Nephrocalcinosis in juvenile farmed Atlantic Salmon (Salmo salar) may be linked to osmoregulatory stress.

Nephrocalcinosis is a widespread challenge in intensive production of salmon smolt. There is however no consensus on its aetiology, which makes it problematic to implement proper measures to limit its development. We performed a survey of nephrocalcinosis prevalence and environmental factors in 11 different hatcheries in Mid-Norway as well as a 6-month monitoring in one of the hatcheries. A multivariate analysis indicated that the most influencing factor for the prevalence of nephrocalcinosis was the supplementation of sea water during smolt production. In the 6-month monitoring, the hatchery introduced salinity in the production water prior to the change in day length. Mismatch in those environmental signals may increase the risk for developing nephrocalcinosis. Salinity fluctuations prior to smoltification can cause osmotic stress and result in unbalanced levels of ions in fish blood. This was clearly illustrated in our study, as the fish experienced chronic hypercalcaemia and hypermagnesaemia. Both magnesium and calcium are excreted over the kidneys and it is possible that their prolonged, elevated levels in plasma resulted in an oversaturation of the urine when finally excreted. This again could have led to the aggregation of calcium deposits within the kidney. This study indicates a relationship between osmotic stress induced by salinity changes in juvenile Atlantic salmon and the development of nephrocalcinosis. Other factors that may affect the severity of nephrocalcinosis are currently subjects for discussion.

Amoebic gill disease increases energy requirements and decreases hypoxia tolerance in Atlantic salmon (Salmo salar) smolts.

Globally, Atlantic salmon (Salmo salar Linnaeus) aquaculture is now routinely affected by amoebic gill disease (AGD; Neoparamoeba perurans). The disease proliferates throughout the summer and is implicated in decreasing tolerance of salmon to environmental perturbations, yet little empirical evidence exists to support these observations. Using salmon acclimated to 15 or 19 °C, our aim was to determine the effects of clinically light-moderate (industry-relevant) AGD on metabolism (MO2rest and MO2max), aerobic scope (MO2max - MO2rest), excess post-exercise oxygen consumption (EPOC), and hypoxia tolerance. An increase in MO2rest (~8% and ~ 13% increase within the 15 and 19 °C acclimation groups, respectively) with increasing disease signs demonstrated an increase in baseline energy requirements as the disease progressed. Conversely, MO2max remained stable at both temperatures (~364 mg O2 kg-1 h-1), resulting in a decline in aerobic scope by 13 and 19% in the 15 and 19 °C groups, respectively. There was evidence of a decrease in hypoxia tolerance as the dissolved oxygen concentrations at loss of equilibrium increased by ~8% with more severe lesion coverage of the gills. These results suggest an increase in basal energy requirements and reduction in hypoxia tolerance as AGD proliferates, lending support to the idea that AGD reduces environmental tolerance. However, the lack of an effect of acclimation temperature indicates that the temperature-disease interaction may be more complicated than currently thought.

microRNA-20-1 and microRNA-101a Suppress the NF-κB-Mediated Inflammation Production by Targeting TRAF6 in Miiuy Croaker.

Upon recognition of pathogen components by pattern recognition receptors, cells could be activated to produce inflammatory cytokines and type I interferons. The inflammation is tightly modulated by the host to prevent inappropriate inflammatory responses. MicroRNAs (miRNAs) are noncoding small RNAs that can inhibit gene expression and participate in various biological functions, including maintaining a balanced immune response in the host. To maintain the balance of the immune response, these pathways are closely regulated by the host to prevent inappropriate reactions of the cells. However, in lower vertebrates, the miRNA-mediated inflammatory response regulatory networks remain largely unknown. Here, we report that two miRNAs, i.e., miR-20-1 and miR-101a, were identified as negative regulators in teleost inflammatory responses. Initially, we found that both miR-20-1 and miR-101a dramatically increased after lipopolysaccharide (LPS) stimulation and Vibrio harveyi infection. Upregulated miR-20-1 and miR-101a inhibited LPS-induced inflammatory cytokine production by targeting tumor necrosis factor receptor-associated factor 6 (TRAF6), thus avoiding excessive inflammation. Moreover, miR-20-1 and miR-101a regulate the inflammatory responses through the TRAF6-mediated NF-κB signaling pathway. Collectively, these data indicate that miR-20-1 and miR-101a act as negative regulators by regulating the TRAF6-mediated NF-κB signaling pathway and participate in host antibacterial immune responses, which will provide new insights into the intricate networks of the host-pathogen interactions in the lower vertebrates.

The Zebrafish Antiapoptotic Protein BIRC2 Promotes Edwardsiella piscicida Infection by Inhibiting Caspases and Accumulating p53 in a p53 Transcription-Dependent and -Independent Manner.

IAPs (inhibitors of apoptosis) are endogenous caspase inhibitors with multiple biological activities. In the present study, we show functional characteristics of antiapoptotic protein BIRC2 (cIAP1) in response to Edwardsiella piscicida infection. Overexpression of BIRC2 in zebrafish larvae promoted the proliferation of E. piscicida, leading to a decreased larvae survival. The expression levels of caspases including casp3, casp8, and casp9 were significantly inhibited by BIRC2 overexpression in the case of E. piscicida infection. Treatment of zebrafish larvae microinjected with BIRC2 with the caspase activator PAC-1 completely blocked the negative regulation of BIRC2 on the E. piscicida infection, with the reduced inhibition on the casp3 and without inhibition on casp8 and casp9. In contrast to the regulation of BIRC2 on the caspases, BIRC2 overexpression significantly induced the expression of p53, especially at 24 hpi. In addition to the cytoplasmic p53 expression, BIRC2 overexpression also induced the expression of the nuclear p53 protein. Further analysis demonstrated that BIRC2 could interact and colocalize with p53 in the cytoplasm. The numbers of E. piscicida in larvae overexpressed with BIRC2 and treated with pifithrin-µ (an inhibitor of mitochondrial p53) or pifithrin-α (an inhibitor of p53 transactivation) were lower than those of larvae without pifithrin-µ or pifithrin-α treatment. Critically, the p53 inactivators pifithrin-µ and pifithrin-α had no significant effect on larval survival, but completely rescued larval survival for zebrafish microinjected with BIRC2 in the case of E. piscicida infection. Collectively, the present study suggest that piscine BIRC2 is a negative regulator for antibacterial immune response in response to the E. piscicida infection via inhibiting caspases, and accumulating p53 in a p53 transcription-dependent and -independent manner.

Interleukin-22 Deficiency Contributes to Dextran Sulfate Sodium-Induced Inflammation in Japanese Medaka, Oryzias latipes.

Mucosal tissue forms the first line of defense against pathogenic microorganisms. Cellular damage in the mucosal epithelium may induce the interleukin (IL)-22-related activation of many immune cells, which are essential for maintaining the mucosal epithelial barrier. A previous study on mucosal immunity elucidated that mammalian IL-22 contributes to mucus and antimicrobial peptides (AMPs) production and anti-apoptotic function. IL-22 has been identified in several teleost species and is also induced in response to bacterial infections. However, the roles of IL-22 in teleost immunity and mucus homeostasis are poorly understood. In this study, Japanese medaka (Oryzias latipes) was used as a model fish. The medaka il22, il22 receptor A1 (il22ra1), and il22 binding protein (il22bp) were cloned and characterized. The expression of medaka il22, il22ra1, and il22bp in various tissues was measured using qPCR. These genes were expressed at high levels in the mucosal tissues of the intestines, gills, and skin. The localization of il22 and il22bp mRNA in the gills and intestines was confirmed by in situ hybridizations. Herein, we established IL-22-knockout (KO) medaka using the CRISPR/Cas9 system. In the IL-22-KO medaka, a 4-bp deletion caused a frameshift in il22. To investigate the genes subject to IL-22-dependent regulation, we compared the transcripts of larval medaka between wild-type (WT) and IL-22-KO medaka using RNA-seq and qPCR analyses. The comparison was performed not only in the naïve state but also in the dextran sulfate sodium (DSS)-exposed state. At the transcriptional level, 368 genes, including immune genes, such as those encoding AMPs and cytokines, were significantly downregulated in IL-22-KO medaka compared that in WT medaka in naïve states. Gene ontology analysis revealed that upon DSS stimulation, genes associated with cell death, acute inflammatory response, cell proliferation, and others were upregulated in WT medaka. Furthermore, in DSS-stimulated IL-22-KO medaka, wound healing was delayed, the number of apoptotic cells increased, and the number of goblet cells in the intestinal epithelium decreased. These results suggested that in medaka, IL-22 is important for maintaining intestinal homeostasis, and the disruption of the IL-22 pathway is associated with the exacerbation of inflammatory pathology, as observed for mammalian IL-22.

CsIL-11, a teleost interleukin-11, is involved in promoting phagocytosis and antibacterial immune defense.

Interleukin (IL)-11 is a multifunctional cytokine belonging to the IL-6 family, which plays essential roles in immune response. However, much less is known about the immunological functions of IL-11 in teleost. In this study, we investigated the immune properties of a teleost IL-11 homologue (CsIL-11) from tongue sole Cynoglossus semilaevis. CsIL-11 possesses four conserved α-helices and conserved CsIL-11 receptor binding residues L86 and R187, and shares 23.3%-80.1% identities with other IL-11 homologues. CsIL-11 expression was constitutive in tissues, with most abundant in blood and least abundant in spleen, and upregulated by bacterial challenge in blood, spleen, and head kidney. Recombinant CsIL-11 (rCsIL-11) in the native form of monomer, could bind to peripheral blood leukocytes (PBLs) membrane and enhance the activation and phagocytosis of PBLs. When administered in vivo, rCsIL-11 could markedly promote the host to defend against microbial infection. Overall, our findings show that CsIL-11 plays a pivotal role in regulating PBLs phagocytosis and antibacterial immunity.

Aquaculture Reuse Water, Genetic Line, and Vaccination Affect Rainbow Trout (Oncorhynchus mykiss) Disease Susceptibility and Infection Dynamics.

Infectious hematopoietic necrosis virus (IHNV) and Flavobacterium psychrophilum are major pathogens of farmed rainbow trout. Improved control strategies are desired but the influence of on-farm environmental factors that lead to disease outbreaks remain poorly understood. Water reuse is an important environmental factor affecting disease. Prior studies have established a replicated outdoor-tank system capable of varying the exposure to reuse water by controlling water flow from commercial trout production raceways. The goal of this research was to evaluate the effect of constant or pulsed reuse water exposure on survival, pathogen prevalence, and pathogen load. Herein, we compared two commercial lines of rainbow trout, Clear Springs Food (CSF) and Troutex (Tx) that were either vaccinated against IHNV with a DNA vaccine or sham vaccinated. Over a 27-day experimental period in constant reuse water, all fish from both lines and treatments, died while mortality in control fish in spring water was <1%. Water reuse exposure, genetic line, vaccination, and the interaction between genetic line and water exposure affected survival (P<0.05). Compared to all other water sources, fish exposed to constant reuse water had 46- to 710-fold greater risk of death (P<0.0001). Tx fish had a 2.7-fold greater risk of death compared to CSF fish in constant reuse water (P ≤ 0.001), while risk of death did not differ in spring water (P=0.98). Sham-vaccinated fish had 2.1-fold greater risk of death compared to vaccinated fish (P=0.02). Both IHNV prevalence and load were lower in vaccinated fish compared to sham-vaccinated fish, and unexpectedly, F. psychrophilum load associated with fin/gill tissues from live-sampled fish was lower in vaccinated fish compared to sham-vaccinated fish. As a result, up to forty-five percent of unvaccinated fish were naturally co-infected with F. psychrophilum and IHNV and the coinfected fish exhibited the highest IHNV loads. Under laboratory challenge conditions, co-infection with F. psychrophilum and IHNV overwhelmed IHNV vaccine-induced protection. In summary, we demonstrate that exposure to reuse water or multi-pathogen challenge can initiate complex disease dynamics that can overwhelm both vaccination and host genetic resistance.

Myo-Inositol Restores Tilapia's Ability Against Infection by Aeromonas sobria in Higher Water Temperature.

Bacterial infection presents severe challenge to tilapia farming, which is largely influenced by water temperature. However, how water temperature determines tilapias' survival to infection is not well understood. Here, we address this issue from the perspective of metabolic state. Tilapias were more susceptible to Aeromonas sobria infection at 33°C than at 18°C, which is associated with differential metabolism of the fish. Compared to the metabolome of tilapia at 18°C, the metabolome at 33°C was characterized with increased an tricarboxylic acid cycle and a reduced level of myo-inositol which represent the most impactful pathway and crucial biomarker, respectively. These alterations were accompanied with the elevated transcriptional level of 10 innate immune genes with infection time, where il-1b, il-6, il-8, and il-10 exhibited a higher expression at 33°C than at 18°C and was attenuated by exogenous myo-inositol in both groups. Interestingly, exogenous myo-inositol inactivated the elevated TCA cycle via inhibiting the enzymatic activity of succinate dehydrogenase and malate dehydrogenase. Thus, tilapias showed a higher survival ability at 33°C. Our study reveals a previously unknown relationship among water temperature, metabolic state, and innate immunity and establishes a novel approach to eliminate bacterial pathogens in tilapia at higher water temperature.

Molecular evidence for homologous strains of infectious spleen and kidney necrosis virus (ISKNV) genotype I infecting inland freshwater cultured Asian sea bass (Lates calcarifer) in Thailand.

Infectious spleen and kidney necrosis virus (ISKNV) is a fish-pathogenic virus belonging to the genus Megalocytivirus of the family Iridoviridae. In 2018, disease occurrences (40-50% cumulative mortality) associated with ISKNV infection were reported in grown-out Asian sea bass (Lates calcarifer) cultured in an inland freshwater system in Thailand. Clinical samples were collected from seven distinct farms located in the eastern and central regions of Thailand. The moribund fish showed various abnormal signs, including lethargy, pale gills, darkened body, and skin hemorrhage, while hypertrophied basophilic cells were observed microscopically in gill, liver, and kidney tissue. ISKNV infection was confirmed on six out of seven farms using virus-specific semi-nested PCR. The MCP and ATPase genes showed 100% sequence identity among the virus isolates, and the virus was found to belong to the ISKNV genotype I clade. Koch's postulates were later confirmed by challenge assay, and the mortality of the experimentally infected fish at 21 days post-challenge was 50-90%, depending on the challenge dose. The complete genome of two ISKNV isolates, namely KU1 and KU2, was recovered directly from the infected specimens using a shotgun metagenomics approach. The genome length of ISKNV KU1 and KU2 was 111,487 and 111,610 bp, respectively. In comparison to closely related ISKNV strains, KU1 and KU2 contained nine unique genes, including a caspase-recruitment-domain-containing protein that is potentially involved in inhibition of apoptosis. Collectively, this study indicated that inland cultured Asian sea bass are infected by homologous ISKNV strains. This indicates that ISKNV genotype I should be prioritized for future vaccine research.

FoxO3 Modulates LPS-Activated Hepatic Inflammation in Turbot (Scophthalmus maximus L.).

In mammals, forkhead box O3 (foxo3) plays important roles in liver immune system. The foxo3 can regulate cell cycle, DNA repair, hypoxia, apoptosis and so on. However, as such an important transcription factor, few studies on foxo3 in fish have been reported. The present study characterized the foxo3 in turbot (Scophthalmus maximus L.). Lipopolysaccharide (LPS) incubated in vitro (hepatocytes) and injected in vivo (turbot liver) were used to construct inflammatory models. The foxo3 was interfered and overexpressed to investigate its functions in liver inflammation. The open reading frame (ORF) of foxo3 was 1998 bp (base pair), encoding 665 amino acids. Sequence analysis showed that foxo3 of turbot was highly homologous to other fishes. Tissue distribution analysis revealed that the highest expression of foxo3 was in muscle. Immunofluorescence result showed that foxo3 was expressed in cytoplasm and nucleus. Knockdown of foxo3 significantly increased mRNA levels of tumor necrosis factor-α (tnf-α), interleukin-1ß (il-1ß), interleukin-6 (il-6), myeloid-differentiation factor 88 (myd88), cd83, toll-like receptor 2 (tlr-2) and protein level of c-Jun N-terminal kinase (JNK) in sifoxo3 + LPS (siRNA of foxo3+ LPS) group compared with NC + LPS (negative control + LPS) group in turbot hepatocytes. Overexpressed foxo3 significantly decreased mRNA levels of tnf-α, il-6, nuclear transcription factor-kappa B (nf-κb), cd83, tlr-2 and the protein level of JNK in vitro. In vivo analysis, foxo3 knockdown significantly increased levels of GOT in serum after LPS injection compared with NC+LPS group. Overexpressed foxo3 significantly decreased levels of GPT and GOT in pcDNA3.1-foxo3+LPS group compared with pcDNA3.1+LPS group in vivo. Foxo3 knockdown significantly increased mRNA levels of tnf-α, il-1ß, il-6, nf-κb, myd88 and protein level of JNK in vivo in sifoxo3+LPS group compared with NC+LPS group in turbot liver. Overexpressed foxo3 significantly decreased mRNA levels of il-1ß, il-6, myd88, cd83, jnk and protein level of JNK in pcDNA3.1-foxo3+LPS group compared with pcDNA3.1+LPS group in turbot liver. The results indicated that foxo3 might modulate LPS-activated hepatic inflammation in turbot by decreasing the proinflammatory cytokines, the levels of GOT and GPT as well as activating JNK/caspase-3 and tlr-2/myd88/nf-κb pathways. Taken together, these findings indicated that FoxO3 may play important roles in liver immune responses to LPS in turbot and the research of FoxO3 in liver immunity enriches the studies on immune regulation, and provides theoretical basis and molecular targets for solving liver inflammation and liver injury in fish.

PRV-1 Infected Macrophages in Melanized Focal Changes in White Muscle of Atlantic Salmon (Salmo salar) Correlates With a Pro-Inflammatory Environment.

Melanized focal changes in white skeletal muscle of farmed Atlantic salmon, "black spots", is a quality problem affecting on average 20% of slaughtered fish. The spots appear initially as "red spots" characterized by hemorrhages and acute inflammation and progress into black spots characterized by chronic inflammation and abundant pigmented cells. Piscine orthoreovirus 1 (PRV-1) was previously found to be associated with macrophages and melano-macrophages in red and black spots. Here we have addressed the inflammatory microenvironment of red and black spots by studying the polarization status of the macrophages and cell mediated immune responses in spots, in both PRV-1 infected and non-infected fish. Samples that had been collected at regular intervals through the seawater production phase in a commercial farm were analyzed by multiplex fluorescent in situ hybridization (FISH) and RT-qPCR methods. Detection of abundant inducible nitric oxide synthase (iNOS2) expressing M1-polarized macrophages in red spots demonstrated a pro-inflammatory microenvironment. There was an almost perfect co-localization with the iNOS2 expression and PRV-1 infection. Black spots, on the other side, had few iNOS2 expressing cells, but a relatively high number of arginase-2 expressing anti-inflammatory M2-polarized macrophages containing melanin. The numerous M2-polarized melano-macrophages in black spots indicate an ongoing healing phase. Co-localization of PRV-1 and cells expressing CD8+ and MHC-I suggests a targeted immune response taking place in the spots. Altogether, this study indicates that PRV-1 induces a pro-inflammatory environment that is important for the pathogenesis of the spots. We do not have indication that infection of PRV-1 is the initial causative agent of this condition.

INFLUENCE OF PATHOGENS, FISH-RELATED CHARACTERISTICS, AND ENVIRONMENTAL FACTORS ON THE DEVELOPMENT OF SKIN ULCERATIONS IN WILD COMMON DAB (LIMANDA LIMANDA) FROM THE NORTH SEA.

Environmental changes or stressors can result in the development of diseases. Through regular fish disease surveys in the Belgian part of the North Sea, attention was drawn to a sudden increase of skin ulceration prevalence between 2011 and 2014 in common dab (Limanda limanda). Information on prevalence, ulceration, bacteriology, fish-related (e.g., length, age, and sex) and (spatial and temporal) environmental factors, and fishing intensity were gathered. This detailed investigation was framed within a long-term monitoring program, executed every spring-autumn from 2000 to present. Ulcerations were observed in 1.3% of fish (n=3,999). Spatial and temporal differences were evident, and highest prevalence was found in summer. Vibrio was the dominant cultivated bacterial genus present in the lesions. Skin ulcerations appeared to be correlated with length and body condition of the fish, as well as with temperature and pH of the seawater and fishing vessel density. Our research suggested the involvement of multiple factors in the development of skin ulcerations in common dab and endorsed the effects of changing environment and human influence on the marine ecosystem through activities such as fishing.

Intussusception caused by intestinal neoplasia in mature rainbow trout (Oncorhynchus mykiss, Walbaum 1792).

The aquaculture industry is growing and includes the farming and breeding of more than 580 aquatic species worldwide. The rainbow trout (Oncorhynchus mykiss, Walbaum 1792) is the most commonly bred trout species in Hungary. As broodfish form the basis of most fish farms, investigation into tumours occurring in trout, although under-researched, has proven to be a valuable and necessary field of study. During our investigation, we examined a broodstock of 3- to 6-year-old rainbow trouts (800) affected with idiopathic intestinal tumours (3%) which had consequentially led to ileus (40%). While performing necropsy, initial pathological observations showed intussusceptions. Tumours were discovered upon opening the body cavity, as well as metastasis forming in the livers and in the vessels of the gills. Histopathological and immunohistochemical tests allowed us to identify the neoplasms. The primary adenocarcinoma was found to have been developed within the intestines of the fish. The tumour tissue broke through the basal membrane and infiltrated the propria, protruding asymmetrically into the lumen of the mid-intestines, causing it to narrow significantly. This subsequently led to passage disorders, invagination of the intestinal segment and finally the emaciation of the fish. Histopathological and immunohistochemical inspection of the tumour cells displayed a high mitotic index, confirming malignancy.

Development of a 3D spheroid cell culture system from fish cell lines for in vitro infection studies: Evaluation with Saprolegnia parasitica.

Understanding the ways in which pathogens infect host cells is essential to improve and develop new treatment strategies. This study aimed to generate a novel in vitro infection model by establishing a reproducible 3D spheroid cell culture system that may lead to a reduced need for animals in fish disease research. 2D models (commonly cell lines) cannot replicate many key conditions of in vivo infections, but 3D spheroids have the potential to provide bridging technology between in vivo and in vitro systems. 3D spheroids were generated using cells from rainbow trout (Oncorhynchus mykiss) cell lines, RTG-2 and RTS-11. The RTG-2 spheroids were tested for their potential to be infected upon exposure to Saprolegnia parasitica spores. Positive infiltration of mycelia into the spheroids was verified by confocal microscopy. As a closer analogue of in vivo conditions encountered during infection, the straightforward model developed in this study shows promise as an additional tool that can be used to further our understanding of host-pathogen interactions for Saprolegnia and possibly a variety of other fish pathogens.

Use of alternative protein sources for fishmeal replacement in the diet of largemouth bass (Micropterus salmoides). Part II: effects of supplementation with methionine or taurine on growth, feed utilization, and health.

Fishmeal has long been a staple protein feedstuff for fish, but its global shortage and high price have prompted its replacement with alternative sustainable sources. In this experiment involving largemouth bass (a carnivorous fish), a new mixture of feedstuffs (45% poultry byproduct meal, 30% soybean meal, 15% blood meal, and 10% krill shrimp meal) was added to low (14.5%) fishmeal diets along with 0.0%, 0.5% taurine, 0.5% methionine, or 0.5% taurine plus 0.5% methionine (dry matter basis). The positive control diet [65.3% fishmeal (46% crude protein on dry matter basis)] and all low-fishmeal diets contained 40% true protein and 10% lipids. There were 3 tanks per treatment group (20 fish/tank). Fish with the mean initial body weight of 16.6 g were fed to satiety twice daily. Compared with the unsupplemented low-fishmeal group, supplementing either 0.5% methionine or 0.5% methionine plus 0.5% taurine to the low-fishmeal diet improved (P < 0.05) the growth, feed utilization, retention of dietary protein and lipids, and health of largemouth bass, reduced (P < 0.05) the occurrence of black skin syndrome from ~ 40 to ~ 10%. Histological sections of tissues from the fish with black skin syndrome showed retina degeneration, liver damage, and enteritis in the intestine. Compared with methionine supplementation, supplementing 0.5% taurine alone to the low-fishmeal diet did not affect the growth or feed efficiency of fish and had less beneficial effects (P < 0.05) on ameliorating the black skin syndrome. These results indicated that: (a) the basal low-fishmeal diet was inadequate in methionine or taurine; and (b) dietary supplementation with methionine was an effective method to improve the growth performance, feed efficiency, and health of largemouth bass. Further studies are warranted to understand the pathogenesis of the black skin syndrome in largemouth bass.

Skeletal deformities and meristic trait variations are common in the intertidal fish Bathygobius cocosensis (Perciformes-Gobiidae).

Extreme environmental conditions, such as temperature, can lead to meristic trait variation and skeletal deformities, which may have major impacts on individual fitness. As intertidal ecosystems experience rapid temperature and physicochemical fluctuations, intertidal fish living and reproducing in these environmental conditions may have phenotypes influenced by such variable environments. The impact of intertidal variability on fish development, however, has not been previously investigated. Skeletal deformities and meristic traits were assessed for Bathygobius cocosensis, a common intertidal fish living across the Indo-Pacific region, using a clearing and staining method on 72 individuals. Over 87% of individuals presented meristic variation and over 70% exhibited at least one type of skeletal deformity, mostly recorded in the caudal fin area. The unexpected prevalence of skeletal deformities among this intertidal fish population suggests that such deformities may be suitable markers to evaluate an individual's stress exposure during development and the subsequent fitness effects.