Juvenile Hormone Titer Research Articles

Molting dynamics and juvenile hormone titer profiles in the nymphal stages of a lower termite, Cryptotermes secundus (Kalotermitidae) – signatures of developmental plasticity

Termites are social cockroaches and this sociality is founded on a high plasticity during development. Three molting types (progressive, stationary and regressive molts) are fundamental to achieve plasticity during alate/sexual development, and they make termites a major challenge to any model on endocrine regulation in insect development. As the endocrine signatures underpinning this plasticity are barely understood, we studied the developmental dynamics and their underlying juvenile hormone (JH) titers in a wood-dwelling termite, Cryptotermes secundus, which is characterized by an ancestral life style of living in dead wood and individuals being totipotent in development. The following general pattern elements could be identified during winged sexual development (i) regressive molts were accompanied by longer intermolt periods than other molting types, (ii) JH titers decreased gradually during the developmental transition from larva (immatures without wing buds), to nymph (immatures with wing buds), to winged adult, (iii) in all nymphal stages, the JH titer rose before the next molt and dropped thereafter within the first week, (iv) considerable variation in JH titers occurred in the midphase of the molting cycle of the 2nd and 3rd nymphal instar, inferring that this variation may reflect the underlying endocrine signature of each of the three molting types, (v) the 4th nymphal instar, the shortest of all, seems to be a switch point in development, as nymphs in this stage mainly developed progressively. When comparing these patterns with endocrine signatures seen in cockroaches, the developmental program of Cryptotermes can be interpreted as a co-option and repetitive use of hormonal dynamics of the post dorsal-closure phase of cockroach embryonic development.

Effects of Long Distance Transportation on Honey Bee Physiology

Despite the requirement of long distance transportation of honey bees used for pollination, we understand little how transportation affects honey bees. Three trials in three different states (CA, GA, and MI) were conducted to study the effects of long distance transportation on honey bee physiology. Newly emerged bees from one colony were split into two groups and introduced into a transported (T) colony or a stationary (S) colony in each trial. Volumes of hypopharyngeal gland acini in T colonies were significantly smaller than S colonies in all three trials. There were no significant differences between S and T colonies in juvenile hormone titers. Protein content in head showed no significant differences between S and T either in 7-day-old or 17-day-old bees of MI trial, but GA trial showed a significant reduction in bees experiencing transportation. Protein content in thorax was only measured in GA trial and was not significantly different between the two groups. Lipid content in abdomen was not significantly different between the S and T colonies in all three trials. This study suggests that bees experiencing transportation have trouble fully developing their food glands and this might affect their ability to nurse the next generation of workers.

Juvenile hormone titre and related gene expression during the change of reproductive modes in the pea aphid

Most aphids show reproductive polyphenism, i.e. they alternate their reproductive modes from parthenogenesis to sexual reproduction in response to short photoperiods. Although juvenile hormone (JH) has been considered a likely candidate for regulating the transition from asexual to sexual reproduction after photoperiod sensing, there are few studies investigating the direct relationship between JH titres and the reproductive-mode change. In addition, the sequencing of the pea aphid genome has allowed identification of the genes involved in the JH pathway, which in turn allows us to examine their expression levels in relation to the reproductive-mode change. Using liquid chromatography-mass spectrometry in the pea aphid, JHIII titre was shown to be lower in aphids producing sexual morphs under short-day conditions than in aphids producing parthenogenetic morphs under long-day conditions. The expression levels of genes upstream and downstream of JH action were quantified by real-time quantitative reverse-transcription-PCR across the reproductive-mode change. The expression level of JH esterase, which is responsible for JH degradation, was significantly higher in aphids reared under short-day conditions. This suggests that the upregulation of the JH degradation pathway may be responsible for the lower JHIII titre in aphids exposed to short-days, leading to the production of sexual morphs.

The influence of drone physical condition on the likelihood of receiving vibration signals from worker honey bees, Apis mellifera

Honey bee workers will perform vibration sig- nals on adult drones, which respond by increasing the time spent receiving trophallaxis. Because trophallaxis provides the proteins for sexual maturation, workers could direct vibration signals towards drones showing certain physical characteristics, potentially influencing drone development and colony reproductive output. We examined the influence of drone condition on the likelihood of receiving vibration signals by comparing body weight, protein concentrations, and hemolymph juvenile hormone (JH) titers between drones that received the vibration signal and same-age, non- vibrated controls. Vibrated and control drones did not differ in total body weight, abdomen weight, abdomen-to-body weight ratio, total protein concentrations, or hemolymph JH titers. In contrast, vibrated drones had significantly lower thorax weight and smaller thorax-to-body weight ratios compared with controls. Because relative thorax weight may affect flight ability and mating success, workers could use the vibration signal to increase the care received by less developed drones, potentially contributing to the production of greater numbers of competitive males. However, the differences in thorax weights, while significant, were very small, and it is unknown how such slight differences might be assessed by workers or affect drone performance. Nev- ertheless, vibration signals performed on drones may pro- vide opportunities for exploring the effect of the quality of reproductive individuals on caste interactions in honey bees.

Relationships between frontal‐gland formation and mandibular modification during JH III‐induced presoldier differentiation in the termite Reticulitermes speratus (Isoptera: Rhinotermitidae)

AbstractTermite soldiers engage in colony defense and they possess weapons in order to attack enemies. Defensive strategies vary among species depending on the morphology of the soldier's weapons. Both the frontal gland and elongated mandibles are formed during soldier differentiation of Reticulitermes speratus workers, which is associated with an increase in juvenile hormone (JH) titer. It was shown that the amount of JH applied and coexistence with soldiers affected mandibular elongation of the induced presoldiers. To determine the relationship between applied JH concentration and frontal‐gland development of R. speratus, mandibular modification and frontal‐gland formation of presoldiers induced by 20, 40 and 80 µg JH III in the presence or absence of soldiers were observed. Both presoldier differentiation rates and their mandibular lengths were affected by the presence of soldiers in treatments with 20 and 40 µg JH III. Especially in the treatment with 20 µg JH III, frontal‐pore malformation was observed in some presoldiers. All presoldiers with a malformed frontal pore had almost the same sized mandibles, compared with those of stationarily molted workers. Interestingly, sections of these presoldiers showed that frontal‐gland invagination occurred incompletely in comparison with presoldiers with relatively elongated mandibles. Consequently, frontal‐gland formation might be affected by JH titer (applied and endogenous JH), and individuals with a malformed frontal pore were probably differentiated by JH titers only slightly above the threshold for presoldier differentiation.

The TUNEL assay suggests mandibular regression by programmed cell death during presoldier differentiation in the nasute termite Nasutitermes takasagoensis

Termite soldiers are the most specialized caste of social insects in terms of their morphology and function. Soldier development requires increased juvenile hormone (JH) titer and the two molts via a presoldier stage. These molts are accompanied by dramatic morphological changes, including the exaggeration and regression of certain organs. Soldiers of the most apical termitid subfamily Nasutitermitinae possess not only a horn-like frontal tube, called the nasus, for the projection of defensive chemicals from the frontal gland reservoir but also regressed mandibles. Although candidate genes regulating soldier mandibular growth were reported in a relatively basal termite species, the regulatory mechanisms of mandibular regression remain unknown. To clarify these mechanisms, we performed morphological and histological examinations of the mandibles during soldier differentiation in Nasutitermes takasagoensis. Mandibular size reduced dramatically during soldier differentiation, and mandibular regression occurred just prior to the presoldier molt. Spotted TUNEL signals were observed in regressing mandibles of presoldiers, suggesting that the regression involved programmed cell death. Because soldiers of N. takasagoensis possess exaggerated organs (nasus and frontal gland), the present results suggest that JH-dependent regressive mechanisms exist in the mandibles without interfering with the formation of the exaggerated organs.

Juvenile Hormone Regulates Extreme Mandible Growth in Male Stag Beetles

The morphological diversity of insects is one of the most striking phenomena in biology. Evolutionary modifications to the relative sizes of body parts, including the evolution of traits with exaggerated proportions, are responsible for a vast range of body forms. Remarkable examples of an insect trait with exaggerated proportions are the mandibular weapons of stag beetles. Male stag beetles possess extremely enlarged mandibles which they use in combat with rival males over females. As with other sexually selected traits, stag beetle mandibles vary widely in size among males, and this variable growth results from differential larval nutrition. However, the mechanisms responsible for coupling nutrition with growth of stag beetle mandibles (or indeed any insect structure) remain largely unknown. Here, we demonstrate that during the development of male stag beetles (Cyclommatus metallifer), juvenile hormone (JH) titers are correlated with the extreme growth of an exaggerated weapon of sexual selection. We then investigate the putative role of JH in the development of the nutritionally-dependent, phenotypically plastic mandibles, by increasing hemolymph titers of JH with application of the JH analog fenoxycarb during larval and prepupal developmental periods. Increased JH signaling during the early prepupal period increased the proportional size of body parts, and this was especially pronounced in male mandibles, enhancing the exaggerated size of this trait. The direction of this response is consistent with the measured JH titers during this same period. Combined, our results support a role for JH in the nutrition-dependent regulation of extreme mandible growth in this species. In addition, they illuminate mechanisms underlying the evolution of trait proportion, the most salient feature of the evolutionary diversification of the insects.

Regulation of Migration inMythimna separata(Walker) in China: A Review Integrating Environmental, Physiological, Hormonal, Genetic, and Molecular Factors

Each year the Mythimna separate (Walker), undertakes a seasonal, long-distance, multigeneration roundtrip migration between southern and northern China. Despite its regularity, the decision to migrate is facultative, and is controlled by environmental, physiological, hormonal, genetic, and molecular factors. Migrants take off on days 1 or 2 after eclosion, although the preoviposition period lasts ≈7 d. The trade-offs among the competing physiological demands of migration and reproduction are coordinated in M. separata by the "oogenesis-flight syndrome." Larvae that experience temperatures above or below certain thresholds accompanied by appropriate humidity, short photoperiod, poor nutrition, and moderate density tend to develop into migrants. However, there is a short window of sensitivity within 24 h after adult eclosion when migrants can be induced to switch to reproductive residents if they encounter extreme environmental factors including starvation, low temperature and long photoperiod. Juvenile hormone (JH) titer is low before migration but high titers are associated with termination of migratory behavior and the switch to reproduction. Early release of JH by the corpora allata in environmentally stressed 1-d old adults, otherwise destined by larval conditions to be migrants, switches them to residents. Offspring inherit parental additive genetic effects governing migratory behavior. However, they also retain flexibility in expression of both flight and reproductive life history traits. The insect neuropeptide, allatotropin, which activates corpora allata to synthesize JH, controls adult flight and reproduction. Future research directions to better understand regulation of migration in this species are discussed.

Intergenerational effect of juvenile hormone on offspring in Pogonomyrmex harvester ants

Parents can influence the phenotypes of their offspring via a number of mechanisms. In harvester ants, whether female progeny develop into workers or daughter queens is strongly influenced by the age and temperature conditions experienced by their mother, which is associated with variation in maternal ecdysteroid deposition in fertilized eggs. In many insects, juvenile hormone (JH) is antagonistic to ecdysteroid release, suggesting that seasonal and age-based variation in maternal JH titers may explain maternal effects on offspring size and reproductive caste. To test this hypothesis, we artificially increased maternal JH titers with methoprene, a JH analog, in laboratory colonies of two Pogonomyrmex populations exhibiting genetic caste determination. Increasing maternal JH resulted in a 50% increase in worker body size, as well as a sharp reduction in total number of progeny reared, but did not alter the genotype of progeny reared to adulthood. The intergenerational effect of JH manipulation was not mediated by a reduction in ecdysteroid deposition into eggs; instead, changes in egg size, trophic egg availability or brood/worker ratio may have altered the nutritional environment of developing larvae. Egg ecdysteroid content was significantly negatively correlated with natural variation in worker body size, however, suggesting that there are multiple independent routes by which queens can modify offspring phenotypes.

Juvenile hormone titer and advertised quality are associated with timing of early spring activity in Polistes dominulus foundresses

Timing of diapause termination has an important influence on individual reproductive success, but relatively little research has explored how individuals differ in their response to diapause termination cues. We tested individual variation in the timing of post-diapause activity in Polistes dominulus paper wasps. Wasps were overwintered in a temperature-controlled chamber. In the spring, ambient temperature was gradually increased and the time each foundress became active was recorded. Timing of post-diapause activity was most strongly associated with the facial patterns that function as a conventional signal of quality. Foundresses with facial patterns indicating high quality became active at lower temperatures than individuals with facial patterns indicating low quality. Early diapause termination is associated with dominance, so the relationship between diapause termination and facial patterns provides a mechanism linking facial patterns with dominance. Body weight and mating status did not influence timing of post-diapause activity. Juvenile hormone (JH) titer at the time of diapause termination was also measured in a subset of foundresses. There was no JH titer threshold for diapause termination. Instead, our results suggest that individuals may have different threshold responses to JH, as individuals that became active at a lower temperature had lower JH titers than individuals that became active at a higher temperature. Overall, there is substantial individual variation in response to diapause termination cues and the variation is likely to have important impacts on the fitness of nest-founding females.

Juvenile hormone changes associated with diapause induction, maintenance, and termination in the beet webworm, Loxostege sticticalis (lepidoptera: pyralidae)

At 22°C and under a long-day photoperiod of L:D 16:8, all the last fifth instar Loxostege sticticalis larvae undergo prepupal stage and pupate without diapause. Under a short-day photoperiod of L:D 12:12, in contrast, they all enter diapause with approximately 36 days diapause maintenance and then terminate diapause spontaneously, although only 44% of the larvae terminated diapause successfully. Changes in hemolymph juvenile hormone (JH I) titers of diapause-destined larvae across diapause induction, maintenance and termination were examined using HPLC, and were compared with those of non-diapause-destined larvae from the fifth instar through pupation. JH I titer of the earliest fifth instar diapause-destined larvae remained at a high level with a peak of 220.4 ng/ml, though it decreased continuously to a minimum of 69.0 ng/ml on day 5 in the fifth instar when the larvae stopped feeding to enter diapause. During the diapause maintenance, JH I titer of the mature larvae increased significantly and maintained a high level until day 31 in prepupae. JH I titer declined and fluctuated at low level from 5 days before pupation. In contrast, JH I titer of both the fifth instar non-diapause-destined larvae and prepupae remained and fluctuated at low level consistently, as well as decreased before pupation. These results indicate that diapause induction and maintenance in this species might be a consequence of high JH, whereas diapause termination can be attributed to low JH titer, which was in agreement with the hormonal regulation observed in many other larval-diapausing insects.

Royalactin induces queen differentiation in honeybees

The honeybee (Apis mellifera) forms two female castes: the queen and the worker. This dimorphism depends not on genetic differences, but on ingestion of royal jelly, although the mechanism through which royal jelly regulates caste differentiation has long remained unknown. Here I show that a 57-kDa protein in royal jelly, previously designated as royalactin, induces the differentiation of honeybee larvae into queens. Royalactin increased body size and ovary development and shortened developmental time in honeybees. Surprisingly, it also showed similar effects in the fruitfly (Drosophila melanogaster). Mechanistic studies revealed that royalactin activated p70 S6 kinase, which was responsible for the increase of body size, increased the activity of mitogen-activated protein kinase, which was involved in the decreased developmental time, and increased the titre of juvenile hormone, an essential hormone for ovary development. Knockdown of epidermal growth factor receptor (Egfr) expression in the fat body of honeybees and fruitflies resulted in a defect of all phenotypes induced by royalactin, showing that Egfr mediates these actions. These findings indicate that a specific factor in royal jelly, royalactin, drives queen development through an Egfr-mediated signalling pathway.

Functional genomics of life history variation in a butterfly metapopulation

In fragmented landscapes, small populations frequently go extinct and new ones are established with poorly understood consequences for genetic diversity and evolution of life history traits. Here, we apply functional genomic tools to an ecological model system, the well-studied metapopulation of the Glanville fritillary butterfly. We investigate how dispersal and colonization select upon existing genetic variation affecting life history traits by comparing common-garden reared 2-day adult females from new populations with those from established older populations. New-population females had higher expression of abdomen genes involved in egg provisioning and thorax genes involved in the maintenance of flight muscle proteins. Physiological studies confirmed that new-population butterflies have accelerated egg maturation, apparently regulated by higher juvenile hormone titer and angiotensin converting enzyme mRNA, as well as enhanced flight metabolism. Gene expression varied between allelic forms of two metabolic genes (Pgi and Sdhd), which themselves were associated with differences in flight metabolic rate, population age and population growth rate. These results identify likely molecular mechanisms underpinning life history variation that is maintained by extinction-colonization dynamics in metapopulations.

Soldier presence suppresses presoldier differentiation through a rapid decrease of JH in the termite Reticulitermes speratus

The regulation of caste differentiation is essential to insect eusociality. Termite soldiers are sterile and cannot eat by themselves because they have specialized mouth morphology. Almost all termite species have a soldier caste, and the soldier ratio per colony is maintained at a low level, probably by elaborate regulatory mechanisms. Although the soldier presence is considered to negatively affect soldier differentiation in all examined species, the detailed mechanism remains unclear. Presoldier differentiation can be induced artificially by juvenile hormone (JH) application to workers, showing that JH is a key factor underlying the regulation of soldier differentiation. In this study, to elucidate physiological changes in workers because of the soldier presence during the molt into presoldiers, JH III applications and JH titer quantifications were carried out in the rhinotermitid termite Reticulitermes speratus. Firstly, the effects of soldier presence before the molt into presoldiers induced by JH III application to workers were investigated. The rates of presoldier molt induced by the treatments with soldiers were significantly lower than those without soldiers. Secondly, worker JH titers in the presence or absence of soldiers were quantified by LC–MS on day 0, 5, 10, and 15 after JH application. Results indicated that the worker JH titers (endogenous+applied JH III) in the presence of soldiers were significantly lower than those without soldiers on day 5 after the JH treatment. On days 10 and 15, such soldier effects were not observed. Finally, the effective duration of soldier presence after the JH application was elucidated. A 4 day period of co-existence with soldiers suppressed presoldier differentiation, suggesting that the soldier presence rapidly decreased the JH titer in other colony members (i.e., workers), resulting in the inhibition of presoldier production.

Ontogenic potentialities of the worker caste in two sympatric subterranean termites in France

In termites, the capacity of workers to differentiate into neotenic reproductives is an important characteristic that deserves particular attention. To gain insight into the differentiation pathway, the potentialities of workers and the endocrinal changes during the formation of neotenics were compared in two sympatric termites, Reticulitermes flavipes and Reticulitermes grassei. After 1 year of development, 100% of R. flavipes worker groups produced neotenics against only 63% of R. grassei groups. The average production of female neotenics was significantly higher in R. flavipes worker groups compared with R. grassei groups and R. flavipes produced a greater proportion of female neotenics. Moreover, R. flavipes produced more offspring, not only because there were more females, but also because R. flavipes females were more productive. Moreover, the offspring produced by R. flavipes grew faster than the offspring of R. grassei. Both ecdysteroid and juvenile hormone (JH) titers varied significantly during the development of neotenics. The two species showed similar ecdysteroid titer variation patterns. However, the JH titer variation patterns strongly differed: in R. grassei, the concentration of JH increased in maturing neotenics then dropped in mature neotenics, whereas in R. flavipes, the level of JH was significantly higher than in R. grassei and remained constantly high in mature neotenics. Overall, these results suggest that these two species differ strongly in many life-history traits as well as in the physiological control of their caste differentiation system. Possible origins and mechanisms of such interspecific variations are discussed, as well as their evolutionary and ecological consequences.

Behavioral and physiological factors associated with juvenile hormone in Polistes wasp foundresses

Although there is increasing interest in the evolution of endocrine systems, relatively little is known about the factors associated with natural endocrine variation in invertebrates. Here, we assess juvenile hormone (JH) titers among nest-founding queens of the wasp Polistes dominulus over 2 years. We allowed unfamiliar wasps to battle for dominance and examined the relationships between dominance rank, JH, ovarian development, and facial patterns. The relationship between JH-titer and dominance varied across years; there was a stronger relationship between JH-titer and dominance in 2006 than in 2008. Across years, wasps that won dominance contests had facial patterns with more broken black spots than wasps that lost dominance contests. There was no relationship between dominance rank and ovarian development. The individual characteristics associated with JH-titer were also tested; JH-titers were correlated with facial pattern brokenness and ovarian development. This study adds to previous work indicating that P. dominulus facial patterns function as a signal of fighting ability. Furthermore, the correlation between JH-titers and facial patterns parallels previous work on testosterone and vertebrate signals and suggests that links between signals of fighting ability and hormones that mediate fighting ability may be common across taxa. Overall, individual JH-titers vary, though they are typically associated with factors related to individual reproductive success, including dominance, fertility, and facial pattern brokenness. Future studies in additional contexts and taxa will be important to test how and why JH-titers vary.

Ovarian Development of Female-Female Pairs in the Termite,Reticulitermes speratus

In the rhinotermitid termite Reticulitermes speratus (Kolbe) (Isoptera: Rhinotermitidae), facultative parthenogenesis is known to occur occasionally and females cooperate with other females to found the colony. To elucidate the ovarian development in these two females, incipient female-female colonies were established under laboratory conditions, and the process of colony development was observed at 0.5, 1.5, 2.5, 3.5, and 7.5 months (stages I–V, respectively) after colony foundation. Ovarian development, vitellogenin gene expression, and juvenile hormone (JH) titers were examined. A precise reproductive cycle in both females was observed, in which the oviposition rate was relatively higher during stages I and II, decreased during stages III and IV, and then increased again at stage V. JH III titer and vitellogenin gene expression changed in parallel throughout the reproductive cycle of these queens. Ovarian maturation and vitellogenesis were similar in both females in a female-female colony at all stages examined, suggesting that no conflicts existed for two females in terms of oviposition.

Differential expression of hypoxia pathway genes in honey bee ( Apis mellifera L.) caste development

Diphenism in social bees is essentially contingent on nutrient-induced cellular and systemic physiological responses resulting in divergent gene expression patterns. Analyses of juvenile hormone (JH) titers and functional genomics assays of the insulin–insulin-like signaling (IIS) pathway and its associated branch, target-of-rapamycin (TOR), revealed systemic responses underlying honey bee ( Apis mellifera) caste development. Nevertheless, little attention has been paid to cellular metabolic responses. Following up earlier investigations showing major caste differences in oxidative metabolism and mitochondrial physiology, we herein identified honey bee homologs of hypoxia signaling factors, HIFα/Sima, HIFβ/Tango and PHD/Fatiga and we investigated their transcript levels throughout critical stages of larval development. Amsima, Amtango and Amfatiga showed correlated transcriptional activity, with two peaks of occurring in both queens and workers, the first one shortly after the last larval molt and the second during the cocoon-spinning phase. Transcript levels for the three genes were consistently higher in workers. As there is no evidence for major microenvironmental differences in oxygen levels within the brood nest area, this appears to be an inherent caste character. Quantitative PCR analyses on worker brain, ovary, and leg imaginal discs showed that these tissues differ in transcript levels. Being a highly conserved pathway and linked to IIS/TOR, the hypoxia gene expression pattern seen in honey bee larvae denotes that the hypoxia pathway has undergone a transformation, at least during larval development, from a response to environmental oxygen concentrations to an endogenous regulatory factor in the diphenic development of honey bee larvae.

The Challenge Hypothesis in an Insect: Juvenile Hormone Increases during Reproductive Conflict following Queen Loss in Polistes Wasps

The challenge hypothesis was proposed as a mechanism for vertebrates to optimize their testosterone titers by upregulating testosterone during periods of aggressive competition. Here we test key predictions of the challenge hypothesis in an independently evolved endocrine system: juvenile hormone (JH) in a social insect. We assess how social conflict influences JH titers in Polistes dominulus wasps. Aggressive conflict was produced by removing the queen from some colonies (experimental) and a low-ranked worker from other colonies (control). Queen removal produced competition among workers to fill the reproductive vacancy. Workers upregulated their JH titers in response to this social conflict; worker JH titers were higher in queenless than in queenright colonies. Furthermore, JH titers were associated with an individual's ability to dominate rivals; the worker that took over as the replacement queen had a substantially higher JH titer than did other workers. Finally, JH titers were positively associated with aggression in queenless colonies, but there was no relationship between JH and aggression in stable, queenright colonies. Overall, these results match key predictions of the challenge hypothesis and parallel much of the work on testosterone in vertebrates. Social modulation of hormone titers is not confined to a particular endocrine system but is likely to be an adaptive feature of endocrine systems across diverse taxa.

The Genetic, Morphological, and Physiological Characterization of a Dark Larval Cuticle Mutation in the Butterfly, Bicyclus anynana

Studies on insect melanism have greatly contributed to our understanding of natural selection and the ultimate factors influencing the evolution of darkly pigmented phenotypes. Research on several species of melanic lepidopteran larvae have found that low levels of circulating juvenile hormone (JH) titers are associated with a melanic phenotype, suggesting that genetic changes in the JH biosynthetic pathway give rise to increased deposition of melanin granules in the cuticle in this group. But does melanism arise through different molecular mechanisms in different species? The present study reports on a Bicyclus anynana (Lepidoptera: Nymphalidae) dark larvae single locus mutation, in which larvae exhibit a darker cuticle relative to wild type. Unlike other lepidopteran melanic larvae mutations, this one is autosomal recessive and does not appear to involve a deficiency in JH titers. Unlike JH deficiency mutants, dark larvae mutants display similar growth rates and sexual behaviors as wild type, and topical application of a JH analogue failed to rescue the wild type cuticular coloration. Finally, transmission electron microscopy showed that sclerotization or deposition of diffuse melanin, rather than deposition of melanin granules, produces the dark coloration found in the cuticle of this species. We conclude that different molecular mechanisms underlie larval melanism in different species of Lepidoptera.