Coping with salinity extremes: Gill transcriptome profiling in the black-chinned tilapia (Sarotherodon melanotheron).

Steeper and sometimes extreme salinity gradients increasingly affect aquatic organisms because of climate change. Hypersalinity habitats demand powerful physiological adaptive strategies. Few teleost species have the capacity to spend their whole life cycle in salinities way over seawater levels. Focusing on the multifunctional gill, we unraveled the tilapia S. melanotheron key strategies to cope with different environmental conditions, ranging from freshwater up to hypersaline habitats. De novo transcriptome assembly based on RNAseq allowed for the analysis of 40,967 annotated transcripts among samples collected in three wild populations at 0, 40 and 80 ‰. A trend analysis of the expression patterns revealed responses across the salinity gradient with different gene pathways involved. Genes linked to ion transport, pH regulation and cell surface receptor signaling were mainly upregulated in the high salinity habitat. We identified tight junction proteins that were critical in high salinity habitats and that were different from the well-known tightening junctional proteins identified and expressed in fresh water. Expression profiles also suggest a change in the vascular tone that could be linked to an osmorespiratory compromise not only in fresh water, but also in high salinity environments. A striking downregulation of genes linked to the immune system and to the heat shock response was observed suggesting an energetic trade-off between immunity and acclimation/adaptation in the hypersaline habitat. The high expression of transcripts coding for immune and heat shock response in the freshwater habitat suggests the establishment of powerful mechanisms to protect gills from environmental threats and to maintain protein integrity. Non-directional expression trends were also detected with an upregulation of genes only in the hypersaline habitat (80 ‰) or only in the marine habitat (40 ‰). Unravel physiological strategies in S. melanotheron populations will help to better understand the molecular basis of fish euryhalinity in salinity-contrasted environments.

Salinity stress in the black-chinned tilapia Sarotherodon melanotheron.

Physiological and morphological acclimation capacities of black-chinned tilapia, Sarotherodon melanotheron were studied from fish to gill cell level when fish are maintained in freshwater, seawater, and hypersaline conditions. Fish osmoregulatory capacity, gill ionocyte morphology, osmo-respiratory compromise, O2 consumption rate, branchial antioxidative defense, and cell apoptosis were considered. Captive juvenile tilapias were maintained in controlled freshwater conditions (FW: 0.4 ppt; 12 mOsm kg-1) or gradually transferred to seawater (SW: 32 ppt; 958 mOsm kg-1) and concentrated SW (cSW: 65 ppt; 1920 mOsm kg-1). After 15 days in these conditions, blood osmolality and chloride ion concentration were determined. Gill ionocyte density and morphology were measured using immunolabelled histological sections to specifically detect the sodium pump (NKA). Gill osmo-respiratory compromise was also calculated along with oxygen consumption rates from normoxic to hypoxic conditions from excised gills (indirect respirometry). Finally, catalase and caspase 3/7activities were recorded from gill extracts. Results indicate that elevated salinity induces an osmotic imbalance and a profound morphological change with proliferating and hypertrophied ionocytes. This thickening of the gill interlamellar cell mass and the shortening of the lamellae induce a reduced osmo-respiratory ratio and reduced respiratory capacity under both normoxic and hypoxic conditions. Although salinity changes do not affect one of the major antioxidative defense mechanism, it strongly affects apoptosis that appears the most elevated in SW. However, in freshwater condition, fish can maintain their osmotic balance with a low ionocyte density, a low apoptotic level and a drastically reduced O2 consumption in normoxic condition that is maintained in hypoxia. Therefore, S. melanotheron presents the typical functional remodeling due to environmental salinity changes ranging from FW to SW. However, elevated seawater induces major cellular stress inducing a profound gill morphofunctional dysfunctioning. While cell apoptosis is reduced, ionocyte proliferation is massively increased with impaired osmotic regulation and reduced O2 consumption both in normoxic and hypoxic conditions.

Modulation of growth performance, feed utilization, and physiological traits in redbelly Tilapia (Tilapia zillii) through environmental and sex-based interactions.

This study explored the combined influence of tank color, stocking density, and gender on Tilapia zillii's performance and well-being. In this 120-day trial, 320 T. zillii, each initially weighing 10.0 ± 0.1 g/fish, were distributed among 24 tanks. The experiment included eight distinct treatment combinations, varying tank color (blue and green), stocking density (10 and 30 fish/m3) and sex (monosexual and mixed). The results showed that blue tanks improved specific growth rate and condition factor, while green tanks were better for feed utilization. Density at 30 fish/m3 showed the highest mean values of final body weight and total length, weight gain (WG), and gain length. Mono-sex outperformed mixed-sex ones in WG and daily growth. Interactions between color, density and sex were significant, affecting growth and feed utilization. Green tanks were best for protein profiles, while blue tanks excelled in glucose. A density of 10 fish/m3 yielded the highest protein profiles, and mono-sex fish had higher protein profiles. For lipid profiles, green tanks were superior, and density affected lipid profiles. Mixed-sex populations were best for certain lipid profile parameters. Interactions between these factors also played a significant role, making the biochemical profiles of T. zillii a complex interplay of various factors. The results explored that tank color, fish density and sex influence the activity of nonspecific immune enzymes in the liver of T. zillii. Blue tanks and lower fish density led to higher nonspecific immune enzymes, while mono-sex fish exhibited more significant nonspecific immune enzymes. Complex interactions between these factors also influenced nonspecific immune enzyme activities. Blue tanks increased malondialdehyde (MDA) levels, while green tanks raised glutathione S-transferases (GST) and catalase (CAT) levels. Lower fish density led to higher MDA, while higher density increased GST and CAT. Mono-sex fish had more MDA and GST, while mixed-sex fish showed greater CAT levels. Complex interactions among these factors affected the antioxidant levels in T. zillii. In summary, our study suggests that rearing T. zillii in green tanks at higher densities (30 fish/m3) and in mono-sex conditions yields the best results in terms of growth and overall performance.

Bile acids as putative social signals in Mozambique tilapia (Oreochromis mossambicus).

Chemical cues provide potential mates with information about reproductive status and resource-holding potential. In the Mozambique tilapia (Oreochromis mossambicus), males can distinguish female reproductive status through chemical cues, and accessibility of males to females depends on their position in the hierarchy, determined in part by chemical cues. Here, we hypothesized that tilapia faecal cues are attractive to conspecifics once released into the water. C18 solid-phase extracts of faeces from dominant males and pre-ovulatory females evoked stronger olfactory epithelium electrical responses (EOG) than, respectively, subordinate males and post-spawning females. Mass spectrometry of the reverse-phase C18 high-performance liquid chromatography fractions of these extracts with highest EOG, identified by amino acids and bile acids. Faeces from pre-ovulatory females contain significantly higher concentrations of cholic acid (CA) and taurocholic acid (TCH) than both post-spawning females and males. A pool of amino acids had no effect on aggression or attraction in males. However, males were attracted to the scent of pre-ovulatory female faeces, as well as CA and TCH, when applied separately. This attraction was accompanied by increased digging behaviour compared to the odour of post-spawning females. CA and TCH exert their action through separate receptor mechanisms. These findings are consistent with a role for faeces - and bile acids therein - in chemical communication in this species, acting as an attractant for males to reproductive females.

Growth acceleration of Nile tilapia at 21 to 31 weeks of age with plasma-treated air-supplied water.

This study aimed to develop a technique to accelerate fish growth without using genetic modification or genome editing. We have prepared a reactor with four pairs of opposed electrodes and a high-voltage power supply for the discharge. An arc discharge generates a plasma-treated gas in the reactor. Plasma-treated gas containing active species such as nitric oxide (NO) was generated via an arc discharge in the atmosphere and inserted into an aquarium containing Nile tilapia. No ozone was detected in the plasma-treated gas. Plasma treatment gas was supplied to the 20 L tank at a flow rate of 10 L per minute for varying supply times. The supply duration of plasma-treated air to the water tank was 0.5, 2, 5, and 15 min. Tanks were prepared for each of these four conditions, and gas was supplied daily at the same time. We observed that on supplying plasma-treated gas to tilapia from the 16th week of age for 5 min daily, the average length of the fish at 31 weeks of age was ∼1.5 times longer than that of the control fish. All other supply time conditions were also found to grow acceleration over the control. In the 15-minute supply time condition, individual differences in body length were more significant. A sample had more growth suppression than controls. In other words, the results suggest that an excess supply of active species can cause growth inhibition. These results suggest that an optimal supply of plasma-treated gas has a growth-promoting effect on fish.Key policy highlightsThe fish growth was accelerated by supplying plasma-treated air to the tank.The amount of ozone in the plasma-treated air was below the detection limit, and a large amount of RNS, such as nitric oxide, was generated.After an experimental period of 16 to 31 weeks, the average length of fish in the most significant growth condition was 1.5 times that of the control fish.

Melanin-concentrating hormone interferes with the hypothalamic-pituitary-gonad axis in the Mozambique tilapia.

This study was conducted to elucidate the influence of melanin-concentrating hormone (MCH) along the reproductive-axis in the female tilapia Oreochromis mossambicus. Administration of MCH (4 µg / 0.1 ml saline) for 22 days resulted in significantly lower gonadosomatic index compared to controls. Significant reduction in the mean numbers of follicles at different stages of development such as previtellogenic (stages I-III), vitellogenic (stage IV) and preovulatory (stage V) follicles was observed in MCH-treated fish compared with controls. On the other hand, the rate of atresia was significantly higher in follicles at stages II, III and IV in MCH-treated fish. In addition, in the pituitary gland, sparsely labelled gonadotropin releasing hormone (GnRH)-immunoreactive fibres were observed in MCH-treated fish in contrast to their intense labelling in controls. The serum level of luteinizing hormone (LH) showed significant decrease, but the serum cortisol level rose significantly following MCH treatment compared to those of controls. Collectively, these results indicate for the first time, that MCH treatment blocks follicular development during the ovarian cycle, possibly through the suppression of GnRH-LH axis in fish. The results also indicate that MCH may activate the stress-axis pathway in fish.

Influence of refrigeration and freezing in Microcystins and Cylindrospermopsin concentrations on fish muscle of tilapia (Oreochromis niloticus) and tench (Tinca tinca).

The consumption of fish contaminated with cyanotoxins is an important public health issue due to their potential adverse effects. The aim of this study was to assess the influence of refrigeration (4 °C) and freezing (-20 °C) on the concentration of Cylindrospermopsin (CYN), Microcystins (MCs) and their combination in tilapia (Oreochromis niloticus) and tench (Tinca tinca). Fish muscle were spiked with a stock solution of each toxin to reach 750 µg/g dry weight (d.w.). Three different periods of time were investigated for each treatment: 24 h, 48 h and 7 days for refrigeration, and 24 h, 7 days and 1 month for freezing. Samples were extracted and quantified by Ultra Performance Liquid Chromatography - Tandem Mass Spectrometry (UPLC-MS/MS). The results showed that freezing for 1 month produced highest decreases of these toxins in both species in comparison to refrigeration, being CYN the most stable cyanotoxin. Moreover, MCs are more stable to storage processes in the mixtures than alone, and fish species is a factor to take into account in their stability. These findings highlight the need to assess the influence of food storage processes on the presence of cyanotoxins in fish species for a more realistic human health risk assessment.

Regulation of thyroid hormones and branchial iodothyronine deiodinases during freshwater acclimation in tilapia.

Euryhaline fishes are capable of maintaining osmotic homeostasis in a wide range of environmental salinities. Several pleiotropic hormones, including prolactin, growth hormone, and thyroid hormones (THs) are mediators of salinity acclimation. It is unclear, however, the extent to which THs and the pituitary-thyroid axis promote the adaptive responses of key osmoregulatory organs to freshwater (FW) environments. In the current study, we characterized circulating thyroxine (T4) and 3-3'-5-triiodothyronine (T3) levels in parallel with the outer ring deiodination (ORD) activities of deiodinases (dios) and mRNA expression of dio1, dio2, and dio3 in gill during the acclimation of Mozambique tilapia (Oreochromis mossambicus) to FW. Tilapia transferred from seawater (SW) to FW exhibited reduced plasma T4 and T3 levels at 6 h. These reductions coincided with an increase in branchial dio2-like activity and decreased branchial dio1 gene expression. To assess whether dios respond to osmotic conditions and/or systemic signals, gill filaments were exposed to osmolalities ranging from 280 to 450 mOsm/kg in an in vitro incubation system. Gene expression of branchial dio1, dio2, and dio3 was not directly affected by extracellular osmotic conditions. Lastly, we observed that dio1 and dio2 expression was stimulated by thyroid-stimulating hormone in hypophysectomized tilapia, suggesting that branchial TH metabolism is regulated by systemic signals. Our collective findings suggest that THs are involved in the FW acclimation of Mozambique tilapia through their interactions with branchial deiodinases that modulate their activities in a key osmoregulatory organ.

The impact of acute cold water stress on blood parameters, mortality rate and stress-related genes in Oreochromis niloticus, Oreochromis mossambicus and their hybrids.

This study aims to evaluated the response of Oreochromissp. to cold stress. Two experiments were conducted involving a total of 1080 juvenile Oreochromis niloticus, O. mossambicus, O. niloticus♂ × O.mossambicus♀=F1♂ × O.mossambicus ♀ (Hybrid 1; H1) and O. mossambicus♂ × O. niloticus♀ (Hybrid 2; H2). In the 1st experiment, fish were exposed to cold water (12 °C) for 24 h and then hematological parameters, serum biochemical variables, innate immune responses, antioxidant status, and liver gene expression responses (hsp70, hsp27, hsp90, hsp40, cat, sod, eef1a1 and calreticulin) were analyzed. Hematological and serum biochemical responses involved species-specific differences. At optimal temperatures (28 °C), respiratory burts activity (RBA) and myeloperoxidase (MPO) values of H1, H2 and O. mossambicus were significantly higher than O. niloticus (p< 0.05). While the RBAvalue of O. mossambicus decreased after exposure to cold water (p< 0.05), lysozyme activities of O. niloticus and H2 and MPO activities of all experimental fish increased significantly (p< 0.05). At control conditions (28 °C), cortisol levels were found to be higher in O. mossambicus than in H1 and O. niloticus (p< 0.05). A significant increase in cat and sod transcripts was observed in liver of fish being very pronounced in O. mossambicus and H2. The highest up-regulation was observed for hsp70 target where the lowest but significant up-regulation was observed for hsp90 gene. In 2nd experiment, water temperature was gradually decrease from 28 °C to 12 °C (average, 1 °C/1 h).Survival rates of H1 and H2 were found to be different compared to O. mossambicus and O. niloticus (p< 0.05) after 20 days of cold water challenge.O. mossambicus was the most cold-sensitive group, followed by the H2, H1 and O. niloticus. Our data should be carfully considered in view of the possible physiological and anti-stressor responses being species-specific in fish.

Enhanced expression of ncc1 and clc2c in the kidney and urinary bladder accompanies freshwater acclimation in Mozambique tilapia.

Euryhaline fishes maintain hydromineral balance in a broad range of environmental salinities via the activities of multiple osmoregulatory organs, namely the gill, gastrointestinal tract, skin, kidney, and urinary bladder. Teleosts residing in freshwater (FW) environments are faced with the diffusive loss of ions and the osmotic gain of water, and, therefore, the kidney and urinary bladder reabsorb Na+ and Cl- to support the production of dilute urine. Nonetheless, the regulated pathways for Na+ and Cl- transport by euryhaline fishes, especially in the urinary bladder, have not been fully resolved. Here, we first investigated the ultrastructure of epithelial cells within the urinary bladder of FW-acclimated Mozambique tilapia (Oreochromis mossambicus) by electron microscopy. We then investigated whether tilapia employ Na+/Cl- cotransporter 1 (Ncc1) and Clc family Cl- channel 2c (Clc2c) for the reabsorption of Na+ and Cl- by the kidney and urinary bladder. We hypothesized that levels of their associated gene transcripts vary inversely with environmental salinity. In whole kidney and urinary bladder homogenates, ncc1 and clc2c mRNA levels were markedly higher in steady-state FW- versus SW (seawater)-acclimated tilapia. Following transfer from SW to FW, ncc1 and clc2c in both the kidney and urinary bladder were elevated within 48 h. A concomitant increase in branchial ncc2, and decreases in Na+/K+/2Cl-cotransporter 1a (nkcc1a) and cystic fibrosis transmembrane regulator 1 (cftr1) levels indicated a transition from Na+ and Cl- secretion to absorption by the gills in parallel with the identified renal and urinary bladder responses to FW transfer. Our findings suggest that Ncc1 and Clc2c contribute to the functional plasticity of the kidney and urinary bladder in tilapia.

Acute social defeat stress upregulates gonadotrophin inhibitory hormone and its receptor but not corticotropin-releasing hormone and ACTH in the Male Nile Tilapia (Oreochromis niloticus).

Stress impairs the hypothalamic-pituitary-gonadal (HPG) axis, probably through its influence on the hypothalamic-pituitary-adrenal (= interrenals in the teleost, HPI) axis leading to reproductive failures. In this study, we investigated the response of hypothalamic neuropeptides, gonadotropin-inhibitory hormone (GnIH), a component of the HPG axis, and corticotropin-releasing hormone (CRH) a component of the HPI axis, to acute social defeat stress in the socially hierarchical male Nile tilapia (Oreochromis niloticus). Localization of GnIH cell bodies, GnIH neuronal processes, and numbers of GnIH cells in the brain during acute social defeat stress was studied using immunohistochemistry. Furthermore, mRNA levels of GnIH and CRH in the brain together with GnIH receptor, gpr147, and adrenocorticotropic hormone (ACTH) in the pituitary were quantified in control and socially defeated fish. Our results show, the number of GnIH-immunoreactive cell bodies and GnIH mRNA levels in the brain and the levels of gpr147 mRNA in the pituitary significantly increased in socially defeated fish. However, CRH and ACTH mRNA levels did not change during social defeat stress. Further, we found glucocorticoid type 2b receptor mRNA in laser captured immunostained GnIH cells. These results show that acute social defeat stress activates GnIH biosynthesis through glucocorticoid receptors type 2b signalling but does not change the CRH and ACTH mRNA expression in the tilapia, which could lead to temporary reproductive dysfunction.

Microalgae-blend tilapia feed eliminates fishmeal and fish oil, improves growth, and is cost viable.

Aquafeed manufacturers have reduced, but not fully eliminated, fishmeal and fish oil and are seeking cost competitive replacements. We combined two commercially available microalgae, to produce a high-performing fish-free feed for Nile tilapia (Oreochromis niloticus)-the world's second largest group of farmed fish. We substituted protein-rich defatted biomass of Nannochloropsis oculata (leftover after oil extraction for nutraceuticals) for fishmeal and whole cells of docosahexaenoic acid (DHA)-rich Schizochytrium sp. as substitute for fish oil. We found significantly better (p < 0.05) growth, weight gain, specific growth rate, and best (but not significantly different) feed conversion ratio using the fish-free feed compared with the reference diet. Fish-free feed also yielded higher (p < 0.05) fillet lipid, DHA, and protein content (but not significantly different). Furthermore, fish-free feed had the highest degree of in-vitro protein hydrolysis and protein digestibility. The median economic conversion ratio of the fish-free feed ($0.95/kg tilapia) was less than the reference diet ($1.03/kg tilapia), though the median feed cost ($0.68/kg feed) was slightly greater than that of the reference feed ($0.64/kg feed) (p < 0.05). Our work is a step toward eliminating reliance on fishmeal and fish oil with evidence of a cost-competitive microalgae-based tilapia feed that improves growth metrics and the nutritional quality of farmed fish.

Spatial distribution, source identification, and risk assessment of organochlorines in wild tilapia from Guangxi, South China.

Seventy-five wild tilapia samples from six rivers (ten sites) in Guangxi province were collected and analyzed for 53 organochlorine compounds. DDTs, endosulfan, and PCBs were the most dominant compounds found in this study. Tiandong County (TD) and Guigang City (GG) sites were found to be heavily contaminated with high levels of endosulfan (385-925 ng/g lw) and/or DDTs (20.1-422 ng/g lw). The diagnostic ratios indicated that the residues of DDTs and endosulfan in wild tilapia are associated with historical applications as well as the recent introduction of technical DDTs and endosulfan at some sampling sites. The correlation between total length, body mass, and organochlorines (OCs) was higher than the correlation between age and lipid content. There was no significant correlation between organochlorine pesticides (OCPs) and lipid content. Therefore, for organisms, the feeding intensity (related to length and mass) of fish could better reflect degree of pollution than exposure time (age) of fish. The hazardous ratios for the 50th and 95th percentile data of OCPs and PCBs in fish were both below 1, suggesting that daily exposure to OCPs and PCBs yields a lifetime cancer risk lower than 1 in 10,000.

Regulation of Female Folliculogenesis by Tsp1a in Nile Tilapia (Oreochromis niloticus).

TSP1 was reported to be involved in multiple biological processes including the activation of TGF-ß signaling pathways and the regulation of angiogenesis during wound repair and tumor growth, while its role in ovarian folliculogenesis remains to be elucidated. In the present study, Tsp1a was found to be expressed in the oogonia and granulosa cells of phase I to phase IV follicles in the ovaries of Nile tilapia by immunofluorescence. tsp1a homozygous mutants were generated by CRISPR/Cas9. Mutation of tsp1a resulted in increased oogonia, reduced secondary growth follicles and delayed ovary development. Expression of the cell proliferation marker PCNA was significantly up-regulated in the oogonia of the mutant ovaries. Furthermore, transcriptomic analysis revealed that expressions of DNA replication related genes were significantly up-regulated, while cAMP and MAPK signaling pathway genes which inhibit cell proliferation and promote cell differentiation were significantly down-regulated. In addition, aromatase (Cyp19a1a) expression and serum 17ß-estradiol (E2) concentration were significantly decreased in the mutants. These results indicated that lacking tsp1a resulted in increased proliferation and inhibited differentiation of oogonia, which in turn, resulted in increased oogonia, reduced secondary growth follicles and decreased E2. Taken together, our results indicated that tsp1a was essential for ovarian folliculogenesis in Nile tilapia.

An osmolality/salinity-responsive enhancer 1 (OSRE1) in intron 1 promotes salinity induction of tilapia glutamine synthetase.

Euryhaline tilapia (Oreochromis mossambicus) are fish that tolerate a wide salinity range from fresh water to > 3× seawater. Even though the physiological effector mechanisms of osmoregulation that maintain plasma homeostasis in fresh water and seawater fish are well known, the corresponding molecular mechanisms that control switching between hyper- (fresh water) and hypo-osmoregulation (seawater) remain mostly elusive. In this study we show that hyperosmotic induction of glutamine synthetase represents a prominent part of this switch. Proteomics analysis of the O. mossambicus OmB cell line revealed that glutamine synthetase is transcriptionally regulated by hyperosmolality. Therefore, the 5' regulatory sequence of O. mossambicus glutamine synthetase was investigated. Using an enhancer trapping assay, we discovered a novel osmosensitive mechanism by which intron 1 positively mediates glutamine synthetase transcription. Intron 1 includes a single, functional copy of an osmoresponsive element, osmolality/salinity-responsive enhancer 1 (OSRE1). Unlike for conventional enhancers, the hyperosmotic induction of glutamine synthetase by intron 1 is position dependent. But irrespective of intron 1 position, OSRE1 deletion from intron 1 abolishes hyperosmotic enhancer activity. These findings indicate that proper intron 1 positioning and the presence of an OSRE1 in intron 1 are required for precise enhancement of hyperosmotic glutamine synthetase expression.

Deleterious impacts of heat stress on steroidogenesis markers, immunity status and ovarian tissue of Nile tilapia (Oreochromis niloticus).

The water temperature of aquacultures is a primary factor of fish welfare, reproductive patterns, and immunity. To elucidate the molecular and biological processes of the temperature modulation of reproduction and immunity, female Nile tilapia (190 ± 10g) were allocated into five groups following acclimatization (150 females, three replicates, each n = 10). Each group was subjected to various temperatures (28 °C, 30 °C, 32 °C, 34 °C, and 37 °C), the group at 28 °C representing the control. Their serum levels of estradiol, cortisol, and vitellogenin were measured as well as serum triiodothyronine (T3) hormone, thyroxine (T4) hormone, and non-specific immunity (phagocytic and lysozyme activity). In addition, steroidogenic acute regulatory protein (STAR), vitellogenin gene receptor, and heat shock protein 70 (HSP70) gene expression were evaluated. The serum levels of estradiol, cortisol, and vitellogenin markedly declined (P < 0.05) in fish group at higher temperatures. In addition to T3, T4 was significantly affected (P < 0.05) in the control group. The expressions of the STAR gene (steroidogenesis) and vitellogenin receptors were also considerably down-regulated. The histopathological photomicrograph of fish subjected to high water temperature revealed injuries in ovary tissues, demonstrating its harmful effects. The experimental results verified the possible role of water temperature as a main stressor on Nile tilapia' physiology through modulation of steroidogenesis-related gene expression and immunity.

Physiological impact and comparison of mutant screening methods in piwil2 KO founder Nile tilapia produced by CRISPR/Cas9 system.

The application of genome engineering techniques to understand the mechanisms that regulate germ cell development opens promising new avenues to develop methods to control sexual maturation and mitigate associated detrimental effects in fish. In this study, the functional role of piwil2 in primordial germ cells (PGCs) was investigated in Nile tilapia using CRISPR/Cas9 and the resultant genotypes were further explored. piwil2 is a gonad-specific and maternally deposited gene in Nile tilapia eggs which is known to play a role in repression of transposon elements and is therefore thought to be important for maintaining germline cell fate. A functional domain of piwil2, PIWI domain, was targeted by injecting Cas9 mRNA and sgRNAs into Nile tilapia embryos at 1 cell stage. Results showed 54% of injected mutant larvae had no or less putative PGCs compared to control fish, suggesting an essential role of piwil2 in survival of PGCs. The genotypic features of the different phenotypic groups were explored by next generation sequencing (NGS) and other mutant screening methods including T7 endonuclease 1 (T7E1), CRISPR/Cas-derived RNA-guided engineered nuclease (RGEN), high resolution melt curve analysis (HRMA) and fragment analysis. Linking phenotypes to genotypes in F0 was hindered by the complex mosacism and wide indel spectrum revealed by NGS and fragment analysis. This study strongly suggests the functional importance of piwil2 in PGCs survival. Further studies should focus on reducing mosaicism when using CRISPR/Cas9 system to facilitate direct functional analysis in F0.

Rising to the challenge? Inter-individual variation of the androgen response to social interactions in cichlid fish.

The Challenge Hypothesis (Wingfield et al. Am. Nat. 136, 829-846) aims to explain the complex relationship between androgens and social interactions. Despite its well acceptance in the behavioral endocrinology literature, several studies have failed to found an androgen response to staged social interactions. Possible reasons for these inconsistencies are the use of single sampling points that may miss the response peak, and the occurrence of inter-individual variability in the androgen response to social interactions. In this study we addressed these two possible confounding factors by characterizing the temporal pattern of the androgen response to social interactions in the African cichlid, Oreochromis mossambicus, and relating it to inter-individual variation in terms of the individual scope for androgen response (i.e. the difference between baseline and maximum physiological levels for each fish) and behavioral types. We found that the androgen response to territorial intrusions varies between individuals and is related to their scope for response. Individuals that have a lower scope for androgen response did not increase androgens after a territorial intrusion but were more aggressive and exploratory. In contrast males with a higher scope for response had fewer aggressive and exploratory behaviors and exhibited two peaks of KT, an early response 2-15 min after the interaction and a late response at 60-90 min post-interaction. Given that the pharmacological challenge of the Hypothalamic-Pituitary-Gonad axis only elicits the late response, we suggest that these two peaks may be regulated by different physiological mechanisms, with the early response being mediated by direct brain-gonad neural pathways. In summary, we suggest that determining the temporal pattern of the androgen response to social interactions and considering inter-individual variation may be the key to understanding the contradictory results of the Challenge Hypothesis.

Physiological and gut microbiome changes associated with low dietary protein level in genetically improved farmed tilapia (GIFT, Oreochromis niloticus) determined by 16S rRNA sequence analysis.

The aim of this study was to determine the effects of different dietary protein levels on the growth, physiological parameters, and gut microbiome of genetically improved farmed tilapia (GIFT, Oreochromis niloticus). Two pellet feed diets with low (25%, LPD) and normal (35%, NPD) protein levels were fed to GIFT in aquaria at 28°C for 8 weeks. The LPD reduced trypsin activity and inhibited the growth of GIFT. The serum alanine amino transferase and aspartate transaminase activities, hepatic malondialdehyde content, and superoxide dismutase, glutathione peroxidase, and catalase activities were significantly higher in LPD GIFT than in NPD GIFT (p < .05). The LPD led to decreased lysozyme activity and increased levels of C3 (p < .05). A 16S rRNA gene profiling analysis showed that the LPD significantly affected the gut microbial composition. Compared with the NPD, the LPD significantly decreased intestinal microbial diversity (p < .05). The macronutrient distribution affected the taxonomic profile of gut bacteria, mainly the phyla Bacteroidetes, Proteobacteria, and Firmicutes. The LPD favored growth of the genus Bacteroides. The NPD appeared to increase the abundance of the genera Lawsonia, Romboutsia, and Sphingomonas. Our results showed that, compared with NPD GIFT, the LPD GIFT had weakened nonspecific immune function, altered microbial community structure, and decreased gut microbial diversity.

The opioid peptide dynorphin suppresses pituitary-ovary axis in the tilapia Oreochromis mossambicus.

The opioid peptides are involved in the regulation of neuroendocrine functions in vertebrates. Nonetheless, the influence of an opioid peptide, dynorphin A (DYN), on reproduction in fish is understudied. The aim of this work was to study the influence of DYN on the pituitary-ovary axis in Oreochromis mossambicus. Daily injections (ip) of 250 µg DYN kg-1 body weight for 22 days during the ovarian cycle caused a reduction in the intensity and the per cent area of luteinizing hormone (LH) immunoreactive content in the proximal pars distalis region of the pituitary gland compared with an intense immunostaining in time-matched controls. In the ovary, DYN treatment caused a decrease in the number of stage I (previtellogenic) follicles compared with time-matched controls. No difference was observed in the number of stage IV (vitellogenic) follicles among different experimental groups, whereas the numbers of stage II and stage III follicles (previtellogenic) were higher in DYN-treated fish than in time-matched controls. Nonetheless, there was a reduction in the number of stage V (preovulatory) follicles in DYN-treated fish compared with time-matched controls. Taken together, these results indicate that DYN exerts an inhibitory effect on follicular recruitment at the late vitellogenic stage, through the suppression of LH secretion in fish.