From the formation of embryonic appendages to the color of wings: Conserved and novel roles of aristaless1 in butterfly development

Highly diverse butterfly wing patterns have emerged as a powerful system for understanding the genetic basis of phenotypic variation. While the genetic basis of this pattern variation is being clarified, the precise developmental pathways linking genotype to phenotype are not well understood. The gene aristaless, which plays a role in appendage patterning and extension, has been duplicated in Lepidoptera. One copy, aristaless1, has been shown to control a white/yellow color switch in the butterfly Heliconius cydno, suggesting a novel function associated with color patterning and pigmentation. Here we investigate the developmental basis of al1 in embryos, larvae and pupae using new antibodies, CRISPR/Cas9, RNAi, qPCR assays of downstream targets and pharmacological manipulation of an upstream activator. We find that Al1 is expressed at the distal tips of developing embryonic appendages consistent with its ancestral role. In developing wings, we observe Al1 accumulation within developing scale cells of white H. cydno during early pupation while yellow scale cells exhibit little Al1 at this timepoint. Reduced Al1 expression is also associated with yellow scale development in al1 knockouts and knockdowns. We also find that Al1 expression appears to downregulate the enzyme Cinnabar and other genes that synthesize and transport the yellow pigment, 3–Hydroxykynurenine (3-OHK). Finally, we provide evidence that Al1 activation is under the control of Wnt signaling. We propose a model in which high levels of Al1 during early pupation, which are mediated by Wnt, are important for melanic pigmentation and specifying white portions of the wing while reduced levels of Al1 during early pupation promote upregulation of proteins needed to move and synthesize 3-OHK, promoting yellow pigmentation. In addition, we discuss how the ancestral role of aristaless in appendage extension may be relevant in understanding the cellular mechanism behind color patterning in the context of the heterochrony hypothesis.

Phenotypic plasticity in chemical defence of butterflies allows usage of diverse host plants

Host plant specialization is a major force driving ecological niche partitioning and diversification in insect herbivores. The cyanogenic defences of Passiflora plants keep most herbivores at bay, but not the larvae of Heliconiu s butterflies, which can both sequester and biosynthesize cyanogenic compounds. Here, we demonstrate that both Heliconius cydno chioneus and H. melpomene rosina have remarkable plasticity in their chemical defences. When feeding on Passiflora species with cyanogenic compounds that they can readily sequester, both species downregulate the biosynthesis of these compounds. By contrast, when fed on Passiflora plants that do not contain cyanogenic glucosides that can be sequestered, both species increase biosynthesis. This biochemical plasticity comes at a fitness cost for the more specialist H. m. rosina , as adult size and weight for this species negatively correlate with biosynthesis levels, but not for the more generalist H. c. chioneus . By contrast, H. m rosina has increased performance when sequestration is possible on its specialized host plant. In summary, phenotypic plasticity in biochemical responses to different host plants offers these butterflies the ability to widen their range of potential hosts within the Passiflora genus, while maintaining their chemical defences.

Neural divergence and hybrid disruption between ecologically isolated Heliconius butterflies

The importance of behavioral evolution during speciation is well established, but we know little about how this is manifest in sensory and neural systems. A handful of studies have linked specific neural changes to divergence in host or mate preferences associated with speciation. However, the degree to which brains are adapted to local environmental conditions, and whether this contributes to reproductive isolation between close relatives that have diverged in ecology, remains unknown. Here, we examine divergence in brain morphology and neural gene expression between closely related, but ecologically distinct, Heliconius butterflies. Despite ongoing gene flow, sympatric species pairs within the melpomene–cydno complex are consistently separated across a gradient of open to closed forest and decreasing light intensity. By generating quantitative neuroanatomical data for 107 butterflies, we show that Heliconius melpomene and Heliconius cydno clades have substantial shifts in brain morphology across their geographic range, with divergent structures clustered in the visual system. These neuroanatomical differences are mirrored by extensive divergence in neural gene expression. Differences in both neural morphology and gene expression are heritable, exceed expected rates of neutral divergence, and result in intermediate traits in first-generation hybrid offspring. Strong evidence of divergent selection implies local adaptation to distinct selective optima in each parental microhabitat, suggesting the intermediate traits of hybrids are poorly matched to either condition. Neural traits may therefore contribute to coincident barriers to gene flow, thereby helping to facilitate speciation.

A novel terpene synthase controls differences in anti-aphrodisiac pheromone production between closely related Heliconius butterflies

Plants and insects often use the same compounds for chemical communication, but not much is known about the genetics of convergent evolution of chemical signals. The terpene (E)-β-ocimene is a common component of floral scent and is also used by the butterfly Heliconius melpomene as an anti-aphrodisiac pheromone. While the biosynthesis of terpenes has been described in plants and microorganisms, few terpene synthases (TPSs) have been identified in insects. Here, we study the recent divergence of 2 species, H. melpomene and Heliconius cydno, which differ in the presence of (E)-β-ocimene; combining linkage mapping, gene expression, and functional analyses, we identify 2 novel TPSs. Furthermore, we demonstrate that one, HmelOS, is able to synthesise (E)-β-ocimene in vitro. We find no evidence for TPS activity in HcydOS (HmelOS ortholog of H. cydno), suggesting that the loss of (E)-β-ocimene in this species is the result of coding, not regulatory, differences. The TPS enzymes we discovered are unrelated to previously described plant and insect TPSs, demonstrating that chemical convergence has independent evolutionary origins.

Divergence of chemosensing during the early stages of speciation

Chemosensory communication is essential to insect biology, playing indispensable roles during mate-finding, foraging, and oviposition behaviors. These traits are particularly important during speciation, where chemical perception may serve to establish species barriers. However, identifying genes associated with such complex behavioral traits remains a significant challenge. Through a combination of transcriptomic and genomic approaches, we characterize the genetic architecture of chemoperception and the role of chemosensing during speciation for a young species pair ofHeliconiusbutterflies,Heliconius melpomeneandHeliconius cydno. We provide a detailed description of chemosensory gene-expression profiles as they relate to sensory tissue (antennae, legs, and mouthparts), sex (male and female), and life stage (unmated and mated female butterflies). Our results untangle the potential role of chemical communication in establishing barriers during speciation and identify strong candidate genes for mate and host plant choice behaviors. Of the 252 chemosensory genes,HmOBP20(involved in volatile detection) andHmGr56(a putative synephrine-related receptor) emerge as strong candidates for divergence in pheromone detection and host plant discrimination, respectively. These two genes are not physically linked to wing-color pattern loci or other genomic regions associated with visual mate preference. Altogether, our results provide evidence for chemosensory divergence betweenH. melpomeneandH. cydno, two rarely hybridizing butterflies with distinct mate and host plant preferences, a finding that supports a polygenic architecture of species boundaries.

Phenotypic plasticity in chemical defence allows butterflies to diversify host use strategies

Hostplant specialization is a major force driving ecological niche partitioning and diversification in insect herbivores. The cyanogenic defences of Passiflora plants keeps most herbivores at bay, but not larvae of Heliconius butterflies, which can both sequester and biosynthesize cyanogenic compounds. Here, we demonstrate that both Heliconius cydno chioneus, a host plant generalist, and H. melpomene rosina, a specialist, have remarkable plasticity in their chemical defence. When feeding on Passiflora species with cyanogenic compounds they can readily sequester, both species downregulate the biosynthesis of these compounds. In contrast, when fed on Passiflora plants that do not contain cyanogenic glucosides that can be sequestered, both species increase biosynthesis. This biochemical plasticity comes at a significant fitness cost for specialist like H. m. rosina, as growth rates for this species negatively correlate with biosynthesis levels, but not for a generalist like H. c. chioneus. In exchange, H. m rosina has increased performance when sequestration is possible as on its specialised hostplant. In summary, phenotypic plasticity in biochemical responses to different host plants offers these butterflies the ability to widen their range of potential host within the Passiflora genus, while maintaining their chemical defences.

Tamaño corporal de cuatro especies de mariposas del género Heliconius (Lepidoptera: Nymphalidae) producidas en zoocriaderos del Caribe de Costa Rica

Introducción: La producción de mariposas es una actividad socioeconómica importante y se ha convertido en una opción para generar ingresos en las microempresas rurales. Objetivo: Observar como los zoocriaderos afectan el tamaño corporal de mariposas Heliconius, producidas en zoocriaderos de Costa Rica. Métodos: Medimos adultos y pupas de Heliconius cydno, H. erato, H. hecale y H. melpomene de 10 zoocriaderos de mariposas, ubicados en el la zona Huetar Atlántica de Costa Rica. Resultados: En algunos zoocriaderos las mariposas adultas de Heliconius erato y H. hecale son significativamente más pesadas (p ≤ 0,05), poseen un abdomen más largo y el ala anterior más grande. El peso no difiere entre zoocriaderos, las relaciones de sexos son cercanas a 0,5. Conclusiones: Todos los zoocriaderos muestreados producen mariposas con calidad para su comercialización.

Inventario de mariposas diurnas en la cuenca de la quebrada Santo Tomás, Pensilvania-Colombia

Se realizó un inventario preliminar con el objetivo, de determinar la diversidad de mariposas diurnas en la cuenca de la quebrada Santo Tomas (Pensilvania-Colombia), durante cuatro meses de muestreo entre abril y julio de 2018. Los especímenes fueron colectados con red entomológica, realizando recorridos en tres coberturas: zonas abiertas, interior de bosque y bosque ribereño, comprendiendo altitudes entre los 1.311 y 1.570 m.s.n.m. Se colectaron 610 mariposas pertenecientes a 6 familias, 15 subfamilias y 83 especies, siendo la más abundante la familia Nymphalidae. Se observaron especies y subespecies endémicas de Colombia como Parides eurimedes, Crocozona pheretima, Heliconius cydno cydnides y Adelpha cytherea despoliata. De igual manera, se encontraron poblaciones abundantes de Anartia amathea amathea, Oressinoma typhla typhla y Hermeneuptychia hermes. El análisis de agrupación por dendrograma de similitud mostró que la cobertura interior de bosque y bosque ribereño tiene una composición similar. La riqueza y abundancia de mariposas colectadas en el presente estudio son un aporte al conocimiento de la biodiversidad en el municipio de Pensilvania (Colombia) y evidencian la importancia de la conservación de quebradas andinas que abastecen acueductos rurales, debido a la variedad de especies que se puede proteger

Pervasive genetic associations between traits causing reproductive isolation in Heliconius butterflies

Ecological speciation proceeds through the accumulation of divergent traits that contribute to reproductive isolation, but in the face of gene flow traits that characterize incipient species may become disassociated through recombination. Heliconius butterflies are well known for bright mimetic warning patterns that are also used in mate recognition and cause both pre- and post-mating isolation between divergent taxa. Sympatric sister taxa representing the final stages of speciation, such as Heliconius cydno and Heliconius melpomene , also differ in ecology and hybrid fertility. We examine mate preference and sterility among offspring of crosses between these species and demonstrate the clustering of Mendelian colour pattern loci and behavioural loci that contribute to reproductive isolation. In particular, male preference for red patterns is associated with the locus responsible for the red forewing band. Two further colour pattern loci are associated, respectively, with female mating outcome and hybrid sterility. This genetic architecture in which ‘speciation genes’ are clustered in the genome can facilitate two controversial models of speciation, namely divergence in the face of gene flow and hybrid speciation.