Evo-devo of wing colour patterns in beetles.

Insects have evolved tremendously diverse wing colour patterns that fulfil ecologically vital functions, including intraspecific sexual signalling, mimesis, mimicry, and detering predators. Beetles, which form the most species-rich order Coleoptera, have amazingly diverse wing colour patterns; however, the molecular mechanisms that give rise to these patterns remain poorly understood. Recently, the gene pannier (pnr), which encodes a transcription factor of the GATA family, was identified as an essential player in the wing patterning of the multi-coloured Asian ladybird beetle Harmonia axyridis. Here we review recent progress in understanding the molecular underpinnings of wing colour pattern formation in H. axyridis.

Precise staging of beetle horn formation in Trypoxylus dichotomus reveals the pleiotropic roles of doublesex depending on the spatiotemporal developmental contexts.

Many scarab beetles have sexually dimorphic exaggerated horns that are an evolutionary novelty. Since the shape, number, size, and location of horns are highly diverged within Scarabaeidae, beetle horns are an attractive model for studying the evolution of sexually dimorphic and novel traits. In beetles including the Japanese rhinoceros beetle Trypoxylus dichotomus, the sex differentiation gene doublesex (dsx) plays a crucial role in sexually dimorphic horn formation during larval-pupal development. However, knowledge of when and how dsx drives the gene regulatory network (GRN) for horn formation to form sexually dimorphic horns during development remains elusive. To address this issue, we identified a Trypoxylus-ortholog of the sex determination gene, transformer (tra), that regulates sex-specific splicing of the dsx pre-mRNA, and whose loss of function results in sex transformation. By knocking down tra function at multiple developmental timepoints during larval-pupal development, we estimated the onset when the sex-specific GRN for horn formation is driven. In addition, we also revealed that dsx regulates different aspects of morphogenetic activities during the prepupal and pupal developmental stages to form appropriate morphologies of pupal head and thoracic horn primordia as well as those of adult horns. Based on these findings, we discuss the evolutionary developmental background of sexually dimorphic trait growth in horned beetles.

Nanopore Formation in the Cuticle of an Insect Olfactory Sensillum.

Nanometer-level patterned surface structures form the basis of biological functions, including superhydrophobicity, structural coloration, and light absorption [1-3]. In insects, the cuticle overlying the olfactory sensilla has multiple small (50- to 200-nm diameter) pores [4-8], which are supposed to function as a filter that admits odorant molecules, while preventing the entry of larger airborne particles and limiting water loss. However, the cellular processes underlying the patterning of extracellular matrices into functional nano-structures remain unknown. Here, we show that cuticular nanopores in Drosophila olfactory sensilla originate from a curved ultrathin film that is formed in the outermost envelope layer of the cuticle and secreted from specialized protrusions in the plasma membrane of the hair forming (trichogen) cell. The envelope curvature coincides with plasma membrane undulations associated with endocytic structures. The gore-tex/Osiris23 gene encodes an endosomal protein that is essential for envelope curvature, nanopore formation, and odor receptivity and is expressed specifically in developing olfactory trichogen cells. The 24-member Osiris gene family is expressed in cuticle-secreting cells and is found only in insect genomes. These results reveal an essential requirement for nanopores for odor reception and identify Osiris genes as a platform for investigating the evolution of surface nano-fabrication in insects.

Development and evolution of color patterns in ladybird beetles: A case study in Harmonia axyridis.

Many organisms show various geometric color patterns on their bodies, and the developmental, evolutionary, genetic, and ecological bases of these patterns have been intensely studied in various organisms. Ladybird beetles display highly diverse patterns of wing (elytral) color and are one of the most attractive model organisms for studying these characteristics. In this study, we reviewed the genetic history of elytral color patterns in the Asian multicolored ladybird beetle Harmonia axyridis from the classical genetic studies led by the pupils of Thomas Hunt Morgan and Theodosius Dobzhansky to recent genomic studies that revealed that a single GATA transcription factor gene, pannier, regulates the highly diverse elytral color patterns in this species. We also reviewed and discussed the developmental and evolutionary mechanisms driven by the pannier locus in H. axyridis. In the development sections, we focused on the following two topics: (a) how the red (carotenoid) and black (melanin) pigmentation of elytra is regulated by the pannier and pigmentation genes and (b) how the diverse color patterns are formed by integrating regulatory inputs from other genes involved in wing development. In the evolution section, we subsequently focused on the highly diversified DNA sequences within the first intron of pannier that are 56-76 kb long and that were generated through recurrent multiple inversions. Furthermore, we discussed how these recurrent inversions have driven the diversification of color patterns throughout evolution.

Repeated inversions within a pannier intron drive diversification of intraspecific colour patterns of ladybird beetles.

How genetic information is modified to generate phenotypic variation within a species is one of the central questions in evolutionary biology. Here we focus on the striking intraspecific diversity of >200 aposematic elytral (forewing) colour patterns of the multicoloured Asian ladybird beetle, Harmonia axyridis, which is regulated by a tightly linked genetic locus h. Our loss-of-function analyses, genetic association studies, de novo genome assemblies, and gene expression data reveal that the GATA transcription factor gene pannier is the major regulatory gene located at the h locus, and suggest that repeated inversions and cis-regulatory modifications at pannier led to the expansion of colour pattern variation in H. axyridis. Moreover, we show that the colour-patterning function of pannier is conserved in the seven-spotted ladybird beetle, Coccinella septempunctata, suggesting that H. axyridis' extraordinary intraspecific variation may have arisen from ancient modifications in conserved elytral colour-patterning mechanisms in ladybird beetles.

The pivotal role of aristaless in development and evolution of diverse antennal morphologies in moths and butterflies.

BACKGROUND: Antennae are multi-segmented appendages and main odor-sensing organs in insects. In Lepidoptera (moths and butterflies), antennal morphologies have diversified according to their ecological requirements. While diurnal butterflies have simple, rod-shaped antennae, nocturnal moths have antennae with protrusions or lateral branches on each antennal segment for high-sensitive pheromone detection. A previous study on the Bombyx mori (silk moth) antenna, forming two lateral branches per segment, during metamorphosis has revealed the dramatic change in expression of antennal patterning genes to segmentally reiterated, branch-associated pattern and abundant proliferation of cells contributing almost all the dorsal half of the lateral branch. Thus, localized cell proliferation possibly controlled by the branch-associated expression of antennal patterning genes is implicated in lateral branch formation. Yet, actual gene function in lateral branch formation in Bombyx mori and evolutionary mechanism of various antennal morphologies in Lepidoptera remain elusive. RESULTS: We investigated the function of several genes and signaling specifically in lateral branch formation in Bombyx mori by the electroporation-mediated incorporation of siRNAs or morpholino oligomers. Knock down of aristaless, a homeobox gene expressed specifically in the region of abundant cell proliferation within each antennal segment, during metamorphosis resulted in missing or substantial shortening of lateral branches, indicating its importance for lateral branch formation. aristaless expression during metamorphosis was lost by knock down of Distal-less and WNT signaling but derepressed by knock down of Notch signaling, suggesting the strict determination of the aristaless expression domain within each antennal segment by the combinatorial action of them. In addition, analyses of pupal aristaless expression in antennae with various morphologies of several lepidopteran species revealed that the aristaless expression pattern has a striking correlation with antennal shapes, whereas the segmentally reiterated expression pattern was observed irrespective of antennal morphologies. CONCLUSIONS: Our results presented here indicate the significance of aristaless function in lateral branch formation in B. mori and imply that the diversification in the aristaless expression pattern within each antennal segment during metamorphosis is one of the significant determinants of antennal morphologies. According to these findings, we propose a mechanism underlying development and evolution of lepidopteran antennae with various morphologies.

Toll ligand Spätzle3 controls melanization in the stripe pattern formation in caterpillars.

A stripe pattern is an aposematic or camouflage coloration often observed among various caterpillars. However, how this ecologically important pattern is formed is largely unknown. The silkworm dominant mutant Zebra (Ze) has a black stripe in the anterior margin of each dorsal segment. Here, fine linkage mapping of 3,135 larvae revealed a 63-kbp region responsible for the Ze locus, which contained three candidate genes, including the Toll ligand gene spätzle3 (spz-3). Both electroporation-mediated ectopic expression and RNAi analyses showed that, among candidate genes, only processed spz-3 induced melanin pigmentation and that Toll-8 was the candidate receptor gene of spz-3 This Toll ligand/receptor set is also involved in melanization of other mutant Striped (pS ), which has broader stripes. Additional knockdown of 5 other spz family and 10 Toll-related genes caused no drastic change in the pigmentation of either mutant, suggesting that only spz-3/Toll-8 is mainly involved in the melanization process rather than pattern formation. The downstream pigmentation gene yellow was specifically up-regulated in the striped region of the Ze mutant, but spz-3 showed no such region-specific expression. Toll signaling pathways are known to be involved in innate immunity, dorsoventral axis formation, and neurotrophic functions. This study provides direct evidence that a Toll signaling pathway is co-opted to control the melanization process and adaptive striped pattern formation in caterpillars.

A genetic mechanism for female-limited Batesian mimicry in Papilio butterfly.

In Batesian mimicry, animals avoid predation by resembling distasteful models. In the swallowtail butterfly Papilio polytes, only mimetic-form females resemble the unpalatable butterfly Pachliopta aristolochiae. A recent report showed that a single gene, doublesex (dsx), controls this mimicry; however, the detailed molecular mechanisms remain unclear. Here we determined two whole-genome sequences of P. polytes and a related species, Papilio xuthus, identifying a single ∼130-kb autosomal inversion, including dsx, between mimetic (H-type) and non-mimetic (h-type) chromosomes in P. polytes. This inversion is associated with the mimicry-related locus H, as identified by linkage mapping. Knockdown experiments demonstrated that female-specific dsx isoforms expressed from the inverted H allele (dsx(H)) induce mimetic coloration patterns and simultaneously repress non-mimetic patterns. In contrast, dsx(h) does not alter mimetic patterns. We propose that dsx(H) switches the coloration of predetermined wing patterns and that female-limited polymorphism is tightly maintained by chromosomal inversion.

The transcription factor Apontic-like controls diverse colouration pattern in caterpillars.

Genetic polymorphisms underlie the convergent and divergent evolution of various phenotypes. Diverse colour patterns on caterpillars, which are ecologically important, are good models for understanding the molecular backgrounds of phenotypic diversity. Here we show that a single evolutionarily conserved gene apontic-like (apt-like) encoding for a putative transcription factor accounts for the silkworm p locus, which causes at least 15 different larval markings involved in branch-like markings and eye-spot formation. The expression of apt-like and melanin synthesis genes are upregulated in association with pigmented areas of marking mutants Striped (p(S)) and normal (+(p)) but not in the non-marking allele plain (p). Functional analyses, ectopic expression, RNAi and TALEN, demonstrate that apt-like causes melanin pigmentation in a cell-autonomous manner. These results suggest that variation in p alleles is caused by the differential expression of the gene apt-like which induces targeted elevation of gene expressions in the melanin synthesis pathway.

Periodic Wnt1 expression in response to ecdysteroid generates twin-spot markings on caterpillars.

Among various pigmentation patterns on caterpillars, sequential spot markings are often observed and used for aposematic colouration. In contrast to adult wings, caterpillar cuticle markings are repeatedly generated at each moult, but little is known about how the patterns are formed and maintained periodically. Here we focus on a silkworm mutant, multi lunar (L), with twin-spot markings on sequential segments. Positional cloning of L and expression analyses reveal that cis-regulatory change in Wnt1 is responsible for the spot patterning. The periodical upregulation of Wnt1 in response to ecdysteroid is detected only in epidermis within spot marking area. We verify by transgenic expression that the ectopic Wnt1 induces the additional pigmentation. Furthermore, the association of Wnt1 expression with spot markings is observed in the wild Bombyx species and swallowtail butterfly Papilio machaon. Taken together, we anticipate that periodic Wnt1 expression may contribute to natural variations of spot patterning on caterpillar cuticle.

Electroporation-mediated somatic transgenesis for rapid functional analysis in insects.

Transgenesis is a powerful technique for determining gene function; however, it is time-consuming. It is virtually impossible to carry out in non-model insects in which egg manipulation and screening are difficult. We have established a rapid genetic functional analysis system for non-model insects using a low-cost electroporator (costing under US$200) designed for somatic transformation with the piggyBac transposon. Using this system, we successfully generated somatic transgenic cell clones in various target tissues (e.g. olfactory neurons, wing epidermis, larval epidermis, muscle, fat body and trachea) of the silkworm Bombyx mori during development. We also induced stable and transient RNA interference (RNAi) using short hairpin RNA (shRNA)-mediating DNA vectors and direct transfer of small interfering RNAs (siRNAs), respectively. We found that these electroporation-mediated approaches could also be applied to the swallowtail butterfly Papilio xuthus and the red flour beetle Tribolium castaneum. Thus, this method could be a powerful genetic tool for elucidating various developmental phenomena in non-model insects.

Dramatic changes in patterning gene expression during metamorphosis are associated with the formation of a feather-like antenna by the silk moth, Bombyx mori.

Many moths use sex pheromones to find their mates in the dark. Their antennae are well developed with lateral branches to receive the pheromone efficiently. However, how these structures have evolved remains elusive, because the mechanism of development of these antennae has not been studied at a molecular level. To elucidate the developmental mechanism of this type of antenna, we observed morphogenesis, cell proliferation, cell death and antennal patterning gene expression in the branched antenna of the silk moth, Bombyx mori. Region-specific cell proliferation and almost ubiquitous apoptosis occur during early pupal stages and appear to shape the lateral branch cooperatively. Antennal patterning genes are expressed in a pattern largely conserved among insects with branchless antennae until the late 5th larval instar but most of them change their expression dramatically to a pattern prefiguring the lateral branch during metamorphosis. These findings imply that although antennal primordium is patterned by conserved mechanisms before metamorphosis, most of the antennal patterning genes are reused to form the lateral branch during metamorphosis. We propose that the acquisition of a new regulatory circuit of antennal patterning genes may have been an important event during evolution of the sensory antenna with lateral branches in the Lepidoptera.

In vivo gene transfer into the honeybee using a nucleopolyhedrovirus vector.

The honeybee Apis mellifera L. is a social insect and one of the most industrially important insects. We examined whether a baculovirus-mediated retrotransposon is applicable to in vivo transfer of exogenous genes to the honeybees. Honeybee larvae and pupae were injected with two types of recombinant Autographa californica nucleopolyhedrovirus (AcNPV) vectors, one that includes the enhanced green fluorescent protein gene (egfp) as a reporter to be inserted into the honeybee genome, and another that includes the reverse transcriptase gene responsible for the insertion. Fluorescence was observed in most of the viral-injected larvae and pupae. Reverse transcription-polymerase chain reaction and immunoblotting confirmed egfp mRNA and eGFP expression in these honeybees, although egfp insertion into the honeybee genome was not confirmed. These results indicate that AcNPV vectors can be used for the transfer and transient expression of an exogenous gene in the larval and pupal honeybees.