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Acoustic communication is linked to fitness traits in many animals, but under the current scenario of global warming, sound signals can be affected by rising temperatures, particularly in ectothermic organisms such as fishes. This study examines the effect of water temperature in acoustic communication in the two-spotted goby, Pomatoschistus flavescens. To address this, we looked at the effect of different temperatures on the acoustic features of drums produced by males during territorial defence and related it with their auditory sensitivity. We also analysed the differences in acoustic features between male agonistic drums and previously reported male courtship sounds, to better understand how acoustic communication may be affected by different temperature conditions. We recorded two-spotted goby males during territorial intrusions for 10 min at 16 °C, 19 °C, and 21 °C in the laboratory. We found that agonistic drums were shorter, had fewer pulses and shorter pulse periods at higher temperature, in contrast with the peak frequency that remained unaffected. Male agonistic and mating drums (recorded in a previous study) at 16 °C only differed in pulse period, which was higher in mating drums. Hearing thresholds obtained with Auditory Evoked Potentials at 16 °C, revealed higher sensitivity below 400 Hz, matching the main energy of agonistic and mating sounds. Our findings suggest that increasing temperature could potentially affect acoustic communication in this species by reducing the duration of agonistic drums, which might hinder effective communication. Nevertheless, the impact may not be significant as there was a good match between the best hearing sensitivity and the peak frequency range of their calls, which was not influenced by temperature. As fish and other organisms are increasingly threatened by multiple anthropogenic stressors, including warming, future research should address how changes in water temperature impact acoustic communication within a more realistic multi-stressor scenario.

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IgM is the major circulating Ig isotype in teleost fish, showing in Antarctic fish unique features such as an extraordinary long hinge region, which plays a crucial role in antibody structure and function. In this work, we describe the replacement of the hinge region of a murine monoclonal antibody (mAb) with the peculiar hinge from Antarctic fish IgM. We use the CRISPR/Cas9 system as a powerful tool for generating the engineered mAb. Then, we assessed its functionality by using an innovative plasmonic substrate based on bimetallic nanoislands (AgAuNIs). The affinity constant of the modified mAb was 2.5-fold higher than that obtained from wild-type mAb against the specific antigen. Here, we show the suitability of the CRISPR/Cas9 method for modifying a precise region in immunoglobulin gene loci. The overall results could open a frontier in further structural modifications of mAbs for biomedical and diagnostic purposes.

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Introduction: Red blood cells (RBCs), also known as erythrocytes, are underestimated in their role in the immune system. In mammals, erythrocytes undergo maturation that involves the loss of nuclei, resulting in limited transcription and protein synthesis capabilities. However, the nucleated nature of non-mammalian RBCs is challenging this conventional understanding of RBCs. Notably, in bony fishes, research indicates that RBCs are not only susceptible to pathogen attacks but express immune receptors and effector molecules. However, given the abundance of RBCs and their interaction with every physiological system, we postulate that they act in surveillance as sentinels, rapid responders, and messengers. Methods: We performed a series of in vitro experiments with Cyprinus carpio RBCs exposed to Aeromonas hydrophila, as well as in vivo laboratory infections using different concentrations of bacteria. Results: qPCR revealed that RBCs express genes of several inflammatory cytokines. Using cyprinid-specific antibodies, we confirmed that RBCs secreted tumor necrosis factor alpha (TNFα) and interferon gamma (IFNγ). In contrast to these indirect immune mechanisms, we observed that RBCs produce reactive oxygen species and, through transmission electron and confocal microscopy, that RBCs can engulf particles. Finally, RBCs expressed and upregulated several putative toll-like receptors, including tlr4 and tlr9, in response to A. hydrophila infection in vivo. Discussion: Overall, the RBC repertoire of pattern recognition receptors, their secretion of effector molecules, and their swift response make them immune sentinels capable of rapidly detecting and signaling the presence of foreign pathogens. By studying the interaction between a bacterium and erythrocytes, we provide novel insights into how the latter may contribute to overall innate and adaptive immune responses of teleost fishes.

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Previous studies show that bisphenol A (BPA) and its analogs induce oxidative stress and promote inflammatory response. However, the key molecules in regulating this process remain unclear. Here, we report significant inductive effects of BPA and bisphenol AF (BPAF) on a newly found long non-coding RNA linc-93.2 accompanied by oxidative stress and activation of pro-inflammatory pathways in treated fish and fish primary macrophages. Silencing linc-93.2 in fish primary macrophages in vitro or fish in vivo significantly promotes the expression of anti-oxidative stress-related genes and anti-inflammatory cytokines. This inhibition of pro-inflammatory cytokine expression, showing cell status disruption towards to M2 polarization. Followed by exposure to BPA or BPAF, silencing linc-93.2 in vitro or in vivo significantly attenuates the increased production of reactive oxygen species and malondialdehyde level aroused by bisphenol treatment, possibly owing to the enhancement of total antioxidant capacity observed in cells and tissue after linc-93.2 knockdown. RNA-sequencing further revealed regulation of nuclear factor-kappa b (NF-κB) in linc-93.2's downstream network, combining with our previous observation on the upstream regulation of linc-93.2 via NF-κB, which together suggest a critical role of linc-93.2 in promoting NF-κB positive feedback loop that may be an important molecular event initiating the immunotoxicity of bisphenols.

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The zebrafish, a widely used model in neurobiology, relies on hearing in aquatic environments. Unfortunately, its auditory pathways have mainly been studied in larvae. In this study, we examined the involvement of the anterior tuberal nucleus (AT) in auditory processing in adult zebrafish. Our tract-tracing experiments revealed that the dorsal subdivision of AT is strongly bidirectionally connected to the central nucleus of the torus semicircularis (TSc), a major auditory nucleus in fishes. Immunohistochemical visualization of the ribosomal protein S6 (pS6) phosphorylation to map neural activity in response to auditory stimulation substantiated this finding: the dorsal but not the ventral part of AT responded strongly to auditory stimulation. A similar response to auditory stimulation was present in the TSc but not in the nucleus isthmi, a visual region, which we used as a control for testing if the pS6 activation was specific to the auditory stimulation. We also measured the time course of pS6 phosphorylation, which was previously unreported in teleost fish. After auditory stimulation, we found that pS6 phosphorylation peaked between 100 and 130 min and returned to baseline levels after 190 min. This information will be valuable for the design of future pS6 experiments. Our results suggest an anatomical and functional subdivision of AT, where only the dorsal part connects to the auditory network and processes auditory information.

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The Actinopterygian and specifically the Teleostean peroxisome proliferator-activated receptors (PPARs) present an impressive variability and complexity in their structures, both at the gene and protein levels. These structural differences may also reflect functional divergence from their mammalian homologs, or even between fish species. This review, taking advantage of the data generated from the whole-genome sequencing of several fish species, highlights the differences in the primary structure of the receptors, while discussing results from the literature pertaining to the functions of fish PPARs and their activation by natural and synthetic compounds.

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Th17 is a lymphocyte T helper (Th) subpopulation relevant in the control and regulation of the immune response characterized by the production of interleukin (IL)-17. This crucial cytokine family acts through their binding to the IL-17 receptors (IL-17R), having up to six members. Although the biology of fish Th17 is well-recognized, the molecular and functional characterization of IL-17 and IL-17R has been limited. Thus, our aim was to identify and characterize the IL-17R repertory and regulation in the two main Mediterranean cultured fish species, the gilthead seabream (Sparus aurata) and the European sea bass (Dicentrarchus labrax). Our in silico results showed the clear identification of six members in each fish species, from IL-17RA to IL-17RE-like, with well-conserved gene structure and protein domains with their human orthologues. All of them showed wide and constitutive transcription in naïve tissues but with highest levels in mucosal tissues, namely skin, gill or intestine. In leucocytes, T mitogens showed the strongest up-regulation in most of the il17 receptors though il17ra resulted in inhibition by most stimulants. Interestingly, in vivo nodavirus infection resulted in alterations on the transcription of il17 receptors. While nodavirus infection led to some increments in the il17ra, il17rb, il17rc and il17rd transcripts in the susceptible European sea bass, many down-regulations were observed in the resistant gilthead seabream. Our data identify the presence and conservation of six coding IL-17R in gilthead seabream and European sea bass as well as their differential regulation in vitro and upon nodavirus infection. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-024-00225-1.

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Fishes of the genus Carassius are useful experimental vertebrate models for the study of evolutionary biology and cytogenetics. Carassius demonstrates diverse biological characteristics, such as variation in ploidy levels and chromosome numbers, and presence of microchromosomes. Those Carassius polyploids with ≥150 chromosomes have microchromosomes, but the origin of microchromosomes, especially in European populations, is unknown. We used cytogenetics to study evolution of tandem repeats (U1 and U2 small nuclear DNAs and H3 histone) and microchromosomes in Carassius from the Czech Republic. We tested the hypotheses whether the number of tandem repeats was affected by polyploidization or divergence between species and what mechanism drives evolution of microchromosomes. Tandem repeats were found in tetraploid and hexaploid Carassius gibelio, and tetraploid Carassius auratus and Carassius carassius in conserved numbers, with the exception of U1 small nuclear DNA in C. auratus. This conservation indicates reduction and/or loss in the number of copies per locus in hexaploids and may have occurred by divergence rather than polyploidization. To study the evolution of microchromosomes, we used the whole microchromosome painting probe from hexaploid C. gibelio and hybridized it to tetraploid and hexaploid C. gibelio, and tetraploid C. auratus and C. carassius. Our results revealed variation in the number of microchromosomes in hexaploids and indicated that the evolution of the Carassius karyotype is governed by macrochromosome fissions followed by segmental duplication in pericentromeric areas. These are potential mechanisms responsible for the presence of microchromosomes in Carassius hexaploids. Differential efficacy of one or both of these mechanisms in different tetraploids could ensure variability in chromosome number in polyploids in general.

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The harlequin sandsmelt (Parapercis pulchella) is a female-to-male sex change fish in which functional females possess ovotestes that consist of both ovarian and testicular tissues. These features indicate that this species could be an excellent model for studying the flexibility of sex differentiation in vertebrates. However, genetic resources in this species have so far been limited. Therefore, in this study, the reference transcriptome of this fish was constructed through RNA-sequencing, de novo transcriptome assembly, superTranscripts construction, and functional annotations. To obtain as many genes as possible, RNA was extracted from various tissues (brains, gills, hearts, livers, guts, and gonads) and various sexual stages (females, individuals during sex change, and males) and then subjected to sequencing and downstream analyses. As a result, 91,884 representative transcripts with 32,627 protein-coding sequences were generated. 72.2% of protein-coding sequences (23,566 sequences) were functionally annotated. Also, our analysis shows that the superTranscripts method effectively removes redundant sequences from raw-assembled data compared with other strategies. The resultant dataset is a valuable resource for future molecular developmental studies on sex change in P. pulchella.

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Germ cell transplantation in fish is a promising technique for surrogate broodstock parents with broader application in aquaculture and conserving endangered and valuable genetic resources. Herein, we describe the establishment of an intrapapillary xenogeneic transplant of germ cells from sexually mature goldfish (C. auratus) males into common carp (C. carpio) males cytoablated with a thermochemical treatment (two doses of busulfan at 40 mg/kg at 35°C). To analyze the presence and development of donor germ cells in recipient testes, donor germ cells were labeled with PKH26, a fluorescent cell membrane dye, before transplantation. Our results demonstrated that thermochemical treatment caused effective spermatogenesis suppression and pronounced germ cell loss. Moreover, transplanted spermatogonial cells were able to colonize the recipients' testes, resume spermatogenesis, and generate spermatozoa within eight weeks after germ cell transplantation. These findings suggested that recipient testes provided suitable conditions for the survival, colonization, proliferation, and differentiation of donor spermatogonia from a related species. This study indicated that recipients' testes exhibited a high degree of plasticity to accept and support xenogeneic donor germ cells, which were able to form sperm in a short time frame. This approach has significant implications for assisted animal reproduction, biotechnology, conservation, and the production of valuable genetic resources and endangered fish species.

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Across vertebrates, the numerous estrogenic functions are mainly mediated by nuclear and membrane receptors, including the G protein-coupled estrogen receptor (GPER) that has been mostly associated with rapid non-genomic responses. Although Gper-mediated signalling has been characterized in only few fish species, Gpers in fish appear to present more mechanistic functionalities as those of mammals due to additional gene duplicates. In this study, we ran a thorough investigation of the fish Gper evolutionary history in light of available genomes, we carried out the functional characterization of the two gper gene duplicates of European sea bass (Dicentrarchus labrax) using luciferase reporter gene transactivation assays, validated it with natural and synthetic estrogen agonists/antagonists and applied it to other chemicals of aquaculture and ecotoxicological interest. Phylogenetic and synteny analyses of fish gper1 and gper1-like genes suggest their duplication may have not resulted from the teleost-specific whole genome duplication. We confirmed that both sbsGper isoforms activate the cAMP signalling pathway and respond differentially to distinct estrogenic compounds. Therefore, as observed for nuclear estrogen receptors, both sbsGpers duplicates retain estrogenic activity although they differ in their specificity and potency (Gper1 being more potent and more specific than Gper1-like), suggesting a more conserved role for Gper1 than for Gper1-like. In addition, Gpers were able to respond to estrogenic environmental pollutants known to interfere with estrogen signalling, such as the phytoestrogen genistein and the anti-depressant fluoxetine, a point that can be taken into account in aquatic environment pollution screenings and chemical risk assessment, complementing previous assays for sea bass nuclear estrogen receptors.

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The hypoxia inducible factor 1 (HIF1) is a central regulator of the molecular responses of animals to low oxygen. While the hypoxia-responsiveness of HIF1 is generally attributed to the stabilization of the alpha protein subunit (HIF1α) at low oxygen, several studies on fish report increased tissue levels of HIF1A mRNA during hypoxia, suggesting transcriptional regulation. In the current study, HIF1α protein and HIF1A mRNA were determined in parallel in tissues of Gulf killifish, Fundulus grandis, exposed to short-term hypoxia (24 h at 1 mg O2 l-1). HIF1α protein was higher in brain, ovary, and skeletal muscle from fish exposed to hypoxia compared with normoxic controls by 6 h, and it remained elevated in brain and ovary at 24 h. In contrast, HIF1A mRNA levels were unaffected by hypoxia in any tissue. Moreover, HIF1α protein and HIF1A mRNA levels in the same tissues were not correlated with one another during either normoxia or hypoxia. Hence, an increase in HIF1α protein does not depend upon an increase in HIF1A mRNA during acute exposure to low oxygen in this species. The results support the widely accepted mechanism of post-translational protein stabilization, rather than new transcription, during the initial response of fish to hypoxia.

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BACKGROUND: The microbiota in fish external mucus is mainly known for having a role in homeostasis and protection against pathogens, but recent evidence suggests it is also involved in the host-specificity of some ectoparasites. In this study, we investigated the influence of seasonality and environmental factors on both fish external microbiota and monogenean gill ectoparasites abundance and diversity and assessed the level of covariations between monogenean and bacterial communities across seasons. To do so, we assessed skin and gill microbiota of two sparid species, Oblada melanura and Diplodus annularis, over a year and collected their specific monogenean ectoparasites belonging to the Lamellodiscus genus. RESULTS: Our results revealed that diversity and structure of skin and gill mucus microbiota were strongly affected by seasonality, mainly by the variations of temperature, with specific fish-associated bacterial taxa for each season. The diversity and abundance of parasites were also influenced by seasonality, with the abundance of some Lamellodiscus species significantly correlated to temperature. Numerous positive and negative correlations between the abundance of given bacterial genera and Lamellodiscus species were observed throughout the year, suggesting their differential interaction across seasons. CONCLUSIONS: The present study is one of the first to demonstrate the influence of seasonality and related abiotic factors on fish external microbiota over a year. We further identified potential interactions between gill microbiota and parasite occurrence in wild fish populations, improving current knowledge and understanding of the establishment of host-specificity.

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Fish are ectothermic and small changes in water temperature could greatly affect reproduction. The two-spotted goby is a small semi-pelagic species that uses visual and acoustic displays to mate. Here, we studied the effect of temperature (16 and 20 °C) on acoustic and visual courtship and associated reproductive success in 39 males. Temperature influenced male visual courtship performed outside the nest, but it did not influence calling rate and the number of laid eggs. Interestingly, the number of sounds (drums) was the sole predictor of spawning success. These findings suggest that exposure to different temperatures within the species' natural range affect courtship behaviour but not its reproductive success. We propose that finding the link between acoustic behaviour and reproduction in fishes offers the opportunity to monitor fish sounds both in the lab and in nature to learn how they respond to environmental changes and human impacts, namely global warming.

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Increasing evidence suggests that long non-coding RNAs (lncRNAs) play pivotal roles in various biological processes. However, current studies on lncRNAs mostly focus on mammalian species, with little research on the functional roles of lncRNAs in teleost fish. Here, we identified a novel intergenic lncRNA (linc-93.2) in the head kidney primary macrophages of common carp (Cyprinus carpio) after exposure to a typical environmental endocrine disrupting chemical, bisphenol A (BPA). As a result, linc-93.2 was more than 3,619 bp in length and predominantly localized to the nucleus of primary macrophages other than cytoplasm, with the highest expression level in spleen followed by head kidney among different organs. Bioinformatic analysis predicted a cis-target gene, dennd1b, and 20 trans-target genes including hsp70, gna13 and rasgap, were potentially regulated by linc-93.2; NFκB and estrogen receptor (ERα) binding sites were located in the promoter region upstream of its transcription start site, which together suggested the involvement of linc-93.2 in immune and neurological functions in fish. Based on that, the expression level of linc-93.2 was determined in macrophages following acute lipopolysaccharide (LPS) and BPA treatments, both of which significantly induced linc-93.2 and IL-1ß expression in cells. Moreover, a NF-κB inhibitor PDTC significantly reduced linc-93.2 expression in macrophages, but co-exposure of macrophages to PDTC with BPA or LPS could significantly rescue linc-93.2 expression, consistent with the observation on that LPS or BPA alone significantly induced both linc-93.2 and its target gene expression. Interestingly, linc-93.2 and its target gene expression was significantly suppressed by an ER antagonist ICI 182,780, however, the co-exposure of macrophages to ICI 182,780 with BPA failed to attenuate their declined expression. Overall, the current study demonstrated that linc-93.2, a novel immune-related lncRNA, may participate in the immune processes of common carp macrophages via the NF-κB and ER pathway. The results presented in this study enhance our understanding of the immunotoxin mechanisms of BPA in teleost fish.

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To identify the key factors influencing the trimethylamine N-oxide (TMAO) content of teleost fishes living in shallow seas and the epipelagic zone of the deep ocean, the muscle TMAO content was measured in 152 teleost fishes (21 species) collected from the marginal seas of China and the epipelagic zone of the northwest Pacific Ocean (NWPO) during May-July 2021. The results showed that the TMAO content in all fishes varied from 4.99 to 82.97 mmol kg-1, and it varied notably among different species, e.g., the highest average content (72.71 ± 8.22 mmol kg-1 in Argyrosomus argentatus) was 1 order of magnitude higher than the lowest one (Scomber japonicus), but the ratios of the highest content to the lowest content in each species varied from 1.29 to 3.28, suggesting that the interspecific variations in TMAO content were obviously greater than the intraspecific variations. Moreover, no correlation was observed between the TMAO content of the 152 fishes and the corresponding environmental factors of seawater depth, salinity and temperature, indicating that species played a more important role than environmental factors in driving TMAO accumulation. To exclude the influence of species, intraspecies correlations between TMAO content and environmental factors were analyzed. In the marginal seas of China, only ∼8 % of the TMAO content of teleost fishes (1 species) showed a positive correlation with salinity and depth, but ∼50 % of the TMAO content (5 species) was negatively correlated with temperature. Moreover, the TMAO content of the fish increased by 4.66 ± 1.38 % compared with their corresponding intraspecific average values for every 1 °C of temperature decrease. A similar phenomenon was also found in the TMAO content of pelagic teleost fishes in the NWPO, suggesting that temperature was a key environmental factor affecting the TMAO content of teleost fishes in shallow seas and the epipelagic zone of the deep ocean.

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There is accumulating evidence demonstrated that long noncoding RNAs (lncRNA) act as gene regulators in various biological processes, including innate immunity, in which lncRNAs could play their regulatory roles by interacting with miRNAs. Compared with mammals, there is little attention paid to the mechanism of the lncRNA-miRNA regulatory network in teleost fish. Herein, we found a long noncoding RNAs LTCONS4500 that could function as a positive regulator of the immune response in miiuy croaker (Miichthys miiuy). Specifically, we found that the expression of LTCONS4500 could be upregulated by gram-negative bacteria, such as Vibrio anguillarum and Vibrio harveyi. Upregulated LTCONS4500 could promote the expression of inflammatory cytokines. Further study showed that LTCONS4500 could act as a competing endogenous RNA (ceRNA) to interact with miR-3570-5p to facilitate MyD88 expression and thus enhance antibacterial immune responses. Our data suggests the function and mechanism of lncRNAs in antibacterial immune responses of teleost fish, which will enrich the gene regulatory network of vertebrates.

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Acute inflammation is crucial to the immune responses of fish. The process protects the host from infection and is central to induction of subsequent tissue repair programs. Activation of proinflammatory signals reshapes the microenvironment within an injury/infection site, initiates leukocyte recruitment, promotes antimicrobial mechanisms and contributes to the resolution of inflammation. Inflammatory cytokines and lipid mediators are primary contributors to these processes. Uncontrolled or persistent induction results in delayed tissue healing. The kinetics by which inducers and regulators of acute inflammation exert their actions is essential for understanding the pathogenesis of fish diseases and identifying potential treatments. Although, a number of these are well-conserved across, others are not, reflecting the unique physiologies and life histories of members of this unique animal group.

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