Evolutionary origin and distribution of amino acid mutations associated with resistance to sodium channel modulators in onion thrips, Thrips tabaci.

In onion thrips Thrips tabaci, reduced sensitivity of the sodium channel caused by several sodium channel mutations have been correlated with pyrethroid resistance. For this study, using mitochondrial cytochrome c oxidase subunit I gene sequences, we examined the phylogenetic relation among a total of 52 thelytokous and arrhenotokous strains with different genotypes of the sodium channel mutations. Then, we used flow cytometry to estimate their ploidy. Results showed that the strains are divisible into three groups: diploid thelytoky, triploid thelytoky, and diploid arrhenotoky. Using 23 whole genome resequencing data obtained from 20 strains out of 52, we examined their genetic relation further using principal component analysis, admixture analysis, and a fixation index. Results showed that diploid and triploid thelytokous groups are further classifiable into two based on the sodium channel mutations harbored by the respective group members (strains). The greatest genetic divergence was observed between thelytokous and arrhenotokous groups with a pair of T929I and K1774N. Nevertheless, they shared a genomic region with virtually no polymorphism around the sodium channel gene loci, suggesting a hard selective sweep. Based on these findings, we discuss the evolutionary origin and distribution of the sodium channel mutations in T. tabaci.

A major endogenous glycoside hydrolase mediating quercetin uptake in Bombyx mori.

Quercetin is a common plant flavonoid which is involved in herbivore-plant interactions. Mulberry silkworms (domestic silkworm, Bombyx mori, and wild silkworm, Bombyx mandarina) take up quercetin from mulberry leaves and accumulate the metabolites in the cocoon, thereby improving its protective properties. Here we identified a glycoside hydrolase, named glycoside hydrolase family 1 group G 5 (GH1G5), which is expressed in the midgut and is involved in quercetin metabolism in the domestic silkworm. Our results suggest that this enzyme mediates quercetin uptake by deglycosylating the three primary quercetin glycosides present in mulberry leaf: rutin, quercetin-3-O-malonylglucoside, and quercetin-3-O-glucoside. Despite being located in an unstable genomic region that has undergone frequent structural changes in the evolution of Lepidoptera, GH1G5 has retained its hydrolytic activity, suggesting quercetin uptake has adaptive significance for mulberry silkworms. GH1G5 is also important in breeding: defective mutations which result in discoloration of the cocoon and increased silk yield are homozygously conserved in 27 of the 32 Japanese white-cocoon domestic silkworm strains and 12 of the 30 Chinese ones we investigated.

Fine mapping of Green a, Ga, on chromosome 27 in Bombyx mori.

Some strains of silkworms produce green cocoons of varying intensities. This results from quantitative and qualitative differences in flavonoid pigments, which are influenced by the environment and genetic background. We discovered that the appearance of a faint green cocoon is regulated by a gene (G27) located on chromosome 27. Through mating experiments, we found that G27 is identical to an essential flavonoid cocoon gene, Ga. This locus has not been previously described. Furthermore, we narrowed down the Ga region to 438 kbp using molecular markers. Within this region, several predicted genes for sugar transporters form a cluster structure, suggesting that Ga is among them.

Highly accurate genome assembly of an improved high-yielding silkworm strain, Nichi01.

The silkworm (Bombyx mori) is an important lepidopteran model insect and an industrial domestic animal traditionally used for silk production. Here, we report the genome assembly of an improved Japanese strain Nichi01, in which the cocoon yield is comparable to that of commercial silkworm strains. The integration of PacBio Sequel II long-read and ddRAD-seq-based high-density genetic linkage map achieved the highest quality genome assembly of silkworms to date; 22 of the 28 pseudomolecules contained telomeric repeats at both ends, and only four gaps were present in the assembly. A total of 452 Mbp of the assembly with an N50 of 16.614 Mbp covered 99.3% of the complete orthologs of the lepidopteran core genes. Although the genome sequence of Nichi01 and that of the previously reported low-yielding tropical strain p50T assured their accuracy in most regions, we corrected several regions, misassembled in p50T, in our assembly. A total of 18,397 proteins were predicted using over 95 Gb of mRNA-seq derived from 10 different organs, covering 96.9% of the complete orthologs of the lepidopteran core genes. The final assembly and annotation files are available in KAIKObase (https://kaikobase.dna.affrc.go.jp/index.html) along with a genome browser and BLAST searching service, which would facilitate further studies and the breeding of silkworms and other insects.

A feasibility trial of genomics-based diagnosis detecting insecticide resistance of the diamondback moth.

BACKGROUND: Insecticide resistance management has been key for crop protection for over 70 years and is increasingly important because the development of new active ingredients has decreased in recent years. By monitoring the development of resistance in a timely manner, we can effectively prolong insecticide efficacy. Genomic-based diagnosis can reliably predict resistance development if information on resistant mutations against major pesticides is available. Here, we developed a feasibility trial of genomics-based diagnosis of insecticide resistance in diamondback moth (Plutella xylostella) populations in Nagano Prefecture, Japan. Amplicon sequencing analyses using a next-generation sequencer (Illumina MiSeq) for major insecticides, including diamides, pyrethroids, Bacillus thuringiensis (Bt) toxin (Cry1Ac), organophosphates, and spinosyns, were conducted. RESULTS: Mutations related to the resistance of pyrethroids, organophosphates, and diamides (flubendiamide and chlorantraniliprole) prevailed, while those of a diamide (cyantraniliprole), Bt (Cry1Ac), and spinosyns were scanty, suggesting that they are still effective. The results of the genomics-based diagnosis were generally concordant with the results of bioassays. Resistance development tendencies were generally uniform across Nagano. CONCLUSION: An insecticide-resistance management campaign can be conducted in Nagano Prefecture with a quick genomic-based diagnosis in early spring while bioassay is the only option for monitoring resistances whose mutations are unavailable. Our study is the first step in the future management of insecticide resistance in all significant pests. © 2022 Society of Chemical Industry.

Origin, selection, and spread of diamide insecticide resistance allele in field populations of diamondback moth in east and southeast Asia.

BACKGROUND: The investigation of molecular mechanisms and evolution of resistance to insecticides is an ongoing challenge, as researchers must provide guidance to manage the resistance to achieve sustainable production in agriculture. Predicting, monitoring, and managing insecticide resistance requires information on the origins, selection, and spread of resistance genes. The resistance of Plutella xylostella (L.) against diamide insecticides is becoming an increasingly severe problem in east and southeast Asia. In this study, the evolution of resistance was investigated using a resistance allele [ryanodine receptor (RyR); G4946E mutation] and its flanking regions, as well as mitochondrial cytochrome c oxidase subunit I (mtCOI). RESULTS: The sequences of the flanking region of the G4946E and mtCOI suggested that the G4946E mutation has a key role in resistance. Furthermore, the G4946E mutation has multiple origins, and congenic resistant mutations have spread across east and southeast Asia, despite substantial geographical barriers. In addition, the susceptibility of field populations partially recovered during winter, based on the observed decrease in the G4946E (resistant allele) frequency. Finally, the resistance level indexed by the frequency of the E4946 allele was significantly lower in non-overwintering regions than in overwintering regions. CONCLUSION: The information of the present study is useful to monitor resistance using molecular markers and to develop strategies to delay the evolution of diamide resistance.

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 SNP marker discovery and phylogenetic analysis of mulberry varieties using double-digest restriction site-associated DNA sequencing.

There has been long taxonomic debate on mulberry species (genus Morus) because the classification of mulberry species has relied on morphological characteristics. Although attempts for classifying mulberry species using molecular markers have been performed, phylogenetic relationships among diploid mulberry species remain unclear. In this study, we aim to investigate the genetic relationship between 54 diploid mulberry varieties belonging to seven different Morus species (M. alba, M. indica, M. bombycis, M. acidosa, M. latifolia, M. kagayamae, and M. rotundiloba) and one unspecified Morus species ('Enbu') using genome-wide SNP discovery and phylogenetic analysis via double-digest restriction site-associated DNA sequencing (ddRAD-seq). Genome-wide 2229 homozygous SNPs of 54 mulberry varieties in the eight species were identified by ddRAD-seq. Results of the phylogenetic analysis identified only three clear monophyletic clades in two Japanese native species, M. acidosa and M. kagayamae, which are found on different geographically isolated islands and a Thai species, M. rotundiloba, whereas the other species were non-monophyletic. Varieties of M. bombycis, another Japanese native species, were roughly classified into three groups. Of these, two M. bombycis groups were monophyletic with M. acidosa and M. kagayamae, respectively, while another M. bombycis group was not monophyletic. Varieties of M. indica, an Indian native species, were classified into two different monophyletic clades. Of these, one clade was clearly monophyletic with an indigenous variety in Kenya, 'Enbu', while another clade was monophyletic with M. rotundiloba and one M. latifolia variety. There were no clear monophyletic clades within M. alba and M. latifolia varieties, which could be a result of several hybridization events after their introductions from China to Japan. Our results suggested that it was difficult to clearly classify the hybridized mulberry varieties even with genome-wide DNA markers. In addition to phylogenetic analysis, we also evaluated morphological characteristics of mulberry leaves for each variety. The results of morphological evaluation indicated that leaf tip ratio may correlate to genetic difference among the two M. bombycis groups in monophyletic clades and another M. bombycis group in non-monophyletic clades. These results suggested that leaf tip ratio might be used for evaluating hybridization of M. bombycis varieties. Over all, our results may provide new insights into taxonomic debate of mulberry species.

Ryanodine receptor mutations (G4946E and I4790K) differentially responsible for diamide insecticide resistance in diamondback moth, Plutella xylostella L.

This study examined diamondback moth (Plutella xylostella) strains showing high-level resistance to cyantraniliprole (KA17 strain) and to flubendiamide and chlorantraniliprole (KU13 strain). The LC50 value of the KA17 strain against cyantraniliprole was ca. 100-fold higher than that of the KU13 strain. The KA17 strain also exhibited high-level resistance to chlorantraniliprole and flubendiamide equivalent to those in the KU13 strain. The KU13 strain showed a higher LC50 value against cyantraniliprole than the susceptible strains. However, the LC50 value of the KU13 strain against cyantraniliprole was below the agriculturally recommended concentration. Subsequent QTL analysis using ddRAD-seq identified the resistance responsible regions of the KA17 and KU13 strains with different diamide resistance profiles. Ryanodine receptor (RyR) gene was included in the identified regions. Single nucleotide polymorphism calling in the RyR gene using RNA-seq found previously reported G4946E (amino acid mutation from glycine to glutamic acid at amino acid position 4946) and novel I4790K (amino acid mutation from isoleucine to lysine at amino acid position 4790) mutations, respectively, in the RyR of the KU13 and KA17 strains. Functional significance of I4790K in the resistance was confirmed in calcium imaging of the human embryonic kidney 293T cell line expressing Bombyx mori RyR with the mutation. This reporting is the first describing I4790K as a fundamental mechanism responsible for the resistance to the diamides including cyantraniliprole. From this study, we also report up-regulated expression of some degradation enzymes and that of the RyR gene in the KA17 and KU13 strains based on results of RNA-seq data analysis.

T929I and K1774N mutation pair and M918L single mutation identified in the voltage-gated sodium channel gene of pyrethroid-resistant Thrips tabaci (Thysanoptera: Thripidae) in Japan.

Pyrethroid-resistance in onion thrips, Thrips tabaci, has been reported in many countries including Japan. Identifying factors of the resistance is important to correctly monitoring the resistance in field populations. To identify pyrethroid-resistance related genes in T. tabaci in Japan, we performed RNA-Seq analysis of seven T. tabaci strains including two pyrethroid-resistant and five pyrethroid-susceptible strains. We identified a pair of single point mutations, T929I and K1774N, introducing two amino acid mutations, in the voltage-gated sodium channel gene, a pyrethroid target gene, in the two resistant strains. The K1774N is a newly identified mutation located in the fourth repeat domain of the sodium channel. Genotyping analysis of field-collected populations showed that most of the T. tabaci individuals in resistant populations carried the mutation pair, indicating that the mutation pair is closely associated with pyrethroid-resistance in Japan. Another resistance-related mutation, M918L, was also identified in part of the resistant populations. Most of the individuals with the mutation pair were arrhenotokous while all individuals with the M918L single mutation were thelytokous. The result of differentially expressed gene analysis revealed a small number of up-regulated detoxification genes in each resistant strain which might be involved in resistance to pyrethroid. However, no up-regulated detoxification genes common to the two resistant strains were detected. Our results indicate that the mutation pair in the sodium channel gene is the most important target for monitoring pyrethroid-resistance in T. tabaci, and that pyrethroid-resistant arrhenotokous individuals with the mutation pair are likely to be widely distributed in Japan.

The R81T mutation in the nicotinic acetylcholine receptor of Aphis gossypii is associated with neonicotinoid insecticide resistance with differential effects for cyano- and nitro-substituted neonicotinoids.

The cotton aphid, Aphis gossypii Glover, is one of the most agriculturally important insect pests. Neonicotinoid insecticides and sulfoxaflor have generally shown excellent control of A. gossypii, but these aphids have recently developed resistance against neonicotinoid insecticides. We previously characterized a field-collected A. gossypii Kushima clone that showed higher resistance to nitro-substituted neonicotinoids, such as imidacloprid, than to cyano-substituted neonicotinoids, such as acetamiprid. This Kushima clone harbors the R81T mutation in the nicotinic acetylcholine receptor (nAChR) ß1 subunit; this mutation is the source of neonicotinoid insecticide resistance. In the present study, electrophysiological analyses and molecular modeling were employed to investigate the differential effects of the R81T mutation on cyano- and nitro-substituted neonicotinoids and sulfoxaflor. We isolated full-length coding sequences of A. gossypii nAChR α1, α2, and ß1 subunits. When co-expressed in Xenopus laevis oocytes with chicken ß2 nAChR, A. gossypii α1 evoked inward currents in a concentration-dependent manner in response to acetylcholine (ACh) and showed sensitivity to neonicotinoid and sulfoxaflor. Additionally, the chicken ß2 T77R+E79V (equivalent double mutant of R81T) mutation resulted in a lower effect to cyano-substituted neonicotinoids and sulfoxaflor than to nitro-substituted neonicotinoids. Electrophysiological data and nAChR homology modeling analysis suggested that the Kushima clone exhibited different levels of resistance to cyano- and nitro-substituted neonicotinoid insecticides.

Feminizing Wolbachia endosymbiont disrupts maternal sex chromosome inheritance in a butterfly species.

Wolbachia is a maternally inherited ubiquitous endosymbiotic bacterium of arthropods that displays a diverse repertoire of host reproductive manipulations. For the first time, we demonstrate that Wolbachia manipulates sex chromosome inheritance in a sexually reproducing insect. Eurema mandarina butterfly females on Tanegashima Island, Japan, are infected with the wFem Wolbachia strain and produce all-female offspring, while antibiotic treatment results in male offspring. Fluorescence in situ hybridization (FISH) revealed that wFem-positive and wFem-negative females have Z0 and WZ sex chromosome sets, respectively, demonstrating the predicted absence of the W chromosome in wFem-infected lineages. Genomic quantitative polymerase chain reaction (qPCR) analysis showed that wFem-positive females lay only Z0 eggs that carry a paternal Z, whereas females from lineages that are naturally wFem-negative lay both WZ and ZZ eggs. In contrast, antibiotic treatment of adult wFem females resulted in the production of Z0 and ZZ eggs, suggesting that this Wolbachia strain can disrupt the maternal inheritance of Z chromosomes. Moreover, most male offspring produced by antibiotic-treated wFem females had a ZZ karyotype, implying reduced survival of Z0 individuals in the absence of feminizing effects of Wolbachia. Antibiotic treatment of wFem-infected larvae induced male-specific splicing of the doublesex (dsx) gene transcript, causing an intersex phenotype. Thus, the absence of the female-determining W chromosome in Z0 individuals is functionally compensated by Wolbachia-mediated conversion of sex determination. We discuss how Wolbachia may manipulate the host chromosome inheritance and that Wolbachia may have acquired this coordinated dual mode of reproductive manipulation first by the evolution of female-determining function and then cytoplasmically induced disruption of sex chromosome inheritance.

Studies on Aphis gossypii cytochrome P450s CYP6CY22 and CYP6CY13 using an in vitro system.

A field-collected Aphis gossypii clone [Kushima resistant (KR) clone] was resistant to neonicotinoid insecticides (23.8- to 394-fold). RNA-seq and next-generation sequence analyses were conducted to identify nine cytochrome P450 (CYP) genes that were significantly upregulated in the KR clone as compared with those in the insecticide-susceptible clone. A. gossypii P450s were transiently and efficiently expressed in S2 cell to show that CYP6CY22 (c21228) and CYP6CY13 (c21368), which were the most upregulated of the nine P450s in the KR clone, did not degrade sulfoxaflor, a new class of insecticides acting on insect nAChRs, but markedly metabolized all of the neonicotinoids tested. Hence, P450s are likely to underpin neonicotinoid resistance in other aphids as well in the future, and the P450 expression protocol established here will prompt studies on P450-medidated insecticide resistance and structural analyses of relevant metabolites.

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.

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.

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.

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 and molecular characterization of a sex chromosome rearrangement causing a soft and pliable (spli) larval body phenotype in the silkworm, Bombyx mori.

We carried out genetic and cytogenetic analyses of X-ray-induced deleterious Z chromosomes that result in a soft and pliable (spli) phenotype in the silkworm, Bombyx mori. In a B. mori strain with a spli phenotype, we found the Z chromosome broken between the sch (1-21.5) and od (1-49.6) loci. We also found a chromosomal fragment bearing a fifth-chromosome locus for egg and eye pigmentation fused to a Z chromosome fragment. By means of fluorescence in situ hybridization using bacterial artificial chromosome clones as probes, we confirmed that the fused chromosome is composed of a fragment of chromosome 5 and a fragment of the Z chromosome. Moreover, a predicted gene, GA002017, the Bombyx ortholog of the Drosophila gene acj6 (Bmacj6), was completely deleted by the Z chromosome breakage event. The relationship between Bmacj6 and the spli phenotype is discussed.

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.

Identification of genes whose expressions are enhanced or reduced in baker's yeast during fed-batch culture process using molasses medium by DNA microarray analysis.

Genes whose expression levels are enhanced or reduced during the cultivation process that uses cane molasses in baker's yeast production were identified in this study. The results showed that baker's yeast grown in molasses medium had higher fermentation ability and stress tolerance compared with baker's yeast grown in synthetic medium. Molasses apparently provided not only sugar as a carbon source but also provided functional components that enhanced or reduced expression of genes involved in fermentation ability and stress tolerance. To identify the genes whose expression is enhanced or reduced during cultivation in molasses medium, DNA microarray analysis was then used to compare the gene expression profile of cells grown in molasses with that of cells grown in synthetic medium. To simulate the commercial baker's yeast production process, cells were cultivated using a fed-batch culture system. In molasses medium, genes involved in the synthesis or uptake of vitamins (e.g., biotin, pyridoxine and thiamine) showed enhanced expression, suggesting that vitamin concentrations in molasses medium were lower than those in synthetic medium. Genes involved in formate dehydrogenase and maltose assimilation showed enhanced expression in molasses medium. In contrast, genes involved in iron utilization (e.g., siderophore, iron transporter and ferroxidase) showed enhanced expression in synthetic medium, suggesting that iron starvation occurred. The genes involved in the metabolism of amino acids also showed enhanced expression in synthetic medium. This identification of genes provides information that will help improve the baker's yeast production process.