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Wetterer & Snelling: Solenopsis invicta in the Virgin Islands


THE RED IMPORTED FIRE ANT, SOLENOPSIS INVICTA,
IN THE VIRGIN ISLANDS (HYMENOPTERA: FORMICIDAE)


JAMES K. WETTERER1 AND ROY R. SELLING2
1Wilkes Honors College, Florida Atlantic University, 5353 Parkside Dr., Jupiter, FL 33458

2Natural History Museum of Los Angeles County, 900 Exposition Blvd., Los Angeles, CA 90007

ABSTRACT

The best known and most destructive exotic ant species in the US is the red imported fire
ant, Solenopsis invicta Buren. Recently, this species has been reported from several islands
in the West Indies, including St. Croix, US Virgin Islands (USVI) and Guana Island, British
Virgin Islands (BVI). In the present study, we report new records of S. invicta on St. Croix
(13 sites) and the first records of S. invicta from 3 other of the Virgin Islands: St. Thomas,
USVI (7 sites), St. John, USVI (2 sites), and Tortola, BVI (6 sites). Solenopsis invicta appears
to be well established in disturbed open environments on all 4 islands. It is important that
people in the Virgin Islands and elsewhere in the West Indies, particularly healthcare pro-
fessionals, are aware of the presence of S. invicta, can recognize the symptoms of S. invicta
stings, and know proper treatments for adverse reactions to the stings, including rare but
potentially deadly anaphylactic shock.

Key Words: exotic species, fire ants, Solenopsis invicta. Virgin Islands, West Indies

RESUME

La especie de la hormiga ex6tica mejor conocida y muy destructive en los EEUU es la impor-
tada hormiga roja del fuego, Solenopsis invicta. Esta especie se ha sido reportado reciente-
mente en varias islas en las Antillas, inclusive S. Croix, las Islas Virgenes de EEUU (USVI)
y la Isla de Guana, las Islas Virgenes inglesas (BVI). En el studio present, nosotros repor-
tamos nuevos registros de S. invicta en el S. Croix (13 sitios) y los primeros registros de S.
invicta de tres otras de las Islas Virgenes: S. Thomas, USVI (7 sitios), S. John, USVI (2 si-
tios), y Tortola, BVI (6 sitios). Solenopsis invicta aparece ser establecido bien en ambientes
abiertos perturbados en las cuatro islas. Es important que personas en las Islas Virgenes y
en otras parties en las Antillas, particulamente profesionales de cuidado medico, estan avi-
sado de la presencia de S. invicta. Estos profesionales deben de reconocer los sintomas de la
picada de S. invicta, y saber los tratamientos para reacciones adversas a la picada.


Translation provided by the authors.


The best known and most destructive exotic
ant species in the US is the red imported fire ant,
Solenopsis invicta Buren, which arrived in Ala-
bama by ship from South America sometime be-
fore 1945 (Buren et al. 1974). Since then, this
predatory ant has spread across the US from
Texas to North Carolina in the southeast and Cal-
ifornia in the west, particularly in open disturbed
areas, causing ecological and economic damage
(e.g., see Tschinkel 1988, 1993; Allen et al. 2004;
Wetterer & Moore 2005). Solenopsis invicta is
well-known for its powerful sting, which causes a
burning sensation in humans, usually followed
within one or two days by the appearance of a
white pustule. These pustules are diagnostic for
the stings of S. invicta and other Solenopsis sae-
vissima complex fire ants from South America (S.
Porter, pers. comm.). The stings of other ants, in-
cluding the widespread tropical fire ant, Solenop-
sis geminata (Fabricius), do not produce pustules.


The venom has hemolytic and neurotoxic proper-
ties and may cause allergic responses and result
in secondary infections, sepsis, anaphylactic
shock, and even death (Prahlow & Barnard 1998;
deShazo et al. 2004).
The earliest known West Indian records of S.
invicta are from Puerto Rico (Buren 1982), where
it is now widespread (Torres & Snelling 1997;
Davis et al. 2001; RRS & JKW, unpublished data).
More recently, S. invicta has been reported from
numerous other islands in the West Indies (Table
1), including the Virgin Islands, which lie to the
east of Puerto Rico. Davis et al. (2001) published
records of S. invicta from St. Croix, US Virgin Is-
lands (in 1997: Fredensborg National Guard facil-
ity, and in 2000; Route 66, 0.8 km east of Route
663) and from Guana Island, a small island north
of Tortola, British Virgin Islands (BVI, in 1996).
Solenopsis invicta closely resembles S. geminata,
both in appearance and in the pain of its sting.







Florida Entomologist 89(4)


TABLE 1. EARLIEST KNOWN SPECIMEN RECORDS FOR SOLENOPSIS INVICTA ON ISLANDS OF THE WEST INDIES. THE AS-
TERISK (*) INDICATES DATE PROVIDED BY M. DEYRUP.

Island Year Source reference

Puerto Rico 1981 Buren 1982
St. Croix, USVI 1988 present study
San Salvador, Bahamas 1993* Deyrup 1994
New Providence, Bahamas 1995* Deyrup et al. 1998
North Andros, Bahamas 1996* Deyrup et al. 1998
Guana Island, BVI 1996 Davis et al. 2001
Gorda Cay, Bahamas 1997 Davis et al. 2001
Antigua 2000 Davis et al. 2001
Abaco, Bahamas 2000 Davis et al. 2001
Trinidad 2000 Davis et al. 2001
Grand Bahama, Bahamas 2000 Davis et al. 2001
Providenciales, Turks & Caicos 2001 Davis et al. 2001
Berry Islands, Bahamas 2005 M. Deyrup, pers. comm.
St. Thomas, USVI 2005 present study
St. John, USVI 2005 present study
Tortola, BVI 2005 present study


Because S. geminata is common throughout the
West Indies, the presence ofS. invicta may be eas-
ily overlooked, even by trained entomologists.
In the present study, we examined museum
specimens and made field collections to evaluate
the distribution ofS. invicta in the Virgin Islands.

METHODS

JKW searched the ant collection at the US Na-
tional Museum (USNM) for Solenopsis invicta spec-
imens from the Virgin Islands. Between Oct 1991
and Oct 2002, RRS collected ants on Guana Island
during several visits (see Snelling 1993, 2003).
From 30 Oct to 21 Nov 2005, JKW collected
ants on the 4 largest of the Virgin Islands, the 3
main islands of the US Virgin Islands (St. Croix -
7 d, St. Thomas 5.5 d, and St. John 4.5 d), and
the main island of the British Virgin Islands (Tor-
tola 4.5 d). Collection sites included a diversity
of disturbed and relatively natural habitats from
the coastlines to the mountaintops. We also made
a number of other observations concerning S. in-
victa in the Virgin Islands.

RESULTS

The USNM collection had Solenopsis invicta
specimens from 2 sites in the Virgin Islands, both
from St. Croix in 1988: Kingshill and Concordia.
These records are earlier than any published
records from the Virgin Islands.
RRS did not find S. invicta on Guana Island
prior to 1996. In Oct 2002, S. invicta was common
on the south side of the island: on the playa be-
hind White Beach and in the "plantation" area.
Forested areas of Guana Island were occupied by
Solenopsis geminata.


In 2005, JKW collected S. invicta from 28 sites
in the Virgin Islands: St. Croix (13 sites), St. Tho-
mas (7 sites), St. John (2 sites), and Tortola (6
sites). All sites were in highly disturbed habitats,
primarily open grassy areas (Table 2). All sites ex-
cept one were low elevation (<100 m above sea
level; the site at Parasol, St. Croix was 200 m
above sea level). JKW collected S. geminata at 83
sites in the Virgin Islands: St. Croix (23 sites), St.
Thomas (19 sites), St. John (23 sites), and Tortola
(18 sites), in a wide variety of disturbed and rela-
tively undisturbed habitats at all elevations.
On St. Croix, Jozef(Jeff) Keularts, an entomol-
ogist with the US Cooperative Extension Service,
was aware of the presence of S. invicta on St.
Croix. Lesley Hoffman, Administrative Director
at the St. George Village Botanical Garden, St.
Croix, related that in Jan 2005, her husband,
Robert Hoffman, was stung by S. invicta while
golfing at the Buccaneer Hotel Golf Course on St.
Croix. He was brought to Juan Luis Hospital,
where he was treated for anaphylactic shock with
adrenaline and antihistamines. He now always
carries an auto-injection charged with epineph-
rine because he was told that a subsequent attack
could cause even more severe anaphylactic shock,
which could be fatal without immediate treat-
ment. Once stung the body builds up antibodies
and subsequent attacks can result in potentially
deadly allergic reactions.
On St. Thomas, George Ralish, the superinten-
dent at Mahogany Run Golf Course knew of the
presence and threat ofS. invicta on the course. He
has been working to control S. invicta on the golf
course through spot treatment of nests using two
insecticides (Extinguish from Wellmark, active
ingredient = 0.5% Methoprene; Varsity from Syn-
genta, active ingredient = 0.011% Abamectin).


December 2006







Wetterer & Snelling: Solenopsis invicta in the Virgin Islands


TABLE 2. NEW COLLECTION SITES OF SOLENOPSIS INVICTA IN THE VIRGIN ISLANDS (30 OCT TO 21 NOV 2005).

N W Island Site Habitat

17.780 64.770 St. Croix Salt River, entrance to Gentle Winds grass lawn
17.759 64.586 St. Croix Cramer's Park grass & weeds
17.757 64.817 St. Croix Parasol; Scenic Dr., 0.5 km E of Rte. 69 grass & weeds
17.740 64.842 St. Croix Montpellier, by church grass lawn
17.732 64.813 St. Croix Upper Love, by church grass lawn
17.729 64.865 St. Croix Little La Grange, by Lawaetz Museum grass lawn
17.720 64.798 St. Croix Kingshill, UVI by parking lot
17.717 64.694 St. Croix Longford, Routes 62 & 85 grass lawn
17.715 64.883 St. Croix Fredriksted, waterfront park plantings
17.715 64.830 St. Croix St George, Botanical Garden grass lawn
17.702 64.885 St. Croix Smithfield, south of Cottages by the Sea grass lawn
17.694 64.891 St. Croix Hesselberg, south end of Shore Drive grass lawn
17.694 64.820 St. Croix Betty's Hope, south of Route 64 scrub forest
18.364 64.923 St. Thomas Magens Bay, end of Route 35 beach weeds
18.359 64.906 St. Thomas Lovenlund, Mahogany Run Golf Course grass green
18.344 64.974 St. Thomas John Brewer's Bay, UVI by parking lot
18.344 64.937 St. Thomas Charlotte Amalie, Griffiths Park grass & weeds
18.344 64.933 St. Thomas Charlotte Amalie, Creques Alleys plantings
18.344 64.930 St. Thomas Charlotte Amalie, Emancipation Garden grass lawn
18.339 64.969 St. Thomas Brewer's Bay, airport plantings
18.348 64.713 St. John Coral Bay baseball field
18.343 64.785 St. John Caneel Bay, resort grass lawn
18.447 64.562 Tortola Josiah's Bay, by hostel grass lawn
18.425 64.619 Tortola Road Town, waterfront weeds
18.425 64.579 Tortola Paraquita Bay, community college grass lawn
18.414 64.589 Tortola Brandy Wine Bay beach weeds
18.412 64.671 Tortola Carrot Bay beach weeds
18.386 64.699 Tortola Sandy Point, boat yard grass & weeds


On St. John, the US quarantine office in the
main harbor at Cruz Bay had no records of any
ants intercepted from in-coming cargo. The per-
sonnel there were unaware of any threat posed by
pest ant species, including S. invicta.
On Tortola, a person visiting a beach com-
plained of white pustules and scars from ant
stings he received while working at a boat yard at
the Sandy Point. JKW found this entire boat yard
heavily infested with S. invicta.

DISCUSSION

In the Virgin Islands, Solenopsis invicta is
now well established on all 4 major islands as
well as on Guana Island. Based on specimen
records, it appears that S. invicta probably ar-
rived in the Virgin Islands in the 1980s, first es-
tablishing itself on St. Croix. The first popula-
tions of S. invicta on the other Virgin Islands
may be quite recent, dating from the 1990s and
later. It is not surprising that S. invicta has
spread to St. Croix and the other Virgin Islands,
given the large amount of commercial ship traf-
fic to these islands from Puerto Rico and ports
in the southeastern US, sites which are heavily


infested with S. invicta. It seems inevitable
that S. invicta will soon spread to most other
populated islands of the West Indies as well.
Solenopsis invicta poses an important threat
not only to terrestrial invertebrates in the Vir-
gin Islands and other West Indian islands, but
also to vertebrates. For example, S. invicta at-
tacks and kills hatchling sea turtles in Florida
(Allen et al. 2001; Parris et al. 2002; Krahe et
al. 2003; Krahe 2005), and may pose a similar
hazard to sea turtles in the Virgin Islands. The
collection site in southwestern Hesselberg, St.
Croix, was adjacent to the Sandy Point Wildlife
Preserve, an important nesting beach for the
endangered leatherback sea turtle, Dermo-
chelys coriacea (Vandelli) (Dutton et al. 2005).
Solenopsis invicta may also represent a threat
to already endangered small vertebrates on
these islands, including many species ofAnolis
lizards. Finally, it is important that people in
the Virgin Islands, particularly healthcare pro-
fessionals, are aware of the threat of S. invicta
to humans, can recognize the symptoms of S. in-
victa stings, and know proper treatments for se-
vere adverse reactions to the stings, including
rare but potentially deadly anaphylactic shock.











ACKNOWLEDGMENTS

We thank A. Wetterer, M. Wetterer, S. Porter, and M.
Lachance for comments on this manuscript; the Na-
tional Science Foundation, Florida Atlantic University,
and the Natural History Museum of Los Angeles
County for financial support.

REFERENCES CITED

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NARTZ, AND R. N. WILLIAMS. 1974. Zoogeography of
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the island of San Salvador, Bahamas, pp. 21-28 In
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BOULON. 2005. Increase of a Caribbean leatherback
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December 2006


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Florida Entomologist 89(4)







Jenkins et al.: Predators and Parasitoids of Plum Curculio


INVERTEBRATE PREDATORS AND PARASITOIDS OF PLUM CURCULIO,
CONOTRACHELUS NENUPHAR (COLEOPTERA: CURCULIONIDAE)
IN GEORGIA AND FLORIDA

DAVID A. JENKINS', RUSS F. MIZELL, III2, DAVID SHAPIRO-ILAN3, TED COTTRELL3 AND DAN HORTON4
'USDA-ARS, Mayaguez, Puerto Rico 00680-5470

2Entomology and Nematology Dept. IFAS, Gainesville, FL 32611

3USDA-ARS, Byron, GA 31008

4University of Georgia, Athens, GA 30602

ABSTRACT

The extent of predation and parasitism on larvae of the plum curculio, Conotrachelus nen-
uphar (Herbst) (Coleoptera: Curculionidae), was measured independently with several dif-
ferent experimental designs at sites in northern Florida and central Georgia. Experimental
manipulation in Monticello, FL, and in Byron, GA, demonstrated equivocal impacts by pre-
dation. However, direct observations in Byron, GA, revealed that ants are the dominant in-
vertebrate predators of plum curculio larvae, causing up to 62% mortality. Primary ant
predators included Solenopsis invicta (Buren) (Hymenoptera: Formicidae) and Dorymyrmex
bureni (Trager) (Hymenoptera: Formicidae). Predation may be more important later in the
season when infested fruit does not abscise and plum curculio larvae must drop to the
ground from the trees and spend a considerable time burrowing into the soil. This contrasts
with the early season when infested fruit abscise and larvae crawl from the fruit directly
into the soil, reducing their exposure to predators. Recorded parasites included Nealiolus
curculionis (Fitch) (Hymenoptera: Braconidae) and Cholomyia inaequipes Bigot (Diptera:
Tachinidae). Parasitism, particularly byN. curculionis, was common in northern Florida but
rare in middle Georgia.

Key Words: Dorymyrmex bureni, Solenopsis invicta, Nealiolus curculionis, Cholomyia
inaequipes

RESUME

El nivel de depredaci6n y parasitismo en contra de Conotrachelus nenuphar (Herbst) (Co-
leoptera: Curculionidae), fue medido independientemente usando diferentes disenos experi-
mentales en lugares como el norte de Florida y la zona central de Georgia. Manipulaci6n
experimental en Monticello, FL, y en Byron, GA, demostr6 que el impact de depredaci6n no
fue precise. Sin embargo, observaciones directs en Byron, GA, revelaron que las hormigas
son el invertebrado dominant en la depredaci6n de la larva de C. nenuphar, causando hasta
62% de mortalidad en las larvas. Entre las principles hormigas depredadoras se encuen-
tran, Solenopsis invicta (Buren) (Hymenoptera: Formicidae) y Dorymyrmex bureni (Trager)
(Hymenoptera: Formicidae). La depredaci6n es mas important en la temporada tardia,
cuando las frutas infestadas no han caido al suelo, por lo tanto las larvas tuvieron que llegar
al suelo desde los arboles y pasaron un tiempo considerable tratando de enterarse en el
suelo. Contrario a esto, en la temporada temprana cuando las frutas infestadas cayeron al
suelo y las larvas pasaron de la fruta al suelo directamente, reduciendo el tiempo que las lar-
vas estuvieron expuestas a los depredadores. Los parasitos reportados incluyen Nealiolus
curculionis (Fitch) (Hymenoptera: Braconidae) y Cholomyia inaequipes (Bigot) (Diptera: Ta-
chinidae). Parasitismo, particularmente porN. curculionis, fue comun en el norte de Florida
pero raro en la zona central de Georgia.

Translation provided by the authors.


The plum curculio, Conotrachelus nenuphar lis 1989). Adult plum curculios migrate in early
(Herbst) (Coleoptera: Curculionidae), an insect spring from overwintering sites in adjacent woods
native to North America (Quaintance & Jenne to infest peach orchards (Snapp 1930; Yonce et al.
1912), is the primary direct insect pest of peaches 1995). The females oviposit on young fruit, often
in the Southeastern United States (Horton & El- causing it to abscise (Quaintance & Jenne 1912;







Florida Entomologist 89(4)


Snapp 1930). Larvae develop in the fruit and
move into the soil to pupate. In the bivoltine
southern strain of plum curculio (Chapman
1938), adults emerge from these pupae the same
summer to continue the infestation, but their off-
spring emigrate from the orchard to overwinter-
ing sites in adjacent woods or other locations with
plenty of leaf litter (Quaintance & Jenne 1912;
Snapp 1930; Yonce et al. 1995).
Plum curculio is currently controlled with
highly efficacious organophosphate insecticides.
The use of these pesticides is being restricted as a
result of the implementation of the Food Quality
Protection Act. Recent insecticide losses, e.g., me-
thyl parathion, have caused the peach industry in
the southeast to seek more sophisticated inte-
grated pest management strategies that take into
account the target pest's natural history and biol-
ogy. These approaches include soil applications of
entomopathogenic nematodes and fungi against
the larval and pupal stages (Shapiro-Ilan et al.
2002; Shapiro-Ilan et al. 2004; Tedders et al. 1982).
Although predators and parasitoids are impor-
tant components of integrated management pro-
grams for other curculionid pests (Stuart et al.
2003; Stuart et al. 2002; McCoy et al. 2000), little
attention has been paid to potential predators or
parasitoids of the plum curculio. Field efficacy tri-
als against plum curculio often have variable
mortality rates (Shapiro-Ilan et al. 2004; Quain-
tance & Jenne 1912; Snapp 1930), suggesting
that natural sources of mortality may be involved
and potentially sources of control. Even though
researchers in the northeastern U.S. concluded
that natural enemies of the plum curculio are in-
efficient (Van Driesche et al. 1987) we were inter-
ested in surveying predators and parasitoids of
plum curculio in central Georgia.
Our objectives were to assess the effects of var-
ious natural enemies on the southern strain of
plum curculio, C. nenuphar, by (1) quantifying the
extent of plum curculio mortality attributable to
predation and parasitism in northern Florida and
middle Georgia, and (2) assaying biological con-
trol organisms, such as the fungus, Beauvaria
bassiana (Bals.) Vuill. (Hyphomycetes) separately
and in conjunction with the application of conven-
tional pesticides (e.g., bifenthrin, thiamethoxam,
and imidacloprid) to the soil targeting late larval
and pupal stages of plum curculio are located. The
impacts of these pesticides on potential natural
enemies also were assessed.

MATERIALS AND METHODS

Experiments Conducted at the Southeastern Fruit and
Tree Nut Research Laboratory, Byron GA: Predation

At the USDA Southeastern Fruit and Tree Nut
Research Laboratory in Byron, GA, (SEFTNRL)
we compared the potential emergence under "op-


timal conditions" to emergence under field condi-
tions in order to estimate overall mortality attrib-
utable to abiotic factors and to predators, parasi-
toids, and pathogens. In May 2004 we exposed 20
virgin female and 20 virgin male plum curculio to
360 green thinning apples (Red Delicious variety)
for 2 weeks. Half of these apples were then ran-
domly selected and distributed equally among 6
tilled locations (30 apples/location) at the base of
peach trees and within the rows of an unsprayed
peach orchard on the grounds of the SEFTNRL.
Each tilled location was 0.6 m2 in area. This or-
chard had not received pesticide applications in
the previous 5 years. Six separate locations in the
same unsprayed orchard were tilled and used as
negative controls, each receiving 30 uninfested
green thinning apples. Each location was covered
with a cone emergence trap (Mulder et al. 2000)
after 3 weeks. This allowed predators to access
the infested apples without interference from the
cages but was not enough time for adults to
emerge from the soil. Cone emergence cages were
monitored daily for the emergence of adults over
60 days. The remaining apples that had been ex-
posed to ovipositing female plum curculio were di-
vided equally among 6 plastic tubs (11.4 L Rub-
bermaidTM storage box). The infested fruit were
placed upon a hardware cloth supported above
the bottom of the tub by four 2-cm long corks. The
tubs were stored in an environmental chamber at
25 + 1C and 50% RH (12:12, L:D) (Amis & Snow
1985). The tubs were monitored daily for the
emergence of larvae which were then placed into
pupation jars. Pupation jars were 950-ml glass
jars 2/3 filled with a moistened mixture of potting
soil and vermiculite (2:1) that had been sifted
with a 10-mesh sieve to ensure that the soil did
not contain insects and covered with a glass Petri
dish. Pupation jars were monitored daily for the
emergence of adult plum curculio. The number of
emerging adult plum curculio was compared be-
tween fruits exposed to predators and fruits not
exposed to predators using a t-test (SAS 2001).
We monitored plum curculio larvae as they bur-
rowed into the soil, recording any predation we ob-
served. Between Mar 15 and Aug 1, 2003, labora-
tory-reared plum curculio larvae (Amis & Snow
1985), within 12 h of emerging from green thinning
apples, were harvested, taken to the field, and
placed singly at random locations on an orchard
floor in Byron, GA, between the hours of 8:00 and
20:00. Each larva was observed until the larva bur-
ied itself or was carried off by predators. The time
interval between setting the larva on the ground
and its complete burial or removal by predators was
noted. Ant abundance (by species) was measured by
counting the number of ants in an area of 0.21 m2 at
random locations in the orchard throughout the
summer. An area of 0.21 m2 was chosen because it
was small enough for researchers to survey it inten-
sively. Results were then converted to ants per m2.


December 2006







Jenkins et al.: Predators and Parasitoids of Plum Curculio


Parasitism

Abscised peaches were collected at Byron, GA,
in 2004 and placed on trays with mesh bottoms
over a large aluminum funnel (0.3 m high with a
slope of 30%). The funnel was positioned over a
collection pan so that larvae emerging from the
infested fruit could be collected. Larvae were col-
lected daily, enumerated and placed in pupation
jars. Pupation jars were monitored daily for 60 d
for the emergence of adult plum curculio or para-
sitoids.
In addition, wild plum fruit, Prunus angustifo-
lia Marshall and P umbellata Elliott, infested
with plum curculio (as denoted by the distinct ovi-
position scar, Quaintance & Jenne 1912) were col-
lected from Peach Co., GA, and placed in plastic
tubs (11.4-L RubbermaidTM storage box) and
stored, as described earlier in the predation stud-
ies. The tubs were monitored daily for the emer-
gence of larvae from fruit. Larvae were collected
and placed into pupation jars and were monitored
for the appearance of adult plum curculio and/or
parasitoids.
One hundred abscised peaches collected from
an unsprayed peach orchard at SEFTNRL were
placed at each of 5 tilled areas (0.6 m2) at the base
of randomly selected peach trees in the orchard.
The peaches were then covered with a cone emer-
gence trap. The cone emergence trap was moni-
tored for 60 d for the emergence of parasitoids his-
torically associated with plum curculio (Krom-
bein et al. 1979).

Experiments Conducted at the North Florida Research
and Education Center, Monticello, FL

One thousand peach fruit that had abscised in
response to infestation by plum curculio were
gathered at the University of Florida, North Flor-
ida Research and Education Center (NFREC) in
Monticello, FL, in 2003. These fruit were distrib-
uted among 10 locations (0.5 m2) on an orchard
floor, so that each location had 100 abscised fruit.
Bifenthrin (Talstar EZ, FMC Corporation, Phil-
adelphia, PA) was applied (1 lb/acre) in a 3.14-m
ring around, but not on, infested fruit at 5 loca-
tions. Five locations were left untreated as con-
trols. The locations were covered with cone emer-
gence traps and monitored daily for the emer-
gence of adult plum curculio or parasitoids
(Krombein et al. 1979).
Parasitoid emergence also was monitored in a
separate field trial in 2003 by assaying 5 pesti-
cides applied to the orchard floor and targeting
plum curculio larvae. Twenty five sites (1 m2)
were selected and treated with imidacloprid (Ad-
mire 2F, Bayer Crop Sciences, Kansas City, MO)
at 1.75 L/ha, bifenthrin (Talstar EZ, FMC Cor-
poration, Philadelphia, PA) at 1.12kg/ha, thia-
methoxam (Platinum, Syngenta, Greensboro,


NC) at 438.07 mL/ha, or Beauvaria bassiana
(GHA strain, supplied by Emerald Bioagricul-
ture, Butte, Montana) applied at a rate of 1014
conidia/ha, or with 2 L of water as a control treat-
ment. All treatments were delivered in 2 L of wa-
ter from a watering can. One hundred abscised
fruit, gathered from the orchard floor, were depos-
ited at each of the 25 sites (5 treatments with 5
replicates).

RESULTS

Southeastern Fruit and Tree Nut Laboratory, Byron
GA: Predation

Significantly more adults emerged from apples
stored in the incubator (7.5 1.3: mean SEM)
than from apples stored in the orchard and ex-
posed to predation, disease, and environmental
factors (3.8 1.0: mean SEM) (t = 2.75; df = 5;
P = 0.022). No adult plum curculio emerged from
the control plots that contained uninfested ap-
ples, indicating that it was likely that all of the
curculio emerging in the cone emergence cages in
the orchard were from the infested apples and not
from plum curculio pupae that were in the soil
prior to the experiment.
In total, 268 m2 were surveyed for ant abun-
dance and 4,038 ants were found. Solenopsis in-
victa Buren (Hymenoptera: Formicidae) com-
prised 77% of the ants found, Dorymyrmex bureni
(Trager) (Hymenoptera: Formicidae) comprised
15%, and a Paratrechina sp. (Hymenoptera: For-
micidae) comprised 8%. There was a mean of 15
(0.88 SEM) ants of any species in a given m2. Of
these, 12 (+0.85 SEM) were S. invicta, 2 (+0.26
SEM) were D. bureni, and 1 (+0.10 SEM) was
Paratrechina sp.
All 3 ant species were observed capturing and
killing larval plum curculio that we had placed on
the ground. In total, 229 last instar plum curculio
larvae were observed on the orchard floor. Of
these, 97 were discovered by S. invicta, 26 were
discovered by Paratrechina sp., and 20 were dis-
covered by D. bureni. Eighty six larvae were able
to bury themselves before being discovered by
ants. On average, all larvae discovered by fire
ants were discovered in 13.94 min (SEM = 1.34).
All larvae discovered by Paratrachina sp. were
discovered on average in 9.35 min (SEM = 1.54).
All larvae discovered by D. bureni were discov-
ered on average in 20.10 min (SEM = 5.67). All
larvae discovered by any species of ant were dis-
covered on average in 13.97 min (SEM = 2.49). All
larvae that successfully buried themselves did so
on average in 15.41 min (SEM = 1.39).
A mean of 4.2 (SEM = 1.2) adult plum curculio
emerged from 100 abscised fruit under the 5 cone
emergence cages. Only 1 specimen of Nealiolus
curculionis (Fitch) (Hymenoptera: Braconidae)
was detected from the cone emergence cages.







438 Florida Entomologist 89(4) December 2006


In total, 930 abscised peaches were collected
from the orchard floor in Byron, GA. We collected
528 larvae from these fruit and placed them into zi i e
8), +1 +1
pupation jars. One specimen each of N. curculio- 8 t
nis and Ci.. ..o.,i i., inaequipes Bigot (Diptera: Ta-
chinidae) was reared from these plum curculio.
In total, 1,146 scarred fruit of P angustifolia
and 269 of P umbellata were collected. These o
fruit yielded a total of 546 plum curculio larvae c" c
(39% of infested fruit yielded larvae). Two N. cur- a
culionis were reared from these larvae. .
qOq Oq
North Florida Research and Education Center, t
Monticello, FL
Sd
A mean of 13.8 (2.4 SEM) adult plum curculio
emerged from 100 abscised fruit in the untreated "
controls and 12.8 (2.8 SEM) adult plum curculio g
emerged from 100 abscised fruit in the locations S 'i 1
that received bifenthrin as a ring treatment to 0
prevent entry of fire ants. These results were not z
different (t = 0.355; df = 4; P = 0.899).
The only parasitoid recovered was N. curculio-
nis. The percent of plum curculio infected with
this parasite, based on the number of adult plum
curculio recovered and the number of adult para-
sitoids, ranged from 30% to 47%, with an average
of 37% (SEM = 2.77) (Table 1). The pesticides as- C +1 +
sayed did not demonstrate significant control of
plum curculio compared to untreated controls, ; C
nor did they appear to significantly impact num-
bers of parasitoids in each treatment (Table 1).

DISCUSSION z

There was a significant reduction in plum cur- S
culio mortality when plum curculio were reared
indoors, in the absence of natural enemies or ad-
verse environmental conditions, as opposed to t s
those on the orchard floor. However, there was no i c
significant difference between the number of
adult plum curculio emerging in areas that had
been surrounded with a treatment of bifenthrin
and areas that had not received a pesticide treat-
ment to preclude foraging ants. These results sug-
gest a number of scenarios, including the follow- E
ing: (1) predation plays a small role in plum cur- <
culio mortality, (2) the pesticide applications used -
did not preclude foraging ants, or (3) differences
in mortality observed between plum curculio "
reared outdoors and those reared indoors may be "
attributed to regulated humidity and tempera-
ture, consistent environment, and fewer patho- .
gens. Direct field observations reveal that preda- ,
tion by ants alone may be responsible for the mor- i S
tality of more than 60% of plum curculio larvae z
attempting to burrow into the soil, with the ca- "
veat that placing lab reared plum curculio larvae
on the orchard floor is not natural and may exag- '"
gerate mortality due to ant predators. Further-
more, there may be a seasonal component to the <







Jenkins et al.: Predators and Parasitoids of Plum Curculio


effect of predation by ants. Peaches infested ear-
lier in the season are small and usually abscise
and drop to the ground when infested with plum
curculio larvae (Quaintance & Jenne 1912; De-
tjen 1938). The larvae continue to develop in the
fruit and can burrow directly from the fruit into
the soil, probably reducing their chances of being
encountered by foraging ants. Peaches infested
later in the season do not abscise and larvae must
drop from the fruit to the ground. Subsequently,
the summer generation of plum curculio in late
season peaches may be more susceptible to ant
predation. The lack of significant difference be-
tween number of adults emerging from infested
fruit that were chemically protected from preda-
tors and infested fruit that was accessible to pred-
ators lends credence to the possibility that larvae
moving directly from infested fruit into the soil
may suffer less predation than larvae that drop
from the tree to the ground.
This is the first quantitative study of the impact
of certain predators on plum curculio, although
many anecdotal observations have been published
(Quaintance & Jenne 1912; Snapp 1930). Plum cur-
culio larvae were monitored in close quarters
(within 1 m) for accurate identification of predators.
Such proximity to the larvae precluded larger pred-
ators, such as birds and carabid beetles, although
these may be additional and important sources of
mortality. Although Solenopsis invicta was not
present in central Georgia at the time, Snapp
(1930) and Quaintance & Jenne (1912) report that
Dorymyrmex bureni (reported as Dorymyrmex
pyramica) was an important predator of larval
plum curculio. Quaintance & Jenne (1912) list
ground beetles (Coleoptera: Carabidae) and a sol-
dier beetle, Chauliognathus pennsylvanicus (De
Geer) (Coleoptera: Cantharidae), as important
predators of larval plum curculio. Unfortunately,
the soldier beetle appears to be in decline in Geor-
gia, perhaps as a result of predation by S. invicta
(Jenkins & Matthews 2003). Dissections of spade-
footed toads, Scaphiopus sp., revealed that they are
consumers of plum curculio (J. Payne, pers. comm.).
Parasitism was of minimal importance as a
mortality factor in middle Georgia but appeared
to contribute significantly to the mortality of
plum curculio in northern Florida. Though we re-
alize the estimates for parasitism in Florida are
necessarily high, the sheer numbers obtained
need no statistical differentiation from those ob-
tained in the Byron, GA, studies, considering that
of more than 1000 larvae collected from peach
and wild plums in central Georgia only 2 yielded
parasitoids. It is possible that the N. curculionis
individuals collected in cone emergence traps in
Monticello, FL, had used hosts other than C. nen-
uphar. Indeed, N. curculionis is known to use
many other hosts (Krombein et al. 1979).
There are a number of parasitoids that have
been recorded from plum curculio but that were


not found in the current study. These include
Nealiolus collaris (Brues), N. rufus (Riley), Trias-
pis kurtogaster Martin, Bracon mellitor Say, B.
politiventris (Cushman), B. variablilis (Pro-
vancher) (Hymenoptera: Braconidae), Tersilochus
conotracheli (Riley) (Hymenoptera: Ichneu-
monidae), Patasson conotracheli (Girault) (Hy-
menoptera: Mymaridae), Myiophasia aenea
Wiedemann, C(. inaequipes Bigot
(Diptera: Tachinidae), and Pegomyia fusciceps
Zett. (Diptera: Anthomyiidae) (Riley 1871; Quain-
tance & Jenne 1912; Snapp 1930; Armstrong
1958; Arnaud 1978; Krombein et al. 1979; Ted-
ders & Payne 1986). All of these species, with the
exceptions of T conotracheli and B. politiventris,
have been recorded in Georgia or Florida (Krom-
bein et al. 1979). The vast majority of these para-
sitoids utilize a variety of other hosts, although
many of their hosts are often found in fruit
(Krombein et al. 1979).
Historically, percent mortality and percent
mortality attributable to parasitism has varied
greatly. Quaintance & Jenne (1912) report that
the percent of adult plum curculio that emerged
from larvae ranged from 9% to 60% with a mean
of 32% and that parasitism ranged from 0.7% to
21% with a mean of 8.1%. Snapp (1930) reported
that the percentage of adults that emerged from
larvae ranged from 1.7% to 18.7% with a mean of
7.4%. Armstrong (1958) reported a range of para-
sitized plum curculio larvae of 7.5 to 26.6 with an
average of 20% parasitized. Even in our study,
parasitism varied greatly between the 2 sites and
presumably does so from year to year. This broad
host range suggests that the abundance of alter-
nate hosts may play an important role in rates of
parasitism of C. nenuphar.
In summary, variation in mortality of plum
curculio is extremely high, as is variation in inci-
dence of parasitism (Quaintance & Jenne 1912;
Snapp 1930). The high levels of parasitism ob-
served in Florida are possibly important sources
of natural control of plum curculio populations.
Further research is needed to elucidate mortality
factors and the causes of this variation. Under-
standing these factors may lead to better pest
management strategies.

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 Betsy Stephens for enduring hours at a time star-
ing at plum curculio larvae and ants and Dr. Lionel
Stange of the Florida Department of Agriculture and
Consumer Services, Division of Plant Industry for iden-
tifying specimens of Nealiolus curculionis. We thank
Drs. Amanda Hodges, Jim Dutcher, Robert Matthews,
and 3 anonymous reviewers for critical reviews of an
earlier version of this manuscript.











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Florida Entomologist 89(4)







Weathersbee et al.: Bacillus thuringiensis Isolates Against Diaprepes abbreviatus 441



ACTIVITY OF BACILLUS THURINGIENSIS ISOLATES AGAINST
DIAPREPES ABBREVIATUS (COLEOPTERA: CURCULIONIDAE)

A. A. WEATHERSBEE III, S. L. LAPOINTE AND R. G. SHATTERS, JR.
USDA, ARS, U.S. Horticultural Research Laboratory, 2001 South Rock Road, Fort Pierce, FL 34945

ABSTRACT

A collection of Bacillus thuringiensis isolates plausibly active against coleopteran insects
was obtained from the Agricultural Research Service Culture Collection. Each isolate was
cultured, spores and 6-endotoxin crystals were pelleted by centrifugation and lyophilized,
and the resulting product was incorporated in insect diet for testing against Diaprepes ab-
breviatus neonates. A bioassay method was developed that utilized small amounts of insect
diet and B. thuringiensis spores and 6-endotoxin to treat single neonates confined to 0.2-mL
clear polymerase chain reaction (PCR) tubes. The method was less expensive in terms of la-
bor and materials as compared to previous methods and reduced control losses due to bur-
rowing and aggressive behaviors of D. abbreviatus larvae confined together. Of 19
B. thuringiensis isolates screened for activity against D. abbreviatus with a discriminating
dose of 250 ppm spores and 6-endotoxin on diet, 5 were selected for further evaluation in
dose-response experiments. Diaprepes abbreviatus larvae demonstrated a significant dose
response to 4 of the 5 isolates tested. The most active isolates were those that expressed
CryET33 and CryET34, or Cyt2Cal proteins. A wild-type B. thuringiensis strain that ex-
pressed Cyt2Cal generated the lowest LC5s value (50.7 gg/ml) and steepest slope (1.11)
based on loglO probit analysis of the data. These B. thuringiensis 6-endotoxins may have
utility in transgenic approaches to citrus rootstock protection from D. abbreviatus.

Key Words: Diaprepes abbreviatus, Bacillus thuringiensis, Cry, Cyt, endotoxin, citrus

RESUME

Una colecci6n de aislamientos de Bacillus thuringiensis posiblemente activos contra insec-
tos del orden Cole6ptera fue obtenido de la Colecci6n de Cultivos del Servicio de Investiga-
ci6n Agricola (USDA, ARS). Cada aislamiento fue criado, las esporas y cristales de
endotoxina-6 fueron sedimentados por una centrifugadora y liofilizado (congelado y secado),
y el product resultante fue incorporado en una dieta de insects para probarlo contra los
neonatos (larvas reci6n nacidas) del Diaprepes abbreviatus. Un m6todo de bioensayo fue de-
sarrollado para utilizar cantidades pequenas de la dieta de insects, esporas de B. thuring-
iensis y la endotoxina-6 para tratar individualmente los neonatos confinados en frascos
claros de 0.2 ml para la reacci6n en cadena por la polimerasa (RCP). Este metodo fue menos
costoso en terminos de mano de obra y materials comparado con los metodos de control usa-
dos anteriormente para reducir las perdidas asociadas al comportamiento minador y agres-
sivo de las larvas juntamente confinadas de D. abbreviatus. De los 19 aislamientos de B.
thuringiensis evaluados con actividad contra D. abbreviatus con una dosis de 250 ppm de es-
poras y endotoxin-6 sobre la dieta, 5 fueron seleccionados para evaluaci6n adicional en ex-
perimentos de respuesta de dosis. Las larvas de Diaprepes abbreviatus demonstraron una
respuesta de dosis significativa en 4 de los 5 aislamientos probados. Los aislados mas activos
fueron los que expresaron las proteinas CryET33 y CryET34, o Cyt2Cal. Una raza de tipo-
silvestre de B. thuringiensis que expreso Cyt2Cal produj6 el valor menor de CLs, (50.7 ig/
ml) y el pendiente empinado (1.11) basado en el andlisis de datos usando el probit de loglO.
Las endotoxinas-6 de B. thuringiensis puede ser tiles en un enfoque transg6nico para la
protecci6n de rizomas citricas contra el D. abbreviatus.


The invasive weevil species, Diaprepes abbre- sidered the most important pest of agriculture, hor-
viatus (L.), has become one of the most damaging ticulture, and silviculture in Puerto Rico (Hantula
insect pests of citrus and nursery crops in Florida et al. 1987). Efforts of researchers and pest manag-
since it was first reported in 1964 (Woodruff 1964). ers to develop an effective long-term management
Prior to its introduction into the continental United strategy for this pest have been unsuccessful. Ap-
States, D. abbreviatus was known to be a serious parently, D. abbreviatus is not under effective bio-
pest of sugarcane in the Lesser Antilles and is con- logical control within its putative native range of
Puerto Rico and the Lesser Antilles (Lapointe
2004). The lack of natural enemies, combined with
Mention of a trademark or proprietary product does not con- the wide host range of this highly polyphagous wee-
stitute a guarantee or warranty of the product by the U.S. De-polyphagous wee-
partment of Agriculture and does not imply its approval to the vil (Simpson et al. 1996) and its slow subterranean
exclusion of other products that may also be suitable, larval development (Lapointe 2000), makes D. ab-







Florida Entomologist 89(4)


breviatus particularly well adapted to semi-perma-
nent, tropical, and subtropical agroecosystems
such as citrus groves. Efforts to identify plant resis-
tance to D. abbreviatus within sexually compatible
citrus germplasm have been only marginally suc-
cessful (Bowman et al. 2001; Lapointe & Bowman
2002; Shapiro et al. 2000).
The bacterial entomopathogen, Bacillus thur-
ingiensis (Berliner), has been recommended for
the control of other insect pests of citrus, particu-
larly those in the order Lepidoptera (Shapiro et al.
1998; Stansly et al. 2006). Although use of
B. thuringiensis as an applied biopesticide can be
an effective control method for some insects, the
subterranean feeding habits ofD. abbreviatus lar-
vae make them difficult to target with a biocontrol
agent, such as B. thuringiensis, that must be in-
gested to be effective. It has been suggested that
the most appropriate and economically viable
method for control of D. abbreviatus will be the
production of transgenic rootstocks engineered to
express exogenous toxins (Lapointe 2004). Al-
Deeb & Wilde (2005) reported that transgenic
corn, expressing the Cry3bbl toxin from B. thur-
ingiensis, was protected from another root-feed-
ing coleopteran, the western corn rootworm, Di-
abrotica virgifera virgifera LeConte. Transgenic
crops that express B. thuringiensis proteins dis-
play resistance to some of the most devastating
pests of agriculture, yet are virtually safe to non-
target organisms (Betz et al. 2000). A transgenic
approach that uses a genetically-engineered cit-
rus rootstock to express a 6-endotoxin active
against D. abbreviatus is a plausible solution.
Currently, the few B. thuringiensis 6-endotox-
ins known to be active against coleopterans are
far outnumbered by known lepidopteran-active
toxins. In order to pursue this paradigm toward
the development of a transgenic citrus rootstock,
B. thuringiensis toxins that are active against
D. abbreviatus larvae must first be identified.
One strain of B. thuringiensis has been reported
to cause mortality of D. abbreviatus larvae
(Weathersbee et al. 2002), but otherwise B. thur-
ingiensis has received minimal attention as a po-
tential biocontrol agent for this pest. We assem-
bled a collection of B. thuringiensis isolates that
expressed novel 6-endotoxins putatively active
against one or more representatives of Cole-
optera. This paper presents the results of experi-
ments that determined if any of these toxins were
active against D. abbreviatus larvae.

MATERIALS AND METHODS

Source and Culture ofB. thuringiensis Isolates

Patent databases at the United States Patent
and Trademark Office were searched to locate
B. thuringiensis isolates potentially active against
species of Coleoptera. Representative samples of


19 isolates, for which patents had issued (Brad-
fisch et al. 2005; Donovan et al. 2005; Narva et al.
2005; Rupar et al. 2003; Rupar et al. 2004; Soares
et al. 1989), were obtained by request from the cu-
rator of the Agricultural Research Service Culture
Collection, National Center for Agricultural Utili-
zation Research, Peoria, Illinois (Table 1). Sam-
ples were received as dried pellets sealed in glass
ampoules. Growing cultures of each isolate were
established following instructions provided with
the samples. Briefly, an ampoule was scored with
a file and broken. The broken end of the ampoule
was flame sterilized, the pellet was removed and
then cultured in Luria-Bertani (LB) broth in an
incubator shaker. Cultures were stored on LB
agar slants at 4C and in LB broth/glycerol stock
solutions at -80C until needed.
Starter cultures of each isolate were prepared
by inoculating 10 mL of LB broth with a loopful of
bacterial cells removed from storage. Cultures
were grown overnight in 125-mL baffled Erlen-
meyer flasks at 27'C and 150 rpm in an incubator
shaker. Then 80 pl of starter culture were added to
100 mL of LB broth in a 500-mL baffled Erlenm-
eyer flask and grown in the incubator shaker until
approximately 90% of the cultured cells had sporu-
lated and autolyzed. Cultures were pelleted by
centrifugation (15,000 G) for 15 min at 4C,
washed 3 times with phosphate buffered saline
(PBS) containing 0.005% Triton X-100, lyophilized,
and weighed. Lyophilized pellets, containing B.
thuringiensis spores and 6-endotoxin crystals,
were stored in 1.5-mL microcentrifuge tubes at -
80C until they were used in the experiments.

Insect Source and Rearing

Neonatal larvae of D. abbreviatus were ob-
tained from a laboratory colony maintained at the
U.S. Horticultural Research Laboratory, Fort
Pierce, FL. Larvae were reared on a commer-
cially-prepared insect diet (Product No. F1675,
Bio-Serv, Frenchtown, NJ). Temperature and
moisture content of the diet were optimized for
larval development according to Lapointe (2000)
and Lapointe & Shapiro (1999). Neonate larvae
were surface sterilized with a solution of 0.31%
sodium hypochlorite and individually inspected
to insure only healthy and active larvae were
used in the experiments.

Bioassay Method

A bioassay method was developed to test the
effectiveness of B. thuringiensis isolates on indi-
vidual D. abbreviatus neonates. A clear poly-
merase chain reaction (PCR) tube containing a
small amount of insect diet in the lid was used to
hold a single D. abbreviatus neonate that could be
visually inspected for response to treatments.
Monitoring of larvae had not been possible with


December 2006







Weathersbee et al.: Bacillus thuringiensis Isolates Against Diaprepes abbreviatus 443



TABLE 1. SCREENING OF B. THURINGIENSIS ISOLATES AGAINST D. ABBREVIATUS NEONATES WITH A DISCRIMINATING
DOSE (250 1JG/ML) OF LYOPHILIZED, SPORULATED CULTURES IN DIET.

% Mortality SE (n=90)
Isolate' Isolate Endotoxinsb Genbank
number description present accession" Treatedd Control

B-21367 recombinant CryET33 AAF76375 91.3 3.0" 20.0 10.1
CryET34 AAF76376
B-21365 wild-type CryET33 AAF76375 90.3 6.7" 20.0 10.1
CryET34 AAF76376
B-21366 recombinant Cry3Bb2 AAA74198 87.7 4.7" 20.0 10.1
CryET33 AAF76375
CryET34 AAF76376
B-21582 wild-type Cyt2Cal AAK50455 81.7 7.5" 11.5 5.9
B-21583 recombinant Cyt2Cal AAK50455 52.3 2.9 0.0 0.0
B-21784 wild-type Cry35Aa2 AAK64561 20.3 8.8 3.0 0.0
Cry34Aa2 AAK64560
Cry38Aal AAK64559
B-21783 wild-type Cry35Aa2 AAK64561 15.7 5.9 2.0 1.0
Cry34Aa2 AAK64560
Cry38Aal AAK64559
B-21915 wild-type Cry35Bal AAK64566 15.7 7.0 4.3 1.3
Cry34Bal AAK64565
CryET84 AAK64564
B-21554 wild-type Cry35Acl AAG50117 14.3 1.3" 3.0 0.0
Cry34Acl AAG50118
B-21787 recombinant Cry36Aal AAK64558 12.0 4.9 1.0 1.0
B-21786 wild-type Cry36Aal AAK64558 11.3 3.0" 2.3 2.3
B-21788 recombinant Cry35Ab2 AAK64563 10.0 4.0 3.0 0.0
Cry34Ac2 AAK64562
B-18765 wild-type Cry5Bal AAA68598 8.7 3.0 1.0 1.0
Cry5Acl P56955
B-21916 recombinant Cry35Bal AAK64566 7.7 2.9 3.0 0.0
Cry34Bal AAK64565
CryET84 AAK64564
B-21785 wild-type Cry35Ab2 AAK64563 6.7 2.0 0.0 0.0
Cry34Ac2 AAK64562
B-18243 wild-type Cry5Aal AAA67694 5.7 1.3 2.0 1.0
Cry5Abl AAA67693
B-21553 wild-type Cry35Abl AAG41672 4.3 1.3 1.0 1.0
Cry34Abl AAG41671
B-18244 wild-type Cryl2Aal AAA22355 1.0 1.0 0.0 0.0
B-18679 wild-type Cryl4Aal AAA21516 1.0 1.0 1.0 1.0
Cry35Aal AAG50342
Cry34Aal AAG50341

Isolate numbers were assigned by curators of the ARS Culture Collection, National Center for Agricultural Utilization Research
(formerly the Northern Regional Research Laboratory), Peoria, Illinois USA.
bEndotoxins labeled CryET## have not yet been assigned names recognized by the B. thuringiensis 5-endotoxin nomenclature
committee.
'Protein accessions can be retrieved from the National Center for Biotechnology Information, Genbank at http://
www.ncbi.nlm.nih.gov/.
Means for D. abbreviatus percent mortality in the treated group marked by an asterisk ( ) were (P < 0.05, paired t-test) greater
than those for the control group.


older bioassay methods that used larger volumes with 75% ethanol, and the procedure was con-
of medium (soil or diet) because D. abbreviatus ducted in a biological safety cabinet. A stock solu-
larvae burrow into the medium, complicating vi- tion was prepared that contained 5% sucrose and
sual inspection. 0.005% Triton X-100 in deionized distilled water.
All items used in the bioassay procedure were The previously prepared lyophilized pellets of
sterilized by autoclaving, filtering, or treating each B. thuringiensis isolate were resuspended in







Florida Entomologist 89(4)


the stock solution and diluted with stock to pro-
vide a discriminating dose of spores and 8-endot-
oxin in diet of 250 ppm (pg/mL) for screening ex-
periments. Dose response assays were conducted
with isolates that caused >50% mortality of neo-
nates at the discriminating dose level. Concentra-
tions of 300, 150, 75, and 32.5 ppm of spores and
8-endotoxin in diet were used in the dose-re-
sponse experiments. Diet treatments for the con-
trols received stock solution only.
Prepared insect diet was liquefied by reheating
and 80 pl of diet were pipetted onto the inside sur-
face of the lid of a 0.2-mL clear PCR tube. The diet
pellets were dried for 15 min to remove approxi-
mately 20 pl of water. Bacillus thuringiensis
treatments were applied in a volume of 20 pl by
pipette to each diet pellet and the pellets were
dried for an additional 5 min. Controls were
treated equally with stock solution only. A #1 fine
camel hair brush was used to place a single D. ab-
breviatus neonate into each PCR tube containing
diet and the lid was affixed. The PCR tubes were
inverted and placed in a tube rack, covered,
placed in a sealed plastic bag with a moist paper
towel, and stored in an incubator at 27C. After 2
weeks, each larva was inspected with the aid of a
dissecting microscope and mortality was re-
corded. There were 3 replications, each contain-
ing 30 larvae, for the initial screening of each iso-
late at 250 ppm. A minimum of 3 replications,
each with 30 larvae, was used for each level of
treatment in the dose-response experiments.

Data Analyses and Statistics

Data collected from the screening experi-
ments were subjected to the Means Procedure
(SAS Institute 1999) to determine means and
standard errors for mortality of D. abbreviatus
neonates exposed to the discriminating dose of
each isolate. Paired t-tests were conducted using
the T-test Procedure (SAS Institute 1999) to de-
termine if means for mortalities in treated
groups differed from those of control groups. A
probability level of 5 percent (P < 0.05) was con-
sidered significant.
Data from the dose-response experiments
were adjusted for control mortality by the Abbott
(1925) formula and transformed (arcsine) before
analyses. Transformed data were analyzed by the
General Linear Models Procedure, and differ-
ences among treatment level means were deter-
mined by Tukey's studentized range test (SAS In-
stitute 1999). Differences among means were con-
sidered significant at a probability level of 5 per-
cent (P < 0.05). Untransformed means are
presented in the data tables. Data from isolates
that elicited a significant response to treatment
were subjected to loglO Probit analyses by the
Probit Procedure (SAS Institute 1999) to gener-
ate LC5o values and slopes of probit lines.


RESULTS

Screening Experiments

Of 19 B. thuringiensis isolates screened in diet
bioassays against D. abbreviatus neonates, 7 caused
significantly greater (P < 0.05, paired t-tests) mor-
tality compared with the controls (Table 1). Iso-
lates B-21365, B-21366, and B-21367 containing
CryET33 and CryET34 toxins caused the highest
observed mortalities (90, 88, and 91%, respec-
tively). Isolates B-21582 and B-21583 containing
the Cyt2Cal toxin provided 82 and 52% mortali-
ties, respectively. These five isolates (B-21365, B-
21366, B-21367, B-21582, and B-21583) provided
meaningful levels of mortality (>50%) and were
further evaluated in dose-response experiments.

Dose-Response Experiments

A significant effect of spore and 8-endotoxin
dose was observed for 4 of the 5 isolates that were
subjected to dose-response experiments against
D. abbreviatus neonates, including B-21365 (F =
15.52; df = 4, 28; P < 0.0001), B-21367 (F = 9.46;
df = 4, 28; P < 0.0001), B-21582 (F = 33.63; df = 4,
8; P < 0.0001), and B-21583 (F = 56.60; df = 4, 16;
P < 0.0001). The highest corrected mortality ob-
served in the dose-response experiments was 81%
provided by the wild-type isolate B-21582 at a
dose of 300 pg spores and 8-endotoxin/ml diet (Ta-
ble 2). Recombinant isolates B-21367 and B-
21583 also elicited good dose-responses with
greater than 60% mortality ofD. abbreviatus lar-
vae observed at the 300 ppm dose. The effect of
spore and 8-endotoxin dose on larval mortality
was not significant for isolate B-21366 (F = 1.71;
df = 4, 8;P = 0.2406). The dose-response obtained
with isolate B-21366 was inconsistent, the re-
sponse data were variable compared to those of
the other isolates, and larval mortality obtained
at the highest dose remained below 50%. Conse-
quently, isolate B-21366 was not included in sub-
sequent probit analyses.
Results obtained for isolates B-21365, B-
21367, B-21582, and B-21583 were examined fur-
ther by loglO probit analyses to model the effects
of spore and 8-endotoxin dose on mortality of D.
abbreviatus larvae (Fig. 1). The calculated LC5s
for larvae exposed to B-21365 in diet was 258.3
(95% FL = 130.5-2779) ppm [AI]. The slope of the
probit line was 0.65 (SE = 0.23) (X2= 8.11; df = 1;
P = 0.0044) (Fig. 1A). The LC50 for larvae exposed
to B-21367 was 115.3 (95% FL = 40.4-269.5) ppm
[AI] and the slope of the probit line was 0.93 (SE
= 0.31) (X2= 8.79; df = 1; P = 0.0030) (Fig. 1B). The
LC5 for larvae exposed to B-21582 was 50.7 (95%
FL = 28.3-72.1) ppm [AI] and the slope was 1.11
(SE = 0.21) (X2= 27.10; df= 1; P < 0.0001) (Fig. 1C).
The calculated LC50 for larvae exposed to B-21583
was 174.1 (95% FL = 114.6-361.2) ppm [AI] and


December 2006







Weathersbee et al.: Bacillus thuringiensis Isolates Against Diaprepes abbreviatus 445


TABLE 2. MORTALITY OF D. ABBREVIATUS NEONATES EXPOSED TO DIET TREATED WITH DIFFERING RATES OF LYO-
PHILIZED, SPORULATED CULTURES OF B. THURINGIENSIS ISOLATES.

% Mortality SE b by isolate&

Dose (lig/ml AI) B-21365 B-21366 B-21367 B-21582 B-21583

0.0 6.7 + 2.2 a 11.1+ 2.2 a 17.5 3.1 a 7.8 2.9 a 1.3 1.3a
32.5 26.3 6.7 b 21.1+ 12.8 a 31.3 7.4 ab 40.6 7.0 b 31.9 3.0 b
75.0 39.4 + 6.8 bc 29.9 3.5 a 40.9 8.7 ab 58.7 6.5 bc 35.0 6.9 b
150.0 40.2 + 6.3 bc 28.6 6.6 a 50.3 10.0 bc 68.2 7.2 c 44.1 + 3.3 bc
300.0 52.9 + 6.8 c 41.8 16.4 a 67.8 7.9 c 80.7 4.3 c 61.6 4.4 c

'AI refers to the active ingredient comprising lyophilized spores and 8-endotoxin ofB. thuringiensis in diet.
bMeans within a column sharing the same letter were not different (P > 0.05, Tukey's studentized range test [SAS Institute
1999]).
'Isolate numbers were assigned by curators of the ARS Culture Collection, National Center for Agricultural Utilization Re-
search, Peoria, Illinois.


the slope was 0.79 (SE = 0.0.19) (X2= 17.96; df= 1;
P < 0.0001) (Fig. 1D).

DISCUSSION

The subterranean habit, and aggressive be-
havior when confined together, complicate the
evaluation of control measures for D. abbreviatus
larvae (Lapointe & Shapiro 1999) and result in
high levels of control mortality (Schroeder & Sie-
burth 1997; Quintella & McCoy 1997; Weathers-
bee et al. 2002). The bioassay method used here
provided an efficient means for screening B. thur-
ingiensis isolates against D. abbreviatus neo-
nates. It was less expensive in terms of labor and
materials than methods used in the past. The cur-
rent method avoids these problems by confining
larvae singly on a nominal amount of diet,
thereby reducing losses in the control group. Con-
trol mortality in these experiments did not exceed
20% and was most often maintained below 10%.
The highest levels of mortality ofD. abbrevia-
tus in the screening experiments (90, 88, and
91%) occurred when neonates were fed diet con-
taining spores and 6-endotoxin of isolates B-
21365, B-21366, and B-21367, respectively. Dono-
van et al. (2005) has shown these isolates to be ac-
tive against the larvae of other species of Co-
leoptera, including the red flour beetle, Tribolium
castaneum (Herbst), and the Japanese beetle,
Popillia japonica Newman. Isolate B-21365 is a
wild-type B. thuringiensis strain that contains
genes for CryET33 and CryET34 toxins. Isolate
B-21367 is a recombinant strain that was engi-
neered to express CryET33 and CryET34 toxins.
This isolate was derived from a parent B. thuring-
iensis strain that was crystal negative (Cry). Iso-
late B-21366 also is a recombinant strain engi-
neered to express CryET33 and CryET34 toxins,
but the parent strain was wild-type and naturally
expressed the Cry3Bb2 toxin. Although B-21366
expressed an additional 6-endotoxin compared to


B-21365 and B-21367, it performed no better
against D. abbreviatus than those isolates in the
screening experiments, and its performance was
inconsistent in the dose-response experiments.
Perhaps expressing the CryET33 and CryET34
toxins in addition to Cry3Bb2 added a burden
that affected overall toxin expression and the vir-
ulence of this strain. Because this isolate pro-
duced erratic results it was not further evaluated.
The wild-type isolate B-21365, expressing the
CryET33 and CryET34 toxins, exhibited the shal-
lowest dose-response curve and highest LC50
value of those tested, probably because the high-
est concentration of B-21365 tested provided 53%
larval mortality. The recombinant isolate B-
21367 expressing the same toxins displayed a
steeper response curve, a lower LC,0 value, and
provided better predictions in the 50% response
range. It is unclear why recombinant strain B-
21367 apparently produced a more virulent prod-
uct than the wild-type B-21365 since both ex-
pressed the same toxins. Perhaps B-21367 invests
less energy into other processes such as spore for-
mation and more into toxin production. Nonethe-
less, the dose responses displayed by the recombi-
nant strain B-21367 and the wild type B-21365
against D. abbreviatus were not statistically dif-
ferent based on overlap of standard errors.
Isolates B-21582 and B-21583 provided 82 and
52% mortalities, respectively, in the screening ex-
periments. Rupar et al. (2004) demonstrated
these isolates were active against the larvae of
representative species of Siphonaptera: including
the cat flea, Ctenocephalides felis (Bouche); and
Coleoptera: including the southern corn root-
worm, Diabrotica undecimpunctata howardi Bar-
ber; the western corn rootworm, Diabrotica vir-
gifera virgifera LeConte; the Colorado potato bee-
tle, Leptinotarsa decemlineata (Say); the red flour
beetle; and the Japanese beetle. Isolate B-21582
is a wild-type B. thuringiensis strain that con-
tains the gene for the Cyt2Cal toxin, while recom-







Florida Entomologist 89(4)


1

0.9

S0.8
t
1 0.7
0
S0.6
C-)
S0.5
cr
2 0.4
LU_
. 0.3

- 0.2

0.1

0


0 200 400 600 800 1000
Dose (ppm)


0 200 400


600 800 1000


Dose (ppm)


Fig. 1. Observed (* SE bars) values and predicted (line) dose response curves for D. abbreviatus neonates ex-
posed to B. thuringiensis spores and 6-endotoxin in diet. LC,, values, slopes of loglO probit lines, and X2 values are
shown for isolates B-21365 (A), B-21367 (B), B-21582 (C), and B-21583 (D).


binant strain B-21583 originally was a Cry B.
thuringiensis that was engineered to express the
Cyt2Cal toxin. The Cyt2Cal protein is a 6-endot-
oxin that fits into a second category, aside from
the Cry toxins, known as cytolitic (Cyt) toxins. The
Cyt proteins, also known as hemolytic toxins,
cause damage to the insect midgut through pore
formation and cell lysis much like the Cry toxins.
Guerchicoff et al. (2001) provides a discussion of
the Cyt gene family, including similarities and
differences with Cry genes.
The wild-type isolate B-21582, expressing the
Cyt2Cal toxin, displayed the steepest dose-re-


sponse curve and lowest LC5s value of those tested
with the highest confidence in predictions. Though
recombinant strain B-21583 also expressed
Cyt2Cal, the probit response was not as steep as
that for B-21582, the LC5s value was greater, and
the confidence in predicted values was lower. It
appeared in this case that the wild-type isolate B-
21582 produced a more virulent product against
D. abbreviatus than did the recombinant strain B-
21583 expressing the same toxin. Perhaps isolate
B-21582 produces another, yet undetected product
that works in conjunction with the Cyt2Cal pro-
tein to induce mortality in D. abbreviatus larvae.


LCso= 258.3 (95% FL = 130.5-2779) A LCso= 115.3 (95% FL = 40.4-269.5) B
Slope= 0.65 (SE = 0.23); Slope = 0.93 (SE = 0.31);
X2 = 8.11; P=0.0044 x2 = 8.79; P = 0.0030
0.8

0.7 -

0.6

0.5 -

0.4

0.3

0.2

1- 0.1

0 200 400 600 800 1000 0 200 400 600 800 1000
0 200 400 600 800 1000 0 200 400 600 800 1000


LCso= 50.7 (95% FL= 28.3-72.1) C
Slope= 1.11 (SE = 0.21);
X2= 27.10; P S 00001


December 2006







Weathersbee et al.: Bacillus thuringiensis Isolates Against Diaprepes abbreviatus 447


Bacillus thuringiesis products have been
widely accepted in agriculture for the control of
many insect pests, but only in recent years have
strains been discovered that control coleopteran
pests. Control strategies that rely on formulated
B. thuringiensis applications are used world-wide
in many crops. Transgenic plant varieties that ex-
press B. thuringiensis 6-endotoxins have been
used in the U.S. for several years and are now
gaining international acceptance (Betz et al.
2000). Unfortunately, transgenic approaches to
plant improvement are not currently being ex-
ploited in some crops that could benefit most from
this technology, such as citrus, where a geneti-
cally engineered rootstock could be used to allevi-
ate damage caused by D. abbreviatus.
These experiments demonstrated that there
are B. thuringiensis 8-endotoxin genes currently
available that could be used to transform citrus
for protection against D. abbreviatus. The
CryET33, CryET34, and Cyt2Cal genes could be
expressed together or separately in a citrus root-
stock. An appropriately engineered citrus root-
stock, if properly managed, has the potential to
offer resistance to D. abbreviatus throughout the
life of the crop.
Because D. abbreviatus has a broad host range
and is known to feed on other plants within and
around citrus groves (Lapointe 2003), the pres-
ence of toxin in citrus roots within a grove may be
expected to result in increased feeding by larvae
on alternative food sources (either wild or inten-
tionally planted) and thus reduce the likelihood of
rapid resistance development to the toxin. The
use of transformed citrus rootstocks would also
avoid concerns associated with the possible effect
of pollen from transformed plants, particularly if
the gene inserted into the rootstock is constructed
to be expressed only in root tissue.
Development of this technology for citrus
should not be delayed since introduction of genet-
ically modified crops requires investments of time
and money, and years to complete, particularly
for slow-maturing crops like citrus. Moreover, an
effective control strategy for D. abbreviatus is
long overdue and alternatives to the proposed ge-
netically-modified citrus rootstock have not been
presented.

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Reinert et al.: Lantana Resistance to Teleonemia scrupulosa


RESISTANCE AMONG LANTANA CULTIVARS TO THE LANTANA
LACE BUG, TELEONEMIA SCRUPULOSA (HEMIPTERA: TINGIDAE)


JAMES A. REINERT, S. W. GEORGE, W. A. MACKAY AND T. D. DAVIS
Texas A&M University Res. & Ext. Center, 17360 Coit Rd, Dallas, TX 75252-6599
e-mail: j-reinert@tamu.edu

ABSTRACT

Lantana lace bug, Teleonemia scrupulosa Stal, (Hemiptera: Tingidae) is a primary insect
pest of lantana, a landscape plant commonly grown across the southern United States.
Twenty-eight cultivars of lantana were evaluated for resistance to lantana lace bug in rep-
licated field plantings. Natural infestations of lantana lace bugs developed in mid-Jul, and
were dispersed across all the replicates within 30 d in Dallas, TX. Populations of nymphs
and adults were sampled bi-weekly from Sep-Nov 1996. Highest mean populations were
present on 'Patriot Desert Sunset' (40.3 nymphs and adults/3-leaf sample/plant), 'Pink
Frolic' (20.6) and'Patriot Sunburst' (19.4). Nineteen of the cultivars exceeded 4 lace bugs per
3-leaf sample. Lace bugs were never detected on 3 cultivars, 'Weeping White', 'White Light-
ning' and 'Weeping Lavender' during the test period, and 'Imperial Purple','Patriot Rainbow'
and 'Denholm Dwarf White' had seasonal means of only 0.1 total lace bugs per sample. Cul-
tivars of L. montevidensis (K. Spreng.) Briq. (mean of 0.02 lace bugs/3 leaf sample) were
highly resistant, whereas many cultivars of L. camera L. and L. hybrida hort (6.73 and 9.54
lace bugs/3 leaf sample, respectively) were susceptible. Cultivars with gold, red, purple, and
white flowers had far fewer lace bugs than did cultivars with either orange/red, yellow, or bi-
colors of yellow with another color. These results indicate that within most flower colors or
bicolors, there exists a range of resistance among the cultivars and usually at least 1 cultivar
per color form with resistance to the lantana lace bug.

Key Words: Lantana montevidensis, Lantana camera, Lantana hybrida, host plant resis-
tance, ornamental plants, herbaceous landscape plants

RESUME

El chinche de encaje de la lantana, Teleonemia scrupulosa Stal, (Hemiptera: Tingidae) es la
plaga insectil principal de lantana, una plant de paisaje sembrada comunmente por todo el
sur de los Estados Unidos. Veinte ocho variedades de lantana fueron evaluadas para su re-
sistencia al chinche de encaje de la lantana en replicaciones de siembras de campos. Infes-
taciones naturales del chinche de encaje de la lantana se desarrollaron a mediados dejulio,
y fueron dispersados por todas las repeticiones de ensayo dentro de 30 dias en Dallas, Texas.
Las poblaciones de las ninfas y adults fueron muestreadas cada dos semanas desde sep-
tiembre hasta el mes de noviembre de 1996. El promedio de las poblaciones mas altas se en-
contraron en el 'Patriot Desert Sunset' (40.3 ninfas y adultos/por muestra de 3 hojas por
planta, el 'Pink Frolic' (20.6) y el 'Patriot Sunburst' (19.4). Diez y nueve de las variedades
sobrepasaron los 4 chinches de encaje por muestra de 3 hojas. Los chinches de encaje no fue-
ron detectados en las siguientes 3 variedades,'Weeping White','White Lightning' y'Weeping
Lavender' durante el period de la prueba; por otro lado'Imperial Purple', 'Patriot Rainbow'
y 'Denholm Dwarf White' tenian un promedio estacional de solamente 0.1 chinche de encaje
por muestra total. Las variedades de L. montevidensis (K. Spreng.) Briq. (promedio de 0.02
chinches de encaje/muestra de 3 hojas) fueron altamente resistentes, mientras muchas de
las variedades de L. camera L. yL. hybrida hort (6.73 y 9.54 chinches de encaje/muestra de
3 hojas, respectivamente) fueron susceptibles. Las variedades con flores de color de oro, rojo,
morado y blanco tenian much menos chinches de encaje que las variedades con flores de co-
lor anaranjado/rojo, amarillo, o los de dos colors de amarillo con otros colors. Estos resul-
tados indican que entire la mayoria de un color de flor o de dos colors, existe un rango de
resistencia entire variedades y usualmente por lo menos una variedad por color se forma con
resistencia al chinche de encaje de la lantana.


Many cultivars of lantana (Verbenaceae) are native to warmer regions of the Old World. As a
used as annuals or as herbaceous perennials in landscape plant, lantana is valued for its profuse
containers and hanging baskets, or as a low hedge show of color throughout a long season, often ev-
or as foundation shrubs in urban landscapes. Most ery month of the year in frost-free areas, its
cultivated species are native to tropical or sub- drought, heat and salt tolerance, aromatic foliage,
tropical North and South America, but some are and attractiveness to butterflies (Arnold 1999;







Florida Entomologist 89(4)


Everett 1981; Welch 1989). Two species are com-
monly used by the landscape industry. Lantana
camera L. is a robust, more or less prickly shrub
that is native to the southern United States and
tropical America, whereas L. montevidensis (K.
Spreng.) Briq. is a trailing or weeping lantana
with slender pubescent stems up to 90 cm long or
longer (Staff, L. H. Bailey Hortorium 1976; Ever-
ett 1981). Lantana hybrida hort cultivars is con-
sidered to be a hybrid between South American,
Mexican, and West Indian species, but some are
probably hybrids between the former two species
(Everett 1981). Lantana hybrida hort cultivars ex-
hibits characteristics ofL. camera but is far more
compact and seldom exceeds 30 cm in height.
Much of the literature on lantana centers on its
introduction around the world as an ornamental
and its unfortunate escape to become a noxious
weed. It has been reported as a weed in 47 coun-
tries competing with 14 crops and infesting mil-
lions of hectares (Holm et al. 1977). Lantana lace
bug, Teleonemia scrupulosa Stal (Hemiptera:
Tingidae), has been introduced for biocontrol of
lantana in over 20 countries, including Australia,
India, many countries in Africa, Hawaii, and many
island nations around the world (Harley and Kas-
sulke 1971; Julien 1987). Most of the literature
(other than taxonomic) on lantana lace bug relates
to its introduction and use for biological control.
Across the southern United States, lantana
lace bug is a late summer and fall annual pest of
lantana cultivated in ornamental plantings. In
Texas, as summer temperatures begin to rise and
most plants begin to suffer from water stress, lan-
tana plants thrive and flower profusely except
where they are under attack by the lantana lace
bug. The insect's behavior has been studied in Fiji
(Simmonds 1929), India (Kahn 1946; Roonwall
1952) and Australia (Fyfe 1937). The nymphs de-
velop on the underside of the leaves first causing a
yellow spotting of the foliage, followed by silver to
white bronzing with the leaves eventually brown-
ing and dropping from the plant. During nymphal
feeding, large patches of black varnish-like drop-
lets of excrement are deposited on the underside of
the leaves and the molted skins of nymphs fre-
quently remain attached. Adults are found on the
leaves but also feed heavily on the flowers and
cause a marked reduction in flowering and seed
set (Wilson 1960). The objective of the present
study was to evaluate 28 cultivars of lantana that
are used in the nursery trade for their resistance
or susceptibility to the lantana lace bug.

MATERIALS AND METHODS

Lantana plants cultivated in 10 x 10 cm pots
were planted =1 m apart in a series of raised field
beds in a randomized complete block design with
6 replications of 1 plant per replicate. Only 3 rep-
licates of several of the cultivars were evaluated


(Table 1) due to a shortage of plant material. The
highly alkaline (~8.0 pH), poorly aerated clay soil
in the beds was amended by thoroughly incorpo-
rating a 5.1-cm-thick layer of sphagnum peat.
Beds were mulched with a layer (7.5 cm thick) of
cottonseed hulls and plants were irrigated thor-
oughly with soaker hoses every 7-10 d. A 21-7-14
(N-P-K) fertilizer, in which half of the N was for-
mulated for slow-release, was incorporated into
the soil mix prior to planting at a rate of 907.2 g /
9.3 m2. A second application at the same rate of
nutrients was applied as a side dressing to the
plants in mid-Jul, ca. 8 wk later.
Most of the lantana cultivars were planted on
either 15 or 16 May 1996. Due to unavailability of
plant material on these dates,'Patriot Dove Wing'
and 'Patriot Honeylove' were not planted until 2
Aug 1996. Cultivars were chosen because of their
popularity with growers across Texas and the
southwestern United States. Only a few of these
cultivars are listed by Howard (1969) in his check-
list of lantana cultivars at the Harvard University
Arboretum, but many of the cultivars evaluated
are recommended for Texas and the Southwest
(Brenzel 1997; Perry 1992; Sperry 1991).
Population counts for lantana lace bug were
taken every 2 wk beginning 11-12 Sep through 11
Nov 1996, by examining each plant. All plants
were examined during a 2-d observation period
for each sample date. The overall plant was exam-
ined by gently lifting each of the terminal
branches and recording the number of nymph and
adult lace bugs on 3 leaves with the heaviest in-
festation. Visual evaluations for leaf bronzing, de-
foliation, overall loss of plant vigor, and the late
summer and fall reduction in flowering were all
good visual indicators of cultivars with high lace
bug populations.

Data Analysis

Data were analyzed by analysis of variance
procedures (ANOVA and GLM) in PC-SAS (SAS
Institute 1990) to determine the differences in
susceptibility among the cultivars at each obser-
vation period. Adult and nymph infestations were
analyzed separately to show colonization levels.
All count data were transformed as square root of
n + 0.001 before analysis to stabilize variances.
Untransformed means are reported. A Resistance
Performance Index = the number of times a culti-
var ranked in the top statistical grouping, was
calculated for each cultivar as a measure of over-
all resistance (Engelke et al. 1994).

RESULTS AND DISCUSSION

A naturally occurring infestation of lantana
lace bugs invaded the replicated lantana planting
in mid Jul 1996, and was first detected on plants
of the cultivar 'Pink Frolic'. By mid Aug, popula-


December 2006









TABLE 1. RESISTANCE AMONG LANTANA CULTIVARS TO THE LANTANA LACE BUG (6 REPLICATED FIELD PLOTS) SUMMER, 1996, DALLAS, TX.

Mean number of nymphs and adults per 3 leaves per plant2

12 Sep 26 Sep 10 Oct 24 Oct 11 Nov Mean Resistance
Total / Performance
Cultivar Species' Nymph Adult Nymph Adult Nymph Adult Nymph Adult Nymph Adult Plant3 Index4

Weeping White Lm 0 a 6'* 0- Oa 0" 0 a 0 a 0 a 0 a 0 a 0 a 0 a 10
White Lightning' Lm Oa 0 Oa 0 Oa Oa Oa Oa Oa Oa Oa 10
Weeping Lavender Lm Oa 0 Oa 0 Oa Oa Oa Oa Oa Oa Oa 10
Imperial Purple Lm 0.2 ab 0.2 Oa 0 Oa Oa Oa Oa Oa Oa 0.1 a 10
Patriot Rainbow' Lc 0 a 0 Oa 0 Oa Oa 0.7 a-c 0 a Oa Oa 0.1 a 10
Denholm Dwarf White Lc Oa 0 Oa 0.2 0.2 a Oa 0.3 ab 0 a Oa Oa 0.1 a 10
Radiation Lc 4.2 a-d 0.2 0.3 a 0.5 0.3 a 0 a 0.3 ab 0 a 0 a 0 a 1.2 ab 10
Dallas Red5 Lc 1.7 a-c 0 2.0 a-d 0.3 1.3 ab 1.0 bc 0.7 a-c 0 a 1.0 a-c 0 a 1.6 ab 9
Gold Mound Lh 5.3 a-d 0.8 1.8 a-c 0 1.8 ab 0.2 ab 0.3 ab 0 a 0.7 ab 0 a 2.2 ab 10
New Gold Lh 5.7 a-d 0.2 7.9 a-e 0.7 3.8 a-d 0.2 ab 1.0 a-d 0 a 0.7 ab 0 a 4.0 a-d 10
Lemon Swirl5 Lc 15.0 d-g 0 7.0 a-e 0.3 0 a 0 a 1.3 a-e 0 a 0 a 0 a 4.7 a-d 9
Patriot Honeylove5 Lc 9.3 a-f 0 1.7 ab 0.3 9.7 d-g 0.3 ab 2.3 b-f 0.7 c 0 a 0 a 4.9 a-d 7
Confetti5 Lc 6.7 a-e 0 10.0 b-f 0 7.3 c-f 0 a 3.7 c-f 0.3 a-c 0.7 ab 0 a 5.7 a-e 7
Samantha Lc 10.2 b-f 0 6.0 a-e 0 6.3 b-e 0 a 4.2 c-f 0.2 ab 4.0 c 0 a 6.2 a-e 6
American Red Bush5 Lc 4.3 a-d 1.3 12.3 d-g 0 13.3 e-h 0 a 8.0 fg 0 a 0.3 ab 0 a 7.9 a-f 7
Patriot Fire Wagon5 Lc 7.0 a-e 0 13.0 d-g 0.3 11.7 e-h 0 a 7.0 fg 0.4 a-c 2.0 bc 0 a 8.3 b-f 6
Miss Huff Lc 14.0 d-g 2.0 10.0 b-f 0.3 8.0 c-f 0 a 7.0 fg 0 a 0 a 0 a 8.3 b-f 6
Pink Caprice Lc 14.5 d-g 0.5 8.0 a-e 0.5 11.2 e-h 0.3 ab 4.5 d-f 0.2 ab 2.7 bc 0 a 8.5 b-f 6
Spreading Sunset Lh 16.8 d-g 0.2 10.3 b-f 0.2 7.3 b-f 0.2 ab 6.5 fg 0 a 1.8 a-c 0 a 8.7 b-g 6
Patriot Dove Wing5 Lc 0 a 0 30.0 f-h 0 15.7 f-h 1.7 c 2.3 b-f 1.0 c 0 a 0 a 10.1 c-g 5
Lemon Drop Lh 21.3 e-g 0.3 16.2 d-g 0.5 9.0 d-g 0.2 ab 2.8 c-f 0 a 1.5 a-c 0 a 10.4 c-g 6
Silver Mound Lh 32.0 f-g 0.3 10.8 b-f 0.5 7.3 c-f 0 a 4.0 c-f 0 a 2.2 bc 0 a 11.4 c-g 5
Golden King Lc 34.2 f-g 1.2 12.3 d-g 0.3 12.3 e-h 0.3 ab 5.2 e-g 0 a 1.8 a-c 0 a 13.5 c-g 6
LSG Red-Orange Lc 37.7 g 0.2 18.2 d-g 0.8 14.7 e-h 0.2 ab 3.8 c-f 0 a 2.3 bc 0 a 15.6 f-i 5
Irene5 Lc 26.7 fg 0.3 36.7 f-h 0 12.3 e-h 0.7 bc 6.0 e-g 0 a 0.7 ab 0 a 16.7 g-i 5
Patriot Sunburst5 Lc 3.0 a-d 0 55.3 h 1.0 22.0 h 0.3 ab 11.3 g 0.6 c 3.3 cd 0 a 19.4 hi 5
Pink Frolic Lc 40.0 g 0.7 28.0 f-h 0.3 20.7 gh 1.0 bc 7.0 fg 2.3 d 2.7 cd 0.3 b 20.6 i 2
Patriot Desert Sunset5 Lc 0 a 0 81.7 i 0.3 74.3 i 8.0 d 24.0 h 2.3 d 9.7 d 1.0 c 40.3j 3

Lantana species in study: Lm = Lantana montevidensis; Lc = L. camera; Lh = L. hybrida.
Mean no. of nymphs or adults per 3-leaf sample per plant for the observation day.
'Mean total / plant is the mean of the total of all nymphs and adults for the 5 observation periods.
Resistance Performance Index is the number of times an entry occurred in the top statistical group (highest possible is 10 for 10).
'These cultivars were only evaluated in 3 reps, all others had 6 reps.
'Analysis was made on square root of n + 0.001 transformation of the data: Untransformed means presented.
*Means in a column not followed by the same letter are significantly different by Waller-Duncan k-ratio t-test (k = 100) (P < 0.05): ns = non significant.







Florida Entomologist 89(4)


tions were also causing damage to foliage of
'Golden King','Irene', 'Lemon Drop','LSG Red-Or-
ange', 'Silver Mound', and 'Spreading Sunset'. By
early Sep, damage was widespread across the
planting and relatively consistent across the rep-
licates of the more susceptible cultivars.
The mean total of lace bugs per plant (Table 1)
represents the average number of lace bugs
(nymph + adult) per 3-leaf sample over the 5 ob-
servation periods. Data for 12 Sep showed
nymphal development on 21 of the 28 cultivars.
Highest populations were present on Pink Frolic
(40.0 nymphs/3-leaf sample/plant) and LSG Red-
Orange (37.7) whereas no populations of either
nymphs or adults were observed on 'Weeping
White', 'White Lightning', 'Weeping Lavender',
'Patriot Rainbow','Denholm Dwarf White', Patriot
Dove Wing, or 'Patriot Desert Sunset'. A signifi-
cantly lower population of only 0.2 nymphs/3-leaf
sample/plant was present on'Imperial Purple'. By
26 Sep, overall populations had decreased but
were still highest on the cultivars that had sup-
ported the high populations throughout the sea-
son. The highest nymphal populations of 81.7 and
55.3 nymphs/3-leaf sample/plant were present on
Patriot Desert Sunset and 'Patriot Sunburst', re-
spectively. Patriot Dove Wing and Patriot Desert
Sunset no longer appeared to be resistant as they
had during the evaluation 2 wk earlier. By 11 Nov,
the populations of lace bugs had declined on most
of the susceptible cultivars. A high and damaging
level, however, had been present on most of these
cultivars throughout the 8-wk evaluation period
and many of the cultivars were severely damaged
with bronzed leaves and a considerable loss of
leaves, flowers, and plant thriftiness. Once a plant
was damaged to the extent that bronzed leaves
were evident, it remained disfigured throughout
the remainder of the growing season.
Lace bugs were never detected on Weeping
White, White Lightning, and Weeping Lavender
during the test period. Imperial Purple, Patriot
Rainbow, and Denholm Dwarf White had mean
populations of 0.1 total lace bugs per sample and
never exceeded <0.7 insects per sample. The Re-
sistance Performance Index shows that in addi-
tion to the aforementioned cultivars, 'Radiation',
'Dallas Red', 'Gold Mound', 'New Gold', and
'Lemon Swirl' also ranked either 9 or 10 (out of


10) times in the top statistical groupings. How-
ever, 'Patriot Honeylove', 'Confetti', 'Samantha'
and 'American Red Bush' were also in the top sta-
tistical group for mean total lace bugs per 3-leaf
plant sample, but these cultivars sustained sig-
nificant lace bug populations during Sep and
early-Oct and only occurred in the top statistical
ranking either 6 or 7 times.
When cultivars are grouped by species and an-
alyzed, the species, L. montevidensis (4 cultivars
with a mean of 0.02 lace bugs/3-leaf sample) is
highly resistant, whereas several of the L. ca-
mara and L. hybrida cultivars were resistant but
most of them were susceptible to the lantana lace
bug (Table 2). Cultivars of L. montevidensis pro-
duce either white or purple flowers.
Cultivars were analyzed separately for flower
color. A cultivar with two predominant flower col-
ors was analyzed as bicolor for the 2 colors. Culti-
vars with purple or white flower color had far
fewer lantana lace bugs (means of 0.03 and 1.73,
respectively) developing on them than did culti-
vars with other flower colors (Table 3). For the 2
white-flowered L. camera, Denholm Dwarf White
is resistant while Patriot Dove Wing is a highly
susceptible cultivar. A cultivar with low infesta-
tions of lace bugs and in the top statistical rank-
ing or resistant was identified for each flower col-
ors except for 2 bicolors, white/yellow and red/yel-
low (Table 3). Overall, it appears that cultivars
with either yellow or yellow bicolor flowers are
among the most susceptible to the lantana lace
bug. Flower color has been implicated as an indi-
cator of resistance in other ornamental plants. In
studies with Canna spp., cultivars with red-, or-
ange-, and scarlet-flowers were more susceptible
to canna leafroller, Calpodes ethlius Stoll than
those with yellow- or rose-flowers (Reinert et al.
1983). Also, in studies with oleander, Nerium ole-
ander L., susceptibility to oleander caterpillar,
Syntomeida epilais jucundissima Dyar, was much
higher on cultivars with certain flower colors than
on those with other flower colors (J. A. Reinert et
al. unpublished data). Resistance may not be de-
termined by flower color, but there appears to be a
relationship to color, although not independent.
Additional work is needed to fully understand
what the relationship is between flower color and
resistance to lantana lace bug and other insects.


TABLE 2. IMPACT OF SPECIES OF LANTANA ON THE INFESTATION LEVEL OF LANTANA LACE BUG.

Lantana spp. No.' Range of means for cultivars Mean total nymphs + adults/3 leaves/plant2

L. monteuidensis 4 0.0-0.1 0.02 a*
L. camera 19 0.1-40.3 6.73 b
L. hybrida 6 2.2-11.4 9.54 b

'No. of cultivars evaluated for each species.
Mean total/plant are the mean of all nymphs and adults per 3 leaves per plant for 5 observation periods.
*Means in column not followed by the same letter are significantly different by Waller- Duncan k-ratio t-test (k = 100) (P < 0.05).


December 2006







Reinert et al.: Lantana Resistance to Teleonemia scrupulosa


TABLE 3. IMPACT OF THE FLOWER COLOR OF LANTANA CULTIVARS ON THE POPULATION LEVELS OF LANTANA LACE BUG.

Lace bugs per Mean total lace bugs/3 leaves/5
observation period3 obs. periods on each flower color4

Flower color (no.)' Cultivars spp2 Highest count Mean5

Purple (2)6 0.03 a
Weeping Lavender Lm 0 0 a*
Imperial Purple Lm 0.4 0.1 a
White (4) 1.73 ab
Weeping White Lm 0 0 a
White Lightning Lm 0 0 a
Denholm Dwarf White Lc 0.3 0.1 a
Patriot Dove Wing Lc 30.0 10.1 c-g
Gold (2) 3.10 bc
Gold Mound Lh 6.1 2.2 ab
New Gold Lh 8.6 4.0 a-d
Red (2) 4.75 bc
Dallas Red Lc 2.3 1.6 ab
American Red Bush Lc 13.3 7.9 a-f
Orange/Red (3) 8.47 cd
Radiation Lc 4.4 1.1 ab
Spreading Sunset Lh 17.0 8.7 b-g
LSG Red-Orange Lc 37.9 15.6 f-i
Pink/Yellow (6) 9.27 d
Patriot Rainbow Lc 0.7 0.1 a
Patriot Honeylove Lc 10.0 4.9 a-d
Confetti Lc 10.0 5.7 a-e
Pink Caprice Lc 15.0 8.5 b-f
Irene Lc 36.7 16.7 g-i
Pink Frolic Lc 40.7 20.6 i
Yellow (6) 10.28 d
Lemon Swirl Lc 15.0 4.7 a-d
Samantha Lc 10.2 6.2 a-e
Miss Huff Lc 16.0 8.3 b-f
Lemon Drop Lh 21.6 10.4 c-g
Golden King Lc 35.4 13.5 c-g
Patriot Sunburst Lc 56.3 19.4 hi
White/Yellow (1) 11.43 d
Silver Mound Lh 32.3 11.4 c-g
Red/Yellow (2) 24.30 e
Patriot Fire Wagon Lc 13.3 8.3 b-f
Patriot Desert Sunset Lc 82.0 40.3 j

Cultivars with two predominant flower colors were analyzed as bicolor for the 2 colors.
'Lantana species in study: Lm = Lantana montevidensis; Lc = L. camera; Lh = L. hybrida.
Plants sampled by counting total nymphs and adults per 3 leaves per plant for each of 5 observation periods; highest count per
any sample period and mean total count during the test period.
'Mean total/plant = mean of the total of all nymphs and adults combined for the 5 observation periods.
5Data taken from Table 1.
'Number of cultivars with the flower color.
*Means in a column not followed by the same letter are significantly different by Waller-Duncan k-ratio t-test
(k =1 00) (P 0.05).


CONCLUSIONS provides resistant purple- (Weeping Lavender
and Imperial Purple) or white-flowered (Weeping
This information on the range of susceptibility White and White Lightning) cultivars. Addition-
among cultivars within each of the flower color ally, Denholm Dwarf White is a resistant white-
groupings should be of considerable value to com- flowered L. camera cultivar with a more upright,
mercial growers, retail nurserymen, landscapers, mounding growth habit. When lantana lace bug
and consumers. The species, L. montevidensis, was being evaluated as a biocontrol agent in Aus-







Florida Entomologist 89(4)


tralia, Haseler (1966) observed that it defoliated
white-flowered lantana (no species or cultivars
given) and caused the plants to die. Our data
show both resistance and susceptibility among
the white-flowered cultivars. For the other flower
color or bicolor groupings, there is a range of sus-
ceptibility among cultivars as well, typically with
at least 1 cultivar ranking in the top statistical
grouping and expressing resistance. For example,
in the pink/yellow-flowered group, Patriot Rain-
bow is resistant, whereas all the other cultivars
are susceptible with Irene and Pink Frolic being
extremely susceptible. Harley & Kassulke (1971)
and Radunz (1971) reported that lantana lace bug
showed a preference for red-flowered lantana spe-
cies, pink-flowered were least preferred, and
white and orange showing intermediate damage,
but they did not identify cultivars or species.
Their statement, and the results presented here,
emphasize the need to understand the potential
genetic resistance of each cultivar regardless of
the flower color. This range of susceptibility
among the cultivars within each color grouping
should allow the consumer to install landscape
plantings of lantana that have an array of flower
color but still provide a high level of natural (ge-
netic) protection against this destructive pest.

ACKNOWLEDGMENTS

Appreciation is extended to Erin Smith for technical
assistance.

REFERENCES CITED

ARNOLD, M. A. 1999. Landscape Plants for Texas and
Environs. Stipes Publ. L.L.C. 596 p.
BRENZEL, K. N. (Ed.). 1997. Sunset National Garden
Book. Sunset Books Inc., Menlo Park, CA.
ENGELKE, M. C., V. G. LEHMAN, AND S. J. MORTON.
1994. A turf performance index to classify varietal
performance in regional and national trials. Agron.
Abstr. 1994: 183.
EVERETT, T. H. 1981. The New York Botanical Garden
Illustrated Encyclopedia of Horticulture. Garland
Publ., Inc., New York Vol. 6.


FYFE, R. V. 1937.The lantana bug, Teleonemia lantanae
Distant. J. Council Sci. and Indian Res. 10(3): 181-
186.
HARLEY, K. L. S., AND R. C. KASSULKE. 1971. Tingidae
for biological control of Lantana camera (Verben-
aceae). Entomophaga 16(4): 389-410.
HOLM, L. G., D. L. PUCKNETT, J. V. PANCHO, AND J. P.
HERBERGER 1977. The World's Worst Weeds. HI
Univ. Press, Honolulu 609 p.
HOWARD, R. A. 1969. A check list of cultivar names used
in the genus Lantana. Arnoldia 29(11): 73-109.
JULIEN, M. H. 1987. Biological Control of Weeds, a
World Catalogue of Agents and their Target Weeds.
2nd ed. Unwin Brothers Ltd., The Gresham Press,
Old Woking, Surrey, Great Britain 144 p.
KAHN, A. H. 1946. On the lantana bug (Teleonemia scru-
pulosa Stal.). Indian J. Entomol. 6 (1-2): 149-161.
PERRY, B. 1992. Landscape Plants for Western Re-
gions, An Illustrated Guide to Plants for Water
Conservation. Dai Nippon Printing Co., Ltd., Hong
Kong.
RADUNZ, L. A. J. 1971. Some Aspects of the Preferential
Behavior of Teleonemia scrupulosa Stal, Towards its
Host Plant Lantana camera. Honours Thesis, Uni-
versity of Queensland, Australia.
REINERT, J. A., T. K BROSCHAT, AND H. M. DONSELMAN.
1983. Resistance of Canna spp. to the skipper but-
terfly, Calpodes ethlius (Lepidoptera: Hesperiidae).
Environ. Entomol. 12(6): 1829-1832.
ROONWALL, M. L. 1952. The natural establishment of an
imported insect in India. The lantana bug, Teleone-
mia scrupulosa Stal (= lantanae Distant: Hemiptera,
Tingidae) with a description of its eggs, nymphs, and
adult. J. Zool. Soc. India 4(1): 1-16.
SAS INSTITUTE. 1990. SAS/STAT User's Guide, version
6.10 ed. SAS Institute, Cary, NC.
SIMMONDS, H. W. 1929. The life history of Teleonemia
lantanae. Agr. J. Dep. Agric., Fiji Islands 2(1): 36-39.
SPERRY, N. 1991. Neil Sperry's Complete Guide to Texas
Gardening. Taylor Publ. Co., Dallas, TX 388 p.
STAFF OF THE LIBERTY HYDE BAILEY HORTORIUM. 1976.
Hortus Third. Macmillan Publ. Co., Inc., New York
1290 p.
WELCH, W. C. 1989. Perennial Garden Color for Texas
and the South. Taylor Publ. Co., Dallas, TX 268 p.
WILSON, F. 1960. A Review of the Biological Control of
Insects and Weeds in Australia and Australian New
Guinea. Commonwealth Agric. Bur., Farnham
Royal, Bucks, England. Technical Communication
No. 1: 91 p.


December 2006







Boyd & Held:Androthrips ramachandrai in the U.S.


ANDROTHRIPS RAMACHANDRAI (THYSANOPTERA: PHLAEOTHRIPIDAE):
AN INTRODUCED THRIPS IN THE UNITED STATES

DAVID W. BOYD, JR.'AND DAVID W. HELD2
'USDA-ARS Southern Horticultural Laboratory P.O. Box 287 Poplarville, MS 39470
e-mail: dboyd@ars.usda.gov

2Mississippi State University Coastal Research and Extension Center 1815 Popps Ferry Road, Biloxi, MS 39532
e-mail: david.held@msstate.edu

ABSTRACT

Androthrips ramachandrai Karny is an exotic thrips, assumed to be predacious, and is as-
sociated with gall-inducing thrips. It was first reported in the U.S. from FL, and intercepted
in CA from Thailand in 2002. We surveyed Ficus spp. with Gynaikothrips-induced galls in
AL, CA, FL, HI, LA, MS, and TX, and document that A. ramachandrai is now established in
CA, FL, HI, and TX. It probably has been spread by the ornamental horticulture industry.
We outline its biology and compare it to a congenerA. flavipes, a documented thrips predator.
Androthrips ramachandrai has the potential to be a beneficial biological control agent and
a hindrance to weed biological control.
Key Words: predator, invasive species, Gynaikothrips, biotic interference

RESUME
Androthrips ramachandrai es un trips ex6tico, que parece ser un depredador, y esta asociado
con trips que produce agallas. El trips fue informado por primera vez en los Estados Unidos
en el estado de la Florida, e interceptado en California en el 2002 de Tailandia. Nosotros
muestreamos las plants de Ficus spp. con agallas inducidas por Gynaikothrips en Alabama,
California, Florida, Hawaii, Louisanna, Mississippi, y Texas y documentamos que A. ram-
achandrai esta ahora establecido en California, Florida, Hawai, y Texas. Probablemente el
trips ha sido dispersado por la industrial de horticulture ornamental. Nosotros tambi6n des-
cribimos su biologia y la comparamos con su congenereA. flavipes, un depredador de trips ya
documentado. Androthrips ramachandrai tiene el potential para ser un agent de control
biol6gico ben6fico y un obstaculo para el control biol6gico de malezas.


Held et al. (2005) predicted that gall-inhabiting
arthropods found in Gynaikothrips-induced galls
on Ficus spp. could be inadvertently transported
within the continental U.S. through the ornamen-
tal plant industry. Androthrips ramachandrai
Karny (Thysanoptera: Phlaeothripidae) is known
to inhabit the galls of both Gynaikothrips uzeli
(Zimmermann) and G. ficorum (Marchal) (Thys-
anoptera: Phlaeothripidae) (Takahashi 1934), and
is probably being moved throughout the U.S. in
shipments of nursery-grown Ficus.
The first record ofA. ramachandrai in the U.S.
was of two specimens collected in March 2002,
Miami-Dade Co., FL from Ficus microcarpa (Mo-
raceae) galled by G. ficorum (Nakahara & Ed-
wards 2002). Another collection ofA. ramachand-
rai was made from Riverside Co., CA on 02 Aug of
the same year (Gaimari 2005). It was intercepted
during a federal foreign-quarantine inspection
along with Gigantothrips elegans Zimmermann
(Thysanoptera: Phlaeothripidae) and Gynaiko-
thrips malabaricus Ramakrishna on Ficus sp. im-
ported from Nong Nooch Tropical Botanical Gar-
den in Thailand (G. Watson, California Depart-
ment of Food and Agriculture, pers. comm.).


Androthrips ramachandrai was described
from India and found in association with the gall
thrips Austrothrips cochinchinensis Karny (Thys-
anoptera: Phlaeothirpidae) on Calycopteris (=Ge-
tonia) floribunda (Combretaceae) (Karny 1926).
Worldwide, Androthrips contains 12 species
(Mound 2005).Androthrips flavipes Schmutz is a
known predator of thrips (Ananthakrishnan &
Varadarasan 1977; Varadarasan & Anan-
thakrishnan 1981), and other Androthrips spe-
cies are assumed to be predators, too, but little or
nothing is known about their biology.
Androthrips ramachandrai (Fig. 1) is dark
brown to black. It can be distinguished from other
dark, large phlaeothripids by its large fore femora
with a strong, cylindrical tooth near the base fol-
lowed by a row of small tubercles (Fig. 2). Its tube
(abdominal segment X) is almost half the length of
Gynaikothrips spp., which is easily detectable. It
can be separated from other Androthrips by the
dark middle and hind tibiae (Karny 1926).
Not much is known about the biology ofA. ra-
machandrai. It is rare in newly formed galls of
Austrothrips cochinchinensis. However, as galls
mature, A. ramachandrai becomes more abun-







Florida Entomologist 89(4)


'''Db.;


r4,


"C


I,











B


Fig. 1. Dorsal view of Androthrips ramachandrai
from Riverside Co., CA. (A) Slide mounted, cleared spec-
imen and (B) wet mounted specimen. Scale bar = 1 mm.


dant as the population of A. cochinchinensis de-
clines (Ananthakrishnan 1978), which might in-
dicate that A. ramachandrai is predacious on the
gall-inducing thrips, similar to A. flavipes (Anan-
thakrishnan & Varadarasan 1977).
Currently, A. ramachandrai is known from
Australia, Costa Rica (L. Mound, pers. comm.),
India (Karny 1926), Taiwan (Takahashi 1934),
and Thailand (Ananthakrishnan 1978). Herein
we report its establishment in the U.S.
The purpose of this paper is to document the
currently known distribution ofA. ramachandrai
in the U.S., provide a brief overview from the lit-
erature of its biology, and increase the awareness
of regulatory and research entomologists in


Fig. 2. Fore femur of Androthrips ramachandrai
from Riverside Co., CA. (A) Wet mounted, right ventral
fore femur and (B) slide mounted, cleared left fore fe-
mur. The white arrow is pointing to the strong, cylindri-
cal tooth near the base and back arrow to the row of
small tubercles. Scale bars = 250 lm.


North American to this thrips, which could be-
come economically and ecologically important.

MATERIALS AND METHODS
We collected and solicited Gynaikothrips-in-
duced galls from the following states in the U.S.:
AL, CA, FL, HI, LA, MS, and TX. Galls and con-
tents were collected in the field, preserved imme-
diately in 95% ethanol, and taken to the lab for
identification of the thrips. Museum records were
requested for A. ramachandrai from CA (Califor-
nia Department of Agriculture), FL (Florida State
Collection of Arthropods), and TX (Texas A & M
University).

RESULTS AND DISCUSSION
Ficus galls collected from South Padre Island,
Cameron Co., TX by DWH on 24 Aug 2005, from
Riverside Co, CA by Chris Hanlon (University of
California, Riverside) on 08 Mar 2005, and from
Oahu Island, HI by Frank Howarth (Bishop Mu-
seum, Honolulu) on 17 Apr 2006 contained speci-
mens ofA. ramachandrai. Galls from TX were ini-
tiated by G. uzeli and collected from 7 Ficus trees
at 2 locations (Table 1). Galls from CA were initi-
ated by G. ficorum and collected at 1 location with
a total of 258 G. ficorum and 21A. ramachandrai
(total number of galls not known). Galls from HI
were initiated by G. ficorum and collected at 1
location with 16 G. uzeli. Voucher specimens of
G. ficorum, G. uzeli, and A. ramachandrai from
CA and TX have been deposited in the USDA,
ARS, Systematic Entomology Laboratory, Belts-
ville, MD; andA. ramachandrai from HI have been
deposited in the Bishop Museum, Honolulu, HI.
The Florida Department of Plant Industry has
at least 44 records of A. ramachandrai (2002-
2006) from 11 southern counties in Florida
(Brevard, Broward, Miami-Dade, Glades, Hills-
borough, Lee, Martin, Monroe, Palm Beach,
Pinellas, and Sarasota) and from the following
plants: Artocarpus heterophyllus (Moraceae); Fi-
cus benjamin and F microcarpa (Moraceae);
Malvaviscus penduliflorus (Malvaceae); Schef-
flera actinophylla (Araliaceae); and Tabebuia het-
erophylla (Bignoniaceae).
The California Department of Food and Agri-
culture has one record of A. ramachandrai col-
lected from F microcarpa originating from Irvine
Co. and intercepted in Santa Clara Co. on 09 Nov
2004. Galls collected or solicited from AL, LA, and
MS did not contain specimens ofA. ramachand-
rai, and no further museum records were avail-
able for TX.
The 2 records from California are new state
records, because the previous record was an inter-
cepted specimen from Thailand (see above). The
records from Texas and Hawaii also are new state
records.


December 2006







Boyd & Held:Androthrips ramachandrai in the U.S.


TABLE 1. GALL INHABITANTS BY SPECIES COLLECTED FROM SOUTH PADRE ISLAND, CAMERON CO., TX, AUGUST 2005.

No. galls Gynaikothrips uzeli Androthrips ramachandrai Montandoniola moraguesi

Site 1
Plant 1 3 15 9 0
Plant 2 3 25 14 0
Plant 3 3 73 3 3 nymphs
Total 9 113 26 3 nymphs
Site 2
Plant 1 4 5 34 0
Plant 2 3 12 1 0
Plant 3 3 24 3 0
Plant 4 4 38 0 0
Total 14 79 38 0

Galls were randomly collected from each site, where site 1 was the Convention Center (large landscape plants) and site 2 a local
restaurant (containerized plants) on the island.


Our findings from TX (Table 1) might indicate
that Gynaikothrips populations decline during an
increased presence ofA. ramachandrai. However,
further data is needed to substantiate this claim.
The trend is consistent with a pattern found by
Anathakrishnan (1978) in which populations of
Austrothrips cochinchinensis decreased as A. ra-
machandrai progressively increased.
Montandoniola moraguesi (Puton) (Hemi-
ptera: Anthocoridae) was present in the galls
from TX and HI. This anthocorid is known to feed
on gall-inducing thrips and also on A. ram-
achandrai and A. flavipes (Dobbs & Boyd 2006).
What impact this anthocorid might have on the
effectiveness ofA. ramachandrai in reducing pest
thrips populations is not known. Another natural
enemy of Gynaikothrips is the wasp Thripasti-
chus gentilei (del Guercio) (Hymenoptera: Eu-
lophidae), which parasitizes species of Andro-
thrips (Loomans et al. 1997). What quantitative
impact these 3 natural enemies (individually or
together) have on pest-thrips populations and on
each other remains unassessed.
Nothing is known about the ecology ofA. ram-
achandrai, but some information may be inferred
from the better-studied congener A. flavipes.
When adults ofA. flavipes enter mature galls of
Arrhenothrips ramakrishnae Hood (Thysan-
optera: Phlaeothripidae), they feed on about 10%
of the available prey and deposit eggs in galls
near their prey eggs. After hatching, the larvae
consume most of the remaining prey and resort to
cannibalism. This behavior can occur whether
prey is abundant or not and is a limiting factor to
its own population growth (Varadarasan & Anan-
thakrishnan 1981). Typically they feed on the
eggs and larvae, but not adults. By the time the
larvae pupate, they have devoured almost 80% of


available prey (Sureshkumar & Ananthakrish-
nan 1987). Androthrips flavipes develops faster
than the galling thrips, which enables the preda-
tor to complete its life cycle more quickly than the
prey (Varadarasan & Ananthakrishnan 1982).
Strangely, the enlarged fore femora of adultA. fla-
vipes are not used in subduing prey (Varadarasan
& Ananthakrishnan 1982; Sureshkumar & Anan-
thakrishnan 1987).
Because A. ramachandrai feeds primarily on
the immature stages of thrips in galls, this does
not preclude it from attacking the immature
stages of surface feeding thrips. In a laboratory
trial,A. flavipes readily consumed thrips prey in a
Petri dish (Varadarasan & Ananthakrishnan
1981). In FL, where A. ramachandrai is well es-
tablished, there may be possible biotic interfer-
ence (Reimer 1988) with thrips, particularly
phlaeothripids such as Pseudophilothrips ichini
(Hood) (Thysanoptera: Phlaeothripidae), used for
biological control of invasive peppertrees (Cuda et
al. 2005). The effectiveness of the weed biocontrol
Liothrips urichi Karny (Thysanoptera: Phlaeo-
thripidae) released for biocontrol of the weed, Cli-
demia hirta (Melastomataceae), in HI has been
reduced by generalist predators (Reimer 1988).
Androthrips ramachandrai is now established
in 3 continental U.S. states and HI, but very little
is known about its biology or ecology. It is as-
sumed to be predacious and could potentially
have an impact on thrips populations, including
pests and weed biological control agents. Its pres-
ence could cause biotic interference (Reimer
1988) or it could prove to be a successful natural
enemy against Gynaikothrips spp. on ornamental
Ficus. We intend this information to increase the
awareness of regulatory agents and facilitate the
identification of this potentially important thrips.











ACKNOWLEDGMENTS

We thank C. Hanlon (University of California, River-
side), Frank Howarth (Bishop Museum, Honolulu, HI),
and R. Messing (University of Hawaii, Kapaa) for pro-
viding material from their respective locations. We
thank G. B. Edwards (Florida Department of Agricul-
ture and Consumer Services) for providing records of
A. ramachandrai in Florida. We thank E. G. Riley
(Texas A&M), G. Watson (California Department of
Food and Agriculture), and D. Nickle (USDA, ARS, SEL)
for checking their respective collections for specimens of
A. ramachandrai. We thank L. Mound (CSIRO Ento-
mology, Canberra, Australia), B. Sampson (Mississippi
State University), and two anonymous reviewers for
providing helpful comments to an earlier draft of the
manuscript. We gratefully acknowledge G. Watson for
providing Fig. 1A.

REFERENCES CITED

ANANTHAKRISHNAN, T. N. 1978. Thrips Galls and Gall
Thrips. Zoological Survey of India Technical Mono-
graph 1: 1-95.
ANANTHAKRISHNAN, T. N., AND S. VARADARASAN. 1977.
Androthrips flavipes Schmutz (Insecta: Thysan-
optera), a predatory inquiline in thrips galls. Ento-
mon 2: 105-107.
CUDA, J. P., J. C. MEDAL, D. H. HABECK, J. H. PEDROSA-
MACEDO, AND M. VITORINO. 2005. Classical Biologi-
cal Control of Brazilian Peppertree (Schinus terebin-
thifolius) in Florida. University of Florida IFAS
Extension Publication ENY-820: 1-7.
DOBBS, T. T., AND D. W. BOYD, JR. 2006. Status and dis-
tribution of Montandoniola moraguesi (Hemiptera:
Anthocoridae) in the continental United States.
Florida Entomol. 89: 41-46.
GAIMARI, S. 2005. California Plant Pest and Disease Re-
port: July 2002 through July 2005. Plant Pest Diag-
nostics Branch, California Department of Food &
Agriculture 22: 1-78.


December 2006


HELD, D. W., D. BOYD, T. LOCKLEY, AND G. B. EDWARDS.
2005. Gynaikothrips uzeli (Thysanoptera: Phlaeo-
thripidae) in the southeastern United States: Distri-
bution and review of biology. Florida Entomol. 88:
538-540.
KARNY, H. H. 1926. Studies on Indian Thysanoptera.
Memoirs of the Department of Agriculture in India
9: 187-239.
LOOMANS, A. J. M., T. MURAI, AND I. D. GREENE. 1997.
Hymenopterous parasitoids and parasitic nema-
todes, pp. 355-397 In T Lewis [ed.], Thrips as Crop
Pests. CAB International, New York.
MOUND, L. A. 2005. Thysanoptera (Thrips) of the
World-A Checklist. http//:www.ento.csiro.au/thysan-
optera/worldthrips.html. Last accessed 22 Sept. 2006.
MOUND, L. A., AND R. MARULLO. 1996. The thrips of
Central and South America: an introduction (In-
secta: Thysanoptera). Memoirs on Entomology, In-
ternational 6: 1-488.
NAKAHARA, S., AND G. B. EDWARDS. 2002. Ornamentals,
woody plants, and palms: Androthrips ramachand-
rai, a thrips, p. 7 In S. E. Halbert [ed.], FDACS, Di-
vision of Plant Industry, Tri-Ology (Entomology
Section) 41(2).
REIMER, N. J. 1988. Predation ofLiothrips urichi Karny
(Thysanoptera: Phlaeothripidae): a case of biotic in-
terference. Environ. Entomol. 17: 132-134.
SURESHKUMAR, N., AND T. N. ANANTHAKRISHNAN. 1987.
Biotic interactions in relation to prey-predator rela-
tionship with special reference to some thrips spe-
cies (Thysanoptera: Insecta). J. Entomol. Res. 11:
192-202.
TAKAHASHI, R. 1934. Association of different species of
thrips in their galls (in Japanese). Botany Zool., To-
kyo 2: 1827-1836.
VARADARASAN, S., AND T. N. ANANTHAKRISHNAN. 1981.
Population dynamics and prey-predator/parasite re-
lationships of gall-forming thrips. Proc. Indian. Nat.
Acad. B 47: 321-340.
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Biological studies on some gall-thrips. Proc. Indian
Acad. Sci. B48: 35-43.


Florida Entomologist 89(4)







Cherry et al.: New Turf Planthopper Pest


LIBURNIA PSEUDOSEMINIGRA (DELPHACIDAE: HOMOPTERA),
A NEW AND UNUSUAL PEST OF ST. AUGUSTINEGRASS

RON CHERRY', PHIL STANSLY2, RUSSELL NAGATA' AND SUSAN HALBERT3
'University of Florida/IFAS, Everglades Research and Education Center
3200 E. Palm Beach Road, Belle Glade, FL 33430

2University of Florida/IFAS, Southwest Florida Research and Education Center
2686 State Road 29 North, Immokalee, FL 34142

3Division of Plant Industry, 1911 SW 34th Street, Gainesville, FL 32608

ABSTRACT

No publications have reported delphacid planthoppers (Family Delphacidae) to be turf pests
in the United States. In March 2005, a large infestation of the delphacid planthopper, Libur-
nia pseudoseminigra (Muir & Gifford), was found infesting St. Augustinegrass, Steno-
taphrum secundatum (Walt.) Kuntze, in a commercial sod farm in southern Florida.
Thereafter, a survey was conducted in commercial sod farms in southern Florida to deter-
mine the extent of the planthopper infestation in different St. Augustinegrass varieties. The
planthoppers were found in low numbers in Floratam, Palmetto, and Seville and were mod-
erately abundant in Bitterblue. However, Classic was clearly the variety supporting large
numbers of the planthopper. Liburnia pseudoseminigra was described from Florida and ap-
parently is a native species. Virtually nothing was known about its biology prior to its ap-
pearance as a pest of St. Augustinegrass in this study.

Key Words: Delphacidae, Liburnia, turf, St. Augustinegrass

RESUME

No se ha reportado publicaciones de delfacidos saltadores de plants (Family Delphacidae)
como plagas de c6sped en los Estados Unidos. En marzo del 2005, se encontr6 una infestaci6n
grande del delfacido saltador de plants, Liburnia pseudoseminigra (Muir y Gifford), infes-
tando el c6sped de San Augustin, Stenotaphrum secundatum (Walt.) Kuntze, en una finca co-
mercial de c6sped en el sur del estado de Florida. Por ello, se realize un muestreo en fincas
comerciales de c6sped en el sur de la Florida para determinar el alcanze de la infestaci6n del
saltador de plants en variedades diferentes de c6sped de San Augustin. Se encontraron los
saltadores de plants en numerous bajos en Floratam, Palmetto y Seville y fueron moderada-
mente abundante en Bitterblue. Sin embargo, la variedad Classic fue claramente la que apoyo
el numero mayor de saltadores de plants. Liburnia pseudoseminigra fue descrito del estado
de Florida y aparentemente es una especie native. Practicamente no se sabia nada acerca de
su biologia antes de que apareciera como plaga de c6sped de San Augustin en este studio.


St. Augustinegrass, Stenotaphrum secunda-
tum (Walt.) Kuntze lawns are grown throughout
the southern United States because of their cli-
matic adaptation and ability to tolerate full sun to
moderate shade. Sod production in Florida is a
large industry with 37,180 ha (92,950 acres)
grown in 2003. Sixty-four percent of the Florida
sod acreage was St. Augustinegrass (Haydu et al.
2005). Numerous insects are pests of St. August-
inegrass (Potter 1998; Vittum et al. 1999). How-
ever, no publications have reported delphacid
planthoppers (Family Delphacidae) to be turf
pests on St. Augustinegrass or any other turf-
grass in the United States. In March 2005, a sod
producer located near Belle Glade, Florida, re-
quested help from the Everglades Research and
Education Center (U. of Florida, IFAS) at Belle


Glade, Florida in identifying and controlling in-
sect pests in his St. Augustinegrass fields. Upon
visiting these fields, large numbers of insects of
an unknown type were observed in fields of Clas-
sic St. Augustinegrass. Sweep net collections
were made and insects sent to Susan Halbert who
identified them as a planthopper, Liburnia pseu-
doseminigra (Muir and Gifford) in the family Del-
phacidae. The identification was confirmed by Dr.
Stephen W. Wilson, Central Missouri State Uni-
versity. Specimens are deposited at the Florida
State Collection of Arthropods (FSCA) in Gaines-
ville, FL (FSCA# E2005-1134). In 2003, a similar
infestation of the planthopper was found in
Gainesville, FL in a lawn planted to Classic St.
Augustinegrass (FSCA# E2003-1394). However,
further study of the insect was not conducted at







Florida Entomologist 89(4)


that time. Because of the novelty of a delphacid as
a turf pest, we pursued research to determine the
extent of the infestation in different St. August-
inegrass varieties.

MATERIALS AND METHODS

The majority of sod production in Florida oc-
curs in southern Florida (Haydu et al. 2005). Our
survey was conducted in 2005 at 8 different sod
farms in 5 counties (Collier, Desoto, Hendry,
Highlands, Palm Beach) in southern Florida to
obtain representative samples. Different variet-
ies also were sampled in different areas to deter-
mine if the planthoppers were responding to vari-
etal differences. Five fields of Bitterblue, 8 fields
of Classic, 10 fields of Floratam, 5 fields of Pal-
metto, and 4 fields of Seville were sampled. All
fields were sampled within a 77-d interval (21
Mar to 6 Jun) to reduce the possibility of seasonal
variation affecting planthopper populations. Only
fields that had not been treated with an insecti-
cide for at least 1 month and had large numbers of
live arthropods present were sampled.
Samples were taken in 5 transects in random
locations in each field. Each transect sample con-
sisted of 100 sweeps in a straight line with a 38-
cm diam. net. After sweeping, arthropods in nets
were bagged and later frozen. Adult and nymphal
L. pseudoseminigra in samples were counted by
microscope examination in a laboratory. Adult
and nymphal leafhoppers (Cicadellidae) also were
counted, since these are known general turf pests
(Potter 1998; Vittum et al. 1999) and this allowed
comparisons of the planthopper versus leafhop-
per abundance in the different varieties. Mean
numbers of planthoppers and leafhoppers in the
different St. Augustinegrass varieties were com-
pared by the Least Significant Difference (LSD)
test (SAS 2005).
Preliminary field observations indicated that
planthopper population densities varied among
St. Augustinegrass varieties, especially between
Classic, with high populations and Floratam,
with low populations. Thus, a laboratory study
was conducted to determine if population growth


on Classic and Floratam corresponded to field ob-
servations. Evaluations were conducted with pot-
ted Floratam and Classic St. Augustinegrass as a
no choice test. Turfgrasses were grown in pots
(6.5 x 6.5 x 9.0 cm deep) filled with a 1:1 mixture
by volume of sand and Fafard #2 potting medium
(Conrad Fafard, Agawam, MA). All test plants
were started from a single double node cutting
and all plants were 8 weeks old at the start of the
experiment on 9 May, 2005.
Individual plants were placed into a holding
cage constructed from two 1 L polypropylene food
storage containers. The containers were held to-
gether top to top by a coupler made from the lids
of the container. The center of the lids was re-
moved and the remaining ring was glued together
to form the coupler. The bottom of the top con-
tainer was removed and replaced with a screen
mesh for ventilation. Ten adult L. pseudosemini-
gra were placed in each cage and maintained in a
plant growth room. After 38 d, adult planthoppers
and nymphs were counted. The 4 treatments were
Classic with and without planthoppers and Flora-
tam with and without planthoppers. Five replica-
tions (individual plants) were tested. The mean
number of planthoppers (nymphs + adults) alive
after 38 d in the treatments was separated by us-
ing LSD analysis (SAS 2005).

RESULTS AND DISCUSSION

Abundance of L. pseudoseminigra in different
St. Augustinegrass varieties is shown in Table 1.
The planthoppers were found in all varieties, but
in low numbers in Floratam, Palmetto, and
Seville. Planthoppers were moderately abundant
in Bitterblue, but not significantly different from
the former 3 varieties. This lack of statistical sep-
aration from those varieties is partly due to very
large variation in planthopper numbers between
the 5 Bitterblue fields. Planthoppers averaged <
10/sample in 3 fields and > 100/sample in 2 fields.
Examination of morphological characteristics for
Bitterblue from the fields showed that the Bitter-
blue was not a homogenous group. Differences in
stigma colors were observed. Preliminary obser-


TABLE 1. ABUNDANCE OF L. PSEUDOSEMINIGRA IN DIFFERENT ST. AUGUSTINEGRASS VARIETIES IN SOUTHERN FLOR-
IDA SOD FIELDS.

Nymphs Adults Total
Variety Mean SD Mean SD Mean SD

Bitterblue 66.9 98.8 b 28.4 49.0 b 95.3 145.2 b
Classic 259.2 289.0 a 121.3 208.0 a 380.5 478.3 a
Floratam 0.2 0.8 b 1.5 4.7 b 1.7 5.3 b
Palmetto 13.4 20.8 b 3.5 5.3 b 16.9 25.7 b
Seville 0 0 b 0.4 + 0.9 b 0.4 + 0.9 b

Means in a column are not significantly different (P > 0.05) when followed by the same letter based on the LSD test (SAS 2005).


December 2006







Cherry et al.: New Turf Planthopper Pest


TABLE 2. ABUNDANCE OF LEAFHOPPERS IN DIFFERENT ST. AUGUSTINEGRASS VARIETIES IN SOUTHERN FLORIDA SOD
FIELDS.

Nymphs Adults Total

Variety Mean SD Mean SD Mean SD

Bitterblue 10.7 16.9 b 124.6 189.2 a 135.2 204.4 a
Classic 22.2 38.7 a 72.7 80.0 b 94.9 107.3 ab
Floratam 10.3 12.6 b 54.0 38.5 bc 64.4 49.3 bc
Palmetto 4.7 4.3 b 28.2 21.8 c 32.8 24.4 c
Seville 2.8 4.0 b 55.0 50.3 bc 57.8 52.5 bc

Means in a column are not significantly different (P > 0.05) when followed by the same letter based on the LSD test (SAS 2005).


vations indicated that the Bitterblue with white
stigma supported more planthoppers than Bitter-
blue with lavender stigma (data not shown). How-
ever, Classic was clearly the variety supporting
large numbers of the planthopper with signifi-
cantly more nymphs, adults, and total numbers
than any other variety.
Field data are corroborated by our potted plant
studies. The mean total number of planthoppers/
plant after 38 days was 375.8 on Classic initiated
with 10 adults versus 0 for the other 3 treat-
ments. Obviously, the 375.8 mean was signifi-
cantly different (P < 0.05) from the other means
and shows the high potential population growth
of the planthoppers on Classic. These data also
show the lack of population growth on Floratam
which corresponds to field observations (Table 1).
In field samples, planthopper nymphs of all
sizes were found in 4 of the varieties with espe-
cially large numbers in Classic and Bitterblue
(Table 1). These data show that the planthoppers
were reproducing in the fields and not just immi-
grating into the fields as adults. The planthoppers
were found in all 5 counties sampled indicating
widespread distribution in southern Florida sod
fields.
Abundance of leafhoppers in different St. Au-
gustinegrass varieties is shown in Table 2. As ex-
pected, some leafhoppers were found in all variet-
ies. Interestingly, Bitterblue and Classic had the
most leafhoppers. These 2 varieties also had the
most planthoppers (Table 1). The presence of leaf-
hopper nymphs indicates that reproduction was
taking place in the fields and not just adults im-
migrating into the fields.
Superimposing data from the 2 tables shows
that the planthoppers were much more respon-
sive to the different varieties than leafhoppers.
For example, planthoppers were 951 times more
abundant in Classic than in Seville. In contrast,
the maximum variation in leafhoppers occurred


between Bitterblue and Palmetto where the
former had only 4 times as many leafhoppers as
the latter variety. Also, superimposing data from
the 2 tables shows that more leafhoppers than the
planthoppers were found in every variety except
Classic. These data again emphasize that Classic
is the variety we tested which is most likely to
have problems with the planthoppers. Lastly, as
noted earlier, leafhoppers are known pests in turf
(Potter 1998; Vittum et al. 1999). Our data show
that 4 times as many L. pseudoseminigra as leaf-
hoppers were found in Classic, showing that
L. pseudoseminigra is potentially a greater pest
than the known leafhopper pests in at least 1
St. Augustinegrass variety.
Liburnia pseudoseminigra was described
(Muir & Gifford 1924) from Florida and appar-
ently is a native species. Virtually nothing was
known about its biology prior to its appearance as
a pest of St. Augustinegrass in this study.
We thank Dr. Stephen W. Wilson, Central Mis-
souri State University, for confirming the identity
of our delphacid and advising us on its proper cur-
rent generic placement.

REFERENCES CITED

HAYDU, J., L. SATTERWAITE, AND J. CISAR 2005. An eco-
nomic and agronomic profile of Florida's sod indus-
try in 2003. University of Florida, Gainesville.
MUIR, F., AND W. M. GIFFORD. 1924. Studies in North
American Delphacidae. Bull. of the Experiment Sta-
tion of the Hawaiian Sugar Planters' Assoc. Ento-
mol. Series. Bull. No. 15. 53 pp.
POTTER, D. 1998. Destructive Turfgrass Insects: Biol-
ogy, Diagnosis, and Control. Ann Arbor Press,
Chelsea, MI. 344 pp.
SAS INSTITUTE. 2005. SAS Systems for Windows. Ver-
sion 6.12. SAS Institute, Cary NC.
VITTUM, P., M. VILLANI, AND H. TASHIRO. 1999. Turf-
grass Insects of the United States and Canada. Cor-
nell Univ. Press. Ithaca, NY. 422 pp.







Florida Entomologist 89(4)


December 2006


HOST SPECIFICITY OF FOUR PSEUDACTEON SPP.
(DIPTERA: PHORIDAE), PARASITOIDS OF FIRE ANTS IN ARGENTINA
(HYMENOPTERA: FORMICIDAE)

CATALINA ESTRADA1, RICHARD J. W. PATROCK1, PATRICIA J. FOLGARAIT AND LAWRENCE E. GILBERT'
'Section of Integrative Biology and Brackenridge Field laboratory, University of Texas, Austin, TX 78712

2Centro de Estudios e Investigaciones, Universidad Nacional de Quilmes
Roque Saenz Pena 180, Bernal B1876BXD, Buenos Aires, Argentina

ABSTRACT

Several South American species of Pseudacteon have been released for biocontrol of red im-
ported fire ants Solenopsis invicta in the U.S. Here we provide additional data from host
specificity tests on 4 additional candidate species, P. nocens, P. nudicornis, P. cultellatus, and
P. obtusus, all of which are components of multi-species complexes that occur within Argen-
tinean Solenopsis populations. All 4 species were tested with sequential, no choice exposures
to the red imported fire ant S. invicta, and the tropical fire ant, S. geminata. Levels of intra-
generic specificity ranged from moderate to high and all 4 species showed greater specificity
than some Pseudacteon species already approved for release.

Key Words: biological control, parasitism, Solenopsis geminata, Solenopsis invicta, Pseudac-
teon

RESUME

Varias species de f6ridos Pseudacteon provenientes de Sur Am6rica han sido introducidas
en los Estados Unidos para utilizar como control biol6gico de la hormiga roja de fuego Sole-
nopsis invicta. En esta publicaci6n mostramos los resultados de pruebas de especificidad de
hospedero realizadas en otros cuatro f6ridos, P. nocens, P. nudicornis, P. cultellatus y P. ob-
tusus que son parte del grupo de species normalmente asociadas con poblaciones de Sole-
nopsis en Argentina. Las pruebas de especificidad consistieron en la exposici6n secuencial de
las moscas a las hormigas de fuego, S. invicta y S. geminata. El grado de especificidad de hos-
pedero en las cuatro species de f6ridos vari6 entire moderado y alto pero fue siempre mayor
que el presentado por algunas de las species aprobadas para liberar como control biol6gico
en los Estados Unidos.


Translation provided by the authors.


After their introduction in the early 20th cen-
tury, the South American fire ants, Solenopsis in-
victa Buren and S. richteri Forel, quickly expanded
their range in the United States (US). Today, S. in-
victa (red imported fire ant) occupies the southern
states of US and Puerto Rico (Callcott & Collins
1996), is spreading south into Mexico (Sanchez et
al. 2005), and is predicted to expand more than
100 km northward in the US due to anticipated cli-
mate changes (Morrison et al. 2005). The success
of the imported fire ants as invasive species is
probably due, in part, to the absence of the natural
enemies that have coevolved with this species in
South America (Porter et al. 1997). Some enemies
left behind include phorid flies of the genus Pseu-
dacteon (Diptera: Phoridae) that parasitize and
eventually kill ant workers and may likely contrib-
ute to decrease population densities to the levels
found in their homeland (Porter et al. 1997).
In the US, 3 Pseudacteon species, P. tricuspis,
P curvatus, and P litoralis, have been released as
part of the biological control program that seeks


to reduce fire ant densities to levels with less eco-
logical and economical impact (Porter & Gilbert
2004). In order to achieve this goal, more species
and specific biotypes (geographic distinctive pop-
ulation) are necessary so phorid communities
could resemble more closely those that exist in
South America. Including more species could, for
example, expose ants throughout the day and
year, increase the range of workers size exposed,
and affect ants in foraging trails as well as dis-
turbed mounds (Pesquero et al. 1996; Orr et al.
1997; Morrison et al. 1997; Folgarait & Gilbert
1999; Folgarait et al. 2003, 2005b). It could offer
the possibility of choosing species or biotypes lo-
cally adapted to ecological or climatic conditions
similar to those where flies are going to be re-
leased (Folgarait et al. 2003; Calcaterra et al.
2005: Folgarait et al. 2005a).
Species and biotypes of Pseudacteon from Bra-
zil and Argentina have been tested for their host
specificity in laboratory conditions (Gilbert &
Morrison 1997; Porter & Alonso 1999; Morrison &







Estrada et al.: Host Specificity of Four Pseudacteon spp.


Gilbert 1999; Vazquez et al. 2004). Their rates of
attack have been low when tested against the
North America-native fire ant S. geminata with
the exceptions of P. curvatus and P borgmeieri
that exhibit moderate and higher rates, respec-
tively (Gilbert & Morrison 1997; Morrison & Gil-
bert 1999). However, in spite of those attacks,
only P curvatus and P. obtusus developed in
S. geminata but with apparent low success (Por-
ter & Gilbert 2004).
Here we evaluated 4 additional species or bio-
types of Pseudacteon for their inclination to at-
tack North America-native fire ants, P nudicornis
Borgmeier, P. nocens Borgmeier, P. cultellatus
Borgmeier, and P. obtusus Borgmeier. We used in-
dividuals for a large biotype ofRP obtusus from the
Corrientes province in Argentina instead of the
small biotype from Campinas (Brazil) tested be-
fore by Morrison & Gilbert (1999). Large females
P obtusus in Argentina have a mean body length
of 1.395 mm (SD = 0.142 mm, n = 10, P.J.F. unpub-
lished data), about 35% larger than the small Ar-
gentinean biotype and the Brazilian P. obtusus
tested in Morrison & Gilbert (1999) (0.90 0.082
mm, n = 10; 0.91 0.087 mm, n = 4, respective
body length of both small biotypes, P.J.F. and
L.E.G. unpublished data). Male morphological
differences between large and small biotypes
have been reported before (Porter & Pesquero
2001). Moreover, recent phylogenetic analysis
with 2 mitochondrial and 1 nuclear gene show
that both biotypes are genetically distinct and
probably constitute different species (Kronforst et
al. 2006). Types used for the description of P ob-
tusus were collected in La Plata (Argentina) and
have a body length of approximately 1.3 mm
(Borgmeier 1925). According to this, flies from the
Argentinean large biotype tested here should
maintain the name P obtusus.

MATERIALS AND METHODS

Individuals of 4 species of Pseudacteon
(Diptera: Phoridae) flies were brought in from Ar-
gentina to the quarantine facility at the Univer-
sity of Texas, Brackenridge Field Laboratories
(BFL), between June 2003 and April 2004. Flies
were either collected directly or were reared as
progeny from females attacking S. invicta in Ar-
gentina. Fly rearing was done in the Centro de
Estudios e Investigaciones at the Universidad
Nacional de Quilmes (Buenos Aires, Argentina).
P nudicornis were collected from the Reserva
Ecol6gica Costanera Sur in Buenos Aires Prov-
ince (34.37'S and 58.22W), P obtusus around
Mercedes in the Corrientes Province (27. 78'S and
58.05W), and P nocens and P. cultellatus near
Brea Pozo in Santiago del Estero province
(28.27'S and 63.95W). Some P. cultellatus came in
February 2006 from collections by S.D. Porter in
Corrientes, Argentina.


Host specificity in these phorid species was
tested with the red imported fire ant S. invicta
and the tropical fire ant S. geminata Fabricius, de-
noted hereafter as exotic and native fire ants, re-
spectively. Exotic and native are adjectives given
with regard to the US ant biota. Polygyne colonies
(multiple queens) for S. invicta were collected
from Travis, Williamson, Wharton, La Salle, and
Bexar counties in central Texas. Polygyne colonies
of S. geminata were obtained in Travis, Lampa-
sas, and Mill counties, also in Texas. Colonies
were transported to BFL, set in a rearing room at
30C and 12:12 (L:D) cycle, and fed with frozen
crickets, sugar water, and water ad libitum.
Female flies were tested upon arrival at the
quarantine facility within 3 or 4 days after field col-
lection. When not used in tests, they were kept hu-
mid and chilled at ca. 10C in the dark to keep
them alive for longer time. Fly specificity tests
were done in plastic flight boxes (15.5 x 9.5 x 5 cm)
(henceforth denoted as arenas), containing either
S. geminata or S. invicta. Arenas were lined inside
with Fluon (Polytetrafluoroethylene) to prevent
ants from escaping and the top was covered with
clear glass. The bottoms of the plastic boxes were
covered with a layer of plaster 1 to 2 cm deep. The
plaster was moistened every day before the begin-
ning of tests. A small hole covered by a rubber
sheet in one of the sides of the arena (about 4 cm
high) allowed the introduction of flies. Plastic boxes
were used only for 1 ant host species to avoid the
occurrence of confounding odor from the other host.
Oviposition rates were recorded in arenas that
held 1 to 5 g of unsieved ants of either species
(=500-5000 ants). Approximately half of the work-
ers used had head widths of 0.8 mm or less (0.49
0.06 mm), which is one of the characteristic
traits of polygyne fire ant colonies (Morrison &
Gilbert 1998). Ants were fed daily with a meal-
worm and had continuous supply of sugar water
and water. One 10 cm diameter x 1.5-cm deep
plastic jar lid with a lateral hole was put inside
each arena to give the ants a place to hide. This
lid was moved manually before introducing the
flies and after 10 min within each test to keep
ants moving constantly around the arena, facili-
tating fly attack behavior. Arenas were placed un-
der fluorescent lamps and their internal temper-
ature fluctuated between 26 and 29C.
Pseudacteon attack behavior and development
has been studied elsewhere (Morrison et al. 1997;
Porter 1998a). Here, two behaviors were observed
as indicators of female attack motivation. 'Ap-
proaches' were recorded when flies hovered over
the dorsum of an ant and followed it for at least a
few seconds. An 'Attack' was designated when fe-
males attempted to oviposit in the ant's thorax,
which usually immobilizes the ant for a short time.
Two types of sequential no choice tests were de-
signed in order to test for Pseudacteon host speci-
ficity and to measure rates of attack on S. invicta







Florida Entomologist 89(4)


and S. geminata. Pseudacteon obtusus, P nocens,
and P cultellatus were tested with the first type of
tests. Here a single female was introduced into a
S. invicta arena for a maximum of 15 min to test
her motivation to oviposit. Behavior in these are-
nas varied among individual flies. Only individu-
als that actively approached and attacked at least
1 ant during those 15 min were considered to be
motivated to oviposit and therefore transferred to
subsequent arenas. The rate of attacks in this and
other arenas was measured as the number of at-
tacks from each female per min during a 5-min pe-
riod, counted from the first attack. After that, the
fly was immediately transferred to an arena con-
taining S. geminata for additional 15 min. There,
the number of approaches and the attack rate were
recorded. In most cases, the fly survived and it was
transferred immediately to a second S. invicta
arena to determine whether it was still motivated
to attack. In this third arena flies were observed
for maximuml5 min or, if they attacked, for 5 min
counted from the first attack. The main purpose of
this was to test for both handling effects and en-
ergy limitations when the flies were transferred
between arenas and after some time of active at-
tacks. Individual females were tested only once.
Pseudacteon nocens and P nudicornis were
tested in a second type of experimental test. In
the former type of tests, some attacks were ob-
served on S. geminata ants. The aim of the second
type of experiments was to test whether the at-
tacks on the native fire ant were either due to
mistakes (after the flies were motivated to attack
S. invicta) or due to low host specificity. Here 1 fe-
male fly was first introduced for 15 min into a S.
geminata arena and the rate of approaches and
attacks recorded. The fly was then transferred to
a S. invicta arena to test for her motivation to ovi-
posit. If the fly did not attack after 15 min the test
was aborted and the female was kept in a cool and
dark environment for later testing. If the fly at-
tacked, the rate of attack was measured for 5 min
and then it was transferred to the next arena. The
final arena for this type of experiments had either
S. geminata or S. invicta. Half of the flies that at-
tacked S. invicta were transferred to a different
S. invicta arena to test for the handling effect and
limitations of energy. The other half were trans-
ferred to S. geminata arenas in order to compare
the rate of approach and attacks on this species of
ants before and after motivation with S. invicta.
The time elapsed between the introduction of
the fly in a S. invicta arena and the time of the first
attack (orientation time) was measured and then
compared between the first and second exposure to
the exotic fire ant with the non-parametric Mann-
Whitney test (STATISTICA for Windows 1999).
The mean attack rates, measured as the mean
number of attacks per female per min, were com-
pared by the non-parametric Wilcoxon matched
pair test (STATISTICA for Windows 1999).


To reduce any effect of minor differences in
light or temperature conditions, individual flies
started the sequential tests in different S. invicta
or S. geminata arenas. Phorid flies where trans-
ferred between arenas with an aspirator similar
to the one described in Gilbert & Morrison (1997).
They were gently aspirated from one arena to a
tube whose bottom was replaced by mesh, and
then released immediately to the next arena.
Weak and disoriented flies usually walked among
the ants after release and were easily killed. To
decrease this handling effect, we let the fly go out
of the tube at their own pace, which often happens
in less than 1 min.
To determine whether flies developed inside
the ants attacked, both species of ants were mon-
itored for more than 90 d after their exposure to
phorids or until all ants died. Forty days is about
the median development period for all the species
of phorids tested (Folgarait et al. 2002a). Dead
ants were examined for pupae 3 times a week and
daily for fly eclosion. Ants were kept in a quaran-
tine room at 27C and 12L:12D cycle.

RESULTS

Females of the Pseudacteon species P obtusus,
P cultellatus, and P nudicornis showed high de-
grees of host specificity for the red imported fire
ant S. invicta. The former two species were tested
with the sequential no choice tests: invicta-gemi-
nata-invicta, whereas P nudicornis females were
exposed to S. geminata before being transferred
to the motivation arenas with the exotic fire ant.
When exposed to S. invicta in the motivation are-
nas, Pseudacteon females of these 3 species at-
tacked ants with a rate of about 3 attacks per min
(Tables 1 and 2). The same flies were then trans-
ferred to S. geminata arenas. There, 13% of P ob-
tusus, 46% P cultellatus, and all P. nudicornis fe-
males were still highly motivated and followed
ants displaying the pursuing behavior called Ap-
proach'. In most cases, flies approached ants after
being moved from the S. invicta arenas for the
first few min, but displayed no activity, after-
wards for the rest of the observation period. In
spite of those approaches only 1 female of P ob-
tusus, 4 P. cultellatus (Table 1) and none ofP. nu-
dicornis (Table 2) attacked S. geminata ants.
Fig. 1 shows the percentage of the females ex-
posed to S. geminata that attacked those ants,
and compares the percentages with those ob-
tained for P. curvatus, P tricuspis, and P litoralis,
species of phorid flies currently used for biocon-
trol of exotic fire ants in the US.
Females of the 2 Pseudacteon species that at-
tacked S. geminata, P. obtusus and P cultellatus,
however did not attempt to oviposit more than
twice, about 10% of the number of attacks per fe-
male on S. invicta. There were not enough fe-
males attacking both S. geminata and S. invicta


December 2006







Estrada et al.: Host Specificity of Four Pseudacteon spp.


TABLE 1. APPROACH AND ATTACK RATES OF PSEUDACTEON NOCENS, P. OBTUSUS, AND P. CULTELLATUS IN THE FIRST
TYPE OF SEQUENTIAL HOST SPECIFICITY TESTS.

P. nocens P. obtusus P. cultellatus

Initial exposure to S. invicta (motivation)
No. of individuals attacking 61 8 24
Attack rate 3.27 3.20 (44) 3.47 + 2.3 4 2.51 (20)
Exposure to S. geminata
No. individuals approaching/No. exposed 48/61 1/8 11/24
Approach rate 8.85 12.88 0.2 1.36 1.58
No. of individuals attacking/No. exposed 22/61 1/8 4/24
Attack rate 0.62 + 0.44 0.4 0.2 0
Final exposure to S. invicta
No. individuals attacking 36/54 4/6 14/21
Attack rate 3.55 3.07 1.76 2.14 3.85 2.18

Attack and approach rates are given as the mean + SD of rates calculated from independent females. Rates are the number of
attacks or approaches per min after 5 or 15-min exposure respectively. In 1 experimental period flies were transferred to S. gemi-
nata arenas immediately after they attacked S. invicta twice. In those cases rates of attack in the motivation arena were not cal-
culated. Therefore when the number of flies used for mean calculations were different than total number of individuals attacking,
this number is written in brackets.


to do statistical tests on this subset of flies. Nev-
ertheless, when all females were considered,
numbers of attacks per female per min to the na-
tive fire ants were always lower than those to
S. invicta (Table 1).
After being exposed to the native fire ants, fe-
males were subsequently transferred to S. invicta
arenas to test for handling effects and energy lim-
itations. Flies attacking in this last step of the
test demonstrated that lack of attraction to
S. geminata was not due to either the disturbance


of being relocated or due to lack of energy. More
than 60% of the P. obtusus, P cultellatus, and
P nudicornis females attacked in the second
S. invicta arenas (Tables 1 and 2), but they did it
in a lower rate than when first exposed to these
ants (Z = 1.98, P = 0.05, Wilcoxon matched pair
test, n = 15, data from the 3 species combined).
Pseudacteon nudicornis first and second exposure
to S. geminata showed that more flies hovered
over ants after being motivated to attack in S. in-
victa than they did otherwise. In addition, rates of


TABLE 2. APPROACH AND ATTACK RATES OF PSEUDACTEON NOCENS AND P. NUDICORNIS IN THE SECOND TYPE OF SE-
QUENTIAL HOST SPECIFICITY TESTS.


P. nocens


Initial exposure to S. geminata
No. individuals approaching/No. exposed
Approach rate
No. of individuals attacking/
Attack rate
Initial exposure to S. invicta
No. of individuals attacking
Attack rate
Subsequent exposure to S. geminata
No. individuals approaching/No. exposed
Approach rate
No. of individuals attacking/No. exposed
Attack rate
Subsequent exposure to S. invicta
No. individuals attacking
Attack rate


2/10
2.17 1.46
0/10
0


10/10
4.92 2.74


4/4
5.05 6.17
1/4
0.2


4/4
2.7 1.83


P. nudicornis


6/6
3.16 1.48


3/3
3.91 2.3
0
0


1/1
2.8


Attack and approach rates are given as the mean + SD of the rates calculated from independent females. Rates are the number
of attacks or approaches per min after 5 or 15-min exposure, respectively.







Florida Entomologist 89(4)


70

60

50

J? 40

30

20

10


0 --


H


n


P. curvatus P. nocens P. cultellatus P. obtusus P. tricuspis P. litoralis P. nudicornis
Fig. 1. Percentage of Pseudacteon females that attacked S. geminata ants in host specificity tests. Pseudacteon
nocens percentage was calculated using both types of sequential tests. Value for P. nudicornis is zero. Percentages
for P. curvatus, P. tricuspis and P. litoralis (bars in black), species currently released in the US for biocontrol of ex-
otic fire ants, are data from Gilbert and Morrison (1997).


approach in both arenas were different, with 1
female approaching the native fire ants 10 times
in the first exposure and 3 females approaching
94, 25, and 57 times in the second trial (Table 2).
Pseudacteon nocens was tested using both the
sequential test: invicta-geminata-invicta and the
tests where flies were exposed to S. geminata first
(Tables 1 and 2). Pseudacteon nocens host speci-
ficity for S. invicta was lower than that seen in
the other Pseudacteon species described above.
From the 61 females exposed to S. geminata in
the first type of tests, 78% approached and 36%
attacked these ants at least once (Table 1).
The behavior of P nocens females was very sim-
ilar in the second type of tests. Those showed that,
once motivated in S. invicta arenas, P nocens fe-
males readily approached and some proportion of
them attacked the native fire ants (Table 2). In
contrast, females exposed first to S. geminata ex-
hibited little motivation to approach and none of
the 10 females exposed attacked (Table 2). The ma-
jority of females first introduced into S. geminata
arenas flew to the top of the arena or stood most of
the time on the box wall ignoring the presence of
ants. The same females, when moved to motiva-
tional S. invicta arenas, were attracted to the ants
as soon as 26 s after introduction (2.19 minutes +
2.54, mean + SD orientation time). Flies that were
then moved to another S. invicta arena started to
attack as soon as 7 s after being transferred (1.74
+ 2.09 mean SD orientation time). In contrast
with the first exposure to the native fire ants, those
flies transferred from motivation S. invicta to
S. geminata arenas started approaching ants very
quickly, and 1 was observed attacking (Table 2).
Rates of approach in both S. geminata arenas were


different, with a lower rate in the first exposure
(although statistic tests were not done because of
the small sample size). Females hovered on top of
ants 17 and 48 times in the first and 34, 107, 7, and
50 times in the second exposure to S. geminata.
Considering both types of tests, out of the 71
P nocens females exposed to S. geminata, 31% at-
tempted to oviposit in those ants (Fig. 1). Never-
theless, the mean number of attacks per female
per min to the native fire ants was very low and
comparable to numbers found for the other Pseu-
dacteon species tested here (Table 1). Females of
P nocens that attacked both species of ants did so
in a significantly reduced rate in S. geminata
than in the exotic fire ant (Z = 3.15, P = 0.002, n =
17, Wilcoxon matched pair test). In contrast, for
this phorid species, neither, the attack rates or
the orientation times differed in their first and
second exposure to S. invicta (Z = 1.62, P = 0.11,
n = 30 and Z = 0.02, P = 0.98, n = 19, Wilcoxon
matched pair test for rates of attack and orienta-
tion time respectively).
In the choice tests only about 40% (17-66%) of
females initially tested attacked at least 1 S. in-
victa ant in the motivation arena and were trans-
ferred to subsequent arenas. We did not use these
percentages to compare to those in subsequent
exposures to S. invicta because several causes be-
sides lack of motivation contributed to the low
percentage of females useful for the specificity
tests. In most cases, for example, after released,
flies were killed by ants in the arena or died pre-
maturely due perhaps to causes related to their
transport from the field to BFL.
From the 69 oviposition attempts observed in
S. geminata by P nocens, only 3 pupae developed.


December 2006







Estrada et al.: Host Specificity of Four Pseudacteon spp.


One pupa was found in the native fire ant at-
tacked by P cultellatus. No adult flies emerged
from these pupae. No pupae were observed in
S. geminata colonies attacked by P. obtusus. Nev-
ertheless, because of the limited sample size, the
possibility of larval development in S. geminata
by the phorid species studied here cannot be elim-
inated. In addition, attempts to rear those phorid
species in S. invicta were largely unsuccessful at
the time when the tests were done. For example,
only 1.5 pupae per female were produced from 349
P nocens that attacked S. invicta repeatedly for
few hours (during a 5-month period), and only
58% of those pupae developed successful to adults.

DISCUSSION

The species of Pseudacteon tested here showed
high and moderate degrees of host specificity. Low
percentages of P obtusus, P cultellatus, and none
of the P nudicornis females attacked S. geminata
even though they were already motivated to at-
tack S. invicta. Those females that attacked
S. geminata, however, did so very infrequently
(no more than 2 attacks in 15 min). In contrast,
31% of P. nocens females attempt to oviposit in
S. geminata after being exposed to exotic fire
ants. These attacks, though, could be attributed
to mistakes because flies only did so after expo-
sure to their usual host, S. invicta, while none of
the females attempted to oviposit on S. geminata
if exposed to this ant first. Even though one third
of the tested P nocens females attacked the native
fire ants, they did so at about 1/6 the frequency
they did in S. invicta arenas. This lower rate of
attack can not be attributed only to handling
effects and energy limitations because the fre-
quencies of attacks in S. invicta arenas before and
after exposure to S. geminata were not different.
Despite of the small sample size of females of
P obtusus, P cultellatus, and P nudicornis used, our
results of host specificity are similar to those for
other species of Pseudacteon reported elsewhere
(Gilbert & Morrison 1997; Porter & Alonso 1999;
Morrison & Gilbert 1999; Folgarait et al. 2002b;
Vazquez et al. 2004). They add to the body of evi-
dence that indicates that Pseudacteon species from
South America are highly specific in the S. saevis-
sima group of fire ants. It is also becoming apparent
that some of these flies may mistakenly approach
and even try to oviposit on other hosts. However, it
seems that Pseudacteon offer a low risk to native
species in the S. geminata group if introduced as
biocontrol agents of red and black imported fire ants
(Porter & Pesquero 2001; Porter & Gilbert 2004).
The species studied here have many character-
istics that made them worth considering for fur-
ther evaluation for classical biocontrol. They
showed high to moderated degrees of host speci-
ficity to S. invicta, in all cases greater than those
exhibited by species already released in the US.


They attacked and developed successfully in both
exotic fire ant species S. invicta and S richteri
(Folgarait et al. 2002a, 2002b, 2005b, 2006) but
with the exception of P obtusus (Porter & Gilbert
2004) none of them have been reported to develop
in native fire ants. In addition, development
times and temperature conditions have been
thoroughly studied for laboratory rearing and
they show high rates of success (Folgarait et al.
2002a,2002b,2005b, 2006).
Finally, these 4 species of Pseudacteon could en-
hance in many ways the effect of the species already
released as biocontrol agents of exotic fire ants.
Pseudacteon obtusus and P nudicornis, for example,
are known to be attracted to workers in foraging
trails in addition to disturbed mounds (Orr et al.
1997; Folgarait et al. 2005b). In contrast, P tricus-
pis, P litoralis, P curvatus (species already intro-
duced to U.S.), P cultellatus, and P nocens are found
more frequently in disturbed mounds, mating
flights or when ants engage in fights, likely cued by
alarm pheromones (Orr et al. 1997; Folgarait et al.
2002a; Morrison & King 2004; Folgarait et al. 2006).
Fly size varies considerably among Pseudac-
teon species and is positively related with the
range of worker sizes they parasitize (Morrison et
al. 1997). Pseudacteon cultellatus, P nudicornis,
and some P nocens are small flies that attack
smaller than average sized workers (Folgarait et
al. 2002a, 2006). This could be especially favorable
in regions were polygyne fire ant colonies with low
worker mean sizes predominate. These Pseudac-
teon species could complement parasitism by the
already introduced small species P curvatus, or
replace it in regions not favorable for this species.
Pseudacteon cultellatus, P. nudicornis, and P cur-
vatus frequently coexist in phorid communities in
Argentina (Folgarait et al. 2005a). Pseudacteon
obtusus (large biotype) and some P nocens, on the
other hand, are big flies that coexist with P tricus-
pis in Argentina and probably will complement
their effect over imported fire ants in the U.S.
Therefore, the species of Pseudacteon studied
here are good candidates for further evaluation
for introduction to the U.S. for fire ant biocontrol.
They (1) show high and moderate specificity for
S. saevissima group species of fire ants, (2) are
widely distributed in their homeland, and proba-
bly locally adapted to several climatic conditions
that can be matched in the southern US, (3) can
be reared in the laboratory, and (4) exhibit differ-
ent sizes and host searching strategies, therefore
complementing the species of phorids already re-
leased as biocontrol.

ACKNOWLEDGMENTS

We thank F. Kozuh, E. Cheng, J. Saunders, C. Watts,
J. Dunn, S. Morgan, C. Britt, A. Hashmi, and B. Dean
(from Brackenridge Field laboratory, University of
Texas), G. A. Montenegro, G. Azzimonti, and R. Carrara











(from Centro de Estudios e Investigaciones, Universidad
Nacional de Quilmes), for lab support and animal collec-
tions in US and Argentina. We thank S. D. Porter for in-
creasing our sample size of P. cultellatus. USDA APHIS
provided import permits. The Lee and Ramona Bass
Foundation, the Robert J. Kleberg and Helen C. Kleberg
Foundation, Texas Fire Ant Research and Management
Project (FARMAAC), CONICET, and the Universidad
Nacional de Quilmes, each contributed to support this
study. The staff of RECS provided permits and logistical
help to collect the phorids. The Direcci6n Nacional de
Fauna y Flora Silvestre from Argentina provided the
necessary permits to export phorids to Texas.

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Florida Entomologist 89(4)







Greenberg et al.: Control of Ants in Organic Citrus


LIQUID BORATE BAIT FOR CONTROL OF THE ARGENTINE ANT,
LINEPITHEMA HUMILE, IN ORGANIC CITRUS
(HYMENOPTERA: FORMICIDAE)

LES GREENBERG, JOHN H. KLOTZ AND MICHAEL K. RUST
Department of Entomology, University of California, Riverside, Riverside, CA 92521

ABSTRACT

A liquid bait delivery system containing borate was evaluated for controlling the Argentine
ant, Linepithema humile (Mayr), in an organic citrus orchard. Two concentrations of diso-
dium octaborate tetrahydrate (1% and 0.5%) were tested in 500-mL capacity bait stations
placed at the base of trees. Both concentrations significantly reduced ant activity over the
1 1-wk duration of the test when compared with controls. However, the 1% concentration of
borate significantly reduced ant activity up to 76 m away from the treatment, whereas the
0.5% did not. Compared to ant control with contact insecticides, the bait delivery system
uses less insecticide and is more target-specific, reducing environmental contamination.

Key Words: Citrus, Argentine ant, borate, liquid bait delivery system, organic, homopterous
pests, bait station

RESUME

Un sistema de distribuci6n de liquidos conteniendo boratos fue evaluado por el control de la
hormiga argentina, Linepithema humile (Mayr), en una huerta de arboles citricos. Dos con-
centraciones de disodium octaborate tetrahydrate (1% y 0.5%) fueron probadas en estaciones
de comida puestas cerca de los troncos de los arboles. Comparado con los controls, las dos
concentraciones redujeron significativamente la actividad de las hormigas durante las 11-
semanas del experiment. No obstante, la concentraci6n 1% del borato produjo una reduc-
ci6n significant en la actividad de las hormigas hasta una distancia de 76 metros, mientras
que la solution 0.5% de borato no tenia ese efecto. Comparado con el control de hormigas con
insecticides de contact, nuestro sistema de distribuci6n usa menos insecticide y es dirigido
especificamente a la hormiga; y de esa manera reduce la contaminaci6n ambiental.


Translation provided by the authors.


Argentine ants became a serious pest in citrus
shortly after their introduction into the United
States in the late 1800s, most likely offloaded
from ships transporting coffee from Brazil into
the port of New Orleans (Newell & Barber 1913).
As early as 1918, a researcher in Louisiana re-
ported trapping 1,307,222 Argentine ant queens
and collecting 1,150 gallons of workers and brood
over a one-year period in a 19-acre citrus grove
(Horton 1918). In 1905 they were reported in
southern California, and by 1908 they had spread
through the citrus growing regions as far north as
San Francisco (Vega & Rust 2001).
Colonies of Argentine ants have tremendous
capacity for growth and expansion due to numer-
ous queens (typically 15 to every 1000 workers)
and their ability to undergo colony multiplication
by fission (Aron 2001; Majer 1993). Colony fission
or budding eventually creates a network of inter-
related nests that form a cooperative unit, which
sometimes extends over an entire habitat. The
flow of food in these supercolonies is decentral-
ized, moving in many directions depending on the
needs of the individual colonies. This behavior is


known as dispersed central place foraging (Hol-
way & Case 2000; McIver 1991).
These large cooperative units channel their
energy into foraging and colony growth, and by
the sheer number of ants produced out-compete
native species for limited resources. Their popula-
tions can reach astronomical proportions, as for
example, in a citrus grove in San Diego County,
California, it was estimated that from 50,000 to
600,000 ants ascended each tree daily in order to
tend homopterans (Markin 1967).
Argentine ants tend a variety of homopterans
in citrus including the citrus mealybug, Planococ-
cus citri Risso, spirea aphid, Aphis spiraecola
Patch, wooly whitefly, Aleurothrixus floccossus
(Maskell), and brown and black soft scales, Coc-
cus hesperidum L. and Saissetia oleae Olivier, re-
spectively. These phloem-feeding homopterans
excrete honeydew, which is the primary food of
Argentine ants (Markin 1970). The ants guard
this resource tenaciously by protecting the ho-
mopterans from parasites and predators and con-
sequently interfere with biological control pro-
grams. The outcome of this trophobiotic associa-







Florida Entomologist 89(4)


tion is an increase in populations of both ants and
homopterans. (See reviews of this topic in Bar-
tlett 1961; Flint et al. 1991; and Gulla 1997).
Moreno et al. (1987) demonstrated that by con-
trolling Argentine ants in citrus, the wooly white-
flies and citrus mealybugs were reduced in num-
ber by their natural enemies. They applied resid-
ual insecticides (chlorpyrifos or diazinon) as a bar-
rier on the trunk or on the ground around skirt-
pruned trees. Recently, however, growers have re-
duced their use of broad spectrum insecticides,
and as a consequence ant populations have in-
creased, and there is a growing demand for selec-
tive pesticide baits (Martinez-Ferrer et al. 2003).
In previous research in commercial citrus
groves, Klotz et al. (2003, 2004) obtained signifi-
cant reductions of Argentine ant populations us-
ing liquid baits (25% sucrose-water) with ultra-
low concentrations (1 x 10-4%) of fipronil or thia-
methoxam.The purpose of this study was to test
borate in a sugary solution for Argentine ant con-
trol in citrus. If effective, then organic growers
would have a means of reducing Argentine ant
populations in citrus. This is especially signifi-
cant considering the limited options for ant con-
trol available to organic growers.

MATERIALS AND METHODS

Test Site and Experimental Design

An organic citrus grower in Fallbrook (Rain-
bow Valley Orchids, San Diego County, Califor-
nia) provided us with an orange grove, which we
partitioned into 21 plots, each consisting of 3 rows
by 5 trees, and measuring 12.2 x 15.2 m. The rows
were 6.1 m apart and trees within rows 3.0 m
apart. Each plot was a minimum of 20 m from ad-
jacent plots. This buffer zone was set up in order
to mitigate any treatment effects from neighbor-
ing plots due to the movement of toxicant through
the ant population.
A randomized block design was used consist-
ing of 7 blocks of 3 treatments. Each block con-
sisted of plots with similar ant activity based on a
pretreatment survey (see monitoring below) in or-
der to reduce variability due to differences in the
initial ant activity.

Monitoring Plots

To estimate ant activity in each plot, we moni-
tored 3 trees in the center row of each plot with
sucrose-water monitors. Monitored trees were
never on the edge of the plot. Due to missing trees
in some plots, several plots had less than 3 trees
to monitor. The monitors consisted of 50-mL plas-
tic centrifuge tubes (Fisher Scientific, Pittsburgh,
PA) filled with 25% sucrose-water. The cap on the
monitor had a 2-cm hole drilled in its center and
was screwed down over a 6-cm square piece of


Weedblock (Easy Gardener, Waco, TX), a perfo-
rated plastic material with many tiny holes. The
monitors were inverted and taped to tree trunks
so that trailing ants could feed on the sucrose-wa-
ter. To correct for evaporative water loss in moni-
tors, a 50-mL tube was filled with 25% sucrose-
water, inverted, and suspended on a string from a
tree branch in the grove. The string was coated
with Stikem Special (Seabright, Emeryville, CA)
to prevent ants from feeding on this tube. The
tube and monitors were left on the trees for 24 h
and consumption of sucrose-water by the ants
was obtained by correcting for evaporative water
loss. Consumption of sucrose-water from these
monitors indicated the number of ant visits, with
each mL consumed corresponding to about 3300
ant-visits (Reierson et al. 1998). Estimates of ant
activity were made in all plots before treatments
and on a weekly basis for 11 wk after treatment
(wk 6 and 10 were skipped).

Monitoring Transects

At the end of the 11-wk study we also monitored
sucrose-water consumption along a series of
transects in order to determine how far the toxic
baits were having an effect. Each transect extended
=76 m out from a baited plot into surrounding un-
treated areas (i.e., some treatment plots were adja-
cent to parts of the grove that we did not use for
plots). Beginning in the middle of the treated plot,
monitors were placed in trees at =6 m intervals
along the transect. Monitors were left out for 24 h,
and then collected to measure the consumption of
sucrose-water by the ants. As described in the pro-
cedure for monitoring plots, a tube was also used to
correct for evaporative water loss.

Treatments

Gourmet Liquid Ant Bait (Innovative Pest
Control Products, Boca Raton, FL) containing 1%
disodium octaborate tetrahydrate (DSOBTH) was
used. One of the treatments consisted of bait ap-
plied at full strength (1% DSOBTH) and the other
diluted with deionized water to half strength
(0.5% DSOBTH). The liquid bait was delivered in
500 mL capacity KM AntPro Stations (KM Ant-
Pro, LLC, Nokomis, FL). Stations were placed on
the ground at the base of every other tree in the
treatment plots, staggering the placement be-
tween rows of trees. In case of a missing tree, the
bait station was placed where the tree should
have been. Thus, there were 7 or 8 stations per
plot, making a total of 105 stations used in the
study. Stations were checked weekly and refilled
when necessary. During the monitoring procedure
the stations were closed to prevent ants from feed-
ing on them and potentially attracting them away
from the monitors, thereby reducing our estimate
of ant numbers at the monitors. In addition to the


December 2006







Greenberg et al.: Control of Ants in Organic Citrus


2 bait treatments we had a third treatment con-
sisting of control plots, which were not baited.

Statistical Analyses

Examination of the plot data with histograms
and probability plots to assess normality showed
that a square root function, rather than a loga-
rithmic transformation, more closely approxi-
mated normality. Therefore, to compare the treat-
ments and controls over time we did a repeated
measures ANOVA (Systat 2004) on the square
root (X + 1) transformation of sucrose-water con-
sumption for the 11 post-treatment wk that we
monitored. In this analysis we were interested in
the Between Subjects (Treatments) effects, which
is equivalent to comparing the grand means of
the treatment profiles over the 11 wk. Each mon-
itor is compared with itself over time, giving a
mean value for each monitor and a grand mean
for each treatment. We also did separate ANOVAs
for each data period with the transformed data.
For all the ANOVAs the blocking variable was
used to remove variability due to differences in
initial ant numbers in the plots and the remain-
der, or MSE, was used for tests of significance.
For the transect data originating in baited
plots, consumption of sucrose-water was plotted
against distance and pooled for each treatment. A
linear regression analysis was performed on these
pooled data for each treatment (Systat 2004).

RESULTS

Table 1 shows a summary of the results. One
wk post-treatments, ant visits to the monitors in
treatment plots were significantly less than in the
controls, with reductions of 54 and 47%, respec-
tively, for the 0.5% and 1% DSOBTH. In the sec-


ond wk the respective reductions were 68% and
70%. However, consumption in the control plots
also began to decline in the second wk and was not
now significantly different from the treatments.
From wk 7 through 11 the consumption of sucrose-
water in the 1% DSOBTH was again significantly
lower than in the control. Consumption of sucrose-
water in the 0.5% DSOBTH treatments was sig-
nificantly lower than controls only in wk 1 and 8.
The grand means of the mean consumption of
sucrose-water for each treatment, ignoring the
pretreatment values, were obtained by finding the
mean of each monitor over the 11 post-treatment
wk and averaging these means within each treat-
ment. These grand means showed overall reduc-
tions from pre-treatment values in sucrose con-
sumption by 76, 52, and 48%, respectively, for the
1% DSOBTH, 0.5% DSOBTH, and the controls.
The differences between the grand means were
tested for significance by looking at the Between
Subjects (Treatments) part of a repeated mea-
sures ANOVA (Systat 2004) for the 11 post-treat-
ment wk on square root (X + 1) transformed grand
means. The treatment (df = 2, 48; F = 12.5) and
blocking (df = 6, 48; F = 4.5) effects were both sig-
nificant (P < 0.001). A follow-up comparison of the
grand means with Tukey's HSD test showed that
sucrose-water consumption for both the 1% and
0.5% DSOBTH bait treatments were lower than
the controls (P < 0.001 and P < 0.01, respectively),
but not different from one another (P > 0.25).
Eleven transects of sucrose-water consump-
tion vs. distance were completed. Five of these re-
ceived the 0.5%, and 6 the 1.0%, DSOBTH treat-
ments. The regression analysis of the 1%
DSOBTH bait transects (Fig. la) was highly sig-
nificant (P < 0.001), whereas it was not significant
for transects from plots treated with 0.5%
DSOBTH bait (P > 0.25, Fig. Ib).


TABLE 1. MEAN' CONSUMPTION OF SUCROSE-WATER (G) AS A MEASURE OF ANT ABUNDANCE.

Pretreat. Wk 1 Wk 2 Wk 3 Wk4 Wk 5 Wk 7 Wk8 Wk 9 Wk 11

0.5% DSOBTH 25.0 11.6 8.0 7.5 3.2 6.9 16.4 6.8 3.4 16.4
(2.48) a (1.77) b (1.28) a (1.20) a (0.73) a (1.27) a (3.23) a (0.76) b (0.60) ab (1.60) a
% reduction 53.5 67.8 70.0 87.1 72.2 34.3 72.9 86.5 34.4
1.0% DSOBTH 29.3 13.8 8.7 7.3 5.1 3.7 6.7 5.6 2.5 6.9
(4.17) a (2.95)b (1.97) a (1.51) a (1.07) a (0.84) b (1.16)b (0.39)b (0.65) b (1.11)b
% reduction 46.8 70.4 75.1 82.5 87.5 77.0 80.8 91.5 76.6
CONTROL 29.4 27.8 16.0 11.4 6.6 5.4 19.0 9.7 6.4 15.2
(3.94) a (5.87) a (3.21) a (2.21) a (1.60) a (0.90) ab (1.82) a (0.96) a (1.53) a (2.74) a
% reduction 9.3 45.4 61.3 77.7 81.4 35.2 67.0 78.1 48.2

Means (+ SE). In each column values followed by the same letter are not significantly different (P > 0.05), with Tukey's HSD
test performed on square root (X + 1) transformed data; untransformed means are shown above. Blocking variable was used in the
ANOVAs to reduce the error variability, thereby increasing the power of the treatment statistics. DSOBTH = disodium octaborate
tetrahydrate. n = 19 for all treatments, except for Wk 1, where n = 16 for the Control and the 1% DSOBTH. % reduction = % reduc-
tion from pretreatment values.







Florida Entomologist 89(4)


I .n


40


30


20


An.


30



20


0 10 20 30 40 50 60 70 80
Distance (m)

Fig. 1. Pooled data of regressions of sucrose-water
consumption (g) vs. distance (m) from treated plots. (a)
1% DSOBTH (6 transects, n = 60), and (b) 0.5%
DSOBTH (5 transects, n = 48).


DISCUSSION

As described above, ant numbers in control
plots began to decline 2 wk after treatments. To
test the hypothesis that the treatments could influ-
ence control plots, we sampled ants along transects
starting at a treatment plot and going into un-
treated parts of the citrus grove. For the treat-
ments with the 1% DSOBTH the significant re-
gression analysis shows an effect at least 70 m
away from the treatment. In spite of the control
plots being within the active space of the treatment
plots, we were still able to show overall differences
between treatments and controls. The regressions
suggest that these differences would be higher if
the control plots were further from the treatments.


y = 7.72 + 2.10 x
-
r2 = 0.51

a
o o



o o


1.0 00
0 o g o 0
i 8


Various non-chemical and chemical methods
have been developed for Argentine ant control in
citrus (Vega & Rust 2001). In the early 1900s in
Louisiana, traps consisting of wooden boxes con-
taining decaying vegetable matter were set out in
groves to attract colonies of ants during the win-
ter (Newell & Barber 1913). The warmth of the
decomposing organic matter was thought to at-
tract the ants, which moved into the boxes where
they were treated with an insecticide such as car-
bon bisulfide. Another early method involved
flooding orchards in order to force the ants into a
concentrated area where they were treated with
scalding water or kerosene (Newell & Barber
1913). Tree banding with a mixture of sulfur and
sticky material was also a recommended treat-
ment (Woglum & Neuls 1917). More modern
banding techniques incorporate Stikem + repel-
lents such as farnesol (Shorey et al. 1992), or con-
trolled-release chlorpyrifos (James et al. 1998).
Although effective, these methods have generally
not been adopted by growers because they are la-
bor intensive (Rust et al. 2003).
A more practical means of control is the appli-
cation of broad-spectrum residual insecticides.
Chlordane, for example, was the standard treat-
ment for ant control in citrus in the mid twentieth
century, until its use was prohibited by the Envi-
ronmental Protection Agency (EPA) in 1980
(Moreno et al. 1987). Organophosphates, such as
chlorpyrifos and diazinon, replaced the chlori-
nated hydrocarbons and are still being used today
for ant control. However, their use is being phased
out in urban environments, and growers are also
reducing their applications of these chemicals for
ant control (Martinez-Ferrer et al. 2003).
Baits offer several advantages over residual
insecticides. First, with regard to efficacy, baits
exploit the recruitment and food-sharing behav-
iors of ants to spread a toxicant throughout the
colony. In the case of Argentine ants, baits have
the added benefit of being spread among nests
due to transfer of foods and movement of ants in
this unicolonial species. For example, Markin
(1968) estimated that >50% of the worker popula-
tion was exchanged among neighboring nests in 5
d. In contrast to baits, residual insecticides kill
ants on contact, mostly the aboveground foragers,
which are readily replaced with colony reserves.
Second, in comparison to residual insecticides
there is far less active ingredient in baits and par-
ticularly when contained, as in bait stations, there
is reduced environmental contamination. Indeed,
the degree of environmental protection provided
by bait stations convinced EPA that certain expen-
sive data requirements could be waived, making
future registration of these innovative technolo-
gies much more likely (Klotz et al. 2004).
In previous tests in urban settings, we reduced
Argentine ant populations by 80% using 0.5%
boric acid in 25% sucrose-water (Klotz et al.


S 0 0


b

oy = 24.49 + 0.32x
r2 = 0.02


S 0 0 o a
0 a 0 o 0
o 1o 01 10 I I I


December 2006







Greenberg et al.: Control of Ants in Organic Citrus


1998). Adopting our techniques, Daane et al.
(2006) used the same bait in grape vineyards and
significantly reduced Argentine ant populations
at one of two sites where it was tested. Over the
course of their 3-year study Daane et al. devel-
oped better dispensers and more effective deploy-
ment patterns for liquid baits leading to more
consistent reduction in ants, and significantly
less mealybugs and crop damage. In comparison,
the standard treatment with chlorpyrifos for ants
in vineyards had little or no long-term impact on
the ant densities (Daane et al. 2006).
We believe that monitoring transects as was
done in this study may provide valuable informa-
tion for determining rates of application as well as
concentration of active ingredient. For example,
after 11 wk of exposure to the 1.0% DSOBTH bait
there was significant reduction of ants up to 76 m
away from the treated plots. On the other hand,
the 0.5% DSOBTH bait did not have an effect over
this same distance. A likely cause for this differ-
ence in efficacy is due to dilution of the bait toxi-
cant by trophallaxis. Rust et al. (2004) showed
that in the case of borates there is a relatively nar-
row range of concentrations that are effective, and
that trophallaxis can readily dilute a toxicant to a
sublethal dose. This dilution effect is magnified in
the high population densities of Argentine ants
that are found in some citrus groves. In lighter in-
festations as in the urban setting mentioned
above, 0.5% boric acid bait was sufficient.
Based on previous research in commercial set-
tings (Klotz et al. 2004), a baiting program for a
heavy infestation of Argentine ants in organic cit-
rus might start with 55 bait stations per hectare
and 1% borate solution. Only half the number of
bait stations would be used the following year,
since there is significant carry-over of reduced
populations from one season to the next (Klotz &
Rust 2002). The number of stations might even be
further reduced, but the amount is yet to be deter-
mined by future research.


ACKNOWLEDGMENTS

We thank Rich Hart of Rainbow Valley Orchids in
Fallbrook for providing the organic citrus grove, and
Edna Diaz for assistance in the field. We also thank Ken
Kupfer for help and providing the KM AntPro bait sta-
tions and Innovative Pest Control Products for provid-
ing the Gourmet Liquid Ant Bait. This research was
funded by the USDA-CSREES Exotic/Invasive Pest
Project.

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Liu et al.: Insects on Native, Invasive, and Non-invasive Eugenia


INSECT HERBIVORE FAUNAL DIVERSITY AMONG INVASIVE,
NON-INVASIVE AND NATIVE EUGENIA SPECIES: IMPLICATIONS
FOR THE ENEMY RELEASE HYPOTHESIS


HONG LIU1'4, PETER STIFLING ROBERT W. PEMBERTON2 AND JORGE PENA3
'Department of Biological Sciences, University of South Florida
4202 East Fowler Ave., SCA 110, Tampa, FL 33620-5200, U.S.A.

2USDA, ARS, Invasive Plant Research Lab, 3225 College Ave., Fort Lauderdale, FL 33314, U.S.A.

3University of Florida, Tropical Research and Education Center, Institute of Food and Agricultural Sciences
18905 SW 280 St., Homestead, FL 33031, U.S.A.

4Correspondence and present address: University of Florida, IFAS, C/O USDA, ARS Invasive Plant Research Lab
3225 College Ave., Fort Lauderdale, FL 33314, U.S.A.
Phone: 954-475-6563; Fax: 954-476-9169; e-mail: hongliuf@ufl.edu

ABSTRACT

The enemy release hypothesis (ERH) frequently has been invoked to explain the naturaliza-
tion and spread of introduced species. One ramification of the ERH is that invasive plants
sustain less herbivore pressure than do native species. Empirical studies testing the ERH
have mostly involved two-way comparisons between invasive introduced plants and their na-
tive counterparts in the invaded region. Testing the ERH would be more meaningful if such
studies also included introduced non-invasive species because introduced plants, regardless
of their abundance or impact, may support a reduced insect herbivore fauna and experience
less damage. In this study, we employed a three-way comparison, in which we compared her-
bivore faunas among native, introduced invasive, and introduced non-invasive plants in the
genus Eugenia (Myrtaceae) which all co-occur in South Florida. We observed a total of 25 in-
sect species in 12 families and 6 orders feeding on the six species of Eugenia. Of these insect
species, the majority were native (72%), polyphagous (64%), and ectophagous (68%). We
found that invasive introduced Eugenia has a similar level of herbivore richness as both the
native and the non-invasive introduced Eugenia. However, the numbers and percentages of
oligophagous insect species were greatest on the native Eugenia, but they were not different
between the invasive and non-invasive introduced Eugenia. One oligophagous endophagous
insect has likely shifted from the native to the invasive, but none to the non-invasive Euge-
nia. In summary, the invasive Eugenia encountered equal, if not greater, herbivore pressure
than the non-invasive Eugenia, including from oligophagous and endophagous herbivores.
Our data only provided limited support to the ERH. We would not have been able to draw this
conclusion without inclusion of the non-invasive Eugenia species in the study.

Key Words: biological invasion, endophagous insect, herbivore fauna, introduced species, in-
vasive species, non-invasive species, oligophagous insects

RESUME

La hip6tesis de escape del enemigo (HEE) ha sido frecuentemente utilizada para explicar la
naturalizaci6n y extension de species introducidas. Una de las ramificaciones de la HEE es
que las plants invasoras soportan un grado de herbivorismo menor que el de las species
nativas. La mayor parte de los studios empiricos para analizar la HEE han implicado com-
pariciones de dos-vias entire la especie invasora y su contraparte native del area de invasion.
Estos andlisis serian de mayor relevancia si los mismos tambi6n incluyeran species no na-
tivas que fueran no invasoras. Estas species, independientemente de su abundancia e im-
pacto, podrian tener una reducido fauna herbivora y por tanto experimentar un grado menor
de dano. En este studio nosotros usamos una comparaci6n de tres vias en la cual se compare
las fauna herbivoras de species nativas, species invasoras introducidas y species introdu-
cidas no invasoras del g6nero Eugenia (Myrtaceae) del Sur de La Florida. Observamos un to-
tal de 25 species de insects en doce families y seis 6rdenes alimentandose sobre seis
species de Eugenia. Entre 6stos, la mayoria son natives (72%) polifagos (64%) y ectofagos
(68%). Nosotros encontramos que species invasoras introducidas de Eugenia tiene niveles
similares de riqueza de herbivoros que los de las species nativas e introducidas no invaso-
ras. Sin embargo el numero y el porcentaje de insects oligofagos fue mayor en las species
nativas, aunque estas diferencias no fueron significativas entire las species introducidas in-







Florida Entomologist 89(4)


vasoras y no invasoras de Eugenia. Uno de los herbivoros oligofago y endofago es probable
que haya cambiado desde la especie native a la invasora, pero ninguno de 6stos a la especie
no invasora de Eugenia. En resume, la especie invasora de Eugenia ha encontrado la
misma, o quizas mayor, presi6n por parte de herbivoros que la especie no invasora de Euge-
nia, incluyendo oligofagos y endofagos. Nuestros datos indican un apoyo muy limitado para
la HEE. Nosotros no habriamos podido llegar a esta conclusion al menos que hubieramos in-
cluido la especie no invasora de Eugenia en nuestro studio.
Translation provided by the authors.


The enemy release hypothesis (ERH) states
that introduced invasive species are successful
because they left their co-evolved natural ene-
mies behind. This idea makes intuitive sense and
is the theoretical foundation of classical biological
control. It is one of the most cited explanations for
the undesired success of introduced invasive spe-
cies worldwide (Williams 1959; Crawley 1997;
Maron & Vila 2001; Keane & Crawley 2002). Al-
though empirical studies testing the ERH on in-
vasive plants are limited in number (Maron &
Vila 2001; Keane & Crawley 2002; Liu & Stiling
2006) and vigor (but see Schierenbeck et al. 1994;
Wolfe 2002; Siemann & Rogers 2003; DeWalt et
al. 2004), there have been several syntheses to
test the predictions stemming from ERH during
the last decade (Maron & Vila 2001; Keane &
Crawley 2002; Colautti et al. 2004; Liu & Stiling
2006). One consensus generated from these syn-
theses and other more recent empirical studies is
that the total number of insect herbivores, and
the numbers of endophagous and oligophagous
herbivores, are all reduced on introduced invasive
species compared with conspecific populations in
the native range or on co-occurring native conge-
ners (Keane & Crawley 2002; Colautti et al. 2004;
Hinz & Schwarzlaender 2004; Torchin & Mitchell
2004; Liu & Stiling 2006). In addition, a modifica-
tion of the ERH, which states that it is the escape
from specialist insects (including endophagous
species) that allow the introduced plants to be
successful, has received increasing support (Wolfe
et al. 2004; Joshi and Vrieling 2005; Stastny et al.
2005; Mitchell et al. 2006).
All the empirical studies reviewed above were
performed in one of two ways: first, insect herbi-
vore diversity, load, or insect herbivore impact ei-
ther on invasive plants in native vs. introduced
ranges was examined (e.g., Wolfe 2002; DeWalt et
al. 2004), or second, the same comparisons were
made between invasive plants and their native
counterparts in the new region (Schierenback et
al. 1994; Agrawal & Kotanen 2003; Siemann &
Rogers 2003). The latter approach is not a direct
test of the ERH. Rather, it tests a ramification of
the ERH that invasive introduced plants sustain
less insect herbivore pressure than their native
counterparts. However, all introduced plants, re-
gardless of their abundance or impact, may sup-
port a reduced insect herbivore fauna and experi-
ence less damage simply because plants tend to


lose their associated insect herbivores during the
introduction (Colautti et al. 2004) and it takes
time, on the ecological and/or evolutionary scale,
for a new population to acquire its insect herbi-
vore fauna (Strong et al. 1984). Testing the ERH
would be more meaningful if such studies also in-
cluded introduced plants which do not become in-
vasive, or so-called innocuous species (Colautti et
al. 2004; Levine et al. 2004). However, few studies
have included introduced non-invasive plants
(but see Mitchell & Power 2003; Cappuccino &
Carpenter 2005; Carpenter & Cappuccino 2005).
A three-way comparison of insect herbivore
faunas in a system in which congeneric native, in-
troduced invasive, and introduced non-invasive
(innocuous) plants that co-occur in the same re-
gion can provide insightful information on the va-
lidity of the ERH. If release from natural enemies
is important in determination of the success of an
introduced plant species, one would expect that
invasive introduced plants escape more from her-
bivore pressure than do non-invasive introduced
plants. One question of particular interest is
whether there have been any shifts of oligopha-
gous and/or endophagous herbivores from the na-
tive to the introduced plant congeners, and if such
shifts occur more onto the non-invasive than to
the invasive congeners. Endophagous herbivores
are of interest because an internal feeding niche
is likely to be correlated with dietary specializa-
tion (Frenzel & Brandl 1998). Plants that are
closely related phylogenetically (i.e., congeners or
confamiliers), as used in many ERH tests, offer a
good chance to detect host shifts by herbivores to
the introduced plants because herbivore host
choice is often determined by plant relatedness.
In this study, we compared insect herbivore
faunas among native (two species), invasive (one
species), and non-invasive (three species) of Euge-
nia growing in South Florida. The Eugenia spp.
studied here are small-medium sized trees native
to Florida and Central-South America (Wunderlin
& Hansen 2003; Ruehle et al. 1958). We predict
that (1) the total number of herbivore species will
be (a) greater on the native Eugenia species than
on the introduced invasive and non-invasive con-
geners; and (b) greater on the introduced non-in-
vasive Eugenia than on the introduced invasive
congener; (2) the number and proportion of oli-
gophagous and endophagous herbivores will be (a)
greater on the native Eugenia species than on the


December 2006







Liu et al.: Insects on Native, Invasive, and Non-invasive Eugenia


introduced invasive and non-invasive congeners;
and (b) greater on the introduced non-invasive
Eugenia than on the introduced invasive conge-
ner, and (3) fewer herbivores, particularly oligoph-
agous and endophagous herbivores, will be shared
between the native Eugenia and the introduced
invasive Eugenia than between the native and in-
troduced non-invasive Eugenia. The first portions
of the first two predictions are comparable to pre-
dictions made by the usual two-way (native vs. in-
troduced invasive plants) comparisons. For ERH
to be supported in the current three-way testing
system, the second portion of the prediction
should be validated. We believe this study repre-
sents the first known comparison of herbivore
funna on native, invasive, and innocuous species
of the same genus in the same geographic location.

MATERIAL AND METHODS

Study Plants

Eugenia uniflora L. (Surinam cherry), E. aggre-
gata Kiaersk. (cherry of the Rio Grande),
E. brasiliensis Lam. (grumichama), and E. lusch-
nathiana Klotzsch (pitomba) are all large shrubs
or small trees with potentially animal-dispersed
fleshy fruits that were introduced to south Flor-
ida from Brazil in the late 1800s or early 1900s for
home garden fruit and ornamental purposes
(Ruehle et al. 1958; Martin et al. 1987). Eugenia
uniflora is a common hedge plant in South Flor-
ida, probably due to its robust and rapid growth.
Since its introduction, E. uniflora has escaped
cultivation and invaded hammocks (evergreen
broad-leaved forests) in South Florida, growing
side by side in some areas with 2 native conge-
ners, E. axillaris (Sw.) Willd. (white stopper) and
E. foetida Pers. (Spanish stopper) (Gann et al.
2001) (Table 1). The other 3 introduced Eugenia
spp. still remain in cultivation in many public and
private gardens and nurseries.

Study Sites

We carried out most of our sampling at two
subtropical hammocks in Broward County where
E. axillaris (native), E. foetida (native), and


E. uniflora (invasive) co-occur: Hugh Taylor Birch
State Park (hereafter referred to as Birch Park),
and the Bonnet House Museum and Garden
(Hereafter referred to as Bonnet House). Subtrop-
ical hammocks in South Florida are evergreen,
broad-leaved forests composed predominantly of
trees common to the Bahamas and Greater Anti-
lles (Snyder et al. 1990). They occupy limestone
outcroppings that are elevated, rarely inundated,
and relatively fire-free. In hammocks of both
Birch Park and Bonnet House, the canopy trees
are primarily composed ofBursera simaruba (L.)
Sarg. (gumbo-limbo), Coccoloba unifera L. (sea-
grape), Krugiodendron ferreum (Vahl) Urb. (black
iron wood), and Ficus aurea Nutt. (strangler fig).
The understory is dominated by E. axillaris,
E. foetida, and E. uniflora. Sandy soil is charac-
teristic of both sites.
For the introduced non-invasive E. aggregata,
E. brasiliensis, and E. luschnathiana, we located
up to 14 individuals per species in 4 research,
public, and private gardens in Miami Dade and
Broward, 2 adjacent counties in South Florida.
These gardens include University of Florida,
Tropical Research and Education Center, the
Fruit and Spice Park, Plantation Heritage Park,
and the Fairchild Tropical Garden. These plants
are referred to as cultivated aggregata, cultivated
brasiliensis, and cultivated lushnathiana (Table
1). In addition, as a control for potential site re-
lated differences between these gardens and the
natural subtropical hammocks, we also sampled
9, 10, and 28 individuals, respectively, of E. axil-
laris (native), E. foetida (native), and E. uniflora
(invasive) at the above gardens. These individuals
were referred to as cultivated axillaris, cultivated
foetida, and cultivated uniflora. Sampling fre-
quencies for the cultivated plants were the same
as for the wild populations mentioned above.

Determination of Insect Herbivore Faunas

Four and two 5 x 3-m2 plots were established at
the Birch Park and the Bonnet House, respec-
tively, for herbivore faunal surveys on wild popu-
lations of E. axillaris, E. foetida, and E. uniflora
(Table 1). We tagged a total of 182, 202, and 97
wild plants of various sizes of E. axillaris, E. foe-


TABLE 1. SUMMARY OF THE STUDY SYSTEM, INCLUDING THE NUMBER OF PLANTS SAMPLED (n). PLANTS THAT GROW IN
GARDENS ARE CULTIVATED.

Plant species Status Growing habitat in south Florida (n)

E. axillaris Native Natural hammocks (182) and garden (9)
E. foetida Native Natural hammocks (202) and garden (10)
E. uniflora Introduced invasive Natural hammocks (97) and garden (28)
E. aggregate Introduced non-invasive Garden (9)
E. brasiliensis Introduced non-invasive Garden (14)
E. lushnathiana Introduced non-invasive Garden (10)







Florida Entomologist 89(4)


tida, and E. uniflora, respectively. All these plants
were visited every other month during the dry
season (Oct to Apr) and monthly during the wet
season (May to Sep) from Jan to Dec 2004. Larval
and adult insects were hand caught and brought
back to the lab for rearing, specimen preparation,
and identification. For fruit and seed feeders, we
collected random fruit samples from 3-10 trees
and 20-100 fruits per tree, depending on availabil-
ity. Some non-rotten fruits on the ground directly
beneath the trees were also included in the sam-
ples. Unidentified fruit/seed feeders were reared
to maturity for identifications. We sent unknown
specimens to specialists in the USA for identifica-
tion. Information on insect immigration status
(i.e., native or exotic) and diet breadth were pro-
vided by these insect specialists when possible. In-
sects were classified as native or exotic, oligopha-
gous or polyphagous, and endophagous or ectoph-
agous feeders. Oligophagous refers to insects
which feed only on plants of 1 family while polyph-
agous indicates herbivores that feed on more than
1 family. Insects were "very important" if they
were seen in every census, or were seen to cause
10% or more of leaf or seed damage on average in
at least 1 census (Liu, unpublished data). Insects
were "important" if they were seen in more than 1
census but caused less than 10% leaf or seed dam-
age. Herbivores were "not important" if they were
seen only once during the entire study period or
caused very little plant damage. Determination of
% damage to plants depended on the nature of the
insect. For example, the % damage by a leaf miner
was determined by counting the % of leaves with
mines, while the % damage by a chewing caterpil-
lar was by counting the % of leaves chewed.

Data Analyses

In addition to the identity of the herbivores,
the number of total insect herbivore species on
each Eugenia species, the number and percentage
of native insect herbivores, the number and per-
centage of endophagous vs. ectophagous feeders,
and the number and percentage of oligophagous
vs. polyphagous feeders were determined. The
differences in these percentages among the na-
tive (average among the 2 species), invasive and
non-invasive (average among the 3 species)
plants were determined with chi-square tests
(Zar 1984) in SPSS 13.0 (SPSS, Chicago, Illinois,
USA). Because there may be differences in the
herbivore fauna between wild and cultivated pop-
ulations of the same species as the latter are in
artificial settings, 2 sets of the chi-square tests
were performed. One was a two-way test that in-
cluded wild native plants and wild introduced in-
vasive plants. The other was a three-way test that
included cultivated native, invasive, and non-in-
vasive plants. We also determined the number of
herbivores, particularly oligophagous and/or en-


dophagous, shared between the native, invasive
and non-invasive plants. Samples from the two
natural area sites were pooled because they had
identical herbivore fauna for the three wild Euge-
nia populations. Samples from the four garden
sites were pooled because all gardens did not have
adequate sample sizes for among site comparisons.

RESULTS

We observed, collected, and reared a total of 25
insect species in 12 families and 6 orders feeding
on the 6 species of Eugenia during the 1-year
sampling period (Table 2). Among them, the ma-
jority were native (72%), polyphagous (64%), and
external feeders (68%). There were 7 additional
uncommon species of Lepidoptera reared from
bagged branches of various Eugenia spp. that
were not included in the results because her-
bivory by these species was not confirmed. The
native wild Eugenia species had higher numbers
of herbivore species than the wild introduced
E. uniflora and most cultivated Eugenia. The only
exception was that the cultivated E. uniflora had
more herbivore species than the native Eugenia
(Fig. 1A).
The introduced invasive and non-invasive Eu-
genia recruited fewer oligophagous insect herbi-
vores than the native Eugenia (Fig. 1A). The dif-
ference in proportions of herbivore diet breadth
(oligophagous vs. polyphagous) among the culti-
vated native, invasive, and non-invasive Eugenia
was marginally insignificant (Pearson x2 = 5.76,
df= 2, P = 0.056). The difference in herbivore diet
breadth was not statistically significant between
the wild native Eugenia and wild invasive Euge-
nia (Pearson x2 = 1.94, df = 1, P = 0.163). In addi-
tion, the proportions of herbivore feeding site (en-
dophagous vs. ectophagous) were not different be-
tween the wild native Eugenia and wild invasive
Eugenia (Pearson X2 = 0.003, df = 1, P = 0.960), or
among the cultivated plants (Pearson X2 = 1.91, df
= 2, P = 0.385) (Fig. 1B). Separate analyses (not
reported here) incorporating the excluded uncom-
mon Lepidoptera yielded similar results. Finally,
all introduced Eugenia species attracted more ex-
otic insect herbivores than the native Eugenia
plants (Fig. 1C). However, the differences in the
proportion of native herbivores were not signifi-
cant between the wild native Eugenia and the
wild invasive Eugenia (Pearson X2 = 1.02, df = 1,
P = 0.311), and among the cultivated native, inva-
sive, and non-invasive Eugenia (Pearson X2 =
0.76, df = 2, P = 0.683) (Fig. 1C).
The native Eugenia shared a total of 6 general-
ist herbivores, 4 with the invasive Eugenia, 4 with
the non-invasive Eugenia, and 2 (the weevil Dia-
prepes abbreviatus L. and a kerriid scale Parata-
chardina lobata Chamberlin) with both kinds (Ta-
ble 2). Among the shared herbivores, only 1 native
weevil (Artipus floridanus Dietz) fed on the inva-


December 2006






Liu et al.: Insects on Native, Invasive, and Non-invasive Eugenia


N I

11 11 8


N


NI
6" '\


A


100%0
So800/-
6so%/o -
40%0
I 200/%
00/0%


* aigophagous
SPolyphagous
o Uhknca


t%. % I, 4t a s c i

Rant species


I NI
D 1 1 N1



In I *
I /om Encophfgous
40% o Ectphagous
206/ --
OD/ o o o o ,
4- 4. \4 .4-

Part Species
SN I N I NI

100%


600/0- Exctic
3 NNtile
406 -1
20%D/ -

/I Iv o c o o ., o,


Plart species
Fig. 1. Percentage of herbivore species found in different categories on six wild and/or cultivated Eugenia species
in South Florida. The vertical dash lines separate wild plants from cultivated ones, with the former on the left.
W_axi = wild E. axillaris, w_foe = wild E. foetida, w_uni = wild E. uniflora, c_axi = cultivated E. axillaris, c_foe =
cultivated E. foetida, c_uni = cultivated E. uniflora, c_agg = cultivated E. aggregata, c_bra = cultivated E. brasil-
iensis, c_lus = cultivated E. lushnathiana. "N" indicate native plants, "I" the introduced invasive plant, and "NI" the
introduced non-invasive plants. Numbers on top of the bars are the total number of herbivore species found.


9 10 12 6 7




- -









TABLE 2. HERBIVOROUS INSECT SPECIES FOUND ON SIX WILD AND/OR CULTIVATED EUGENIA SPECIES IN SOUTH FLORIDA. NATIVE EUGENIA SPECIES ARE IN BOLD AND IN-
VASIVE EUGENIA ARE IN ITALICS. W_AXI = WILD E. AXILLARIS, W_FOE = WILD E. FOETIDA, W_UNI = WILD E. UNIFLORA, C_AXI = CULTIVATED E. AXILLARIS, C_FOE
= CULTIVATED E. FOETIDA, C_UNI = CULTIVATED E. UNIFLORA, C_AGG = CULTIVATED E. AGGREGATA, C_BRA = CULTIVATED E. BRASILIENSIS, C_LUS = CULTIVATED
E. LUSHNATHIANA, POLY = POLYPHAGOUS OR GENERALIST. OLIGO = OLIGOPHAGOUS OR SPECIALIST. ENDO = ENDOPHAGOUS, ECTO = ECTOPHAGOUS. DOES NOT
OCCUR, + NOT IMPORTANT, ++ IMPORTANT, +++ VERY IMPORTANT. UNID = UNIDENTIFIED. ? INDICATES UNKNOWN OR UNCERTAIN INFORMATION.

Occurrence on Eugenia species
Diet Feeding Guild/plant
Insect species Originsb breadth nichb parts w_axi w_foe w uni caxi cfoe c uni c_agg cbra clus

Coleoptera
Curculionidae
Anthonomus alboannulatus Boheman Native Oligo Endo Seed ++ +++
Anthonomus irroratus Dietz Native Oligo Endo Seed -+++ ++
Atractomerus punctipennis Gyllenhal Native Oligo Ecto Leaf +
Artipus floridanus Horn Native Poly Ecto Leaf, root? + + + + + + -
Diaprepes abbreviatus L. Exotic Poly Ecto Leaf, root? ++ ++ ++ + + + + + +
Myctides imberbis Lea Exotic Oligo Ecto Leaf, fruit? ++ +
Myllocerus undatus Marshall Exotic Poly Ecto Leaf, root? + + +++ ++ ++ ++ -
Pheloconus hispidus LeConte Native Poly Endo Seed ++ ++ ++ ++ ++
Nitidulidae
Lobiopa insularis Castlenaua Native Poly Ecto Fruit flesh ++ ++ ++ ++
Epuraea luteolus Erichsona Native Poly Ecto Fruit flesh ++ ++ ++ ++
Diptera
Cecidomyiidae
Dasineura eugeniae Felt Native Oligo Endo Leaf, fruit +++ +++ ++ ++
gallery (fruit only)
Stephomyia eugeniae Felt Native Oligo Endo Leaf gallery +++
Tephritidae
Anastrepha suspense Loew Exotic Poly Endo Fruit flesh +++ +++ ++ ++ +++

Hemiptera
Coccidae
Pulvinaria psidii Maskell Native Poly Ecto Stem and leaf ++
Flatidae
Melormenis basalis Walker Exotic Poly Ecto Leaf -- +
Kerriidae
Paratachardina lobata Chamberlin Exotic Poly Ecto Stem ++ + + ++ ++ ++ + ++ ++

Herbivores with little fitness consequences because they only consume fleshy parts of the fruit without damaging the seed.
unknown cases are assumed to be native, polyphagous, and external feeders for the chi-square tests.













TABLE 2. (CONTINUED) HERBIVOROUS INSECT SPECIES FOUND ON SIX WILD AND/OR CULTIVATED EUGENIA SPECIES IN SOUTH FLORIDA. NATIVE EUGENIA SPECIES ARE IN
BOLD AND INVASIVE EUGENIA ARE IN ITALICS. W_AXI = WILD E. AXILLARIS, W_FOE = WILD E. FOETIDA, W_UNI = WILD E. UNIFLORA, C_AXI = CULTIVATED E. AXIL-
LARIS, C_FOE = CULTIVATED E. FOETIDA, C_UNI = CULTIVATED E. UNIFLORA, C_AGG = CULTIVATED E. AGGREGATA, C_BRA = CULTIVATED E. BRASILIENSIS, C_LUS
= CULTIVATED E. LUSHNATHIANA, POLY = POLYPHAGOUS OR GENERALIST. OLIGO = OLIGOPHAGOUS OR SPECIALIST. ENDO = ENDOPHAGOUS, ECTO = ECTOPHAGOUS.
DOES NOT OCCUR, + NOT IMPORTANT, ++ IMPORTANT, +++ VERY IMPORTANT. UNID = UNIDENTIFIED. ? INDICATES UNKNOWN OR UNCERTAIN INFORMATION.

Occurrence on Eugenia species
Diet Feeding Guild/plant
Insect species Originsb breadth nichb parts w_axi w_foe w uni caxi cfoe c uni c_agg cbra clus

Psyllidae
Katacephala tenuipennis Tuthill Native Oligo Ecto Leaf +++ +++

Lepidoptera
Gracillariidae
Chilocampyla dyariella Busck Native Oligo Endo Leaf miner ++ ++ +? ++ +
Tortricidae
Ancylis sp. Native Poly Ecto Leaf tier +++ +++ -+++
young leaves
Platynota flavedana Clemens Native Poly Ecto Leaf tier + + -
Sparganothis lentiginosana Walsingham Native Poly Ecto Leaf tier + -
young leaves
Strepsicrates smithiana Walsingham Native poly Ecto Leaf tier +++ +++ +++ +++
young leaves

Orthoptera
Acrididae
Stenacris vitreipennis Marshall Native Poly Ecto Leaf + -
Unid. Acrididae Native? Poly Ecto Leaf +

Thysanoptera
Phlaeothripidae
Elaphrothrips sp. Native Poly? Endo Leaf gallery ++ ++

Herbivores with little fitness consequences because they only consume fleshy parts of the fruit without damaging the seed.
unknown cases are assumed to be native, polyphagous, and external feeders for the chi-square tests.







Florida Entomologist 89(4)


sive Eugenia, while two native insects (Ancylis sp.
and Elaphrothrips sp.) fed on the non-invasive
Eugenia. The insect that caused substantial dam-
age on the invasive Eugenia was an exotic weevil
(Myllocerus undatus Marshall), while the insect
that caused substantial damage on the non-inva-
sive Eugenia was a native moth (Ancylis sp.). The
native Eugenia also likely shared a specialist in-
sect (a leaf blotch mining moth, Ci.i!..... .....i. .
dyariella Busck) with the invasive congener (Ta-
ble 2). However, it was not clear if the leaf miners
were able to complete their development in E. uni-
flora leaves, because these incidents were rare
and we were not able to rear any adults.

DISCUSSION

Prediction 1-there will be greater numbers of
herbivore species on native Eugenia than on in-
troduced species.
There is limited evidence supporting our first
prediction in relation to herbivore species rich-
ness on native vs. introduced non-invasive Euge-
nia because the cultivated native species had
more insect herbivore species than 2 of the 3 in-
troduced non-invasive species. This is consistent
with the results found in a study comparing in-
sect herbivore fauna between a native Pinus and
a co-occurring introduced non-invasive congener
(Lindeldw & Bjorkman 2001). There also was only
limited support for the prediction in relation to
the native vs. introduced invasive species in this
study because the native Eugenia species had
more insect herbivore species than the introduced
invasive Eugenia in the wild, but not in cultiva-
tion. In the only other similar study (Birki &
Nentwig 1997), comparing the herbivore fauna
between populations of the native Heracleum
sphonylium L. and the co-occurring introduced
invasive congeners, H. mantegazzianum Simmier
& Levier, there was an equal number of insects
associated with both plant species.
Furthermore, contrary to the second part of
our first prediction that the invasive Eugenia
should have a smaller number of herbivore spe-
cies than the non-invasive congeners, the inva-
sive Eugenia (E. uniflora), wild or in cultivation,
had greater numbers of insect herbivore species
than all 3 non-invasive Eugenia. This result is the
opposite to that reported in a study on plant
pathogens (Mitchell & Power 2003), in which the
authors found that more invasive plants tended
to have fewer pathogens. Nevertheless, differ-
ences in herbivore richness were small among the
Eugenia species studied here. In addition, there is
always the possibility that high number of herbi-
vore species may not translate into high damage
level (Liu, unpublished data).
Prediction 2-There will be greater numbers of
oligophagous and endophagous herbivore species
on native Eugenia than on introduced species.


The data support the first part of our second
prediction that native Eugenia species should
have the highest number and percentage of oli-
gophagous insect herbivores. However, the statis-
tical results should be interpreted with caution
due to the small number of insect species on each
Eugenia species. Our result is consistent with 1
congeneric native vs. introduced species compari-
son (Birki & Nentwig 1997), but differs from an-
other (Lindeldw & Bjorkman 2001). In addition,
the native plants had higher number of internal
feeders even though the percentage of endopha-
gous herbivore species was not different between
the native and introduced Eugenia. However, in
contrast to the second part of our second predic-
tion, the invasive Eugenia had as many or more
oligophagous and/or endophagous feeders than
non-invasive introduced Eugenia. No other stud-
ies were found to compare the number of oligoph-
agous and endophagous insects between invasive
and non-invasive plants.
Prediction 3-Fewer herbivores will be shared
between native Eugenia and invasive Eugenia
than between native Eugenia and non-invasive
Eugenia.
The third prediction that native Eugenia
should share fewer specialist and endophagous
herbivores with invasive Eugenia than with non-
invasive Eugenia was not supported by the data.
While native Eugenia shared no oligophagous or
endophagous herbivores with non-invasive Euge-
nia, they likely shared a leaf miner with E. uni-
flora (the invasive introduced Eugenia). However,
because the blotch mines were only found on the
wild individuals, it is possible that the host shift
occurred after E. uniflora had invaded the natu-
ral areas. In addition, because the mines occurred
at such a low rate the biotic resistance from this
miner should be small. Host sharing by oligopha-
gous herbivores largely depends on the taxonomic
closeness of the host plants (Strong et al. 1984). A
phylogeny of the genus Eugenia may help to ex-
plain and predict the shifts of specialists from the
native to the introduced congeners.
No leaf galls were observed on any of the intro-
duced Eugenia species in this study whereas one
specialist galling fly, Eugeniamyia dispar Maia et
al. (Diptera, Cecidomyiidae) (Maia et al. 1996)
was found on E. uniflora in its native range. All in-
troduced Eugenia studied here have probably es-
caped specialist insects that may be found in their
native ranges. The lack of specialist insect attack
may lead to a shift in plant resource allocation to
growth (Blossey & Ndtzold 1995; Siemann & Rog-
ers 2001, Wolfe et al. 2004) and/or defense to gen-
eralist herbivores (Joshi & Vrieling 2005).
Native Eugenia plants in cultivation have a
less diverse insect fauna than those in the wild,
probably due to the differences in time since pop-
ulation establishment (Strong et al. 1984). Culti-
vated populations tend to be much younger and


December 2006







Liu et al.: Insects on Native, Invasive, and Non-invasive Eugenia


have less time to acquire insect fauna. Pesticide
treatment in some horticulture or agriculture sit-
uations also may cause a decrease in herbivore
fauna. However, all cultivated Eugenia individu-
als sampled in this study were not treated di-
rectly with pesticides (Jonathan H. Crane of
TREC, Micheal Davenport of FTBG, Chris Rollins
of FSP, personal communications). Nevertheless,
our analyses and discussions are mostly limited
to faunal comparisons among different species of
the same source (wild or cultivated).
A result that is not related to the ERH testing
but nonetheless interesting is the composition of
native vs. exotic herbivores on the 3 categories of
Eugenia plants. The native herbivores consti-
tuted about half of the insect herbivore fauna ac-
quired by the introduced Eugenia. The numbers
of exotic insects attacking the native, invasive,
and non-invasive plants are similar (3-5 on each
plant species). Since most of these exotic herbi-
vores came from continents other than Central
and South America, where the introduced Euge-
nia are native, it is unlikely that these exotic her-
bivores were associated with the exotic Eugenia
in its native range. We did not observe any native
insect herbivores having more importance on the
introduced than on the native Eugenia plants. In
contrast, it appeared that an exotic weevil (M. un-
datus), a new comer from Sri Lanka (Schall 2000),
fed more heavily on E. uniflora (the invasive Eu-
genia) than on other congeners (Liu, personal ob-
servations). In addition, the only exotic oligopha-
gous weevil (Myctides imberbis, an Australian na-
tive) found in this study also was observed on E.
uniflora more than on the native or the non-inva-
sive Eugenia. Together, our data suggested that
the exotic herbivores provided as much, if not
more, herbivore pressure as the native insects to
the introduced Eugenia. Our finding was differ-
ent from that of a recent study which found that
the native herbivores, mostly vertebrates, sup-
pressed introduced plants, whereas exotic herbi-
vores, also mostly vertebrates, promoted exotic
plants (Parker et al. 2006).
In summary, data on herbivore faunal diver-
sity of Eugenia species provided limited support
to the ERH. It is likely that other factors contrib-
ute to the success of E. uniflora. If we did not in-
clude the non-invasive Eugenia species in the
study and only compared the herbivore fauna be-
tween the native Eugenia and invasive Eugenia,
we would have thought that release from the in-
sect herbivores was an important factor in the
success of E. uniflora. We did not include patho-
gens, also recognized as natural enemies, in this
study. Future study should take advantage of this
unique three-way system to examine the effects of
pathogens and other competing but non-exclusive
hypotheses to help explain the success of E. uni-
flora. For example, competitive interactions of in-
troduced invasive Eugenia vs. native co-occurring


plants and non-invasive introduced Eugenia vs.
native plants could be examined. The three- way
comparison could also be used to examine the im-
portance of relative seed numbers (the propagule
pressure hypothesis (Williamson 1996), which
states that the species with the greater number of
propagules will be the most invasive). Eugenia
uniflora, is much more abundant than the non-in-
vasive Eugenia because it has long been used as a
hedge plant, and probably produces more poten-
tially invasive seeds.

ACKNOWLEDGMENTS
Rita Duncan is acknowledged for helping and teaching
Hong Liu insect preparation prior to identifications. Jose
Allegria helped with field sampling. The following people
helped with specimen identifications: M. C. Thomas (Co-
leoptera), S. E. Halbert (Homoptera: Flatidae), J. B. Hep-
pner (Lepidoptera), G. S. Hodges (Coccidae), G. B.
Edwards (Thysanoptera), and S. E. Halbert (Psyllidae),
all at the Division of Plant Industry, Florida Department
of Agriculture and Consumer Services; Don Davis (Gracil-
laridae and Yponomeutidae), Department of Entomology,
Smithsonian Institute; David Adamski (Blastobasidae),
USNM, C/O Department of Entomology, Smithsonian In-
stitute; Ray Gagne (Cecidomyiidae), Systematic Entomol-
ogy Laboratory, Agriculture Research Service, US
Department of Agriculture. Support for this project came
from NSF grant DEB 03-15190 to Peter Stiling.

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Florida Entomologist 89(4)







Homziak & Homziak: P. demoleus record in Puerto Rico


PAPILIO DEMOLEUS (LEPIDOPTERA: PAPILIONIDAE): A NEW RECORD
FOR THE UNITED STATES, COMMONWEALTH OF PUERTO RICO

NICHOLAS T. HOMZIAK1 AND JURIJ HOMZIAK2
'29 Crescent Terrace, Burlington, VT 05401
jhomziak@sover.net

2Rubenstein School of Environment and Natural Resources
317 Aiken, University of Vermont, Burlington, VT 05405
jurij.homziak@uvm.edu

ABSTRACT
We report the first record of the citrus pest Papilio demoleus Linnaeus collected near
Guanica in the United States, Commonwealth of Puerto Rico, in March 2006.

RESUME
Reportamos la primera ocurrencia de la mariposa asidtica Papilio demoleus en Puerto Rico
qua fue coleccionado alrededor de Guanica, Puerto Rico, en marzo, 2006.

Translation provided by the authors.


Papilio demoleus L., commonly known as the
lime or citrus swallowtail, is found throughout
southern Asia (Corbet & Pendlebury 1992, cited
in Guerrero et al. 2004) where it is a commercially
important pest of citrus. In recent times it has ex-
panded its range into new areas of the Old World
following the introduction and cultivation of cit-
rus (Matsumoto 2002). More recently, Guerrero et
al. (2004) documented the presence of P demoleus
in the eastern Dominican Republic on the island
of Hispaniola; the first confirmed report of this
species in the Americas. Eastwood et al. (2006)
subsequently reported that P demoleus had
spread across much of the Dominican Republic
and, using molecular data, were able to trace its
provenance and confirm the pest status of the in-
troduced population. To date it has not been re-
corded from any other locality in the Western
Hemisphere (although there is a dubious record
from California (Tilden 1968, cited in Guerrero et
al. 2004). Here, we report the collection of 1 spec-
imen (female) of P. demoleus in a residential en-
clave within the Guanica Dry Forest Reserve in
Puerto Rico (Fig. 1). It was 1 of 3 specimens ob-
served alighting and possibly ovipositing on an
ornamental lime tree (Citrus aurantifolia Swin-
gle) in a residential garden.
Collection data are UNITED STATES: COM-
MONWEALTH OF PUERTO RICO, Municipality
of Guanica. 4.III.2006. Nicholas T Homziak. 7.1
km south and east of town of Guanica on Rte 333
(from junction with Rte 116) to right turn at Hoya
Hondo, then 1 km south. (17 degrees, 57.0 minutes
North; 66 degrees, 52.6 minutes West). Elevation:
near sea level. The identity of the specimen was
confirmed by Rod Eastwood (Griffith University,


Brisbane, Australia, personal communication). The
residential area is located along a slight coastal
ridge; largely cleared of the original Subtropical
Dry Forest. The low area behind the ridge is domi-
nated by introduced drought-tolerant legumes on
poorly draining saline soils. There is commercial
citrus production in the nearby region ofYauco.
Guerrero et al. (2004) suggested that the lime
swallowtail was likely to disperse rapidly away
from its initial point of introduction in the eastern
Dominican Republic. Papilio demoleus is recog-
nized as a major pest of citrus throughout most of
its Old World range, causing significant economic
losses (Agribusiness Information Centre of India,
2005; Malaysian Tropical Fruit Information Sys-
tem 2004; Pakistan Agricultural Research Coun-
cil 2003). Based on its dispersal and life history
characteristics documented in Asia, P demoleus
is likely to expand and become a serious citrus
pest throughout the Caribbean and adjacent
mainland locations. Our collection of a specimen
from Puerto Rico indicates that it is expanding its
range across a much wider area, with potentially
serious economic implications for regional citrus
production in the Caribbean and Florida.
Papilio demoleus has a history of successful
dispersal and range extensions throughout Asia.
Found throughout Southeastern Asia (Common-
wealth Institute of Entomology 1979), it has ex-
tended its range across mountain ranges, deserts,
and other inhospitable terrain to become a major
citrus pest in India (Agribusiness Information
Centre of India, 2005), Pakistan (Pakistan Agri-
cultural Research Council 2003), Iraq (Larsen
1977, cited in Eastwood et al. 2006) and the Mid-
dle East (Farid 1987; Badawi 1981). From South







Florida Entomologist 89(4)


Fig. 1. Papilio demoleus from Guanica, Puerto Rico, (a) dorsal view, (b) ventral view.


December 2006







Homziak & Homziak: P. demoleus record in Puerto Rico


and East Asia it has extended its range into the
Indo-Pacific, dispersing throughout the islands of
Indonesia (Dunn 1999; Matsumoto 2002; Moonen
1991) to New Guinea (Moonen 1999) and Austra-
lia (Smithers 1978; Williams et al. 1998). With
this capacity for successful migration and range
extension, P demoleus is likely to rapidly expand
its range beyond Hispaniola to include most is-
lands in the Caribbean and adjacent mainland ar-
eas, including Florida.
Papilio demoleus has the potential to become a
pest because it shows rapid population growth un-
der favorable circumstances (Bhan & Singh 1997;
Chatterjee et al. 2000; Pathak & Rizvi 2003; Radke
& Kandalkar 1988). Papilio demoleus can have 5
broods per year in warm temperate China (Chen et
al. 2004). Under ideal experimental conditions in
India, Pathak & Rizvi (2003) reported generation
time for P demoleus to be just over 30 d.
Dispersal ability and the capacity for rapid
population growth make P. demoleus a potentially
serious pest throughout the Caribbean with sig-
nificant economic impact. Citrus is an important
agricultural commodity in most of the Caribbean.
It is already in decline in several countries be-
cause of pests and diseases (Donovan 2002). In a
review of the Caribbean citrus industry, Donovan
(2002) reports that citrus production contributes
significantly to income generation, foreign ex-
change earnings, employment, food security, eco-
nomic diversification and growth in the region.
Citrus production and associated manufacture
are important contributors to the GDP of most
Caribbean island nations.
Based on estimates from the Caribbean Cooper-
ative Citrus Association, 52,000 persons are em-
ployed in the industry across CARICOM, generat-
ing over US$61 million dollars in foreign exchange
earnings. Instead of crop value, production data
may be more informative because most Caribbean
citrus is produced for domestic markets. Production
for CARICOM countries in 2001 was estimated to
be 510,000 metric tons, 520,000 metric tons for
Cuba, 70,200 metric tons for the Dominican Repub-
lic and 27,000 metric tons for Haiti. Countries of
the Organization of Eastern Caribbean States
(OECS) also produce significant amounts of citrus.
Most production is by small farmers for domestic
consumption. Small citrus farms (less than 5000
boxes per year) make up between 93 and 98 percent
of all CARICOM farms and supply 42 percent of cit-
rus fruits. Because the citrus industry is critical to
the economic survival of many small farmers, this
group would be most affected by the spread of P
demoleus in the Caribbean.
In 2002, the value of the Florida citrus crop ex-
ceeded $1.5 billion; the U.S. total (Florida, Cali-
fornia, Arizona, and Texas) was more than $2.6
billion (National Agricultural Statistics Service
2004). The introduction of P demoleus could have
a significant economic impact on production and


profitability of the industry. While advanced pest
management tools are more readily available in
the US than in the wider Caribbean region, grow-
ing insect resistance to microbial and other con-
trol strategies (Narayanan 2005) may leave the
industry vulnerable to this new pest species.

ACKNOWLEDGMENTS

We thank Dr. Rod Eastwood for identification of the
specimen and for encouragement in getting this article
published.

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Park & Park: Two New Species of Ceratophysella from Korea



TWO NEW SPECIES OF CERATOPHYSELLA
(COLLEMBOLA: HYPOGASTRURIDAE) FROM KOREA

KYUNG-HWA PARK AND NAM-YEE PARK
Department of Biology Education, Chonbuk National University, Jeonju 561-756, Korea

ABSTRACT

Two new species of the genus Ceratophysella from Korea, Ceratophysella biclavata n. sp.
and Ceratophysella platyna n. sp. are described and illustrated. Ceratophysella biclavata
differs from the closely related species Ceratophysella sigillata (Uzel 1891) by the shape of
antennal bulb on antennal segment IV, the number of clavate tenent hairs and the number
of granules between p, upon abdominal segment V. Ceratophysella platyna resembles Cer-
atophysella denticulata (Bagnall 1941) and Ceratophysella communis (Folsom 1898), but
distinctly differs from the latter by the shape of tenent hairs. A key to the identification of
the Korean species of Ceratophysella is included. In addition, the known species Hypogas-
trura gracilis (Folsom 1899) is described and recorded for the first time from Korea.

Key Words: Hypogastrura, Poduromorpha, Arthropleona, springtail, Apterygota, South Korea

RESUME

Dos nuevas species del g6nero Ceratophysella de Korea, Ceratophysella biclavata sp. n. y
Ceratophysella platyna sp. n. son descritas e ilustradas. Ceratophysella biclavata se distingue
de la especie cercana Ceratophysella sigillata (Uzel, 1891) por la forma del bulbo antenal en el
segment IV de la antena, el numero de setas adhesivas clavadas y el numero de los granulos
entire p, en el segment V del abdomen. Ceratophysella platyna se parece a Ceratophysella den-
ticulata (Bagnall, 1941) y Ceratophysella communis (Folsom, 1898), pero difiere claramente de
estos por la forma de las setas adhesivas. Una clave para la identificaci6n de las species ko-
reanas de Ceratophysella es incluida. Tambi6n se adjunta la especie conocida Hypogastrura
gracilis (Folsom, 1899) la cual es descrita y registrada por primera vez en Korea.


The family Hypogastruridae is common, wide-
spread, and has cosmopolitan distribution con-
taining approximately 659 world species in about
40 genera. The genus Ceratophysella also with
worldwide distribution is one of the largest gen-
era in the family, with more than 108 known spe-
cies (Bellinger et al. 2006). Their habits were
noted by Hopkin (2002), who stated that they of-
ten form enormous swarms on roads, glaciers,
snow, and on the surfaces of puddles. Individuals
in the swarms all leap together in the same direc-
tion using the orientation of the sun to navigate.
They have small expandable sticky sacs on their
antennae that help them adhere to the substrate
when they land after a jump to stabilize them
(Hopkin 2002).
Eight species of the genus Ceratophysella oc-
cur in Korea. Yosii & Lee (1963) recorded C. com-
munis (Folsom 1897), Lee (1974) added 4 species,
C. liguladorsi Lee, 1974, C. sinetertiaseta Lee,
1974, C. armata (Nicolet 1841) and C. duplicispi-
nosa Yosii, 1954. Later Thibaud & Lee (1994)
added the species, C. bengtssoni (Agren 1904),
and Lee & Kim (1995, 2000) recorded 2 species,
C. dolsana Lee & Kim, 1995, C. denticulata (Bag-
nall 1941). We add here 2 new species of the genus
Ceratophysella and 1 species of the genus Hypo-
gastrura as additions to the Korean fauna.


The purpose of this paper is to describe 2 new
species and to provide an identification key to the
species of Ceratophysella from Korea. Lee & Kim
(1995) described C. dolsana as a new species, but
there is no description of the genus in their work.
Most authors regarded dolsana as belonging in the
genus Hypogastrura (Bellinger et al. 2006; Thibaud
et al. 2004). However, we include it in the key of
Ceratophysella, primarily on the basis of long p2
seta on thoracic segments II-III and on the shape of
mucro in holotype and paratypes. Morphological
abbreviations used in this paper are as follows: Ant.
I-IV: antennal segments I-IV; Th. I-III: thoracic seg-
ments I-III; Abd. I-VI: abdominal segments I-VI;
seta a and b: seta a and b among the 7 dorsal sen-
sory setae of Ant. IV; al, .... : setae 1, 2 ... of the
anterior row counted from the "middle line"; m1, 2..
.: setae 1, 2 ... of the middle row, counted from the
"middle line"; p,, ... : setae 1, 2 ... of the posterior
row, counted from the "middle line".

MATERIALS AND METHODS

Material was collected from 3 localities in Ko-
rea. Either an aspirator for direct collection or a
Tullgren apparatus for extracting specimens was
used. Collembola were fixed in 90% ethanol. Marc
Andr6 I and II solutions were used to clear and







Florida Entomologist 89(4)


prepare specimen slides (Massoud 1967). KOH
solution (10%) was used for rapid de-coloration.
To prepare permanent slides, glycerine was
placed along the cover glass edge to prevent the
slide medium from drying. All type specimens are
deposited in the Insect Collection of Biology Edu-
cation Department, Chonbuk National Univer-
sity, Jeonju, Korea.

Ceratophysella biclavata,
new species

Description (Fig. 1). Body length 1,110-1,400
pm (1,200 pm long in holotype). Color dark brown
or blackish brown on whole body except inter-seg-
mental portions and the ventral side. Body cylin-
drical, being narrower abruptly at Abd.V (Fig.
1A). Head length 220 pm in holotype. Antenna
shorter than head, 0.9 in ratio to head; ratio of
length of antennal segments I:II:III:IV is 5:5:6:4.
Ant. IV with a simple apical bulb and a closely as-
sociated small papilla, a socket seta and some
weak setae (Fig. 1B), and with 7 dorsal sensory
setae of which seta a and b thickened. Eversible
sac between Ant. III and Ant. IV distinctly devel-
oped. Ant. III organ with 2 short sensory and 2
guard sensilla (Fig. 1D). Mandible with 4 apical
teeth (Fig. 1F). Eyes 8 + 8, eye patch with 3 setae.
Postantennal organ (PAO) consists of 4 periph-
eral tubercles, about 1.2-1.5 times as long as the
diameter of the nearest ocelli, with anterior lobes
distinctly larger than posterior and with a small
accessory tubercle (Fig. 1C). Tenent hairs 2, 2, 2
with distal end weakly clavate. Unguis elongate,
with an inner tooth and a pair of lateral teeth.
Unguiculus setaceous and with broad, rounded
basal lamella (Figs. II-K). Ventral tube with 4 se-
tae on each half. Tenaculum with 4 + 4 barbs
without setae (Fig. 1E). Dens dorsally finely gran-
ulated and with 7 setae, 4 of them thicker than
the others, about twice as long as mucro. Mucro
apically rounded and with well developed outer
lobe, anterior margin modified to form a tooth-
like thickening from which a thin lamella extends
basally (Fig. 1H). Abd.V with a granulated medial
stripe, granules not modified, but arranged
rather regularly. Mostly 11-13 granules lying be-
tween the p, seta on Abd.V (Fig. 1L). Anal spines
1/2-2/3 as long as inner unguis and about 2-2.5
times as long as papillae. On Abd. VI, a1 shorter
than anal spine including anal papilla (Fig. 1G).
Chaetotaxy. Area verticalis confluent with
area occipitalis and with 2 + 2 setae. Th. I with 3
+ 3 setae in a row. Th. II and III composed of 3
rows of setae, lacking m2, p2 a macrosetae and p4
the sensory seta. Abd. I-III with 2 rows of setae, p2
a macroseta and p5 the sensory seta. Abd. IV with
3 rows of setae, lacking a m, and m3; p, longer
than p, and p3. Abd. IV setae often asymmetric in
position. Abd. V with 2 rows of setae, p, longer
than p2, a lacking and p, sensory seta (Fig. 1M).


Type Materials

Holotype: Female, Temple Jeongamsa, Ga-
cheon-ri Dongmyeong-myeon Chilgok-gun, Gyeo-
ngsangbuk-do Province, collected from litter soil
layer of the forest near stream. 24-X-2004, collec-
tion no. 204-21. Paratypes: 2 males and 3 females,
same data as holotype.
Etymology. The specific name is derived from
the number and shape of tenent hairs in each leg.
Remarks. The present species is very similar
to C. sigillata (Uzel 1891), and redescribed by Ba-
benko et al. (1994), in chaetotaxy of thorax and
abdomen, in shape of mucro and basal lamella of
unguiculus and in shape of seta on dens. How-
ever, they can be separated easily by differences
in the shape of antennal bulb on Ant. IV, the num-
ber of tenent hairs on each leg and in the number
of granules between p, upon Abd.V. Number of
granules between p, of Abd. V is 20-25 in C. sig-
illata and 11-13 in the present new species. Also,
the present species differs from C. sigillata by the
strongly developed eversible sac (weakly devel-
oped in C. sigillata) and the absence of hook-like
sensilla upon fourth antennal segment (Table 1).

Ceratophysella platyna,
new species

Description (Fig. 2). Body length 1,200-1,400
pm (1,200 pm long in holotype). Body dark brown
with blue pigment scattered over dorsum of seg-
ments in the form of irregular transverse bands
(Fig. 2A). Head length 270 pm in holotype. An-
tenna shorter than head, 0.8 length of head; ratio
of length of antennal segments I:II:III:IV is
3:4:5:6. Fourth antennal segment with a simple
apical bulb and a closely associated protective pa-
pilla, giving a bilobed appearance to the antenna
apex; lacking ventral file, but with 11-13 rela-
tively long straight setae and seven clear blunt
setae (Figs. 2B, E). Eversible sac between Ant. III
and IV distinctly differentiated. Left mandible
with 5 apical teeth and right with 4 apical teeth
(Figs. 2D, H). Postantennal organ with 4 periph-
eral tubercles, a small accessory tubercle, ante-
rior lobes strikingly larger than posterior and
about 1.5 times as long as nearest ocelli. Eye
patch with 8 ocelli on each side (Fig. 2C). Unguis
slender, slightly curving distally, with 1 inner
tooth on internal lamella. Unguiculus pointed
and with a basal lamella tapering into a filament,
almost 1/2 as longer internal lamella of unguis. Te-
nent hairs 1, 1, 1 almost as long as outer unguis
and truncate to feebly clavate (Fig. 2G). Ventral
tube with 3 + 3 setae. Tenaculum with 4 + 4 barbs.
Dens about twice as long as mucro, with 7 poste-
rior setae, without basally enlarged angled setae
(Fig. 2F). Outer unguis 1.5 times as long as mu-
cro. Mucro 0.8-0.9 times as long as anal spines.
Body setae all smooth and slender. Integument


December 2006







Park & Park: Two New Species of Ceratophysella from Korea


B-L soA
IM I g0W&


Fig. 1. Ceratophysella biclavata n. sp. A. Habitus. B. Apical view of antenna IV segment. C. Postantennal organ
(PAO) and 8 ocelli. D. Dorsal view of antenna III, IV segments and the expandable sac between antennal segment
III and IV. E. Tenaculum. F. Mandible. G. Anal spine. H. Dorsal view of mucro and dens. I. First leg. J. Second leg.
K. Third leg. L. Abdomen V segment. M. Dorsal chaetotaxy of body.


moderately granular. Granular stripe on Abd. V
arranged regularly, 9-12 granules lying between
the p, setae on Abd. V (Fig. 21). Fovea lying be-
tween the pl. Anal spines slender, on unusually


large contiguous papillae. On Abd. VI, a, nearly as
long as anal spine including anal papilla (Fig. 2J).
Chaetotaxy. Area verticalis confluent with
area occipitalis and with 2 + 2 setae. Th. I with 3







Florida Entomologist 89(4)


TABLE 1. DIAGNOSTIC CHARACTERS FOR CERATOPHYSELLA SIGILLATA AND CERATOPHYSELLA BICLAVATA N. SP.

Species/Character C. sigillata C. biclauata n. sp.

The number of clavate tenent hairs 1, 1, 1 2, 2, 2
Ant. IV antennal bulb a simple apical bulb a simple apical bulb and a closely
associated small papilla
The number of granules between 20-25 grains 11-13 grains
p, upon Abd. V
Eversible sac weakly developed strongly developed
hook-like sensilla upon Ant. IV Present absent


+ 3 setae in a row. Th. II and III with 3 rows of se-
tae, m2 and m3 absent, p2 a macroseta and P4 the
sensory seta. Abd. I-III with 2 rows of setae, with-
out m-seta, with a,', p2 a macroseta and p, the sen-
sory seta. Abd. IV with 3 rows of setae, a1 slightly
laterally dislocated, a,, m2 and m, absent, p2
longer than p, and p, the sensory seta. Abd. V
with 2 rows of setae, without a,', p1 longer than p,,
a, lacking and p, the sensory seta (Fig. 2K).

Type Materials

Holotype: Male, 700 m a.s.l., Mt. Moacksan,
Gui-myeon, Wanju-gun, Jeollabuk-do Province,
collected from the leaf litter under snow, 14 Feb
2004, collection no. 204-01-1. Paratypes: 2 males
and 2 females, same data as holotype.
Etymology: The specific name, platyna, refers
to the shape of body in this species.
Remarks: This species is characterized by the
presence of an antennal bulb and the shape of te-
nent hairs. In many respects this species resem-
bles C. pratorum of C. boletivora-group from
North America (Christiansen & Bellinger 1998),
but they differ in chaetotaxy. The present species
is a member of Gisin's A type (Gisin 1947) with p,
seta longer than p, seta on Abd. IV (p, > p2 in
C. pratorum). The antennal bulb clearly sepa-
rates C. platyna n. sp. from C. boletivora and
C. biloba of C. boletivora-group. Also, the present
species is closely related to palaearctic species
C. annae described by Babenko (1994), but is dis-
tinguished by the darker body colour, the pres-
ence of eversible sac and having 7 dorsal sensilla
setae on Ant. IV (C. annae has 6). Chaetotaxy of
the present species is similar to C. communis (Fol-
som) from Korea (Lee 1974; Lee & Thibaud 1975)
by the presence of the a,' seta on Abd. I-III, the
absence of the a,' seta on Abd. V, but it is sepa-
rated from the latter in the shape of tenent hairs
and the number of granules between p, upon Abd.
V. It also has the same number of granules be-
tween p, upon Abd. V with cosmopolitan C. dentic-
ulata (Bagnall 1941) (Yosii 1962; Lee & Kim
2000). However, this new species is distinctly dif-
ferent from C. denticulata and C. communis in the
shape of tenent hairs (Table 2).


Hypogastrura gracilis (Folsom, 1899),
new record

Diagnosis (Fig. 3). Body length 1,500-1,900
pm (1,700 pm long in holotype). Color grey or
blackish brown on whole body except only inter-
segmental portions and the ventral side. Body
laterally swollen at Abd. II and III, being gradu-
ally narrower toward posterior end (Fig. 3A).
Head length 310 pm in holotype. Antenna longer
than head, ratio 1.1 to head length; ratio of
length of antennal segments I:II:III:IV is
12:13:18:30. Fourth antennal segment with a
distal, slightly trilobed end-bulb and a number
of socket setae, with 3 weak setae each on a
slightly differentiated, small subapical papillae
(Figs. 3C, E). Third antennal segment organ of 2
small rods in a shallow groove accompanied by 2
curved setae. Labrum with 4/5, 5, 4 setae, their
distal row very weak. Labral margin with 4
rounded tubercles (Fig. 3H). Postantennal organ
of 4 peripheral tubercles, with or without a small
accessory tubercle, subequal to nearest ocelli
(Figs. 3B, D). Eyes 8 + 8, on black patches. Un-
guis of all legs subequal, relatively small, dor-
sally carinate and with 1 inner tooth near the
distal end. Unguiculus setaceous and reaching
three-quarters of the distance from base to apex
of unguis. Basal half with lamella on the inner
side apically arcuate. Tenent hairs 2, 3, 3 rather
thick and conspicuously swollen at apex. Median
tenent hairs larger than others and above the
level of others on the second and third legs (Fig.
3F). Ventral tube with 4+4 setae. Tenaculum
with 3 + 3 barbs. Dens almost smooth dorsally
with 7 setae, about 4 times as long as mucro. Mu-
cro strongly compressed bilaterally and some-
what blade-shaped (Fig. 3G). Mucro 3.7-5.5
(mostly 4) times as long as anal spines. Outer
unguis 1.3-1.8 times as long as mucro. Anal
spines 0.25 times as long as inner unguis and
subequal to anal papillae. All body setae short
and fine.
Chaetotaxy. Th. I with 3+3 setae in a row. Th.
II and III composed of 3 rows of setae, p4 a little
longer than others, sensory seta on Th. II without
m, seta and Th. III without m,, m,, a, setae. Abd.


December 2006





Park & Park: Two New Species of Ceratophysella from Korea


K. 1Iowa- B

















s i j ,j^- ,lH ^t < s


















Fig. 2. Ceratophysella platyna n. sp. A. Habitus. B. Ventral view of antenna IV segment. C. Postantennal organ
(PAO) and 8 ocelli. D. Left mandible. E. Dorsal view of antenna IV segment. F. Dorsal view of mucro and dens. G.
First leg. H. Right mandible. I. Abdomen V segment. J. Anal spine. K. Dorsal chaetotaxy of body.







Florida Entomologist 89(4)


TABLE 2. DIAGNOSTIC CHARACTERS FOR CERATOPHYSELLA PLATYNA N. SP.

Species/Character C. denticulata C. communis C. platyna n. sp.

Shape of tenent hair acuminate acuminate clavate
The number of granules between p, upon Abd. V 9-12 grains 20 grains 9-12 grains
a2' seta on Abd. V present absent absent


I-III bearing two rows of setae, p2 a macroseta and
p, the sensory seta. Abd. IV with three rows of se-
tae and P4 sensory seta. Abd. V bearing 2 rows of
setae, p, longer than p2, and p, the sensory seta
(Fig. 31).
Material Examined. Numerous specimens col-
lected from soil samples taken from mixed forest
floor at Bisugumi, Dongchon, Hwacheon-eup,
Hwacheon-gun, Gangwon-do Province. 15 Nov
2003, collection no. 203-27. Numerous specimens
collected from litter of natural mixed forest con-
sisting of coniferous and broad-leaved trees 300 m
a.s.l., at the foot of Mt. Obongsan Gui-myeon
Wanju-gun Jeollabuk-do Province. 10 Dec 2005,
collection no. 205-33.
Remarks. This specimen generally correlates
with the descriptions by Yosii (1960) from Ja-
pan. Some minor differences are observed, how-
ever, in the fourth antennal segment setae, in
the presence or absence of accessory tubercle, in
the position of the median tenent hair on the
second and third legs. In addition, the present
material is shown to have some local variation
as compared to the original description. More
extensive collections must be examined to de-
termine whether this is a geographically vari-
able species or a group of several similar spe-
cies. The present species resembles H. bulba
Christiansen & Bellinger 1980 of the viatica
group in the trilobed antennal bulb. But it dif-
fers somewhat from H. bulba in the length ratio
of mucro and dens, the number of tenent hairs
on each leg (2, 3, 3 or 3, 3, 3 in H. bulba), and
relative length of anal spine to anal papilla.
Also, this species is similar to H. tullbergi
(Schaffer 1900), but differs in the absence of
spine-like setae on the apex of the third anten-
nal segment.
Distribution. Japan, Korea (new record).


DISCUSSION

The species of Ceratophysella are character-
ized by having a well developed unguiculus and a
spoon-shaped mucro with a lateral lamella. Poste-
rior arms of postantennal organ are large, and
seta m, on thoracic segment II is absent. In Ja-
pan, about 12 species are recorded (Furuno et al.
2000; Tamura 2001). Three species are known to
occur in China (Zhao et al. 1997).
The taxonomic status of the members of genus
Ceratophysella have been described by several re-
searchers world-wide (Yosii 1960, 1962; Bourgeois
& Cassagnau 1972; Bonet et al. 1973; Christiansen
& Bellinger 1998; Babenko et al. 1994; Thibaud
2004). According to Yosii (1960, 1962), 3 species-
groups are recognized in the genus Ceratophysella:
communis, armata, and denisana-groups. The
communis-group has the chaetotaxy of Gisin's A
type (1947), which seta pon Abd. IV larger than p,
and is represented by C. denticulata Bagnall 1941
in Europe. The chaetotaxy of armata-group repre-
sents Gisin's B type (1947), which seta p, on Abd.
IV smaller than p,. Chaetal arrangement of Cerato-
physella biclavata n. sp. is typical for the armata-
group in the chaetotaxy of Abd. IV. Ceratophysella
platyna n. sp. is clearly different from armata-
group in the chaetotaxy of Abd. IV, where seta p, is
longer than p, and p3. Microsetae and macrosetae
of the species weakly differentiated, but some setae
as p2 on Th. II and III, p, on Abd. I-IV and p, on Abd.
V are longer than others, thus indicating the com-
munis-group of chaetotaxy, that is Gisin's A type.
Ceratophysella platyna n. sp., commonly forms
enormous swarms under leaves covered with snow.
In the present study, 2 new species and 1 newly
recorded species are recognized in Korea. As re-
sult of this study, the Korean faunal list of Hypo-
gastruridae consists of 28 species in 6 genera.


KEY TO 10 SPECIES OF CERATOPHYSELLA FROM KOREA

1. Fourth abdominal segment with seta p, longer than seta P2 ................ ....................... 2
-. Fourth abdominal segment with seta p, shorter than seta P2 ....................................... .8
2. Fourth abdominal segment with seta P2 and seta p, short, sensory seta p, .................. ........... 3
-. Fourth abdominal segment with seta P2 short and seta p, long, sensory seta p, .......................... 7
3. Fifth abdominal segment, an integumentary process "languette" present ....................... liguladorsi
-. Fifth abdominal segment, an integumentary process "languette" absent .............................. 4
4. Dens with bladder-like swelling ........................................................ bengtssoni


December 2006







Park & Park: Two New Species of Ceratophysella from Korea


IB S jamtr
C.F 60a"M
20c00 w
G.H .50&m


Fig. 3. Hypogastrura gracilis. A. Habitus. B. Postantennal organ (PAO) and 8 ocelli. C. Dorsal view of antenna
IV segment. D. Various types of postantennal organ (PAO). E. Various types of fourth antennal segment apical bulb.
F. Second leg. G. Dorsal view of mucro and dens. H. Labrum. I. Dorsal chaetotaxy of body.



- Dens without bladder-like swelling ............................................................. 5
5. Fourth antennal segment with conspicuous ventral "file", tenent hair acuminate ................... armata
-. Fourth antennal segment without conspicuous ventral "file", tenent hair clavate or truncate .............. 6

6. P6, p5 and p, sensory setae upon Abd. I-III, Abd. IV and Abd. V, respectively. Tenent hairs 1, 1, 1
and apical bulb of fourth antennal segment trilobed ..................................... dolsana







Florida Entomologist 89(4)


December 2006


- ps, p, and p, sensory setae upon Abd. I-III, Abd. IV and Abd. V, respectively. Tenent hairs 2, 2, 2
and apical bulb of fourth antennal segment unilobed ............................... biclavata n. sp.
7. Two spines present in the position of p, setae on Abd. V .................................. duplicispinosa
-. Two spines absent in the position of p, setae on Abd. V .................. ................ sinetertiaseta
8. Abd. V with a,' setae; tenent hair acuminate; 9-12 granules between pupon fifth abdominal
segm ent .................................................................... denticulata
- .Abd. V without a2' setae ............................................................... ....... 9
9. Tenent hair acuminate; 20 granules between p,upon fifth abdominal segment ................... communis
-. Tenent hair clavate or truncate; 9-12 granules between p,upon fifth abdominal segment ...... platyna n. sp.


ACKNOWLEDGMENTS

We are grateful to Professor B.-H. Lee for reviewing
the draft, and adding many helpful comments. Particu-
lar thanks due to Professor P. Greenslade of Australian
National University for critical review and reading
through the manuscript. We express our gratitude to
Professor J. H. Shim for assisting with the collection the
materials. This research was supported by a grant (No.
052-052-040) from the Core Environmental Technology
Development Project for Next Generation funded by the
Ministry of Environment of the Korean Government.

REFERENCES CITED

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S. K. STEBAEVA. 1994. Collembola of Russia and ad-
jacent countries: Family Hypogastruridae. N. M.
Chernova (ed.), Moscow: Nauka.
BELLINGER, P. F., K. A. CHRISTIANSEN, AND F. JANS-
SENS. 2006. Checklist of the Collembola of the World.
http://www.collembola.org
BONET, L., A. BOUGEOIS, AND P. CASSAGNAU. 1973.
Valeur et limits des crit6res ch6totaxiques chez les
Collemboles Hypogastruridae: analyse biom6trique
des soies axiales chez les Ceratophysella. Bulletin de
la Societe d'Histoire Naturelle de Toulouse 109: 35-51.
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CHRISTIANSEN, K., AND P. BELLINGER 1998. The Col-
lembola of North America, North of the Rio Grande.
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FURUNO, K., M. HASEGAWA, M. HISAMATSU,
K. ICHISAWA, R. ITOH, K. NIIJIMA, Y. SUMA,
H. TAMURA, AND S. TANAKA. 2000. List of Collembola
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names. Edaphologia 66: 75-88.
GISIN, H. 1947. Notes taxonomiques sur quelques es-
peces suisses des genres Hypogastrura et Xenylla.
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HOPKIN, S. P. 1997. Biology of the Springtails (Insecta:
Collembola). Oxford University Press. 30 pp.


HOPKIN, S. P. 2002. The Biology of the Collembola
(Springtails): The Most Abundant Insects in the
World. Http://www. fathom.com/feature/122603/.
LEE, B.-H. 1974. Etude de la faune Cor6enne des In-
sectes Collemboles II. Description de quatre 6speces
nouvelles de la Famille Hypogastruridae. Nov. Rev.
Entomol. 4(2): 89-102.
LEE, B.-H., AND J.-M. THIBAUD. 1975. Etude de la faune
Cor6enne des Insectes Collemboles VII. Hypogastru-
ridae de Cor6e du Nord. Nov. Rev. Entomol. 5(1): 3-
11.
LEE, B.-H., AND J.-T. KIM. 1995. Two new species of Col-
lembola (Insecta) from Korea. Korean J. Entomol.
25(2): 135-138.
LEE, B.-H., AND J.-T. KIM. 2000. Systematic Study on
Aquatic Collembola (Insecta) from Mankyung River
System Korea. Korean J. Entomol. 30(3): 179-185.
MASSOUD, Z. 1967. Monographie des Neanuridae Col-
lemboles Poduromorphes a Pieces Buccales Modi-
fi6es. C.N.R.S. 399 pp.
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the Ou Mountains, northeast Japan. II. A new spe-
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logia. 68: 11-14.
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of interstitial Collembola (Insecta) from sand dunes
of South Korea. Korean J. Syst. Zool. 10(1): 39-46.
THIBAUD, J.-M., H.-J. SCHULZ, and M. M. DA GAMA.
2004. Synopses on Palaearctic Collembola, Volume 4,
Hypogastruridae. Staatliches Museum fur
Naturkunde Gdrlitz 1-287 pp.
YOSII, R. 1960. Studies on the Collembolan Genus Hy-
pogastrura. American Midland Naturalist 64(2):
257-281.
YOSII, R. 1962. Studies on the Collembolan Genus Hy-
pogastrura II. Contr. Biol. Lab. Kyoto Univ. 13: 1-25.
YOSII, R., AND C. E. LEE. 1963. On some Collembola of
Korea with notes on the Genus Ptenothrix. Contr.
Biol. Lab. Kyoto Univ. 15: 1-37.
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Checklist of Collembolan Species from China (In-
secta). Publ. Itako Hydrobiol. Stn. 9: 15-40.







Welch: Competition by Ormia depleta


INTRASPECIFIC COMPETITION FOR RESOURCES
BY ORMIA DEPLETA (DIPTERA: TACHINIDAE) LARVAE

C. H. WELCH
USDA/ARS/cmave, 1600 SW 23'd Drive, Gainesville, FL 32608

ABSTRACT
Ormia depleta is a parasitoid of pest mole crickets in the southeastern United States. From
2 to 8 larvae of 0. depleta were placed on each of 368 mole cricket hosts and allowed to de-
velop. The weights of the host crickets, number of larvae placed, number of resulting pupae,
and the weights of those pupae were all factored to determine optimal parasitoid density per
host under laboratory rearing conditions. Based on larval survival and pupal weight, this
study indicates that 4-5 larvae per host is optimal for laboratory rearing.

Key Words: biocontrol, Scapteriscus, parasitoid, superparasitism

RESUME

Ormia depleta es un parasitoide de grillotopos en el sureste de los Estados Unidos. Entre 2
y 8 larvas de 0. depleta se colocaron en 368 grillotopos hu6spedes y se dejaron madurar. El
peso de los hu6spedes, el numero de larvas de 0. depleta colocadas, el numero de pupas re-
sultantes y el peso de las pupas fueron usados para determinar la densidad optima de para-
sitoides en cada hu6sped para ser usadas en la reproducci6n de este parasitoide en el
laboratorio. Nuestros resultados muestran que entire 4 y 5 larvas por cada grillotopo es la
densidad optima para la reproducci6n en el laboratorio de este parasitoide.


Translation provided by the author.


Ormia depleta (Wiedemann) is a parasitoid of
Scapteriscus spp. mole crickets, imported pests of
turf and pasture grasses in the southeastern
United States (Frank et al. 1998). Female flies are
phonotactic to the call of the male Scapteriscus
spp. crickets (Fowler 1987; Fowler & Garcia 1987;
Walker et al. 1996). Ormia depleta was originally
collected from Piracicaba, Brazil, for use as a bio-
control agent against Scapteriscus spp. mole
crickets and was first released in 1988 (Frank et
al. 1996). Since then, it has established in at least
38 counties in Florida, and in some it has sup-
pressed mole cricket populations (Parkman et al.
1996).
Ormia depleta can be a difficult organism to
maintain in a laboratory colony. One of the factors
that makes it difficult to rear lies in the variable
and generally low proportion of gravid females
obtained under the current laboratory rearing
protocol. For example, a colony of 100 individuals
may in 1 generation produce 20 gravid females
and in the next only 1 or 2 or even zero (R. He-
menway, Dept. Entomology and Nematology, Uni-
versity of Florida, personal communication).
Therefore, it is necessary to determine the best
way to use the number of planidia available in
any 1 generation to produce the maximum num-
ber of healthy pupae to start the next generation.
This must also be balanced with the expense of
rearing the mole cricket hosts, which are very la-
bor intensive to maintain. Current laboratory


protocol requires hand inoculation of 3 planidia
under the posterior margin of the pronotum of
each host (R. Hemenway, Dept. Entomology and
Nematology, University of Florida, personal com-
munication). Fewer planidia per host may in-
crease the chances of survival by reducing compe-
tition and subsequently producing larger pupae.
This would, however, require more hosts to pro-
duce enough pupae to maintain the colony. Inocu-
lating hosts with more planidia may increase the
number of pupae and reduce the cost associated
with host rearing, but superparasitism should be
avoided to minimize consequences associated
with production of pupae and adults with reduced
fitness.
Previous research with 0. depleta showed that
there was no relationship between the number of
planidia used to inoculate the host and the num-
ber of pupae produced (Fowler 1988), but my pre-
liminary research suggested that higher numbers
of pupae could be produced than previously re-
corded. Additionally, Fowler & Martini (1993)
found a weak correlation between host size and
the weights of the flies produced. In the present
experiment, the host-parasitoid relationship was
also examined to determine (1) whether host
weight should be a selecting factor and (2) to de-
termine whether an increase in the number of pu-
pae produced per host could be achieved without
sacrificing the survivability or vigor of the larvae
due to superparasitism. In addition to varying the







Florida Entomologist 89(4)


number of planidia applied to each host, the
weights of the host mole crickets were measured
during inoculation to see whether larger hosts
could provision more parasitoids. These factors
were examined to determine their effect on the
number of pupae produced, the mean weight of
those pupae, and the survivability of the larvae to
the pupal stage.

MATERIALS AND METHODS

During the maintenance of the laboratory col-
ony of 0. depleta, S. abbreviatus Scudder from
the University of Florida mole cricket rearing
lab were individually weighed and inoculated
with varying numbers of O. depleta planidia. The
weights of the hosts ranged from 0.54-1.59 g and
the weights of the hosts were not considered in
determining the number of planidia used to inoc-
ulate each individual. The number of planidia
per host ranged from 2-8, with most of the mole
crickets being inoculated with 3, 4, or 5 planidia.
These numbers were favored because they are
the numbers most frequently used in the routine
maintenance of the colony. Host crickets were
randomly assigned to particular numbers of
planidia. The numbers of mole crickets inocu-
lated with 2, 3, 4, 5, 6, 7, and 8 planidia were 12,
108, 110, 52, 43, 32, and 11, respectively. Each
mole cricket was then returned to an individual
20-dram (90-mL) plastic vial filled with moist
sand, and the larvae were allowed to develop for
12 d at a room temperature of ~26C. At that
time, the pupae were collected and weighed. Sta-
tistical analysis was performed with the general
linear model procedure (SAS Institute 2001). Re-
gression analysis was used to determine the ef-
fect of the number of planidia on pupal produc-
tion, the mean pupal weight, and the survivabil-
ity. Additionally, the weights of the host mole
crickets were analyzed to determine their effect
on the survivability of the planidia used. Where
applicable, the differences between the means
were determined by Duncan's multiple range
test (SAS Institute 2001). Regression analyses
were conducted to determine the relationships
between each of these factors (SAS Institute
2001).
To determine the effect that host mole cricket
weight had on planidia survival, the number of
pupae produced and the mean weights of those
pupae and mole cricket weights were rounded to
the nearest 0.1 g to place them into weight
classes. Additionally, weight classes which had
only 2 or fewer samples were eliminated. In this
case, the smallest weight class, 0.70 g (n = 2) and
the 2 largest weight classes, 1.5 g (n = 2) and 1.6
g (n = 2) were eliminated from the statistical
analysis. The survival of the planidia on hosts in
the remaining weight classes were analyzed by
ANOVA PROC GLM (SAS Institute 2001).


RESULTS

The mean number of pupae produced relative
to the number of planidia used is shown in Fig. 1.
There is an increase in the number of pupae pro-
duced as the number of planidia increases (F =
15.77; df= 360; P < 0.0001) and significant differ-
ences between the means of the treatments. The
regression analysis (Fig. 2) supports this trend
and indicates an increase of 0.41 pupae for each
increase in planidia (F = 83.77; P < 0.0001; r2 =
0.19).
Fig. 3 shows the survival of planidia grouped
by the number of planidia placed on each host.
ANOVA is significant for the model (F = 2.57; df=
360; P < 0.02). Fig. 4 is the regression analysis of
the same data set (F = 9.16; P < 0.002; r2 = 0.03),
indicating an approximate 3% reduction in sur-
vival for each increase in the level of planidia den-
sity.
The analysis of the number of planidia used as
it affected the mean weight of the pupae produced
was found to be significant at the 0.10 level, but
not at the 0.05 level by ANOVA (F = 2.06; df= 360;
P = 0.06). There was some significance among the
means as indicated by the letters over the bars in
Fig. 5. The regression analysis for the mean
weights of pupae produced as a function of num-
ber of planidia inoculated per host is in Fig. 6 (F =
8.33; P < 0.004; r2 = 0.02) and indicates a reduc-
tion in the mean weight of the pupae of 2.2 mg for
each additional planidium.
The effect that host mole cricket weight had on
the number of pupae produced was not significant
when analyzed by ANOVA (F = 1.06; df= 361; P =
0.39). The effect of host mole cricket weight on the
survivability of the larvae was significant (F =
2.12; df = 361; P = 0.05). The effect of host mole
cricket weight on mean weight of the pupae pro-
duced was highly significant (F = 3.49; df= 361; P
< 0.002) (Fig. 7). The regression analysis can be
seen in Fig. 8 (F = 20.62; P < 0.0001; R2 = 0.05).


6


'
3

i ,


4
ed d
Zc
a a b e



Ii_ ||||


2 3 4 5 6 7 8
Number of planidi inoculated per host
Fig. 1. The effect of planidia density used to inocu-
late mole crickets on the number of pupae produced (er-
ror bars indicate standard deviation, significantly
different means indicated by letters over bars as deter-
mined by Duncan's procedure, a = 0.05).


December 2006







Welch: Competition by Ormia depleta


1 .oe
1.0
SO..

E 0.6
0.4-
0.2
n-l-


y-0.4105x+0.6515
r= 0.19. P <0.0001


1 2 3 4 5 6 7 8 9
Number ofphnida inaculted per host

Fig. 2. The effect of number of planidia used to inoc-
ulate mole crickets on the number of pupae produced;
regression analysis with 95% confidence intervals.




DISCUSSION

The number of planidia used to inoculate host
mole crickets as well as the weight of those mole
crickets are important factors to the rearing of
0. depleta in the laboratory. Although these data
do not clearly dictate a specific protocol that
should be used, they do provide a framework that
would allow anyone rearing 0. depleta to struc-
ture an inoculation protocol specific to their
needs. When large numbers of planidia are avail-
able with only a few possible hosts, the data sug-
gest that inoculating mole crickets with more
planidia would increase the production of pupae.
Too many, however, would result in reduced pupal
size. At times when fewer planidia are available
and maximum survivability is required, inoculat-
ing 2 or 3 planidia per host would be more effec-
tive. Alternatively, if larger pupae are desired, re-
ducing the number of planidia per host along with
using larger hosts would achieve the desired goal.
Therefore, the current method of inoculating 3
planidia per host is less efficient than inoculating
4 or 5, because there is no significant reduction in
pupal size, but there is a significant increase in
the number of pupae produced. The reduction in


70 -

I4
60


40

30
2 3 4 5 6 7 8


Phnidi
Fig. 3. The effect of number of planidia used to inoc-
ulate mole crickets on the survival rate of the larvae to
the pupal stage (error bars indicate standard deviation;
significantly different means indicated by letters over
bars as determined by Duncan's procedure, a = 0.05).


y=-0.0317x+0.7129
r = 0.03, P <0.002


1 2 3 4 5 6 7 8 9
Number ofphnida inocuhied per host

Fig. 4. The effect of number of planidia used to inoc-
ulate mole crickets on the survival rate of the larvae to
the pupal stage; regression analysis with 95% confi-
dence intervals.


size that results from the use of 8 planidia, or pos-
sibly more, would likely be detrimental to the col-
ony of flies. Furthermore, these data only show a
reduction in larval survival, they do not indicate
other negative factors that may be associated
with reduced size. Future research may be
needed to determine whether individuals devel-
oping from heavily parasitized hosts show any re-
duction in longevity, ability to mate, or in fecun-
dity as well as how the reduction in size of a gen-
eration may affect the size or fitness of future gen-
erations of flies.
Due to the flies' phonotactic search method for
hosts and the solitary nature of the adult mole
crickets, it would seem advantageous for the flies
to maximize the number of offspring per host. Un-
der field conditions, however, the mean number of
0. depleta larvae found within trapped Scap-
teriscus hosts is less than 2 (Amoroso 1990).
The closely related 0. ochracea Bigot, a parasi-
toid of Gryllus spp. crickets, has an optimal labora-
tory clutch size of 4-5 larvae per host, but under
field conditions only deposit 1.7 1.0 (SD). larvae
(Adamo et al. 1995). There must be some ecological
advantage to depositing fewer larvae than what
would appear to be the optimal number.


ab
ab ab ab ab






2 3 4 5 6 7 8


Phnida
Fig. 5. The effect of number of planidia used to inoc-
ulate mole crickets on the mean weight of the pupae
produced (error bars indicate standard deviation; sig-
nificantly different means indicated by letters over bars
as determined by Duncan's procedure, a = 0.05)


III I I I I







Florida Entomologist 89(4)


y = -2.2456x + 62.649
- 0.02, P < 0.004


0 2 4 6 8
Number ofplanidu nocuated per host

Fig. 6. The effect of number of planidia used to inoc-
ulate mole crickets on the mean weight of the pupae
produced; regression analysis with 95% confidence in-
tervals.


It may be that 0. depleta does not suffer from
any shortage of hosts. Mole crickets are certainly
abundant and calling during certain times of the
year, but at other times seemingly unavailable.
Ormia depleta may be able to find non-calling
mole crickets in other ways, or there may be alter-
native hosts. Adamo et al. (1995) concluded that
host availability was not a likely factor in deter-
mining the number of larvae deposited on hosts
by 0. ochracea. Another possibility is that O. de-
pleta is responding to a factor in the field that is
greatly reduced in the laboratory, such as mortal-
ity of the hosts. Under laboratory conditions, mole
crickets suffer little disease and no predation.
Higher host mortality in the field may make it ad-
vantageous for parasitoid offspring to be located
in multiple hosts and subsequently reduce the ef-
fects on their population due to host predation.
This hypothesis is somewhat strengthened by the
fact that 0. depleta does not deposit eggs, but
planidia larvae, so the female's investment in
parasitizing a host is already greater than that of
an egg layer. Another laboratory factor that
should be considered is hand-inoculating. The
mole crickets that are hand-inoculated are unable
to protect themselves in any way and have no op-
portunity to use any natural defenses such as
brushing of planidia or retreating underground.


SO ab ab abc bc
S a T
S60
40o
20

0.8 0.9 t.0 1.1 1.2
Mole Cricket Weight Class (grams]

Fig. 7. The effect of host cricket weight
pal weight (error bars indicate standard de
nificantly different means indicated by lette
as determined by Duncan's procedure, a =


y=31.346x + 20.163
=0.05, P < 0.0001


0.7 0.9 1.1 1.3 1.5
Host cricket weight cbss (g)

Fig. 8. The effect of host cricket weight class on the
mean weight of the pupae produced; regression analysis
with 95% confidence intervals.


This type of grooming has been observed in Gryl-
lus spp. crickets after an encounter with 0. ochra-
cea (Adamo et al. 1995).
The final reason for the low numbers of larvae
found in field-captured hosts may be that there is
a reduction in fitness caused by the high numbers
of larvae used in this experiment. Reduced size is
the easiest type of fitness reduction to observe,
but many others may be at work. It may be that,
due to competition, certain key resources are not
available in sufficient amounts for the flies reared
under superparasitoid conditions for the result-
ing adult flies to develop, mate, locate hosts, or re-
produce properly. Many physiological deficiencies
may result from superparasitoidism, and they
may not be obvious either externally, or immedi-
ately (Waage & Ng 1984). These possibilities still
remain for future research.

ACKNOWLEDGMENTS

I thank Dr. J. Howard Frank and Dr. Robert Hemen-
way for help in this project as well as for their pioneer-
ing role in Ormia research. I also thank Alejandro
Arevalo for assistance in the Spanish translation of the
abstract.

REFERENCES CITED


ADAMO, S. A., D. ROBERT, J. PEREZ, AND R. R. HOY.
b c 1995. The response of an insect parasitoid, Ormia
ochracea (Tachinidae), to the uncertainty of larval
success during infestation. Behav. Ecol. Sociobiol.
36: 111-118.
AMOROSO, J. 1990. Ormia depleta parasitism of Mana-
tee Co. trapped Scapteriscus borellii. Department of
Entomology & Nematology, University of Florida,
Annu. Report Mole Cricket Res. 12: 190-191.
FOWLER, H. G. 1987. Field confirmation of the phono-
1.3 1.4 taxis ofEuphasiopteryx depleta (Diptera: Tachinidae)
to calling males of Scapteriscus vicinus (Orthoptera:
Gryllotalpidae). Florida Entomol. 70: 409-410.
on mean pu- FOWLER, H. G. 1988. Suitability of Scapteriscus mole
aviation; sig- crickets (Ort.: Gryllotalpidae) as hosts ofEuphasiop-
rs over bars teryx depleta (Dip.: Tachinidae). Entomophaga 33:
).05). 397-401.


December 2006







Welch: Competition by Ormia depleta


FOWLER, H. G., AND A. V. MARTINI. 1993. Influ6ncia do
tamanho hospedeiro (Scapteriscus borellii: Gryllo-
talpidae: Orthoptera) sobre a producao experimental
do parasit6ide (Ormia depleta:Tachinidae: Diptera).
Cientifica 21: 339-343.
FOWLER H. G., AND C. R. GARCIA. 1987. Attraction to
synthesized songs and experimental and natural
parasitism of Scapteriscus mole crickets (Ortho-
ptera: Gryllotalpidae) by Euphasiopteryx depleta
(Diptera: Tachinidae). Rev. Bras. Biol. 47: 371-374.
FRANK, J. H., T. R. FASULO, D. E. SHORT, AND A. S.
WEED. 2005. MCRICKET, Alternative Methods of
Mole Cricket Control. Published on world wide web
at: http://molecrickets.ifas.ufl.edu/.


FRANK, J. H., T. J. WALKER, AND J. P. PARKMAN. 1996.
The introduction, establishment and spread of
Ormia depleta in Florida. Biol. Control 6: 368-377.
PARKMAN, J. P., J. H. FRANK, T. J. WALKER, AND D. J.
SCHUSTER 1996. Classical biocontrol of Scapteriscus
spp. (Orthoptera: Gryllotalpidae) in Florida. Envi-
ron. Entomol. 25: 1415-1420.
SAS INSTITUTE, INC. 2001. SAS system for Windows
Rel. 8.2. SAS Institute, Inc. Cary, NC, USA.
WAAGE, J. K., AND S. M. NG. 1984. The reproductive
strategy of a parasitic wasp. I. Optimal progeny allo-
cation in Trichogramma evanescens. J. Anim. Ecol.
53: 401-415.
WALKER, T. J., J. P. PARKMAN, J. H. FRANK, AND D. J.
SCHUSTER 1996. Seasonality of Ormia depleta and
limits to its spread. Biol. Control 6: 378-383.







Florida Entomologist 89(4)


December 2006


PESTICIDE SUSCEPTIBILITY OF CYBOCEPHALUS NIPPONICUS
AND RHYZOBIUS LOPHANTHAE (COLEOPTERA:
CYBOCEPHALIDAE, COCCINELLIDAE)

TREVOR RANDALL SMITH 1AND RONALD D. CAVE2
'Department of Entomology and Nematology, University of Florida, P.O. Box 110620, Gainesville, FL 32611, U.S.A.
e-mail: trsmith@ufl.edu

2Indian River Research & Education Center, University of Florida, 2199 S Rock Rd., Ft. Pierce, FL 34945-3138, U.S.A
e-mail: rdcave@ifas.ufl.edu

ABSTRACT

The susceptibility of the predatory beetles Cybocephalus nipponicus Endr6dy-Younga and
Rhyzobius lophanthae Blaisdell to 6 pesticides commonly used for treating cycad aulacaspis
scale, Aulacaspis yasumatsui Takagi, was tested. Three concentrations (half field rate, field
rate, and twice field rate) of each pesticide were tested against both beetle species with a
coated glass vial bioassay. Nearly 100% mortality in both beetle species occurred at all con-
centrations when treated with methidathion, dimethoate, and malathion. Insecticidal soap,
fish oils, and imidacloprid were much less toxic. At one-half the field rate, C. nipponicus had
66% mortality with insecticidal soap, 76% mortality with imidacloprid, and 83% mortality
with fish oil. At one-half the field rate, R. lophanthae had 43% mortality with insecticidal
soap, 63% mortality with imidacloprid, and 46% mortality with fish oil. Mortality rate for
each beetle species rose with increasing concentration of each pesticide and the soap and oil
were the least toxic of all pesticides tested.

Key Words: biocontrol, coated glass vial bioassay, predatory beetle, toxicity test

RESUME

Se investig6 la susceptibilidad de los escarabajos depredadores Cybocephalus nipponicus
Endr6dy-Younga y Rhyzobius lophanthae Blaisdell a seis pesticides comunmente usados en
el control de la escama de las cicadas, Aulacaspis yasumatsui Takagi. Se probaron tres con-
centraciones (mitad de la tasa recomendada en el campo, la tasa recomendada en el campo,
y double la tasa recomendada en el campo) de cada pesticide contra cada species de escara-
bajo, usando frascos de vidrio aplicado para bioensayos. La mortalidad en ambas species de
escarbajos fue casi 100% a todas las concentraciones de metidati6n, dimetoato, y malati6n.
Jab6n insecticide, aceite de pescado, e imidacloprid fueron much menos t6xicos. A la mitad
de la tasa recomendada en el campo, los niveles de mortalidad de C. nipponicus fueron 66%
con jab6n insecticide, 76% con imidacloprid, y 83% con aceite de pescado. A la mitad de la
tasa recomendada en el campo, los niveles de mortalidad de R. lophanthae fueron 43% con
jab6n insecticide, 63% con imidacloprid, y 46% con aceite de pescado. La tasa de mortalidad
por cada especie de escarabajo aument6 con mayores concentraciones de cada pesticide y el
jab6n y aceite de pescado fueron los menos t6xicos de todos los pesticides probados.


Translation provided by the authors.


Beetles of the families Coccinellidae and Cybo-
cephalidae are the most economically important
groups of predators of diaspidid scales in the world
(Blumberg & Swirski 1982). Cybocephalus nip-
ponicus Endrddy-Younga (Cybocephalidae) and
Rhyzobius lophanthae Blaisdell (Coccinellidae)
are commonly used as biological control agents for
many armored scale pests. Rhyzobius lophanthae
has been established in Florida since the 1930s
(according to specimen label data in the Florida
State Collection ofArthropods). Cybocephalus nip-
ponicus, misidentified as Cybocephalus binotatus
Grouvelle, was recently released in south Florida
in an effort to control the cycad aulacaspis scale


(CAS), Aulacaspis yasumatsui Takagi (Homop-
tera: Diaspididae) (Anon. 1998; Howard et al.
1999; Howard & Weissling 1999). CAS is the most
economically damaging scale to cycads that the
state of Florida has ever seen (Hodges et al. 2003).
Although C. nipponicus is present in Hawaii (Heu
& Chun 2000), R. lophanthae is usually suggested
as the better control agent of CAS (Heu et al. 2003;
A. Hara, personal communication). In both places,
CAS has continued to spread and multiply. A more
promising approach to controlling CAS would be
one using integrated pest management (IPM). In
this manner, a combination of pesticides and bio-
logical control would be used to combat CAS.







Smith & Cave: Pesticide Susceptibility of two Predatory Beetles


There has been some success controlling CAS
with various pesticides. Oils, either an ultra-fine
horticultural oil or a product containing fish oils,
seem to be the most effective chemical control
method (Hodges et al. 2003). This is not surprising
given that oils have long been used to control ar-
mored scale insects. The oil not only covers the in-
sects and suffocates them but also covers the sur-
face of the plant making it difficult for crawlers to
settle onto the plant (Howard & Weissling 1999).
Soaps are quite popular with homeowners; but they
must be applied frequently, in some cases once a
week (personal observation). The effective applica-
tion of pesticides for control of CAS is difficult due to
the scale's tendency to heavily infest the abaxial
surface of leaves, a site difficult to spray (Howard &
Weissling 1999). In the case of Cycas revoluta
Thunberg (Cycadaceae), the architecture of the
plant itself, with the margins of the leaflets curling
down and inward to form an arch on the abaxial
surface of the leaflet, makes foliar treatments inef-
ficient (Hodges et al. 2003). Frequent or "as needed"
applications of oils seems to be the most effective
technique for controlling CAS, and by mixing oil
with contact pesticides such as malathion, even
greater scale mortality can be achieved (Hodges et
al. 2003). Systemic pesticides such as dimethoate
and contact pesticides like methidathion have
yielded mixed results, being very effective in some
instances and completely ineffective in other cases
(Hodges et al. 2003). Imidacloprid used as a soil
drench can be very effective, but Howard &
Weissling (1999) found that this product had to be
mixed at very high concentrations to be effective.
This product can also be used as a foliar spray.
The reproductive biology of C. nipponicus
makes it a good biological control agent. Alvarez &
Van Driesche (1998) found that, at low scale densi-
ties, C. nipponicus was able to maintain its popu-
lations and maintain populations of euonymus
scale, Unaspis euonymi (Comstock), and San Jose
scale, Quadraspidiotus perniciosus (Comstock), in
check. In the presence of greater scale densities,
C. nipponicus will increase egg production accord-
ingly. With a total life cycle from egg to adult only
taking around 44 days (Smith & Cave 2006), it is
conceivable that 5-6 generations could be pro-
duced every year in Florida. Cybocephalus nippon-
icus is available commercially in the U.S. market.
Rhyzobius lophanthae is an exceptional biolog-
ical control agent because of its high fecundity,
lack of parasitoids, absence of diapause, and re-
sistance to low temperatures especially in the im-
mature stages (Rubstov 1952; Smirnoff 1950;
Stathas 2000). Female R. lophanthae are able to
lay hundreds of eggs in a lifetime (Stathas 2000).
Rhyzobius lophanthae also seems to be able to re-
sist extreme heat, but Atkinson (1983) found that
adult R. lophanthae could not survive for long at
420C. Rhyzobius lophanthae is also available
commercially in the U.S. market.


This study was conducted to determine the
susceptibility of C. nipponicus and R. lophanthae
to 6 pesticides commonly used in the control of
CAS. Given the established presence of both pred-
ators on cycads in south Florida and their com-
mercial availability, it is very important to learn
what effects the commonly used pesticides
against CAS will have on them. This information
is vital for development of IPM programs aimed
at controlling CAS.

MATERIALS AND METHODS

Insects

Adult R. lophanthae were reared at, and pur-
chased from, Rincon-Vitova Insectaries (Ventura,
California). Adult C. nipponicus also were pur-
chased from Rincon-Vitova but were reared by
Philip Alampi Beneficial Insect Laboratory, New
Jersey Department of Agriculture. Both beetle spe-
cies were maintained in Plexiglas cages at 25C
and 80% relative humidity prior to testing. All life
stages of CAS were provided as a food source.
Food was not provided during testing because
of the very small size of the beetles (1 mm in width
and 2.5 mm in length). The beetles could have con-
ceivably perched on the food source for long peri-
ods of time, never coming into contact with the
walls of the treated vial. Preliminary studies indi-
cated that a 24-h period without food would not
unduly stress the beetles. On average, untreated
C. nipponicus survived for 8-9 d (n = 30) and un-
treated R. lophanthae lived for 5-6 d (n = 30) be-
fore dying of starvation. Cotton used to stopper the
vials was soaked in water to prevent dehydration.

Bioassays with the Coated Glass Vial Method

A coated glass vial method (Plapp 1971; Ama-
lin et al. 2000; Snodgrass 1996; Snodgrass et al.
2005) was used to determine the chemical suscep-
tibility of adult R. lophanthae and C. nipponicus
to 6 pesticides used to control CAS (Howard et al.
1997; Howard & Weissling 1999; Weissling et al.
1999; Hodges et al. 2003; Emshousen & Mannion
2004). This is a very effective method for testing
the chemical susceptibility of small arthropods
(Amalin et al. 2000) such as R. lophanthae and es-
pecially C. nipponicus because of its extremely
small size. The 6 pesticides tested were fish oil
emulsion (Organocide), insecticidal soap (Garden
Safe, Inc.), imidacloprid (Provado), malathion
(Spectracide, Inc.), methidathion (Supracide),
and dimethoate (Cygon). The fish oil and insec-
ticidal soap were purchased as commercial grade,
while the imidacloprid (99% purity), malathion
(98% purity), methidathion (98.6% purity), and
dimethoate (98.7% purity) were purchased as the
technical grade from Chem Service (West Ches-
ter, PA).







Florida Entomologist 89(4)


All pesticides were dissolved in acetone, except
the insecticidal soap, which does not dissolve in
acetone. Instead, the insecticidal soap was dis-
solved in 95% ethanol. The fish oil was shaken in
a paint shaker after being placed in acetone in or-
der to break the oil into fine globules. Each pesti-
cide was separated into 3 dilutions: field rate,
twice field rate, and one-half field rate. The field
rate was taken from label data for each pesticide
as directed for use against scale insects. A small
amount (0.5 mL) of the pesticide working solution
was dispensed into 20-mL scintillation vials. Con-
centrations of active ingredient for the working
solution and the amount of active ingredient res-
idue within the vials can be seen in Table 1. Vials
were hand rotated until the acetone or ethanol
completely evaporated leaving an insecticidal res-
idue on the inner surface. Vials treated with only
acetone or ethanol, as well as untreated vials,
were used as controls. A single beetle was placed
into a treated vial. All beetles had emerged from
pupae within the previous 14 d. Vials were sealed
with cotton soaked in water allowing the beetles
to drink. Vials were placed upright in a ventilated
cabinet with a fume hood and at a constant tem-
perature of 25C and 80% relative humidity for 24
h. For each treatment of 10 beetles, 5 females and
5 males were used. Each treatment of 10 beetles
was replicated 3 times for each dosage. All trials
were carried out the same day that the pesticide
was applied to the vials.
Mortality of beetles was determined immedi-
ately after the 24-h period. A beetle was consid-
ered dead if it was not moving or could not right
itself. Percent mortality was measured as the pro-
portion of 30 beetles dead after a 24-h exposure to
the pesticides.

Statistical Analyses

All descriptive statistics were generated in EX-
CEL (Microsoft 2000). The mortality rates for each
pesticide were compared by the Student-Newman-
Keuls mean separation test (SAS Institute 2001).


TABLE 1. FIELD RATES (IX) FOR EACH PESTICIDE USED.

Working solution Insecticide residue
Insecticide (ig*AI/mL) (ig*AI/cm2)

Organocide@ 47000 8.29
Insecticidal Soap@ 512300 27.71
Imidacloprid 106 2.40
Methidathion 233 5.26
Dimethoate 305 6.91
Malathion 1990 45.07

*AI = Active Ingredient.


RESULTS

Of the 6 pesticides tested on adult C. nipponicus
and R. lophanthae, 3 (methidathion, dimethoate,
and malathion) caused >90% mortality at all con-
centrations, while the other 3 (fish oil, insecticidal
soap, and imidacloprid) were less toxic but still
caused very high mortality (Tables 2 and 3).

Effects of Pesticides on C. nipponicus

Cybocephalus nipponicus was extremely sus-
ceptible to all pesticides. The three least toxic pes-
ticides were imidacloprid, insecticidal soap, and
fish oil (Table 2). There were significant differ-
ences (P < 0.05; Table 4) in mortality between con-
centrations among these 3 pesticides. Fish oil was
not only toxic to the beetle, but due to its very
small size, C. nipponicus would often get trapped
in small globules of oil, eventually dying from
suffocation.

Effects of Pesticides on R. lophanthae

Rhyzobius lophanthae was more tolerant than
C. nipponicus to the experimental pesticides, al-
though mortality rates were high for this species,
too. The 3 least toxic pesticides to R. lophanthae
were imidacloprid, insecticidal soap, and fish oil
(Table 3). There were significant differences in


TABLE 2. PERCENT MORTALITY OF CYBOCEPHALUS NIPPONICUS PER 30 INDIVIDUALS EXPOSED. X = FIELD RATE.

% Beetle mortality

Pesticide at OX at 0.5X at 1X at 2X

Organocide@ 83 100 96
Insecticidal Soap@ 66 86 96
Imidacloprid 76 93 100
Methidathion 100 100 100
Dimethoate 100 96 100
Malathion 93 100 100
Control (Acetone) 0 -
Control (Ethanol) 0 -
Control (No coating) 0 -


December 2006







Smith & Cave: Pesticide Susceptibility of two Predatory Beetles


TABLE 3. PERCENT MORTALITY OF RHYZOBIUS LOPHANTHAE PER 30 INDIVIDUALS EXPOSED. X = FIELD RATE.

% Beetle mortality

Pesticide at OX at 0.5X at 1X at 2X

Organocide@ 46 83 100
Insecticidal Soap@ 43 76 96
Imidacloprid 63 80 100
Methidathion 100 100 100
Dimethoate 100 96 100
Malathion 93 90 96
Control (Acetone) 0 -
Control (Ethanol) 6 -
Control (No coating) 0 -


survivorship between concentrations of these 3
pesticides (Table 4). Rhyzobius lophanthae, about
twice the size of C. nipponicus, had much less
difficulty traversing oil globules on the surface of
the vials.

DISCUSSION

In the present study, a significant difference
(P < 0.05) was observed between mortality in the
control and that of even the lowest pesticide con-
centration. This sensitivity to pesticides makes
an IPM approach to the control of CAS quite diffi-
cult. Unfortunately, most of the success in chemi-
cally controlling CAS has involved very toxic pes-
ticides often being used at higher than recom-
mended doses (Howard & Weissling 1999;
Weissling et al. 1999).
The high mortalities experienced by C. nip-
ponicus and R. lophanthae are not unexpected.
Nakao et al. (1985) found that all 18 species of
Coccinellidae inhabiting Japanese citrus groves
were severely affected by the application of pesti-
cides, including methidathion and dimethoate.
They also found that Cybocephalus gibbulus


Erichson, one of the most common scale predators
found in Japanese citrus groves, was virtually
eliminated by long-term pesticide use. Oils have
proven to be the most effective pesticides used
against many plant-sucking pests, while main-
taining the natural enemy populations. Erkilic &
Uygun (1997) found that oils were much less toxic
to Cybocephalus fodori minor (Endrddy-Younga)
and Chilocorus bipustulatus (Linnaeus) than was
methidathion. In fact, they went as far as saying
that methidathion should not be used in IPM pro-
grams.
In natural conditions, the predatory beetles
may not be in contact with the pesticide for as
long as the exposures in this experiment. How-
ever, C. nipponicus and R. lophanthae are
uniquely suited for life in chemically-treated en-
vironments. Both beetle species place their eggs
underneath the scale cover and at least part of
larval development takes place beneath the ar-
mored scale, allowing the beetles some protection
from both the elements and pesticides (Smirnoff
1950; Alvarez & Van Driesche 1998; Stathas
2001). In Greece, Katsoyannos (1984) found that
C. fodori was able to survive in pesticide-treated


TABLE 4. STUDENT-NEWMAN-KEULS TEST SHOWING RANKED VALUES OF MORTALITY OF ADULT CYBOCEPHALUS NIP-
PONICUS AND RHYZOBIUS LOPHANTHAE BY 4 DOSES OF IMIDACLOPRID, INSECTICIDAL SOAP, AND ORGANO-
CIDE.1

Dose Imidacloprid Organocide Insecticidal soap

C. nipponicus 0.OX 2.0 a 2.0 a 2.0 a
0.5X 5.5 b 5.3 b 5.3 b
1.OX 8.5 c 8.6 c 8.3 c
2.0X 10.0 c 10.0 c 10.3 c
R. lophanthae 0.OX 2.0 a 2.0 a 2.0 a
0.5X 5.6 b 5.0 b 5.3 b
1.OX 7.3 b 8.0 c 7.8 c
2.0X 11.0 c 11.0 d 10.8 d

'Means within columns with the same letter are not significantly different based on Student-Newman-Keuls mean separation
test, P = 0.05.











fruit orchards. In date palm plantations in Israel,
Kehat et al. (1974) found that, while all coccinel-
lids in a chemically-treated plantation died, spe-
cies of Cybocephalus survived.
For some pesticides, it is apparent that from
these tests, the lower the concentration of the pes-
ticide, the lower the mortality. However, these
tests were conducted in a laboratory environment
wherein the test subjects were in constant contact
with the pesticide for 24 h. A whole host of factors,
such as humidity, UV degradation, evaporation,
and precipitation, will influence pesticide activity
in the field. Nevertheless, whenever possible, in-
secticidal soaps and fish oils should be used.
While many homeowners use various types of
soaps to treat CAS, this method requires treat-
ment every 7 to 10 d, thus increasing exposure of
the beetles to the pesticide. If more toxic pesti-
cides must be used, then applying them to "hot
spots" rather than broadcast spraying may pro-
tect the scale predators from complete annihila-
tion. This type of selective spraying may also pro-
tect other entomophagous insect populations
from being decimated (Kuznetsov 1997). The re-
sults of these laboratory experiments yield some
baseline data from which more research in the
field can be conducted.

ACKNOWLEDGMENTS

We thank Simon Yu for help and advice on experi-
mental design. We thank Howard Frank and Michael
Thomas for reviews of the manuscript. This research
was supported in part by a grant from the Florida De-
partment of Agriculture and Consumer Services (DACS
7276186-12) and approved for publication as IRREC-
Journal Series No. 020601.

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Florida Entomologist 89(4)


December 2006


HYMENOPTERAN PARASITOIDS OF ANASTREPHA FRUIT FLIES (DIPTERA:
TEPHRITIDAE) REARED FROM DIFFERENT HOSTS IN YUCATAN, MEXICO


VICENTE HERNANDEZ-ORTIZ 1, HUGo DELFIN-GONZALEZ 2, ANDRES ESCALANTE-TIO 2AND PABLO MANRIQUE-SAIDE2
'Instituto de Ecologia A.C., Km 2.5 carretera antigua a Coatepec No. 351, Congregaci6n El Haya
Apartado Postal 63, C.P. 91000, Xalapa, Veracruz, M6xico

2Universidad Aut6noma de Yucatan, Facultad de Medicina Veterinaria y Zootecnia, Depto. de Zoologia
Apartado Postal 4-116, M6rida, Yucatan, Mexico

ABSTRACT

In order to carry on the detection and species inventory of hymenopteran parasitoids asso-
ciated with fruit flies, we examined various tropical fruits growing at the Southern region of
Yucatan. During a yearly cycle (Jun 2000 to Jun 2001), 9 host fruit species (including some
varieties) were collected by 2 different methods. The first method involved weekly collection
of ripened fruits that were transported to the laboratory ("Fruit-Lab"); and the second
method was collection of fruits placed on the ground below the tree canopy ("Fruit-Beds"),
and which remained in the field for two weeks, after which they were transported to the lab-
oratory. Fruits obtained were counted and weighed, and the recovered pupae were quanti-
fied for each sample. As a whole, we sampled 4,470 fruits (850.8 Kg) from the 9 host plant
species and varieties, which were infested by 5 fruit fly species: Anastrepha ludens (Loew),
A. obliqua (Macquart), A. serpentina (Wiedemann), A. striata Schiner, and A. fraterculus
(Wiedemann). The average parasitism in all samples was 3.69% represented by 11 hy-
menopteran species as follows: Braconidae, Doryctobracon areolatus (Sz6pligeti), and Opius
bellus (Gahan); Figitidae, Aganaspis pelleranoi (Brethes), Aganaspis sp., Odontosema anas-
trephae Borgmeier and Odontosema sp.; Diapriidae, Coptera haywardi (Oglobin); Chalcid-
idae, Dirhinus sp.; Pteromalidae, Spalangia endius Walker; Eurytomidae, Sycophila sp.;
and Perilampidae, Euperilampus sp. On the basis of results in differences among samples
for parasitism rates, fruit fly parasitoid, and fruit fly host plant, parasitoid assemblages are
analyzed and discussed.

Key Words: parasitism, fruit flies, host plants, natural enemies

RESUME

Con el prop6sito de realizar la detecci6n e inventario de species de parasitoides asociados
con moscas de la fruta, se examinaron diversos frutos tropicales cultivados en la region Sur
del estado de Yucatan. Durante el ciclo annual comprendido entire junio de 2000 a junio de
2001, se estudiaron nueve species y variedades de frutos de la region, empleando dos m6to-
dos de colecta: el primero se realize por medio de la colecta semanal de frutos maduros trans-
portados al laboratorio ("Fruit-Lab"); y el segundo mediante la recolecci6n de camas de frutos
("Fruit-Beds") colocados en el suelo bajo la cobertura de los arboles, los cuales permanecieron
por dos semanas, y posteriormente trasladados al laboratorio. En ambos casos, los frutos fue-
ron contados y pesados, ademas de la cuantificaci6n de pupas recuperadas en cada muestra.
En total se recolectaron 4,470 frutos (850.8 Kg) de las nueve species y variedades de plants
hospederas, las cuales resultaron infestadas por cinco species de moscas de la fruta: Anas-
trepha ludens (Loew),A. obliqua (Macquart),A. serpentina (Wiedemann),A. striata Schiner,
y A. fraterculus (Wiedemann). La proporci6n de parasitismo en todas las muestras fue de
3.69% representado por 11 species de himen6pteros de las siguientes families: Braconidae,
Doryctobracon areolatus (Sz6pligeti), y Opius bellus (Gahan); Figitidae,Aganaspis pelleranoi
(Brethes), Aganaspis sp., Odontosema anastrephae Borgmeier, and Odontosema sp.; Diaprii-
dae, Coptera haywardi (Oglobin)); Chalcididae, Dirhinus sp.; Pteromalidae, Spalangia en-
dius Walker; Eurytomidae, Sycophila sp.; and Perilampidae, Euperilampus sp.. Con base en
estos resultados, se analizan y discuten las diferencias entire los indices de parasitismo, asi
como entire los ensambles mosca- parasitoide y plant hospedera-parasitoide.

Translation provided by the authors.



Diverse regional studies in Latin America Guatemala (Eskafi 1990), Costa Rica (Jir6n &
have addressed the incidence of native parasi- Mexzon 1989), Colombia (Yepes & V1lez 1989;
toids of the genusAnastrepha in countries such as Carrejo & Gonzalez 1999), Venezuela (Katiyar et







Herndndez-Ortiz et al.: Hymenopteran Parasitoids ofAnastrepha


al. 1995; Boscan & Godoy 1996; Garcia & Mon-
tilla 2001), Brazil (Canal et al. 1995; Leonel et al.
1995; GuimarAes et al. 1999; Aguiar-Menezes et
al. 2001), and Argentina (Ovruski 1995; Ovruski
et al. 2004, 2005).
Previous studies have stated that as many as
18 parasitoid species of Anastrepha have been
recorded in Mexico, including the exotic species
Diachasmimorpha longicaudata (Ashmead) and
Aceratoneuromyia indica (Silvestri), both of
which have been considered as established
(Ovruski et al. 2000). However, at least 6 other
exotic species have been introduced into Mexico
for control ofA. ludens and A. obliqua (Jimenez-
Jimenez 1955, 1956, 1963).
Inventories of native parasitoids of Anas-
trepha fruit flies have been conducted in commer-
cial orchards at Morelos and Chiapas (McPhail &
Bliss 1933; Baker et al. 1944; Aluja et al. 1990),
but also in wild environments associated with
native fruit fly hosts in Nuevo Le6n (Plummer &
McPhail 1941; Gonzalez-Hernandez & Tejada
1979), Veracruz (Hernandez-Ortiz et al. 1994; L6-
pez et al. 1999), and Chiapas (Aluja et al. 2003).
Inventories have not been done in many other
fruit growing regions of Mexico.
Anastrepha ludens (Loew), A. obliqua (Mac-
quart), A. serpentina (Wiedemann), A. striata
Schiner, A. fraterculus (Wiedemann), A. ampliata
Hernandez-Ortiz, andA. pallens (Coquillett) have
been recorded from the state of Yucatan (Hernan-
dez-Ortiz et al. 2002). The first 4 species are sig-
nificant pests in fruit crops in Mexico and most of
the Neotropics (Hernandez-Ortiz & Aluja 1993).
Fruit fly control in Yucatan has generally involved
use of pesticides (CESVY 2000), and very little is
known of the native hymenopteran parasitoid
communities. An earlier regional study showed
the presence in Yucatan of certain Opiinae (Bra-
conidae) that potentially parasitize Anastrepha
species, including Doryctobracon Ender, Utetes
Foerster, and Opius Wesmael (Delfin-Gonzalez &
Le6n 1997), although sampling methods in that
study were not focused on host collection. Thus,
specific relationships between Anastrepha and
braconid species remain unknown.
The present study focuses on the search for
and inventory of parasitoids that attack Anas-
trepha species, as well as determination of the
relationships between fruit flies, host plants,
and parasitoids in the fruit growing region of
southern Yucatan, which mainly consists of
mixed orchards of citrus, mango, sapodilla,
guava, and red mombin.

MATERIALS AND METHODS

The study was carried out in mixed commer-
cial orchards in the Yaax-Hom Fruit Unit, 5 km
from the Lol-Tun archaeological site, Oxkutzcab
municipality, in southern Yucatan (2018'N,


89042'W). Surrounding native vegetation is semi-
evergreen tropical forest (Flores & Espejel 1994).
Collection of fruit samples occurred from Jun
2000 to Jun 2001, and included 9 host plant spe-
cies during their fruit-growing seasons: sour or-
ange, Citrus aurantium L. (Aug 2000 to Jan
2001); Valencia orange, C. sinensis (L.) Osbeck
var. valenciana (Oct 2000 to Mar 2001); Ruby
grapefruit, C. paradise MacFad (Jul 2000 to Jan
2001); star apple, Chrysophyllum cainito L. (Jan
to Mar 2001); mango, Mangifera indica L. c.v. cor-
doba, criollo, pico de loro, and manglova (Mar to
Jul 2001); sapodilla, Manilkara zapota (L.) P.
Royen (Sept to Dec 2000); mamey sapote, Poute-
ria sapota (Jacq.) H. Moore & Steam (Apr to Jul
2001); guava, Psidium guajava L. (Jun to Sep
2000; Febr April to Jun 2001); and red mombin,
Spondias purpurea L. c.v. San Juan, tuxpana, and
chi-abal (Apr to May 2001).
Fruits were sampled during the fruiting sea-
son of each host plant, according to availability of
mature fallen fruits under the trees by means of 2
different methods as follows:

(1) Fruit-Lab Samples. Fruits were weekly sam-
pled, placed in 20-liter containers with a sub-
strate of soil from the collection site, covered
with wire mesh and topped with a fine-mesh
screen to prevent contamination. Samples
were taken to the laboratory where they were
counted, weighed, and reviewed daily. The re-
covered pupae were separated in small plastic
containers for adult fly and parasitoid emer-
gence.

(2) Fruit-Bed samples. This method was imple-
mented once a sufficient amount of fruits were
available. Collected fruits were arranged in
"fruit-beds" under the tree canopy, consisting
of a plastic tarp covered with soil, containing a
known number of fruits previously weighed.
"Fruit-beds" remained in the field for 2 weeks
and were observed. All pupae recovered were
taken to the laboratory in small plastic con-
tainers for adult fly and parasitoid emergence.

Percent of parasitism (PP) was recorded as PP
= a/(a + b) 100, where a = Number of recovered
parasitoids; and b = Number of emerged adult
flies in each sample (Steck et al. 1986). Correla-
tion analysis (Statistica 1999) was used to com-
pare mean fruit weight of host sampled (calcu-
lated as the Log,,,, of fruit weight), infestation in-
dex (calculated as the number of larvae/Kg fruit),
and percentage of parasitoids recovered in each
sample.
Specimens of fruit flies and parasitoids were
determined by VHO and HDG, respectively.
Voucher specimens are deposited in the Insect Col-
lections (IEXA) of the Instituto de Ecologia (Xal-







Florida Entomologist 89(4)


apa, Veracruz), and in the Regional Entomological
Collections (CERUY) of the Universidad Au-
tonoma de Yucatan (Merida, Yucatan). Botanical
samples were identified by personnel of the Botan-
ical Department of the UADY and deposited in the
Herbarium of this institution. Botanical nomen-
clature is based on Terrel et al. (1986), and parasi-
toid nomenclature follows Ovruski et al. (2000).

RESULTS

Altogether, 4,470 fruits (850.8 kg) from 9 host
species (including 4 mango varieties and 3 red
mombin varieties) were examined and found to be
infested by 5 Anastrepha species. All the citrus
hosts (C. aurantium, C. sinensis, and C. paradisi)
were infested by A. ludens, and 1 specimen of
A. serpentina was recovered from ruby grapefruit
and 2 from sour orange. A single specimen of
A. fraterculus was found in sour orange. The
hosts of the family Sapotaceae (C. cainito, P sa-
pota and M. zapota) were only infested byA. ser-
pentina, and all S. purpurea varieties were in-
fested by A. obliqua. The mango varieties (M. in-
dica) were infested by A. ludens (53.4%) and
A. obliqua (45.9%), and 2 specimens ofA. serpen-
tina were recovered. The guava fruits (Psidium
guajava) were infested by A. fraterculus (84.2%)
andA. striata (15.8%).
In total, 12,929 larvae and pupae were recov-
ered from the sampled fruits. Although the num-
ber of fruits collected by each sampling method
were equivalent, the "Fruit-Lab" samples exhib-
ited a higher degree of infestation (2,227 fruits,
with 8,511 recovered pupae), than that left in the
"Fruit-Bed" samples (2,243 fruits, with 4,418 re-
covered pupae). The highest infestation indices
per host were observed in P guajava (103.2 lar-
vae/Kg), S. pupurea (all varieties with 83.3 to 44
larvae/Kg), C. cainito (40.4 larvae/Kg), P sapota
(29.6 larvae/Kg), and C. aurantium (22.5 larvae/
Kg). The lowest infestation rates occurred in
M. indica (all varieties with 15.9 to 0.3 larvae/
Kg), M. zapota (15.7 larvae/Kg), C. sinensis (4.2
larvae/Kg), and C. paradise (3.1 larvae/Kg). Sam-
ple sizes in some of these low-infestation hosts
were relatively small. In total, 9,223 fruit fly via-
ble pupae were recovered during the study, which
produced 8,883 adult flies and 340 parasitoid
specimens. Average parasitism of all fruit flies
was 3.69% (Table 1).
The recovered parasitoids included the follow-
ing 11 species: the larval-pupal parasitoids D. ar-
eolatus (Szepligeti) and Opius bellus (Gahan)
(Braconidae); Aganaspis pelleranoi (Brethes),
Aganaspis sp., Odontosema anastrephae Borg-
meier and Odontosema sp. (Figitidae); and the
pupal parasitoids Coptera haywardi (Oglobin)
(Diapriidae), Dirhinus sp. (Chalcididae), and
Spalangia endius Walker (Pteromalidae). In ad-
dition, 2 other parasitoid species in the genera


Sycophila sp. (Eurytomidae) and Euperilampus
sp. (Perilampidae) were recorded for the first time
in Anastrepha.
Relationships between fruit fly-parasitoids
among samples showed that A. ludens was
attacked in Citrus spp. by 5 parasitoids, which
accounted for 29.3% of overall species, while in
M. indica only 2 parasitoid species were recorded
with 0.6%. In this sense, A. obliqua was parasit-
ized in Spondias purpurea by 5 parasitoid species
(16.7%); A. serpentina was attacked by 5 parasi-
toids (25.6%) infesting 3 hosts of the family Sapo-
taceae; and the Psidium guajava fruits infested
byA. striata/A. fraterculus were parasitized by 8
species (27.8%).
Odontosema anastrephae was found in 7 host
plant species representing 43.2% of all recovered
parasitoids with highest proportions in Psidium
guajava and Citrus aurantium. Coptera hay-
wardi represented by 16.2% of parasitoids was
found in 6 hosts; Doryctobracon areolatus (14.2%)
was present in 4 hosts, particularly in C. cainito;
and Spalangia endius only accounted for 6.5% of
the overall recorded parasitism, but it was found
in 4 different fruit hosts (Table 2).
Parasitism observed between 2 sampled collec-
tions revealed that specimens recovered from
"Fruit-Bed" samples were higher than those re-
covered from the "Fruit-Lab" samples with 68.5%
and 31.5%, respectively. In this sense, species as
C. haywardi, 0. anastrephae, S. endius, and Dirhi-
nus sp. were dominant in "Fruit-Beds" accounting
for 65% of all parasitoid specimens. On the con-
trary, the dominant species observed in "Fruit-
Lab" samples were D. areolatus, Sycophila sp. and
Euperilampus sp., which accounted for 21.2%. Ta-
ble 3 shows the proportions of parasitoids by hosts
obtained from each sampling method.
Correlation analysis between average fruit
weight (Log Fruit Weight) and the infestation in-
dex (Mean Larvae/kg Fruit) were significant (r =
-0.695; P = 0.005), indicating that as average
weight increased in the different fruit species, the
degree of infestation in the sample decreased. In
contrast, there was not a significant correlation
between the average fruit weight and the per-
centage of parasitism (r = -0.090; P = 0.758), and
no correlation between infestation index and the
percentage of parasitism among samples (r =
0.270; P = 0.350).

DISCUSSION

All parasitoid species reported here are first
records for Anastrepha in Yucatan. No previous
published records exist in literature of the genera
Sycophila sp. (Eurytomidae) and Euperilampus
sp. (Perilampidae) as parasitoids in Anastrepha
(Ovruski et al. 2000). In this sense, Eurytoma siv-
inskii Gates & Grissell (Eurytomidae) was re-
cently described attacking field populations of


December 2006



















TABLE 1. HOST PLANT SAMPLED AND RECOVERED FRUIT FLY PUPAE AND PARASITOIDS OFANASTREPHA SPECIES IN YUCATAN MEXICO. MANGIFERA INDICA: 1 = VAR. CORDOBA;
2 = VAR. CRIOLLO; 3 = VAR. PICO DE LORO; 4 = VAR. MANGLOVA; SPONDIAS PURPUREA: 1 = VAR. SAN JUAN; 2 = VAR. TUXPANA, 3 = VAR. CHI-ABAL.

Total fruit Mean fruit Infestation Total pupae Parasitoids
Host plant Fruit sampled weight (Kg) weight (Kg) (larvae/Kg) recovered Pupae viable Flies emerged emerged Parasitism %

C. aurantium 558 90.40 0.162 22.50 2037 1630 1578 52 3.19
C. sinensis 732 138.60 0.189 4.24 587 438 414 24 5.48
C. paradisi 251 124.40 0.496 3.15 392 269 245 24 8.92
Ch. cainito 200 22.80 0.114 40.40 918 514 447 67 13.04
M. indica 1 325 64.70 0.199 2.80 179 110 108 2 1.82
M. indica 2 29 4.60 0.159 15.90 73 67 67 0 0.00
M. indica 3 225 87.00 0.387 0.33 29 23 23 0 0.00
M. indica 4 234 105.90 0.453 1.20 127 81 81 0 0.00
Ma. zapota 454 83.60 0.184 15.70 1310 1084 1072 12 1.11
Po. sapota 92 72.80 0.791 29.60 2157 1940 1932 8 0.41
Ps. guajava 442 26.80 0.061 103.20 2765 1773 1679 94 5.30
S. purpurea 1 716 26.80 0.037 83.30 2232 1188 1133 55 4.63
S. purpurea 2 138 1.75 0.013 44.00 77 73 72 1 1.36
S. purpurea 3 74 0.65 0.009 70.80 46 33 32 1 3.03

All samples 4470 850.8 0.232 31.2 12929 9223 8883 340 3.69












TABLE 2. PARASITOID SPECIES EMERGED BY HOST FRUIT SPECIES UNDER 2 DIFFERENT SYSTEMS OF COLLECTION. M. INDICA; 1 = VAR. CORDOBA; S. PURPUREA; 1 = VAR. SAN
JUAN; 2 = VAR. TUXPANA; 3 = VAR. CHI-ABAL. ACRONYMS FOR PARASITOID SPECIES ARE AS FOLLOWS: DAR = D. AREOLATUS; OBEL = 0. BELLUS; CHAY = C. HAY-
WARDI; APELL = A. PELLERANOI; ASP = AGANASPIS SP.; OANAS = 0. ANASTREPHAE; OSP = ODONTOSERNA SP.; SPEND = S. ENDIUS; DSP = DIRHINUS SP.; SYSP =
SYCOPHILA SP.; ESP = EUPERILAMPUS SP.

% by
Hosts Parasitoids Oar Obel Chay Apell Asp Oanas Osp Spend Dsp Sysp Esp Totals sample

C. aurantium Fruit Lab 0 0 0 1 0 1 1 0 0 0 0 3 0.9
Fruit beds 0 0 8 0 0 38 0 3 0 0 0 49 14.4
C. sinensis Fruit Lab 0 0 0 0 0 1 0 0 0 0 0 1 0.3
Fruit beds 0 0 6 0 0 10 0 7 0 0 0 23 6.8
C. paradisi Fruit Lab 0 0 0 0 0 0 0 0 0 0 0 0 0.0
Fruit beds 0 0 1 0 0 23 0 0 0 0 0 24 7.1
Ch. cainito Fruit Lab 30 0 0 0 0 12 0 0 0 0 0 42 12.4
Fruit beds 5 0 7 0 0 13 0 0 0 0 0 25 7.3
M. indica 1 Fruit Lab 0 0 0 0 0 1 0 0 0 1 0 2 0.6
Fruit beds 0 0 0 0 0 0 0 0 0 0 0 0 0.0
Ma. zapota Fruit Lab 0 0 0 0 0 0 0 0 0 0 0 0 0.0
Fruit beds 0 0 9 0 0 3 0 0 0 0 0 12 3.5
S. purpurea 1 Fruit Lab 5 0 0 0 0 0 0 0 0 8 16 29 8.5
Fruit beds 0 0 0 0 0 0 0 10 15 1 0 26 7.7
S. purpurea 2 Fruit Lab 0 0 0 0 0 0 0 0 0 1 0 1 0.3
Fruit beds 0 0 0 0 0 0 0 0 0 0 0 0 0.0
S. purpurea 3 Fruit Lab 1 0 0 0 0 0 0 0 0 0 0 1 0.3
Fruit beds 0 0 0 0 0 0 0 0 0 0 0 0 0.0
Po. sapota Fruit Lab 0 0 0 0 0 0 0 0 4 0 4 8 2.3
Fruit beds 0 0 0 0 0 0 0 0 0 0 0 0 0.0
Ps. guajava Fruit Lab 6 2 0 7 1 3 1 0 0 0 0 20 5.9
Fruit beds 1 0 24 5 0 42 0 2 0 0 0 74 21.7
Total specimens Fruit Lab 42 2 0 8 1 18 2 0 4 10 20 107 31.5
Fruit beds 6 0 55 5 0 129 0 22 15 1 0 233 68.5

% parasitism FLab + FBeds 14.1 0.6 16.2 3.8 0.3 43.2 0.6 6.5 5.6 3.2 5.9 340 100







Hernandez-Ortiz et al.: Hymenopteran Parasitoids ofAnastrepha


TABLE 3. RELATIONSHIP FRUIT FLY-PARASITOID SPECIES RECOVERED FROM ALL SAMPLED HOSTS EXPRESSED IN PER-
CENTAGES.

A. ludens I A. striatal
Fruit fly A. ludens A. obliqua A. obliqua A. serpentina A. fraterculus Parasitism %

Ch. cainito,
Spondias P. sapota,
Hosts Citrus spp. M. indica purpurea M. zapota P. guajava All hosts

Parasitoids

Odontosema anastrephae 21.4 0.3 0.0 8.2 13.3 43.2
Odontosema sp. 0.3 0.0 0.0 0.0 0.3 0.6
Aganaspis pelleranoi 0.3 0.0 0.0 0.0 3.5 3.8
Aganaspis sp. 0.0 0.0 0.0 0.0 0.3 0.3
Doryctobracon areolatus 0.0 0.0 1.8 10.3 2.1 14.2
Opius bellus 0.0 0.0 0.0 0.0 0.6 0.6
Coptera haywardi 4.4 0.0 0.0 4.7 7.1 16.2
Spalangia endius 2.9 0.0 2.9 0.0 0.6 6.4
Dirhinus sp. 0.0 0.0 4.4 1.2 0.0 5.6
Sycophila sp. 0.0 0.3 2.9 0.0 0.0 3.2
Euperilampus sp. 0.0 0.0 4.7 1.2 0.0 5.9
Parasitism % 29.3 0.6 16.7 25.6 27.8 100.0


A. obliqua in Mexico (Gates & Grissell 2004). The
eurytomids also occur as parasites in Cynipidae,
Pteromalidae, Eurytomidae, Tanaostigmatidae,
and Agaonidae (Grisell & Schauff 1990; DiGiulio
1997), and members of the family Perilampidae
are hyperparasitoids of Ichneumonidae (Darling
1997). However, since the tephritid pupae were
separated from the fruit and counted before adult
emergence, these may be cases of hyperparasit-
ism. These results should be further investigated.
The genus Dirhinus (Chalcididae) has been re-
ported as a pupal parasite in Brachycerous
Diptera widely distributed throughout the world
tropics, with 3 known species in the USA (Burks
1947), and about 15 native species yet to be stud-
ied in regions ranging from Indiana (USA) to cen-
tral Argentina (Boucek 1992). Unpublished data
for Mexico indicate the presence of at least D. bus-
cki (Crawford), D. schwarzi (Crawford), D. texa-
nus (Ashmead), and D. giffardii (Silvestri) (data
provided by Alejandro Gonzalez-Hernandez and
Serguei Triapitsyn), although there are probably
1 or 2 more species with cosmopolitan distribu-
tion (Robert A. Wharton, Texas A & M University,
personal communication).
Dirhinus giffardi is the unique species re-
ported attacking fruit flies in the Neotropics, a na-
tive western African species introduced in Israel
around 1950 (Podoler & Mazor 1981), and in Latin
American countries of Puerto Rico (1935-1937),
Costa Rica (1955), Peru (1960), Colombia (1970),
and Bolivia (1971), and in Florida, USA (1977-
1979) (Ovruski et al. 2000). In Mexico, it has been
introduced in the states of Morelos and Oaxaca
(Jimenez-Jimenez 1956), however there is no evi-


dence that it is established in these regions. The
Dirhinus species reported in this paper is very
similar to D. schwarzi and D. giffardii, represent-
ing an undescribed species, and a new record of a
native parasitoid forA. obliqua andA. serpentina.
The exotic species Diachasmimorpha longi-
caudata and Aceratoneuromyia indica were not
recorded during the present study, but both have
been documented as established and as having
significant parasitism indices in Costa Rica
(Wharton et al. 1981) and Mexico (Aluja et al.
1990) respectively, though both these studies
were only concerned with coffee and mango or-
chards. Spalangia endius is a remarkable record,
since it has been recorded from Anastrepha in
Florida, though rarely reared from tephritids
(Ovruski et al. 2000).
The majority of the published papers onAnas-
trepha parasitoids indicate that D. areolatus
(Braconidae) is the most important native parasi-
toid species, having the highest parasitism indi-
ces in the Neotropical region in countries such as
Mexico (Hernandez-Ortiz et al. 1994; L6pez et al.
1999), Guatemala (Eskafi 1990), Costa Rica
(Jir6n & Mexzon 1989), Colombia (Yepes & Velez
1989; Carrejo & Gonzalez 1999), Venezuela (Kati-
yar et al. 1995), Brazil (Canal et al. 1995; Leonel
et al. 1995; Aguiar-Menezes & Menezes 1997;
Aguiar-Menezes et al. 2001), and Argentina
(Ovruski et al. 2004, 2005).
On the basis of our results, 0. anastrephae
(Figitidae) is the dominant species occurring in 7
host plants attacked by 5 Anastrepha species.
This species is considered a koinobiont parasitoid
of Anastrepha larvae (Ovruski et al. 2000),







Florida Entomologist 89(4)


though most of the recovered specimens were
found in the Fruit-Bed samples, particularly from
Citrus species and guava accounting for 87.7%.
Such differences in the parasitism indices may be
related to parasitoid biological factors, such as
the ability of 0. anastrephae to reach their host
larvae by entering wounds in fruit located on the
ground (Sivinski et al. 1997, 2000).
Comparisons between fruit weight and infesta-
tion rates among different hosts showed that the
number of larvae was larger in small fruits but de-
creased as fruit size increased. This coincides with
results observed for A. suspense in Florida (USA),
when fruit sizes and infestation indices were com-
pared for 6 host species (Sivinski 1991).
Previous hypothesis on parasitism levels have
been attributed in part to physical difficulties in
locating immature stages within large fruits (Siv-
inski 1991). However, our comparisons between
fruit weight of 14 hosts and the parasitism rates
of the 11 parasitoid species showed no correla-
tion. This may be due to the fact that more sample
sizes are needed in order to test this hypothesis,
or that the native parasitoid community has only
become recently adapted to certain exotic fruit
species included in our analysis, such as Citrus
spp. and M. indica.
The low level of parasitism (3.69%) observed in
this study is probably due to orchard manage-
ment practices, in which destruction of fallen
fruit and periodic pesticide use (CESVY 2000),
could have a negative impact on parasitoid popu-
lations. Similar studies carried out in Brazil re-
ported similar species diversity and levels of par-
asitism (Uch6a-Fernandes et al. 2003). Based on
the parasitoid species diversity that attack the
Anastrepha fruit flies in Yucatan, further studies
need to be focused on the biology and ecology of
certain native parasitoids such as 0. anastrephae,
C. haywardi, and D. areolatus as promising bio-
logical control agents.

ACKNOWLEDGMENTS

The authors thank Luis Hernandez Puch for allow-
ing access to his family's orchard for this research.
Thanks are due to 2 anonymous reviewers for helpful
suggestions on the earlier version of the manuscript.
This study was partially supported by the project "Rel-
aciones hu6sped-parasitoide y caracterizaci6n de la co-
munidad de brac6nidos (Hymenoptera: Parasitica) en el
estado de Yucatan" funded by the CONACYT, Mexico
(Ref: 25016-N), and by the project "Ecologia y
sistematica de insects fit6fagos y sapr6fagos", funded
by the Instituto de Ecologia, A.C. (Ref: 902-08/128).

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Florida Entomologist 89(4)


INCIDENCE OF LYTTA UNGUICULARIS (COLEOPTERA: MELOIDAE)
ON HYBRID AZALEAS, RHODODENDRON SPP, IN THE
GREAT SMOKY MOUNTAINS NATIONAL PARK

ADRIEAN MAYOR*, JEROME F. GRANT AND PARIS L. LAMBDIN
Department of Entomology and Plant Pathology, The University of Tennessee, Knoxville, TN 37996-4560

'Current Address: 107 Park Headquarters Road, Great Smoky Mountains National Park, Gatlinburg, TN 37738


The genus Lytta (Coleoptera: Meloidae) con-
tains approximately 69 species found in the Ne-
arctic (Pinto & Bologna 2002), primarily in the
United States and Mexico. Although this genus
contains approximately 17% of all known species
of meloids in the Nearctic (n = 410) (Pinto & Bolo-
gna 2002), little is known about many of these
species of blister beetles. Several Lytta species are
extremely uncommon in collections and have not
been seen in decades (Pinto & Bologna 1999). One
colorful species, Lytta unguicularis (LeConte,
1866), has a metallic green or blue body and or-
ange legs and has been documented in Illinois
(type locality), Alabama, North Carolina, and
Tennessee (Downie & Arnett 1996; Selander
1960). More specifically, its known distribution
ranges from eastern Alabama to Illinois and
northeast to the Smoky Mountains in eastern
Tennessee and western North Carolina. This
large beetle, with a maximum length of 25 mm, is
uncommon in museum collections and is known
from fewer than 70 specimens from 9 documented
locations (Selander 1960). The larval hosts of
L. unguicularis are unknown; however, other spe-
cies of Lytta are known to parasitize the nests of
native bees, where larvae feed on provisions and
possibly on immature bees. Larval hosts include
immatures of Apoidea, particularly Anthophori-
dae, Megachilidae, Halictidae, and Colletidae
(Pinto & Bologna 1999; Bologna & Pinto 2002).
Adults reportedly feed on the flowers and foliage
of Rosaceae and Ericaceae, including peach, rose,
and mountain laurel, and have been collected on
azalea (Selander 1960).
During a study to identify pollinators of a hy-
brid swarm of azalea, Rhododendron arborescens
(Pursh) Torrey, R. viscosum (L.) Torrey, and R.
cumberlandense Braun, in the Great Smoky
Mountains National Park, a small population
(<50) of adult L. unguicularis was observed on
flowers and foliage of hybrid azaleas. This report
is the first documentation of this species in the
Great Smoky Mountains National Park since the
1950s and early 1960s based on museum and
park collections and other records (Selander
1960). In 1958, adult L. unguicularis were col-
lected previously on azalea in the Park (Selander
1960). Sherman (1913) reported thousands of bee-
tles on peach, rose, and mountain laurel at a site
in Blowing Rock, North Carolina, from 8 to 25 Jun


1901. He stated that they consumed the blossoms
of the mountain laurel and leaves of peach. Se-
lander (1960) suggested that the somewhat gre-
garious nature of Meloidae, including Lytta spe-
cies, serves to maintain the adult beetles near
nesting sites of host bees.
In our study, L. unguicularis was found on aza-
lea plants growing along the northern margin of
one of the balds in the Great Smoky Mountains
National Park. The origin of the grassy balds may
have been natural; however, their present flora is
partially an artifact of human interference, such
as animal grazing, lumber harvesting, and fire
prevention (Lindsay 1977; Lindsay & Bratton
1979). The specific identity and location of this
bald cannot be provided because of low numbers
of individuals and the sensitivity of the site, but
interested persons can contact the Inventory and
Monitoring Coordinator of the Great Smoky
Mountains National Park for additional informa-
tion. This bald, similar to one of the many grassy
balds that occur only in the Southern Appala-
chian Mountains, is currently maintained by per-
sonnel with the Great Smoky Mountains Na-
tional Park. The bald is home to a hybrid swarm
of multicolored azaleas, with flowers ranging in
color from red, orange, pink, yellow, to white, and
many of these flower colors and forms are not
found on other balds. Most of the insects visiting
flowers of these hybrid azaleas were bees in the
families Andrendidae, Halictidiae, and Apidae.
The nests of some of these families of Hy-
menoptera are hosts of larvae of other species of
Lytta (Pinto & Bologna 1999).
Adult beetle activity was observed on only 5 or
6 azaleas located on the north side of the bald on
15 and 18 Jun 2000 between 10 AM and 3 PM. This
observed activity coincides with Selander (1960),
who reported that the seasonal incidence of this
species was from 2 May to 4 Jul. Adults were ob-
served to feed on the blossoms and foliage of aza-
lea on each sampling date, but the extent of this
feeding was not quantified. Mating also was ob-
served on each date. Representative male and fe-
male specimens were collected into individual 31/2
dram vials and taken to the laboratory, where
they were sexed, pinned, labeled and identified.
Specimens included 1 6 and 1 Y collected on 15
Jun 2000 and 11 6 and 6 Y collected on 18 Jun
2000. Voucher specimens were deposited in the


December 2006







Scientific Notes


University of Tennessee Insect Museum, the Uni-
versity of California Riverside Museum, the Flor-
ida State Collection of Arthropods (Gainesville),
the Museum of the Great Smoky Mountains Na-
tional Park, and with the Coleoptera Taxonomic
Working Group at the Louisiana State Arthropod
Museum.
Only 15 species of meloids including two
species of Lytta (L. unguicularis and L. aenea
Say) are recorded in the Checklist of Co-
leoptera Known from Great Smoky Mountains
National Park (http://www.lsuagcenter.com/
Inst/research/departments/arthropodmuseum/
smokieschecklist.htm, 25 Jan 2006); this data-
base is maintained in support of the ATBI (All
Taxa Biological Inventory) Project in the Great
Smoky Mountains National Park. Although L.
unguicularis is listed as previously found in
the Park, no detailed source collection informa-
tion was provided (Selander 1960). Thus, this
research contributes to the known distribution
and host records of this uncommonly collected
species and may encourage researchers to
learn more about the biology and life history of
this little known species.

SUMMARY

This report documents the occurrence ofL. un-
guicularis on hybrid azaleas in the Great Smoky
Mountains National Park, representing the first
time it has been recorded in the Park since the late
1950s and early 1960s. The infrequent collections
of a relatively large and conspicuous beetle in a
reasonably well-known and visited area suggests
that its populations may be limited. Certain other
species of Lytta in the western U.S. have been
identified as 'species of concern' by the U.S. Fish


and Wildlife Service (Halstead & Haines 1992),
and L. unguicularis in the Great Smoky Moun-
tains National Park may represent a similar case.
Additional research is necessary to more fully de-
fine the population density, dynamics, and status
ofL. unguicularis in this geographical area.

REFERENCES CITED

BOLOGNA, M. A., AND J. D. PINTO. 2002. The Old World
genera of Meloidae (Coleoptera): a key and synopsis.
J. Nat. Hist. 36: 2013-2102.
DOWNIE, N. M., AND R. H. ARNETT. 1996. The Beetles of
North America, Vol. II: Polyphaga, pp. 891-1721. The
Sandhill Crane Press, Gainesville, Florida.
HALSTEAD, J. A., AND R. D. HAINES. 1992. New distribu-
tional records for some candidate species of Lytta in
California (Coleoptera: Meloidae). Pan-Pacific Ento-
mologist 68: 68-69.
LINDSAY, M. M. 1977. Management of grassy balds in
Great Smoky Mountains National Park. Natl. Park
Serv. Mgmt. Rpt. No. 17, 67 pp.
LINDSAY, M. M., AND S. P. BRATTON. 1979. The vegeta-
tion of grassy balds and other high elevation dis-
turbed areas in the Great Smoky Mountains
National Park. Bull. Torrey Botanical Club 106: 264-
275.
PINTO, J. D., AND M. A. BOLOGNA. 1999. The New World
genera of Meloidae (Coleoptera): a key and synopsis.
J. Nat. Hist. 33: 569-620.
PINTO, J. D., AND M. A. BOLOGNA. 2002. Family 111. Me-
loidae, pp. 522-529 In R. H. Arnett, Jr., M. C. Tho-
mas, P. E. Skelley, and J. H. Frank (eds.), American
Beetles, Vol. 2. CRC Press, Boca Raton, FL, 861 pp.
SELANDER, R. B. 1960. Bionomics, systematics, and phy-
logeny of Lytta, a genus of blister beetles (Co-
leoptera, Meloidae). Illinois Biol. Monographs, No.
28, 295 pp.
SHERMAN, F., JR. 1913. The Meloidae (Blister-beetles) of
North Carolina (Col.). Entomol. News 24: 245-247.







Florida Entomologist 89(4)


December 2006


FECUNDITY OF THE SISAL WEEVIL, SCYPHOPHORUS ACUPUNCTATUS
(COLEOPTERA: CURCULIONIDAE), ON POLIANTHES TUBEROSA
(LILIALES: AGAVACEAE)

MARIA C. HERNANDEZ R., MIRNA GUTIERREZ O., LUCILA ALDANA LL AND MA. ELENA VALDES E.
Departamento de Interacciones Planta-Insecto, Centro de Desarrollo de Productos Bi6ticos (CEPROBI)
Institute Polit6cnico Nacional, Carr. Yautepec-Jojutla Km. 8.5 Col. San Isidro Yautepec, Morelos M6xico. C.P. 62731


The sisal weevil Scyphophorus acupunctatus
Gyllenhal breeds in economically important
agave varieties in M6xico, including Agave tequi-
lana Weber, Agave fourcroides Lemaire, and
Agave salmiana Otto ex Salm Dick. These weevils
also are associated with tuberose, Polianthes
tuberosa L. in Morelos, M6xico, (Camino et al.
2002). Although not fully understood, mating and
oviposition apparently occur in the subterranean
bulbs of the plants. Larvae develop inside the
bulb where they make galleries. The last instars
migrate to the fibrous periphery of the bulb and
construct cocoons from fiber and mud.
Camino et al. (2002) reported tuberose as a
new host and outlined damage the weevil causes
in cultivated P tuberosa. Solis et al. (2001) men-
tion that S. acupunctatus is active throughout the
year with overlapping populations. This is consis-
tent with Waring and Smith (1986), who point out


that it is a multivoltine species associated with
wild and cultivated agaves. Adults drill holes in
the base of the plant, causing mechanical damage
and facilitating the entry of microorganisms that
decompose the plant tissues. Ramirez (1993) re-
ported that the adults of S. acupunctatus were
most frequently found between the base of the
leaves and the main root of the henequen. The
weevil prefers mature plants and abandoned
plantations. Adults can be detected every month
of the year, but are more abundant in the rainy
season. The adult's favorite habitat is the inferior
stratum of the agave, with oviposition occurring
in moist tissues of rotten leaves or in the base of
the leaves (Lock 1969). The adults are rarely
found on recently planted specimens. Copulation
usually occurs on the rotten shafts of plants (Lock
1969; Hill 1983). In the field larvae ofS. acupunc-
tatus feed on P. tuberosa bulbs until completing


-4-without male --with male


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Weeks after mating

Fig. 1 Mean (SEM) weekly egg production by newly-emerged S. acupunctatus female (n = 20) confined with and
without males.







Scientific Notes


their larval development (pers. obs.). They pupate
inside the bulb, from which the adult emerges.
There are no published references about the fe-
cundity and fertility of S. acupunctatus: However,
we did find a report for Rhynchophorus cruentatus
indicating that the average fecundity of the field
female is of 26 + 15 eggs (Giblin-Davis et al. 1989).
In this study we made observations on fecun-
dity of Scyphophorus acupunctatus females con-
fined with or without males, using tuberose bulbs
as an ovipositional substrate. In Oct 2001, larvae
and cocoons were harvested in the field from in-
fested tuberose or in the laboratory from tuberose
bulbs (P. tuberosa). Larvae and cocoons were
placed individually in covered 100-mL plastic
cups with moistened tissue paper (Giblin-Davis et
al. 1989) and were stored at 29C until adult
emergence. One male and one female at 14 days
post-emergence were placed in a 60-mL covered
plastic container with moistened tissue and were
stored at 29C, 60% RH. One test was with con-
fined females and males, and in a second test
males were removed after 24 h and a thin slice
(5-10 mm; 5-15 g wet weight) of tuberose was
added. All containers were placed in an environ-
mental chamber (Presicion, incubator 818, mod.


FFU20FCACWO18, Electrolux home products,
USA) at 29C with photoperiod of 11:13 (L:D)
60% RH. Tuberose slices usually were replaced
every day. The tests were repeated 4 times with 5
females per test (20 females total). Slices re-
moved from containers were carefully dissected
and eggs were removed. The tuberose bulb slices
were inspected and changed daily for the dura-
tion of the experiment (Oct 2001-Feb 2002) until
mortality began. During 2 tests, eggs were sepa-
rated from tuberose bulb slices and placed in
petri dishes (60 x 15 mm) with wet filter paper,
sealed with parafilm, and stored at 29C. Neo-
nate larvae were inspected daily and dead ones
were removed. The number of eggs and of larvae
that emerged were recorded daily and were con-
verted to eggs laid per female per week. The data
were analyzed by Student-Newman-Keul means
separation procedure in the Sigma Stat program.
The Pearson's correlation test was applied to de-
termine if the male's presence influenced the fe-
cundity of females.
As an index of fecundity, the number of eggs
oviposited and egg viability was recorded (Figs. 1
and 2). The two curves parallel each other, with
greater oviposition and egg viability in the first


---with male -4-without male


1 2 3


4 5 6


7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Weeks after mating


Fig. 2 Percent hatching (eclosion) of eggs produced by newly-emerged S. acupunctatus females (n = 20).







Florida Entomologist 89(4)


weeks. Fecundity was analyzed by ANOVA. We
were unable to demonstrate a significant differ-
ence between the number of eggs for the group of
females with males and that of females without
males (P = 0.429). Nor were we able to demon-
strate a significant difference between the two
groups with regard to the viability of the eggs.
The effect of the different treatments doesn't
depend on the time it is presented. We were not
able to detect a statistically significant interac-
tion between treatment and time (week) (P =
0.055). These results suggest that the presence of
males does not significantly affect oviposition or
egg viability, but these two factors are affected by
the age of the adults, resulting in a general trend
of diminishing number of eggs and decline in via-
bility over time, with an intervening cyclical in-
crease and decrease.
Figs. 1 and 2 suggest the existence of a cyclical
pattern of oviposition and egg viability with a
variable periodicity. This could be due to the re-
productive physiology of the females or the exist-
ence of a mechanism of population self-regula-
tion, as described by Padmanaban and Sathia-
moothy (2001) for the banana tree borer Odoi-
porus longicollis Olivier. This reduces the number
of eggs deposited as the borers become more fre-
quent on their host, indicating the existence of a
spacer pheromone, which may deter oviposition
by females of the same species. Koppenhofer
(1993) observed that females of the banana wee-
vil, Cosmopolites sordidus Germar, laid an aver-
age of 2.7 eggs/week in rhizome and 0.7 eggs/week
in banana pseudo stem in the laboratory, and ovi-
position declined in high populations. However,
Gold and Messiaen (2000) found that the oviposi-
tion rate of C. sordidus is one egg per week. Adair
et al. (1999) found that, under laboratory condi-
tions, females ofDiaprepes abbreviatus L., a wee-
vil pest of citrus fruits, deposited approximately
60 masses of between 30 and 260 eggs each, with
an average of 5000 eggs during their lifetime. The
results suggest that females store enough sperm
in their spermatheca to fertilize eggs for 20 wk,
making multiple copulation unnecessary. The fe-
males may have the capacity to select the sperm
to fertilize the eggs, as mentioned by (C6rdoba
2000). In some cases the female is discriminatory
in fertilization of her eggs, and can even avoid us-
ing the last male's sperm (Siva & Hooper 1996).

SUMMARY

As an index of fecundity, the number of eggs
deposited by females, both with and without


males, and egg viability (proportion of eggs
hatched) on tuberose bulbs were measured. The
results showed that the presence of males does
not affect the number of eggs deposited or the vi-
ability of eggs.

REFERENCES CITED

ADAIR, R., H. N. NIGG, S. SIMPSON, AND L. LE FEVRE.
1999. Observations on the oviposition process of Di-
aprepes abbreviatus (Coleoptera: Curculionidae).
Florida Entomol. 82(2): 362-365.
CAMINO, L. M., V. CASTREJON, R. FIGUEROA., L. AL-
DANA, AND M. E. VALDES. 2002. Scyphophorus acu-
punctatus Gyllenhall, (Coleoptera: Curculionidae)
Attacking Polianthes tuberosa (Liliales: Agavaceae)
in Morelos, M6xico. Florida Entomol. 85(2): 392-393.
CORDOBA, A. A. 2000. Evoluci6n y diversidad de la mor-
fologia de los genitales masculinos en insects. Folia
Entomol6gica Mexicana. 110: 95-111.
GIBLIN-DAVIS, R. M., K. GERBER, AND R. GRIFFITH.
1989. Laboratory rearing ofRhynchophorus cruenta-
tus and R. palmarum (Coleoptera: Curculionidae).
Florida Entomol. 72(3): 480-488.
GOLD, C., AND S. MESIAEN. 2000. El picudo negro del ba-
nano Cosmopolites sordidus. INIBAP. Red Interna-
cional para el mejoramiento del banano y el platano.
Plagas de Musa. Hoja divulgativa. No. 4.
HILL, D. S. 1983.Agricultural Insect Pests ofthe Tropics
and Their Control. Cambridge University Press.
New York. pp. 393-394.
KOPPENHOFER, A. M. 1993 Observations of egg-laying
behaviour of the banana weevil, Cosmopolites sordi-
dus (Gennar). Entomol. Exp. Appl. 68: 187-192.
LOCK, G. W. 1969. Sisal. Thirty Years' Sisal Research in
Tanzania, 2nd ed. Tanganyika Sisal Growers Associ-
ation. Longmans Green and Co. Ltd. London. 365 pp.
PADMANABAN, B., AND S. SATHIAMOOTHY. 2001. El bar-
renador del tallo del banano Odoiporus longicollis.
INIBAP. Red Internacional para el mejoramiento del
banano y el platano. Plagas de Musa. Hoja divulga-
tiva. No. 5 pp. 1-5.
RAMIREZ, CH. J. L. 1993. Max del henequ6n Scyphopho-
rus interstitialis bioecologia y control. Serie libro t6c-
nico. Centro de investigaci6n Regional del Sureste.
INIFAP- SARH. M6rida, Yucatan, M6xico.
SIVA-JOTHY, M. T., AND R. E. HOOPER 1996. Differen-
tial use of stored sperm during oviposition in the
damselfly Calopteryx splendens xanthostoma (Char-
pentier). Behavioral Ecol. and Sociobiol. 39: 389-391.
SOLIS, A. J. F., H. GONZALEZ, J. L. LEYVA V., A. EQUIUA,
M. F. J. FLORES, AND G. A. MARTINEZ. 2001. Scypho-
phorus acupunctatus Gyllenhal plaga del agave te-
quilero en Jalisco, M6xico. Agrociencia. 35(6): 663-
670.
WARING, G. L., G. A. R. L. SMITH. 1986. Natural history
and ecology of Scyphophorus acupunctatus (Co-
leoptera: Curculionidae) and its associated microbes
in cultivated and native agaves. Ann. Entomol. Soc.
Am. 79(2): 334-340.


December 2006







Scientific Notes


GENETIC ANALYSIS OF BREEDING STRUCTURE
IN LABORATORY-REARED COLONIES OF RETICULITERMES FLAVIPES
(ISOPTERA: RHINOTERMITIDAE)

CATHERINE E. LONG', EDWARD L. VARGO2, BARBARA L. THORNE3 AND THOMAS R. JUBA2
'American Pest Management, Inc., 6460 New Hampshire Avenue, Takoma Park, MD 20912, USA

2Department of Entomology, 3312 Gardner Hall, North Carolina State University, Raleigh, NC 27695, USA

3Department of Entomology, 4112 Plant Science Bldg., University of Maryland, College Park, MD 20742, USA


Primary reproductive, or kings and queens,
within Reticulitermes flavipes (Kollar) (Isoptera:
Rhinotermitidae) colonies suppress sexual matura-
tion of their offspring (Liischer 1961). In the ab-
sence of this influence, immature individuals may
differentiate into replacement reproductive (neo-
tenics) (Pickens 1932; Esenther 1969; Howard &
Haverty 1980; Thorne 1996). Snyder (1920) specu-
lated that these neotenic individuals may leave the
main nesting area with a small group of workers in
order to establish distinct bud nests. To evaluate
whether colonies containing neotenics would estab-
lish distinct daughter or bud nests within a network
of physically separated but linked food resources,
we provided laboratory colonies with 3, equal-vol-
ume food resources linked by 1-m sections of tubing.
Termites were permitted to forage and move among
the locations. After 20 months, workers were sam-
pled from each of the 3 resources. Microsatellite
analyses were performed to determine whether
subpopulations within the resources exhibited dis-
tinct genotypic frequencies.
In 1993, incipient R. flavipes colonies were es-
tablished in the laboratory with pairs of sibling
alates collected from dispersal flights in Prince
George's County, Maryland, USA (Thorne et al.
1997). In 2000, 13 of these colonies were trans-
ferred to their own three-resource feeding net-
works (Long et al. 2006 in press). All of these col-
onies retained their kings; 9 "queenright" colonies
also contained a queen. In 4 queenlesss" colonies,
the founding queen had been replaced by at least
1 neotenic female 2-6 years prior to this experi-
ment (Long et al. 2003).
Here we present data from Colony 1, a queen-
right colony (for simplicity, a single, representa-
tive sample is discussed), and the 4 queenless col-
onies (Colonies 2-5). DNA was extracted from 60
workers per colony, with 20 workers pulled from
each food resource. Preparation and analysis of
DNA followed Vargo (2003). Individuals were gen-
otyped at seven microsatellite loci: Rs 16, Rs 33,
Rs 62, Rf 1-3, Rf 5-10, Rf 15-2, and Rf 24-2.
Twenty-one alleles were identified (Table 1); loci
contained an average of 3 alleles. Average het-
erozygosity was 0.54 (0.31-0.90), a value compa-
rable with those observed in North Carolina field
populations (Vargo 2000; DeHeer & Vargo 2004).


Worker genotypes in the queenright colony
and 3 of the 4 queenless colonies (Colonies 1-4)
were consistent with those from simple families.
However, locus Rf 24-2 in Colony 5, which con-
tained 14 neotenic females, contained 3 alleles in
5 genotypic classes; 4 homozygous genotypes
were scored at Rs 33. Both scenarios are possible
only if at least 3 and 4 parents, respectively, con-
tribute to the offspring. Genotype frequencies
alone cannot indicate exactly how many parents
contribute.
Significant deviation from expected, homoge-
neous genotype frequencies for each locus were
evaluated by a G-based test of differentiation
among the subpopulations and then summed for
an overall estimate of significance (Genepop
2004; Raymond & Rousset 2004). Only Colony 5
showed evidence of significant differentiation in
genotype frequencies among the resources (P <
0.0001, df = 12).
The non-uniform distribution of Colony 5's al-
leles across the three-resource network suggests
that differentiation may have a spatial compo-
nent, either in offspring production or preferred
distribution (i.e., associations of closest kin). At 2
loci, alleles or genotypes were not observed in all
resources: at Rs 33, alleles 259 and 267 were
missing in two resources, and the genotype 196/
106 at locus Rf24-2 was absent from 1 of the sites.
In Colony 5, the resource in which workers
harbored 2 unique alleles also contained the
king, all 14 neotenic sisters, and all of colony's
eggs and instars 1-3. Travel and mark-recapture
data indicate that worker exchange occurred
among all 3 sites throughout the colony's tenure
in the three-resource network (Long 2005). Al-
though the co-habitation of all reproductive
does not suggest nest budding in this case, ge-
netic isolation of a subset of workers that main-
tain constant contact with less genetically differ-
entiated individuals lends support to the hy-
pothesis that physical or functional budding can
occur without complete isolation from nestmates
(Thorne et al. 1999).
Our results provide a rare opportunity to eval-
uate the response of queenless colonies to a forag-
ing arena consisting of physically separated but
linked food resources. Even after 20 months, 3 of







Florida Entomologist 89(4)


TABLE 1. NUMBERS OF EACH GENOTYPE FOUND AMONG
R. FLAVIPES WORKERS SAMPLED FROM 5 COLO-
NIES. COLONY 1 WAS QUEENRIGHT; THE OTH-
ERS WERE HEADED BY AT LEAST 1 NEOTENIC
FEMALE. TWENTY-ONE ALLELES WERE IDENTI-
FIED AT 7 LOCI. MISSING DATA (-) INDICATE EI-
THER NON-SCORABLE PCR PRODUCT FOR THAT
LOCUS OR THAT THE LOCUS WAS NOT SE-
QUENCED FOR THAT COLONY.

Colony

Locus genotypes 1 2 3 4 5


Rs 16
305/305
305/295
295/295
Rs 33
259/259
267/259
255/255
263/255
263/263
267/267
Rs62
315/315
319/319
319/315
Rf 1-3
236/221
224/224
224/221
236/224
224/218
236/218
221/218
221/221
218/218
245/245
245/224
245/218
Rf 5-10
153/153
153/147
Rf 15-2
235/232
235/235
232/232
Rf 24-2
106/106
196/106
169/106
169/169
196/169


"Sixty workers were examine
dividual samples to yield read
between these totals and the nu


27 60 60 38
30 30 17
29


39 16 60 60 4
32 5
21
17
7
18 10 1


57 60 21 19 55
9 9
29 32


TABLE 1. (CONTINUED) NUMBERS OF EACH GENOTYPE
FOUND AMONG R. FLAVIPES WORKERS SAMPLED
FROM 5 COLONIES. COLONY 1 WAS QUEEN-
RIGHT; THE OTHERS WERE HEADED BY AT LEAST
1 NEOTENIC FEMALE. TWENTY-ONE ALLELES
WERE IDENTIFIED AT 7 LOCI. MISSING DATA (-
) INDICATE EITHER NON-SCORABLE PCR PROD-
UCT FOR THAT LOCUS OR THAT THE LOCUS WAS
NOT SEQUENCED FOR THAT COLONY.

Colony

Locus genotypes 1 2 3 4 5

199/106 21
196/196 28
199/169 16

"Sixty workers were examined from each colony. Failure of in-
dividual samples to yield readable data account for discrepancies
between these totals and the number of genotypes presented.


the 4 queenless colonies were genetically homoge-
neous. The genotypes sampled from the fourth
queenless colony, which contained 14 female neo-
tenics, indicate that genetic differentiation had
begun to develop among the resources.


SUMMARY


Thirteen laboratory-reared R. flavipes colonies
16 22 were housed in 3-resource foraging arenas for 20
17 months. Four of these colonies were queenless,
16 having lost their founding queen 2-6 years prior.
8 Microsatellite analysis performed on workers
4 sampled from each resource allowed each colony
7 to be classified as either a simple or an extended
25 13 family and to examine the queenless colonies for
17 evidence of genetic differentiation among the 3
33 linked feeding resources.
2 F-statistics (Wright 1921) and relatedness co-
8 efficient (b) (Pamilo 1984) were generated with
7 Genetic Data Analysis software (Lewis & Zaykin
2001) with notational conventions ofThorne et al.
(1999) and Bulmer et al. (2001). Among the 4
59 60 31 24 queenless colonies, F, = 0.52 (c.i. 0.37-0.65), FC, =
25 33 0.59 (c.i. 0.48-0.69), Fc = -0.17 (c.i. -0.27-0.08),
and b = 0.78. These results are not significantly
31 30 36 23 different from values predicted for an inbred col-
57 29 24 22 ony with 2 female neotenics and a single male
29 10 (Thorne et al. 1999). FT = 0.52 and b = 0.78 indi-
cate marked inbreeding in this laboratory popula-
tion. The founding of these colonies by probable
12 30 1 10 siblings undoubtedly accounted for a portion of
31 30 6 this observed loss of heterozygosity, but regional
12 15 20 variation in levels of inbreeding may have also
12 27 6 contributed (Reilly 1987; Bulmer et al. 2001;
16 Vargo 2003). Fc = -0.17 suggests an intermediate
loss of heterozygosity within each colony, but not
ed from each colony. Failure of in- existence of differentiated bud nests. F, = 0.59 in
ble data account for discrepancies this laboratory population indicates relatively
mber of genotypes presented. high contrast between colonies.


December 2006







Scientific Notes


REFERENCES CITED

BULMER, M. S., E. S. ADAMS, AND J. F. A. TRANIELLO.
2001. Variation in colony structure in the subterra-
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Florida Entomologist 89(4)


December 2006


EFFICIENCY OF HETERORHABDITIS BACTERIOPHORA
(NEMATODA: HETERORHABDITIDAE) ON ANASTREPHA SERPENTINA
(DIPTERA: TEPHRITIDAE) LARVAE UNDER LABORATORY CONDITIONS

JORGE TOLEDO1, ROMEO ROJAS' AND JORGE E. IBARRA2
'Departamento de Entomologia Tropical, El Colegio de la Frontera Sur
Apartado Postal 36. 30700 Tapachula, Chiapas, Mexico
jtoledo@tap-ecosur.edu.mx

2Departamento de Biotecnologia y Bioquimica, Centro de Investigaciones y de Estudios Avanzados, IPN
Apartado Postal 629, 36500 Irapuato, Guanajuato, Mexico


The sapote fruit fly, Anastrepha serpentina
(Wiedemann), sometimes called the tropical fruit
fly, is an important species in Mexico because its
larvae infest sapote (Calocarpum spp.), mammee
[Pouteria sapota (Jacq.) Moore & Steam], sapo-
dilla (Achras zapota L.), willowleaf lucuma (Lu-
cuma salicifolia Hbk.) and related fruits (Aluja
1994). Infestations in tree-ripe fruits frequently
are so high that in parts of the country where
these fruits are grown, especially in Veracruz, the
growers do not allow them to mature on the trees,
but pick them green and ripen them artificially to
avoid infestation. Mammee tree is native to Cen-
tral America and southern Mexico and it is becom-
ing important as an exotic fruit in international
commerce. For this reason, the sapote fruit fly is
part of the pest management program of the
National Campaign Against Fruit Flies (CNCMF,
after its Spanish acronym) (Reyes et al. 2000). Un-
fortunately, its control is mostly based on the use
of chemical insecticides, applied either on the foli-
age to control adults or on the soil to control larvae
or newly emerged adults. Consequently, new con-
trol alternatives are being explored, such as natu-
ral products and biological control agents, which
may at least partially substitute for the chemical
insecticides. This is an important strategy due to
the growing interest in organic agriculture.
The entomopathogenic nematode Heterorhab-
ditis bacteriophora (Poinar) is a natural soil
dweller that parasitizes a number of insect spe-
cies. Infection occurs through the insect's natural
apertures such as the mouth, spiracles, or anus
(Woodring & Kaya 1988). Once in the host hemo-
coel, the nematode releases its symbiotic bacte-
rium Photorhabdus spp., which causes a rapid
and lethal septicemia. This allows the growth and
reproduction of the nematode for one or more gen-
erations. Due to its lethal efficiency, H. bacterio-
phora may become an important regulation factor
for several insect populations whose larvae co-ex-
ist within the soil. This includes several species of
fruit fly larvae (Tephritidae) whose susceptibility
to nematode infection has been demonstrated
previously (Beavers & Calkins 1984; Lindegren &
Vail 1986; Lindegren et al. 1990; Lezama-Gutier-
rez et al. 1996; Gazit et al. 2000; Toledo et al.


2001, 2005, 2006). In this report we present evi-
dence on the infectivity of H. bacteriophora to
third instars of A. serpentina under laboratory
conditions.
Sapote fruit fly larvae were obtained from the
mass rearing facility at Moscafrut Plant (SA-
GARPA-IICA), located in Metapa de Dominguez,
Chiapas, Mexico. They were reared on artificial
diet, following the procedure and conditions de-
scribed by Dominguez et al. (2000). The nematode
was originally collected in Costa Rica with wax
moth (Galleria mellonella L.) soil traps from a
warm, rainy region, described by Castillo & Mar-
ban-Mendoza (1996). The nematode was reared
by infecting wax moth larvae, and infective juve-
niles (IJ) were collected in White traps (Woodring
& Kaya 1988). IJs were quantified and working
concentrations were adjusted to 800 IJ/mL in
sterile, distilled water. Suspensions were stored
at 10 2C until further use (Woodring & Kaya
1988).
Bioassays were performed on late, mature
third instars of the sapote fruit fly with infection
units made from PVC pipes 5 cm long and 5 cm in
diameter (19.63 cm2 surface). Each unit was filled
with 70 g of sandy soil (96% sand, 3% clay, 1%
lime, 0.18% organic matter, and adjusted to 6.6
pH), previously sieved (mesh 18), autoclaved, and
adjusted to 15% mixture (weight/volume). A total
of 25 larvae was added to each unit. Larvae im-
mediately crawled into the soil (<10 min). The
nematode IJ concentrations tested were 0, 6, 13,
25, 51, 76, 102, 127, and 178 IJ/cm2 soil, added in
1 mL suspension and uniformly distributed on
the soil surface. Infection units were incubated at
26 1C, 70 + 5% RH, and L12:D12 photoperiod
for 7 d. After this period, soil was sieved to sepa-
rate larvae and pupae, and mortality was quanti-
fied under a dissecting microscope to verify nem-
atode infection. To estimate an LC5s, a total of five
replicates was performed and data were subjected
to Probit analysis (SAS Institute 1992), in which
statistical requirements were fulfilled as de-
scribed by Ibarra & Federici 1987.
Once an LC5s was estimated, a simple test on
the dispersion of mortality was performed by test-
ing the LC5o and three times the LC50, under the







Scientific Notes


same bioassay conditions. A total of five replicates
was carried out and statistical difference was an-
alyzed by Student's t test (Steel & Torrie 1993).
The LC,, of H. bacteriophora infective juve-
niles tested on late third instars of the sapote
fruit fly was estimated at 36.0 5.4 IJ/cm2 (n =
491; 2 = 3.6;Y = 3.00 + 1.28 X), within highly pre-
cise fiducial limits (26.7-46.4). The LC95 was esti-
mated at 686 IJ/cm2. The negative control never
showed infection and the natural mortality was
always around 2%, with >90% adult emergence.
In the dispersion of mortality test, although mor-
tality caused by the LC5, and three times the LC5,
(108 IJ/cm2) showed a statistically significant dif-
ference (t = -3.5; df = 4; P = 0.001), actual mortal-
ity barely increased, ranging only from 42.4 +
2.0% at LC5, to 54.5 2.7% at three times the
LC,. These results indicate that, in spite of the
low number of IJs required to kill 50% of the lar-
val population, a much larger number of nema-
todes is required to kill a significant proportion of
the insect population. According to these results,
approximately 700 IJ/cm2 are necessary to obtain
significant control levels, which is close to the es-
timated LC95.
This is the first report on the susceptibility of
sapote fruit fly larvae to H. bacteriophora under
laboratory conditions. Based on our finding this
nematode can be considered a potential biological
control agent for this pest, and the results should
be corroborated under field conditions. The test
was conducted on third instars because it is the
only larval stage that may be in contact with the
soil under natural conditions. The highest toler-
ance to nematode infection occurs in the 3rd in-
star, as observed in otherAnastrepha species (To-
ledo et al. 2005). However, the invasive ability of
the nematode varies not only among the different
species but also between strains of the same spe-
cies, as observed when different species and
strains of nematodes were tested against A. sus-
pensa (Beavers & Calkins 1984).
Laboratory tests on the infectivity of nema-
todes are important because they are performed
under controlled, optimum conditions. The inter-
action host/parasite is tested without the influ-
ence of other factors that may be found in the
field. It is known that H. bacteriophora moves
easily in the soil, showing a high ability to find
hosts at different soil depths (Campbell et al.
1996). However, its performance can be severely
hampered by some soil factors such as texture,
pH, humidity, and possibly other factors. In gen-
eral, these factors influence the failure or success
of these control agents when tested under field
conditions (Portillo-Aguilar et al. 1999). Fruit fly
larvae also are influenced by these factors (Eskafi
& Fernandez 1990; Jackson et al. 1998; Alyokhin
et al. 2001), and especially the soil compactness
(Aluja 1994), which can influence nematode infec-
tivity (Portillo-Aguilar et al. 1999).


Moisture is another important factor. IJs of
H. bacteriophora are more infective in sandy-clay
soils with 15% moisture (Toledo et al. 2006), while
in sandy soils the optimum is at 10% moisture
(Toledo et al. 2005). Slightly higher or lower mois-
ture levels drastically decrease its infective effi-
ciency. High moisture content in a sandy soil may
slow down the IJ's movement, due to an excess of
water between the particles, while a low moisture
content may limit the search for hosts. The effect
of humidity may vary among the nematode spe-
cies. The nematode Steinernema riobrave kept its
infectivity to the Mediterranean fruit fly larvae in
a sandy-clay soil at humidity levels ranging from
3 to 20% (Gazit et al. 2000).
Heterorhabditis bacteriophora has shown its
potential as a biological control agent in the field,
against other fruit fly larvae (Toledo et al. 2006).
A field test on the sapote fruit fly is feasible, and
should be followed by an analysis of economical
and practical viability.
The authors are grateful for the technical sup-
port of Azucena Oropeza and Gustavo Rodas
(ECOSUR, Mexico); for the taxonomic support of
Patricia Stock (University of Arizona, USA); and
for the larval supply by Moscafrut Plant (SA-
GARPA-IICA, Mexico). This project was partially
supported by project A-024, Sistema de Investi-
gaci6n Benito Juarez (SIBEJ-CONACYT, Mexico).

SUMMARY

The infectivity of the entomopathogenic nema-
tode Heterorhabditis bacteriophora was tested on
third instars of the tropical fruit fly, Anastrepha
serpentina, under laboratory conditions. An LC5,
was estimated at 36.0 5.4 IJ /cm2 of sandy soil,
adjusted to 15% humidity, with 5-cm-deep infec-
tivity units. Significant amounts of nematodes
are required to obtain satisfactory control levels,
as shown by 3x the LC5,value. This is the first re-
port on the susceptibility of the tropical fruit fly
larvae to H. bacteriophora. Potential of this nem-
atode as a biological control agent of this pest
should be corroborated under field conditions.

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December 2006


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Florida Entomologist 89(4)







Scientific Notes


SURVEY FOR POTENTIAL PREDATORS OF THE ELONGATE
HEMLOCK SCALE IN TENNESSEE AND NORTH CAROLINA

CHRISTINE LYNCH', PARIS LAMBDIN', JEROME GRANT', RICHARD REARDON2 AND RUSTY RHEA3
'Department of Entomology and Plant Pathology, University of Tennessee, Knoxville, TN 37996

2USDA Forest Service, 180 Canfield St., Morgantown, WV 26505

3USDA Forest Service, Forest Health Protection, 200 Weaver Boulevard, Asheville, NC 28804


The elongate hemlock scale, Fiorinia externa
Ferris (Hemiptera: Diaspididae) (EHS), is an inva-
sive insect from Japan (Takagi 1963) that feeds on
the needles of eastern hemlock, Tsuga canadensis
(L.) Carriere. This diaspidid often co-exists with
the exotic hemlock woolly adelgid, Adelges tsugae
Annand (Hemiptera: Adelgidae), throughout the
northern United States (McClure 2002), where
they cause extensive damage to eastern hemlock
and threaten to disrupt forest composition. EHS
invaded eastern hemlocks in the southern Appala-
chian range and has become well established in
western North Carolina and eastern Tennessee.
Heavily-infested trees were discovered in Mar
2004 in two urban areas located at Tyson Park and
Lynnhurst Cemetery, Knoxville, TN (Buck et al.
2005; Lambdin et al. 2005), where little is known
about the natural enemies of this introduced pest.
Several species of predators including Atractoto-
mus magnicornis buenoi (Mercet) and Phytocorus
sp. (Heteroptera: Miridae), Conwentzia pineticola
Enderlin (Neuroptera: Coniopterygidae), and
Chilocorus stigma (Say) and C. kuwanae Silvestri
(Coleoptera: Coccinellidae) have been recorded to
feed on EHS in other areas of the United States
(Davidson & McComb 1958; McClure 1977). In
addition, the parasitoid Encarsia citrina (Craw)
(Hymenoptera: Aphelinidae) has been reported to
be an important mortality factor of EHS (McClure
1978, 1979, 2002). To better understand the popu-
lation dynamics of EHS, a survey was designed to
identify its potential predators in eastern Tennes-
see and western North Carolina.
Populations of potential predators were sam-
pled with beat sheets and branch extractions of
eastern hemlock at 2 urban and 2 forest sites.
Urban sites were located at Lynnhurst Cemetery
(Knoxville, TN) and at Biltmore Estate
(Asheville, NC). Forest sites were located at Bays
Mountain Park (Kingsport, TN) and at Biltmore
Estate (Asheville, NC). All sites were sampled
from Sep 15, 2004 to Apr 28, 2006 except at the
Bays Mountain Park site, which was sampled
from Aug 29, 2005 to Apr 10, 2006. Each urban or
forest site was arranged into 5 blocks with 3 trees
sampled monthly per block for predators.
Predators were collected with a beat sheet (75
cm x 75 cm) by striking one branch 3 times from
each of the 4 cardinal directions per tree. The beat
sheet was scanned for predators, and when dis-


covered, they were placed into a glass vial (6
dram), labeled (date, site, block, tree, and direc-
tion) and transported to the laboratory for pro-
cessing. Data (date, site, tree number, species,
number of specimens per species, location and de-
velopmental stage of the predators collected) re-
corded from the 4,380 beat sheet samples were
subjected to Kruskal-Wallis Test with SPSS 14.0
for Windows. We extracted 2 branch samples (30
cm) from each of 5 trees per block, and placed
them into separate, labeled "Ziploc" bags to ob-
serve predators and the impact of their feeding
upon the scale insect in the laboratory. Data re-
corded included the date of collection, site, num-
ber of EHS from 100 needles per sample, and the
number and location of EHS damaged by preda-
tors. Predator damage (defined as any injury or
mutilation to the scale test or body) was deter-
mined from EHS specimens taken from 3,600
branch samples throughout the study period.
Six predaceous species (C. stigma, Conwentzia
nr. pineticola Enderlein, Harmonia axyridis Pal-
las, Rhyzobius lophanthae (Blaisdell), Scymnillus
horni (Gordon), and Scymnus loweii Mulsant)
were collected and identified from EHS-infested
eastern hemlock. A total of 504 specimens consist-
ing of 347 adults and 157 larvae was obtained
from beat sheet sampling. Experiments are un-
derway to assess the ability of these field-col-
lected predators to feed on EHS. The only other
scale insect species encountered was the native
hemlock scale, Abgrallaspis ithacae (Ferris)
(Hemiptera: Diaspididae), comprising less than
0.02% of the scale insect fauna at the sites. Two of
the coccinellid species (S. horni and R. lophan-
thae) represent new state records for Tennessee,
while collections of S. loweii represent new county
records for eastern Tennessee. Except for S. horni,
the remaining predators were previously col-
lected and identified in North Carolina (Kathleen
Kidd, personal comm.). The native species S. horni
is common to the forests of the eastern United
States (Robert Gordon, personal comm.). The coc-
cinellids C. stigma, R. lophanthae, and S. horni
are primary predators of scale insects, while S. lo-
weii, H. axyridis, and Coniopterix sp. appear to be
more generalist predators feeding primarily on
aphids, scale insects, and mites.
Lowest numbers of specimens were collected
for the species S. loweii, Conwentzia sp., and







Florida Entomologist 89(4)


H. axyridis. Percent damage to EHS field-
collected samples was 9.8% for Biltmore Estate
urban, 9.7% for Biltmore Estate forest, 6.2% for
Lynnhurst Cemetery urban, and 4.7% for Bays
Mountain Park forest sites, respectively. Al-
though predators were found at urban and forest
sites, the number of species and species combina-
tions differed per site.
More predator specimens for each species were
found in urban areas (n = 55, df = 3, H < 0.05)
with R. lophanthae, C. stigma, and S. horni com-
prising the dominant species. Scymnillus horni
was the only species found at the Bays Mountain
Park site, and S. loweii was only found at the
Lynnhurst Cemetery site. Although more speci-
mens of S. horni were found on the south side of
the tree and higher numbers of R. lophanthae
were found on the north side of the tree, no differ-
ences (n = 80, df = 3, H > 0.05) for direction were
noted for direction preference among the species.
We thank Robert Gordon (Northern Plains
Entomology, Willow City, ND 58384) for kind
assistance in identifying the coccinellids collected
during this survey. We also appreciate the assis-
tance of Bill Hatcher (Director, Biltmore Estates,
Asheville, NC 28801), Charles Limebarger (Su-
perintendent, Lynnhurst Cemetery, Knoxville,
TN 37918), and Ken Childress (Bays Mountain
Park, Kingsport, TN 37660) for providing the
study sites, and to the USDA Forest Service for
financial support of this project.

SUMMARY

Six predators from EHS-infested eastern hem-
locks were collected in eastern Tennessee and
western North Carolina. Rhyzobius lophanthae
and S. horni are known to feed on scale insects as
their primary food source, and both represent
new state records for Tennessee. The most domi-
nant species collected were S. horni, R. lophan-


thae, and C. stigma, respectively. The natural
predator abundance does appear sufficient to sig-
nificantly reduce the heavy populations of EHS
now present on eastern hemlocks within the re-
gion. However, augmentation of their numbers
along with the use of the parasitoid E. citrina or
other more host specific parasitoids offer the po-
tential of suppressing pest populations in both
forests and urban landscapes.

REFERENCES CITED

BUCK, S. E., P. LAMBDIN, D. PAULSEN, J. GRANT, AND A.
SAXTON. 2005. Insect species associated with eastern
hemlock in the Great Smoky Mountains National
Park and environs. J. Tennessee Acad. Sci. 80: 60-69.
DAVIDSON, J., AND C. MCCOMB. 1958. Notes on the biol-
ogy and control ofFiorinia external. J. Econ. Entomol.
51: 405-406.
LAMBDIN, P., C. LYNCH, J. GRANT, R. REARDON, B.
ONKEN, AND R. RHEA. 2005. Elongate hemlock scale
and its natural enemies in the southern Appala-
chians, pp. 145-154 In B. Onken and R. Reardon
[Compilers], Third Symposium on Hemlock Woolly
Adelgid in the Eastern United States. USDA Forest.
Serv., Asheville, NC.
MCCLURE, M. S. 1977. Resurgence of the scale, Fiorinia
external (Homoptera: Diaspididae), on hemlock fol-
lowing insecticide application. Environ. Entomol. 6:
480-484.
MCCLURE, M. S. 1978. Two parasitic wasps have poten-
tial for controlling hemlock scales. Frontier Plant
Sci. 30: 2-3.
MCCLURE, M. S. 1979. Self-regulation in populations of
the elongate hemlock scale, Fiorinia external (Ho-
moptera: Diaspididae). Oecologia 39: 25-36.
MCCLURE, M. S. 2002. The elongate hemlock scale, Fior-
inia external Ferris (Homoptera: Diaspididae): a new
look at an old nemesis, pp. 248-253 In B. Onken, R.
Reardon, and J. Lashomb [eds.], Proceedings, Hem-
lock Woolly Adelgid in the Eastern United States.
USDA Forest Serv., East Brunswick, NJ.
TAKAGI, S. 1963. Discovery of Fiorinia external Ferris in
Japan. Insecta Matsumurana 26: 115-117.


December 2006







Scientific Notes


GEOGRAPHIC RANGE EXPANSION OF BOREIOGLYCASPIS MELALEUCAE
(HEMIPTERA: PSYLLIDAE) TO PUERTO RICO

PAUL D. PRATT1, MIN B. RAYAMAJHI1, LOURDES S. BERNIER2 AND TED D. CENTER1
'USDA-ARS, Invasive Plant Research Laboratory, 3225 College Ave., Ft. Lauderdale, FL 33314

2Departamento de Recursos Naturales y Ambientales
P.O. Box 9066600, Puerto de Tierra Station San Juan, Puerto Rico, 00906-6600


The Australian tree Melaleuca quinquenervia
(Cav.) S.T. Blake (Myrtaceae) was introduced into
South Florida (U.S.) by horticulturists during the
late 1800s (Dray 2003). Nearly 100 years later,
M. quinqueneruia was widely recognized as a per-
nicious invader of wetland systems in the Florida
Everglades (Browder & Schroeder 1981; Woodall
1981, 1982), due in part to the tree's competitive
superiority over most native vegetation (Turner
et al. 1998). Current estimates of geographic dis-
tribution suggest that the invasive tree now occu-
pies approximately 200,000 ha of graminoid/her-
baceous wetlands, including portions of the Ever-
glades National Park (Turner et al. 1998).
A classical weed biological control program
targeting M. quinqueneruia was initiated in 1986,
with expectations that introduced herbivores
would limit invasion and complement conven-
tional control tactics (Balciunas et al. 1994). The
curculionid weevil Oxyops vitiosa Pascoe (Cole-
optera: Curculionidae) was the first candidate
selected for quarantine-based host specificity
testing (Purcell & Balciunas 1994) and, once
deemed environmentally safe, was released in
South Florida during 1997 (Center et al. 2000;
Pratt et al. 2003).
The second herbivore introduced for biological
control of M. quinqueneruia in Florida was the
melaleuca psyllid, Boreioglycaspis melaleucae
Moore. Host range studies demonstrated that the
insect completes its development only on a small
group of species in the Melaleuca genus (Winer-
iter et al. 2003), of which there are no native rep-
resentatives in the New World. Based on this nar-
row host range, the psyllid was permitted for re-
lease in South Florida during the spring of 2002
(Pratt et al. 2004). Both adults and nymphs feed
on expanding buds and leaves but nymphs also
exploit mature, fully expanded leaves as competi-
tion for preferred feeding sites increases. Initial
field data indicate that feeding by psyllids in-
duces leaf senescence, eventually resulting in
mortality of coppicing stumps and seedlings
(Morath et al. 2006; Franks et al. 2006). Psyllids
also rapidly disperse from release points, spread-
ing on average 4.7 km/yr but ranging as high as
10 km/yr (P. D. Pratt, unpublished data). Follow-
ing establishment, common garden experiments
confirmed that feeding and development by the
melaleuca psyllid was restricted to Melaleuca
species, as predicted in quarantine-based host


range testing, and so it posed no threat to native
or economically important species (P. D. Pratt un-
published data). In response to observed impacts
of the psyllid, federal, state, and county agencies
initiated a redistribution campaign for B. mela-
leucae in 2003. Over 1 million individuals have
been redistributed to nearly 100 locations in
South Florida since 2002.
In addition to its occurrence in Florida,
M. quinqueneruia has been planted throughout
much of the Caribbean (Serbesoff-King 2003). In
Puerto Rico, for instance, it was planted island-
wide in public parks, promenades, and along cer-
tain highway medians and green areas from the
1970-90s (Angler6 1960; Pratt et al. 2005). Not
surprisingly, the extensive use of M. quinquen-
eruia as an ornamental in Puerto Rico enabled it
to naturalize in ecologically sensitive wetlands,
including the Tortuguero Lagoon Natural Re-
serve (Pratt et al. 2005). The implementation of
chemical controls for invasive populations of the
tree on the island is currently underway. The use
of biological controls, which have been very effec-
tive in Florida, were considered less suitable for
Puerto Rico due to the small size of the infested
areas and possible conflicts of interest. Conserva-
tionists in Puerto Rico are interested in halting
continued invasion of the tree in wetlands, al-
though public policy as to how to address orna-
mentally planted trees has yet to be determined.
More importantly, the biological control agents
approved for introduction into Florida have not
been evaluated as to their propensity to oviposit
and develop on Caribbean species of Myrtaceae.
Liogier (1994) cites 30 species in the family Myrt-
aceae that are native to the island of Puerto Rico
and these were not included in initial host testing
for the biological control agents described above.
Pratt et al. (2005) indicated that additional repre-
sentatives from the Puerto Rican Myrtaceae and
closely related economically important flora must
be tested as possible hosts prior to introducing
the natural enemies.
In Jan 2006, however, the psyllid B. melaleu-
cae was observed on leaves of M. quinquenervia
trees growing near the San Juan Airport, Puerto
Rico. A survey of the island was conducted in Apr
2006 to determine the geographical distribution
of B. melaleucae on the island. This was accom-
plished by traveling E, W, and S on primary roads
while stopping every 10-20 km to search for







Florida Entomologist 89(4)


M. quinquenervia trees. Once encountered, trees
were examined by 3 observers for 15 min each to
detect psyllid presence and estimate feeding dam-
age and proportion of trees infested. Feeding
damage was assessed on a 5-point scale based on
a visual estimation of percentage of the suitable
foliage destroyed by psyllid feeding as follows: 0 =
no damage; 1 = <25% destroyed; 2 = 26 to 50%; 3
= 51 to 75%; 4 = 76 to 100% destroyed.
Identification of B. melaleucae was confirmed
by Susan Halbert (Florida Department of Agri-
culture and Consumer Services) and voucher
specimens were deposited in the Florida State
Collection of Arthropods (E2006-2142-201). Sur-
veys indicated that B. melaleucae was distributed
widely on the island, except for the west coast
where no psyllids were found on M. quinquen-
eruia trees near Aguadilla and Cabo Rojo (Fig. 1).
Damage was greatest (level 3) near the San Juan
Airport and Rio Piedras but decreased with in-
creasing distance from the greater San Juan area
(ANOVA df= 3, 14; F = 3.71; P = 0.0460). The pro-
portion of trees infested exhibited a similar trend,
with fewer trees per site harboring psyllids as the
distance from San Juan increased (linear regres-
sion df = 1, 14; F = 1.94; P = 0.0742).
The discovery of B. melaleucae in Puerto Rico
raises several questions regarding pathways of
introduction. First, where was the point of intro-
duction on the island? If we assume that in-
creased damage and infestation levels are posi-
tively correlated with time, then B. melaleucae
was likely established in the greater San Juan
area prior to other locations. The subsequent dis-
persal and its current distribution underscores
the long range host-finding abilities ofB. melaleu-
cae under highly fragmented populations of its
host. The psyllid had successfully located isolated
M. quinquenervia trees, for instance, <30 m from
the ocean (Arecibo) as well as within canopies of 3
trees growing at 800 m elevation.


Florida, as compared to Australia, is the most
logical origin of the Puerto Rican psyllid popula-
tion based on proximity and frequency of trans-
portation. On-going genetic analyses may help
elucidate the country of origin for the Puerto
Rican populations. Considering the widespread
occurrence of M. quinquenervia among the Carib-
bean islands, one introduction pathway may in-
clude unassisted inter-island dispersal from Flor-
ida, through the Bahamas or Greater Antilles to
Puerto Rico. Hurricanes may facilitate the long
range dispersal of insects through the Caribbean
(Drake & Farrow 1988). This line of reasoning,
however, is not supported by recent surveys of
M. quinqueneruia in the northern Bahamian is-
lands (Grand Bahama, New Providence, and An-
dros) where M. quinqueneruia is abundant but
where B. melaleucae was not detected despite a
recent hurricane (Hurricane Wilma, Oct. 2005)
that crossed South Florida prior to making land-
fall on Grand Bahama (Pratt unpublished data).
San Juan lies approximately 1660 km southeast
of Miami whereas New Providence is about 300
km east and Grand Bahama is only about 130 km
northeast. Thus, if the psyllid were dispersing on
its own or through the agency of hurricanes, it
should reach the more proximate Bahama Is-
lands first. A more probable explanation is that
B. melaleucae was introduced, either accidentally
or intentionally, to Puerto Rico. Human activities
play an important role in accidental insect inva-
sions, with the most common introduction path-
ways including international transportation of
airplane luggage and cargo (Kiritani & Yama-
mura 2003). Considering the frequent transport
of tourists and cargo between South Florida and
Puerto Rico, the premise that B. melaleuca was
inadvertently carried or "hitchhiked" to the is-
land remains a plausible explanation. Of greater
concern, however, is the possibility that the
B. melaleucae may have been intentionally smug-


Fig. 1. Geographical distribution of B. melaleucae in Puerto Rico.


December 2006




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