Control of reproduction and a survival cost to mating in female Bicyclus anynana butterflies

Abstract.  1. Given that sexual conflict is all pervasive, investigating potential costs to mating and the control of female reproduction are important issues.
2. Here, female reproductive output and longevity are investigated in relation to mating status (virgin vs. once-mated females) and host-plant availability in the tropical butterfly Bicyclus anynana .
3. Both factors significantly affected realised fecundity in a manner demonstrating that female reproduction is not entirely under control of the female herself, but is rather subject to additive contributions of the female, her male partner, and intrinsic physiological processes. As evident from the deposition of significant egg numbers by virgin, host-deprived females, B. anynana is effectively unable to completely inhibit oogenesis.
4. Mated females suffered a reduction in adult life span, which cannot be explained as a side-effect of variation in egg size, lifetime, or early fecundity.
5. Such detrimental effects of mating per se are indicative of the cooperation–conflict balance between sexes being shifted towards conflict in B. anynana .     

Microsatellite markers associated with genes expressed in developing wings of Bicyclus anynana butterflies

Deriving useful microsatellite markers in lepidopterans has been challenging when relying on scans of genomic DNA libraries, presumably due to repetitiveness in their genomes. We assayed 96 of 320 microsatellites identified in silico from a collection of Bicyclus anynana ESTs, in 11 independent individuals from a laboratory population. From the 68 successful assays, we identified 40 polymorphic markers including 22 with BLAST-based annotation. Nine of 12 selected polymorphic markers tested in a panel of 24 wild-caught individuals converted to successful assays and were all polymorphic. We discuss how microsatellite discovery in ESTs is an efficient strategy with important attendant advantages.     

Energetics of reproduction: consequences of divergent selection on egg size, food limitation, and female age for egg composition and reproductive effort in a butterfly

Using lines artificially selected on egg size and being subjected to a restricted and an unrestricted feeding treatment, we examined the relationships between egg size, egg number, egg composition, and reproductive investment in the butterfly Bicyclus anynana . Despite a successful manipulation of egg size, correlated responses to selection in larval time, pupal mass, pupal time, longevity, fecundity, or the amount of energy allocated to reproduction were virtually absent. Thus, there was no indication for an evolutionary link between offspring size and reproductive investment. Egg composition, in contrast, was affected by selection, with larger eggs containing relatively more lipid and water, but less protein and energy compared to smaller eggs. Hence, females producing large eggs did not have to sacrifice fecundity due to adjustments in egg composition. Food limitation per se caused only minor changes in egg composition, and there was no general reduction in egg provisioning with female age. The latter was restricted to food-limited females, whereas egg quality remained remarkably similar throughout the females' life in control groups. We conclude that neglecting changes in biochemical egg composition, depending on genetic background, food availability, and female age, may introduce substantial error when estimating reproductive effort, and may ultimately lead to invalid conclusions.  © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 91 , 403–418.     

Effects of temperature on reproductive output, egg provisioning, juvenile hormone and vitellogenin titres in the butterfly Bicyclus anynana

Environmentally induced phenotypic plasticity is common in nature. Hormones, affecting multiple traits and signaling to a variety of distant target tissues, provide a mechanistic link between environments, genes and trait expression, and may therefore well be involved in the regulation phenotypic plasticity. Here, we investigate whether in the tropical butterfly Bicyclus anynana temperature-mediated plasticity in egg size and number, with fewer but larger eggs produced at lower temperatures and vice versa, is under control of juvenile hormone, and whether different temperatures cause differences in egg composition. Female B. anynana butterflies showed the expected response to temperature, however, we found no evidence for an involvement of juvenile hormone. Neither haemolymph JH II and JH III titres nor vitellogenin levels differed across temperatures. The smaller eggs produced at the higher temperature contained relatively higher amounts of water, free carbohydrates and proteins, but relatively lower amounts of lipids. While these smaller eggs had a lower absolute energy content, total reproductive investment was higher at the higher temperature (due to a higher fecundity). Overall, our study indicates that temperature-mediated plasticity in reproduction in B. anynana is mechanistically related to a biophysical model, with oocyte production (differentiation) and oocyte growth (vitellogenesis) having differential temperature sensitivities.     

Formulation of pyriproxyfen, a juvenile hormone mimic, for tsetse control

A topical dose, in 1 microliter acetone, of 0.02 microgram-2-[1-methyl-2-(4-phenoxyphenoxy) ethoxy] pyridine, the juvenile hormone mimic pyriproxyfen (S-31183, Sumitomo Chemical Co.), caused an adult female tsetse, Glossina morsitans morsitans Westwood, to produce non-viable offspring for the whole of her life. Using 14C labelled pyriproxyfen it was determined that as little as 0.001 microgram transferred to the in utero larva was sufficient to arrest development in the pupal stage. A formulation in vegetable oil was prepared for treating black cotton cloth targets which caused females to pick up 0.1 microgram active ingredient (a.i.) by tarsal contact during 1 min of exposure. Males exposed similarly for between 1 and 5 min transferred up to 0.016 microgram a.i. to females if they mated immediately after treatment. Doses as low as 0.01 micrograms in 10 microliters oil cm-2 on black cotton cloth targets caused females to produce non-viable offspring for at least two reproductive cycles following exposure. However, a dose of 0.1 microgram in 10 microliters oil cm-2 was necessary for an exposed male to cause disruption of the reproductive potential of his mate. This juvenile hormone mimic has potential to induce sterility via both sexes of tsetse using treated targets or traps under field conditions.     

Pedigree analysis on small laboratory populations of the butterfly Bicyclus anynana: The effects of selection on inbreeding and fitness

The effect of small population size and gene flow on the rate ofinbreeding and loss in fitness in Bicyclus anynana populationswas quantified by means of a pedigree analysis. Laboratorymetapopulations each consisted of four subpopulations with breeding sizeof N = 6 or N = 12 and migration rate of m = 0 or m= 0.33. Pedigrees were established by individually marking about35,000 butterflies. The increase in inbreeding coefficients(F-coefficients) over time was compared to that of simulated populationswith similar N and m. In the seventh generation, the level of inbreedingin larger subpopulations did not deviate significantly from the expectedvalues, but smaller subpopulations were less inbred than expected.Individuals in the small populations still showed considerableinbreeding depression, indicating that only a small proportion of therecessive deleterious alleles had been purged by selection. Two opposingprocesses potentially affected the rate of inbreeding and fitness: (1)Inbreeding depression increased the variance in family size and reducedthe effective population size. This will accelerate the rate ofinbreeding and is expected to selectively purge deleterious recessivealleles. (2) Variance in reproductive success of families was reducedbecause individuals which had a large number of siblings in thepopulation were more likely to mate with a full-sib than individualswith a smaller number of siblings. Subsequent inbreeding depressionreduced the number of viable offspring produced by these full-sibmatings. As a consequence, natural selection purged only some of thedeleterious alleles from the butterfly populations during sevengenerations with inbreeding. These findings emphasise the potentialproblems of using only small numbers of breeding individuals (N10) incaptive populations for conservation purposes.     

Effects of bottlenecks on quantitative genetic variation in the butterfly Bicyclus anynana

The effects of a single population bottleneck of differing severity on heritability and additive genetic variance was investigated experimentally using a butterfly. An outbred laboratory stock was used to found replicate lines with one pair, three pairs and 10 pairs of adults, as well as control lines with approximately 75 effective pairs. Heritability and additive genetic variance of eight wing pattern characters and wing size were estimated using parent-offspring covariances in the base population and in all daughter lines. Individual morphological characters and principal components of the nine characters showed a consistent pattern of treatment effects in which average heritability and additive genetic variance was lower in one pair and three pair lines than in 10 pair and control lines. Observed losses in heritability and additive genetic variance were significantly greater than predicted by the neutral additive model when calculated with coefficients of inbreeding estimated from demographic parameters alone. However, use of molecular markers revealed substantially more inbreeding, generated by increased variance in family size and background selection. Conservative interpretation of a statistical analysis incorporating this previously undetected inbreeding led to the conclusion that the response to inbreeding of the morphological traits studied showed no significant departure from the neutral additive model. This result is consistent with the evidence for minimal directional dominance for these traits. In contrast, egg hatching rate in the same experimental lines showed strong inbreeding depression, increased phenotypic variance and rapid response to selection, highly indicative of an increase in additive genetic variance due to dominance variance conversion.     

Juvadecene: Discovery of a juvenile hormone mimic in the plant,Macropiper excelsum

A compound with significant insect juvenile hormone activity was isolated from the plant, Macropiper excelsum. The chemical structure was determined by spectral methods to be 1-(3,4-methylenedioxyphenyl)-trans-3-decene(l), and confirmed by synthesis. The hormonally active substance applied topically to last (fifth)-instar nymphs of the milkweed bug (Oncopeltus fasciatus) induced a supernumerary metamorphosis at 30 μg. Higher doses were toxic.     

The use of chemical and visual cues in female choice in the butterfly Bicyclus anynana

Investigating the relative importance of multiple cues for mate choice within a species may highlight possible mechanisms that led to the diversification of closely related species in the past. Here, we investigate the importance of close-range pheromones produced by male Bicyclus anynana butterflies and determine the relative importance of these chemical cues versus visual cues in sexual selection by female choice. We first blocked putative androconial organs on the fore- and hindwings of males, while also manipulating the ability of females to perceive chemical signals via their antenna. We found that male chemical signals were emitted by both fore- and hindwing pairs and that they play an important role in female choice. We subsequently tested the relative importance of these chemical cues versus visual cues, previously identified for this species, and found that they play an equally important role in female choice in our laboratory setting. In addition, females will mate with males with only one signal present and blocking both androconial organs on males seems to interfere with male to male recognition. We discuss the possible functions of these signals and how this bimodal system may be used in intra- and interspecific mate evaluation.     

Maternal body size as an evolutionary constraint on egg size in a butterfly

Genetic and developmental constraints have often been invoked to explain patterns of existing morphologies. Yet, empirical tests addressing this issue directly are still scarce. We here set out to investigate the importance of maternal body size as an evolutionary constraint on egg size in the tropical butterfly Bicyclus anynana, employing an artificial two-trait selection experiment on simultaneous changes in body and egg size (synergistic and antagonistic selection). Selection on maternal body size and egg size was successful in both the synergistic and the antagonistic selection direction. Yet, responses to selection and realized heritabilities varied across selection regimes: the most extreme values for pupal mass were found in the synergistic selection directions, whereas in the antagonistic selection direction realized heritabilities were low and nonsignificant in three of four cases. In contrast, for egg size the highest values were obtained in the lines selected for low pupal mass. Thus, selection on body size yielded a stronger correlated response in egg size than vice versa, which is likely to bias (i.e., constrain), if weakly, evolutionary change in body size. However, it seems questionable whether this will prevent evolution toward novel phenotypes, given enough time and that natural selection is strong. Correlated responses to selection were overall weak. Egg and larval development times tended to be associated with changes in maternal size, whereas variation in pupal development times weakly tended to follow variation in egg size. Lifetime fecundity was similar across selection regimes, except for females simultaneously selected for large body mass and small egg size, exhibiting increased fecundity. Multiple regressions showed that lifetime fecundity and concomitantly reproductive investment were primarily determined by longevity, as expected for an income breeder, whereas egg size was primarily determined by pupal mass. Evidence for a phenotypic trade-off between egg size and number was weak.     

Effects of adult-derived carbohydrates, amino acids and micronutrients on female reproduction in a fruit-feeding butterfly

It is generally believed that butterflies (and other holometabolous insects) rely primarily on reserves accumulated during the larval stage for reproduction, whereas the carbohydrate-rich adult diet is thought to mainly cover energy requirements. In at least some species though, realization of the full reproductive potential is extensively affected by post-eclosion nutrition. While the importance of carbohydrates is fairly well understood, the role of adult-derived amino acids and micronutrients is controversial and largely unknown, respectively. We here focus on the effects of different adult diets on female reproduction in the tropical, fruit-feeding butterfly Bicyclus anynana (Nymphalidae). Carbohydrates were the most important adult-derived nutrients affecting reproduction. Adding amino acids, vitamins or minerals to sucrose-based solutions did not yield a reproductive output equivalent to that of fruit-fed females, which showed the highest performance throughout. This suggests that either not yet identified compounds of fruit substantially contribute to reproduction, or that resource congruence (the use of nutrient types in a specified ratio) rather than any specific nutrient component is of key importance. Apart from adult income, realized fecundity depended on egg size and longevity, with the former dominating when dietary quality was low, but the latter when quality was high. Thus, the egg size-number trade-off seems to be affected by female nutrition.     

The effect of C18 juvenile hormone and Altosid on the efficiency of egg production in Rhodnius prolixus

The product (R) of the weight of the blood meal and the initial weight of the insect is shown to be a reliable predictor of egg production. The egg production efficiency (E), defined as the number of eggs produced per R, has a value characteristic of virgin females, and another, higher, value characteristic of mated females. Topical applications of C18 JH or Altosid to virgin females increase the value of E to the mated level in a fashion which suggests that these compounds act via a trigger mechanism. These compounds do not affect the rate at which oviposition occurs.     

Effects of pyriproxyfen on ovarian development and fecundity ofRhagoletis pomonella flies

Three-day old female apple maggot flies,Rhagoletis pomonella (Walsh), were topically exposed to different doses (0.1, 1, 10, and 100 μg per fly) of a juvenile hormone mimic, pyriproxyfen, in the laboratory. Pyriproxyfen had little lethal effect on females except at the extremely high dose of 100 μg. It also had no significant effect on egg viability of treated females. A non-lethal dose of 1 μg per fly did, however, enhance significantly the fecundity (egg production) as well as the ovarian development (number of eggs in ovaries and length of egg folicles) of treated flies. We conclude that pyriproxyfen could be a useful aid in exploring endocrine regulation of feeding and reproductive physiology behavior inR. pomonella, about which current knowledge is scant.     

Effects of delayed mating on the fecundity,fertility and longevity of females of diamondback moth,Plutella xylostella

Abstract The effects of delayed mating on the copulation duration, female fertility, fecundity, egg fertility, longevity and the number days alive after mating of females of diamondback moth (DBM), Plutella xylostella, were studied. When male mating was delayed, the female fertility, fecundity, egg fertility, longevity and number days alive after mating of DBM decreased, and there was a negative correlation between the age of the moth with those variables except copulation duration. When female mating was delayed, the female fertility, fecundity, percent egg fertility and number days alive after mating of DBM also decreased, but the longevity increased, which also showed a negative relationship between the age of the moth with the variables except copulation duration and longevity. When both males and females delayed mating, the female fertility and fecundity decreased; egg fertility was affected marginally, and the longevity of females increased. The moth age was negatively correlated with those variables.     

Within- and between-generation effects of temperature on life-history traits in a butterfly

Non-genetic parental effects may largely affect offspring phenotype, and such plasticity is potentially adaptive. Despite its potential importance, little is known about cross-generational effects of temperature, at least partly because parental effects were frequently considered a troublesome nuisance, rather than a target of experimental studies. We here investigate effects of parental, developmental and acclimation temperature on life-history traits in the butterfly Bicyclus anynana. Higher developmental temperatures reduced development times and egg size, increased egg number, but did not affect pupal mass. Between-generation temperature effects on larval time, pupal time, larval growth rate and egg size were qualitatively very similar to effects of developmental temperature, and additionally affected pupal mass but not egg number. Parental effects are important mediators of phenotypic plasticity in B. anynana, and partly yielded antagonistic effects on different components of fitness, which may constrain the evolution of cross-generational adaptive plasticity.     

Genetic and environmental sources of egg size variation in the butterfly Bicyclus anynana

By dividing families of the tropical butterfly, Bicyclus anynana, among different larval (including early pupal) and adult (including late pupal) temperatures, we investigate the genetic and environmental effects on egg size. Both sources of variation affected egg size to similar extents. As previously found in other arthropods, egg size tended to increase at lower temperatures. Our data suggest that the plastic response in egg size can be induced during the pupal stage. Females reared as larvae at the same high temperature tended to lay larger eggs when transferred to a lower temperature, either as prepupae or pupae, compared to those remaining at the high temperature. Additionally, females reared as larvae at different temperatures, but maintained at the same temperature from the early pupal stage onwards, laid larger eggs after larval growth at a low temperature. Heritability estimates for egg size were about 0.4 (parent-offspring regression) and 0.2 (variance component estimates using the full-sib families). Although there seemed to be some variation in the plastic response to temperature among families, genotype-environment interactions were nonsignificant.     

The evolutionary genetics of egg size plasticity in a butterfly

Abstract The evolution of phenotypic plasticity requires that it is adaptive, genetically determined, and exhibits sufficient genetic variation. For the tropical butterfly Bicyclus anynana there is evidence that temperature-mediated plasticity in egg size is an adaptation to predictable seasonal change. Here we set out to investigate heritability in egg size and genetic variation in the plastic response to temperature in this species, using a half-sib breeding design. Egg size of individual females was first measured at a high temperature 4 days after eclosion. Females were then transferred to a low temperature and egg size was measured after acclimation periods of 6 and 12 days respectively. Overall, additive genetic variance explained only 3-11% of the total phenotypic variance, whereas maternal effects were more pronounced. Genotype-environment interactions and cross-environmental correlations of less than unity suggest that there is potential for short-term evolutionary change. Our findings strengthen the support for the adaptive nature of temperature-mediated plasticity in egg size.     

Feeding responses and food preferences in the tropical, fruit-feeding butterfly, Bicyclus anynana

In the tropical butterfly Bicyclus anynana (Nymphalidae) essential components of fitness (such as fecundity and longevity) depend to a large degree on exogenous adult-derived nutrients, particularly carbohydrates. We investigated which of the nutrients/compounds found in the adult diet act as feeding stimuli, and whether butterflies show preferences for particular nutrients or combinations. Only sugars and alcohols acted as feeding stimuli, the highest responses being found for sucrose, glucose, ethanol, butanol and propanol. Various other compounds (e.g. amino acids, acetic acid, vitamins, lipids, salts, and yeast) did not elicit any probing or feeding responses. Behavioural tests revealed a clear preference hierarchy for sugars (sucrose > glucose > fructose > maltose), but not for alcohols. Butterflies did not discriminate between sucrose solutions enriched with different nutrients and plain sucrose solutions, although they showed a preference for acetic acid and an aversion to salts and ascorbic acid when offered in combination with sucrose. Throughout, both sexes showed very similar patterns. We conclude that locating carbohydrate sources seems sufficient to cover all the butterflies’ nutritional needs, while alcohols function primarily as long range signals, guiding the butterflies to food sources. Thus, fruit-feeding butterflies, in contrast to nectar-feeding butterflies, appear not to have distinctive preferences for e.g. amino acids or salts, but do share a common primary preference for sucrose.     

Testing the beneficial acclimation hypothesis: temperature effects on mating success in a butterfly

Traditionally, it has been assumed that all acclimation changesto the phenotype enhance the performance of an individual organismin the environment in which those changes were induced (beneficialacclimation hypothesis [BAH]), a theory that has been repeatedlychallenged in recent years. We here use a full-factorial designwith 2 developmental and 2 acclimation temperatures to testtheir effects on reproductive performance in the tropical butterfly,Bicyclus anynana. Competition experiments among virgin malesfrom different thermal groups revealed that, at 20 °C, bothgroups acclimated to 20 °C achieved more than twice as manymatings as those acclimated to 27 °C, whereas at 27 °C,only one group (acclimated to 27 °C) outperformed all others.Chill-coma recovery times were also longer for butterflies thatdeveloped at higher temperatures, indicating that butterfliesresponded physiologically to the temperatures at which theywere reared. Our results support the BAH at least in part, anddo not support any alternative hypotheses.