Oxidative stress and cytotoxicity elicited lipid peroxidation in hemocytes of Bombyx mori larva infested with dipteran parasitoid, Exorista bombycis.

Parasitization of silkworm, Bombyx mori by invasive larva of dipteran parasitoid Exorista bombycis caused upto 20% revenue loss in sericulture. The parasitism was successful by suppressing host immune system however mechanism of immune suppression induced by E. bombycis is unknown which is unravelled here. The infestation induced cytotoxic symptoms in host hemocytes, such as vacuolated cytoplasm, porous plasma membrane, indented nuclei with condensed chromatin and dilated RER. One of the markers of necrosis is cell permeabilization, which can be measured as released lactate dehydrogenase (LDH). LDH level showed significantly (P<0.01) high release into extracellular medium in vitro after exposure of hemocytes to parasitoid larval tissue protein compared with control revealing membrane permeability and loss of cell integrity. At five minutes after exposure, cytotoxicity was 43% and was increased to 99% at 3h. The cytotoxicity is signalled by increased content of hydrogen peroxide (H2O2) causing lipid peroxidation followed by porosity in plasma membrane. A test for lipid peroxidation by measurement of lipid peroxidation breakdown product, malondialdehyde (MDA) revealed significant increase in peroxidation from one to 24 h post-invasion, with maximum at 12 h (P<0.008). Level of reactive oxygen species measured as H2O2 production increased from 6 to 12 h post-invasion and continued to increase significantly (P<0.03) reaching maximum at 48 h. These observations reveal that dipteran endoparasitoid invasion induced H2O2 production in the hemocytes causing cytotoxicity, lipid peroxidation and membrane porosity that suppressed both humoral- and cell-mediated immune responses of hemocytes in B. mori.

Attacin gene sequence variations in different ecoraces of tasar silkworm Antheraea mylitta.

Attacin gene exists as paralogous conversion and is being used for identification of strain variations in insects based on the sequence variation. Hence, a study was undertaken to analyze the sequence variation of the attacin gene isoforms in the tasar silkworm Anthereae mylitta that exists in the form of different ecoraces depending upon the environment, food plant and location. Comparison of the previously reported attacin sequences with the DNA sequences of attacin A and B genes revealed six amino acid substitutions among the sequences of the ecoraces which however did not affect the functional domain of Attacin. The generated dendrogram clearly indicated unique branches for each ecorace with two separate gene clusters for attacin A and B. The Sarihan ecorace formed a separate sub-group under both the gene clusters. The present study also revealed the presence of Attacin_N Superfamily domain exclusively in Exon I separated from the Attacin_C Superfamily domain that was present in Exon II and part of Exon III, a prominent character of attacin gene. The phylogenetic reconstruction analysis of attacin gene in A.mylitta supported the common evolutionary origin of attacin genes belonging to the Lepidoteran and Dipteran families that formed two separate clusters.

Genome wide microarray based expression profiles associated with BmNPV resistance and susceptibility in Indian silkworm races of Bombyx mori.

The molecular mechanism involved in BmNPV resistance was investigated using a genome wide microarray in midgut tissue of Indian silkworm Bombyx mori. In resistant race (Sarupat), 735 genes up-regulated and 589 genes down-regulated at 12 h post BmNPV infection. Similarly, in case of susceptible race (CSR-2), 2183 genes up-regulated and 2115 genes down-regulated. Among these, nine up-regulated and eight down-regulated genes were validated using real-time qPCR analysis. In Sarupat, vacuolar protein sorting associated, Xfin-like protein and carboxypeptidase E-like protein genes significantly up-regulated in infected midgut; prominently down-regulated genes were glutamate receptor ionotropic kainite 2-like, BTB/POZ domain and transferrin. Considerably up-regulated genes in the CSR-2 were peptidoglycan recognition protein S6 precursor and rapamycin while the conspicuous down-regulated genes were facilitated trehalose transporter and zinc transporter ZIP1-like gene. The up-regulation of genes in resistant race after BmNPV infection indicates their possible role in antiviral immune response.

Coregulation of host-response genes in integument: switchover of gene expression correlation pattern and impaired immune responses induced by dipteran parasite infection in the silkworm, Bombyx mori.

The activation of host response proteins against parasitic infection is dependent on the coregulation of immune gene expression. The infection of commercially important silkworm Bombyx mori through endoparasite Exorista bombycis enhanced host-response gene expression in integument early in the infection and was lowered asymptotically. Principal component analysis (PCA) showed heterogeneity while explaining ∼80 % variance among expression timings. PCA showed positive and negative correlation with gene expression and differentiated transcriptional timings, and revealed cross talk within the immune system. Pearson correlation analysis showed significant linear correlation (mean R (2) = >0.7; P < 0.004) between the expression of 16 pairs of genes in control, while the relation switched over to curvilinear due to parasitism. The genes showed pleiotropic interaction among them, with four genes each for prophenoloxidase activating enzyme (PPAE) and caspase. Besides, after parasitism, exclusive correlation of five gene pairs including PPAE-Spatzle pair (R (2) = 0.9; P < 0.011) was observed in the integument. In integument, the phenol oxidase (PO) activity showed a positive correlation with the tyrosine level (R (2) = 0.410; P < 0.002) and a curvilinear relation (R (2) = 0.745; P < 0.0002) with the expanding lysis area. The PO activity was positively correlated with BmToll expression and negatively correlated with paralytical peptide expression, revealing polygenic influence. Caspase expression was tightly regulated by signal genes in control integument, whereas they were deregulated after infection. Switchover from linear to curvilinear correlation and the appearance of new gene correlations in parasitized integument revealed deviation from gene coregulation, leading to impaired immune responses, characterized by lowered gene expression and varied phenotypic consequences.

Activation of autophagic programmed cell death and innate immune gene expression reveals immuno-competence of integumental epithelium in Bombyx mori infected by a dipteran parasitoid.

In insects, the integument forms the primary barrier between the environment and internal milieu, but cellular and immune responses of the integumental epithelium to infection by micro- and macro-parasites are mostly unknown. We elucidated cellular and immune responses of the epithelium induced through infection by a dipteran endoparasitoid, Exorista bombycis in the economically important silkworm Bombyx mori. Degradative autophagic vacuoles, lamella-like bodies, a network of cytoplasmic channels with cellular cargo, and an RER network that opened to vacuoles were observed sequentially with increase in age after infection. This temporal sequence culminated in apoptosis, accompanied by the upregulation of the caspase gene and fragmentation of DNA. The infection significantly enhanced the tyrosine level and phenol oxidase activity in the integument. Proteomic analysis revealed enhanced expression of innate immunity components of toll and melanization pathways, cytokines, signaling molecules, chaperones, and proteolytic enzymes demonstrating diverse host responses. qPCR analysis revealed the upregulation of spatzle, BmToll, and NF kappa B transcription factors Dorsal and BmRel. NF kappa B inhibitor cactus showed diminished expression when Dorsal and BmRel were upregulated, revealing a negative correlation (R = (-)0.612). During melanization, prophenol oxidase 2 was expressed, a novel finding in integumental epithelium. The integument showed a low level of melanin metabolism and localized melanism in order to prevent the spreading of cytotoxic quinones. The gene-encoding proteolytic enzyme, beta-N-acetylglucosaminidase, was activated at 24 h post-infection, whereas chitinase, was activated at 96 h post-infection; however, most of the immune genes enhanced their expression in the early stages of infection. Thus the integument contributes to humoral immune responses that enhance resistance against macroparasite invasion.

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.

In vitro antiviral activity of an alkaline trypsin from the digestive juice of Bombyx mori larvae against nucleopolyhedrovirus.

Alkaline trypsin protein of molecular mass 25,436 Da purified from the digestive juice of Bombyx mori larvae indicated strong antiviral activity against Bombyx mori nucleopolyhedrovirus (BmNPV) under in vitro conditions. Partial N-terminal amino acid sequence of the protein was determined and the cDNA was cloned based on the amino acid sequence. A homology search of the deduced amino acid sequence of the cDNA showed 55% identity with Helicoverpa armigera trypsin and the active site of this protein was completely conserved. Hence, the protein was designated B. mori trypsin (Bmtryp). The results suggest that Bmtryp, an insect digestive enzyme, can be a potential antiviral factor against BmNPV at the initial site of viral infection.

Differential gene expression during early embryonic development in diapause and non-diapause eggs of multivoltine silkworm Bombyx mori.

Quantification of the differential expression of metabolic enzyme and heat-shock protein genes (Hsp) during early embryogenesis in diapause and non-diapause eggs of the silkworm B. mori was carried out by semi-quantitative RT-PCR. Data analysis revealed that, the phosphofructokinase (PFK) expression started at a higher level in the early stage (6 h after oviposition) in non-diapause eggs, while in diapause induced eggs, it started at a lower level. However, the PFK gene expression in diapause eggs was comparatively higher than in non-diapause eggs. PFK facilitates use of carbohydrate reserves. The lower level of PFK gene expression in the early stage of diapause induced eggs but comparatively higher level of expression than in non-diapause eggs is due to enzyme inactivation via protein phosphorylation during early embryogenesis followed by de-phosphorylation in later stage. The sorbitol dehydrogenase-2 (SDH-2) gene was down regulated in diapause induced eggs up to 24 h and its expression levels in diapause induced eggs coincided with that of PFK gene at 48h in non-diapause eggs. During carbohydrate metabolism, there is an initial temporary accumulation of sorbitol which acts as protectant. The down regulation of SDH-2 gene during the first 24 hours in diapause induced eggs was due to the requirement of sorbitol as protectant. However, since the diapause process culminates by 48 h, the SDH-2 gene expression increased and coincided with that of PFK gene expression. The trehalase (Tre) gene expression was at a lower level in diapause induced eggs compared to non-diapausing eggs. The induction of Tre activity is to regulate uptake and use of sugar by the tissues. The non-diapause eggs revealed maximum expression of GPase gene with major fluctuations as well as an overall higher expression compared to diapause induced eggs. The diapause process requires less energy source which reflects lower activity of the gene. Heat shock protein (Hsp) genes (Hsp20.4, 40, 70, and 90) revealed differential levels of expression in both the eggs at all stages of embryonic development. The present study thus provides an overview of the differential expression levels of metabolic enzyme and Hsp genes in non-diapause and diapause induced eggs of multivoltine silkworm B. mori within 48 h after oviposition, confirming the major role of in early embryogenesis.

Genetic diversity and relationships in mulberry (genus Morus) as revealed by RAPD and ISSR marker assays.

BACKGROUND: The genus Morus, known as mulberry, is a dioecious and cross-pollinating plant that is the sole food for the domesticated silkworm, Bombyx mori. Traditional methods using morphological traits for classification are largely unsuccessful in establishing the diversity and relationships among different mulberry species because of environmental influence on traits of interest. As a more robust alternative, PCR based marker assays including RAPD and ISSR were employed to study the genetic diversity and interrelationships among twelve domesticated and three wild mulberry species. RESULTS: RAPD analysis using 19 random primers generated 128 discrete markers ranging from 500-3000 bp in size. One-hundred-nineteen of these were polymorphic (92%), with an average of 6.26 markers per primer. Among these were a few putative species-specific amplification products which could be useful for germplasm classification and introgression studies. The ISSR analysis employed six anchored primers, 4 of which generated 93 polymorphic markers with an average of 23.25 markers per primer. Cluster analysis of RAPD and ISSR data using the WINBOOT package to calculate the Dice coefficient resulted into two clusters, one comprising polyploid wild species and the other with domesticated (mostly diploid) species. CONCLUSION: These results suggest that RAPD and ISSR markers are useful for mulberry genetic diversity analysis and germplasm characterization, and that putative species-specific markers may be obtained which can be converted to SCARs after further studies.