Epinecidin-1 is an antimicrobial peptide and plays a vital role in protecting fish against pathogenic infection. As a mimic of a grouper epinecidin-1 peptide, it has tertiary structures that closely resemble those of pleurocidin found in the winter flounder (Pleuronectes americanus). The tissue-specific, lipopolysaccharide (LPS)–stimulation-specific, and poly(I):poly(C)-stimulation-specific expressions of the grouper (Epinephelus coioides) epinecidin-1 antimicrobial peptide were determined using a comparative reverse-transcription polymerase chain reaction. Results of the tissue distribution analysis revealed high levels of epinecidin-1 messenger RNA (mRNA) in the head kidneys, intestines, and skin. Expression of epinecidin-1 mRNA was dose-dependently stimulated by both LPS and poly(I):poly(C). Immunohistochemical analysis with the polyclonal antiserum of a grouper epinecidin-1 peptide (rabbit polyclonal antibody) showed that the peptide was localized with the epinecidin-1 antibody in the gills and intestines. Two synthetic peptides of the grouper epinecidin-1 peptide (g-ple 22–51 and g-ple 22–42) and one winter flounder pleurocidin as a control exhibited high antimicrobial activities against gram-negative or gram-positive bacteria. In addition, peptide treatment was effective in promoting a significant increase in fish survival after the injection of Vibrio vulnificus in tilapia (Oreochromis mossambicus) and grouper. These results are relevant to the design of prophylactic and therapeutic strategies to counter bacterial infections, especially for preventing or ameliorating immune defects in fish during bacterial infections.

Grouper iridovirus in Taiwan (TGIV) infection in the Epinephelus hybrid is a major problem in the grouper industry. ATPase gene sequences indicate that this virus is closely related to cell hypertrophy iridoviruses. Histologically, the appearance of basophilic or eosinophilic enlarged cells in internal organs is the most characteristic feature of this disease. These cells are acid-phosphatase positive and are able to phagocytose injected carbon particles. In our study, TGIV infection inhibited normal phagocytic ability in these cells in vivo after 4 d post-infection (p.i.) but not before 2 d p.i. Their staining properties and phagocytic ability suggested a monocyte origin of enlarged cells, which appeared in high numbers in the trunk kidney, head kidney, spleen and gill. After infection, the enlarged cells first appeared in the spleen, with an abundance peak at 64 h p.i. (Peak 1); at 120 h p.i., a second peak (Peak 2) occurred in the spleen, head kidney, trunk kidney and gill. Lower numbers of enlarged cells were observed in the liver, muscle, heart, eye, intestine, but no enlarged cells were found in the brain. A TGIV-specific DNA probe labeled most of the basophilic but not eosinophilic enlarged cells. Nuclei of infected cells were labeled during an early stage of the infection; at later stages, both nuclei and cytoplasms were labeled. Ultrastructurally, heterochromatins of the infected cells were marginated or aggregated to one side of the nuclei during the early stages of infection. Damage and rupture of the nuclear membrane started before formation of the viromatrix. Capsids were assembled in ring-shaped or disc-shaped structures. Bullet-shaped electron-dense material was present near the incomplete virus particles, and is speculated to be inserted into the capsids later.

Caenorhabditis elegans has been used for studying host-pathogen interactions since long, and many virulence genes of pathogens have been successfully identified. In several studies, fluorescent pathogens were fed to C. elegans and fluorescence observed in the gut was considered an indicator for bacterial colonization. However, the grinder in the pharynx of these nematodes supposedly crushes the bacterial cells, and the ground material is delivered to the intestine for nutrient absorption. Therefore, it remains unclear whether intact bacteria pass through the grinder and colonize in the intestine. Here we investigated whether the appearance of fluorescence is indicative of intact bacteria in the gut using both fluorescence microscopy and transmission electron microscopy. In wild-type N2 C. elegans, Escherichia coli DH5α, and Vibrio vulnificus 93U204, both of which express the green fluorescence protein, were found intact only proximal to the grinder, while crushed bacterial debris was found in the post-pharyngeal lumen. Nevertheless, the fluorescence was evident throughout the lumen of worm intestines irrespective of whether the bacteria were intact or not. We further investigated the interaction of the bacteria with C. elegans phm-2 mutant, which has a dysfunctional grinder. Both strains of bacteria were found to be intact and accumulated in the pharynx and intestine owing to the defective grinder. The fluorescence intensity of intact bacteria in phm-2 worms was indistinguishable from that of crushed bacterial debris in N2 worms. Therefore, appearance of fluorescence in the C. elegans intestine should not be directly interpreted as successful bacterial colonization in the intestine.

Starvation is a common stress experienced by bacteria living in natural environments and the ability to adapt to and survive intense stress is of paramount importance for any bacterial population. A series of starvation experiments were conducted using V. vulnificus 93U204 in phosphate-buffered saline and seawater. The starved population entered the death phase during the first week and approximately 1% of cells survived. After that the population entered a long-term stationary phase, and could survive for years. Starvation-induced diversification (SID) of phenotypes was observed in starved populations and phenotypic variants (PVs) appeared in less than 8 days. The cell density, rather than the population size, had a major effect on the extent of SID. SID was also observed in strain YJ016, where it evolved at a faster pace. PVs appeared to emerge in a fixed order: PV with reduced motility, PV with reduced proteolytic activity, and PV with reduced hemolytic activity. All of the tested PVs had growth advantages in the stationary phase phenotypes and increased fitness compared with 93U204 cells in co-culture competition experiments, which indicates that they had adapted to starvation. We also found that SID occurred in natural seawater with a salinity of 1%–3%, so this mechanism may facilitate bacterial adaptation in natural environments.

Metabolic-associated fatty liver disease (MAFLD) is the most common chronic liver disease. Gut dysbiosis is considered a significant contributing factor in disease development. Increased intestinal permeability can be induced by gut dysbiosis, followed by the entry of lipopolysaccharide into circulation to reach peripheral tissue and result in chronic inflammation. We reviewed how microbial metabolites push host physiology toward MAFLD, including short-chain fatty acids (SCFAs), bile acids, and tryptophan metabolites. The effects of SCFAs are generally reported as anti-inflammatory and can improve intestinal barrier function and restore gut microbiota. Gut microbes can influence intestinal barrier function through SCFAs produced by fermentative bacteria, especially butyrate and propionate producers. This is achieved through the activation of free fatty acid sensing receptors. Bile is directly involved in lipid absorption. Gut microbes can alter bile acid composition by bile salt hydrolase-producing bacteria and bacterial hydroxysteroid dehydrogenase-producing bacteria. These bile acids can affect host physiology by activating farnesoid X receptor Takeda G protein-coupled receptor 5. Gut microbes can also induce MAFLD-associated symptoms by producing tryptophan metabolites kynurenine, serotonin, and indole-3-propionate. A summary of bacterial genera involved in SCFAs production, bile acid transformation, and tryptophan metabolism is provided. Many bacteria have demonstrated efficacy in alleviating MAFLD in animal models and are potential therapeutic candidates for MAFLD.