maintained at the USDA-ARS-IBPMRL at
Tifton, GA. Larvae were introduced into the feed-
ing chambers as neonates within a few hours after
hatching in Experiment 1. In Experiment 2, they
were introduced as 4-d-old larvae that had been
developed on fresh tissue of 'Laser' rough blue-
grass (Poa trivialis L.). This grass serves as an ex-
cellent host, usually with near 100% survival.
For the first experiment, 3 neonate larvae were
randomly selected after egg hatch and placed on
each grass in the feeding chambers in each repli-
cate (Table 1), and dishes were arranged in a ran-
domized complete block design with 6 replicates
on the laboratory bench. Since FAW egg masses
are usually laid on some structure or debris adja-
cent to the turf area and the larvae then migrate
to the turf setting to feed, we also evaluated 4-d-
old larvae on each of the test genotypes. For the
second experiment, neonate larvae were allowed
to develop for 4 d on leaf clipping of Laser rough
bluegrass. When larvae were 4-d-old, 3 larvae
were randomly selected and placed in the feeding
chambers with the respective Poa genotypes (Ta-
ble 2) in a randomized complete block design with
6 replicates.
For both experiments, survivorship was re-
corded when clippings were added either daily or
every-other-day until pupation and at adult emer-
gence. All surviving larvae were weighed when
12-d-old, well before any pupation occurred and
all pupae were weighed within 24 h after pupa-
tion. Days from egg hatch to pupation and adult
emergence were recorded.

Statistical Analysis

Data were analyzed by analysis of variance
procedures (ANOVA and GLM) and means sepa-
rated by Tukey's studentized range (HDS) test (P
= 0.05) (SAS Institute 2008). Mortality data were
transformed to arcsine (x + 0.001) before each
ANOVA was performed, but the actual percent-
age for mortality is presented.















TABLE 1. RESISTANCE IN BLUEGRASSES TO FEEDING BY NEONATE FALL ARMYWORM LARVAE (MEANS OF 6 REPLICATES).


7-d-old 12-d-old 12-d-old Pupa Days Pupa Adult
Mort wt (mg)b % mort wt (mg)c to pupad % mort days


Cultivar or Genotype


Kentucky bluegrass (Poa pratensis)
Delwood Fine
Baron

TX x KY hybrids (Poa pratensis x Poa arachnifera)
Reveille
TXKY90-13-16
TXKY90-13-8

Texas bluegrass (Poa arachnifera)
TBPC15-10
TBPC20-16
TBPC27-7
Syn3
Syn4
Syn5
Tejas 1
Syn2

Rough bluegrass (Poa trivialis)
Laser


100 a'
72.2 a


100 a
100 a
83.3 a


100 a
100 a
100 a
100 a
100 a
94.4 a
94.4 a
83.3 a


- 9 100 a
22.8 a' 72.2 a


100 a
100 a
66.9 ab 83.3 a


3.2 a
6.0 a
28.7 a


5.6 b 149.0 b


100 a
100 a
100 a
100 a
100 a
94.4 a
94.4 a
83.3 a


100 a
100 a


237.1 a 23.3 b


184.6 ab
208.4 ab
149.7 b


35.0 a
26.0 b
25.0 b


5.6 b 212.9 ab 19.5 c


100 a
100 a
83.3 a


100 a
100 a
100 a
100 a
100 a
94.4 a
94.4 a
88.9 a


5.6 b


100 a
100 a


100 a
100 a
37.3 b 83.3a


47.0 a
31.0 c
30.0 c


33.6 bc


100 a
100 a
100 a
100 a
100 a
94.4 a
94.4 a
94.4 a


Mean % accumulated mortality at 7 d old, 12 d old, pupation, and at adult emergence on each grass genotype.
bMean weight of 12-d-old larvae after 8 d of feeding.
Mean pupa weight taken within 1 d of pupation.
d Mean number of days from egg hatch to pupation.
SMean number of days from egg hatch to adult emergence.
SMeans in a column followed by the same letter are not significantly different by
Tukey's Studentized Range (HSD) test (P = 0.05)
'No larvae survived to this growth stage.


Adult
% mort















TABLE 2. RESISTANCE IN BLUEGRASSES TO FEEDING BY 4-DAY-OLD FALL ARMYWORM LARVAE (MEANS OF 6 REPLICATES)

7-d-old 12-d-old 12-d-old Pupa Days Pupa
Cultivar or Genotype Mort' wt (mg)b % mort wt (mg), to pupad % mort


Kentucky bluegrass (Poa pratensis)
Delwood Fine
Baron

TX x KY hybrids (Poa pratensis x Poa arachnifera)
Reveille
TXKY90-13-16
TXKY90-13-8

Texas bluegrass (Poa arachnifera)
TBPC15-10
TBPC20-16
TBPC27-7
Syn3
Syn4
Syn5
Tejas 1
Syn2

Rough bluegrass (Poa trivialis)
Laser


16.7 be' 22.1 a'
32.2 bc 25.4 a


94.4 a
44.4 b
11.1 bc


11.1 bc
5.6 bc
5.6 bc
11.1 bc
5.6 bc
5.6 bc
Oc
5.9 bc


5.6 a
66.6 b


14.5 a
20.1 a
17.1 a
18.5 a
16.4 a
15.6 a
18.4 a
16.8 a


0 c 139.4 c


22.2 c
50.0 b


100 a
83.3 ab
11.1 c


11.1 c
5.6 c
5.6 c
11.1 c
16.7 c
5.6 c
5.6 c
17.8 c


100 a
100 a


229.1 c 24.4 d


187.4 a
193.1 a
182.9 a
181.3 a
172.6 a
179.9 a
201.7 b
197.1 ab


33.8 a
29.3 c
30.8 bc
30.9 bc-
33.6 a
32.1 ab
29.1 c
31.5 abc


0 c 229.9 c 21.9 e


100 a
100 a
16.7 bcd


27.8 bcd
16.7 bcd
5.6 cd
44.4 b
33.3 bcd
33.3 bcd
5.6 cd
38.9 bc


38.1 d


48.3 a
43.2 c
45.0 bc
45.6 abc
27.6 ab
46.3 ab
43.2 c
45.6 abc


"Mean % accumulated mortality at 7 d old, 12 d old, pupation, and at adult emergence on each grass genotype.
bMean weight of 12-d-old larvae after 8 d of feeding.
'Mean pupa weight taken within 1 d of pupation.
dMean number of days from egg hatch to pupation.
'Mean number of days from egg hatch to adult emergence.
MVeans in a column followed by the same letter are not significantly different by Tukey's Studentized Range (HSD) test (P = 0.05)
'No larvae survived to this growth stage.


Adult
days


Adult
% mort'


100 a
100 a


100 a
100 a
16.7 bcd


27.8 bcd
16.7 bcd
5.6 cd
44.4 b
33.3 bcd
33.3 bcd
5.6 cd
38.9 bc


0d 35.7 d







Florida Entomologist 91(4)


RESULTS

Neonate Larvae

Each Poa genotypes, except P trivialis, was
highly antibiotic to the neonate FAW larvae. All
Texas bluegrass, Kentucky bluegrass, and Texas
bluegrass x Kentucky bluegrass hybrids produced
>61% mortality within 4 d of feeding (Table 1).
Five of the grasses, 'Reveille' (TXKY90-16-1) a
bluegrass hybrid, Syn4, TBPC15-10 and TXPC27-
7 Texas bluegrasses, and'Delwood Fine' Kentucky
bluegrass each provided 100% mortality within 4
d. After 7 d of feeding, TXPC20-16 and Syn3 pro-
vided 100% mortality, and after 12 d, TXKY90-13-
16 also provided 100% mortality of the confined
larvae. 'Baron' Kentucky bluegrass did not pro-
duce 100% mortality until after larvae had fed for
17 d. All of the grasses except Laser rough blue-
grass provided >80% and statistically significant
mortality before larvae were able to pupate. Laser
was an excellent host for the FAW with only 5.6%
mortality of the larvae started as neonates, which
may be within the expected mortality in nature.
Differences in larval weights at 12 d were also sig-
nificantly different (Table 1). Larvae that fed on
Laser were much heavier than those that fed on
any of the other Poa genotypes. The larvae that
did survive and mature to adults on TXKY90-13-
8 bluegrass hybrid, however, were only about one
half the sizes of those that developed on Laser
(Table 1). Larvae that pupated after developing
on TXKY90-13-8 were the largest and produced
significantly heavier pupae than those that devel-
oped on Syn2. The larvae feeding on Laser pu-
pated in the shortest feeding period (19.5 d) while
those feeding on the other Poa genotypes took 4.0
to 15.5 d longer. Individuals that successfully
emerged as adults on Syn5 took 47 d in contrast to
only 33.6 d for the larvae that developed on Laser.
Even though 1 to 3 individuals per genotype were
able to pupate and reach adult emergence on
TXKY90-13-8, Tejasl, Syn2 or Syn5, these
grasses should be considered as poor hosts for ne-
onate larvae of FAW because of the sublethal an-
tibiosis exhibited.

4-day-old Larvae

The survival of larvae was much higher if they
first fed for 4 d on the very palatable host, Laser
before being confined in the no-choice feeding
study with each of the 14 Poa genotypes (Table 2).
'Reveille' hybrid bluegrass produced highly signif-
icant antibiosis (94%) of the larvae after only 3 d
of feeding (7-d-old) and all larvae were dead
within 12 d. Another hybrid, TXKY90-13-16, pro-
vided 100% mortality after 13 d of feeding (17-d-
old), whereas, the third hybrid, TXKY90-13-8 was
one of the most susceptible Poa genotypes evalu-
ated and only caused 11.1% mortality of larvae af-


ter 13 d of feeding and only 16.7% mortality by
pupation and adult emergence. The 2 cultivars of
Kentucky bluegrass, Baron and Delwood Fine,
provided only 50 and 22.2%, respectively, mortal-
ity after 8 d of feeding (12-d-old) and neither pro-
duced 100% mortality until pupation. None of the
P arachnifera genotypes produced <25% mortal-
ity of larvae and <45% maximum mortality at pu-
pation or adult emergence (Table 2). The cultivar
'Tejasl' and 2 other Texas bluegrass genotypes
(TBPC27-7, TBPC20-16) and 1 hybrid (TXKY90-
13-8) showed no resistance (<17% mortality) and
should be considered highly susceptible to the
FAW. No mortality was recorded on Laser rough
bluegrass during this study.
Similar to the results in the previous experi-
ment that started with neonate larvae, the larvae
in this study developing on TXKY90-13-8 were
also ca. 3 times larger than those developing on
the other Poa genotypes, except for those on the
highly susceptible Laser rough bluegrass which
were about 7 times larger (Table 2). Additionally,
the 12-d-old larvae feeding on Laser in both stud-
ies were about the same weight and required sim-
ilar time periods to reach adult emergence. Even
though survivorship was relatively high on most
of the Poa genotypes, the significantly reduced
larval and pupal weights are an indication of sub-
lethal (antibiotic) resistance. Individuals confined
on most of these genotypes also required 7 d or
longer before pupation and adult emergence com-
pared to those developing on Laser which re-
quired an average of only 21.9 d to pupation and
only 35.7 d before adult emergence.
Another mechanism of resistance was ob-
served in larvae feeding on 'Baron' and 'Delwood
Fine' Kentucky bluegrass and on 'Reveille' and
TXKY90-13-16 hybrids. Larvae feeding on these
genotypes would develop normally to either the
second or third instar, when they would begin
swelling during ecdysis and appear to freeze in
this bloated state and they were unable to com-
plete the molt or the shedding of the old larval
skin. Since this phenomenon was only observed
with larvae feeding on genotypes of Kentucky
bluegrass and hybrids between Kentucky blue-
grass and Texas bluegrass, it is assumed that this
mechanism of resistance is inherited from the
Kentucky bluegrass parents and was transferred
to'Reveille' and TXKY90-13-16 during hybridiza-
tion.

DISCUSSION

Results of this study indicate that a strong
level of resistance to the FAW is present in Ken-
tucky bluegrass cultivars, Baron and Delwood
Fine. The resistance level to FAW, however, can
vary among Kentucky bluegrass cultivars. Wa-
bash, Adelphi, Eagleton, and Monopoly each pro-
duced >90% mortality after only 7 d of feeding,


December 2008







Reinert & Read: Fall Armyworm Resistance in Bluegrass


while Kenblue, PTDF22B2, and Glade did not
produce grater than 30% mortality even at adult
emergence (Reinert 2004b). The level of suscepti-
bility varied considerably among the Texas blue-
grass genotypes with Syn3 providing the higher
level of resistance (44.4% mortality) to the 4-d-old
larvae. Two of the Texas bluegrass x Kentucky
bluegrass hybrids ('Reveille' and TXKY90-13-16)
inherited the high level of resistant (100% mortal-
ity) while the third hybrid, TXKY90-13-8, is sus-
ceptible. Laser rough bluegrass is highly suscep-
tible to FAW and should be used as a standard for
comparison in other studies with turfgrasses to
document their levels of susceptibility or resis-
tance. Additional feeding studies with FAW lar-
vae on Kentucky bluegrass are needed to charac-
terize the true nature of the ecdysis mechanism of
resistance.
There appears to be 2 different mechanisms for
antibiosis in the bluegrasses. One mechanism
was observed in Baron and Delwood Fine Ken-
tucky bluegrass and in the 2 Texas bluegrass x
Kentucky bluegrass hybrids, Reveille and
TXKY90-13-16. In this case during the second or
third instar, the larvae begin to swell during
ecdysis and appear to freeze in the bloated state
thus causing death. This antibiosis would most
likely be due to some toxin associated with Ken-
tucky bluegrass. The other antibiosis mechanism
observed in Texas bluegrass and the hybrid
TXKY90-13-8 may to be related to poor quality
diet. In this case, the antibiosis appears related to
poor digestibility or palatability of the plant ma-
terial as opposed to some toxic compound. Fur-
ther studies with FAW resistance will address
this hypothesis.

ACKNOWLEDGMENTS
This study was supported in part by grants from
Gardner Turfgrass, Inc., O. J. Noer Research Founda-
tion, Inc. and the U.S. Golf Association. Appreciation is
extended to S. J. Maranz for technical assistance.

REFERENCES CITED
CHANG, N. T., B. R. WISEMAN, R. E. LYNCH, AND D. H.
HABECK. 1985. Fall armyworm expressions of antibi-
osis in selected grasses. J. Entomol. Sci. 20: 179-188.
JAMJANYA, T., AND S. S. QUISENBERRY. 1988. Fall army-
worm (Lepidoptera: Noctuidae) consumption and
utilization of nine bermudagrasses. J. Econ. Ento-
mol. 81: 697-704.


LEUCK, D. B., C. M. TALIAFERRO, G. W. BURTON, R. L.
BURTON, AND M. C. BOWMAN. 1968. Resistance in
bermudagrass to the fall armyworm. J. Econ. Ento-
mol. 61: 1321-1322.
LUGINBILL, P. 1928. The Fall Armyworm. U.S. Dep. Ag-
ric. Tech. Bull. 34, 92 p.
LYNCH, R. E., W. G. MORSON, B. R. WISEMAN, AND G. W.
BURTON. 1983. Bermudagrass resistance to the fall
armyworm (Lepidoptera: Noctuidae). Environ. Ento-
mol. 12: 1837-1840.
QUISENBERRY, S. S. 1990. Plant resistance to insects
and mites in forage and turf grasses. Florida Ento-
mol. 73: 411-421.
QUISENBERRY, S. S., AND H. K. WILSON. 1995. Con-
sumption and utilization ofbermudagrass by fall ar-
myworm (Lepidoptera: Noctuidae) larvae. J. Econ.
Entomol. 78: 820-824.
READ, J. C. 1994. Potential of Texas bluegrass x Ken-
tucky bluegrass hybrids for turf in north central Tex-
as. TX Turfgrass Res. 1993, Consolidated Prog.
Rep. PR-5108: 11-12.
READ, J. C. 2001. Utilization of apomictic and dioecious
method of reproduction in breeding of Poa sp. Int.
Turfgrass Soc. Res. J. 9: 202-205.
REINERT, J. A. 1982. A review of host resistance in turf-
grasses to insects and acarines with emphasis on the
southern chinch bug, pp. 3-12 In H. D. Niemczyk and
B.G. Joiner [eds.], Advances in Turfgrass Entomolo-
gy. Hammer Graphics, Inc., Piqua, OH. 150 p.
REINERT, J. A., M. C. ENGELKE, AND J. C. READ. 2004a.
Host resistance to insects and mites, a review A
major IPM strategy in turfgrass culture. 1" Int. Soc.
Hort. Sci. Conf. Turfgrass Manage. Sci. Sports
Fields. Athens, Greece. Acta Hort. 661: 463-486.
REINERT, J. A., M. C. ENGELKE, J. C. READ, S. J. MA-
RANZ, AND B. R. WISEMAN. 1997. Susceptibility of
cool and warm season turfgrasses to fall armyworm,
Spodoptera frugiperda. Int. Turfgrass Soc. Res. J. 8:
1003-1011.
REINERT, J. A., J. C. READ, AND R. MEYER 2004b. Resis-
tance to fall armyworm (Spodoptera frugiperda)
among Kentucky bluegrass (Poa pratensis) cultivars.
1" Int. Soc. Hort. Sci. Conf. Turfgrass Manage. Sci.
Sports Fields, Athens, Greece. Acta Hort. 661: 525-
530.
REINERT, J. A., J. C. READ, M. C. ENGELKE, P. F. COL-
BAUGH, S. J. MARANZ, AND B. R. WISEMAN. 1999. Fall
armyworm, Spodoptera frugiperda, resistance in
turfgrass. Mededelingen, Faculteit Landbouwkundi-
ge en Toegepaste Biologische Wetenschappen. Proc.
50th Inter. Sym. Crop Protection, Gent, Belgium,
64(3a): 241-250.
SAS INSTITUTE. 2008. SAS system for Windows, release
9.1. SAS Institute, Cary, NC
WISEMAN, B. R., R. C. GUELDNER, AND R. E. LYNCH.
1982. Resistance in common centipedegrass to the
fall armyworm. J. Econ. Entomol. 75: 245-247.







Florida Entomologist 91(4)


December 2008


BIOLOGY OF EURYTOMA SIVINSKII, AN UNUSUAL EURYTOMID
(HYMENOPTERA) PARASITOID OF FRUIT FLY
(DIPTERA: TEPHRITIDAE) PUPAE

J. MENA-CORREA,1, J. SIVINSKI2, M. GATES3, R. RAMIREZ-ROMERO1 AND M. ALUJA1'4
'Instituto de Ecologia, A.C., Apartado Postal 63, 91000 Xalapa, Veracruz, M6xico

2Center for Medical, Agricultural and Veterinary Entomology,
USDA-ARS, P.O. Box 14565, Gainesville, FL 32604, USA

3Systematic Entomology Laboratory, PSI, ARS, USDA, c/o Smithsonian Institution,
National Museum of Natural History, PO Box 37012, Washington, DC 20013-7012, USA

4Corresponding author: martin.aluja@inecol.edu.mx

ABSTRACT

The recently described Mexican parasitic wasp Eurytoma sivinskii Gates and Grissell (Hy-
menoptera: Eurytomidae), attacks Anastrepha obliqua (Diptera: Tephritidae) pupae in the
soil. The life cycle (egg to adult) is completed in 23.1 ( 2.1) d (mean S.E.) at 27 2 C. Fe-
males were capable of superparasitism and laid 1-8 eggs per host (2.59 1.56, mean S.E.),
but invariably only 1 adult parasitoid emerged. Oviposition occurred primarily in the medial
and posterior portions of the host. Eurytoma sivinskii is ectoparasitic since 100% of the eggs
are laid within the internal cavity of the puparium and on the surface of the pupa of the host
fly. In no case were first and second instars parasitized. However, 1 third-instar out of 625
fly pupae exposed, yielded a single parasitoid per host. Eight-day-old pupae yielded the most
parasitoids although females laid eggs in 1-d- to 14-d-old pupae. There were no significant
differences in rates of parasitism among female E. sivinskii of different ages. Adults derived
from eggs laid in the posterior region developed more rapidly, but adult sex ratio and percent
of emergence were the same in both posterior and medially laid eggs. Regardless of oviposi-
tion location, adults were more likely to emerge through the middle of the puparium.

Key Words: Anastrepha, Eurytoma, Eurytomidae, Hymenoptera, natural history, parasitoid,
Tephritidae

RESUME

La avispa parasitoide Eurytoma sivinskii Gates y Grissell (Hymenoptera: Eurytomidae), re-
cientemente descrita en M6xico, ataca pupas de Anastrepha obliqua (Diptera: Tephritidae)
en el suelo. Este parasitoide complete su ciclo de vida (huevo a adulto) en 23.1 ( 2.1) d (me-
dia E.E.) a 27 2 C. Las hembras son capaces de superparasitar a sus hu6spedes, ovipo-
sitando 1-8 huevos por hu6sped (2.59 1.56, media + E.E.), aunque invariablemente solo
emerge 1 parasitoide adulto. La oviposici6n ocurre principalmente en las porciones media y
posterior del hu6sped. Eurytoma sivinskii es claramente ectoparasitico, ya que 100% de los
huevos fueron puestos dentro de la cavidad internal del pupario y sobre la superficie de la
pupa de la mosca hu6sped. En ningun caso hubo parasitismo del primer y segundo instar de
la larva. Sin embargo, de 1 larva de tercer instar expuesta a hembras de E. sivinskii (de un
total de 625 larvas expuestas), emergi6 1 parasitoide. De las pupas de 8 d de edad se obtu-
vieron la mayoria de los parasitoides, aunque las hembras pusieron huevos en pupas de 1 a
14 d de edad. No hubieron diferencias significativas en la tasa de parasitismo entire hembras
de E. sivinskii de diferentes edades. Los adults que provinieron de huevos puestos en la re-
gi6n posterior de la pupa se desarrollaron mas rdpidamente, pero tanto la proporci6n sexual
como el porcentaje de emergencia fueron similares entire los huevos colocados en las parties
posterior y media de la pupa. Independientemente de la localizaci6n de la oviposici6n, los
adults emergieron principalmente de la parte media del pupario.

Translation provided by the authors.


Eurytoma sivinskii Gates and Grissell, an from pupae of the West Indian fruit fly, Anas-
unusual eurytomid pupal parasitoid of Diptera, trepha obliqua (Macquart) (Diptera: Tephriti-
was recently discovered in the vicinity of Te- dae) (Gates & Grissell 2004). Most species of
jeria, Veracruz, Mexico, where it was recovered the large and widespread parasitoid genus Eu-







Mena-Correa et al.: Biology ofEurytoma sivinskii


rytoma attack gall-forming Cynipidae (Hy-
menoptera) and Diptera (Tephritidae and Ceci-
domyiidae) (DiGiulio 1997), and numerous
other arthropod taxa and plants. In this case,
instead of attacking hosts hidden within a plant
structure, E. sivinskii forages for hosts (i.e., pu-
pae) in and on the soil (Mena-Correa 2005).
Because ofE. sivinskii's peculiar foraging be-
havior for tephritid hosts of economic impor-
tance, its potential as a biological control agent
is under investigation (J. Mena-Correa, J. Siv-
inski, R. Ramirez-Romero, M. Gates & M. Aluja,
unpublished). Some basic parameters of the
parasitoid oviposition behavior and develop-
ment in pupae of the Mexican fruit fly Anas-
trepha ludens (Loew) are determined herein.
Type of parasitism (ecto- or endoparasitism),
number of emerged adults per host, duration of
immature stages, host stage attacked, parasit-
ism related to female age, and propensity to su-
perparasitize were studied. The locations of
eggs laid by the parasitoid on the host pupae, lo-
cation of adult parasitoid emergence through
the host puparium, and how these locations are
related to survival, developmental rate, compe-
tition among ovipositing females, and sex of the
resulting adult were analyzed.

MATERIALS AND METHODS

Insect Cultures and Experimental Conditions

This study was carried out at the Instituto de
Ecologia A.C., Xalapa, Veracruz, Mexico. Envi-
ronmental conditions were kept at 27 + 2C, 75 +
5% RH, with a 12:12 h photoperiod. The E. sivin-
skii colony was maintained on 2-d-old pupae
from anAnastrepha ludens colony kept for > 200
generations (Aluja et al. 2008). Pupae were
placed on the surface of a layer of unsterilized
clay soil and exposed to parasitoids for 6-8 d.
This soil was gathered from the area where E.
siuinski was originally discovered in Tejeria, Ve-
racruz. Adults of E. sivinskii were placed in
Plexiglas cages (30 x 30 x 30 cm) after emergence
and fed ad libitum with honey and water. To
avoid prior oviposition experience, parasitoids to
be used in experiments were not exposed to host
pupae.
Parasitoids were transferred from emer-
gence cages to experimental cages (23 x 23 x 23
cm) by capturing them in glass vials. The exper-
imental cages were prepared 24 h before the be-
ginning of tests to minimize insect stress. Host
pupae were always manipulated with flexible
forceps to avoid damage. After pupae were par-
asitized, they were placed in plastic cups (200
mL) containing vermiculite moistened with wa-
ter mixed with sodium benzoate at 2 g / L which
prevented fungal contamination. Cups were
covered with a lid.


Duration of Immature Stages

Development stages of E. sivinskii have been
described elsewhere (Gates et al. 2008). Recorded
images with Image Pro-Pl'hi software were ex-
amined to determine duration of each stage. All
specimens were monitored until d 23 when adult
females began to emerge as described by (Mena-
Correa 2005).
Specimens used for the determination of the
duration of immature stages stemmed from the
47-49th generations of our colony. To rear the par-
asitoids, we exposed 300 mL ofA. ludens pupae
(ca. 7000 pupae) to 3 E. sivinskii cohorts kept in
30 x 30 x 30 Plexiglas cages (200 females and 100
males per cage). Twenty-four h after exposure to
parasitoids, pupae were removed and placed in
500 mL plastic vials with humidified vermiculite
and covered with a lid. That same day (i.e., 24 h
after exposure to parasitism) and from then on ev-
ery 24 h over a 23-d period pupae were dissected
individually in search of immature stages of the
parasitoid. Twenty six eggs, 169 larvae (all stages
represented), 40 prepupae, and 127 pupae were
recovered. All specimens were placed in recently
prepared Carnoy fixing solution (60 mL absolute
alcohol, 30 mL chloroform and 10 mL acetic acid)
for 24 h. Subsequently, they were washed and pre-
served in 70% EtOH in a hermetic glass flask un-
til needed for evaluations. All dissections were
made in a physiological solution to avoid tissue
contraction (Martinez 2002).

Determination of the Host Stage Attacked

First-, second- and third-instars, as well as 3,
8, and 14-d-oldA. ludens pupae at different devel-
opmental stages were exposed to E. sivinskii
adults inside 200-mL plastic cups. Twenty-fiveA.
ludens larvae or pupae were presented to E. siu-
inskii cohorts of 10 females and 5 males (8-d-old)
for 5 h. Each treatment was replicated 5 times.
Rates of parasitism among treatments were
compared via a one-way analysis of variance (Zar
1998) with developmental stage as the indepen-
dent factor. Consecutively, we compared the rates
of parasitism in host pupae alone with a one-way
analysis of variance (Zar 1998) with pupal age as
the independent factor. Data were arc-sin /x-
transformed prior to analysis (Zar 1998).

Parasitism Related to Female Age

To obtain E. sivinskii adults of every age, 150
mL ofA. ludens pupae (3-to 5-d-old) were intro-
duced daily into colony cages for 24 h. After this
period, each batch of pupae was placed in a sepa-
rate cage and newly emerged E. sivinskii adults
were recovered every day and age-classified.
Then, we tested 1-to 27-d-old females to deter-
mine if there was an age effect on ability to para-







Florida Entomologist 91(4)


sitize pupae. Individuals (10 females and 5 males)
within each age class were exposed to twenty 3-d-
oldA. ludens pupae over a 15-d-period. Each age-
treatment was replicated 5 times, and the num-
ber of parasitoids recovered from each treatment
was recorded.
Rates of parasitism among treatments were
compared via a one-way analysis of variance (Zar
1998) with female age as the independent factor.
When significant differences were found, a
Tukey's test was performed (Zar 1998). Data were
arc-sin m x-transformed before analysis (Zar
1998).

Determination of Superparasitism

Different host pupal densities (i.e., 1, 5, and 10
pupae) were exposed for 5 h to adult parasitoid co-
horts of 10 females and 5 males (6-to 9-d-old). Af-
ter exposure, hosts were dissected with saline so-
lution and a stereo-microscope and the number of
eggs per host was recorded. Dissections were per-
formed either the same day or 24 h after parasi-
toid exposure. Each treatment had 5 replicates.
We analyzed with a two-way ANOVA test the
mean number of eggs per parasitized pupae, the
rate of parasitized pupae (attacked pupae / ex-
posed hosts), and the rate of pupae with more
than 1 parasitoid egg (pupae with more than 1
egg / attacked pupae), with host density and expo-
sure time as independent factors. The rates of
parasitized pupae and of pupae with more than 1
egg were arc-sin transformed prior to analysis
(Zar 1998). In addition, the randomness of egg
distributions in 5- and 10-pupae lots was deter-
mined by Chi-square tests, following the genera-
tion of expected egg/pupae frequencies through
Poisson distributions (Zar, 1998). The nature of
the distributions, random, dispersed or clumped,
were then identified by dividing the variances by
the means and comparing these ratios to a ran-
dom distribution where the ratio = 1.0 (Zar, 1998).

Locations of Eggs and Adult Emergence from the Pu-
parium

After determining that multiple eggs were de-
posited in a single host, we ran an additional ex-
periment to detect if the initial position of eggs in
the host body influenced the parasitoid emer-
gence, rate of parasitism, and sex ratios. Four-d-
oldA. ludens pupae were exposed to 2 E. sivinskii
cohorts (total of 50 and 150 six- to 9-d-old females
and males, respectively). Exposed host pupae
were recovered after 24 h and examined under sa-
line solution 24 h later to identify the number of
parasitoid eggs and their position in the host
body. Based on the number of parasitoid eggs and
their position, host pupae were classified and
then placed individually in plastic containers (di-
ameter: 4 cm; height: 2 cm) with humidified ver-


miculite and covered with a lid that allowed air-
flow. Pupae were observed daily for 30 d and the
number of parasitoids that emerged per host,
their sex and the site of emergence on the host
body (anterior, medium, or posterior) recorded.
The number of days it took adults to emerge
was compared by a two-way ANOVA, with the
number of eggs per host and the initial location of
the eggs in the pupae as independent factors.
Prior to this, data were mean-rank transformed.
We compared the rates of emergence among
emergence positions (i.e., location of emergence
hole), considering the initial position of eggs and
the number of eggs per host by chi-square tests.
When significant differences were found, multiple
two-by-two comparisons were performed with a
significant threshold level, which was corrected
according to the Dunn-Sidak method (Sokal &
Rholf 1995). We compared parasitoid sex-ratios
against a 1:1 ratio with a chi-square goodness-of-
fit test. In addition, for each set of experiments,
parasitoid sex-ratios were compared among pu-
pae with different numbers of eggs and egg posi-
tions in the pupae, based on a chi-square test (Zar
1998). Finally, a possible correlation between the
location of adult emergence (i.e., emergence hole),
egg location, and number of eggs was tested by an
r Spearman test (Zar, 1998).

RESULTS

Duration of Immature Instars and Stages

The life cycle (egg to adult) was completed in
23.1 ( 2.1) d (mean S.E.) at 27 2C. Minimum
and maximum durations for the egg stage were 0
(where 0 < 24 h) and 4 d. The larval stage lasted
between 9 and 18 d. Minimum and maximum du-
rations for the prepupal stage were 0 and 13 d, re-
spectively, and those for the pupal stage were 8
and 14 d.
Minimum and maximum periods for larval in-
stars were 1 and 4 d (first instar), 2 and 7 d (sec-
ond and third instars), 0 and 8 d (fourth instar)
and 4 and 13 d (fifth instar), respectively. The
minimum and maximum periods of the pupal in-
stars were 0 and 9 d (first instar), 8 and 14 d (sec-
ond instar), 0 and 6 d (third instar), 4 and 7 d
(fourth instar) and 0 and 3 d (fifth instar). Male
emergence began on the 20th d following parasi-
toid exposure. Females started to emerge 23 d af-
ter exposure.

Host Stage Attacked

Parasitism of E. sivinskii in pupae was higher
than in larvae (F = 839.31; df= 1;P < 0.001). In no
case were first and second instar larvae parasit-
ized. However, 1 third-instar larva out of 625 ex-
posed yielded a single parasitoid. When rates of
parasitism at different pupal ages were com-


December 2008







Mena-Correa et al.: Biology ofEurytoma sivinskii


pared, significant differences were observed (F =
24.43; df= 2;P < 0.001). Eight-d-old pupae exhib-
ited the highest rate of parasitism (98.1 4)
(Fig. 1).

Parasitism Related to Female Age

There were no differences in rates of parasit-
ism by female E. sivinskii of different ages (F =
1.284; df= 26; P = 0.181) (Fig. 2).

Type of Parasitism

Eurytoma sivinskii was clearly ectoparasitic
since 100% of the eggs were laid within the inter-
nal cavity of the puparium and on the surface of
the pupa.

Determination of Superparasitism

Eurytoma sivinskii will lay more than 1 egg
per host. In the first experiment, of the total par-
asitized pupae, 25.42% contained 1 egg, 32.20%
had 2, 16.94% had 3, 13.56% had 4, 6.77% had 5,
3.39% had 6, and 1.70% had 8 eggs. Host density
did not affect the number of eggs per pupae (F =
0.277; df = 2; P = 0.763), the rates of parasitism (F
= 3.062; df = 2; P = 0.084), or the rates of pupae
with more of 1 egg (F = 0.205; df = 2; P = 0.817)
(Table 1). The numbers of eggs / pupae were not
randomly distributed in either the 5 or 10 pupae
lots (x2 (5)= 15.9, df= 2, P < 0.005; x2 (10)= 12.6, df
= 4, P < 0.01). These non-random distributions
were due to clumping as evidenced by variance/
mean rations of 1.9 and 1.6, respectively. Eggs
were oviposited on the posterior and middle re-


T
-


-I


First Second Thrd Three Elghl Fouween
Larval Insar Age of Pupae (Days)
Fig. 1. Parasitism by Eurytoma sivinskii related to
Anastrepha ludens immature stage. Rates of parasitism
were compared among the different host stages. In no
case were first and second-instar larvae parasitized, but
notably one third-instar larva out of 625 exposed yielded
a single parasitoid. Significant differences were observed
when comparing rates of parasitism among pupal ages (F
= 24.43; df= 2;P < 0.001). Eight-day old pupae exhibited
the highest rate of parasitism (98.1 4).


100

so

w80



20


1-3 4-6 7-9 10-12 13-15 16-18 19-21 22-24 25-27
Female age (days)
Fig. 2. Parasitism of Anastrepha ludens by female
Eurytoma sivinskii related to female age. No significant
differences in rates of parasitism were observed (F =
1.284; df = 26; P = 0.181).


gions of the host. No oviposition was ever detected
in the anterior region.

Locations of Eggs and Adult Emergence through the
Puparium

In these experiments, the maximum number of
eggs per host was 5. Thus, the following combina-
tions of egg number vs. location in host were sta-
tistically compared: (a) 1 egg-posterior, (b) 2 eggs-
posterior, (c) 3 eggs-posterior, (d) 4 eggs-posterior,
(e) 5 eggs-posterior, (f) 1 egg-middle, (g) 2 eggs-
middle, (h) 3 eggs-middle, (i) 4 eggs-middle and (j)
5 eggs-middle.
The number of eggs (F = 6.468; df = 4; P <
0.001) and their location (F = 3.958; df = 1; P =
0.048) had a significant influence of on the time of
adult emergence. In pupae containing 5 eggs,
adult emergence was significantly delayed when
compared to pupae containing 1, 2, or 3 eggs. In
addition, eggs oviposited in the middle of the pu-
pae resulted in more rapid development than eggs
in the posterior (Table 2). Peak female and male
eclosion were observed at 23.9 (+1.8) and 21.4
(1.6) d, respectively. Adults were significantly
more likely to emerge from the middle of the host
puparium, independent of the initial position of
the eggs (i.e., medium or posterior) or the number
of eggs (i.e., 1, 2, or 5 eggs).
When sex-ratios were compared against a 1:1
distribution, we observed a female-bias when host
pupae contained 2, 4, and 5 eggs (X2 = 4.00, 6.760,
and 18.614; df = 1; P < 0.05) but not when host pu-
pae contained 1 and 3 eggs (X2 = 1.666 and 0.104; df
= 1; P > 0.05). Regarding the initial position of ovi-
posited eggs, sex-ratio was female-biased in both
middle (X2 = 4.312, df= 1;P < 0.05) and posterior (X2
= 14.720, df = 1; P < 0.001) positions. No relation-
ship was observed between the emergence location
and the number of eggs (R = 0.047;P = 0.558) or the
location of eggs (R = 0.005; P = 0.948).







Florida Entomologist 91(4)


TABLE 1. MEAN NUMBER ( S.E.) OF OVIPOSITED EGGS PER PUPAE, RATES OF PARASITISM, AND OF PUPAE CONTAINING
MORE THAN 1 EGG AFTER EXPOSURE OF DIFFERENT DENSITIES OF ANASTREPHA LUDENS PUPAE TO EURY-
TOMA SIVINSKII FEMALES.

Mean number Mean parasitism Mean number of pupae
Host density of eggs per pupaea rate (%) with more of one egg (%)

1 2.20 1.3 100 0.0 60.0 54.8
5 1.96 1.5 60.0 6.9 56.0 51.8
10 2.54 0.9 78.0 16.4 77.8 15.7

aNon significant differences (F=0.277; 2 df; P=0.763).
bNon significant differences (F =3.062; 2 df; P =0.084).
cNon significant differences (F =0.205; 2 df; P =0.817).


DISCUSSION

Eurytoma sivinskii is a solitary ectoparasitoid
of fruit fly pupae, particularly those midway in
development. Its females could superparasitize,
under laboratory conditions, by ovipositing up to
8 eggs per host. Adults emerged in a shorter pe-
riod of time when hosts originally contained a sin-
gle parasitoid egg, although there was no lower
likelihood of adult emergence when multiple eggs
were present. However, longer development could
inflict other costs such as increased exposure to
pathogens, predators, or hyperparasitoids (Ben-
rey & Denno 1997).
Eggs were commonly laid in the middle and
posterior region of the host, but not in the ante-
rior third. Adult parasitoids derived from eggs
laid in the posterior region developed more rap-
idly, but adult sex ratio and percent of emergence
were the same in both posterior and medially laid
eggs. Oviposition sites might be influenced both
by local differences in the resistance offered by
the puparium and by advantageous placement of
the egg on the pupa itself. Spalangia endius
Walker (Pteromalidae) females are as likely to
drill in the anterior or posterior portions of young


Musca domestic L. pupae, but as the puparium
toughens with age, parasitoids are more likely to
drill in the posterior, where the vulnerable anal
spiracles are located, and these drilling are more
likely to be successful (King 2001). For S. endius
larvae, there appears to be no developmental ad-
vantage to an initially posterior or anterior place-
ment, which may be due to the capacity of the
hatchlings to wander to optimal feeding sites
(King 2001). However, initial location does seem
to matter in another pupal parasitoid of Diptera,
Nasonia vitripenis Walker (Pteromalidae) (Rivers
& Yoder 1996), where oviposition in the posterior
portion of Sarcophaga bullata Parker (Sarcoph-
agidae) puparia results in increased larval oxy-
gen consumption, weight, and lipid content. Para-
sitized S. bullata have elevated lipid levels and
the effect on the parasitoid was most pronounced
when eggs were laid in the posterior portion (Riv-
ers & Yoder 1996). At the same time, envemona-
tion in the anterior parts of host leads to more
rapid death and greater developmental disrup-
tion (Rivers & Denlinger 1994), which might also
have nutritional consequences for the parasitoid.
Unlike E. sivinskii, oviposition position in N. vit-
ripenis has no effect on developmental rate.


TABLE 2. LIFE HISTORY PARAMETERS OF EURYTOMA SIVINSKII DEVELOPED IN ANASTREPHA LUDENS PUPAE CORRE-
LATED WITH THE INITIAL POSITION OF OVIPOSITED EGGS AND THE NUMBER OF OVIPOSITED EGGS.

Proportion (%) of adult emergence
Mean ( S.E.) in relation to position in pupae
Initial position time to adult Total adult Sex-ratio (%
of eggs emergence Anterior Medium Posterior emergence (%) males)

Medium 23.52.6 a 25.4 a 49.3 b 25.4 a 41.9 a 33.3 a
Posterior 22.71.6 b 14.0 a 65.6 b 20.4 a 58.1 b 30.1 a
No of eggs
1 23.41.7 a 8.6 b 74.3 a 17.1 b 21.9 a 42.9 a
2 22.61.7 a 16.7 b 75.0 a 8.3 b 22.5 a 33.3 ab
3 22.82.0 a 28.9 a 28.9 a 42.1 a 23.8 a 41.2 a
4 23.62.4 ab 24.0 a 52.0 a 24.0 a 15.6 a 24.0 ab
5 24.42.2 b 15.4 b 65.4 a 19.2 b 16.3 a 7.7 bc

Different letters for each parameter within treatment indicate significant differences at (P < 0.05).


December 2008







Mena-Correa et al.: Biology ofEurytoma sivinskii


The exit sites of adult E. sivinskii are concen-
trated in the middle of the A. ludens puparium.
This contrasts strongly with the exit sites ofS. en-
dius which are mostly in the anterior end of the
host (King 2001), where the puparium is likely to
have weak cleavage lines that would have allowed
the fly host itself to emerge. The reasons that E.
siuinskii leaves from the middle are not entirely
clear, but other parasitoids concentrate their pen-
etration activities on the middle of the tephritid
puparium. In the fruit fly specialist Coptera hay-
wardi (Ogloblin) (Diapriidae), nearly all oviposi-
tions are in the middle regions ofAnastrepha sus-
pensa (Loew) puparia, while the generalist chal-
cid, Dihrinus himalayanus Westwood, frequently
oviposits in both the middle and the posterior
(Sivinski et al. 1998). Perhaps the relatively
strong preference for the middle by the tephritid
specialist reflects a structural weakness in the
Anastrepha puparial design that is also exploited
by emerging adults.

ACKNOWLEDGMENTS

Larissa Guill6n, Andrea Birke, and Guadalupe
C6rdova (Instituto de Ecologia, A.C., Xalapa, Ver-
acruz, Mexico (INECOL) helped organize experi-
ments and provided critical advice along the way. We
gratefully acknowledge the technical help of Armando
Torres Anaya, Cecilia Martinez Arcos, Jovita Mar-
tinez Tlapa, and Sandi M6ndez Trejo (INECOL) who
helped rear the parasitoids and their hosts. We grate-
fully acknowledge the useful suggestions for improve-
ment by James L. Nation, Alberto Anzures-Dadda
and 2 anonymous referees that helped us produce a
better quality paper. Alberto Anzures-Dadda also pro-
vided invaluable help while preparing the revised
version of this paper. Financial support for this study
was principally furnished by the Mexican Campaia
Nacional contra las Moscas de la Fruta (Secretaria de
Agriculture, Ganaderia, Desarrollo Rural y Pesca -
Instituto Interamericano de Cooperaci6n para la Ag-
ricultura (SAGARPA-IICA). Additional funds were
provided by the United States Department of Agricul-
ture Agricultural Research Service (USDA-ARS)
and INECOL. We acknowledge partial financial sup-
port from the Comisi6n Nacional para el Cono-
cimiento y Uso de la Biodiversidad (CONABIO;
Proyect No. FB325/H296/96) and the Consejo Nacio-
nal de Ciencia y Tecnologia (CONACyT)/Fondo del
Sistema de Investigaci6n del Golfo de M6xico
(SIGOLFO, Project No. SIG96001/ 96-01-003-V). In-
formation reported here forms part of the undergrad-
uate thesis of JMC (Universidad Nacional Aut6noma
de M6xico [UNAM]), directed by MA.


REFERENCES CITED

ALUJA, M., J. SIVINSKI, S. OVRUSKI, L. GUILLEN, J. CAN-
CINO, M. LOPEZ, A. TORRES-ANAYA, G. GALLEGOS-
CHAN, AND L. RUIZ. 2008. Colonization and domesti-
cation of seven species of native new world hy-
menopterous larval-prepupal and pupal fruit fly
(Diptera: Tephritidae) parasitoids. Biol. Sci. Tech.
(In Press).
BENREY, B., AND R. F. DENNO. 1997. The slow-growth-
high-mortality hypothesis: a test using the cabbage
butterfly. Ecology 78: 987-999.
DIGUILIO, J. 1997. Eurytomidae, pp. 477-495 In G. Gib-
son, J. Huber, and J. Wooley [eds], Annotated Key to
the Genera of Nearctic Chalcidoidea (Hymenoptera).
NRC Research Press, Ottawa, CA.
GATES, M. W., AND E. E. GRISSELL. 2004. A new species
ofEurytoma (Hymenoptera: Eurytomidae) attacking
the mango fruit fly, Anastrepha obliqua (Macquart)
(Diptera: Tephritidae), pp. 147-159 In K. Rajmoha-
na, K. Sudheer, P. Girish-Kumar, and S. Santhosh
[eds.], Perspectives on Biosystematics and Biodiver-
sity. Harvest Ledia Services, Calicut, India.
GATES, M., J. MENA-CORREA, J. SIVINSKI, R. RAMIREZ-
ROMERO, G. CORDOVA-GARCIA, AND M. ALUJA. 2008.
Description of the immature stages ofEurytoma siu-
inskii Gates and Grissell (Hymenoptera: Eurytomi-
dae), an ectoparasitoid of Anastrepha (Diptera: Te-
phritidae) pupae. Entomol. News (In Press).
KING, B. 2001. Parasitization site on the host of the par-
asitoid wasp Spalangia endius (Hymenoptera: Pter-
omalidae). Environ. Entomol. 30: 346-349.
MARTINEZ, I. 2002. Metodologias. T6cnicas basicas de
anatomia microsc6pica y de morfometria para estu-
diar los insects. Bol. Soc. Entomol. Mexicana 30:
187-195.
MENA-CORREA, J. 2005. Biologia Basica de Eurytoma
siuinskii (Hymenoptera: Chalcidoidea: Eurytomi-
dae). Tesis, Universidad Nacional Aut6noma de
M6xico. M6xico, D.F., M6xico.
RIVERS, D., AND D. DENLINGER 1994. Developmental
fate of the flesh fly, Sarcophaga bullata envemonat-
ed by the pupal ectoparasitoid, Nasonia vitripennis.
J. Insect Physiol. 40: 121-127.
RIVERS, D., AND J. YODER 1996. Site-specific effects of
parasitism on water balance and lipid content of the
parasitic wasp Nasonia vitripennis (Hymenoptera:
Pteromalidae). Europ. J. Entomol. 93: 75-82.
SIVINSKI, J., K. VULINEC, E. MENEZES, AND M. ALUJA.
1998. The bionomics of Coptera haywardi (Ogloblin)
(Hymenoptera: Diapriidae) and other pupal parasi-
toids of tephritid fruit flies (Diptera). Biol. Control.
11: 193-202.
SOKAL, R. R., AND F. J. ROHLF. 1995. Biometry: The
Principles and Practice of Statistics in Biological Re-
search. W. H. Freeman and Co. New York.
ZAR, J. H. 1998. Biostatistical Analysis. Pearson Educa-
tion. New Jersey.







Florida Entomologist 91(4)


December 2008


SEISMIC BEHAVIORS OF A LEAFMINER, ANTISPILA NYSAEFOLIELLA
(LEPIDOPTERA: HELIOZELIDAE)


CANDACE LOW
Department of Ecology, Evolution, and Marine Biology, University of California,
Santa Barbara, California 93106 USA
E-mail: c low@lifesci.ucsb.edu


ABSTRACT

This paper presents the first descriptions of 2 distinct behaviors of the Tupelo leafminer,
Antispila nysaefoliella Clemens (Lepidoptera: Heliozelidae). Through the use of special-
ized morphological structures, the leafminers use these behaviors to generate substrate-
borne vibrations that can be emitted as audible sounds to humans. Scanning electron mi-
crographs of these structures are presented. In 1 behavior, the larvae "tick" their abdo-
mens back and forth rhythmically; and in the other, they "rattle" their abdomens in short
rapid pulses. These are named for the sounds produced. Previous studies have shown
that parasitoids emit substrate vibrations while walking and probing during their
search for hosts from the leaf surface. The experimental results show that the larvae of
A. nysaefoliella are sensitive to vibrational stimuli in general by "wriggling to all fre-
quencies", but "ticked" only to some frequencies which are characteristic of parasitoid
probing behavior, and "rattled" rarely.

Supplementary material (videos) online at http://www.fcla.edu/FlaEnt/fe914.htm

Key Words: anti-parasitoid, defense, host-parasitoid, plant-insect, signals, sound playback,
vibrations


RESUME

Se described dos comportamientos distintos observados en Antispila nysaefoliella Cle-
mens (Lepidoptera: Heliozelidae), insecto minador de hoja especializado en arboles de
goma negra Nyssa sylvatica Marsh (Cornaceae). Ambos comportamientos son realizados
utilizando estructuras morfol6gicas especializadas que general vibraciones en la super-
ficie de las hojas. En el primer tipo de vibraci6n, las larvas del tupelo utilizan movimien-
tos "pendulares" de sus abd6menes hacia atras y adelante de forma ritmica. En la
segunda vibraci6n, los movimientos son producidos por una series rdpida de sonidos rui-
dosos cortos originados en el abdomen, los que son denominados movimientos de "casca-
beleo". Se presentan microfotografias obtenidas por escaneo de electrones de las
estructuras especializadas involucradas en la creaci6n de vibraciones. Los resultados de-
mostraron que las larvas de tupelo son sensitivas a todo tipo de vibraciones, pero respon-
den de manera diferente. Las larvas utilizan "serpenteos" como respuesta general,
movimientos "pendulares" solo a determinadas frecuencias, y raramente utilizan "casca-
beleos".


Translation provided by the author.


The use of substrate-borne vibrations for com-
munication is prevalent among many insects
(Roces & Holldobler 1996; Cummings et al. 1999;
Cokl & Doberlet 2003; Virant-Doberlet & Cokl
2004; Cocroft 2005; Cocroft & Rodriguez 2005;
Casas & Magal 2006; Castellanos & Barbosa
2006; Hoch et al. 2006; Casas et al. 2007). For in-
sects living on plants, vibrations are especially ef-
fective for signal transmission because signalers
typically have small body sizes and use short-
range communication (within individual plants)
(Bennet-Clark 1998; Cokl & Doberlet 2003; Cokl


et al. 2004; Cocroft et al. 2006). The majority of
studies on vibrational communication, thus far,
have been aimed at understanding its utility in
mate selection (Cokl et al. 2004; Rodriguez et al.
2004; Moraes et al. 2005; Virant-Doberlet & Ze-
zlina 2007) or complex social interactions (Roces
& Holldobler 1996; Cocroft 1999, 2005; Cum-
mings et al. 1999; Cocroft & Rodriguez 2005).
However, researchers are beginning to uncover
the role of vibrations in food-finding and mediat-
ing interactions between prey and predators (or
hosts and parasitoids) (Bacher et al. 1996; Mey-







Low: Seismic Behaviors of a Leafminer


h6fer et al. 1997; Djemai et al. 2004; Evans et al.
2005; Castellanos & Barbosa 2006). Some preda-
tory wasps that search for concealed prey have
evolved specialized traits, such as modified anten-
nae to tap on substrates to detect prey position
through vibrational sounding (Meyh6fer & Casas
1999; Broad & Quicke 2000; Djemai et al. 2004),
and likewise, their prey have evolved sensory ap-
paratus to detect the presence of their potential
attackers and respond with defensive or evasive
behaviors (Connor & Cargain 1994; Bacher et al.
1996; Bacher et al. 1997; Djemai et al. 2000, 2001;
Castellanos & Barbosa 2006).
The adaptations of leaf-mining larvae to detect
and evade attack by their parasitoids are likely to
be under intense selection. Those parasitoids that
hunt for concealed prey have been found to re-
lease vibrations that provide information for the
host hiding beneath the leaf surface (See Video 1),
which subsequently can trigger host evasive re-
sponses (Meyh6fer et al. 1994; Bacher et al. 1996;
Meyh6fer & Casas 1999). In this paper, I provide
the first descriptions of vibration-producing be-
haviors and associated morphology of a leaf-min-
ing moth, Antispila nysaefoliella Clemens (Lepi-
doptera: Heliozelidae), which specializes on black
gum, Nyssa sylvatica Marsh (Cornaceae).

MATERIALS AND METHODS

Natural History and Observations

In general, the shape and design of a leaf mine
depends on both the feeding morphology and be-
havior of the species, as well as, the leaf morphol-
ogy of the host plant (Needham et al. 1928; Her-
ing 1951). The mines ofA. nysaefoliella always be-
gin as solitary mines (1 larva per mine) beside a
leaf vein and expand radially to form blotches
that become more oblong-shaped at later instars
(Fig. 1). By the time of pupation, mines are 2.48 +
0.56 cm2in size, and larvae are 3.18 0.07 mm in
length and weigh up to 0.472 0.22 mg (dry
weight) (mean + 1 SD, n = 30). For 7 consecutive
field seasons (2001-07), I have observed the be-
haviors of the larvae of A. nysaefoliella in my
study population, which is located within a mixed
deciduous forest in Clarke County, VA (390 00.85'
N, 780 03.88' W). I have observed the same behav-
iors in larvae upon random encounters in loca-
tions throughout northern Virginia and Washing-
ton, D.C. where there are black gum trees.
During the peak of larval activity in 2001 (25
Aug to 3 Sep) at my study site in Clark Co., I set
a mini-digital video camera on a tripod in front of
leaves (with larvae) for 1-h intervals throughout
the day (0900-1800 h) to record larval behaviors
and other activity at the leaf (e.g., visitations by
parasitoids). Because A. nysaefoliella feed on both
upper and lower tissue layers, mines are translu-
cent and larvae can be observed easily inside


Fig. 1. Larvae of Antispila nysaefoliella in their
mines on black gum, Nyssa sylvatica, pictured here
from the lower leaf surface. These 6 mines are the result
of approximately 1-6 d (smallest to largest) of feeding.
Note: The dark sclerotized band (dorsal side) is visible
because larvae feed dorsal-side down. Scale (1 mm) is in
top left corner.


their mines, especially when sunlight illuminates
the background (Fig. 1 and Videos 2-4, online). In
total, I recorded 20 h of observation on 20 leaves
with variable number of larvae per leaf (total =
144 larvae). All activity and behaviors were de-
scribed and scored on computer monitors, based
on frame-by-frame enhancements whenever nec-
essary to score rapid movements and activity. The
resolution of the video images did not allow for
identification of visiting insects. However, a best
guess at whether the insect might be a searching
parasitic wasp could be made based on the behav-
ioral (search) pattern of the insect. See Video 2 for
an example of a visit by a potential wasp.

Recording Vibrations

In a subsequent field season in 2004, leaves
with mines (and larvae) were collected from the
field and brought to the lab of Prof R. B. Cocroft
at the University of Missouri, Columbia. Record-
ings of the vibrations caused by A. nysaefoliella
larvae were taken under controlled conditions
(enclosed room, ~23 C) by a laser-vibrometer
(Polytec Compact Laser Vibrometer CLV 1000
with a CLV M030 decoder module, at a sensitivity
of 25 mm s-' V-; Polytec Inc., Auburn, MA). Tiny
(~1-2 mm square) pieces of reflective tape were
placed roughly in the center of the leaf on the leaf-
midrib, and the width of the laser beam was ~0.5
mm. Signals were high-pass filtered at 80 Hz by a
Krohn-Hite 3202 filter and low-pass filtered at
5000 Hz (44100 Hz sampling rate, 16 bit resolu-
tion). Data output was recorded with a Dell desk-







Florida Entomologist 91(4)


top computer with a national Instruments acqui-
sition board (44, 100 Hz sampling rate, 16-bit res-
olution) and a custom program written in Lab-
view (v. 6.0; national Instruments, Austin, TX).
Files were then exported as .wav files on a Macin-
tosh G3 computer with OS 9.2.2.

Scanning Electron Microscopy

Larvae in late-instars were fixed in glutaralde-
hyde and post-fixed in osmium tetroxide for mor-
phological observations. Specimens were dehy-
drated in acetone of varying concentrations (i.e.,
50%, 70%, 80%, 90%, 100%) prior to critical point
drying. They were then glued to mounting stubs
in various orientations and sputter coated with
gold.

RESULTS

Wriggles, Ticks, and Rattles

Four different behaviors were identified from
the field observations and video recordings. These
are "wriggling without" and "wriggling with" dis-
placement (i.e., where the body travels a measur-
able distance within the mine), "ticking" (rhyth-
mic movement of abdomens back and forth like a
clock pendulum), and "rattling" (rapid pulse of ab-
domen like the rattle of a rattlesnake). Ticks are
slow and rhythmic (Video 3, online), and rattles
are very rapid and occur in short bursts that often
punctuate bouts of ticking (Video 4, online). All of
these described behaviors are distinct from gen-
eral feeding activity where, during feeding, larvae
remain motionless and with their heads pointed
toward the green leaf tissue at the mine periph-
ery. When they wriggle, tick, or rattle, they typi-
cally pull away from the edge of the mine and
move towards the center.
The average time that an individual larva
spent wriggling, ticking, or rattling during the 60-
min observation was, respectively, 3.00 0.45,
1.10 + 0.25, and 0.01 0.00 min on leaves without
any insect (i.e., potential parasitoid) visitations,
and 2.28 0.27, 1.19 + 0.18, and 0.02 0.01 min
(mean 1 SE) on leaves with an insect visitation.
Flying insects appeared in 11 of the 20 observa-
tions; and there were typically 1 (n = 7) or 2 insect
visits (n = 4) per 1-h observational period for those
11 leaves that did receive a visit. The average
time that an insect stayed on a leaf was 4.8 0.3
min (1 SD). Multivariate analysis of variance of
the time spent by each larva performing each be-
havior (wriggle, tick, rattle) in the presence or ab-
sence of an insect visitation showed that an indi-
vidual spent a significantly greater amount of
time ticking when there was an insect visitation
than when there was not (F1,113 = 5.182, P = 0.025).
However, there were no effects on the amount of
time spent wriggling (F,,,3 = 0.336, P = 0.563) or


rattling (F, 113 = 2.245, P = 0.137). Leaf, group size,
and the time that a visiting insect stayed on a leaf
were entered as covariates.
The tick and rattle behaviors are named for
the sounds that they emit, and are distinct in
their spectral qualities (Fig. 2).Antispila nysaefo-
liella larvae appear to be able to create substrate-
borne vibrations with specialized sclerotized mor-
phological structures which line the dorsal side of
the abdominal segments and occur as points on
the last caudal segment and on the tip (Figs. 1, 3).
All such morphology and sclerotization is lost at
pupation. The mine epidermis may be acting as a
tympanic membrane, and hence, may be trans-
mitting the vibrations that are produced by the
larval movements into sounds that can be clearly
audible to the human ear (even when standing 2
m below the canopy). Larvae that have been re-
moved from their mines do not generate these
sounds, despite their ticking and rattling motions
in response to being removed from their mines.

DISCUSSION

To the best of my knowledge, no other leaf-min-
ing species has been reported to exhibit the tick-
ing and rattling behaviors or the associated mor-
phological structures that I have observed inAn-
tispila nysaefoliella. However, the use of vibra-
tions, acoustic signals, and body-waving
behaviors in defensive or aggressive interactions
has been reported for the larvae of other Lepi-
doptera (Yack et al. 2001; Fletcher et al. 2006;
Brown et al. 2007). Support also comes from ob-
servations in other insect taxa, where the vibra-
tions caused by the knocking and scraping of
gasters on substrate by several species of ants
(Formicidae: Dolicherinae) and the head-drum-
ming of the damp-wood termite, Zootermopsis ne-
vadensis (Isoptera: Termopsidae) appear to func-


2 4 a
Frequency (kHz)


10 12


Fig. 2. The fast Fourier transforms of the frequency
spectra of the tick and rattle signals.


December 2008







Low: Seismic Behaviors of a Leafminer


Fig. 3. Scanning electron micrographs and 100X images of specialized structures for producing substrate vibra-
tions. Top panels: (A) Interdigitating sclerotized ridges along dorsal surface. Bottom panels: (B) Sclerotized
pointed bumps and sclerotized caudal tip. Note also the sensory hairs (h) that outline the body. Scale is provided in
the lower right of each SEM, and distance between tick marks represents 200 ym.


tion as alarm calls (Kirchner et al. 1994; Rohe &
Rupprecht 2001). The results presented here sug-
gest that the ticking and rattling behaviors in A.
nysaefoliella are important for generating vibra-
tions, which has a well-documented adaptive role
in mediating insect interactions (Meyh6fer et al.
1997; Cokl & Doberlet 2003; Djemai et al. 2004;
Cocroft & Rodriguez 2005; Drosopoulos & Clar-
idge 2006).
Several lines of evidence support the hypothe-
sis that vibration production in A. nysaefoliella
operates as a defense against parasitoids. First,
parasitism is the most important mortality source
of leaf-mining insects (Hawkins et al. 1997; Low
2008), which suggests that selection by parasi-
toids is intense and may drive the evolution of de-
fense mechanisms in leafminers that match the
mode(s) of attack of their enemies (i.e., vibra-
tions). Second, because parasitoids of leafminers
use vibrations to search for hosts from the leaf
surface after detecting the mines from flight, gen-
erating seismic waves (substrate-borne vibra-


tions) can be an effective tactic for disrupting or
increasing the costs of the search process of para-
sitic wasps (Djemai et al. 2004). Last, the loss of
the specialized morphology associated with vibra-
tion production at pupation suggests a function
specialized for the larval stage (a time during
which they are most vulnerable to parasitism).
Although this study supports the importance
of vibrations in A. nysaefoliella, more work is
clearly needed to fully understand the context(s)
and functions) of their vibration-generating be-
haviors. Future studies will require sophisticated
equipment and more experimentation to test al-
ternative hypotheses relating to the adaptive
value of transmitting substrate vibrations and
whether the behaviors are functioning as direct
(individual) evasive responses, conspecific warn-
ing signals, or honest signals that may cause par-
asitoids to give up their search more quickly
(Maynard-Smith & Harper 2004). Because most
leaf-mining species are faced with similar con-
straints and natural enemies, there may be other











leaf-mining species with similar behaviors and
morphologies that have yet to be discovered or
recognized. This would be an exciting area of re-
search for future studies.

ACKNOWLEDGMENTS

I am especially grateful to Ed Connor for introducing
me to the Tupelo Leafminer and for first noticing its
unique behaviors. Al Uy provided friendship, help, and
support throughout this project. I thank Rex Cocroft for
providing lab space, equipment, and his expertise, along
with Gabe McNett and Gregg Tully for help on vibra-
tions. Greg Lum and Rick Harris provided help with the
SEM images, Don Davis provided his expertise of micro-
lepidopterans, and Jorge Luis Hurtado-Gonzales trans-
lated the abstract into Spanish. Al Uy, John Endler,
Roger Nisbet, Rex Cocroft, and 2 anonymous reviewers
provided helpful comments on the manuscript. The Pro-
fessor Hering Memorial Research Fund (British Ento-
mological and Natural History Society) and Blandy
Experimental Farm (University of Virginia) provided
funds and logistical support. Red Gate Farms permitted
use of their property for this study.

REFERENCES CITED

BACHER, S., J. CASAS, AND S. DORN. 1996. Parasitoid vi-
brations as potential releasing stimulus of evasive
behaviour in a leafminer. Physiol. Entomol. 21: 33-
43.
BACHER, S., J. CASAS, F. WACKERS, AND S. DORN. 1997.
Substrate vibrations elicit defensive behaviour in
leafminer pupae. J. Insect Physiol. 43: 945-952.
BENNET-CLARK, H. C. 1998. Size and scale effects as
constraints in insect sound communication. Phil.
Trans. Roy. Soc. London Series B-Biol. Sci. 353: 407-
419.
BROAD, G. R., AND D. L. J. QUICKE. 2000. The adaptive
significance of host location by vibrational sounding
in parasitoid wasps. Proc. Roy. Soc. London Series B-
Biol. Sci. 267: 2403-2409.
BROWN, S. G., G. H. BOETTNER, AND J. E. YACK. 2007.
Clicking caterpillars: acoustic aposematism in An-
theraea polyphemus and other Bombycoidea. J. Exp.
Biol. 210: 993-1005.
CASAS, J., AND C. MAGAL. 2006. Mutual eavesdropping
through vibrations in a host-paraitoid interaction:
from plant biomechanics to behavioral ecology, pp.
263-271 In S. Drosopoulos and M. F. Claridge [eds.],
Insect Sounds and Communication: Physiology, Be-
haviour, Ecology and Evolution. CRC Press, Boca
Raton.
CASAS, J., C. MAGAL, AND J. SUEUR 2007. Dispersive
and non-dispersive waves through plants: implica-
tions for arthropod vibratory communication. Proc.
Roy Soc. B-Biol. Sci. 274: 1087-1092.
CASTELLANOS, I., AND P. BARBOSA. 2006. Evaluation of
predation risk by a caterpillar using substrate-borne
vibrations. Animal Behaviour 72: 461-469.
COCROFT, R. B. 1999. Parent-offspring communication
in response to predators in a subsocial treehopper
(Hemiptera: Membracidae: Umbonia crassicornis).
Ethology 105: 553-568.
COCROFT, R. B. 2005. Vibrational communication facili-
tates cooperative foraging in a phloem-feeding in-
sect. Proc. Roy. Soc. B-Biol. Sci. 272: 1023-1029.


December 2008


COCROFT, R. B., AND R. L. RODRIGUEZ. 2005. The behav-
ioral ecology of insect vibrational communication.
Biosci. 55: 323-334.
COCROFT, R. B., H. J. SHUGART, K. T. KONRAD, AND
K. TIBBS. 2006. Variation in plant substrates and its
consequences for insect vibrational communication.
Ethology 112: 779-789.
COKL, A., AND M. V. DOBERLET. 2003. Communication
with substrate-borne signals in small plant-dwelling
insects. Annu. Rev. Entomol. 48: 29-50.
COKL, A., J. PRESERN, M. VIRANT-DOBERLET, G. J. BAG-
WELL, AND J. G. MILLAR. 2004. Vibratory signals of
the harlequin bug and their transmission through
plants. Physiol. Entomol. 29: 372-380.
CONNOR, E. F., AND M. J. CARGAIN. 1994. Density-relat-
ed foraging behaviour in Closterocerus tricinctus, a
parasitoid of the leaf-mining moth, Cameraria ha-
madryadella. Ecol. Entomol. 19: 327-334.
CUMMINGS, D. L. D., G. J. GAMBOA, AND B. J. HARDING.
1999. Lateral vibrations by social wasps signal lar-
vae to withhold salivary secretions (Polistes fusca-
tus, Hymenoptera: Vespidae). J. Insect Behavior 12:
465-473.
DJEMAI, I., J. CASAS, AND C. MAGAL. 2001. Matching
host reactions to parasitoid wasp vibrations. Proc.
Roy Soc. Biol. Sci. Series B 268: 2403-2408.
DJEMAI, I., J. CASAS, AND C. MAGAL. 2004. Parasitoid
foraging decisions mediated by artificial vibrations.
Animal Behaviour 67: 567-571.
DJEMAI, I., R. MEYHOFER, AND J. CASAS. 2000. Geomet-
rical games between a host and a parasitoid. Ameri-
can Naturalist 156: 257-265.
DROSOPOULOS, S., AND M. F. CLARIDGE [Eds.]. 2006. In-
sect Sounds and Communication: Physiology, Be-
haviour, Ecology, and Evolution. CRC Press.
EVANS, T. A., J. C. S. LAI, E. TOLEDANO, L. MCDOWELL,
S. RAKOTONARIVO, AND M. LENZ. 2005. Termites as-
sess wood size by using vibration signals. Proc. Nat'l.
Acad. Sci. USA 102: 3732-3737.
FLETCHER, L. E., J. E. YACK, T. D. FITZGERALD, AND R.
R. HoY. 2006. Vibrational communication in the
cherry leaf roller caterpillar Caloptilia serotinella
(Gracillarioidea: Gracillariidae). J. Insect Behavior
19: 1-18.
GATES, M. W., J. M. HERATY, M. E. SCHAUFF, D. L.
WAGNER, J. B. WHITFIELD, AND D. B. WAHL. 2002.
Survey of the parasitic hymenoptera on leafminers
in California. J. Hymenoptera Res. 11: 213-270.
HAWKINS, B. A., H. V. CORNELL, AND M. E. HOCHBERG.
1997. Predators, parasitoids, and pathogens as mor-
tality agents in phytophagous insect populations.
Ecol. 78: 2145-2152.
HERING, E. M. 1951. Biology of Leaf Miners. 's-Graven-
hage, Berlin.
HOCH, H., J. DECKERT, AND A. WESSEL. 2006.Vibration-
al signalling in a Gondwanan relict insect (Hemi-
ptera: Coleorrhyncha: Peloridiidae). Biol. Letters 2:
222-224.
KIRCHNER, W. H., I. BROECKER, AND J. TAUTZ. 1994. Vi-
brational alarm communication in the damp-wood
termite Zootermopsis nevadensis. Physiol. Entomol.
19: 187-190.
KROMBEIN, K. V., J. P. D. HURD, AND D. R. SMITH. 1979.
Catalog of Hymnoptera in America North of Mexico.
Smithsonian Institution Press, Washington, D. C.,
U.S.A.
Low, C. 2008. Group size increases visual detection risk
by specialist parasitoids. Behav. Ecol. 19: 532-538.


Florida Entomologist 91(4)







Low: Seismic Behaviors of a Leafminer


MAYNARD-SMITH, J., AND D. HARPER 2004. Animal Sig-
nals. Oxford University Press.
MEYHOFER, R., AND J. CASAS. 1999. Vibratory stimuli in
host location by parasitic wasps. J. Insect Physiol.
45: 967-971.
MEYHOFER, R., J. CASAS, AND S. DORN. 1994. Host loca-
tion by a parasitoid using leafminer vibrations:
Characterizing the vibrational signals produced by
the leaf-mining host. Physiol. Entomol. 19: 349-359.
MEYHOFER, R., J. CASAS, AND S. DORN. 1997. Vibration-
mediated interactions in a host-parasitoid system.
Proc. Roy Soc. London Series B Biol. Sci. 264: 261-266.
MORAES, M. C. B., R. A. LAUMANN, A. COKL, AND
M. BORGES. 2005. Vibratory signals of four Neotropi-
cal stink bug species. Physiol. Entomol. 30: 175-188.
NEEDHAM, J. S., S. W. FROST, AND B. H. TOTHILL. 1928.
Leaf-mining Insects. Balliere, London.
ROCES, F., AND B. HOLLDOBLER. 1996. Use of stridula-
tion in foraging leaf cutting ants: Mechanical sup-
port during cutting or short range recruitment sig-
nal? Behav. Ecol. Sociobiol. 39: 293-299.


RODRIGUEZ, R. L., L. E. SULLIVAN, AND R. B. COCROFT.
2004. Vibrational communication and reproductive
isolation in the Enchenopa binotata species complex
of treehoppers (Hemiptera: Membracidae). Evolu-
tion 58: 571-578.
ROHE, W., AND R. RUPPRECHT. 2001. Knocking and
scraping as alarm signals in Dolichoderinae ants
from the Malay Peninsula (Hymenoptera: Formi-
cidae : Dolichoderinae). Entomologia Generalis 25:
81-96.
VIRANT-DOBERLET, M., AND A. COKL. 2004. Vibrational
communication in insects. Neotrop. Entomol. 33:
121-134.
VIRANT-DOBERLET, M., AND I. ZEZLINA. 2007. Vibration-
al communication of Metcalfa pruinosa (Hemiptera:
Fulgoroidea: Flatidae). Ann. Entomol. Soc. America
100: 73-82.
YACK, J. E., M. L. SMITH, AND P. J. WEATHERHEAD.
2001. Caterpillar talk: Acoustically mediated terri-
toriality in larval Lepidoptera. Proc. Nat'l. Acad. Sci.
USA 98: 11371-11375.







Florida Entomologist 91(4)


December 2008


TOXICITY OF ORGANOSILICONE ADJUVANTS
AND SELECTED PESTICIDES TO THE ASIAN CITRUS
PSYLLID (HEMIPTERA: PSYLLIDAE) AND ITS PARASITOID
TAMARIXIA RADIATA (HYMENOPTERA: EULOPHIDAE)

ARTURO COCCO AND MARJORIE A. HOY
Department of Entomology and Nematology, University of Florida, Bldg 970,
Natural Area Drive, Gainesville, FL, 32611-0629, USA
Corresponding author, e-mail: acocco@uniss.it

ABSTRACT
The acute toxicity of the adjuvants Silwet L-77 and Kinetic, alone and in combination with
petroleum oil and copper hydroxide, to the Asian citrus psyllid Diaphorina citri Kuwayama
was evaluated in screenhouse bioassays. In addition, the acute and residual toxicity of Sil-
wet L-77 and Kinetic, alone and in combination with petroleum oil, copper hydroxide, imi-
dacloprid, and abamectin, to the parasitoid Tamarixia radiata (Waterston) were evaluated
under laboratory conditions. In screenhouse trials, Silwet L-77 (0.05%) was more insecti-
cidal than Kinetic (0.05%) and increased the toxicity of both petroleum oil and copper hy-
droxide to D. citri. Petroleum oil at reduced rates (0.5 and 1%) in combination with Silwet
L-77 or Kinetic was less effective in reducing D. citri populations than petroleum oil at 2%
in combination with these adjuvants. Petroleum oil at 2% plus Silwet L-77 was the most
toxic combination to D. citri eggs, young (first- and second- instars) and mature nymphs
(third- to fifth-instars), and adults (81, 83, 74, and 55% mortality, respectively). Copper hy-
droxide was only toxic to young nymphs when combined with Silwet L-77 (64.9% mortality).
Under laboratory conditions, survival of T radiata was reduced by the residual effects of im-
idacloprid (>95% mortality) and by the acute toxicity of abamectin (>91% mortality). Silwet
L-77 and Kinetic alone, and petroleum oil and copper hydroxide alone or in combination with
these adjuvants, had low residual and acute toxicity to the parasitoid and appear to be com-
patible with the biological control of D. citri by T radiata. The results of this study suggest
that Silwet L-77 may be used in a citrus IPM program in combination with petroleum oil or
copper hydroxide to increase psyllid control while spraying to suppress other insect pests or
plant diseases. Field trials should be conducted to evaluate the effectiveness of these prod-
ucts against D. citri and their impact on T radiata populations.

Key Words: Silwet L-77, Kinetic, Diaphorina citri, Tamarixia radiata, petroleum oil, copper,
imidacloprid, abamectin, acute toxicity, residual effects

RESUME
La toxicidad aguda de los adyuvantes Silwet L-77 y Kinetic, solo o en combinaci6n con aceite pe-
tr6leo y hidr6xido de cobre, al silido asidtico de los citricos, Diaphorina citri Kuwayama fue eva-
luada en bioensayos hechos en la casa de tamizado. Ademas, la toxicidad aguda y residual de
Silwet L-77 y Kinetic, solos o en combinaci6n con aceite de petr6leo, hidr6xido de cobre, imida-
cloprid y abamectin, al parasitoide Tamarixia radiata (Waterston) fueron evaluadas baja condi-
ciones del laboratorio. En las pruebas en la casa de tamizado, Silwet L-77 (0.05%) fue mas como
un insecticide que Kinetic (0.05%) y aumento la toxicidad del aceite de petr6leo y hidr6xido de
cobre a D. citri. Tasas reducidas (0.5 y 1%) del de aceite petr6leo en combinaci6n con Silwet L-77
o Kinetic fueron menos efectivas en reducir poblaciones de D. citri que el aceite de petr6leo al 2%
en combinaci6n con estos adyuvantes. El aceite de petr6leo al 2% mas Silwet L-77 fue la combi-
naci6n mas toxica a los huevos, las ninfas inmaduras (j6venes) del primero y segundo estadio, las
ninfas inmaduras (maduras) del tercer a quinto estadio y los adults (con una mortalidad 81, 83,
74 y 55%, respectivamente). El hidr6xido de cobre solamente fue toxico a los inmaduros j6venes
cuando fue combinado con Silwet L-77 (mortalidad de 64.9%). Bajo condiciones de laboratorio, el
sobrevivencia de T radiata fue reducida por los efectos residuales de imidacloprid (mortalidad
>95%) y por la toxicidad aguda de abamectin (mortalidad >91%). El Silwet L-77 y Kinetic solo,
y el aceite de petr6leo y el hidr6xido de cobre solo o en combinaci6n con estos adyuvantes, tenian
una baja toxicidad residual y aguda al parasitoide y aparece ser compatible con el control biol6-
gico de D. citri por T radiata. Estos resultados sugirieron que Silwet L-77 puede ser usado en un
program de MIP en los citricos en combinaci6n con el aceite de petr6leo o hidr6xido de cobre
para aumentar el control del silido mientras rocian el cultivo para suprimir las otras plagas de
insects o enfermedades. Se debe realizar pruebas del campo para evaluar la efectividad de estos
products contra D. citri y su impact sobre poblaciones de T radiata.







Cocco & Hoy: Toxicity of Organosilicone Adjuvants and Pesticides to D. citri


The Asian citrus psyllid Diaphorina citri Ku-
wayama (Hemiptera: Psyllidae) is the vector of
citrus greening disease (also known as huan-
glongbing or HLB) caused by the bacterium Can-
didatus Liberibacter asiaticus. Greening disease
is one of the most important citrus diseases in the
world (Bove 2006), causing mottling and leaf chlo-
rosis, twig dieback, reduced production, and even-
tual death of the trees in 5-8 years. Infected trees
produce misshapen, poorly colored, and bitter-
tasting fruits, not usable for consumption (Hal-
bert & Manjunath 2004). Diaphorina citri was
first found in Florida in Jun 1998 (Hoy & Nguyen
1998) and is now established in Florida and Texas
(French et al. 2001). Although the Asian citrus
psyllid causes direct feeding damage to citrus
(Mead 1976), its economic importance is due to
transmission of the bacterium. Citrus greening
was detected in Florida during 2005 and is now
found in 30 counties (Florida Department of Agri-
culture and Consumer Services 2008). An eradi-
cation program for citrus greening disease was
not developed because of its wide distribution
when detected.
Current management measures for greening
disease in Florida citrus groves include soil appli-
cation of systemic insecticides (imidacloprid and
aldicarb) and multiple applications (up to 8-18) of
broad-spectrum foliar insecticides (including fen-
propathrin, imidacloprid, abamectin, dimethoate,
carbaryl, and chlorpyrifos). Insecticides are ap-
plied during the dormant foliar season, when new
flushes required for psyllid female oviposition and
nymphal development are rare, with the goal of
reducing adult psyllid populations prior to the
first flush cycle and during the flushing season
(Rogers 2008; University of Florida-IFAS Exten-
sion 2008). Trees are visually inspected and re-
moved if infected. These approaches to managing
greening disease have increased the costs of cit-
rus production, may lead to development of insec-
ticide resistance in D. citri, and are likely to be
disruptive to natural enemies of psyllids (and
other citrus pests), such as ladybeetles, lacew-
ings, spiders (Michaud 2004), and parasitic wasps
including the specialist parasitoid of the Asian
citrus psyllid, Tamarixia radiata (Waterston)
(Hymenoptera: Eulophidae) (Hoy & Nguyen
1998; Hoy et al. 1999). Repeated sprays of these
broad-spectrum insecticides over several years
could cause secondary outbreaks of whiteflies,
aphids, armored scales, and mealybugs previ-
ously held below damage thresholds by a complex
of beneficial insects (University of Florida-IFAS
Extension 2008). Tamarixia radiata was im-
ported as a part of a classical biological control
program and is now established in Florida (Hoy et
al. 1999; Skelley & Hoy 2004).
Insecticides are often used in combination
with organosilicone adjuvants to facilitate the
wetting and the spread of droplets on leaves, re-


sulting in a more uniform distribution of active
ingredients (Foy 1989; Pollicello et al. 1995). Ad-
juvants may also show insecticidal activity to
several pests, although the mechanism by which
organosilicones are toxic to insects and mites has
not been adequately determined (Srinivasan et
al. 2008). Petroleum oil and organosilicone adju-
vants might be suitable for citrus IPM programs
if they are effective in suppressing the pests
while allowing their natural enemies to persist.
Treatments with petroleum oil are compatible
with the IPM program developed for the citrus
leafminer Phyllocnistis citrella Stainton (Lepi-
doptera: Gracillariidae), because petroleum oil
has short residual activity and allows survival of
its parasitoid Ageniaspis citricola Logvi-
novskaya (Hymenoptera: Encyrtidae) (Villan-
ueva-Jimenez & Hoy 1998).
Recent laboratory, greenhouse, and small-
scale field studies demonstrated the effective-
ness of reduced rates of imidacloprid and abam-
ectin in combination with Silwet L-77, a non-
ionic organosilicone surfactant, to control D. citri
eggs, young nymphs, and adults (Srinivasan et
al. 2008). However, they did not evaluate toxicity
of petroleum oil in combination with Silwet L-77
or Kinetic to psyllid immatures and adults and
did not evaluate the effects of these products on
any natural enemies ofD. citri. Because imida-
cloprid and abamectin are toxic to several bene-
ficial arthropods, multiple applications could
disrupt biological control of citrus pests and fre-
quent applications could select for resistant pop-
ulations (Villanueva-Jimenez & Hoy 1998;
Grafton-Cardwell et al. 2001; Liburd et al. 2004;
Toscano et al. 2004). To further investigate D.
citri management strategies that are compatible
with biological control by T. radiata (and, poten-
tially, other natural enemies), we investigated
the acute toxicity of 3 rates of petroleum oil alone
and in combination with Silwet L-77 and Kinetic
to all life stages ofD. citri (eggs, young or mature
nymphs, and adults). We tested petroleum oil be-
cause it is considered more compatible with IPM
programs than imidacloprid and abamectin, due
to its moderate or low toxicity to beneficial ar-
thropods and its short residual effects (Beattie &
Hardy 2004; Erkilic & Uygun 1997). Further-
more, petroleum oil applications do not increase
the likelihood of pesticide resistance (Thomson
2001). Bioassays using D. citri were conducted
inside a screenhouse to evaluate the effective-
ness of treatments with natural photoperiods
and temperatures. In addition, we investigated,
for the first time, the susceptibility of T. radiata
adults when parasitoids were sprayed directly
(acute toxicity) or exposed to the residues (resid-
ual toxicity) of these adjuvants alone and in com-
bination with petroleum oil, copper hydroxide,
abamectin, and imidacloprid under laboratory
conditions.







Florida Entomologist 91(4)


MATERIALS AND METHODS

Rearing ofD. citri and T radiata

Diaphorina citri and T radiata were reared ac-
cording to a method modified from Skelley & Hoy
(2004). The psyllid colony was maintained with
potted citrus trees, approximately 30-50 cm tall.
The trees were pruned, fertilized, watered, and
placed in PVC (Poly Vinyl Chloride) frame cages
(60 x 60 x 60 cm) covered with organdy cloth in a
greenhouse maintained at 21-42C, 20-79% RH,
under a 16L:8D photoperiod. After 10-14 d, the
trees had new tender growths (= flushes) 0.5-1 cm
long, and were exposed to ovipositing D. citri fe-
males (approximately 5 per tree). Females were
aspirated out with a vacuum pump after 72 h.
The parasitoid T radiata was reared on potted
citrus trees as described above for the D. citri col-
ony. Ten trees were placed in wooden-framed
cages covered with organdy cloth (114 x 94 x 76
cm) held in a greenhouse at 20-38C, 22-81% RH,
under a 16L:8D photoperiod. Approximately 50
ovipositing D. citri females were released in the
cage and allowed to oviposit for 3-5 d, then re-
moved with a vacuum pump. Adults of T radiata
(50 females and 25 males) were collected and re-
leased into the cage when the psyllid population
reached the third instar. Adults of D. citri that
emerged from unparasitized nymphs were col-
lected as a by-product and were used to maintain
the psyllid colony.

Effects of Adjuvants With and Without Petroleum Oil
and Copper Hydroxide on D. citri

Screenhouse experiments were undertaken to
evaluate the acute toxicity of Silwet L-77 (99.5%
polyalkyleneoxide modified heptamethyltrisilox-
ane; Helena Chemical Co., Collierville, TN) and
Kinetic (99% proprietary blend of polyalkyleneox-
ide modified polydimethylsiloxane and nonionic
surfactants; Helena Chemical Co., Collierville,
TN) alone and in combination with agrochemicals
to psyllid eggs, young or mature nymphs, and
adults. The screenhouse bioassays described here
were conducted under variable environmental
conditions between May and Oct 2007 to mimic
grove conditions. Fifteen treatments were tested:
water; Silwet L-77 at 0.05% (v:v); Kinetic at
0.05% (v:v); petroleum oil 435 (Growers 435,
Growers Fertilizer Co., Lake Alfred, FL) at 0.5, 1,
and 2% (v:v); copper hydroxide (Kocide 2000, Du-
Pont, Wilmington, DE) at the recommended low-
est label rate (LLR) (2.4 g/L of water) for suppres-
sion of citrus canker in Florida; petroleum oil 435
at 0.5% + Silwet L-77 at 0.05%; petroleum oil 435
at 1% + Silwet L-77 at 0.05%; petroleum oil 435 at
2% + Silwet L-77 at 0.05%; petroleum oil 435 at
0.5% + Kinetic at 0.05%; petroleum oil 435 at 1%
+ Kinetic at 0.05%; petroleum oil 435 at 2% + Ki-


netic at 0.05%; copper hydroxide at the LLR+ Sil-
wet L-77 at 0.05%; and copper hydroxide at the
LLR + Kinetic at 0.05%. The recommended LLR
was calculated for 935 L per ha (100 gals/acre).
Petroleum oil was evaluated at rates lower than
2% (Childers & Rogers 2005) to determine
whether the adjuvants improved its toxicity to D.
citri. Because an unnamed organosilicone surfac-
tant tested at 0.2% (v:v) was reported to enhance
the spread of citrus bacterial spot caused byXan-
thomonas axonopodis pv. citrumelo (Gottwald et
al. 1997), questions were raised as to whether Sil-
wet L-77 might enhance the rate of infection with
citrus canker (Xanthomonas axonopodis pv. citri).
Because copper hydroxide is often used to protect
citrus trees from citrus canker, it was combined
with these adjuvants to evaluate the toxicity of
this formulation to D. citri. All products were
mixed with purified water (Barnstead Nanopure
II system, Dubuque, IA) at a pH of 6.8 at 24.80C.
The experiments were carried out at the Depart-
ment of Entomology and Nematology, University
of Florida, Gainesville.
The protocol to evaluate the acute toxicity of
the pesticide solutions on D. citri eggs, young or
mature nymphs, and adults was similar to that of
Srinivasan et al. (2008), except that each shoot
was treated separately, and the untreated canopy
was shielded with a plastic bag. The experimental
design was modified because they did not test
treatments on mature nymphs ofD. citri and they
evaluated only the residual toxicity of imidaclo-
prid and abamectin to psyllid adults. To investi-
gate the toxicity of treatments to psyllid eggs, pot-
ted citrus trees with at least 5 flushes 0.5-1 cm
long that were infested with eggs were selected.
On each tree, 5-6 shoots were sprayed with a dif-
ferent Preval gun sprayer (Precision Valve
Corp., Yonkers, NY) for each treatment. Shoots
were sprayed from a distance of 70-80 cm for 10 s
around each shoot, which covered approximately
90% of the leaf surface. Each shoot had 28-369
eggs (mean SD = 110 57) and was considered
a replicate. The trees were placed into cages in-
side a covered screenhouse for 9 d at 13-32C, 27-
94% RH, under a natural photoperiod during May
2007. The numbers of live nymphs and the num-
bers of unhatched eggs were scored with use of a
dissecting microscope. Dead first-instars were
considered as uneclosed eggs, because hatching
larvae might be intoxicated if the treatment pen-
etrated the egg shell or the larvae encountered a
lethal concentration on the egg shell.
To assess the acute toxicity of the treatments
to first- and second-instars (= young) D. citri, the
potted citrus trees were infested as described for
eggs. After 72 h, the trees were placed into cages
inside the same screenhouse and left undisturbed
for 6 d at 11-30C, 19-96% RH, under a natural
photoperiod during May 2007 to allow develop-
ment to the nymphal stage. Shoots were sprayed


December 2008







Cocco & Hoy: Toxicity of Organosilicone Adjuvants and Pesticides to D. citri


using the same method as for the eggs. Each shoot
had 33-390 nymphs (mean + SD = 143 72). After
4 d at 12-31C and 18-94% RH under a natural
photoperiod, live and dead nymphs were scored
under a dissecting microscope. Motion, posture,
presence of waxy excretions, dehydration of the
body were considered to determine whether the
nymphs were dead or live. To estimate the per-
centage distribution of the different instars at the
time of the treatment, 6 flushes were sampled
separately from shoots used to evaluate the treat-
ments. The nymphs in each instar were scored
separately to assess the instar densities across
the tree. Each flush had 55-164 nymphs (mean +
SD = 113 45).
Another experiment was conducted with third-
to fifth-instars to verify whether the treatments
were effective on 'large nymphs'. The same meth-
odology as for young nymphs was used, except
that the trees were left undisturbed for 10 d after
the oviposition period at 18-34C and 27-95% RH
under a natural photoperiod during Jun 2007.
Each shoot had 20-184 nymphs (mean + SD = 79
+ 34). The treatments were based on the results of
the toxicity bioassay on young nymphs, and cho-
sen on the assumption that ineffective treatments
for young nymphs were likely to be ineffective
against larger nymphs. The 9 treatments were:
water; Silwet L-77 at 0.05%; Kinetic at 0.05%; pe-
troleum oil 435 at 2%; copper hydroxide at the
LLR; petroleum oil 435 at 2% + Silwet L-77 at
0.05%; petroleum oil 435 at 2% + Kinetic at
0.05%; copper hydroxide at the LLR+ Silwet L-77
at 0.05%; and copper hydroxide at the LLR + Ki-
netic at 0.05%. Two days after treatment, alive
and dead nymphs were recorded with use of a dis-
secting microscope, based on the same criteria as
described above for young nymphs (post-treat-
ment conditions: 21-36C, 26-94% RH, natural
photoperiod). The shoots sampled to estimate the
percentage distribution of nymphs had 63-163
nymphs (mean + SD = 96 36).
Acute toxicity to adults was assessed on potted
citrus trees with only young flushes approx 3-5 cm
long that fit inside Plexiglas cylinders (45.5 cm
tall, 12.6 cm outside diameter). The top of the cyl-
inder and 4 side holes (5.4 cm in diameter) were
covered with mesh for air circulation. The soil
surface was lined with plastic wrap and 2 paper
coffee filters. The coffee filters were taped to the
pot to facilitate the count of psyllid adults. Psyllid
adults approximately 3 weeks old were exposed to
the trees for 4-6 h within a cage in the green-
house. Trees were infested with 20-98 adults
(mean psyllids per tree SD = 51 19). Different
sprayers for each treatment were used to spray
the trees containing adults; sprayers were held
70-80 cm from the trees and sprays applied for 15
s around the canopy The trees were set inside the
cylinders and placed into a roofed screenhouse.
Alive and dead adults inside cylinders were


counted after 72 h. Motion and maintenance of
their typical posture (with the head touching the
surface and the abdomen raised to a 30-45 degree
angle) were considered to determine whether the
adults were dead or live. The experiment was con-
ducted 8 times on 4 different dates during Jul,
Aug, and Oct 2007. During the experiments, the
average minimum and maximum temperatures
(mean SD) were 22 3.1C and 33 2.9C, re-
spectively. The mean RH ( SD) averaged 42
8.6% (minimum) and 92 4.5% (maximum). Daily
weather data were obtained from the weather
station maintained by the Department of Agricul-
tural and Biological Engineering, University of
Florida, Gainesville, approximately 800 m away.
Temperature and RH inside and outside the cyl-
inders were recorded with a Traceable@ Digital
Thermometer (Fisher Electronics, Pittsburgh,
PA). The mean minimum and maximum temper-
atures inside the cylinders were higher (0.18C
and 0.480C, respectively) than the environment
temperature. The minimum and maximum RHs
inside the cylinder were higher by 7.8 and 0.2%,
respectively. Data expressed as percentages were
evaluated with one-way analysis of variance with
Proc GLIMMIX (SAS Institute 2002), and a
Tukey-Kramer test was used to separate means
(a < 0.05).

Acute and Residual Toxicity of Adjuvants and Selected
Pesticides to T radiata Adults

The acute and residual toxicities of adjuvants
alone and in combination with petroleum oil, cop-
per hydroxide, abamectin, and imidacloprid were
tested in the laboratory with clip cage bioassays
(Villanueva-Jimenez & Hoy 1998).
Residual Toxicity. Potted citrus trees with
leaves 1 month old were sprayed to cover approx-
imately 90% of the leaf surface. Silwet L-77 and
Kinetic were tested alone (0.05%) and in combina-
tion with petroleum oil 435 at 2%; copper hydrox-
ide at the LLR; abamectin (Agrimek 0.15 EC;
Syngenta Crop Protection, Greensboro, NC) at
the LLR; and imidacloprid (Provado 1.6 Flowable;
Bayer Cropscience, Research Triangle Park, NC)
at the LLR. Water was used as control. Trees were
allowed to dry for about 30 min. Two treated
leaves were pruned randomly from each treat-
ment and placed at the top (with the abaxial sur-
face downward) and the bottom (with the adaxial
surface upward) of an acrylic ring (38 mm outside
diameter, 10 mm tall). Foam tape (M-D Building
Products, Oklahoma City, OK) 6 mm wide was
placed on both edges of the ring to seal the clip
cage. The treated surface of the clip cage chamber
represented ca. 64.3% of the internal area. The
rings had 2 mesh-covered windows 5 mm in diam-
eter to reduce RH inside the chamber and pro-
vided a method for feeding T radiata by placing a
water-soaked rolled paper outside the mesh win-







Florida Entomologist 91(4)


dow and a honey-soaked strip inside the chamber.
The clip cage was held together by 2 pieces of cir-
cular cardboard on the top and bottom (40 mm
diam) with 4 hair clips (Villanueva-Jimenez &
Hoy 1998). To place T radiata into the clip cage,
10 adult females 3-4 d old were chilled inside a 50-
mL conical centrifuge tube in crushed ice for 5
min and gently tapped inside the clip cage. The
clip cage was placed inside a plastic bag with a
wet paper towel to ensure survival of the parasi-
toids throughout the experiment (McFarland &
Hoy 2001). The plastic bags were placed inside a
growth chamber at 24 + 1C under a 16L:8D pho-
toperiod. Adult mortality was evaluated after 24
h. The insects were touched gently with an insect
pin and considered alive if they walked, jumped,
or flew away, and dead if they did not move or only
moved antennae, wings, or legs. When the mortal-
ity in the water control was >15%, the entire rep-
licate was eliminated. The bioassay was repli-
cated 6 times on 3 different dates. The percentage
of mortality data were arcsine IP transformed be-
fore statistical analysis to approximate a normal
distribution. Data were subjected to one-way
analysis of variance (ANOVA) with Proc GLM
(SAS Institute 2002), and significant differences
among means were evaluated by Fisher's Least
Significance Difference (LSD) test (a = 0.05%).
When water-control mortality was detected, data
were corrected by Abbott's formula for control re-
sponse (Abbott 1925).
The position of T radiata adults inside the clip
cage chamber was observed to determine whether
the treatments were repellent, and the parasitoid
preferred to move or settle on the untreated sur-
faces. The relative position (inner surface of the
ring or treated leaves) of the parasitoid was re-
corded after 2 and 6 h, in 3 replicates per treat-
ment. Data expressed as number of T radiata on
the leaves over total parasitoids inside the clip
cage were evaluated with analysis of variance
(Proc GLIMMIX) (SAS Institute 2002).
Acute (Direct) Toxicity. The bioassays were
performed as for the residual toxicity test, except
that untreated foliage was used and T radiata
were treated directly by placing 10 females 3-4 d
old into a Petri dish upon crushed ice for 5 min
(Villanueva-Jimenez & Hoy 1998). The adult fe-
males then were sprayed with a gun sprayer from
a distance of 1 m for 5 s and placed inside the clip
cage chamber. Twelve treatments was tested: wa-
ter, oil 435 at 2%, abamectin at the LLR, copper
hydroxide at the LLR, Silwet L-77 (0.05%) and Ki-
netic (0.05%) alone and in combination with pe-
troleum oil 435 at 2%, abamectin at the LLR, and
copper hydroxide at the LLR. Because imidaclo-
prid alone and in combination with both adju-
vants killed almost all parasitoids in the residual
toxicity bioassay (see Results), acute toxicity of
these products was not tested. Data were ana-
lyzed as for the residual toxicity test.


RESULTS

Effects of Adjuvants With and Without Petroleum Oil on
D. citri

Eggs. Silwet L-77 and Kinetic alone caused
only 20.4 and 16.5% mortality, respectively, to D.
citri eggs (Table 1). Mortality of psyllid eggs
sprayed with petroleum oil at 0.5, 1, and 2%
ranged from 61.5 to 66.7%, but the differences
were not significant. When petroleum oil at 1 and
2% was combined with 0.05% Silwet L-77, the tox-
icity increased from 66.3 to 78.7%, and from 61.5
to 81.3%, respectively, (F = 122.37; df = 11, 57; P
< 0.0001) (Table 1). Petroleum oil at 0.5, 1, and 2%
in combination with Kinetic was less effective
(52.6, 57.7, and 52.3%, respectively) in killing
psyllid eggs than petroleum oil alone at the same
rates. Mortality rates of eggs sprayed with petro-
leum oil at 1 and 2% plus Silwet L-77 were signif-
icantly higher than when petroleum oil in combi-
nation with Kinetic was applied.
Young Nymphs. The bioassay on young nymphs
was conducted when nymphs were predominantly
first or second instars (20.9 and 64.7%, respec-
tively). Silwet L-77 sprayed on trees infested with
D. citri nymphs killed 69.7%, while Kinetic alone
was significantly less effective (46.2% mortality)
(F = 101.09; df= 11, 60; P < 0.0001) (Table 1). Tox-
icity of petroleum oil to young nymphs increased
with the rate applied. Petroleum oil at 0.5%
caused 34.5% mortality, which was significantly
lower than at 1% (56.5%) or at 2% (84.7%). Silwet
L-77 increased significantly the toxicity of petro-
leum oil to young D. citri nymphs when applied at
0.5% from 34.5 to 57.2%, and at 1% from 56.5 to
68.2%. However, adding Silwet L-77 or Kinetic did
not improve the efficacy of petroleum oil at 2% be-
cause it was effective by itself to young nymphs
(84.7% mortality). When Kinetic was combined
with 0.5% petroleum oil, mortality of nymphs in-
creased significantly from 34.5 to 56.2%, but not at
the other rates tested. Petroleum oil at 2% was the
most effective treatment in suppressing young
nymphs (84.7%).
Mature Nymphs. Infested trees used to test
the toxicity of petroleum oil and adjuvants to ma-
ture nymphs were sprayed when 94.5% of the
nymphs were either third, fourth or fifth instars.
Mortality of these larger D. citri nymphs on Sil-
wet L-77-treated trees was 64.2%, which was
higher than on Kinetic-treated trees (35.2%) (F =
56.95; df = 5, 28; P < 0.0001) (Table 1). When pe-
troleum oil at 2% was applied in combination with
an adjuvant, only Silwet L-77 significantly in-
creased the mortality of mature nymphs from
44.8 to 74.3%. These tests indicate that Silwet L-
77 is better than Kinetic in suppressing young
and mature nymphs, and that Silwet L-77 signif-
icantly improves the efficacy of petroleum oil at
2% against large nymphs.


December 2008







Cocco & Hoy: Toxicity of Organosilicone Adjuvants and Pesticides to D. citri 615



TABLE 1. ACUTE TOXICITY OF PETROLEUM OIL 435 AT DIFFERENT RATES, ALONE AND IN COMBINATION WITH SILWET
L-77 OR KINETIC, TO IMMATURES AND ADULTS OF D. CITRI UNDER SCREENHOUSE CONDITIONS IN GAINES-
VILLE, FL, DURING MAY-OCT 2007.

Mean mortality ofD. citri (% SE)

Treatments" Eggs" Young nymphs' Mature nymphs' Adults"

Water 7.1 + 0.8 f 3.1 + 0.5 g 7.1 + 1.0 e 0.8 + 0.8 e
Silwet L-77 (0.05%) 20.4 1.7 e 69.7 1.9 c 64.2 8.7 b 30.6 5.3 b
Kinetic (0.05%) 16.5 1.6 e 46.2 5.8 ef 35.2 3.8 d 5.9 1.2 de
Oil 0.5% 66.7 4.8 b 34.5 5.6 f -2.7 0.9 e
Oil 1% 66.3 + 4.3 b 56.5 5.9 d -4.9 1.3 de
Oil 2% 61.5 4.3 bc 84.7 2.6 a 44.8 2.6 c 17.5 3.4 c
Oil 0.5% + Silwet L-77 (0.05%) 60.3 7.8 bcd 57.2 4.6 d -38.4 5.0 b
Oil 1% + Silwet L-77 (0.05%) 78.7 4.7 a 68.2 5.3 c 35.2 3.8 b
Oil 2% + Silwet L-77 (0.05%) 81.3 4.9 a 83.0 3.1 ab 74.3 5.3 a 54.7 + 6.1 a
Oil 0.5% + Kinetic (0.05%) 52.6 5.1 cd 56.2 6.0 ed -4.6 0.6 de
Oil 1% + Kinetic (0.05%) 57.7 4.8 cd 49.3 5.0 e -8.2 2.1 d
Oil 2% + Kinetic (0.05%) 52.3 5.0 cd 76.0 + 3.1 bc 41.5 5.5 cd 34.5 5.4 b

"Screenhouse conditions: eggs (13-32C, RH 27-94%, during May 2007); young nymphs (11-31C, RH 18-96%, during Jun 2007);
mature nymphs (18-36C, RH 26-95%, during Jul 2007); adults (22 3.1 33 2.9C, RH 42 8.6 92 4.5%, during Jul, Aug, and
Oct 2007), all under a natural photoperiod.
'Significant differences compared with Proc GLIMMIX (P < 0.0001), treatment means with the same letter within a column are
not different by Tukey-Kramer test (a < 0.05).


Adults. Silwet L-77 applied to D. citri adults
caused 30.6% mortality, which was more effective
than Kinetic (5.9% mortality) (F = 58.35; df = 11,
84; P < 0.0001) (Table 1). Mortality of adults
sprayed with petroleum oil at 0.5, 1, and 2% was
2.7, 4.9, and 17.5%, respectively, indicating that
petroleum oil alone is not effective in suppressing
D. citri adults. Combining Silwet L-77 with petro-
leum oil at 0.5, 1, and 2% significantly increased
the mortality of adults to 38.4, 35.2, and 54.7%,
respectively. Kinetic applied in combination with
petroleum oil improved the toxicity of that insec-


ticide only at the rate of 2%, from 17.5 to 34.5%.
These data suggest that Silwet L-77 is better than
Kinetic at increasing the efficacy of petroleum oil
to D. citri adults.

Effects of Adjuvants With and Without Copper Hydrox-
ide on D. citri

Mortality ofD. citri eggs on trees sprayed with
copper hydroxide at the LLR alone and in combi-
nation with Kinetic was not different, ranging
from 14.1 to 14.7% (Table 2). However, when Sil-


TABLE 2. ACUTE TOXICITY OF COPPER HYDROXIDE, ALONE AND IN COMBINATION WITH SILWET L-77 AND KINETIC, TO
IMMATURES AND ADULTS OF D. CITRI UNDER SCREENHOUSE CONDITIONS IN GAINESVILLE, FL, DURING MAY-
OCT 2007.

Mean mortality ofD. citri (% SE)

Young Mature
Treatments" Eggs" nymphs' nymphs' Adults"

Water 7.1+ 0.8 d 3.1 + 0.5 f 7.1 + 1.0 e 0.8 0.8 c
Silwet L-77 (0.05%) 20.4 1.7 b 69.7 1.9 a 64.2 8.7 a 30.6 5.3 a
Kinetic (0.05%) 16.5 1.6 bc 46.2 5.8 c 35.2 3.8 c 5.9 1.2 bc
Copper hydroxide (LLR) 14.1 2.2 c 18.8 3.6 e 15.1 3.6 e 0.4 0.4 c
Copper hydroxide (LLR) + Silwet L-77 (0.05%) 37.3 6.3 a 64.9 4.4 b 46.7 4.8 b 34.5 + 5.1 a
Copper hydroxide (LLR) + Kinetic (0.05%) 14.7 2.1 cd 30.1 + 6.1 d 22.6 2.8 d 6.6 + 2.1 b

"LLR indicates the lowest label rate. Screenhouse conditions: eggs (13-32C, RH 27-94%, during May 2007); young nymphs (11-
31C, RH 18-96%, during Jun 2007); mature nymphs (18-36C, RH 26-95%, during Jul 2007); adults (22 3.1 33 2.9C, RH 42
+ 8.6 92 4.5%, during Jul, Aug, and Oct 2007), all under a natural photoperiod.
'Significant differences compared with Proc GLIMMIX (P < 0.0001), means with the same letter within a column are not differ-
ent by Tukey-Kramer test (a < 0.05).







Florida Entomologist 91(4)


wet L-77 was added to copper hydroxide at the
LLR, mortality increased from 14.1 to 37.3% (F =
31.45; df= 5, 29; P < 0.0001). Copper hydroxide at
the LLR caused 18.8 and 15.1% mortality of
young and mature nymphs, respectively. When
Kinetic was added to copper hydroxide, mortality
rates of young (F = 172.52; df = 5, 30; P < 0.0001)
and mature nymphs (F = 69.70; df = 5, 29; P <
0.0001) increased significantly to 30.1 and 22.6%,
respectively. However, these rates were signifi-
cantly lower than mortality of nymphs exposed to
copper hydroxide at the LLR plus Silwet L-77,
which were 64.9 and 46.7%, respectively
(Table 2).
Copper hydroxide at the LLR killed very few
D. citri adults (0.4%). When it was combined
with Kinetic the mortality rate increased signif-
icantly to 6.6%, which was significantly lower
than the mortality caused by copper hydroxide
at the LLR in combination with Silwet L-77
(34.5%) (F = 45.69; df = 5, 42; P < 0.0001) (Table
2). However, the 34.5% mortality observed with
the Silwet L-77 plus copper hydroxide combina-
tion was caused primarily by Silwet L-77 be-
cause there were no significant differences in
mortality between copper hydroxide at the LLR
plus Silwet L-77 and Silwet L-77 alone. Adding
Silwet L-77 to copper hydroxide or petroleum
oil caused these pesticides to uniformly cover
the leaf surface rather than forming small sep-
arated droplets.


Acute and Residual Toxicity of Adjuvants and Selected
Pesticides to T radiata Adults

Residual Toxicity. Tamarixia radiata adult
females did not appear to show any preference
between treated and untreated surfaces inside
the clip cages sprayed with water, Silwet L-77,
and Kinetic alone and in combination with cop-
per hydroxide at the LLR, petroleum oil at 2%,
and abamectin at the LLR (data not shown).
There was no significant difference in the pro-
portion of T. radiata settling or walking on the
leaf surface (treated with water, Silwet L-77,
and Kinetic alone and in combination with pe-
troleum oil, copper hydroxide, and abamectin)
or on the inner surface of the untreated ring (F
= 1.07; df = 11, 59; P = 0.4039). However, after
2 h, parasitoids exposed to leaves previously
treated with imidacloprid alone and in combi-
nation with Silwet L-77 or Kinetic had died;
thus these data were not evaluated by analysis
of variance to determine the preferred position
ofT. radiata within the clip cages.
Mortality of T. radiata exposed to treated fo-
liage was different among treatments (F =
55.05; df = 14, 75; P < 0.0001) (Table 3). Silwet
L-77 and water did not affect the survival of the
parasitoid, but Kinetic was significantly more
toxic, resulting in the death of 13.3% of adults
after 24 h. Leaves treated with 2% petroleum
oil in combination with Silwet L-77 or Kinetic


TABLE 3. RESIDUAL AND ACUTE TOXICITY OF PETROLEUM OIL 435, ABAMECTIN, IMIDACLOPRID, AND COPPER HYDROX-
IDE ALONE AND IN COMBINATION WITH SILWET L-77 AND KINETIC, TO ADULT FEMALES OF T. RADIATA USING
CLIP CAGES.

Mortality of T radiata (mean % SE)

Treatments" Residual toxicity" Acute toxicity"

Water 0 e 1.7 + 1.7 b
Silwet L-77 (0.05%) 0 e 0 b
Kinetic (0.05%) 13.3 + 4.9 c 1.9 1.9 b
Oil 2% 11.7 + 4.0 c 1.7 1.7 b
Oil 2% + Silwet L-77 (0.05%) 8.3 3.1 cd 1.7 1.7 b
Oil 2% + Kinetic (0.05%) 15.0 5.6 c 0 b
Abamectin (LLR) 23.3 11.2 c 93.1 5.0 a
Abamectin (LLR) + Silwet L-77 (0.05%) 15.0 4.3 c 96.7 3.3 a
Abamectin (LLR) + Kinetic (0.05%) 91.7 3.1 b 96.7 2.1 a
Imidacloprid (LLR) 100 a
Imidacloprid (LLR) + Silwet L-77 (0.05%) 95.0 3.4 ab
Imidacloprid (LLR) + Kinetic (0.05%) 100 a
Copper hydroxide (LLR) 5.0 2.2 cde 1.7 1.7 b
Copper hydroxide (LLR) + Silwet L-77 (0.05%) 1.7 3.1 de 3.3 3.3 b
Copper hydroxide (LLR) + Kinetic (0.05%) 6.7 5.6 cde 0 b

"LLR indicates the lowest label rate. Laboratory conditions: 24 + 1 C, 16L:8D photoperiod
'Significant differences compared with Proc GLM (P < 0.0001), treatment means with the same letter within a column are not
different by Fisher's LSD test (a < 0.05). Imidacloprid caused high residual mortality and was not tested for acute toxicity.


December 2008




University of Florida Home Page
© 2004 - 2010 University of Florida George A. Smathers Libraries.
All rights reserved.

Acceptable Use, Copyright, and Disclaimer Statement
Last updated October 10, 2010 - Version 2.9.7 - mvs