Onthophagus Taurus Research Articles

Offensive and defensive sperm competition roles in the dung beetle Onthophagus taurus (Coleoptera: Scarabaeidae)

Sperm competition is predicted to generate opposing selection pressures on males. On one hand, selection should favour ‘defensive’ adaptations that protect a male’s ejaculate from displacement, while, on the other hand, selection should favour ‘offensive’ adaptations that overcome paternity assurance mechanisms of rivals. Here, we use the sterile male technique to assess sperm precedence when a male dung beetle Onthophagus taurus mates in both a defensive (first male) and an offensive (second male) role. Significant variation in a male’s sperm precedence (both P1 and P2) was detected, and an individual’s defensive (P1) and offensive (P2) abilities were positively correlated. Thus, it appears that sexual selection simultaneously selects for ‘defensive’ and ‘offensive’ adaptations in O. taurus. We discuss a variety of male traits in O. taurus that potentially contribute to a male’s ability to be successful when mating in an ‘offensive’ and a ‘defensive’ role.

The Contribution of Tunneling Dung Beetles to Pasture Soil Nutrition

The effect of dung beetle activity on soil nutrition was studied in three distinct soil types under laboratory conditions. Two tunneling dung beetles, Onthophagus gazella (Fabricius) and Onthophagus taurus (Schreber), were allowed to incorporate cattle dung, for brood production, into a piedmont Cecil clay soil, a coastal plain sandy-loam soil and commercially available play sand. Controlled treatments included soil alone and soil exposed to dung only. Soils were tested for primary nutrients (P and K), secondary nutrients (Ca and Mg), and micronutrients (Mn, Zn, and Cu), as well as other soil characteristics (pH, exchangeable acidity, etc.). Both O. gazella and O. taurus produced the most offspring in the piedmont clay soil; variable numbers of brood were produced in other soil types. Soils exposed to dung beetle activity were generally higher in nutrient content than both soils left untreated, and those that had been exposed to cattle dung only. In this manner, tunneling dung beetles can be considered vital to nutrient recycling and plant health in pasture systems.

The evolution of male genitalia: patterns of genetic variation and covariation in the genital sclerites of the dung beetle Onthophagus taurus

Three main hypotheses, have been invoked to explain divergent genital evolution, the lock and key, pleiotropy, and sexual selection hypotheses, each of which make different predictions about how genital traits are inherited. Here we used a half-sib breeding design to examine the patterns of genetic variation and covariation between male genital sclerites, and their covariance with general body morphology in the dung beetle Onthophagus taurus. We found CV(A)'s and CV(P)'s were similar for both genital and general morphological traits and that CV(R)'s were large for both trait types. We found that male genital sclerites were negatively genetically correlated with general morphological traits. Variation in male genital morphology has direct implications for a male's fertilization success and the resulting sexual selection acting on male genitalia is predicted to maintain high levels of additive genetic variance. Contrary to this prediction, we found that individual genital sclerites all had low levels of additive genetic variance and large maternal and environmental sources of variation. Our data suggest that the genital sclerites in O. taurus are not inherited independently but as a genetically integrated unit. More importantly, the way the different sclerites function to influence male fertilization success reflects this genetic integration. Even though levels of V(A) in individual genital sclerites may be low, there may still be sufficient V(A) in multivariate trait space for selection to generate evolutionary change in the overall morphology of male genitalia.

Relative influence of male and female genital morphology on paternity in the dung beetle Onthophagus taurus

Studies of fertilization success have demonstrated that male effects are often a strong and important source of variation in P2 (the proportion of offspring that are fertilized by the second male to mate). More recently there has been emphasis on female processes that occur during and after copulation that might bias the outcome of male-male interactions. Here we used the sterile male technique to evaluate whether female genital morphology influences the repeatability of P2 when the same pair of male dung beetles, Onthophagus taurus, copulated with a series of full-sib females or unrelated females that were all unrelated to the male pair. Repeatability estimates of measures of female genital morphology showed that full-sib females varied less in their genital morphology than did unrelated females. Therefore, if female genital traits are an important source of variation in male fertilization success, P2 should be more repeatable across full-sib than unrelated females. Contrary to this prediction, we show that the repeatability of P2 did not differ between female groups. Moreover, specific dimensions of the female genitalia (sclerotized vagina and bursa) did not contribute significantly to variance in P2. In contrast, male effects had consistent and repeatable influences on paternity across females. These were partly explained by variation in the morphology of male genital sclerites.

Seasonal Activity and Species Composition of Dung Beetles (Coleoptera: Scarabaeidae and Geotrupidae) Inhabiting Cattle Pastures in North Carolina

Species composition and seasonal distribution of dung beetles were studied on dairy and beef cattle pastures in North Carolina. Study sites included a dairy located in the piedmont region (North Carolina Department of Agriculture Piedmont Research Station, Salisbury, NC) and a combined dairy/beef facility in the coastal plain (North Carolina Department of Agriculture Center for Environmental Farming Systems, Goldsboro, NC). Dung beetles were trapped in cattle pastures from March 2002 through September 2003 by using dung-baited pitfall traps. Trapping yielded 4,111 beetles representing 14 species from the piedmont dairy, including Aphodius prodromus Brahm, a new record for North Carolina. Totals of 57,026 beetles representing 28 species and 28,857 beetles representing 26 species were trapped from the dairy unit and beef unit in the coastal plain site, respectively. Onthophagus gazella (F.), a second new record for North Carolina, was collected from the coastal plain. Beetles common to all collection sites include Aphodius erraticus (L.), Aphodius fimetarius (L.), Aphodius granarius (L.), Aphodius pseudolividus Balthasar, Onthophagus taurus Schreber, Onthophagus hecate hecate Panzer, and Onthophagus pennsylvanicus Harold. The introduced beetle O. taurus dominated the dung beetle population, accounting for >50% of the total beetles caught at either site. Beetle activity was greatest from March until November, with activity declining during the winter. Nine exotic species in the genera Onthophagus and Aphodius represented nearly 95% of the beetles trapped.

Matters of Scale: Positive Allometry and the Evolution of Male Dimorphisms

The developmental independence of alternative phenotypes is key to evolutionary theories of phenotypic plasticity and the origins of diversity. Male dimorphisms associated with alternative reproductive tactics are widely cited examples of such facultative expression of divergent fitness optima. Current models for the evolution of male dimorphisms invoke a size-dependent threshold at which the phenotype is reprogrammed. We use predictions derived from allometric modeling to test for the existence of reprogramming thresholds in two species of beetle, Onthophagus taurus and Onthophagus binodis, and the European earwig Forficula auricularia. We also compare the allometry of a number of morphological traits to determine whether minor males suppress their secondary sexual traits. The intercept of the horn allometry was suppressed, but there was no evidence of reprogramming of horn growth in either beetle species. There was reprogramming in the earwig. In the beetles, the horn length in all males can be explained largely in terms of exponential horn growth following an extraordinarily steep power function. The asymptote in O. taurus can be explained by exponential growth meeting the constraint of resource exhaustion. These findings question the currently held view that beetle horn dimorphisms showcase the importance of developmental independence in the evolution of diversity.

Genetic analysis of parentage within experimental populations of a male dimorphic beetle, Onthophagus taurus, using amplified fragment length polymorphism

We used the multilocus DNA fingerprinting technique, amplified fragment length polymorphism, to examine parentage of 902 offspring from eight experimental populations of the dung beetle, Onthophagus taurus. The males of this species exhibit a suit of morphological and behavioural traits that characterise alternative mating tactics. Hornless minor males sneak copulations with females that are guarded by horned major males. Our aims were to provide a prospective assessment of the potential role of frequency dependence in the maintenance of alternative mating tactics, to assess the levels of polyandry, and to determine the patterns of sperm usage by multiply mated females. The average proportion of offspring sired by major and minor males did not co-vary with the relative frequency of each morph present in experimental populations. However, there was some indication that the effective mating frequency (number of females producing offspring sired by a given male) of major and minor males may exhibit frequency dependence. Mating success of both male types declined with increasing numbers of major males. Paternity was positively associated with effective mating frequency. Females produced offspring sired from between one and eight males and, on average, paternity was distributed equally amongst a female’s mates, regardless of the number of males mated. Differences in fertilisation success among males were not associated with alternative male phenotypes. Neither did a males fertilisation success depend on his genetic dissimilarity with the female. These results are discussed in the context of the evolution of alternative mating strategies, and mechanisms of postcopulatory sexual selection.

Optimal maternal investment in the dung beetle Onthophagus taurus ?

Optimal investment theory is based on the assumption that the proximate constraint acting on parental investment is resource based. A trade-off between per offspring investment and total investment seems intuitive. Consequently, a parent’s investment strategy is expected to represent a trade-off between the benefits of investment for current offspring and the costs to future reproduction for parents. In this study, we provide clear evidence that the costs and benefits of maternal provisioning in the dung beetle Onthophagus taurus influence the amount of provisions provided by the mother. Horse dung is typically of a higher nutritional value than cow dung and females were shown to provide 20% less dung to offspring when provisioning with horse dung. By reducing their investment per offspring and exhibiting a clear preference to provision offspring with horse dung, females were able to produce significantly more offspring. Females provisioning with horse dung received greater fitness returns per unit of investment and experienced lower provisioning costs, in terms of the minimum amount of dung required to produce a surviving offspring, than females provisioning with cow dung. Females provisioning in soil of low moisture content were found to have higher tunneling costs than those provisioning in soil of high moisture content, while the fitness returns per unit of investment did not differ. We adopted a marginal value theorem (MVT) approach to calculate the theoretical optimal level of investment for each dung type and for each soil moisture. Predicted levels of provisioning were lower for horse dung than for cow dung and for moist soil than for dry soil. Therefore, the results of this study are in qualitative agreement with MVT predictions and provide empirical support for the proposal that females can adaptively adjust their level of investment in response to resource and/or habitat quality. However, the theoretically predicted optimal investment yielded a poor quantitative fit with our observed levels of investment, with females providing over twice the investment predicted by the MVT approach. We suggest that this difference may reflect either our inability in directly quantifying all the necessary costs and benefits of investment in O. taurus and/or the applicability of the underlying assumptions of MVT.

The behavioral ecology of threshold evolution in a polyphenic beetle

Facultative expression of alternative male morphologies is thought to allow individual males to select the phenotype with the highest fitness gain given their competitive status relative to other males with which they compete for females. Choice of, or switching between, morphs commonly relies on developmental threshold responses. Evolutionary changes in developmental threshold responses are thought to provide an important avenue for phenotypic diversification and the evolution of morphological and behavioral novelties. However, the extent to which alternative male phenotypes and their underlying threshold responses actually evolve in natural populations is unclear. Likewise, the ecological factors that shape the evolution of threshold responses in natural populations are unexplored for most organisms, as are the consequences of such modifications for patterns of morphological diversity. I examined the ecological basis of rapid threshold evolution in exotic populations of the horn-polyphenic dung beetle Onthophagus taurus. Male O. taurus vary continuously in body size as a function of larval feeding conditions. Only males that exceed a critical threshold body size develop a pair of long horns on their heads, whereas males below this threshold remain hornless. Populations in two exotic ranges of this species, the eastern United States and western Australia, have diverged in the mean threshold body size, which has resulted in the evolution of highly divergent and novel horn length–body size allometries in these populations. Populations in a third and previously unstudied exotic range of O. taurus in eastern Australia exhibit threshold body sizes roughly intermediate between the eastern U.S. and western Australian populations. I tested three hypothesis to explain how differences in ecological and demographic factors can drive allometric divergences between populations, using data derived from comparative, standardized sampling of a large number of populations in each exotic range. Results suggest that differences in the intensity of both intra- and interspecific competition have contributed to the evolution of divergent thresholds in these populations. My results do not support the hypothesis that shifts in threshold body sizes to larger body sizes are a consequence of increases in the mean body size of competing males. I discuss my results in the context of Onthophagus mating systems and the evolutionary implications of threshold evolution.

Males influence maternal effects that promote sexual selection: a quantitative genetic experiment with dung beetles Onthophagus taurus.

Recently, doubt has been cast on studies supporting good genes sexual selection by the suggestion that observed genetic benefits for offspring may be confounded by differential maternal allocation. In traditional analyses, observed genetic sire effects on offspring phenotype may result from females allocating more resources to the offspring of attractive males. However, maternal effects such as differential allocation may represent a mechanism promoting genetic sire effects, rather than an alternative to them. Here we report results from an experiment on the horned dung beetle Onthophagus taurus, in which we directly compare genetic sire effects with maternal effects that are dependent on sire phenotype. We found strong evidence that mothers provide more resources to offspring when mated with large-horned males. There were significant heritabilities for both horn length and body size, but when differential maternal effects were controlled, the observed estimates of genetic variance were greatly reduced. Our experiment provides evidence that differential maternal effects may amplify genetic effects on offspring traits that are closely related to fitness. Thus, our results may partly explain the relatively high coefficients of additive genetic variation observed in fitness-related traits and provide empirical support for the theoretical argument that maternal effects can play an important role in evolution.

Genital morphology and fertilization success in the dung beetle Onthophagus taurus: an example of sexually selected male genitalia.

In animals with internal fertilization and promiscuous mating, male genitalia show rapid and divergent evolution. Three hypotheses have been suggested to explain the evolutionary processes responsible for genital evolution: the lock-and-key hypothesis, the pleiotropy hypothesis and the sexual-selection hypothesis. Here, we determine whether variation in male genital morphology influences fertilization success in the dung beetle Onthophagus taurus, as predicted by the sexual-selection hypothesis. Variation in four out of five genital sclerites of the endophallus influenced a male's fertilization success, supporting the general hypothesis that male genitalia can evolve under sexual selection. Furthermore, different genital sclerites were found to enhance first versus second male paternity, indicating that different sclerites serve offensive and defensive roles. Genital-trait variability was comparable to that in other species but was less variable than a non-genital sexually selected trait (head horns). We suggest that directional selection for genital elaboration may be countered by natural selection, which should favour genitalia of a size and shape necessary for efficient coupling and sperm transfer.

Sexual selection and condition dependence of courtship display in three species of horned dung beetles

Sexual selection has traditionally been divided into competition over mates and mate choice. Currently, models of sexual selection predict that sexual traits are expressed in proportion to the condition of their bearer. In horned beetles, male contest competition is well established, but studies on female preferences are scarce. Here I present data on male mating success and condition dependence of courtship rate in three species of horn-dimorphic dung beetles, Onthophagus taurus, Onthophagus binodis, and Onthophagus australis. I found that in the absence of male contest competition, mating success of O. taurus and O. australis was unrelated to their horn length and body size, whereas in O. binodis horn size had a negative effect but body size had a positive effect on male mating success. Overall, in O. binodis major morph males had greater mating success than minor morph males. In all three species male mating success was affected by courtship rate, and the courtship rate was condition dependent such that when males were manipulated to be in poor condition they had lower courtship rates than males that were manipulated to be in good condition. My findings provide new insight into the mating systems of horned dung beetles and support an important assumption in indicator models of sexual selection.

Confidence of paternity and paternal care: covariation revealed through the experimental manipulation of the mating system in the beetle Onthophagus taurus

Abstract Theoretical models of paternal care predict that facultative reductions in male care may occur under certain conditions. One important parameter that has been shown to influence the outcome of these models is a male's confidence of paternity. In this study, we tested whether the amount of care provided by horned males in the dimorphic beetle, Onthophagus taurus, varied with his confidence of paternity. Male care results in an increased weight of dung provided in the brood masses produced by the pair. Using the sterile male technique we showed that a horned male's paternity declined with the number of sneak males in the population. The relationship was nonlinear, with paternity declining most rapidly between a frequency of one and three sneaks, and stabilizing thereafter at about 50%. A horned male's paternity was directly related to the number of copulations with the female, relative to the number of copulations achieved by sneaks. Horned males were shown to reduce their care in relation to their declining paternity. Video analysis demonstrated that reductions in male care occurred through a combination of male desertion and a trade-off between caring and paternity assurance behaviours. The number of fights with sneak males was negatively related to the amount of care provided by a horned male. These results suggest that by gauging his expected paternity through the number of fights with sneaks, a horned male is able to assess his paternity and reduce his investment accordingly. Our data thus provide strong empirical support for the proposed link between paternity and paternal care.

Evolution of ejaculates: patterns of phenotypic and genotypic variation and condition dependence in sperm competition traits.

Sperm competition is widely recognized as a potent force in evolution, influencing male behavior, morphology, and physiology. Recent game theory analyses have examined how sperm competition can influence the evolution of ejaculate expenditure by males and the morphology of sperm contained within ejaculates. Theoretical analyses rest on the assumption that there is sufficient genetic variance in traits important in sperm competition to allow evolving populations to move to the evolutionarily stable equilibrium. Moreover, patterns of genotypic variation can provide valuable insight into the nature of selection currently acting on traits. However, our knowledge of genetic variance underlying traits important in sperm competition is limited. Here we examine patterns of phenotypic and genotypic variation in four sperm competition traits in the dung beetle Onthophagus taurus. Testis weight, ejaculate volume, and copula duration were found to have high coefficients of additive genetic variation (CV(A)S), which is characteristic of fitness traits and traits subject to sexual selection. Heritabilities were high, and there was some evidence for Y-linked inheritance in testis weight. In contrast, sperm length had a low CV(A), which is characteristic of traits subject to stabilizing selection. Nevertheless, there was little residual variance so that the heritability of sperm length exceeded 1.0. Such a pattern is consistent with Y-linked inheritance in sperm length. Interestingly, we found that testis weight and sperm length were genetically correlated with heritable male condition. This finding holds important implications for potential indirect benefits associated with the evolution of polyandry.

Developmental mechanisms of threshold evolution in a polyphenic beetle.

Polyphenic development is thought to play a pivotal role in the origin of morphological novelties. However, little is known about how polyphenisms evolve in natural populations, the developmental mechanisms that may mediate such evolution, and the consequences of such modification for patterns of morphological variation. Here we examine the developmental mechanisms of polyphenism evolution in highly divergent natural populations of the dung beetle, Onthophagus taurus. Males of this species express two alternative morphologies in response to larval feeding conditions. Favorable conditions cause males to grow larger than a threshold body size and to develop a pair of horns on their heads. Males that encounter relatively poor conditions during larval life do not reach this threshold size and remain hornless. Exotic populations of O. taurus have diverged dramatically in body size thresholds in less than 40 years since introduction to new habitats, resulting in the expression of highly divergent and novel horn length-body size scaling relationships in these populations. Here we show that larvae of populations that have evolved a larger threshold body size (1) have to accumulate greater mass to become competent to express the horned morph, (2) require more time to complete the final instar, (3) are less sensitive to the juvenile hormone (JH) analogue methoprene, and (4) exhibit a delay in the sensitive period for methoprene relative to other developmental events. JH has been shown previously to control horn expression in this species. Our results show that threshold evolution may be mediated via changes in the degree and timing of sensitivity to JH and may result in correlated changes in the dynamics and duration of larval development. Strain-specific differences in JH sensitivity have previously been demonstrated in other insects. However, to the best of our knowledge this is the first demonstration that changes in the timing of the sensitive period for JH may play an equally important role in the evolution of novel thresholds. We discuss our findings in the context of the developmental regulatory mechanisms that underlie polyphenic development and use our results to explore the consequences of, and constraints on, polyphenism evolution in nature.

Behavioural dynamics of biparental care in the dung beetle Onthophagus taurus

In the dimorphic dung beetle Onthophagus taurus major males provide assistance during offspring provisioning. We examined the behavioural dynamics of biparental care to quantify directly how males and females allocate time to parental and nonparental behaviours and to determine whether parents adjust their level of investment relative to their partner's contribution. Females allocated more of their time budget to parental behaviours than males. The proportion of time females allocated to parental behaviours increased after oviposition while that of a male decreased. Male paternity assurance behaviours were negatively associated with male and female parental behaviours. Theoretical models predict that the investment provided by the members of a cooperative pair should be negatively correlated and that any shortfall of one parent should be partially compensated for by the other. In the absence of a male, unassisted females allocated more time to parental care, and performed more parental behaviours. However, compensation was incomplete as unassisted females performed fewer parental behaviours than pairs, resulting in significantly lighter brood masses (the egg and its associated dung supply). Males performed more parental behaviours when paired with small females, and small females more than large females. Contrary to prediction, the investments provided by males and females in a cooperative pair were positively correlated. Males coordinated their parental behaviours with the females rather than acting independently. Since parental behaviours were directly related to the weight of brood masses, the observed parental interactions will have important fitness consequences in this species. Copyright 2002 The Association for the Study of Animal Behaviour. Published by Elsevier Science Ltd. All rights reserved.

The genetics of maternal care: direct and indirect genetic effects on phenotype in the dung beetle Onthophagus taurus.

While theoretical models of the evolution of parental care are based on the assumption of underlying genetic variance, surprisingly few quantitative genetic studies of this life-history trait exist. Estimation of the degree of genetic variance in parental care is important because it can be a significant source of maternal effects, which, if genetically based, represent indirect genetic effects. A major prediction of indirect genetic effect theory is that traits without heritable variation can evolve because of the heritable environmental variation that indirect genetic effects provide. In the dung beetle, Onthophagus taurus, females provide care to offspring by provisioning a brood mass. The size of the brood mass has pronounced effects on offspring phenotype. Using a half-sib breeding design we show that the weight of the brood mass females produce exhibits significant levels of additive genetic variance due to sires. However, variance caused by dams is considerably larger, demonstrating that maternal effects are also important. Body size exhibited low additive genetic variance. However, body size exerts a strong maternal influence on the weight of brood masses produced, accounting for 22% of the nongenetic variance in offspring body size. Maternal body size also influenced the number of offspring produced but there was no genetic variance for this trait. Offspring body size and brood mass weight exhibited positive genetic and phenotypic correlations. We conclude that both indirect genetic effects, via maternal care, and nongenetic maternal effects, via female size, play important roles in the evolution of phenotype in this species.

A cost of maternal care in the dung beetle Onthophagus taurus?

Abstract Parental care theory assumes that investment in current offspring will trade against future investment. A number of field studies on birds have used clutch size manipulations to demonstrate a survival cost to chick rearing. However, such studies do not account for costs accrued during earlier stages of reproduction because not all aspects of reproductive effort are manipulated by varying the number of nestlings. In this study, we investigate the effect of reproductive effort on female survival in the dung beetle, Onthophagus taurus. By experimentally manipulating mating status and dung availability, we demonstrate that virgin females survive longer than mated females and that the survival of mated females was negatively associated with the number of brood masses produced. Using a novel manipulation of the mating system, we separated the effects of egg production and maternal care on female survival. Previously, we have shown that females provisioning with the assistance of a major male provide relatively less care than unassisted females. However, paternal assistance did not alter the number of brood masses produced and hence the amount of reproductive effort that was allocated to egg production. Therefore, our finding that female survival was increased when receiving paternal assistance provides, to our knowledge, the first definitive evidence that maternal care reduces female lifespan. These results are of major importance to theoretical models on the evolution of parental care.

EVOLUTION OF EJACULATES: PATTERNS OF PHENOTYPIC AND GENOTYPIC VARIATION AND CONDITION DEPENDENCE IN SPERM COMPETITION TRAITS

Sperm competition is widely recognized as a potent force in evolution, influencing male behavior, morphology, and physiology. Recent game theory analyses have examined how sperm competition can influence the evolution of ejaculate expenditure by males and the morphology of sperm contained within ejaculates. Theoretical analyses rest on the assumption that there is sufficient genetic variance in traits important in sperm competition to allow evolving populations to move to the evolutionarily stable equilibrium. Moreover, patterns of genotypic variation can provide valuable insight into the nature of selection currently acting on traits. However, our knowledge of genetic variance underlying traits important in sperm competition is limited. Here we examine patterns of phenotypic and genotypic variation in four sperm competition traits in the dung beetle Onthophagus taurus. Testis weight, ejaculate volume, and copula duration were found to have high coefficients of additive genetic variation (CVAs), which is characteristic of fitness traits and traits subject to sexual selection. Heritabilities were high, and there was some evidence for Y-linked inheritance in testis weight. In contrast, sperm length had a low CVA, which is characteristic of traits subject to stabilizing selection. Nevertheless, there was little residual variance so that the heritability of sperm length exceeded 1.0. Such a pattern is consistent with Y-linked inheritance in sperm length. Interestingly, we found that testis weight and sperm length were genetically correlated with heritable male condition. This finding holds important implications for potential indirect benefits associated with the evolution of polyandry.

Hormonal control of male horn length dimorphism in Onthophagus taurus (Coleoptera: Scarabaeidae): a second critical period of sensitivity to juvenile hormone

Male dung beetles ( Onthophagus taurus) facultatively produce a pair of horns that extend from the base of the head: males larger than a threshold body size develop long horns, whereas males that do not achieve this size develop only rudimentary horns or no horns at all. Using topical applications of methoprene, we identified a sensitive period during the feeding stage of third (final) instar larvae when application of methoprene shifted the threshold body size for horn expression. Male larvae that received methoprene at this time delayed horn production until they attained a larger threshold body size than acetone-treated control larvae. This new sensitive period occurs earlier than a sensitive period previously reported for male horn regulation, and it coincides with a morph-specific pulse of ecdysteroid secretion described for this species. It appears that male horn expression is influenced by endocrine events at two different periods of larval development. We incorporate these results into an expanded model for the endocrine regulation of male horn expression.