Transcriptome analysis of Nosema assamensis infecting muga silkworms (Antheraea assamensis) reveals insights into candidate pathogenicity related genes and molecular pathways required for pathogenesis.

Muga silkworms are often prone to many diseases since, these are non-domesticated and are reared outdoors. Microsporidia, an obligate intracellular pathogen with spore as its active form, causes pebrine disease in these silkworms. The study has attempted to categorise the transcript data of the Nosema obtained from the infected muga silkworm using gene ontology and KEGG pathway studies. A total of 2850 unigene sets were identified out of which 2739 unigenes were placed under biological, cellular as well as molecular function categories based on the gene ontology (GO) terms. 1620 out of these unigenes sets found their orthologous partner in the corresponding Nosema bombycis transcriptome. The unigenes were found to be enriched under organic substance metabolic process, organic cyclic compound binding and intracellular anatomical structure for biological process, molecular function and cellular components respectively. The KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis indicated majority of the enzymes were found to be mapped under purine and thiamine metabolic pathways, indicating an increase in the energy metabolism required to establish the infection in the silkworms. The putative virulence genes identified in this study are PTP2, PTP3, SWP12 and SWP26 which were found to be expressed in other Nosema species indigenous to India, indicating a probable conservation of these genes, which are primarily involved in establishing host pathogen interactions. The expression of these genes was in detectable levels in the infected silkworm samples. These genes may be validated further through bioassay in order understand their roles in establishing the infection and propagation of the spores. The identified virulence genes may be further targeted to develop diagnostic tools for identification of the pathogen at early stages of infection.

Genetic diversity and population structure of Indian golden silkmoth (Antheraea assama).

BACKGROUND: The Indian golden saturniid silkmoth (Antheraea assama), popularly known as muga silkmoth, is a semi-domesticated silk producing insect confined to a narrow habitat range of the northeastern region of India. Owing to the prevailing socio-political problems, the muga silkworm habitats in the northeastern region have not been accessible hampering the phylogeography studies of this rare silkmoth. Recently, we have been successful in our attempt to collect muga cocoon samples, although to a limited extent, from their natural habitats. Out of 87 microsatellite markers developed previously for A. assama, 13 informative markers were employed to genotype 97 individuals from six populations and analyzed their population structure and genetic variation. METHODOLOGY/PRINCIPAL FINDINGS: We observed highly significant genetic diversity in one of the populations (WWS-1, a population derived from West Garo Hills region of Meghalaya state). Further analysis with and without WWS-1 population revealed that dramatic genetic differentiation (global F(ST) = 0.301) was due to high genetic diversity contributed by WWS-1 population. Analysis of the remaining five populations (excluding WWS-1) showed a marked reduction in the number of alleles at all the employed loci. Structure analysis showed the presence of only two clusters: one formed by WWS-1 population and the other included the remaining five populations, inferring that there is no significant genetic diversity within and between these five populations, and suggesting that these five populations are probably derived from a single population. Patterns of recent population bottlenecks were not evident in any of the six populations studied. CONCLUSIONS/SIGNIFICANCE: A. assama inhabiting the WWS-1 region revealed very high genetic diversity, and was genetically divergent from the five populations studied. The efforts should be continued to identify and study such populations from this region as well as other muga silkworm habitats. The information generated will be very useful in conservation of dwindling muga culture in Northeast India.

Genetic analysis of scattered populations of the Indian eri silkworm, Samia cynthia ricini Donovan: Differentiation of subpopulations.

Deforestation and exploitation has led to the fragmentation of habitats and scattering of populations of the economically important eri silkworm, Samia cynthia ricini, in north-east India. Genetic analysis of 15 eri populations, using ISSR markers, showed 98% inter-population, and 23% to 58% intra-population polymorphism. Nei's genetic distance between populations increased significantly with altitude (R(2) = 0.71) and geographic distance (R(2) = 0.78). On the dendrogram, the lower and upper Assam populations were clustered separately, with intermediate grouping of those from Barpathar and Chuchuyimlang, consistent with geographical distribution. The Nei's gene diversity index was 0.350 in total populations and 0.121 in subpopulations. The genetic differentiation estimate (Gst) was 0.276 among scattered populations. Neutrality tests showed deviation of 118 loci from Hardy-Weinberg equilibrium. The number of loci that deviated from neutrality increased with altitude (R(2) = 0.63). Test of linkage disequilibrium showed greater contribution of variance among eri subpopulations to total variance. D('2)IS exceeded D('2)ST, showed significant contribution of random genetic drift to the increase in variance of disequilibrium in subpopulations. In the Lakhimpur population, the peripheral part was separated from the core by a genetic distance of 0.260. Patchy habitats promoted low genetic variability, high linkage disequilibrium and colonization by new subpopulations. Increased gene flow and habitat-area expansion are required to maintain higher genetic variability and conservation of the original S. c. ricini gene pool.

Genetic evaluation of eri silkworm Samia cynthia ricini: ISSR loci specific to high and low altitude regimes and quantitative attributes.

Genetic structure of populations is under constant pressure from varying geographical conditions that induce phenotypic plasticity in insects. Spatial distribution of 15 populations of Indian eri silkworm, Samia cynthia ricini originated at various altitudes of sub Himalayas based on Euclidean distance realized from yield attributes showed two population clusters irrespective of their place of origin and altitude. However, DNA amplification profile by inter SSR (ISSR) markers showed genetic variations among the populations depend on low and high altitudes. One ISSR locus each specific to high and low altitude population was identified. The locus from high altitude showed deviation from Hardy-Weinberg equilibrium but that from low altitude was in neutrality suggests that the high altitude loci could be under pressure from the altitudinal variations. In association with different yield traits, 18 loci were identified. Of which, three markers showed association with more than one trait indicative of pleiotropic influence. Stepwise addition of markers enhanced the correlation between markers and the associated trait pointed to polygenic influence. Association of markers with altitude and yield traits suggests an imperative relation of rare genetic loci with gene-environment interaction and phenotypic variability in S. c. ricini.

Genetic analysis of scattered populations of the Indian eri silkworm, Samia cynthia ricini Donovan: differentiation of subpopulations

Deforestation and exploitation has led to the fragmentation of habitats and scattering of populations of the economically important eri silkworm, Samia cynthia ricini, in north-east India. Genetic analysis of 15 eri populations, using ISSR markers, showed 98 percent inter-population, and 23 percent to 58 percent intra-population polymorphism. Nei's genetic distance between populations increased significantly with altitude (R² = 0.71) and geographic distance (R² = 0.78). On the dendrogram, the lower and upper Assam populations were clustered separately, with intermediate grouping of those from Barpathar and Chuchuyimlang, consistent with geographical distribution. The Nei's gene diversity index was 0.350 in total populations and 0.121 in subpopulations. The genetic differentiation estimate (Gst) was 0.276 among scattered populations. Neutrality tests showed deviation of 118 loci from Hardy-Weinberg equilibrium. The number of loci that deviated from neutrality increased with altitude (R² = 0.63). Test of linkage disequilibrium showed greater contribution of variance among eri subpopulations to total variance. D'2IS exceeded D'2ST, showed significant contribution of random genetic drift to the increase in variance of disequilibrium in subpopulations. In the Lakhimpur population, the peripheral part was separated from the core by a genetic distance of 0.260. Patchy habitats promoted low genetic variability, high linkage disequilibrium and colonization by new subpopulations. Increased gene flow and habitat-area expansion are required to maintain higher genetic variability and conservation of the original S. c. ricini gene pool.

Molecular Markers for Biomass Traits: Association, Interaction and Genetic Divergence in Silkworm Bombyx mori.

Improvement of high yielding, disease resistant silkworm strains became imminent to increase production of silk, which is a major revenue earner for sericulturists. Since environment interacts with phenotype, conventional breeding did not result in commendable yield improvement in synthetic strains of silkworm, Bombyx mori. Identification of DNA markers associated with different economically important biomass traits and its introgression could assist molecular breeding and expression of stabilized high yielding characters, but genetic basis of most quantitative traits in silkworm is poorly understood due to its polygenic control. Correlation analysis (R = 0.9) revealed significant interrelation among biomass traits viz., larval duration (TLD), larval weight (LWT), cocoon weight (CWT), shell weight (SWT), shell ratio (SR) and floss content. PCR using inter simple sequence repeat (ISSR) primers revealed 92% polymorphism among 14 tropical and temperate strains of B. mori, with average diversity index of 0.747. Stepwise multiple regression analysis (MRA) selected 35 ISSR markers positively or negatively correlated with different biomass traits, illustrated polygenic control. ISSR marker 830.8(1050bp) was significantly associated with LWT, CWT, SWT, SR and floss content, indicated its pleiotropic role. Two ISSR markers, 835.5(1950bp) and 825.9(710bp) showed significant association with floss content and TLD. These markers were segregated in F(2) generation and Chi-square test confirmed (chi(2) = ~45; P < 0.05) its genetic contribution to the associated biomass traits. Strains, with both positively and negatively correlated markers, had intermediate mean value for biomass traits (eg. SWT = 0.17 +/- 0.014 g in GNM and Moria) indicated interaction of loci in natural populations. Low yielding Indian strains grouped together by Hierarchical clustering. Chinese and Japanese strains were distributed in the periphery of ALSCAL matrix indicated convergence of genetic characters in Indian strains. Average genetic distance between Chinese strains and Indian strains (0.193) significantly (P < 0.01) varied from that between Chinese and Japanese strains. Interaction of loci and allelic substitutions induced phenotypic plasticity in temperate B. mori populations on tropic adaptation in India. These outcomes show possibility to combine favorable alleles at different QTL to increase larval, cocoon and shell weight.