Genetic analysis of Indian tasar silkmoth (Antheraea mylitta) populations.

Indian tasar silkmoth, Antheraea mylitta is an economically important wild silkmoth species distributed across India. A number of morphologically and ethologically well-defined ecotypes are known for this species that differ in their primary food plant specificity. Most of these ecotypes do not interbreed in nature, but are able to produce offspring under captive conditions. Microsatellite markers were developed for A. mylitta, and out of these, ten well-behaved microsatellite loci were used to analyze the population structure of different ecoraces. A total of 154 individual moths belonging to eight different ecoraces, were screened at each locus. Hierarchical analysis of population structure using Analysis of MOlecular VAriance (AMOVA) revealed significant structuring (FST = 0.154) and considerable inbreeding (FIS = 0.505). A significant isolation by distance was also observed. The number of possible population clusters was investigated using distance method, Bayesian algorithm and self organization maps (SOM). The first two methods revealed two distinct clusters, whereas the SOM showed the different ecoraces not to be clearly differentiated. These results suggest that although there is a large degree of phenotypic variation among the different ecoraces of A. mylitta, genetically they are not very different, and the phenotypic differences may largely be a result of their respective ecology.

Review of methods for the detection and quantification of adulteration of rice: Basmati as a case study.

Rice is a staple and widely grown crop endowed with rich genetic diversity. As it is difficult to differentiate seeds of various rice varieties based on visual observation accurately, the harvested seeds and subsequent processed products are highly prone to adulteration with look-alike and low quality seeds by the dishonest traders. To protect the interests of importing countries and consumers, several methods have been employed over the last few decades for unambiguous discrimination of cultivars, accurate quantification of the adulterants, and for determination of cultivated geographical area. With recent advances in biotechnology, DNA based techniques evolved rapidly and proved successful over conventional non-DNA based methods to purge the problem of adulteration at commercial level. In the current review, we made an attempt to summarize the existing methods of adulteration detection and quantification in a comprehensive manner by providing Basmati as a case study to enable the traders to arrive at a quick resolution in choosing the apt method to eliminate the adulteration practice in the global rice industry.

bmnpv-miR-3 facilitates BmNPV infection by modulating the expression of viral P6.9 and other late genes in Bombyx mori.

During the last decade, microRNAs (miRNAs) have emerged as fine tuners of gene expression in various biological processes including host-pathogen interactions. Apart from the role of host encoded miRNAs in host-virus interactions, recent studies have also indicated the key role of virus-encoded miRNAs in the regulation of host defense responses. In the present study, we show that bmnpv-miR-3, a Bombyx mori nucleopolyhedrovirus (BmNPV) encoded miRNA, regulates the expression of DNA binding protein (P6.9) and other late genes, vital for the late stage of viral infection in the host, Bombyx mori. We have performed both cell culture and in vivo experiments to establish the role of bmnpv-miR-3 in the infection cycle of BmNPV. Our findings showed that bmnpv-miR-3 expresses during early stage of infection, and negatively regulates the expression of P6.9. There was an upregulation in P6.9 expression upon blocking of bmnpv-miR-3 by Locked Nucleic Acid (LNA), whereas overexpression of bmnpv-miR-3 resulted in a decreased expression of P6.9. Besides, a remarkable enhancement and reduction in the viral loads were observed upon blocking and overexpression of bmnpv-miR-3, respectively. Furthermore, we have also assessed the host immune response using one of the Lepidoptera-specific antimicrobial proteins, Gloverin-1 upon blocking and overexpression of bmnpv-miR-3, which correlated viral load with the host immune response. All these results together; clearly imply that bmnpv-miR-3-mediated controlled regulation of BmNPV late genes in the early stage of infection helps BmNPV to escape the early immune response from the host.

Characterization of antiviral and antibacterial activity of Bombyx mori seroin proteins.

Lepidopterans as other insects have a very potent innate immune system, which basically comprises cellular and humoral defence mechanisms against bacterial and fungal infections. In lepidopterans, not much is known about the defence mechanisms against viral pathogens, such as baculoviruses. Here we show that small silk proteins of the domesticated silkworm, Bombyx mori, called seroins, act as antiviral agents against a baculovirus pathogen, Bombyx mori nucleopolyhedrovirus (BmNPV). Involvement of these proteins in the inhibition of baculovirus infection was revealed by estimating the viral load upon their dsRNA-mediated knockdown. Additionally, we found through antimicrobial assays that seroins are potent inhibitors of bacterial growth. Binding competition assays followed by antimicrobial assays showed that seroins bind to peptidoglycan, a cell wall component of bacteria. Analysis of bacterial load upon knockdown of seroins resulted in higher proliferation of bacteria. Phylogenetic analysis showed the recent origin of seroins in a few moth species and duplication only in Bombycids. The antiviral and antibacterial activity of seroins shown in this study using several biochemical and molecular biological assays provide strong evidence to characterize them as antimicrobial proteins. Hence, we hypothesize that seroins are potent candidates for use in development of transgene-based disease resistant silkworm strains.

Lepidopteran sex determination: a cascade of surprises.

Sex determination is a developmental pathway that fixes the sexual fate (male or female) of an individual at early stages of embryonic development. This pathway is ideally suited for evolutionary studies given the astoundingly diverse mechanisms found in the animal kingdom. In particular, insects use multiple different cues to specify the sexual fate of an individual. In this review, we focus on genes and genetic interactions involved in the sex determination of insect species belonging to the order Lepidoptera. Unique features of the lepidopteran sex determination system are discussed.

A MATLAB-Based Boundary Data Simulator for Studying the Resistivity Reconstruction Using Neighbouring Current Pattern.

Phantoms are essentially required to generate boundary data for studying the inverse solver performance in electrical impedance tomography (EIT). A MATLAB-based boundary data simulator (BDS) is developed to generate accurate boundary data using neighbouring current pattern for assessing the EIT inverse solvers. Domain diameter, inhomogeneity number, inhomogeneity geometry (shape, size, and position), background conductivity, and inhomogeneity conductivity are all set as BDS input variables. Different sets of boundary data are generated by changing the input variables of the BDS, and resistivity images are reconstructed using electrical impedance tomography and diffuse optical tomography reconstruction software (EIDORS). Results show that the BDS generates accurate boundary data for different types of single or multiple objects which are efficient enough to reconstruct the resistivity images for assessing the inverse solver. It is noticed that for the BDS with 2048 elements, the boundary data for all inhomogeneities with a diameter larger than 13.3% of that of the phantom are accurate enough to reconstruct the resistivity images in EIDORS-2D. By comparing the reconstructed image with an original geometry made in BDS, it would be easier to study the inverse solver performance and the origin of the boundary data error can be identified.

PhiC31 integrase-mediated cassette exchange in silkworm embryos.

To construct an effective site-specific integration system in the silkworm, we examined if phiC31 integrase works in silkworm embryos. As an assay system, we constructed an extrachromosomal cassette exchange reaction system between two attP sites of an acceptor plasmid and two attB sites of a donor plasmid. To evaluate the activity, integrase mRNAs synthesized from three different plasmids were used. We injected a mixture of the acceptor and donor plasmids with the mRNA synthesized in vitro from one of the three plasmids into silkworm embryos at 4-6 h after oviposition and recovered plasmid DNAs from the embryos 3 days after injection. The resultant plasmids were transformed into Escherichia coli and spread on selection medium plates containing the appropriate antibiotics. A colony-forming assay and restriction enzyme digestion of the plasmids purified from the colonies showed that the phiC31 integrase worked very efficiently in the silkworm embryos. Notably, a phiC31 integrase mRNA synthesized from two of the plasmids produced cassette exchange plasmids at a high frequency, suggesting that the mRNA can be used to construct a targeted integration system in silkworms.

A baculovirus-encoded MicroRNA (miRNA) suppresses its host miRNA biogenesis by regulating the exportin-5 cofactor Ran.

MicroRNAs have emerged as key players in the regulation of various biological processes in eukaryotes, including host-pathogen interactions. Recent studies suggest that viruses encode miRNAs to manipulate their host gene expression to ensure their effective proliferation, whereas the host limits virus infection by differentially expressing miRNAs that target essential viral genes. Here, we demonstrate that an insect virus, Bombyx mori nucleopolyhedrosis virus (BmNPV), modulates the small-RNA-mediated defense of its host, B. mori, by encoding an miRNA (bmnpv-miR-1) that downregulates the expression of the host GTP-binding nuclear protein Ran, an essential component of the exportin-5-mediated nucleocytoplasmic transport machinery mainly involved in small-RNA transport from the nucleus to the cytoplasm. We demonstrate the sequence-dependent interaction of bmnpv-miR-1 with Ran mRNA using cell culture and in vivo assays, including RNA interference (RNAi) of Ran. Our results clearly show that bmnpv-miR-1 represses Ran, leading to reduction in the host small-RNA population, and consequently, the BmNPV load increases in the infected larvae. Blocking of bmnpv-miR-1 resulted in higher expression levels of Ran and a decrease in BmNPV proliferation. In contrast, blockage of host miRNA, bmo-miR-8, which targets the immediate-early gene of the virus and whose production was repressed upon bmnpv-miR-1 and Ran dsRNA administration, resulted in a significant increase in the virus load in the infected B. mori larvae. The present study provides an insight into one of the evasion strategies used by the virus to counter the host defense for its effective proliferation and has relevance to the development of insect virus control strategies.

Drosophila intersex orthologue in the silkworm, Bombyx mori and related species.

Intersex (ix), a gene required for female sexual development in Drosophila, acts in concert with doublesex (dsx) at the end of the sex determination pathway. In the present study a homologue of ix was identified in Bombyx mori. Expression analysis of this gene by RT-PCR and RNase protection assay revealed a diagnostic alternative splice form present only in testis, whereas the most common splice form was found to express in all other tissues from early embryonic developmental stages. The present study provides evidence for the presence of an alternative splice form of ix in three species of silkmoths examined. Taken together with the results of an earlier study on ix in piralid moth, Maruca vitrata (Cavaliere et al. 2009), the present study suggests that the testis-specific splice form may be a characteristic feature of lepidopterans. Though ix lacks a conserved splicing pattern it appears to have retained its functional conservation in terminal sexual differentiation. We speculate that the presence of an additional splice form, perhaps encoding non-functional protein only in testis, may prevent the feminizing effects exerted by the functional IX protein.

Doublesex: a conserved downstream gene controlled by diverse upstream regulators.

Sex determination, an integral precursor to sexual reproduction, is required to generate morphologically distinct sexes. The molecular components of sex-determination pathways regulating sexual differentiation have been identified and characterized in different organisms. The Drosophila doublesex (dsx) gene at the bottom of the sex-determination cascade is the best characterized candidate so far, and is conserved from worms (mab3 of Caenorhabditis elegans) to mammals (Dmrt-1). Studies of dsx homologues from insect species belonging to different orders position them at the bottom of their sex-determination cascade. The dsx homologues are regulated by a series of upstream regulators that show amazing diversity in different insect species. These results support the Wilkin's hypothesis that evolution of the sex-determination cascade has taken place in reverse order, the bottom most gene being most conserved and the upstream genes having been recruited at different times during evolution. The pre-mRNA of dsx is sex-specifically spliced to encode male or female-specific transcription factors that play an important role in the regulation of sexually dimorphic characters in different insect species. The generalization that dsx is required for somatic sexual differentiation culminated with its functional analysis through transgenesis and knockdown experiments in diverse species of insects. This brief review will focus on the similarities and variations of dsx homologues that have been investigated in insects to date.

Discovering microRNAs from Bombyx mori nucleopolyhedrosis virus.

MicroRNAs (miRNAs) have emerged as key players in host-pathogen interaction. Recently, many virus-encoded miRNAs have been identified from different mammalian species. However, the large family of invertebrate viruses of Baculoviridae, which infects diverse species of beneficial insects and agriculture pests, has hardly been investigated for elucidating the role of miRNAs in host-pathogen interaction. In the study reported here, we have identified four Bombyx mori nucleopolyhedrosis virus (BmNPV)-encoded miRNAs using a combination of in silico and experimental methods. Unlike other reported viral miRNAs, the BmNPV-encoded miRNAs identified in the present study were found to be evolutionarily conserved among many closely related baculoviruses. Besides, we have computationally predicted 8 viral and 64 cellular targets of these virus-encoded miRNAs and the putative functions of these targets suggest a key role of viral miRNAs in insect-pathogen interactions by modulating several viral replication genes as well as those involved in host immune defense machinery.

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 (DSX(F1) and DSX(F2)) and one male-specific (DSX(M)) 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 (DSX(F1) and DSX(F2)) 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.

The silkworm Z chromosome is enriched in testis-specific genes.

The role of sex chromosomes in sex determination has been well studied in diverse groups of organisms. However, the role of the genes on the sex chromosomes in conferring sexual dimorphism is still being experimentally evaluated. An unequal complement of sex chromosomes between two sexes makes them amenable to sex-specific evolutionary forces. Sex-linked genes preferentially expressed in one sex over the other offer a potential means of addressing the role of sex chromosomes in sexual dimorphism. We examined the testis transcriptome of the silkworm, Bombyx mori, which has a ZW chromosome constitution in the female and ZZ in the male, and show that the Z chromosome harbors a significantly higher number of genes expressed preferentially in testis compared to the autosomes. We hypothesize that sexual antagonism and absence of dosage compensation have possibly led to the accumulation of many male-specific genes on the Z chromosome. Further, our analysis of testis-specific paralogous genes suggests that the accumulation on the Z chromosome of genes advantageous to males has occurred primarily by translocation or tandem duplication.

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.

Polymorphic microsatellite loci for primitively eusocial wasp Ropalidia marginata.

We report here development and characterization of 48 novel microsatellite markers for Ropalidia marginata, a tropical, primitively eusocial polistine wasp from peninsular India. Thirty-two microsatellites showed polymorphism in a wild population of R. marginata (N = 38) collected from Bangalore, India. These markers will facilitate answering some interesting questions in ecology and evolutionary biology of this wasp, such as population structure, serial polygyny, intra-colony genetic relatedness and the pattern of queen succession.

WildSilkbase: an EST database of wild silkmoths.

BACKGROUND: Functional genomics has particular promise in silkworm biology for identifying genes involved in a variety of biological functions that include: synthesis and secretion of silk, sex determination pathways, insect-pathogen interactions, chorionogenesis, molecular clocks. Wild silkmoths have hardly been the subject of detailed scientific investigations, owing largely to non-availability of molecular and genetic data on these species. As a first step, in the present study we generated large scale expressed sequence tags (EST) in three economically important species of wild silkmoths. In order to make these resources available for the use of global scientific community, an EST database called 'WildSilkbase' was developed. DESCRIPTION: WildSilkbase is a catalogue of ESTs generated from several tissues at different developmental stages of 3 economically important saturniid silkmoths, an Indian golden silkmoth, Antheraea assama, an Indian tropical tasar silkmoth, A. mylitta and eri silkmoth, Samia cynthia ricini. Currently the database is provided with 57,113 ESTs which are clustered and assembled into 4,019 contigs and 10,019 singletons. Data can be browsed and downloaded using a standard web browser. Users can search the database either by BLAST query, keywords or Gene Ontology query. There are options to carry out searches for species, tissue and developmental stage specific ESTs in BLAST page. Other features of the WildSilkbase include cSNP discovery, GO viewer, homologue finder, SSR finder and links to all other related databases. The WildSilkbase is freely available from http://www.cdfd.org.in/wildsilkbase/. CONCLUSION: A total of 14,038 putative unigenes was identified in 3 species of wild silkmoths. These genes provide important resources to gain insight into the functional and evolutionary study of wild silkmoths. We believe that WildSilkbase will be extremely useful for all those researchers working in the areas of comparative genomics, functional genomics and molecular evolution in general, and gene discovery, gene organization, transposable elements and genome variability of insect species in particular.

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.

Genetic evidence from Indian red jungle fowl corroborates multiple domestication of modern day chicken.

BACKGROUND: Domestication of chicken is believed to have occurred in Southeast Asia, especially in Indus valley. However, non-inclusion of Indian red jungle fowl (RJF), Gallus gallus murghi in previous studies has left a big gap in understanding the relationship of this major group of birds. In the present study, we addressed this issue by analyzing 76 Indian birds that included 56 G. g. murghi (RJF), 16 G. g. domesticus (domestic chicken) and 4 G. sonneratii (Grey JF) using both microsatellite markers and mitochondrial D-loop sequences. We also compared the D-loop sequences of Indian birds with those of 779 birds obtained from GenBank. RESULTS: Microsatellite marker analyses of Indian birds indicated an average FST of 0.126 within G. g. murghi, and 0.154 within G. g. domesticus while it was more than 0.2 between the two groups. The microsatellite-based phylogenetic trees showed a clear separation of G. g. domesticus from G. g. murghi, and G. sonneratii. Mitochondrial DNA based mismatch distribution analyses showed a lower Harpending's raggedness index in both G. g. murghi (0.001515) and in Indian G. g. domesticus (0.0149) birds indicating population expansion. When meta analysis of global populations of 855 birds was carried out using median joining haplotype network, 43 Indian birds of G. g. domesticus (19 haplotypes) were distributed throughout the network sharing haplotypes with the RJFs of different origins. CONCLUSION: Our results suggest that the domestication of chicken has occurred independently in different locations of Asia including India. We found evidence for domestication of Indian birds from G. g. spadiceus and G. g. gallus as well as from G. g. murghi, corroborating multiple domestication of Indian and other domestic chicken. In contrast to the commonly held view that RJF and domestic birds hybridize in nature, the present study shows that G. g. murghi is relatively pure. Further, the study also suggested that the chicken populations have undergone population expansion, especially in the Indus valley.