Temporal expression profiling of novel Spodoptera litura nucleopolyhedrovirus-encoded microRNAs upon infection of Sf21 cells.

Baculoviruses are arthropod-specific pathogens, and find extensive applications in pest control strategies and recombinant protein expression. Spodoptera litura nucleopolyhedrovirus (SpltNPV) infects the tropical armyworm Spodoptera litura, which is an important polyphagous crop pest widely distributed in regions of Asia and Oceania. Using next-generation sequencing, we report stage-specific profiling of SpltNPV-encoded microRNAs (miRNAs) at different time intervals post-infection (p.i.) of Sf21 cells. Sequence length distribution analysis of the small RNA libraries revealed a significant increase in 20 nt reads and a reduction of other size fractions during late phases of infection. In silico miRNA prediction tools identified 48 novel SpltNPV-encoded miRNAs, of which 10 were validated experimentally in Sf21 cells using Northern blot analysis and TaqMan quantitative real-time (qRT)-PCR. The viral miRNAs were also found to be expressed in fat-body and mid-gut tissues of infected fifth-instar S. litura larva. qRT-PCR analysis confirmed that expression of most viral miRNAs was triggered 12 h p.i. and continued thereafter. Gene Ontology and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway annotation of computationally predicted targets of the reported miRNAs suggested a major impact of these miRNAs on cell signalling, protein translation and metabolic processes.

Molecular characterization of brinjal shoot and fruit borer, Leucinodes orbonalis (Guenée) (Lepidoptera: Crambidae) based on mitochondrial marker cytochrome oxidase I and their phylogenetic relationship.

Shoot and fruit borer, Leucinodes orbonalis is an important insect pest infesting brinjal or eggplant in India. Molecular characterization of nine different populations belonging to various brinjal growing regions was done using Cytochorome C Oxidase I (COI) gene. Nucleotide analysis of genetic diversity and phylogenetic analysis of the COI indicate that the L. orbonalis from different geographical regions are homogenous. The results showed less nucleotide diversity (π = 0.007895) and overall mean distance (0.008 ± 0.003). Topologies of neighbour-joining (NJ) trees indicate all the populations belong to single major clade. Therefore, it is inferred that there was no significant molecular diversity within L. orbonalis of different geographical locations of India with respect to COI.

Influence of experimental methods on the mechanical properties of silk fibers: A systematic literature review and future road map.

Spider silk fibers are of scientific and industrial interest because of their extraordinary mechanical properties. These properties are normally determined by tensile tests, but the values obtained are dependent on the morphology of the fibers, the test conditions, and the methods by which stress and strain are calculated. Because of this, results from many studies are not directly comparable, which has led to widespread misconceptions in the field. Here, we critically review most of the reports from the past 50 years on spider silk mechanical performance and use artificial spider silk and native silks as models to highlight the effect that different experimental setups have on the fibers' mechanical properties. The results clearly illustrate the importance of carefully evaluating the tensile test methods when comparing the results from different studies. Finally, we suggest a protocol for how to perform tensile tests on silk and biobased fibers.

Development of endosulfan tolerant strain of an egg parasitoid Trichogramma chilonis Ishii (Hymenoptera: Trichogrammatidae).

A strain of T. chilonis, an egg parasitoid of lepidopteran pests tolerant to the most commonly used cyclodiene insecticide--endosulfan was developed in the laboratory. Tolerance to endosulfan was induced by exposing adult parasitoids sequentially from a sub-lethal concentration (0.004%) to the field recommended concentration (0.09%). The strain acquired tolerance to the insecticide after 341 generation of continuous exposure with LC50 values of 1074.96 ppm as compared to LC50 of (70.91 ppm) in susceptible strain. The genetical study showed that F1 crosses exhibited a semi-dominant response to endosulfan with degree of dominance value (D) of 0.58. The resistant factor of tolerant strain was 15.1 folds and of F1 cross were 8.53 folds over susceptible strain. Under net house conditions, the tolerant strain parasitised 56% Helicoverpa armigera eggs on potted cotton plants immediately after an insecticide spray, compared to 3% by the susceptible strain. High percentage survival of the immature stages of the tolerant strain proved their ability to withstand the insecticide load. Breakdown of insecticide tolerance in the strain occurred after four generations in absence of insecticide load. Use of the tolerant strain as a component of bio-intensive IPM in various crops where insecticide use is higher is discussed.

Expression and purification of virus like particles (VLPs) of foot-and-mouth disease virus in Eri silkworm (Samia cynthia ricini) larvae.

Foot-and-mouth disease (FMD) is a highly contagious viral disease, which causes severe economic loss to livestock. Virus like particles (VLPs) produced by recombinant DNA technology are gaining importance because of their immunogenic properties and safety in developing a new vaccine for FMD. In the present study, a practical and economically feasible approach of expression, purification and characterization of VLPs of FMDV in Eri silkworm (Samia cynthia ricini) larvae was described. Although three lepidopteran insect larvae (Helicoverpa armigera, Spodoptera litura and Samia cynthia ricini) were tested for production of VLPs, expression was obtained only in Eri silkworm larvae. High titred recombinant baculovirus encoding the polyprotein P1-2A-3C of FMDV was prepared in Sf9 cells. Injection of recombinant baculovirus into hemocoel of Eri silkworm larvae resulted in increasing levels of expression of VLPs in the hemolymph from 3 to 7 days post infection (dpi) compared to low level expression by oral feeding. The VLPs reacted in Sandwich ELISA with serum raised against whole virus particles of FMDV type O/IND/R2/75 and protein banding pattern of 26, 37 and 47 kDa in Western blotting demonstrated their antigenic resemblance to native virus. Sucrose density gradient purified VLPs were used for immunization of rabbits and guinea pigs for assessing immunogenicity. Further, the reactivity of serum samples of rabbits and guinea pigs in Indirect-ELISA with titres (1.30-2.81 Log10) indicated that the VLPs were antigenic and immunogenic in nature. We demonstrate that Eri silkworm larvae could be used for production of VLPs of FMDV type O/IND/R2/75 for the first time. This approach could be useful for large scale production of recombinant VLPs for vaccine or diagnostic use in FMD control programme.

Molecular characterization of nucleopolyhedrovirus of three lepidopteran pests using late expression factor-8 gene.

A PCR based method for detection of viral DNA in nucleopolyhedrovirus of three lepidopterans, Spodoptera litura, Amsacta albistriga and Helicoverpa armigera, was developed by employing the late expression factor-8 (lef-8) gene of three NPV using specific primers. The amplicons of 689, 699 and 665 bp were amplified, respectively, and the nucleotide sequences were submitted to GenBank and the accession numbers were obtained. The sequences of lef-8 gene of S. litura NPV and H. armigera NPV matched with those of their respective references in the GenBank database, thereby confirming their identity, however, the sequence of A. albistriga NPV was the first sequence submitted to the GenBank database. The sequence similarity analysis between the three lef-8 gene of NPV sequenced in the present study revealed that there was no significant similarity between them, however A. albistriga NPV and S. litura NPV were found to be closely related. CLUSTAL alignment of the sequences generated revealed general relatedness among NPVs lef-8 gene. The study confirmed that lef-8 gene can be used for quick and correct discriminatory identification of insect viruses.

Identification and analysis of host plant resistance in leading maize genotypes against spotted stem borer, Chilo partellus (Swinhoe) (Lepidoptera: Pyralidae).

Twenty six popular varieties of maize in Karnataka state (India) were screened for resistance to Chilo partellus (Swinhoe). In field trial, varieties CM132, CM137 and PMZ103 showed the highest level of resistance, respectively. While the varieties GK3014, HY4642 and DK984 had the lowest level of resistance, respectively. In glasshouse trial, the varieties CM137 and HY4642 had the highest and lowest level of resistance. A positive correlation equal to +0.111 (in field) and +0.521 (in glasshouse) was found between the Leaf Injury Score (LIS) and Stem Diameter (SD). Meanwhile, negative correlation equal to -0.432 (in field) and -0.896 (in glasshouse) was found between Leaf Injury Score (LIS) and Internodal Distance (InD). Results of the studies on major nutritional elements on resistant variety CM137 and susceptible variety HY4642 indicated higher percentage of P, K, Fe and Si in stem tissues of the variety CM137, while the variety HY4642 had higher percentage of N and Sugar, highlighting the role of these elements in conferring resistance and susceptibility to C. partellus. Studies on the impact of resistance factors on biological events of C. partellus revealed significant differences between percentage survival of larvae, percentage of pupation, pupal weight and pupal period (females only) on two varieties CM137 and HY4642. But there was no significant difference in the percentage of moth emergence on two varieties.

Toxicity analysis of N- and C-terminus-deleted vegetative insecticidal protein from Bacillus thuringiensis.

A vegetative insecticidal protein (VIP)-encoding gene from a local isolate of Bacillus thuringiensis has been cloned, sequenced, and expressed in Escherichia coli. The expressed protein shows insecticidal activity against several lepidopteran pests but is ineffective against Agrotis ipsilon. Comparison of the amino acid sequence with those of reported VIPs revealed a few differences. Analysis of insecticidal activity with N- and C-terminus deletion mutants suggests a differential mode of action of VIP against different pests.