Temperature-based phenology model for predicting the present and future establishment and distribution of recently invasive Spodoptera frugiperda (J. E. Smith) in India.

Fall armyworm, Spodoptera frugiperda (J. E. Smith) is a polyphagous and highly destructive invasive insect pest of many crops. It was recently introduced into India and widely reported in almost all parts of India. Development of a temperature-based phenology model for predicting its rate of development and distribution will help in understanding the establishment and further spread of introduced invasive insect pests. Development, survival and reproduction parameters of S. frugiperda at six constant temperature conditions (15, 20, 25, 27, 30 and 35°C) were investigated and further validated with data generated under fluctuating temperature conditions. The estimated lower developmental threshold temperatures were 12.1°C for eggs, 11°C for larvae, 12.2°C for pupae, 15.13°C for males and 12.66°C for females. Degree-day (DD) requirements for the development of the different stages of S. frugiperda were 50, 250 and 200 DD for egg, larva and pupa, respectively. The best-fitted functions were compiled for each life stage to yield a phenology model, which was stochastically simulated to estimate the life table parameters. The developed phenology model predicted temperature ranges between 27 and 30°C as favourable for S. frugiperda development, survival and reproduction. The results revealed that maximum net reproductive rate (215.66 females/female/generation) and total fecundity (981.08 individuals/female/generation) were attained at 30°C constant temperature. The mean length of generations decreased from 74.29 days at 15°C to 38.74 days at 30°C. The maximum intrinsic rate of increase (0.138 females/female/day) and shortest doubling time (4.9 days) were also observed at 30°C. Results of simulated life table parameters showed high temperature-dependent development of S. frugiperda and complete development within all the tested constant temperature ranges (15-35°C). Simulated life table parameters for predicting risk indices of S. frugiperda in India indicated a significant increase in activity indices and establishment risk indices with a higher number of generations during future (2050 and 2070) climatic change scenarios compared to present conditions. Our results indicate that India will be highly suitable for the establishment and survival of S. frugiperda in future time periods.

Heavy Metals Scavenging Potential of Trichoderma asperellum and Hypocrea nigricans Isolated from Acid Soil of Jharkhand.

Trichoderma asperellum (NAIMCC-F-03167) and Hypocrea nigricans (NAIMCC-F-03168) were isolated from the acidic soil of the vicinity of Litchi orchard, Ranchi, Jharkhand and were characterized on the basis of morphological, molecular and biochemical features. Both strains are fast growing, light to dark green, highly sporulative and have ability to cover 90 mm Petri dish within 96 h of inoculation. Biochemcial estimation of both isolates indicated significant cellulase and phosphate solubilisation activity. Highest cellulase activity was observed in T. asperellum (5.63 cm) followed by H. nigricans (5.10 cm) and phosphate solubilisation index was observed maximum in T. asperellum (1.93) followed by H. nigricans (1.39). Moreover, these isolates were molecularly identified on the basis of ribosomal DNA based sequences database and phylogenetic analysis in NCBI GenBank as T. asperellum (NCBI-KM 438015) and H. nigricans (NCBI-KJ910335). Negetive effect on sporulation of Lead (Pb) and Cadmium (Cd) was observed while in heavy metal scavenging potential, T. asperellum (88.9% Cd) showed highest scavenging potential followed by H. nigricans (87.2% Cd) while in Pb scavenging potential, H. nigricans (88% Pb) followed highest scavenging potential followed by T. asperellum (81.30% Pb) after 21 days of inoculation from 30 µg/ml heavy metals concentrated broth medium. If both potential bioagents can apply in Cd and Pb affected soil/water will be helpful in scavenging of heavy metals as well as management of phosphorus deficiency and soilborne fungal diseases.

Population genetic structure of cotton pink bollworm, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae) using mitochondrial cytochrome oxidase I (COI) gene sequences from India.

Pink bollworm (PBW), Pectinophora gossypiella is one of the most destructive pest's globally inflicting huge economic losses in cotton even during later stages of crop growth. In the present investigation, the population genetic structure, distribution, and genetic diversity of P. gossypiella in cotton growing zones of India using partial mitochondrial DNA cytochrome oxidase-I (COI) gene was addressed. The overall haplotype (Hd), number of nucleotide differences (K), and nucleotide diversity (π) were 0.3028, 0.327, and 0.00047, respectively which suggest that entire population exhibited low level of genetic diversity. Zone-wise clustering of population revealed that central zone recorded low level of Hd (0.2730) as compared to north (0.3619) and south (0.3028) zones. The most common haplotype (H1) reported in all 19 locations could be proposed as ancestral/original haplotype. This haplotype with one mutational step formed star-like phylogeny connected with 11 other haplotypes. The phylogenetic relationship studies revealed that most haplotypes of populations are closely related to each other. Haplotype 5 was exclusively present in Dharwad (South zone) shared with populations of Hanumangarh and Bathinda (North zone). The result indicated that there is no isolation by distance effect among the Indian populations of PBW. The present study reports a low genetic diversity among PBW populations of India and H1, as ancestral haplotype from which other haplotypes have evolved suggests that the migration and dispersal over long distance and invasiveness are major factors.

SynGAP isoforms exert opposing effects on synaptic strength.

Alternative promoter usage and alternative splicing enable diversification of the transcriptome. Here we demonstrate that the function of Synaptic GTPase-Activating Protein (SynGAP), a key synaptic protein, is determined by the combination of its amino-terminal sequence with its carboxy-terminal sequence. 5' rapid amplification of cDNA ends and primer extension show that different N-terminal protein sequences arise through alternative promoter usage that are regulated by synaptic activity and postnatal age. Heterogeneity in C-terminal protein sequence arises through alternative splicing. Overexpression of SynGAP α1 versus α2 C-termini-containing proteins in hippocampal neurons has opposing effects on synaptic strength, decreasing and increasing miniature excitatory synaptic currents amplitude/frequency, respectively. The magnitude of this C-terminal-dependent effect is modulated by the N-terminal peptide sequence. This is the first demonstration that activity-dependent alternative promoter usage can change the function of a synaptic protein at excitatory synapses. Furthermore, the direction and degree of synaptic modulation exerted by different protein isoforms from a single gene locus is dependent on the combination of differential promoter usage and alternative splicing.

Interrogating the human genome using uninterpreted mass spectrometry data.

The public availability of a draft assembly of the human genome has enabled us to demonstrate, for the first time, the feasibility of searching a complete, unmasked eukaryotic genome using uninterpreted mass spectrometry data. A complex LC-MS/MS data set, containing peptides from at least 22 human proteins, was searched against a comprehensive, nonidentical protein database, an expressed sequence tag (EST) database, and the International Human Genome Project draft assembly of the human genome. The results from the three searches are compared in detail, and the merits of the different databases for this application are discussed. In the case of the EST database, the UniGene index provided a method of simplifying and summarising the search results. In the case of the genomic DNA, the presence of introns prevented matching of roughly one quarter of the spectra, but the technique can provide primary experimental verification of predicted coding sequences, and has the potential to identify novel coding sequences.

Matching peptide mass spectra to EST and genomic DNA databases.

The use of mass spectrometry data to search molecular sequence databases is a well-established method for protein identification. The technique can be extended to searching raw genomic sequences, providing experimental confirmation or correction of predicted coding sequences, and has the potential to identify novel genes and elucidate splicing patterns.

Proteomics characterization of abundant Golgi membrane proteins.

A mass spectrometric analysis of proteins partitioning into Triton X-114 from purified hepatic Golgi apparatus (84% purity by morphometry, 122-fold enrichment over the homogenate for the Golgi marker galactosyl transferase) led to the unambiguous identification of 81 proteins including a novel Golgi-associated protein of 34 kDa (GPP34). The membrane protein complement was resolved by SDS-polyacrylamide gel electrophoresis and subjected to a hierarchical approach using delayed extraction matrix-assisted laser desorption ionization mass spectrometry characterization by peptide mass fingerprinting, tandem mass spectrometry to generate sequence tags, and Edman sequencing of proteins. Major membrane proteins corresponded to known Golgi residents, a Golgi lectin, anterograde cargo, and an abundance of trafficking proteins including KDEL receptors, p24 family members, SNAREs, Rabs, a single ARF-guanine nucleotide exchange factor, and two SCAMPs. Analytical fractionation and gold immunolabeling of proteins in the purified Golgi fraction were used to assess the intra-Golgi and total cellular distribution of GPP34, two SNAREs, SCAMPs, and the trafficking proteins GBF1, BAP31, and alpha(2)P24 identified by the proteomics approach as well as the endoplasmic reticulum contaminant calnexin. Although GPP34 has never previously been identified as a protein, the localization of GPP34 to the Golgi complex, the conservation of GPP34 from yeast to humans, and the cytosolically exposed location of GPP34 predict a role for a novel coat protein in Golgi trafficking.

Proteomic analysis of NMDA receptor-adhesion protein signaling complexes.

N-methyl-d-aspartate receptors (NMDAR) mediate long-lasting changes in synapse strength via downstream signaling pathways. We report proteomic characterization with mass spectrometry and immunoblotting of NMDAR multiprotein complexes (NRC) isolated from mouse brain. The NRC comprised 77 proteins organized into receptor, adaptor, signaling, cytoskeletal and novel proteins, of which 30 are implicated from binding studies and another 19 participate in NMDAR signaling. NMDAR and metabotropic glutamate receptor subtypes were linked to cadherins and L1 cell-adhesion molecules in complexes lacking AMPA receptors. These neurotransmitter-adhesion receptor complexes were bound to kinases, phosphatases, GTPase-activating proteins and Ras with effectors including MAPK pathway components. Several proteins were encoded by activity-dependent genes. Genetic or pharmacological interference with 15 NRC proteins impairs learning and with 22 proteins alters synaptic plasticity in rodents. Mutations in three human genes (NF1, Rsk-2, L1) are associated with learning impairments, indicating the NRC also participates in human cognition.

Applications of protein mass spectrometry in cell biology.

Advances in mass spectrometry combined with accelerated progress in genome sequencing projects have facilitated the rapid identification of proteins by enzymatic digestion, mass analysis, and sequence database searching. Applications for this technology range from the surveillance of protein expression in cells, tissues, and whole organisms, to the identification of proteins and posttranslational modifications. Here we consider practical aspects of the application of mass spectrometry in cell biology and illustrate these with examples from our own laboratories.