Rare Minnow Research Articles

Effects of Sublethal Exposure to Three Water Pollutants on Scototaxis in Rare Minnow (Gobiocypris rarus)

The biological early warning system with fish behavior as the detection index is an efficient and rapid early warning technology for the ecological damage caused by water pollutants. However, the attempt to apply the scototaxis (dark preference) behavior of fish to biological early warning is still relatively lacking. In this study, we delved into the dark and light preferences of the rare minnows (Gobiocypris rarus), employing three distinct tank configurations. Additionally, we systematically examined the modulating effects of environmental illumination, nutritional status, and the number of test subjects on this behavior, aiming to establish optimal experimental parameters for its observation. Furthermore, cadmium ions [Cd2+], tricaine methanesulfonate [MS222], and p-chloroaniline were employed as representative heavy metal ions, neuroactive agents, and organic toxicants, respectively, to test the impact of chemicals on scototaxis in gradient concentrations. The results demonstrated that the rare minnow exhibited a clear scototaxis (dark preference), and this behavior was not affected by the nutritional status of the test fish, the illumination, or the number of subjects. While the dark chamber was consistently the preferred location of rare minnows during the chemical exposure tests, the degree of scototaxis by the rare minnow significantly decreased at Cd2+ ≥ 3 mg/L, MS222 ≥ 11 mg/L, and p-chloroaniline ≥ 29 mg/L, suggesting a potential disruption of their innate behavioral patterns by these chemicals. These findings underscore the sensitivity of rare minnows to water pollutants. Therefore, the scototaxis behavior of rare minnows can be a potential and useful behavioral indicator for biological early warning, which can be used for early biological warning of sudden water pollution caused by chemicals such as Cd2+, MS222, and p-chloroaniline.

Escherichia coli heat-labile enterotoxin B subunit as an adjuvant of mucosal immune combined with GCRV-II VP6 triggers innate immunity and enhances adaptive immune responses following oral vaccination of grass carp (Ctenopharyngodon idella)

The grass carp reovirus (GCRV) is the most major pathogen that has threatened the grass carp (Ctenopharyngodon idella) industry of China for years. Though the oral vaccine has many advantages, the current vaccines still do not provide complete protection. Therefor the exploration of new preventive strategies is urgently needed. In this study, heat-labile enterotoxin B subunit of Escherichia coli (LTB) was combined with VP6 from GCRV type II (GCRV-II) via Lactococcus lactis expression system to form a potent oral vaccine and determines if fusion of LTB to the protective vaccine antigen can enhance protection in the fish. The expression of recombinant protein was confirmed by Western-blotting and enzyme-linked immunosorbent assay. The rare minnow was set as the model for the evaluation of the experiment administrated orally. The immune response including the antibody titer and the immune-related gene expression, and the protective efficacy which included the virus loaded and the relative protection, were thoroughly investigated after the trial. The results indicated that LTB can significantly elicit a higher neutralizing antibody responses and enhanced T-cell priming, activities and proliferation in mononuclear cells from intestine, spleen and kidney tissues when compared to the VP6 vaccine alone. Moreover, the combined adjuvant can significantly up-regulate type I interferon signaling in different immune organs, especially the mucosa associated lymphoidtissue which could not be induced by VP6 along, result in the contribution of the improvement in adaptive immune responses of the fish. In addition, challenge study showed that LTB combined VP6 could greatly improve the relative percent survival of the fish during the virus infection. These results highlight that LTB has the potential value to be a mucosal adjuvant of the fish, approaching for improving the efficacy of vaccination against GCRV-II, which does elicit both non-specific and specific immune responses.

Enhancing Cryopreserved Sperm Quality in Chinese Rare Minnow Gobiocypris rarus: The Impact of Antifreeze Proteins.

The Chinese rare minnow (Gobiocypris rarus), an important model fish in China, faces endangerment in the wild. Sperm cryopreservation facilitates the development of new strains and germplasm conservation, but the quality of its cryopreserved sperm remains low. This study evaluates the protective effects of different concentrations of antifreeze proteins (AFP I and AFP III) on the cryopreservation of Chinese rare minnow sperm. Cryopreserved sperm showed significant declines in progressive motility, curvilinear velocity (VCL), average path velocity (VAP), and lifespan compared to fresh sperm, except for straight-line velocity (VSL). The cryomedium containing 10 μg/mL AFP I improved these parameters to their highest levels. However, no significant difference was found in progressive motility and kinetic parameters between cryopreserved sperm with and without AFPs. Cryopreserved sperm with 10 μg/mL AFP I showed the highest plasma membrane integrity, mitochondrial activity, and DNA integrity, significantly better than without AFPs; importantly, the fertilization rate of cryopreserved sperm with 10 μg/mL AFP I was not significantly different from that of fresh sperm. These results indicate that the addition of 10 μg/mL AFP I to the cryomedium for Chinese rare minnow sperm does not improve kinetic parameters but significantly enhances sperm quality, aiding in its new strain development and germplasm conservation.

Zinc oxide nanoparticles cause hepatotoxicity in rare minnow (Gobiocypris rarus) via ROS-mediated oxidative stress and apoptosis activation and inhibition of lipid peroxidation

Zinc oxide nanoparticles (ZnO NPs) measuring 30 ± 10 nm in diameter are utilized in various applications, including batteries, plastics, ceramics, cosmetics, flame retardants, and food. However, their potential impact and risk on aquatic ecosystems remain unclear. This study examined the hepatotoxic effects of dietary ZnO NPs in rare minnow. Three hundred rare minnows were fed diets containing 0 mg/kg, 20 mg/kg and 60 mg/kg of ZnO NPs for 60 days, with hepatotoxicity assessments at 15-day intervals. The histological data showed that ZnO NPs severely damage liver tissues, causing cytoplasmic vacuolization and irregular or missing nuclei. The treatment groups showed a significant rise in the liver injury index (P < 0.05). Moreover, there was a notable increase in zinc accumulation in the ZnO NPs groups compared to the control group (P < 0.05). ZnO NPs also reduced body weight and hepato-somatic index (HSI) in rare minnows. The enzyme activity results showed elevated levels of reactive oxygen species (ROS), total cholesterol (T-CHO), triglyceride (TG), acetyl Coa carboxylase (ACC), and fatty acid synthase (FAS) in the ZnO NPs fed groups, while total antioxidant capacity (T-AOC) and malondialdehyde (MDA) decreased. Further investigation found that accumulation of ZnO NPs in the liver tissues up-regulated the levels of genes related to antioxidant (mn-sod, cat and nf-κb), pro-apoptotic (bax) and lipogenesis (gpat3, dgat1a and dgat1b), while down-regulating genes associated with lipid catabolism (cpt1 and tpi1). These findings suggest that dietary ZnO NPs cause hepatotoxicity by inducing oxidative stress through ROS, triggering apoptosis, and inhibiting lipid peroxidation. The results indicate that ZnO NPs may pose a significant threat to aquatic animals and ecosystems.

Embryonic Exposure to Organophosphate Flame Retardants (OPFRs) Differentially Induces Cardiotoxicity in Rare Minnow (Gobiocypris rarus).

Organophosphorus flame retardants (OPFRs) such as triphenyl phosphate (TPHP) and tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) were reported to impair cardiac function in fish. However, limited information is available regarding their cardiotoxic mechanisms. Using rare minnow (Gobiocypris rarus) as a model, we found that both TPHP and TDCIPP exposures decreased heart rate at 96 h postfertilization (hpf) in embryos. Atropine (an mAChR antagonist) can significantly attenuate the bradycardia caused by TPHP, but only marginally attenuated in TDCIPP treatment, suggesting that TDCIPP-induced bradycardia is independent of mAChR. Unlike TDCIPP, although TPHP-induced bradycardia could be reversed by transferring larvae to a clean medium, the inhibitory effect of AChE activity persisted compared to 96 hpf, indicating the existence of other bradycardia regulatory mechanisms. Transcriptome profiling revealed cardiotoxicity-related pathways in treatments at 24 and 72 hpf in embryos/larvae. Similar transcriptional alterations were also confirmed in the hearts of adult fish. Further studies verified that TPHP and TDCIPP can interfere with Na+/Ca2+ transport and lead to disorders of cardiac excitation-contraction coupling in larvae. Our findings provide useful clues for unveiling the differential cardiotoxic mechanisms of OPFRs and identifying abnormal Na+/Ca2+ transport as one of a select few known factors sufficient to impair fish cardiac function.

Effect of Heat Shock Treatment on the Virulence of Grass Carp Reovirus in Rare Minnow Gobiocypris rarus

The mode and outcome of fish–virus interactions are influenced by many abiotic factors, among which water temperature is especially important in poikilothermic fish. Rare minnow Gobiocypris rarus is a eurythermal small cyprinid fish that is sensitive to infection with genotype II grass carp reovirus (GCRV). HSP70, a conservative and key player in heat shock response, is previously identified as an induced pro-viral factor during GCRV infection in vitro. Here, rare minnow was subjected to heat shock treatment (HST), 1 h treatment at 32 °C followed by reverting to a normal temperature of 24 °C, and subsequently challenged with GCRV-II at a dosage of 1 × LD50. The effect of HST on GCRV virulence in vivo was evaluated by calculating virus-associated mortality and viral load in both dead and survival fish. The results revealed that HST enhanced the mortality of rare minnow infected with GCRV; the fact that viral loads in the tissue samples of HST-treated fish were significantly higher than those in samples of the control group at 6, 8 d p.i. reflected a faster infection process due to HST. Quantitative gene expression analysis was further employed to show that the expression levels of Hsp70 in intestine and liver tissues from the HST group declined faster than muscle tissue after HST. HST W/O GCRV challenge upregulated proinflammatory cytokines such as MyD88 and Nf-κB, which was in consistence with the inflammation observed in histopathological analysis. This study shed light on the complexity of the interaction between fish abiotic and biotic stress response, which suggested that HST, an abiotic stress, could enhance the virulence of GCRV in Gobiocypris rarus that involved modulating the gene expression of host heat shock, as well as a pro-inflammatory response.

Effects of Different Tank Sizes on Laboratory Rare Minnow (Gobiocypris rarus)-Under the Same Density

ABSTRACT Laboratory fish play an important role in many research fields, and they are also raised and bred in many laboratories around the world. While many studies have suggested suitable feeding densities for various laboratory fish, significant variations exist in the sizes of rearing tanks across different laboratories. Moreover, there is limited research on the effects of different rearing tank sizes. In this study, we set up the same rearing density (one liter of water corresponding to two fishes) and four treatments with different tank sizes (5 L, 10 L, 15 L, 20 L). We found that different sizes of rearing tanks had no significant effect on the growth and shoaling of laboratory rare minnow. However, the rare minnow in 10 L tank had lower cortisol levels, while the rare minnow in 20 L tank had higher levels of dopamine (DA), serotonin (5-HT) and their metabolites (DOPAC and 5-HIAA). These results show that even under the same rearing density, different sizes of rearing tanks will still have a physiological effect on the rare minnow, and the tank size suitable for laboratory fish should be considered.

The toxicity of polystyrene micro- and nano-plastics on rare minnow (Gobiocypris rarus) varies with the particle size and concentration

How the particle size and concentration of microplastics impact their toxicity is largely unknown. Herein, the effects of polystyrene microplastics (1 μm, MPs) and nanoplastics (100 nm, NPs) exposed at 1 mg/L (L) and 10 mg/L (H), respectively, on the growth, histopathology, oxidative stress, gut microbiome, and metabolism of rare minnow (Gobiocypris rarus) were investigated by chemical analysis and multi-omics. MPs and NPs inhibited the growth, induced histopathological injury and aggravated oxidative stress markedly with contrasting significance of particle size and concentration. The composition of core gut microbiota changed dramatically especially for the MPs-H. Similarly, gut bacterial communities were reshaped by the MPs and NPs but only NPs-H decreased both richness and Shannon indexes significantly. Co-occurrence network analysis revealed that the potential keystone genera underwent great changes in exposed groups compared to the control. MPs-H increased the network complexity and the frequency of positive interactions which was opposite to other exposed groups. Moreover, the metabolomic profiles associated with amino acid, lipid, unsaturated fatty acid and hormone metabolism were disturbed significantly especially for MPs-H and NPs-H. In conclusion, the toxicity of MPs depends on both the particle size and concentration, and varies with the specific indicators as well.

The effect of isolation on laboratory rare minnow (Gobiocypris rarus): Growth, behavior and physiology

Laboratory fish play an important role in modern scientific research. More and more researchers have begun to pay attention to the other living environment except the physical and chemical factors of the water body, because it is not only related to animal welfare, but also related to the validity of the experimental results. Unfortunately, laboratory fish are sometimes raised alone, primarily because the impact of isolation of fish is still poorly understood. This study assessed the effects of long-term social isolation (lasting 90 days) on the growth, clustering, anxiety-like behavior, and physiological parameters (cortisol, dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA)) of laboratory rare minnow. We found that isolation did not affect the growth, clustering behavior, and anxiety-like behavior of the laboratory rare minnow. However, we found a significant decrease in the levels of cortisol, DOPAC and 5-HT in isolated fish. Our results suggested that isolated environments might impact the responsive ability of rare minnows, and future researchers should thus provide appropriate living environments for them.

Effects of environmentally relevant polystyrene microplastics and cadmium on the development and reproduction of rare minnow (Gobiocypris rarus)

Microplastic pollution is widespread in the natural environment, and its enrichment, inherent adsorbability, and toxicity are key concerns in society. However, few studies of the effects of microplastic toxicity on the reproduction of fish have been reported. In this study, we investigated the effects of exposure to environmentally relevant concentrations of polystyrene microplastics (PS) and cadmium (Cd) alone and in combination for 28 days on the gonad development and reproduction of rare minnow (Gobiocypris rarus). Decreased condition factor, number of spawning events, number of eggs per spawning, and average hatching rate were detected in fish in the treatment groups. These changes were likely due to long-term oxidative stress and inflammation, which inhibited the development of gonads and further affected the reproduction function of rare minnow. The effects of treatment on oxidative stress, inflammation, and expression of genes related to gonad development differed between female and male fish in the treatment groups. PS alone exposure resulted in a significant reduction in the number of eggs per spawning and average hatching rate, but these effects were not observed in the Cd and PS + Cd groups. These results provide new insights into the ecotoxicological effects of microplastics in the natural environment.

The Developmental Toxicity and Endocrine-Disrupting Effects of Fenpropathrin on Gobiocypris rarus during the Early Life Stage.

In the present study, the developmental toxicity and endocrine-disrupting effects of fenpropathrin on Gobiocypris rarus during the early life stage were studied using a semi-static water exposure method. The results showed that the LOEC (lowest observed effect concentration) of fenpropathrin on the incubation of rare minnow embryos was above 2.5 μg·L-1. The LOEC and NOEC (no observed effect concentration) of fenpropathrin on the developmental malformations and death indicators were 2.0 and 1.5 μg·L-1, respectively. After exposure to 1.5 μg·L-1 of fenpropathrin for 31 days, the expressions of androgen receptor genes (AR) and sex hormone-synthesis-related genes (CYP17 and CYP19a) were significantly decreased and the expressions of thyroid hormone receptor genes (TRβ) and aryl hydrocarbon receptor genes (AhR1a and AhR2) were significantly increased in juvenile Gobiocypris rarus. The expression levels of the androgen receptor gene (AR), estrogen receptor gene (ER1), and the sex hormone-synthesis-related genes (HMGR, CYP17, and CYP19a) were significantly decreased, while the estrogen receptor gene (ER2a), thyroid hormone receptor gene (TRβ), and aromatic hydrocarbon receptor genes (AhR1a and AhR2) were upregulated in juvenile Gobiocypris rarus under exposure to 2.0 μg·L-1 of fenpropathrin. Relatively low concentrations of fenpropathrin can affect the expression of sex hormone receptor genes, genes related to sex hormone synthesis, thyroid hormone receptor genes, and aromatic hydrocarbon receptor genes, thus interfering with the reproductive system, thyroid system, and metabolic level in Gobiocypris rarus. Therefore, more attention should be paid to the endocrine-disrupting effect caused by the pyrethroid insecticides in the water environment. Furthermore, studies on the internal mechanism of the endocrine-disrupting effect of pyrethroid insecticides on fish is needed in the future.

Parental exposure to environmental cadmium causes developmental defects in the rare minnow (Gobiocypris rarus) embryos: The association between ROS-mediated oxidative stress and developmental dysfunction

Cadmium is widely distributed in aquaculture systems. The exposure of parental fish species to cadmium is known to retard of growth and cause death in offspring. However, the potential mechanisms responsible for how parental cadmium exposure effects embryonic development yet to be elucidated. Herein, we investigated the effects of parental cadmium exposure on embryonic developmental abnormalities in rare minnow. Our results demonstrated that significantly increased rates of malformation were recorded in the parental cadmium exposure groups. The most significant impairments and morphological changes were egg condensation and spine curvature malformations. Parental cadmium exposure significantly increased the cadmium content of rare minnow embryos, thus increasing the production of reactive oxygen species (ROS) and malondialdehyde (MDA). Further investigation found that parental cadmium exposure reduced superoxide dismutase (SOD) activity and significantly up-regulated the expression of antioxidative genes (gpx-1, cat and mn-sod) and detoxifying metallothionein (MT) in rare minnow embryos. In addition, stress-related genes (hsp70, ccyp1α and jnk) were significantly upregulated while developmental-related genes (wnt8α, vezf1 and mstn) were significantly downregulated in rare minnow embryos. Our analysis revealed that parental cadmium exposure in rare minnow caused developmental abnormalities in embryos via the accumulation of cadmium content which induced oxidative stress and developmental dysfunction. Collectively, our analysis suggested that environmental cadmium may pose a severe threat to the continuation of certain fish species.

Analyzing the impact and mechanism of bisphenol A on testicular lipid metabolism in Gobiocypris rarus through integrated lipidomics and transcriptomics

Bisphenol A (BPA) is one of the most common environmental endocrine chemicals, known for its estrogenic effects that can interfere with male spermatogenesis. Lipids play crucial roles in sperm production, capacitation, and motility as important components of the sperm plasma membrane. However, limited research has explored whether BPA affects lipid metabolism in the testes of male fish and subsequently impacts spermatogenesis. In this study, we employed Gobiocypris rarus rare minnow as a research model and exposed them to environmentally relevant concentrations of BPA (15 μg/L) for 5 weeks. We assessed sperm morphology and function and analyzed changes in testicular lipid composition and transcriptomics. The results demonstrated a significant increase in the sperm head membrane damage rate, along with reduced sperm motility and fertilization ability due to BPA exposure. Lipidomics analysis revealed that BPA increased the content of 11 lipids while decreasing the content of 6 lipids in the testes, particularly within glycerophospholipids, glycerolipids, and sphingolipid subclasses. Transcriptomics results indicated significant up-regulation in pathways such as cholesterol metabolism, peroxisome proliferator-activated receptor signaling, and fat digestion and absorption, with significant alterations in key genes related to lipid metabolism, including apolipoprotein A-I, apolipoprotein C-I, and translocator protein. These findings suggest that BPA exposure can induce testicular lipid metabolism disruption in rare minnows, potentially resulting in abnormalities in rare minnow spermatogenesis.

Metabolomics in rare minnow (Gobiocypris rarus) after infection by attenuated and virulent grass carp reovirus genotype Ⅱ

Grass carp reovirus genotype Ⅱ (GCRV Ⅱ) causes hemorrhagic disease in a variety fish, seriously affecting the aquaculture industry in China. However, the pathogenesis of GCRV Ⅱ is unclear. Rare minnow is an ideal model organism to study the pathogenesis of GCRV Ⅱ. Herein, we applied liquid chromatography-tandem mass spectrometry metabolomics to investigate metabolic responses in the spleen and hepatopancreas of rare minnow injected with virulent GCRV Ⅱ isolate DY197 and attenuated isolate QJ205. Results indicated that marked metabolic changes were identified in both the spleen and hepatopancreas after GCRV Ⅱ infection, and the virulent DY197 strain induced more significantly different metabolites (SDMs) than the attenuated QJ205 strain. Moreover, most SDMs were downregulated in the spleen and tend to be upregulated in hepatopancreas. The Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that tissue-specific metabolic responses were identified after viruses infection, and the virulent DY197 strain induced more SDMs involved in amino acid metabolism in the spleen, especially the tryptophan metabolism, cysteine and methionine metabolism, which were essential for immune regulation in host; Meanwhile, nucleotide metabolism, protein synthesis and metabolism related pathways were enriched in the hepatopancreas by both virulent and attenuated strains. Our findings revealed the large scale metabolic alterations in rare minnow in response to attenuated and virulent GCRV Ⅱ infection, which will lead to a better understanding of the pathogenesis of viruses and host-pathogens interactions.

Transcriptomics in Rare Minnow (Gobiocypris rarus) towards Attenuated and Virulent Grass Carp Reovirus Genotype II Infection.

Grass carp reovirus genotype Ⅱ (GCRV Ⅱ) causes a variety of fish hemorrhagic disease, which seriously affects the sustainable development of grass carp aquaculture in China. Rare minnow (Gobiocypris rarus) is an ideal model fish to study the pathogenesis of GCRV Ⅱ. To investigate the involved molecular responses against the GCRV Ⅱ infection, we performed comparative transcriptomic analysis in the spleen and liver of rare minnow injected with virulent strain DY197 and attenuated strain QJ205. Results showed that the virulent DY197 strain induced more differently expressed genes (DEGs) than the attenuated QJ205 strain, and tissue-specific responses were induced. In the spleen, the attenuated and virulent strains induced different DEGs; the attenuated QJ205 infection activated steroid synthesis pathway that involved in membrane formation; however, virulent DY197 infection activated innate immunity and apoptosis related pathways while suppressing cell proliferation and migration related pathways that are important for damage tissue repair, as well as hemorrhage related pathways. In the liver, the attenuated and virulent strains infection induced similar DEGs; both strains infection activated immunity and apoptosis related pathways but suppressed metabolism-related pathways; virulent DY197 infection especially activated protein digestion and absorption-related pathways and suppressed steroid synthesis pathway. To conclude, virulent strain infection especially induced tissue-specific alterations and caused severe suppression of hemorrhage-related pathways in spleen. Our findings will contribute to better understanding of the interactions between host and GCRV II.

A study on the mechanism of Indium phosphide/zinc sulfide core/shell quantum dots influencing embryo incubation of rare minnow (Gobiocypris rarus)

Quantum dots (QDs) inhibit fish hatching, but the mechanism is still unclear. In this study, the effect of Indium phosphide/zinc sulfide quantum dots (InP/ZnS QDs) on the embryo incubation of rare minnow was investigated. Five experimental concentration groups were set up according to the preliminary experimental results, which were 0, 50, 100, 200 and 400 nM. A direct exposure method was adopted to expose embryos to InP/ZnS QDs solution. The results showed that InP/ZnS QDs significantly inhibited the embryo hatching rate, delayed embryo emergence, affected the expression of genes associated with hatching gland cells and hatching enzymes. InP/ZnS QDs also destroy the structure of the embryo chorion. In addition, QDs can cause oxidative stress in embryos. Transcriptional sequencing analysis showed that InP/ZnS QDs InP/ZnS QDs may have induced the production of a hypoxic environment and triggered induce abnormal cardiac muscle contraction, inflammatory response and apoptosis process in embryos. In conclusion, QDs influences embryo hatchability largely through egg chorion mediation.

Effects of cadmium exposure during the breeding period on development and reproductive functions in rare minnow (Gobiocypris rarus).

Cadmium is a common reproductive toxin in aquatic systems. Cd exposure of fish species at high concentrations can severely affect the reproductive function of fish. However, the underlying toxicity of cadmium exposure at low concentrations on the reproductive function in parental fish remains unclear. To investigate the impacts of cadmium exposure on reproductive capability, eighty-one male and eighty-one female rare minnows (Gobiocypris rarus) were exposed to cadmium at 0 (control group), 5 and 10μg/L for 28days, and then transferred into clean water to pair spawn. The results showed that cadmium exposure at 5 or 10μg/L for 28days in rare minnows could reduce the success rates of pair spawning in parent rare minnows, lessen no-spawning activities, and prolong the time for first spawning. Furthermore, the mean egg production of the cadmium exposure group increased. The fertility rate of the control group was significantly higher than that of the 5μg/L cadmium exposure group. Anatomical and histological data further revealed that the intensity of atretic vitellogenic follicles significantly increased and spermatozoa vacuolated after cadmium exposure (p < 0.05), but slightly increased the condition factor (CF), and relatively stable gonadosomatic index (GSI) values were also observed in the cadmium exposure groups. These observed results indicated that cadmium exposure at 5 or 10μg/L affected the reproductive activity of paired rare minnow by accumulating Cd in the gonads, and the effect diminished over time. The reproductive risk of low-dose cadmium exposure to fish species remains a cause for concern.

Effects of Dietary Cholesterol Regulation on Spermatogenesis of Gobiocypris rarus Rare Minnow.

Cholesterol is an important component of cell membranes, and also a precursor for the synthesis of sex hormones, playing an important role in reproduction. However, few studies have focused on cholesterol and reproductive health. To investigate the toxic effects of different cholesterol levels on the spermatogenesis of rare minnows, we regulate the cholesterol content in fish by feeding them a high-cholesterol diet and cholesterol inhibitor pravastatin, and cholesterol levels, sex hormone (T and 11KT) levels, testis histology, sperm morphology and function, and the expression of genes related to sex hormone synthesis were investigated. The research findings indicate that increasing cholesterol levels significantly increases the liver weight and hepatic-somatic index, as well as the total cholesterol and free cholesterol levels in the testis, liver, and plasma of rare minnow, while inhibiting cholesterol has the opposite effect (p < 0.05). However, both increasing and decreasing cholesterol levels can suppress rare minnow testicular development, as evidenced by a decrease in testis weight, lowered gonadosomatic index, suppressed sex hormone levels, and reduced mature sperm count. Further exploration revealed that the expression of sex hormone synthesis-related genes, including star, cyp19a1a, and hsd11b2, was significantly affected (p < 0.05), which may be an important reason for the decrease in sex hormone synthesis and consequent inhibition of testicular development. At the same time, the fertilization ability of mature sperm in both treatment groups significantly decreased. Scanning electron microscopy and fluorescence polarization tests showed that reducing cholesterol levels significantly increased the rate of sperm head cell membrane damage, while both increasing and decreasing cholesterol levels led to a reduction in sperm cell membrane fluidity, which may be the main reason for the decrease in sperm fertilization ability. This study demonstrates that both increasing and decreasing the levels of cholesterol are detrimental to the fish spermatogenesis, providing fundamental information for the study of fish reproduction and also a reference for the causes of male reproductive dysfunction.

Identification of fish spermatogenic cells through high-throughput immunofluorescence against testis with an antibody set.

Image-based identification and quantification of different types of spermatogenic cells is of great importance, not only for reproductive studies but also for genetic breeding. Here, we have developed antibodies against spermatogenesis-related proteins in zebrafish (Danio rerio), including Ddx4, Piwil1, Sycp3, and Pcna, and a high-throughput method for immunofluorescence analysis of zebrafish testicular sections. By immunofluorescence analysis of zebrafish testes, our results demonstrate that the expression of Ddx4 decreases progressively during spermatogenesis, Piwil1 is strongly expressed in type A spermatogonia and moderately expressed in type B spermatogonia, and Sycp3 has distinct expression patterns in different subtypes of spermatocytes. Additionally, we observed polar expression of Sycp3 and Pcna in primary spermatocytes at the leptotene stage. By a triple staining of Ddx4, Sycp3, and Pcna, different types/subtypes of spermatogenic cells were easily characterized. We further demonstrated the practicality of our antibodies in other fish species, including Chinese rare minnow (Gobiocypris rarus), common carp (Cyprinus carpio), blunt snout bream (Megalobrama amblycephala), rice field eel (Monopterus albus) and grass carp (Ctenopharyngodon idella). Finally, we proposed an integrated criterion for identifying different types/subtypes of spermatogenic cells in zebrafish and other fishes using this high-throughput immunofluorescence approach based on these antibodies. Therefore, our study provides a simple, practical, and efficient tool for the study of spermatogenesis in fish species.

Construction of hybrid constructed wetlands for phosphorus chemical industry tailwater treatment in the middle Yangtze river basin: Responses of plant growth and root-associated microbial communities

Constructed wetlands are commonly utilized to treat industrial wastewater due to their effectiveness, affordability, and environmental benefits. Many phosphorus chemical enterprises in the middle Yangtze River basin are facing high pollution load challenges and efforts are needed to improve removal efficiency of pollutants. This work was the first to use constructed wetlands for phosphorus chemical industry tailwater (PCITW) treatment. The new hybrid constructed wetlands (CWs) proposed were the aeration horizontal subsurface flow constructed wetland (AHSCW) and the integrated vertical flow constructed wetland (IVCW), which were constructed on a pilot scale. Here, the effectiveness of pollutant removal along hybrid CWs, wetland plant growth, and root-associated microbial community responses to the PCITW were investigated. The results showed that there was spatial variation in removal of carbon, nitrogen, and phosphorus in the hybrid CWs, and that the AHSCW and the IVCW played synergistic roles in the removal of pollutants. Compared with influent, the toxic effect of effluent to embryos of a rare minnow (Gobiocypris rarus) was alleviated. Exposure to the effluent resulted in no malformation or death for embryos. Plants and microorganisms in the wetland system of the phosphate chemical tailwater were then compared with the control system of municipal tailwater. Plants in the former system had lower root density, and higher average root diameter, root shoot ratio, specific root length, and specific surface area. Plant roots also had decreased NH4+ uptake ability but increased Ca2+ uptake to adapt to the high load and complex pollution stress. Unlike the control system, stochastic mechanisms had a more important role than deterministic processes in shaping the microbial community assembly associated with the PCITW. Meanwhile, analysis of microbial network-level topological characteristics demonstrated substantial reduction in network interactions complexity and microbiome stability in the treatment system. Findings from this study suggest wetlands will be helpful for efficient purification of phosphorus chemical industry wastewater in the Yangtze River basin.