Abstract:MyomiRs, a class of muscle-specific microRNAs (miRNAs), play an important role in the proliferation and differentiation of myoblasts. This study investigated the spatio-temporal expression characteristics of four myomiRs (miR-1a,miR-133a-3p, miR-206, and miR-499) in Chinese perch (Siniperca chuatsi), as well as their expression features under short-term starvation stress, and predicted their regulation of Pax7. The expressions of the four myomiRs in various tissues of Chinese perch, induding white muscle at different stages of post embryonic development and white muscle after 5 days of starvation, were examined by real-time fluorescence quantitative PCR. Then, the targeting sites of the four myomiRs on the 3′-untranslated region (UTR) of Pax7 mRNA were predicted using RNAhybrid. The results showed that miR-1a,miR-133a-3p, and miR-206 were highly expressed at D60 (60 days post hatching), while miR-499 was highly expressed at D100. Furthermore, miR-1a andmiR-133a-3p were highly expressed in red muscle, white muscle, and the heart, yet low expressed in other tissues. miR-206 was highly expressed in red muscle and white muscle, while miR-499 was highly expressed in heart, followed by red muscle and white muscle, and was low expressed in other tissues without a significant difference observed. The expressions of all four myomiRs increased significantly after 5 days of starvation, suggesting that skeletal muscle may respond to starvation stress by upregulating the expression of the four myomiRs to regulate physiological processes such as metabolism. Target gene prediction analysis showed that miR-1a,miR-133a-3p, and miR-206 had binding sites on the 3′-UTR of Pax7. Further verification by real-time fluorescence quantitative PCR showed that the expression of miR-206 and Pax7 at various post embryonic developmental stages and the circadian rhythms of the Chinese perch were in opposite trends. These results suggest that miR-1a andmiR-133a-3p are muscle-specific, while miR-206 and miR-499 are enriched in muscle-derived tissues. In addition, miR-206 may directly regulate the expression of Pax7, thus affecting the differentiation and development of muscle tissue in juvenile Chinese perch.

Abstract:China has abundant saline-alkaline water resources, mainly including the North China coastal composite type, Northwest sulfate type, and Northeast carbonate type, which differ in their ion composition, being mainly composed of Cl^– , SO₄ ^2–, and HCO₃ ^– . The toxic effects of saline-alkaline water on aquatic organisms are mainly caused by factors such as high ionic concentration, osmotic effect, and high pH. Simulating the saline-alkaline habitat in the laboratory has practical and guiding significance for the rational development of corresponding taming and screening schemes based on the toxic effects of different saline-alkaline water types. To investigate the toxic effects of different salts on mandarin fish (Siniperca chuatsi), acute stress tests were conducted with NaCl, Na₂SO₄, and NaHCO₃ at equal cation concentrations (Na⁺ =210 mmol/L). Blood and gill tissues were collected at 0, 12, and 24 hours after the NaCl and Na₂SO₄ stress tests, and at 24 and 72 hours after freshwater recovery. Blood and gill tissues were also collected at 0.5 hours after the NaHCO₃ stress test and at 2 and 4 hours after freshwater recovery. The serum osmotic pressure, electrolyte concentrations, and enzyme activities of Na⁺ /K⁺ -ATPase (NKA), Na⁺ /K⁺ /2Cl^– cotransporter (NKCC), Na⁺ /HCO₃ ^– cotransporter (SLC4A4), and Cl^– /HCO₃ ^– ion exchanger (SLC26A6) were analyzed. The expression levels of immune-related genes (Ccl20, γ-IFN, Dapk2) were also examined. The results showed that under 210 mmol/L Na⁺ stress, the Na⁺ concentration in the serum of the mandarin fish fluctuated significantly in all the three salt stress groups, while the changes in Cl^– and K⁺ concentration were weak. The NKA enzyme activity in gill tissues was upregulated, and the NKCC and SLC26A6 enzyme activities were inhibited. Further, γ-IFN and Dapk2 were downregulated. Unlike the fish in the NaCl and Na₂SO₄ stress test groups, the mandarin fish had a stronger stress response in the NaHCO₃ stress test group, and the serum osmotic pressure could not be restored after transferring to freshwater. The SLC4A4 enzyme activity was inhibited, and the expression levels of Ccl20 were downregulated. Therefore, the NaHCO₃ stress had a stronger toxic effect on mandarin fish than either NaCl or Na₂SO₄ stress. NaHCO₃ stress inhibited immune function and significantly inhibited the activity of most ion transporters, leading to the failure of serum osmotic regulation after being transferred to fresh water, while the osmotic regulation was functional under NaCl and Na₂SO₄ stress. The research results have practical significance for the rational development of taming and screening schemes of mandarin fish for different saline-alkaline water types.

Abstract:The aim of this study was to identify functional genes associated with the oviposition of Mytilus galloprovincialis and to elucidate the underlying mechanisms of oviposition. Transcriptomic analysis of female Mytilus galloprovincialis gonads was conducted before and after spawning. Pairwise comparisons of sequencing libraries from tissue samples were also performed. Before and after spawning, there were 4060 differential genes, with 1488 up-regulated and 2572 down-regulated genes. The GO functional enrichment terms are grouped into three functional categories: biological process, cellular component, and molecular function. Some genes were annotated by GO terms such as growth, developmental process, reproduction, and reproductive process. KEGG enrichment analysis revealed signaling pathways associated with oviposition and reproduction, including the TCA cycle, GnRH signaling pathway, ovarian steroidogenesis, estrogen signaling pathway, oxytocin signaling pathway, and steroid biosynthesis. In conjunction with GO and KEGG enrichment results, we identified five potential genes associated with reproduction: gonadotropin-releasing hormone receptor (GnRHR), luteinizing hormone β (LHβ), matrix metalloproteinase 14 (MMP14), cytochrome P4503A enzyme (CYP3A), and cytochrome P45017A enzyme (CYP17A). The expression levels of LHβ and MMP14 genes increased, whereas the expression levels of GnRHR, CYP3A, and CYP17A genes decreased. These findings were validated using real-time quantitative PCR (RT-qPCR). The differential expression of the five genes mentioned above is crucial in regulating the synthesis of sex hormones and reproductive process during the spawning of Mytilus galloprovincialis, which is consistent with the results of transcriptome sequencing. In this study, we utilized transcriptome sequencing technology to analyze the differences in the gonad transcriptome of female Mytilus galloprovincialis before and after spawning. The aim was to identify the key pathways and candidate genes involved in spawning, which could provide valuable data for the breeding of Mytilus galloprovincialis.

Abstract:Asynchrony among the various biological clock systems is considered one of the primary reasons for the occurrence and development of adverse diseases such as physiological and metabolic disorders. Changes in feeding frequency can affect the expression rhythms of the biological clock system, thereby affecting biological growth and development. To investigate the effect of feeding frequency on the growth of Exopalaemon carinicauda and determine the appropriate feeding frequency for its cultivation, this study analyzed the rhythmic changes in biological clock gene expression in the eyestalk and liver of E. carinicauda under varying feeding regimens, evaluated the impact of different feeding frequencies on the synchronization of biological clock systems in E. carinicauda, and simultaneously verified growth and muscle composition changes through cultivation. The results showed that when the feeding frequency was less than 3 times per day, the expression rhythm of biological clock genes did not change in response to changes in feeding frequency. Similar expression cycles and peak phases were observed in both the eyestalk and liver, with no significant difference in their respective expression levels. However, when the feeding frequency increased to 4 times per day, there were significant changes in the expression rhythm in the liver, which manifested as changes in the expression cycle, phase shifts, and expression levels themselves. Notably, when the feeding frequency is 3 times per day, the synchronization of clock gene expression rhythm between the eyestalk and the liver is at its highest. Furthermore, the results also showed that the growth of E. carinicauda increased with feeding frequency from 1 to 3 times/d. Indeed, the specific growth rate (SGR) and weight gain rate (WGR) were (6.65±0.98)%/d and (199.46±5.42)%, respectively. Concurrently, as the feeding frequency increased, so did the crude protein and crude fat content in the muscle tissue, reaching a maximum in the treatment C with (19.98±0.04)% and (1.88±0.23)%, respectively. In summary, the feeding frequency has a significant impact on the expression rhythm of circadian clock genes in the liver of E. carinicauda, indicating its modulatory effect on resetting or regulating the peripheral circadian clock system.

Abstract:In recent years, many innovative achievements have been made in the artificial culture of Chinese perch Siniperca chuatsi, but many gaps exist in the study of the effects of light environment on behavior, physiology, and biochemistry. Herein, the effects of light intensity and spectrum on behavior, feeding and growth performance of Chinese perch were studied. The feeding rates at 10, 25, and 50 lx were significantly higher than those at 5, 300 and 500 lx (P＜0.05). The feeding ratio was the highest (P＜0.05), under green light conditions. The results showed that the growth rate and specific growth rate in the 10 lx group were significantly higher than those in other groups (P＜0.05), and the feed coefficient was significantly lower (P＜0.05). There was no significant improvement in growth performance at 200 or 500 lx (P＞0.05). In addition, the expression level of igf1 gene in muscle was significantly higher than that in other groups under 10 lx exposure; the appetite-promoting genes npy and agrp were significantly up-regulated (P＜0.05), and the appetite-suppressing genes cart and pomc were down-regulated. At 200 and 500 lx, the expression levels of npy and agrp were not significantly different from those in the control group (P＞0.05). At the same time, the expression levels of pomc at 500 lx were significantly higher than those at 10 and 200 lx (P＜0.05). In conclusion, the growth performance of Chinese perch was significantly improved under the green light, and the appetite-promoting factors including npy and agrp and their interactions with growth-related factors promoted feeding and growth under the green light stimulation.

Abstract:This study aimed to investigate the effects of feeding diets supplemented with Lactobacillus plantarum LP-37 and Pediococcus pentosaceus PP-23 on the growth and immune performance of Nile tilapia. Seven experimental groups were used in this study, namely, control group C0: feeding basic feed; L1-L3 groups: adding three different concentrations of Lactobacillus plantarum LP-37 (1.0×10⁶ CFU/g, 1.0×10⁷ CFU/g, 1.0×10⁸ CFU/g) to basic feed; P1-P3 groups: adding three different concentrations of Pediococcus pentosaceus PP-23 (1.0×10⁶ CFU/g, 1.0×10⁷ CFU/g, 1.0×10⁸ CFU/g) to basic feed. The results showed that except for those in the L1 group, the final body weights of the fish in the experimental groups (123.22~158.60 g) were significantly higher than those of the C0 group fish (118.16±4.88) g (P＜0.05). Except for those in the L1 and P1 groups, the weight gain rates in the other experimental groups (128.69~156.20 g) were significantly higher than that in the C0 group (10⁸.16±0.45)% (P＜0.05). The final body weight and weight gain rate of the fish in the L3 [(150.00±6.66) g, (152.69±3.51)%] and P2 [(153.24±5.36) g, (143.12±5.23)%] groups were significantly higher than those of the C0 group fish (P＜0.01). The addition of different concentrations of LP-37 and PP-23 was able to improve activities of three digestive enzymes of Nile tilapia. Among various groups, the greatest increases appeared in the L3 and P2 groups. In L3, protease (PRS) was (1.39±0.06) U/mg, α-amylase (AMS) was (55.20±3.39) U/dL, and alkline phosphatase (AKP) was (3.28±0.28) U/L. In P2, PRS was (1.40±0.04) U/mg, α-amylase (AMS) was (65.28±11.50) U/dL, and alkaline phosphatase (AKP) was (3.50±0.36) U/L. Compared with the expression in the C0 group, IL-1β and IFN-γ genes in the spleen of the fish in the experimental groups were significantly down-regulated (P＜0.05), and the highest down-regulation occurred in the L3 and P2 groups. IL-1β gene in the head kidney of fish of various experimental groups was also significantly down-regulated (P＜0.05), and the highest down-regulation occurred in the L3 and P2 groups. In terms of intestinal tissue morphology, the villous height in various experimental groups was significantly higher than that in the C0 group (P＜0.05). The crypt depth, the ratio of villous height to crypt depth, and muscle layer thickness in the L3 and P2 groups were significantly higher than those in the other experimental and control groups (P＜0.05), whereas there was no significant difference in these parameters between the other experimental and control groups (P＞0.05). In summary, adding 1.0×10⁸ CFU/g Lactobacillus plantarum LP-37 and (1.0×10⁷ ) CFU/g Pediococcus pentosaceus PP-23 to the diet can significantly increase the growth rate, digestive enzyme activity, and intestinal tissue structure, and alter the expression of certain immune related genes in Nile tilapia.

Abstract:The integrated rice-fish farming mode is an environment-friendly ecological cycle agricultural production mode, which effectively improves the utilization rate of land and water resources. Stocking density is an important factor affecting fish growth. Excessive stocking density has been found to cause eutrophication and deterioration of the water quality, leading to abnormal physiological functions in fish. It also leads to competition among cultured fish for feed and living space, which inhibits growth performance and immune function, and increases the risk of disease outbreaks. However, it remains unknown whether high stocking density affects the antioxidant status, nutritional composition, and metabolic function in the muscle of Micropterus salmoides during rice-fish integrated farming. Therefore, the aim of the study was to investigate the effects of stocking density on the antioxidant capacity, muscle nutritional composition and metabolic functions of Micropterus salmoides in integrated rice-bass farming systems. The largemouth bass with an initial body weight of 40.63±0.13 g were reared at low density (L, 40 g/m³ ) and high density (H, 120 g/m³ ) for 90 days. After culturing, muscle tissue was collected to determine biochemical indexes and perform metabolome and transcriptome sequencing. The results showed that muscle total antioxidant capacity (T-AOC) in the H group was significantly lower than that in the L group. The catalase (CAT) activity decreased and malondialdehyde (MDA) content increased, but the differences were not significant, indicating that a high stocking density caused moderate oxidative stress, and its effects may be within the tolerance range of muscle antioxidant systems. The content of alanine and histidine free amino acids decreased significantly, whereas the content of glycine, aspartic acid, glutamic acid, and umami amino acids increased significantly. With respect to fatty acids, the content of ∑SFA, ∑MUFA, ∑PUFA, and n-6 PUFA increased significantly, whereas the ratio of n-3 PUFA to n-6 PUFA decreased. These results indicate that the nutritional quality of largemouth bass muscle decreased under high density, and the flavor was affected. Metabolomic results showed that 186 significantly different metabolites were detected between the L and H groups, including 136 up-regulated and 50 down-regulated metabolites. These metabolites were mainly related to the biosynthesis of unsaturated fatty acids, phenylalanine metabolism, nicotinate and nicotinamide metabolism, pentose and glucuronate interconversions, which indicated that high density stocking may cause metabolic disorders. Transcriptomic results showed that 688 significantly differentially expressed genes (DEGs) were detected between the L and H groups, including 508 up-regulated and 180 down-regulated genes, which were mainly enriched in cellular processes, metabolic processes, and protein digestion and absorption. Meanwhile, the key signaling pathways, including ECM-receptor interaction and PI3K-Akt, were up-regulated in the H group. In conclusion, high stocking density in integrated rice-bass farming systems affected antioxidant capacity, muscle nutrition and flavor and metabolic functions of lipids, carbohydrates, and proteins in M. salmoides. Additionally, M. salmoides could adapt to environmental stress by metabolic function regulation and intracellular signaling pathway activation. The results of this study provide a new premise for the molecular mechanism for the response of farmed fish to environmental stress in rice-fish integrated farming system.

Abstract:Dam construction alters natural river flows and hydrological regimes, including water temperature regimes, and this is a key environmental factor influencing fish reproduction. The aim of this study was to investigate the distribution and replenishment mechanism of the early population of silver cap, Hypophthalmichthys molitrix under the influence of the cascade hydropower station on Jinsha River Dam. Based on the early resource survey data of three sections [Yibin (YB), Luzhou (LZ) and Jiangjin (JJ)] in the upper Yangtze River reaches taken from May to July of 2017 to 2019, generalized additive models with Tweedie distribution were used to analyze the major environmental factors influencing their temporal and spatial patterns. The results revealed that the density of silver carp eggs showed a consistent trend from 2017 to 2019. Spatially, no H. molitrix egg was collected at YB, which was closest to the dam in the three river sections, whereas the density of H. molitrix eggs was highest at JJ, which was the farthest from the dam with the highest flow and water temperature and the lowest water transparency. Temporally, the density of fish eggs in June was significantly higher than that in May and July, suggesting that June was the peak breeding period of H. molitrix in the upper reaches of the Yangtze River. Generalized additive model analysis indicated that water discharge, distance from the dam, water transparency, and water temperature were the main environmental factors significantly affecting the spatial distribution of fish egg density with different relative contributions, together explaining more than 48.9% of total deviance observed (P＜0.05). The highest interpretation rate of the deviation of the flow was 29.4%, followed by 8.7% by the distance from the dam. Density distribution of H. molitrix egg were mainly in the range of water temperature 21–23 ℃, flow rate 6000‒14000 m³ /s, and transparency of less than 50 cm. Comprehensive analysis indicates that the low-temperature, low-flow, and high-transparency water released by the dam operation impact the natural reproduction of H. molitrix in the river section near the dam, while the degree of dam impact on fish reproduction is gradually weakened by increasing distance on the dam and the inflow of tributaries along the river, thus forming the longitudinal recovery gradients. The correlation analysis between spawning yield and hydrological indexes showed that the quantity of fish eggs was positively correlated with the duration of water rise and the increase of discharge (P＜0.05). The natural reproduction of H. molitrix in the upper reaches of the Yangtze River benefits when the duration of water rise lasts for more than four days and the cumulative increase of flow exceeds 5030 m³ /s. Additionally, the spawning activities of H. molitrix showed a tendency to migrate upstream in the post-dam of the Three Gorges Dam, and the upper reaches of the Yangtze River from Baisha Town to Rongshan Town were the main spawning grounds of H. molitrix. Hence, the river sections further away from the dam, especially the Chongqing-Luzhou section above the reservoir tail, are suggested to be protected as the key breeding area for the growth and sustenance of H. molitrix population.

Abstract:The slender top-mouth gudgeon Pseudorasbora elongata is a rare fish belonging to the family Cyprinidae. This species is endemic to China and distributed in restricted regions around the tributaries of the middle and lower Yangtze and Xijiang Rivers. P. elongata has suffered an increasing decline and has been vulnerable to extinction in the wild due to illegal and overfishing, water project construction, and other anthropogenic factors that destroyed their natural habitat and spawning grounds over the last few decades. Therefore, it is vital to assess the genetic diversity and structure of P. elongata for conserving and utilizing this fish effectively. In this study, we analyzed three P. elongata populations from Shanli (SL), Likou (LK), and Shitai (ST) in the Anhui section along the Yangtze River based on mitochondrial Cyt b gene and D-loop region sequences. The results showed that the total haplotypes defined by Cyt b gene and D-loop region in all populations were 18 and 27, respectively; the corresponding overall haplotype diversity (H_d) and nucleotide diversity (π) were 0.792 and 0.01332 and 0.777 and 0.01140, respectively. The data based on Cyt b gene and D-loop region showed that the genetic diversity of the ST population was lowest among those of the three populations. The genetic distances among the population were 0.00173–0.03615 (Cyt b) and 0.00193–0.02639 (D-loop). Obvious genetic differentiation and limited gene exchange were observed between the ST and SL and the ST and LK populations based on fixation indexes (F_st) and the gene flow value (N_m). Analysis of molecular variance (AMOVA) indicated that the source of variation between population (94.60%, 90.69%) was much higher than that within populations (5.40%, 9.31%). The maximum likelihood phylogenetic tree and haplotype network showed that the genetic distance between SL and LK populations was short and formed a branch, while the other branch only consisted of ST haplotypes. A neutral test and mismatch distribution analysis suggested that P. elongata has not undergone a recent population expansion. For ST population, neutral test Tajima’s D and Fu’s F_s values were negative (P＜0.05), and the mismatch distribution was represented by a single peak curve, which revealed that P. elongata populations in the ST area had possibly experienced a population expansion or bottleneck effect during evolution. Overall, P. elongata belongs to the category that includes populations with a high haplotype diversity index and low nucleotide diversity index, whereas obvious genetic differentiation among the three populations were observed with the ST population having the lowest genetic diversity. Thus, we recommend that ST populations should be regarded as a management protection unit to prioritize their conservation, while, SL and LK populations should be managed and protected as a whole. Our results could provide scientific evidence for the conservation, sustainable development, and commercial utilization of germplasm resources of P. elongata.

Abstract:In October 2022, an infectious disease outbreak was observed in farmed Portunus trituberculatus in Zhoushan, China. Samples of diseased crabs showing unspecific signs, such as anorexia, slow activity, and gill edema, were analyzed using parasitology, microbiology, histopathology, electron microscopy, and molecular identification. Parasitological and microbiological assays indicated that the diseased crabs were not infected with parasites or bacteria. The crab samples were tested by PCR methods recommended by World Organization for Animal Health and demonstrated to be free of Hematodinium, white spot syndrome virus (WSSV), mud crab discistrovirus-1 (MCDV-1), and Scylla serrata reovirus (SSRV). Histopathological examination revealed eosinophilic inclusions in hematopoietic tissue and hemocytes in gills, hepatopancreas, and the myocardial tissue of diseased crabs. Meanwhile, transmission electron microscopy showed that the virus in diseased crab tissues exhibited a typical icosahedral structure with a mean diameter of 150 nm, which was similar to the morphological characteristics of Decapoda iridescent virus 1 (DIV1). Nested PCR detection of DIV1 result showed that the first step of the PCR produced a 457 bp amplicon and the second step of the PCR produced a 129 bp amplicon. Phylogenetic analyses using gene sequences of major capsid protein (MCP) and ATPase revealed amplified sequences, DS.MCP201010 and DS.ATP202210 sequences, had the highest homology with the MCP gene and ATPase gene of DIV1, 100%. Therefore, it was determined that the pathogen of the disease was DIV1. In addition, specific probes directed to the MCP and ATPase gene of DIV1 were designed to obtain the distribution of virus particles in the hepatopancreas, heart, muscles, and gills of diseased crabs by fluorescence in situ hybridization, which was consistent with the results of electron microscopy and nested PCR. This study presents the first report of DIV1 infection in cultured Portunus trituberculatus, which will provide a strong reference for the prevention and control of the disease.