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Title: Florida Entomologist
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Publication Date: 2003
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Insects -- Florida -- Periodicals
Insects -- Periodicals
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Florida Entomologist 86(2)


ON SEXUAL SELECTION IN FLORIDA'S PYRACTOMENA BOREALIS
(COLEOPTERA: LAMPYRIDAE)

ERIN GENTRY
Department of Entomology and Nematology, University of Florida, Gainesville, Florida 32611

ABSTRACT

To what extent can a small animal with limited mobility use behavior to take advantage of
its environment and how might this influence the population as a whole? This was examined
in a firefly species Pyractomena borealis (Randall), by looking at the features of the micro-
habitat where larvae pupate, how developmental rates are influenced by extrinsic factors,
and how the population's spatial distribution differed according to sex. In two populations of
P. borealis in Gainesville Florida, larvae pupated at the warmest locations on trees, poten-
tially causing a faster development rate than individuals in cooler areas. In these popula-
tions males pupated sooner and in warmer areas than females, suggesting males chose their
pupation locations in order to have a shorter development period and an earlier emergence
date than females. This is the first evidence of protandry being experimentally linked with
behavioral usage of habitat.

Key words: protandry, Microhabitat, Microclimate, Pupation Duration, Ectotherm, Behavior

RESUME

Hasta que punto puede un animal pequeio con mobilidad limitada usar el comportamiento
para aprovecharse de su ambiente y como esto puede influenciar la poblaci6n complete? Esto
fu6 investigado en una especie de luci6rnaga Pyractomena borealis (Randall), al observer las
caracteristicas del microhabitat donde se empupan las larvas, como las tasas de desarrollo
son influenciadas por factors extrinsicos, y como la distribuci6n espacial de la poblaci6n va-
ria de acuerdo al sexo. En dos poblaciones de P. borealis en Gainesville Florida, las larvas se
empuparon en las localidad mas calidas de los arboles, potencialmente causando una tasa de
desarrollo mas rdpido que en los individuos en areas mas heladas. En estas poblaciones los
machos empuparon mas pronto en las areas mas calidas que las hembras, sugeriendo que los
machos escogen las localidades donde van a empupar para tener un period de desarrollo
mas corto y una fecha de emerg6ncia de las hembras mas temprana. Esta es la primera evi-
dencia de protandria que experimentalmente conecta el comportamiento del uso del habitat.


Virtually all aspects of the life history of an ec-
totherm (physiology, development, activity levels,
reproduction, etc.) are strongly influenced by am-
bient temperature (Fagerstrom & Wiklund 1982;
Branson 1986; Zonneveld & Metz 1991; Wiklund
et al. 1996; Olsson et al. 1999; Hemptinne et al.
2001). Behavioral responses to the limitations of
being an ectotherm may be an important factor in
the evolution of a species. There is no clearer ex-
ample of this than the behavior that is involved in
pupation.

Arboreal Pupation

Unlike most lampyrids, which pupate under-
ground, members of the genus Pyractomena (and
perhaps all of the fireflies in the tribe Cratomor-
phini) pupate above ground, mostly on vegetation
(Lloyd 1997). Pyractomena borealis (Randall) lar-
vae climb up tree trunks and glue the holdfast or-
gan (at the tip of their abdomens) to the tree
trunk (Lloyd 1997; Archangelsky & Branham
1998). Pupae hang upside down, generally with
their ventral surface against the tree, the same


position they use during ecdysis between larval
instars (Archangelsky & Branham 1998).
There are many potential costs associated with
arboreal pupation that are not as extreme for spe-
cies that pupate underground. An underground
burrow buffers environmental temperature fluc-
tuation while arboreal pupation provides little
shelter from such extremes. Similarly, burrows
are moist environments, whereas arboreal pupa-
tion presents a greater risk of desiccation. Finally,
underground pupae are less exposed to predation
and parasitism compared to the often highly visi-
ble P borealis pupae. Given these additional costs
of arboreal pupation, why should this unusual
mode of pupation exist at all?
Lloyd (1997) suggested that Pyractomena
evolved aerial pupation as a way to avoid floodwa-
ters, since the habitats they are found in are
prone to flooding. While arboreal pupation may
also expose the firefly to extremes of temperature,
they may be exposed to much warmer average
temperatures than species that pupate in the
ground; thus, there is a potential for more rapid
development and earlier eclosion (Regniere et al.


June 2003







Symposium: Insect Behavioral Ecology-2001: Gentry


1981; Fagerstrom & Wiklund 1982; Branson
1986; Wagner et al. 1987; Leather 1990; Wiklund
et al. 1996; Hemptinne et al. 2001). P borealis is
unique in Florida because adults can emerge as
early as mid-February.

Protandry

Protandry (males maturing to a reproductive
stage earlier than females) occurs commonly in
ectotherms and has been found in many insect
species (Wiklund & Fagerstrom 1977; Wiklund &
Solbreck 1982; Regniere et al. 1981; Fagerstrom
and Wiklund 1982; Bulmer 1983a, b; Parker &
Courtney 1983; Branson 1986; Zonneveld & Metz
1991; Wedell 1992; Wiklund et al. 1992; Nylin et
al. 1993; Wiklund et al. 1996; Zonneveld 1996;
Bradshaw et al. 1997; Carvalho et al. 1998;
Harari et al. 2000). Protandrous systems have
been shown to have sexual advantages for males
(Fagerstrom & Wiklund 1982; Zonneveld & Metz
1991; Wedell 1992; Nylin et al. 1993; Harari et al.
2000). Emerging early gives males the advantage
of having virgin females to mate with, increased
time to produce sperm, and assurance that they
will not emerge after the female population be-
gins to decline resulting in either no or low qual-
ity females remaining (Wiklund & Fagerstrom
1977; Wiklund & Solbreck 1982; Fagerstrom &
Wiklund 1982; Wiklund et al. 1992; Wiklund et al.
1996; Zonneveld 1996b; Carvalho et al. 1998; Ols-
son et al. 1999). It has also been suggested that fe-
males may not merely be passive participants in
protandry, but may actually benefit from emerg-
ing after males and therefore be selected to do so
(Wiklund & Solbreck 1982; Zonneveld & Metz
1991; Wedell 1992; Wiklund et al. 1996). Protan-
dry could reduce the chances of pre-reproductive
mortality in females (Wiklund & Solbreck 1982;
Zonneveld & Metz 1991; Wedell 1992; Wiklund
et al. 1996; Harari et al. 2000) and also act as a
mechanism for passive female choice by assuring
that females mate with old and therefore, by way
of longevity, the fittest males (Wedell 1992).
Protandry has not been reported in any Pyracto-
mena species (Buschman 1977), though this may
be because it has not been specifically looked for.
However it has been suggested that protandry
may occur in the firefly Photinus knulli (Cicero
1983) and in other Photinus species (Lewis &
Wang 1991).
P borealis is vulnerable to extreme tempera-
ture variation during pupation. Therefore it is
possible that the microhabitat of a pupation site
influences the developmental rate of individuals,
and if there is a sex difference in microhabitat us-
age, it is possible this may influence the dynamics
of protandry across a population (Regniere et al.
1981; Bulmer 1983a; Fagerstrom & Wiklund
1982; Parker & Courtney 1983; Branson 1986;
Leather 1990; Zonneveld & Metz 1991; Wiklund


et al. 1992; Nylin et al. 1993; Wiklund et al. 1996;
Harari et al. 2000; Hemptinne et al. 2001).
Behavioral manipulation of emergence timing
has been suggested by Regniere et al. (1981) for
the Japanese beetle (Scarabaeidae: Popillia
japonica). Since the duration of pupation is dic-
tated by temperature, males might pupate at dif-
ferent soil depths according to surface
temperature to decrease pupation duration and
to emerge before females. Similarly, there is high
variation in the arboreal microhabitats of P. bore-
alis and thereby the potential to exploit certain
microhabitats. This study looks at how behavioral
manipulation of emergence timing by individuals
could potentially impact the population dynamics
through protandry.

MATERIALS AND METHODS

The Study Areas

This study was performed in mid-January,
2001 at two locations in Gainesville, Alachua
County Florida (Latitude = 29041'N, Longitude =
8216'W). Study area A was a flood plain forest lo-
cated in a residential area between Blues Creek
and Devil's Millhopper Geological Site. Study
area B was located in Possum Creek, also a flood
plain forest. Deciduous trees dominated both hab-
itats. The specific plots were 30.5 m by 30.5 m ar-
eas with high concentrations of P borealis larvae.
All of the trees in these plots were numbered and
categorized according to bark roughness on a
scale of 1-5 (1 = the smoothest, 5 = the roughest).
Similarly, tree calipers were used to measure all
the tree's width (east to west axis) and depth
(north to south axis) at 1.22 m from the ground.

Collection Techniques and Measurements

Between 18th of January 2001 (day-of-year 18)
and 18th of February 2001 (day-of-year 49) all
trees at both plots were scanned daily for at-
tached P borealis larvae, pupae, and adults. Lar-
val collection date was also their attachment date
because of the daily scans. Trees were scanned be-
tween ground level and up to nine meters. Once
an individual was located, I assigned a number to
it and recorded the stage (larvae or pupae), tree
number it was found on, height above ground,
and the aspect of the individual using a Suunto
compass. For this study, aspect is considered the
compass direction the individual was facing (i.e.
the face of the tree it was on). These data were
used to describe the microhabitat, that is, the ap-
parent key features of the specific location at the
point of attachment described at the scale that is
relevant to that individual. If the individual was
within reach, I collected the firefly by scoring the
bark approximately 2.5 cm around the individual
with a contractor grade Stanley utility blade; the







Florida Entomologist 86(2)


section of bark was then pried from the tree with
a wood-carving chisel. The specimen was placed
in a semi-opaque plastic film canister covered
with netting secured with a rubber band.

Rearing Temperatures

The specimens were immediately taken back
to the laboratory and randomly and evenly dis-
tributed amongst three rearing chambers. Two of
the chambers were Florida Reach-in Chambers@
set at a constant temperature of 13 and 24C re-
spectively. The other chamber was an Environa-
tor set at a constant temperature of 18C. All
three chambers maintained a constant humidity
of 70% and nine hours of light (8 am-5 pm) simu-
lating the natural hours of daylight at the start of
the field season. I monitored the fireflies every
day and recorded their date of pupation and eclo-
sion, sex, and adult weight.

Field Temperature Monitoring

At study area A the ambient temperature was
monitored on eleven trees randomly selected
within the marked plot. I refer to these data as
the microclimate measurements, not to be con-
fused with the microhabitat data collected for in-
dividual pupation locations. Microhabitat is
defined by the features of a specific location (tree
size, aspect, height, bark roughness); microcli-
mate in this study is considered the temperature
regime for a specific point on a tree.
I used four Optic StowAway@ Temp loggers
(Onset Computer Corporations, Bourne, MA) on
each tree to measure microclimate. The loggers
were placed at 0N and 0.61 m above ground, 0N
and 2.44 m above ground, 180S and 0.61 m above
ground, and 180S and 2.44 m above ground. The
loggers recorded the temperature every 30 sec-
onds for 66 hours.
I used two approaches to analyze the tempera-
ture data. The first was to find the mean hourly
temperature and standard deviation (as a mea-
sure of temperature variability within hours) for
each location. As successive temperature read-
ings are not truly independent, for the second
method of analysis, I randomly selected five per-
cent of the total recorded data. The random selec-
tion increased the independence of the individual
temperature readings. This process was repeated
ten times to ensure accurate representation of the
data by the random selection. In this case, there
was no measurement of standard deviation.

Statistics

I conducted the statistical analyses using SPSS
version 9.0 (SPSS Inc., Chicago, Illinois). All data
sets were examined for normality using a Kolmog-
orov-Smirnov test. When data were normally dis-


tribute, or could be transformed to be normally
distributed, I utilized parametric tests for subse-
quent analyses. I analyzed non-normal data using
appropriate non-parametric tests. The specific
tests used are detailed in the results section.

RESULTS

The Habitat Data

To ensure equality of tree distribution between
the two sites, I first had to compare the size of the
trees. Tree width and depth were not normally
distributed at either study area. There was no dif-
ference in tree width between study area A and B
(Mann-Whitney U = 6099, Z = -1.191, p = 0.234)
and no difference in depth (Mann-Whitney U =
6148.5, Z = -0.998, p = 0.318).
In order to find physical characteristics of a
microhabitat that would influence the microcli-
mate, I analyzed the mean and the standard devi-
ation of the hourly temperature for each
microhabitat. The mean and standard deviation
of the hourly temperature were not normally dis-
tributed. I developed a stepwise linear regression
model using the mean hourly temperature as the
independent variable to examine the potential
causes of temperature variation. The putative ex-
planatory variables entered were the vertical
height up the tree, the side on the tree (North =
0, South = 180), the tree width (representing the
tree's girth), the bark roughness, the day of the
year, how many hours from noon it was, and
whether it was AM or PM. I split these data into
the two latter variables for analysis to reduce the
circular nature of time.
All significant variables had positive correla-
tions with the mean hourly temperature. Begin-
ning with the most significant, these variables
were: The time of day according to the number of
hours from noon (Adjusted R2 = 0.285, Pearson
Correlation = 0.533, F Change = <0.001), the day
of the year (Adjusted R2 = 0.184, Pearson Correla-
tion = 0.434, F Change = <0.001), if the sample
was taken in the AM or the PM (Adjusted R2 =
0.101, Pearson Correlation = 0.357, F Change =
<0.001. A positive correlation means that it was
warmer in the PM), the size of the tree (Adjusted
R2= 0.061, Pearson Correlation = 0.138, F
Change = <0.001), or if the microhabitat was fac-
ing north or facing south (Adjusted R2= 0.003,
Pearson Correlation = 0.053, F Change = <0.001.
The positive correlation meaning that the south
was warmer than the north). These variables ex-
plained a total of 63.2% of the variation in the
mean hourly temperature.
I repeated the same regression model, but
used data from 5% randomly selected tempera-
tures as the dependent variable for all ten repli-
cates. The mean adjusted R2 value for these ten
trials was 0.622, and the standard deviation


June 2003







Symposium: Insect Behavioral Ecology-2001: Gentry


0.003. In all ten cases the same variables oc-
curred in the same order as the hourly mean val-
ues. However, in four out of ten trials the height
up the tree was included as the last variable in
addition to the other five variables. The mean ad-
justed R2 change when adding the height variable
was less than 0.001.
To examine the potential causes of the varia-
tion in the fluctuation of temperature, I con-
ducted a stepwise linear regression using the
square root of the standard deviation of the mean
hourly temperature. The square root of the stan-
dard deviation was used as the dependent vari-
able to make the data more normally distributed.
The independent variables included for analysis
were the same as the stepwise linear regression of
the mean temperatures.
In order of significance, the variables with a
positive correlation to the variance of the hourly
mean temperature were: The time of day accord-
ing to the number of hours from noon (Adjusted
R2 = 0.353, Pearson Correlation = 0.594, F Change
= <0.001), if the microhabitat was facing north or
facing south (Adjusted R2 = 0.017, Pearson Corre-
lation = 0.135, F Change = <0.001. The positive
correlation means the south was more fluctuating
than the north), if the sample was taken in the
AM or the PM (Adjusted R2 = 0.016, Pearson Cor-
relation = 0.195, F Change = <0.001) A positive
correlation means that it was more fluctuating in
the PM), the day of the year (Adjusted R2 = 0.004,
Pearson Correlation = 0.061, F Change = <0.001),
and the bark roughness (Adjusted R2 = 0.003,
Pearson Correlation = 0.024, F Change = <0.001).
The size of the tree was negatively correlated with
the variance of the mean hourly temperature (Ad-
justed R2 = 0.013, Pearson Correlation = -0.041, F
Change = <0.001). These variables explained a to-
tal of 40.4% of the variation in the variance of the
mean hourly temperature.

Distribution of Fireflies at Study Areas A and B

I compared the physical characteristics of
those trees with and without fireflies to deter-
mine any differences between the trees fireflies
"chose" to pupate on and those they did not. Trees
with fireflies were larger than trees without fire-
flies (Width: Mann-Whitney U = 2777.5, Z =
-7.224, p < 0.001; Depth: Mann-Whitney U =
2814.5, Z = -7.101, p < 0.001). Trees with fireflies
were also rougher than trees without fireflies (Chi
square = 12.7, p < 0.01, df = 3).
To examine differences between the distribu-
tion of males and females, I analyzed height,
girth, and aspect of pupation locations with re-
spect to sex. Males were found higher up the trees
than females (Mann-Whitney U = 2230, Z =
-2.148, p = 0.032). Males were also found on larger
trees than females (Width: Mann-Whitney U =
2301, Z = -1.993, p = 0.046; Depth: Mann-Whitney


U = 2287.5, Z = -2.044, p = 0.041). Females devi-
ated more from 180 than males did, i.e. males
were more clustered on the south side of the trees
than females (Mann-Whitney U = 2250.500, Z =
-2.071, p = 0.038) (see Fig. 1 for females, and Fig.
2 for males). The descriptive statistics for the dis-
tribution of female and male P borealis can be
found in Tables 1 and 2, respectively.

Attachment Timing

I looked at the population wide pattern of de-
velopment in order to begin examining protandry
in P. borealis. The collection dates of the larvae
(i.e. attachment dates, expressed as Day-of-Year
or DY January 1st is 1 DY, February 1st is 32 DY)
were not normally distributed. Overall, females
were collected and therefore had attached later
than males (Females: N = 70, Mean = 28.21 DY,
Median = 27 DY, SD = 7.13; Males: N = 81, Mean
= 23.84 DY; Median = 22 DY, SD = 5.36; Mann-
Whitney U = 1464, Z = -3.915, p < 0.001).

Developmental Timing According to Ambient Tempera-
ture

I compared the development rates for individ-
uals reared under the three different tempera-
ture regimes to determine temperature effect on
pupation. None of the developmental parameters
that were measured were normally distributed.
The duration of the attached larval stage, pupa-
tion, and emergence all decreased with increasing
temperature (see Tables 3 and 4). The general de-
scriptive statistics for all variables at 13C, 18C,
and 24C can be found in table 4.
In all three temperature regimes females pu-
pated and also emerged as adults on later dates


0 . I EII .J
o 0 0 0 0 0 0 0

Female's Aspect Difference From 180
Fig. 1. The Female's Aspect Deviation from 180. On
the X axis 0 represents south, because it is the differ-
ence from 180.







Florida Entomologist 86(2)


,,


Fig. 2. The Male's Aspect Deviation from 180 On
the X axis 0 represents south, because it is the differ-
ence from 180.


than males (see Tables 5, 6, and 7). At 13C and
24C the length of time it took from attachment to
pupation was longer in females (see Tables 5 and
7). However, at 18C and 24C the length of pupa-
tion was longer for males than for females (see Ta-
bles 6 and 7). At 13 the total length of time from
collection to emergence was significantly longer in
females (see Table 5). The descriptive statistics for
all of the significant results are found in Table 8.
To examine the potential causes of variation in
the total duration of development, from attached
larvae to eclosion, I developed a stepwise linear
regression model using the total number of days
from collection to emergence as the independent
variable. Date of collection, rearing temperature,
sex, and adult weight were entered as the possi-
ble explanatory variables. The temperature the
individual was reared at was negatively corre-
lated with the duration of development (Adjusted
R2 = 0.748, Pearson Correlation = -0.865, F Change
= <0.001). The sex of the individual was positively
correlated with the duration of the development;
meaning that individuals with longer develop-
ment times tended to be female (Adjusted R2 =
0.016, Pearson Correlation = 0.287, F Change =


Microhabitat Features

The largest features in the variation of temper-
ature were not surprisingly associated with time.
The first three features were related to the time of
day and the day of the year. However, tree size and
the aspect of attachment were also significantly
important contributors to the variation of mean
hourly temperature. Larger trees were warmer
than smaller trees; large trees retain absorbed
heat from the sun more than smaller trees. This
was also shown by Lloyd (1997) through his phys-
ical model experiment that simulated different
microhabitats that P borealis might encounter. In
addition, the south side of the tree was warmer
than the north side. This result is also expected
because the south side of the tree receives direct
sunlight (and therefore solar radiation) where the
north side does not. This also corresponds with the
results of Lloyd's physical models (1997).
Interestingly, height was not a feature that in-
fluenced the variation of mean hourly temperature
between microhabitats. This seemingly contradicts
the results of Lloyd's (1997) model trees that found
height to be positively correlated with tempera-
ture. This result may also be due to half of the data
coming from the north side, therefore the data with
significant differences in height from the south
side would had less of an influence on the data set
as a whole. However, upon closer examination,


TABLE 1. DESCRIPTIVE STATISTICS OF FEMALE DISTRIBUTION.

Percentiles
Std.
N Mean deviation 25th 50th (median) 75th

Deviation from 180 aspect 70 49.971 44.559 14.50 37.00 74.50
Height up tree (m) 70 1.625 0.574 1.120 1.646 2.073
Tree width (m) 70 0.203 0.137 0.086 0.180 0.318
Tree depth (m) 70 0.200 0.133 0.086 0.180 0.326


20

o 15
t--

10

5

0


June 2003


0.003). These two variables explained 76.0% of
the total variation of development times.

DISCUSSION

In this study I have shown that P borealis
tends to pupate in the warmest microhabitats
and that warmer temperature leads to faster pu-
pation rates. In addition there were temporal and
spatial differences between males and females.
Males not only attach earlier than females, but
they also pupate in warmer areas than females.
These two behaviors would lead to males emerg-
5- mm ing earlier than females; this suggests that
S o 8 protandry is found in P borealis and the degree of
protandry in a population may be influenced by
Males Aspect Difference From 180 the behavior of individuals.







Symposium: Insect Behavioral Ecology-2001: Gentry


TABLE 2. DESCRIPTIVE STATISTICS OF MALE DISTRIBUTION.

Percentiles
Std.
N Mean deviation 25th 50th (median) 75th

Deviation from 180 aspect 80 34.738 34.060 5.250 26.500 52.250
Height up tree (m) 80 1.818 0.592 1.379 1.905 2.240
Tree width (m) 81 0.245 0.139 0.131 0.216 0.318
Tree depth (m) 81 0.244 0.137 0.127 0.218 0.326


when viewed at a tree-by-tree basis, Lloyd's find-
ings are in fact corroborated by this study. Height
was important in four out of the ten trials examin-
ing 5% of the randomly selected data. This may re-
flect the inconsistent nature of solar exposure to
trees in the same forest. Not all trees are in areas
of uniform solar exposure; therefore on some trees
height is an important feature for maximizing
heat. The randomly selected data would not con-
tain an equal representation of all trees, so those
trees in areas of patchy sunlight where height was
important may have had a larger representation in
the four trials where height was important.
Time of day also plays a key role in the fluctu-
ation of temperature, but the second most impor-
tant feature is the aspect. Areas on the south side
of the tree fluctuate much more than areas on the
north side; the north side continuously being in
shadow, and the south side receiving more or less
solar radiation depending on cloud cover, shad-
ows, etc. ... Finally, tree size is negatively corre-
lated with temperature fluctuation; larger trees
have more stable microclimates than smaller
trees. This is corroborated by Lloyd's study of
model trees (1997). This is probably for similar
reasons as to why large trees are warmer, because
large trees have a smaller surface to volume ratio,
they can maintain absorbed heat longer than
small trees, therefore making them more stable.
The contribution of microhabitat features on
the microclimate may appear to be minor, but it is
important nonetheless (Ohsaki 1986). All fireflies
are exposed to the same daily and seasonal effects
of temperature, but aspect, tree size, and height
are all features that individuals can control
through behavioral decisions. An individual that
has selected to pupate on the south side of a large


tree will, over the course of several days, have the
advantage because of the cumulative effect of the
warmer temperature throughout development. If
this behavior were genetically based, it would be a
source of selectable variation among individuals.

Pupation Site Selection Behavior Based on Microhabitat

It is important to note that there was no signif-
icant difference of tree characteristics between
the two sites, therefore we may assume the micro-
climate data collected for study area A can also be
applied to study area B. The overall distribution of
P borealis suggests that the fireflies are taking
advantage of the best microhabitats to maximize
the temperature of their microclimate. Trees with
fireflies were larger and had rougher bark than
trees without fireflies. This study also confirms
Lloyd's findings in 1997 that P. borealis prefer the
south side of the tree, but does not support his
findings that individuals preferred smoother
trees; this difference may be due to differences in
habitats and the availability of bark types. Height
also seemed to be an influencing factor; as sug-
gested by Lloyd (1997), P borealis pupate higher
than is necessary to avoid floodwaters. On some
trees this may take advantage of areas with more
direct sunlight. The features that determined the
distribution of P. borealis were also the same fea-
tures that maximized the mean temperature.
The distribution of P borealis stands in stark
contrast to that of P limbicollis. P. limbicollis pu-
pate low to the ground on the northeastern side of
small trees; they also emerge several weeks after
P borealis (Lloyd 1997). The distribution ofP. lim-
bicollis suggests that these fireflies are in fact
taking advantage of the cooler more stable envi-


TABLE 3. COMPARISONS OF DEVELOPMENT AMONG INDIVIDUALS REARED AT 13, 18, AND 24 C. IN ALL CASES, THE VAL-
UES FOR 13 C ARE LARGER THAN 18 C, WHICH IS LARGER THAN 24 C.

Kruskal-Wallis df p

Pupation date 31.081 2 <0.001
Emergence date 94.496 2 <0.001
Attached larvae duration 67.659 2 <0.001
Pupa duration 105.868 2 <0.001
Attached larvae to emergence duration 110.300 2 <0.001







Florida Entomologist 86(2)


TABLE 4. DESCRIPTIVE STATISTICS OF DEVELOPMENT AT THREE DIFFERENT TEMPERATURES. THE PUPATION AND EMER-
GENCE DATES ARE IN UNITS OF THE DAY-OF-YEAR (DY). THE ATTACHED LARVAE DURATION, PUPA DURATION,
AND LARVAE TO PUPA DURATION ARE IN UNITS OF NUMBER OF DAYS (D).

Percentiles
Std.
Temperature N Mean deviation 25th 50th (median) 75th

Pupation date (DY) 13 C 40 42.38 11.60 32.00 45.00 51.00
18 C 45 34.44 8.22 26.50 36.00 42.00
24 C 54 29.67 6.97 24.00 28.50 33.25
Emergence date (DY) 13 C 37 76.57 15.60 67.00 78.00 88.00
18 C 49 48.57 7.62 42.00 49.00 55.50
24 C 56 36.68 6.90 31.00 35.00 41.00
Attached larvae 13 C 40 16.93 7.24 10.25 17.50 24.00
Duration (D) 18 C 45 8.07 4.45 5.00 8.00 11.00
24 C 54 4.33 2.07 3.00 4.00 6.00
Pupa duration (D) 13 C 35 35.83 6.23 35.00 36.00 37.00
18 C 44 15.93 7.06 13.00 14.00 16.00
24 C 53 7.36 0.56 7.00 7.00 8.00
Larvae to emergence 13 C 37 51.22 11.60 45.00 54.00 60.00
Duration (D) 18 C 49 21.41 5.42 17.50 22.00 25.00
24 C 56 11.38 2.40 10.00 12.00 13.00


ronments (the lower stability of small trees is with the warmer microclimates will result in re-
probably counterbalanced by the preference for duced developmental durations. The laboratory
the north side). In this case, they would also not conditions P borealis were reared in were conser-
need to pupate high up the trees to maximize vative compared to the actual field sites. This sug-
light, but merely high enough to avoid flood wa- gests there may be more highly variable
ters (Lloyd 1997). P. limbicollis is considerably development rates based on microclimate in the
smaller than P borealis, so it may be that P. lim- field than were seen in the laboratory.
bicollis is too small to overcome the potentially
desiccating effects of direct sunlight. Protandry

Developmental Timing According to Ambient Tempera- Protandry is evident in P. borealis. In the field
tures males attach before females. In the laboratory
males pupate and emerge earlier than females.
All insects have a temperature threshold However, there are differences at each of the
above which they can develop, and warmer tem- three temperatures, suggesting that the patterns
peratures cause faster development rates in in- of development of the sexes are not consistent
sects (Regniere et al. 1981; Branson 1986;Wagner throughout a wide range of temperatures. At 13C
et al. 1987; Leather 1990; Miller 1992; Wiklund et females have a longer development time, but at
al. 1996; Hemptinne et al. 2001); P borealis is no 18 and 24C there is no difference in the total de-
exception. In this study, pupal development was velopment time between the sexes at p < 0.05.
shown to be shorter at warmer temperatures, and However, sex was a determinant of developmen-
temperature was the largest influence on devel- tal duration in the linear regression; meaning an
opment time. This suggests that the selective be- individual with a long developmental time would
havior of P borealis to pupate in microhabitats most likely be a female.


TABLE 5. COMPARISONS OF THE DEVELOPMENTAL STAGES BETWEEN MALES AND FEMALES REARED AT 13 C.

Value higher for
Mann-Whitney U Z p (see real numbers in Table 8)

Pupation date 54.50 -3.44 0.001 Female
Emergence date 57.50 -3.34 0.001 Female
Attached larvae duration 54.00 -3.46 0.001 Female
Pupation duration 94.00 -1.72 0.086 Not significant
Larvae to emergence duration 62.00 -3.19 0.001 Female


June 2003







Symposium: Insect Behavioral Ecology-2001: Gentry


TABLE 6. COMPARISONS OF THE DEVELOPMENTAL STAGES BETWEEN MALES AND FEMALES REARED AT 18 C.

Value higher for
Mann-Whitney U Z p (see real numbers in Table 8)

Pupation date 74.5 -2.67 0.008 Female
Emergence date 113.0 -2.70 0.007 Female
Attached larvae duration 109.5 -1.55 0.121 Not significant
Pupa duration 70.0 -3.07 0.002 Male
Larvae to emergence duration 176.0 -1.11 0.266 Not significant


This latter result is consistent with studies of
P lucifera in which females have a longer dura-
tion of the larval stage and therefore males pu-
pate sooner than females (Buschman 1977).
Interestingly, in both of these systems, the actual
duration of the pupal stage is longer for males
than for females (Buschman 1977). The explana-
tion for this extended pupation duration is un-
known. However, regardless of the developmental
differences there has been no suggestion of
protandry in P lucifera (Buschman 1977).
Protandry may be limited in this system because
the male's slow pupation duration negates any
time advantage they gained by attaching early.

Microhabitat Influences on Protandry

When looking at all the individuals collected,
there is a significant difference between the pupa-
tion locations of males and females. Overall,
males were found on larger trees and were lo-
cated on the south side more often and were
higher up the trees. From what we know about
microhabitat, the males appear to be maximizing
developmental rates through microclimate more
than the females.
It is unclear whether the females are "inten-
tionally" choosing smaller trees, lower down and
deviating from the south more than males in or-
der to slow their development or are simply choos-
ing a "large enough" tree without using up time
looking for the largest tree to pupate on. It may
also take more effort to find the southern most
part of a tree, and so females may not be that spe-
cific in their site selection to save time and energy.
Similarly, it was shown on some trees that height
positively influences microclimate and so it is also


unclear if females are specifically selecting low
pupation sites on the trees or if they are pupating
just high enough for successful development. In
contrast, the behavior of males seems to have an
obvious result. By pupating on large trees on the
southern-most part and pupating significantly
higher than females males can take advantage of
microhabitat to decrease their development time.
In P borealis there is an obvious benefit to
males that emerge early, it gives them more time
to search for adult females and more time to
"tend" pupae and mate with closing females
(Lloyd 1997). The benefits for females are not as
evident. Many have suggested that females can
benefit from protandry through reducing pre-
mating mortality (Wiklund & Solbreck 1982; Fag-
erstrom & Wiklund 1982; Zonneveld & Metz
1991; Wedell 1992; Wiklund et al. 1996; Harari et
al. 2000). However, females seem more vulnera-
ble as immobile pupa than as mobile adults and
so it is unclear why they would want to prolong
this stage. It has also been suggested that females
benefit from protandry through passive female
choice (Wedell 1992). However, because the pupal
"tending" behavior by males is greatly enabled by
protandry, the benefits of female passive choice
must be considered in light of the costs associated
with being "tended" as a pupa.
Previously published models that discuss
protandry suggest that developmental timing is
primarily under physiological control (Wiklund &
Fagerstrom 1977; Wiklund & Solbreck 1982; Reg-
niere et al. 1981; Parker & Courtney 1983; Bran-
son 1986; Zonneveld & Metz 1991; Nylin et al.
1993; Bradshaw et al. 1997). In the case ofP. bo-
realis, developmental timing is influenced by be-
havior with regard to the choice of pupation site.


TABLE 7. COMPARISONS OF THE DEVELOPMENTAL STAGES BETWEEN MALES AND FEMALES REARED AT 24 C.

Value higher for
Mann-Whitney U Z p (see real numbers in Table 8)

Pupation date 12.500 -2.617 0.009 Female
Emergence date 14.500 -2.599 0.009 Female
Attached larvae duration 17.500 -2.307 0.021 Female
Pupa duration 22.000 -2.288 0.022 Male
Larvae to emergence duration 26.500 -1.887 0.059 Not Significant







Florida Entomologist 86(2)


June 2003


TABLE 8. DESCRIPTIVE STATISTICS OF DEVELOPMENT AT THREE DIFFERENT TEMPERATURES BETWEEN MALES AND FE-
MALES. THE PUPATION AND EMERGENCE DATES ARE IN UNITS OF THE DAY-OF-YEAR (DY). THE ATTACHED LAR-
VAE DURATION, PUPA DURATION, AND LARVAE TO PUPA DURATION ARE IN UNITS OF NUMBER OF DAYS (D).

Percentiles
Std.
Temperature Sex N Mean deviation 25th 50th (median) 75th

Pupation date (DY) 13C Female 9 40.00 9.54 32.50 36.00 51.00
Male 12 32.92 8.35 26.25 31.00 39.00
18C Female 21 38.10 7.74 32.50 38.00 44.00
Male 15 30.87 7.04 25.00 30.00 37.00
24C Female 21 33.10 7.06 27.00 33.00 37.50
Male 33 27.49 6.063 23.00 25.00 29.50
Emergence date (DY) 13C Female 9 74.78 9.50 67.00 71.00 85.50
Male 12 65.50 15.85 62.00 67.00 76.25
18 C Female 22 51.96 7.43 48.25 51.50 57.25
Male 20 45.60 6.68 41.00 45.50 51.75
24C Female 21 40.19 6.88 35.00 40.00 44.50
Male 35 34.57 6.07 31.00 32.00 37.00
Attached larvae 13C Female 9 15.11 5.47 11.50 14.00 19.50
Duration (D) Male 12 11.42 6.27 7.00 9.50 17.00
18C Female 21 9.10 4.70 5.50 9.00 13.00
Male 15 6.67 3.60 5.00 6.00 9.00
24C Female 21 5.62 1.75 4.00 6.00 7.00
Male 33 3.52 1.86 2.00 4.00 5.00
Pupa duration (D) 18C Female 21 13.62 1.36 13.00 13.00 14.00
Male 16 19.69 10.76 14.00 16.00 16.75
24C Female 21 7.10 0.44 7.00 7.00 7.00
Male 32 7.53 0.57 7.00 7.50 8.00
Larvae to emergence 13C Female 9 49.89 5.30 46.00 49.00 54.50
Duration (D) Male 12 44.25 14.25 42.25 45.50 53.75


It is clear that future models should also consider
behavior as a mechanism for protandry.
This study is the first to experimentally link
protandry with behavioral usage of the environ-
ment. The variation in microhabitat and its po-
tential effects on individual success provide a
basis upon which selection can occur (Regniere et
al. 1981; Parker & Courtney 1983). This suggests
that fine scale variations in the environment can
influence the dynamics of protandry and sexual
selection in the population as a whole.

ACKNOWLEDGMENTS

I owe many thanks to Dr. James Lloyd for advice on
this project; to Dr. John Sivinski, Dr. Jonathan Reis-
kind, Dr. Tim Forrest, Dr. Roger Gentry, and Dr. Sean
Twiss for suggestions on early drafts of this paper; to Dr.
Skip Choate for the use of his property as a field site;
and to University of Florida's Department of Entomol-
ogy & Nematology for the use of equipment.

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Florida Entomologist 86(2)


June 2003


THE EVOLUTION OF INSECT MATING STRUCTURES
THROUGH SEXUAL SELECTION

LAURA KING SIROT
Department of Zoology, University of Florida, 223 Bartram Hall, Gainesville, FL 32611


ABSTRACT

Mating structures are of interest to a wide range of biologists because, in many taxa, mating
structures are incredibly diverse and range widely in elaboration even between closely re-
lated species. As a result of this diversity, mating structures have been useful in species
identification. Historically, the evolution of diverse mating structures has been attributed to
post-zygotic selection for pre-zygotic isolation to avoid production of hybrid offspring. More
recently, sexual selection has been proposed as an alternative explanation for the rapid di-
versification of mating structures. Mating structures could diversify between populations
through sexual selection if sexual selection acted differently on mating structures in differ-
ent populations. Eberhard (1985) wrote a comprehensive book explaining how sexual selec-
tion could result in the diversification of mating structures and providing examples to
support the hypothesis, but none of the examples were experimental tests of the hypothesis.
Since 1985, a few studies have experimentally tested this hypothesis. However, there have
been no empirical studies that connect intraspecific selection with interspecific diversifica-
tion. In this paper, I review the reproductive isolation and sexual selection hypotheses and
two recent experimental tests of the sexual selection hypothesis. Then, I provide a descrip-
tion of a system that may allow one to establish a connection between sexual selection on
mating structures within a species and diversification of mating structures between species.

Key Words: genitalia, diversification, sexual selection, Melanoplus

RESUME

Las estructuras de apareamiento son de interest de una amplia variedad de biologos por que,
en muchos taxa, las estructuras de apareamiento son increiblemente diversas y se extiende
ampliamente en elaboraci6n aun entire species estechamente relacionadas. Como resultado
de esta diversidad, las estructuras de apareamiento han sido tiles en la identificaci6n de es-
pecies. Hist6ricamente, la evoluci6n de las estructuras de apareamiento diversas ha sida
atribuida a la selecci6n poscig6tico para el aislamiento precig6tico para evitar la producci6n
de descendientes hibridos. Mas recientemente, la selecci6n sexual ha sido propuesta como
una explicaci6n alternative para la diversificaci6n rdpida de las estructuras de aparea-
miento. Las estructuras de apareamiento puede diversificar entire poblaciones por medio de
la selecci6n sexual si la selecci6n sexual actua diferentement en las estructuras de aparea-
miento en poblaciones diferentes. Eberhard (1985) escribio un libro comprehensive expli-
cando como la selecci6n sexual puede resultar en la diversificaci6n de las estructuras de
apareamiento y proveyendo ejemplares para apoyar su hip6tesis, pero ninguno de los ejem-
plares fueron pruebas experimentales de la hip6tesis. Desde 1985, unos pocos studios han
probados experimentalmente esta hip6tesis. No obstante, no han habido studios empiricos
que relacionan la selecci6n intraspecifica con la diversificaci6n interspecifica. En este papel,
examine las hip6tesis del aislamiento reproductive y la selecci6n sexual y dos pruebas expe-
rimentales recientes de la hip6tesis de la selecci6n sexual. Despu6s, proveo una descripci6n
de un sistema que puede permitir establecer una conecci6n entire la selecci6n sexual de las
estructuras sexuales dentro de una especie y la diversificaci6n de las estructuras de aparea-
miento entire species.


Morphological structures involved in coupling, seem necessary for mating. For example, in the
and in transferring and receiving sperm have long damselfly genus Argia, male genitalia vary from
been of interest to taxonomists because of their rather simple structures to extremely complex
utility in distinguishing between species (e.g., structures (Fig. 1). It seems unlikely that the diffi-
Hubbell 1932; Kennedy 1919). These structures culty of transferring sperm would differ enough
(hereafter called "mating structures") are also of between species in this genus to account for the
interest to evolutionary biologists for two reasons differences in complexity in genitalia. Second,
(e.g., Alexander & Otte 1967; Arnqvist 1998; Eber- mating structures show much more rapid diversi-
hard 1985; Tatsuta & Akimoto 2000). First, they fiction than structures that are not involved in
are much more complex in appearance than would mating. This is again exemplified by the diversity






Symposium: Insect Behavioral Ecology-2001: Sirot


.-- )



--_ 2


-lc~ r~ci



-- -54


---- -,


-Ilk9


E':9


Fig. 1. Diversity of male genitalia in the damselfly genus Argia. Figure from Eberhard 1985, reprinted with per-
mission of author and courtesy of Harvard University Press.


of forms of genitalia within the genus, Argia
(Kennedy 1919; Fig. 1). Similar diversification oc-
curs in female structures that receive and store
sperm (e.g., grasshoppers: Slifer 1943; water strid-


ers, Gerris: Andersen 1993; Fig. 2). Further, we
also see this diversification in other structures
that are involved in matings such as modified an-
tennae and legs that males use to grasp females


~-- I









-


c-IJ2.


~


_7





126 Florida Entomologist 86(2) June 2003


va
cca










fc
















A.. F n6 F
:ca \



C












D E
Fig. 2. Diversity of female structures that receive and store sperm in the water strider genus Gerris. Drawings
from Andersen 1993, reprinted courtesy of the canadian Journal of Zoology.







Symposium: Insect Behavioral Ecology-2001: Sirot


during copulation in some water strider species.
This diversification is seen across many taxonomic
groups (Eberhard 1985). Two main hypotheses
have been proposed to explain the diversification
of genitalia: the reproductive isolation hypothesis
and the sexual selection hypothesis.

Reproductive Isolation Hypothesis

Historically, diversification of mating structures
has been attributed to selection for prezygotic iso-
lating mechanisms that prevent hybridization. Ac-
cording to this "reproductive isolation" hypothesis
(a.k.a. "lock-and-key"), there is strong selection on
females to avoid mating with heterospecific males.
As a result, females evolve complicated reproduc-
tive structures that allow them to discriminate be-
tween conspecific and heterospecific males and to
avoid heterospecific fertilizations. The occurrence
of this process with each speciation event would re-
sult in a pattern of rapid diversification of genitalia
across closely related species.
The reproductive isolation hypothesis has two
main predictions. First, if the diversification and
elaboration of mating structures results from se-
lection for reproductive isolation, there should be
species-specific fits of male and female mating
structures. Second, there should be more diversi-
fication of mating structures in sympatry than in
allopatry. Certain systems are consistent with
these predictions (Eberhard 1985). However,
there are many systems for which we do not see a
species-specific fit between male and female mat-
ing structures; in these species, female structures
do not prevent intromission by males of other spe-
cies (Eberhard 1985; Shapiro & Porter 1989). This
finding alone is not sufficient to reject the repro-
ductive isolation hypothesis because it is possible
that (1) reproductive isolation is achieved not
through a mechanical fit but through a sensory fit
such that the male reproductive parts stimulate
females in a species-specific manner or (2) the
genitalia no longer serve as reproductive isolating
mechanisms because other mechanisms have
evolved (e.g., behavioral).
Data from many taxa also do not support the
second prediction of the reproductive isolation hy-
pothesis. In several cases, rapid diversification of
mating structures appears to have occurred in al-
lopatry. There are patterns of extreme diversifica-
tion of mating structures of species that are
geographically isolated from any morphologically
similar species. For example, certain species of
the homopteran genus Oliarus appear to have
evolved separately on different islands of the Gal-
apagos (Fig. 3). The male intromittent organs of
species on different islands have diverged sub-
stantially (Eberhard 1985; Fennah 1967). In sum,
there are many cases of apparent rapid diversifi-
cation of mating structures that the reproductive
isolation hypothesis cannot explain.


Fig. 3. Male genitalia of species of the homopteran
genus Oliarus found on different islands of the Galapa-
gos. Figure from Eberhard 1985, reprinted with permis-
sion of author and courtesy of Harvard University
Press. Genitalia drawings from Fennah 1967, reprinted
courtesy of the California Academy of Sciences.


Sexual Selection Hypothesis

An alternative to the reproductive isolation
hypothesis is that the diversification of mating
structures is a result of sexual selection. Sexual
selection results from differential access to mates
based on differences in phenotypic traits. How-
ever, in the last twenty years, it has become abun-
dantly clear that sexual selection does not end
once coupling has begun. Within the female re-
productive tract, there are battles between sperm
of different males and differential use of sperm by
females (Birkhead & Moller 1998; Eberhard
1996). Sexual selection could act on mating struc-
tures if differences in the shape or size of these
structures resulted in differential coupling and
fertilization success (Lloyd 1979; Short 1979).
The sexual selection hypothesis is that sexual se-
lection acting on mating structures differently in
different populations could result in diversifica-
tion of mating structures between populations.
There are three mechanisms by which sexual
selection can act, and all have been invoked in ex-
plaining the evolution of elaborate mating struc-
tures. First, sexual selection could act on mating
structures through mate choice. Male mating
structures may evolve through cryptic female
choice in which females preferentially use sperm
from males based on characteristics of the male
structures. Selection could also act on females, fa-
voring those that have structures that enable
them to be more selective amongst males.
Second, sexual selection could act on mating
structures through intrasexual competition. For
example, selection could act if certain character-
istics of male reproductive structures made them
better able to deliver sperm or remove or other-
wise compete with the sperm of other males.







Florida Entomologist 86(2)


Third, sexual selection could act on mating
structures through intersexual conflict over fertil-
ization. If male quality varies, then females
should be selected to choose sperm of high quality
males. Males should be selected to overcome the
female choice mechanisms and to manipulate fe-
male behavior to their advantage (Gavrilets et al.
2001; Holland & Rice 1999; Rice 1996) and selec-
tion should act on females to avoid this manipula-
tion (at least to some degree; Alexander et al.
1997; Cordero & Eberhard 2003) leading to an in-
tersexual arms race involving the mating struc-
tures of males and females.
Diversification of mating structures between
populations through sexual selection is most
likely to occur through female choice because fe-
male choice can act on arbitrary traits (Andersson
1994). Advances in the study of the evolution of
mating structures through sexual selection have
taken two forms: investigations of the form and
function of mating structures (e.g., Arnqvist 1998;
Arnqvist & Thornhill 1998; Eberhard 1992, 2001;
Eberhard & Pereira 1993; Fritz & Turner, 2002;
Robinson & Novak 1997; Waage 1979) and exper-
imental tests of selection acting on these struc-
tures (e.g., Arnqvist and Danielsson 1999;
Arnqvist et al. 1997; Cordoba-Aguilar 1999).

Studies of the Form and Function of Mating Structures

Investigations into the form and function of
male and female mating structures support the
hypothesis that sexual selection is acting on mat-
ing structures. For example, in Waage's (1979)
classic work on jewelwinged damselflies, Calop-
teryx maculata, he concluded that the intricate
structures of the damselfly penis were used not
only to transfer sperm to females but also to re-
move sperm of other males from the female repro-
ductive tract. Waage (1979) came to this
conclusion based on four lines of evidence. 1. Fe-
males who had previously mated had more sperm
in their reproductive tract before and after a sec-
ond mating than when mating was interrupted. 2.
When copulating pairs were dissected (after be-
ing killed), male genitalia were found in the fe-
male sperm storage organs. 3. Males have
backward-pointing spines on the parts of their
genitalia that reach the sperm storage organs. 4.
Clumps of sperm were found on the male genita-
lia after the male withdrew from the female. To-
gether, these results suggest that selection could
be acting on the size and shape of male genitalia
in Calopteryx. Subsequent studies suggest that
similar processes occur in other odonate species.
More recently, investigations into the form and
function of female reproductive structures have
supported the cryptic female choice hypothesis for
the diversification and elaboration of mating
structures. Mechanisms have been found by which
females could control the use of sperm (Eberhard


1996). This appears to be the case in the Carib-
bean fruit fly, Anastrepha suspense. In this spe-
cies, females have multiple spermathecae and
store different amounts of sperm in each sper-
matheca (Fig. 4). Females have thin spermathecal
ducts leading to the bursa copulatrix. Each of the
spermathecae has a separate valve that could po-
tentially be used by females to control the storage
and release of sperm. These data suggest that fe-
male A. suspense have the ability to discriminate
between the sperm of different males by control-
ling the storage and release of the sperm. Whether
they use this ability has not been established.
These studies of form and function of mating
structures are important for understanding how
selection might act on these structures, but they
are not actual tests of the sexual selection hypoth-
esis. To demonstrate sexual selection, one must
show that differences in the mating structures re-
sult in differential access to gametes. Very few
studies have actually tested this. In fact, in the in-
sect literature, I am aware of only four studies
that actually test for differential fertilization suc-
cess based on differences in mating structures, al-
though there are other studies that relate
differences in mating structures to differences in
access to mates (e.g., Arnqvist et al. 1997). I will
review two recent studies that test for sexual se-
lection on mating structures.

Case Study I: Gerris lateralis

The first case is a recent study by Arnqvist and
Danielsson (1999) on the water strider, Gerris lat-
eralis. They studied the effect of variation in re-
productive and non-reproductive structures on


Spermatheca


Fig. 4. Female reproductive structures ofAnastrepha
suspense. Drawing by A. Fritz, printed with permission.


June 2003







Symposium: Insect Behavioral Ecology-2001: Sirot


sperm precedence of the first and second males to
mate with a female. There was evidence for sex-
ual selection acting on sclerites that are found in
the distal portion of the aedeagus. Although the
function of these sclerites is not known, they ap-
pear to play a role in the placement of the aedea-
gus within the female reproductive tract and/or
stimulation of the female.
Arnqvist and Danielsson (1999) found that the
shape of the lateral sclerites of the first male to
mate and the dorsal and ventral sclerites of the
second male to mate affect sperm precedence. In
addition, the degree of the effect of the ventral
sclerite of the second male on sperm precedence
depended on the size of the female. Together,
these results suggest that selection acts on male
mating structures in G. lateralis and that the
strength of selection depends on the distribution
of female phenotypes in the population. However,
two questions remain unanswered about the se-
lection process. First, it is unclear whether selec-
tion is acting directly or indirectly on the
sclerites. It is possible that selection is actually
acting on a trait that is correlated with the shape
of the sclerites and not on the sclerites them-
selves. The authors controlled for many possible
correlates, but, without manipulating the struc-
tures and randomly assigning males to treatment
groups with differently shaped structures, it is
difficult to infer causal relationships. Second, the
mechanism by which selection is acting is also
still unclear. It could be that (1) the shape of the
sclerites allow males to position their own sperm
or the sperm of other males in such a way that
they have an advantage or (2) females use sperm
of certain males preferentially depending on the
shape of their sclerites.

Case Study 2: Calopteryx haemorrhoidalis

A study of damselfly reproduction provides
more evidence of selection acting directly on a
mating structure. This study is on a species of ca-
lopterygid damselflies, the same group in which
Waage (1979) found sperm removal by males.
Cordoba-Aguilar (1999) found patterns of sperm
storage in Calopteryx haemorrhoidalis similar to
those that Waage (1979) found in C. maculata,
suggesting that sperm removal was also occur-
ring in C. haemorrhoidalis. However, in C. haem-
orrhoidalis, the male genitalia could not get into
the spermatheca, ruling out the possibility of di-
rect sperm removal by males. Instead, Cordoba-
Aguilar (1999) proposed that males stimulate fe-
males to eject sperm. Females have two sclero-
tized plates in their reproductive tract each
bearing sensilla. When eggs pass by these plates,
the plates are distorted and this distortion sends
a stimulus through an abdominal ganglion to the
sperm storage organs. The sperm storage organs
respond by ejecting sperm for fertilization. Dur-


ing copulation, the male genitalia distort these
plates in a manner similar to that of eggs passing
through. Females with more sensilla store less
sperm when their copulations are interrupted
than females with fewer sensilla.
Cordoba-Aguilar predicted that males with
wider genitalia would stimulate the sensilla more
and stimulate the females to eject more sperm.
He tested this prediction experimentally by simu-
lating copulations using genitalia that he had re-
moved from males. He used only the portion of the
genitalia that normally makes contact with the
plates to control for the effect of any correlated
characters and to ensure that no sperm was re-
moved directly by the male genitalia. Females
mated with males with wider genitalia stored less
sperm after simulated mating than females
mated with males with narrower genitalia. How-
ever, the mechanism of sperm ejection is still
poorly understood. It is very difficult to distin-
guish whether this is a case of female choice,
male-male competition, or sexual conflict.

Connecting Intraspecific Selection with Interspecific
Diversification

These two case studies are among the first to
demonstrate sexual selection on mating struc-
tures. However, no studies have yet connected se-
lection on mating structures within a species to
diversification of mating structures between spe-
cies. A group of grasshoppers found in Florida of-
fers an excellent opportunity to study this
connection (Fig. 5) These are the brachypterous
(short-winged) species of the genus Melanoplus
(Capinera et al. 1999; Deyrup 1996; Hubbell
1932, 1984; Squitier et al. 1998). In Florida, most
of these species are found only in sandhill and
scrub habitat. Because much of this habitat oc-
curs in patches in Florida (Myers 1990; White
1970), some of the species are effectively isolated
from other similar species (Fig. 6). This group is


Fig. 5. Melanoplus ordwaye pair in copula at Gold-
head Branch State Park, FL.







Florida Entomologist 86(2)


2.K,
K.I


Fig. 6. Cerci of male Melanoplus rotundipennis from four sites in Florida. Map from Myers & Ewel 1990, re-
printed courtesy of University Press of Florida. A. Goldhead Branch State Park; B. Welaka State Forest; C. Ocala
National Forest; D. University of Florida's Thomas Farm (Gilchrist Co.).


characterized by extraordinary diversification of
both internal and external male mating struc-
tures. For example, the cerci of different species
form what Lloyd (1979) predicted as "a veritable
Swiss Army Knife of gadgetry" (Fig. 7). The inter-
nal genitalia are similarly complex and diverse.
In addition to the interspecific variation in
mating structures, there is also much intraspe-
cific variation. For example, the cerci of Melano-
plus rotundipennis vary both within and between
populations. Figure 6 shows cerci from four popu-
lations of M. rotundipennis. The cerci differ both
in curvature and in the width of the head relative
to the rest of the cercus.
During copulation, the cerci appear to be used
by males to gain access to the genital chambers of
females (Fig. 8). The cerci squeeze against a flap
that lies flat against the female's ventral surface,
just below her ovipositor blades. This flap, called


the egg guide, encloses the genital chamber,
which is attached to the spermathecal tube. Dur-
ing coupling, the male's cerci appear to pinch ei-
ther side of the egg guide (pers. obs.). Pressure on
the sides of the egg guide results in the egg guide
popping open, exposing the genital chamber. Sex-
ual selection could act on the shape and size of the
cerci through female choice in which females
mate only with males whose cerci fit into the
grooves of their egg guides (Eberhard 1998).
The shape of the cerci differ between popula-
tions of M. rotundipennis (Fig. 6). This variation
suggests that selection could be acting differently
in different populations. One could test this hy-
pothesis in M. rotundipennis because it is possible
to manipulate the shape and size of cerci (e.g.,
Krieger & Krieger-Loibl 1958), thus, removing the
effect of correlated traits on reproductive success.
It is possible to manipulate the shape and size of


June 2003






Symposium: Insect Behavioral Ecology-2001: Sirot


Fig. 7. "Veritable Swiss Army Knife" of cerci of differ-
ent species of brachypterous grasshoppers of the genus
Melanoplus found in Florida. Drawings of cerci from
Capinera et al. 2001, with permission of author.

cerci by cutting them with microscissors. A similar
method was used to test for sexual selection on
male genitalia in the beetle, C'i/..! .. ..,l., alter-
nans (Rodriguez 1995). In this species, males with
longer genitalic structures (called flagellaa") sire
more offspring. This pattern could indicate direct
selection on flagellum length or indirect selection


A


Fig. 8. SEM photo of external mating structures of M. rotundipennis pair in copula.


on a correlated trait. Rodriguez distinguished be-
tween these possibilities by manipulating the
length of males' flagella. Males with longer manip-
ulated flagella sired more offspring, demonstrat-
ing direct selection on flagellum length. By using
this method in M. rotundipennis, one could test
whether and how cerci size or shape affected male
reproductive success. Cerci size or shape could af-
fect male reproductive success in a number of
ways including increasing a male's sperm prece-
dence or the female's oviposition rate or decreas-
ing the likelihood that the female will remate
(Eberhard 1996; Simmons 2001). Demonstration
of sexual selection for different sized or shaped
cerci in different populations would provide a con-
nection between sexual selection on mating struc-
tures within a species and diversification of
mating structures between species.
In conclusion, recent studies have established
that sexual selection is acting on male mating
structures. However, more work is needed in three
main areas for us to have a better understanding
of the evolution of mating structures through sex-
ual selection. 1. We need to investigate and at-
tempt to distinguish the processes by which sexual
selection is acting on mating structures. As exem-
plified by Cordoba-Aguilar's (1999) research on
C. haemorrhoidalis, it is often difficult to distin-
guish whether sexual selection on mating struc-
tures is a result of female choice, male competition,
or intersexual conflict. More than one of these pro-







Florida Entomologist 86(2)


cesses could be acting simultaneously. We can un-
derstand sexual selection on mating structures
more thoroughly by determining which of these
processes are occurring. 2. We need to study the
form and function of female mating structures and
how selection acts on these structures. Female
mating structures are a part of the selective envi-
ronment in which male mating structures evolve,
and vice versa. Understanding the biology of fe-
male mating structures will allow us to under-
stand the sensory and physical environment in
which male mating structures evolve. 3. We need
to connect the process of intraspecific sexual selec-
tion on mating structures with interspecific diver-
sification of mating structures. Current research
on sexual selection on mating structures is focused
predominantly on intraspecific processes. We must
conduct studies across populations of the same
species and closely related species to extrapolate
how intraspecific sexual selection can result in
interspecific diversification.

ACKNOWLEDGMENTS

I thank John Sivinski for inviting me to participate
in the Behavioral Ecology Symposium and to Jim Lloyd
for helping to organize this symposium. I am grateful to
Bill Eberhard and to my colleagues in University of
Florida's Department of Zoology for their helpful feed-
back on the material presented in this paper. I would
also like to thank the Department of Zoology and the
University Women's Club for research funding.

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Florida Entomologist 86(2)


ON WATER BEARS

FRANK A. ROMANO, III
Jacksonville State University, Biology Department, Jacksonville, AL 36265

ABSTRACT

Tardigrades, or "water bears" are microscopic invertebrates that require water in their en-
vironment and are found in freshwater, marine and terrestrial habitats. The morphology
and phylogeny of this "little known phylum" is described as are ways the naturalist might
collect water bears. Examples of species distributions in different locations in the southeast-
ern USA are given.

Key Words: tardigrade, taxonomy, phylogeny, ecology, collecting

RESUME

Los tardigrados, u "osos de agua" son invertebrados microsc6picos que requieren agua en su
ambiente y son encontrados en ambientes marines de agua fresca y ambientes terrestres. Se
describe la morfologia y la filogenia de este filoo poco conocido" y tambi6n las maneras que
los naturalistas pueden recolectar los osos de agua. Se proven ejemplares de la distribuci6n
de species en lugares diferentes en el sureste de los Estados Unidos.


WATER BEARS: AT LAST, IN A PERSONAL
REAL-LIFE INTRODUCTION

A line drawing in an already-classic entomol-
ogy text book (Fig. 0) and a comment from a pro-
fessor that "you are unlikely to ever see one" was
my introduction to the idea of"tardigrades". Forty
years later when visiting Jacksonville State Uni-
versity in northeastern Alabama a sign on a lab
door said "Beware of the Bears", and was my invi-
tation to actually see a living water bear. Frank
Romano, the bear-room caretaker and propri-
etor-and now the Head of the Biology Depart-
ment-said that bears were as near as the large
tree on the lawn and that he could show me a liv-
ing specimen in ten minutes!-not an idle boast,
for in minutes and out of accumulated muck from
the crotch of the tree he produced actual living
water bears! For those who have never heard of
tardigrades, nor the legend of their enigmatic and
"by default" position in the Animal Kingdom, an
"all about them and how to find them" encyclope-


Fig. 0. The tardigrade line drawing in Herbert Ross's
1948 entomology text (p. 47), with the attribution "After
U.S.D.A., B.E.P.A".


dia entry may not mean much, but for some of us
it is an invitation to a twilight zone where mythol-
ogy becomes reality. (JEL)
Tardigrades are microscopic invertebrates
that belong to the Phylum Tardigrada (proposed
by Ramazzotti in 1962). Active tardigrades re-
quire water in their environments and can be
found in three main habitats; marine water, fresh
water and terrestrial habitats (Kinchin 1994;
Nelson 1991; Ramazzotti & Maucci, 1983). First
described by Goeze in 1773. Commonly recog-
nized as "Water Bears" (Wassar Bar) by observ-
ers, tardigrades are best classified as one of the
"lesser-known phyla" (Nelson 1991). Tardigrades,
the current name in use since the 18th century
(adopted by Spallanzini in 1776) is also a descrip-
tive name based on the animals lumbering gait
(tardi-slow, grade-walker).
Tardigrades are generally considered cosmopol-
itan in their distribution and are commonly found
in a variety of marine, freshwater, and terrestrial
habitats: sand, algae, rooted aquatic vegetation,
soil, leaf litter, mosses, lichens, and liverworts.
These bilaterally symmetrical micrometazoans
are generally flattened on their ventral side and
convex on their dorsal side and average 250-500
pm in length as adults (see Dewel et al. 1993 for
detailed morphology). Their body is composed of 5
somewhat indistinct body segments including a
cephalic segment and four trunk segments each
supporting a pair of legs that terminate in either
claws and/or digits. The first 3 pairs of legs are di-
rected ventrolaterally and are the primary means
of locomotion, while the 4th pair is directed poste-
riorly and is used primarily for grasping the sub-
strate (Fig. 1). Tardigrades feed by piercing the


June 2003







Symposium: Insect Behavioral Ecology-2001: Romano


cells of bacteria, algae, plants (mosses, liverworts,
and lichens) or animals protozoanss, rotifers, nem-
atodes, larvae, and other small invertebrates) with
hardened stylets and sucking out their contents
using their muscular pharynx (Fig. 2). In some
cases, the whole organism is ingested. Detritus
may also be a major nutrient source of some spe-
cies. Regardless of their specific habitat (marine,
freshwater, or terrestrial), all tardigrades are
aquatic, since they require a film of water sur-
rounding the body to be active. Some, those that
are limno-terrestrial, can undergo cryptobiosis
when environmental conditions become unfavor-
able (e.g., loss of the film of water) creating an en-
vironmentally resistant state. Thus, this latent
state has a significant impact on the ecological role
of limnoterrestrial tardigrades.
Despite their overall abundance and presumed
cosmopolitan distribution (McInnes 1994), tardi-
grades have been relatively neglected by inverte-
brate zoologists. Because of the difficulty in
collecting and culturing these organisms and
their apparent lack of economic importance to hu-
mans, our knowledge of tardigrades has re-
mained in a relatively nascent state since their
discovery over 200 years ago (Nelson 1991).

Tardigrade Taxonomy

Marcus (1929) established the major taxa
within the phylum Tardigrada splitting the group
in two, forming the classes: Heterotardigrada (ar-
moured tardigrades) and Eutardigrada (naked
tardigrades). "Naked" and "armoured" refer to the
cuticular dorsal plates found in terrestrial hetero-
tardigrades, that are absent in eutardigrades.
Morphological and anatomical differences are the
only characters used to identify organisms to spe-
cies. Within the heterotardigrades (armoured) the
main features are cephalic appendages, cuticular
extensions, claws and the pattern of dorsal cuti-
cular plates (Fig. 3). Within the eutardigrade
(naked) the more important morphological char-
acteristics are the claws, the buccopharyngeal ap-
paratus; and the cuticle structure (smooth,
granulated or bearing tubercles) (Fig. 4).
Tardigrade taxonomy stems from a number of
papers but primarily from Thulin (1928) who re-
vised the systematics of the taxon, Marcus (1929)
who wrote a chapter on tardigrades in "Classes
and orders in the animal realm" (Vol. 5) and a
book entitled "The animal realm" (1936), Ramaz-
zotti (1962, 1972) who published monographs on
the phylum tardigrada, and Ramazzotti and
Maucci (1983) who collaborated to produce the
monograph entitled "The phylum tardigrada"
(English translation by Beasley 1993).
Marcus named the classes Eutardigrada
(meaning 'true' tardigrades) and Heterotardi-
grada (meaning 'other' tardigrades). The genus
Macrobiotus was described in 1834 (a eutardi-


grade) and the genus Echiniscus was described in
1840 (a heterotardigrade). A third class, Mesotar-
digrada (meso = middle), was established by
Rahm 1937 for Thermozodium esakii discovered
in a hot spring near Nagasaki, Japan. Neither
type material nor type locality have survived and
no other mesotardigrade have been discovered a
consensus from the last symposium on tardi-
grades (Eighth International Symposium on Tar-
digrada 2000) was that this should be removed
from the classification.

Tardigrade Phylogeny

Tardigrades have been closely aligned with ar-
thropods and were described as primitive arthro-
pods by Plate 1889 (from Kinchin 1994). The
morphological characters that align them with
arthropods are: vermiform animals with a cuticle,
lobopodia (poorly articulated limbs) that termi-
nate in claws, terminal mouths, caudal end (seg-
ment) terminating into the last pair of legs.

Ecology of Tardigrades

Active tardigrades require water in their envi-
ronment and as noted can be found in three main
habitats: marine, freshwater, and terrestrial hab-
itats (Ramazzotti & Maucci 1983; Nelson 1991).
Bryophytes, which hold water within the inter-
stices of their cushions (mats), and leaf axils, pro-
vide ideal sites for terrestrial tardigrades. Species
living in these wet terrestrial habitats are classi-
fied as limnoterrestrial, a useful term to distin-
guish the moss inhabiting species from the
marine and freshwater species (Kinchin 1994).
Ramazzotti and Maucci (1983) identified three
common conditions that make the terrestrial hab-
itat, such as mosses, suitable for tardigrades: (1)
a structure that allows sufficient oxygen diffu-
sion, (2) the ability to undergo alternate periods
of wetting and drying, mainly through solar radi-
ation and wind, and (3) one that contains suffi-
cient food. In reference to moist-dry mosses,
Kinchin (1994) stated that they share a drought
tolerant pattern of adaptation to dehydration
with animal groups, including tardigrades, named
poikilohydry. This adaptation is advantageous to
both the bryophyte and the bryofauna. Both the
moss and the tardigrade can survive adverse con-
ditions in a dormant state called a tun (Fig. 5)
(anhydrobiosis for tardigrades).
Although Bertolani (1983) found that some
tardigrade species were related to specific coastal
dune mosses, other authors did not find enough
evidence to support a direct correlation between
particular moss species and particular tardigrade
species (Nelson 1975; Kathman & Cross 1991).
Hunter (1977) found no relationship between ep-
iphyte species and species of tardigrades nor did
Kathman and Cross (1991).







Florida Entomologist 86(2)


Figs. 1-6. 1) Whole mount of a Eutardigrade showing the indistinct segmentation and 4 pairs of legs. 2) Bucco-
pharyngeal apparatus of a Eutardigrade showing the muscular pharynx, pharyngeal tube, stylet supports, and
mouth. 3) Whole mount of a limnoterrestrial Heterotardigrade (Echiniscus sp.). 4) Whole mount of a limnoterres-
trial Eutardigrade (Hypsibius sp.). 5) Tun formation in a Eutardigrade (Milnesium tardigradum). 6) Whole mount
of a marine Heterotardigrade (Batillipes sp.).


In an effort to better understand when and
where which tardigrades are abundant or rare,
three ecological surveys (2 terrestrial and 1 ma-
rine) were conducted in Alabama, one on Dugger
Mountain (Nichols et al. 2001), Alabama's second
highest peak, one along Choccolocco Creek (Ro-
mano et al. 2001) within the riparian zone, and one
on Dauphin Island. Five trees (Quercus alba) with
cryptogams, three on north-facing slopes and two
on south-facing slopes, were sampled seasonally at
three sites (headwaters, midwaters, mouthwaters)
along an unnamed tributary of the South Fork of
Terrapin Creek. Trees were chosen based on their
location outside the riparian zone at the peak, mid-
point, or base of the north-facing and south-facing
slopes along the creek. Seasonal and altitudinal


variations in the distribution of the populations on
the north- and south-facing slopes were deter-
mined. Significant seasonal and altitudinal differ-
ences were found in tardigrade abundance from
samples collected at specific sites and between
north- and south-facing slopes. Pooled data showed
no differences in the overall abundance or number
of species at each altitude. However, significant
seasonal differences in both abundance and num-
ber of species were seen in pooled samples. Six sites
along Choccolocco Creek were selected and 3 trees
with mosses within each were surveyed for an 18
month period. No significant difference was found
between tardigrade occurrence (total number of in-
dividuals) and season, moss genera, or tree species.
However, there was a significant relationship be-


June 2003







Symposium: Insect Behavioral Ecology-2001: Romano


tween the number of tardigrades and site, indicat-
ing the need for additional replicate samples. A
marine meiofauna survey of subtidal regions of
Dauphin Island, AL in the northeast region of the
Gulf of Mexico was initiated 1999. Samples were
taken at mile intervals from the Mobile Bay side
(north) and the Gulf of Mexico side (south). A sam-
ple consisted of 500 cc's of sand collected from the
subtidal zone. Meiofauna were counted and tardi-
grades extracted from samples. A total of 20,846
meiofaunal organisms have been observed from 11
samples. Nematodes account for 69.1%, harpacto-
copepods account for 13.5%, and tardigrades ac-
count for 11.1% of the collection. Miscellaneous
organisms make up the remainder of the collec-
tions (5.8%) containing organisms such as foramin-
iferans, bivalves, cnidarians, polycheates, and
kinorhynchs. The genus Batillipes dominated the
tardigrade collection (Fig. 6).

Tardigrade Collecting

The best source of tardigrades is within moss
growing on the bark of live trees or leaf litter.
Moss on rocks is okay but contains a lot of dirt,
making the animals even more difficult to find.
Moss on soil is even worse, although you will find
tardigrades in about 50% of the samples. Moss on
rotten logs has very few, if any tardigrades, and
you might skip that habitat. Lichens on trees and
rocks are sometimes fruitful.
Following the procedure of Nelson (1975) soak
the moss sample in a stoppered funnel in tap water
(a bucket for leaf litter) for at least 3 hours (3-24
hours). You can leave the samples overnight in wa-
ter. Realize that you are trying to induce anoxybio-
sis so that the tardigrades release their hold of the
moss plants and are more easily removed. Remove
the moss and squeeze the remaining water out into
a clean beaker or jar. Some samples require vigor-
ous shaking and squeezing to remove a sufficient
quantity oftardigrades. Let the water and collected
materials in the jar settle and then decant the top
water. Pour the bottom layer of water and debris
into a collecting jar. If too much debris, especially
dirt, has been collected, the material may be sieved
through a nested series. Tardigrades, and eggs, will
be trapped on a #325 (45 pm) screen. Be sure to col-
lect material from 2-3 different sized sieves, since
larger tardigrades may be trapped by these. Each
piece of leaf litter should be rinsed and the water in
the bucket poured into a nested sieve series and
collected as above.


REFERENCES CITED

BERTOLANI, R. 1983. Tardigardi muscicoli delle dune
costiere Italiane, con descrizione di una nuova
specie. Atti. Soc. Tosc. Sci. Nat. Mem., Serie B, 90:
139B148. Eighth International Symposium on Tar-
digrada, Copenhagen, Denmark. 2000.
DEWEL, R. A., D. R. NELSON, AND W. C. DEWEL. 1993.
Tardigrada. In Microscopic Anatomy of Inverte-
brates, volume 12: Onychophora, Chilopoda, and
Lesser Protostomata. Wiley-Liss, Inc.
HUNTER, M. A. 1977. A study of tardigrada from a farm
in Montbomery County, Tennessee. MS Thesis, Aus-
tin Peay State University, 61 p.
KATHMAN, R. D., AND S. F. CROSS. 1991. Ecological dis-
tribution of moss-dwelling tardigrades on Vancouver
Island, British Columbia, Canada. Can. J. Zool. 69:
122-129.
KINCHIN, I. M. 1994. The Biology of Tardigrades. Black-
well Publishing Co., London.
MARCUS, E. 1929. Tardigrada. In H. G. Bronn (ed.) Klas-
sen und Ordnungen des tierreichs. Vol 5, Seciont 4,
Part 3: 1-608.
MARCUS, E. 1936. Tardigrada. In F. Schultze (ed.). Das
Tierreich. Walter de Gruyter, Berlin. 340 p.
MCINNES, S. J. 1994. Zoogeographic distribution of ter-
restrial/freshwater tardigrades from current litera-
ture. J. Nat. Hist. 28: 257B352.
NELSON, D. R. 1975. Ecological distribution of Tardi-
grada on Roan Mountain, Tennessee-North Caro-
lina. In R. P. Higgins (ed). Proceedings of the first
international symposium on tardigrades. Mem. Ist.
Ital. Idrobiol., Suppl. 32: 225-276.
NELSON, D. R. 1991. Tardigrada. In J. H. Thorp and A. P.
Covich (eds). Ecology and Classification of North
American Freshwater Invertebrates. London, Aca-
demic Press. Pp. 501-521.
NICHOLS, P. B., F. A. ROMANO, III, AND D. R. NELSON.
2001. Seasonal and altitudinal variation in the dis-
tribution and abundance of Tardigrada on Dugger
Mountain, Alabama. Zool. Ang. 240: 501-504.
RAHM, G. 1937. A new order of tardigrades from the hot
springs of Japan (Furu-Section, Unzen).Annot. Zool.
Japon. 16: 345-352.
RAMAZZOTTI, G. 1962. Phylum Tardigrada. Mem. Ist.
ital. Idrobiol. 14: 1-595.
RAMAZZOTTI, G. 1972. Il Phylum Tardigrada (Seconda
edizione aggionata). Mem. Ist. ital. Idrobiol. 28: 1-732.
RAMAZZOTTI, G., AND W. MAUCCI. 1983. The phylum
Tardigrada-3rd edition, English translation by CW
Beasley. Mem. Ist. Ital. Idrobiol. Dott. Marco de Mar-
chi 41: 1B680.
ROMANO, F. A., III, B. BARRERAS-BORRERO, AND D. R.
NELSON. 2001. Ecological distribution and commu-
nity analysis ofTardigrada from Choccolocco Creek,
Alabama. Zool. Ang. 240: 535-541.
THULIN, G. 1928. Uber die phylogenie und das system
der Tardigraden. Hereditas 11: 207-266.







Florida Entomologist 86(2)


June 2003


ADAPTATIONS OF NEMATODES TO ENVIRONMENTAL EXTREMES

ROBERT MCSORLEY
Department of Entomology and Nematology, University of Florida, Gainesville, FL 32611-0620

ABSTRACT

Nematodes are a highly diverse group of organisms that show a variety of adaptations to ex-
tremes in soil and plant environments. Developmental dormancy and diapause are impor-
tant for seasonal survival and long-term longevity of eggs in some species, whereas changing
sex ratios may improve survival chances of the next generation in some instances. More di-
rect and immediate responses to environmental conditions include aggregation or the for-
mation of relatively resistant dauer larvae. Many nematodes can undergo temporary
quiescence in response to environmental stress, and entry into anhydrobiosis or other ex-
treme states allows long-term survival in unusually stressful environments. These inactive
survival stages may make up a substantial proportion of the nematode population in some
terrestrial environments.

Key Words: anhydrobiosis, dormancy, nematode survival, plant-parasitic nematodes, soil
ecology

RESUME

Los nematodos son un grupo de organismos sumamente divers que demuestra una varie-
dad de adaptaciones a los ambientes extremes de suelo y plants. La latencia desarrollada
y la diapausa son importantes para la sobrevivencia estacional y la larga longevidad de hue-
vos en algunas species, mientras que cambiando la proporci6n de sexos puede mejorar la
probabilidad para sobrevivir de la proxima generaci6n en algunos casos. Las respuestas mas
directs e inmediatas a la condiciones ambientales incluyen la agregaci6n o la formaci6n de
larvas del estadio "dauer" (etapa alternative adaptada para su supervivencia) relativamente
resistentes. Muchos nematodos pueden pasar por una quiescencia temporaria en respuesta
al estres ambiental, y entrar a la anhidrobiosis u otros estados extremes permit la sobrevi-
viencia de largo plazo en ambientes extraordinariamente severos. Estos estadios inactivos
de sobrevivencia pueden representar una proporci6n substantial de la poblaci6n de nemato-
dos en algunos ambientes terrestres.


Nematodes are a diverse group of inverte-
brates abundant as parasites or free-living forms
in soil, freshwater, and marine environments. The
more than 15,000 described species probably rep-
resent only a small portion of the total members
in the Phylum Nematoda (Barker 1998). The soil
is a particularly rich habitat for nematodes, with
about 26% of described genera inhabiting soil as
bacterivores, fungivores, omnivores, predators, or
plant parasites (Wharton 1986). Added to this are
soil-dwelling stages of parasites on insects or
other animals, as well as freshwater genera that
colonize soil to varying degrees. A moisture film is
necessary for normal nematode activity (Wallace
1973), and therefore soil moisture, relative hu-
midity, and related environmental factors directly
affect nematode survival.
The soil environment offers varying degrees of
protection for nematodes from dehydration. Para-
sites that are inside plant roots or insects enjoy op-
timal moisture and protection from desiccation as
long as the health of the host persists. Life stages
or species that do not live inside a host find protec-
tion in moist soil, but risk increasing exposure to
dehydration as soils dry. Hazards increase as the


soil-air interface is approached (Womersley 1987).
A few unusual genera of plant parasites, such as
Anguina, Ditylenchus, and Aphelenchoides, risk
exposure in air as they climb (under humid condi-
tions) to infect aerial plant parts. Risk may in-
crease further as above-ground plant parts (leaves,
seeds, etc.) dry up or die along with the nematode
parasite inside. This overview introduces some of
the strategies that soil-inhabiting nematodes use
to cope with deteriorating environmental condi-
tions and with particularly severe conditions.

ADAPTATIONS IN THE LIFE CYCLE

The most generalized life cycle of a nematode
involves an egg, four juvenile stages (referred to as
J1 to J4), and the adult. In many species, the ap-
pearance of juveniles and adults are similar, but
great diversity exists in the life cycles of this large
group (Wharton 1986). The life cycle of some nem-
atodes offers built-in opportunities for resisting
environmental stresses, such as a protective cyst
that covers the eggs of some species. Many nema-
todes undergo the first molt in the egg, retaining
the protection of the eggshell for the developing J2.







Symposium: Insect Behavioral Ecology-2001: McSorley


Developmental Dormancy and Diapause

Diapause and other delays in development that
are common in insects (Chapman 1971; Romoser
& Stoffolano 1998) occur in some nematodes as
well (Evans & Perry 1976; Wharton 1986). Al-
though diapause is not necessarily a result of ad-
verse environmental conditions nor ended by
favorable conditions (Chapman 1971; Evans &
Perry 1976; Wharton 1986), it is nonetheless a
critical survival mechanism during cold seasons
and in the absence of a host. The stimulation of
egg hatching in Meloidogyne naasi Franklin by
chilling is a well-known example of diapause in a
nematode (Van Gundy 1985). In some species of
root-knot (Meloidogyne spp.) and cyst (Heterodera
spp., Globodera spp.) nematodes, a portion of the
eggs hatch quickly while others hatch slowly over
time (DeGuiran 1979; Zheng & Ferris 1991;
Huang & Pereira 1994). The distribution of egg
hatch over time may be quite complex. Zheng &
Ferris (1991) recognized four types of dormancy in
eggs of Heterodera schachtii Schmidt. Some eggs
hatched rapidly in water, some required host-root
diffusate for rapid hatch, while others hatched
slowly in water or in host-root diffusate. Stimuli
for hatching and ending of dormancy in various
species include such factors as temperature (Van
Gundy 1985) or the presence of host plant or root
leachate (Huang & Pereira 1994; Sikora & Noel
1996). The quality of the latter depended on crop
cultivar, phenology, and other factors (Sikora &
Noel 1996). Interpretation of dormancy and dia-
pause in nematode eggs is further complicated in
that the induction of dormancy in cyst nematodes
varies seasonally, and may be dependent on tem-
perature or host phenology (Yen et al. 1995; Sikora
& Noel 1996). Diapause and developmental dor-
mancy seem to apply mostly to the egg stage and
to juvenile stages within eggs, although instances
of diapause in later juvenile stages or adults are
known, mainly in a few animal-parasitic nema-
todes (Evans & Perry 1976).

Sex Ratios

Sex ratios are environmentally determined in
many nematodes, including amphimictic species
and those that are primarily parthenogenetic (Tri-
antaphyllou 1973). The production of males has
been especially well-studied in the root-knot nem-
atodes. In this group, the nematode hatches from
the egg as a mobile J2, which migrates through soil
and into plant root tissue, where it establishes a
permanent feeding site. Once the J2 begins to feed,
it becomes immobile, increases its body size, and
progresses through subsequent molts, developing
into a female that can reproduce parthenogeneti-
cally. Males can be very rare in root-knot nematode
populations, but in some instances may comprise
more than 60% of the population (Papadopoulou &


Triantaphyllou 1982). A variety of stresses may
lead to increased production of males. These in-
clude nutritional deficiency or reduced photosyn-
thesis in the host plant, age of the host plant, plant
growth regulators or inhibitors, increased nema-
tode population density, presence of plant patho-
gens, level of host plant resistance, and even
temperature or irradiation (Bird 1971; Trianta-
phyllou 1973). If stress is imposed during develop-
ment, second-stage juveniles developing as females
can undergo sex reversal, producing intersexes or
males (Triantaphyllou 1973; Papadopoulou & Tri-
antaphyllou 1982). Aside from the obvious advan-
tage of producing fertilized eggs with perhaps a
better chance of surviving adverse conditions, in-
creased male production in root-knot nematodes
results in the production of a mobile form that can
leave an area or plant under stress (Bird 1971).

RAPID RESPONSES
TO ENVIRONMENTAL STRESS

Protective strategies built into the life cycles of
nematodes help to ensure survival of the current
or subsequent generation. Some physiological
and behavioral responses allow nematodes to re-
act more quickly to environmental stresses. For
example, Steinernema carpocapsae (Weiser)
Wouts, Mracek, Gerdin, & Bedding can cope with
changing levels in soil 02 by alternating between
aerobic and anaerobic metabolism (Shih et al.
1996). Many species of nematodes will coil in re-
sponse to drying (Bird & Bird 1991).

Dauer Larvae

Many nematodes form a temporary stage
called a "dauer larva" in response to various types
of environmental or nutritional stresses. Depend-
ing on the nematode species, dauer larvae can be
formed in J2, J3, or J4 stages (Bird & Bird 1991).
They undergo modifications in the cuticle struc-
ture to decrease permeability (Bird & Bird 1991),
and some forms retain the cuticle from the previ-
ous molt as additional protection (Evans & Perry
1976). Dauer larvae are relatively inactive, but
can react if stimulated, and revert to the normal
juvenile stage if conditions improve. Desiccation,
depletion of food supply, crowding, or deteriora-
tion of an insect host are factors that can stimu-
late formation of dauer larvae (Wharton 1986;
Bird & Bird 1991; Womersley 1993). The forma-
tion of dauer larvae in Caenorhabditis elegans
(Maupas) Dougherty as the food supply declines
is mediated by pheromones (Huettel 1986).
The abilities of dauer larvae to resist environ-
mental stress and to recover quickly to normal
stages vary from species to species. The J4, or pre-
adult, ofDitylenchus dipsaci (Kuhn) Filipjev as well
as the J3 can control water loss to such an extent
that both stages could be considered as forms of







Florida Entomologist 86(2)


dauer larvae (Bird & Bird 1991). The J3 ofS. carpoc-
apsae is a relatively resistant infective stage that
may be exposed on vegetation or the soil surface as
it actively searches for insect hosts (Poinar 1979).

Aggregation

Occasionally, individuals of some nematode
species will mass together forming large aggrega-
tions. Probably the best known example is the ac-
cumulation of large numbers ofD. dipsaci on the
surface of stored flower bulbs (Christie 1959). The
nematode clumps may be so large that they are
actually visible to the naked eye as whitish
masses referred to as "nema wool." The masses
probably offer some protection against desiccation
(Cooper et al. 1971), and nematodes in the masses
may exhibit other low moisture adaptations such
as coiling and anhydrobiosis. In contrast, swarm-
ing, which refers to large coordinated population
movements of nematodes, is believed to function
more in dispersal and migration than in moisture
conservation (Croll 1970). Nematode aggregation
is difficult to study since large masses of nema-
todes building up in laboratory culture may not be
typical of those found in nature.

RESPONSES TO EXTREME ENVIRONMENTS

Quiescence refers to a dormant state in which
metabolism and activity are slowed down in re-
sponse to environmental stress. Unlike diapause,
the dormant state ends when the environmental
stress is relieved, and nematodes then return to
normal activity. A variety of environmental
stresses may trigger quiescent states (Table 1). In
extreme cases of prolonged quiescence, the meta-
bolic rate may fall below detectable levels and ap-
pear to cease. This extreme dormant condition is
referred to as anabiosis (Wharton 1986) or alter-
natively as cryptobiosis (Cooper et al. 1971). The
term "anhydrobiosis" is used most often to refer to
quiescent and anabiotic states, probably because
desiccation is the most frequent and most studied
cause of quiescence. The degree of quiescence ob-


TABLE 1. TERMINOLOGY APPLIED TO QUIESCENT STATES
OF NEMATODES.

Quiescent state
Environmental stress in response to stress'

Desiccation Anhydrobiosis
Low temperature Cryobiosis
Osmotic stress Osmobiosis
Low oxygen Anoxybiosis

1These terms used in response to specific environmental
stresses. The terms quiescence (least extreme) and anabiosis
(most extreme) refer to the intensity of the quiescent state.
Cryptobiosis is a synonym for anabiosis.


served among nematodes varies along a contin-
uum from mild quiescence to anabiosis, depending
on the nematode species involved and even within
the same species (Wharton 1986). Most nema-
todes can show quiescence at some point, but rel-
atively fewer species are capable of anabiosis.
Anabiosis is not restricted to nematodes, but is
common in some other invertebrate groups such
as rotifers and tardigrades (Barnes 1980).
Nematodes in anhydrobiosis (including ex-
treme anabiosis) can survive under remarkably
severe conditions (Table 2). Filenchus polyhypnus
(Steiner & Albin) Meyl was revived from a dry
herbarium specimen after 39 years (Steiner & Al-
bin 1946). Important observations and insights
into the unusual phenomenon of anhydrobiosis
have been provided by several reviews (Cooper et
al. 1971; Demeure & Freckman 1981; Wharton
1986; Womersley 1987; Barrett 1991). During en-
try into anhydrobiosis, a gradual water loss oc-
curs over time, as water content falls from 75-80%
in active nematodes to 2-5% in anhydrobiotic
forms (Demeure & Freckman 1981). Survival is
best if nematodes dry slowly; most species are
killed if drying occurs too quickly (Barrett 1991;
Demeure & Freckman 1981). Anhydrobiotic nem-
atodes will rehydrate in water, but there is a lag
time between immersion and their return to nor-
mal activity (Barrett 1991). The lag time is nor-
mally a few hours, but can vary from less than an
hour to several days, increasing with the inten-
sity of anhydrobiosis (Cooper et al. 1971; Wharton
1986; Barrett 1991). Recovery is improved if rehy-
dration is slow, and if nematodes are exposed to
high relative humidity before being immersed in
water. Repeated cycles of drying and rehydration
decrease viability (Barrett 1991).
The mechanisms responsible for anhydrobiosis
are not well understood, but decreased cuticular
permeability and the condensation or packing to-
gether of tissues and organelles are often ob-
served, and in some species, increased levels of
glycerol or trehalose are noted (Demeure &
Freckman 1981; Wharton 1986; Womersley 1987;
Barrett 1991). Coiling is a typical behavioral re-
sponse observed in anhydrobiotic nematodes, and
in most anabiotic forms since they enter anabiosis
through anhydrobiosis. However, the behavioral
response seems to depend on the factor inducing
anabiosis, since Aphelenchus avenae Bastian coils
in response to drying but relaxes in a straight po-
sition in response to low 02 (Cooper et al. 1971).
Many of the extreme examples of anhydrobio-
sis (Table 1) are foliar nematodes that venture
above ground or bacterivorous and fungivorous
nematodes from dry soils. But anhydrobiosis is
probably common in many types of nematodes, in-
cluding plant parasites living in soil (Womersley
1987), entomopathogenic nematodes (Womersley
1990), and possibly even freshwater forms inhab-
iting temporary ponds (Wharton 1986; Womers-


June 2003







Symposium: Insect Behavioral Ecology-2001: McSorley


TABLE 2. EXAMPLES OF NEMATODE SURVIVAL AFTER LENGTHY TIME IN ANHYDROBIOSIS.

Anhydrobiosis Time in
Nematode Normal active habits conditions' anhydrobiosis Reference

Anguina agrostis Foliar plant parasite Dried plant material 4 yr Fielding 1951
A. tritici Foliar plant parasite Dried plant material 9-30 yr Fielding 1951
Ditylenchus dipsaci Foliar plant parasite Dried plant material 16-23 yr Fielding 1951
D. dipsaci Foliar plant parasite -80C 5 yr Cooper et al. 1971
Filenchus polyhypnus Foliar in moss Dried plant material 39 yr Steiner & Albin 1946
Acrobeloides nanus Bacterivore in soil Dry soil 6.5 yr Nicholas & Stewart 1989
Panagrolaimus sp. Bacterivore in soil Dry soil 8.7 yr Aroian et al. 1993
Plectus sp. Bacterivore in soil -190C 125 hr Cooper et al. 1971
Plectus sp. Bacterivore in soil -270 C 8 hr Cooper et al. 1971
Dorylaimus keilini Freshwater nematode Dry mud 10 yr Cooper et al. 1971
Helicotylenchus dihystera Plant parasite in soil Dry soil 250 d Aroian et al. 1993
Pratylenchus penetrans Plant parasite in soil, Dry soil 770 d Townshend 1984
roots

Most at room temperature except as noted. Subzero exposures in laboratory, free of dry plant material or soil.


ley & Ching 1989). Plant-parasitic nematodes
living in soil or roots, such as Rotylenchulus reni-
formis Linford & Oliveira or Pratylenchus pene-
trans (Cobb) Filipjev & Schuurmans Stekhoven,
are able to undergo rather extreme states of an-
hydrobiosis, but in general are not considered as
successful at this strategy (e.g., less extreme an-
hydrobiosis, shorter time in anhydrobiosis) as
some of the more extreme examples such as D.
dipsaci (Townshend 1984; Womersley & Ching
1989), and their long-term survival under anhy-
drobiosis is lower (Wharton 1986).

CONCLUSIONS

Varying degrees of quiescence, particularly an-
hydrobiosis, enable nematodes to survive a vari-
ety of extreme conditions, including desert soils
(Freckman et al. 1977), Antarctic climates
(Pickup & Rothery 1991), dry fallow soils without
hosts (Womersley & Ching 1989), or dispersal in
dry seed, plant debris, or dust (Barrett 1991). The
phenomenon may be more common in nature
than formerly thought, if we consider that many
common soil nematodes may use this strategy to
some extent (Womersley 1987). In the plant para-
site P penetrans, for example, 22-31% of the pop-
ulation was in an anhydrobiotic state in soils
dried quickly, while 58-70% of the population was
in anhydrobiosis in soils dried slowly (Townshend
1984). It is likely that substantial portions of a
nematode population in soil may be overlooked,
since commonly used methods for extracting
nematodes from soil may miss anhydrobiotic
forms (McSorley 1987), for which specialized ex-
traction methods are required (Freckman et al.
1977). Extreme states of anhydrobiosis appear to
be more common in nematodes in water-stressed
environments such as drying, above-ground plant
parts, but nematodes active at the soil-air inter-


face are also vulnerable to desiccation and would
benefit from such strategies (Womersley 1987).
The fungivorous genus Aphelenchoides comprised
65-75% of the nematode fauna in pine litter in
Florida (McSorley 1993), and the capability of
Aphelenchoides spp. and the closely related Aph-
elenchus spp. for anhydrobiosis is well known (De-
meure & Freckman 1981; Wharton 1986). The
bacterivores and fungivores living in litter envi-
ronments are relatively unstudied compared to
economically important plant parasites. However,
it is possible that anhydrobiosis is a common phe-
nomenon and that a high proportion of the nema-
tode population may be in an anhydrobiotic state
in extreme environments such as those at the
soil-air interface, litter, above ground, or in very
cold or dry climates. Anhydrobiosis is fairly typi-
cal among Antarctic nematodes, for example
(Pickup & Rothery 1991; Wharton & Barclay
1993). Our ability to investigate and understand
nematode ecology in these environments will re-
main limited unless the anhydrobiotic portion of
the community is considered. Studies of such
marginal and stressful environments have and
will continue to yield more information on anhy-
drobiosis and other nematode survival strategies.

ACKNOWLEDGMENTS
The author thanks Drs. Kooh-hui Wang and Khuong
B. Nguyen for their reviews of this manuscript, and
Nancy Sanders for manuscript preparation. This work
was supported by the Florida Agricultural Experiment
Station, and approved for publication as Journal Series
No. R-08633.

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and C. C. Carter [eds.], An Advanced Treatise on Me-
loidogyne. Volume I. Biology and Control. North
Carolina State University Graphics, Raleigh, NC.
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WHARTON, D. A. 1986. A Functional Biology of Nema-
todes. The Johns Hopkins University Press, Balti-
more, MD.
WHARTON, D. A., AND S. BARCLAY. 1993. Anhydrobiosis
in the free-living antarctic nematode Panagrolaimus
davidi (Nematoda: Rhabditida). Fundam. Appl.
Nematol. 16: 17-22.
WOMERSLEY, C. 1987. A reevaluation of strategies em-
ployed by nematode anhydrobiotes in relation to
their natural environment, pp. 165-173. In J. A.
Veech and D. W. Dickson [eds.], Vistas on Nematol-
ogy. Society of Nematologists, Hyattsville, MD.
WOMERSLEY, C. Z. 1990. Dehydration survival and anhy-
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K. Kaya [eds.], Entomopathogenic Nematodes in Bio-
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Nematodes and the Biological Control of Insect Pests.
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YEN, J. H., T. L. NIBLACK, AND W. J. WIEBOLD. 1995.
Dormancy of Heterodera glycines in Missouri. J.
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ZHENG, L., AND H. FERRIS. 1991. Four types of dormancy
exhibited by eggs of Heterodera schachtii. Revue
Nematol. 14: 419-426.


June 2003







Symposium: Insect Behavioral Ecology-2001: Sivinski & Aluja


THE EVOLUTION OF OVIPOSITOR LENGTH
IN THE PARASITIC HYMENOPTERA AND THE SEARCH
FOR PREDICTABILITY IN BIOLOGICAL CONTROL

JOHN SIVINSKI AND MARTIN ALUJA
USDA-ARS, Center for Medical, Agricultural, and Veterinary Entomology, P.O. Box 14565, Gainesville, FL 32605

Institute de Ecologia, A.C., Km 2.5 Antigua Carretera a Coatepec
Apartado Postal 63, 91000 Xalapa, Veracruz, Mexico

ABSTRACT

Ovipositor lengths are thought to reflect the egg-laying and host-searching behaviors of par-
asitoids. For example, parasitoids that attack exposed foliage feeders often have short ovi-
positors compared to species that must penetrate a substrate to reach a host. However, the
relationship between host accessibility and ovipositor length is not apparent in a guild of
braconids that oviposits in the larvae of frugivorous Mexican tephritids. While the longest
ovipositors are up to 5x longer than the shortest, all attack roughly the same stages of their
shared hosts, often in the same fruits. Nor is there any evidence that the shorter ovipositors
represent a saving of metabolic resources and energy that is redirected toward egg produc-
tion or greater ability to move. It has been suggested that if the ovipositor length of an in-
troduced parasitoid is substantially different from the ovipositors of species already present,
then it is more likely to find an empty niche in its new environment, become established, and
add to the control of its host. However, with the present lack of a simple explanation for the
variety of ovipositor lengths within the Mexican guild it is not clear how predictive oviposi-
tor length would be in this instance. Until the evolution and maintenance of the various
lengths is better understood it may be more circumspect to practice fruit fly biological control
through the conservation and augmentation of parasitoid species already present.

Key Words: Hymenoptera, Diptera, Ichnuemonoidea, Braconidae, Opiinae, Chalcidoidea,
Tephritidae

RESUME

Se piensa que la longitud del ovipositor refleja el comportamiento de los parasitoides para
ovipositar y buscar el hospedero. Por ejemplo, los parasitoides que atacan hospederos que es-
tan expuestos sobre el follaje de que se alimentan a menudo tienen ovipositores cortos com-
parados con las species que tienen que penetrar un sustrato para alcanzar al hospedero. Sin
embargo, la relaci6n entire la accesibilidad al hospedero y la longitud del ovipositor en un
gremio de braconidos que oviposita en larvas de tefritidos mexicanos fruteros no es evidence.
Mientras que los ovipositores mas largos son hasta 5 veces mas largos que el mas corto, todos
atacan mas o menos las mismas etapas del hospedero compartido, a menudo en la misma
fruta. Tampoco hay evidencia que los ovipositores mas cortos representan un ahorro de los
recursos metab6licos y de energia que es redirijido hacia la producci6n de huevos o ha una
mayor mobilidad. Se ha sugerido que si la longitud del ovipositor de un parasitoide introdu-
cido es significativamente diferente de los ovipositores de las species ya presents, luego es
mas probable encontrar en ese nuevo ambiento un nicho vacio, establecerse, y aiadir para
el control de su hospedero. No obstante, con la falta de una explicaci6n sencilla para la va-
riedad en la longitud de los ovipositores en el gremio mexicano, no es claro cuan predicible
la longitud del ovipositor puede ser en este caso. Hasta que se entienda mejor la evoluci6n y
mantenimiento de las diferentes longitudes puede ser mas prudent practicar el control bio-
l6gico de la mosca de la fruta a trav6z de la conservaci6n y aumento de las species de para-
sitoides ya presents.


The extended-piercing ovipositor is perhaps needed to position the ovipositor/stinger at the
the key innovation that led to the diversity and most appropriate angle to reach the host or pene-
abundance of the parasitic Hymenoptera. It al- trate a cuticle (e.g., Quicke 1997).
lows feats of carnivory that are difficult or even While in essence a tube attached to a mobile
impossible for the other great parasitoid group, "delivery system", it is an over simplification to
the Diptera, and underlies the evolution of the imagine ovipositors as just biotic hypodermic nee-
distinctive "wasp" morphology. The wasp-waist dles (Quicke et al. 1999). They have external and
for instance, is a pivot that provides the flexibility internal structures that help steer them along







Florida Entomologist 86(2)


their course, serrations hardened with heavy
metal-protein complexes, internal channels that
deliver venoms, and microsculpturing to help
move eggs along often considerable distances
(Quicke et al. 1999; Vincent & King 1996). How-
ever, one of their seemingly simplest properties,
their length, has a number of complex ecological
and behavioral implications.
Even a passing familiarity with the parasitic
Hymenoptera reveals the considerable variety of
ovipositor lengths within the group. Why do these
egg-laying tools exist in all these various lengths?
The obvious answer is "to do their job by reaching
their hosts", recognizing that hosts have different
types of bodies and cuticles, and occur in a diver-
sity of environments, surrounded by different
depths and forms of materials, from unobstructed
air to solid wood. Price (1972; LeRalec et al. 1996)
accounted for the differences in ovipositor length
among the parasitoids of the Swaine jack pine
sawfly, Neodiprion swainei Middleton, by consid-
ering the tasks facing the different species. Some
attack buried pupae and others oviposit in larvae
exposed on leaf surfaces (Price 1972). Those that
lay eggs in pupae have long ovipositors, designed
to reach through leaf litter, while those that attack
foliage-feeding larvae have short ovipositors just
long enough to penetrate the host's integument.
But, will ovipositors be lengthened to deal with
every contingency the wasp might face? Or, as-
suming there are tradeoffs to ever increasing size,
will selection favor a length for every species that
is just sufficient to undertake the typical piercing-
depositing job it is likely to face? Might there be
an optimal length, neither a "deluxe" nor "econ-
omy" model? And if there is an optimal length, are
the only factors of any importance in its evolution
the type of host being exploited and the environ-
ment where the host occurs? The answer to the
last question seems to be no-at least not all the
time or in any straightforward manner. Consider
for example the braconids attacking Mexican
fruit flies (L6pez et al. 1999).
In the state of Veracruz 10 species of Hy-
menoptera attack tephritid flies of the genus
Anastrepha (e.g., L6pez et al. 1999). Among these
parasitoids are a suite of native opiine braconids:
Utetes anastrephae (Viereck), Doryctobracon are-
olatus (Szepligeti), Doryctobracon crawfordi (Vi-
ereck), and Opius hirtus (Fisher). An exotic
opiine, Diachasmimorpha longicaudata (Ash-
mead) originally from the Indo-Philippine region,
was established in the region over 30 years ago
(Ovruski et al. 2000). All are solitary, endopara-
sitic koinobionts (parasitoids whose hosts con-
tinue to develop after being attacked) that
oviposit only in frugivorous tephritids and com-
plete development within the host's puparium.
These species, both native and exotic, are geo-
graphically widespread and attack a wide range
of fruit flies in a diversity of fruits (L6pez et al.


1999; Sivinski et al. 2000). It is not unusual for
several to occur in any particular locale, or even
for more than one species to emerge from flies in-
festing a single piece of fruit; e.g., U. anastrephae
and D. areolatus are commonly found attacking
Anastrepha obliqua (Macquart) in the same
Spondius mombin L. fruits (Sivinski et al. 1997)
and up to 5 species of parasitoids have been recov-
ered from a single piece of fruit (Lopez et al.
1999). But while they have many similarities
with respect to host range, distribution, and life
histories, there are substantial differences in
their ovipositor lengths (Fig. 1). These range from
being less than the length of the abdomen in
U. anastrephae to several times the abdominal
length in D. crawfordi.
While these sympatric parasitoids share over-
lapping opportunities for oviposition, it appears
they are not able to take equal advantage of the
pool of hosts (Sivinski et al. 2001).Anastrepha lar-
vae infest fruits over a large range of sizes, from
little tropical "plums" weighing a few grams to
commercial mangos more than half a kilo in
weight (L6pez et al. 1999). All the braconids attack
flies in the smaller fruits, but only those with
longer ovipositors are common in larger fruits (Fig.
1). How do the short-ovipositor species persist, and
even flourish? Could there be a cost to having a
long ovipositor, one so great that an insect with
fewer options for oviposition, but investing in
"cheaper" equipment, is still able to compete?
There are certainly problems inherent in hav-
ing a very long ovipositor. Occasionally, species
such as the Peruvian ichnuemonid Dolichomitus
hypermenses Townes and the Japanese braconid
Euurobracon yakohamae Dalla Torre carry prodi-
gious external ovipositors, up to 8 times as long as
their bodies (e.g., Townes 1975; Fig. 2). Some Afri-
can Torymidae (or perhaps aberrant Pteromal-
idae) with ovipositors between 5 and 6 times their
body lengths, e.g., Ecdamura sp. and Eukoebelea
sp., are the likely record-holders among the chal-
cidoids (Compton & Nefdt 1988). However, these
are rare exceptions to the rule, and few oviposi-
tors exceed the more modest relative length of 1.3
times the body (Townes 1975). One reason is that
the greatest force can be applied to the ovipositor
when it is held perpendicular to the cuticle of a
host or to the surface of the surrounding medium,
and to accomplish this the abdominal tip must be
held at least an "ovipositor-length" above the sur-
face (van Achterberg 1986). Females wielding
moderately long ovipositors often assume a head
down/abdomen in the air/tip toe position to gain
the greatest possible elevation. But even if the op-
timal position can be attained, too great a force on
too-thin an ovipositor can cause it to bend (termed
Euhler buckling), and prevent effective penetra-
tion (Vincent & King 1996; Quicke et al. 1999). All
other things being equal the danger of this buck-
ling is greater the longer the ovipositor.


June 2003







Symposium: Insect Behavioral Ecology-2001: Sivinski & Aluja


\ThN _


Log fruit weight


Fig. 1. The relationship between the mean size (weight) of a fruit sample containing tephritid larvae and the
mean lengths of the ovipositors of the various parasitoids that attacked these particular larvae (see Sivinski et al.
2001). In general only parasitoids with longer ovipositors can exploit hosts in large fruits. The species, from top to
bottom, are Doryctobracon crawfordi, Diachasmimorpha longicaudata, Doryctobracon areolatus, Opius hirtus, and
Utetes anastrephae.


There are means of mitigating the positioning
and buckling difficulties caused by extreme
length. In Megarhyssa spp. ovipositors several
times their owner's length can be effectively
shortened by initially looping the shaft into a
membranous sac at the tip of the abdomen
(Townes 1975). The very long ovipositor of the
parasitic orussid sawflies is coiled within the ab-
domen, and gripped by apodemes as it is extruded
a bit at a time during drilling towards wood bor-
ing hosts. In this way the length of the exposed
portion of the ovipositor is minimized, as is the
problem of buckling (Cooper 1953; Quicke et al.
1999). In other instances, very long ovipositors
are not used to penetrate tough substrates, but
follow fissures or previously excavated tunnels
through the medium surrounding the host. Under
these circumstances, force and perpendicularity
are not as critical and the ovipositor may meet the
substrate at an angle of 120 degrees or less (van
Achtenberg 1986).
In addition to exacerbating the penetration
problems facing the ovipositor itself, increasing
length can strain the "delivery system", the body
of the wasp, by restricting movement, increasing
wind resistance in flight, and making the insect
more vulnerable to predators. Long ovipositors in


a number of parasitoid taxa are held internally,
e.g., that of the previously mentioned orussids is
looped several times within the abdomen (Cooper
1953). All cynipoids and some chalcidoids carry
the bulk of the ovipositor concealed in an internal
pouch (Fergusson 1988; Quike et al. 1999). In
chrysidids, platygasterids, and some scelionids
the terminal abdominal segments telescope the
ovipositor outward when in use and retract it
when at rest (Kimsey 1992; Felid & Austin 1994).
Even if not strictly internalized, the ovipositor is
sometimes held out of the way by doubling its
length back on the body. In the Vanhornidae it
bends forward to rest in a groove on the ventral
surface of the abdomen (Deyrup 1985). Leu-
cospids carry the ovipositor curved over the dorsal
surface of the abdomen, and in some platygas-
terids, such as Inostemma, the receptacle contain-
ing the internal portion of the ovipositor projects
forward, "handle-like", from the base of the abdo-
men over the thorax (e.g., Goulet & Huber 1993).
No matter how useful it would be to have an
ovipositor that could reach every host under the
most difficult circumstances, it would seem that
with all the problems, additional expenses and
modifications that go along with size, the maxi-
mum length ovipositor may not be the optimal for







Florida Entomologist 86(2)


Fig. 2. A female Megarhyssa atrata (Fab.), a large ichnuemonid parasitoid with a very long ovipositor. The ovi-
positor can loop into a membranous pouch at the tip of the abdomen which shortens its exposed length. Such short-
ening prevents the ovipositor from buckling as it penetrates wood to reach the wasp's host.


the insect design as a whole. In terms of the Mex-
ican braconids with the variety of ovipositor
lengths, what might be the costs that prevent
D. crawfordi (long) from displacing D. areolatus
(medium) from displacing U. anastrephae (short)?
The energy and materials used to construct,
maintain, and move an extended ovipositor could
presumably have been spent elsewhere, perhaps in
the production of more eggs, or in bigger flight
muscles and better searching capacity. Of course,
some fly larvae-hosts might be too deep in large
fruits for the short-cheap ovipositor parasitoid to
exploit, but access to these could be the benefit that
makes it worthwhile for a competing species to
continue to invest in a long-expensive ovipositor.
That is, disruptive selection might result in a re-
source being shared by species with long and short
ovipositors with few and many eggs, respectively.
The original prediction that fecundity should
drop as ovipositors become longer, was made by
Price (1973), who argued that if species with
longer ovipositors deal with less accessible hosts,
then, all other things being equal, handling time
per oviposition should be greater and oviposition
opportunities/unit of time should be fewer. In ad-
dition, since less accessible hosts are typically


more mature, and because inevitable mortality
occurs over the developmental period of the host,
older, less accessible hosts should not be as abun-
dant as younger, more accessible hosts. Both of
the factors, longer handling time and fewer hosts,
would contribute to lower potential rates of para-
sitism in species with long ovipositors. His hy-
pothesis was supported by a strong negative
correlation among species of Ichnuemonidae be-
tween ovipositor lengths and the numbers of ova-
rioles per ovary (which reflects the potential for
egg production).
Is there a relationship between ovipositor
length and fecundity in the Mexican braconids?
No, there is not. The number of eggs does not sig-
nificantly increase or decrease with ovipositor
length. If there is a trend at all, it is in the oppo-
site direction. The longer the ovipositor, the rela-
tively more of the body is taken up by egg volume
(No. of eggs*size of eggs) (Sivinski et al. 2001).
Though the "longer the ovipositor the lower the
fecundity argument" is broadly supported when
many species of Ichnuemonidae attacking a vari-
ety of host stages are considered, it is not as suc-
cessful when looking at the one small guild of
Braconidae attacking similar aged fruit flies un-


June 2003







Symposium: Insect Behavioral Ecology-2001: Sivinski & Aluja


der what seem to be similar circumstances. But
are circumstances really so comparable after all?
Despite overlaps in host ranges, each species has
one or more specialized foraging areas within its
niche. If the fruits within these specialized areas
differ in size or penetrability, then the hosts they
contain differ in accessibility, and this difference
in host accessibility might lead to differences in
ovipositor length. Maybe ovipositor lengths have
evolved in a variety of unrelated situations, and
each length is so well suited to this core ecological
"stronghold" that whatever advantage or disad-
vantage it faces with competing species exerts a
relatively trifling selection pressure. For example,
the short-ovipositored 0. hirtus attacks the
monophagous tephritid Anastrepha cordata Ald-
rich as it develops in Tabernaemontana alba Mill.
(Hernandez-Ortiz et al. 1994). For unknown rea-
sons it is the only parasitoid to commonly do so,
and since the pulp of this fruit is relatively shal-
low there may be no selection for a longer ovipos-
itor in this particular, and arguably important,
tritrophic interaction. There are any number of
other such "specializations" such as greater toler-
ance for heat or ability to flourish at high alti-
tudes (Sivinski et al. 2000).
While the diversity of ovipositors can form en-
gaging intellectual puzzles, their different
lengths also have broad practical, agricultural
implications. These arise from the argument by
Price (1972) that ovipositor length might be a
means of predicting which newly introduced par-
asitoids will be able to avoid competition within
an already existing guild of natural enemies, and
so have the best chance of successful establish-
ment and the provision of additional control.
At this point, let us make a somewhat lengthy
digression to discuss the history of prediction in
biological control. Predictability is a supreme vir-
tue in an applied science such as entomology
where we strive to find some way of saying that
this good thing will happen and this bad thing will
not. The search for biological predictability has
become an issue of increasing importance in
terms of both invasive species that arrive in new
locations by accident and potentially beneficial ar-
thropods deliberately moved from one place to an-
other. As the world becomes more homogeneous
through the spread of weedy species, the aesthetic
appreciation of biological diversity increases
along with greater awareness of its economic and
ethical implications (widespread similarity miti-
gates the evolutionary potential of life). There is a
growing cultural mandate to prevent the accumu-
lation of potential pests and extraneous biological
control agents (Simberloff & Stiling 1996; Thomas
& Willis 1998). The latter always present some
risk, no matter how small, of attacking nontarget
insects or plants. In cases where nontargets have
relatively slow rates of increase, "apparent compe-
tition", where an organism harbors a natural en-


emy that also attacks a more vulnerable species
and as a result becomes a superior competitor, can
be potentially devastating (Bonsall & Hassell
1997; Hudson & Greenman 1998). Even some-
thing that is initially safe may have the capacity
to adapt to a more diverse environment and in-
crease its host range (Willamson 1996).
A means of judging the present predictability
of biological control is to compare the rates of es-
tablishment and resulting abundances of deliber-
ately introduced natural enemies with the fates of
"invasive" organisms that arrive in new areas
largely by chance. It seems that establishing a
beachhead is a long shot for an invading organ-
ism, and can be described by the "Rule of 10s".
Willamson (1996) estimated that only 1 acciden-
tally introduced animal or plant in 10 becomes es-
tablished and only 1 out of 10 of these becomes
abundant and pestiferous. Interestingly, the odds
of a deliberately introduced biological control or-
ganism becoming common enough to exert an eco-
nomic impact are only somewhat better, perhaps
3 in 10 become established and 3 of those effect
control (depending on how success is measured).
Apparently, there is often a far from complete un-
derstanding of the relevant ecology of natural en-
emies and their prey, and hence a long standing
interest in why some natural enemies "work" and
others do not.
Among practitioners of biological control there
have been several attempts to collect and synthe-
size the attributes of successful natural enemies
in order to focus explorations and make establish-
ments more effective and environmentally safe.
Propagule pressure, the size of the released co-
hort, is important to the outcome of natural en-
emy establishment. In a survey of Canadian
programs, increases in the numbers of released
insects, from <5000 to >30,000, improved success
rates from 9% to 79% (Beirne 1975; Willamson
1996). If fewer than 800 individuals were in-
cluded in individual releases success occurred
15% of the time compared to 65% if more than 800
insects were involved, and more than 10 releases
gave 70% success compared to 10% for programs
using fewer releases. When Goeden (1983) exam-
ined the insects introduced for weed control he
found long attack season, gregarious feeding, and
ease of colonization to be the most important con-
tributors to success. The last of these has implica-
tions for propagule pressure.
In addition to how the craft of biological con-
trol is practiced there are some ecological gener-
alizations concerning the vulnerability of insects
to their natural enemies that might result in
more predictable control. For example, biocontrol
has tended to be more efficacious when applied
against specialist herbivores rather than general-
ists and against exposed rather than concealed
feeders (Gross 1991). Hosts that suffer high max-
imum parasitism rates, and by implication have







Florida Entomologist 86(2)


fewer or less effective refuges to shelter within,
are more likely to be successfully controlled
(Hawkins & Gross 1992; Hawkins 1994). Within
particular host taxa there are a number of even
more specific correlations between vulnerability
and type of natural enemy, and these relation-
ships could be used to direct future establishment
attempts. For example, Dyer and Gentry (1999)
have examined the categories of predators and
parasitoids that typically inflict high or low mor-
talities on Lepidoptera larvae with different
morphological characteristics and defensive be-
haviors. Brightly colored larvae were likely to be
rejected by wasps and bugs, but were attacked by
ants and parasitoids, generalists were more likely
to succumb to predation than to parasitoids,
while hairy species were relatively immune to
ants and bugs but fell victim to wasps and parasi-
toids, and so on. On the basis of their analysis
they suggest that the generalist feeding habits of
the infamous pest caterpillars of the genus
Spodoptera (Noctuidae) are the reason they have
not been successfully controlled by parasitoids,
despite considerable efforts, and argue that in the
future, predators, such as carabid beetles, might
be more profitably employed.
In addition to morphology and ecology, the his-
tory of a pest and parasitoid interaction might be
used to predict successful biological control. Hok-
kanen and Pimentel (1984) proposed that new as-
sociations between insects and natural enemies
resulted in substantially greater mortality and a
higher degree of pest suppression. The basis of
their thesis was that long standing interactions
will tend to be more benign since a prey species
will have had ample opportunities to adapt to its
hunterss, but that it will be relatively defenseless
when confronted with a novel set of weapons and
hunting tactics. There are at least two criticisms of
this theory. One is that the data used to substanti-
ate the greater vulnerability of prey to new para-
sitoids can be reinterpreted to reach the opposite
conclusion (Waage 1991). The second is that there
is accumulating evidence that long term associa-
tions are typically more virulent than new ones:
i.e., it is the natural enemies, including pathogens
and parasites, that are ahead in arms races with
their victims, and that familiarity has resulted in
increasingly effective weapons and hunting tactics
(e.g., Herre 1993; Ebert 1994; Kraaijeveld et al.
1998). While the opposite of earlier thinking, this
emerging generalization of familiarity breeding le-
thality can be used as a predictive tool. It suggests
that the closest possible match between the origi-
nal populations of exotic pests and the populations
of natural enemies that attacked them would tend
to be most efficacious. However, as noted by Waage
(1991), there seem to numerous exceptions to this
rule of thumb, and in a practical sense one should
not ignore any potential natural enemy regardless
of origin.


There are also population characteristics, i.e.,
the distribution and abundance of a parasitoid in
nature that might predict usefulness in a biologi-
cal control program. Rare species on the periph-
ery of host populations may be less competitive
than other natural enemies, but be better forag-
ers at low host densities. Such a species might do
very well indeed if it could be introduced by itself
into a new environment to deal with an exotic
pest (e.g., Force 1974).
Now let us return to Price (1972) who reasoned
that ovipositor length could be yet another means
of estimating the likelihood that an exotic species
would become established and whether it would
disrupt the composition of an already existing na-
tive guild. He followed Hutchinson (1959) and
Schoener (1965) who found that a trophicc appara-
tus", such as a bird's beak or an ovipositor, typi-
cally differs in size among sympatric species at the
same trophic level, and that these differences in
size are related to the differences in foraging be-
haviors that allow the species to coexist. A ratio of
the larger to the smaller apparatus of 1.15 indi-
cates sufficient niche separation in terms of the re-
source the apparatus is used to exploit. When
ovipositor length ratios were examined in the
guild of parasitoids attacking the Swaine jack
pine sawfly, Price found that this threshold ratio
was exceeded in comparisons among native spe-
cies, but that the introduction of a European ex-
otic had created a too close pairing of lengths
between itself and a native species, and that there
was already evidence of competitive displacement.
In the spirit of Price's search for predictability
through ovipositor length, what do the various
ovipositors of the Mexican braconids reveal about
the potential for expansion through new introduc-
tions of this fruit fly parasitoid guild where it is al-
ready established, and about the use of its
constituent species in future tephritid biological
control programs elsewhere? There is the well-es-
tablished relationship between ovipositor length
and the size of the fruit a parasitoid can effectively
forage upon. One might prefer to introduce a long
ovipositored species such as D. crawfordi into new
habitats dominated by large fruits. Other than
this, there is little that can be said with certainty.
There are obvious differences in ovipositor
lengths, much as there are in Price's sawfly para-
sitoids. But, while the sawfly-parasitoid oviposi-
tors are clearly due to distinct differences in
foraging for different host stages, the same cannot
be easily said for the Mexican tephritid-parasi-
toids. At this point it is difficult to say with any
conviction how the various parasitoids manage to
coexist in sympatry, and what role the differences
in their ovipositors play in their coexistence.
If attempts were made to improve fruit fly bio-
logical control in Mexico are there "empty" niche
spaces where exotic parasitoids would fit? Given
our lack of understanding how the present diver-


June 2003







Symposium: Insect Behavioral Ecology-2001: Sivinski & Aluja


sity of tephritid parasitoids is maintained this is
a troubling question to address. There is some cir-
cumstantial evidence of displacement of the long-
ovipositored native D. crawfordi by the long-ovi-
positored exotic D. longicaudata (Sivinski et al.
1997), but the way in which this may have oc-
curred remains obscure.
What is the best response to ignorance of the
consequences of projected parasitoid introduc-
tions? More study is an obvious answer, but what
if the sort of study that could predict success or
dangerous failure requires time, and that during
that period of study inactivity is impractical? We
suggest that one way to deal with the present con-
fusion and to best adhere with the applied-biology
dictum of "do no harm" is to fully exploit what is
already there; i.e., to conserve the existing guild
and enhance its effectiveness through habitat
manipulation.
For example, only 3.5% of the >200 species of
Anastrepha are of any economic importance, yet a
number of benign, generally monophagous, spe-
cies developing in native fruits harbor the same
parasitoids that attack notorious pests such as
the West Indian fruit fly, A. obliqua, or Mexican
fruit fly,A. ludens (Loew) (Aluja 1999). By encour-
aging the replanting of these sometimes endan-
gered fruit trees in the vicinity of orchards it may
be possible to support large numbers of parasi-
toids that will suppress pests that threaten crops
destined for local consumption or markets (Aluja
1999). In addition to insect control and the con-
servation of disappearing plants and the flies and
other arthropods associated with them, replanted
fruit trees can be managed as timber and har-
vested for a profit. Tapirira mexicana Marchand,
a tree that supports A. obliqua but also large
numbers of 4 species of braconid parasitoids, has
a wood equal in quality to mahogany (Terrazas &
Wendt 1995).

ACKNOWLEDGMENTS

Gina Posey prepared the illustrations and Valerie
Malcolm the manuscript. Rob Meagher, James Lloyd,
and Laura Sirot all made valuable criticisms of an ear-
lier draft. The research described was supported in part
by grants from USDA-OICD, the Campaia Nacional
Contra Moscas de la Frutas (SAGARPA-IICA), the
Sistema de Investigati6n del Golofo de M6xico
(SIGOLFO-CONACyT Project 96-01-003-V), and the
Comision Nacional para el Conocimiento y Uso de la
Biodiversidad (CONABIO Project H-296).

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June 2003







Thomas et al.: African Cluster Bug


THE AFRICAN CLUSTER BUG, AGONOSCELIS PUBERULA
(HETEROPTERA: PENTATOMIDAE), ESTABLISHED IN THE NEW WORLD

DONALD B. THOMAS1, JOSEPH E. EGER2, WALKER JONES' AND GUILLERMINA ORTEGA-LEON3
'Kika de la Garza Subtropical Agricultural Research Center, United States Department of Agriculture
Agricultural Research Service, 2413 E. Hwy 83, Weslaco, TX 78596

2Dow AgroSciences, 2606 S. Dundee St. Tampa, FL 33629

'Instituto de Biologia, Universidad Nacional Autonoma de Mexico A.P. 70-153, Mexico D.F. 04510

ABSTRACT

An African species of Pentatomidae, Agonoscelis puberula Stal, is reported for the first time
from Mexico, the southern United States and the islands of Jamaica and Hispaniola, where
it has now established. The oldest Western Hemisphere record dates from 1985. This species
has gone unrecognized probably because of its close resemblance to species of the New World
genus Trichopepla Stal. The primary host plant ofA. puberula is the introduced weed, com-
mon horehound, Marrubium vulgare L. It has also been reported damaging winter fruits in
South Africa.

Key Words: cluster bug, horehound, stink bug, invasive species

RESUME

Una especie Africana de Pentatomini, Agonoscelis puberula Stal, es reportada por primera
vez para M6xico, sur de Estados Unidos y las islas de Jamaica y Espaiola, en donde se ha
establecido. Los registros en el hemisferio oeste mas antiguos son de 1985. Esta especie no
habia sido reconocida probablemente por su gran parecido a las species del g6nero del
Nuevo Mundo Trichopepla Stal. La plant hospedera primaria deA. puberula es la hierba co-
nocida como marrubio, Marrubium vulgare L. Tambi6n ha sido reportada daiando frutos de
invierno en Sudafrica.


Translation provided by author.


In this paper we give the first report of the Af-
rican pentatomid bug, Agonoscelis puberula Stal,
in the New World. Established populations of this
stink bug have been discovered in the United
States, Mexico, and the islands of Jamaica and
Hispaniola. The species was first found among a
series of specimens collected in 1991 near the
town of Yautla in the state of Morelos, Mexico.
These specimens were tentatively identified by
one of us (GOL) as an undescribed species of the
north-temperate genus Trichopepla Stal, to which
they keyed in Rolston and McDonald's (1984)
treatment of Western Hemisphere Pentatomini.
Its discovery in the Greater Antilles led another
of us (JEE) to recognize this stink bug as an intro-
duced species of the genus Agonoscelis Spinola,
one referred to in the economic literature as a
"cluster bug" (Haines 1935).

Taxonomy and Recognition

As in Trichopepla, species of Agonoscelis are
generally yellowish, often with a red tinge and
with black punctures arranged in a pattern of ir-
regular dark stripes, and a distinctly hirsute dor-


sum (Fig. 1). Other shared characteristics include
the scent gland orifice attended by a short auricle;
the post-frenal scutellum more than half the
width of the scutellar base, and base of the abdo-
men lacking a tubercle. In both genera the head is
elongate compared with other pentatomines. Our
specimens range from 8-10 mm in body length. In
spite of their similarity, Trichopepla and Agono-
scelis have been split into different tribes. This
anomaly arises largely because there is no con-
sensus classification for the Pentatominae. Amer-
ican workers, such as Rolston & McDonald (1984),
follow the tribal arrangement in Kirkaldy's Cata-
logue (1909) which places both genera in the Pen-
tatomini. Asian workers, such as Ahmad et al.
(1974), follow the arrangement of Distant (1921)
which places Agonoscelis in the Eurydemini
(=Strachiini). African workers, such as Cachan
(1952), include Agonoscelis with the tribe Carpo-
corini. Inasmuch as there are no external mor-
phological characters to distinguish Agonoscelis
from Trichopepla, their placement in different
tribes is problematic. According to McDonald
(1966), the female spermatheca of Trichopepla is
unique in lacking a sclerotized supporting rod







Florida Entomologist 86(2)


r'A


Fig. 1.Agonoscelis puberula, specimen from Morelos,
Mexico.


and pumping region that is present in all other
Pentatomines, including Agonoscelis (illustrated
by Gross 1976). Such being the case, the separa-
tion of the two genera can be sustained, although
the tribal-level separation seems dubious. Ago-
noscelis puberula has a distinctively marked
hemelytral membrane featuring dark radiating
stripes. This character is variable among species
ofAgonoscelis, but the membrane is unmarked in
all species of Trichopepla (McDonald 1976); thus,
the striped membrane allows quick recognition of
this adventive stink bug.
The genus Agonoscelis has not been revised, al-
though regional faunal treatments (Horvath 1904;
Jensen-Haarup 1920; Cachan 1952; Yang 1962;
Ahmad et al. 1974; Linnavuori 1975, 1982; Hsaio
et al. 1977) provide means to diagnose many of the
species. There are 22 nominal species ofAgonosce-
lis including those of uncertain validity. Among
the determined specimens available to us for
study, representing six species, we noted that the
male genitalia are distinctive to a given species,
and it is on these characters that our determina-
tion relies. The New World invader is conspecific


with specimens from South Africa identified by D.
A. Rider and others asA. puberula Stal. The male
genitalia of this particular species are illustrated
by Linnavuori (1975) whose material was com-
pared to Stal's types in Stockholm. We have depos-
ited voucher specimens in the United States
National Museum, the Canadian National Collec-
tion, the Instituto de Biologia of the Universidad
Nacional Autonoma de Mexico, at Texas A&M
University, in the Florida State Collection of Ar-
thropods, and in the collections of the authors.

Distribution

Agonoscelis puberula is native to southern and
eastern Africa extending northward to the Ara-
bian peninsula (Linnavuori 1982). Our oldest
New World record dates from 1985 on the island
of Jamaica. The first records for the United States
are from Arizona in 1990. U.S. records include Ar-
izona, New Mexico, and Texas. It is also well es-
tablished in Mexico with records from Yucatan in
the south to Nuevo Leon in the north covering the
years 1988 to 2001. Our collection data includes
the following specific localities and dates:
JAMAICA: St. Andrew Parish, 2 mi. S. New-
castle, 2-VIII-1985, C.B. & H.V. Weems Jr. & G.B.
Edwards.
DOMINICAN REPUBLIC: La Vega: 21 Km S
Jarabacoa, 18-26-VI-1994, C. & K. Messenger.
MEXICO: Guanajuato: San Miguel de Allende,
7-11-VIII-1988, G. B. Edwards; Chipicuaro, Presa
Solis, 12-III-1997, E. Barrera & H. Brailovsky;
Ojo Seco, 12-III-1997, E. Barrera & H.
Brailovsky; San Antonio Emenguaro, 12-III-1997,
H. Brailovsky, E. Barrera & G. Ortega-Leon. Mo-
relos: Yautla, 3-V-1991, H. Brailovsky & E. Bar-
rera. Distrito Federal: Piramides de Cuicuilco, 2-
IV-1992, E. Gonzalez; Delegacion Iztapalapa, 1-
VIII-1999, J. Contreras; Colonia Irrigacion, 18-
VI-2001, H. Brailovsky. Mexico: Ixtapan de la Sal,
4-X-2000, H. Brailovsky & E. Barrera; Malinalco,
VII-1996, E. Barrera. Guerrero: Tuxpan, 25-X-
2001, H. Brailovsky, E. Barrera & G. Ortega-
Leon. Hidalgo: Huichapan, 5-VI-1999, H.
Brailovsky & E. Barrera; Huasca, 4-VIII-1995, H.
Brailovsky. Michoacan: San Lorenzo, 24-X-2001,
H. Brailovsky & E. Barrera. Oaxaca: Domin-
guillo, 18-II-1998, H. Brailovsky, E. Barrera & G.
Ortega-Leon; Tehuacan-Oaxaca Km 140, 11-III-
2000, H. Brailovsky & E. Barrera. Puebla: Teca-
machalco-Tehuacan Km 1, 12-VI-1993, H.
Brailovsky & E. Barrera; La Trinidad, 3-II-1994,
E. Barrera & G. Ortega-Leon; La Trinidad, 13-II-
1994, E. Barrera & G. Ortega-Leon; La Trinidad,
21-III-1994, E. Barrera & G. Ortega-Leon; Atlixco
23-IV-1994, E. Barrera & G. Ortega-Leon;
Atlixco-La Trinidad, 29-V-1994, H. Brailovsky &
E. Barrera; La Trinidad, 15-VI-1994, E. Barrera
& G. Ortega-Leon; 5 Km SE Atlixco, 23-IV-1994,
15-VI-1994, H. Brailovsky, E. Barrera & G. Or-


June 2003







Thomas et al.: African Cluster Bug


tega-Leon; 2 Km W. La Trinidad, 19-III- 1994, G.
Ortega-Leon & E. Barrera; Atexcal, 11-III-1994,
E. Barrera & G. Ortega-Leon; Nicolas Bravo, 20-
III-1993, H. Brailovsky, E. Barrera & G. Ortega-
Leon; Tecamachalco, 6-1-1993, 27-1-1993, 12-VI-
1992, 20-VII-1992, H. Brailovsky & E. Barrera;
Atlixco, 18-VIII-1996, H. Brailovsky, E. Barrera
& G. Ortega-Leon; Portezuelo, 10-II-1995, E. Bar-
rera & G. Ortega-Leon. Queretaro: Pinal de
Amoles, 27-IV-1998, 1-III-1998, E. Barrera & G.
Ortega-Leon. Yucatan: Temax, 24-V-1995, E. Bar-
rera & H. Brailovsky. Nuevo Leon: El Pinito, 3-IX-
1995, D.B. Thomas & J. Burne.
UNITED STATES: Arizona: Pinal Co., Pepper-
sauce Canyon, Santa Catalina Mtns., 9-IV-1991,
C. Olson; Santa Cruz Co., Madera Canyon, 16-IV-
1990, 17-VII-1990, 27-VII-1990, W. Jones; Patago-
nia, 8-VII-1994, B. Brown & E. Wilk; Santa Rita
Mtns., Florida Canyon, 1-VIII-1992, W. Jones; Ar-
rivaca Springs 1-VIII-1992, W. Jones. New Mex-
ico: Hidalgo Co., 11 mi. NE Lordsburg, 31-VIII-
2000, J. Huether. Texas: Concho Co., Eden, 29-
XII-1999 [no collector].

Host Plants

At four separate Arizona localities a total of 26
adults was collected by one of us (WJ) on the pan-
demic weed, common horehound, Marrubium
vulgare L. (Labiatae). At one of these sites
nymphs were also present. This is also a known
host plant for the Australian horehound bug, Ag-
onoscelis rutila (F.) (Gross 1976). In South Africa,
Haines (1935) reported that A. puberula breeds
on its natural host plant in the summer, but over-
winters in buildings and on fruit trees, sometimes
clustering on the fruits and causing "considerable
damage." Unfortunately, Haines neglected to
state the species of the host plant or the fruit
damaged in South Africa. Our specimens from
Concho County, Texas, were found on December
29 among stems and leaves of live oak, suggesting
that they were overwintering in this habitat.
Our colleague Thomas J. Henry (USDA-ARS) in-
forms us that he has frequently identified Ago-
noscelis versicolor (F.), intercepted on cut flowers
shipped to the United States from South Africa via
the Netherlands. This suggests a plausible route for
the entry of A. puberula which may have estab-
lished because of the ready availability of an accept-
able host plant. According to Correll and Johnston
(1970), common horehound is widely distributed in
North America, flowers throughout the year, and is
a weed typical of waste places and roadsides.

ACKNOWLEDGMENTS

The authors are grateful to Thomas J. Henry, Sys-
tematic Entomology Laboratory, USDA-ARS, Washing-
ton D.C. for the loan of determined specimens of


Agonoscelis in the National Museum of Natural History,
Smithsonian Institution, Washington D.C., and infor-
mation on intercepted stink bugs. We also extend thanks
to the curators of the following institutions for access to
material in their collections: Carl Olson, University of
Arizona, Tucson; Edward G. Riley, Texas A&M Univer-
sity, College Station; Susan Halbert, Florida State Col-
lection of Arthropods, Gainesville; and, Michael D.
Schwartz, Canadian National Collection, Ottawa. Chris
Mari Van Dyck produced the photograph in Figure 1 and
Rene Davis enhanced the photo with Adobe Photoshop.

LITERATURE CITED

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and supergeneric keys with reference to a checklist
of Pentatomid fauna of Pakistan (Heteroptera: Pen-
tatomoidea) with notes on their distribution and
food plants. Entomol. Soc. Karachi, Supplement No.
1. 103 p.
CACHAN, P. 1952. Les Pentatomidae de Madagascar
(Hemipteres, Heteropteres). Memoires de L'Institute
Scientifique de Madagascar. Serie E. vol. 1, Fasc. Pp.
231-462.
CORRELL, D. S., AND M. C. JOHNSTON. 1970. Manual of
the vascular plants of Texas. Texas Research Foun-
dation, Renner, Texas.
DISTANT, W. L. 1921. The Heteroptera of Indo-China,
Family Pentatomidae. The Entomologist 54: 3-6.
GROSS, G. 1976. Plant-feeding and other bugs (Hemip-
tera) of South Australia. Heteroptera-Part I. A.B.
James, South Australia.
HAINES, G. C. 1935. Cluster Bugs. Farming South Africa
10(109): 182, 188.
HORVATH, G. 1904. Pentatomidae novae Africanae. Ann.
Mus. Nat. Hungarici 2: 253-271.
HSIAO, T., S. REN, L. ZHENG, H. JING, H. ZOU, AND S.
LIU. 1977. A handbook for the determination of the
Chinese Hemiptera-Heteroptera. Academia Sinica,
Beijing (in Chinese).
JENSESN-HAARUP, A. C. 1920. Hemipterological Notes
and descriptions I. Entomol. Medd. 13: 209-224.
KIRKALDY, G.W. 1909. Catalogue of the Hemiptera (Het-
eroptera) with biological and anatomical references,
lists of food plants and parasites, etc. Vol. I. Cimi-
cidae. Berlin. 392 p.
LINNAVUORI, R. 1975. Hemiptera of the Sudan, with re-
marks on some species of the adjacent countries, 5.
Pentatomidae. Bol. Soc. Port. Cienc. Nat. 15: 5-128.
LINNAVUORI, R. 1982. Pentatomidae and Acanthosomi-
dae (Heteroptera) of Nigeria and the Ivory Coast, with
remarks on species of the adjacent countries in West
and Central Africa. Acta Zool. Fennica 163: 1-176.
McDONALD, F. J. D. 1966. The genitalia of North Amer-
ican Pentatomoidea (Hemiptera: Heteroptera).
Quaest. Entomol. 2: 7-150.
McDONALD, F. J. D. 1976. Revision of the genus Tri-
chopepla (Hemiptera: Pentatomidae) in N. America.
J. New York Entomol. Soc. 84: 9-22.
ROLSTON, L. H., AND F. J. D. McDONALD. 1984. A con-
spectus of Pentatomini of the Western Hemisphere.
Part 3. (Hemiptera: Pentatomidae). J. New York En-
tomol. Soc. 92: 69-86.
YANG, W. I. 1962. Economic insect fauna of China: Fasc.
2. Hemiptera, Pentatomidae. Academia Sinica. Sci-
ence Press, Beijing.







Florida Entomologist 86(2)


June 2003


FEEDING AND SURVIVAL OF CITRUS SHARPSHOOTERS
(HEMIPTERA: CICADELLIDAE) ON HOST PLANTS

JOSE M. MILANEZ1, JOSE R. P. PARRA2, ISABEL A. CUST6DIO2, DENISE C. MAGRI2,
CHRISTINA CERA2 AND JOAO R. S. LOPES2
'Centro de Pesquisa para Pequenas Propriedades-EPAGRI, CP.791, Chapec6, SC, 89801-970, Brazil

2Departamento de Entomologia, Fitopatologia e Zoologia Agricola, ESALQ/Universidade de Sao Paulo
CP.9, Piracicaba, SP 13418-900, Brazil

ABSTRACT

The liquid excretion and survival of the sharpshooters Dilobopterus costalimai Young and
Oncometopia facialis (Signoret), vectors ofXylella fastidiosa in citrus, were measured on
various host plants as an indirect approach to assess their feeding and performance on these
hosts and determine suitable plants for laboratory rearing. Adult females of D. costalimai
showed the highest excretion rate on Vernonia condensata (Asteraceae). 0. facialis excreted
larger volumes on three species of Vernonia and on Lantana camera (Verbenaceae). On av-
erage, single D. costalimai females excreted a liquid volume equivalent to 292 times its body
volume per day when feeding on V condensata, whereas 0. facialis females excreted 430
times their body volume on the same host. In contrast, the excretion rates of D. costalimai
and 0. facialis females on Citrus sinensis did not exceed 248 and 140 times their body vol-
ume per day, respectively. The mortality of adults after 96 h was lower on hosts upon which
higher liquid volumes were excreted; therefore, there is a positive relationship between the
excretion rate by the sharpshooters and their nutritional adequacy to hosts. V condensata
is a suitable host to maintain adult populations of both sharpshooters in the laboratory.

Key Words: leafhopper vectors, host plant suitability, honeydew excretion, citrus variegated
chlorosis

RESUMO

A taxa de excrecao de liquidos e a sobreviv6ncia das cigarrinhas Dilobopterus costalimai
Young e Oncometopia facialis (Signoret) (Hemiptera: Cicadellidae), vetoras de Xylella fas-
tidiosa em citros, foram quantificadas em diferentes esp6cies vegetais, como forma indireta
de se avaliar a adequacao dessas plants como hospedeiras das cigarrinhas para estudos
ecol6gicos e de criacgo em laborat6rio. O maior volume de excrecgo liquida por f6meas de D.
costalimai foi observado em Vernonia condensata (Asteraceae). 0. facialis excretou maiores
volumes em tr6s esp6cies de Vernonia e em Lantana camera (Verbenaceae). Alimentando-se
em V condensata, uma inica f6mea de D. costalimai excretou, em m6dia, o equivalent a 292
vezes seu volume corp6reo por dia, enquanto que as f6meas de 0. facialis excretaram 430
vezes seu volume corp6reo no mesmo hospedeiro. Em Citrus sinensis, as taxas de excrecao
de D. costalimai e 0. facialis nao excederam em 248 e 140 vezes o volume corp6reo por dia,
respectivamente. A mortalidade dos adults ap6s 96 h foi menor naqueles hospedeiros onde
houve maior excrecgo, havendo, portanto, uma relacao direta entire a taxa de excrecao pelas
cigarrinhas e sua adequacao nutricional aos hospedeiros. V. condensata 6 um hospedeiro ad-
equado para manter populacoes de adults de ambas as cigarrinhas em laborat6rio.
Translation provided by author


The leafhoppers Dilobopterus costalimai Young
and Oncometopia facialis (Signoret) (Hemiptera:
Cicadellidae: Cicadellinae) are vectors of the bacte-
rium Xylella fastidiosa (Roberto et al. 1996), the
causal agent of citrus variegated chlorosis (CVC), a
disease reported in Brazil in the late 1980s (Ros-
setti & De Negri 1990), which currently affects
38% of citrus trees in the state of Sao Paulo, Brazil
(68 million plants) (Anonymous 2002).
Cicadellinae leafhoppers, commonly named
sharpshooters, are usually found on plant
branches, feeding in the xylem vessels of young


shoots. They have well developed suction cham-
bers that allow fluid intake even under strong
negative pressure of the xylem (Purcell 1989).
They extract most of the nutrients present in the
ingested sap, mainly amino acids and organic ac-
ids (Andersen et al. 1989), and excrete the liquid
excess through the anus. To make up for the low
concentration of amino acids in the xylem sap of
the plants, these insects usually ingest a large
amount of liquids (Raven 1983; Purcell 1989).
Studies on insects feeding directly from the xy-
lem fluid, e.g., the sharpshooters, rarely provide







Milanez et al.: Host Preference of Citrus Sharpshooters


direct results of the assimilated nutrients due to
the fact that this liquid has a low chemical diver-
sity in comparison with other plant tissues and is
little likely to contain compounds of secondary
metabolism (Raven 1983). Previous studies with
the glassy-winged sharpshooter, Homalodisca co-
agulata Say revealed that its adaptation to the
host includes high rates of assimilation of organic
compounds (above 98%) and excretion of ammo-
nia as a primary product (Andersen et al. 1989;
Andersen et al. 1992). The requirement of plant
nutrients varies according to the development
stage of H. coagulata, which rarely completes its
development on a single host (Andersen et al.
1989; Brodbeck et al. 1993; Brodbeck et al. 1995).
According to Paiva et al. (1996) and Gravena et
al.(1998), there is a clear difference between leaf-
hopper species occurring on citrus trees and those
on invasive vegetation of orchards; nevertheless,
some sharpshooters that occur predominantly on
the weeds are eventually trapped in the citrus
canopy. Likewise, citrus sharpshooters have been
found on a wide range of trees and shrubs in
woody habitats adjacent to citrus orchards
(J. R. S. Lopes et al., unpublished data).
The goal of this work was to develop a method
to collect and measure the liquid excretion of
sharpshooters, in order to evaluate feeding and
survival rates of 0. facialis and D. costalimai on
various host plants, as an indirect approach to de-
termine host suitability and understand the nu-
tritional ecology of these important vectors.

MATERIALS AND METHODS

The experiment was performed in a green-
house at the Dept. of Entomology, Plant Pathol-
ogy and Agricultural Zoology, University of Sao
Paulo, Brazil. The liquid excretion ofD. costalimai
and 0. facialis was measured on three plant spe-
cies of the family Asteraceae (Vernonia sp.,
V condensata, V polyanthes), two of Verbenaceae
(Lantana camera and Aloysia virgata), and one of
Rutaceae (Citrus sinensis; sweet orange), which
are field hosts of these sharpshooters (J.R.S.
Lopes et al., unpublished data). Six-month old
potted citrus trees were used. The other host
plants were 3-4 months old.
Sharpshooters used in the experiment were
reared on plants of V condensata in a greenhouse.
For collecting the liquid excretion, 1-week old
adults of D. costalimai and 0. facialis males and
females were individually placed inside 100-ml
plastic cages with lids containing ventilation
holes covered by a fine fabric (Fig. 1A). The cages
were attached with adhesive tape to the young
branches of the plants. Feeding was allowed for
96 h; the liquid excretion accumulated in the bot-
tom of the cages was collected daily by a 1-ml sy-
ringe, and the volume was measured (Fig. 1B).
The data were transformed into liquid excretion


Fig. 1. A) Transparent plastic cage (100 ml) used for
confinement of sharpshooters on plant stems. Lid with
ventilation holes covered by a fine fabric. B) Collection
and measurement of liquid volume excreted in the bot-
tom of the cage by a 1-ml syringe.


volume produced in relation to the body volume of
the insects. The body volume was determined by
plunging sharpshooter adults into a known vol-
ume of liquid excretion and measuring the vol-
ume of liquid displaced. Feeding trials were run
until 12 replicates were completed. Only repli-
cates in which the insect was alive throughout the
96-h feeding period were considered for the anal-
yses of excretion rates.
The experimental design was in blocks com-
pletely randomized with six treatments and 12
replications. The data were analyzed using anal-
ysis of variance (ANOVA) followed by the Tukey
test (P < 0.05).

RESULTS AND DISCUSSION

The method developed was efficient to estimate
the excretion rates of the sharpshooters.
D. costalimai adults excreted a higher liquid vol-
ume when fed on V condensata (male and female)
Vernonia sp.(male) and V polyanthes, which are
plants of the family Asteraceae. On V condensata,







Florida Entomologist 86(2)


a single D. costalimai female excreted up to 620
times its own body volume in a 24-h period. D. cos-
talimai males and females nearly did not feed on L.
camera, and the liquid excretion of males was null
(Table 1). The same trend of higher liquid excretion
on Asteraceae was verified for 0. facialis, except for
L. camera (Verbenaceae), upon which the excretion
was equivalent to that observed on Vernonia sp.
and V condensata. In 24 h, 0. facialis females ex-
creted up to 900 and 990 times their body volume
when fed on V condensata and L. camera, respec-
tively (Table 1). It should be pointed out that under
field conditions L. camera is frequently visited by
adults of 0. facialis (Gravena et al. 1998).
The mortality ofD. costalimai adults after 96 h
was higher onA. virgata, L. camera and C. sinen-
sis, and null for V condensata and Vernonia sp.
For 0. facialis the mortality was also null when
fed on V condensata, Vernonia sp. and V polyan-
thes (Table 2). Therefore, the host plants of the
family Asteraceae appear to be nutritionally more
adequate for both sharpshooters because a lower
mortality and a higher liquid excretion occurred
on those plants, even though the xylem sap nutri-
ents (amino acids and sugars) considered impor-
tant to the adults were not measured in this
research. Milanez et al. (2001) showed that V con-
densata is more adequate than Citrus limonia for
the nymphal development ofD. costalimai and 0.
facialis, because it shortens the nymphal period
and increases the viability of these sharpshoot-


ers. In the present study, male and female adults
of D. costalimai and 0. facialis excreted much less
on C. sinensis than on V condensata, which seems
to be an optimum feeding host (Table 1).
The feeding preferences of the sharpshooters
might influence their competency as vectors of
X. fastidiosa. The low rates of sap ingestion and
survival on citrus by sharpshooters may theoreti-
cally reduce the chances of acquisition of
X. fastidiosa from or inoculation to the xylem of
citrus plants. This might explain in part the low
transmission efficiency ofX. fastidiosa by sharp-
shooters reported in citrus (Lopes 1999; Yama-
moto et al. 2002); other possible factors are related
to pathogen-plant or pathogen-vector interactions.
Overall, this study shows a relationship be-
tween the liquid volume excreted by D. costalimai
and 0. facialis adults and the nutritional adequacy
of host plants. It was observed that some plants
promote higher feeding and survival rates than
the others. Among these hosts, V condensata ap-
pears to be the most suitable to maintain adult
populations of both sharpshooters in the labora-
tory. Further studies on oviposition and develop-
ment of these sharpshooters on various host plants
are necessary to understand their nutritional ecol-
ogy and improve the rearing system. Previous
studies showed that H. coagulata, a sharpshooter
with similar habits, requires different hosts to
complete its development (Andersen et al. 1989;
Brodbeck et al. 1993; Brodbeck et al. 1995).


TABLE 1. DAILY AVERAGE AND VARIATION INTERVAL (IN PARENTHESIS) OF THE RATIO OF LIQUID VOLUME EXCRETED
PER BODY VOLUME OF DILOBOPTERUS COSTALIMAI AND ONCOMETOPIA FACIALIS, WHEN FED ON DIFFERENT
HOST PLANTS.

D. costalimai 0. facialis

Host plant Male Female Male Female

Vernonia condensata 196 42 a' 292 60 a 128 51 ab 430 99 a
(80 520) (160 620) (10 630) (40 900)
V polyanthes 134 20 a 84 + 38 bc 164 70 ab 70 + 10 b
(42-246) (4-380) (4-340) (10-530)
Vernonia sp. 98 40 bc 48 20 bc 337 58 a 255 + 81 a
(32-366) (40-184) (50-630) (170-680)
Aloysia uirgata 24 3 d 56 20 bc 31 12 c 44 + 24 b
(20 180) (20 520) (2 140) (1- 260)
Lantana camera Od 8 2 c 291+ 84 a 411+ 67 a
(0 12) (50 810) (80 990)
Citrus sinensis 46 2 cd 120 30 b 36 + 20 c 32 + 18 b
(18- 128) (20- 248) (2- 230) (16- 140)
ANOVA2
F 4.94 9.87 8.10 11.11
df 5,66 5,66 5,66 5,66
P <0.05 <0.05 <0.05 <0.05

Means (+SEM) within columns followed by the same letter do not differ by Tukey test (P > 0.05).
Analysis of variance (ANOVA) statistics; df = degrees of freedom (treatment, residue).


June 2003







Milanez et al.: Host Preference of Citrus Sharpshooters


TABLE 2. MORTALITY (%) OF DILOBOPTERUS COSTALIMAI AND ONCOMETOPIA FACIALIS ADULTS WHEN CONFINED ON
DIFFERENT HOST PLANTS FOR 96 H.

D. costalimai 0. facialis

Host plant Male Female Male Female

Vernonia condensata 0 0 0 0
V polyanthes 25 40 0 0
Vernonia sp. 0 0 0 0
Aloysia virgata 77 50 57 40
Lantana camera 55 50 25 25
Citrus sinensis 50 57 62 40


The information obtained in this work should
be useful for development and application of new
vector control strategies involving trap plants or
vegetation management in citrus groves.


ACKNOWLEDGMENTS

Research supported by Fundo de Desenvolvimento da
Citricultura (FUNDECITRUS). First author received a
post-doctoral scholarship from Conselho Nacional de De-
senvolvimento Cientifico e Tecnol6gico (CNPq).

REFERENCES CITED

ANDERSEN, P. C., B. V. BRODBECK, AND R. F. MIZELL.
1989. Metabolism of amino acids, organic acids and
sugars extracted from the xylem fluid of four host
plants by adults Homalodisca coagulata. Entomol.
Exp. Appl. 50: 149-159.
ANDERSEN, P. C., B. V. BRODBECK, AND R. F. MIZELL.
1992. Feeding by leafhopper, Homalodisca coagu-
lata, in relation to xylem fluid chemistry and ten-
sion. Journal of Insect Physiology 38: 611-622.
ANONYMOUS. 2002. CVC diminui nas plants novas. Re-
vista do Fundecitrus, Araraquara, SP, Brazil, 15
(111): 14-15.
BRODBECK, B. V, P. C. ANDERSEN, AND R. F. MIZELL.
1995. Differential utilization of nutrients during de-
velopment by the xylophagous leafhopper, Homa-
lodisca coagulata. Entomologia Experimentalis et
Applicata 75: 279-289.
BRODBECK, B. V, R. F. MIZELL, AND P. C. ANDERSEN.
1993. Physiological and behavioral adaptations of
three species of leafhoppers in response to the dilute
nutrient content of xylem fluid. Journal of Insect
Physiology 39: 73-81.
GRAVENA, S., J. R. S. LOPES, P. E. B. PAIVA, P. T. YAMA-
MOTO, AND S. R. ROBERTO. 1998. The Xylella fastid-
iosa vectors, pp. 36-53. In L. C. Donadio and C. S.


Moreira (eds.), Citrus Variegated Chlorosis. Fun-
decitrus, Araraquara, SP, Brazil.
LOPES, J. R. S. 1999. Estudos com vetores deXylella fas-
tidiosa e implicapoes no manejo da clorose variegada
dos citros. Laranja, Cordeir6polis, SP, Brazil, 20:
329-344.
MILANEZ, J. M., J. R. P. PARRA, AND D. C. MAGRI. 2001.
Alternation of host plants as a survival mechanism
of leafhoppers Dilobopterus costalimai and Oncome-
topia facialis (Hemiptera Cicadellidae), vectors of
Citrus Variegated Chlorosis (CVC). Scientia Agri-
cola, Piracicaba, SP, Brazil, 58: 699-702.
PAIVA, P. E. B., J. L. DA SILVA, S. GRAVENA, AND P. T.
YAMAMOTO. 1996. Cigarrinhas do xilema em
pomares de laranja do Estado de Sao Paulo, Laranja,
Cordeir6polis, SP, Brazil 17: 41-54.
PURCELL, A. P. 1989. Homopteran transmission of xy-
lem-inhabiting bacteria, pp. 243-266. In H. K. Harris
(ed.), Advances in Virus Vector Research, v.6.
Springer-Verlag, New York.
PURCELL, A. H., AND A. H. FINLAY. 1979. Evidence for
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69: 393-395.
RAVEN, J. A. 1983. Phytophages of xylem and phloem: a
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MIRANDA, AND E. F. CARLOS. 1996. Transmissao de
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Florida Entomologist 86(2)


June 2003


RELATIVE EFFECTS OF CLIMATE AND CROWDING
ON WING POLYMORPHISM IN THE SOUTHERN GROUND CRICKET,
ALLONEMOBIUS SOCIUS (ORTHOPTERA: GRYLLIDAE)

ALEXANDER E. OLVIDO3, ELIZABETH S. ELVINGTON2 AND TIMOTHY A. MOUSSEAU1
'Department of Biological Sciences, University of South Carolina at Columbia

2South Carolina Governor's School for Science and Mathematics

3Current address: Department of Biology, Morehouse College, 830 Westview Drive S.W., Atlanta, GA 30314, U.S.A.
(email: alex_olvido@yahoo.com)

ABSTRACT

Many factors determine the formation of flight wings in wing-polymorphic insects. Earlier
studies on a cricket (Gryllus firmus) population producing spring and summer generations
showed a declining frequency of macropterous, or long-winged, adults towards the end of a
growing season. Numerous confounding factors can explain this seasonal decline, one of
which is increasing mortality rates of juveniles that may otherwise emerge as macropterous
adults. To test this hypothesis, we measured rates of juvenile mortality and adult macrop-
tery in Allonemobius socius Scudder (Orthoptera: Gryllidae), an organism with a seasonal
phenology similar to that of G. firmus. After rearing A. socius juveniles exclusively under
"spring" versus "summer" conditions and at different population densities, we found that
crickets reared in groups under "summer" conditions tended to emerge as macropters, with
females being more likely than males to emerge long-winged. Juvenile mortality did not ad-
equately explain the emergence pattern of macropters. Surprisingly, variation among fami-
lies accounted for <1% of total variation in frequency of long-winged adults. Thus, seasonal
climate, followed by population density, and then their interaction with each other appear to
be the three major determinants of wing morph frequencies inA. socius. We discuss the pos-
sible adaptive significance of wing polymorphism in insects with respect to habitat persis-
tence and mating success.

Key Words: migration, habitat persistence, polyphenism, crowding, wing dimorphism, plas-
ticity

RESUME

Muchos factors determinan la formaci6n de las alas de vuelo en insects de alas polimorfi-
cas. Estudios anteriores sobre una poblaci6n del grillo (Gryllus firmus) produciendo genera-
clones en la primavera y en el verano mostraron una frecuencia diminuyendo de adults
macr6pteros, o de alas largas, acercandose al final de la estaci6n de crecimiento. Numerosos
factors components pueden explicar esta declinaci6n estacional, uno de ellos es el aumento
en la tasa de mortalidad de los juveniles que de otra manera emergiran como macr6pteros
adults. Para probar esta hip6tesis, nosotros medimos las tasas de mortalidad juvenile y la
macroteria (el estado de alas largas) de los adults enAllonemobius socius Scudder (Orthop-
tera: Gryllidae), un organismo con una fenologia estacional similar a la de G. firmus. Des-
pu6s de criar los juveniles de A. socius exclusivamente bajo condiciones de "primavera"
versus "verano" y en diferentes densidades de poblacion, nosotros encontramos que los gri-
llos criados en grupos bajo condiciones de "verano" tendian a merger como macr6pteros, y
fue mas probable que las hembras emergen con alas largas que los machos. La mortalidad
juvenile no explic6 adecuadamente el patron de emergencia de los macr6pteros. Sorprenden-
temente, la variaci6n entire las families contaba por <1% de la variaci6n total en la frecuen-
cia de adults con alas largas. Asi, el clima estacional, seguido por la densidad de la
poblaci6n, y despu6s la interacci6n entire ellos parecen ser los tres mayores determinantes en
la frecuencia de las diferentes formas de alas enA. socius. Nosotros discutimos el possible sig-
nificado adaptive derivado del polimorfismo de alas en insects al respect de la persistencia
de habitat y el 6xito en el apareamiento.


The independent evolution of wings among ripheral areas of their current habitat, as well as
several animal taxa (Kingsolver & Koehl 1994) is migrate to more distant and, perhaps, novel envi-
due, in part, to the apparent benefits of flight. ronments in search of food and mates. By enhanc-
Flight-capable organisms can easily colonize pe- ing mobility and dispersal, flight undoubtedly







Olvido et al.: Climate and Density-Dependent Wing Polymorphism


contributed to the remarkable diversification of
insects (Roff & Fairbairn 1991; Rankin & Burch-
sted 1992; Kingsolver & Koehl 1994).
Developing and maintaining the flight appara-
tus, however, often carries a cost. For example,
long-winged (or macropterous) females of the
sand cricket, Gryllus firmus, reach reproductive
age at a later date and have lower lifetime fecun-
dities when compared to their short-winged coun-
terparts (Roff 1990a). In the brown planthopper,
Nilaparvata lugens, macroptery is associated
with longer egg-to-adult development time and
lowered male mating success (Novotny 1995).
Such trade-offs between flight wings and other
traits closely associated with fitness allows one to
view flight ability as itself a fitness-determining
trait along the same lines as growth rate and fe-
cundity. Thus, not only is wing polymorphism in-
teresting for its own sake, but also for its
apparent ties to life history and life cycle evolu-
tion (Roff & Fairbairn 1991; Rankin & Burchsted
1992; Kingsolver & Koehl 1994; reviewed exten-
sively in Dingle 1996).
Apart from being genetically determined
(Masaki & Walker 1987; Mousseau & Roff 1989;
Roff 1990a, 1990b), flight wings can develop in re-
sponse to numerous environmental factors. For ex-
ample, warm temperatures and long-day
conditions typical of summer tend to produce mac-
ropterous adults, e.g., in crickets and grasshoppers
(Tanaka 1978; see also Masaki & Walker [1987],
and references therein) and in Gerris species (Ve-
psalainen 1978, in Dingle 1996). Conditions of
crowding and food shortage also contribute to vari-
ation in the frequency of wing morphs in insect
populations (Tauber et al. 1986; Walker 1987).
In their exhaustive survey of one population of
the sand cricket, Gryllus firmus, Veazey et al.
(1978) found that the frequency of macropterous
adults caught in pitfall traps was lower in the
summer than in the spring brood. However, such
a pattern can be attributed to a number of con-
founding and interacting variables, including: (1)
migration of flight-capable, macropterous adults
away from the sampling site; (2) differential mor-
tality of presumptive macropters and micropters,
due perhaps to intraspecific competition for food
or space; (3) increased predation by insectivores
maturing later in the growing season; (4) shed-
ding of flight wings by individuals that emerged
earlier in the season as macropterous adults
(though this phenomenon has not been reported
for G. firmus); (5) a genetically fixed seasonal phe-
nology for macropters and micropters, e.g. mac-
ropterous adults from only the spring brood
always producing offspring that always emerge a
year later (in the following spring) as long-winged
adults; (6) the emergence of 2nd-generation, or
summer brood, juveniles as short-winged rather
than macropterous adults; and (7) a deficiency in
the sampling methods used by Veazey et al.


(1978). Investigations since then have elaborated
some of the genetic and physiological mecha-
nisms that contribute to variation in G. firms
wing morph frequencies (Roff 1990a, 1990b; Zera
et al. 1998). But surprisingly, very little empirical
work has been done to tease out which ecological
factors are most responsible for this pattern of
naturally occurring wing polymorphism (cf. Roff
1994a; Crnokrak & Roff 1998).
The current study addresses the hypothesis
that differential juvenile mortality explains vari-
ation in wing morph frequency in wing polymor-
phic insects (see Factor 2 above). Both spring and
summer broods of the southern ground cricket,
Allonemobius socius Scudder (Orthoptera: Gryl-
lidae), occur at high densities throughout the
southeastern region of North America, and expe-
rience a variety of seasonal temperatures and
day-lengths associated with their widespread lat-
itudinal and altitudinal distribution (Howard &
Furth 1986; Mousseau & Roff 1989). Moreover,
like G. firmus, both field and laboratory popula-
tions ofA. socius produce a mixture of short- and
long-winged adults, with the latter form also ex-
hibiting variation in flight propensity (A.E.O.,
personal observation). Thus,A. socius is useful for
investigating the genetic and environmental fac-
tors responsible for variation in wing morph fre-
quencies in natural insect populations.

Materials and Methods

Cricket Stocks

All individuals used in this experiment were
first-generation, laboratory-reared descendents
of crickets caught as juveniles from a wet, grassy
field adjacent to a greenhouse on the University
of South Carolina-Columbia. Before the start of
the experiment, all crickets and their eggs were
incubated, reared, and maintained under condi-
tions simulating a hot, summer day (31C, 15 h
day-length, >60% relative humidity) in Columbia,
South Carolina, U.S.A. (Wood 1996). Voucher
specimens have been sent to J. C. Morse of the
Clemson University Arthropod Collection.

Experimental Design

Individually reared juveniles from 23 full-sib-
ling families were housed in clear plastic petri
plates (diameter = 100 mm) each provisioned ad li-
bitum with pulverized cat chow, chopped carrots,
water, and shredded unbleached paper towels for
cover. Group-reared juveniles from 31 full-sibling
families were housed in 9 x 9 x 8 cm clear plastic
cages that were similarly provisioned. Left-over cat
chow and carrots were changed every 2 to 3 days.
We used a double split-brood design, in which
one-half of a cohort of newly hatched juveniles
from each family was randomly assigned to a






Florida Entomologist 86(2)


"summer" (31C, 15 h day-length) rearing environ-
ment, while the other half was reared in a "spring"
(24C, 11 h day-length) environment. Within a
seasonal environment, group-reared juveniles
were then assigned to either a high population
density (14 to 21 juveniles per cage) or low popu-
lation density (3 to 6 juveniles per cage) treat-
ment. Among-family variation in maternal
fecundity, egg-hatching rate, and juvenile survi-
vorship precluded assignment of exactly equal
numbers of individuals to replicate cages (in each
population-density treatment). Because A. socius
will consume dead conspecifics when available, we
minimized scavenging by removing dead individu-
als without replacement. All surviving juveniles
were reared to adulthood exclusively in the envi-
ronment to which they were initially assigned.

Scoring Macroptery and Juvenile Survivorship
Only macropterous adults possess the ivory-
colored flight wings that extend from beneath the
beige-black tegmina, or outer wings (Fig. 1). We
scored adults <3 d after the final molt as mac-
ropterous if they emerged with flight wings in-
tact. In group-reared crickets, incidence of
macroptery was then calculated as the number of
macropterous adults divided by the total number
of adults from a given replicate cage. Similarly,









I' / /


incidence of macroptery in crickets reared in iso-
lation was calculated as the number of macropter-
ous adults divided by the total number of adults
that were reared in petri plates within a given
seasonal environment.
Juvenile survivorship of group-reared crickets
was calculated as the number of emerging adults
(regardless of wing morph) divided by the total
number of nymphs from a given replicate cage.
Similarly, juvenile survivorship for crickets
reared in isolation was calculated as the total
number of petri plates that yielded (long- or
short-winged) adults divided by the original num-
ber of petri plates used to rear cricket nymphs in-
dividually.

Statistical Analyses
Because the macroptery data did not satisfy
the normality assumptions for valid parametric
analyses, we turned to a nonparametric, van der
Waerden normal scores analysis to test for sex-
specific differences in proportion of macropters
for each treatment. We performed the
NPAR1WAY procedure in SAS For Windows, Ver-
sion 6.12 (SAS 1989). As discussed fully in
Conover (1999), a van der Waerden analysis
achieves asymptotic relative efficiency, or A.R.E.
(~statistical power), comparable to that of para-


49/I


Fig. 1. Wing polymorphism in A. socius. Females are distinguished from males by the presence of a sword-like
ovipositor protruding from the posterior end of the abdomen. Note the flight wings in long-winged adults-the two
individuals flanking the vertical black bar-extend from beneath the darker tegmina (i.e., outer wings). Length of
vertical black bar is 20 mm.


June 2003


_1







Olvido et al.: Climate and Density-Dependent Wing Polymorphism


metric statistical tests, e.g., F-test, when data sat-
isfy normality assumptions, and greater A.R.E.
when data are non-normal. We adjusted the re-
sulting probability values using the sequential
Bonferroni method in order to maintain an exper-
iment-wide Type I error rate of <5% for all pair-
wise comparisons (Rice 1989).
We partitioned total variance in both the fre-
quency of macropters and juvenile survivorship
via the restricted maximum likelihood (REML)
method available in the VARCOMP procedure of
SAS for Windows 6.12 (SAS 1989). Although the
VARCOMP procedure does not provide P-values
for estimated variance components, it allows a
quantitative description of importance of each bi-
otic and abiotic factor to observed macroptery
patterns in A. socius. The choice of REML as a
method for estimating variances is justified by its
common use in modern quantitative genetic stud-
ies, e.g., Shaw (1987), Meyer and Hill (1991), and
Ferreira et al. (1999).

RESULTS

Compared to the spring rearing treatment, the
summer rearing environment tended to produce a
higher proportion of long-winged adults (Fig. 2).
Differences in macroptery rates between the
sexes appeared only at high population densities
(Table 1), with more females than males emerg-
ing as long-winged adults (Fig. 2).
The largest contributing factor to variance in
macroptery rates was juvenile rearing climate,
followed by the interaction of rearing season with
population density (Table 2). Family origin
(nested within population density since not all of
the families were represented in the population-
density manipulations) contributed <1% to vari-
ance in macroptery rates (Table 2).
Juvenile survivorship was high across all rear-
ing treatments. As expected, crickets reared in
isolation had higher survival rates (~85% in both
spring and summer rearing environments) than
those reared in groups, with high population den-
sities resulting in the lowest survival rates (be-
tween 58-78%). At a given population density,
juvenile survival rates were similar for both sum-


O Alone E Low Density E High Density


(a) MALES


(b) FEMALES

""w;50X
"""/'* $~


Spring


Summer


Rearing Season

Fig. 2. The effects of rearing season and population
density during juvenile development on mean (SE) in-
cidence of macroptery in A. socius males and females.
All juveniles reared in isolation and under spring-like
conditions emerged as short-winged adults.


mer and spring rearing conditions (Fig. 3), sug-
gesting no effect of seasonal climate and no
interaction between seasonal climate and popula-
tion density on juvenile survivorship (Table 2).
We found that the largest contributors to vari-
ation in juvenile survivorship (aside from experi-
mental error) were population density and family
origin (Table 2). There were no significant inter-
actions between either of these factors with rear-
ing season.

DISCUSSIoN

Both seasonal climate and population density
during juvenile development affect frequency of
macropters inA. socius. In our study, high temper-
atures and long day-lengths typical of the summer


TABLE 1. EFFECT OF REARING ENVIRONMENT ON THE DIFFERENCE IN THE PROPORTION OF LONG-WINGED FEMALES TO
LONG-WINGED MALES.

Spring-reared" Summer-reared

Reared aloneb N/A 4.739
Reared at low population density 1.569 0.001
Reared at high population density 13.692* 13.139*

"Numbers are test statistics, Tl, from a nonparametric van der Waerden one-way normal scores analysis (Conover 1999) with
one degree of freedom.
bAll juveniles reared alone under spring-like conditions emerged as short-winged adults.
*Statistically significant at experiment-wide a = 5%.


~







Florida Entomologist 86(2)


TABLE 2. RESTRICTED MAXIMUM-LIKELIHOOD VARIANCE-COMPONENTS ESTIMATION OF RELATIVE CONTRIBUTION TO IN-
CIDENCE OF LONG-WINGED, EMERGING ADULTS AND TO JUVENILE SURVIVORSHIP.

Observed variance component Incidence of macroptery Juvenile survivorship

(1) Rearing season 40.1% 0.9%
(2) Population density 12.2% 38.8%
(3) Family (population density)b 0.1% 21.5%
(4) Sex (population density, family)c 0.8% N/A
Interaction of 1 and 2 25.5% 0.0%
Interaction of 1 and 3 1.1% 0.0%
Interaction of 1 and 4 1.1% N/A
Error 19.1% 38.8%
Total 100% 100%

"Sex of newly hatched juveniles could not be determined non-invasively because secondary sexual traits appear only in the mid-
dle-to-late stages of development. Hence, variance in juvenile survivorship due to sex and to interaction between sex and rearing
season cannot be estimated.
bFamilies experienced both rearing seasons, but not all population-density treatments.
'Because a few families produced single-sex progeny, sex is nested within family, which in turn is nested within population den-
sity.


season led to greater numbers of macropterous
males and females (Fig. 2; see comparable results
in Tanaka 1978). Population density greatly com-
pounded the effect of the summer rearing environ-
ment in producing macropterous adults, especially
with crickets reared in groups (Fig. 2). At low pop-
ulation densities, the proportion of macropterous
adults increased as climate changed from spring-
to summer-like (Fig. 2; but see Walker 1987). At


1o
0
0
4-
C 0.75
0
'4

0.50

U-
2 0.25
a-
0
0.
a.


o Alone
B 0 Low Density
[I High Density


Spring Summer
Rearing Season

Fig. 3. Juvenile survivorship ofA. socius at different
rearing densities and seasonal climates. Column height
represents the mean (SE) proportion of newly hatched
juveniles that eventually emerged as adults. Numbers
in parentheses above each clear column indicates total
number of individually rearedA. socius juveniles scored
for that treatment.


high population densities, we found a reduced pro-
portion of macropterous adults (Fig. 2), a result
that may be due to intraspecific competition for
space and nutrients (discussed in Walker [1987]
and Zera & Tiebel [1988]).
Differences in juvenile survivorship among
our rearing treatments do not adequately explain
the variation in incidence of macroptery. Though
juvenile survivorship appeared inversely related
to population density in the rearing cages, the dif-
ferences in juvenile survivorship among the pop-
ulation-density treatments were similar between
spring- and summer-rearing treatments (Fig. 3),
suggesting that seasonal climate had little effect
on A. socius juvenile survivorship. More impor-
tantly, the pattern of juvenile survivorship did not
parallel that of adult macroptery rates among
treatment groups (Fig. 2). Thus, wing length vari-
ation in A. socius appears to be a response to sea-
sonal climate and population rearing density
(Fig. 2), and does not appear to reflect differences
in juvenile survivorship.
The extent to which population density affects
the timing of flight wing removal in A. socius is
not known. It is possible that macropterousA. so-
cius adults shed their flight wings <3 d post-eclo-
sion, which would have introduced a downward
bias in our method of scoring macroptery. In other
words, the incidence of long-winged morphs in
our high cage-density treatment may actually be
higher than was observed in this study (Fig. 2).
Notwithstanding this potential bias, the observed
pattern of juvenile survivorship does not parallel
that of macroptery rates.
Probabilistic models, e.g. in Roff (1975), "adap-
tive coin-flipping" of Kaplan and Cooper (1984),
and "stochastic polyphenism" of Walker (1986),
have been proposed to explain wing polymor-
phism in crickets and other flight-capable insects.


June 2003







Olvido et al.: Climate and Density-Dependent Wing Polymorphism


One simple prediction under probabilistic theory
is that the proportion of long-winged adults will
reflect the probability of those individuals experi-
encing a future environment that selects for
flight. Unfortunately, the present results cannot
confirm such predictions, nor can they validate
probabilistic models in general because environ-
mental variation in the wild (as experienced by
A. socius juveniles of the parental generation in
this study) would have been impossible to mimic
in a laboratory setting. Critical tests of probabilis-
tic models about macroptery may inevitably in-
volve the use of more closely monitored rearing
environments and isogenic lines.
The greater propensity of females than males
to possess flight wings (Fig. 2) may be attributed,
in part, to sex-specific reproductive behavior. In
many crickets, sexually receptive females often
travel some distance to locate the stationary, call-
ing male (Loher & Dambach 1989). Presumably,
the fitness gain from flight-aided mate-locating
behavior more than compensates for the cost of
developing the necessary flight apparatus in A.
socius females.
Macroptery in male crickets, on the other hand,
can impose a tremendous fitness cost in that mac-
ropterous males tend to be less successful than
their short-winged counterparts in attracting fe-
males (Crnokrak & Roff 1995), although this has
not been tested forA. socius. As well, male crickets
probably have greater mating success when they
remain in their natal habitat rather than fly to un-
known destinations where mating opportunities
may be scarce or absent (Walker 1986).
The greater incidence of macroptery in A. so-
cius females than males might also reflect the role
of habitat persistence in maintaining wing dimor-
phism (Denno et al. 1991; Roff 1994b). To the best
of our knowledge, the field from which we col-
lected crickets was watered daily and mowed ev-
ery 3-6 wk during late-spring and throughout the
summer months by campus grounds crew. The en-
vironmental disturbance caused by mowing could
have augmented selection for late-summer dis-
persal, especially in female A. socius, since fe-
males can store sperm from previous matings. In
such case, a female cricket has little reproductive
"need" for males once she has reached the less af-
fected periphery of her natal habitat. Thus, in
patchy and temporary environments, natural se-
lection might have acted to maintain the flight
apparatus in a flight-capable female just long
enough for her to escape a disturbed or deteriorat-
ing patch of habitat, and to colonize areas more
conducive to oviposition and optimal development
of her offspring during the regular growing sea-
son (Southwood 1962; Dingle 1996; Denno et al.
1991; Roff 1994b).
The apparent synergy between summer cli-
mate and moderate-to-high rearing density in
producing long-wingedA. socius suggests that, in


bivoltine populations, long-wingedess is more
common in the 2nd generation than in the first. In
the bivoltine life cycle ofA. socius, the 1st genera-
tion is comprised of individuals that had survived
the past winter as diapausing eggs and then
hatched out in spring. These 1st generation juve-
niles develop to adulthood through spring and
early summer, by which time they mate and pro-
duce non-diapausing eggs that hatch out immedi-
ately (Walker & Masaki 1989; Mousseau & Dingle
1991; Olvido et al. 1998). Thus, 2nd generation ju-
veniles appear likely to experience the macrop-
tery-inducing summer season, as well as higher
population density resulting from the presence of
1st generation adults and other newly hatched
2nd generation juveniles. Experiments are under
way to assess this prediction.
On the other hand, Veazey et al. (1976) showed
that macroptery rates declined from summer
through autumn in a Florida population of G. fir-
mus. Proximate mechanisms that can explain
such a pattern, e.g. predation pressure and mi-
gration of macropters from field sites, have yet to
be fully explored in this and other insects.
In this current study, we found no evidence
that would suggest differential juvenile survival
affects macroptery rates in A. socius. That is, dif-
ferences in wing morph frequencies between
spring- and summer-reared full-siblings ofA. so-
cius are not likely due to differences in juvenile
mortality, but instead may result from a response
to seasonal climate and population density dur-
ing juvenile growth. However, the generality of
emergence patterns inA. socius, like in G. firmus,
will require further and more detailed investiga-
tion of seasonal phenology in other wing polymor-
phic organisms.

ACKNOWLEDGMENTS

We thank Ken Fedorka for providing photogenic
crickets. Comments from Tom Walker, Dave Reznick,
Andy McCollum, Charles Henry, Bill Evans, John Sivin-
ski, and several anonymous referees improved the
manuscript considerably. This study was supported by
the National Science Foundation (N.S.F.) grant IBN-
9604226 to Bill Wagner Jr., a South Carolina University
Research and Education fellowship to E.S.E., N.S.F.
grant DEB-9409004 to T.A.M., and Ford predoctoral and
N.S.F. minority postdoctoral fellowships to A.E.O.

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Cricket Behavior and Neurobiology. Cornell Univer-
sity Press, Ithaca, NY.
WOOD, R. A. 1996 (ed.). Weather of U.S. Cities, 5th ed.
Gale Research, Detroit, MI.
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fect of group rearing, juvenile hormone III, and me-
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rubens. J. Insect Physiol. 34: 489-498.


June 2003







Mannion et al.: Oviposition and Survival of Diaprepes


OVIPOSITION AND LARVAL SURVIVAL OF DIAPREPES ABBREVIATUS
(COLEOPTERA: CURCULIONIDAE) ON SELECT HOST PLANTS


CATHARINE MANNION1, ADRIAN HUNSBERGER2, JORGE E. PENA AND LANCE OSBORNE3
'University of Florida, Tropical Research and Education Center, 18905 SW 280th Street, Homestead, FL 33031

2University of Florida, Miami-Dade Cooperative Extension Service, 18710 SW 288th Street, Homestead, FL 33030

3University of Florida, Mid-Florida Research and Education Center, 2725 Binion Road, Apopka, FL 32703-8504

ABSTRACT
In a preliminary survey in four commercial ornamental nurseries in south Florida (1998),
Diaprepes abbreviatus (L.) (Coleoptera: Curculionidae) egg masses, feeding damage, or
adults occurred on numerous field-grown ornamental plant species. Live oak (Quercus vir-
giniana Mill.), silver buttonwood (Conocarpus erectus L. variety sericeus Fors. Ex DC), and
black olive (Bucida buseras L.) had the highest percentage of plants with egg masses. Adult
feeding damage was found on all examined plants of dahoon holly (Ilex cassine L.), cocoplum
(Chrysobalanus icaco L.), black olive, live oak, Bauhinia sp., and Cassia sp. Oviposition of
D. abbreviatus was evaluated in no-choice, two-choice, three-choice and multiple-choice
caged tests. In no-choice tests, silver buttonwood had the highest mean number of egg
masses. In two-choice tests, egg masses were laid on all plant species tested but there were
significantly more egg masses on silver buttonwood than the alternate choice. The number
of egg masses in the three-choice tests was low and there were no significant differences
among the plant species tested. As in the no-choice and two-choice tests, significantly more
egg masses were found on silver buttonwood in multiple-choice tests. Survival of larvae and
their effect on plant growth was examined on several commonly grown plant species in
southern Florida. Larval survival was highest on silver buttonwood and Sorghum sudan-
ense Pers (sorghum-sudan) compared with other plant species. Root and/or total biomass
was significantly reduced on green bean (Phaseolus vulgaris), silver buttonwood, Tahiti lime
(Citrus aurantifolia), and sorghum-sudan.
Key Words: root weevil, oviposition, host preference, larval survival

RESUME
En un muestreo preliminary realizado en 1998 en cuatro viveros de plants ornamentals lo-
calizados en el sur de Florida, se demostr6 que Diaprepes abbreviatus (L.) (Coleoptera: Cur-
culionidae) estaba asociado oviposici6n, daio al follaje, o en base a la presencia de adults
en varias species de plants ornamentals. Las species de plants que presentaron el ma-
yor porcentaje de oviposici6n fueron el roble, (Quercus virginiana Mill), el arbol plateado del
bot6n (Conocarpus erectus L.,) variedad sericeus y la bucida (Bucida buseras L). Se observe
consume del follaje por adults en todas las plants muestreadas de las siguientes species,
acebo (Ilex cassine L.), icaco (Chrysobalanus icaco L.), bucida, roble, casco de vaca Bauhinia
sp., y Cassia sp. Se evalu6 la oviposicion de Diaprepes mediante experiments en jaulas
donde se ofrecieron una opci6n de plant, dos opciones de plants, tres opciones de plants,
y varias opciones de plants. En los experiments con una sola opci6n de plants, el arbol
plateado del boton obtuvo el mayor numero de huevos. En experiments con dos opciones de
plants, se encontraron posturas en todas las plants, pero mas en el arbol plateado del bo-
ton comparado con otras plants. Cuando se ofrecieron 3 opciones de plants el numero de
posturas por plant fue bajo, y no hubo diferencias entire las species expuestas. En las prue-
bas de opciones multiples de plants hospederas, el arbol plateado del bot6n tuvo mayor ovi-
posici6n que las otras species. La supervivencia de las larvas y su efecto en el crecimiento
de examinado en varias plants cultivadas en el Sur de la Florida. La supervivencia de las
larvas fue mayor en raices del arbol plateado del bot6n, y sorgo sudan6s comparado con la
supervivencia en las raices de otras species de plants. Diaprepes abbreviatus redujo signi-
ficatinamente el peso de las races y el peso total de frijol verde (Phaseolus vulgaris), arbol
plateado del bot6n, lima acida (Citrus aurantifolia) y sorgo sudan6s.
Translation provided by author.


The root weevil, Diaprepes abbreviatus (L.), na- ornamental plants (Woodruff 1985). It was first re-
tive to the Caribbean Islands, is believed to have ported in an Apopka nursery in 1964 (Woodruff
entered Florida from Puerto Rico on a shipment of 1968) and has spread throughout many counties







Florida Entomologist 86(2)


in Florida. Diaprepes abbreviatus is associated
with at least 270 plant species including several
important and economic crops grown in Florida
such as citrus, ornamentals, and sugar cane (Sim-
pson et al. 1996). In citrus groves the cost of con-
trol and losses incurred by Diaprepes root weevils
exceed $1,200 per acre. This pest infests approxi-
mately 60,000 acres of citrus at an annual cost of
about $72 million to the Florida citrus industry
(Stanley 1996). Diaprepes is currently attacking
many ornamental nursery plants, which has re-
sulted in restrictions on the movement of plant
material from areas infested with the weevil.
Quarantine treatments can be expensive, labor in-
tensive and time-consuming. Sometimes there are
losses in sales and customers because there are no
known treatments acceptable for quarantine and
plants cannot be shipped to a particular location.
Twenty-four Florida counties were known to be in-
fested as of April 2001 (Michael C. Thomas, Flor-
ida Department of Agriculture and Consumer
Services, pers. comm.).
Adult weevils feed on plant foliage, often leav-
ing a characteristic pattern of notches around leaf
edges. Female weevils lay clusters of eggs be-
tween leaves and protect them by secreting a
sticky substance that cements the leaf surfaces
together (Fennah 1942; Woodruff 1968; Adair et
al. 1998). The number of eggs per egg mass varies
but on average is approximately 50 eggs. One fe-
male may lay as many as 5,000 to 29,000 eggs
during her three to four month lifespan (Wolcott
1936; Beavers 1982). Neonates hatch from the
eggs in 7 to 10 days, fall to the soil surface, and
burrow into the soil seeking out plant roots on
which to feed. The larvae remain in the soil 8 to 12
months where they complete development to the
adult stage. Adult weevils live 4 to 5 months, but
often half of this time is spent below the surface of
the ground (Wolcott 1936). In Florida, there are
overlapping generations with two peak adult
emergence periods in the spring (May-June) and
fall (August-September) (Beavers & Selhime 1976).
Adult weevils can cause moderate to severe de-
foliation of host plants; larval feeding can kill
hosts. In some plants, larvae girdle the taproot,
which reduces nutrient uptake, and ultimately
kill the plant (Quintela et al. 1998). Additionally,
larval root-feeding injury also provides an avenue
for microbial infections such as Phytophthora and
Fusarium (Knapp et al. 2000; Nigg et al. 2001a).
Several larvae can cause serious decline of estab-
lished citrus trees and it has been speculated by
researchers that one larva is capable of killing a
young citrus tree.
The objectives of this research were to evaluate
host preferences for oviposition and to determine
the presence ofD. abbreviatus eggs, adults, or adult
feeding damage in a preliminary survey of field-
grown ornamentals, and to evaluate larval survival
and root consumption of various plant species.


MATERIALS AND METHODS

Field Survey

A preliminary survey for the presence of adult
D. abbreviatus, feeding damage and egg masses
was conducted over a 2-day period in four com-
mercial, field-grown ornamental nurseries in Mi-
ami-Dade County, Florida. Three plant rows were
randomly selected from each field and two people
inspected all plants within the three rows for 5
minutes per plant. The presence or absence of
adults, feeding damage, and egg masses on each
plant were recorded. All sites contained a diver-
sity of ornamental plants with moderate to high
populations ofD. abbreviatus.

No-Choice Oviposition Tests

Three tests were conducted to compare ovipo-
sition on four plant species when there was no
choice in host plant. In each test, eight plants of
the same species were placed in a screen cage (1.8
x 3.7 x 1.8 m). Each plant was planted in a 7.6 li-
ter container with Pro-Mix'BX' (peat-based grow-
ing medium) potting media and exhibited new
leaves at the time of the experiment. In Test 1, the
plants evaluated were Conocarpus erectus L.vari-
ety sericeus Fors. Ex DC (silver buttonwood),
Manihot esculenta Krantz (cassava), Carica pa-
paya L. (papaya), and Xanthosoma sp. (malanga).
In Test 2, the plants evaluated were silver button-
wood, Sorghum sudanense Pers (sorghum-
sudan), Persea americana Mill. (avocado), and
C'i., i.../..i!!,.'... oliviforme L. satinleaff tree). In
Test 3 the plants evaluated were silver button-
wood, Solanum tuberosum (white potato), Pennis-
etum purpureum Schumach (elephant grass), and
Zea mays L. (sweet corn). Fifty male and fifty fe-
male, adult D. abbreviatus were released in each
cage. The number of adults used was selected to
ensure some oviposition. Adult weevils were field
collected from mixed plant species. The adult
weevils were maintained in cages with Conocar-
pus erectus L. (green buttonwood) before use for
approximately 24 hours. Each treatment was rep-
licated four times. The number of egg masses per
plant was recorded 7 days after the adults had
been released into the cages, which was sufficient
time for the weevils to lay eggs. The number of
eggs per egg mass were not counted.

Two-Choice Oviposition Tests

Two tests were conducted in which adults were
given a choice of silver buttonwood or another
plant host (each planted in a 7.6 liter containers
with ProMix potting media) on which to oviposit.
In the first test the alternate plant was Citrus au-
rantifolia (Christm.) Tahiti lime, and in the sec-
ond test the alternate plant was sorghum-sudan.


June 2003







Mannion et al.: Oviposition and Survival of Diaprepes


In each test, eight plants (four of each species)
were placed in a screen cage (2 x 4 x 2 m). Each
test was replicated four times. Plant species
within a cage were placed in an alternating pat-
tern. One hundred field-collected adult D. abbre-
viatus (50 female; 50 male) were released into
each cage. Each plant was examined for number
of egg masses as above.

Three-Choice Oviposition Tests

Three plant species were evaluated in a choice
test for oviposition preference by D. abbreviatus.
The plant species included Citrus sinensis L. sour
orange, Tahiti lime, and silver buttonwood each
grown in a 7.6 liter containers in a Pro-Mix 'BX'
(peat-based growing medium) potting soil. The
containers were placed on five raised beds in a
screen house (12.2 x 18.3 m). Beds were 16.5 x 0.7
m with 1.3 m between beds. Twenty-four silver
buttonwood were evenly spaced on each of the two
outside beds with 0.61 m between plants. On the
three inner beds, ten orange and ten lime trees
were alternated with two buttonwood plants at
the end of each bed totaling 24 plants per bed. Five
hundred field-collected adult weevils from plant
species not included in the test were released in-
side the screen house in the late afternoon on day
1. The male-female ratio was 1:1. The adult wee-
vils were released along the centerline of the
screen house perpendicular to the beds. The num-
ber of egg masses per plant was recorded on day 8.

Multiple-Choice Oviposition Test

Plants of seven species were each planted in a
7.6 liter container with Pro-Mix 'BX' (peat-based
growing medium) potting soil. The plants in-
cluded silver buttonwood, lime, elephant grass,
sorgham-sudan, sweet corn, malanga, and white
potato. Five replications of each plant were placed
in a randomized complete block design in a
screenhouse (12.2 m x 18.3 m). Five hundred
adult D. abbreviatus were collected from the field
from hosts other than those in the test and re-
leased inside the screen house. Ten days after the
release, plant leaves were checked for egg masses.

Larval Survival and Root Consumption

Survival of larvae on different host plants and
their effect on the plant was measured. Treat-
ment containers were infested with 50 neonate
D. abbreviatus. Neonates were collected from
eggs produced by field-collected adults held in
cages. For each plant species, a paired comparison
of infested and not infested plants was conducted.
Eight plant species were tested; Phaseolus vul-
garis L. (green bean), silver buttonwood, lime,
malanga, satinleaf, sorghum-sudan, cassava and
Ilex cassine L. (dahoon holly). Each plant was


planted in a 7.6 liter container with Pro-Mix 'BX'
(peat-based growing medium) and maintained in
a greenhouse. Replications for each plant species
varied between 5 and 10. Green bean and sweet
corn were evaluated 2 months after infestation.
Silver buttonwood, Tahiti lime, malanga, satin-
leaf, and sorghum-sudan were evaluated 3
months after infestation. Larval survival was also
evaluated on silver buttonwood, Tahiti lime and
malanga 6 months after infestation. Plant height,
fresh and dry weight of roots and total biomass,
and the number and weight of surviving larvae
were recorded. Comparisons were made between
infested plants and not infested plants.
Data in all of the oviposition choice tests except
the two-choice test and the larval survival tests
were subjected to analysis of variance with the
means compared by the Student-Newman-Keuls
Range Test (SAS 1999-2001). Data from the two-
choice oviposition test and all the larval feeding
tests were subjected to a t-test (SAS 1999-2001).

RESULTS AND DISCUSSION

Field Survey

Numerous field-grown ornamental plant spe-
cies were examined for the presence of adults,
feeding damage, or egg masses (Table 1). The re-
sults of this survey are preliminary but are con-
sistent with previous surveys or plant host lists
(Simpson et al. 1996; Knapp et al. 2000) that in-
dicate that Diaprepes will feed and lay eggs on a
wide host range of ornamental plants. The survey
conducted by Simpson et al. (1996) is a compila-
tion of specimen identification reports submitted
to the Florida Department of Agriculture and
Consumer Services, Division of Plant Industry,
from 1964 through 1995 in addition to any host
records listed in the scientific literature from
1898 to 1995. Knapp et al. (2000) also compiled a
list of plant hosts from the scientific literature but
there is no information about the type of associa-
tion with the host plant (i.e., adult host, larval
host, etc.). The current survey is a snapshot of
adult weevil presence, host plant damage, and
presence of egg masses in four commercial, field-
grown ornamental nurseries in south Florida.
The plant species in this survey with the highest
percentage of plants with egg masses were live
oak, Quercus virginiana (46.2%), silver button-
wood (41.2%), and black olive, Bucida buseras
(33.3%). All of the dahoon holly, cocoplum,
C'i I.i../,!..,.. icaco, black olive, live oak, Bau-
hinia sp. and Cassia sp. plants evaluated had
adult feeding damage. Plant species with the
highest number of adults per plant include da-
hoon holly, black olive, and Bauhinia sp. All plant
species in the survey with egg masses, adults or
feeding damage, except Jacaranda mimosiflia
(jacaranda), Clusia rosea (autograph tree), and







Florida Entomologist 86(2)


TABLE 1. PRELIMINARY SURVEY OF ADULTS, FEEDING DAMAGE, AND EGG MASSES OF Diaprepes abbreviatus ON CULTI-
VATED NURSERY PLANTS IN MIAMI-DADE COUNTY, FL, SEPTEMBER 3-4, 1998.

No. No.
No. plants plants
plants with with
No.plants withegg feeding adults
Plant family Plant species (common name) examined masses damage present

Agavaceae Cordyline terminalis (ti plant) 3 0 2 0
Dracaena marginata (Dracena) 2 0 0 0
Aquifoliaceae Ilex cassine (dahoon holly) 8 2 8 7
Bignonaceae Tabebuia heterophylla (pink trumpet tree) 5 0 0 0
Tabebuia caraiba (silver trumpet tree) 2 0 1 0
Jacaranda mimosifolia (jacaranda) 5 0 1 1
Boraginaceae Cordia sebestena (geiger tree) 1 0 1 0
Burseracea Bursera simaruba (gumbo limbo) 8 0 5 4
Chrysobalanaceae Chrysobalanus icaco (cocoplum) 10 0 10 2
Combretaceae Conocarpus erectus var. sericeus (silver buttonwood) 17 7 10 6
Bucida buseras (black olive) 9 3 9 7
Cycadaceae Cycas revolute (king sago) 5 0 0 0
Fagaceae Quercus virginiana (live oak) 13 6 13 4
Guttiferae Calophyllum braziliense (Brazilian beauty leaf) 25 3 11 1
Clusia rosea (autograph tree) 11 0 10 1
Leguminosae Bauhinia sp. 7 0 7 5
Cassia sp. 31 1 31 12
Lythraceae Lagerstroemia sp. (crape myrtle) 11 0 6 0
Meliaceae Swietenia mahogany (mahogany) 6 1 4 1
Myrtaceae Eugenia sp. 5 0 0 0
Musaceae Strelitzia nicolai (bird-of-paradise) 5 0 0 0
Oleaceae Ligustrum sp. (privet) 1 0 1 0
Palmae Cocos nucifera (coconut palm) 9 2 1 0
Phoenix roebelinii (pygmy date palm) 38 4 20 1
Acoelorraphe wrightii (Everglade palm) 1 0 0 0
Livistona chinensis (Chinese fan palm) 1 0 0 0
Vetchia merrillii (Christmas palm) 18 0 0 1
Sapindaceae Litchi chinensis (lychee) 2 0 2 0
Sapotaceae Chrysophyllum olivivorme satinleaff) 2 0 1 0


Cocos nucifera (coconut palm), have previously
been reported as being associated with D. abbre-
viatus (Simpson et al. 1996; Knapp et al. 2000).

No-Choice Oviposition Tests

In two of the three tests conducted, silver but-
tonwood had the highest mean number of egg
masses ranging from 1.75 to 4.12 egg masses per
plant (Table 2). Only two plant species (malanga
and satinleaf) had no egg masses.
In Test 1, significantly more egg masses were
found on cassava and silver buttonwood leaves
compared with papaya and malanga (F = 6.10; df
= 3, 28; P = 0.0025) (Table 2). A similar result was
seen in Test 2. More egg masses were found on sil-
ver buttonwood and sorghum-sudan compared
with avocado (West Indies cultivar) and satinleaf
(F = 8.26; df = 3, 28; P = 0.0004) (Table 2). In Test
3, the mean number of egg masses did not signif-
icantly differ among plant species (F = 1.90; df =
3, 28; P = 0.1525). However, there were approxi-


mately twice as many egg masses on silver but-
tonwood as on the alternate hosts (Table 2).

Two-Choice Oviposition Tests

Egg masses were found on all plant species
tested, however, there were significantly more egg
masses on silver buttonwood than the alternate
choice, sorghum-sudan (t = 3.39; df= 29;P = 0.002)
or lime (t = -2.83; df = 30; P = 0.008) (Table 3).

Three-Choice Oviposition Tests

The numbers of egg masses per plant were low.
There were no significant differences among the
three host plants, silver buttonwood, Tahiti lime
and sour orange, although the highest mean
number of egg masses occurred on silver button-
wood (F = 1.06; df = 2, 117; P = 0.3510) (Table 4).
There were significantly more adults per plant on
silver buttonwood compared with sour orange (F
= 4.83; df = 2, 117; P = 0.0096) 8 days after the


June 2003







Mannion et al.: Oviposition and Survival of Diaprepes


TABLE 2. MEAN NUMBER OF EGG MASSES FROM 50 FEMALE DIAPREPES ABBREVIATUS IN A NO-CHOICE TEST.


Host plant


Mean egg masses per plant (SE)'


Test 1 Manihot esculenta (cassava) 4.75 1.21 a
Conocarpus erectus (silver buttonwood) 2.87 1.25 ab
Carica papaya (papaya) 0.50 0.38 bc
Xanthosoma sp. (malanga) 0.00 0.00 c
Test 2 Conocarpus erectus (silver buttonwood) 1.75 0.36 a
Sorghum sudanense (sorghum-sudan) 1.25 0.45 a
Persea americana (avocado) 0.13 0.12 b
Chrysophyllum oliviforme satinleaff) 0.00 0.00 b
Test 3 Conocarpus erectus (silver buttonwood) 4.12 1.24 a
Solanum tuberosum (white potato) 2.25 0.31 a
Pennisetum purpureum (elephant grass) 2.25 0.70 a
Zea mays (sweetcorn) 1.75 0.41 a

'Means within a column for each test followed by different letters are significantly different (P < 0.05).


adults were released into the screen house (Table
4). The number of female adults on silver button-
wood was significantly greater than on the other
two plant species (F = 6.79; df = 2, 117; P =
0.0016), while the number of male adults was not
(F = 2.15; df= 2, 117; P = 0.1209).

Multiple-Choice Oviposition Tests

Significantly more egg masses were found on
the foliage of silver buttonwood compared with all
other plants in the test (F = 26.31; df= 6, 326; P =
0.0001) (Table 5). No eggs were found on the foli-
age of malanga.
Overall, silver buttonwood appeared to be the
preferred host for oviposition in all the choice
tests. Although differences were not always sig-
nificant, the highest mean numbers of egg masses
per plant were on silver buttonwood in all tests
but one. In the latter test, the second highest
mean number of egg masses was found on silver
buttonwood. Silver buttonwood is very common in
nursery production, and in the landscape in
southern Florida. The preference for silver but-
tonwood, however, did not preclude oviposition on
other hosts. No choice tests were conducted with-
out silver buttonwood but should be considered in
future studies to help better understand host se-


election by adult weevils. In southern Florida or-
namental nurseries, mixed species of plants are
commonly planted within a row. Thus, female
D. abbreviatus may lay eggs on the foliage of sev-
eral species, despite the presence of a more pre-
ferred host, such as silver buttonwood.
There were other factors inherent in the choice
bioassays that may have influenced the outcome.
First, the egg-laying potential of the weevils was
unknown because they were field collected. All
weevils used in a given test were all collected at
the same time, however, the choice tests were not
conducted concurrently. Therefore, there could be
differences in oviposition due to female age, con-
dition, etc. Additionally, plant phenology could
also influence the level of oviposition. Although
plant phenology was not controlled for, all plants
exhibited foliage that appeared suitable for ovipo-
sition. Lastly, all adult weevils collected from the
field were caged and provided green buttonwood
as a food source. Although the time the weevils
here held before use was relatively short (24 h),
feeding on green buttonwood prior to the test may
have increased their preference for oviposition on
silver buttonwood. Also, no tests were conducted
without silver buttonwood. More tests are neces-
sary to evaluate these influences as well as when
no preferred hosts are available for oviposition.


TABLE 3. MEAN NUMBER OF EGG MASSES OF 50 FEMALE DIAPREPES ABBREVIATUS IN A TWO-CHOICE TEST.

Host plant Mean egg masses per plant (SE)'

Test 1 Conocarpus erectus (silver buttonwood) 7.00 1.22 a
Sorghum sudanense (sorghum-sudan) 2.38 0.65 b
Test 2 Conocarpus erectus (silver buttonwood) 0.88 0.27 a
Citrus aurantifolia (lime) 0.19 0.03 b

Means within a column for each test followed by different letters are significantly different (P < 0.05).







Florida Entomologist 86(2)


TABLE 4. THE MEAN NUMBER OF DIAPREPES ABBREVIATUS ADULTS AND EGG MASSES ON THREE PLANT SPECIES 8 DAYS
AFTER RELEASE OF 500 ADULTS (1:1 MALE:FEMALE) INTO THE SCREEN HOUSE.

Mean egg masses Mean adults Mean males Mean females
per plant' per plant' per plant' per plant'
Plant species (common name) (+SE) (+SE) (+SE) (+SE)

Conocarpus erectus (silver buttonwood) 0.30 0.12 a 1.53 0.34 a 0.67 0.20 a 0.90 0.19 a
Citrus aurantifolia (Tahiti lime) 0.10 0.05 a 0.60 0.30 ab 0.33 + 0.19 a 0.27 + 0.13 b
Citrus sinensis (sour orange) 0.13 + 0.13 a 0.17 0.08 b 0.13 + 0.06 a 0.03 0.03 b

'Means within a column followed by different letters are significantly different (P < 0.05).


Larval Survival and Root Consumption

Survival of larvae and their effect on plant
growth was examined on several commonly
grown plant species in southern Florida. Both
fresh and dry root weight (fresh: t = 3.68; df = 18;
P = 0.001; dry: t = 3.85; df = 18; P = 0.001) and
plant biomass (fresh: t = 4.71; df = 18;P = 0.0002;
dry: t = 3.58; df = 18; P = 0.002) were significantly
reduced on green bean as a result of larval feed-
ing 2 months after infestation (Table 6). However,
the measured traits of sweet corn were not al-
tered (Table 6). Almost no larvae survived on the
sweet corn but an average of 2.6 larvae survived
per green bean plant (Table 7).
Larvae survived on silver buttonwood, lime,
and sorghum-sudan 3 months after infestation
(Table 7). Larvae did not survive on malanga or
satinleaf (Table 7), and therefore, there was no ef-
fect on plant height, root weight and biomass of
malanga or satinleaf (Table 6). The highest mean
number of larvae survived on silver buttonwood.
The fresh root weight and fresh biomass weight
were significantly reduced in silver buttonwood
plants infested with larvae with a 13.1 percent re-
duction in the dry biomass (root: t = 3.30; df = 20;
P = 0.003; biomass: t = 3.04; df = 20; P = 0.006)
(Table 6). On Tahiti lime, an average of 1.8 larvae
per plant survived (Table 7), and both the fresh
and dry root weight and biomass weight were sig-
nificantly reduced (fresh root: t = 3.33; df = 42; P
= 0.001; fresh biomass: t = 8.02; df = 42; P =
0.0001; dry root: t = 3.07; df = 42; P = 0.004; dry


biomass: t = 6.60; df = 41; P = 0.0001) (Table 6).
The net reduction of dry biomass was 42.9%. At
the time of evaluation, the lime plants were dead
or dying. An average of 5.5 larvae per plant sur-
vived on sorghum-sudan (Table 7). Both fresh and
dry root weights (fresh: t = 3.09; df = 18; P =
0.0063; dry: t = 3.83; df = 18; P = 0.003) and fresh
and dry biomass weights (fresh: t = 2.71; df = 18;
P = 0.014; dry: t = 2.71; df= 10; P = 0.02) were sig-
nificantly reduced as a result of larval feeding
(Table 6). The overall reduction in biomass of the
sorghum-sudan was 41.9%.
Larval survival was low on silver buttonwood,
Tahiti lime and malanga 6 months after infesta-
tion and there were no significant differences
among host plants (F = 2.41; df = 3, 28; P = 0.08).
Nevertheless, silver buttonwood supported the
highest mean number of larvae and these larvae
had the highest weights (Table 7).
Regardless of the host plant infested, the num-
ber of larvae per plant that survived was low rel-
ative to the number of neonates initially used to
inoculate (50). Neonates are highly mobile (Wol-
cott 1936), and some of them may actually leave
the containers at the time of infestation. Neo-
nates have been shown to move over the tops of
containers as well as through holes in the bottoms
of containers (Mannion, unpublished data). It is
very difficult to prevent this movement. Mean
weights of larvae varied with the infestation time
and the host plant species. The highest mean lar-
val weight 3 months after infestation was that of
larvae feeding on silver buttonwood. Average


TABLE 5. MEAN NUMBERS OF EGG MASSES OF DIAPREPES ABBREVIATUS IN A MULTIPLE-HOST CHOICE TEST.

Plant species (common name) Mean no. egg masses per plant (SE)'

Conocarpus erectus (silver buttonwood) 1.66 0.27 a
Zea mays (sweetcorn) 0.33 0.06 b
Solanum tuberosum (white potato) 0.23 0.07 b
Sorghum sudanense (sorghum-sudan) 0.18 0.06 b
Pennisetum purpureum (elephant grass) 0.04 0.03 b
Citrus aurantifolia (lime) 0.04 0.03 b
Xanthosoma sp. (malanga) 0.00 0.00 b

Means within a column followed by different letters are significantly different (P < 0.05).


June 2003








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weights of larvae feeding on the other hosts 3
months after infestation were relatively low.
Numerous plant hosts are suitable as a food
source and for oviposition by adult D. abbreviatus
as well as supporting larvae. In our study, silver
buttonwood, a common landscape plant in south
Florida, was generally preferred. More larvae
survived and more egg masses were found on this
host plant. However, it is important to note that
in the absence of silver buttonwood, other plant
species still provide suitable sites for oviposition
and larval survival. Schroeder et al. (1979) found
9 species of ornamental plants and one native
plant species other than citrus and sugarcane to
be suitable for larval development. Simpson et al.
(1996) identified nine plant species that support
oviposition and larval development. More than 40
plant species were associated with larval feeding.
The host plants identified as having some associ-
ation with D. abbreviatus are diverse belonging to
59 plant families. Eggs may be present without
feeding adults and larvae may be present without
evidence of oviposition. The survival of larvae and
subsequent damage from root feeding for most
plant host is not known. Dispersion of this pest is
likely by the movement of plant material infested
with any of the life stages ofD. abbreviatus. Cur-
rently, this pest is considered a regulatory risk
and any plant associated with any life stage of
D. abbreviatus is considered a regulatory host.
Growers in known infested counties are required
to follow strict guidelines of treatments, which
are time-consuming, expensive, and disruptive to
natural enemies, before shipping plant material
to non-infested areas.
Florida Agricultural Experiment Station Jour-
nal Series No. R-08259.



REFERENCES

ADAIR, R. C., H. N. NIGG, S. E. SIMPSON, AND L. LEFE-
VRE. 1998. Ovipositional preferences of Diaprepes
abbreviatus (Coleoptera: Curculionidae). Florida
Entomol. 81: 225-234.
BEAVERS, J. B. 1982. Biology of Diaprepes abbreviatus
(Coleoptera: Curculionidae) reared on an artificial
diet. Florida Entomol. 65: 263-269.
BEAVERS, J. B., AND A. G. SELHIME. 1976. Population dy-
namics ofDiaprepes abbreviatus in an isolated citrus
grove in central Florida. J. Econ. Entomol. 69: 9-10.
FENNAH, R. G. 1942. The citrus pest's investigation in the
Windward and Leeward Islands, British West Indies
1937-1942. Agr. Advisory Dept., Imp. Coll. Tropical
Agr. Trinidad, British West Indies. Pp. 1-67.
KNAPP, J. L., S. E. SIMPSON, J. E. PENA, AND H. N. NIGG.
2000. Diaprepes root weevil host list. Fla. Coop. Ext.
Serv. ENY-641 (http://edis.ifas.ufl.edu).
NIGG, H. N, S. E. SIMPSON, N. E. EL-GHOLL, AND F. G.
GMITTER, JR. 2001a. Response of citrus rootstock
seedlings to Diaprepes abbreviatus L. (Coleoptera:
Curculionidae) larval feeding. Proc. Fla. State Hort.
Soc. 114: 57-64.


June 2003


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Mannion et al.: Oviposition and Survival of Diaprepes


QUINTELA, E. D., J. FAN, AND C. W. McCOY. 1998. De-
velopment of Diaprepes abbreviatus (Coleoptera:
Curculionidae) on artificial and citrus root sub-
strates. J. Econ. Entomol. 91: 1173-1179.
SAS INSTITUTE. 1999-2001. SAS Proprietary Software.
Release 8.02. Cary, NC.
SCHROEDER, W. J., R. A. HAMLEN, AND J. B. BEAVERS.
1979. Survival of Diaprepes abbreviatus larvae on
selected native and ornamental Florida plants. Flor-
ida Entomol. 62: 309-312
SIMPSON, S. E., H. NIGG, N. COILE, AND R. ADAIR. 1996.
Diaprepes abbreviatus (Coleoptera: Curculionidae):
Host plant associations. Environ. Entomol. 25: 333-
349.


STANLEY, D. 1996. Suppressing a serious citrus pest. Ag-
ric. Res. 44: 22.
WOLCOTT, G. N. 1936. The life history of Diaprepes ab-
breviatus at Rio Piedras, Puerto Rico. J. Agr. Univ.
Puerto Rico 20: 883-914.
WOODRUFF, R. E. 1968. The present status of a West In-
dian weevil (Diaprepes abbreviatus (L.)) in Florida
(Coleoptera: Curculionidae). Florida Department of
Agriculture Division of Plant Industry Entomology
77, Gainesville, FL.
WOODRUFF, R. E. 1985. Citrus weevils in Florida and
the West Indies: Preliminary report on systematics,
biology, and distribution (Coleoptera: Curculion-
idae). Florida Entomol. 68: 370-379.







Florida Entomologist 86(2)


June 2003


OVIPOSITION BY METAMASIUS HEMIPTERUS SERICEUS
(COLEOPTERA: DRYOPHTHORIDAE: RHYNCHOPHORINAE)

THOMAS WEISSLING1, ROBIN GIBLIN-DAVIS2, BARBARA CENTER2, ROBERT HEATH' AND JORGE PENA3
'Subtropical Horticultural Research Station, United States Department of Agriculture
Agricultural Research Service, 13601 Old Cutler Road, Miami, FL 33158, U.S.A.

2University of Florida, Fort Lauderdale Research and Education Center, 3205 College Ave., Davie, FL 33314, U.S.A.

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

ABSTRACT

Metamasius hemipterus sericeus (Olivier) is a widely distributed weevil in Central and
South America, as well as the West Indies. It was introduced into Florida, Miami-Dade
County, in 1984. This insect generally is regarded as a secondary pest of sugarcane, bananas,
palms and several other tropical plants grown as ornamentals. Larvae bore into stems and
petioles, thus weakening the plant and providing a pathway for penetration by fungi or other
pests. In addition to investigating the biology, this study was conducted to gather basic in-
formation to help optimize culturing efforts for large numbers of M. h. sericeus to be used for
mass rearing of potential biological control organisms. After pairing males and females, it
took an average of 27.0 days for females to begin oviposition. The oviposition period lasted
56.8 days. Females lived 142.3 days and laid an average of 51.6 eggs. Mean generation time
was 63 days. Mean egg production during the oviposition period was 1.1 eggs/day. Egg eclo-
sion averaged 81.3% during the oviposition period.

Key Words: Dryophthoridae, Rhynchophorinae, silky cane weevil, fertility, fecundity

RESUME

El picudo rayado Metamasius hemipterus sericeus (L.) esta ampliamente distribuido en Cen-
tro y Suramerica, asi como tambi6n en las Indias Occidentales. Fu6 introducido en el con-
dado de Miami-Dade, Florida en 1984. Se le consider una plaga de segunda importancia en
cana de azucar, banano, palmas y en varias plants ornamentales tropicales. La larva per-
fora los tallos y peciolos, debilitando la plant y brindando un puerto de entrada a hongos y
otras plagas. Ademas de investigar la biologia basica durante este studio, hemos generado
informaci6n con el fin de ayudar a la optimizacion de metodos de crianza masiva de M. h.
sericeus. Las hembras comienzan a ovipositar en un promedio de 27 dias despues de aparear
con los machos. El period de oviposici6n dura 56.8 dias. Las hembras viven 142.3 dias y ovi-
positan un promedio de 51.6 huevos. El promedio de production diaria de huevos por hembra
fu6 de 1.1. El porcentaje promedio de eclosi6n de huevos durante el period de oviposici6n fue
de 81.3%.


Translation provided by author.


Metamasius hemipterus sericeus (Olivier), a
weevil that is widely distributed in Central and
South America, as well as the West Indies, was in-
troduced into Florida and reported there for the
first time in Miami-Dade County in 1984 (Wood-
ruff & Baranowski 1985). Generally it is regarded
as a secondary pest of sugarcane, bananas, palms
and many other tropical plants grown as orna-
mentals. The larvae bore into stems and petioles,
thus weakening the plant and providing a path-
way for penetration by fungi or other pests.
According to the literature, M. h. sericeus
adults live for 60 days and females lay 500 eggs
(Castrillon & Herrera 1986). Females are at-
tracted to and oviposit in damaged or stressed
host tissues (Giblin-Davis et al. 1994). Eggs hatch


in about 4 days and larvae begin to feed. In sug-
arcane, larvae feed in the pith, sometimes boring
into healthy tissue. Larval tunneling in palms
starts in the petioles, crown, or stem, usually in
wounds, and extend into healthy tissue. After
about 7 weeks, larvae construct a fibrous pupal
case. After 10 days, pupae transform to adults,
which may immediately break free of the cocoon,
or may remain within the cocoon until conditions
are favorable for emergence. The mean genera-
tion time is 63 days (Woodruff & Baranowski
1985). Adults of M. h. sericeus are free living, and
often are found on or within banana pseudostems,
palm fronds, sugarcane sheaths, and leaf litter.
Metamasius hemipterus sericeus poses a signif-
icant threat to the economic establishment of the







Weissling et al.: Oviposition by M. h. sericeus


sugarcane cultivar 'CP-85-1382' (Sosa et al. 1997)
and nursery grown palms. The expense of control
with traditional insecticides or with biopesticides
can increase production costs substantially. Bio-
logical control, however, offers the potential of
long term, relatively inexpensive control of M. h.
sericeus. A logical candidate for classical biological
control of M. h. sericeus in Florida is Lixophaga
sphenophori (Villeneuve), a tachinid parasitoid
used to manage a sugarcane weevil species, Rhab-
doscelus obscurus (Boisduval), in New Guinea
(Waggy & Beardsley 1972) and Hawaii that is
closely related to M. h. sericeus. Assuming L.
sphenophori will parasitize M. h. sericeus, large
numbers of host larvae need to be reared.
This study was conducted to gather basic infor-
mation to help optimize culturing efforts for large
numbers of M. h. sericeus to be used for mass-
rearing L. sphenophori or other potential biologi-
cal control organisms. In addition, we are study-
ing the biology of this pest as part of a long-range
objective leading to management.

MATERIALS AND METHODS

Metamasius hemipterus sericeus adults were
collected December 1998-March 1999 in Broward
Co., FL using optimized pheromone bucket traps
described by Giblin-Davis et al. (1996). As they be-
came available, trapped weevils were placed in 68-
1 plastic storage tubs (usually 30 or more weevils
were caged per tub). Each tub was provisioned
with 3 kg of sugarcane stem cut into 0.2 m length
pieces, and covered with screening to provide ven-
tilation and prevent weevil escape. Tubs were left
outdoors in a location sheltered from rainfall and
sunlight. Metamasius hemipterus sericeus cocoons
were periodically collected from sugarcane stem
pieces and held in an incubator set at 27C until
adults emerged. Upon emergence, adults were
sexed by the presence of tufts of hair on the apical
segment of the abdomen (males) or the absence of
these tufts (females) (Vaurie 1966). Males and fe-
males were paired and placed in 200 ml cups cov-
ered by lids with a 4.5 cm diam. hole covered by
aluminum screen (14 mm opening). To prepare an
ovipositional substrate, sugarcane stems were
peeled and thinly sliced (1.4-1.8 mm thick). Pre-
liminary observations demonstrated that eggs
laid in thin slices could be removed easily and
counted. Cane slices much thicker than this re-
sulted in poor or inaccurate egg recovery. Cane
slices (1 per oviposition cup) were placed over the
screened cup opening and covered with a water-
moistened piece of filter paper. Cups were in-
verted and placed outdoors [mean low 24.5 1.4C
(SD); mean high 29.4 1.5C (SD); range 20-33C]
in a location protected from sunlight and rain.
Cane slices were replaced daily, and old slices
were dissected for eggs. Males were removed from
oviposition cups the first day after eggs were


found on cane slices. Eggs were carefully removed
from the cane and individually placed in petri
dishes (15 x 100 mm) lined with water-moistened
filter paper. Individual confinement was deter-
mined to be necessary as a preventative measure
against possible larval cannibalism. Petri dishes
containing eggs were placed in an incubator set at
27C and checked daily for eclosion. A total of 29
cups were monitored daily for oviposition, and for
female longevity. Measurements were made on 14
eggs to determine dimensions.
Descriptive statistics (means, standard devia-
tion and range) were calculated and used to help
describe observed parameters. Linear regression
was used to determine if eclosion changed
through time.

RESULTS AND DISCUSSION

Eggs are oblong and measure 1.31 mm ( 0.08
[SD]; range = 1.17-1.44 mm) in length and 0.44
mm ( 0.05 [SD]; range = 0.39-0.51 mm) in width.
Twenty-two of the 29 weevils (76%) observed laid
eggs. After pairing newly emerged males with fe-
males, it took an average of 27.0 d ( 11.3 [SD];
range = 7-95 days) for females to begin laying
eggs. Of the females that laid eggs, the oviposition
period lasted an average of 59.4 d ( 5.7 [SD];
range = 12-128 days). Females lived on average
142.3 d ( 7.8 [SD]; range = 40-204 days) and laid
an average of 51.6 eggs ( 1.4 [SD]; range = 0-192
eggs). Mean egg production per female during the
oviposition period was 1.1 eggs/day ( 0.02 [SD];
range = 0.08-3.3 eggs/days).
Fecundity observed in this study was consider-
ably less than that reported by Castrillon & Her-
rera (1986) who observed up to 500 eggs laid per
M. h. sericeus female. However, it is unclear how
these data were collected. Observed fecundity be-
tween this work and that of Castrillon & Herrera
(1986) may be due to differences in ovipositional
substrates offered to weevils. For example, Giblin-
Davis et al. (1989) using pineapple as an oviposi-
tion medium estimated fecundity ofR. cruentatus
to be substantially less than fecundity observed
when weevils were allowed to oviposit in apples
(Weissling & Giblin-Davis 1994). In preliminary
experiments, M. h. sericeus females were offered
many ovipositional substrates, including apple
slices, banana stem, agar, and various arrange-
ments of sugarcane chunks and slices. Resulting
oviposition was poor and results were variable.
However, females readily oviposited in thin sugar-
cane slices (1.4-1.8 mm thick) and the eggs were
easy to locate and remove from the tissue.
Newly-emerged M. h. sericeus females laid few
if any eggs during the first two-weeks of confine-
ment with males. The greatest number of eggs
were laid during the third through eleventh weeks,
after which egg production generally declined (Fig.
1). There was a slight increase in oviposition 19






Florida Entomologist 86(2)


0.8
S- A.
0.7





gX 0.4



1 0.2

S0.1








S20 ------------ --
r,10









0 1 2 3 4 5 6 7 8 9 101112131415161718192021


Weeks after paired with male
Fig. 1. Mean weekly egg production by M. h. sericeus females (A) and number of females observed (B) confined
individually on sugarcane slices.


and 20 weeks after pairing. A similar trend in late- Fertility of eggs laid by M. h. sericeus varied
oviposition period productivity was observed with through time but remained at a fairly high level
R. cruentatus (Weissling & Giblin-Davis 1994). during the 15 weeks of observation (Fig. 2).
Reasons are unclear for this observation. Regression analysis over time revealed no clear
4 -1 ^------





o 31 2 --4------------------------------ 9 20
(U -**


June 2003







Weissling et al.: Oviposition by M. h. sericeus


100too S S

90 *

80

70

60 "

50 p

40

30
0 y =-1.3707x + 87.908
20 R = 0.0495
10

0 I I I I I I I ? i-

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15


Weeks After Mating

Fig. 2. Percent eclosion of eggs produced by M. h. sericeus females through time.


linear trend (R2 < 0.05). Throughout the experi-
mental period, eclosion averaged 81.3%. These
results indicate that mating during the preovipo-
sitional period resulted in the transfer of ade-
quate quantities of sperm to fertilize eggs without
subsequent mating. In contrast, fertility of R.
cruentatus eggs declined to zero 9 weeks after
mating (Weissling and Giblin-Davis 1994).
The observed mean fecundity of M. h. sericeus,
although lower than that reported by Castrillon
& Herrera (1986), is at levels high enough to
make mass rearing for the production of parasi-
toids feasible. Based on specifics of this study,
weevil culture can be optimized by providing fe-
males with thin sugarcane slices for oviposition.
These slices could then be transferred to large
sugarcane pieces for larval feeding.

ACKNOWLEDGMENTS
This work was supported in part by funding from the
Florida Sugar Cane League, Inc. Helpful comments to
improve the manuscript were provided by Drs. Nancy
Epsky and Bill Howard.

REFERENCES CITED
CASTRILLON, C., AND J. G. HERRERA. 1986. Los picudos
negro y rayado del platano y banano. Ica-Informa,
Abril-Mayo-Junio, 4 p.
GIBLIN-DAVIS, R. M., AND F. W. HOWARD. 1989. Vulner-
ability of stressed palms to attack by Rhynchophorus


cruentatus (Fab.) (Coleoptera: Curculionidae) and
insecticidal control of the pest. J. Econ. Entomol. 72:
480-488.
GIBLIN-DAVIS, R. M., J. E. PENA, AND R. E. DUNCAN.
1994. Lethal pitfall trap for evaluation of semio-
chemical mediated attraction of Metamasius hemi-
pterus sericeus (Coleoptera: Curculionidae). Florida.
Entomol. 77: 247-255.
GIBLIN-DAVIS, R. M., J. E. PENA, A. C. OEHLSCHLAGER,
AND A. L. PEREZ. 1996. Optimization of semiochemi-
cal-based trapping of Metamasius hemipterus seri-
ceus (Olivier) (Coleoptera:Curculionidae). J. Chem.
Ecol. 22: 1389-1410.
SOSA, O., J. SHINE, AND P. TAI. 1997. West Indian cane
weevil (Coleoptera: Curculionidae): a new pest of
sugarcane in Florida. J. Econ. Entomol. 90: 634-638.
WAGGY, S. L., AND J. W. BEARDSLEY. 1972. Biological
studies on two sibling species of Lixophaga (Diptera:
Tachinidae) parasites of the New Guinea sugarcane
weevil, Rhabdoscelus obscurus (Boisduval) Proc. Ha-
waiian Entomol. Soc. 21: 485-494.
WEISSLING, T. J., AND R. M. GIBLIN-DAVIS. 1994. Fecun-
dity and fertility of Rhynchophorus cruentatus (Co-
leoptera: Curculionidae). Florida Entomol. 77: 373-
376.
WOODRUFF, R. E., AND R. M. BARANOWSKI. 1985. Meta-
masius hemipterus (Linnaeus) recently established
in Florida (Coleoptera: Curculionidae). Florida Dept.
Agric. & Consumer Serv. Division of Plant Industry,
Entomology Circular No. 272. 4 p.
VAURIE, P. 1966. A revision of the Neotropical genus
Metamasius (Coleoptera: Curculionidae, Rhyn-
chophorinae). Species groups I and II. Bull. Ameri-
can Mus. Nat. Hist. 131: 213-337.







Florida Entomologist 86(2)


June 2003


EFFECTS OF INSECTICIDES ON ORIUS INSIDIOSUS
(HEMIPTERA: ANTHOCORIDAE), MEASURED BY FIELD,
GREENHOUSE AND PETRI DISH BIOASSAYS

GLENN E. STUDEBAKER1 AND TIMOTHY J. KRING2
'Northeast Research & Extension Center, University of Arkansas, P.O. Box 48, Keiser, AR 72351

2Department of Entomology, University of Arkansas, Fayetteville, AR 72701


ABSTRACT

Orius insidiosus (Say) is an important predator of several economic pests in cotton. Labora-
tory-reared males, females and third instar nymphs were exposed to residues of nine insec-
ticides applied to cotton plants in the field, in potted plants in the greenhouse and glass Petri
dishes in the laboratory. Insects were exposed for 24-hours and then removed to determine
mortality. Insecticides tested were spinosad, indoxacarb, imidacloprid, tebufenozide, meth-
oxyfenozide, abamectin, emamectin benzoate, fipronil and X-cyhalothrin. Differences were
observed in mortality as measured by different methods. Spinosad, imidacloprid and indox-
acarb induced significantly higher mortality with treated Petri dishes than on treated cotton
plants in the field or greenhouse. No differences in mortality were observed between meth-
ods with fipronil or X-cyhalothrin, and in only one instance with abamectin. Spinosad was
not toxic in the field or greenhouse bioassays, but was highly toxic in the Petri dish bioassay.
Imidacloprid was moderately toxic in the field and greenhouse, but was highly toxic in the
Petri dish bioassay. Indoxacarb had variable toxicity, with low to moderate toxicity in the
field and greenhouse, and high toxicity in the Petri dish bioassay. It is apparent that multi-
ple testing methods should be used in evaluating the effects of pesticides on beneficial ar-
thropods.

Key Words: insidious flower bug, pesticides, mortality

RESUME

Orius insidiosus (Say) es un depredador important de diferentes plagas econ6micas en el
algod6n. Machos criados en el laboratorio, hembras y ninfas del tercer estadio fueron ex-
puestos a residues de nueve insecticides aplicados a plants de algod6n en el campo, en plan-
tas en masetas en el inveradero, y en plates de Petri de vidrio en el laboratorio. Los insects
fueron expuestos por 24-horas y despu6s sacados para determinar la mortalidad. Los insec-
ticidas probados fueron spinosad, indoxacarb, imidacloprid, tebufenozide, methoxyfenozide,
abamectin, emamectin benzoate, fipronil X-cyhalothrin. Se observaron diferencias en la mor-
talidad media por m6todos diferentes. Los spinosad, imidacloprid e indoxacarb inducian
una mortalidad significativamente mas alta en los plates tratados en los Petri tratados que
en las plants de algod6n en el campo y en el invernadero. Ninguna diferencia en la morta-
lidad fu6 observada entire los m6todos con fipronil X-cyhalothrin, y solamente en una ocasi6n
con abamectin. El spinosad no fue t6xico en los bioensayos del campo o del inveradero, pero
fu6 altamente t6xico en el bioensayo en el plato Petri. Imidacloprid fu6 moderadamente
t6xico en el campo y en el inveradero, pero fu6 altamente t6xico en el bioensayo en el plato
de Petri. Indoxacarb tenia una t6xicidad variable, con una t6xicidad de baja a moderada en
el campo y en el invernadero, y altamente t6xico en el bioensayo en el plato de Petri. Es evi-
dente que se debe usar m6todos de pruebas multiples para evaluar los efectos de pesticides
en artr6podos beneficos.


An increasing number of scientists are evalu-
ating the toxicity of new pesticide chemistries on
beneficial arthropods. Although a considerable
number of studies have been published, it is
sometimes difficult to compare results among re-
searchers. The variety of methods used in bioas-
says is as varied as the number of individuals
conducting the work. Scientists have used direct
topical applications of the pesticide to the insect
(Yu 1988; De Cock et al. 1996; Trisyono et al.


2000) or injected the insect with insecticide (Yu
1988), or fed treated prey (De Cock et al. 1996;
Trisyono et al. 2000; Elzen 2001). These methods
insure that a specific insecticide dose makes con-
tact with the insect, either topically or internally,
and will give an accurate indication of the actual
toxicity of the pesticide to the insect in question.
It is likely that if the insect species survives the
topical or injection application, it will also survive
any exposure in the field. However, the reverse







Studebaker & Kring: Effect of Insecticides on Orius


may not always be true. High mortality resulting
from a topical or injection bioassay may not be re-
lated to mortality observed under field conditions.
Researchers commonly use an inert substrate
such as glass vials, Petri dishes or slides to test
the toxicity of various insecticide residues to
predatory or parasitic insects (Plapp & Bull 1978;
Mizell & Schiffhauer 1990; Bayoun et al. 1995;
Getting & Latimer 1995; De Cock et al. 1996) or
may use treated plastic cups (Mizell & Schiff-
hauer 1990). There are several possible errors
that could occur in using treated substances such
as glass or plastic to evaluate the toxicity of any
insecticide to an insect. While data from these
bioassays will give an indication of the toxicity of
a pesticide to an insect, relating this toxicity to
that which may be encountered in the field is dif-
ficult. The activity of a pesticide may be affected
by the substrate upon which it is deposited (Cog-
burn 1972; White 1982). Jain and Yadav (1989)
found that some insecticides persisted much
longer when applied to a plastic substrate as com-
pared with glass or painted wood.
Potentially more realistic testing methods use
treated excised leaves (Samsoe-Petersen 1985;
Getting & Latimer 1995; Jones et al. 1997; Elzen
& Elzen 1999), or treated potted plants grown in
the greenhouse (Brown & Shanks 1976; Pietran-
tonio & Benedict 1999). These methods should
provide a more realistic picture of actual toxicity
from contact with residues on a natural substrate.
Environmental effects (e.g., solar radiation or in-
sect movement within the plant canopy) which
may effect actual toxicity are not addressed.
Numerous researchers have evaluated insecti-
cide effects on beneficial arthropods by making
evaluations in the field from treated field plots
(Brown & Shanks 1976; Stoltz & Stern 1978;
Young et al. 1997; Simmons & Jackson 2000).
Generally, the results from field studies express
toxicity as the resulting presence or absence of
the insect from a treated plot in comparison with
an untreated plot or with pretreatment counts.
These data are often taken within a few days to a
week after treatment, depending on the re-
searcher and the experimental design. In many
instances the studies were designed to evaluate
mortality induced in the target pest, with benefi-
cial arthropod counts being made as a secondary
goal to the study. The test plots in this latter case
are not designed to accurately evaluate the in-
duced mortality in the beneficial arthropods nat-
urally present in the study.
In evaluating the effects of pesticides on any
insect, the method used may have an effect on the
final results. Confounding factors include solar
radiation, rainfall, substrate treated, tempera-
ture, etc. Under field conditions, the effectiveness
of a properly-applied insecticide may be dimin-
ished by high temperatures, sunlight and rainfall
events. Similarly, the same tests may underesti-


mate mortality caused by those insecticides that
are systemic in the plant tissue, particularly on
plant feeding insects. Therefore, it would be im-
portant to compare these effects as measured
through various methodologies.

MATERIALS AND METHODS

Orius insidiosus (Say) were collected from host
plants (crimson clover, vetch and corn) early in the
season of each year and used to start a lab colony
maintained on green bean pods and Helicoverpa
zea (Boddie) eggs. H. zea pupae were obtained
from a colony maintained at the University ofAr-
kansas Agricultural Research and Extension Cen-
ter, Fayetteville, AR. Once adult moths emerged,
they were placed in aquariums covered with a
layer of cheesecloth onto which the females could
oviposit. Wild adult moths were also collected and
added to the colony during the growing season
when they were abundant. 0. insidiosus were
reared at a photoperiod of 14:10 (L:D) at 25C in
an illuminated incubator (Precision Scientific
model 818, Winchester, VA). Green bean pods were
not only a source of food and moisture, but also
served as a substrate into which females would
readily oviposit. Green beans and H. zea eggs were
replaced daily. Pods with 0. insidiosus eggs were
placed into separate containers to allow nymphs
to hatch. Fresh bean pods and H. zea eggs were
provided to nymphs as well.

Field Plots

Plots of SureGrow 125 cotton were planted at
the University of Arkansas Northeast Research
and Extension Center, Keiser, AR during the
growing seasons of 2000 and 2001. Fertility and
weed control recommendations outlined by the
University of Arkansas Cooperative Extension
Service were followed (Baldwin et al. 2001). No
insecticides were applied to plots with the excep-
tion of the insecticide treatments outlined in this
study (Table 1). Also, no in-furrow insecticides
were applied at planting to insure insecticide-free
plants. Plots were 4 rows by 7.6-m long arranged
in a RCB design with 4 replications. Insecticides
were applied using a CO2 powered backpack
sprayer. The sprayer was calibrated to deliver 10
gpa at a pressure of 40 psi through 2-TX8 hollow-
cone nozzles per row. Water alone was applied to
the untreated control plots. Only the center 2
rows of each plot were treated to give a buffer of 2
rows between each pair of treated rows. Treat-
ments were applied early in the morning, just af-
ter sunrise, when wind conditions were negligible
to avoid spray drift. The spray boom was cleaned
between each treatment by rinsing with a water
and bleach solution, followed by water.
0. insidiosus were caged on plants as soon as
sprays had dried (approximately 1-h after applica-







Florida Entomologist 86(2)


TABLE 1. PERCENT MORTALITY IN O. INSIDIOSUS MALES MEASURED BY THREE METHODS IN 2000.

Insecticide Rate kg ai/ha Field' Greenhouse' Petri dish'

Untreated 21.3 cAB 12.5 dB 26.3 cA
Spinosad 0.09 18.8 cB 13.8 dB 98.8 aA
Spinosad 0.199 23.8 cB 21.3 dB 92.5 aA
Indoxacarb 0.078 25.0 cB 20.0 dB 100.0 aA
Indoxacarb 0.123 21.3 cB 16.3 dB 100.0 aA
Imidacloprid 0.027 42.5 bB 46.3 bcB 100.0 aA
Imidacloprid 0.053 53.8 bB 53.3 bB 100.0 aA
Methoxyfenozide 0.28 23.8 cA 18.8 dA 25.0 cA
Methoxyfenozide 0.84 27.5 cAB 36.3 cA 18.3 cB
Tebufenozide 0.14 23.8 cA 21.3 cA 28.8 cA
Tebufenozide 0.28 23.8 cA 23.8 cA 20.0 cA
Emamectin benzoate 0.005 100.0 aA 100.0 aA 91.3 aA
Emamectin benzoate 0.01 100.0 aA 100.0 aA 78.8 bB
Abamectin 0.01 100.0 aA 100.0 aA 100.0 aA
Abamectin 0.02 98.8 aA 100.0 aA 100.0 aA
Fipronil 0.042 100.0 aA 100.0 aA 100.0 aA
Fipronil 0.056 100.0 aA 100.0 aA 100.0 aA
X-cyhalothrin 0.014 100.0 aA 100.0 aA 100.0 aA
X-cyhalothrin 0.028 100.0 aA 100.0 aA 100.0 aA

Means within a column followed by the same lower case letter and means within a row followed by the same upper case letter
do not significantly differ (P < 0.05, LSD).
A total of 80 individuals were used per treatment.


tion). Cages were placed on the fourth leaf down
from the plant's terminal. Insects were caged on
the plants for 24 h and then removed to evaluate
mortality. Cages were constructed from 11.5 cm
hair clips that were bent to fit around 6 cm diam-
eter polystyrene Petri dishes. Each cage was con-
structed of either 2 Petri dish bases or 2 Petri dish
tops so that the edges would meet forming an en-
closure. Strips of foam were glued to the edges of
each dish so that a seal would form when the cage
was closed. A hole 3.2-cm in diameter was cut in
each side of the cage and a piece of organdy cloth
was glued over the opening to allow for air flow
through the cage. Males, females and third instar
nymphs were evaluated separately to determine
the effects on gender and insect stage. Data were
arcsine transformed and means from all bioassays
were subjected to analysis of variance and sepa-
rated by least significant difference test (LSD, P <
0.05). Detransformed means are reported.

Greenhouse

SureGrow 125 cotton was grown in pots in the
greenhouse at the University of Arkansas North-
east Research and Extension Center, Keiser, AR.
Potted plants were treated in a DeVries model
SB8 spray chamber. The chamber was calibrated
to deliver 11.5 gpa through a single TX8 hollow-
cone nozzle. Potted plants were treated individu-
ally with insecticide and then placed back into the
greenhouse. The spray chamber nozzle was


cleaned between each treatment by rinsing with
a water and bleach solution, followed by pure
water. 0. insidiosus were caged on plants as soon
as sprays had dried (approximately 1 h after
application). Insects were caged on the plants for
24 h and then removed to evaluate mortality.
Cages were the same as those used in the field
study (20 per replicate). Males, females and third
instar nymphs were evaluated separately to de-
termine the effects on gender and insect stage.
Data were arcsine transformed and means from
all bioassays were subjected to analysis of vari-
ance and separated by least significant difference
test (LSD, P < 0.05). Detransformed means are
reported.

Laboratory

Glass Petri dishes 6-cm in diameter were
treated with the insecticides listed in Table 1.
Dishes were treated in the same spray chamber
as the potted plants at the same rate (20 dishes
per replicate, 4 replications). Individual 0. insidi-
osus were placed in each dish as soon as sprays
had dried (approximately 1-h after application),
which was then covered with a piece ofparafilm to
keep insects from escaping. Mortality was
checked after 24 h. Data were arcsine trans-
formed and means from all bioassays were sub-
jected to analysis of variance and separated by
least significant difference test (LSD, P < 0.05).
Detransformed means are reported.


June 2003







Studebaker & Kring: Effect of Insecticides on Orius


RESULTS AND DISCUSSION

Abamectin, emamectin benzoate, fipronil and
X-cyhalothrin were consistently the most toxic of
the tested insecticides to 0. insidiosus as mea-
sured by all three methods during 2000 (Tables 1-
3) and 2001 (Tables 4-6). Mortality from X-cyhal-
othrin ranged from 95% to 100%, fipronil 77% to
100%, emamectin benzoate 61% to 100% and ab-
amectin 56.3% to 100%. No differences in mortal-
ity were observed for any of the three methods
with fipronil or X-cyhalothrin.
In all instances, mortality induced by abamec-
tin and emamectin benzoate was significantly
higher than that in the untreated control. In
three instances, the mortality measured after
treatment with these two products using the Petri
dish bioassay was significantly lower than that in
the field and greenhouse bioassays. In two in-
stances mortality measured in the Petri dish bio-
assay was significantly higher than that in the
field bioassay. In all other instances mortality was
not significantly different among methods with
these two pesticides.
Mortality induced by tebufenozide and meth-
oxyfenozide was not significantly different from
that of the untreated control when measured by
field or Petri dish bioassays. However, in a few in-
stances, mortality was significantly higher with
these two insecticides when measured by the
greenhouse bioassay.
Differences in mortality measured between
methods was most consistent with spinosad, imi-


dacloprid and indoxacarb. In every instance, mor-
tality was much higher in the Petri dish bioassay
compared with both the field and greenhouse bio-
assays. This was most pronounced with spinosad.
While no significant mortality was observed in
the field and greenhouse bioassays, mortality was
very high in the Petri dish bioassay with spi-
nosad. Mortality was also quite high in the Petri
dish bioassay with imidacloprid and indoxacarb,
but the difference was not as great because mor-
tality was approximately 50% in the field and
greenhouse bioassays.
Overall, there were few differences in mortal-
ity as measured by the field and greenhouse bio-
assays. The majority of significant differences
were between these two plant bioassays and the
Petri dish bioassay. The field and greenhouse bio-
assays would be expected to be similar in the fact
that both use the same substrate, treated cotton
leaves. The only differences between the two
would be environmental conditions (e.g., solar ra-
diation, temperature, relative humidity).
Croft (1990) defines that mortality or sublethal
effects of pesticides occur through three avenues:
1) direct contact with the insecticide, 2) residual
uptake (contacting pesticide residues on another
surface), and 3) food chain uptake (consuming
prey or host plants containing the pesticide). In
this study, obviously 0. insidiosus could only take
up pesticide through the residual uptake avenue
in the Petri dish bioassay. However, because of
this insect's omnivorous habit, the possible up-
take on treated plants used in the field and green-


TABLE 2. PERCENT MORTALITY IN O. INSIDIOSUS FEMALES MEASURED BY THREE METHODS IN 2000.

Insecticide Rate kg ai/ha Field' Greenhouse' Petri dish'

Untreated 15.0 dA 8.8 eA 11.3 dA
Spinosad 0.09 20.0 dB 15.0 eB 92.5 aA
Spinosad 0.199 17.5 dB 21.3 deB 100.0 aA
Indoxacarb 0.078 18.8 dB 16.3 deB 81.3 bcA
Indoxacarb 0.123 28.8 cdB 18.8 deB 92.5 aA
Imidacloprid 0.027 36.3 cB 36.3 bcB 100.0 aA
Imidacloprid 0.053 52.5 bB 46.3 bB 100.0 aA
Methoxyfenozide 0.28 20.0 dA 21.3 deA 23.8 dA
Methoxyfenozide 0.84 22.5 dA 17.5 deA 22.0 dA
Tebufenozide 0.14 13.8 dA 15.0 eA 18.8 dA
Tebufenozide 0.28 27.5 cdA 28.8 cdA 21.3 dA
Emamectin benzoate 0.005 100.0 aA 100.0 aA 68.8 cB
Emamectin benzoate 0.01 100.0 aA 100.0 aA 90.0 aA
Abamectin 0.01 100.0 aA 100.0 aA 81.3 bcB
Abamectin 0.02 97.5 aA 100.0 aA 87.5 abA
Fipronil 0.042 92.5 aA 97.5 aA 92.5 aA
Fipronil 0.056 96.3 aA 98.8 aA 96.3 aA
X-cyhalothrin 0.014 95.0 aA 100.0 aA 100.0 aA
X-cyhalothrin 0.028 100.0 aA 100.0 aA 100.0 aA

Means within a column followed by the same lower case letter and means within a row followed by the same upper case letter
do not significantly differ (P < 0.05, LSD).
A total of 80 individuals were used per treatment.







Florida Entomologist 86(2)


June 2003


TABLE 3. PERCENT MORTALITY IN 0. insidiosus NYMPHS MEASURED BY THREE METHODS IN 2000.

Insecticide Rate kg ai/ha Field' Greenhouse' Petri dish'

Untreated 20.0 dA 12.5 deA 25.0 bcA
Spinosad 0.09 21.3 dB 25.0 cdB 91.3 aA
Spinosad 0.199 30.0 dB 21.3 cdeB 95.0 aA
Indoxacarb 0.078 17.5 dB 16.3 deB 91.3 aA
Indoxacarb 0.123 23.8 dB 25.0 cdB 100.0 aA
Imidacloprid 0.027 23.8 dB 26.3 cdB 100.0 aA
Imidacloprid 0.053 52.5 cB 51.3 bB 100.0 aA
Methoxyfenozide 0.28 26.3 dA 32.5 cA 27.5 bcA
Methoxyfenozide 0.84 18.8 dA 16.3 deA 16.5 bcA
Tebufenozide 0.14 13.8 dB 8.8 eB 32.5 bA
Tebufenozide 0.28 20.0 dA 26.3 cdA 15.0 cA
Emamectin benzoate 0.005 100.0 aA 100.0 aA 93.8 aA
Emamectin benzoate 0.01 100.0 aA 100.0 aA 97.5 aA
Abamectin 0.01 100.0 aA 100.0 aA 98.8 aA
Abamectin 0.02 97.5 aA 98.8 aA 100.0 aA
Fipronil 0.042 100.0 aA 100.0 aA 100.0 aA
Fipronil 0.056 80.0 bB 95.0 aA 100.0 aA
X-cyhalothrin 0.014 100.0 aA 100.0 aA 100.0 aA
X-cyhalothrin 0.028 100.0 aA 100.0 aA 100.0 aA

Means within a column followed by the same lower case letter and means within a row followed by the same upper case letter
do not significantly differ (P < 0.05, LSD).
A total of 80 individuals were used per treatment.


house bioassays could be through residual uptake bioassays as compared with the Petri dish bio-
and/or food chain uptake. With uptake possibly assay. Although this was not true in the majority
occurring through two avenues, one would expect of cases in this study. Imidacloprid, indoxacarb
mortality to be higher in the greenhouse and field and spinosad had much higher mortality in the


TABLE 4. PERCENT MORTALITY IN O. INSIDIOSUS MALES MEASURED BY THREE METHODS IN 2001.

Insecticide Rate kg ai/ha Field' Greenhouse' Petri dish'

Untreated 12.5 dA 7.5 eA 16.3 cA
Spinosad 0.09 13.8 dB 15.0 eB 78.8 bA
Spinosad 0.199 27.5 dB 33.8 cB 80.0 abA
Indoxacarb 0.078 52.5 cB 67.5 bB 91.3 abA
Indoxacarb 0.123 47.5 cB 53.3 bcB 97.5 abA
Imidacloprid 0.027 48.8 cB 31.3 cdB 97.5 abA
Imidacloprid 0.053 51.3 cB 47.5 bcB 96.3 abA
Methoxyfenozide 0.28 7.5 dA 12.5 eA 11.3 cA
Methoxyfenozide 0.84 12.5 dA 10.0 eA 8.8 cA
Tebufenozide 0.14 8.8 dA 7.5 eA 8.8 cA
Tebufenozide 0.28 18.8 dA 22.5 deA 10.0 cA
Emamectin benzoate 0.005 82.5 abA 96.3 aA 97.5 abA
Emamectin benzoate 0.01 68.8 bcB 90.0 aA 97.5 abA
Abamectin 0.01 88.8 abA 96.3 aA 78.8 bA
Abamectin 0.02 90.0 aA 95.0 aA 86.3 abA
Fipronil 0.042 91.3 aA 90.0 aA 97.5 abA
Fipronil 0.056 93.8 aA 91.3 aA 98.8 abA
X-cyhalothrin 0.014 100.0 aA 100.0 aA 100.0 aA
X-cyhalothrin 0.028 100.0 aA 100.0 aA 100.0 aA

Means within a column followed by the same lower case letter and means within a row followed by the same upper case letter
do not significantly differ (P < 0.05, LSD).
A total of 80 individuals were used per treatment.







Studebaker & Kring: Effect of Insecticides on Orius


TABLE 5. PERCENT MORTALITY IN O. INSIDIOSUS FEMALES MEASURED BY THREE METHODS IN 2001.

Insecticide Rate kg ai/ha Field' Greenhouse' Petri dish'

Untreated 16.3 gA 8.8 eA 15.0 dA
Spinosad 0.089 12.5 gB 11.3 eB 67.5 bcA
Spinosad 0.178 11.3 gB 12.5 eB 92.5 abA
Indoxacarb 0.07 43.8 efB 53.8 bB 82.5 abA
Indoxacarb 0.11 18.8 fgB 26.3 cdeB 88.8 abA
Imidacloprid 0.024 53.8 eB 38.8 bcdB 92.5 abA
Imidacloprid 0.047 58.8 cdeB 48.8 bcB 95.0 aA
Methoxyfenozide 0.25 20.0 fgAB 31.3 b-eA 5.0 dB
Methoxyfenozide 0.75 17.5 gA 17.5 deA 15.0 dA
Tebufenozide 0.125 20.0 fgA 15.0 deA 7.5 dA
Tebufenozide 0.25 22.5 fgA 20.0 deA 12.5 dA
Emamectin benzoate 0.0045 61.3 cdeB 82.5 aA 88.8 abA
Emamectin benzoate 0.009 82.5 abcA 91.3 aA 81.3 abcA
Abamectin 0.009 70.0 bcdA 85.0 aA 56.3 cA
Abamectin 0.018 85.0 abA 91.3 aA 75.0 abcA
Fipronil 0.038 83.8 abcA 90.0 aA 91.3 abA
Fipronil 0.05 78.8 a-dA 86.3 aA 77.5 abcA
X-cyhalothrin 0.012 95.0 ab 97.5 aA 97.5 aA
X-cyhalothrin 0.025 100.0 aA 98.8 aA 100.0 aA

Means within a column followed by the same lower case letter and means within a row followed by the same upper case letter
do not significantly differ (P < 0.05, LSD).
A total of 80 individuals were used per treatment.


Petri dish bioassay. Possibly, 0. insidiosus did not exposure time used in this study. Another expla-
receive a toxic dose in every instance in the nation offered is that the plant surface somehow
treated plant bioassays (field and greenhouse) by altered or bound the pesticide deposits making
not feeding on the treated plant during the 24-h them less available for uptake by the test insects.


TABLE 6. PERCENT MORTALITY IN O. INSIDIOSUS NYMPHS MEASURED BY THREE METHODS IN 2001.

Insecticide Rate kg ai/ha Field' Greenhouse' Petri dish'

Untreated 13.8 cA 17.0 cdA 12.5 cA
Spinosad 0.09 16.3 cB 26.3 cdB 85.0 abA
Spinosad 0.199 15.0 cB 23.8 cdB 75.0 bA
Indoxacarb 0.078 26.3 cB 42.5 bcB 70.0 bA
Indoxacarb 0.123 27.5 cB 40.0 bcB 93.8 abA
Imidacloprid 0.027 61.3 bB 31.3 bcC 100.0 aA
Imidacloprid 0.053 77.5 abA 48.8 bB 97.5 abA
Methoxyfenozide 0.28 22.5 cA 31.3 bcA 17.5 cA
Methoxyfenozide 0.84 18.8 cA 13.8 dA 12.5 cA
Tebufenozide 0.14 17.5 cA 22.5 cdA 13.8 cA
Tebufenozide 0.28 17.5 cA 18.8 cdA 25.0 cA
Emamectin benzoate 0.005 85.0 aA 93.8 aA 96.3 abA
Emamectin benzoate 0.01 90.0 aA 95.0 aA 100.0 aA
Abamectin 0.01 86.3 aA 97.5 aA 78.8 abA
Abamectin 0.02 87.5 aA 90.0 aA 96.3 abA
Fipronil 0.042 87.5 aA 93.8 aA 98.8 aA
Fipronil 0.056 95.0 aA 90.0 aA 100.0 aA
X-cyhalothrin 0.014 100.0 aA 100.0 aA 98.8 aA
X-cyhalothrin 0.028 100.0 aA 100.0 aA 100.0 aA

Means within a column followed by the same lower case letter and means within a row followed by the same upper case letter
do not significantly differ (P < 0.05, LSD).
A total of 80 individuals were used per treatment.







Florida Entomologist 86(2)


Imidacloprid and indoxacarb are known to have
good translaminar movement into the leaf and
would therefore move the pesticide away from di-
rect contact to the insect in the plant bioassays.
Because glass is an inert substance, it is not likely
that the pesticide deposits would be altered or
somehow bound to the substrate, leaving them
free for uptake by an insect. Also, the entire in-
side surface of the dish was treated, making the
parafilm cover the only area in which the insects
could avoid the pesticide. In this study, test in-
sects were observed on the inside of the parafilm
cover only occasionally. In both the field and
greenhouse bioassays, the clip cages offered a
greater surface area on which the insects could
avoid the pesticide. Even if the insect was not at-
tempting to avoid the pesticide deposits, the
chances of picking up a lethal dose would have
been greater in the Petri dish. The most interest-
ing results from this study were with spinosad. In
both the field and greenhouse bioassays, mortal-
ity was not significantly different from that found
in the untreated control, indicating that this pes-
ticide is not toxic to 0. insidiosus. However, mor-
tality was very high in the Petri dish bioassay
with this pesticide (100% in some instances). This
leads one to think that the plant surface somehow
makes this compound unavailable to this insect.
Although spinosad is reported to have some
translaminar movement into the leaf (Bret et al.
1997), this does not adequately explain the low
toxicity in the plant bioassays.
Obviously, experimental design can have a pro-
nounced effect on the outcome of a study and may
offer some explanation on the disparity of results
sometimes observed in the literature. In this
study, particularly with spinosad, one would come
to the conclusion that this pesticide would not be
a good fit in a cotton IPM program with 0. insid-
iosus when looking at the Petri dish bioassay
alone. However, no effects were observed in the
caged field and greenhouse studies, leading one to
the opposite conclusion. It is apparent that merely
evaluating mortality of pesticides on beneficial ar-
thropods by only one method does not give an ac-
curate depiction on how those pesticides would fit
into IPM programs. This study concurs with Ban-
ken and Stark (1998) and Croft (1990) in that
multiple testing methods should be used in evalu-
ating pesticide effects on beneficial arthropods.
However, this may not always be feasible. Often,
lab studies utilizing an artificial substrate, are
the quickest and least expensive means of obtain-
ing data. However, particularly when working
with omnivorous predators such as 0. insidious,
utilizing field studies or potted plants grown in
the greenhouse would be the preferred method for
bioassays. Bioassays utilizing artificial sub-
strates, while providing important information,
should not be the sole means of evaluating the ef-
fects of pesticides on beneficial arthropods.


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ELZEN, G. W. 2001. Lethal and sublethal effects of insec-
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Florida Entomologist 86(2)


THE GENUS RHOPALOSYRPHUS (DIPTERA: SYRPHIDAE)

HOWARD V. WEEMS, JR.1, F. CHRISTIAN THOMPSON2, GRAHAM ROTHERAY3 AND MARK A. DEYRUP4
'(retired) Florida State Collection of Arthropods, P.O. Box 2309, Hawthorne, FL 32640-2309

2Systematic Entomology Laboratory, USDA, NHB-168 Smithsonian Institution, Washington, D.C. 20560

3Department of Natural History, Royal Museum of Scotland, Chambers Street, Edinburgh, EH1 1JF Scotland

4Archbold Biological Station, P.O. Box 2057, Lake Placid, FL 33862

ABSTRACT
The flower fly genus Rhopalosyrphus Giglio-Tos (Diptera: Syrphidae) is revised. The genus
is redescribed; a key to species is presented; the phylogenetic relationships of the genus and
species are hypothesized; the included species are described; with new species, R. ramu-
lorum Weems & Deyrup, described from Florida (type) and Mexico; R. australis Thompson
from Brazil and Paraguay (type); and the critical characters are illustrated.

Key Words: taxonomy, identification key, neotropics, nearctic

RESUME
El g6nero de la mosca de la flor del g6nero, Rhopalosyrphus, (DIPTERA: Syrphidae) es revi-
sada y es redescrito; se present una clave para las species; la relaci6n filogen6tica del g6-
nero y las species es formulada; las species incluidas son descritas; con las nuevas
species, R. ramulorum Weems & Deyrup, descrita de Florida (tipo) y M6xico; R. australis
Thompson de Brasil y Paraguay (tipo); y los caracteres critics son ilustrados.

Translation provided by author.


Rhopalosyrphus Giglio-Tos is a small group of
microdontine flower flies restricted to the New
World subtropics and tropics, ranging from south-
ern United States to northern Argentina. These
flies are rarely collected, only some two dozen
specimens are known. The adults mimic eume-
nine vespids, such as Zethus, which nest in twigs
(Bohart & Stange 1965). The immature stages are
known for only one species. These were found in
ant nests (Pseudomyrmex) in twigs and grass
culms, where the larvae probably prey on ant
brood. The genus contains only three species. One
wide ranging species, R. guentherii, is found from
southwestern United States to northern Argen-
tina. The others are more restricted in their
ranges; R. ramulorum, presently known from a
few specimens from Florida and Mexico, and R.
australis from southeastern Brazil, Peru and Par-
aguay. The genus is here revised, with complete
synonymies, descriptions, and distributional and
biological data given for all taxa. Adult terminol-
ogy follows Thompson (1999), larval terminology
follows Hartley (1961) and Rotheray (1991).

GENUS RHOPALOSYRPHUS GIGLIO-TOS

Rhopalosyrphus Gilgio-Tos 1891: 3. Type spe-
cies, Holmbergia guentherii Lynch Arribalzaga
(subsequent monotypy, Giglio-Tos 1892a: 2). Willis-
ton 1892: 78 (catalog citation, descriptive note);


Giglio-Tos 1892b: 34 [journal (1893: 130] (descrip-
tion); Aldrich 1905: 347 (catalog citation); Kert6sz
1910: 360 (catalog citation); Hull 1949: 312, figs.
(description, figures of habitus, head, abdomen,
hind leg); Capelle 1956 (review, key); Cole &
Schlinger 1969: 307 (descriptive notes); Thomp-
son et al. 1976: 60 (catalog citation); Vockeroth &
Thompson 1987: 729 (key reference).
Holmbergia Lynch Arribalzaga 1891: 195.
Type species, guentherii Lynch Arribalzaga
(monotypy). Synonymy by Giglio-Tos (1892a).
Head: face convex, produced anteroventrally,
pilose; gena small, linear, pilose; frontal promi-
nence absent, antenna inserted above middle of
head; frons short, about 14 as long as face, as wide
as face (2) or slightly narrowed dorsally (6), pi-
lose; vertex broad, about 3 times as long as frons,
as wide as frons, not swollen, pilose and punctuate;
ocellar triangle small, equilateral, well separated
from eye margins; occiput broad on dorsal 13; eye
bare, dichoptic in male. Antenna elongate, longer
than face; scape and basoflagellomere elongate, at
least 4 times as long as pedicel; scape about 6
times as long as broad; arista bare, inserted baso-
laterally on mesal surface, about as long as scape.
Thorax: longer than broad; postpronotum pi-
lose; meso-anepisternum with anterior portion not
differentiated, uniformly pilose; meso-katepister-
num completely pilose; meso-anepimeron with
posterior portion bare; meropleuron with barrette


June 2003







Weems et al.: Genus Rhopalosyrphus (Diptera: Syrphidae)


pilose; metasternum developed, pilose (although
reduced in some species); scutum punctuate, with
appressed pile; metatibia expanded apically;
scutellum with or without small apical calcar,
without distinct ventral pile fringe. Wing: brown
on anterior 1/3, extensively microtrichious; mar-
ginal cell broadly open; stigmatic crossvein
present; vein Ml with apical portion straight,
joining vein R4+5 perpendicularly; vein M2
present or absent; vein R4+5 with spur.
Abdomen: petiolate; 1st segment short; 2nd
segment as broad as thorax basally, but con-
stricted, cylindrical apically; 3rd segment cylin-
drical; 4th and 5th segments forming a compact
club; aedeagus bifid.
Puparium: elongate with broader ventral than
dorsal surface; marginal band of variously-sized
setae; dorsal surface flat; ventral surface convex;
marginal band notched anteriorly; prothorax and
mesothorax hidden beneath metathorax; mandi-
ble with serrate ventral margin.
Rhopalosyrphus belongs to the subfamily Micro-
dontinae and is the sister group of Ceriomicrodon
Hull, together these taxa are the sister group of
Microdon Meigen, sensu lato. Rhopalosyrphus is
defined (synapomorphy) by its 1) abdominal struc-
ture and 2) pilose meropleuron. Other diagnostic
characters are 3) antenna elongate, longer than
face, usually about twice as long; 4) scape and ba-
soflagellomere elongate; 5) face produced ven-
trally; 6) occiput greatly developed on dorsal 1/3; 7)
metasternum developed, not reduced; and 8)
metatibia flared apically. The relationship to Micr-


odon, sensu lato is unresolved: Rhopalosyrphus
shares with the Microdon clade the bifid aedeagus
and appears closely related to Ceriomicrodon. Ce-
riomicrodon shares characters 3, 4, 5, 6, 7, 8 and
its abdominal shape could be considered derived
from that of Rhopalosyrphus. The two differ only
by the presence of pile on the meropleuron.
Microdon aurcinctus, described by Sack (1921:
138) in Rhopalosyrphus, belongs to the Pseudomi-
crodon group of Microdon. The species of this
group differ from Rhopalosyrphus in the charac-
ters listed above and in having the vertex swollen
and shiny.
Based on puparial characters, the immature
stages of Rhopalosyrphus closely resemble Micro-
don. They both have the anterior end consisting of
the metathorax, with the prothorax and mesotho-
rax hidden beneath it, a marginal band of setae
which surrounds the puparium except for a notch
at the anterior end, sharply-pointed antennomax-
illary organs and mandibles with a serrated ven-
tral margin.
The very distinctive shape of the puparium of
Rhopalosyrphus separates it from that of Micro-
don: it has a curved ventral surface that is
broader than the narrow, flat dorsal surface. Also,
the whole structure is elongate rather than oval
in outline. The reverse appears in Microdon, with
the dorsal surface being broader and curved and
the ventral surface narrower and flat. These dif-
ferences in shape suit the larva to life in hollow
twigs and grass culms in which its prey, larvae
and pupae of the ant, Pseudomyrmex, live.


KEY TO SPECIES OF RHOPALOSYRPHUS

1. 3rd tergum short, about 1/3 as long as 2nd; 2nd tergum elongate (Fig. 9); eye with an area of enlarged om-
matidia medially and posterior to antenna ............... ......................... australis
3rd tergum elongate, as long as 2nd; 2nd tergum not greatly elongate posteriorly (Fig. 10); eye without en-
larged ommatidia ................. ...................................... .......... 2
2. Alula completely microtrichose; cell R extensively microtrichose, bare only on basoposterior 1/4 or less;
metasternum appearing bare, with pile greatly reduced; face and anepisternum partially black pilose
...... ................................................................... ram u loru m
Alula bare basomedially; cell R completely bare behind spurious vein; metasternum with long, distinct pile,
not reduced; face and anepisternum entirely white pilose guentherii


Rhopalosyrphus guentherii Lynch Arribalzaga
Figs. 10-13

Holmbergia giintherii Lynch Arribalzaga
1891: 198, Fig. 3 (habitus) Argentina, Buenos
Aires (T Y MACN lost?). Giglio-Tos 1892a: 2
(notes), 1893: 131 [sep. 35], pl. 1, Figs. 10, 10a-b
(description, figures of abdomen, wing); Aldrich
1905: 347 (catalog citation); Kertesz 1910: 360
(catalog citation); Fluke 1957: 36 (catalog cita-
tion); Capelle 1956: 172, Fig. 2 (description, syn-
onymy, key reference, figure of head); Thompson
et al. 1976: 60 (catalog citation).


Rhopalosyrphus carolae Capelle 1956: 174 A*
S 2 Arizona, Huachuca Mts., Sunnyside Canyon
(HT Y UKaL). Byers et al. 1962: 168 (HT UKaL);
Wirth et al. 1965: 599 (catalog citation); Cole &
Schlinger 1969: 307 (descr. note, distr. western
N.A.); Thompson et al. 1976: 60. NEW SYNONYM
Wing length: 8.8 mm (6)-10.5 mm (2). Head:
brownish black, yellowish white pilose; occiput
grayish white pollinose on ventral 2/3, shiny dor-
sally; eye with ommatidia of more or less equal
size; antenna brownish black except orange basal
1/3 of scape, about twice as long as face; antenna
ratio 5: 1: 8.







Florida Entomologist 86(2)


Thorax: brownish black; pleuron silvery-white
pilose; scutum brown pilose medially, silvery
white pilose anteriorly, along transverse suture,
laterally and posteriorly; scutellum silvery white
pilose; calypter white, with brown margin and
fringe; halter orange; wing brown anteriorly, hya-
line posteriorly, microtrichose except bare cell R
& BM, anterobasal 1/2 of cell CuP and basomedi-
ally on alula; legs reddish brown except yellow
basal 1/2 of metafemur; pro- and mesofemora
black pilose anteriorly, yellow pilose posteriorly;
pro- and mesotibia yellow pilose; tarsi black pi-
lose; metafemur black pilose with a few yellow
pili intermixed; metatibia yellow pilose basally,
black pilose apically.
Abdomen: brownish black except yellow basal
1/3 of 3rd tergum and reddish apically on 2nd ter-
gum; 1st tergum white pilose; 2nd tergum con-
stricted on apical 1/3, yellowish white pilose; 3rd
tergum about twice as broad apically as basally,
yellow pilose; 4th tergum brown pilose basome-
dial 2/3, yellow-white pilose apically, about as long
as 2nd tergum; 5th tergum yellowish-white pilose.
Distribution: Texas (Cameron, Harris,
Hidalgo, Kenedy and Kleberg counties); Arizona;
Mexico (Chiapas, Colima, Michoacan, Morelos);
Guatemala, Costa Rica, Peru, Brazil, Paraguay,
Argentina (Lynch Arribalzaga).
Material examined: PARAGUAY: Colonia
Nueva Italia, X-XI-1940, Pedro Willim (1 Y
AMNH). BRAZIL: Amazonas: Parana do Xiboren-
inho, 0315.S 6000.W, mixed water, Canopy fog-
ging project, TRS #60 Tray 392, 7-VIII-1979,
Erwin, Adis & Montgomery (1 6 USNM ENT
00032864 USNM). PERU: Lambayeque, 1 km S
Lambayeque, 24, 26-27-VII-1975, C. Parker & L.
Stange (1 Y USNM ENT 0003863 FSCA). COSTA
RICA: Alajuela, Cerro La Lana, San Ram6n, 1200
m, LN 221750_481050, 17-1-1997, Betty Thomp-
son, lot# 45327 (1 Y INBIOCRI002499628 INBIO);
Guanacaste, Estaci6n Exper. Enrique Jimenez
Nuiex, 20 km SW Caias, 5-17-XI-1991, Malaise
Trap, A. S. Menke (1 Y USNM ENT 0003862
USNM); Puntarenas, Coto Brus. Sabalis, Estaci6n
El Progresso, Sector Fila Pizote, 1400 m, LS
317700_597800, 11-V-2001, M. Alfaro Libre, lot#
63200 (1 Y INB0003331118 INBIO). GUATE-
MALA: Alta Vera Paz, Trece Aguas, "Cacao," XI-
1905, "GPColl" (1 S USNM). MEXICO: [label just
as "Mex."], (1 S ANSP); Chiapas, Gutierrez, 20
miles S Tuxtla, 12-VIII-1963, F. D. Parker & L. A.
Stange (1 S USNM ENT 00032859 UCDavis);
Colima, 6 km NE Tepames, 23-IX-Sept 1986, R.
Miller & L. Stange (1 S 1 2 USNM ENT 0003865-
6 USNM, FSCA); Michoacan, Hidago, 12-VII-1963,
F. D. Parker & L. A. Stange (1 Y USNM ENT
00032858 UCDavis); Morelos, 3 miles N Alpuyeka,
3400', 5-VI-1959, HE Evans (1 S Cornell); ..., Hua-
jitlan, 27-IX-1957, R. & K. Dreisbach (1 Y USNM
ENT 00032867 FSCA); Puebla, Chinantla, Salle (1
Y UTOR). USA. ARIZONA: [Cochise/Santa Cruz


Counties], Huachuca Mts, Sunnyside Canyon, 9-
VII-1940, DE Hardy (allotype S, UKaL). TEXAS:
Hidalgo Co: Pharr, 23-VI-1947 (1 Y USNM); La
Joya, 19-III-1970, J, O'Grady (1 Y USNM), Rio
Grande Park, 10 July 1981, A. Hook (1 Y
USNM);... 12-VII-1981, A. Hook (1 Y USNM);
McAllen, Valley Botanical Garden, 28-III-1975 (2
S USNM ENT 0003868-9 FSCA),... 20-III-1976 (1
Y USNM ENT 0003870 FSCA),... 3-IV-1975 (1 Y
USNM ENT 0003871 FSCA),... 5-IV-1975 (1 Y
USNM ENT 003872 FSCA),... 2-IX-1975 (1 S
USNM ENT 0003873 FSCA); Relampago, 17-X-
1986, FC Fee (1 S 1 2 Fee) flying around flowering
shrub, Schinus sp.; Santa Ana N.W.R., 17-X-1984
(1 Y Fee); Madero, 18-X-1995, FD Fee (1 S Fee), 11-
XI-1995, FD Fee (1 Y Fee) collected on flowers of
composite shrub, Gochnatia hypoleuca DC. Kenedy
Co.: 2710.N 9740.W, 8-X-1975, J.E. Gillaspy (1 S
USNM). Cameron Co.: Brownsville, Los Palamos
Mgt Area, 17-X-1976, FD Fee (1 Y USNM, 2 S
Fee); Brownsville, VI, ("Cata 1439" Brooklyn Mus
Coll 1929 (1 Y USNM); Brownsville, 23-X-1976,
FD Fee (1 S 2 Y Fee); Sabal Palm Grove Sanctuary,
9-X-1986, FD Fee (1 Y Fee),... 20-X-1986, FD Fee
(4 S 1 2 Fee), all individuals fly about, attracted to,
or feeding on exudate from glands at base of leaves
of sapling trees ofEhretia anagua (Teran & Berl.)
I. M. Johson; ... 27-III-1988 (1 S Fee), 5-IV-1988 (1
S Fee) flying about or attracted to flowers ofZan-
thoxylum fagara (L.) Sarg.; ... 21-XI-1995, FD Fee
(1 S Fee). Harris Co.: Houston, 45 W Virginia Str.,
28-VIII-1969, at black light trap, TJ Henry (1 S
USNM). Kleberg Co.: Kingsville, South Pasture,
26-IX-1976, at Baccharis, JE Gillaspy (1 S USNM)
The traditional nomenclature and taxonomy of
Rhopalosyrphus are maintained. Giglio-Tos es-
tablished that there was a single widespread spe-
cies, ranging from Mexico to Argentina, and that
the appropriate name for that taxon was Rhopal-
osyrphus guentherii. The specimens studied sup-
port the single widespread taxon concept of
Giglio-Tos. That the appropriate name for the
taxon is quentherii is not as certain, as the holo-
type of quentherii has not been found and nothing
mentioned in the original description will un-
equivocally allow the assignment of that name to
either the widespread taxon or australis. The
types of Rhopalosyrphus carolae Capelle were ex-
amined and are representative of the widespread
taxon (new synonym).

Rhopalosyrphus australis Thompson, new species
Fig. 9

Wing length: 10 mm (6) 11 mm (9). Head:
Face reddish to brownish black (except holotype
broadly yellowish dorsolateral), always black me-
dially, white pilose except for a few black pili ven-
trally; gena small, linear, white pilose; frons and
vertex black, white pilose; occiput reddish brown,
white pollinose and pilose on ventral 1/3, shiny


June 2003







Weems et al.: Genus Rhopalosyrphus (Diptera: Syrphidae)


dorsally; eye with a medial fasciate area of en-
larged ommatidia; antenna about 1.5 times as
long as face, antennal ratio 5:1:10.
Thorax: brownish black; pleuron white pilose;
mesonotum punctate, very short appressed pi-
lose, white pilose in males, more extensively black
pilose medially in females; metasternum with
short, appressed pile; calypter white, with brown
margin and fringe; halter orange with brown
head; wing brown anteriorly, hyaline posteriorly,
microtrichose except bare cell R & BM, anter-
obasal 1/2 of cell CuP and basomedially on alula;
legs reddish brown except yellow basal 2/3 of
metatibia, pale pilose except black pilose dorso-
medially on pro- and mesotibiae and tarsi, with
dense black spinose pile on ventrolateral 2/3 of
metafemur, with ventromedial appressed black
spinose pile on basal 2/3 of metatibia.
Abdomen: black except yellow 3rd tergum and
reddish apically on 2nd tergum, mainly short ap-
pressed white pilose, except black pilose medially
on 4th tergum; 1st short, as long as 3rd; 2nd ter-
gum half as long as entire abdomen, constricted
and cylindrical on apical 2/3, as wide as thorax
basally; 3rd tergum short, as long as 1st; 4th ter-
gum oval, as long as 2nd, forming with 3rd a dis-
tinct club in 6; 5th tergum elongate, about 1/2 as
long as 2nd, forming with 3rd and 4th a distinct
club in Y.
Distribution: Peru, southern Brazil and Para-
guay
Holotype Y: PARAGUAY, Villarica, 1-1939, F.
Schade, deposited in the American Museum of
Natural History, New York. Paratypes: BRAZIL:
Ceara, Russa, s., 11-1940, D. C. Alves (1 Y
USNM); Ceara, Limoeiro, X-1938, R. C. Shannon
(1 6 USNM); Ceara, Luixeramobug, XI-1940, D.
C. Alves (1 6 MZUSP); Minas Gerais, Belo Hori-
zonte, 800 m, Estacao Ecol6gica, UFMG Campus,
clear trail 60 m in from road, near swamp, Mal-
aise trap, S. D. Gaimari, 25-29-V-1993 (1 6
USNM ENT 00032860 USNM),... 15-18-VI-1993
(1 6 USNM ENT 00032860 USNM). PERU: Ju-
nin, Colonia Perene, Rio Perene, 18 miles NE La
Merced, 3-1-1955, E. I. Schlinger & E. S. Ross (1 Y
USNM ENT 00030699 CAS).
Rhopalosyrphus austalis is readily distin-
guished from the other two species of Rhopalosyr-
phus by its distinctive abdominal shape. The
epithet, australis, refers to the southern distribu-
tion of the species and is an adjective.

Rhopalosyrphus ramulorum Weems & Deyrup,
new species
Figs. 1-8, 14

Wing length: 6 mm (6)-7 mm (9). Head: black;
face silvery white pilose, with a few dark pili me-
dially; frons sparsely white pilose, with a distinct
bare fascia dorsally and separating off vertex;
gena white pilose; vertex silvery white pilose,


with a few dark pili medially; eye with ommatidia
of more or less equal size; occiput white pilose on
ventral 2/3, shiny dorsally; antenna brownish
black except orange basal 1/3 of scape, about 1.5
times as long as face; antennal ratio 5: 1: 7 6 4: 1:
5 .
Thorax: black, silvery white appressed pilose
on pleuron except with some dark pili on anepis-
ternum; scutum dark appressed pilose except sil-
very white pilose anteriorly, along transverse
suture and anterior to scutellum; calypter white,
with brown margin and fringe; halter yellow; legs
dark brownish black, except yellow basal 1/3 of
pro- and mesotibia and basal 2/3 of metatibia,
black pilose except pale pilose on pale areas.
Wing: extensively dark fumose, except paler on
posterior 1/3, microtrichose except bare basal 1/3
of cell R.
Abdomen: black except very narrowly yellow
basolaterally on 3rd tergum; 1st tergum black pi-
lose laterally, white pilose medially; 2nd tergum
constricted on apical 1/3, black pilose on basal 2/
3, white apically; 3rd tergum about twice as broad
apically as basally, black pilose on basal 1/2, yel-
lowish white pilose apically; 4th tergum about as
long as 2nd tergum, black pilose on basal 1/2 and
extending to posterior 1/3 laterally, yellowish
white pilose on apicomedial 1/2 and apicolater-
ally; 5th tergum yellowish white pilose.
Puparium: 7.5 mm, width 2.5 mm; semi-circu-
lar in cross-section with a ventral surface about
twice as broad as dorsal surface; elongate, nearly
3 times as long as broad; antennomaxillary or-
gans (extracted from puparium) sharply pointed;
prothorax and mesothorax retracted into met-
athorax so that structures associated with
mouthparts not visible; anterior margin of pupar-
ium consisting of metathorax; cephalopharyngeal
skeleton (extracted from puparium, Fig. 14) simi-
lar to Microdon (Garnett et al. 1990); mandible
blade-like, with serrated ventral margin, with
rounded tip; abdominal segments with 5 dorsal
(about marginal band of setae) groups of sensilla,
each group with 2-3 terminal setae, 4 ventral
groups which lack terminal setae, with some sen-
silla group on two separate papillae; marginal
band composed of 2 types of papillae alternating
with each other: larger papillae with 3 terminal
setae and smaller papillae with 2 terminal setae
(Fig. 8); papillae comprising marginal band
longer and packed close together on anterior and
posterior ends of puparium; above these alternat-
ing bands of papillae 2 rows of short papillae,
about as long as those bearing sensilla; marginal
band interrupted only on anterior margin of met-
athorax; dorsal surface coated in small, dot-like
papillae aggregated into vague reticulate pattern;
ventral surface smooth, lacking setae and papil-
lae; mid-dorsal region of abdominal segments 2-7
with 6 longitudinal rows of larger papillae, be-
tween outer 2 rows dot-like papillae densely ag-







Florida Entomologist 86(2)


Figs. 1-8. Puparium of Rhopalosyrphus ramulorum. 1-3, habitus, 1, dorsal, 2, lateral, 3, anterior. 4, anterior spi-
racular process, lateral. 5, 6, posterior respiratory process. 5, dorsal view and surrounding papillae, anterior end
uppermost; 6, lateral view, anterior end to the left. 7-8. Papillae from marginal band.


June 2003







Weems et al.: Genus Rhopalosyrphus (Diptera: Syrphidae)


9 K 10 \v

4 / N ri '-f




14_. 13 ^t ^T

Figs. 9-14. Features of Rhopalosyrphus. 9, abdomen, australis, dorsal; 10. Habitus guentherii, dorsal; 11, hind
leg, guentherii, lateral; 12. head, guentherii, lateral; 13. abdomen, guentherii, lateral; 14. cephalopharyngeal skel-
eton, ramulorum, lateral. Figures 10-13 from Hull (1949).


gregated creating impression of vague pair of
vittae running along dorsal surface (Fig. 1). Pos-
terior respiratory process (Fig. 6): 0.3 mm long,
0.2 mm high, oval, nodulate with mid-dorsal pro-
jection, surrounded by papillae, with 4 pairs of in-
terspiracular setae and 3 pairs of spiracular
openings (Fig. 5).
Distribution: USA (Florida) south to Mexico
(Chiapas).
Holotype 6: USA: Florida, Highlands Co.,
Lake Placid, Archbold Biological Station, Trail 1
SSo, 22-V-1985, Malaise Trap, M. Deyrup, depos-
ited in the National Museum of Natural History
(USNM), Washington. Paratypes: USA. FLOR-
IDA, same locality as holotype, 5-V-1986, reared
from nest of Pseudomyrmex simplex in twig of
Carya floridana, M. Deyrup (1 Y USNM); ..., L. L.
Lampert, Jr. & H. W. Weems, Jr., 8-IV-1978 (2 6
USNM ENT 0003891-2 FSCA); ..., 11-IV-1978 (1
2 USNM ENT 0003893 FSCA); ..., 18-11-1975,
H. W. Weems, Jr. (1 6 USNM ENT 0003894
FSCA);..., 17-IX-1979, T. A. Webber & H. W.
Weems, Jr. (1 Y USNM ENT 0003895 FSCA); F.
E. Lohrer, H. W. Weems, Jr., 11-15-IV-1980 (1 Y
USNM ENT 0003896 FSCA),..., 21-22-IV-1980 (1
S USNM ENT 0003897 FSCA); ..., 14-15-V-1980


(1 6 USNM ENT 0003898 FSCA); ..., 18-20-V-
1980 (1 6 USNM ENT 0003899 FSCA); ..., High-
lands Hammock State Park, 3-IV-1965, H.
Weems, Jr. (1 Y USNM ENT 0003877 FSCA);...,
27-III-1966 (1 Y USNM ENT 0003878 FSCA);
Collier County, SR94, 1.8 miles south of US 41,
25-II-1992, M. Deyrup & B. Ferster, reared from
nest of Pseudomyrmex ejectus in culm of Cladium
jamaicense (ABS); Liberty Co., Torreya State
Park, 14-V-1964, H. Weems, Jr. (1 Y USNM ENT
0003875 FSCA); ..., 30-IV-5-V-1973, C. R. Artaud
& H. Weems, Jr., Malaise trap, (1 6, 4 Y Y USNM
ENT 0003879-83 FSCA, USNM); Alachua Co.,
Gainesville, Beville Heights, L. A. Stange, Black-
light, 2-VII-1980 (2 Y Y USNM ENT 0003886-7
FSCA); ..., 1-VII-1980 (1 Y USNM ENT 0003888
FSCA); ..., 5-VII-1980 (1 6 USNM ENT 0003889
FSCA);..., 30-VII-1979 (1 Y USNM ENT
0003890 FSCA); Dade Co., Ross & Castello Ham-
mock, 30-III-1963, C. F. Zeiger (1 6 USNM ENT
0003884 FSCA);..., Fuch's Hammock, near
Homestead, 27-29-VII-1978, Terhune S. Dickel &
H. Weems, Jr. (1 Y USNM ENT 0003885 FSCA);
Chekika State Recreation Area, 10-XI-1982, FD
Fee (1 6 Fee). MEXICO. Morelos, 3 miles N
Alpuyeka, 3400', 5 June 1959, HE Evans, 14-V-







Florida Entomologist 86(2)


1959, Biol. Note 601 (1 6 USNM ENT 0003876
FSCA); Chiapas, 28 miles west Cintalpa, 9-IV-
1962, F. D. Parker (1 6 USNM). Another broken
S specimen is in the Canadian National Collec-
tion and is labelled "Letitia, Colombia? (or Flor-
ida)." According to Vockeroth (pers. comm.) this
specimen was found in a Malaise trap which had
been used both in the Florida Keys and Colombia.
Rhopalosyrphus ramulorum is similar toguen-
therii, but is smaller, narrower, not as robust, and
has much more extensive black pile on face,
scutum and anepisternum. The metasternal pile
is greatly reduced and closely appressed, so the
metasternum appears bare at low magnifications.
Although many specimens of R. ramulorum
have been collected, two of these are reared spec-
imens and provide most of our insights into the
natural history of the genus Rhopalosyrphus.
When alive, the reared adults, like many other
syrphids, bore a strong resemblance to stinging
Hymenoptera. The wasp-like features of elongate
antennae, narrow abdominal "petiole," pale bands
and spots, and dark wings are enhanced by the
wasp-like habit of holding the wings out from the
body. The wings are also partially folded, so that
they appear long and narrow. The general impres-
sion is of a very small individual of the twig-nest-
ing eumenid genus Zethus.
One specimen was found in a nest of the ant
Pseudomyrmex simplex in a small twig (hence the
species epithet "ramulorum," the epithet to be
treated as a noun in the genitive case) of Carya
floridana in long unburned Florida scrub habitat.
The adult emerged from its pupa the day after the
twig was opened. A second specimen was in a nest
of P ejectus in a culm of Cladiumjamaicense; this
adult also emerged a day after the nest was opened.
The nests of these two species of Pseudomyrmex
are kept clean and free of debris and fungi, and it
is probable that the fly larvae are not scavengers,
but predators feeding on ant brood. This would fit
well with the known larval habits of the closely re-
lated genus Microdon, (Duffield 1981; Garnett et
al. 1985). This is apparently the only known exam-
ple of a predatory inquiline attacking members of
the large neotropical ant genus Pseudomyrmex,
though there must be others, especially among the
Eucharitidae. Pseudomyrmex species are less sus-
ceptible to inquilines than most ants because the
nests are in plant cavities with access by only one
or a few small, well-guarded holes, and the nests
themselves are bare, with minimal edible detritus
and no hiding places for inquilines.
The holes used by Pseudomyrmex simplex and
P ejectus are much too small to permit the adult fly
to escape, and it seems probable that emergence
from the puparium is delayed until the nest has
been broken open. Since the term "strategy" has
been used extensively in discussing Microdon
(Duffield 1981), the problem of adult egress is a
major flaw in the strategy ofR. ramulorum. It may


be, however, that there are ways an ovipositing fe-
male can increase the likelihood that her offspring
will be freed. Small, dead, exposed twigs are more
likely to get broken off than larger twigs in the in-
terior of the tree crown. Culms on the edge of a tus-
sock of sedge or grass are more likely to get broken
off than culms in the interior. If there is some spe-
cial site selection by the female, this may explain
why only two pupae were found in a 10-year study
of Florida ants, a study that involved opening hun-
dreds of colonies of Pseudomyrmex.
This brings up the topic of apparent rarity of
Rhopalosyrphus species, especially in Florida. At
the Archbold Biological Station, two Townes traps
running continuously for 3 years captured only
one specimen. If the adults spend their time in
the tops of trees or in extensive open marshes,
this would explain why so few specimens appear
in Malaise traps, which are usually set-up in un-
derstory flyways. IfR. ramulorum is actually de-
pendent on chance events to release the adults,
actual populations would need to be quite high for
the sexes to meet, even if there were a mechanism
for adult aggregation, and even if some synchro-
nous emergence were provided by wind storms.
Whatever the actual abundance of Rhopalosyr-
phus species, the rarity of specimens in collec-
tions suggests that there could be additional
undiscovered species, especially in the neotropics,
where the fauna of arboreal ants is large.

ACKNOWLEDGMENTS
This was a cooperative project: the larval taxonomy
was done by Rotheray; nomenclature, literature review
and adult taxonomy by Thompson; and biology by Dey-
rup. After the manuscript was submitted for publication
and during the review process, it was discovered that
Weems was about to also describe the Florida species.
Hence, Weems was added to this manuscript as a co-au-
thor and his material was incorporated into it. We thank
Drs. Paul Arnaud, Jr., California Academy of Sciences,
San Francisco (CAS); George Byers, Snow Entomological
Museum, University of Kansas (UKaL), Lawrence; Peter
Cranston, University of California, Davis (UCD); Brian
Pitkin, Entomology, The Natural History Museum (for-
merly the British Museum (Natural History)), London
(BMNH); Mauro Daccordi, Museuo Regionale di Storia
Natural, Torino (UTOR); John Gelhaus and Douglas
Azuma, Academy of Natural Sciences, Philadelphia;
David A. Grimaldi, the American Museum of Natural His-
tory, New York (AMNH);Adriana Oliva, Museo Argentino
de Ciencias Naturales Bernardino Rivadavia, Buenos
Aires (MACN); Nelson Papavero, Museum of Zoology,
Universidade de Sao Paulo, Sao Paulo (MZSP); J. R. Voc-
keroth, the Canadian National Collection, Ottawa (CNC);
Manuel Zumbado, Instituto Nacional de Biodiversidad,
Santo Domingo (INBIO), for permission to study material
in their care. Suzanne Mangan assembled the plates, etc.
We also thank Chris T. Maier, Connecticut Agricul-
tural Experimental Station, New Haven; Neal Even-
huis, B. Bishop Museum, Honolulu (BPBM); Frank D.
Fee, State College (Fee); Gary Steck, Florida State Col-
lection of Arthropods, Gainesville (FSCA); Nat Vander-


June 2003







Weems et al.: Genus Rhopalosyrphus (Diptera: Syrphidae)


berg, James Pakaluk and Manya B. Stoetzel of the
Systematic Entomology Laboratory, USDA, Washing-
ton; and Marion Kotrba and Wayne N. Mathis of the
Smithsonian Institution (USNM), Washington, for their
critical review of the manuscript.

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BOHART, R. M., AND L. A. STANGE. 1965. A revision of
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sphere (Hymenoptera: Eumenidae). Univ. California
Publ. Ent. 40, 208 p.
BYERS, G. W., F. BLANK, W. J. HANSON, D. F. BENEWAY,
AND R. W. FREDRICHSON. 1962. Catalogue of the
types in the Snow Entomological Museum. Part III
(Diptera). Univ. Kansas Sci. Bull. 43: 131-181.
CAPELLE, K. J. 1956. The genus Rhopalosyrphus, with a
description of a new species from Arizona (Diptera,
Syrphidae). J. Kansas Ent. Soc. 29: 170-175.
COLE, F. R., AND E. I. SCHLINGER. 1969. The flies of
Western North America. xii + 693 p. Berkeley & Los
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DUFFIELD, R. M. 1981. Biology of Microdon fuscipennis
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GIGLIO-TOS, E. 1892a. Sui due generi Sirfidi Rhopalo-
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(Syrphidae). An. Soc. Cien. Argentina 32: 80-99; 118-
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poorly known British Hoverflies (Diptera: Syrphi-
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87: 127-149.
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mol. Inst. 3: 321-378.
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1976. Family Syrphidae. Catalog. Dipt. Amer. S.
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Syrphidae. Pp. 713-743. In J. F. McAlpine (ed.), Man-
ual of Nearctic Diptera. Res. Br., Agric. Canada,
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ruary 1892). In F. D. Godman and 0. Salvin (eds.),
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1965. Family Syrphidae. Pp. 557-625. In A. Stone,
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Florida Entomologist 86(2)


June 2003


EFFECT OF SCREENING METHODS ON EXPRESSION OF ROMAINE
LETTUCE RESISTANCE TO ADULT BANDED CUCUMBER BEETLE,
DIABROTICA BALTEATA (COLEOPTERA: CHRYSOMELIDAE)

JUAN HUANG1', GREGG S. NUESSLY'2, HEATHER J. MCAUSLANE' AND RUSSELL T. NAGATA2'3
'Department of Entomology & Nematology, University of Florida
P.O. Box 110620, Gainesville, FL 32611-0620

2Everglades Research and Education Center, University of Florida
3200 E. Palm Beach Rd., Belle Glade, FL 33430-4702

3Department of Horticultural Sciences, University of Florida
P.O. Box 110690, Gainesville, FL 32611-0620

4Current Address: Center for Medical, Agricultural and Veterinary Entomology
USDA, ARS, P.O. Box 14565, Gainesville, FL 32604

ABSTRACT

Resistance in lettuce, Lactuca sativa L., to feeding by adult banded cucumber beetle, Di-
abrotica balteata (LeConte), was evaluated using three screening methods: leaf disks, ex-
cised leaves and intact leaves attached to plants. Dual-choice and no-choice bioassays were
used to evaluate each method based on leaf area consumption. Methods of testing had a sig-
nificant effect on the level of feeding damage by D. balteata on two lettuce cultivars, Tall
Guzmaine and Valmaine. Valmaine expressed a significant degree of resistance to D. bal-
teata damage when intact leaf and excised leaf methods were used in dual-choice bioassays
between Tall Guzmaine and Valmaine, but the latter failed to show resistant characteristics
in no-choice tests when excised leaves were used. Furthermore, there was no significant dif-
ference in D. balteata feeding between Tall Guzmaine and Valmaine in the leaf disk tests.
Therefore, whole plants are the best method to evaluate lettuce cultivars for resistance to D.
balteata. Reduction or cessation of resistance characters in excised Valmaine whole leaves
and disks are discussed with references to potential changes in concentration of feeding
stimulants and deterrents and changes in latex pressure.

Key Words: Lactuca sativa, leaf disk, excised leaves, intact leaves

RESUME

La resistencia en la lechuga, Lactuca sativa L., hacia la alimentaci6n de adults del escara-
bajo rayado del pepino, Diabrotica balteata (LeConte), fue evaluada usando tres m6todos de
seleccionar: hojas cortadas en forma de disco, hojas cortadas, y hojas intactas pegadas a la
plant. Se utilizaron un bioensayo de una prueba de double opci6n y de una prueba sin opci6n
para evaluar cada m6todo basado en el consume del area de la hoja. Los m6todos de prueba
tienen un efecto significativo sobre el nivel de dano causado por la alimentaci6n de D. bal-
teata en dos variedades cultivadas de lechuga, la "Tall Guzmaine" y la "Valmaine". La varie-
dad Valmaine express un grado de resistencia significativo al dano de D. balteata cuando
fueron usados los m6todos de las hojas intactas y de las hojas cortadas en los bioensayos de
double opci6n entire las variedades Tall Guzmaine y Valmaine, pero la ultima no mostr6 ca-
racteristicas de resistencia en pruebas de una sola opci6n cuando se usaron hojas cortadas.
Ademas, no habia una diferencia significativa entire la Tall Guzmaine y la Valmaine en
cuanto de la alimentaci6n de D. balteata en pruebas de hojas cortadas en forma de discos. Por
lo tanto, las plants enteras son el mejor m6todo para evaluar las variedad de lechuga para
su resistencia al D. balteata. Se discuten la reducci6n o el paro de las caracteristicas resis-
tentes en hojas cortadas de la Valmaine en hojas enteras y en de hojas cortadas en forma de
discos con referencia a los cambios potenciales en la concentraci6n de estimulantes y disua-
civos de alimentaci6n y cambios en la presi6n de latex.


Host plant resistance is recognized as an effec- screening techniques are essential for accurately
tive component of IPM (Panda & Khush 1995), be- evaluating resistance levels. Excised leaves or
cause it has low impact on non-target organisms leaf disks are often used for evaluating plants for
and the environment and is usually compatible resistance to leaf feeding insects. For example,
with other control tactics. Reliable and efficient Sams et al. (1975) found that the use of excised







Huang et al.: Screening Methods for Lettuce Resistance


leaflets was an efficient method for evaluating re-
sistance to green peach aphid (Myzus persicae
(Sulzer)) in tuber-bearing Solanum germplasm.
Excised leaves were found to be quite reliable for
screening bean cultivars for Mexican bean beetle
(Epilachna varivestis Mulsant) feeding prefer-
ence as long as large mature leaves were used
(Raina et al. 1980). The leaf disk and whole leaf
techniques worked equally well for screening re-
sistance to two-spotted spider mites (Tetranychus
urticae Koch) on muskmelon leaves (Cucumis
melo L.) (East et al. 1992). However, some screen-
ing methods may have a significant impact on the
test outcome by altering natural resistance mech-
anisms. Risch (1985) found that screening meth-
ods affected the expression of resistance and the
order of feeding preference among corn (Zea mays
L.), bean (Phaseolus vulgaris L.) and squash (Cu-
curbita pepo L.) to specialist and generalist chry-
somelid beetles.
In this study, we evaluated the effect of three
screening methods (leaf disks, excised leaves and
intact leaves attached to plants) on the expres-
sion of resistance in romaine lettuce cultivars to
feeding by adult banded cucumber beetles (Dia-
brotica balteata). Leaf area consumed by beetles
was evaluated in dual-choice and no-choice bio-
assays to compare results for both leaf disks and
detached leaves against intact leaves.

MATERIALS AND METHODS

Plants and Insects

A previous study showed that Valmaine (Val)
was the most resistant cultivar and Tall Guz-
maine was the most susceptible one toD. balteata
feeding among four lettuce cultivars (Huang et al.
2002). Therefore, Valmaine and Tall Guzmaine
were used for this experiment. Seeds of each cul-
tivar were kept overnight in the laboratory in sep-
arate petri dishes lined with wet filter paper for
better germination. Germinated seeds were
planted in a transplant tray filled with a commer-
cial soil mix (MetroMix 220, Grace Sierra, Milpi-
tas, CA) and grown for 2 wk in a greenhouse with
natural light. Seedlings were transplanted to 10-
cm diameter plastic pots filled with MetroMix
220. Each plant was watered daily and fertilized
weekly with 10 ml of a 10 g/L solution of a soluble
fertilizer (Peters 20-20-20, N-P-K, W. R. Grace,
Fogelsville, PA) from transplantation until the
end of the experiment. Fully expanded leaves
from the seventh position (counting from the first
true leaf) were used in all the assays and selected
plants had seven to eight fully expanded leaves.
Adult D. balteata for feeding bioassays were
obtained from a laboratory colony originally col-
lected from the field in Belle Glade, Florida in
June 1996. Adults were fed lima bean leaves and
sweetpotato tubers and larvae were reared on


corn seedling roots as previously described
(Huang et al. 2002). Only unfed adults which had
emerged within 48 h were used for the assays. All
tests were conducted in a rearing room at 25 +
1C, 14:10 (L:D) h photoperiod.

Intact Leaves (Experiment 1)

Dual-choice tests were first conducted using
intact leaves of Tall Guzmaine and Valmaine
plant pairs. Feeding arenas made from plastic
petri dishes (8.9 cm diameter) were attached us-
ing hair clips to the upper leaf surface of a pair of
leaves from each cultivar. Two round holes (2.9
cm diameter) that were 65 mm apart provided ac-
cess to the leaves, and a 5.8 cm diameter hole that
was covered with gauze material at the top of the
dish provided ventilation. One pair (female plus
male) of beetles was placed in each feeding arena
and allowed to feed for 48 h. Each test was repli-
cated 11 times. The extent of feeding was evalu-
ated by scanning the leaf material (JADE 2,
Linotype-Hell, Taiwan) and importing the result-
ing images into an imaging program (ImagePC
beta version 1, Scion Corporation, Frederick,
Maryland) where leaf area consumed (mm2) was
determined. The difference in leaf area consump-
tion between cultivars was analyzed by paired t-
test using Proc MEANS (SAS Institute 1999).

Excised Versus Intact Leaves (Experiment 2)

Resistance to beetle feeding was next com-
pared between excised and intact leaves. The pet-
ioles of individual leaves excised at their base
were immediately immersed in separate beakers
filled with tap water and maintained therein for
the duration of testing. Dual-choice feeding are-
nas as described above were used to expose single
pairs of adults (female plus male) for 48 h to pairs
of either excised or intact Tall Guzmaine and Val-
maine leaves. Each test was replicated 17 times
in each bioassay. The difference in leaf area con-
sumption between cultivars was estimated and
analyzed as described above.
Since a significant difference was found in leaf
area consumption between the two cultivars in
dual-choice tests, the resistance level of excised
and intact leaves was further evaluated using no-
choice bioassays. Excised and intact leaves were
chosen and prepared as above. One pair of female
and male beetles was confined on individual ex-
cised or intact leaves of a single cultivar using a
modified feeding area with only a single 4 cm di-
ameter hole through which beetles accessed the
upper leaf surface. The adults were allowed to
feed for 48 h. This study was arranged as a ran-
domized complete block design with excised and
intact leaves from each cultivar in each block.
Each block was replicated 18 times. Leaf area
consumed was estimated as described above and







Florida Entomologist 86(2)


analyzed by Proc GLM (SAS Institute 1999).
Means with significant ANOVA were separated
using Tukey's HSD test with a significance level
of a = 0.05 (SAS Institute 1999).

Leaf Disks

Leaf disks for the bioassays were harvested
from freshly excised Valmaine or Tall Guzmaine
leaves. Two 380 mm2 disks were punched out from
non-midrib areas of each leaf using a No. 15 cork-
borer. The bioassay consisted of four leaf disks (two
from Tall Guzmaine and two from Valmaine)
placed an equal distance apart on two layers of
moistened paper towel inside a 8.9 cm diameter
plastic petri dish. A female and male beetle was
placed in each petri dish and allowed to feed for
48 h. Bioassays were replicated 15 times. Leaf area
consumed was estimated as described above, but
the remaining leaf area was subtracted from the
mean disk area of 10 disks not offered to beetles for
two days in order to account for shrinkage during
the assay. The difference in leaf area consumption
between cultivars was analyzed by paired t-test us-
ing Proc MEANS (SAS Institute 1999).

RESULTS

Valmaine was strongly resistant to beetle feed-
ing compared with susceptible Tall Guzmaine
when intact or excised leaves were presented in
dual-choice tests (experiments 1 and 2, Table 1).
However, beetles ate significantly more from ex-
cised leaves of both cultivars compared to intact
leaves (experiment 2, t = 3.88, df = 32, p < 0.0001).
Beetles on intact leaves consumed 12-fold less
from Valmaine compared to Tall Guzmaine, while
on excised leaves consumption was only 4-fold
less. When leaf disks were used as test materials,
there was no significant (p > 0.05) difference in
feeding between Tall Guzmaine and Valmaine.
Adults ate up to 23 times as much Valmaine on
leaf disks as on intact leaves (experiment 1).
Mean leaf area consumed on leaf disks and intact


leaves of Tall Guzmaine was similar, and both
were over 318 mm2.
A significant difference among treatments in
leaf area consumed per pair of adults in no-choice
tests was also found (F = 21.78; df = 3, 51; P =
0.0001) (Fig. 1). Beetle pairs consumed signifi-
cantly less (81%) Valmaine than Tall Guzmaine
on intact leaves, but consumed similar leaf areas
from excised leaves. Feeding was significantly in-
creased on excised leaves compared to intact
leaves, irrespective of cultivar. Intact Valmaine
leaves were the least damaged by adult D. bal-
teata with mean leaf area consumption of 66.3
mm2, which was only 12% of that on excised Val-
maine leaves.

DISCUSSION

Although excised leaves or leaf disks are often
used for evaluating plants for resistance to leaf
feeding insects, biochemical and physiological
changes in such plant tissue may affect the feed-
ing of the test insects (Raina et al. 1980; Risch
1985; van Emden & Bashford 1976). In our case,
Valmaine expressed a higher degree of resistance
to adult feeding on intact leaves than on excised
leaves in choice tests. Moreover, Valmaine failed
to show significant resistance to D. balteata feed-
ing in either the leaf disk choice test (Table 1) or
the excised leaf no-choice test (Fig. 1). Therefore,
methods of testing for resistance had a significant
effect on relative leaf consumption of Tall Guz-
maine and Valmaine by adult D. balteata. The in-
tact leaf method was the most suitable and
reliable of the tested methods used to evaluate
lettuce cultivars for resistance to D. balteata.
Risch (1985) also reported that testing method
(i.e., whole plants, excised leaves, and leaf disks)
had a significant effect on preferences of chry-
somelid beetles, including D. balteata, when corn,
bean and squash were tested. In his tests, differ-
ences in resistance ratios between leaf disk and
whole plant tests were much greater than those
between excised leaves and whole plant tests.


TABLE 1. MEAN SEM LETTUCE LEAF AREA CONSUMED PER PAIR (FEMALE PLUS MALE) OF ADULT D. BALTEATA IN 48
H WHEN PRESENTED A CHOICE BETWEEN TWO CULTIVARS USING DIFFERENT SCREENING METHODS

Methods Cultivar" N Leaf area (mm2) Pr> t I

Intact leaf (expt 1) TG 11 318.1 29.3 0.0001
Val 11 14.4 2.1
Intact leaf (expt 2) TG 17 350.5 28.7 0.0001
Val 17 28.8 4.8
Excised leaf TG 17 532.2 57.8 0.0004
Val 17 125.9 53.8
Leaf disk TG 15 382.8 36.3 0.0941
Val 15 334.6 37.4

"TG = Tall Guzmaine, Val = Valmaine.


June 2003







Huang et al.: Screening Methods for Lettuce Resistance


E-TG E-Val I-TG I-Val
Leaf treatment
Fig. 1. Mean leaf area consumed per pair of adult D.
balteata within 48 h during no-choice test using excised
(E) and intact (I) leaves from susceptible Tall Guzmaine
(E-TG, I-TG) and resistant Valmaine (E-Val, I-Val). Bars
topped with the same letter are not significantly differ-
ent by Tukey's HSD test at the 0.05 level. Vertical lines
indicate + 1 SEM.


Furthermore, the feeding preferences of the two
specialist species, Acalymona thiemei (Baly) and
Ceratoma ruficornis (Olivier) were less affected
by test method than were the more generalist
species, D. balteata and D. adelpha (Harold). In
another example of different results between in-
tact and excised leaf tissue, lettuce cultivars nor-
mally resistant to the lettuce aphid, Nasonovia
ribisnigri (Mosley), lost their resistance when leaf
fragments were given in a leaf disk test
(Schoonhoven et al. 1998).
Leaf disk size is another variable that could af-
fect the outcome of insect feeding preferences.
The ratio of cut edge to overall leaf disk surface
area can influence the chance of encountering in-
ternal attractants and stimulants (Jones & Cole-
man 1988).
The fact that leaves of both Valmaine and Tall
Guzmaine were consumed much more when ex-
cised from the plants suggests a change in the
chemical profile inside leaves or a reduced capac-
ity to deliver deterrents effectively after cutting.
Latex in some laticiferous plants has been re-
ported as a natural defense system against cer-
tain herbivores. In many laticiferous plants,
including L. sativa, latex is stored under pressure
within laticifers, which results in rapid release of
latex upon cutting (Fahn 1979; Data et al. 1996;
Dussourd 1995). The secretions often contain sec-
ondary metabolites known to be toxic or deterrent
to animals (Farrell et al. 1991). Data et al. (1996)
found that young vine material of sweet potato
produced more latex and had fewer sweetpotato
weevils, Cylas formicarius (F.), than older and
more mature portions of the vine. Several insects
have been observed immobilized in exudates,
such as caterpillars (Dussourd 1993), ants (Dillon


et al. 1983), aphids and whiteflies (Dussourd
1995). Many different organic compounds have
been identified in latex of Lactuca sp., including
organic acids, phenolics and a triterpene alcohol
(Crosby 1963; Gonzalez 1977; Cole 1984). Like
many plant secondary compounds these organic
compounds may act as deterrents or toxins to po-
tential herbivores. Both Valmaine and Tall Guz-
maine produce latex upon cutting, but latex flows
from Valmaine longer after cutting than from Tall
Guzmaine (Huang et al. 2003). Beetles may have
eaten more on excised than on intact leaves be-
cause latex flow from injured tissue on excised
leaves placed in water may be decreased and di-
luted compared to intact leaves. No latex emis-
sion was observed from leaf disks which may be
the major reason why no feeding difference was
found between Valmaine and Tall Guzmaine.
However, Tall Guzmaine was preferred over Val-
maine by beetles when excised leaves were used
in choice tests and when intact leaves were used
in both choice and no-choice tests. Therefore, the
observed feeding preferences may be due to differ-
ences in the composition or concentration of sec-
ondary compounds within the latex between Tall
Guzmaine and Valmaine.

ACKNOWLEDGMENTS

The authors are grateful to D. Boyd (Department of
Entomology & Nematology, University of Florida,
Gainesville, Florida) for technical assistance. We also
thank Ron Cherry (Everglades Research and Education
Center, University of Florida, Belle Glade, FL) and
Cameron Lait (Center for Medical, Agricultural and
Veterinary Entomology, ARS-USDA, Gainesville, FL)
for reviewing this manuscript. The Wedgworth Family
of Florida Vegetable and Sugar Producers provided fi-
nancial support for this research. This research was
partially supported by the Florida Agricultural Experi-
ment Station, and approved for publication as Journal
Series No. R-08859.

REFERENCES CITED

CROSBY, D. G. 1963. The organic constituents of food. 1.
Lettuce. J. Food. Science 28: 347-355.
COLE, R. A. 1984. Phenolic acids associated with the re-
sistance of lettuce cultivars to the lettuce root aphid.
Ann. Appl. Biol. 105: 129-145.
DATA, E. S., S. F. NOTTINGHAM, AND S. J. KAYS. 1996.
Effect of sweetpotato latex on sweetpotato weevil
(Coleoptera: Curculionidae) feeding and oviposition.
J. Econ. Entomol. 89: 544-549.
DILLON, P. M., S. LOWRIE, AND D. MICKEY. 1983. Dis-
arming the "Evil woman": petiole constriction by a
sphingid larvae circumvents mechanical defenses of
its host plant, Cnidoscolus urens (Euphorbiaceae).
Biotropica 15: 112-116.
DUSSOURD, D. E. 1993. Foraging with fitness: caterpil-
lar adaptations for circumventing plant defenses,
pp. 92-131. In N. E. Stamp and R. M. Casey [eds.],
Caterpillars: Ecological and Evolutionary Con-
straints on Foraging. Chapman and Hall, New York.







Florida Entomologist 86(2)


DUSSOURD, D. E. 1995. Entrapment of aphids and
whiteflies in lettuce latex. Ann. Entomol. Soc. Am.
88: 163-172.
EAST, D. A., J. V. EDELSON, E. L. COX, AND M. K. HAR-
RIS. 1992. Evaluation of screening methods and search
for resistance in muskmelon, Cucumis melo L., to the
two-spotted spider mite, Tetranychus urticae Koch.
Crop Protection 11: 39-44.
FAHN, A. 1979. Secretory Tissues in Plants. Academic
Press, London.
FARRELL, B. D., D. E. DUSSOURD, AND C. MITTER. 1991.
Escalation of plant defense: do latex/resin canals
spur plant diversification? Am. Nat. 138: 891-900.
GONZALEZ, A. G. 1977. Lactuceae-Chemical review, pp
1081-1095. In V. H. Heywood and J. B. Harborne
[eds.], The Biology and Chemistry of the Composi-
tae. Academic Press, New York.
HUANG, J., G. S. NUESSLY, H. J. MCAUSLANE, AND F.
SLANSKY. 2002. Resistance to adult banded cucum-
ber beetle, Diabrotica balteata (Coleoptera: Chry-
somelidae), in romaine lettuce. J. Econ. Entomol. 95:
849-855.
HUANG, J., H. J. MCAUSLANE, AND G. S. NUESSLY. 2003.
Resistance in lettuce to Diabrotica balteata (Co-
leoptera: Chrysomelidae): the role of latex and in-
ducible defense. Environ. Entomol. 32: 9-16.


JONES, G. C., AND J. S. COLEMAN. 1988. Leaf disc size
and insect feeding preference: implications for as-
says and studies on induction of plant defense. Ento-
mol. Exp. Appl. 47: 167-172.
PANDA, N., AND G. S. KHUSH. 1995. Host Plant Resis-
tance to Insects. CAB International, Wallingford,
Oxon, UK.
RAINA, A. K., P. S. BENEPAL, AND A. Q. SHEIKH. 1980.
Effects of excised and intact leaf methods, leaf size,
and plant age on Mexican bean beetle feeding. Ento-
mol. Exp. Appl. 27: 303-306.
RISCH, S. J. 1985. Effects of induced chemical changes
on interpretation of feeding preference tests. Ento-
mol. Exp. Appl. 39: 81-84.
SAMS, D. W., F. I. LAUER, AND E. B. RADCLIFFE. 1975.
Excised leaflet test for evaluating resistance to
green peach aphid in tuber-bearing Solanum germ-
plasm. J. Econ. Entomol. 68: 607-609.
SCHOONHOVEN, L. M., T. JERMY, AND J. J. A. VAN LOON.
1998. Chapter 3: Plant chemistry: endless variety.
Insect-Plant Biology. Chapman & Hall, London, UK.
SAS INSTITUTE. 1999. Guide for Personal Computers,
Version 6, SAS Institute, Cary, NC.
VAN EMDEN, H. F., AND M. A. BASHFORD. 1976. The effect
of leaf excision on the performance of Myzus persicae
and Brevicoryne brassicae in relation to the nutrient
treatment of the plants. Physiol. Entomol. 1: 67-71.


June 2003







Adjei et al: Mole Cricket Infestation on Pasture


SURVEY OF PEST MOLE CRICKETS (ORTHOPTERA: GRYLLOTALPIDAE)
ACTIVITY ON PASTURE IN SOUTH-CENTRAL FLORIDA


M. B. ADJEI', J. H. FRANK2, AND C. S. GARDNER3
'Range Cattle Research and Education Center, University of Florida, Ona, FL 33865-9706

2Department of Entomology and Nematology, University of Florida, Gainesville, FL 32611-0630

3Center for Cooperative Agricultural Programs, Florida A&M University, Tallahassee, FL 32307-4100


ABSTRACT

Histories of pest mole cricket activity (Scapteriscus spp. Scudder) (Orthoptera: Gryllotalpi-
dae) on bahiagrass (Paspalum notatum Fluegge) pastures were needed to provide baseline
data for evaluating on-going biological control with Steinernema scapterisci (Nguyen and
Smart) nematodes. Seven ~4-ha pastures were selected from five county sites for the survey.
These consisted of one mole cricket-infested bahiagrass pasture each from two ranches in
Polk county and from one ranch each in Manatee and Pasco counties. The rest were two ren-
ovated and uninfested pastures located at the Range Cattle Research and Education Center,
Ona, in Hardee county and a third in Desoto county. Six linear pitfall traps (each 12.2 m to-
tal) were installed on equal subdivisions (0.67 ha) of each of the seven pastures in July 1997
and labeled 1 to 6 at each site according to a visually-determined decreasing slope of terrain.
Traps were cleaned weekly from the time of installation through December 1999, and the to-
tal weekly-captures per trap of tawny, S. vicinus (Scudder) and southern, S. borellii, (Giglio-
Tos) mole crickets were recorded along with weekly rainfall for each site. The mean, weekly
mole cricket capture on heavily-infested bahiagrass pastures increased exponentially over
time beginning with the early summer rains. Mean weekly-count on these pastures peaked
at 20-60 juveniles per trap, depending on site, in June-July and then declined sharply
through September and October as mole crickets matured. The annual mean weekly-cap-
ture on heavily-infested pastures was 10 to 12 juveniles per trap. There was very little sur-
face activity by overwintering adult mole crickets during December and January. Mean
weekly-capture on uninfested new bahiagrass pasture was erratic and usually less than 2
juveniles per trap. The data suggest that peak weekly mole cricket pitfall trap captures be-
tween June and August in excess of 20 juveniles per trap and a total seasonal capture in ex-
cess of 43 m1 of trap were indicative of a serious infestation problem.

Key Words: Scapteriscus vicinus, Scapteriscus borellii, Paspalum notatum, Linear pitfall
trap, Pasture grasses

RESUME

Se necesita la historic de la actividad de plagas de los grillotopos (Scapteriscus spp.) (Or-
thoptera: Gryllotalpidae) en pastizales de grama de bahia (Paspalum notatum Fluegge) para
proveer los datos iniciales para evaluar el control biol6gico en march con los nematodos,
Steinernema scapterisci (Nguyen and Smart). Siete pastizales de ~4-ha fueron seleccionadas
de sitios en cinco condados para el sondeo. Estas consistian en una pastizal de grama de
bahia infestada con grillotopos cada una de dos haciendas el condado de Polk y de una ha-
cienda en cada uno de los condados de Manatee y de Pasco. Los restos fueron dos pastizales
renovados y no infestados situados en el Range Cattle Research and Education Center, Ona
(Centro de Investigaci6n y Educaci6n de Ganado de Rangos) en el condado de Hardee y una
tercera en el condado de De Soto. Seis trampas de "pitfall" (trampas donde la presa cae en
un hoyo en el suelo) en linea (cada 12.2 m total) fueron instaladas en subdivisions iguales
(de 0.67 ha) en cada una de los siete pastizales enjulio de 1997 y marcadas 1 a 6 en cada sitio
segun la inclinaci6n de terreno visualmente determinada. Se limpiaron las trampas sema-
nalmente desde el tiempo de instalaci6n hasta el diciembre de 1999, y se registrar6n el nu-
mero total del grillotopo aleonado, S. vicinus (Scudder) y del grillotopo sureio, S. borellii,
(Giglio-Tos) capturados por trampa por cada semanajunto con la cantidad de lluvia que cay6
en cada sitio por semana. El promedio del numero de los grillotopos capturados cada semana
en pastizales de grama de bahia infestadas altamente aument6 exponencialmente sobre el
tiempo empezando con las lluvias en el principio de verano. El promedio del numero captu-
rado semanalmente en estas pasturas lleg6 a lo mas alta de 20-60 juveniles por tampa, de-
pendiendo del sitio, en junio-julio y luego baj6 agudamente en septiembre y octubre cuando
los grillotopos maduraron. El promedio annual del numero capturado en pasturas altamente
infestadas fu6 10 a 12 juveniles por trampa. Habia muy poca actividad sobre la superficie por







Florida Entomologist 86(2)


adults de grillotopos invernando durante diciembre y enero. El promedio del numero cap-
turado semanalmente en pastizales de grama de bahia no infestadas fu6 6rratico y usual-
mente menos de 2 juveniles por trampa. Estos datos sugieren que el numero mas alto de
grillotopos capturados semanalmente en las trampas "pitfall" entire junio y agosto en exceso
de 20 juveniles por trampa y una cantidad en exceso de 43 m' por trampa por toda la esta-
ci6n fueron indicativos de un problema de infestaci6n seria.


Adventive mole crickets Scapteriscus spp.
(Scudder) cause serious damage to pasture, lawn
and crops in Florida. It is estimated from a survey
(South Florida Beef and Forage Extension Pro-
gram 1999) that nearly $45 million-revenue is lost
annually to cattle producers in south central Flor-
ida as a result of reduction in hay and forage pro-
duction as a result of mole cricket damage and an
extra $10 millon/year spent on pasture renovation.
All three pest mole crickets found in Florida:
tawny, S. vicinus (Scudder), southern, S. borellii
(Giglio-Tos) and short-winged, S. abbreviatus
(Scudder); were inadvertently introduced from
South America in ship's ballast into ports of Geor-
gia, South Carolina, Alabama and Florida in the
early 1900s (Walker & Nickle 1981). From these
points of arrival, the tawny and southern mole
crickets spread westwards and southwards, and
by 1960 had covered and become serious pests
throughout Florida (Walker & Nickle 1981). Due
to its inability to fly, the short-winged is largely
restricted to point of introduction in coastal areas.
Mole crickets spend nearly all their year-long
life cycle underground (Walker 1984), which makes
population sampling very difficult. Eggs are laid in
clutches in underground chambers. Nymphs tun-
nel to the surface and feed in the upper soil and lit-
ter. Juveniles and adults make and occupy
extensive gallery and tunnel systems. In south and
central Florida, tawny mole cricket has one gener-
ation per year, but the southern mole cricket has
two generations annually (Walker 1984). It is only
during their peak mating flights in early-spring
and to a lesser extent in the fall that pest mole
crickets are conspicuous to the casual observer.
Mole cricket damage to pasture and turfgrass
is principally due to feeding by tawny mole crick-
ets (Walker & Dong 1982; Hudson 1984). At night,
mole crickets usually leave their tunnels to bite
off stems and leaves, which are dragged into their
burrows to be eaten. Roots are eaten at any time
from within tunnels. Mechanical damage to
plants is caused by the tunneling activity of mole
crickets and this is the principal detrimental ef-
fect of southern mole crickets on pasture. Damage
in pasture first appears in yellow patches which
die and turn brown. In areas of high mole cricket
population density, the surface 20 to 25-cm soil
layer is honeycombed with numerous galleries
and the ground feels spongy when stepped on.
Heavily-damaged pasture has virtually no root
system and is easily pulled from the soil by cattle
or foot traffic in a pasture.


The most direct way to evaluate classical bio-
control agents is to compare the population levels
of target species before and after establishment of
natural enemies. Three basic sampling tech-
niques for monitoring mole crickets have been de-
scribed (Hudson 1988) although none has
overcome the basic obstacle of showing good cor-
relation with true population density. These sam-
pling techniques are sound traps for flying adults
(Walker 1982), linear pitfall traps for monitoring
the activity of immature mole crickets (Lawrence
1982) and soil flushing for both juveniles and
adults (Short & Koehler 1979). More than 20 yr
data have been collected around suburban
Gainesville and Bradenton on adult pest mole
crickets' flight with sound traps (Walker et al.
1995). However, adult mole crickets can fly over
long distances, and sound trap captures may not
reflect the level of mole cricket infestation on spe-
cific ranches. Hudson (1989) developed an equa-
tion for soap flushing from repeated sampling
that predicted mole cricket population estimates
within 25% of the true population, however, soap
detergent is lethal to S. scapterisci nematodes
(Grover Smart, Jr., pers. comm.). Lawrence (1983)
used linear pitfall traps to monitor the activity of
juvenile mole crickets on pasture. Data on sea-
sonal activity of mole crickets in Florida pastures
are lacking.
This study was designed to use permanently
set pitfall traps to monitor the long-term seasonal
abundance of immature mole crickets on pasture
in relation to rainfall and pasture damage. Abun-
dance histories developed on specific ranches was
to be used as baseline information for future eval-
uation of biocontrol with nematodes.

MATERIALS AND METHODS

The survey was conducted on five ranches in
south-central Florida and the Range Cattle Re-
search and education Center, Univ. of Florida, fol-
lowing a severe mole cricket outbreak on pastures
in central Florida in 1996. These sites repre-
sented two extremes of initial mole cricket infes-
tation. In July 1997, six pitfall traps were
installed on one 4-ha bahiagrass pasture at each
of two ranches (A. D. Combee and George Clark)
which had heavy mole cricket damage in the
Green Swamp area of Polk county and one similar
bahiagrass pasture each in Manatee (Harlee
ranch) and Pasco (Mary Nutt ranch) counties. Six
traps were also installed on two 4-ha, renovated


June 2003







Adjei et al: Mole Cricket Infestation on Pasture


pastures which appeared to be lightly infested
with mole crickets at the Range Cattle REC, Ona,
in Hardee county and another similar pasture in
Desoto county.
The 4-ha bahiagrass pasture at each site was
divided into six equal blocks (reps) each installed
with one trap. Traps at each site were labeled 1 to
6 in decreasing slope of terrain and were cleaned
weekly from July 1997 through December 1999.
At cleaning, weekly-captured tawny and southern
mole crickets in each trap were counted together.
Body decomposition of nymphs during summer
rainfall made differentiation between remaining
tiny heads of tawny and southern mole crickets
very difficult and unreliable. However, the heads
were resistant to decomposition and used as
markers for the weekly counts whenever body de-
composition occurred. Development of immature
mole crickets was monitored at one site in the
Green Swamp area by measuring the length of
the pronotum of 20 trapped mole crickets monthly
from June 1998 to April 1999. Amount of weekly
rainfall was recorded for the two Green Swamp
sites in Polk county, the Manatee, and the Pasco
sites. Bahiagrass pasture on each site was rated
as to percentage green, yellow, dead or brown,
bare ground, and weed cover, every spring using a
subdivided m2 quadrat. The quadrat had 100 divi-
sions, each representing a percentage unit, and
was thrown randomly to twenty-four locations (4
on each subdivision) on the 4-ha pasture.
Data on weekly trapped mole crickets were
subjected to statistical analysis of variance (SAS
1999) with site as main plot and year and week as
split and split-split plots in time, respectively, and
traps as replicated blocks. Due to significant site
by week (P < 0.0008) and year by week (P <
0.0076) interactions, weekly abundances of
trapped mole crickets were fitted to week of the
year, separately for each site, using SigmaPlot re-
gression software (SPSS, Inc. 1997) that maxi-
mized regression R2and minimized standard error
(SE). Ratings of pasture condition were analyzed


as a split plot with site as main plot, year as split
plot in time and pasture subdivision as replicates.

RESULTS

The analysis of variance of weekly trapped im-
mature mole cricket numbers on bahiagrass pas-
tures from July 1997 to June 1999 is shown in
Table 1. There was a distinct migration of pasture
mole crickets from low-lying, inundated soils to
drained soils at the peak of summer rains and
vice versa during the dry spring period. This re-
sulted in large trap location and trap location x
week effect. As expected, trapped mole cricket
numbers also varied depending on site of bahia-
grass pasture, year and week of the year. The site
x week, year x week interactions were significant
and the site x year x week interaction approached
significance (P < 0.10).
The 3-yr mean weekly trapped mole crickets
ranged from 10.1 to 12.4/trap for the heavily mole
cricket-infested pastures in the Green Swamp in
Polk county, and the sites in Pasco and Manatee
counties (Table 2). From 0.7 to 1.7 mole crickets/
trap were found in the lightly-infested bahiagrass
pastures in Desoto and Hardee counties.
Level of mole cricket infestation was highly
correlated with pasture damage (r = 0.89). Sites
where seasonal mean weekly mole cricket trap
captures >10, such as Combee, Clark, Nutt, and
Harlee ranches, had severe pasture damage (yel-
low + dead/weeds) ranging from 49 to 72% (Table
2). Conversely, the lightly-infested mole cricket
sites including pastures at the Range Cattle REC
and Desoto county stayed green in spring and
showed little sward damage.
Mean weekly capture of nymph and adult pest
mole crickets on damaged bahiagrass pastures
within the year was best described by an expo-
nential curve (Gaussian 3 Parameters) (Figs. 1-4).
Most mole cricket eggs normally hatch in May
and June (Walker 1984), and the number of
trapped nymphs on pasture reached a peak after


TABLE 1. SOURCES OF VARIATION, DEGREES OF FREEDOM, F-TEST VALUE, AND THE PROBABILITY OF A GREATER F-VALUE
FOR WEEKLY DISTRIBUTION OF PEST MOLE CRICKETS ON BAHIAGRASS PASTURES IN SOUTH-CENTRAL FLORIDA.

Variable df F Pr > F

Trap 5 6.86 0.0001
Site 6 3.08 0.0255
Site x Trap (Error a) 30
Year 2 7.44 0.0001
Site xYear 12 0.64 0.8979
Site x Year x Trap (Error b) 60
Week 51 3.72 0.0001
Site x Week 306 1.84 0.0008
Year x Week 102 1.72 0.0075
Site x Year x Week 612 0.86 0.0998
MSE (Error c) 4105








Florida Entomologist 86(2)


June 2003


TABLE 2. THE EFFECT OF BAHIAGRASS PASTURE SITE ON 3-YR MEAN WEEKLY PEST MOLE CRICKET COUNT/TRAP AND
CORRESPONDING PASTURE DAMAGE.


Weekly Damage estimate
mole cricket
County Ranch count/trap Green Yellow Dead/weeds

No. ------------------- % cover ------------------

Polk A.D. Combee 10.1 45 4 51
Polk George Clark 12.4 50 12 38
Manatee Harlee Farm 11.2 28 10 62
Pasco Mary Nutt 11.0 51 37 12
Hardee RCREC-71A1 0.7 98 1.5 0.5
Hardee RCREC-871 1.7 85 5 10
Desoto Steven Houk 1.6 97 2 1.0

LSD P = 0.05 5.7 12 8 10

Range Cattle Research and Education Center, pastures 71A and 87.


the first major summer rainfall in June or July.
For the 2.5 yr study, week 30 (Xo) had the highest
mean peak weekly mole cricket trap count ('a'
value) of 23 on Combee ranch (Fig. 1), week 25 of


1997
v 1998
1999
Regression

Y= aep[-0.5((X-Xo)lb)2]
a =23.1
b=8.1
X = 29.67
R2 0.72
P 0.0001
SE = 5.24


0 5 10 15 20 25 30 35 40 45 50 55
Feb Mar Apr May June July Sep Oct Nov Dec
6

5 \v 1998



4 .







0 5 10 15 20 25 30 35 40 45 50 55
Feb Mar Apr May June July Sep Oct Nov Dec
Week of year

Fig. 1. Seasonal distribution (1997-1999) of mean
weekly pitfall trap captures of immature Scapteriscus
mole crickets (a) in relation to weekly rainfall pattern
(b) on bahiagrass pasture at A.D. Combee Ranch in Polk
county.


Juvenile & Adult


26 peak trap count on Clark ranch (Fig. 2), and
week 25 of 40 peak trap count on Nutt ranch
(Fig. 3). On all these severely-damaged fields,
there was at least one weekly-spike episode that


(a)


"1


1997
Rers19
(a) 80~ 1999
S- Regression
40
y = a.exp[-0.5((X-X0)ll
S35 a 25.5
b=8.3
30 X= 25.42
SR2 =0.66
ts. 25 SE=6.8
20 P<0.0001
20

S15

10
5
0 LSE*:


0 5 10 15 20 25 30 35 40 45 50 55
Feb Mar Apr May June July Sep Oct Nov Dec
6
-- 1997 (b)
5 1, 1998

5 4

.! 3-







0 5 10 15 20 25 30 35 40 45 50 55
Feb Mar Apr May June July Sep Oct Nov Dec
Week of year

Fig. 2. Seasonal distribution (1997-1999) of mean
weekly pitfall trap captures of immature Scapteriscus
mole crickets (a) in relation to weekly rainfall pattern
(b) on bahiagrass pasture at George Clark Ranch in
Polk county.








Adjei et al: Mole Cricket Infestation on Pasture


100
90 1'99
S1998
80 1999

A,


40-

O 30-
E
S20

10

0-


Y = aexp[-0.5((X-X0)/b)21
a = 39.5
b = 3.71
X = 24.5
R2= 0.63
SE=9.16
P < 0.0001


*/v


S


0 5 10 15 20 25 30 35 40 45 50 55
Feb Mar Apr May June July Sep Oct Nov Dec
10
9 19~7 (b)
1998
c0 8 -* 199
7 V
6
54


2 %



0 5 10 15 20 25 30 35 40 45 50 55
Feb Mar Apr May June July Sep Oct Nov Dec
Week of year

Fig. 3. Seasonal distribution (1997-1999) of mean
weekly pitfall trap captures of immature Scapteriscus
mole crickets (a) in relation to weekly rainfall pattern
(b) on bahiagrass pasture at Mary Nutt Ranch in Pasco
county.




exceeded 50 nymphs/trap during the 2.5-yr moni-
toring. On Harlee ranch, also a heavily infested
pasture, we experienced variable patterns of
weekly nymph abundance over the years and a
single exponential curve did not provide a good fit
across years (Fig. 4). A weekly peak >100 nymphs/
trap occurred in early July of 1997. A bimodal
peak (June and September) averaging 45
nymphs/trap was observed in 1998, and a weekly
peak of only 20 nymphs/trap in 1999 (Fig. 4).
Weekly spikes of trapped nymphs on heavily in-
fested pastures usually coincided with rainfall
that saturated the soil early in the summer, forc-
ing the nymphs to relocate within a pasture. Sub-
sequent heavy rainfall within the season was not
generally accompanied by similar hikes in nymph
activity because they had already dispersed to
higher grounds that were less likely to become
saturated. There was also a lack of fit of trapped
mole cricket data to week of sampling on the
lightly-infested pastures in Hardee sites (Fig. 5)
and mole cricket infestation on DeSoto site was
low ('1.6 nymphs/trap/week) but uniform
throughout the year (plot not shown).


180
160 997
140 v 1998
80, 1999


60o

S50-

40-
E
I 30
Sn


* u
9


NVV m
00"'sj -,


(a)


meo


'7 =. & u


0 5 10 15 20 25 30 R5 40 .45 50 55
Feb Mar Apr May June July Sep Oct ov Dec
12
11 -- 199 (b)




6-






0 5 10 15 20 25 30 35 40 45 50 55
Feb Mar Apr May June July Sep Oct Nov Dec
Week of year

Fig. 4. Seasonal distribution (1997-1999) of mean
weekly pitfall trap captures of immature Scapteriscus
mole crickets (a) in relation to weekly rainfall pattern
(b) on bahiagrass pasture at Harlee Ranch in Manatee
county. Year x week interaction P < 0.05.



The mean pronotal length of 20 pitfall trap-
captured mole crickets was 3.1 0.3 mm in June;
4.8 0.2 mm in July; 5.2 0.3 in August; 5.8 0.4
mm in September; 6.9 0.5 mm in October; 7.3 +
7 in November; and 11.6 1.0 in April. When dis-
cernible, the ratio of numbers of tawny:southern
mole crickets on pasture was approximately 3:1
but there was no difference in pronotal length be-
tween tawny and southern mole crickets. The in-
crease in pronotal length and standard deviation
of pronotal length over time was due to an in-
creasing adult component in the population later
in the season.
DISCUSSION

The extent of damage observed on pastures was
dependent on the level of pest mole cricket infesta-
tion. A seasonal mean-weekly mole cricket nymph
trap capture >10 was associated with more than
50% pasture damage. A mean-weekly trap capture
of 10 mole crickets amounts to 520 mole crickets
per trap yearly. A mean-weekly trap capture of 1.7
mole crickets or 88 mole crickets per year was as-
sociated with moderate pasture damage (15%).







Florida Entomologist 86(2)


60 ----
50 1997 Pasture 71A
so 5

. 25

E
10


o .. .f.. ."
0 5 10 15 20 25 30 35 40 45 50 55


* 1997
S1998
* 1999


Pasture 87


0 6


U0* I
gM 0 0V *W 0
m *
0 7 .V . . V * v W. . . V V 7 7 7
0 5 10 15 20 25 30 35 40 45 50 55
Feb Mar Apr May June July Sep Oct Nov Dec

Fig. 5. Seasonal distribution (1997-1999) of mean
weekly pitfall trap captures of immature Scapteriscus
mole crickets on bahiagrass for Pastures 71A and 87 at
the Range Cattle REC, Ona, in Hardee county.


Each trap had a total length of 12.2 m which sug-
gests that the seasonal damage threshold falls
somewhere between 7 and 43 mole crickets m-1 on
bahiagrass pasture. In a 1-year study (1 May 1982
to 30 April 1983), Lawrence (1983) installed three
linear pitfall traps (5.5 m total length) on bahia-
grass turf in Palm Beach within 4.6 m of a sound
trap station (Walker 1982) and captured 609 im-
mature mole crickets of which 89 were S. borellii,
139 were S. vicinus and 381 were S. abbreviatus.
Within the same 12 month period, 13,496 adult S.
borellii and 197 S. vicinus were captured in the
sound station. It is known that many crickets at-
tracted to sound callers miss the sound trap, there-
fore, Lawrence's (1983) total capture of 111
Scapteriscus nymphs m-' of pitfall trap could have
been inflated by egg deposition in the vicinity from
adult females which missed the sound traps. We
did not encounter any S. abbreviatus in our study
on ranches in central Florida probably because
that species was restricted to coastal areas where
it was first introduced because it cannot fly. Simi-
lar to our data, 85% of Lawrence's (1983) mole
cricket nymphs were captured between June and
August, and the nymphs increased in pronotal
length from 2 to 9 mm between June and April.
Mole cricket buildup on Hardee and DeSoto
sites following pasture renovation was slow. Re-
sults seem to agree with the general producers'
perception that it takes >3 yr after a successful
pasture renovation before mole cricket popula-
tions in bahiagrass fields build up to damaging
thresholds.


Decline of mole cricket pitfall captures in late
summer and fall has been attributed to marked
differences between nymph and adult mole
cricket behavior (Hudson 1989) and also to the ac-
tion by generalist native natural enemies (Hud-
son et al. 1988). Available evidence (Hudson 1985;
Hudson & Shaw 1987) suggests that nymphs are
largely nomadic, with no "home" burrow, and so
are more likely to seek escape on the surface
whereas adults often have an established and
deep burrow system into which they retreat
rather than coming to the surface. Adult females
in the fall tend to dig a permanent burrow system
and stay there, with little apparent foraging.
Males are more active on the surface but not as
active as nymphs and they also dig extensive bur-
row systems (Nickerson et al. 1979).
Both the natural decline in surface activity
and the action by generalist natural enemies
have been inadequate to prevent heavy damage
in pastures and turf during fall, for which reason
a program was begun to import South American
specialist natural enemies (Sailer 1984). Four
specialist natural enemies have been imported to
Florida, three of them released and established,
and their future in integrated pest management
of pest mole crickets has been outlined (Frank &
Parkman 1999). The nematode, S. scapterisci
Nguyen & Smart (1990), received research em-
phasis in the late 1980s, attracted attention from
industry as a biopesticide, and perhaps appeals to
most ranchers because it can be purchased and
applied as a pesticide (a method familiar to
them). Our preliminary analysis did not show
nematode infection of mole crickets trapped in
any of the test sites. Other biocontrol agents such
as Ormia depleta Wiedemann and Larra bicolor
F. may yet prove to be more effective and less
costly, but data on their efficacy are incomplete,
and methods for management of their field popu-
lations are inadequately researched because of
lack of funds.
Development of mole cricket seasonal activity
history on bahiagrass pasture is critical for eval-
uation of a successful outcome of any biocontrol
agent. Spikes of nymphs exceeding 100 per pitfall
trap may be captured following the early summer
rains, but a mean seasonal capture >10 nymphs
m-1 of trap may indicate the start of a mole cricket
infestation problem and may represent a working
threshold for future studies.

ACKNOWLEDGMENTS

This paper was based on work partly supported by
the Center for Cooperative Agricultural Programs be-
tween the University of Florida A & M University and
the University of Florida and is Florida Agric. Exp. Stn.
Journal Series No. R-08333. The authors are heavily in-
debted to Dr. T. J. Walker, Univ. of Florida, Gainesville,
for technical assistance and helpful suggestions to im-
prove the manuscript.


June 2003







Adjei et al: Mole Cricket Infestation on Pasture


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management of pest mole crickets with emphasis on
southeastern USA. IPM Reviews. 4: 39-52.
HUDSON, W. G. 1984. Other behavior, damage and sam-
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Florida. Agr. Exp. Sta. Bull. 846. IFAS, Univ. Florida,
Gainesville.
HUDSON, W. G. 1985. Ecology of tawny mole cricket,
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population estimation, spatial distribution, move-
ment, and host relationships. Ph.D. dissertation,
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HUDSON, W. G. 1988. Field sampling of mole crickets
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HUDSON, W. G. 1989. Field sampling and population es-
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Gryllotalpidae). Florida Entomol. 72: 337-343.
HUDSON, W. G., J. H. FRANK, AND J. L. CASTNER. 1988.
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HUDSON, W. G., AND J. G. SAW. 1987. Spatial distribu-
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LAWRENCE, K. L. 1982. A linear pitfall trap for mole
crickets and other soil arthropods. Florida Entomol.
65: 376-377.
LAWRENCE, K. L. 1983. One year pitfall captures of im-
mature mole crickets in Palm Beach Co. Florida. An-
nual report Mole Cricket Research. 5: 11-12.
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Gainesville.
NICKERSON, J. C., D. E. SNYDER, AND C. C. OLIVER
1979. Acoustical burrows constructed by mole crick-
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NGUYEN, K. B., AND G. C. SMART, JR. 1990. Steinernema
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SAILER, R. I. 1984. Biological control of mole crickets.
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SAS INSTITUTE, INC. 1999. SAS/STAT user's guide, Ver-
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SHORT, D. E., AND D. P. KOEHLER 1979. A sampling
technique for mole crickets and other pests in turf-
grass and pasture. Florida Entomol. 62: 282-283.
SPSS, INC. 1997. Transforms & regressions. SigmaPlot
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Ext. Serv., Gainesville.
WALKER, T. J. 1982. Sound traps for sampling mole
cricket flights (Orthoptera: Gryllotalpidae: Scap-
teriscus). Florida Entomol. 65: 105-110.
WALKER, T. J. 1984. Biology of pest mole crickets. Sys-
tematics and life cycles. Pp. 3-10. In T. J. Walker
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WALKER, T. J., AND NGO DONG. 1982. Mole crickets and
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WALKER, T. J., AND D. A. NICKLE. 1981. Introduction
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nus and S. acletus reexamined. Ann. Entomol. Soc.
America. 74: 158-163.
WALKER, T. J., J. P. PARKMAN, AND D. J. SCHUSTER
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Florida, Gainesville.







Florida Entomologist 86(2)


June 2003


COMPARISON OF BIODEGRADABLE, PLASTIC AND WOODEN
IMIDACLOPRID-TREATED SPHERES FOR CONTROL OF
RHAGOLETIS MENDAX (DIPTERA: TEPHRITIDAE) FLIES

JON E. HAMILL, OSCAR E. LIBURD AND STEVEN R. ALM1
Department of Entomology and Nematology, Natural Area Drive, University of Florida, Gainesville, FL, 32611

'Department of Plant Sciences, Woodward Hall, University of Rhode Island, Kingston, RI 02881

ABSTRACT

In experiments comparing biodegradable, plastic and wooden imidacloprid-treated spheres for
control of Rhagoletis mendax Curran, the mean number of flies caught on plexiglas panes be-
low each sphere type was not significantly different for the entire season. However, the mean
time spent by R. mendax flies alighting on biodegradable imidacloprid-treated spheres was
significantly greater (2.6x) than plastic imidacloprid-treated spheres. During 2001, signifi-
cantly fewer larvae were found in blueberries harvested from bushes that had wooden imida-
cloprid-treated spheres hung within the canopy compared with bushes where biodegradable
and plastic imidacloprid-treated spheres were deployed. There was no significant difference
between the number of larvae found in berries picked from bushes where biodegradable or
plastic spheres were deployed. All imidacloprid-treated sphere treatments were found to sig-
nificantly reduce blueberry maggot larval infestation in fruit compared with the control.

Key Words: attractant, imidacloprid-treated sphere, blueberry maggot

RESUME

En experiments comparando las esferas de plastico y de madera biodegradable y tratadas
con imidacloprid para el control de Rhagoletis mendax Curran, el promedio del numero de
las moscas atrapadas sobre la superficie de "plexiglas" debajo cada clase de esfera no fu6 sig-
nificativamente diferente para la estaci6n complete. No obstante, el promedio del tiempo pa-
sado por mosca de R. mendax encima de las esferas biogradables tratadas con imidacloprid
fu6 significativamente mayor (2.6 veces) que en las esferas plasticas tratadas con imidaclo-
prid. Durante 2001, fueron significativamente encontradas menos larvas sobre las moras
(Vaccinium sp.) cosechadas de arbustos que tenian las esferas de madera tratadas con imi-
dacloprid colgadas dentro del dosel comparados con arbustos donde pusieron esferas de plas-
tico biodegradable y tratadas con imidacloprid. No habia una diferencia significativa entire
el nmmero de larvas encontradas en la frutas cortadas de los arbustos donde habian puestas
las esferas de plastico biodegradable y tratadas con imidacloprid. Se encontraron que todos
los tratamientos de las esferas tratadas con imidacloprid redujieron significativamente la in-
festaci6n de larvas en las frutas comparados con el control.


The potential for using a lure and toxicant sys-
tem to control fruit flies has been examined by sev-
eral researchers. Hanotakis et al. (1991) combined
a food attractant, a phagostimulant, a male sex
pheromone, a female aggregation pheromone, a hy-
groscopic substance (glycerin), and two insecticides
(deltamethrin and dichlorvos) with a trap to control
the olive fruit fly, Bactrocera oleae (Gmelin). Duan
and Prokopy (1995) and Hu et al. (2000) tested
dimethoate, abamectin, phloxine B, diazinon, imi-
dacloprid, azinphosmethyl, methomyl, tralom-
ethrin, malathion, fenvalerate, and carbaryl on
wooden spheres and found that only dimethoate,
malathion and imidacloprid were viable candidates
for incorporation into spheres to suppress apple
maggot, Rhagoletis pomonella (Walsh), activity.
Dimethoate, malathion, and imidacloprid did not
reduce the time of visitation by R. pomonella flies
on treated spheres in field cage studies.


Recently, Ayyappath et al. (2000) evaluated thi-
amethoxam at 2-4% Al in sugar/starch spheres and
found this insecticide to be significantly less effec-
tive than spheres treated with 2% AI imidacloprid.
Wright et al. (1999) determined that regardless of
trap design and pesticide incorporation, several
conditions must exist for spheres to become a via-
ble alternative for control of Rhagoletis flies.
Spheres must be: 1) easy and safe to deploy, 2) as
effective as insecticide sprays, 3) able to endure
throughout the growing season, and 4) maintain
fly-killing power with a very low dose of toxicant.
A recent trap design is a biodegradable sphere
consisting of water, gelatinized corn flour, corn
syrup, sugar, cayenne pepper, and sorbic acid
(Liburd et al. 1999; Stelinski & Liburd 2001). The
biodegradable sphere is coated with a mixture of
70% paint, 20% sucrose solution (wt:vol), 4% imi-
dacloprid (AI), and 6% water. Biodegradable







Hamill et al.: Imidacloprid Spheres for Control of R. mendax


spheres were developed as alternatives to broad-
spectrum insecticides for management of key
Rhagoletis spp. in the northeastern United
States. The benefits of using insecticide-treated
spheres include the reduction of pesticide resi-
dues on crops as well as reduced environmental
and worker hazards.
The purpose of this study was to compare biode-
gradable, plastic, and wooden imidacloprid-treated
spheres to determine the most efficacious sphere
type for preventing blueberry maggot injury. All
previous trap designs, with the exception of the
biodegradable sphere, had focused on using
wooden spheres brush painted with enamel paint
mixed with an insecticide. Using a plastic sphere,
either dipping it into an insecticide/sugar solution
or coating it with a mixture of paint and insecticide
presents a third alternative to previous designs.

MATERIALS AND METHODS

Research plots were located in Rhode Island
and Michigan. In Rhode Island plots were located
at two locations during 2000, a 0.5 ha highbush
blueberry, Vaccinium corymbosum L., planting of
'Patriot', 'Blueray', and 'Jersey' located in North
Kingstown and a 2 ha planting of 'Berkley' and
'Collins' located in West Kingston. In 2001, re-
search was conducted at a 0.3 ha planting of 6 cul-
tivars; 'Bluecrop', 'Bluetta', 'Darrow', 'Earliblue',
'Herbert' and'Lateblue' in Kingston, RI and at a 2
ha planting of'Jersey' located in Holland, MI.

Sphere preparation (2000)

Biodegradable spheres were obtained from the
USDA, National Center for Agricultural Utilization
Research Laboratory in Peoria, Illinois and pre-
pared as described in Liburd et al. (1999). Spheres
were brush painted with a mixture containing 70%
enamel paint (Shamrock Green 197A111, ACE
Hardware, Kensington, IL.), 20% (wt:vol) sucrose
solution, 2% (AI) imidacloprid (Provado 1.6 F, Bayer,
Kansas City, MO), and 8% water.
Plastic spheres (Great Lakes IPM, Vestaburg,
MI) were dipped in a solution containing 946 ml
water, 28 g of Merit 75 WP (imidacloprid) (Bayer,
Kansas City, MO), 189 ml of 20% sucrose solution
(wt:vol), and 22 ml (2 ml of product in 20 ml water)
finished additive of Turbo spreader (Bonide,
Yorkville, NY). This mixture represents 81.6% wa-
ter, 2.4% Merit 75WP (1.8% AI imidacloprid), and
16% (wt:vol) sucrose solution. Spheres were dipped
a total of three times during the growing season.

2000

Three treatments were evaluated in two high-
bush blueberry plantings for control ofR. mendax
in a completely randomized block design with
four replicates. Each block consisted of ten 9-cm


diameter green biodegradable imidacloprid-
treated spheres (treatment 1), ten 9-cm diameter
green plastic imidacloprid-treated spheres (treat-
ment 2), and a section of the block consisting of 30
bushes was left untreated (treatment 3, control).
Spheres, approximately one per three bushes,
were hung about 15-cm from the uppermost bush,
which is the most effective position (Liburd et al.
2000), and baited with ammonium acetate (1 g in
4 ml of water) in a 5 ml scintillation vial (National
Diagnostics, Atlanta, GA). A 45 cm x 45 cm square
of plexiglas spray-coated with Tangletrap (The
Tanglefoot Co., Grand Rapids, MI) was hung 30
cm beneath each of the imidacloprid-treated
spheres and supported by four tie-wires (Liburd
et al. 1999).
During each sampling period, R. mendax flies
that landed on treated spheres were observed for
30 minutes; a total of 54 flies were observed. R.
mendax flies captured on plexiglas panes were
counted and removed twice weekly. In addition to
monitoring fly populations with Plexiglas panes,
R. mendax fly populations were also monitored
twice weekly using 9-cm diameter unbaited green
plastic spheres coated with Tangletrap. Towards
the end of the season, an 8-liter sample of'Patriot'
and 'Blueray' was taken on July 13 and 'Blueray'
and 'Jersey' taken on July 27 from North Kings-
town, RI and placed on screens (0.5 cm mesh) over
clear plastic containers to determine the number
of maggots in fruit (Liburd et al. 1998). The num-
ber of maggots collected into the containers was
counted twice a week to determine the effective-
ness of the sphere treatments. Fruit was not sam-
pled from West Kingston, RI because deer had
damaged the majority of the biodegradable
spheres.

Sphere preparation (2001)

During 2001, sphere preparation methods dif-
fered from those used in 2000 because additional
research data were available on the deployment
of insecticide-treated spheres.
Biodegradable imidacloprid-treated spheres
were prepared as described in 2000. However, the
active ingredient (AI) was increased to 4% because
Stelinski et al. (2001) had shown that the effec-
tiveness of field-exposed imidacloprid-treated
spheres with 2% AI was significantly reduced over
a 12 wk period whereas spheres treated with 4%
AI were not significantly affected.
Plastic imidacloprid-treated spheres were first
painted with a mixture of 26 ml Provado 1.6F (4%
AI imidacloprid) (Bayer, Kansas City, MO), 87 ml
'Bell Pepper' paint (Pittsburgh Paints, Pitts-
burgh, PA) and 20 ml sucrose solution (5.5 g per
20 ml water = ca. 4.8% of the total mixture). In ad-
dition, a newly developed sucrose cap (Prokopy et
al. unpublished data) was attached to the spheres
to act as a feeding stimulant.







Florida Entomologist 86(2)


In Rhode Island, the same three treatments
(biodegradable and plastic imidacloprid-treated
spheres and control) evaluated in 2000 were re-
evaluated in 2001. In Michigan, a fourth treat-
ment, wooden imidacloprid-treated spheres, was
included in the experimental design. Wooden im-
idacloprid-treated spheres (9-cm) were brush
painted with a mixture of DevFlex latex green
paint (ICI Paints, Cleveland, OH) (70%), sucrose
feeding stimulant (20%), water (6%), and imida-
cloprid (4% AI). Like plastic spheres, wooden
spheres had the sucrose cap attached to act as a
feeding stimulant.
The experimental design was similar to 2000
and consisted of randomized block with four rep-
licates. The placement and position within the
canopy of imidacloprid-treated spheres were the
same as 2000. However, spheres were baited with
polycon dispensers containing 5 g of ammonium
carbonate (Great Lakes IPM, Vestaburg, MI). The
dispensers were attached to the strings used for
hanging spheres. Flies were monitored using the
same Plexiglas pane system used in 2000.
In Michigan, four samples of 100 berries per
replicate (totaling 400 berries per treatment)
were taken July 31 and August 1, 3, and 8. Ber-
ries were then placed over 0.5 cm mesh hardware
cloth to allow larvae to exit the fruit and drop into
containers filled with vermiculite (Liburd et al.
1998). The vermiculite was then sifted and blue-
berry maggot fly puparia were collected and
counted to quantify fruit infestation.


14
4-,
S12 -
4J
0 Biodegradable
10
S*Plastic
a)





0


2
a -
VT2
.4 H
O B ^, ^


Statistical Analysis

Data were analyzed by analysis of variance.
(SAS Institute 1989).

RESULTS

2000

The population ofR. mendax flies at the North
Kingstown, RI site was small, and captures on
plexiglas panes for biodegradable and plastic im-
idacloprid-treated spheres were not significantly
different, except on July 18th (Fig. 1). However,
Plexiglas panes placed under biodegradable
spheres consistently captured more flies than
panes placed beneath plastic spheres. The time
spent by R. mendax flies on biodegradable imida-
cloprid-treated spheres (62.6 12.0 sec) was sig-
nificantly greater (F = 32.5, df = 26, 53; P < 0.01)
than the time spent by flies on dipped plastic
spheres (24.2 24.2 sec).
Data collected using 9-cm diameter, unbaited,
green plastic sticky spheres coated with Tangle-
trap indicated that peak flight activity occurred
on July 4th. No maggots were found in 32 liters of
berries harvested on July 13th and 27th from any
of the treatment blocks including the control. The
data at the West Kingston, RI site could not be an-
alyzed due to the high incidence of deer damage.

2001

In Rhode Island, the mean number of flies col-
lected on plexiglas panes below plastic (35.6


4-Jul 6-Jul 11-Jul 13-Jul 18-Jul 20-Jul 25-Jul 27-Jul Season
Date
Fig. 1. Mean number ofR. mendax flies trapped on plexiglas panes beneath imidacloprid-treated biodegradable
and plastic spheres, North Kingston, RI July 4-27, 2000.


June 2003







Hamill et al.: Imidacloprid Spheres for Control of R. mendax


16.0) and biodegradable (24.6 11.0) imidaclo-
prid-treated spheres was not significantly differ-
ent for the entire season. Similarly, in Michigan
the mean number of flies collected on Plexiglas
panes below biodegradable (33.8 2.98), wooden
(31.3 4.71), and plastic (26.0 9.06) spheres was
not significantly different for the entire season.
Again, plexiglas panes placed below biodegrad-
able spheres consistently captured more R.
mendax flies than plastic and wooden spheres.
In our fruit infestation counts, significantly
fewer (F = 24.63, df = 3,6, P < 0.01) larvae were
collected from berries that had wooden spheres
deployed in blocks compared with plastic and bio-
degradable spheres (Fig. 2). Overall, the mean
number of larvae found in berries treated with
biodegradable, plastic, or wooden spheres was
significantly lower (F = 24.63, df = 3,6, P < 0.01)
than untreated checks. Berries collected from un-
treated (control) plots had 1.8 times as many lar-
vae compared with other treated plots (Fig. 2). Six
biodegradable imidacloprid-treated spheres were
lost to deer feeding during the 6 weeks of experi-
mentation in Michigan. Peak flight activity for
R. mendax occurred on July 24 as measured with
yellow unbaited sticky boards.

DISCUSSION

Experiments comparing the effectiveness of
biodegradable, wooden, and plastic imidacloprid-
treated spheres showed no significant differences
in the number of flies trapped on Plexiglas panes.
This is the first study showing the effectiveness of
plastic imidacloprid-treated spheres for suppres-

80 -


sion of R. mendax. Previous studies have focused
on the efficacy of wooden and biodegradable
insecticide-treated spheres. Currently, wooden
spheres are not commercially available. Also, pro-
duction of wooden and biodegradable spheres
may be prohibitive since the cost may range be-
tween $2-4 per sphere for either sphere type. In
blueberries, depending on infestation of R.
mendax, it may take as many as 100 spheres per
hectare for effective control.
The variation in sphere preparation through-
out 2000 and 2001 was done to optimize insecti-
cide concentration and formulation as well as to
further develop the feeding stimulant system so
that flies will alight for longer time on treated
spheres. Our data showed that flies spent much
longer time on biodegradable imidacloprid-
treated spheres compared with plastic imidaclo-
prid-treated spheres. However, the fact that lar-
val infestation was not significantly affected
between sphere types may indicate that the dura-
tion of stay on treated spheres to deliver a lethal
dose may not be as important as previously
thought. Insecticide compatibility with treated
spheres and susceptibility of the insect may be
the key factors regulating the effectiveness of in-
secticide-treated spheres.
Because data from our green sticky sphere
monitoring traps indicated that R. mendax flies
were active throughout the season and flies were
trapped continuously with our Plexiglas trapping
device, it was rather surprising that no larvae
were found in treated and untreated plots at our
North Kingston, Rhode Island site. However, be-
cause the plots were relatively small, it is possible


Wooden Plastic Biodegradable Control
Treatment
Fig. 2. Mean number of maggots in four samples of 100 berries, Holland, MI (2001).







Florida Entomologist 86(2)


that the ammonium acetate attractant used for
baiting imidacloprid-treated spheres may have
attracted flies from treated and untreated areas
resulting in a high mortality and subsequently
preventing infestation in both treated and un-
treated plots. Liburd et al. (1999) also found that
ammonium lures were effective in attracting R.
mendax flies from within a 5 m radius to insecti-
cide-treated sphere traps.
The biodegradable imidacloprid-treated spheres
used in our study may be more appealing to grow-
ers than the plastic spheres used in 2000. Bio-
degradable spheres did not require additional
maintenance after initial deployment in the field.
However, some of these spheres needed to be re-
placed because rodents and deer frequently ate
them. As Stelinski et al. (2001) stated, prevention
of deer feeding and inhibition of mold growth are
needed before these spheres can be recommended
to growers.
The plastic spheres used in 2000 needed suc-
cessive dipping in pesticide solution to maintain
their effectiveness in killing R. mendax flies. De-
pending on the insecticide used, the risks of re-
peated exposure to the applicator may not justify
the use of plastic spheres in this manner. The su-
crose caps (Prokopy et al. unpublished) used on
plastic and wooden spheres in 2001 may make
these spheres more appealing to growers.
Wooden pesticide-treated spheres deployed
with a sucrose cap may be another alternative.
We noted that the resulting fruit injury from plots
treated with wooden imidacloprid-treated spheres
was lower than plastic and biodegradable spheres.
The major problem with wooden spheres is that
they are no longer commercially available; a prob-
lem that can be rectified if their usefulness in the
cropping system exceeds production costs.
The sucrose cap included in our experiments in
2001 was designed to last for a longer duration in
the field compared with earlier versions of sucrose
caps. An increase in the duration of available
sugar may have lead to an increase in fly kill over
time. As Stelinski et al. (2001) noted, pesticide-
treated spheres require a constant supply of sugar
to act as a feeding stimulant and be effective.
Further research is needed to determine how
many spheres are needed to treat different fields
possessing varying fly densities. Our results show
that plastic spheres may be a viable option to con-
trol blueberry maggot. However, there should be a
system for releasing a constant supply of sugar
such as the sugar caps used in 2001. In addition
plastic spheres must maintain the residual effi-
cacy of the pesticide.


ACKNOWLEDGMENTS

We thank Charles Dawson, Marsha Browning, Jason
Koopman, Rhiannon O'Brien, and Erin Finn for assis-
tance in collection of data. We also thank Lukasz Stelin-
ski (Michigan State University) for critical review of the
manuscript. In addition we would like to thank David
Bloomberg (Fruitspheres, Inc., Macomb, IL) for provid-
ing biodegradable spheres. This research was supported
by USDA-CSREES Grant No. 721495612. This manu-
script is Florida Agricultural Experiment Station Jour-
nal Series R-09081.

REFERENCES CITED

AYYAPPATH, R., S. POLAVARAPU, AND M. R. McGUIRE.
2000. Effectiveness ofthiamethoxam-coated spheres
against blueberry maggot flies (Diptera: Tephriti-
dae). J. Econ. Entomol. 93: 1473-1479.
DUAN, J. J., AND R. J. PROKOPY. 1995. Control of apple
maggot flies (Diptera: Tephritidae) with pesticide-
treated red spheres. J. Econ. Entomol. 88: 700-707.
HANIOTAKIS, G., M. KOZYRAKIS, T. FITSAKIS, AND A. AN-
TONIDAKI. 1991. An effective mass trapping method
for the control ofDacus oleae (Diptera: Tephritidae).
J. Econ. Entomol. 84: 564-569.
HU, X. P., R. J. PROKOPY, AND J. M. CLARK. 2000. Toxic-
ity and residual effectiveness of insecticides on in-
secticide-treated spheres for controlling females of
Rhagoletis pomonella (Diptera: Tephritidae). J.
Econ. Entomol. 93: 403-411.
LIBURD, O. E., S. R. ALM, AND R. A. CASAGRANDE. 1998.
Susceptibility of highbush blueberry cultivars to lar-
val infestation by Rhagoletis mendax (Diptera: Te-
phritidae) flies. Environ. Entomol. 27: 817-821.
LIBURD, 0. E., L. J. GUT, L. L. STELINSKI, M. E. WHALON,
M. R. McGUIRE, J. C. WISE, J. L. WILLETT, X. P. Hu,
AND R. J. PROKOPY. 1999. Mortality ofRhagoletis spe-
cies encountering pesticide-treated spheres (Diptera:
Tephritidae). J. Econ. Entomol. 92: 1151-1156.
LIBURD, O. E., S. POLAVARAPU, S. R. ALM, AND R. A.
CASAGRANDE. 2000. Effect of trap size, placement, and
age on captures of blueberry maggot flies (Diptera:
Tephritidae). J. Econ. Entomol. 93: 1452-1458.
PROKOPY, R. J., S. WRIGHT, AND J. BLACK. 1999. Com-
mercial orchard trials of attracticidal spheres for
controlling apple maggot flies. Fruit Notes 64: 14-17.
STELINSKI, L. L, AND O. E. LIBURD. 2001. Evaluation of
various deployment strategies of Imidacloprid
treated spheres in highbush blueberries for control
of Rhagoletis mendax (Diptera: Tephritidae). J.
Econ. Entomol. 94: 905-910.
STELINSKI, L. L., O. E. LIBURD, S. WRIGHT, R. J.
PROKOPY, R. BEHLE, AND M. R. McGUIRE. 2001.
Comparison of Neonicotinid insecticides for use with
biodegradable and wooden spheres for control of key
Rhagoletis species (Diptera: Tephritidae). J. Econ.
Entomol. 94: 1142-1150.
WRIGHT, S., B. CHANDLER, AND R. J. PROKOPY. 1999.
Comparison of Provado and Actara as toxicants on
pesticide-treated spheres. Fruit Notes 64: 11-13.


June 2003







Eitam et al.: Fruit Fly Parasitoid and Host Fruit Cues


USE OF HOST FRUIT CHEMICAL CUES FOR LABORATORY REARING
OF DORYCTOBRACONAREOLATUS (HYMENOPTERA: BRACONIDAE),
A PARASITOID OF ANASTREPHA SPP. (DIPTERA: TEPHRITIDAE)

AVI EITAM1, TIM HOLLER2, JOHN SIVINSKI3 AND MARTIN ALUJA4
'Department of Entomology and Nematology, University of Florida, Gainesville, FL 32611, USA
Current address: Institute of Evolution, University of Haifa, 31905 Haifa, Israel

2USDA-APHIS, Florida Division of Plant Industry, P.O. Box 147100, 1911 SW 34th St., Gainesville, FL 32614, USA

3Center for Medical, Agricultural and Veterinary Entomology, USDA-ARS, PO. Box 14565, Gainesville, FL 32604, USA

4Instituto de Ecologia, A.C., Apartado Postal 63, 91000 Xalapa, Veracruz, Mexico

ABSTRACT

Doryctobracon areolatus (Szepligeti) (Hymenoptera: Braconidae) is a common parasitoid of
Anastrepha spp. (Diptera: Tephritidae). An efficient method of laboratory rearing incorpo-
rates chemicals from pear fruits into oviposition units. Production for the F, and F2 genera-
tions was 12.1 and 9.3 progeny per female, respectively. Mean daily progeny production by
F, females was between 1-2 progeny per female for almost all ages from 9 to 22 days. A bio-
assay was designed to determine the source of chemical cues used for host location. Parasi-
toids were given a choice between two oviposition units: a positive control containing all
possible cues, and a treatment unit with cues derived from either the host fly, host fruit,
both, or none. The number of females active on each oviposition unit was recorded. This ex-
periment demonstrated that chemical cues derived from the host fruit, probably the peel, are
involved in host location.

Key Words: biological control, fruit fly, host location, oviposition

RESUME

Doryctobracon areolatus (Szepligeti) (Hymenoptera: Braconidae) es un parasitoide comun
de Anastrepha spp. (Diptera: Tephritidae). Un m6todo eficiente de criarlos en el laboratorio
incorpora unos quimicos de la fruta de la pera en las unidades de oviposici6n. La producci6n
en las generaciones F, y F2 fueron 12.1 y 9.3 descendientes por hembra, respectivamente. El
promedio de la producci6n diaria de los descendientes para las hembras de F2 fu6 entire 1-2
descendientes por hembra para casi todas las edades de 9 a 22 dias. Un bioensayo fue dise-
iado para determinar la fuente de las seiales quimicas usadas para la ubicaci6n del hospe-
dero. Los parasitoides podian escoger entire dos unidades de oviposicion: un control positive
que tenia todas las seiales posibles, y una unidad de tratamiento con las seiales derivadas
ya sea de la mosca hospedera, de la fruta hospedera, 6 ambas, 6 ninguna de las dos. Se re-
gistr6 el numero de hembras activas sobre cada unidad de oviposici6n. Este experiment de-
mostr6 que las seiales quimicas derivadas de la fruta hospedera, probablemente la cascara,
estan envueltas en la localizaci6n del hospedero.


Doryctobracon areolatus (Szepligeti) (Hymenop-
tera: Braconidae) is a widespread Neotropical
parasitoid of Anastrepha Schiner spp. (Diptera:
Tephritidae), ranging from Mexico to Argentina
(Wharton & Marsh 1978). In Brazil, it is the dom-
inant species, constituting between 62% and 89%
of all Anastrepha parasitoids in various surveys
(Canal et al. 1994, 1995; Leonel et al. 1995;
Araujo et al. 1996; Aguiar-Menezes & Menezes
1997; Aguiar-Menezes et al. 2001). Furthermore,
D. areolatus represented 43-59% of all parasitoids
collected in the State of Veracruz, Mexico (Her-
nandez-Ortiz et al. 1994; L6pez et al. 1999), and
accounted for 33% of the parasitism in Venezuela
(Katiyar et al. 1995).


Doryctobracon areolatus was introduced into
Florida in 1969 for the control of the Caribbean
fruit fly, Anastrepha suspense (Loew) (Bara-
nowski & Swanson 1970). It is currently the dom-
inant parasitoid in the interior region of south-
central Florida. In a recent study we found that it
parasitized up to 36% of the host larvae and con-
stituted 61-100% of all parasitoids at various
sites (unpublished data).
Due to the importance ofD. areolatus as a par-
asitoid ofAnastrepha spp., there is much interest
in establishing laboratory cultures of this species.
Rearing of several fruit fly parasitoids has been
facilitated by the use of 'oviposition units' in
which host larvae are presented to the females







Florida Entomologist 86(2)


within an artificial apparatus (Wong & Ramadan
1992). This is based on the finding that female Di-
achasmimorpha longicaudata (Ashmead) (Hy-
menoptera: Braconidae) exhibit an ovipositional
response to vibrations of the host larvae
(Lawrence 1981). However, D. areolatus females
show no response to hosts in such an apparatus,
and rearing has been successful only through the
presentation of fruit fly larvae within host fruit.
Several studies have demonstrated the impor-
tance of fruit-associated chemicals in host loca-
tion by parasitoids of fruit flies. Greany et al.
(1977) found that chemicals released by fungi as-
sociated with rotten fruits are attractive toD. lon-
gicaudata females. Messing & Jang (1992), using
chopped ripe fruits placed in traps, demonstrated
attraction of D. longicaudata females to various
host fruits. Messing et al. (1996) demonstrated
similar responses by Psyttalia fletcheri (Silvestri)
(Hymenoptera: Braconidae) to odors of fresh cu-
cumber and decaying pumpkin.
Parasitoids may also respond to cues associ-
ated with the host fly. Prokopy & Webster (1978)
found that Utetes canaliculatus (=Opius lectus)
(Gahan) (Hymenoptera: Braconidae) responds
primarily to the host-marking pheromone of
Rhagoletis pomonella (Walsh) (Diptera: Tephriti-
dae). Similarly, Halticoptera rosae Burks (Hy-
menoptera: Pteromalidae) was found to respond
to the pheromone deposited by Rhagoletis basiola
(Osten Sacken) (Diptera: Tephritidae) (Roitberg
& Lalonde 1991).
In this paper we describe an efficient method
of D. areolatus rearing by incorporating host
chemicals into oviposition units. We demonstrate
that the ovipositional response is to chemicals de-
rived from the host fruit, and not the fly.

MATERIALS AND METHODS

Laboratory Rearing

Insects. A parent generation ofD. areolatus, a
total of 128 females and 41 males, was reared
from cattley guava, Psidium cattleianum Sabine,
fruit collected mostly at LaBelle, Florida. Larvae
ofA. suspense were obtained from a laboratory
colony maintained for approximately 150 genera-
tions at the Florida Department of Agriculture
and Consumer Services, Division of Plant Indus-
try, Gainesville, Florida.
Cage Setup. Adult parasitoids were main-
tained in 20 cm3 metal-framed cages, the top and
two side panels with 16-mesh (per inch) screens,
and other panels Plexiglas. One of the side Plexi-
glas panels included a cloth sleeve. A brown paper
towel was taped to the outside of the opposing
Plexiglas panel, in order to reduce light intensity.
Each cage was stocked over a period of several
days (depending on the emergence rate) with up
to 100 females and 100 males. Food was supplied


daily in the form of a fresh block of honey agar set
on an inverted 30 ml plastic cup, and a strip of
honey on the Plexiglas side panel. Water was sup-
plied in a 100 ml plastic cup with a cloth wick in-
serted through a hole in the lid; the external part
of the wick was split in half and laid upon the lid.
Cages were maintained at 25 + 0.5C, 45% R.H.,
and a light-dark cycle of 14:10.
Oviposition Unit. Oviposition units were com-
posed of A. suspense larvae in diet (Burns 1995)
between two layers of cloth, topped with a layer of
parafilm, all maintained within a 7.6 cm diameter
plastic embroidery hoop. Before exposure to the
parasitoids, the parafilm had been wrapped over-
night on a fresh'Anjou' pear (chosen because pears
were commercially available throughout the year),
previously placed for several hours in a cage with
adult A. suspense. The parafilm was placed in the
unit with the side previously in contact with the
host fruit facing out. This procedure, allowing
transfer of fruit chemicals to the oviposition unit,
was previously used by Papaj & Prokopy (1986) for
fruit fly bioassays. Each sheet of parafilm was
used on two consecutive days and, when not in use,
was kept in a sealed and refrigerated plastic cup.
Approximately 40 cm3 diet containing several
hundred host larvae were placed in each oviposi-
tion unit. The larvae-diet mixture was selected
from areas of the larval trays containing high
densities of larvae, so that at least 50% of the vol-
ume was larvae. This was done to increase the
chance of successful probing by the parasitoids. A
greater amount of diet would have allowed larvae
to migrate away from the oviposition surface and
avoid parasitism. Less diet would have left parts
of the unit devoid of hosts, thus decreasing the
chance of a successful probing. Host larvae were
usually 4 or 5 days old, corresponding to late sec-
ond and/or early third instar; occasionally 3 or 6
day old larvae were used.
The oviposition unit was elevated onto an in-
verted 100 ml plastic cup to set it closer to the
center of the cage, thus improving access of the
parasitoids to it. Hosts were exposed to parasi-
toids for approximately 8 h daily. However, when
high activity (15 or more parasitoids simulta-
neously on the oviposition unit) was observed,
two successive exposures were performed, with
units being replaced after 4 h. This was done to
reduce the chance of superparasitism.
Parasitoids were first provided with hosts within
several days of emergence. Exposure continued
daily, depending on availability of suitable hosts,
until the last female in the cage died. Because cages
were stocked over several days, the exact age of ovi-
positing females could not be determined. Age was
estimated as the difference between the exposure
date and the median emergence date of all females
in a particular cage. This age estimate for F2 fe-
males was subsequently related with the number
and sex ratio of their progeny.


June 2003







Eitam et al.: Fruit Fly Parasitoid and Host Fruit Cues


Immature Stages and Adult Emergence. Upon
completion of exposure, host larvae were trans-
ferred to 30 ml plastic cups, which were filled to
the top with fresh diet. These cups were then
placed upon moist fine vermiculite (15-20 ml wa-
ter per 100 cm3 vermiculite) in 500 ml plastic cups.
Fully developed larvae emerged from the diet,
dropping to the vermiculite in which they pu-
pated. After allowing larvae to pupate for several
days, the vermiculite was sieved, and host puparia
transferred into fresh moist vermiculite within
100 ml plastic cups. These cups were covered with
a solid lid, which was replaced after one week with
a cloth lid. This procedure allowed the vermiculite
to remain moist while minimizing development of
fungi. Immature stages were maintained at the
same environmental conditions as adults.
Number and sex of adult parasitoids were de-
termined upon emergence, and adults were trans-
ferred to screened cages. Cups were discarded
when no emergence was observed for several days.

Bioassay of Chemical Cues

Doryctobracon areolatus were reared success-
fully from host larvae in oviposition units with
parafilm that had contained possible chemical
cues from both adult fruit flies and host fruit
(described above). A subsequent study was con-
ducted to confirm that chemicals from the host
fruit and/or adult fly were used as cues for host
location and to further determine the source of
these cues.
Insects. Adult D. areolatus used in the bioassay
were F3 individuals from the laboratory culture
described above. Larvae ofA. suspense were ob-
tained from the laboratory culture at the Division
of Plant Industry described above.
Experimental Design. Cages were 30 cm long x
20 cm wide x 20 cm high. The bottom and two
longer sides were Plexiglas, with a cloth sleeve in
the middle of one of the side panels. The top panel
was 52-mesh (per inch) screen, and the two
smaller sides 16-mesh screen. Each of 6 cages was
stocked with 100 female and 70 maleD. areolatus.
Dead females were replaced daily. Before experi-
mentation, females were provided at least once
with an oviposition unit containing both host fruit
and fly chemicals. Oviposition units were as de-
scribed above for the laboratory culture, except
that the embroidery hoops were made of wood
and not plastic.
Parasitoids in each cage were allowed to
choose between two oviposition units, both placed
upon inverted plastic containers. One unit ('Posi-
tive control') contained parafilm wrapped over-
night on unwaxed 'Anjou' pears exposed to
ovipositingA. suspense females for several hours.
This unit contained all possible chemical cues de-
riving from the host fruit and adult host fly, simi-
lar to units used in rearing the laboratory culture.


The second oviposition unit ('Treatment') con-
tained parafilm with chemical cues from either
the host fruit or fly, a combination of both, or with-
out added cues. This treatment unit presumably
represented a subset of the positive control unit,
and response was expected to be either equal to or
less than response to the positive control. Treat-
ments were: (1) Untreated parafilm; (2) 'Intact
fruit'-wrapped on fresh undamaged pear; (3)
'Punctured fruit'-wrapped on pear punctured
approximately 200 times with a no. 0 insect pin
(to simulate puncturing by ovipositing flies); (4)
'Damaged fruit'-wrapped on pear from which
sections of pulp had been cut out (to simulate ver-
tebrate damage); (5) 'Fly cues'-placed for several
hours within a cage containing ovipositingA. sus-
pensa females (flies oviposited through the para-
film from several to several hundred times); (6)
'Fly cues + punctured fruit'-as treatment (5) but
subsequently wrapped on punctured pear.
The experiment was replicated on 12 of 13 con-
secutive days. On each day, each of the 6 cages
contained a different treatment. Each treatment
was replicated twice in each cage, placed alter-
nately on the left and right side of the cage; the
placement on any given day was random.
Response Variable and StatisticalAnalysis. The
number of females active on each oviposition unit
was recorded at 1, 4 and 8 h following placement of
the units in the cage. An active female was defined
as an individual either probing into the unit with
its ovipositor, or one standing on the unit with ovi-
positor at a horizontal or below horizontal posi-
tion; when the female is not reproductively active
the ovipositor is curved slightly upward.
The difference between the 'Positive control'
and 'Treatment' units ('diff') was calculated for
each cage at each hour. This variable was submit-
ted to the MIXED procedure of the SAS statistical
software package (Verbeke & Molenberghs 1997),
with the hourly observations treated as repeated
measurements. This procedure produced t-statis-
tics for each treatment, testing whether the vari-
able 'diff' was different than zero, i.e., whether
there was a significant difference between'Positive
control' and 'Treatment'. It further produced t-val-
ues comparing'diff' among the various treatments.

RESULTS

Laboratory Rearing

Lifetime progeny production averaged 2.4,
12.1 and 9.3 for P1, F, and F2 females, respectively.
Mean daily production by surviving F2 females
was between 1-2 progeny per female for almost all
ages from 9 to 22 days (Fig. 1).
The sex ratio was 44.7, 62.5 and 48% males for
the progeny of P1, F, and F2 females, respectively.
The sex ratio of the progeny of F2 females was rel-
atively stable over time, averaging close to 50%




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