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

BACKGROUND: Vibriosis 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. RESULTS: Comparative 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. CONCLUSIONS: Differentially 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

Chronic wounds alter the proteome profile in skin mucus of farmed gilthead seabream

Abstract Background Skin and its mucus are known to be the first barrier of defence against any external stressors. In fish, skin wounds frequently appear as a result of intensive culture and also some diseases have skin ulcers as external clinical signs. However, there is no information about the changes produced by the wounds in the mucosae. In the present paper, we have studied the alterations in the proteome map of skin mucus of gilthead seabream during healing of experimentally produced chronic wounds by 2-DE followed by LC-MS/MS. The corresponding gene expression changes of some identified skin proteins were also investigated through qPCR. Results Our study has successfully identified 21 differentially expressed proteins involved in immunity and stress processes as well as other metabolic and structural proteins and revealed, for the first time, that all are downregulated in the skin mucus of wounded seabream specimens. At transcript level, we found that four of nine markers (ighm, gst3, actb and krt1) were downregulated after causing the wounds while the rest of them remained unaltered in the wounded fish. Finally, ELISA analysis revealed that IgM levels were significantly lower in wounded fish compared to the control fish. Conclusions Our study revealed a decreased-expression at protein and for some transcripts at mRNA levels in wounded fish, which could affect the functionality of these molecules, and therefore, delay the wound healing process and increase the susceptibility to any infection after wounds in the skin of gilthead seabream

Ubiquitous presence of piscidin-1 in Atlantic cod as evidenced by immunolocalisation

Abstract Background Antimicrobial peptides (AMPs), the natural antibiotics bestowed upon all forms of life, consist of small molecular weight proteins with a broad spectrum antimicrobial activity against a variety of pathogenic microorganisms. Piscidins are one of the AMP families that are imperative for the innate defence mechanisms of teleosts. Atlantic cod, a basal fish belonging to the superorder Paracanthopterygii also possesses multiple piscidin peptides. Two piscidin paralogues (pis1 and pis2) and a novel alternative splice variant of pis2 of this fish were previously described by us. To shed light on other potent roles of these molecules, now we have mapped the distribution of piscidin 1 (Pis1), in different tissues and organs of cod through immunohistochemistry (IHC) employing an affinity purified polyclonal antibody specific to Pis1. Results Various cell types and tissues of Atlantic cod including those from the immune organs of naïve fish are armed with Pis1 peptide. Different types of the blood leucocytes and phagocytic cells among the leucocytes examined gave a relatively strong indication of Pis1 immunopositivity. In addition, other cell types such as hematopoietic cells, epithelial cells and multi-granular cells located in the mucosal and hematopoietic tissues were also Pis1-immunoreactive. More interestingly, chondrocytes appear to produce Pis1 and this is the first report on the presence of an AMP in cartilage tissue of fish. Furthermore, Pis1 immunopositivity was detected in other tissues and organs of naïve fish including neural tissues, exocrine and endocrine glands, compound gland cells, excretory kidney, intestinal and respiratory epithelial cells, swim bladder, skin and hypodermis layer, myosepta, liver, heart, eye and oocytes. Conclusions Pis1 peptide is produced by various cell types located in different tissues and organs of Atlantic cod. It is present in all immune-related organs of naïve fish and the elevated peptide expression following phagocytosis strongly suggest their involvement in innate defence. Further, its widespread occurrence in non-immune tissues and organs of apparently healthy fish implies that piscidin may have other functions in addition to its role as an immune effector molecule.</p

The concept map as a substitute for lectures: Effects on student performance and mental health

AbstractIn comparison with traditional lectures, active learning methods have the potential to foster learner agency and consequently raise academic performance. However, these methods are often applied with little knowledge about their effectiveness. In this study, we introduced two concept maps in an undergraduate cell biology course, to investigate their effect on student performance, but also mental health, in a quasi-experimental setup. By implementing group tasks into the work, we aimed to reduce feelings of loneliness among the participants, a common phenomenon among students in the study country. We evaluated the effects of the concept maps via exam grades and in an anonymous student survey. Here, we found no increase in student performance after introduction of the active learning task. Students also did not report improvements in their feelings of loneliness. We conclude that this active learning method did not detectably improve student performance or mental health, a result which does not conform to the popular perception of positive effects from active learning. The survey revealed that students do not have a preference for either active learning or traditional lectures, however, the students experienced that the concept map helped them to describe, explain and hypothesize about the topics. Overall, this shows that the concept map method is a valid alternative to traditional lectures, since students perform equally between both learning approaches

Mucosal barrier status in Atlantic salmon fed rapeseed oil and Schizochytrium oil partly or fully replacing fish oil through winter depression

The study was designed to investigate the effects of replacing fish oil by algal oil and rapeseed oil on histomorphology indices of the intestine, skin and gill, mucosal barrier status and immune-related genes of mucin and antimicrobial peptide (AMP) genes in Atlantic salmon (Salmo salar). For these purposes, Atlantic salmon smolts were fed three different diets. The first was a control diet containing fish oil but no Schizochytrium oil. In the second diet, almost 50% of the fish oil was replaced with algal oil, and in the third diet, fish oil was replaced entirely with algal oil. The algal oil contained mostly docosahexaenoic acid (DHA) and some eicosapentaenoic acid (EPA). The study lasted for 49 days in freshwater (FW), after which some fish from each diet group were transferred to seawater (SW) for a 48-hour challenge test at 33 ppt to test their ability to tolerate high salinity. Samples of skin, gills, and mid intestine [both distal (DI) and anterior (AI) portions of the mid intestine] were collected after the feeding trial in FW and after the SW-challenge test to assess the effects of the diets on the structure and immune functions of the mucosal surfaces. The results showed that the 50% VMO (Veramaris® algal oil) dietary group had improved intestinal, skin, and gill structures. Principal component analysis (PCA) of the histomorphological parameters demonstrated a significant effect of the algal oil on the intestine, skin, and gills. In particular, the mucosal barrier function of the intestine, skin, and gills was enhanced in the VMO 50% dietary group after the SW challenge, as evidenced by increased mucous cell density. Immunolabelling of heat shock protein 70 (HSP70) in the intestine (both DI and AI) revealed downregulation of the protein expression in the 50% VMO group and a corresponding upregulation in the 100% VMO group compared to 0% VMO. The reactivity of HSP70 in the epithelial cells was higher after the SW challenge compared to the FW phase. Immune-related genes related to mucosal defense, such as mucin genes [muc2, muc5ac1 (DI), muc5ac1 (AI), muc5ac2, muc5b (skin), and muc5ac1 (gills)], and antimicrobial peptide genes [def3 (DI), def3 (AI), and cath1 (skin)] were significantly upregulated in the 50% VMO group. PCA of gene expression demonstrated the positive influences on gene regulation in the 50% VMO dietary group. In conclusion, this study demonstrated the positive effect of substituting 50% of fish oil with algal oil in the diets of Atlantic salmon. The findings of histomorphometry, mucosal mapping, immunohistochemistry, and immune-related genes connected to mucosal responses all support this conclusion.publishedVersio

A Novel Beta-Defensin Antimicrobial Peptide in Atlantic Cod with Stimulatory Effect on Phagocytic Activity

<div><p>A novel defensin antimicrobial peptide gene was identified in Atlantic cod, <i>Gadus morhua</i>. 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 (<i>defb</i>). 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 <i>situ</i> hybridisation revealed that <i>defb</i> was specifically expressed by cells located in the swim bladder submucosa and the oocytes. During embryonic development, <i>defb</i> gene transcripts were detectable from the golden eye stage onwards and their expression was restricted to the swim bladder and retina. <i>Defb</i> was differentially expressed in several tissues following antigenic challenge with <i>Vibrio anguillarum</i>, 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 <i>Planococcus citreus</i> and <i>Micrococcus luteus</i>, respectively. In addition, Defb stimulated phagocytic activity of cod head kidney leucocytes <i>in</i><i>vitro</i>. These findings imply that beta-defensins may play an important role in the innate immune response of Atlantic cod.</p></div

Three dimensional structures of cod Defb (A), crotamine (B), mBD8 (C) and zebrafish Defb1 (D).

<p>The presence of an α-helix (red), three antiparallel β-strands (blue), and the disulphide linkages of 1–5, 2–4 and 3–6 pattern (yellow) were found in all species. Homology modelling of cod Defb was performed using the PBD structure of crotamine, a neurotoxin from rattlesnake, <i>Crotalus durissus trrificus</i> (PDB ID: 1H5O), and mouse beta-defensin 8 (mBD8, PDB ID: 1E4R) as templates.</p

List of primers employed and their application in this study.

<p>T = annealing temperature; E = qPCR efficiency.</p

Beta-defensins from teleost fish available at the NCBI repository.

a<p>Predicted from EST sequences.</p