Predominant contribution of an endogenous cellulase (OlCel) to the cellulolysis in the digestive system of larvae of banana pseudostem weevil, Odoiporus longicollis (Coleoptera: Curculionidae).

Insects have evolved with effective strategies to utilize cellulose as an energy source by possessing cellulolytic enzymes which can be used as an optimal resource in the bioenergy sector. The study was aimed at evaluating the cellulolytic enzyme in the larval gut of the banana pseudostem weevil, Odoiporus longicollis Olivier (Coleoptera: Curculionidae). Primarily, cellulase activity was localized along the gut, in which the midgut showed the highest activity (2858 U/mg). The thermo-tolerance of cellulase activity was found to be up to 80°C (highest at 60°C), and the enzyme was stable at a pH between 5 and 6. Various concentrations of divalent cations (CaCl2 , MgCl2 , and CuCl2 ) have differential enhancing and inhibitory effects on cellulase activity. The cellulase (OlCel) was purified using anion exchange chromatography. The molecular weight of the cellulase was determined to be 47 kDa. The physicochemical parameters of the purified enzyme were similar to that of enzyme activity of whole gut extract. Mass spectrometry results identified sequence similarities of purified cellulase to the glycosyl hydrolase family 5 (GHF5) family. The gut microbial cellulase activity as exogenous source showed no competence compared with the endogenous activity.

Cloning, expression and characterization of arcelin and its impact on digestive enzymes of the stored product insect pest, Callosobruchus maculatus (F.).

The pulse beetle Callosobruchus maculatus causes potential damage to legume crops by infesting the seeds, leading to a reduction of total protein content. Arcelin found in the wild accessions of the common bean, is an insecticidal protein that has the potency to hamper the metabolism of the bruchid beetle. The arcelin gene from the wild accession of Phaseolus lunatus was isolated and the ORF encoding 158 amino acids was cloned in pET-45b (+) vector. The recombinant clones were transformed in BL21 STAR (DE3) pLysS cells, and the expressed arcelin was purified using Ni-NTA column. The recombinant protein was used in preparing an artificial diet, and the insecticidal activity was elucidated against the bruchid pest C. maculatus. Adult emergence and seed damage were drastically reduced in the treated groups. The response towards ingested diet by digestive enzymes involved in metabolism was elucidated through quantitative gene expression. The highest expression was observed in the aminopeptidase, followed by upregulation of alpha-amylase, glycoside hydrolase family 31 and cathepsin D-like aspartic protease, and downregulation of cathepsin L-like cysteine protease. The recombinant arcelin demonstrates effective insecticidal activity against the bruchid beetle. The changes in digestive enzymes to counteract the anti-nutritional nature of the protein were the strategies of the insect defense mechanism.

Molecular genotypic diversity of populations of brinjal shoot and fruit borer, Leucinodes orbonalis and development of SCAR marker for pesticide resistance.

BACKGROUND: The brinjal shoot and fruit borer, Leucinodes orbonalis is a destructive pest of Solanum melongena. The control of L. orbonalis with extensive application of synthetic chemical insecticides resulted in the development of resistance with known genetic heterogeneity among populations. Understanding the genetic diversity of their populations is important in developing strategies for their management. The present investigation was performed to characterize populations of L. orbonalis for their genetic diversity in the entire region of Tamil Nadu, South India using random amplified polymorphic DNA (RAPD) primers as a tool of the molecular marker. METHODS AND RESULTS: Among 60 random 10-mer primers, only ten primers generated reproducible and scorable banding profile. Among the ten different random primers, the primers namely OPG 7, OPG 8, OPS 2 and OPS 7 generated the highest genetic variation with over 80% genetic polymorphism. Phylogram analysis produced 18 clusters with eight major and ten minor clusters. Cluster analysis, statistical fitness, population structure and analysis of molecular variance confirmed the significant genetic variation among different populations. A trait specific marker obtained through RAPD was cloned, sequenced and used to develop a stable diagnostic SCAR marker for DNA fingerprinting to distinguish the populations. Amplification of this locus in the samples of 20 different populations indicated recognition of the trait for pesticide resistance in 12 populations. CONCLUSIONS: The results suggest that the biochemical nature of host plant varieties of this insect pest and variation in the application of different insecticides are essential contributing factors for the genotypic variations observed among populations of L. orbonalis.

Analysis on the arcelin expression in bruchid pest resistant wild pulses using real time RT-qPCR.

Arcelin, the antimetabolic protein from wild pulses is a known natural insecticidal molecule. Wild pulses with high arcelin content could serve as potential source to. increase the levels of insect resistance in cultivated pulse crops. In this study, arcelin (Arl) gene expression was screened in seven stored product insect pest resistant wild pulse varieties using real time RT-qPCR. Arcelin gene specific real time PCR primers were synthesized from arcelin mRNA sequence of the wild pulse variety, Lablab purpureus. The results revealed different levels of arcelin gene expression in the tested varieties. Canavalia virosa registered significantly high content indicating its suitability for utilization of arcelin gene in developing stored product insect pest resistance with other cultivated pulses.

In silico analysis of carbohydrate-binding pockets in the lectin genes from various species of Canavalia.

Legumes are endowed with an opulent class of proteins called lectins that can detect tenuous variations in carbohydrate structures and bind them reversibly with high affinity and specificity. The genus Canavalia, in the family of Leguminosae, is considered to be an affluent source of lectin. An effort has been made to analyse the sequences encoded by the lectin gene and its carbohydrate binding pockets from three species of Canavalia, including C. virosa, C. rosea, and C. pubescens. Crude seed extract showed highest haemagglutination titer against buffalo RBCs and has high affinity to mannose and trehalose. Amplification of the lectin gene by gene-specific primers showed the presence of an 870 bp amplicon. Physicochemical characterization using various bioinformatic tools showed that the isoelectric point was below 7, suggesting that lectin molecules were acidic. A high aliphatic index and high instability index were observed, which indicated that lectin molecules were stable towards a wide range of temperatures. The occurrence of N-glycosylation sites at two sites was also identified in all three species. Prediction of secondary structure showed that approximately 59.05 %, 56.76 % and 54.88 % of the elements were random coils in the case of C. virosa, C. pubescens and C. rosea, respectively. Comparative modelling of the proteins and docking of hypothetical models with sugar moieties that inhibited the agglutination activity suggested that asparagine, serine, alanine, valine, tyrosine and threonine were the major residues involved in hydrogen bonding and other stacking interactions. This can further provide insights on its prospective antibiosis property.