Comprehensive microarray-based analysis for stage-specific larval camouflage pattern-associated genes in the swallowtail butterfly, Papilio xuthus

<p>Abstract</p> <p>Background</p> <p>Body coloration is an ecologically important trait that is often involved in prey-predator interactions through mimicry and crypsis. Although this subject has attracted the interest of biologists and the general public, our scientific knowledge on the subject remains fragmentary. In the caterpillar of the swallowtail butterfly <it>Papilio xuthus</it>, spectacular changes in the color pattern are observed; the insect mimics bird droppings (mimetic pattern) as a young larva, and switches to a green camouflage coloration (cryptic pattern) in the final instar. Despite the wide variety and significance of larval color patterns, few studies have been conducted at a molecular level compared with the number of studies on adult butterfly wing patterns.</p> <p>Results</p> <p>To obtain a catalog of genes involved in larval mimetic and cryptic pattern formation, we constructed expressed sequence tag (EST) libraries of larval epidermis for <it>P. xuthus</it>, and <it>P. polytes </it>that contained 20,736 and 5,376 clones, respectively, representing one of the largest collections available in butterflies. A comparison with silkworm epidermal EST information revealed the high expression of putative blue and yellow pigment-binding proteins in <it>Papilio </it>species. We also designed a microarray from the EST dataset information, analyzed more than five stages each for six markings, and confirmed spatial expression patterns by whole-mount <it>in situ </it>hybridization. Hence, we succeeded in elucidating many novel marking-specific genes for mimetic and cryptic pattern formation, including pigment-binding protein genes, the melanin-associated gene <it>yellow-h3</it>, the ecdysteroid synthesis enzyme gene <it>3-dehydroecdysone 3b-reductase</it>, and <it>Papilio</it>-specific genes. We also found many cuticular protein genes with marking specificity that may be associated with the unique surface nanostructure of the markings. Furthermore, we identified two transcription factors, <it>spalt </it>and ecdysteroid signal-related <it>E75</it>, as genes expressed in larval eyespot markings. This finding suggests that <it>E75 </it>is a strong candidate mediator of the hormone-dependent coordination of larval pattern formation.</p> <p>Conclusions</p> <p>This study is one of the most comprehensive molecular analyses of complicated morphological features, and it will serve as a new resource for studying insect mimetic and cryptic pattern formation in general. The wide variety of marking-associated genes (both regulatory and structural genes) identified by our screening indicates that a similar strategy will be effective for understanding other complex traits.</p

Catalogue of epidermal genes: Genes expressed in the epidermis during larval molt of the silkworm Bombyx mori

<p>Abstract</p> <p>Background</p> <p>The insect cuticle is composed of various proteins and formed during the molt under hormonal regulation, although its precise composition and formation mechanism are largely unknown. The exhaustive catalogue of genes expressed in epidermis at the molt constitutes a massive amount of information from which to draw a complete picture of the molt and cuticle formation in insects. Therefore, we have catalogued a library of full-length cDNAs (designated epM) from epidermal cells during the last larval molt of <it>Bombyx mori</it>.</p> <p>Results</p> <p>Of the 10,368 sequences in the library, we isolated 6,653 usable expressed sequence tags (ESTs), which were categorized into 1,451 nonredundant gene clusters. Seventy-one clusters were considered to be isoforms or premature forms of other clusters. Therefore, we have identified 1,380 putative genes. Of the 6,653 expressed sequences, 48% were derived from 92 cuticular protein genes (RR-1, 24; RR-2, 17; glycine-rich, 29; other classes, 22). A comparison of epM with another epidermal EST data set, epV3 (feeding stage: fifth instar, day 3), showed marked differences in cuticular protein gene. Various types of cuticular proteins are expressed in epM but virtually only RR-1 proteins were expressed in epV3. Cuticular protein genes expressed specifically in epidermis, with several types of expression patterns during the molt, suggest different types of responses to the ecdysteroid pulse. Compared with other <it>Bombyx </it>EST libraries, 13 genes were preferentially included in epM data set. We isolated 290 genes for proteins other than cuticular proteins, whose amino acid sequences retain putative signal peptides, suggesting that they play some role in cuticle formation or in other molting events. Several gene groups were also included in this data set: hormone metabolism, P450, modifier of cuticular protein structure, small-ligand-binding protein, transcription factor, and pigmentation genes.</p> <p>Conclusion</p> <p>We have identified 1,380 genes in epM data set and 13 preferentially expressed genes in epidermis at the molt. The comparison of the epM and other EST libraries clarified the totally different gene expression patterns in epidermis between the molting and feeding stages and many novel tissue- and stage-specifically expressed epidermal genes. These data should further our understanding of cuticle formation and the insect molt.</p

Chromatin-induced Spindle Assembly Plays an Important Role in Metaphase Congression of Silkworm Holocentric Chromosomes

The kinetochore plays important roles in cell cycle progression. Interactions between 4 chromosomes and spindle microtubules allow chromosomes to congress to the middle of the 5 cell and to segregate the sister chromatids into daughter cells in mitosis. The chromosome 6 passenger complex (CPC), composed of the Aurora B kinase and its regulatory subunits 7 INCENP, Survivin, and Borealin, plays multiple roles in these chromosomal events. In the 8 genome of the silkworm, Bombyx mori, which has holocentric chromosomes, the CPC 9 components and their molecular interactions were highly conserved. In contrast to 10 monocentric species, however, the silkworm CPC co-localized with the chromatin-driven 11 spindles on the upper side of prometaphase chromosomes without forming bipolar mitotic 12 spindles. Depletion of the CPC by RNAi arrested the cell cycle progression at prometaphase 13 and disrupted the microtubule network of the chromatin-driven spindles. Interestingly, 14 depletion of mitotic centromere-associated kinesin (MCAK) recovered formation of the 15 microtubule network but did not overcome the cell cycle arrest at prometaphase. These 16 results suggest that the CPC modulates the chromatin-induced spindle assembly and 17 metaphase congression of silkworm holocentric chromosomes

End-sequencing and characterization of silkworm (Bombyx mori) bacterial artificial chromosome libraries

<p>Abstract</p> <p>Background</p> <p>We performed large-scale bacterial artificial chromosome (BAC) end-sequencing of two BAC libraries (an <it>Eco</it>RI- and a <it>Bam</it>HI-digested library) and conducted an <it>in silico </it>analysis to characterize the obtained sequence data, to make them a useful resource for genomic research on the silkworm (<it>Bombyx mori</it>).</p> <p>Results</p> <p>More than 94000 BAC end sequences (BESs), comprising more than 55 Mbp and covering about 10.4% of the silkworm genome, were sequenced. Repeat-sequence analysis with known repeat sequences indicated that the long interspersed nuclear elements (LINEs) were abundant in <it>Bam</it>HI BESs, whereas DNA-type elements were abundant in <it>Eco</it>RI BESs. Repeat-sequence analysis revealed that the abundance of LINEs might be due to a GC bias of the restriction sites and that the GC content of silkworm LINEs was higher than that of mammalian LINEs. In a BLAST-based sequence analysis of the BESs against two available whole-genome shotgun sequence data sets, more than 70% of the BESs had a BLAST hit with an identity of ≥ 99%. About 14% of <it>Eco</it>RI BESs and about 8% of <it>Bam</it>HI BESs were paired-end clones with unique sequences at both ends. Cluster analysis of the BESs clarified the proportion of BESs containing protein-coding regions.</p> <p>Conclusion</p> <p>As a result of this characterization, the identified BESs will be a valuable resource for genomic research on <it>Bombyx mori</it>, for example, as a base for construction of a BAC-based physical map. The use of multiple complementary BAC libraries constructed with different restriction enzymes also makes the BESs a more valuable genomic resource. The GenBank accession numbers of the obtained end sequences are <ext-link ext-link-type="gen" ext-link-id="DE283657">DE283657</ext-link>–<ext-link ext-link-type="gen" ext-link-id="DE378560">DE378560</ext-link>.</p

Identification of 20-hydroxyecdysone-inducible genes from larval brain of the silkworm, bombyx mori, and their expression analysis

The insect brain secretes prothoracicotropic hormone (PTTH), which stimulates the prothoracic gland to synthesize ecdysone. The active metabolite of ecdysone, 20-hydroxyecdysone (20E), works through ecdysone receptor (EcR) and ultraspiracle (USP) to initiate molting and metamorphosis by regulating downstream genes. Previously, we found that EcR was expressed in the PTTH-producing neurosecretory cells (PTPCs) in larval brain of the silkworm Bombyx mori, suggesting that PTPCs function as the master cells of development under the regulation of 20E. To gain a better understanding of the molecular mechanism of the 20E control of PTPCs, we performed a comprehensive screening of genes induced by 20E using DNA microarray with brains of day-2 fifth instar silkworm larvae. Forty-one genes showed greater than twofold changes caused by artificial application of 20E. A subsequent semiquantitative screening identified ten genes upregulated by 20E, four of which were novel or not previously identified as 20E-response genes. Developmental profiling determined that two genes, UP4 and UP5, were correlated with the endogenous ecdysteroid titer. Whole-mount in situ hybridization showed exclusive expression of these two genes in two pairs of cells in the larval brain in response to 20E-induction, suggesting that the cells are PTPCs. BLAST searches revealed that UP4 and UP5 are Bombyx homologs of vrille and tarsal-less, respectively. The present study identifies 20E-induced genes that may be involved in the ecdysone signal hierarchies underlying pupal-adult development and/or the 20E regulation of PTPCs. © 2012 Zoological Society of Japan.発行後1年より全文公

Identification of novel bombyxin genes from the genome of the silkmoth bombyx mori and analysis of their expression

Insulin family peptide members play key roles in regulating growth, metabolism, and reproduction. Bombyxin is an insulin-related peptide of the silkmoth Bombyx mori. We analyzed the full genome of B. mori and identified five novel bombyxin families, V to Z. We characterized the genomic organization and chromosomal location of the novel bombyxin family genes. In contrast to previously identified bombyxin genes, bombyxin-V and -Z genes had intervening introns at almost the same positions as vertebrate insulin genes. We performed reverse transcription-polymerase chain reaction and in situ hybridization in different tissues and developmental stages to observe their temporal and spatial expression patterns. The newly identified bombyxin genes were expressed in diverse tissues: bombyxin-V, -W, and -Y mRNAs were expressed in the brain and bombyxin-X mRNA in fat bodies. Bombyxin-Y gene was expressed in both brain and ovary of larval stages. High level of bombyxin-Z gene expression in the follicular cells may suggest its function in reproduction. The presence of a short C-peptide domain and an extended A chain domain, and high expression of bombyxin-X gene in the fat body cells during non-feeding stages suggest its insulin-like growth factor-like function. These results suggest that the bombyxin genes originated from a common ancestral gene, similar to the vertebrate insulin gene, and evolved into a diverse gene family with multiple functions. © 2011 Zoological Society of Japan

Role of the silkworm argonaute2 homolog gene in double-strand break repair of extrachromosomal DNA

The argonaute protein family provides central components for RNA interference (RNAi) and related phenomena in a wide variety of organisms. Here, we isolated, from a Bombyx mori cell, a cDNA clone named BmAGO2, which is homologous to Drosophila ARGONAUTE2, the gene encoding a repressive factor for the recombination repair of extrachromosomal double-strand breaks (DSBs). RNAi-mediated silencing of the BmAGO2 sequence markedly increased homologous recombination (HR) repair of DSBs in episomal DNA, but had no effect on that in chromosomes. Moreover, we found that RNAi for BmAGO2 enhanced the integration of linearized DNA into a silkworm chromosome via HR. These results suggested that BmAgo2 protein plays an indispensable role in the repression of extrachromosomal DSB repair

W-derived BAC probes as a new tool for identification of the W chromosome and its aberrations in Bombyx mori

We isolated four W chromosome-derived bacterial artificial chromosome (W-BAC) clones from Bombyx mori BAC libraries by the polymerase chain reaction and used them as probes for fluorescence in situ hybridization (FISH) on chromosome preparations from B. mori females. All four W-BAC probes surprisingly highlighted the whole wild-type W sex chromosome and also identified the entire original W-chromosomal region in W chromosome-autosome translocation mutants. This is the first successful identification of a single chromosome by means of BAC-FISH in species with holokinetic chromosomes. Genomic in situ hybridization (GISH) by using female-derived genomic probes highlighted the W chromosome in a similar chromosome-painting manner. Besides the W, hybridization signals of W-BAC probes also occurred in telomeric and/or subtelomeric regions of the autosomes. These signals coincided well with those of female genomic probes except one additional GISH signal that was observed in a large heterochromatin block of one autosome pair. Our results support the opinion that the B. mori W chromosome accumulated transposable elements and other repetitive sequences that also occur, but scattered, elsewhere in the respective genome. Edited by: E.R. Schmid

Genome-wide annotation and comparative analysis of cuticular protein genes in the noctuid pest \u3cem\u3eSpodoptera litura\u3c/em\u3e

Insect cuticle is considered an adaptable and versatile building material with roles in the construction and function of exoskeleton. Its physical properties are varied, as the biological requirements differ among diverse structures and change during the life cycle of the insect. Although the bulk of cuticle consists basically of cuticular proteins (CPs) associated with chitin, the degree of cuticular sclerotization is an important factor in determining its physical properties. Spodoptera litura, the tobacco cutworm, is an important agricultural pest in Asia. Compared to the domestic silkworm, Bombyx mori, another lepidopteran whose CP genes have been well annotated, S. litura has a shorter life cycle, hides in soil during daytime beginning in the 5th instar and is exposed to soil in the pupal stage without the protection of a cocoon. In order to understand how the CP genes may have been adapted to support the characteristic life style of S. litura, we searched its genome and found 287 putative cuticular proteins that can be classified into 9 CP families (CPR with three groups (RR-1, RR-2, RR-3), CPAP1, CPAP3, CPF, CPFL, CPT, CPG, CPCFC and CPLCA), and a collection of unclassified CPs named CPH. There were also 112 cuticular proteins enriched in Histidine residues with content varying from 6% to 30%, comprising many more His-rich cuticular proteins than B. mori. A phylogenetic analysis between S. litura, M. sexta and B. mori uncovered large expansions of RR-1 and RR-2 CPs, forming large gene clusters in different regions of S. liturachromosome 9. We used RNA-seq analysis to document the expression profiles of CPs in different developmental stages and tissues of S. litura. The comparative genomic analysis of CPs between S. litura and B. moriintegrated with the unique behavior and life cycle of the two species offers new insights into their contrasting ecological adaptations