Jep Lokesh

Transcriptional regulation of cytokines in the intestine of Atlantic cod fed yeast derived mannan oligosaccharide or β-Glucan and challenged with Vibrio anguillarum

Immunomodulatory feed additives are expected to exert their primary influence at the intestinal level through the expression of cytokines, which in turn affect the immune responses in fish. In two separate experiments a yeast-derived mannan oligosaccharide product (YM) or a purified β-glucan (BG) product were fed to Atlantic cod (Gadus morhua L.) for 5 weeks, after which they were bath-challenged with a bacterial pathogen--Vibrio anguillarum. The transcription of selected cytokines (proinflammatory--il1b, il8, ifng; anti-inflammatory--il10) in different intestinal segments was analysed using qPCR. In the case of YM study, the effect of the compound was observed in both the posterior intestine and rectum of Atlantic cod, upon challenge with the pathogen. iIl1b expression in the posterior intestine and rectum of post-challenge fish was significantly higher than that of pre-challenge fish. In the case of il8 the difference was confined to rectum. The expression of ifng was altered only in the anterior intestine upon YM feeding. In the BG trial, the additive had a differential effect on the expression of the cytokine genes. In anterior intestine and rectum, the purified β-glucan additive significantly elevated the expression of il1b when challenged with V. anguillarum. An effect of BG on the anti-inflammatory cytokine il10 was visible in the rectum after the pathogen challenge. The differential responses of cytokines in the intestine of fish upon exposure to V. anguillarum suggest that both mannan oligosaccharides and β-glucans impact the ability of Atlantic cod to respond to the pathogen.

Differentially expressed proteins in the skin mucus of Atlantic cod ( Gadus morhua ) upon natural infection with Vibrio anguillarum

BackgroundVibriosis caused by V. anguillarum is a commonly encountered disease in Atlantic cod farms and several studies indicate that the initiation of infection occurs after the attachment of the pathogen to the mucosal surfaces (gut, skin and gills) of fish. Therefore it is necessary to investigate the role of different mucosal components in fish upon V. anguillarum infection. The present study has two parts; in the first part we analyzed the differential expression of skin mucus proteins from Atlantic cod naturally infected with V. anguillarum using two dimensional gel electrophoresis coupled with mass spectrometry. In the second part, a separate bath challenge experiment with V. anguillarum was conducted to assess the mRNA levels of the genes in skin tissue, corresponding to the selected proteins identified in the first part.ResultsComparative proteome analysis of skin mucus of cod upon natural infection with V. anguillarum revealed key immune relevant proteins like calpain small subunit 1, glutathione-S-transferase omega 1, proteasome 26S subunit, 14-kDa apolipoprotein, beta 2-tubulin, cold inducible RNA binding protein, malate dehydrogenase 2 (mitochondrial) and type II keratin that exhibited significant differential expression. Additionally a number of protein spots which showed large variability amongst individual fish were also identified. Some of the proteins identified were mapped to the immunologically relevant JNK (c-Jun N-terminal kinases) signalling pathway that is connected to cellular events associated with pathogenesis. A bath challenge experiment with V. anguillarum showed differential expression of beta 2-tubulin, calpain small subunit 1, cold inducible RNA binding protein, flotillin1, and glutathione S-transferase omega 1 transcripts in the skin tissue of cod during early stages of infection.ConclusionsDifferentially expressed proteins identified in the cod skin mucus point towards their possible involvement in V. anguillarum pathogenesis. The role of some of these proteins in vibriosis in cod described in this paper can be considered unconventional with respect to their established functions in higher vertebrates. Based on the differential expression of these proteins they are possibly important components of fish defence against bacteria and innate immunity at large. The feasibility of utilizing these proteins/genes as markers of bacterial infection or stress in cod needs to be explored further.

Transition from freshwater to seawater reshapes the skin-associated microbiota of Atlantic salmon

Knowledge concerning shifts in microbiota is important in order to elucidate the perturbations in the mucosal barrier during the transitional life stages of the host. In the present study, a 16S rRNA gene sequencing technique was employed to examine the compositional changes and presumptive functions of the skin-associated bacterial communities of Atlantic salmon reared under controlled laboratory conditions and transferred from freshwater to seawater. Proteobacteria was the dominant phylum in salmon from both freshwater (45%) and seawater (above 89%). Bacteroidetes, Actinobacteria, Firmicutes, Cyanobacteria and Verrucomicrobia were the most abundant phyla in salmon from freshwater. The transition to seawater influenced the OTU richness and evenness. The high abundance (~62%) of the genus Oleispira made Proteobacteria the most significantly abundant phylum in salmon from seawater. The predictive functional profile suggested that the communities had the ability to extract energy from amino acids in order to maintain their metabolism and scavenge and biosynthesise compounds to make structural changes and carry out signalling for their survival. These findings need to be further explored in relation to metabolic processes, the fish genotype, and the environment.

A novel beta-defensin antimicrobial peptide in Atlantic cod with stimulatory effect on phagocytic activity.

A novel defensin antimicrobial peptide gene was identified in Atlantic cod, Gadus morhua. This three exon/two intron defensin gene codes for a peptide precursor consisting of two domains: a signal peptide of 26 amino acids and a mature peptide of 40 residues. The mature cod defensin has six conserved cysteine residues that form 1–5, 2–4 and 3–6 disulphide bridges. This pattern is typical of beta-defensins and this gene was therefore named cod beta-defensin (defb). The tertiary structure of Defb exhibits an α/β fold with one α helix and β1β2β3 sheets. RT-PCR analysis indicated that defb transcripts were present mainly in the swim bladder and peritoneum wall but could also be detected at moderate to low levels in skin, head- and excretory kidneys. In situ hybridisation revealed that defb was specifically expressed by cells located in the swim bladder submucosa and the oocytes. During embryonic development, defb gene transcripts were detectable from the golden eye stage onwards and their expression was restricted to the swim bladder and retina. Defb was differentially expressed in several tissues following antigenic challenge with Vibrio anguillarum, being up-regulated up to 25-fold in head kidney. Recombinant Defb displayed antibacterial activity, with a minimal inhibitory concentration of 0.4–0.8 µM and 25–50 µM against the Gram-(+) bacteria Planococcus citreus and Micrococcus luteus, respectively. In addition, Defb stimulated phagocytic activity of cod head kidney leucocytes in vitro. These findings imply that beta-defensins may play an important role in the innate immune response of Atlantic cod.

A Microbial Feed Additive Abates Intestinal Inflammation in Atlantic Salmon

The efficacy of a microbial feed additive (Bactocell®) in countering intestinal inflammation in Atlantic salmon was examined in this study. Fish were fed either the additive-coated feed (probiotic) or feed without it (control). After an initial 3-week feeding, an inflammatory condition was induced by anally intubating all the fish with oxazolone. The fish were offered the feeds for 3 more weeks. Distal intestine from the groups was obtained at 4 h, 24 h, and 3 weeks, after oxazolone treatment. Inflammatory responses were prominent in both groups at 24 h, documented by changes in intestinal micromorphology, expression of inflammation-related genes, and intestinal proteome. The control group was characterized by edema, widening of intestinal villi and lamina propria, infiltration of granulocytes and lymphocytes, and higher expression of genes related to inflammatory responses, mul1b, il1b, tnfa, ifng, compared to the probiotic group or other time points of the control group. Further, the protein expression in the probiotic group at 24 h after inducing inflammation revealed five differentially regulated proteins – Calr, Psma5, Trp1, Ctsb, and Naga. At 3 weeks after intubation, the inflammatory responses subsided in the probiotic group. The findings provide evidence that the microbial additive contributes to intestinal homeostasis in Atlantic salmon.

Recognition of purified beta 1,3/1,6 glucan and molecular signalling in the intestine of Atlantic salmon.

Atlantic salmon was orally intubated with a highly purified β-glucan product (MacroGard(®)) to study the recognition of the molecule by the receptor genes, the regulation of the downstream signalling genes and global proteins, and the micromorphological changes in the intestine. The β-glucan receptor genes of Atlantic salmon, sclra, sclrb, sclrc and cr3, seem to recognize the molecule, and initiate the downstream ITAM-motif signalling, as evident from the significantly high mRNA levels of ksyk, mapkin2, il1b and mip2a levels. Among the altered proteins, the Apoa4 (involved in carbohydrate and lipid metabolism); Tagln, Actb (uptake of β-glucan); Psma2 (associated with substrate recognition); and Ckt (energy metabolism-related) were the overexpressed ones. The underexpressed proteins included the Uk114, Rpl9, Ctsb and Lgal that are connected to proliferation, LPS-stimulation, Il1b and lactose recognition, respectively. Furthermore, the mRNA levels of igt and the number of immune cells in the distal intestine were found to increase upon β-glucan uptake by the fish. This study provides some clues on the mechanisms by which the β-glucan evokes response in Atlantic salmon, particularly at the intestinal level.

Succession of embryonic and the intestinal bacterial communities of Atlantic salmon (Salmo salar) reveals stage-specific microbial signatures

Host‐associated microbiota undergoes a continuous transition, from the birth to adulthood of the host. These developmental stage‐related transitions could lead to specific microbial signatures that could impact the host biological processes. In this study, the succession of early‐life and intestinal bacterial communities of Atlantic salmon (starting from embryonic stages to 80‐week post hatch; wph) was studied using amplicon sequencing of 16S rRNA. Stage‐specific bacterial community compositions and the progressive transitions of the communities were evident in both the early life and the intestine. The embryonic communities showed lower richness and diversity (Shannon and PD whole tree) compared to the hatchlings. A marked transition of the intestinal communities also occurred during the development; Proteobacteria were dominant in the early stages (both embryonic and intestinal), though the abundant genera under this phylum were stage‐specific. Firmicutes were the most abundant group in the intestine of late freshwater; Weissella being the dominant genus at 20 wph and Anaerofilum at 62 wph. Proteobacteria regained its dominance after the fish entered seawater. Furthermore, LEfSe analysis identified genera under the above ‐ mentioned phyla that are significant features of specific stages. The environmental (water) bacterial community was significantly different from that of the fish, indicating that the host is a determinant of microbial assemblage. Overall the study demonstrated the community dynamics during the development of Atlantic salmon.

The Intestinal Mycobiota in Wild Zebrafish Comprises Mainly Dothideomycetes While Saccharomycetes Predominate in Their Laboratory-Reared Counterparts

As an integral part of the resident microbial community of fish intestinal tract, the mycobiota is expected to play important roles in health and disease resistance of the host. The composition of the diverse fungal communities, which colonize the intestine, is greatly influenced by the host, their diet and geographic origin. Studies of fungal communities are rare and the majority of previous studies have relied on culture-based methods. In particular, fungal communities in fish are also poorly characterized. The aim of this study was to provide an in-depth overview of the intestinal mycobiota in a model fish species (zebrafish, Danio rerio) and to determine differences in fungal composition between wild and captive specimens. We have profiled the intestinal mycobiota of wild-caught (Sharavati River, India), laboratory-reared (Bodø, Norway) and wild-caught-laboratory-kept (Uttara, India) zebrafish by sequencing the fungal internal transcribed spacer 2 region on the Illumina MiSeq platform. Wild fish were exposed to variable environmental factors, whereas both laboratory groups were kept in controlled conditions. There were also differences in husbandry practices at Bodø and Uttara, particularly diet. Zebrafish from Bodø were reared in the laboratory for over 10 generations, while wild-caught-laboratory-kept fish from Uttara were housed in the laboratory for only 2 months before sample collection. The intestine of zebrafish contained members of more than 15 fungal classes belonging to the phyla Ascomycota, Basidiomycota, and Zygomycota. Fungal species richness and diversity distinguished the wild-caught and laboratory-reared zebrafish communities. Wild-caught zebrafish-associated mycobiota comprised mainly Dothideomycetes in contrast to their Saccharomycetes-dominated laboratory-reared counterparts. The predominant Saccharomycetes in laboratory-reared fish belonged to the saprotrophic guild. Another characteristic feature of laboratory-reared fish was the significantly higher abundance of Cryptococcus (Tremellomycetes) compared to wild fish. This pioneer study has shed light into the differences in the intestinal fungal communities of wild-caught and laboratory-reared zebrafish and the baseline data generated will enrich our knowledge on fish mycobiota.