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.

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.

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.

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 two 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 sub-fertile and sex ratio of offspring is female-biased. By analyzing all aspects of male reproduction with 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 non-existent, 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 three 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.

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.

The physiological significance of plasma-accessible carbonic anhydrase in the respiratory systems of fishes.

Carbonic anhydrase (CA) activity is ubiquitously found in all vertebrate species, tissues and cellular compartments. Most species have plasma-accessible CA (paCA) isoforms at the respiratory surfaces, where the enzyme catalyzes the conversion of plasma bicarbonate to carbon dioxide (CO2) that can be excreted by diffusion. A notable exception are the teleost fishes that appear to lack paCA at their gills. The present review: (i) recapitulates the significance of CA activity and distribution in vertebrates; (ii) summarizes the current evidence for the presence or absence of paCA at the gills of fishes, from the basal cyclostomes to the derived teleosts and extremophiles such as the Antarctic icefishes; (iii) explores the contribution of paCA to organismal CO2 excretion in fishes; and (iv) the functional significance of its absence at the gills, for the specialized system of O2 transport in most teleosts; (v) outlines the multiplicity and isoform distribution of membrane-associated CAs in fishes and methodologies to determine their plasma-accessible orientation; and (vi) sketches a tentative time line for the evolutionary dynamics of branchial paCA distribution in the major groups of fishes. Finally, this review highlights current gaps in the knowledge on branchial paCA function and provides recommendations for future work.

The evolution of olfactory sensitivity, preferences, and behavioral responses in Mexican cavefish is influenced by fish personality.

Animals are adapted to their natural habitats and lifestyles. Their brains perceive the external world via their sensory systems, compute information together with that of internal states and autonomous activity, and generate appropriate behavioral outputs. However, how do these processes evolve across evolution? Here, focusing on the sense of olfaction, we have studied the evolution in olfactory sensitivity, preferences, and behavioral responses to six different food-related amino acid odors in the two eco-morphs of the fish Astyanax mexicanus. To this end, we have developed a high-throughput behavioral setup and pipeline of quantitative and qualitative behavior analysis, and we have tested 489 six-week-old Astyanax larvae. The blind, dark-adapted morphs of the species showed markedly distinct basal swimming patterns and behavioral responses to odors, higher olfactory sensitivity, and a strong preference for alanine, as compared to their river-dwelling eyed conspecifics. In addition, we discovered that fish have an individual 'swimming personality', and that this personality influences their capability to respond efficiently to odors and find the source. Importantly, the personality traits that favored significant responses to odors were different in surface fish and cavefish. Moreover, the responses displayed by second-generation cave × surface F2 hybrids suggested that olfactory-driven behavior and olfactory sensitivity is a quantitative genetic trait. Our findings show that olfactory processing has rapidly evolved in cavefish at several levels: detection threshold, odor preference, and foraging behavior strategy. Cavefish is therefore an outstanding model to understand the genetic, molecular, and neurophysiological basis of sensory specialization in response to environmental change.

Fish antifreeze protein origin in sculpins by frameshifting within a duplicated housekeeping gene.

Antifreeze proteins (AFPs) are found in a variety of marine cold-water fishes where they prevent freezing by binding to nascent ice crystals. Their diversity (types I, II, III and antifreeze glycoproteins), as well as their scattered taxonomic distribution hint at their complex evolutionary history. In particular, type I AFPs appear to have arisen in response to the Late Cenozoic Ice Age that began ~ 34 million years ago via convergence in four different groups of fish that diverged from lineages lacking this AFP. The progenitor of the alanine-rich α-helical type I AFPs of sculpins has now been identified as lunapark, an integral membrane protein of the endoplasmic reticulum. Following gene duplication and loss of all but three of the 15 exons, the final exon, which encoded a glutamate- and glutamine-rich segment, was converted to an alanine-rich sequence by a combination of frameshifting and mutation. Subsequent gene duplications produced numerous isoforms falling into four distinct groups. The origin of the flounder type I AFP is quite different. Here, a small segment from the original antiviral protein gene was amplified and the rest of the coding sequence was lost, while the gene structure was largely retained. The independent origins of type I AFPs with up to 83% sequence identity in flounder and sculpin demonstrate strong convergent selection at the level of protein sequence for alanine-rich single alpha helices that bind to ice. Recent acquisition of these AFPs has allowed sculpins to occupy icy seawater niches with reduced competition and predation from other teleost species.

The evolution of exceptional diversity in parental care and fertilization modes in ray-finned fishes.

Among vertebrates, ray-finned fishes (Actinopterygii) display the highest diversity in parental care, and their diversification has been hypothesized to be related to phylogenetic changes in fertilization modes. Using the most comprehensive, sex-specific data from 7600 species of 62 extant orders of ray-finned fishes, we inferred ancestral states and transitions among care types and caring episodes (i.e. the stage of offspring development). Our work has uncovered three novel findings. First, transitions among different care types (i.e. male-only care, female-only care, biparental care and no care) are common, and the frequencies of these transitions show unusually diverse patterns concerning fertilization modes (external, or internal via oviduct, mouth or brood pouch). Second, both oviduct and mouth fertilization select for female-biased care, whereas fertilization in a brood pouch selects for male-biased care. Importantly, internal fertilization without parental care is extremely unstable phylogenetically. Third, we show that egg care in both sexes is associated with nest building (which is male-biased) and fry care (which is female-biased). Taken together, the aquatic environment, which supports considerable flexibility in care, facilitated the diversification of parenting behavior, creating the evolutionary bases for more comprehensive parenting to protect offspring in semiterrestrial or terrestrial environments.

Characterization of CD83 homologs differently expressed during monocytes differentiation in ginbuna crucian carp, Carassius auratus langsdorfii.

CD83 is a costimulatory molecule of antigen-presenting cells (APCs) that plays an important role in eliciting adaptive responses. It is also a well-known surface protein on mature dendritic cells (DCs). Furthermore, monocytes have been reported to differentiate into macrophages and monocyte-derived dendritic cells, which play an important role in innate immunity. CD83 expression affects the activation and maturation of DCs and stimulates cell-mediated immune responses. This study aims to reveal the CD83 expression during monocyte differentiation in teleosts, and the CD83 homologs evolutionary relationship. This study found two distinct CD83 homologs (GbCD83 and GbCD83-L) in ginbuna crucian carp (Gb) and investigated the evolutionary relationship among GbCD83 homologs and other vertebrates and the gene and protein expression levels of the homologs during 4 days of monocyte culture. The phylogenetic tree showed that the two GbCD83 homologs are classified into two distinct branches. Interestingly, only ostariophysians (Gb, common carp, rohu, fathead minnow and channel catfish), but not neoteleosts, mammals, and others, have two CD83 homologs. Morphological observation and colony-stimulating factor-1 receptor (CSF-1R), CD83, CD80/86, and CCR7 gene expressions illustrated that there is a differentiation of monocytes isolated from peripheral blood leukocytes after 4 days. Specifically, gene expression and immunocytochemistry revealed that GbCD83 is mainly expressed on monocytes at the early stage of cell culture, whereas GbCD83-L is expressed in the latter stage. These findings provided the first evidence of differential expression of CD83 homologs during monocytes differentiation in teleost.

A novel long non-coding RNA linc-93.2 participates in bisphenol induced oxidative stress and macrophage polarization in red common carp (Cyprinus carpio).

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.

The Dorsal Part of the Anterior Tuberal Nucleus Responds to Auditory Stimulation in Zebrafish (Danio rerio).

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.

Ace Deficiency Induces Intestinal Inflammation in Zebrafish.

Inflammatory bowel disease (IBD) is a nonspecific chronic inflammatory disease resulting from an immune disorder in the intestine that is prone to relapse and incurable. The understanding of the pathogenesis of IBD remains unclear. In this study, we found that ace (angiotensin-converting enzyme), expressed abundantly in the intestine, plays an important role in IBD. The deletion of ace in zebrafish caused intestinal inflammation with increased expression of the inflammatory marker genes interleukin 1 beta (il1b), matrix metallopeptidase 9 (mmp9), myeloid-specific peroxidase (mpx), leukocyte cell-derived chemotaxin-2-like (lect2l), and chemokine (C-X-C motif) ligand 8b (cxcl8b). Moreover, the secretion of mucus in the ace-/- mutants was significantly higher than that in the wild-type zebrafish, validating the phenotype of intestinal inflammation. This was further confirmed by the IBD model constructed using dextran sodium sulfate (DSS), in which the mutant zebrafish had a higher susceptibility to enteritis. Our study reveals the role of ace in intestinal homeostasis, providing a new target for potential therapeutic interventions.

Identification and functional characterization of fish IL-17 receptors suggest important roles in the response to nodavirus infection.

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.

Molecular dating of the teleost whole genome duplication (3R) is compatible with the expectations of delayed rediploidization.

Vertebrate evolution has been punctuated by three whole genome duplication (WGD) events that have been implicated causally in phenotypic evolution, from the origin of phenotypic novelties to explosive diversification. Arguably the most dramatic of these is the 3R WGD event associated with the origin of teleost fishes which comprise more than half of all living vertebrate species. However, tests of a causal relationship between WGD and teleost diversification have proven difficult due to the challenge of establishing the timing of these phenomena. Here we show, based on molecular clock dating of concatenated gene alignments, that the 3R WGD event occurred in the early-middle Permian (286.18-267.20 Million years ago; Ma), 52.02-12.84 million years (Myr) before the divergence of crown-teleosts in the latest-Permian-earliest Late Triassic (254.36-234.16 Ma) and long before the major pulses of teleost diversification in Ostariophysi and Percomorpha (56.37-100.17 Myr and at least 139.24-183.29 Myr later, respectively). The extent of this temporal gap between putative cause and effect precludes 3R as a deterministic driver of teleost diversification. However, these age constraints remain compatible with the expectations of a prolonged rediploidization process following WGD which, through the effects of chromosome rearrangement and gene loss, remains a viable mechanism to explain the evolution of teleost novelties and diversification.

Meningitis caused by Aeromonas hydrophila in Oreochromis niloticus: Proteomics and druggability of virulence factors.

Meningitis caused by Gram-negative bacteria is a serious public health problem, causing morbidity and mortality in both children and adults. Here, we propose a novel experimental model using Nile tilapia (Oreochromis niloticus) to study neuroinflammation. The fish were infected with Aeromonas hydrophila, and the course of infection was monitored in the peripheral blood. Septicemia was obvious in the blood, while in the brain tissue, infection of the meninges was present. The histopathological examination showed suppurative meningitis, and the cellular immune response in the brain tissue during infection was mediated by microglia. These cells were morphologically characterized and phenotyped by MHC class II markers and CD68. The increased production of TNF-α, IL-1ß and iNOS supported the infiltration of these cells during the neuroinflammatory process. In the proteomic analysis of A. hydrophila isolated from brain tissue, we found chemotactic and transport proteins, proteolytic enzymes and enzymes associated with the dismutation of nitric oxide (NO), as well as motor proteins and those responsible for cell division. After characterizing the most abundant proteins during the course of infection, we investigated the druggability index of these proteins and identified promising peptide sequences as molecular targets that are similar among bacteria. Thus, these findings deepened the understanding of the pathophysiology of meningitis caused by A. hydrophila. Moreover, through the proteomics analysis, important mechanisms and pathways used by the pathogen to subvert the host response were revealed, providing insights for the development of novel antibiotics and vaccines.

The Peroxisome Proliferator-Activated Receptors of Ray-Finned Fish: Unique Structures, Elusive Functions.

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.

Developing rainbow trout (Oncorhynchus mykiss) lose branchial plasma accessible carbonic anhydrase expression with hatch and the transition to pH-sensitive, adult hemoglobin polymorphs.

Salmonids possess a unique respiratory system comprised of three major components: highly pH-sensitive hemoglobins, red blood cell (RBC) intracellular pH (pHi) protection, and a heterogeneous distribution of plasma accessible carbonic anhydrase (paCA), specifically with absence of paCA at the gills. These characteristics are thought to have evolved to enhance oxygen unloading to the tissues while protecting uptake at the gills. Our knowledge of this system is detailed in adults, but little is known about it through development. Developing rainbow trout (Oncorhynchus mykiss) express embryonic RBCs containing hemoglobins that are relatively insensitive to pH; however, availability of gill paCA and RBC pHi protection is unknown. We show that pre-hatch rainbow trout express gill paCA, which is lost in correlation with the emergence of highly pH-sensitive adult hemoglobins and RBC pHi protection. Rainbow trout therefore exhibit a switch in respiratory strategy with hatch. We conclude that gill paCA likely represents an embryonic trait in rainbow trout and is constrained in adults due to their highly pH-sensitive hemoglobins.

Organisation of the Atlantic salmon (Salmo salar) thymus and its content of Ig-expressing cells.

The thymus of fishes is located as a dual organ in a rostrodorsal projection within the gill chamber and is covered by the operculum. The histological organization of the teleost fish thymus displays considerable diversity, particularly in salmonids where a clear distinction between the thymus cortex and medulla is yet to be defined. Recent interest has focused on the role of B cells in thymic function, but the presence of these cells within the salmon thymus remains poorly understood. In this morphological study, we applied in situ hybridization to investigate developing Atlantic salmon thymi for the expression of recombination activating (Rag) genes 1 and 2. We identified the location of the cortex, aligning with the previously described inner zone. Expression of IgM and IgD transcripts was predominantly observed in cells within the outer and subcapsular zones, with lesser expression in the cortex and inner zone. IgT expression was confined to a limited number of cells in the inner zone and capsule. The location of the thymus medulla could not be established. Our results are discussed in the context of the recently identified lymphoid organs, namely the intrabranchial lymphoid tissue (ILT) and the salmon bursa.

The decision of male medaka to mate or fight depends on two complementary androgen signaling pathways.

Adult male animals typically court and attempt to mate with females, while attacking other males. Emerging evidence from mice indicates that neurons expressing the estrogen receptor ESR1 in behaviorally relevant brain regions play a central role in mediating these mutually exclusive behavioral responses to conspecifics. However, the findings in mice are unlikely to apply to vertebrates in general because, in many species other than rodents and some birds, androgens-rather than estrogens-have been implicated in male behaviors. Here, we report that male medaka (Oryzias latipes) lacking one of the two androgen receptor subtypes (Ara) are less aggressive toward other males and instead actively court them, while those lacking the other subtype (Arb) are less motivated to mate with females and conversely attack them. These findings indicate that, in male medaka, the Ara- and Arb-mediated androgen signaling pathways facilitate appropriate behavioral responses, while simultaneously suppressing inappropriate responses, to males and females, respectively. Notably, males lacking either receptor retain the ability to discriminate the sex of conspecifics, suggesting a defect in the subsequent decision-making process to mate or fight. We further show that Ara and Arb are expressed in intermingled but largely distinct populations of neurons, and stimulate the expression of different behaviorally relevant genes including galanin and vasotocin, respectively. Collectively, our results demonstrate that male teleosts make adaptive decisions to mate or fight as a result of the activation of one of two complementary androgen signaling pathways, depending on the sex of the conspecific that they encounter.