A common method for analysis of 17 amino acids from various insect species and plant parts was standardized using HPLC-PDA. Prior to hydrolysis, lyophilization of test samples was found indispensible to remove excess moisture, which interferes in hydrolysis and separation of amino acids. For the hydrolysis of plant and insect samples, 500 and 100 µL of boiling HCl, respectively, and 20 µL of hydrolyzed samples used for derivatization, provided best results. Gradient profile of mobile phase and run time up to 65 min were standardized to (i) encompass the problems related to eluting underivatized sample part, (ii) optimize the use of mobile phase and run time, and (iii) get better separation of different amino acids. Analysis of Chilo partellus larvae reared on sorghum seedling powder based artificial diet indicated that arginine and histidine quantities were on par in both samples. However, methionine was higher, and leucine, isoleucine, lysine, phenylalanine, threonine and valine were lower in sorghum seedlings than in C. partellus larvae, suggesting compensation of these amino acids by the insect through voracious feeding, as is being expected from artificial diet. This method was found highly sensitive, reproducible and useful for the analysis of amino acids for better understanding of insect-plant interactions.
The mode of inheritance of resistance to Bt toxins Cry1Ac and Cry2Ab was studied in the Pectinophora gossypiella (Saunders). Both strains, Jalg-R showing 605-fold resistance to Cry1Ac and another Jal-Sc showing 37.7-fold resistance to Cry2Ab were mated with susceptible insects to study inheritance of Cry1Ac and Cry2Ab resistance. The resistance in both cases was semi-dominant and autosomal in nature. Further, association of alkaline phosphatase with Cry2Ab resistance was studied. ALP ranged from 60-65 to 115-120 kDa in resistant strains and 60 to 105 kDa in susceptible strains. ALP activity was more in resistant insects (26.35 µM/min/µg) as compared to susceptible insects (6.45 µM/min/µg) and untreated control (9.95 µM/min/µg).
Chitin has to be degraded during the development of the insects periodically and hence chitinase plays an important role during the insect growth and development. Chitinase expression therefore coincides with the molting process and is developmentally regulated in insects. Putative chitinase genes from S. litura and H. armigera respectively were identified through genome-wide search. siRNAs were designed and synthesized from the EST region of chitinase gene of H. armigera as well as S. litura. Feeding and injection assay of SiRNA as well as dsRNA against neonates and 10 day old larvae respectively showed inter-molt mortality. This provides proof of applying SiRNA to knockdown of chitinase gene.
The effect of chitinase specific dsRNA was studied on larval growth and development of H. armigera and S. litura with feeding of neonates and last instar larvae @ 0.50 µg/g and 0.50 µg/insect, respectively. The mortality of larvae occurred during molting due to interference with chitinase that plays an important role prior to deposition of new cuticle. The phenotypic effects involved larval-larval, larval-pupal, pupal-adult deformities. Further studies also showed interference with fecundity and fertility in eggs of next generation. RT-PCR studies showed inhibition of chitinase. Expression profiling of chitinase gene on 1.2% agarose gel (Fig.3) showed an enormous reduction in levels of chitinase transcripts in dsRNA treated samples as compared to chitinase transcripts in untreated control, internal andnon-target siRNA control (ApSuc2). Besides that decrease in titres was also detected in malformed pupae, malformed adults as well as in next generation (1st instar larvae) showing later on persistent effect of siRNA and dsRNA treatment after feeding for four days in neonates and one time single dose feeding in last instar of both the test insects.
Generic identities of about 150 gut bacterial isolates from three lepidopteran insects H. armigera, Plutella xylostella and Antherarea assamensis were established by using 16s rRNA probes. The analysis revealed the presence of diverse group of gut bacterial genera including Delftia, Stenotrophomonasp, Pseudomonas, Bacillus, Acetobacter, Acinetobacter, Gamma proteobacterium, Lysinibacillus and Enterobacter which have potential role in insecticide detoxification, nitrification, amide metabolism and other biological activity in insects. Metagenomic analysis of gut homogenates of these lepidopteran insect species led to the identification of about 5846 Operational Taxonomic Units (OTUs) in the three species. Alpha and beta diversities of gut bacterial strains were found to be significantly higher in P. xylostella in comparison to other two species.
The Gas Chromatography (GC) profiling of selected maize genotypes viz., CPM 2, CPM 8, CPM 13, CPM 15, and CPM 18 along with resistant check CML 334 vis-à-vis the C. partellus larvae fed on these genotypes for different fatty acids revealed that the contents of myristic acid, palmitic acid, margaric acid, linoleic acid, stearic acid, methyl 11-eicosenoate, and eicosanoic acid were comparatively lower in the test maize genotypes as compared to the C. partellus larvae fed on them. However reverse was the trend for methyl 3-methoxy tetradecanoate. Furthermore, the fatty acids viz., cinnamic, linolenic, bhenic, and lignoceric acids were present in the maize seedlings, while absent from the C. partellus larvae fed on them. Conversely, the fatty acids viz., palmitoleic acid, methyl 14-methyl hexadecanoate, oleic acid and erucic acid in spite of absence from the maize seedlings were found present in the C. partellus larvae.