Reduction of embryonic E93 expression as a hypothetical driver of the evolution of insect metamorphosis.

The early embryo of the cockroach Blattella germanica exhibits high E93 expression. In general, E93 triggers adult morphogenesis during postembryonic development. Here we show that E93 is also crucial in early embryogenesis in the cockroach, as a significant number of E93-depleted embryos are unable to develop the germ band under maternal RNAi treatment targeting E93. Moreover, transcriptomic analysis indicates that E93 depletion results in important gene expression changes in the early embryo, and many of the differentially expressed genes are involved in development. Then, using public databases, we gathered E93 expression data in embryo and preadult stages, finding that embryonic expression of E93 is high in hemimetabolan species (whose juveniles, or nymphs, are similar to the adult) and low in holometabolans (whose juveniles, or larvae, are different from the adult). E93 expression is also low in Thysanoptera and in Hemiptera Sternorrhyncha, hemimetabolans with postembryonic quiescent stages, as well as in Odonata, the nymph of which is very different from the adult. In ametabolans, such as the Zygentoma Thermobia domestica, E93 transcript levels are very high in the early embryo, whereas during postembryonic development they are medium and relatively constant. We propose the hypothesis that during evolution, a reduction of E93 expression in the embryo of hemimetabolans facilitated the larval development and the emergence of holometaboly. Independent decreases of E93 transcripts in the embryo of Odonata, Thysanoptera, and different groups of Hemiptera Sternorrhyncha would have allowed the development of modified juvenile stages adapted to specific ecophysiological conditions.

Knock-Out of ACY-1 Like Gene in Spodoptera litura Supports the Notion that FACs Improve Nitrogen Metabolism.

Volicitin [N-(17-hydroxylinolenoyl)-L-glutamine] and N-linolenoyl-L-glutamine were originally identified in the regurgitant of Spodoptera exigua larvae. These fatty acid amino acid conjugates (FACs) are known to be elicitors that induce plants to release volatile compounds which in turn attract natural enemies of the larvae such as parasitic wasps. FAC concentrations are regulated by enzymatic biosynthesis and hydrolysis in the intestine of Lepidoptera larvae. It has been proposed that FAC metabolism activates glutamine synthetase and plays an important role in nitrogen metabolism in larvae. In this study, we identified candidate genes encoding a FACs hydrolase in Spodoptera litura using genomic information of various related lepidopteran species in which FACs hydrolases have been reported. We analyzed the importance of FAC hydrolysis on caterpillar performance with CRISPR/Cas9 knock outs. Larvae of strains with an inactive FACs hydrolase excreted FACs in their feces. They absorbed 30% less nitrogen from the diet compared to WT caterpillars resulting in a reduction of their body weight of up to 40% compared to wild type caterpillars. These results suggest that the hydrolysis of FACs is an important metabolism for insects and that FACs are important for larval growth.

Dimorphic sperm formation by Sex-lethal.

Sex is determined by diverse mechanisms and master sex-determination genes are highly divergent, even among closely related species. Therefore, it is possible that homologs of master sex-determination genes might have alternative functions in different species. Herein, we focused on Sex-lethal (Sxl), which is the master sex-determination gene in Drosophila melanogaster and is necessary for female germline development. It has been widely shown that the sex-determination function of Sxl in Drosophilidae species is not conserved in other insects of different orders. We investigated the function of Sxl in the lepidopteran insect Bombyx mori In lepidopteran insects (moths and butterflies), spermatogenesis results in two different types of sperm: nucleated fertile eupyrene sperm and anucleate nonfertile parasperm, also known as apyrene sperm. Genetic analyses using Sxl mutants revealed that the gene is indispensable for proper morphogenesis of apyrene sperm. Similarly, our analyses using Sxl mutants clearly demonstrate that apyrene sperm are necessary for eupyrene sperm migration from the bursa copulatrix to the spermatheca. Therefore, apyrene sperm is necessary for successful fertilization of eupyrene sperm in B. mori Although Sxl is essential for oogenesis in D. melanogaster, it also plays important roles in spermatogenesis in B. mori Therefore, the ancestral function of Sxl might be related to germline development.

Involvement of the Clock Gene Period in the Photoperiodism of the Silkmoth Bombyx mori.

We established a knockout strain of a clock gene, period (per), by using TALEN in a bivoltine strain (Kosetsu) of Bombyx mori (Insecta, Lepidoptera), and examined the effect of per knockout on the circadian rhythm and photoperiodism. The generated per knockout allele was considered to be null, because a new stop codon was present in the insertion allele. The wild type (Kosetsu) showed clear circadian rhythms in eclosion and hatching, whereas the per knockout strain showed arrhythmic eclosion and hatching under constant darkness. In this strain, moreover, temporal expression changes of clock genes per and timeless were disrupted. The wild type showed a clear long-day response for induction of embryonic diapause: when larvae were reared under long-day and short-day conditions at 25°C, adults produced nondiapause and diapause eggs, respectively. However, the per knockout strain lost the sensitivity to photoperiod and laid nondiapause eggs under both conditions. We conclude that per plays an important role both in circadian rhythms and in photoperiodism of B. mori, indicating the involvement of the circadian clock consisting of per in the photoperiodism.

Mutations in cardinal are responsible for the red-1 and peach eye color mutants of the red flour beetle Tribolium castaneum.

Ommochromes are the major pigments found in the eyes, eggs, wings and epidermis of insects. Here, we report the identification and characterization of the gene responsible for red-1 locus of Tribolium, whose mutants have white eyes due to lack of ommochrome pigments in the eyes. Using a candidate gene approach, we demonstrated that red-1 and peach mutants have molecular defects in the cardinal gene, which encodes a haem peroxidase that is considered to convert 3-hydroxykynurenine into ommochromes in pigment granules. Our experiments showed that the expression pattern of cardinal correlates well with the progression of eye pigmentation during pupal stages. We performed gene editing experiments using the Receptor-Mediated Ovary Transduction of Cargo (ReMOT) Control technique to disrupt the cardinal gene by adult injection, and were able to establish a novel cardinal mutant line. Our complementation test provided definitive genetic evidence that cardinal is located at the red-1 locus. The present study will lead to a greater understanding of the function and diversity of ommochrome pathway genes in insects. Our successful use of ReMOT Control in beetles will facilitate the development of more efficient and versatile systems for insect genome editing by simple adult injection.

A mitochondrial phosphatase PTPMT1 is essential for the early development of silkworm, Bombyx mori.

Juvenile hormone (JH) plays important roles in the control of many biological processes in insects, such as development, reproduction, and polyphenism. JH is primarily produced in the corpora allata (CA) by specific JH biosynthetic enzymes under strict temporal regulation. In a previous study, we identified a novel putative JH biosynthetic gene, protein tyrosine phosphatase, mitochondrial 1 (PTPMT1), from silkworm, Bombyx mori, whose expression is nearly exclusive in the CA and is correlated with JH synthetic activities during late larval development. In this study, to reveal the function of PTPMT1 in vivo, we generated PTPMT1 knockout silkworms using TALEN. In the knockout mutants, no signs indicating defects in JH activity were observed. Instead, PTPMT1 knockout silkworms showed embryonic lethality, developmental arrest, and 3rd-instar lethality not only in mutants lacking total enzymatic activity but also in mutants lacking mitochondrial translocation signals. Moreover, in PTPMT1 knockout embryos, the expression of two genes encoded by the mitochondrial genome, CYTB and ND3, was decreased, indicating a mitochondrial disorder. These results suggested that PTPMT1 plays conserved vital role(s) reported in vertebrates in insect mitochondria.

Knockout silkworms reveal a dispensable role for juvenile hormones in holometabolous life cycle.

Insect juvenile hormones (JHs) prevent precocious metamorphosis and allow larvae to undergo multiple rounds of status quo molts. However, the roles of JHs during the embryonic and very early larval stages have not been fully understood. We generated and characterized knockout silkworms (Bombyx mori) with null mutations in JH biosynthesis or JH receptor genes using genome-editing tools. We found that embryonic growth and morphogenesis are largely independent of JHs in Bombyx and that, even in the absence of JHs or JH signaling, pupal characters are not formed in first- or second-instar larvae, and precocious metamorphosis is induced after the second instar at the earliest. We also show by mosaic analysis that a pupal specifier gene broad, which is dramatically up-regulated in the late stage of the last larval instar, is essential for pupal commitment in the epidermis. Importantly, the mRNA expression level of broad, which is thought to be repressed by JHs, remained at very low basal levels during the early larval instars of JH-deficient or JH signaling-deficient knockouts. Therefore, our study suggests that the long-accepted paradigm that JHs maintain the juvenile status throughout larval life should be revised because the larval status can be maintained by a JH-independent mechanism in very early larval instars. We propose that the lack of competence for metamorphosis during the early larval stages may result from the absence of an unidentified broad-inducing factor, i.e., a competence factor.

Importance of juvenile hormone signaling arises with competence of insect larvae to metamorphose.

Juvenile hormone (JH) postpones metamorphosis of insect larvae until they have attained an appropriate stage and size. Then, during the final larval instar, a drop in JH secretion permits a metamorphic molt that transforms larvae to adults either directly (hemimetaboly) or via a pupal stage (holometaboly). In both scenarios, JH precludes metamorphosis by activating the Kr-h1 gene through a JH receptor, Methoprene-tolerant (Met). Removal of Met, Kr-h1, or JH itself triggers deleterious precocious metamorphosis. Although JH is thought to maintain the juvenile status throughout larval life, various methods of depleting JH failed to induce metamorphosis in early-instar larvae. To determine when does JH signaling become important for the prevention of precocious metamorphosis, we chose the hemimetabolous bug, Pyrrhocoris apterus, and the holometabolous silkworm, Bombyx mori. Both species undergo a fixed number of five larval instars. Pyrrhocoris larvae subjected to RNAi-mediated knockdown of Met or Kr-h1 underwent precocious adult development when treated during the fourth (penultimate) instar, but younger larvae proved increasingly resistant to loss of either gene. The earliest instar developing minor signs of precocious metamorphosis was the third. Therefore, the JH-response genes may not be required to maintain the larval program during the first two larval instars. Next, we examined Bombyx mod mutants that cannot synthesize authentic, epoxidized forms of JH. Although mod larvae expressed Kr-h1 mRNA at severely reduced levels since hatching, they only entered metamorphosis by pupating after four, rarely three instars. Based on findings in Pyrrhocoris and Bombyx, we propose that insect postembryonic development is initially independent of JH. Only later, when larvae gain competence to enter metamorphosis, JH signaling becomes necessary to prevent precocious metamorphosis and to optimize growth.

Precocious metamorphosis in the juvenile hormone-deficient mutant of the silkworm, Bombyx mori.

Insect molting and metamorphosis are intricately governed by two hormones, ecdysteroids and juvenile hormones (JHs). JHs prevent precocious metamorphosis and allow the larva to undergo multiple rounds of molting until it attains the proper size for metamorphosis. In the silkworm, Bombyx mori, several "moltinism" mutations have been identified that exhibit variations in the number of larval molts; however, none of them have been characterized molecularly. Here we report the identification and characterization of the gene responsible for the dimolting (mod) mutant that undergoes precocious metamorphosis with fewer larval-larval molts. We show that the mod mutation results in complete loss of JHs in the larval hemolymph and that the mutant phenotype can be rescued by topical application of a JH analog. We performed positional cloning of mod and found a null mutation in the cytochrome P450 gene CYP15C1 in the mod allele. We also demonstrated that CYP15C1 is specifically expressed in the corpus allatum, an endocrine organ that synthesizes and secretes JHs. Furthermore, a biochemical experiment showed that CYP15C1 epoxidizes farnesoic acid to JH acid in a highly stereospecific manner. Precocious metamorphosis of mod larvae was rescued when the wild-type allele of CYP15C1 was expressed in transgenic mod larvae using the GAL4/UAS system. Our data therefore reveal that CYP15C1 is the gene responsible for the mod mutation and is essential for JH biosynthesis. Remarkably, precocious larval-pupal transition in mod larvae does not occur in the first or second instar, suggesting that authentic epoxidized JHs are not essential in very young larvae of B. mori. Our identification of a JH-deficient mutant in this model insect will lead to a greater understanding of the molecular basis of the hormonal control of development and metamorphosis.

Anatomical and functional analysis of domestication effects on the olfactory system of the silkmoth Bombyx mori.

The silkmoth Bombyx mori is the main producer of silk worldwide and has furthermore become a model organism in biological research, especially concerning chemical communication. However, the impact domestication might have had on the silkmoth's olfactory sense has not yet been investigated. Here, we show that the pheromone detection system in B. mori males when compared with their wild ancestors Bombyx mandarina seems to have been preserved, while the perception of environmental odorants in both sexes of domesticated silkmoths has been degraded. In females, this physiological impairment was mirrored by a clear reduction in olfactory sensillum numbers. Neurophysiological experiments with hybrids between wild and domesticated silkmoths suggest that the female W sex chromosome, so far known to have the sole function of determining femaleness, might be involved in the detection of environmental odorants. Moreover, the coding of odorants in the brain, which is usually similar among closely related moths, differs strikingly between B. mori and B. mandarina females. These results indicate that domestication has had a strong impact on odour detection and processing in the olfactory model species B. mori.

Recent progress in genome engineering techniques in the silkworm, Bombyx mori.

Rapid advances in genome engineering tools, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced palindromic repeats/CRISPR-associated (CRISPR/Cas) system, have enabled efficient gene knockout experiments in a wide variety of organisms. Here, we review the recent progress in targeted gene disruption techniques in the silkworm, Bombyx mori. Although efficiency of targeted mutagenesis was very low in an early experiment using ZFNs, recent studies have shown that TALENs can induce highly efficient mutagenesis of desired target genes in Bombyx. Notably, mutation frequencies induced by TALENs can reach more than 50% of G0 gametes. Thus, TALENs can now be used as a standard tool for gene targeting studies, even when mutant phenotypes are unknown. We also propose guidelines for experimental design and strategy for knockout experiments in Bombyx. Genome editing technologies will greatly increase the usefulness of Bombyx as a model for lepidopteran insects, the major agricultural pests, and lead to sophisticated breeding of Bombyx for use in sericulture and biotechnology.

Antennal lobe organization and pheromone usage in bombycid moths.

We investigated the neuroanatomy of the macroglomerular complex (MGC), which is involved in sex pheromone processing, in five species in the subfamily Bombycinae, including Ernolatia moorei, Trilocha varians, Rondotia menciana, Bombyx mandarina and Bombyx mori. The glomerulus located at the dorsal-most part of the olfactory centre shows the largest volume in moth species examined to date. Such normal glomerular organization has been observed in E. moorei and T. varians, which use a two-component mixture and includes the compound bombykal as a mating signal. By contrast, the other three species, which use another component as a single attractant, exhibited a modified arrangement of the MGC. This correlation between pheromone usage and neural organization may be useful for understanding the process of speciation.

Direct parental CRISPR gene editing in the predatory bug Orius strigicollis, a biocontrol agent against small arthropods.

BACKGROUND: The predatory flower bug Orius strigicollis serves as a valuable biocontrol agent against small arthropods; however, its effectiveness can vary, especially when population establishment fails due to low prey/pest densities. A promising approach to improve the efficacy of O. strigicollis as a biocontrol agent is through gene editing. However, as females lay their eggs in plant tissue, the conventional embryo injection approach is challenging in this species. RESULTS: In this study, we aimed to develop an efficient and practical gene editing technique for O. strigicollis using direct parental CRISPR (DIPA-CRISPR). Female bugs at various postemergence stages received Cas9 ribonucleoprotein injections, with subsequent genotyping of their offspring (G0) using PCR and a heteroduplex mobility assay. We targeted the kynurenine 3-monooxygenase gene (cinnabar), pivotal for insect ommochrome pigment biosynthesis. Through experimental optimization, we achieved a peak gene editing efficiency of 52%, i.e., 52% of G0 progeny carried gene-edited alleles when injecting 1 day postemergence. Notably, some gene-edited G0 adults exhibited a red-eye mosaic phenotype, in contrast to the black-eyed wild type. Crossing experiments confirmed the heritability of the introduced mutations in the subsequent generation (G1), enabling the establishment of a cinnabar-knockout line with bright red eyes. CONCLUSION: We demonstrate that our DIPA-CRISPR gene editing method tailored for O. strigicollis is efficient and practical. Our findings highlight the potency of DIPA-CRISPR as a tool for O. strigicollis genetic engineering and suggest broader applications for enhancing other biocontrol agents. © 2024 Society of Chemical Industry.

Reduced expression of the dysbindin-like gene in the Bombyx mori ov mutant exhibiting mottled translucency of the larval skin.

The ov (mottled translucent of Var) mutant, an oily mutant of Bombyx mori, exhibits mottled translucent skin with a varying degree of transparency among individuals. By linkage analysis of 2112 backcross individuals using polymorphic DNA markers, we successfully mapped a 179-kb region of chromosome 20 responsible for the ov phenotype. This region contains nine predicted genes. We compared the mRNA expression of these nine genes between the wild type and mutants and found that the expression of one of them, Bmdysb, was strikingly decreased in the epidermis of ov as well as its allelomorph, ov(p). Moreover, its expression level was well correlated with the degree of transparency among individuals. Bmdysb was homologous to DTNBP1 encoding human dysbindin, a subunit of the biogenesis of lysosome-related organelles complex-1. Our results suggest that the translucent skin may be due to repression of Bmdysb in the ov mutants and that Bmdysb plays an important role in the formation and accumulation of urate granules in the silkworm epidermis.

Function, diversity, and application of insect juvenile hormone epoxidases (CYP15).

Juvenile hormones (JHs) represent a family of sesquiterpenoid hormones in insects, and they play a key role in regulating development, metamorphosis, and reproduction. The last two steps of the JH biosynthetic pathway, epoxidation and methyl esterification of farnesoic acid to JH, are insect specific, and thus have long been considered a promising target for biorational insecticides. Recently, the enzymes involved in the last two steps have been molecularly identified: JH acid methyltransferase catalyzes the esterification step and the cytochrome P450 CYP15 enzyme catalyzes the epoxidation step. In this review, we describe the recent progress on the characterization of JH biosynthetic enzymes, with special focus on the function and diversity of the CYP15 family. CYP15 genes have evolved lineage-specific substrate specificity and regulatory mechanisms in insects, which appear to be associated with the lineage-specific acquisition of unique JH structure and function. In addition, the lack of CYP15 genes in crustacean (Daphnia pulex) and arachnid (Tetranychus urticae) species, whose genomes have been fully sequenced, may imply that CYP15 enzymes are an evolutionary innovation in insects to use the epoxide forms of methylated farnesoid molecules as their principal JHs. Molecular identification and characterization of CYP15 genes from broad taxa of insects have paved the way to the design of target-specific, biorational anti-JH agents.

The silkworm Green b locus encodes a quercetin 5-O-glucosyltransferase that produces green cocoons with UV-shielding properties.

In the silkworm Bombyx mori, dietary flavonoids are metabolized and accumulate in cocoons, thereby causing green coloration. Classical genetic studies suggest that more than seven independent loci are associated with this trait; however, because of the complex inheritance pattern, none of these loci have been characterized molecularly, and a plausible and comprehensive model for their action has not been proposed. Here, we report the identification of the gene responsible for the Green b (Gb) locus involving the green cocoon trait. In +(Gb) animals, glucosylation at the 5-O position of dietary quercetin did not occur, and the total amount of flavonoids in tissues and cocoons was dramatically reduced. We performed positional cloning of Gb and found a 38-kb deletion in a UDP-glucosyltransferase (UGT) gene cluster associated with the +(Gb) allele. RT-PCR and biochemical studies suggested that deletion of Bm-UGT10286 (UGT) is responsible for Gb and Bm-UGT10286 is virtually the sole source of UGT activity toward the 5-O position of quercetin. Our data show that the regiospecific glucosylation of flavonoids by the quercetin 5-O-glucosyltransferase can greatly affect the overall bioavailability of flavonoids in animals. Furthermore, we provide evidence that flavonoids increase the UV-shielding activity of cocoons and thus could confer an increased survival advantage to insects contained in these cocoons. This study will lead to greater understanding of mechanisms for metabolism, uptake, and transport of dietary flavonoids, which have a variety of biological activities in animals and beneficial effects on human health.

Redundant actions of neuropeptides encoded by the dh-pban gene for larval color pattern formation in the oriental armyworm Mythimnaseparata.

The pyrokinin (PK)/pheromone biosynthesis-activating neuropeptide (PBAN) family, which is defined by a conserved C-terminal pentapeptide (FXPRLamide), is involved in many physiological processes in insects. In the oriental armyworm Mythimna separata, the larvae exhibit a variety of color patterns in response to changes in population density, which are caused by melanization and a reddish coloration hormone (MRCH), which is a member of the FXPRLamide neuropeptides. Interestingly, in some lepidopteran insects, MRCH is known as a PBAN, which activates the pheromone gland to produce sex pheromones. PBAN is encoded by a single gene, dh-pban, which encodes additional FXPRLamide neuropeptides, such as the diapause hormone (DH) and subesophageal ganglion neuropeptides (SGNPs). To determine the roles of the dh-pban gene, which produces multiple types of FXPRLamide neuropeptides after post-transcriptional cleavage of the precursor protein, we performed CRISPR/Cas9-mediated targeted mutagenesis in M. separata. We demonstrated that knockout armyworm larvae lost density-dependent cuticular melanization and retained yellow body color, even when reared under crowded conditions. Moreover, our rescue experiments using the synthetic peptides showed that not only PBAN but also ß- and γ-SGNPs significantly induce the cuticular melanization in a dose dependent manner. Taken together, our results provide genetic evidence that neuropeptides encoded by the single dh-pban gene act redundantly to control density-dependent color pattern formation in M. separata.

Positional cloning of silkworm white egg 2 (w-2) locus shows functional conservation and diversification of ABC transporters for pigmentation in insects.

The white, scarlet and brown genes of Drosophila melanogaster encode three half-type ATP-binding cassette (ABC) transporters. In Drosophila, precursors of ommochromes and pteridines are transported by White/Scarlet and White/Brown heterodimers, respectively. The white egg 2 (w-2) mutant of the silkworm, Bombyx mori, has white eggs and eyes because of lack of ommochrome granules in the serosa and eyes. Here, we report that the silkworm w-2 locus encodes an ortholog of Drosophila scarlet. Our results indicate that Bombyx Scarlet forms a heterodimer with Bombyx White to transport ommochrome precursors, suggesting that formation of a White/Scarlet heterodimer and its involvement in the transport of ommochrome precursors are evolutionarily ancient and widely conserved traits in insects. Contrary to dipteran insects, white and scarlet were juxtaposed in a head-to-tail orientation in the silkworm genome, suggesting that the origin of white and scarlet was a tandem duplication of their ancestral transporter gene. In Bombyx, White is also essential for the transport of uric acid in larval epidermis. However, our results suggest that a Bombyx White/Scarlet heterodimer is not involved in this process. Our results emphasize the functional conservation and diversification of half-type ABC transporter families in insects, which may contribute to their extremely diverse color patterns.

Female sex pheromone and male behavioral responses of the bombycid moth Trilocha varians: comparison with those of the domesticated silkmoth Bombyx mori.

Analysis of female sex pheromone components and subsequent field trap experiments demonstrated that the bombycid moth Trilocha varians uses a mixture of (E,Z)-10,12-hexadecadienal (bombykal) and (E,Z)-10,12-hexadecadienyl acetate (bombykyl acetate) as a sex pheromone. Both of these components are derivatives of (E,Z)-10,12-hexadecadienol (bombykol), the sex pheromone of the domesticated silkmoth Bombyx mori. This finding prompted us to compare the antennal and behavioral responses of T. varians and B. mori to bombykol, bombykal, and bombykyl acetate in detail. The antennae of T. varians males responded to bombykal and bombykyl acetate but not to bombykol, and males were attracted only when lures contained both bombykal and bombykyl acetate. In contrast, the antennae of B. mori males responded to all the three components. Behavioral analysis showed that B. mori males responded to neither bombykal nor bombykyl acetate. Meanwhile, the wing fluttering response of B. mori males to bombykol was strongly inhibited by bombykal and bombykyl acetate, thereby indicating that bombykal and bombykyl acetate act as behavioral antagonists for B. mori males. T. varians would serve as a reference species for B. mori in future investigations into the molecular mechanisms underlying the evolution of sex pheromone communication systems in bombycid moths.

Reinvestigation of the sex pheromone of the wild silkmoth Bombyx mandarina: the effects of bombykal and bombykyl acetate.

Sex pheromone investigations of the domesticated silkmoth, Bombyx mori (Lepidoptera: Bombycidae), helped elucidate the molecular and physiological fundamentals of chemical communication in moths, yet little is known about pheromone evolution in bombycid species. Therefore, we reexamined the sex pheromone communication in the wild silkmoth, Bombyx mandarina, which is considered ancestral to B. mori. Our investigations revealed that (a) B. mandarina females produce (E,Z)-10,12-hexadecadienol (bombykol), but not (E,Z)-10,12-hexadecadienal (bombykal) or (E,Z)-10,12-hexadecadienyl acetate (bombykyl acetate), which are pheromone components in other bombycid moths; (b) antennae of male B. mandarina respond strongly to bombykol as well as to bombykal and bombykyl acetate; and (c) bombykal and bombykyl acetate strongly inhibit attraction of B. mandarina males to bombykol in the field. The present study clarifies the evolution of pheromone communication in bombycid moths.