A proteomic view on the developmental transfer of homologous 30 kDa lipoproteins from peripheral fat body to perivisceral fat body via hemolymph in silkworm, Bombyx mori

<p>Abstract</p> <p>Background</p> <p>A group of abundant proteins of ~30 kDa is synthesized in silkworm larval peripheral fat body (PPFB) tissues and transported into the open circulatory system (hemolymph) in a time-depended fashion to be eventually stored as granules in the pupal perivisceral fat body (PVFB) tissues for adult development during the non-feeding stage. These proteins have been shown to act anti-apoptotic besides being assigned roles in embryogenesis and defense. However, detailed protein structural information for individual PPFB and PVFB tissues during larval and pupal developmental stages is still missing. Gel electrophoresis and chromatography were used to separate the 30 kDa proteins from both PPFB and PVFB as well as hemolymph total proteomes. Mass spectrometry (MS) was employed to elucidate individual protein sequences. Furthermore, 30 kDa proteins were purified and biochemically characterized.</p> <p>Results</p> <p>One- and two-dimensional gel electrophoresis (1/2D-PAGE) was used to visualize the relative changes of abundance of the 30 kDa proteins in PPFB and PVFB as well as hemolymph from day 1 of V instar larval stage to day 6 of pupal stage. Their concentrations were markedly increased in hemolymph and PVFB up to the first two days of pupal development and these proteins were consumed during development of the adult insect. Typically, three protein bands were observed (~29, 30, 31 kDa) in 1D-PAGE, which were subjected to MS-based protein identification along with spots excised from 2D-gels run for those proteomes. Gas phase fragmentation was used to generate peptide sequence information, which was matched to the available nucleotide data pool of more than ten highly homologous insect 30 kDa lipoproteins. Phylogenetic and similarity analyses of those sequences were performed to assist in the assignment of experimentally identified peptides to known sequences. Lipoproteins LP1 to LP5 and L301/302 could be matched to peptides extracted from all bands suggesting the presence of full length and truncated or modified protein forms in all of them. The individual variants could not be easily separated by classical means of purification such as 2D-PAGE because of their high similarity. They even seemed to aggregate as was indicated by native gel electrophoresis. Multistep chromatographic procedures eventually allowed purification of an LP3-like protein. The protein responded to lipoprotein-specific staining.</p> <p>Conclusions</p> <p>In <it>B. mori </it>larvae and pupae, 30 kDa lipoproteins LP1 to LP5 and L301/302 were detected in PPFB and PVFB tissue as well as in hemolymph. The concentration of these proteins changed progressively during development from their synthesis in PPFB, transport in hemolymph to storage in PVFB. While the 30 kDa proteins could be reproducibly separated in three bands electrophoretically, the exact nature of the individual protein forms present in those bands remained partially ambiguous. The amino acid sequences of all known 30 kDa proteins showed very high homology. High-resolution separation techniques will be necessary before MS and other structural analysis can shed more light on the complexity of the 30 kDa subproteome in <it>B. mori</it>. A first attempt to that end allowed isolation of a <it>B. mori </it>LP3-like protein, the complete structure, properties and function of which will now be elucidated in detail.</p

Life Cycle and Secondary Production of Four Species from Functional Feeding Groups in a Tropical Stream of South India

This study focused on life strategies of species from functional feeding groups (FFGs) found in a tropical stream of the Sirumalai hills, South India. We examined the life cycle and secondary production of species of shredders (Lepidostoma nuburagangai), scrapers (Baetis sp.), collectors (Choroterpes alagarensis), and predators (Neoperla biseriata). In addition, we studied the assemblage structure of functional feeding groups. We found the collectors occupied the highest percentage, followed in turn by scrapers, predators, and shredders. The diversity of FFGs was higher at riffle areas and assemblage with stream substrates differing in each functional group. An asynchronous life cycle was observed for Baetis, C. alagarensis, and N. biseriata, while L. nuburagangai was found in four to five generations per year. We acquired data on secondary production of scraper species of Baetis, which reached the highest values among all investigated species. This observation stresses the importance of scrapers as playing a key role in converting coarse particulate organic matter to fine particulate organic matter with low or high abundances of shredder population and maintaining the food chain in tropical streams

Assessment of electron beam irradiation induced proteomic changes and its effect on the development of silkworm, Bombyx mori (Bombycidae: Lepidoptera)

The present study has been designed to determine the hemolymph protein changes induced by EBI using Microtron (ranges from 20 to 100 Gy) on the 5th instar larvae of silkworm, Bombyx mori using SDS–PAGE. The EBI did not caused any impact on 5th instar larval hemolymph proteins, however a significant reduction of pupal hemolymph proteins such as lipophorin (250 kDa), vitellogenin (180 kDa), storage protein (76–80 kDa) and a 30 kDa protein was observed through SDS–PAGE and densitometry analysis. These proteins are known to play a crucial role in various developmental processes including transport of lipids, as amino acid reservoir for providing precursors for egg and cuticle formation and immunity of B. mori. Further, a decrease of antioxidant enzymes such as Superoxide Dismutase (SOD) and Catalase (CAT) in the EBI treated larval and pupal hemolymph was observed. In addition, a negative influence on growth characteristics and appearance of pupal deformity was noted. This may be due to damage in hemolymph proteins, when the larvae were exposed to EBI at >80 Gy

Species diversity of black flies (Diptera: Simuliidae) in Oriental region and molecular phylogeny of the subgenus Gomphostilbia members

Background & objectives: Black flies (Diptera: Simuliidae) are ecologically and medically important insects. Female adults of black flies are the solitary vectors of river blindness (onchocerciasis) and their larvae play a vital role in stream ecosystem. This study examined the distribution of black flies in the Oriental region and analyzed the phylogenetic relationship of the subgenus Gomphostilbia members based on two molecular loci. Methods: The distribution data of black fly species in different countries of Oriental region were obtained from world black flies geographic inventory. The two gene sequences, COI and ITS1 were used to study the phylogenetic relationships of the members of subgenus Gomphostilbia members. Results: The distribution analysis revealed that out of the 16 subgenera in the genus Simulium Latreille s., the species-level diversity of three subgenera (Gomphostilbia, Nevermannia and Simulium) contributes about thrice of total black fly species diversity. The highest diversity of species was found in the subgenus Simulium. The strict consensus of Tree analysis using New Technology (TNT) and Maximum Likelihood (ML) recovered similar topologies for Gomphostilbia members and they formed as monophyly. The overall sequence identities of the 19 species of subgenus Gomphostilbia were high and shared 55–60% similarity. Interpretation & conclusion: Results of this study highlighted that eight subgenera of Simulium Latreille s. str are commonly distributed in different parts of Oriental region. Among these the subgenera of Simulium, Gomphostilbia and Nevermannia are most common with high diversity in China, Pakistan, Thailand and Vietnam. The phylogenetic analysis of Gomphostilbia members demonstrates the inter-specific divergence, indicating the centre of origin (India) or the recipient of ancestral migrant lineages in Oriental region

A new species of Simulium (Gomphostilbia) (Diptera: Simuliidae) from South India

Anbalagan, Sankarappan, Vijayan, Suruliyandi, Dinakaran, Sundaram, Krishnan, Muthukalingan (2019): A new species of Simulium (Gomphostilbia) (Diptera: Simuliidae) from South India. Zootaxa 4551 (4): 479-486, DOI: https://doi.org/10.11646/zootaxa.4551.4.

Two new species of Simulium (Gomphostilbia) (Diptera: Simuliidae) from Peninsular India with keys to Peninsular Indian members of the genus Simulium

Anbalagan, Sankarappan, Prasanna, Vimalanathan Arun, Dinakaran, Sundaram, Krishnan, Muthukalingan (2014): Two new species of Simulium (Gomphostilbia) (Diptera: Simuliidae) from Peninsular India with keys to Peninsular Indian members of the genus Simulium. Zootaxa 3861 (5): 451-465, DOI: 10.11646/zootaxa.3861.5.

Phylogeographical Structure in Mitochondrial DNA of Legume Pod Borer (Maruca vitrata) Population in Tropical Asia and Sub-Saharan Africa.

This study was undertaken to assess the genetic diversity and host plant races of M. vitrata population in South and Southeast Asia and sub-Saharan Africa. The cytochrome c oxidase subunit 1 (cox1) gene was used to understand the phylogenetic relationship of geographically different M. vitrata population, but previous studies did not include population from Southeast Asia, the probable center of origin for Maruca, and from east Africa. Extensive sampling was done from different host plant species in target countries. Reference populations from Oceania and Latin America were used. An amplicon of 658 bp was produced by polymerase chain reaction, and 64 haplotypes were identified in 686 M. vitrata individuals. Phylogenetic analysis showed no difference among the M. vitrata population from different host plants. However, the results suggested that M. vitrata has formed two putative subspecies (which cannot be differentiated based on morphological characters) in Asia and sub-Saharan Africa, as indicated by the high pairwise FST values (0.44-0.85). The extremely high FST values (≥ 0.93) of Maruca population in Latin America and Oceania compared to Asian and African population seem to indicate a different species. On the continental or larger geographical region basis, the genetic differentiation is significantly correlated with the geographical distance. In addition, two putative species of Maruca, including M. vitrata occur in Australia, Indonesia and Papua New Guinea. The negative Tajima's D and Fu's FS values showed the recent demographic expansion of Maruca population. The haplotype network and Automatic Barcode Gap Discovery analyses confirmed the results of phylogenetic analysis. Thus, this study confirmed the presence of three putative Maruca species, including one in Latin America, one in Oceania (including Indonesia) and M. vitrata in Asia, Africa and Oceania. Hence, the genetic differences in Maruca population should be carefully considered while designing the pest management strategies in different regions

Use of Random Amplified Polymorphic DNA (RAPD) to study genetic diversity within a population of blackfly, Simulium gravelyi from Palni hills, peninsular India

Abstract: The genetic variation of local subpopulations (Palni hills, India) of the blackfly Simulium gravelyi was surveyed at different altitudinal locations using random amplified polymorphic DNA (RAPD) analysis. By comparing the similarity of the bands produced by RAPDs, it may be concluded that there is variation within this population. These data suggest that the heterogeneity of band pattern of S. gravelyi steadily increased from low elevation to high elevation. Site 5 (1590 m altitude) was more polymorphic and it differed most from site 1 (290 m). This is established by the ecological diversity and statistical analyses (PCA) and predicting two major ecological factors of conductivity and current velocity. The speciation hypothesis is consistent with the molecular evidence, postulates a high elevational site 5 (1590 m) and a subsequent site 4 (1250 m)

A new black fly species of Simulium (Nevermannia) (Simuliidae: Diptera) from the Southern Eastern Ghats, India

Anbalagan, Sankarappan, Rekha, Kalimuthu, Vijayan, Suruliyandi, Balachandran, Chellapandian, Dinakaran, Sundaram, Krishnan, Muthukalingan (2020): A new black fly species of Simulium (Nevermannia) (Simuliidae: Diptera) from the Southern Eastern Ghats, India. Zootaxa 4768 (3): 374-382, DOI: https://doi.org/10.11646/zootaxa.4768.3.