Inouye & Johnson: Aggregation Affects Feeding Rate
LARVAL AGGREGATION AFFECTS
FEEDING RATE IN CHLOSYNE POECILE (LEPIDOPTERA: NYMPHALIDAE)
BRIAN D. INOUYE1 AND DEREK M. JOHNSON2
'Department of Biological Science, Florida State University, Tallahassee, FL 32306-1100, USA
2Department of Entomology, Pennsylvania State University, 501 ASI Building, University Park, PA 18602, USA
Larvae of Chlosyne poecile (Felder) (Lepidoptera: Nymphalidae: Melitaeini) on Razisea sp.
(Acanthaceae) feed in large aggregations as early instars but disperse and feed in small
groups or as solitary caterpillars in later instars. The effect of group size on per capital feed-
ing rate was tested by manipulating the number of larvae on a leaf and measuring the leaf
area eaten in short-term feeding trials. Feeding rate increased significantly with group size
for first instars but decreased with group size for all larger instars. Although feeding rate de-
creased significantly with group size for second instars, second instars in the field were usu-
ally found in large groups and did not begin to disperse until the third instar. Variance-to-
mean ratios indicate that larval aggregation was lower in later instars, slowly approaching
a random distribution. The distributions of larger instars may therefore be the result of ran-
dom dispersal to food and not the active avoidance of other larvae. If the observed lag be-
tween the predicted optimal time to disperse and the observed pattern is adaptive, then it
may be due to unmeasured benefits of aggregation, such as lower predation rates and un-
measured costs of dispersal. Egg clustering and aggregation of larvae may be more common
for butterflies in the Neotropics than in other areas.
Key Words: caterpillars, feeding facilitation, group defense, Costa Rica, Neotropical Lepi-
Las larvas de Chlosyne poecile (Lepidoptera: Nymphalidae) comen hojas se Razisea sp.
(Acanthaceae). Durante sus primeros estadios se formen agregados grandes, pero se disper-
san y alimentan en grupos pequenos o solitariamente en los ultimos estadios. El efecto del
tamano de las agregaciones en las tasas de alimentaci6n per capital fue puesto a prueba ma-
nipulando el numero de larvas por hoja y metiendo el area de la hoja que a sido comida. La
tasa de alimentaci6n aument6 significativamente junto con el numero de individuos presen-
tes en la hoja para el primer estadio, pero disminuy6 en relaci6n al tamano del grupo para
todo los demas estadios. Aunque la tasa de alimentaci6n disminuy6 significativamente con
tamano del grupo para las larvas del segundo estadio, estas larvas usualmente se encuenta-
ron en grandes grupos en el campo, y su dispersion ocurri6 solo en el tercer estadio. Las tazas
promedio de varaza indicate que la agregaci6n larval fue menor en los ultimos estadios,
aproximandose lentamente a una distribuci6n tipo Poisson. Esto sugiere que las distribucio-
nes de los estadios mas grandes son el resultado de la dispersion al azar hacia el alimento y
no un meanismo active para evitar a otras larvas. Se propone que el retraso en la predicci6n
del tiempo 6ptimo para la dispersion y el patron observado es debido de beneficio obtenidos
al agregarse que no he sido cuantificado, como una reducci6n en las tasas de depredaci6n y
costs de dispersion. Las agregaciones de larvas y huevos de mariposas pueden ser mas fre-
cuentes en el Neotropico que en otras areas.
Translation provided by Authors.
Although most Lepidoptera lay single eggs and
develop as solitary larvae, 5% to 15% of butterfly
species are reported to lay eggs in large clusters
and have larvae that feed gregariously during
early instars (Stamp 1980). This life history has
arisen independently in many different lineages
but, it is especially common among the Nympha-
lidae and is common for species in the genus Chlo-
syne (e.g., Scott 1986; DeVries 1987; Clark & Faeth
1997; Denno & Benrey 1997). Proposed selective
advantages of larval aggregation include in-
creased feeding efficiency (Clark & Faeth 1997;
Denno & Benrey 1997), enhanced group defense
against predators (Stamp 1980; Chew & Robbins
1984; Vulinec 1990), and improved thermoregula-
tion (Tsubaki 1981; Stamp & Bowers 1990; Casey
1993; Bryant et al. 2000). Selective pressures may
lead females to oviposit in large clutches (Stamp
1980; Courtney 1984; Heard & Remer 1997),
causing at least the first instars to be aggregated
by default until they disperse to feed individually.
Aggregation may increase feeding efficiency by
allowing early instars to overcome leaf toughness
by a "group attack" in one spot (Ghent 1960), by
Florida Entomologist 88(3)
overwhelming plant defenses (Storer et al. 1997),
or by laying down an architecture of silk that aids
the larvae in feeding (Rathcke & Poole 1975;
Fitzgerald 1995). Higher larval feeding rate can
reduce development time, potentially both raising
intrinsic rate of increase and decreasing the dura-
tion of larval exposure to parasitoids and abiotic
mortality sources (Clancy & Price 1987; Benrey &
Denno 1997). Higher larval feeding rates also can
result in larger adults, a characteristic that is
positively correlated with fecundity in many in-
sects (e.g., Juliano 1998).
Aggregating caterpillars often exhibit apose-
matic coloration and distastefulness (Sillen-Tull-
berg & Leimar 1988; Vulinec, 1990). Aggregation
may therefore enhance the effects of caterpillar
defenses because predators learn to avoid larvae
after eating a few distasteful individuals (Sillen-
Tullberg & Leimar 1988). Alternatively, the ad-
vantage of aggregation may be not to the larvae
but to the ovipositing female or the eggs. Fresh fe-
males in the genus (C'., ....i are often so heavy
with eggs that they can barely fly (DeVries 1987),
so the female may need to unload the eggs quickly
because of energetic costs of carrying so much
weight in flight or higher predation risk due to re-
duced evasion ability. Moreover, "dumping" eggs
quickly may be advantageous to the female, espe-
cially when adult mortality risk is high, even if it
lowers the average fecundity of the offspring
(Courtney 1984). Egg clusters may also suffer a
lower intensity of parasitoid attack than solitary
eggs (Morrison & Strong 1981).
Many species with gregarious larvae, and most
if not all in the genus Chlosyne, only feed in large
groups as early instars, becoming increasingly
solitary through later instars (Clark & Faeth
1997; Benrey & Denno 1997). Several hypotheses,
which are not mutually exclusive, can explain this
across-instar decrease in aggregation. If larvae
are initially aggregated for increased feeding effi-
ciency, these benefits may disappear in later in-
stars as the larvae increase in size and are able to
penetrate the leaf cuticle and/or overwhelm plant
defenses without assistance. Moreover, effects of
competition for leaf area may be greater in larger
larvae and thus depress the feeding efficiency of
groups. Group advantages for defense against
predators may diminish in later instars if larger
larvae are exposed to fewer potential predators or
if later instars have more effective defense mech-
anisms (Stamp 1986). For example, later instars
may have sequestered defensive compounds that
were unavailable earlier, the aposematic colora-
tion of individuals may be more visible, or larger
larvae may deliver a more potent dosage of defen-
sive compounds. If larvae were initially aggre-
gated only because of advantages to the oviposit-
ing female or the eggs, then larvae could be less
aggregated as later instars merely because of ran-
dom dispersal patterns.
Our objective was to characterize the effect of
group size on the short-term larval feeding rate
for different instars of ('i.!. .... poecile (Felder).
This species is locally abundant in northwestern
Costa Rica, where it feeds on shrubs in the family
Acanthaceae in second-growth habitats and in
light gaps. We hypothesize that any benefit to
feeding in larger groups will be largest for the
first instar and decrease for later instars, perhaps
even becoming a cost of group feeding in the later
instars. Finally, a review of the natural history of
Costa Rican Lepidoptera suggests that aggrega-
tive behaviors (egg clustering and gregarious
feeding) are more common in the Neotropics than
in other regions of the world. Whether the differ-
ence is caused by differences in selective pres-
sures among the regions deserves further re-
C'i./.. I ... poecile is found from Costa Rica to
Venezuela (DeVries 1987). In Costa Rica it is lo-
cally abundant from sea level to 900 m on the Pa-
cific slope in dry forest and semideciduous forest.
Casual observations by the authors in multiple
years suggest that C. poecile is abundant during
the rainy season and rare or absent in the dry
season, a pattern typical for this genus in the
Neotropics (DeVries 1987). DeVries (1987) re-
ports the eggs, larval stages, and host plants for
C. poecile as unknown. We observed females ovi-
positing on, and larvae of all instars feeding on, a
woody shrub in the family Acanthaceae, which we
identified to the genus Razisea on the basis of
vegetative characters. All egg clusters we found
were on the undersides of leaves near the top of
the plant. Newly laid eggs were yellow and
turned tan and then brown shortly before hatch-
ing on their fifth day.
The study was conducted in the forest immedi-
ately adjacent to the Estaci6n Biol6gica San
Miguel (EBSM) within Cabo Blanco National
Park, Puntarenas Province, Costa Rica. The mar-
itime forest around EBSM is mostly 35-year-old
second growth (C. Castrillo, pers. comm.). Razisea
sp. grows commonly in the understory near
EBSM, especially along trails and at the edges of
gaps. Caterpillars of C. poecile were extremely
abundant on the Razisea sp. shrubs growing near
the station. Razisea sp. and C. poecile caterpillars
also were found less abundantly at the edges of
light gaps along streams near EBSM at 50-100 m
elevation. All of the plants and larvae in the cen-
suses and experiments were located along the
"beach trail" at the EBSM. The experiments de-
scribed below were initiated during the Organiza-
tion for Tropical Studies course 2000-3 and were
conducted from 16 to 22 July 2000.
Inouye & Johnson: Aggregation Affects Feeding Rate
Larval Group Size and Aggregation
We used two types of censuses to quantify lar-
val aggregation. In the first census we searched
for C. poecile larvae and recorded the instar and
number of larvae in a group. These data were
used to estimate the average group-size for each
instar and revealed the instar at which larvae
switched to feeding individually. In the second
census we searched every leaf of 14 Razisea sp.
plants and recorded the number of empty leaves
as well as the group size and instar for all larvae
we encountered. We used these data to estimate
the degree of aggregation of larvae for each instar
by calculating the variance-to-mean ratio of the
number of larvae per leaf, including unoccupied
leaves on the same plant. The degree of aggrega-
tion of the larvae was compared with the expected
variance-to-mean ratio (equal to one) for a ran-
dom (Poisson) distribution with a chi-square test
Effects of Group Size and Instar on Feeding Rate
We estimated larval feeding rates for different
instars by placing an individual or a group of sib-
ling larvae on the underside of a single, large, un-
damaged leaf and measuring the leaf area eaten
within a given amount of time. Leaves are lan-
ceolate and approximately 15-25 cm long and 7 -
10 cm wide, and larvae were corralled on a leaf by
a band of Tanglefoot Tangle-Trap@ smeared
around the petiole. No larvae were observed try-
ing to cross the band of Tanglefoot. Larvae were
left to feed for 4 to 25 h, and all replicates of the
same instar started and stopped at approximately
the same time. Smaller instars were left to feed
longer than larger instars. The leaf area eaten
was estimated with gridded transparencies. Re-
sults are expressed as the leaf area eaten (in
square millimeters) per hour per larva or per lar-
val volume, based on the average volume mea-
sured for 10 individuals of each instar. Average
volumes were estimated to be 0.72 mm3 for first,
4.56 mm3 for second, and 36.82 mm3 for third in-
stars and 149.92 mm3 for the fourth and fifth in-
stars, which differed more in head capsule size
and coloration than in estimated volume. The re-
sults were analyzed by single classification
ANOVA and regressions. By using as wide a
range of group sizes as possible, instead of repli-
cating only a few group sizes, we were better able
to characterize the effects of group size and look
for nonlinear effects of group size, including an in-
termediate optimum. All analyses were done with
S-Plus 6.1 (Insightful Corporation 2001).
The C. poecile larvae fed both day and night,
and on 12 leaves the groups of larvae ate too much
of the leaf to permit accurate estimation of the
leaf area removed. These leaves were excluded
from further analyses, leaving 19 groups of first
instars, 19 groups of second instars, 9 groups of
third instars, and 16 groups of fourth/fifth instars.
Group sizes in the final data ranged from 1 to 100
larvae, but instars differed in the maximum num-
ber of larvae in a group, because fewer late in-
stars would fit on a leaf. Fourth and fifth instars
were lumped together to increase the sample size
of these late instars. The largest group of fourth
and fifth instars that we used was 10 caterpillars,
which consumed most of a large leaf in a few
The mean group size for egg masses and each
instar counted in the first census are shown in
Fig. 1. The variance-to-mean ratio calculated
from the second census was high in the second in-
stars, intermediate for first and third instars, and
near one for fourth and fifth instars. First instars
were consistently highly aggregated in large
groups. The larvae began to disperse in the second
instar and continued in the third, so some of these
larvae were found in large groups whereas others
were found as solitary larvae, thus inflating the
variance-to-mean ratio. Fourth and fifth instars
were mostly found as solitary caterpillars and
data suggest their distributions were not signifi-
cantly different from a random distribution
among all possible leaves, although sample sizes
for these instars were too low to warrant a formal
_, ^1 II ^1P r4i *i
Eggs 1 2 3 4 5
Fig. 1. Group sizes observed for eggs and for instars
1-5. The box contains 50% of the data, and the median is
indicated by a line. The whiskers contain approximately
95% of the data, and outliers beyond the whiskers are
shown as points. Observed group size decreased with in-
creasing instar. The sample size for each instar is shown
beside its box.
Florida Entomologist 88(3)
Effects of Group Size and Instar on Feeding Rate
Instars differed tremendously in feeding rate.
Larger larvae ate more leaf area per hour than
small larvae (F1,, = 58.227, P < 0.0001), but when
feeding rate was expressed as leaf area consumed
per hour per unit larval volume, the effect of in-
star on feeding rate was no longer significant (F159
= 2.087, P = 0.154). Feeding rate increased signif-
icantly with group size for the first instars (Table
1, Fig. 2). Nine of the 12 groups of first instars
with fewer than 25 individuals never began feed-
ing, whereas all 7 larger groups of first instars
fed. Solitary first instars either never attempted
to feed or were unable to break the leaf cuticle.
The feeding rates decreased with group size for
the second, third, and fourth/fifth instars, but this
decrease was statistically significant only for the
second instars (Table 1, Fig. 2). The effect of group
size was therefore smaller in later instars. For all
instars the effects of group size were linear; no
higher-order terms were significant. Because the
much smaller range of group sizes tested for the
fourth and fifth instars would obscure interac-
tions involving group size, we used data only for
first, second, and third instars in a single-classifi-
cation ANOVA. The interaction between group
size and instar was significant (F143 = 4.526, P =
0.039); that is, these smaller instars differ signif-
icantly in the slopes of the relationships between
feeding rate and group size.
Our results clearly support the hypothesis that
larval group size positively affects C. poecile feed-
ing rate for first instars. First instars construct a
sparse network of silk that appears to help them
feed, and they appear less likely to deposit silk
and begin feeding successfully when in small
groups. Surprisingly, larval aggregation becomes
a disadvantage for the second instars ofC. poecile.
These results are contrary to those of Denno &
Benrey (1997), who found a positive effect of
group size on larval growth rate in second instars
of a congener, C. janais (Drury). The maximum
group size that these authors tested was less than
half of the mean group size in the field, however,
so they would not have detected effects that be-
came apparent only with larger feeding groups.
Clark & Faeth (1997) concluded that feeding facil-
itation at least partly explained shorter develop-
ment time from hatching to third instar in larger
groups ofC. lacinia larvae, but they did not sepa-
rate group-size effects among instars. Our study
supports their finding of feeding facilitation, but
also shows large across-instar differences in feed-
ing rate, suggesting different benefits and costs
for different instars.
Larval aggregation and average group size de-
creased with increasing instar number, approach-
ing a random distribution among available leaves
by fourth instars. This behavior is consistent with
the lack of significant effect of group size on feed-
ing rate in the third through fifth instars. If in-
traspecific competition reduced the feeding rate of
larger instars in groups, then we would expect the
larvae to become overdispersed by avoiding other
larvae. Instead, later instars seem to take random
walks to search for available leaves and thus only
slowly approach a random distribution among
available leaves. In contrast, when the first and
second instars disperse, they follow silk trails
from their old leaves to new leaves, as do those of
C. lacinia Geyer (Bush 1969), retaining most of
their original feeding groups. Dispersal from ag-
gregations in the congener C. janais occurs when
the caterpillars reach a certain body length, often
in the middle of an instar rather than at an instar
transition (Denno & Benrey 1997). A similar pat-
tern in C. poecile could explain the high level of
variation observed in second and third instar
Our results clearly indicate that on the basis of
feeding rate alone, larvae should disperse as soon
as they molt into the second instar. Why do many
groups remain aggregated through the second
and into the third instar when earlier group dis-
persal might increase feeding rate? Group ther-
moregulation is probably not an important factor
because larvae were observed feeding through the
night in this warm tropical climate. Unmeasured
predation, parasitism, or travel costs may drive
the delay in larval dispersal. Because early in-
stars move much more slowly than the later in-
stars (personal observation), the costs of travel
TABLE 1. SUMMARY OF LINEAR RELATIONSHIPS BETWEEN FEEDING RATE [LEAF AREA EATEN (MM2) PER LARVA PER DAY
PER AVERAGE LARVA VOLUME (MM3)] AND GROUP SIZE FOR DIFFERENT INSTARS. THE POSITIVE EFFECT OF
GROUP SIZE ON FEEDING RATE DECREASED WITH INCREASING INSTAR.
Instar Slope F,, 1) P-value n
First 2.5224 28.328 <0.0001 19
Second -1.9494 8.770 0.009 19
Third -2.6172 3.889 0.089 9
Fourth/fifth -0.0968 0.002 0.965 16
Inouye & Johnson: Aggregation Affects Feeding Rate
0 20 40 60 80 100
0 20 40 60 80 100
0 20 40 60 80 100 0
Fig. 2. The relationship between feeding rate [leaf area eaten (mm2) per larva per hour per average larva volume
(mm3)] and group size (number of individuals) for different instars. The lines are linear regressions. Note that the
range of group sizes for fourth/fifth instars is much smaller than that for the other instars. Feeding rate increased
significantly with group size for the first instars then decreased significantly with group size for the second instars,
and tended to decrease with group size with third and fourth/fifth instars.
time to a new leaf and risk of falling off the host
plant may be substantial for even second instars.
Moving to new feeding areas may also increase a
caterpillar's risk of predation (Bernays 1997).
The changes in appearance of larvae in succes-
sive instars suggest that larger caterpillars are
also better able to defend against natural ene-
mies. The early instars have few spines and are
tan to brown in color. Late instars have typical
aposematic coloration; they are black with red
heads and occasional orange markings on their
backs. The spines on late instars are larger, more
numerous, and mildly urticating, causing an itch-
ing rash on sensitive skin (personal observation).
Although we do not have data on the palatability
of the different instars, we noted that the most
aposematic larvae fed individually, whereas the
least aposematic larvae fed in large groups. In-
creased feeding efficiency could select for the ag-
gregation of first instars, regardless of their dis-
tastefulness, and might be a more important fac-
tor than predation for this species. Changes in the
risk of parasitism across instars also could con-
tribute to their distributions.
Despite the short duration of the experiments,
we were able to find significant effects of group
size on the larval feeding efficiency of C. poecile.
The large feeding groups of first instars and soli-
tary feeding of late instars appear to maximize
their feeding rates, but second instars present a
conundrum because they were commonly ob-
served in relatively large groups even though
their feeding rate declines with group size. We did
not examine the costs and benefits of group size
for predator defense, which may help to explain
the behavior of these caterpillars. We encourage
further, more detailed studies of the costs and
benefits of insect group-feeding behaviors for dif-
ferent instars, because these costs and benefits
are likely to change with instar.
Here we present the first report of gregarious
feeding in C. poecile larvae, although the behavior
was previously known in congeners (DeVries
1987; Clark & Faeth 1997; Denno & Benrey
1997). Egg-laying behavior and gregarious feed-
ing are nearly always coupled. For example,
nearly all lepidopterans, including Chlosyne, that
exhibit gregarious feeding also lay eggs in clus-
ters (and vice versa) in species for which complete
data are available-29 out of 30 in Costa Rica
(DeVries 1987, 1997; present study) and 22 out of
23 in North America (Stamp 1980). To determine
how common these aggregative behaviors are in
Neotropical Lepidoptera, we reviewed the natural
history of Costa Rican lepidopterans (DeVries
1987, 1997) and found that, of 234 species for
which information is available, 26% lay eggs in
clusters. This figure is much higher than esti-
mates for other regions of the world: 5% in North
American, 13% in Great Britain, 6% in Australia-
New Guinea, and 3% in India (see Stamp 1980 for
review). Moreover, although Stamp (1980) found
that egg clustering is generally predominant in
just one butterfly group per region, families in
Costa Rica show consistently high levels of egg
clustering: 26% in Papilionidae, 44% in Pieridae,
22% in Nymphalidae, and 32% in Riodinidae.
These results raise the question of whether selec-
tive pressures acting on Lepidoptera in the Neo-
tropics differ from those in other regions of the
world. We suggest further research that compares
top-down (predators and parasitoids) and bottom-
up (leaf toughness and feeding-induced resis-
tance) effects on gregarious feeding by larvae in
the Neotropics with those in other regions of the
We thank the excellent staff of P.N. Cabo
Blanco, especially C. Castrillo, and the Organiza-
tion for Tropical Studies for making this work
possible. E. Bruna, J. Fordyce, N. Underwood, and
anonymous reviewers provided helpful comments
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Florida Entomologist 88(3)
Ferkovich & Shapiro: Orius insidiosus and Nutritional Factor
ENHANCED OVIPOSITION IN THE INSIDIOUS FLOWER BUG, ORIUS
INSIDIOSUS (HEMIPTERA: ANTHOCORIDAE) WITH A PARTIALLY PURIFIED
NUTRITIONAL FACTOR FROM PREY EGGS
STEPHEN M. FERKOVICH AND JEFFREY P. SHAPIRO
Center for Medical, Agricultural, and Veterinary Entomology, USDA, ARS, 1700 SW 23rd Dr.,
P. O. Box 14565, Gainesville, FL 32604
The insidious flower bug, Orius insidiosus (Say), can be maintained on a minimal artificial
diet composed of brewers yeast, soy protein hydrolysate and chicken yolk. However, egg pro-
duction is poor even though the level of protein in the diet exceeds the amount consumed by
adults that are fed insect eggs and have higher levels of egg production. We therefore frac-
tionated eggs of the almond moth, Ephestia kuehniella Zeller by preparative isoelectric fo-
cusing and bioassayed the resultant fractions in test diets. Ovipositional rates were
evaluated using a short 1-week bioassay. Adult predators were placed on the diets the third
day after eclosion, allowed to feed for six days, and then provided with an oviposition sub-
strate for 24 h on day seven. Egg production significantly increased only in a fraction with
an isoelectric point of pH 5. SDS-PAGE revealed the presence of several Commassie blue-
stained bands; however, the nature of the factor is unknown. These results point to a fecun-
dity factor required by females of 0. insidiosus for egg laying that potentially may be used
to supplement artificial diets for Orius species by commercial producers of beneficial insects.
Key Words Orius insidiosus, Ephestia kuehniella, predator, artificial diet, oviposition, prey
El chinche insidiador de flores, Orius insidiosus (Say), puede ser mantenido sobre una dieta
artificial minima compuesta de levadura de cerveza, hidrolisado de protein de soya e yema
de huevo de gallina. Sin embargo, la producci6n de huevos es pobre a pesar de que el nivel
de protein en la dieta excede la cantidad consumida por los adults alimentados con huevos
de insects y con un nivel de producci6n de huevos mas alto. Por eso, nosotros fraccionamos
los huevos de la polilla de almendra, Ephestia kuehniella Zeller utilizando el enfoque prepa-
rativo isoelectrico y por el bioensayo de las fracciones resultantes de las dietas probadas. Las
tasas de oviposici6n fueron evaluadas utilizando un bioensayo corto de una semana. Los
adults depredadores fueron sujetos a las dietas el tercer dia despu6s de eclosionar, se los
permitio la alimentaci6n por seis dias y en el septimo dia fueron proveidos con un substrato
para la oviposici6n por 24 h. La producci6n de huevos aumento significativamente una frac-
ci6n solamente con un punto isoelectrio de pH 5. La PAGINA-SDS revel6 la presencia de va-
rias bandas de 'Commassie' de tinte azul; sin embargo, la naturaleza del factor es
desconocida. Estos resultados indican que hay un factor de fecundidad requerido por las
hembras de 0. insidiosus para la oviposici6n de los huevos que potencialmente puede ser
usados para suplementar las dietas artificiales para las species de Orius por los producto-
res comerciales de insects beneficos.
The insidious flower bug, Orius insidiosus
(Say) (Hemiptera: Anthocoridae) is a generalist
feeder of thrips, aphids, mites and whiteflies, and
eggs of other insects in the field (Barber 1936; Mc-
Caffrey & Horsburgh 1986; van der Veire & De-
gheele 1992; van Lenteren et al. 1997; Funder-
burk et al. 2000). This predator has been reared in
the laboratory on an artificial diet devoid of any
insect host components (Weiru & Ren 1989). How-
ever, predators reared on this artificial diet had
reduced fecundity (Ferkovich & Shapiro 2004a). A
general problem associated with a number of
other species of predators reared on artificial di-
ets has been a reduction in reproductive rate (De
Clercq & Degheele 1992, 1993a & b; De Clercq et
al. 1998; Wittmeyer & Coudron 2001). The reason
for the reduced fecundity observed in predators
fed artificial diets is not clear.
Since adult females of 0. insidiosus, as with
other heteropteran predators such as Podisus ma-
culiventris (Say) (Heteroptera: Pentatomidae)
(Shapiro et al. 2000), exhibited higher yolk con-
tent in developing oocytes as well as higher egg
production when fed prey versus artificial diet
(Shapiro & Ferkovich 2002), we surmised that
natural prey may contain a specific nutritional
factor needed by the predator for egg production.
When adult 0. insidiosus were fed an artificial
Florida Entomologist 88(3)
diet, the females exhibited poor egg production
even though the level of protein in the diet ex-
ceeded the amount consumed per day by adults
fed eggs of the Indian meal moth, Plodia inter-
punctella (Hiibner) (Lepidoptera: Pyralidae)
When a protein extract from Plodia eggs was
tested as a supplement to the Orius diet, it signif-
icantly increased egg production at concentra-
tions of protein that were 8.3-, 55.7-, and 83.7-
times lower than the concentrations needed for
beef liver, bovine serum albumin, and chicken egg
albumin, respectively (Ferkovich & Shapiro
2004a). Subsequently, Ferkovich & Shapiro
(2004b) found that the egg protein extract could
be replaced in the diet with cells from an embry-
onic cell line (PIE) derived from P interpunctella
eggs to enhance oviposition of 0. insidiosus.
In view of some of the positive effects on the
rate of oviposition of 0. insidiosus fed diet con-
taining prey egg-extracted protein (Ferkovich &
Shapiro 2004a), we fractionated the proteins in
prey eggs to determine if the increased rate of ovi-
position could be attributed to a specific fraction
MATERIALS AND METHODS
Preparation of Egg Protein Extract
Soluble egg proteins were isolated from 5 g of
Ephestia kuehniella (Zeller) (Lepidoptera: Pyral-
idae) eggs (1.25 x 106 eggs) as described by Ferk-
ovich & Shapiro (2004a). Briefly the eggs were ho-
mogenized in ammonium acetate buffer (pH 7.5)
and the soluble proteins were separated by cen-
trifugation. The soluble proteins were then run
through a desalting column, freeze-dried, and
stored at -80C. The freeze-dried desalted powder
(352 mg) was then added to 58 ml of distilled wa-
ter; and the soluble protein concentration of the
solution was determined to be 174 mg/total vol.
The Lowry procedure (Protein Assay Kit,
Sigma, St. Louis, MO) was used to assay the
quantity of soluble proteins in the egg protein so-
lution and in the fractions after isoelectric focus-
Preparative Isoelectric Focusing
Five ml of the soluble protein solution and 3 ml
of ampholyte solution (pH range 3-10, Bio-Rad,
Hercules, CA) were mixed in 42 ml of 1 M urea to
prevent loss of proteins due to excessive precipita-
tion. The protein solution was run in a Rotofor
Cell@ isoelectric focusing unit (Bio-Rad instruc-
tion manual) for 2.5 h at 12 W constant power and
4C. The initial conditions were 408 V and 38 mA
and 668 V and 23 mA at equilibrium. Twenty frac-
tions were collected and their volumes (approx.
2.0 ml each) and pH values measured. Fractions
12-17 were cloudy and contained precipitates.
Ampholytes were used to form the pH gradient in
which the egg proteins were separated. These am-
pholytes were then removed due to interference
with the subsequent protein assay and SDS-gel
electrophoresis. They were removed by adding
NaCl to each fraction to a final concentration of 1
M for 15 min and dialyzing against water. Ali-
quots of 10 or 20pl of each fraction were analyzed
for protein content. After the fractions were ana-
lyzed for protein, they were combined based on
the protein profile. Fractions with low protein lev-
els (1-8, 9-12, 18-20) were combined, and ones
with higher protein concentrations (13-17) were
kept as individual fractions. Each combined or in-
dividual fraction was then concentrated to 0.5 ml
in a Centriprep@ concentrator (10k molecular
weight (MW) cutoff; Millipore, Bedford, MA) and
10 or 20pl of each fraction were used to analyze
for soluble protein.
Assay of Isoelectric Focusing Fractions in Diet
The 0.5 ml-fractions obtained from the isoelec-
tric focusing of the Ephestia egg proteins were
each added to 0.5 ml of diet and encapsulated
(20pl vol.). Artificial diet was prepared under
aseptic conditions in a clean room and encapsu-
lated in stretched Parafilm@ with a diet encapsu-
lation apparatus (Analytical Research Systems,
Gainesville, FL) described earlier (Carpenter &
Greany 1998, Ferkovich et al. 1999). Artificial diet
was prepared as described for rearing 0. sauteri
(Weiru & Ren 1989), and consisted of 0.33 g brew-
ers yeast, 0.03 g sucrose, 0.18 g soy protein acid
hydrolysate, 3.8 mg of 99% palmitic acid (all from
Sigma, St. Louis, MO), 0.04 g chicken egg yolk,
and 0.08 g honey in 1.0 ml of distilled water con-
taining the concentrated fractions from isoelectric
focusing. Palmitic acid was mixed with the egg
yolk component before adding it to the diet.
Newly emerged adults of a Florida strain of O.
insidiosus (<24 h after eclosion) were obtained
from a commercial producer of beneficial insects
(Entomos, Gainesville, FL) and placed on the di-
ets on the third day after eclosion. At the end of
the sixth day, one 7-cm section of green bean pod,
as a substrate for oviposition, was placed in each
jar for 24 h. Eggs deposited in the green beans
were then counted under a microscope. The in-
sects were held in a growth chamber at 25.5
1C, with 75 5% RH and a photoperiod of 15:9
(L:D) h. The treatment diets were (1) Eggs (stan-
dard)-jars contained 150 Ephestia eggs (approx.
3 mg) each as a reference standard; (2) Diet (con-
trol)-jars contained artificial diet with no addi-
tional substances as control diet; and (3) Diet
(amended)-jars contained artificial diet supple-
mented with each of combined fractions 1-8, 9-12,
Ferkovich & Shapiro: Orius insidiosus and Nutritional Factor
18-20, and individual fractions 13 through 17 as
Fractions resulting from the separation of the
Ephestia egg proteins by isoelectric focusing were
analyzed by SDS polyacrylamide gel electro-
phoresis (SDS-PAGE). Gradient SDS-PAGE (4-
20%) was carried out in minivertical gels (Bio-
Rad) as described by Shapiro et al. (2000).
Each treatment was replicated four times
with six females and four males per replicate. The
egg counts were adjusted for female mortality
within each treatment. Data were analyzed by
ANOVA with StatMost software (Dataxiom Soft-
ware Inc.). Dunnett's test was used to determine
if the number of eggs oviposited per female on
each of the diet treatments supplemented with
the isoelectric focusing fractions was significantly
greater than the number of eggs oviposited per fe-
male on the control diet. Since insectaries gener-
ally produce 0. insidiosus on eggs of E. kueh-
niella, we used them as a reference standard but
did not include the treatment in the ANOVA.
Figure 1 shows the protein profile versus pH of
the Ephestia egg protein extract separated on in a
pH gradient of 3-10. Average rate of eggs ovipos-
ited per female was significantly increased rela-
tive to the control Diet in only one Diet (amended)
treatment, a fraction with an isoelectric point of
pH 5 fractions (Fig. 2). The active fraction con-
tained 6.8 mg or 16% of the total protein recov-
Eggs Dit 1-B 9-12 13 14 15 16 17 18-20
Fig. 2. Average number of eggs (SE) oviposited by
females of 0. insidiosus after being fed artificial diet
supplemented with protein in fractions from isoelectric
focusing separation of Ephestia egg protein shown in
Fig. 1. Eggs (standard)-whole eggs of E. kuehniella;
Diet (control)-diet with no additional substances; and
Diet (amended)-supplemented with each of combined
fractions 1-8, 9-12 and 18-20 and individual fractions 13
through 17 in separate diet treatments. Dunnet's test
was used to compare the amended treatment diets
against the Diet (control); asterisk indicates that the
treatment means were significantly different from Diet
(control) (P <0.05).
ered in all the fractions (43.3 mg). The active frac-
tion (#16) shown in the diet bioassay of the frac-
tions in Fig. 2 contained one major band at 47,000
MW that also appeared to be present as a lighter
band in fractions 17 and 18-20 (Fig. 3). Other
faint bands at 163k, 51k, 39k, 31k, 27k, and 23k
MW were present in fraction 16 (Fig. 3). Recovery
of the total protein applied to the gradient was
1 2 3 4 5 6 7 8 1011 1213141516 1718 19 20
Fig. 1. Protein profile ofEphestia kuehniella egg pro-
tein separated by isoelectric focusing on a pH gradient
of 3-10. Fractions that were combined for bioassay in ar-
tificial diet are shown by vertical dotted lines. Arrow in-
dicates the fraction that stimulated the rate of
Fig. 3. SDS-PAGE analysis of fractions separated as
shown in Fig. 1. Lane 1 (Std) MW standards; lane 2
(CP)-crude protein; lane 3-combined fractions 1-8;
lane 4-combined fractions 9-12; lanes 5-9-individual
fractions 13-17; and lane 10-combined fractions 18-20.
Florida Entomologist 88(3)
24.8% (174 mg applied; 43.2 mg recovered); heavy
precipitates in fractions 12-17 after isoelectric fo-
cusing resulted in a loss of protein in these frac-
tions when the samples were dialyzed and con-
The fecundity of females was increased with
the addition of a specific fraction of Ephestia egg
proteins that were separated by isoelectric focus-
ing. The effect was not dependent on the concen-
tration of egg proteins in the fraction as other
fractions that contained higher levels of protein
did not stimulate the ovipositional rate of Orius
females. SDS-PAGE analysis of the fractions re-
vealed major proteins with relative molecular
weights ranging between 100k and 150k and less
than 40k MW which were not present in the ac-
tive fraction. Although we did not identify the
yolk proteins in Ephestia kuehniella, Shirk (1984)
identified four major yolk proteins ranging in mo-
lecular weight from 33k to 150k MW in a closely
related pyralid species, the Indian meal moth, P.
interpunctella. Proteins extracted from eggs of P.
interpunctella by the same procedure described in
this study stimulated egg production in 0. insid-
iosus fed artificial diet; however, a chloro-
form:methanol extract of the egg lipids had no ef-
fect (Ferkovich & Shapiro 2004a). Furthermore,
in a separate study with 0. insidiosus, in which
total protein, RNA, and DNA were extracted from
Ephestia eggs and bioassayed in diet, only the egg
proteins stimulated the rate of oviposition when
the three egg-extracted components were bioas-
sayed in artificial diet (unpublished data).
Fecundity could be improved further with the
addition of a higher concentration of the fraction
that increases the rate of oviposition. Wheeler
(1996) indicated that oogenesis is typically a nu-
trient-limited process and is initiated only if suf-
ficient nourishment is taken for egg production.
Adequate nourishment for 0. insidiosus egg pro-
duction is apparently not acquired during the
nymphal stage because newly emerged adults
that fed on the control Diet oviposited fewer eggs
that those fed on whole Ephestia eggs. Only fe-
males that fed on Diet supplemented with frac-
tion #16 oviposited significantly more eggs than
the control Diet. This indicated that a specific nu-
trient or factor is required for egg production and
is found in the protein component of the prey egg.
The nature of factor, however, is unknown and
awaits further purification and characterization.
Protein extracts from whole eggs ofP. interpunc-
tella and a cell line derived from eggs of P inter-
punctella (Ferkovich & Shapiro 2004 a & b) stim-
ulated egg production of 0. insidiosus females,
but this is the first report of a specific protein frac-
tion from prey eggs having oviposition-stimulat-
In view of a specific fraction having a positive
effect on oviposition in 0. insidiosus, we suggest
that future research should focus on identifying
the oviposition-enhancing materials) from Ephe-
stia eggs so that it can be more easily tested at
various concentrations in the diet. Moreover, once
the identity of the material is known, it may be
possible to obtain it from a commercial source for
supplementing the diet of 0. insidiosus.
We appreciate the excellent technical assis-
tance in this study of Delaine Miller and Jan
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Florida Entomologist 88(3)
COURTSHIP OF THE TWO FEMALE
MORPHS OF MELITTOBIA DIGITATA (HYMENOPTERA: EULOPHIDAE)
JORGE M. GONZALEZ'AND ROBERT W. MATTHEWS
The University of Georgia, Department of Entomology, Athens, GA 30602 USA
1Current address: Department of Entomology, Texas A & M University, College Station, TX 77843-2475
Courtship of sib-mating Melittobia digitata Dahms, a parasitoid of solitary wasps and bees,
is reviewed, described, and quantified for 125 virgins of the non-dispersing brachypterous fe-
male (BF) morph paired with 24 experienced males, and for 158 virgins of the dispersing
macropterous female (MF) morph paired with 21 males. Males performed 1-5 courtship
bouts with both morphs; about half of all successful matings in both morphs occurred after
a single bout. Depending on number of bouts performed, mean courtship durations ranged
from 47-268 sec for MFs and 59-277 sec for BFs. Courtship success rates were greater for BF
couples (80%) than for MF couples (57%). Compared to BF couples, MF couples were more
apt to undergo multiple bouts. Results are interpreted in the context of the morphs' life his-
tory and the costs/benefits of alternatives. Failure to initiate any courtship during the 15-
min observation period (22% for MF pairs, 21% for BF pairs) appeared to be due to apparent
lack of interest or to occasional male violence toward females. Possible explanations for the
latter, including mistaken identity, odor contamination, and nutritional stress are discussed.
Key Words: polymorphism, sexual selection theory, alternative reproductive strategies, re-
productive isolation, aggression, sib mating
El cortejo de Melittobia digitata Dahms, parasitoide de avispas y abejas solitarias que se
aparea con sus hermanos, se revisa, se describe, y se cuantifica para 125 virgenes de la forma
hembra braquiptera que no dispersa (HB) apareadas con 24 machos experimentados, y para
158 virgenes de la forma hembra macr6ptera que dispersa (HM) apareadas con 21 machos.
Los machos ejecutaron 1-5 sesiones de cortejo con las dos formas femenias; aproximada-
mente la mitad de las unions exitosas en las dos formas ocurri6 despu6s de una sola sesi6n.
Dependiente del numero de sesiones implementadas, las duraciones promedias para el cor-
tejo duraron entire 47-268 segundos para HMs y 59-277 segundos para HBs. La tasa de cor-
tejo exitoso fue mas alta para parejas HB (80%) que para parejas HM (57%). Comparadas
con las parejas HB, las parejas HM solian ejecutar sesiones multiples. Los resultados se in-
terpretan en el context de la historic vital de los morfos y los costos/beneficios de las alter-
nativas. La falta de iniciar cortejo durante el period de observaci6n de 15 minutes (22%
para parejas HM, 21% para parejas HB) pareciera ser por falta de interns o por violencia oca-
sional de los machos hacia las hembras. Explicaciones posibles para 6ste, incluyendo identi-
dad err6nea, contaminaci6n de olor y estr6s nutricional se discuten.
Translation provided by the authors.
Melittobia are small (ca. 1 mm) eulophid wasps
that are ectoparasitic upon prepupae or pupae of
various larger insects, particularly solitary wasps
and bees. Upon discovering a potential host, a fe-
male stings it, then feeds on host hemolymph em-
anating from the woundss; this enables her to de-
velop and then lay dozens to hundreds of eggs on
that host (Dahms 1984b). Extreme inbreeding
characterizes this genus; sib mating is the rule,
and as a result of haplodiploid sex determination
(arrhenotoky), virgin females produce sons with
whom they can mate (Dahms 1984b).
Melittobia are unusual in having polymorphic
female forms (Fig. 1), as first described by
Schmieder (1933). Under certain conditions (ap-
parently nutritional- see C6nsoli & Vinson 2002,
2004), a small number of females (<30) develop
more quickly than the rest, and emerge as short-
winged, stout-bodied individuals. Each of these
brachypterouss" (BF) females (termed "second
form" by Schmieder, 1933) is born with a clutch of
about 30 mature eggs (C6nsoli & Vinson 2002)
that they immediately lay on their natal host soon
after mating with an early-emerging brother. All
later-developing females on the same host possess
functional wings. These "macropterous" (MF) fe-
males (termed "type form" by Schmieder, 1933)
have incompletely developed eggs that mature
only after they have fed on a new host after dis-
persing (C6nsoli & Vinson 2002).
Gonzalez and Matthews.: Melittobia digitata Courtship
Fig. 1. Female morphs of M. digitata. Above: Brac-
hypterous form [BF] (= second form of Schmieder 1933);
Below: Macropterous form [MF] (= type form of
Schmieder 1933). Body length of MF, 1.2mm.
Aspects of the courtship behavior of 10 of the
14 known Melittobia species have been reported
by Assem (1975, 1976); Assem et al. (1982); Assem
& Maeta (1978, 1980); Dahms (1973); Doroshina
(1989); Evans & Matthews (1976); Gonzalez
(1985, 1994); Gonzalez et al. (1996); Lapp (1994);
and Varanda et al. (1984). Guided largely by
chemical and tactile cues, the blind males per-
form an elaborate sequence of leg, wing, and an-
tennal movements that vary from one species to
another but are always surprisingly complex.
Males of several Melittobia species appear to re-
lease a pheromone to which virgin MFs are
strongly attracted (C6nsoli et al. 2002; Hermann
et al. 1974; Gonzalez et al. 1985; Gonzalez et al.
1996; Matthews et al. 1985), often to the extent of
forming a queue around a courting couple (Assem
1976). The male pheromone of M. digitata has
been identified as trans-bergamotene (C6nsoli et
Further knowledge of the nature of courtship
in Melittobia is desirable for a number of reasons.
With few exceptions (Gonzalez 1985, 1994;
Gonzalez et al. 1996; Lapp 1994), previous studies
have involved only MF. As a practical matter,
study of Melittobia mating rituals might identify
behavioral characters useful for understanding
species relationships where morphological traits
alone leave some uncertainty about species
boundaries (Assem et al. 1982; Gonzalez et al.
1996); in one case, such information already has
been pivotal (Evans & Matthews 1976). Because
Melittobia increasingly are being used as model
organisms to teach various concepts in the life sci-
ences curriculum (e.g., Guinan et al. 2000; Mat-
thews 1997, 1982; Matthews & Matthews 2003;
Matthews et al. 1996; Pyle et al. 1997), basic bio-
logical information such as that in this study also
will help teachers and students by supporting and
underpinning curriculum materials.
The objectives of this study were to quantify
and compare the courtship interactions for both
morphs (MF and BF) of M. digitata, and to relate
findings to other aspects of the life history.
MATERIALS AND METHODS
Laboratory cultures of Melittobia digitata
were started with individuals that were reared
from field-parasitized nests of mud dauber wasps
(Trypoxylon politum Say, Hymenoptera; Sphe-
cidae). The parasitoids were maintained in con-
tinuous culture at 25C and 75% RH on T politum
To obtain virgin females for the courtship tri-
als, groups of female pupae (distinguished from
males by the presence of eyes) were isolated. Be-
cause BFs develop somewhat faster than MFs
(C6nsoli & Vinson 2002), to obtain them we iso-
lated the first 20-30 pupae to develop on each par-
asitized mud dauber prepupa. All later-develop-
ing female pupae were of the macropterous
At 24-48 h after eclosion, each female was
placed with a randomly chosen male removed di-
rectly from laboratory stock cultures. Unlike fe-
males, males of M. digitata are not known to be
polymorphic. By continually monitoring the stock
cultures, we knew that the selected males were 1
to 3 days old; because males are well documented
to mate readily and repeatedly (e.g., Assem et al.
1982; Dahms 1984), we presumed them all to be
Each male-female pair was placed in a deep
well slide (8 mm diameter, 3 mm depth), capped
with a glass cover slip, and illuminated with a fi-
ber optic lamp. Interactions were observed at am-
bient temperatures (23C + 1) and were recorded
with a Sony digital video camera with an attached
Macro-Zoom lens (18-108 mm). Data on pairing
outcomes and durations of selected courtship
components were subsequently transcribed from
the video recordings. If no courtship activity oc-
curred within 15 min, a trial was terminated. Be-
tween trials, slides were washed with 95% etha-
nol; new cover slips were used for each trial.
We recorded 158 pairings with MF and 125
pairings with BF. Individual males were used for
1-5 successive pairings; because females mate but
Florida Entomologist 88(3)
once, every trial used a different female. Statisti-
cal comparisons of various parameters for the two
morphs used the student's t-test.
RESULTS AND DISCUSSION
Overall, 124/158 (78%) ofMF and 99/125 (79%)
of BF began courting within 15 min after being
placed with an experienced male. Among the 124
MF courting pairs, 71 females (57%) mated;
among the 99 BF courting pairs, 79 females (80%)
mated (Table 1). The difference in proportion of
overall mating success was significant (P < 0.001).
Bout-by-bout comparisons of mating success rates
showed similar significantly greater success rates
for BF pairs in bouts 1 (P = 0.011) and 2 (P =
0.034), but no differences between the proportions
of each morph succeeding in bouts 3, 4, and 5. Ev-
ery randomly assigned male mated at least once.
When a female responds to the male's odor by
touching the side of his abdomen with her anten-
nae, the male typically responds by turning toward
her body and touching her side with his antennae.
This is a female's first decision point. If she is not
receptive, she attempts to move away, with her an-
tennae held downward. If she is receptive, she be-
comes still. Approaching either posteriorly or later-
ally, the male then mounts her dorsum and usually
turns first toward her posterior, then reverses and
ultimately orients his body in a plane parallel to
hers. He then moves forward and makes initial
contact with his antennae. At this point, we de-
fined a courtship bout as having begun.
Single- and Multiple-Bout Courtship
Qualitatively, courtship was essentially the
same for both morphs and our description thus
applies to both. During a courtship bout, the male
holds the female by placing his front tarsi just be-
hind her head, his middle tarsi on the sides of her
metathorax aligned with her middle and hind
legs, and his hind tarsi on her dorsal anterior ab-
domen with wings and abdomen raised, he begins
rhythmically opening and closing his antennae in
a more or less lateral plane. As he does so, his an-
tennae contact the clubs of the female's antenna
flagellae. During each closing stroke, he maneu-
vers her clubs into the ventral grooves on his
scapes, where they are briefly embraced by the
modified pedicel and a finger-like scape projection
(the "digit" that inspired this wasp's specific epi-
thet). During each opening stroke, her antenna
clubs are released, thereby completing an anten-
nal stroking cycle.
Concurrently, the male begins a rhythmic kick-
ing-lifting-swinging motion with his hind legs. At
the end of each antennal cycle, the male's hind legs
kick rapidly outwards, lift upward and forward,
and slowly swing around return to their original
position on the female's abdomen. Concurrently,
the male also lowers his wings slightly and begins
to flutter them, and rests the tip of his arched ab-
domen on the dorsum of the female's abdomen.
Initial alternations of antennal stroking and
leg kicking appear leisurely, but the pace of the al-
ternation soon accelerates. Kicking becomes less
vigorous and more like continuous quivering, as
antennal movements tighten in scope and in-
crease in tempo. During each succeeding anten-
nal phase, the male antennae open less widely,
and ultimately do not appear to open at all. Con-
currently, wing fluttering becomes more intense.
In a final convulsive motion, the male
stretches his abdomen backward and straightens
his hind legs, effectively lengthening his body in a
plane parallel to the female's body axis, and
swings his middle legs forward to hit the back of
the female's head, concluding the bout.
First-bout conclusion represents a second dis-
tinct female decision point. The female's behavior
at this time directs the courtship's subsequent di-
rection. If she stretches lengthwise, flattens her
abdomen into a wedge-like profile, and exposes
her genital aperture, the male immediately un-
dertakes a distinctive "backing-up" behavior with
an easily measurable duration. He comes into po-
sition, bends his abdomen forward under hers, in-
serts his aedeagus, and copulation ensues.
TABLE 1. DURATION OF SUCCESSFUL COURTSHIPS BETWEEN MELITTOBIA DIGITATA MAKES AND FEMALES OF THE TOW
MORPHOLOGICAL FORMS IN RELATION TO NUMBER OF BOUTS PERFORMED. TOTAL COURTSHIP DURATION IS
SUM OF INDIVIDUAL BOUT LENGTHS PLUS BACKUP AND COPULATION TIME. VALUES ARE MEANS S.D.
Total Courtship Duration (sec)
Female All successful
Morphology 1 bout 2 bouts 3 bouts 4 bouts 5 bouts courtships
Macropterous 47.2 18.3 85.6 17.9 146.2 33.2 201.5 20.8 267.8 79.6 50.2
(long winged) (n = 36) (n = 24) (n = 7) (n = 3) (n = 1) (n = 71)
Brachypterous 59.1 24.2 106.8 27.6 133.0 24.1 169.2 31.2 276.5 84.0 43.6
(short winged) (n = 45) (n = 29) (n = 2) (n = 2) (n = 1) (n = 79)
Gonzalez and Matthews.: Melittobia digitata Courtship
If the female does not stretch and flatten (i.e.,
fails to signal receptivity), two possibilities arise.
In the commonest outcome, a 'persistent' male
does not back up nor dismount; instead, after a
brief pause (<5 sec) he begins a second courtship
bout (bout 2) with renewed slow and exaggerated
antennal stroking. The other outcome occurs with
a 'non-persistent' male. In this case, when the fe-
male fails to indicate receptivity, the male simply
dismounts rather than beginning anew. In three
cases ofBF pairs a dismounted male immediately
remounted the same female and began a new bout
1, but most often dismounted males moved away
from the female without further interaction (com-
pare flow charts at bout 1 in Figs. 2 and 3).
The fact that the females' behavior seems to
determine the outcome of courtship is not surpris-
ing. However, the fact that Melittobia females
wait until the conclusion of a complete bout se-
quence by the male (termed the "finale" by Assem
et al. 1982) prior to indicating whether they are
receptive or not is thought to be unique among the
chalcidoid wasps. In other chalcidoid species so
far studied, female receptivity may be indicated
at varying points during the male's display, obvi-
ating the need for males to complete a full stereo-
typed display (Assem et al. 1982).
About half of all courtships that ultimately re-
sulted in successful copulation occurred after only
a single courtship bout (MF, 36/71; BF, 45/79); the
other half required additional courtship, most
commonly 1 more bout, rarely as many as 4 more
(Figs. 2 and 3). With 2 bouts, the cumulative cop-
ulation success rate for both morphs increased
dramatically (84.5% for MF pairings, 93.7% for
Assem and colleagues (1982) are the only other
researchers to have described the courtship of M.
digitata (their M. species 4). They used only mac-
ropterous females and did not systematically
track pairing outcomes, nor did they record fre-
quencies or outcomes of unsuccessful courtship
interactions. For comparisons with unsuccessful
courtship, they paired males with previously
mated females, because Melittobia females will
usually allow only a single copulation. Their de-
scriptive data for 25 MF pairs differ slightly in
terminology, but agree with our observations in
all essential respects. Their average of 76.6 9.6
sec for courtship duration and average of 25 leg-
kick-lift (swing) cycles are both similar to our MF
findings. They do not present data for courtships
having more than one bout, and although we reg-
ularly observed multiple-bout courtships (Figs. 2
and 3), it is not clear whether they ever saw any.
Although essentially the same proportion of
both morphs began courting, virgins of the BF
morph that courted were more initially receptive
to mating than were courting MF virgins. As the
numbers above indicate, BF were more likely to
require only a single courtship bout, and had a
higher copulation success rate overall. Under-
standably, as the number of courtship bouts re-
quired for inducing receptivity in M. digitata goes
up, courtship duration does also (Table 1). De-
pending upon the number of courtship bouts that
preceded female receptivity signaling, a success-
ful courtship with MF required 47-268 sec. With
BF, again depending upon number of bouts, suc-
cessful courtship lengths ranged from 59-277 sec.
Comparison of duration of first bouts of suc-
cessful (female displays receptivity posture) ver-
sus unsuccessful (female fails to display receptiv-
ity) courting couples revealed that unsuccessful
courtship durations were significantly shorter in
BF couples (P = 0.035, t = -2.238, 14 df versus P =
0.657, t = 0.446, 64 df for MF couples). Thus for
BFs, decisions about whether to copulate may re-
late to male bout duration, but not for MFs. As-
sem et al. (1982) mention that there was no differ-
ence between successful and unsuccessful court-
ship durations in any species they studied except
for M. clavicornis, however, they did not compare
the two morphs. Interestingly, successful and un-
successful couples of both morphs did not differ in
leg-kick-lift-swings/min in their first courtship
bouts (P = 0.76, t = 0.313, 10 dffor BF couples; P
= 0.613, t = 0.658, 60 dffor MF couples). Thus dif-
ferences in first bout duration of successful and
unsuccessful couples of the two morphs was not
related to the rate of leg-kick-lift-swings.
Overall, successful courtship durations aver-
aged slightly longer for the BF pairings (Table 1),
but the difference was not statistically significant
(P = 0.26, t = -0.572, 140 df). However, bout-by-
bout comparisons showed that for BF pairs aver-
age successful bout durations were significantly
longer for the first two bouts (bout 1 P = 0.007;
bout 2 P = 0.001), but considerably shorter for the
third and forth bouts (Table 1).
The longer overall average courtship durations
for successful BF pairs may simply reflect the fact
that BFs are demonstrably thickset and slow-
moving in comparison to their slimmer, livelier
MF siblings (see Fig. 1). This difference in female
shape and agility, and male compensation for it,
may also account for the finding that average cop-
ulation time for all successful BF couples was
longer than for MF couples (6.6 2.5 sec versus
5.0 1.4 sec, P < 0.001, t = 4.668, 121 df) and all
back-up times were less for males with BFs (4.6
2.9 sec versus 6.6 + 1.6 sec, P < 0.001, t = -5.051,
124 df). The longer average duration of successful
BF courtships may also reflect a disparity in fe-
male receptivity thresholds. Males successfully
copulating with BFs used on average 3 more leg
kick-lift-antennal stroke cycles per bout than suc-
cessful males courting MFs (27.5 versus 24.5). In
addition, the relative pace of the leg-kick-lift-
Florida Entomologist 88(3)
swings/min was greater in successful MF couples
(BF mean =36.3 + 6.2, MF mean =39.5 + 7.5; P =
0.005, t = -2.889, 148 df). Thus, males courting
BFs used a greater number of leg-kick-lift cycles
and performed them at a somewhat slower pace
compared to males courting Mfs. This in combina-
tion with the longer average copulation time for
BF couples resulted in the longer average court-
ship durations for BF couples compared to MF
In the course of their shared courtship reaction
chain, both sexes have opportunities for choice.
Females can signal decisions about male accep-
tance both at the initial encounter and after the
male has mounted and completed a courtship dis-
play bout. Likewise, a male can decide whether to
respond to a female's initial touch, and whether to
persist or leave when a given bout does not result
in female receptivity.
As noted above, the two morphs began court-
ship at about the same rate, but overall, BF cou-
ples had significantly higher courtship success
rates over all bouts combined and for each of the
first two bouts analyzed separately. Among all ini-
tial courtship pairings that failed to result in cop-
ulation, MF pairs quit about twice as often as BF
pairs (43% versus 19%, Figs. 2 and 3). Of 124 MF
pairs that began a first bout, only 36 successfully
copulated at the conclusion of that bout (29%),
compared to 45 of the 99 BF pairs (46%).
These differences may relate to the differing
ecological roles of each female morph (Freeman &
Ittyeipe 1976, 1982; Gonzalez & Teran 2001). As
the dispersive portion of the population, MFs
emerge with undeveloped eggs. Their options in-
clude (1) to mate with their brothers (rarely with
unrelated males, see below) and then disperse; (2)
to disperse as a virgin to a new host and produce
sons with which they can and do mate (e.g., As-
sem 1976; Dahms 1984; Schmieder 1933); or (3) to
find both a new host and an unrelated male with
which to mate. Options 2 and 3 likely are very un-
common in nature, since parasitized hosts nor-
mally always yield progeny of both sexes, and Abe
et al. (2003a) has confirmed that all dispersing M.
australica females are fertilized. Moreover, even
if a virgin female somehow rejects one male's ini-
tial courtship attempt, she is likely to have other
mating opportunities with the same or other sib-
ling males in the same clutch.
In contrast, as the nondispersive portion of the
population, each BF lays her eggs upon the rem-
nants of the natal host, a limited resource that is
already declining in nutritional quality due to
feeding pressure (C6nsoli & Vinson 2002). De-
spite attempts to facilitate BF dispersal on foot to
neighbor hosts in the laboratory, we have never
observed a BF female to leave her natal host (un-
publ. observe ) and we doubt that it ever occurs in
nature. Thus BFs are in resource- and time-
driven competition with each other for the success
of their own offspring. Without the option of dis-
persing to new hosts, readily mating with clutch
mates and rapidly ovipositing on the natal host
would be strongly favored. Thus, it is perhaps not
surprising that BFs mate more readily than MFs.
The difference in sex ratios of the early and
later emerging M. digitata may also have rele-
vance for the higher receptivity of BF. The very
first progeny to emerge from a single female M.
digitata-parasitized mud dauber consist of an av-
erage of 26.7 BF and 12.1 males (R. W. Matthews,
unpubl.). Thus the initial sex ratio is much less fe-
male-biased than the final sex ratio will be after
all the MFs have emerged. From a lone male's
standpoint, additional mating opportunities with
virgin BFs are far fewer than for MFs and the
competition from brothers is relatively greater.
From a BF's perspective there are far more poten-
tial male mating partners than needed since she
will only mate one time, and has a host immedi-
ately at hand. Taken together, these life history
variables also may help to explain why males
paired with BFs performed both longer duration
bouts and displayed a higher level of persistence
into the second bout than males paired with MFs.
BFs under these circumstances may require more
"proof" of a male's genetic worth.
As noted above, courtship durations for the
two morphs were not statistically different. How-
ever, there was a trend for males to perform more
leg-kick-lift-swing cycles with BFs but BFs were
more likely to require only a single courtship
bout. Conversely, males performed fewer leg-kick-
lift-swing cycles but more bouts with MFs. A pos-
sible scenario to explain these differences as-
sumes that originally females appeared as only
the macropterous morph. (While no phylogenetic
analyses exist for Melittobia species, this assump-
tion seems reasonable since macroptery is the
most prevalent condition in the Chalcidoidea.)
Males attempting to mate with MFs were (and
are) under intense time pressure. Not only are
they in fierce competition with their brothers (see
Abe et al. 2005), but they are also racing the clock
because any unmated females will begin to dis-
perse as virgins after they are a few days old (un-
publ. observ.). Due to natural variation, some
MFs likely would be willing to copulate sooner
than the average; the problem for a male is that
he has no way to know in advance which females
these might be.
Male courtship behavior might also be ex-
pected to vary, with some males "cheating" by at-
tempting to reduce the number of leg-kick-lift-
swing cycles, or inserting a finale partway along
the series of leg-kick-lift-swing cycles. If the re-
duced effort was genetically based and proved ac-
ceptable to the female, such males would gain ad-
Gonzalez and Matthews.: Melittobia digitata Courtship
ditional time in which to court others; when it was
not acceptable, they would lose only a few sec-
onds, and could quickly resume the courtship.
Such variation could lead to fewer leg-kick-lift-
swing cycles per bout and an increase in the num-
ber of bouts required for success.
Compared to MF couples, time and perfor-
mance pressures are reversed for BF couples. As
noted above, for BFs time is of the essence. On the
other hand, pressures to hurry along or "cheat"
were/are much weaker for the males, due to a less
extreme sex ratio and the lack of dispersal by
BFs. Thus, in BF courtships, a gradual increase in
the number of leg-kick-lift-swing cycles (resulting
in an increased bout length) rather than fewer
leg-kick-lift-swing cycle and more bouts might be
Male Stability, Persistence, and Life Strategies
The world of the blind, flightless male of M.
digitata is closed, violent, and highly competitive
(see Abe et al. 2003a, 2003b, 2005; Hartley & Mat-
thews 2003; and references therein), and he faces
very real risks to life and limb from other males
throughout his brief life. As a greatly outnum-
bered (average sex ratio, 3 males: 97 females
[Gonzalez & Matthews 2002]) male hurries to
out-compete his brothers in the race to insemi-
nate 500+ potential mates, success might be en-
hanced in many ways, but by any measure a male
may gain an advantage through any reduction in
Assem et al. (1982) assert that successive
courtships by a single male have very stable aver-
age durations (though they provide no data on the
matter). Neither our study nor Assem's quantita-
tively compared virgin males with more experi-
enced ones, but examination of our data subset for
males with 2 successive single-bout courtships
shows that for MF pairings (n = 9), the average
duration of 42.7 sec with the first female was not
different from the 43.4 sec duration with the sec-
ond female (P = 0.835, t = -0.215, 8 df). Likewise,
for BF pairings (n = 13), the average duration of
55.7 sec with the first female did not differ from
the 68.1 sec duration with the second female (P =
0.133, t = -1.612, 12 df). These results suggest
that experience does not increase the males'
Persistence may play an important role in the
eventual success of a male. According to invest-
ment theory we can imagine that at the end of
each unsuccessful bout, a male must choose be-
tween two alternatives: an investment in persis-
tence in which he continues additional bouts with
a so-far unreceptive female, or an investment in
"playing the field" in which he always moves on
after one bout. As noted earlier, although about
half of all ultimately successful courtships with
both morphs were consummated after a single
bout, males paired with MFs succeeded in a single
bout at a considerably lower rate than males
paired with BFs. Males that continued through a
second bout with initially unreceptive females
dramatically increased their success rate, by 60%
for males paired with MFs and 41% for males
paired with BFs.
Pursuing the two alternatives, we assume a
situation where virgin females are already
queued up, so that searching time is minimized
and courtship with a new individual can begin al-
most immediately after leaving the previous one.
A successful single bout courtship with a MF
takes about 47 s (Table 1), but in an initial en-
counter, a "field-playing" male has only a 51%
chance of ultimately copulating. Since such a
male cannot know in advance which "first date"
will ultimately give rise to success, he would need
to court 2 females (requiring an average of 94 s) to
achieve an average of 1 copulation. On the other
hand, a persistent male who does not leave after a
single bout more than doubles his chances, and
successful 2-bout courtship only requires an aver-
age of 86 s, including copulation. Thus for a male
courting a MF, persistence is superior to playing
the field. In addition, if a male stays in a "commit-
ted relationship" with a MF no matter how long it
takes rather than leaving, his cumulative chance
of ultimate success rises steadily. In contrast, the
non-persistent field-player's chances of success
remain at the initial 51%. Moreover, by persisting
with a single female, a male's probability of en-
countering another aggressive male is nil, com-
pared to what could ensue if he dismounted to
play the field.
With BF pairs, a different picture emerges. A
single-bout courtship takes somewhat longer
than for a MF pair, about 59 s, but carries a higher
potential success rate, so that a field-playing male
on average will need to court fewer than two BF
per copulation, at most 108 s. A persistent BF-
courting male with success after 2 bouts requires
107 s, and increases his chances of success by less
than 50%. Thus, time investment for the two al-
ternative strategies is more similar in BF pair-
ings than in MF pairings, relaxing selection for
male persistence with the former. On the other
hand, because BFs do not queue around males
(unpubl. observe) a male may require extra time
and encounter increased risks from competing
males in a search for another receptive female,
factors that might favor persistence. It would be
interesting to manipulate such factors experi-
mentally to gain further insight into the evolution
of alternative strategies.
When Courtship Goes Awry
All previous work on Melittobia courtship (in-
cluding our own earlier work) ignores data for un-
successful courtship pairings. However, it may be
Florida Entomologist 88(3)
instructive to examine the cases in which court-
ship goes wrong. As is evident from our data and
the flow charts for both female morphs (Figs. 2
and 3), most instances of failure to court appeared
to be a matter of non-attraction, at least during
our relatively short observation period.
Among those 34 MF pairs that failed to court,
29 showed no sign of interest or receptivity on the
part of one or the other sex during the trial (Fig.
2). Similarly, of those 26 BF pairs that did not
court, 23 displayed no apparent interest (Fig. 3).
The proportion of pairings in which females ac-
tively rebuffed the male's attempt to mount and
appeared to refuse to cooperate was about the
same for each morph, 26/158 (17%) for MF and 19/
125 (15%) for BF.
Reasons for apparent lack of interest by the
virgin females are unknown. Although it is possi-
ble that a deficiency in male pheromone produc-
tion might have accounted for some of the female
disinterest, we feel this is unlikely. Our males
were 1-3 days old, and C6nsoli et al. (2002) found
that male pheromone production peaked at 2 days
post-emergence. Furthermore, each male used in
our pairings successfully attracted and copulated
with a virgin female on at least one occasion.
Alternatively, these cases might simply have
been an artifact of the experimental situation. In
our study, as in those before us, single individuals
of each sex were confined together within a com-
paratively large, lighted space, whereas in their
natural context, the sexes would emerge inside a
crowded and dark host cocoon and have essentially
unlimited time to get together. The relatively brief
time allotted in our trials, plus possible physiolog-
ical stress as a result of handling and manipula-
tion, may have been contributing factors.
The remaining failures to court in our experi-
ments involved cases of overt male aggression to-
ward females (5/34 for MF pairs and 3/26 for BF
pairs). In 2 of the trials with MFs, males killed
their partner. With BFs, males also killed twice.
Fig. 2. Flow chart of the outcomes of 158 pairings of MF of M. digitata.
Gonzalez and Matthews.: Melittobia digitata Courtship
Fig. 3. Flow chart of the outcomes of 125 pairings of BF of M. digitata.
These attacks typically occurred after the male
had mounted and appeared ready to begin a
courtship bout. However, instead of moving for-
ward to contact the female's antennae, the male
instead would begin to chew at her neck or ante-
rior thorax region. Our "killer males" mated nor-
mally on other occasions, but because each male
in our study was used for only a limited number of
trials, the question is left open as to whether
these aggressive incidents represent an isolated
(and perhaps environmentally influenced) fluke.
Longer-term studies involving larger numbers of
pairings with males that show such behavior
would be valuable.
As noted above, male-male aggression often re-
sulting in death is a characteristic of the genus
Melittobia. However, the occurrence of aggressive
actions toward conspecific virgin females is sel-
dom mentioned in the literature and appears to
vary between species. Balfour-Browne (1922) re-
ported that males of M. acasta and M. chalybii
(=M. australica) commonly killed females, but at-
tributed this behavior to experimental conditions.
Dahms (1984b) noted similar female killing be-
havior occasionally in his observations on M. aus-
tralica. Neither previous study quantified the in-
cidence of male violence to females.
The basis for such seemingly maladaptive be-
havior is unclear. Dahms (1984b) postulated that
remnants of male odor might remain in the court-
ship chamber, stimulating male aggression that
mistakenly became directed toward females.
However, such a "laboratory artifact" is in fact the
natural situation inside a host cocoon where, be-
cause males continually fight with one another,
male odor is likely to be a constant part of the ol-
Mistaken identity and/or inappropriate signal-
ing might also be a factor. On occasion we have ob-
served females behaving atypically. For example,
sometimes upon being antennated by a male, a fe-
male will retract her legs and assume an inert
pupa-like form (unpubl. observ.). Such visually
apparent weirdness would have little impact on
sightless males within a dark host cocoon, but it
might be accompanied by relevant (but as yet un-
known) chemical, auditory, or tactile cues.
Nutritional stress provides a third not mutu-
ally exclusive explanation. Hermann (1971) men-
tioned that males ofM. chalybii (=M. australica) 8
or more days old would "grasp and feed on" a re-
ceptive female. Matthews (1975) also noted males
of this species chewing on the female victim dur-
ing or after attack. Whether any nutritional ben-
efit accrues to the male in these cases awaits fur-
Why Such Complex Courtship?
The elaborate courtship rituals observed in
Melittobia parasitoids are reportedly some of the
most intricate known in this large group of insects
(Assem 1975). Since it is generally believed that
Melittobia males never leave their natal host, and
that all females are fertilized by their brothers
(Dahms 1984b), the existence and maintenance of
such complexity is somewhat perplexing.
Assem et al. (1982) hypothesized that the
courtship might serve to prevent sperm depletion
in males by spacing out copulations. However,
this would not seem particularly relevant for
males courting BFs, since there are rarely more
than 30 females in a clutch of BF offspring. Addi-
tionally, Assem et al. (1982) raised the possibility
that the leg-raising component may have arisen
as a result of male-male competition and the need
to fend off intruders, particularly other females
attracted to the queue surrounding courting cou-
ples. Their argument would not apply particu-
larly well to courtships with BFs, which behave
sluggishly and show little tendency to queue
around males. However, if, as suggested above,
ancestral females only existed as the macropter-
ous morph, the competitive nature of males, once
evolved, may have persisted even after the BF
The role of courtship behavior in the mainte-
nance of reproductive isolation may be of more
importance to Melittobia than researchers have
previously appreciated. Superparasitism in some
Melittobia has been reported. Molumby (1996)
discovered up to 5 M. femorata females (mean,
1.84) per host cell in a sample of 53 parasitized
cells from 28 Trypoxylon politum hosts nests in
Mississippi. Whether multiple species colonize a
single host in nature is not known. In part this
may reflect the fact that prior to 1984 it was be-
lieved that only one species (M. chalybii) existed
in North America and another in the Old World
(M. acasta). However, Schmeider (1933) men-
tioned possibly having more than one species in
his studies and Dahms (1984a) recognized 8 spe-
cies from North America. Indeed, on one occasion
3 different Melittobia species were found within a
single field-collected host cell of Trypoxylon poli-
tum in Georgia (J. M. Gonzalez, unpubl.). Fur-
thermore, Matthews et al. (1985) note that Melit-
tobia females are sometimes attracted to odors of
males of other species. Thus the potential exists
for multiple species to occur and interact in some
localities. Even if relatively rare, cases of inter-
and intraspecific host settling could provide a se-
lective context favoring development and mainte-
nance of elaborate courtship.
We thank Janice Matthews, Lu Ann Brown,
and Christian Torres for insightful observations
and discussions throughout the progress of this
project. This study was supported by a grant from
the National Science Foundation, R.W. Matthews,
ABE J., Y. KAMIMURA, N. KONDO, AND M. SHIMADA.
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ABE J., Y. KAMIMURA, H. ITO, H. MATSUDA, AND M. SHI-
MADA. 2003b. Local mate competition with lethal
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Florida Entomologist 88(3)
FEEDING AND SURVIVORSHIP OF BLUEBERRY MAGGOT FLIES (DIPTERA:
TEPHRITIDAE) ON PROTEIN BAITS INCORPORATED WITH INSECTICIDES
JAMES D. BARRY1 AND SRIDHAR POLAVARAPU2
Blueberry and Cranberry Research and Extension Center, Rutgers University
125A Lake Oswego Road, Chatsworth, NJ 08019
1Current Address: DuPont Crop Protection, Stine-Haskell Research Center, 1090 Elkton Road, Newark DE 19714;
2Deceased, 7 May 2004
Laboratory feeding trials evaluated fly survivorship on six insecticides (acetamiprid,
clothianidin, deltamethrin, fipronil, imidacloprid, and spinosad) incorporated at 4, 40, and
400 ppm in protein baits. Higher concentrations of insecticides resulted in increased fly mor-
tality. At all concentrations of insecticides in baits, except those on deltamethrin, there was
a significantly higher mortality 4 d after the initial feeding, compared with flies that fed on
a control bait. The presence of clothianidin or imidacloprid in baits led to significantly less
feeding compared with a control bait without insecticide. There were no feeding deterrent ef-
fects of bait containing either fipronil or spinosad compared with a control bait without in-
secticide. Exposure of flies to fresh bait containing 40 ppm of acetamiprid, clothianidin, or
imidacloprid, resulted in significantly more flies becoming knocked down than the control.
Baits containing 40 ppm of fipronil or spinosad resulted in higher levels of fly mortality than
baits containing either neonicotinoids (acetamiprid, clothianidin, or imidacloprid) or no in-
secticide for trials with fresh and 1-d-old bait with unlimited exposure. At the rates tested
baits containing deltamethrin resulted in no fly knockdown and always had the lowest mor-
tality of any insecticide treatment. The tradeoffs between insecticides capable of knockdown
and mortality are discussed as they relate to management of R. mendax.
Key Words: Rhagoletis mendax, acetamiprid, clothianidin, fipronil, imidacloprid, spinosad
La sobrevivencia de la mosca, Rhagoletis mendax, contra seis insecticides (acetamiprid, clo-
thianidin, deltamethrin, fipronil, imidacloprid, y spinosad) incorporados a 4, 40, y 400 ppm
en cebos de protein fue evaluada en pruebas de alimentaci6n en el laboratorio. Las concen-
traciones mas altas de insecticides resultaron en un aumento de la mortalidad de las mos-
cas. En todas las concentraciones de los insecticides en cebo, menos aquellas tratadas con
deltamethrin, hubo una mortalidad significativamente mas alta 4 d despu6s de la alimenta-
ci6n inicial, comparada con las moscas que se alimentaron sobre el cebo de control. La pre-
sencia de clothianidin o imidacloprid en el cebo result en una alimentaci6n
significativamente menor comparada con el cebo de control sin insecticide. No hubo ningun
efecto detrimental en la alimentaci6n del cebo que tenia fipronil o spinosad comparado con
el cebo de control sin insecticide. La exposici6n de las moscas al cebo fresco con 40 ppm de
acetamiprid, clothianidin o imidacloprid, result en significativamente mas moscas derriba-
das que en el control. Los cebos con 40 ppm de fipronil o spinosad resultaron en un nivel mas
alto de la mortalidad de moscas que en los cebos con neonicotinoids (acetamiprid, clothiani-
din, o imidacloprid) o sin insecticide para las pruebas con cebo fresco y de cebo de un dia con
exposici6n sin limited. A las tasas de insecticides probadas, ningun mosca fue afectada en la
prueba de cebo con deltamethrin y siempre tenian la menor mortalidad que cualquier otro
tratamiento de insecticides. Se commentan sobre los factors de los insecticides con la capa-
cidad para un efecto de noqueo de las moscas versus un insecticide que mata las moscas en
relaci6n al manejo de R. mendax.
The blueberry maggot fly, Rhagoletis mendax ern United States and Atlantic Provinces of Can-
Curran, is a serious pest oflowbush and highbush ada. In areas not infested with R. mendax there is
blueberries, Vaccinium angustifolium Aiton and zero-tolerance for maggot presence. As a result,
V corymbosum L., respectively, in the northeast- growers exporting fruit to non-infested areas of
Scientific names italics only
Canada must participate in a Blueberry Certifica-
tion Program (Canadian Food Inspection Agency
This certification program mandates following
either a calendar-based or an integrated pest
management (IPM) spray program. A calendar-
based approach requires growers to start spray-
ing insecticides within 10 d of the first detection of
an adult fly in the area, and continue spraying at
7- to 10-d intervals until the end of harvest. An
IPM-spray program requires growers to monitor
the presence of adults with traps baited with am-
monium acetate. A recommended insecticide
should be applied within 5 d of the date of capture
of a single fly in any one of the monitoring traps,
followed by a second spray 7-10 d later. This spray
sequence should be repeated for each subsequent
fly detection until the end of harvest. Many blue-
berry growers use one of these spray regimens,
but there are several alternative strategies that
have been investigated.
Rhagoletis flies can be controlled and managed
by a variety of insecticides and application meth-
ods. Broad-spectrum insecticides, such as organo-
phosphates and carbamates, have been applied in
ultra low volume sprays, where contact and feed-
ing toxicity of small droplets can cause fly mortal-
ity (Mohammad & Aliniazee 1989; Hu et al. 2000).
The enactment of the Food Quality Protection Act
(FQPA) (1996) has placed severe restrictions on
the use of these broad-spectrum insecticides, and
future management of Rhagoletis flies will in-
volve the use of insecticides that are not impacted
by FQPA reassessment. Many of these new com-
pounds have little or no contact toxicity; there-
fore, they are often incorporated into a bait sta-
tion or bait spray, in which mortality results after
flies ingest significant quantities of insecticide.
Painted spheres baited with ammonia com-
pounds are highly attractive to tephritids and
have been developed as bait stations. Spheres
were first coated with a sticky material to trap
flies (Prokopy 1975), but the need for decreased
deployment and handling time necessitated find-
ing an insecticide replacement (Duan & Prokopy
1995b). Studies evaluating the effects of insecti-
cides, which were incorporated into the paint and
sugar matrix that coated the surface of spheres,
have been performed for Anastrepha ludens
(Loew) (Prokopy et al. 2000b); Ceratitis capitata
(Wiedemann) (Hu et al. 1998); andR. mendax and
R. pomonella (Walsh) (Duan & Prokopy 1995b;
Liburd et al. 1999; Ayyappath et al. 2000; Stelin-
ski et al. 2001). Comparisons between baited
spheres and azinphos-methyl sprays in a com-
mercial apple orchard showed similar reductions
in populations of R. pomonella (Prokopy et al.
2000a). However, in commercial blueberry fields
insecticidal spheres are not currently used be-
cause of the deployment density, lack of attractive
selective lure, associated costs of products (i.e.,
spheres and residue extending agents), and labor
requirements (i.e., monitoring and applying in-
secticides to spheres) (Barry et al. 2004).
Another alternative to broad-spectrum sprays
are protein bait sprays which contain ammonia-
based attractants, a feeding stimulant such as su-
crose, and an insecticide. Protein bait sprays have
been used to control outbreaks of Anastrepha
ludens, Bactrocera dorsalis (Hendel), and Cerati-
tis capitata since the 1950s in the United States
(Steiner 1952; Moreno & Mangan 2003). However,
development and evaluation of protein bait
sprays on R. mendax has begun only recently. Pro-
tein and ammonia-based attractants have been
evaluated onAnastrepha spp. (Moreno & Mangan
2003), B. cucurbitae (Coquillett) (Fabre et al.
2003), B. dorsalis (Cornelius et al. 2000), R. cerasi
(L.) (Katsoyannos et al. 2000), and R. pomonella
(Duan & Prokopy 1992). Different concentrations
of sugar feeding stimulants have been tested on
A. suspense (Loew) (Sharp & Chambers 1984), R.
pomonella (Duan & Prokopy 1993), and R.
mendax (Barry & Polavarapu 2004).
Dowell (1994) outlined alternatives to a
malathion bait spray, which had been the pre-
ferred method in eradicating incipient infesta-
tions of C. captitata. Further development of a re-
placement has led to evaluation of insecticides
classified as reduced risk. One compound that has
already been incorporated into a bait spray for
tropical and sub-tropical tephritid pests is spi-
nosad, which was developed from the bacterium
Saccharopolyspora spinosa Mertx and Yao. Feed-
ing on baits containing spinosad has resulted in
high mortality for A. ludens (Prokopy et al.
2000b), A. suspense (King & Hennessey 1996), B.
cucurbitae (Prokopy et al. 2003), and C. capitata
(Peck & McQuate 2000; Vargas et al. 2001; Barry
et al. 2003). Trials assessing toxicity have oc-
curred for several of the non-organophosphate
and non-carbamate compounds, such as delta-
methrin, imidacloprid, and spinosad, on R.
pomonella (Duan & Prokopy 1995a; Hu et al.
2000; Bostanian & Racette 2001; Reissig 2003)
and acetamiprid, deltamethrin, fipronil, and imi-
dacloprid on R. mendax (Barry et al. 2004). The
reduced risk insecticide clothianidin, a neonicoti-
noid, has not been evaluated on any tephritid spe-
Our goal was to identify the most effective con-
centrations of insecticides present in bait that re-
sulted in knockdown, mortality, and had the least
feeding deterrence on R. mendax.
MATERIALS AND METHODS
Infested blueberries were collected near Chat-
sworth, NJ, in the summer of 2002 and 2003. The
rearing procedures of Ayyappath et al. (2000)
Florida Entomologist 88(3)
were used to obtain adult R. mendax. Briefly, in-
fested berries were placed over moist sand for lar-
vae to drop and pupate. Puparia were sifted from
sand three-five weeks later and kept in a screen-
house. Puparia were transferred to an incubator
on 1 November 2002 and 2 November 2003, at 6C
with a photoperiod of 12:12 (L:D) to complete dia-
pause. On 27 March 2003 and 30 March 2004 pu-
paria were placed at 8C. Periodically groups of
puparia were transferred from 8 to 15C for ap-
proximately 8 d and then transferred to an incu-
bator at 25C with a photoperiod of 16:8 (L:D) un-
til adult emergence, which occurred 25-45 d later.
Adult flies were kept at 22C and were provided a
diet of sucrose and water (i.e., protein-starved).
Flies used in assays were 7-13 d-old and allowed
to acclimatize to experimental conditions in the
laboratory for several hours before trials com-
Feeding Assay-Feeding for 10 s
In the laboratory (21-23C), a no-choice feeding
test was used to evaluate survivorship of R.
mendax on a control bait with baits containing
three concentrations (4, 40, and 400 ppm or
0.0004, 0.004, and 0.04% [AI], respectively) of six
insecticides: acetamiprid (technical, 30% [AI];
Cerexagri, King of Prussia, PA), clothianidin
(technical, 49.17% [AI]; Arvesta, San Francisco,
CA); deltamethrin and imidacloprid (technical
99.1, and 98.9% [AI], respectively; Bayer, Kansas
City, MO); fipronil (technical 88% [AI]; Aventris
Crop Science, Research Triangle Park, NC); and
spinosad (technical, 90.4% [AI]; Dow Agro-
Sciences, Indianapolis, IN). Solutions of each in-
secticide concentration were prepared by weigh-
ing the appropriate amount of technical and then
adding it to the corresponding 1:3-mixture of Sol-
Bait (prepared as a 2x concentrate, USDA-ARS,
Weslaco, TX) and water. (A 1:3-mixture corre-
sponds to a 1:4 mixture of GF-120 Fruit Fly Bait
[Dow AgroSciences] to water.) After preparing the
highest concentration, serial dilutions with a 1:3-
mixture of SolBait were used to obtain mixtures
with lower concentrations of insecticides. The con-
trol was a 1:3-mixture of SolBait to water contain-
ing no insecticide.
One 10-pl droplet of bait was placed on a white
plastic lid (5.5 cm in diameter) located on top of a
plastic cylinder (4 cm in diameter, 4 cm in height)
in the center of a Plexiglas cage (30 cm x 30 cm x
30 cm). A fly was transferred to this lid and placed
next to the droplet. After feeding on a droplet for
10 seconds, the fly was removed from the lid and
placed inside a plastic cylinder (5 cm in diameter,
8.5 cm in height) containing water and sucrose.
Flies that fed less than 10 seconds were dis-
carded, unless it was determined that after the
initial feeding a fly became incapable of feeding as
a result of the insecticide (i.e., knockdown). A total
of 30 flies were evaluated with the control and for
each of three concentrations of insecticide (except
deltamethrin which was not evaluated at 4 ppm).
Flies were assessed for knockdown (i.e., immo-
bile or incapable of walking) 1 h after the 10-s
feeding. The number of dead, active, and incapac-
itated flies was recorded after 1, 2, 3 and 4 d. Flies
were characterized as dead if there was no pres-
ence of visible body movement (i.e., no leg twitch),
active if able to walk, and incapacitated if incapa-
ble of walking (Hu et al. 2000; Reissig 2003). The
number of living flies is represented by the sum of
active and incapacitated flies.
Feeding Assay-Feeding for 5 min
In the laboratory (21-23C), a no-choice test
was used to evaluate feeding propensity of female
R. mendax on protein bait containing 40 ppm of
insecticide. Treatments were prepared by the
methods described in the 10-s assay and included
a control bait (without insecticide), clothianidin,
fipronil, imidacloprid, and spinosad. One 10-pl
droplet of a treatment was placed on a silk ficus
leaf (Michaels, Irving, TX) that was placed on top
of a plastic cylinder (4 cm in diameter, 4 cm in
height) in the center of a Plexiglas cage (30 x 30 x
30 cm). Silk leaves were preferred to blueberry
leaves because of the presence of chemical cues in
the latter. One fly was transferred to the leaf
within 1 cm of the droplet. Each feeding trial
ended after 5 min if a fly was still present on a leaf
or when a fly left a leaf after 5 s. (Flies that left a
leaf in less than 5 s were not counted because they
were believed to be in an agitated state.) In addi-
tion, all flies had to feed a minimum of 1 s on the
The amount of time that a fly spent feeding on
a droplet was recorded. Flies were assessed for
knockdown 1 h after feeding. Mortality was re-
corded 1 and 4 d after feeding. Each fly was tested
only once. A total of 28 replicates were completed
for fly feeding and 20 replicates were completed
for knockdown and mortality. One replicate was
completed after a female fly had been tested on
four protein baits incorporated with different in-
secticides and the control bait.
Survivorship and knockdown of flies was as-
sessed to blueberry bushes treated with insecti-
cidal baits. A control bait and four baits contain-
ing 40 ppm insecticide of clothianidin, fipronil,
imidacloprid, and spinosad, were prepared by the
methods described in the 10-s Feeding Assay. Bait
was applied with a handheld sprayer (30 Gunjet;
Spraying Systems Co., Wheaton, IL) to deliver
three 1-ml squirts at 30 psi to each of four three-
yr-old blueberry bushes. This rate is equivalent to
9 liters/ha (0.95 gallons/acre). Three branches
Scientific names italics only
(10-15 cm in length) were removed from each
bush and placed inside a 250-ml Erlenmeyer flask
in a Plexiglas cage (30 x 30 x 30 cm) that con-
tained 20 flies (10 male, 10 female). The flask and
branches were removed after 4 h. Flies were as-
sessed for knockdown 3 h after introduction of
treated branches and for mortality after 24 and
48 h. A total of 4 replicates were completed.
Unlimited Access-Fresh and 1-d-old bait
A no-choice assay evaluated fly mortality to
bait containing the following insecticides: ace-
tamiprid, clothianidin, fipronil, imidacloprid, and
spinosad. Bait was prepared by adding enough
technical insecticide to obtain 40 ppm [AI] in a
mixture with GF-120 Fruit Fly Bait blank that
did not contain spinosad (Dow AgroSciences). The
control contained bait without the addition of in-
secticide. For each treatment, one 10-pl droplet of
bait was applied to 60 highbush blueberry leaves.
Half of these leaves were removed within 10 min
of application for use in fresh assays and the other
half remained on bushes for 24 h before being col-
lected. Three treated leaves of the same bait were
placed inside each often 1-liter plastic containers
with a screened lid, which contained a moist cot-
ton ball. Five flies were then placed in each con-
tainer, which constituted a replicate. Flies were
assessed for knockdown after 1 h and mortality 24
and 48 h after the start of exposure. Ten replicates
were completed for fresh and 1-d-old bait. This ex-
periment occurred in the laboratory where tem-
perature was 21-23C.
Knockdown and survivorship data from the 10-
s Feeding Assay are presented in tabular and
graphical form, respectively. For this feeding as-
say, comparisons also were made between the
control and each treatment with multiple chi-
square tests after Bonferroni correction for the
number of flies living versus dead after 4 d. In the
5-min assay feeding, duration was log trans-
formed and analyzed by Fisher's least significant
different (LSD) tests (P = 0.05). Knockdown and
mortality were analyzed by multiple chi-square
tests after Bonferroni correction. Prior to analysis
of variance (ANOVA), mortality and knockdown
were arcsine-square root transformed in both the
4-h exposure assay and the unlimited access as-
says (SAS Institute 1999). Means were separated
by Fisher's LSD tests (P = 0.05).
Feeding Assay-10 s
Insecticide type and concentration resulted in
different survivorship of living (active + incapaci-
tated) and active flies (Figs. 1 and 2, respectively).
After 4 d, 97% of flies fed the control bait were liv-
ing, which was significantly higher than all treat-
ments except 4 ppm of acetamiprid, clothianidin,
and imidacloprid, and 40 and 400 ppm of delta-
methrin (X2, with Bonferroni correction, P = 0.05).
Greater than 90% of flies were living 4 d after
feeding on bait containing 4 ppm of acetamiprid,
clothianidin, and imidacloprid (Fig. 1A-C);
whereas less than 10% were living after feeding
on baits with the same concentration of fipronil
and spinosad (Fig. 1E, F).
Four days after feeding on bait containing 400
ppm of insecticide, there were 13, 43, and 87%
flies categorized as living for treatments of
clothianidin, acetamiprid, and deltamethrin, re-
spectively (Fig. 1A, B, D). At 400 ppm of fipronil,
spinosad, and imidacloprid in baits it took 1, 3,
and 4 d after treatment to reach 0% survivorship,
respectively (Fig. 1C, E, F). Large decreases in
survivorship occurred between 1 and 4 d for all
concentrations of spinosad and 4 ppm fipronil.
Comparison of survivorship curves of living
(active + incapacitated) with active flies appeared
similar for treatments of clothianidin, delta-
methrin, and fipronil (compare Fig 1B with 2B,
1D with 2D, 1E with 2E, respectively), but dif-
fered for the other three insecticides. The number
of active flies increased from 1 to 4 d after feeding
on bait containing 400 ppm acetamiprid, which
indicated that some flies which had been incapac-
itated were now active (Fig. 2A). The percent of
living flies compared with active flies was 40 and
3%, respectively, 1 d after feeding on bait contain-
ing 400 ppm imidacloprid, indicating that most
(>90%) living flies were incapacitated (Fig. 1C
and 2C, respectively). A large proportion of flies
that fed on bait containing 40 and 400 ppm of spi-
nosad were incapacitated, resulting in signifi-
cantly fewer active than living flies 1-2 d after
feeding (compare Fig. 1F with 2F).
More than 80% of flies were knocked down af-
ter 1 h on baits containing 400 ppm of acetami-
prid, clothianidin, and imidacloprid, with 30%
knocked down for fipronil and spinosad (Table 1).
Flies exposed to treatments of deltamethrin and
control bait were not affected. Compared with the
control bait there were significant higher knock-
down effects after 1 h for baits containing 40 ppm
of acetamiprid (20%), clothianidin (80%), and im-
Feeding Assay-5 min
Protein baits containing insecticide had a sig-
nificant effect on feeding duration (F = 65.79; df=
4, 135;P < 0.0001; Fig. 3). Compared with the con-
trol bait flies fed significantly less on baits con-
taining imidacloprid and clothianidin, and fly
feeding was not significantly different for baits
containing fipronil and spinosad. Bait containing
Florida Entomologist 88(3)
100 yX VX yX vX
j 60 -- ---- -
20 A acetamiprid
100 X- - - X - - -TX
* E fipronil
Fig. 1. Percent of flies that were living (active + incapacitated flies) after feeding for 10 s on a droplet of a given
insecticide concentration. A) Acetamiprid, B) Clothianidin, C) Imidacloprid, D) Deltamethrin, E) Fipronil, F) Spi-
nosad; (V = 4 ppm, 0 = 40 ppm, 0 = 400 ppm insecticide; X = control bait without insecticide) (Deltamethrin was
not evaluated at 4 ppm.)
imidacloprid was the only treatment that resulted
in knockdown after 1 h that was significantly
higher than the control (X2 with Bonferroni correc-
tion, P = 0.05; Table 2). Flies that fed on fipronil
were dead after one day and flies that fed on spi-
nosad were all dead after four days; and both re-
sults were significantly higher than the fly mor-
tality in the control (X2 with Bonferroni correction,
P = 0.05; Table 2).
All insecticide treatments resulted in signifi-
cantly higher knockdown than the control except
spinosad after 3 h (F = 4.47; df = 4, 15; P = 0.014;
Table 3). After 24 h, treatments had a significant
effect on fly mortality (F = 3.58; df = 4, 15; P =
0.031; Table 3), with all insecticide treatments re-
sulting in significantly higher mortality than the
. . -
Scientific names italics only
1X rX VX --
x x x
X X X
0 0 0
Fig. 2. Percent of flies that were active (with incapacitated flies excluded) after feeding for 10 s on a droplet of a
given insecticide concentration. A) Acetamiprid, B) Clothianidin, C) Imidacloprid, D) Deltamethrin, E) Fipronil, F)
Spinosad; (V = 4 ppm, O = 40 ppm, 0 = 400 ppm insecticide; X = control bait without insecticide) (Deltamethrin
was not evaluated at 4 ppm.)
control except imidacloprid. After 48 h, there were
no differences among the treatments including
the control (F = 1.12; df = 4, 15; P = 0.385;
Survivorship Unlimited Access-Fresh bait
Feeding on fresh bait containing insecticide re-
sulted in significant fly knockdown after 1 h (F =
7.45; df= 5, 54; P < 0.0001; Table 4). Significantly
more flies were knocked down on treatments of
bait containing acetamiprid, clothianidin, and im-
idacloprid compared with the control or treat-
ments containing fipronil and spinosad. After 24
and 48 h, treatments had a significant effect on fly
mortality (F = 14.86; df = 5, 54; P < 0.0001; and F
= 15.65; df = 5, 54; P < 0.0001, respectively). After
24 h, fly mortality was significantly higher on
fipronil and spinosad baits compared with the
other three insecticide treatments, all of which
x x Ox
------- --_ --X
X X X
x x x
Florida Entomologist 88(3)
TABLE 1. KNOCKDOWN OF FLIES 1 H AFTER FEEDING FOR
10 S ON INSECTICIDAL BAIT.
Fly knockdown (%)
Treatment 400 ppm 40 ppm 4 ppm
Acetamiprid 83 a 20 b 0
Clothianidin 96 a 79 a 0
Deltamethrin 0 c 0b -
Fipronil 30 b 0 b 0
Imidacloprid 100 a 63 a 3
Spinosad 30 b 0 b 0
Control 0 c 0 b 0
Values in the same column having the same letter are not
significantly different (multiple chi-square tests after Bonfer-
roni corrections; P = 0.05).
n = 600 flies.
were significantly higher than the control. These
relative treatment relationships were the same
after 48 h.
Survivorship Unlimited Access-1-d old bait
Treatments of 1-d old bait containing insecti-
cides had a significant effect on fly knockdown (F
= 8.49; df = 5, 54;P < 0.0001; Table 4). The highest
numbers of flies were knocked down on acetami-
prid, followed by imidacloprid, with both signifi-
cantly higher than the control. The other three in-
secticides were not different from the control in
fly knockdown. After 24 and 48 h, treatments had
a significant effect on fly mortality (F = 8.88; df =
5, 54; P < 0.0001; and F = 9.9; df = 5, 54; P <
0.0001, respectively). After 24 h, fly mortality was
100 b ab
Fipronll Spinosd Cloltianidin Imidaloprid Control
Fig. 3. Duration of fly feeding (mean SE) on a bait
containing 40 ppm insecticide. Flies were allowed to
feed a maximum of 5 min on a 10-pl droplet. The control
was bait without insecticide. Vertical bars with the
same letter are not significantly different. (Fisher's LSD
test with log transformed data). (F = 65.79; df = 4, 135;
P < 0.0001).
TABLE 2. FLY MORTALITY AND KNOCKDOWN AFTER 5 MIN
EXPOSURE TO BAIT CONTAINING 40 PPM INSEC-
Knockdown (%) Mortality (%)
Treatment 1 h ld 4d
Clothianidin 55 ab 15 ab 25 ab
Fipronil 5 ab 100 a 100 a
Imidacloprid 80 a 20 ab 50 ab
Spinosad 5 ab 40 ab 100 a
Control Ob Ob 5 b
Values in the same column having the same letter are not
significantly different (multiple chi-square tests after Bonfer-
roni corrections; P < 0.05).
n = 100 flies.
significantly higher on fipronil and spinosad baits
compared with the control and the other insecti-
cide baits. After 48 h, baits containing fipronil and
spinosad resulted in significantly higher mortal-
ity than baits containing either acetamiprid or
clothianidin, with the latter two baits resulting in
significantly higher mortality than either the con-
trol bait or bait containing imidacloprid.
Novel compounds were initially evaluated to
find replacements for organophosphates and car-
bamates. Results of several insecticides warrant
future field trials to determine the efficacy of dif-
ferent bait spray formulations for controlling R.
mendax. Compounds differed in their ability to in-
capacitate and kill flies. Depending on the insec-
ticide chosen for inclusion in protein baits, the
modes of action can be predominantly knockdown
(acetamiprid, clothianidin, and imidacloprid) or
kill (fipronil and spinosad).
TABLE 3. FLY KNOCKDOWN AND MORTALITY AFTER 4 H
EXPOSURE TO BAIT CONTAINING 40 PPM INSEC-
Treatment 3 h 24 h 48 h1
Clothianidin 11.3 1.3 a 13.8 3.8 a 25.0 6.5
Fipronil 8.8 4.3 a 26.3 9.4 a 37.5 14.4
Imidacloprid 5.0 2.0 a 12.5 + 4.3 ab 23.8 7.2
Spinosad 3.8 1.3 ab 16.3 2.4 a 35.0 7.4
Control 0.0 0.0 b 3.8 2.4 b 16.3 5.9
Values in the same column having the same letter are not
significantly different (Fisher's LSD test, P = 0.05).
'NS, ANOVA, P > 0.05.
n = 400 flies.
Scientific names italics only
TABLE 4. FLY KNOCKDOWN AND MORTALITY AFTER EXPOSURE TO BLUEBERRY LEAVES CONTAINING BAIT DROPLETS
WITH 40 PPM INSECTICIDE.
Knockdown (%) Mortality (%)
Experiment Treatment 1 h 24 h 48 h
Fresh' Acetamiprid 12.0 4.4 a 42.0 6.9 b 68.0 4.4 b
Clothianidin 24.0 8.3 a 58.0 8.6 b 78.0 4.6 b
Fipronil 0.0 0.0 b 80.0 4.2 a 96.0 2.6 a
Imidacloprid 16.0 4.0 a 44.0 4.9 b 68.0 8.5 b
Spinosad 0.0 0.0 b 80.0 6.6 a 94.0 3.0 a
Control 0.0 0.0 b 14.0 2.1 c 30.0 9.1 c
1-d-old1 Acetamiprid 16.0 4.0 a 30.0 7.4 b 56.0 10.2 b
Clothianidin 2.0 2.0 bc 28.0 8.0 b 58.0 9.1 b
Fipronil 0.0 0.0 c 60.0 6.6 a 86.0 5.2 a
Imidacloprid 6.0 3.0 b 10.0 4.4 b 30.0 7.4 c
Spinosad 0.0 0.0 c 68.0 6.8 a 88.0 4.4 a
Control 0.0 0.0 c 16.0 10.2 b 22.0 10.5 c
For each experiment, values in the same column having the same letter are not significantly different (Fisher's LSD test, P=
'n = 300 flies.
Bait sprays containing feeding stimulants (e.g.,
sucrose) have several advantages to conventional
sprays. Lower concentrations of insecticide are
needed in bait sprays than conventional sprays be-
cause mortality is primarily from oral toxicity,
which has lower LC50 thresholds than dermal tox-
icity, and more insecticide is consumed because of
the presence of feeding stimulants (e.g., sucrose)
(Hu et al. 2000; Reissig 2003; Barry & Polavarapu
2004). Therefore, baits sprays can be applied at a
lower rate of active ingredient per hectare than
conventional sprays. The attraction and feeding re-
sponses of flies to bait sprays have led to evalua-
tions assessing their potential use as border sprays
(Prokopy et al. 2003; Prokopy et al. 2004).
Fly survivorship differed based on concentra-
tion and type of insecticide used. As expected,
higher concentrations of insecticide resulted in
higher mortality of flies. At 400 ppm the shortest
lag time between feeding and 100% mortality re-
sulted from bait containing fipronil, followed by
bait containing spinosad. The pyrethroid delta-
methrin did not result in fly knockdown or mor-
tality that was significant enough to warrant fur-
ther evaluation on R. mendax, which was also the
finding of Barry et al. (2004) investigating insec-
ticidal coatings for spheres used in attract and
kill of R. mendax. The neonicotinoids (acetami-
prid, clothianidin, and imidacloprid) resulted in
intermediate survivorship, performing better
than deltamethrin, but not as well as spinosad or
fipronil. Our findings are in agreement with Reis-
sig (2003), who found the LC50 (with flies unable
to walk considered dead) of imidacloprid and spi-
nosad for R. pomonella to be approximately 11
ppm and between 3-10 ppm, respectively.
Many published insecticide assays involve ex-
posing flies to an insecticide treatment for several
days in a small container to determine mortality.
These conditions are likely to underestimate the
concentration of insecticide needed for fly mortal-
ity in the field. The importance of such studies is
to determine the suitable type and range of activ-
ity for insecticides to be further evaluated. In the
current study we used three types of assays to
evaluate the effects of insecticides: a variable
feeding duration (up to 5 min), a fixed short dura-
tion (Feeding Assay-10 s), and a fixed long dura-
tion (Survivorship Unlimited Access). Each of
these assays has limitations, but taken together
supports the findings of the other assays.
Sub-lethal effects of insecticides are known to
manifest as a reduction in fecundity, measured in-
directly from oviposition punctures by R.
pomonella (Reissig 2003). In most of the assays in
the current study, observations for knockdown oc-
curred 1 h after a fly fed, but flies feeding on the
neonicotinoids were often in that state much ear-
lier and later, as evidenced by some flies being un-
able to feed for the duration of the 10-s trial from
becoming incapacitated. Liburd et al (2003) found
insecticide-fed flies have lower levels of activity
compared with a control. In our study flies that
were knocked down often died, but some of the
flies in this condition appeared no different than
control flies after 1-2 d, apparently recovering
from exposure to the insecticide. This finding
leads us to suggest that there may be an optimal
concentration for consumption to achieve the de-
Measuring fly mortality in the context of field
evaluations of insecticides contained in bait
sprays is one way to determine the effectiveness
of knockdown. This would provide a realistic set-
ting in which the effects of natural enemies could
be evaluated on flies that are not completely dead,
as well as other sub-lethal effects associated with
a reduction in oviposition and larval presence.
The results of future field trials can determine the
effectiveness of bait sprays containing insecti-
cides with different modes of action.
We thank Elizabeth Bender for rearing flies;
Linda Tran-Barry for assisting with laboratory
work; Rob Holdcraft, Andy Kyryczenko, and Jenn
Hall for collecting flies, the late Daniel Moreno for
supplying SolBait; and Arvesta, Bayer and Dow
AgroSciences for providing insecticides. This re-
search was funded in part by a USDA-CS-
REES-Risk Avoidance and Mitigation Program
and Hatch grant.
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border area sorghum plants for control of melon
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Florida Entomologist 88(3)
ECOLOGY OF CRABRONID WASPS FOUND IN TRAP NESTS FROM SPAIN
J. TORMOS, J. D. Asis, S. F. GAYUBO, J. CALVO AND M. A. MARTIN
Unidad de Zoologia, Facultad de Biologia, Universidad de Salamaca, 37071-Salamanca. Spain
We report data obtained concerning the occupation of trap nests by xylicolous Crabronidae
(sensu Melo 1999) in a study carried out in central Spain between 1992 and 1995. In partic-
ular, we analyze the data on the occupation of the nests for Psenulus concolor (Dahlbom),
Trypoxylon attenuatum F. Smith, and Trypoxylon beaumonti Antropov. All three species use
pre-existing cavities of 2-4 mm to establish their nests. The mortality rates varied between
33% and 55%, and of special interest was the variation between the two species of Trypoxy-
lon L. and the absence of mortality due to natural enemies in P. concolor. In the three spe-
cies, mortality was similar along the nests, with no increase in the innermost or outermost
cells. Trichrysis cyanea (L.) was the most abundant natural enemy in the nests analyzed.
Sex distribution was not random in any of the species studied: in P. concolor and T attenua-
turn, the males developed in the outermost cells, while in T beaumonti they appeared in the
innermost ones. The sex ratio did not deviate from 0.5 in P. concolor and T attenuaun, al-
though in T beaumonti, the number of females was significantly higher than that of males.
Key Words: trap-nests, xylicolous, kleptoparasitoids, Psenulus, Trypoxylon, Trichrysis
Se presentan los datos obtenidos sobre la ocupaci6n de nidos trampa por Crabronidae (sensu
Melo 1999) xilicolas, en un studio llevado a cabo en el centro de Espana, entire 1992 y 1995.
Se analizan especificamente los datos de ocupaci6n y contenido de los nidos para Psenulus
concolor (Dahlbom), Trypoxylon attenuatum F. Smith, y Trypoxylon beaumonti Antropov. Las
tres species utilizan cavidades preexistentes de 2-4 mm para establecer sus nidos. Las tasas
de mortalidad obtenidas varian entire un 33% y un 55%, destacando la variaci6n observada
entire las dos species de Trypoxylon L. y la ausencia de mortalidad ocasionada por enemigos
naturales en P. concolor. En las tres species la mortalidad es similar a lo largo del nido, no
incrementandose en las celdas mas externas o internal. Trichrysis cyanea (L.) es el enemigo
natural mas abundante en los nidos analizados. La distribuci6n de sexos no es aleatoria en
ninguna de las tres species estudiadas: en P. concolor y T attenuatum, los machos se desar-
rollan en las celdas mas externas, mientras que en T beaumonti aparecen en las celdas mas
internal del nido. El sex ratio no se aparta de 0.5 en P. concolor y T attenuaum, aunque en
T beaumonti, el numero de hembras es significativamente mayor que el de machos.
Translation provided by the authors.
The Crabronidae include a large number of
xylicolous species. These wasps build their nests
either in soft core or hollow stems and even in soft
pieces of wood. They may excavate their own nests
(such that they function as true constructors) or
may occupy pre-existing nests or empty holes.
The nests may be linear, with cells located one
after the other, or branched; the latter are never
found in hollow stems. In both cases, the cells are
divided by a septum of mud, resin, or wood parti-
cles. According to Krombein (1967) the septa
"serve to protect against parasites, parasitoids,
and predators; they ensure the nutrition of the
larvae and prevent cannibalism; they serve as ori-
entation for the exit of the adult". In some cases,
there is also a plug at the end of the gallery that
forms a "vestibular cell" in front of the last provi-
Here we report data obtained in a study with
trap-nests on 9 species of crabronids (sensu Melo
1999): Passaloecus gracilis (Curtis, 1834), P. sin-
gularis (Dahlbom, 1844), Pemphredon lethifer
(Suckard, 1837), Psenulus concolor (Dahlbom,
1843), Spilomena troglodites (Van der Linden,
1829), Stigmus solskyi Morawitz, 1864; Trypoxy-
lon attenuatum F. Smith, 1851, T beaumonti
Antropov, 1991, and T minus Beaumont, 1945.
However, we only analyze the data from those
species whose samples can be considered repre-
sentative: Psenulus concolor, Trypoxylon attenua-
tum and T beaumonti.
Psenulus Kohl includes about 160 species, of
which 10 have been found in Europe. The biology
and ecology of the European species have been de-
scribed by Freeman (1938); Grandi (1961); Janvier
(1962, 1975); Danks (1970, 1971a); Jacob-Remacle
Tormos et al.: Crabronidae in Trap Nests
(1976, 1985, 1986); and Bonelli (1988), who pro-
vided data concerning nest structure, prey, and
parasitoids. Lomholdt (1976); Bohart & Menke
(1976); and Dolfuss & Bitsch (2001) compiled data
published by other authors, among which the ref-
erences to natural enemies are outstanding.
Trypoxylon L. is a cosmopolitan genus with
around 700 species. The presence of 17 species
has been recorded in Europe (Antropov 2001).
Many works have addressed the biology of the Eu-
ropean species, most of them referring to nest
structure, prey identification, and parasitoids.
Some works are purely descriptive or just compi-
lations, such as those by Hamm & Richards
(1930); Marechal (1936); Freeman (1938); Bris-
towe (1948); Binaghi (1956); Grandi (1961); Abra-
ham (1982); and Antropov (2001). Danks (1970,
1971a, b); Jacob-Remacle (1976, 1985, 1986,
1987); and Asis et al. (1994) conducted surveys
that, unlike previous works, quantified data ob-
tained about nests, prey, natural enemies, and
mortality. Of the species addressed in the present
work, T attenuatum has been studied by Danks
(1970) and Asis et al. (1994), while T beaumonti
biology is almost unknown.
MATERIALS AND METHODS
One thousand and seventeen trap-nests were
placed in 19 localities of Burgos, Cuenca, Le6n,
Segovia, Soria, Teruel, Valladolid, and Zamora
provinces, from central-western Spain, and 931
were collected at the end of the study. The trap-
nests, made from stems of Ailanthus altissima
Swingle (Simaroubaceae) (1 = 20-30 cm; d = 2-14
mm) and Phragmites australis (Cav.) (Poaceae)
(1 = 20-30 cm; d = 1-8 mm) (cane) were grouped in
sheaves with four stems each. The sheaves repre-
sented one of the following models: a) four cane
stems of different diameters (>2 mm); b) two
stems ofAilanthus and two of cane; c) four stems
of cane of 1-2 mm. These sheaves were placed in
the field at the beginning of the spring of 1992,
1993, 1994 and 1995, attached to the branches of
trees with insulating tape, and were withdrawn
in the autumn of each of the above years. Thus,
they remained in place for 6-7 months. Once
collected, the sheaves were placed in an lar
(model CF 85) refrigerator at 6-8C. The stems
were opened later, any occupation by aculeates
was determined, and stems with nests inside
The data gathered were primarily diameter
and length of the built nest, cell numbers and con-
tents, and presence of vestibular cells and septa.
The cells were numbered from the exterior to the
interior (cell 1 being the outermost one), although
chronologically the innermost cell was the one
constructed first. The contents of each cell were
transferred to glass vials, which were held in a re-
frigerator (6-8C) until the following spring, when
they were transferred to a Heraeus culture cham-
ber (28C) to promote the emergence of adults. It
was then possible to identify the occupants and
some of their parasitoids. The total number of
nests studied was 511. Between 15% and 25% of
the mature larvae were conserved for a possible
later study of the preimaginal stages. This means
that the mortalities, calculated as a function of
the adults obtained, are slightly overestimated
(because mortality affecting immature larvae
could not act on mature larvae).
The stems of each class and diameter that
were occupied, unoccupied, or abandoned were
counted. The calculations regarding occupancy
were carried out based on the number of "nesting
sites" (approximately double the number of
stems) since two nests could have been placed in
each of them: one on each side. Possible reoccupa-
tion (stems occupied in the first and second gener-
ation) was not taken into account in determining
global occupation index.
Analyses were limited to the wasps Psenulus
concolor, Trypoxylon attenuatum, and T beau-
monti (Crabronidae), and kleptoparasitoid
Trichrysis cyanea (L., 1758) (Chrysididae). Data
refer to individuals of the second generation, be-
cause only one collection was made, at the end of
the summer. Abandoned nests (i.e., nests occupied
during the first generation and abandoned by the
adults) were considered to be susceptible to occu-
pation during the second generation.
The sex ratio was calculated as the "number of
males/total number of adults obtained". The fol-
lowing abbreviations are employed: Ml = mortal-
ity in the egg stage, including the possible absence
of oviposition; M2 = mortality of the different lar-
val stages (with the exception of the mature
larva); M3 = mortality due to natural enemies; M4
= mortality in the mature larval stage or in meta-
morphosis giving rise to the adult.
Trap Nest Occupation
The occupation index was 19.34%, slightly
higher for the Ailanthus stems (19.75%) as com-
pared with those of cane (19.28%) (Table 1).
In cane stems (Table 1), the differences in occu-
pation, as a function of diameter, were significant
(X23 = 31.37; P < 0,0001), those with a diameter of
3-4 mm being the ones most used. This is because
these diameters are better adapted to the size of
Trypoxylon attenuatum and T beaumonti, the
species that established the greatest number of
nests (Table 2). In Ailanthus stems, a preference
as a function of diameter was not observed (23 =
0.57; P = 0.90) (Table 1).
Trypoxylon was the most abundant genus (272
nests, 72.9%), while Pemphredon L. (43 nests) and
Psenulus (12 nests) had much lower percentages
Florida Entomologist 88(3)
TABLE 1. STEMS COLLECTED AS A FUNCTION OF DIAMETER AND TYPE (A = ABANDONED, NO = NOT OCCUPIED, O = OC-
CUPIED, T = TOTAL).
Cane A (%) NO (%) O (%) T
Total 239(14.1) 1126(66.6) 326(19.3) 1691
1 mm 0 (0.0) 107 (99.1) 1(0.9) 108
2 mm 30 (5.6) 409 (76.9) 93 (17.5) 532
3 mm 183 (20.0) 537 (58.6) 197 (21.5) 917
4 mm 20 (16.5) 69 (57.0) 32 (26.4) 121
5-8 mm 6 (46.2) 4 (30.8) 3 (23.1) 13
total 32(13.4) 159(66.8) 47(19.7) 238
2-4 mm 1(9.1) 8(72.7) 2 (18.2) 11
5-7 mm 11(9.3) 82 (69.5) 25 (21.2) 118
8-10 mm 16 (18.2) 55 (62.5) 17 (19.3) 88
12-14 mm 4 (19.0) 14 (66.7) 3 (14.3) 21
of occupation (11.5% and 3.2%, respectively). The data obtained for Spilomena and Stigmus (Table
presence of Spilomena Shuckard, Passaloecus 2), although also scarce, point towards a preferen-
Shuckard, and Stigmus Panzer was very reduced, tial use of soft-cored stems.
with scarcely 2.9% of the nests among the three
genera (Table 2). Wasp Biology
Differences among the genera were observed
in the number of nests employed by each species Psenulus concolor. Twelve nests were ob-
in the different types of stems (Ailanthus-cane). trained, 11 in cane stems of 3-4 mm and one inAil-
Trypoxylon females established their nests exclu- anthus (Table 2). The number of cells varied be-
sively in pre-existing cavities, its reported pres- tween 2 and 16 (x = 9.5 cells). The observed mor-
ence inAilanthus stems (14 nests, 5.1% of the to- tality was 48.4%, representing that undergone by
tal found for the genus) probably is anecdotal and mature larvae or 37.7% of those in the process of
undoubtedly due to the occupation of pre-existing metamorphosis that gave rise to adults (Table 3).
galleries in stems excavated by various Apoidea No parasitoids were found attacking this species.
or Pemphredon. Although data reported for Upon analyzing the mortality in cells as a
Psenulus and Passaloecus are scarce, the data function of the position they occupied from the ex-
also indicate the use of pre-existing cavities. Pem- terior, we observed that this was similar in all of
phredon spp. (Table 2) occupied both types of them (X24 = 1.90; NS). The outermost cells did not
stems (22 nests in Ailanthus, 21 in cane). Thus, show a higher mortality index.
the females excavated their nests in stems with The sex ratio obtained was 0.78 /1 Y, not sig-
soft cores, or occupied pre-existing cavities. The nificantly different from 0.5 (binomial test, z =
TABLE 2. CRABRONID NESTS FOUND IN THE DIFFERENT MODELS OF STEMS.
1 mm 2 mm 3 mm 4 mm 5 mm 5 mm Total Ailanthus Total
Total (%) 1(0.3) 93 (28.5) 197 (60.4) 32(9.8) 1(0.3) 2 (0.6) 326 47 373
Passaloecus gracilis 0 2(100) 0 0 0 0 2 0 2
Passaloecus singularis 0 1(100) 0 0 0 0 1 0 1
Pemphredon lethifer 0 0 5(83.3) 1(16.7) 0 0 6 14 20
Pemphredon sp. 0 0 14(93.3) 1(6.7) 0 0 15 8 23
Psenulus concolor 0 0 7 (63.6) 4 (36.4) 0 0 11 1 12
Trypoxylon attenuatum 0 67(37.8) 95(53.7) 15(8.5) 0 0 177 4 181
Trypoxylon beaumonti 0 3(10.7) 20(71.4) 3 (10.7) 0 2 (7.1) 28 0 28
Trypoxylon minus 0 0 1(100) 0 0 0 1 0 1
Trypoxylon sp. 0 15(28.8) 32(61.5) 4 (10.7) 1(1.9) 0 52 10 62
Spilomena troglodytes 0 0 0 0 0 0 0 6 6
Stigmus solskyi 0 0 0 0 0 0 0 2 2
Unknown 1(3.0) 5(15.2) 23(69.7) 4(12.1) 0 0 33 2 35
Tormos et al.: Crabronidae in Trap Nests
TABLE 3. CONTENTS OF NESTS OF PSENULUS CONCOLOR, TRYPOXYLON ATTENUATUM, AND T. BEAUMONTI. M1, M2,
M3, M4 = TYPE OF MORTALITY.
P. concolor (%) T attenuatum (%) T beaumonti (%)
Preserved 19 153 32
y y 27 209 34
66 21 188 10
Adults 48 (51.6) 397 (66.8) 44 (45.4)
Total mortality 45 (48.4) 197 (33.2) 53 (54.6)
M1 3(3.2) 44 (7.4) 17 (17.5)
M2 6 (6.5) 26 (4.4) 5 (5.2)
M3 0 14 (2.4) 6(6.2)
M4 36 (38.7) 113 (19.0) 25 (25.8)
Total cells 112 747 129
0.72; NS). The distribution of males and females
inside the nests was not similar (x24 = 15.99; P <
0.01); the sex ratio follows a negative exponential
model (sex ratio = -0.396 In (cell position) + 1.040;
R2 = 0.825) (F13 = 92.12; P < 0,0001), and hence
the males are increasingly less abundant towards
the innermost cells.
Trypoxylon. At least three species nested in the
stems provided (Table 2): T attenuatum, T beau-
monti and T minus, a total of 210 nests being
counted. Moreover, 62 nests were detected that
could not be assigned to any given species with
certainty because the larvae had not developed
Trypoxylon attenuatum. With 181 nests (177 in
cane and 4 in Ailanthus) T. attenuatum was the
most abundant species. Occupation is shown in
Table 2 as a function of stem diameter. No prefer-
ence for any specific diameter was observed
within the 2-4 mm range (X2 = 1,82; NS), al-
though no nests in canes of other diameters were
The number of cells per nest varied between 1
and 11 (x = 4.12). The observed mortality was
33.2%. M4 accounted for the greater part of the
mortality (almost 60%), while the incidence of
natural enemies was very low, and only 14 cells
out of 594 (2.4%) were parasitized (Table 3).
Mortality as a function of the position occupied
by the cells from the exterior was found to be
quite similar in all of them (X2, = 11.33; NS). Thus,
mortality was not higher in the cells closer to the
exterior. Neither were there any significant differ-
ences as a function of the number of cells in the
nests. Mortality in the nests with few cells was of
the same order as in those with more cells (26 =
The number of males and females obtained
was similar (0.96/1 ), and did not depart signifi-
cantly from a sex ratio of 0.5 (binomial test, z =
1.42; NS). However, the distribution of males and
females within the nests was not the same (23 =
46.35;P < 0.0001): the males developed in the out-
ermost cells, and the sex ratio follows a negative
exponential model (sex ratio= -0.314 In (cell posi-
tion) + 0.743; R2 = 0.941) (F1,,9 = 76.80;P < 0.0001).
Trypoxylon beaumonti. Twenty-eight nests
were obtained, all of them from cane stems (Table
2). Although the data are scarce, there seemed to
be a significant preference towards the occupation
of galleries of 3-4 mm (X2 = 8.17; P < 0.05). The
number of cells varied between 2 and 7 (x = 4.6).
The observed mortality was 54.6% (Table 3),
with six cells attacked by Trichrysis cyanea. Mor-
tality varies depending on the position occupied
by the cell from the exterior (X2 = 10.48; P < 0.01),
being higher for the most exterior cell (80%) and
smaller for inner cells (around 40%).
Important differences were seen between the
number of males and females obtained (0.296/1 ),
and the sex ratio departed significantly from 0.5
(binomial test, z = 3,47; P < 0.001). Sex distribu-
tion inside the nests was not similar (X2, = 13.96;
P < 0.01), the males being found in the innermost
nests and the females being progressively more
abundant towards the exterior.
Trichrysis cyanea. This kleptoparasitoid was
found in 32 nests, belonging to different species of
Pemphredon and Trypoxylon, and parasitized 44
(37%) of the cells (Table 4). The mean number of
cells parasitized per nest was 1.38. Eleven of the
nests affected, with two or more cells, had more
than 50% of the cells parasitized. No significant
differences were observed as a function of the posi-
tion of the cell. The outermost cells did not exhibit
a greater probability of being parasitized than
those located more to the interior of the nest (X25 =
9.77; NS). In three of the 44 cells the kleptoparasi-
toid did not complete its development, although
the larvae managed to become pupae. Of the 41
adults obtained, 23 were male and 18 female, and
the sex ratio (1.27 6:19) was not significantly dif-
ferent from 0.5 (binomial test, z = 0.63; NS).
In Psenulus concolor, the mortality value
(48.4%) was higher than that reported in Great
Florida Entomologist 88(3)
TABLE 4. NESTS AND CELLS OF DIFFERENT HOSTS AFFECTED BY TRICHRYSIS CYANEA.
Total number Nests with 2 or more
Nests affected of cells Parasitized cells cells parasitized
Pemphredon sp. 2 10 3 1
Trypoxylon attenuatum 4 23 7 2
Trypoxylon beaumonti 8 29 10 3
Trypoxylon sp. 18 57 24 5
Total 32 119 44 11
Britain for the same species by Danks (1970,
1971b) (29.3% and 26%) (X22 = 10.72; P < 0.01).
However, the mean number of adults produced
per nest was greater in this study (5 adults/nest)
than in those of Danks (1970, 1971b) (4.0-4.2
adults/nest), due to the higher number of cells in
the nests analyzed (9.3 in this study as compared
with 5.4-5.9 in those of Danks 1970, 1971b). It is
striking, nevertheless, that no parasitoids were
found attacking this species; all mortality derived
from the interruption of development during lar-
val and pupal stages. The same observation was
reported and attributed to a paucity of data by
The mortality rates found for European species
of Trypoxylon vary between 33.2% (for T attenua-
tum in this study) and 63.8% (for T attenuatum
and T figulus in Danks 1971b). The observed dif-
ferences are significant (x25 = 144.25; P < 0.001).
Mortality in T attenuatum is lower, both in this
study (33.2%) and in that of Asis et al. (1994)
(44.0%). Furthermore, the values reported by
Danks (1971b) for T attenuatum and T figulus
(63.8%) and by Jacob Remacle (1986) for T clavi-
cerum and T minus (58.2%) are clearly higher,
while the value found for T beaumonti (54.6%) and
that given by Danks (1970) for T attenuatum and
T figulus (54.6%) do not deviate from the mean
values. This shows that the populations of these
wasps are subject to important fluctuations, al-
though it seems that T attenuatum in the Iberian
Peninsula could have appreciably lower rates than
the rest of the species or than other populations of
this species present in more northern areas.
It is also possible to observe an important vari-
ability in mortality due to different agents, and of
special interest is the low mortality attributable
to the action of natural enemies in the species an-
alyzed here (2.4-6.4%), whereas the rates found in
other works are between 15% and 25%.
In Trypoxylon, cases have been described in
which the distribution of the individuals of each
sex in the nest seems to be irregular (see Cross et
al. 1975), although in many cases it follows a de-
fined trend. Thus, in some species the males are
found in the innermost cells and the females in
the outermost ones (IMOF, inner males outer fe-
males) while in other species the inner cells har-
bor females (IFOM). The IMOF model is the main
one among species of the subgenus Trypargilum,
in which the male remains inside the nest during
its provisioning, copulating with the female when
she returns to it (Krombein 1967; Medler 1967;
Coville & Coville 1980; Coville & Griswold 1983,
1984; Camillo et al. 1993, 1994). In wasps of the
subgenus Trypoxylon, the model seems to be
IFOM (Krombein 1967; Asis et al. 1994; Oku &
Nishida 1999). Trypoxylon attenuatum, as re-
ported by Asis et al. (1994), follows this model.
However, the data obtained for Trypoxylon beau-
monti seem to reflect the IMOF model. This could
be due to a lack of data, although it might also be
a reflection of differences in the behavior of this
species, an aspect that deserves further attention.
In some species of Psenulus, sex ratios that
clearly deviate towards a greater production of
males have been described (0.86 in Krombein
1967, for Psenulus pallipes (Panzer), z = 5.04, P <
0,0001; 0.68 in Matthews 2000, for P interstitialis
Cameron, z = 2.19, P < 0.05). Nevertheless, the
data obtained by us and those reported by Danks
(1970, 1971b) point to a different situation for
P concolor, with a sex ratio that does not depart
significantly from 0.5. Regarding the species of
Trypoxylon studied, the sex ratio does not depart
significantly from 0.5 in T attenuatum, while it
does deviate from that figure, and towards a
greater production of females, in T beaumonti. Ac-
cording to the theory of parental inversion pro-
posed by Fisher (1999), in large randomly breed-
ing populations selection will result in equal in-
vestment in sons and daughters (hence a sex ratio
of 0.5). Since the males are smaller than the fe-
males in most solitary wasps and require a lower
investment, one would expect a greater produc-
tion of males than of females (i.e., a sex ratio >0.5).
However, in situations such as those that affect
certain wasps, with fragmented populations in
which there is local competition for mates among
brothers, the females will be selected to produce
female-biased sex investment ratios (Hamilton
1967; Cowan 1991). This could explain the devia-
tion, towards the production of females, observed
in T beaumonti, whereas T attenuatum, with
larger and less isolated populations on the Iberian
Peninsula, would show sex ratios close to 0.5.
It is also necessary to take into account the in-
fluence that can be exerted by the nesting sub-
Tormos et al.: Crabronidae in Trap Nests
state on the sex ratio since different studies have
shown that cavities with smaller diameters shift
the sex ratio towards the production of males
(Krombein 1967; Charnov et al. 1981). The diam-
eters offered do not seem the limit the nesting
possibilities of the species of Trypoxylon studied
because mainly the cavities with a medium-sized
diameter were those occupied. However, if the
cavities offered are larger or smaller than those
usually available in nature, a bias towards a
greater production of one of the sexes could arise,
as long as there is sexual dimorphism as regards
size (Trivers & Hare 1976). In any case, Oku &
Nishida (2001) have demonstrated that the use of
small samples could lead to mistaken conclusions
concerning the sex ratio, so caution should be ex-
ercised on drawing conclusions from few data.
Financial support for this paper was provided from
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Entomological Website Usage Patterns
ENTOMOLOGICAL WEBSITE USAGE PATTERNS
R. W. MANKIN
United States Department of Agriculture, Agricultural Research Service
Center for Medical, Agricultural, and Veterinary Entomology, P.O. Box 14565, Gainesville, FL 32604
Usage patterns of entomological research websites were examined to assess their current
roles as information resources. A 5-year review of logfiles at three Florida entomological re-
search websites indicated that usage has increased since 1999 and that visitors increasingly
have taken advantage of Internet search engines to find pages with high information con-
tent. Websites provide opportunities for dissemination of information (for example, in sound
files or databases) that is difficult to include in traditional refereed publications. Given the
rapid growth of website usage, research organizations may wish to consider formal proce-
dures for vetting such information.
Key Words: Internet, world wide web, search engine
Los patrons de uso de paginas electr6nicas para investigaci6n entomol6gica fueron exami-
nados para determinar sus papeles actuales como recursos de informaci6n. Una revision de
un period de 5-aios de los registros de archives de tres paginas electr6nicas de investiga-
ci6n entomologica indica que su uso ha aumentado desde 1999 y los visitantes han tomado
una ventaja creciente de sistemas de busqueda de la Red-electr6nica para encontrar paginas
con un alto contenido de informaci6n. Las paginas electr6nicas proven oportunidades para
la diseminaci6n de informaci6n (por ejemplo, en un archivo de sonido o una base de datos)
que son dificiles para incluir en publicaciones tradicionales reguladas. Tomando en cuenta
el crecimiento rdpido del uso de paginas electr6nicas, las organizaciones de investigaci6n tal
vez quieren considerar los procedimientos formales para evaluar esta informaci6n.
Entomological institutions began establishing
websites in the early 1990s to disseminate
research and provide membership services
(VanDyk 2000). The Florida Entomological Soci-
ety (FES) was one of the pioneers in this effort,
beginning online publication of new issues of the
Florida Entomologist at the Florida Center for
Library Automation (FCLA) in 1994 (Zenger &
Walker 2000). Online journals at society and in-
stitutional websites have since become important
resources for researchers, but few studies have
been published on scientific website usage pat-
terns (Davis 2004) or the roles of institutional
websites as information resources (Treise et al.
2003; Lederbogen & Trebbe 2003).
In addition to the original online issues of Flor-
ida Entomologist in .pdf format, FES launched a
membership website in 1998 which hosted a
search engine for online issues, FESsite (Table 1),
and began offering an .html version of new arti-
cles at BioOne (www.bioone.org) in 2002. A search
engine for all Florida Entomologist issues was
initiated at FCLA in 2004. To evaluate usage of
current FES website resources in the context of
activity at similar websites, a review was con-
ducted of activity between 2000 and 2004 at FES-
site, the United States Department of Agricul-
ture, Agricultural Research Service, Center for
Medical, Agricultural, and Veterinary Entomol-
ogy website, CMAVEsite, and a personal research
site at CMAVE, Perssite. The usage was compared
with activity at the Florida Entomologist journal
homepage, the American Chemical Society jour-
nal server (Davis 2004), and two entomological
websites at the University of Florida (UF). The
two UF sites were the Entomology Department
Newsletter and the highly visited, Featured Crea-
tures site (Table 1), hosted jointly with the Flor-
ida Division of Agriculture and Consumer Ser-
MATERIALS AND METHODS
Daily logs from FESsite were analyzed from 5-
Sep-99 to 31-Dec-04, using Mediahouse or
LiveSTATS (Deepmetrix Corp., Gatineu, Quebec,
CA). Daily logs from CMAVEsite were analyzed
from 14-Jan-00 to 24-Dec-04, with Analog
(www.analog.cx, Cambridge, UK). Additional
analyses were conducted on a subset of the
CMAVE daily activity in a personal research site,
Perssite (Table 1). File-editing records were ex-
amined and directory searches were conducted to
count the numbers of pages on the sites at the be-
Florida Entomologist 88(3)
TABLE 1. DESCRIPTIONS AND INTERNET ADDRESSES (URLS) OF FREQUENTLY ACCESSED CMAVE, FES AND UF ENTO-
MOLOGY WEB PAGES, WITH ASSOCIATED SITE/PAGE NAMES.
Description URL Site/Page name
CMAVE website cmave.usda.ufl.edu CMAVEsite
CMAVE home cmave.usda.ufl.edu/index.html CMAVEhpage
IFAHI Unit home' cmave.usda.ufl.edu/ifahi/index.html
Formis database2 cmave.usda.ufl.edu/formis/
CMAVE publ. list cmave.usda.ufl.edu/publications.html
Personal website cmave.usda.ufl.edu/~rmankin Perssite
Personal home cmave.usda.ufl.edu/~rmankin/index.html Pershpage
Sound library cmave.usda.ufl.edu/~rmankin/soundlibrary.html Perssound
FES website flaentsoc.org FESsite
FES home flaentsoc.org/index.html FEShpage
FlaEnt search3 flaentsoc.org/FEASearch.cfm
FlaEnt home link4 flaentsoc.org/fe.html
FES pests' flaentsoc.org/fespestweblinks2.html
FES Fla. insects6 flaentsoc.org/arthropdiversity/
UF/FDAC featured creatures creatures.ifas.ufl.edu/
UF Entomology newsletter entnews.ifas.ufl.edu/
CMAVE Imported Fire Ant and Household Insects Research Unit home.
'Bibliography of ant literature.
Link to original search engine for 1993-2004 Florida Entomologist online issues.
FES page with information and links to Florida Entomologist journal homepage (Fig. 1B).
5Links to information about Florida pest insects.
'Florida Arthropod Conservation homepage.
innings and ends of the analysis periods. The
Florida Entomologist home page (http:/ /
www.fcla.edu/FlaEnt/index.htm) was monitored
with Net.Data (IBM, Armonk, NY).
Requests for FES, CMAVE, and personal re-
search website pages were also compared with
records of monthly page views of the UF, IFAS En-
tomology and Nematology Newsletter and the UF,
IFAS/FDACS Featured Creatures websites ana-
lyzed by LiveSTATS from 1-Jan-02 to 31-Dec-04.
Logfile Analysis Procedures
Logfile software typically provides information
about the originators of requests, and the num-
bers of successful and unsuccessful requests for
pages (files with .htm or .html extensions), as well
as graphics, .pdf, .wav, or other files embedded in
the pages (Srivastava et al. 2000). Unless other-
wise specified below, the counts listed in this re-
port refer only to successful requests for (.htm
and .html) web pages and not directly to requests
for embedded files.
CMAVE and FES website page monthly totals
were estimated by summing the counts from all
successful views of pages at the website in the 30
days preceding the count. To evaluate the contri-
bution of specific pages to website totals, monthly
page views also were counted individually for sev-
eral frequently accessed FES, CMAVE, and per-
sonal research pages (Table 1). The Featured
Creatures and Newsletter monthly totals were
determined from daily counts.
Monthly totals were counted for page queries
initiated by easily identifiable search engines, in-
cluding Google, MSN, Yahoo, Ask Jeeves, Alta
Vista, HotBot, and Lycos. Potential relationships
between website page-view rates and search en-
gine query rates were tested with Proc GLM (SAS
Website and Home Page View Rates
Between 2000 and 2005, the rates of page
views of the FES (FESsite), CMAVE (CMAVE-
site), and personal research (Perssite) websites in-
creased 2.8-, 6.7-, and 9.5-fold, respectively (Fig.
1A), or 0.6-, 1.3-, and 1.9-fold per year, respec-
tively. The greatest rate of increase was seen at
the Featured Creatures website which increased
6.3-fold between January 2002 and December
2004, or 3.1-fold per year. The activity at FES and
personal research website pages in 2003-2004
was similar in magnitude to the 3316 monthly
queries of the American Chemical Society Server
by chemists at Cornell University in 2002-03
(Davis 2004). Apart from the homepage, the Flor-
ida Entomologist search engine was the most fre-
quently viewed page at FESsite. The insect sound
library (Leslie 2002) was the most frequently
viewed page at CMAVEsite. These two pages are
considered in greater detail below.
The general trend of increasing rates of page
views was modulated by a yearly cycle. As ex-
pected with smaller audiences at holidays, the
Entomological Website Usage Patterns
,- .- ,- , 0- =.- ,
Fig. 1. A) Numbers of Featured Creatures (dot-dashed line), CMAVE (dotted line), FES (solid line), UF Entomol-
ogy newsletter (dot-dot-dashed line), and Personal website (dashed line) pages viewed monthly between September
1999 and December 2004 (page addresses in Table 1); B) Numbers of CMAVE, FES, Personal, and FlaEnt journal
(dot-dashed line) homepages viewed per month between September 1999 and December 2004 (page addresses in
view rates at all of the entomological web sites de-
creased briefly each year in December and Janu-
ary. Increased rates of page views were observed
at FESsite in each year in June and July, probably
due to activities involving the annual meeting. In-
creased rates of viewing at the Featured Crea-
tures website occurred in August and September,
near the beginning of the elementary and second-
ary school year.
Part of the increased activity between 2000
and 2005 at the FES and CMAVE websites could
have been due to an increase in the numbers of
web pages at each site, but the percentage in-
crease in page-view rates exceeded the percent-
TABLE 2. REGRESSION EQUATION STATISTICS FOR RELATIONSHIPS BETWEEN PAGE-VIEW RATES AND SEARCH ENGINE
QUERY RATES, LISTED BY DECREASING r2.
Name F1 r2 Root MSE2
Perssite 161.06 0.797 2586.48
FESsite 143.13 0.777 1722.06
CMAVEsite 131.37 0.762 8032.98
Perssound 60.89 0.596 672.64
Pershpage 48.24 0.541 68.14
FEShpage 23.10 0.360 275.28
CMAVEhpage 13.42 0.247 686.24
ldf= 1,41, P < 0.001 for all variables except for CMAVEhpage, with P = 0.007.
Mean Square Error.
Florida Entomologist 88(3)
TABLE 3. REGRESSION COEFFICIENT ESTIMATES AND STANDARD ERRORS (SE) FOR RELATIONSHIPS BETWEEN PAGE-
VIEW RATES AND SEARCH ENGINE QUERY RATES.
Name (unit) Estimate SE P
ACMAe, (pages) 13550.130 2299.41 <0.0001
BCMA.,, (pages/query) 9.750 0.85 <0.0001
ACMAih,, (pages) 2202.080 196.43 <0.001
BCMAVEhpag (pages/query) 0.267 0.073 0.007
AFES (pages) 1497.300 626.68 0.0216
BFE,,, (pages/query) 6.700 0.56 <0.001
AFEShpg (pages) 589.100 100.10 <0.0001
BFE, (pages/query) 0.430 0.09 <0.0001
A,,,,,, (pages) 2141.320 722.57 0.0050
B,_,, (pages/query) 12.830 1.01 <0.0001
A,,,_o, (pages) 590.070 187.92 0.0031
Bso (pages/query) 2.050 0.26 <0.0001
A,,h,, (pages) 203.070 19.04 <0.0001
B,,,, (pages/query) 0.180 0.03 <0.0001
age increase in web pages. The numbers of web
pages increased only from 136 to 619 (455%) at
CMAVEsite, 85 to 188 (221%) at FESsite, and 32
to 84 (262%) at Perssite.
In contrast with the website activity, there was
less evidence of a trend in the rates of views of
FES, CMAVE, and personal research homepages
(Fig. 1B). However, there was an increased rate of
A t 0) ...'
#.*" # 6-
o Personal 2-
0 i FES
... .... :.... .
15 25 35
Search Engine Query Rate (102 queries/mo)
Fig. 2. Comparison of A) website and B) homepage view rates with search engine query rates for CMAVE
(hashes, dotted lines), Personal (open circles, dashed lines), FES (dots, solid lines), and Insect sound library (trian-
gles, dot-dashed line), websites.
Entomological Website Usage Patterns
0 FFlaEnt home
Florida pests i
Insect diversity .
Fig. 3. Rates of viewing of frequently accessed pages at FESsite: Florida Entomologist text-search engine (solid
line), Florida Entomologist information page (dotted line), Florida pest control methods (dot-dashed line), insect di-
versity (dot-dot-dashed line).
viewing of the Florida Entomologist journal
homepage during 2004 (Fig. 1B) similar to the
website trend in Fig. 1A. The difference between
the usage patterns for CMAVEsite, FESsite, Pers-
site, and the corresponding homepages suggests
that the behavior of visitors at those three sites
(but not at the journal) may have changed over
time. If a typical user enters a website through
the homepage and then visits five or fewer pages
altogether (Cooper 2001), about 15% or more of
the pages viewed would be expected to be homep-
ages. Instead, the percentage of homepage views
at the CMAVE, FES, and personal websites had
declined below 8% by August 2004. Perhaps, expe-
rienced visitors were bookmarking pages of inter-
est in one session and then returning to the book-
marked pages directly rather than through the
homepage. Alternatively, visitors may have ac-
cessed multiple pages directly from a search en-
gine, in which case the rates of website page views
might be reflected in search-engine query rates.
Comparisons of Page-Views and Search Engine Queries
The hypothesis that the rates of viewing of
pages at CMAVE, FES, and personal research
websites between 2000 and 2005 were propor-
tional to search engine query rates was tested un-
der the model:
PageViewRate = A + B QueryRate,
where PageViewRate is the number of page views
per month at a specified website and QueryRate
is the number of search engine requests per
month for pages at that site. The regressions for
the CMAVE, FES, and personal websites, the cor-
responding home pages, and the insect sound li-
brary page are compared in Table 2. The regres-
sion coefficients are listed in Table 3 and the rela-
tionships are graphed in Fig. 2. The coefficients of
determination for website page-view rates and
search engine query rates were larger than for
the home pages, but all of the regressions in Table
2 were statistically significant.
The slopes of the regression equations for
CMAVEsite, FESsite and Perssite (Table 3, Fig.
2A) were all greater than 6 views per query, while
the slopes of the regression equations for the cor-
responding home pages (Fig. 2B) were all less
than 0.5 views per query. The large difference in
the slopes of the website and homepage page-
Florida Entomologist 88(3)
c- cJ -, C~' C'~ ~' *t -t -t *t -t .
0 C9 fl O C 0 P C 00 0 C
Fig. 4. Rates of viewing of frequently accessed pages at CMAVEsite: Insect sound library (dashed line), IFAHI
research unit home (solid line), Formis database of ant literature (dot-dashed line), CMAVE publications list (dot-
view-rate regressions suggests that many of the
viewers had queries in search for specific infor-
mation present at the site rather than for general
information about the hosting institution. Such
information most likely would be present in high-
content pages rather than homepages. An analy-
sis of frequently viewed pages was conducted to
determine whether they were characterized by
high levels of information content.
Analysis of Frequently Viewed Pages
Page-view rates for the four most frequently
viewed pages other than the website homepages
in 2004 are shown for FES in Fig. 3 and CMAVE
in Fig. 4. It should be noted that the Florida En-
tomologist search engine page was replaced with
an improved version at the FCLA website, which
reduced the rate of website page views (Fig. 1B)
after September 2004. The introductory page to
the Florida Entomologist has been viewed fre-
quently since the FES website began, and two
pages initiated in 2003 that contain information
about Florida pests and Florida arthropod ecology
have been viewed at increasing rates in 2004.
These, together with the annual meeting program
and abstract pages, contain a large fraction of the
scientific information available at FESsite.
The most frequently accessed pages at
CMAVEsite other than the homepage in 2004
were the insect sound library, the Formis data-
base, the IFAHI Research Unit home page, and
the CMAVE publication list (Fig. 4). The sound
library contained information that is not easy to
include in a traditional refereed publication,
although increasing numbers of journals allow
for online posting as supporting online material.
The Formis database is a popular source of bibli-
ographic information on ant literature. The
CMAVE website has a larger number of high-con-
tent pages than FESsite, which possibly contrib-
uted to its higher rate of page-views in 2004
The patterns described above suggest that in-
creased usage of Internet search engines has se-
CMAVE publication list \
Entomological Website Usage Patterns
lectively increased the rates of viewing of specific
FES and CMAVE web pages. Overall, page-view
rates have increased, but the greatest increase
has occurred for sites and pages with high infor-
mation content. Search engines typically assign
high visibility to research pages from scientific in-
stitutions (Jepsen et al. 2004). This has enabled
entomological databases, associated software
(Byers 2002), and sound files to become impor-
tant, easily accessible research tools along with
the refereed literature. The popularity of the Fea-
tured Creatures website may also have benefited
from its high ranking in search engines.
The trend of increasingly data-rich scientific
websites is not confined to entomology. Research
in bioinformatics (Eiden 2004), biodiversity
(Maddison & Schulz 2004) and astrophysics
(Kurtz et al. 2005), for example, has become
highly dependent on digital libraries and online
databases. The rapid growth of information in-
dexed by Internet search engines has enabled re-
searchers to modify their search strategies and
accelerate their rate of gathering information
(Davis 2004). It has also enabled them to dissem-
inate information faster to wider audiences. The
improved visibility of Internet-accessible re-
search has resulted already in increased relative
effect of open-access research articles published
online (Antelman 2004). If such trends continue
unabated, researchers could benefit if institutions
developed new procedures to vet website content,
perhaps similar to the peer review process of
scholarly journals. One of the challenges to such
development is that web content is frequently up-
dated and, unlike a journal article, would need to
be revetted after a major change.
Tom Fasulo (University of Florida) provided
LiveSTATS records for monthly page views of the Fea-
tured Creatures and Entomology Department Newslet-
ter websites. Tom Walker (University of Florida), and
Pat Greany, Paul Shirk, Eric Daniels, and Dianne Un-
derwood (CMAVE) have provided assistance and advice
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Florida Entomologist 88(3)
STRIDULATION OF GRYLLOTALPA AFRICANA
(ORTHOPTERA: GRYLLOTALPIDAE) ON TURF GRASS IN SOUTH AFRICA
J. DE GRAAF1, A. S. SCHOEMAN1 AND R. L. BRANDENBURG2
'Department of Zoology and Entomology, University of Pretoria, Pretoria 0002, Republic of South Africa
2North Carolina State University, Department of Entomology, Box 7613, Raleigh, NC 27695-7613
During spring to autumn, Gryllotalpa africana males stridulate and produce phonotactic
calling songs from specially constructed acoustical burrows. Songs start just after dusk and
continue for several hours. The characteristics of the trilling song and sound pressure levels
produced were investigated by near field digital recordings made during autumn 2002 and
spring 2002 with soil temperatures noted by measuring sound pressures beyond the near
field with a sound level meter in spring 2002, respectively. The carrier frequency (2.161-
2.477 kHz) and syllable duration (7.340-12.078 ms) of calls showed no significant relation-
ship with soil temperature and no significant differences between autumn and spring with
soil temperature constant. Syllable period (10.455-17.221 ms) and inter syllable interval
(1.912-9.607 ms) were significantly negatively correlated with soil temperature, and with
the latter constant, significantly longer in spring than in autumn. The syllable repetition
rate (0.058-0.096 syllables/ms) and duty cycle (43.31-81.72%) showed a significant positive
relationship with soil temperature and significant decrease in values with soil temperature
constant in spring relative to autumn. Sound pressure levels (re. 20 pPa) at 200 mm from the
burrow varied from 77.6 to 89.8 dB.
Key Words: male song characters, seasonal variance, soil temperature, sound pressure level,
Desde la primavera hasta el otofio, los machos de Gryllotalpa africana vibran producen can-
ciones de llamado fonotacticos) de madrigueras acusticas construidas especialmente. Las
canciones empiezan un poco despu6s del atardecer y continuan por varias horas. Las carac-
teristicas de las canciones vibradas y los niveles de presi6n del sonido producido fueron in-
vestigados por medio de grabaciones digitales en un campo cercano durante el otono de 2002
y la primavera de 2002 (con la temperature del suelo anotada) por medio de la media de la
presi6n de los sonidos (mas alla del campo cercano) con un medidor de nivel de sonido en la
primavera de 2002, respectivamente. La frecuencia aportada (2.161-2.477 kHz) y la dura-
ci6n de la silaba (7.340-12.078 ms) de las llamadas no mostraron una relaci6n significativa
con la temperature del suelo y ningun diferencia significativa entire el otoio y la primavera
(con la temperature del suelo constante. El period de silaba (10.455-17.221 ms) y el inter-
valo entire las silabas (1.912-9.607 ms) fueron negativamente significativas correlacionadas
con la temperature del suelo, y con la constant Tiltima, significativamente mas largo en la
primavera que en el otofio. La tasa de repetici6n de silaba (0.058-0.096 silabas/ms) y el ciclo
obligatorio (43.31-81.72%) mostraron una relaci6n positive significativa con la temperature
del suelo y una diminuci6n significativa en valores (con la temperature del suelo constant)
en la primavera (en relative al otofio). Los niveles de la presi6n del sonido (re. 20 pPa) a 200
mm de la madriguera varian de 77.6 a 89.8 dB.
Numerous insect species produce stereotyped
acoustic signals that are important in intraspe-
cific communication (Kavanagh 1987). In most
species that communicate by sound, the male's
calling song, which appears to attract conspecific
females, is the most obvious and imperative com-
ponent of the repertoire (Kavanagh 1987).
Male African mole crickets differ morphologi-
cally from females by having a pair of large cells
(anterior of which is the harp) on each forewing,
known as the stridulatory area (Townsend 1983)
(Figs. 1 and 2). Males usually stridulate at night,
using the entrance of borrows as sound amplifiers
(De Villiers 1985). Singing position of Gryllotalpa
sp. appears to be very similar, although acoustic
burrows may have two (G. vineae, G. gryllotalpa,
and G. africana) (Bennet-Clarke 1970a; Branden-
burg et al., 2002) to four horn-shaped openings (G.
australis) (Kavanagh & Young 1989). The division
between openings may collapse over time, produc-
ing fewer openings (Bennet-Clarke 1970a & Ka-
vanagh and Young 1989).
Variation between temporally segregated
songs of chirping and trilling mole crickets may
DeGraaf et al.: Stridulation of Gryllotalpa africana in South Africa
Fig. 1. Ventral view of right male tegmen, showing stridulatory area. A = Stridulatory area, B = File (pars
stridens) and C = Scraper (plectrum).
be caused by environmental factor dependence.
Chirp rate and syllable or pulse repetition rate in
crickets and mole crickets increase linearly with
soil temperature over an intermediate tempera-
ture range (Bennet-Clark 1970a; Bennet-Clarke
1989; Kavanagh & Young 1989; Doherty & Callos
1991; Ciceran et al. 1994; Hill 1998, 2000). Inter
syllable interval is usually negatively correlated
with temperature in the Gryllotalpinae and car-
rier frequency appears to be temperature inde-
pendent in mole crickets (Bennet-Clark 1989). In
the Oecanthinae (Gryllidae), however, the carrier
frequency is positively correlated to temperature,
but with a smaller slope than for syllable rate
(Bennet-Clarke 1989). Walker (1962) also re-
ported carrier frequency to be a regression func-
tion of air temperature at low and moderate tem-
peratures for crickets in three genera and three
subfamilies. Another potential factor contributing
to variation may be physiological, such as size,
condition etc. In the Gryllidae, song structure
does not, however, appear to vary with male mass
or age (Souroukis et al. 1992; Ciceran et al. 1994).
In trilling Gryllotalpa, the song differences ap-
pear to be of fundamental frequency (Bennet-
Clark 1970a), while in gryllids, the interval be-
tween syllables may be more important (Walker
1962). Male song characteristics in mole crickets
are species specific (Bennet-Clark 1970a; Bennet-
Clark 1970b; Otte & Alexander 1983; Nickle &
Castner 1984; Kavanagh & Young 1989; Walker &
Figg 1990; Broza et al. 1998) and provide a key to
determine the validity of reports of G. africana
Sound pressure levels measured just beyond
the near field (15-20 cm in line with the burrow,
re. 20 pPa) may vary from 65 to 97 dB between
trilling mole cricket species, with highest in-
traspecific sound pressure level variation of 67 to
91 dB (Ulagaraj 1976; Forrest 1983; Bennet-
Clarke 1987; Kavanagh & Young 1989; Walker &
Forrest 1989). Song intensity of trilling species is
positively correlated to male size and usually to
temperature, rainfall, and soil moisture (Bennet-
Clarke 1970a; Ulagaraj 1976; Forrest 1980; For-
rest 1983; Forrest 1991).
Some song characteristics reported for the Af-
rican mole cricket include a phonotaxis study by
Kim (1993) in Hwaseong-gun, Kyonggi-do Korea,
who found intensities of calling songs vary be-
tween 77 and 80 dB at 150 mm above the open-
ings of calling chambers. The study of Kim (1993)
probably does not refer to the "true" G. africana.
Other song characters ofG. africana are based on
four recordings (Townsend 1983) and vary be-
tween reports (Nickle & Castner 1984). Calling
Florida Entomologist 88(3)
Fig. 2. Ventral view of male tegmen, showing stridulatory teeth arrangement on the file or pars stridens of G.
song intensities of G. africana from Africa have
not been measured.
MATERIALS AND METHODS
Field recordings (n = 20) of the calling song of
G. africana males chosen at random but not over-
lapping were made in a kikuyu grass area of ap-
proximately 300 m2 between and surrounding the
putting green and green no. 18 at the Pretoria
Country Club from March 2002 to April 2002. Soil
temperatures were measured at a vertical depth
of 100 mm in the soil profile immediately after
recordings were made. Recordings were made
between 19h30 and 21h15, local time (GMT + 2
hours). Due to the relative homogeneity including
irrigation program, turf grass and soil of the ex-
perimental area and relatively short temporal
measurement period, soil moisture was assumed
to be constant. During October 2002 and Novem-
ber 2002, 20 stridulating males were recorded ac-
cording to a similar methodology, but at a nearby
site comprising a kikuyu grass area (300 m2) be-
tween and surrounding the chipping and bowling
green at Pretoria Country Club with a similar ir-
rigation program than the previous site. Record-
ings between and within the two periods were as-
sumed to be of different males, as no recording
sites overlapped. The calls were recorded with a
Nomad DAP-3201 digital recorder (Creative
Technology Ltd.), with the self-contained micro-
phone held 50 mm from the burrow opening, lon-
gitudinal to the long axis of the burrow. Recording
distance was within the near field, or distance
covered by one wavelength at the carrier fre-
quency of this call (s/2300 cycles x 343 m/s at 20C
= 149.13 mm) (Hill 2000).
All the recordings were analyzed with the com-
puter software program "Canary" V1.2.4 (Cornell
Laboratory of Ornithology 1998). A power spec-
trum (Fig. 3) and oscillogram (Fig. 4) were used to
measure three different call characteristics for
nine syllables (three successive syllables ran-
domly selected at the beginning, middle and end
of each recording, respectively) per recording:
Carrier frequency (Fig. 3), syllable duration (Fig.
4) and syllable period (Fig. 4). The inter syllable
interval (syllable period syllable duration),
mean syllable repetition rate (inverse of syllable
period), and duty cycle ((syllable duration/syllable
period) x 100) were calculated from the measured
The sound pressure level of 20 different calling
males which were all assumed to be G. africana
was also measured according to the methodology
for each recording, but at a distance of 200 mm be-
yond the near field from the burrow opening and
longitudinal to the long axis on a night between
DeGraaf et al.: Stridulation of Gryllotalpa africana in South Africa
0 1 2 3 4 5 6
annual calibration. The sound level meter was
used in LAeq mode, which records the time-
weighted average of a series of fast root mean
square (RMS) recordings (time constant 125 ms).
This gave the A-weighted sound pressure level
(dB A scale) (at re. 20 pPa) that was the equiva-
lent continuous level as the fluctuating signal be-
ing recorded. A period of approximately 20 s was
sufficient to provide a stable level for G. africana.
7 8 9 10
Fig. 3. The power spectrum of a field recorded G. af-
ricana call (up to 10 kHz), indicating a carrier frequency
of approximately 2.3 kHz.
20h00 and 20h30, local time (GMT + 2 hours) in
late November 2002. A kikuyu grass area of
approximately 300 m2 including and surrounding
of the first tee at Pretoria Country Club was used
for measurements. The area sampled had an irri-
gation program similar to the previous recording
sites. Sound level measurements were made with
a precision integrating sound level meter (Rion
Type NL-14), calibrated by a Rion Type NC-73
sound level calibrator. The equipment was within
The relationship of call characteristics mea-
sured in autumn (March/April) and spring (Octo-
ber/November) of 2002 with soil temperature at
100 mm in the soil profile is represented in Table
1. Soil temperature ranged from 20.7C to 24.8C
(23.2 1.24C, mean SD) in March/April 2002
recordings and 22.3C to 26.8C (23.5 1.16C,
mean SD) in October/November 2002 record-
ings. The data of all the sound characters except
syllable period fitted a normal distribution (Kol-
mogorov-Smirnov test, P > 0.05, "Statistica" Ver-
sion: 5, Statsoft, Inc., 1995) without transforma-
tion (Sokal & Rohlf 1997). The syllable period
data for both sampling periods was not signifi-
cantly different from a normal distribution only
S I ISDI I I I
I..-- 15 MS -4I
0 6 90 240 270 300 45 40 510
0. 306090'' 240270300 450480510
l Time (ms)
Fig. 4. Oscillogram of a field recorded G. africana trilling call over 510 ms. The thickened red lines indicate an
approximate eight times shorter temporal scale with the different measurements made. SD = Syllable duration, ISI
= Inter syllable interval and SP = Syllable period.
Florida Entomologist 88(3)
after logarithmic transformation (Sokal & Rohlf
1997) (Kolmogorov-Smirnov test, P > 0.05, "Sta-
tistica" Version: 5, Statsoft, Inc., 1995).
The multiple regression parametric test ("Sta-
tistica" Version: 5, Statsoft, Inc., 1995) showed a
highly significant relationship of syllable period,
inter syllable interval, syllable repetition rate
and duty cycle with soil temperature for both re-
cording periods (Table 1). Carrier frequency vari-
ation of G. africana males was not significantly
related to the tested temperature range. The re-
sults show a negative relationship between sylla-
ble period and soil temperature for both sampling
periods after data were transformed to linear
scale before presentation, with soil temperature
constantly explaining more than 80% of the vari-
ation in the former. The rate of decline in the syl-
lable period was slightly higher in the spring re-
cordings. The syllable duration had no significant
relationship with soil temperature. Inter syllable
interval was negatively correlated with soil tem-
perature, with R2 values under 0.50. The rate of
decline, however, was slightly higher for the
spring recordings relative to those in autumn.
The syllable repetition rate was positively related
to soil temperature during spring and autumn. In
the latter season recordings, the rate of syllable
increase was lower than during the spring record-
ings over a similar range of soil temperatures.
Soil temperature was a relatively good predictor
(R2 approximately 0.80) of syllable repetition rate
in both recording periods. The duty cycle in-
creased significantly with soil temperature, but
with relatively low R2 values, during both record-
ing periods, respectively. The rate of increase with
soil temperature was higher in spring relative to
autumn values. Slopes of regression lines should
be compared with caution, as they are dependant
on the measurement scale.
The values for the different measured and calcu-
lated sound characteristics at variable soil tempera-
tures and differences between autumn 2002 and
spring 2002 recordings with soil temperature con-
stant are summarized in Table 2. Only syllable rep-
etition rate needed to be arcsine-transformed (Sokal
& Rohlf 1997) for all the dependant variables to be
normally distributed (Kolmogorov-Smirnov test, P
> 0.05, "Statistica" Version: 5, Statsoft, Inc., 1995).A
multi analysis of variance (MANCOVA, parametric
test, Sokal & Rohlf 1997, "Statistica" Version: 5,
Statsoft, Inc., 1995), with soil temperature entered
as a covariate, was used to determine significant
song character differences between the two tempo-
rally segregated field recordings.
The results showed that the carrier frequency
of G. africana males was constant between au-
tumn and spring at approximately 2340 cycles per
second (Table 2). The power spectrum (Fig. 3, rep-
resentative for most songs) graphically represents
the carrier frequency and shows a low frequency
component and no clear harmonics for G. africana
males. The spectrogram (Fig. 5) of a general sound
recording shows the sound structure during and
between syllables. Fig. 5 shows the low frequency
observed in the power spectrum was also present
between syllables and therefore when no mole
cricket sound was produced (Fig. 5).
Syllable duration did not vary significantly be-
tween seasons and was usually just longer than
nine milli-seconds (Table 2). The syllable period,
TABLE 1. RELATIONSHIP BETWEEN MALE G. AFRICANA SONG CHARACTERS AND SOIL TEMPERATURES OF 23.2 1.24C
(MEAN SD) AND 23.5 1.16 C (MEAN SD) AT A VERTICAL DEPTH OF 100 MM PROFILE IN THE SOIL FOR
MARCH/APRIL 2002 (RECORDING 1) AND OCTOBER/NOVEMBER 2002 (RECORDING 2), RESPECTIVELY, AT PRE-
TORIA COUNTRY CLUB.
Data Regression variable
Song character Recording Slope Intercept R2 F P
Carrier frequency (kHz) 1 0.001 2.310 0.0004 0.019 0.891
2 -0.009 2.569 0.0228 0.931 0.340
Syllable period (ms) 12 -1.067 63.826 0.8092 195.174 0.0000001
22 -1.127 41.089 0.8139 174.960 0.0000001
Syllable duration (ms) 1 -0.079 11.104 0.0118 0.552 0.461
2 -0.198 13.702 0.0412 1.7205 0.197
Inter syllable interval (ms) 12 -0.874 25.395 0.4521 37.968 0.0000001
22 -0.929 27.387 0.3913 25.712 0.000009
Syllable repetition rate (Syllable ms 1) 12 0.004 -0.031 0.7926 175.781 0.0000001
22 0.007 -0.084 0.8406 210.990 0.0000001
Duty cycle (%) 12 3.485 -15.920 0.2755 17.495 0.000128
22 4.245 -37.130 0.2581 13.915 0.000593
'P < 0.05.
'P < 0.001.
DeGraaf et al.: Stridulation of Gryllotalpa africana in South Africa
TABLE 2. VALUES OF MALE G. AFRICANA SONG CHARACTERISTICS RECORDED AT PRETORIA COUNTRY CLUB AT SOIL TEM-
PERATURES OF 23.2 1.24C (MEAN SD) AND 23.5 1.16C (MEAN SD) AT A VERTICAL DEPTH OF 100 MM
IN THE SOIL FOR MARCH/APRIL 2002 (RECORDING 1) AND OCTOBER/NOVEMBER 2002 (RECORDING 2), RESPEC-
TIVELY. SIGNIFICANT DIFFERENCES BETWEEN RECORDINGS WITH SOIL TEMPERATURES CONSTANT ARE SHOWN.
Data Value MANCOVA variable
Song character (Unit) Recording Range Mean SD F P
Carrier frequency (kHz) 1 2.198-2.476 2.34 0.067
2 2.161-2.477 2.34 0.075
Syllable period (ms)2 1 12.031-17.061 14.3 1.09
2 10.455-17.221 14.6 1.45
Syllable duration (ms) 1 7.340-10.959 9.3 + 0.91
2 7.372-12.078 9.1 1.13
Inter syllable interval (ms)1 1 2.979-9.607 5.1 1.62
2 1.912-7.779 5.6 1.72
Syllable repetition rate (Syllable ms-1)2 1 0.059-0.083 0.070 0.0061
2 0.058-0.096 0.069 0.0082
Duty cycle (%)' 1 43.31-78.15 64.9 + 8.25
2 48.66-81.72 62.44 0.097 7.276 0.00845
P < 0.05.
P < 0.001.
inter syllable interval, syllable repetition rate
and duty cycle were significantly different, with
soil temperature constant, between the autumn
and spring recordings. The syllable period and in-
ter syllable interval were significantly longer and
the syllable repetition rate and duty cycle signifi-
cantly shorter in spring than in autumn, respec-
During the spring recordings, one individual
was recorded at a soil temperature of 21.9C with
the following sound characters (mean SD): car-
rier frequency: 2.638 0.0068 kHz, syllable pe-
riod: 17.89 0.085 ms, syllable duration: 7.9 +
0.30 ms. Inter syllable interval, syllable repeti-
tion rate, and duty cycle was calculated as (mean
+ SD) 10.00 + 0.217 ms, 0.0559 0.00026 sylla-
bles/ms and 44.1 1.47%, respectively.
The sound pressure levels (re. 20 pPa) ofG. af-
ricana varied from 77.6 to 89.8 dB at 200 mm
from the burrow. The ambient and soil tempera-
ture (average of five measurements) at the onset
of the experiment were 21.5 0.30C and 23.24 +
0.112C, respectively. At the end of the experi-
ment, ambient and soil temperatures (average of
five measurements) were 21.15 0.263C and
Gryllotalpa africana males constructed acous-
tical burrows with one or two horn-shaped open-
ings observed. Two openings may initially have
been constructed, but one opening may have col-
lapsed over time. Male African mole crickets
started calling just after sunset and, especially
during the warm summer months, called until
midnight, attracting flying conspecifics and even
walking nymphs. Calling activity was generally
limited to soil temperatures exceeding 14C dur-
ing late August to late May, when conspecifics
flew. Initial calling was characterized by a dis-
tinctive warm up period. The sound matured from
the initial slow erratic trill to a constant trilling
call. Some male callers exploited microclimatical
conditions near brick walls and concrete slabs.
These spatial orientations, which artificially in-
creased soil temperatures, were especially uti-
lized during times of relatively low soil tempera-
tures. Males called singularly, but were usually
observed in calling groups as individuals sepa-
rated by a few meters during stridulation.
Males randomly selected from the field in
spring and autumn and acclimatized for one week
at L:D 12 h:12 h, which was a relative shorter
daily light cycle, and 28 1C, did not call in the
laboratory, suggesting photoperiod as a factor
contributing to stridulation activity. This observa-
tion may have been biased by the fact that mole
crickets were kept in containers, which have been
found to influence their behavior (Walker 1979 &
Songs of G. africana males were produced at
sound pressure levels of 77.6 to 89.8 dB and charac-
terized by a carrier frequency of approximately 2.34
kHz with some variation between males. The latter
did not vary significantly between autumn and
spring and with soil temperature. If the song had a
low frequency component, it could not be distin-
Florida Entomologist 88(3)
Fig. 5. The spectrogram presenting two complete syllables of a field recorded G. africana call (up to approxi-
mately 2.7 kHz).
guished from background noise in the current
study. Harmonics, which were generally not clearly
visible, are usually at a relatively low level in the
family (Bennet-Clark 1987). African mole cricket
males usually stopped calling, usually less than one
minute, when the burrow opening was approached
within a one meter radius (personal observation),
and the mole cricket was deemed to show some seis-
mic sensitivity. Males in full song were usually less
sensitive. Trilling species are generally not very
sensitive to substrate vibrations (Bennet-Clarke
1970a; Forrest 1991), although Bennet-Clark
(1970a) reported G. gryllotalpa to be highly sensi-
tive. Sensitivity may be related to sound pressure
level, which may saturate mechano-receptors at
high intensities (Bennet-Clark 1970a).
The syllable duration of male G. africana calls
did not vary significantly between autumn and
spring nor with soil temperature, but did show
some variation between males. Syllable period
was negatively related with soil temperature and
varied significantly with soil temperature con-
stant between autumn and spring. Additional
sound characters calculated from the syllable pe-
riod or syllable period and syllable duration, re-
flected their relationships with the tendencies of
the measured variables.
Townsend (1983) reported a mean syllable rep-
etition rate of 49.1-57.8 per second and a mean
carrier frequency of 2.1-2.4 kHz for the calling
song of G. africana based on four recordings. No
temperature values or other variables were anno-
tated during these recordings. The calling song
frequency of G. africana reported from Hawaii is
3.3 kHz, with a syllable repetition rate of 56 per
second (Nickle & Castner 1984). Although sylla-
ble repetition rates were similar between the two
reports, it is not comparable without any temper-
ature information. The carrier frequency values
of the present study correspond with that re-
ported by Townsend (1983). Differences in calling
song carrier frequency have been used to distin-
guish between Gryllotalpa species (Bennet-Clark
1970b; Nevo & Blondheim 1972). These stridula-
tory character differences support reports that
the Hawaiian species is in fact not G. africana.
Frank et al. (1998) also stated that the immigrant
mole cricket to Hawaii was misidentified as Gryl-
lotalpa africana. According to Frank et al. (1998),
the species occurring in Hawaii is G. orientalis, a
species originating from Asia, not Africa.
It appears that a mole cricket species, other
than G. africana, also inhabited Pretoria Country
Club in spring 2002. The distinction of the species
was in its higher carrier frequency values. Gryllo-
talpa robusta has a carrier frequency of 1.6 kHz,
based on one recording, and G. parva has a carrier
frequency of 2.9-3.3 kHz, based on two recordings
DeGraaf et al.: Stridulation of Gryllotalpa africana in South Africa
(Townsend 1983). Hence, the carrier frequency of
the unidentified species does not correspond to
known values of species occurring in South Africa.
Thanks to K. Drews, South African Bureau of Stan-
dards (SABS), who assisted in sound pressure level mea-
surements; J. W. H. Ferguson, University of Pretoria,
who assisted with the interpretation of sound results,
statistical analysis, and allowed use of his laboratory; P.
Kryger, University of Pretoria; M. Ferreira, University
of Pretoria; and L. Verburgt, University of Pretoria, who
assisted with some technical aspects.
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Florida Entomologist 88(3)
DEVELOPMENT OF THE MOST EFFECTIVE TRAP TO MONITOR
THE PRESENCE OF THE CACTUS MOTH CACTOBLASTIS CACTORUM
STEPHANIE BLOEM1, STEPHEN D. HIGHT2, JAMES E. CARPENTER3 AND KENNETH A. BLOEM4
'Center for Biological Control, Florida A&M University, Tallahassee, FL 32308
2USDA-ARS-CMAVE at Florida A&M University, Center for Biological Control, Tallahassee, FL 32308
3USDA-ARS-CPMRU, Tifton, GA 31794
4USDA-APHIS-PPQ-CPHST, at Florida A&M University, Center for Biological Control, Tallahassee, FL 32307
Various trap specifications were evaluated to identify the most effective trap for capturing
wild male Cactoblastis cactorum (Berg). All traps were baited with virgin female C. cac-
torum and, except for the first comparison of trap type, a standard wing trap was used in all
experiments. Although wing traps captured more males than did the other trap types (delta
or bucket), the differences were not significant. However, significantly higher numbers of
males were captured in wing traps placed 2 m above ground than traps at 1 m or 0.5 m, and
wing traps baited with four virgin females caught significantly more males than wing traps
baited with a single female. Differences in number of males captured by young and old fe-
males were not significant, but more than twice as many males were captured in traps
baited with one-day-old females than traps baited with four day old females. In addition,
there were no significant differences in number of males caught in unpainted, white, wing
traps and wing traps painted one of eight different colors (flat white, black, dark green, flu-
orescent green, yellow, fluorescent yellow, orange, or blue), although, more males were cap-
tured in the unpainted wing traps. The results presented here suggest that the best trap
currently available to monitor C. cactorum is a standard (unpainted) wing trap, placed at a
height of 2.0 m aboveground, and baited with four newly emerged females.
Key Words: population monitoring, trapping, trap design, invasive species, opuntia
Se evaluaron various tipos de trampas con el objetivo de identificar la tampa mas efectiva
para la capture de machos silvestres de Cactoblastis cactorum (Berg). Todas las trampas fu-
eron evaluadas utilizando hembras virgenes de C. cactorum como cebo atractivo. Todos los
ensayos a excepci6n del primero (donde se evaluaron tipos distintos de trampas) emplearon
la trampa "wing". A pesar de que las trampas wing capturaron un mayor numero de machos
que ninguna de las otras trampas evaluadas (delta o cubeta) las diferencias en capture de
machos no fueron estadisticamente significativas. Tambien, se capturaron un numero mayor
de machos en las trampas wing colocadas a una altura de 2 metros que en trampas colocadas
a una altura de 1 metro o 0.5 metros, y las trampas con 4 hembras como cebo atractivo cap-
turaron un mayor numero de machos que las trampas con solamente una hembra. Las difer-
encias en el numero de machos capturados en trampas con hembras de 1 dia de edad en
comparaci6n con trampas con hembras de 4 dias de edad no fueron estadisiticamente signif-
icativas, pero se capturaron mas del double del numero de machos en trampas con hembras
jovenes. No se encontraron diferencias estadiaticas en el numero de machos capturados en
trampas wing de color standard o en trampas wing de ocho colors diferentes (blanco mate,
negro, verde oscuro, verde fluorescent, amarillo, amarillo fluorescent, naranja o azul),
aunque se capturaron mas machos en las trampas wing de color standard (blanco). Nues-
tros resultados sugieren que la mejor trampa que se tiene al moment para uso en el moni-
toreo de C. cactorum es la trampa wing de color standard colocada a una altura de 2.0
metros y con 4 hembras jovenes como cebo atractivo.
Translation provided by the authors.
The cactus moth, Cactoblastis cactorum (Berg), rapid spread along the Atlantic and Gulf Coasts
was accidentally introduced into Florida in 1989 has heightened concerns about its imminent im-
(Habeck & Bennett 1990; Dickel 1991), and its pact on native Opuntia cacti in the southern
Bloem et al.: Trap development for Cactoblastis cactorum
United States and Mexico (Johnson & Stiling 1998;
Zimmermann et al. 2001). Recent publications sug-
gest that C. cactorum is dispersing over a distance
of about 50-75 km per year in North America (Stil-
ing 2002; based on data reported in Johnson & Stil-
ing 1998; and Hight et al. 2002). Recent data sug-
gest that the dispersal rate for C. cactorum is closer
to 160 km per year (S. D. Hight, unpublished data).
Establishment of the cactus moth in the south-
western United States and Mexico would likely
have serious detrimental effects on the landscape,
biodiversity, and stability of native desert ecosys-
tems, and on the vegetable, fruit and forage Opun-
tia industries in these areas (Sober6n et al. 2001;
Zimmermann et al. 2001, 2004).
The ability to quickly detect new pest infesta-
tions, accurately delineate their size and bound-
aries, and assess the pest's seasonal population
trends is of critical importance in the successful ap-
plication of any pest control strategy. The objective
of this research is to develop an optimum monitor-
ing system for adult C. cactorum by evaluating var-
ious trapping parameters. An efficient adult trap-
ping system is necessary for the development and
application of the Sterile Insect Technique (SIT), a
control strategy that may be used to study and
manage the spread ofC. cactorum (Carpenter et al.
2001a, b) in North America. SIT is a species-spe-
cific pest control tactic that could be used to estab-
lish a barrier to prevent further geographic expan-
sion of C. cactorum into the western states and
Mexico, eradicate new or localized infestations
when and where they occur, and/or protect environ-
mentally sensitive areas from attack by the cactus
moth. Unfortunately, although female C. cactorum
produce a pheromone that attracts males (Hight et
al. 2003), no synthetic pheromone is currently
available to monitor populations ofC. cactorum.
Hight et al. (2002) were the first to report on
the use of sticky traps baited with virgin females
to corroborate field damage and better under-
stand the current distribution of C. cactorum in
Florida and Georgia. More recently, Bloem et al.
(2003) showed that sticky traps could be baited
with reproductively sterilized females (treated
with 200 Gy of gamma radiation) to monitor pop-
ulations beyond the leading edge without the con-
cern of accidentally establishing a breeding popu-
lation if females escaped into the environment.
The objective of our study was to conduct further
field evaluations to ensure that the best monitor-
ing trap is being used to detect the presence of
C. cactorum and accurately assess its geographi-
cal expansion. In this paper we report the results
of a series of field experiments conducted during
2003-2004 to evaluate trap types, trap placement
heights, and trap colors, as well as the age and
number of females for their ability to capture wild
male cactus moths. Our results are discussed in
the context of developing an area-wide control
strategy for C. cactorum in North America.
MATERIALS AND METHODS
Cactoblastis cactorum used in these experi-
ments came from a laboratory colony kept at the
USDA-ARS Crop Protection and Management
Research Unit in Tifton, GA. Insects were reared
on cladodes of Opuntia stricta (Haworth)
Haworth inside rectangular plastic boxes and
maintained at 26C + 1C, a 14:10 (L:D) photo-
period and 70% relative humidity as described in
Carpenter et al. (2001b). Cocoons with pupae
were collected every two to three days, and pupae
were extracted from the cocoons and sorted by
gender. Female pupae were placed in a screened
cage (30.5 by 30.5 by 30.5 cm) and allowed to
emerge at the above mentioned conditions.
Emerged adult females were placed inside modi-
fied translucent, plastic, film canisters (35 mm,
with two 2 by 2-cm screen windows, perforated
tops and Velcro fasteners to attach the canister to
the top of an insect trap) with a small (1 by 1 cm)
piece of 0. stricta. A moistened cotton dental wick
was placed through the perforated canister top to
provide moisture to the caged females. Canisters
with females were transported to the field in a
small cooler and used to bait all treatments in
these experiments. All experiments were con-
ducted to coincide with peak flight activity of
adult moths (Zimmermann et al. 2004).
Evaluation of Trap Type
Three commercially available insect traps-
Pherocon 1-C Wing trap, Pherocon-VI Delta trap
(both Trece Incorporated, Salinas, CA), and Uni-
versal moth trap (Unitrap, Great Lakes IPM,
Vestaburg, MI)-were evaluated for their effec-
tiveness at capturing wild C. cactorum males. Ex-
periments were conducted during the summer of
2003 at coastal locations in Florida and Georgia.
In Florida, three replicates (three traps, one of
each type = one replicate) were placed at each of
two different coastal locations: Alligator Point (N
2954', W 8423') and St. Marks National Wildlife
Refuge (NWR) (N 3004', W 8410'). Abundant
naturally occurring patches of native 0. strict
that were heavily damaged by C. cactorum were
present at both sites. Infested 0. ficus-indica (L.)
Miller was also common at Alligator Point as a
planted and naturalized species. Hollow metal
stakes were placed in the ground in groups of
three in the vicinity of infested plants. Within
each replicate, metal stakes were separated by no
less than 3.0 m from one another and buried so
that the top of each stake was at a height of
approximately 1.0 m. Each trap was mounted to
the top of a stake. Distance between replicates at
each location was no less than 50 m. A single vir-
gin female (<48 h post emergence) was used to
Florida Entomologist 88(3)
bait each trap type. The experiment was initiated
on 10 July 2003 and ended on 31 July 2003. Traps
were checked every 72 h at which time the num-
ber of male C. cactorum caught per trap was re-
corded, traps were re-baited with new virgin fe-
males, and the position of each trap was changed
in a clockwise manner.
In Georgia, six additional replicates were
placed in the proximity of a salt marsh estuary at
the southern banks of the Brunswick River in
Glynn County, Georgia, west of US Highway 17
(N 31005', W 8131'). Within the estuary, a large
area of naturally occurring patches of 0. strict
plants was chosen. One replicate was assigned to
each of six patches with cactus plants between
0.5-1.5 m in height. Traps were arranged and ser-
viced as previously described. The experiment
was conducted from 2-26 May 2003.
Evaluation of Trap Height
Based on the results from the evaluation of
trap type, Pherocon 1-C Wing traps were used in
all subsequent experiments. Traps placed at
three different heights above ground (2.0, 1.0, and
0.5 m) and were evaluated for their effectiveness
at capturing wild C. cactorum males. As above,
experiments were conducted during the summer
of 2003 at coastal locations in Florida and Geor-
gia. In Florida, six replicates (three traps, one
trap at each height = one replicate) were placed at
St. Marks NWR. Hollow metal stakes were placed
in the ground in groups of three close to infested
0. stricta cactus plants and buried so that the top
of each stake was at a height of approximately 1.0
m. Plastic (PVC) poles of a slightly smaller diam-
eter than the hollow metal stakes were cut to the
appropriate height and slipped inside each metal
stake. A Pherocon 1-C trap was mounted to the
top of each plastic pole. Within each replicate,
traps were separated by no less than 3.0 m from
one another, and distance between replicates at
each location was 25-75 m. A single virgin female
(<48 h post emergence) was used to bait each
trap. The experiment was initiated on 15 July
2003 and ended on 2 August 2003. Traps were
checked every 72 h at which time the number of
male C. cactorum caught at each height was re-
corded, the positions within replicates of PVC
poles with traps were rotated in a clockwise man-
ner, and the traps were re-baited with new virgin
females. Six additional replicates were placed at
the salt marsh estuary location in Glynn Co.,
Georgia described above. Six patches with cactus
plants between 0.5-1.5 m in height were selected
and three hollow metal stakes were placed in the
ground within the patches separated by no less
than 3.0 m from one another at a height of ap-
proximately 1.0 m. As above, one trap was placed
on a pole and one pole of each height was placed
per patch and checked, serviced, and rotated ev-
ery 72 h. The experiment was initiated on 29 July
2003 and ended on 28 August 2003.
Evaluation of One Versus Four Females
Pherocon 1-C Wing traps were baited with ei-
ther one or four virgin female C. cactorum (<48 h
post emergence) and evaluated for their effective-
ness at capturing wild C. cactorum males. The ex-
periment was conducted at St. Marks NWR be-
tween 24 July 2003 and 5 August 2003. Seven
replicates (two traps, baited either with one or
four females = one replicate) were completed. Hol-
low metal stakes were placed in groups of two in
close proximity to infested 0. stricta. Poles were
separated by no less than 3.0 m from one another
and buried so that the top of each stake was at a
height of approximately 1.0 m. A Pherocon 1-C
Wing trap was placed on the top of each stake.
Distance between replicates at each location was
no less than 50 m. Traps were checked every 72 h
and the number of male C. cactorum caught per
trap was recorded, traps re-baited with new vir-
gin females, and trap positions rotated.
Evaluation of Females of Different Ages
Pherocon 1-C Wing traps were baited with sin-
gle virgin C. cactorum females that were either 24
h or 120 h post emergence and evaluated for their
effectiveness at capturing wild males. Experi-
ments were conducted at St. Marks NWR from 16-
23 April 2003 and at Alligator Point from 28 July-
1 August 2003. A total of eleven replicates (each
replicate consisted of two traps, one baited with a
24-h-old female and one with a 120-h-old female)
were completed: six replicates at St. Marks NWR
and five at Alligator Point. As above, hollow metal
stakes with a trap on top were placed in the
ground in groups of two in the vicinity of infested
0. stricta and 0. ficus-indica plants. Within each
replicate, metal stakes were separated by no less
than 3.0 m and buried so that the top of each stake
was at a height of approximately 1.0 m. Distance
between replicates at each location was no less
than 65 m. Traps were checked at 24 h intervals at
which time the number of male C. cactorum
caught per trap was recorded, traps re-baited with
new females, and trap positions rotated.
Evaluation of Trap Color
The outside top and bottom of Pherocon 1-C
Wing traps were either left unpainted (controls)
or painted with two coats of the following com-
mercially available paints: Gloss White (#7792),
Gloss Black (#7779), Dark Hunter Green (#7733),
Fluorescent Green Marking (#207464), Sunburst
Yellow (#7747), Fluorescent Yellow (#1942) (Rust-
Oleum Corp., Vernon Hills, IL), Pumpkin Orange
Gloss (#2411), and True Blue Gloss (#1910) (Kry-
Bloem et al.: Trap development for Cactoblastis cactorum
lon Products Group, Cleveland, OH). Color selec-
tions were based on reported evaluations of trap
colors influencing the attraction of various Lepi-
doptera (Hendricks et al. 1972; Mitchell et al.
1989; Pair et al. 1989; Hendrix & Showers 1990;
Lopez 1998; Meagher 2001). Trap surfaces were
given two coats of Plastic Primer (Rust-Oleum
Corp., Vernon Hills, IL) before painting with ex-
perimental colors to increase coverage and adher-
ence of the paint. Experiments were conducted
from 14-26 April 2004 on a dike located at St.
Marks NWR. Hollow metal stakes were placed in
the ground along the dike, separated by no less
than 4 m from one another, at a height of approx-
imately 1.5 m, and in the proximity of infested
O. strict. Two traps of each color plus two un-
painted traps (18 traps total) were deployed ran-
domly in two separate groups (= replicates) on 14
April 2004. All trap bodies were oriented with
trap openings in the direction of the prevailing
wind. Traps were first baited with two virgin fe-
males (<48 h post emergence) on 15 April 2004.
Thereafter, traps were serviced, cotton wicks re-
wetted, and trap location re-randomized every
24 h until 26 April 2004. Females were changed
every 48 h. At each trap servicing we noted the
status of the female (alive or dead) and the num-
ber of male C. cactorum captured per trap.
The spectral reflectance of the painted traps,
the standard unpainted trap, and of healthy
0. strica pads (<1 year-old) was measured with a
FieldSpec Handheld spectroradiometer (Analyt-
ical Spectral Devices, Inc., Boulder, CO-spectral
range of 325-1075 nm). Two readings were taken,
one before the traps were placed in the field (15
April 2004 at 1400 h) and one after the experi-
ment was completed (25 May 2004 at 1415 h).
Cactus pad reflectance also was measured twice
at the same times as the traps.
All statistical analyses were performed with
PROC ANOVA (SAS Institute 1989). The effect of
trap type (Wing trap, Delta trap, and Universal
moth trap) on the number of C. cactorum males
captured was examined using analysis of vari-
ance with trapping date, field site (Brunswick
River, Alligator Point and St. Marks), replication,
and trap type as sources of variation. Because a
significant three-way interaction was found be-
tween date, field site, and trap type, the number
of males captured was sorted by field site and an-
alyzed with trap type as the main effect, replica-
tion as a blocking effect, date as a superblock, and
interaction between the trap type and trapping
date as an error term. The effect of trap height on
the number of male cactus moths captured in
Pherocon 1-C Wing traps was analyzed with trap
height (2.0, 1.0, and 0.5 m) as the main effect, rep-
lication as a blocking effect, field site as a super-
block, and interaction between the trap height
and field site as an error term. Because no inter-
action was detected, the data were pooled and
ANOVA was conducted by orthogonal contrasts to
compare the response of males to the different
trap heights. The number of C. cactorum males
captured in traps baited with one or four virgin fe-
males was analyzed with the number of females
as the main effect, replication as a blocking effect,
date as a superblock, and interaction between the
number of females and replication as an error
term. The number of C. cactorum males captured
in traps baited with females that were either 24
or 120 h post emergence was analyzed with trap-
ping date, field site (St. Marks and Alligator
Point), and female age as sources of variation.
Finally, the effect of trap color on the number of
C. cactorum males captured in traps was ana-
lyzed with trap color as the main effect, replica-
tion as a blocking effect, date as a superblock, and
interaction between the trap color and date as an
error term. All data meet the assumptions for the
ANOVA model and were not transformed. Esti-
mates of central tendencies are reported as mean
+ standard deviation (SD).
All three trap types (Wing, Delta, and Univer-
sal) were successful in capturing males of C. cac-
torum (Table 1). Although the highest mean num-
ber of males was captured in the Pherocon 1-C
TABLE 1. INFLUENCE OF TRAP TYPE ON THE NUMBER OF MALE CACTOBLASTIS CACTORUM CAPTURED AT THREE DIF-
FERENT SITES. EACH TRAP WAS BAITED WITH A SINGLE VIRGIN C. CACTORUM FEMALE.
Mean ( SD) number of males captured at each trap site**
Trap type Alligator Point, FL St. Marks NWR, FL Brunswick River, GA Pooled means
Universal moth trap 0.83 1.4 1.67 3.2 0.53 1.2 0.89 1.9
Delta trap 0.39 0.9 1.33 1.7 0.92 1.7 0.92 1.6
Wing trap 1.11 1.6 1.06 2.5 1.33 2.4 1.12 2.2
*Differences among means are not significant, P > 0.05 (PROC ANOVA, SAS Institute 1989).
Florida Entomologist 88(3)
Wing traps tested at Alligator Point, FL and
Brunswick River, GA, this type of trap recorded
the lowest mean number of captures at St. Marks
NWR, FL. Overall, Pherocon 1-C Wing traps cap-
tured more males than did the other trap types.
However, differences among the means for each
trap type were not significant overall or at any of
the three field sites. The height at which Phero-
con 1-C wing traps were placed above ground (F =
2.73; df = 2, 272; P < 0.0352) influenced the num-
ber of male C. cactorum captured per trap (Table
2). The number of males captured at a height of
2 m was almost twice as many as those captured
at 0.5 m. When Pherocon 1-C Wing traps were
baited with one or four virgin females, the num-
ber of cactus moth males captured was higher
(F = 8.18; df = 1, 22; P < 0.0091) in traps baited
with four females (1.88 2.4) than when baited
with single females (0.62 1.1). Furthermore, the
age of the female (24 or 120 h post emergence) in-
fluenced the number of males captured in Phero-
con 1-C Wing traps. Traps baited with a young fe-
male (0.84 1.63) captured more than twice the
number of males than traps baited with an older
female (0.36 0.90). Although the difference be-
tween means was not significant, analysis of the
data suggests that female age influenced the
number of males captured (F = 3.22; df = 1, 72;
P < 0.0768).
Finally, Pherocon 1-C Wing traps left un-
painted (controls) or painted white, black, dark
green, fluorescent green, yellow, fluorescent yel-
low, orange, or blue, and baited with two females
were all successful at capturing C. cactorum
males. Overall, most males were captured in the
unpainted Pherocon 1-C Wing traps (1.42 1.8)
and the fewest were captured in traps painted
gloss white (0.50 0.7), though the differences
were not significant (F = 0.71; df = 8, 214; P <
0.6819). In addition, we found no difference in the
spectral reflectance readings made on unpainted
or painted traps at the beginning compared with
the end of the field test. Peak wavelengths for
traps of different colors ranged between 0 and 638
nm, and cladode readings were 552 and 759 nm.
TABLE 2. INFLUENCE OF TRAP HEIGHT ON THE NUMBER
OF CACTOBLASTIS CACTORUM MALES CAP-
TURED IN PHEROCON 1-C WING TRAPS BAITED
WITH A SINGLE VIRGIN FEMALE.
Mean ( SD) number
Trap height (m) of males captured**
0.5 1.13 2.3a
1.0 1.92 3.3b
2.0 2.14 3.3c
**Means followed by a different letter are significantly dif-
ferent, P < 0.05 (PROC ANOVA using orthogonal contrasts,
SAS Institute 1989).
Nevertheless, traps varied widely in their indi-
vidual spectral reflectance (Table 3). Traps that
were painted black, dark green and blue had the
lowest reflectance values (all below 50%), as did
the cladodes of 0. stricta. Traps painted orange,
fluorescent green, yellow, white and fluorescent
yellow, as well as the unpainted control, had high
reflectance values (above 50%). Even though the
white and the unpainted traps had the same peak
wavelength (425 nm) the unpainted trap had the
highest spectral reflectance.
Trap optimization is of vital importance in de-
veloping useful and reliable monitoring systems
for pest insects. Habeck and Bennett (1990) were
the first to report the presence of C. cactorum in
the Florida Keys. That initial finding of C. cac-
torum consisted of one adult female collected in a
mercury vapor lamp and larvae from infested
O. stricta. From 1989-2002 cactus moth popula-
tions were recorded at different locations along
both coasts and at inland sites as far north as Folly
Island, SC and as far west as St. Vincent Island,
FL (Dickel 1991; Johnson & Stiling 1998; Hight et
al. 2002). All of these reported infestations were
based on finding damaged cactus plants and/or the
presence of immature stages (eggsticks, larvae,
pupae) of C. cactorum at these locations.
Our group deployed the first virgin female-
baited sticky traps used to detect C. cactorum in
May 2002. As a result of this work, we determined
that this insect completes three non-overlapping
generations per year in Florida. Each generation
has distinct periods of flight activity followed by
periods of larval development during which no
adults are flying (Zimmermann et al. 2004). In
2003, Hight et al. (2003) reported that the new
western limit of C. cactorum was at Pensacola
Beach, FL, based on visual inspection of highly
infested prickly pear plants found during a larval
development period. Positive confirmation of
C. cactorum infestation is relatively easy when
larval stages are present and causing heavy dam-
age to Opuntia plants. However, determination of
the presence of C. cactorum can be easily over-
looked when infestations are new or small, or the
immature stages are not active. Traps baited with
adult sexual attractants are effective when the
target species is at a low population level (Hanula
et al. 1984), the adults are expanding into new ar-
eas (Walters et al. 2000), and at times when im-
mature stages are not present (Lalone 1980).
Therefore, we wanted to conduct evaluations of
our female virgin-baited sticky traps to ensure
that the best monitoring tool is being used to de-
tect the presence of C. cactorum. While three of
the characteristics tested did not reveal signifi-
cant differences (trap color, trap type, and female
age), the trend for each characteristic indicated
Bloem et al.: Trap development for Cactoblastis cactorum
TABLE 3. MEAN PEAK WAVELENGTHS (IN NM) AND SPECTRAL REFLECTANCE (IN %) FOR FRESH CLADODES OF OPUNTIA
STRICTA AND FOR PHEROCON 1-C TRAPS EITHER UNPAINTED (STANDARD) OR PAINTED WITH TWO COATS OF
EIGHT DIFFERENT COLORS.
Trap color Mean peak wavelength (nm) Reflectance
Gloss Black 0 0.025
Dark Hunter Green 537 0.095
True Blue Gloss 462 0.382
0. stricta cladode 552-759 0.386
Pumpkin Orange Gloss 638 0.667
Fluorescent Green Marking 515 0.926
Sunburst Yellow 557 0.985
Gloss White 425 1.088
Fluorescent Yellow 501 1.219
Unpainted 425 1.286
that unpainted, wing traps baited with 24 h post
emergence females resulted in the highest mean
capture rate. Considering the overall trap design,
the results presented here suggest that the best
trap currently available to monitor C. cactorum is
a standard (unpainted) Pherocon 1-C Wing trap,
placed at a height of 2.0 m aboveground, and
baited with four newly emerged (24 h post emer-
Trapping studies on other Pyralidae report re-
sults similar to ours. For example, Ahmad (1987)
and Hanula et al. (1984) found that Pherocon 1-C
Wing traps captured the highest number of male
almond moth Cadra cautella (Walker) and male
coneworm Dioryctria spp., respectively, when
field tested against other trap designs. It is inter-
esting to note that traps placed at a height of
2.0 m captured significantly higher numbers of
male C. cactorum than did traps placed at 0.5 or
1.0 m. This trap height (2.0 m) is about 0.5-1.0 m
higher than the tallest 0. stricta host plant
present at Brunswick River, GA and St. Marks
NWR, FL. For some economically important
Pyralidae the most effective trap heights are
those at or just above host canopy level. This is
true for male coneworm captured in pheromone-
baited traps (Hanula et al. 1984) and male pickle-
worm Diaphania nitidalis (Stoll) captured in
virgin female-baited traps (Valles et al. 1991), but
it is not true for almond moth males (Ahmad
1987). One possible explanation for our results is
that traps placed at 2.0 m might be "escaping" the
competition from pheromone emitting virgin
females present in the vegetation. The pheromone
plume emitted above the layer of vegetation may
be more distinct and attractive to male
Our results suggest that female C. cactorum
are ready to mate within 24 h of emergence. In re-
cent field experiments Hight et al. (2003) used
virgin female C. cactorum that were <24 h post
emergence in mating tables and observed females
emitting pheromone and forming mating pairs. In
our Wing traps, the concentration of pheromone
from four females is likely to be higher and more
attractive to males than the pheromone concen-
tration from a single female C. cactorum. Further-
more, the advantage of using more than one fe-
male per trap might be that each female actively
emits pheromone at slightly different times, thus
extending the attractiveness of traps baited with
multiple rather than with single females.
Trap color had no influence on male cactus
moth captures. However, more males were cap-
tured in the unpainted trap which had the high-
est spectral reflectance of all traps tested. Trap
color has been shown to improve trapping effi-
ciency in other economically important Lepi-
doptera. Knight and Miliczky (2003) found that
painted Delta traps captured significantly more
male codling moths (Cydia pomonella L., Lepi-
doptera: Tortricidae) than did unpainted traps. In
addition, Meagher (2001) showed that traps that
had contrasting colors captured more fall army-
worm Spodoptera frugiperda (J.E. Smith) males
than did traps of only one color.
The results presented here give us confidence
that we are currently using the most effective
trap to detect the presence of cactus moth infes-
tations in North America. Using the best avail-
able trapping tool is crucial to determining the
best location to deploy a barrier of sterile insects
to prevent further westward spread of C. cac-
torum. In addition, and as suggested by Bloem et
al. (2003), this same monitoring tool can be de-
ployed beyond the leading edge of infestation by
baiting the traps with reproductively sterilized
females. While progress is being made on the
identification and synthesis of the cactus moth
sexual pheromone by colleagues at the USDA-
ARS laboratories in Miami and Gainesville FL,
no commercial lure is currently available. When
experimental pheromone blends do become
available, the data presented here will be ex-
tremely useful in the field-testing of synthetic
lures for cactus moth.
We thank Nathan Herrick, John Mass, Carla Evans,
Stephen McLean, and Melany Coombs (USDA-ARS-
CMAVE, Tallahassee, FL); Robert Caldwell, Susan
Drawdy, and Robert Giddens (USDA-ARS-CPMRU, Tif-
ton, GA) for technical assistance. We thank Dr.
Katherine Milla (Florida A&M University) for the use of
the spectrophotometer and Richard Layton (University
of Georgia) for assistance with the statistical analysis of
the data. We also thank Nathan Herrick and Dr. Stuart
Reitz (USDA-ARS, Tallahassee, FL) and Dr. Russ Mizell
(University of Florida) for helpful reviews of this manu-
script. Mention of trade names or commercial products
in this publication is solely for the purpose of providing
specific information and does not imply recommenda-
tion or endorsement by the U.S. Department of Agricul-
AHMAD, T. R. 1987. Effects of pheromone trap design
and placement on capture of almond moth Cadra
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BLOEM, S., J. E. CARPENTER, AND K. A. BLOEM. 2003.
Performance of sterile Cactoblastis cactorum (Lepi-
doptera: Pyralidae) females in luring males to traps.
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Florida Entomologist 88(3)
Shelly et al.: Mating success ofB. dorsalis males
INFLUENCE OF DIET AND METHYL EUGENOL ON THE
MATING SUCCESS OF MALES OF THE ORIENTAL FRUIT FLY,
BACTROCERA DORSALIS (DIPTERA: TEPHRITIDAE)
TODD E. SHELLY, JAMES EDU AND ELAINE PAHIO
USDA-APHIS, 41-650 Ahiki Street, Waimanalo, HI 96795 USA
The chief objective of this study was to determine whether the inclusion of protein in the
adult diet influences male mating success in the oriental fruit fly, Bactrocera dorsalis (Hen-
del). Previous studies on this species have shown that ingestion of methyl eugenol (ME)
greatly enhances male mating performance. Accordingly, we also examined the interaction
between adult diet and ME and investigated whether this chemical boosts the male mating
success independent of diet. In trials conducted in field tents, we compared the mating fre-
quency of control, protein-fed males (no ME) versus males (1) deprived of protein during the
entire adult life, the pre-maturation period, or the post-maturation period and (2) not pro-
vided ME or provided ME 1 day before testing. Males deprived of protein completely or when
immature (1-12 days old) obtained very few matings (<5% total matings) with or without ME
feeding. Males provided protein as immature adults but deprived of protein as mature
adults (>12 days old; no ME) also were competitively inferior to control males but achieved
a significantly higher proportion (37%) of total matings than males in the preceding treat-
ments. ME exposure boosted the mating success of these males slightly (40% of total mat-
ings) such that their mating frequency was not significantly different from control males.
Additional tests showed that for treated males fed protein their entire adult life short-term
(30 h) food deprivation resulted in a significant decrease in mating success, and feeding on
ME did not boost the mating frequency of the food-deprived males. The implications of our
findings for controlling B. dorsalis via sterile male releases are discussed.
Key Words: Diptera, Tephritidae, oriental fruit fly, diet, methyl eugenol, mating behavior
El objetivo principal de este studio fue el determinar si la inclusion de protein en la dieta
del adulto tiene una influencia en el 6xito de apareamiento de los macho en la mosca oriental
de fruta Bactrocera dorsalis (Hendel). Estudios anteriores sobre esta especie han mostrado
que la ingestion de eugenol metil (EM) mejora sustancialmente la capacidad del aparea-
miento del macho. De consiguiente, nosotros tambien examinamos la interaci6n entire la
dieta del adulto y EM e investigamos si esta quimica aumentard el exito de apareamiento
del macho independiente de la dieta. En pruebas llevadas a cabo en tiendas de campana en
el campo, nosotros comparamos la frecuencia de apareamiento del grupo control, machos ali-
mentados con pr6teina (no EM) versus machos 1) privados de pr6teina durante su vida de
adulto entera, el period de pre-maduraci6n, o el period del pos-maduraci6n y 2) no provei-
dos con EM o no proveidos con EM un dia antes de la prueba. Los machos completamente
privados de protein o que eran inmaduros (1-12 dias de edad) conseguieron muy pocos apa-
reamientos (<5% de los apareamientos total) con o sin alimentar de EM. Los machos provei-
dos con protein en el estadio inmaduro pero privados de protein como adults maduros
(>12 dias de edad, sin EM) fueron inferiores en competir con los macho del grupo control pero
lograron obtener una proporci6n significativamente mas alta (37%) de apareamientos total
que los machos en los tratamientos anteriores. La exposici6n a EM aument6 el 6xito del apa-
reamiento de estos machos ligeramente (40% de todos los apareamientos) de tal manera que
la frecuencia de apareamiento no fue significativamente diferente de la frecuencia de los ma-
chos en el grupo de control. Pruebas adicionales mostraron que, para los machos tratados
con alimentos con pr6teina para toda su vida del adulto, la privaci6n de alimentos a un corto
plazo (30 h) resultaron en una diminuci6n significativa en el 6xito de apareamiento, y la ali-
mentaci6n de EM no aument6 la frecuencia de apareamiento de los machos privados de ali-
mentos. Se discuten las implicaciones de nuestros hallazgos para controlar B. dorsalis por la
via de liberaci6n de machos est6riles.
The quality of the adult diet, particularly the and Diptera (Stoffolano et al. 1995; Droney 1998).
inclusion of protein, may influence the ability of Recent data from two economically important
male insects to attract females and obtain copula- genera of tephritid fruit flies (Diptera: Tephriti-
tions as shown for certain species of Orthoptera dae) likewise reveal an important effect of dietary
(Andrade and Mason 2000; Scheuber et al. 2003) protein on male signaling and mating success (for
Florida Entomologist 88(3)
protein effects on female tephritids, see Cangussu
& Zucoloto 1995; Jacome et al. 1999). Working
with a wild population of Mediterranean fruit
flies (medflies), Ceratitis capitata (Wiedemann),
in Israel, Yuval et al. (1998) found that sexually
active males (i.e., those participating in leks) con-
tained more sugar and protein than sexually inac-
tive (resting) males. In a follow-up study, Kaspi et
al. (2000) observed wild medflies on field-caged
host trees and found that protein-fed males spent
more time emitting pheromone (see Papadopoulos
et al. 1998 for a similar finding) and achieved
more matings than protein-deprived males. In a
study of wild medflies in Hawaii, Shelly et al.
(2002) observed no effect of dietary protein on the
frequency of pheromone calling but found that
protein-fed males attracted more females than
protein-deprived males and had a significant ad-
vantage in mating competition over protein-de-
prived males (see also Shelly & Kennelly 2002).
In addition to this focus on dietary protein, sev-
eral studies (Papadopoulos et al. 1998; Shelly &
Kennelly 2003) have demonstrated the adverse
effect of short-term food deprivation (18-24 h) on
signaling activity and mating success of wild
The effect of diet on the mating behavior of
wild males also has been examined in several
Anastrepha species. Aluja et al. (2001) reared wild
males of 4Anastrepha species on sugar, a mixture
of sugar and protein, open fruit, or a mixture of
sugar and bird feces and then compared males in
the different diet treatments with respect to sig-
naling activity and mating frequency. In 3 of the
species, males fed the combination of sugar and
protein displayed the highest frequency of phero-
mone-calling, and in two of these, males fed only
sugar called significantly less frequently than
conspecifics fed other diets. In the same 3 species,
males fed the sugar and protein diet obtained sig-
nificantly more matings than males fed other di-
ets. In only 1 species, A. ludens (Loew), did diet
have no effect on male sexual activity.
The chief objective of the present study was to
determine whether dietary protein has a simi-
larly strong effect on male mating success in the
Oriental fruit fly, Bactrocera dorsalis (Hendel).
Previous studies on B. dorsalis (e.g., Shelly &
Dewire 1994) have demonstrated that ingestion
of methyl eugenol (ME), a powerful male attrac-
tant occurring naturally in many plant species
(Tan & Nishida 1996), dramatically increases
male mating success, apparently owing to the in-
corporation of ME metabolites in the sex phero-
mone (Nishida et al. 1987) and the subsequently
heightened attractiveness of the olfactory signal
(Shelly & Dewire 1994). The effect of ME feeding
on male reproductive performance has been in-
vestigated only within the context of protein-rich,
adult diets. Consequently, we also examined the
interaction between diet and ME feeding to deter-
mine whether ME feeding similarly boosts the
mating performance of protein-deprived males.
Finally, in light of the aforementioned results for
the medfly, we measured the effect of food depri-
vation on the mating success ofB. dorsalis males.
Implications of our findings for controlling B. dor-
salis via sterile male release are discussed. For
background information on the mating behavior
ofB. dorsalis, see Fletcher (1987) and Shelly and
Materials and Methods
All flies used in the present study were from a
laboratory colony started with 600-800 adults
reared from papayas (Carica papaya L.) collected
near Hilo, HI. The colony was maintained in a
screen cage (l:w:d, 1.2 x 0.6 x 0.6 m) and provided
a mixture (3:1, wt:wt) of sugar (sucrose) and hy-
drolyzed protein (enzymatic yeast hydrolysate)
and water ad libitum and papayas for oviposition.
Infested papayas were held over vermiculite, and
the pupae were sifted from vermiculite 16-18 days
later. Adults used in mating trials were separated
by sex within 48 h of eclosion, well before reach-
ing sexual maturity at = 15 days of age (TES, un-
published data), and held in screen-covered, plas-
tic buckets (volume 5 liters with a cloth sleeve to
allow transfer of flies, food, and water; 100-125 in-
dividuals per bucket) with ample food and water.
Flies were held at 24-28C and 60-90% RH and re-
ceived natural and artificial light under a 12:12
(L:D) photoperiod. When used in the experiments,
the flies were 3-7 generations removed from the
Competitive Mating Tests
Four mating experiments were conducted in
which males subject to different rearing regimes
competed for copulations. All tests used mature
individuals of both sexes (males: 21-25 days old;
females: 21 29 days old). In each experiment, the
control males were fed the sugar-protein mixture
(hereafter termed the SP diet) and water continu-
ously during their entire adult life (as were all fe-
males tested) and never given access to ME. The
food mixture was placed in a small Petri dish (5
cm diameter), and water was provided via a cot-
ton wick emerging from a covered, plastic cup.
Both the food and water were changed every 5-7
Treated males were subject to the following
Experiment la: Treated males were fed sugar-
agar exclusively throughout their entire adult life
(i.e., these males were protein-deprived) and were
not given access to ME. No water cup was pro-
vided. The sugar-agar diet (hereafter termed the
Shelly et al.: Mating success ofB. dorsalis males
S diet) was prepared following the recipe of the
California Preventative Release Program and in-
cluded water (84.66% by weight), sugar (14.57%),
agar (0.76%), and methyl parabin (0.01%). A block
of the sugar-agar (l:w:h, approximately 6 x 3 x 2
cm) was placed directly on the screen-cover of the
bucket and was replaced every 2-3 days.
Experiment lb. Same as above, except that
treated males were given access to ME (supplied
by FarmaTech Intl. Corp., Fresno, CA). In this,
and all subsequent experiments involving ME ex-
posure, treated males were given access to ME on
the day before testing. Using a micropipette, we
applied 100 pl of ME to a cotton wick (held verti-
cally by insertion through a hole in the lid of a
plastic cup), which was then placed in a bucket
holding 80-100 males. The chemical was intro-
duced between 1000-1300 hours and removed 1 h
after placement. Feeding activity was not moni-
tored in this study, but in a previous study (Shelly
1997) over 90% of mature males were found to
feed on ME within a 1-h interval.
Experiment 2a. Treated males were fed the S
diet from 1-12 days of age and were then provided
the SP diet until tested (hereafter termed the S-
SP diet). As males do not attain sexual maturity
until about 15 days of age (TES, unpublished
data), this treatment provided sugar only to im-
mature males but sugar and protein to sexually
mature males. Treated males were not given ac-
cess to ME.
Experiment 2b. Same as experiment 2a, except
that treated males were given access to ME fol-
lowing the above protocol.
Experiment 3a. Treated males were fed the SP
diet and water from 1-12 days of age and were
then provided the S diet until tested (hereafter
termed the SP-S diet). Here, we reversed the
treatment of experiment 2a and provided sugar
and protein to immature males but sugar only to
mature males. Treated males were not given ac-
cess to ME.
Experiment 3b. Same as experiment 3a, except
that treated males were given access to ME fol-
lowing the above protocol.
Experiment 4a. Treated males were fed the SP
diet during their entire life but were starved for
approximately 30 h prior to testing. Treated
males were not given access to ME. As noted
above, B. dorsalis is sexually active at dusk, and
consequently we removed the food (but not the
water cup) at noon on the day before testing.
Experiment 4b. Same as experiment 4a, except
that treated males were fed ME. In this case, ME
was introduced at 1100 hours, and starvation was
initiated upon termination of the exposure period.
Competitive mating trials were conducted be-
tween August 2004-January 2005, in field cages (3
m diameter; 2.5 m height) at the USDA-ARS labo-
ratory, Honolulu (air temperature: 25-30C; RH: >
60%). The tents each contained two artificial trees
whose leaves resembled those ofFicus benjamin
L. Each tree was approximately 2 m tall and bore
700-800 leaves. Males perform the normal suite of
reproductive behaviors on these trees, and mat-
ings occur as frequently as on potted host trees
(TES, unpublished data). Groups of 75 control
males, 75 treated males, and 75 females were re-
leased approximately 2 h before sunset (between
1600-1700 hours depending on the test date). For
a given trial, we marked both control and treated
males 1 day prior to testing by cooling them for
several minutes and placing a dot of enamel paint
on the thorax. The cages were monitored from 1 h
before sunset until approximately 30 min after
sunset with a flashlight. Mating pairs were col-
lected in vials and returned to the laboratory
where the males were identified. Eight replicates
were conducted for experiments 1-3, and 10 repli-
cates were conducted for experiment 4.
Non-Competitive Mating Tests
Because adult diet had a profound effect on
male mating success (see below), we conducted a
series of non-competitive mating tests to assess
the mating propensity of males exposed to differ-
ent dietary regimes. To expedite data collection,
these tests were run in our laboratory by placing
10 females (maintained on the SP diet) and 10
males of a given treatment in plexiglass cages
(l:w:h, 40 x 30 x 30 cm) approximately 2 h before
sunset and scoring the number of matings 2-3 h
after sunset. Cages were placed near a west-fac-
ing window and exposed to natural light (room
lights were extinguished when flies were placed
in the cages). When tested, males were 21-24 days
old, and females were 23-29 days old. For a given
male treatment, 6-10 cages were run per day over
3-5 different days for a total of 30 replicates
(cages) per treatment.
Non-competitive mating tests were conducted
with males subject to treatments identical to
those described above for the competitive mating
trials and included control males and treated
males as described for experiments 1-3. Test
males were subject to the following conditions.
Experiment 5. Males were fed the SP diet dur-
ing their entire life and were not given access to
Experiment 6a. Males were fed the S diet dur-
ing their entire life and were not given ME.
Experiment 6b. Same as above, but males were
given access to ME.
Experiment 7a. Males were fed the S-SP diet
and were not given access to ME.
Experiment 7b. Same as above, but males were
given access to ME.
Experiment 8a. Males were fed the SP-S diet
and were not given access to ME.
Experiment 8b. Same as above, but males were
given access to ME.
Diet and Male Survival
Survival was compared among males main-
tained on the SP, S, S-SP, or SP-S diets. For each
diet, 20 males (1 day old) were placed in screen
cages (30 cm cubes) with the appropriate diet un-
der the laboratory conditions described above. All
diets were presented in small Petri dishes; water
was provided with all diets except the S diet. Food
and water were changed every 2-3 days. Cages
were checked midday every day for 40 days, and
dead males were removed during the daily obser-
vations. Ten cages were observed for each diet
For the competitive mating experiments, we
first compared the number of matings obtained
per replicate between control and treated males
using a t-test as the assumptions of normality and
equal variances were met in nearly all cases (the
exceptions were experiments la, Ib, and 2a, re-
spectively, and here data were normalized via log10
[x + 1] transformation). Because there was signif-
icant variation in the total number of matings ob-
served per replicate among the different experi-
ments (F7,60 = 5.2, P < 0.001, ANOVA; presumably
owing to slight variation in weather conditions),
we compared mating performance among the dif-
ferent treatments using the proportion (arc sine
transformed) of the total matings obtained per
replicate in an ANOVA. For the noncompetitive
mating experiments, mating numbers were com-
pared among male treatments by the Kruskal-
Wallis test (a logarithmic transformation failed to
normalize the data). For male survivorship data,
the number of survivors was plotted against time
for the different treatments, and slopes of the re-
gression lines were compared by ANCOVA follow-
ing Zar (1996). As described below, multi-group
comparisons involving mating frequency or survi-
vorship revealed significant variation in all cases,
and consequently the Tukey test was run to iden-
tify significant differences in specific pair wise
comparisons. For survivorship, the test statistic q
was calculated according to Zar (1996).
Competitive Mating Tests
Results of the competitive mating trials are
presented in Table 1. The most striking finding
was the dramatic, negative effect ofprotein-depri-
TABLE 1. EFFECTS OF ADULT DIET, METHYL EUGENOL (ME), AND STARVATION ON THE MATING SUCCESS OF B. dorsalis
MALES IN COMPETITIVE MATING TRIALS.
Number of % Total
Experiment Diet ME Starvation Male type Matings1 t2 Matings3
1 a S no no Control 20.4 (3.7) 12.3***
Treated 0.8(1.1) 2.8
1 b S yes no Control 21.0 (3.4) 16.6***
Treated 0.7 (0.7) 3.2
2 a S-SP no no Control 17.1 (2.4) 13.2***
Treated 0.9 (1.0) 4.7
2 b S-SP yes no Control 13.6 (3.4) 0.2N
Treated 13.0 (6.2) 47.0"
3 a SP-S no no Control 18.9 (4.6) 3.3**
Treated 11.2 (4.5) 37.5"
3 b SP-S yes no Control 16.8 (4.8) 1.2N
Treated 13.2 (5.2) 40.5b
4 a SP no yes Control 12.8 (4.5) 2.8*
Treated 7.3 (3.8) 35.6b
4 b SP yes yes Control 17.1(8.9) 2.5*
Treated 8.7 (5.5) 35.9b
Values represent average numbers (1 sd) of matings per replicate and average proportion of total matings obtained by treated
males; 8-10 replicates were conducted per experiment.
Tests compare control and treated groups for a given experiment, where significance levels are: ***P < 0.001; **P < 0.01; *P <
0.05; "not significant.
'Proportions followed by the same letter are not significantly different at P = 0.05, tukey test.
Florida Entomologist 88(3)
Shelly et al.: Mating success ofB. dorsalis males
vation on male mating success. Males fed the S
diet their entire life obtained, on average, less
than 1 mating per replicate without (experiment
la) or with (experiment Ib) prior access to ME.
Males maintained on the S-SP diet and not pro-
vided ME (experiment 2a) had a similarly low
mating success. Treated males in these experi-
ments accounted for a similar proportion (3%-5%)
of the total matings. In contrast to the S diet,
males fed the S-SP diet and then exposed to ME
(experiment 2b) displayed a significant increase
in mating success and were, in fact, competitively
equivalent to control males.
Males maintained on the opposite dietary re-
gime, SP-S, and denied access to ME (experiment
3a) were competitively inferior to control males,
but they obtained, on average, a significantly
higher proportion of matings per replicate than S-
SP males denied ME (experiment 2a; 38% vs. 5%,
respectively). Exposure to ME increased the mat-
ing success of males reared on the SP-S diet (ex-
periment 3b), and the mean number of matings
obtained by these males was similar to that ob-
served for the control males. Relative to the S-SP
diet used in experiment 2, however, the effect of
ME exposure with the SP-S diet was slight: males
fed the SP-S diet and given ME or denied ME ac-
counted for a similar proportion of the total mat-
ings (40% versus 37%, respectively).
Starvation had a strong negative effect on mat-
ing frequency. Control males had a mating advan-
tage over starved males (previously maintained
on the SP diet) independent of ME feeding (exper-
iments 4a and 4b). Starved males denied or pro-
vided ME obtained the same proportion (36%) of
the total matings.
Non-Competitive Mating Tests
Results from the non-competitive mating tests
mirrored those described above for the competi-
tive tests (Table 2). Significant variation in mat-
ing frequency was observed among male treat-
ments (H = 64.8, df = 6, P < 0.001). Males on the
S and S-SP diets exhibited mating frequencies
that were significantly lower than those observed
for the SP or SP-S diets. Over the 60 replicates in-
volving the S diet (i.e., experiments 6a and 6b
combined), we observed a total of only 3 matings.
Similarly, only 5 matings were recorded for males
fed the S-SP diet independent of ME exposure.
Males on the SP diet displayed the highest mat-
ing frequency, although this was not significantly
different from males on the SP-S diet after ME ex-
Significant variation in slope existed among
the different diet treatments (F3, 2 = 5.9, P =
0.007), with survival rate being greatest for the
TABLE 2. EFFECTS OF ADULT DIET AND METHYL EUGENOL
(ME) ON THE MATING FREQUENCY OF B. DORSA-
LIS MALES IN NON-COMPETITIVE MATING TRIALS.
Experiment Diet ME Mean Median'
5 SP no 3.8(1.5) 4
6 a S no 0.1(0.2) 0W
6b S yes 0.1(0.2) 0W
7 a S-SP no 0.2 (0.5) 0W
7 b S-SP yes 0.2 (0.4) 0W
8 a SP-S no 2.6 (1.3) 3b
8b SP-S yes 2.9 (1.4) 3ab
'Mean (1 sd) and median numbers of matings are given
over 30 replicates per experiment. Medians followed by a com-
mon letter are not significantly different atP = 0.05, Tukey test.
SP diet, lowest for the S diet, and intermediate for
the SP-S and S-SP diets (Fig. 1). Multiple compar-
ison tests revealed that survival rate for the SP
diet was significantly greater than for the S or SP-
S diets but not significantly different from the S-
SP diet. No significant differences were detected
in pair wise comparisons among the S, SP-S, and
S-SP diets. The simple linear regression equa-
tions were: SP: y = 18.5-0.16x, r2 = 0.89; S: y =
18.6-0.31x, r2 = 0.97; S-SP: y = 18.1-0.22x, r2 =
0.90; SP-S: y = 18.8- 0.26x, r2 = 0.94.
The present study demonstrates a strong effect
of diet quality on the mating success ofB. dorsalis
males. The inclusion of hydrolyzed protein in the
0 10 20 30 40
Fig. 1. Survivorship of males maintained on differ-
ent diet regimes. Points represent means of 10 cages per
Florida Entomologist 88(3)
adult diet was very important for mating; males
fed the sugar-agar gel exclusively achieved very
few matings in competitive or even non-competi-
tive conditions. The low number of copulations ob-
served in the non-competitive situation further
suggests that males were not sexually active at
all or were producing signals unattractive to fe-
males. Interestingly, males reared on the S diet
until day 12 and then switched to the SP diet for
8-12 days before testing (and not given ME) per-
formed as poorly as males reared on the S diet ex-
clusively, and this was evident in both competitive
and noncompetitive situations. Males subject to
the opposite treatment (SP-S) had a lower mating
success than control males in the competitive tri-
als but nonetheless accounted for a much higher
proportion of the total matings than did the S or
S-SP males. Results from the starvation treat-
ment further revealed that males deprived of the
PS diet for a single day had reduced mating suc-
cess relative to males continuously fed the same
Dietary protein appears to have a greater ef-
fect on mating success for wild males ofB. dorsa-
lis than for wild males of C. capitata. Whereas
sugar-fed males of B. dorsalis rarely mated,
sugar-fed males of C. capitata accounted for ap-
proximately 33% (Kaspi et al. 2000) and 40%
(Shelly et al. 2002) of the total matings in compe-
tition with protein-fed males. The impact of di-
etary protein varied greatly among Anastrepha
species (Aluja et al. 2001). As in B. dorsalis, males
of A. serpentina (Wiedemann) and A. striata
Schiner that were fed a sucrose solution only
mated very infrequently (<10% total matings). In
contrast, based on non-competitive mating trials,
males ofA. obliqua (Macquart) fed sugar only still
mated at nearly half the rate as protein-fed
males, and no diet effect at all was detected forA.
The present findings provide only a broad de-
scription of the impact of adult nutrition on male
mating success, and additional tests are required
to determine more specifically the effect of inter-
mittent protein-feeding on male performance. In
particular, future studies should address the
question of whether, among sexually mature
males, mating success declines with time since
the last protein meal. In other words, while the
present study showed that both (i) a sugar-only,
post-maturation diet and (ii) starvation from a
protein-rich diet reduced male mating success, it
did not investigate the impact of more realistic
feeding regimes, where, for example, males may
locate sugar-rich foods more or less continuously
but protein-rich foods only irregularly.
In addition to demonstrating the importance
of dietary protein, our study also provided infor-
mation regarding the interaction between diet
and ME on male mating success. Among contin-
uously fed males, ME had no effect on mating
performance for males that were fed the S diet
exclusively and only a slight effect on males fed
the SP-S diet. However, ME dramatically
boosted mating success under the S-SP diet: ME-
fed males obtained an average of 47% of all mat-
ings per replicate (experiment 2b) compared to
only 5% for ME-deprived males (experiment 2a).
Although ME exposure under the S-SP diet
yielded equivalent mating success between
treated and control males, prior studies (Shelly
& Dewire 1994; Shelly & Nishida 2004) have
shown that, among males fed the SP diet exclu-
sively, individuals provided with ME obtain ap-
proximately 2/3 of all matings in competition
against ME-deprived individuals. Thus, ME ex-
posure under the S-SP diet apparently compen-
sated for a low quality diet, but it did not confer
a mating advantage as evident under the SP di-
etary regime. Interestingly, ME feeding did not
offset the adverse effect of starvation (from the
SP diet; experiment 4b) on male mating success.
It thus appears that the ability to locate protein-
rich food is essential for B. dorsalis males, be-
cause temporary deprivation of the SP diet not
only reduced mating performance but also elim-
inated the potential benefit associated with ME
In conclusion, our study is potentially relevant
to control programs, such as that ongoing in Thai-
land, that utilize the sterile insect technique
(SIT) to suppress or eradicate infestations of B.
dorsalis. Our tests were performed exclusively on
flies from a relatively "young" laboratory colony,
and additional work is required to determine
whether the composition of the adult diet simi-
larly influences the mating competitiveness of
males from long-established, mass-reared strains
of B. dorsalis. In the Mediterranean fruit fly, for
example, inclusion of protein in the adult diet in-
variably results in increased mating success of
wild males (Yuval et al. 1998; Kaspi et al. 2000;
Shelly et al. 2002), whereas the results for mass-
reared males have been inconsistent (compare
Blay & Yuval 1997; Kaspi & Yuval 2000 with
Shelly and Kennelly 2002; Shelly and McInnis
2003). Clearly, however, if adult diet similarly af-
fects mass-reared B. dorsalis males, an effective
use of the SIT would require inclusion of protein
in the pre-release diet.
We thank Mike McKenney for kindly supply-
ing the wild pupae, Mindy Teruya for laboratory
assistance, and Don McInnis for helping with the
field cage mating trials. Comments by B. Yuval
improved the manuscript. The study was sup-
ported, in part, with funds from the U.S.-Israel Bi-
national Agricultural Research and Development
Fund (BARD Project No. US-3256-01) to TES and
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Florida Entomologist 88(3)
INFESTATION OF STORED SAW PALMETTO BERRIES
BY CADRA CAUTELLA (LEPIDOPTERA: PYRALIDAE) AND
THE HOST PARADOX IN STORED-PRODUCT INSECTS
R. T. ARBOGAST1, S. R. CHINI1 AND P. E. KENDRA2
'Center for Medical, Agricultural and Veterinary Entomology, ARS, USDA, P. O. Box 14565, Gainesville, FL 32604
2Subtropical Horticulture Research Station, ARS, USDA, 13601 Old Cutler Road,
Miami, FL 33158
The almond moth, Cadra cautella (Walker), is a common storage pest known to infest a wide
range of dried plant materials, and it has been recorded from a warehouse in Florida during
storage of dried passion-flower (Passiflora incarnata L.) and dried saw palmetto berries Ser-
enoa repens (Bartram) Small. Its status as a pest of stored saw palmetto was confirmed by
trapping in a second warehouse used solely for storage of this commodity. The moth occurred
in high numbers, captures were closely associated with stacks of bagged berries, and trap
catch was very low after the stacks were consolidated under a tarp and fumigated. Yet the
results of laboratory rearing on saw palmetto suggested that C. cautella has little ability to
infest this commodity-development was protracted and highly variable in duration, mor-
tality was high, and pupal weight was low. This sort of contradiction in host suitability,
which we refer to as the "host paradox," may be widespread among stored-product insects
but has seldom been reported and almost never studied. Published reports suggest that the
solution lies partly in dietary supplementation through fungivorous, saprophagous, or car-
nivorous feeding, although more subtle factors also are suggested. Even cursory observa-
tions of the host paradox should be reported to document frequency of occurrence and
perhaps stimulate studies directed toward solutions. Such studies would inevitably provide
better understanding of population dynamics, which would, in turn, lend support to better
management of insects in commercial storage situations.
Key Words: development, saw palmetto, stored-product insects, almond moth
La polilla de la almendra, Cadra cautella (Walker), es una plaga comun de almac6n que es co-
nocida por infestar un amplio rango de material de plants secas, y ha sido registrada de una
bodega en Florida durante el almacimiento de la flor seca de maracuya (Passiflora incarnata
L.) y las moras secas del palmito Serenoa repens (Bartram) Small. Su estado como una plaga
del palmito fue confirmado por medio de trampas puestas en una segunda bodega usadas so-
lamente para el almacimiento de esta material. La polilla ocurre en altos numerous, las cap-
turadas fueron asociadas estrechamente con los apilados de moras embolsadas y el numero
de polillas capturadas en trampas fue muy bajo despu6s de que los estantes fueron cubiertos
bajo una tarpa y fumigados. Aun asi los results de criar la polilla sobre el palmito en el la-
boratorio sugiere que C. cautella tiene poca abilidad de infestar este material-el desarrollo
fue prolongado y altamente variable en duraci6n, la mortalidad fue alta, y el peso de la pupa
fue muy bajo. Esta clase de contradicci6n en el mantenimiento desarrollo sobre el hospedero,
lo cual referimos como la "paradoja del hospedero", puede ser ampliamente distribuida entire
los insects de products almacenados pero raramente ha sida reportada y casi nunca estu-
diada. Los informes publicados sugieron que la soluci6n consiste parcialmente en la suple-
mentaci6n de dieta por medio de la alimentaci6n fungivora, saprofaga, o carnivora, aunque
se sugiere que hay unos factors mas sutiles. Las observaciones precipitadas de la "paradoja
del hospedero deben ser reportadas para documentary la frecuencia de ocurrencia y tal vez con
ello estimular studios dirigidos hacia el encuentro de soluciones. Tales studios definitive-
mente proveeran un mejor entendimento de la dinamica de la poblaci6n, lo cual en cambio,
dara apoyo para el mejor manejo de insects en products comerciales almacenados.
The almond moth, Cadra cautella (Walker), is has been reported from dried passion-flower and
a common, often serious pest of dried plant mate- dried saw palmetto berries (Arbogast et al. 2002).
rials. It has been recorded from cereal grains and Saw palmetto, Serenoa repens (Bartram)
their products, dried fruit, nuts, oilseeds, pulses, Small, is one of many plants that provide botani-
and cacao (Richards & Thomson 1932), and also cals for the production of pharmaceuticals and
Arbogast et al.: Infestation of Saw Palmetto Berries
herbal supplements. It occurs in the southeastern
coastal plain of the United States from South
Carolina to Mississippi, where it is a common el-
ement of pine flatwoods, mesic hammocks, prai-
ries, and scrubs (Bennett & Hicklin 1998). An ex-
tract of the berries, or the ground berries them-
selves, are reported to be useful in maintaining
prostate and urinary tract well-being (Koch
2001), and they are sold for this purpose as a di-
etary supplement. The berries are harvested by
hand from their native habitats during late sum-
mer and fall. They are dried to a moisture content
of 8-14%, bagged in burlap, and stored pending
shipment to end processors. During storage, they
are subject to infestation by a variety of stored-
Arbogast et al. (2002) described the species
composition and spatial distribution of an insect
population infesting a botanicals warehouse at
Mascotte, FL, which was used alternately for
storage of dried saw palmetto berries and dried
passion-flower maypopp), Passiflora incarnata L.
Cadra cautella was the dominant species, com-
prising 47% of the insect population when saw
palmetto was in storage. To confirm the status of
C. cautella as a pest of stored saw palmetto, we
studied the spatial relationship between adult
moths and stacks of bagged berries in a second
warehouse used solely for storage of this commod-
ity. We also conducted laboratory experiments to
test the hypothesis that successful infestation of
the dried berries can be explained entirely by the
suitability of this host for growth and develop-
ment of the moth. The present paper reports the
results of these studies and examines the results
in the context of host range in stored-product in-
sects, especially the utilization of marginal host
MATERIALS AND METHODS
The warehouse, located in La Belle, FL, re-
cently had been acquired for storage of saw pal-
metto berries and previously had been used for
processing and storage of fresh peppers (green
bell peppers, jalapeno peppers, etc.). The building
was a modern steel structure 40 m wide by 55 m
long with a covered dock along the east side
(Fig. 1). The only walled areas within the building
were restrooms and a small office in one corner
and a large cold storage room in another. There
were sixteen propane-fired batch driers and a belt
drier adjacent to the dock, which served as a work
area during drying operations and also for stor-
age of crates and bagged saw palmetto debris,
which is used as mulch. In addition to saw pal-
metto, the building itself contained processing
equipment and a few pallets, crates, and burlap
bags. The dried and bagged berries were stacked,
usually six bags high, on wooden pallets, and the
pallets were in turn stacked one on top of another.
In January 2001, when we first visited the ware-
house, most of the stacks were inside the cold
storage room, but there were several small stacks
elsewhere (Figs. 1 and 2A). The door to the cold
storage room was kept closed, even though the re-
frigeration was no longer used. In late April, all of
the stacks were moved and consolidated in prepa-
ration for a commercial fumigation with phos-
phine (Fig. 2 B-C), which was done under a tarp
covering the stacked bags. The fumigation began
on April 27, and the warehouse was closed until
We made three, 24-h trapping runs, one in Jan-
uary, one in late April immediately before the fu-
migation, and one in early May immediately after
the fumigation. The traps were pheromone-baited
sticky traps (SP-Locator traps with SP Minimoth
pheromone dispensers, AgriSense-BCS Ltd., Pon-
typridd, Mid Glamorgan, UK). Trap locations
were specified in rectangular coordinates with the
origin at the southwest corner of the warehouse.
The number and configuration of trap locations
(Fig. 2A-C) varied slightly. Trap density was in-
creased in areas with stacked commodity, but the
spacing between traps was always > 4 m, the mea-
sured active space of the lures (Mankin et al.
1999). When trap locations were not occupied by
stacks, moth traps were placed 1.2 m above the
floor, attached by means of Velcro either to the
walls of the warehouse or to the tops of wooden
stakes supported by stands on the floor. Other-
wise, moth traps were placed on top of the stacked
bags or attached to walls slightly above the
stacks. Placement of traps under these circum-
stances was necessarily imprecise, but we esti-
mated that all were within <1 m of their desig-
nated coordinates. The stacks ranged in height
from slightly over 1 m to about 4.5 m, and trap
height varied accordingly.
To determine the spatial distribution of trap
catch for each trapping run, the x, y-coordinates
of the trap positions and the corresponding num-
bers of moths captured were entered in Surfer 7
(Golden Software, Golden, CO) for contour analy-
sis. This software posted observed trap catch to
the appropriate coordinates on a floor plan of the
warehouse, which had been entered as a base
map, and then created a denser grid of trap catch
values by interpolation, using radial basis func-
tions (with the multiquadric function). This
method of interpolation produces good represen-
tation of most small data sets (<250 observations)
(Golden Software 1999).
Laboratory cultures of C. cautella were estab-
lished in January 2001 with adults collected from
the La Belle warehouse. The moths were reared on
Florida Entomologist 88(3)
RAMP W *|
Fig. 1. Floor plan of a botanicals warehouse at La Belle, FL used for drying, bagging, and storing saw palmetto
berries. The light gray areas represent docks outside the building. The dark gray areas are stacks of bagged berries
a standard laboratory diet (Silhacek and Miller
1972) consisting of ground Gaines dog meal (10%),
rolled oats (4%), white cornmeal (26%), whole
wheat flour (23%), wheat germ (2%), brewers'
yeast (5%), glycerol (16%), and honey (14%). Cul-
tures were maintained in a walk-in environmental
chamber (EGC, Chagrin Falls, OH) at 27 1C
and 60 5% RH under a 12:12 h light-dark cycle.
All experimental procedures, except moisture de-
terminations, were conducted within the chamber.
To compare developmental time, pupal weight,
and survivorship on saw palmetto with that on
the laboratory diet, larvae were reared individu-
ally on 8 ml of ground saw palmetto in 16 ml
Wheaton sample bottles (Wheaton Scientific,
Millville, NJ). Ventilation was achieved by a
screen-covered opening (2 cm diam) in each bottle
cap. The screen consisted of disks cut from extra
fine phosphor-bronze cloth (Hillside Wire Cloth
Co., Bloomfield, NJ) to fit inside the caps.
Dried saw palmetto berries from the La Belle
warehouse were coarsely ground in a blender.
These ground berries and the laboratory diet
were equilibrated to the relative humidity of the
environmental chamber, for which purpose a
quantity of each diet sufficient to complete all ex-
periments, as well as provide 6 samples for mois-
ture determination, was placed in clear polysty-
rene boxes (19.5 x 14.0 x 7.0 cm) (Tri-State Plas-
tics, Dixon, KY) with screened holes (7 cm diam)
in the lids. A shallow layer of diet was placed in
each box and held in the environmental chamber
for one week, during which time it was stirred
daily. The diets were then retained in the boxes
until they were used for rearing or moisture de-
termination. Moisture content of the diets was de-
termined by an air-oven method, AACC Method
44-15A (American Association of Cereal Chemists
2003), when the first experiment was set up and
again when the last experiment was completed.
Arbogast et al.: Infestation of Saw Palmetto Berries
(A) 24-25 January 2001
(B) 25-26 April 2001
(C) 02-03 May 2001
C1 I 1 I i 4 """ 'I I 4 0 1 I i ;
0 10 20 30 0 10 20 30 0 10 20 30
METERS FROM SW CORNER
Fig. 2. Spatial distribution of C. cautella in a botanicals warehouse as indicated by trap catch of adult males.
Trap locations are indicated by dots and locations of stacked bags by rectangular outlines. Contour lines represent
total trap catch during a 24-h trapping period. Following fumigation (C), there were too few captures for contour
analysis, so trap catch is indicated by numerals adjacent to trap locations. The total number of moths captured dur-
ing each of the three trapping runs was (A) 65, (B) 78, and (C) 11.
About 8 ml of moisture-equilibrated diet were
placed in each of 100 bottles, 50 bottles per diet,
and one newly hatched larva was added to each
bottle. This procedure was repeated three times
at weekly intervals. To obtain larvae, eggs were
collected over a 6-8 hr period, confined in petri
dishes without food, and held in the environmen-
tal chamber. After 2 days, the eggs were checked
for hatching every 2-3 hr during the workday, and
newly hatched larvae were transferred with a
camel hair brush to the bottles with diets. Enough
larvae were obtained in this manner during a sin-
gle day to set up one experiment.
The bottles with larvae were allowed to remain
undisturbed for 2 wk and then examined daily for
pupation and adult eclosion. When a pupa became
fully tanned (light brown), it was gently removed
from its cocoon with soft forceps, weighed on an
ultra-microbalance (Type UMT2, Mettler Instru-
ment Corp., Hightstown, NJ) with readability to
0.0001 mg, and then returned to the bottle. Obser-
vations were continued until all the insects either
emerged or died.
Combined data for males and females were
used in running statistical tests. Differences in
mean survivorship and in pupal weight were an-
alyzed by t-tests, the latter after square-root
transformation of the data. The data for develop-
mental period failed tests for normality and equal
variance, which could not be corrected by any of
the common transformations, so the data were
analyzed by the Mann-Whitney test (SigmaStat
3.0, Systat Software, Inc., Richmond, CA), which
compares median values.
The spatial distribution of adult C. cautella, as
indicated by trap catch, showed a close associa-
tion of the moths with the stacks of berries (Fig.
2). In late January 2001, most of the berries were
stored inside the closed cold room, and the stacks
covered much of the floor space (Fig. 2A). The
highest trap catch occurred within this room
along the east wall. The remaining captures were
concentrated on two stacks near the office and
rest rooms with captures extending from there to
three very small stacks just outside the cold room.
Again in late April the highest trap catch oc-
curred inside the cold room (Fig. 2B), from which
the berries had been removed a few days earlier.
Following fumigation (Fig. 2C), only 11 moths
were trapped, 5 inside the cold room and the re-
mainder widely scattered about the warehouse.
None were trapped on the stack and only one im-
mediately adjacent to the stack.
The moisture content of the laboratory diet
(mean SE) was 15.2 ( 0.04)% at the beginning
of the experiment and 13.9 ( 0.05)% at the end.
Florida Entomologist 88(3)
The moisture content of the saw palmetto ranged
from 10.2 ( 0.07)% initially to 10.1 ( 0.08)% at
Development of C. cautella on saw palmetto
was protracted and highly variable in duration,
mortality was high, and pupal weight was low
(Table 1). The median developmental period on
saw palmetto was significantly longer (by 60 d)
than on laboratory diet (Mann-Whitney test; P <
0.01) and pupal weight was significantly lower (t
= 21.60; df = 153; P < 0.01). Survival on saw pal-
metto also was lower than on laboratory diet (t
=11.75, df = 4, P < 0.01). The mean survival rate
(SE) based on three groups of 50 insects was 89.3
+ 4.4 on laboratory diet and 16.7 (4.4) on saw
palmetto. Although no adult moths were weighed
or otherwise measured, those that developed on
saw palmetto were obviously smaller than those
reared on the laboratory diet, as would be ex-
pected given the difference in pupal weight.
The high numbers of C. cautella in the La Belle
warehouse, the close association of trap captures
with stacks of bagged berries, and the very low
trap catch after fumigation of the consolidated
stack, provide conclusive evidence that C. cautella
infests saw palmetto during storage and is, in fact,
a major pest. Yet in the laboratory, C. cautella dis-
played little ability to attack saw palmetto. Its
protracted development, with high larval mortal-
ity and low pupal weight, suggest that popula-
tions would increase little, or even decline, on saw
palmetto. This contradiction, a phenomenon we
term the "host paradox," may be quite widespread
among stored-product insects, but has seldom
been reported and almost never studied.
Storage insects typically have broad host
ranges that include dry commodities of both plant
and animal origin, but these commodities differ
markedly in their ability to support population
growth (Arbogast 1991). Variation in host suit-
ability is well illustrated by studies of the ciga-
rette beetle, Lasioderma serricorne (F.), which is
an extremely polyphagous stored product insect
with reported hosts including tobacco, spices, ce-
reals, pulses, seeds, nuts, dried fruit, dried vege-
tables, cocoa beans, coffee beans, yeast, bamboo,
copra, ginger, licorice root, herbarium specimens,
dried fish, fish meal, and meat meal (Howe 1957).
In a comparative study of selected plant products,
Howe (1957) found a range of 80 d in mean devel-
opmental period and 97% in survival rate (at
30C). In a study of spices, LeCato (1978) ob-
served a range of 100 d in median developmental
period and 88% in survivorship (at 28C). Prod-
ucts such as whole peas, curry powder and chili
powder are relatively poor diets for L. serricorne
and do not support rapid population growth. An
additional example of variation in host suitability
is provided by a study of three stored-product
moths on dried fruits, almonds, and carobs (Cox
1975). The mean developmental period of C. cau-
tella, for instance, ranged from 35 d on almonds to
84 d on raisins (30C). The developmental period
of C. cautella on the standard laboratory diet in
the present study was much shorter than on any
of the commodities reported by Cox (1975), even
though temperature was lower (Table 1).
Clearly, some commodities are only marginally
suitable as hosts for storage insects. They barely
support development, but yet these same com-
modities may become seriously infested in com-
mercial storage. Awareness of this paradox is im-
portant in evaluating the potential pest status of
stored-product insects; host studies in the labora-
tory may not reveal true pest potential under
commercial storage conditions.
The reasons for this discrepancy and the occur-
rence of the host paradox may lie partly in dietary
supplementation through fungivorous, sapropha-
gous, and carnivorous feeding, which are known
to enhance development and population growth,
especially on poor host commodities. The foreign
grain beetle, Ahasverus advena (Waltl), for exam-
ple, feeds on a wide variety of stored products, in-
cluding grain and cereal products, but usually oc-
TABLE 1. DEVELOPMENT OF C. CAUTELLA FROM EGG HATCH TO ADULT ECLOSION ON LABORATORY DIET AND ON DRIED
SAW PALMETTO BERRIES AT 27 1 C AND 60 5% RH.
Laboratory diet Saw palmetto
Development n Mean SE n' Mean SE
Period (d) 55 23.1 0.2 18 84.7 2.9
Pupal wt (mg) 55 14.5 0.25 16 5.3 0.32
Period (d) 72 23.4 0.2 7 91.6 7.9
Pupal wt (mg) 74 19.9 0.30 7 6.0 0.45
n, the number of insects on which the mean is based.
Arbogast et al.: Infestation of Saw Palmetto Berries
curs in large numbers only when a commodity is
moldy. Woodroffe (1962) found thatA. advena can-
not breed successfully on rolled oats or whole
wheat flour in the absence of visible mold unless
yeast or wheat germ are added, suggesting that
some cereal products are deficient in an essential
nutrient that can be provided by molds, yeast, or
wheat germ. The Indianmeal moth, Plodia inter-
punctella (Hiibner), may also require fungal sup-
plementation for development on some of its
hosts. This moth is a pest of stored raisins, but
fails to develop on raisins in the laboratory unless
the grapes used to produce the raisins are inocu-
lated before drying, with a particular fungus
known to infect grapes in the field (Charles
Burks, personal communication). Burks hypothe-
sized that the conidia of this fungus support neo-
nate larval development, while raisins alone do
not. Other storage insects are known to supple-
ment their diet by saprophagous feeding. Thus,
population growth of the red flour beetle, Tibo-
lium castaneum (Herbst), on several cereal grains
increases when dead eggs or adults of P inter-
punctella are added to the grain (LeCato & Fla-
herty 1973; LeCato 1975a). Supplementation
with dead moth eggs and adults also increases
population growth of the sawtoothed grain beetle,
Oryzaephilus surinamensis (L.), on peanuts, but
not on more suitable diets such as corn, wheat, or
rice (LeCato 1973). Facultative predation also has
been shown to enhance population growth of stor-
age insects. Population growth of T castaneum,
for instance, is increased on some commodities by
predation on the immature stages of 0. surina-
mensis (LeCato 1975b).
Fungus feeding, saprophagy, and predation
may sometimes provide the answer to the host
paradox, but less obvious factors also can be in-
volved. This is evidenced by the association be-
tween field infestation of carobs by the pyralid
moth Ectomyelois ceratoniae (Zeller) and the abil-
ity of C. cautella to infest carobs in storage (Dobie
1978). Carob pods crack as they are ripening on
the tree and become infested by E. ceratoniae,
which is attracted by a mold growing in the
cracked pods. After harvest, carobs may become
infested by a variety of stored-product insects, in-
cluding C. cautella. Dobie (1978) found that previ-
ous field infestation has a marked effect on the
suitability of carobs as a food for C. cautella, an ef-
fect that cannot be attributed to mechanical dam-
age, because the carobs used in his experiments
were coarsely ground. Ovipositing females and
first instars of C. cautella show a preference for
previously infested carobs, on which survival is
much higher. Dobie postulated that factors di-
rectly associated with field infestation must ren-
der carobs attractive to C. cautella and aid in lar-
Development of storage insects in commercial
settings on commodities that fail to support devel-
opment in the laboratory may occur more com-
monly than suggested by the meager number of
references in the literature. Under-reporting of
this phenomenon may arise from a reluctance to
publish data perceived to be negative. An incisive
discussion on the importance of negative data and
the potential consequences for scientific theory of
withholding publication can be found in an essay
by Gould (1993). Even cursory observations of the
host paradox should be reported to document fre-
quency of occurrence and perhaps stimulate stud-
ies directed toward solutions. Such studies would
inevitably provide better understanding of popu-
lation dynamics, which would in turn lend sup-
port to better management of insects in commer-
cial storage situations.
We are indebted to the management of U. S.
Nutraceuticals and especially to Paul Cowin,
manager of the La Belle warehouse, for making
facilities available and for cooperation in the re-
search. Melanie Gray assisted with many aspects
of the study, and we appreciate her untiring ef-
forts in rearing insects, setting up experiments,
making observations, and tabulating data. We are
especially indebted to Charles Burks (USDA-
ARS, Fresno, CA) for sharing unpublished data
from his studies of P interpunctella on raisins. Fi-
nally, we thank Stephen Ferkovitch (USDA-ARS,
Gainesville, FL), Paul Flinn (USDA-ARS, Man-
hattan, KS), and two anonymous reviewers for
critical review of an earlier version of the manu-
script and for helpful suggestions. The use of
trade, firm, or corporation names in this publica-
tion is for the information and convenience of the
reader. Such use does not constitute an official en-
dorsement or approval by the United States de-
partment of Agriculture or the Agricultural Re-
search Service of any product or service to the ex-
clusion of others that may be suitable.
AMERICAN ASSOCIATION OF CEREAL CHEMISTS. 2003.
Approved methods of the American Association of Ce-
real Chemists (AACC). AACC, St. Paul, MN. 1, 200 pp.
ARBOGAST, R. T. 1991. Beetles: Coleoptera, pp.131-176
In J. R. Gorham [ed.], Ecology and Management of
Food Industry Pests. FDA Bulletin 4. Association of
Official Analytical Chemists, Arlington, VA. 595 pp.
ARBOGAST, R. T., P. E. KENDRA, R. W. MANKIN, AND R.
C. MCDONALD. 2002. Insect infestation of a botani-
cals warehouse in north-central Florida. J. Stored
Prod. Res. 38: 349-363.
BENNETT, B. C., AND J. R. HICKLIN. 1998. Uses of saw
palmetto (Serenoa repens, Arecaceae) in Florida.
Econ. Bot. 52: 381-393.
Cox, P. D. 1975. The suitability of dried fruits, almonds,
and carobs for the development of Ephestia figu-
lilella, E. calidella and E. cautella. J. Stored Prod.
Res. 11: 229-233.
DOBIE, P. 1978. The effects of previous field infestation
upon Ephestia cautella (Walker) and Lasioderma
serricorne (F.) infesting carobs. J. Stored Prod. Res.
GOLDEN SOFTWARE. 1999. Surfer 7 user's guide. Golden
Software, Inc., Golden, CO. 619 pp.
GOULD, S. J. 1993. Cordelia's Dilemma. Nat. Hist. 2/93:
HOWE, R. W. 1957. A laboratory study of the cigarette
beetle, Lasioderma serricorne (F.) (Col., Anobiidae)
with a critical review of the literature on its biology.
Bull. Entomol. Res. 48: 9-56 + 2 plates.
KOCH, E. 2001. Extracts from fruits of saw palmetto
(Sabal serrulata) and roots of stinging nettle (Urtica
dioica): viable alternatives in the medical treatment
of benign prostatic hyperplasia and associated lower
urinary tract symptoms. Planta Med. 67: 489-500.
LECATO, G. L. 1973. Sawtoothed grain beetle: Popula-
tion growth on peanuts stimulated by eating eggs or
adults of the Indianmeal moth. Ann. Entomol. Soc.
Amer. 66: 1365.
LECATO, G. L. 1975a. Red flour beetle: Population
growth on diets of corn, wheat, rice, or shelled pea-
nuts supplemented with eggs and adults of the Indi-
anmeal moth. J. Econ. Entomol. 68: 763-765.
LECATO, G. L. 1975b. Predation by red flour beetle on saw-
toothed grain beetle. Environ. Entomol. 4: 504-506.
LECATO, G. L. 1978. Infestation and development by the
cigarette beetle in spices. J. Georgia Entomol. Soc.
LECATO, G. L., AND B. R. FLAHERTY. 1973. Tribolium
castaneum progeny production and development on
diets supplemented with eggs or adults of Plodia in-
terpunctella. J. Stored Prod. Res. 9: 199-203.
MANKIN, R. W., R. T. ARBOGAST, P. E. KENDRA, AND
D. K. WEAVER 1999. Active spaces of pheromone
traps for Plodia interpunctella (Lepidoptera: Pyral-
idae) in enclosed environments. Environ. Entomol.
RICHARDS, O. W., AND W. S. THOMSON. 1932. A contri-
bution to the study of the genera Ephestia Gn. (in-
cluding Strymax Dyar), and Plodia Gn. (Lep.
Phycitidae), with notes on parasites of the larvae.
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Silhacek, D. L., AND G. L. Miller. 1972. Growth and de-
velopment of the Indian meal moth. Plodia inter-
punctella (Lepidoptera: Phycitidae), under
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Florida Entomologist 88(3)
Sivinski & Pereira: Wing Markings in Fruit Flies
DO WING MARKINGS IN FRUIT FLIES
(DIPTERA: TEPHRITIDAE) HAVE SEXUAL SIGNIFICANCE?
JOHN SIVINSKI' AND RUI PEREIRA2
1USDA-ARS, CMAVE, 1600/1700 SW 23rd Dr., Gainesville, FL 32604
2Entomology and Nematology Dept., University of Florida, Gainesville, FL 32611
The patterned wings of tephritid fruit flies often are moved in complex manners during sex-
ual encounters. However, there are few cases of sexual dimorphism, and wing movements
also may occur in non-sexual contexts. There was no evidence that enhancing or obliterating
the patterns on the wings of male Caribbean fruit flies, Anastrepha suspense (Loew), had
any effect on their sexual success. There is convergence in wing patterns with another
Dipteran family, the distantly related Bombyliidae. Additional studies of mating systems
with this family might illuminate the significance of similar wing patterns in tephritids.
Key Words: sexual selection, mate choice, sexual signal, insect vision, Bombyliidae, court-
Las alas moteadas de las moscas tefritidas de fruta a menudo son movidas de una manera
compleja durante los encuentros sexuales. Sin embargo, hay pocos casos de dimorfismo
sexual, y los movimientos de las alas pueden occurir en un context no sexual. No hubo evi-
dencia que el incremento o eliminaci6n de los patrons sobre las alas de los machos de la
mosca de la fruta del Caribe, Anastrepha suspense (Loew), tuvo un efecto sobre el exito
sexual. Hay una convergencia de los patrons de alas con otra familiar en el orden Diptera,
la familiar Bombyliidae, que esta relacionada lejanamente. Estudios adicionales de los siste-
mas de apareamiento con esta familiar podrian exclarecer el significado de los patrons simi-
lares de alas en los tefritidos.
The wings of tephritid fruit flies, often intri-
cately patterned with spots, stripes, and blotches,
are both lovely and mysterious. Within the super-
family Tephritoidea, only the wings of the Lon-
chaeidae are typically unmarked (Sivinski 2000),
and in the Tephritidae, relatively few species,
such as some Bactrocera spp. and Neospilota spp.,
have largely hyaline wings (e.g., Foote et al.
1993). Yet the significance(s) of these common and
complex colorations is obscure.
In many tephritids, specialized wing move-
ments occur in a sexual context (Sivinski et al.
2000). Wings are moved rapidly to create acoustic
signals and perhaps to waft pheromones (e.g., Siv-
inski et al. 1984), but are also more slowly tilted
and/or held away from the body in a variety of mo-
tions and postures (Headrick & Goeden 1994).
These have been described as: (1) arching- the
wings are held over the dorsum, slightly spread,
and arched from the base to the apex such that
the tips nearly touch the substrate; (2) enanation-
the extension of both wings away from the body
simultaneously; (3) hamation- the movement of
the wings together over the dorsum or while they
are extended away from the body; (4) lofting- both
wings are extended upward 90 degrees above the
substrate and supinated up to 90 degrees; and 5)
supination- bringing the wing forward perpendic-
ular to the long axis of the body while the ventral
surface of the wing is turned to face anterior such
that the costal margin of the wing is dorsal (White
et al. 2000).
It is tempting to hypothesize that elaborate
wing patterns and complex wing movements con-
tribute to visual sexual signals (e.g., Bush 1969),
and wing coloration, movements and mating sys-
tems frequently are correlated in Californian te-
phritid genera (Headrick & Goeden 1994). If pat-
terns are sexual signals, it may be no coincidence
that clear-winged lonchaeids are the only tephri-
toid family that appears frequently to mate in
aerial swarms where wing patterns are unlikely
to serve a communicative function (McAlpine &
Munroe 1968; Sivinski 2000). However, there are
several inconsistencies in the wing pattern and
movement as sexual signal argument. Sexual di-
morphisms might be predicted in courtship sig-
nals directed by males to females, but differences
in visible-light wing patterns are relatively rare,
although there are some striking exceptions. For
example, Aciurina idahoensis Steyskal females
have striped wings and males spotted, and in the
related A. semilucida (Bates), female wings are
striped and male wings fully infuscated (Headrick
Florida Entomologist 88(3)
& Goeden 2000). In some instances, e.g., Trupa-
nea spp., dimorphisms are the opposite of expec-
tation with male wings fainter or having fewer
markings (Foote et al. 1993). Only the wings of
two species, the Caribbean fruit fly, Anastrepha
suspense (Loew) and the Mediterranean fruit fly,
Ceratitis capitata (Wiedemann) have been exam-
ined for ultra-violet reflectance and transmit-
tance (Sivinski et al. 2005). There is little UV
transparency in the wings, and there is no sexual
dimorphism when placed against a non-UV reflec-
tive background such as the leaves from which
males are likely to be sexually signaling.
Other objections against patterned wings per-
forming simple sexual signals include the com-
plex wing movements of females, and the wing
motions by males and females in apparently non-
sexual situations. For example, in the genus
Goedenia both sexes exhibit hamation throughout
the day while grooming, resting, and feeding (e.g.,
Goeden 2002), although in such species there are
often male wing movements unique to courtship
(Headrick & Goeden 1994). In addition, markings
are occasionally known to serve non-sexual pur-
poses. When seen from behind, the wing patterns
of Zonostemata vittigera (Coquillett) and Rhago-
letis zephyria Snow create the illusion of a salticid
spider seen face on and the resemblance deters
spider attacks (Greene et al. 1987, Mather & Roit-
In order to test the hypothesis that wing pat-
terns have been sexually selected and contribute
to sexual success, we first quantified the design of
wing markings among North American Tephrit-
dae and contrasted these markings to those of a
distantly related brachyceran fly family, the Bom-
byliidae. The latter family was chosen for compar-
ison and contrast because of the large number of
species bearing wing patterns and its distant phy-
logenetic relationship to fruit flies. We then per-
formed an experiment designed to test the impor-
tance of wing patterns in maleA. suspense sexual
MATERIALS AND METHODS
The Nature and Distribution of Wing Patterns
We quantified wing marking patterns in the
Tephritidae and Bombyliidae in the following
manner. First, illustrations of wings were roughly
divided into quadrants: frontal-distal, frontal-
proximal, trailing-distal, and trailing-proximal.
Then, the markings in each quadrant were char-
acterized as either clear, dark, spotted, striped, or
stellate (clear spots on a dark background) and
given a numeric value depending on the patterns
location on the wing (i.e., a lack of markings in the
frontal-distal section would be given the numeric
value of 1, in the frontal-proximal the same condi-
tion would be characterized as 6, in the trailing-
distal it would be 11 and in the trailing-proximal
16). Wings were then categorized by the combined
nature of the markings in each quadrant, e.g.,
dark in frontal proximal and clear in all others or
striped in all quadrants but trailing-proximal.
Thus a completely hyaline wing would be de-
scribed by the combined numbers listed above
and have the designation 161116.
The samples of wings were obtained from large
taxonomic works (Tephritidae; Foote et al. 1993
and Bombyliidae; Hull 1973). The tephritid sam-
ples included a species from every North Ameri-
can genus (n = 57 in 3 subfamilies), and multiple
species if there was diversity of wing pattern
within the genus. Although we attempted to cap-
ture pattern diversity at the generic level, this
method did not quantify the actual proportions of
any particular pattern at the species level. For ex-
ample, if genus X has 10 species, 9 of which have
stripped wings and one spotted, both stripes and
spots would be included in the data by a single ex-
ample. The bombyliid sample contained for the
most part single species from each of the 193 gen-
era in 14 subfamilies, but multiple species were
included when divergent wing patterns were ap-
parent. However, we did not have access to the
wing patterns of every species and as a result we
were more likely to have underestimated the di-
versity in wing pattern in this family than in the
Tephritidae. Because of the shortcomings in the
samples, the results should be viewed as illustrat-
ing possible qualitative examples of convergence
and divergence in wing patterns.
Sexual Success Following Wing Pattern Manipulation
The role of wing pattern in male sexual success
was investigated by either obliterating or enhanc-
ing wing markings. First, virgin female A. sus-
pensa, 15-21 d old, were transferred from 20 x 20
x 20-cm screen cages to smaller cylindrical screen
cages (6.3 cm x 8.8 cm) prior to the experiment.
Temperatures throughout the maturation and ex-
perimental periods were 25 1C and relative hu-
midity 55% 5%. Three mature males 15-21 d old
that had been treated in the three different man-
ners described below were then added to the cage
and their sexual successes noted. The three treat-
ments were: (1) males removed from larger hold-
ing cage, chilled and then placed on a plastic sheet
that had been stretched over ice; (2) dark wing
markings on similarly treated males painted over
with a brown India ink artists pen (Faber Castell,
Pitt artist pen, medium point, brown, Cleveland,
OH 44125); and (3) the hyaline spaces between
dark wing markings filled in with the same ink. It
was difficult to obtain a marking substance that
would adhere to tephritid wings. India ink was
the best of several alternatives, but even this cov-
erage deteriorated rapidly over time. Because of
this, males were marked the morning prior to sex-
Sivinski & Pereira: Wing Markings in Fruit Flies
ual exposure in the afternoon (during the last 4 h
of the photoperiod). There were 100 replicates and
the sexual successes of the various treatments
were compared by contingency X2test (Zar 1974).
Male characteristics other than wing pattern,
specifically large male size, are known to influ-
ence sexual success (e.g., Sivinski et al. 1984). Be-
cause of this, we measured the wing lengths of all
three males in each cage and they were given a
relative rank. The summed ranks of successful
males were then compared through a X2 test to an
expected mean rank n replicates (expected prod-
uct of rank = 2 x 100 = 200) had mating occurred
regardless of size. There might also have been an
interaction of pattern and size, so that a small
male that suffered in competition with a larger ri-
val overcame this disadvantage with a more at-
tractive wing pattern. This possibility was exam-
ined with a Mann-Whitney nonparametric U-test
(Zar 1974) by comparing the rank-sizes of mating
males that had their patterns emphasized with
ink and those whose patterns had been obliter-
ated. Specifically, we looked to see if a male with
one painted treatment was more likely to mate
when smaller than was a male with the other
The Nature and Distribution of Wing Patterns
Keeping in mind the differences in the te-
phritid and bombyliid samples, there are some
suggestive similarities in the types of wing pat-
terns and interesting differences in their pur-
ported distribution within their respective fami-
lies (Fig. 1). Unmarked wings were more common
in the Bombyliidae (36%), as were fully infuscated
patterns. Pattern diversity appeared to be greater
in the bombyliids with 42 patterns other than all
hyaline displayed by 126 species (0.33 patterns /
species) as opposed to 12 patterns in 61 species of
Tephritidae (0.20 patterns / species). The majority
of genus-level wing patterns in the Tephritidae
were stellate or barred, with a smaller number of
spotted and darkened-costal region patterns. Cer-
tain patterns were typical of different tephritid
subfamilies: 87% of Trypetinae wings patterns
could be characterized as barred, while the diver-
sity in the Dacinae and Tephritinae was greater.
The Dacinae is relatively species-poor in North
America and excluded from further discussion.
There were nine different wing patterns found in
the Tephritinae, but the most common were stel-
late (29%) and, again, barred (36%).
Sexual Success Following Wing Pattern Manipulation
There were no significant differences in sexual
success among wing treatments: Mated (un-
treated) = 37; (pattern enhanced) = 32; (pattern
0 10 20 30
% of Wng Pattems
Fig. 1. The more common wing patterns found in Te-
phritidae (from Foote et al. 1993) and Bombyliidae (Hull
1973) and their proportions of the total number of pat-
tered wings sampled.
obliterated) = 31 (X2 = 0.62; P > 0.50). There was
no evidence that male size by itself played a role
in male sexual success (mean size rank of success-
ful males= 1.97; expected value from random
mating= 2.0; x2 = 0.09; P > 0.95). While males who
mated and whose patterns had been enhanced
tended to be relatively larger than males that
mated and had their patterns obliterated (mean
rank= 1.69 and 2.11, respectively), the difference
was not significant (U = 252; Z = 1.56, P = 0.12).
Given these results, wing markings remain
lovely and mysterious; there was no evidence that
markings played a role in the abilities of males to
mate. However, we do not wish to overstate our
results and conclude that markings have no com-
municative or sexual importance. Negative evi-
dence is often difficult to interpret and, given the
limitations of small cage experiments in the labo-
ratory such as restricted movement and atypi-
cally high densities, and the likely inexact match
of the brown ink to the color of the wing markings,
a different experiment may well yield different re-
sults. That being said, the present result of find-
ing no diminution of mating success following
rather gross manipulation of the markings sug-
gests that alternative explanations for the evolu-
tion of wing patterns in the Diptera should at
least be considered (see True 2003).
One alternative, spider mimicry, was men-
tioned in the introduction. Also, the distinctive
outline of an animal may be obscured by a disrup-
tive pattern of stripes and spots (Cott 1940) and a
resting fly with patterned wings might be thus
camouflaged. Beside sexual signaling and adap-
tive coloration, another hypothesis is that pig-
ments such as melanin are structural components
of the wings and that any resulting visual effect is
fortuitous and without significance. For example,
melanin pigmented surfaces warm up faster and
cool down more quickly in a variety of insects, and
melanic cuticle can be more resistant to abrasion
than unpigmented cuticle (Majerus 1998). The
numerous instances of coloration along the lead-
ing edge of the wing in the vicinity of the costal
vein might be consistent with pigments strength-
ening a region that receives unusual stress. It
may be that all of these factors play a role, and
that ". .. wing displays and patterns are part of a
dynamic system involving reproductive behavior,
crypsis, and thermoregulation" (Headrick &
The seeming convergence in wing patterns be-
tween the Tephritidae and the Bombyliidae, at
least in type if not frequency of design, might offer
an opportunity for illuminating comparisons. Lit-
tle has been published on the mating systems of
bee flies. Males in Comptosia sp. near latealis
Newman, perch in clearings and dart at nearby
flying insects (Yeates & Dodson 1990). The wings
in this genus are typically darkly pattered (Hull
1973), but male-male interactions occur in flight,
as do at least some of the matings, which may ar-
gue against wing markings having any signifi-
cance as courtship signals. Males ofLordotus pul-
chrissimus Williston form mating swarms (Toft
1989). The wings of this genus are generally hya-
line (Hull 1973) and so are consistent with an
aerial lack of signaling opportunity.
We thank Hoa Nguyen for his skills in painting
fly wings, Gina Posey for preparing the illustra-
tion, and Valerie Malcolm for preparing the
manuscript. James Lloyd, David Headrick, and
an anonymous reviewer made many valuable
comments on an earlier draft. Comstock Publish-
ing Associates and the Smithsonian Institution
Press kindly allowed the reproduction of wing il-
BUSH, G. 1969. Mating behavior, host specificity, and
the ecological significance of sibling species in fru-
givorous flies of the genus Rhagoletis (Diptera: Te-
phritidae). Amerian Nat. 103: 669-672.
COTT, H. 1940. Adaptive Coloration in Animals. Meth-
uen & Co., London.
FOOTE, R., F. BLANC, AND A. NORRBOM. 1993. Handbook
of the Fruit Flies (Diptera: Tephritidae) of America
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GOEDEN, R. 2002. Life history and descriptions of adults
and immature stages of Goedenia stenoparia (Stey-
skal) (Diptera: Tephritidae) on Gutierrezia califor-
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californica (Nuttal) (Asteraceae) in southern Cali-
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GREENE, E., L. ORSACK, AND D. WHITMAN. 1987. A te-
phritid fly mimics the territorial displays of its jump-
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HEADRICK, D., AND R. GOEDEN. 1994. Reproductive be-
havior of California fruit flies and the classification
and evolution of Tephritidae (Diptera) mating sys-
tems. Studia Dipterol. 1: 194-252.
HEADRICK, D., AND R. GOEDEN. 2000. Behavior of flies
in the subfamily Tephritinae, pp. 671-707 In M.
Aluja and A. Norrbom [eds.] Fruit Flies (Tephriti-
dae): Phylogeny and the Evolution of Behavior. CRC
Press, Boca Raton, FL.
HULL, F. 1973. Bee flies of the World: the Genera of the
Family Bombyliidae. Smithsonian Institution Press,
MCALPINE, J., AND D. MUNROE. 1968. Swarming of lon-
caeid flies and other insects, with descriptions of new
species of Lonchaeidae (Diptera). Canadian Ento-
mol. 100: 1154-1178.
MAJERUS, M. 1998. Melanism: Evolution in Action. Ox-
ford University Press. Oxford, UK
MATHER, M., AND B. ROITBERG. 1987. A wolf in sheep's
clothing: tephritid fruit flies mimic spider predators.
Science 236: 308-310.
SIVINSKI, J. 2000. Breeding habits and sex in families
closely related to the Tephritidae: opportunities for
comparative studies of the evolution of fruit fly be-
havior, pp. 23-37 In M. Aluja and A. Norrbom [eds.],
Fruit Flies (Tephritidae): Phylogeny and the Evolu-
tion of Behavior. CRC Press, Boca Raton, FL.
SIVINSKI, J., M., ALUJA, G. DODSON, A. FREIDBERG, D.
HEADRICK, K. KANESHIRO, AND P. LANDOLT. 2000.
Topics in the evolution of sexual behavior in the
Tephritidae, pp. 751-792 In M. Aluja and A. Norr-
bom [eds.], Fruit Flies (Tephritidae): Phylogeny
and the Evolution of Behavior. CRC Press, Boca
SIVINSKI, J., T. BURK, AND J. C. WEBB. 1984. Acoustic
courtship signals in the Caribbean fruit fly, Anas-
trepha suspense (Loew). Anim. Behav. 32: 1011-
SIVINSKI, J., H. KLUG, J. SHAPIRO, J. LANE, AND R. MAN-
KIN. 2005. Ultraviolet reflectance on the heads and
wings of Anastrepha suspense (Loew) and Ceratitis
capitata (Wiedemann) (Diptera: Tephritidae). Stu-
dia Dipterol. 11: 313-322.
TOFT, C. 1989. Population structure and mating system
of a desert bee fly (Lordotus pulchrissimus; Diptera:
Bombyliidae). 1. Male demography and interactions.
Oikos 54: 345-358.
TRUE, J. 2003. Insect melanism: the molecules matter.
Trends Ecol. and Evol. 18: 640-647.
WHITE, I., D. HEADRICK, A. NORRBOM, AND L. CARROLL.
2000. Glossar, pp. 881-924 In M. Aluja and A. Norr-
bom [eds.], Fruit Flies (Tephritidae): Phylogeny and
Evolution of Behavior. CRC Press, Boca Raton, FL.
YEATES, D., AND G. DODSON. 1990. The mating system
of a bee fly (Diptera: Bombyliidae). I. non-resource-
based hilltop territoriality and a resource-based al-
ternative. J. Insect Behav. 3: 603-617.
ZAR, J. 1974. Biostatistical Analysis. Prentice Hall Inc.
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Florida Entomologist 88(3)
PARTITIONING NATIVE AND AUGMENTATIVE TRICHOGRAMMA
PLATNERI (HYMENOPTERA: TRICHOGRAMMATIDAE) PARASITISM OF
AMORBIA CUNEANA (LEPIDOPTERA: TORTRICIDAE) EGG MASSES IN
SOUTHERN CALIFORNIA AVOCADO ORCHARDS
JEFFREY Y. HONDA
Department of Biological Sciences, San Jose State University, One Washington Square, San Jose, CA 95192-0100
The objective of this experiment was to test the
efficacy of augmentative releases of Trichogramma
platneri Nagarkatti against the avocado pest,
Amorbia cuneana Walsingham, and to determine
natural parasitism rates concurrently in the field by
examining parasitism of sentinel A. cuneana egg
masses placed in avocado orchards. The released
wasps used in the experiments were marked with a
unique phosphoglucose mutase (PGM) allele from T
minutum Riley that is absent in the coastal, native
populations of the closely related T platneri (Pinto
et al. 1992). Wasps were produced by introducing
the unique PGM marker into native T platneri par-
asitoids by repeated backcrossing (>20 genera-
tions), to ensure that released lines were different
only at the PGM locus. Wasps from each generation
were electrophoresed for the unique PGM locus by
the methods ofKazmer & Luck (1995) to ensure cul-
ture purity and quality assurance.
Studies were done in three blocks of'Haas' av-
ocado on ranches located at Temecula (Riverside
Co.), Vista (San Diego Co.), and Moorpark (Ven-
tura Co.), California. Orchard characteristics
were quite variable between the Moorpark site
and the Temecula/Vista sites. Temecula and Vista
orchards were categorized as 'mature' and pos-
sessed trees that were at least 25 years in age,
over 12.0 m in height and spaced between 7.5 and
10.5 m apart. In contrast the Moorpark orchard
was categorized as 'immature' and possessed
trees that were approximately 10 years old, less
than 4.5 m in height, and spaced between 4.5 and
6.0 m apart. Parasitism and dispersal was evalu-
ated by monitoring sentinelA. cuneana egg mass
traps placed in 10 trees per orchard. Egg-masses
were placed in nine avocado trees (30 egg masses
per tree) arranged in the shape of a cross. Thus,
there were two trees per arm of the cross and one
tree at the center beneath which wasps were re-
leased. An additional tree located 10 rows east of
the release tree containing egg masses served as a
control. Within each tree, 15 egg-masses were ar-
ranged in the tree canopy 0.5-1.5 m above the
ground and 15 egg-masses were placed 3.0-6.0 m
above the ground in the tree canopy.
Egg-masses were placed in the ten experimen-
tal trees on day one. These egg masses were used
to determine the amount of parasitism by resi-
dent T platneri prior to release of the marked
line. These egg-masses were replaced with fresh
egg-masses on day four and followed immediately
by the release of marked T platneri in the center
tree of each plot. The second egg-mass group was
collected on day seven of the experiment and re-
placed with fresh egg-masses that were then col-
lected on day 10 of the experiment. Thus, a total of
900 egg masses were placed in each orchard over
a 10 d sampling period.
Approximately 40,522 wasps were released
under the centrally located release tree. Egg-
mass cards were collected and blackened eggs in-
dicating parasitism were separated to collect par-
asitoids. Egg parasitism was calculated as the
percentage of egg masses with at least one egg
parasitized. Emerging parasitoids were snap fro-
zen in liquid nitrogen and electrophoresed for
PGM to determine if they were from the resident
or released populations. Differences in the num-
ber of egg-masses parasitized between canopy
treatments within trees, parasitism rate differ-
ences between adjacent and non-adjacent trees
within an orchard, and comparisons between plot
sites (mature vs. immature orchards) were ana-
lyzed by ANOVA (SAS Institute Inc. 1988).
Egg-masses placed in the experimental groves
prior to release of the marked wasps remained
unparasitized. Released wasps constituted al-
most all of the recorded parasitism as indicated
by the presence of the unique PGM allele collected
from wasps emerging from the sentinel egg-
masses. A total of three sentinel egg-masses from
a single tree at the Moorpark site were collected
which contained wasps that had PGM alleles dif-
fering from those of the released T platneri, how-
ever, they were similar to those of resident coastal
T platneri (Pinto et al. 1992), indicating that
these wasps were the progeny of resident T plat-
neri. This represents only a small percentage
(0.33%) of the observed parasitism in this plot.
None of the 1,800 sentinel egg-masses placed in
10 trees over the three monitoring periods in ei-
ther the Temecula or Vista plots showed T plat-
neri allozyme patterns typical of resident wasp
Augmentative parasitism rates of the sentinel
egg-masses in the upper and lower portions of the
trees for all three locations were not significant
(F,108 = 3.31 P > 0.05). Parasitism rates of egg
masses collected on day seven were significantly
higher than those collected on day 10 for all three
orchards studied with 78%, 89%, and 94% of the
total parasitism observed occurring within three
days of release for the Temecula, Vista, and Moor-
park sites, respectively (Table 1). At each site,
Florida Entomologist 88(3)
TABLE 1. NUMBERS OF EGG MASSES PARASITIZED BY TRICHOGRAMMA PLATNERI BEFORE, DURING, AND AFTER RELEASE
IN AVOCADO TREES IN THREE ORCHARDS.
Treatment Release (eggs collected Post Release (eggs collected
Release Site Pre Release three days after release) six days after release)
Vista 0/270 71/270 9/270*
(0%) (26.3%) (3.3%)
Temecula 0/270 64/270 18/270*
(0%) (23.7%) (6.6%)
Moorpark 0/270 150/270 10/270*
(0%) (55.6%) (3.7%)
*Each row indicates significance at the 0.05 level for chi-square tests of independence between release and post release treat-
ments for each of the three orchards.
parasitism rates between the outermost trees and
trees adjacent to the release tree decreased signif-
icantly (F1,,, = 41.02 P < 0.05). Thus, parasitoid
searching efficiency appeared to be limited to the
few trees in close proximity to the release tree and
only for a brief period (<3 days) after release.
Parasitism rates appear to be affected by plant
complexity and interplant distance based on this
experiment as the Moorpark plot with smaller
and closely spaced trees had higher levels of par-
asitism (F2,108 = 11.79 P < 0.05). Larger, more com-
plex trees may cause parasitoids to search more
area per host encounter than in smaller less com-
In conclusion, although indigenous T platneri
were extremely scarce, the use of unique alloz-
ymes incorporated into wasps released in the field
may be an effective tool to accurately determine
indigenous and augmentative parasitism rates
concurrently. Moreover, augmentative releases
are most effective three days after release and
only in those trees immediately adjacent to para-
sitoid release points. Thus, point releases of T
platneri should not be spaced more than a few
trees apart and should be performed every 3-4
days againstA. cuneana in avocado.
The use of a unique PGM allozyme marker was
introduced into a culture of Trichogramma plat-
neri used for augmentative field releases in an ef-
fort to distinguish between native and augmenta-
tive parasitism against Amorbia cuneana in avo-
cado orchards. Although native parasitism rates
were extremely low, the marker was useful in dis-
tinguishing native parasitoids from those released
in the field. Augmentative releases were most ef-
fective up to three days post release and in those
trees adjacent to the release trees. Orchard com-
position appears to affect parasitoid efficiency.
KAZMER, D. J., AND R. F. LUCK. 1995, Size fitness rela-
tionships in a field population of the egg parasitoid
Trichogramma pretiosum: Ecol. 76: 412-425.
PINTO, J. D., KAZMER, D. J., G. R, PLATNER, AND C.A.
SASSAMAN. 1992, Taxonomy of the Trichgramma
minutum complex (Hymenoptera: Trichogramma-
tidae): allozymic variation and its relationship to re-
productive and geographic data: Ann. Entomol. Soc.
Am. 85: 413-422.
SAS INSTITUTE INC. 1988, SAS User's Guide: Statistics,
Version 6 Edition. SAS Institute Inc., Cary, NC.
OCCURRENCE OF LARRA BICOLOR
(HYMENOPTERA: SPHECIDAE), ECTOPARASITE OF
MOLE CRICKETS (SCAPTERISCUS SPP), IN COASTAL MISSISSIPPI
DAVID W. HELD
Mississippi State University, Coastal Research & Extension Ctr., 1815 Popps Ferry Road, Biloxi, MS 39532
Larra bicolor Fabricius (Hymenoptera: Sphe-
cidae) is an immigrant species native to South
America but introduced into Florida, Hawaii, and
Puerto Rico for control of pest mole crickets
(Frank et al. 1995; Frank & Sourakov 2002).
Larra wasps are black with wings that are brown
to blue with light-colored markings on the head.
Larra analis Fabricius, which has a black abdo-
men with red typically at the tip, is native to Mis-
sissippi and the Gulf Coast region. Larra bicolor,
in contrast, has a solid red abdomen (Frank &
Sourakov 2002). The biology and ecology of this
species has been reviewed recently (Frank et al.
199; Frank & Sourakov 2002) and will not be dis-
The first successful relocation ofL. bicolor into
North America was made between 1981-1983,
when wasps were collected from Puerto Rico and
released into five sites in Florida. From these re-
leases, only wasps at the southernmost release
site (Ft. Lauderdale) became established (Frank
et al. 1995). Subsequent releases were made be-
tween 1988-1989 when presumably three species
of wasps, Larra bicolor, L. praedatrix, and L. god-
mani, were collected from Bolivia and released
along with parasitized hosts in three sites near
Gainesville (Frank et al. 1995). There was no evi-
dence that these releases were successful until L.
bicolor was observed feeding on Spermacoce verti-
cillata on the UF-Gainesville campus in 1993.
Based on morphological characteristics of these
wasps, they were presumably of Bolivian origin
(Frank et al. 1995).
Larra bicolor has been released in Tifton GA
and near Baton Rouge LA. Of these releases, only
those released in GA have become established (W.
Hudson & H. Frank, personal communication).
Apart from these sites, Florida, Puerto Rico, and
South America are the only other sources ofLarra
bicolor. This paper represents the first record of
the natural expansion ofL. bicolor outside of Flor-
ida, and the first record of this species in Missis-
On 29-IX-2004, hybrid bermudagrass (C. dac-
tylon x C. transvaalensis 'Tifway') plots were be-
ing evaluated for mole cricket damage at Great
Southern Golf Course, Gulfport, Harrison County,
MS when a digger wasp resembling Larra was ob-
served. This individual was not collected but its
presence prompted a subsequent survey of dam-
aged grass on that golf course. Three areas at
Great Southern with fresh mole cricket damage
were surveyed. Surveys were conducted by walk-
ing across mole cricket damaged areas of grass
while looking for wasps resting on the turf. A soap
solution, 30 ml of dishwashing soap per liter of
water, was prepared in a 900-ml spray bottle with
a trigger and used to collect wasps. When at rest
on the turf, a wasp was shot with the soap solu-
tion repeatedly until dead, at which time it was
collected and preserved in alcohol. Two digger
wasps, L. analis and L. bicolor, were collected that
day using this method. The University of Florida
Insect Taxonomy Laboratory confirmed the iden-
tities of both species, and these were deposited as
voucher specimens in the Mississippi Entomolog-
ical Museum at Mississippi State University.
The same three damaged sites at Great South-
ern Golf Course were surveyed again on 1-X-2004.
When a wasp was spotted, it was collected with
the soap solution as before. Two L. bicolor were
collected; of which one was exiting a mole cricket
burrow. Two mole crickets also were observed
moving across the grass. This is consistent with
the previously described hunting behavior of
Larra spp. (Frank & Sourakov 2002).
Because this was a first record of this species
in Mississippi, it seemed important to determine
whether L. bicolor was present on other golf
courses in coastal Mississippi. On 11-X-2004,
three additional courses in Harrison County were
surveyed. The first, Bay Breeze Golf Course, also
is located on the Mississippi Sound in Biloxi. Hy-
brid bermudagrass 'Tifway' tees, greens, and fair-
ways that had fresh damage from mole crickets
were surveyed for L. bicolor as previously de-
scribed. Five wasps were observed on four differ-
ent holes, but only three, all L. bicolor, were col-
The second course surveyed that day was the
President-Broadwater Golf Course in Biloxi. This
course had abundant damage from mole crickets,
but no wasps were observed or collected. Unlike
Bay Breeze and Great Southern, this course had
no naturalized areas where blooming wildflowers
were present, nor were there any plantings of
blooming woody or herbaceous ornamentals. The
last course in Harrison County, Sunkist Country
Club, had only one area with mole cricket damage
and no Larra were observed or collected.
On 12-X-2004, the Bridges Golf Course at the
Casino Magic resort in Bay St. Louis, Hancock
County, MS was surveyed. This course was chosen
because it borders the Mississippi Sound and has
Florida Entomologist 88(3)
abundant naturalized areas of wildflowers. Areas,
primarily greens and tees, on each hole had some
mole cricket damage. No Larra were observed or
The last site surveyed was St. Andrews Golf
Course, Ocean Springs, Jackson County, MS on
18-X-2004. This course is located on the Missis-
sippi Sound and has a perennial mole cricket pop-
ulation. The three areas surveyed were on close
roughs of Bermuda grass near tees or greens, and
all had extensive damage caused by mole crickets.
One L. bicolor was collected from this course.
The results of these surveys indicate that
while L. bicolor is present in coastal Mississippi,
it is not abundant. Only those courses that were
adjacent to the Mississippi Sound had L. bicolor.
Perhaps these sites, being buffered by the coastal
waters, provide a suitable microclimate where
this wasp can successfully over winter.
I thank the staff of Great Southern Golf
Course, Sunkist Country Club, Bay Breeze Golf
Club, President Golf Course, St. Andrews Golf
Course, and The Bridges at Casino Magic for co-
operation with the survey. I thank Lionel Stange,
Lyle Buss, and Howard Frank for identification of
collected specimens. Richard Brown, Linda An-
drews, and Jianzhong Sun (MS State University)
provided helpful comments on an earlier draft of
this manuscript. This paper is No. J10668 of the
Mississippi State Agricultural Experiment Sta-
Larra bicolor (Hymenoptera: Sphecidae) is an
ectoparasite of exotic mole crickets (Scapteriscus
spp., Orthoptera: Gryllotalpidae). This wasp was
introduced into Florida as a biological control
agent, but natural spread of this insect has not
been reported outside of that state. In 2004, spec-
imens of Larra bicolor were collected from three
golf course sites in coastal Mississippi. This find is
the first record of this species in Mississippi and
represents the first record of natural movement of
this wasp outside of Florida.
FRANK, J. H., AND A. SOURAKOV. 2002. Larra wasps;
mole cricket hunters. University of Florida pub. No.
FRANK, J. H., J. P. PARKMAN, AND F. D. BENNETT. 1995.
Larra bicolor (Hymenoptera: Sphecidae), a biologi-
cal control agent of Scapteriscus mole crickets (Or-
thoptera: Gryllotalpidae), established in Northern
Florida. Florida Entomol. 78: 619-623.
ENHANCED EGG LAYING IN ADULT PREDATORS FED ARTIFICIAL DIET
SUPPLEMENTED WITH AN EMBYONIC CELL LINE DERIVED FROM EGGS
OF EPHESTIA KUEHNIELLA ZELLER (LEPIDOPTERA: PYRALIDAE)
STEPHEN M. FERKOVICH1 AND DWIGHT E. LYNN2
1USDA, ARS, CMAVE, 1700 SW 23rd Drive, Gainesville, FL 32608
2USDA, ARS, Insect Biocontrol Laboratory, Bldg. 011A, BARC-West, 10300 Baltimore, MD 20705-2350
Suboptimal fecundity in entomophagous in-
sects reared on artificial diet is a common prob-
lem and barrier in implementing cost-effective
large-scale production of beneficial insects for
augmentative biological control (Grenier et al.
1994). The artificial diets of a number of ento-
mophagous insects can only be improved by add-
ing insect tissues such as hemolymph to the diet
(Nettles 1990). The use of insect materials, how-
ever, is not feasible because of the labor required
and associated problems (e.g., melanization), and
other means of improving the diets are needed.
One obvious approach is to identify the com-
pounds in insect materials that are responsible
for improving fecundity, however, this is difficult
and none have been identified yet (Ferkovich &
Shapiro 2004a). Another approach is to use insect
cells to improve artificial diets for predators. In-
sect cell lines have been tested on several parasi-
toids with promising results (Rotundo et al. 1988;
Ferkovich et al. 1994; Hu et al. 1999; Heslin et al.
2005). The fecundity of the insidious flower bug,
Orius insidiosus (Say), reared on artificial diet
was improved with IPLB-PiE, a cell line derived
from eggs of the Indian meal moth, Plodia inter-
punctella (Hiibner) (Ferkovich & Shapiro 2004b).
Because insectaries generally produce the preda-
tor on eggs of the Mediterranean flower moth,
Ephestia kuehniella Zeller (Association of Natu-
ral Bio-control Producers), a cell line (Ek-x4V)
was recently developed from eggs ofE. kuehniella
(Lynn & Ferkovich 2004). In this study, we inves-
tigated the potential of using the Ephestia cell
line (Ek-x4V) to promote egg production of adults
of 0. insidiosus maintained on artificial diet.
The IPLB-PiE and the Ek-x4V cell lines were
cultured as described earlier (Lynn 1996; Lynn &
Ferkovich 2004). Briefly, the IPLBPiE cells were
grown in modified TNM-FH insect medium
(Sigma, St. Louis, MO) in 25-cm2 culture flasks for
7 days. For larger-scale culture of the cell lines,
the PiE cells were grown in 250 ml of medium in
500-ml magnetic spinner flasks (Bellco Glass,
Vineland, NJ) at 24.9E for 14 days. The Ek-x4V
cells were grown in SF900II medium (Invitrogen
Corp., Grand Island, NY) in 25-cm2 culture flasks
for 14 days but could not be cultured in the larger
spinner flasks because they grew as cellular ag-
gregates and the spinning motion of the flasks in-
terfered with their growth. Both cell lines were
centrifuged (1370 g for 3 min) in graduated coni-
cal glass tubes to obtain a pellet of cells. The pel-
lets were resuspended in distilled water and
washed 2x. Cells from each line were then bioas-
sayed in two tests.
The objective of the first bioassay was to deter-
mine if the Ek-x4V cells would affect the oviposi-
tion rate of females in a dose-response manner.
Washed cells were centrifuged to obtain 0.25, 0.5,
0.75, and 1.0 ml of soft pellets of cells which were
each homogenized with a hand-held homogenizer.
Aliquots (201l) of the homogenates were removed
and assayed for protein by the Lowry procedure
(Protein Assay Kit, Sigma, St. Louis, MO). Diet in-
gredients (0.33 g brewers yeast, 0.03 g sucrose,
0.18 g soy protein acid hydrolysate, 3.8 mg of 99%
palmitic acid (all from Sigma, St Louis, MO), 0.04
g fresh chicken egg yolk, and 0.08 g honey) were
added to each of the tubes to give a final volume of
1.2 ml. The diet was then encapsulated in Para-
film@ (25pl capsules) and bioassayed as described
earlier (Ferkovich & Shapiro 2004b). Adults (three
days after eclosion) were fed 3 mg ofE. kuehniella
eggs (Eph Eggs), two capsules of artificial diet
(AD), and two capsules of artificial diet + Ek cells
(Ek Cells) for six days. Each treatment diet con-
sisted of six females and four males, two Para-
film@ capsules of water (25pl each) and two cap-
sules (25pl each) of treatment diet with four repli-
cates per treatment. Diets were replaced daily and
mortality was recorded. At the end of the sixth day,
a green bean pod was placed in each jar as an ovi-
position substrate and the number of eggs ovipos-
ited during a 24-h period were recorded.
The objective of the second bioassay was to
compare artificial diet fortified with Ek-x4V cells
(Ek Cells) against diet fortified with the IPLB-
PiE cells (PiE Cells). Cells (0.74 ml, 52 mg pro-
tein) from each cell line were added to diet. Artifi-
cial diet (AD) and Ephestia eggs (Eph Eggs) treat-
ments were also included in the bioassay.
Data were analyzed by one-way ANOVA with
Dunnet's test for comparison of treatment means
with control and Newman-Keuls post test for
multiple mean comparisons (GraphPad Software,
San Diego, CA).
Females fed on the Ek Cells diet oviposited sig-
nificantly more eggs at the 0.75 ml-, and 1.0- ml
doses of cells per 1.2 ml of diet than those that fed
on the AD (F = 3.8, df=4, P = 0.02) (Fig. 1). In com-
paring the PiE Cells diet with the Ek Cells diet,
egg production on both diets approached that of
females fed Eph Eggs and both diets significantly
increased the average rate of oviposition relative
Florida Entomologist 88(3)
Eph Eggs AD 0.25 0,5 0.75 1.0
Volume of Cellular Pellet (ml)
Fig. 1. Comparison of mean oviposition by females
fed whole Ephestia eggs (Eph Eggs), artificial diet (AD)
and artificial diet supplemented with aliquots of Ek-
x4V h cells per 1.2 ml of diet. Dunnet's test was used to
compare the treatment means against the artificial diet;
asterisk indicates that the treatment means are signif-
icantly different from the artificial diet, (P < 0.05); error
bars = standard error.
to the control AD. Neither of the cell line-supple-
mented diets, however, was better than the other
in improving egg production (Fig. 2). The IPLB-
PiE cell line is easier to culture since it grows as a
suspension and lends itself to culture in spinner
flasks and higher densities of cells can be
achieved in less time. In contrast, the Ek-x4V line
grows as aggregates of cells composed of orga-
nized vesicles that did not grow well in spinner
flasks to attain the same level of cell densities as
the PiE line during the 14 day culture period.
EphEg AD PE Cd EXCU
Fig. 2. Comparison of mean oviposition by females
fed whole Ephestia eggs (Eph Eggs), artificial diet (AD),
artificial diet + Pie cells (PiE Cells), and artificial diet +
Ek cells (Ek Cells); bars with the same letter are not sig-
nificantly different (Newman-Keuls method, P > 0.05);
error bars = standard error.
25 Consequently, our present findings do not indicate
that the EK-x4V cell line affords an advantage
over the IPLB-PiE cell line as a diet supplement
20 for improving the fecundity of 0. insidiosus. The
PiE cell line appears to be a better candidate for
Future studies directed at adapting the PiE line to
15 E grow in cheaper, serum-free cell culture medium
S for use in a large scale fermentation system and
0 methods of preserving and packaging the cells for
10 evaluation in artificial diets by insectaries.
E We thank Drs. Terry Arbogast and Simon Yu
for helpful comments. We also appreciate the ex-
cellent technical assistance in this study of De-
Artificial diet supplemented with the Ek-x4V
cell line significantly enhanced the average rate
of oviposition relative to the control diet but was
not better than diet augmented with the IPLB-
PiE cell line. The Ek-x4V line grew as aggregates
of hollow vesicles of cells in contrast to the IPBL-
PiE line which grew as a suspension of unat-
tached cells and did not produce sufficient cell
growth in spinner flasks for large-scale produc-
tion of the cells. Therefore, the EK-x4V cell line
does not afford an advantage over the IPLB-PiE
cell line as a diet supplement for improving the fe-
cunduty of 0. insidiosus.
ASSOCIATION OF NATURAL BIO-CONTROL PRODUCERS.
2001. Supplier members, www.anbp.org, Santa Ana,
FERKOVICH, S. M., AND J. SHAPIRO. 2004a. Comparison
of prey-derived and non-insect supplements on egg-
laying of Orius insidiosus (Hemiptera: Anthoc-
oridae) maintained on artificial diet as adults. Biol.
Control 31: 57-64.
FERKOVICH, S. M., AND J. SHAPIRO. 2004b. Increased
egg-laying in Orius insidiosus (Hemiptera: Anthoc-
oridae) fed artificial diet supplemented with an em-
bryonic cell line. Biol. Control. 31: 11-15.
FERKOVICH, S. M., H. OBERLANDER, C. DILLARD, AND E.
LEACH. 1994. Embryonic development of an en-
doparasitoid, Microplitis croceipes (Hymenoptera:
Braconidae) in cell line-conditioned media. In Vitro
Cell Dev. Biol. 30A: 279-282.
HESLIN, L. M., R. A. KOPITTKE, AND D. J. MERRITT.
2005. The role of insect cell lines in an artificial diet
for the parasitoid wasp, Trichogramma pretiosum.
Biol. Control 33: 186-193.
GRENIER, S., P. D. GREANY, AND A. C. COHEN. 1994. Po-
tential for mass release of insect parasitoids and pred-
ators through development of artificial culture
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and J. L. Capinera [eds.], Pest Management in the
Subtropics: Biological Control-a Florida Perspective.
Intercept Publishers, Andover, Hampshire, England.
Hu, J. S., D. B. GELMAN, R. A. BELL, AND D. E. LYNN.
1999. In vitro rearing of Edovum puttleri, an egg
parasitoid of the Colorado potato beetle, on artificial
diets: effects of insect cell line-conditioned medium.
Arch Insect Biochem Physiol. 40: 173-182.
LYNN, D. E. 1996. Development and characterization of
insect cell lines. Cytotechnology 20: 3-11.
LYNN, D. E., AND S. M. FERKOVICH. 2004. New cell lines
from Ephestia kuehniella: characterization and sus-
ceptibility to baculoviruses, 5 pp. Journal of Insect
Science 4:9. Available online: insectscience.org/4.9.
NETTLES, W. C. 1990. In vitro rearing of parasitoids:
Role of host factors in nutrition. Arch. Insect Bio-
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Florida Entomologist 88(3)
IN VITRO PRODUCTION OF HYPHAE OF THE GRASHOPPER PATHOGEN
ENTOMOPHAGA GRYLLI (ZYGOMYCOTA: ENTOMOPHTHORALES):
POTENTIAL FOR PRODUCTION OF CONIDIA
SERGIO R. SANCHEZ PENA
Departamento de Parasitologia, Universidad Aut6noma Agraria Antonio Narro, Saltillo, Coahuila 25315, Mexico
Fungi of the Entomophaga grylli pathotype or
species complex (Zygomycota: Entomophthorales,
Entomophthoraceae) are ecologically obligate
parasites of grasshoppers and locusts (Ortho-
ptera); their host range includes many economi-
cally important orthopteran species worldwide
(Carruthers et al. 1994; Bidochka et al. 1995). The
species of the E. grylli complex (heretofore collec-
tively called E. grylli) are highly pathogenic and
can cause spectacular field epizootics. They have
attracted interest as insect control agents, in both
classical and augmentative biological control of
Orthoptera (Carruthers et al. 1994; Sawyer et al
1994; Bidochka et al. 1995). Penetration through
insect cuticle by germinating conidia is the uni-
versal invasion route of entomopathogenic fungi,
including E. grylli. In the E. grylli species com-
plex, no production of walled cells hyphaee or
conidia) has been reported to occur on the few ar-
tificial media that support its stable growth.
These conidia must be produced on hyphal conid-
Upon landing on a host, E. grylli conidia can
penetrate the cuticle with germ tubes and reach
the hemocoel; once there, the fungus produces
amoeboid protoplasts. These fragile, wall-less,
amoeboid vegetative cells are the invasive phase
in the orthopteran haemocoel (Ramoska et al.
1988; Carruthers et al. 1994). The wall-less condi-
tion of protoplasts seems to allow them to remain
undetected and escape the insect's immune sys-
tem (Roberts & Humber 1982). After proliferating
and killing the host, protoplasts produce cell
walls. This results, in some members of the E.
grylli species complex, in walled hyphae that
emerge through the host cuticle and produce con-
idiophores and infective conidia externally on the
insect. A latent intermediate stage (resting
spores) inside the host can also result after pro-
duction of cell walls by protoplasts. External,
aerial spores conidiaa), the only infective stage in
nature, are responsible for the often-rapid hori-
zontal transmission leading to epizootics (Carru-
thers et al. 1994).
Unfortunately, E. grylli is fastidious regarding
its artificial culture; these fungi will rarely grow
on solid media. In practice, they can only be prop-
agated either in vivo in their orthopteran hosts, or
in complex liquid media such as Graces's insect
tissue culture medium (Ramoska et al. 1988; Bi-
dochka et al. 1995). Further, in Grace's and simi-
lar media these fungi have so far been reported to
grow only as protoplasts, not hyphae. Protoplasts
cannot be distributed directly as a biological con-
trol tool. Their lack of cell wall renders them very
fragile, and they are neither infective upon appli-
cation to the insect's cuticle nor upon ingestion.
Thus, manipulation of E. grylli usually requires
that considerable amounts of appropriate live in-
sect hosts are available.
Entomophaga grylli protoplasts are infective
upon injection in their hosts, and they induce typ-
ical lethal mycoses. In classical biological control
projects, live, protoplast-injected orthopterans
have been released into populations free of these
diseases (Carruthers et al. 1994; Bidochka et al.
1995; Sanchez Pefa et al.1996). Production of E.
grylli infective conidia on artificial media would
facilitate its use as a bioinsecticide, analogous to
other entomopathogenic fungi. Manipulation of
fungi in biocontrol is possible if stable, resistant
walled cells (such as infective conidia, or at least
hyphae) can be produced. Hyphae of ento-
mopathogenic fungi such as Beauveria, Hirsu-
tella, and Metarhizium spp. (Deuteromycota: Hy-
phomycetes), and Zoophthora and Pandora (Zygo-
mycota: Entomophthorales) can be massively pro-
duced in artificial media. These hyphae
subsequently produce infective walled cells
conidiaa) that cause lethal disease in insects in
the field (Rombach et al. 1986; Wraight et al.
1986; Sanchez-Pefia and Thorvilson 1991; McCoy
In the E. grylli species complex, no production
of walled cells hyphaee or conidia) has been re-
ported to occur on the few artificial media that
support its stable growth (i.e., Humber & Hansen
2004). Herein I report the observation of the in
vitro transition from protoplasts to walled, myce-
liar (hyphal) vegetative phase in E. grylli patho-
Entomophaga grylli pathotype I (informally
called E. macleodii) was isolated from Camnula
pellucida collected in Alpine, Arizona, passed
through Melanoplus bivittatus (Orthoptera: Acri-
didae), and reisolated in Grace's insect tissue cul-
ture medium plus 1% fetal bovine calf serum
(FBS) (Invitrogen-GIBCO, Carlsbad, CA) and
stored in liquid nitrogen for 13 years as strain AR-
SEF 770 (Humber & Hansen 2004). The author
passed ARSEF 770 through Camnula pellucida
and reisolated it, originating ARSEF strains 4948,
4949, 4950, 4952, and 4953 (strains deposited at
PPRU, USDA, ARS, Ithaca, NY). All in vitro cul-
Fig. 1. Entomophaga grylli pathotype 1 cells from in vitro liquid culture: a, amoeboid and moniliform, motile pro-
toplasts; s, spherical protoplasts; t, transition between amoeboid and spherical protoplasts; h, walled, septate hy-
phae. Notice typical entomophthoralean hyphae with apical cytoplasm and empty, septated spaced behind the tip.
Bar = 20 microns.
tures were grown in 8-10 ml of Graces's medium
plus FBS as described, in vented tissue culture
flasks (9 x 5 x 2.5 cm) (TPP-MIDSCI, St. Louis,
MO), under diffuse fluorescent and natural light.
After at least four weeks on artificial medium,
numerous irregularly shaped, amoeboid as well
as spherical protoplasts were observed in flasks
from all these isolates (Fig. 1). Protoplasts are the
propagules normally observed in such cultures of
E. grylli. Some of the protoplasts assumed fila-
mentous growth as walled hyphae. Hyphal
growth was observed originating from both amoe-
boid and spherical protoplasts (Fig. 1 and 2). As
described, E. grylli pathotype I changes from pro-
toplasts to hyphae in its hosts. The hyphae then
can produce the infective units, conidia, on conid-
iophores externally on the insect's surface. The
transitions reported herein show that the first
part of development leading to production of in-
fective conidia (i.e., transition from protoplasts to
hyphae) can be obtained in artificial media. It is
possible that further differentiation to conidia
can be obtained from artificial medium-grown
biomass. This would facilitate the deployment of
E. grylli fungi as biological control tools of Ortho-
ptera. I expect that this report will stimulate
searches towards completing the life cycle of these
fungi in vitro, and towards the production of
conidia from fungal biomass grown in artificial
The logistic support of R. Carruthers, J. Cor-
rea, M-L Cummings, and S. P. Wraight is ac-
The species of the Entomophaga grylli complex
that are fastidious grasshopper and locust patho-
gens have not been reported to complete their life
_S "- .
-- ..c_ .:.+d
Fig. 2. Entomophaga grylli pathotype 1 walled, sep-
tate, empty hypha originating from now empty spheri-
cal cell. Bar = 35 microns.
cycles in vitro. The production of true hyphae in
semisynthetic, liquid medium is described for E.
grylli pathotype 1, a pathogen of banded-winged
grasshoppers (Orthoptera: Oedipodinae). It is
possible that induction of further differentiation
of E. grylli hyphae grown on artificial medium
might lead to production of infective conidia for
biocontrol of pest Orthoptera.
BIDOCHKA, M. J., S. R., WALSH, M. E. RAMOS, R. J. ST.
LEGER, J. C. SILVER, AND D. W. ROBERTS. 1995. Fate
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North America. Proc. Nat. Acad. Sci. USA, 93 (2),
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HOSTETTER, AND R. S. SOPER. 1997. The Entomoph-
aga grylli (Fresenius) Batko species complex: Its bi-
ology, ecology, and use for biological control of pest
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Strains. Plant Protection Research Unit, USDA-
ARS, Ithaca, New York.
McCoY, C. W. 1996. Pathogens of eriophyoids, pp. 481-
490 In E. E. Lindquist, M. W. Sabelis, and J. Bruin
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mies and Control. Elsevier Science B.V., Amsterdam.
RAMOSKA, W. A., A. E. HAJEK, M. E. RAMOS, AND R. S.
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species complex (Zygomycetes: Entomopthorales). J.
Invertebr. Pathol. 52: 309-313.
ROBERTS, D. W., AND R. A. NUMBER. 1984. Ento-
mopathogenic fungi, In D. W. Roberts, and J. R Aist
[eds], Infection Processes of Fungi. Rockefeller Foun-
dation. New York.
ROMBACH, M. C., R. M. AGUDA., B. M SHEPARD, AND D.
W. ROBERTS. 1986. Entomopathogenic fungi (Deu-
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J. CORREA, AND S. WRAIGHT. 1996. Entomopat6-
genos como agents de control biol6gico clasico: expe-
riencias con Entomophaga grylli en el control
biol6gico de saltamontes y langostas. Memorias XIX
Congress, Sociedad Mexicana de Control Biol6gico,
Culiacan, Sinaloa. p. 8-10.
SANCHEZ PENA, S. R., AND H. G. THORVILSON. 1991. The
entomopathogenic fungus Beauveria bassiana for
fire ant control in nursery stock, pp. 94-105 In M.
Mispagel [ed.], Proc. 1991 Imported Fire Ant Confer-
ence, U. of Georgia, Atlanta, GA.
SAWYER, A. J., M. E. RAMOS, T. J. POPRAWSKI, R. S.
SOPER, AND R. I. CARRUTHERS. 1997. Seasonal pat-
terns of cadaver persistence and sporulation by the
fungal pathogen Entomophaga grylli (Entomophtho-
rales: Entomophthoraceae) infecting Camnula Pel-
lucida (Orthoptera: Acrididae). Memoirs Can.
Entomol. Soc. 171: 835-855.
WRIGHT, S. P., S. GALAINI-WRAIGHT, R. I. CARRUTH-
ERS, AND D. W. ROBERTS. 1986. Field transmission of
Erynia radicans to Empoasca leafhoppers in alfalfa
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Florida Entomologist 88(3)
A COMPARISON OF FECAL PROTEIN CONTENT IN
MALE AND FEMALE CAT FLEAS, CTENOCEPHALIDES FELIS (BOUCHE')
JEFFREY A. SHRYOCK AND RICHARD M. HOUSEMAN
Department of Entomology, University of Missouri-Columbia, 1-87 Agriculture Building,
Columbia, MO 65211
The diet of the larval cat flea, Ctenocephalides
felis (Bouche'), has been the focus of study by a
number of researchers (Bruce 1948; Hsu et al.
2002; Moser et al. 1991; Richman et al. 1999). The
consensus is that adult flea feces are the essential
nutritional requirement for developing cat flea
larvae. Various other organic materials found
within the micro-habitat of larvae, previously
thought to be of importance, have proven to have
no significance in the diet (Strenger 1973).
There is an incomplete utilization of host blood
imbibed by adult cat fleas (Hinkle et al. 1991; Sil-
verman & Appel 1994). Hinkle et al. (1991) re-
ported that the protein content of cat flea feces
was actually higher than the bovine blood upon
which they fed, while Silverman & Appel (1994)
found only a slight difference. The fact that adult
flea feces are nutritionally necessary for larval de-
velopment has led to the suggestion that there
may be a unique form of parental investment ex-
hibited in cat fleas (Hinkle et al. 1991; Silverman
& Appel 1994).
The objectives of this study were to compare
protein content in the feces of adult male and fe-
male C. felis over a 10-day feeding period and to
examine the extent to which male fleas may pro-
vide protein for developing flea larvae.
Adult male and female cat fleas were reared
from eggs at the Missouri Research Center Labora-
tory in Fulton, Missouri. Fleas were held in an in-
cubator at 28C and 85% RH and exposed to a 12:12
light/dark regime until used for study purposes.
At 25 days following the egg collection date,
fleas were sorted to sex on a vacuum stage under
a stereomicroscope for identification. Males and
females were aspirated from the stage separately
and placed individually into test tubes.
A total of 190 female and 285 male fleas were
fed bovine blood containing a 20% solution of so-
dium citrate in an artificial membrane system
(FleaData, Inc., Freeville, NY) similar to the one
described by Wade & Georgi (1988). Blood was ob-
tained from a Holstein calf that had no history of
exposure to parasiticides. Blood in the feeding
sleeves was maintained at approximately 38C to
simulate blood temperature of the live host. Fleas
were provided with fresh blood daily from a con-
tainer refrigerated at 4C. After six days, fresh
blood was obtained from the same calf.
Flea feeding cages were 6 mm in diameter and
1.5 mm deep and consisted of two chambers. The
upper chamber containing fleas had a fine nylon
mesh top through which fleas imbibed the blood
and a bottom of coarse mesh that allowed feces to
fall through but which also prevented fleas from
escaping. A very thin layer of clean cat hair was
placed in the upper chamber with the fleas to facil-
itate movement onto the feeding screen. The lower
chamber was fastened to the upper chamber but
was removable. The lower chamber had a very fine
mesh bottom, and its sole purpose was to collect fe-
ces that fell from fleas in the upper chamber. Every
24 h for a 10-day period, the lower chamber was re-
moved from each feeding cage and the feces were
transferred to /2-dram glass vials. Flea feeding
cages were shaken vigorously before removal of the
lower chamber to make sure all feces were cap-
tured for each particular day. All vials were imme-
diately placed in a freezer at -20C after collection.
One milligram aliquots of adult male and fe-
male feces from each of 10 consecutive feeding
days were dissolved in 1 ml of de-ionized water,
vortexed, and centrifuged for five minutes at 3000
rpm to move any foreign material to the bottom of
the test tube. Thirty microliters of supernatant
from each sample were combined with 1.5 ml Coo-
massie Blue reagent (Pierce) in a modified Brad-
ford (1976) total protein assay. Samples were vor-
texed again and then transferred to square dis-
posable 10-mm cuvettes (Elkay Products) where
they were allowed to incubate at room tempera-
ture for 10 minutes.
Six replicate samples from each of the 10 days
for male and female fleas were measured by ab-
sorbance for total protein content with a spectro-
photometer (Shimadzu UV-1601) at a wavelength
of 595 nm. Protein concentrations were estimated
with reference to absorbance values obtained for
a series of standard protein dilutions of known
concentration, which were assayed along with the
flea fecal samples.
A standard curve was prepared by plotting the
average blank-corrected 595 nm measurement for
each standard versus its concentration in (pg/
mg). Total fecal protein concentrations for male
and female fleas on each of the 10 days was ana-
lyzed with a t-test (a = 0.05).
In comparing total protein concentration be-
tween adult male and female feces over a 10-day
period, a significant difference was found only on
day 1 of the study with males having a higher to-
tal protein concentration (Table 1). The mean to-
336 Florida Entomologist 88(3) September 2005
.o C.i tal protein concentration between males and fe-
o a males for the other 9 days of the feeding period re-
+ +1 suited in no significant difference between the
C.i two sexes.
O C This study determined that male feces contain
+1 +1 the same amount of protein as female feces when
(o d measured as total protein. Protein content in
male feces was as high as, or significantly higher
than, female feces throughout the entire period of
this study. These results demonstrate that male
Xo C. felis are equally capable of providing protein
c 1i for developing larvae, as are females.
Whether or not the inefficient use of host blood
1 C warrants a form of parental investment is debat-
able. An alternate hypothesis is that adult fleas
i are imbibing large volumes of blood to glean nu-
1 +1 trients that may be at low levels in the blood. A
e 1 +1
comparison of a wider range of host blood nutri-
Sents to the feces excreted could support such a hy-
hC C1 pothesis.
o .- The significance of the differences in fecal pro-
cz tein content observed on day 1 can only be conjec-
1 +1 +tured without further investigation. While the
; (0i data presented here indicate that there is equal
C1 s qualitative investment between the sexes, the
1 C1 greater volume of feces produced by the female
5 o may be indicative of a greater quantitative invest-
o eL- ment.
+1 +15 In conclusion, these data demonstrate that
male C. felis, while providing a smaller volume of
i1 1 food, are providing a food source as equally rich in
S total protein as the female of the species.
+ +1 ACKNOWLEDGMENTS
Sc The authors wish to express their gratitude to
C1 1 ( Tom Malinski for assistance in the protein deter-
Sminations and Merial Limited for the use of facil-
S 4 Q ities and supplying test insects.
PL C1 REFERENCES CITED
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o 0 -v ments of the cat flea. Ann. Entomol. Soc. Amer. 41:
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t Hematophagous strategies of the cat flea (Sipho-
6 't ^ naptera: Pulicidae). Florida Entomol. 74(3): 377-
+1 +1 +1 Hsu, M. H., Y. C. Hsu, AND W. J. WU. 2002. Consump-
ti tion of flea feces and eggs by larvae of the cat flea,
Sin Ctenocephalides felis. Medical and Veterinary Ento-
C1 mol. 16: 445-447.
MOSER, B. A., P. G. KOEHLER, AND R. S. PATTERSON.
S1991. Effect of larval diet on cat flea (Siphonaptera:
Pulicidae) development times and adult emergence.
J. Econ. Entomol. 84(4): 1257-1261.
RICHMAN, D. L., P. G. KOEHLER, AND R. J. BRENNER
1999. Spray-dried bovine blood: An effective labora-
tory diet for Ctenocephalides felis felis (Sipho-
naptera: Pulicidae). J. Med. Entomol. 36(3): 219-
SILVERMAN, J., AND A. G. APPEL. 1994. Adult cat flea
(Siphonaptera: Pulicidae) excretion of host blood
proteins in relation to larval nutrition. J. Med. Ento-
mol. 31(2): 265-271.
STRENGER, A. 1973. Zur Ernahrungsbiologie der Larve
von Ctenocephalides felis felis. B. Zool. Jb. Syst. 100:
WADE, S. E., AND J. R. GEORGI. 1988. Survival and re-
production of artificially fed cat fleas, Ctenocepha-
lides felis Bouche' (Siphonaptera: Pulicidae). J. Med.
Entomol. 25(3): 186-190.
Florida Entomologist 88(3)
TRAPPING OF SCYPHOPHORUS ACUPUNCTATUS
(COLEOPTERA:CURCULIONIDAE) WITH TWO NATURAL BAITS
IN A FIELD OF POLIANTHES TUBEROSA (LILIALES:AGAVACEAE)
IN THE STATE OF MORELOS, MEXICO
MA. ELENA VALDES E.1, LUCILA ALDANA LL.1, RODOLFO FIGUEROA B.1, MIRNA GUTIERREZ O.',
MARIA C. HERNANDEZ R AND TOMASA CHAVELAS M.2
'Departamento de Interacciones Planta-Insecto. Centro de Desarrollo de Productos Bi6ticos (CEPROBI)
Institute Polit6cnico Nacional. Carr. Yautpec-Jojutla Km. 8.5. Col. San Isidro, Yautepec, Morelos M6xico. C.P. 62731
2Instituto Profesional de la Regi6n Oriente, UAEM. Nicolas Bravo S/N Parque Industrial Cuautla
The plant locally called "tuberose", Polianthes
tuberosa L. (Liliales: Agavaceae) is endemic to
Mexico. It is used to make flower ornaments and
to extract volatile compounds for perfume manu-
facturing (Conzatti 1981; Watson & Dallwitz
1999). The black weevil Scyphophorus acupuncta-
tus Gyllenhal (Coleoptera: Curculionidae) is a
pest of P tuberose. The highest percentage of
plants damaged by this weevil, observed in
Coatlan del Rio, was reported as 69% (Camino et
al. 2002a,b). It also attacks the Agave salmiana
Otto ex. Salm-Dyck "pulque" and A. fourcroydes
Lemaire "henequen" (MacGregor & Gutierrez
1983; Mor6n & Terr6n 1988). Ramirez-Choza
(1993) found it in all regions where agave is culti-
vated. It is the main pest of sisal, causing damage
of up to 50% of this crop. It has caused damage in
"tequila" agave (A. tequilana Wever var. Blue), ac-
counting for 10% loss of crops (Valenzuela 1994;
Solis et al. 1999). Solis et al. (2001) reported up to
24.5% damage by S. acupunctatus in the A. tequi-
lana heads. Recently in Morelos state "tequila"
agave culture was introduced and Cabrera &
Orozco (2002) reported that Counter (terbufos)
controlled this insect in tuberose culture. Camino
et al. (2000a, b) reported the use of 20 different
bait types consisting of fruits and plant residues
in two trap types (different from the ones used in
the present work), where fermented agave, ripe
pineapple, banana, and guava apple captured the
highest number of adult S. acupunctatus; no nat-
ural baits were reported previously as attractive
for this insect species.
This study was conducted to assess the re-
sponse of S. acupunctatus towards two natural
baits (Camino et al. 2002a), and the effectiveness
of two trap types (a commercial one plus a funnel-
type homemade one) for capturing adult weevils.
Fieldwork was conducted from August to Octo-
ber, 2001, in Morelos, Mexico (1853'N-9911'W)
at an altitude of 1350 m. A 1.2-ha parcel planted
with offshoots of P tuberosa was used as an exper-
imental field and was divided into 18 plots (28 x
35m), with one trap placed in the center of each
plot. A two-factor design (two trap types and two
bait types) including six treatments and three
replicates was used. The traps evaluated were the
Victor (V) trap made of transparent plastic mea-
suring 9 x 16 x 7 cm (depth:height:width) with a
black cap having four 1 cm-diameter orifices at its
base and with a yellow umbrella-shaped cap mea-
suring 7 cm in diameter (Fig. la) and a yellow
Fig. 1. Traps used for capturing the tuberoses black weevil S. acupunctatus: a) Victor trap, and b) Funnel trap.
funnel (F) trap consisting of a cylindrical 20 x 32
x 20-cm (depth:height:width) plastic container,
with a plastic funnel measuring 25 x 27 x 22 cm
(depth:height:width) (Fig. Ib). The bait types
were ripe chopped pineapple (Ananas comosus)
and fermented maguey (A. salmiana); addition-
ally, water was used as control. The traps were
randomly distributed. Baited traps were rotated
at one-week intervals to avoid any position-re-
lated bias, for a total period of three months.
The nine V and F traps were baited as follows:
three, with 300g of ripe, chopped pineapple plus
250 ml of water, three with 500 ml of fermented
maguey, and three traps contained 250 ml of wa-
ter only. Traps were rotated, checked every 8 days
at 0900 h to collect adult weevils, and baits were
replaced at each sampling interval. There were 10
sampling events. Captured weevils were placed in
separate plastic containers and taken to the labo-
ratory for counting.
The numbers of weevils captured were com-
pared during the 3- month test by a two-way
ANOVA. Means were separated by a Student
Newman-Keuls test. Data were analyzed with the
computer software SigmaStat for windows, ver-
sion 2.03 (SigmaStat 1995).
Trap V captured significantly more weevils (a
total of 1726 specimens) than trap F (F = 7.620, df
= 1,114, P = 0.007) (Fig. 2) In August, the V trap
captured 277 organisms, and the number in-
creased to 385 in September, with maximum cap-
ture of 2232 in October. The V-trap design in-
cludes 4 orifices in the cap, which may lead to bet-
ter release of volatiles. Weevils captured by the F
trap were always lower than those for the V trap;
however, the trend in counts over the course of the
investigation were similar to those observed for V
traps, namely less than 50 specimens captured in
August and September followed by an increase in
early October, with a peak totaling 2894 insects
captured with both bait types, then decreasing
and remaining at low numbers until the end of
the experiment. This may be because of trap de-
sign, which only presents one opening for the vol-
atilization of fermentation products. No captures
were recorded in the control treatments through-
out the experiment.
The fermented maguey was the most attrac-
tive bait for S. acupunctatus in August, but pine-
apple accounted for the highest counts in Septem-
ber-October. No statistical differences were ob-
served between baits (F = 0.106, df = 2,114, P >
0.05) (Fig. 3). This might be explained by the fact
that some of the fermentation products are iden-
tical or similar in both bait types; according to
Figueroa et al. (2001) the major component in
both pineapple and fermented maguey is ethanol,
with minor components including acetaldehyde,
acetic acid, and ethyl acetate. A difference be-
tween sampling events was detected (P < 0.001),
with sampling 7 being the most significant one
(748 insects). Data obtained for the sugar cane
weevil M. hemipterus sericeus (Giblin-Davis et al.
1994a) and palm weevils R. palmarum and R.
cruentatus (Camino et al. 1992; Oehlschlager et
al. 1993; Giblin-Davis et al. 1994b; Oehlschlager
et al. 1995) suggest that fragrances and fermenta-
tion products (ethyl acetate, ethyl lactate, ethyl
isobutyrate, ethanol, butanol, acetic acid, hex-
anoic acid, and lactic acid) derived from a variety
of plants or fruits are attractive for these insects.
The statistical comparison of traps and baits
revealed no significant differences; however, sub-
stantial capture may contribute to decreasing the
size of populations feeding on and damaging tube-
The Victor type was the most effective trap for
capturing S. acupunctatus, suggesting that this
f victor 60
Fig. 2. Capture of S. acupunctatus adults in the field
with two natural baited traps in Morelos Mexico (F =
7.620, df= 1,114, P > 0.007). Bars show SE.
D fermented maguey
Fig. 3. Capture of S. acupunctatus adults in the field
using two natural baits in Morelos Mexico. There is not
statistical difference observed between fermented
maguey and pineapple (F = 0.606, df = 2,114, P > 0.05).
Bars show SE.
trap design allows a better volatile dissemination.
Hence, this trap type can be recommended to
monitor weevil populations, or as an aid to reduce
the population size of this pest as part of an Inte-
grated Pest Management program.
The results presented in this research work
are part of the project CGPI-IPN 200091. The au-
thors thank the Fundaci6n Produce Morelos A.C.
economic support received to carry out this inves-
Tests were conducted to assess the attraction
of Scyphophorus acupunctatus to two natural
baits (fermented maguey and pineapple) in two
trap designs (Victor and funnel) in 2001 in fields
planted with offshoots of Polianthes tuberosa in
Emiliano Zapata, Morelos, Mexico. There was a
statistically significant difference between traps,
with the Victor-type trap giving the largest
CABRERA, R. J., AND M. R. OROZCO. 2002. Diagn6stico de
ornamentales en el Estado de Morelos. INIFAP, CE
Zacatepec. Zacatepec, Morelos. Publicaci6n especial
CAMINO, L. M., A. JIMENEZ, AND F. CASTREJON. 1992.
Pruebas de atrayentes quimicos para la capture de
Rhychophorus palmarum (L.). Revista Latinoameri-
cana de Quimica 23 (1): 11-13.
CAMINO, L. M., Y. Rios, R. FIGUEROA, M. GUTIERREZ,
MA. C. HERNANDEZ, J. MARTINEZ, L. ALDANA, AND
MA. E. VALDES. 2000a. "Manejo integrado de plagas
del nardo Polianthes tuberosa (Liliflorae: Ama-
rillidae) en el estado de Morelos." Informe T6cnico.
Fundaci6n Produce Morelos A. C.
CAMINO, L. M., M. GUTIERREZ, R. FIGUEROA, L. ALDANA,
MA. E. VALDES, MA. C. HERNANDEZ, AND R. URIBE.
2000b. Avances para la propuesta del program MIP
del picudo negro del nardo y agave en el estado de
Morelos. En: Memorias VII Encuentro de Entom61-
ogos del IPN CIIDIR-Oaxaca, Oaxaca, M6xico, 31
October-1 November 2000: 25 p.
CAMINO, L. M., L. ALDANA, MA. E. VALDES, R.
FIGUEROA, MA. C. HERNANDEZ, M. GUTIERREZ, AND
J. MARTINEZ. 2002a. Bases para un program MIP
en el cultivo de nardo (Polianthes tuberosa) y agave
(Agave tequilana var. azul) en el estado de Morelos,
M6xico. En: Resumenes VIII Congreso Latinoameri-
cano y del Caribe de Manejo Integrado de Plagas.
Panama, Panama, 22-24 November 2002: 31 p.
CAMINO, L. M., V. CASTREJON, R. FIGUEROA, L. ALDANA,
AND MA. E. VALDES. 2002b. Scyphophorus acupunc-
tatus (Coleoptera: Curculionidae) attacking Polian-
thes tuberosa (Liliales: Agavaceae) in Morelos,
M6xico. Florida Entomol. 85 (2): 392-393.
CONZATTI, C. 1981. Flora taxon6mica Mexicana II. Cen-
eti; Guadalajara, M6xico. (see p. 87-88).
FIGUEROA, B. R., M. CAMINO, J. MARTINEZ, E. LAGUNA,
T. HERNANDEZ, Y. RIOS, L. ALDANA, AND MA. E.
VALDES E. 2001. Evaluation of natural products in
traps for the capture of the Scyphophorus acupunc-
tatus (Coleoptera: Curculionidae) in tuberose Po-
lianthes tuberosa (Liliiflorae: Amaryllidaceae) from
Morelos, M6xico. 42nd Annual Meeting of the Amer-
ican Society of Pharmacognosy. Oaxaca, M6xico, 14-
18 July 2001: 243 p.
GIBLIN-DAVIS, R. M., J. PENA, AND R. DUNCAN. 1994a.
Lethal pitfall trap for evaluation of semiochemical
mediated attraction of Metamasius hemipterus seri-
ceus (Coleoptera: Curculionidae). Florida Ento-
mol.77 (2): 247-255.
GIBLIN-DAVIS, R. M., T. WEISSLING, A. OEHLSCHLAGER,
AND L. GONZALEZ. 1994b. Field response of Rhyn-
chophorus cruentatus (Coleoptera: Curculionidae) to
its aggregations pheromone and fermenting plant
volatiles. Florida Entomol.77 (1): 164-177.
MACGREGOR, R., AND Y. O. GUTIERREZ. 1983. Guia de
insects nocivos para la agriculture en M6xico. Ed.
Alhambra Mexicana. 166 p.
MORON, M. A., AND Y. R. TERRON. 1988. Entomologia
practice. Institute de Ecologia. Jalapa, M6xico. (see
OEHLSCHLAGER, A. C., C. CHINCHILLA-LOPEZ, C.
GONZALEZ, L. JIRON-PORRAS, L MEXSON-VARGAS,
AND R. MORGAN. 1993. Development of a phero-
mone-based trapping system for Rhynchophorus
palmarum (Coleoptera: Curculionidae). J. Econ.En-
tomol.86 (5): 1381-1392.
OEHLSCHLAGER, A. C., R. MCDONALD, C. CHINCHILLA-
LOPEZ, AND S. PATSCHKE. 1995. Influence of a pher-
omone-based mass-trapping system on the distribu-
tion of Rhynchophorus palmarum (Coleoptera:
Curculionidae) in oil palm. Environ. Entomol. 24 (5):
RAMIREZ-CHOZA, J. L. 1993. Max del henequ6n Scypho-
phorus interstitialis bioecologia y control. Serie libro
t6cnico. Centro de Investigaci6n Regional del Sur-
este. INIFAP-SARH. M6rida, Yucatan, M6xico.
SIGMASTAT. 1995. Version 2.03. Access, Soften Inc., San
SOLIS, A. J., H. GONZALEZ, AND F. FLORES. 1999. Insec-
tos asociados con Agave tequilana var. azul en cinco
localidades de Jalisco, M6xico. En: Memorias XXXIV
Congress Nacional de Entomologia, Aguascalientes,
Aguascalientes. 23-26 May 1999: 455-457.
SOLiS, A. J. 2001. El picudo del agave tequilero Scypho-
phorus acupunctatus Gyllenhal (Coleoptera: Curcu-
lionidae) en Jalisco, M6xico. Tesis Doctoral. Colegio
de Postgraduados. Institute de Fitosanidad. Mon-
tecillo, Texcoco, Edo. de M6xico. 93 p.
VALENZUELA, Z. A. G. 1994. El agave tequilero. Ed. Lit-
teris. (See p. 21-137).
WATSON, L., AND M. DALLWITZ. 1999. The families of
flowering plants: descriptions, illustrations, identifi-
cation and information retrieval. (On line) Available:
http: /biodiversity.uno.edu/delta (4 November
Florida Entomologist 88(3)
PEDARIDIUM MAYA (COLEOPTERA: SCARABAEIDAE):
FIRST RECORD IN YUCATAN, MEXICO
CUAUHTEMOC DELOYA AND LIZANDRO N. PERAZA-FLORES
Departamento de Entomologia, Instituto de Ecologia, A.C., Km 2.5 carretera antigua a Coatepec 351, Congregaci6n
El Haya, 91070 Xalapa, Veracruz, MEXICO
Pedaridium maya Vaz de Melo, Halffter &
Halffter (2004) was described recently from Mex-
ico (Quintana Roo, Chiapas, and Campeche) and
Guatemala and is known nowhere else. It is iso-
lated geographically and morphologically from
other species of the genus. During a year of
monthly systematic samplings (August 2001 to
July 2002) in Tigre Grande, Tzucacab, Yucatan
(19042'36" N, 89002'28"W), three permanent
'necro' traps and three temporary 'copro' traps
(Mor6n & Terr6n 1984) captured five adult speci-
mens of P maya. Collection data are: MEXICO:
YUCATAN, Tzucacab, Tigre Grande, 20-VIII-17-
IX-2001, NTP80, L.N. Peraza, col. (If); MEXICO:
YUCATAN, Tzucacab, Tigre Grande, 15-V-12-VI-
2002, NTP80, L.N. Peraza, col. (2m, If); MEXICO:
YUCATAN, Tzucacab, Tigre Grande, 10-12-VI-
2002, copro trampa temporal, L.N. Peraza, col.
The specimens are deposited in the entomolog-
ical collection of the Instituto de Ecologia, A.C.
(IEXA), Xalapa, Veracruz. They represent the
first records for the State of Yucatan and were
captured in May, June, and August in tropical me-
dium subperennifolious forest at 70 m altitude,
with annual precipitation between 1000 and 1200
mm. In Tigre Grande, P maya was captured with
26 other species of Scarabaeidae of the genera
Canthon Hoffmansegg (3 species), Deltochilum
Eschscholtz (3), Pseudocanthon Bates (1), Sisy-
phus Latreille (1), Eurysternus Dalman (1 spe-
cies), Onthophagus Latreille (6 species), Copris
Miller (2 species), Dichotomius Hope (1 species),
Canthidium Erichson (2 species),Ateuchus Weber
(1 species), Coprophanaeus Olsoufieff (1 species),
Phanaeus MacLeay (2 species), and Uroxys West-
wood (2 species).
This communication is a contribution to the
project "Systematics and ecology of phytophagous
and saprophagous insects" of the Departamento
de Entomologia (902-08), Instituto de Ecologia,
Pedaridum maya Vaz de Melo, Halffter &
Halffter is recorded for the first time for the State
of Yucatan with five adult specimens captured in
Tigre Grande, Tzucacab, Yucatan, Mexico in
monthly systematic sampling during one year
(August 2001 to July 2002).
MORON, M. A., AND R. A. TERRON. 1984. Distribuci6n al-
titudinal y estacional de los insects necr6filos de la
Sierra Norte de Hidalgo, M6xico. Acta Zool. Mexi-
cana (n.s.) 3: 1-47.
VAZ DE MELO, F. Z., G. HALFFTER, AND V. HALFFTER.
2004. A new species of Pedaridium Harold from
Mexico and Guatemala (Coleoptera: Scarabaeidae:
Scarabaeinae: Coprini: Ateuchina). Coleopterist's.
Bull. 58: 247-252.
Florida Entomologist 88(3)
NOTES ON A NATIVE AND AN EXOTIC SCARAB COLLECTED IN GUERRERO,
MEXICO (COLEOPTERA: SCARABAEIDAE)
Institute de Ecologia, A.C., Departamento de Entomologia, Km 2.5 carretera antigua a Coatepec 351, Congregaci6n
El Haya, C.P. 91070, Xalapa, Veracruz, MEXICO
During collections carried out in Guerrero,
Mexico in 2001, Euoniticellus intermedius (Re-
iche 1849), a scarab not previously known from
Guerrero was captured. In addition, Eurysternus
magnus Castelnau 1840 was collected in
Acahuizotla on September 4, 2001 at an altitude
of 650 m, a range extension for this native species.
Euoniticellus intermedius (Reiche 1849) was
introduced into Texas in the 1970s. The first
record from Mexico was from the Reservation of
the Biosphere "La Michilia", in the state of Du-
rano by Montes de Oca et al. (1994). Three years
later, Montes de Oca & Halffter (1997) reported it
from Baja California Sur, Guanajuato, Micho-
acan, Chihuahua, Tamaulipas, Hildago, and Ver-
acruz. Deloya (2000) recorded it from the south of
the state of Morelos. In monthly collections car-
ried out in the Sierra Madre del Sur between
1,400-1,670 between January and June, 2001 at
Ocotito (1714'17" N, 99030'43"W), Guerrero, a fe-
male specimen ofE. intermedius was collected. It
is the first record for the state of Guerrero. The
specimen is labelled "MEXICO: Guerrero,
Chilpancingo, Ocotito, 30-III-2001, altitude 680
m, bovine excrement, 12:30 pm, E. Ramirez, col.
(1)". It is deposited in the entomological collection
of the Instituto de Ecologia, A.C. (IEXA). The lo-
cality is a Pinus-Quercus forest with annual pre-
cipitation of 1650 mm and annual half tempera-
ture of 24C.
The native scarab Eurysternus magnus has a
wide distribution between Panama and Mexico in
montane rain forest between the 900 and 2,200 m
(Howden & Young 1981; Jessop 1985; Mor6n
1994). In Mexico, it has been reported from Chia-
pas, Guerrero Hidalgo, Oaxaca, Queretaro,
Tamaulipas, and Veracruz. In the Sierra Madre
del Sur, E. magnus had been recorded from Ju-
quila, Oaxaca to 1,550 m (Bates 1888) and from
the Sierra del Alquitran, Guerrero, in a Quercus-
Pinus forest between 1,400 and 1,670 m (Delgado
Delgado Castillo (1989) carried out a study be-
tween 1985 and 1987 on the Scarabaeoidea fauna
of Acahuizotla, between 650-850 m altitude. He
mentions Pseudocanthon Bates, Deltochilum
Eschscholtz, Canthon Hoffmansegg, Copropha-
naeus Olsoufieff, Phanaeus MacLeay, Copris
Miller, Dichotomius Hope, Canthidium Erichson,
Ateuchus Weber, Scatimus Erichson, Uroxys
Westwood, and Onthophagus Latreille. In that
study, intensive sampling with pitfall traps baited
with carrion, bovine dung, and equine dung failed
to reveal the presence of Eurysternus.
A specimen of Eurysternus now deposited in
the entomological collection of the Instituto de
Ecologia, A.C. (IEXA), is labelled MEXICO: Guer-
rero, Achauizotla, 650 m msm, 4-IX-2001, Juarez
Pineda, A.J. col. (1)." This specimen was collected
at substantially lower altitude (650m) than spec-
imens reported by previous collectors, who col-
lected at 1400-1670 m.
This communication is a contribution to the
project "Systematic and ecology of insects phy-
tophagous and saprophagous" of the Departa-
mento Entomologia (912-044), Instituto de
Euoniticellus intermedius (Reiche) is reported
for the first time from the State of Guerrero, Mex-
ico, with a female specimen captured at Ocotito on
30 March 2001. The altitudinal range of Euryster-
nus magnus Castelnau in the Sierra Madre del
Sur (1,400-1,670 m), is greatly extended with a
male specimen captured at 650 m at Acahuizotla,
Guerrero, on September 4, 2001.
BATES, H. W. 1886-1890. Pectinicornia & Lamellicornia.
Biologia Centrali-Americana, Insecta Coleoptera
DELGADO CASTILLO, L. L. 1989. Fauna de cole6pteros
lamelicornios de Acahuizotla, Guerrero, M6xico. Te-
sis Licenciatura, Facultad de Ciencias, UNAM, M6x-
ico, 154 pp.
DELGADO, L. 1997. Distribuci6n estatal de la diversidad
y nuevos registros de Scarabaeidae (Coleoptera)
mexicanos. Folia Entomol6gica Mexicana 99: 37-56.
DELOYA, C. 2000. Escarabajos ex6ticos (Coleoptera:
Scarabaeidae) para la fauna de los estados de More-
los y Oaxaca, M6xico. Folia Entomol. Mexicana
HOWDEN, H. F., AND 0. P. YOUNG. 1981. Panamian
Scarabaeinae: Taxonomy, distribution and habitats
(Coleostera, Scarabaeidae). Contrib. American Ento-
mol. Inst. 18(1): 1-204.
JESSOP, L. 1985. An identification guide to Eurystern-
ine dung beetles (Coleoptera, Scarabaeidae). J. Nat.
Hist. 19: 1087-1111.
MONTES DE OCA, E., S. ANDUAGA, AND E. RIVERA. 1994.
Presence of the exotic dung beetle Euoniticellus in-
termedius (Reiche) in northern Mexico. The Coleop-
terist's Bulletin 48(3): 244.
MONTES DE OCA, E., AND G. HALFFTER 1997. Invasion
of Mexico by dung beetles previously introduced into
the United States. Stud. Neotrop. Fauna Environ.
Vol. 33: 37-45.
MORON, M. A. 1994. Fauna de Coleoptera Lamellicornia
en las montanas del Noreste de Hidalgo. Acta
Zool6gica Mexicana (n.s.) 63: 7-59.
Florida Entomologist 88(3)
IFKOVIC, E. 2004. The Life and Works of Writer Annie Trumbull Slosson a Connecticut Local Color-
ist. Studies in American Literature Vol. 68. Edwin Mellen Press; Lewiston, NY. viii + 481 pp. ISBN 0-
7734-6396-8. Hardback. $139.95
Female entomologists were a rarity in the
nineteenth century. In his biography of Annie
Trumbull Slosson, Edward Ifkovic describes the
times and circumstances that led to the entomo-
logical pursuits of one of the greatest. Born in
1838 to a wealthy New England family that en-
couraged writing, women's education, and the
study of science, she became a keen naturalist
with the means to follow good weather and collect
from New Hampshire to southern Florida, often
accompanied by fellow botanists and entomolo-
gists. Her accomplishments as an "amateur" ento-
mologist are astonishing, made more so by her en-
try into the field at the advanced age of 48. De-
spite continual donations of insect specimens to
collectors and taxonomists, she amassed a collec-
tion of 35,000 specimens, which she donated to
the American Museum of Natural History. She
described several species, and over a hundred
species were named for her. She was a founding
member and financial supporter of the New York
Entomological Society, and was instrumental in
launching its journal. Her many publications con-
tributed to insect taxonomy and systematics, life
histories, faunistic surveys, and philosophy. Her
extensive collection of entomological correspon-
dence was catalogued. A year before her death in
1926, the Brooklyn Entomological Society elected
her an Honorary Member.
Using a wealth of correspondence, personal
journals, published works, and archival material,
Ifkovic provides a richly detailed account of Slos-
son's life and accomplishments. He deftly resur-
rects the character of an eccentric woman who, at
middle age, was poised on the brink of greatness
as a local color writer but fell instead under the
spell of entomology, severely curtailing her output
of fiction. In the introductory chapters he de-
scribes the literary style called local color that
thrived in the United States after the Civil War
and lingered through the turn of the century. The
term "local color" certainly applies to Slosson's
work. The style centered on rural life and close-
ness to nature, capturing the rough dialects of vil-
lage folk and details of the fauna and flora of the
story setting. In stark contrast to the romantic
sentimentalist style that it was supplanting, local
color, especially from New England, often cele-
brated single older women whose lives revolved
not around men but on their own strengths. Not
surprisingly, the best of the local colorists were
single women, including the widowed Slosson.
And not unexpectedly, the style became popular
during the second half of the nineteenth century,
when women were allowed to pursue higher edu-
cation and the suffrage movement was in full
Within the introductory chapters, Ifkovic de-
scribes the social, economic, literary and religious
matrix of Hartford, Connecticut, during the
1800s, and the activities of the influential Trum-
bulls and their extended family. Together they
form a backdrop for Slosson's development as a
writer and scientist. Annie's parents were pro-
gressive Calvinists who advocated women's edu-
cation and sent her to the Hartford Female Semi-
nary, which had a strong curriculum in science,
unusual in a girls' school at that time. She re-
mained religious all her life, but promulgated in
her fiction and spiritual essays a shift toward
more liberal and sensible Calvinism, reflecting
the currents of the day.
In subsequent chapters, Ifkovic analyzes Slos-
son's major works of fiction in chronological con-
text. Many of her popular stories first appeared in
'The Atlantic Monthly' and'Harper's Bazaar', and
were later collected and published in book form.
Some were written as novels. They ranged, for in-
stance, from a loose conglomeration of essays and
stories on a club of women who collect china, to
the story of a woman who decides to forgo her
place in heaven because her beloved animals are
unfairly excluded from it. Ifkovic points out that
the seemingly sentimental stories were satires
and parables, reflecting mainstream changes in
spirituality and Slosson's belief in the redeeming
influence of woman's sensibility. After an evalua-
tion of one of Slosson's later works, a story titled
Dumb Foxglove, Ifkovic offers an eloquent sum-
mary that can serve for most of her local color
writing: "Slosson shows that nature-this time
the flower called Dumb Foxglove-is symbolic
metaphor for human experience. The story exhib-
its what Slosson does best, meshing her natural-
istic vision with the eccentricities of the isolated
New England village, seen through a glass darkly
illuminated by the peculiarities of religious zeal".
Slosson's interest in insects began around the
time of her first successful fiction publications,
and probably stemmed from her well-established
passion for plant collecting and floral surveys. In
the manner of the day, she called herself an "am-
ateur botanizer", but was well versed in methods
of identification and curation, and regularly cor-
responded with eminent botanists such as Asa
Gray. She was actually an accomplished botanist
who published floral surveys and species descrip-
tions. Ifkovic suggests that Slosson was coaxed to
study insects by her brother-in-law and compan-
ion, William C. Prime, a well-known writer, editor,
trout fisherman, and lepidopterist. After the
deaths of their spouses, the two established a rou-
tine of travel and collecting, summering in the
White Mountains of New Hampshire, and winter-
ing in the rich collecting grounds of Florida, with
interludes in New York and Hartford. Slosson's
interest in insects was encouraged also by an-
other close friend, the actor and entomologist
Henry Edwards, who fueled her growing passion
by naming a moth for her, a species she collected
in Florida. She was hooked. "You know what an
insidious, enthralling, captivating habit it is...
What are drugs to bugs!" She established corre-
spondence and friendships with a veritable Who's
Who of early entomology, among them W. A. Ash-
mead, Philip P. Calvert, D. W. Coquillet, Edward
P. van Duzee, Harrison G. Dyar, A. S. Packard,
and Henry Skinner. Her passion for what she in-
formally called "bugology" was so intense that she
apparently carried a cyanide jar even to church,
and admitted to using it to collect a small moth
from the pew in front of her one Sunday while on
her knees in prayer. This was recounted in a light-
hearted essay published by Bradford Torrey, an
ornithologist and another close friend. Ifkovic
provides several humorous excerpts by and about
Slosson that highlight her thrill with the eccen-
tricity of being an entomologist, and a female one
Slosson's literary contributions to entomology
include philosophical essays on a variety of topics,
such as the use of common versus scientific
names, and the meshing of entomology and liter-
ature. Her descriptive articles were written in a
more literary style, to, in her words, "relieve the
heaviness of the masculine articles." The lively
pieces, says Ifkovic, were "filled with engaging an-
ecdote and wry observation", and were well re-
ceived by her colleagues. He sums up her articles
as "... always filled with human-instinct vignette,
her fusion of the drama of literature with the cir-
cumstances of entomology".
Beside the introduction and eleven chapters of
Slosson's biography, Ifkovic presents notes, an ex-
tensive bibliography of Slosson's publications and
other sources, an index, and a selection of photo-
graphs and illustrations. Ronna Coffey Privett,
an authority on British and American literature,
ably wrote the preface. The book offers a thorough
and engaging account of one of America's most
colorful early entomologists, a woman who was
once listed as a tourist attraction because of her
habit of flailing a butterfly net in public places,
whose wit and knowledge entertained many, and
who contributed generously to the field and to the
advancement of the careers of both young and es-
tablished entomologists. Combined with a well-
textured description of the times and places in
which Slosson lived, wrote, and collected, the
book has an offering for a wide range of readers,
from biologists to sociologists and historians of lit-
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