Download Relative fat content and age in the tropical butterfly Bicyclus anynana

Relative fat content and age in the tropical butterfly Bicyclus
anynana
Bas J. Zwaan, Annelies Burgers, Fanja Kesbeke & Paul M. Brakefield
Evolutionary Biology, Institute of Evolutionary and Ecological Sciences, Leiden University, PO Box 9516, 2300 RA
Leiden, The Netherlands ([email protected])
The butterfly Bicyclus anynana has to survive the six-month dry season in which food is scarce and
patchily available. To determine the role of genetic and environmental variation in fat content and
starvation resistance in survival, we studied the age-specific dynamics of relative fat content in
captive populations of virgin butterflies. Dry weight and fat-free dry weight decreased with age in
both sexes, indicating that non-fat substances were used by the butterflies (e.g. protein). Relative fat
content increased with age in female, but not in male, butterflies. Analysis showed that this is only
partly due to the decrease in fat-free dry weight, suggesting that virgin female butterflies may
increase their fat content because of reduced reproduction. Developmental time had a small effect
on these age-related changes, but, interestingly, fat content increased with age more rapidly in
animals with a longer developmental time.
Keywords: life history, fat content, age, Bicyclus anynana
The butterfly Bicyclus anynana lives in tropical seasonal environments with starkly different
ecological demands on the phenotype. Between the wet (high temperature) and dry (low
temperature) seasons the morphology of the wing patterns is strikingly different, and has been
shown to be mainly induced by the environmental cue temperature (Brakefield, 1997; Kooi &
Brakefield, 1999). This plasticity in wing pattern is thought to be an adaptive response to the
seasonal changes in climate and resting background (Brakefield, 1997; Brakefield & Kesbeke,
1997). In concordance, the life history shows a remarkable contrast between the seasons for traits
including weight, fecundity, fat content, longevity and developmental time. In the wet season, food
is abundant and phenotypes with fast developmental time and rapid and abundant reproduction are
favoured by selection. In contrast, in the dry season food is scarce or completely absent and slow
development and large size at maturity, long adult life span and dormant reproduction are
beneficial.
Especially in the dry season, fat content is one of the key traits in the Bicyclus life history
(Kooi et al., 1997). Fat provides an internal energy source in times of food shortage, as is indicated
by the strong dependence of starvation resistance on fat content (e.g. Zwaan et al., 1995a). In
Drosophila melanogaster this was shown in both phenotypic (Zwaan et al., 1991) as well as
genetic (Zwaan et al., 1995) manipulation experiments. Moreover, fat content appeared to trade off
with reproduction in this species (Zwaan et al., 1995b) and when we apply this to the Bicyclus
situation, antagonistic selection on fat content is expected in the two seasons, which may result in
the maintenance of (additive) genetic variation for fat content. We therefore plan experiments to
genetically manipulate fat content in Bicyclus anynana by selecting on starvation resistance to, (i)
establish the expected relatively high levels of additive genetic variation for starvation resistance
and fat content, and (ii) to determine the genetic covariance matrix with other life history traits.
The latter will, together with our research on other key life history traits, such as developmental
time, (pupal) weight and egg size, shed light on the evolutionary history of, and constraints on, the
life history configuration of Bicyclus anynana.
One potential complicating aspect for a selection experiment on starvation resistance is the fact
that fat content, at least in females, can increase with age in some species (Fairbanks & Burch,
1970; Fairbanks & Burch, 1974; Service, 1987; Zwaan et al., 1991). This is especially evident in
virgin animals and since the starvation time phenotype will be determined on virgin butterflies,
this needs to be done at an age when fat content is stable or increasing only slightly with age.
Hence, in this paper we determined the age-specific dynamics of relative fat content with age.
PROC. EXPER. APPL. ENTOMOL., NEV AMSTERDAM – VOLUME 12 – 2001
25
26
LIFE-HISTORY STUDIES
MATERIAL AND METHODS
Butterfly rearing and handling
Eggs were collected at 27°C on two-week-old maize plants for 24 h from about 500 females of our
large outbreeding stock. This plant was put in a rearing cage (50x50x50 cm) at 27°C and hatched
larvae were fed maize plants until pupation. Pupae were collected and butterflies were allowed to
eclose in cylindrical hanging cages (30x40 cm). Over an emerging period of 8 days, butterflies
were marked with a felt-tipped pen on their left forewing on the day of eclosion (first day was
marked ‘0’ up until ‘7’ at day 8). Butterflies were stored in groups of 25 as separate sexes in the
aforementioned hanging cages at 27°C. The butterflies were fed on slices of banana on moist
cotton wool and food was refreshed twice a week.
Fat content
We measured fat content of virgin male and female butterflies over a range of 0 to 37 days. We
focussed on the following time-points, 0-2, 5, 12-14, 20-27 and 31-37 days, with the aim of
collecting at least 15 female and 15 male individuals per time-point. In order to remove potential
confounding effects of developmental time, each time-point sample consisted of a mixture of
individuals from at least two eclosion days (e.g. for time-point 0 days, individuals were used from
eclosion day 2, 4 and 5). At the appropriate time points, individuals were removed from the cages
and frozen and stored at –20°C for later analysis. Fat content was determined on the butterflies
after removal of the wings, legs and antennae. The bodies were transferred to 5-ml glass vials and
dried for 48 h at 40°C. The bodies were then weighed to the nearest 0.01 mg to determine the dry
weight (DW) and transferred back into their vials, which were subsequently filled with 2 ml of
dichloromethane/methanol (2:1) solution, sealed and placed on a shaker (100 rpm) at room
temperature for 48 h. After this period, the dissolvent was refreshed and the fat extraction
continued in the same way for another 48 h. After removing the dissolvent, the bodies were dried
for 24 h at 40°C, after which they were weighed again to determine the fat-free dry weight
(FFDW). Fat content (FAT in mg) was calculated as (FAT=DW-FFDW), and relative fat content
(%FAT) as (%FAT=FAT/FFDW).
Analysis
All analyses were performed in JMP version 4.02 and %FAT was arcsine transformed before
statistical analysis. For females and males, DW, FFDW and %FAT were normally distributed
(Wilk-Shapiro test, ns) and variances were equal for females between the different age classes
(Levene’s test, ns). For males, the variances were not equal for the three traits (Levene’s test), but
no correlation between variance and age or sample size was found (analysis not shown). For all
traits analysed, the variances were not equal between the sexes (Levene’s test), therefore all
analysis are given for separate sexes and sex differences were tested with the Wilcoxon rank-sum
test. Effects of age and developmental time on DW, FFDW and %FAT were determined with an
analysis of covariance. In all cases, we present the minimum adequate models that resulted from
reduction of the full factorial model (Crawley, 1993). We performed weighted linear regression on
the age-class means in all our graphs to test for specific patterns for all three traits analysed.
RESULTS
Females had significantly higher DW and FFDW than males, but males had higher relative fat
contents than females (Figs. 1 and 2; Wilcoxon rank-sum test). DW and FFDW significantly
decrease with age in both females and males (Table 1; Fig. 1), indicating that the butterflies use
non-fat bodily resources in the course of their life. In addition, DW as well as FFDW decreased
with increasing developmental time in females. A significant interaction was found between
developmental time and age for DW in males: males with a longer developmental time did not lose
weight as fast as fast developing males (Table 1). Relative fat content increased significantly with
age in females but not in males (Table 1, Fig. 2). Analysis of covariance with age, developmental
time and DW as factors, indicated that this increase was not only due to the decrease in FFDW
with age (analysis not shown). Interestingly, for both sexes, fat content increased more slowly for
B.J. ZWAAN, A. BURGERS, F. KESBEKE & P.M. BRAKEFIELD
27
butterflies with a short developmental time (Table 1, DT*A interaction). This suggests, at least for
females, that the lower relative fat content of long developing butterflies is compensated by the
faster increased of fat content with age of this group (Table 1).
Table 1. Results of analysis of covariance with developmental time and age as factors for both
females and males reduced to the minimum adequate model
Females
Males
MS
Parameter sign
MS
Parameter sign
ns
Trait
DW
Developmental time (DT)
110.45**
Age (A)
773.94***
161.47***
+
DT*A
r
17.57*
FFDW
Developmental time (DT)
43.05*
r
93.82***
Age (A)
722.85***
DT*A
r
r
ns
%FAT
Developmental time (DT)
0.0116^
+
ns
Age (A)
0.2448***
+
0.2110***
+
DT*A
0.0254**
r, removed from model; ns, not significant; ^P<0.054, *P<0.05; **P<0.01;***P<0.0001
25
Dry weight
females
males
mg
20
15
10
5
0
5
10
15
20
25
30
35
40
age (days)
20
females
Fat free dry weight
males
mg
15
10
5
0
0
5
10
15
20
25
30
35
40
age (days)
Figure 1. Relation between dry weight (top) and fat-free dry weight (bottom)
and age for females and males. Lines are shown for significant linear weighted
regression on the age-class means (females, DW, y=−0.160x+18.61, P<0.0001;
FFDW, y=−0.157x+15.56, P<0.001; males, DW, y=−0.070x+10.74, P<0.001;
FFDW, y=−0.055x+8.05, P<0.001)
28
LIFE-HISTORY STUDIES
0.5
females
males
Relative fat content
fraction
0.4
0.3
0.2
0.1
0
5
10
15
20
25
30
35
40
age (days)
Figure 2. Relation between relative fat content and age for females and
males. The line is shown for significant linear weighted regression on the
age-class means (females, %Fat, y=0.0030619x+0.1887, P<0.0001)
DISCUSSION
In the course of their life, females and males use non-fat resources as indicated by the decrease of
dry weight and fat-free dry weight decreased with age. They may use this for survival and/or
reproduction, but since the butterflies are virgins and live in single-sex groups, the emphasis will
be on survival. Relative fat content significantly increased with age in virgin females, which is in
close agreement with results from Drosophila melanogaster (e.g. Zwaan et al., 1991). This
probably reflects a general mechanism of increases in fat storage relative to reproductive output,
corroborated by the finding that relative fat content does not increase in mated females (Service,
1989) as well as by the observations that fat content and starvation resistance can be enhanced at
the expense of decreasing fecundity in phenotypic (Chippindale et al., 1993) and genetic
manipulation experiments (Zwaan et al., 1995b).
Increasing fat content is probably energetically costly but enhances survival under scarcity of
food and can be traded in for higher reproductive output under favourable conditions. Hence, the
higher relative fat content at eclosion for dry season relative to wet season butterflies (Kooi et al.,
1997) may well be an adaptation to the dry season environment. We will test this hypothesis using
classical and molecular quantitative genetic tools and plan a selection experiment on starvation
resistance. Our results show that, although fat content is increasing with age, it mainly does so
between the age of 10 to 37 days. Hence, we will establish the starvation time phenotype for 3-7
day old butterflies.
REFERENCES
Brakefield, P.M. 1997. Phenotypic plasticity and fluctuating asymmetry as responses to environmental stress
in the butterfly Bicyclus anynana. In: R. Bijlsma & Loeschcke, V. (Eds.), Environmental stress,
adaptation and evolution, pp. 66-78. Birkhauser Verlag, Basel.
Brakefield, P.M. & Kesbeke, F. 1997. Genotype-environment interactions for insect growth in constant and
fluctuating temperature regimes. Proc. R. Soc. Lond. Ser. B 264: 717-723.
Chippindale, A.K., Leroi, A.M., Kim, S.B. & Rose, M.R. 1993. Phenotypic plasticity and selection in
Drosophila life history evolution. I. Nutrition and the cost of reproduction. J. Evol. Biol. 6: 171-193.
Crawley, M.J. 1993. GLIM for ecologists. Blackwell Science, Oxford.
Fairbanks, L.D. & Burch, G.E. 1970. Rate of water loss and water and fat content of adult Drosophila
melanogaster of different ages. J. Ins. Physiol. 16: 1429-1436.
Fairbanks, L.D. & Burch, G.E. 1974. Changes with age in the ability of adult Drosophila melanogaster to
respond to yeast feeding. Physiol. Zool. 47: 190-197.
B.J. ZWAAN, A. BURGERS, F. KESBEKE & P.M. BRAKEFIELD
29
Kooi, R.E., Bergshoeff, C., Rossie, W.E.M-T. & Brakefield, P.M. 1997. Bicyclus anynana (Lepidoptera:
Satyrinae) comparison of fat content and egg-laying in relation to dry and wet season temperatures.
Proc. Exper. Appl. Entomol., NEV Amsterdam 8: 17-22.
Kooi, R.E. & Brakefield, P.M. 1999. The critical period for wing pattern induction in the polyphenic tropical
butterfly Bicyclus anynana (Satyrinae). J. Ins. Physiol. 45: 201-212.
Service, P.M. 1987. Physiological mechanisms of increased stress resistance in Drosophila melanogaster
selected for postponed senescence. Physiol. Zool. 60: 321-326.
Service, P.M. 1989. The effect of mating status on lifespan, egg laying, and starvation resistance in
Drosophila melanogaster in relation to selection on longevity. J. Ins. Physiol. 35: 447-452.
Zwaan, B., Bijlsma, R. & Hoekstra, R.F. 1995a. Artificial selection for developmental time in Drosophila
melanogaster in relation to the evolution of aging - direct and correlated responses. Evolution 49: 635648.
Zwaan, B., Bijlsma, R. & Hoekstra, R.F. 1995b. Direct selection on life-span in Drosophila melanogaster.
Evolution 49: 649-659.
Zwaan, B.J., Bijlsma, R. & Hoekstra, R.F. 1991. On the developmental theory of ageing. I. Starvation
resistance and longevity in Drosophila melanogaster in relation to preadult breeding conditions.
Heredity 68: 123-130.