Long-read de novo genome assembly of Gulf toadfish (Opsanus beta).

BACKGROUND: The family Batrachoididae are a group of ecologically important teleost fishes with unique life histories, behavior, and physiology that has made them popular model organisms. Batrachoididae remain understudied in the realm of genomics, with only four reference genome assemblies available for the family, with three being highly fragmented and not up to current assembly standards. Among these is the Gulf toadfish, Opsanus beta, a model organism for serotonin physiology which has recently been bred in captivity. RESULTS: Here we present a new, de novo genome and transcriptome assemblies for the Gulf toadfish using PacBio long read technology. The genome size of the final assembly is 2.1 gigabases, which is among the largest teleost genomes. This new assembly improves significantly upon the currently available reference for Opsanus beta with a final scaffold count of 62, of which 23 are chromosome scale, an N50 of 98,402,768, and a BUSCO completeness score of 97.3%. Annotation with ab initio and transcriptome-based methods generated 41,076 gene models. The genome is highly repetitive, with ~ 70% of the genome composed of simple repeats and transposable elements. Satellite DNA analysis identified potential telomeric and centromeric regions. CONCLUSIONS: This improved assembly represents a valuable resource for future research using this important model organism and to teleost genomics more broadly.

Expression and regulation of the CXCL9-11 chemokines and CXCR3 receptor in Atlantic salmon (Salmo salar).

Chemokines are cytokines that mediate leukocyte traffic between the lymphoid organs, the bloodstream, and the site of tissue damage, which is essential for an efficient immune response. In particular, the gamma interferon (IFN- γ) inducible chemokines CXCL9, CXCL10, and CXCL11, and their receptor CXCR3, are involved in T cell and macrophage recruitment to the site of infection. The nature and function of these chemokines and their receptor are well-known in mammals, but further research is needed to achieve a similar level of understanding in fish immunity. Thus, in this study, we seek to identify the genes encoding the components of the Atlantic salmon (Salmo salar) CXCL9, CXCL10, CXCL11/CXCR3 axis (CXCL9-11/CXCR3), predict the protein structure from the amino acid sequence, and explore the regulation of gene expression as well as the response of these chemokines and their receptor to viral infections. The cxcl9, cxcl10, cxcl11, and cxcr3 gene sequences were retrieved from the databases, and the phylogenetic analysis was conducted to determine the evolutionary relationships. The study revealed an interesting pattern of clustering and conservation among fish and mammalian species. The salmon chemokine sequences clustered with orthologs from other fish species, while the mammalian sequences formed separate clades. This indicates a divergent evolution of chemokines between mammals and fish, possibly due to different evolutionary pressures. While the structural analysis of the chemokines and the CXCR3 receptor showed the conservation of critical motifs and domains, suggesting preserved functions and stability throughout evolution. Regarding the regulation of gene expression, some components of the CXCL9-11/CXCR3 axis are induced by recombinant gamma interferon (rIFN-γ) and by Infectious pancreatic necrosis virus (IPNV) infection in Atlantic salmon cells. Further studies are needed to explore the role of Atlantic salmon CXCL9-11 chemokines in regulating immune cell migration and endothelial activation, as seen in mammals. To the best of our knowledge, there have been no functional studies of chemokines to understand these effects in Atlantic salmon.

Turning up the heat: Effects of temperature on agonistic acoustic communication in the two-spotted goby (Pomatoschistus flavescens).

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.

ScRNA-seq reveals trained immunity-engaged Th17 cell activation against Edwardsiella piscicida-induced intestinal inflammation in teleost.

Mucosal immunity typically involves innate and adaptive immune cells, while the cellular mechanism of teleost's intestinal immune cells that engages gut homeostasis against bacterial infection remains largely unknown. Taking advantage of the enteric fish pathogen (Edwardsiella piscicida) infection-induced intestinal inflammation in turbot (Scophthalmus maximus), we find that ß-glucan training could mitigate the bacterial infection-induced intestinal inflammation. Through single-cell transcriptome profiling and cellular function analysis, we identify that E. piscicida infection could tune down the activation of intestinal Th17 cells, while ß-glucan-training could preserve the potential to amplify and restore the function of intestinal Th17 cells. Moreover, through pharmacological inhibitor treatment, we identify that Th17 cells are essential for ameliorating bacterial infection-induced intestinal inflammation in teleost. Taken together, these results suggest a new concept of trained immunity activation to regulate the intestinal Th17 cells' function, which might contribute to better developing strategies for maintaining gut homeostasis against bacterial infection.

Ecological filtering drives rapid spatiotemporal dynamics in fish skin microbiomes.

Skin microbiomes provide vital functions, yet knowledge about the drivers and processes structuring their species assemblages is limited-especially for non-model organisms. In this study, fish skin microbiome was assessed by high throughput sequencing of amplicon sequence variants from metabarcoding of V3-V4 regions in the 16S rRNA gene on fish hosts subjected to the following experimental manipulations: (i) translocation between fresh and brackish water habitats to investigate the role of environment; (ii) treatment with an antibacterial disinfectant to reboot the microbiome and investigate community assembly and priority effects; and (iii) maintained alone or in pairs to study the role of social environment and inter-host dispersal of microbes. The results revealed that fish skin microbiomes harbour a highly dynamic microbial composition that was distinct from bacterioplankton communities in the ambient water. Microbiome composition first diverged as an effect of translocation to either the brackish or freshwater habitat. When the freshwater individuals were translocated back to brackish water, their microbiome composition converged towards the fish microbiomes in the brackish habitat. In summary, external environmental conditions and individual-specific factors jointly determined the community composition dynamics, whereas inter-host dispersal had negligible effects. The dynamics of the microbiome composition was seemingly non-affected by reboot treatment, pointing towards high resilience to disturbance. The results emphasised the role of inter-individual variability for the unexplained variation found in many host-microbiome systems, although the mechanistic underpinnings remain to be identified.

MIPs: multi-locus intron polymorphisms in species identification and population genomics.

The study of species groups in which the presence of interspecific hybridization or introgression phenomena is known or suspected involves analysing shared bi-parentally inherited molecular markers. Current methods are based on different categories of markers among which the classical microsatellites or the more recent genome wide approaches for the analyses of thousands of SNPs or hundreds of microhaplotypes through high throughput sequencing. Our approach utilizes intron-targeted amplicon sequencing to characterise multi-locus intron polymorphisms (MIPs) and assess genetic diversity. These highly variable intron regions, combined with inter-specific transferable loci, serve as powerful multiple-SNP markers potentially suitable for various applications, from species and hybrid identification to population comparisons, without prior species knowledge. We developed the first panel of MIPs highly transferable across fish genomes, effectively distinguishing between species, even those closely related, and populations with different structures. MIPs offer versatile, hypervariable nuclear markers and promise to be especially useful when multiple nuclear loci must be genotyped across different species, such as for the monitoring of interspecific hybridization. Moreover, the relatively long sequences obtained ease the development of single-locus PCR-based diagnostic markers. This method, here demonstrated in teleost fishes, can be readily applied to other taxa, unlocking a new source of genetic variation.

Involvement of vimentin- and BLBP-positive glial cells and their MMP expression in axonal regeneration after spinal cord transection in goldfish.

In goldfish, spinal cord injury triggers the formation of a fibrous scar at the injury site. Regenerating axons are able to penetrate the scar tissue, resulting in the recovery of motor function. Previous findings suggested that regenerating axons enter the scar through tubular structures surrounded by glial elements with laminin-positive basement membranes and that glial processes expressing glial fibrillary acidic protein (GFAP) are associated with axonal regeneration. How glia contribute to promoting axonal regeneration, however, is unknown. Here, we revealed that glial processes expressing vimentin or brain lipid-binding protein (BLBP) also enter the fibrous scar after spinal cord injury in goldfish. Vimentin-positive glial processes were more numerous than GFAP- or BLBP-positive glial processes in the scar tissue. Regenerating axons in the scar tissue were more closely associated with vimentin-positive glial processes than GFAP-positive glial processes. Vimentin-positive glial processes co-expressed matrix metalloproteinase (MMP)-14. Our findings suggest that vimentin-positive glial processes closely associate with regenerating axons through tubular structures entering the scar after spinal cord injury in goldfish. In intact spinal cord, ependymo-radial glial cell bodies express BLBP and their radial processes express vimentin, suggesting that vimentin-positive glial processes derive from migrating ependymo-radial glial cells. MMP-14 expressed in vimentin-positive glial cells and their processes might provide a beneficial environment for axonal regeneration.

IL-18 signaling is regulated by caspase 6/8 and IL-18BP in turbot (Scophthalmus maximus).

Interleukin (IL)-18 is synthesized as a precursor that requires intracellular processing to become functionally active. In human, IL-18 is processed by caspase 1 (CASP1). In teleost, the maturation and signal transduction mechanisms of IL-18 are unknown. We identified two IL-18 variants, IL-18a and IL-18b, in turbot. IL-18a, but not IL-18b, was processed by CASP6/8 cleavage. Mature IL-18a bound specifically to IL-18 receptor (IL-18R) α-expressing cells and induced IL-18Rα-IL-18Rß association. Bacterial infection promoted IL-18a maturation in a manner that required CASP6 activation and correlated with gasdermin E activation. The mature IL-18a induced proinflammatory cytokine expression and enhanced bacterial clearance. IL-18a-mediated immune response was suppressed by IL-18 binding protein (IL-18BP), which functioned as a decoy receptor for IL-18a. IL-18BP also functioned as a pathogen pattern recognition receptor and directly inhibited pathogen infection. Our findings revealed unique mechanism of IL-18 maturation and conserved mechanism of IL-18 signaling and regulation in turbot, and provided new insights into the regulation and function of IL-18 related immune signaling.

Investigating Development and Defense Systems in Early Reproductive Stages of Male and Female Gonads in Black Scorpionfish Scorpaena porcus (Linnaeus, 1758).

One of the most crucial biological indicators in tracking long-term variations in the reproductive cycle is sexual development. Scorpaena porcus (Linnaeus, 1758), commonly known as the black scorpionfish, is a small teleost from the family Scorpaenidae. Much is known about its ecology, but data on its reproductive and defense systems are still lacking. Antimicrobial peptides (AMPs), such as piscidins, are integral components of the innate immune system in fish. These peptides exhibit a wide range of activity against bacteria, fungi, viruses, and protozoa and act as the first line of host defense. This study aims to investigate the primary sexual development stages in male and female gonads of black scorpionfish, providing additional knowledge on the reproductive biology of this teleost while evaluating concomitant changes in the expression of a Piscidin-1 antimicrobial peptide. The results show a histological, morpho-structural change from the immature stage to the developing virgin stage. Immunohistochemical analyses show that germinal and somatic cells are strongly reactive to Piscidin-1 in both gonads at an early ontogeny stage. These data suggest that Piscidin-1 may play a key role in the local defense system of scorpionfish gonads at this delicate stage, which is critical for the continuation and maintenance of the species. The present findings are potentially useful for a better understanding of the reproductive cycle of this fish, improving our knowledge of the interaction between the immune system and reproduction.

A structural peculiarity of Antarctic fish IgM drives the generation of an engineered mAb by CRISPR/Cas9.

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.

MicroRNAs are involved in ovarian physiology of greater amberjack (Seriola dumerili) under captivity.

Gonad maturation is critical for the reproductive success of any organism, and in fish, captivity can significantly affect their reproductive performance, leading to maturation incompetence and spawning failure. The greater amberjack (Seriola dumerili), a fish species recently introduced to aquaculture fails to undergo oocyte maturation, ovulation, and spawning when reared in aquaculture facilities. Since confinement has been shown to influence gonad maturation and completion of the reproductive cycle, investigations into epigenetic mechanisms may shed light on the reasoning behind the reproductive dysfunctions of fish under captivity. Among the known important epigenetic regulators are small non-coding RNAs (sncRNAs), and in particular microRNAs (miRNAs). In this study, immature, maturing (late vitellogenesis), and spent ovaries of captive greater amberjack were collected, and the differential expression of miRNAs in the three different ovarian development stages was examined. Expression patterns of conserved and novel miRNAs were identified, and potential targets of highly differentially expressed miRNAs were detected. Additionally, read length distribution showed two prominent peaks in the three different ovarian maturation stages, corresponding to miRNAs and putative piwi-interacting RNAs (piRNAs), another type of ncRNAs with a germ-cell specific role. Furthermore, miRNA expression patterns and their putative target mRNAs are discussed, in relevance with the different ovarian maturation stages of captive greater amberjack. Overall, this study provides insights into the role of miRNAs in the reproductive dysfunctions observed in fish under captivity and highlights the importance of epigenetic mechanisms in understanding and managing the reproductive performance of economically important fish species.

Control of fluid intake in dehydrated rats and evolution of sodium appetite.

The objective of the present work is to examine from a new perspective the existence of causal factors not predicted by the classical theory that thirst and sodium appetite are two distinct motivations. For example, we ask why water deprivation induces sodium appetite, thirst is not "water appetite", and intracellular dehydration potentially causes sodium appetite. Contrary to the classical theory, we suggest that thirst first, and sodium appetite second, designate a temporal sequence underlying the same motivation. The single motivation becomes an "intervenient variable" a concept borrowed from the literature, fully explained in the text, between causes of dehydration (extracellular, intracellular, or both together), and respective behavioral responses subserved by hindbrain-dependent inhibition (e.g., lateral parabrachial nucleus) and forebrain facilitation (e.g., angiotensin II). A corollary is homology between rat sodium appetite and marine teleost thirst-like motivation that we name "protodipsia". The homology argument rests on similarities between behavior (salty water intake) and respective neuroanatomical as well as functional mechanisms. Tetrapod origin in a marine environment provides additional support for the homology. The single motivation hypothesis is also consistent with ingestive behaviors in nature given similarities (e.g., thirst producing brackish water intake) between the behavior of the laboratory rat and wild animals, rodents included. The hypotheses of single motivation and homology might explain why hyperosmotic rats, or eventually any other hyperosmotic tetrapod, shows paradoxical signs of sodium appetite. They might also explain how ingestive behaviors determined by dehydration and subserved by hindbrain inhibitory mechanisms contributed to tetrapod transition from sea to land.

Loss of Function of Vasoactive-intestinal Peptide Alters Sex Ratio and Reduces Male Reproductive Fitness in Zebrafish.

Vasoactive-intestinal peptide (Vip) is a pleiotropic peptide with a wide range of distribution and functions. Zebrafish possess 2 isoforms of Vip (a and b), in which Vipa is most homologous to the mammalian form. In female zebrafish, Vipa can stimulate LH secretion from the pituitary but is not essential for female reproduction, as vipa-/- females display normal reproduction. In contrast, we have found that vipa-/- males are severely subfertile and sex ratio of offspring is female-biased. By analyzing all aspects of male reproduction with wild-type (WT) males, we show that the testes of vipa-/- are underdeveloped and contain ∼70% less spermatids compared to WT counterparts. The sperm of vipa-/- males displayed reduced potency in terms of fertilization (by ∼80%) and motility span and duration (by ∼50%). In addition, vipa-/- male attraction to WT females was largely nonexistent, indicating decreased sexual motivation. We show that vipa mRNA and protein is present in Leydig cells and in developing germ cells in the testis of WT, raising the possibility that endogenous Vipa contributes to testicular function. Absence of Vipa in vipa-/- males resulted in downregulation of 3 key genes in the androgen synthesis chain in the testis, 3ß-hsd, 17ß-hsd1, and cyp11c1 (11ß-hydrogenase), associated with a pronounced decrease in 11-ketotestosterone production and, in turn, compromised reproductive fitness. Altogether, this study establishes a crucial role for Vipa in the regulation of male reproduction in zebrafish, like in mammals, with the exception that Vipa is also expressed in zebrafish testis.

Soluble adenylyl cyclase is an acid-base sensor in rainbow trout red blood cells that regulates intracellular pH and haemoglobin-oxygen binding.

AIM: To identify the physiological role of the acid-base sensing enzyme, soluble adenylyl cyclase (sAC), in red blood cells (RBC) of the model teleost fish, rainbow trout. METHODS: We used: (i) super-resolution microscopy to determine the subcellular location of sAC protein; (ii) live-cell imaging of RBC intracellular pH (pHi) with specific sAC inhibition (KH7 or LRE1) to determine its role in cellular acid-base regulation; (iii) spectrophotometric measurements of haemoglobin-oxygen (Hb-O2) binding in steady-state conditions; and (iv) during simulated arterial-venous transit, to determine the role of sAC in systemic O2 transport. RESULTS: Distinct pools of sAC protein were detected in the RBC cytoplasm, at the plasma membrane and within the nucleus. Inhibition of sAC decreased the setpoint for RBC pHi regulation by ~0.25 pH units compared to controls, and slowed the rates of RBC pHi recovery after an acid-base disturbance. RBC pHi recovery was entirely through the anion exchanger (AE) that was in part regulated by HCO3 --dependent sAC signaling. Inhibition of sAC decreased Hb-O2 affinity during a respiratory acidosis compared to controls and reduced the cooperativity of O2 binding. During in vitro simulations of arterial-venous transit, sAC inhibition decreased the amount of O2 that is unloaded by ~11%. CONCLUSION: sAC represents a novel acid-base sensor in the RBCs of rainbow trout, where it participates in the modulation of RBC pHi and blood O2 transport though the regulation of AE activity. If substantiated in other species, these findings may have broad implications for our understanding of cardiovascular physiology in vertebrates.

Erythrocytes of the common carp are immune sentinels that sense pathogen molecular patterns, engulf particles and secrete pro-inflammatory cytokines against bacterial infection.

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.

The Chromosome-Scale Genome of Chitala ornata Illuminates the Evolution of Early Teleosts.

Teleosts are the most prolific vertebrates, occupying the vast majority of aquatic environments, and their pectoral fins have undergone remarkable physiological transformations throughout their evolution. Studying early teleost fishes, such as those belonging to the Osteoglossiformes order, could offer crucial insights into the adaptive evolution of pectoral fins within this group. In this study, we have assembled a chromosomal-level genome for the Clown featherback (Chitala ornata), achieving the highest quality genome assembly for Osteoglossiformes to date, with a contig N50 of 32.78 Mb and a scaffold N50 of 40.73 Mb. By combining phylogenetic analysis, we determined that the Clown featherback diverged approximately 202 to 203 million years ago (Ma), aligning with continental separation events. Our analysis revealed the intriguing discovery that a unique deletion of regulatory elements is adjacent to the Gli3 gene, specifically in teleosts. This deletion might be tied to the specialized adaptation of their pectoral fins. Furthermore, our findings indicate that specific contractions and expansions of transposable elements (TEs) in teleosts, including the Clown featherback, could be connected to their adaptive evolution. In essence, this study not only provides a high-quality genomic resource for Osteoglossiformes but also sheds light on the evolutionary trajectory of early teleosts.

Elemental Composition and Health Risk Assessment of Deep-Sea Teleost's of the Levantine Basin.

The determination of metal(loid) (As, Fe, Al, Sr, Zn, Pb, Mn, Cu, Cr, and Cd) levels in the muscle tissue of 23 different deep-sea bony fish sampled off Mersin Bay (NE Levantine Basin) and the assessment of health risks for human consumption were aimed. Tissue metal(loid) concentrations were determined as dry weight and analyzed by inductively coupled plasma mass spectrometry (ICP-MS). The tissue metal(loid) concentrations (µg g dw) were converted to wet weight prior to health risk assessment calculations. Standard mathematical formulas were used to determine the health risk assessment. There was a statistically significant difference between the fish species in terms of tissue metal(loid) levels (p < 0.05). The highest metal(loid) level was found in C. sloani among other species. As and Fe had the highest and Cd the lowest tissue concentrations in the examined species (p < 0.05). The relationships between the metal(loid)s analyzed in the tissue were significant (p < 0.01;0.05). Fe had an antagonistic effect with Cd, while other metal(loid)s had a synergetic effect with each other. Risk assessment analyses were performed for the consumable species, and it was found that the estimated daily and weekly intakes were below the tolerable limits established by the Food and Agriculture Organization (FAO) and the World Health Organization (WHO). The target hazard quotient (THQ) values exceeded the threshold of 1 (THQ > 1) only for As. The target cancer risk (TCR) was below the tolerable limits (> 10-5) except for As, Cd, and Al.

Androgen receptor alpha deficiency impacts aromatase expression in the female cichlid brain.

Steroid hormones bind to specific receptors that act as transcription factors to modify gene expression in the brain to regulate physiological and behavioural processes. The specific genes controlled by steroid hormones in the brain are not fully known. Identifying these genes is integral to establishing a comprehensive understanding of how hormones impact physiology and behaviour. A popular organism for answering this question is the cichlid fish Astatotilapia burtoni. Recently, CRISPR/Cas9 was used to engineer A. burtoni that lack functional androgen receptor (AR) genes encoding ARα. ARα mutant male A. burtoni produced fewer aggressive displays and possessed reduced expression of the gene encoding brain-specific aromatase, cyp19a1, in the ventromedial hypothalamus (VMH), an aggression locus. As a follow-up, we investigated whether ARα deficiency affected cyp19a1 expression in female A. burtoni using the same genetic line. We find that female A. burtoni possessing one or two non-functional ARα alleles had much higher expression of cyp19a1 in the preoptic area (POA), while females with one non-functional ARα allele possessed lower expression of cyp19a1 in the putative fish homologue of the bed nucleus of the stria terminalis (BNST). Thus, ARα may have a sex-specific role in modifying cyp19a1 expression in the teleost POA and BNST, regions that underlie sex differences across vertebrates.

A leukocyte immune-type receptor specific polyclonal antibody recognizes goldfish kidney leukocytes and activates the MAPK pathway in isolated goldfish kidney neutrophil-like cells.

Leukocyte immune-type receptors (LITRs) belong to a large family of teleost immunoregulatory receptors that share phylogenetic and syntenic relationships with mammalian Fc receptor-like molecules (FCRLs). Recently, several putative stimulatory Carassius auratus (Ca)-LITR transcripts, including CaLITR3, have been identified in goldfish. CaLITR3 has four extracellular immunoglobulin-like (Ig-like) domains, a transmembrane domain containing a positively charged histidine residue, and a short cytoplasmic tail region. Additionally, the calitr3 transcript is highly expressed by goldfish primary kidney neutrophils (PKNs) and macrophages (PKMs). To further investigate the immunoregulatory potential of CaLITR3 in goldfish myeloid cells, we developed and characterized a CaLITR3-epitope-specific polyclonal antibody (anti-CaL3.D1 pAb). We show that the anti-CaL3.D1 pAb stains various hematopoietic cell types within the goldfish kidney, as well as in PKNs and PKMs. Moreover, cross-linking of the anti-CaL3.D1-pAb on PKN membranes induces phosphorylation of p38 and ERK1/2, critical components of the MAPK pathway involved in controlling a wide variety of innate immune effector responses such as NETosis, respiratory burst, and cytokine release. These findings support the stimulatory potential of CaLITR3 proteins as activators of fish granulocytes and pave the way for a more in-depth examination of the immunoregulatory functions of CaLITRs in goldfish myeloid cells.

Single-cell atlas of rainbow trout peripheral blood leukocytes and profiling of their early response to infectious pancreatic necrosis virus.

The recent development of single cell sequencing technologies has revolutionized the state-of-art of cell biology, allowing the simultaneous measurement of thousands of genes in single cells. This technology has been applied to study the transcriptome of single cells in homeostasis and also in response to pathogenic exposure, greatly increasing our knowledge of the immune response to infectious agents. Yet the number of these studies performed in aquacultured fish species is still very limited. Thus, in the current study, we have used the 10x Genomics single cell RNA sequencing technology to study the response of rainbow trout (Oncorhynchus mykiss) peripheral blood leukocytes (PBLs) to infectious pancreatic necrosis virus (IPNV), an important trout pathogen. The study allowed us to obtain a transcriptomic profile of 12 transcriptionally distinct leukocyte cell subpopulations that included four different subsets of B cells, T cells, monocytes, two populations of dendritic-like cells (DCs), hematopoietic progenitor cells, non-specific cytotoxic cells (NCC), neutrophils and thrombocytes. The transcriptional pattern of these leukocyte subpopulations was compared in PBL cultures that had been exposed in vitro to IPNV for 24 h and mock-infected cultures. Our results revealed that monocytes and neutrophils showed the highest number of upregulated protein-coding genes in response to IPNV. Interestingly, IgM+IgD+ and IgT+ B cells also upregulated an important number of genes to the virus, but a much fainter response was observed in ccl4 + or plasma-like cells (irf4 + cells). A substantial number of protein-coding genes and genes coding for ribosomal proteins were also transcriptionally upregulated in response to IPNV in T cells and thrombocytes. Interestingly, although genes coding for ribosomal proteins were regulated in all affected PBL subpopulations, the number of such genes transcriptionally regulated was higher in IgM+IgD+ and IgT+ B cells. A further analysis dissected which of the regulated genes were common and which were specific to the different cell clusters, identifying eight genes that were transcriptionally upregulated in all the affected groups. The data provided constitutes a comprehensive transcriptional perspective of how the different leukocyte populations present in blood respond to an early viral encounter in fish.