Honggang Zhao

Physiology and immunology of mucosal barriers in catfish (Ictalurus spp.)

The mucosal barriers of catfish (Ictalurus spp) constitute the first line of defense against pathogen invasion while simultaneously carrying out a diverse array of other critical physiological processes, including nutrient adsorption, osmoregulation, waste excretion, and environmental sensing. Catfish depend more heavily on mucosal barriers than their terrestrial counterparts as they are continuously interacting with the aquatic microbiota. Our understanding of these barriers, while growing, is still limited relative to that of mammalian model systems. Nevertheless, a combination of molecular and cellular studies in catfish over the last few decades, and particularly within the last few years, has helped to elucidate many of the primary actors and pathways critical to their mucosal health. Here we describe aspects of innate and adaptive immune responses in the primary mucosal tissues (skin, gill, and intestine) of catfish, focusing on mucus-driven responses, pathogen recognition, soluble mediators, and immunoglobulin and T-cell derived immunity. Modulation of mucosal barriers will be critical moving forward for crafting better diets, improving vaccine delivery, enhancing water quality, and ensuring sustainable production practices in catfish.

Impact of feed additives on surface mucosal health and columnaris susceptibility in channel catfish fingerlings, Ictalurus punctatus

One of the highest priority areas for improvement in aquaculture is the development of dietary additives and formulations which provide for complete mucosal health and protection of fish raised in intensive systems. Far greater attention has been paid to dietary impact on gut health than to protective effects at other mucosal surfaces such as skin and gill. These exterior surfaces, however, are important primary targets for pathogen attachment and invasion. Flavobacterium columnare, the causative agent of columnaris disease, is among the most prevalent of all freshwater disease-causing bacteria, impacting global aquaculture of catfish, salmonids, baitfish and aquaria-trade species among others. This study evaluated whether the feeding of a standard catfish diet supplemented with Alltech dietary additives Actigen(®), a concentrated source of yeast cell wall-derived material and/or Allzyme(®) SSF, a fermented strain of Aspergillus niger, could offer protection against F. columnare mortality. A nine-week feeding trial of channel catfish fingerlings with basal diet (B), B + Allzyme(®) SSF, B + Actigen(®) and B + Actigen(®)+Allzyme(®) SSF revealed good growth in all conditions (FCR < 1.0), but no statistical differences in growth between the treatments were found. At nine weeks, based on pre-challenge trial results, basal, B + Actigen(®), and B + Allzyme(®) SSF groups of fish were selected for further challenges with F. columnare. Replicated challenge with a virulent F. columnare strain, revealed significantly longer median days to death in B + Allzyme(®) SSF and B + Actigen(®) when compared with the basal diet (P < 0.05) and significantly higher survival following the eight day challenge period in B + Actigen(®) when compared with the other two diets (P < 0.05). Given the superior protection provided by the B + Actigen(®) diet, we carried out transcriptomic comparison of gene expression of fish fed that diet and the basal diet before and after columnaris challenge using high-throughput RNA-seq. Pathway and enrichment analyses revealed changes in mannose receptor DEC205 and IL4 signaling at 0 h (prior to challenge) which likely explain a dramatic divergence in expression profiles between the two diets soon after pathogen challenge (8 h). Dietary mannose priming resulted in reduced expression of inflammatory cytokines, shifting response patterns instead to favor resolution and repair. Our results indicate that prebiotic dietary additives may provide protection extending beyond the gut to surface mucosa.

Mucosal expression signatures of two Cathepsin L in channel catfish (Ictalurus punctatus) following bacterial challenge.

The mucosal surfaces of fish are the first line of host defense against various pathogens. The mucosal immune responses are the most critical events to prevent pathogen attachment and invasion. Cathepsins are a group of peptidases that involved in different levels of immune responses, but the knowledge of the roles of Cathepsin in mucosal immune responses against bacterial infection are still lacking. Therefore, in the present study we characterized the Cathepsin L gene family in channel catfish, and profiled their expression levels after challenging with two different Gram-negative bacterial pathogens. Here, two Cathepsin L genes were identified from channel catfish and were designated CTSL1a and CTSL.1. Comparing to other fish species, the catfish CTSL genes are highly conserved in their structural features. Phylogenetic analysis was conducted to confirm the identification of CTSL genes. Expression analysis revealed that the CTSL genes were ubiquitously expressed in all tested tissues. Following infection, the CTSL genes were significantly induced at most timepoints in mucosal tissues. But the expression patterns varied depending on both pathogen and tissue types, suggesting that CTSL genes may exert disparate functions or exhibit distinct tissue-selective roles in mucosal immune responses. Our findings here, clearly revealed the key roles of CTSL in catfish mucosal immunity; however, further studies are needed to expand functional characterization and examine whether CTSL may also play additional physiological roles in catfish mucosal tissues.

Expression profile analysis of two cathepsin S in channel catfish (Ictalurus punctatus) mucosal tissues following bacterial challenge

Cathepsin S belongs to the papain family of cysteine protease, and is considered to play key roles in immune responses after bacterial challenge. However, despite the recognized importance of Cathepsin S in immunity, no studies have systematically characterized Cathepsin S in catfish. In this regard, here, we characterized the Cathepsin S gene family in channel catfish, and investigated their expression patterns following two different Gram-negative bacterial challenge. In the present study, two Cathepsin S genes (ctss and ctssa) were captured in channel catfish. In comparison to other species, the catfish Cathepsin S genes are highly conserved in their structural features. Phylogenetic analysis indicated the strongest phylogenetic relationship with zebrafish, which is consistent with their evolutional relationships. Tissue distribution analysis revealed that Cathepsin S genes were ubiquitously expressed in catfish tissues. Following bacterial infection, the Cathepsin S genes were significantly up-regulated at most time-points in mucosal surfaces, with an acute response post Edwardsiella ictaluri infection. Obviously, the expression profiles were quite distinct between two Cathepsin S genes, across the tissues and between pathogens, suggesting that Cathepsin S genes may exert disparate roles in mucosal immune responses. Our findings here, provide early insight into the immune functions of Cathepsin S in catfish; however, further studies are needed to determine the mechanisms of Cathepsin S for antigen presentation during inflammatory processes and innate host defense.

Identification and mucosal expression analysis of cathepsin B in channel catfish (Ictalurus punctatus) following bacterial challenge

The mucosal surfaces of fish (skin, gill and intestine) constitute the primary line of defense against pathogen invasion. Although the importance of fish mucosal surfaces as the first barriers against pathogens cannot be overstated, the knowledge of teleost mucosal immunity are still limited. Cathepsin B, a lysosomal cysteine protease, is involved in multiple levels of physiological and biological processes, and playing crucial roles for host immune defense against pathogen infection. In this regard, we identified the cathepsin B (ctsba) of channel catfish and investigated the expression patterns of the ctsba in mucosal tissues following Edwardsiella ictaluri and Flavobacterium columnare challenge. Here, catfish ctsba gene was widely expressed in all examined tissues with the lowest expression level in muscle, and the highest expression level in trunk kidney, followed by spleen, gill, head kidney, intestine, liver and skin. In addition, the phylogenetic analysis showed the catfish ctsba had the strongest relationship to zebrafish. Moreover, the ctsba showed a general trend of up-regulated in mucosal tissues following both Gram-negative bacterial challenge. Taken together, the increased expression of ctsba in mucosal surfaces indicated the protective function of ctsba against bacterial infection, and the requirement for effective clearance of invading bacteria. Further studies are needed, indeed, to expand functional characterization and examine whether ctsba may play additional physiological and biological roles in catfish mucosal tissues.

Expression profiling analysis of immune-related genes in channel catfish (Ictalurus punctatus) skin mucus following Flavobacterium columnare challenge

Fish are covered by a watery gel-mucus, mainly secreted by the goblet cells, serving as the physical and biochemical barrier between the external environment and the interior milieu, playing more important roles in fish that without scale. Despite the important roles of mucus in fish immunity, the knowledge of detailed molecular events happened during infection process is still limited. While most studies were focused on characterizing the protein and enzyme activities in the mucus following challenge, no studies have examined the gene expression profiles in fish mucus. In this regard, herein we carried out the first gene profiling analysis in catfish mucus using real-time PCR. Ten important immune-related genes were selected according to our previous studies. Their expression levels were examined in the early timepoints (namely, 1 h, 2 h, 4 h, 8 h, and 24 h) following Flavobacterium columnare challenge. Notably, expression levels of most of the selected genes were rapidly altered by the challenge. Seven genes were down-regulated, while only three genes were up-regulated. In addition, the gene expression patterns in mucus were very different from the mucosal surfaces (skin, gill and intestine) and the classical immune organs (liver, spleen and kidney). The unique expression patterns obtained here may be resulted from the great advantage of the large amount of attached bacteria in the mucus than the internal tissues, and resulted from the bacteria virulent actors to suppress the host immune response. Taken together, our results can expand our knowledge of fish mucosal immunity, and the un-lethal mucus sampling can provide early insight for developing the strategies for selection of disease resistant families and strains in catfish as well as other fish species.

Galectins in channel catfish, Ictalurus punctatus: Characterization and expression profiling in mucosal tissues.

Galectins, a family of β-galactoside-binding lectins with conserved CRDs, which can recognize the glycans on the surface of viruses, bacteria and protozoan parasites, are emerging as key players in many important pathological processes, including acute and chronic inflammatory diseases, autoimmunity and apoptosis. Although galectins have attracted great interest in mammals, they are still poorly-characterized in teleost. Previously, several studies have reported their high expression levels in mucosal tissues before and post infection. Given the important roles for galectins in mucosal immunity, therefore, we characterized the galectin gene family and profiled family member expression after challenge with two different Gram-negative bacterial pathogens. Here, twelve galectins genes were captured in channel catfish (Ictalurus punctatus), and phylogenetic analysis showed the strongest relationship to zebrafish and salmon, which is consistent with their phylogenetic relationships. Furthermore, the galectin genes were widely expressed in catfish tissues, while most of the galectin genes were strongly expressed in mucosal tissues (skin, gill and intestine). In addition, the expression profiles of galectins after bacterial infection varied depending on both pathogen and tissue type, suggesting that galectins may exert disparate functions or exhibit distinct tissue-selective roles in the host immune response to bacterial pathogens. Further studies are needed, however, to expand functional characterization and examine whether galectins may also play additional physiological roles in catfish immunity.

More than just antibodies: Protective mechanisms of a mucosal vaccine against fish pathogen Flavobacterium columnare.

Abstract A recently developed attenuated vaccine for Flavobacterium columnare has been demonstrated to provide superior protection for channel catfish, Ictalurus punctatus, against genetically diverse columnaris isolates. We were interested in examining the mechanisms of this protection by comparing transcriptional responses to F. columnare challenge in vaccinated and unvaccinated juvenile catfish. Accordingly, 58 day old fingerling catfish (28 days post‐vaccination or unvaccinated control) were challenged with a highly virulent F. columnare isolate (BGSF‐27) and gill tissues collected pre‐challenge (0 h), and 1 h and 2 h post infection, time points previously demonstrated to be critical in early host‐pathogen interactions. Following RNA‐sequencing and transcriptome assembly, differential expression (DE) analysis within and between treatments revealed several patterns and pathways potentially underlying improved survival of vaccinated fish. Most striking was a pattern of dramatically higher basal expression of an array of neuropeptides (e.g. somatostatin), hormones, complement factors, and proteases at 0 h in vaccinated fish. Previous studies indicate these are likely the preformed mediators of neuroendocrine cells and/or eosinophilic granular (mast‐like) cells within the fish gill. Following challenge, these elements fell to almost undetectable levels (>100‐fold downregulated) by 1 h in vaccinated fish, suggesting their rapid release and/or cessation of synthesis following degranulation. Concomitantly, levels of pro‐inflammatory cytokines (IL‐1b, IL‐8, IL‐17) were induced in unvaccinated fish. In contrast, in vaccinated catfish, we observed widespread induction of genes needed for collagen deposition and tissue remodeling. Taken together, our results indicate an important component of vaccine protection in fish mucosal tissues may be the sensitization, proliferation and arming of resident secretory cells in the period between primary and secondary challenge. HighlightsVaccinated catfish had a higher basal expression of many preformed mediators.Proteases and neuropeptide level in vaccinated fish fell rapidly following challenge.Collagen deposition and tissue remodeling processes were induced in vaccinated fish.Pro‐inflammatory genes were upregulated in unvaccinated fish post challenge.

Impact of oral and waterborne administration of rhamnolipids on the susceptibility of channel catfish (Ictalurus punctatus) to Flavobacterium columnare infection

ABSTRACT Flavobacterium columnare is the causative agent of columnaris disease and causes tremendous morbidity and mortality of farmed fish globally. Previously, we identified a potential lectin‐mediator (a rhamnose‐binding lectin; RBL1a) of F. columnare adhesion and showed higher RBL1a expression in susceptible channel catfish under basal conditions and following infection. Exposure of challenged fish to the carbohydrate ligand l‐rhamnose just prior to a challenge substantially decreased columnaris mortality and pathogen adherence via the down‐regulation of RBL1a. While highly effective in protecting fish from columnaris, l‐rhamnose is prohibitively expensive, underscoring the need for alternative cost‐effective sources of rhamnose for disease control. One such alternative may be microbially produced glycolipid compounds termed rhamnolipids (RLs), which feature abundant l‐rhamnose moieties and are readily available from commercial sources. In the present study, we examined whether commercially available RLs (administered either by immersion or via feed) would function similarly to l‐rhamnose in affording host protection against F. columnare. A four‐week feeding trial with basal and RL top‐coated diets (basal diet + RLs) was conducted in channel catfish fingerlings. Surprisingly, columnaris challenges revealed significantly lower survival following the 10 d challenge period in RL diet fed fish when compared with the basal treatment group (p < 0.001). In fish fed RLs, we observed a rapid and large‐scale upregulation of RBL1a immediately after challenge combined with a suppression of mucin and lysozyme transcripts. Similarly, fish that were briefly pre‐exposed to RLs by immersion and then challenged exhibited lower survival as compared to unexposed fish during a 4 d trial. In conclusion, RLs do not represent an alternative to rhamnose as an experimental treatment for protecting catfish from columnaris mortality. Further research is needed to find other affordable and efficacious alternative sources of l‐rhamnose. HIGHLIGHTSRhamnolipid exposure (via feed or immersion) heightened columnaris susceptibility.Rhamnolipid exposure modulated mucosal immune gene expression.Rhamnolipids do not appear to be appropriate substitutes for l‐rhamnose.

Identification, expression, and innate immune responses of two insulin-like peptide genes in the razor clam Sinonovacula constricta.

Insulin-like peptide (ILP) has emerged as a cell regulatory factor with multiple functions in vertebrates and invertebrates. In the present study, we identified and characterized two ILP genes, ILP1 and ILP2, in the razor clam Sinonovacula constricta. Both ILPs have a signal peptide and a mature domain consisting of six strictly conserved cysteines. The tertiary structure is divided into three main α-helices with a C-domain loop that separates helix 1 from helix 2. Both of ILPs were found to be regulated according to tissue type and developmental stage. After challenge with Vibrio anguillarum, Vibrio parahaemolyticus and Micrococcus lysodeikticus, the expression of two ILP genes was significantly up-regulated in the liver, hemocytes and mantle tissues, suggesting that the ILPs may play roles in the innate immunity in the razor clam Sinonovacula constricta.