Project description:Background: Germ-free or axenic organisms are valuable tools for studying immunity, digestion, and development in different hosts. Although most of these studies have been conducted on mice, recently, germ-free invertebrate models (e.g. Drosophila and Apis) are used due to their easy husbandry, low cost for production, maintenance and the high number of individuals per generation they produce. However, a limitation of using these insects is the simple bacterial community present in their guts. The gut of the American cockroach Periplaneta americana displays a complex gut bacterial community composed of hundreds of species. Using P. americana, we developed a germ-free omnivorous invertebrate model to investigate how gut bacteria stimulate and shape normal gut development and metabolism. To determine if the insect host is directly affected by the presence of specific members of their bacterial community, gnotobiotic cockroaches were generated by inoculating a set of various P. americana gut-endemic Gram-negative (Bacteroidetes; n=11) and Gram-positive (Firmicutes; n=2) bacterial strains into germ-free insects. Additionally, we were able to recover the ‘normal’ bacterial-induced gut phenotype by co-housing germ-free cockroaches with wildtype P. americana to produce gut-bacteria conventionalized insects. Changes in gene expression profiles from two distinct regions (midgut and hindgut) of P. americana guts were quantified by RNA-Seq analysis of the germfree, gnotobiotic and conventionalized insects. Basic transcriptomics description: High-resolution transcriptome profiling of germ-free, gnotobiotic, and conventionalized treated P. americana midgut and hindguts. Ca. 43 million reads were obtained for each treatment. A de-novo assembly of all sequence reads was performed by Trinity assembler. Transcriptome assembly yielded 369,082 gene models and 554,155 isoforms. After running Trinotate pipeline, 65,047 (12 %) these transcripts matched an annotated product in at least one of the reference databases used (Uniprot, pfam, KEGG, COG). Additionally, 1,008 putative bacterial genes were annotated in the P. americana genome and ultimately excluded from these analyses. After bacteria decontamination, 553,147 assembled isoforms were used for transcript quantification and differential expression analysis using the DESeq2 pipeline. DESeq2 analysis detected 6,730 and 3,958 differentially expressed transcripts among the germ-free, gnotobiotic and conventionalized treatments in P. americana hindgut and midgut, respectively.
Project description:Streptococcal disease results in major mortality events of both marine and freshwater fishes worldwide. Streptococcus iniae is among the prominent causative bacterial strains as it has been found to cause a higher incidence of mortality and act as a zoonotic pathogen. Here we examine the susceptibility of two important aquaculture species in the United States, striped bass (Morone saxatilis) and white bass (Morone chrysops), to S. iniae. A high incidence of mortality was observed in both species, although striped bass succumbed more rapidly than white bass. Spleen gene expression at three time points following infection was analyzed to further elucidate the mechanisms underlying these observations. The down-regulation of gene transcripts associated with pathogen detection (tlr1, tlr8, tlr9), antigen processing (cd74a), immune cell recruitment and migration (ccl44, ccr6b, ccr7), macrophage function (csf1ra), T-cell signaling and NF-kB activation (card11, fyna, tirap) was detected in both species. These findings potentially indicate impairment in these critical early immune system processes such that both species were ultimately highly susceptible to S. iniae infection despite the detected up-regulation of transcripts typically associated with effective immune response, such as cytokines (il1β, il8, il12b2, il17rc, tnfb) and hepcidins (hamp, hamp2). The presented results collectively identify several candidate genes and associated pathways for further investigation to characterize the vulnerability of striped bass and white bass to S. iniae and that may be considered for selective breeding efforts, biotechnological intervention, and/or exploitation in the development of vaccines and alternative treatments.