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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.