Enhanced neurotoxic effect of PCB-153 when co-exposed with polystyrene nanoplastics in zebrafish larvae.

Nanoplastics (NPs) and persistent organic pollutants such as polychlorinated biphenyls (PCBs) are ubiquitous aquatic pollutants. The coexistence of these pollutants in the environment emphasises the need to study their combined toxicity. NPs can cross biological membranes and act as vectors for other pollutants, whereas PCBs are known for their ability to bioaccumulate and biomagnify. The present work aimed to study the combined toxicity of polystyrene NPs and PCB-153 using physiological (development, heart rate, respiration), behavioural (swimming behaviour) and molecular (transcriptome) endpoints in zebrafish larvae. The results show that exposure to NPs, PCB and their mixture significantly affected the development and respiration in zebrafish larvae. Larvae co-exposed to NPs and PCB exhibited significant hyperlocomotion, whereas no such effect was observed after exposure to NPs or PCB alone. The transcriptomic results revealed that NPs exposure significantly affected several pathways associated with DNA compaction and nucleosome assembly, whereas PCB exposure significantly affected critical neurogenic pathways. In contrast, co-exposure to NPs and PCB generated multi-faceted toxicity and suppressed neurobehavioural, immune-related and detoxification pathways. The study highlights the complex interplay between NPs and PCBs, and documents how the two toxicants in combination give a stronger effect than the single toxicants alone. Understanding the mixture toxicity of these two pollutants is important to assess the environmental risks and developing effective management strategies, ultimately safeguarding ecosystems and human health.

Mixture toxicity of 6PPD-quinone and polystyrene nanoplastics in zebrafish.

Plastic pollution, including micro- and nanoplastics, is a growing concern. Tyre-wear particles (TWPs) are the second largest source of microplastics in the ocean following abrasion of synthetic fibres. In addition to the particles themselves, TWPs contain many harmful chemicals, including 6PPD. This chemical reacts with atmospheric ozone and forms the toxic compound 6PPD-quinone (6PPDq), which poses a danger to aquatic life. There is a knowledge gap in understanding risks associated with the combined toxicity of nanoplastics (NPs) and 6PPDq. The present study aimed to investigate the toxicity of NPs and 6PPDq on adult zebrafish using phenotypic (behaviour, histology) and transcriptomic endpoints. Zebrafish were exposed to four treatments: control (contaminant-free), 50 µg/L 6PPDq, 3 mg/L polystyrene (PS)-NPs, and a combination of 50 µg/L 6PPDq and 3 mg/L PS-NPs. We did not observe locomotory dysregulation in zebrafish exposed to NPs. However, we found significant hyperlocomotion in zebrafish exposed to 6PPDq and this effect was even more substantial after co-exposure with PS-NPs. This study explores the molecular mechanisms behind these effects, identifying genes associated with neurotransmitters and fatty acid metabolism that were dysregulated by the co-exposure. Transcriptomic analysis further showed that both 6PPDq and PS-NPs impacted cellular processes associated with sterol biosynthesis, cholesterol metabolism, and muscle tissue development. The effects on these mechanisms were stronger in co-exposed zebrafish, indicating a heightened risk to cellular integrity and mitochondrial dysfunction. These results highlight the significance of mixture toxicity when studying the effects of NPs and associated chemicals like 6PPDq.

Gill epithelial cell line ASG-10 from Atlantic salmon as a new research tool for solving water quality challenges in aquaculture.

Here we evaluated the gill epithelial cell line ASG-10 from Atlantic salmon, as an in vitro model for research on known water quality challenges in aquaculture. Ammonia/ammonium (NH3/NH4+), a recognized challenge in water-intensive recirculating aquaculture systems (RAS), induced lysosomal vacuolization, reduced protein degradation and cell migration of the ASG-10 cells. Aluminium (Aln+), another challenge in freshwater aquaculture facilities had only minor effects. Next, we investigated the tolerance for direct water exposure of ASG-10. The cells tolerated water with osmolarity between 169 and 419 mOsmol/kg for 24 h. However, cells exposed for 3 h to water at 863 mOsmol/kg changed cellular morphology and induced gene expression related to stress (gpx1, casp3, hsp70), and after 24 h exposure cellular viability was severely reduced. Nevertheless, when the cells were grown in transwell inserts, they tolerated 863 mOsmol/kg for 3 h and induction of stress response associated genes was considerably reduced. Lastly, the ASG-10 cells were exposed to water samples, with no known quality issues, from different aquaculture facilities. The cells showed no differences in viability or morphology compared to their representative control. In conclusion, the ASG-10 cell line is a promising in vitro model to study water quality challenges and whole water samples.

Unravelling the temporal and spatial variation of fungal phylotypes from embryo to adult stages in Atlantic salmon.

Early microbial colonization has a profound impact on host physiology during different stages of ontogeny. Although several studies have focused on early bacterial colonization and succession, the composition and role of fungal communities are poorly known in fish. Here, we sequenced the internal transcribed spacer 2 (ITS2) region of fungi to profile the mycobiome associated with the eggs, hatchlings and intestine of Atlantic salmon at various freshwater and marine stages. In most of the stages studied, fungal diversity was lower than bacterial diversity. There were several stage-specific fungal phylotypes belonging to different stages of ontogeny but some groups, such as Candida tropicalis, Saccharomyces cerevisiae, Alternaria metachromatica, Davidiella tassiana and Humicola nigrescens, persisted during successive stages of ontogeny. We observed significant changes in the intestinal fungal communities during the first feeding. Prior to first feeding, Humicola nigrescens dominated, but Saccharomyces cerevisiae (10 weeks post hatch) and Candida tropicalis (12 weeks post hatch) became dominant subsequently. Seawater transfer resulted in a decrease in alpha diversity and an increase in Candida tropicalis abundance. We also observed notable variations in beta diversity and composition between the different farms. Overall, the present study sheds light on the fungal communities of Atlantic salmon from early ontogeny to adulthood. These novel findings will also be useful in future studies investigating host-microbiota interactions in the context of developing better nutritional and health management strategies for Atlantic salmon farming.

Ecotoxicological assessment of Cu-rich acid mine drainage of Sulitjelma mine using zebrafish larvae as an animal model.

Acid mine drainage (AMD) is widely acknowledged as a substantial threat to the biodiversity of aquatic ecosystems. The present study aimed to study the toxicological effects of Cu-rich AMD from the Sulitjelma mine in zebrafish larvae. The AMD from this mine was found to contain elevated levels of dissolved metals including Mg (46.7 mg/L), Al (20.2 mg/L), Cu (18.3 mg/L), Fe (19.8 mg/L) and Zn (10.6 mg/L). To investigate the toxicological effects, the study commenced by exposing zebrafish embryos to various concentrations of AMD (ranging from 0.75% to 9%) to determine the median lethal concentration (LC50). Results showed that 96 h LC50 for zebrafish larvae following AMD exposure was 2.86% (95% CI: 2.32-3.52%). Based on acute toxicity results, zebrafish embryos (<2 hpf) were exposed to 0.1% AMD (Cu: 21.7 µg/L) and 0.45% AMD (Cu: 85.7 µg/L) for 96 h to assess development, swimming behaviour, heart rate, respiration and transcriptional responses at 116 hpf. Light microscopy results showed that both 0.1% and 0.45% AMD reduced the body length, eye size and swim bladder area of zebrafish larvae and caused phenotypic abnormalities. Swimming behaviour results showed that 0.45% AMD significantly decreased the locomotion of zebrafish larvae. Heart rate was not affected by AMD exposure. Furthermore, exposure caused a significant increase in oxygen consumption indicating vascular stress in developing larvae. Taken altogether, the study shows that even heavily diluted AMD with environmentally relevant levels of Cu caused toxicity in zebrafish larvae.

Host habitat rather than evolutionary history explains gut microbiome diversity in sympatric stickleback species.

Host-associated microbiota can influence host phenotypic variation, fitness and potential to adapt to local environmental conditions. In turn, both host evolutionary history and the abiotic and biotic environment can influence the diversity and composition of microbiota. Yet, to what extent environmental and host-specific factors drive microbial diversity remains largely unknown, limiting our understanding of host-microbiome interactions in natural populations. Here, we compared the intestinal microbiota between two phylogenetically related fishes, the three-spined stickleback (Gasterosteus aculeatus) and the nine-spined stickleback (Pungitius pungitius) in a common landscape. Using amplicon sequencing of the V3-V4 region of the bacterial 16S rRNA gene, we characterised the α and ß diversity of the microbial communities in these two fish species from both brackish water and freshwater habitats. Across eight locations, α diversity was higher in the nine-spined stickleback, suggesting a broader niche use in this host species. Habitat was a strong determinant of ß diversity in both host species, while host species only explained a small fraction of the variation in gut microbial composition. Strong habitat-specific effects overruled effects of geographic distance and historical freshwater colonisation, suggesting that the gut microbiome correlates primarily with local environmental conditions. Interestingly, the effect of habitat divergence on gut microbial communities was stronger in three-spined stickleback than in nine-spined stickleback, possibly mirroring the stronger level of adaptive divergence in this host species. Overall, our results show that microbial communities reflect habitat divergence rather than colonisation history or dispersal limitation of host species.

CircPrime: a web-based platform for design of specific circular RNA primers.

BACKGROUND: Circular RNAs (circRNAs) are covalently closed-loop RNAs with critical regulatory roles in cells. Tens of thousands of circRNAs have been unveiled due to the recent advances in high throughput RNA sequencing technologies and bioinformatic tools development. At the same time, polymerase chain reaction (PCR) cross-validation for circRNAs predicted by bioinformatic tools remains an essential part of any circRNA study before publication. RESULTS: Here, we present the CircPrime web-based platform, providing a user-friendly solution for DNA primer design and thermocycling conditions for circRNA identification with routine PCR methods. CONCLUSIONS: User-friendly CircPrime web platform ( http://circprime.elgene.net/ ) works with outputs of the most popular bioinformatic predictors of circRNAs to design specific circular RNA primers. CircPrime works with circRNA coordinates and any reference genome from the National Center for Biotechnology Information database).

The novel circular RNA CircMef2c is positively associated with muscle growth in Nile tilapia.

Muscle growth in teleosts is a complex biological process orchestrated by numerous protein-coding genes and non-coding RNAs. A few recent studies suggest that circRNAs are involved in teleost myogenesis, but the molecular networks involved remain poorly understood. In this study, an integrative omics approach was used to determine myogenic circRNAs in Nile tilapia by quantifying and comparing the expression profile of mRNAs, miRNAs, and circRNAs in fast muscle from full-sib fish with distinct growth rates. There were 1947 mRNAs, 9 miRNAs, and 4 circRNAs differentially expressed between fast- and slow-growing individuals. These miRNAs can regulate myogenic genes and have binding sites for the novel circRNA circMef2c. Our data indicate that circMef2c may interact with three miRNAs and 65 differentially expressed mRNAs to form multiple competing endogenous RNA networks that regulate growth, thus providing novel insights into the role of circRNAs in the regulation of muscle growth in teleosts.

Polystyrene nanoplastics enhance the toxicological effects of DDE in zebrafish (Danio rerio) larvae.

Anthropogenic releases of plastics, persistent organic pollutants (POPs), and heavy metals can impact the environment, including aquatic ecosystems. Nanoplastics (NPs) have recently emerged as pervasive environmental pollutants that have the ability to adsorb POPs and can cause stress in organisms. Among POPs, DDT and its metabolites are ubiquitous environmental pollutants due to their long persistence. Despite the discontinued use of DDT in Europe, DDT and its metabolites (primarily p,p'-DDE) are still found at detectable levels in fish feed used in salmon aquaculture. Our study aimed to look at the individual and combined toxicity of NPs (50 mg/L polystyrene) and DDE (100 µg/L) using zebrafish larvae as a model. We found no significant morphological, cardiac, respiratory, or behavioural changes in zebrafish larvae exposed to NPs alone. Conversely, morphological, cardiac and respiratory alterations were observed in zebrafish larvae exposed to DDE and NPs + DDE. Interestingly, behavioural changes were only observed in zebrafish larvae exposed to NPs + DDE. These findings were supported by RNA-seq results, which showed that some cardiac, vascular, and immunogenic pathways were downregulated only in zebrafish larvae exposed to NPs + DDE. In summary, we found an enhanced toxicological impact of DDE when combined with NPs.

Toxicological effects of 6PPD and 6PPD quinone in zebrafish larvae.

N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD) is the most widely used antioxidant in automobile tyres and many rubber products. We investigated the impact of 6PPD and 6PPD quinone on acute toxicity, morphology, swimming behaviour, heart rate, and oxygen consumption in zebrafish larvae. Zebrafish embryos were exposed to 6PPD and 6PPD quinone at concentrations of 1, 10, and 25 µg/L during the development period of 1-96 hpf. In the present study, 6PPD quinone was found to be toxic to zebrafish larvae with a 24 h LC50 of 308.67 µg/L. No significant mortality was observed at any of the tested concentrations. A dose-dependent reduction in swimming performance was observed in the exposed larvae at 116 hpf for both toxicants. Overall, our study shows that exposure of zebrafish embryos to 6PPD and 6PPD quinone at environmentally relevant concentrations (1 µg/L) does not affect its behaviour. However, exposure to higher but still sublethal concentrations of 6PPD and 6PPD quinone (10 and 25 µg/L) can affect behavioural endpoints. These findings reveal the toxicity of 6PPD and 6PPD quinone to early life stages of fish.

Structural Identification of the Pacemaker Cells and Expression of Hyperpolarization-Activated Cyclic Nucleotide-Gated (HCN) Channels in the Heart of the Wild Atlantic Cod, Gadus morhua (Linnaeus, 1758).

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are proteins that contain highly conserved functional domains and sequence motifs that are correlated with their unique biophysical activities, to regulate cardiac pacemaker activity and synaptic transmission. These pacemaker proteins have been studied in mammalian species, but little is known now about their heart distribution in lower vertebrates and c-AMP modulation. Here, we characterized the pacemaker system in the heart of the wild Atlantic cod (Gadus morhua), with respect to primary pacemaker molecular markers. Special focus is given to the structural, ultrastructural and molecular characterization of the pacemaker domain, through the expression of HCN channel genes and the immunohistochemistry of HCN isoforms, including the location of intracardiac neurons that are adjacent to the sinoatrial region of the heart. Similarly to zebrafish and mammals, these neurons are immunoreactive to ChAT, VAChT and nNOS. It has been shown that cardiac pacemaking can be modulated by sympathetic and parasympathetic pathways, and the existence of intracardiac neurons projecting back to the central nervous system provide a plausible link between them.

Zebrafish intestinal transcriptome highlights subdued inflammatory responses to dietary soya bean and efficacy of yeast ß-glucan.

Anti-nutritional factors in dietary components can have a negative impact on the intestinal barrier. Here, we present soya bean-induced changes in the intestine of juvenile zebrafish and the effect of yeast ß-glucan through a transcriptomic approach. The inclusion of soya bean meal affected the expression of several intestinal barrier function-related genes like arl4ca, rab25b, rhoub, muc5ac, muc5d, clcn2c and cltb in zebrafish. Several metabolic genes like cyp2x10.2, cyp2aa2, aldh3a2b, crata, elovl4, elovl6, slc51a, gpat2 and ATP-dependent peptidase activity (lonrf, clpxb) were altered in the intestinal tissue. The expression of immune-related genes like nlrc3, nlrp12, gimap8, prdm1 and tph1a, and genes related to cell cycle, DNA damage and DNA repair (e.g. spo11, rad21l1, nabp1b, spata22, tdrd9) were also affected in the soya bean fed group. Furthermore, our study suggests the plausible effect of yeast ß-glucan through the modulation of several genes that regulate immune responses and barrier integrity. Our findings indicate a subdued inflammation in juvenile zebrafish fed soya bean meal and the efficacy of ß-glucan to counter these subtle inflammatory responses.

Study on pathogenicity and characterization of disease causing fungal community associated with cultured fish of Kashmir valley, India.

Cultured fisheries of developing countries are continously challenged by a number of pathogenic microbes. Among microbial diseases, fungal and fungal like pathogen outbreaks lead to negative social and economic impacts on stakeholders. The cultured fisheries of Kashmir valley are also facing challenge from fungal pathogens, leading to tremendous socio-economic lossess to the fish farmer community hence, yearns to boost the sector with efficient management strategy. Our study was aimed at investigating the diversity of fungal communities infecting cultured rainbow trout and carp fish species. We employed classical microbiology, macro and micro morphological characteristics, and molecular analysis (multilocus typing) for fungal identification. Also histopathological approach was used to examine the pathogenicity patterns of diverse fungal groups. The study revealed that the infection in fish was predisposed to both superficial as well as visceral organs. However, skin, gills and head were predominantly infected compared to internal organs. The microbiological investigation of infected fish by culture dependent approach helped us to obtain the total of 250 fungal isolates. Out of these isolates, 21 different species were identified belonging to three diverse fungal groups which mostly included 14 species among Ascomycetes, 03 species of Oomycetes and 04 species of Zygomycetes. The majority of fungi which were infectious to cultured fish of valley are biotrophic or opportunistic soil fungi, and some of them being exclusive pathogens of fish.

Management of Hypercholesterolemia Through Dietary ß-glucans-Insights From a Zebrafish Model.

Consumption of lipid-rich foods can increase the blood cholesterol content. ß-glucans have hypocholesterolemic effect. However, subtle changes in their molecular branching can influence bioactivity. Therefore, a comparative investigation of the cholesterol-lowering potential of two ß-glucans with different branching patterns and a cholesterol-lowering drug, namely simvastatin was undertaken employing the zebrafish (Danio rerio) model of diet-induced hypercholesterolemia. Fish were allocated to 5 dietary treatments; a control group, a high cholesterol group, two ß-glucan groups, and a simvastatin group. We investigated plasma total cholesterol, LDL and HDL cholesterol levels, histological changes in the tissues, and explored intestinal transcriptomic changes induced by the experimental diets. Dietary cholesterol likely caused the suppression of endogenous cholesterol biosynthesis, induced dysfunction of endoplasmic reticulum and mitochondria, and altered the histomorphology of the intestine. The two ß-glucans and simvastatin significantly abated the rise in plasma cholesterol levels and restored the expression of specific genes to alleviate the endoplasmic reticulum-related effects induced by the dietary cholesterol. Furthermore, the distinct patterns of transcriptomic changes in the intestine elicited by the oat and microalga ß-glucans impacted processes such as fatty acid metabolism, protein catabolic processes, and nuclear division. Oat and microalgal ß-glucans also altered the pattern of lipid deposition in the liver. Our study provides insights into the effectiveness of different ß-glucans to alleviate dysfunctions in lipid metabolism caused by dietary cholesterol.

Intestinal Transcriptome Analysis Reveals Soy Derivative-Linked Changes in Atlantic Salmon.

Intestinal inflammation in farmed fish is a non-infectious disease that deserves attention because it is a major issue linked to carnivorous fishes. The current norm is to formulate feeds based on plant-derived substances, and the ingredients that have antinutritional factors are known to cause intestinal inflammation in fishes such as Atlantic salmon. Hence, we studied inflammatory responses in the distal intestine of Atlantic salmon that received a feed rich in soybean derivatives, employing histology, transcriptomic and flow cytometry techniques. The fish fed on soy products had altered intestinal morphology as well as upregulated inflammation-associated genes and aberrated ion transport-linked genes. The enriched pathways for the upregulated genes were among others taurine and hypotaurine metabolism, drug metabolism-cytochrome P450 and steroid biosynthesis. The enriched gene ontology terms belonged to transmembrane transporter- and channel-activities. Furthermore, soybean products altered the immune cell counts; lymphocyte-like cell populations were significantly higher in the whole blood of fish fed soy products than those of control fish. Interestingly, the transcriptome of the head kidney did not reveal any differential gene expression, unlike the observations in the distal intestine. The present study demonstrated that soybean derivatives could evoke marked changes in intestinal transport mechanisms and metabolic pathways, and these responses are likely to have a significant impact on the intestine of Atlantic salmon. Hence, soybean-induced enteritis in Atlantic salmon is an ideal model to investigate the inflammatory responses at the cellular and molecular levels.

Pseudozyma Priming Influences Expression of Genes Involved in Metabolic Pathways and Immunity in Zebrafish Larvae.

Fungi, particularly yeasts, are known essential components of the host microbiota but their functional relevance in development of immunity and physiological processes of fish remains to be elucidated. In this study, we used a transcriptomic approach and a germ-free (GF) fish model to determine the response of newly hatched zebrafish larvae after 24 h exposure to Pseudozyma sp. when compared to conventionally-raised (CR) larvae. We observed 59 differentially expressed genes in Pseudozyma-exposed GF zebrafish larvae compared to their naïve control siblings. Surprisingly, in CR larvae, there was not a clear transcriptome difference between Pseudozyma-exposed and control larvae. Differentially expressed genes in GF larvae were involved in host metabolic pathways, mainly peroxisome proliferator-activated receptors, steroid hormone biosynthesis, drug metabolism and bile acid biosynthesis. We also observed a significant change in the transcript levels of immune-related genes, namely complement component 3a, galectin 2b, ubiquitin specific peptidase 21, and aquaporins. Nevertheless, we did not observe any significant response at the cellular level, since there were no differences between neutrophil migration or proliferation between control and yeast-exposed GF larvae. Our findings reveal that exposure to Pseudozyma sp. may affect metabolic pathways and immune-related processes in germ-free zebrafish, suggesting that commensal yeast likely play a significant part in the early development of fish larvae.

Dietary inclusion of plant ingredients induces epigenetic changes in the intestine of zebrafish.

Epigenetic modifications, such as DNA methylation, can be regulated by nutrition and dietary factors. There has been a large increase in the use of sustainable plant-based protein sources in fish feed due to limitations of fishmeal resources, which are needed to sustain a rapidly growing aquaculture industry. With this major transition from marine ingredients to plant-based diets, fish are abruptly introduced to changes in dietary composition and exposed to a variety of phytochemicals, some of which known to cause epigenetic changes in mammals. However, the effect of plant ingredients on the epigenome of fish is barely understood. In the present study, the nutriepigenomic effects of the addition of pea, soy, and wheat gluten protein concentrate to aquafeeds were investigated using zebrafish as a model. A genome-wide analysis of DNA methylation patterns was performed by reduced representation bisulphite sequencing to examine global epigenetic alterations in the mid intestine after a 42-day feeding trial. We found that inclusion of 30% of wheat gluten, pea and soy protein concentrate in the diet induced epigenetic changes in the mid intestine of zebrafish. A large number of genes and intergenic regions were differentially methylated with plant-based diets. The genes concerned were related to immunity, NF-κB system, ubiquitin-proteasome pathway, MAPK pathway, and the antioxidant defence system. Epigenetic regulation of several biological processes, including neurogenesis, cell adhesion, response to stress and immunity was also observed. Ultimately, the observed epigenetic changes may enable zebrafish to rapidly regulate inflammation and maintain intestinal homoeostasis when fed plant protein-based diets.

Macroalga-Derived Alginate Oligosaccharide Alters Intestinal Bacteria of Atlantic Salmon.

Prebiotics are substrates intended to sculpt gut microbial communities as they are selectively utilized by the microorganisms to exert beneficial health effects on hosts. Macroalga-derived oligosaccharides are candidate prebiotics, and herein, we determined the effects of Laminaria sp.-derived alginate oligosaccharide (AlgOS) on the distal intestinal microbiota of Atlantic salmon (Salmo salar). Using a high-throughput 16S rRNA gene amplicon sequencing technique, we investigated the microbiota harbored in the intestinal content and mucus of the fish offered feeds supplemented with 0.5 and 2.5% AlgOS. We found that the prebiotic shifts the intestinal microbiota profile; alpha diversity was significantly reduced with 2.5% AlgOS while with 0.5% AlgOS the alteration occurred without impacting the bacterial diversity. Beta diversity analysis indicated the significant differences between control and prebiotic-fed groups. The low supplementation level of AlgOS facilitated the dominance of Proteobacteria (including Photobacterium phosphoreum, Aquabacterium parvum, Achromobacter insolitus), and Spirochaetes (Brevinema andersonii) in the content or mucus of the fish, and few of these bacteria (Aliivibrio logei, A. parvum, B. andersonii, A. insolitus) have genes associated with butyrate production. The results indicate that the low inclusion of AlgOS can plausibly induce a prebiotic effect on the distal intestinal microbiota of Atlantic salmon. These findings can generate further interest in the potential of macroalgae-derived oligosaccharides for food and feed applications.

Exposure to Yeast Shapes the Intestinal Bacterial Community Assembly in Zebrafish Larvae.

Establishment of the early-life gut microbiota has a large influence on host development and succession of microbial composition in later life stages. The effect of commensal yeasts - which are known to create a conducive environment for beneficial bacteria - on the structure and diversity of fish gut microbiota still remains unexplored. The present study examined the intestinal bacterial community of zebrafish (Danio rerio) larvae exposed to two fish-derived yeasts by sequencing the V4 hypervariable region of bacterial 16S rRNA. The first stage of the experiment (until 7 days post-fertilization) was performed in cell culture flasks under sterile and conventional conditions for germ-free (GF) and conventionally raised (CR) larvae, respectively. The second phase was carried out under standard rearing conditions, for both groups. Exposure of GF and CR zebrafish larvae to one of the yeast species Debaryomyces or Pseudozyma affected the bacterial composition. Exposure to Debaryomyces resulted in a significantly higher abundance of core bacteria. The difference was mainly due to shifts in relative abundance of taxa belonging to the phylum Proteobacteria. In Debaryomyces-exposed CR larvae, the significantly enriched taxa included beneficial bacteria such as Pediococcus and Lactococcus (Firmicutes). Furthermore, most diversity indices of bacterial communities in yeast-exposed CR zebrafish were significantly altered compared to the control group. Such alterations were not evident in GF zebrafish. The water bacterial community was distinct from the intestinal microbiota of zebrafish larvae. Our findings indicate that early exposure to commensal yeast could cause differential bacterial assemblage, including the establishment of potentially beneficial bacteria.

The Intestinal Mycobiota in Wild Zebrafish Comprises Mainly Dothideomycetes While Saccharomycetes Predominate in Their Laboratory-Reared Counterparts.

As an integral part of the resident microbial community of fish intestinal tract, the mycobiota is expected to play important roles in health and disease resistance of the host. The composition of the diverse fungal communities, which colonize the intestine, is greatly influenced by the host, their diet and geographic origin. Studies of fungal communities are rare and the majority of previous studies have relied on culture-based methods. In particular, fungal communities in fish are also poorly characterized. The aim of this study was to provide an in-depth overview of the intestinal mycobiota in a model fish species (zebrafish, Danio rerio) and to determine differences in fungal composition between wild and captive specimens. We have profiled the intestinal mycobiota of wild-caught (Sharavati River, India), laboratory-reared (Bodø, Norway) and wild-caught-laboratory-kept (Uttara, India) zebrafish by sequencing the fungal internal transcribed spacer 2 region on the Illumina MiSeq platform. Wild fish were exposed to variable environmental factors, whereas both laboratory groups were kept in controlled conditions. There were also differences in husbandry practices at Bodø and Uttara, particularly diet. Zebrafish from Bodø were reared in the laboratory for over 10 generations, while wild-caught-laboratory-kept fish from Uttara were housed in the laboratory for only 2 months before sample collection. The intestine of zebrafish contained members of more than 15 fungal classes belonging to the phyla Ascomycota, Basidiomycota, and Zygomycota. Fungal species richness and diversity distinguished the wild-caught and laboratory-reared zebrafish communities. Wild-caught zebrafish-associated mycobiota comprised mainly Dothideomycetes in contrast to their Saccharomycetes-dominated laboratory-reared counterparts. The predominant Saccharomycetes in laboratory-reared fish belonged to the saprotrophic guild. Another characteristic feature of laboratory-reared fish was the significantly higher abundance of Cryptococcus (Tremellomycetes) compared to wild fish. This pioneer study has shed light into the differences in the intestinal fungal communities of wild-caught and laboratory-reared zebrafish and the baseline data generated will enrich our knowledge on fish mycobiota.