Diet choice: The two-factor host acceptance system of silkworm larvae.

Many herbivorous insects are mono- or oligophagous, having evolved to select a limited range of host plants. They specifically identify host-plant leaves using their keen sense of taste. Plant secondary metabolites and sugars are thought to be key chemical cues that enable insects to identify host plants and evaluate their quality as food. However, the neuronal and behavioral mechanisms of host-plant recognition are poorly understood. Here, we report a two-factor host acceptance system in larvae of the silkworm Bombyx mori, a specialist on several mulberry species. The first step is controlled by a chemosensory organ, the maxillary palp (MP). During palpation at the leaf edge, the MP detects trace amounts of leaf-surface compounds, which enables host-plant recognition without biting. Chemosensory neurons in the MP are tuned with ultrahigh sensitivity (thresholds of attomolar to femtomolar) to chlorogenic acid (CGA), quercetin glycosides, and ß-sitosterol (ßsito). Only if these 3 compounds are detected does the larva make a test bite, which is evaluated in the second step. Low-sensitivity neurons in another chemosensory organ, the maxillary galea (MG), mainly detect sucrose in the leaf sap exuded by test biting, allowing larvae to accept the leaf and proceed to persistent biting (feeding). The two-factor host acceptance system reported here may commonly underlie stereotyped feeding behavior in many phytophagous insects and determine their feeding habits.

Imaginal disc growth factor maintains cuticle structure and controls melanization in the spot pattern formation of Bombyx mori.

The complex stripes and patterns of insects play key roles in behavior and ecology. However, the fine-scale regulation mechanisms underlying pigment formation and morphological divergence remain largely unelucidated. Here we demonstrated that imaginal disc growth factor (IDGF) maintains cuticle structure and controls melanization in spot pattern formation of Bombyx mori. Moreover, our knockout experiments showed that IDGF is suggested to impact the expression levels of the ecdysone inducible transcription factor E75A and pleiotropic factors apt-like and Toll8/spz3, to further control the melanin metabolism. Furthermore, the untargeted metabolomics analyses revealed that BmIDGF significantly affected critical metabolites involved in phenylalanine, beta-alanine, purine, and tyrosine metabolism pathways. Our findings highlighted not only the universal function of IDGF to the maintenance of normal cuticle structure but also an underexplored space in the gene function affecting melanin formation. Therefore, this study furthers our understanding of insect pigment metabolism and melanin pattern polymorphisms.

Unpredictable recombination of PB transposon in Silkworm: a potential risk.

The piggyBac (PB) transposon is the most widely used vector for generating transgenic silkworms. The stability of the PB transposon in the receptor is a serious concern that requires attention because of biosafety concerns. In this study, we found that the transgene silkworm developed loss of reporter gene traits. To further investigate the regularity, we traced the genes and traits of this silkworm. After successful alteration of the silkworm genome with the MASP1 gene (named red-eyed silkworm; RES), silkworm individuals with lost reporter genes were found after long-term transgenerational breeding and were designated as the white-eyed silkworm (WES). PCR amplification indicated that exogenous genes had been lost in the WES. Testing was conducted on the PB transposons, and the left arm (L arm) did not exist; however, the right arm (R arm) was preserved. Amino acid analysis showed that the amino acid content of the WES changed versus the common silkworm and RES. These results indicate that the migration of PB transposons in Bombyx mori does occur and is unpredictable. This is because the silkworm genome contains multiple PB-like sequences that might influence the genetic stability of transgenic lines. When using PB transposons as a transgene vector, it is necessary to fully evaluate and take necessary measures to prevent its re-migration in the recipient organism. Further experiments are needed if we want to clarify the regularity of the retransposition phenomenon and the direct and clear association with similar sequences of transposons.

Identification of an aldehyde oxidase involved in indole-3-acetic acid synthesis in Bombyx mori silk gland.

Auxin is thought to be an important factor in the induction of galls by galling insects. We have previously shown that both galling and nongalling insects synthesize indole-3-acetic acid (IAA) from tryptophan (Trp) via two intermediates, indole-3-acetaldoxime (IAOx) and indole-3-acetaldehyde (IAAld). In this study, we isolated an enzyme that catalyzes the last step "IAAld → IAA" from a silk-gland extract of Bombyx mori. The enzyme, designated "BmIAO1", contains two 2Fe-2S iron-sulfur-cluster-binding domains, an FAD-binding domain, and a molybdopterin-binding domain, which are conserved in aldehyde oxidases. BmIAO1 causes the nonenzymatic conversion of Trp to IAAld and the enzymatic conversion of IAOx to IAA, suggesting that BmIAO1 alone is responsible for IAA production in B. mori. However, a detailed comparison of pure BmIAO1 and the crude silk-gland extract suggested the presence of other enzymes involved in IAA production from Trp. Abbreviations: BA: benzoic acid; CE: collision energy; CXP: collision cell exit potential; DP: declustering potential; IAA: indole-3-acetic acid; IBI1: IAA biosynthetic inhibitor-1; IAAld: indole-3-acetaldehyde; ICA: indole-3-carboxylic acid; IAOx: indole-3-acetaldoxime; IEtOH: indole-3-ethanol; LC-MS/MS: liquid chromatography-tandem mass spectrometry; Trp: tryptophan.

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.

Morphology of visual projection neurons supplying premotor area in the brain of the silkmoth Bombyx mori.

Sex pheromones orient male moths toward conspecific female moths; the presence of visual information modulates this behavior. In the current study, we explore candidate neuronal pathways for the interaction between vision and the locomotor signal for pheromone orientation. We describe the connectivity between visual neuropils and brain premotor centers, the posterior slope (PS) and the lateral accessory lobe (LAL), in the silkmoth Bombyx mori. Using a single-cell labeling technique, we analyze visual projection neurons supplying these areas. Neurons from both the medulla and lobula complex projected to the PS but only the neurons originating in the lobula complex had additional processes to the LAL. Further, we identified populations of putative feedback neurons from the premotor centers to the optic lobe. Neurons originating in the PS were likely to project to the medulla, whereas those originating in the LAL were likely to project to the lobula complex. The anatomical study contributes to further understanding of integration of visual information on the locomotor control in the insect brain.

Proteomic Analysis of Larval Integument in a Dominant Obese Translucent (Obs) Silkworm Mutant.

The dominant obese translucent (Obs) mutant of the silkworm (Bombyx mori) results in a short and stout larval body, translucent phenotype, and abnormal pigmentation in the integument. The Obs mutant also displays deficiency in ecdysis and metamorphosis. In the present study, to gain an understanding of multiple Obs phenotypes, we investigated the phenotypes of Obs and performed a comparative analysis of the larval integument proteomes of Obs and normal silkworms. The phenotypic analysis revealed that the Obs larvae were indeed short and fat, and that chitin and uric acid content were lower but melanin content was higher in the Obs mutant. Proteomic analysis revealed that 244 proteins were significantly differentially expressed between Obs and normal silkworms, some of which were involved in uric acid metabolism and melanin pigmentation. Twenty-six proteins were annotated as cuticular proteins, including RR motif-rich cuticular proteins (CPR), glycine-rich cuticular protein (CPG), hypothetical cuticular protein (CPH), cuticular protein analogous to peritrophins (CPAPs), and the chitin_bind_3 motif proteins, and accounted for over 84% of the abundance of the total significantly differentially expressed proteins. Moreover, 22 of the 26 cuticular proteins were downregulated in the Obs mutant. Comparative proteomic analysis suggested that the multiple phenotypes of the Obs mutant might be related to changes in the expression of proteins that participate in cuticular formation, uric acid metabolism, and melanin pigmentation. These results could lay a basis for further identification of the gene responsible for the Obs mutant. The data have been deposited to ProteomeXchange with identifier PXD010998.

Comparative Analysis of the Integument Transcriptomes between Stick Mutant and Wild-Type Silkworms.

In insects, the integument provides mechanical support for the whole body and protects them from infections, physical and chemical injuries, and dehydration. Diversity in integument properties is often related to body shape, behavior, and survival rate. The stick (sk) silkworm is a spontaneous mutant with a stick-like larval body that is firm to the touch and, thus, less flexible. Analysis of the mechanical properties of the cuticles at day 3 of the fifth instar (L5D3) of sk larvae revealed higher storage modulus and lower loss tangent. Transcriptome sequencing identified a total of 19,969 transcripts that were expressed between wild-type Dazao and the sk mutant at L5D2, of which 11,596 transcripts were novel and detected in the integument. Differential expression analyses identified 710 upregulated genes and 1009 downregulated genes in the sk mutant. Gene Ontology (GO) enrichment analysis indicated that four chitin-binding peritrophin A domain genes and a chitinase gene were upregulated, whereas another four chitin-binding peritrophin A domain genes, a trehalase, and nine antimicrobial peptides were downregulated. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that two functional pathways, namely, fructose and mannose metabolism and tyrosine metabolism, were significantly enriched with differentially-expressed transcripts. This study provides a foundation for understanding the molecular mechanisms underlying the development of the stiff exoskeleton in the sk mutant.

Sequential steps of macroautophagy and chaperone-mediated autophagy are involved in the irreversible process of posterior silk gland histolysis during metamorphosis of Bombyx mori.

To elucidate the degradation process of the posterior silk gland during metamorphosis of the silkworm ITALIC! Bombyx mori, tissues collected on the 6th day after entering the 5th instar (V6), prior to spinning (PS), during spinning (SP) and after cocoon formation (CO) were used to analyze macroautophagy, chaperone-mediated autophagy (CMA) and the adenosine triphosphate (ATP)-dependent ubiquitin proteasome. Immediately after entering metamorphosis stage PS, the levels of ATP and phosphorylated p70S6 kinase protein decreased spontaneously and continued to decline at SP, followed by a notable restoration at CO. In contrast, phosphorylated AMP-activated protein kinase α (AMPKα) showed increases at SP and CO. Most of the Atg8 protein was converted to form II at all stages. The levels of ubiquitinated proteins were high at SP and CO, and low at PS. The proteasome activity was high at V6 and PS but low at SP and CO. In the isolated lysosome fractions, levels of Hsc70/Hsp70 protein began to increase at PS and continued to rise at SP and CO. The lysosomal cathepsin B/L activity showed a dramatic increase at CO. Our results clearly demonstrate that macroautophagy occurs before entering the metamorphosis stage and strongly suggest that the CMA pathway may play an important role in the histolysis of the posterior silk gland during metamorphosis.

Marker-assisted selection in breeding silkworm strains with high tolerance to fluoride, scaleless wings, and high silk production.

In the silkworm (Bombyx mori), tolerance to fluoride and scaleless wings are controlled by the dominant gene Dtf (dominant tolerance to fluoride) and recessive gene nlw (no Lepidoptera wings), respectively, and these genes have been mapped by using simple sequence repeat and sequence tag site markers. Marker-assisted evaluation and selection of silkworms with fluoride tolerance and scaleless wings were used for predicting fluoride resistance and scaleless wings in backcrossed animals. A silkworm strain was bred using this method, and its economic characteristics were found to be similar to those of commercial silkworms. These methods will therefore be useful for silkworm breeding programs and in screening for two or more characteristics of interest for segregating populations.

Transcriptomic analysis of developmental features of Bombyx mori wing disc during metamorphosis.

BACKGROUND: Wing discs of B. mori are transformed to pupal wings during the larva-to-pupa metamorphosis with dramatic morphological and structural changes. To understand these changes at a transcriptional level, RNA-seq of the wing discs from 6-day-old fifth instar larvae (L5D6), prepupae (PP) and pupae (P0) was performed. RESULTS: In total, 12,254 transcripts were obtained from the wing disc, out of which 5,287 were identified to be differentially expressed from L5D6 to PP and from PP to P0. The results of comprehensive analysis of RNA-seq data showed that during larvae-to-pupae metamorphosis, many genes of 20E signaling pathway were up-regulated and those of JH signaling pathway were down-regulated. Seventeen transcription factors were significantly up-regulated. Cuticle protein genes (especially wing cuticle protein genes), were most abundant and significantly up-regulated at P0 stage. Genes responsible for the degradation and de novo synthesis of chitin were significantly up-regulated. There were A and B two types of chitin synthases in B. mori, whereas only chitin synthase A was up-regulated. Both trehalose and D-fructose, which are precursors of chitin synthesis, were detected in the hemolymph of L5D6, PP and P0, suggesting de novo synthesis of chitin. However, most of the genes that are related to early wing disc differentiation were down-regulated. CONCLUSIONS: Extensive transcriptome and DGE profiling data of wing disc during metamorphosis of silkworm have been generated, which provided comprehensive gene expression information at the transcriptional level. These results implied that during the larva-to-pupa metamorphosis, pupal wing development and transition might be mainly controlled by 20E signaling in B. mori. The 17 up-regulated transcription factors might be involved in wing development. Chitin required for pupal wing development might be generated from both degradation of componential chitin and de novo synthesis. Chitin synthase A might be responsible for the chitin synthesis in the pupal wing, while both trehalose and D-fructose might contribute to the de novo synthesis of chitin during the formation of pupal wing.

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.

[Genetic analysis and gene mapping of two novel quail-like mutants from the silkworm (Bombyx mori)].

Two novel body marking mutants were discovered during silkworm (Bombyx mori) breeding. The mutants have no obvious eye-spots compared with normal marking (+) individuals, but their star spots and semilunar markings on dorsal sides are normal, and there are dots and lines with longitudinal wave markings on dorsal sides of the 6th to 7th abdominal segments which consist quail markings in between star spots and semilunar markings. The whole body markings are very similar to that of quail mutant (q); thus these mutants are named as quail-like mutants (q-l). Young larvae of one mutant are in brown color, and develop normally. Their cocoons are regular and uniform in size. Thus, this mutant is designated as brown quail-like (q-lb). Another mutant's larvae are in light purple skin; thus this mutant is named as purple quail-like (q-lp). They take little amount of mulberry leaves, and are weak and develop slowly and unevenly. Their larval bodies and cocoons are small. Genetic analysis revealed that both q-lb and q-lp were recessive genes, and they were allelic, with q-lb recessive to q-lp. These genes are different from quail mutant (q) and located on the chromosome 8 after tested by the morphological markers, P3(2), p(2), Ze(3), L(4), re(5), E(6), q(7), I-a(9), ms(12), ch(13), oa(14), cts(16), mln(18), msn(19), rb(21) and so(26) and SSR markers.

Fine mapping of a supernumerary proleg mutant (E(Cs) -l) and comparative expression analysis of the abdominal-A gene in silkworm, Bombyx mori.

Patterning and phenotypic variations of appendages in insects provide important clues on developmental genetics. In the silkworm Bombyx mori, morphological variations associated with the E complex, an analogue of the Drosophila melanogaster bithorax complex, mainly determine the shape and number of prolegs on abdominal segments. Here, we report the identification and characterization of the allele responsible for the supernumerary crescents and legs-like (E(Cs) -l) mutant, a model derived from spontaneous mutation of the E complex, with supernumerary legs and extra crescents. Fine mapping with 1605 individuals revealed a ∼68 kb sequence in the upstream intergenic region of B. mori abdominal-A (Bmabd-A) clustered with the E(Cs) -l locus. Quantitative real-time PCR (qRT-PCR) and Western blotting analyses disclosed a marked increase in Bmabd-A expression in the E(Cs) -l mutant at both the transcriptional and translational levels, compared to wild-type Dazao. Furthermore, we observed ectopic expression of the Bmabd-A protein in the second abdominal segment (A2) of the E(Cs) -l mutant. Our results collectively suggest that the 68 kb region contains important regulatory elements of the Bmabd-A gene, and provide evidence that the gene is required for limb development in abdominal segments in the silkworm.

Detailed investigation of the sequential pathological changes in silkworm larvae infected with Bombyx densovirus type 1.

Bombyx mori densovirus type 1 (BmDNV-1) is a pathogen causing flacherie disease in silkworms. BmDNV-1 multiplies only in the nuclei of the columnar cells of larval midgut epithelium. Although several immunohistochemical studies using anti-BmDNV-1 antibody have been reported to date, sequential pathological changes in BmDNV-1-infected larvae have not been completely elucidated. In this paper, sequential investigations were performed on the pathological features of BmDNV-1-infected larvae and BmDNV-1 propagation. Oral infection experiments using newly ecdysed 4th instar larvae revealed that the larvae began to die 9 days post infection (dpi), and the remaining died 10 dpi. Histological observations revealed phenotypic alterations in the midgut cells from 4 dpi, and complete disruption of the midgut structure at 9 dpi. Quantitative RT-PCR of two BmDNV-1 genes indicated that BmDNV-1 began to propagate from 4 dpi, and gradually increased until the larvae died. These expression patterns revealed marked correlation with the histological changes observed in the virus-infected midgut cells. Moreover, bioassays using larvae at various developmental stages clearly indicated that the pathogenicity of this virus is not dependent on the larval stage or the molting process.

Hierarchical, multilayered cell walls reinforced by recycled silk cocoons enhance the structural integrity of honeybee combs.

We reveal the sophisticated and hierarchical structure of honeybee combs and measure the elastic properties of fresh and old natural honeycombs at different scales by optical microscope, environmental scanning electron microscope, nano/microindentation, and by tension and shear tests. We demonstrate that the comb walls are continuously strengthened and stiffened without becoming fragile by the addition of thin wax layers reinforced by recycled silk cocoons reminiscent of modern fiber-reinforced composite laminates. This is done to increase its margin of safety against collapse due to a temperature increase. Artificial engineering honeycombs mimic only the macroscopic geometry of natural honeycombs, but have yet to achieve the microstructural sophistication of their natural counterparts. The natural honeycombs serve as a prototype of truly biomimetic cellular materials with hitherto unattainable improvement in stiffness, strength, toughness, and thermal stability.

Biological and molecular characterization of silkworm strains from the Brazilian germplasm bank of Bombyx mori.

Brazil has only one public genetic pool of Bombyx mori strains, which was established in 2005 at Universidade Estadual de Maringá, Maringá, Paraná State. This genetic bank has been maintained, and the strains have been characterized using genetic and morphological tools. The quantitative and qualitative traits, directly or indirectly related to productivity, were evaluated in 14 silkworm strains. In addition to biological and productivity analyses, DNA markers related to susceptibility to the B. mori nucleopolyhedrovirus (BmNPV) were analyzed. BmNPV is a major cause of production loss and is a serious problem for Paraná sericulture. The silkworm strains from diverse geographic origins were found to have different characteristics, including body weight, larval stage duration, cocoon weight, and other biological traits. In terms of productivity, the raw silk percentages were almost uniform, with an overall average of 16.28%. Overall, the Chinese strain C37 gave the best performance in many of the quantitative traits, and it surpassed the other strains in productivity traits. Therefore, it can be used as one of the strains that compose the elite germplasm for silkworm breeding programs. Additionally, genetic molecular markers were efficient in discriminating between B. mori strains that had been identified based on their geographical origin. We found that all Japanese strains produced a 400-bp molecular marker that has been associated with susceptibility to BmNPV.

Evidence of selection at melanin synthesis pathway loci during silkworm domestication.

The domesticated silkworm (Bombyx mori) was domesticated from wild silkworm (Bombyx mandarina) more than 5,000 years ago. During domestication, body color between B. mandarina and B. mori changed dramatically. However, the molecular mechanism of the silkworm body color transition is not known. In the present study, we examined within- and between-species nucleotide diversity for eight silkworm melanin synthesis pathway genes, which play a key role in cuticular pigmentation of insects. Our results showed that the genetic diversity of B. mori was significantly lower than that of B. mandarina and 40.7% of the genetic diversity of wild silkworm was lost in domesticated silkworm. We also examined whether position effect exists among melanin synthesis pathway genes in B. mandarina and B. mori. We found that the upstream genes have significantly lower levels of genetic diversity than the downstream genes, supporting a functional constraint hypothesis (FCH) of metabolic pathway, that is, upstream enzymes are under greater selective constraint than downstream enzymes because upstream enzymes participate in biosynthesis of a number of metabolites. We also investigated whether some of the melanin synthesis pathway genes experienced selection during domestication. Neutrality test, coalescent simulation, as well as network and phylogenetic analyses showed that tyrosine hydroxylase (TH) gene was a domestication locus. Sequence analysis further suggested that a putative expression enhancer (Abd-B-binding site) in the intron of TH gene might be disrupted during domestication. TH is the rate-limiting enzyme of melanin synthesis pathway in insects. Real-time polymerase chain reaction assay did show that the relative expression levels of TH gene in B. mori were significantly lower than that in B. mandarina at three different developmental stages, which is consistent with light body color of domesticated silkworm relative to wild silkworm. Therefore, we speculated that expression change of TH gene may contribute to the body color transition from B. mandarina to B. mori. Our results emphasize the exceptional role of gene expression regulation in morphological transition of domesticated animals.

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.

A CD36-related transmembrane protein is coordinated with an intracellular lipid-binding protein in selective carotenoid transport for cocoon coloration.

The transport pathway of specific dietary carotenoids from the midgut lumen to the silk gland in the silkworm, Bombyx mori, is a model system for selective carotenoid transport because several genetic mutants with defects in parts of this pathway have been identified that manifest altered cocoon pigmentation. In the wild-type silkworm, which has both genes, Yellow blood (Y) and Yellow cocoon (C), lutein is transferred selectively from the hemolymph lipoprotein to the silk gland cells where it is accumulated into the cocoon. The Y gene encodes an intracellular carotenoid-binding protein (CBP) containing a lipid-binding domain known as the steroidogenic acute regulatory protein-related lipid transfer domain. Positional cloning and transgenic rescue experiments revealed that the C gene encodes Cameo2, a transmembrane protein gene belonging to the CD36 family genes, some of which, such as the mammalian SR-BI and the fruit fly ninaD, are reported as lipoprotein receptors or implicated in carotenoid transport for visual system. In C mutant larvae, Cameo2 expression was strongly repressed in the silk gland in a specific manner, resulting in colorless silk glands and white cocoons. The developmental profile of Cameo2 expression, CBP expression, and lutein pigmentation in the silk gland of the yellow cocoon strain were correlated. We hypothesize that selective delivery of lutein to specific tissue requires the combination of two components: 1) CBP as a carotenoid transporter in cytosol and 2) Cameo2 as a transmembrane receptor on the surface of the cells.