Embryonic stage selection for cryopreservation of the silkworm Bombyx mori and the effects of cryopreservation on embryo tissues.

Successful cryopreservation of the important silkworm bioresource, Bombyx mori, is essential. In this study, we aimed for successful cryopreservation using vitrification of silkworm embryos. Furthermore, the embryos were assessed for the most appropriate sampling stage. We found that vitrified embryos developed to the serosa ingestion stage when they were vitrified at embryonic stage 24-25. The most suitable stage for vitrification was around a 5-10 h period when the tracheal fibers were elongating in stage 25. None of the vitrified embryos developed into larvae, although some did develop to the pre-hatching stage. From histological analysis, we found that several small cracks formed on the cuticle covering the hypodermis in the vitrified embryos. Additionally, the midgut epithelium was detached from the midgut wall and mixed with the yolk in the midgut lumen. We speculate that the vitrified embryos died from a rapid loss of body water from the small cracks formed in the cuticle. We also suggest that the vitrified embryos may have resulted in dysfunction of the midgut.

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 reexamination on the deficiency of riboflavin accumulation in Malpighian tubules in larval translucent mutants of the silkworm, Bombyx mori.

A variety of insects accumulate high contents of riboflavin (vitamin B2) in their Malpighian tubules (MTs). Although this process is known to be genetically controlled, the mechanism is not known. In the 1940s and the 1950s, several studies showed that riboflavin contents were low in the MTs of some Bombyx mori (silkworm) mutants with translucent larval skin mutations (e.g., w-3, od, oa, and otm) and that genes responsible for these translucent mutations also affected riboflavin accumulation in the MTs. Since the 2000s, it has been shown that the w-3 gene encodes an ABC transporter, whereas genes responsible for od, oa, and otm mutations encode for the biogenesis of lysosome-related organelles. These findings suggest that some genes of ABC transporters and biogenesis of lysosome-related organelles may control the accumulation of riboflavin in MTs. Therefore, we reexamined the effects that translucent mutations have on the accumulation of riboflavin in MTs by using the translucent and wild-type segregants in mutant strains to measure the specific effect that each gene has on riboflavin accumulation (independent of genomic background). We used nine translucent mutations (w-3oe, oa, od, otm, Obs, oy, or, oh, and obt) even though the genes responsible for some of these mutations (Obs, oy, or, oh, and obt) have not yet been isolated. Through observation of larval MTs and measurements of riboflavin content using high-performance liquid chromatography, we found that the oa, od, otm, and or mutations were responsible for low contents of riboflavin in MTs, whereas the Obs and oy mutations did not affect riboflavin accumulation. This indicates that the molecular mechanism for riboflavin accumulation is similar but somewhat different than the mechanism responsible for uric acid accumulation in epidermal cells. We found that the genes responsible for oa, od, and otm mutations were consistent with those already established for uric acid accumulation in larval epidermis. This suggests that these three genes control riboflavin accumulation in MTs through a mechanism similar to that of uric acid accumulation, although we do not yet know why the or mutation also controls riboflavin accumulation.

Discovery of a New Species of Telenomus (Hymenoptera: Scelionidae) Parasitic on Eggs of Bombyx mandarina and Bombyx mori (Lepidoptera: Bombycidae) in Japan and Taiwan.

We reared a Telenomus species from eggs of Bombyx mandarina (Moore) (Lepidoptera: Bombycidae) and Bombyx mori (Linnaeus) (Lepidoptera: Bombycidae) in Japan, and from eggs of B. mandarina in Taiwan. Morphological examination revealed that this Telenomus species is new to science. In this article, we describe it as Telenomus moricolus Matsuo et Hirose, sp. nov. Because B. mandarina is considered to be an ancestor of B. mori, a domestic insect, it is reasonable to assume that B. mandarina is an original host of T. moricolus. This is the second discovery of an egg parasitoid attacking wild and domesticated silkworms, following the first discovery of T. theophilae, a Chinese species. The significance of the discovery of T. moricolus is discussed in relation to examining the effects of host-insect domestication on egg parasitism.

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.

Long-term preservation of eri and ailanthus silkworms using frozen gonads.

Cryopreservation of eri and ailanthus silkworms using frozen gonads was investigated. First, we evaluated the freeze tolerance of ovary and testis in the eri silkworm, which showed high tolerance. Mating between frozen ovary-transplanted females and frozen testis-transplanted males produced 163.0 eggs, yielding 105.7 larvae per moth. In a second experiment, we tested the use of the eri silkworm as a host insect for gonad transplantation from ailanthus silkworm donors. A high success ratio for laid and hatched eggs was demonstrated for ovary transplantation (97.8 and 51.3 eggs per moth, respectively). For testis transplantation, however, the average number of hatched larvae was low (12.0). Mating between host eri females and males in which both frozen ovary and testis of the ailanthus silkworm had been transplanted produced 6.4 fertilized eggs per host moth. Our success in using cross subspecies cryopreservation between these wild silkworms could lead to the alternative use of hosts between species in other insects.

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.

Vitellogenin receptor mutation leads to the oogenesis mutant phenotype "scanty vitellin" of the silkworm, Bombyx mori.

BACKGROUND: The vitellogenin receptor (VgR) mediates the uptake of vitellogenin (Vg) from the hemolymph by developing oocytes. RESULTS: VgR with the mutational EGF1 domain can bind ligand proteins but cannot be dissociated under acidic conditions. The mutant is lethal in embryos. CONCLUSION: Bombyx mori VgR (BmVgR) has an important role in egg formation and embryonic development. SIGNIFICANCE: BmVgR is a potential target for pest control. In insects, the vitellogenin receptor (VgR) mediates the uptake of vitellogenin (Vg) from the hemolymph by developing oocytes. The oogenesis mutant scanty vitellin (vit) of Bombyx mori (Bm) lacks vitellin and 30-kDa proteins, but B. mori egg-specific protein and BmVg are normal. The vit eggs are white and smaller compared with the pale yellow eggs of the wild type and are embryonic lethal. This study found that a mutation in the B. mori VgR gene (BmVgR) is responsible for the vit phenotype. We cloned the cDNA sequences encoding WT and vit BmVgR. The functional domains of BmVgR are similar to those of other low-density lipoprotein receptors. When compared with the wild type, a 235-bp genomic sequence in vit BmVgR is substituted for a 7-bp sequence. This mutation has resulted in a 50-amino acid deletion in the third Class B region of the first epidermal growth factor (EGF1) domain. BmVgR is expressed specifically in oocytes, and the transcriptional level is changed dramatically and consistently with maturation of oocytes during the previtellogenic periods. Linkage analysis confirmed that BmVgR is mutated in the vit mutant. The coimmunoprecipitation assay confirmed that mutated BmVgR is able to bind BmVg but that BmVg cannot be dissociated under acidic conditions. The WT phenotype determined by RNA interference was similar to that of the vit phenotype for nutritional deficiency, such as BmVg and 30-kDa proteins. These results showed that BmVgR has an important role in transporting proteins for egg formation and embryonic development in B. mori.

A novel third chromosomal locus controls susceptibility to Autographa californica multiple nucleopolyhedrovirus in the silkworm, Bombyx mori.

Baculovirus demonstrates specific infection spectrums and thus one certain host exhibits particular response to single baculovirus isolate. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is considered to be not an innate pathogen to Bombyx mori, but some silkworm strains have been identified to be permissive to AcMNPV, indicating the positive or negative involvement of certain host factors in baculovirus replications in vivo. To provide a fundamental knowledge of this process, we performed large-scale screening to investigate the responses of 448 silkworm strains against recombinant AcMNPV inoculation. By genetic analysis between permissive and resistant strains identified, we further confirmed that a potential corresponding locus on chromosome 3 regulates host responses to AcMNPV in silkworm. Additionally, we found that it is available for AcMNPV-silkworm baculovirus expression vector system to produce proteins of interest.

Purification and characterization of cocoonase from the silkworm Bombyx mori.

Cocoonase (CCN) which facilitates the degradation of a cocoon is recognized as a trypsin-like serine protease. In this study, CCN from the silkworm Bombyx mori was purified and comprehensively characterized. Its activity was maximal at about pH 9.8. It was stable above pH 3.4 at 4 °C and below 50 °C at pH 7.5. CuSO4, FeSO4, and ZnSO4 showed inhibitory effects on CCN, but other salts improved activity. Typical trypsin inhibitors inhibited CCN, but the relative inhibitory activities were much lower than those against bovine trypsin. An extract of cocoon shells inhibited trypsin, but it was only slightly inhibitory against CCN. There were significant differences in catalytic efficiencies and substrate specificities as between CCN and bovine trypsin.

CD36 homolog divergence is responsible for the selectivity of carotenoid species migration to the silk gland of the silkworm Bombyx mori.

Dietary carotenoids are absorbed in the intestine and delivered to various tissues by circulating lipoproteins; however, the mechanism underlying selective delivery of different carotenoid species to individual tissues remains elusive. The products of the Yellow cocoon (C) gene and the Flesh (F) gene of the silkworm Bombyx mori determine the selectivity for transport of lutein and ß-carotene, respectively, to the silk gland. We previously showed that the C gene encodes Cameo2, a CD36 family member, which is thought to function as a transmembrane lipoprotein receptor. Here, we elucidated the molecular identity of the F gene product by positional cloning, as SCRB15, a paralog of Cameo2 with 26% amino acid identity. In the F mutant, SCRB15 mRNA structure was severely disrupted, due to a 1.4 kb genomic insertion in a coding exon. Transgenic expression of SCRB15 in the middle silk gland using the binary GAL4-UAS expression system enhanced selective ß-carotene uptake by the middle silk gland, while transgenic expression of Cameo2 enhanced selective lutein uptake under the same GAL4 driver. Our findings indicate that divergence of genes in the CD36 family determines the selectivity of carotenoid species uptake by silk gland tissue and that CD36-homologous proteins can discriminate among carotenoid species.

Identification of the Bombyx red egg gene reveals involvement of a novel transporter family gene in late steps of the insect ommochrome biosynthesis pathway.

Ommochromes are one of the major pigments involved in coloration of eggs, eyes, and body surface of insects. However, the molecular mechanisms of the final steps of ommochrome pigment synthesis have been largely unknown. The eggs of the silkworm Bombyx mori contain a mixture of ommochrome pigments, and exhibit a brownish lilac color. The recessive homozygous of egg and eye color mutant, red egg (re), whose eggs display a pale orange color instead of normal dark coloration, has been long suggested to have a defect in the biosynthesis of the final ommochrome pigments. Here, we identify the gene responsible for the re locus by positional cloning, mutant analysis, and RNAi experiments. In the re mutants, we found that a 541-bp transposable element is inserted into the ORF of BGIBMGA003497-1 (Bm-re) encoding a novel member of a major facilitator superfamily transporter, causing disruption of the splicing of exon 9, resulting in two aberrant transcripts with frameshifts yielding nonfunctional proteins lacking the C-terminal transmembrane domains. Bm-re function in pigmentation was confirmed by embryonic RNAi experiments. Homologs of the Bm-re gene were found in all insect genomes sequenced at present, except for 12 sequenced Drosophila genomes, which seemed to correlate with the previous studies that have demonstrated that eye ommochrome composition is different from other insects in several Dipterans. Knockdown of the Bm-re homolog by RNAi in the red flour beetle Tribolium castaneum caused adult compound eye coloration defects, indicating a conserved role in ommochrome pigment biosynthesis at least among holometabolous insects.

Non-molting glossy/shroud encodes a short-chain dehydrogenase/reductase that functions in the 'Black Box' of the ecdysteroid biosynthesis pathway.

In insects, the precise timing of molting and metamorphosis is strictly guided by a principal steroid hormone, ecdysone. Among the multiple conversion steps for synthesizing ecdysone from dietary cholesterol, the conversion of 7-dehydrocholesterol to 5beta-ketodiol, the so-called 'Black Box', is thought to be the important rate-limiting step. Although a number of genes essential for ecdysone synthesis have recently been revealed, much less is known about the genes that are crucial for functioning in the Black Box. Here we report on a novel ecdysteroidgenic gene, non-molting glossy (nm-g)/shroud (sro), which encodes a short-chain dehydrogenase/reductase. This gene was first isolated by positional cloning of the nm-g mutant of the silkworm Bombyx mori, which exhibits a low ecdysteroid titer and consequently causes a larval arrest phenotype. In the fruit fly, Drosophila melanogaster, the closest gene to nm-g is encoded by the sro locus, one of the Halloween mutant members that are characterized by embryonic ecdysone deficiency. The lethality of the sro mutant is rescued by the overexpression of either sro or nm-g genes, indicating that these two genes are orthologous. Both the nm-g and the sro genes are predominantly expressed in tissues producing ecdysone, such as the prothoracic glands and the ovaries. Furthermore, the phenotypes caused by the loss of function of these genes are restored by the application of ecdysteroids and their precursor 5beta-ketodiol, but not by cholesterol or 7-dehydrocholesterol. Altogether, we conclude that the Nm-g/Sro family protein is an essential enzyme for ecdysteroidogenesis working in the Black Box.

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.

Salt-induced changes in the subunit structure of the Bacillus stearothermophilus lipoate acetyltransferase.

The Bacillus stearothermophilus lipoate acetyltransferase (E2), composed of sixty identical, subunits is the core component of the pyruvate dehydrogenase complex (PDC). E2 polypeptide is composed of LD, PSBD, and CD domains. Most studies had focused on a truncated E2 that is deficient in LD and PSBD, because CD mainly contributes to maintaining the multimeric structure. We examined salt-induced changes in E2 without truncation and constructed reaction models. We speculate that in the presence of KCl, E2 is dissociated into a monomer and then assembled into an aggregative complex (C(A)) and a quasi-stable complex (C(Q)). C(A) was larger than C(Q), but smaller than intact E2. C(A) and C(Q), were dominant complexes at about neutral pH and at basic pH respectively. PDC, in which PSBD is occupied by other components, and a truncated E2 undergo dissociation only. LD-PSBD region besides CD might then contribute to the partial association of dissociated E2.

Development of a method for long-term preservation of Bombyx mori silkworm strains using frozen ovaries.

Development of long-term preservation is essential for conservation of stocks of silkworm genetic resources. Thus far, a few methods have been reported, but more improvement is required for practical use. We have developed two effective modifications of a method for long-term preservation using frozen ovaries. One was slow cooling (1 °C per min) until -80 °C of the donor ovaries made possible by use of a BICELL freezing vessel. Using donor ovaries of 4th instar larvae, the average number of eggs laid per moth increased significantly from 110.7 ± 53.4 eggs per moth by slow cooling with the BICELL vessel vs 12.3 ± 10.3 eggs per moth by direct cooling in liquid nitrogen. A second improvement was connecting the thread bodies of the donor ovaries with those of the host in the transplantation step. Females operated on with the new method yielded a significantly higher percentage of moths that laid fertilized eggs than those transplanted with the standard procedure (70.4 ± 21.6% vs 22.9 ± 9.3%).

Repression of tyrosine hydroxylase is responsible for the sex-linked chocolate mutation of the silkworm, Bombyx mori.

Pigmentation patterning has long interested biologists, integrating topics in ecology, development, genetics, and physiology. Wild-type neonatal larvae of the silkworm, Bombyx mori, are completely black. By contrast, the epidermis and head of larvae of the homozygous recessive sex-linked chocolate (sch) mutant are reddish brown. When incubated at 30 degrees C, mutants with the sch allele fail to hatch; moreover, homozygous mutants carrying the allele sch lethal (sch(l)) do not hatch even at room temperature (25 degrees C). By positional cloning, we narrowed a region containing sch to 239,622 bp on chromosome 1 using 4,501 backcross (BC1) individuals. Based on expression analyses, the best sch candidate gene was shown to be tyrosine hydroxylase (BmTh). BmTh coding sequences were identical among sch, sch(l), and wild-type. However, in sch the approximately 70-kb sequence was replaced with approximately 4.6 kb of a Tc1-mariner type transposon located approximately 6 kb upstream of BmTh, and in sch(l), a large fragment of an L1Bm retrotransposon was inserted just in front of the transcription start site of BmTh. In both cases, we observed a drastic reduction of BmTh expression. Use of RNAi with BmTh prevented pigmentation and hatching, and feeding of a tyrosine hydroxylase inhibitor also suppressed larval pigmentation in the wild-type strain, pnd(+) and in a pS (black-striped) heterozygote. Feeding L-dopa to sch neonate larvae rescued the mutant phenotype from chocolate to black. Our results indicate the BmTh gene is responsible for the sch mutation, which plays an important role in melanin synthesis producing neonatal larval color.

The biological role of core 1ß1-3galactosyltransferase (T-synthase) in mucin-type O-glycosylation in Silkworm, Bombyx mori.

O-glycosylation of secreted and membrane-bound proteins is an important post-translational modification that affects recognition of cell surface receptors, protein folding, and stability. However, despite the importance of O-linked glycans, their biological functions have not yet been fully elucidated and the synthetic pathway of O-glycosylation has not been investigated in detail, especially in the silkworm. In this study, we aimed to investigate O-glycosylation in silkworms by analyzing the overall structural profiles of mucin-type O-glycans using LC-MS. We found GalNAc or GlcNAc monosaccharide and core 1 disaccharide (Galß1-3-GalNAcα1-Ser/Thr) were major components of the O-glycan attached to secreted proteins produced in silkworms. Furthermore, we characterized the 1 b1,3-galactosyltransferase (T-synthase) required for synthesis of the core 1 structure, common to many animals. Five transcriptional variants and four protein isoforms were identified in silkworms, and the biological functions of these isoforms were investigated. We found that BmT-synthase isoforms 1 and 2 were localized in the Golgi apparatus in cultured BmN4 cells and functioned both in cultured cells and silkworms. Additionally, a specific functional domain of T-synthase, called the stem domain, was found to be essential for activity and is presumed to be needed for dimer formation and galactosyltransferase activity. Altogether, our results elucidated the O-glycan profile and function of T-synthase in the silkworm. Our findings allow the practical comprehension of O-glycosylation required for employing silkworms as a productive expression system.

Molecular characterization of an insecticide-induced novel glutathione transferase in silkworm.

BACKGROUND: The glutathione transferase (GST) superfamily is involved in the detoxification of various xenobiotics. We have identified a GST mRNA that was induced in the fat bodies of a silkworm strain exhibiting diazinon resistance and have investigated the enzyme properties of this GST. METHODS: A soluble recombinant protein was overexpressed in Escherichia coli. Amino acid residues of interest were changed to alanine by site-directed mutagenesis. RESULTS AND CONCLUSIONS: Phylogenetic analysis of the deduced amino acid sequence indicates that this GST belongs to an unclassified group previously reported in mosquitoes. This enzyme, named bmGSTu, has highly conserved amino acid residues, including Tyr7, Ser12 and Asn50. A recombinant bmGSTu was able to catalyze the biotranslation of glutathione with 1-chloro-2,4-dinitrobenzene, a synthetic substrate of GST. Kinetic analysis of bmGSTu mutants indicated that Tyr7, Ser12 and Asn50 are involved in enzyme function. GENERAL SIGNIFICANCE: These results support the hypothesis that bmGSTu may play a role in insecticide resistance in Bombyx mori.

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.