Project description:Spider silk research has largely focused on spidroins, proteins that are the primary components of spider silk fibers. Although a number of spidroins have been characterized, other types of proteins associated with silk synthesis are virtually unknown. Previous comparison of tissue-specific RNAseq libraries identified 647 predicted genes that were differentially expressed in silk glands of the Western black widow, Latrodectus hesperus. Only ~5% of these transcripts encode spidroins and the remaining predicted genes presumably encode other proteins associated with silk production. Here, we used proteomic analysis of multiple silk glands and dragline silk fiber to investigate the translation of the differentially expressed genes. We find 48 proteins encoded by the differentially expressed transcripts in L. hesperus major ampullate, minor ampullate, and tubuliform silk glands, and detect 16 SST encoded proteins in major ampullate silk fibers. The observed proteins include known silk-related proteins, but most are uncharacterized, with no annotation. These unannotated proteins likely include novel silk associated proteins. Major ampullate and minor ampullate glands have the highest overlap of identified proteins, consistent with their shared, distinctive ampullate shape and the overlapping functions of major ampullate and minor ampullate silks. Our study substantiates and prioritizes predictions from differential expression analysis of spider silk gland transcriptomes.
Project description:Spider silk research has largely focused on spidroins, proteins that are the primary components of spider silk fibers. Although a number of spidroins have been characterized, other types of proteins associated with silk synthesis are virtually unknown. Previous comparison of tissue-specific RNAseq libraries identified 647 predicted genes that were differentially expressed in silk glands of the Western black widow, Latrodectus hesperus. Only ~5% of these transcripts encode spidroins and the remaining predicted genes presumably encode other proteins associated with silk production. Here, we used proteomic analysis of multiple silk glands and dragline silk fiber to investigate the translation of the differentially expressed genes. We find 48 proteins encoded by the differentially expressed transcripts in L. hesperus major ampullate, minor ampullate, and tubuliform silk glands, and detect 16 SST encoded proteins in major ampullate silk fibers. The observed proteins include known silk-related proteins, but most are uncharacterized, with no annotation. These unannotated proteins likely include novel silk associated proteins. Major ampullate and minor ampullate glands have the highest overlap of identified proteins, consistent with their shared, distinctive ampullate shape and the overlapping functions of major ampullate and minor ampullate silks. Our study substantiates and prioritizes predictions from differential expression analysis of spider silk gland transcriptomes.
Project description:Although the salivary transcriptome of adult mosquitoes has been thoroughly described in several recent papers, very little information exists regarding the biological role of larval salivary glands in the Culicidae. We used whole-transcriptome Affymetrix® chips to compare the transcriptional profiles of Anopheles gambiae larval (L4) salivary glands and whole larvae. We identified a total of 277 transcripts as being significantly enriched in the salivary glands. Based on available annotation for the known or predicted protein sequences encoded by these transcripts, 41 were identified as corresponding to secreted proteins, 233 as corresponding to non-secreted (housekeeping) proteins, and 3 as encoding proteins of unknown function. Based on functional annotation of the putatively secreted gene products, we propose that larval salivary secretions have roles in nutrient digestion, detoxification, immunity, and mouthpart lubrication. Interestingly, several components of the larval saliva (e.g. apyrase and serine proteases) have also been reported to exist in adult female saliva, where they are though to help regulate a vertebrate hostâs immune response to bloodfeeding. In conclusion, our results suggest that the salivary glands are important components of both the digestive and immune systems of larval mosquitoes, and that their study might provide clues about the evolution of adaptations to bloodfeeding observed in adults. Experiment Overall Design: We attempted to identify transcripts that are both present and significantly enriched in the salivary glands (SG) of the 4th instar Anopheles gambiae larva. Transcripts were considered as present in the SG if the built-in Affymetrix detection call scored them as such in all three biological replicates of the experiment. Expression values (i.e. signal intensities) of transcripts from the salivary glands were compared to those of a matching group of whole 4th instar larvae using a t-test. Transcripts showing an expression value two-fold or higher (p < 0.01; false discovery rate=1%) in the SG as compared to whole larvae were considered to be significantly enriched.