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The pearlspot, Etroplus suratensis is a climate resilient cichlid fish that exhibits unusual adaptation to salinity. The fish is able to complete full life cycle in diverse salinity habitats ranging from fresh water to marine environments. High-quality primary and phased genome assemblies were generated for pearlspot fish using PacBio HiFi and Arima HiC sequencing technologies, for the first time. The primary assembly is highly contiguous with contig N50 length of 36 Mb. The final assembly is of 1.247 Gb with N50 length of 51.57 Mb and 98% of the genome length anchored to 24 chromosomes. The genome was assessed to be 99.9% complete based on BUSCO evaluation and was predicted to contain 52.96% repeat elements. We have predicted 27,192 protein encoding genes, of which 21,580 were functionally annotated. The genome offers an invaluable resource to understand adaptation of pearlspot fish to diverse salinity habitats., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Haemoglobin is a key molecule for oxygen transport in vertebrates. It exhibits remarkable gene diversity in teleost fishes, reflecting adaptation to various aquatic environments. In this study, we present the dynamic evolution of haemoglobin subunit genes based on a comparison of high-quality genome assemblies of 24 vertebrate species, including 17 teleosts (of which six are cichlids). Our findings indicate that teleost genomes contain a range of haemoglobin genes, from as few as five in fugu to as many as 43 in salmon, with the latter being the largest repertoire found in vertebrates. We find evidence that the teleost ancestor had at least four Hbα and three or four Hbβ subunit genes, and that the current gene diversity emerged during teleost radiation, driven primarily by (tandem) gene duplications, genome compaction, and rearrangement dynamics. We provide insights into the genomic organisation of haemoglobin clusters in different teleost species. We further show that the evolution of paralogous rhbdf1 genes flanking both teleost clusters (LA and MN) supports the hypothesis for the origin of the LA cluster by rearrangement within teleosts, rather than by the teleost specific whole-genome duplication. We specifically focus on cichlid fishes, where adaptation to low oxygen environment plays role in species diversification. Our analysis of six cichlid genomes, including Pungu maclareni from the Barombi Mbo crater lake, for which we sequenced a representative genome, reveals 18-32 copies of the Hb genes, and elevated rates of non-synonymous substitutions compared to other teleosts. Overall, this work facilitates a deeper understanding of how haemoglobin genes contribute to the adaptive potential of teleosts., (© 2024 The Author(s). Molecular Ecology published by John Wiley & Sons Ltd.)

Perforin, produced by natural killer (NK) cells and cytotoxic T lymphocytes (CTLs), is one of the effectors of cell-mediated cytotoxicity (CMC) in vertebrates, playing a paramount role in killing target cells. However, whether and how perforin is involved in adaptive immune responses in early vertebrates remains unclear. Using Nile tilapia (Oreochromis niloticus) as a model, we investigated the characteristics of perforin in early vertebrates. Oreochromis niloticus perforin (OnPRF) possesses 2 conserved functional domains, membrane attack complex/perforin (MACPF) and protein kinase C conserved region 2 (C2) domains, although they share low amino acid sequence similarity with other homologs. OnPRF was widely expressed in various immune tissues and could respond to lymphocyte activation and T-cell activation in vitro at both the transcriptional and protein levels, indicating that it may be involved in adaptive immune responses. Furthermore, after infection with Edwardsiella piscicida and Aeromonas hydrophila, the mRNA and protein levels of OnPRF were significantly up-regulated within the adaptive immune response period. Additionally, we revealed that many transcription factors were involved in the transcriptional regulation of OnPRF, including p65, c-Fos, c-Jun, STAT1 and STAT4, and there was a synergy among these transcription factors. Overall, these findings demonstrate the involvement of OnPRF in T-cell activation and adaptive immune response in tilapia, thus providing new evidence for comprehending the evolution of immune response in early vertebrates., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Neural crest (NC) is a vertebrate-specific embryonic progenitor cell population at the basis of important vertebrate features such as the craniofacial skeleton and pigmentation patterns. Despite the wide-ranging variation of NC-derived traits across vertebrates, the contribution of NC to species diversification remains underexplored. Here, leveraging the adaptive diversity of African Great Lakes' cichlid species, we combined comparative transcriptomics and population genomics to investigate the evolution of the NC genetic program in the context of their morphological divergence. Our analysis revealed substantial differences in transcriptional landscapes across somitogenesis, an embryonic period coinciding with NC development and migration. This included dozens of genes with described functions in the vertebrate NC gene regulatory network, several of which showed signatures of positive selection. Among candidates showing between-species expression divergence, we focused on teleost-specific paralogs of the NC-specifier sox10 (sox10a and sox10b) as prime candidates to influence NC development. These genes, expressed in NC cells, displayed remarkable spatio-temporal variation in cichlids, suggesting their contribution to interspecific morphological differences, such as craniofacial structures and pigmentation. Finally, through CRISPR/Cas9 mutagenesis, we demonstrated the functional divergence between cichlid sox10 paralogs, with the acquisition of a novel skeletogenic function by sox10a. When compared with teleost models zebrafish and medaka, our findings reveal that sox10 duplication, although retained in most teleost lineages, had variable functional fates across their phylogeny. Altogether, our study suggests that NC-related processes-particularly those controlled by sox10s-are involved in generating morphological diversification between species and lays the groundwork for further investigations into the mechanisms underpinning vertebrate NC diversification., Competing Interests: Conflict of Interest The authors declare no competing interests., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

In teleosts, GnRH1 neurons stand at the apex of the Hypothalamo-Pituitary-Gonadal (HPG) axis, which is responsible for the production of sex steroids by the gonads (notably, androgens). To exert their actions, androgens need to bind to their specific receptors, called androgen receptors (ARs). Due to a teleost-specific whole genome duplication, A. burtoni possess two AR paralogs (ARα and ARβ) that are encoded by two different genes, ar1 and ar2, respectively. In A. burtoni, males stratify along dominance hierarchies, in which an individuals' social status determines its physiology and behavior. GnRH1 neurons have been strongly linked with dominance and circulating androgen levels. Similarly, GnRH3 neurons are implicated in the display of male specific behaviors. Some studies have shown that these GnRH neurons are responsive to fluctuations in circulating androgens levels, suggesting a link between GnRH neurons and ARs. While female A. burtoni do not naturally form a social hierarchy, their reproductive state is positively correlated to androgen levels and GnRH1 neuron size. Although there are reports related to the expression of ar genes in GnRH neurons in cichlid species, the expression of each ar gene remains inconclusive due to technical limitations. Here, we used immunohistochemistry, in situ hybridization chain reaction (HCR), and spatial transcriptomics to investigate ar1 and ar2 expression specifically in GnRH neurons. We find that all GnRH1 neurons intensely express ar1 but only a few of them express ar2, suggesting the presence of genetically-distinct GnRH1 subtypes. Very few ar1 and ar2 transcripts were found in GnRH2 neurons. GnRH3 neurons were found to express both ar genes. The presence of distinct ar genes within GnRH neuron subtypes, most clearly observed for GnRH1 neurons, suggests differential control of these neurons by androgenic signaling. These findings provide valuable insight for future studies aimed at disentangling the androgenic control of GnRH neuron plasticity and reproductive plasticity across teleosts., (© 2024 British Society for Neuroendocrinology.)

Temperature is a preeminent factor in the regulation of fish reproduction and hinders gonadal development beyond a specific threshold. To comprehend the molecular mechanism responsible for reproductive suppression at different temperature, expression of the genes encoding kisspeptin (kiss2), gonadotropin-releasing hormone (gnrh1) and their receptors (gpr54, gnrh1r) in the brain, and the gonadotropin (GTH) subunits (fshb and lhb) in the pituitary were studied in juvenile Nile tilapia (Oreochromis niloticus) along with gonadal histology. Fish were acclimatized to three distinct temperatures, including 31 °C, 34 °C and 37 °C for 14 days. The mRNA levels of kiss2, gpr54, gnrh1, and gnrh1r were significantly decreased at 37 °C compared to 31 °C and 34 °C in the both sexes. In parallel, the expression level of fshb in the both sexes and lhb in the female were significantly lower at 37 °C in the pituitary. Histologically, the gonads of both sexes had normal growth of gametes at control temperature (31 °C), whereas the spermatogenesis and oocyte maturation were slowed down and atretic oocytes were found in the ovary at 37 °C acclimation temperature. Taken together, the results imply that elevated temperature beyond the specific threshold may have a negative impact on reproduction by suppressing the gene expressions of kisspeptin/GnRH1/GTH system and eventually restrains normal growth and maturation of gametes in the both sexes of Nile tilapia., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Hypoxia stress resulted in mortality during the fish aquaculture program, affecting the sustainable development of the aquaculture industry. The Egyptian strain of O. niloticus showed a strong ability to hypoxia. In this study, a Nile tilapia strain that was kept and selected for 45 years by the author's team was used to elucidate the mechanism of the hypoxia response in the liver, including the identification of metabolic pathways and genes, involved in the hypoxia response of this strain., Results: The effects of hypoxia stress were detected at 0-hour, 6-hour, and 72-hour time points (0 h, 6 h, 72 h) on tilapia liver at 1 mg/L dissolved oxygen conditions. The blood triglyceride, blood glucose and cholesterol values exhibited significantly different change trends, but the hemoglobin content showed no significant differences between 0 h, 6 h and 72 h (P > 0.05). The activities of catalase (CAT), glutathione peroxidase (GSH-PX), total antioxidant capacity (T-AOC), lactate dehydrogenase (LDH), and acid phosphatase (ACP) in the liver tissue gradually increased at 0 h, 6 h and 72 h (P < 0.05). Histological analyses revealed structural changes in intracellular lipid droplets, nuclear migration and dissolution, and cell vacuolization in liver tissues. Six pathways were identified as the main enriched metabolic pathways according to the transcriptome profiling analysis, which were protein processing in endoplasmic reticulum, steroid biosynthesis, peroxisome, PPAR signaling pathway, glycolysis/gluconeogenesis and Insulin signaling pathway. The expressions of the important differentially expressed genes were verified by qPCR analysis, including erola, LOC100692144, sqle, cratb, pipox, cpt1a2b, hik and acss2l, ehhadh, prkcz, fasn and plaa, which showed the same expressions trends as those of RNA-Seq., Conclusions: The Nile tilapia strain improves the abilities of hypoxia response through energy metabolism. Antioxidant enzyme measurements in the liver indicate that these five antioxidant enzymes play important roles in protecting the body from hypoxic damage. The histological changes in liver cells indicate that the damage caused by hypoxia stress. The immune-related metabolic pathways and energy metabolism-related pathways were obtained by transcriptome profiling, and these metabolic pathways and the differentially expressed genes selected from these metabolic pathways may be involved in the mechanism of hypoxia tolerance in this strain. These findings provide a better understanding of the hypoxia response mechanism of fish, and represent a useful resource for the genetic breeding of O. niloticus., (© 2024. The Author(s).)

Secreted by natural killer cells and cytotoxic T lymphocytes, Granzyme B is involved in regulating the adaptive immune response in vertebrates and plays a pivotal role in resisting virus invasion and removing pathogens. Although it had been extensively studied in mammals, the involvement of Granzyme B in adaptive immune response of early vertebrates remained elusive. In this study, we investigated the Granzyme B in Oreochromis niloticus (OnGrB), found that its function domain was conserved. Additionally, OnGrB was widely expressed in various tissues and could respond to T-cell activation in vitro at the transcriptional level. Furthermore, we prepared the recombinant OnGrB (rOnGrB) as an immunogen to develop a mouse anti-OnGrB monoclonal antibody (mAb). Using this anti-OnGrB mAb as a tool, we explored the expression of OnGrB in the adaptive immune response of tilapia. Our findings revealed that T cell was a significant source of OnGrB production, the expression of OnGrB at the protein level and the proportion of OnGrB  +  T cells increased after both T cell activation in vitro and infection with Edwardsiella piscicida in vivo. More importantly, our findings also preliminarily illuminated that p65 could regulate the transcriptional activity of OnGrB. These results indicated that OnGrB was involved in the adaptive immunity of tilapia and played a critical role in T cell function in teleost. Our study provided theoretical support and new perspectives for understanding adaptive immunity in teleost., (Copyright © 2024. Published by Elsevier Ltd.)

The Nile Tilapia (Oreochromis niloticus), a gonochoristic teleost fish with a XX/XY sex-determination system, is an ideal model for investigating gonadal sex differentiation. During gonadal differentiation, the expression of cyp19a1a in XX gonads and dmrt1 in XY gonads are required for undifferentiated tissues to develop into ovary or testis. In this study, quantitative real-time RT-PCR assessed the expression of cyp19a1a and dmrt1 genes in gonads and tail fin tissues. Differences in gene expression mean among sexually differentiated fish were analyzed using two-way analysis of variance (ANOVA) and validation of mixed model using discriminant analysis (DA) for morphometric traits and the gene expression in gonads and tail fin tissues used to validate and utilize them in discriminating sexes in sex-differentiated Nile Tilapia fish. The results revealed that, cyp19a1a gene expression in female ovaries was more significant than dmrt1 in male testis. In the other hand, the dmrt1 gene expression in the tail fin was higher in males than females. Both, cyp19a1a and dmrt1 genes, can discriminate fish sexes by 100% by using their expression in tail fin tissues. In conclusion, the cyp19a1a and dmrt1 genes could be used as a genetic marker to discriminate between the Nile Tilapia sexes, whereas used as an indicator for ovarian or testis differentiation in sexually differentiated Nile Tilapia using tail fin tissues. It is worth mentioning that this is the first investigation for using cyp19a1a and dmrt1 genes from Nile Tilapia tail fin tissues in sex determination., (© 2024. The Author(s).)

Innate social behaviors like aggression are modulated by sex steroid hormones such as androgens and estrogens. However, we know little about how the same hormone regulates similar behaviors in both sexes. We investigated the role of androgenic signaling in the regulation of aggression in Astatotilapia burtoni, a social fish in which males and females perform similar aggressive behaviors. We used androgen receptor (AR) α knockout (KO) animals for this study since this gene was recently shown to be required for male-typical aggression and mating. Surprisingly, ARα KO females did not show deficits in aggression. We also determined that females lacking the other AR, ARβ, showed normal levels of aggression. Blocking both ARs pharmacologically confirmed that neither AR is necessary for aggression in females. However, ARα KO males showed clear deficits in attacks. Thus, in A. burtoni there appears to be a sexual dimorphism in the role of ARα in the control of aggression., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The daily variations of temperature are one of the main synchronizers of the circadian rhythms. In addition, water temperature influences the embryonic and larval development of fish and directly affects their metabolic processes. The application of thermocycles to fish larvae has been reported to improve growth and the maturation of the digestive system, but their effects on metabolism are poorly understood. The aim of the present study was to evaluate the effect of two different temperature regimes, cycling versus constant, on the daily rhythms of metabolic factors of Nile tilapia (Oreochromis niloticus) larvae. For this purpose, fertilized eggs were divided into two groups: one reared in a 31 °C:25 °C day:night thermocycle (TCY) and another group maintained in a constant 28 °C temperature (CTE). The photoperiod was set to a 12:12 h light/dark cycle. Samples were collected every 4 h during a 24-h cycle on days 4, 8 and 13 post fertilization (dpf). The expression levels of alanine aminotransferase (alt), aspartate aminotransferase (ast), malic enzyme, glucose-6-phosphate dehydrogenase (g6pd), phosphofructokinase (pfk) and pyruvate kinase (pk) were analyzed by qPCR. Results showed that, in 13 dpf animals, most of the genes analyzed (alt, ast, malic, g6pd and pfk) showed daily rhythms in TCY, but not in the group kept at constant temperature, with most acrophases detected during the feeding period. An increase in nutrient metabolism around feeding time can improve food utilization and thus increase larval performance. Therefore, the use of thermocycles is recommended for tilapia larviculture., Competing Interests: Declaration of competing interest The authors whose names are listed immediately below certify that they have NO affi liations with or involvement in any organization or entity with any fi nancial interest (such as honoraria; educational grants; participation in speakers' bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-fi nancial interest (such as personal or professional relationships, affi liations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript., (Copyright © 2024 Elsevier Inc. All rights reserved.)

MicroRNAs play crucial regulatory roles in various aspects of development and physiology, including environmental adaptation and stress responses in teleosts. RT-qPCR is the most commonly used method for studying microRNA expression, with the accuracy and reliability of results depending on the use of an appropriate reference gene for normalization. This study aimed to evaluate seven miRNAs (U6, Let-7a, miR-23a, miR-25-3, miR-103, miR-99-5, and miR-455) expression stability in different tissues of Nile tilapia subjected to osmotic stress. Fish were divided into two groups: a control and an experimental group, raised in 0 and 12 ppt salinity water respectively. After 21 days, brain, gills, liver, and posterior intestine were collected for analysis. Different mathematical algorithms (geNorm, NormFinder, BestKeeper, and the comparative ΔCt method) were employed to identify the most suitable reference miRNAs. The results indicate that the miR-455/miR-23a combination is a robust reference for normalizing miRNA expression levels in studies of osmotic stress responses in Nile tilapia. The stability of miRNA expression can vary depending on specific stress conditions and biological processes, underscoring the necessity of selecting appropriate normalizing miRNAs for each experimental context. This study identifies reliable reference genes for future RT-qPCR analyses of miRNA expression, thereby enhancing our understanding of molecular responses in fish to environmental challenges. These insights are fundamental to the development of new technologies for the improved management and sustainability of aquaculture practices., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

High mobility group protein B2 (HMGB2) is an abundant chromatin-associated protein with pivotal roles in transcription, cell proliferation, differentiation, inflammation, and tumorigenesis. However, its immune function in Nile tilapia (Oreochromis niloticus) remains unclear. In this study, we identified a homologue of HMGB2 from Nile tilapia (On-HMGB2) and investigated its functions in the immune response against streptococcus infection. The open reading frame (ORF) of On-HMGB2 spans 642 bp, encoding 213 amino acids, and contains two conserved HMG domains. On-HMGB2 shares over 80 % homology with other fish species and 74%-76 % homology with mammals. On-HMGB2 was widely distributed in various tissues, with its highest transcript levels in the liver and the lowest in the intestine. Knockdown of On-HMGB2 promoted the inflammatory response in Nile tilapia, increased the bacterial load in the tissues, and led to elevated mortality in Nile tilapia following Streptococcus agalactiae infection. Taken together, On-HMGB2 significantly influences the immune system of Nile tilapia in response to streptococcus infection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

17α-Methyltestosterone (MT) is a widely used androgen for all-male fish production in aquaculture. However, the molecular mechanism underlying MT-induced masculinization remains unclear. In this study, we aim to identify the key gene responsible for MT-induced masculinization using the Nile tilapia ( Oreochromis niloticus ) amhy , dmrt1 , and gsdf mutants, which exhibit male-to-female sex reversal. Nile tilapia fry from these three mutant lines were treated with 50 μg/g MT from 5 to 30 days after hatching (dah). The results showed that amhy and gsdf mutants, but not dmrt1 mutants, were masculinized by the MT treatment. Gonadal transcriptome analysis revealed that genes involved in steroidogenesis and germ cell development in MT-treated dmrt1 mutants exhibited a similar expression pattern to that of the wild type (WT) XX. In addition, the dmrt1 mutants cannot be masculinized by co-treatment with MT and the aromatase inhibitor fadrozole. The MT treatment completely blocked early steroidogenic enzyme (Star2, Cyp17a2, and Cyp19a1a) expression independent of amhy , gsdf , and dmrt1 . A luciferase analysis showed that MT directly suppressed basal and Sf-1-activated cyp19a1a promoter activity through ara and arb in cultured HEK293 cells. Furthermore, MT treatment inhibited germ cell proliferation in amhy and gsdf mutants but not in dmrt1 mutants. Consistently, dmrt1 expression was induced in MT-treated WT XX, - amhy , and - gsdf mutants. Taken together, these results suggest that dmrt1 is indispensable for MT-induced masculinization in Nile tilapia and that MT functions by inhibiting early steroid synthesis and activating dmrt1 to promote testis development.

Climatic and geomorphological changes during the Quaternary period impacted global patterns of speciation and diversification across a wide range of taxa, but few studies have examined these effects on African riverine fish. The lower Congo River is an excellent natural laboratory for understanding complex speciation and population diversification processes, as it is hydrologically extremely dynamic and recognized as a continental hotspot of diversity harboring many narrowly endemic species. A previous study using genome-wide SNP data highlighted the importance of dynamic hydrological regimes to the diversification and speciation in lower Congo River cichlids. However, historical climate and hydrological changes (e.g., reduced river discharge during extended dry periods) have likely also influenced ichthyofaunal diversification processes in this system. The lower Congo River offers a unique opportunity to study climate-driven changes in river discharge, given the massive volume of water from the entire Congo basin flowing through this short stretch of the river. Here, we, for the first time, investigate the impacts of paleoclimatic factors on ichthyofaunal diversification in this system by inferring divergence times and modeling patterns of gene flow in four endemic lamprologine cichlids, including the blind cichlid, Lamprologus lethops. Our results suggest that Quaternary climate changes associated with river discharge fluctuations may have impacted the diversification of species along the system and the emergence of cryptophthalmic phenotype in some endemic species. Our study, using reduced representation sequencing (2RADseq), indicates that the lower Congo River lamprologines emerged during the Early-Middle Pleistocene transition, characterized as one of the earth's major climatic transformation periods. Modeling results suggest that gene flow across populations and between species was not constant but occurred in temporally constrained pulses. We show that these results correlate with glacial-interglacial fluctuations. The current hyper-diverse fish assemblages of the lower Congo River riverscape likely reflect the synergistic effects of multiple drivers fueling complex evolutionary processes through time., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology.)

Fish sex is largely influenced by steroid hormones, especially sex hormones. Here, we established a steroid hormone-free genetic model by mutation of cyp11a1 in Nile tilapia, which was confirmed by EIA assay. Gonadal phenotype and transcriptome analyses showed that the XX mutants displayed sex reversal from female to male but with defective spermatogenesis. Despite the sex reversal, the aromatase encoding gene cyp19a1a was continuously expressed in the gonads of the XX mutants, which might be caused by androgen deficiency. Whole-mount fluorescence in situ hybridization and transcriptome analysis showed that the gonads of the XX mutants firstly developed towards ovary but shifted to testis between 10 to 15 days after hatching. Detailed expression analysis of key sex differentiation pathway genes foxl3 and dmrt1 combined with apoptosis analysis revealed transdifferentiation of germ cells from female to male during sex reversal. Rescue experiments showed that both P5 and E2 treatment rescued the sex reversal of cyp11a1 mutant XX fish. Overall, our results revealed a transient ovary-like stage and transdifferentiation of germ cells from female to male in the early gonads of the steroid hormone-deprived cyp11a1 mutant XX fish., (© 2024. The Author(s).)

Reproductive behaviors differ across species, but the mechanisms that control variation in mating and parental care systems remain unclear. In many animal species, pheromones guide mating and parental care. However, it is not well understood how vertebrate pheromone signaling evolution can lead to new reproductive behavior strategies. In fishes, prostaglandin F 2α (PGF 2α ) drives mating and reproductive pheromone signaling in fertile females, but this pheromonal activity appears restricted to specific lineages, and it remains unknown how a female fertility pheromone is sensed for most fish species. Here, we utilize single-cell transcriptomics and CRISPR gene editing in a cichlid fish model to identify and test the roles of key genes involved in olfactory sensing of reproductive cues. We find that a pheromone receptor, Or113a, detects fertile cichlid females and thereby promotes male attraction and mating behavior, sensing a ligand other than PGF 2α . Furthermore, while cichlid fishes exhibit extensive parental care, for most species, care is provided solely by females. We find that males initiate mouthbrooding parental care if they have disrupted signaling in ciliated sensory neurons due to cnga2b mutation or if or113a is inactivated. Together, these results show that distinct mechanisms of pheromonal signaling drive reproductive behaviors across taxa. Additionally, these findings indicate that a single pheromone receptor has gained a novel role in behavior regulation, driving avoidance of paternal care among haplochromine cichlid fishes. Lastly, a sexually dimorphic, evolutionarily derived parental behavior is controlled by central circuits present in both sexes, while olfactory signals gate this behavior in a sex-specific manner., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Oreochromis niloticus has great economic value and potential for farming and development. Transportation of fish was done for breeding or trading purpose and it is a challenging aspect of aquaculture. This study aimed to investigate the effect of transportation in freshwater and brackish water on the resistance of O. niloticus as well as transportation stress mitigation effect of NaCl. Four equal groups were used; each of 50 fish, the 1st group served as the control (P 1), while the 2nd group (PT 2) was transported in water without salt, the 3rd (PT 3) and 4th (PT 4) groups were transported in water containing 5 gL - 1 and 10 gL - 1 salt respectively. PT 2, PT 3 and PT 4 were transported for 5 h without any rest or sedative drugs., Results: The serum cortisol of O. niloticus significantly increased at 0 h and then decreased at 12 and 24 h post transportation in the PT 2 group and non-significantly increased at all point times in the PT 3 and PT 4 groups comparing to P 1 group. Mucin2 gene (MUC2) expression was non-significantly up regulated in the PT 2 group and down regulated in the PT 3 and PT 4 groups at 0 h comparing with P 1 group, but at 12 and 24 h it was significantly up regulated in the PT 2, PT 3 and PT 4 groups. The β Defensin-1 (β D1) and 2 (β D2) genes expression was non-significantly down-regulated in the PT 2 group and significantly up regulated in the PT 3 and PT 4 groups at 0 h., while at 12 and 24 h was significantly down regulated in the PT 2 group and non-significantly down regulated in the PT 3 and PT 4 groups, it significantly down regulated in the PT 2 and PT 3 group and non-significantly down regulated in the PT 4 group at 24 h. Non-significant up regulation in interleukin - 1β (IL-1β) gene expression was reported in the PT 2 group and non-significant down regulation in the PT 3 and PT 4 groups at 0 h. However, significant up regulation was recorded in the PT 2, PT 3 and PT 4 groups at 12 and 24 h. The Tumor necrosis factor-alpha (TNF-α) gene expression was non-significantly up regulated in the PT 2 group and non-significantly down regulated in the PT 3 and PT 4 groups at 0 h. However, it was significantly up regulated in the PT 2, PT 3 and PT 4 groups at 12 and 24 h., Conclusion: The results of this study confirmed the stressful effect of transportation on O. niloticus as well as the transportation stress mitigation effect of NaCl., (© 2024. The Author(s).)

Type I IFNs are a subset of cytokines exerting their antiviral effects mainly through the JAK-STAT signalling. Immunogenetic studies have shown that fish possess key components of IFN-JAK-STAT cascade, but the information about the distinct responses of STAT1 and STAT2 to different IFNs is rather limited in fish. Here, we identified and cloned STAT1 and STAT2 genes (named as On-STAT1 and On-STAT2) from tilapia, Oreochromis niloticus. On-STAT1 and On-STAT2 genes were detected in all orangs/tissues examined, and were rapidly induced in spleen, head kidney, and liver following the stimulation of poly(I:C). In addition, the stimulation of poly(I:C), poly(A:T), and different subgroups of recombinant IFNs could induce the expression of On-STAT1 and On-STAT2 in TA-02 cells with distinct induction levels. Importantly, On-STAT2 was rapidly phosphorylated by all three subgroups of IFNs, but the phosphorylation of On-STAT1 was only observed in IFNc- and IFNh-treated TA-02 cells, reflecting the distinct activation of STAT by different subgroups of fish IFNs. The present results thus contribute to better understanding of the JAK-STAT signalling mediated by different subgroups of IFNs in fish., Competing Interests: Declaration of competing interest The authors have no financial conflicts of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Genetic relationships were examined for a total of 90 individuals of 90 species or species var. from 13 genera of Malawian cichlids based on the sequences of an amplified 991-bp fragment of the mtDNA control region (mtDNA-CR). In the network analysis, no exclusive clades were made by all the members of any genera in this study. However, congeneric clades were observed by genera Buccochromis, Copadichromis, Protomelas, and Sciaenochromis, whereas no congeneric clades were observed by genera Mylochromis, Nimbochromis, and Otopharynx. In non-mbuna, an Aulonocara-Lethrinops group was divided into two groups, and the mean genetic distance of the larger group was much lower from mbuna than from other non-mbuna. Overall mean genetic distance within a genus was generally low in mbuna, whereas it was relatively high in non-mbuna. In the genetic tree of each genus, two or more large clades were observed, and some clades, such as those of Aulonocara hansbaenschi and Aulonocara nyassae in genus Aulonocara, Lethrinops micrentodon and Lethrinops sp. "gold harbor" in Lethrinops, and Otopharynx ovatus and Otopharynx brooksi in Otopharynx, were very deeply differentiated. Besides, a mbuna species, Pseudotropheus crabro, was extremely deeply differentiated from other members of this genus. These results suggest a widespread morphological convergence across the taxa in parallel with deep genetic differentiation in the long evolutionary story and some possibility of generation of the species of Aulonocara-Lethrinops group by hybridization of small non-mbuna and mbuna species. Furthermore, taxonomical reexamination is necessary based on a strong support by genetic connection., (© 2024 Fisheries Society of the British Isles.)

AbstractAdaptive radiations highlight the mechanisms by which species and traits diversify and the extent to which these patterns are predictable. We used 1,110 high-speed videos of suction feeding to study functional and morphological diversification in 300 cichlid species from three African Great Lake radiations of varying ages (Victoria, Malawi, and Tanganyika) and an older, spatially dispersed continental radiation in the Neotropics. Among African radiations, standing diversity was reflective of time. Morphological and functional variance in Lake Victoria, the youngest radiation, was a subset of that within Lake Malawi, which itself was nested within the older Tanganyikan radiation. However, functional diversity in Neotropical cichlids was often lower than that in Lake Tanganyika, despite being much older. These two radiations broadly overlapped, but each diversified into novel trait spaces not found in the youngest lake radiations. Evolutionary rates across radiations were inversely related to age, suggesting extremely rapid trait evolution at early stages, particularly in lake radiations. Despite this support for early bursts, other patterns of trait diversity were inconsistent with expectations of adaptive radiations. This work suggests that cichlid functional evolution has played out in strikingly similar fashion in different radiations, with contingencies eventually resulting in lineage-specific novelties.

Steroidogenic acute regulatory protein (Star) plays an essential role in the biosynthesis of corticosteroids and sex steroids by mediating the transport of cholesterol from the outer to the inner membrane of mitochondria. Two duplicated Star genes, namely star1 and star2, have been identified in non-mammalian vertebrates. To investigate the roles of star genes in fish steriodogenesis, we generated two mutation lines of star1 -/- and star1 -/- /star2 -/- in Nile tilapia (Oreochromis niloticus). Previous studies revealed that deficiency of star2 gene caused delayed spermatogenesis, sperm apoptosis and sterility in male tilapia. Our present data revealed that mutation of star genes impaired male fertility. Disordered seminiferous lobules and spermatic duct obstruction were found in the testis of both types of mutants. Moreover, significant decline in semen volume, sperm abnormality and impaired fertility were also detected in star1 -/- and star1 -/- /star2 -/- males. In star1 -/- male fish, lipid accumulation, up-regulation of steroidogenic enzymes, and significant decline of androgens were found. Additionally, hyperplasic interrenal cells, elevated steroidogenic gene expression level and decline of serum glucocorticoids were detected in star1 mutants. Intriguingly, either 11-KT or cortisol supplementation successfully rescued the impaired fertility of the star1 -/- mutants. Taken together, these results further indicate that Star1 might play critical roles in the production of both 11-KT and glucocorticoids, which are indispensable for the maintenance of male fertility in fish., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Speciation is familiar in radiations, but personality is not. In a recent article, Sommer-Trembo et al. linked exploratory behavior in African cichlids to a SNP in the promoter of a gene, the homolog of which is associated with human personality disorders, offering clues about the first fish of this radiation, with implications for vertebrate evolution., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

This study aimed to investigate the toxicity and endocrine disrupting potential of a complex mixture of polycyclic aromatic hydrocarbons (PAHs) in the estrogen pathway using hepatocytes of Nile tilapia Oreochromis niloticus, the hepatocytes were exposed to various concentrations of the PAH mixture, and multiple endpoints were evaluated to assess their effects on cell viability, gene expression, oxidative stress markers, and efflux activity. The results revealed that the PAH mixture had limited effects on hepatocyte metabolism and cell adhesion, as indicated by the non-significant changes observed in MTT metabolism, neutral red retention, and crystal violet staining. However, significant alterations were observed in the expression of genes related to the estrogen pathway. Specifically, vitellogenin (vtg) exhibited a substantial increase of approximately 120% compared to the control group. Similarly, estrogen receptor 2 (esr2) showed a significant upregulation of approximately 90%. In contrast, no significant differences were observed in the expression of estrogen receptor 1 (esr1) and the G protein-coupled estrogen receptor 1 (gper1). Furthermore, the PAH mixture elicited complex responses in oxidative stress markers. While reactive oxygen species (ROS) and reactive nitrogen species (RNS) levels remained unchanged, the activity of catalase (Cat) was significantly reduced, whereas superoxide dismutase (Sod) activity, glutathione S-transferase (Gst) activity, and non-protein thiols levels were significantly elevated. In addition, the PAH mixture significantly influenced efflux activity, as evidenced by the increased efflux of rhodamine and calcein, indicating alterations in multixenobiotic resistance (MXR)-associated proteins. Overall, these findings, associated with bioinformatic analysis, highlight the potential of the PAH mixture to modulate the estrogen pathway and induce oxidative stress in O. niloticus hepatocytes. Understanding the mechanisms underlying these effects is crucial for assessing the ecological risks of PAH exposure and developing appropriate strategies to mitigate their adverse impacts on aquatic organisms., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Tugstenio Lima de Souza, Francisco Filipak Neto, Ciro Alberto Oliveira Ribeiro reports financial support was provided by Copel. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Heme oxygenase-1 (HO-1), an inducible rate-limiting metabolic enzyme, exerts critical immunomodulatory functions by potential anti-oxidant, anti-inflammatory, and anti-apoptotic activities. Although accumulative studies have focused on the immune functions of HO-1 in mammals, the roles in fish are poorly understood, and the reports on involvement in the defensive and immune response are very limited. In this study, On-HO-1 gene from Oreochromis niloticus was successfully cloned and identified, which contained an open reading frame (ORF) of 816 bp and coded for a protein of 271 amino acids. The On-HO-1 protein phylogenetically shared a high homology with HO-1 in other teleost fish (76.10%-98.89 %) and a lowly homology with HO-1 in mammals (38.98%-41.55 %). The expression levels of On-HO-1 were highest in the liver of healthy tilapias and sharply induced by Streptococcus agalactiae or Aeromonas hydrophila. Besides, On-HO-1 overexpression significantly increased non-specific immunological parameters in serum during bacterial infection, including LZM, SOD, CAT, ACP, and AKP. It also exerted anti-inflammatory and anti-apoptotic effects in response to the immune response of the infection with S. agalactiae or A. hydrophila by upregulating anti-inflammatory factors (IL-10, TGF-β), autophagy factors (ATG6, ATG8) and immune-related pathway factors (P65, P38), and down-regulating pro-inflammatory factors (IL-1β, IL-6, TNF-α), apoptotic factors (Caspase3, Caspase9), pyroptosis factor (Caspase1), and inflammasome (NLRP3). These results suggested that On-HO-1 involved in immunomodulatory functions and host defense in Nile tilapia., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024. Published by Elsevier Ltd.)

Nanoplastics (NPs) are one of the most hazardous marine litters, having the potential to cause far-reaching impacts on the environment and humankind. The effect of NPs on fish health has been studied, but their impact on the subcellular organelles remains unexplored. The present investigation studied the possible implications of polystyrene-nanoplastics (PS-NPs) on the hematology, tissue organization, and endoplasmic reticulum (ER) stress-related proteins in Nile tilapia (Oreochromis niloticus). Fish were exposed to ∼100 nm PS-NPs at environmentally relevant (0.1 mg/L), and sublethal (1, 10 mg/L) concentrations for 14 days through water exposure. The growth performance and hematological parameters such as erythrocytes, hemoglobin, hematocrit, and leucocytes decreased, while thrombocytes increased with PS-NPs dose-dependently. The gills, liver, kidney, and heart tissues displayed increasing degrees of pathology with increased concentrations of PS-NPs. The gills showed severe epithelial hyperplasia and lamellar fusion. The liver had an abstruse cellular framework, membrane breakage, and vacuolation. While glomerular and tubular atrophy was the most prominent pathology in the kidney tissue, the heart displayed extensive myofibrillar loss and disorderly arranged cardiac cells. The ER-stress-related genes such as bip, atf6, ire1, xbp1, pkr, and apoptotic genes such as casp3a, and bax were over-expressed, while, the anti-apoptotic bcl2 was under-expressed with increasing concentrations of PS-NPs. Immunohistochemistry and blotting results of GRP78, CHOP, EIF2S, and ATF6 in gills, liver, kidney, and heart tissues affirmed the translation to ER stress proteins. The results revealed the sub-lethal adverse effects and the activation of the ER-stress pathway in fish with sub-chronic exposure to PS-NPs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The first author is an Editorial Board Member for Cardiovascular Toxicology and was not involved in the editorial review or the decision to publish this article., (Copyright © 2023. Published by Elsevier Inc.)

The transforming growth factor beta-activated kinase 1 (TAK1)/c-Jun N-terminal kinase (JNK) axis is an essential MAPK upstream mediator and regulates immune signaling pathways. However, whether the TAK1/JNK axis harnesses the strength in regulation of signal transduction in early vertebrate adaptive immunity is unclear. In this study, by modeling on Nile tilapia (Oreochromis niloticus), we investigated the potential regulatory function of TAK1/JNK axis on lymphocyte-mediated adaptive immune response. Both OnTAK1 and OnJNK exhibited highly conserved sequences and structures relative to their counterparts in other vertebrates. Their mRNA was widely expressed in the immune-associated tissues, while phosphorylation levels in splenic lymphocytes were significantly enhanced on the 4th day post-infection by Edwardsiella piscicida. In addition, OnTAK1 and OnJNK were significantly up-regulated in transcriptional level after activation of lymphocytes in vitro by phorbol 12-myristate 13-acetate plus ionomycin (P + I) or PHA, accompanied by a predominant increase in phosphorylation level. More importantly, inhibition of OnTAK1 activity by specific inhibitor NG25 led to a significant decrease in the phosphorylation level of OnJNK. Furthermore, blocking the activity of OnJNK with specific inhibitor SP600125 resulted in a marked reduction in the expression of T-cell activation markers including IFN-γ, CD122, IL-2, and CD44 during PHA-induced T-cell activation. In summary, these findings indicated that the conserved TAK1/JNK axis in Nile tilapia was involved in adaptive immune responses by regulating the activation of lymphocytes. This study enriched the current knowledge of adaptive immunity in teleost and provided a new perspective for understanding the regulatory mechanism of fish immunity., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

In mammals, glial cell derived neurotrophic factor (GDNF) plays a critical role in the self-renewal and maintenance of spermatogonial stem cells (SSCs) in testis and oogenesis in ovary, whilst retinoic acid (RA), the key factor of meiosis initiation, can downregulate its expression. Unlike mammals, two Gdnf replication genes are widely present in teleost fishes, however, our understanding of them is still poor. In the present study, two paralogous gdnf from Nile tilapia (Oreochromis niloticus), namely as Ongdnfa and Ongdnfb, were characterized, and then their cellular expression profiles in testis and ovary and responsiveness to RA treatment at the tissue and cellular levels were investigated. In phylogenetic tree, the Gdnfa and Gdnfb from teleost fishes were clustered into two different subclasses, respectively, and then clustered with the homologs from cartilaginous fish and tetrapods, suggesting that OnGdnfa and OnGdnfb are orthologous to GDNF and paralogous to each other. Ongdnfa is expressed in Sertoli cells and Leydig cells in testis and oocytes in ovary. The expression pattern of Ongdnfb is similar to Ongdnfa. In the ex vivo testicular organ culture, RA down-regulated the expression of Ongdnfa, whereas up-regulated the expression of Ongdnfb (P < 0.05), suggesting that they have differential responsiveness to RA signaling. RA treatment of the cultured cells derived from adult Nile tilapia testis which have the expression of RA receptors (RAR), Ongdnfa and Ongdnfb further confirmed the above result. Collectively, our study suggests that Ongdnfa and Ongdnfb have non-germline expression patterns in testis and germline expression patterns in ovary; furthermore, they have differential responsiveness to RA signaling, implying that they might have differential biological functions. This study broadens and enriches our understanding of fish GDNF homologs and lays foundation for the study of their biological functions in the future., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Relaxin3 (rln3) has been associated with various emotional and cognitive processes, including stress, anxiety, learning, memory, motivational behavior, and circadian rhythm. Notably, previous report revealed that Rln3a played an indispensable role in testicular development and male fertility in Nile tilapia (Oreochromis niloticus). However, the underlying molecular mechanisms remain largely unknown. We found that Rln3a is expressed exclusively in the diencephalon* (Di*) of the brain. Deficiency of Rln3a resulted in a significant increase in serum dopamine level and an upregulation of gene expression of gnrh1 and kisspeptin2. To further elucidate the role of Rln3a in fish fertility, we collected two different regions of Di* and hypothalamus (Hyp) tissues for subsequent RNA-seq analysis of both wild-type (rln3a +/+ ) and rln3a -/- male tilapia. Upon the transcriptomic data, 1136 and 755 differentially expressed genes (DEGs) were identified in the Di* and Hyp tissues, respectively. In Di*, the up-regulated genes were enriched in circadian rhythm, chemical carcinogenesis, while the down-regulated genes were enriched in type II diabetes mellitus, dopaminergic synapse, and other pathways. In Hyp, the up-regulated genes were enriched in circadian rhythm, pyrimidine metabolism, while the down-regulated genes were enriched in type I diabetes mellitus, autoimmune thyroid disease, and other pathways. Subsequently, the results of both qRT-PCR and FISH assays highlighted a pronounced up-regulation of core circadian rhythm genes, cry1b and per3, whereas genes such as clocka, clockb, and arntl exhibited down-regulation. Furthermore, the genes associated with dopamine biosynthesis were significantly increased in the Hyp. In summary, the mutation of rln3a in male tilapia resulted in notable changes in circadian rhythm and disease-linked signaling pathways in the Di* and Hyp. These changes might account for the fertility defects observed in rln3a -/- male mutants in tilapia., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

To evaluate Tribulus terrestris and Mucuna pruriens for inducing all-male tilapia, mixed-sex Nile tilapia, Oreochromis niloticus, (mean weight 0.025 ± 0.009 g; mean length 1.25 ± 0.012 cm), were given a meal supplemented with either T. terrestris powder (commercial fish feed, 40% crude protein) (TT group), M. pruriens seed extract (MP group), MP + TT (mixed group), 17α-methyl testosterone (MT, control positive), or without supplements (control negative). The MP extracts significantly increased (P < 0.05) the final weight, weight gain, weight gain rate, and specific growth rate while feed conversion ratio was significantly decreased (P < 0.05). Plant extracts markedly improved (P < 0.05) the survival rate, proportion of males, and total testosterone compared to control and MT. Estrogen levels were lower in groups with plant extract than other groups. Fifteen days post-feeding, the Amh gene was expressed in the brain of O. niloticus fries with higher levels in MP, TT, and MT groups. Additionally, the expression of the Sox9 and Dmrt1 genes as a male related genes in fish fry gonads revealed significantly (P < 0.05) higher levels in groups fed on MP, TT, and MT compared to control after 30-day post-feeding, whereas; Foxl2 gene expression as a female related gene was significantly (P < 0.05) lower in fish fed on MP, TT, and MT compared to other groups after 30 days post feeding. Histologically, MT, MP, TT, and the mixture all exhibited solely male reproductive traits without noticeable abnormalities. This study concluded that each of the TT or MP extracts can induce sex reversal in tilapia while having no negative health impact compared to MT as the growth and survival rate in the treated groups with TT and MP were higher than control and group treated with MT., (© 2024. The Author(s).)

Pigment patterns are incredibly diverse across vertebrates and are shaped by multiple selective pressures from predator avoidance to mate choice. A common pattern across fishes, but for which we know little about the underlying mechanisms, is repeated melanic vertical bars. To understand the genetic factors that modify the level or pattern of vertical barring, we generated a genetic cross of 322 F2 hybrids between two cichlid species with distinct barring patterns, Aulonocara koningsi and Metriaclima mbenjii. We identify 48 significant quantitative trait loci that underlie a series of seven phenotypes related to the relative pigmentation intensity, and four traits related to patterning of the vertical bars. We find that genomic regions that generate variation in the level of eumelanin produced are largely independent of those that control the spacing of vertical bars. Candidate genes within these intervals include novel genes and those newly-associated with vertical bars, which could affect melanophore survival, fate decisions, pigment biosynthesis, and pigment distribution. Together, this work provides insights into the regulation of pigment diversity, with direct implications for an animal's fitness and the speciation process., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Brandon et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Cichlid fishes of the genus Oreochromis (tilapia) are among the most important fish for inland capture fisheries and global aquaculture. Deliberate introductions of non-native species for fisheries improvement and accidental escapees from farms have resulted in admixture with indigenous species. Such hybridization may be detrimental to native biodiversity, potentially leading to genomic homogenization of populations and the loss of important genetic material associated with local adaptation. By contrast, introgression may fuel diversification when combined with ecological opportunity, by supplying novel genetic combinations. To date, the role of introgression in the evolutionary history of tilapia has not been explored. Here we studied both ancient and recent hybridization in tilapia, using whole genome resequencing of 575 individuals from 23 species. We focused on Tanzania, a natural hotspot of tilapia diversity, and a country where hybridization between exotic and native species in the natural environment has been previously reported. We reconstruct the first genome-scale phylogeny of the genus and reveal prevalent ancient gene flow across the Oreochromis phylogeny. This has likely resulted in the hybrid speciation of one species, O. chungruruensis. We identify multiple cases of recent hybridization between native and introduced species in the wild, linked to the use of non-native species in both capture fisheries improvement and aquaculture. This has potential implications for both conservation of wild populations and the development of the global tilapia aquaculture industry., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

African cichlid fishes are known for their high rates of phenotypic evolution. A rapid rate of diversification is apparent also in the diversity of their sex chromosomes. To date, sex determiners have been identified on 18 of 22 chromosomes in the standard karyotype. Here, we use whole-genome sequencing to characterize the sex chromosomes of seven populations of basal haplochromines, focusing on the genus Pseudocrenilabrus. We identify six new sex chromosome systems, including the first report of a cichlid sex-determining system on linkage group 12. We then quantify the rates and patterns of sex chromosome turnover in this clade. Finally, we test whether some autosomes become sex chromosomes in East African cichlids more often than expected by chance., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Determining how internal and external stimuli interact to determine developmental trajectories of traits is a challenge that requires the integration of different subfields of biology. Internal stimuli, such as hormones, control developmental patterns of phenotypic changes, which might be modified by external environmental cues (e.g. plasticity). Thyroid hormone (TH) modulates the timing of opsin gene expression in developing Midas cichlid fish (Amphilophus citrinellus). Moreover, fish reared in red light accelerate this developmental timing compared to fish reared in white light. Hence, we hypothesized that plasticity caused by variation in light conditions has coopted the TH signaling pathway to induce changes in opsin gene expression. We treated Midas cichlids with TH and crossed this treatment with two light conditions, white and red. We observed that not only opsin expression responded similarly to TH and red light but also that, at high TH levels, there is limited capacity for light-induced plasticity. Transcriptomic analysis of the eye showed that genes in the TH pathway were affected by TH, but not by light treatments. Coexpression network analyses further suggested that response to light was independent of the response to TH manipulations. Taken together, our results suggest independent mechanisms mediating development and plasticity during development of opsin gene expression, and that responses to environmental stimuli may vary depending on internal stimuli. This conditional developmental response to external factors depending on internal ones (e.g. hormones) might play a fundamental role in the patterns of phenotypic divergence observed in Midas cichlids and potentially other organisms., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

A key challenge in animal behavior is disentangling the social stimuli that drive conspecific behaviors. For some species, like teleost fish, putative sexual signaling cues are inextricably linked to others, making it difficult to parse the precise roles distinct signals play in driving conspecific behaviors. In the African cichlid Astatotilapia burtoni, males are either dominant or subordinate, wherein bright coloration, territoriality, and courtship behavior inextricably correlate positively with rank. Here, we leveraged androgen receptor (AR) mutant male A. burtoni that lack dominance-typical coloration but not behavior to isolate the role of male coloration in driving female mating behaviors in this species. We found in independent behavioral assays that females behave aggressively towards AR mutant but not WT males, yet still mated with both types of males. Females showed enhanced activation of esr2b + cells in the hypothalamus when housed with either mutant or WT males and this activation scaled with spawning activities. Therefore, there is not a simple relationship between male coloration and female mating behaviors in A. burtoni, suggesting independent sensory mechanisms converge on hypothalamic esr2b cells to coordinate behavioral output., Competing Interests: Declaration of competing interest The authors declare no competing or financial interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The intestinal microbiota is increasingly recognized as playing an important role in aquatic animals. To investigate the functional roles and mechanisms of the intestinal microbial genes/enzymes responding to salinity stress or osmotic pressure in fish, metagenomic analysis was carried out to evaluate the response of intestinal microbiota and especially their functional genes/enzymes from freshwater (the control group) to acute high salinity stress (the treatment group) in Nile tilapia. Our results showed that at the microbial community level, the intestinal microbiota in Nile tilapia generally underwent significant changes in diversity after acute high salinity stress. Among them, the shift in the bacterial community (mainly from Actinobacteria to Proteobacteria) dominated and had a large impact, the fungal community showed a very limited response, and other microbiota, such as phages, likely had a negligible response. At the functional level, the intestinal bacteriadecreased the normal physiological demand and processes, such as those of the digestive system and nervous system, but enhanced energy metabolism. Furthermore, at the gene level, some gene biomarkers, such as glutathione S-transferase, myo-inositol-1(or 4)-monophosphatase, glycine betaine/proline transport system permease protein, and some families of carbohydrate-active enzymes (GT4, GT2), were significantly enriched. However, GH15, GH23 and so on were significantly reduced. Exploring the functional details of the intestinal microbial genes/enzymes that respond to salinity stress in Nile tilapia sheds light on the mechanism of action of the intestinal microbiota with respect to the salinity adaptation of fish., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Monitoring heavy metal accumulation is essential for assessing the viability of aquatic ecosystems. Our methodology involved integrating analysis of immunological, stress, inflammatory, and growth-related gene expression in male and female Nile tilapia with on-site recordings of physicochemical parameters. Additionally, we assessed the effect of different physicochemical parameters on heavy metal bioavailability and residual concentration in fish and water. Samples of fish and water were gathered from three different localities: Lake Brullus, a brackish lake sited in northern Egypt; Lake Nasser, an artificial freshwater reservoir located in southern Egypt; and El-Qanater El-Khayria, a middle-freshwater location belonging to the Rashid branch of the river Nile. The assessment of heavy metal residues (Fe, Cu, Zn, Mn, and Ni) revealed that their concentrations were higher in fish specimens compared to their counterparts in water (except for Ni). In addition, Lake Brullus emerges as the most polluted area, exhibiting elevated levels of heavy metals concentrations in water and fish specimens. In contrast, Lake Nasser showed the least degree of heavy metals pollution. Gene expression analysis revealed gender-specific responses to heavy metal exposure at the three investigated water bodies. The expression of hepatic antioxidant genes (GST and MT) and inflammatory-related genes (CC-chemokine and TNFα) increased in males compared to females. In females, the immune and pro-inflammatory-related genes (IgM and CXC2-chemokine) transcripts were upregulated. Additionally, growth-related genes were downregulated in both Lake Brullus and El-Qanater; on the contrary, fish samples from Lake Nasser exhibited a normal expression pattern of growth-related genes. Stress-related genes (HSP70 and HSP27) showed significant downregulation in gills of both genders from Lake Brullus. The minimal presence of heavy metal contaminants in Lake Nasser seems to endorse the normal patterns of gene expression across all gene categories. A potential gender-specific gene expression response towards pollution was noticed in genes associated with inflammation and antioxidant activities. This highlights the importance of considering gender-related responses in future environmental assessments., (© 2024. The Author(s).)

The inflammasome is a multimeric protein complex that plays a vital role in the defence against pathogens and is therefore considered an essential component of the innate immune system. In this study, the expression patterns of inflammasome genes (NLRC3, ASC, and CAS-1), antiviral genes (IFNγ and MX), and immune genes (IL-1β and IL-18) were analysed in Oreochromis niloticus liver (ONIL) cells following stimulation with the bacterial ligands peptidoglycan (PGN) and lipopolysaccharide (LPS) and infection with TiLV. The cells were stimulated with PGN and LPS at concentrations of 10, 25, and 50 µg/ml. For viral infection, 10 6 TCID 50 of TiLV per ml was used. After LPS stimulation, all seven genes were found to be expressed at specific time points at each of the three doses tested. However, at even higher doses of LPS, NLRC3 levels decreased. Following TiLV infection, all of the genes showed significant upregulation, especially at early time points. However, the gene expression pattern was found to be unique in PGN-treated cells. For instance, NLRC3 and ASC did not show any response to PGN stimulation, and the expression of IFNγ was downregulated at 25 and 50 µg of PGN per ml. CAS-1 and IL-18 expression was downregulated at 25 µg of PGN per ml. At a higher dose (50 µg/ml), IL-1β showed downregulation. Overall, our results indicate that these genes are involved in the immune response to viral and bacterial infection and that the degree of response is ligand- and dose-dependent., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

The adaptative responses and divergent evolution shown in the environments habited by the Cichlidae family allow to understand different biological properties, including fish genetic diversity and structure studies. In a zone that has been historically submitted to different anthropogenic pressures, this study assessed the genetic diversity and population structure of cichlid Caquetaia kraussii, a sedentary species with parental care that has a significant ecological role for its contribution to redistribution and maintenance of sedimentologic processes in its distribution area. This study developed de novo 16 highly polymorphic species-specific microsatellite loci that allowed the estimation of the genetic diversity and differentiation in 319 individuals from natural populations in the area influenced by the Ituango hydroelectric project in the Colombian Cauca River. Caquetaia kraussii exhibits high genetic diversity levels (Ho: 0.562-0.885; He: 0.583-0.884) in relation to the average neotropical cichlids and a three group-spatial structure: two natural groups upstream and downstream the Nechí River mouth, and one group of individuals with high relatedness degree, possibly independently formed by founder effect in the dam zone. The three genetic groups show recent bottlenecks, but only the two natural groups have effective population size that suggest their long-term permanence. The information generated is relevant not only for management programs and species conservation purposes, but also for broadening the available knowledge on the factors influencing neotropical cichlids population genetics., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Cataño Tenorio et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Nile tilapia (Oreochromis niloticus) is valued in aquaculture because of its quick development and ability to thrive in various environments. Myxosporeans are among the fish parasites that affect fish productivity, as they impact fish growth and reproduction, resulting in large fish deaths in farms and hatcheries. This study has been focused on morpho-molecular identification for the myxosporean parasites infecting Nile tilapia from three governorates in Egypt and assessment of gene expression of different cytokines (Interleukin-1βeta (IL-1β), major histocompatibility complex class II (MHC-II), and clusters of differentiation 4 (CD-4) and 8 (CD-8)) in tissues. Additionally, this work aimed to correlate the developed histopathological alterations and inflammatory reactions in gills with immunohistochemical expression of inducible nitric oxide synthase (iNOS) and tumor necrosis factor-alpha (TNF-α). Finally, the infected fish's cortisol levels and blood glucose were assessed. Results of BLAST sequence analysis of the 18S rRNA for the collected protozoans confirmed Myxobolus agolus, M. brachysporus, M. tilapiae, and Henneguya species. The molecular characterization of the immunological status of gills revealed marked upregulation of different inflammatory cytokines in the gills of infected fish. There was a significantly increased serum cortisol and glucose level in infected fish compared with control, non-infected ones. Severe histopathological alterations were observed in the infected fish gills, associated with increased expression of iNOS and TNF-α and related to myxosporean infection. The present study provides new insights into oxidative stress biomarkers in Nile tilapia infected with Myxosporeans and elucidates the gill's immune status changes as a portal of entry for protozoa that contribute to tissue damage., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Ramadan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The effects of long-term dietary supplementation with sandalwood (Santalum album L.) essential oil (SEO) was investigated on hemato-biochemical biomarkers, immune status, antioxidant capacity, and resistance against Staphylococcus aureus in Nile tilapia (Oreochromis niloticus). Five groups (with four replicates) of O. niloticus (12.60 ± 0.20 g) were fed diets supplemented with SEO at doses of 0, 0.5, 1.0, 2.0, and 4.0 mL/kg diet for 60 days. Results indicated a substantial increase in blood protein levels and lower serum cholesterol, cortisol, glucose, urea, creatinine levels and, transaminase activities of fish fed a 2.0-mL SEO/kg diet. Serum lysozyme activity, nitric oxide, complement-3 levels, and phagocytic activity were significantly improved in O. niloticus after 60 days of feeding SEO-supplemented diets. Dietary SEO at level of 2.0-mL SEO/kg diet increased the activities of SOD, CAT, and GPx, and decreased MDA levels in liver homogenate. In addition, dietary 2.0-mL SEO/kg diet significantly upregulated antioxidant genes expression (CAT, SOD, GPx, GST, and GSR) with downregulation of apoptotic genes (HSP70, TLR2, caspase-3, and PCNA) in the liver. Furthermore, SEO-enriched diets significantly down-regulated pro-inflammatory (TNF-α, IL-1β, and IL-8) and up-regulated anti-inflammatory cytokine genes (TFG-β and IL-10) in the spleen. Moreover, SEO fortification increased the relative percentage of survival against S. aureus challenge and regulated immune-antioxidant genes in the spleen after the challenge. Overall, the results revealed that long-term using SEO might strengthen the physiological performance, hepatic oxidant/antioxidant balance, innate immune response, and resistance of O. niloticus against bacterial infections., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Objective: During Egypt's hot summer season, Aeromonas veronii infection causes catastrophic mortality on Nile Tilapia Oreochromis niloticus farms. Egypt is ranked first in aquaculture production in Africa, sixth in aquaculture production worldwide, and third in global tilapia production. This study aimed to investigate, at the molecular level, the early innate immune responses of Nile Tilapia to experimental A. veronii infection., Methods: The relative gene expression, co-expression clustering, and correlation of four selected immune genes were studied by quantitative real-time polymerase chain reaction in four organs (spleen, liver, gills, and intestine) for up to 72 h after a waterborne A. veronii challenge. The four genes studied were nucleotide-binding oligomerization domain 1 (NOD1), lipopolysaccharide-binding protein (LBP), natural killer-lysin (NKL), and interleukin-1 beta (IL-1β)., Result: The four genes showed significant transcriptional upregulation in response to infection. At 72 h postchallenge, the highest NOD1 and IL-1β expression levels were recorded in the spleen, whereas the highest LBP and NKL expression levels were found in the gills. Pairwise distances of the data points and the hierarchical relationship showed that NOD1 clustered with IL-1β, whereas LBP clustered with NKL; both genes within each cluster showed a significant positive expression correlation. Tissue clustering indicated that the responses of only the gill and intestine exhibited a significant positive correlation., Conclusion: The results suggest that NOD1, LBP, NKL, and IL-1β genes play pivotal roles in the early innate immune response of Nile Tilapia to A. veronii infection, and the postinfection expression profile trends of these genes imply tissue-/organ-specific responses and synchronized co-regulation., (© 2024 The Authors. Journal of Aquatic Animal Health published by Wiley Periodicals LLC on behalf of American Fisheries Society.)

Non-specific cytotoxic cells (NCCs) are vital immune cells involved in teleost's non-specific immunity. As a receptor molecule on the NCCs' surface, the non-specific cytotoxic cell receptor protein 1 (NCCRP-1) is known to play a crucial role in mediating their activity. Nevertheless, there have been limited studies on the signal molecule that transmits signals via NCCRP-1. In this study, a yeast two-hybrid (Y2H) library of tilapia liver and head kidney was constructed and subsequently screened with the bait vector NCCRP-1 of Oreochromis niloticus (On-NCCRP-1) to obtain a C-type lectin (On-CTL) with an interacting protein sequence. Consequently, the full-length sequence of On-CTL was cloned and analyzed. The expression analysis revealed that On-CTL is highly expressed in the liver and is widely distributed in other tissues. Furthermore, On-CTL expression was significantly up-regulated in the brain, intestine, and head kidney following a challenge with Streptococcus agalactiae. A point-to-point Y2H method was also used to confirm the binding between On-NCCRP-1 and On-CTL. The recombinant On-CTL (rOn-CTL) protein was purified. In vitro experiments demonstrated that rOn-CTL can up-regulate the expression of killer effector molecules in NCCs via its interaction with On-NCCRP-1. Moreover, activation of NCCs by rOn-CTL resulted in a remarkable enhancement in their ability to eliminate fathead minnow cells, indicating that rOn-CTL effectively modulates the killing activity of NCCs through the NCC receptor molecule On-NCCRP-1. These findings significantly contribute to our comprehension of the regulatory mechanisms governing NCC activity, paving the way for future research in this field., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Within 15,000 years, the explosive adaptive radiation of haplochromine cichlids in Lake Victoria, East Africa, generated 500 endemic species. In the 1980s, the upsurge of Nile perch, a carnivorous fish artificially introduced to the lake, drove the extinction of more than 200 endemic cichlids. The Nile perch predation particularly harmed piscivorous cichlids, including paedophages, cichlids eat eggs and fries, which is an example of the unique trophic adaptation seen in African cichlids. Here, aiming to investigate past demographic events possibly triggered by the invasion of Nile perch and the subsequent impacts on the genetic structure of cichlids, we conducted large-scale comparative genomics. We discovered evidence of recent bottleneck events in 4 species, including 2 paedophages, which began during the 1970s to 1980s, and population size rebounded during the 1990s to 2000s. The timing of the bottleneck corresponded to the historical records of endemic haplochromines" disappearance and later resurgence, which is likely associated with the introduction of Nile perch by commercial demand to Lake Victoria in the 1950s. Interestingly, among the 4 species that likely experienced bottleneck, Haplochromis sp. "matumbi hunter," a paedophagous cichlid, showed the most severe bottleneck signatures. The components of shared ancestry inferred by ADMIXTURE suggested a high genetic differentiation between matumbi hunter and other species. In contrast, our phylogenetic analyses highly supported the monophyly of the 5 paedophages, consistent with the results of previous studies. We conclude that high genetic differentiation of matumbi hunter occurred due to the loss of shared genetic components among haplochromines in Lake Victoria caused by the recent severe bottleneck., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Tilapia lake virus (TiLV) is an emerging virus that seriously threatens the tilapia industries worldwide. Interferon regulatory factors (IRFs), which are the crucial mediators regulating the response of interferon (IFN) to combat invading viruses, have not yet been reported in tilapia during TiLV infection. Here, six IRF (IRF1, IRF2, IRF4, IRF7, IRF8, and IRF9) homologs from tilapia were characterized and analyzed. These IRFs typically shared the conserved domains and phylogenetic relationship with IRF homologs of other species. Tissue distribution analysis showed that all six IRF genes were expressed in various tissues, with the highest expression in immune-related tissues. Furthermore, overexpression of IRFs in tilapia brain (TiB) cells significantly inhibited TiLV propagation, as evidenced by decreased viral segment 8 gene transcripts and copy numbers of viral segment 1. More importantly, all six IRF genes significantly enhanced the promoter activity of type I interferon-a3 (IFNa3) in TiB cells, suggesting that tilapia IRF genes serve as positive regulators in activating IFNa3. Surprisingly, the promoter activity of IFNa3 mediated by IRF genes was markedly inhibited post-TiLV infection, indicating that TiLV antagonized IRF-mediated IFN immune response. Taken together, six IRF genes of tilapia are highly conserved transcription factors that inhibit TiLV infection by activating the promoter of IFNa3, which is in turn restrained by TiLV. These findings broaden our knowledge about the functionality of IRF-mediated antiviral immunity in tilapia against TiLV infection and host-TiLV interaction, which lays a foundation for developing antiviral strategies in tilapia cultural industries., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Elongation of very long-chain fatty acids protein 6 (ELOVL6) plays a pivotal role in the synthesis of endogenous fatty acids, influencing energy balance and metabolic diseases. The primary objective of this study was to discover the molecular attributes and regulatory roles of ELOVL6 in male Nile tilapia, Oreochromis niloticus. The full-length cDNA of elovl6 was cloned from male Nile tilapia, and was determined to be 2255-bp long, including a 5'-untranslated region of 193 bp, a 3'-untranslated region of 1252 bp, and an open reading frame of 810 bp encoding 269 amino acids. The putative protein had typical features of ELOVL proteins. The transcript levels of elovl6 differed among various tissues and among fish fed with different dietary lipid sources. Knockdown of elovl6 in Nile tilapia using antisense RNA technology resulted in significant alterations in hepatic morphology, long-chain fatty acid synthesis, and fatty acid oxidation, and led to increased fat deposition in the liver and disrupted glucose/lipid metabolism. A comparative transcriptomic analysis (elovl6 knockdown vs. the negative control) identified 5877 differentially expressed genes with significant involvement in key signaling pathways including the peroxisome proliferator-activated receptor signaling pathway, fatty acid degradation, glycolysis/gluconeogenesis, and the insulin signaling pathway, all of which are crucial for lipid and glucose metabolism. qRT-PCR analyses verified the transcript levels of 13 differentially expressed genes within these pathways. Our findings indicate that elovl6 knockdown in male tilapia impedes oleic acid synthesis, culminating in aberrant nutrient metabolism., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Heterosis has been utilized in aquaculture for many years, yet its molecular basis remains elusive. Therefore, a comprehensive analysis of heterosis was conducted by comparing growth, digestion and biochemistry indices, as well as the intestinal gene expression profiles of Nile tilapia, blue tilapia and their hybrids. The results revealed that hybrid tilapia demonstrated an enhanced growth traits and elevated digestive enzyme activity compared to Nile and blue tilapia. Additionally, the hybrid tilapia displayed superior antioxidants and non-specific immune levels, with increased levels of catalase (CAT), alkaline phosphatase (AKP), acid phosphatase (ACP), glutathione (GSH), superoxide dismutase (SOD), total antioxidant capacity (TAOC), lysozyme, and immunoglobulin M (IgM) relative to Nile and blue tilapia. Moreover, 3392, 2470 and 1261 differentially expressed genes (DEGs) were identified in the intestinal tissues when comparing Nile tilapia to blue tilapia, hybrid tilapia to blue tilapia, and hybrid tilapia to Nile tilapia. Upon classifying the differentially expressed genes (DEGs), non-additively expressed DEGs accounted for 68.1 % of the total DEGs, with dominant and over-dominant expressed DEGs comprising 63.7 % and 4.4 % in the intestines, respectively. These non-additively expressed DEGs were primarily associated with metabolic, digestive, growth, and developmental pathways. This enrichment enhances our comprehension of the molecular underpinnings of growth heterosis in aquatic species., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Recombination activating genes (RAGs) mediates the process of rearrangement and somatic recombination (V(D)J) to generate different antibody repertoire. Studies on the expression pattern of adaptive immune genes during ontogenic development are crucial for the formulation of fish immunization strategy. In the present study, Nile tilapia was taken to explore the relative expression profile of RAG genes during their developmental stages. The developmental stages of Nile tilapia, i.e., unfertilized egg, 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 and 30 days post-hatch (dph) and kidney, blood, gill, liver and spleen tissues from adult fish were collected and the cDNA synthesis was carried out. Gene specific primers for RAG-1 and RAG-2 of Nile tilapia were designed and their annealing temperature (Tm) was optimized by gradient PCR. Consequently, PCR was performed to confirm the specific amplification of RAG-1 and RAG-2 genes. Quantitative real-time PCR (qRT-PCR) gene expression of RAG-1 and RAG-2 were noticed in all the developmental stages; however, a significant increase was observed after 12 dph and peaked at 24 dph, followed by a gradual decrease until 30 dph. Tissue-specific gene expression profiling revealed that the highest expression of RAG-1 and RAG-2 was observed in the kidney, followed by spleen, gill, liver and blood. The findings of the study explored the suitable timing of lymphoid maturation that could be technically used for the adoption of strategies to improve disease resistance of fish larvae for mitigating larval mortality., Competing Interests: Declaration of competing interest The authors declare that they do not have any conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The telencephalon has undergone remarkable diversification and expansion throughout vertebrate evolution, exhibiting striking variations in structural and functional complexity. Nevertheless, fundamental features are shared across vertebrate taxa, such as the presence of distinct regions including the pallium, subpallium, and olfactory structures. Teleost fishes have a uniquely "everted" telencephalon, which has confounded comparisons of their brain regions to other vertebrates. Here we combine spatial transcriptomics and single nucleus RNA-sequencing to generate a spatially-resolved transcriptional atlas of the Mchenga conophorus cichlid fish telencephalon. We then compare cell-types and anatomical regions in the cichlid telencephalon with those in amphibians, reptiles, birds, and mammals. We uncover striking transcriptional similarities between cell-types in the fish telencephalon and subpallial, hippocampal, and cortical cell-types in tetrapods, and find support for partial eversion of the teleost telencephalon. Ultimately, our work lends new insights into the organization and evolution of conserved cell-types and regions in the vertebrate forebrain., (© 2024. The Author(s).)

Background: Chlorpyrifos (CPF) is a widely used pesticide in the production of plant crops. Despite rapid CPF biodegradation, fish were exposed to wastewater containing detectable residues. Recently, medicinal plants and algae were intensively used in aquaculture to replace antibiotics and ameliorate stress impacts., Methods and Results: An indoor experiment was conducted to evaluate the deleterious impacts of CPF pollution on Nile tilapia health and the potential mitigation role of Chlorella vulgaris algae. Firstly, the median lethal concentration LC 50 - 72 h of CPF was determined to be 85.8 µg /L in Nile tilapia (35.6 ± 0.5 g body weight) at a water temperature of 27.5 °C. Secondly, fish were exposed to 10% of LC 50 - 72 h for six weeks, and tissue samples were collected and examined every two weeks. Also, Nile tilapia were experimentally infected with Streptococcus agalactiae. Exposed fish were immunosuppressed expressed with a decrease in gene expressions of interleukin (IL) 1β, IL-10, and tumor necrosis factor (TNF)-α. Also, a decline was recorded in glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase (CAT) gene expression in the head kidney tissue. A high mortality rate (MR) of 100% was recorded in fish exposed to CPF for six weeks and challenged with S. agalactiae. Fish that received dietary C. vulgaris could restore gene expression cytokines and antioxidants compared to the control. After six weeks of CPF exposure, fish suffered from anemia as red blood cell count (RBCs), hemoglobin (Hb), and packed cell volume (PCV) significantly declined along with downregulation of serum total protein (TP), globulin (GLO), and albumin (ALB). Liver enzymes were significantly upregulated in fish exposed to CPF pollution, alanine aminotransferase (ALT) (42.5, 53.3, and 61.7 IU/L) and aspartate aminotransferase (AST) (30.1, 31.2, and 22.8) after 2, 4, and 6 weeks, respectively. On S. agalactiae challenge, high MR was recorded in Nile tilapia exposed to CPF (G3) 60%, 60%, and 100% in week 2, week 4, and week 6, and C. vulgaris provided a relative protection level (RPL) of 0, 14.29, and 20%, respectively., Conclusions: It was concluded that CPF pollution induces immunosuppressed status, oxidative stress, and anemic signs in Nile tilapia. In contrast, C. vulgaris at a 50 g/kg fish feed dose could partially ameliorate such withdrawals, restoring normal physiological parameters., (© 2024. The Author(s).)