Genome-wide assessment and development of molecular diagnostic methods for imidacloprid-resistance in the brown planthopper, Nilaparvata lugens (Hemiptera; Delphacidae).

BACKGROUND: The brown planthopper, Nilaparvata lugens (Stål), is one of the most notorious pests of rice throughout Asia. The brown planthopper has developed high resistance to imidacloprid, a member of neonicotinoid insecticides. Several genes and mutations conferring imidacloprid resistance in N. lugens, especially in eastern and southeastern Asia populations, have been reported. Thus, the key mechanisms of imidacloprid resistance need to be examined. RESULTS: RNA-seq analyses revealed that only one cytochrome P450 monooxygenase gene, CYP6ER1, was commonly upregulated in the five resistant strains tested. Sequences of CYP6ER1, which were highly expressed in the imidacloprid-resistant strains, contained a three-nucleotide deletion in the coding region, and amino acid substitutions and deletion, compared to that in an imidacloprid-susceptible strain. RNAi-mediated gene knockdown of CYP6ER1 increased imidacloprid susceptibility in a resistant strain. Further, we established two simple and convenient PCR-based molecular diagnostic methods to detect the CYP6ER1 locus with the three-nucleotide deletion. Using these methods, the resistance of F2 progenies derived from the crosses of F1 siblings from susceptible and resistant parents was analyzed, showing that the imidacloprid resistance had a relationship to the CYP6ER1 locus with the three-nucleotide deletion. CONCLUSION: The overexpression of a variant CYP6ER1 with amino acid substitutions and deletion was involved in imidacloprid resistance in N. lugens. Based on these findings, molecular diagnostic methods have been developed and are promising tools for monitoring imidacloprid resistance in paddy fields. © 2020 Society of Chemical Industry.

Genome-wide Identification of Tebufenozide Resistant Genes in the smaller tea tortrix, Adoxophyes honmai (Lepidoptera: Tortricidae).

The smaller tea tortrix, Adoxophyes honmai, has developed strong resistance to tebufenozide, a diacylhydrazine-type (DAH) insecticide. Here, we investigated its mechanism by identifying genes responsible for the tebufenozide resistance using various next generation sequencing techniques. First, double-digest restriction site-associated DNA sequencing (ddRAD-seq) identified two candidate loci. Then, synteny analyses using A. honmai draft genome sequences revealed that one locus contained the ecdysone receptor gene (EcR) and the other multiple CYP9A subfamily P450 genes. RNA-seq and direct sequencing of EcR cDNAs found a single nucleotide polymorphism (SNP), which was tightly linked to tebufenozide resistance and generated an amino acid substitution in the ligand-binding domain. The binding affinity to tebufenozide was about 4 times lower in in vitro translated EcR of the resistant strain than in the susceptible strain. RNA-seq analyses identified commonly up-regulated genes in resistant strains, including CYP9A and choline/carboxylesterase (CCE) genes. RT-qPCR analysis and bioassays showed that the expression levels of several CYP9A and CCE genes were moderately correlated with tebufenozide resistance. Collectively, these results suggest that the reduced binding affinity of EcR is the main factor and the enhanced detoxification activity by some CYP9As and CCEs plays a supplementary role in tebufenozide resistance in A. honmai.

Transcriptome analysis of the anhydrobiotic cell line Pv11 infers the mechanism of desiccation tolerance and recovery.

The larvae of the African midge, Polypedilum vanderplanki, can enter an ametabolic state called anhydrobiosis to overcome fatal desiccation stress. The Pv11 cell line, derived from P. vanderplanki embryo, shows desiccation tolerance when treated with trehalose before desiccation and resumes proliferation after rehydration. However, the molecular mechanisms of this desiccation tolerance remain unknown. Here, we performed high-throughput CAGE-seq of mRNA and a differentially expressed gene analysis in trehalose-treated, desiccated, and rehydrated Pv11 cells, followed by gene ontology analysis of the identified differentially expressed genes. We detected differentially expressed genes after trehalose treatment involved in various stress responses, detoxification of harmful chemicals, and regulation of oxidoreduction that were upregulated. In the desiccation phase, L-isoaspartyl methyltransferase and heat shock proteins were upregulated and ribosomal proteins were downregulated. Analysis of differentially expressed genes during rehydration supported the notion that homologous recombination, nucleotide excision repair, and non-homologous recombination were involved in the recovery process. This study provides initial insights into the molecular mechanisms underlying the extreme desiccation tolerance of Pv11 cells.

Identification of allatostatins in the brown-winged green bug Plautia stali.

Juvenile hormone (JH) biosynthesis is inhibited under short-day conditions in the brown-winged green bug Plautia stali. We investigated allatostatic molecules in the brain of P. stali. Methanol brain extracts strongly inhibited JH biosynthesis. The allatostatic activities of the brain extracts were heat stable but gently suppressed by trypsin treatment, indicating that the allatostatic molecules were peptides. Grybi-MIP1, found in Gryllus bimaculatus as an allatostatic molecule, inhibited JH biosynthesis in P. stali. In contrast, peptides such as Dippu-AST2, 8, and 9, found in Diploptera punctata, did not affect JH biosynthesis in P. stali. We found a cDNA sequence encoding a peptide precursor of myoinhibitory peptides (MIPs), which we named Plast-MIP. Three synthetic peptides, AWKDLSKAW-NH2 (Plast-MIP1), GWSDLQSAGW-NH2 (Plast-MIP5), and AADWGSFRGSW-NH2 (Plast-MIP8), deduced from the precursor sequence, showed clear inhibition of JH biosynthesis in P. stali. Analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and tandem mass spectrometry showed that Plast-MIP8 resides in the brain. Expression of the Plast-MIP mRNA precursor was detected in the brain of insects reared under short- and long-day conditions. These results suggest that Plast-MIP is an allatostatic molecule and that MIPs are synthesized irrespective of photoperiod. To our knowledge, this is the first study to identify Plast-MIP as a functional allatostatin in hemipteran insects.

Genome-wide microarray screening for Bombyx mori genes related to transmitting the determination outcome of whether to produce diapause or nondiapause eggs.

The bivoltine silkworm Bombyx mori (Lepidoptera: Bombycidae) exhibits a maternally controlled embryonic diapause. Maternal silkworms decide whether to lay diapause or nondiapause eggs depending on environmental factors such as the temperature and photoperiod during the egg and larval stages, and then induce diapause eggs during the pupal stage. However, little is known about the molecular mechanism that conveys the outcome of whether to produce diapause or nondiapause eggs from the egg or larval stages to the pupal stage. This study used microarray analysis to investigate differentially expressed genes in the larval brains of diapause- and nondiapause-egg producers, to which bivoltine silkworms were destined by thermal or photic stimulation during the egg stage. The cytochrome P450 18a1 and Krüppel homolog 1 genes were upregulated in producers of diapause eggs compared with those of nondiapause eggs under both experimental conditions. Cytochrome P450 18a1 encodes a key enzyme for steroid hormone inactivation and Krüppel homolog 1 is an early juvenile hormone-inducible gene that mediates the repression of metamorphosis. The upregulation of these genes during the larval stage might be involved in the signaling pathway that transmits information about the diapause program from the egg stage to the pupal stage in the silkworm.

Genomic Analysis of an Ascomycete Fungus from the Rice Planthopper Reveals How It Adapts to an Endosymbiotic Lifestyle.

A number of sap-sucking insects harbor endosymbionts, which are thought to play an important role in the development of their hosts. One of the most important rice pests, the brown planthopper (BPH), Nilaparvata lugens (Stål), harbors an obligatory yeast-like symbiont (YLS) that cannot be cultured in vitro. Genomic information on this YLS would be useful to better understand its evolution. In this study, we performed genome sequencing of the YLS using both 454 and Illumina approaches, generating a draft genome that shows a slightly smaller genome size and relatively higher GC content than most ascomycete fungi. A phylogenomic analysis of the YLS supported its close relationship with insect pathogens. We analyzed YLS-specific genes and the categories of genes that are likely to have changed in the YLS during its evolution. The loss of mating type locus demonstrated in the YLS sheds light on the evolution of eukaryotic symbionts. This information about the YLS genome provides a helpful guide for further understanding endosymbiotic associations in hemiptera and the symbiotic replacement of ancient bacteria with a multifunctional YLS seems to have been a successful change.

Superoxide dismutases, SOD1 and SOD2, play a distinct role in the fat body during pupation in silkworm Bombyx mori.

One way that aerobic biological systems counteract the generation of reactive oxygen species (ROS) is with superoxide dismutase proteins SOD1 and SOD2 that metabolize superoxide radicals to molecular oxygen and hydrogen peroxide or scavenge oxygen radicals produced by the extensive oxidation-reduction and electron-transport reactions that occur in mitochondria. We characterized SOD1 and SOD2 of Bombyx mori isolated from the fat body of larvae. Immunological analysis demonstrated the presence of BmSOD1 and BmSOD2 in the silk gland, midgut, fat body, Malpighian tubules, testis and ovary from larvae to adults. We found that BmSOD2 had a unique expression pattern in the fat body through the fifth instar larval developmental stage. The anti-oxidative functions of BmSOD1 and BmSOD2 were assessed by exposing larvae to insecticide rotenone or vasodilator isosorbide dinitrate, which is an ROS generator in BmN4 cells; however, exposure to these compounds had no effect on the expression levels of either BmSOD protein. Next, we investigated the physiological role of BmSOD1 and BmSOD2 under environmental oxidative stress, applied through whole-body UV irradiation and assayed using quantitative RT-PCR, immunoblotting and microarray analysis. The mRNA expression level of both BmSOD1 and BmSOD2 was markedly increased but protein expression level was increased only slightly. To examine the differences in mRNA and protein level due to UV irradiation intensity, we performed microarray analysis. Gene set enrichment analysis revealed that genes in the insulin signaling pathway and PPAR signaling pathway were significantly up-regulated after 6 and 12 hours of UV irradiation. Taken together, the activities of BmSOD1 and BmSOD2 may be related to the response to UV irradiation stress in B. mori. These results suggest that BmSOD1 and BmSOD2 modulate environmental oxidative stress in the cell and have a specific role in fat body of B. mori during pupation.

Transcriptome analysis of the salivary glands of Nephotettix cincticeps (Uhler).

The green rice leafhopper (GRH), Nephotettix cincticeps, is one of the most important pests of rice in temperate Asian countries. GRH, a vascular feeder, secretes watery and gelling saliva in the process of feeding on phloem and xylem sap. It is known that GRH saliva contains several bioactive proteins, including enzymes such as laccase and beta-glucosidase. In this study, we performed transcriptome analysis of salivary glands of GRH using Illumina paired-end sequencing. Of 51,788 assembled contigs, 16,017 (30.9%) showed significant similarity to known proteins in the NCBI nr database, while 34,978 (67.5%) could not be annotated by similarity search, Pfam, or gene ontology (GO). Contigs (905) with predicted signal peptides and no putative transmembrane domains are suggested to represent secreted protein coding genes. Among the 76 most highly expressed putative secretory protein contigs, 68 transcripts were found to be salivary gland-specific or at least -dominant, but not expressed in stomach or Malpighian tubules. However, 45 of the 68 transcripts were unknown proteins. These findings suggest that most of the GRH transcripts encoding secreted proteins expressed in salivary glands are species and/or tissue specific. Our results provide a fundamental list of genes involved in GRH-Poaceae host plant interactions including successful feeding and plant pathogen transmission.

Comparative genome sequencing reveals genomic signature of extreme desiccation tolerance in the anhydrobiotic midge.

Anhydrobiosis represents an extreme example of tolerance adaptation to water loss, where an organism can survive in an ametabolic state until water returns. Here we report the first comparative analysis examining the genomic background of extreme desiccation tolerance, which is exclusively found in larvae of the only anhydrobiotic insect, Polypedilum vanderplanki. We compare the genomes of P. vanderplanki and a congeneric desiccation-sensitive midge P. nubifer. We determine that the genome of the anhydrobiotic species specifically contains clusters of multi-copy genes with products that act as molecular shields. In addition, the genome possesses several groups of genes with high similarity to known protective proteins. However, these genes are located in distinct paralogous clusters in the genome apart from the classical orthologues of the corresponding genes shared by both chironomids and other insects. The transcripts of these clustered paralogues contribute to a large majority of the mRNA pool in the desiccating larvae and most likely define successful anhydrobiosis. Comparison of expression patterns of orthologues between two chironomid species provides evidence for the existence of desiccation-specific gene expression systems in P. vanderplanki.

Genetic mapping of the rice resistance-breaking gene of the brown planthopper Nilaparvata lugens.

Host plant resistance has been widely used for controlling the major rice pest brown planthopper (BPH, Nilaparvata lugens). However, adaptation of the wild BPH population to resistance limits the effective use of resistant rice varieties. Quantitative trait locus (QTL) analysis was conducted to identify resistance-breaking genes against the anti-feeding mechanism mediated by the rice resistance gene Bph1. QTL analysis in iso-female BPH lines with single-nucleotide polymorphism (SNP) markers detected a single region on the 10th linkage group responsible for the virulence. The QTL explained from 57 to 84% of the total phenotypic variation. Bulked segregant analysis with next-generation sequencing in F2 progenies identified five SNPs genetically linked to the virulence. These analyses showed that virulence to Bph1 was controlled by a single recessive gene. In contrast to previous studies, the gene-for-gene relationship between the major resistance gene Bph1 and virulence gene of BPH was confirmed. Identified markers are available for map-based cloning of the major gene controlling BPH virulence to rice resistance.

Complete genome sequences of two iflaviruses from the brown planthopper, Nilaparvata lugens.

The complete genome sequences of two new iflaviruses (genus Iflavirus, family Iflaviridae) were determined. These viral sequences were first identified in RNA-seq contig sequences of Nilaparvata lugens in two distinct colonies: Izumo and Kagoshima. The accuracy of the contig sequences of the two viruses was verified by restriction enzyme digestion of RT-PCR products from viruliferous insects. RT-PCR of RNA extracted from honeydews after viruliferous insect feeding detected the expected viral products, which suggested that viruses were excreted into the honeydews by the insects. Since we previously designated a similar iflavirus as "Nilaparvata lugens honeydew virus 1", the two new viruses have been tentatively named "Nilaparvata lugens honeydew virus 2" and "Nilaparvata lugens honeydew virus 3". The identity of the putative amino acid sequences of the capsid proteins of these viruses met the criterion for iflavirus species demarcation. Therefore, these two viruses are suggested to be members of distinct species in the genus Iflavirus.

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.

Transcriptome analysis of neuropeptides and G-protein coupled receptors (GPCRs) for neuropeptides in the brown planthopper Nilaparvata lugens.

The genes encoding neuropeptides, neurohormones and their putative G-protein coupled receptors were identified in the brown planthopper (BPH), Nilaparvata lugens (Stål) by transcriptome analysis (RNA-seq). Forty-eight candidate genes were found to encode neuropeptides or peptide hormones. These include all known insect neuropeptides and neurohormones, with the exception of neuropeptide-like precursor 2 (NPLP2) and trissin. The gene coding for prothoracicotropic hormone (PTTH) was first identified from hemimetabolous insect. A total of 57 putative neuropeptide GPCR genes were identified and phylogenetic analysis showed most of them to be closely related to insect GPCRs. A notable finding was the occurrence of vertebrate hormone receptors, thyrotropin-releasing hormone receptor (TRHR)-like GPCR and parathyroid hormone receptor (PTHR)-like GPCRs. These results suggest that N. lugens possesses the most comprehensive neuropeptide system yet found in insects. Moreover, our findings demonstrate the power of RNA-seq as a tool for analyzing the neuropeptide-related genes in the absence of whole genome sequence information.

Genomes of the rice pest brown planthopper and its endosymbionts reveal complex complementary contributions for host adaptation.

BACKGROUND: The brown planthopper, Nilaparvata lugens, the most destructive pest of rice, is a typical monophagous herbivore that feeds exclusively on rice sap, which migrates over long distances. Outbreaks of it have re-occurred approximately every three years in Asia. It has also been used as a model system for ecological studies and for developing effective pest management. To better understand how a monophagous sap-sucking arthropod herbivore has adapted to its exclusive host selection and to provide insights to improve pest control, we analyzed the genomes of the brown planthopper and its two endosymbionts. RESULTS: We describe the 1.14 gigabase planthopper draft genome and the genomes of two microbial endosymbionts that permit the planthopper to forage exclusively on rice fields. Only 40.8% of the 27,571 identified Nilaparvata protein coding genes have detectable shared homology with the proteomes of the other 14 arthropods included in this study, reflecting large-scale gene losses including in evolutionarily conserved gene families and biochemical pathways. These unique genomic features are functionally associated with the animal's exclusive plant host selection. Genes missing from the insect in conserved biochemical pathways that are essential for its survival on the nutritionally imbalanced sap diet are present in the genomes of its microbial endosymbionts, which have evolved to complement the mutualistic nutritional needs of the host. CONCLUSIONS: Our study reveals a series of complex adaptations of the brown planthopper involving a variety of biological processes, that result in its highly destructive impact on the exclusive host rice. All these findings highlight potential directions for effective pest control of the planthopper.

Large scale full-length cDNA sequencing reveals a unique genomic landscape in a lepidopteran model insect, Bombyx mori.

The establishment of a complete genomic sequence of silkworm, the model species of Lepidoptera, laid a foundation for its functional genomics. A more complete annotation of the genome will benefit functional and comparative studies and accelerate extensive industrial applications for this insect. To realize these goals, we embarked upon a large-scale full-length cDNA collection from 21 full-length cDNA libraries derived from 14 tissues of the domesticated silkworm and performed full sequencing by primer walking for 11,104 full-length cDNAs. The large average intron size was 1904 bp, resulting from a high accumulation of transposons. Using gene models predicted by GLEAN and published mRNAs, we identified 16,823 gene loci on the silkworm genome assembly. Orthology analysis of 153 species, including 11 insects, revealed that among three Lepidoptera including Monarch and Heliconius butterflies, the 403 largest silkworm-specific genes were composed mainly of protective immunity, hormone-related, and characteristic structural proteins. Analysis of testis-/ovary-specific genes revealed distinctive features of sexual dimorphism, including depletion of ovary-specific genes on the Z chromosome in contrast to an enrichment of testis-specific genes. More than 40% of genes expressed in specific tissues mapped in tissue-specific chromosomal clusters. The newly obtained FL-cDNA sequences enabled us to annotate the genome of this lepidopteran model insect more accurately, enhancing genomic and functional studies of Lepidoptera and comparative analyses with other insect orders, and yielding new insights into the evolution and organization of lepidopteran-specific genes.

KONAGAbase: a genomic and transcriptomic database for the diamondback moth, Plutella xylostella.

BACKGROUND: The diamondback moth (DBM), Plutella xylostella, is one of the most harmful insect pests for crucifer crops worldwide. DBM has rapidly evolved high resistance to most conventional insecticides such as pyrethroids, organophosphates, fipronil, spinosad, Bacillus thuringiensis, and diamides. Therefore, it is important to develop genomic and transcriptomic DBM resources for analysis of genes related to insecticide resistance, both to clarify the mechanism of resistance of DBM and to facilitate the development of insecticides with a novel mode of action for more effective and environmentally less harmful insecticide rotation. To contribute to this goal, we developed KONAGAbase, a genomic and transcriptomic database for DBM (KONAGA is the Japanese word for DBM). DESCRIPTION: KONAGAbase provides (1) transcriptomic sequences of 37,340 ESTs/mRNAs and 147,370 RNA-seq contigs which were clustered and assembled into 84,570 unigenes (30,695 contigs, 50,548 pseudo singletons, and 3,327 singletons); and (2) genomic sequences of 88,530 WGS contigs with 246,244 degenerate contigs and 106,455 singletons from which 6,310 de novo identified repeat sequences and 34,890 predicted gene-coding sequences were extracted. The unigenes and predicted gene-coding sequences were clustered and 32,800 representative sequences were extracted as a comprehensive putative gene set. These sequences were annotated with BLAST descriptions, Gene Ontology (GO) terms, and Pfam descriptions, respectively. KONAGAbase contains rich graphical user interface (GUI)-based web interfaces for easy and efficient searching, browsing, and downloading sequences and annotation data. Five useful search interfaces consisting of BLAST search, keyword search, BLAST result-based search, GO tree-based search, and genome browser are provided. KONAGAbase is publicly available from our website (http://dbm.dna.affrc.go.jp/px/) through standard web browsers. CONCLUSIONS: KONAGAbase provides DBM comprehensive transcriptomic and draft genomic sequences with useful annotation information with easy-to-use web interfaces, which helps researchers to efficiently search for target sequences such as insect resistance-related genes. KONAGAbase will be continuously updated and additional genomic/transcriptomic resources and analysis tools will be provided for further efficient analysis of the mechanism of insecticide resistance and the development of effective insecticides with a novel mode of action for DBM.

The genome sequence and transmission of an iflavirus from the brown planthopper, Nilaparvata lugens.

A previously unknown iflavirus has been identified in a laboratory colony of the brown planthopper, Nilaparvata lugens. The iflavirus-like sequence was first identified in contig sequences obtained from transcriptome sequencing (RNA-seq) of the brown planthopper. The complete viral genome was resequenced using the Sanger method. The positive-strand RNA genome was 10,937 nucleotides excluding the 3' poly(A) tail, and contained a single large open reading frame encoding coat proteins in the 5' region and replicases in the 3' region. Conserved motifs for coat proteins, helicase, cysteine protease, and RNA-dependent RNA polymerase were identified in the deduced amino sequence, and the estimated molecular mass of the large polyprotein was 358.6kDa. RT-PCR detection of the viral genome indicated that viral shedding occurred through the honeydews of insects in the infected colony. To test transmission, the collected honeydews were used to feed insects in a non-viruliferous colony. After 7 days, the expected RT-PCR fragment was detected in the insects, indicating that the virus can be transmitted horizontally. This is the first iflavirus identified in planthoppers; thus, we propose the name of the virus as Nilaparvata lugens honeydew virus-1.

A simple sequence repeat- and single-nucleotide polymorphism-based genetic linkage map of the brown planthopper, Nilaparvata lugens.

In this study, we developed the first genetic linkage map for the major rice insect pest, the brown planthopper (BPH, Nilaparvata lugens). The linkage map was constructed by integrating linkage data from two backcross populations derived from three inbred BPH strains. The consensus map consists of 474 simple sequence repeats, 43 single-nucleotide polymorphisms, and 1 sequence-tagged site, for a total of 518 markers at 472 unique positions in 17 linkage groups. The linkage groups cover 1093.9 cM, with an average distance of 2.3 cM between loci. The average number of marker loci per linkage group was 27.8. The sex-linkage group was identified by exploiting X-linked and Y-specific markers. Our linkage map and the newly developed markers used to create it constitute an essential resource and a useful framework for future genetic analyses in BPH.

KAIKObase: an integrated silkworm genome database and data mining tool.

BACKGROUND: The silkworm, Bombyx mori, is one of the most economically important insects in many developing countries owing to its large-scale cultivation for silk production. With the development of genomic and biotechnological tools, B. mori has also become an important bioreactor for production of various recombinant proteins of biomedical interest. In 2004, two genome sequencing projects for B. mori were reported independently by Chinese and Japanese teams; however, the datasets were insufficient for building long genomic scaffolds which are essential for unambiguous annotation of the genome. Now, both the datasets have been merged and assembled through a joint collaboration between the two groups. DESCRIPTION: Integration of the two data sets of silkworm whole-genome-shotgun sequencing by the Japanese and Chinese groups together with newly obtained fosmid- and BAC-end sequences produced the best continuity (~3.7 Mb in N50 scaffold size) among the sequenced insect genomes and provided a high degree of nucleotide coverage (88%) of all 28 chromosomes. In addition, a physical map of BAC contigs constructed by fingerprinting BAC clones and a SNP linkage map constructed using BAC-end sequences were available. In parallel, proteomic data from two-dimensional polyacrylamide gel electrophoresis in various tissues and developmental stages were compiled into a silkworm proteome database. Finally, a Bombyx trap database was constructed for documenting insertion positions and expression data of transposon insertion lines. CONCLUSION: For efficient usage of genome information for functional studies, genomic sequences, physical and genetic map information and EST data were compiled into KAIKObase, an integrated silkworm genome database which consists of 4 map viewers, a gene viewer, and sequence, keyword and position search systems to display results and data at the level of nucleotide sequence, gene, scaffold and chromosome. Integration of the silkworm proteome database and the Bombyx trap database with KAIKObase led to a high-grade, user-friendly, and comprehensive silkworm genome database which is now available from URL: http://sgp.dna.affrc.go.jp/KAIKObase/.

yellow and ebony are the responsible genes for the larval color mutants of the silkworm Bombyx mori.

Many larval color mutants have been obtained in the silkworm Bombyx mori. Mapping of melanin-synthesis 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.