Development of a SNP linkage map and genome-wide association study for resistance to Aeromonas hydrophila in pacu (Piaractus mesopotamicus).

BACKGROUND: Pacu (Piaractus mesopotamicus) is one of the most important Neotropical aquaculture species from South America. Disease outbreaks caused by Aeromonas hydrophila infection have been considered significant contributors to the declining levels of pacu production. The current implementation of genomic selection for disease resistance has been adopted as a powerful strategy for improvement in fish species. This study aimed to investigate the genetic architecture of resistance to A. hydrophila in pacu via Genome-Wide Association Study (GWAS), the identification of suggestive Quantitative Trait Loci (QTLs) and putative genes associated with this trait. The genetic data were obtained from 381 juvenile individuals belonging to 14 full-sibling families. An experimental challenge was performed to gain access to the levels of genetic variation for resistance against the bacteria using the following trait definitions: binary test survival (TS) and time of death (TD). RESULTS: The analyses of genetic parameters estimated moderate heritability (h2) for both resistance traits: 0.20 (± 0.09) for TS and 0.35 (± 0.15) for TD. A linkage map for pacu was developed to enable the GWAS, resulting in 27 linkage groups (LGs) with 17,453 mapped Single Nucleotide Polymorphisms (SNPs). The length of the LGs varied from 79.95 (LG14) to 137.01 (LG1) cM, with a total map length of 2755.60 cM. GWAS identified 22 putative QTLs associated to A. hydrophila resistance. They were distributed into 17 LGs, and were considered suggestive genomic regions explaining > 1% of the additive genetic variance (AGV) for the trait. Several candidate genes related to immune response were located close to the suggestive QTLs, such as tbk1, trim16, Il12rb2 and lyz2. CONCLUSION: This study describes the development of the first medium density linkage map for pacu, which will be used as a framework to study relevant traits to the production of this species. In addition, the resistance to A. hydrophila was found to be moderately heritable but with a polygenic architecture suggesting that genomic selection, instead of marker assisted selection, might be useful for efficiently improving resistance to one of the most problematic diseases that affects the South American aquaculture.

Fish Skin and Gut Microbiomes Show Contrasting Signatures of Host Species and Habitat.

Teleost fish represent an invaluable repertoire of host species to study the factors shaping animal-associated microbiomes. Several studies have shown that the phylogenetic structure of the fish gut microbiome is driven by species-specific (e.g., host ancestry, genotype, or diet) and habitat-specific (e.g., hydrochemical parameters and bacterioplankton composition) factors. However, our understanding of other host-associated microbial niches, such as the skin mucus microbiome, remains limited. The goal of our study was to explore simultaneously the phylogenetic structure of the fish skin mucus and gut microbiome and compare the effect of species- and habitat-specific drivers on the structure of microbial communities in both tissues. We sampled 114 wild fish from 6 populations of 3 ecologically and phylogenetically contrasting Amazonian teleost species. Water samples were collected at each site, and 10 physicochemical parameters were characterized. The skin mucus, gut, and water microbial communities were characterized using a metabarcoding approach targeting the V3-V4 regions of the 16S rRNA. Our results showed a significant distinction between the phylogenetic profile and diversity of the microbiome from each microbial niche. Skin mucus and bacterioplankton communities were significantly closer in composition than gut and free-living communities. Species-specific factors mostly modulated gut bacterial communities, while the skin mucus microbiome was predominantly associated with environmental physicochemistry and bacterioplankton community structure. These results suggest that the variable skin mucus community is a relevant target for the development of microbial biomarkers of environmental status, while the more conserved gut microbiome is better suited to study long-term host-microbe interactions over evolutionary time scales.IMPORTANCE Whether host-associated microbiomes are mostly shaped by species-specific or environmental factors is still unresolved. In particular, it is unknown to what extent microbial communities from two different host tissues from the same host respond to these factors. Our study is one of the first to focus on the microbiome of teleost fish to shed a light on this topic as we investigate how the phylogenetic structure of microbial communities from two distinct fish tissues are shaped by species- and habitat-specific factors. Our study showed that in contrast to the teleost gut microbiome, skin mucus communities are highly environment dependent. This result has various implications: (i) the skin mucus microbiome should be used, rather than the gut, to investigate bacterial biomarkers of ecosystem perturbance in the wild, and (ii) the gut microbiome is better suited for studies of the drivers of phylosymbiosis, or the coevolution of fish and their symbionts.

Physiological activity of Aloe vera in pacu (Piaractus mesopotamicus) inoculated with Aeromonas hydrophila.

Aloe vera is a traditional medicinal plant; however, its use in fish is fairly recent. We evaluated the effects of dietary A. vera on stress, innate immunity, and energy metabolism in pacu inoculated with Aeromonas hydrophila. For 7 days, 192 fish were fed with diets supplemented with 0% (control), 0.5%, 1.0%, and 2.0% of the plant extract and then inoculated with bacteria and sampled 3, 6, and 24 h later. All concentrations of A. vera reduced basal levels of cortisol, and 1.0% reduced cortisol levels more intensely 3 h after inoculation. A. vera increased the basal respiratory activity of leukocytes/RAL (0.5 and 1.0%), increased the serum levels of lysozyme (1.0 and 2.0%) 6 h after inoculation, and increased the activity of the complement system after 3 h. Spleen somatic index/SSI increased with 1.0 and 2.0% A. vera. A. vera also promoted metabolic effects. It increased basal levels of lipids in the liver and muscle, as well as hepatosomatic index (1.0%) and, 3 h after inoculation, prevented the reduction of serum triglyceride (1.0%) and reduced the mesenteric fat (1.0%). Bacterial inoculation increased RAL from 3 to 24 h and lysozyme levels at 24 h, increased serum cholesterol at 24 h, and decreased serum triglyceride from 3 to 24 h, regardless of A. vera. We concluded that A. vera offered for only 7 days had stress-reducing effects, stimulated innate immunity, protected triglyceride levels in blood, lipid depots in the liver and muscle, and directed the energy mobilization to visceral depots.

Role of neuroendocrine modulation and biochemistry in the sepsis in Piaractus mesopotamicus.

Sepsis is a systemic process with multifactorial pathophysiology that affects most animal species. It is responsible for high rates of morbidity and mortality. This work aimed to study the biochemical and neuroendocrine changes of the sepsis process in Piaractus mesopotamicus after Aeromonas hydrophila inoculation analyzing changes in blood leukocyte and differences in neuroendocrine-biochemical modulation using RNA-seq. Fish showed hypercortisolemia, inhibition of glucose absorption, followed by hypocortisolemia and then hyperglycemia. Thyroid hormones (T3 and T4) showed immediate decrease in serum and T4 increased 6 h post-inoculation (HPI). Sepsis-induced hormonal alterations triggered changes in the metabolic pathways increasing protein and lipid catabolism, use of transient anaerobic glycolysis and liver injury. A reference transcriptome was constructed based on blood leukocytes from P. mesopotamicus. The assembly resulted in total 266,272 contigs with a N50 of 2786 bp. There was a reorganization of plasma membrane of leukocytes at the beginning of the septic process with increased expression of neuroendocrine receptors and with continuous flow of neurotransmitters, hormones and solutes with compensatory regulation at 6 HPI. Three and nine HPI seemed to be critical, the expression of a number of transcription factors was increased, including the modulatory DEGs related to glucocorticoid and thyroid hormones induced and suppressed (FDR < 0.05). Neuroendocrine modulation can regulate leukocytes and biochemical parameters of peripheral blood, being important sources for the study of the pathophysiology of sepsis. These finding highlights the importance of further studies focusing on biochemical-neuroendocrine changes in blood leukocytes and systemic sepsis.

Morphological, microbiological and ultrastructural aspects of sepsis by Aeromonas hydrophila in Piaractus mesopotamicus.

Aeromonas bacteria can cause an infection characterized by septicemia and is one of the most common pathogens in tropical fish. This disease is responsible for high morbidity and mortality rates, causing considerable losses in aquaculture. Thus, the understanding of its pathophysiology is crucial to develop control strategies of this bacterial infection in farmed fish. This study aimed to characterize early pathological aspects of acute sepsis in pacu (Piaractus mesopotamicus) experimentally infected with Aeromonas hydrophila. A total of 160 juvenile pacus were inoculated intraperitoneally with A. hydrophila (1.78 x 109 CFU/mL) and at 0 (control), 1, 3, 6, and 9 hours post-inoculation (hpi), animals were anesthetized and samples were collected for microbiological, light microscopy and transmission electron microscopy (TEM) analyzes. The results showed the occurrence of hemodynamic alterations, such as hemorrhage and congestion, which were observed mainly after 6 and 9 hpi. It was possible to re-isolate Aeromonas at all sampling times except in control group. However, just after 9 hpi it was possible to find the bacteria in all fish and tissues. Light microscopy analyses revealed a degenerative process, necrosis and vascular damage mainly at 6 and 9 hpi. According to the ultrastructural examination, areas of cellular death were identified in all examined tissues, especially at 6 and 9 hpi. However, the most severe, related to necrosis, were observed after 6 and 9 hpi. The findings suggested that this bacterium spreads in the first hpi through the fish organs, mainly affecting spleen, liver and kidney, causing irreversible lesions at the molecular level.

Oral administration of antibiotics increased the potential mobility of bacterial resistance genes in the gut of the fish Piaractus mesopotamicus.

BACKGROUND: Aquaculture is on the rise worldwide, and the use of antibiotics is fostering higher production intensity. However, recent findings suggest that the use of antibiotics comes at the price of increased antibiotic resistance. Yet, the effect of the oral administration of antibiotics on the mobility of microbial resistance genes in the fish gut is not well understood. In the present study, Piaractus mesopotamicus was used as a model to evaluate the effect of the antimicrobial florfenicol on the diversity of the gut microbiome as well as antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) using a metagenomic approach. RESULTS: The total relative abundance of ARGs and MGEs significantly increased during the antibiotic exposure. Additionally, phage integrases, transposases, and transposons flanking ARGs accumulated in the gut microbiome of P. mesopotamicus because of the antibiotic exposure. MGEs co-occurring with ARGs showed a significant positive correlation with the total ARGs found. Furthermore, shifts in the gut microbiome towards well-known putative pathogens such as Salmonella, Plesiomonas, and Citrobacter were observed following florfenicol treatment. Mainly Plesiomonas and Citrobacter harbored genes that code for multidrug and phenicol efflux pumps. Moreover, several genes related to RNA processing and modification, cell motility, SOS response, and extracellular structure were enriched due to the antibiotic application. The observed effects were visible during the complete application phase and disappeared at the post-exposure phase. CONCLUSIONS: Our findings suggest that the oral administration of antibiotics increases the potential for MGE-mediated exchange of ARGs in the gut of fish and could contribute to the enrichment and dispersion of ARGs in aquaculture systems. Importantly, this increase in the potential for ARGs exchange could be an effect of changes in community structure and/or ARG mobilization.

Hematological and immune changes in Piaractus mesopotamicus in the sepsis induced by Aeromonas hydrophila.

The pathogenesis of sepsis involves complex systems and multiple interrelationships between the host and pathogen producing high mortality rates in various animal species. In this study, hematological disturbances, innate immunity and survival during the septic process in Piaractus mesopotamicus inoculated with Aeromonas hydrophila were studied. For this aim, fish blood samples were taken from control and infected groups 1, 3, 6, and 9 h post-inoculation (HPI). Leukogram showed reduction in the number of leukocytes and thrombocytes, followed by cessation of leukocyte chemotaxis 6 HPI and severe morphological changes in leukocytes and erythrocytes. At 3 HPI production of reactive oxygen species increased and at 6 HPI decreased. There was no change in serum lysozyme concentration and lytic activity of the complement system, despite the progressive increase in serum lytic activity and bacterial agglutination. Finally, the changes in clinical signs due to aeromonosis and increasing septicemia resulted in a reduction in survival to 57.14% after 36 HPI. It was possible concluded that these hematological and immune are crucial event in the worsening of sepsis in P. mesopotamicus, and these findings are utility for diagnosing and understanding the pathophysiology sepsis in pacu induced by A. hydrophila.

Conservation status and bio-ecology of Brycon orbignyanus (Characiformes: Bryconidae), an endemic fish species from the Paraná River basin (Brazil) threatened with extinction

Brycon orbignyanus is an endemic species from La Plata basin whose stocks have been presenting significant reductions throughout the Paraná River. Brycon orbignyanus is categorized as an endangered species. This study evaluated aspects of the bio-ecology of this species that may be related to this threat, highlighting its distribution, abundance, and diet as well as the corresponding relationships between its recruitment and flood regimes. Data were obtained from different parts of the upper Paraná River (stretches free and regulated by dams) from 1986 to 2010 with more detailed data collected from the free remnant of this basin. The results indicate that no records for species exist at more than half of the sampling points located in dam-regulated sections of the Paraná River, whereas specimens were collected from 75% sites in the free plain remnant. We observed a remarkable effect of the hydrological regime on recruitment as well as distinct food demands during ontogenetic development, with adults almost exclusively consuming fruits and seeds, revealing that these individuals are supported by riparian vegetation. Thus, it is concluded that changes in the natural flood regime as well as riparian vegetation removal threaten B. orbignyanus populations in the Paraná River basin.(AU)

Brycon orbignyanus é uma espécie endêmica da bacia do rio da Prata, cujos estoques vêm apresentando redução relevante em todo o rio Paraná, e está classificada como espécie ameaçada. Este estudo avaliou aspectos da bio-ecologia da espécie, relacionados a essa ameaça, destacando sua distribuição, abundância, dieta e relação entre o recrutamento e o regime de inundação. Os dados foram obtidos em diferentes partes da bacia do alto rio Paraná (trechos regulados por barragens e trechos livres) de 1986 a 2010, com detalhes no remanescente livre desse segmento (planície de inundação do alto rio Paraná). Os resultados indicam que não existem registros da espécie em mais da metade dos pontos de amostragem localizados nos trechos da bacia do rio Paraná reguladas por barragens, enquanto a espécie foi capturada em 75% dos locais amostrados na planície (remanescente livre de barragem). Foi observado um efeito marcante do regime hidrológico sobre o recrutamento de B. orbignyanus, bem como o uso de diferentes alimentos durante seu desenvolvimento ontogenético, com adultos consumindo quase exclusivamente frutos e sementes, revelando que esses indivíduos são sustentados pela vegetação ripária. Assim, conclui-se que mudanças no regime natural de inundação, bem como a remoção da vegetação ripária, ambas decorrentes de ações antropogênicas, ameaçam B. orbignyanus na bacia do rio Paraná.(AU)

Evaluation antibacterial and antibiofilm activity of the antimicrobial peptide P34 against Staphylococcus aureus and Enterococcus faecalis.

The adhesion ability of bacteria to abiotic surfaces has important implications in food industries, because these organisms can survive for long periods through the biofilm formation. They can be transferred from one place to another in the industry causing contamination of the food processing environment. In this study, the antibacterial and antibiofilm activities of the antimicrobial peptide P34, characterized as a bacteriocin-like substance (BLS P34) were tested against planktonic and sessile cells of Staphylococcus aureus and Enterococcus faecalis isolated from foods. The BLS P34 showed inhibitory effect against all planktonic cells of E. faecalis. The inhibition of biofilm formation and the eradication of pre-formed biofilm were evaluated with the crystal violet assay and with the reduction of 3-bromide [4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium. The BLS P34 promoted a reduction of percentage of adhered microbial cells on the surface, not being able to perform the complete elimination of biofilm formation. The metabolic activity of S. aureus biofilms decreased considerably between 41-95%. However, E. faecalis cells showed up metabolically stimulated. The BLS P34 has the potential antibiofilm for the species S. aureus. Studies suggest more detailed approaches to a better understanding of the interactions between the antimicrobial and bacterial cells within the biofilm structure.

An Unexpected Fish Bite.

We report a case of a 22-month-old boy who suffered a piranha bite, amputating his fourth distal phalanx. His finger was irrigated and closed with a flap under sedation, but we could find little evidence during a brief literature search of what prophylactic antibiotics, if any, to provide. Because reports of infections from piranha bites are lacking, we examined studies evaluating oral flora from fish, as well as flora cultured from aquariums. In conclusion, if infection is present or prophylaxis is strongly desired, a broad spectrum oral antibiotic that covers Gram-positive and Gram-negative bacteria including Pseudomonas and Aeromonas species, such as ciprofloxacin, would be recommended; however, there is little evidence to suggest that prophylactic antibiotics provide benefit over appropriate wound management.

Microbiological quality of trahira fish (Hoplias malabaricus) from Baixada Maranhense, municipality of São Bento, MA / Qualidade microbiológica de peixe traíra (Hoplias malabaricus) proveniente da Baixada Maranhense, município de São Bento, MA

Fish are considered rich sources of nutrients. Health care throughout its production chain aims to ensure quality, minimizing the risks of transmission of foodborne diseases. In order to evaluate the microbiological quality of trahira fish (H. malabaricus), 40 samples were analysed for Most Probable Number (MPN) of coliforms at 45ºC, counts of aerobic mesophilic bacteria and Staphylococcus spp., identification of E. coli, Salmonella spp. and Aeromonas spp.. Analyses were conducted according to official methods, procedures, and recommendations. Microbiological results showed coliform values at 45 °C ranging from <3 to > 1.1 × 103 MPN/g, presence of E. coli in 14 (35%) samples, counts of mesophilic aerobic bacteria from 9 × 102 to 109 CFU/g and absence of coagulase positive Staphylococcus aureus. Salmonella spp. was detected in 2 (5%) samples, which is in disagreement with the standards required by the RDC Nº 12 of January, 2001 (ANVISA) regarding Aeromonas spp. In total, 36 (90%) samples were contaminated, 7 (19.4%) by A. cavie and 29 (80.6%) by A. hidrophila. The results of this research showed unsatisfactory hygienic and sanitary conditions of fish from the municipality of São Bento (MA), exposing consumers to the risk of foodborne diseases.(AU)

Os peixes são considerados fontes ricas de nutrientes. Cuidados sanitários durante toda a sua cadeia produtiva visam garantir a qualidade, minimizando os riscos de transmissão de doenças alimentares. Com objetivo de avaliar a qualidade microbiológica de peixes traíra (H. malabaricus), foram analisadas 40 amostras quanto à determinação do Número Mais Provável (NPM) de coliformes a 45 ºC, contagem de bactérias aeróbias mesófilas e Staphylococcus spp. e identificação de E. coli; Salmonella spp. e Aeromonas spp. As análises foram processadas conforme métodos, procedimentos e recomendações oficiais. Os resultados microbiológicos mostraram valores de coliformes a 45 ºC variando de < 3 a > 1,1 × 103 NMP/g, presença de E. coli em 14 (35%) amostras, contagens de bactérias aeróbias mesófilas de 9 × 102 a 109 UFC/g e ausência de Staphylococcus aureus coagulase positivo. Detectou-se Salmonella spp. em 2 (5%) amostras, portanto, em desacordo com os padrões exigidos pela RDC Nº 12 de janeiro de 2001 (ANVISA) e para Aeromonas spp. No total, 36 (90%) amostras estavam contaminadas, sendo 7 (19,4%) por A. cavie e 29 (80,6%) por A. hidrophila. Os resultados da pesquisa demonstram que os peixes provenientes do município de São Bento (MA) apresentaram condições higiênico-sanitárias insatisfatórias, expondo os consumidores a risco de doenças veiculadas por alimentos.(AU)

pH drop impacts differentially skin and gut microbiota of the Amazonian fish tambaqui (Colossoma macropomum).

Aquatic organisms are increasingly exposed to lowering of environmental pH due to anthropogenic pressure (e.g. acid rain, acid mine drainages). Such acute variations trigger imbalance of fish-associated microbiota, which in turn favour opportunistic diseases. We used the tambaqui (Colossoma macropomum), an Amazonian fish tolerant to significant pH variation in its natural environment, to assess the response of fish endogenous microbiota to acute short-term acid stress. We exposed 36 specimens of tambaquis to acidic water (pH 4.0) over 2 consecutive weeks and sampled cutaneous mucus, feces and water at 0, 7 &14 days. The 16S RNA hypervariable region V4 was sequenced on Illumina MiSeq. After two weeks of acidic exposure, fecal and skin microbiota taxonomic structures exhibited different patterns: skin microbiota was still exhibiting a significantly disturbed composition whereas fecal microbiota recovered a similar composition to control group, thus suggesting a stronger resilience capacity of the intestinal microbiota than cutaneous microbiota.

Impact of stress on Aeromonas diversity in tambaqui (Colossoma macropomum) and lectin level change towards a bacterial challenge.

Tambaqui (Colossoma macropomum) is among the most cultivated fish species in tropical countries. Stress is the main cause of disease in fish farms. The genus Aeromonas is a common causative agent of fish diseases. This work reports the identification of Aeromonas species colonizing gills of C. macropomum submitted or not to a confinement stress. We also evaluated changes in serum levels of lectins (carbohydrate-binding proteins that are components of fish immune system) in tambaqui submitted to a challenge using two isolated Aeromonas strains. Gill tissues from stressed and unstressed fishes were used to isolate Aeromonas. Then 72 Aeromonas strains were isolated, 97% being from stressed fishes. Among these, 63 were identified at species level and 6 were classified as atypical Aeromonas strains. The most prevalent species were Aeromonas bestiarum and Aeromonas caviae and their strains were used in bacterial challenges. The lectin serum levels significantly increased after 24 h of infection with A. bestiarum; however, no significant increase was found for infection with A. caviae. In conclusion, C. macropomum gills are susceptible to colonization by different Aeromonas species, mainly at confinement stressful conditions, and serum lectins may have a role in the acute immunological response towards infection by A. bestiarum.