Microbiome and epigenetic variation in wild fish with low genetic diversity.

Non-genetic sources of phenotypic variation, such as the epigenome and the microbiome, could be important contributors to adaptive variation for species with low genetic diversity. However, little is known about the complex interaction between these factors and the genetic diversity of the host, particularly in wild populations. Here, we examine the skin microbiome composition of two closely-related mangrove killifish species with different mating systems (self-fertilising and outcrossing) under sympatric and allopatric conditions. This allows us to partition the influence of the genotype and the environment on their microbiome and (previously described) epigenetic profiles. We find the diversity and community composition of the skin microbiome are strongly shaped by the environment and, to a lesser extent, by species-specific influences. Heterozygosity and microbiome alpha diversity, but not epigenetic variation, are associated with the fluctuating asymmetry of traits related to performance (vision) and behaviour (aggression). Our study identifies that a proportion of the epigenetic diversity and microbiome differentiation is unrelated to genetic variation, and we find evidence for an associative relationship between microbiome and epigenetic diversity in these wild populations. This suggests that both mechanisms could potentially contribute to variation in species with low genetic diversity.

Genome sequencing and annotation of multi-virulent Aeromonas veronii XhG1.2 isolated from diseased Xiphophorus hellerii.

The present study was intended to elucidate the genomic basis of antibiotic resistance and hyper-virulence of the fish pathogen Aeromonas veronii XhG1.2 characterized in our previous work. The identity of XhG1.2 was confirmed through 16S rDNA sequence analysis and whole genome sequence analysis. The top-hit species distribution analysis of XhG1.2 sequence data revealed major hits against the Aeromonas veronii. The identification of virulence genes using the VFDB showed the genome of XhG1.2 to have the genes coding for the virulence factors viz. aerolysin, RtxA, T2SS, T3SS and T6SS. The presence of antibiotic resistance predicted through the CARD database analysis showed it to have the CephA3, OXA-12, adeF and pulvomycin resistance genes. By the phylogenetic and comparative genomic analysis, A. veronii species were found to have genes for toxin production. This also confirmed the pathogenicity and drug resistance of A. veronii XhG1.2 and also its potential to cause disease in diverse ornamental fishes.

Examination of Host Phenotypes in Gambusia affinis Following Antibiotic Treatment.

The commonality of antibiotic usage in medicine means that understanding the resulting consequences to the host is vital. Antibiotics often decrease host microbiome community diversity and alter the microbial community composition. Many diseases such as antibiotic-associated enterocolitis, inflammatory bowel disease, and metabolic disorders have been linked to a disrupted microbiota. The complex interplay between host, microbiome, and antibiotics needs a tractable model for studying host-microbiome interactions. Our freshwater vertebrate fish serves as a useful model for investigating the universal aspects of mucosal microbiome structure and function as well as analyzing consequential host effects from altering the microbial community. Methods include host challenges such as infection by a known fish pathogen, exposure to fecal or soil microbes, osmotic stress, nitrate toxicity, growth analysis, and measurement of gut motility. These techniques demonstrate a flexible and useful model system for rapid determination of host phenotypes.

Pathological alterations due to motile Aeromonas infection in red swordtail fish (Xiphophorus helleri).

A herd of red swordtail fish (Xiphophorus helleri) was reared in outdoor concrete ponds and suffered from occasional mortality. Moribund fishes showing abdominal dropsy and fin rots were sent for diagnosis. Gross necropsy findings showed enlargement of liver, spleen, and kidney in concurrence with congestion, and a severe accumulation of peritoneal fluid. Histopathological findings revealed an alteration of hepatocytes, with a severe diffuse accumulation of fat vacuoles in the cytoplasm. In the trunk kidney, severe accumulation of mononuclear cells together with cloudy swelling of the renal tubular epithelium was observed. From internal organs of the fish motile Aeromonas spp. were identified. The pathological findings might be associated with a long-term infection of affected fish fostered by common stressors such as improper feeding and poor pond environment condition (water temperature). Effective therapeutic measures comprised an advancement of keeping conditions and appropriate feeding to improve the health status in combination with the application of antibiotic substances.

The host effects of Gambusia affinis with an antibiotic-disrupted microbiome.

While serving as critical tools against bacterial infections, antimicrobial therapies can also result in serious side effects, such as antibiotic-associated entercolitis. Recent studies utilizing next generation sequencing to generate community 16S gene profiles have shown that antibiotics can strongly alter community composition and deplete diversity. However, how these community changes in the microbiota are related to the host side effects is still unclear. We have used the freshwater Western mosquitofish (Gambusia affinis) as a tractable vertebrate model system to study host effects following exposure to a broad spectrum antibiotic, rifampicin. After 3days of exposure, the bacterial communities of the mucosal skin and gut microbiomes lost diversity and shifted composition. Compared to unexposed controls, treated fish were more susceptible to a specific pathogen, Edwardsiella ictaluri, yet displayed no survival differences when subjected to a polymicrobial water challenge of soil or feces. Treated fish were more susceptible to osmotic stress from NaCl, but not to the toxin nitrate. Treated fish failed to gain weight as well as controls over one month when fed a matched diet. Because of small sample sizes, pathogen susceptibility and weight gain differences were not statistically significant. This study provides supporting evidence in an experimental laboratory system that an antibiotic can have significant and persistent negative host effects, and provides for future study into the mechanisms of these effects.

Biomarkers involved in energy metabolism and oxidative stress response in the liver of Goodea gracilis Hubbs and Turner, 1939 exposed to the microcystin-producing Microcystis aeruginosa LB85 strain.

Goodea gracilis is an endemic fish that only habitats in some water bodies of Central Mexico that are contaminated with cyanobacteria-producing microcystins (MC); however, a lack of information on this topic prevails. With the aim to generate the first approximation about the physiological changes elicited by cyanobacterium that produce MC congeners in this fish species, specimens born in the laboratory was exposed for 96 h to cell densities of 572.5, 1145, 2290, 4580, and 9160 × 10(6) cells of Microcystis aeruginosa strain LB85/L, and a set of novel endpoint related to hepatic gluconeogenesis (ADH/LDH) and pro-oxidant forces O2., H2 O2 ) in addition to biomarkers of oxidative damage and antioxidant response was evaluated in the liver. Results suggest that high inhibition of protein serine/threonine phosphatase (PP) may trigger many metabolic processes, such as those related to hepatic gluconeogenesis (ADH/LDH) and pro-oxidant O2⋅, H2 O2 , TBARS, ROOH, RC=O) as well as antioxidant (SOD, CAT, GPx) response to oxidative stress. Particularly, we observed that inhibition of LDH and PP, and H2 O2 increase and TBARS production were the key damages induced by high densities of M. aeruginosa. However, changes between aerobic and anaerobic metabolism related with ROS metabolism and ADH/LDH balance are apparently an acclimation of this fish species to exposure to cyanobacteria or their MCs. Fish species living in environments potentially contaminated with cyanobacteria or their MCs possess mechanisms of acclimation that allow them to offset the damage induced, even in the case of fish that have never been exposed to MCs.

Determinación enzimática y metales pesados en cerebro e hígado del modelo ecotoxicológico Gambusia punctata (Poeciliidae) / Enzymatic determination and heavy metals in the brain and liver in ecotoxicological model Gambusia punctata (Poeciliidae)

Se realizó un estudio durante el 2011, con vistas a evaluar los niveles de actividad enzimática y metales pesados bioacumulados en cerebro e hígado de la especie Gambusia punctata, que habita en los ecosistemas San Juan y Filé de Santiago de Cuba, para lo cual se seleccionaron 3 estaciones, correspondientes a la parte alta, media y baja de ambos sistemas. Se escogieron ejemplares que midieron biométricamente de 2,1-3,0 cm de longitud total. Se determinó la enzima acetilcolinesterasa en el cerebro y la glutation-S-transferasa y catalasa, respectivamente, en el hígado. En ambos órganos se analizaron concentraciones de cobre, cinc, plomo y cadmio, tratados por vía húmeda y cuantificados por espectroscopia de plasma inductivamente acoplado con vista axial. En la especie del San Juan, los niveles enzimáticos variaron entre las estaciones, siendo mayores y estadísticamente diferentes para las hembras (p<0,05); sin embargo, en la del Filé, fueron similares entre las partes y sexos. En cuanto a los metales, no se detectaron concentraciones en el cerebro, ni plomo ni cadmio en el hígado. Las concentraciones de cobre y cinc fueron mayores en la parte baja y menores en la alta, de manera que hubo diferencias con respecto a la especie del Filé. Se concluyó que las enzimas variaron desde una estación ambientalmente diferente a otra y que los metales cobre y cinc, a pesar de ser esenciales, representan un riesgo ecotoxicológico en la especie del San Juan, debido a sus elevadas capacidades bioacumulativas(AU)

A study during 2011 was carried out in order to evaluate the levels of enzymatic activity and heavy metals bioaccumulated in brain and liver of the species Gambusia punctata, inhabiting San Juan and Filé ecosystems in Santiago de Cuba. For this purpose 3 stations were selected, corresponding to the upper, middle and lower parts of both systems. Specimens biometrically measuring 2.1-3.0 cm in total length were chosen. Acetylcholinesterase enzyme in the brain and glutathione-S-transferase and catalase in the liver were determined, respectively. Copper, zinc, lead and cadmium concentrations were analyzed in both organs, wet processed and quantified by axial view inductively coupled plasma spectroscopy. In San Juan species the enzymatic levels varied between the stations, being higher and statistically different for females(p<0.05), but in that from Filé they were similar between the parts and genders. Regarding metals, concentrations were not detected in brain, neither lead nor cadmium in the liver. Copper and zinc concentrations were higher in the lower part and lower in the upper one, so that there were differences regarding the Filé species. It was concluded that the enzymes varied from an environmentally different station to another and copper and zinc metals, in spite of being essential, pose an ecotoxicological risk in the San Juan species, due to their high bioaccumulative capabilities(AU)

Heat shock proteins protect platyfish (Xiphophorus maculatus) from Yersinia ruckeri induced mortality.

The significant disadvantages accompanied with the use of antibiotics in aquaculture, emphasize the need for developing alternative disease control strategies, like novel vaccine approaches and immunostimulating measures. Several studies have already pointed out the ability of heat shock proteins (HSPs) to modulate innate and adaptive immune responses, what makes them potent candidates for the development of a new disease prevention method. In this study, the use of self and non-self heat shock proteins as a new prophylactic treatment against bacterial diseases in freshwater aquaculture was investigated. Therefore, an infection model was developed with platyfish as a host for Yersinia ruckeri infections. In this infection model, the effect of different treatments with HSPs on the survival of the fish after bacterial infection was tested: non-lethal heat shock, intracoelomal injection with two recombinant bacterial HSPs, GroEL and DnaK, and a combination of a non-lethal heat shock and an injection with bacterial HSPs. The results show that a non-lethal heat shock could not protect fish against a subsequent infection with Y. ruckeri. However, when the fish received an injection with bacterial HSPs, Y. ruckeri induced mortality was reduced. This effect became significant when the administration of bacterial HSPs was combined with a non-lethal heat shock. These data suggest a possible role for heat shock proteins as an immunostimulating treatment in fish against bacterial infections.

Preliminary investigations of hydrogen peroxide treatment of selected ornamental fishes and efficacy against external bacteria and parasites in green swordtails.

The objectives of these preliminary studies were to evaluate the use of hydrogen peroxide (H2O2) for the treatment of selected species of ornamental fishes and its efficacy in treating external bacteria and parasites. In the first part of the study, fish of five species (serpae tetra Hyphessobrycon eques (also known as Serpa tetra H. serpae), tiger barb Puntius tetrazona, blue gourami Trichogaster trichopterus, suckermouth catfish Hypostomus plecostomus, and green swordtail Xiphophorus hellerii) were exposed to H2O2 for 1 h at concentrations between 6 and 34 mg/L or for 24 h at concentrations between 1 and 6 mg/L. The results were species specific: green swordtails tolerated all of the treatments, serpae tetras and tiger barbs were sensitive only to the highest concentration, and mortalities of suckermouth catfish and blue gourami were recorded in every treatment. In the second part of the study, clinically healthy green swordtails and fish infested with external motile rod-shaped bacteria (i.e., Ichthyobodo spp., Trichodina spp., and Gyrodactylus spp.) were treated with several concentrations of H2O2. A single H2O2 treatment of 3.1 mg/L or more for 1 h effectively eliminated external bacteria, concentrations of 6.5 mg/L or more appeared to effectively kill Ichthyobodo spp., and none of the treatments tested was effective against Trichodina spp. or Gyrodactylus spp. These preliminary findings suggest that H2O2 is effective for treating certain external bacterial infections and flagellate infestations in some species of ornamental fish at the dosages tested. Other treatment regimens may need to be tested for effectiveness against Trichodina spp. and Dactylogyrus spp.

Mycobacteriosis in mangrove killifish (Rivulus magdalenae) caused by living fish food (Tubifex tubifex) infected with Mycobacterium marinum.

Mycobacterium marinum was demonstrated in the mud tube worm Tubifex tubifex. These worms served as live fish food for aquarium fishes. The ingestion of Tubifex tubifex by the mangrove killifish Rivulus magdalenae, a neotropical species in the order Cyprinodontiformes, was the source of an atypical mycobacteriosis of these fishes.

[Detection of pathogenic Edwarsiella tarda].

25 Edwardsiella tarda(Et) strains had been detected both on their viruslent factor Excellular Product (ECP), including the hemolysin and extracellular protease (ECPase), and on their pathogenicity to mice and Xiphophorus helleri. ECP was detected by Dot-ELISA with rabbit antiserum against ECP of reference strain JEL4. The results showed that the animal pathogenicity of Et had good correlation with its hemolysin other than with ECPase. The agreement between Dot-ELISA of JEL4 ECP and pathogenicity to animal was up to 100%. It was desirable to establish a detecting method, which only need detect the ECP with plate assay (PA) and Dot-ELISA, but needn't have animal experiment. Furthermore it is possible to develop a diagnosis kit of application to simplify the detecting procedure of pathogenic Et.

Using a new inbred fish model and cultured fish tissue cells to study Aeromonas hydrophila and Yersinia ruckeri pathogenesis.

An inbred strain of the southern platyfish, Xiphophorus maculatus, was used as a host for Aeromonas hydrophila and Yersinia ruckeri infections. The infections were initiated by holding the platyfish in inoculation baths containing dilutions of virulent A. hydrophila or Y. ruckeri strains. Inoculating the platyfish in this manner resulted in a dose-dependent mortality over a range of bacterial input from 10(5) to 10(8) A. hydrophila and 10(6) to 10(8) Y. ruckeri/ml. Clinical manifestations of A. hydrophila infections were noted in infected platyfish that eventually died, but not in platyfish that survived. In this model, the Y. ruckeri infected fish died before obvious signs of infection were detected. The A. hydrophila strain used to establish the infections was recovered from the kidney and intestine of infected fish that died, but not from survivors receiving the same inoculation dose. Both infective bacteria were tested for the ability to invade a number of different fish and human cultured cells. A hydrophila strain TF7 did not invade of the cells tested, whereas the Y. ruckeri strain invaded fish derived cultured cells, but not human derived Hep-2 cells.

Isolation and characterization of a retrovirus from the fish genus Xiphophorus.

A cell line (BsT) established from neoplastic embryonal tissues of the platyfish (Xiphophorus maculatus) released spontaneously retrovirus-like particles. The particles have a buoyant density of 1.16 g/ml, a mean diameter of 100 nm and the morphology of immature retroviruses. The particle-associated proteins p70, p65, and p28 react with an antiserum directed against the major internal feline leukemia virus structural protein p27. The particles are associated with a reverse transcriptase. The purified enzyme has a molecular weight of about 70 kDa and prefers the template primers poly(rA):oligo(dT), poly(dC):oligo(dG), and poly(rC):oligo(dG) in the presence of Mn2+. The enzyme activity is inhibited by antibodies directed against the reverse transcriptase of feline leukemia virus and simian sarcoma virus. The particles contain a ribonucleic acid of about 70 S. In an endogenous reverse transcriptase reaction nucleic acids in the range of 0.2 to 0.4 kb were synthesized. In Northern blots with these nucleic acids as probe, three transcripts of about 8.5, 4.2, and 1.5 kb were detected in BsT cells. Southern blot analysis with the same probe demonstrates related sequences in the DNA of BsT cells and the platyfish and swordtail (Xiphophorus helleri). Hybridization experiments with the LTR-gag region of the feline leukemia virus show homologous sequences in the Xiphophorus genome.

Retrovirus-like particles in embryonic kidney tissue of the platyfish, Xiphophorus maculatus.

Ultrastructural studies were performed on embryos of an inbred strain (163A) of the platyfish Xiphophorus maculatus, and a pooled stock of the Rio Sarabia strain of the swordtail, X. helleri. Viral-like particles were found in the kidney tissue of platyfish embryos. Exposure of pregnant platyfish to 5-bromodeoxyuridine during the time of differentiation of embryonic melanocytes enhanced the production of viral-like particles in platyfish embryos. Viral-like particles were not found in untreated or drug-treated swordtail embryos. The morphology of these particles corresponds to type C retroviruses.