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Aquaculture now provides half of all aquatic protein consumed globally—with most current and future production occurring in low- and middle-income countries (LMICs). Concerns over the availability and application of effective policies to deliver safe and sustainable future supply have the potential to hamper further development of the sector. Creating healthy systems must extend beyond the simple exclusion of disease agents to tackle the host, environmental, and human drivers of poor outcomes and build new policies that incorporate these broader drivers. Syndemic theory provides a potential framework for operationalizing this One Health approach.

Abstract Background Rainbow trout (Oncorhynchus mykiss) is a salmonid species with a complex life-history. Wild populations are naturally divided into freshwater residents and sea-run migrants. Migrants undergo an energy-demanding adaptation for life in seawater, known as smoltification, while freshwater residents display these changes in an attenuated magnitude and rate. Despite this, in seawater rainbow trout farming all fish are transferred to seawater. Under these circumstances, weeks after seawater transfer, a significant portion of the fish die (around 10%) or experience growth stunting (GS; around 10%), which represents an important profitability and welfare issue. The underlying causes leading to GS in seawater-transferred rainbow trout remain unknown. In this study, we aimed at characterising the GS phenotype in seawater-transferred rainbow trout using untargeted and targeted approaches. To this end, the liver proteome (LC-MS/MS) and lipidome (LC-MS) of GS and fast-growing phenotypes were profiled to identify molecules and processes that are characteristic of the GS phenotype. Moreover, the transcription, abundance or activity of key proteins and hormones related to osmoregulation (Gill Na+, K + –ATPase activity), growth (plasma IGF-I, and liver igf1, igfbp1b, ghr1 and ctsl) and stress (plasma cortisol) were measured using targeted approaches. Results No differences in Gill Na+, K + –ATPase activity and plasma cortisol were detected between the two groups. However, a significant downregulation in plasma IGF-I and liver igf1 transcription pointed at this growth factor as an important pathomechanism for GS. Changes in the liver proteome revealed reactive-oxygen-species-mediated endoplasmic reticulum stress as a key mechanism underlying the GS phenotype. From the lipidomic analysis, key observations include a reduction in triacylglycerols and elevated amounts of cardiolipins, a characteristic lipid class associated with oxidative stress, in GS phenotype. Conclusion While the triggers to the activation of endoplasmic reticulum stress are still unknown, data from this study point towards a nutritional deficiency as an underlying driver of this phenotype.

ABSTRACT The major aquatic interface between host and environment in teleost finfish species is the gill. The diversity of this infraclass, high complexity of the organ, and its direct exposure to the surrounding environment make it an ideal candidate for furthering our understanding of the intertwined relationships between host and microbiome. Capturing the structure and diversity of bacterial communities from this low-biomass, inhibitor-rich tissue can, however, prove challenging. Lessons learned in doing so are directly applicable to similar sample types in other areas of microbiology. Through the development of a quantitative PCR assay for both host material and 16S rRNA genes, we tested and developed a robust method for low-biomass sample collection which minimized host DNA contamination. Quantification of 16S rRNA facilitated not only the screening of samples prior to costly library construction and sequencing but also the production of equicopy libraries based on 16S rRNA gene copies. A significant increase in diversity of bacteria captured was achieved, providing greater information on the true structure of the microbial community. Such findings offer important information for determining functional processes. Results were confirmed across fresh, brackish, and marine environs with four different fish species, with results showing broad homology between samples, demonstrating the robustness of the approach. Evidence presented is widely applicable to samples similar in composition, such as sputum or mucus, or those that are challenging due to the inherent inclusion of inhibitors. IMPORTANCE The interaction between the fish gill and surrounding bacteria-rich water provides an intriguing model for examining the interaction between the fish, free-floating bacteria, and the bacterial microbiome on the gill surface. Samples that are inherently low in bacteria, or that have components that inhibit the ability to produce libraries that identify the components of microbial communities, present significant challenges. Gill samples present both of these types of challenges. We developed methods for quantifying both the bacterial and host DNA material and established a sampling method which both reduced inhibitor content and maximized bacterial diversity. By quantifying and normalizing bacteria prior to library construction, we showed significant improvements with regards to the fidelity of the final data. Our results support wide-ranging applications for analyzing samples of similar composition, such as mucus and sputum, in other microbiological spheres.

Abstract Background In humans the stress response is known to be modulated to a great extent by psychological factors, particularly by the predictability and the perceived control that the subject has of the stressor. This psychological dimension of the stress response has also been demonstrated in animals phylogenetically closer to humans (i.e. mammals). However, its occurrence in fish, which represent a divergent vertebrate evolutionary lineage from that of mammals, has not been established yet, and, if present, would indicate a deep evolutionary origin of these mechanisms across vertebrates. Moreover, the fact that psychological modulation of stress is implemented in mammals by a brain cortical top-down inhibitory control over subcortical stress-responsive structures, and the absence of a brain cortex in fish, has been used as an argument against the possibility of psychological stress in fish, with implications for the assessment of fish sentience and welfare. Here, we have investigated the occurrence of psychological stress in fish by assessing how stressor controllability modulates the stress response in European seabass (Dicentrarchus labrax). Results Fish were exposed to either a controllable or an uncontrollable stressor (i.e. possibility or impossibility to escape a signaled stressor). The effect of loss of control (possibility to escape followed by impossibility to escape) was also assessed. Both behavioral and circulating cortisol data indicates that the perception of control reduces the response to the stressor, when compared to the uncontrollable situation. Losing control had the most detrimental effect. The brain activity of the teleost homologues to the sensory cortex (Dld) and hippocampus (Dlv) parallels the uncontrolled and loss of control stressors, respectively, whereas the activity of the lateral septum (Vv) homologue responds in different ways depending on the gene marker of brain activity used. Conclusions These results suggest the psychological modulation of the stress response to be evolutionary conserved across vertebrates, despite being implemented by different brain circuits in mammals (pre-frontal cortex) and fish (Dld-Dlv).

Abstract Background Consistent individual differences in behaviour, known as animal personalities, have been demonstrated within and across species. In fish, studies applying an animal personality approach have been used to resolve variation in physiological and molecular data suggesting a linkage, genotype-phenotype, between behaviour and transcriptome regulation. In this study, using three fish species (zebrafish; Danio rerio, Atlantic salmon; Salmo salar and European sea bass; Dicentrarchus labrax), we firstly address whether personality-specific mRNA transcript abundances are transferrable across distantly-related fish species and secondly whether a proactive transcriptome signature is conserved across all three species. Results Previous zebrafish transcriptome data was used as a foundation to produce a curated list of mRNA transcripts related to animal personality across all three species. mRNA transcript copy numbers for selected gene targets show that differential mRNA transcript abundance in the brain appears to be partially conserved across species relative to personality type. Secondly, we performed RNA-Seq using whole brains from S. salar and D. labrax scoring positively for both behavioural and molecular assays for proactive behaviour. We further enriched this dataset by incorporating a zebrafish brain transcriptome dataset specific to the proactive phenotype. Our results indicate that cross-species molecular signatures related to proactive behaviour are functionally conserved where shared functional pathways suggest that evolutionary convergence may be more important than individual mRNAs. Conclusions Our data supports the proposition that highly polygenic clusters of genes, with small additive effects, likely support the underpinning molecular variation related to the animal personalities in the fish used in this study. The polygenic nature of the proactive brain transcriptome across all three species questions the existence of specific molecular signatures for proactive behaviour, at least at the granularity of specific regulatory gene modules, level of genes, gene networks and molecular functions.

The external mucosal surfaces of the fish harbor complex microbial communities, which may play pivotal roles in the physiological, metabolic, and immunological status of the host. Currently, little is known about the composition and role of these communities, whether they are species and/or tissue specific and whether they reflect their surrounding environment. Co-culture of fish, a common practice in semi-intensive aquaculture, where different fish species cohabit in the same contained environment, is an easily accessible and informative model toward understanding such interactions. This study provides the first in-depth characterization of gill and skin microbiomes in co-cultured Nile tilapia (Oreochromis niloticus) and grey mullet (Mugil capito) in semi-intensive pond systems in Egypt using 16S rRNA gene-based amplicon sequencing. Results showed that the microbiome composition of the external surfaces of both species and pond water was dominated by the following bacterial phyla: Proteobacteria, Fusobacteriota, Firmicutes, Planctomycetota, Verrucomicrobiota, Bacteroidota, and Actinobacteriota. However, water microbial communities had the highest abundance and richness and significantly diverged from the external microbiome of both species; thus, the external autochthonous communities are not a passive reflection of their allochthonous communities. The autochthonous bacterial communities of the skin were distinct from those of the gill in both species, indicating that the external microbiome is likely organ specific. However, gill autochthonous communities were clearly species specific, whereas skin communities showed higher commonalities between both species. Core microbiome analysis identified the presence of shared core taxa between both species and pond water in addition to organ-specific taxa within and between the core community of each species. These core taxa included possibly beneficial genera such as Uncultured Pirellulaceae, Exiguobacterium, and Cetobacterium and opportunistic potential pathogens such as Aeromonas, Plesiomonas, and Vibrio. This study provides the first in-depth mapping of bacterial communities in this semi-intensive system that in turn provides a foundation for further studies toward enhancing the health and welfare of these cultured fish and ensuring sustainability.

The absence of MHC class II antigen presentation and multiple pathogen recognition receptors in the Atlantic cod has not impaired its immune response however how underlying mechanisms have adapted remains largely unknown. In this study, ex vivo cod macrophages were challenged with various bacterial and viral microbe-associated molecular patterns (MAMP) to identify major response pathways. Cytosolic MAMP-PRR pathways based upon the NOD-like receptors (NLRs) and RIG-I-like receptors (RLRs) were identified as the critical response pathways. Our analyses suggest that internalization of exogenous ligands through scavenger receptors drives both pathways activating transcription factors like NF-kB (Nuclear factor-kappa B) and interferon regulatory factors (IRFs). Further, ligand-dependent differential expression of a unique TLR25 isoform and multiple NLR paralogues suggests (sub)neofunctionalization toward specific immune defensive strategies. Our results further demonstrate that the unique immune system of the Atlantic cod provides an unprecedented opportunity to explore the evolutionary history of PRR-based signaling in vertebrate immunity.

The burden of disease is a major challenge in aquaculture production. The fish gill characterized with a large surface area and short route to the bloodstream is a major environmental interface and a significant portal of entry for pathogens. To investigate gill responses to viral infection the salmonid gill cell line RTgill-W1 was stimulated with synthetic dsRNA and the salmonid alphavirus subtype 2 (SAV-2). Epithelial integrity in polarized cells can be measured as transepithelial electrical resistance (TEER) which is defined as the electrical resistance across a cell monolayer. TEER is a widely accepted quantitative measure of cellular integrity of a cell monolayer. TEER increased immediately after stimulation with the synthetic dsRNA, polyinosinic:polycytidylic acid (poly(I:C)). In parallel, tight junction and gene expression of innate immune activation markers was modulated in response to poly(I:C). The SAV-2 virus was found to replicate at a low level in RTgill-W1 cells where TEER was disturbed at an early stage of infection, however, gene expression related to tight junction regulation was not modulated. A strong poly(I:C)-driven antiviral response was observed including increases of Rig-like receptors (RLRs) and interferon stimulating genes (ISGs) mRNAs. At the level of signal transduction, poly(I:C) stimulation was accompanied by the phosphorylation of 671 proteins, of which 390 were activated solely in response to the presence of poly(I:C). According to motif analysis, kinases in this group included MAPKs, Ca2+/calmodulin-dependent kinase (CaMK) and cAMP-dependent protein kinase (PKA), all reported to be activated in response to viral infection in mammals. Results also highlighted an activation of the cytoskeletal organization that could be mediated by members of the integrin family. While further work is needed to validate these results, our data indicate that salmonid gill epithelia has the ability to mount a significant response to viral infection which might be important in disease progression. In vitro cell culture can facilitate both a deeper understanding of the anti-viral response in fish and open novel therapeutic avenues for fish health management in aquaculture.

The sea-run phenotype of rainbow trout (Oncorhynchus mykiss), like other anadromous salmonids, present a juvenile stage fully adapted to life in freshwater known as parr. Development in freshwater is followed by the smolt stage, where preadaptations needed for seawater life are developed making fish ready to migrate to the ocean, after which event they become post-smolts. While these three life stages have been studied using a variety of approaches, proteomics has never been used for such purpose. The present study characterised the blood plasma proteome of parr, smolt and post-smolt rainbow trout using a gel electrophoresis liquid chromatography tandem mass spectrometry approach alone or in combination with low-abundant protein enrichment technology (combinatorial peptide ligand library). In total, 1,822 proteins were quantified, 17.95% of them being detected only in plasma post enrichment. Across all life stages, the most abundant proteins were ankyrin-2, DNA primase large subunit, actin, serum albumin, apolipoproteins, hemoglobin subunits, hemopexin-like proteins and complement C3. When comparing the different life stages, 17 proteins involved in mechanisms to cope with hyperosmotic stress and retinal changes, as well as the downregulation of nonessential processes in smolts, were significantly different between parr and smolt samples. On the other hand, 11 proteins related to increased growth in post-smolts, and also related to coping with hyperosmotic stress and to retinal changes, were significantly different between smolt and post-smolt samples. Overall, this study presents a series of proteins with the potential to complement current seawater-readiness assessment tests in rainbow trout, which can be measured non-lethally in an easily accessible biofluid. Furthermore, this study represents a first in-depth characterisation of the rainbow trout blood plasma proteome, having considered three life stages of the fish and used both fractionation alone or in combination with enrichment methods to increase protein detection.

Antibiotics play a vital role in aquaculture where they are commonly used to treat bacterial diseases. However, the impact of antibiotic treatment on the gut microbiome and the development of antimicrobial resistance in Nile tilapia (Oreochromis niloticus) over time remains to be fully understood. In this study, fish were fed a single treatment of oxytetracycline (100 mg/kg/day) for eight days, followed by a 14-day withdrawal period. Changes in the distal gut microbiome were measured using 16S rRNA sequencing. In addition, the abundance of antimicrobial resistance genes was quantified using real-time qPCR methods. Overall, the gut microbiome community diversity and structure of Nile tilapia was resilient to oxytetracycline treatment. However, antibiotic treatment was associated with an enrichment in Plesiomonas, accompanied by a decline in other bacteria taxa. Oxytetracycline treatment increased the proportion of tetA in the distal gut of fish and tank biofilms of the treated group. Furthermore, the abundance of tetA along with other tetracycline resistance genes was strongly correlated with a number of microbiome members, including Plesiomonas. The findings from this study demonstrate that antibiotic treatment can exert selective pressures on the gut microbiome of fish in favour of resistant populations, which may have long-term impacts on fish health.

Ectotherms choose the best thermal conditions to mount a successful immune response, a phenomenon known as behavioral fever. The cumulative evidence suggests that behavioral fever impacts positively upon lymphocyte proliferation, inflammatory cytokine expression, and other immune functions. In this study, we have explored how thermal choice during infection impacts upon underpinning molecular processes and how temperature increase is coupled to the immune response. Our results show that behavioral fever results in a widespread, plastic imprint on gene regulation, and lymphocyte proliferation. We further explored the possible contribution of histone modification and identified global associations between temperature and histone changes that suggest epigenetic remodeling as a result of behavioral fever. Together, these results highlight the critical importance of thermal choice in mobile ectotherms, particularly in response to an infection, and demonstrate the key role of epigenetic modification to orchestrate the thermocoupling of the immune response during behavioral fever.

Individual differences in animal personality and external appearance such as colouration patterns have both been extensively studied separately. A significant body of research has explored many of pertinent ecological and biological aspects that can be affected by them and their impact upon fitness. Currently little is known about how both factors interact and their effect on reproductive success. In this study, we evaluated two major parameters contributing to the fitness of the species: reproduction and offspring survival. We selected two different phenotypes of the zebrafish (Danio rerio) selected by their colouration patterns: phenotype 1) named Wild type, UAB line (WT-UAB) with a homogeneous colouration pattern (clear and defined lateral stripes) and phenotype 2) Wild type indefinite (WT-I) had a heterogeneous colouration pattern and different degrees of lateral stripe definition. All animals were also screened for personality. We then compared their reproductive success (spawning rate) and offspring survival at different stages, from egg to larvae, and for 2 successive generations (parental generation was G0; First and second generations were G1 and G2 respectively). Our results show that personality traits were the main source of variability between the fitness components measured: both personalities had similar total numbers of eggs spawned but proactive animals, for both colour phenotypes, had higher reproductive success. This was reflected in a higher percentage of spawning viability at 1day post fertilization (dpf), higher total survival and growth rates at larval stages. Proactive phenotypes from WT-UAB population had a higher overall fitness in comparison to the other phenotypes studied. Our findings imply that fitness of this species when kept under similar husbandry conditions is significantly influenced by parental personality and not by their external appearance. Under these conditions the reproductive success is enhanced. The implications of this study are important for zebrafish breeding and husbandry in captivity and are relevant toward understanding the underlying drivers of trait selection in natural environments.

Dominance is defined as the preferential access to limited resources. The present study aimed to characterise dominance in a non-aggressive flatfish species, the Senegalese sole (Solea senegalensis) by 1) identifying dominance categories and associated behaviours and 2) linking dominance categories (dominant and subordinate) with the abundance of selected mRNA transcripts in the brain. Early juveniles (n = 74, 37 pairs) were subjected to a dyadic dominance test, related to feeding, and once behavioural phenotypes had been described the abundance of ten selected mRNAs related to dominance and aggressiveness was measured in the brain. Late juveniles were subjected to two dyadic dominance tests (n = 34, 17 pairs), related to feeding and territoriality and one group test (n = 24, 4 groups of 6 fish). Sole feeding first were categorized as dominant and sole feeding second or not feeding as subordinate. Three social behaviours (i. "Resting the head" on another fish, ii. "Approaching" another fish, iii. "Swimming above another" fish) were associated with dominance of feeding. Two other variables (i. Total time occupying the preferred area during the last 2 hours of the 24 h test, ii. Organisms occupying the preferred area when the test ended) were representative of dominance in the place preference test. In all tests, dominant fish compared to subordinate fish displayed a significantly higher number of the behaviours "Rest the head" and "Approaches". Moreover, dominant sole dominated the sand at the end of the test, and in the group test dominated the area close to the feed delivery point before feed was delivered. The mRNA abundance of the selected mRNAs related to neurogenesis (nrd2) and neuroplasticity (c-fos) in dominant sole compared to subordinate were significantly different. This is the first study to characterise dominance categories with associated behaviours and mRNA abundance in Senegalese sole and provides tools to study dominance related problems in feeding and reproduction in aquaculture.

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This study describes the development and validation of an enriched oligonucleotide-microarray platform for Sparus aurata (SAQ) to provide a platform for transcriptomic studies in this species. A transcriptome database was constructed by assembly of gilthead sea bream sequences derived from public repositories of mRNA together with reads from a large collection of expressed sequence tags (EST) from two extensive targeted cDNA libraries characterizing mRNA transcripts regulated by both bacterial and viral challenge. The developed microarray was further validated by analysing monocyte/macrophage activation profiles after challenge with two Gram-negative bacterial pathogen-associated molecular patterns (PAMPs; lipopolysaccharide (LPS) and peptidoglycan (PGN)). Of the approximately 10,000 EST sequenced, we obtained a total of 6837 EST longer than 100 nt, with 3778 and 3059 EST obtained from the bacterial-primed and from the viral-primed cDNA libraries, respectively. Functional classification of contigs from the bacterial- and viral-primed cDNA libraries by Gene Ontology (GO) showed that the top five represented categories were equally represented in the two libraries: metabolism (approximately 24% of the total number of contigs), carrier proteins/membrane transport (approximately 15%), effectors/modulators and cell communication (approximately 11%), nucleoside, nucleotide and nucleic acid metabolism (approximately 7.5%) and intracellular transducers/signal transduction (approximately 5%). Transcriptome analyses using this enriched oligonucleotide platform identified differential shifts in the response to PGN and LPS in macrophage-like cells, highlighting responsive gene-cassettes tightly related to PAMP host recognition. As observed in other fish species, PGN is a powerful activator of the inflammatory response in S. aurata macrophage-like cells. We have developed and validated an oligonucleotide microarray (SAQ) that provides a platform enriched for the study of gene expression in S. aurata with an emphasis upon immunity and the immune response.

DNA vaccines encoding the viral G glycoprotein show the most successful protection capability against fish rhabdoviruses. Nowadays, the molecular mechanisms underlying the protective response remain still poorly understood. With the aim of shedding light on the protection conferred by the DNA vaccines based in the G glycoprotein of viral haemorrhagic septicaemia virus (VHSV) in turbot (Scophthalmus maximus) we have used a specific microarray highly enriched in antiviral sequences to carry out the transcriptomic study associated to VHSV DNA vaccination/infection. The differential gene expression pattern in response to empty plasmid (pMCV1.4) and DNA vaccine (pMCV1.4-G860) intramuscular administration with regard to non-stimulated turbot was analyzed in head kidney at 8, 24 and 72 hours post-vaccination. Moreover, the effect of VHSV infection one month after immunization was also analyzed in vaccinated and non-vaccinated fish at the same time points. Genes implicated in the Toll-like receptor signalling pathway, IFN inducible/regulatory proteins, numerous sequences implicated in apoptosis and cytotoxic pathways, MHC class I antigens, as well as complement and coagulation cascades among others were analyzed in the different experimental groups. Fish receiving the pMCV1.4-G860 vaccine showed transcriptomic patterns very different to the ones observed in pMCV1.4-injected turbot after 72 h. On the other hand, VHSV challenge in vaccinated and non-vaccinated turbot induced a highly different response at the transcriptome level, indicating a very relevant role of the acquired immunity in vaccinated fish able to alter the typical innate immune response profile observed in non-vaccinated individuals. This exhaustive transcriptome study will serve as a complete overview for a better understanding of the crosstalk between the innate and adaptive immune response in fish after viral infection/vaccination. Moreover, it provides interesting clues about molecules with a potential use as vaccine adjuvants, antiviral treatments or markers for vaccine efficiency monitoring.

Stenotrophomonas maltophilia is a Gram-negative pathogen with emerging nosocomial incidence. Little is known about its pathogenesis and the genomic diversity exhibited by clinical isolates complicates the study of pathogenicity and virulence factors. Here, we present a strategy to identify such factors in new clinical isolates of S. maltophilia, incorporating an adult-zebrafish model of S. maltophilia infection to evaluate relative virulence coupled to 2D difference gel electrophoresis to explore underlying differences in protein expression. In this study we report upon three recent clinical isolates and use the collection strain ATCC13637 as a reference. The adult-zebrafish model shows discrimination capacity, i.e. from very low to very high mortality rates, with clinical symptoms very similar to those observed in natural S. maltophilia infections in fish. Strain virulence correlates with resistance to human serum, in agreement with previous studies in mouse and rat and therefore supporting zebrafish as a replacement model. Despite its clinical origin, the collection strain ATCC13637 showed obvious signs of attenuation in zebrafish, with null mortality. Multilocus-sequence-typing analysis revealed that the most virulent strains, UV74 and M30, exhibit the strongest genetic similitude. Differential proteomic analysis led to the identification of 38 proteins with significantly different abundance in the three clinical strains relative to the reference strain. Orthologs of several of these proteins have been already reported to have a role in pathogenesis, virulence or resistance mechanisms thus supporting our strategy. Proof of concept is further provided by protein Ax21, whose abundance is shown here to be directly proportional to mortality in the zebrafish infection model. Indeed, recent studies have demonstrated that this protein is a quorum-sensing-related virulence factor.

BACKGROUND: Individuals of many vertebrate species show different stress coping styles and these have a striking influence on how gene expression shifts in response to a variety of challenges. PRINCIPAL FINDINGS: This is clearly illustrated by a study in which common carp displaying behavioural predictors of different coping styles (characterised by a proactive, adrenaline-based or a reactive, cortisol-based response) were subjected to inflammatory challenge and specific gene transcripts measured in individual brains. Proactive and reactive fish differed in baseline gene expression and also showed diametrically opposite responses to the challenge for 80% of the genes investigated. SIGNIFICANCE: Incorporating coping style as an explanatory variable can account for some the unexplained variation that is common in gene expression studies, can uncover important effects that would otherwise have passed unnoticed and greatly enhances the interpretive value of gene expression data.