In silico prediction and characterization of microRNAs from red flour beetle (Tribolium castaneum)

MicroRNAs (miRNAs), are endogenous, ~22-nucleotide-long RNA molecules. They bind to the complementary sites on target mRNAs and regulate protein production of the target transcript by unknown mechanisms. Since the discovery of first miRNA in Caenorhabditis elegans, different approaches have been pursued for the prediction of miRNAs and their target(s). Because of many difficulties and limitations involved in the experimental identification of spatially and temporally expressed miRNAs, many computational approaches have been successfully employed for prediction of miRNAs and their target(s). In the present study, we demonstrate a genome-wide computational approach to predict miRNAs and their target(s) in the red flour beetle, Tribolium castaneum. We have predicted and characterized 45 miRNAs by genome-wide homology search against all the reported miRNAs. These miRNAs were further validated by statistical and phylogenetic analyses. In addition, we have also attempted to predict the putative targets of these miRNAs, by making use of 3' untranslated regions of mRNAs from T. castaneum. These miRNAs and their targets in T. castaneum will serve as useful resources for initiating studies on their experimental validation and functional analyses of miRNA-regulated phenotypes in T. castaneum through gene knockdown and transgenesis

How is sex determined in insects? Preface.

insects provide vivid examples of an astonishing diversity of primary signals of sex determination that not only vary between species but even within species, in contrast to terminal genes which are conserved across taxa. Accordingly this special issue of Journal of Genetics is dedicated to provide an update of the data available from genetic studies of sex determination and of sexual differentiation in a wide range of insect specie

How is sex determined in insects?

This article does not have an abstract

Two female-specific DSX proteins are encoded by the sex-specific transcripts of dsx, and are required for female sexual differentiation in two wild silkmoth species, Antheraea assama and Antheraea mylitta (Lepidoptera, Saturniidae)

doublesex (dsx) is the bottom most gene of the sex-determination cascade of Drosophila melanogaster. The pre-mRNA of dsx splices to produce male- and female-specific transcripts which code for the male- and female-specific proteins, respectively. dsx homologues have been characterized from different (many in Diptera, two in Hypmenoptera and only one in Lepidoptera) insect species. Sex-specific splice forms of dsx pre-mRNA in all these species code for one male- and one female-specific DSX proteins, which regulate the downstream target genes responsible for sex-specific characters. In the present study we have cloned and characterized the dsx homologues from two saturniid silkmoths, Antheraea assama and Antheraea mylitta. The divergence time between Saturniidae and Bombycidae to which the domesticated silkworm, Bombyx mori belongs is estimated to be around 160.9 MY. Interestingly, the dsx pre-mRNA of these wild silkmoths sex-specifically splices to generate multiple splice variants. On the basis of their open reading frame (ORF) and conceptual translation, two female-specific (DSXF1 and DSXF2) and one male-specific (DSXM) proteins could be inferred, in both the moths. Presence or absence of a 15 bp stretch within the ORF of the two groups of female-specific transcripts resulted in the production of two distinct female-specific DSX proteins. The sex-specific DSX proteins have common amino-terminal sequence but sex-specific carboxy termini. The two female-specific DSX proteins (DSXF1 and DSXF2) share common DNA binding domain (DM domain) and oligomerization domain (OD domain) and differ only at their extreme C-termini by 21aa. Functional analysis of dsx transcripts in A. assama by dsRNA mediated knock-down resulted in complete abolition of expression of vitellogenin and hexamerin genes, the direct targets of the DSX proteins, irregular differentiation of gonads, and drastic reduction in fecundity and hatchability. Together, these results suggest the involvement of both the female-specific DSX proteins in the process of female sexual differentiation. Further, conservation of the 4th exon sequence, especially the PESS sequence responsible for the sex-specific splicing of Bmdsx in the female-specific transcripts of Aadsx and Amydsx, indicated the existence of a common mechanism of sex-specific splicing of dsx homologues in silkmoths. To our knowledge this is the first report of existence of multiple splice forms of dsx pre-mRNA encoding two female-specific DSX proteins

A comparative phylogenetic analysis of full-lengthmariner elements isolated from the Indian tasar silkmoth, Antheraea mylitta (Lepidoptera: saturniidae)

Mariner like elements (MLEs) are widely distributed type II transposons with an open reading frame (ORF) for transposase. We studied comparative phylogenetic evolution and inverted terminal repeat (ITR) conservation of MLEs from Indian saturniid silkmoth,Antheraea mylitta with other full length MLEs submitted in the database. Full length elements fromA. mylitta were inactive with multiple mutations. Many conserved amino acid blocks were identified after aligning transposase sequences. Mariner signature sequence, DD(34)D was almost invariable although a few new class of elements had different signatures.A. mylitta MLEs(Anmmar) get phylogenetically classified under cecropia subfamily and cluster closely with the elements from other Bombycoidea superfamily members implying vertical transmission from a common ancestor. ITR analysis showed a conserved sequence of AGGT(2-8N)ATAAGT for forward repeat and AGGT(2-8N)ATGAAAT for reverse repeat. These results and additional work may help us to understand the dynamics of MLE distribution inA. mylitta and construction of appropriate vectors for mariner mediated transgenics

Purification and characterization of digestive amylase from the tasar silkworm, Antheraea mylitta (Lepidoptera: Saturniidae)

Digestive amylase was purified from larvae of Indian tasar silkworm, Antheraea mylitta using ammonium sulphate precipitation, glycogen complex precipitation and gel filtration chromatography. Specific activity increased from 0.673 AU/mg in the crude digestive juice to 94.80 AU/mg in the final purified sample. Activity of the purified enzyme was 15-fold less than that of the digestive amylase of silkworm. Bombyx mori. The zymogram pattern of the purified amylase was similar to that of crude digestive juice on 7.5% native PAGE. The purified enzyme exhibited five bands on native PAGE. IEF of the purified enzyme also revealed five bands with pls of 6.5, 6.15, 5.9, 5.8 and 4.7, respectively. The purified enzyme is a single polypeptide chain with a Mr of 58 kDa. The amylase is most active at pH 9.5 and is a Ca2+ dependent endoenzyme which hydrolyses starch into maltose, maltotriose and maltotetrose and hence behaves as an α-amylase (EC 3.2.1.1). The enzyme was unaffected by the presence or absence of CI-, with Km for soluble starch of 0.113%

Silkworm genomics - progress and prospects

The biology and genetics of silkworm, Bombyx mori, is the most advanced of any lepidopteran species. Its rich repertoire of genetic resources and potential applications in sericulture and as a model for other Lepidoptera led to the initiation of genomics research. During the past decade much effort has been made in the areas of marker development, and molecular maps have been constructed in standard strains with the use of RFLPs, RAPDs, ISSRs, STSs, and microsatellites. The potential applications of molecular markers and linkage maps include stock identification, Marker Assisted Selection (MAS), identification of Quantitative Trait Loci (QTL), and, ultimately, positional cloning of visible mutations and QTL. To these ends, BAC libraries have been constructed and are being used to make large-scale physical maps, with markers based on ESTs as framework anchors. Altogether this work provides a foundation for identification of gene function, gene and chromosome evolution, and comparative genomics

Microsatellite markers for the Indian golden silkmoth, Antheraea assama (Saturniidae: Lepidoptera)

Antheraea assama, an economically important and scientifically unexplored Indian wild silkmoth, is unique among saturniid moths. For this species, a total of 87 microsatellite markers was derived from 35 000 expressed sequence tags and a microsatellite-enriched sub-genomic library. Forty individuals collected from Tura and West Garo Hills region of Northeast India were screened for each of these loci. Ten loci from expressed sequence tags and one from genomic library were found to be polymorphic. These microsatellite markers will be useful resources for population genetic studies of A. assama and other closely related species of saturniids. This is the first report on development of microsatellite markers for any saturniid species

Molecular phylogeny of silkmoths reveals the origin of domesticated silkmoth, Bombyx mori from chinese Bombyx mandarina and paternal inheritance of Antheraea proylei mitochondrial DNA

Molecular phylogeny of some of the economically important silkmoths was derived using three mitochondrial genes, 12S rRNA, 16S rRNA, and COI, and the control region (CR). Maximum likelihood (ML) analyses showed two distinct clades, one consisting of moths from Bombycidae family and the other from Saturniidae family. The mitochondrial CR showed length polymorphisms with indels. The ML analyses for complete mitochondrial genome sequences of Bombyx mori (strains Aojuku, C108, Backokjam, and Xiafang), Japanese and Chinese strains of B. mandarina (Japanese mandarina and Chinese mandarina) and, Antheraea pernyi revealed two distinct clades, one comprising of B. mori strains and the other with B. mandarina, and A. pernyi forming an outgroup. Pairwise distances revealed that all of the strains of B. mori studied are closer to Chinese than to Japanese mandarina. Phylogenetic analyses based on whole mitochondrial genome sequences, the finding of a tandem triplication of a 126 bp repeat element only in Japanese mandarina, and chromosome number variation in B. mandarina suggest that B. mori must have shared its recent common ancestor with Chinese mandarina. Another wild species of the Bombycidae family, Theophila religiosa, whose phylogenetic status was not clear, clustered together with the other bombycid moths in the study. Analysis of the interspecific hybrid, A. proylei gave evidence for paternal inheritance of mitochondrial DNA

Genetic characterization of the silkworm Bombyx mori by simple sequence repeat (SSR)-anchored PCR

Thirteen diverse strains of the silkworm Bombyx mori were analysed using the simple sequence repeat anchored polymerase chain reaction (SSR-anchored PCR) or Inter-SSR-PCR (ISSR-PCR). A set of four 5'-anchored and two 3'-anchored repeat primers amplified a total of 239 bands out of which 184 (77%) were polymorphic. The 5'-anchored primers revealed more distinct polymorphic markers than the 3'-anchored primers and the ISSR-PCR method showed greater variability than RAPDs. The strain-specific pattern was shown to be inherited and segregated in a Mendelian fashion. A dendrogram constructed using the UPGMA method revealed two distinct groups, one comprising nondiapausing and one comprising diapausing strains. These results suggest that the ISSR-PCR method is potentially useful for genetic fingerprinting of silkworm genotypes and as a mapping tool in the silkworm