Aquaculture

Selection for disease resistance forms one of the most highlighted areas of aquaculture breeding. A breeding program for Arctic charr has been operating in Sweden for over 40 years, making it the oldest of its kind worldwide for this species. However, the lack of available genomic resources prevented selection for any disease-resistance traits. A 72k Axiom SNP array was produced in this study and used to assess the potential to select for charr resistant to bacterial kidney disease (BKD), which is currently a major threat to the industry. Following a challenge experiment with Renibacterium salmoninarum, the causative agent of BKD, relevant phenotypic proxies were collected from approximately 2,000 charr. Thereafter, those animals were genotyped with the new 72k SNP array. The magnitude of the estimated variance components suggested potential for breeding for BKD resistance in charr, with relevant heritabilities ranging from 0.05 to 0.56 depending on the resistance proxy used. In addition, GWAS suggested that BKD resistance is a polygenic trait. Furthermore, genomic prediction approaches indicated that BKD-resistant animals can be identified using their SNP genotypes. Accuracies, expressed as Pearson correlation coefficients, when BKD resistance was analysed as a continuous trait, ranged from 0.42 to 0.52. In the scenario where BKD resistance was treated as a binary trait, the efficiency of genomic prediction was assessed using ROC curves, with an area under the curve of 0.72. Finally, no unfavourable correlations were found with growth traits. The developed 72k SNP array has the potential of being a pivotal tool for the Swedish Arctic charr breeding program. Moreover, our data support the use of genomic prediction in breeding BKD-resistant Arctic charr. As a critical next step, further validations in actual industry conditions would be required.

Penaeid shrimp farming plays a pivotal role in ensuring future food security and promoting economic sustainability. Compared to the extensive long history of domestication observed in terrestrial agriculture species, the domestication and selective breeding of penaeids are relatively recent endeavors. Selective breeding aimed at improving production traits holds significant promise for enhancing efficiency and reducing the environmental impact of shrimp farming, thereby contributing to its long-term sustainability. Assessing genotype-by-environment (G-by-E) interactions is essential in breeding programs to ensure that improved penaeid shrimp strains perform consistently across different production environments, with genomic selection proving more effective than sib-testing alone in mitigating environmental sensitivity. Genome editing tools like CRISPR/Cas9 offer significant potential to accelerate genetic gains in penaeid shrimp by enabling rapid introduction of desired genetic changes, with recent advancements showing promising results in achieving high transfection efficiency in shrimp embryos. Additionally, artificial intelligence and machine learning are being leveraged to streamline phenotyping and enhance decision-making in shrimp breeding and farming, improving efficiency and accuracy in managing traits and predicting disease outbreaks. Herein, we provide an overview and update on the domestication of penaeid shrimp, including the current status of domestication for principal farmed species, key milestones in domestication history, targeted breeding traits in selective breeding programs, the advantages of integrating genomeic selection for enhancing production traits, and future directions for selective breeding of penaeid shrimp.

Survival is an old trait in animal breeding, yet commonly neglected nowadays for its simple binary records and low levels of heritability in aquaculture species. These traits however, can provide valuable field data when selecting for robustness in genetic improvement programs. In the current study, linear multivariate animal model (LMA) was used for the genetic analysis of survival records from two-year classes (BL2019 and BL2020) of white-leg shrimp (Penaeus vannamei) breeding lines with a total number of 52, 248 individuals from 481 fullsib families recorded for data collection. During grow-out test period, 10 day intervals of survival data were considered as independent traits. Two survival definitions, binary survivability (S) and continuous survival in days (SL), were used for the genetic analysis of survival records to investigate; i) whether adding more survival time information could improve estimation of genetic parameters; ii) the trajectory of survival heritability across time, and iii) patterns of genetic correlations of survival traits across time. Levels of heritability estimates for both S and SL were low (0.005 to 0.076), while heritability for survival day number was found to be similar with that of binary records at each observation time and were highly genetically correlated (rg >0.8). Heritability estimates of body weight (BW) for BL2019 and BL2020 were 0.486 and 0.373, respectively. Trajectories of survival heritability across time showed a gradual increase across the grow-out test period but slowed or reached a plateau during the later grow-out test period. Genetic correlations among survival traits in the grow-out tests were moderate to high, and the closer the times were between estimates, the higher were their genetic correlations. In contrast, genetic correlations between both survival traits and body weight were low but positive. Here we provide the first report of the trajectory of heritability estimates for survival traits across time in aquaculture. Results will be useful for developing robust improved white-leg shrimp culture strains in selective breeding programs based on field survival data.

The pacific whiteleg shrimp (Litopenaeus vannamei) and the crayfish (Procambarus clarkii) are the most productive aquatic animals in the world. The prevalence of the microsporidium Enterocytozoon hepatopenaei (EHP), an intracellular spore-forming unicellular parasite which leading retarded growth of L. vannamei, has caused severe economic losses in most shrimp farming country. In this study, we found that the wild P. clarkii, living in shrimp ponds with EHP outbreak, excreted white faeces after temporary laboratory culture. The hepatopancreas (HP) of symptomatic crayfish exhibited a lighter color and severely atrophied. H&E-stain showed tissue lesions in both hepatopancreas and intestine, and clustered microsporidian spores were filled in the cytoplasm of the cells. PCR using EHPptp2 and two microsporidian-universal primers sets demonstrated the existence of EHP in the hepatopancreas, intestine and the white feces of P. clarkii. The EHP loads of 103-104 copies of EHPptp2/50 ng HPgDNA were detected by qPCR. Developing stages and mature spores of EHP were observed in hepatopancreas of P. clarkii through indirect-immunofluorescence assay (IFA) and transmission electron microscopy (TEM). Considering the large-scale cultivation of L. vannamei and P. clarkii, overlap farming areas between these two species as well as the ability of crayfish to crawl on land and water, our finding indicates the potential role of P. clarkii in the transmission of EHP, and it is an early warning for crayfish and shrimp farming.

From the Asia-Pacific region, the suspected TPD/GPD specimens of postlarvae (PL) preserved in Davidsons fixative and 95% ethanol were sent to our laboratory to prepare for PCR and histological analysis. Interestingly, PCR tests with the specimens were negative with the VPHLVD(vtcc2 and vtcc3 gene) primers, but positive with the VPTPD(vhvp-2 gene) primers. There were no characteristic lesions of acute hepatopancreatic necrosis disease detected in these specimens. However, in the sections of several specimens from two affected tanks, the midgut was cut tangentially in the abdominal region, revealing a novel histopathology characterized by severe necrosis specifically in the thin, smooth muscle tissues underlying the midgut lumen epithelium. This comprised nuclear pyknosis and karyorrhexis limited to the smooth muscle and occasionally affecting the midgut epithelium but not involving the surrounding skeletal muscles. The pathology is called midgut smooth muscle necrosis (MSMN). Such pathology would undoubtedly lead to loss of midgut peristalsis and cessation of feeding, but it can be easily overlooked without careful histological examination using a high magnification microscope objective. No bacterial cells were evident, suggesting that the lesions arose from a virus or from a specific toxin response. This report is an urgent plea for colleagues studying TPD/GPD to review their samples histologically to help in determining whether MSMN is pathognomonic for TPD/GPD.

A systematic review and meta-analysis of genetic parameters underlying inheritance and complex biological relationships for quantitative traits are not available for aquatic animal species. I synthesised and conducted a comprehensive meta-analysis of the published information from 1985 to 2017 on heritability, common full-sib effects and genetic correlations for quantitative characters of biological importance (growth, carcass and flesh quality, disease resistance, deformity and reproduction) for aquaculture species. A majority of the studies (73.5%) focussed on growth related traits (body weight), followed by those on disease resistance (15.9%), whereas only a limited number of studies (10.6%) reported heritability estimates for carcass and flesh quality, deformity or reproduction characteristics. The weighted means of heritability for growth (weight, food utilisation efficiency, maturity) and carcass (fillet weight and yield) traits were moderate. Resistance against various bacteria, virus and parasites were moderately to highly heritable. Across aquatic animal species, the weighted heritability for a range of deformity measures and reproductive traits (fecundity, early survival) was low and not significantly different from zero. The common full-sibs (c2) accounted for a large proportion of total variance for body traits but it was of smaller magnitude in later phase of the growth development. The c2 effects however were not significant or in many cases they were not reported for carcass and flesh quality attributes as well as survival and deformity. The maternal genetic effects were not available for all traits studied especially for reproductive and early growth characters. Genetic correlations between body and carcass traits were high and positive, suggesting that selection for rapid growth can improve fillet weight, a carcass trait of paramount importance. Body weight, the most commonly used selection criterion in aquatic animals, showed non-significant genetic correlation with disease resistance, likely because both positive and negative genetic associations between the two types of traits. Interestingly the genetic associations between growth and reproductive performance (fecundity) and fry traits (fry weight, fry survival) were favourable. To date, there are still no published data on genetic relationships of carcass and flesh quality with disease resistance or reproductive performance in any aquaculture species. Additionally, the present study discussed new traits, including functional, immunological, behavioural and social interaction as well as uniformity that are emerging as potential selection criteria and which can be exploited in future genetic improvement programs for aquatic animals.

Nile tilapia (Oreochromis niloticus) is one of the most important aquaculture species in the world. When bacteria are present in cultured tilapia but do not cause a declared disease, it makes them asymptomatic carrier organisms. Once environmental or nutritional conditions change, an outbreak may occur. This is why it is so important to detect pathogens before outbreaks occur. This is the first study that use molecular techniques based on PCR to estimate prevalence of fish pathogens in southwest Mexico. During 2018, 2019, 2020 and 2022 samples of internal organs and lesions of Nile tilapia were taken and analyzed for detection of the main bacterial tilapia pathogens using one-step PCR or qPCR. A total of 2396 samples from the internal organs of Nile tilapia pond and cage cultured come from the Mexican Pacific southwest states of Guerrero, Oaxaca and Chiapas were analyzed. Most of the sampled tilapias were apparently healthy and had no relation between the clinical signs and the pathogens detection was found. No Francisella sp. was detected in any sample, Staphylococcus sp. was the most prevalent bacterial genus from the three states over time (from 0 to 64 %). Prevalence of Aeromonas sp. was from 0 to 4.3 %, although the fish pathogen A. dhakensis was not detected. Meanwhile, S. iniae was only detected in Chiapas in 2019 at low prevalence (1.4 %) and S. agalactiae was detected in the three sites at high prevalence (from 0 to 59 %). Both Streptococcus can cause streptococcosis, the most dangerous re-emergent disease to cultured tilapia, which means a great risk for tilapia farming in Mexico.

Japanese medaka (Oryzias latipes) are a teleost fish and an emerging vertebrate model for ecotoxicology, developmental, genetics, and physiology research. Medaka are also used extensively to investigate vertebrate reproduction, which is an essential biological function as it allows a species to perpetuate. Sperm quality is an important indicator of male fertility and thus reproduction success. Techniques for extracting sperm and sperm analysis are well documented for many species, including for teleost fish. Collecting semen is relatively simple in larger fish but can be more complicated in small model fish as they produce less sperm and are more delicate. This article therefore describes two methods of sperm collection in the small model fish Japanese medaka: testes dissection and abdominal massage. We demonstrate that both approaches are viable for medaka and show that abdominal massage can be performed a repeated number of times as the fish quickly recover from the procedure. We also describe a protocol for computer-assisted sperm analysis in medaka to objectively assess several important indicators of medaka sperm quality (motility, progressivity, duration of motility, relative concentration). The use of these procedures combined with the other advantages of using this small teleost model will greatly improve the understanding of the environmental, physiological, and genetic factors influencing fertility in vertebrate males. SUMMARYThis article describes two quick and efficient methods to collect semen from the small model fish medaka (Oryzias latipes), as well as a protocol to reliably assess sperm quality using computer-assisted sperm analysis (CASA).

Enterocytozoon hepatopenaei (EHP) is a shrimp pathogen that causes huge economic losses. In the present study, the hosts of EHP were investigated using polymerase chain reaction (PCR) in an aquaculture farm located in Maoming, China. EHP was detected in Litopenaeus vannamei, Penaeus monodon, crab, false mussel, and three dragonfly species (Anax parthenope, Pantala flavescens, and Ischnura senegalensis). In the histopathological examination using hematoxylin-eosin staining, EHP spores were found in nymphs and adult dragonflies naturally infected with EHP that were collected from the shrimp farm. Fluorescence in situ hybridization results showed a positive signal for EHP infection in the fat body of dragonfly nymphs. Immature and mature microsporidian spores and late sporogonial plasmodium were observed in the cytoplasm of dragonfly nymphs using transmission electron microscopy. The transmission of EHP from shrimp to dragonfly nymphs was confirmed via cohabitation challenge experiments in which EHP-free dragonfly nymphs were cohabited with EHP-infected shrimp, and the transmission of EHP from dragonfly nymphs to shrimp was demonstrated via the cohabitation of EHP-infected dragonfly nymphs with EHP-free shrimp and oral administration challenge experiments. This study confirms that dragonflies can act as natural EHP hosts, and a novel EHP horizontal transmission route exists between dragonflies and shrimp. Author summaryTo the authors knowledge, this study presents the first report of microsporidia (EHP) infecting both crustaceans and insects (A. parthenope, P. flavescens, and I. senegalensis). The horizontal transmission of EHP between dragonfly nymphs and shrimp was confirmed through cohabitation and oral administration challenge experiments. EHP has become a globally significant threat to shrimp aquaculture. The findings of the present study will help to design prevention strategies, such as the use of nets to prevent dragonflies from entering shrimp ponds.

Marine medaka (Oryzias melastigma) animal models play critical roles in environmental and human health by facilitating evaluation of pollutant toxicity and building of disease models. The fish gut microbiota contributes to host health and physiological metabolism, especially special bacterial strains and their functions in marine organisms. However, the distribution of the gut microbiota during medaka growth and development is still unclear, and successful generation of a germ-free (GF) marine medaka model has not been reported to date. In this study, we investigated the microbial composition with the major phyla and genera of marine fish at different life stages, as well as the isolated culturable intestinal bacteria, and then identified them by sequencing of the16S rRNA V3-V4 region. Importantly, the early stage model (larvae) of GF marine medaka without feeding and long-term (from juvenile to early adult stages) GF fish fed GF brine shrimp (Artemia sp.) were first generated. Moreover, the basic indexes and behavioral ability of GF fish showed weaker and delayed developmental changes compared to conventionally raised (CR) marine medaka at the same life stages. Notably, the significant differences in the histopathological characteristics of immune organs, intestinal tissues and the reproductive system were observed between GF and CR early-adult and adult fish. Furthermore, the transcriptomic profiles of the screened critical genes in signaling pathways in GF and CR marine medaka were also explored to illustrate the developmental impacts of the absence of the intestinal microbiota during the host growth. Comprehensively, our study provided novel insights into the intestinal microbiota distribution of CR fish during growth, and GF marine medaka from the larval to adult stages via GF fish food preparation. The histopathological and transcriptomic differences indicated the potential microbial regulation on growth, and application prospects of GF medaka fish models to clarify the relationships of intestinal bacterial functions to host health in the future. SignificanceThe generation and application of germ-free (GF) fish models are mostly limited to the early life stages with innate immunity and without feeding. Marine medaka (Oryzias melastigma) is a critical animal for evaluating environmental toxicity and human disease models. The gut microbiota contributes to host growth and development, but GF model of this organism has not been successfully generated. In this study, we revealed for the first time the distribution of the gut microbiota in marine medaka during growth and generated GF fish from the larval to adult stages with GF Artemia provided daily as food. According to the basic indexes, weaker behavioral ability, smaller immune organs, reproductive system, intestinal tissues, and transcriptome, the delayed development and differences indicated the negative influences of the absence of the microbiota in GF medaka, compared to conventionally raised (CR) fish at the same life stages. All these results provided novel insights into the application of GF medaka models to define intestinal bacterial functions in the host. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=86 SRC="FIGDIR/small/536225v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@15625b5org.highwire.dtl.DTLVardef@c5cf62org.highwire.dtl.DTLVardef@b264c5org.highwire.dtl.DTLVardef@1f5e7d3_HPS_FORMAT_FIGEXP M_FIG C_FIG This work revealed the distribution of the gut microbiota in marine medaka during growth, and successfully generated GF marine medaka models from larvae to adults with GF Artemia as food, which indicated the delayed development in the absence of the microbiota in GF fish. Moreover, the histopathological analysis presented further evidence of developmental differences in immune organs, intestinal villi, goblet cells, gonad tissues and cell maturation between GF and CR fish at various life stages. Finally, the transcriptomic profile showed the significantly differentially regulated genes, which combined with the major bacteria can be potential "biomarkers" to explore the inner mechanisms or signaling pathways of GF fish models for studying host development and health.

BackgroundCryopreservation is a valuable tool in aquaculture and conservation programs, yet it exposes spermatozoa to physiological and molecular stresses that may impair motility, fertilisation capacity, and genomic stability. In fishes, where sperm motility is brief and easily activated, post-thaw separation of high-quality sperm remains technically challenging and poorly understood. This study evaluated whether density-gradient centrifugation can isolate a functionally superior subpopulation of sterlet (Acipenser ruthenus) spermatozoa with enhanced motility, fertilising ability, and reduced cryopreservation-induced epigenetic alterations. We further examined whether the use of this selected fraction influences DNA methylation patterns in resulting embryos. ResultsCryopreservation substantially reduced the proportion of motile spermatozoa, while density-gradient centrifugation consistently enriched motile cells both before and after freezing. Motility enhancement reflected a higher proportion of cells capable of activation rather than changes in kinematic behaviour. Fertilisation trials confirmed that the selected post-thaw fraction produced fewer malformed embryos compared with unselected cryopreserved sperm. Cryopreservation and post-thaw selection induced small but significant methylation changes in sperm, predominantly in intergenic regions and promoter-proximal elements. However, these epigenetic differences were not maintained in embryos. Embryo methylomes showed minimal variation between treatments, no distinct clustering by sperm origin, and negligible numbers of differentially methylated regions. Thus, although cryopreservation and sperm selection influenced sperm DNA methylation, these alterations did not translate into measurable changes in embryo methylation patterns. ConclusionsDensity-gradient centrifugation effectively isolates a motile, functionally improved sterlet sperm fraction after cryopreservation, enhancing fertilisation outcomes and reducing developmental abnormalities. Cryopreservation and sperm selection introduce detectable but limited methylation variation in spermatozoa; however, these changes are not inherited by embryos. The findings highlight the utility of post-thaw sperm selection in aquaculture practice and indicate that cryopreservation-associated epigenetic variation in sterlet sperm does not propagate to early developmental stages.

Changes in growth, survival, maturation and health in Atlantic salmon post-smolts were observed under moderate and more extreme heat-wave conditions for the west coast of Scotland. A Scottish Atlantic salmon breeding programme population of 518 salmon of age 13-14 months, was observed for 4 weeks in summer, in tanks with ambient water temperature or [~] 4 degrees and [~] 8 degrees above ambient temperature. Data were recorded for the fish before and after the challenge. All fish were genotyped using a custom SNP (single nucleotide polymorphism) array (8,978 SNP genotypes after quality control). Temperature-dependant genotype-by-environment interactions and the potential for selective breeding to improve resilience were investigated. Statistical analyses revealed significant differences between thermal environments for body weight, average daily weight gain, and survival, while higher temperature induced earlier maturation, and an increase of gill health scores. QPCR (quantitative polymerase chain reaction) analyses revealed the presence of Aeromonas hydrophila, a pathogen typically more prevalent in warmer climates. Using genomic relationships and animal mixed models, body weight and average daily weight gain provided moderate heritabilities, while between-tank genetic correlations were close to 1, indicating no significant re-ranking of genotypes between the different thermal environments. These findings suggest that even short-term exposure to heat stress may be sufficient to negatively impact survival and gill health, and induce earlier maturation. However, observations took place within a commercial farm where having replicates for each thermal environment was not possible, hence further experiments with larger populations, exposed to more prolonged heat stress are needed.

As a promising biotechnology, fish germ cell transplantation shows potentials in conservation germplasm resource, propagation of elite species, and generation of transgenic individuals. In this study, we successfully transplanted the Japanese flounder (P. olivaceus), summer flounder (P. dentatus), and turbot (S. maximus) spermatogonia into triploid Japanese flounder larvae, and achieved high transplantation efficiency of 100%, 75-95% and 33-50% by fluorescence tracking and molecular analysis, respectively. Eventually, donor-derived spermatozoa produced offspring by artificial insemination. We only found male and intersex chimeras in inter-family transplantations, while male and female chimeras in both intra-species and intra-genus transplantations. Moreover, the intersex chimeras could mature and produce turbot functional spermatozoa. We firstly realized inter-family transplantation in marine fish species. These results demonstrated successful spermatogonial stem cells transplantation within Pleuronectiformes, suggesting the germ cells migration, incorporation and maturation within order were conserved across a wide range of teleost species.

Knowledge about paternal-effect genes (the expression of which in progeny is controlled by the paternal genome) in fish is very limited. To explore this issue, we used semen cryopreservation as a specific challenge test for sperm cells, thus enabling selection amidst cryo-sensitivity. We created two groups of Eurasian perch (Perca fluviatilis) as a model - eggs fertilized either with fresh (Fresh group) or cryopreserved (Cryo group) semen from the same male followed by zootechnical-transcriptomic examination of consequences of cryopreservation in obtained progeny (at larval stages). Most of the zootechnical observations were similar in both groups, except the final weight was higher in the Cryo group. Semen cryopreservation appeared to act as a "positive selection" factor, upregulating most paternal-effect genes in the Cryo group. Transcriptomics profile of freshly hatched larvae sourced genes involved in the development of visual perception as paternal-effect genes. Consequently, larvae from the Cryo group exhibited enhanced eyesight, potentially contributing to more efficient foraging and weight gain compared to the Fresh group. This study unveils, for the first time, the significant influence of the paternal genome on the development of the visual system in fish, highlighting pde6g, opn1lw1, and rbp4l as novel paternal-effect genes.

Viral haemorrhagic septicaemia (VHS) is a severe disease affecting rainbow trout (Oncorhynchus mykiss) and a wide range of wild freshwater and marine fish species. VHSV threatens rainbow trout aquaculture, as it may cause 100% mortality in fry. Previous studies identified a quantitative trait locus (QTL) on chromosome 3 associated with resistance to VHSV waterborne challenge and reduced viral replication in fin explants, although these findings were obtained using limited genetic diversity. The objective of this study was to validate and extend the identification of genomic regions associated with resistance to VHSV in the genetically diverse rainbow trout line designated "synthetic." A genome-wide association study (GWAS) was conducted using whole-genome sequences from parents of progeny classified as resistant or susceptible to a VHSV waterborne challenge. While the QTL on chromosome 3 was not validated in the synthetic line, four novel suggestive SNPs associated with survival following VHSV waterborne challenge were identified on chromosomes 6, 8, 17, and 32. Notably, one SNP on chromosome 17 was located within a gene potentially involved in antiviral defence, a paralog of lrp1 (low-density lipoprotein receptor-related protein 1). To further investigate its role, lrp1 function was analysed in vitro using CRISPR-Cas9 genome editing. Three independent lrp1-/- CHSE-EC cell lines were generated and challenged with VHSV. The results showed that lrp1 is not essential for viral entry but may modulate the inflammatory response during VHSV infection in epithelial cell lines.

Recombinant gonadotropins, follicle stimulating (rFSH) and luteinizing hormone (rLH), offer the potential to induce gametogenesis in prepubertal fish. This study aimed to determine the in vivo effect of Argyrosomus regius rFSH (arrFSH) and Argyrosomus regius rLH (arrLH) administered to prepubertal meagre juveniles at the initial stages of sex differentiation. Meagre single-chain recombinant gonadotropins, arrFSH and arrLH were produced with the CHO expression system by Rara Avis Biotec, S. L. Juvenile meagre, 9-months old with mean weight of 222 {+/-} 36 g (mean {+/-} SD) were randomly distributed into seven experimental groups (n = 8 per group) that were treated weekly for three weeks with an acute injection of 6, 12 or 18 g kg-1 of arrFSH (groups, 6-arrFSH, 12-arrFSH and 18-arrFSH) or 6, 12 or 18 g kg-1 of arrLH (groups, 6-arrLH, 12-arrLH and 18-arrLH) or saline solution (Control group). Two more groups (n=8) were set up and treated for 6 weeks, with 12 g kg-1 of arrFSH or saline Control. The fish were held in a 10 m3 tank with natural photoperiod (Feb. - March) and temperature 16.1 {+/-} 0.4{degrees}C. At the start of the experiment (n = 8) and end of the three week experiment all fish were sacrificed and gonads dissected. Gonads were weighed, fixed in Bouins solution and processed for histological analysis. Blood was sampled from all fish at the start and end of the experiment (week 3 and 6) for 17{beta}-estradiol (E2) and 11-ketotestosterone (11-KT) analysis. Juvenile meagre at the start of the experiment were in the initial stages of sexual differentiation, indicated by the presence of the ovarian or testes lumen that was surrounded by undifferentiated embryonic germ stem cells and somatic cells. At the end of the experiment, there was no significant difference in gonadosomatic index (GSI) amongst control (initial and saline treated) and the experimental groups. After three weeks of application of arrFSH, arrLH or saline all fish presented a similar gonadal structure as at the start of the experiment. However, the incidence of isolated developing germ cells (principally spermatogonia, spermatocytes, spermatids, but also perinucleolar stage oocytes) generally increased in arrGTH treated meagre. A mean of 44 % of meagre treated with arrFSH or arrLH presented isolate developing germ cells, mainly male cells. Plasma steroid levels of E2 decreased significantly from the start of the experiments to the end. At the end of the experiment there were no differences in plasma E2 amongst Control fish and rGTH treated fish. Plasma 11-KT showed no change from the start of the experiment to week 3. However, a significant increase was observed in the arrFSH group after six weeks of treatment compared to the start of the experiment and the control group on week 6. The application of arrFSH or arrLH to meagre at the initial stages of sex differentiation did not stimulate steroid production until week six and had a limited, but evident effect on the development of isolated germ cells. The rGTHs, arrFSH or arrLH did not stimulate large developmental changes in undifferentiated gonads. HIGHLIGHTSExogenous recombinant gonadotropins administered to meagre did not advance sexual differentiation. Exogenous recombinant gonadotropins administered to meagre did not affect plasma estradiol. Exogenous recombinant FSH increased the proportion of meagre with isolated male germ cells. Exogenous recombinant FSH increased plasma 11-ketotestosterone in meagre treated for 6 weeks.

MicroRNAs (miRNAs) are small, non-coding RNAs that are involved in post-transcriptional gene regulation in many cellular functions and are highly conserved throughout evolution. In teleost fish species, miRNAs are believed to play a role in the reproductive system, but more research is needed to better understand the functions of miRNAs in fish gonads. Furthermore, miR-210 has previously been described to be involved in many processes, such as hypoxia response, angiogenesis, cell proliferation and male infertility. The aim of this study was, first, to develop an in vitro model in fish to study the functions of miRNAs and second, to identify target genes of dre-miR-210-5p by establishing a primary ovarian cell culture in zebrafish (Danio rerio). The cell culture was performed by isolating ovaries from adult female fish (n=4) which were incubated with dre-miR-210-5p mimic or scramble miRNA mimic. Cell survival was studied by flow cytometry analysis and fluorescent microscopy. Following, the effect of dre-miR-210-5p on ovarian cells was uncovered by RNA-sequencing, identifying [~]6,000 targeted-genes differentially expressed (DEGs), of which [~]2,600 downregulated and [~]3,400 upregulated. GO term and KEGG pathway analyses showed downregulated genes involved in cell cycle processes and reproduction-related pathways while, in contrast, immune-related pathways were upregulated after miR-210 mimic treatment in the ovarian cells. To provide the molecular mechanisms triggered by miRNA-210, seed regions of targeted genes were identified and in silico analysis classified DEGs as potential biomarkers in reproduction or immune cell types. These results support the crosstalk between the reproductive and immune system in which dre-miR-210-5p plays a key role in the transcriptomic alteration in the fish ovaries. Take home messagemiR-210 promotes the immune response and suppresses oocyte meiosis in the ovarian cells in zebrafish

Pacific oysters (Crassostrea or Magallana gigas) are one of the most economically important aquaculture species globally. Over the past two decades, ostreid herpesvirus (OsHV-1), has become a major pathogen of cultured Pacific oysters resulting in widespread mortality with a global distribution. Experimental use of OsHV-1 is challenging for many reasons, including both complexity and relative obscurity of host pathogen dynamics, and a lack of functioning model systems. The goal of this study was to improve the tools available for working with OsHV-1 in both whole animals and in tissue explants established ex vivo from oysters and maintained in controlled laboratory conditions. Tissue explants were taken from oysters originating from two different sources that have different levels of mortality in OsHV-1 challenges and were used in disease challenges alongside whole animals for comparison. Quantitative PCR, histology and electron microscopy were used to confirm that the explants were capable of replicating OsHV-1. Furthermore, the quantitative PCR results suggests that the source of the oysters was significant in determining the outcome of infection in the explants, supporting the validity of the explant model for OsHV-1 infection. This approach for studying OsHV-1 allows for the control of confounding factors in disease outcome that is not possible in whole animal challenges, providing a new tool for studying OsHV-1 in Pacific oysters.

Multiple PCR analyzes were performed using 19 different primer sets to open and broaden the search spectrum for shrimp pathogens. In addition, multiple primer pairs for 10 pathogens were compared to see if there were differences in selectivity or sensitivity among them. Some pathogens that did not present histological lesions were detected. The most important outcome was that selection of appropriate primers was the most critical factor in obtaining reliable results. We found high variability in results among primers and we learned it was prudent to seasonally assess among them for the best set selection. It is important to understand that a PCR positive test result alone does not confirm the presence of a viable pathogen or a disease state. Nor, as might be expected, does it mean that the positive PCR test results will be necessarily accompanied by histological lesions characteristic of the targeted pathogen. However, the use of appropriately selected primers sets can reveal whether there is an evolution in the result spectrum over time and if some pathogens disappear or reappear or new ones emerge. In general, most shrimp presented coinfections that consisted of the presence of WzSV8, DHPV, chronic midgut inflammation and tubule distension/epithelial atrophy consistent with Pir A/B toxicity. Also included were RLB/NHPB, microsporidia, striated muscle necrosis, gregarines in the hindgut caecum (gametocyte stage, and not associated with tegumental glands but glands that line the mouth and anus) and encysted, presumed nematode larvae. WzSV8 was newly discovered in gonads. Histological changes and the presence of spheroids in the lymphoid organ were considered as healthy host responses of often unidentified cause.

An understanding of the reproductive biology of aquatic organisms is crucial for the efficient conservation and management of species and/or populations. Nevertheless, conventional spawning surveys such as visual- and capture-based monitoring generally require laborious, time-consuming work and are subject to monitoring biases such as observer bias, as well as miscounts due to false spawning. In addition, direct capture often damages eggs or individuals. Thus, an efficient non-invasive approach for monitoring spawning activity on aquatic species would be a valuable tool to understand their reproductive biology and conserving biodiversity. Here, we proposed an environmental DNA (eDNA)-based approach for monitoring and understanding spawning activity by observing spikes in eDNA concentration after spawning activity. We found in cross experiments using two medaka species (Oryzias latipes and O. sakaizumii, 1:1 individual per tank) that an eDNA spike occurred in only male species after spawning activity. In addition, the magnitude of the eDNA spike was dependent on the number of spawning activities with egg and sperm release. In the field survey during the reproductive season, eDNA concentration after spawning were 3-25 times higher than before spawning. On the other hand, there was no increase in eDNA concentration during the non-reproductive season. Therefore, our results demonstrated that spikes in the eDNA concentration are mainly caused by the release of sperm during spawning activity, and it can be used as evidence of spawning in field survey. The presented approach could be a practical tool for studying reproductive biology and provides an opportunity to design effective conservation and environmental management actions.