Title: Florida Entomologist
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Title: Florida Entomologist
Physical Description: Serial
Creator: Florida Entomological Society
Publisher: Florida Entomological Society
Place of Publication: Winter Haven, Fla.
Publication Date: 2002
Copyright Date: 1917
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Subject: Florida Entomological Society
Entomology -- Periodicals
Insects -- Florida
Insects -- Florida -- Periodicals
Insects -- Periodicals
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McCoy et al.: Entomopathogenic Nematode


FIELD EFFICACY OF TWO COMMERCIAL PREPARATIONS OF
ENTOMOPATHOGENIC NEMATODES AGAINST LARVAE OF DIAPREPES
ABBREVIATUS (COLEOPTERA: CURCULIONIDAE) IN ALFISOL TYPE SOIL

C. W. MCCoY,1 R. J. STUART,1 L. W. DUNCAN'AND K. NGUYEN2
'University of Florida, IFAS, 700 Experiment Station Road, Lake Alfred, FL 33850

2Department of Entomology and Nematology, University of Florida, Gainesville, FL 32611

ABSTRACT

Spring and fall field trials were conducted to determine the efficacy of two species of ento-
mopathogenic nematodes for the control of larvae of Diaprepes abbreviatus in a citrus grove
with alfisol type soil (sandy clay loam). Both Steinernema riobrave (Bio Vector 355) as a wa-
ter-dispersible granule and Heterorhabditis indica (GrubstakeTM 100) as a paste on sponge
at rates from 22-108 IJ's/cm2 failed to reduce larval populations in the tree rhizosphere at 25
d post-treatment. Larval parasitism by entomopathogenic nematodes in baited screen cages
was sporadic over time, with the only significant treatment effect occurring at the highest
rate (108 IJ's/cm2) ofS. riobrave in the fall at 7 d post-treatment. Possible constraints to nem-
atode efficacy are discussed.

Key Words: biological control, citrus root weevils, Diaprepes, entomopathogenic nematodes,
Pachnaeus

RESUME

Se llevaron a cabo pruebas de campo en la primavera y el otono para determinar la eficacia
de dos species de nematodos entomopat6genos para el control de larvas de Diaprepes ab-
breviatus en huertos de citricos con suelo de tipo alfisol (marga arcilla arenosa). Las dos es-
pecies, Steinernema riobrave (Bio Vector 355) en la forma granular dispersable en agua, y
Heterorhabditis indica (GrubstakeTM 100) en la forma de una pasta puesta encima de una es-
ponja al porcentaje de 22-108 IJ's/cm2, no redujeron la poblaci6n de larvas en la rizosfera a
25 d despu6s del tratamiento. El parasitismo de larvas por nematodos entomopat6genos en
jaulas de tela metalica con cebo fu6 esporadico durante el tiempo del studio, el unico efecto
significativo del tratamiento sucedi6 en la concentraci6n mas alta (108 IJ's/cm2) de S. rio-
brave en el otono a los 7 d despu6s del tratamiento. Se discuten las posibles restricciones a
la eficacia de nematodos.


Several species of polyphagous root weevils,
particularly Diaprepes abbreviatus L. and Pach-
naeus spp., are important field and nursery pests
of citrus, ornamentals, and some agronomic crops
in Florida (McCoy 1999). Larval feeding injury to
roots byD. abbreviatus can have a devastating ef-
fect on citrus trees since all stages feed on the
roots for most of the year. Root injury appears to
be cumulative, and most importantly, feeding
sites can serve as infection courts for root rot dis-
eases (Graham et al. 1996), thereby exacerbating
economic loss. Tree decline can be particularly
bad in poorly drained groves when water stress
affects root health. There is no estimate of the to-
tal economic loss to the growers from larval root
injury to citrus, but the end result can frequently
be tree death. Current integrated pest manage-
ment (IPM) strategies for weevil control include
sound horticultural practices, fungal disease con-
trol, adult weevil monitoring, and a mix of sup-
pressive tactics to control larvae and adults
(McCoy & Duncan 2000).


Native and introduced entomopathogenic nem-
atodes are infectious to all larval stages and possi-
bly adults (Adair 1994, Beavers et al. 1983,
Schroeder 1990). Naturally occurring species
within the genera Heterorhabditis and Stein-
ernema have been found in citrus groves through-
out Florida infecting as much as 38-68% of caged
D. abbreviatus larvae in the summer in deep
sandy soils found on the central ridge and the
sandy clay loam soils of the coastal and interior
flatwoods (Beavers et al. 1983, McCoy et al. 2000).
The density and distribution of endemic nematode
populations, regardless of species, vary from grove
to grove, within a grove and within a season. Inun-
dative releases of mass-produced entomopatho-
genic nematodes (EPN) for larval control have
been pursued by private industry for about 15 yrs
(Duncan et al. 1999, Schroeder 1987). During this
time, four nematode species have been sold in
Florida to control D. abbreviatus in citrus: Heter-
orhabditis bacteriophora Poinar; Heterorhabditis
indica Poinar, Karunaker and David; Steinernema







Florida Entomologist 85(4)


carpocapsae (Weiser); and Steinernema riobrave
(= riobravis) Cabanillas, Poinar and Raulston
(Shapiro & McCoy 2000a). Currently, H. indica
(GrubstakeTM 100, Integrated BioControl Sys-
tems, Aurora, IN) and S. riobrave (Bio Vector 355,
Certis Corporation, Columbia, MD) are sold com-
mercially for use on Florida citrus.
The label rate for Bio Vector 355 is 4.9 x 108 vi-
able IJ's/treated hectare at 250 trees/grove hect-
are, that is, 2,000,000 IJ's/tree (Knapp 2000). The
label rate for GrubstakeTM 100 is one-half the Bio-
Vector rate. These field rates can be highly vari-
able since the area of soil treated per hectare can
change according to tree size and method of appli-
cation. To circumvent this problem, private indus-
try suggests that growers apply GrubstakeTM 100
at 11 IJ's/cm2 and Bio Vector 355 at twice that
rate. To date, no published field research has
shown that these rates are effective against Di-
aprepes. Field trials by Bullock & Miller (1994),
Bullock et al. (1999), and Schroeder (1990),
showed that rates of 2-5 million IJ's of S. carpo-
capsae or S. riobrave per tree applied within a
~0.3 m2 area surrounding the base of the tree in
the spring significantly reduced adult emergence
of both D. abbreviatus and Pachnaeus litus (Ger-
mar). In addition, field trials in groves on the cen-
tral ridge using S. riobrave and H. bacteriophora
showed that rates of 120-250 IJ's/cm2 reduced lar-
val populations significantly within 4 weeks post-
treatment (Downing et al. 1991, Duncan et al.
1996, Duncan & McCoy 1996). In three separate
trials, McCoy et al. (2000) showed that nematode
parasitism by either S. riobrave or H. indica at 22
IJ's/cm2 or less was no different than parasitism
in the untreated control in a flatwoods grove. In
fact, 108-216 IJ's/cm2 of S. riobrave were required
to increase parasitism to 40-60%.
Although published data cited above clearly
show that higher rates of entomopathogenic nem-
atodes result in (i) higher parasitism, (ii) greater
suppression of larvae in the soil, and (iii) reduced
adult emergence from the soil, data also suggest
that efficacy is influenced by other unknown fac-
tors relating to nematode, host and/or environ-
ment (Kaya & Gaugler 1993). For example, S.
carpocapsae at rates greater than 100 IJ's/cm2
gave no control in central ridge and coastal flat-
woods groves (Adair 1994; Bullock et al. 1999;
Duncan et al. 1996). Laboratory studies have
shown that S. riobrave is more effective at
warmer soil temperatures, and host age also af-
fects susceptibility ofD. abbreviatus larvae to the
nematode (Shapiro & McCoy 2000b; Shapiro et al.
1999). In addition, soil type can affect virulence
and persistence of S. riobrave and H. bacterio-
phora (Shapiro et al. 2000), whereas culture and
formulation method have no effect on larval mor-
tality for S. riobrave (Shapiro & McCoy 2000a).
The objectives of this study were to further test
the efficacy of a commercial formulation of S. rio-


brave (Bio Vector 355) in a flatwoods-like grove
with alfisol type (sandy clay loam) soil at different
rates per soil surface area and, for the first time,
test the field efficacy of high rates of H. indica
(GrubstakeTM 100) in the same soil. Tree destruc-
tion and baited traps were used to assess larval
population survival and nematode parasitism, re-
spectively.

MATERIALS AND METHODS

Experimental Site

Two field trials were conducted near Poinci-
ana, FL, (Osceola County) in a declining mature
planting of Hamlin oranges grafted to Swingle
citrumelo rootstock. The grove was planted on
two row beds with a setting pattern of 6.1 x 8.5 m.
The grove was equipped with under-tree micro-jet
sprinkler irrigation. The alfisol soil type for the
grove was classified as Floridana fine sand 68.8%
sand, 11.8% silt, 19.4% clay. The surface layer
was about 35.6 cm loam and the subsurface layers
about 76.2 cm grey fine sand followed by clay. The
soil was poorly drained with a low to moderate or-
ganic content and a pH of 4.8. The trials were con-
ducted within 40 m of each other. Adult weevil
injury to the leaf (McCoy 1999) was evident in all
trees throughout the grove.
In trial one, 40 single tree plots were arranged
on two row beds in a completely randomized de-
sign with four experimental treatments and 10
replications. Beds were separated by a drainage
ditch ~7.6 m in width. In trial two, 48 single tree
plots were arranged on two row beds in a com-
pletely randomized block design with eight exper-
imental treatments and six replications. In both
trials, an in-row variable tree buffer was also es-
tablished to prevent treatment interference.

Nematode Viability and Application

Two species of entomopathogenic nematodes,
S. riobrave and H. indica, formulated as a water-
dispersible granule (WDG) and a paste, respec-
tively, were used in these field trials. Regardless
of formulation, nematodes were kept cool (~20C)
both in storage and in the field prior to tank mix-
ing. Within 2-3 h of field application, the viability
(nematode mobility) of each preparation was de-
termined microscopically by counting the number
of mobile and dead infective juveniles (IJ's) in a
fixed number of fields at 60x magnification. Sam-
ples of nematode preparations used for viability
determination were held for a minimum of 2 h
with and without aeration before counting. In
both trials, viability of the water-dispersible gran-
ules of S. riobrave averaged only 57.1 and 46.2%,
respectively, and therefore, an adjustment in
quantity of preparation was made to achieve the
desired field rate for experimentation. In trial


December 2002







Scientific names italics only


two, the viability of the paste formulation ofH. in-
dica averaged 94.7% and no adjustments were
necessary to achieve the desired field rate.
Since the WDG formulation of S. riobrave used
in trial one had poor viability, infectivity (viru-
lence) of the preparation was compared to an in
vivo laboratory culture that was passed through
Diaprepes six times and an untreated control. The
bioassay procedure was identical to that de-
scribed by Shapiro & McCoy (2000a). Eighth in-
star D. abbreviatus were obtained from the
USDA-ARS Horticultural Laboratory (Fort
Pierce, FL). One laboratory assay was performed
in 50-dram plastic containers filled with Candler
sand with soil moisture of about 8% by weight. A
single larva was placed on the bottom of each con-
tainer prior to adding sand, then 500 IJ's were ap-
plied to the soil surface. The experiment was
arranged in a randomized design with each treat-
ment replicated 10 times. The experiment was
conducted at 24C for 10 d post-inoculation. Nem-
atode parasitism was confirmed via microscopy.
Control parasitism was 0%, in vivo culture para-
sitism 40%, and WDG parasitism 80%, suggesting
that the viable nematodes in the preparation
were highly infectious.
For nematodes used in trial two, a similar lab-
oratory assay was conducted and compared the
WDG formulation of S. riobrave, the paste formu-
lation ofH. indica, an in vivo laboratory culture of
S. riobrave that had been passed through Di-
aprepes 16 times, and an untreated control. In
this case, 30 replicates were conducted per treat-
ment. Larval mortality in the control was 3.3%,
whereas it was 63.3% for the paste formulation,
50.0% for the WDG, and 53.3% for the in vivo cul-
ture.
In trial one, S. riobrave was applied to the soil
beneath 10 trees at rates 0, 22, 54, and 108 IJ's/
cm2 on 16 April 2000, from 3:30-6:00 p.m. under
overcast skies. Water-dispersible granules were
pre-mixed in 1 liter of water and the appropriate
volume of nematode suspension then added to the
tank of a 50-liter electric field sprayer (Chemical
Containers, Lake Wales, FL) equipped with a
hand-held spray boom with two flat-fan nozzles
set apart by 30.5 cm (R and D Sprayers, Opelou-
sas, LA). Nematodes were uniformly applied in
1.9 liters of water per tree at 20 psi. Irrigation
was applied to all plots prior to nematode applica-
tion to assure adequate soil moisture to a depth of
30 cm. Soil temperature ranged from 22-24C at a
depth of 10 cm. Irrigation was applied again for 3
h the following day.
In trial two, S. riobrave and H.indica were ap-
plied at 0, 11, 54, and 108 IJ's/cm2 on 5 October
2000, from 6:00-9:00 p.m. under clear skies. Soil
moisture and temperature fell within the range of
those for trial one. Both WDG and paste on
sponge formulations of S. riobrave and H. indica,
respectively, were pre-mixed in 1 liter of water


and the appropriate volume of nematode suspen-
sion then added to the spray tank. Prior to and af-
ter nematodes were applied to the soil, irrigation
was applied for about 3 h to assure soil moisture
in the top 30 cm and again for 1 h the following
day.

Field Efficacy

In trial one, larval suppression in the soil
rhizosphere was determined at 23 + 1 d post-
treatment using a tree removal-soil sampling pro-
cedure. Initially, trees were topped using a chain
saw, then the roots along with the surrounding
soil were removed using a backhoe. Most of the
soil adhering to the roots was removed by shaking
and/or probing with a shovel. The soil within the
root crown was generally wet and compact, while
the surrounding soil was moist and easy to pro-
cess. Using a shovel, soil from the roots and be-
neath the tree was then placed in buckets for
subsequent sieving. Approximately 0.4 m3 of soil
was collected per tree to a depth of 30 cm accord-
ing to the procedures of Duncan & McCoy (1996).
All developmental stages ofD. abbreviatus except
larvae less than fifth instar were visually detect-
able and recovered from the soil using a motor-
driven shaker and 0.64-cm mesh sieve. The num-
ber of larvae, pupae, and adults were recorded per
tree. All larvae exhibiting normal behavior were
recorded as live. Each dead larvae was placed in a
disposable Petri dish (50 x 9 mm) on a moistened
filter paper. Cadavers were examined microscopi-
cally every other day for 7 d to detect characteris-
tic changes typical of bacterial, fungal, or
nematode infection. Differences in mean larval
population density between treatments were
tested on square root transformed data by analy-
sis of variance.
In trial two, larval suppression in the soil
rhizosphere was determined at 26 1 d post-
treatment using the previously described sam-
pling procedure. Soil moisture in relation to the
tree was similar to trial one, except where the
sandy layer appeared at the surface in Block F
causing low soil moisture within a small area
within the grove. The experiment was analyzed
using a three-way analysis of variance for nema-
tode species, application rate, and block (SAS In-
stitute, Cary, NC). Larval counts were
transformed using a square root transformation
prior to analysis. Because of the obvious differ-
ence in soil texture for Block F, data were ana-
lyzed with and without the block included.
In addition to estimating differences in native
populations of Diaprepes among treatments by
tree removal, larval-baited traps were used to
measure post-treatment parasitism by nema-
todes. Our intention here was to determine if lev-
els of larval parasitism by nematodes were
comparable to changes in wild larval populations.







Florida Entomologist 85(4)


In the field, a hand held auger was used to make
a circular hole in the soil beneath the tree canopy
midway between the trunk and canopy margin to
a depth of 30 cm for insertion of a baited cylindri-
cal cage (McCoy et al. 2000). The cage made from
an in-line liquid filter (7 x 3 cm diam.) with stain-
less steel screen (mesh size 225) was filled par-
tially with excavated soil. Then a single 8th instar
larva of Diaprepes produced on synthetic diet in
the laboratory was placed in the cage in the soil.
Additional soil was then added to fill the cage be-
fore capping. In trial one, four traps per tree were
buried at the compass points beneath 10 trees (n =
40) where they remained for 7 d. Within 12 h of re-
trieval from the soil, each cage was opened and re-
covered larvae examined for nematode infection.
Healthy and dead larvae were processed and diag-
nosed for parasitism in the manner described for
wild larval collections. This procedure was con-
ducted at 1, 2, and 3 weeks post-treatment. In
trial two, two traps per tree (n = 12) were buried in
the above manner and the procedure was con-
ducted at 1 week pre-treatment and 1, 2, and 3
weeks post-treatment. For each field test, a con-
tingency table analysis using Chi-square test
(SAS Institute, Cary, NC) was performed to com-
pare statistically the effect of field rates of nema-
todes on larval survival and parasitism in the soil.

RESULTS

Trial One

In the spring trial, the density of larvae (> 5th
instar) recovered from the soil (0.4 m3/tree) via
sieving was variable but quite high, ranging from
11 to 73 with a mean of 35.8 + 20.5in the control
(Table 1). A few scattered pupae, usually encased
in soil, and adults were also recovered at the time
of tree extraction. Although larval location in the
soil was not quantified, they were more prevalent
in the root crown in close proximity to the roots,
often lodged in compact soil surrounding the


crown roots or in association with moist soil at
any depth to 30-40 cm. However, all developmen-
tal stages were recovered from dry soil. Dead and
diseased larvae and adults were rarely observed.
However, the sieving process was most likely de-
structive to cadavers. Arthropod predators were
rare with only an occasional fire ant mound de-
tected. The total root system on all trees was se-
verely damaged by larval feeding over time, and
many trees were infected with root rot diseases.
Trees were virtually devoid of fibrous roots.
As shown in Table 1, there was no significant
difference (P = 0.316) in larval density between
the different rates of Bio Vector 355 and the con-
trol according to a one-way analysis of variance.
The greatest number of late instar larvae were re-
covered from the highest rate of nematodes. Lar-
val density among trees was variable (pooled S.D.
= 1.337).
No difference in larval survival of caged 8th in-
stars of D. abbreviatus was found among treat-
ments after exposure for 1 week at 7 d (P = 0.24)
(Table 2), 14 d (P = 0.09), or 21 d (P = 0.31) post-
application. From all treatments, 34, 20, and 9
larval cadavers, respectively, were recovered from
baited cages buried for 7 d at 4, 14, and 21 d post-
application. All remaining larvae recovered from
the different treatments were healthy. Nematodes
recovered from cadavers were identified as bacte-
rial feeding rhabditids only.

Trial Two

The density of larvae (> 5th instar) recovered
from the soil via sieving in the fall trial was also
high, but numerically lower than the spring. Lar-
val recovery from all treatments ranged from 5 to
50 per tree with a mean of 17.8 10.1 (n = 50). In-
terestingly, the different developmental stages of
D. abbreviatus recovered from the soil in the fall
trial were similar to those found in the spring,
that is, mostly mid-instar larvae, a few scattered
pupae, and adults. Root systems of all trees sam-


TABLE 1. LARVAL, PUPAL, AND ADULT RECOVERY OF DIAPREPES ABBREVIATUS FROM THE RHIZOSPHERE AFTER 25 D EX-
POSURE FOLLOWING TREATMENT WITH DIFFERENT RATES OF STEINERNEMA RIOBRAVE (BIO VECTOR 355) IN
THE SPRING AT POINCIANA, FLORIDA.

Live insects recovered from all trees

Treatment Rate IJ'/scm2 Larvae Pupae Adults Mean no larvae/0.4cm3 + SDA

Control 358 7 7 35.8 + 20.5
Bio Vector 22 443 17 8 44.3+ 14.1
Bio Vector 54 375 13 7 37.5+ 14.1
Bio Vector 108 485 5 4 48.5 + 12.3
F = 1.22,
P = 0.316

Means based on 10 single tree replication/treatment.


December 2002







Scientific names italics only


TABLE 2. CONTINGENCY TABLES, CHI-SQUARE ANALYSIS FOR SURVIVING CAGED 8TH INSTAR LARVAE OF DIAPREPES AB-
BREVIATUS AFTER 1 WEEK EXPOSURE TO FIELD SOIL TREATED WITH DIFFERENT RATES OF STEINERNEMA RI-
OBRAVE (BIO VECTOR 355) IN THE SPRING AT POINCIANA, FLORIDA.

Mean % larval survival, days post-treatmenta

Treatment Rate IJ's/cm2 7 14 21

Control 50.0 72.4 90.4
Bio Vector 22 62.1 85.5 78.3
Bio Vector 54 45.6 64.9 85.5
Bio Vector 108 45.0 70.7 80.0

Chi-square value 4.20 6.42 3.59
Probability 0.24 0.09 0.31
n.s. n.s. n.s.

Means based on 15 single tree replicates per treatment; 4 cages/replicate.


pled were severely damaged by weevils and
showed root rot symptoms.
The effect of the different rates of Bio Vector
355 and GrubstakeTM 100 on the reduction of lar-
vae of D. abbreviatus is presented in Fig. 1. The
overall analysis of variance produced highly sig-
nificant results (3-way ANOVA, F = 3.56, df = 12,
35, P = 0.0016). However, neither the main effect
for nematode species nor for application rate was
significant (nematode species, F = 2.83, df = 1, 35,
P = 0.1013; application rate, F = 1.43, df = 3, 35, P


30 ]


25 -2


S 20

J +
15
0

aO- 10
E
z
Z


= 0.2510), whereas the main effect for blocks was
highly significant (F = 6.98, df= 5, 35, P = 0.0001).
The species by dose interaction was also not sig-
nificant (F = 0.24, df = 3, 35, P = 0.8707).
The data were also analyzed after pooling the
results for the three application rates within spe-
cies. Again, the overall analysis of variance pro-
duced a highly significant result (3-way ANOVA,
F = 5.08, df = 8, 39, P = 0.0002). However, the
main effects for nematode species and for applica-
tion rate failed to reach significance (nematode



H Steinernema riobrave

U Heterorhabditis indica


I


Nematode rate/cm 2
Fig. 1. Live larval recovery of Diaprepes abbreviatus from the rhizosphere after 26 d exposure following treat-
ment with different rates of Steinernema riobrave (Bio Vector 355) and Heterorhabditis indica (GrubstakeM 100)
in the fall at Poinciana, FL.


-


-







Florida Entomologist 85(4)


species, F = 3.06, df = 1, 39, P = 0.0882; applica-
tion rate, F = 1.68, df = 1, 39, P = 0.2021), whereas
the main effect for blocks was highly significant
(F = 7.15, df = 5, 39, P = 0.0001). The species by
dose interaction was also not significant (F = 0.30,
df = 1, 39, P= 0.5874).
As previously mentioned, Block F produced ex-
tremely low numbers of larvae (range, 0-26;
mean, 6.5) in nearly all treatments. Therefore, we
repeated the original analysis but omitted the ex-
treme replicate. In this case, the overall analysis
of variance failed to achieve significance (3-way
ANOVA, F = 1.88, df = 11, 28, P = 0.0862). How-
ever, repeating this analysis without this extreme
replicate and after pooling the three lowest appli-
cation rates produced a significant result (3-way
ANOVA, F = 2.98, df = 7, 32, P = 0.0158). None-
theless, again, the main effect for nematode spe-
cies was not significant (F = 0.81, df = 1, 32, P =
0.3738) and the main effect for application rate
narrowly missed significance (F = 3.45, df = 1, 32,
P = 0.0723). The main effect for blocks was highly
significant (F = 4.15, df = 4, 32, P = 0.0080). The
species by dose interaction were also not signifi-
cant (F = 0.16, df = 1, 32, P = 0.6960).
In the fall field trial, larval survival of caged
8th instar larvae of D. abbreviatus following 7 d
exposure to pre-treated soils ranged from 81.8 to
100% (Table 3) and from 66.7 to 100% (Table 4).
No significant difference was found among treat-
ments receiving either S. riobrave (P = 0.58) or H.
indica (P = 0.115). Larval survival of D. abbrevia-
tus after 7 d exposure to soil treated with differ-
ent rates of S. riobrave at 7, 14, and 21 d post-
treatment was significantly lower for the highest
rate of S. riobrave at 7 d (P = 0.016), while no dif-
ference between treatments was found at 14 (P =
0.363) and 21 d (P = 0.279) (Table 3). Larval sur-
vival ofD. abbreviatus following exposure for 7 d
to soil treated with different rates ofH. indica at
7, (P = 0.279) 14, (P = 0.271), and 21 d (P = 0.271)
post-treatment was not significantly different


from the control (Table 4). Nematodes recovered
from cadavers were identified by the junior au-
thor. Twelve infected larvae produced Stein-
ernema sp. close to S. riobrave, 10 infected larvae
had H. bacteriophora, 6 infected larvae had Het-
erorhabditis sp., and several Cephalobus sp. (bac-
terial feeders) were also recovered.

DISCUSSION

Spring and fall applications of S. riobrave and
H. indica at commercial rates (10-20 IJ's/cm2) and
higher (54-108 IJ's/cm2) failed to reduce larval
populations and increase nematode parasitism
appreciably in the soil. When compared to previ-
ous field trials, these data further substantiate
the broad variability in field efficacy experienced
by previous researchers when applying ento-
mopathogenic nematodes as biopesticides to dif-
ferent citrus groves (Duncan et al. 1999, McCoy &
Duncan 2000).
Variation in efficacy can be caused by multiple
factors relating to the nematode, its host, and the
environment (Kaya & Gaugler 1993). A number of
contributing factors such as larval age, soil tem-
perature, nematode virulence, culture method,
and soil characteristics have been recognized in
experimentation with entomopathogenic nema-
todes as biological control agents of Diaprepes in
citrus soils (Shapiro & McCoy 2000a, 2000b,
2000c; Shapiro et al. 1999). In addition, sampling
methods for assessing nematode efficacy in the
field have differed widely among researchers and
no doubt have contributed somewhat to the vari-
ation in larval control (Bullock & Miller 1994,
Duncan & McCoy 1996, McCoy et al. 2000).
Recent field and microcosm experiments, de-
signed to determine the effect of soils of different
composition and texture on nematode efficacy,
strongly suggest that field failures reported
herein were soil-related (Duncan et al. 2001).


TABLE 3. CONTINGENCY TABLE, CHI-SQUARE ANALYSIS FOR SURVIVING CAGED 8TH INSTAR LARVAE OF DIAPREPES AB-
BREVIATUS AFTER 1 WEEK EXPOSURE TO FIELD SOIL TREATED WITH DIFFERENT RATES OF STEINERNEMA RI-
OBRAVE (BIO VECTOR 355) IN THE FALL AT POINCIANA, FLORIDA.

Mean % larval survival, day post-treatmenta

0 7 14 21

Control 100.0 100.0 a 90.9 83.3
Bio Vector 11 81.8 100.0 a 83.3 100.0
Bio Vector 54 91.7 91.7 a 75.0 100.0
Bio Vector 108 90.9 63.6 b 60.0 83.3

Chi-square value 1.97 10.26 3.19 3.84
Probability 0.58 0.016* 0.363 0.279
n.s. n.s. n.s.

Means based on 12 cages/treatment.


December 2002







Scientific names italics only


TABLE 4. CONTINGENCY TABLE, CHI-SQUARE ANALYSIS FOR SURVIVING CAGED 8TH INSTAR LARVAE OF DIAPREPES AB-
BREVIATUS AFTER 1 WEEK EXPOSURE TO FIELD SOIL TREATED WITH DIFFERENT RATES OF HETERORHABDITIS
INDICA (GRUBSTAKETM 100) IN THE FALL AT POINCIANA, FLORIDA.

Mean % larval survival, day post-treatmenta

Treatment Rate IJ's/cm2 0 7 14 21

Control 66.7 83.3 100.0 100.0
Grubstake 11 100.0 91.7 100.0 100.0
Grubstake 54 90.9 83.3 83.3 83.3
Grubstake 108 72.7 60.0 91.7 91.7

Chi-square value 5.92 3.74 3.91 3.911
Probability 0.115 0.291 0.271 0.271
n.s. n.s. n.s. n.s.

Means based on 12 cages/treatment.


When soil (entisol type) from a deep sandy ridge
grove (Lake Alfred) with a percent sand:silt:clay
ratio of 97.6:1.5:0.9 was compared to sandy clay
loam soil (alfisol type) from our experimental site
(Poinciana) with a ratio of 68.8:11.8:19.4, S. rio-
brave applied at 20 IJ's/cm2 killed 70-80% of the
larvae ofD. abbreviatus buried to a depth of 30 cm
in the sandy soil (Lake Alfred), but only 4-17% of
the larvae in sandy clay loam soil (Poinciana) sug-
gesting that higher clay soil with finer texture re-
duced host contact or affected the infection
process.
This marked difference in nematode efficacy
between sandy and sandy clay loam soils is sug-
gested in the published literature. For example,
in two groves on the central ridge with a deep
sandy soil (entisol type), S. riobrave applied at
108 IJ's/cm2 reduced larval populations by 75-
90% after three weeks (Duncan & McCoy 1996,
Duncan et al. 1996). Both tests were evaluated in
the same manner as these trials using the tree re-
moval/soil sieve methodology. In another series of
field trials conducted in a flatwoods grove near Ft.
Pierce with Pineda sandy clay loam soil, larval
parasitism by S. riobrave ranged from 40-45%
when rates of 54-108 IJ's/cm2 were applied, sug-
gesting soil-related inhibition (McCoy et al.
2000).
Shapiro et. al. (2000) measured virulence and
persistence of S. riobrave and H. bacteriophora in
Marl (high silt + clay), ridge (entisol, sandy), and
coastal flatwoods (spodosol, sandy clay loam) soils
in the laboratory. Although both nematode spe-
cies were virulent to D. abbreviatus larvae in all
soils, both virulence and persistence were greater
in the heavier Marl soil and virulence was greater
in spodosol compared to entisol soil. These labora-
tory data appear contradictory to field results.
However, physical properties of the soil were not
a factor. Shapiro et al. (2000) suggest that the
chemical composition of the soils is not a deter-
rent to parasitism but physical properties of the


soil, such as structure, compaction, etc. might
contribute to the variation in field efficacy.
In recent microcosm studies comparing the ef-
ficacy ofS. riobrave at a rate of 20 IJ's/cm2 in eight
autoclaved soils from citrus groves including
Poinciana, Duncan et al. (2001) found that larval
mortality of D. abbreviatus was positively corre-
lated with the proportion of sand in the soils, but
was inversely related to the percentage of fine
sand. The strongest correlation with efficacy was
with percentage of medium and coarse sand in
soils. Both nematode emergence from the cadaver
and recycling in cadavers were favored coarse
sandy soils.
The importance of soil texture in relation to
soil compaction as determined by Duncan et al.
(2001) is supported by field observations we made
on soil compaction within the tree rhizosphere at
the time of tree removal. Soil surrounding the
roots was very fine in texture resulting in extreme
compaction on the roots and within the rhizo-
sphere. Soil was so compact within the root crown
of the tree, it was virtually impossible to remove
with a probe. When larvae adjoining the roots
were removed with a probe, invariably they were
healthy suggesting the soil was so compact nem-
atode penetration of the soil was infrequent.
As previously mentioned in the methods, nem-
atode mobility and viability of WDG formulation
of S. riobrave was generally lower in these studies
compared to formulations from earlier field stud-
ies (Duncan & McCoy 1996; Duncan et al. 1996).
It might be argued that nematode vigor was a fac-
tor in explaining poor field efficacy in these trials.
Although this could be true for S. riobrave, it can-
not explain our results with H. indica, where
nematode viability was excellent.
Both larvae used in baited cages to measure
nematode parasitism (8th instar) and the larval
instars recovered from the native soil (6th-10th
instar) fall within the age group (i.e., 100 d old)
reported by Shapiro et al. (1999) as being least







Florida Entomologist 85(4)


susceptible to nematode infection by both S. rio-
brave and H. indica. In view of their findings, it is
reasonable to assume that host age can influence
nematode parasitism in the field.
The results of these field studies supported by
the studies of Duncan et al. (2001) pose important
implications relating to the biological control of
larvae of D. abbreviatus with entomopathogenic
nematodes in Florida citrus. Foremost, soil char-
acteristics appear to be important determinants
of field efficacy. Current nematode products ap-
pear most efficacious in deep sandy soils common
to the central ridge of Florida; however, efficacy is
affected substantially by different soils, particu-
larly the sandy clay loams. The issue of optimal
rate appears variable and is likely influenced by
host age and edaphic factors. Finally, further re-
search is warranted on nematode species selec-
tion and the dynamics of edaphic conditions in
relationship to field performance.


ACKNOWLEDGMENTS

This research was supported by the Florida Agricul-
tural Experiment Station, a grant from the Florida Cit-
rus Production Research Advisory Council Grant No.
942-18E and approved for publication as Journal Series
No. R-08331.


REFERENCES CITED

ADAIR, R. C. 1994. Four year field trial of entomopatho-
genic nematodes for control ofDiaprepes abbreuiatus
in a flatwoods citrus grove. Proc. Florida State Hort.
Soc. 107: 63-68.
BEAVERS, J. B., D. T. KAPLAN, AND C. W. McCOY. 1983.
Natural enemies of subterranean Diaprepes abbre-
viatus (Coleoptera: Curculionidae) larvae in Florida.
Environ. Entomol. 12: 840-843.
BULLOCK, R. C., AND R. W. MILLER. 1994. Suppression
of Pachnaeus litus and Diaprepes abbreviatus (Co-
leoptera: Curculionidae) adult emergence with
Steinernema carpocapsae (Rhabditida: Steinerne-
matidae) soil drenches in field evaluations. Proc.
Florida State Hort. Soc. 107: 90-92.
BULLOCK, R. C., R. R. PELOSI, AND E. E. KILLER 1999.
Management of citrus root weevils (Coleoptera: Cur-
culionidae) on Florida citrus with soil-applied ento-
mopathogenic nematodes (Nematoda: Rhabditida)
Florida Entomol. 82: 1-7.
DOWNING, A. S., S. G. ERICKSON, AND M. J. KRAUS.
1991. Field evaluation of entomopathogenic nema-
todes against citrus root weevils in Florida citrus.
Florida Entomol. 74: 584-586.
DUNCAN, L. W., AND C. W. MCCOY. 1996. Vertical distri-
bution in soil, persistence, and efficacy against citrus
root weevil of two species of entomogenous nema-
todes. Environ. Entomol. 25: 174-178.
DUNCAN, L. W., C. W. MCCOY, AND A. C. TERRANOVA.
1996. Estimating sample size and persistence of en-
tomogenous nematodes in sandy soils and their effi-
cacy against the larvae of Diaprepes abbreviatus in
Florida. J. Nematol. 28: 56-67.


DUNCAN, L. W., D. I. SHAPIRO, C. W. MCCOY, AND J. H.
GRAHAM. 1999. Entomopathogenic nematodes as a
component of citrus root weevil IPM, pp. 69-78. In S.
Polavarapu [ed], Optimal Use of Insecticidal Nema-
todes in Pest Management. Rutgers University
Press, New Brunswick, NJ.
DUNCAN, L. W., J. G. GENTA, AND J. ZELLERS. 2001. Ef-
ficacy of Steinernema riobrave against larvae of Di-
aprepes abbreviatus in Florida soils of different
texture. Nematropica 31: 130.
GRAHAM, J. H., C. W. MCCOY, AND J. S. ROGERS. 1996.
Insect-plant pathogen interactions: Preliminary
studies of Diaprepes root weevils injuries and Phy-
tophthora infections. Proc. Florida State Hort. Soc.
109: 57-62.
KAYA, H. K., AND R. GAUGLER 1993. Entomopathogenic
nematodes. Ann. Rev. Entomol. 38: 181-206.
KNAPP, J. L. 2000. Florida Citrus Pest Management
Guide. Florida Cooperative Extension Service SP 43,
pp. 15.1.4. University of Florida, Institute Food and
Agricultural Sciences, Gainesville, FL.
McCOY, C. W. 1999. Arthropod pests of citrus roots, pp.
149-156. In L. W. Timmer and L. W. Duncan [eds.],
Citrus Health Management. APS Press, St. Paul, MN.
McCOY, C. W., AND L. W. DUNCAN. 2000. IPM: An
emerging strategy for Diaprepes in Florida citrus,
pp. 90-104. In Diaprepes Short Course. Florida Agri-
cultural Experiment Station, Lake Alfred, FL.
McCoY, C. W., D. I. SHAPIRO, L. W. DUNCAN, AND
N. KHUONG. 2000. Entomopathogenic nematodes
and other natural enemies as mortality factors for
larvae of Diaprepes abbreviatus (Coleoptera: Curcu-
lionidae). Biol. Control 19: 182-190.
SCHROEDER, W. J. 1987. Laboratory bioassays and
field trials of entomogenous nematodes for control
of Diaprepes abbreviatus. Environ. Entomol. 16:
987-989.
SCHROEDER, W. J. 1990. Suppression ofDiaprepes abbre-
viatus (Coleoptera: Curculionidae) adult emergence
with soil application of entomopathogenic nema-
todes (Nematoda: Rhabditida). Florida Entomol. 73:
680-683.
SHAPIRO, D. I., AND C. W. MCCOY. 2000a. Effects of cul-
ture method and formulation on the virulence of
Steinernema riobrave (Rhabditida: Steinernema-
tidae) to Diaprepes abbreviatus (Coleoptera: Curcu-
lionidae). J. Nematol. 32: 281-288.
SHAPIRO, D. I., AND C. W. MCCOY. 2000b. Susceptibility
of Diaprepes abbreviatus (Coleoptera: Curculionidae)
larvae to different rates of entomopathogenic nema-
todes in the greenhouse. Florida Entomol. 83: 1-9.
SHAPIRO, D. I., AND C. W. MCCOY. 2000c. Virulence
of entomopathogenic nematodes to Diaprepes abbre-
viatus in the laboratory. J. Econ. Entomol. 93: 1090-
1095.
SHAPIRO, D. I., J. R. CATE, J. PENA, A. HUNSBERGER,
AND C. W. MCCOY. 1999. Effects of temperature and
host age on suppression of Diaprepes abbreviatus
(Coleoptera: Curculionidae) by entomopathogenic
nematodes. J. Econ. Entomol. 92: 1086-1092.
SHAPIRO, D. I., C. W. MCCOY, A. FARES, T. OBREZA, AND
H. DOU. 2000. Effects of soil type on virulence and
persistence of entomopathogenic nematodes in re-
lation to control of Diaprepes abbreviatus (Coleop-
tera: Curculionidae). Environ. Entomol. 29: 1083-
1087.


December 2002







Shelly & Pahio: Trapping Male Medflies


RELATIVE ATTRACTIVENESS OF ENRICHED GINGER ROOT OIL
AND TRIMEDLURE TO MALE MEDITERRANEAN
FRUIT FLIES (DIPTERA: TEPHRITIDAE)

TODD E. SHELLY AND ELAINE PAHIO
USDA-APHIS, P.O. Box 1040, Waimanalo, HI 96795

ABSTRACT

This study describes field experiments that compare the relative attraction of male Mediter-
ranean fruit flies (or medfly), Ceratitis capitata (Wiedemann), to trimedlure and ginger root
oil, which contains the natural attractant a-copaene. The ginger root oil was embedded in a
paste-like matrix, and the concentration of a-copaene was enhanced 20-fold above natural
levels (hence the term "enriched" ginger root oil or EGRO). In tests conducted in a mixed
fruit orchard in Waimanalo, Hawaii, 8 Jackson traps (4 baited with trimedlure, 4 baited with
enriched ginger root oil) were placed in a circle (40 m radius) about a central point from
which 500 males were released per replicate. Trap catches were scored 48 h after male re-
lease. In experiments using fresh (non-aged) lures, the amount of trimedlure used per trap
was constant (1 ml), but the amount of EGRO-containing paste used in traps was 1, 10, or
20 drops. Significantly more males were captured in the trimedlure traps than the EGRO
traps over all doses of EGRO. Similar experiments conducted in a small citrus grove yielded
the same results. Additional experiments revealed that female medflies showed no attrac-
tion to either trimedlure- or EGRO-baited traps and that immature and mature males
showed equal, short-range attraction to trimedlure and EGRO-baited traps.

RESUME

Este studio describe los experiments de campo para comparar la atracci6n relative de los
machos de la mosca mediterranea de la fruta, Ceratitis capitata (Wiedemann) al trimedlure
y al aceite de la raiz de jengibre los cuales contienen un atrayente natural a-copaene. El
aceite de la raiz de jengibre fu6 embutido en una pasta a manera de matriz y la concentra-
ci6n de a-copaene fu6 mejorada 20-veces por encima del nivel natural (de aqui el t6rmino
"aceite de la raiz dejengibre "enriquecido" o EGRO por su sigla en ingles). En pruebas lleva-
das a cabo en huertos de frutas mezcladas en Waimanalo, Hawaii, 8 trampas de tipo Jackson
(4 con el cebo de trimedlure, 4 con el aceite de la raiz de jengibre enriquecido) fueron coloca-
das en un circulo (40 m de radio) alrededor de un punto central de donde liberaron 500 ma-
chos por cada replica. El contenido de las trampas fu6 contabilizado 48 h despu6s de liberar
los machos. En experiments usando atrayentes frescos (no viejos), la cantidad de trime-
dlure usado por trampa fu6 constant (1 ml), pero la cantidad de pasta embutida con EGRO
usada en trampas fu6 1, 10, 6 20 gotas. Una cantidad significativamente mayor de machos
fueron capturados en trampas de trimedlure que en las trampas de EGRO con las diferentes
dosis de EGRO. Experimentos similares llevados a cabo en huertos pequenos de citricos pro-
dujeron los mismos resultados. Experimentos adicionales revelaron que las hembras de la
mosca mediterranea no mostraron ninguna atracci6n a ninguna de las trampas con cebo de
trimedlure- o de EGRO y los machos maduros e inmaduros mostraron una atracci6n de corto
alcance igual hacia las trampas con cebo de trimedlure como las de EGRO.


Females of the Mediterranean fruit fly, Ceratitis
capitata (Wied.) (medfly), may lay several hundred
eggs over their lifetime and infest a wide variety of
fruits and vegetables (Christenson & Foote 1960).
Given this high intrinsic rate of population growth,
early detection of incipient medfly outbreaks is crit-
ical for successful suppression or eradication. Fol-
lowing intensive screening of potential attractants
in the 1950's (Beroza & Green 1963), trimedlure,
tert-butyl 4 (and 5)-chloro-trans-2-methylcyclohex-
ane-1-carboxlyate, emerged, and still prevails, as
the standard attractant used to detect and monitor
medfly populations (Beroza et al. 1961).
Despite its wide use, however, trimedlure is
not the most attractive compound known for male


medflies. In the aforementioned screening efforts,
botanically derived compounds were tested, and
oil from the seeds of the angelica flower, Angelica
archangelica L. was found to be highly attractive
to C. capitata males. The active components were
subsequently identified as a-copaene and a-ylan-
gene, two structurally related tricyclic sesquiter-
penes (Guitto et al. 1972). Field tests (Flath et al.
1994a,b) revealed that male medflies displayed
greater attraction to a-copaene than a-ylangene
and that a-copaene was actually more attractive
than trimedlure (when presented in equal vol-
umes). a-copaene was subsequently identified as
a minor component in the essential oils of many
host plants of C. capitata, including orange, Cit-







Florida Entomologist 85(4)


rus sinensis L. MacLeod et al. 1988, Nishida et al.
2000), guava, Psidium guajava L. (MacLeod & De
Troconis 1982, Oliveros-Belardo et al. 1986), and
mango, Mangifera indica L. (Koulibaly et al.
1992).
Although attractive, a-copaene is not used in
medfly detection programs, because 1) it occurs in
very low concentrations in plants, making extrac-
tion impractical, and 2) it has a complex chemical
structure, making synthesis laborious and expen-
sive (Flath et al. 1994b). Ginger root, Zingiber of-
ficinalis Roscoe, oil is an inexpensive and readily
available source of a-copaene, and a recently de-
veloped distillation procedure can greatly in-
crease the concentration of this compound. a-
copaene comprises 8% of this so-called enriched
ginger root oil (hereafter EGRO) compared to only
0.4% in commercially available oil (Citrus and Al-
lied Essences, Ltd., Lake Success, NY; F. Webster,
personal communication).
The primary purpose of the present study was
to compare trap captures of male medflies to tri-
medlure- and EGRO-baited Jackson traps in field
tests. Similar tests were conducted in a mixed
fruit orchard and a citrus grove to assess potential
inter-habitat differences. At both sites, compari-
sons were made using different relative amounts
of the attractants. As noted below, the amount of
trimedlure used per trap was typical of that used
in ongoing survey and detection programs. As
such, the field experiments were designed to com-
pare attractiveness, not between equal amounts
of the active constituents of trimedlure and
EGRO, but rather between presently used doses
of trimedlure and a range of'realistic' (in terms of
practical application) doses of EGRO. In addition
to field tests, we also examined potential age-de-
pendent response of male medflies to trimedlure-
and EGRO-baited traps using field-caged host
trees.

MATERIALS AND METHODS

Medflies

The flies used in this study were from a labora-
tory colony started with 300-400 adults reared
from coffee, Coffea arabica L., berries collected
near Haleiwa. The colony was 5-7 generations re-
moved from the wild when used for the Waim-
analo experiments and 11 generations removed
when used for the Pearl City experiments (see be-
low). Standard rearing procedures were employed
(Tanaka et al. 1969), and prior to release adults
were fed a sugar/protein (yeast hydrolysate) mix-
ture (3:1 by volume). Adults were separated
within 24 h of emergence, well before they at-
tained sexual maturity at 6-8 d of age (T. E. Shelly,
unpublished data). When used in the field experi-
ments, males were 9-15 d old, and females were 9-
14 d old.


Field Experiments Waimanalo

Most of the field experiments were conducted
at the University of Hawaii Agricultural Experi-
ment Station in Waimanalo in a mixed fruit or-
chard that contained orange, guava, and mango
trees. Jackson traps, containing a sticky insert,
were placed singly in the canopies of 8 trees (2 m
above ground) arranged in a circle (radius 40 m)
about a central tree, which served as the release
point. The Jackson traps contained trimedlure in
4 trees and EGRO in the remaining 4 trees (see
below). The lures were applied to the traps at the
laboratory (4 km from the study site), and the
traps were then immediately transported to and
placed in the test trees. At a given tree, the bait
used was alternated between successive repli-
cates, and for a given replicate, adjacent test trees
contained different baits.
For the trimedlure-baited traps in all experi-
ments, 1 ml of the lure was applied to a cotton
wick (2.5 cm long), which was then placed in a
perforated, plastic basket that was, in turn, sus-
pended within the Jackson trap. This dose is V2 of
that used in area-wide survey and detection pro-
grams (California Department of Food and Agri-
culture 1995). Trimedlure is composed primarily
of eight isomers of which one (designated C) is the
most attractive (McGovern et al. 1987), and 1 ml
of trimedlure contains approximately 0.358 g of
isomer C (trimedlure purity x concentration of the
C isomer x weight of 1 ml trimedlure = 0.965 x
0.399 x 0.929g; J. Knapp, personal communica-
tion). For the EGRO-baited traps, the amount of
lure used varied between experiments (see below)
and was placed on a small piece of aluminum foil
directly on the sticky insert within the Jackson
trap. The oil was embedded within a paste-like
matrix (Last CallTM, IPM Technologies, Inc.), com-
prised primarily of tinuvin, that was applied with
a calibrated pump (1 drop of paste = 50 pl or
0.05g). EGRO comprised 20% of the paste (by
weight), a-copaene constituted 8% of the EGRO
(by weight), and thus one drop of paste contained
0.0008 g of a-copaene (J. McLaughlin, personal
communication). It should be noted that the dis-
tillation procedure used to increase the concen-
tration of a-copaene increases the concentration
of other sequiterpenes as well (e.g., a- and p-ylan-
gene; F. Webster, personal communication) and
that the combination of all sesquiterpenes is more
attractive to male medflies than a-copaene alone
(T. W. Phillips, personal communication).
Five hundred males were released per repli-
cate for all experiments. As wild flies were rare at
the study site, released flies were not marked, and
we assumed that all captured flies were from the
releases. Successive replicates were run a mini-
mum of 2 d apart to allow previously released flies
time to disperse from the test area. Ten replicates
were performed per experiment. Flies were re-


December 2002







Shelly & Pahio: Trapping Male Medflies


leased between 1000-1200 hours by placing 4
buckets (volume 5 liters and each containing 125
flies) on the ground beneath the release tree and
gently removing the screen cover from the bucket.
The buckets were not tapped or shaken, and the
flies exited the bucket on their own volition. Trap
catches were counted 2 d later. Field work at
Waimanalo was conducted during September-De-
cember, 2000, and daily minimum and maximum
temperatures ranged from 17-21 C and 24-28C,
respectively.
The number of males captured at trimedlure-
baited traps was compared with traps baited with
1 (50 pl), 10 (0.5 pl), or 20 (1 ml) drops of the paste
containing EGRO. When multiple drops of EGRO-
bearing paste were used, the individual drops
were placed close together but were not overlap-
ping. To investigate temporal decline in lure at-
tractiveness, male catches were compared
between trimedlure-baited traps and traps baited
with 1 drop of the EGRO-containing paste using
baits that had been aged 5 d prior to use (lures
were placed in Jackson traps and placed outdoors
in a covered area several km from the study site).
In a final experiment, the female attraction to tri-
medlure-baited traps and traps baited with 1 drop
of the paste containing EGRO was compared fol-
lowing the procedures outlined above for males.
Following the trimedlure-EGRO comparisons,
we ran 2 additional experiments (using the proto-
col described above) that examined the potential
attractiveness and repellency, respectively, of the
paste in which the EGRO was delivered. In the
first case, we compared male captures in 4 traps
baited with 1 drop of the EGRO-containing paste
versus traps baited with the one drop of paste to
which no EGRO was added. In the second, we
placed 0.5 ml of ginger root oil (Citrus and Allied
Essences, Ltd., Lake Success, NY) on wicks for all
8 traps. In 4 of the traps, we also placed 10 drops
of paste (lacking EGRO) on a piece of aluminum
foil fastened to the sticky insert, while in the re-
maining 4 traps, we placed the foil only and no
paste. Six replicates were run of each of these 2
experiments.

Field Experiments-Pearl City

To examine potential habitat differences in
lure attractiveness, a second set of field experi-
ments was conducted in a small citrus grove (1 ha)
at the University of Hawaii's Urban Garden Cen-
ter in Pearl City. Fieldwork at this site was con-
ducted during June-July, 2001, and daily
minimum and maximum temperatures ranged
from 22-26C and 30-33C, respectively. The pro-
tocol used was identical to that described above,
except that 1) trimedlure plugs containing 2 g of
trimedlure (Farma Tech International, Fresno,
CA), were used in place of liquid trimedlure and 2)
only 4 traps, 2 baited with trimedlure and 2


baited with EGRO, were used per replicate. Tri-
medlure -baited traps were compared only with
traps baited with 1 or 10 drops of the EGRO-con-
taining paste, and no experiments were run in-
volving aged baits or females. Eight replicates
were performed per experiment.

Field-Cage Experiments Waimanalo

Four experiments were conducted during June
- September, 2001, at Waimanalo using field-cages
(height 2.5 m, diameter: 3.0 m) that contained
rooted guava trees. In the first, we investigated
age-dependent attraction of males to trimedlure
by comparing trap catches of immature (1 d old)
and mature (9-13 d old) males. Groups of 100 im-
mature and 100 mature males were released be-
tween 0900-1100 hours in a field-cage containing
1 baited (treated) and 1 unbaited (control) trap,
and trap catches were recorded 24 h later. For the
purpose of identification, males were marked by
age group by cooling them for several minutes and
then placing a small dot of enamel paint on the
thorax. Immature males were marked in the late
afternoon of the day of adult emergence to allow
hardening of the exoskeleton; mature males were
marked 1-2 d before testing. Traps were rectangu-
lar pieces of white cardboard (9 by 16 cm) coated
on both sides with TanglefootTM and suspended in
the canopy (1.5 m above ground) with wire hooks.
A wick containing 1 ml of trimedlure was placed
in the center of one side of the treated trap, and a
cotton wick (without trimedlure) was applied to
the control trap. The trimedlure used had a deep
red color, and consequently we added a small
amount of diluted red food coloring (McCormick &
Co., Inc.) to the wick on the control trap to equal-
ize visual stimuli. The same trap sites were used
over all replicates, but the positions of baited and
control traps were alternated between successive
replicates. The second experiment examined age-
dependent response of males to EGRO following
the same procedures described above. In this case,
1 drop of EGRO-containing paste was placed on a
small piece of aluminum foil, which was then
placed in the center of one side of the trap. For the
control trap, aluminum foil was placed on the
trap, but no paste was applied. In the third and
fourth experiments, we investigated close-range
attraction of females to trimedlure and EGRO, re-
spectively. The same protocol was followed except
that only 1 group of 100 females (9-13 d old) was
released per replicate, and their numbers on
baited and control traps were compared. Seven
replicates were conducted for each of the 4 field-
cage experiments.

Statistical Analyses

Pairwise comparisons of trap catches were
made using the Mann-Whitney test (test statistic







Florida Entomologist 85(4)


T), a nonparametric equivalent of the Students t-
test. Multiple comparisons were made using the
Kruskal-Wallis (test statistic H), and if significant
variability was detected, the multiple comparison
Tukey test (test statistic q) was used to assess
pairwise differences. Nonparametric tests were
employed to avoid assumptions of normality and
equal variance for the sampled populations. Anal-
yses were conducted using SigmaStat Statistical
Software (Version 2.0).

RESULTS

Field Experiments Waimanalo

On average, the trimedlure-baited traps cap-
tured more males than the EGRO-baited traps in
all experiments (Table 1A). With fresh (non-aged)
baits, the trimedlure-baited traps captured ap-
proximately 1.6-2.6 times more males, on aver-
age, than did the EGRO-baited traps. When aged
baits were used, the trimedlure-baited traps
caught 19 times as many males as the EGRO
traps (experiment 4). The number of males cap-
tured in trimedlure-baited traps did not differ sig-
nificantly across the 4 experiments (H = 2.7, df =
3, P > 0.05), indicating that the 5-d aging period
did not reduce trimedlure's attractiveness. In con-
trast, male numbers in EGRO-baited traps varied
significantly among experiments (H = 23.4, df= 3,
P < 0.001), with the male catch for aged EGRO-


baited traps being significantly lower than those
recorded for any of the freshly-baited traps (P <
0.001 in all cases). Among traps having fresh
EGRO-containing paste, the number of males
captured varied independently of the amount of
paste used (H = 3.5, df = 2, P > 0.05).
Neither trimedlure nor GRO was attractive to
females. Not a single female was caught in any
trap over 10 replicates.
The paste used to deliver EGRO was not at-
tractive or repellant to male medflies. Similar to
experiment 1, traps baited with 1 drop of EGRO-
containing paste caught an average of 11.1
(range: 5-18) males per replicate. However, traps
baited with paste that lacked EGRO captured no
males at all over 6 replicates. In testing for poten-
tial repellency of the paste, we found no signifi-
cant difference in captured males between traps
with paste (x = 11.3; range: 8-18) versus traps
without paste (x = 10.7; range: 7-14; T = 40.0, P >
0.05) adjacent to the wick containing ginger root
oil.

Field Experiments Pearl City

Results obtained at Pearl City were similar to
those reported above for Waimanalo (Table 1B).
The trimedlure-baited traps, on average, cap-
tured significantly more males than did the
EGRO-baited traps. In relative terms, the differ-
ence between the baits was even more pronounced


TABLE 1. NUMBERS OF MALES CAPTURED IN TRIMEDLURE- OR EGRO-BAITED JACKSON TRAPS AT WAIMANALO AND
PEARL CITY STUDY SITES'.

Males/trap
Amount of
Experiment Baits aged? EGRO-containing paste Trimedlure GRO T

A. Waimanalo
1 no 50 l1 21.7A,a 8.3B,b 152.0***
(11-27) (3-17)
2 no 0.5 ml 18.5A,a 11.2B,b 143.0**
(11-26) (4-18)
3 no 1.0 ml 18.4A,a 11.2B,b 141.0*
(10-26) (4-16)
4 yes 50 pl 19.2A,a 1.8B,c 155.0***
(6-30) (0-4)

B. Pearl City
1 no 50 l1 24.1A,a 6.6B,b 98.0***
(11-37) (4-17)
2 no 0.5 ml 19.4A,a 7.7B,b 92.0**
(6-27) (2-9)

'In all experiments, 1 ml of trimedlure was used in the trimedlure-baited traps. Values represent mean numbers of males captured per replicate;
ranges are given in parentheses. Ten and 8 replicates were conducted for experiments conducted at Waimanalo and Pearl City, respectively. T values were
computed for Mann-Whitney tests comparing male captures for the 2 attractants within a given experiment (row). For a given location, values in the
same row followed by the same uppercase letter were not significantly different, and values in the same column followed by the same lowercase letter
were not significantly different following the Kruskal-Wallis or Tukey test (P = 0.05). Significance levels: *P < 0.05, **P < 0.01, ***P < 0.001.


December 2002







Shelly & Pahio: Trapping Male Medflies


at Pearl City, where the average trap catches were
3-4 times higher for trimedlure-baited traps than
EGRO-baited traps. Additionally, no difference in
male captures was detected between traps having
1 or 10 drops of the EGRO-bearing paste over the
two experiments (T = 75.0, P > 0.05). Male cap-
tures were also similar for the trimedlure-baited
traps across the 2 experiments (T = 78.0, P >
0.05).
Inter-habitat comparisons revealed that, for
trimedlure-baited traps, the number of males cap-
tured per trap did not differ significantly between
Waimanalo and Pearl City (T = 431.5, n,= 30, n2 =
16, P > 0.05). For the EGRO-baited traps, how-
ever, trap catches were significantly greater at
Waimanalo (x = 10 males/trap) than at Pearl City
(x = 6.5 males/trap; T = 245.5, n, = 30, n2 = 16, P <
0.05).

Field-Cage Experiments Waimanalo

No difference was detected in the number of
immature and mature males captured on treated
versus control traps for the experiments involving
trimedlure or EGRO (Table 2). Data pooled for im-
mature and mature males revealed that, on aver-
age, treated traps captured approximately 13
times as many males as control traps in a given
replicate for both trimedlure (80/6) and EGRO
(66/5). Consistent with the field experiments, the
average number of males captured per replicate
(age groups combined) was higher for trimedlure-
than EGRO-baited, although this difference was
not statistically significant (T = 64.0, P > 0.05). In
the third and fourth experiments, females showed
no short-range attraction to trimedlure or EGRO.
On average, the trimedlure-baited trap captured


2.7 (range: 0-6) females per replicate compared to
2.4 (range: 1-5) for the control trap (T = 54.5, P >
0.05). Similarly, the EGRO-baited trap captured
3.7 females per replicate compared to 2.7 females
for the control trap (T = 63.5, P > 0.05).

DISCUSSION

Based on field trapping tests conducted in 2
habitats, trimedlure-baited traps were more at-
tractive to male medflies than EGRO-baited
traps. This difference was evident over a wide
range of EGRO dosage: at Waimanalo, traps
baited with 1 ml of (non-aged) trimedlure caught
1.6 -2.6 more males than traps baited with 1-20
drops of EGRO-containing paste. Likewise, at
Pearl City traps baited with trimedlure plugs cap-
tured 4.0 and 2.6 times as many males as traps
baited with 1 and 10 drops of EGRO-containing
paste, respectively. At both sites, the relative con-
sistency in trap catches across a wide range of
EGRO doses was unexpected. Among EGRO-
baited traps, the number of males captured per
trap did not vary significantly among traps with
1-20 drops of paste at Waimanalo or between
traps with 1 or 10 drops of paste at Pearl City.
The difference in trap capture between the 2
attractants was even more dramatic when aged
baits were compared. For trimedlure, the number
of males trapped using aged baits did not differ
significantly from that trapped with fresh baits.
This result is consistent with previous studies
(McGovern et al. 1966; Nakagawa et al. 1981; Rice
et al. 1984) showing trimedlure maintains attrac-
tiveness for 1-6 weeks depending on local weather
conditions. In contrast, traps with EGRO-bearing
paste aged 5 days caught only about 10% as many


TABLE 2. NUMBERS OF 1-D OLD (IMMATURE) AND 9-13 D-OLD (MATURE) MALES CAPTURED IN CONTROL AND TREATED
(A-TRIMEDLURE; B-EGRO) PANEL TRAPS IN FIELD-CAGES AT WAIMANALO1.

Male Age

Trap type 1 day 9-13 days T
A. Trimedlure
Control 2.6A 3.0A 54.5
(0-5) (0-8)
Treated 42.8A 37.0A 59.5
(29-54) (21-58)

B. EGRO
Control 2.2A 3.1A 54.5
(0-7)
Treated 32.5A 34.6A
(24-42) (27-42)

Values represent mean numbers of males captured per replicate; ranges are given in parentheses. Seven replicates were performed for all field-cage
experiments. T values were computed for Mann-Whitney tests comparing captures for the 2 age groups within a given experiment (row). Values in the
same row followed by the same uppercase letter were not significantly different (P = 0.05).











males as traps baited with fresh paste. Thus, data
from both fresh and aged baits indicate that tri-
medlure is superior to EGRO as a detection and
monitoring tool in medfly control programs.
Although only 2 sites were involved, the field
experiments indicate, preliminarily at least, that
the attractancy of EGRO-baited traps may vary
more among different habitats than that of tri-
medlure-baited traps. Trap catches were similar
between the 2 sites for trimedlure-baited traps,
whereas a significantly higher number of males
was captured at Waimanalo than Pearl City for
the EGRO-baited traps. While this difference
could reflect the small sample of sites included, it
may also indicate differences in the 'aromatic' en-
vironment at the 2 sites that differentially af-
fected (interfered with) the effectiveness of the 2
baits. For example, volatiles emanating from the
citrus trees at Pearl City may have more closely
resembled (or mimicked) those given off from
EGRO-baited traps than trimedlure-baited traps.
If so, the citrus trees might have effectively
"swamped" or outcompeted the olfactory stimuli
of EGRO-baited traps and thus effectively less-
ened the attractiveness of this bait to male med-
flies.
Females were not attracted to either bait in the
field trials or the field-cage tests. The lack of fe-
male response has been documented previously
for trimedlure (Delrio & Zumreoglu 1983, Howse
& Knapp 1996) and a-copaene (Nishida et al.
2000). As with pure a-copaene, the non-attraction
of females to EGRO was unexpected, because a-
copaene, which is present in many medfly hosts
(see aforementioned references), has been consid-
ered a rendezvous stimulus that brings the sexes
together for mating (Nishida et al. 2001). Al-
though not attractive to females by itself, a-co-
paene may still affect the mating system of the
medfly by enhancing female response to the male
pheromone. Dickens et al. (1990) reported that
green leaf volatiles boost female response to male
medfly pheromone, and it seems likely that par-
ticular plant compounds, such as a-copaene, may
act in a similar way (see examples in Landolt &
Phillips 1997).
Although differing in their overall attractive-
ness, trimedlure and EGRO were both attractive
to immature and mature males. Initial experi-
ments (Shelly 2001) showed that mature male
medflies exposed to the aroma of ginger root oil
had a mating advantage over non-exposed males
in tests conducted 2 days after the exposure. Fol-
low-up tests (Shelly 2001) further revealed that 1-
day old males exposed to ginger root oil had a
mating advantage over non-exposed males in
tests conducted 8-10 days after exposure. Given
these findings, it was not surprising that male at-
traction to EGRO was age independent: attrac-
tion to an a-copaene source appears to confer an
immediate benefit to mature males and a delayed


December 2002


benefit to immature males. This explanation,
however, does not appear applicable for trimed-
lure. Although exposing mature males to trimed-
lure boosts their mating success, the effect is
short-lived, and mating enhancement was evi-
dent only within 24 h of exposure (Shelly et al.
1996). Although we have not exposed 1-day old
males to trimedlure, it appears unlikely that such
early exposure would affect mating performance a
week following exposure. Consequently, the at-
traction of immature males to trimedlure cannot
apparently be explained in the context of sexual
selection.

ACKNOWLEDGMENTS

We thank Roger Coralis and Dale Sato for permis-
sion to work at the Waimanalo and Pearl City sites, re-
spectively. We are also grateful to John McLaughlin,
Tom Phillips, and Fran Webster for information on gin-
ger root oil and a-copaene and Charmian Dang, Susan
Kennelly, Courtney Ishimura, Eric Rutka, and Mindy
Teruya for assistance in rearing and maintaining the
flies. The EGRO-containing paste was kindly supplied
by IPM Technologies, Inc., Portland, Oregon. Comments
by Don McInnis and Grant McQuate improved the pa-
per. We gratefully acknowledge the financial support of
the California Citrus Research Board for this research
(Agreement No. 5510-144).

REFERENCES CITED

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insect attractants. USDA-ARS, Agricultural Hand-
book No. 239. Washington, D.C.
BEROZA, M., N. GREEN, S. I. GERTLER, L. F. STEINER,
AND D. H. MIYASHITA. 1961. Insect attractants: new
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CHRISTENSON, L. D., AND R. H. FOOTE. 1960. Biology of
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DELRIO, G., AND A. ZUMREOGLU. 1983. Attractability
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DICKENS, J. C., E. B. JANG, D. M. LIGHT, AND A. R. AL-
FORD. 1990. Enhancement of insect pheromone re-
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FLATH, R. A., R. T. CUNNINGHAM, T. R. MON, AND J. O.
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FLATH, R. A., R. T. CUNNINGHAM, T. R. MON, AND J. O.
JOHN. 1994b. Male lures for Mediterranean fruitfly
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GUITTO, A., U. FORNASIERO, AND F. BACCICHETTI. 1972.
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HOWSE, P. E., AND J. J. KNAPP. 1996. Pheromones of
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Fly Pests: A World Assessment of their Biology and
Management. St. Lucie Press, Delray Beach, Florida.
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LANDOLT, P. J., AND T. W. PHILLIPS. 1997. Host plant in-
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HARDT. 1987. Attractiveness of trans-trimedlure and
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NAKAGAWA, S., E. J. HARRIS, AND I. KEISER 1981. Per-
formance of capilure in capturing Mediterranean
fruit flies in Steiner plastic or cardboard sticky
traps. J. Econ. Entomol. 74: 244-245.
NISHIDA, R., T. E. SHELLY, T. S. WHITTIER, AND K. Y.
KANESHIRO. 2000. a-copaene, a potential rendezvous
cue for the Mediterranean fruit fly, Ceratitis capi-
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AND V. ALBANO. 1986. A chemical study of the essen-
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1984. Weathering and efficacy of trimedlure dispens-
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paene-containing oils enhances mating success of
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1996. Trimedlure affects mating success and mate
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Florida Entomologist 85(4)


December 2002


POTENCY, SPECTRUM AND RESIDUAL ACTIVITY OF FOUR NEW
INSECTICIDES UNDER GLASSHOUSE CONDITIONS


JOSEPH A. ARGENTINE'2, RICHARD K. JANSSON34, W. ROSS HALLIDAY1'6, DOUGLAS RUGG36 AND CHRISTINE S. JANY37
'Ricerca, Inc., P.O. Box 1000, 7528 Auburn Road, Painesville, OH 44077

2Current Address: FMC Corportation, Box 8, Princeton, NJ 08088

3Merck Research Laboratories, Agricultural Research and Development, P.O Box 450
Hillsborough Road, Three Bridges, NJ 08887

4Current Address: Aventis Behring, 1020 First Avenue, King ofPrussia, PA 19406

5Current Address: Technology Partnership Practice, Battelle Memorial Institute
20445 Emerald Parkway Drive, Cleveland, OH 44135

6Current Address: Fort Dodge., P.O. Box 400, Princeton, NJ 08543

7Current Address: 563 Rusell Street, #40W, Elgin, IL 60120


ABSTRACT

The toxicities of four classes of insecticides, emamectin benzoate (avermectin), chlorfenapyr
(pyrrole), fipronil (phenylpyrazole), and tebufenozide (benzoylhydrazide) were compared us-
ing an artificial diet assay and a residual efficacy assay against several species of Lepi-
doptera. Emamectin benzoate was consistently the most toxic insecticide; it was 20- to
64,240-times more toxic than the other compounds tested. The LC,, values for emamectin
benzoate ranged from 0.0050 to 0.0218 ug/ml for six species of Lepidoptera. Similarly, chlo-
rfenapyr displayed consistent toxicity to all species, with LC,, values ranging from 1.9 to 4.6
ug/ml. The toxicities of fipronil and tebufenozide varied among the species tested. Fipronil
LC,, values varied 501-fold (range, 0.64 to 321.3 ug/ml), while tebufenozide toxicity varied
113-fold (range, 0.24 to 27.1 ug/ml) among species tested. In residual efficacy tests conducted
in the glasshouse, all compounds were effective (i.e., >90% mortality) at controlling Heliothis
uirescens on garbanzo bean at projected field rates and at 1/10 of projected field rates with
fipronil and emamectin benzoate. Emamectin benzoate, chlorfenapyr and tebufenozide were
effective at controlling Spodoptera exigua on sugar beet at projected field rates. However,
mortality with fipronil was reduced to 20% or less at 7 to 14 days after treatment. All com-
pounds at projected field use rates were effective against Trichoplusia ni on cabbage, al-
though tebufenozide was the only compound effective at 1/10 of projected field rate for 14
days after treatment. However, tebufenozide was ineffective against Plutella xylostella at
projected field use rates on cabbage while emamectin benzoate, chlorfenapyr, and fipronil
were effective. The potential of these compounds for arthropod pest management are dis-
cussed.

Key Words: Chlorfenapyr, emamectin benzoate, fipronil, tebufenozide, residual assay

RESUME

La toxicidad de cuatro classes de insecticides, emamectin benzoate (avermectin), chlorfena-
pyr (pyrrole), fipronil (phenylpyrazole), y tebufenozide (benzoylhydrazide)fueron compara-
das utilizando un ensayo con dieta artificial y un ensayo de eficacia de residue en contra de
various species de Lepidoptera. Emamactin benzoate fu6 consistentemente el insecticide
mas t6xico, (fu6 de 20- a 64,240 veces mas t6xico que los otros compuestos probados). Los va-
lores de LC0,para emamectin benzoate fueron de 0.0050 hasta 0.0218 ug/ml para seis espe-
cies de Lepidoptera. Similarmente, chlorfenapyr mostr6 toxicidad consistent para todos las
species, con valores de LC,,desde 1.9 hasta 4.6 ug/ml. Las toxicidades de fipronil y tebufe-
nozide varian entire las species probadas. Los valores de LC,, del Fipronil variaron 501-ve-
ces (de 0.64 hasta 321.3 ug/ml), mientras que la toxicidad de tebufenozide vario 113 veces (de
0.24 hasta 27.1 ug/ml), entire las species probadas. En pruebas de eficacia de residue lleva-
das a cabo en invernaderos, todos los compuestos fueron efectivos (i.e. >90% mortalidad) en
el control de Heliothis virescens en el garbanzo aplicados a la taza proyectada en el campo (o
sea a la misma concentraci6n estipulada para el area del campo) y fueron efectivos al 1/10







Argentine et al.: Potency of Four New Insecticides


de la taza proyectada del campo en el fipronil y el emamectic benzoate. Emamectin benzoate,
chlorfenapyr y tebufenozide fueron efectivos en controlar Spodoptera exigua en remolacha a
la taza proyectada del campo. Sin embargo, la mortalidad con fipronil fu6 reducida a 20% o
menos desde los 7 hasta los 14 dias despu6s del tratamiento. Todos los compuestos aplicados
a la taza proyectada del campo fueron efectivos contra Trichoplusia ni en repollo, aunque te-
bufenozide fu6 el unico compuesto efectivo a 1/10 de la taza proyectada del campo durante
los 14 dias despu6s del tratamiento.
Sin embargo, tebufenozide no fu6 efectivo contra Plutella xylostella a la taza proyectada del
campo en repollo mientras que emamectin benzoate, chlorfenapyr y fiprionil fueron efecti-
vos. Se discuten el potential de estos compuestos para el manejo de plagas artr6podos.


The insecticide market has been dominated by
the organophosphate, carbamate, and pyrethroid
classes of insecticides. Recently, a number of new
insecticide classes have been discovered and com-
mercialized. Chlorfenapyr, a mitochondrial un-
coupler (Black et al. 1994), is effective against
both Acarina and Lepidoptera (Lovell et al. 1990,
Wier et al. 1994, Ahn et al. 1996) in laboratory
and field tests. Fipronil, an antagonist of the
GABA-gated chloride channel (Bloomquist 1994),
has efficacy against a number of insect pests (Col-
liot et al. 1992, Burris et al. 1994, Hoy & Dunlap
1995). Emamectin benzoate is a second genera-
tion avermectin with superior activity against
lepidopterans compared with abamectin (Dybas
et al. 1989, Jansson & Dybas 1997). Tebufenozide,
an ecdysone-receptor agonist (Retnakaran et al.
1995), has demonstrated activity against many
lepidopterans (Chandler 1994, Smagghe & De-
gheele 1994, Ishaaya et al. 1995).
The purpose of this research was to compare
the potencies, spectrum, and residual effective-
ness of these compounds against a broad panel of
lepidopteran pests. These comparisons will help
to provide information on the potential strengths
and weaknesses of each compound in crop protec-
tion.

MATERIAL AND METHODS

Chemicals

Emamectin benzoate (Proclaim@ 0.16 EC) was
obtained from Merck & Co., Inc. (Rahway, NJ).
Tebufenozide (Confirm 2F) was obtained from
Rohm & Haas (Spring House, PA). Chlorfenapyr
(Rampage@ 10% EC) and fipronil (Ascend@ 5%
SC) were obtained commercially. These formula-
tions were marketed for use in cotton and were
presumed to be optimized.
Insect Strains. Tobacco budworm, Heliothis
virescens (F.), and soybean looper, Pseudoplusia
includes (Walker) were obtained from the
USDA, ARS, Southern Insect Management Labo-
ratory, Jamie Whitten Research Center, Stonev-
ille, MS. Diamondback moth, Plutella xylostella
(L.), beet armyworm, Spodoptera exigua (Hubner)
and cabbage looper, Trichoplusia ni (Hubner)
were obtained from Ecogen Co. (Langhorne, PA).


Fall armyworm, Spodoptera frugiperda (J. E.
Smith) were obtained from the USDA ARS, Insect
Biology and Population Management Research
Laboratories, Tifton, GA. Plutella xylostella eggs
were shipped on artificial diet to Ricerca, Inc.
(Painesville, OH) and held at 24 + 2C and 50 +
20% RH until needed. Heliothis virescens and S.
exigua eggs were surface sterilized upon arrival
at Ricerca, Inc. with sodium hypochlorite solution
(0.2%), dried, and then held at 11.0 + 0.2C until
needed. All other eggs were shipped to Ricerca,
Inc. and held at 11.0 + 0.2C until needed. Eggs
were placed in disposable plastic cups with
clipped foliage at 28 2C and 50 + 20% RH two
d before use. Larvae were tested as neonates (12 -
24 h) except P xylostella, which were 6 d old due
to the small size and delicacy of neonate P xylos-
tella.

Diet Assay

Methods were similar to those described previ-
ously (Jansson et al. 1998). Serial dilutions were
made from formulated products in combination
with a surfactant (0.01% Triton X-155) and deion-
ized H20. Controls consisted of 0.01% Triton X-
155 in deionized H20. Plutella xylostella diet was
obtained from Southland Products (Lake Village,
AR). Artificial diet for all Lepidopterans, except P.
xylostella, was prepared using established meth-
ods (King and Hartley 1985). Agar was heated in
an autoclave (121C) until dissolved and then
added to a blender (3.8 liter) containing the dry
ingredients. The agar and dry ingredients were
blended for 1 min and transferred to a steam-
jacketed kettle maintained at 70C. The diet was
dispensed (500 pl per well) into diet trays (C-D In-
ternational, Inc., Pitman, NJ) using a semi-auto-
mated diet filler (Model MDF-100, C-D
International, Inc., Pitman, NJ). Diet trays were
cooled, wrapped in plastic, and used within 48 h
after preparation.
An 50 pl aliquot of each dose of each test con-
centration was pipetted onto the surface of the
diet in each of 16 individual wells per dose. Trays
were shaken slightly to ensure that the aliquot
evenly covered the surface of the diet. After
treated diet was air dried, neonates were trans-
ferred onto the diet (one per well) and the wells







Florida Entomologist 85(4)


sealed using plastic adhesive strips. The tops of
the plastic strips were pierced for ventilation. The
criterion for death was the ability of the larvae to
right itself. Mortality was recorded 6 d after ap-
plication (Jansson et al. 1998).

Residual Efficacy Assays

Methods were similar to those described previ-
ously (Jansson et al. 1996, 1997). A custom built
track sprayer system was used to apply insecti-
cides. Treatments were applied using a calibrated
double-nozzle (TJ8001E, Sprayer Systems,
Wheaton, IL) track sprayer that delivered 100 ml
of spray solution to 7-10 plants over 2 meters at
3.5 kg/cm2. Plants 14-20 days old were sprayed
with chlorfenapyr, emamectin benzoate, fipronil,
and tebufenozide at estimated field use rates
(224.2, 8.42, 56.0 and 140.0 g ai/ha, respectively)
and 10% of these rates. Insecticides were applied
in combination with the nonionic surfactant Leaf-
Act 80 (PureGro, W. Sacramento, CA) at a rate of
0.58 1/ha (0.0625%). Controls consisted of the sur-
factant treatment alone. Plants were held in a
glasshouse at 24 + 6C after treatment. Five rep-
licate leaf cuttings from different plants were in-
fested with larvae on 0, 4, 7, 10 and 14 days after
treatment (DAT). Foliage was clipped and placed
in Petri plates containing 20 ml of 1.8% water
agar. Clippings were infested with 10-12 larvae
and mortality was assessed after 4 d. Garbanzo
bean, Cicer arietinum (L.) cv. Burpee Garbanzo
5024, and sugar beet, Beta vulgaris L. cv. USH-
11, were used to test residual effectiveness at con-
trolling H. virescens and S. exigua, respectively.
Cabbage, Brassica oleracea var. capitata L. cv.
Jersey Wakefield, was used to assess residual ef-
fectiveness against T ni and P. xylostella. A com-
pound was considered effective if mortality
remained above 90%.

Data Analysis

Data from the diet assays were analyzed using
probit analysis models in the POLO-PC program
(Russell et al. 1977). Significant difference be-
tween LC values was based on overlap of 95% fi-
ducial limits. The percentage mortality data of
the residual efficacy assays was arcsine trans-
formed and analyzed by ANOVA. Means within
each rate range were separated by the Waller-
Duncan K-ratio t-test (SAS Institute, 1993).

RESULTS

Diet Assay

Emamectin benzoate was the most toxic com-
pound tested. The Lepidopteran species tested
were 20- to 64,260-times more sensitive to ema-
mectin benzoate than to the other three com-


pounds (i.e., T ni was 20-times more sensitive to
emamectin benzoate than tebufenozide, and S. ex-
igua was 64,240-times more sensitive to emamec-
tin benzoate than fipronil} (Table 1). Fiducial
limits for LC90 values against most Lepidoptera
overlapped, indicating that emamectin benzoate
was equally potent against most Lepidoptera
tested. Spodoptera exigua and P xylostella were
the most sensitive species to emamectin benzoate
(LC90 = 0.005 and 0.0053 ug/ml, respectively),
while T ni and P includes were the least sensi-
tive (LC90 = 0.0125 0.0218 ug/ml, respectively).
There was a 4-fold difference in LC90 values be-
tween the least sensitive and most sensitive spe-
cies.
Chlorfenapyr toxicity was consistent among
species. The LC90 values had a narrow range (1.9-
4.6 ug/ml) with fiducial limits ranging from 1.6-
6.5 ug/ml (Table 1). The slopes of the chlorfenapyr
concentration responses against Lepidoptera
were the steepest (3.9-8.2) among the four com-
pounds tested.
In contrast to chlorfenapyr and emamectin
benzoate, there was wide variation in sensitivity
to fipronil among the six species tested. Plutella
xylostella was over 10 to 502-times more sensitive
to fipronil than the other five species tested (Table
1). LC90 values for H. virescens, P includes, T ni
and S. frugiperda ranged between 6.4 and 18.8
ug/ml and had overlapping fiducial limits.
Spodoptera exigua was the least sensitive species
to fipronil (LC90 = 321.3 ug/ml).
There also was wide variation in sensitivity of
Lepidoptera to tebufenozide with Trichoplusia ni
being the most sensitive species (LC90 = 0.24 ug/
ml, Table 1). Pseudoplusia includes and S. fru-
giperda (LC90 = 2.6 and 2.lug/ml, respectively)
were almost equally sensitive to tebufenozide.
Spodoptera exigua and P xylostella were approx-
imately 33-times and 51-times more tolerant, re-
spectively, of tebufenozide than T ni. The
Lepidopteran most tolerant of tebufenozide in
these tests was H. virescens, which was 113-times
more tolerant of tebufenozide than T ni.

Residual Efficacy Assays

All compounds at projected field rates were ef-
fective at controlling H. virescens up to 14 DAT
except tebufenozide, which was effective up to 10
DAT (Table 2). Emamectin benzoate also caused
100% mortality on all evaluation dates when ap-
plied at low rate (0.84 g AI/ha). Fipronil was effec-
tive at controlling H. virescens at all evaluation
dates up to 14 DAT at low rate (5.6 g AI/ha). Mor-
tality in these treatments was comparable to that
produced by emamectin benzoate. The low rate of
chlorfenapyr (22.4 g AI/ha) had lower percentage
mortality on 7 DAT than the corresponding rates
of emamectin benzoate and fipronil, but mortality
increased to 100.0% on 14 DAT. This may have


December 2002









Argentine et al.: Potency of Four New Insecticides


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Argentine et al.: Potency of Four New Insecticides


been due, in part, to the nutritional quality of the
older leaves used on these DAT, as control mortal-
ity ranged from 30.1 to 36.9% on evaluations from
7 to 14 DAT. At the low rate (14.0 g ai/ha)
tebufenozide caused mortality ranging from 68.7
to 90.0%, although mortality only differed from
other insecticide treatments on 10 DAT. It should
be noted that a number of dead and live
tebufenozide-treated H. virescens larvae showed
molting deformities characteristic of tebufenozide
toxicity (Retnakaran et al. 1995).
The results of the residual efficacy tests on
sugar beet using S. exigua were different from
those on garbanzo bean. Chlorfenapyr, emamec-
tin benzoate and tebufenozide resulted in com-
plete or nearly complete control of S. exigua on
sugar beet for up to 14 DAT when applied at the
high rates (Table 3). Emamectin benzoate also
caused 100% mortality for up to 14 DAT when ap-
plied at the low rate. Chlorfenapyr was not signif-
icantly different from emamectin benzoate up to
14 DAT. However, at 14 DAT chlorfenapyr treat-
ments caused only 66.8% S. exigua mortality.
Tebufenozide was effective for up to 4 DAT when
applied at the low rates (14.0 g AI/ha). Phytotox-
icity (i.e., chlorosis) was noted in sugar beets
treated with chlorfenapyr at the projected field
rate. As in the case of H. virescens, a number of
dead and alive tebufenozide-treated S. exigua lar-
vae showed molting deformities characteristic of
tebufenozide toxicity. Fipronil at the projected
field rate (56 g AI/ha) was effective at controlling
S. exigua for up to 4 DAT. Mortality dropped
markedly by 7 DAT and was similar to controls at
10 and 14 DAT (Table 3). Mortality caused by
fipronil at the low rate was comparable to control
mortality.
The high rates of chlorfenapyr, emamectin
benzoate and fipronil resulted in 100% mortality
of P xylostella on cabbage for the duration of the
test (Table 4). Emamectin benzoate and fipronil
at the low rates (0.84 and 5.6 g AI/ha, respec-
tively) caused 100% mortality up to 4 DAT Ema-
mectin benzoate also caused 95% mortality at 7
DAT. At the low rate, chlorfenapyr was ineffective
from 4 to 14 DAT.
Tebufenozide was the only compound that was
ineffective against P xylostella when applied at
the high rate (Table 4). The highest level of mor-
tality caused by tebufenozide during the course of
the test was 82.5% on 7 DAT. Mortality at the low
rate of tebufenozide was comparable to controls
between 4 and 14 DAT.
All compounds were effective against T ni in
the residual efficacy tests up to 10 DAT when ap-
plied to cabbage at their high rates. At 14 DAT,
fipronil and tebufenozide caused less than 90%
mortality, although these treatments were not
significantly different from chlorfenapyr or ema-
mectin benzoate. Emamectin benzoate was the
only insecticide that caused 100% mortality of


T ni for the duration of the test (Table 5). How-
ever, differences among compounds were more
apparent at their low rates. The low rate of
tebufenozide (14.0 g AI/ha) caused 82.0-96.4%
mortality of T ni between 0-14 DAT. At 14 DAT,
tebufenozide ranked higher than any of the other
compounds tested (Table 5). At the low rate (0.84
g AI/ha), the efficacy of emamectin benzoate for
control of T ni started to diminish at 4 DAT. Con-
trol with emamectin benzoate at the low rate
ranked lower than that from tebufenozide at its
low rate (14.0 g AI/ha) on 10 and 14 DAT, and
ranked lower than that from chlorfenapyr at its
corresponding rate (22.4 g AI/ha) at 10 DAT. At
the low rate, chlorfenapyr was ranked lower than
tebufenozide at 14 DAT. Fipronil at low rate (5.6 g
AI/ha) was only effective on 0 DAT, and at 4 DAT
all the other insecticides outperformed fipronil.
At 14 DAT only tebufenozide was different from
control at the low rate.

DISCUSSION

Emamectin benzoate was consistently the
most potent compound tested. It was at least 1-5
orders of magnitude more potent than all other
compounds evaluated. Emamectin benzoate was
potent against a wide spectrum of Lepidoptera
species; toxicity differed by only 4-fold among the
Lepidoptera tested.
Chlorfenapyr was the second most potent com-
pound against most Lepidoptera, followed by
tebufenozide and fipronil. Like emamectin ben-
zoate, chlorfenapyr demonstrated broad spectrum
activity, and was equally effective against all Lep-
idoptera tested. The spectrum oftebufenozide and
fipronil were more variable. Of these three com-
pounds, chlorfenapyr was the most potent to H.
virescens, S. exigua and S. frugiperda, while
tebufenozide was the most potent to T ni and
fipronil the most potent to P xylostella.
Residual efficacy data under glasshouse condi-
tions correlated with the spectrum and potency
data. Emamectin benzoate and fipronil were par-
ticularly effective at controlling H. virescens
when applied at high rates and at 10% of these
rates. Tebufenozide and chlorfenapyr were effec-
tive at the field rate against H. virescens for 10
and 14 DAT, respectively. Emamectin benzoate
and chlorfenapyr were more effective at control-
ling S. exigua than tebufenozide and, particularly,
fipronil, which agreed with the diet bioassay data.
It should be noted that a number of larvae treated
with tebufenozide had molting deformities char-
acteristic of tebufenozide toxicity. Some of these
deformed larvae would have probably succumbed
within a few days after the 4-day mortality as-
sessment used in the residual efficacy test (Jans-
son et al. 1998). Tebufenozide was the least
effective compound at controlling P. xylostella,
which also concurred with the diet bioassay data.









Florida Entomologist 85(4)


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Argentine et al.: Potency of Four New Insecticides


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Argentine et al.: Potency of Four New Insecticides


Emamectin benzoate was the only compound
that resulted in complete control (i.e., 100% mor-
tality) of T ni through 14 DAT when applied at
the high rates, although control achieved with the
other insecticides at equivalent rates was also ac-
ceptable (Table 5). Tebufenozide was superior to
all other compounds at controlling T ni when ap-
plied at the low rates.
A number of factors, such as photostability,
translaminar uptake, and leaf nutritional status,
will effect residual efficacy (Verkerk & Wright
1996). In our studies the concentration-response
appeared to correlate with residual efficacy in the
two cabbage pests. At field rate, emamectin ben-
zoate was equally effective at controlling T ni and
P xylostella, causing 96.7-100.0% mortality to both
insects for the duration of the test. Emamectin
benzoate was only effective up to 4 DAT against T
ni at the low rate (Table 5), while against P xylos-
tella it was effective for up to 7 DAT (Table 4). In
diet assays, T ni was approximately 2-times more
tolerant of emamectin benzoate compared with P.
xylostella (Table 1). This may be the reason for the
different response in the residual efficacy assays at
the low rate between the two species.
Fipronil was effective against both species at
the field rate. At the low rate, fipronil was effec-
tive for up to 7 DAT against P xylostella (Table 4),
but it was ineffective against T ni after 0 DAT
(Table 5). Again, these data confirm diet assay re-
sults. Plutella xylostella was the most sensitive
species to fipronil based on LC values, while T ni
was less sensitive to fipronil (Table 1).
Chlorfenapyr was effective against both spe-
cies for the duration of the test when applied at
field rate (Tables 4 and 5). These data agree with
results from the diet assay, which showed no dif-
ference in LCgvalues between these two species.
Tebufenozide was efficacious against T ni and
remained effective at controlling this insect at both
rates for the duration of the test (Table 5). How-
ever, tebufenozide was ineffective at both rates
against P xylostella, even on 0 DAT. These data
confirm diet assay results. T ni was the most sen-
sitive species to tebufenozide based on LCg, values,
whereas P xylostella was 51-times more tolerant of
tebufenozide compared with T ni (Table 1).
Collectively, these data show that all four com-
pounds have potential for controlling Lepidoptera
pests. Emamectin benzoate and chlorfenapyr con-
trolled a broader spectrum of lepidopteran pests
and for this reason should have a broader utility
in crop protection. Tebufenozide and fipronil con-
trolled a narrower range of lepidopteran pests,
but have already demonstrated utility under field
conditions against certain lepidopteran pests.

ACKNOWLEDGMENTS

This is Merck Research Laboratories literature
clearance no. 97-MS- We thank L. Limpel, G. Misich,


and M. Poling for assistance with this project. We thank
Rohm and Haas Co., Spring House, PA, for supplying
tebufenozide.


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Wuellner et al.: Phorid Fly Behavior


ECLOSION, MATING, AND GROOMING BEHAVIOR OF THE PARASITOID
FLY PSEUDACTEON CURVATUS (DIPTERA: PHORIDAE)

CLARE T. WUELLNER1, SANFORD D. PORTER2 AND LAWRENCE E. GILBERT1
'Section of Integrative Biology, Brackenridge Field Laboratory, Fire Ant Laboratory
University of Texas, Austin, TX 78712

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

ABSTRACT

Phorid flies from the genus Pseudacteon are parasitoids of Solenopsis ants. Recent efforts of
controlling imported fire ants in the United States have focused on rearing and releasing
these flies as biocontrol agents. We studied eclosion, mating, and grooming behavior ofPseu-
dacteon curvatus Borgmeier in an effort to increase understanding of its biology. The sex ra-
tio of closing flies in the lab was 1:1. The flies emerged only in the morning and were
protandrous. Mating in the lab occurred on the substrate and did not require disturbed ants.
Males and probably also females mated multiply.

Key Words: Biocontrol, Solenopsis invicta, Phoridae, Mass Rearing

RESUME

Las moscas del g6nero Pseudacteon (Phoridae) son parasitoides de las hormigas Solenopsis.
Esfuerzos recientes para controlar a la hormiga de fuego importada (Solenopsis invicta) en
los Estados Unidos han estado enfocados en la cria y liberaci6n de estas moscas como agen-
tes de control biol6gico. Nosotros estudiamos la eclosi6n, apareamiento y el comportamiento
de acicalamiento de Pseudacteon curvatus Borgmeier en un esfuerzo para aumentar nuestro
entendimiento de su biologia. La proporci6n de nacimiento de hembras y machos en el labo-
ratorio fu6 1:1. Las moscas emergieron solamente en la manana y fueron protandrosas (los
machos nacen mas temprano que las hembras). El apareamiento en el laboratorio sucedi6
sobre el substrato y no requeria que las hormigas fueran perturbadas. Los machos y proba-
blemente las hembras se aparearon varias veces.


Until recently, attempts at controlling the red
and black imported fire ants, Solenopsis invicta
Buren and Solenopsis richteri Forel, respectively,
have been almost entirely by chemical means. The
consequences of early practices were quite disas-
trous in some cases (Carson 1962). Fortunately,
more ecologically sound and sustainable methods
have been developed (Drees et al. 1996). One of
the methods currently under investigation is the
use ofPseudacteon phorid flies as agents ofbiolog-
ical control, first suggested for the control of at-
tine ants in Weber (1972).
The female Pseudacteon phorid fly attacks and
lays an egg in an adult worker ant. The larva
ecloses, consumes its host's internal organs, and
then pupates in the ant's head capsule (e.g., Por-
ter 1998). The supposed biological control effect
lies not so much in direct mortality, for each ma-
ture Solenopsis nest contains hundreds of thou-
sands of worker ants, but in the indirect effect the
fly's presence has on normal fire ant behavior: fire
ants stop foraging and defending resources in the
presence of phorid flies, which potentially has a
detrimental effect on the colony's fitness (e.g., Orr
et al. 1995, 1997).


Progress has been made in development and
evaluation of 2 species (Pseudacteon tricuspis
Borgmeier and P curvatus Borgmeier) for mass-
rearing and release (Porter & Alonso 1999, Porter
2000, Porter 1998a, Porter 1998b, Morrison et al.
1997, Gilbert & Morrison 1997, Porter et al. 1997,
Pesquero et al. 1996, Porter et al. 1995a), and ini-
tial investigations into suitability of several other
species have also been published (Porter 2000,
Porter & Alonso 1999, Morrison & Gilbert 1999,
Porter 1998a, Porter 1998b, Gilbert & Morrison
1997, Morrison et al. 1997, Orr et al. 1997,
Pesquero et al. 1996, Orr et al. 1995, Pesquero et
al. 1995, Porter et al. 1995a, Porter et al. 1995b).
There are 18 described species of phorid flies
that attack fire ants in South America (Porter &
Pesquero 2001). Such diversity can only be sup-
ported if the species of parasitoids have differing
habitat preferences, foraging ecologies, and host
preferences. Indeed, this has been shown to be the
case (Porter & Briano 2000, Orr et al. 1997, Gil-
bert & Morrison 1997, Pesquero et al. 1996, Porter
1998a, Porter et al. 1995a, Fowler et al. 1995,
Borgmeier 1922). For instance, some phorid flies
attack fire ant minors, whereas others attack the







Florida Entomologist 85(4)


majors; some attack during the heat of the day,
and others attack during the cooler parts of the
day; and some attack at fire ant foraging trails,
whereas others attack at disturbed mounds.
Because the ecology of the various species of
parasitoids differ, the introduction of more than
one species is likely to have a greater effect than
if a single species is introduced. Further, to have
the greatest effect, the suite of species released
should have complementary biologies. This paper
adds to what is known about the basic biology of P.
curvatus. Much of this information will assist
mass rearing and release efforts.

MATERIALS AND METHODS

A general description of eclosion behavior was
obtained by observing five newly-eclosed flies
from the time that the pupal cap opened until the
fly was able to fly. For these observations, flies
were kept in individual containers (capped 1.2 x
7.5 cm test tubes) and observed through a dissect-
ing microscope.
A general description of mating was generated
from observations of >20 matings. The mean du-
ration of mating was timed from mounting until
dismounting. Actual duration of copulation was
probably less than "mating duration," but we were
not able to discern when actual intromission oc-
curred.
To determine if multiple mating occurred, five
virgin females and one virgin male were put into
a container and left together for 30 h (n = 3). At
the end of the 30 hours, all living flies were
squashed and a phase contrast microscope was
used to look for sperm.
The description of grooming was generated
from observations of flies as they groomed while
in the mating observation arena or during eclo-
sion. We observed >20 grooming bouts.

RESULTS

In the lab, P. curvatus flies have been observed
to emerge between 0300 and 1000 h. Emergence
in the late morning was probably due to exposure
to artificial light the evening before they emerged.
In the field, emergence probably occurs within a
few hours of dawn depending on the temperature
and light cycle. In the lab, flies emerged over a pe-
riod of about 2 h with males emerging about 30 +
5 (SE) min earlier on average than females
(ANOVA, F1,2 = 35.3,p < 0.0001, n = 45 and 59, re-
spectively). Emerging flies required about 10 min
to expand their wings. When temperatures were
warm (26C) flight can occur within an hour of
emergence and mating about an hour later. How-
ever, cool temperatures (<22C) can delay flight
and mating until late morning or even into the af-
ternoon. The sex ratio of males to females did not
differ significantly from 1:1 (Porter 2000).


Average duration of mating was 22.3s + 8.6
(mean SD, n = 10). There was considerable varia-
tion in mating time, ranging from 12 to 39 s. Be-
cause mating was terminated by the female kicking
the male off of her back, the variation was probably
not due to actual time required for mating, but to
changes in female motivation during mating.
The following is a detailed description of eclo-
sion and post-eclosion behavior of one female fly.
The emerging fly popped open the pupal cap and
climbed out of the ant head capsule onto the sub-
strate, a process that usually only requires a few
seconds. On first emerging, the fly's abdomen was
distended and elongated, and the ovipositor was
extended. The cuticle of the legs, head and thorax
was a dusty tan, the abdomen was a pale whitish
color, and the wings were white and dull. The eyes
and ovipositor appeared to be sclerotized. Almost
immediately, the fly started pumping her abdo-
men to fill and expand her wings. After about 10
min, the wings appeared to be fully expanded and
the abdomen had reduced considerably in size but
was still elongated. About this time, the abdomi-
nal pumping stopped. Over the next several min-
utes, fluid returned to her abdomen, which
became distended again, and she began pumping
her abdomen again. About 15 min after eclosion,
the costal margins of her wings began to sclerotize
and her entire body began darkening about 10
min later. She continued to pump her abdomen
from time to time and her cuticle became darker
and darker until about 75 min after eclosion,
when she finally retracted her ovipositor. Al-
though her body seemed sclerotized and she be-
gan walking about 10 min later, her wings were
still somewhat opaque, and she did not fly until
almost 2h 15 min after eclosion.
Before mating, the females remained station-
ary for the most part, usually moving only if dis-
turbed by another fly. Females groomed
themselves while sitting. Males were also station-
ary and groomed some of the time, but were much
more mobile than females; often walking about on
the substrate and flying around the arena. While
males were in flight, they sometimes tussled
briefly with one another, but there were no dis-
cernible territories, nor were males particularly
aggressive.
Visual cues and probably chemical cues were
important to males for locating a mate. Males in-
vestigated small bumps in the substrate and were
attracted to empty head capsules. One male vigor-
ously attempted to copulate with an empty ant
head capsule.
Females that had sat unnoticed for some time
suddenly became attractive to males. We could not
discern any difference in behavior of females that
were attractive versus those that were not, so pre-
sumably they emitted some mate attraction chem-
ical. There did not seem to be any mate choice by
females. Mating was determined by scramble com-


December 2002







Wuellner et al.: Phorid Fly Behavior


petition. Often, 2 or 3 males simultaneously at-
tempted to copulate with the same female.
The male flew toward the female, approaching
her posteriorly. The male sat on top of the female
and struggled to hold onto the back of the female's
abdomen with his hind legs. Once the male had a
hold on the female's abdomen, the adeagus was
extended and curved around the back of the fe-
male's abdomen to one side of the ovipositor. The
female's cloaca was just in front of the ovipositor.
Toward the end of the mating, the female began to
walk around on the substrate and sometimes
pushed off the male with her hind legs.
Following mating, the male and female sat
within a few cm of each other and groomed, first
the abdomen, then the hind legs, thorax, head,
and legs. They did not groom their wings. After
grooming, the female became more active and
flew about the arena. The male also sometimes
flew about, but tended to become less active.
Squashes of single virgin males kept with five
virgin females clearly showed multiple mating by
males. Of the female flies recovered alive after 30
h, 5/5, 3/3 and 2/4 were mated. All 3 males were
still positive for sperm at the end of the test.
While making observations for mating dura-
tion, it was common to see more than one male at-
tempting to mount a female both simultaneously
and sequentially. However, whether females func-
tionally mate more than once is not clear.
The abdomen, ovipositor, and wings are all
groomed with the third pair of legs. To groom the
abdomen, the inside surfaces of the hind tibiae
and fibiae are drawn over the ventral and lateral
surfaces of the abdomen. The wings are drawn to
the side of the abdomen with the hind legs and the
ventral surface of the hind leg is rubbed over the
dorsal surface of the wing. The underside of the
wing is groomed by rubbing the dorsal surface of
the third leg along the ventral surface of the wing.
The posterior portion of the thorax is groomed
using the femur of the third leg, whereas the fore-
legs are used to groom the posterior portion of the
thorax and the head. The head is turned to each
side and ventrally in order for each of the surfaces
to be rubbed. Much more care is given to grooming
the aristae than to any other part of the body. The
fly often goes over each one with its forelegs sev-
eral times.
The legs are groomed by rubbing against other
legs. The forelegs are rubbed together, starting at
the base, and moving toward the tarsi. The hind
legs are rubbed together in similar fashion. To
groom the middle legs, the forelegs are drawn
back to the side to be groomed and rubbed over
the middle leg. The hind legs are also used to
groom the middle legs in similar fashion. It is pos-
sible that these grooming movements also groom
the fore and hind legs respectively. Similar leg-
cleaning movements are described and illustrated
for D. melanogaster in Szebenyi (1969).


DISCUSSION

Our observations revealed that P curvatus
flies mate on the ground in the morning several
hours after eclosion from the puparium. This in-
formation will be useful to researchers interested
in releasing P. curvatus flies for biocontrol of im-
ported fire ants because field releases of this fly
should be timed for late morning or afternoon, as
mating does not occur in the air while females are
trying to oviposit.
Males readily and successfully mated multiple
times in our tests, indicating that lab-reared fe-
males will rarely lack sufficient mates for mating.
Very little is known about mating behavior of
other Pseudacteon flies. Published reports of mat-
ing behavior for this genus are primarily field
studies reporting male Pseudacteon presence at
aggregations or trails of Solenopsis ants (Will-
iams 1980, Feener 1987, Feener & Brown 1992,
Porter et al. 1995a). The most detailed account is
for P tricuspis (Porter et al. 1997). From this, we
know that mating behavior differs considerably
between P tricuspis and P curvatus. Although
males of P tricuspis are attracted to disturbed fire
ants, males of P curvatus are not. Mating for P.
curvatus takes place on the ground and takes
more than 20 seconds on average; whereas for P.
tricuspis, mating is initiated on the wing and is
extremely brief ( Mating in other kinds of phorids often occurs
in mating swarms (reviewed in Disney 1994). Not
surprisingly, the most common type of mating
swarm was composed of lekking males. However,
there are species in which there are both sexes in
one swarm, another with both sexes forming sep-
arate swarms, and one species with female mat-
ing swarms to which males were attracted
(reviewed in Disney 1994). The preponderance of
species using swarms for mating probably only in-
dicates the ease with which swarms can be ob-
served versus other mating strategies that are
less likely to be stumbled upon.
The duration of mating among phorids ranges
from utes (Dohrniphora cornuta, Barnes 1990). How
this interspecific variation relates to sperm com-
petition and/or mate selection by females would
be an interesting research direction.

ACKNOWLEDGMENTS

We thank D. Kelly, B. Mayfield, C. Vann, and J. Saun-
ders for help in completing this project. The State of Texas
Fire Ant Research and Management Committee (FAR-
MAAC) provided partial funding for this research.

REFERENCES CITED
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(Bigot) (Diptera: Phoridae), a filth-inhabiting hump-
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BORGMEIER, T. 1922 (1921). Zur Lebensweise von Pseu-
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GILBERT, L. E., AND L. W. MORRISON. 1997. Patterns of
host specificity in Pseudacteon parasitoid flies
(Diptera: Phoridae) that attack Solenopsis fire ants
(Hymenoptera: Formicidae). Environ. Entomol. 26:
1149-1154.
MORRISON, L. W., AND L. E. GILBERT. 1999. Host speci-
ficity in two additional Pseudacteon spp. (Diptera:
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menoptera: Formicidae). Florida Entomol. 82: 1-6.
MORRISON, L. W., C. G. DALL'AGLIO-HOLVERCEM, AND
L. E. GILBERT. 1997. Oviposition behavior and devel-
opment ofPseudacteon flies (Diptera: Phoridae), par-
asitoids of Solenopsis fire ants (Hymenoptera:
Formicidae). Environ. Entomol. 26: 716-724.
ORR, M. R., S. H. SEIKE, W. W. BENSON, AND L. E. GIL-
BERT. 1995. Flies suppress fire ants. Nature. 373:
292-293.
ORR, M. R., S. H. SEIKE, AND L. E. GILBERT. 1997. Forag-
ing ecology and patterns of diversification in dipteran
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teon (Phoridae). Ecol. Entomol. 22: 305-314.
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CAMPIOLO. 1995. Rearing ofPseudacteon spp. (Dipt.,
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December 2002


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PESQUERO. 1995a. Host specificity of several Pseu-
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Florida Entomologist 85(4)







Yocum & Evenson: Auxiliary Diet for Perillus bioculatus


A SHORT-TERM AUXILIARY DIET FOR THE PREDACEOUS STINK BUG,
PERILLUS BIOCULATUS (HEMIPTERA: PENTATOMIDAE)


GEORGE D. YOCUM AND PATSY L. EVENSON
RRVARC, USDA, ARS, BRL,1605 Albrecht Boulevard, Fargo, ND 58105-5674

ABSTRACT

Perillus bioculatus (F.) can be maintained in the laboratory on a diet of Heliothis virescens
F. larvae if supplemented with Colorado potato beetle (CPB) eggs. Here we demonstrate that
an artificial diet can replace the CPB eggs and maintain the colony for at least three gener-
ations. This enables us to maintain our Perillus colonies at high numbers independent of the
normal fluctuations in our CPB colony. Survival of nymphs, adult longevity, start of oviposi-
tioning, total number of eggs per female, total number of clutches per female, and percent
hatch were equivalent between the two rearing regimes for three generations. Fecundity on
the artificial diet was greatly reduced by the sixth generation, leading to the collapse of the
colony during the seventh generation.

Key Words: Perillus bioculatus, artificial diet

RESUME

Perillus bioculatus (F.) puede ser mantenido en el laboratorio con una dieta de larvas de He-
liothis virescens F. si son suplementados con huevos del escarabajo de papa de Colorado
(CPB), (Leptinotarsa decemlineata Say), Aqui demostramos que una dieta artificial puede
reemplazar los huevos de CPB y mantener la colonia por lo menos en tres generaciones. Este
nos permit mantener poblaciones altas de nuestra colonia de Perillus independientemente
de las fluctuaciones normales en nuestra colonia de CPB. La sobrevivencia de las ninfas, la
longevidad de los adults, el inicio de la oviposici6n, el numero total de huevos por hembra,
el numero total de grupos de huevos por hembra, y el porcentaje de eclosi6n fueran equiva-
lentes entire los dos regimenes de cria por tres generaciones. La fecundidad de las hembras
alimentadas con la dieta artificial fu6 grandemente reducida en la sexta generaci6n, cau-
sando la caida total de la colonia durante la septima generaci6n.


Perillus bioculatus (F.) is a predaceous pen-
tatomid endemic to North America. P. bioculatus
feeds on a number of insect orders under both
field and laboratory conditions, but has an intrin-
sic preference for the Colorado potato beetle
(CPB), Leptinotarsa decemlineata (Say) (Saint-
Cyr & Cloutier 1996). CPB is the major defoliator
of potato (reviewed Ferro 1985, Hare 1990,
Cloutier et al. 1996), tomato (Schalk & Stoner
1979), and eggplant (Cotty & Lashomb 1982;
Hamilton & Lashomb 1996). With its strong pref-
erence for CPB, P. bioculatus has drawn atten-
tion as a possible biocontrol agent for CPB in
North America (Biever & Chauvin 1992) and Eu-
rope (Jermy 1980).
Currently there is no published optimized arti-
ficial diet for P. bioculatus. Therefore, P. biocula-
tus must be reared on CPB or a secondary host
such as Trichoplusia ni (Biever & Chauvin 1992)
or Heliothis virescens supplemented with CPB
(Adams 2000). Currently we are maintaining our
P bioculatus colony on H. uirescens larvae, and
feeding only first and second instar nymphs on
CPB egg masses (Adams 2000). Even with de-
creased dependence on CPB, problems with the
CPB colony have led to loss of our P bioculatus


colonies, or to insufficient numbers of CPB egg
masses to undertake planned experiments. We in-
vestigate here whether a simple, inexpensive diet
for another predaceous insect, C(' i, .. 7.'..' i. rufila-
bris (Cohen & Smith 1998), could replace the CPB
eggs to maintain P bioculatus for several genera-
tions. This would serve as a bridging diet when
CPB is unavailable, while avoiding the cost of in-
corporating an additional prey insect colony into
our research program.

MATERIALS AND METHODS

Rearing

P bioculatus were reared as described by Ad-
ams (2000). Stock colonies were reared in a walk-
in environmental chamber set to 16 h light:8 h
dark, 25 + 2C and 65% relative humidity. Both
nymphs and adults were fed 5th instar H. uire-
scens larvae. The H. uirescens larvae were frozen
and stored at -25C until needed. Approximately
300 P. bioculatus eggs were used to start each
cage of nymphs. First and second instars received
three CPB egg masses and one H. uirescens larva
daily. Third through fifth instar nymphs received







Florida Entomologist 85(4)


seven larvae per day. All nymphal colonies also
received two water wicks with a potato leaf in-
serted in each.
Adults were removed daily from the nymphal
colonies and placed into egging cages which had
three water wicks with an inserted potato leaf.
Each egging cage contained 40 females and 10
males, and 10 H. uirescens larvae were provided
daily. The females deposited their eggs on any
hard substrate in the cage. Eggs were collected on
Monday, Wednesdays and Fridays and placed
into hatching tubs.

Diet Preparation

The diet was prepared according to Cohen &
Smith (1998), except that antibiotics were not in-
cluded. The diet contained: 100 g 70% lean ground
beef, 100 g beef liver, 100 g hen's eggs, 15 g su-
crose, 5 g honey dissolved in 20 ml water, 10 g
brewer's yeast, 5 ml 10% acetic acid and 45 ml wa-
ter. Because we were not able to get the diet mix-
ture to solidify as described by Cohen & Smith
(1998), the diet was fed to stink bugs in Parafilm
domes (Adams 2000).

Experimental Treatment

In the colony-hatching containers the first in-
star nymphs aggregated on the water wicks and
fed minimally, if at all, on the CPB egg masses.
Therefore, for ease of handling, all experiments
were set up using unfed (water only) second in-
star nymphs. The nymphs were set up in plastic
containers (25 cm diameter by 7 cm high, Pioneer
Packaging, Dixon, KY) with a 5 cm hole in the lid
covered by nylon screen (Sefar America, Kansas
City, MO). The nymphs were reared at 16 h light:8
h dark and 30 + 0.5C in a reach-in environmental
chamber (Conviron, model I25L, Winnipeg, Can-
ada). Three replicates with 30 nymphs each were
used for each treatment group.

Developmental Experiments

The control nymphal colony was given three
CPB egg masses per day for the first three days,
and three water wicks with potato leaves. The
diet treatment nymphs received 2 parafilm domes
of artificial diet and three water wicks without po-
tato leaves. The artificial diet domes were
changed every other day. All treatment groups re-
ceived two H. uirescens larvae per day for the first
eight days, after which the number was increased
to three per day.

Fecundity Experiments

Potato leaves were not used, because the devel-
opmental experiment showed no significant dif-
ference in development of P. bioculatus with or


without potato leaves. The control adults received
H. uirescens larvae, three CPB egg masses per
day for the first three days and three water wicks.
The artificial diet-treatment group received the
artificial diet, H. uirescens larvae and three water
wicks throughout their life cycle. Adults were re-
moved daily from the rearing containers, sexed
and weighed. Individual mating pairs were
placed into plastic wide-mouth containers (10 x
8.5 x 9.5 cm) (Consolidiated Plastics, Twinsburg,
OH), with a 6.5 cm hole in the lid covered by Nitex
screen (Tetko, Lancaster, NY). Each mating con-
tainer had a roll of paper (6 x 64 cm) for oviposi-
tioning and one water wick. The adults were fed
one H. uirescens larvae per day. The cups were
checked daily for eggs, which were subsequently
removed, counted and placed in 1 oz. plastic cups
(Fill-Rite, Newark, NJ) with lids (Stan-Pac,
Lewiston, NY) to measure percent hatch.

Statistical Analysis

Proportions of adult emergence were com-
pared using constant statements in PROC LO-
GISTIC, (SAS Institute 1989). Longevity, start of
oviposition, total number of eggs per female, total
number of clutches per female, and percent hatch
were analyzed using ANOVA with the means be-
ing separated using Student-Newman-Keuls test,
SAS Institute (1989). The level of significance in
all tests was 0.05. The developmental rate for
each treatment group was estimated by using the
median adult emergence time computed from Ka-
plan-Meier estimates of survival produced by the
surfvit function of S-Plus (S-Plus 2001). For the
Fecundity Experiments the initial experimental
design called for collecting data from the first,
third, sixth, ninth and twelfth generations.

RESULTS

Preliminary experiments demonstrated that
the Cohen-Smith artificial diet alone could not
support nymphal development (results not
shown). There was a significant decrease in sur-
vival from 65% to 36% for P. bioculatus nymphs
fed only H. uirescens larvae (X2= 9.6747, n = 6, df
= 1, P < 0.0019), as compared to control nymphs,
which received both H. uirescens larvae and CPB
eggs. Substituting the artificial diet for CPB eggs
had no significant effect upon survival of the
nymphs to adult emergence, compared to controls
at the 0.05 level (Table 1). Increasing the avail-
ability of the artificial diet from the first 3 days to
6 days or for all nymphal instars did not increase
survival of the nymphs compared to the controls
at the 0.05 level (Table 2). Nymphs fed the artifi-
cial diet had median adult emergence times
equivalent to the controls (Table 3). Of nymphs
receiving only Heliothis larvae, insufficient num-
bers survived to analyze their median adult emer-


December 2002







Yocum & Evenson: Auxiliary Diet for Perillus bioculatus


TABLE 1. EFFECT OF NYMPHAL DIET ON SURVIVAL OF
NYMPHS TO ADULT EMERGENCE.*

Diet Number of Adults

Larvae only 10.8 + 2.1
Larvae + CPB eggs (control) 19.5 + 1.4b
Larvae + artificial diet 17.3 + 2.0b

*Data are expressed as means +SE of six replicates of 30 nymphs each.
Nymphs were fed H. virescens larvae only (Larvae only), or H. virescens
larvae supplemented during the 2nd instar with either Colorado potato
beetle eggs (Larvae + CPB eggs) or artificial diet (Larvae + artificial diet).
Means followed by the same letter are not significantly different at theP
< 0.05 level (PROC LOGISTIC).


gence times. It is clear from these data that the
artificial diet can support a generation of
nymphal development as effectively as can Colo-
rado potato beetle eggs.
There was no significant difference in the
weights of day 1 adults for the first generation.
However, by the third generation there was a sig-
nificant increase in the weight of females receiv-
ing the artificial diet (F = 3.95, n = 66, df = 2, P <
0.0239) (Table 4). There was no observed differ-
ence in weight of males in the course of these ex-
periments. Females that were fed H. virescens
larvae supplemented with the artificial diet
throughout nymphal development showed no re-
duction in fecundity for three generations, com-
pared to the controls (Table 5). There were no
significant differences in female longevity, onset
of oviposition, total number of eggs, number of
clutches per female, or percent hatch at the 0.05
level. There was a marked decrease in the num-
ber of eggs laid by the females during the sixth
generation of continuous rearing on the artificial
diet plus larvae, and loss of the colony during the
seventh generation.

DISCUSSION

The data presented here clearly demonstrate
that a diet regime of the modified Cohen-Smith
artificial diet for C. rufilabris (Cohen & Smith
1998) plus H. virescens larvae is as efficient as
CPB eggs and H. virescens larvae for supporting


TABLE 2. EFFECT OF DURATION OF FEEDING ON ARTIFICIAL
DIET UPON NYMPH SURVIVAL TO ADULT EMER-
GENCE.*

Treatment Number of Adults

Control 23.0 + 2.5
3D 22.7 + 1.7
6D 24.5 + 1.4
15D 24.0 + 1.8

*Data are expressed as mean +SE of six replicates of 30 nymphs each.
Nymphs were fedH. virescens larvae supplemented during the 2nd instar
with Colorado potato beetle eggs (Control) for the first 3 days (3D), six
days (6D) or throughout nymphal development (15D) with artificial diet.
PROC LOGISTIC analysis revealed no significant effects of diet on the
number of adults obtained.


P bioculatus development for at least three gen-
erations. Supplementing H. virescens larvae with
the Cohen-Smith artificial diet (Cohen & Smith
1998) produced three generations of P bioculatus
phenotypically indistinguishable (except for in-
creased weight of female adults) from those on
the control diet of larvae plus CPB eggs. This
demonstrates that an artificial diet and a subop-
timal secondary prey, which by themselves can-
not support normal development, can be used in
combination as a bridging diet to maintain a re-
search colony during periods when the primary
prey is unavailable.
However, while early generations seemed
healthy, the experimental colony suffered from re-
duced fecundity by the sixth generation, and com-
pletely collapsed during the seventh. This raises
intriguing questions as to what biochemical and
molecular changes have occurred over the seven
generations. Insects will express a particular sub-
set of genes in order to survive environmental
stresses such as heat shock (Roberts & Feder
1999), cold shock (Yocum 2000), and desiccation
(Tammariello et al. 1999). As with other stresses,
low food quality or quantity will induce a number
of physiological and behavioral changes in insects
(reviewed, Slansky & Scriber 1985; Wheeler
1996). We are currently investigating unique
gene expression associated with feeding on sub-
optimal diets. Our bridging diet fed to P. biocula-


TABLE 3. EFFECT OF NYMPHAL DIET ON THE MEDIAN EMERGENCE TIME OF ADULT P. BIOCULATUS.*

Treatment Nymphs' Adults2 Median adult emergence (D) 0.95 LCL 0.95 UCL

Control 360 255 21 20 21
3D 360 243 22 21 22
6D 180 147 20 20 20
15D 180 144 21 21 22

*Data are expressed as median adult emergence times (days) + lower (LCL) and upper (UCL) 95% confidence limits. Nymphs were fedH. virescens
larvae supplemented during the 2nd instar with Colorado potato beetle eggs (Control) or for the first 3 days (3D), six days (6D) or throughout nymphal
development (15D) with artificial diet.
'Total number of 2nd instar nymphs at the start of the experiment.
'Total number of adults that emerged.







Florida Entomologist 85(4)


December 2002


TABLE 4. EFFECTS OF PROLONGED FEEDING ON THE ARTIFICIAL DIET ON DAY 1 ADULT P. BIOCULATUS WEIGHT.*

Males Females

Treatment Generations N Weight (mg) N Weight (mg)

Control Fl 18 47.8 + 1.5 27 60.1 + 1.1
Diet-fed Fl 18 47.8 + 1.3 24 63.8 + 1.2"b
Diet-fed F3 14 48.9 + 1.9 18 65.2 + 2.1b

*Data are expressed as means +SE of the weights of N number of day 1 adults. Means followed by different letters are significantly different from
each other at P < 0.05 as determined by Student-Newman-Keuls test. Nymphs were fed H. virescens larvae supplemented with either Colorado potato
beetle eggs during the 2nd instar (Control) or the artificial diet throughout development (Diet-fed).


TABLE 5. EFFECTS OF PROLONGED FEEDING ON THE ARTIFICIAL DIET ON P. BIOCULATUS FECUNDITY.*

Initiation
Longevity Oviposition Eggs/ Clutch/ %
Treatment Generation N (D) (D) female female Hatch

Control Fl 17 17.4 + 2.4 4.5 + 0.2 109.1 17.5 9.5 + 1.0 39.1 +5.4
Diet-fed Fl 14 16.1 +2.0 4.6 + 0.4 95.5 + 13.8 11.4 + 1.0 25.7 + 5.4
Diet-fed F3 9 21.7 + 2.3 4.4 + 0.3 156.6 + 22.7 11.7 + 1.0 39.1 +4.0

*Data are expressed as means +SE of N number of females each paired with a single male. Nymphs were fedH. virescens larvae supplemented with
either Colorado potato beetle eggs during the 2nd instar (Control) or artificial diet throughout development (Diet-fed). ANOVA analysis revealed no sig-
nificant effects of diet on female longevity (days), start of oviposition, total number of eggs per female, number of clutches per female, or percent hatch
at the P < 0.05.


tus will also be an excellent model system to
examine how gene expression is altered over gen-
erations in response to feeding on a suboptimal
diet. Our ultimate goal is to identify molecular
markers to use in diagnosing deficiencies in other
artificial diets under development.


ACKNOWLEDGMENTS

We want to thank Drs. Terry Adams and Mark West
for their assistance in statistical analysis of the data,
and Dr. Adams for his helpful comments on the draft of
this manuscript.


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the Colorado potato beetle (Coleoptera: Chrysomel-
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CLOUTIER, C., C. JEAN AND F. BAUDUIN. 1996. More bi-
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into Europe to control the Colorado potato beetle.
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perthermia and expression of the heat shock protein
Hsp70 affect developmental abnormalities in Droso-
phila melanogaster. Oecologia 121: 323-329.
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ence by the stinkbug Perillus bioculatus, a predator of
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puters, version 6, 4th ed. SAS Institute, Cary, NC.
SCHALK, J. M., AND A. K. STONER. 1979. Tomato produc-
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vae and adults of the Colorado potato beetle. J. Econ.
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SLANSKY, F., JR., AND J. M. SCRIBER. 1985. Food Con-
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Florida Entomologist 85(4)


December 2002


INFLUENCE OF MALE DIET ON MALE MATING SUCCESS
AND LONGEVITY AND FEMALE REMATING IN THE MEDITERRANEAN
FRUIT FLY (DIPTERA: TEPHRITIDAE) UNDER LABORATORY CONDITIONS

TODD E. SHELLY'2 AND SUSAN KENNELLY2
'USDA-APHIS, P.O. Box 1040,Waimanalo, HI 96795

2Hawaiian Evolutionary Biology Program, University of Hawaii, Honolulu, HI 96822

ABSTRACT

The purpose of the present study was to investigate the effect of dietary protein on the mat-
ing behavior and survival of male Mediterranean fruit flies (medflies), Ceratitis capitata
(Wied.), as a means of enhancing the effectiveness of mass-reared males in sterile release
programs to suppress wild populations. Conducted in the laboratory, our study addressed
three main questions: 1) Does the inclusion of protein in the adult diet affect mating success
of wild and mass-reared males? 2) Are copulation duration and remating tendency of wild fe-
males affected by the strain (wild versus mass-reared) and diet (protein-fed versus protein-
deprived) of their initial mating partner? 3) Does the inclusion of protein in the adult diet af-
fect the longevity of mass-reared males? In mating trials involving wild flies, protein-fed
males had a mating advantage over protein-deprived males. However, the addition of pro-
tein to the diet did not boost the mating success of mass-reared males in competition with
wild or mass-reared males for wild females. The inclusion of protein in the male diet had no
apparent effect on female remating tendency, copulation duration, or male longevity. Inde-
pendent of male diet, we found no difference between wild and mass-reared males in the du-
ration of copulations with wild females, and wild females mated initially to wild and mass-
reared males displayed similar remating propensity. The implications of these findings for
SIT are discussed.

RESUME

El prop6sito del present studio fu6 para investigar el efecto de la protein diet6tica en el
comportamiento del apareamiento y la sobrevivencia de machos de la mosca mediterranea,
Ceratitis capitata (Wied.), como una manera de mejorar la eficacia de la producci6n en masa
de los machos en programs de liberaciones de machos esteriles para suprimir las poblacio-
nes silvestres. Nuestro studio, conducido en el laboratorio, se enfoc6 en tres preguntas prin-
cipales: 1) Si la inclusion de protein en la dieta del adulto afecta el 6xito en el apareamiento
de los machos silvestres y de los machos criados en masa? 2) Si la duraci6n de la c6pula y la
tendencia de reaparearse las hembras silvestres con la pareja inicial son afectadas por la
raza (silvestres versus criados en masa) y la dieta (alimentadas con protein versus privadas
de proteina? 3) Si la inclusion de protein en la dieta del adulto afecta la longevidad de los
machos criados en masa?
En pruebas de apareamiento con moscas silvestres, los machos alimentados con protein tu-
vieron una ventaja de apareamiento sobre los machos privados de protein. Sin embargo, la
adici6n de protein a la dieta no aument6 el 6xito de aparearse en los machos criados en
masa en competencia con machos silvestres con las hembras silvestres. La inclusion de pro-
teina en la dieta del macho no tuvo un efecto aparente sobre la tendencia de las hembras
para reaparearse, la duraci6n de la c6pula, o la longevidad del macho. Independientemente
de la dieta del macho, no encontramos una diferencia en la duraci6n de la c6pula entire ma-
chos silvestres y machos criados en masa al aparearse con hembras silvestres, y las hembras
silvestres apareadas inicialmente con machos silvestres o con machos criados en masa mos-
traron una tendencia similar para aparearse de nuevo. Se discuten las implicaciones de es-
tos resultados para t6cnica del insecto est6ril (SIT).


The sterile insect technique (SIT) is widely males derived from large-scale, production facili-
used in suppression or eradication programs ties are hereafter referred to as 'mass-reared' re-
against the Mediterranean fruit fly, Ceratitis cap- gardless of whether they were irradiated
itata (Wied.) (Hendrichs et al. 1995). To a large de- (sterilized) prior to study or release). Unfortu-
gree, the success of SIT depends on the ability of nately, the mass-rearing environment, and, in
mass-reared, sterile males to compete success- particular, the high density at which adults are
fully against wild males in obtaining copulations held, imposes strong selection factors that may al-
with wild females (unless otherwise indicated, ter courtship behavior and subsequently lessen







Shelly & Kennelly: Dietary Effects on Mating in the Medfly


the competitive ability of sterile males in the wild
(Leppla & Ozaki 1991; Cayol 2000). For example,
Briceno and Eberhard (1998) observed that mass-
reared males displayed shorter courtships than
wild males and suggested that accelerated court-
ship evolved in response to frequent disturbances
resulting from dense crowding.
Genetically based changes in sexual behavior
and life history traits appear to be an inherent
consequence of mass-rearing and are therefore
difficult (if not impossible) to avoid or mitigate.
Because of this, there is a persistent need to de-
velop procedures that boost the performance of
mass-reared males via simple, easily incorpo-
rated, and inexpensive modifications to the stan-
dard mass-rearing protocol. For example, a recent
study on the medfly (Shelly & McInnis 2001) dem-
onstrated that exposure to the odor of ginger root
oil dramatically enhanced the mating success of
mass-reared males. In the absence of chemical ex-
posure, wild males outcompeted mass-reared
males and obtained 74% of all matings. However,
following exposure to ginger root oil, the mating
frequencies were reversed, and mass-reared
males achieved 75% of all matings.
Modification of the pre-release, adult diet may
represent another simple approach to increase the
effectiveness of male medflies in SIT. Current pro-
grams (e.g., California, Israel) feed newly emerged
adult medflies with sucrose-containing agar ex-
clusively. However, recent studies showed that the
addition of protein hydrolysate to the diet resulted
in a significant increase in male mating success.
In noncompetitive conditions, mass-reared male
medflies fed a protein-sugar mixture mated more
frequently than males fed sugar only (Blay &
Yuval 1997). Likewise, in direct competition for fe-
males, mass-reared males given the sugar-protein
diet signaled (pheromone-called) and mated more
frequently than mass-reared males that were pro-
tein-deprived (Kaspi & Yuval 2000; Taylor &
Yuval 1999). Similar findings were reported for
tests involving wild medflies as well (Kaspi et al.
2000; see also Papadopoulos et al. [1998]).
Although these findings clearly support the ad-
dition of protein to the pre-release diet, this mod-
ification may have costs in terms of reduced male
survivorship after release. Following 24 h of star-
vation on the fifth day of life, mass-reared males
fed a protein-containing diet for the preceding 4 d
were far more likely to die than were males fed
sugar only during the first 4 d of life (Kaspi &
Yuval 2000). Thus, development of a pre-release,
protein-containing diet that optimizes male effec-
tiveness in SIT programs requires additional in-
formation on the balance between heightened
sexual competitiveness and reduced survivorship.
The purpose of the present study was to inves-
tigate further the effect of dietary protein on the
mating behavior and survival of male medflies.
Based on laboratory observations, our study ad-


dressed three main questions: 1) Does the inclu-
sion of protein in the adult diet affect mating
success of wild and mass-reared males? 2) Are
copulation duration and remating tendency of
wild females affected by the strain (wild versus
mass-reared) and diet (protein-fed versus protein-
deprived) of their initial mating partner? 3) Does
the inclusion of protein in the adult diet affect the
longevity of mass-reared males? Although field
cages would have provided a more natural envi-
ronment, we chose to conduct this study in the
laboratory, because we were able to measure cop-
ulation duration, female remating and male lon-
gevity with greater accuracy and obtain larger
sample sizes than possible from field tests.

MATERIALS AND METHODS

Mating Experiments

Wild flies were reared from the fruits of Jeru-
salem Cherry (Solanum capsicum L.) collected in
the Hawaii Volcanoes National Park, HI. Fruits
were held over vermiculite, and larval develop-
ment proceeded in situ. Pupae were sifted from
the vermiculite 7-9 d after fruit collection, and
adults were separated by sex within 2 d of eclo-
sion, well before reaching sexual maturity (T E. S.,
unpublished data). Mass-reared males were ob-
tained from the Hawaii Fruit Fly Rearing Facility,
Waimanalo, HI. Pupae were exposed in air to 150
Gy of gamma radiation from a 137Cs source 2 d be-
fore eclosion and then delivered to our laboratory.
Males were collected within 12 h of eclosion. Both
wild and mass-reared adults were held in plastic
buckets covered with nylon screening (volume 5
liters; 100-200 flies per bucket). Room tempera-
ture was maintained at 22-25C and relative hu-
midity at 65-90%, and flies were exposed to
natural and artificial light in an approximately
12: 12 h light:dark cycle. Wild and mass-reared
males were separated into 2 dietary regimes:
"protein-deprived" males were given only sugar
(sucrose) plus water, and "protein-fed" males were
given a 3:1 mixture (by volume) of sugar and pro-
tein hydrolysate plus water. Wild females were
given the sugar-protein mixture plus water. Sam-
ples (n = 30 individuals) of wild and mass-reared
males maintained on the two diets and wild fe-
males were weighed (wet weight) to the nearest
0.1 mg with an electronic balance. In all cases,
adults were weighed when 5 d old.
Three experiments were performed that com-
pared the mating frequency of 1) protein-fed ver-
sus protein-deprived wild males, 2) protein-fed
versus protein-deprived mass-reared males, and
3) protein-fed or protein-deprived mass-reared
males versus protein-fed wild males. Wild females
were used in all cases. Mating tests were con-
ducted in the same manner for all experiments.
Sixty males of each diet type (or strain type for ex-







Florida Entomologist 85(4)


periment 3) and 60 females were placed in trans-
parent plexiglass cages (30 x 30 x 40 cm with
screen-covered openings on the top and sides). Al-
though this density far exceeds natural levels
(Shelly et al. 1994), females nonetheless had am-
ple space for movement and were clearly capable
of moving away from (i.e., rejecting) courting
males. Wild flies were used at 10-14 d of age, and
mass-reared males were 6-9 d old when tested
(the different ages reflect differing maturation
rates between wild and mass-reared males).
Males were marked 1 d before testing by cooling
them for several minutes and placing a dot of
enamel paint on the thorax. This procedure had
no adverse effects, and males resumed normal ac-
tivities within minutes of handling.
Flies were placed in the cages between 0730-
0800 hours and monitored continuously for the
next 5 h. Cages were placed adjacent to east-fac-
ing windows and thus received both natural and
artificial light. Mating pairs were collected by
gently coaxing couples into vials, which were then
labeled and placed on holding trays. Vials were
monitored continuously, and times of collection
(i.e., mating start) and break-up (i.e., mating stop)
were recorded for each pair. When mating ceased,
males were removed and discarded, and females
were held in plastic buckets for the remating tri-
als (see below; data on copulation duration and fe-
male remating were collected only for
experiments 1 and 2). All unmated flies were dis-
carded. Two or 3 cages were run on each of 5 days
for all experiments. Eleven replicates (cages)
were run for each experiment.
Mated females were provided with food and wa-
ter as above and a perforated, plastic vial (contain-
ing a sponge soaked with lemon juice) for
oviposition. Two days after their initial mating,
groups of females that mated with males of the
same diet type were placed in glass cages (30 cm
cubes with a cloth sleeve covering one side) with an
equal number of protein-fed, wild males between
0730-0800 hours. Cages were monitored for 5 h,
and copulation durations were recorded as noted
above. Females that remated 2 d after the initial
mating were discarded, and the remaining females
were held and re-tested in the same manner 4 d af-
ter the initial mating. Females that failed to mate
at this time were held and tested a final time 7 d af-
ter the initial mating. Males used in the different
remating trials were 11-19 d old. Female mortality
was relatively low and varied independently of the
strain or diet of the initial mate. Over all females,
95% survived from the initial mating to the 2-d
test, 96% survived from the 2-d test to the 4-d test,
and 91% survived from the 4-d to the 7-d test.

Male Longevity

Two experiments were performed to investi-
gate the effect of adult diet on the longevity of


mass-reared males. First, the probability of sur-
viving the first week of adult life was compared
between protein-fed and protein-deprived males.
Emerging males were collected between 0600-
0900 hours, and groups of 50 individuals were
placed in screen cages (30 cm cubes) and given
continuous access to water plus sugar only or the
protein-sugar mixture. Dead flies were removed
daily. Twenty-one cages were run for each diet
type. The second experiment examined the effect
of dietary protein on male longevity following food
removal. Newly emerged males were protein-fed
or protein-deprived for 4 d following eclosion. On
the morning of the fifth day, groups of 50 males
from a given diet were placed in screen cages with
water only, and deaths were recorded every morn-
ing over the next 4 d (by which time all individu-
als in both treatments were dead; see below).
Nine cages were run for each diet type.

Statistical Analyses

The Mann-Whitney test (test statistic T) was
used for pairwise comparisons involving male
weights, the number of matings achieved by com-
peting male types, and the number of surviving
males from the two diet groups. With respect to
sexual competition, this test does not explicitly
test for deviation from random mating (i.e., each
male type accounts for 50% of the total matings),
so a binomial test (using the normal approxima-
tion and Z scores with Yates correction for conti-
nuity) was performed with data pooled over all
replicates. Remating frequencies were compared
using the log-likelihood ratio for contingency ta-
bles (test statistic x2, where df = (rows-1) X (col-
umns-1)) with Yates correction for continuity.
Because sample sizes were relatively small (espe-
cially for females initially mated to mass-reared
males) and because remating was a relatively
rare event, comparisons were made using data
pooled over all groups of females initially mated
to the same male type. Copulation durations were
compared among male types using the Kruskal-
Wallis test (test statistic H). Statistical proce-
dures followed Zar (1996).

RESULTS

Adult Body Weight

Among wild males, diet had no apparent effect
on weight; protein-fed males had an average
weight of 7.9 mg (SD = 1.2; range: 5.4-9.8 mg)
compared to 7.5 mg (SD = 1.0; 5.4-9.4 mg) for pro-
tein-deprived males (T = 799, P > 0.05, n,= n = 30
in this and subsequent weight comparisons).
Among mass-reared males, however, protein-fed
males (x = 7.8 mg; SD = 0.9; range: 6.0-9.7 mg)
were significantly heavier, on average, than pro-
tein-deprived males (x = 7.3; SD = 0.7; range: 6.0-


December 2002







Shelly & Kennelly: Dietary Effects on Mating in the Medfly


8.6 mg). Wild and mass-reared males fed protein-
containing diets did not differ significantly in
body weight (T = 926.5, P > 0.05), but protein-fed,
wild males were significantly heavier than pro-
tein-deprived, mass-reared males (T = 1078.0, P <
0.05). The average weight of wild females was 8.2
mg (SD = 1.3; range: 5.0-10.7), which did not dif-
fer significantly from that of protein-fed, wild (T =
986; P > 0.05) or mass-reared (T = 999.0; P > 0.05)
males.

Male Mating Success

In the experiment using wild males exclu-
sively, diet had a marked effect on male mating
success, and protein-fed males obtained an aver-
age of 16.5 matings per replicate compared to only
10.2 for protein-deprived males (Table 1). Protein-
fed, wild males obtained 61% (182/296) of the to-
tal matings observed over all replicates (Z = 4.0; P
< 0.001).
Diet had a lesser effect on the outcome of mat-
ing competition involving mass-reared males only
(Table 1). On average, protein-fed males obtained
a greater number of matings per replicate than
protein-deprived males (6.6 versus 4.5, respec-
tively), but this difference was not statistically
significant. However, pooling data over all repli-
cates, we found that protein-fed, mass-reared
males obtained a disproportionately larger num-
ber of matings than expected by chance alone (73/
123 = 59%; Z = 2.2; P < 0.05). Combined over both
diet types, the total number of matings recorded
per replicate was significantly higher for wild
than mass-reared males (26.7 vs. 11.2, respec-
tively; T = 184.0; n = n2 = 11; P < 0.001).
In the final experiment, where wild and mass-
reared males competed directly, diet had no de-
tectable influence on the relative mating success
of mass-reared males (Table 1).When both strains
were protein-fed, wild males obtained an average
of 19.0 matings per replicate compared to 5.4 for
the mass-reared males. In this case, wild males
obtained 78% (209/269) of the matings over all
replicates (Z = 10.9, P < 0.001). When the mass-


reared males were protein-deprived, the wild
males obtained an average of 18.4 matings per
replicate compared to only 4.1 for the mass-reared
males. Wild males obtained 82% (202/247) of the
total matings recorded over all replicates (Z =
13.2, P < 0.001). In competition with protein-fed
wild males, there was no significant difference in
the proportion of total matings obtained by pro-
tein-fed and protein-deprived mass-reared males
(2 = 1.4, P > 0.05).

Female Remating

In tests involving wild males, female remating
tendency was independent of the diet of the mat-
ing partner in tests conducted 2, 4, or 7 d after the
initial mating. Approximately 10% of the females
that mated with protein-fed (18/173) or protein-
deprived (11/107) males remated 2 d after the ini-
tial mating (X2 = 0.3; df = 1 in this and all subse-
quent pairwise comparisons of frequency; P >
0.05). In tests conducted 4 d after the first mating,
4% (6/150) and 2% (2/95) of females mated to pro-
tein-fed and protein-deprived males remated, re-
spectively (x2 = 0.4; P > 0.05). In tests conducted 7
d after the first mating, 7% (9/131) and 6% (5/79)
of females mated to protein-fed and protein-de-
prived males remated, respectively (X2 = 0.2; P >
0.05). Combining data over diet types, we found
significant variation in the incidence of remating
over the different intervals tested (X2 = 10.6, P <
0.01), reflecting primarily the large difference in
remating probability between 2 d (29/280 = 10%)
and 4 d (8/245 = 3%) after the initial mating.
In tests involving mass-reared males, female
remating tendency was also independent of the
diet of the mating partner. Remating frequencies
2 d after the initial mating were 3% (2/67) for fe-
males that mated with protein-fed males and 6%
(3/46) for females that mated with protein-de-
prived males (X2 = 0.6; P > 0.05). In tests con-
ducted 4 d after the first mating, none of the
females that initially mated to protein-fed or pro-
tein-deprived males remated (X2 = 0; P > 0.05). In
tests conducted 7 d after the first mating, 7% (4/


TABLE 1. INFLUENCE OF MALE DIET AND STRAIN ON MATING SUCCESS. MATING VALUES REPRESENT AVERAGE (+1 SD)
NUMBER OF MATINGS PER REPLICATE (N = 11 IN ALL CASES).

Experiment Male strain Male diet Matings T Significance

1 Wild Protein-fed 6.5 (3.8) 161.0 P <0.05
Wild Protein-deprived 10.2 (3.1)
2 Mass-reared Protein-fed 6.6 (2.6) 147.0 P > 0.05
Mass-reared Protein-deprived 4.5 (2.0)
3a Wild Protein-fed 19.0 (4.2) 187.0 P < 0.001
Mass-reared Protein-fed 5.4 (2.2)
3b Wild Protein-fed 18.4 (5.2) 189.0 P < 0.001
Mass-reared Protein-deprived 4.1 (2.2)











TABLE 2. SURVIVAL AND REMATING OF WILD FEMALE
MEDFLIES FIRST MATED TO WILD VERSUS LABO-
RATORY MALES FED PROTEIN-CONTAINING (+)
OR PROTEIN-DEPRIVED (-) DIETS.

Identity of first mate

Wild male Laboratory male
Female
event Protei + Protein Protei + Protein -

Mate initially 182 114 73 50
Die prior 2 d 9 7 6 4
Test at 2 d 173 107 67 46
Mate at 2 d 18 11 2 3
Die prior 4 d 5 1 6 3
Test at 4 d 150 95 59 40
Mate at 4 d 6 2 0 0
Die prior 7 d 13 14 4 1



55) and 5% (2/39) of females mated to protein-fed
and protein-deprived males remated, respectively
(X2 = 0.2; P > 0.05). Combining data over diet
types, we found that the probability of female re-
mating varied significantly over the intervals
tested (X2 = 9.0; P < 0.05), owing chiefly to the ab-
sence of any remating in tests conducted 4 d after
the initial mating.
Combining data over diet types, we found no
difference in remating frequency between fe-
males mated to wild or mass-reared males for any
time interval tested (2 d: wild 29/280, mass-
reared 5/113, x2 = 3.4; P > 0.05; 4 d: wild 8/245,
mass-reared 0/99, X2 = 3.0; P > 0.05; 7 d: wild 14/
210, mass-reared -6/94, X2 = 0.2, P > 0.05). Using
data combined across male diet and male strain,
we found significant variation in the incidence of
female remating over the intervals tested (X2 =
14.8, P < 0.001). Over all females, remating fre-
quencies were 9% (34/393) at 2 d, 2% (8/344) at 4
d, and 6% (20/304) at 7 d after the initial mating.

Copulation Duration

For initial matings, copulation duration varied
independently of male strain and diet (H = 6.1; df
= 3; P > 0.05; Fig. 1). Among wild males, mating
times averaged 144.3 min (SD = 40.8; range: 31 -
275) and 141.5 min (SD = 46.3; range: 21 239) for
the protein-fed (n = 182) and protein-deprived (n
= 114) males, respectively. Among mass-reared
males, mating times averaged 135.6 min (SD =
41.5; range: 11 256) and 134.4 min (SD = 39.8;
range: 67 223) for the protein-fed (n = 73) and
protein-deprived (n = 50) males, respectively. In-
dependent of diet, there was no significant differ-
ence in copulation duration for initial matings
with wild (x = 143.3 min; SD = 42.3; n = 296) and
mass-reared (x = 135.1; SD = 40.6; n = 123) males
(T = 22,054.0; P > 0.05).


30

25

20-

15

10 *

5

0






30

25

Z 20

15

10
u lo



o
0


SProtein-fed
[ Protein-deprived


December 2002


Wild males


0 30 60 90 120 150 180 210 240
Copulation duration (min)


Protein-fed
E Protein-deprived


0 30 60 90


Laboratory males


120 150 180 210 240


Copulation duration (min)

Fig. 1. Frequency distributions of duration of initial
copulations between wild females and protein-fed (n =
182) and protein-deprived (n = 114) wild males (top) and
protein-fed (n = 73) and protein-deprived (n = 50) mass-
reared males (bottom).


For females mated initially with wild males
(independent of diet), the duration of second mat-
ings did not vary significantly with the number of
days elapsed since the initial mating (H = 1.4; n,
= 29, n2 = 8, n3 = 14; df = 3; P > 0.05). Likewise,
among females first mated with a mass-reared
male (independent of diet), there was no signifi-
cant difference in the length of rematings that oc-
curred 2 d or 7 d after the initial mating (T = 29.0;
n, = 5, n, = 6; P > 0.05). Rematings (x = 114.4 min;
SD = 45.1) were significantly shorter than initial
matings for females first mated to wild males (T =
6107.0; n, = 296, n, = 51; P < 0.01). For females
first mated to mass-reared males, rematings (x =
121.5; SD = 23.6) were similar in length to initial
matings (T = 633.5; n, = 123, n, = 11; P > 0.05).

Male Longevity

Among mass-reared males, survival probabil-
ity to 7 d of age was independent of diet type. On
average, 38.5 (SD = 4.5) protein-fed males per


Florida Entomologist 85(4)







Shelly & Kennelly: Dietary Effects on Mating in the Medfly


cage survived the first week of life compared to
37.4 (SD = 4.0) protein-deprived males (T = 494.0;
n, = n, = 11; P > 0.05).
Survivorship following food removal was like-
wise independent of diet type. On average, 45.0
(SD = 3.7) protein-fed males per cage were alive 1
d following food removal compared to 46.7 (SD =
2.2) of the protein-deprived males (T = 97.0; n, =
n, = 9; P > 0.05). Two days after food removal, an
average of 4.4 (SD = 3.6) protein-fed and 5.4 (SD
= 5.0) protein-deprived males were alive per cage
(T = 89.0; n, = n, = 9; P > 0.05). Of the 900 males
observed in this experiment, only 4 protein-fed
and 3 protein-deprived males survived for 3 d af-
ter food removal, and none survived to 4 d in ei-
ther treatment.

DISCUSSIoN

Consistent with previously conducted field-
cage trials (Shelly et al. 2001), the laboratory data
reported here revealed that, among wild male
medflies in Hawaii, protein-fed individuals
achieved significantly more matings than protein-
deprived individuals in direct competition for wild
females. In the field-cage trials, we found no differ-
ence in levels of pheromone-calling between pro-
tein-fed and protein-deprived, wild males (Shelly
et al. 2001), and consequently it appears that the
difference in mating frequency reported here does
not simply reflect a diet-mediated difference in
sexual signaling. This situation differs from other
studies reporting increased calling and mating for
mass-reared (Kaspi & Yuval 2000) and wild (Kaspi
et al. 2000) male medflies fed protein as adults. Al-
though diet-mediated variation in signaling activ-
ity may not be evident among wild males in
Hawaii, the inclusion of protein in the adult diet
may have resulted in qualitative differences in the
composition, and hence attractiveness, of the male
sex pheromone. In comparing female arrivals to
artificially established leks in the field, we found
that, while signaling activity did not vary notice-
ably with diet, approximately twice as many fe-
male sightings were made at leks composed of
protein-fed males than at leks composed of pro-
tein-deprived males (Shelly et al. 2001).
In contrast to wild males, mating success was
independent of adult diet among mass-reared
males whether the competition involved mass-
reared males only (experiment 2, although a mat-
ing advantage for protein-fed males was sug-
gested when data were pooled over all replicates)
or wild and mass-reared males (experiment 3). Al-
though the results are preliminary, ongoing field-
cage tests (T. E. S., unpublished data) similarly
indicate that, relative to wild males, the inclusion
of protein in the adult diet has no influence on the
mating success of mass-reared males from a ge-
netic sexing (temperature sensitive lethal, Franz
& McInnis 1995) strain. Thus, contrary to other


studies (Blay & Yuval 1997; Taylor & Yuval 1999;
Kaspi & Yuval 2000), our results do not support
the notion that the addition of protein to the pre-
release diet would, in general, enhance the mat-
ing competitiveness of mass-reared medfly males
in SIT programs (but see below).
Male body weight had no obvious impact on the
outcome of mating competition in any of the ex-
periments. Among wild males, protein-fed and
protein-deprived males were similar in body
weight, yet protein-fed males enjoyed a signifi-
cant mating advantage (experiment 1). Con-
versely, protein-fed, mass-reared males were
heavier, on average, than protein-deprived, mass-
reared males, yet there was no difference in mat-
ing frequency between them (experiment 2).
When wild and mass-reared males competed di-
rectly, the protein-fed and protein-deprived,
mass-reared males had similar mating success
relative to wild males despite the fact that wild
males were similar in weight to protein-fed, mass-
reared males but significantly heavier than pro-
tein-deprived, mass-reared males (experiment 3).
The low level of mating observed in the exper-
iment involving mass-reared males exclusively
(experiment 2) suggests that mass-reared males
were generally unacceptable to wild females inde-
pendent of their diet. This interpretation, in turn,
suggests that females of C. capitata select males
largely on the basis of "absolute" criteria and not
on a relative or "best-of-n" basis (Janetos 1980).
That is, females may accept only males above a
certain threshold level for a particular sexual sig-
nal(s) and reject other males. If true, C. capitata
females are not choosing mates on the basis of be-
tween-male comparisons made over a sampling
interval, and consequently the relative abun-
dance of males of varying quality does not affect
female choice. This, in turn, implies that mass-
rearing facilities for SIT should concentrate, not
simply on increasing production of males, but on
maintaining sexual competitiveness of the males
as well (Calkins 1984).
The frequency of female remating varied inde-
pendently of male diet regardless of whether the
initial mate was a wild or mass-reared male. This
finding differs from that of Blay and Yuval (1997),
who, in their study of mass-reared flies, found
that females that first mated with a protein-fed
male were less likely to remate (on the day follow-
ing the initial mating) than females that first
mated with a protein-deprived male. In that
study, protein-fed males were significantly
heavier than protein-deprived males, a difference
that may have affected renewal of female recep-
tivity (Blay & Yuval 1997; see also Bloem et al.
1993a). Although diet-related differences in male
weight were detected in our study, the incidence of
female remating was similar following initial
mating to males of different sizes (e.g., protein-fed
and protein-deprived, mass-reared males).











We also noted that, when data were combined
over male diet types, wild females initially mated
to wild or mass-reared males remated at approxi-
mately the same frequency. To our knowledge, no
prior studies have drawn this comparison despite
its potential importance for SIT programs. Sev-
eral studies, however, have examined the effect of
irradation per se on female remating, and the re-
sults obtained are contrary to our finding. Work-
ing exclusively with laboratory flies, Katiyar and
Ramirez (1970) and Bloem et al. (1993b) both
found that females first mated to irradiated males
were more likely to remate than females whose
initial mate was non-irradiated.
Within wild and mass-reared strains of the
medfly, male diet had no apparent effect on copu-
lation duration. Taylor et al. (2000) likewise found
no effect of male diet on copulation duration for
matings involving flies from a wild population in
Israel. However, male diet has been found to af-
fect copulation duration in matings involving
mass-reared flies (Taylor & Yuval 1999; Field &
Yuval 1999), with copulations involving protein-
fed males being shorter than those involving pro-
tein-deprived males. Independent of male diet, we
also found no difference in copulation duration be-
tween matings (with wild females) involving wild
or mass-reared males (see also Orozco and Lopez
1993). Using laboratory flies exclusively, several
studies have examined the influence of irradia-
tion on males on copulation duration with incon-
sistent results: Katiyar and Ramirez (1973) found
no effect of irradiation, whereas Seo et al. (1990)
reported that copulations involving irradiated
males (and non-irradiated females) were shorter
than those involving non-irradiated males.
Data from the present study contribute further
evidence for global variation in copulation dura-
tion in the medfly. For example, copulation dura-
tion is similar between wild flies from Hawaii (x
= 143 min) and Israel (median = 145 min; Taylor
et al. 2000) but is appreciably longer for wild flies
in Argentina (x = 172 min; Cayol et al. 1999).
Conversely, copulation duration between mass-
reared males and wild females is similar between
Hawaii (x = 135 min) and Argentina (x = 134 min
calculated using from Table 1 in Cayol et al. 1999)
but is much longer for mass-reared flies in Israel
(median = 160 180 min; Field et al. 1999; Taylor
et al. 2001).
We also failed detect an effect of diet on the lon-
gevity of mass-reared males. In contrast, Kaspi
and Yuval (2000) found that, after 4 d of feeding,
protein-fed males were less likely to survive a 24
h period of starvation (on day 5) than protein-de-
prived males. Following the same protocol, we
found that the number of males surviving the
starvation period was nearly identical between
protein-fed and protein-deprived males. Survivor-
ship of protein-deprived males was similar be-
tween the two studies (approximately 10%), but


December 2002


protein-fed males had much higher mortality in
Kaspi and Yuval's (2000) study than in our exper-
iment (approximate mortality after 1-d starva-
tion: 50% versus 10%, respectively). Protein
hydrolysate comprised 25% of the protein-con-
taining diet in our study but only 9% in Kaspi and
Yuval (2000), but whether this difference was re-
sponsible for the differential mortality is un-
known.
In conclusion, the finding that dietary protein
affected mating frequency in wild males indicates
an important role for adult nutrition in mating
competition in the Mediterranean fruit fly, a con-
clusion consistent with other studies (Blay &
Yuval 1997; Taylor & Yuval 1999; Kaspi & Yuval
2000; Shelly et al. 2001). The absence of the same
effect in Hawaiian mass-reared males might indi-
cate inter-strain differences in the behavioral and
physiological responses of males to dietary compo-
sition. However, it appears more likely that it de-
rived from the low overall quality of the Hawaiian
mass-reared strain tested, which essentially'over-
whelmed' any positive effect resulting from the in-
clusion of dietary protein. This, in turn, suggests
that the potential benefits to SIT of including pro-
tein in the pre-release, adult diet will vary with
the quality of the mass-reared strain, being great-
est for males that compete relatively well against
wild males independently of diet composition.

ACKNOWLEDGMENTS

We thank Charmian Dang, Erik Rutka, and Mindy
Teruya for assistance in rearing the flies and Emma and
Miranda Shelly for helping with field collections of in-
fested fruits. Don McInnis and Boaz Yuval made valu-
able comments on an earlier draft. This work was
supported in part by the Binational Agricultural Re-
search and Development Fund to T.E.S. and Boaz Yuval
(Bard Project No. US-3256-01).

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TAYLOR, P. W., R. KASPI, S. MOSSINSON, AND B. YUVAL.
2001. Age-dependent insemination success of sterile
Mediterranean fruit flies. Entomol. Exp. Appl. 98:
27-33.
ZAR, J. H. 1996. Biostatistical analysis. Prentice-Hall,
Upper Saddle River, NJ.







Florida Entomologist 85(4)


December 2002


CURRENT DISTRIBUTION OF THE FORMOSAN
SUBTERRANEAN TERMITE AND OTHER TERMITE SPECIES
(ISOPTERA: RHINOTERMITIDAE, KALOTERMITIDAE) IN LOUISIANA


MATTHEW T. MESSENGER', NAN-YAO SU2 AND RUDOLF H. SCHEFFRAHN2
1City of New Orleans Mosquito and Termite Control Board,6601 Stars and Stripes Blvd., New Orleans, LA 70126

2Ft. Lauderdale Research and Education Center, University of Florida, Institute of Food & Agricultural Sciences,
3205 College Avenue, Ft. Lauderdale, FL 33314

ABSTRACT

A statewide survey in Louisiana on the current distribution of the Formosan subterranean
termite, Coptotermes formosanus Shiraki, and other termite species was conducted with 91
pest control companies, city and state agencies, and the New Orleans Mosquito and Termite
Control Board from January 1999 to August 2002. A total of 812 samples were used in the
survey constituting all eight known termite species from Louisiana. The subterranean ter-
mite species identified were Reticulitermes flavipes (Kollar), R. virginicus (Banks), R. hageni
Banks, and C. formosanus. The drywood termite species identified were Incisitermes snyderi
(Light), I. minor (Hagen), Cryptotermes brevis (Walker), and Kalotermes approximatus (Sny-
der). Incisitermes minor was also collected in Mississippi and is a new record in that state.
The collective data on the flight season of each species was also recorded.

Key Words: Coptotermes formosanus, Rhinotermitidae, Kalotermitidae

RESUME

Un reconocimiento de la distribuci6n actual de la termita subterranea formosana, Coptoter-
mes formosanus Shiraki y de otras species de termitas fue llevado a cabo en Louisiana,
EE.UU. con la colaboraci6n de 91 compafifas de control de plagas, las agencies estatales y
municipales, y el Bur6 de Control de Mosquitos y Termitas de Nueva Orleans desde enero de
1999 hasta agosto de 2002. Un total de 812 muestras fueron usados en el reconocimiento
constituyendo las ocho species de termitas conocidas de Louisiana. Las termitas subterra-
neas identificadas fueron Reticulitermes flavipes (Kollar), R. virginicus (Banks), R. hageni
Banks, y C. formosanus. Las termitas de madera seca identificadas fueron Incisitermes sny-
deri (Light), I. minor (Hagen), Cryptotermes brevis (Walker), y Kalotermes approximatus
(Snyder). Incisitermes minor fue tambien colectada en Mississippi y es un nuevo registro en
aquel estado. Los datos colectivos sobre la temporada de vuelos para cada especie tambi6n
fueron registrados.
Translation provided by author.


The Formosan subterranean termite (FST),
Coptotermes formosanus Shiraki (Isoptera: Rhino-
termitidae), was first identified in Lake Charles,
Louisiana, in 1966 and in New Orleans in 1967
(Spink 1967). It is widely believed that this exotic
species was introduced into the continental U.S.
after infested material was brought over from
Asia after World War II (Su and Tamashiro 1987).
For the past 30 years, FST infestations have been
found in other cities and various small communi-
ties throughout Louisiana. The main source of in-
troduction to these other areas is caused, in part,
by the transportation of infested building materi-
als, utility poles, and railroad ties used in land-
scaping (La Fage 1987). Then, natural spread has
occurred via alate dispersal flights.
The last statewide survey involving the pest
control community for all termite species was con-
ducted around the time of the first confirmed re-


port of the FST (Weesner 1965). During the last
survey, species and flight data were only recorded
from Rapides Parish, which includes the city of
Alexandria. Reticulitermes flavipes (Kollar), R.
virginicus (Banks), R. hageni Banks (Isoptera:
Rhinotermitidae), and Incisitermes snyderi
(Light) (Isoptera: Kalotermitidae) were collected
from this region of the state. Previously, Light
(1934) and Snyder (1954) listed five species in
Louisiana. They included R. flavipes, R. virginicus
(Isoptera: Rhinotermitidae), Kalotermes (=In-
cisitermes) snyderi, Kalotermes approximatus
(Snyder), and Cryptotermes brevis (Walker)
(Isoptera: Kalotermitidae). Recently, Messenger
et al. (2000) discovered established populations of
Incisitermes minor (Hagen) (Isoptera: Kaloter-
mitidae) in New Orleans.
Since the last national survey in 1965, individ-
ual statewide termite surveys have been con-







Messenger et al.: Louisiana Termite Survey


ducted in Georgia (Scheffrahn et al. 2001), Florida
(Scheffrahn et al. 1988), Texas (Howell et al.
1987), and South Carolina (Hathorne et al. 2000).
These surveys significantly contributed to our un-
derstanding of the current distribution of the eco-
nomically important FST.
Because there have been many unconfirmed re-
ports of the FST throughout the state, the main ob-
jective of this survey was to identify and confirm
the current distribution of the FST in Louisiana
with the help of the pest control industry, the Loui-
siana Department of Agriculture and Forestry, and
mosquito control districts. In addition, the New Or-
leans Mosquito and Termite Control Board concur-
rently conducted a separate statewide survey for all
subterranean and drywood termite species.

MATERIALS AND METHODS

Pest Management Professional (PMP) Survey

Beginning in January 1999, letters asking for
participation in the survey were mailed to 589
PMPs and mosquito control districts throughout
Louisiana, including a few pest control companies
operating near the state line in Mississippi and
Texas. Termite collecting packets were then pre-
pared and sent to each company who returned the
postcard with a response of willingness to partici-
pate. Each packet included individually num-
bered collection vials (13 ml polypropylene Snap-
Seal@, Corning Brand) containing 85% ethanol,
corresponding vial data sheets, return padded en-
velopes, and a hand-held aspirator. Each partici-
pant was encouraged to collect termite alates and
soldiers during routine inspections and treat-
ments of residential and commercial structures.
They were also encouraged to include any rele-
vant information from each collection on the data
sheet, which included date and location of collec-
tion, flight date (if applicable), and any additional
comments and requests for more collection vials.

N. O. Mosquito and Termite Control Board (NOMTCB)
Survey

The senior author and other coworkers con-
ducted a deliberate survey throughout Louisiana
from 1999 to 2001. Termites were collected from live
and dead trees, state parks, railroad ties, highway
rest areas, private and public buildings, and any
other type of wood found along highways and parish
roads. We also traveled to addresses throughout the
state to verify FST infestations and conduct further
surveys in the surrounding areas. In addition, sam-
ples and FST locations were received from J.
McPherson, Program Coordinator, Pesticide and
Environmental Programs, Louisiana Department
of Agriculture and Forestry, Baton Rouge, LA.
For both surveys, termite alates and soldiers
were identified to species using termite keys de-


veloped by Banks & Snyder (1920), Miller (1949),
Snyder (1954), Weesner (1965), Scheffrahn & Su
(1994), and Hostettler et al. (1995). Samples con-
taining only workers (Reticulitermes spp.) or
pseudergates were identified to the family and/or
genus level. Data from both surveys was entered
into a computer database (FileMaker Pro 3.0,
Claris@ Corporation). Longitude and latitude co-
ordinates from the NOMTCB survey were re-
corded at each sample site using a Garmin GPS
model 12 CX (Garmin International, Inc., Olathe,
KS) hand-held global positioning receiver. Loca-
tions of each collection were plotted using Arc-
View GIS version 3.1 software (Environmental
Systems Research Institute, Inc., Redlands, CA).

RESULTS

PMP Survey

Out of the original 589 survey letter mailings,
91 (15%) companies and individuals agreed to
participate by collecting any type of termite they
encountered during routine inspections and treat-
ments of urban structures and trees. There was no
response from 453 (77%) companies and 45 (8%)
responded, but declined to participate. The major-
ity of the companies who declined indicated that
they do not conduct termite treatments.
As a result, 52 of the 91 participants returned
collection vials for a total of 426 samples. All eight
known termite species were collected (Table 1).
The majority of these samples were collected from
separate addresses. Reticulitermes flavipes was
the most commonly collected species throughout
the state (Table 1). The FST was the second most
commonly collected species; however, the majority
of the FST samples were collected from the New
Orleans and Lake Charles areas (Table 1).
Each participant also included an exact or ap-
proximate date of disperal flight whenever they
collected alates. For the subterranean species,

TABLE 1. TOTAL NUMBER OF IDENTIFIED TERMITE SPE-
CIES FROM VIALS COLLECTED DURING THE PMP
SURVEY.

Number
Termite species of vials

Reticulitermes flavipes 204
Coptotermes formosanus 118
Reticulitermes virginicus 40
Incisitermes snyderi 9
Cryptotermes brevis 7
Incisitermes minor 3
Kalotermes approximatus 3
Reticulitermes hageni 3
Workers/pseudergates only 39
TOTAL 426







Florida Entomologist 85(4)


TABLE 2. TOTAL NUMBER OF TERMITE SPECIES AND SAM-
PLES COLLECTED DURING THE NOMTCB SUR-
VEY.

Number of
Termite species collections

Reticulitermes flavipes 177
Reticulitermes hageni 65
Reticulitermes virginicus 64
Coptotermes formosanus 40
Incisitermes snyderi 21
Incisitermes minor 9
Cryptotermes brevis 8
Kalotermes approximatus 2
TOTAL 386


R. flavipes alates were recovered from Jan. 17 to
April 19, R. virginicus alates from March 1 to May
17, R. hageni on Dec. 17, 2001 (single record), and
the FST from April 12 to May 9. For the kaloter-


mid species, I. snyderi alates were recovered from
May 10 to July 22, C. brevis from May 9 to July 25,
and K. approximatus from Oct. 10 to Nov. 1. Alate
samples of minor were collected from Sept. 10 to
Dec. 4 in Rayne, Cameron, and Le Moyeu, LA;
however, monitoring of dispersal flights by the se-
nior author in the New Orleans metro area oc-
curred each year from late April to early June.


NOMTCB Survey

Reticulitermes flavipes was by far the most
commonly collected termite species throughout
Louisiana (Table 2). Reticulitermes hageni and R.
virginicus were the two second most commonly
collected species (Table 2). The number of FST col-
lections only represents a few selected, confirmed
sites throughout the state and does not include
any samples taken from New Orleans. The distri-
bution of FST infestations in Louisiana has signif-
icantly increased since 1966 (Table 3).


TABLE 3. LOCATION OF COPTOTERMES FORMOSANUS INFESTATIONS IN LOUISIANA, 1966-2001.

Year Parish City


1966* Orleans


Calcasieu
Orleans
Calcasieu
Jefferson
La Fourche
Orleans
Calcasieu
Jefferson
La Fourche
St. Tammany
Lafayette
East Baton Rouge
Orleans
Calcasieu
Jefferson
La Fourche
St. Tammany
Lafayette
East Baton Rouge
Ascension
St. Charles
Assumption
Terrebonne
St. Bernard
Plaquemines
Iberia
Vermilion
St. Landry
Sabine
Ouachita
Acadia
St. Mary


New Orleans, Algiers
Lake Charles
New Orleans, Algiers
Lake Charles
Grand Isle
Raceland
New Orleans, Algiers
Lake Charles, Westlake
Metairie, Gretna, Grand Isle
Raceland
Slidell, Covington
Lafayette
Baton Rouge
New Orleans, Algiers
Lake Charles, Westlake, Moss Bluff, Sulphur
Metairie, Gretna, Grand Isle, Kenner, Harahan, Westwego, Marrero
Raceland, Thibodaux, Larose, Cut Off, Galliano
Slidell, Covington
Lafayette
Baton Rouge
Prairieville
Norco
Pierre Part
Houma, Montegut
Chalmette
Belle Chase
New Iberia
Abbeville
Sunset
Noble
Monroe, West Monroe
Rayne
Amelia


1968*



1986*







2001



















2002


*La Fage 1987.


December 2002







Messenger et al.: Louisiana Termite Survey


In New Orleans, FST flight activity was moni-
tored by the senior author using glue traps (TRAP-
PER LTD, Bell Laboratories, Inc., Madison, WI)
installed under lights near the French Quarter.
Nightly observations and the number of FST alates
recovered from glue traps reveal peak flight activity
usually occurs from mid-May to early June, with
some activity through mid-July (Table 4).
The majority of the I. minor and C. brevis sam-
ples were received from J. McPherson and local
residents of New Orleans.
Location data from both surveys for the FST
(Fig. 1), Reticulitermes species (Fig. 2), and kalo-
termid species (Fig. 3) are presented on ArcView-
generated maps.


DISCUSSION

The distribution of the FST in Louisiana has
increased dramatically since the first confirmed
reports in the mid-60s. However, many of these
newer, confirmed infestations have remained rel-
atively localized, and state officials have begun to
target these areas for immediate treatment. Most
of these localized introductions have occurred
around structures, such as churches, or parks and
campsites where FST-infested railroad ties were
used as landscaping and/or building material. Fu-
ture monitoring and confirmation of any new FST
reports throughout the state is the first step to
controlling human-aided spread.


TABLE 4. COMBINED ALATE FLIGHT DATES FOR COPTOTERMES FORMOSANUS IN NEW
1998 TO 2001.


ORLEANS, LOUISIANA, FROM


Date Year Date Year Date Year


May 4
May 11
May 16
May 18*
May 19
May 22
May 26
May 29*
June 6
June 8
June 18
June 21
June 23
June 28
2nd week of Feb.
April 6
April 26
April 27
April 28
May 6
May 7
May 12*
May 14
May 17
May 18
May 19
May 25
May 27
May 29*
June 6
June 9
June 10
June 22
June 27
1st week of July
July 7
July 13
3rd week of July


2nd week of Jan.
2nd week of Feb.
March 26
April 18
April 25
April 29
May 3
May 4
May 5
May 8
May 10
May 14
May 15 *
May 21
May 22
May 26
May 29
June 4
June 15
June 16
June 17


*Largest dispersal flight (s) each year.


2000


April 8
April 13
April 14
April 15
April 16
April 24
May 1
May 4
May 5
May 7
May 9
May 12
May 13
May 14
May 15*
May 17
May 20
May 21
May 22
May 23
May 24
May 25
May 26
May 31
June 1*
June 4
June 7
June 11
June 14
June 18
June 21
June 24
June 28







Florida Entomologist 85(4)


Fig. 1. Current distribution of Coptotermes formosanus in Louisiana.


Outside the New Orleans and Lake Charles
areas, R. flavipes and R. virginicus are the two
most economically important subterranean ter-
mite species, with R. flavipes being the most com-
mon. The spatial distribution of all three
Reticulitermes species is consistent statewide;
however, R. flavipes seems to be more common in
the extreme southern portions of the state. For ex-
ample, samples of R. flavipes were collected from
house pilings directly in the sand at Holly Beach
on the Gulf of Mexico and from fishing camps
around the Mississippi River delta basin.
During the PMP survey, R. hageni was rarely
encountered in structures. In addition, K. approx-
imatus was only collected from dead portions of
trees and from alates flying into the vehicles of
participants on two separate occasions. For both
species, this confirms their general status as very
limited structural pests (Weesner 1970, Schef-
frahn et al. 1988).
Incisitermes snyderi and C. brevis are the two
most economically important kalotermid species
in Louisiana, with I. snyderi being the most com-


mon. Cryptotermes brevis is a non-endemic spe-
cies and has only been recovered from structural
lumber and furniture. Incisitermes snyderi is an
endemic species commonly found in structural
lumber and in dead portions of live trees through-
out the southern half of the state.
The overall number of I. minor collections
throughout the state was unexpected. Another in-
teresting discovery was the number of public
schools throughout the state with very active I
minor infestations, particularly in window frame-
work. Incisitermes minor is endemic to CA, AZ,
and Mexico, but has been introduced to many ar-
eas in the state, and in most cases, inside furni-
ture. For example, a sample was taken from an
infested pool table in Natchez, MS. In New Or-
leans, I minor alates are usually collected from
mid-April to mid-June during midday flights.
However, alates were recovered after swarming
from a window frame in an elementary school in
Rayne, LA, during the second week of September
2001. In addition, I minor alates were collected
after swarming in a high school in Cameron, LA,


December 2002







Messenger et al.: Louisiana Termite Survey


Fig. 2. Combined distribution data of Reticulitermes spp. in Louisiana from PMP and NOMTCB surveys.


in late September 2001. Historical records reveal
the flight season of I. minor usually occurs from
July to December, and as early as May in the lab-
oratory (Harvey 1934). In addition, I. minor flight
records in California (Snyder 1954), Florida
(Scheffrahn et al. 1988), and Georgia (Scheffrahn
et al. 2001) revealed swarming usually occurs
from September to November. An alarming dis-
covery revealed I. minor alates swarming in a
lumberyard near Le Moyeu, LA, in December
2001. This could lead to future introductions
throughout the state.
In addition to the overall survey, a pictorial ter-
mite identification key was developed in 2001 to
help PMPs, state officials, and termite researchers
identify the FST and other economically important
subterranean and drywood termite species cur-
rently present in Louisiana (Messenger 2002).

ACKNOWLEDGMENTS
We are grateful to E. S. Bordes, M. K. Carroll, and J.
C. McAllister (NOMTCB) for reviewing the manuscript.
Special thanks to John McPherson, Louisiana Depart-


ment of Agriculture and Forestry, for providing samples
and FST locations. The senior author would like to
thank the following NOMTCB employees, Mike Schultz,
Perry Ponseti, and Gus Ramirez, for their help in col-
lecting termites. We would also like to thank the follow-
ing individuals for advice, information, and collecting
support: Dan Foster and Chris Castalano (Terminix-
Houma), Eddie Martin and Vincent Palumbo (Ter-
minix-Metairie), Zack Lemann (Audubon Institute),
and Claudia Riegel (Dow AgroSciences LLC). We are
very grateful to the following pest control companies for
submitting termite samples: Al's Pest Control Service,
Inc.; Louisiana Bug Doctors; Beasley Pest Control, Inc.;
Pest Aid Co. of Alexandria, Inc.; Dial One Franklynn
Pest Control; American Exterminating Co.; McKenzie
Pest Control; Absolute Termite Control; Foti Extermi-
nating Co.; Edgewood Pest Control, Inc.; Johnny Jones
Pest Control Co.; Joyner's Pest Control; Fischer Envi-
ronmental Services, Inc.; Terminix-Gretna; Al Lati-
olais Exterminating Co.; International Rivercenter;
Responsible Pest Management LLC.; Sikes Pest Con-
trol, Inc.; J & R Pest Control, Inc.; Hubbards Pest Con-
trol; Tri-Parish Pest Control Co., Inc.; David Carter
Exterminating Co., Inc.; Denney Exterminating Co.;
Environmental Termite and Pest Control; Orkin Exter-
minating-Baton Rouge; E.A. Redd Pest Control, Inc.;


* Reticulitermes flavipes
o Reticulitermes virginicus
A Reticulitermes hageni







Florida Entomologist 85(4)


December 2002


Fig. 3. Combined distribution data of kalotermid species in Louisiana from PMP and NOMTCB surveys.


Richard L. Robards Termite Services; Hookfin Pest Con-
trol Co., Inc.; Sugarland Exterminating Co., Inc.; Cou-
hig Southern Environmental; Terminix-Slidell; Anti-
Pest & Veitch, Inc.; Kevin's Pest Control, Inc.; Slug-A-
Bug Exterminating Co.; E & G Pest Control, Inc.; Jer-
ome Williams Pest Control Co.; Woods Pest Control;
Sears Termite & Pest Control Inc.; Billiot Industries,
Inc.; Vexcon Inc.; Stetler Pest Control; A Plus Extermi-
nators, Inc.; Brent's Pest Control Services; Guardian
Pest Control; Arceneaux Consulting; Calcasieu Parish
Mosquito Control; East Baton Rouge Mosquito and Ro-
dent Control; Mosquito Control, Inc.; St. Bernard Parish
Mosquito Control; Louisiana Department of Agriculture
and Forestry; and USDA-ARS SRRC. Partial funding
for this project was provided by USDA-ARS under the
grant agreement No. 58-6435-8-108. This article is Flor-
ida Agricultural Experiment Station Journal Series No.
R-08828.


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Florida Entomologist 85(4)


December 2002


DISTRIBUTION AND NATURAL PARASITISM OF SPODOPTERA
FRUGIPERDA (LEPIDOPTERA: NOCTUIDAE) EGGS AT DIFFERENT
PHENOLOGICAL STAGES OF CORN


EDUARDO B. BESERRA1, CARLOS T. DOS S. DIAS2 AND JOSi R. P. PARRA3
1Univ. Estadual da Parafba, Depto. de Farmncia e Biologia, ex 781/791, 58100-000 Campina Grande, PB, Brazil

2ESALQ-USP, Depto. de Ci6ncias Exatas, ex 09, 13418-900, Piracicaba, SP, Brazil Entomol

3ESALQ-USP, Fitop e Zool. Agricola, ex 09,13418-900, Piracicaba-SP, Brazil

ABSTRACT

The oviposition behavior of Spodoptera frugiperda (J. E. Smith) and natural parasitism of
this pest by Trichogramma spp. at different phenological stages of corn were evaluated un-
der field conditions. The distribution of S. frugiperda eggs varied according to the phenolog-
ical stage of the corn. The preferred site for oviposition was the lower region of the plant and
the abaxial leaf surface during the early development stages of the crop (4-6 leaves), chang-
ing to the middle and upper regions of the plant and the adaxial leaf surface at subsequent
stages (8-10 and 12-14 leaves). A larger number of egg masses, and, therefore, of eggs was
collected at the 4-6 and 8-10 leaf stages compared to plants in the 12-14 leaf stages. Natural
parasitism was low, with a maximum of 2.21% eggs parasitized, especially on the lower and
middle parts of the plant. The distribution and degree of parasitism by Trichogramma spp.
on different regions of the plant were independent of the developmental stage of the crop.
Trichogramma pretiosum Riley was the most frequent parasitoid, found in 93.79% of the
parasitized eggs, followed by Trichogramma atopovirilia Oltman & Platner, with 2.07%.

Key Words: fall armyworm, Trichogramma, egg parasitoid, oviposition behavior, Zea mays

RESUME

Se evalu6 el comportamiento de la oviposici6n de Spodoptera frugiperda (J. E. Smith) y en
el parasitismo natural de esta plaga por Trichogramma spp. a diferentes tapas fenol6gicas
de maiz bajo condiciones del campo. La distribuci6n de los huevos de S. frugiperda vari6 se-
gin la 6tapa fenol6gica del maiz. El sitio preferida para la oviposici6n fu6 la region inferior
de la plant y la superficie abaxial (dorsal o del envez) de la hoja durante las tapas tempra-
nas de desarrollo del cultivo (4-6 hojas), cambiando a las regions medianas y superiores de
la plant y la superficie adaxial (ventral o del haz) de la hoja en las tapas subsecuentes (8-
10 y 12-24 hojas). Un mayor nmiero de masas de huevos, y, por lo tanto, un mayor numero
de huevos fueron recolectados en las tapas de 4-6 y de 8-10 hojas en comparaci6n con plan-
tas en la 6tapa de 12-14 hojas. El parasitismo natural fu6 bajo, con un maximo de 2.21% de
huevos parasitados, especialmente en las parties bajas y medias de la plant. La distribuci6n
y el grado de parasitismo por Trichogramma spp.en diferentes regions de la plant fu6 in-
dependiente de la 6tapa de desarrollo del cultivo. Trichogramma pretiosum Riley fu6 el pa-
rasitoide mas frecuentemente encontrado con el 93.79% de los huevos parasitados, seguido
por Trichogramma atopovirilia Oltman & Platner, con el 2.07% de los huevos parasitizados.


In Brazil, Spodoptera frugiperda (J. E. Smith)
is a common species in corn plantations. It is a
pest of great economic importance, with produc-
tion losses reaching 34% (Carnevalli & Florcovski
1995). It is found in corn from plant emergence up
to the ear stage, within which it is often sheltered,
and in many cases has become as important a pest
as the corn earworm, Helicoverpa zea (Boddie)
(Parra et al. 1995).
Morphological and physiological variations oc-
cur during the development and maturation of
plants, often provoking changes in S. frugiperda
egg distribution and egg mass characteristics,


such as number of egg layers and scale density.
These may be due to nutritional factors that vary
as a consequence of physiological changes in the
plants. S. frugiperda egg mass distribution on the
host plant is influenced by the phenological stage
of the crop, and can be concentrated within the
lower, middle or upper plant regions and on differ-
ent parts of the leaves and fruiting structures (Ali
et al. 1989, Sifontes et al. 1988, Meneses et al.
1991). Sifontes et al. (1988) found that as the rice
plant host grew older, S. frugiperda egg masses
had a higher scale density, as well as a larger
number of layers and eggs.







Beserra et al.: Parasitism of S. frugiperda


A possible control for S. frugiperda is the use of
egg parasitoids like Trichogramma. These spe-
cies, however, have difficulties in parasitizing egg
masses of this pest because they are covered in
scales and the eggs are deposited in layers
(Toonders & Sanchez 1987, Cortez & Trujillo
1994). Thus, optimal use of Trichogramma spp. to
control S. frugiperda requires information on egg
placement by S. frugiperda during development of
the corn crop, especially egg distribution in layers
and the presence of scales, as well as the degree of
natural parasitism by Trichogramma spp. The
aim of this research was to evaluate the egg dis-
tribution and the natural parasitism of S. fru-
giperda eggs at different phenological stages of
corn.

MATERIALS AND METHODS

The trials were conducted at the Areao Farm,
in Piracicaba, state of Sao Paulo, Brazil, at
22o42'00" South latitude, 47o38'00" West longi-
tude and 546 meters altitude. Data were collected
from March-July, 1998 and 1999, October-De-
cember, 1998, and from February-April, 2000.
Each time a 2-hectare field, with approximately
50,000 corn plants per hectare was studied. The
crop received conventional cultural practices rec-
ommended for corn, except that no insecticides
were applied after the first leaves appeared.
Weekly, 250 plants were sampled, randomly
distributed among 10 sampling spots, approxi-
mately 30 meters apart. In each spot, 25 plants in
an "X" distribution were evaluated, with 5 plants
in the center and 5 at each end of the "X". Each
group thus included 5 plants, approximately 5
meters from the next group. The plants were eval-
uated thoroughly and the plant height, the num-
ber of leaves per plant and the egg-laying sites
were recorded. The eggs were grouped according
to the plant region (lower, middle or upper) and
leaf surface abaxiall or adaxial) and distribution
and natural parasitism were studied at the corn
plant phenological stages of 4-6, 8-10 and 12-14
leaves (Cruz & Turpin 1982). The eggs were col-
lected and placed in labeled plastic tubes (3.5 x
1.0 cm), taken to the laboratory, and then kept in
glass tubes (8.5 x 2.5 cm). Data were collected on
the number of egg layers, the presence or absence
of scales and the number of eggs, based on the
number of larvae that hatched. Natural parasit-
ism by Trichogramma spp. was determined by
counting the number of parasitized eggs. The S.
frugiperda eggs were placed in glass tubes (8.5 x
2.5 cm) in the laboratory for the observation of
parasitoid emergence.
The results on the distribution and number of
eggs found, the number of eggs and egg layers in
each egg mass, and the number of parasitized
eggs and their distribution at different phenolog-
ical stages and on different parts of the corn plant


were subjected to an analysis of variance with the
means compared by the PLSD (Protected Least
Significant Difference) test (alpha = 0.05). The
treatments were arranged in a factorial scheme
with two factors: plant age and plant region; the
number of parasitized eggs and percentage S. fru-
giperda egg parasitism at different phenological
stages were compared by the Tukey test (alpha =
0.05) with a completely randomized design, using
SAS software for analysis.

RESULTS

The S. frugiperda eggs were found at every
phenological stage of the corn plant, and over 99%
were found on leaves with only two egg masses
found on the stem. A significant interaction was
found between plant phenological stage and re-
gion (lower, middle and upper) (F = 2.79; P = 0.02)
and corn leaf surface abaxiall and adaxial) (F =
7.79; P = 0.009). That is, the egg distribution
among the different parts of the plant varied with
the age and development of the corn plant.
At the early developmental stages of the crop
(4-6 leaves), the majority of oviposition occurred
on the 1st and 2nd leaves (60.4%) and on the
abaxial surface of the leaves (83.4%). There were
significant differences between the upper, me-
dium and lower regions and the abaxial versus
the adaxial leaf surface at the same developmen-
tal stage and among the various developmental
stages (4-6, 8-10 and 12-14 leaves) (Table 1). As
the plant developed to the 8-10 leaf stage, the pre-
ferred oviposition site changed and the eggs be-
came more concentrated on the middle and upper
plant regions (73.5%) and on the adaxial leaf sur-
face (66.9% of the egg masses). At the 12-14-leaf
stage, the greatest concentration of egg masses
(61.4%) was observed on the middle region of the
plant, significantly greater than on the lower and
upper regions. However, there was no significant
difference between the abaxial and adaxial leaf
surfaces in terms of egg distribution. The middle
part of the plant was, to a degree, constant as an
oviposition site, with no significant difference be-
tween the phenological stages (Table 1).
The mean number of egg masses was signifi-
cantly (F = 4.09; P = 0.01) different among the
three developmental stages of the corn, with the
highest concentration of egg masses at the early
developmental stages (4-6 leaves) until approxi-
mately 60 days after planting, when the plants
had 8-10 leaves, decreasing at the 12-14 leaf
stage. However, the mean number of eggs did not
differ among the three stages (F = 2.76; P = 0.01)
(Table 2).
A significant interaction was found between
plant-age and the number of egg layers in each S.
frugiperda egg mass (F = 2.72; P = 0.01). At every
phenological stage, most egg masses had more than
one layer; the most frequent configuration was









Florida Entomologist 85(4)


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Beserra et al.: Parasitism of S. frugiperda


three layers, with 91.56% covered by scales. At the
4-6-leaf stage the eggs predominantly had 2 or 3
layers and at the following stages 8-10 and 12-14
leaves, those with 3 or 4 layers prevailed (Table 2).
With respect to parasitism, no significant in-
teraction was observed between plant phenology
and plant region (F = 1.49; P = 0.21), and no sig-
nificant differences were found among the three
developmental stages in the number (F = 0.3; P =
0.74) and percentage (F = 1.37; P = 0.25) ofS. fru-
giperda egg masses parasitized by Trichogramma
spp. (Table 3). However, significant differences in
parasitism were detected among the three parts
of the plant (F = 2.91; P = 0.05); the greatest num-
ber of parasitized eggs occurred on the lower and
middle regions of the plant (Table 3). Also the
number (F = 0.98; P = 0.38) and percentage (F =
1.84; P = 0.17) of parasitized S. frugiperda eggs
did not differ among the three phenological stages
of the corn (Table 4), showing that the distribu-
tion and degree of parasitism by Trichogramma
spp. on different regions of the plant were inde-
pendent of the developmental stage of the crop.
Overall natural parasitism was very low with a
maximum of 2.21% of the eggs parasitized (Table
4), despite the fact that S. frugiperda eggs were
available at every plant phenological stage. Para-
sitism remained constant over all densities of
eggs observed at the 4-6 and 8-10 leaf stages (Ta-
ble 2). Few Trichogramma species were found par-
asitizing the S. frugiperda eggs. Trichogramma
pretiosum Riley was most abundant, comprising
93.79%. Trichogramma atopovirilia Oatman &
Platner occurred in 2.07% of the samples. Multi-
parasitism-a frequent phenomenon in Tri-
chogramma-was observed, as T pretiosum and
T atopovirilia appeared simultaneously in vari-
ous egg masses. The remaining infested eggs
(4.14%) were parasitized by other species of the
genus Trichogramma. Only one individual of an-
other genus of the family Trichogrammatidae was
found. It was not identified because only one fe-


male emerged (the identification is based on
males). In addition, this female failed to repro-
duce in eggs of the factitious hostAnagasta kueh-
niella Zeller.

DISCUSSION

Changes in the oviposition behavior of S. fru-
giperda occurred as the host plant developed. Egg
masses were most abundant on the lower region
of the plant, and on the abaxial leaf surface, at the
early 4-6-leaf stage; and on the middle and upper
regions and the adaxial leaf surface at the subse-
quent stages (8-10 and 12-14 leaves). Pitre et al.
(1983) showed that while in grasses such as sor-
ghum and corn, S. frugiperda egg masses are
more numerous and larger than in other hosts
such as soybean and cotton. They also showed
that the abaxial leaf surface, and the plant re-
gions with greatest leaf mass, are preferred ovipo-
sition sites since they constitute a protected
environment. The oviposition strategy of S. fru-
giperda on corn may focus on protection from nat-
ural enemies or suitable feeding sites for progeny,
since females oviposit in areas near the feeding
sites of the larvae. As the plant develops, egg lay-
ing shifts to the upper region of the plant, close to
the spindle. When this site is not available after
bolting, the middle region of the plant, close to the
ear, becomes the main oviposition site. Labatte
(1993) found that larvae migrate towards the up-
per region of the corn plant to feed at the 4-6 leaf
stage. He indicated that up to the 10-leaf stage
feeding occurs preferably among the whorl of
young leaves, where the worms remain until the
end of their development. After the 12-14-leaf
stage, the worms migrate to the tassel and the
middle region of the plant, during tasselling and
ear formation, remaining in the leaves and ears.
This egg laying and larva distribution pattern
was also observed for Ostrinia nubilalis (Hiib.) by
Shelton et al. (1986) and Labatte (1991), who


TABLE 3. MEAN NUMBER AND PERCENTAGE OF SPODOPTERA FRUGIPERDA EGG MASSES PARASITIZED BY TRI-
CHOGRAMMA SPP. AT THREE DIFFERENT STAGES AND ON THREE DIFFERENT PARTS OF THE CORN PLANT, DUR-
ING THE 1998 2000 GROWING SEASONS IN PIRACICABA, SAO PAULO STATE.

Number of parasitized Percentage
Phenological stage n egg masses' parasitism

4-6 leaves 21 7.0 + 3.01a 43.7 + 11.29a
8-10 leaves 42 2.9 + 1.14a 18.4 + 4.67a
12-14 leaves 36 2.8 + 1.15a 27.7 + 6.52a
Plant region
Lower 33 2.0 + 0.44a 26.0 + 5.39a
Middle 38 1.5 + 0.13a 18.6 + 3.52a
Upper 29 0.6 + 0.05b 15.4 + 5.33a
means followed by the same letter do not differ by the F test among phenological stages or the PLSD (Protected Least Significant Difference) and F
tests, respectively among plant regions (alpha = 0.05).







Florida Entomologist 85(4)


TABLE 4. PARASITISM OF SPODOPTERA FRUGIPERDA EGGS BY TRICHOGRAMMA SPP. AT DIFFERENT PHENOLOGICAL
STAGES OF THE CORN PLANT DURING THE 1998 2000 GROWING SEASONS IN PIRACICABA, SAO PAULO STATE.
Average number Average
of parasitized parasitism
Phenological stage n Total egg masses Total eggs eggs' percentage2

4-6 leaves 8 114.0 18,752.0 51.8 + 21.85a3 2.2 + 0.91a
8-10 leaves 16 243.0 58,364.0 33.2 + 12.66a 0.9 + 0.60a
12-14 leaves 15 116.0 31,293.0 38.8 + 16.05a 1.8 + 0.57a

'Data transformed into (x)-0.1 and 1/ x, respectively.
'Means followed by the same letter do not differ by the F test (alpha = 0.05).


stated that the phenological stage of the corn is
the main factor affecting the pest's behavior.
The decreased number of egg masses observed
in our study as the plant grew older coincides with
the population fluctuation of S. frugiperda adults
and larvae observed by Mitchell et al. (1984) and
Gutierrez-Martinez et al. (1989). Surveys con-
ducted by Gutierrez-Martinez et al. (1989)
showed that the first adults appear right after
plant emergence, with the highest frequency from
the 10th through the 41st day of emergence, when
the plant is more susceptible, decreasing consid-
erably up to the 72nd day, when fewer attacks on
the corn crop occur. This phase also coincides with
the period of greatest production losses, which
may reach 19% when the plant achieves the 8-10
leaf stage (Cruz & Turpin 1982).
Although the number of egg masses decreased
with plant age, the number of eggs found on the 8-
10 and 12-14 leaf stages was higher than at the 4-
6-leaf stage (Table 4). Meneses et al. (1991) also
observed a high frequency of egg masses with
three layers and an average of 385.6 eggs/egg
mass in rice. Sifontes et al. (1988) found S. fru-
giperda with a higher number of layers and eggs
in egg masses, and a higher scale density on older
rice plants.
Studies by Garcia & Sifontes (1987), Toonders
& Sanchez (1987) and Hoffmann et al. (1995) re-
vealed that Trichogramma spp. have difficulty in
parasitizing S. frugiperda eggs, which is in accor-
dance with our results. Garcia & Sifontes (1987)
reported that 46.9% of 47 egg masses parasitized
by Telenomus and 4.3% parasitized by Tri-
chogramma spp. together with Telenomus. More-
over, they considered only the number of egg
masses parasitized and neglected the number of
eggs parasitized, which obviously would give an
even lower percentage of parasitism. Toonders &
Sanchez (1987), on the other hand, counted the
number of parasitized eggs, and observed that
natural parasitism by Trichogramma spp. varied
from 0 to 10% in samples examined in six different
fields and when 30,000 parasitoids were released
in 1.5 ha of corn the parasitism rate was only 4%.
The differences found in the degree of parasit-
ism observed among the three parts of the plant
(upper, middle and lower) does not indicate a pref-


erence of Trichogramma spp. for parasitizing the
host eggs in the lower and middle portions, rather
it may be associated with the oviposition behavior
of S. frugiperda which overall oviposited more in
these regions (average 5.37, 5.64 and 3.29 egg
masses for the lower, middle and upper regions,
respectively), allowing an increase in the number
of parasitized eggs, although there were no differ-
ences in the percentage of parasitism (Table 3).
These results are in accordance with those re-
ported by Wang et al. (1997), who observed a
greater preference of T ostriniae for parasitizing
0. nubilalis eggs on the lower and middle regions
of the corn plant, as these are the sites with the
greatest concentration of the host's egg masses.
However, a greater availability of eggs in a given
region of the plant does not always result in in-
creased parasitism by Trichogramma. For exam-
ple, Romeis et al. (1998, 1999) observed that
Trichogramma chilonis Ishii parasitized more
Helicoverpa armigera eggs on pigeonpea leaves
than on flowers and pods, even though more H. ar-
migera eggs were available on the latter struc-
tures. These differences, in this case, can be
explained by the presence of trichromes, which
damage the parasite.
Records of parasitism by Trichogramma on S.
frugiperda eggs are scarce and usually indicate
low intensities, as found by Garcia & Sifontes
(1987) in the Sancti Spiritus region of Cuba, with
4.3% parasitized egg masses, similar to our data.
Knowledge about changes in the oviposition
behavior of S. frugiperda at the various phenolog-
ical stages could help in the planning of field-sam-
pling methods, location of ovicide applications,
and releases of Trichogramma spp. in augmenta-
tive biological control. However, doubts remain on
the effectiveness of using Trichogramma to con-
trol this lepidopteran pest as natural infestation
rates are quite low. This problem needs to be ad-
dressed before this parasitoid can be used as a vi-
able alternative to chemical controls.

ACKNOWLEDGMENTS
To Coordenacao de Aperfeicoamento de Pessoal de
Nivel Superior (CAPES) for the scholarship grant which
enabled this research, and Agricultural Engineer Ran-
ise Barbosa for identifying the Trichogramma species.


December 2002







Beserra et al.: Parasitism of S. frugiperda


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Appl. 83: 337-345.







Florida Entomologist 85(4)


December 2002


QUALITY ASSESSMENT OF CHRYSOPERLA RUFILABRIS
(NEUROPTERA: CHRYSOPIDAE) PRODUCERS IN CALIFORNIA

CRESSIDA S. SILVERS', JOSEPH G. MORSE2 AND ELIZABETH E. GRAFTON-CARDWELL3
'USDA-ARS, Invasive Plant Research Laboratory, 3205 College Avenue, Fort Lauderdale, FL 33314

2Department of Entomology, University of California, Riverside, CA 92521

3University of California Kearney Agricultural Research Center, 9240 South Riverbend Drive, Parlier, CA 93648

ABSTRACT

Chrysoperla rufilabris (Burmeister) egg shipments from three commercial California insec-
taries were evaluated during a nine-month period. All three insectaries shipped similar
numbers of eggs per unit weight (range 301.0 + 10.3 to 315.4 7.8 eggs/25 mg). Estimated
total number of eggs per shipment for all three insectaries was between 1.80 and 3.30 times
the number ordered (1,000). The estimated number of dead eggs per shipment ranged from
76.0 to 418.0 and the estimated number of larvae per shipment ranged from 0 to 9.9. Final
hatch rates for all three insectaries were between 70.9% and 73.9%. Hatch began on the
third day after shipment receipt and 70% of total hatch had occurred by the fourth day. Im-
plications for timing of egg and larval releases are discussed.

Key Words: Chrysoperla rufilabris, lacewing, quality control, insectary rearing

RESUME

Los envios de huevos de Chrysoperla rufilabris (Burmeister) de tres insectarios comerciales
de California fueron evaluados durante un period de nueve meses. Todos los tres insectarios
enviaron cantidades similares de huevos por unidad de peso (de 301.0 10.3 hasta 315.4 +
7.8 huevos/25 mg). El numero total estimado de huevos por cada envio de los tres insectarios
fu6 entire 1.83 y 3.31 veces mayor del numero solicitado (1,000). El numero estimado de hue-
vos muertos por envio fu6 de 75.98 hasta 418 y el numero de larvas estimadas por envio fue
de 0 hasta 9.9. La taza de eclosi6n final para los tres insectarios fu6 entire 70.9% y 73.9%. La
eclosi6n empez6 en el tercer dia despu6s de recibir el envio y 70% de la eclosi6n total ocurri6
en el cuarto dia. Se discuten las implicaciones para el tiempo de liberar los huevos y las lar-
vas.


Inundative and augmentative releases of natu-
ral enemies are widely used as non-disruptive al-
ternatives to chemical control of arthropod pests.
Ci.,, .' .... /. (Neuroptera: Chrysopidae) species,
commonly known as green lacewings, are among
the most commonly marketed generalist insect
predators (Tauber et al. 2000). Lacewings are ap-
plied in home gardens, row crops, orchards, and
greenhouses on a variety of crops (Ridgway &
Murphy 1984, Daane et al. 1998). Although
Ci., .' .. /.... adults are not predaceous, all three
larval stages are voracious eaters of soft-bodied
arthropods and therefore are the desired stages
for release (Tauber et al. 2000). High production
costs, however, often make high volume larval
purchases prohibitive. Alternatively, lacewing
eggs can be as much as 17 times less expensive
than larvae (Cranshaw et al. 1996). Eggs may be
released upon receipt, or held until hatch and re-
leased as larvae. Substantial work has been done
recently to develop, evaluate, and improve lacew-
ing egg and larva field application methods
(Morisawa & Giles 1995, Gardner & Giles 1996,


Daane & Yokota 1997, Giles & Wunderlich 1998,
Wunderlich & Giles 1999). Few studies, however,
have evaluated the quality of commercial insec-
tary egg shipments. The performance of natural
enemy releases is only as high as the quality of or-
ganisms shipped by suppliers. In a self-regulated
industry such as natural enemy mass-production,
external product evaluations are important to
maintain quality, which, in turn, positively pro-
motes natural enemies as a potential tool for pest
management.
O'Neil et al. (1998) evaluated post-shipment
quality of four natural enemy species, including
C(i 1 ... I i. carnea (Stephens). They found differ-
ences among C. carnea suppliers in the ratio of or-
dered eggs/received eggs, the number of larvae in
egg shipments, survivorship of starved first instar
larvae, and the sex ratio of reared adults. In addi-
tion, lacewing larvae reared to adulthood were
identified as C'i, .. ...i / rufilabris (Burmeister),
not C. carnea. For the consumer planning lacew-
ing releases, another important aspect of egg
shipments, which O'Neil's group did not test, is







Silvers et al.: Quality Assessment of Chrysoperla rufilabris


when and how many eggs hatch into larvae. If
consumers know what to expect in terms of ship-
ment hatch, then they may be better able to coor-
dinate release rates and timings to optimally
target pest phenology and environmental condi-
tions.
In this study, we evaluated C. rufilabris egg
shipments from three California producers. Num-
ber of eggs per unit weight, estimated total num-
ber of eggs per shipment, and developmental
stages of eggs upon receipt were measured and
compared across the three insectaries. To com-
pare egg quality among producers, timing and fi-
nal percentage of eggs hatched were also
determined. Among possible chrysopids, C. rufila-
bris was chosen for study because it was the only
species produced by all three insectaries.

MATERIALS AND METHODS

Three insectaries in California were identified
as producers of C. rufilabris: Beneficial Insectary
(Oak Glen), Buena Biosystems (Ventura), and
Rincon Vitova (Ventura). Throughout the experi-
ment, larvae reared from egg shipments were
identified as C. rufilabris by markings on head
capsules and by setal patterns as described in
Tauber (1974). For the sake of anonymity, each in-
sectary was randomly assigned a unique number
between one and three. Between March and No-
vember, 1999, ten shipments each of 1,000 eggs
each were ordered and shipped overnight to our
laboratory at the University of California, River-
side from Insectary 1 and Insectary 2. Four over-
night shipments of 1,000 eggs each were received
from Insectary 3 between July and November,
1999. Insectaries were aware that shipments
were to be used in experimental evaluations of C.
rufilabris, but were not told specifically that they
were being evaluated on shipment quality.
Upon arrival, egg shipments were opened and
the method of packaging noted. Total weight of
each 1,000-egg shipment was recorded (Sartorius
1212 MP digital scale, Brinkman Instrument,
Inc., Westbury, NY). A 25 mg sample was taken
from each shipment, with the following excep-
tions: two samples were taken from the second
shipment from Insectary 1, four from the ninth
shipment from Insectary 2, and six and two from
the third and fourth shipments, respectively, from
Insectary 3. Using a dissecting microscope, the
number of eggs per 25 mg sample was counted
and eggs were categorized as either dead (rup-
tured or desiccated), green (indicating recent ovi-
position), or partially to completely brown with
abdominal striping of the developing embryo visi-
ble (Gepp 1984). The number of hatched larvae, if
any, was also recorded. Data for each category
were analyzed for differences among insectaries
using an unbalanced, nested ANOVA with un-
equal numbers of shipments per source and un-


equal numbers of samples per shipment. The
specified model treated source as a fixed effect
and shipments within source as a random effect
(Sokal & Rohlf 1995, SAS Institute 1999). Tukey-
Kramer's test was used for comparison of least
square means. The level of significance for all
tests was p = 0.05.
From each of the 25 mg samples taken from
each egg shipment upon arrival, between one and
four subsamples of 40 randomly selected eggs
each were used in hatch rate determinations.
Eggs were placed, one egg per well, in uncoated,
plastic assay plates with rounded bottoms (96-
well Assay Plates, Corning, Inc., Science Products
Division, Acton, MA). Strips of clear adhesive tape
were placed over the wells containing a single egg.
To maintain relative humidity at a level (>70%)
conducive to C. rufilabris development (Tauber
1974), plates were placed on wet sponges and
loosely enclosed in plastic boxes. Plastic boxes
were kept under ambient laboratory temperature
and light conditions, resulting in a temperature of
24 + 1.5C inside the boxes. Temperature and hu-
midity were measured by HOBO Temp and
HOBO RH, respectively (Onset Computer Corpo-
ration, Pocasset, MA). The number of emerged
larvae, i.e. those completely separated from the
chorion, were counted daily until the number of
hatched larvae remained unchanged for two con-
secutive days. Cumulative percentage of hatched
larvae was calculated daily. Thirty-six 40-egg sub-
sample replicates were hatched out for Insectary
1, 33 replicates for Insectary 2, and eight repli-
cates for Insectary 3. Arcsine (square-root) trans-
formation was applied to daily cumulative hatch
rates. Transformed data were analyzed for differ-
ences in daily cumulative percentage hatch
among insectaries using a one-way ANOVA. Sig-
nificant differences were further separated with
Tukey's test for comparison of means. The level of
significance for all tests was p = 0.05.

RESULTS

All egg shipments arrived on time and were
packaged in small plastic cups with tight-fitting
lids, wrapped in paper. Both Insectaries 1 and 2
placed each paper-wrapped plastic cup into a sty-
rofoam cooler with artificial ice packs, and the
cooler was in turn packaged in a cardboard box for
shipping. Insectary 3 shipped each paper-
wrapped plastic cup in a cardboard box without
styrofoam insulation or ice packs.
The mean weight of shipments from Insectary 2
(145.4 9.2 mg) was significantly (F = 5.20; df = 2,
21; p = 0.015) less than the mean shipment weights
from Insectaries 1 (247.5 37.7 mg) and 3 (274.8
47.6 mg). Variation in shipment weight for Insec-
tary 2 (range 100 mg to 203 mg) was also less than
that for either Insectary 1 (range 130 mg to 451
mg) or Insectary 3 (range 155 mg to 369 mg).







Florida Entomologist 85(4)


The mean number of eggs within a 25 mg sam-
ple was similar for all three insectaries, at just
over 300 eggs (Table 1). Based on these counts,
the estimated mean number of eggs per shipment
was 3,046 for Insectary 1, 1,834 for Insectary 2,
and 3,309 for Insectary 3.
Table 1 shows the composition of the 25 mg
samples taken from each insectary's egg ship-
ments. For all insectaries, the majority of eggs
(65%) in each sample had reached the striped
stage, indicating imminent hatching. The esti-
mated number of larvae per shipment ranged
from 0 to 9.9 and the estimated number of dead
eggs per shipment ranged from 76.0 to 418.0.
Timing of egg hatch was similar for all three
insectaries (Fig. 1). There was little to no hatch on
the first and second days. On day three, however,
hatch for eggs from Insectary 1 and 3 were 13.2%
and 21.6%, respectively, whereas a significantly
lower percentage of eggs from Insectary 2 had
hatched (3.7%). On days four through seven, per-
centage hatch for all three insectaries did not dif-
fer statistically. Final mean percentages of eggs
hatched ranged from 70.9 to 73.9.

DISCUSSION

The similarity in hatch rates observed for all
three insectaries suggests similar quality of eggs
received from each. The approximately 30% of
eggs from which larvae did not emerge was com-
pensated for by the fact that shipments, on aver-
age, exceeded the ordered amount by between


83% and 231%. However, implications for ship-
ments to the average consumer are not clear be-
cause the university shipping address may have
biased insectaries to include extra eggs as a cour-
tesy. This is underscored by comparing our results
to those of O'Neil et al. (1998), who used a "blind
ordering" system and found fewer lacewings than
ordered in a majority of shipments received.
In addition to some level of egg mortality ex-
pected during the shipping and handling pro-
cesses, the risk of egg mortality in lacewing
shipments is higher due to the cannibalistic na-
ture of their larvae (New 1975). Eggs held until re-
lease tend to be confined at high densities,
increasing exposure to predation by newly emerg-
ing larvae as holding time increases (Daane &
Yokota 1997, O'Neil et al. 1998). Without alterna-
tive prey provided, O'Neil et al. (1998) suggested
immediate release of lacewing eggs to prevent can-
nibalism. Our findings suggest that losses to can-
nibalism may be minimized by releasing lacewing
eggs on or before the third day after receipt.
Even with the development of efficient egg re-
lease technologies (Gardner & Giles 1996,
Wunderlich & Giles 1999), high post-release egg
mortality due to environmental conditions
(Daane & Yokota 1997) and intraguild predation
(Tauber et al. 2000) reduce efficacy of egg releases
as compared with larval releases. Our results in-
dicate that for larval releases, holding eggs for
four days allows a majority (approximately 70%)
of total hatch to occur while limiting larval hold-
ing time to 24 hours. Waiting an additional day


TABLE 1. COMPOSITION OF 25 MG SAMPLES TAKEN FROM CHRYSOPERLA RUFILABRIS EGG SHIPMENTS.

Mean (SEM)'in each stage

Insectary 1: Insectary 2: Insectary 3:
10 shipments, 10 shipments, 4 shipments, ANOVA
total of 11 samples total of 13 samples total of 10 samples (num. df = 2)

Green Egg 63.5 a 15.8 a 52.3 a F = 2.52
(15.4) (15.4) (24.3) p = 0.105
den. df= 20.9
Striped Egg 204.7 a 288.3 b 209.9 ab F = 4.90
(20.1) (20.1) (31.4) p = 0.018
den. df= 20.6
Emerged Larvae 1.0 b 0.0 a 0.7 ab F = 4.92
(+0.2) (0.2) (0.3) p = 0.025
den. df= 13.8
Dead Egg 36.6 b 13.1 a 38.0 b F = 760
(+5.3) (4.9) (5.6) p = 0.002
den. df= 31
Total 307.7 a 315.4 a 301.0 a F = 0.65
(8.0) (7.8) (10.3) p = 0.536
den. df= 13.3

'Means (+SEM) listed are estimated least square means based on a nested ANOVA model with unequal sample size. Means within a row followed by
the same letter did not differ statistically (Tukey-Kramer's test, p < 0.05).


December 2002







Silvers et al.: Quality Assessment of Chrysoperla rufilabris



OINSECTARY 1 INSECTARY 2 WINSECTARY 3


F = 1.27
P = 0.288


F = 6.98
P = 0.002
b

b I


F = 0.01 F 0.01
F 0.26 P = 0.988 P = 0.994
P 0.772 a
aa a a a a


2 3 4 5 6 7


DAYS POST RECEIPT
Fig. 1. Cumulative mean percentage (SEM) of Chrysoperla rufilabris eggs hatched each day after shipment re-
ceipt from three insectaries. Within days, means labeled with the same letter are not statistically different (Tukey's
test, df= 2,74, p< 0.05).


may increase hatch, but that may be countered by
an accompanying increase in cannibalism (O'Neil
et al. 1998).
The few reports ofC. rufilabris egg hatch rates
found in the literature are for untreated controls
within studies of egg release methodologies. Our
observed hatch rates of 70.9 to 73.9% after seven
days fall within the 64.1% after five days reported
by Gardner & Giles (1996) and the 91.2% after
seven days reported by Daane & Yokota (1997),
but direct comparison of these rates is difficult be-
cause specific holding conditions were not re-
ported for each. The question should be addressed
in future egg hatch studies in which effects of
temperature, relative humidity and light regime
are compared and related to conditions an aver-
age consumer may be able to replicate.
Whereas the development stages present in
egg shipments differed slightly among the three
insectaries, all contained mostly eggs that were
close to emergence and few contained larvae. This
is an improvement on the average percentage lar-
vae in shipments ranging up to 52.6% reported by
O'Neil et al. (1998). Although sources were not
specified in that study, perhaps the wider range of
larval emergence they observed was a result of in-
cluding both producers and distributors. Eggs
shipped through distributors may be older and
therefore more likely to hatch before arrival than
eggs shipped directly from the producer.


Shipments from all three insectaries contained
a similar number of eggs per unit weight, indicat-
ing little difference in contamination levels. One of
the insectaries, however, had less variation in ship-
ment weight, significantly fewer damaged and des-
iccated eggs, and slightly more uniformity in egg
developmental stage as indicated by shipment
composition and hatch data. Uniform age struc-
ture could contribute to a more synchronous and
predictable larval hatch, equally useful for timing
of egg releases as for larval releases. These obser-
vations suggest slightly better handling tech-
niques and precision in egg collection that perhaps
could be employed in the other two insectaries.

ACKNOWLEDGMENTS

The authors thank Beneficial Insectary (Oak Glen),
Buena Biosystems (Ventura), and Rincon Vitova (Ven-
tura) for providing C. rufilabris used in this study. Re-
search assistance was provided by P. Watkins and A.
Urena, and statistical assistance by H. Schweizer. We
also thank two anonymous reviewers and the editor for
their invaluable comments. Funding was provided in
part by grants from the California Citrus Research
Board and California Avocado Commission.

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view of 1994 pricing and marketing by suppliers of











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DAANE, K. M., K. S. HAGEN, AND N. J. MILLS. 1998. Pre-
daceous insects for insect and mite management, pp.
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scoe, and C. S. Glenister [eds.] Mass-reared Natural
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DAANE, K. M., AND G. Y. YOKOTA. 1997. Release strate-
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GEPP, J. 1984. Morphology and anatomy of the preimag-
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ogy of Chrysopidae. W. Junk, The Hague.
GILES, D. K., AND L. R. WUNDERLICH. 1998. Electroni-
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Agric. Eng.) 41: 839-847.
MORISAWA, T., AND D. K. GILES. 1995. Effects of me-
chanical handling on green lacewing larvae
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obiidae (Neuroptera), with reference to their usage
as biocontrol agents: a review. Trans. R. Entomol.
Soc. London 127: 115-140.
O'NEIL, R. J., K. L. GILES, J. J. OBRYCKI, D. L. MAHR, J.
C. LEGASPI, AND K. KATOVICH. 1998. Evaluation of
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Florida Entomologist 85(4)







Cuda et al.: Acceptability of Exotic Solanum spp. to Native Leptinotarsa Beetles 599



EVALUATION OF EXOTIC SOLANUM SPP. (SOLANALES: SOLANACEAE)
IN FLORIDA AS HOST PLANTS FOR THE LEAF BEETLES LEPTINOTARSA
DEFECTA AND L. TEXANA (COLEOPTERA: CHRYSOMELIDAE)

J. P. CUDA,1 P. E. PARKER,2 B. R. COON,1 F. E. VASQUEZ1 AND J. M. HARRISON3
1Entomology and Nematology Department, Institute of Food and Agricultural Sciences
University of Florida, Gainesville, Florida 32611-0620

2USDA, APHIS Mission Plant Protection Center, Mission, Texas 78572

I ..i ni.. l Department of Statistics, Institute of Food and Agricultural Sciences
University Florida, Gainesville, Florida 32611-0339


ABSTRACT

Tropical soda apple, Solanum viarum Dunal, wetland nightshade, S. tampicense Dunal, and
turkey berry, S. toruum Swartz, are considered three of Florida's most invasive plant species.
These nonnative perennial broadleaf weeds are disrupting native plant communities in ag-
ricultural areas and natural ecosystems. The lack of natural enemies in Florida is thought
to be an important factor contributing to their invasiveness. The North American leaf bee-
tles Leptinotarsa defecta (Stal) and L. texana (Schaeffer) that attack silverleaf nightshade,
Solanum elaeagnifolium Cav, a native congener of the three nonnative solanums, were eval-
uated for their potential as biological control agents. The suitability of tropical soda apple,
wetland nightshade and turkey berry as host plants for the native Leptinotarsa beetles was
studied in a quarantine laboratory using single plant and paired plant tests. Neonate larvae
of L. defecta developed to the pupal stage only on their natural host plant silverleaf night-
shade. Feeding damage on turkey berry and wetland nightshade was negligible and no feed-
ing occurred on tropical soda apple. In contrast, development and reproduction ofL. texana
on the nonnative turkey berry were comparable with silverleaf nightshade. These results
suggest the nonnative turkey berry may be included in the potential host range of the native
silverleaf nightshade beetle L. texana.

Key Words: Biological control, weeds, Solanum viarum, S. tampicense, S. toruum, S. elae-
agnifolium, risk assessment

RESUME

Solanum viarum Dunal, S. tampicense Dunal, y S. toruum Swartz se consideran como tres
de las species de plants mas invasoras en Florida. Estas malezas perennes no nativas de
hoja ancha estan perturbando las comunidades de plants en areas agricolas y ecosistemas
naturales. Se piensa que la falta de enemigos naturales en Florida es un factor important
que contribute a su habilidad para ser invasoras. Se evaluaron los escarabajos norteameri-
canos, Leptinotarsa defecta (Stal) y L. texana (Schaeffer) que atacan las hojas de Solanum
elaeagnifolium Cav., una plant ndtiva en el mismo g6nero de los tres solanums no natives,
para determinar su potential como agents de control biol6gico. Se estudi6 si las plants de
Solanum viarum, S. tampicense y S. toruum podrian ser hospederos adecuados de los esca-
rabajos nativos Leptinotarsus en el laboratorio de la cuarentena usando pruebas de plants
individuals y en pares. Se desarrollaron las larvas reci6n nacidas de L. defecta hasta la
6tapa de pupa solamente en su plant hospedera natural Solanum elaeagnifolium. El daio
de alimentaci6n en el S. toruum y S.tampicense fu6 insignificant y no se aliment6 de Sola-
num viarum. Al contrario, el desarrollo y la reproduci6n de L. texana sobre S. toruum no na-
tivo, fu6 similar con los de S. elaeagnifolium. Estos resultados suguieron que se puede incluir
S. torvum no native entire los hospederos potenciales del escarabajo de Solanum elaeagnifo-
lium no native, L. texana.


Tropical soda apple, Solanum viarum Dunal, in Florida in 1988 (Mullahey et al. 1993, Mulla-
wetland nightshade, S. tampicense Dunal, and hey et al. 1998), and by 1995 infested between
turkey berry, S. torvum Swartz, are perennial 0.25 and 0.5 million ha of prime agricultural and
nonnative invasive weeds that have been identi- nonagricultural lands (Mullahey 1996a, Mulla-
fied as candidates for biological control (Cuda et hey et al. 1998). This invasive weed infests a vari-
al. 2002). Tropical soda apple was first discovered ety of habitats including improved pastures,







Florida Entomologist 85(4)


natural areas, citrus (Citrus spp.), sugar cane
(Saccharum officinarum L.), sod fields, ditch
banks, and roadsides. After establishing in Flor-
ida, tropical soda apple continued to expand its
range into Alabama, Georgia, Louisiana, Missis-
sippi, North Carolina, South Carolina, Pennsyl-
vania, Puerto Rico and Tennessee (Westbrooks &
Eplee 1996, Mullahey et al. 1998). The Pennsyl-
vania infestation has since been eradicated
(Westbrooks 1998).
The foliage and stems of tropical soda apple
are prickly and unpalatable to livestock. How-
ever, cattle and wildlife readily ingest the fruits
and spread the seeds in their droppings. If left un-
controlled, pasture production declines and stock-
ing rates are drastically reduced (Mullahey et al.
1993). In 1994, production losses to Florida cattle
ranchers attributed to tropical soda apple infesta-
tions were estimated at US $11 million annually
(Cooke 1997). Tropical soda apple also serves as a
reservoir for various diseases and insect pests of
solanaceous crop plants (McGovern et al. 1994a,
1994b, Medal et al. 1999). A special symposium
devoted entirely to various aspects of tropical
soda apple and to a lesser extent wetland night-
shade's biology, ecology, environmental effects
and control strategies was held in Florida in 1996
to address these emerging weed problems (Mulla-
hey 1996b).
Wetland nightshade is a bramble-like plant
with spiny tangled stems and leaves that was
first reported in Florida in 1983 (Wunderlin et al.
1993, Fox & Bryson 1998). In contrast to tropical
soda apple, which dominates upland sites, regu-
larly flooded wetlands are particularly vulnerable
to invasion by wetland nightshade (Wunderlin et
al. 1993, Fox & Bryson 1998). The largest infesta-
tion, approximately 60 ha, occurs in southwest
Florida (Fox & Wigginton 1996, Wunderlin &
Hansen 2000). The ability of wetland nightshade
to form dense thickets that are difficult for other
species to penetrate suggests this noxious weed
has the potential to invade and alter many of the
state's wetland habitats thus impeding access to
and use of water resources (Fox & Wigginton
1996, Fox & Bryson 1998).
Turkey berry is a large, prickly shrub that can
attain heights of up to 3 m (Ivens et al. 1978). Tur-
key berry was first collected in Columbia Co.,
Florida, in 1899, and has been reported in at least
nine counties throughout the state (Wunderlin &
Hansen 2000, Cuda et al. 2002). This noxious
solanum invades disturbed sites such as pas-
tures, crop fields, roadsides, damp waste areas
and forest clearings where it competes with desir-
able plants for moisture, light and nutrients. Al-
though it is frequently cultivated as a yard plant
in south Florida (Westbrooks & Eplee 1989), tur-
key berry is potentially poisonous to animals
(Chadhokar 1976, Abatan et al. 1997), and possi-
bly carcinogenic to humans (Balachandran & Si-


varamkrishnan 1995). Turkey berry has been
reported as a reservoir for Alternaria solani Sor-
auer (Deuteromycetes: Dematiaceae), the caus-
ative agent of wilt disease in potatoes and
tomatoes (Mune & Parham 1967), and is consid-
ered one of the most invasive weeds on other con-
tinents, particularly in parts of Australia and
South Africa that are climatically similar to Flor-
ida (Holm et al. 1979). In the Pacific region, tur-
key berry was identified as a possible target for
classical biological control (Waterhouse & Norris
1987). The occurrence of this plant as an invasive
weed in other countries is perhaps the most com-
pelling evidence for predicting its eventual effect
on Florida's native plant communities.
Tropical soda apple, wetland nightshade and
turkey berry are currently recognized as three of
Florida's most invasive nonnative plant species
(FLDACS 1999, FLEPPC 1999, Langeland 2001).
Although it is unclear why these exotic solana-
ceous plants have become weeds, the lack of host-
specific natural enemies in Florida (the intro-
duced range) may have afforded these plants a
competitive advantage over native species (Cuda
et al. 2002). Tropical soda apple and wetland
nightshade are native to South America (and pos-
sibly the West Indies), and Mexico, respectively
(Wunderlin et al. 1993), whereas turkey berry is
thought to have originated in West Africa (Ivens
et al. 1978), Central or South America and the
Caribbean region (Morton 1981, Waterhouse &
Norris 1987), or Asia (Medal et al. 1999).
Silverleaf nightshade, Solanum elaeagnifo-
lium Cav., is a close relative of tropical soda apple,
wetland nightshade, and turkey berry that is na-
tive to the southern United States, Mexico and
possibly Argentina (Goeden 1971, Boyd et al.
1983), and belongs to the same subgenus Leptos-
temonum as the three nonnative Solanum spp.
(D'Arcy 1972, Nee 1991). Silverleaf nightshade is
attacked by many insect herbivores in the south-
western United States and Mexico (Goeden 1971).
Two of the most damaging insects attacking sil-
verleaf nightshade in its native range are the de-
foliating beetles Leptinotarsa defecta (Stal) and L.
texana (Schaeffer) (Jacques 1988). Both L. defecta
and L. texana were released recently in South Af-
rica for biological control of silverleaf nightshade
(Olckers et al 1999), and their biologies were sum-
marized by Olckers et al. (1995).
Silverleaf nightshade is considered the natural
host plant of L. defecta and L. texana (Goeden
1971, Neck 1983, Jacques 1988). This solanum de-
fines the actual, realized or field host range of the
beetles (Kogan & Goeden 1970, Cullen 1990, van
Klinken 2000). Host range encompasses those
plants on which an insect completes normal devel-
opment in nature (Hanson 1983). However, the
study by Olckers et al. (1995) demonstrated that
under laboratory conditions these two beetles also
developed and reproduced on other solanums that


December 2002







Cuda et al.: Acceptability of Exotic Solanum spp. to Native Leptinotarsa Beetles 601


do not occur in the insects' native ranges. Simi-
larly, Hsiao (1981) observed L. texana developed
and reproduced to some extent on eggplant as
well as three native plant species-S. dulcamara
L., S. carolinense L. and S. rostratum Dunal.
These solanaceous plants are not typically ex-
ploited by the beetles in nature but are capable of
supporting some development and reproduction,
and comprise what is considered the insects' po-
tential, physiological or fundamental host range
(Kogan & Goeden 1970, Cullen 1990, van Klinken
2000). Horsenettle (S. carolinense) and presum-
ably Florida horsenettle (S. carolinense L var.
floridanum Chapm.) are the only potential host
plants of L. texana that are native to Florida
(Wunderlin & Hansen 2000). In spite of its native
status in Florida, horsenettle is listed as a trou-
blesome weed by Hall & Vandiver (1991).
Silverleaf nightshade is adventive in Florida,
occurring sporadically from the Panhandle to the
Keys (Wunderlin 1982, Wunderlin & Hansen
2000). Its natural enemies L. defecta and L. tex-
ana have not spread to Florida (Jacques
1985,1988), presumably because the Gulf of Mex-
ico is an effective barrier to insects like L. texana
that are incapable of long range aerial dispersal
(see Hoffmann et al. 1998). However, a computer
model (CLIMEX) that uses various climatic fac-
tors to determine whether insects can colonize
and persist in new geographic areas (Sutherst &
Maywald 1985) predicted that Leptinotarsa bee-
tles collected from silverleaf nightshade in the
Brownsville area of south Texas could establish
and persist in peninsular Florida if tropical soda
apple, wetland nightshade or turkey berry were
suitable host plants.
The purpose of this research was to determine
whether the nonnative and invasive tropical soda
apple, wetland nightshade or turkey berry are ca-
pable of supporting normal development and con-
tinuous reproduction of the North American
silverleaf nightshade leaf beetles L. defecta and L.
texana. If these native insects are capable of es-
tablishing 'new associations' with the exotic
solanums (Hokkanen & Pimentel 1984), they
could be introduced into Florida for biological con-
trol of these weeds after preintroduction host
specificity tests demonstrated they were safe to
release.

MATERIALS AND METHODS

Collections of the silverleaf nightshade leaf
beetles L. defecta and L. texana were made during
the months of June-October 1997 and May 2001
in Starr County, TX, USA, by personnel affiliated
with the USDA-Animal and Plant Health Inspec-
tion Service, Mission Plant Protection Center,
Mission, TX. Parasitoid-free colonies of L. defecta
and L. texana were maintained on potted silver-
leaf nightshade plants held in screen cages at the


laboratory in Mission, TX. Egg masses of L. de-
fecta and L. texana deposited on silverleaf night-
shade were shipped via overnight mail to the
Quarantine Laboratory, Entomology & Nematol-
ogy Department, University of Florida after
USDA, APHIS, PPQ issued an importation per-
mit. A shipment of 138 eggs ofL. defecta and 310
eggs of L. texana was received on 8 September
1997. The eggs were deposited in small masses on
individual silverleaf nightshade leaves separated
by species in petri dishes sealed with Parafilm
to prevent desiccation. The eggs were removed
from the silverleaf nightshade leaves with a
camel hair brush and transferred to moistened fil-
ter paper placed inside another petri dish. This
procedure ensured that neonate larvae were not
preconditioned by feeding on silverleaf night-
shade prior to the host acceptability tests, which
would bias the results of the feeding trials.
Percent survival, development time, and
amount of feeding for the larval stages of both leaf
beetles were measured on each test plant species.
Single plant (no-choice) and paired plant (choice)
host suitability tests with three replications were
conducted with neonate larvae in a quarantine
room maintained at a temperature of 24.0 3.1C,
relative humidity of 66.8 6.8% and a 16-h photo-
phase. Leaves used in the experiments were ob-
tained from potted plants fertilized with Peters
20-20-20 (N: P: K) solution and maintained in a
glasshouse or an outdoor shade house. In the sin-
gle plant tests, five neonate larvae were trans-
ferred directly to a freshly excised leaf of each test
plant. The leaf was placed inside a large covered
petri dish (25.0 cm diam. by 9.0 cm depth) lined
with a Seitz filter disk (25 cm diam.). The filter
disk was routinely moistened with deionized wa-
ter to prevent the leaf from desiccating, and the
leaf was replaced each day or every other day un-
til the larvae pupated or died. Leaf consumption
was measured by scanning the leaves photometri-
cally before and after exposure to the larvae. The
difference in leaf areas was assumed to be the
amount eaten by the developing larvae. The sin-
gle plant larval feeding and development tests
were initiated in early September and completed
in late November 1997.
Paired plant (choice) tests of the feeding prefer-
ences ofL. texana larvae were conducted with sil-
verleaf nightshade as the control. Four leaf disks
(30 mm diam.) were punched from the base of
freshly detached leaves of silverleaf nightshade
and turkey berry, the test plant species that sup-
ported larval development ofL. texana in the sin-
gle plant trials (See Results). The leaf disks were
positioned alternately by species and equidis-
tantly around the perimeter of the same container
used in the single plant trials. Ten neonate larvae
were placed in the center of the container and al-
lowed to select their food source when presented
with a choice of silverleaf nightshade or turkey







Florida Entomologist 85(4)


berry leaf disks. The amount of feeding on each
test plant species in the paired comparison tests
was measured by the same procedure used in the
single plant trials. The paired plant (choice) larval
feeding trials with three replications were initi-
ated in mid-September and were completed by the
end of December 1997 when the last larva pu-
pated or died.
On 9 May 2001, a final shipment of 72 adults of
L. texana (48 males, 24 females) was received
from Texas to compare the beetle's reproductive
performance on turkey berry with silverleaf
nightshade, and larval feeding and development
on potato tree, Solanum donianum Walpers. Po-
tato tree is a state listed threatened species (Coile
1998), and a critical non-target plant that would
be vulnerable to attack by L. texana if this insect
were approved for release in Florida for biological
control of turkey berry
The beetles were equally divided among whole
plants of either silverleaf nightshade or turkey
berry in 3.8 liter (1 gal.) pots covered with acrylic
cylinders (41 cm height x 14 cm diam.). The tops
of the cylinder cages were covered with Nitex
(41 x 42 in. mesh) to prevent the beetles from es-
caping. Individual leaves with the egg masses in-
tact were removed from the plants daily, and
placed in standard petri dishes with moistened
filter paper to incubate. When the larvae hatched,
a maximum of 10 larvae was transferred to a
plastic rectangular container (20 cm x 14 cm x 10
cm) provisioned with leaves of the same host
plant from which they originated, and a piece of
paper toweling to collect the frass produced by the
developing larvae. Each plastic container also
had a hardware cloth insert (16 cm x 10 cm x 5
cm) that served as a platform to keep the leaves
from coming in contact with the frass at the bot-
tom of the container. By elevating the leaves in
this manner, disease problems were avoided.
When the larvae stopped feeding, they were al-
lowed to pupate in the same plastic containers
filled to a depth of 5 cm with vermiculite.
New adults (F1 generation) that emerged in
the containers were sexed, and exposed to the
same species of potted plant silverleaff night-
shade or turkey berry) on which they completed
their development. In total, 12 cages of silverleaf
nightshade and 12 of turkey berry, each contain-
ing 2 males and 1 female ofL. texana, were main-
tained inside the quarantine room under the
same environmental conditions. Survival of the F,
females as well as the number of egg masses pro-
duced, eggs per mass, and percent larval eclosion
on each test plant species were recorded.
A final single plant (no-choice) feeding and de-
velopment test was conducted to determine the
acceptability of potato tree as a host plant for L.
texana. The experimental procedures and condi-
tions were the same as those described above for
the other single plant tests except the neonates


used in this test were F2 generation larvae of L.
texana obtained from F, adults reared on turkey
berry, the control plant in this experiment. The
adult reproduction and potato tree risk assess-
ment experiments were completed in late Decem-
ber 2001.

Data Analysis

The data on larval development time and leaf
consumption were analyzed by ANOVA (SAS
1990). Leaf consumption means were compared
with Tukey's Studentized Range (HSD) test. Non-
parametric estimates of larval survival data were
analyzed using the LIFETEST procedure (SAS
1990), and were compared with chi-square. The
TTEST procedure (SAS 1990) was used to com-
pare the effect of plant species silverleaff night-
shade or turkey berry) on adult female
reproductive performance, and plant species (tur-
key berry or potato tree) on larval feeding and de-
velopment of L. texana. Data obtained on larval
eclosion (%) were arcsine transformed prior to
analysis.

RESULTS

Larval Feeding and Development

Single plant tests. As expected, larvae of both
Leptinotarsa beetles completed development on
their natural host plant silverleaf nightshade
(Figs. 1 and 2). The durations of the first, second,
third and fourth stadia for L. defecta on silverleaf
nightshade were 3.7 + 0.3, 3.7 0.3, 3.7 0.3, and
9.0 + 1.5 days, respectively (Fig. 3). However, L.
defecta was unable to develop on any of the non-
native solanum species tested (Fig. 1). All larvae
on turkey berry, tropical soda apple, and wetland
nightshade died by day 7 and none developed to
the second instar. The likelihood ratio test for ho-
mogeneity of the survival curves was significant
(Chi square = 7.9413, df = 3, p < 0.05), indicating
that differences in survival occurred among lar-
vae fed the different host plant leaves.
In contrast, development ofL. texana larvae on
turkey berry was comparable to that on silverleaf
nightshade (Figs. 2 and 4). Durations of the first,
second, third and fourth stadia for L. texana
reared on silverleaf nightshade were 3.0 + 0.0, 2.0
0.0, 3.0 + 0.0, and 8.7 + 1.9 days compared to 2.7
0.3, 3.0 + 0.0, 3.0 1.0, and 9.5 + 0.5 days for tur-
key berry, respectively. Host plant diets of either
silverleaf nightshade or turkey berry in the single
plant trials did not affect total larval development
time. Likewise, the test for equality of the sur-
vival curves for L. texana reared on silverleaf
nightshade or turkey berry was not significant
(Chi square = 5.942, df = 4, p > 0.05), suggesting
that no differences in survival could be detected
on these two solanum species.


December 2002







Cuda et al.: Acceptability of Exotic Solanum spp. to Native Leptinotarsa Beetles


20 -

10



1 3 5 7 9 11 13 15 17 19 21 23 25 27
AGE (DAYS)

Fig. 1. Survival of larvae of Leptinotarsa defecta on four species of the genus Solanum in single plant (no-choice)
feeding tests in the laboratory. Lines end at larval death or adult emergence. SLN, silverleaf nightshade; TBY, tur-
key berry; TSA, tropical soda apple; and WLN, wetland nightshade.


The amount of feeding observed on the four
solanums by larvae ofL. defecta and L. texana in
the single plant feeding trials is presented in Ta-
ble 1. Larvae of L. defecta consumed on average
64.0 + 9.2 cm2 of silverleaf nightshade leaf tissue,
and mean survival to the pupal stage (= day 18)
on its natural host plant was 46.7 24.0% (Fig. 1).
Although a small amount of feeding occurred on
turkey berry and wetland nightshade, all larvae
died as first instars. Furthermore, newly hatched
larvae confined on tropical soda apple leaves did
not feed at all and died within a few days. In con-
trast, larvae of L. texana readily accepted turkey
berry leaves as a food source. Larvae ingested
104.5 + 26.4 cm2 of turkey berry leaf tissue com-
pared to only 52.3 + 7.7 cm2 for silverleaf night-
shade (Table 1). Also, larval survival on both
plant species was the same for L. texana. Survi-
vorship to the pupal stage (= day 18) was 40.0 +
23.1% and 40.0 11.5% for turkey berry and sil-
verleaf nightshade, respectively (Fig. 2).
Potato tree, which is considered a threatened
species in Florida, was not an acceptable host
plant for L. texana. Although the leaves sustained
some feeding damage, average leaf consumption
by the larvae was significantly lower on potato


tree (17.8 17.8 cm2) compared to turkey berry
(98.06 22.33 cm2) (t = 2.81, df= 4,p < 0.05). More
importantly, no larvae of L. texana restricted to a
diet of potato tree leaves survived beyond the sec-
ond instar on this high risk species whereas seven
out of 15 larvae, or 47%, experienced normal de-
velopment and pupation exclusively on a diet of
turkey berry leaves. The amount of turkey berry
leaf tissue consumed by larvae in this test was not
statistically different (t = 0.239, df = 4, p > 0.05)
from that observed for turkey berry in the earlier
single plant test shown in Table 1.
Paired plant tests: Paired comparison tests
were conducted only with L. texana because the
single plant trials demonstrated this insect was
capable of completing its development to the pu-
pal stage on turkey berry in the absence of its nat-
ural host plant silverleaf nightshade. When
offered a choice between leaf disks of silverleaf
nightshade and turkey berry as a food source, the
larvae did not exhibit a clear preference for silver-
leaf nightshade over turkey berry (Table 1). Al-
though average leaf consumption on silverleaf
nightshade was 76.2 + 6.69 cm2 compared to 40.0
+ 12.9 cm2 for turkey berry, the observed differ-
ences were not significant (t = 2.49, df = 4, p >







Florida Entomologist 85(4)


30 -

20 -

10

0
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29
AGE (DAYS)

Fig. 2. Survival of larvae of Leptinotarsa texana on four species of the genus Solanum in single plant (no-choice)
and paired plant (choice) feeding tests in the laboratory. Lines end at death or pupation of larvae. SLN, silverleaf
nightshade; TBY, turkey berry; TSA, tropical soda apple; WLN, wetland nightshade; and SLN + TBY, silverleaf
nightshade + turkey berry.


0.05). Survival and development of L. texana to
the pupal stage (= day 18) in the choice tests were
virtually identical (40.0 5.8%) to that observed
in the single plant trials (Fig. 2).

Adult Female Survival and Reproduction

In total, eight out of 12 females (67%) of the F,
generation survived and reproduced on silverleaf
nightshade compared to only three F, females
(25%) on turkey berry. However, the surviving fe-
males on average lived as long on turkey berry
(58.0 18.3 days) as they did on their natural host
plant silverleaf nightshade (58.1 22.4 days) (t =
0.00, df= 9,p > 0.05) (Table 2).
Adults of L. texana caged on potted turkey
berry plants exhibited an unusual feeding behav-
ior not observed on the silverleaf nightshade
plants in this study. Beetles often completely
stripped the turkey berry plants of their leaves by
feeding on the petioles where they were attached
to the stem. This feeding behavior resulted in
complete defoliation of the turkey berry plants
even at the low adult densities (1 to 3 beetles per


plant) maintained in this study. Hoffmann et al.
(1998) observed a similar phenomenon on silver-
leaf nightshade but only when L. texana reached
high densities following its release and establish-
ment in South Africa for biological control of this
weed.
The reproductive performance of female L. tex-
ana on potted turkey berry plants was similar to
silverleaf nightshade in this study (Table 2). The
number of egg masses deposited by the surviving
females on silverleaf nightshade was 18.9 + 3.8
compared to 9.7 + 6.7 on turkey berry, but the dif-
ference was not significant (t = 1.24, df = 9, p >
0.05). Also, the number of eggs laid in each mass
by females confined to each of these test plants
was similar. The number of eggs per mass aver-
aged 22.6 + 1.8 for silverleaf nightshade com-
pared to 16.0 + 3.0 for turkey berry (t = 1.96, df =
9, p > 0.05). More importantly, the viability of the
eggs produced by the F, females reared exclu-
sively on a diet of either silverleaf nightshade or
turkey berry leaves was the same. Average per-
cent eclosion of F generation larvae from eggs de-
posited on silverleaf nightshade and turkey berry


December 2002







Cuda et al.: Acceptability of Exotic Solanum spp. to Native Leptinotarsa Beetles


MSLN


2 -


0
I II III IV

INSTAR

Fig. 3. Average stadial length (in days) of each larval instar of Leptinotarsa defecta on Solanum elaeagnifolium
silverleaff nightshade, SLN) in single plant (no-choice) feeding tests in the laboratory.


I II III IV

INSTAR

Fig. 4. Average stadial length (in days) of each larval instar of Leptinotarsa texana on Solanum elaeagnifolium
silverleaff nightshade, SLN) and Solanum torvum (turkey berry, TBY) in single plant (no-choice) tests in the labo-
ratory.







Florida Entomologist 85(4)


TABLE 1. FEEDING (CM2) BY LARVAE OF LEPTINOTARSA DEFECTA AND L. TEXANA ON SOLANUM SPP. IN THE LABORA-
TORY.

L. defecta L. texana

Test/Plant Species' Mean (SEM) Mean (SEM)

Single Plant2
SLN 64.00 (9.2) a4 52.30 (7.7) b4
TBY 0.02 (0.01) c 104.50 (26.4) a
TSA 0.00 (0.0) c 0.00 (0.0) d
WLN 0.17 (0.04) b 0.08 (0.4) c

Paired Plant3
SLN 76.20 (6.69) b
TBY -40.00 (12.9) b

SLN = silverleaf nightshade; TBY = turkey berry; TSA = tropical soda apple; WLN = wetland nightshade.
Amount of feeding per n = 3 groups of 5 larvae; each group of larvae exposed to only one test plant species. Values for leaf consumption were based
on the number of larvae surviving in each trial.
'Amount of feeding per n = 3 groups of 10 larvae; each group of larvae exposed to both plant species simultaneously. Values for leaf consumption were
based on the number of larvae surviving in each trial.
'Means followed by the same letters within columns are not statistically different (p > 0.05) according to Tukey's Studentized Range (HSD) test.
"Not tested.


was 78.9 6.4% versus 78.0 7.1%, respectively (t
= 0.08, df= 9,p > 0.05). Taken together, these data
strongly suggest that L. texana is capable of con-
tinuous reproduction on turkey berry.

DISCUSSION

Risk assessment has been a cornerstone of the
practice of weed biological control since its incep-
tion because of safety concerns for crop species
(Strong & Pemberton 2000). Clearly, any insect
introduced for the biological control of a weed
must not itself become a plant pest. The rigorous
screening process ensures that non-specialist in-
sects capable of reproducing on economically im-
portant, or environmentally sensitive species that
are close relatives of the target weed, are dropped
from further consideration. In recent years, risk
assessment has focused less on crop species and
more on native plant species related to the target
weed, and the ecological consequences of "envi-
ronmental spillover"-when a non-target species
is attacked by the insect after its introduction
(Tisdell et al. 1984). The ecological risks associ-
ated with releasing an insect for weed biological
control with a host range that includes non-target
native species (especially those threatened with
extinction) are high, and it is unlikely that the ef-
fects will be reversible once the insect is intro-
duced (Strong 1997, Louda et al. 1997, Strong &
Pemberton 2000, Louda & O'Brien 2002).
Environmental risks can be reduced by select-
ing weed targets for classical biological control
that (a) are nonnative invasive plant species, and
(b) have few native relatives in the United States
that could become host plants of the introduced
insects (Center et al. 1997, Strong & Pemberton


2000). From this premise, it follows that selecting
the nonnative solanum species tropical soda ap-
ple, wetland nightshade and turkey berry as can-
didates for classical biological control raises
questions about the potential effects of imported
insect herbivores on the numerous nontarget cul-
tivated and native representatives of the genus
Solanum in North America.
The genus Solanum contains over 30 species
that are indigenous to the United States, 27 of
these occurring in the southeast (Soil Conserva-
tion Service 1982). Two native species that are es-
pecially vulnerable to attack are the potato tree in
Florida (Coile 1998), and S. pumilum Dunal, a di-
minutive species once thought to be extinct yet
persists in a few sites in Alabama and Georgia
(C. T. Bryson, personal communication). In this
study, the potato tree was found to be an unac-
ceptable host plant for L. texana.
The genus and family (Solanaceae) also con-
tain economically important crop plants closely
related to tropical soda apple, wetland night-
shade, and turkey berry (Bailey 1971). Species
such as bell pepper (Capsicum), tomato (Lycoper-
sicon), tobacco (Nicotiana), eggplant and potato
(both Solanum spp.) contribute significantly to
Florida's economy. For example, the combined
economic value for Florida's solanaceous crop
plants in 1998 was reported to be over US $920
million (FLDACS 1998).
To reduce the risk of non-target damage, insect
natural enemies imported from the native range
of the nonnative solanaceous plants should use
only the target weeds as host plants. However,
the high degree of host specificity that must be
demonstrated in order to obtain federal and state
approval for release of these insects in the United


December 2002







Cuda et al.: Acceptability of Exotic Solanum spp. to Native Leptinotarsa Beetles 607


TABLE 2. LABORATORY SURVIVAL AND REPRODUCTIVE PERFORMANCE OF FEMALE LEPTINOTARSA TEXANA ON SOLANUM
TORVUM COMPARED TO ITS NATURAL HOST PLANT S. ELAEAGNIFOLIUM.

SLN2 TBY

Parameter' Mean (+SEM)3 Mean (SEM)

Longevity (days) 58.1(22.4) 58.0 (18.3)
Egg Masses 18.9 (3.8) 9.7 (6.7)
Eggs/Mass 22.6 (1.8) 16.0 (3.0)
% Larval Eclosion 78.9 (6.4) 78.0 (7.1)

'Data in each category derived from 8 ovipositing females in the S. elaeagnifolium tests, and 3 females in the tests with S. toruum. Adults were ob-
tained from neonate larvae reared through one generation on each of the test plants before the experiment was initiated.
'SLN silverleaf nightshade, S. elaeagnifolium; TBY turkey berry, S. torvum.
'Means within a row compared by t-test; none were statistically different (p > 0.05, df= 9).


States may be an unrealistic expectation. For ex-
ample, Leptinotarsa undecemlineata Stal, a con-
gener of the two leaf beetles whose host plant
relationships were examined in this study, is pur-
ported to be monophagous on turkey berry in
Cuba (Ballou 1928, Pospisil 1972). In reality, L.
undecemlineata is actually oligophagous, attack-
ing several different host plants in the genus
Solanum (Hsiao and Hsiao 1983, Jacques 1985).
This particular example is relevant not only be-
cause it concerns the same group of insects and
one of the plants that were the subject of this
study, but clearly illustrates that most plant-feed-
ing insects feed on a small group of closely related
plants instead of a single species (Pemberton
1996).
The risk assessment process is further compli-
cated by the fact that herbivorous insects that are
screened as candidates for weed biological control
projects often exhibit expanded host ranges under
confined laboratory conditions (Cullen 1990, Blos-
sey 1995, Olckers et al. 1999). For example, sev-
eral candidates for classical biological control of
tropical soda apple and other solanaceous weeds
usually developed in laboratory studies on egg-
plant, Solanum melongena L., potato, Solanum
tuberosum L. and tomato, Lycopersicon esculen-
tum Mill., and other solanums that were not at-
tacked in nature (Olckers et al. 1995, Hill &
Hulley 1996, Olckers 1996, 1999, Gandolfo 1997,
Medal et al. 1999, 2002).
An alternative to classical biological control-
the importation of natural enemies from the na-
tive range of the target weed-is to select native
insects from North American congeners, and at-
tempt to establish 'new associations' between
these native insects and the nonnative Solanum
spp. (Hokkanen & Pimentel 1984). This approach
differs from classical biological control in that the
natural enemies have not played a major role in
the evolutionary history of the host plant, and are
therefore considered "new associates" (Hokkanen
& Pimentel 1984). In theory, insect natural ene-
mies from closely related plant species growing in
similar climates but different geographical areas


from the target plant are potentially more damag-
ing than co-evolved natural enemies. The target
weed is more likely to experience greater damage
by the "new associates" because it lacks the ap-
propriate defense mechanisms to resist attack
(Hokkanen & Pimentel 1984). The 'new associa-
tion' approach for selecting plant-feeding insects
as biological control agents has been critically ex-
amined and supported by some practitioners of
biological control of weeds (Dennill & Moran
1989, DeLoach 1995), but has been criticized as
being based on faulty data by other specialists
(Goeden & Kok 1986).
Although there are risks associated with re-
leasing an insect in Florida from a congener of the
nonnative Solanum spp. that occurs in another
geographical region of North America ecoclimati-
cally similar to Florida (e.g., south Texas), the
risk of collateral attack on non-target species may
be acceptable. The only known potential host
plants for L. texana in Florida are eggplant and
horsenettle. In the unlikely event that eggplant
were to be attacked by L. texana, insecticides
used for crop production in Florida would be an
effective feeding deterrent (Nesheim & Vulinec
2001). Likewise, minor damage to horsenettle
could be viewed as beneficial as this native
solanum is regarded as a weed in Florida (Hall &
Vandiver 1991). More importantly, the 'new asso-
ciation' approach has been attempted in the
United States against Eurasian watermilfoil,
Myriophyllum spicatum L. (Haloragaceae),
(Buckingham 1994, Sheldon and Creed 1995) and
more recently English cordgrass, Spartina an-
glica Lois. (Poaceae) (Wu et al. 1999) without
harming native plant communities.
The results of this study indicate that the na-
tive leaf beetle L. texana, which attacks silverleaf
nightshade, is capable of using the nonnative tur-
key berry as a host plant whereas none of the non-
native solanums supported development in the
laboratory of its congener L. defecta. The inclu-
sion of turkey berry in the potential host range of
L. texana was not entirely unexpected. Studies by
Hsiao (1981) and Olckers et al. (1995) showed the







Florida Entomologist 85(4)


potential host ranges of L. defecta and L. texana
are much broader than their actual host ranges
would indicate. In these laboratory studies, both
beetles exhibited limited reproduction on several
native Solanum spp. as well as on cultivated egg-
plant. However, the study by Olckers et al. (1995)
also showed these beetles would not attack other
members of the plant family Solanaceae that are
vital to Florida agriculture, including potato, to-
mato, or bell pepper, and would not survive on
plants outside the genus Solanum.
The acceptance of eggplant as a host plant in
laboratory tests by candidate natural enemies of
solanaceous weeds appears to be the rule rather
than the exception (Olckers 1996, Medal et al.
1999, 2002). Eggplant apparently is devoid of cer-
tain feeding deterrents (chemical or physical)
that normally play a role in host plant selection,
and often produces false positives in a laboratory
setting. However, L. texana never has been re-
corded on eggplant in south Texas even though
this economically important solanum is often cul-
tivated extensively in the vicinity of its natural
host silverleaf nightshade. Furthermore, egg-
plant crops in Florida would be chemically pro-
tected from attack by L. texana. Thus, the risk to
eggplant from damage by L. texana would be low
if the insect were approved for released in Florida
for biological control of turkey berry.
If L. texana were approved for release, this
"new associate" might provide substantial control
of one of Florida's most invasive solanaceous
weeds. Sustained defoliation by L. texana could
severely stress turkey berry and perhaps make it
less competitive with native plants. More impor-
tantly, the ecological risks associated with the re-
lease in Florida of L. texana may be acceptable
because of the behavior exhibited by the beetle
following its introduction and establishment on
silverleaf nightshade in South Africa. Hoffmann
et al. (1998) reported that L. texana attained high
densities and had well-developed wings, but was
unable to fly or reluctant to do so. The beetle re-
mained in the release area until the food supply
was exhausted and only dispersed by crawling en
masse to adjacent plants. Because it appears that
L. texana is incapable of flight, the beetle could be
confined to a small area during the initial release
and establishment phase where appropriate mit-
igation procedures would be implemented if post
release surveys indicated that non-target plants
were vulnerable to attack.
Although L. texana is native to North America,
and would be exempt from the rigorous screening
and approval process required by the federal
Technical Advisory Group on the Introduction of
Weed Biological Control Agents (TAG) (Lima
1990), other nonweedy members of the genus
Solanum that are native to Florida could be at-
tacked. The risk to these non-target species
should be thoroughly assessed and the appropri-


ate state agencies consulted to obtain their ap-
proval before releasing L. texana in Florida for
biological control of turkey berry.

ACKNOWLEDGMENTS

We thank John Capinera and Howard Frank for re-
viewing an earlier version of the manuscript. We also
thank Lucy Treadwell for technical assistance. This
project was funded by grants from the Florida Depart-
ment of Environmental Protection, Bureau of Invasive
Plant Management Contract No. ERP039, and the Of-
fice of the Dean for Research, UF/IFAS. Florida Agricul-
tural Experiment Station Journal Series No. R-07585.

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Florida Entomologist 85(4)







Triapitsyn & Berezovskiy: Revision of Kalopolynema (Mymaridae)



REVISION OF KALOPOLYNEMA, WITH NOTES ON PLATYPOLYNEMA
(HYMENOPTERA: MYMARIDAE)

SERGUEI V. TRIAPITSYN AND VLADIMIR V. BEREZOVSKIY
Entomology Research Museum, Department of Entomology, University of California
Riverside, California 92521

ABSTRACT
The New World fairyfly genus Kalopolynema is divided into two subgenera, Kalopolynema s.
str., the nominal subgenus with the type species K. (Kalopolynema) discrepans Ogloblin, and
Floripolynema S. Triapitsyn and Berezovskiy, subgen. nov. with the type species K. (Flori-
polynema) mizelli S. Triapitsyn and Berezovskiy, sp. nov., described from Florida, USA. The
four known species of Kalopolynema are keyed and illustrated, including the North Ameri-
can K. (Kalopolynema) ema (Schauff and Grissell), comb. nov. from Polynema, and K. (Kalo-
polynema) poema S. Triapitsyn and Berezovskiy, sp. nov. from Argentina. Both Kalopolynema
and its relative Platypolynema, a monotypic genus from Argentina also treated in this re-
view, are characterized by the presence of subantennal grooves on the face.
Key Words: Hymenoptera, Mymaridae, Kalopolynema, Floripolynema, Platypolynema, tax-
onomy
RESUME
El g6nero de avispas mimaridos Kalopolynema del Nuevo Mundo es dividido en dos subg6-
neros, Kalopolynema s. str., el subg6nero nominal con la especie tipica K. (Kalopolynema)
discrepans Ogloblin, y Floripolynema S. Triapitsyn y Berezovskiy, subgen. nov. con la especie
tipica K. (Floripolynema) mizelli S. Triapitsyn y Berezovskiy, sp. nov., descrita de la Florida,
los EE.UU. Una clave ilustrada para las cuatros species conocidas de Kalopolynema es pre-
sentado, incluyendo la especie norteamericana K. (Kalopolynema) ema (Schauff y Grissell),
comb. nov. desde Polynema, y K. (Kalopolynema) poema S. Triapitsyn y Berezovskiy, sp. nov.
de Argentina. Ambos Kalopolynema y su relative Platypolynema, un g6nero monotipico de
Argentina que esta tambi6n tratado en este revision, se caracterizan por la presencia de sur-
cos subantenales sobre el frente.
Translation provided by senior author.


The little known mymarid genus Kalopoly-
nema was described by Ogloblin (1960) from two
females belonging to the type species, K discrep-
ans Ogloblin. Both the holotype and the paratype
were collected in the Province of Buenos Aires,
Argentina, and until recently this genus has not
been recorded outside that country. Our interest
in studying Kalopolynema was sparked by the re-
sult of a comparison of the type material of K. dis-
crepans with the very distinctive North American
species Polynema ema Schauff and Grissell,
which we found to better fit in Kalopolynema,
based on several morphological features dis-
cussed below.
Kalopolynema species have been practically
unrecognizable although the genus was included
in the key to females of New World Mymaridae by
Yoshimoto (1990). However, Platypolynema
Ogloblin would also key to the same couplet with
Kalopolynema as both genera share two impor-
tant morphological features, i.e., a short, sub-
quadrate, petiole and the presence of two
converging subantennal grooves on the face, ex-
tending from toruli to margin of clypeus. The lat-
ter character was not noticed in Kalopolynema by
Ogloblin (1960) because of the way he slide-
mounted the specimens: the position of the head


prevents one from seeing the lower face and al-
lows only the vertex and the occiput to be ob-
served easily; however, at high magnification we
noticed the presence of these grooves just below
the toruli in the paratype of K. discrepans. The
same happened with the original description of
Platypolynema (Ogloblin 1960) but later, when
more specimens of P. cautum Ogloblin became
available to him, the presence of subantennal
grooves on the face was added to the generic diag-
nosis (Ogloblin 1967).
Kalopolynema was not treated by Annecke and
Doutt (1961) nor was it included in the key to the
Nearctic genera of Mymaridae (Huber 1997),
where it would key in the same couplet together
with Polynema Haliday. To facilitate recognition
of Kalopolynema from related genera in the New
World, a key is provided that also includes Platy-
polynema and Polynema s. 1.
Our interest in the Polynema group of genera
in a broad sense, which corresponds roughly to
the tribe Mymarini of Annecke and Doutt (1961)
and where both Kalopolynema and Platy-
polynema belong, was further instigated by the
recent discovery of a specimen from Monticello,
Florida, that we first thought to represent a new
mymarid genus from that group. However, after a







Florida Entomologist 85(4)


second female of the same species was discovered
and both specimens were carefully studied, we de-
cided that they would be better placed in Ka-
lopolynema as a new subgenus and species,
described here respectively as Floripolynema and
K. (Floripolynema) mizelli. Inclusion of this taxon
in Kalopolynema requires broadening of its diag-
nosis compared to that given by Ogloblin (1960).
Terms for morphological features are those of
Gibson (1997). Measurements are given in mi-
crometers (pm) as length or, where appropriate,
as length/width. Abbreviations (codens) for depos-
itories of specimens are as follows: CNCI, Cana-
dian National Collection of Insects, Ottawa,
Ontario, Canada; FSCA, Florida State Collection
of Arthropods, Gainesville, Florida, USA; MLPA,
Museo de La Plata, La Plata, Buenos Aires, Ar-
gentina; UCRC, Entomology Research Museum,
University of California, Riverside, California,
USA; USNM, National Museum of Natural His-
tory, Washington, D.C., USA. An abbreviation
used in the text is: F = funicle segment.

GENUS KALOPOLYNEMA OGLOBLIN
(Figs. 1-10)

Kalopolynema Ogloblin, 1960: 3, 6;Yoshimoto, 1990: 72-
73.
Type species: Kalopolynema discrepans Ogloblin, by
monotypy and original designation.

Diagnosis. Face (Figs. 3, 7) with two converg-
ing subantennal grooves extending from toruli to
margin of clypeus; prosternum "open", not closed
by propleura anteriorly; mesoscutum and scutel-
lum with cellulate sculpture; forewing (Figs. 2, 6,


9) narrow, marginal vein relatively short and with
1 (in Floripolynema subgen. nov.) or 2 (in the
nominal subgenus) dorsal macrochaetae; petiole
in dorsal view subquadrate, subrectangular, or
cross-shaped (Fig. 10), at most 2 x as long as wide,
attached posteriorly to gastral tergum; female
with ovipositor usually longer than body and
strongly exserted beyond apex of gaster.
Comments. The previous diagnoses of Kalo-
polynema are incomplete because they failed to
indicate perhaps the most important morpho-
logical feature that distinguishes Kalopoly-
nema, including the new subgenus described
below, and its sister genus Platypolynema from
all other described genera belonging to the
Polynema group: the presence of well-developed
subantennal grooves on the face. The diagnoses
by Ogloblin (1960) and Yoshimoto (1990) empha-
sized the peculiar shape of the petiole in Ka-
lopolynema, which is also almost as short in
Platypolynema; however, such a character is not
unique among the Polynema-group genera (e.g.,
it is also found in the Australian Polynema
quadripetiolatum Girault), although is very
rare. Kalopolynema and Platypolynema are
closely related but can be separated from each
other by the combination of several morphologi-
cal features given in the key.
Known host associations of Kalopolynema in-
clude two species of the planthopper genus Mega-
melus Fieber (Delphacidae) that reproduce on
plants in or near water. The females of Kalo-
polynema species are equipped with a long ovi-
positor, apparently to be able to reach their hosts'
eggs imbedded in the host plant tissue, which
could be the aerenchyma in some water plants.


KEY TO THE SUBGENERA AND SPECIES OF KALOPOLYNEMA, FEMALES

1. Marginal vein of forewing with two dorsal macrochaetae (Figs. 2, 6); propodeum without dorsal elevation
in the middle (subgenus Kalopolynema Ogloblin s. str.) ........................................ 2
Marginal vein of forewing with only one (the distal) dorsal macrochaeta (Fig. 9); propodeum with dorsal el-
evation in the middle (Floripolynema S. Triapitsyn and Berezovskiy, subgen. nov.) ..................
................................ K. (Floripolynema) mizelli S. Triapitsyn and Berezovskiy, sp. nov.
2. Fl almost as long as pedicel; F4 almost as long as F1 ........... K. (Kalopolynema) discrepans Ogloblin
- Fl less than 0.5 x length of pedicel; F4 longer than Fl ......................................... 3
3. Petiole in dorsal view longer than wide ..... K. (Kalopolynema) ema (Schauff and Grissell), comb. nov.
- Petiole in dorsal view about as long as wide ...................................................
.............................. K. (Kalopolynema) poema S. Triapitsyn and Berezovskiy, sp. nov.

SUBGENUS KALOPOLYNEMA OGLOBLIN S. STR. rior to frenal line notably less pronounced than on
(Figs. 1-6) mesoscutum and frenal area of scutellum; propo-
Kalopolynema Ogloblin, 1960: 3,6; Yoshimoto, 1990: 72- deum smooth or with a median carina, without a
73. dorsal elevation in the middle; marginal vein of
Type species: Kalopolynema discrepans Ogloblin. forewing with 2 dorsal macrochaetae; petiole in
dorsal view subquadrate or subrectangular; female
Diagnosis. Female clava with 7 or 9 longitudinal with ovipositor usually much longer than body and
sensilla (Figs. 1, 5); sculpture on scutellum ante- very strongly exserted beyond apex of gaster.


December 2002







Triapitsyn & Berezovskiy: Revision of Kalopolynema (Mymaridae)


KALOPOLYNEMA (KALOPOLYNEMA)
DISCREPANS OGLOBLIN
(Figs. 1, 2)

Kalopolynema discrepans Ogloblin, 1960: 6-7, figs. 5-11.
Type locality: Tigre, Buenos Aires, Argentina.
Types. Holotype female (MLPA), examined. On slide, la-
beled: 1. "Kalopolynema discrepans Ogl. HOLOTIPO 9
Bs. As. Tigre IV-1942, A.O."; 2. "Kalopolynema dictynn[a
- crossed out]um [this is a manuscript name] A. Ogl. Y
Tigre, B. A. IV.1942. A. O.". Paratype female (MLPA), ex-
amined. On slide, labeled: 1. "Kalopolynema discrepans
Ogl. PARATIPO 9 Bs. As. Bella Vista X.1946, A. O." 2.
"Kalopolynema dictynna A. O. 9 [an illegible word fol-
lows] Bella Vista X.1942. A. O.".

Diagnosis. This species is easy to separate from
the other two known species in this genus by char-
acters given in the key. Other distinguishing fea-
tures of K. discrepans females include the
presence of two longitudinal sensilla on both F5
and F6 and nine longitudinal sensilla on the
clava.
Male. Unknown.

Distribution. Known only from the type local-
ities, Tigre and Bella Vista, in the Province of
Buenos Aires, Argentina.
Host. Unknown.


Comments. The original description of this
species is adequate and well-illustrated. Here we
provide drawings of the antenna (Fig. 1) and
forewing (Fig. 2) of K. discrepans because Oglob-
lin's illustrations are incomplete.

KALOPOLYNEMA (KALOPOLYNEMA) EMA (SCHAUFF AND
GRISSELL), COMB. NOV.
(Figs. 3, 4)
Polynema ema Schauff and Grissell, 1982: 530-533.
Type locality: Etherton Pond, 3 mi. N. of Pomona, Jack-
son Co., Illinois, USA.
Types. Holotype female and numerous paratypes
(USNM and CNCI), examined.

Other material examined. CANADA, Ontario,
Ottawa, Montfort hospital wood, VII-1994, J. R.
Vockeroth (1 2, CNCI). USA. Florida: Alachua
Co., Gainesville: 1-5-1-1976, E. E. Grissell (1 2,
FSCA); 26-IX-5.XII-1986, D. B. Wahl (2 2,
CNCI); 14-IV-1987, CNC Hym. Team (1 9, CNCI);
23-II-2.VI-1988, D. B. Wahl (2 92 CNCI). Citrus
Co., 30 km N Homosassa, Ozello Trail, 14-IV-
1992, L. Masner (1 2, CNCI). Collier Co., Faka-
hatchee Strand State Park, 13-XI-1998-15-I-1999,
M. Owen (1 2, CNCI). St. Johns Co., Fort Caro-
line, 13-X-1980, L. Masner, B. Bowen (1 2, CNCI).
Georgia, Liberty Co., St. Catherines I., 22-27-
VIII-1995, A. Sharkov (1 2, UCRC). Massachu-
setts, Hampden Co., Westfield, 30-IX-1992, J. R.


------------------7







2
Figs. 1 and 2. Kalopolynema (Kalopolynema) discrepans Ogloblin, female. Fig. 1. Antenna (paratype). Fig. 2.
Forewing holotypee). Scale bars = 0.1 mm.







Florida Entomologist 85(4)


Fig. 3. Kalopolynema (Kalopolynema) ema (Schauff
and Grissell). Head (frontal view), female (Gainesville,
Florida, USA).

Vockeroth, pan trap in Typha sp. (1 2, CNCI).
Maryland, Prince Georges Co., Laurel, Patuxent
Wildlife Research Center, 22-25-VI-1980, L. Mas-
ner (1 Y, 1 6, CNCI). Virginia, Louisa Co., 4 mi. S
Cuckoo, 28-VII-11-VIII-1988, J. Kloke, D. R.
Smith (1 Y, USNM).
Diagnosis. This species can be distinguished
from the type species of the genus, K discrepans,
and from the newly described K. poema by char-


Fig. 4. Kalopolynema (Kalopolynema) ema (Schauff
and Grissell). Genitalia, male (Laurel, Maryland, USA).
A. Dorsal view. B. Lateral view. Scale bars = 0.1 mm.


acters given in the key. Other distinguishing
characters of both K ema and K poema females
include the absence of longitudinal sensilla on F5
and F6 and the presence of 7 longitudinal sensilla
on the clava. The mesosoma of K ema is very
short and compact, shorter than the gaster. The
propodeum in this species has a complete median
carina in the male and a broken carina in the fe-
male (Illinois specimens); in the female speci-
mens from Florida and Georgia the propodeum is
either almost smooth or with a weak trace of a
broken median carina only. The petiole is about
2 x as long as wide; subrectangular in dorsal view
and produced into a tooth ventrally (best seen in
lateral view).
Here we provide an illustration of the head in
frontal view (Fig. 3) to show the presence and con-
figuration of subantennal grooves on the face.
Also illustrated are the male genitalia (Fig. 4)
which are very similar to those in many Polynema
species. These illustrations supplement the fig-
ures in Schauff and Grissell (1982).
Distribution. As seen from "Other material
examined" above, new distribution records since
Schauff and Grissell (1982) are from Canada (On-
tario) and USA (Florida, Georgia, Massachusetts,
Virginia); it probably will be found to occur
throughout the range of its host (Schauff and
Grissell 1982).
Host. The lily (water-lily) planthopper, Mega-
melus davisi Van Duzee (Homoptera: Delphaci-
dae).
Comments. This species was introduced in
1941 from Michigan into Honolulu, Oahu Island,
Hawaii, under the incorrect name Polynema cili-
ata (Say) (its nomenclatural history was discussed
by Schauff and Grissell (1982)), and successfully
established there on local populations of the wa-
ter-lily planthopper (Zimmerman 1948). The like-
lihood that K ema is also able to parasitize eggs of
other Megamelus species is very high.

KALOPOLYNEMA (KALOPOLYNEMA) POEMA S. TRIAPITSYN
AND BEREZOVSKIY, SP. NOV.
(Figs. 5, 6)

Types. Holotype female (CNCI). On card, labeled:
1."ARGENTINA: Buenos Aires Prov., Hurlingham Lab.,
18.xi. 1999. Ex. Megamelus sp. on Eichhornia crassipes";
2. "Kalopolynema (Kalopolynema) poema S. Triapitsyn
& Berezovskiy HOLOTYPE Y ". Paratype female
(CNCI) on slide, same data as the holotype except the
date is 19-XI-1999.

Description. Female. Color. Brown except
scape, pedicel, legs, and petiole light brown; distal
tarsomeres slightly darker than other leg seg-
ments; eye pink.
Head. Width 223, round in frontal view; face
with distinct, narrow subantennal grooves and
with several symmetrical rows of small setae;
torulus slightly above mid level of eye. Vertex


December 2002







Triapitsyn & Berezovskiy: Revision of Kalopolynema (Mymaridae)


7-;= i rY /


Figs. 5 and 6. Kalopolynema (Kalopolynema) poema S. Triapitsyn and Berezovskiy, sp. nov., female (paratype).
Fig. 5. Antenna. Fig. 6. Forewing. Scale bars = 0.1 mm.


rounded, with fine sculpture, ocelli in very obtuse
triangle. Mandible tridentate.
Antenna (Fig. 5) shorter than body, sparsely
setose except clava more densely setose. Radicle
not fused with scape, the scape smooth, 3.4 x as
long as wide; pedicel pear-shaped, longer than
wide, much longer than Fl; all funicle segments
longer than wide, Fl the shortest and F2 the long-
est, F3 longer than F4, F5 markedly shorter than
F4 and slightly shorter than F6, all funicle seg-
ments without longitudinal sensilla; clava 2.6 x
as long as wide, with 7 longitudinal sensilla, all of
them subapical.
Mesosoma. Pronotum very short, divided me-
diolongitudinally; pronotum, mesoscutum, axilla,
and frenal area of scutellum with conspicuous cel-
lulate sculpture; mesoscutum wider than long;
scutellum about as long as wide and as long as
mesoscutum, scutellar sensilla close to anterior
margin of scutellum, frenal line with small
foveae; metanotum strap-like; propodeum
smooth, without median carina.
Wings. Forewing (Fig. 6) 7.3 x as long as wide;
venation reaching slightly less than 14 length of
wing; longest marginal cilia almost 2 x greatest
width of blade; disc hyaline, more or less uni-
formly setose beyond venation. Hind wing disc
hyaline, with setae only along margins; longest
marginal cilia 6 x maximum width of blade.
Legs. Coxae smooth, metacoxa longer than
petiole. Protibia with 5 conical sensilla.


Metasoma. Petiole subquadrate in dorsal view.
Gaster longer than mesosoma. Ovipositor broadly
rounded anteriorly, occupying more than 4/5 length
of gaster, markedly exserted beyond its apex (ex-
serted part of ovipositor about 0.6 x its total length
in paratype); ovipositor/metatibia ratio 3.4:1.
Measurements (n = 1, taken from paratype ex-
cept body and head lengths from holotype): Body:
792. Head: 117; mesosoma: 300; mesoscutum:
106; scutellum: 101; gaster: 546; ovipositor: 1010
(1123 in holotype). Antenna: scape (including
radicle): 120; pedicel: 63; Fl: 31; F2: 75; F3: 61;
F4: 51; F5: 43; F6: 47; clava: 129. Forewing: 983/
146; longest marginal cilia: 259. Hind wing: 792/
16. Legs (given as coxa, femur, tibia, tarsus): fore:
92, 233, 233, 254; middle: 80, 194, 288, 292; hind:
118,202,318,391.
Etymology. The new species name means "a
poem" in Russian; the sole reason for choosing it is
the fact that it rhymes with the name of the
closely related species, K. ema.
Male. Unknown.
Diagnosis. This species is similar to K. ema. It
differs mainly in the shape of the petiole, as indi-
cated in the key, as well as in the shape of the mar-
ginal vein which is relatively shorter in K. poema.
The female clava in K. poema is about 2.6 x as long
as wide (2.1-2.2 x as long as wide in K. ema).
Distribution. Known only from the type local-
ity in Hurlingham, Buenos Aires, Argentina.


q







Florida Entomologist 85(4)


Host. Megamelus scutellaris Berg (Homop-
tera: Delphacidae).
Comments. The material of the new species
was sent to John T. Huber (CNCI) by Livy Will-
iams, III (USDA-ARS, Stoneville, Mississippi).
More detailed information on the identity of the
host planthopper was provided to me recently, as
a personal communication, by Alejandro Sosa
(South American Biological Control Laboratory,
USDA-ARS, Hurlingham, Buenos Aires, Argen-
tina) who apparently was the actual collector of
the two type specimens of K. poema. Megamelus
scutellaris lives on water-hyacinth, Eichhornia
crassipes (C. Martins) Solms-Loubach, in Argen-
tina and has been studied there as a potential bi-
ological control agent against this aquatic weed.
Should M. scutellaris ever be considered for es-
tablishment beyond its native range, caution
must be applied to avoid an inadvertent introduc-
tion of its egg parasitoid, K. poema.

FLORIPOLYNEMA S. TRIAPITSYN AND BEREZOVSKIY,
SUBGEN. NOV.
(Figs. 7-10)
Type species: Kalopolynema (Floripolynema) mizelli
S. Triapitsyn and Berezovskiy, sp. nov. Monobasic.
Diagnosis. Female clava with 7 longitudinal
sensilla (Fig. 8); sculpture on scutellum anterior
to frenal line as pronounced as on mesoscutum
and frenal area of scutellum; propodeum with an
incomplete median carina and with a dorsal ele-
vation in the middle; marginal vein of the forew-
ing with one (the distal) dorsal macrochaeta (Fig.
9); petiole in dorsal view cross-shaped, almost
subquadrate (Fig. 10); female with ovipositor al-
most as long as body.
Description. Female. Head in dorsal view
about as wide as mesosoma, oval in lateral view.


Figs. 7. Kalopolynema (Floripolynema) mizelli S. Tri-
apitsyn and Berezovskiy, sp. nov., female (paratype).
Head (frontal view). Scale bars for Figs. 7-10 = 0.1 mm.


Face (Fig. 7) with narrow, distinct subantennal
grooves; torulus slightly above mid level of eye, al-
most touching preorbital trabecula. Vertex
rounded, with fine sculpture, ocelli in very obtuse
triangle. Mandible tridentate.
Antenna (Fig. 8). Scape much longer than
wide; pedicel longer than wide, funicle 6-seg-
mented, all segments more or less cylindrical;
clava entire, with 7 longitudinal sensilla.
Mesosoma. Pronotum divided mediolongitu-
dinally, neck strongly wrinkled transversely, lobes
of pronotal collar with fine cellulate sculpture.
Mesoscutum, axilla, scutellum, and metanotum
with conspicuous cellulate sculpture; mesoscu-
tum a little longer than wide, with prominent no-
tauli; scutellum shorter than mesoscutum,
scutellar sensilla almost in the middle and far
apart from each other, frenal line with small
foveae; metanotum strap-like. Propodeum
smooth, in dorsal view elevated posteriorly in the
middle to form a ridge projecting beyond posterior
margin and in lateral view forming almost a right
angle (somewhat as in Polynema (Dorypolynema)
mendeli Girault), with an incomplete median car-
ina in distal half of propodeum; propodeal seta
strong, near posterior margin; propodeal spiracle
rounded.
Wings. Forewing (Fig. 9) relatively narrow; ve-
nation short, extending about 14 length of wing,
hypochaeta reaching posterior margin, marginal
vein with one (the distal) dorsal macrochaeta and
one short ventral seta at apex; disc hyaline, more
or less uniformly setose beyond venation; longest
marginal cilia about as long as greatest width of
blade. Hind wing much shorter than forewing,
typical for Polynema-group of genera.
Legs. Tarsi 4-segmented.
Metasoma. Petiole (Fig. 10) in dorsal view
cross-shaped, almost subquadrate, attached pos-
teriorly to gastral tergum; gaster projecting for-
ward ventrally, almost reaching base of mesocoxa
(best seen in lateral view); ovipositor long, almost
as long as body, markedly exserted beyond its
apex.

Male. Unknown.

Etymology. An arbitrary use of the first part
of the word Florida, referring to the state where
the new subgenus was found, combined with the
generic name Polynema. Gender: neuter.

KALOPOLYNEMA (FLORIPOLYNEMA) MIZELLI
S. TRIAPITSYN AND BEREZOVSKIY, SP. NOV.
(Figs. 7-10)
Types. Holotype female (CNCI). On point, labeled: 1.
"USA: Florida, Alachua Co., Gainesville, AEI, 23.ii-
2.vi.1988, D. B. Wahl, FIT"; 2. "Floripolynema mizelli S.
Triapitsyn & Berezovskiy HOLOTYPE Y ". Paratype fe-
male (UCRC). On slide, labeled: 1. "USA, Florida, Jef-
ferson Co., Monticello, University of Florida Research &


December 2002







Triapitsyn & Berezovskiy: Revision of Kalopolynema (Mymaridae)


9


Fig. 8. Antenna. Fig. 9. Forewing.


Education Center, 25.vii-12.viii.2000, R. Mizell, III. MT
at forest edge"; 2. "Floripolynema mizelli S. Triapitsyn
& Berezovskiy PARATYPE Y ".

Description. Female. Color. Head and mesos-
oma black; flagellum and gaster dark brown;
scape, pedicel, wing venation, petiole, ovipositor
sheath and external plate of ovipositor brown;
legs light brown except base of metacoxa, apical
half of mesotibia, metatibia, last tarsomeres of
fore- and middle legs and metatarsus darker. Eye
dirty pink.
Head. Width 241, face (Fig. 7) with several
symmetrical rows of small setae. Ocellar setae
small, inconspicuous.
Antenna (Fig. 8) much shorter than body,
sparsely setose except clava more densely setose.
Radicle almost fused with scape, scape smooth,
about 3 x as long as wide; pedicel slightly longer
than Fl; F2 longest of funicle segments, F3 much
longer than following funicle segments; F4 and
F6 subequal in length (F5 slightly shorter); all fu-
nicle segments without longitudinal sensilla; F6
slightly wider than preceding funicle segments;
clava 2.7 x as long as wide, with 7 longitudinal
sensilla, 6 of them subapical.
Mesosoma. Lobe of pronotal collar with 5 se-
tae; axilla small, with one weak seta; scutellum
about as wide as long.
Wings. Forewing (Fig. 9) 6.5 x as long as wide;
marginal + stigmal vein with 4 placoid sensilla at
apex; longest marginal cilia 1.15 x greatest width
of blade. Hind wing disc hyaline, with a few setae


in an incomplete row in distal half; longest mar-
ginal cilia 5 x maximum width of blade.
Legs. Coxae smooth, metacoxa much longer
than gastral petiole. Protibia with 3 or 4 conical
sensilla.
Metasoma. Petiole (Fig. 10) slightly wrinkled
transversely in basal third and with a transverse


Fig. 10. Petiole (dorsal view).


10







Florida Entomologist 85(4)


carina ventrally. Ovipositor occupying the whole
length of gaster, markedly exserted beyond its
apex (by about 13 total length of ovipositor); ovi-
positor/metatibia ratio 2.1:1.
Measurements (n = 1, paratype): Body (length
of the dry-mounted specimen taken before slide-
mounting): 1121. Head length (taken before slide-
mounting): 168; mesosoma: 482; mesoscutum:
153; scutellum: 118; petiole: 92; gaster: 692; ovi-
positor: 1037. Antenna: scape (including radicle):
129; pedicel: 66; Fl: 59; F2: 115; F3: 99; F4: 56;
F5: 52; F6: 54; clava: 153. Forewing: 1200/185;
longest marginal cilia: 212. Hind wing: 1001/21.
Legs (given as coxa, femur, tibia, tarsus): fore:
128, 281, 321, 277; middle: 113, 256, 464, 307;
hind: 153, 255, 491,428.
Male. Unknown.
Etymology. The new species is named in
honor of Russell F. Mizell, III, collector of one of
the type specimens.
Distribution. Known only from the type lo-
calities in Florida.
Host. Unknown.
Comments. This species is one of the most
beautiful of North American Mymaridae.


GENUS PLATYPOLYNEMA OGLOBLIN
(Figs. 11, 12)

Platypolynema Ogloblin, 1960: 7; Ogloblin, 1967: 192;
Yoshimoto, 1990: 73.
Type species: Platypolynema cautum Ogloblin, by
monotypy and original designation.

Diagnosis. Head very large and high, mark-
edly wider than mesosoma, with well-defined sub-
antennal grooves; prosternum anteriorly "closed"
by propleura; mesoscutum smooth, much longer
than scutellum; forewing (Fig. 12) long and nar-
row, with a constriction of blade beyond venation;
marginal vein long and with 2 dorsal macrochae-
tae; petiole in dorsal view subquadrate; female
with ovipositor very long, acutely elbowed and
strongly produced forward anteriorly beneath
mesosoma.
Comments. Yoshimoto (1990) apparently
overlooked the earlier description of the female of
Platypolynema by Ogloblin (1967) and therefore
it is included only in his key to the males of the
New World genera of Mymaridae.
The biology of the single known species of Platy-
polynema is unknown. Like Kalopolynema species,
it is quite possible that it is associated with some
Auchenorrhyncha on plants near water.


-- -- ^, -, -.-- 777777">


Figs. 11 and 12. Platypolynema cautum Ogloblin, female (allotype). Fig. 11. Antenna. Fig. 12. Forewing. Scale
bars = 0.1 mm.


December 2002







Triapitsyn & Berezovskiy: Revision of Kalopolynema (Mymaridae)


PLATYPOLYNEMA CAUTUM OGLOBLIN
(Figs. 11, 12)

Platypolynema cautum Ogloblin, 1960: 8-9, figs. 12-16;
Ogloblin, 1967: 192-194.
Type locality: Chacra "Yabebiri", San Ignacio, Misiones,
Argentina.

Types. Holotype male (MLPA), examined; in
good condition, mounted dorso-ventrally, with part
of the left antenna missing. On slide, labeled: 1.
"Platypolynema cautum [dictynnum-crossed out,
this is a manuscript name] A. Ogl. S Chacra Yabe-
biri, S. Ignacio, Mis. 11.III.1951 A. O.". Allotype fe-
male (MLPA), examined; pedicel and flagellum of
the right antenna are missing. On slide, labeled: 1.
"Platypolynema cautum A. Ogloblin 9, Misiones, 2
de Mayo 20-XI-1964"; 2. (Mostly illegible, in pen-
cil) "Platypolynema 2 de Mayo A. O. 15.XII.1964".
We added the word "Allotype" to the first label in
order to clearly mark this specimen as such.
Other material examined. ARGENTINA, Mi-
siones: Dos de Mayo, 6-XII-1964, A. A. Ogloblin
(1 6, MLPA). Parque Nacional Iguazui, Cantera,


200 m, 8-XII-1990-6-I-1991, S. & J. Peck (1 6,
CNCI).

Distribution. Province of Misiones, Argentina.
Host. Unknown.

Comments. The holotype male is labeled
slightly differently on the slide from what was in-
dicated by Ogloblin (1960): the date of the collec-
tion is 11-III-1951 instead of 12-III-1953.
However, there is no doubt that this specimen is
indeed the holotype as it perfectly matches Oglob-
lin's illustrations. In fact, many type specimens
from Ogloblin's collection of Mymaridae are not
marked as such and often the label data on the
slides contradict the published information.
The original description of the holotype male of
this species and the follow-up description of the
allotype female are sufficient for its recognition.
Here we provide drawings of the antenna (note
that the clava is collapsed) (Fig. 11) as well as of
the forewing (Fig. 12), taken from the allotype fe-
male of P cautum.


KEY TO THE NEW WORLD GENERA RELATED TO KALOPOLYNEMA

1. Face without distinct subantennal grooves converging from toruli to margin of clypeus, at most with slight
depressions; petiole more or less cylindrical, usually much longer than wide. ...... Polynema Haliday s. 1.
Face with distinct subantennal grooves converging from toruli to margin of clypeus (Figs. 3, 7); petiole sub-
quadrate, subrectangular, or cross-shaped (Fig. 10), at most 2 x as long as wide ..................... 2
2. Head typical in size for the group, about as wide as mesosoma; forewing without narrowing of the blade be-
yond venation, marginal vein relatively short (Figs. 2, 6, 9) ................... Kalopolynema Ogloblin
Head very large and high, markedly wider than mesosoma; forewing with a narrowing of the blade beyond
venation, marginal vein relatively long (Fig. 12). ........................... Platypolynema Ogloblin


ACKNOWLEDGMENTS

We thank John T. Huber (CNCI) for the loan of spec-
imens, valuable advice, and review of the manuscript,
Russell F. Mizell, III (University of Florida, North Flor-
ida Research and Education Center, Quincy, Florida) for
maintaining a Malaise trap in Monticello, Florida, and
providing us with material, Patricio Fidalgo (CONICET,
San Miguel de Tucuman, Tucuman, Argentina) for mak-
ing specimens of the type species of Kalopolynema and
Platypolynema available for study, and Michael E.
Schauff (USNM) for access to the type series of
Polynema ema.


REFERENCES CITED

ANNECKE, D. P., AND R. L. DOUTT. 1961. The genera of
the Mymaridae Hymenoptera: Chalcidoidea. Ento-
mol. Mem., Rep. South Africa Dept. Agric. Tech.
Serv., 5: 1-71.
GIBSON, G. A. P. 1997. Chapter 2. Morphology and ter-
minology, pp. 16-44. In G. A. P. Gibson, J. T. Huber
and J. B. Woolley (eds.). Annotated keys to the gen-


era of Nearctic Chalcidoidea (Hymenoptera). NRC
Research Press, Ottawa, Ontario, Canada. 794 pp.
HUBER, J. T. 1997. Chapter 14. Mymaridae, pp. 499-530.
In G. A. P. Gibson, J. T. Huber and J. B. Woolley
(eds.). Annotated keys to the genera of Nearctic
Chalcidoidea (Hymenoptera). NRC Research Press,
Ottawa, Ontario, Canada. 794 pp.
OGLOBLIN, A. 1960. Los representantes nuevos de la
tribu Polynematini de la Repiblica Argentina (Hy-
menoptera, Mymaridae). Neotropica, 6 (19): 1-11.
OGLOBLIN, A. A. 1967. Mimaridos nuevos de Argentina
(Hymenopt. Mymaridae). Acta Zool. Lilloana, 22:
183-196.
SCHAUFF, M. E., AND E. GRISSELL. 1982. Nomenclatural
notes on Polynema (Hymenoptera: Mymaridae),
with description of a new species. Proc. Entomol.
Soc. Washington, 84 (3): 529-534.
YOSHIMOTO, K. 1990. A review of the genera of New
World Mymaridae (Hymenoptera: Chalcidoidea).
Flora & Fauna Handbook No. 7, Sandhill Crane
Press, Inc., Gainesville, Florida. 166 pp.
ZIMMERMAN, E. C. 1948. Insects of Hawaii. Volume 4.
Homoptera: Auchenorhyncha. University of Hawaii
Press, Honolulu. 268 pp.







Florida Entomologist 85(4)


December 2002


A NEW PARASITOID OF WHITEFLIES FROM MEXICO, WITH A KEY
TO NEW WORLD SPECIES OF THE GENUS ENCARSIELLA
(HYMENOPTERA: APHELINIDAE)


S. N. MYARTSEVA 12 AND J. M. CORONADO-BLANCO '
1UAM Agronomia y Ciencias, Universidad Aut6noma de Tamaulipas, 87149 Ciudad Victoria
Tamaulipas, M6xico

2National Institute of Deserts, Flora, and Fauna, Ministry of Nature Protection of Turkmenistan
744000 Ashgabat, Turkmenistan

ABSTRACT

A new species, Encarsiella tamaulipeca Myartseva and Coronado-Blanco sp. nov., from Mex-
ico is described and illustrated. A new combination is proposed, Encarsiella narroi (Gomez
& Garcia) from Encarsia. A key to the species of Encarsiella (females) of the New World is
given.

Key Words: Encarsiella sp. nov., distribution

RESUME

Se describe e ilustra una nueva especie: Encarsiella tamaulipeca Myartseva et Coronado-
Blanco sp. nov. de M6xico. Se propone una nueva combinaci6n de Encarsia narroi G6mez &
Garcia a Encarsiella narroi (G6mez & Garcia). Se incluye la clave de las species de Encar-
siella (hembras) del Nuevo Mundo.
Translation provided by author.


Among the parasitic Hymenoptera, species of
the family Aphelinidae (Chalcidoidea) are among
the most important biological control agents of in-
sect pests. Aphelinid species play a significant
role in ecosystems as natural enemies of many ho-
mopteran hosts and have been used successfully
as biological control agents in Mexico and in many
parts of the world (Clausen 1978).
The genus Encarsiella Hayat (1983) belongs to
the subfamily Coccophaginae sensu De Santis
(1948) and Hayat (1985), tribe Pteroptricini Ash-
mead, that also includes the genera Encarsia Foe-
rster, Dirphys Howard, Bardylis Howard,
Coccophagoides Girault and Pteroptrix Westwood


(Hayat 1998). Relationships within the family
Aphelinidae have been studied by many taxono-
mists, but the classification of the aphelinid gen-
era into subfamilies and tribes is still in formative
stages (Hayat 1994, Yasnosh 1976, Shafee & Rizvi
1991 and Hayat 1998).
Encarsiella is characterized by having an 8-
segmented antenna in both sexes, the third seg-
ment of the club oblique or transverse at apex,
linea calva absent, stigmal vein narrow, submar-
ginal vein with 2-4 long setae, mesoscutum with a
variable number of setae but always more than 6
and the axilla elongate and strongly projecting
forward.


ENCARSIELLA IS CLOSELY TO THE GENERA DIRPHYS AND ENCARSIA.
THE DIFFERENCES AMONG THESE GENERA ARE SHOWN IN THE FOLLOWING KEY:

1. Axillae small and separated medially by more than the maximal length of an axilla. Mid lobe of mesoscu-
tum with reduced number of setae arranged in bilateral symmetry. Scutellum distinctly wider than long
........................................... ................... Encarsia Foerster
Axillae large and separated medially by less than the maximal length of an axilla. Mid lobe of mesoscutum
with many scattered setae, not arranged in bilateral symmetry .................. ............... 2
2. Side lobes divided; sculpture of mesoscutum aciculate. Scutellar placoid sensilla closely placed, separated
by about diameter of a sensillum .............................................. Dirphys Howard
Side lobes not divided; sculpture of mesoscutum imbricate-reticulate. Scutellar placoid sensilla widely
placed, separated by distance distinctly longer than diameter of a sensillum ......... Encarsiella Hayat







Myartseva & Coronado-Blanco: A New Parasitoid of Whiteflies


Most Encarsiella species are solitary endopara-
sitoids of whiteflies belonging to the subfamily
Aleurodicinae (Homoptera, Aleyrodidae). However,
Encarsiella boswelli (Girault) is known to attack
eggs of Heteroptera (Polaszek & Hayat 1990), and
an undescribed species from India was reared from
nymphs of Psyllidae (Huang & Polaszek 1996).
Nine species of Encarsiella are known world-
wide; of these, four are recorded in the New World:
E. aleurodici (Girault), E. magniclava (Girault),
E. pithecura Polaszek, and E. noyesi Hayat
(Huang and Polaszek 1996; Martin and Polaszek
1999). The latter species is widely distributed in
Central America and has been used in biological
control programs of Aleurodicus cocois (Curtis)
(Cock 1985). Some undescribed species are known
from the New World. The correct identification of
the parasitoids reared from pests species is essen-
tial to the success of biocontrol programs.
G. Viggiani (1986) stated new combinations for
Encarsiella aleurodici from Encarsia and En-
carsiella magniclava from Coccophagus. We pro-
pose a new combination-Encarsiella narroi
(G6mez & Garcia), comb.n. from Encarsia. This
species was reared from Aleurodicus sp. collected
on Bauhinia variegata L. and Hibiscus sp. in Mex-
ico, Coahuila State (G6mez and Garcia 2000). The
description and illustrations of this species show
characteristics belonging to Encarsiella, espe-
cially the number of setae on the mesoscutum (42
pairs according to the authors) and the structure
of the antennal club. Thus, Encarsiella narroi
(G6mez & Garcia), comb.n. is the fifth species of
this genus known from the New World.
Encarsiella noyesi was described from Mexico,
reared from Aleurodicus dugesii Cockerell in the
State of Guanajuato, and from Aleurothrixus floc-
cosus (Maskell) on Citrus aurantifolia (Christm.)
Swingle, in the State of Yucatan (Polaszek and
Hayat 1992). We reared E. noyesi from Aleurodic-
inae whiteflies in the State of San Luis Potosi
(new record for this State), and from an aleyrodid
species in the State of Tamaulipas (new record for
this State). In addition, a new species of En-
carsiella was reared from an undetermined spe-
cies of Aleurodicinae on Psidium guajava L. in the
State of Tamaulipas.
The abbreviations R = radicle, S = scape, P =
pedicel and F = funicle segment are used in the fol-
lowing description of the new species and key to the
species of Encarsiella (females) of the New World.

ENCARSIELLA TAMAULIPECA
MYARTSEVA AND CORONADO-BLANCO SP. NOV.
(Figs. 1-3)

Description

FEMALE (Figs. 1-2). Length: 0.75-0.82 mm (N
= 8 specimens on points, 2 on slides); holotype -
0.75 mm.


Coloration

Head black, face ferrugineous from anterior oc-
ulus to interantennal prominence and whitish be-
low (except upper margin of mouth, hind part of
cheeks and antennal scrobes). Pedicel and anten-
nal club brown, scape (except distal half dorsally
brown) and F3 whitish, F1-F2 pale brown. Meso-
soma and metasoma black. Legs yellowish-white,
middle and hind coxae, hind femur black, middle
femur and hind tibia infuscate. Wings hyaline.
Sheaths of ovipositor whitish.

Head

Wider than high and as wide as mesosoma.
Frontovertex 2x as wide as long, about 0.5x head
width. Occipital margin slightly rounded and con-
cave. Ocelli in slightly obtuse triangle; lateral
ocelli close to occipital margin, at a distance of
less than diameter of an oculus, and about 2 diam-
eters of an oculus from eye margins. Eyes about
2x longer than cheeks. Malar sulcus present. An-
tenna (Fig. 1) inserted immediately under lower
margin of eyes, closer to mouth margin than to
eye margins. Antennal segments R-F3 and club
(3-jointed) with the following ratios, length/
width: R-15:9, S-60:15, P-22:13, F,-18:12, F,-
25:14, F,-20:15, club-67:20. Pedicel slightly
longer than F1; club slightly longer than funicle
and scape. F,, F, and club joints with two longitu-
dinal sensilla each, sensilla absent on F,. A very
thin anellus is also present.

Mesosoma

Sculpture of dorsum with more or less hexago-
nal cells, sides of mesoscutum and scutellum with
longitudinal cells. Mesoscutum slightly wider
than long, with many setae varying in number
from 54 to 64. Scutellum about 2x wider than
long, with 2 pairs of long setae. Axilla with one
seta, lateral lobes with three setae. Fore wing
more than 2x longer than wide, marginal fringe
about 0.14x wing width. Length of marginal vein
equal to submarginal vein, postmarginal vein ab-
sent, stigmal vein very short. Strong setae in two
rows on anterior margin form narrow bare band,
interrupted near vein by a few setae (Fig. 2). Base
of wing with 7-10 setae. Marginal vein with 10-13
setae, marginal fringe 2.5x maximum wing width,
discal setae uniformly distributed, hind wing
more than 4.5x as long as wide. Tibial spur of mid-
dle leg slightly shorter than basitarsus.
Metasoma: rounded at apex, about 0.67 times
length of mesosoma (in dry specimens). Oviposi-
tor exserted, its exserted part 0.5x length of
gaster (in dry specimens); ovipositor longer than
middle tibia (14:11), sheaths about 0.5x inner
plates.








Florida Entomologist 85(4)


December 2002


antenna, male (x 200).
Figs. 4-6. Encarsiella noyesi Hayat: 4- antenna, female (x 200), 5- marginal part of fore wing (x 280), 6- antenna,
male (x 200).


~

~ooy ,
L- Cc

,, --
4,







Myartseva & Coronado-Blanco: A New Parasitoid of Whiteflies


MALE (Fig. 3). Coloration Similar to female
in color, but face brown; legs black, except apices
of fore and middle femora, apices of fore and hind
tibiae, and the apical half of mid tibia and tarsi
which are whitish.
Antenna (Fig. 3) inserted at level of lower eye
margin, at equal distance from margins of eye and
mouth. Funicle 4-segmented, club 2-segmented.
Antennal segments with the following ratios
length/width: R-11:8, S-50:13, P-17:13, F1-
31:14, F,-36:15, F,-35:15, F,-34:16, F5-35:16, F6
34:13. Pedicel slightly less than 0.5x F,; club as
long as the two proceeding segments together. F,
- F6 with 3 longitudinal sensilla each. Forewing
with bare base. Mesoscutum wider than long;
scutellum about 1.5x wider than long.
Diagnosis. Using the key and the revision of
Encarsiella species of the world provided by Po-
laszek and Hayat (1992), E. tamaulipeca sp. nov.
is close to E. aleurodici in coloration of body and
antenna, and also the following morphological
features: long pedicel (P > F,) and club (>F,- F,),
absence of sensilla on F1, and setaceous wing base.
It differs from E. aleurodici in the following: fe-
male with antennal anellus and anterior margin
of fore wing with bare band; length of marginal
vein equal to submarginal vein, and marginal
fringe longer (0.14x maximum wing width) (in
aleurodici it is very short), ovipositor slightly
longer than mid tibia (14:11); male antennal club
equal to lengths of two proceeding segments.
Material Examined. Holotype, female: Mex-
ico, Tamaulipas, Ciudad Victoria, ex Aleyrodidae
on Psidium guajava, 7-8-XII-1995, E. Chouva-
khina; paratypes: same data as holotype, 6 fe-
males (all on points); 27-X-1999, S. Myartseva, 1
female, 1 male, on slides.
The holotype and one paratype are deposited
in the National Museum of Natural History,
Washington, D.C., USA; two paratypes in the De-
partment of Zoology, Institute of Biology, National
Autonomous University of Mexico, Mexico City,
D.F., Mexico; two paratypes in the Zoological In-


stitute, Russian Academy of Sciences, St. Peters-
burg, Russia; one paratypeon point and one
female and one male on slides in the Insect Mu-
seum, Universidad Aut6noma de Tamaulipas,
Ciudad Victoria, Tamaulipas, Mexico.
Etymology. Encarsiella tamaulipeca is named
after the State of Tamaulipas where it was discov-
ered.

ENCARSIELLA NOYESI HAYAT, 1983

Material Examined.

Mexico, San Luis Potosi, Xilitla, ex Aley-
rodidae, 10-XI-1999, S. Myartseva, 16 females, 17
males, MIFA (UAT); Tamaulipas, Jaumave, ex
Aleyrodidae, 36 females, 2 males, 30-IV-2000 (S.
Hernandez-Aguilar); USA, California, Riverside,
UCR Quarantine culture, A. Briones, emerged 3-
IV-2000 from Aleurodicus dugesii Cockerell, 7 fe-
males, 1 male, orig. from Mexico, Jalisco, Guad-
alajara, 5-V-1997 (D. Headrick).
Morphological differences were observed
among populations ofEncarsiella noyesi reared in
several regions. For example, Mexican specimens
are different from those described by Hayat, who
studied specimens reared from June to August
from Trinidad, St. Vincent and Tobago. The Mexi-
can specimens are smaller (female body length
0.52-0.67 mm, male 0.45-0.62 mm) and some fe-
males have only the F, pale yellow (also observed
in specimens from California). Living female
specimens have a violet-bluish face and pearlish-
bluish-white scutellum. Dry female specimens
are lighter yellow than the Californian specimens
and the basal third of the mesopleurum is black; P
equal to F1; and F, usually has longitudinal sen-
silla (Figs. 4-6) (also observed in specimens from
California); lateral lobes with three setae; mar-
ginal fringe of forewing longer (0.14x wing width);
and ovipositor exserted and slightly longer than
midtibia. Male specimens have F2-F4 more pallid
and pedicel length about 0.5x F,.


KEY TO FEMALES OF ENCARSIELLA SPECIES OF THE NEW WORLD

1. Scutellum entirely black ................ ................... ........................... 2
- Scutellum pallid...................................................................3
2. Discal setae on forewing uniformly distributed; marginal vein longer than submarginal vein. Ovipositor as
long as mid tibia ..................................................... E. aleurodici (Girault)
Distribution: Barbados, Ecuador, Trinidad (Polaszek and Hayat 1992).
Forewing with a long band bare of setae along anterior margin (Fig. 3); marginal vein equal to submarginal
vein in length. Ovipositor 1.3x longer than mid tibia ......................... E. tamaulipeca sp. nov.
Distribution: Mexico.
3. Mesoscutum entirely dark. F, less than 2x as long as wide. Club slightly less than 3x as long as wide. Forew-
ing with 2 large setae on submarginal vein ................ ................................ 4
Mesoscutum pale, excluding the anterior edge and notauli. F, 2.4x as long as wide. Club slightly less than
2x as long as wide. Forewing with 2 large setae and 2-4 smaller setae on submarginal vein
................................................................. E. magniclava (Girault)







Florida Entomologist 85(4)


December 2002


Distribution: Guyana, Panama (De Santis 1979).
4. Base of forewing with an infuscated area. Antennal scrobes and clypeus entirely pale; pedicel and scape en-
tirely pale ............................................................. E. pithecura Polaszek
Distribution: Belize (Martin and Polaszek 1999).
Base of fore wing hyaline. Antennal scrobes and clypeus completely dark; pedicel and scape partly or en-
tirely dark ................... ........................................................ 5
5. Forewing with a long band bare of setae along anterior margin and without asetose area below stigmal
vein. Fl without sensillum and F2 somewhat longer than Fl and F3 ................. E. noyesi Hayat
Distribution: Anguilla, Antigua, Barbados, Costa Rica, Grenada, Mexico, Peru, St. Vincent, Tobago (Polas-
zek and Hayat 1992).
Forewing without a long band bare of setae along anterior margin and with asetose area below stigmal
vein. Fl with one sensillum, F1-F3 of about equal length .......... E. narroi (G6mez & Garcia), comb.n.
Distribution: Mexico (G6mez and Garcia 2000).


ACKNOWLEDGMENTS

The authors thank VA. ATrjapitzin (Universidad Au-
tonoma de Tamaulipas, Ciudad Victoria, Mexico and
Zoological Institute, Russian Academy of Sciences, St.
Petersburg, Russia) for valuable consultations; M.
Hayat (Department of Zoology, Aligarh Muslim Univer-
sity, Aligarh, India) and G. A. Evans (Entomology and
Nematology Department, University of Florida, Gaines-
ville, Florida, USA) for sending important articles on
the Aphelinidae and also for reviewing the manuscript;
Mrs. E. Ya. Chouvakhina, who collected specimens of
this new species; S.V. Triapitsyn (University of Califor-
nia, Riverside) who supplied specimens of Encarsiella
noyesi and for critically reviewing an earlier version of
this manuscript. CONACyT, Mexico, through the "Cate-
dras Patrimoniales de Excelencia" Program, and Re-
search Project 31620-B, provided funding.


REFERENCES CITED

CLAUSEN, C. P. 1978. Introduced parasites and preda-
tors of arthropod pests and weeds: A world review.
Agriculture Handbook 480, U.S. Department of Agri-
culture. Washington, D.C. 545 pp.
COCK, M. J. W. (ed.). 1985. A review of biological con-
trol of pests in the Commonwealth Caribbean and
Bermuda up to 1982. Technical Communication,
Commonwealth Institute of Biological Control 9:
1-218.
DE SANTIS, L. 1948. Studio monografico de los afelini-
dos de la Republica Argentina (Hymenoptera, Chal-
cidoidea). Revista del Museo de la Plata (Nueva
Serie), 5 (Secci6n Zoologia): 23-280.
DE SANTIS, L. 1979. Catalogo de los Himen6pteros Cal-
cidoideos de Am6rica al Sur de los Estados Unidos.
Publicaci6n especial, Comisi6n de Investigaciones
Cientificas de la Provincia de Buenos Aires. La
Plata, Argentina. 488 pp.
GOMEZ, J., AND O.GARCIA. 2000.A new species ofEncar-
sia (Hymenoptera: Aphelinidae), a parasitoid of
whitefly Aleurodicus sp. (Homoptera: Aleyrodidae)
in Mexico. Pan-Pacific Entomologist 76: 49-51.


HAYAT, M. 1983. The genera of Aphelinidae (Hy-
menoptera) of the world. Systematic Entomology 8:
63-102.
HAYAT, M. 1985. Family Aphelinidae. pp. 226-232. In
Subba Rao, B. R. and M. Hayat (eds.). The Chalci-
doidea of India and the adjacent countries. Part 1.
Review of families and keys to families and genera.
Oriental Insects 19: 163-310.
HAYAT, M. 1994. Notes on some genera of the Aphelin-
idae (Hymenoptera: Chalcidoidea), with comments
on the classification of the family. Oriental Insects
28:81-96.
HAYAT, M. 1998. Aphelinidae of India (Hymenoptera:
Chalcidoidea): a taxonomic revision. Memoirs on En-
tomology, International, Publ. Assoc. Publishers,
Gainesville, Florida, 13. 416 pp.
HUANG, J., AND A. POLASZEK. 1996. The species of En-
carsiella Hayat (Hymenoptera: Aphelinidae) from
China. Journal of Natural History 30: 1649-1659.
MARTIN, J., AND A. POLASZEK. 1999. A new genus of
Neotropical whitefly, secreting blue-iridescent wax
(Sternorrhyncha, Aleyrodidae, Aleurodicinae), and
its parasitoids (Hymenoptera, Aphelinidae). Journal
of Natural History 33: 1545-1559.
POLASZEK, A., AND M. HAYAT. 1990. Dirphys boswelli
(Hymenoptera: Aphelinidae), an egg-parasitoid of
Plataspidae (Heteroptera). Journal of Natural His-
tory 24: 1-5.
POLASZEK, A., AND M. HAYAT. 1992. A revision of the
genera Dirphys Howard and Encarsiella Hayat (Hy-
menoptera: Aphelinidae). Systematic Entomology
17: 181-197.
SHAFEE, S. A., AND S. RIZVI. 1991 (1990). Classification
and phylogeny of the family Aphelinidae (Hy-
menoptera: Chalcidoidea). Indian Journal of Sys-
tematic Entomology 7: 103-115.
VIGGIANI, G. 1986. Notes on some species of Coccopha-
gus Westwood, Coccophagoides Girault, Encarsia
Foerster and Encarsiella Hayat (Hymenoptera: Aph-
elinidae), mainly from the Nearctic and Neotropical
regions. Bolletino del Laboratorio di Entomologia
Agraria "Filippo Silvestri" Portici 43: 59-78.
YASNOSH, V. A. 1976. Classification of the parasitic Hy-
menoptera of the family Aphelinidae (Chalcidoidea).
Entomological Review 55: 114-120.







Melika & Buss: Description of Two Cynipids


DESCRIPTION OF THE SEXUAL GENERATION OF CALLIRHYTIS
QUERCUSCORNIGERA AND A NEW INQUILINE
(HYMENOPTERA: CYNIPIDAE)

GEORGE MELIKA1 AND EILEEN A. BUSS2
'Systematic Parasitoid Laboratory, Kelcz-Adelffy Str. 6, Koszeg 9730, Hungary

2Entomology & Nematology Department, University of Florida, Bldg. 970 Natural Area Drive
Gainesville, Florida 32611-0620

ABSTRACT

The alternate, sexual generation of Callirhytis quercuscornigera (Osten Sacken), comb. rev.
was found by experimental rearing and field observations. Descriptions of the adult, gall, bi-
ology, and host plants of the sexual generation are given. A new species of cynipid inquiline,
Ceroptres cornigera Melika & Buss, reared from galls of the sexual and asexual generations
of Callirhytis quercuscornigera (Osten Sacken) of the eastern United States is also described
and illustrated. This is the first known species of Ceroptres to inhabit galls from alternating
generations of its host cynipid. Descriptive data, diagnostic characters, distribution, and bio-
logical information are given.

Key Words: Taxonomy, morphology, inquiline, Ceroptres cornigera, horned oak gall

RESUME

Por observaciones de campo y cria experimental, se descubri6 la generaci6n alterna sexual
de Callirhytis quercuscornigera (Osten Sacken), combinaci6n revisada. Se described el
adulto, la agalla, el desarrollo y las plants huesped de esta generaci6n sexual. Se describe
e ilustra tambi6n una especie nueva de cynipido inquilino, Ceroptres cornigera Melika y
Buss, criada de las agallas producidas por las generaciones sexuales y asexuales de Callir-
hytis quercuscornigera en el este de los Estados Unidos. Esta es la primera especie de Cerop-
tres descrita que habitat las agallas de ambas generaciones de su huesped. Se presentan
datos, caracteres descriptivos, distribuci6n e informaci6n biol6gica.


The greatest diversity of cynipid gall wasps
(Hymenoptera: Cynipidae, Cynipini) in the world
is found in the Nearctic region, especially in the
United States and Mexico, with more than 600 de-
scribed species. Burks (1979) listed 485 species of
Cynipini in the United States, but several addi-
tional species have since been described (Melika
& Abrahamson 1997a, b, 2000; Abrahamson et al.
1998a, b). However, the alternate generations are
known for only a few species (Doutt 1959, 1960;
Dailey & Sprenger 1973a, b; Dailey et al. 1974;
Evans 1967, 1972; Lyon 1959, 1963, 1964, 1969a,
b, 1970, and others).
Diagnosis. The galls of the asexual generation
of Callirhytis quercuscornigera (Osten Sacken)
are similar to those of C. pomiformis (Bassett), C.
punctata (Osten Sacken), C. quercusclavigera
(Ashmead), C. quercuspunctata (Bassett), C. quer-
cussuttonii (Bassett), and C. seminosa (Bassett)
(McCracken & Egbert 1922, Weld 1959, Lyon
1969b). However, the only other gall in which the
larval chambers protrude externally is induced by
C. pomiformis (McCracken & Egbert 1922).
Wasps from the tiny, blister-like leaf galls were
previously known as the alternate generation
(Felt 1940). These galls resemble the leaf galls in-


duced by C. pomiformis (McCracken & Egbert
1922), but leaf galls induced by C. quercuscornig-
era occur on the midveins, large lateral veins, and
infrequently on petioles and tiny lateral veins
(Eliason & Potter 2000).
Taxonomic comments. Osten Sacken (1862)
first described the gall-maker, based on stem gall
characteristics, as Cynips quercus cornigera. He
later reared two specimens, a female and possibly
a male, from the same gall and named the wasp
Cynips cornigera based on the female (Osten
Sacken 1865). Because species names based on
gall descriptions before 1930 are valid, the appro-
priate name for this species is Callirhytis quercus-
cornigera (Osten Sacken 1862), comb. rev.
Description. Sexual generation. Female.
Head, except mandible, scutum, and scutellum
black; mesopleuron, propodeum, metasoma dark
brown; antenna, mandible, and legs uniformly yel-
low brown. Head as broad as mesosoma, rounded,
as high or very slightly higher than broad in front
view; gena not broadened behind eye; malar space
without sulcus, 3.5-3.7 times as short as eye height.
Ocelli small, ocellar-ocular distance shorter than
post-ocellar distance; distance between antenna
sockets nearly equal to diameter, shorter than dis-










tance to inner margin of eye. Head finely coria-
ceous, except rugose lower face; clypeus separated
from face by deep depression. Antenna 14-seg-
mented (sometimes suture which indicated F12
indistinct); scape as long as pedicel, nearly twice
as long as broad; F1-F4 filiform, subsequent
flagellomeres broadened (Fig. 3). Mesosoma longer
than high in lateral view (Fig. 1), scutum slightly
broader than long in dorsal view, alutaceous,
shiny, without setae, with complete notauli, me-
dian dorsal line absent or present in a form of very
short triangular depression; anterior parallel and
parapsidal lines absent. Mesopleuron uniformly
finely coriaceous. Scutellum elongate, longer than
broad in dorsal view, finely coriaceous anteriorly,
with more dull sculpture posteriorly (Fig. 2);
scutellar foveae narrow with distinct carina sepa-
rating them (Fig. 2). Forewing 1.4 times as long as
body, pubescent, with cilia on margin, veins brown
and with areolet closed; radial cell narrow, elon-
gate, nearly 6.0 times as long as broad; cubitalis
(Rs+M) joint basalis in upper 13 (Fig. 4). Tarsal
claws without basal lobe, simple. Central portion
of propodeum smooth, shiny, limited by distinct
lateral longitudinal carinae, which only slightly
bend outward posteriorly; side of propodeum cori-
aceous. Metasoma smooth, shiny, slightly longer
than high in lateral view, ventral spine of hypopy-
gium short, with a few sparse white setae, its
prominent part only slightly longer than broad
(Fig. 1). Length 1.6-1.9 mm. Male differs from fe-
male in that head higher than broad in front view,
eye larger, antenna 15-segmented, filiform
throughout length; forewing darker with more
dense pubescence; metasoma shorter than head
and mesosoma together. Length 1.5-1.7 mm.
Material examined. The description of the
sexual female and male was made on the basis of
nine females and 11 males which were reared
from leaf galls and labeled as "USA, Fayette Co.,
Lexington, Kentucky, Q. palustris, 6-8.06.1999,
leg. E. A. Eliason". Voucher specimens were de-
posited in the insect collection of the Department
of Entomology, University of Kentucky and in the
cynipid collection of the Systematic Parasitoid
Laboratory, Koszeg, Hungary.
Distribution. Canada: Ontario; USA: New
York, Massachusetts, Connecticut, District of Co-
lumbia, Maryland, west to Illinois, Indiana, Mon-
tana, Iowa, Arkansas, Tennessee, Colorado,
Texas, south to Virginia, North Carolina (Croatan
National Forest, Carperet Co. and Camp Sea
Gull, near Arapahoe, Pamlico Co., on Q.
hemisphaerica Bartr. ex Willd. and Q. falcata
Michaux), Alabama, Georgia. New for Florida
(Archbold Biological Station, Highlands Co.; Lake
Manatee State Recreation Area, Manatee Co.;Eg-
lin Air Force Base, Okaloosa and Walton Cos.;
Winegarner's property in the vicinity of De Fu-
niak Springs, Walton Co.; Wakulla Spring State
Park, Wakulla Co.-all were on Q. hemisphaerica.


December 2002


Trees in May 1995 were heavily infested; entire
branches were killed by the galls and inducers).
The asexual generation develops on Q. incana
Bartr., Q. imbricaria Michaux, Q. ilicifolia Wan-
genh., Q. laevis Walt., Q. nigra L., Q. palustris
Muenchhausen, Q. rubra L., and Q. velutina
Lamarck (Weld 1959). This species was very com-
mon on Q. hemisphaerica, Q. laurifolia Michaux,
Q. phellos L., and Q. myrtifolia Willd. in Florida,
and on Q. falcata in North Carolina. Sexual leaf
galls were observed on Q. palustris and Q. phellos.
Biology. The biology of both generations of
C. quercuscornigera was recently described in
Lexington, Kentucky (Eliason & Potter 2000).
Sexual females lay eggs into young shoots, and
the subsequent stem galls, in which the asexual
generation develops, begin visibly growing the
following spring. Twelve-month-old galls appear
as slight swellings on small diameter branches.
Twenty-two month old stem galls are smooth in
texture, green-colored externally, and the inter-
nal gall tissue is pale yellow. The larval chambers,
or horns, begin to push through the thin gall epi-
dermis when galls are ~24 months old. Horns
project up to 6 mm from the rounded, succulent
stem galls, and harden after about a month. One
asexual C. quercuscornigera larva develops at the
base of the larval chamber. Horns break off sev-
eral months after adults chew a circular exit hole
and leave the galls at bud burst in the spring.
Asexual females exit ~33-month-old stem galls
and oviposit into swelling buds (Eliason & Potter
2000). Eggs are deposited next to the midvein or
large lateral veins, and are slightly embedded
into the leaf tissue on the abaxial leaf surface.
One larva lives in each leaf gall, but two or more
galls may develop next to each other. Maximum
leaf gall length (~2.0 mm) occurs by late May. Lar-
vae completely consume the moist nutritive tis-
sue, leaving only a thin gall layer around the final
instar. Pupation occurs in May and adults exit in
late May or early June.

CEROPTRES CORNIGERA MELIKA AND BUSS,
NEW SPECIES
(Figs. 5-11)

We describe a new species ofinquiline, Ceroptres
cornigera, reared from galls of the sexual and asex-
ual generations of a cynipid gall wasp, Callirhytis
quercuscornigera, on stem and leaf galls of pin oak,
Quercus palustris. We also include relevant biologi-
cal information potentially useful in distinguishing
this species from other Ceroptres species.
Diagnosis. Similar to Ceroptres quercusarbos
(Fitch, 1859) and C. quercustuber (Fitch, 1859),
both reared from stem-swelling like galls of Calli-
rhytis clavula (Osten Sacken). In Ceroptres corni-
gera submedian pits on the pronotum are distinct
and large, separated with a strong carina; the en-
tire body is black, antenna and legs are bright


Florida Entomologist 85(4)







Melika & Buss: Description of Two Cynipids


-,


Figs. 1-4. Callirhytis quercuscornigera, sexual female. 1, Body, lateral view. 2, Scutum and scutellum, dorsal
view. 3, Antenna. 4, Forewing (without indication of pubescence).


brown. In C. quercusarbos and C. quercustuber
submedian pits of the pronotum are indistinct
and separated by a weak, narrow carina; the me-


sosoma only is black, gaster brown, face and
mouthparts lighter than the rest of the head; an-
tennae and legs are yellow.







Florida Entomologist 85(4)


Description. Female. Head, mesosoma, and
gaster uniformly black; antenna and legs bright
brown. Head broader than thorax, broader than
high in front view, gena very slightly broadened
behind eye, indistinct striae converging toward
clypeus, malar space 0.37 times eye length (Fig.
5); entire head very finely punctate, with dense


short white setae on face. Antenna 12-segmented
(suture between F10 and F11 invisible), filiform;
Fl very slightly longer than F2, F3 equal F2, sub-
sequent flagellomeres progressively shorter, but
F10 longer than F8+F9 (Fig. 7). Mesosoma
shorter than gaster (Fig. 8); pronotum with two
elliptical submedian pits with smooth shiny bot-


Figs. 5-8. Ceroptres cornigera. 5, Head of female, front view. 6, Mesosoma of female, dorsal view. 7, Antenna of
female. 8, Side view of female, general.


December 2002







Melika & Buss: Description of Two Cynipids


toms, broadly separated medially. Scutum nearly
as long as broad in dorsal view, finely transversely
rugose, with notauli distinct in posterior 23; me-
dian line short, anterior parallel lines distinct in
anterior 13 (Fig. 6). Scutellum rounded, rugose,
with much rougher sculpture than scutum (Fig.
6). Mesopleuron smooth and shiny. Fore wing hy-
aline, pubescent, with cilia on margin; radial cell
closed, 2.62.7 times as long as broad; veins pale
yellow (Fig. 9). Tarsal claws with strong teeth.
Metasoma nearly as long as mesosoma and head
together, as high as long; terga 2 and 3 fused into
one large segment occupying nearly whole of
gaster, smooth, shiny, with white setae at the
base; indistinct fine suture present between terga
2 and 3. Subsequent terga are finely punctate
(Fig. 8). Ventral spine of hypopygium short, slen-
der. Length 1.62.1 mm.
Male. Differs from female in having 15-seg-
mented antenna, Fl slightly curved and extended
proximally (Fig. 10); petiole distinct, longer than
in female (Fig. 11); legs and antenna lighter.
Length 1.5-1.8 mm.
Types. Holotype female from Lexington, Fay-
ette Co., Kentucky, from asexual galls of Callirhy-
tis quercuscornigera, collected on Quercus
palustris (Muenchhausen), 21 April 1998,
emerged 18 May 1998. Also 17 female and 18 male
paratypes from the same locality, from sexual and
asexual galls of C. quercuscornigera on Q. palus-
tris. Holotype, 5 female and 5 male paratypes in
the National Museum of Natural History, Smith-
sonian Institution, Washington, D.C. (USNM); 5
female and 5 male paratypes in the American Mu-
seum of Natural History, New York City (AMNH);
2 female and 3 male paratypes in the Department
of Entomology, University of Kentucky, and 5 fe-
male and 5 male paratypes in the collection of Sys-
tematic Parasitoid Laboratory, Koszeg, Hungary.
Material examined. We examined two simi-
lar Ceroptres species from the eastern USA,
Ceroptres quercusarbos and C. quercustuber,
known as inquilines in stem swelling-like galls of
Callirhytis clavula. The type material for the first
species, deposited in the USNM, was destroyed
with only Fitch's original label remaining without
an insect on the pin. Barnes (1988) mentioned
only one type locality for this species: "Salem, NY,
28.iii.1857, irregular knobs from tips of twigs of
white oak in brother Harvey's field, south of Bat-
tle Hill, placed in vial, small Cynips found dead,
May 27th". Five females and three males of C.
quercusarbos from the USNM, labelled as "Ag.
Coll. Mich.", "Collection Ashmead" were exam-
ined. Syntypes of C. quercustuber, deposited in the
USNM, were examined. They were mounted on
one pin (1 female and 1 male, the third insect to-
tally destroyed) and labeled as "Fitch's Type",
Fitch's Coll.," red "Type No 1809 USNM," and a
handwritten label "Cynips Quercus tuber, Fitch."
Barnes (1988) gave the next type locality "Salem,


NY, 19.iv.1859, find several of these galls on Ti-
tus's Hill." Five females and one male from the
USNM were also examined. Two females are la-
beled as "Accession No 6032d AD Hopkins W. Va.,
"Ceroptres tuber Fitch;" 1 female as "N. Brunsw.
NJ", "Coll. Ashmead", "Ceroptres tuber Fitch;" 1
female "Portsmouth, 6.28.97;" 1 female "Riley
Coll., Marlat", "Ceroptres tuber Fitch," and 1 male
"Toronto, Ont.", "Ceroptres tuber Fitch".
Ceroptres quercuarbos was described by Fitch
(1859) on the basis of one male as "a small black
gall-fly having all its legs and antennae of a bright
pale yellow color." This is the only description of
the wasp given by the author. Such a coloration is
typical for nearly all Ceroptres species. Fitch
(1859) described Ceroptres quercustuber as "a
small black gall-fly with dull pale yellow anten-
nae, mouth and legs, its hind shanks and its an-
tennae towards their tips being dusky, its length
0.08 and to the tips of its wings 0.13". This species
was described on the basis of both the female and
male. Fitch (1859) thought that he had described
the gall-inducing insects, but they were inquilines.
Other Ceroptres species known from the east-
ern USA differ strongly from this new species not
only in the morphology of the adults, but also in
their distribution and trophic associations. Thus,
we did not compare the new species to them.
Distribution. Kentucky (Fayette Co., Lexing-
ton).
Etymology. Named from the host gall, Calli-
rhytis quercuscornigera, from which it was
reared.
Biology. Members of this species are in-
quilines in galls of the asexual and sexual gener-
ations of Callirhytis quercuscornigera, which
associates with Q. palustris and other red oaks
(Burks 1979).
Ceroptres cornigera is one of several inquilines
and opportunistic insects living within stem galls,
but it is the only inquiline of the leaf galls. It is the
first known species of Ceroptres to inhabit galls
from both alternating generations of its host cyn-
ipid. Ceroptres cornigera larvae develop in the
succulent tissue below a Callirhytis quercuscorni-
gera larval chamber in young stem galls. It is un-
certain whether or not this inquiline contributes
to gall-maker mortality. Twenty-four female and
17 male Ceroptres cornigera adults emerged from
stem galls collected in spring 1998. Most galls
from which Ceroptres adults emerged (219 adults
from 86 galls reared in 1999) lacked external
horns and were of small diameter (2.5 0.1 cm).
However, horns typically protrude when galls are
25 months old. Adults may live as many as 6 days
without a water or sugar source. Mean female
body length is 2.2 0.1 mm, and mean ovipositor
length, when dissected and uncoiled, is also 2.2 +
0.1 mm (Eliason & Potter 2000). When adults
emerge in May, female ovaries are not fully ma-
ture, and may contain >100 eggs per female.






Florida Entomologist 85(4)


December 2002


--b
-'3 ''- 'C





9


10


Figs. 9-11. Ceroptres cornigera. 9, Forewing of female. 10, Antenna of male. 11, Side view of male, general.


Individual Ceroptres cornigera develop in each
leaf gall, and kill the gall-maker larva while feed-
ing on the succulent gall tissue. In 1998, more
than 1000 leaf galls were placed in individual


transparent gelatin capsules in the laboratory. Of
the 713 galls from which wasps emerged, 37.3%
were Ceroptres cornigera (138 female, 128 male).
Adults may live as many as 4 days without a wa-







Melika & Buss: Description of Two Cynipids


ter or sugar source. When adults emerge in June
and July, female ovaries are not fully mature
(Eliason & Potter 2001).

ACKNOWLEDGMENTS

We are obliged to D. Smith and C. Anderson for send-
ing us the types and some Ceroptres specimens from the
National Museum of Natural History, Smithsonian In-
stitution, Washington, DC. We also thank E. Foki (Sys-
tematic Parasitoid Laboratory) for the illustrations, and
M. Bechtold (Systematic Parasitoid Laboratory) for
mounting and labeling the type material. J. H. Frank
kindly translated the abstract. This is Journal Article
No. R-09002 of the Florida Agricultural Experiment
Station.

REFERENCES CITED

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sights into plant systematic relationships. American
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ABRAHAMSON, W. G., G. MELIKA, R. SCRAFFORD, AND G.
CS6Ka. 1998b. Host plant associations and specific-
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ology of three Callirhytis gall wasps from Pacific slope
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DAILEY, D. C., AND C. M. SPRENGER 1973a. Synonymy
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dricus crenatus (Hymenoptera: Cynipidae). Pan-Pa-
cific Entomol. 49: 188-191.
DAILEY, D. C., AND C. M. SPRENGER 1973b. Unisexual
generation of Andricus atrimentus (Hymenoptera:
Cynipidae). Pan-Pacific Entomol. 49: 171-173.
DIXON, N. W. 1992. Spined or horned galls on oaks in
Florida induced by gall wasps, Callirhytis quercus-
claviger (Ashmead) and Callirhytis cornigera (Osten
Sacken) (Hymenoptera: Cynipidae). Florida Dept. of
Agric. and Consumer Services, Division of Plant In-
dustry, Entomology Circular No. 355, 2 pp.
DOUTT, R. L. 1959. Heterogony in Dryocosmus (Hy-
menoptera: Cynipidae). Ann. Entomol. Soc. America
52: 69-74.
DOUTT, R. L. 1960. Heterogony in Andricus crystallinus
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mol. 36: 167-170.
ELIASON, E. A., AND D. A. POTTER 2000. Biology of Cal-
lirhytis cornigera (Hymenoptera: Cynipidae) and its


associated gall community in Kentucky. Environ.
Entomol. 29: 551-559.
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bution and parasitism of leaf galls induced by Calli-
rhytis cornigera (Hymenoptera: Cynipidae) on pin
oak in Kentucky. Environ. Entomol. 30: 280-287.
EVANS, D. 1967. The bisexual and agamic generations of
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EVANS, D. 1972. Alternate generations of gall cynipids
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Florida Entomologist 85(4)


December 2002


OLD TRAPS FOR NEW WEEVILS: NEW RECORDS FOR
CURCULIONIDS (COLEOPTERA: CURCULIONIDAE), BRENTIDS
(COLEOPTERA: BRENTIDAE) AND ANTHRIBIDS (COLEOPTERA:
ANTHRIBIDAE) FROM JEFFERSON CO., FLORIDA


STEPHANIE BLOEM'3, RUSSELL F. MIZELL, II2 AND CHARLES W. O'BRIEN3
1USDA-APHIS-PPQ-CPHST-NBCI at University of Florida NFREC, Quincy, FL 32351

2University of Florida NFREC, Quincy, FL 32351

3Florida A&M University, CESTA, Center for Biological Control, Tallahassee, FL 32307-4400

ABSTRACT

Trapping studies using three different types of traps placed in several wild and cultivated
habitats in North Florida (Jefferson Co.) produced 97 different species of adult weevils in the
family Curculionidae and the closely related primitive families Anthribidae, Brentidae and
Ithyceridae. Sixty-one of 97 species trapped have not been reported previously from Jeffer-
son Co. Furthermore, seven species of Curculionidae are first records for the state, with four
of these being important agricultural pests (Hypera meles, H. nigrirostris, H. punctata and
Sitona lineatus). In addition, some weevils taken in our traps represent undescribed species
ofApteromechus, Cercopeus and Conotrachelus. Herein we include: (1) an alphabetical list-
ing of species collected in these traps, (2) a description and illustration of the traps used, and
(3) months of the year and generalized habitats where traps were placed and specimens
were collected. Information on host associations is provided for many species.

Key Words: Tedders trap, Stinkbug trap, Circle trap, Florida, invasive weevils

RESUME

Studios de trampeo utilizando tres diferentes tipos de trampas colocadas en areas cultiva-
das y silvestres en el norte de Florida (Jefferson Co.) resultaron en la colecta de 97 diferentes
species de curculi6nidos pertenecientes a la familiar Curculionidae y a las families primiti-
vas de curculi6nidos Anthribidae, Brentidae e Ithyceridae. Sesenta y una de las 97 species
capturadas constituyen primeros reports para estas species en el condado de Jefferson.
Asimismo, siete de las species capturadas nunca habian sido reportadas en el estado de Flo-
rida y cuatro de ellas son plagas agriculturales de suma importancia (Hypera meles, H. ni-
grirostris, H. punctata y Sitona lineatus). Adicionalmente, algunas de las species
capturadas representan nuevas species pertenecientes a los g6nerosApteromechus, Cerco-
peus y Conotrachelus. En este articulo incluimos: (1) un listado alfab6tico de las species cap-
turadas en las trampas, (2) la descripci6n e ilustraci6n de las trampas utilizadas, y (3) los
meses del aio y descripci6n de las areas donde las trampas fueron colocadas y los especime-
nes colectados. Tambi6n hemos incluido informaci6n sobre las plants hospederas para mu-
chas de las species capturadas.
Translation provided by author.


Weevils (Coleoptera: Curculionidae) are ex-
tremely important insects as plant pests and as
beneficial biological control agents for noxious
weeds. More than 863 genera and 7,000 species
currently are recognized in North America
(O'Brien & Wibmer 1982). Native species such as
the plum curculio, Conotrachelus nenuphar
(Herbst), and the cosmopolitan maize weevil, Si-
tophilus zeamais (Motschulsky), feed on every-
thing from agronomic and fruit crops to stored
products. Thousands of foreign species, particu-
larly from the Caribbean and southeast Asia, are


potential invaders of the United States, following
the pattern of such destructive exotic pests as the
boll weevil, Anthonomus grandis grandis Bohe-
man, and the recently detected Myllocerus unda-
tus Marshall in Florida (C. W. O., unpublished
data).
The Florida beetle fauna is one of the most di-
verse in North America, given the many tropical
species entering south Florida from the West In-
dies (Peck & Thomas 1998). In the most recent
distributional checklist for the Coleoptera of Flor-
ida (Peck & Thomas 1998) the authors indicate







Bloem et al.: New Weevil Records from Jefferson Co., FL


that close to 18% (or = 825 species) belong to the
superfamily Curculionoidea (as defined in Alonso-
Zarazaga & Lyal 1999). Some of the species can be
very abundant at particular sites and times of the
year and, as such, can be collected with relative
ease. An example of this is the Fuller rose beetle,
Naupactus cervinus (Boheman) (R. F. M., unpub-
lished data). However, many Curculionoidea are
cryptic and nocturnally active, and collecting
them can prove difficult. As a result, exotic pest
weevils entering the state may remain undetec-
ted for many years until their population builds-
up to economically important levels.
We conducted a series of trapping studies over
a period of nine years (1993-2001) in several wild
and cultivated habitats in North Florida near the
town of Monticello. Three different types of traps
previously reported as effective for capturing a
variety of economically important weevils (Ted-
ders & Wood 1994, Mizell & Tedders 1999) were
tested for their ability to capture other species.
Ninety-seven different species of adult weevils,
Curculionidae, and the closely related primitive
families Anthribidae, Brentidae and Ithyceridae
(as treated in Alonso-Zarazaga & Lyal 1999) were
collected. In this paper we include: (1) a table
with information on all of the species collected, (2)
months of the year when specimens were col-
lected, (3) descriptions and photographs of the
traps used, and (4) information on host associa-
tions for most species. Our results are discussed
in the context of the importance of visual cues for
weevils and the use of traps in early detection of
pest weevil introductions.

MATERIALS AND METHODS

Sites

Traps were placed in various wild and culti-
vated habitats on the grounds of the University of
Florida, North Florida Research and Education
Center in Monticello, FL, which is located in Jef-
ferson County about 16 km S of the Florida-Geor-
gia state line. Different traps or trap
combinations were placed in pecan (PCN) (Carya
illinoiensis) and peach (PCH) (Prunus persica) or-
chards and in small plantings of Japanese per-
simmon (PSN) (Diospyros kaki) (Table 1). These
cultivated areas are interspersed with several
wet woodland sites (WD) containing tree species
that are typical for North Florida, including slash
pine (Pinus elliottii), loblolly pine (P taeda),
longleaf pine (P palustris), tupelo or black gum
(Nyssa sylvatica), sweet gum (Liquidambar
styraciflua), water oak (Quercus nigra), red oak
(Quercus rubra) and shagbark hickory (Carya
ovata) (USDA 1989). In addition, other weedy, for-
age and ornamental plant species were present in
the surrounding habitats. Among these were
mixed bahia-grass (Paspalum notatum), vetch


(Vicia sativa), red clover (Trifolium pratense),
rabbit-eye blueberries (Vaccinium ashei), Lan-
tana camera, and Sesbania spp.
Traps. Three types of traps were tested for
their ability to capture weevils. We used two ac-
tive pyramidal-shaped traps (Figs. la and Ib)
similar (equal dimensions) to those described in
Tedders and Wood (1994) and Mizell and Tedders
(1999). Trap bodies were manufactured from 1.3
cm thick masonite and painted black (Ace
acrylic flat latex house paint #103A105) or safety
yellow (Glidden alkyd industrial formula
#4540). The screen cone and collecting cylinder
components of the boll weevil trap (Anonymous
1990) were used to capture the adult weevils in
the black traps, while the window screen top col-
lection device described by Mizell and Tedders
(1995) was used to capture the weevils in the yel-
low pyramidal trap. In this manuscript we refer
to the black pyramidal trap as a Tedders trap (T
in Table 1) and the yellow pyramidal trap as a
Stinkbug trap (SB) (after Mizell & Tedders 1995).
The third trap used was a passive Circle trap (C)
(Fig. Ic), made of aluminum insect screening,
similar to the one described by Mulder et al.
(2000) to capture the pecan weevil and the plum
curculio in Oklahoma. As above for the Tedders
trap, a boll weevil trap top was modified to fit the
top of the circle trap to capture adult weevils.
The total number of traps of each type varied
from year to year, but at no time were fewer than
20 traps of each type present in the field. Tedders,
Stinkbug and Circle traps were placed in all hab-
itats except for peach orchards, which did not re-
ceive Circle traps. All traps were unbaited and
were serviced 1-3 times per week throughout the
course of the study. Trap tops were emptied,
cleaned of debris and replaced. Captured insects
were preserved in 95% ethyl alcohol and brought
back to the laboratory, where a representative se-
ries of all weevils from each trap were mounted
and labeled. Taxonomic determinations to species
were made by C.W.O. Trap(s), habitat(s) and
months) of the year when each species of weevil
was collected were summarized, and the informa-
tion is presented in Table 1. Species for which
host associations are provided in the text are in-
dicated in Table 1, as are the species that have
been introduced into North America.

RESULTS AND DISCUSSION

Ninety-seven species of adult Curculionidae
and the closely related primitive families Anthri-
bidae, Brentidae and Ithyceridae were collected in
our traps from 1993-2001 (Table 1). The prepon-
derance of species collected was advanced weevils
(89 species), with only six species of Anthibidae,
one species of Brentidae and the one species of
Ithyceridae being captured. Table 1 indicates the
species for which host associations are provided in








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