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GRAVIMETRIC METHOD FOR THE MEASUREMENT OF SUGAR CONSUMPTION
BY ADULT VELVETBEAN CATERPILLAR (LEPIDOPTERA: NOCTUIDAE)
Xikui Wei1 and Seth J. Johnson
Department of Entomology
1Current address: Texas A&M University
Abstract
Determination of food consumption by adult Lepidoptera,
especially noctuid species, has not been well studied, possibly
because of a lack of appropriate methodology. In order to measure
sugar consumption by velvetbean caterpillar, Anticarsia gemmatalis
Hübner, a gravimetric method was developed and is presented
here together with results obtained using this method. After 24-h
exposure to a moth, liquid consumption by the moth was determined
by weighing the remaining solution on an electronic balance. The
amount of solution consumed was calculated by converting the difference
between the weight of the remaining solution and that of the unfed
control to a volumetric value. Velvetbean caterpillar moths consumed
a significantly greater volume of solution at lower sugar concentrations
than at higher concentrations. Female moths from larvae reared
on soybean foliage in field cages consumed three times that of
moths from larvae reared on artificial diet in the laboratory.
This method was simple, accurate, and required minimum handling
of test insects. It was suitable for the measurement of food consumption
of velvetbean caterpillar moths and could also be suitable for
other lepidopteran species.
Key Words: Anticarsia gemmatalis, adult feeding,
sugar consumption, compensatory feeding
Resumen
La determinación del consumo de alimento por
lepidópteros adultos, especialmente por especies de noctuidos,
no ha sido bien estudiada, posiblemente debido a la falta de una
metodología adecuada. A fin de medir el consumo de azúcar
por la oruga del frijol de terciopelo, Anticarsia gemmatalis Hübner,
fue desarrollado un método gravimétrico que es presentado
aquí junto a los resultados obtenidos mediante su uso.
Luego de 24 horas de exposición a la polilla, el consumo
del líquido fue determinado mediante el pesaje en una balanza
electrónica de la solución remanente. La cantidad
de solución consumida fue calculada mediante la conversión
a un valor volumétrico de la diferencia entre el peso de
la solución remanente y el del testigo donde las polillas
no se alimentaron. Las polillas consumieron un volumen significativamente
mayor de las soluciones con baja concentración de azúcar.
Las hembras de larvas criadas con follaje de frijol de soya en
jaulas de campo consumieron tres veces más que las polillas
de larvas criadas en dieta artificial en el laboratorio. Este
método fue simple, preciso, y requirió una mínima
manipulación de los insectos. El método fue adecuado
para la medida del consumo de alimento de A. gemmatalis y podría
además ser adecuado para otras especies de lepidópteros.
The adult velvetbean caterpillar, Anticarsia gemmatalis
Hübner (Lepidoptera: Noctuidae), like many other noctuid
species, feeds on floral and extrafloral nectars (Lukefahr &
Rhyne 1960). The moths have also been observed feeding on crushed
grapes (Greene et al. 1973), cut apples (X. W., personal observation),
whitefly honeydew on hemp sesbania, Sesbania exaltata (Raf.) (Collins
& Johnson 1985), and honeydew on seed heads of a few species
of grasses infected with ergot fungi including Bahiagrass, Paspalum
notatum Flugge (Greene et al. 1973) and Dallisgrass, Paspalum
dilatatum Poiretat (Collins & Johnson 1985, Wei & Johnson
1995).
The importance of nectar sources to adult Lepidoptera
has been demonstrated in studies that have shown increased numbers
of eggs (Collins & Johnson 1985), increased egg-laying (Wales
1983, Adler 1989, Mason et al. 1989), increased population density
(Lukefahr & Rhyne 1960, Collins 1984), insect pest outbreaks
in certain agroecosystems (Burleigh 1972), and probably enhanced
long-distance dispersal (Mason et al. 1989). However, comprehensive
knowledge of adult food consumption is needed before we can understand
how adult Lepidoptera utilize nectars in nature, and how important
adult feeding is to reproduction and population dynamics, especially
noctuids of economic importance. This knowledge could improve
IPM programs or adult control technologies. However, adult feeding
of most lepidopteran species, including noctuids, has been poorly
studied (Adler 1989). One of the reasons appears to have been
a lack of appropriate experimental methodology.
In measuring liquid consumption by adult Lepidoptera,
the following methods have been explored: an artificial flower
consisting of graduated pipette and small funnel (Alm et al. 1990),
hollow plastic stoppers covered with Parafilm® to serve as
a reservoir for artificial nectar (Pivnick & McNeil 1985),
weighing the test insect before and after feeding (Pivnick &
McNeil 1985, Adler 1989), and using a 50 or 100 ul microcapillary
to hold sugar solution to feed test insects (May 1985a, b). Adler
(1989) adopted a microcapillary method combined with spring-action
clothes pins to bind the wings of Helicoverpa zea (Boddie) moths
to restrain them while they were feeding. While the above mentioned
techniques were obviously acceptable for measuring consumption
in the studies cited, each one has some problem or problems which
would preclude its use in measuring daily or lifetime sugar consumption
by a noctuid moth. Also, previous studies with Lepidoptera, except
for Alm et al. (1990) with the white cabbage butterfly, have used
uptake rate as the consumption variable rather than daily consumption.
In uptake rate measurement, test insects were given food once
or twice a day and the liquid ingested per unit time was then
calculated (May 1985a, Pivnick & McNeil 1985, Adler 1989).
However, because daily consumption was not determined, the estimates
to predict daily and total consumption were not suited to our
objectives which were to determine the effects of sugar consumption
on activity, feeding pattern, lipid accumulation, and fecundity
of the velvetbean caterpillar. To reach this objective we needed
a technique that would give us absolute values of sucrose consumed
on a daily basis. In the present study, we developed a new method
to measure the daily and lifetime total nectar consumption by
velvetbean caterpillar adults.
Materials and Methods
Insect
Two sources of test insects were used in this study,
lab-reared and field-reared moths. Laboratory rearing procedures
were a modification of the techniques of Greene et al. (1976)
and King & Hartley (1985) which had been used for maintaining
the velvetbean caterpillar colony in our laboratory from 1991
to 1994 (Wei 1995). Field-reared moths were obtained from larvae
reared in field cages in a soybean field at the Ben Hur Experiment
Farm of Louisiana State University Agricultural Center, Baton
Rouge, LA. A 1.5-ha field was planted with a soybean variety,
‘Pioneer 9791’, which is susceptible to velvetbean caterpillar.
During the summer of 1993, six cages, 1.8 × 1.8 × 1.8
m, were set-up in the field when the soybeans were mostly at developmental
stage R2 (Fehr & Caviness 1979). For ease of collection and
reduction of damage, the procedures of Wei & Johnson (1994)
for rearing velvetbean caterpillar larvae and harvesting their
pupae from field cages were followed. The ground inside the cages
was covered with one layer of saran screen (32 × 32 mesh)
over which a 2-cm layer of coarse vermiculite (STRONG-LITE®
Products Corp., Seneca, IL) was evenly placed at time of cage
set-up. Forty lab-reared adults (20 males and 20 females) were
released in each cage and removed three days later. After the
larvae emerged, densities were monitored to ensure they were below
levels which would result in total defoliation of soybean foliage
prior to pupation. After most larvae had pupated, pupae were collected
and transported to the laboratory. Pupae from either field cages
or laboratory sources were sexed, and placed in individual 155
ml plastic diet cups (SOLO® Cup Company, Urbana, IL). They
were held in a walk-in environmental chamber at 27 ± 1°C,
85 ± 5% RH, and a photoperiod of 14:10 (L:D) until emergence.
We performed three different experiments to examine
sugar consumption by velvetbean caterpillar moths using the gravimetric
method. First, we examined total liquid consumption for 10 female
and 10 male field-reared moths over their entire adult life span
when the insects were provided with both 30% sucrose solution
and distilled water. Second, we measured daily consumption over
14 days for 10 lab-reared female moths and 10 field-reared female
moths when the insects were provided with both 30% sucrose solution
and distilled water. Third, we measured consumption of five concentrations
of sucrose solutions (0%, 5%, 10%, 20% and 40%) in the 48 h after
adult emergence. We tested 10 field-reared female moths at each
of these concentrations. In each test, only moths that emerged
within a 24-h period were randomly selected and transferred singly
from the diet cups to 1.8 liter paper cartons (16 cm high ×
13 cm in diam, Fonda® Group Inc., Union, NJ), covered with
cheese cloth secured with a rubber band.
Experimental Conditions and Evaporation Monitoring
The experiments were conducted in a walk-in environmental
chamber at 27 ± 1°C, 85 ± 5% RH, and a photoperiod
of 14:10 (L:D). To monitor evaporation under such conditions,
we measured the evaporation of distilled water and 30% sucrose
solution over a 24-h period using the gravimetric method described
in the present study. We also examined the effects of location
(horizontal and vertical locations inside the walk-in environmental
chamber) on evaporation.
Development of Methods for Measuring Sugar Consumption
When we were investigating methods to use for measuring
sugar consumption by adult moths, we tested most of the available
methods. We modified the method used by Pivnick & McNeil (1985)
(using hollow plastic stoppers covered with Parafilm® ) by
weighing the plastic stoppers instead of weighing the test insects
before and after feeding. However, the moths could not easily
locate the food source. Weighing test insects before and after
feeding not only was too time-consuming, but some test insects
were damaged. More importantly, weighing the insect before and
after feeding would certainly not be suitable for the velvetbean
caterpillar moth which has the ability to quickly excrete liquid
from both proboscis and anus during or shortly after feeding (X.
W., S. J. J., & Abner M. Hammond, unpublished data). We also
tested the method of using a 100 ul microcapillary to hold the
sugar solution to feed test insects (May 1985a, b). This method
was not suitable for our experiment because threading the proboscis
manually into the openings of the microcapillary was not practical
when measuring food consumption for 24 h per day over an extended
period of time and with multiple test insects involved. We did
not use the method described by Adler (1989) (using a microcapillary
to hold the artificial nectar and spring-action clothes pins to
bind the wings of moths to restrain them while they were feeding)
because we felt that refrigerating, weighing, and pinning the
wings of moths each time they were fed would cause an unacceptable
amount of damage to the moths.
The method of Alm et al. (1990) (which involved the
use of a small funnel connected to a graduated pipette though
plastic tubing) was designed to measure the daily nectar consumption
by white cabbage butterfly. However, this method was not sensitive
enough to measure consumption by a velvetbean caterpillar moth.
Using a similar apparatus, the difference in the liquid levels
could not be detected before and after 200 ul of liquid were removed
from a 5 ml funnel connected to a 0.1 ml pipette. Also, evaporation
associated with this method would be problematic because of evaporation
from the relatively large surface area of the funnel although
no dimensions of the “funnel” or “graduated pipette”
were given in their method.
During the development of our method, we also experimented
with several kinds of containers as a reservoir for the sugar
solution, including clear plastic micro tubing, plastic cells
cut from blister cards (used for packing medicine by pharmacies),
hollow plastic stoppers, porcelain liquid holders, and polystyrene
wells. However, none of them, except for the small polystyrene
wells, allowed for easy and accurate measurement without the risk
of spill. The polystyrene wells, which were chosen for our method,
were big enough for moths to locate the food source easily but,
at the same time, small enough to minimize evaporation.
Feeding Platform
Feeding solutions were held in the 360 ul individual
polystyrene wells (10.7 mm high × 6.9 mm in top diam) cut
from EIA/RIA 8-well strips (Costar Corporation, Cambridge, MA).
Two polystyrene wells were embedded 2-cm apart in a piece of styrofoam
(5 × 4 × 2 cm) which formed a feeding platform.
In the first and second tests, 300 ul of distilled
water and 300 ul of 30% sucrose solution (wt:vol) were separately
provided in the 2 wells in each feeding platform. A 30% sugar
solution was selected based on experimental results that indicated
it was an appropriate concentration for velvetbean caterpillar
moths (X. W., S. J. J., & Abner M. Hammond, unpublished data).
For the third test, only sucrose solution of the designated concentration
was added to the 2 wells in a platform with 300 ul in each well.
A feeding platform containing measured solution was then placed
in each paper carton in the morning (0800, CST), and the moths
were allowed to feed for 24 h. Each moth received new wells and
solutions daily for the designated experimental period.
Weighing Procedure
At the end of each 24-h exposure period, wells from
each feeding platform were transferred to a 32-cell weighing board
made of a 10 × 15 × 2 cm piece of styrofoam with rows
of holes punched about the size of the outside diameter of the
well. The following steps were carried out: (1) the weighing board
(with wells and residual liquid) was placed on an electronic balance
(precision to 0.001 g), and the balance was zeroed; (2) the liquid
was pipetted from one well and the inside of that well dried with
a paper towel; (3) the negative number then displayed indicated
the weight of the liquid left in the well by the moth after feeding.
This three-step procedure was repeated for each well in succession
until all wells in a board were weighed. One of the advantages
of the weighing procedure was that handling of each individual
well was unnecessary, reducing the risk of spill while weighing
and also increasing handling efficiency. The residual weight was
subtracted from the weight of the appropriate control (distilled
water or sugar solution) included for monitoring evaporation.
The volumetric estimate of sugar solution or distilled water ingested
was calculated by converting the solution weight to volume.
Statistical Analysis
All statistical analyses were performed using SuperANOVA
version 1.11. Tukey-Kramer’s test (Gagnon et al. 1989) was used
for means separation.
Results and Discussion
Evaporation Monitoring
The monitoring of evaporation was very important
to the accuracy of the consumption measurement. We found: (1)
there was no significant difference (F= 0.0514; df= 1, 46; P =
0.8217) between distilled water and 30% sucrose solutions in the
volume evaporated [see Alm et al. (1990)]; (2) the environmental
chamber was uniform in effects on evaporation when relative humidity
was high (85 ± 5%) and air circulation was maintained as
evidenced by the finding that there was no significant difference
in evaporation among various locations (F= 0.1209; df= 1, 22;
P = 0.7314); (3) the average daily evaporation from a well was
24.87 ± 1.7 ul (8.3% water evaporated). Sugar concentration
increased about 3% due to water loss by evaporation during a 24-h
period, which was not considered significant in altering the sucrose
concentration [velvetbean caterpillar moths were observed to feed
on an extremely wide range of sugar solutions, ranging from 1%
concentration to even sugar crystals (X. W., S. J. J., & Abner
M. Hammond, unpublished data)]. However, in order to monitor evaporation
closely, we included 2 controls (same as treatment except with
no test insect) for each treatment group (usually 10 cartons)
during all experiments. The mean weight of the liquid of these
unfed controls served as the post-evaporation reference from which
we subtracted the residual liquid weight obtained after feeding
by a moth for 24 h. Volumetric consumption data were then obtained
by converting the resulting weight (the difference in weight between
fed and unfed) to a volumetric value.
Consumption
Using this gravimetric method, we obtained the daily
food consumption pattern of male and female velvetbean caterpillar
moths. Both male and female moths consumed heavily during the
first 2 days after emergence, with peak consumption occurring
on the first day. Females consumed 314.1 ± 72.8 ul in the
initial 2 days when provided with 30% sucrose solution and distilled
water, which represents 37% of the total consumption over their
adult lifetime (19.8 ± 8.0 days at 27°C). Males consumed
356.9 ± 69.8 ul in the initial 2 days representing 36.9%
of their total food consumption. After day 2 they consumed a nearly
constant daily volume of 25.6 ± 8.1 ul for females and 25.8
± 5.1 ul for males. The differences in consumption between
day 2 and day 3 were found significant for both male (F= 16.6798;
df= 1, 18; P= 0.007) and female moths (F= 6.7824; df= 1, 18; P=
0.0179). On average, the total liquid consumption for the entire
life span of a female moth was 849.3 ± 328.9 ul. Males consumed
a total of 964.0 ± 189.6 ul of liquid during their entire
life of 27.7 ± 6.4 d. There was no significant difference
(F= 0.9128; df= 1, 18; P= 0.3520) in total liquid consumption
between sexes. Even with the presence of 30% sucrose solution,
distilled water was still taken up daily by moths until death,
at an almost constant volume: 8.1 ± 4.0 ul for females and
7.6 ± 3.7 ul for males. A total of 150.4 ± 68.2 ul and
211.0 ± 57.4 ul distilled water was consumed per female or
male moth, representing respectively, 17.6% and 21.9% of their
total liquid consumption. Total sucrose consumed in dry weight
was 0.21 ± 0.08 g for a female and 0.23 ± 0.05 g for
a male moth when they were provided with both 30% sucrose solution
and distilled water.
The daily total liquid consumption of field- versus
lab-reared velvetbean caterpillar female moths is compared in
Fig. 1. Females from larvae reared on soybean foliage in field
cages consumed 644.2 ± 176.3 ul liquid in the initial 2 weeks
compared with 204.8 ± 93.1 ul for females from the laboratory.
The consumption by field-reared moths was 3 times that of lab-reared
moths, showing a highly significant difference (F = 48.58; df=
1, 18; P= 0.0001) in consumption between the 2 sources, although
there was no statistical difference in pupal weights between these
2 groups (F = 1.767; df= 1, 398; P= 0.1844). The lab-reared moths
consumed much less, even during the peak consumption period which
occurred in the first 2 days after emergence. Furthermore, distilled
water constituted 22.5% of the total liquid consumption by the
lab-reared moths while distilled water represented only 16.8%
of the total liquid consumption by field moths. These results
suggest that laboratory insects may have more stored energy carried
over from larval to the adult stage and thus do not depend as
heavily on feeding for their reproductive needs. Mason et al.
(1989) found much more adult lipid when larvae of soybean loopers,
Pseudoplusia includens (Walker), were reared on artificial medium
in the laboratory than in adults from larvae reared on plants
in the field. Also, this finding suggests that laboratory insects
may be inappropriate experimental subjects for food consumption
studies because their consumption is not representative of the
consumption of natural populations. This could be especially important
in research on adult feeding stimulants.
Moths consumed significantly more solution when they
fed at low concentrations than at high concentrations (Fig. 2;
F= 61.54; df = 4, 44; P= 0.0001). As the sugar concentration increased,
the volume consumed decreased dramatically. In the initial 48-h
feeding period after emergence, they consumed 657.5 ± 151.4;
526.4 ± 134.4; 346.6 ± 88.3; and 193.2 ± 36.8 ul,
respectively, when they were fed with 5, 10, 20, or 40% sucrose
solutions. Only 35 ± 16.6 ul of distilled water was taken
up during the same period when the insects were provided with
only distilled water. All pairwise comparisons of consumption
between concentrations were highly significantly different from
each other. When moths were fed on 5% sucrose, they consumed 3
times as much as they did on 40%. This is an example of compensatory
feeding by which moths consumed a greater volume of solution at
a lower sugar concentration than at a higher concentration.
Acknowledgments
We are grateful to Zhuofei Song for contributions
to the weighing procedure development and assistance throughout
the study and to A. M. Hammond, R. N. Story, J. R. Fuxa (Louisiana
State University), R. L. Crocker, W. T. Nailon (Texas A&M
University), J. D. Lopez, Jr. (USDA-ARS, Area-Wide Pest Manage.
Res. Unit, College Station, TX), and unknown reviewers for critical
reviews of the manuscript. We also thank Terrie R. Thomas for
assistance in insect rearing. Approved for publication by the
director, Louisiana Agricultural Experiment Station, as manuscript
number 94-17-8234.
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Fig. 1. Daily liquid consumption by 10 females from
larvae reared on soybean foliage in field cages and 10 females
from larvae reared on artificial diet in the laboratory. Moths
were provided with both 30% sucrose solution and distilled water.
Fig. 2. Consumption by female velvetbean caterpillar
moths during the initial 48-h period after emergence when provided
with various concentrations of sucrose solution. Ten field-reared
moths were tested at each concentration. Different letters shown
by each solid and standard error bar indicates significant difference
(a=0.05, Tukey-Kramer’s test) in consumption.
Louisiana Agricultural Experiment Station
Louisiana State University Agricultural Center
Baton Rouge, LA 70803
Research & Extension Center
17360 Coit Road
Dallas, Texas 75252