Dietary Protein Quality Affects the Interplay between Gut Microbiota and Host Performance in Nile Tilapia.

Dietary protein quality plays a key role in maintaining intestinal mucosal integrity, but also modulates the growth of luminal microorganisms. This work assessed the effect of dietary protein sources on the performance, gut morphology, and microbiome in Nile tilapia. Four isonitrogenous and isolipidic diets comprising equivalent amounts of the protein supply derived from either PLANT, ANIMAL, INSECT, or BACTERIAL (bacterial biomass) sources were fed to triplicate groups of fish (IBW: 12 g) for 46 days. Fish fed the ANIMAL and BACTERIAL diets showed significantly higher weight gains than those fed the PLANT and INSECT diets (p < 0.05). Relative abundance at the phylum level showed that Bacteroidetes, Fusobacteria, and Proteobacteria were the more abundant phyla in tilapia's intestine, while Cetobacterium was the most representative genus in all treatments. Interesting patterns were observed in the correlation between amino acid intake and genus and species abundance. Metabolism prediction analysis showed that BACTERIAL amine and polyamine degradation pathways are modulated depending on diets. In conclusion, different protein sources modulate the relationship between bacteria functional pathways and amino acid intake.

Modulatory effect of Gracilaria gracilis on European seabass gut microbiota community and its functionality.

Seaweeds are an important source of nutrients and bioactive compounds and have a high potential as health boosters in aquaculture. This study evaluated the effect of dietary inclusion of Gracilaria gracilis biomass or its extract on the European seabass (Dicentrarchus labrax) gut microbial community. Juvenile fish were fed a commercial-like diet with 2.5% or 5% seaweed biomass or 0.35% seaweed extract for 47 days. The gut microbiome was assessed by 16S rRNA amplicon sequencing, and its diversity was not altered by the seaweed supplementation. However, a reduction in Proteobacteria abundance was observed. Random forest analysis highlighted the genera Photobacterium, Staphylococcus, Acinetobacter, Micrococcus and Sphingomonas, and their abundances were reduced when fish were fed diets with algae. SparCC correlation network analysis suggested several mutualistic and other antagonistic relationships that could be related to the predicted altered functions. These pathways were mainly related to the metabolism and biosynthesis of protective compounds such as ectoine and were upregulated in fish fed diets supplemented with algae. This study shows the beneficial potential of Gracilaria as a functional ingredient through the modulation of the complex microbial network towards fish health improvement.

Physiological Response of Atlantic Salmon (Salmo salar) to Long-Term Exposure to an Anesthetic Obtained from Heterosigma akashiwo.

Despite the invaluable role of anesthetics as a tool for ensuring animal welfare in stressful situations, there is currently a lack of anesthetic drugs that meet the requirements of intensive aquaculture. In response to the growing interest in anesthetic substances of natural origin, this study evaluated the physiological and health impact of an anesthetic based on an extract of the microalga Heterosigma akashiwo on juvenile salmon (Salmo salar) exposed for a period of 72 h. To simulate a condition closer to reality where fish are subjected to stimuli (e.g., transport), the animals were exposed to 50 mg L-1 of algal extract and to physical stress. Functional, physiological, and histological parameters were evaluated in blood and tissues at different sampling periods (0, 24, and 72 h). There was no mortality and the induction and recovery times observed were within the established criteria for anesthetic efficacy. The anesthetic extract did not induce any side effects, such as stress or metabolic damage, indicating that this extract is a viable option for supporting fish welfare during deleterious events. This study provides information to support that the anesthetic extract tested, derived from H. akashiwo, is a promising candidate drug for operations requiring sedation (e.g., Salmonid transport).

Algae as Food in Europe: An Overview of Species Diversity and Their Application.

Algae have been consumed for millennia in several parts of the world as food, food supplements, and additives, due to their unique organoleptic properties and nutritional and health benefits. Algae are sustainable sources of proteins, minerals, and fiber, with well-balanced essential amino acids, pigments, and fatty acids, among other relevant metabolites for human nutrition. This review covers the historical consumption of algae in Europe, developments in the current European market, challenges when introducing new species to the market, bottlenecks in production technology, consumer acceptance, and legislation. The current algae species that are consumed and commercialized in Europe were investigated, according to their status under the European Union (EU) Novel Food legislation, along with the market perspectives in terms of the current research and development initiatives, while evaluating the interest and potential in the European market. The regular consumption of more than 150 algae species was identified, of which only 20% are approved under the EU Novel Food legislation, which demonstrates that the current legislation is not broad enough and requires an urgent update. Finally, the potential of the European algae market growth was indicated by the analysis of the trends in research, technological advances, and market initiatives to promote algae commercialization and consumption.

Alternative Proteins for Fish Diets: Implications beyond Growth.

Aquaculture has been challenged to find alternative ingredients to develop innovative feed formulations that foster a sustainable future growth. Given the most recent trends in fish feed formulation on the use of alternative protein sources to decrease the dependency of fishmeal, it is fundamental to evaluate the implications of this new paradigm for fish health and welfare. This work intends to comprehensively review the impacts of alternative and novel dietary protein sources on fish gut microbiota and health, stress and immune responses, disease resistance, and antioxidant capacity. The research results indicate that alternative protein sources, such as terrestrial plant proteins, rendered animal by-products, insect meals, micro- and macroalgae, and single cell proteins (e.g., yeasts), may negatively impact gut microbiota and health, thus affecting immune and stress responses. Nevertheless, some of the novel protein sources, such as insects and algae meals, have functional properties and may exert an immunostimulatory activity. Further research on the effects of novel protein sources, beyond growth, is clearly needed. The information gathered here is of utmost importance, in order to develop innovative diets that guarantee the production of healthy fish with high quality standards and optimised welfare conditions, thus contributing to a sustainable growth of the aquaculture industry.

Proximity ligation strategy for the genomic reconstruction of microbial communities associated with the ectoparasite Caligus rogercresseyi.

The sea louse Caligus rogercresseyi has become one of the main constraints for the sustainable development of salmon aquaculture in Chile. Although this parasite's negative impacts are well recognized by the industry, some novel potential threats remain unnoticed. The recent sequencing of the C. rogercresseyi genome revealed a large bacterial community associated with the sea louse, however, it is unknown if these microorganisms should become a new focus of sanitary concern. Herein, chromosome proximity ligation (Hi-C) coupled with long-read sequencing were used for the genomic reconstruction of the C. rogercresseyi microbiota. Through deconvolution analysis, we were able to assemble and characterize 413 bacterial genome clusters, including six bacterial genomes with more than 80% of completeness. The most represented bacterial genome belonged to the fish pathogen Tenacibacullum ovolyticum (97.87% completeness), followed by Dokdonia sp. (96.71% completeness). This completeness allowed identifying 21 virulence factors (VF) within the T. ovolyticum genome and four antibiotic resistance genes (ARG). Notably, genomic pathway reconstruction analysis suggests putative metabolic complementation mechanisms between C. rogercresseyi and its associated microbiota. Taken together, our data highlight the relevance of Hi-C techniques to discover pathogenic bacteria, VF, and ARGs and also suggest novel host-microbiota mutualism in sea lice biology.

Immune Status and Hepatic Antioxidant Capacity of Gilthead Seabream Sparus aurata Juveniles Fed Yeast and Microalga Derived ß-glucans.

This work aimed to evaluate the effects of dietary supplementation with ß-glucans extracted from yeast (Saccharomyces cerevisiae) and microalga (Phaeodactylum tricornutum) on gene expression, oxidative stress biomarkers and plasma immune parameters in gilthead seabream (Sparus aurata) juveniles. A practical commercial diet was used as the control (CTRL), and three others based on CTRL were further supplemented with different ß-glucan extracts. One was derived from S. cerevisiae (diet MG) and two different extracts of 21% and 37% P. tricornutum-derived ß-glucans (defined as Phaeo21 and Phaeo37), to give a final 0.06% ß-glucan dietary concentration. Quadruplicate groups of 95 gilthead seabream (initial body weight: 4.1 ± 0.1 g) were fed to satiation three times a day for 8 weeks in a pulse-feeding regimen, with experimental diets intercalated with the CTRL dietary treatment every 2 weeks. After 8 weeks of feeding, all groups showed equal growth performance and no changes were found in plasma innate immune status. Nonetheless, fish groups fed ß-glucans supplemented diets showed an improved anti-oxidant status compared to those fed CTRL at both sampling points (i.e., 2 and 8 weeks). The intestinal gene expression analysis highlighted the immunomodulatory role of Phaeo37 diet after 8 weeks, inducing an immune tolerance effect in gilthead seabream intestine, and a general down-regulation of immune-related gene expression. In conclusion, the results suggest that the dietary pulse administration of a P. tricornutum 37% enriched-ß-glucans extract might be used as a counter-measure in a context of gut inflammation, due to its immune-tolerant and anti-oxidative effects.

Chromosome-scale genome assembly of the sea louse Caligus rogercresseyi by SMRT sequencing and Hi-C analysis.

Caligus rogercresseyi, commonly known as sea louse, is an ectoparasite copepod that impacts the salmon aquaculture in Chile, causing losses of hundreds of million dollars per year. In this study, we report a chromosome-scale assembly of the sea louse (C. rogercresseyi) genome based on single-molecule real-time sequencing (SMRT) and proximity ligation (Hi-C) analysis. Coding RNAs and non-coding RNAs, and specifically long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) were identified through whole transcriptome sequencing from different life stages. A total of 23,686 protein-coding genes and 12,558 non-coding RNAs were annotated. In addition, 6,308 lncRNAs and 5,774 miRNAs were found to be transcriptionally active from larvae to adult stages. Taken together, this genomic resource for C. rogercresseyi represents a valuable tool to develop sustainable control strategies in the salmon aquaculture industry.

Search for potential biomarkers for saxitoxin detection.

The Neuro-2a cell assay has been a promising in vitro alternative for the detection of saxitoxin (STX)-like toxins. However, its application is problematic in samples with complex matrices containing different toxins, whose mechanisms of action could be antagonistic. In the search of alternative procedures that reduce or avoid this interference, we evaluated the transcriptional modulation produced by a 24-h exposure to STX in Neuro-2a cells under three conditions: exposure to STX (33 nM), a mussel meat matrix (12.5 mg meat/mL) and a fortified sample (STX-fortified matrix). Differential gene expression was evaluated by RNA-seq after Illumina high-throughput sequencing, and data were analyzed to identify genes differentially expressed regardless of the matrix. From the 9487 identified genes, 213 were differentially expressed of these, 10 genes were identified as candidate markers for STX detection due to their regulation by STX regardless of the matrix interference. Expression dynamics of 7 of these candidate genes (Fgf-1, Adgrb2, Tfpt, Zfr2, Nup 35, Fam195a, and Dusp7) was further evaluated by qRT-PCR analysis of cells exposed to different concentrations of STX for up to 24 h. A downregulation of some markers expression was observed, namely Nup35 (involved in nucleo-cytoplasmic transporter activity) and Zfr-2 (involved in nucleic acids binding), whereas Fgf-1 (apoptosis signaling) was significantly upregulated. Markers' expression was not influenced by the matrix, suggesting that gene expression variations are directly related to STX response. These results support the potential of these genes as biomarkers for the development of an alternative STX-like toxins screening method.

Host Defense Effectors Expressed by Hemocytes Shape the Bacterial Microbiota From the Scallop Hemolymph.

The interaction between host immune response and the associated microbiota has recently become a fundamental aspect of vertebrate and invertebrate animal health. This interaction allows the specific association of microbial communities, which participate in a variety of processes in the host including protection against pathogens. Marine aquatic invertebrates such as scallops are also colonized by diverse microbial communities. Scallops remain healthy most of the time, and in general, only a few species are fatally affected on adult stage by viral and bacterial pathogens. Still, high mortalities at larval stages are widely reported and they are associated with pathogenic Vibrio. Thus, to give new insights into the interaction between scallop immune response and its associated microbiota, we assessed the involvement of two host antimicrobial effectors in shaping the abundances of bacterial communities present in the scallop Argopecten purpuratus hemolymph. To do this, we first characterized the microbiota composition in the hemolymph from non-stimulated scallops, finding both common and distinct bacterial communities dominated by the Proteobacteria, Spirochaetes and Bacteroidetes phyla. Next, we identified dynamic shifts of certain bacterial communities in the scallop hemolymph along immune response progression, where host antimicrobial effectors were expressed at basal level and early induced after a bacterial challenge. Finally, the transcript silencing of the antimicrobial peptide big defensin ApBD1 and the bactericidal/permeability-increasing protein ApLBP/BPI1 by RNA interference led to an imbalance of target bacterial groups from scallop hemolymph. Specifically, a significant increase in the class Gammaproteobacteria and the proliferation of Vibrio spp. was observed in scallops silenced for each antimicrobial. Overall, our results strongly suggest that scallop antimicrobial peptides and proteins are implicated in the maintenance of microbial homeostasis and are key molecules in orchestrating host-microbiota interactions. This new evidence depicts the delicate balance that exists between the immune response of A. purpuratus and the hemolymph microbiota.

Nanopore sequencing of microbial communities reveals the potential role of sea lice as a reservoir for fish pathogens.

Caligus rogercresseyi is a copepod ectoparasite with a high prevalence in salmon farms in Chile, causing severe welfare and economic concerns to the sector. Information on the parasite's underpinning mechanisms to support its life strategy is recently being investigated. Due to the critical role of microbiota, this study aimed to characterize the microbiota community associated with C. rogercresseyi from different regions with salmon aquaculture in Chile. Using third-generation sequencing with Nanopore technology (MinION) the full 16S rRNA gene from sea lice obtained from 8 areas distributed over the three main aquaculture regions were sequenced. Microbiota of the parasite is mainly comprised of members of phyla Proteobacteria and Bacteroidetes, and a core microbiota community with 147 taxonomical features was identified, and it was present in sea lice from the three regions. This community accounted for 19% of total identified taxa but more than 70% of the total taxonomical abundance, indicating a strong presence in the parasite. Several taxa with bioactive compound secretory capacity were identified, such as members of genus Pseudoalteromonas and Dokdonia, suggesting a possible role of the lice microbiota during the host infestation processes. Furthermore, the microbiota community was differentially associated with the salmon production, where several potential pathogens such as Vibrio, Tenacibaculum, and Aeromonas in Los Lagos, Aysén, and Magallanes region were identified. Notably, the Chilean salmon industry was initially established in the Los Lagos region but it's currently moving to the south, where different oceanographic conditions coexist with lice populations. The results originated by this study will serve as foundation to investigate putative role of sea lice as vectors for fish pathogens and also as reservoirs for antibiotic-resistant genes.

Catching the complexity of salmon-louse interactions.

The study of host-parasite relationships is an integral part of the immunology of aquatic species, where the complexity of both organisms has to be overlayed with the lifecycle stages of the parasite and immunological status of the host. A deep understanding of how the parasite survives in its host and how they display molecular mechanisms to face the immune system can be applied for novel parasite control strategies. This review highlights current knowledge about salmon and sea louse, two key aquatic animals for aquaculture research worldwide. With the aim to catch the complexity of the salmon-louse interactions, molecular information gleaned through genomic studies are presented. The host recognition system and the chemosensory receptors found in sea lice reveal complex molecular components, that in turn, can be disrupted through specific molecules such as non-coding RNAs.

Transcriptome mining of apoptotic mechanisms in response to density and functional diets in Oncorhynchus mykiss and role in homeostatic regulation.

Husbandry conditions often expose fish to several stressors, compromising organism's global homeostasis, which has consequences on aquaculture production. In order to depict intestinal homeostatic responses to deleterious conditions and the potential beneficial effects of functional diets in aquaculture fish, the effects of chronic suboptimal density condition on the neuro-immune endocrine system of rainbow trout was investigated through a transcriptomic survey. After 30 days under high stocking density, 67% of genes involved in homeostasis were found down-regulated whereas when fed with a diet supplemented with the probiotic Saccharomyces cerevisiae, <1% of these genes were found down-regulated. Genes involved in apoptotic mechanisms were found to be particularly responsive to both density and diets. At high density, several genes involved in the intrinsic and extrinsic apoptotic pathway (Fadd, Fas, Bcl-2 and Bax) as well as caspases (Casp8 and Casp3) were found down-regulated (Fold change <-7). However, fish fed with a diet supplemented with the probiotic yeast had an opposite response, indicating mitigation of stress effects. Overall, these results indicate the activation of homeostatic machinery and particularly genes involved in the apoptotic pathway as a result of probiotic feeding, counteracting apoptotic molecular repression derived from chronic stress.

Coding/non-coding cross-talk in intestinal epithelium transcriptome gives insights on how fish respond to stocking density.

Fish respond to increasing stocking density as a stressor, adjusting physiological functions to increase energy supply for coping with deleterious effects and adapting. These responses are complex and systemic, and the molecular mechanisms involved remain undetermined. One of the most sensitive organs to environmental and homeostatic disruptions is the intestine, and since it plays several vital functions, understanding the molecular underpinnings of this organ under deleterious conditions is imperative for health improvement in aquaculture systems. This study aimed to understand how different stocking densities of rainbow trout (Oncorhynchus mykiss) modulates the coding and non-coding RNAs profiling, that in turn, play key roles to maintain the fish homeostasis. For this, the intestine tissue of juvenile trout stocked for 30 days either at low (LD: 3 kg m-3) or high density (HD: 40 kg m-3) were sampled to isolate total RNA and then construct cDNA libraries for an illumina sequencing platform. Differential gene expression analysis revealed a generalized downregulation of transcripts, including coding and long non-coding RNAs (lncRNAs). Notably, significant differences in transcripts involved in metabolic pathways, as well as immune and epithelium integrity and stability related pathways were found. A high number of downregulated transcripts enriched these pathways, and a strong correlation was observed between the most differentially expressed transcripts and the highly expressed lncRNAs. This study suggests a cross-talk between coding and non-coding RNAs in the intestine of fish exposed to suboptimal conditions, providing new insights into the regulatory role of the lncRNAs on fish response to stressors.

Functional diets modulate the acute phase protein response in Oncorhynchus mykiss subjected to chronic stress and challenged with Vibrio anguillarum.

The acute phase response to pathogens alters the production of proinflammatory cytokines that, in turn, activate the synthesis of acute phase proteins. These proteins neutralize, prevent, and indicate tissue damage, thereby influencing the specific immune response and allowing the organism to regain homeostasis. Functional diets based in pre- and probiotics are used in aquaculture to improve fish health and resistance to diseases, but there is an information gap on the mechanisms involved in these effects and if these diets are efficient when fish are raised under high stocking densities. This study aimed an evaluation of the acute phase response in Oncorhynchus mykiss fed functional diets supplemented with pre- and probiotics (i.e. mannan-oligosaccharides and Saccharomyces cerevisiae, respectively) and challenged by either Vibrio anguillarum or chronic stress via maintenance under high stocking densities. For this, the relative expression of acute phase response related genes in liver, and of inflammatory response related genes in head kidney was evaluated by RT-qPCR. The supplemented diets differentially modulated the acute phase protein response to the assessed challenge conditions, specifically evidencing an overexpression of the genes HAPT, SAA, LECT2, and IL-1ß under chronic stress and of HAPT, IL-1ß, IL8, and LECT2 at 24 h post-challenge with V. anguillarum. The observed early-stage regulation of acute phase proteins and of the immune response by the probiotic S. cerevisiae and by prebiotic mannan-oligosaccharides suggests that both supplements have high immunostimulatory potentials for fish farmed under high stocking densities.

Functional Diets Modulate lncRNA-Coding RNAs and Gene Interactions in the Intestine of Rainbow Trout Oncorhynchus mykiss.

The advent of functional genomics has sparked the interest in inferring the function of non-coding regions from the transcriptome in non-model species. However, numerous biological processes remain understudied from this perspective, including intestinal immunity in farmed fish. The aim of this study was to infer long non-coding RNA (lncRNAs) expression profiles in rainbow trout (Oncorhynchus mykiss) fed for 30 days with functional diets based on pre- and probiotics. For this, whole transcriptome sequencing was conducted through Illumina technology, and lncRNAs were mined to evaluate transcriptional activity in conjunction with known protein sequences. To detect differentially expressed transcripts, 880 novels and 9067 previously described O. mykiss lncRNAs were used. Expression levels and genome co-localization correlations with coding genes were also analyzed. Significant differences in gene expression were primarily found in the probiotic diet, which had a twofold downregulation of lncRNAs compared to other treatments. Notable differences by diet were also evidenced between the coding genes of distinct metabolic processes. In contrast, genome co-localization of lncRNAs with coding genes was similar for all diets. This study contributes novel knowledge regarding lncRNAs in fish, suggesting key roles in salmons fed with in-feed additives with the capacity to modulate the intestinal homeostasis and host health.

Intestinal transcriptome modulation by functional diets in rainbow trout: A high-throughput sequencing appraisal to highlight GALT immunomodulation.

Functional ingredients such as pre- and probiotics are used in aquaculture to improve fish condition, modulating microbiota and promoting a healthy intestinal functioning. They also exert an active effect on the gut associated lymphoid tissue (GALT), stimulating the immune system. However, the molecular underpinnings of pre- and probiotics effect on intestinal mucosa are still unknown. This study investigated the intestinal mucosa transcriptome modulation when fish were fed functional diets and kept at different stocking densities. Juvenile rainbow trout were kept at low (LD-3Kgm-3) and high density (HD-40 kgm-3) and fed for 30 days functional diets with the prebiotic mannanoligosaccharide (PRE-0.6%), the probiotic Saccharomyces cerevisiae (PRO-0.5%), the mixture of both (MIX) and a control diet (CTRL). Intestinal transcriptome was evaluated by high-throughput sequencing and blood plasma for biochemical parameters. Fish fed functional diets presented better condition regardless density, and that functional diets modulate intestinal transcriptome in different manner depending on the stocking density. At LD, fish from PRO presented stronger modulation with the majority of transcripts being down-regulated, including the immune related ones, whereas at HD both PRO and MIX groups were more modulated, when comparing to the respective CTRL groups. Density had an overwhelming suppressive effect on the immune-related genes, but this effect was counteracted by feeding functional diets, especially in fish fed with probiotics. This study shows for the first time the intestinal transcriptomic modulation when fish are fed functional diets at different stocking densities, and it shows the mitigating effect of these diets against deleterious conditions such as high density.

Transcription expression of immune-related genes from Caligus rogercresseyi evidences host-dependent patterns on Atlantic and coho salmon.

The transcriptomic response of the sea louse Caligus rogercresseyi during the infestation on Atlantic salmon (Salmo salar) and coho salmon (Oncorhynchus kisutch) was evaluated using 27 genes related to immune response, antioxidant system and secretome. Results showed early responses of TLR/IMD signaling pathway in sea lice infesting Atlantic salmon. Overall, genes associated with oxidative stress responses were upregulated in both host species. This pattern suggests that reactive oxygen species emitted by the host as a response to the infestation, could modulate the sea louse antioxidant system. Secretome-related transcripts evidenced upregulation of trypsins and serpins, mainly associated to Atlantic salmon than coho salmon. Interestingly, cathepsins and trypsin2 were downregulated at 7 days post-infection (dpi) in coho salmon. The principal component analysis revealed an inverse time-dependent pattern based on the different responses of C. rogercresseyi infecting both salmon species. Here, Atlantic salmon strongly modulates the transcriptome responses at earlier infection stages; meanwhile coho salmon reveals a less marked modulation, increasing the transcription activity during the infection process. This study evidences transcriptome differences between two salmon host species and provides pivotal knowledge towards elaborating future control strategies.

Transcriptome immunomodulation of in-feed additives in Atlantic salmon Salmo salar infested with sea lice Caligus rogercresseyi.

One of the most significant threats to the Chilean salmon aquaculture industry is the ectoparasitic sea louse Caligus rogercresseyi. To cope with sea lice infestations, functional diets have become an important component in strengthening the host immune response. The aim of this study was to evaluate molecular mechanisms activated through immunostimulation by in-feed plant-derived additives in Atlantic salmon infected with sea lice. Herein, a transcriptome-wide sequencing analysis was performed from skin and head kidney tissues, evidencing that the immune response genes were the most variable after the challenge, especially in the head kidney, while other genes involved in metabolism were highly expressed individuals fed with the immunostimulants. Interestingly, defensive enzymes such as Cytochrome p450 and serpins were down-regulated in infested individuals, especially in skin tissue. Additionally, MHC-I and MHC-II genes were differentially expressed after the incorporation of the in-feed additives, giving some cues about the protection mechanisms of plant-derived compound as immunostimulants for infested salmons. This is the first published study that evaluates the transcriptomic response of sea lice-infested Atlantic salmon fed with in-feed additives.