Identification of the odorant binding proteins of Western Flower Thrips (Frankliniella occidentalis), characterization and binding analysis of FoccOBP3 with molecular modelling, molecular dynamics simulations and a confirmatory field trial.

Olfactory systems are indispensable for insects as they, including Western Flower Thrips (Frankliniella occidentalis), use olfactory cues for ovipositing and feeding. F. occidentalis use odorant binding proteins (OBPs) to transport semiochemicals to odorant receptors to induce a behavioural response from the sensillum lymph of the insect's antennae. This study identifies four OBPs of F. occidentalis and analyses their expression at three stages of growth: larvae, adult males and adult females. Further, it investigates the presence of conserved motifs and their phylogenetic relationship to other insect species. Moreover, FoccOBP3 was in silico characterized to analyse its structure along with molecular docking and molecular dynamics simulations to understand its binding with semiochemicals of F. occidentalis. Molecular docking revealed the interactions of methyl isonicotinate, p-anisaldehyde and (S)-(-)-verbenone with FoccOBP3. Moreover, molecular dynamics simulations showed bonding stability of these ligands with FoccOBP3, and field trials validated that Lurem TR (commercial product) and p-anisaldehyde had greater attraction as compared to (S)-(-)-verbenone, given the compound's binding with FoccOBP3. The current study helps in understanding the tertiary structure and interaction of FoccOBP3 with lures using computational and field data and will help in the identification of novel lures of insects in the future, given the importance of binding with OBPs.Communicated by Ramaswamy H. Sarma.

Odorant Binding Proteins (OBPs) and Odorant Receptors (ORs) of Anopheles stephensi: Identification and comparative insights.

Anopheles stephensi is an important vector of malaria in the South Asia, the Middle East, and Eastern Africa. The olfactory system of An. stephensi plays an important role in host-seeking, oviposition, and feeding. Odorant binding proteins (OBPs) are globular proteins that play a pivotal role in insect olfaction by transporting semiochemicals through the sensillum lymph to odorant receptors (ORs). Custom motifs designed from annotated OBPs of Aedes aegypti, Drosophila melanogaster, and Anopheles gambiae were used for the identification of putative OBPs from protein sequences of the An. stephensi Indian strain. Further, BLASTp was also performed to identify missing OBPs and ORs. Subsequently, the presence of domains common to OBPs was confirmed. Identified OBPs were further classified into three sub-classes. Phylogenetic and syntenic analyses were carried out to find homology, and thus the evolutionary relationship between An. stephensi OBPs and ORs with those of An. gambiae, Ae. aegypti and D. melanogaster. Gene structure and physicochemical properties of the OBPs and ORs were also predicted. A total of 44 OBPs and 45 ORs were predicted from the protein sequences of An. stephensi. OBPs were further classified into the classic (27), atypical (10) and plus-C (7) OBP subclasses. The phylogeny revealed close relationship of An. stephensi OBPs and ORs with An. gambiae homologs whereas only five OBPs and two ORs of An. stephensi were related to Ae. aegypti OBPs and ORs, respectively. However, D. melanogaster OBPs and ORs were distantly rooted. Synteny analyses showed the presence of collinear block between the OBPs and ORs of An. stephensi and An. gambiae as well as Ae. aegypti's. No homology was found with D. melanogaster OBPs and ORs. As an important component of the olfactory system, correctly identifying a species' OBPs and ORs provide a valuable resource for downstream translational research that will ultimately aim to better control the malaria vector An. stephensi.

Design, synthesis, and delivery studies of organotin(IV) based HCV inhibitor.

Tributylstannic[3-(3,5 -dimethylphenylamido)propionate] is synthesized and characterized by elemental analysis, FT-IR, multinuclear NMR ((1)H, (13)C and (119)Sn) and mass spectrometry. The organic anion was found to act as monodentate O-bound ligand in solution. The compound was screened for the anti-HCV potency by the Gaussia luciferase Assay using infected Huh 7.5 cells (human hepatocellular cell) and is found active against HCV with logIC50 1.2nM in the cell-based assay. Cationic surfactant cetyl N,N,N-trimethylammoniumbromide (CTAB) was used to study the interactions of the organotin(IV) complex with positively charged micelles of the surfactant acting as a model cell membrane. The thermodynamics parameters of complex- CTAB interaction concluded that the complex is located in the palisade layer of CTAB micelles. The increase in absorbance of visible spectra of the compound confirmed its solubilization into micelles. The two carbonyl oxygen's were found to be binding sites of the complex with CTAB.

Repeated in vitro subculturing alters spore surface properties and virulence of Metarhizium anisopliae.

Repeated subculturing caused rapid changes in the spore surface properties and virulence of Metarhizium anisopliae. Of the two strains evaluated, M. anisopliae V245 attenuated more rapidly than V275. Electrophoretic mobility and Radial Flow Chamber assays were used for the first time to generate qualitative and quantitative information on the adhesive forces of M. anisopliae conidia. Independent of strain, adhesion, hydrophobicity and spore-bound Pr1 declined after the first subculture; however, spore surface charge decline was erratic. Adhesion and hydrophobicity stabilized after the third subculture, whereas spore-bound Pr1 continues to decline following repeated subculturing. Decline in spore bound Pr1 was directly correlated with decline in virulence, however, such correlation with adhesion, hydrophobicity or surface charge could not be established. Because spore-bound Pr1 activities were directly correlated with M. anisopliae virulence; it could be used as a quality-control marker to monitor changes in virulence.

Effect of nutrition on growth and virulence of the entomopathogenic fungus Beauveria bassiana.

Three isolates of the entomopathogen Beauveria bassiana along with one strain of Metarhizium anisopliae were cultured on seven media with different carbon/nitrogen (C/N) ratios. The effect of nutrition on virulence of the isolates was evaluated via measurement of colony growth, spore yield, germination speed, conidial C/N ratio and Pr1 (a serine protease) activity. 'Osmotic stress' medium produced the lowest colony growth with low numbers of conidia in all isolates. However, these conidia showed a high germination rate and virulence. However, conidial Pr1 activity was low in some isolates. In most but not in all cases conidia from 1% yeast extract, 2% peptone and low (10 : 1) C/N medium had higher Pr1 activity compared with conidia from other media. However, in some instances we could not conclude that there was a relationship among germination rate, conidial Pr1 activity and virulence. C/N ratio of conidia was statistically different among various media and fungal isolates. Conidia with lower C/N ratio generally produced lower LT(50) (lowest median lethal time) values (more virulent). Insect-passaged conidia from different media had lower C/N ratio compared with similar conidia from artificial cultures. Therefore, they should be more virulent than in vitro produced conidia. As germination rate, conidial Pr1 activity and C/N ratio are independent of host, it seems that host-related determinants such as insect cuticle and physiology and environmental conditions may influence host susceptibility and therefore fungal isolate virulence towards host insects.

Nutrition influences growth and virulence of the insect-pathogenic fungus Metarhizium anisopliae.

Nutrition influenced growth, sporulation and virulence of the insect pathogenic fungus, Metarhizium anisopliae. Virulent conidia were produced on susceptible insect hosts, 1% yeast extract, 2% peptone, osmotic stress medium (OSM) and CN 10:1 medium. Several strain independent markers were identified that could be used to predict the virulence of M. anisopliae conidia. Virulent conidia typically had high levels of spore bound Pr1, an important cuticle degrading protease, and high germination rates. We also show for the first time that virulent conidia have an endogenous CN ratio below 5.2:1. Real Time PCR revealed that virulent conidia from insects contained significantly higher levels of transcripts of pr1 A and other pathogenicity-related genes than inoculum from artificial media. Of the artificial media studied, 1% yeast extract medium yielded the most virulent conidia, these had higher levels of transcripts of these pathogenicity-related genes than the least virulent conidia from the high conidia yielding CN 35:1 medium (=SDA), however, the levels were significantly lower than those in insect-derived conidia. Our study shows for the first time that the passaged inoculum is virulent irrespective of the original culture medium or insect host. Virulent conidia were consistently produced on OSM even though growth and sporulation were poor. We postulate that starvation conditions, whether in vivo or in vitro, results in de-repression of Pr1 and that elevated levels of this enzyme enhance fungal virulence.

Influence of nutrition on the production and physiology of sectors produced by the insect pathogenic fungus Metarhizium anisopliae.

Metarhizium anisopliae strains V245 and V275 differed in their stability when grown on different nutrient media. V275 produced fewer sectors than V245 irrespective of the cultural conditions. Both strains produced more sectors on nutrient rich media. At least four distinct types of sectors were produced in vitro. Most sectors were sterile or sporulated poorly and produced significantly lower quantities of virulence determining enzymes like Pr1. Real-time PCR confirmed differential expression of the pathogenicity-related genes pr1 A, ste 1, try 1, and chy 1 encoding for the subtilisin Pr1A, esterase, trypsin and chymotrypsin, respectively. API-ZYM revealed that the enzyme profiles of sectors differed from those of the parent cultures and also from other sectors. Sectors of M. anisopliae also produced less destruxins than the parent cultures independent of the strain.

Molecular investigation on strain genetic relatedness and population structure of Beauveria bassiana.

Triplicate molecular methods, i.e. polymerase chain reaction-restriction fragment length polymorphism of the pr1 gene, microsatellite markers and 28S rDNA haplotyping by detecting the presence or absence of group I introns, were used for population study of the entomopathogenic fungus, Beauveria bassiana. The findings showed that the average genetic diversity index of geographical populations was significantly smaller than that of populations derived from insect host orders, indicating that the genetic relatedness of B. bassiana strains was highly associated with geographical locality rather than insect host species. The reproductive style of all the B. bassiana populations was found to be non-clonal. Population structure analysis revealed that the average divergent coefficient among populations of B. bassiana was far below 1 (0.1112), which indicated that there was no significant genetic differentiation between populations, and that the overall genetic diversity mainly resulted from the genetic variations within geographical populations. Statistically, genetic distances between populations were positively correlated with geographical distances, suggesting that geographical separation poses an obstacle to the possibility and frequency of genetic exchanges between populations. On the other hand, gene flow was indirectly established to occur between B. bassiana populations.