Examining the Effectiveness of Discriminant Function Analysis and Cluster Analysis in Species Identification of Male Field Crickets Based on Their Calling Songs

Traditional taxonomy based on morphology has often failed in accurate species identification owing to the occurrence of cryptic species, which are reproductively isolated but morphologically identical. Molecular data have thus been used to complement morphology in species identification. The sexual advertisement calls in several groups of acoustically communicating animals are species-specific and can thus complement molecular data as non-invasive tools for identification. Several statistical tools and automated identifier algorithms have been used to investigate the efficiency of acoustic signals in species identification. Despite a plethora of such methods, there is a general lack of knowledge regarding the appropriate usage of these methods in specific taxa. In this study, we investigated the performance of two commonly used statistical methods, discriminant function analysis (DFA) and cluster analysis, in identification and classification based on acoustic signals of field cricket species belonging to the subfamily Gryllinae. Using a comparative approach we evaluated the optimal number of species and calling song characteristics for both the methods that lead to most accurate classification and identification. The accuracy of classification using DFA was high and was not affected by the number of taxa used. However, a constraint in using discriminant function analysis is the need for a priori classification of songs. Accuracy of classification using cluster analysis, which does not require a priori knowledge, was maximum for 6-7 taxa and decreased significantly when more than ten taxa were analysed together. We also investigated the efficacy of two novel derived acoustic features in improving the accuracy of identification. Our results show that DFA is a reliable statistical tool for species identification using acoustic signals. Our results also show that cluster analysis of acoustic signals in crickets works effectively for species classification and identification.

Psychoacoustic sampling as a reliable, non-invasive method to monitor orthopteran species diversity in tropical forests

We evaluated trained listener-based acoustic sampling as a reliable and non-invasive method for rapid assessment of ensiferan species diversity in tropical evergreen forests. This was done by evaluating the reliability of identification of species and numbers of calling individuals using psychoacoustic experiments in the laboratory and by comparing psychoacoustic sampling in the field with ambient noise recordings made at the same time. The reliability of correct species identification by the trained listener was 100% for 16 out of 20 species tested in the laboratory. The reliability of identifying the numbers of individuals correctly was 100% for 13 out of 20 species. The human listener performed slightly better than the instrument in detecting low frequency and broadband calls in the field, whereas the recorder detected high frequency calls with greater probability. To address the problem of pseudoreplication during spot sampling in the field, we monitored the movement of calling individuals using focal animal sampling. The average distance moved by calling individuals for 17 out of 20 species was less than 1.5 m in half an hour. We suggest that trained listener-based sampling is preferable for crickets and low frequency katydids, whereas broadband recorders are preferable for katydid species with high frequency calls for accurate estimation of ensiferan species richness and relative abundance in an area.

Azospirillum lipoferum tRNAs: Fractionation and identification of tRNA species and the nucleotide sequence of tRNA(Asn) (QUU)

Transfer RNAs of Azospirillum lipoferum were separated by two- dimensional gel electrophoresis and identified by aminoacylation. Thirty-six tRNA spots were resolved by this technique and twenty-six tRNA species have been identified. There are five tRNAs for Leu, four for Val, three for Pro, two each for Arg, Ile, Lys and Tyr, and one each for Ala, Asp, His, Phe, Ser and Thr. The tRNA(Asn) (QUU) was purified and its nucleotide sequence was determined. The A. lipoferum tRNA(Asn) (QUU) is 92% similar to B. subtilis tRNA(Asn) gene and two hypermodified nucleosides, queuosine (Q) and N-(9-beta-D Ribofuranosylpurine-6-YL) carbamoyl)-threonine (t(6)A) are present in this tRNA.

Molecular Identification and Phylogeny of Bactrocera Species (Diptera: Tephritidae)

Fruit flies that belong to the genus Bactrocera (Diptera: Tephritidae) are major invasive pests of agricultural crops in Asia and Australia. Increased transboundary movement of agricultural produce has resulted in the chance introduction of many invasive species that include Bactrocera mainly as immature stages. Therefore quick and accurate species diagnosis is important at the port of entry, where morphological identification has a limited role, as it requires the presence of adult specimens and the availability of a specialist. Unfortunately when only immature stages are present, a lacunae in their taxonomy impedes accurate species diagnosis. At this juncture, molecular species diagnostics based on COX-I have become handy, because diagnosis is not limited by developmental stages. Yet another method of quick and accurate species diagnosis for Bactrocera spp. is based on the development of species-specific markers. This study evaluated the utility of COX-I for the quick and accurate species diagnosis of eggs, larvae, pupae and adults of B. zonata Saunders, B. tau Walker, and B. dorsalis Hendel. Furthermore the utility of species-specific markers in differentiating B. zonata (500bp) and B. tau (220bp) was shown. Phylogenetic relationships among five subgenera, viz., Austrodacus, Bactrocera, Daculus, Notodacus and Zeugodacus have been resolved employing the 5' region of COX-I (1490-2198); where COX-I sequences for B. dorsalis Hendel, B. tau Walker, B. correcta Bezzi and B. zonata Saunders from India were compared with other NCBI-GenBank accessions. Phylogenetic analysis employing Maximum Parsimony (MP) and Bayesian phylogenetic approach (BP) showed that the subgenus Bactrocera is monophyletic.

Evolution, Homology Conservation, and Identification of Unique Sequence Signatures in GH19 Family Chitinases

The discovery of GH (Glycoside Hydrolase) 19 chitinases in Streptomyces sp. raises the possibility of the presence of these proteins in other bacterial species, since they were initially thought to be confined to higher plants. The present study mainly concentrates on the phylogenetic distribution and homology conservation in GH19 family chitinases. Extensive database searches are performed to identify the presence of GH19 family chitinases in the three major super kingdoms of life. Multiple sequence alignment of all the identified GH19 chitinase family members resulted in the identification of globally conserved residues. We further identified conserved sequence motifs across the major sub groups within the family. Estimation of evolutionary distance between the various bacterial and plant chitinases are carried out to better understand the pattern of evolution. Our study also supports the horizontal gene transfer theory, which states that GH19 chitinase genes are transferred from higher plants to bacteria. Further, the present study sheds light on the phylogenetic distribution and identifies unique sequence signatures that define GH19 chitinase family of proteins. The identified motifs could be used as markers to delineate uncharacterized GH19 family chitinases. The estimation of evolutionary distance between chitinase identified in plants and bacteria shows that the flowering plants are more related to chitinase in actinobacteria than that of identified in purple bacteria. We propose a model to elucidate the natural history of GH19 family chitinases.

Identification of Specific DNA Binding Residues in the TCP Family of Transcription Factors in Arabidopsis

The TCP transcription factors control multiple developmental traits in diverse plant species. Members of this family share an similar to 60-residue-long TCP domain that binds to DNA. The TCP domain is predicted to form a basic helix-loop-helix ( bHLH) structure but shares little sequence similarity with canonical bHLH domain. This classifies the TCP domain as a novel class of DNA binding domain specific to the plant kingdom. Little is known about how the TCP domain interacts with its target DNA. We report biochemical characterization and DNA binding properties of a TCP member in Arabidopsis thaliana, TCP4. We have shown that the 58-residue domain of TCP4 is essential and sufficient for binding to DNA and possesses DNA binding parameters comparable to canonical bHLH proteins. Using a yeast-based random mutagenesis screen and site-directed mutants, we identified the residues important for DNA binding and dimer formation. Mutants defective in binding and dimerization failed to rescue the phenotype of an Arabidopsis line lacking the endogenous TCP4 activity. By combining structure prediction, functional characterization of the mutants, and molecular modeling, we suggest a possible DNA binding mechanism for this class of transcription factors.

Identification of corona discharge-induced SF6 oxidation mechanisms using SF6/18O2/H216O and SF6/16O2/H218O gas mixtures

The absolute yields of gaseous oxyfluorides SOF2, SO2F2, and SOF4 from negative, point-plane corona discharges in pressurized gas mixtures of SF6 with O2 and H2O enriched with18O2 and H2 18O have been measured using a gas chromatograph-mass spectrometer. The predominant SF6 oxidation mechanisms have been revealed from a determination of the relative18O and16O isotope content of the observed oxyfluoride by-product. The results are consistent with previously proposed production mechanisms and indicate that SOF2 and SO2F2 derive oxygen predominantly from H2O and O2, respectively, in slow, gas-phase reactions involving SF4, SF3, and SF2 that occur outside of the discharge region. The species SOF4 derives oxygen from both H2O and O2 through fast reactions in the active discharge region involving free radicals or ions such as OH and O, with SF5 and SF4.

Identification of tropomyosin as the major shrimp allergen and characterization of its IgE-binding epitopes

The major heat-stable shrimp allergen (designated as Sa-II), capable of provoking IgE-mediated immediate type hypersensitivity reactions after the ingestion of cooked shrimp, has been shown to be a 34-kDa heat- stable protein containing 300 amino acid residues. Here, we report that a comparison of amino acid sequences of different peptides generated by proteolysis of Sa-II revealed an 86% homology with tropomyosin from Drosophila melanogaster, suggesting that Sa-II could be the shrimp muscle protein tropomyosin. To establish that Sa-II is indeed tropomyosin, the latter was isolated from uncooked shrimp (Penaeus indicus) and its physicochemical and immunochemical properties were compared with those of Sa-II. Both tropomyosin and Sa-II had the same molecular mass and focused in the isoelectric pH range of 4.8 to 5.4. In the presence of 6 M urea, the mobility of both Sa-II and shrimp tropomyosin shifted to give an apparent molecular mass of 50 kDa, which is a characteristic property of tropomyosins. Shrimp tropomyosin bound to specific IgE antibodies in the sera of shrimp-sensitive patients as assessed by competitive ELISA inhibition and Western blot analysis. Tryptic maps of both Sa-II and tropomyosin as obtained by reverse phase HPLC were superimposable. Dot-blot and competitive ELISA inhibition using sera of shrimp-sensitive patients revealed that antigenic as well as allergenic activities were associated with two peptide fractions. These IgE-binding tryptic peptides were purified and sequenced. Mouse anti-anti-idiotypic antibodies raised against Sa-II specific human idiotypic antibodies recognized not only tropomyosin but also the two allergenic peptides, thus suggesting that these peptides represent the major IgE binding epitopes of tropomyosin. A comparison of the amino acid sequence of shrimp tropomyosin in the region of IgE binding epitopes (residues 50-66 and 153-161) with the corresponding regions of tropomyosins from different vertebrates confirmed lack of allergenic cross-reactivity between tropomyosins from phylogenetically distinct species.

Phage Displayed Short Peptides against Cells of Candida albicans Demonstrate Presence of Species, Morphology and Region Specific Carbohydrate Epitopes

Candida albicans is a commensal opportunistic pathogen, which can cause superficial infections as well as systemic infections in immuocompromised hosts. Among nosocomial fungal infections, infections by C. albicans are associated with highest mortality rates even though incidence of infections by other related species is on the rise world over. Since C. albicans and other Candida species differ in their susceptibility to antifungal drug treatment, it is crucial to accurately identify the species for effective drug treatment. Most diagnostic tests that differentiate between C. albicans and other Candida species are time consuming, as they necessarily involve laboratory culturing. Others, which employ highly sensitive PCR based technologies often, yield false positives which is equally dangerous since that leads to unnecessary antifungal treatment. This is the first report of phage display technology based identification of short peptide sequences that can distinguish C. albicans from other closely related species. The peptides also show high degree of specificity towards its different morphological forms. Using fluorescence microscopy, we show that the peptides bind on the surface of these cells and obtained clones that could even specifically bind to only specific regions of cells indicating restricted distribution of the epitopes. What was peculiar and interesting was that the epitopes were carbohydrate in nature. This gives insight into the complexity of the carbohydrate composition of fungal cell walls. In an ELISA format these peptides allow specific detection of relatively small numbers of C. albicans cells. Hence, if used in combination, such a test could help accurate diagnosis and allow physicians to initiate appropriate drug therapy on time.

Vitellins and vitellogenins of Dysdercus koenigii (Heteroptera : Pyrrhocoridae) - identification, purification and temporal pattern

Two vitellins, VtA and VtB, were purified from the eggs of Dysdercus koenigii by gel filtration and ion exchange chromatography. VtA and VtB have molecular weights of 290 and 260 kDa, respectively. Both Vts are glycolipoproteinaceous in nature. VtA is composed of three polypeptides of M-r 116, 92 and 62 kDa while VtB contained an additional subunit of M-r 40 kDa. All subunits except the 116-kDa subunit are glycolipopolypeptides. Polyclonal antibody raised against VtA (anti-VtA antibody) cross-reacted with VtB and also with vitellogenic haemolymph and ovaries and pre-vitellogenic fat bodies, but not with haemolymph from either adult male, fifth instar female, or pre-vitellogenic females demonstrating sex and stage specificity of the Vts. Immunoblots in the presence of anti-VtA revealed two proteins (of 290 and 260 kDa) in both vitellogenic haemolymph and pre-vitellogenic fat bodies that are recognised as D. koenigii Vgs. In newly emerged females, Vgs appeared on day 1 in fat bodies and on day 3 in haemolymph and ovaries. Vg concentration was maximum on day 2 in fat body, day 4 in haemolymph and day 7 in ovary. Although the biochemical and temporal characteristics of these proteins show similarity to some hemipterans, they are strikingly dissimilar with those of a very closely related species. (C) 1999 Elsevier Science Inc. All rights reserved.

Assessment of genetic diversity and identification of core collection in sandalwood germplasm using RAPDS

Sandalwood is an economically important aromatic tree belonging to the family Santalaceae. The trees are used mainly for their fragrant heartwood and oil that have immense potential for foreign exchange. Very little information is available on the genetic diversity in this species. Hence studies were initiated and genetic diversity estimated using RAPD markers in 51 genotypes of Santalum album procured from different geographcial regions of India and three exotic lines of S. spicatum from Australia. Eleven selected Operon primers (10mer) generated a total of 156 consistent and unambiguous amplification products ranging from 200bp to 4kb. Rare and genotype specific bands were identified which could be effectively used to distinguish the genotypes. Genetic relationships within the genotypes were evaluated by generating a dissimilarity matrix based on Ward's method (Squared Euclidean distance). The phenetic dendrogram and the Principal Component Analysis generated, separated the 51 Indian genotypes from the three Australian lines. The cluster analysis indicated that sandalwood germplasm within India constitutes a broad genetic base with values of genetic dissimilarity ranging from 15 to 91 %. A core collection of 21 selected individuals revealed the same diversity of the entire population. The results show that RAPD analysis is an efficient marker technology for estimating genetic diversity and relatedness, thereby enabling the formulation of appropriate strategies for conservation, germplasm management, and selection of diverse parents for sandalwood improvement programmes.

Identification and characterization of starvation induced msdgc-1 promoter involved in the c-di-GMP turnover

C-di-GMP Bis-(3'-5')-cyclic-dimeric-guanosine monophosphate], a second messenger is involved in intracellular communication in the bacterial species. As a result several multi-cellular behaviors in both Gram-positive and Gram-negative bacteria are directly linked to the intracellular level of c-di-GMP. The cellular concentration of c-di-GMP is maintained by two opposing activities, diguanylate cyclase (DGC) and phosphodiesterase (PDE-A). In Mycobacterium smegmatis, a single bifunctional protein MSDGC-1 is responsible for the cellular concentration of c-di-GMP. A better understanding of the regulation of c-di-GMP at the genetic level is necessary to control the function of above two activities. In this work, we have characterized the promoter element present in msdgc-1 along with the + 1 transcription start site and identified the sigma factors that regulate the transcription of msdgc-1. Interestingly, msdgc-1 utilizes SigA during the initial phase of growth, whereas near the stationary phase SigB containing RNA polymerase takes over the expression of msdgc-1. We report here that the promoter activity of msdgc-1 increases during starvation or depletion of carbon source like glucose or glycerol. When msdgc-1 is deleted, the numbers of viable cells are similar to 10 times higher in the stationary phase in comparison to that of the wild type. We propose here that msdgc-1 is involved in the regulation of cell population density. (C) 2013 Elsevier B.V. All rights reserved.

Genome-wide methylation profiling identifies an essential role of reactive oxygen species in pediatric glioblastoma multiforme and validates a methylome specific for H3 histone family 3A with absence of G-CIMP/isocitrate dehydrogenase 1 mutation

Background. Pediatric glioblastoma multiforme (GBM) is rare, and there is a single study, a seminal discovery showing association of histone H3.3 and isocitrate dehydrogenase (IDH) 1 mutation with a DNA methylation signature. The present study aims to validate these findings in an independent cohort of pediatric GBM, compare it with adult GBM, and evaluate the involvement of important functionally altered pathways. Methods. Genome-wide methylation profiling of 21 pediatric GBM cases was done and compared with adult GBM data (GSE22867). We performed gene mutation analysis of IDH1 and H3 histone family 3A (H3F3A), status evaluation of glioma cytosine-phosphate-guanine island methylator phenotype (G-CIMP), and Gene Ontology analysis. Experimental evaluation of reactive oxygen species (ROS) association was also done. Results. Distinct differences were noted between methylomes of pediatric and adult GBM. Pediatric GBM was characterized by 94 hypermethylated and 1206 hypomethylated cytosine-phosphate-guanine (CpG) islands, with 3 distinct clusters, having a trend to prognostic correlation. Interestingly, none of the pediatric GBM cases showed G-CIMP/IDH1 mutation. Gene Ontology analysis identified ROS association in pediatric GBM, which was experimentally validated. H3F3A mutants (36.4%; all K27M) harbored distinct methylomes and showed enrichment of processes related to neuronal development, differentiation, and cell-fate commitment. Conclusions. Our study confirms that pediatric GBM has a distinct methylome compared with that of adults. Presence of distinct clusters and an H3F3A mutation-specific methylome indicate existence of epigenetic subgroups within pediatric GBM. Absence of IDH1/G-CIMP status further indicates that findings in adult GBM cannot be simply extrapolated to pediatric GBM and that there is a strong need for identification of separate prognostic markers. A possible role of ROS in pediatric GBM pathogenesis is demonstrated for the first time and needs further evaluation.

Differences in host species relationships and biogeographic influences produce contrasting patterns of prevalence, community composition and genetic structure in two genera of avian malaria parasites in southern Melanesia

1. Host-parasite interactions have the potential to influence broadscale ecological and evolutionary processes, levels of endemism, divergence patterns and distributions in host populations. Understanding the mechanisms involved requires identification of the factors that shape parasite distribution and prevalence. 2. A lack of comparative information on community-level host-parasite associations limits our understanding of the role of parasites in host population divergence processes. Avian malaria (haemosporidian) parasites in bird communities offer a tractable model system to examine the potential for pathogens to influence evolutionary processes in natural host populations. 3. Using cytochrome b variation, we characterized phylogenetic diversity and prevalence of two genera of avian haemosporidian parasites, Plasmodium and Haemoproteus, and analysed biogeographic patterns of lineages across islands and avian hosts, in southern Melanesian bird communities to identify factors that explain patterns of infection. 4. Plasmodium spp. displayed isolation-by-distance effects, a significant amount of genetic variation distributed among islands but insignificant amounts among host species and families, and strong local island effects with respect to prevalence. Haemoproteus spp. did not display isolation-by-distance patterns, showed marked structuring of genetic variation among avian host species and families, and significant host species prevalence patterns. 5. These differences suggest that Plasmodium spp. infection patterns were shaped by geography and the abiotic environment, whereas Haemoproteus spp. infection patterns were shaped predominantly by host associations. Heterogeneity in the complement and prevalence of parasite lineages infecting local bird communities likely exposes host species to a mosaic of spatially divergent disease selection pressures across their naturally fragmented distributions in southern Melanesia. Host associations for Haemoproteus spp. indicate a capacity for the formation of locally co-adapted host-parasite relationships, a feature that may limit intraspecific gene flow or range expansions of closely related host species.