Conservation and lineage-specific rearrangements in the GOBP/PBP gene complex of distantly related ditrysian Lepidoptera.

General odorant binding proteins (GOBPs) and pheromone binding proteins (PBPs) form a monophyletic subfamily of insect odorant binding proteins (OBPs) specific for Lepidoptera, butterflies and moths. The GOBP/PBP genes include six subgroups (GOBP1-2, PBP-A-D) previously reported to form a complex arrayed in a conserved order in representative moths (superfamily Bombycoidea) and butterflies (Nymphalidae). Although our knowledge of lepidopteran genomes has increased greatly recently, the structure of the GOBP/PBP complex has been studied only for species that represent limited lineages of the highly diverged Ditrysia. To understand the evolution of this functionally important gene complex, we determined 69-149 kb genomic sequences that include GOBP2 and five PBP genes in three Ostrinia moths (Pyraloidea), O. nubilalis, O. furnacalis, and O. latipennis, using bacterial artificial chromosome (BAC) and fosmid clones. The structure of the GOBP2/PBP gene cluster was well conserved despite the different sex pheromone composition utilized by the three moths. Five expressed PBP genes in Ostrinia moths were the result of two duplications of PBP-A genes. Surprisingly, an allele containing a fusion gene between tandemly arrayed PBP-A genes was observed in O. nubilalis. We also revealed duplication and intra-chromosomal translocation of the GOBP1 gene in P. xylostella by fluorescence in situ hybridization (FISH) analysis. Additionally, we compared the structure of the GOBP/PBP gene complex of seventeen species covering six superfamilies and twelve families of the lepidopteran clade, Ditrysia, and found the gene order was basically conserved despite the frequent occurrence of lineage-specific gains, losses, inversions and translocations of these genes, compared with their neighboring genes. Our findings support the hypothesis that the structure of the GOBP/PBP gene complex was already established in the common ancestor of Ditrysia.

Multiple Δ11-desaturase genes selectively used for sex pheromone biosynthesis are conserved in Ostrinia moth genomes.

Regulation of the expression of fatty acyl-CoA desaturases, which introduce a double bond into the fatty acid moiety of the substrate, is crucial for the production of species-specific sex pheromones in moths. In Ostrinia moths, two distinct Δ11-desaturases and a Δ14-desaturase are known to be selectively used in the biosynthesis of sex pheromones. Of the two Δ11-desaturases, one identified from Ostrinia nubilalis and Ostrinia scapulalis, Z/EΔ11, forms the Z and E isomers of a double bond at position 11, whereas the other identified from Ostrinia latipennis, LATPG1(=EΔ11), exclusively forms an E double bond at position 11. Since the retroposon(ezi)-fused, non-functional Δ11-desaturase gene, ezi-Δ11α, in the genomes of O. nubilalis and O. furnacalis was previously suggested to be an orthologue of latpg1, we here explored Z/EΔ11 orthologues in the genome of O. latipennis. We newly identified two Δ11-desaturase genes, latpg2 and latpg3, which were orthologous to ezi-Δ11ß and Z/EΔ11, respectively. We found that an ezi-like element was integrated in intron 1 of latpg1, and confirmed that only latpg1 was expressed in the pheromone gland of O. latipennis. Thus, at least three Δ11-desaturase genes are present in the genome of O. latipennis, and latpg1 is selectively transcribed in the pheromone gland of this moth. The non-functionality of ezi-inserted desaturase genes in O. nubilalis and O. furnacalis may not be a direct consequence of the insertion of an ezi- or ezi-like element into the gene.

Embryonic thermosensitive TRPA1 determines transgenerational diapause phenotype of the silkworm, Bombyx mori.

In the bivoltine strain of the silkworm, Bombyx mori, embryonic diapause is induced transgenerationally as a maternal effect. Progeny diapause is determined by the environmental temperature during embryonic development of the mother; however, its molecular mechanisms are largely unknown. Here, we show that the Bombyx TRPA1 ortholog (BmTrpA1) acts as a thermosensitive transient receptor potential (TRP) channel that is activated at temperatures above ∼ 21 °C and affects the induction of diapause in progeny. In addition, we show that embryonic RNAi of BmTrpA1 affects diapause hormone release during pupal-adult development. This study identifying a thermosensitive TRP channel that acts as a molecular switch for a relatively long-term predictive adaptive response by inducing an alternative phenotype to seasonal polyphenism is unique.

FISH identification of Helicoverpa armigera and Mamestra brassicae chromosomes by BAC and fosmid probes.

Since the Bombyx mori genome sequence was published, conserved synteny between B. mori and some other lepidopteran species has been revealed by either FISH (fluorescence in situ hybridization) with BAC (bacterial artificial chromosome) probes or linkage analysis. However, no species belonging to the Noctuidae, the largest lepidopteran family which includes serious polyphagous pests, has been analyzed so far with respect to genome-wide conserved synteny and gene order. For that purpose, we selected the noctuid species Helicoverpa armigera and Mamestra brassicae, both with n = 31 chromosomes. Gene-defined fosmid clones from M. brassicae and BAC clones from a closely related species of H. armigera, Heliothis virescens, were used for a FISH analysis on pachytene chromosomes. We recognized all H. armigera chromosomes from specific cross-hybridization signals of 146 BAC probes. With 100 fosmid clones we identified and characterized all 31 bivalents of M. brassicae. Synteny and gene order were well conserved between the two noctuid species. The comparison with the model species B. mori (n = 28) showed the same phenomenon for 25 of the 28 chromosomes. Three chromosomes (#11, #23 and #24) had two counterparts each in H. armigera and M. brassicae. Since n = 31 is the modal chromosome number in Lepidoptera, the noctuid chromosomes probably represent an ancestral genome organization of Lepidoptera. This is the first identification of a full karyotype in Lepidoptera by means of BAC cross-hybridization between species. The technique shows the potential to expand the range of analyzed species efficiently.

EST sequencing and fosmid library construction in a non-model moth, Mamestra brassicae, for comparative mapping.

Genome data are useful for both basic and applied research; however, it is difficult to carry out large-scale genome analyses using species with limited genetic or genomic resources. Here, we describe a cost-effective method to analyze the genome of a non-model species, using the cabbage moth, Mamestra brassicae (Lepidoptera: Noctuidae). First, we conducted expression sequence tag (EST) analysis. In this analysis, we performed PCR-based prescreening of a non-normalized embryonic cDNA library to eliminate already sequenced cDNAs from further sequencing, which significantly increased the percentage of unique genes. Next, we constructed a fosmid library of M. brassicae and isolated 120 clones containing 119 putative single copy genes by PCR-based screening with primer sets designed from the ESTs. Finally, we showed that the isolated fosmid clones could be used as probes for multicolor fluorescence in situ hybridization (FISH) analysis against an M. brassicae chromosome and confirmed conserved gene order between M. brassicae and the silkworm, Bombyx mori. Thus, we developed new genomic resources for comparative genome analysis in M. brassicae using robust and relatively low cost methods that can be applied to any non-model organism.

The silkworm W chromosome is a source of female-enriched piRNAs.

In the silkworm, Bombyx mori, the W chromosome plays a dominant role in female determination. However, neither protein-coding genes nor transcripts have so far been isolated from the W chromosome. Instead, a large amount of functional transposable elements and their remnants are accumulated on the W chromosome. PIWI-interacting RNAs (piRNAs) are 23-30-nt-long small RNAs that potentially act as sequence-specific guides for PIWI proteins to silence transposon activity in animal gonads. In this study, by comparing ovary- and testis-derived piRNAs, we identified numerous female-enriched piRNAs. Our data indicated that female-enriched piRNAs are derived from the W chromosome. Moreover, comparative analyses on piRNA profiles from a series of W chromosome mutant strains revealed a striking enrichment of a specific set of transposon-derived piRNAs in the putative sex-determining region. Collectively, we revealed the nature of the silkworm W chromosome as a source of piRNAs.

Sex-linked pheromone receptor genes of the European corn borer, Ostrinia nubilalis, are in tandem arrays.

BACKGROUND: Tuning of the olfactory system of male moths to conspecific female sex pheromones is crucial for correct species recognition; however, little is known about the genetic changes that drive speciation in this system. Moths of the genus Ostrinia are good models to elucidate this question, since significant differences in pheromone blends are observed within and among species. Odorant receptors (ORs) play a critical role in recognition of female sex pheromones; eight types of OR genes expressed in male antennae were previously reported in Ostrinia moths. METHODOLOGY/PRINCIPAL FINDINGS: We screened an O. nubilalis bacterial artificial chromosome (BAC) library by PCR, and constructed three contigs from isolated clones containing the reported OR genes. Fluorescence in situ hybridization (FISH) analysis using these clones as probes demonstrated that the largest contig, which contained eight OR genes, was located on the Z chromosome; two others harboring two and one OR genes were found on two autosomes. Sequence determination of BAC clones revealed the Z-linked OR genes were closely related and tandemly arrayed; moreover, four of them shared 181-bp direct repeats spanning exon 7 and intron 7. CONCLUSIONS/SIGNIFICANCE: This is the first report of tandemly arrayed sex pheromone receptor genes in Lepidoptera. The localization of an OR gene cluster on the Z chromosome agrees with previous findings for a Z-linked locus responsible for O. nubilalis male behavioral response to sex pheromone. The 181-bp direct repeats might enhance gene duplications by unequal crossovers. An autosomal locus responsible for male response to sex pheromone in Heliothis virescens and H. subflexa was recently reported to contain at least four OR genes. Taken together, these findings support the hypothesis that generation of additional copies of OR genes can increase the potential for male moths to acquire altered specificity for pheromone components, and accordingly, facilitate differentiation of sex pheromones.

Samia cynthia versus Bombyx mori: comparative gene mapping between a species with a low-number karyotype and the model species of Lepidoptera.

We performed gene-based comparative FISH mapping between a wild silkmoth, Samia cynthia ssp. with a low number of chromosomes (2n=25-28) and the model species, Bombyx mori (2n=56), in order to identify the genomic components that make up the chromosomes in a low-number karyotype. Mapping of 64 fosmid probes containing orthologs of B. mori genes revealed that the homologues of either two or four B. mori chromosomes constitute the S. c. ricini (Vietnam population, 2n=27♀/28♂, Z0/ZZ) autosomes. Where tested, even the gene order was conserved between S. c. ricini and B. mori. This was also true for the originally autosomal parts of the neo-sex chromosomes in S. c. walkeri (Sapporo population, 2n=26♀/26♂, neo-Wneo-Z/neo-Zneo-Z) and S. cynthia subsp. indet. (Nagano population, 2n=25♀/26♂, neo-WZ1Z2/Z1Z1Z2Z2). The results are evidence for an internal stability of lepidopteran chromosomes even when all autosomes had undergone fusion processes to form a low-number karyotype.

Isolation of BAC clones containing conserved genes from libraries of three distantly related moths: a useful resource for comparative genomics of Lepidoptera.

Lepidoptera, butterflies and moths, is the second largest animal order and includes numerous agricultural pests. To facilitate comparative genomics in Lepidoptera, we isolated BAC clones containing conserved and putative single-copy genes from libraries of three pests, Heliothis virescens, Ostrinia nubilalis, and Plutella xylostella, harboring the haploid chromosome number, n = 31, which are not closely related with each other or with the silkworm, Bombyx mori, (n = 28), the sequenced model lepidopteran. A total of 108-184 clones representing 101-182 conserved genes were isolated for each species. For 79 genes, clones were isolated from more than two species, which will be useful as common markers for analysis using fluorescence in situ hybridization (FISH), as well as for comparison of genome sequence among multiple species. The PCR-based clone isolation method presented here is applicable to species which lack a sequenced genome but have a significant collection of cDNA or EST sequences.

A eukaryotic (insect) tricistronic mRNA encodes three proteins selected by context-dependent scanning.

Eukaryotic mRNAs are generally considered monocistronic and encode only one protein. Although dicistronic mRNAs encoding two proteins were found in fungi, plants, and animals, polycistronic mRNAs encoding more than two proteins have remained elusive so far in any eukaryote. Here we demonstrate that a single mRNA from silkworm encodes the precursor of an insect cytokine paralytic peptide (PP) and two new cytokine precursor-like proteins, uENF1 and uENF2. RT-PCR analysis showed that this mRNA is widely conserved in moths. Western blot analyses and reporter assays using its modified mRNAs, created by replacing each one of the three ORFs with the firefly luciferase ORF, showed that all three proteins were translated from this mRNA in cell lines, larval tissues, and cell-free systems. Insertion experiments using the Renilla luciferase ORF or a stem loop ruled out the possible involvement of internal ribosome entry site in the three protein translation. On the other hand, systematic mutation analysis of the translation initiation sequence of the 5'-proximal uENF1 ORF suggested that the context-dependent leaky-scanning mechanism is involved in translation of the downstream uENF2 and PP ORFs. In vitro, a synthetic peptide corresponding to the putative mature form of uENF1 stimulated spreading of hemocytes as did the synthetic PP, whereas that of uENF2 antagonized the stimulating activities of PP and the uENF1 peptide, suggesting that the three proteins control cellular immunity interactively. Thus, eukaryotes have a cellular tricistronic mRNA that encodes three functionally related proteins as in an operon.

Molecular cloning and expression profile analysis of a novel beta-D-N-acetylhexosaminidase of domestic silkworm (Bombyx mori).

Lepidoptera such as the domestic silkworm (Bombyx mori) produce proteins modified with unsialylated, mannose-rich moieties known as 'high mannose-type'N-glycans. However, we observed that, under intrinsic acetylglucosaminidase (GlcNAcase)-inhibited conditions, moth cells tend to synthesize different types of glycoform with sialic acid modification. To identify molecules essential to assemble Lepidoptera-specific N-glycans, we performed BLAST analysis on the silkworm genetic database and isolated the entire coding sequence of novel Bombyx GlcNAcase, BmGlcNAcase 2. This enzyme showed weak homology to currently known, lysosome-associated eukaryotic hexosaminidases, but it revealed remarkable similarity with recently reported glycosyl hydrolases of Spodoptera and Bombyx. Interestingly, BmGlcNAcase 2 was found to be expressed in embryos and in certain tissues of molting larvae (i.e. ovary, fat bodies, mid-intestine, skin), but not in pupae, suggesting its unique function in the carbohydrate metabolism of juvenile silkworm.

Extensive conserved synteny of genes between the karyotypes of Manduca sexta and Bombyx mori revealed by BAC-FISH mapping.

BACKGROUND: Genome sequencing projects have been completed for several species representing four highly diverged holometabolous insect orders, Diptera, Hymenoptera, Coleoptera, and Lepidoptera. The striking evolutionary diversity of insects argues a need for efficient methods to apply genome information from such models to genetically uncharacterized species. Constructing conserved synteny maps plays a crucial role in this task. Here, we demonstrate the use of fluorescence in situ hybridization with bacterial artificial chromosome probes as a powerful tool for physical mapping of genes and comparative genome analysis in Lepidoptera, which have numerous and morphologically uniform holokinetic chromosomes. METHODOLOGY/PRINCIPAL FINDINGS: We isolated 214 clones containing 159 orthologs of well conserved single-copy genes of a sequenced lepidopteran model, the silkworm, Bombyx mori, from a BAC library of a sphingid with an unexplored genome, the tobacco hornworm, Manduca sexta. We then constructed a BAC-FISH karyotype identifying all 28 chromosomes of M. sexta by mapping 124 loci using the corresponding BAC clones. BAC probes from three M. sexta chromosomes also generated clear signals on the corresponding chromosomes of the convolvulus hawk moth, Agrius convolvuli, which belongs to the same subfamily, Sphinginae, as M. sexta. CONCLUSIONS/SIGNIFICANCE: Comparison of the M. sexta BAC physical map with the linkage map and genome sequence of B. mori pointed to extensive conserved synteny including conserved gene order in most chromosomes. Only a few rearrangements, including three inversions, three translocations, and two fission/fusion events were estimated to have occurred after the divergence of Bombycidae and Sphingidae. These results add to accumulating evidence for the stability of lepidopteran genomes. Generating signals on A. convolvuli chromosomes using heterologous M. sexta probes demonstrated that BAC-FISH with orthologous sequences can be used for karyotyping a wide range of related and genetically uncharacterized species, significantly extending the ability to develop synteny maps for comparative and functional genomics.

Reprobing multicolor FISH preparations in lepidopteran chromosome.

Multicolor fluorescence in-situ hybridization (FISH) and subsequent reprobing of chromosome preparations increase the number of chromosomes and/or anchor loci on the chromosomes simultaneously identified. Reprobing techniques have been widely applied to chromosomes of vertebrates and plants. We have developed a novel reprobing protocol that utilizes multicolor FISH and bacterial artificial chromosome (BAC) probes to examine chromosome preparations in a model lepidopteran species, the silkworm, Bombyx mori. With standard two-color BAC-FISH, routinely used to map genes on B. mori chromosomes, we could localize only two probes on one preparation, whereas our new protocol combining five-color BAC-FISH and preparation reprobing enabled us to simultaneously map 10 probes, as demonstrated with the Bombyx Z chromosome. The improved BAC-FISH technique will facilitate karyotyping and synteny analysis in Lepidoptera.

Conserved synteny of genes between chromosome 15 of Bombyx mori and a chromosome of Manduca sexta shown by five-color BAC-FISH.

The successful assignment of the existing genetic linkage groups (LGs) to individual chromosomes and the second-generation linkage map obtained by mapping a large number of bacterial artificial chromosome (BAC) contigs in the silkworm, Bombyx mori, together with public nucleotide sequence databases, offer a powerful tool for the study of synteny between karyotypes of B. mori and other lepidopteran species. Conserved synteny of genes between particular chromosomes can be identified by comparatively mapping orthologous genes of the corresponding linkage groups with the help of BAC-FISH (fluorescent in situ hybridization). This technique was established in B. mori for 2 differently labeled BAC probes simultaneously hybridized to pachytene bivalents. To achieve higher-throughput comparative mapping using BAC-FISH in Lepidoptera, we developed a protocol for five-color BAC-FISH, which allowed us to map simultaneously 6 different BAC probes to chromosome 15 in B. mori. We identified orthologs of 6 B. mori LG15 genes (RpP0, RpS8, eIF3, RpL7A, RpS23, and Hsc70) for the tobacco hornworm, Manduca sexta, and selected the ortholog-containing BAC clones from an M. sexta BAC library. All 6 M. sexta BAC clones hybridized to a single M. sexta bivalent in pachytene spermatocytes. Thus, we have confirmed the conserved synteny between the B. mori chromosome 15 and the corresponding M. sexta chromosome (hence provisionally termed chromosome 15).

The Pitx homeobox gene in Bombyx mori: regulation of DH-PBAN neuropeptide hormone gene expression.

The diapause hormone-pheromone biosynthesis activating neuropeptide gene, DH-PBAN, is expressed exclusively in seven pairs of DH-PBAN-producing neurosecretory cells (DHPCs) on the terminally differentiated processes of the subesophageal ganglion (SG). To help reveal the regulatory mechanisms of cell-specific DH-PBAN expression, we identified a cis-regulatory element that regulates expression in DHPCs using the recombinant AcNPV-mediated gene transfer system and a gel-mobility shift assay. Bombyx mori Pitx (BmPitx), a bicoid-like homeobox transcription factor, binds this element and activates DH-PBAN expression. The BmPitx was expressed in various tissues, including DHPCs in the SG. Suppression of DH-PBAN expression by silencing of the BmPitx successfully induced non-diapaused eggs from a diapause egg producer. To the best of our knowledge, this report is the first to identify a neuropeptide-encoding gene as a target of the Pitx transcriptional regulator in invertebrates. Thus, it is tempting to speculate that functional conservation of Pitx family members on neuropeptide gene expression occurs through a "combinational code mechanism" in both vertebrate and invertebrate in neuroendocrine systems.

Detection of gamma-tubulin in spermatogonial cells of Bombyx mori (Lepidoptera) and Chortophaga viridifasciata (Orthoptera).

We detected a putative gamma-tubulin gene in silico and detected BACs containing the gene from a Bombyx mori BAC library. BAC-FISH mapping revealed that the gene is located on chromosome 5. To observe the distribution of gamma-tubulin, we employed antibodies against mammalian gamma-tubulin peptides. Western blot analysis disclosed a band very similar in size to gamma-tubulin protein in other species (approximately 48 kDa). In mitotic metaphase of B. mori spermatogonial cells, gamma-tubulin is exclusively localized in the spindle poles, where the centrosomes occur. We applied the same system to the grasshopper Chortophaga viridifasciata, as a representative of insect orders in which the gamma-tubulin distribution had not previously been studied. Gamma-tubulin was also found in the spindle poles during metaphase of spermatogonial cells in the grasshopper.

Molecular cloning and chromosomal localization of the Bombyx Sex-lethal gene.

We cloned Bm-Sxl, an orthologue of the Drosophila melanogaster Sex-lethal (Sxl) gene from embryos of Bombyx mori. The full-length cDNAs were of 2 sizes, 1528 and 1339 bp, and were named Bm-Sxl-L and Bm-Sxl-S, respectively. Bm-Sxl-L consists of 8 exons and spans more than 20 kb of genomic DNA. The open reading frame (ORF) codes for a protein 336 amino acids in length. Bm-Sxl-S is a splice variant that lacks the second exon. This creates a new translation start 138 nucleotides downstream and an ORF that codes for 46 amino acids fewer at the N-terminus. Linkage analysis using an F2 panel mapped Bm-Sxl to linkage group 16 at 69.8 cM. We isolated 2 BACs that include the Bm-Sxl gene. With BAC-FISH we located Bm-Sxl cytogenetically on the chromosome corresponding to linkage group 16 (LG16) at position >68.8 cM.

A second-generation integrated map of the silkworm reveals synteny and conserved gene order between lepidopteran insects.

A second-generation linkage map was constructed for the silkworm, Bombyx mori, focusing on mapping Bombyx sequences appearing in public nucleotide databases and bacterial artificial chromosome (BAC) contigs. A total of 874 BAC contigs containing 5067 clones (22% of the library) were constructed by PCR-based screening with sequence-tagged sites (STSs) derived from whole-genome shotgun (WGS) sequences. A total of 523 BAC contigs, including 342 independent genes registered in public databases and 85 expressed sequence tags (ESTs), were placed onto the linkage map. We found significant synteny and conserved gene order between B. mori and a nymphalid butterfly, Heliconius melpomene, in four linkage groups (LGs), strongly suggesting that using B. mori as a reference for comparative genomics in Lepidotera is highly feasible.

Simple sequence repeat-based consensus linkage map of Bombyx mori.

We established a genetic linkage map employing 518 simple sequence repeat (SSR, or microsatellite) markers for Bombyx mori (silkworm), the economically and culturally important lepidopteran insect, as part of an international genomics program. A survey of six representative silkworm strains using 2,500 (CA)n- and (CT)n-based SSR markers revealed 17-24% polymorphism, indicating a high degree of homozygosity resulting from a long history of inbreeding. Twenty-nine SSR linkage groups were established in well characterized Dazao and C108 strains based on genotyping of 189 backcross progeny derived from an F(1) male mated with a C108 female. The clustering was further focused to 28 groups by genotyping 22 backcross progeny derived from an F(1) female mated with a C108 male. This set of SSR linkage groups was further assigned to the 28 chromosomes (established linkage groups) of silkworm aided by visible mutations and cleaved amplified polymorphic sequence markers developed from previously mapped genes, cDNA sequences, and cloned random amplified polymorphic DNAs. By integrating a visible mutation p (plain, larval marking) and 29 well conserved genes of insects onto this SSR-based linkage map, a second generation consensus silkworm genetic map with a range of 7-40 markers per linkage group and a total map length of approximately 3431.9 cM was constructed and its high efficiency for genotyping and potential application for synteny studies of Lepidoptera and other insects was demonstrated.

Integration of molecular and classical linkage groups of the silkworm, Bombyx mori (n = 28).

Previously published linkage groups (LGs) composed of molecular markers were assigned to classical LGs in the silkworm, Bombyx mori (n = 28). Four markers from the classical linkage map, og, w-1, Lp, and Pfl, were assigned to the molecular linkage maps using sequence tagged sites. In addition, linkage analysis was carried out using BF1 progeny between wild-type and mutant stocks carrying morphological phenotypic markers. As a result, the counterparts for 26 of 28 molecular LGs were identified with their counterparts of the classical LGs. Two visible markers, Sel and Xan, representing different classical LGs, were found to be linked.