Title: Florida Entomologist
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Permanent Link: http://ufdc.ufl.edu/UF00098813/00009
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Title: Florida Entomologist
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Creator: Florida Entomological Society
Publisher: Florida Entomological Society
Place of Publication: Winter Haven, Fla.
Publication Date: 2007
Copyright Date: 1917
Subject: Florida Entomological Society
Entomology -- Periodicals
Insects -- Florida
Insects -- Florida -- Periodicals
Insects -- Periodicals
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Meagher et al.: Caterpillar Feeding on Pasture Grasses


'Center for Medical, Agricultural and Veterinary Entomology, Agricultural Research Service
U.S. Department of Agriculture, 1700 SW 23rd Drive, Gainesville, FL 32608

2University of Florida, Range Cattle Research and Education Center, Ona, FL

Stargrasses (Cynodon nlemfuensis Vanderyst var. nlemfuensis) and bermudagrasses (C. dac-
tylon (L.) Persoon) are important warm-season forage grasses, with several cultivars devel-
oped for conditions found in central and southern Florida. Major insect pests of these grasses
include grass loopers (Mocis spp.) and fall armyworm (Spodoptera frugiperda (J. E. Smith)),
which annually may impose economic losses for beef cattle and hay producers. Population
studies conducted during a 3-year period showed that both species had similar profiles with
respect to larval population seasonality but not abundance. Plot studies with 4 stargrass
and 4 bermudagrass lines showed that higher grass looper populations were found in star-
grasses than bermudagrasses. Laboratory studies found grass loopers and fall armyworm
larvae generally developed faster with larger weights on lines of stargrass than lines of ber-
mudagrass. The two fall armyworm host strains also can differ substantially in their larval
weight, developmental time, and survivability when grown on different lines of grasses.
These results indicate that the selection of pasture grasses made by growers can signifi-
cantly and differentially affect the population densities of these grass defoliators.

Key Words: Mocis latipes, Mocis disseverans, Mocis marcida, Spodoptera frugiperda, larval

Los pastos, Cynodon nlemfuensis Vanderyst var. nlemfuensis) y C. dactylon (L.) Persoon, son
pastos importantes para las estaciones calidas, con varias variedades desarrolladas para las
condiciones encontradas en el centro y sur de la Florida. Las plagas insectiles mayores de es-
tos pastos incluyen: gusanos medidores de pastos (Mocis spp.) y el cogollero, Spodoptera fru-
giperda (J. E. Smith), las cuales puedan imponer perdidas econ6micas para los ganaderos y
productores de pastos de corte. Estudios de poblaci6n realizados durante un period de tres
aios mostraron que ambas species tuvieron perfiles similares con respect al period de es-
taci6n en que se encontraban las poblaciones de larvas pero no de su abundancia. Estudios
de parcelas con 4 lineas de C. nlemfuensis y 4 lineas de C. dactylon mostraron que se encuen-
tran poblaciones mas altas del gusano medidor de past en C. nlemfuensis que en C. dacty-
lon. Estudios del laboratorio mostraron que las larvas del medidor y cogollero generalmente
se desarrollaron mas rdpidamente con peso mayores en las lines de C. nlemfuensis que en
las lines de C. dactylon. Las dos variedades de hospederos para el cogollero pueden variar
substancialmente en el peso de la larva, el tiempo de desarrollo, y su capacidad para sobre-
vivir cuando estan criados sobre diferentes lines de pastos. Estos resultados indican que la
selecci6n de pastos para pasturas hecha por los agricultores puede afectar significativa-
mente y diferentialmente la densidad de estos defoliadores de pastos.

Several Cynodon species are used in the south-
eastern United States as the base forage by beef
and dairy producers. These grasses yield more
than bahiagrasses (Paspalum notatum Flugge)
during short daylength periods (cool season), and
depending on temperature and soil fertility, can
produce considerable forage during Jan and Feb
(Mislevy & Martin 1997). Improved bermudagrass
(C. dactylon (L.) Persoon) and stargrass (C. nlem-
fuensis var. nlemfuensis) (Mislevy 2002) cultivars
have been developed and production practices op-
timized for beef cattle growers in central Florida
for many years, and new germplasm lines are con-

tinuously screened under grazing conditions (Mis-
levy et al. 1991; Mislevy et al. 1996).
Mocis spp. larvae or grass loopers are pests of
Cynodon forage grasses in the southeastern
United States (Watson 1933; Ogunwolu & Ha-
beck 1975; Koehler et al. 1977). Meagher & Mis-
levy (2005) found three Mocis species (dissever-
ans (Walker), latipes (Guenee) (striped grass
looper), and marcida (Guenee)) in central Florida
when developing attractants for adults. Mocis
spp. also are important pests of both pasture and
cultivated grasses in Central America, South
America, and the Caribbean (Gibbs 1990; Portillo

Florida Entomologist 90(2)

et al. 1991; Cave 1992). Determination of life his-
tory, biology, and geographic information for
Mocis spp. has been hampered by misuse of scien-
tific names in the literature and misidentification
in the field (Dean 1985; Gregory et al. 1988).
Another lepidopteran pest of pasture grasses
is the fall armyworm, Spodoptera frugiperda
(J. E. Smith). Differential susceptibility to fall ar-
myworm of grasses grown for hay production and
grazing has been shown in various trials con-
ducted with bermudagrass lines developed in
Georgia, Louisiana, and Oklahoma (Leuck et al.
1968; Lynch et al. 1983; Lynch et al. 1986; Jam-
janya et al. 1990). None of the grasses tested in
the earlier studies is used in central and southern
Florida, but the parents of 'Tifton 85' (an F, hy-
brid pentaploid between the bermudagrass PI
290884 (in the literature as 'Tifton 292') from
South Africa and the stargrass (C. nlemfuensis
Vanderyst) 'Tifton 68', a highly digestible but
cold-susceptible hybrid released in 1983 (Burton
et al. 1993)) have been compared for resistance.
'Tifton 68' was shown to be susceptible, with high
larval weights and high larval survival in feeding
trials (Lynch et al. 1983). The other parent, 'Tif-
ton 292', was shown to be highly resistant to lar-
val feeding (Leuck et al. 1968; Lynch et al. 1983)
and larvae exhibited nonpreference resistance in
comparative tests (Chang et al. 1985).
Research in Louisiana, Georgia, and Florida
has shown that there are two host strains (corn
strain and rice strain) of fall armyworm (Pashley
1986; Lu et al. 1992; Lu et al. 1994; Levy et al.
2002; Meagher & Gallo-Meagher 2003). In Flor-
ida, corn plants are invaded by both host strains,
while forage and turf grasses are infested pre-
dominately by rice strain larvae (Meagher &
Gallo-Meagher 2003; Nagoshi et al. 2006a; Na-
goshi et al. 2006b). Pashley et al. (1987) compared
feeding of'Tifton 292' by larvae from a rice strain
and a corn strain culture and found that the grass
was resistant to corn strain individuals but sus-
ceptible to rice strain larvae. Further testing clas-
sified 'Tifton 292' as intermediately resistant
when fed to rice strain larvae (Jamjanya &
Quisenberry 1988), but other factors such as arti-
ficial diet (Quisenberry & Whitford 1988) and
whether the plants were grown in the field or in
the greenhouse affected larval response (Jam-
janya et al. 1990; Pitman et al. 2002). Research
conducted to improve 'Tifton 292' by producing a
bermudagrass with both high quality and fall ar-
myworm resistance, led to the creation of 'Tifton
85' (Burton 2001). Although there have been
many published reports on agronomic and graz-
ing attributes of'Tifton 85', there are no reports
comparing fall armyworm feeding on this grass
with other forage grasses.
Field sampling and larval feeding studies with
Mocis spp. or fall armyworm have not been con-
ducted on the grasses grown and developed in

Florida. We conducted studies to determine the
population densities of Mocis spp. and fall army-
worm supported by different grass lines in the
subtropical environment of central Florida. The
results of these field surveys were compared to
laboratory studies examining the capacity of the
different grasses to support larval development of
these species. The grass lines were selected based
on their popularity with growers or on field obser-
vations that certain lines were highly susceptible
to feeding by caterpillars.


Field Site and Population Density

Field experiments were conducted at the Uni-
versity of Florida, Range Cattle Research and Ed-
ucation Center (RCREC), Ona (2726'N, 8155'W;
26 m elevation). This subtropical center contains
over 1150 hectares of natural and improved
grasses divided into large pastures and small
plots for multi-discipline research in beef cattle
and forage grass production.
Sampling of Mocis spp. and fall armyworm lar-
val populations was done with sweep nets and
was conducted in various bermudagrass and star-
grass pastures at the RCREC during 2001, 2002,
and 2003.

Grass Lines

This study was designed to compare popula-
tions of Mocis spp. and fall armyworm larvae on
various Cynodon spp. Mocis spp. larvae can be
found in large numbers but separation of larvae
by species is difficult. Ogunwolu & Habeck (1979)
separated latipes/disseverans from marcida/tex-
ana using the shape and length of the anal setae,
but no characters were found to separate individ-
ual pairs of species. Therefore, Mocis larvae were
not identified to species.
The grass lines used in this study are impor-
tant to growers who raise beef cattle in central
Florida. They include cultivars and ecotypes de-
veloped at the RCREC, cultivars developed in
other locations but are popular with beef cattle
growers, or lines that are being considered for use
in central Florida. Grass lines (cultivars,
ecotypes, and ecotypes released as cultivars (Ka-
raca et al. 2002; Taliaferro et al. 2004)) included
the bermudagrasses 'Jiggs', a common bermuda-
grass selection found growing along the Texas
Gulf Coast (Redmon 2002), 'World Feeder', a mu-
tant of'Alicia' bermudagrass released by Agricul-
ture Enterprises, Inc. in Bethany, OK (Gordon
1989), 'Tifton 85', and a locally-derived ecotype
known as Bermudagrass 2000, a daylength-in-
sensitive bermudagrass found growing at the
RCREC during the cool season of 1999-2000 (PM,
unpublished data). The stargrasses were 'Flo-

June 2007

Meagher et al.: Caterpillar Feeding on Pasture Grasses

rona', found growing in a 'Pensacola' bahiagrass
pasture in Ona in 1973 (Mislevy et al. 1989; Mis-
levy et al. 1993), 'Okeechobee', a local stargrass
ecotype that was originally found growing with
'Callie' bermudagrass in Okeechobee Co., FL
(PM, unpublished data), and two locally-derived
ecotypes known as Stargrass 2000, a highly di-
gestible coarse grass found growing in Hemar-
thria altissima (Poiret) Stapf & C. E. Hubbard, at
Ona in 1999 and Ona Pasture #2 (believed to be a
natural hybrid developed from a seed from 'Ona'
stargrass hay fed to cattle in the middle of a bahi-
agrass pasture).
Grasses were planted beginning the week of 23
Jul 2001. The experiment was designed as a ran-
domized complete block with 3 blocks and 4 repli-
cations of the 8 grass entries arranged in plots (81
m2). Tilled ground separated plots (1 m) and
blocks (10 m) from each other. Lepidopteran lar-
vae can be located either at the ground surface or
spatially within the grass canopy (Dean 1985).
Therefore, larvae were sampled by either search-
ing a 0.2787 m2 area of grass (ground samples) or
by using a sweep net (38.1 cm diameter) (sweep
net samples, 30 sweeps per plot). Ground samples
and sweep samples were taken in the experimen-
tal plots on 16 Oct, 30 Oct, and 1 Nov, 2001. Anal-
ysis of variance of square root (x + 0.5)-trans-
formed data (PROC MIXED, Contrasts, Littell et
al. 1996) was used to examine variation among
grass plots.

Larval Feeding

This study was designed to compare larval
feeding on the different grass lines grown in the
field study (except Bermudagrass 2000). Striped
grass looper larvae (M. latipes) were colonized
from individuals collected, reared, and identified
from the RCREC in 2002. Larvae were reared on
greenhouse- and field-grown grasses in the labo-
ratory. Neonates were placed in plastic tubs, 35 (1)
x 24 (w) x 13 (h) cm, containing bermudagrass
('NuMex Sahara', Pennington Seeds, Madison,
GA). The tubs were lined with paper towels (Spar-
kleTM, Georgia-Pacific, Atlanta, GA) and the grass
was placed on top of a plastic grate (holes at 1.5
cm). After 1 week a metal screen (holes at 0.7 cm)
was placed on top of the grate. New grass ('Flo-
rona' stargrass, original material from the
RCREC) was placed under the screen while the
"old" grass was placed on top of the screen. In this
way, larvae feeding on the "old" grass could mi-
grate down to the "new" grass. The "old" grass was
removed the next day and the larval rearing pro-
cedure repeated. This technique slowed the devel-
opment of mold in the rearing tubs. Pupae were
harvested from the grass and paper toweling,
sexed, and 8 to 12 pairs of adults were placed in
screen cages that were 24 x 24 x 24 cm. Paper
towels were attached to 3 sides of the cage for ovi-

position and adults were supplied distilled water
and a 2% sugar-honey solution for nourishment.
Larvae and adults were reared in incubators or
large rearing units at =23C, 70% RH, and 14:10
Fall armyworm larvae were from the same cul-
tures described previously (Meagher et al. 2004).
Larvae shown to carry the mitochondrial marker
of corn strain (Tifton) were from a culture pro-
vided by Dr. James Carpenter, USDA-ARS, Tif-
ton, GA. This culture was maintained on a pinto
bean artificial diet according to the procedures of
Guy et al. (1985). Larvae shown to carry the mito-
chondrial marker of rice strain (Ona) were from a
culture of individuals collected from the RCREC
in Jul 2002 (Nagoshi & Meagher 2003), and were
maintained on bermudagrass and stargrass
grown in Gainesville.
Grass line plants (except Bermudagrass 2000)
were grown in 3.8-L pots in a greenhouse at am-
bient temperature, and were fertilized weekly
with Miracle-Gro 15-30-15 plant food. No pesti-
cides were applied to the plants. New leaf growth
was placed on filter paper discs (Whatman, 90
mm) moistened with =1 mL deionized water in a
9-cm diameter polystyrene petri dish (Thomas
Scientific, catalog #3488-B32). One neonate larva
was placed on plant foliage, and the petri dishes
were placed in an incubator at 23.9 + 2C with a
14:10 photoperiod. The filter paper in each petri
dish was moistened daily with = 1 mL of deionized
water for the first 10 d. Larvae were supplied
with fresh plant material until time of pupation.
Larval weights were measured at 10 d. Develop-
ment time (in d) from neonate to pupa was calcu-
lated and pupal weight was recorded at pupation.
For both M. latipes and fall armyworm, 15 lar-
vae were arranged in 3 replications on different
dates, and mortality on each host plant was re-
corded. Analysis of variance of loglO-transformed
data (PROC MIXED, Contrasts, Littell et al.
1996) was used to examine variation among grass


Population Density

Larval populations were variable both within
and across years. In 2001, populations were low
until early Sep, when Mocis spp. peaked at 90 and
fall armyworm peaked at 30 larvae per 30 sweeps
on 11 Sep. Larval populations of both species de-
clined to 19.6 and 2.2, respectively, in early Nov
(Fig. la). In 2002, the increase in larval popula-
tions of both species occurred about one month
earlier, with the highest number ofMocis spp. lar-
vae collected in mid-Aug and comparatively low
numbers found through early Nov (Fig. Ib). Pop-
ulations of fall armyworm were low with fewer
than 5 larvae per 30 sweeps collected in mid-Aug.

Florida Entomologist 90(2)

a Jt s -

12 2001

*. A2


1SO 17 200 2 25 0 275 300 325 350
Julin o.

12 2003 2

2 :: /* :
S. 17S 200 2 50 275 3 00 32 5
Julian ODa

stargrass pastures in 2001, 2002, and 2003, Ona, FL.

C. oo 1 2 117 20 175 2 0 225 2 0 275 m 325
Julbn 0D1

Fig 1. Populations overocis spp. and fall armyworm larvae ywoper 30
larvae sampled with sweep nets from beudagrass and
stargrass pastures in 2001, 2002, and 2003, Ona, FL.

Substantially different population dynamics was
observed in 2003. Momis spp. populations were
low throughout the sampling period, not reaching
more than 4 larvae per 30 sweeps (Fig. Ic). Fall
armyworm larval numbers were high in mid-Jun
(12 per 30 sweeps) and very high in early Oct,
with over 100 fall armyworm larvae per 30
sweeps collected.

Grass Lines

The effects of different grass germplasm on lar-
val populations were examined by sweep net and
ground sampling. Significant differences were

found among grasses in number of Mocis spp. lar-
vae collected by sweep net and ground samples
(Fig. 2). Stargrass plots ('Florona', 'Okeechobee',
Ona Pasture #2, and Stargrass 2000) contained
more larvae than bermudagrass plots (Bermuda-
grass 2000, 'Jiggs', 'Tifton 85', and 'World Feeder')
(sweep net samples (mean number of larvae per
30 sweeps SE), stargrass 25.5 2.3 vs. bermuda-
grass 13.7 + 1.7; F = 14.3, df = 1, 14, P = 0.0020;
ground samples (mean number of larvae per m2
SE), stargrass 75.6 4.6 vs. bermudagrass 55.6
3.8, F = 11.3, df= 1, 14, P = 0.0046).
Compared to Mocis spp., about a 10-fold lower
number of fall armyworm larvae was collected
and stargrass and bermudagrass plots showed
similar numbers of larvae. For sweep net sam-
ples, stargrass plots contained 2.1 0.55 larvae
per 30 sweeps compared to bermudagrass plots
which contained 2.4 0.63 (F = 0.07, df= 1, 14, P
= 0.8001). Ground sample stargrass plots had 4.3
+ 0.7 larvae per m2 vs. bermudagrass plots which
had 3.4 + 0.6 (F = 0.87, df = 1, 14, P = 0.3665). Se-
lected larvae were returned to the laboratory and
all were shown to carry the mitochondrial marker
for rice strain (Meagher & Gallo-Meagher 2003).

Larval Feeding

There was no difference in striped grass looper
larval weights among grass lines (F = 0.7, df = 6,
12, P = 0.6326), however there was a trend for lar-
vae fed stargrasses (31.9 mg 4.5) to be heavier
than those fed bermudagrasses (21.3 3.7; F =
3.1, df = 1, 12, P = 0.1069). Development time to
pupation differed among grass lines, and larvae
fed 'Florona' stargrass developed 4 days faster
than those fed 'World Feeder' bermudagrass (Ta-
ble 1). Overall, larvae fed stargrasses developed
2.4 days faster than those fed bermudagrasses.
Pupal weights were not different among lines (F =
1.9, df = 6, 12, P = 0.1687), however larvae fed
stargrasses (234.8 mg 7.6) produced larger pu-
pae than those fed bermudagrasses (206.2 + 7.0;F
= 6.8, df = 1, 12, P = 0.0228). There was no differ-
ence in neonate survival among lines (F = 1.3, df
= 6, 12, P = 0.3373) or between grass species (F =
2.1, df = 1, 12, P = 0.1777), as survival averaged
0.793 0.03.
Feeding by fall armyworm larvae provided dif-
ferences between insect cultures (host strains),
between grass species, and among grass lines.
Rice strain (Ona culture) larvae were heavier and
developed faster than corn strain (Tifton culture)
larvae (Table 2). Pupal weights and survival were
similar between host strains. However, there was
a significant insect culture x grass line interaction
with larval weight (F = 4.1, df = 6, 26, P = 0.0049),
therefore host strains were compared among each
grass line, and grass lines were compared within
both host strains. The insect culture x grass line
interactions for the other variables were not sig-

June 2007

Meagher et al.: Caterpillar Feeding on Pasture Grasses 2!



Ona Pasture #2

Stargrass 2K

Bermudagrass 2K


Tifton 85'

'World Feeder'



Ona Pasture #2

Stargrass 2K

Bermudagrass 2K


Tifton 85'

'World Feeder'

0 4 8 12 16 20 24 28 32 36 40
Number per 30 sweeps







I I I I l I I I I 1 I I

0 10 20 30 40 50 60 70 80 90 100 110 120
Number per m 2

Fig. 2. Number of Mocis spp. larvae collected in sweep net (a) and ground samples (b) from different Cynodon
spp. grasses, Ona, FL, 2001. Means ( SE) with the same letter are not significantly different (P > 0.05). The top 4
lines are stargrasses (C. nlemfuensis var. nlemfuensis); the bottom 4 lines are bermudagrasses (C. dactylon).


SI, abc

S lab


j bc


i i I I ic

Florida Entomologist 90(2)


Grass line Development time

'Florona' 21.6 0.3 a
'Okeechobee' 22.6 1.2 ab
Ona Pasture #2 23.8 0.7 abc
Stargrass 2000 22.4 0.1 ab
'Jiggs' 25.0 1.6 bc
'Tifton 85' 24.4 1.0 bc
'World Feeder' 25.6 0.5 c
F = 2.9; df= 6, 12; P = 0.0561
Stargrasses 22.6 0.38 A
Bermudagrasses 25.0 0.57 B
F = 13.0; df= 1, 12; P = 0.0036

nificant (development time, F = 2.5, df = 6, 26, P =
0.0521; pupal weight, F = 0.7, df = 6, 26, P =
0.6276; survival, F = 1.4, df = 6, 26, P = 0.2363).
Differences in larval feeding parameters were
found between stargrasses and bermudagrasses
when host strains were analyzed separately. Both
rice strain and corn strain larvae showed signifi-
cantly enhanced growth and development when
fed stargrass leaves (Table 3). Larval survival for
rice strain larvae was not different between grass
species, but there was a trend for corn strain lar-
vae to have higher survival on stargrasses.
Rice strain larvae fed Stargrass 2000 and 'Flo-
rona' were heavier and developed more quickly
than the other lines (Table 4). Pupal weights av-
eraged 138.9 mg 3.8 and did not differ among
lines (F = 1.8, df = 6, 12, P = 0.1859). Larval sur-
vival also did not differ among lines, averaging
0.607 0.04 (F = 2.4, df = 6, 12, P = 0.0896). Corn
strain larvae were heavier, developed faster, and
had larger pupal weights when fed Stargrass
2000 leaves (Table 5). Survival was not different
among lines (F = 2.4, df = 6, 12, P = 0.0896), aver-
aging 0.505 0.04. However, there was a trend for
corn strain larvae placed on 'Tifton 85' (0.333 +
0.067) and 'World Feeder' (0.361 0.02) to have
very low survival.


Surveys of bermudagrass and stargrass pas-
tures showed similar population profiles for Mocis
spp. and fall armyworm with respect to larval pop-
ulation dynamics. The peaks of larval abundance
occurred at different times in each of the 3 years
and were typically associated with sharp increases
and sudden declines in numbers. In each case the
timing of the changes in Mocis populations coin-
cided with that of fall armyworm larvae, suggest-
ing that these species were possibly responding to
the same environmental factors with respect to
oviposition and development on their plant hosts.
However, the level of infestation between species
was more variable. In the first 2 years, approxi-
mately 2-3 fold higher Mocis larval numbers were
observed than fall armyworm. This changed dra-
matically in 2003 when high fall armyworm larval
density coincided with low Mocis infestation. Lit-
tle is known about what factors influence the se-
verity of infestations in these species and why con-
ditions suitable for high fall armyworm density in
2003 were apparently less for Mocis.
Several lines of bermudagrasses and star-
grasses were tested for their ability to support
M. latipes and fall armyworm populations. This
was the first examination of M. latipes feeding of
Cynodon spp. germplasm developed or isolated in
Florida, and both bermudagrasses and star-
grasses are important forage grasses for the beef
cattle industry in the central and southern parts
of the state. Bermudagrasses as a group were
generally associated with lower densities ofMocis
larvae than stargrasses when tested in field set-
tings. This observation compared well with the
results of laboratory feeding studies showing that
M. latipes larvae were smaller and developed
slower on bermudagrasses. M. latipes larvae took
approximately 2.4 d longer to develop to pupa-
tion, which prolongs the period of larval exposure
to natural enemies and disease.
Fall armyworm showed a similar preference
for a subset of stargrasses over bermudagrasses.
Larvae reared on Stargrass 2000 or 'Florona'
were heavier and developed 2-4 d quicker than
those reared on bermudagrasses. However, this
difference was not reflected in changes in popula-
tion densities in the field. It may be that localized
variations between plant hosts in their ability to


Variable Rice strain Corn strain F-value df P-value

Larval weight (mg) 28.5 2.9 23.9 3.7 12.20 1, 26 0.0018
Development (days) 20.6 0.4 23.2 0.6 47.20 1, 26 <0.0001
Pupal weight (mg) 138.9 3.8 140.8 3.2 0.23 1, 26 0.6335
Survival (prop.) 0.607 0.04 0.505 0.04 3.60 1, 26 0.0706

June 2007

Meagher et al.: Caterpillar Feeding on Pasture Grasses


Variable Stargrass Bermudagrass F-value df P-value

Rice strain
Larval weight (mg) 33.4 4.5 22.0 1.9 25.70 1, 12 0.0003
Development (d) 19.9 0.5 21.7 0.4 20.80 1, 12 0.0006
Pupal weight (mg) 143.8 4.7 132.5 5.8 4.30 1, 12 0.0594
Survival (prop.) 0.600 0.064 0.615 0.044 0.05 1, 12 0.8194
Corn strain
Larval weight (mg) 32.9 5.0 11.9 1.9 69.10 1, 12 <0.0001
Development (d) 21.3 0.5 25.9 0.5 64.30 1, 12 <0.0001
Pupal weight (mg) 149.5 2.4 129.2 4.3 17.00 1, 12 0.0014
Survival (prop.) 0.568 0.053 0.42 0.069 4.60 1, 12 0.0541

support fall armyworm are substantially masked
by the mobility of this species, requiring larger
plot size and/or sample size to detect statistical
differences between grass species.
Fall armyworm strain-specific differences
were observed for larvae reared on the various
grass lines. In most cases, rice strain larvae were
heavier, had better survival, and developed faster

than corn strain larvae. Previous studies showed
that rice strain also produced larger larvae, faster
development times, and higher survival even
when grown on plant hosts favored by the corn
strain (Meagher et al. 2004). This suggests that
the observed differences reflected general charac-
teristics of the strains rather than specific re-
sponses to the plant hosts tested.


Grass line Larval weight Development time

'Florona' 38.5 4.6 b 18.9 0.5 a
'Okeechobee' 22.7 2.9 cd 21.5 0.5 b
Ona Pasture #2 18.7 4.3 d 21.1 + 0.6 b
Stargrass 2000 53.4 4.6 a 18.0 0.4 a
'Jiggs' 20.8 1.7 d 22.0 1.0 b
'Tifton 85' 27.7 3.6 c 20.7 0.5 b
'World Feeder' 17.4 0.7 d 22.3 0.2 b
F = 22.7, df= 6, 12, P < 0.0001 F = 9.8, df= 6, 12, P = 0.0005

FERENT (P > 0.05).

Grass line Larval weight Development time Pupal Weight

'Florona' 24.7 8.1 b 21.3 0.2 ab 146.5 5.3 a
'Okeechobee' 28.8 8.5 b 22.8 1.2 b 151.7 3.8 a
Ona Pasture #2 29.7 8.5 b 21.3 0.4 ab 148.8 4.4 a
Stargrass 2000 48.2 13.5 a 19.7 0.9 a 151.1+ 7.3 a
'Jiggs' 13.6 5.5 c 26.1 1.1 c 137.5 2.3 ab
'Tifton 85' 12.0 2.6 c 25.7 0.8 c 124.6 4.9 b
'World Feeder' 10.0 2.1 c 25.8 0.7 c 125.4 11.7 b
F= 13.6, df= 6, 12, P < 0.0001 F= 12.5, df= 6, 12, P = 0.0002 F = 3.4, df= 6, 12, P = 0.0346

Florida Entomologist 90(2)


We thank C. Dillard, C. Stuhl, and N. Novello
(USDA-ARS, Gainesville) and T. J. Mitchell and C. Neu-
hofer for technical support. We thank K. Flanders (Au-
burn University), J. H. Frank (University of Florida),
and S. Reitz (USDA-ARS-CMAVE, Gainesville, FL) for
review of an earlier manuscript. Voucher specimens
were placed in the USDA-ARS-CMAVE Behavior and
Biocontrol Entomology Collection, Gainesville, FL.
The use of trade, firm, or corporation names in this
publication is for the information and convenience of
the reader. Such use does not constitute an official en-
dorsement or approval by the United States Depart-
ment of Agriculture or the Agricultural Research
Service of any product or service to the exclusion of oth-
ers that may be suitable.


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Florida Entomologist 90(2)

June 2007


'Center for Medical, Agricultural and Veterinary Entomology, USDA-ARS, Gainesville, FL 32608

2Subtropical Horticulture Research Station, USDA-ARS, 13601 Old Cutler Rd., Miami, FL 33158

3Everglades Research and Education Center, University of Florida/IFAS
3200 E. Palm Beach Road, Belle Glade, FL 33430


Research was initiated to develop a pheromone-based monitoring system for the tropical sod
webworm, Herpetogramma phaeopteralis (Guen6e). A laboratory rearing procedure was de-
veloped to produce individuals for field tests and behavioral bioassays. Virgin females placed
in Unitraps in the field attracted and captured males for 8 d, while no males were captured
in unbaited traps. Total male capture ranged from 1 to 24, and there was a slight decrease
in capture as females aged. Laboratory mating behavior studies suggested that mating oc-
curs later in the scotophase. Males responded to virgin females in a linear olfactometer
throughout the dark period (scotophase), although there was a trend for higher male activity
late in scotophase. There was no observed calling behavior, and adults exhibit simple mating
behavior. Lack of both calling posture among virgin females and periodicity of male response
will make it difficult to determine the optimal time periods for pheromone production, which
would facilitate the collection and subsequent identification of pheromone components.

Key Words: Herpetogramma phaeopteralis, female calling, male attraction, turf


Se inicio una investigaci6n para desarrollar un sistema de monitoreo en base de feromonas
contra el gusano tropical de c6sped Herpetogramma phaeopteralis (Guen6e). Se desarroll6
un procedimiento de cria en el laboratorio para producer individuos para pruebas del campo
y de bioessayos de comportamiento. Hembras virgenes puestas en "Unitraps" en el campo
atrayeron y capturaron machos por 8 dias, mientras que ningun macho fue capturado en
trampas sin cebo. El numero total de machos capturados fue de 1 a 24, y hubo una menor dis-
minuci6n en el numero de machos capturados cuando las hembras envejecieron. Los estu-
dios de comportamiento del apareamiento en el laboratorio sugerieron que el apareamiento
ocurre mas tarde en la sc6tofase. Los machos respondieron a las hembras virgenes en un ol-
factometro linear a trav6s del period oscuro (sc6tofase), aunque hubo una tendencia para
un aumento en la actividad del macho en la parte final de la sc6tofase. No fue observado el
comportamiento de llamar, y los adults exhibieron un comportamiento del apareamiento
sencillo. La falta del la postura de llamar entire las hembras virgenes y la periodicidad de la
respuesta de los machos hace dificil el poder determinar el period del tiempo optimo para
la producci6n de feromonas, con la cual facilitaria la identificaci6n y recolecci6n de los com-
ponentes de la feromona.

Tropical sod webworm (TSW), Herpeto-
gramma phaeopteralis (Guenee) (Lepidoptera:
Crambidae), is a pest of Florida turfgrasses. Spe-
cies within Herpetogramma attack grasses in the
continental United States, Australia, Hawaii, and
Guam, and H. licarsisalis (Walker) is the most se-
rious turfgrass pest in Hawaii (Davis 1969; Mur-
doch & Tashiro 1976; Tashiro 1976). TSW also has
a wide tropical distribution and occurs through-
out the Caribbean (Wolcott 1936). In the U.S.,
TSW occurs across the Gulf Coast from Texas to
Florida (Kerr 1955).
TSW feeds on a variety of grasses including
bermudagrass Cynodon dactylon (L.) Persoon,

centipedegrass Eremochloa ophiuroides (Munro)
Hackel, seashore paspalum Paspalum vaginitium
Swartz, St. Auginegrass Stenotaphrum secunda-
tum (Walter) Kuntze, and zoysiagrass Zoysia
japonica Steudel (Kerr 1955; Reinert 1983; Korn-
dorfer et al. 2004). TSW populations are managed
by using primarily chemical and cultural controls
(Reinert 1973, 1974, 1976, 1983; Buss & Meagher
2005), although resistant cultivars have been re-
ported (Reinert & Busey 1983).
Few studies have been conducted on the gen-
eral biology of TSW (Kerr 1955; Reinert & Busey
1983). More recently, Cherry & Wilson (2005) de-
termined that more adults of both sexes rested in

Meagher et al.: Tropical Sod Webworm Mating Behavior

unmowed grass versus mowed grass, and that
when disturbed, adults flew only a short distance.
Seasonally, more TSW adults were attracted to
light traps in the fall (Sep-Nov) than summer,
spring, or winter in southern Florida. TSW larvae
are sampled by locating damaged turf and con-
ducting soap flushes (Buss & Meagher 2005).
Adult monitoring is accomplished by either sweep
nets or light traps (Cherry & Wilson 2005), both of
which have limitations for turfgrass consultants
or homeowners. Studies were initiated in 2004 to
develop a pheromone-based monitoring system
for this pest. Pheromone monitoring could allow
turfgrass managers to predict future damaging
populations and prescribe insecticide applica-
tions based on action levels. Field studies were
conducted to confirm production of female-pro-
duced volatile chemicals that could be used for at-
traction of male moths. Laboratory studies were
conducted to evaluate the mating behavior of
TSW and periodicity of pheromone release. This
study provides information on the biology of this
important pest, which will be needed for identifi-
cation and formulation of a synthetic pheromone
lure for field use.


TSW Colonies

TSW were colonized at 2 locations from larvae
collected from the Everglades Research and Edu-
cation Center (EREC), Belle Glade, FL. At the
USDA-ARS Center for Medical, Agricultural and
Veterinary Entomology (CMAVE), Gainesville,
FL, adults were placed in screen cages (24 x 24 x
24 cm) and supplied with distilled water and 2%
sugar-honey solution for nourishment. After 2 d, a
237-mL plastic cup containing greenhouse-grown
bermudagrass ('NuMex Sahara', Pennington
Seeds, Madison, GA) was placed in a cage for ovi-
position. Cups were left for 2 d, removed, and re-
placed with new cups of bermudagrass. This ovi-
position cycle was repeated until adults died.
Cups containing grass with TSW eggs were
placed on top of a plastic grate within plastic tubs,
35 (1) x 24 (w) x 13 (h) cm, lined with paper towels
(SparkleTM, Georgia-Pacific, Atlanta, GA). When
egg hatch was complete, the grass in the cups was
cut and the soil and cup were removed from the
tub. Bermudagrass was added daily for 7 d. After
7 d field-grown 'Florona' stargrass (Cynodon
nlemfuensis Vanderyst var. nlemfuensis) was
added by placing it on a metal screen that was
placed on top of the grate. Each day new grass
was placed under the screen while the old grass
was placed on top of the screen. In this way, lar-
vae feeding on the old grass could move down to
the new grass. The old grass was removed the
next day. This technique slowed mold develop-
ment in the rearing tubs. Pupae were harvested

from the grass and paper toweling, and the rear-
ing procedure repeated. Larvae and adults were
reared in incubators or large rearing units at
=26C, 70% RH, and 14:10 (L:D) photoperiod.
Rearing procedures were similar at EREC except
that larvae were reared on St. Auginegrass and
adults were fed 0.25 M sucrose solution.

Field Study

The attraction of male moths to females was
tested with Standard Universal Moth Traps,
'Unitraps' (Great Lakes IPM, Vestaburg, MI)
baited with virgin females obtained from the
EREC colonies. Unitraps are comprised of a green
top with a 2.0-cm hole, yellow funnel, and white
collecting bucket. The trap is designed to have the
attractant placed within an insert (1.8 cm W x 5.0
cm L) that is put in the hole in the top. Moths that
are attracted to the lure become excited and fall
through the funnel into the collecting bucket. For
our experiments, the traps were modified by plac-
ing fine-meshed window screen around the insert
and attaching a small vial (2 dram, 1.5 cm W x 5.5
cm L) with a cotton dental wick to the bottom of
the insert. A virgin female (<24 h old) was placed
in the insert and had access to the vial, which con-
tained 0.25 M sucrose solution.
The experiment was conducted at the EREC
during Jul and Aug 2004. Wild TSW adults were
observed at the EREC during this time and were
present for testing pheromone attraction in the
field. Pairs of baited and unbaited traps (10 m
apart) were attached to metal poles (1.5 m) placed
in mowed grass of different species. Traps were
placed in the shade to avoid exposure to the sun.
Two to 5 pairs of traps were deployed per week 9
Jul through 25 Aug. Each trap pair was >10 m
apart and there were 11 trap pairs. Survival of
the females and number of males captured was
observed daily until females died. Baited trap
capture numbers were compared against number
of males captured in unbaited traps (Paired t-test,
SigmaStat, Systat Software, Richmond, CA). Ad-
ditionally, the length of time that females contin-
ued to attract males was tested 2 to 3 d, 4 to 6 d,
or 7 to 8 d later (One-way analysis of variance,
ANOVA, SigmaStat).

Laboratory Mating Behavior

Pupae from CMAVE were shipped to the
USDA-ARS Subtropical Horticulture Research
Station, Miami, FL (SHRS) for laboratory tests.
Newly emerged adults were collected each day
and maintained in single-sex cages in separate
holding rooms at 25C and 70% RH until time of
testing. The holding rooms had windows to pro-
vide natural lighting and were supplemented
with room lights set to a photoperiod of 12:12
(L:D) h, with lights off at 2000 h and lights on at

Florida Entomologist 90(2)

0800 h. Adults were provided with water and a
sucrose solution.
Tests were conducted to document aspects of
mating behavior under laboratory conditions. All
tests were conducted in rooms with windows so
that natural light was available and room lights
were set to same photoperiod so that bioassay
rooms had the same light conditions as the adult
holding rooms. Observations on adult mating be-
havior (e.g., female "calling" or pheromone release
posture, periodicity of calling, and time of mating)
were made by filming adults under an infra-red
light with a low light CCTV camera (BP330, Pa-
nasonic Corp., Secaucus, NJ). Moths were placed
in clear plastic 140-mL vials (8.6 cm length x 4.8
cm ID) with removable snap-top lids (Thornton
Plastics, Salt Lake City, UT). A piece of aluminum
window screen (4 cm diam) was attached to the
bottom of the clear vial with hot glue, and a piece
of filter paper (Whatman #1, 7.62 cm x 7.62 cm)
was placed along the back wall of the vial to pro-
vide foot-holds for the moths. The vial was in-
verted with the snap-lid becoming the floor and a
moistened piece of cotton wick (~2 cm long) was
added to provide water to the adults. Moths were
placed in the vials prior to the start of the dark
period (scotophase), video output was recorded on
a VCR throughout scotophase, and the tapes were
reviewed for observations of mating behavior. All
moths were dissected at the end of the observa-
tion period to confirm sex, mating status, and
presence of mature eggs in females. Initial stud-
ies evaluated behavior of 10 sets of male and fe-
male adults that were 0-5 d old at time of testing.
Linear olfactometers (Analytical Research
Systems, Inc., Gainesville, FL) were used to eval-
uate female behavior during time periods of male
response. Individual virgin females were placed
in small glass chambers (~12.7 cm x 2.2 cm ID)
with a downwind screen that was attached to a
glass tube (35.5 cm total length x 2.54 cm ID). A
moistened piece of cotton wick (~1.5 cm long) was
added to provide water to the females and to add
humidity to the air. Purified air was delivered to
the chamber containing the female and then into
the olfactometer through connectors made from
Teflon tubing. A single male was released at the
downwind end of the tube prior to the start of
scotophase, and there were 4 linear olfactometers
used per test. Tests were conducted on 5 different
nights, for a total of 20 samples. Activity of fe-
males in the small glass chambers and of males in
~5 cm of the upwind ends of the glass tubes (re-
sponse window) during 0000-0800 h (ET) was re-
corded by video capture on a VCR. Video tapes
were reviewed, entrance and exit times for males
into the response windows were recorded, and the
difference in entrance and exit time was used to
determine number of min that males spent per in-
dividual visit. Min per visit were summed for
each h to quantify sum total time per h per male.

Sum total time per h per male was used as the
response variable and effect of time period was
analyzed by one-way ANOVA in Proc GLM (SAS
Institute 2001). Tapes also were reviewed to eval-
uate posture and activity of the females during
time periods of male response as indications of
female calling behavior.


Field Study

Females survived in the traps for up to 8 d. Sig-
nificantly more males were collected in virgin fe-
male-baited traps than in traps with no females
(baited traps, mean + SEM = 9.3 2.5 males per
female; unbaited, 0.0 + 0, t = 3.76 with 10 df, P =
0.004). Total male capture per trap ranged from 1
to 24, with a total of 102 males collected. Males
were collected in baited traps up to 8 d after fe-
males were placed in traps. Females aged 2-3 d
attracted 5.7 1.9 males, those aged 4-6 d at-
tracted 3.6 1.5 males, and those aged 7-8 d at-
tracted 3.3 1.7 males to the traps. Although
there was a decline in the number of males cap-
tured as females aged, this difference was not sig-
nificant (F = 0.6; df= 2, 21; P = 0.555).

Laboratory Mating Behavior

Mating occurred in only 3 of the 10 pairs video-
taped. Of these, mating occurred at 0218, 0234,
and 0310 h, which was 3-4 h before sunrise. Two of
these 3 pairs completed mating during the video-
taped time period and they remained paired for 78
and 98 min. Eight of the 10 females videotaped
were sexually mature, as indicated by presence of
mature eggs, and all 3 females that mated were
sexually mature. There was no obvious calling pos-
ture observed among females that either did or did
not eventually mate, and there were no obvious
differences in behaviors of successful versus un-
successful males. TSW used a simple courtship
pattern, with behavioral steps most similar to
those described for Amyelois transitella (Walker)
and Laetilia coccidivora (Comstock) (Phelan &
Baker 1990), although females for both of those
species displayed calling behavior. TSW females
that eventually mated tended to be positioned
close to the bottom of the vial, remained stationary
or moved a short distance away when approached
by the male but then remained stationary until
copulation was successful. As described forA. tran-
sitella and L. coccidivora, the male approached the
female from behind, with rapid wing-fanning and
walking. The male faced the same direction as the
female and attempted copulation with a ventrolat-
eral thrust. If the initial attempt was unsuccessful
and the female moved away, the male would follow
and make additional attempts. After a successful
copulation attempt, the pair moved to a tail-to-tail

June 2007

Meagher et al.: Tropical Sod Webworm Mating Behavior

position and remained stationary for the duration
of the copulation. This behavioral sequence was
observed for all 3 successful matings.
Sixteen of the 20 males tested in the linear ol-
factometer were observed in the upwind end of
the glass tube of the linear olfactometer at some
time during the sample period. Total time spent
in the upwind end of the glass tube over the 8-h
test period ranged from 7.4 min to 368 min, with
the overall average ( SEM) of 125.2 27.0 min.
The highest sum total number of min per h that
males were observed in the upwind end of the
glass tube occurred from 0600-0700 h (Fig. 1), but
males were observed during all time periods and
there were no significant differences among time
periods (F = 1.15; df = 7, 120; P = 0.3352; square-
root x + 0.5 transformed data). Females were ac-
tive periodically throughout scotophase and
again there were no obvious calling postures ob-
served throughout scotophase. Females tended to
be quiescent during the time periods of greatest
male response, but quiescence and male response
were not always concurrent.


Sex attractants have been identified for other
crambid (Crambini) sod webworms including the
bluegrass webworm, Parapediasia teterrella

10 -


0000 0100 0200 0300 0400 0500 0600 0700
Time of day (hours)

Fig. 1. Number of min (mean SE) that virgin males
appeared in response to volatile chemicals from virgin fe-
male tropical sod webworms (n = 16). Number of min per
h was determined from videotape recordings of male activ-
ity in linear olfactometers 4 h after the start of scotophase.

(Zincken) (Clark & Haynes 1990), the cranberry
girdler, C'i ..r....... ..i., topiaria (Zeller) (Kamm &
McDonough 1979; McDonough & Kamm 1979;
Kamm & McDonough 1980), and the western
lawn moth, Tehama bonifatella Hulst (McDon-
ough et al. 1982). Pheromones also have been
identified for several moth species in the same
tribe (Spilomelini) as TSW, including Cnapha-
locrocis medinalis Guenee (Ramachandran et al.
1990; Ganeswara Rao et al. 1995; Kawazu et al.
2000) and 3 species of Diaphania. One chemical,
(E)-11-hexadecenal, was a major component in
the pheromone blends of D. indica (Saunders)
(Wakamura et al. 1998), melonworm D. hyalinata
(L.) (Raina et al. 1986), and pickleworm D. niti-
dalis (Stoll) (Klun et al. 1986).
Our linear olfactometer results suggested that
both males and females are active throughout sc-
otophase, with a trend for higher male response 4
to 5 h after the onset of scotophase and at the end
of scotophase. Research with other crambids/
pyralids has shown variable results relating call-
ing behavior, pheromone production, and mating.
In some species, the relationship between female
calling and pheromone production was weak,
where pheromones appeared to be produced with-
out apparent calling behaviors (Coffelt et al.
1978; Kawazu & Tatsuki 2002). In other species,
however, females initiated calling, males re-
sponded and mating occurred within a relatively
short time period either in early scotophase (El-
sey 1982; Valles et al. 1992) or in late scotophase
(Hight et al. 2003).
In summary, these results show that female
moths release pheromone that is attractive to
males and that Unitraps are a suitable trapping
system for this species under field conditions. Our
field and laboratory studies confirm that female
tropical sod webworms use a sex pheromone for
chemical communication and if available, a syn-
thetic pheromone lure could be used for trapping
males. However, preliminary tests have found that
TSW females release very small amounts of pher-
omone (P.E.A. Teal, personal communication). The
lack of calling posture among virgin females and
flexibility in the periodicity in the time period of
male response to volatile chemicals make it diffi-
cult to determine if there is a specific calling period
for optimal chemical collection, which would facil-
itate pheromone chemical identification.


We thank C. Dillard and B. Dueben (USDA-ARS,
Gainesville), A. Wilson (Univ. of Florida, EREC, Belle
Glade), P. Anderson and N. Theresias (USDA-ARS
SHRS, Miami) for technical support. We thank P.E.A.
Teal (USDA-ARS, Gainesville) and E. Buss (Univ. of
Florida) for review of an earlier manuscript.
The use of trade, firm, or corporation names in this
publication is for the information and convenience of
the reader. Such use does not constitute an official en-

Florida Entomologist 90(2)

dorsement or approval by the United States Depart-
ment of Agriculture or the Agricultural Research
Service of any product or service to the exclusion of oth-
ers that may be suitable.


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Y. YOSHIYASU, AND S. TATSUKI. 2000. Geographical
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mone of the cranberry girdler, Chrysoteuchia topi-
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tant for the western lawn moth, Tehama bonifatella
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study of courtship in twelve Phycitine moths (Lepi-
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1990. Pheromone components of rice leaffolders (LF)
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June 2007

de Freitas et al.: Biology of Diatraea flavipennella


'Laborat6rio de Ecologia Quimica, Instituto de Quimica e Biotecnologia, Universidade Federal de Alagoas
Campus A.C. Simoes, 57072-970, Macei6, AL, Brazil

2Assist6ncia Fitossanitaria e Controle Biol6gico Ltda-FITOSSAN-Fazenda Jequid
BR 101 Sul km 155. Jequid da Praia, AL, Brazil

Aspects of the biology of the sugarcane pest Diatraea flavipennella (Box 1931) (Lepidoptera:
Crambidae), locally named broca-pequena da cana-de-acucar, reared and maintained under
laboratory conditions and fed on an artificial diet have been investigated. The larval stage,
which involved 7 instars, continued for a mean period of 34.87 d. Each instar could be char-
acterized by the size of the cephalic capsule, which increased 1.28-fold on average between
instars. The mean duration of the pupal stage was 12.75 d. The pupae exhibited sexual di-
morphism in that the females were larger than the males, while the latter exhibited a gen-
ital pore that was absent in the females. In adult insects, the female/male ratio was 1:1.3.
Adult females were on average 28.73 mm in size while the mean value for adult males was
only 20.80 mm. Females commenced oviposition on the first d of their adult life and were
able to oviposit until d 6. On average each female produced 431.05 eggs during her lifetime,
although the majority of eggs were deposited during the first 2 d after emergence.

Key Words: artificial diet, sugarcane borer, biological aspects, lepidoptera, Diatraea flavipen-
nella, Crambidae

Aspectos da biologia da broca-pequena da cana-de-acgcar, Diatraea flavipennella (Lepidop-
tera: Crambidae), foram investigados em condigoes de laborat6rio (26 + 1 C; 80 + 10% U.R.;
fotoperiodo de 12h) e alimentada em dieta artificial. O estagio larval apresentou um period
m6dio de 34,87 dias e 7 instares, sendo cada estadio separado pela largura da capsula cefa-
lica, a qual aumentou a cada instar numa razao de 1,28. O estagio pupal durou um period
m6dio de 12,75 dias. As pupas apresentaram dimorfismo sexual, onde as f6meas foram maio-
res do que os machos, os quais exibiram um poro genital, ausente em f6meas. A razao sexual
entire adults foi de 1:1,3. As f6meas adults apresentaram-se maiores que os machos, com
envergadura, em m6dia, de 28,73 cm e 20,80 cm para machos. A oviposicao iniciou-se no pri-
meiro dia de vida da f6mea e estendeu-se at6 o sexto dia, com uma m6dia geral de 431,05
ovos/f6mea, apresentando uma maior producao de ovos nos dois primeiros dias de vida.

Translation provided by the authors.

Within the world economy, sugarcane (Saccha-
rum officinarum) constitutes one of the most im-
portant crops in terms of annual production and
as a major source of employment. Sugarcane bio-
mass is the raw material for the production of al-
cohol (for beverages and fuel) and animal feed, as
well as for sugar and various derived products.
However, the production of sugarcane is not
straightforward by virtue of the considerable
problems caused by numerous pests that can dev-
astate the crop and diminish the yield. Insects of
the genus Diatraea (Lepidoptera: Crambidae)
cause the most damage to sugarcane crops result-
ing in significant losses of revenue.
Commercially available pesticides are, unfor-
tunately, not efficient for the control of Diatraea

spp. on sugarcane for a variety of reasons mainly
associated with the continuous presence of the
host plant in the field throughout the whole year,
the simultaneous occurrence of mature and im-
mature forms of the insect, and the feeding habits
of the insect. An alternative strategy is that of in-
tegrated pest management (IPM), which involves
biological control of the insect together with a
range of tactics including manual collection of the
larvae, introduction of resistant varieties of sug-
arcane, and the use of pheromone baits. So far,
IPM has been the most efficient method of con-
trolling Diatraea spp. infestation.
In a number of regions of Brazil, Diatraea fla-
vipennella (Box, 1931), popularly known as broca
pequena da cana-de-aqucar, is considered to be

Florida Entomologist 90(2)

the main sugarcane pest. This insect can not only
kill a plant directly by damaging the apical buds,
but it can also cause indirect damage through in-
filtration of the larvae into the culms, leading to
the ingress of phytopathogenic organisms into the
plant (Mendonca 1996). The duration of complete
metamorphosis of D. flavipennella is very irregu-
lar and depends on numerous factors such as the
climate and the host plant (Guagliumi 1972/73).
Studies concerning the morphology, physiol-
ogy, and biology of insect pests are very important
since they provide valuable insights into aspects
of pest management including damage potential,
population dynamics and fluctuation, growth
rate, and spatial distribution. Such knowledge
permits the establishment of appropriate control
measures. However, most studies have focused on
overall understanding of the genus Diatraea. The
objective of the present investigation was, there-
fore, to examine the specific biology ofD. flavipen-
nella through determination of defined parame-
ters including the number of ovipositions per fe-
male, number of eggs per oviposition, viability
and incubation time of eggs, development of lar-
vae and pupae, male/female ratio, and longevity
of adults.


Initiation and Maintenance of the Insect Population

Larvae ofD. flavipennella were obtained from
infested sugarcane plants located in commercial
plantations in the State of Alagoas, Brazil, and
transported to the Laborat6rio de Quimica Ento-
mologica at the Universidade Federal de Alagoas.
Eggs, pupae, and adults were maintained in the
laboratory at 22 + 1C, 70 10% relative humidity
and 12 h photoperiod, while larvae were main-
tained at 26 + 1C, 80 10% relative humidity
and 12 h photoperiod. Larvae received an artifi-
cial diet developed by Hensley & Hammond
(1968) and modified in collaboration with the
Laborat6rio de Assistancia Fitossanitaria e Con-
trole Biologico (FITOSSAN Macei--AL, Brazil),
according to the following description: ascorbic
acid (7.0 g); agar-carrajeenate (26.0 g: 12.0 g), vi-
tamin solution (60 mL), sucrose (162 g), and sug-
arcane culms in powder (40 g). Adult insects were
fed with 10% sucrose solution. All of the described
experiments were conducted with insects that
had been reared and maintained under labora-
tory conditions.

Incubation of Eggs, Emergence of Larvae, Pupae
and Adults, and Viability of the Immature Forms

The number of eggs in each of 10 newly depos-
ited egg masses was determined with use of a
Wild Leica model M3B stereomicroscope. Each
egg mass was placed in a separate glass tube (8.5

length x 2.5 cm diam) containing artificial diet
and observed daily until larvae emerged. Newly
emerged larvae (n = 110; 0 to 24 h old) were
placed individually into acrylic dishes (1.5 cm
depth x 6.0 cm diameter) containing artificial diet
and observed daily. The following aspects relating
to the development of larvae were recorded: oc-
currence of pupation, presence of a cephalic cap-
sule, the size of the cephalic capsule (measured
with a stereomicroscope containing an ocular mi-
crometer), and the number of dead larvae. Pupae
(n = 140) originating from the egg masses men-
tioned above, were grouped by sex, measured
with a calliper and maintained in acrylic dishes
(1.5 cm depth x 6.0 cm diameter) until emergence
of adult insects. The following aspects relating to
the development of pupae were recorded daily: oc-
currence of metamorphosis, the number of males
and females that emerged, and the number of
dead pupae.

Longevity and Reproduction of Adult Insects

Twenty four newly emerged adults originating
from the egg masses mentioned above were
placed in pairs (1 male with 1 female) in glass
cages (15 cm x 30 cm x 20 cm). Each group was ob-
served daily and the numbers of dead males and
females were recorded. Measurements of adult
size were performed at this stage.

Measurements of Adult Size

Twenty two pairs (1 male with 1 female) of
newly emerged adults were placed in PVC tubes
(10 cm length x 10 cm diameter) that had been
lined with greaseproof paper. The paper lining
was removed each day and the number of eggs
present was determined.

Statistical Analysis

All experiments were conducted in a random-
ized design. The results concerning the sizes of
male and female pupae and the longevity of male
and female adults were submitted to analysis of
variance, and differences between mean values of
each sex were determined by Tukey's test at the
5% probability level.


Number of Eggs per Oviposition, Incubation Period,
and Viability of Eggs

Females of D. flavipennella deposited egg
masses containing between 3 and 58 (mean 33.2
2.53 SEM) elliptical-shaped, milky-white eggs
per oviposition. The eggs gradually became dark
yellow in color as the embryos matured, and even-
tually turned black at the stage when the larvae

June 2007

de Freitas et al.: Biology of Diatraea flavipennella

Fig. 1. Stages of the life cycle of Diatraea flavipennella reared and maintained under laboratory conditions: A
an egg mass (160-fold increased); B-pupae; C-a larva; and D-male and female adults.

emerged (Fig. 1A). The viability of the eggs per
mass varied between 54 and 100% (mean 87.8%),
and the average incubation period was 8.35 d (+
0.17) (Table 1).

Development and Viability of Larvae and Pupae

The larvae ofD. flavipennella were yellowish in
color with brown spots that did not appear to form
any uniform pattern along the dorsal surface of
the insect (Fig. 1B). The cephalic capsule was yel-
low or brownish color. The mean duration of the
larval stage was 34.87 d ( 0.41) and the average
viability of larvae was 75.46% (Table 1). Assuming
that metamorphosis from one instar to another is
indicated by the release of the cephalic capsule,
the total number of instars was determined to be
7 (Fig. 2). The mean durations of the instars (Ta-
ble 2) varied between 6.20 0.37 d (1st instar) and
2.89 0.65 d (7th instar), while the average sizes
of the cephalic capsules ranged between 0.32 +
0.01 mm (1st instar) and 1.50 0.04 mm (7th in-
star). The mean size of the last instars was 26.0 +
0.4 mm although some were as large as 32 mm.
The average duration of the pupal stage (Fig.
1C) was 12.75 d 0.42 and the mean viability was
77.63% (Table 1). Male and female pupae had 8
tergites and could be identified from the differ-
ence in the external genitalia because males had
a distinct pore that was absent in females. The
size of the pupae varied between 12 and 21 mm,


Number of
specimens Duration Viability
Phase examined (n) of stage (d) (%)

Eggs 110 (masses) 8.35 0.17 87.80
Larvae 110 34.87 0.41 75.46
Pupae 140 12.75 0.42 77.63
Adults 24 9.17 0.69 -

Mean values + SEM are shown.

with a mean value of 16.13 0.17 mm; however,
female pupae were larger (mean 17.90 0.22 mm)
than males (mean 14.77 0.11 mm), and the dif-
ference was significant (P < 0.05).

Longevity of Adult Insects and Number of Eggs
Deposited per Female

The female/male ratio in adult insects was
1:1.3. Adults were milky white in color and varied
in size from 18-33 mm, with average dimensions
of 28.73 ( 0.25) mm for females and 20.80 ( 0.86)
mm for males. The wings were striated and the
central part of the frontal wings bore a black spot
(Fig. 1D). The average life span of adult insects
was 9.17 0.69 d. The lifespan of male insects was
not different statistically (P > 0.05) from female
insects. Females began to oviposit on d 1 after
emergence and continued until the d 6. The max-
imum production of eggs occurred during the first
2 d (Fig. 3). The lifetime-number of eggs laid by
the females varied between 96 and 585, with an
average of 431.05 30.28.


Diatraea flavipennella was able to complete its
life cycle successfully within a population reared
and maintained under experimental conditions.
The number of eggs laid by adult females of this


20 *
o L -AiiAt t
3 l. 1. 2.0

Width ofcephalkcapsule (am)

Fig. 2. Frequency distribution of the sizes of cephalic
capsules during the larval stage of Diatraea flavipen-
nella. The 7 instars were characterized by the most fre-
quently occurring sizes of cephalic capsules (arrowed).

Florida Entomologist 90(2)


Duration of Size of cephalic
Instar instar (d) capsule (mm)

1st instar 6.20 0.37 0.32 0.01
2nd instar 5.80 0.94 0.46 0.01
3rd instar 4.93 0.40 0.61 0 02
4th instar 5.56 1.04 0.91 0.03
5th instar 4.95 0.45 1.22 0.03
6th instar 4.54 0.72 1.44 0.04
7th instar 2.89 0.65 1.50 0.04

Mean values + SEM are shown.

species was similar to that found forD. saccharalis
(Holloway et al. 1928). The 7 instars detected dur-
ing the larval stage ofD. flavipennella were within
the range expected for the order Lepidoptera,
which is normally 5 to 6 but which can vary be-
tween 3 and 11 owing to intrinsic and extrinsic fac-
tors for each species (Parra & Haddad 1989). Vari-
ations in the number of instars have been previ-
ously reported for a number of species including
D. saccharalis, Lacanobia oleracea, Delterollyta
majuscule, and Copitarsia incommoda (Guagliumi
1972/73; Corbitt et al. 1996; Acatitla-Trejo et al.
2004; Nava et al. 2004). The size of the cephalic
capsule varied with each instar and increased be-
tween instars by a mean ratio of 1.28 in agreement
with Dyar (1980), who reported that the size ratio
between instars can vary between 1.1 and 1.9. The
size of the cephalic capsule can thus be used as a
precise indication of each instar.
The duration of the pupal stage in D. flavipen-
nella was longer than that previously reported for
D. saccharalis (Holloway et al. 1928; Guagliumi
1972/73). The pupae ofD. flavipennella exhibited
sexual dimorphism that was characterized
mainly by differential size in which the females
were significantly larger than the males. This fea-
ture is typical of insects of the order Lepidoptera
(Slansky & Scriber 1985), and has been observed

250 -
I soo

z o-
1 2 3 4 5 6
Day of adult female life

Fig. 3. Mean ( SEM) numbers of eggs deposited per
d by individual females of D. flavipennella during their
adult life span.

in other species of the same genus, i.e., D. saccha-
ralis (Holloway et al. 1928) and D. grandiosella
(Chippendale & Sorenson 1997).
Adult females ofD. flavipennella were able to
oviposit for 6 d, although most eggs were depos-
ited during the first 2 d of adult life. In contrast,
oviposition in D. saccharalis is reported to last for
only 4 d (Holloway et al. 1928).
The viabilities of larvae, pupae, and adults of
D. flavipennella were found to be satisfactory, pro-
viding this species with a high reproductive po-
tential and permitting the facile maintenance of
an insect population both under laboratory and
natural conditions. These findings are very simi-
lar to those previously reported forD. saccharallis
(Filho & Lima 2001). However, for D. flavipen-
nella, the periods necessary for the development
of eggs, larvae, and pupae subjected to an artifi-
cial diet were different from those reported for in-
sects fed on natural diet (Guagliumi 1972/73).
The present results contribute to the under-
standing of the biology ofD. flavipennella and will
be of value in the context of further studies con-
cerning the reproductive behavior, survival rate,
feeding habits, and pheromone production in this
species, as well as in the establishment of biolog-
ical control programs for this detrimental pest.


The authors thank the Coordenacio de AperfeiCoa-
mento de Pessoal de Nivel Supeior (CAPES), the Con-
selho Nacional de Desenvolvimento Cientifico e
Tecnol6gico (CNPq) and the Fundacio de Amparo a Pes-
quisa do Estado de Alagoas (FAPEAL) for financial sup-
port. Our thanks are extended to the Laborat6rio de
Fitossanidade e Controle Biol6gico for collaboration in
the development of the artificial diet used in the present


CAC-CORTERO. 2004. Ciclo biol6gico y tasas de super-
vivencia y reproducci6n de Copitarsia incommoda
Walker (Lepidoptera: Noctuidae) en cinco dietas ar-
tificiales. Agrociencia 38: 355-363.
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borer In E. B. Radcliffe and W. D. Hutchison [eds.],
Radcliffe's IPM World Textbook. University of Min-
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Florida Entomologist 90(2)

June 2007


'USDA-ARS, Subtropical Horticulture Research Station,13601 Old Cutler Road, Miami, FL 33158

2USDA-APHIS-PPQ-CPHST, Plant Epidemiology and Risk Analysis Laboratory
1730 Varsity Drive, Suite 300, Raleigh, NC 27606


Composting plant waste is a beneficial practice commonly used by American gardeners, but
disposal of infested fruit directly into the environment creates a potential pathway for intro-
duction of insect pests. This study estimates the likelihood of adult emergence for exotic fruit
flies (Tephritidae) from residential composting in south Florida. Ripe grapefruits, Citrus x
paradisi Macfad., were infested with the Caribbean fruit fly, Anastrepha suspense (Loew).
Half of the infested fruit was placed onto outdoor compost piles and half was maintained un-
der controlled laboratory conditions. Adult fly emergence was recorded daily for 30 d from
both the compost piles and control bins. Compost temperature, air temperature, relative hu-
midity, and precipitation were monitored, and the study was repeated 4 times under differ-
ent seasonal conditions. Despite high mortality of flies from the composted fruit relative to
control fruit, the overall risk of a potentially mated female emerging from composted fruit
was calculated to be -10%. Of the environmental factors evaluated, compost temperature
was found to have a significant effect on adult emergence. Mortality approached 100% in
piles with maximum compost temperatures >48C. This report provides experimental data
in support of quantitative risk analysis for a tephritid-compost pathway.

Key Words: Caribbean fruit fly, risk assessment, pathway analysis, quarantine pest


Amontonar la desperdici6n de las plants para convertirla en abono es una practice benefi-
ciosa y comun usada por los jardineros americanos, pero disponer de fruta infestada direc-
tamente en el ambiente crea un pasaje potential para la introducci6n de insects pestilentes.
Este studio estima la probabilidad de la aparici6n de las moscas de fruta ex6ticas (Tephri-
tidae) del abonamiento residential en el sur de Florida. Toronjas maduras, Citrus xparadisi
Macfad., fueron infestadas con la mosca de fruta del Caribe, Anastrepha suspense (Loew).
Mitad de la fruta fu6 desechada en los montones de abono afuera y la otra mitad fu6 mante-
nida en condiciones controladas en el laboratorio. La aparici6n de las moscas adults fu6 re-
gistrada diariamente por 30 dias en los montones y en los recipients de control. La
temperature dentro del abono, la temperature del aire, la humidad relative, y la precipita-
ci6n fueron vigiladas, y el studio fu6 repetido cuatro veces en diferentes condiciones en va-
rias 6pocas del afo. Aunque la mortalidad de las moscas en las frutas abonadas fu6 muy alta
en comparaci6n a la mortalidad de las moscas en las frutas de los controls, el riesgo de en-
trada de una hembra pareada de la fruta abonada fu6 calculado a ~10%. La temperature del
abono fu6 el factor con un efecto significant asociado con la aparici6n de las moscas adults.
La mortalidad aproxim6 100% en montones con temperature maxima >48C. Este report
provee data experimental para soportar un andlisis cuantitativo del riesgo de un pasaje de
introducci6n de moscas tefritidas por medio de abonamiento.

Translation provided by the authors.

In pest risk analysis, a pathway is any means congressional appropriation of $57 million to fruit
that allows the entry or spread of a pest species. fly risk management programs in 2005 (USDA-
Pathway risk analysis entails identification of via- APHIS 2006a). This threat is pronounced in Flor-
ble pathways, assignment of probabilities to the se- ida due to a favorable climate, availability of hosts,
quence of events involved (e.g., entry, reproduction, and the large volume of foreign produce entering
establishment, etc.), and assessment of the conse- the state's ports. From Dec 2003 to Sep 2006, there
quences of pest introduction (Hennessey 2004). In- were over 1200 interceptions of live tephritids at
vasive fruit flies, family Tephritidae, constitute a the Miami International Airport, primarily from
serious threat to U.S. agriculture, as evidenced by a larval-infested fruit concealed within baggage

Kendra et al.: Compost Pathway for Anastrepha Fruit Flies

(2048 larvae, 40 puparia, 15 adults; USDA-APHIS
2006b). The Mediterranean fruit fly, Ceratitis capi-
tata (Wiedemann), is one of the most destructive
pests worldwide. Several outbreaks of C. capitata
have occurred in Florida, with the most recent in-
vasion in 1997-1998 (Silva et al. 2003). Of the 198
recognized species ofAnastrepha fruit flies (USDA-
ARS 2007), only 1 species of economic importance
is established in Florida, the Caribbean fruit fly,
A. suspense (Loew). Restricted to the Bahamas
and Greater Antilles until the 1960s (reviewed in
Weems et al. 2001),A. suspense is now common in
the southern half of the peninsula where it impacts
production of citrus, guava (Psidium guajava L.),
and other subtropical fruits (Greany & Riherd
1993). Several other Anastrepha species pose a
threat to Florida, including A. ludens (Loew),
A. obliqua (Macquart),A fraterculus (Wiedemann),
A. striata (Schiner), and A. grandis (Macquart).
Anastrepha ludens represents a special concern be-
cause it has an affinity for grapefruit (Citrus x par-
adisi Macfad.) and Florida is one of the world's
leading producers (Weems et al. 2004).
Anastrepha females have well-developed ovi-
positors, inserting their eggs beneath the skin of
host fruits, where larvae feed and develop within
the flesh (White & Elson-Harris 1992). Conse-
quently, larval infestations are difficult to detect
through visual inspections of intact fruit. At ports
of entry, quarantine inspectors check incoming
shipments by cutting open a small sample of fruit
(typically no more than 2 percent) and searching
for eggs or larvae (USDA-APHIS 2006c). The effi-
cacy of this procedure has been evaluated in lab-
oratory tests, and Gould (1995), using trained ag-
ricultural inspectors, concluded that only about
35% of grapefruits infested with A. suspense
could be detected through manual dissection. A
comparison of 6 different inspectors revealed con-
siderable variability, with the percentage of lar-
vae detected ranging from 8% to 49% (Gould
1995). Thus, it is highly likely that some infested
fruit may evade detection and, if not subjected to
appropriate quarantine treatments, contain via-
ble eggs or larvae when distributed to consumers.
Subsequent to entry as immature stages within
host fruit, pest Anastrepha may spread if that
fruit is discarded directly into the environment.
Two risk assessment studies have estimated
the amount of infested fruit discarded by consum-
ers into the environment. Wearing et al. (2001), re-
viewing potential spread of codling moth (Cydia
pomonella L.) via imported cherries, determined
that up to 5% was discarded in suburban New
Zealand. For urban Japan, Roberts et al. (1998) cal-
culated 0.5% disposal of apples infected with Er-
winia amylovora (Burr.), the pathogen of fire
blight. Although no data are available for the
amount of fruit infested with fruit flies that is dis-
carded by Americans, the USDA-APHIS (2004) has
estimated it to be 5% for avocados imported from

Mexico, choosing the higher proportion from the 2
former studies so as not to underestimate the risk.
With today's emphasis on environmentally-
friendly practices there is increased interest in or-
ganic gardening, including composting of plant
waste. A quick internet search will yield much in-
formation on residential composting, dissemi-
nated to the public by federal, state, and local
agencies (e.g., EPA 2006, USDA-NRCS 2004, Sa-
rasota County 2006, UF-IFAS 2004). But unlike
commercial organic recycling facilities which are
regulated in Florida, composting in backyard, mi-
cro-scale, and farm sites is exempt from state reg-
ulation, Florida Administrative Code 62-
709.300(10). As a result, compost practices vary
widely among consumers, creating a potential
means for spread of pests into susceptible areas.
This study simulated disposal of Anastrepha-in-
fested grapefruit on backyard compost piles in
south Florida to estimate the likelihood of emer-
gence of mated females. The results provide exper-
imental data to facilitate quantitative risk analy-
sis for a tephritid-compost pathway, information
currently lacking in the scientific literature.



Anastrepha suspense were obtained from a lab-
oratory colony maintained at the USDA-ARS, Sub-
tropical Horticulture Research Station, Miami,
FL. Insects were reared at 25C (+2), 70% RH, and
a photoperiod of 12:12 h (L:D), by methods previ-
ously described (Kendra et al. 2006). Approxi-
mately 3500 sexually mature (10-12 d old), mated
females were placed in each of 2 infestation cages
(94 x 51 x 51 cm) constructed from PVC frames
covered with mesh pollination bags (Delstar Tech-
nologies, Middletown, DE). Each cage contained 60
ripe, Florida-grown grapefruit (Citrus x paradise
Macfad., cv White Marsh), arranged in a single
layer, and oviposition was allowed for 7 d under the
same environmental conditions used for rearing.
Infested fruit (120 total) was then removed from
the cages and randomly divided into 3 groups:
Compost fruit (50), Control fruit (50), and Inspec-
tion fruit (20). All grapefruits were held in the lab-
oratory while the Inspection fruits were cut open
at 2-3 d intervals (5 fruits per sample) to monitor
progress of larval development and to approximate
the level of infestation. When the majority of the
larvae had reached the third (final) instar, typi-
cally 9 d after oviposition, field tests were initiated.

Field Tests

Ten replicate compost piles (~1.5 m3 each) were
constructed for each field test. Piles were con-
tained within square wooden frames (1.2 x 1.2 m),
spaced 5 m apart, located under partial shade.

Florida Entomologist 90(2)

Compost material consisted of a 1:1 mixture of
wood chips and fresh grass clippings, applied in
alternating layers, according to the guidelines
provided by the USDA Natural Resources Con-
servation Service (USDA-NRCS 2004). Piles were
turned weekly to promote active decomposition,
then left undisturbed once grapefruit had been
added. Compost piles received no manual water-
ing during the study, only natural rainfall.
For each of the 10 compost replicates, 5 infested
fruit were placed on top of the pile, nestled 4-5 cm
deep, and left in place for 30 d. For the first 5 d, the
piles were left exposed to allow access of potential
predators and competitors. Then each pile was cov-
ered with a pyramidal screen cage (modified from
Raney & Eikenbary 1969) fitted on top with the up-
per assembly from a boll weevil trap (Great Lakes
IPM, Vestaburg, MI). The cages were sealed with
hook-and-loop tape (Velcro Industries, Manchester,
NH), and the screen at the base of each cage was
pulled tightly over the wooden frame and secured
with bricks and soil, creating a closed system for
the remainder of the field test. Adult fly emergence
(number and sex) was recorded daily for the next
25 d. Temperature of compost beneath the fruit (15
cm depth) was monitored throughout the 30-d pe-
riod, using compost thermometers (Reotemp, San
Diego, CA). Air temperature, relative humidity,
and rainfall data were obtained from the SHRS
weather station. At the completion of the tests, the
upper layer of compost and any remaining fruit
was collected for sampling with Berlese funnels.
To accompany each field test, 10 replicate con-
trols were set up in polyethylene bins (51 x 31 x 15
cm; U.S. Plastic Corp., Lima, OH) maintained in the
laboratory under the same conditions used for rear-
ing the Anastrepha colony. Each bin contained 5 in-
fested fruit placed above vermiculite (8 cm deep),
and bins were enclosed in clear plastic cages (Bug-
Dorm-2; BioQuip Products, Rancho Dominquez,
CA). Vermiculite was sifted weekly to collect wan-
dering larvae and puparia, which were held for
adult emergence for the same 30-d period monitored
in the field. The controls showed that 30 d was suf-
ficient to allow all larvae to complete development.
The study consisted of 4 field tests (and con-
trols), conducted from the late summer through
early spring of 2004-2005. The 30-d monitoring
periods were as follows: Summer test from 23 Aug
to 22 Sep 2004; Early Fall test from 26 Sep to 26
Oct 2004; Late Fall test from 29 Oct to 28 Nov
2004; and Spring test from 28 Feb to 30 Mar 2005.
This time period allowed us to evaluate compost-
ing of infested fruit under a variety of environ-
mental conditions, with the first 2 tests conducted
during the wet season, and the second 2 tests con-
ducted during the dry season in south Florida.
Statistical Analysis
The levels of infestation (number of larvae per
fruit) varied among the 4 field tests, therefore

adult emergence counts were converted to percent
emergence from compost fruit relative to control
fruit prior to statistical analysis. Differences in
percent emergence among the tests were ana-
lyzed by one-way analysis of variance (ANOVA)
followed by Tukey-Kramer HSD test for mean
separation (P = 0.05), with JMP (SAS Institute
2006). To evaluate environmental factors during
the 4 tests, ANOVA followed by mean separation
was performed initially. If significant differences
were found among tests, then regression analysis
was used to explore the relationship with percent
fly emergence, which was then graphed with Sig-
maPlot 10 (Systat Software, Inc. 2006). Addition-
ally, two-way ANOVA was used to assess potential
interaction effects of environmental factors on
percent emergence. Actual fly counts were ana-
lyzed in 1 instance, to determine if there was a dif-
ference in adult emergence based on sex. Two-way
ANOVA with interaction, followed by Student's t-
test, was performed separately on compost and
control treatments to evaluate the number of
males and females emerging in each test.


Manual grapefruit dissections indicated the
following levels of infestation at the start of the 4
field tests: 19.0, 22.4, 34.8, and 8.2 larvae per
fruit, respectively. In light of Gould's (1995) con-
clusions regarding the reliability of this detection
procedure, these values were used only as relative
indicators of the degree of infestation among the
tests. Infestation was considered to be moderate
for the Summer and Early Fall tests, high for the
Late Fall test, and low for the Spring test. Differ-
ences in levels of infestation were not unexpected
since Marsh grapefruit susceptibility to A. sus-
pensa infestation has been shown to vary season-
ally in relation to fruit senescence (Greany et al.
1985). All 3 instars were represented in the in-
fested fruit of each test, but on average, 62.4% of
the larvae were in the third instar when field tests
were initiated. The percentages for the 4 tests
were 75.8, 50.9, 66.8, and 56.1, respectively. This
developmental stage was chosen to provide condi-
tions comparable to those used in previous inves-
tigations ofAnastrepha-infested grapefruits (e.g.,
Hallman et al. 1990; Hallman 1994; Gould 1995),
but more significantly, use of fruit infested with
third instars represented a scenario that would
maximize the likelihood of adult emergence and
thereby estimate the greatest potential risk of
pest escape and establishment via a compost
pathway. Shortly after infested fruit was placed
on the piles, third instars would exit the fruit,
burrow into the compost, and pupate. By restrict-
ing the field tests to this time window, any reduc-
tion in adult emergence would result primarily
from conditions in the compost environment. Also,
a third instar/wandering larva scenario would

June 2007

Kendra et al.: Compost Pathway for Anastrepha Fruit Flies 317

represent the point at which consumers would
most likely notice an insect and discard the fruit.
Overall, the mean emergence of adult flies L
(males and females) from composted fruit was 11% +1 +1 +1 +1 +
of that observed emerging from the control treat- -
ments (Table 1). In terms of risk assessment, the
critical component is the percentage of females
that survive the composting process, successfully c c 3
mate, and escape into the environment. For the C1
purposes of this study, 2 criteria were adopted to 2 +1+ +1 +1 +1
address that component. First, risk was defined as C .
the presence of a single mated female emerging
from compost. Second, a female was considered po-
tentially mated if at least 1 male emerged from the
same compost pile. These are valid assumptions H Ci 6 C-
since a single A. suspense female (wild strain) has o +1 +1 +1 +1 +1
been shown to lay an average of 1.9 (+0.3) eggs/day s
over a lifespan of 73.6 (4.9) days (Sivinski 1993), 8
and males ofA. suspense have been documented to
engage in multiple matings (Teal et al. 2000). X C 1
Based on these criteria, percent emergence of po- -
tentially mated females was calculated for each + +1 +l +1 +1
field test, and presented in Table 2. The probabil- o -
ity of a mated female emerging from compost was c
estimated to be10% for the study, with the greatest
risk (22% emergence) observed during the Spring r U c c
2005 test. The lowest risk was seen with the Early & 0 6 ec 6 4
Fall 2004 test, when no mated females emerged. In +1 +1 +1 +1 +1
addition, the compost treatments had a significant s L C6 L :
difference in mean number of adults emerging u
based on sex (F = 6.27; df = 1, 79; P = 0.014). The 0
average number of females emerging from com-
post piles was 1.15 times greater than the number 6 ) c
of males (t = 2.50; df = 75; P = 0.014), suggesting | +1 + + +1 +1
differential survival of females within the com- 5
posting environment. This bias was not observed m
in fly emergence from the control fruits (F = 3.43;
df = 1, 79; P = 0.068).
Adult emergence data indicated approximately '
90% mortality ofAnastrepha in the late larval and +1 +1 +1 +1 +1
pupal stages when exposed to composting condi- .
tions. Factors in the physical environment during
the 4 seasonal tests (Table 3) were assessed for po-
tential effects on fly emergence. The Spring 2005 < t
test, which had significantly higher percent emer- rP P i
gence than the other replicates (F = 6.40; df = 3, +1 +1 +1 +1 +1
39; P = 0.002, Table 1), was characterized by hav- c& c "
ing the lowest compost temperatures and the low- z
est precipitation. Conversely, the test conducted in a
Early Fall 2004 had the lowest fly emergence, and a ,
the highest values for both compost temperature o C
and precipitation. Therefore, these 2 environmen- 1 1 +1
tal factors were analyzed further. For compost c. L-- .
temperature, the mean maximum values were
used for analysis, since maximum temperatures
were more likely to contribute to fruit fly mortal-
ity. The interaction between maximum compost
temperature and precipitation had no effect on the
percentage of flies that emerged (F = 1.66; df = 1, 1
39; P = 0.205), but there was a significant effect s 5 da
due to maximum compost temperature (F = 6.52; C
cc ccl W 5-10 0

Florida Entomologist 90(2)


Compost pile replicate Percent
emergence' of
Number mated females2
Season emerged 1 2 3 4 5 6 7 8 9 10 (Mean+ SE)

Summer Male 0 0 2 2 5 3 2 1 3 4
Female 0 0 3 3 4 6 5 5 6 1
Mated female2 0 0 3 3 4 6 5 5 6 1 5.7 1.3 ab
Early Fall Male 0 1 0 0 0 0 0 0 0 0
Female 0 0 1 0 0 0 0 0 0 0
Mated female2 0 0 0 0 0 0 0 0 0 0 0.0 0.0 a
Late Fall Male 0 0 0 0 0 2 2 0 0 3
Female 0 0 0 0 0 10 4 2 0 8
Mated female2 0 0 0 0 0 10 4 0 0 8 12.5 + 6.9 ab
Spring Male 0 2 4 0 0 0 5 3 1 0
Female 3 5 6 0 3 1 8 3 4 0
Mated female2 0 5 6 0 0 0 8 3 4 0 22.2 8.2 b

Overall 10.1 2.9

Percent emergence from compost relative to controls. Means followed by the same letter are not significantly different (Tukey-
Kramer HSD test, P = 0.05).
'Females were considered potentially mated if at least 1 male emerged from the same compost pile.

df= 1, 39;P = 0.015). The mean maximum temper-
ature of compost piles was significantly different
for each of the 4 seasons (F = 137.6; df = 3, 39; P <
0.0001; Table 3). The relationship between maxi-
mum compost temperature and percent emer-
gence (Fig. 1) showed a marked clustering of the
compost treatments: the Spring 2005 test with the
highest emergence was at the lower end of the
temperature scale, and the Early Fall test with
the lowest emergence was at the upper end. Adult
emergence decreased with increasing compost
temperature, predicting mortality to approach
100% as the temperature rises above 48C. Since
the host fruits were placed 5 cm into the compost,
andA. suspense larvae typically pupate at a depth
of 1-3 cm into the substrate (Hennessey 1994), the
actual temperatures experienced by the insects
were probably slightly less than that recorded by
the compost thermometers inserted to 15 cm. It
should also be noted that the heat of the compost,
since generated by biologically active decompos-
ing organic matter, is relatively stable and not
subject to rapid changes due to fluctuations in
weather conditions. Thus, the insects within an
active compost environment are exposed to long
periods of sustained high temperatures. Under
"hot composting" conditions, as defined by the
USDA-NRCS (2004), internal compost can reach
43-71C, temperatures sufficient to kill most in-
sects, weed seeds, and plant pathogens. The com-
post temperature of 480C estimated for fly mortal-
ity in this study is consistent with the range of

temperatures used in quarantine heat treatments
of commercial fruit, 43-48C (Hallman 1994), and
documented in A. suspense laboratory studies for
mortality of isolated third instars and pupae, 43C
(Hallman 1996), or for mortality of third instars in
host grapefruits, 43-46C (Hallman et al. 1990).
In addition to the abiotic conditions of the com-
post system, several factors contributing to mor-
tality were identified from Berlese sampling of the
arthropod community. Competitors on the host
fruit included adult and larval sap beetles, Lo-
biopa insularis (Cast.) and Carpophilus spp.,
which were often quite abundant. Predators and
parasitoids detected in the compost included spe-
cies known to feed on dipteran larvae, such as a
macrochelid mite, Glyptholaspis fimicola
(Sellnick) (Krantz 1998) and a rove beetle, Belonu-
chus pallidus Casey (Frank 2004); plus several
species which have been documented to attack
A. suspense larvae and pupae, including a parasi-
toid wasp, Diachasmimorpha longicaudata (Ash-
mead) (Lawrence et al. 1976), the ringlegged ear-
wig, Euborellia annulipes (Lucas) (Hennessey
1997), and the red imported fire ant, Solenopsis in-
victa Buren (Hennessey 1997). Predation by ants
has been shown to be an important biotic mortality
factor forAnastrepha larvae during the wandering
prepupal stage (Aluja et al. 2005). Fungal growth,
common on the surface of host fruits, was another
potential mortality factor since it contributed to
rapid breakdown of fruit on compost piles, espe-
cially in tests conducted during the wet season.

June 2007

Kendra et al.: Compost Pathway for Anastrepha Fruit Flies


Weather data Compost temperature (oC)

Season Air temp. (oC) Rel. humidity (%) Precip. (cm) Mean Minimum Maximum

Summer 28.6 0.2 a 83.6 0.7 a 0.53 + 0.18 a 38.3 0.3 a 34.2 + 0.2 a 42.9 0.6 a
Early Fall 26.8 0.3 b 81.8 1.lab 0.61 0.36 a 41.5 0.5 b 30.7 0.4 b 50.0 0.8 b
Late Fall 24.3 0.3 c 78.4 1.3 b 0.12 + 0.08 a 34.8 0.8 c 30.6 0.5 b 39.3 1.1 c
Spring 21.5 0.7 d 79.3 1.5 ab 0.08 0.03 a 25.9 0.2 d 21.2 0.2 c 28.3 0.2 d

Means within a column followed by the same letter are not significantly different (Tukey-Kramer HSD test,P = 0.05).

In summary, this study estimates a 10% likeli-
hood for emergence of a mated femaleAnastrepha
through a residential composting pathway. This
risk applies to the spread of the established ex-
otic, A. suspense, into areas of Florida or other
states that are currently fly-free. It applies
equally to spread of exotic Anastrepha species
should infested fruit evade detection and quaran-
tine measures, as was the case in 2003 with man-
zano peppers imported from Mexico (Thomas
2004). Peppers infested with A. ludens cleared
customs in Texas in Apr, were distributed to sev-
eral U.S. locations, including 2 in Florida, and an
adult A. ludens was captured in Orlando in May
(Thomas 2004). Should pest entry occur despite
current safeguards, the risk of spread via com-
posting can be reduced by burying produce deep
and promoting high internal compost tempera-
tures (e.g., keeping piles moist and turning them
often). Gould & Maldonado (2006) recently re-
ported data on the likelihood of escape of Copitar-

go i-------------
Summer 2004
SA Early Fall 2004
S60 a Late Fall 2004
Spring 2005


I 20

r* n
o A A
25 30 35 40 45 50 55
Maximum Temperature of Compost (OC)

Fig. 1. Relationship between maximum compost tem-
perature and percent emergence of adult A. suspense
from infested grapefruits placed on outdoor compost piles
in Miami, FL. Each point represents 1 compost pile (5
fruit/pile, n = 40 piles); percent emergence and compost
temperature (recorded at 15 cm depth) were monitored
for 30 days. (Regression with arcsin ( of percent emergence: y = -0.02x + 1.09; r2 = 0.41. Non-
transformed data depicted in graph.)

sia decolora (Lepideptera: Noctuidae) larvae from
disposal of infested asparagus, estimating that
~1.2% of first instars were able to escape from a
commercial garbage dumpster during a 1-week
period. These data were critical components of a
pathway risk assessment estimating the likeli-
hood of establishment of C. decolora. To our
knowledge, this is the only other experimental
study which quantifies risk of insect pest escape
as a result of discarded produce. Comparable
studies assessing tephritid emergence from fruit
discarded in dumpsters or landfills are needed to
support comprehensive pathway risk analysis for
invasive fruit flies in Florida.


The authors are grateful to Monica Schiessl and Paolo
Gonzalez for technical assistance; to Tomas Ayala-Silva,
Will Bergstrom, and Steve Tally for supplying the com-
post materials; to J. B. Heppner, G. J. Steck, M. C. Tho-
mas, and W. C. Welbourn (FDACS-DPI, Gainesville, FL)
for identification of insect specimens; to Thomas L. Skar-
linsky (USDA-APHIS-PPQ, Miami, FL) for pest intercep-
tion records; and to Pansy Vazquez-Kendra and Elena
Schnell for translation of the abstract. We also acknowl-
edge Amy L. Roda (USDA-APHIS-PPQ, Miami, FL), Juli
R. Gould (USDA-APHIS-PPQ, Otis ANGB, MA), Guy J.
Hallman (USDA-ARS, Weslaco, TX), and 2 anonymous
reviewers for helpful suggestions with the manuscript.
This article reports the results of research only. Mention
of a proprietary product does not constitute an endorse-
ment or recommendation for its use by the USDA.


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for International Agriculture Research), Canberra,

June 2007

Ferkovich & Shapiro: Improved Fecundity of Orius insidiosus


Center for Medical, Agricultural, and Veterinary Entomology, USDA, ARS
1700 SW 23rd Dr., Gainesville, FL 32608


A specific factor that stimulates egg production in the predator Orius insidiosis (Say) was
earlier shown to be present in eggs of the Indian meal moth, Plodia interpunctella (Htibner).
We investigated whether the embryonic cell line IPLB-PiE, derived from eggs of the Indian
meal moth P. interpunctella also produces a specific factor that improves fecundity of the
predator. We fractionated cells by preparative isoelectric focusing in a pH gradient of 3-10
and bioassayed the resultant fractions in test diets to determine their effects on egg produc-
tion. Rates of oviposition were determined by placing adult predators on the test diets the
third d after eclosion, allowing them to feed for 3 d, and then providing them with oviposi-
tional substrates for 24 h on d 7. Six out of 20 fractions with isoelectric points between pH
5.2 and 7.3 had significant activity relative to the control diet. The nature of the factors) is
unknown but corresponds to a partially purified fecundity factor from eggs ofEphestia kueh-
niella Zeller with an isoelectric point of pH 5 in an earlier study. The results indicate that the
cell line, which was originally derived from embryos of P. interpunctella, has retained a dif-
ferentiated function in culture by producing products similar to those produced in the
P. interpunctella egg.

Key Words: artificial diet, factor, insidious flower bug, Plodia interpunctella, cell line, Orius
insidiosus, Ephestia kuehniella, eggs


Un factor especifico que estimula la producci6n de huevos del depredador Orius insidiosis
(Say) anteriormente se mostro el estar present en los huevos de la palomilla, Plodia inter-
punctella (Hibner). Nosotros investigamos si la linea cellular IPLB-PiE de los embriones de-
rivada de huevos de la palomilla, P. interpunctella tambi6n produce un factor especifico que
mejore la fecundidad del depredador. Desde entonces hemos distribuido las clulas por medio
de un preparativo isoel6ctrico enfocandose en un gradiente de pH de 3-10 y realizamos un bio-
ensayo sobre las fracciones en pruebas de dieta para determinar sus efectos sobre la produc-
ci6n de huevos. Las tasas de oviposici6n fueron determinadas por medio de la alimentaci6n de
los depredadores adults con las dietas de prueba al tercer dia despu6s de la eclosi6n, permi-
tiendo que ellos se alimentaran por 3 dias, y luego proveyendolos con sustratos oviposiciona-
les por 24 h en el dia 7. Seis de los 20 fracciones con puntos isoel6ctricos entire pH 5.2 y 7.3
tenian una actividad significativa mayor relacionada con la dieta de control. La naturaleza
del factor(es) es desconocida pero se corresponde a un factor de fecundidad purificado parcial-
mente de los huevos de Ephestia kuehniella Zeller con un punto isoel6ctrico de pH 5 en un es-
tudio anterior. Estos resultados indican que la linea cellular que fuera derivada originalmente
de los embriones de P. interpunctella ha retenido una funci6n diferenciada en el cultivo por la
producci6n de products similares a los producidos en el huevo de P. interpunctella.

Reduced fecundity is a general problem associ-
ated with the insidious flower bug, Orius insidio-
sus (Say) (Hemiptera: Anthocoridae) (Ferkovich &
Shapiro 2004a), and a number of other species of
predators reared on artificial diets without insect
components (Cohen 1985a, 1985b, 1992; 2000; De
Clercq & Degheele 1992, 1993a, 1993b; Cohen &
Staten 1994; De Clercq et al. 1998; Adams 2000a,
2000b; Rojas et al. 2000; Wittmeyer & Coudron
2001; Coudron et al. 2002). Insect hemolymph and
tissue extracts have been used to improve artifi-
cial diets (Grenier et al. 1994). The use of estab-
lished insect cell lines as replacements is a rela-

tively recent approach (Rotundo et al. 1988; Ferk-
ovich & Oberlander 1991; Ferkovich et al. 1994;
Hu et al. 1999; Ferkovich & Shapiro 2004b; Ferk-
ovich & Lynn 2005; Heslin et al. 2005a, 2005b).
The advantages of using cell lines in developing
and/or improving artificial diets will be realized
when the technology for large-scale cell produc-
tion with cost-effective serum-free media is avail-
able. Cell lines could substitute for hemolymph or
other insect materials (e.g., an embryonic cell line
could substitute for insect egg homogenates),
where those materials are critical for optimal de-
velopment of insects. Also, the use of cell lines in

Florida Entomologist 90(2)

chemically defined media could simplify down-
stream purification and identification of growth-
inducing factors, fecundity-inducing factors, and
others found naturally in insect hosts and prey.
In an earlier work, we addressed the fecundity
problem by supplementing artificial diet for 0. in-
sidiosus with 2 embryonic cell lines. One line,
Ek-x4 was derived from eggs of Ephestia kueh-
niella Zeller (Lynn & Ferkovich 2004), which are
generally used to rear Orius species by commer-
cial insectaries, and 1 line, IPLB-PiE was derived
from eggs of Plodia interpunctella (Hiibner)
(Tsang et al. 1985). Although the resultant fecun-
dity was comparable with both cell lines, the
growth characteristics of the IPLB-PiE cell line
were conducive to larger scale production of those
cells. In a recent study, we fractionated P. inter-
punctella egg proteins and bioassayed the frac-
tions in diet, demonstrating the existence of a
specific factor that stimulates egg production.
Correspondingly, in this study we have examined
the IPLB-PiE line derived from whole egg em-
bryos to determine if a similar fecundity factor is
also produced by the IPLB-PiE cells.


Orius Rearing

A colony of 0. insidiosus, originating from a
Florida strain collected in Bronson, FL in 2002,
was maintained on eggs of E. kuehniella Zeller
(received frozen from Beneficial Insectary, Red-
ding, CA). Briefly, freshly laid eggs of 0. insidio-
sus (about 500 eggs in 1-3 green beans) were
placed in 400-mL canning jars, each covered with
a 15 x 15-cm square of nylon ripstop cloth. Each
jar received 0.3 mL ofE. kuehniella eggs, 1.25 mL
of Hydrocapsules (1-2 mm dia.; Analytical Re-
search Systems, Gainesville, FL), and 2 granules
of local pollen, (Buzzn Bee, Inc., West Palm
Beach, FL). After the 0. insidiosus eggs hatched
(approximately 5 d), the green beans were re-
placed with fresh beans, E. kuehniella eggs, and
additional pollen every other day. Adults started
to emerge in approximately 3 weeks and, when
adults began to oviposit in the green beans, new
jars were set up with green beans containing
eggs. The insects were held at 25.5 1C, with 70
+ 5% RH, and light: dark cycle of 16:8 h.

Artificial Diet

Artificial diet was prepared under aseptic con-
ditions in a clean room and encapsulated in
stretched Parafilm@ domes (25 uL) with a diet en-
capsulation apparatus (Analytical Research Sys-
tems, Gainesville, FL) described by Ferkovich et
al. (1999). Diet ingredients were 330 mg brewers
yeast, 30 mg sucrose, 180 mg soy protein acid hy-
drolysate, 3.8 mg of 99% palmitic acid (all from

Sigma, St. Louis, MO), 40 mg chicken egg yolk,
and 80 mg honey in 1.2 mL of distilled water.
Palmitic acid was mixed with the egg yolk compo-
nent before adding it to the diet.


The embryonic cell line (IPLB-PiE), originally
derived from embryonated eggs of P. interpunc-
tella by Tsang et al. (1985), was cultured in TNM-
FH insect medium (Sigma, St. Louis, MO) in 25-
cm2 culture flasks for 7 d as described by Lynn
(1996). For large-scale production of the cells,
they were cultured in 250-mL magnetic spinner
flasks (Bellco Glass, Vineland, NJ) at 24.9C for
14 d. The cell suspension was centrifuged (1370 x
g for 3 min) in a graduated conical centrifuge tube
to obtain a soft pellet of cells. The pellet was re-
suspended in 1.0 mL purified water, washed twice
and homogenized with a hand-held homogenizer
in 1.0 mL purified water. The homogenate was
then sonicated for 60 s with a Polytron@ unit
(model W-375, Heat Systems-Ultrasonic, Inc.,
Plain View, NY). Twenty pL were removed and as-
sayed for protein by the Lowry procedure (Protein
Assay Kit, Sigma, St. Louis) and the remainder of
the cell suspension was saved for the isoelectric

Electrophoresis and Protein Assay

The Lowry procedure (Protein Assay Kit,
Sigma, St. Louis, MO) was used to assay the solu-
ble proteins in the egg protein solution and in the
fractions after isoelectric focusing. Gradient SDS-
PAGE (4-20%) was carried out in vertical mini-
gels (Bio-Rad) as described by Shapiro et al.

Preparative Isoelectric Focusing

The cell homogenate (approx 0.98 mL) and 3
mL of ampholyte solution (pH range 3-10, Bio-
Rad, Hercules, CA) were mixed in 58 mL of dis-
tilled water. The protein solution was run in a
Rotofor Cell@ isoelectric focusing unit (Bio-Rad,
Hercules, CA) for 2.5 h at 12 W constant power at
4C. Twenty fractions were collected and their vol-
umes (approx. 2 mL each) and pH values mea-
sured. Ampholytes were removed by bringing each
fraction to 1 M NaCl for 15 min and then aliquots
of 10-20 pL of each fraction were used for protein
analysis. After the fractions were analyzed for pro-
tein, they were combined based on the protein pro-
file. Fractions with low protein levels were com-
bined, and ones with higher protein concentra-
tions were kept as individual fractions. The frac-
tions were combined as follows: 1-5, 12-16 and 17-
20. Fractions 6-11, which were cloudy and noted to
contain minor precipitates, were kept as individual
fractions. The combined and individual fractions

June 2007

Ferkovich & Shapiro: Improved Fecundity of Orius insidiosus

were then concentrated to 0.5 mL in Centriprep@
concentrators (10k molecular weight (MW) cutoff;
Millipore, Bedford, MA) and 10-20 pL of each frac-
tion were used to analyze for soluble protein.

Proteinase K Digestion of Cells

One mL of pelleted PiE cells was homogenized
in 1 mL of PBS (0.15 M sodium chloride/0.1 M so-
dium phosphate, pH 7.0), centrifuged for 2 min,
and the supernatant was applied to 5 mL- Zebra@
desalt spin columns (Pierce, Rockford, IL). The re-
sultant desalted protein solution was incubated
with 20 mg proteinase K (immobilized on agarose
beads) per 30 mg cell protein and held on a
shaker-bath at 37C for 18 h. A second 1-ml sam-
ple without proteinase K was incubated at the
same temperature. The proteinase K sample was
centrifuged to remove the beads and 40 pL were
removed for the protein assay and PAGE analy-
sis. Dry diet ingredients were then added to both
of the samples for the diet bioassay.

Diet Bioassay of IEF Fractions

Newly emerged (24 h after eclosion) adults of
mixed sexes were collected with a camel hair
brush and maintained on E. kuehniella eggs for
3 d before they were placed on the diet capsules
containing the IEF fractions. Each replicate con-
sisted of 6 females and 4 males in a 100-mL plant
tissue culture jar (Sigma, St Louis, MO) with 4
jars per treatment. Each jar contained 0.6 mL of
Hydrocapsules, 2 capsules of treatment diet
(each 25 pL), and 3 crumpled strips of wax paper
(5 x 80 mm) as substrates. Ephestia kuehniella
eggs and beads of water and diet were replaced
daily and mortality was recorded. At the end of
d 6, one 7-cm section of green bean pod, used as a
substrate for oviposition, was placed in each jar
for 24 h. Eggs deposited in the green beans were
then counted under a microscope. The insects
were held in a growth chamber at 25.5 + 1C, with
75 + 5% RH, and a photoperiod of 15:9 (L:D) h.
Diet treatments consisted of the following:
(1) Eggs (standard)-jars each contained whole
E. kuehniella eggs (3 mg, approx. 150 eggs), which
were used as a standard in the bioassay because
they are widely used by commercial insectaries
for rearing predators; (2) Diet (control)-jars con-
tained artificial diet with no additional sub-
stances; and (3) Diet (amended)-jars contained
artificial diet supplemented with combined frac-
tions 1-5, 12-16, or 17-20, or individual fractions 6
through 11 as separate treatments.

Data Analysis

Each treatment was replicated 4 times. Egg
counts (eggs/female) were adjusted for female
mortality within each treatment. Data were ana-

lyzed by ANOVA with StatMost software (Datax-
iom Software, Inc.). Dunnett's test was used to de-
termine if the number of eggs laid per female on
each of the diet treatments supplemented with
the isoelectric focusing fractions was significantly
greater than the number of eggs laid per female
on the Diet (control).


Figure 1 shows the protein profile versus pH of
the IPLB-PiE proteins separated in a pH gradient
of 3-10. The average rate of eggs oviposited per fe-
male was highly significant relative to the Diet
(control) (P < 0.01) in fractions 7 through 9 with
isoelectric points ranging from pH 5.2 to 6.1 and
significant (P < 0.05) in fractions 10 and 11, pH
6.8 and 7.3 (Fig. 2). The active fractions contained
12.6 mg or 40.3% of the total protein (31.4 mg) re-
covered in all the fractions; the remaining frac-
tions contained 18.8 mg of the total protein and
no associated activity. Recovery of the total pro-
tein applied to the gradient was 82.6% (38 mg ap-
plied; 31.4 mg recovered); light precipitates in
fractions 7-11 after isoelectric focusing resulted
in a loss of protein in these fractions when the
samples were dialyzed and concentrated. SDS-
PAGE analysis of the fractions is shown in Fig. 3.
Of the active fractions (fractions 7-11), fractions
10 and 11 had fewest bands and displayed 4 eas-
ily discernible bands in a relative molecular
weight range of 34-133k. Treatment of the cell ho-
mogenate with proteinase K revealed a loss of the
cellular proteins on an SDS-PAGE gel (Fig. 4);
however, females fed proteinase K-treated cells
showed oviposition rates similar to those fed un-
treated cells (Fig. 5).

0 -H 10


It- i 4

1 2 3 4 5 S 7 8 9 1011121314 15117181920
Fig. 1. Protein profile of IPLB-PiE cellular homoge-
nate separated by isoelectric focusing on a pH gradient
of 3-10. Fractions that were combined for bioassay in ar-
tificial diet are shown by vertical dotted lines. Arrows
indicate range of fractions with ovipositional stimulat-
ing activity and pH values indicate isoelectric points.

Florida Entomologist 90(2)

10 Mr

S 97k
0 45k



- *e

Fig. 2. Average number of eggs oviposited by females
of 0. insidiosus after being fed artificial diet supple-
mented with fractions from isoelectric focusing separa-
tion of IPLB-PiE cellular homogenate shown in Fig. 1.
Eggs (standard), whole eggs of E. kuehniella; Diet (con-
trol); Diet (amended), combined and individual frac-
tions were each biossayed in separate diet treatments;
error bars refer to standard error; ** indicates P < 0.01.


The fecundity of 0. insidiosis females was im-
proved when fed diet supplemented with 6 of the
20 fractions from isoelectric focusing separation
of the homogenate of IPLB-PiE cells. This indi-
cated that the effect was not due to a protein defi-
ciency, since all of the fractions contained protein.
Moreover, the activity was limited to fractions in
a pH range of 5.2-7.3 and was associated with
40% of the total protein recovered. Since multiple
bands were present in all the active fractions, the
fecundity-promoting activity could not be attrib-
uted to a specific polypeptide. However, active

116k am k-1

21k 4 W

Std 1-5 6 7 8 9 10 11 bk 3bk CP
Fig. 3. SDS-PAGE analysis of fractions separated as
shown in Fig. 1. MW standards (Std); combined frac-
tions 1-5; individual fractions 6-11; blank (bk); com-
bined fractions 12-16; blank (bk); combined fractions
17-20; blank; crude protein (CP). Twenty micrograms of
protein applied per lane.



1 4 k q

Std 1 2 3 4

Fig. 4. SDS-PAGE analysis of fractions separated as
shown in Fig. 1. MW standards (Std); lanes: 1) blank, 2)
untreated, 3) without proteinase K but incubated at 37
for 18 h, and 4) with proteinase K at 37C for 18 h. Ten
micrograms of protein applied per lane.

fractions 10 and 11 contained fewer bands, with 2
densely staining bands at MW 156k, 69k, 42, and
34k. A recent study that used whole eggs of
E. kuehniella and the same procedure employed
in this study found only 1 active fraction associ-
ated with 16% of the total recovered protein at a
pH of 5 (Ferkovich & Shapiro 2005). In addition,
SDS-PAGE analysis of the whole egg fractions re-
vealed the presence of 1 major protein band with
MW 47k and other faint bands at 163k, 51k, 39k,
31k, and 27k. Other egg components such as ex-
tracts of egg lipids and nucleic acids (DNA and
RNA) had no effect on the ovipositional rate nor
did other non-insect proteins such bovine serum
albumin and hen egg albumin tested at similar
The results of this study suggest that the
IPLB-PiE cell line, originally derived from em-
bryos of P interpunctella, has retained a differen-
tiated function in culture and produces products
similar to those synthesized in the P interpunc-
tella egg. We now know that the active material is
at least associated with cellular proteins. Diges-
tion of the proteins did not result in a loss of ac-
tivity, indicating that none of the basic nutritional
components of the cell homogenate were de-
stroyed by the enzyme. The activity could be asso-
ciated with a peptide fragment resulting from the

June 2007

Ferkovich & Shapiro: Improved Fecundity of Orius insidiosus

2 6.0.
* 5.5*
'- 4.5.
o 3.5-
o 2.5-
, 2.0-
-0 1.5.
E 1.0.
Z 0.5.

** **



Fig. 5. Average number of eggs oviposited by females
of 0. insidiosus after being fed artificial diet alone
(Diet), and diet supplemented with fresh PiE cells (PiE),
PiE cells incubated at 37C for 18 h (PiE heat-treated),
and PiE cells incubated with proteinase K at 37C for 18
h (PiE Pk-treated). Error bars refer to standard error;
** indicates P < 0.01.

proteolytic digestion or a ligand carried by one
the proteins. Proteins in diets not only provide
amino acids as nutrients, but also serve to func-
tionally bind lipids, ions, enzyme co-factors, and
flavors, act as emulsifiers and film-formers be-
tween diet components, and have buffering and
stabilizing effects on diet components (Cohen
2004), none of which was addressed in this study.
An alternative possibility for the enhanced fecun-
dity that was observed is that proteins in insect
diets may provide other needed nutrients or fac-
tors such as the "token stimuli" described by
Cohen (2004), which stimulate predators to feed
on diets. Although the nature of active material is
unknown, this information provides a new ave-
nue for isolation and identification of the fecun-
dity-enhancing substance.


We appreciate the excellent technical assistance of
Delaine Miller in this study.


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June 2007

Hall et al.: Trapping Asian Citrus Psyllid


'USDA, ARS, U. S. Horticultural Research Laboratory, Subtropical Insects Research Unit
2001 South Rock Road, Fort Pierce, FL 34945

2USDA APHIS PPQ CPHST, Pest Detection Diagnostics and Management Laboratory
22675 N. Moorefield Rd., Bldg. 6414, Edinburg, TX 78541-9398


Studies were conducted at 2 different field sites to compare yellow sticky card traps, blue
sticky card traps, Multi-Lure traps, and CC traps (red, blue, black, white, yellow, and dark
green bases) for monitoring adult Asian citrus psyllid, Diaphorina citri Kuwayama, in cit-
rus. The Multi-Lure and CC traps were charged with either ethylene glycol or a dichlorvos
kill strip to kill psyllids entering the trap. We also investigated a stem tapping method for
monitoring adult D. citri. Yellow sticky card traps captured significantly more adults than
blue sticky card traps over a 4-week period in one study but not the other. Over all sample
weeks, each of these traps captured significantly greater numbers of adults than any of the
other traps. Yellow and blue sticky traps were equally effective in detecting the presence of
adults in trees given the infestation levels present at the 2 study sites. The CC and Multi-
Lure traps captured so few adult psyllids and provided such poor detection of trees infested
by adults that they appeared to have no value for monitoring D. citri. Tap sampling was easy
to conduct and provided relatively good detection of trees infested by adults given the infes-
tation levels present at the 2 groves. An advantage to stem tap sampling over sticky trap
sampling is that tap sampling provides information on the presence and relative abundance
of adult D. citri during a single visit to a block of trees while sticky trap sampling requires
2 visits. Research to develop standard protocols for sticky trap and stem tap sampling for
adult D. citri in citrus would be advantageous.

Key Words: trapping, sampling, sticky traps, Multi-Lure, CC trap


Se realizaron studios en dos campos diferentes para comparar las trampas de tarjetas pega-
josas del color amarillo, trampas de tarjetas pegajosas de color azul, trampas "Multi-lure" y
trampas "CC" (con bases de color rojo, azul, negro, blanco, amarillo y verde oscuro) para el mo-
nitoreo de adults del psilido Asidtico de los citricos, Diaphorina citri Kuwayama en citricos.
Las trampas Multi-Lure y CC fueron cargadas con un "kill strip" (una plancha para matar)
que contenia ya sea etilenglicol o diclorovos para matar los psilidos que entraban a las tram-
pas. Tambi6n, investigamos un m6todo de trampa de pegado al tallo para el monitoreo de los
adults de D. citri. Las trampas de tarjetas pegajosas de color amarillo capturaron significa-
tivamente mas adults que las trampas de tarjetas pegajosas de color azul durante el period
de 4 semanas en uno de los studios pero no en el otro. En todas las semanas de muestreo,
cada una de estas trampas amarillas capturaron significativamente un numero mayor de
adults que cualquiera de las otras trampas. Las trampas pegajosas de los colors amarillo y
azul fueron igualmente efectivas en detectar la presencia de adults en arboles teniendo en
cuenta el nivel de infestaci6n present en los dos sitios del studio. Las trampas de "CC" y
"Multi-lure" capturaron muy pocos adults de psilidos y proveyeron una detecci6n tan pobre
de arboles infestados que pareciera indicar que no tienen ningun valor para realizar un mo-
nitoreo de D. citri. El muestreo de pega a los tallos fue facil de realizar y provey6 una detecci6n
relativamente buena de arboles infestados por adults teniendo en cuenta el nivel de infesta-
ci6n present en los 2 huertos. Una ventaja del muestreo de pega de tallos sobre el muestreo
usando trampas pegajosas es que el muestreo de pega de tallos provee informaci6n sobre la
presencia y la abundancia relative de los adults de D. citri durante una sola visit al bloque
de arboles mientas que el muestreo usando trampas pegajosas require 2 visits mas. Inves-
tigaciones para desarrollar protocolos estandardizados para el muestreo de adults de D. citri
usando trampas pegajosas y de pega de tallos de los citricos serian de gran provecho.

The Asian citrus psyllid, Diaphorina citri Ku- dispersed throughout the state (Michaud 2004).
wayama, was first discovered in Florida during D. citri has a wide host range within the plant
Jun 1998 (Tsai et al. 2000), and it subsequently family Rutaceae, including citrus and citrus rela-

Florida Entomologist 90(2)

tives such as orange jasmine, Murraya panicu-
lata (L.) Jack (Halbert & Manjunath 2004). Ma-
ture citrus plants fed upon by D. citri can sustain
damage to growing shoots, while young plants
can suffer death during high psyllid populations
(Aubert 1987; Michaud 2004). Additionally, D.
citri vectors the causative bacterial agents (Can-
didatus Liberibacter asiaticus, C. L. africanus,
and C. L. americanus) of citrus greening disease
(huanglongbing), one of the world's most serious
diseases of citrus (McClean & Schwartz 1970;
Bove 2006). Trees infected by this devastating
disease may only live 5 to 8 years, during which
time they produce misshapen, inedible, and un-
marketable fruit (Bove 2006). Halbert & Manju-
nath (2004) provide a comprehensive overview of
citrus greening disease and D. citri biology. Citrus
greening was discovered in southern Florida dur-
ing late Aug 2005 and has since been detected at
a number of locations across the state's citrus
growing region (FDACS 2006). This sets the stage
for the spread of the disease into other citrus-pro-
ducing areas in North America.
A simple and efficient sampling procedure for
D. citri is vital to the development of a successful
IPM program aimed at controlling citrus green-
ing disease. The presence and relative abundance
of adult D. citri in a planting of citrus or orange
jasmine can be determined by counting adults on
plant samples (Tsai et al. 2000; Tsai et al. 2002).
Adults can be observed by tapping an infested
branch with a stick, which promotes adults to
drop onto a surface (e.g., a board or pan) held be-
neath the branch. A similar stem-tapping method
has been shown useful for monitoring pear psylla,
Cacopsylla pyricola (Foerster) in pear (Horton &
Lewis 1997). Sticky traps can be used to detect
and gauge the relative abundance ofD. citri (Aub-
ert & Quilici 1988; Aubert & Hua 1990). Prelimi-
nary research by Quilici & Trahais (1990) and
Aubert & Hua (1990) indicated D. citri was more
attracted to yellows than other colors, but specific
information on the attractiveness of other colors
was not presented. Working with sticky traps
hung 50 cm above the canopy of an orange jas-
mine planting, Aubert & Hua (1990) tested sticky
Rebell traps (similar to those marketed by Great
Lakes IPM, Vestaburg, MI) that were uniformly
Saturn yellow, bright yellow, orange yellow,
brown yellow, black or white in color and traps
that were checkered brown-yellow and bright yel-
low. These authors did not clarify the difference
between Saturn and bright yellow. Brown-yellow
colored traps performed best during cloudy
weather conditions, while bright yellow func-
tioned best during sunny conditions (Aubert &
Hua 1990). A plastic cup trap referred to as the
CC trap (named after C. Chu who developed the
trap) has been shown to be useful for monitoring
thrips, whiteflies and leafhoppers (Chu et al.
2000; Chu et al. 2006), and unidentified adult

psyllids have occasionally been caught in these
traps in St. Vincent (M. Ciomperlik, unpub-
lished). The Multi-Lure trap (Better World Manu-
facturing, Inc., Fresno, CA) has been useful in cit-
rus for monitoring fruit flies (Diptera: Tephriti-
dae) (Hall et al. 2005), but its efficacy for monitor-
ing adult D. citri is not known.
Although published information indicated yel-
low to be the most attractive color to adult D. citri
(Aubert & Hua 1990), quantitative data on the
relative attractiveness of blue sticky cards were
lacking. Therefore, the purpose of the research
presented here was to compare the efficacy of yel-
low and blue sticky traps, the Multi-Lure trap,
and the CC trap along with a stem tapping tech-
nique for monitoring adult D. citri in citrus.


The following traps were compared with re-
spect to numbers of adult D. citri trapped weekly:
yellow sticky cards, blue sticky cards, Multi-Lure
trap (clear top with standard yellow and white
base), and 6 CC traps (clear top with a blue, yel-
low, white, dark green, black, or red base). The
yellow sticky cards (7.62 x 12.7 cm) (a bright yel-
low hue similar to S-G-390 by Behr Process Corp.,
Santa Ana, CA), blue sticky cards (trimmed to
7.62 x 12.7 cm) (hue similar to 550B-6 by Behr
Process Corp.), and Multi-Lure traps were ob-
tained from Great Lakes IPM (Vestaburg, MI).
The CC traps were supplied by the Pest Detection
Diagnostics and Management Laboratory, Edin-
burg, TX (USDA, APHIS, Plant Protection and
Quarantine, Center for Plant Health Science and
Technology). Information on the spectral reflec-
tance of the CC trap colors is provided by Chu et
al. (2000). The colors of the CC trap bases were
similar to the following Behr Process Corp. hues:
blue 3C-20; yellow 310B-6; and red S-G-170. The
dark green CC trap base was a hue similar to
green 07GG 08/244 by Glidden (Cleveland, OH).

Experiment 1

The study was conducted in a USDA-ARS
grove near Ft. Pierce in St. Lucie County, Florida.
The block of trees chosen for the study contained
'Hamlin' orange trees (Citrus sinensis L.) (4 yr
old, ~2 m tall, row spacing 8 m, tree spacing 3 m).
No systemic or foliar hard insecticides were ap-
plied prior to the study during 2006 or during the
course of the study. Each trap was hung near the
exterior of a tree canopy about 1.5 m above
ground, 1 type of trap per tree. Sixteen trees
along each of 5 rows replicationss) were randomly
assigned one of the traps. Each row consisted of
21 to 40 trees. The test followed a randomized
complete block design with 5 replications. The
traps were deployed on May 11, 2006, and
checked weekly for 4 weeks. At the beginning of

June 2007

Hall et al.: Trapping Asian Citrus Psyllid

each week, the traps along each row were re-ran-
domized. One set of CC and Multi-Lure traps was
charged with 15 mL of a 50% pre-mixed solution
of ethylene glycol and water (Super Tech anti-
freeze, Bentonville, AR) as an entrapment and
preservative fluid for adult psyllids, and one set
was charged with Hercon Vaportape (10% dichlor-
vos, 0.229 g ai/cm2, 2.54 x 4.5-cm strip) (obtained
from Great Lakes IPM, Inc., Vestaburg, MI) as a
toxicant to kill adults entering a trap. A hole at
the center of the top of each CC trap allowed a
string to be attached to hang the trap from a
branch. No kick plates (Chu et al. 2006) were used
with the CC traps in this experiment. Sticky
cards were suspended from branches near the
outer edge of the canopy with a twist tie. When
the CC and Multi-Lure traps were checked for
psyllids, all the contents from the traps were
emptied into vials and transported to a labora-
tory. The number of psyllid adults per trap was
tabulated weekly. New sticky card traps were de-
ployed each week. The CC and Multi-Lure traps
were washed with soap and water each week be-
fore they were redeployed in the field.
Data on number of adults per trap per week
were analyzed by a multi-observation (measure-
ments over time) analysis of variance, and
Tukey's studentized range (honestly significant
difference, HSD) was used to determine signifi-
cant differences (a = 0.05) among traps. Prior to
these analyses, Levene's test was used to verify
homogeneity of variances (a = 0.05), and the data
were log-transformed where appropriate. The
percentage of trees in which adult D. citri was de-
tected with each type of trap was computed each
week. An analysis of variance over all weeks was
conducted on percentage detection (on arcsine
square-root-transformed data where appropriate
based on Levene's test), and Tukey's studentized
range (HSD) was used to determine significant
differences (a = 0.05) among traps. All analyses of
variance were conducted in PROC GLM (SAS In-
stitute, 2002) with the Levene and Tukey options.
In addition to trapping psyllids, adult D. citri
were monitored with stem tap samples in the
same trees in which the above traps were de-
ployed. This allowed a measure of adult abun-
dance in each tree based on both trap and tap
samples. A white metal pan (20.32 x 20.32 x 10.16
cm; length, width, and depth, respectively) was
held several cm under a haphazardly-chosen
branch (1.0-1.5 m above ground), and a polyvinyl
chloride (PVC) pipe (0.6 m length, 1.27 cm i.d.,
2.13 cm o.d.) was used to tap the branch 3 times.
All adult psyllids falling in the pan were counted.
Tap sampling was conducted on May 11 when the
above traps were deployed and at the end of each
week when traps were checked for adult psyllids.
The mean number of adult D. citri per tap sample
was computed for each tree from samples taken at
the beginning and end of each sample week. Data

were subjected to analyses of variance, and
Tukey's studentized range (HSD) was used to in-
vestigate for significant differences (a = 0.05) in
numbers of adults per tap sample among trees as-
signed the different trap types and to evaluate
dispersion of adults among trees. Prior to these
analyses, Levene's test was used to verify homo-
geneity of variances (a = 0.05), and the data were
log-transformed where appropriate. For trees as-
signed to each trap type, the percentage of trees
in which adult D. citri was detected each week
with tap sampling was computed. An analysis of
variance over all weeks was conducted among
trees assigned each type of trap on the percentage
of trees in which adult D. citri were detected by
tap sampling (on arcsine square-root-trans-
formed data where appropriate based on Levene's
test). Tukey's studentized range (HSD) was used
to determine significant differences (a = 0.05)
among trees assigned each type of trap with re-
spect to the percentage infested based on tap
sampling. All analyses of variance were con-
ducted in PROC GLM (SAS Institute, 2002) with
the Levene and Tukey options.

Experiment 2

A second study was conducted near Vero Beach
in Indian River County, Florida in a block of'Tem-
ple' orange trees [C. reticulate Blanco x C. sinensis
(L.) Osbeck] (36 yr old, ~3.4 m tall, row spacing 9
m, tree spacing 5 m). No systemic or foliar hard
pesticides were applied prior to the study during
2006 or during the course of the study. An appli-
cation of a nutritional spray including 470 oil (71
L per ha) was applied ~1 h before the traps were
placed in the field during the first week of the
study. However, the intent of the experiment was
to judge relative numbers of adult D. citri col-
lected at traps and during tap sampling, not to as-
sess the effects of the treatment against the psyl-
lid. This study was similar to Experiment 1 in all
respects except each of the 5 replicates consisted
of 3 rows of trees (21 to 26 trees per replicate), and
a yellow CC trap with a kick plate (Chu et al.
2006) and charged with ethylene glycol was
added to the study. This study was initiated Jun
29, 2006, and ran for 4 weeks.


Experiment 1

Heterogeneity in variances was detected in
numbers of adult D. citri per trap per week for
data over all sample weeks (F = 3.39, Pr > F =
<0.0001, 15 df) and for data from week 3 (F = 2.70,
Pr > F = 0.0030, 15 df) (analyses on other weeks
not presented). Heterogeneity was detected in
mean numbers of adults captured from week-to-
week for data from blue sticky card traps (F =

Florida Entomologist 90(2)

3.30, Pr > F = 0.05, 3 df) but not for data from yel-
low sticky card traps (F = 1.76, Pr > F = 0.20, 3 df)
(analyses on other traps not presented). Vari-
ances were homogeneous with respect to numbers
of adults per stem tap sample for data over all
sample weeks (F = 0.92, Pr > F = 0.54, 15 df) and
for data from week-to-week (F = 0.78, Pr > F =
0.52, 3 df). Data analyses on percentages of trees
in which adult D. citri were detected with traps
and stem tap sampling indicated heterogeneity in
variances associated with traps (F = 3.32, Pr > F
= 0.001, 15 df) but not tap sampling (F = 1.47, Pr
> F = 0.16, 15 df).
There was no significant difference over the 4-
week study with respect to numbers of adults cap-
tured at yellow and blue sticky card traps (Table
1, F = 4.73, Pr > F < 0.0001, 318 df). Each of these
sticky card traps captured significantly more
adult D. citri than any of the other traps. There
were no significant differences from week-to-week
in captures of adults at yellow sticky traps (F =
1.67, Pr > F = 0.21, 19 df) or blue sticky traps (F =
1.60, Pr > F = 0.23, 19 df) (data for other traps not
presented). Low numbers of adults were captured
in the CC and Multi-Lure traps, and there were
no significant differences among any of these
traps with respect to numbers of adults captured.
There was no evidence of any difference with re-

spect to charging CC and Multi-Lure traps with
ethylene glycol or dichlorvos strips. Means SEM
of 1.6 0.1, 1.2 + 0.1, 1.1 0.1, and 1.3 + 0.1 adults
per stem tap sample were observed across all
trees during sample weeks 1, 2, 3 and 4, respec-
tively. There were no significant differences
among these weekly means (F = 1.45, Pr > F =
0.22, 39 df). Means of 1.6 0.4, 1.0 + 0.3, 0.7 0.4
and 0.9 0.2 adults per tap sample were observed
in trees with yellow sticky traps during weeks 1,
2, 3 and 4, respectively, and means of 2.4 0.7, 1.0
+ 0.3, 2.1 0.5 and 0.6 0.2 adults per tap sample
were observed in trees with blue sticky traps dur-
ing the same respective weeks. Mean numbers of
adults per tap sample did not differ significantly
from week-to-week in trees with yellow sticky
traps (F = 1.67, Pr > F = 0.21, 19 df) nor in trees
with blue sticky traps (F = 2.55, Pr > F =0.09, 19
df) (analyses for tap samples taken in trees with
other trap types not presented). No significant dif-
ferences (F = 1.07, Pr > F = 0.34, 319 df) were ob-
served in mean numbers of adult psyllids per tap
sample among trees assigned the different types
of traps (Table 1). Adult D. citri were collected on
yellow and blue sticky card traps in every tree
sampled (Table 2). Percentage detection of trees
infested by adults with the other trap types
ranged from 10 to 40% (no significant differences).


Mean number
(SEM) per tap
Mean number (SEM) adults per trap per treeb sample per tree"

Type of trap in treed Week 1 Week 2 Week 3 Week 4 Overall' Overall

Yellow sticky card 9.2 (2.4) a 10.8 (4.3) a 3.6 (1.4) a 5.2 (0.9) a 7.2 (1.4) a 1.1 (0.2) a
Blue sticky card 6.2 (0.6) a 6.0 (1.3) ab 4.2 (1.4) a 2.8 (0.7) a 4.8 (0.6) a 1.5 (0.3) a
Yellow CC EG 0.4 (0.4) b 0.8 (0.6) bc 0.6 (0.2) b 0.4 (0.4) b 0.6 (0.2) b 1.0 (0.2) a
Multi-Lure DC 0.8 (0.4) b 1.0 (0.5) bc 0.2 (0.2) b 0.2 (0.2) b 0.6 (0.2) b 1.0 (0.2) a
Blue CC DC 0.6 (0.4) b 1.0 (0.3) bc 0.2 (0.2) b 0.0 (0.0) b 0.5 (0.2) b 1.5 (0.2) a
Red CC EG 0.8 (0.4) b 0.6 (0.2) bc 0.2 (0.2) b 0.0 (0.0) b 0.4 (0.1) b 1.3 (0.3) a
Green CC DC 0.6 (0.2) b 1.0 (0.5) bc 0.0 (0.0) b 0.0 (0.0) b 0.4 (0.2) b 1.1(0.1) a
Green CC EG 0.6 (0.6) b 0.2 (0.2) c 0.2 (0.2) b 0.0 (0.0) b 0.3 (0.2) b 1.4 (0.2) a
Black CC DC 0.0 (0.0) b 0.2 (0.2) c 0.6 (0.2) b 0.2 (0.2) b 0.3 (0.1) b 1.2 (0.2) a
Black CC EG 0.2 (0.2) b 0.0 (0.0) c 0.0 (0.0) b 0.4 (0.2) b 0.2 (0.1) b 1.2 (0.2) a
White CC EG 0.4 (0.4) b 0.4 (0.2) bc 0.0 (0.0) b 0.0 (0.0) b 0.2 (0.1) b 1.5 (0.2) a
White CC DC 0.0 (0.0) b 0.4 (0.2) bc 0.0 (0.0) b 0.2 (0.2) b 0.2 (0.1) b 1.6 (0.2) a
Yellow CC DC 0.2 (0.2) b 0.2 (0.2) c 0.0 (0.0) b 0.2 (0.2) b 0.2 (0.1) b 1.3 (0.2) a
Red CC DC 0.2 (0.2) b 0.4 (0.4) bc 0.0 (0.0) b 0.0 (0.0) b 0.2 (0.1) b 1.3 (0.2) a
Blue CC EG 0.2 (0.2) b 0.2 (0.2) c 0.0 (0.0) b 0.0 (0.0) b 0.1(0.1) b 1.3 (0.2) a
Multi-Lure EG 0.2 (0.2) b 0.0 (0.0) c 0.2 (0.2) b 0.0 (0.0) b 0.1 (0.1) b 1.2 (0.2) a

"Means in the same column followed by the same letter are not significantly different (a = 0.05), Tukey's test.
bFor traps-1 trap per tree, 16 trees with traps per replication, 5 replications. Tap sampling was conducted weekly in each tree
with a trap.
'Weekly mean number of adult D. citri observed in tap samples taken in the trees assigned to each specific type of trap.
CC = CC trap; CC and Multi-Lure traps were charged with either EG (ethylene glycol) (15 ml of a 50% solution) or DC (dichlo-
rvos kill strip).
Analyses on log-transformed data, raw means presented.

June 2007

Hall et al.: Trapping Asian Citrus Psyllid


Mean (SEM) percentage trees
in which adults were detectedb

Type of trap in treec Trapsd Tap samples

Yellow sticky card 100.0 (0.0) a 80.0 (8.2) a
Blue sticky card 100.0 (0.0) a 90.0 (5.8) a
Multi-Lure DC 40.0 (4.2) b 80.0 (8.2) a
Yellow CC EG 35.0 (9.6) b 80.0 (8.2) a
Blue CC DC 35.0 (17.1) b 100.0 (0.0) a
Red CC EG 35.0 (15.0) b 85.0 (5.0) a
Green CC DC 30.0 (17.3) b 100.0 (0.0) a
Black CC EG 25.0 (12.6) b 100.0 (0.0) a
Green CC EG 15.0 (5.0) b 85.0 (9.6) a
Black CC DC 15.0 (9.6) b 90.0 (10.0) a
White CC EG 15.0 (9.6) b 95.0 (5.0) a
White CC DC 15.0 (9.6) b 95.0 (5.0) a
Yellow CC DC 15.0 (5.0) b 85.0 (9.6) a
Red CC DC 10.0 (5.8) b 90.0 (10.0) a
Blue CC EG 10.0 (5.8) b 85.0 (9.6) a
Multi-Lure EG 10.0 (5.8) b 80.0 (11.5) a

aFor traps-1 trap per tree, 16 trees with traps per replica-
tion, 5 replications. Tap sampling was conducted weekly in
each tree with a trap.
bMeans in the same column followed by the same letter are
not significantly different (a = 0.05), Tukey's test.
'CC = CC trap; CC and Multi-Lure traps were charged with
either EG (ethylene glycol) (15 ml of a 50% solution) or DC
(dichlorvos kill strip).
dAnalyses on arcsine square-root transformed percentages
(raw percentages presented).
'Percentage of trees in which adult D. citri were detected in
tap samples taken in the trees assigned to each specific type of

Overall, tap sampling indicated 88.8% of the trees
studied were infested by adults. There were no
significant differences among trees with each type
of trap with respect to the percentage identified as
being infested by tap sampling (Table 2). Stem tap
samples failed to detect a small percentage of in-
fested trees that were identified as being infested
by yellow and blue sticky traps.

Experiment 2

Heterogeneity in variances was detected in
numbers of adult D. citri per trap per week for
data over all sample weeks (F = 4.12, Pr > F =
<0.0001, 16 df) and for data from each of the 4
weeks separately (analyses on individual weeks
not presented). Heterogeneity was detected in
mean numbers of adults captured from week-to-
week for data from both blue (F = 44.19, Pr > F =
<0.0001, 3 df) and yellow sticky traps (F = 8.73, Pr
> F = 0.001, 3 df) (analyses on other traps not pre-
sented). Variances were homogeneous with re-
spect to numbers of adults per stem tap sample for

data over all sample weeks (F = 0.72, Pr > F = 0.77,
16 df) and for data from week-to-week (F = 0.3.1,
Pr > F = 0.06, 3 df). Data analyses on percentages
of trees in which adult D. citri were detected indi-
cated no heterogeneity in variances associated
with either traps (F = 0.86, Pr > F = 0.61, 16 df) or
tap sampling (F = 1.56, Pr > F = 0.12, 16 df).
Yellow sticky card traps captured significantly
more adult D. citri over the 4-week study than
blue sticky card traps, and each of these traps
captured significantly more adult D. citri over the
4-week study than any of the other traps (Table 3,
F = 2.78, Pr > F <0.0001, 338 df). There were no
significant differences in numbers of adults col-
lected each week at yellow and blue sticky traps.
One blue sticky card trap was found to be missing
when the traps were checked on Jul 20. There
were no significant differences from week-to-
week in captures of adults at blue sticky traps (F
= 2.14, Pr > F = 0.13, 18 df), but significantly
greater numbers of adults were collected at yel-
low sticky traps during weeks 1 and 4 than during
weeks 2 and 3 (F = 3.97, Pr > F = 0.02, 19 df) (data
for other traps not presented). Numbers of adults
captured at the CC and Multi-Lure traps were
consistently low, and there were no significant dif-
ferences among any of these traps with respect to
numbers of adults captured. There was no evi-
dence of any difference with respect to charging
CC and Multi-Lure traps with ethylene glycol or
dichlorvos strips. Means of 2.36 + 0.2, 1.2 + 0.1,
0.8 0.1, and 2.2 + 0.3 adults per stem tap sample
were observed across all trees during sample
weeks 1, 2, 3 and 4, respectively, and significant
differences were found between these weekly
means (F = 2.55, Pr > F = 0.03, 39 df). Means of
2.2 0.3, 0.5 + 0.2, 0.9 + 0.3 and 2.2 1.0 adults
per tap sample were observed in trees with yellow
sticky traps during weeks 1, 2, 3 and 4, respec-
tively, and means of 2.9 + 0.7, 1.1 + 0.4, 1.6 0.9
and 2.1 0.8 adults per tap sample were observed
in trees with blue sticky traps during the same re-
spective weeks. Week-to-week means varied sig-
nificantly for data from trees with yellow sticky
traps (F = 4.38, Pr > F = 0.02, 19 df) but not for
data from trees with blue sticky traps (F = 2.11,
Pr > F = 0.14, 18 df) (analyses for tap samples
taken in trees with other trap types not pre-
sented). No significant differences (overall F =
1.35, Pr > F = 0.04, 339 df; main effect trap F =
0.40, Pr > F = 0.98, 16 df) were observed in mean
numbers of adult psyllids per tap sample among
trees assigned the different types of traps (Table
3). Adult D. citri were collected on yellow and blue
sticky card traps in 95 and 85%, respectively, of
the trees sampled (Table 4). Percentage detection
of trees infested by adults with the other trap
types ranged from 15 to 50% (no significant differ-
ences). Overall, tap sampling indicated 81.5% of
the trees studied were infested during the study.
There were no significant differences among trees

Florida Entomologist 90(2)


Mean number
(SEM) per tap
Mean number (SEM) per trap per treeb sample per tree"

Type of trap in treed Week 1 Week 2 Week 3 Week 4 Overall Overall

Yellow sticky card 20.6 (4.7) a 5.8 (2.9) a 4.0 (1.6) a 29.0 (13.7) a 14.8 (4.2) a 1.5 (0.3) a
Blue sticky card 10.3 (4.2) ab 4.0 (1.9) ab 2.8 (1.2) ab 5.0 (0.8) ab 5.3 (1.6) b 1.9 (0.4) a
Yellow CC KP EG 3.2 (1.2) bc 0.2 (0.2) c 0.4 (0.2) bc 2.4 (2.2) bc 1.6 (0.6) c 1.6 (0.4) a
Yellow CC EG 1.8 (0.6) cd 0.6 (0.4) bc 0.2 (0.2) bc 1.6 (1.6) c 1.1 (0.4) c 1.7 (0.3) a
Multi-Lure DC 0.8 (0.2) cd 0.4 (0.2) c 0.2 (0.2) bc 1.6 (0.8) bc 0.8 (0.2) c 1.4 (0.2) a
White CC EG 1.0 (0.6) cd 0.2 (0.2) c 0.8 (0.6) bc 0.2 (0.2) c 0.6 (0.2) c 1.8 (0.3) a
Blue CC EG 1.6 (0.6) cd 0.2 (0.2) c 0.2 (0.2) bc 0.2 (0.2) c 0.6 (0.2) c 1.5 (0.4) a
Green CC DC 0.6 (0.4) cd 0.4 (0.2) c 0.2 (0.2) bc 0.8 (0.5) c 0.5 (0.2) c 1.8 (0.5) a
Multi-Lure EG 0.8 (0.2) cd 0.4 (0.2) c 0.2 (0.2) bc 0.4 (0.2) c 0.5 (0.1) c 1.3 (0.3) a
Black CC EG 1.4 (0.9) cd 0.0 (0.0) c 0.2 (0.2) bc 0.2 (0.2) c 0.5 (0.2) c 1.4 (0.3) a
Red CC EG 0.6 (0.4) cd 0.2 (0.2) c 0.2 (0.2) bc 0.4 (0.2) c 0.4 (0.1) c 1.7 (0.4) a
Green CC EG 0.2 (0.2) d 0.8 (0.4) bc 0.0 (0.0) c 0.2 (0.2) c 0.3 (0.1) c 1.4 (0.3) a
Yellow CC DC 0.6 (0.2) cd 0.2 (0.2) c 0.2 (0.2) bc 0.0 (0.0) c 0.3 (0.1) c 1.4 (0.3) a
White CC DC 0.0(0.0) d 0.4 (0.2) c 0.0 (0.0) c 0.6 (0.4) c 0.3 (0.1) c 1.7 (0.7) a
Blue CC DC 0.4 (0.4) cd 0.0 (0.0) c 0.0 (0.0) c 0.6 (0.4) c 0.3 (.01) c 1.7 (0.4) a
Black CC DC 0.6 (0.2) cd 0.0 (0.0) c 0.0 (0.0) c 0.4 (0.4) c 0.3 (0.1) c 2.0 (0.4) a
Red CC DC 0.2 (0.2) d 0.2 (0.2) c 0.0 (0.0) c 0.2 (0.2) c 0.2 (0.1) c 2.0 (0.4) a

*Means in the same column followed by the same letter are not significantly different (a = 0.05), Tukey's test.
For traps-1 trap per tree, 17 trees with traps per replication, 5 replications. Tap sampling was conducted weekly in each tree
with a trap. Analyses on log-transformed data, raw means presented.
Weekly mean number of adult D. citri observed in stem tap samples taken in the trees assigned to each specific type of trap.
dCC = CC trap; KP = kickplate attached; CC and Multi-Lure traps were charged with either EG (ethylene glycol) (15 ml of a 50%
solution) or DC (dichlorvos kill strip).

with traps with respect to the percentage identi-
fied as being infested by tap sampling (Table 4).
Stem tap samples failed to detect a small percent-
age of infested trees that were identified as being
infested by yellow sticky traps. Blue sticky traps
failed to identify a small percentage of trees that
were identified as being infested by tap sampling.


Numerically greater numbers of adult D. citri
were usually captured each week with yellow
sticky card traps than blue sticky card traps, but
statistical differences in numbers captured were
only found during the second study across all 4
weeks of the study. Significant differences over all
study weeks during the first study and during the
individual weeks of each study might have been
found had we used more than 5 replications of
each type of trap. Previous studies indicated yel-
low sticky traps capture more adult D. citri than
sticky traps of other colors, and traps of a bright
yellow hue captured more adults than traps of a
brown yellow hue under sunny conditions (Aubert
& Hua 1990). We did not investigate the occur-
rence of clouds during our studies, but sunlight
may have contributed to increased captures of
adults at yellow sticky traps during some weeks.

Yellow and blue sticky traps were equally effec-
tive in detecting the presence of adult D. citri in
trees given the infestation levels present. The CC
and Multi-Lure traps studied captured so few
adult psyllids and provided numerically such low
levels of percentage detection of trees infested by
adults that they appeared to have no value for
monitoring D. citri. Additional advantages for
sticky cards to detect psyllids were that they were
inexpensive, readily available, and relatively
easy to work with.
Significant fluctuations from week-to-week
were observed in numbers of adult D. citri col-
lected at yellow sticky traps during the second
study, and these fluctuations were reflected in
stem tap samples across all trees with traps. We
attributed these fluctuations to suppression of
psyllids by the spray oil treatment. By the fourth
week, developing nymphs had matured to adults,
thus contributing to the increased adult popula-
tion. No significant fluctuations from week-to-
week were observed in numbers of adults cap-
tured at blue sticky traps during the second
study. Reasons were unknown why increased
numbers of adult D. citri were observed at the end
of the second study both at yellow sticky traps
and during tap sampling but not at blue sticky
traps. These differences may have been related to

June 2007

Hall et al.: Trapping Asian Citrus Psyllid


Mean (SEM) percentage trees
in which adults were detectedb

Type of trap in treec Traps Tap samples

Yellow sticky card 95.0 (5.0) a 85.0 (5.0) a
Blue sticky card 85.0 (9.6) ab 95.0 (5.0) a
Yellow CC KP EG 50.0 (17.3) abc 75.0 (15.0) a
Multi-Lure DC 50.0 (12.9) abc 80.0 (11.5) a
Yellow CC EG 40.0 (14.1) bc 80.0 (8.2) a
Multi-Lure EG 45.0 (12.6) bc 75.0 (9.6) a
Blue CC EG 35.0 (15.0) c 70.0 (19.1) a
Green CC DC 35.0 (5.0) c 85.0 (15.0) a
White CC EG 30.0 (5.8) c 85.0 (9.6) a
Red CC EG 30.0 (5.8) c 75.0 (15.0) a
Black CC EG 25.0 (12.6) c 80.0 (8.2) a
Green CC EG 25.0 (12.6) c 75.0 (12.6) a
Yellow CC DC 25.0 (12.6) c 85.0 (9.6) a
White CC DC 20.0 (11.5) c 90.0 (5.8) a
Blue CC DC 20.0 (9.6) c 75.0 (15.0) a
Black CC DC 20.0 (14.1) c 85.0 (15.0) a
Red CC DC 15.0 (5.0) c 90.0 (5.8) a

*For traps-1 trap per tree, 17 trees with traps per replica-
tion, 5 replications. Tap sampling was conducted weekly in
each tree with a trap.
bMeans in the same column followed by the same letter are
not significantly different (a = 0.05), Tukey's test.
'CC = CC trap; KP = kickplate attached; CC and Multi-Lure
traps were charged with either EG (ethylene glycol) (15 ml of a
50% solution) or DC (dichlorvos kill strip).
dPercentage of trees in which adult D. citri were detected in
tap samples taken in the trees assigned to each specific type of

sunlight or other environmental factors that af-
fect the attractancy of the yellow traps more than
blue traps. Additionally, adults may actually be
less attracted to blue traps but subject to being
accidentally captured at these traps during their
movement within trees, as supported by the fact
that the blue sticky traps caught similar numbers
of adults at each study site. However, it remained
possible that significant differences might have
been found from week-to-week in numbers of
adult D. citri on blue sticky traps had we studied
more than 5 blue traps each week.
We observed some non-target insect species on
both yellow and blue sticky traps but did not iden-
tify or quantify these. Various species of Diptera
including the love bug, Plecia nearctica Hardy,
have sometimes been captured in large numbers
on yellow sticky card traps during other trapping
studies in citrus (D. G. Hall, unpublished). The
presence of other insects on sticky traps can inter-
fere with finding and counting adult D. citri on
the traps and may also interfere with captures of
D. citri. Whether blue traps might have less im-
pact on non-target insects in citrus than yellow

traps remains to be investigated. Other research-
ers have reported that color influences captures of
non-target insects. For example, Knight & Milic-
zky (2003) reported that the choice of trap color
affected numbers of honeybee (Aphis mellifera L.)
and non-target muscoid flies captured at sticky
delta traps used to monitor codling moth (Cydia
pomonella L.).
Our traps were hung directly in citrus trees.
Other researchers working with D. citri have
placed sticky traps on poles near plants or sus-
pended them above plants (Aubert & Hua 1990).
Where traps are placed in a citrus tree or grove
may affect their relative efficiency for monitoring
adult D. citri as well as other insects. This was
demonstrated by Dowell & Cherry (1981), who re-
ported that the location of sticky traps in citrus
trees affected captures of parasitoids and preda-
tors of citrus blackfly, Aleurocanthus woglumi
(Ashmead). Research to establish guidelines for
using sticky traps to detect and monitor adult
D. citri, including numbers of traps to operate
and how these traps should be allocated within
trees and across an area of trees, would be benefi-
cial to both growers and researchers.
Stem tap sampling was easy to conduct and
provided relatively good detection of trees in-
fested by adults, at least at the infestation levels
present at the 2 groves. Data from tap sampling
indicated adult psyllids were uniformly dispersed
among the trees studied, supporting the conclu-
sion that differences in numbers of adults col-
lected at the various types of traps were due to
differences in trap efficiency. An obstacle to stem
tap sampling was defining the force at which a
branch should be hit. Also, some adults flew be-
fore falling to the pan, and it was sometimes diffi-
cult to count all adults in the pan before they took
flight. Although week-to-week fluctuations in
mean numbers of adult D. citri per tap sample fol-
lowed the same trend in trees with yellow and
blue sticky traps during the second study, differ-
ences were only significant for data from trees
with the yellow traps. Larger numbers of trees
and tap samples per tree may be required for
mean estimates with less variability than were
obtained with a sample size of 1 tap sample per
week in 5 trees. Overall, however, the stem tap
sampling method appeared to provide a good rel-
ative measure of the presence and abundance of
adult D. citri and might be improved by placing a
cloth (e.g., see Horton & Lewis 1997) or sticky
card in the pan. An advantage to stem tap sam-
pling over sticky trap sampling is that tap sam-
pling provides information on the presence and
relative abundance of adult D. citri during a sin-
gle visit to a block of trees. Sticky trap sampling
requires 2 visits to a block of trees with a period of
time between visits (7 d in our study). Captures of
non-target insects was less an issue with stem tap
sampling to monitor adults than sticky trap sam-

Florida Entomologist 90(2)

pling. Research to develop formal protocols for tap
sampling would be advantageous. Of interest
would be optimum numbers of tap samples to
take across an area of trees and how these sam-
ples should be allocated within and among trees.
The ultimate decision of whether to use sticky
traps or stem tap sampling for adult D. citri in cit-
rus may depend on the intent of sampling and
cost. If one is simply interested in whether or not
adults are present in trees, stem tap sampling
may be preferable, at least at the infestation den-
sities of adults observed during these studies.


The authors thank and acknowledge the following
individuals for their assistance in this research: Kath-
ryn Moulton, Chris Knox, and Paula Hall (USDA-ARS,
U.S. Horticultural Research Laboratory, Fort Pierce,
FL); Robert Adair (Florida Research Center for Sustain-
able Agriculture, Vero Beach, FL); and Tom Higgins
(South Fork High School, Stuart, FL). Mention of trade
names or commercial products in this publication is
solely for the purpose of providing specific information
and does not imply recommendation or endorsement.


AUBERT, B. 1987. Trioza erytreae Del Guercio and Dia-
phorina citri Kuwayama Homoptera: Psylloidea),
the two vectors of citrus greening disease: biological
aspects and possible control strategies. Fruits 42:
AUBERT, B., AND X. Y. HUA. 1990. Monitoring flight ac-
tivity ofDiaphorina citri on citrus and Murraya can-
opies, pp. 181-187 In B. Aubert, S. Tontyaporn, and
D. Buangsuwon [eds.], Rehabilitation of citrus in-
dustry in the Asia Pacific Region. Proc. 4th Interna-
tional Asia Pacific Conference on Citrus
Rehabilitation, Chiang Mai, Thailand, 4-10 Febru-
ary 1990. FAO-UNDP, Rome.
AUBERT B., AND S. QUILICI. 1988. Monitoring adult psyl-
las on yellow traps in Reunion Island, pp. 249-254 In
L. W. Timmer, S. M. Garnsey, and L. Navarro [eds.],
Proc. 10th Conference of the International Organi-
zation of Citrus Virologists, Valencia, Spain, 17-21
November 1986. University of California, Riverside.
BoVE, J. M. 2006. Huanglongbing: a destructive, newly-
emerging, century-old disease of citrus. J. Plant
Pathol. 88: 7-37.
SHREPATIS. 2000. Use of CC traps with different trap
base colors for silverleaf whiteflies (Homoptera:

Aleyrodidae), thrips (Thysanoptera: Thripidae), and
leafhoppers (Homoptera: Cicadellidae). J. Econ. En-
tomol. 93: 1329-1337.
ARDS, AND T. J. HENNEBERRY. 2006. Developing and
evaluating traps for monitoring Scirtothrips dorsalis
(Thysanoptera: Thripidae). Florida Entomol. 89: 47-55.
DOWELL, R. V., AND R. H. CHERRY. 1981. Survey traps
for parasitoids and coccinellid predators of the citrus
blackfly, Aleurocanthus woglumi. Ent. Exp. & Appl.
29: 356-361.
SERVICES (FDACS). 2006. http://www.doacs.state.fl.
HALBERT, S. E., AND K. L. MANJUNATH. 2004. Asian cit-
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ing disease of citrus: a literature review and
assessment of risk in Florida. Florida Entomol. 87:
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for Caribbean fruit :i. A-..r.i repha suspense (Loew)
(Diptera: Tephritidae), in Florida citrus. J. Econ. En-
tomol. 98: 1641-1647.
HORTON, D. R., AND T. M. LEWIS. 1997. Quantitative re-
lationship between sticky trap catch and beat tray
counts of pear psylla (Homoptera: Psyllidae): sea-
sonal, sex, and morphotypic effects. J. Econ. Ento-
mol. 90: 170-177.
KNIGHT, A. L., AND E. MILICZKY. 2003. Influence of trap
colour on the capture of codling moth (Lepidoptera:
Tortricidae), honeybees, and non-target flies. J. En-
tomol. Soc. Brit. Columbia. 100: 65-70.
MCCLEAN, A. P. D., AND R. E. SCHWARTZ. 1970. Green-
ing of blotchy-mottle disease in citrus. Phytophylac-
tica 2: 177-194.
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QUILICI, S., AND B. TRAHAIS. 1990. Experiments on color
attractivity for the adults of Diaphorina citri Ku-
wayama, pp. 198-202 In Proc. 4th International Asia
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Mai, Thailand 4-10 February 1990. FAO-UNDP,
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June 2007

Yee et al: Insecticide Aging Effects on R. pomonella


'Yakima Agricultural Research Laboratory, United States Department of Agriculture
Agricultural Research Service, 5230 Konnowac Pass Road, Wapato, WA 98951

2Washington State University, Research and Extension Center, 7612 Pioneer Way E, Puyallup, WA 98371


The effects of field-aged residues of the new semi-synthetic spinosyn insecticide spinetoram
(Dow AgroSciences, Indianapolis, IN) and the spinosad bait GF-120 (Dow AgroSciences, In-
dianapolis, IN) on mortality of apple maggot fly, Rhagoletis pomonella (Walsh), were deter-
mined in Washington State. Mortality caused by spinetoram (100 g a.i./ha) sprayed on
apples and aged 7 d was significantly lower than that caused by fresh spinetoram. Spineto-
ram (100 and 75 g a.i./ha) aged for 7 d caused less than or as much mortality as spinosad (100
g a.i./ha) aged for 7 d. Fresh spinetoram and azinphos-methyl aged 7 or 14 d caused similar
mortality, but aged spinetoram and spinosad caused lower mortality than azinphos-methyl.
Apples treated with spinetoram (75 g a.i./ha) and with spinosad aged 7 d and exposed to flies
produced a few larvae. However, even though spinetoram (100 g a.i./ha) aged 14 d did not kill
all female flies, no larvae emerged from apples sprayed with this treatment. In separate
tests, 0-d, 3-d, and 7-d old GF-120 on apple leaves caused greater mortality than 14-d old
GF-120. Results show that spinetoram and GF-120 when fresh are highly toxic to R.
pomonella, but that both have relatively short residual activities under the hot, dry condi-
tions typical of central Washington in summer. Ingredients that prolong their toxicities or
make their toxins available to flies longer may be needed to optimize their performance.
Also, results suggest that adult fly mortality caused by spinetoram is not an accurate pre-
dictor of larval emergence from apples, and that possible non-lethal effects caused by spine-
toram need to be examined.

Key Words: apple maggot, bait sprays, spinosyn formulations, organophosphate, residual ac-


Se determinaron los efectos de residues envejecidos en el campo del insecticide spinosyn
semi-sint6tico spinetoram (Dow AgroSciences, Indianapolis, IN) y el cebo de spinosad GF-120
(Dow AgroSciences, Indianapolis, IN), sobre la mortalidad de la mosca de la manzana, Rhago-
letis pomonella (Walsh) en el estado de Washington (EEUU). La mortalidad causada por spi-
notoram (100 g i.a./ha) aplicada en manzanas y madurado por 7 dias fue significativamente
mas baja que la mortalidad causada por spinetoram fresco. Spinetoram (100 y 75 g i.a./ha)
madurado por 7 d caus6 una mortalidad menor o igual que spinosad (100 g i.a../ha) madurado
por 7 d. Spinetoram fresco y azinofosmetil madurado por 7 o 14 d caus6 una mortalidad simi-
lar, pero el spinetoram madurado y spinosad causaron una mortalidad mas baja que azino-
fosmetil. Las manzanas tratadas con spinetoram (75 g i.a./ha) y con spinosad madurado por
7 d y expuestas a moscas producieron muy pocas larvas. Sin embargo, aunque el spinetoram
(100 g i.a./ha) madurado por 14 d no mat6 todas las hembras de moscas, ninguna larva emer-
gi6 de manzanas rociadas con este tratamiento. En pruebas separadas de GF-120 madurado
por 0-d, 3-d y 7-d, en hojas de manzana caus6 una mayor mortalidad que en GF-120 madu-
rado por 14-d. Los resultados muestran que el spinetoram y GF-120 cuando esten frescos son
altamente t6xicos al R. pomonella, pero ambos tienen una actividad residual relativamente
corta bajo condiciones de alta temperature y secas tipicas del centro del estado de Washington
en el verano. Ingredientes que prolongan su toxicidad o hagan que sus toxinas sean disponi-
bles a las moscas por mas tiempo puede ser requeridos para optimizar su desempefio. Tam-
bien, los resultados sugerieron que la mortalidad de las moscas adults causada por
spinetoram no es un pronosticador precise para la emergencia de larvas de las manzanas, y
por otro lado se necesita examiner los posibles efectos no letales causados por spinetoram.

The apple maggot fly, Rhagoletis pomonella an emerging pest in residential apple and haw-
(Walsh), is a major pest of apple, Malus domestic thorn trees in central Washington State. The fly
(Borkh.) Borkh., in eastern North America and is has established in low numbers in this region,

Florida Entomologist 90(2)

based on larvae found in hawthorn and apple on
residential trees in 2003 and 2004. These findings
resulted in export quarantines in areas within
Kittitas and Yakima counties (Washington State
Department of Agriculture 2005), which are part
of the major apple-growing region in central
Washington. The establishment of the fly in this
region has major economic implications and
threatens the export of commercial apples from
Washington. The apple industry in Washington
was estimated at US$1.11 billion in 2004
(Garibay 2005). There is a zero tolerance for
R. pomonella larvae in apples transported within
the state and to many overseas markets (Wash-
ington State Department of Agriculture 2001).
Trap captures of R. pomonella adults in central
Washington result in the spraying of host trees to
suppress fly populations and reduce chances that
flies will move into commercial apple orchards.
Commercial apple orchards in central Washing-
ton to date have been free ofR. pomonella.
Conventional insecticides are considered the
leading candidates for controllingR. pomonella in
central Washington. Currently, the organophos-
phate imidan (phosmet) is being used in residen-
tial trees in this region. Similarly, the organo-
phosphates malathion and azinphos-methyl have
been used for many years to control R. pomonella
in the eastern (Neilson & Maxwell 1964; Neilson
& Sanford 1974) and western U.S. (Mohammad &
AliNiazee 1989). Despite the effectiveness of orga-
nophosphates, alternative materials are increas-
ingly important because of the impending phase-
out of organophosphate use due to the federal
Food Quality & Protection Act (FQPA) (1996). Be-
cause of their relatively high mammalian toxicity,
organophosphate insecticides may be hazardous
to use, especially around homes or near water
where fly-infested apple or hawthorn trees can oc-
cur. Effective organophosphates for controlling
R. pomonella are not available for residential use.
Newer and safer insecticides need to be tested
against R. pomonella. Laboratory bioassays with
the newer insecticides imidacloprid, indoxacarb,
pyriproxyfen, spinosad (85% spinosyn A and 15%
spinosyn D), thiacloprid, and thiamethoxam
showed that imidacloprid reduced oviposition the
most and that imidacloprid and spinosad were
the most toxic (Reissig 2003). However, these ma-
terials were not aged, and none of them appears
to equal the organophosphates in toxicity. The re-
sidual toxicity after field aging for any material
needs to be studied for several reasons. Some ma-
terials may be highly toxic initially when fresh
but lose that toxicity quickly. This affects the fre-
quency of insecticide applications and spray costs.
No study has determined the effects of aging
newer materials under the hot, dry central Wash-
ington conditions during summer on the toxicity
of these materials to R. pomonella. In addition,
even though resistance has never been docu-

mented in R. pomonella, overuse of one material
invites the potential for increased tolerance to in-
secticides among non-target pests of apple, such
as leafrollers and codling moth. Finally, the nega-
tive effects of insecticides on beneficial insects,
which probably are exacerbated by frequent in-
secticide use, are well documented (e.g., Williams
et al. 2003).
In this study, the objectives were to determine
the effects of aging new or newer spinosyn insec-
ticides, one incorporated into a bait mix, on the
mortality ofR. pomonella. Effects of aging insec-
ticides on damage to apples caused by the flies
were also determined. Tests focused on spineto-
ram, a new semi-synthetic spinosyn insecticide
developed by Dow AgroSciences (Indianapolis,
IN) that was accepted for expedited review under
the United States Environmental Protection
Agency's Reduced Risk Pesticide Program. Spine-
toram is derived from fermentation products of
the soil bacterium Saccharopolyspora spinosa
Mertz and Yao, has a high safety profile, may have
relatively long residual effects, and has never
been tested against R. pomonella. Tests were also
conducted with GF-120 Fruit Fly Bait (Dow Agro-
Sciences, Indianapolis, IN), which contains spi-
nosad, an insecticide that also has a high safety
profile (Dow AgroSciences 2002). Results are dis-
cussed with respect to residual toxicities of these
materials and their potential use in the manage-
ment ofR. pomonella in central Washington.


Effects of Field-Aging Spinetoram and Other
Insecticides on Fly Mortality and Apple Fruit Damage

Experiment 1 compared a control and spineto-
ram and 2 other insecticides at various rates and
ages: (1) untreated control, (2) a 100-g a.i./L sus-
pension concentrate (SC) formulation of spineto-
ram at 172 mL/935 L water/ha (100 g a.i/ha), aged
0 d, (3) spinetoram at 100 g a.i./ha, aged 7 d, (4)
spinetoram at 100 g a.i./ha, aged 14 d, (5) spineto-
ram at 127 mL/935 L water/ha (75 g a.i./ha), aged
7 d, (6) spinosad (Entrust 80 WP, Dow Agro-
Sciences, Indianapolis, IN) at 50.6 g/935 L water/
ha (100 g a.i./ha), aged 7 d, (7) azinphos-methyl
(Guthion 50 WP, Gowan Company, Yuma, AZ) at
908 g/935 L water/ha (1,121 g a.i./ha), aged 7 d,
and (8) azinphos-methyl at 1,121 g a.i./ha, aged 14
d. Spinosad and azinphos-methyl rates fell within
recommended field rates. Materials were used
within 1 year of receipt from the manufacturer.
'Fuji' apple trees at the United States Depart-
ment of Agriculture, Agricultural Research Ser-
vice (USDA, ARS) experimental orchard in
Moxee, WA (4633.23'N, 12023.50'W) were
sprayed with the various insecticide treatments
in Jul and Aug 2004 at 7 or 14 d before exposure
to flies. Control apples were from 1 tree and 0-d

June 2007

Yee et al: Insecticide Aging Effects on R. pomonella

treatment apples were from another tree. One
treatment was sprayed on apples on each of 7
other trees with 1.18 liter RL Flo-Master pres-
surized sprayers (Root-Lowell Manufacturing
Co., Lowell, MI) until thorough coverage was
achieved visually. Three different sets of trees
were used or sprayed for 3 tests: test 1A, insecti-
cides aged 13-27 Jul; test 1B, aged 3-17 Aug; and
test 1C, aged 31 Aug to 14 Sep. Mean high and low
temperatures and precipitation for tests 1A, 1B,
and 1C were (1) 33.5C and 12.4C and 8 mm, (2)
31.5C and 13.0C and 9 mm (over 2 days), and (3)
25.7C and 8.9C and 2 mm (on 1 day), respec-
tively. In tests 1A and 1B, rain occurred before 7-
d sprays were made and in test 1C, it occurred af-
ter 7-d sprays were made. Most days were sunny,
with the mean low humidity being -30% and the
daily mean being -50%.
Three to 6 apples, each from a different branch,
were removed from each tree -5-6 h before test-
ing. Apples were inserted through the calyxes
into single upright nails on a board to keep them
from rolling and contacting other surfaces and
then transported to the Washington State Uni-
versity Research and Extension Center
(WSUREC) in Puyallup in western Washington
for the experiment. Quarantine restrictions pre-
cluded the maintenance of R. pomonella in cen-
tral Washington for testing.
Flies were collected from feral and unmanaged
apple trees in Puyallup in glass vials. Flies were
maintained on dry 20% yeast extract (EZ Mix,
Sigma, St. Louis, MO) and 80% sucrose (wt/wt) on
paper strips ('food' hereafter) and on water in cot-
ton wicks inside 3.8-liter cylindrical paper con-
tainers (17.5 cm high x 17.0 cm diameter) for up
to 2 weeks before tests. This amount of time was
needed to accumulate enough flies for tests. For
testing, 1 control or treated apple was placed ca-
lyx end down on a shallow plastic dish inside a
3.8-liter container with food and water. For the 0-
d treatment (fresh spinetoram at 100 g a.i./ha),
apples were hung from a tree branch, sprayed,
and dried for 1 h before being placed inside con-
tainers. Ten flies-6 males and 4 females in test
1A and 5 males and 5 females in tests 1B and
1C-were then transferred into a test container.
Adult mortality was recorded from 1 to 10 d after
exposure. Flies were recorded as dead if they
could not walk when prodded or within 30 s of ob-
servation. Water vials were refilled every 2 d. Ap-
ple damage was measured as numbers of stings
on fruit (from probing or oviposition) and larval
emergence from fruit (an indication of larval in-
festation). Numbers of stings were counted under
a microscope and apples were weighed and their
circumferences measured at d 10. Each apple was
then placed in a clear plastic 550-ml capacity con-
tainer covered with organdy cloth. Numbers of
larvae that emerged from the apples over 4 weeks
were recorded. There were 3 replicates in test 1A,

6 in test 1B, and 3 in test IC. Laboratory test con-
ditions were 20-27C and 40-50% RH under a 16
h L: 8 h D cycle.

Effects of Field-Aging GF-120 on Fly Mortality

Experiment 2 tested GF-120 Fruit Fly Bait,
which is composed of 0.02% spinosad (wt/vol)
(Dow AgroSciences 2002) mixed in a bait of Solu-
lys corn protein, sugar, ammonium acetate, propy-
lene glycol, and other ingredients (Thomas &
Mangan 2005). An (1) untreated control and four
age treatments of 40% GF-120 (vol/vol) (recom-
mended rate, Dow AgroSciences 2002) were com-
pared: (2) 0-d old, (3) 3-d old, (4) 7-d old, and (5) 14-
d old. Trees used were 'Fuji' apple trees at the
USDA, ARS orchard in Moxee. One tree provided
control leaves and another tree provided 0-d treat-
ment leaves. One age treatment was sprayed on
each of 3 other trees. Leaves on 60-90 cm lengths
of 5 branches of each tree were sprayed with -10
mL of GF-120 in Jul and Aug 2004 with RL Flo-
Master pressurized sprayers. Two different sets
of trees were used or sprayed for 2 tests: test 2A
used GF-120 aged from 12-26 Jul and test 2B used
GF-120 aged from 2-16 Aug. Mean high and low
temperatures and precipitation during the 14 d of
aging for tests 2A and 2B were (1) 29.8C and
14.1C and 8 mm (1 d), and (2) 31.90C and 13.1C
and 9 mm (over 2 d), respectively. Most days were
sunny, and humidity values were similar to those
during aging for experiment 1. Three or 5 leaves,
each from a different branch, were removed from
each tree and placed inside plastic bags -5-6 h be-
fore testing at the WSUREC in Puyallup.
Flies were held and tested inside 473-ml paper
cartons (7 cm high x 5 cm diameter) (Neptune Pa-
per Products, Newark, NJ) covered with organdy
cloth and provided with food and water in the lab-
oratory. Flies used in test 2A were reared from
hawthorn fruit collected in 2003. Pupae had been
chilled for 6 months at 4C and then transferred
to 20-27C for adult emergence. Adults were held
for 2-4 weeks before testing. Flies used in test 2B
were collected from apple trees in Puyallup and
held for 2-3 weeks before testing. For testing, a
single control or treated leaf was placed inside a
carton. The 0-d old treatment was a leaf sprayed
with 1 mL of GF-120 1 h before testing. Ten flies
(5 of each sex) were then introduced. Mortality
was recorded daily, except for d 5 and 6 for both
tests (weekends), up until d 10. There were 3 rep-
licates of the control and treatments in test 2A
and 5 replicates of each in test 2B. Laboratory
test conditions were 25-27C and -40-50% RH
under a 16 h L: 8 h D cycle.

Data Analyses and Statistics

Repeated-measures analysis of variance
(ANOVA) was conducted on mortality data. One-

Florida Entomologist 90(2)

way ANOVA also was conducted on mortality data
within days and on apple injury data. Data from d
1-4, 7, and 10 and from d 1-4 and 7-10 in experi-
ments 1 and 2, respectively, are presented and an-
alyzed. Because there were few replicates in tests
1A and 1B and because weather conditions during
insecticide aging in these tests were similar, data
from these were pooled for analyses. Test 1C was
kept separate because of weather differences.
Tests 2A and 2B also were pooled because there
were few replicates per test and because weather
conditions during GF-120 aging were similar. Per-
centages were square-root and arcsine-trans-
formed before analyses. Sting and larval counts +
1 were subjected to square-root transformation.
Means in one-way ANOVA were separated by us-
ing the Tukey test (SAS Institute 2001). This con-
servative test was chosen because of the high
numbers of pairwise comparisons made.


Effects of Field-Aging Spinetoram and Other
Insecticides on Fly Mortality and Apple Fruit Damage

Repeated-measures ANOVA indicated there
were treatment and day differences, but there
were also significant treatment x day interactions
(tests 1A and 1B, F = 16.5, df= 7, 408, P < 0.0001;
test 1C, F = 17.0, df= 7, 126, P < 0.0001), indicat-
ing patterns of mortality among the treatments
differed on various days, even though mortality
increased over time across all treatments. In tests
1A and 1B (Table 1), one-way ANOVA of data
within days showed mortality caused by spineto-
ram (100) aged 7 d was significantly lower than

that caused by fresh spinetoram (100), up until d
7. There were no differences in mortalities caused
by spinetoram (100) aged 7 or 14 d until d 4-10,
when 7-d old residues caused greater mortality.
Spinetoram (75) aged 7 d was similar to spineto-
ram (100) aged 7 d but caused greater mortality
than spinetoram (100) aged 14 d at d 4-10. Spino-
sad (100) aged 7 d caused greater mortality than
spinetoram (100 and 75) aged 7 d at d 2 and 3, but
caused similar mortality on other days. Fresh
spinetoram usually was not different from aged
azinphos-methyl within days. Mortality in tests
1A and 1B and in test 1C differed on 2 of the 6 d,
and test number x treatment interactions oc-
curred on 3 of the days (two-way ANOVA, P <
0.05). In test 1C (Table 2), similar to tests 1A and
1B, mortality caused by spinetoram (100) aged 7 d
was significantly lower than that caused by fresh
spinetoram, up until d 4-10. Also similar, in test
1C there were significantly greater mortalities
caused by spinetoram (100) aged 7 than 14 d, this
time on all 6 d of exposure, and spinetoram (75)
aged 7 d caused lower mortality than spinetoram
(100) aged 7 d on all days except d 10. Similar to
tests 1A and 1B, spinetoram (75) aged 7 d caused
greater mortality than spinetoram (100) aged 14 d
at d 3 and 10. Unlike in tests 1A and 1B, however,
in test 1C, spinosad (100) aged 7 d caused lower
mortality than spinetoram (100) aged 7 d at d 1
and not other days, and greater mortality than
spinetoram (75) aged 7 d at d 3-7, but not on other
days. Fresh spinetoram usually was not different
from aged azinphos-methyl within days.
Apples were smaller in tests 1A and 1B than in
test 1C, but similar differences were seen in each
analysis with respect to numbers of stings in ap-


Insecticides field-aged 13-27 Jul and 3-7 Aug 2004

Treatment D1 D2 D3 D4 D7 D 10

Control 0.0 + 0.0 c 0.0 + 0.0 d 0.0 + 0.0 d 0.0 + 0.0 d 2.2 + 2.2 d 6.7 + 5.5 c
Spinetoram (100) 0 d 36.7 7.8 b 96.7 + 1.7 a 100.0 + 0.0 a 100.0 + 0.0 a 100.0 + 0.0 a 100.0 + 0.0 a
Spinetoram (100) 7 d 6.7 + 2.4 c 14.4 + 3.4 c 36.7 + 8.7 c 47.8 + 10.8 c 73.3 11.9 ab 85.6 + 9.3 a
Spinetoram (100) 14 d 5.6 + 2.4 c 5.6 + 2.4 cd 7.8 + 4.3 cd 10.0 + 4.4 d 24.4 + 5.8 c 36.7 7.1 b
Spinetoram (75) 7 d 5.6 + 1.8 c 14.4 + 5.0 c 28.9 + 9.2 c 42.2 + 9.8 c 67.8 10.9 b 82.2 + 6.8 a
Spinosad (100) 7 d 11.1 + 4.8 c 43.3 8.7 b 67.8 11.5 b 72.2 11.5 be 91.1 7.7 ab 95.6 + 4.4 a
(1,121) 7 d 76.7 + 2.4 a 92.2 + 1.5 a 93.3 1.7 ab 95.6 1.8 ab 98.9 + 1.1 a 100.0 + 0.0 a
(1,121) 14 d 73.3 + 3.7 a 94.4 + 2.4a 95.6 + 2.4a 98.9 + 1.1a 100.0 + 0.0 a 100.0 + 0.0 a
One-Way ANOVA F 47.1 77.6 41.1 39.0 34.1 44.9
df= 7, 64 P <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001

Tests pooled, 9 replicates of 10 flies each (test 1A: 6 males, 4 females, 3 replicates; test 1B: 5 of each sex, 6 replicates); 100 = 100
g a.i./ha; 75 = 75 g a.i./ha; 1,121= 1,121 g a.i./ha.
Means followed by the same letter within days (columns) are not significantly different (Tukey test, P > 0.05).

June 2007

Yee et al: Insecticide Aging Effects on R. pomonella


Insecticides field-aged 31 Aug-14 Sep 2004

Treatment D1 D2 D3 D4 D7 D 10

Control 0.0 + 0.0 c 0.0 + 0.0 e 0.0 + 0.0 e 0.0 + 0.0 c 0.0 + 0.0 c 3.3 + 3.3 c
Spinetoram (100) 0 d 66.7 + 8.8 a 96.7 + 3.3 a 100.0 + 0.0 a 100.0 + 0.0 a 100.0 + 0.0 a 100.0 + 0.0 a
Spinetoram (100) 7 d 20.0 10.0 b 53.3 8.8 be 86.7 3.3 b 90.0 + 0.0 a 100.0 + 0.0 a 100.0 + 0.0 a
Spinetoram (100) 14 d 0.0 + 0.0 c 0.0 + 0.0 e 10.0 + 0.0 d 33.3 8.8 b 46.7 13.3 b 53.3 17.6 b
Spinetoram (75) 7 d 0.0 + 0.0 c 16.7 + 12.0 de 33.3 + 3.3 c 56.7 12.0 b 73.3 12.0 b 90.0 + 10.0 a
Spinosad (100) 7 d 0.0 + 0.0 c 43.3 + 3.3 cd 90.0 + 0.0 b 100.0 + 0.0 a 100.0 + 0.0 a 100.0 + 0.0 a
Azinphos-methyl 80.0 + 10.0 a 100.0 + 0.0 a 100.0 + 0.0 a 100.0 + 0.0 a 100.0 + 0.0 a 100.0 + 0.0 a
(1,121) 7 d
Azinphos-methyl 56.7 + 6.7 a 86.7 3.3 ab 93.3 3.3 b 93.3 + 3.3 a 100.0 + 0.0 a 100.0 + 0.0 a
(1,121) 14 d
One-way ANOVA F 45.1 49.5 190.0 70.6 69.7 27.3
df= 7, 16 P <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001

Three replicates of 10 flies each (5 of each sex); 100 = 100 g a.i./ha; 75 = 75 g a.i./ha; 1,121 = 1,121 g a.i./ha.
Means followed by the same letter within days (columns) are not significantly different (Tukey test, P > 0.05).

ples and numbers of larvae that emerged from ap-
ples (Tables 3 and 4). In tests 1A and 1B (Table 3),
spinetoram and other insecticides significantly
reduced numbers of stings and numbers of larvae
per apple by 90 to 100%. No larvae emerged from
the spinetoram (100) treatments, including the
14-d old treatment, even though it killed only
36.7% of flies by d 10, with some females alive at
that time. One larva emerged from an apple
treated with spinetoram (75) and a total of 3 lar-
vae emerged from 2 apples treated with spinosad.
In test 1C (Table 4), there was a tendency toward
reduced numbers of stings in all treatments (al-

though not significant, according to the Tukey
test) and all treatments prevented larval emer-
gence from apples, including again from the spin-
etoram (100) aged 14 d treatment, even though it
killed only 53.3% of flies.

Effects of Field-Aging GF-120 on Fly Mortality

Mortalities caused by 0-7 d old GF-120 were
high, especially as days after exposure to flies in-
creased (Table 5). Repeated-measures ANOVA re-
sulted in a significant treatment x day interaction
(F = 22.0, df= 4, 303, P < 0.0001), indicating the


Insecticides field-aged 13-27 Jul and 3-7 Aug 2004

Apple size Apple damage

Treatment Weight (g) Circ. (cm) No. stings/apple No. larvae/apple

Control 70.6 + 5.8 16.9 + 0.4 18.3 + 6.0 a 3.9 + 2.4 a
Spinetoram (100) 0 d 78.4 + 8.7 17.3 + 0.7 0.3 + 0.2 b 0.0 0.0 b
Spinetoram (100) 7 d 74.2 + 7.5 17.1 + 0.6 1.0 + 0.5 b 0.0 0.0 b
Spinetoram (100) 14 d 77.8 + 6.8 17.5 + 0.6 1.9 + 0.3 b 0.0 0.0 b
Spinetoram (75) 7 d 61.6 + 5.8 16.2 + 0.5 0.4 + 0.2 b 0.1 0.1 b
Spinosad (100) 7 d 67.3 + 5.0 16.8 + 0.5 0.9 + 0.7 b 0.3 0.2 ab
Azinphos-methyl (1,121) 7 d 82.5 + 4.7 17.9 + 0.4 0.1 + 0.1 b 0.0 0.0 b
Azinphos-methyl (1,121) 14 d 72.4 + 4.4 17.2 + 0.3 0.0 + 0.0 b 0.0 0.0 b
One-way ANOVA F -- 14.8 2.7
df= 7, 64 P -<0.0001 0.0159

Tests pooled, 9 replicates of 10 flies each (test 1A: 6 males, 4 females, 3 replicates; test 1B: 5 of each sex, 6 replicates); 100 = 100
g a.i./ha; 75 = 75 g a.i./ha; 1,121 = 1,121 g a.i./ha. Circ., circumference.
Means followed by the same letter within apple damage measures (columns) are not significantly different (Tukey test,P > 0.05).

Florida Entomologist 90(2)

June 2007


Insecticides field-aged 31 Aug to 14 Sep 2004

Apple size Apple damage

Treatment Weight (g) Circ. (cm) No. stings/apple No. larvae/apple

Control 112.8 + 5.8 20.4 + 0.1 24.3 + 19.8 8.3 + 6.3 a
Spinetoram (100) 0 d 111.7 + 3.0 19.9 + 0.2 0.0 + 0.0 0.0 0.0 b
Spinetoram (100) 7 d 113.5 + 9.1 20.0 + 0.5 0.0 + 0.0 0.0 0.0 b
Spinetoram (100) 14 d 119.1 + 7.0 20.4 + 0.4 0.0 + 0.0 0.0 + 0.0 b
Spinetoram (75) 7 d 111.5 + 4.7 19.8 + 0.3 0.0 + 0.0 0.0 0.0 b
Spinosad (100) 7 d 101.5 + 5.7 19.4 + 0.5 1.0 + 0.6 0.0 0.0 b
Azinphos-methyl (1,121) 7 d 123.0 + 3.2 20.5 + 0.3 0.0 + 0.0 0.0 0.0 b
Azinphos-methyl (1,121) 14 d 127.3 + 6.2 20.8 + 0.3 0.0 + 0.0 0.0 0.0 b
One-Way ANOVA F 2.8 3.0
df= 7, 16 P -- 0.0412 0.0312

Three replicates of 10 flies each (5 of each sex); 100 = 100 g a.i./ha; 75 = 75 g a.i./ha; 1,121 = 1,121 g a.i./ha. Circ., circumference.
Means followed by the same letter within apple damage measures (columns) are not significantly different (Tukey test,P > 0.05).

pattern of mortality among treatments differed at
various days after exposure. There was almost no
control mortality, large increases in mortality in
0-7 d old treatments, and small increases in mor-
tality in the 14-d old treatment over time. One-
way ANOVA indicated that the 14-d old GF-120
was not any more effective than the control until
d 7 after exposure, and was never as effective as
the 0-3 d old GF-120 during the 10 d (Table 5).


In experiment 1, we showed that the new insec-
ticide spinetoram is highly toxic to R. pomonella
when fresh, on a similar level to that of azinphos-

methyl aged 7 to 14 d, but that its toxicity de-
creases rapidly after 7 d of aging in the field un-
der the sunny, hot, and dry conditions typical of
central Washington in Jul and Aug. By 14 d,
spinetoram was no longer toxic to R. pomonella,
except after 3 to 7 d of continuous exposure. Thus,
spinetoram at 100 g a.i./ha would probably need
to be applied at < 7-d intervals to be effective. The
relatively short period of activity (compared with
azinphos-methyl) suggests that spinetoram
breaks down quickly in central Washington con-
ditions and that ultraviolet light blocking or other
agents need to be improved to prolong its toxicity.
Also, it is possible that spinetoram was absorbed
into plant tissue which occurs with spinosad (Dow


GF-120 Field-Aged 12-26 Jul and 2-16 Aug 2004

Age of GF-120 on apple leaves One-way ANOVA
Days after
Exposure Control OD 3D 7 D 14 D F (df = 4, 35) P

1 0.0 + 0.0 b 47.5 + 5.9 a 27.5 + 4.9 a 25.0 + 3.3 a 8.8 4.0 b 23.3 <0.0001
2 0.0 + 0.0 b 77.5 8.2 a 55.0+ 13.2 a 50.0 + 9.6 a 12.5 5.3 b 15.6 <0.0001
3 0.0 + 0.0 b 86.3 5.0 a 70.0 + 9.1 a 75.0 + 6.3 a 16.3 6.3 b 30.6 <0.0001
4 0.0 + 0.0 b 88.8 4.8 a 78.8 + 6.7 a 83.8 + 5.6 a 17.5 5.9 b 43.2 <0.0001
7 0.0 + 0.0 c 100.0 + 0.0 a 93.8 + 3.2 a 96.3 + 1.8 a 38.8 12.0 b 73.1 <0.0001
8 0.0 + 0.0 c 100.0 + 0.0 a 98.8 + 1.3 a 97.5 + 1.6 a 42.5 11.6 b 114.3 <0.0001
9 0.0 + 0.0 c 100.0 + 0.0 a 100.0 + 0.0 a 97.5 + 1.6 a 50.0 14.4 b 68.3 <0.0001
10 1.3 + 1.3 c 100.0 + 0.0 a 100.0 + 0.0 a 100.0 + 0.0 a 53.8 15.8 b 47.6 <0.0001

Tests pooled, 8 replicates of 10 flies each (5 of each sex).
Means followed by the same letter within days after exposure (rows) are not significantly different (Tukey test, P > 0.05).

Yee et al: Insecticide Aging Effects on R. pomonella

AgroSciences 2004), making it unavailable to flies
over time. If so, ingredients that prevent rapid
absorption into leaves may help prolong its effec-
tiveness. Spinosad in other studies shows de-
creased residual activity at 3-7 d after application
(Williams et al. 2003), apparently similar to spin-
etoram. This suggests the different formulations
of these 2 spinosyn insecticides do not affect the
durations of their toxicity. Spinetoram used at
rates described in this study might also be more
effective if incorporated into a bait mix, similar to
spinosad in GF-120, so that flies ingest more of it.
Also, if a fly ingests degraded toxin, it may lead to
quicker mortality than a fly that repeatedly con-
tacts the degraded toxin.
The increases in mortality in spinetoram and
other insecticide treatments over the 10 d of the
tests suggest flies repeatedly contacted the apples
as days progressed or that there was a delayed ef-
fect from 1 or a few initial contacts with the insec-
ticide. Repeated contacts inside a cage may result
in an overestimate of expected mortality under
field conditions (Barry & Polavarapu 2005). Re-
peated contacts or a delayed effect may explain
why fresh spinetoram was more toxic than spine-
toram aged 7 d at d 1-4 and not 7 and 10 (tests 1A
and 1B) or d 1-3 and not d 4-10 (test 1C).
Other results of tests 1A and 1B and of 1C were
similar, with one key difference being the relative
effectiveness of spinetoram versus spinosad. Over-
all results suggest spinetoram at 75 g a.i./ha is less
effective than at 100 g a.i./ha, perhaps because the
smaller amount is broken down more quickly than
the larger amount, and that 75 g a.i./ha aged 7 d is
more effective than 100 g a.i./ha aged 14 d, so ag-
ing may be more critical to effectiveness than the
amount. In the one key difference, in tests 1A and
1B, spinosad at 100 g a.i./ha aged 7 d was more ef-
fective at d 2 and 3 than spinetoram at 100 g a.i./
ha aged 7 d, but in test 1C, spinetoram was more
effective than spinosad at d 1 and similar on all
other days. The difference could be a result of
warmer temperatures or ultraviolet light having a
greater negative impact on spinetoram than spi-
nosad over 7 d (5.8-7.8 C warmer in tests 1A and
1B than in test 1C). Another conclusion is that 100
g a.i./ha of spinosad, like this amount of spineto-
ram, is more effective than 75 a.i./ha spinetoram.
Azinphos-methyl seemed less affected by warm
temperatures or ultraviolet light than spinetoram
and spinosad, based on its high effectiveness even
after 14 d of aging.
The insecticides in this study did not kill R.
pomonella adults quickly enough to prevent them
from stinging apples. This included azinphos-me-
thyl, albeit only one sting was detected in azin-
phos-methyl-treated fruit in all 3 tests combined.
The inability of insecticides to prevent stinging or
oviposition by R. pomonella agrees with earlier
work with azinphos-methyl (Reissig et al. 1983)
and indicates that insecticides must be applied

before females are reproductively mature. Protec-
tion of apples from stings will therefore be prob-
lematic if treatment trees are surrounded by in-
fested trees. However, overall results show that,
despite the inability of spinetoram (100 g a.i./ha)
to prevent stings, mortality and apple injury data
did not lead to the same conclusions concerning
effectiveness. Even though 14-d old spinetoram
killed only 36.7 and 53.3% of flies after 10 d of ex-
posure, the numbers of stings on the 14-d old spin-
etoram-treated apples were reduced 90 and 100%
compared with controls, and no larvae emerged
from any of these apples. This suggests there were
non-lethal effects that reduced oviposition (some
females were still alive at 10 d), including a repel-
lent effect, or that flies stung the apples but did
not oviposit. Eggs or larvae in fruit also may have
been affected by spinetoram if it was absorbed
into fruit tissue. Thus, mortality of adults is not
the only variable to consider when evaluating the
ability of spinetoram and the other insecticides to
control R. pomonella. We did not cut apples and
examine them for larvae. This could have resulted
in the detection of internal damage in apples that
had stings. It is possible some larvae died inside
the fruit and did not emerge. If so, our data under-
estimated larval infestation rates.
In experiment 2 with GF-120, spinosad, simi-
lar to spinetoram, was highly toxic when fresh,
but appeared to break down or was absorbed into
leaves between 7-14 d under sunny, hot, and dry
conditions. Whether bait components of GF-120
can be modified to protect spinosad against ultra-
violet rays or to prevent rapid absorption into
leaves under these conditions needs study. Re-
sults suggest that GF-120 in its present form
needs to be applied every 7 d to be effective.
Against the walnut husk fly, Rhagoletis complete
Cresson, 20% GF-120 aged for only 3 d in hot
weather in California lost toxicity (Van Steenwyk
et al. 2003). The inconsistency of GF-120 in con-
trolling R. pomonella in the eastern U.S.-ineffec-
tive in New York (Reissig 2003) and effective in 1
of 2 years in Michigan (Pelz et al. 2005)-may be
caused in part by its short residual activity or by
the high precipitation in the regions where it was
tested. Residual toxicity of GF-120 in the eastern
U.S. may more likely be reduced by rainfall than
by dry conditions and high temperatures.
Our overall results indicate spinetoram and GF-
120 when fresh are highly toxic to R. pomonella but
that their residual toxicities need to be prolonged
to optimize their performance against flies in cen-
tral Washington. Direct comparisons of spineto-
ram and GF-120 are needed to determine whether
one holds more promise than the other for fly con-
trol. Tests with both at higher rates, in different
bait formulations, and in different spray volumes
are needed to determine if they can be used in a
management program for R. pomonella. Aged
spinetoram is less toxic against adult flies than

Florida Entomologist 90(2)

azinphos-methyl, but it and GF-120 are more be-
nign to the environment. Also, results suggest that
adult fly mortality caused by spinetoram is not an
accurate predictor of larval emergence from ap-
ples, and that possible non-lethal effects caused by
spinetoram need to be examined.


We thank John Stark (Washington State University,
Puyallup) for providing laboratory space, Pete Chap-
man (USDA, ARS) for field assistance, Jim Mueller and
Harvey Yoshida (Dow AgroSciences) for providing spine-
toram 100 g a.i./L SC and advice during this study, Jim
Dripps (Dow AgroSciences), Michael Bush (Washington
State University, Yakima), and Jim Hansen (USDA,
ARS) for reviewing the manuscript, and Dow Agro-
Sciences and the Washington Tree Fruit Research Com-
mission for funding.


BARRY, J. D., AND S. POLAVARAPU. 2005. Feeding and
survivorship of blueberry maggot flies (Diptera:
Tephritidae) on protein baits incorporated with in-
secticides. Florida Entomol. 88: 268-277.
Dow AGROSCIENCES. 2002. Specimen Label, revised 11-
27-02. GF-120 Naturalyte Fruit Fly Bait. Indianap-
olis, IN.
Dow AGROSCIENCES. 2004. Spinosad Technical Bulle-
tin. Indianapolis, Indiana. 7 pp.
sional Record 142: 1489-1538.
GARIBAY, R. 2005. Washington's 2004 apple crop ap-
proaches record level. Washington agricultural
statistics service. USDA, Olympia, Washington.
www.nass.usda.gov/wa/apples.pdf. accessed 25 Jan-
uary 2005.
Malathion bait sprays for control of apple maggot
(Diptera: Tephritidae). J. Econ. Entomol. 82: 1716-
NEILSON, W. T. A., AND C. W. MAXWELL. 1964. Field
tests with a malathion bait spray for control of the

apple maggot, Rhagoletis pomonella. J. Econ. Ento-
mol. 57: 192-194.
NEILSON, W. T. A., AND K. H. SANFORD. 1974. Apple
maggot control with baited and unbaited sprays of
azinphos-methyl. J. Econ. Entomol. 67: 556-557.
PELZ, K. S., R. ISAACS, J. C. WISE, AND L. J. GUT. 2005.
Protection of fruit against infestation by apple mag-
got and blueberry maggot (Diptera: Tephritidae) us-
ing compounds containing spinosad. J. Econ.
Entomol. 98: 432-437.
STEEN, AND J. B. BOURKE. 1983. Effects of surface
residues of azinphosmethyl on apple maggot behav-
ior, oviposition, and mortality. Environ. Entomol.
12: 815-822.
REISSIG, W. H. 2003. Field and laboratory tests of new
insecticides against the apple maggot, Rhagoletis
pomonella (Walsh) (Diptera: Tephritidae). J. Econ.
Entomol. 96: 1463-1472.
SAS INSTITUTE. 2001. SAS/STAT user's guide, version
8. Cary, NC.
THOMAS, D. B., AND R. L. MANGAN. 2005. Nontarget im-
pact of spinosad GF-120 Bait sprays for control of
the Mexican fruit fly (Diptera: Tephritidae) in Texas
citrus. J. Econ. Entomol. 98: 1950-1956.
MOTO. 2003. Walnut husk fly control with reduced
risk insecticides, In B. Beers [ed.], Proceedings of the
77th Annual Western Orchard Pest & Disease Man-
agement Conference, Portland, Oregon. Washington
State University, Pullman. 5 pp. http://entomol-
accessed 19 October 2006.
2001. Washington Administrative Code 16-470-108.
Distribution of infested or damaged fruit is prohib-
ited. http://agr.wa.gov/ accessed 15 August 2005.
2005. Washington Administrative Code 16-470-105.
Area under order for apple maggot-Pest free area-
Quarantine areas. http://agr.wa.gov/ accessed 15
August 2005.
naturally derived insecticide spinosad compatible
with insect natural enemies? Biocontrol Sci. Tech-
nol. 13: 459-475.

June 2007

Torres & Hallman: Medfly Phytosanitary Irradiation Treatment


'San Borja, Lima, Peru

2USDA, ARS, Weslaco, TX 78596


The Mediterranean fruit fly, Ceratitis capitata (Wiedemann), is one of the most important
quarantine pests in the world. Host commodities shipped from infested parts of the world to
non-infested parts that might be susceptible to infestation should undergo a phytosanitary
measure to render negligible the risk of shipping viable flies. Ionizing irradiation is a prom-
ising phytosanitary treatment that is tolerated by the great majority of hosts of the Medi-
terranean fruit fly. The current dose in the US is 150 Gy. This research conducted with cage-
infested 'Haden' mangoes in Peru showed that 100 Gy is sufficient to provide a high level of
quarantine security against this important pest. That dose did not affect pupation when ap-
plied to late 3rd instars, but it did prevent any from emerging as adults. A dose of 100 Gy
might allow for irradiation of avocados, one of the few fruits that does not tolerate more than
100-200 Gy

Key Words: Ceratitis capitata, ionizing irradiation, quarantine treatment, disinfestation


La mosca mediterranea de la fruta, Ceratitis capitata (Wiedemann), es una de las plagas
cuarentenarias mas importantes en el mundo. Las materials primas hospederos embarcadas
desde regions del mundo infestadas con destino a zonas no infestadas que podrian ser sus-
ceptibles de infestaci6n deberian ser sometidas a una media fitosanitaria que permit eli-
minar el riesgo de estos embarques. La radiaci6n ionizante es un tratamiento fitosanitario
prometedor y tolerable por la gran mayoria de hospederos de la mosca mediterranea de la
fruta. La dosis corriente en los Estados Unidos es 150 Gy. La present investigaci6n condu-
cida con mangos infestados enjaulas en Pern mostr6 que 100 Gy son suficientes para pro-
porcionar un nivel alto de seguridad cuarentenaria contra esta important plaga. La
mencionada dosis no afect6 el process de empupar cuando se aplic6 en larvas del tercer ins-
tar, pero si evit6 la emergencia a adults. Una dosis de 100 Gy podria adecuarse para la irra-
diaci6n de aguacates, una de las pocas frutas que no tolera mas de 100-200 Gy.

Translation provided by the authors.

The Mediterranean fruit fly, Ceratitis capitata
(Wiedemann), may be considered the premier
quarantine pest in the world. It occurs in much of
Africa and southern Europe, most of Latin Amer-
ica from southern Mexico to Argentina, parts of
Australia and the Middle East, and Hawaii. Non-
infested countries from the tropics into temperate
regions place quarantines on a broad range of
fresh fruit hosts from infested regions. The host
list includes most commercial sweet tree-fruits,
avocados, tomatoes, peppers, cotton bolls, walnut
fruits, and coffee berries. Over 250 hosts have
been listed. Even poor hosts are quarantined be-
cause they may carry enough Mediterranean fruit
fly individuals for the pest to reproduce and be-
come established.
Much effort and money are spent preventing,
managing, and eradicating the Mediterranean
fruit fly throughout the world. Millions of sterile
males per week are released in Florida, California,

and other areas as a preventative measure. Thou-
sands of survey traps are maintained in many
countries. The pest has been found and eradicated
7 times in Florida and individuals are found almost
every year in California. The United States (US),
Mexico, and Guatemala collaborate in trying to
eradicate the fly from Mexico and parts of Guate-
mala. The Mediterranean fruit fly was officially
eradicated from Chile in 1995 and cooperation with
Peru aims to prevent its reintroduction into Chile
as well as achieve eradication in southern Peru.
Phytosanitary treatments may be required to
export hosts from Mediterranean fruit fly-in-
fested areas to non-infested areas that could sup-
port establishment of the pest. Several treatment
options are available. Holding at 1.1-2.2C for 14-
18 d is used to disinfest tangerines shipped from
Spain to the US. Methyl bromide fumigation is
used for various fruits. Immersion of mangoes in
water at 46.1C for 65-110 min (depending on

Florida Entomologist 90(2)

shape, mass, and origin of the mangoes) is used to
facilitate shipment of mangoes from Latin Amer-
ica to several countries. Heated air is used to fa-
cilitate shipment of papayas from Hawaii to Ja-
pan and the mainland US. Ionizing irradiation is
used for shipment of several fruits from Hawaii to
the mainland US.
From the standpoint of fruit quality, irradia-
tion is the most broadly applicable commercial
treatment at the doses for tephritid fruit flies
(Hallman 2007). It also has advantages over
other treatments; e.g., very few external variables
affect treatment efficacy and it can be applied af-
ter packing and palletizing.
The chief disadvantage of irradiation is that,
unlike all other commercially-applied treat-
ments, it does not cause acute mortality but ren-
ders insects unable to complete development and/
or reproduce. Although preventing development
or reproduction is sufficient to prevent the estab-
lishment of invasive species, it does not provide
inspectors with a simple and reliable independent
verification of treatment efficacy, i.e., dead in-
sects. Correct and complete conduction of the re-
search supporting the treatment, robust certifica-
tion that treatments are done adequately, and
careful protection of the treated lots from re-in-
festation are necessary to ensure commercial via-
bility of phytosanitary irradiation treatments. Ir-
radiation has been used commercially since 1995
for interstate disinfestation of perishable com-
modities of several pests including tephritids
within the US and since 2004 to disinfest man-
goes shipped from Australia to New Zealand of
tephritids without insurmountable incident.
In the US a minimum absorbed dose of 150 Gy
is allowed for any fruit against any tephritid fruit
fly (APHIS 2006). Very few fruits do not tolerate
that dose applied on a commercial scale, which
could be up to 2.5 times the minimum absorbed
dose (Hallman & Loaharanu 2002). The reason
commercial doses may be up to 2.5 times the min-
imum prescribed dose is that commercial irradia-
tion facilities may treat pallet-loads and irradia-
tion diminishes as the distance from the source
increases. In order to get the minimum absorbed
dose required to the farthest fruit in a pallet-load,
the nearest fruit may receive a much higher dose.
Because it is such a significant quarantine
pest Mediterranean fruit fly has received much
phytosanitary attention. For example in the book,
Invasive Arthropods in Agriculture: Problems
and Solutions (Hallman & Schwalbe 2002), Med-
iterranean fruit fly is mentioned more than any
other arthropod. It is also the most studied pest
regarding irradiation phytosanitary treatments.
Thirteen studies provide sufficient data to esti-
mate doses required for quarantine security with
varying levels of security (Table 1), the most for
any quarantine pest. Reasoning for many of the
doses listed in Table 1 is given in Hallman (1999).


Dose (Gy) Fruit Reference

225 Papaya Seo et al. (1973)
>200 Orange F6stis et al. (1981)
-80 Mango Potenza et al. (1989)
-80 Mango Raga (1990)
-80 Peach Arthur et al. (1993a,b)
-70 Grapefruit Raga (1996)
-200 Orange Adamo et al. (1996)
401 Peach, orange Mansour & Franz (1996)
150 Mango Bustos et al. (2004)
100' Papaya Follett & Armstrong (2004)

Fruit infestation involved rearing larvae in diet and insert-
ing them into fruit 24-30 h before treatment.

The literature suggests 2 conflicting peak
doses for providing quarantine security against
Mediterranean fruit fly, one at 70-100 Gy and the
other at 200-225 Gy (Table 1). Hallman & Loaha-
ranu (2002) argue that the upper peak is not well
supported and could be dismissed. The currently
accepted dose for this pest in the US is 150 Gy
(APHIS 2006), but this may not be the minimum
absorbed dose that could prevent adult emer-
gence of 3rd instar Mediterranean fruit fly in fruit
as evidenced by several studies in Table 1.
Two large-scale studies used Mediterranean
fruit fly 3rd instars reared in diet and then in-
serted in fruit 24-30 h before treatment. Mansour
& Franz (1996) obtained no adult emergence
when >100,000 3rd instars were reared in diet
and then placed in peaches and oranges 30 h prior
to irradiation with 40 Gy. Follett and Armstrong
(2004) found no adult emergence when 31,920 3rd
instars were reared in diet and placed in papayas
24 h before irradiation with 100 Gy. Follett and
Armstrong obtained 0.47% emergence of normal-
looking adults when 3rd instars were irradiated
with 40 Gy and 0.07% at 50 Gy, doses that pro-
vided complete prevention of adult emergence in
the similar study by Mansour & Franz (1996).
For any phytosanitary treatment, infestation
that differs significantly from the natural situa-
tion should be tested for relative tolerance to the
natural situation. If the semi-artificial technique
results in increases in pest tolerance, it would not
be of phytosanitary concern, although the treat-
ment may be harsher on the commodity than it
need be. But if the semi-artificial infestation in-
creases susceptibility, phytosanitary security will
be at risk. Hypoxia reduces radiosusceptibility of
organisms (Hallman & Hellmich 2007), and te-
phritid immatures inside the hypoxic atmosphere
of fruit seem to show increased tolerance (Hall-

June 2007

Torres & Hallman: Medfly Phytosanitary Irradiation Treatment

man & Worley 1999). Lack of hypoxic protection
may explain why 40 Gy prevented Mediterranean
fruit fly adult emergence in >100,000 third in-
stars reared in diet and placed in peaches and or-
anges 30 h before irradiation (Mansour & Franz
1996). A higher dose was required using the same
techniques with papayas (Follett & Armstrong
2004). Perhaps a hypoxic atmosphere was easier
to achieve and maintain in papayas after artifi-
cial infestation compared with peaches and or-
anges making work with papayas more akin to
natural conditions, at least in this case.
The most rigorous standard used for confirm-
ing the efficacy of a phytosanitary treatment is
"probit 9" at the 95% confidence level (Hallman &
Loaharanu 2002). Probit 9 represents the effec-
tive dose (ED) to achieve a result at the 99.9968
percentile (ED999968). This entails treating 93,600
individuals with no survivors when done to a con-
fidence level of 95% (Couey & Chew 1986). Effi-
cacy of an irradiation phytosanitary treatment
against tephritids is measured by the prevention
of the emergence of adults capable of flight when
irradiated as 3rd instars inside fruit (Hallman &
Loaharanu 2002).
Although Follett & Armstrong (2004) found
that 100 Gy would probably control Mediterra-
nean fruit fly in the system that they studied
(diet-reared larvae placed in papaya a day before
irradiation), they did not do a "probit 9"-level
study because their goal was to find a single dose
that would control all quarantined tephritid fruit
flies in Hawaii. They did not try to make the dose
for all fruit flies in Hawaii lower than 150 Gy be-
cause their studies indicated that one tephritid,
melon fly, Bactrocera cucurbitae (Coquillet),
would not be controlled to the "probit 9" level with
<150 Gy
To save resources and reduce the potential
negative effect of a phytosanitary treatment on
commodities the effective dose for any treatment
should be made as low as possible. In the case of
ionizing irradiation and the Mediterranean fruit
fly Hallman & Loaharanu (2002) observed that
doses to control all Anastrepha fruit flies studied
were similar and about 70 Gy achieved a high
level of control. They argued that enough phy-
tosanitary research had been done with the neo-
tropical genus to permit a dose of 70 Gy for all
Anastrepha. Four studies that included both
Anastrepha spp. and Mediterranean fruit fly
showed the latter to require about 1.4x the dose to
achieve the same effect against Anastrepha.
Therefore, if 70 Gy is sufficient for Anastrepha
then 70 Gy x 1.4 or ~100 Gy should provide a high
level of control of Mediterranean fruit fly.
The objective of the research was to determine
the dose at the 95% confidence level, beginning at
100 Gy that would prevent the emergence of
adults capable of flight when irradiated as 3rd in-
star Mediterranean fruit flies inside fruit.


The flies used in this research were from the
colony in the sterile insect release program in La
Molina, Peru. About 16,500 Mediterranean fruit
fly adults (sex ratio about 1:1) were maintained in
each of 3 cages (0.6 x 2 x 0.35 m). They were fed a
mixture of sugar, protein, and water. About 60-80
mature green, freshly picked 'Haden' mangoes
(0.3-0.5 kg) were placed in each cage for 24 h at
about 27C with constant lighting. The resulting
mean infestation rate was about 45 larvae/fruit.
Upon removal from the infestation cages mangoes
were kept at 27C until the flies developed to late
3rd instars (9-10 days); mangoes were periodically
opened to observe fly development. The test was
repeated until at least 93,600 third instars were ir-
radiated at 100 Gy, which would satisfy "probit 9"
at the 95% confidence level (Couey & Chew 1986).
When most Mediterranean fruit flies had de-
veloped to the 3rd instars 90% of the infested
mangoes were irradiated with a cobalt-60 source
(model Gammabeam 127, Nordion, Kanata, Can-
ada) that was delivering a dose rate of 2.8 Gy/
minute. Dosimetry was done with the Fricke sys-
tem (ASTM 2006). Timing of irradiation was set
so that the maximum dose measured did not ex-
ceed the target dose. At that timing the minimum
absorbed dose was about 87 Gy when the maxi-
mum was 100 Gy The 10% non-irradiated man-
goes were held as controls. After irradiation the
mangoes were held at 25C on a bed of moist saw-
dust to absorb fluids leaking from the fruit and
serve as a burrowing and pupariation medium for
emerging larvae. After 5-6 d the mangoes were
examined for remaining larvae, both dead and
alive. All larvae and puparia were collected,
counted, and held at 25C until after adults
emerged. Tests continued until a minimum of
96,400 third instars were irradiated with no
adults emerging. If adults capable of flight (fully
extended wings) were found the dose would be
raised depending on the failure rate and the test-
ing begun anew.
It is expected that Mediterranean fruit fly 3rd
instars will largely pupariate at 100 Gy, although
adult emergence should be extremely low. Statis-
tical significance of pupariation between irradi-
ated and control 3rd instars was tested via a two-
tailed, paired t-test (Prism 4, GraphPad Software,
San Diego, CA).


After 9 tests consisting of 7-12 thousand irra-
diated 3rd instars each, a total of 99,562 Mediter-
ranean fruit fly 3rd instars were irradiated in
mangoes with no adults emerging; 88.5% of these
3rd instars pupariated. Pupariation rate of the
control was 90.7%, but it was not significantly dif-
ferent from the irradiated 3rd instars (t = 0.98, df

Florida Entomologist 90(2)

= 8, P = 0.36) showing that 100 Gy did not affect
the ability of Mediterranean fruit fly late 3rd in-
stars to pupariate. Adult emergence from control
puparia was 86.9%.
This research shows that phytosanitary irradi-
ation at an absorbed minimum dose of 100 Gy
provides quarantine security against Mediterra-
nean fruit fly to the highest degree demanded of a
commercial phytosanitary treatment, ED99 996 at
the 95% confidence level. Almost all hosts of the
pest would tolerate this treatment applied on a
commercial scale. Even avocado, which has low
tolerance to any phytosanitary treatment, such
as those based on fumigation, heat, or cold (Mc-
Donald & Miller 1994), might have a viable treat-
ment against Mediterranean fruit fly with 100
Gy. Avocado tolerates about 100-200 Gy (Thomas
2001), and the dose uniformity ratio (maximum
absorbed dose divided by minimum absorbed
dose) expected in commercial irradiation facilities
can vary anywhere from 1.2-2.5, depending on the
source and arrangement of irradiated product.


This research was supported by the Joint United Na-
tions Food and Agriculture Organization/International
Atomic Energy Agency Program, Nuclear Techniques in
Food and Agriculture, Food and Environmental Protec-
tion Section in Vienna, Austria, and the Instituto Peru-
ano de Energia Nuclear in Lima, Peru. We thank the
sterile Mediterranean fruit fly program in La Molina,
Peru, for use of the irradiation facility.


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June 2007

Sivinski et al.: Thermal Environment of Immature Anastrepha suspense


'USDA-ARS, Center for Medical, Agricultural and Veterinary Entomology
1600-1700 SW 23'd Dr., Gainesville, FL 32604

2USDA-APHIS/PPQ 1600-1700 SW 23rd Dr., Gainesville, FL 32605

3Department of Entomology and Nematology, University of Florida, Gainesville, FL 32611


Because many plants regulate their internal temperatures, there is no a priori reason to be-
lieve air temperature accurately reflects the temperatures faced by tephritid larvae inhabit-
ing fruit interiors. Larvae also move across and burrow into soil to pupate, and immature
flies at this point are also likely to encounter temperatures that might be less than or exceed
air temperature. Using thermocouples and a computerized data logger we measured a range
of temperatures in the 4 major hosts ofAnastrepha suspense (Loew), the Caribbean fruit fly:
(Surinam cherry, Eugenia uniflora L., Cattley guava, Psidium cattleianum Sabine, guava,
Psidium guajava L., and loquat, Eriobotrya japonica (Thunb.)), and in grapefruit, Citrus par-
adisi Macf, an economically important secondary host. Generally, temperatures were higher
in the southwestern portions of tree canopies relative to those in the northeastern interiors.
Fruit on the ground was warmer than in the tree, but there was no significant pattern of
maximum fruit core temperatures being warmer than subcutaneous pulp. Soil temperatures
were also higher than fruit-in-tree temperatures, and decreased and displayed less variance
with increasing depth. Fruit in trees seldom reached temperatures 0.05 of air tempera-
tures, but fruit on the ground could be more than 0.25 the adjacent air temperature. There
were positive relationships between the ratio of mean and minimum fruit temperature/adja-
cent air temperature and fruit diameter. Information on the temperatures confronted by im-
mature fruit flies can be used to model population dynamics, and to design temperature
sensitive strains through conditional gene expression for mass-rearing and release.

Key Words: larvae, pupae, heat, cold, conditional gene expression


Debido a que muchas plants regulan su temperature internal no hay una raz6n a priori
para career que la temperature ambiental refleja es precisamente la temperature enfrentada
por las moscas tefritidos que habitan el interior de las frutas. Las larvas a su vez cruzan y
escavan en el suelo para empupar, y las moscas inmaduras en este punto tambi6n son mas
propicias para encontrar temperatures que pueden ser menos o mas alta que la temperature
ambiental. Usando un termoel6ctrico y una grabadora de datos computerizados, nosotros
medimos el rango de temperatures en 4 de los hospederos mas importantes de Anastrepha
suspense (Loew): (Eugenia uniflora L., Psidium cattleianum Sabine, Psidium guajava L., y
Eriobotrya japonica (Thunb.)), y en toronja, Citrus paradise Macf que es un hospedero se-
gundario de importancia econ6mica. En general las temperatures mas altas fueron en las
areas suroeste de las copas de los arboles en relaci6n con las de la parte interior de los arbo-
les en el noreste. Las frutas en el suelo estaban mas calidas que las frutas en el arbol, pero
no hubo un patron significativo en la temperature maxima del interior de la fruta siendo
mas caliente que la pulpa subcutanea. Las temperatures del suelo tambi6n fueron mas altas
que las temperatures del fruto en el arbol, y diminuyeron y mostraron menos variaci6n con
el aumento de la profundidad. Las frutas en el arbol raramente alcanzaron temperatures de
0.05 de temperature ambiental, pero la temperature de la fruta en el suelo pudo ser mas
alta de 0.25 que la temperature ambiental adyacente. Hubo una relaci6n positive entire la
raz6n del promedio y la temperature minima de la fruta/la temperature ambiental adya-
cente y el diametro de la fruta. Informaci6n sobre las temperatures enfrentadas por los es-
tados inmaduros de las moscas de la fruta puede ser usada para hacer un model de la
dinamica de la poblaci6n, y para disenar razas sensibles a la temperature por medio de la ex-
presi6n gen6tica condicional para la cria y liberaci6n masiva.

Plants are relatively sessile and exposed to their parts, such as shapes that minimize surface
whatever light falls upon them. Characteristics of areas to volumes and high moisture contents,

Florida Entomologist 90(2)

might evolve for a number of reasons, but can also
result in internal temperatures quite different
from the surrounding air. This includes fruit, par-
ticularly larger species such as apples, which in
full sunlight, can be 14C warmer than an ambient
temperature of 27C (Thorpe 1974). Nor are plants
necessarily passive in terms of heat and cold, but
rely on several non-behavioral mechanisms to reg-
ulate their temperatures. These commonly include
(1) emission of infra-red radiation, (2) heat conduc-
tion and convection, and (3) evaporative cooling
(e.g., Jones 1992; Nobel 1999; Roth-Neblesick
2001). The thermal consequences of various adap-
tations can be substantial. For example, the alpine
cushion plant, Silene acaulis spp. excapa [All.]
J. Braun, and its relatives exploit, among other
things, a small, prostrate growth form to avoid
heat-loss into the atmosphere and reach tempera-
tures 15-25C above ambient (Neuner et al. 2000).
Flowers of the sacred lotus, Nelumbo nucifera (L.)
Druce, can maintain temperatures up to 10C be-
low effective ambient through evaporative cooling
(Seymor & Shultze-Motel 1998), as can the leaves
of the perennial Phragmites communis (Cav.) Trin.
(Percy et al. 1972). Fig fruit, Ficus spp., in sunlight
have temperatures no more than 2-3C above am-
bient, but reach temperatures 3-8C above ambi-
ent when an experimentally applied oil coating
prevents their evaporative cooling through tran-
spiration (Patiio et al. 1994).
Organisms that inhabit the interior tissues of
plants, such as the eggs and larvae of frugivorous
tephritid fruit flies, are also relatively limited in
their ability to move to different environments to
regulate their body temperatures. To a substantial
degree they must tolerate the temperature they
encounter within the confines of the fruit they in-
fest. However, given the capacity of some plants to
maintain temperatures different from the ambi-
ent and the variety of lighting that exists within
most tree canopies (Aluja & Birke 1993; Aluja et
al. 2000; Sivinski et al. 2004), the range of thermal
environments encountered by fruit fly larvae may
be considerable and is largely undescribed.
The difficulty in simply estimating the temper-
ature faced by immature fruit flies through ex-
trapolation from air temperature is further com-
plicated by the pupation behavior of the larvae
that typically exit fallen fruit to pupate in the soil
at depths of near-surface to more than 5 cm (e.g.,
Hodgson et al. 1998). Soil temperatures are
known to vary with depth (e.g., Hillel 1982), sea-
son and microhabitat (Thomas 1993, 1995).
A better description of tephritid thermal envi-
ronments would yield several benefits. Tempera-
ture is a critical component in modeling popula-
tion dynamics (e.g., Meats 1981). In addition, pro-
posed new autocidal techniques for tephritid con-
trol and eradication rely on temperature
sensitivity in offspring (Handler 2002, 2004).
Mass-reared and released males would carry

genes that, when expressed in immature off-
spring, result in death after a certain tempera-
ture is reached. Such a scheme would avoid the
sterilizing radiation believed to diminish male
sexual success and which may compromise the
Sterile Insect Technique (=SIT) (Lux et al. 2003).
The success of the Conditional Gene Expression
Technique (=CGE) could be optimized by predict-
ing the minimum and maximum temperatures
eggs and larvae are likely to encounter in differ-
ent locations within the canopies of different
hosts fruiting at different times of the year.
The model tephritid we considered was the Car-
ibbean fruit fly, Ananstrepha suspense (Loew).
Originally from the Greater Antilles, it was acci-
dentally introduced into southern Florida during
the mid-1960s and subsequently spread over ~2/3
of the state's peninsular region (Baranowski et al.
1993). Larvae develop in over 90 species of fruit
(Norrbom & Kim 1988), but a smaller number of
roughly sequentially-fruiting hosts are character-
istically the most highly infested. These include:
Surinam cherry, Eugenia uniflora L. (typically late
spring-early summer), Cattley guava,Psidium cat-
tleianum Sabine (typically mid-late summer),
guava, Psidium guajava L. (typically late summer-
early autumn), and loquat, Eriobotrya japonica
(Thunb.) (typically late winter-early spring) (Sivin-
ski et al. 1999). In addition, a number of citrus spe-
cies are attacked, including grapefruit, Citrus par-
adisi Macf. (Simpson 1993). The temporal distribu-
tion and size differences among these fruit suggest
that larvae confront considerable within-year vari-
ance in temperature (Sivinski et al. 2004).
The present study documented the tempera-
tures near the surfaces and at the cores of the pri-
mary hosts (+ grapefruit) in and under tree cano-
pies as they occurred in several geographical loca-
tions within the range of the fly. In addition, the
temperatures of mature and fallen fruit were
measured in the field, as were soil temperatures
at several likely pupation depths. Particular at-
tention was given to the minimum and maximum
temperatures since these may be important in the
distribution/abundance of the fly and its parasi-
toids (Eitam et al. 2004) and in the design of CGE
systems. Finally the relationship of fruit temper-
atures to air temperature was determined so that
the temperature of larval habitats might be esti-
mated by making relatively simple air tempera-
ture measurements.


Sampling Procedure

Four sets of fruit and 4 sets of soil tempera-
tures were obtained from each host tree, this
number determined by the capacity of the mea-
suring and data logging device. Ripe intact fruit
on the tree were chosen from what would typi-

June 2007

Sivinski et al.: Thermal Environment of Immature Anastrepha suspense

cally be those portions of the canopy most and
least exposed to sunlight, one on the southwest-
ern exterior and one in the northeastern interior,
respectively. Intact, fallen fruit were placed on
the soil under the southwest portion of the canopy
along an imaginary line extending down from the
canopy margin in order to maximize exposure to
sunlight. Intact, fallen fruit under the northeast
portion of the canopy were placed <0.5 the dis-
tance between the canopy margin and the trunk
in order to minimize exposure to sunlight.
Thermocouple devices to measure tempera-
ture were placed in 2 locations in each piece of
fruit, 1 directly under the skin/rind and another
as close as possible to the center. For fruit in trees,
thermocouple wires were supported by 1 or more
twists of wire attached to branches. In some
cases, relatively large seeds prevented absolute-
center measurements, but regardless, the range
of locations potentially occupied by larvae within
the fruit pulp was taken into account. A drop of
cyanoacrylate gel glue was used to hold thermo-
couples in the fruit, and cover the wound. Soil
temperature measurements were taken within 10
cm of the fruit at 5 depths: on the surface directly
under the fruit, at 5 mm, 15 mm, 25 mm, and 50
mm. Air temperature was obtained from within 2
cm above each piece of fruit examined. Tabular
data describing actual temperatures in various
microhabitats consist of first 24 h of data alone
when fruit condition presumably most closely re-
sembled the undisturbed state.
To compare the temperature of larval fruit-mi-
crohabitats to local air temperatures, ratios of
fruit temperature over air temperature were cal-
culated as follows. Minimum, maximum, and av-
erage fruit temperatures from a particular micro-
habitat (subcutaneous or core, tree canopy or
ground, southwest canopy, or northeast canopy)
were divided by the minimum, maximum, or av-
erage air temperatures recorded directly above
the fruit for the same period of time. Because fruit
size might influence the thermal dynamics of
fruit, these ratios are presented graphically in re-
lation to the log of fruit diameter. In order to bal-
ance the needs of maximizing the data set while
at the same time minimizing deterioration of the
fruit, only the first 3 days of data were considered
for comparisons with air temperature regardless
of how long the thermocouples were in place.

Temperature Measuring Device

Temperatures were measured by 32 Type T
thermocouples 18.29 m in length which were in-
serted into fruit and soil at the depths described
above. The thermocouple consist of shielded ther-
mocouple wire with factory manufactured mea-
suring junctions 1 mm in diameter and covered in
Omega Bond (OB-101), a high thermally conduc-
tive epoxy to prevent corrosion due to fruit acids.

All the thermocouples measuring air temperature
were shielded from the effect of thermal radiation
by a small sheet of highly reflective aluminum foil.
The thermocouples were connected to a Camp-
bell Scientific CR-10 Datalogger through a Camp-
bell Scientific AM 416 Relay Multiplexer. A ther-
mocouple reference thermistor was wired to the
CR-10 datalogger to provide temperature compen-
sation and power was provided by a 12-V car bat-
tery. A fifth-order polynomial, resident in the data-
logger, converts the EMF to temperature in Cel-
sius. Although calibrated by the manufacturer
with an accuracy of +0.5C, the 32 thermocouples
connected to the Multiplexer and datalogger were
left to acclimate in the lab and their readings com-
pared the reference thermistor and the internal
datalogger temperature. All the readings were
within the accuracy provided by the manufacturer.
The datalogger and multiplexer were housed
in UV protected-plastic box (45 cm x 30 cm) to
protect them from the elements. Temperature
data were obtained every min, and averaged and
stored every 30 min.

Fruit Tree Locations

Trees were chosen on the basis of being as iso-
lated as possible so that sunlight on the canopy
was unimpeded by neighboring plants. The addi-
tional necessity of being secure enough to leave
unattended computer equipment resulted in the
use of different numbers of trees of the various
species. In several cases, as noted in the individual
descriptions of the sites, different fruit were later
sampled on the same tree to obtain a second data
set. All sites were within the perennially-occurring
range ofA. suspense (Baranowski et al. 1993).
Citrus paradisi. (Two trees, 2 sets of tempera-
ture measurements/tree for a total of 4 sets of
measurements, dates started: 30-Oct-03, 6-Nov-
03, 20-Nov-03, 2-Dec-03): Near Dundee, Florida,
Polk County, Florida (2817'1"N, 8162'2"W; soil
in the area is described as Candler-Tavares-
Apopka: excessively drained, moderately drained
and well drained, sandy soils underlain by loamy
or clayey material; USDA 1990a).
Eriobotrya japonica. (Two trees, 2 sets of tem-
perature measurements on one tree and 1 set on
the other for a total of 3 sets of measurements,
dates started: 2-Mar-04, 11-Mar-04, 26-Mar-04):
Ft. Pierce, Florida, St. Lucie County, (2744'6"N,
8032'5"W; soil is described as Waveland-Lawn-
wood: poorly drained soil, sandy throughout with
dark subsoil weakly cemented; USDA 1980).
Eugenia uniflora. (Two trees with 2 sets of
temperature measurements on each tree for a to-
tal of 4 sets of measurements, dates started: 9-
Apr-04, 16-Apr-04, 21-Apr-04, 5-May-04): LaBelle,
Florida, Hendry County (2644'6"N, 8032'5"W; soil
is described as Holopaw-Basinger association:
poorly drained or very poorly drained, sandy,

Florida Entomologist 90(2)

loamy and organic soils that have a loamy subsoil;
USDA 1990b).
Psidium cattleianum. (Three trees, 1 set of
temperature measurements/tree for a total of 3
sets of measurements, starting dates: 17-July-03,
2-Aug-03, 22-Aug-03): LaBelle, Florida, Hendry
County (soil described as Oldsmar-Wabasso asso-
ciation: poorly drained, sandy soils that have a
sandy and loamy subsoil with organic staining in
the sandy layers; USDA 1990b) and Clewiston,
Florida, Hendry County (2645'12"N, 8056'1"W;
soil is described as Margate association: poorly
drained, sandy soils that are underlain by lime-
stone; USDA 1980).
Psidium guajava. (One tree, 1 set of tempera-
ture measurements, starting date: 29-Aug-03):
LaBelle, Florida, Hendry County (see P. cattli-
eanum above).

Statistical Analyses

Mean, minimum, and maximum temperatures
were initially and individually compared on the
basis of species, location (within the canopy and
on the ground), and the interaction of these 2
variables with SAS (proc GLM) (SAS Inst., Inc.,
Raleigh, NC). Where applicable, means were com-
pared through analysis of variance followed by
the Waller separation of means test (proc
ANOVA). Paired comparisons of temperatures
fruit in and under particular portions of tree can-
opies were made by the nonparametric Wilcoxon
paired-sample test (Zar 1974). Regressions of
fruit diameter to minimum, maximum and mean
temperatures were performed with SAS (proc
GLM) (SAS Inst., Inc., Raleigh, NC).


As suspected, our "thermal snapshots" demon-
strated that immature A. suspense within fruit
and in the soil confront a range of temperatures
over both a seasonal and spatial scale (Tables 1
and 2). The following are some noteworthy points
about this thermal diversity.

Effect of Location In and Under the Canopy
on Temperature Maxima and Minima

The maximum fruit temperatures were signifi-
cantly higher in and under the southwest portion of
the canopies. Fruit in the southwest portions of the
canopies reached significantly higher tempera-
tures than those in the northeast (Table 3) as did
fallen fruit along the southwest margins of the can-
opy. However, there were no significant differences
in the mean and minimum temperatures of fruit in
or under the southwestern and northeastern por-
tions of the canopies. This was probably due to
maxima occurring during daylight hours with more
light striking fruit on the margins of the southwest

canopy, while minima occurring during the night
when location was relatively unimportant.
Tree species consistently and significantly in-
fluenced mean, minimum, and maximum fruit
temperatures, but any interspecific differences in
fruit and canopy morphology co-occurred with
seasonal variation in temperature. However, be-
cause there were no significant interactions be-
tween tree species and the sites of the fruit within
their canopies it is reasonable to assume that tree
morphologies were homogeneous relative to sea-
sonal temperature differences.

Within Fruit Differences in Temperature

There was relatively little difference in mean,
minimum, or maximum temperatures measured
under the surface of fruit and at their cores and no
significant pattern in those temperature differ-
ences that did occur (Tables 1, 2 and 3). Concen-
trating on the southwest portion of the trees where
temperatures were consistently more extreme,
neither fruit still in the tree or on the ground had
warmer subcutaneous than core temperatures.

Effect of Remaining on the Tree and Falling on the
Ground on Maximum and Minimum Temperatures

In the southwest portion of the canopy, maxi-
mum core fruit temperatures were higher in
fallen fruit on the ground than, in some case as
much as 15C hotter (Tables 1; T = 0, P < 0.001).
This was also the case in the northeast (T = 16.5,
P = 0.05), although the mean temperature differ-
ences between fruit cores in the tree and on the
ground was much less in the northeast (6.8C
[southwest] vs. 2.8C [northeast]; T = 12, P<
0.01). There are at least 2 reasons for the warmer
temperature of the fallen fruit: (1) less effective
evaporative cooling after leaving the parent
plant, and (2) the higher temperature of the
ground surface relative to the air. The later is par-
ticularly plausible given the relative insignifi-
cance of a ground-effect in the more shaded areas
in and under the canopy.

Relationship of Fruit to Air Temperature

As might be expected from the above, the rela-
tionship of fruit temperature to the air tempera-
ture immediately above the fruit differed in re-
gards to fruit on the tree and on the ground (Figs.
1 and 2). The temperatures of tree-fruits were sel-
dom +0.05 of the air temperature. However,
ground fruit were sometimes 0.25 of the air tem-
perature. There were no relationships between
fruit size and the maximum temperatures fruits
reach relative to air temperature. However, there
was a consistent pattern of fruit size being posi-
tively correlated to minimum and mean tempera-
tures and this pattern held regardless of location

June 2007

Sivinski et al.: Thermal Environment of Immature Anastrepha suspense


Fruit SW ts SW tc NE ts NE tc SW gs SW gc NE gs NE gc

Cattley 28.4 4.4 28.5 4.8 27.7 3.1 27.7 3.2 31.0 7.6 31.0 7.7 28.8 4.6 28.9 4.5
Guava 1 (23.7-39.2) (23.4-40.5) (23.8-33.5) (23.9-33.8) (24.4-48.0) (24.2-48.1) (23.7-41.1) (23.7-41.1)
Cattley 28.5 3.7 24.3 3.6 28.6 3.9 28.0 3.3 31.5 8.1 31.1 6.7 29.1 4.6 28.8 3.9
Guava 2 (24.2-36.4) (23.7-35.5) (23.6-36.5) (23.7-34.8) (24.6-48.6) (25.2-44.5) (24.0-40.7) (24.4-37.8)
Cattley 27.5 4.7 27.8 5.3 27.0 4.3 27.1 4.4 29.3 6.1 29.0 5.9 29.1 5.7 29.1 5.3
Guava 3 (22.2-37.6) (22.0-39.8) (22.1-35.8) (22.2-37.2) (23.4-47.5) (23.0-45.9) (23.0-41.5) (23.4-40.0)
Guava 26.4 3.5 26.6 3.6 26.0 2.9 26.0 2.8 27.8 4.7 27.6 4.5 26.3 2.6 26.2 2.2
(22.6-33.8) (22.6-34.5) (22.6-31.5) (22.6-31.3) (23.0-38.1) (22.9-37.4) (23.3-31.0) (23.5-30.3)
Surinam 19.9 5.8 20.0 5.9 18.8 4.6 18.9 4.7 22.1 9.1 22.6+ 9.1 18.6 3.9 18.7 3.7
Cherry 1 (12.9-28.6) (12.9-28.7) (12.8-25.2) (12.9-25.2) (13.0-40.4) (13.7-40.8) (13.4-24.5) (13.8-24.5)
Surinam 24.9 3.3 25.0 3.4 24.5 2.8 24.5 2.7 27.5 7.4 27.8 7.7 24.5 2.4 24.4 2.3
Cherry 2 (21.3-32.0) (21.3-32.4) (21.3-29.9) (21.4-29.8) (20.8-46.0) (20.9-47.6) (21.6-29.0) (21.6-28.9)
Surinam 22.2 5.7 20.0 5.4 22.0 5.3 24.8 9.5 24.5 8.7 21.9 4.4 21.9 4.4
Cherry 3 (14.1-30.6) (14.0-29.9) (14.0-29.5) (14.6-49.1) (15.0-46.0) (15.3-29.5) (15.5-30.8)
Surinam 22.2 7.1 22.1 7.0 21.4 6.0 21.4 5.9 23.7 10.6 22.8 9.1 21.3 5.8 21.3 6.2
Cherry 4 (12.9-33.3) (12.9-33.2) (13.1-29.2) (13.1-29.2) (13.0-45.4) (12.7-39.1) (13.7-31.9) (13.1-32.4)
Loquat 1 23.4 5.3 23.3 5.1 22.6 4.2 22.6 4.5 23.8 7.8 23.8 7.8 22.4 5.4 22.5 5.7
(17.8-36.7) (17.8-35.5) (17.6-30.2) (17.4-31.2) (16.8-45.8) (16.8-44.8) (16.6-32.5) (16.4-33.6)
Loquat 2 16.7 + 8.1 16.9 8.3 16.2 7.5 17.1 9.3 18.2 10.2 18.6 10.8 16.4 8.9 16.6 + 9.1
(7.6-29.5) (7.6-29.8) (7.4-28.1) (7.2-34.4) (8.6-42.6) (8.5-43.4) (7.2-34.8) (7.3-34.9)
Loquat 3 22.5 3.5 22.6 3.8 21.6 2.2 21.5 2.2 24.8 7.5 24.4 6.7 21.4 2.6 21.7 3.3
(17.1-28.8) (16.8-29.6) (17.2-25.3) (17.3-25.6) (17.5-41.6) (17.7-39.4) (18.2-41.3) (17.7-36.6)
Grapefruit 23.9 6.7 24.2 7.5 21.8 3.7 21.9 3.7 24.2 6.4 25.5 7.8 22.0 3.4 22.0 3.4
1 (16.8-37.7) (17.0-37.9) (17.6-28.7) (17.8-28.7) (17.4-37.1) (17.5-40.5) (18.1-28.4) (18.1-28.2)
Grapefruit 23.1 2.7 22.8 1.7 22.5 1.3 22.5 1.3 23.7 3.0 23.7 2.2 22.7 1.3 22.9 1.2
2 (21.3-33.3) (21.4-27.5) (21.2-26.3) (21.3-26.1) (21.7-33.8) (22.0-30.6) (21.6-26.7) (21.8-26.3)
Grapefruit 20.5+ 8.1 20.3 7.9 16.3 3.5 16.3 3.4 20.0 10.2 20.2 8.8 16.7 3.3 16.6 3.3
3 (11.4-34.9) (11.4-33.8) (12.1-25.0) (12.1-23.2) (9.8-37.9) (10.9-36.6) (12.7-26.7) (12.7-28.0)
Grapefruit 18.3+ 8.1 18.1 7.5 15.3 4.0 15.4 4.0 18.5 9.5 19.8 9.5 15.4 4.0 15.5 3.4
4 (9.1-32.0) (9.4-29.3) (10.1-21.8) (10.2-21.9) (7.9-35.9) (9.3-36.5) (10.3-21.9) (11.0-21.0)

(tree NE mean int.= 1.0 b = 0.002; tree NE mini-
mum int. = 0.99 b = 0.006; tree SW mean int. =
0.99 b = 0.005; tree SW minimum int. = 1.0 b =
0.001; ground NE mean int. = 0.99 b = 0.002;
ground NE minimum int. = 1.007 b = 0.005;
ground SW mean int.= 1.01 b = 0.007; ground SW
minimum int. = 1.009 b = 0.009). There was con-
siderable variance in many of relative tempera-
ture relationships (see r2 values in Figs. 1 and 2),
due presumably to a complex set of factors that
differed under individual circumstances (Tables 1
and 2; Figs. 1 and 2). All other things being equal,
larger fruits should retain greater amounts of
heat derived from sunlight. However, it should be
noted that while fruit size and temperatures were
sometimes correlated, the different sized fruit
also had a variety of morphologies, and that it is
possible that it was these morphological differ-
ences that were related to temperature. If so, the

size relationship was coincidental, and particular
attention might be focused on the thermodynam-
ics of grapefruit, the largest fruit measured.

Relationship of Soil Depth to Temperature

As in fruit, maximum soil temperatures were
higher under the southwestern margin of the can-
opy than under the northeastern interior (Table
2; surface temperature: T = 0, P < 0.001). Maxi-
mum temperatures declined with depth on the
southwestern canopy margin, but there was no
relationship in northeast soils (Table 3).


In general, fruit temperatures were higher in
the southwestern portions of tree canopies rela-
tive to those in the northeastern interiors. Fruit

Florida Entomologist 90(2)

June 2007


Fruit SW s SW 5 SW 25 SW 50 NE s NE 5 NE 25 NE 50

Guava 1
Guava 2

30.9 6.3 30.7 4.5 30.4 3.4 30.2 2.6 28.7 3.1 29.0 3.4 28.6 2.3 28.6 2.0
(25.3-45.5) (26.4-42.4) (26.7-38.5) (27.2-35.8) (25.5-37.7) (25.5-38.9) (26.0-33.8) (26.2-32.8)
31.0 5.9 30.2 4.2 30.1 3.2 29.9 2.7 28.5 2.3 28.5 2.4 28.4 2.0 28.3 1.7
(25.6-44.7) (26.2-39.2) (26.6-36.6) (26.9-35.8) (25.8-33.7) (25.7-34.7) (25.9-33.3) (26.2-31.6)

Cattley 30.2 6.0 29.2 2.7 29.3 2.3 29.3 1.7 29.8 4.1 29.8 4.0 29.5 2.8 29.4 2.3
Guava 3 (24.6-48.2) (26.2-35.8) (26.6-34.8) (27.1-32.5) (25.0-37.7) (25.1-36.8) (25.9-34.4) (26.3-33.2)
Guava 28.3 4.9 27.8 3.9 27.7 3.2 27.6 2.7 26.0 1.2 26.0 1.3 26.0 1.1 26.1 0.8
(23.6-41.7) (23.7-37.4) (24.1-34.7) (24.5-33.4) (24.5-28.5) (24.3-28.5) (24.6-28.2) (24.8-27.6)
Surinam 22.6 7.2 22.8 4.5 23.0 4.2 23.2 4.0 18.1 2.3 18.1 1.8 17.9 1.2 17.8 1.1
Cherry 1 (15.3-37.9) (17.8-31.7) (18.5-31.3) (18.6-31.2) (14.9-21.2) (15.4-20.6) (15.9-19.6) (16.1-19.3)
Surinam 26.7 6.3 28.2 5.9 27.7 4.7 27.5 3.1 24.4 2.1 24.3 1.9 24.0 1.4 23.7 1.2
Cherry 2 (20.6-42.7) (22.7-43.3) (23.2-39.5) (24.3-34.3) (21.9-28.3) (22.0-28.1) (22.2-26.5) (22.2-25.9)
Surinam 24.4 6.0 24.8 4.7 24.7 3.6 24.8 3.5 21.0 2.1 21.1 2.1 20.8 1.5 20.6 1.2
Cherry 3 (17.3-39.7) (19.2-35.8) (20.3-32.3) (20.4-32.0) (17.5-24.1) (17.8-24.1) (18.2-22.9) (18.6-22.3)

Cherry 4
Loquat 1

23.9 10.7 24.2 4.6 24.0 3.1 24.0 2.8 21.0 4.4 21.4 4.1 20.9 3.1 21.4 1.6
(13.4-46.1) (19.2-33.7) (20.3-30.2) (20.7-29.3) (14.9-27.2) (14.1-28.3) (16.6-27.8) (19.0-26.0)
23.6 6.2 22.5 3.0 22.3 2.4 22.1 1.8 22.4 5.4 22.0 3.5 22.0 2.9 21.6 1.7
(18.0-40.9) (19.3-30.3) (19.7-28.3) (20.0-26.2) (16.7-33.1) (18.1-28.4) (18.8-28.1) (19.6-25.0)

Loquat 2 19.1 10.2 19.9 6.2 19.9 4.5 20.0 2.8 17.3 8.3 17.8 6.3 18.8 5.0 19.3 3.1
(9.7-45.6) (13.7-34.9) (15.1-30.0) (16.6-26.0) (8.8-33.9) (11.0-29.4) (13.2-28.8) (15.5-24.7)
Loquat 3 25.6 8.0 25.2 6.7 24.8 5.8 24.7 4.5 21.6 3.4 21.4 2.7 21.0 1.6 21.1 1.6
(18.1-45.0) (18.4-41.3) (18.4-36.6) (19.6-33.9) (17.9-33.0) (18.3-29.5) (18.8-24.0) (18.9-23.6)
Grapefruit 25.8 4.9 26.0 4.9 25.0 4.4 25.9 3.4 21.8 2.1 21.6 1.9 21.6 1.4 21.6 1.1
1 (20.4-35.5) (20.1-36.1) (20.5-34.7) (21.8-32.6) (19.2-25.5) (19.3-25.3) (19.8-24.1) (20.2-23.5)
Grapefruit 24.4 2.3 24.2 1.9 24.0 1.7 24.4 1.5 22.5 1.2 23.0 0.7 23.1 0.7 23.3 0.5
2 (22.7-30.3) (22.4-31.9 (22.8-29.8) (23.1-28.7) (21.2-26.4) (22.3-25.0) (22.4-25.0) (22.7-24.7)
Grapefruit 21.9 7.8 21.9 7.3 22.3 7.0 20.2 6.2 16.7 2.7 16.9 1.9 17.1 2.0 18.1 1.7
3 (13.4-36.6) (13.3-35.1) (14.1-35.0) (14.7-30.1) (14.0-30.5) (14.4-23.8) (15.1-27.9) (16.7-28.3)
Grapefruit 20.8 7.7 20.6 7.5 20.9 6.5 19.1 6.2 15.5 2.1 15.3 1.8 15.5 1.5 16.2 1.0
4 (12.1-35.1) (11.5-34.9) (12.1-34.9) (13.1-31.1) (12.1-19.7) (12.8-17.8) (13.3-17.7) (14.7-17.5)

on the ground were warmer than those in the
tree, but there was no significant pattern of max-
imum fruit core temperatures being warmer than
subcutaneous pulp. Soil temperatures were also
higher than fruit-in-tree temperatures, and de-
creased and displayed less variance with increas-
ing depth. Fruit in trees seldom reached temper-
atures +0.05 of adjacent air temperatures, but
fruit on the ground could be more than 0.25 the
adjacent air temperature. There were significant
relationships between the ratio of minimum and
mean fruit temperatures/adjacent air tempera-
ture and fruit diameter. Typically, air tempera-
ture in various portions of the canopy are unlikely
to grossly underestimate the minimum tempera-
tures faced by the local immature tephritids, but
maximum temperatures encountered by larvae in

fallen fruit can be substantially higher than sug-
gested by air temperatures. Thus, air tempera-
ture could generally be a useful tool in estimating
many fruit fly thermal environments.

Fruit Temperature Relative to Ambient
and the Function of Cooling

Moist spherical objects in sunlight, sheltered
from winds that increase heat flux, will retain so-
lar energy and reach temperatures well above
ambient (Thorpe 1974). However, certain fruit,
e.g., Ficus spp. evaporatively cool by transpiring
water through stomata on their surface (Patiio et
al. 1994). Fruit in the canopy examined in the
present study, even the more exposed southwest
portion, tended to show little deviation from sur-

Sivinski et al.: Thermal Environment of Immature Anastrepha suspense


Canopy site Mean Minimum Maximum

SW fruit surface a 23.3 (0.98) a 17.2 (1.5) a 33.8 (0.91)
SW fruit core a 22.8 (0.94) a 17.0 (1.4) a 33.2 (1.0)
NE fruit surface a 22.0 (1.1) a 17.1 (1.4) b 29.1 (1.1)
NE fruit core a 22.2 (1.0) a 17.1 (1.4) b 29.5 (1.2)

Ground Site Mean Minimum Maximum

SW fruit surface a 24.7 (1.1) a 17.0 (1.5) a 42.5 (1.3)
SW fruit core a 24.8 (1.0) a 17.4 (1.4) a 41.8 (1.3)
NE fruit surface a 22.0 (1.2) a 17.5 (1.4) b 32.1 (1.7)
NE fruit core a 22.5 (1.5) a 17.6 (1.4) b 31.6 (1.5)

SW soil depth Mean Minimum Maximum

surface a 25.3 (0.93) a 18.7 (1.3) a 41.0 (1.3)
5 mm a 25.2 (0.88) a 20.0 (1.2) b 36.3(1.0)
25 mm a 25.0 (0.85) a 20.7 (1.9) bc 33.8 (0.87)
50 mm a 25.9 (0.92) a 21.2 (1.1) c 31.5 (0.79)

NE soil depth Mean Minimum Maximum

surface a 22.4 (1.2) a 18.7 (1.4) a 29.9 (1.3)
5 mm a 22.4 (1.1) a 19.1 (1.3) a 27.9 (1.5)
25 mm a 22.3 (1.1) a 19.8 (1.2) a 27.5 (1.4)
50 mm a 22.5 (1.1) a 20.5 (1.0) a 25.7 (1.2)

rounding maximum air temperature. This sug-
gests the possibility of adaptive cooling. Patio et
al. (1994) argued that cooling inFicus spp. was re-
quired to protect mutualist pollinators, since figs
prevented from transpiration reached tempera-
tures fatal to the agaonid wasps harbored inside
the fruit. It is difficult to propose such a hypothe-
sis in the present case since most of the insects lo-
cated inside the fruit are frugivores, or parasi-
toids of frugivores that would disperse and be un-
likely to protect the subsequent fruit of any par-
ticular individual fruit tree (see discussion of
larval behavior below). Perhaps such high tem-
peratures damage seeds as well, and fruits are
sometimes designed and located to cool and pro-
tect plant genetic material.

Temperature and Population Dynamics

The distributions ofAnastrepha spp. and other
tephritids are believed to be influenced by abiotic
environmental factors (e.g., Messenger & Flitters
1954; Meats 1981; Drew & Hooper 1983; Sivinski
et al. 2000), and temperature is also a principal
factor in the distribution ofAnastrepha spp. par-
asitoids. For example, the relative abundance of 2
introduced braconid parasitoids ofA. suspense in
Florida is related to temperature and the effects

of temperature on host fruit diversity and avail-
ability (Eitam et al. 2004).
On a smaller spatial scale, Aluja & Birke
(1993) found fewer Anastrepha obliqua (Mac-
Quart) ovipositing in exposed as opposed to
shaded host trees. While females might avoid the
warmer and drier microenvironment of the ex-
posed trees for their own wellbeing, they could
also be seeking more suitable larval habitats in
the shade. As to the distribution of subtropical
and tropical fruit fly and parasitoid larvae within
tree canopies, several studies have yielded some-
what mixed results with the emergence of rela-
tively weak patterns (Sivinski et al. 1997, 1999,
2004). Perhaps the multitudinous combinations
of microhabitat-abiotic effects, local natural ene-
mies and competitors make it difficult to general-
ize about the role of any particular variable. Tho-
mas (1993) found similarly weak correlations be-
tween temperature and moisture extremes and
the survival ofA. ludens pupae in the field, and
argued that the effects of weather variables were
probably masked by predation.
As previously noted, the present work sug-
gests that larval-environment temperatures vary
with microhabitat, but are relatively similar to
the air temperatures in the same vicinities. How-
ever, there are significant relationships between

Florida Entomologist 90(2)

SW Portion of Canopy

lnnumwr.i 3 pCAl S01c .
MAnkllm r0.l. paO.710, t.'002
o Avnrios t.ll. 4 8P0.. rO0.i

0 2 4 I I 10 12
Fruit Diameter (cm)

NE Portion of Canopy

0 2 4

I I 10

Fruit Diameter (cm)

Fig. 1. (A) The ratio of fruit temperature to air tem-
perature in the south west portions of the canopies for
each of the first 3 d of monitoring in relation to the di-
ameter of the fruit. The minimum, maximum, and mean
ratios of fruit on the southwest margins of the canopies
are considered. Because there were no significant differ-
ences between subcutaneous and core temperatures,
only core temperatures are considered. (B) As above in
the north east portions of the canopies.

minimum and mean temperatures relative to air
temperature and fruit size.

Temperature and Larval Behavior

Heating through "forced air" or in water baths
has long been used to disinfest fruit destined for
export (e.g., Hawkins 1932). In general, but with
some variance depending on species, tempera-
tures in excess of 45C will quickly kill fruit fly
eggs and larvae (e.g., Armstrong 1992). At 43C,
the exposure time required for 95% of 3rd instar
A. suspense larvae to perish depends on both the
medium in which the insects were reared and
that in which they are heated (Hall 1996). The
adult "L(ethal)T(ime)95" of larvae reared in grape-
fruit and exposed in grapefruit juice, the most
natural of the tested regimens, was 24 min. Using
temperature probes inserted into olive (Olea euro-

1.1 -

2 -

0.; -

2 0. -

Ground under SW Portion of the Canopy

a Mi nmumn r-o.zrp".. r-o.oN
S Mmum r-0.23, p=0.12. r- 0.05
O *Avre -0. 41.1p0.004, r 0.17 a

0 2 4 4 8 10 12
Fruit Olameter (cm)

Ground under NE Portion of the Canopy
a Minimum r40.57, pc0.0001, r'0.32
0 Maxim .0, 3. p-O.3, 0.04 00
0 Avrag r0S,, p-0.0001, r'0.35

---- o


P 2 4 5 a 10
Fruit Diameter (cm)

Fig. 2. (A) The ratio of fruit temperature / air temper-
ature for fruit on the ground at the south west margin of
the canopies for each of the first 3 d of monitoring in re-
lation to the diameter of the fruit. The minimum, maxi-
mum, and mean ratios of fallen fruit on the southwest
margins of the canopies are considered. Because there
were no significant differences between subcutaneous
and core temperatures, only core temperatures are con-
sidered. (B) As above, but under the north east-interior
portions of the canopies.

paea L.) drupes, Pucci et al. (1981) correlated
mortality in immature olive fruit flies Bactrocera
oleae (Gmelin) to temperature. Eighty-five per-
cent of eggs and first-instar larvae and 95% of ma-
ture larvae died when daily maximum tempera-
tures reach just 36C for a period of a week. Given
that fruit in the present study, particularly fallen
fruit on the southwest margin of tree canopies, of-
ten reached temperatures in excess of 43C and
sometimes temperatures that approached 50C, it
would seem that larvae could frequently find
themselves in danger of overheating.
Once on the soil surface, a fruit-exiting larva
could still face lethal temperatures. Even 5 mm
below the surface temperatures sometimes
reached 43C, and it was only at depths of 25 mm
that no temperatures >40C were recorded. At a

June 2007

S Mirnu.nm t. 68. p ManximLm e.t25. pa.t, t/s 6.
0 --p
0 Avnmgo r0.40, p<.Mi. r'-.o.1


I --4--A _


Sivinski et al.: Thermal Environment of Immature Anastrepha suspense

site inhabited by fruit flies in northern Mexico,
Thomas (1993) measured temperatures as high as
38C at depths of 30-40 mm, and noted that ex-
posed soils were 6-7 warmer at than those under
shade. In addition to harmful temperatures just
beneath the surface, ant predators and pupal par-
asitoids tend to be more efficient at lesser pupa-
tion depths (Hogdson et al. 1998; Baeza et al.
2002; Guillen et al. 2002). Not surprisingly, in one
Mexican field survey ofAnastrepha spp. pupations
depths no pupae were found on the surface, 56%
were uncovered at depths up to 20 mm and most of
the rest at depths of 20-50 mm. Only one occurred
deeper than 50 mm (Hogdson et al. 1998).
Perception of soil surface temperature appears
to influence the speed with whichAnastrepha lar-
vae begin to burrow. Under warm condition in
Mexico larvae quickly burrow directly beneath, or
close to, the fruit they developed within (Aluja
et al. 2006). However, under cooler conditions,
Thomas (1995) describes A. ludens wandering on
the surface to find suitable pupations sites.

Temperature and Conditional-effect-lethal Strains

Sterilization through irradiation often harms
the performance of released insects and, as a con-
sequence, SIT sometimes fails to reach its theoret-
ical potential (e.g., Proshold 1993; Barry et al.
2003b). Autocidal strains that result in offspring
death or sterility and also avoid radiation may be
more effective (Alphey 2002). Such strains, based
on the conditional regulation of genes that encode
lethal products, might be most easily produced
through genetic transformation (Robinson &
Franz 2000; Handler 2002; Handler & Atkinson
2006). A variety of mutant and normal genes af-
fecting cell viability can be used, including mu-
tant lethal genes affecting vital processes, normal
genes involved in programmed cell death (White
et al. 1994), and genes for toxin subunit molecules
(Kalb et al. 1993). A critical component to the use
of these genes is the ability to regulate their ex-
pression in terms of developmental stage, tissue,
and sex-specificity for the desired phenotype so
that breeding populations can be maintained.
This can be achieved by conditional regulation
where lethal gene expression is determined by
manipulation of temperature, chemical treat-
ment, or by interbreeding 2 independent strains.
Model systems have already been tested inDroso-
phila spp. with temperature-sensitive lethal alle-
les and by creating female lethals and steriles by
tetracycline-dependent transcriptional repression
(Heinrich & Scott 2000; Horn & Wimmer 2003).
Among the temperature regulated lethal sys-
tems developed in Drosophila is the inclusion of a
cold-sensitive allele that kills both heterozygous
and homozygous individuals when the tempera-
ture falls below 18C (Fryxell & Miller 1995). Thus
the offspring of homozygous individuals reared

and released at higher temperatures would die as
temperatures fell. In the A. suspense habitats ex-
amined minimum temperatures were frequently
well above 18C (e.g., Psidium spp.) and this par-
ticular scheme, if transferable to A. suspense,
would require an upward temperature adjustment
to have an immediate effect. However, the propor-
tion of individuals carrying such a gene could be
increased by repeated releases during warm sea-
sons of the year and the population would then
crash with the onset of winter. Alternatively, con-
ditional systems under consideration/develop-
ment would release fruit flies reared at relatively
low temperatures whose offspring would perish af-
ter encountering warmer temperatures in the field
(Handler & Atkinson 2006).
In summary, immature Caribbean fruit flies
faced a variety of temperatures, but with the ex-
ception of fallen fruit exposed to strong sun light,
these temperatures are similar to ambient air
temperatures. While fruit size was correlated to
the mean and minimum temperatures reached, it
did so to a relatively minor extent. If other host
fruit of other tephritid species have similar ther-
mal properties, then air temperature should be a
useful tool to estimate the thermal environments
of immature fruit flies outside of Florida. It
should be kept in mind that not all subtropical
pest tephritids face temperatures identical to
those recorded in the present study. For example,
A. ludens in northern Mexico sometimes encoun-
ter and survive below freezing temperatures
(Thomas 1993).


Martin Aluja and Nancy Epsky made many useful
criticisms of the manuscript as did two anonymous re-
viewers. Gina Posey and Charlie Stuhl made the fig-
ures, and Valerie Malcolm prepared the manuscript for


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(Diptera: Tephritidae) in an agricultural and nonag-
ricultural situation. J. Econ. Entomol. 28: 350-362.
THOMAS, D. 1995. Predation on the soil inhabiting
stages of the Mexican fruit fly. Southwest. Entomol.
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Englewood Cliffs, NJ.

Florida Entomologist 90(2)

June 2007


'Department of Entomology and Nematology
P.O. Box 110620, University of Florida, Gainesville, FL 32611-0620 USA

2University of Florida, Institute of Food and Agricultural Sciences, Citrus Research & Education Center
700 Experiment Station Road, Lake Alfred, FL 33850


After the citrus leafminer (CLM), Phyllocnistis citrella Stainton (Lepidoptera: Gracillarii-
dae), invaded Florida in 1993, the endoparasitoid Ageniaspis citricola Logvinovskaya (Hy-
menoptera: Encyrtidae) was introduced in 1994 in a classical biological control program.
Subsequent to its establishment, only limited information has been obtained regarding the
seasonal abundance ofA. citricola and its host in central Florida citrus groves. During 2005,
we monitored replicated plots treated with oil or imidacloprid once on 23 Jun 2005, along
with untreated control trees, in a Polk County commercial Valencia orange grove on a weekly
basis when tender new growth (= flush) was available. As expected, CLM abundance in the
early spring flush was nearly undetectable due to the lack of suitable flush during winter
when CLM populations decline nearly to zero. Also as expected, A. citricola was not found
during this time. During the second flush (Jun through Jul) CLM populations increased and
A. citricola appeared, parasitizing up to 39% of the pupae in the untreated controls and up to
33% in the blocks treated with oil. Imidacloprid did not significantly reduce the number of
CLM larvae but did reduce Asian citrus psyllid, Diaphorina citri Kuwayama, nymphal den-
sities. Peak abundance of the CLM occurred during the third flush cycle on 5 Oct from trees
treated once with oil, with a mean (SD) of 1.3 (0.8) CLM mines per leaf. Parasitism by A. cit-
ricola increased through the season, peaking at 56% of the CLM that had pupated prior to the
16 and 23 Nov samples in the untreated control trees and at 37% in the oil-treated trees;
A. citricola was not found in imidacloprid-treated trees on those dates. During the growing
season, a high proportion (up to 100% in some samples) of the CLM mines were empty, pre-
sumably due to predation. The data confirmed, for the first time, that A. citricola is an impor-
tant natural enemy of those CLM larvae that escaped predation in this citrus-growing area
in Florida. Nymphs of the Asian citrus psyllid were significantly reduced for 3 weeks after the
imidacloprid treatment. However, shoots on trees treated with imidacloprid were signifi-
cantly shorter than shoots on untreated trees and the number of shoots produced in imida-
cloprid-treated trees was reduced, raising concerns that imidacloprid might affect growth of
citrus flush. Brown citrus aphids were nearly absent throughout the growing season.

Key Words: citrus leafminer, Ageniaspis citricola, population dynamics, imidacloprid, oil,


Despu6s de que el minador de la hoja de los citricos (MHC), Phyllocnistis citrella Stainton
(Lepidoptera: Gracillariidae), invadi6 el estado de la Florida en 1993, el endoparasitoide
Ageniaspis citricola Logvinovskaya (Hymenoptera: Encyrtidae) fue introducido durante
1994 para un program de control biol6gico clasico. Subsiguiente a su establecimiento, in-
formaci6n obtenida en cuanto a la abundancia estacional deA. citricola y sus hospederos en
huertos de citricos en Florida central fue muy limitada. Durante el ano del 2005, nosotros
realizamos un monitoreo en parcelas replicadas tratadas con aceite o imidacloprid por una
vez en el 23 de junio de 2005, incluidos con arboles no tratados para control, en un huerto co-
mercial de naranjas "Valencia" en el condado de Polk todo ello revisado semanalmente
cuando los brotes de nuevas hojas fueron disponibles. Como fue esperado, la abundancia de
MHC en los brotes de nuevas hojas en el principio de la primavera fue casi no detectable de-
bido a la falta de brotes apropiados durante el invierno cuando la poblaci6n de MHC baj6 a
casi cero. A su vez, como era de esperar, A. citricola no fue encontrado durante este tiempo.
Durante el segundo brote de hojas (junio ajulio) la poblaci6n de MHC aument6 yA. citricola
apareci6, parasitizando hasta 39% de la pupas en los bloques no tratados de control y hasta
33% de los bloques tratados con aceite. Imidacloprid no reduj6 significativamente el nmmero
de larvas de MHC pero si reduj6 la densidad de las ninfas del psila de citrico Asidtico, Dia-
phorina citri Kuwayama. El pico de la abundancia de MHC ocurri6 durante el ciclo del tercer

Hoy et al.: CLM and Natural Enemy Dynamics in Central Florida

brote de las hojas nuevas en el 5 de octubre en arboles tratados una vez con aceite, con un
promedio (DS) de 1.3 (0.8) minas de MHC por hoja. El parasitismo porA. citricola aument6
a travez de la estaci6n, llegando a un climax de 56% de los MHC que han empupado antes
de las muestras de 16 y 23 de noviembre en arboles no tratados y 37% en arboles tratados
con aceite; A. citricola no fue encontrado en arboles tratados con imidacloprid en estas fe-
chas. Durante la estaci6n de crecimiento, una alta proporci6n (hasta 100% en algunas mues-
tras) de las minas de MHC fueron vacias, propuestamente debido a la depredaci6n. Los datos
confirmaron, por primera vez, que A. citricola es un enemigo natural important de las lar-
vas de de MHC que escapan a los depredadores en esta area en Florida donde se siembra ci-
tricos. Las ninfas del psila de citrico Asidtico fueron reducidas significativamente por 3
semanas despu6s del tratamiento con imidaloprid. Sin embargo, los brotes en los arboles tra-
tados con imidacloprid fueron significativamente mas cortos que en los arboles no tratados
y el nmmero de los brotes producidos en arboles tratados con imidacloprid fue reducido, au-
mentando la preocupaci6n de que el imidacloprid posiblemente puede estar afectando el cre-
cimiento de los brotes de los citricos. El afido pardo de los citricos [Toxoptera citricida]
durante la estaci6n de crecimiento estuvo casi ausente.

The citrus leafminer (CLM), Phyllocnistis cit-
rella Stainton (Lepidoptera: Gracillariidae), was
discovered in Florida in May of 1993 and quickly
spread through >800,000 acres of citrus, attack-
ing tender new growth (= flush) and, occasionally,
fruits and stems when densities were particularly
high (Heppner 1993). Shortly after the invasion,
native parasitoids (primarily eulophids) attacked
this pest, as well as generalist predators (Brown-
ing & Peia 1995; Peia et al. 1996; Evans 1999;
Amalin et al. 1996), but growers still considered
CLM densities too high and treated both mature
and young groves multiple times per season
(Heppner 1995; Knapp et al. 1996). The host-spe-
cific endoparasitoid Ageniaspis citricola Logvi-
novskaya (Hymenoptera: Encyrtidae) was im-
ported from Australia (Neale et al. 1995) and first
released in Florida in May 1994 in a classical bio-
logical control program (Hoy & Nguyen 1994a;
1997; Smith & Hoy 1995). A subsequent importa-
tion ofA. citricola from Taiwan also was released
(Hoy et al. 2000). Although the Australian (which
was originally from Thailand) and Taiwan popu-
lations appeared morphologically identical, sub-
sequent molecular studies indicated that they
were, in fact, cryptic species (Hoy et al. 2000;
Alvarez & Hoy 2002). Both populations were re-
leased in Florida, but a subsequent analysis
failed to show that the Taiwan population had es-
tablished (Alvarez 2000).
Establishment and spread ofA. citricola, pre-
sumably the Australian population, in Florida
was rapid and high rates of parasitism of CLM pu-
pae were observed (Hoy & Nguyen 1994a; 1994b;
1997; Hoy et al. 1995; 1997; Pomerinke & Stansly
1998; Amalin et al. 1996; 2002). However, after es-
tablishment and dispersal were documented,
funding for monitoring the phenology and dynam-
ics of the CLM and A. citricola was unavailable
because the CLM'problem' appeared to have been
solved, at least temporarily. As a result, informa-
tion about the abundance and phenology ofA. cit-
ricola in Florida's citrus groves remained anec-
dotal. Concerns about CLM population densities

in Florida resurfaced during the citrus canker
eradication program, because mines produced by
CLM larvae allow the canker bacteria access to
ideal growing conditions (Sohi & Sandhu 1968;
Chagas et al. 2001; Gottwald et al. 2001; Graham
et al. 1996; Christiano et al. 2007). In addition,A.
citricola appeared less effective during 2000-2002
than in previous years because Florida was un-
dergoing a drought and this parasitoid performs
poorly when relative humidity is low (Yoder &
Hoy 1998). Despite the fact that natural enemies
cannot eliminate all CLM in a grove and even a
single CLM can cause damage to foliage that in-
creases the susceptibility of a tree to canker infec-
tion, consideration was given to importing addi-
tional parasitoids of the CLM, with the goal of fur-
ther reducing CLM densities and, hopefully, can-
ker incidence. Semielacher petiolatus Girault
(Hymenoptera: Eulophidae) was imported and
evaluated in quarantine, but not released because
the potential risk of disrupting biological control
byA. citricola was considered higher than the po-
tential benefit of establishing S. petiolatus in Flor-
ida (Lim & Hoy 2005: Lim et al. 2006).
During 2005, we monitored a commercial cit-
rus block in central Florida (Polk County) near
Haines City each week during the major flush cy-
cles to evaluate the phenology and relative abun-
dance of the CLM and A. citricola in Valencia or-
anges that were untreated or treated with oil or
with imidacloprid. We also evaluated additional
mortality factors of the CLM. Relative abun-
dances of the Asian citrus psyllid, Diaphorina
citri Kuwayama, and the brown citrus aphid, Tox-
optera citricida Kirkaldy, on the flush also were


Plots were established in a Valencia orange
grove near Haines City, Florida (GPS coordinates:
N 2803.656, W 08134.937). The trees were 4-5
years old and spaced 7.3 m apart between the
rows and 3 m within the rows. During 2004, the

Florida Entomologist 90(2)

year prior to this study, the only pesticide applied
in this grove was petroleum oil (470 weight oil,
(Petro-Canada, Calgary, Alberta), which was ap-
plied 3 times (May, Jul, and Sep) at a rate of 7 gal
acre (26.5 L/ 0.4 ha), which should not have had
significant negative effects on A. citricola in the
grove because oil has little residual toxicity (Vil-
lanueva-Jimenez & Hoy 1998). Trees were drip
irrigated as needed and fertilized in 2004 with
Nutri 5 at 1 qt/acre (0.95 L/0.4 ha) and with 3
Key-Plex foliar sprays at a rate of 2 qt/acre (1.89
L/0.4 ha) each. During 2005, a foliar application
of potassium nitrate (N:P:K at a rate of 13.75-0-
46) was added on 18 Oct 2005 and an organic
amendment from poultry houses was added at a
rate of 1000 lbs/acre (453.6 kg/0.4 ha) on 1 Sep
2005. During 2005 no sprays, other than those re-
quired by the experiment, were applied.
Randomized complete blocks with 4 treat-
ments and 3 replicates of each treatment were set
up in Mar, with each replicate consisting of 3 ad-
jacent rows of 10 trees each with 3 buffer rows be-
tween each of the treatments. Trees were left un-
treated between replicates to reduce any effect of
spray drift. Out of 30 trees in an experimental
unit, 6 central, uniform and healthy trees were la-
beled and 4 young shoots per tree were collected,
when present, at weekly intervals throughout the
2005 growing season from Mar until the end of
Nov. Each week the percentage of terminals hav-
ing new flush was estimated to determine the
flushing patterns.
The 4 planned treatments consisted of (A) un-
treated control, (B) 3 sprays at 6-week intervals
starting in Jun when the flush was about 3 cm in
length with petroleum oil 455 (Petro-Canada,
Calgary, Alberta) at 2% (20 mL/L of water), (C) 1
application of imidacloprid (Provado 1.6 F, Bayer
CropScience, North Carolina) at the lowest rec-
ommended foliar application rate of 10 oz/acre
(295 mL/0.4 ha) when the flush was 3 cm long,
and (D) 2 sprays of petroleum oil 455 in weeks 1
and 3 of the Jun flush cycle at 2%. Treatment B
was planned because many growers were using
this spray schedule and Treatment D was
planned to determine whether 2 treatments dur-
ing the Jun flush cycle (second cycle) would allow
A. citricola to 'catch up' with the citrus leafminer
population and eliminate the need for additional
sprays. Because populations of the citrus leaf-
miner and its host-specific parasitoidA. citricola
decline to very low levels during the winter in
Florida when very little tender new growth is
available to support reproduction of the leafminer
(Lim & Hoy 2006), populations of both species
may be nearly undetectable during the first flush
cycle in Feb or Mar (Villanueva-Jimenez et al.
2000). Treatment C was considered the standard
to which the 2 oil treatments would be compared.
However, only 1 application of oil was applied on
23 Jun to treatments B, C, and D because so few

CLM were present during Jun through Nov that
additional sprays could not be justified.
When flush was present, 4 shoots longer than
0.5 cm were collected from each of 6 trees in each
replicate and placed in a labeled plastic bag con-
taining a paper towel to soak up any moisture
that could cause the leaves to begin to rot prior to
scoring. Plastic bags were placed in an ice chest
with ice packs and shipped to the University of
Florida, Department of Entomology and Nema-
tology in Gainesville by FedEx overnight delivery.
Samples were scored with the aid of a dissect-
ing microscope. Shoot length and numbers of
leaves per shoot were recorded, as well as the
number of CLM mines (>0.5 cm) per leaf, the
number of CLM larvae in mines that were alive,
parasitized, absent, or dead. Larvae missing from
the mines were assumed to be dead from preda-
tion if no pupal chambers were associated with
the mine. Pupal chambers were opened and the
number of CLM pupae that were alive, parasit-
ized by A. citricola (including number of A. citri-
cola pupae) or by other parasitoids, or dead due to
unknown causes was recorded. Relative abun-
dance per shoot of Asian citrus psyllids was re-
ported as 0 = none, 1 = 1-20, 2 = 21-50, 3 = 51-80,
and 4 = >80 and the relative abundance of brown
citrus aphids was reported as 0 = none, 1 = <10, 2
= 10-50, and 3 = >50. Data were analyzed by
ANOVA and means separated by Fisher's least
significant difference (LSD) test, based on 5%
level of significance (SAS Institute, Cary, NC,
Weather data were obtained from the nearest
weather station at the Citrus Research and Edu-
cation Center, Lake Alfred (http://fawn.ifas.ufl.
edu/scripts/reportrequest.asp), and averaged
each week over the experiment; the weather sta-
tion is approximately 12.8 km from the experi-
mental site.


During 2005, the study trees produced flush
suitable for CLM, Asian citrus psyllid, and brown
citrus aphid populations 3 times (Fig. 1). The first
flush cycle began by 22 Feb and ended by 18 Apr;
the second cycle began around 13 Jun and ended
around 2 Aug, and the third began 27 Sep and
ended by 30 Nov 2005. Tender new growth suit-
able for oviposition by CLM females was present
during the first week of each flush cycle and the
flush continued to grow and harden off during the
subsequent 5 or 6 weeks. The proportion of tree
branches that were flushing during the first 2
flush cycles was not different, but there were
fewer (F = 3.26, df = 3, P = 0.025) flushes in the
imidacloprid-treated trees during the third flush
cycle. The average weekly temperature (C), rela-
tive humidity (% RH), and rainfall (cm) during
the trial are shown in Fig. 2.

June 2007

Hoy et al.: CLM and Natural Enemy Dynamics in Central Florida

18 .-.- GOIL
16 --OIL

14 Flush One Flush Two Flush Three

. 10



Fig.1 Mea s t lg (c o cf rom M D i o n ame Ci For i

Fig. 1. Mean shoot length (cm) of flushes from a Valencia orange grove near Haines City, Florida in 2005.

First Flush

During the 7 weeks of the first flush cycle there
were no differences among the treatments in tim-
ing of flush (Fig. 1). Likewise, there were no sig-
nificant differences in shoot length among the
treatments when the data were analyzed weekly,
or over the entire flush cycle (Fig. 1) (F = 0.11, df
= 3, P = 0.95). No CLM mines were observed ex-
cept for 1 sample date (1 Mar), when an average
of 0.01 (SD + 0.12) mines/leaf were observed in
the untreated control trees (Table 1). NoA. citri-
cola or other mortality factors were observed dur-
ing this flush.
Densities of Asian citrus psyllid nymphs dur-
ing the first flush were in categories 0 and 1, with
category 0 indicating no psyllids and category 1
indicating 1-20 psyllids/shoot were present (Table
2). During weeks 1 through 5, there were no sig-
nificant differences in psyllid densities among the
4 treatments when data were analyzed on a
weekly basis (P = 0.20 to 0.95). When psyllid den-
sities were analyzed over the entire flush cycle, no
significant differences occurred among treat-
ments (F = 0.03, df = 3, P = 0.99). No brown citrus
aphids were observed during this flush.

Second Flush

On 22 Jun, prior to the application of sprays,
very low numbers of CLM mines (0.04 to 0.07
CLM mines/leaf) were present (Table 1). The pro-
portion of living CLM larvae in the mines on 22
Jun ranged from 79% (in the block to be treated

with imidacloprid) to 100% (all other blocks) (Fig.
3). The mortality observed in the imidacloprid-
treated trees was probably due to predation, be-
cause the mines were empty and no pupal cham-
bers were present (Fig. 4).
After the sprays were applied on 23 Jun, the
number of mines remained low throughout the
subsequent 5 weeks, with mean densities during
week 6 ranging from 0.10 to 0.15 CLM mines/leaf
(Table 1). After the trees in treatments B, C, and
D were sprayed with oil or imidacloprid, the num-
ber of living CLM larvae dropped to 12% in the 28
Jun sample and to zero in the 5 Jul sample in the
imidacloprid-treated trees while 28, 36, and 16%
of the larvae in the control and 2 oil treatments
remained alive, respectively (Fig. 3). However,
the number of live CLM larvae in the 4 treat-
ments was not significantly different over the en-
tire second flush when densities were combined
over the 6 weeks (F = 1.25, df = 3, P = 0.37). This
indicates that neither oil nor imidacloprid signif-
icantly reduced CLM larval feeding damage dur-
ing this flush cycle. The maximum densities of
CLM mines in this flush occurred on 28 Jun and
ranged from 0.05 (0.03) CLM mines per leaf to
0.29 (0.12) mines per leaf (Table 1). Thus, densi-
ties of CLM larvae remained low during the en-
tire second flush.
The proportion of CLM mines that were empty,
presumably due to predation, during the second
flush ranged from 21 to 97% (Fig. 4). There were
no differences (F = 1.58, df = 3, P = 0.29) in the
proportion of empty mines among the 4 treat-
ments over the flush cycle, suggesting that the

Florida Entomologist 90(2)


" 40



1.4 *

1.2 ;







Fig. 2. Average weekly temperature (oC), relative ] .... i lI 'and rainfall (cm) in a Valencia orange grove near
Haines City, Florida in 2005.

pesticides applied had no impact on predators,
perhaps because oil has a very short residual and
imidacloprid is a systemic.
Parasitism by A. citricola, as determined by
evaluating the pupal chambers of those CLM lar-
vae that survived to the pupal stage, ranged from
0 to 39% over this flush (Fig. 5). There were no dif-
ferences in parasitism among the 4 treatments
(F = 0.74, df= 3, P = 0.53), indicating that oil and
imidacloprid did not have a negative effect on
A. citricola densities in this trial.
Psyllid density categories during the second
flush varied by treatment (Table 2). Prior to treat-
ment on 23 Jun, there were no differences in psyl-
lid densities, but after treatment with imidaclo-
prid, a significant reduction in psyllid densities
was seen for 3 weeks (28 Jun, 5 and 12 Jul) com-
pared to the untreated control (F = 25.66, df = 3,
P = 0.001) (Table 2). By contrast, the growers' oil
treatment (treatment B) reduced psyllid densities
for only 1 week (7 Jul) compared to the untreated
control trees, and the other oil-treated trees
(treatment D) did not show a significant differ-
ence from the untreated control trees (A). By 19
Jul, there were no differences in psyllid densities
among the treatments and again, no differences
were observed in densities during the last sample
on 26 Jul. No brown citrus aphids were observed
in these trees during the second flush.
Third Flush
During the third flush cycle, CLM densities re-
mained relatively low, ranging from 0.05 to 1.27

mines per leaf (Table 1). The number of CLM lar-
vae in the 4 treatments was not different over the
entire third flush cycle when densities were com-
bined over the 8 weeks (F = 0.32, df= 3, P = 0.80).
Parasitism by A. citricola increased compared
to the second flush, ranging from 56% in the un-
treated control trees to 33% in the oil-treated
trees and 22% in the imidacloprid-treated trees
(Fig. 5), but these rates were not different among
the treatments over this flush cycle (F = 2.36, df=
3, P = 0.08).
Parasitism of the CLM by eulophid parasitoids
was not observed during flush cycles 1 or 2, but
during flush cycle 3 some pupal chambers con-
tained an unidentified parasitoid. For example,
during week 1 of this flush cycle, a total of 7, 34,
and 4 pupal chambers were produced in the un-
treated, imidacloprid- and oil-treated trees, re-
spectively. Of these pupal chambers, 100% of 7
pupal chambers in the untreated control trees,
59% of 34 pupal chambers in the imidacloprid-
treated trees, and 25% of 4 pupal chambers in the
oil-treated trees contained this unidentified para-
sitoid; none were found in the growers' oil treat-
ment. During week 2, trees in 2 treatments (un-
treated and imidacloprid-treated), had 8% (of 12)
of the pupal chambers and 67% (of 3) of the pupal
chambers, respectively, with an unidentified par-
asitoid. During weeks 3 and 4, no parasitoids
other than A. citricola were observed. During
week 5, 28% of 18 pupal chambers in the growers'
oil treatment contained the unidentified parasi-
toid. No parasitoids other thanA. citricola or this

June 2007

Hoy et al.: CLM and Natural Enemy Dynamics in Central Florida


Mean ( SD) no. of CLM mines per leaf during sample dates

First flush 1 Mar 7 Mar 14 Mar 21 Mar 28 Mar 4 Apr 11 Apr

A) Untreated control 0.01 0 0 0 0 0 0
B) Grower's oil 0 0 0 0 0 0 0 -
C) Imidacloprid 0 0 0 0 0 0 0 -
D) Oil 0 0 0 0 0 0 0 -

Second flush 22 Jun 28 Jun 7 Jul 12 Jul 19 Jul 26 Jul

A) Untreated control 0.04 0.13 a 0.04 0.05 0.09 0.10 -
(0.01) (0.08) (0.03) (0.05) (0.02) (0.02)
B) Grower's oil 0.07 0.05 a 0.04 0.06 0.09 0.13 -
(0.03) (0.03) (0.03) (0.03) (0.05) (0.08)
C) Imidacloprid 0.06 0.10 a 0.02 0.12 0.06 0.10 -
(0.04) (0.01) (0.03) (0.04) (0.03) (0.02)
D) Oil 0.04 0.29 b 0.13 0.03 0.06 0.15 -
(0.02) (0.12) (0.13) (0.03) (0.02) (0.04)
P value 0.41 0.001 0.14 0.08 0.45 0.43

Third flush 5 Oct 12 Oct 19 Oct 26 Oct 2 Nov 9 Nov 16 Nov 23 Nov

A) Untreated control 0.25 1.17 0.55 0.50 0.39 0.32 0.38 0.54
(0.25) (0.17) (0.28) (0.30) (0.07) (0.15) (0.17) (0.10)
B) Grower's oil 0.05 0.52 0.48 0.33 0.46 0.32 0.36 0.59
(0.04) (0.49) (0.37) (0.10) (0.14) (0.12) (0.08) (0.30)
C) Imidacloprid 0.29 0.60 0.49 0.33 0.30 0.30 0.40 0.44
(0.17) (0.22) (0.15) (0.16) (0.06) (0.16) (0.06) (0.11)
D) Oil 1.27 0.65 0.42 0.31 0.28 0.19 0.29 0.44
(0.84) (0.08) (0.16) (0.07) (0.03) (0.10) (0.14) (0.18)
P value 0.26 0.15 0.93 0.60 0.12 0.71 0.70 0.63

Means were analyzed weekly in each flush cycle by ANOVA and means separated by Fisher's LSD, withP < 0.05. Means within
a flush cycle within a column with the same letters are not significantly different.

unidentified parasitoid were observed in any
During the third flush cycle, psyllid densities
were not different among the 4 treatments during
the entire flush cycle when the data were ana-
lyzed on a weekly basis (Table 2) or over the en-
tire flush cycle (F =0.59, df= 3, P = 0.62).

Shoot Length and Shoot Numbers

The number of shoots that could be sampled
each week was not different among the treat-
ments during flush cycles 1 (F = 1.23, df = 3, P =
0.31, data not shown) and 2 (F = 0.77, df = 3, P =
0.52). However, during flush cycle 3, there were
fewer shoots in the imidacloprid-treated trees
over the entire flush cycle (F = 3.26, df = 3, P =
The length of each shoot sampled was mea-
sured weekly throughout the growing season to
document when flush cycles began and ended and

to determine whether there were differences in
growth rates among the treatments (Fig. 1). Dur-
ing the first flush cycle, there were no differences
in shoot lengths among the treatments. During
the second flush cycle, there were no significant
differences in shoot lengths among the treat-
ments, except that treatment B had significantly
longer shoots (P = 0.03) on 22 Jun, prior to appli-
cation of the spray. After that, there were no dif-
ferences among the treatments, although there
was a trend for the trees treated with imidaclo-
prid to have shorter shoots. During flush cycle 3,
there were significant differences in shoot lengths
on 2 dates when the data were analyzed weekly.
On 26 Oct, the imidacloprid-treated trees had
shorter shoots (mean = 6.3 cm) compared to the
untreated trees (9.6 cm) (P = 0.008). Further-
more, when the combined shoot lengths were
compared for flush 2 and 3, (post spray), differ-
ences were found (F = 6.29, df = 3, P = 0.03), with
the imidacloprid-treated shoots significantly

Florida Entomologist 90(2)

DURING 2005.

Mean ( SD) score (range 0-4)* each sample date

First flush 1 Mar 7 Mar 14 Mar 21 Mar 28 Mar 4 Apr 11Apr

A) Untreated 0.90 0.98 1.04 1.04 0.89 0.26 0.09 -
(0.13) (0.02) (0.04) (0.08) (0.11) (0.13) (0.10)
B) Grower's oil 0.86 1.00 1.11 1.02 0.79 0.48 0.12 -
(0.10) (0.04) (0.07) (0.05) (0.11) (0.08) (0.13)
C) Imidacloprid 1.01 0.98 1.07 1.01 0.75 0.23 0.11 -
(0.02) (0.02) (0.12) (0.02) (0.11) (0.09) (0.09)
D) Oil 0.96 0.98 0.96 1.02 0.86 0.33 0.14 -
(0.08) (0.05) (0.04) (0.05) (0.09) (0.09) (0.07)
P value 0.20 0.94 0.26 0.94 0.15 0.11 0.95

Second flush 22 Jun 28 Jun 7 Jul 12 Jul 19 Jul 26 Jul

A) Untreated 1.08 0.72 a 0.82 a 0.96 a 0.81 0.44 -
(0.08) (0.16) (0.10) (0.04) (0.17) (0.28)
B) Grower's oil 1.05 0.47 a 0.43 b 0.97 a 0.73 0.38 -
(0.01) (0.13) (0.16) (0.05) (0.17) (0.19)
C) Imidacloprid 1.28 0.09 b 0 c 0.25 b 0.68 0.46 -
(0.35) (0.12) (0.11) (0.17) (0.17)
D) Oil 1.17 0.77 a 0.78 a 1.07 a 0.75 0.51 -
(0.12) (0.18) (0.21) (0.12) (0.05) (0.10)
P value 0.52 0.006 0.0008 0.001 0.79 0.86

Third flush 5 Oct 12 Oct 19 Oct 26 Oct 2 Nov 9 Nov 16 Nov 23 Nov

A) Untreated 0 0.79 0.92 0.88 0.82 0.96 0.49 0.74
(0.40) (0.14) (0.12) (0.05) (0.03) (0.15) (0.24)
B) Grower's oil 0 0.97 0.73 0.93 0.89 0.91 0.58 0.45
(0.21) (0.23) (0.07) (0.10) (0.15) (0.23) (0.21)
C) Imidacloprid 0.03 0.50 0.88 0.57 0.89 0.77 0.18 0.67
(0.05) (0.50) (0.03) (0.25) (0.12) (0.18) (0.17) (0.58)
D) Oil 0 0.81 0.68 0.95 0.89 0.97 0.62 0.27
(0.23) (0.26) (0.05) (0.15) (0.05) (0.29) (0.24)
P value 0.45 0.67 0.28 0.08 0.66 0.35 0.09 0.28

*Psyllid densities were scored as: 0 = none, 1 = 1-20, 2 = 21-50, 3 = 51-80 and 4 = >80. Data were analyzed weekly in each flush
cycle by ANOVA and means separated by Fisher's LSD, with P < 0.05. Means within a flush cycle within a column with the same
letters are not significantly different.

shorter. Interpretation of these results is difficult,
because there were no differences over the season
in CLM densities among the treatments and
there were fewer psyllids in the imidacloprid-
treated trees during flush cycles 2 and 3 after
treatment. The higher psyllid densities in the un-
treated or oil-treated trees could have caused re-
ductions in growth. Thus, the data suggest that
imidacloprid might have detrimental effects on
shoot growth and the number of shoots.
Others have found that imidacloprid may have
detrimental effects on growth or yield of crops
when there are no pest pressures. Obviously, imi-
dacloprid can result in increased crop growth and
yield when pest populations exceed the economic
injury level and Oosterhuis & Brown (2003) sug-

gested that imidacloprid might promote plant
health, stress recovery, and yield increases in cot-
ton. However, McGuire (2005) evaluated imidaclo-
prid for 2 years and failed to find evidence that im-
idacloprid enhances growth and/or yield in cotton.
By contrast, Wu et al. (2004) and Qiu et al. (2004)
found that imidacloprid reduced the size of rice
grains in treated plants. Hurley & Patel (2003)
found that imidacloprid reduced the growth ofEu-
calyptus nitens Deane & Maiden (Maiden) tree
seedlings after a root drench at 2 concentrations
by 13 and 8%, respectively. Wallace et al. (2000)
found that imidacloprid was phytotoxic to cucum-
bers in the greenhouse and Ebel et al. (2000)
found it was toxic to tomatoes and cucumbers in
the greenhouse. Dewar et al. (1997) found that

June 2007

Hoy et al.: CLM and Natural Enemy Dynamics in Central Florida

M O UO N 0) ( N4 r- tO CWJ "1 r` W 0) w0 CQ 0
coQco Q Q Z Q a o c ; Q

Fig. 3. Mean percentage living citrus leafminer larvae in mines in a Valencia orange grove near Haines City,
Florida in 2005.

sugar beet seeds in pellets containing higher rates
of imidacloprid had a slower germination rate,
and the total number of seedlings emerging was
reduced, but different cultivars affected the de-
gree of these effects by imidacloprid. Bhagwat &
Lane (2003) found that imidacloprid caused chlo-
rosis of in vitro shoot cultures of apples at the end
of the 6-week treatment. By contrast, Thielert
(2006) reported that imidacloprid protects crops
against abiotic stresses such as drought. Our data
suggest that imidacloprid could be reducing shoot
length in Valencia oranges over the season after a
single treatment, but these experiments were not
designed to evaluate these effects and there is a
possibility that the differences observed are by
chance alone. Thus, additional research is needed
to confirm any negative effects on growth by imi-
dacloprid in Florida's citrus cultivars. Such re-
search is relevant to developing an IPM program
for managing citrus leafminers and Asian citrus
psyllids in Florida as a means of reducing the
spread of citrus canker and citrus greening dis-
ease, respectively, because increased use of imida-
cloprid to control these disease vectors could have
growth or yield costs, as well as benefits.


There were essentially no brown citrus aphids
and relatively low densities of psyllid nymphs
and CLM larvae in this grove in Polk County,
Florida throughout the growing season during
2005. The density of psyllids was estimated by an

abundance score, with only nymphs being esti-
mated, because previous experience in monitor-
ing psyllid populations in a grapefruit grove in
the Ft. Pierce area during 2004 had found that
high rates of predation occurred on eggs and
newly hatched nymphs (Hoy et al., unpubl.).
Prior to this study, no information was avail-
able on the phenology ofA. citricola and CLM in
this citrus-growing area ('the Ridge') of Florida.
These results indicate that A. citricola is an im-
portant natural enemy of the CLM, as shown by
the proportion of those CLM larvae that survived
to the pupal stage during 2005 in both treated
and control trees. As expected,A. citricola popula-
tions lagged behind their CLM host during flush
cycle 2.
A large number of empty mines were observed
in all 4 treatments. Empty mines are often due to
predation by ants (Amalin et al. 2002; Zappala et
al., 2007). Some dead larvae appeared to have
been fed on by lacewing larvae or spiders. Previ-
ous work by Browning & Peia (1995) and Amalin
et al. (1996, 2002) found that green lacewing lar-
vae (C'i .. .'.,/' rufilabris (Burmeister)), ants
(especially the red imported fire ant, Solenopsis
invicta Buren), thrips, hunting spiders (Chira-
canthium inclusum (Hentz), Hibana velox
(Becker) and Trachelas volutes (Gertsch)), and
mirid bugs are predators of CLM larvae in lime or-
chards in south Florida, causing approximately 34
to 39% of the mortality observed. Villanueva-
Jimenez et al. (2000) found that total morality of
the CLM in a Gainesville, FL grapefruit grove var-

Florida Entomologist 90(2)

-- *. GOIL
-A-IMIDA 100
Flush Two -e-OIL Flush Three


FI 40


2005 r .
Mean percentage empty citrus leafminer mines in a Valencia orange grove near Haines City, Florida in

ied throughout the season, but was greater than
70% after the first flush cycle, with "unexplained
larval mortality" that was as high as 70.4% during
the fourth flush during 1997. In that study, 32 to
80% of the mortality was caused byA. citricola on
those CLM that managed to reach the pupal stage
(Villanueva-Jimenez et al. 2000), but empty mines
also were observed and could have been due to
predation by red imported fire ants, which were
abundant in the grove. Zappala et al. (2007) found
that red imported fire ants removed CLM larvae
that had been parasitized by A. citricola from
mines in laboratory and field trials.
From about Jul through Nov in this Valencia
orange grove in central Florida, a substantial pro-
portion of the few CLM larvae that survived to
the pupal stage were parasitized by A. citricola;
this mortality factor would help to reduce the
number of adults entering the winter. However,
the proportion of CLM pupae that were parasit-
ized by A. citricola was lower than expected, for
unknown reasons. Previous samples during Aug
through Oct in multiple sites in Florida had found
up to 99% of the pupae parasitized byA. citricola
(Hoy et al. 1995; Hoy & Nguyen 1997; Pomerinke
& Stansly 1998; Villanueva-Jimenez et al. 2000).
The reasons) for the relatively lower parasitism
rates by A. citricola in this citrus grove near
Haines City is unknown.
We are unable to conclude that the combined
action of natural enemies and pesticide applica-
tions suppressed CLM densities below the eco-
nomic threshold, because it is not known what an

economic injury level is in Valencia oranges grown
in the 'Ridge' area of Florida, especially now that
the canker eradication program has ended (in
2006) and canker is considered established, al-
though there was no canker in the grove at the
time of this study. When the CLM attacks trees in
nurseries and young trees in groves, direct dam-
age by the CLM can delay growth and alter cano-
pies as well as open the mines to infection by the
canker bacterium. The economic impact of CLM
on mature orchards in areas where citrus canker
is now endemic in Florida is not yet known, and
depends on canker bacterial density, weather con-
ditions, tree age, and timing of the damage. It is
unclear if there is a surplus of leaf area in citrus
grown in central Florida, although Knapp et al.
(1995) suggested that a 10% leaf area loss due to
CLM mines did not affect yield (prior to canker es-
tablishment). In Florida, CLM densities typically
are very low during winter and during the first
spring flush, but increase during the growing sea-
son to peak in the fall, and this pattern was fol-
lowed in the Valencia grove studied. In China,
Huang & Li (1989) found that leaf area loss of less
than 20% did not affect yield, and suggested that
a loss of 15% of leaf area, or about 0.74 CLM lar-
vae per leaf, was the economic threshold. Garcia-
Marf et al. (2002) evaluated the economic injury
level of CLM in the Valencia area of eastern Spain
from 1996 to 1999 and found that 5-15% of the an-
nual new leaf area of mature trees could be dam-
aged without affecting yield, primarily because
the production of new shoots was concentrated



Fig. 4.

June 2007

Hoy et al.: CLM and Natural Enemy Dynamics in Central Florida


Fig. 5. Mean percentage citrus leafminer pupae parasitized by Ageniaspis citricola near Haines City, Florida in

early in the spring when CLM densities were very
low but high CLM populations occurred in sum-
mer and fall so the citrus leafminer's effect on bio-
mass, yield and fruit quality was minimal. If this
damage level is used to assess potential growth or
yield loss for the Valencia grove in this study, then
none of the trees reached this level of infestation
during either the first or second flush. During the
second flush, the maximum density of CLM
mines/leaf averaged 0.29 (SD 0.12) in the trees
treated with oil, while the maximum number of
CLM mines/leaf was 0.13 (0.08) in the untreated
control. During the third flush, CLM densities
peaked in the untreated control and in one set of
oil-treated trees at 1.17 (0.22) and 1.27 (0.80)
mines/leaf, respectively, but these densities were
found only during 1 week. Thus, there is no evi-
dence that the treatments (oil or imidacloprid)
significantly reduced CLM densities. The imida-
cloprid treatment did reduce psyllid densities for
3 weeks, but may have reduced shoot length. The
possibility of a detrimental effect by imidacloprid
on shoot growth and shoot number should be in-
vestigated, particularly if multiple applications of
imidacloprid are applied to suppress psyllid popu-
lations in an effort to reduce transmission of
greening disease in Florida.


This work was supported in part by the Davies, Fis-
cher and Eckes Endowment in Biological Control, the
University of Florida Institute of Food and Agricultural
Sciences, and a TSTAR Caribbean Special Research

Grant to M. A. Hoy. We thank Harry Anderson and
Michael Simms for assistance with the fieldwork and
Dr. Linda Young of the Department of Statistics, Uni-
versity of Florida, for statistical advice.


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Florida Entomologist 90(2)

June 2007


'University of Florida, IFAS, Department of Entomology and Nematology
Citrus Research and Education Center, 700 Experiment Station Road, Lake Alfred, FL 33850

2Florida Department of Agriculture and Consumer Services, Division of Plant Industry
1911 SW 34th Street, Gainesville, FL 32608


Nu-Lure and other protein solutions were presented to Anastrepha suspense in J-tubes and
consumption was quantified spectrophotometrically. In choice comparisons, flies consumed
more or equal water compared to Nu-Lure and more Nu-Lure compared to Bragg's Liquid
Aminos, corn steep liquor, NZ case, pepticase, Solulys, soy hydrolysate, Torula yeast, whey,
and yeast enzymatic hydrolysate. Consumption of protein solutions was one-half or less than
0.2 M sucrose, the positive control. The addition of 0.2 M sucrose or 0.2 M fructose to Nu-Lure
did not increase the consumption of Nu-Lure compared to the consumption of sucrose alone,
suggesting that Nu-Lure negates the phagostimulant properties of sucrose and possibly fruc-
tose forA. suspense. If higher consumption rates of a bait/toxicant mixture is a goal, 0.2 M
sucrose would be a better choice than the protein solutions tested, including Nu-Lure.


Se suministr6 Nu-Lure asi como otras soluciones prot6icas a individuos de Anastrepha sus-
pensa en tubos "J" y se cuantific6 por via fotom6trica el consume de estas proteinas durante
un intervalo adecuado de tiempo. En las comparaciones seleccionadas se observe que el con-
sumo de agua se mantuvo igual o superior al de Nu-Lure; asimismo, el consume de esta pro-
teina fue superior al observado para Amino Liquido de Bragg, licor de maiz, NZ case,
pepticase, Solulys, hidrosilato de soya, levadura de Torula y suero e hidrosilato enzimatico
de levadura. En general, el consume de soluciones prot6icas se mantuvo por debajo de la mi-
tad del correspondiente al control positive de sacarosa 0.2 M. La adici6n de sacarosa o fruc-
tosa amboss a la concentraci6n de 0.2 M), a Nu-Lure no increments el consume de dicha
protein en comparaci6n con el consume de azucar, lo que sugiere que Nu-Lure podria elimi-
nar las propiedades fagoestimulantes de la sacarosa y posiblemente tambien de la fructosa
enA. suspense. En aquellos casos en que se desea alcanzar velocidades de consume mas ele-
vadas de agents t6xicos mezclados con el sebo correspondiente por parte deA. suspense, la
soluci6n de sacarosa 0.2 M podria constituir una mejor alternative que las soluciones prot6i-
cas prepadas, incluido el Nu-Lure.

Translation provided by the authors.

Nu-Lure7, a commercially available, corn pro-
tein hydrolysate (Miller Chemical and Fertilizer
Corp., P.O. Box 333, Hanover, PA 17331) is com-
bined with malathion for the management of
Anastrepha suspense (Loew) in Florida (Nigg et
al. 2004a). The 20% malathion/80% Nu-Lure mix-
ture is described as a bait/pesticide and may be
applied by air or by ground equipment (Nigg et al.
2004a). We attempted to attract and kill approxi-
mately 20,000 A. suspense in the greenhouse with
this mixture without success (H. N. Nigg & S. E.
Simpson, personal observation).
Others have studied the attractiveness of Nu-
Lure to fruit flies under various conditions in trap-
ping studies (Epsky et al. 1993, 1999; Heath et al.
1994; Katsoyannos et al. 1999; Fabre et al. 2003).
Although consumption was not determined, Nu-
Lure appeared to be an attractant toA. suspense
and other tephritidae in those studies.

In A. suspense management programs, Nu-
Lure/malathion is applied as a droplet to sur-
faces. There is an assumption by scientists, grow-
ers, and the public that these pesticide-laden bait
droplets are consumed by the fly with resultant
mortality. Our greenhouse observation appears to
be the sole contrary observation to this supposi-
If we could increase the consumption of Nu-
Lure, the amount of pesticide added to NuLure
could be reduced on a 1:1 basis. That is, if con-
sumption were doubled, pesticide concentration
could be halved. Our initial efforts on bait im-
provement were feeding requirements (Nigg et al.
2004c), development of an individual fly con-
sumption method (Nigg et al. 2004b), and deter-
mination of sugar consumption (Nigg et al. 2006).
With our development of an accurate method for
monitoring individual A. suspense consumption

Nigg et al.:A. suspense Bait Consumption

(Nigg et al. 2004b), the premise that Nu-Lure was
consumed byA. suspense could be evaluated.
The purpose of this study was to quantify the
consumption of Nu-Lure and other protein solu-
tions by adultA. suspense.



Anastrepha suspense pupae were shipped
overnight from the Florida Department of Agri-
culture and Consumer Services (Division of Plant
Industry, Gainesville, FL) fly-rearing facility. The
ziplock bags in which they were shipped were
opened, the pupae were gently manipulated by
hand, and the bags were resealed and placed in a
refrigerator at 4C. This procedure allowed for
gas exchange and resulted in better adult emer-
gence. Flies destined to be tested at 24 h were
held in the refrigerator as pupae for 48 h before
being placed in emergence cages. Flies destined to
be tested at 6 d of age were held in the refrigera-
tor as pupae for 24 h. This procedure allowed co-
ordination of fly emergence so experiments could
be conducted Monday through Friday. Flies were
allowed to emerge into cages that were 30 x 30 x
30 cm (Bioquip, Inc., Gardena, CA) and were
tested as immature (24-h) and sexually mature
(6-d) flies. Flies were fed yeast, sugar, and water
according to Nigg et al. (1994, 1995) in their
emergence cages. Once adult emergence began,
the pupae were removed to an empty cage, emer-
gence was allowed to continue for 12 h, and all re-
maining pupae were discarded. This procedure
resulted in flies 1-2 and 6-7 d old on the day of an
experiment. Twenty-four h prior to an experi-
ment, flies were selected directly from their emer-
gence cage. Only active flies with normal wings
were transferred by grasping one wing and plac-
ing the fly into a 950 mL translucent plastic con-
tainer. Flies were provided only on agar patty for
water for 16 h prior to an experiment.
The consumption of solutions by flies was stud-
ied in cages by allowing flies to feed for 45 min
(Nigg et al. 2004a). Each cage contained 5 males
and 5 females and was treated as a replicate. Five
positive control cages, presented with 0.2 M su-
crose plus 0.1% cresol red in a J-tube, were in-
cluded in each trial (Nigg et al. 2006). If the flies
in the positive control did not average 2.5 pL or
greater consumption over 45 min, the entire data
set for that week was discarded. This procedure
eliminated 1 data set during these experiments.
Nu-Lure was obtained from Miller Chemical
and Fertilizer Corp. (P.O. Box 333, Hanover, PA
17331); whey protein (W-1500) from bovine milk,
pepticase (P1192), N-Z-Case M (C7585), and soy
protein acid hydrolysate (S-1674) were from
Sigma Chemical Company (P.O. Box 14508, St.
Louis, MO 63178); sodium caseinate (spray dried)

and hydrolyzed casein (HCA411) from American
Casein Company (Burlington, NJ 08016-4123);
yeast hydrolysate enzymatic (103304), corn glu-
ten meal (960015), and Torula yeast (903085)
from MP Biomedicals, LLC (1263 South Chillico-
the Road, Aurora, OH 44202); soy protein (Pro-
lisse) from Cargill Health & Food Technologies
(15407 McGinty Road W., Wayzata, MN 55391);
and Bragg Liquid Aminos (Live Food Products,
Inc., Box 7, Santa Barbara, CA 93102) from a lo-
cal supermarket. Solulys was from Roquette
America, Inc. (1417 Exchange St., P.O. Box 6647,
Keokuk, IA 52632-6647).

Consumption Quantification

Flies were allowed to feed for 45 min as this is
the time for maximum initial consumption (Nigg
et al. 2004b). Quantification of consumption was
according to Nigg et al. (2004b). Briefly, flies were
presented with protein solutions containing 0.1%
fluorescein or 0.1% cresol red in 5-mL J-tubes. So-
lutions containing 0.1% cresol red or 0.1% fluores-
cein are consumed equally by these flies (Nigg et
al. 2006). Different dyes allowed the direct com-
parison of two solutions in the same fly (Nigg et
al. 2004a). Consumption was measured by ex-
tracting each fly in 0.1 M NaOH and quantifying
the dye spectrophotometrically, cresol red at 573
nm and fluorescein at 491 nm (Nigg et al. 2004b).

Experiment One

This experiment was designed to directly com-
pare the consumption of NuLure with other pro-
tein solutions. Two J-tubes with different solu-
tions were presented in each treatment cage for
45 min. Consumption of 10% Nu-Lure was com-
pared to distilled water and to 10% solutions of
the proteins listed above except for Solulys which
was tested as packaged. There were 5 replicates
of each treatment. All flies were included in the
statistical analysis of this experiment whether
they had fed or not. To calculate the mean for each
replicate, the sum of each solution by sex and cage
(replicate) was divided by the number of that sex
in the cage.

Experiment Two

Our previous work showed that A. suspense
readily consumed 0.2 M sucrose so we compared
its consumption to consumption of NuLure (Nigg
et al. 2006). This experiment indirectly compared
the consumption of NuLure, water, and 0.2 M su-
crose. A single J-tube was presented in each cage
for 45 min. Treatments were 10% Nu-Lure plus
0.1% cresol red or glass-distilled deionized water
plus 0.1% fluorescein or 0.2 M sucrose plus 0.1%
fluorescein, or 0.2 M sucrose in 10% Nu-Lure plus
0.1% fluorescein. After 45 min, flies were pro-

Florida Entomologist 90(2)

cessed and consumption was quantified as de-
scribed above. There were 5 replicates of each

Experiment Three

Sugars are phagostimulants for many insects
(Hagen & Finney 1950; Peacock & Fisk 1970;
Sutherland 1971; Ma & Kubo 1977; Friend 1981;
Cobbinah et al. 1982; Doss & Shanks, Jr. 1984;
Mochizuki et al. 1985; Shanks & Doss 1987; Ladd
1988; Schmidt & Friend 1991; Allsop 1992;
Sharma 1994; Soetens & Pasteels 1994; Shields &
Mitchell 1995; Yazawa 1997; Saran & Rust 2005),
including A. suspense (Nigg et al. 2006). This ex-
periment examined the influence on the con-
sumption of NuLure by the addition of sucrose,
fructose, valine, or sodium tetraborate to 10% Nu-
Lure. Two J-tubes containing different solutions
were presented in each treatment cage for 45 min.
The choice comparisons for experiment 3 were as
follows: (1) 10% Nu-Lure plus 0.1% cresol red vs.
distilled water plus 0.1% fluorescein; (2) 10% Nu-
Lure plus 0.1% cresol red vs. 0.2 M sucrose plus
0.1% fluorescein; (3) 10% Nu-Lure plus 0.1%
cresol red vs. 10% Nu-Lure in 0.2 M sucrose plus
0.1% fluorescein; (4) 0.2 M sucrose plus 0.1%
cresol red vs. 10% Nu-Lure in 0.2 M sucrose plus
0.1% fluorescein; (5) 10% Nu-Lure plus 0.1% fluo-
rescein vs. 10% Nu-Lure in 0.2 M fructose plus
0.1% cresol red; (6) 10% Nu-Lure plus 0.1% fluo-
rescein vs. 10% Nu-Lure plus 0.05 M valine plus
0.1% cresol red; (7) 10% Nu-Lure in 0.2 M sucrose
plus 0.1% cresol red vs. 10% Nu-Lure in 0.2 M su-
crose plus 0.05 M valine plus 0.1% fluorescein;
and (8) 10% Nu-Lure plus 0.1% fluorescein vs.
10% Nu-Lure in 5% sodium tetraborate plus 0.1%
cresol red. There were 5 replicates of each com-
parison except there were 10 replications for 10%
Nu-Lure vs. distilled water and for 10% Nu-Lure
vs. 10% Nu-Lure in 0.2 M sucrose. We compared
statistically the percent of flies that did not feed,
flies that fed only on one of the solutions, and flies
that fed on both solutions. We examined in detail
the consumption of flies that fed on both solu-


A replicate for all experiments is the mean of a
cage by sex. For example, a five-replicate experi-
ment is 5 cages. The means of the 5 cages by sex
are the basis for the means and variation of each
treatment. Standard deviation is used through-
out. Means in Table 2 were compared with paired
t-tests a = 0.05, 0.01, or 0.001 (Microsoft Office
Excel 2003). Means in Tables 3, 4, and 5 were sta-
tistically compared by analysis of variance
(ANOVA) followed by Tukey's honestly significant
difference (HSD) test at a = 0.05 (SAS Institute


The means and standard deviations of the con-
sumption of the sucrose positive controls by males
were 2.50 0.31 pL (range 2.07-3.08 pL) and by
females 3.27 0.74 pL (range 2.15-4.43 pL). There
were no statistical differences week to week in the
consumption of sucrose by the sucrose control
flies except for one week with less than 2.5 pL/fly;
that data set was discarded.
The pH of the protein solutions ranged from a
low of 3.79 (Nu-Lure in 0.2 M sucrose + 0.05 M va-
line) to 7.12 (EZ Case M), a factor that may affect
attractiveness (Flath et al. 1989; Heath et al.
1994), and ranged from completely soluble to in-
soluble (Table 1). The 10% Nu-Lure sugar and
Nu-Lure valine solutions pHs ranged from 3.80 to
3.78. The pH of 10% Nu-Lure + 5% sodium tetrab-
orate was 8.01. Materials that were insoluble and
unsuitable for a liquid bait were Torula yeast,
Prolisse, and sodium caseinate (Table 1).

Experiment One

No fly consumed Nu-Lure only. The percentage
of flies feeding ranged from 36-100% compared to
sucrose controls at 98-100%. Male and female
flies consumed about 5x more water compared to
Nu-Lure, although 24-h fly consumption was low
(Table 2). There was no difference in the con-
sumption of Braggs liquid amino acids vs. Nu-
Lure for 6-d flies (Table 2); more NuLure was con-
sumed by 24-h flies. Six-day flies preferred Nu-
Lure compared to corn steep liquor; there were no
differences for 24-h flies (Table 2). Nu-Lure was
preferred to NZ Case and pepticase by 24-h and 6-
d flies (Table 2). Nu-Lure was preferred over So-
lulys by 6-d males only (Table 2). Nu-Lure was
preferred over soy protein hydrolysate only by 24-
h females (Table 2). Nu-Lure was preferred over
Torula yeast except by 6-d males (Table 2). Whey
protein was consumed less than Nu-Lure by 24-h
males and 6-d females (Table 2). Nu-Lure was
preferred over yeast hydrolysate by 24-h flies, but
not by 6-d flies (Table 2). The important point
about Table 2 data is the less than 2.0 pL average
consumption of protein solutions, actually most
below 1.0 pL, compared to an average sucrose
control consumption of 2.50 pL for males and 3.27
pL for females.

Experiment Two

With the discovery in Experiment 1 that the
consumption of protein solutions was low com-
pared to the sucrose controls, we designed Exper-
iment 2 to examine a no-choice comparison of Nu-
Lure, sucrose, and water. Experiment 2 no-choice
consumption data are presented in Table 3. For
males, the percent feeding was not different
across solutions (Table 3). For 6-d females, the

June 2007

Nigg et al.:A. suspense Bait Consumption


10% type Solubility pH

Corn steep liquor Slight sediment 3.98
EZ Case M Soluble 7.12
Hydrolyzed casein Stable suspension 5.02
Liquid amino acids Soluble 5.58
Nu-Lure Soluble 3.82
Nu-Lure in 0.2 M fructose Soluble 3.82
Nu-Lure + 5% sodium tetraborate Soluble 8.01
Nu-Lure in 0.2 M sucrose Soluble 3.80
Nu-Lure in 0.2 M sucrose + 0.05 M valine Soluble 3.79
Nu-Lure plus 0.05 M valine Soluble 3.78
Pepticase Soluble 6.96
Prolisse Thick suspension 6.96
Sodium caseinate Not soluble 6.22
Solulys Slight sediment 3.98
Soy protein acid hydrolysate Some sediment 5.70
Torula yeast Not soluble 6.31
Whey Some sediment 5.38
Yeast hydrolysate enzymatic Some sediment 5.60

percent feeding on water was lower than the
other solutions, but the amount of water con-
sumed was not different than Nu-Lure or Nu-
Lure plus sucrose. For males and females, the
amount of 0.2 M sucrose consumed was 2x to 5x
greater than water, Nu-Lure, or Nu-Lure plus su-
crose (Table 3). The addition of sucrose to Nu-
Lure did not enhance its consumption compared
to Nu-Lure alone (Table 3).

Experiment Three

Experiment 3 examined the choices flies made
in their consumption of the solutions in Experi-
ment 2 (Table 2) and the possible improvement of
Nu-Lure consumption. The percentage of flies
that fed ranged from 36 to 100% (data not pre-
sented). For most experiments, the percent feed-
ing was 70% or more (data not presented). Only
the flies that fed on both solutions were included
in these analyses.
When comparing the quantities consumed,
there was no difference between Nu-Lure and wa-
ter (line 1, Tables 4 and 5). This is the same result
as in Table 3, that is, no difference between the
consumption of Nu-Lure and the consumption of
Flies fed more on sucrose than on 10% Nu-
Lure; this reached statistical significance with 6-
d females (line 2, Tables 4 and 5). The addition of
sucrose to Nu-Lure led to more consumption of
sucrose/Nu-Lure compared to Nu-Lure alone for
24-h females only (line 3, Tables 4 and 5).
Valine improved Nu-Lure and Nu-Lure in 0.2
M sucrose consumption by 6-d, but not 24-h males
and females (line 6, Tables 4 and 5). Although
more NuLure plus 0.2 M sucrose was consumed

when valine was added, this reached statistical
significance only with 6-d females (line 7, Tables 4
and 5). The addition of 5% borax to 10% Nu-Lure
did not improve its consumption (line 8, Tables 4
and 5),a combination known to increase Nu-Lure
attractiveness to Anastrepha spp. (Heath et al.
1994). Our interpretation of these data is that the
addition of NuLure to 0.2 M sucrose decreased
the consumption of sucrose and the inclusion of
NuLure in a comparison decreased the consump-
tion of solutions in general. If we total the con-
sumption of flies in Tables 4 and 5, we can com-
pare these totals to the consumption of sucrose
controls. Overall, male sucrose controls averaged
2.5 pL/fly; females 3.27 pL/fly. By comparison, 24-
h males consumed 3.41 pL/fly; females 5.81 pL for
the Nu-Lure/sucrose comparison (line 2, Table 4).
This is the only set of totals for Table 4 that meet
or exceed the control average. Sucrose control
consumption was exceeded by males in Table 5
(line 2); females consumed 3.28 pL/fly (line 2, Ta-
ble 5). In some cases, sucrose may overcome a de-
terrent effect of a substance (Shields & Mitchell
1995), but apparently not with Nu-Lure and
A. suspense.
Ninety-eight to 100% of the sucrose positive
controls fed over the 10 weeks of these experi-
ments (data not presented). There were no differ-
ences in the consumption of water and 10% Nu-
Lure by 24-h and 6-d males and females (Table 3).
In the water-Nu-Lure comparison, an average of
92% of males and 96% of females in the sucrose
checks fed with a mean consumption of 3.24
0.21 pL (males) and 3.89 0.33 pL (females). The
consumption of the protein solutions was gener-
ally less than one-half of the consumption of the
0.2 M sucrose controls. The addition of Nu-Lure to


Comparison 24-h 6-d

% feeding Male % feeding Female % feeding Male % feeding Female

1. Nu-Lure 87 12 0.14 0.12*** 53 23 0.04 0.04* 56 26 0.17 0.07* 92 11 0.37 0.11*
Water 0.59 0.19 0.16 0.12 1.10 0.56 1.75 0.84
2. Nu-Lure 52 34 0.33 + 0.21* 88 18 0.67 0.26** 100 0 0.45 0.28 NS 96 9 0.42 0.33 NS
Braggs Liquid Aminos 0.05 0.04 0.28 0.16 0.24 0.20 0.49 0.24
3. Nu-Lure 64 17 0.10 0.11 NS 72 17 0.27 0.24 NS 100 0 0.65 0.32* 100 0 0.63 0.24**
Corn Steep Liquor 0.11 + 0.03 0.10 + 0.03 0.06 0.06 0.04 0.03
4. Nu-Lure 56 9 0.23 + 0.14 NS 60 32 0.62 0.50* 91 12 0.99 0.22*** 100 0 1.76 0.45***
NZ Case 0.03 0.04 0.03 0.05 0.02 0.04 0.33 + 0.15
5. Nu-Lure 100 0 0.40+ 0.13*** 96 9 0.83 0.30** 84 26 0.61 0.29** 100 0 1.34 0.41**
Pepticase 0.01 0.03 0.12 0.05 0.11 0.07 0.26 0.17
6. Nu-Lure 56 26 0.11 + 0.10 NS 100 0 0.49 0.41 NS 100 0 0.72 + 0.18*** 96 9 0.52 0.34 NS
Solulys 0.10 0.10 0.46+ 0.10 0.03 0.03 0.20 0.21
7. Nu-Lure 48 11 0.38 0.32 NS 76 17 1.39 0.90* 64 9 0.38 0.20 NS 76 17 0.84 0.47 NS
Soy Protein Hydrolysate 0.09 0.07 0.09 0.09 0.13 0.09 0.28 0.31
8. Nu-Lure 100 0 0.88 + 0.21** 100 0 1.07 0.47* 36 22 0.28 0.18 NS 88 18 1.91 0.31***
Torula Yeast 0.04 0.09 0.33 + 0.11 0.07 0.08 0.20 0.09
9. Nu-Lure 36 22 0.21 + 0.18 NS 48 27 0.95 0.69* 93 12 0.91 0.36** 80 20 0.86 0.82 NS
Whey 0.03 0.02 0.05 0.03 0.07 0.11 0.09 0.07
10. Nu-Lure 56 33 0.14 + 0.09* 100 0 0.84 0.23*** 88 18 0.34 0.15 NS 88 18 0.50 0.24 NS
Yeast Hydrolysate 0.01 + 0.01 0.004 0.01 0.37 + 0.12 0.59 0.48

Mean standard deviation, n = 5; means are different at *0.05, **0.01, and

*0.001 by paired t-tests (Microsoft Office Excel 2003) or NS = not significantly different.

Nigg et al.:A. suspense Bait Consumption


Mean SD Mean SD
pL per fly pL per fly
% feeding consumed % feeding consumed

24-h Male 24-h Female

DDI water 66 24 a* 0.51 0.44 b 48 39 b 0.70 0.52 b
10% Nu-Lure plus 0.2 M sucrose 80 20 a 0.45 0.12 b 74 19 ab 0.44 0.20 b
10% Nu-Lure 68 30 a 0.78 0.23 b 96 9 a 1.11 0.34 b
0.2 M sucrose 76 17 a 2.10 0.33 a 84 17 ab 2.68 0.77 a

6-d Male 6-d Female

DDI water 52 33 a 0.69 + 0.36 b 20 0 b 0.13 0.07 b
10% Nu-Lure plus 0.2 M sucrose 96 9 a 0.78 0.29 b 100 0 a 0.80 0.13 b
10% Nu-Lure 65 25 a 1.61 0.37 b 82 10 a 0.84 0.59 b
0.2 M sucrose 76 33 a 2.57 0.70 a 84 17 a 2.79 1.43 a

*Means by age and sex followed by the same letter are not statistically different by ANOVA followed by Tukey's HSD test, + =
0.05, n = 5. SD = standard deviation.

a consumption comparison appears to decrease
the total consumption of both solutions (Tables 4
and 5). One possibility for our data is that
A. suspense self-selected an optimal diet (Hagen
& Finney 1950; Waldbauer & Friedman 1991).
Anastrepha suspense seems to prefer sugar as an
immature fly and protein when sexually mature
(Nigg et al. 1995). Our previous data suggested

that 6-d-old females would have preferentially
consumed protein (Nigg et al. 1995). However, in
the present study, both sexually mature and im-
mature flies preferentially consumed 0.2 M su-
crose (Table 3). This said, the goal here was an in-
crease in consumption so that pesticide quantity
might be reduced. The mechanism of the increase
might be studied in the future.


Mean SD Mean SD
pL per fly consumed pL per fly 6-d

Feeding category 24-h male 24-h female

1. 10% Nu-Lure 0.46 0.21 NS 0.82 + 0.13 NS
Water 0.59 0.29 0.67 0.13
2. 10% Nu-Lure 1.19 + 0.34 NS 1.93 0.33 NS
0.2 M sucrose 2.22 2.82 3.88 1.92
3. 10% Nu-Lure 0.36 0.13 NS 0.48 + 0.15*
10% Nu-Lure in 0.2 M sucrose 1.39 0.85 1.40 0.52
4. 0.2 M sucrose 1.13 + 0.95 NS 0.67 0.29 NS
10% Nu-Lure in 0.2 M sucrose 0.14 + 0.05 0.24 0.16
5. 10% Nu-Lure 0.36 0.16 NS 0.48 0.26 NS
10% Nu-Lure in 0.2 M fructose 1.37 1.03 2.19 1.62
6. 10% Nu-Lure 0.17 0.06 NS 0.44 0.20 NS
10% Nu-Lure in 0.05 M valine 1.16 1.14 1.30 0.76
7. 10% Nu-Lure in 0.2 M sucrose 0.35 + 0.16 NS 0.66 0.28 NS
10% Nu-Lure in 0.2 M sucrose + 0.05 M valine 1.79 0.88 2.32 1.92
8. 10% Nu-Lure 0.31 + 0.12** 0.52 0.38 NS
10% Nu-Lure in 5% borax 0.54 + 0.18 0.77 0.43

Means are significantly different at *0.05, **0.01, ***0.001. Rows with the same number were compared statistically. SD = stan-
dard deviation.

Florida Entomologist 90(2)

June 2007


Mean SD Mean SD
pL per fly consumed pL per fly 6-d

Feeding category 6-d Male 24-h Female

1. 10% Nu-Lure 0.75 0.22 NS 1.03 0.27 NS
Water 1.39 0.73 1.41 0.46
2. 10% Nu-Lure 0.80 0.49 NS 1.21 0.51*
0.2 M sucrose 2.20 0.76 2.07 0.57
3. 10% Nu-Lure 0.70 0.55 NS 1.15 0.73 NS
10% Nu-Lure in 0.2 M sucrose 1.32 0.95 0.89 0.78
4. 0.2 M sucrose 0.30 0.47 NS 0.88 1.09 NS
10% Nu-Lure in 0.2 M sucrose 0.37 0.07 0.80 0.30
5. 10% Nu-Lure 0.30 0.20 NS 0.51 0.14 NS
10% Nu-Lure in 0.2 M fructose 1.13 0.61 1.96 1.21
6. 10% Nu-Lure 0.20 0.12** 0.56 0.15**
10% Nu-Lure in 0.05 M valine 2.09 0.55 3.71 0.71
7. 10% Nu-Lure in 0.2 M sucrose 0.31 0.17 NS 0.61 0.36*
10% Nu-Lure in 0.2 M sucrose + 0.05 M valine 0.50 0.47 2.34 0.95
8. 10% Nu-Lure 0.65 0.16 NS 0.84 0.48 NS
10% Nu-Lure in 5% borax 1.00 0.25 0.62 0.20

Means are significantly different at *0.05, **0.01, ***0.001. Rows with the same number were compared statistically. SD = stan-
dard deviation.

A bait must be both attractive and readily con-
sumed. Maximum consumption is desirable in or-
der to reduce pesticide while maintaining effec-
tiveness. For consumption, our data suggest that
Nu-Lure and other tested protein solutions are
inappropriate as consumed baits for A. suspense
and could be replaced by 0.2 M sucrose.


This research was supported by the Florida Agricul-
tural Experiment Station and a grant from the Florida
Citrus Production Research Advisory Council. We thank
the Florida citrus growers for support of this research.


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organophosphate insecticides to Caribbean fruit fly
(Diptera: Tephritidae). J. Econ. Entomol. 87: 589-
E. BURNS, AND R. C. LITTELL. 1995. Age-related re-
sponse of Anastrepha suspense (Diptera: Tephriti-
dae) to protein hydrolysate and sucrose. J. Econ.
Entomol. 88: 669-677.
NIGG, H. N., S. E. SIMPSON, AND J. L. KNAPP. 2004a. The
Caribbean fruit fly-free zone programme in Florida,
U.S.A. pp. 179-182 In Proc. 6th Intl. Fruit Fly Symp.
RIS, AND S. FRASER. 2004b. Quantifying individual
fruit fly consumption with Anastrepha suspense
(Diptera: Tephritidae). J. Econ. Entomol. 97: 1850-
BERRIA, AND E. B. JANG. 2004C. Kairomones for the
management ofAnastrepha spp. fruit flies, pp. 335-
347 In Proc. 6th Intl. Fruit Fly Symp.
BERRIA, J. J. YANG, AND S. FRASER 2006. Consump-
tion of sugars by Anastrepha suspense Loew
(Diptera: Tephritidae). Ann. Entomol. Soc. America
(in press).

PEACOCK, J. W., AND F. W. FISK. 1970. Phagostimulants
for larvae of the Mimosa webworm, Homadaula
anisocentra. Ann. Entomol. Soc. Amer. 63: 1755-
SARAN, R. K., AND M. K. RUST. 2005. Feeding, uptake,
and utilization of carbohydrates by western subter-
ranean termite (Isoptera: Rhinotermitidae). J. Econ.
Entomol. 98: 1284-1293.
SAS INSTITUTE. 2001. SAS version 8.2. SAS Institute,
Cary, NC.
SCHMIDT, J. M., AND W. G. FRIEND. 1991. Ingestion and
diet destination in the mosquito Culiseta inornata:
effects of carbohydrate configuration. J. Insect Phys-
iol. 37: 817-828.
SHANKS, C. H., JR. AND R. P. DOSS. 1987. Feeding re-
sponses by adults of five species of weevils (Co-
leoptera: Curculionidae) to sucrose and sterols. Ann.
Entomol. Soc. Amer. 80: 41-46.
SHARMA, H. C. 1994. Phagostimulant activity of su-
crose, sterols, and soybean leaf extractables to the
cabbage looper Trichoplusia ni (Lepidoptera: Noctu-
idae). Insect Sci. App. 15: 281-286.
SHIELDS, V. D. C., AND B. K. MITCHELL. 1995. The effect
of phagostimulant mixtures on deterrent receptor(s)
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SOETENS, PH., AND J. M. PASTEELS. 1994. Synergistic ef-
fect of secondary compounds and nutrients in the
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Phrator vitellinae (Coleoptera: Chrysomelidae).
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SUTHERLAND, O. R. W. 1971. Feeding behaviour of the
grass grub Costelytra zealandica (White) (Cole-
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Florida Entomologist 90(2)


'College of Earth Sciences, Jilin University, Changchun 130061, China
e-mail: wudhyang@yahoo.com.cn
2Institute of Plant Physiology & Ecology, Shanghai Institutes for Biological Sciences
Chinese Academy of Sciences, Shanghai 200032, China

Two new species of Xenylla from Jilin Province, Northeast China are described and illus-
trated. Xenylla changlingensis, new species clearly differs from the closely related species
X. piceeta Stebaeva & Potapov, 1994 in the presence of dorsal la2 of thoracic segments II and
III, the absence of ventral setap2 on abdominal segment II, 1 median ventral seta above the
retinaculum on abdominal segment III, and lack of teeth on the mucro. Xenylla changchun-
ensis, new species is similar to the species X. osetica Stebaeva & Potapov, 1994. However,
it is separable from the latter by the presence of a furca, a tenaculum, and the ventral chae-
totaxy on abdominal segment III.
Key words: Collembola, Hypogastruridae, Xenylla, new species, China

Se describe e ilustran dos species del g6nero Xenylla de la Provincia de Jilin, en el noreste
de China.Xenylla changlingensis nueva especie claramente se distingue de su especie cer-
canaX. piceeta Stebaeva & Potapov, 1994 por la presencia de la2 dorsal de los segments to-
racicos II y III, la ausencia de la seta ventral p2 en el segment abdominal II, una seta
median ventral arriba del retinaculum en el segment abdominal III, y la falta de dientes
sobre el mucro. Xenylla changchunensis nueva especie es parecida aX. osetica Stebaeva &
Potapov, 1994. Sin embargo, se distingue de la especie posterior por la presencia de una
furca, el tenaculum y la chaetotaxia ventral del segment abdominal III.

The genus Xenylla was established by Tullberg
for X. maritima Tullberg, 1869 as type species. It
is one of the largest and most widespread genera
in the family of Hypogastruridae. According to
Thibaud et al. (2004), species in the genus Xe-
nylla are mainly characterized by (1) 5+5, rarely
4+4 ommatidia, (2) postantennal organ absent,
(3) mandible short with a well developed molar
plate, maxillary head with normal lamellae, (4)
furca rarely absent, showing a diverse morphol-
ogy, if mucro separated from the dens, which nor-
mally bears 2 setae; mucro, however, fused with
the dens or mucro absent, the dens has 1 or 2 se-
tae, (5) empodium absent, and (6) abdominal seg-
ment V tergite with p3 as sensilla.
So far, about 126 species of the genus Xenylla
have been described worldwide (Christiansen
2006). However, only one, X. boerneri Axelson,
1905, has been reported from East China (Zhao et
al. 1997). The taxonomy of the fauna of many Chi-
nese habitats is poorly known, especially those of
soil. In the present paper, two new species of the
genus Xenylla that were found from Northeast
China are described.


al, 2, ... setae 1, 2, ... of the anterior row,
counted from the "middle line", ml, 2, ... setae

1, 2, ... of the middle row, counted from the "mid-
dle line", pl, 2, ... setae 1, 2, ... of the posterior
row, counted from the "middle line", cl, 2, ... -
cervical setae 1, 2, ... of area occipitalis, counted
from the "middle line", Lal, 2, ...- setae 1, 2, ...
of the lateral anterior row in thoracic segments,
L1, 2, ...- lateral setae 1, 2, .... in head (Yosii
1960; Gama 1988).

Xenylla changlingensis, new species
(Figs. 1-10)
Type Materials
Holotype: Female, from the grassland of Ley-
mus chinensis, 44035'N, 123030'E, 141 m altitude,
Changling county, Jilin Province, Northeast
China, 6-5-2005, collected by Dr. Donghui Wu.
Paratypes: Two females, 3 males, same data as
holotype. Holotype and paratypes deposited in
Shanghai Institute of Plant Physiology & Ecology.


Body length up to 0.95 mm. Body color in alco-
hol dark blue-violet. With 5+5 ommatidia in head
(Fig. 1). Antennal segment I with 7 setae, anten-
nal segment II with 12 setae. Sensory organ of
antennal segment III consists of 2 microsensilla
which are embedded in a tegumentary fold and

June 2007

Wu & Yin: Two New Species ofXenylla

..) 45gm

.. ........- /
/ aS
(~j/ %.Jf '

32? ~&7


* *.. ...... .:r ...:. .... "



Fig. 1-7. Xenylla changlingensis, new species. 1. Dorsal chaetotaxy of the head. 2. Dorsal chaetotaxy of Th. I -
III. 3. Dorsal chaetotaxy of Abd. III VI. 4. Ventral chaetotaxy of the head. 5. Ventral chaetotaxy of Th. III, Abd. II
and III, ventral tube, and retinaculum. 6. Ventral chaetotaxy of Abd. IV and V female genital plate, and anal plate.
7. Male genital plate.

Florida Entomologist 90(2)


Fig. 8-10. Xenylla changlingensis, new species. 8. Antenna, dorsal view. 9. Tibiotarsi III with claw. 10. Furca,
posterior view.

flanked by 2 longer guard sensillum. Antennae IV
with a simple apical bulb and 4 weakly thickened
sensillum, of which 3 are dorso-external and 1
dorso-internal, and 2 internal sensillum, which
are thinner and shorter than the others (Fig. 8).
External maxillary lobe with 2 sublobal hairs.
Tibiotarsi each with 2 capitate, dorsal tenent
hairs, which are longer than the inner edge of the
claws. Claws with a small, distal internal tooth, 2/
3 as long as tibiotarsal hairs (Fig. 9). Mucro well
separated from the dens with 2 posterior setae, 1/
2 as long as the dens but dens and mucro partic-
ularly slender, width of dens at distal seta about
an eighth its length, mucro straight and without
teeth (Fig. 10). Ventral tube with 4+4 setae. Reti-
naculum with 3+3 teeth (Fig. 5). Female genital
(Fig. 6) and male genital plate (Fig. 7) normal.
Anal spines small, on weakly developed papillae
separated at the base, 1/4 as long as claws (Fig. 3).
Chaetotaxy, consisting of short setae and
longer and fine sensorial setae. Dorsally head
without seta c2 (a2 a/c to Babenko), cephalic se-
tae 11 and 13 subequal (Fig. 1), thoracic segments
II-III with central setae in 3 rows, on thoracic seg-
ments II-III p2 displaced apically relative to pl,
and on thoracic segment III a2 displaced distally
compared with a 1(Fig. 2), on abdominal segments
I-III p5 present, abdominal segments IV with a3.
Abd. V with a2 (Fig.3). Ventrally head without
seta pi (Fig. 4), thoracic segments II and III with
a pair of medial setae, abdominal segments II
without p2 and a5, abdominal segments III with 1
median seta above the retinaculum (Fig. 5).

The new species is distinguished from all the
known species of the genus Xenylla by the ab-

sence of mucronal teeth, dorsal side of head with-
out c2 seta, seta p2 on tergite of thoracic seg-
ments II-III set in front of pl seta, head without
ventral seta pi, thoracic segments II and III with
a pair of ventral medial setae, abdominal seg-
ment II without ventral setae p2 and a5, abdomi-
nal segment III with 1 median ventral seta above
the retinaculum, abdominal segment IV with
ventral seta ml, unguis with 1 internal tooth.


This species is named after the type locality.

Taxonomic Remarks

This species keys out to X. piceeta Stebaeva &
Potapov, 1994 (Babenko et al. 1994), from Far East,
southern maritime province, Russia, which was col-
lected in litter of a fir forest (Babenko et al. 1994),
but the new species clearly differs fromX. piceeta by
the presence of dorsal la2 of thoracic segments II
and III, which is stable on the tergites, and the ab-
sence of ventral seta p2 on abdominal segment II.
On abdominal segment III, X. changlingensis has
only 1 median ventral seta above the retinaculum,
while X. piceeta has a pair of medial setae. In addi-
tion, the mucro ofX. changlingensis is straight and
thin, but lacking teeth.

Xenylla changchunensis, new species
(Figs. 11-19)

Type Materials
Holotype: Female, from the deciduous-conifer-
ous mixed forest of Jingyuetan Park, 4345'N,
12527'E, 242 m altitude, Changchun city, Jilin

June 2007

Wu & Yin: Two New Species ofXenylla



Fig. 11-13. Xenylla changchunensis, new species. 11. Antenna, dorsal view. 12. Tibiotarsi III with claw. 13.
Furca, posterior view.

Province, Northeast China, 3-9-2003, collected by
Dr. Donghui Wu. Paratypes: Two females, same
data as holotype. Holotype and paratypes depos-
ited in Shanghai Institute of Plant Physiology &


Body length up to 0.81 mm. Body color in alco-
hol red-brown. Antennal segment I with 7 setae,
antennal segment II with 12 setae. Sensory organ
of antennal segment III consists of 2 microsen-
silla, embedded in a tegumentary fold and
flanked by 2 longer guard sensillum. Antennae IV
with a simple apical bulb and 4 weakly thickened
sensillum, of which 3 dorso-external and 1 dorso-
internal, and 2 internal sensillum, thinner and
longer than the others (Fig. 11). External maxil-
lary lobe with 3 sublobal hairs.
Tibiotarsi each with 2 capitate, dorsal tenent
hairs, longer than the inner edge of the claws.
Claws toothless, 11/15 as long as tibiotarsal hairs
(Fig. 12). Dens with 2 posterior setae. Mucro well
separated from the dens, straight and fine with-
out teeth, dens broad, at level of distal setae
length of dens about 3 times breadth, ratio mucro:
dens = 1/2 (Fig. 13). There are 4+4 setae on ven-
tral tube. Retinaculum with 3+3 teeth (Fig. 18).
Female genital plate (Fig. 19). Anal spines short,
inserted on poorly developed papillae, 1/4 as long
as claws (Fig. 16).
Chaetotaxy, consisting of short setae and
longer and fine sensorial setae. Dorsally head
without seta cl with both pl and p2, 11 longer
than 13 (Fig. 14), thoracic segments II-III with
medial setae in 3 rows, seta p2 on tergite of tho-

racic segments II-III set in front ofpl, on thoracic
segments III a2 displaced distally relative to al
(Fig. 15), on abdominal segments I-III, p5
present, abdominal segments IV without a3, ab-
dominal segments V without a2 (Fig. 16). Ven-
trally head with seta pl (Fig. 17), thoracic seg-
ments II and III with a pair of medial setae, ab-
dominal segment II without p2 and a5, abdomi-
nal segment III with 1 median seta above the
retinaculum (Fig. 18), abdominal segment IV
without ml (Fig. 19).


The new species is distinguished from other
species ofXenylla by possessing a mucro without
teeth, dorsal side of head with cl (pl a/c Babenko
et al. 1994) seta absent, seta p2 on tergite of tho-
racic segments II-III set in front of pl seta, tho-
racic segments II and III with a pair of ventral
medial setae, abdominal segment II without ven-
tral setae p2 and a5, abdominal segment III with
1 median ventral seta above the retinaculum, ab-
dominal segment IV without ventral seta ml, un-
guis lacking teeth.


Named changchunensis alluding to Chang-
chun, the city where the species was found.

Taxonomic Remarks

The new species resemblesX. osetica Stebaeva
& Potapov, 1994 in general shape, antenna, tibio-
tarsi, and claws, especially in dorsal chaetotaxy,

Florida Entomologist 90(2)

14 17
45Km 1 45pm

. .. I ol

T ; 15 I

..8. .,;,. ,.-....-. 1, .
.'... .... ...........

1- ...... ......

.r ..q..i 1^ 9 .

... .... rr rr-: ..... .. .
y 4 --** ^-L^......... .... ....

anal plate.
\r pr M1/


SOpm 5Opm

Fig. 14-19. Xenylla changchunensis, new species. 14. Dorsal chaetotaxy of the head. 15. Dorsal chaetotaxy of
Th. I-III. 16. Dorsal chaetotaxy ofAbd. III-VI. 17. Ventral chaetotaxy of the head. 18. Ventral chaetotaxy ofTh. III,
Abd. II and III, ventral tube, and retinaculum. 19. Ventral chaetotaxy of Abd. TV and V, female genital plate, and
anal plate.

June 2007

Wu & Yin: Two New Species ofXenylla

but distinctly differs fromX. osetica in the follow-
ing characters: (1) furca and tenaculum present,

and (2) ventral chaetotaxy on abdominal segment


1. Furca without mucro, retinaculum with 2+2 teeth ............................. .X. boerneri Axelson, 1905
-Mucro separated from dens that has 2 setae, retinaculum with 3+3 teeth ........................ 2
2. Dorsal side of head without c2 seta, abdominal segment IV with ventral seta ml,
unguis with 1 internal tooth .......................................... X. Changlingensis sp. nov
Dorsal side of head with cl seta absent, abdominal segment IV without
ventral seta ml, unguis lacking teeth .................................. X. Changchunensis sp. nov


Thanks to Mr. Rongdong Xie, Mr. Yiming Yang, Dr.
Yunxia Luan, Dr. Yun Bu, and Dr. Yan Gao for help in
our taxonomic work. Thanks also to two anonymous re-
viewers for excellent suggestions. This study is sup-
ported by the National Natural Sciences Foundation of
China (No. 40601047, 30370169), and China Postdoc-
toral Science Foundation (20060390643).


S. K. STEBAEVA. 1994. Family Hypogastruridae, pp.

259-305, 329 In N. M. Chernova [ed.], Collembola of
Russia and Adjacent Countries.
CHRISTIANSEN, K. A. 2006. Http://www.collembola.org.
GAMA, M. M. DA. 1988. Filogenia des esp6cies deXenylla
a escala mundial (Insecta, Collembola). Evoluci6n
boil6gia 2: 139-147.
2004. Hypogastruridae, In W. Dunger [ed.], Synop-
ses on Palaearctic Collembola 4: 7-10, 217-250.
YosII, R. 1960. Studies on the Collembolan genus Hypo-
gastrura. The American Midland Naturalist 64: 257-
ZHAO, L., A. H. TAMUR, AND X. KE. 1997. Tentative
Checklist of Collembolan Species from China (In-
sect). Publications of the Itako Hydrobiological Sta-
tion 9: 15-40.

Florida Entomologist 90(2)

June 2007


USDA-ARS, Mayaguez, Puerto Rico 00680-5470


The authors evaluated the host status of mamey sapote, Pouteria sapota (Sapotaceae) to
Anastrepha obliqua by collecting mature fruits and monitoring them for the emergence of
larval Tephritidae. Fruits were also scarred and placed in cages with female A. obliqua and
monitored for the emergence of larvae and adults. Multi-lure traps baited with putrescine
and ammonium acetate were used to compare the number of flies in orchards of mamey sa-
pote to the number of flies in nearby orchards of carambola (Averrhoa carambola: Oxali-
daceae). There are a number of references citing mamey sapote as a host ofA. obliqua in
different countries. However, we only found two unidentified tephritid larva from 1,160 ma-
mey sapote fruits collected in the field and these fly larvae did not survive to adulthood. We
were not able to rear adultA. obliqua on scarred, mature fruit of mamey sapote, whereas we
were able to do so on mango under identical conditions. Abundance in orchards based on
trapping indicates that flies are very rarely encountered in orchards of mamey sapote com-
pared with orchards of carambola. We conclude that in Puerto Rico mamey sapote has a very
low (undetectable) rate of infestation by fruit flies in the family Tephritidae.

Key Words: Mamey sapote, Pouteria sapota,Anastrepha, hosts


Se evalu6 si el mamey sapote, Pouteria sapota (Sapotaceae) puede ser hospedero de la mosca
de las frutasAnastrepha obliqua. Con este fin se colectaron frutas maduras las cuales fueron
monitoreadas para detectar la presencia de larvas Tephritidae. Las frutas fueron tambi6n
rasgadas y colocadas en jaulas conteniendo moscas hembras deA. obliqua e inspeccionadas
regularmente para determinar si larvas y adults emergian de las frutas. Trampas con cebo
de putrescina y acetato de amonio fueron colocadas en los predios de mamey sapote y huer-
tos cercanos de carambola (Averrhoa carambola: Oxalidaceae) para comparar la poblaci6n
de moscas de las frutas en estos huertos. Aunque various escritos citan el mamey sapote como
un hospedero de A. obliqua, los autores solo pudimos encontrar dos larvas en various cente-
nares de frutas y estas larvas no se desarrollaron a su estado adulto. Tampoco se pudo indu-
cir oviposicion de moscas fruteras en frutas de mamey sapote con la superficie rasgada. Los
datos obtenidos de las trampas indicaron una poblaci6n insignificant de moscas de las fru-
tas en huertos de mamey sapote en comparaci6n con aquellos de carambola. Concluimos que
en Puerto Rico el mamey sapote tiene un nivel de infestacion extremadamente bajo (indetec-
table) para las moscas de las frutas de la familiar Tephritidae.

Translation provided by the authors.

Mamey sapote, Pouteria sapota (Jacq.) H.E.
Moore & Steam (Sapotaceae), is native to Central
America (Morton 1987) and its fruits are prized
throughout Central America and the Caribbean
for their sweetness. It is currently cultivated and
sold in Puerto Rico, but some growers would like
to expand their market to include Latin American
populations in the continental US. However, the
possible introduction of new insect pests, includ-
ing fruit flies in the genus Anastrepha, precludes
importation of this fruit crop into the continental
US. Gould & Hallman (2001) concluded that ma-
mey sapote presents no discernible risk of trans-
porting Anastrepha suspense (Loew). However, a
second species of economic importance,A. obliqua

(Marquart), is present in Puerto Rico and the
host-status of mamey sapote with respect to this
fly is unclear. Cowley et al. (1992) defined a host
as a fruit or vegetable that fruit flies oviposit in
under field conditions and that these eggs subse-
quently develop into larvae, pupae and adults.
Anastrepha obliqua is not thought to occur in
Florida (Steck 2001) so the importation of any
fruit that may serve as a host for this tephritid
poses a serious risk for agriculture in Florida and,
potentially, elsewhere in the subtropical main-
land. At least 13 reports indicate that the West
Indian fruit fly, A. obliqua, does indeed use ma-
mey sapote as a host (Emmart 1933; Stone 1942;
Aczel 1950; Oakley 1950; Gonzalez Mendoza 1952;

Jenkins & Goenaga: Host Status of Pouteria sapota to Anastrepha obliqua

Blanchard 1961; Korytkowski & Ojeda Pena
1970; Weems 1970; Wasbauer 1972; Kandybina
1977; Norrbom & Kim 1988; White & Elson-Har-
ris 1992; Fernandez et al. 1998; Norrbom 2004).
However, these reports are based on unreliable
identifications of host and insect species (confu-
sion remains about the plant or insect species
names used in these reports), or are citations of
unreliable literature.
Our objective was to estimate the likelihood of
infestation of mamey sapote byA. obliqua by sur-
veying the incidence of infestation in fruits col-
lected from the field, assessing the incidence of in-
festation when A. obliqua females have no other
host options, and by monitoring fruit fly popula-
tions in orchards of mamey sapote with baited
traps. We used the principles outlined in Cowley
et al. (1992) as guidelines for our investigation.


From Jun 2005 to Jun 2006 mature mamey sa-
pote fruits of cultivars Magaia, Mayapan, Pantin
(Key West), Tazumal, Pace, and Copan from or-
chards in Isabela and Corozal, PR, were scarred
by removing approximately 20 cm2 of skin from
each fruit. Fruit were scarred in order to give ac-
cess to the pulp, in case the fruit flies could only
oviposit in damaged fruit. Mature fruits reveal a
deep orange color when a thin layer of the coarse
skin of the mamey sapote is removed. At this
stage fruits are typically very hard and yield
sticky latex when cut. Harvested mature fruits
will ripen and soften over the next 3-6 days and
no longer yield sticky latex. We decided to harvest
fruit in this stage because that is the prevailing
practice in PR: fruit left on the tree typically do
not abscise until they have mummified, so we
could not collect dropped fruit, as is often done
with other species of fruits when surveying for
fruit fly infestations. Different varieties of mam-
eys were harvested as they were available.
The Isabela location is on the north coast on
the west side of the island (1828'18.97"N;
6702'49.66"W) and is 15.24 meters above sea
level. The mean rainfall for 2005 was 14.58 cm
with a range per month of 0-21.59 cm. The mean
rainfall for 2006 was 10.68 cm with a range per
month of 0-23.42 cm. The mean temperatures for
2005 and 2006 were 24.58C and 24.780C, respec-
tively. The Corozal location is on the north-central
portion of the island (18'19'39.05" N; 6621'38.04"
W) and is 212.14 meters above sea level. The
mean rainfall for 2005 was 20.6 cm with a range
per month of 1.12-42.82 cm. The mean rainfall for
2006 was 13.69 cm with a range per month of
2.34-29.36 cm. The mean temperatures for 2005
and 2006 were 24.84C and 25.29C, respectively.
One week after scarring, all scarred fruits
were harvested, weighed, and placed on a wire
mesh over vermiculite. Harvested fruits were

stored in a room at 25-27C and approximately
60% RH (never less than 50% RH). The vermicu-
lite was monitored weekly for the presence of fruit
fly larvae or pupae. Any recovered larvae or pu-
pae were collected and placed in a plastic Petri-
dish with a small amount of moistened vermicu-
lite and stored at 25C and 85% RH in an environ-
mental chamber (12:12 D:L) (White & Elson-
Harris 1992). Petri-dishes containing pupae were
monitored daily for the emergence of adults.
Between 1 and 4 mature mamey sapote fruits
of each variety were scarred as described above
and placed in collapsible mesh cages (60 x 60 x 60
cm) (Bioquip, Rancho Dominguez, CA) with 20
female and 20 maleAnastrepha obliqua flies, 12 d
post emergence. Fruit was exposed to flies for
48 h, removed and stored as described above to
collect emerging larval tephrititids. Concomi-
tantly, mature naturalized Mayaguezano variety
mangoes that had been covered with brown paper
bags when they were green (preventing infesta-
tion by fruit flies) were exposed to male and fe-
male A. obliqua under conditions identical to
those described for the mamey sapote fruit. The
exposure described was conducted for all 6 variet-
ies of mamey and replicated 3 times for each vari-
ety. To ensure that the mangoes used in the cage-
trials were not infested prior to the trials, mature
mangoes that had been bagged were stored over
vermiculite as described above and monitored for
the emergence of larvae and pupae.
To demonstrate that fruit flies occurred at the
experimental sites, 5 plastic multilure traps (A
Better World, Inc.) baited with ammonium ace-
tate and putrescine were placed in each mamey
sapote orchard and monitored weekly. Five traps
were also placed in nearby (120 m) carambola or-
chards (Averrhoa carambola: Oxalidaceae) and
monitored weekly. All adult flies obtained from
traps or from fruit were identified by the author
(D.J.) and voucher specimens were deposited in
the Entomological Laboratory of the Tropical Ag-
riculture Research Station, Mayaguez, PR.


A total of 1160 mamey sapote fruits weighing
777 kg were collected from orchards in Corozal
and Isabela, PR (Table 1). Of the fruit collected,
only 1 fruit of the Tazumal variety, harvested on
21 Nov 2005, yielded 2 larval tephritids and these
did not become adults. None of the 15 mamey
sapote fruits exposed to colonies of A. obliqua
yielded larvae, while mango fruit similarly ex-
posed yielded an average of 5.8 0.7 (mean SE)
larvae (Table 2). Control mangoes that had been
bagged when green and not exposed toA. obliqua
in cage-trials did not produce any larvae or
pupae. The number of adult Anastrepha obliqua
observed per trap per week in mamey sapote
orchards was always 2 or less (Fig. 1). Traps in

Florida Entomologist 90(2)

June 2007


Variety Dates collected Number of fruit Kg of fruit Total fruit per variety Total kg per variety

Magania 18-Aug-05 14 17.5 48 56.8
23-Aug-05 12 15.0
13-Sep-05 22 24.3
Mayapan 24-Jun-05 15 12.3 369 259.6
15-Jul-05 15 11.6
16-Aug-05 15 13.6
13-Apr-06 242 155.5
5-Jun-06 82 66.6
Tazumal 28-Jul-05 8 3.9 238 102.7
30-Aug-05 2 1.5
6-Sep-05 2 0.9
18-Oct-05 80 29.5
21-Nov-05 146 66.9
Pantin 16-Aug-05 5 4.2 97 83.7
13-Sep-05 10 5.6
20-Sep-05 2 1.8
2-Jun-06 80 72.1
Pace 24-Jun-05 15 10.2 156 103.6
15-Jul-05 15 10.4
30-Aug-05 4 3.1
20-Sep-05 24 14.9
22-Feb-06 98 65.0
Copan 3-Jun-05 6 3.4 252 170.2
28-Jul-05 10 6.1
29-Sep-05 6 3.7
21-Nov-05 42 61.2
13-Dec-05 64 31.0
19-Jan-06 124 64.8
Total 1160 776.6

nearby carambola orchards indicated that adult
A. obliqua were present and, at times, abundant
(1 trap in Corozal had 103 flies in it one week) in
the area being surveyed (Fig. 1). Traps in the car-

ambola orchards also caught A. suspense adults
in numbers similar to those reported for A. obli-
qua, but these data will be published in a future


Pupae recovered

Date Fruit/rep Rep 1 Rep 2 Rep 3 Mean + SE

13-Apr-06 Mango 5 5 7 3 5.0 + 1.2
Mayapan 2 0 0 0 0.0 +0.0
3-May-06 Mango 5 8 2 4 4.7 + 1.8
Tazumal 3 0 0 0 0.0 +0.0
8-May-06 Mango 5 3 3 7 4.3 + 1.4
Magania 1 0 0 0 0.0 +0.0
15-May-06 Mango 5 13 5 8 8.7 + 2.4
Pace 4 0 0 0 0.0+0.0
2-Jun-06 Mango 5 5 6 9 6.7 + 1.2
Pantin 3 0 0 0 0.0 + 0.0
5-Jun-06 Mango 5 9 4 3 5.3 + 1.9
Copan 2 0 0 0 0.0 + 0.0

Jenkins & Goenaga: Host Status of Pouteria sapota to Anastrepha obliqua




0 ^ ^^
Sy #

Traps in

20 -
/o /g I/
^^/^/ /a"
^ ^ <^ ^

Traps in
mamey sapote

P y

Fig. 1. Mean number ofAnastrepha obliqua per trap per week ( SEM) in orchards of mamey sapote and caram-
bola at Isabela and Corozal, Puerto Rico (n = 5 traps per orchard).


Our data show that the likelihood of infesta-
tion of mamey sapote by A. obliqua is extremely
small. Similar methods have been used to demon-
strate the non-host status of litchi and longan
(Litchi chinensis Sonn. and Euphoria longana
(Lour.), respectively: Sapindaceae) and mamey
sapote toA. suspense (Gould et al. 1999; Gould &
Hallman 2001). We collected more than 1000 ma-
ture mamey sapote fruits and reared only 2 lar-
vae from these. These larvae did not survive to
adulthood. Fruit exposed to fecund female fruit
flies did not yield any larvae or pupae. This indi-
cates that eitherA. obliqua females refuse to ovi-
posit in mamey sapote fruit or that eggs put in
mamey sapote are unlikely to develop.
We noted that the process of scarring mamey
sapotes yields a sticky latex, in common with
many sapotaceaous plants (Morton 1987). This la-
tex persists for up to 24 h. After this time the scar
is healed, resulting in a rough, corky tissue. We
suspect that the latex and the corky tissue may
preclude oviposition byA. obliqua.Anastrepha ser-
pentina is known to oviposit in sapotaceous hosts

that release sticky latex upon being punctured and
presumably have adapted their oviposition behav-
ior to deal with this defense system (Aluja et al.
2000). Anastrepha obliqua often infest mangoes
and other anacardiaceous fruits (White & Elson-
Harris 1992) that exude sticky polyphenolic resins
(Morton 1987; Zomlefer 1994). Females may ovi-
posit when the fruit is ripe and the resin levels in
the peel are reduced. There is also reason to be-
lieve that mango cultivars vary in their suscepti-
bility toA. obliqua and that the level of infestation
is correlated with the density of resin canals in the
fruit peel (Alex Segarra, unpubl.).
Mamey sapote has the potential to become an
important tropical fruit crop in Southern Florida
and Puerto Rico. We have compiled evidence that
mamey sapote is an extremely unlikely host of
A. obliqua in Puerto Rico and that the threat of
transporting larval fruit flies of this species in
fruit of mamey sapote is not likely.

Mention of trade names or commercial products in
this article is solely for the purpose of providing specific

Florida Entomologist 90(2)

information and does not imply recommendation or en-
dorsement by the U.S. Department of Agriculture. We
thank Yadzaida Garcia and Elkin Vargas for excellent
field work. We also thank Drs. Guy Hallman and Jorge
Peia and two anonymous reviewers for critiquing an
earlier version of this manuscript.


ACZEL, M. L. 1950. Catalogo de la familiar 'Trypetidae'
(Dipt. Acalypt.) de la region neotropical. Acta Zool.
Lilloana (1949) 7: 177-328.
AND J. SIVINSKI. 2000. Behavior of flies in the genus
Anastrepha (Trypetinae: Toxotrypanini), p. 384 In
M. Aluja and A. L. Norrbom [eds.], Fruit Flies (Te-
phritidae): Phylogeny and Evolution of Behavior.
CRC Press, Boca Raton, FL. 963 pp.
BLANCHARD, E. E. 1961. Species argentinas del g6nero
Anastrepha Schiner (sens. lat.) (Diptera, Try-
petidae). Rev. Invest. Agric. 15 (2): 281-342.
COWLEY, J. M., R. T. BAKER, AND D. S. HARTE. 1992.
Definition and determination of host status for mul-
tivoltine fruit fly (Diptera: Tephritidae) species. J.
Econ. Entomol. 85: 312-317.
EMMART, E. W. 1933. The eggs of four species of fruit
flies of the genus Anastrepha. Proc. Entomol. Soc.
Wash. 35: 184-191.
ORTIZ. 1998. Notas sobre el genero Anastrepha
Schiner en Cuba con description de una nueva espe-
cie (Diptera: Tephritidae). Folia Entomol. Mex.
(1997) No. 99: 29-36.
GONZALEZ MENDOZA, R. 1952. Contribucion al studio
de las moscas Anastrephas en Colombia. Rev. Fac.
Nac. Agron. Medellin 12: 423-549.
RAS, R. NGUYEN, AND J. CRANE. 1999. Nonhost status
of lychess and longans to Caribbean fruit fly (Diptera:
Tephritidae). J. Econ. Entomol. 92: 1212-1216.
GOULD, W. P., AND G. HALLMAN. 2001. Host status of
mamey sapote to Caribbean fruit fly (Diptera: Te-
phritidae). Florida Entomol. 84: 730-375.

KANDYBINA, M. N. 1977. Lichinki plodovykh mukh-pes-
trokrylok (Diptera, Tephritidae). [Larvae of fruit-in-
festing fruit flies (Diptera, Tephritidae)]. Opred.
Faune SSSR No. 114: 1-210.
pecies del genero Anastrepha Schiner 1868 en el nor-
oeste peruano. Rev. Peru. Entomol. (1968) 11: 32-70.
MORTON, J. F. 1987. Fruits of Warm Climates. Media In-
corporated, Greensboro, NC. 506 pp.
NORRBOM, A. L., AND K. C. KIM. 1988. A list of the re-
ported host plants of the species of Anastrepha
(Diptera: Tephritidae). U.S. Dep. Agric. Animal Plant
Health Insp. Serv. APHIS 81-52: 114 p.
NORRBOM, A. L. 2004. Host plant database for Anas-
trepha and Toxotrypana (Diptera: Tephritidae: Tox-
otrypanini). Diptera Data Dissemination Disk (CD-
ROM) Volume 2, Systematic Entomology Laboratory,
OAKLEY, R. G. 1950. Part III Fruit Flies (Tephritidae),
pp. 169-246 In Manual of Foreign Plant Pests.
United States Department of Agriculture, Agricul-
tural Research Administration, Bureau of Entomol-
ogy and Plant Quarantine, Division of Foreign Plant
STECK, G. J. 2001. Concerning the occurrence of Anas-
trepha obliqua (Diptera: Tephritidae), in Florida.
Florida Entomol. 84: 320-321.
STONE, A. 1942. The Fruitflies of the Genus Anastrepha.
U.S. Dep. Agric. Misc. Publ. 439: 112 p.
WASBAUER, M. S. 1972. An Annotated Host Catalog of
the Fruit Flies of America North of Mexico (Diptera:
Tephritidae). Occas. Pap. Calif. Dep. Agric. Bur. En-
tomol. 19: [i] + 172 p.
WEEMS, H. V., JR. 1970. West Indian fruit fly Anas-
trepha mombinpraeoptans Sein (Diptera: Tephriti-
dae). Florida Dept. Agric. Consum. Serv., Div. Plant
Ind. Entomol. Circ. 101: 2 p.
WHITE, I. M., AND M. M. ELSON-HARRIS. 1992. Fruit
Flies of Economic Significance: Their Identification
and Bionomics. CAB International, Wallingford. xii
+ 601 p.
ZOMLEFER, W. B. 1994. Guide to Flowering Plant Fami-
lies. University of North Carolina Press, Chapel Hill
& London. 430 pp.

June 2007

Scientific Notes


'Entomology and Nematology Department, University of Florida, 970 Natural Area Drive
P.O. Box 110620 Gainesville, FL 32611-0620

2Center for Medical, Agricultural and Veterinary Entomology (USDA-ARS)
1600 SW 23rd Drive, P.O.Box 14565 Gainesville, FL 32604

3Florida Department of Agriculture and Consumers Services, Division of Plant Industry
P.O. Box 147100, Gainesville, FL 32614-7100

4Subtropical Horticultural Research Station, USDA-APHIS, 13641 Old Cutler Road, Miami, FL 33158

Florida's best known member of the genus
Anastrepha is an introduced pest, the Caribbean
fruit fly, A. suspense (Loew). However, Florida is
also home to other innocuous species, including
Anastrepha interrupta Stone. The latter was first
reported as an undescribed "species E" collected
during an early fruit fly survey program in south
Florida (Brown 1937) and eventually described by
Stone (1942). It is also known from the Bahamas,
Virgin Islands, and Dominica (Steyskal 1977),
Cuba (Fernandez et al. 1997), and Puerto Rico
(A. L. Norrbom, personal communication). The
larvae infest fruits of Schoepfia schreberi J. F.
Gmel. ("gulf greytwig", family Olacaceae; previ-
ously known as Schoepfia chrysophylloides (A.
Rich.) Planch. (McClahanan & Merrill 1951); be-
cause this is its only known host, the fly has been
called the "Schoepfia fruit fly" (Heppner 1990). In
Florida, A. interrupta has been trapped since
1934, and many museum specimens exist up to
the early 1960s, but far fewer were collected dur-
ing later years. The literature contains only a few
bits of information concerning this species: Brown
(1937)-detection; Stone (1942), Shaw (1962),
Weems (1967)-adult identification and taxon-
omy; McClanahan & Merrill (1951)-host plant;
Marsh (1970), Wharton & Marsh (1978)-parasi-
toids; Steck & Wharton (1988), Steck et al. (1990),
Heppner (1990)-larval identification.
In early 2003, a population ofA. interrupta was
discovered through adult trapping and fruit col-
lections in Miami-Dade County near Homestead,
Florida at Camp Owaissa Bauer. This population
offered an unusual opportunity to make addi-
tional observations on A. interupta. At the same
time, an effort was made to detect other popula-
tions of the fly where the hosts were abundant.
Field work was conducted at the Deering Es-
tate at Cutler (Miami) and Camp Owaissa Bauer,
both Miami-Dade County Parks. Schoepfia fruit-
ing phenology was followed in both parks, and
two distinct patterns were observed. There was
one fruiting season (Mar-Apr) at Deering Estate
at Cutler, and two fruiting seasons at Camp
Owaissa Bauer (Mar-Apr and Oct-Nov). Even

though they are near each other (32 km apart),
Camp Owaissa Bauer is in the interior (11 km
from the Atlantic coast), unlike Deering Estate at
Cutler, which is directly on the Atlantic coast. It
has been observed that populations of Schoepfia
in the Florida Keys flower and fruit at different
times than the mainland populations (R. Ham-
mer, personal communication). Phenological dif-
ferences may be due to climatic differences among
locations, or, alternatively, differences in soil com-
position and nutrients or plant variety may result
in different capacities to produce fruit or different
adaptive fruiting schedules.
Adult trapping was conducted during Mar and
Apr, 2004 at Deering Estate at Cutler in 3 loca-
tions within the park where S. schreberi occurred.
Five multi-lure traps (Better World, Fresno. CA)
with torula yeast pellets dissolved in water were
deployed and serviced every 2 weeks. Only one
adult female specimen was trapped in Apr. At
Camp Owaissa Bauer we did not conduct any
trapping activities for fear of reducing the resi-
dent fly population.
Two hundred fruit were collected at the Deer-
ing Estate at Cutler in Mar and Apr, 2004, and
150 fruits in Mar, 2005, but no larvae were ob-
tained. At Camp Owaissa Bauer, fruits were col-
lected in Oct 2003, Mar, Oct, and Nov 2004, and
Mar 2005 (about 100 fruits on each occasion). The
fruits ranged in fresh weight from 61.1 mg to
316.0 mg and averaged 146.9 54.9 mg (n = 25).
More than 50% of the collected fruit was infested.
Anastrepha interrupta apparently oviposits
into the fruit of S. schreberi while they are still
green and not yet full size. We observed only 1
larva per fruit based on field observation of
about 100 infested fruits, in which the seed had
been entirely consumed leaving most of the
space inside the fruit occupied by the third in-
star. Normal fruits became purple as they ma-
tured, but infested fruits did not appear to fully
mature and change color. The oviposition punc-
ture was clearly evident to the naked eye, and
the larval exit hole was relatively large, obvious,
and circular.

Florida Entomologist 90(2)

The samples of collected fruit were maintained
in the laboratory (temperature of 25 1C and
relative humidity of 55 5%), in a container with
vermiculite until larvae emerged and pupation
occurred. The mean weight ofA. interrupta pupae
was 13.6 4.8 mg (n = 25), with a range of 4.9 to
22.7 mg. Anastrepha interrupta's closest relative,
Anastrepha spatulata Stone (Foote et al. 1993),
infests the same host plant in Mexico, where the
fruit are slightly larger (mean = 185.0 mg), but
the pupae are slightly smaller (12.0 mg) (Aluja et
al. 2000).
The adult flies emerged 20-24 d after pupation
at 25 1C and RH of 55 5%. Under the same
conditions, adults took about 20 d to mature sex-
ually; i.e., the start of male pheromone produc-
tion, sexual signaling, and mating. Maximum
adult longevity in the laboratory was 177 d (fe-
male). This is similar to the lifespan reported for
other Anastrepha species such as A. suspense
(Sivinski 1994). No diapause in the pupal stage
was observed; all pupae that developed from the
fall collections and the spring collections closed
as adults within a few weeks of pupation.
We searched museum collections and records
of the Florida State Collection of Arthropods
(FSCA) and the U.S. National Museum of Natural
History (NMHN) (Norrbom 2006) for information
on flight time ofA. interrupta (Fig. 1). Almost all
records are based on specimens captured in fruit
fly detection traps (McPhail traps). We excluded
those few records based on adults reared from
fruits. As previously noted (Weems 1967), adults
have been collected in every month of the year.
Both the number of collection records and the
number of flies collected clearly indicate elevated
population levels in the months from Dec to May,







which overlaps with the primary fruiting seasons
of its host. During the winter months of 2004/
2005, A. interrupta clearly produced at least 2
generations at Camp Owaissa Bauer. Herbarium
records at the Fairchild Tropical Botanic Garden
(http://www.virtualherbarium.org/, accessed 20
Oct 2006) show that Schoepfia schreberi fruits at
least sporadically during other months than ob-
served here, e.g. Jan and Feb; and fruiting has
also been observed during late Jul, Aug and Sep
(R. Hammer, personal communication). The op-
portunistic availability of fruit throughout the
year probably allows additional generations to be
produced. An adult life span of several months as
observed in the laboratory would allow this spe-
cialist species to persist as adults from one fruit-
ing period to another as occurs at Camp Owaissa
Bauer. A population may not be sustainable at
sites with only a single fruiting season, such as
Deering Estate at Cutler, and such sites might be
repopulated only by immigration.
After emergence the flies were separated by sex
and maintained with food (3 parts sugar and 1 part
yeast) and water ad libitum in separate cages. At
20 d of adult age, flies from both sexes were placed
in the same cage and their sexual behaviors ob-
served. During the mid-afternoon males per-
formed the following peculiar behavior: after ap-
proaching a female, the male faced her and then
moved laterally, back and forth, in a half circle pat-
tern. It is not known if this behavior is confined to
interactions between males and females. Male
pheromone calling, recognized by the extrusion of
anal membranes and pleural glands, as seen in
other tephritids (Aluja 1994), took place only in
complete darkness, followed by mating. Male pher-
omone components were identified and will be de-

SNunber of records
o Nrrter of flies

Jan Feb Mar Apr May Jun Jy

Aug Sep Oct Nv Dec

Fig. 1. Phenology ofAnastrepha interrupta based on Florida State Collection ofArthropods and the U.S. National
Museum of Natural History specimens and records, 1934-2003.

June 2007

Scientific Notes

scribed elsewhere (B. D. Dueben, personal commu-
nication). Male-male interaction was not observed
during the behavioral observations in laboratory.
Two females of the parasitoid Utetes anas-
trephae (Viereck) (Hymenoptera: Braconidae)
were reared from fly pupae derived from fruit
sampled in Mar 2005. This is the first record of
this parasitoid attacking A. interrupt. The only
other parasitoid previously reported was another
braconid, Doryctobracon anastrephilus (Marsh)
(Marsh 1970; Wharton & Marsh 1978). Utetes
anastrephae is widespread in South Florida,
where it also parasitizes Anastrepha suspense
(Loew) (Eitam et al. 2004).
This study was conducted under permit #061
from Miami-Dade County Park and Recreation
Department. We offer special thanks to Alice War-
ren-Bradley (Manager of Deering Estate Park)
and Pam Rose (Manager of Camp Owaissa Bauer)
for their cooperation and Robert A. Wharton
(Texas A&M University, College Station, TX) for
identification of the parasitoids. Roger Hammer
(Miami-Dade Parks Department) provided com-
ments on phenology of Schoepfia schreberi in south
Florida. We thank Allen L. Norrbom (Systematic
Entomology Laboratory, USDA-ARS, Washington
DC) and Robert A. Wharton for critical reviews of
an earlier version of this manuscript. Financial
support was provided to RP by the Centro de Ciin-
cia e Tecnologia da Madeira through the BD I/
2002-004 grant. This is Entomology Contribution
No. 1049, Bureau of Entomology, Nematology, and
Plant Pathology, FDACS-DPI.


Observations on the phenology, larval host
feeding, adult longevity, behavior, and parasites
ofAnastrepha interrupta Stone (Diptera: Tephri-
tidae) are presented. A population of this fly was
bivoltine in Homestead, Florida where its host
plant fruited twice per year. Anastrepha inter-
rupta larvae were found singly in fruits of Schoe-
pfia schreberi (Olacaceae) in which they con-
sumed the seed. Adult flies lived up to 177 d, and
there was no evidence of diapause in the imma-
ture stages. Male calling behavior begins after
dusk and continues in darkness. Utetes anas-
trephae (Viereck) (Hymenoptera: Braconidae)
was reared from pupae, the first observation of
this parasitoid onA. interrupta.


ALUJA, M. 1994. Bionomics and management of Anas-
trepha. Annu. Rev. Entomol. 39: 155-178.

2000. First host plant and parasitoid record for
Anastrepha spatulata Stone (Diptera: Tephritidae).
Proc. Entomol. Soc. Washington 102: 1072-1073.
BROWN, A. C. 1937. Report of the grove inspection de-
partment. State Plant Board of Florida, Report for
the period July 1, 1934-June 30, 1936 (Eleventh Bi-
ennial Report). 37 pp.
Biogeography of braconid parasitoids of the Carib-
bean fruit fly (Diptera: Tephritidae) in Florida. Ann.
Entomol. Soc. America 97: 928-939.
ORTIZ. 1997. Notas sobre el genero Anastrepha
Schiner en Cuba con description de una nueva espe-
cie (Diptera: Tephritidae). Folia Entomol. Mexicana
99: 29-36.
Handbook of the Fruit Flies (Diptera: Tephritidae) of
America North of Mexico. Comstock Publishing As-
sociates, Ithaca, NY.
HEPPNER, J. B. 1990. Larvae of Fruit Flies. 6. Anas-
trepha interrupta (Schoepfia fruit fly) (Diptera: Te-
phritidae). Florida Dept. Agric., Div. Plant Ind.
Entomol. Circ. 327.
MARSH, P. M. 1970. A new species of fruit fly parasite
from Florida (Hymenoptera: Braconidae). Florida
Entomol. 53: 31-32.
MCCLANAHAN, H. S., AND G. B. MERRILL. 1951. Reports
of the Grove Inspection and Entomological Depart-
ments, Florida State Plant Board Biennial Report
NORRBOM, A. L. (ed.). F. Louie Blanc New World Fruit Fly
Specimen Database (http://www.sel.barc.usda.gov:591/
diptera/Tephritidae/TephIntro.html, accessed on 16
Oct. 2006).
SHAW, J. G. 1962. Species of the spatulata group of
Anastrepha. J. Kansas Entomol. Soc. 35: 408-414.
SIVINSKI, J. 1994. Longevity in the Caribbean fruit fly;
effects of sex, strain and sexual experience. Florida
Entomol. 76: 635-644.
STECK, G. J., AND R. A. WHARTON. 1988. Description of
immature stages ofAnastrepha interrupta, A. limae,
and A. grandis (Diptera: Tephritidae). Ann. Ento-
mol. Soc. Am. 81: 994-1003
AND J. GUILLEN-AGUILAR 1990. Methods for identi-
fication of Anastrepha larvae (Diptera: Tephritidae),
and key to 13 species. Proc. Entomol. Soc. Washing-
ton 92: 333-346.
STEYSKAL, G. C. 1977. Pictorial Key to Species of the Ge-
nusAnastrepha (Diptera: Tephritidae). Special Publ.
Entomol. Soc. Washington. 35 pp.
STONE, A. 1942. The Fruit Flies of the Genus Anas-
trepha. U.S. Dep. Agric. Misc. Publ. 439: 1-112.
WHARTON, R. A., AND P. M. MARSH. 1978. New World
Opiinae (Hymenoptera: Braconidae) parasitic on Te-
phritidae (Diptera). J. Washington Acad. Sci. 68:
WEEMS, H. V. JR. 1967. Anastrepha interrupta Stone
(Diptera: Tephritidae). Florida Dept. Agric., Div.
Plant Ind. Entomol. Circ. 61.

Florida Entomologist 90(2)


1USDA-APHIS, Beltsville, MD 20705

2Florida Department of Agriculture and Consumer Services, Division of Plant Industry
1911 SW 34th Street, Gainesville, FL 32608

The grasses (Poaceae) are some of the most im-
portant agricultural plants in the world with
corn, sugarcane and wheat all being widely used
as food crops. In addition to agricultural impor-
tance, grasses also are used as ornamentals in
landscapes and other horticultural situations, as
well as being the major plants used in turf for
lawns and recreational activities. Because these
plants are so widely used in a variety of monocul-
tural plantings, they attract a wide array of pests.
One of the most common and taxonomically diffi-
cult groups of insects that diagnosticians have to
identify are scale insects (Hemiptera: Coccoidea).
Twenty-one families of scale insects are known
worldwide with the most commonly encountered
pest species found within the following 3 families:
(1) Diaspididae (armored scales), (2) Pseudococ-
cidae mealybugss) and (3) Coccidae (soft scales).
The most common scale insects associated with
grasses are armored scales and mealybugs, and
several species of armored scales are commonly
collected on grasses in Florida. The most common
of these are Odonaspis species, Aspidiella sac-
chari (Cockerell), Haliaspis species, in addition to
Frogattiella and Kuwanaspsis species, which are
found on bamboo.
More recently, Duplachionaspis divergens
(Green) has steadily become one of the most com-
monly encountered grass infesting armored
scales in Florida. This armored scale has been re-
ported throughout much of the Eastern Hemi-
sphere including Algeria, Australia, China,
Egypt, India, Japan, Sri Lanka, Taiwan, and
Thailand, and is the only species of the 35 de-
scribed species of the genus known to occur in the
United States. Lastra and Gomez (1997) reported
the first occurrence in the Western Hemisphere
from collections on sugarcane in Colombia in
1996. However, specimens collected by Fred Ben-
nett in Venezuela on sugarcane confirmed its
presence in the Western Hemisphere as early as
1991. Its occurrence in Florida and the continen-
tal United States was first recorded from speci-
mens on a grass in Seminole Co., Florida in 2002.
However, a re-examination of specimens collected
in Manatee Co., Florida on Miscanthus species in
2000 is the earliest record of this species occur-
ring in Florida. Since the initial finds of D. diver-
gens in Florida, interceptions have occurred in
both Alabama (Charles Ray, Auburn University,

pers. comm.) and Texas (Scott Ludwing, Texas
A&M University, pers. comm.).
The biology of D. divergens was studied by Las-
tra and Gomez (1997) in Colombia. They reported
that adult females lay an average of 130 eggs and
that 9 generations/year occurred with an average
generation time of 39 days. The scale cover of the
adult female (Fig. 1) resembles that of false olean-
der scale (Pseudaulacaspis cockerelli (Cooley) in
that they appear as small white "tear drops" that
are about 3 mm long. Male covers are much smaller
(1 mm) and appear as white tricarinate tubes.
The economic importance of D. divergens in
Florida is not clearly defined. However, Pruthi &
Rao (1942) reported it as a minor pest of sugar-
cane in India, and Lastra & Gomez (1997) noted it
as a pest of sugarcane in Colombia. Therefore, it
is a potential pest of sugarcane in Florida where
about 450,000 acres are grown annually
(Meagher 2003). This species has also been found
on St. Augustine grass (Stenatophrum secunda-
tum (Walter) Kuntze), a common lawn grass and
Bahia grass (Paspalum notatum Flugge), an im-
portant pasture grass. Sugarcane growers usu-
ally implement natural control strategies to con-
trol pests and seldom use pesticides. Natural en-
emies are known for D. divergens (Sankaran
1984; Shafee et al. 1975; Lastra & Gomez 1997);
however, we have reared a species in the Aphytis
lingnanesis group and Encarsia citrina (Craw)
(Hymenoptera: Aphelinidae) from specimens col-
lected in Florida.

Fig. 1. Field specimen, adult female cover of Dupla-
chionaspis divergens. Photograph credit: Avas Hamon,

June 2007

Scientific Notes


Duplachionaspis divergens is established in
Florida and has been intercepted in both Ala-
bama and Texas, but its overall economic impact
is yet unknown. However, due to its potential as a
pest of sugarcane and other grasses, it warrants
close observation to ensure that this insect does
not become a major pest of grasses in the south-
eastern United States.


LASTRA, L. A., AND L. A. GOMEZ. 1997. Observaciones
del ciclo devida de la escama blanca, Duplachionas-
pis divergens (Green) (Homoptera: Diaspididae) y

reconcimiento de enemigos naturals, pp. 41-51 In IV
Congress Colombiana de la Asociaci6n de t6cnios de
la can'a de azucar. Cali, Colombia 24-26 de Sept. de
1997, 473 pp.
MEAGHER, R. L. 2003. Sugarcane IPM. http://ipm-
PRUTHI, H. S., AND V. P. RAO. 1942. Coccids attacking
sugarcane in India. Indian J. Entomol. 4: 87-88.
SANKARAN, T. 1984. Survey for natural enemies of Dia-
spine Scale Insects in South India: Final Technical
Report for the Period November 5, 1980 to November
4, 1983. Commonwealth Institute of Biological Con-
trol, Bangalore India. 87 pp.
Taxonomic survey of encyrtid parasites (Hy-
menoptera: Encyrtidae) in India. Publications (Ali-
garh Muslim University) 10: 123 pp.

Florida Entomologist 90(2)


University of Florida, Department of Entomology and Nematology
Building 970, P.O. Box 110620, Gainesville, FL 32611-0620

Key Words: Diaphorina, Huanglongbing, Asian citrus psyllid, Candidatus Liberibacter asi-

Citrus greening disease or Huanglongbing
(HLB) is caused by the gram-negative bacterium
Candidates Liberibacter asiaticus (Ca. L. asiati-
cus) (Garnier et al. 2000) and was confirmed in
southern Florida in 2005 (Halbert 2005; Bouffard
2006). This disease is vectored by Diaphorina
citri Kuwayama (Hemiptera: Psyllidae), which
colonized the citrus-growing regions of Florida af-
ter it was discovered in 1998 (Knapp et al. 1998;
Halbert 1998; Halbert et al. 2000). Diaphorina
citri acquires the greening bacterium while feed-
ing on infected phloem (Hung et al. 2004). HLB
ultimately is fatal to susceptible citrus trees, so
early detection and removal of infected trees is
important for disease management. Unfortu-
nately, citrus trees often are asymptomatic for
years before the common signs of HLB, including
yellowing and mottling of leaf veins and mis-
shapen green-colored fruit, are noticeable (da
Graca 1991). Current chemical and biological con-
trols reduce D. citri populations (Rae et al. 1997;
Hoy et al. 1999; Hoy & Nguyen 2000; Michaud
2004; Browning et al. 2006), but may not be suffi-
cient to eliminate all HLB transmission.
It will be important to understand the epide-
miology of HLB to control the spread of this dis-
ease. The regions of Florida with citrus showing
symptoms of HLB currently are being mapped
cgsit_map.pdf). However, little currently is
known about infection rates and transmission
frequency of HLB by the psyllid vector. We sur-
veyed the vector, D. citri, for the greening bacte-
rium in 11 citrus-growing counties in Florida (Ta-
ble 1). In most of the counties sampled, citrus
trees did not show signs of HLB infection, so we
anticipated a low incidence of the greening bacte-
rium (perhaps <1-2%) in these psyllid popula-
tions. However, we hypothesized that if citrus
trees had acquired HLB recently and did not
show disease symptoms, HLB could still be
spread in these regions and detected in vector
populations by molecular analyses.
Adult psyllids collected in this survey were
killed in 95% ethanol in the field and placed on ice
during transit to the Department of Entomology
and Nematology at the University of Florida,

Gainesville FL. Adult and immature D. citri were
separated, counted, and stored in fresh 95% etha-
nol or acetone at -80C (Fukatsu 2005). Tools used
to separate insect specimens were washed with
bleach, which degrades DNA, to avoid cross-con-
tamination between samples. A maximum of 10
D. citri were pooled for DNA isolation by PURE-
GENE reagents according to the manufacturer's
instructions (Gentra Systems, Minneapolis, MN).
DNA pellets were re-suspended in 50 pL of sterile
water or TE buffer and stored at -80C. High-
fidelity PCR was used to analyze each sample for
the 16S rRNA (Subandiyah et al. 2000) and nusG-
rplK (Villechanoux et al. 1993; Hoy et al. 2001)
gene sequences of Ca. L. asiaticus, which would
yield DNA bands 0.5 kb and 0.6 kb in length, re-
spectively. The samples also were screened for a
0.6-kb portion of the wsp gene ofWolbachia (Braig
et al. 1998), an endosymbiotic bacterium found in
D. citri (Subandiyah et al. 2000), to control for
DNA quality.
A positive control was obtained from Vernon
Damsteegt at the USDA-ARS quarantine facility
in Beltsville, MD, where adult D. citri fed on cit-
rus trees positive for HLB. A total of 3 DNA ex-
tractions from these adult D. citri, including 2 ex-
tractions from single adults and 1 extraction from
10 pooled adults, was conducted by Micki Kuhl-
mann with the methods described above. The
DNA was shipped from Beltsville, MD to the Uni-
versity of Florida and used in a high-fidelity PCR
assay. Amplification products were detected in
each of the 3 samples with primers for the nusG-
rplK gene of Ca. L. asiaticus and for the wsp gene
To quantify the sensitivity of our high-fidelity
PCR assay, a dilution series of plasmid DNA con-
taining the nusG-rplK gene of Ca. L. asiaticus,
mixed with DNA from adult D. citri from a labo-
ratory colony that previously had tested negative
Ca. L. asiaticus, was amplified with high-fidelity
PCR (Fig. 1). As little as 1 fg of the target tem-
plate could be detected 100% of the time, which is
approximately equivalent to 100 copies of the
nusG-rplK gene sequence (Fig. 1), while as few as
10 copies could be detected 50% of the time (Hoy
et al. 2001). Control reactions containing no DNA

June 2007

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