Bodies are often made of repeated units, or serial homologs, that develop using the same core gene regulatory network. Local inputs and modifications to this network allow serial homologs to evolve different morphologies, but currently we do not understand which modifications allow these repeated traits to evolve different levels of phenotypic plasticity. Here we describe variation in phenotypic plasticity across serial homologous eyespots of the butterfly Bicyclus anynana, hypothesized to be under selection for similar or different functions in the wet and dry seasonal forms. Specifically, we document the presence of eyespot size and scale brightness plasticity in hindwing eyespots hypothesized to vary in function across seasons, and reduced size plasticity and absence of brightness plasticity in forewing eyespots hypothesized to have the same function across seasons. By exploring the molecular and physiological causes of this variation in plasticity across fore and hindwing serial homologs we discover that: 1) temperature experienced during the wandering stages of larval development alters titers of an ecdysteroid hormone, 20-hydroxyecdysone (20E), in the hemolymph of wet and dry seasonal forms at that stage; 2) the 20E receptor (EcR) is differentially expressed in the forewing and hindwing eyespot centers of both seasonal forms during this critical developmental stage; and 3) manipulations of EcR signaling disproportionately affected hindwing eyespots relative to forewing eyespots. We propose that differential EcR expression across forewing and hindwing eyespots at a critical stage of development explains the variation in levels of phenotypic plasticity across these serial homologues. This finding provides a novel signaling pathway, 20E, and a novel molecular candidate, EcR, for the regulation of levels of phenotypic plasticity across body parts or serial homologs.

Experimental infections of Arabidopsis thaliana (Arabidopsis) with genomically characterized plant pathogens such as Pseudomonas syringae have facilitated dissection of canonical eukaryotic defense pathways and parasite virulence factors. Plants are also attacked by herbivorous insects, and the development of an ecologically relevant genetic model herbivore that feeds on Arabidopsis will enable the parallel dissection of host defense and reciprocal resistance pathways such as those involved in xenobiotic metabolism. An ideal candidate is Scaptomyza flava, a drosophilid fly whose leafmining larvae are true herbivores that can be found in nature feeding on Arabidopsis and other crucifers. Here we describe the eukaryotic life cycle of S. flava on Arabidopsis, and use multiple approaches to characterize the response of Arabidopsis to S. flava attack. Oviposition choice tests and growth performance assays on different Arabidopsis ecotypes, defense-related mutants, and hormone and chitin-treated plants revealed significant differences in host preference and variation in larval performance across Arabidopsis accessions. The jasmonate (JA) and glucosinolate pathways in Arabidopsis are important in mediating quantitative resistance against S. flava, and priming with JA or chitin resulted in increased resistance. Expression of xenobiotic detoxification genes was reduced in S. flava larvae reared on Arabidopsis JA signaling mutants, and increased in plants pre-treated with chitin. These results and future research directions are discussed in the context of developing a genetic model system to analyze insect/ plant interactions.

Delta opioid agonists can selectively enhance the antinociceptive effects of mu opioid agonists without enhancing some other, potentially undesirable mu agonist effects. However, the degree of delta receptor efficacy required to produce this profile of interactions is unknown. To address this issue, the present study examined interactions produced by the mu agonist fentanyl and the intermediate-efficacy delta opioid MSF61 in rhesus monkeys. For comparison, interactions were also examined between fentanyl and the relatively high-efficacy delta agonist SNC243A and the delta antagonist naltrindole, which has neglible efficacy at delta receptors. Two different behavioral procedures were used: (a) a warm-water tail-withdrawal assay of thermal nociception, and (b) an assay of schedule-controlled responding for food reinforcement. Drug interactions within each procedure were evaluated using dose-addition analysis to compare experimental results with expected additivity. Drug interactions across procedures were evaluated using dose-ratio analysis to assess relative potencies to produce antinociception vs. response-rate suppression. As expected, doseaddition analysis found that fentanyl/SNC243A interactions were superadditive in the assay of antinociception but additive in the assay of schedule-controlled responding. Conversely, fentanyl/ MSF61 interactions were generally additive in both procedures, and fentanyl/naltrindole interactions were additive or subadditive in both procedures. Dose-ratio analysis found that fentanyl alone produced antinociception and rate suppression with similar potencies. Some fentanyl/SNC243A mixtures produced antinociception with up to 4-fold greater potency than rate-suppression. However, fentanyl/MSF61 and fentanyl/naltrindole mixtures produced antinociception with lower potency than rate suppression. These results suggest that relatively high delta receptor efficacy is required for mu/ delta antinociceptive synergy.

Environmental cues can act to initiate alternative developmental trajectories that result in different adult phenotypes, including behavioral phenotypes. The developmental period when an organism is sensitive to the cue is often described as a critical period. Here we investigated the critical period for temperature-sensitive courtship rate plasticity in the butterfly Bicyclus anynana. We performed a series of temperature-shift experiments in which larvae, pupae, or adults were shifted for blocks of time from one temperature to an alternative temperature, and then we quantified the courtship rate exhibited by adult males. We discovered that the critical period begins during pupal development and extends into adulthood, but temperature experienced during larval development does not affect male courtship rate. This finding allows us to develop hypotheses that address how developmental and physiological factors may have influenced the evolution of behavioral plasticity in this species.

In contrast to the important role of hormones in the development of sexual traits in vertebrates (Cox RM, Stenquist DS, Calsbeek R. 2009. Testosterone, growth and the evolution of sexual size dimorphism. J Evol Biol. 22(8):1586–1598.), the differentiation of these traits in insects is attributed almost exclusively to cell-autonomous mechanisms controlled by members of the sex determination pathway (Verhulst EC, van de Zande L. 2015. Double nexus – doublesex is the connecting element in sex determination. Brief Funct Genomics 14(6):396–406.), such as doublesex. Although hormones can shape the development of sexual traits in insects, variation in hormone levels are not conclusively known to cause dimorphism in these traits (Prakash A, Monteiro A. 2016. Molecular mechanisms of secondary sexual trait development in insects. Curr Opin Insect Sci. 17:40–48.). Here, we show that butterflies use sex-specific differences in 20-hydroxyecdysone hormone titers to create sexually dimorphic wing ornaments. Females of the dry season (DS) form of Bicyclus anynana display a larger sexual ornament on their wings than males, whereas in the wet season form both sexes have similarly sized ornaments (Prudic KL, Jeon C, Cao H, Monteiro A. 2011. Developmental plasticity in sexual roles of butterfly species drives mutual sexual ornamentation. Science 331(6013):73–75.). High levels of circulating 20-hydroxyecdysone during larval development in DS females and wet season forms cause proliferation of the cells fated to give rise to this wing ornament, and results in sexual dimorphism in the DS forms. This study advances our understanding of how the environment regulates sex-specific patterns of plasticity of sexual ornaments and conclusively shows that hormones can play a role in the development of secondary sexual traits in insects, just like they do in vertebrates.

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.

It is well established that steroid hormones regulate sexual behavior in vertebrates via organizational and activational effects. However, whether the organizational/activational paradigm applies more broadly to the sexual behavior of other animals such as insects is not well established. Here we describe the hormonal regulation of a sexual behavior in the seasonally polyphenic butterfly Bicyclus anynana is consistent with the characteristics of an organizational effect. By measuring hormone titer levels, quantifying hormone receptor gene expression in the brain, and performing hormone manipulations, we demonstrate steroid hormone signaling early in pupal development has a latent effect on adult male sexual behavior in B. anynana. These findings suggest the organizational/activational paradigm may be more highly conserved across animal taxa than previously thought.

The differentiation of male and female characteristics in vertebrates and insects has long been thought to proceed via different mechanisms. Traditionally, vertebrate sexual development was thought to occur in two phases: a primary and a secondary phase, the primary phase involving the differentiation of the gonads, and the secondary phase involving the differentiation of other sexual traits via the influence of sex hormones secreted by the gonads. In contrast, insect sexual development was thought to depend exclusively on cell-autonomous expression of sexspecific genes. Recently, however, new evidence indicates that both vertebrates and insects rely on sex hormones as well as cell-autonomous mechanisms to develop sexual traits. Collectively, these new data challenge the traditional vertebrate definitions of primary and secondary sexual development, call for a redefinition of these terms, and indicate the need for research aimed at explaining the relative dependence on cell-autonomous versus hormonally guided sexual development in animals.