Gene silencing through RNA interference (RNAi) has revolutionized the study of gene 98 function, particularly in non-model insects. However, in Lepidoptera (moths and butterflies) 99 RNAi has many times proven to be difficult to achieve. Most of the negative results have been 100 anecdotal and the positive experiments have not been collected in such a way that they are 101 possible to analyze. In this review, we have collected detailed data from more than 150 102 experiments including all to date published and many unpublished experiments. Despite a 103 large variation in the data, trends that are found are that RNAi is particularly successful in the 104 family Saturniidae and in genes involved in immunity. On the contrary, gene expression in 105 epidermal tissues seems to be most difficult to silence. In addition, gene silencing by feeding 106 dsRNA requires high concentrations for success. Possible causes for the variability of success 107 in RNAi experiments in Lepidoptera are discussed. The review also points to a need to further 108 investigate the mechanism of RNAi in lepidopteran insects and its possible connection to the 109 innate immune response. Our general understanding of RNAi in Lepidoptera will be further 110 aided in the future as our public database at http://insectacentral.org/RNAi will continue to 111 gather information on RNAi experiments.

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Dragonflies are colorful and large-eyed animals strongly dependent on color vision. Here we report an extraordinary large number of opsin genes in dragonflies and their characteristic spatiotemporal expression patterns. Exhaustive transcriptomic and genomic surveys of three dragonflies of the family Libellulidae consistently identified 20 opsin genes, consisting of 4 nonvisual opsin genes and 16 visual opsin genes of 1 UV, 5 short-wavelength (SW), and 10 long-wavelength (LW) type. Comprehensive transcriptomic survey of the other dragonflies representing an additional 10 families also identified as many as 15-33 opsin genes. Molecular phylogenetic analysis revealed dynamic multiplications and losses of the opsin genes in the course of evolution. In contrast to many SW and LW genes expressed in adults, only one SW gene and several LW genes were expressed in larvae, reflecting less visual dependence and LW-skewed light conditions for their lifestyle under water. In this context, notably, the sand-burrowing or pit-dwelling species tended to lack SW gene expression in larvae. In adult visual organs: (i) many SW genes and a few LW genes were expressed in the dorsal region of compound eyes, presumably for processing SW-skewed light from the sky; (ii) a few SW genes and many LW genes were expressed in the ventral region of compound eyes, probably for perceiving terrestrial objects; and (iii) expression of a specific LW gene was associated with ocelli. Our findings suggest that the stage-and region-specific expressions of the diverse opsin genes underlie the behavior, ecology, and adaptation of dragonflies.dragonfly | opsin | color vision | molecular evolution

Beetles, representing the majority of the insect species diversity, are characterized by thick and hard cuticle, which plays important roles for their environmental adaptation and underpins their inordinate diversity and prosperity. Here, we report a bacterial endosymbiont extremely specialized for sustaining beetle’s cuticle formation. Many weevils are associated with a γ-proteobacterial endosymbiont lineage Nardonella, whose evolutionary origin is estimated as older than 100 million years, but its functional aspect has been elusive. Sequencing of Nardonella genomes from diverse weevils unveiled drastic size reduction to 0.2 Mb, in which minimal complete gene sets for bacterial replication, transcription, and translation were present but almost all of the other metabolic pathway genes were missing. Notably, the only metabolic pathway retained in the Nardonella genomes was the tyrosine synthesis pathway, identifying tyrosine provisioning as Nardonella’s sole biological role. Weevils are armored with hard cuticle, tyrosine is the principal precursor for cuticle formation, and experimental suppression of Nardonella resulted in emergence of reddish and soft weevils with low tyrosine titer, confirming the importance of Nardonella-mediated tyrosine production for host’s cuticle formation and hardening. Notably, Nardonella’s tyrosine synthesis pathway was incomplete, lacking the final step transaminase gene. RNA sequencing identified host’s aminotransferase genes up-regulated in the bacteriome. RNA interference targeting the aminotransferase genes induced reddish and soft weevils with low tyrosine titer, verifying host’s final step regulation of the tyrosine synthesis pathway. Our finding highlights an impressively intimate and focused aspect of the host–symbiont metabolic integrity via streamlined evolution for a single biological function of ecological relevance.

Like the adult wing, butterfly larvae are unique in their coloring. However, the molecular mechanisms underlying the formation of insect larval color patterns are largely unknown. The larva of the swallowtail butterfly Papilio xuthus changes its color pattern markedly during the 4th ecdysis. We investigated its cuticular color pattern, which is thought to be composed of melanin and related pigments derived from tyrosine. We cloned three enzymes involved in the melanin-synthesis pathway in P. xuthus: tyrosine hydroxylase (TH), dopa decarboxylase (DDC), and ebony. Whole-mount in situ hybridization showed that the expression of both TH and DDC is strongly correlated with the black markings. ebony is strongly expressed only in the reddish-brown area. The expression pattern of each enzyme coincides with the cuticular color pattern of the subsequent instar. We also investigated the uptake of melanin precursors into cultured integument. Inhibition of either TH or DDC activity prevents in vitro pigmentation completely. Addition of dopamine to integuments in the presence of TH inhibitor causes overall darkening without specific markings. From these results, specific larval cuticular color patterns are regulated by stage-specific colocalization of enzymes in epidermal cells rather than by the differential uptake of melanin precursors into individual epidermal cells. Epidermal cells expressing TH and DDC, but not ebony, produce the black cuticle, and epidermal cells expressing TH, DDC, and ebony produce the reddish-brown cuticle.

Many larval color mutants have been obtained in the silkworm Bombyx mori. Mapping of melaninsynthesis genes on the Bombyx linkage map revealed that yellow and ebony genes were located near the chocolate (ch) and sooty (so) loci, respectively. In the ch mutants, body color of neonate larvae and the body markings of elder instar larvae are reddish brown instead of normal black. Mutations at the so locus produce smoky larvae and black pupae. F 2 linkage analyses showed that sequence polymorphisms of yellow and ebony genes perfectly cosegregated with the ch and so mutant phenotypes, respectively. Both yellow and ebony were expressed in the epidermis during the molting period when cuticular pigmentation occurred. The spatial expression pattern of yellow transcripts coincided with the larval black markings. In the ch mutants, nonsense mutations of the yellow gene were detected, whereas large deletions of the ebony ORF were detected in the so mutants. These results indicate that yellow and ebony are the responsible genes for the ch and so loci, respectively. Our findings suggest that Yellow promotes melanization, whereas Ebony inhibits melanization in Lepidoptera and that melanin-synthesis enzymes play a critical role in the lepidopteran larval color pattern.

In insects, antimicrobial humoral immunity is governed by two distinct gene cascades, IMD pathway mainly targeting Gram-negative bacteria and Toll pathway preferentially targeting Gram-positive bacteria, which are widely conserved among diverse metazoans. However, recent genomic studies uncovered that IMD pathway is exceptionally absent in some hemipteran lineages like aphids and assassin bugs. How the apparently incomplete immune pathways have evolved with functionality is of interest. Here we report the discovery that, in the hemipteran stinkbug Plautia stali , both IMD and Toll pathways are present but their functional differentiation is blurred. Injection of Gram-negative bacteria and Gram-positive bacteria upregulated effector genes of both pathways. Notably, RNAi experiments unveiled significant functional permeation and crosstalk between IMD and Toll pathways: RNAi of IMD pathway genes suppressed upregulation of effector molecules of both pathways, where the suppression was more remarkable for IMD effectors; and RNAi of Toll pathway genes reduced upregulation of effector molecules of both pathways, where the suppression was more conspicuous for Toll effectors. These results suggest the possibility that, in hemipterans and other arthropods, IMD and Toll pathways are intertwined to target wider and overlapping arrays of microbes, which might have predisposed and facilitated the evolution of incomplete immune pathways.

The larval color patterns in Lepidoptera exhibit splendid diversity, and identifying the genes responsible for pigment distribution is essential to understanding color-pattern evolution. The swallowtail butterfly, Papilio xuthus, is a good candidate for analyzing marking-associated genes because its body markings change dramatically at the final molt. Moreover, the silkworm Bombyx mori is most suitable for identification of lab-generated color mutants because genome information and many color mutants are available. Here, we analyzed the expression pattern of 10 melanin-related genes in P. xuthus, and analyzed whether these genes were responsible for Bombyx larval color mutants. We found that seven genes correlated strongly with the stage-specific larval cuticular markings of P. xuthus, suggesting that, compared with Drosophila, more genes showed marking specificity in lepidopteran larvae. We newly found that the expression of both tan and laccase2 is strongly correlated with the larval black markings in both P. xuthus and B. mori. The results of F2 linkage analysis and mutant analysis strongly suggest that tan is the responsible gene for Bombyx larval color mutant rouge, and that tan is important in emphasizing black markings of lepidopteran larvae. Detailed comparison of temporal and spatial expression patterns showed that larval cuticular markings were regulated at two different phases. Marking-specific expression of oxidizing enzymes preceded the marking-specific expression of melanin synthesis enzymes at mRNA level, which is the reverse of the melanin synthesis step.