Rare Minnow Research Articles

Establishment of a rare minnow (Gobiocypris rarus) model for evaluation of experimental vaccines against a disease induced by grass carp reovirus genotype II

Vaccination is the most effective way to control the grass carp haemorrhagic disease (GCHD) with the primary pathogen grass carp reovirus genotype II (GCRV-II). However, due to the large difference in breeding conditions and unclear genetic background of grass carp, the results of the experiment were not reliable, which further hinders the effective prevention and control of GCHD. The rare minnow (Gobiocypris rarus) is highly sensitive to GCRV. Its small size, easy feeding, transparent egg membrane, and annual spawning are in line with the necessary conditions for an experimental aquatic animals culture object. In this study, immunogenicity and protective effects of attenuated and inactivated viruses for grass carp and rare minnow were evaluated in parallel. The expression of immune-related genes increased statistically significant after immunization. With the rise of specific serum antibody titers, the results of rare minnow and grass carp were consistent. In addition, there was no significant residue of adjuvant observed in both fish species injected with an adjuvanted and inactivated virus. Challenge of immunized grass carp and rare minnow with the isolate HuNan1307 resulted in protection rates of 95.8% and 92.6% for attenuated virus, 81.4% and 77.7% for inactivated virus, respectively, as well as the viral load changed consistently. The results indicated that rare minnow can be used as a model for evaluation of experimental vaccines against GCHD.

Quercetin protects rare minnow Gobiocypris rarus from infection of genotype II grass carp reovirus

Plant-derived quercetin is a major dietary flavonoid and known mammalian transcriptional repressor for heat shock factor 1 (HSF-1), which is responsible for the transcriptional activation of heat shock proteins upon stress stimulation. Our previous studies indicated that Hsp70, the major heat shock protein during heat shock response, is induced to facilitate the infection of grass carp reovirus (GCRV), and quercetin could reduce the replication efficiency of both genotype I and III GCRVs in vitro. Here, we provide in vivo evidence to show that quercetin could effectively increase the survival rate of rare minnow Gobiocypris rarus from genotype II GCRV (GCRV-JX02) infection. Quercetin is shown to be safe for rare minnow in a dosage range of 30–480 μg/fish. Infection of rare minnow with GCRV-JX02 results in typical haemorrhagic symptom and tissue damage in pathological examination, and the semi-lethal dose (LD50) of GCRV-JX-02 is determined to be 6.0 × 102 virus genome copy number through a quantitative real-time RT-PCR assay. Quercetin dose-dependently protects rare minnow from GCRV-JX02 challenge through decreasing progeny virus production and increasing fish survival rate. Thus, quercetin demonstrates anti-aquareovirus activity in vivo and should act as a promising environmentally friendly therapeutic agent for aquaculture.

The feasibility of the zebrafish embryo as a promising alternative for acute toxicity test using various fish species: A critical review

With the European Union's restrictions on toxicity tests using vertebrates and the issue of the Organization for Economic Co-operation and Development test guideline 236, the fish embryo test (FET) has become a promising alternative to acute fish toxicity test (AFT), with zebrafish embryos being used the most. A large number of studies showed zebrafish FET correlated well with zebrafish AFT, yet its representativeness for other fish species is still under evaluation. In this review, toxicity data of zebrafish FET were summarized and compared with AFT using zebrafish and other test species in various countries, such as fathead minnow (Pimephales promelas), bluegill sunfish (Lepomis macrochirus), and Chinese rare minnow (Gobiocypris rarus). Previous findings of good relationship between zebrafish FET and AFT were confirmed, with a significant relationship between the median lethal and effect concentrations (LC50 and EC50) in FET and AFT LC50 for 87 chemicals (log (LC50AFT) = 1.00 × log (LC50FET) − 0.0829, p < 0.0001). Since embryo lethality generally occurs at chemical concentrations much higher than their environmentally relevant concentrations and involves in multiple modes of action, the EC50s gradually become the preferred FET parameters. Therefore, the use of FET EC50 for predicting fish toxicity was also evaluated. Interestingly, we found that the zebrafish FET toxicity data well predicted AFT of G. rarus that is native AFT model species in China, yet its corresponding FET is still immature. This highlights the zebrafish embryo test would serve as a good alternative for AFT across fish species.

Environmentally relevant concentrations of clozapine induced lipotoxicity and gut microbiota dysbiosis in Chinese rare minnow (Gobiocypris rarus)

Clozapine (CLZ) is a neuroactive pharmaceutical that is frequently detected in aquatic environments. Although the cardiotoxicity, developmental toxicity, and neurotoxicity of CLZ in aquatic non-target organisms have been reported, its lipotoxicity and underlying mechanism are unknown. Therefore, in this study, 2-month-old Chinese rare minnows were exposed to 0, 0.1, 1, and 10 μg/L CLZ for 90 days. Overt dyslipidemia was observed after CLZ exposure, whereas the body weights of females significantly increased after CLZ exposure (p < 0.05). In addition, obvious hepatocyte vacuolization and hepatic lipid droplet accumulation were observed at all treatment groups (p < 0.05). The activities of sterol regulatory element binding proteins 1 (SREBP1) and fatty acid synthase (FAS) were significantly upregulated at the 1 and 10 μg/L CLZ treatment groups (p < 0.05). Moreover, evident cell boundary disintegration of the intestinal villi and increasing mucus secretion were observed at all treatment groups (p < 0.05). Furthermore, the diversity of the gut microbiota increased, whereas the relative abundances of Proteobacteria, Firmicutes and Bacteroidetes significantly increased after CLZ exposure (p < 0.05). Furthermore, significantly increased bacterial secondary bile acid biosynthesis activity in Chinese rare minnows was observed after 1 μg/L CLZ exposure (p < 0.05). Therefore, our findings confirmed that CLZ induced lipotoxicity by stimulating SREBP1 and affecting the bacterial secondary bile acid biosynthesis activity in Chinese rare minnows.

Chronic exposure to Bisphenol A resulted in alterations of reproductive functions via immune defense, oxidative damage and disruption DNA/histone methylation in male rare minnow Gobiocypris rarus.

Bisphenol A (BPA) is a widely used chemical that represents a reproductive hazard in fish. However, the molecular pathways mediating reproductive toxicity under chronic BPA exposure remain unclear. To study the reproductive hazards associated with chronic BPA exposure, adult male rare minnows (Gobiocypris rarus) were treated with 15μg L-1 and 225μg L-1 BPA for 90 days. Results showed that chronic BPA treatment induced reproductive impairments with decreased fertilization capacity and movement time of sperm. Transcriptome analysis indicated 1421 transcripts that were differentially expressed in response to BPA exposure, which are involved in the biological process of oxidative stress, immune responses and DNA/histone methylation. BPA caused the oxidative stress via significantly increasing hydrogen peroxide (H2O2) levels and inhibiting the activities of antioxidant-related enzymes (Catalase, CAT). BPA caused an inflammatory response in the testes by significantly increasing IL-1β levels and inducing infiltration of inflammatory cells. Moreover, exposure to 15μg L-1 BPA significantly decreased the genomic DNA methylation level. These data revealed that chronic BPA exposure had adverse effects on male reproduction. Oxidative stress, inflammatory response and DNA/histone methylation might account for the decreased sperm quality.

Identification, Virulence, and Molecular Characterization of a Recombinant Isolate of Grass Carp Reovirus Genotype I.

The hemorrhagic disease of grass carp (HDGC) caused by grass carp reovirus (GCRV) still poses a great threat to the grass carp industry. Isolation and identification of the GCRV genotype I (GCRV-I) has been rarely reported in the past decade. In this study, a new GCRV was isolated from diseased fish with severe symptoms of enteritis and mild hemorrhages on the body surface. The isolate was further identified by cell culture, transmission electron, indirect immunofluorescence, and SDS-PAGE electrophoretic pattern analysis of genomic RNA. The results were consistent with the new isolate as a GCRV-I member and tentatively named GCRV-GZ1208. Both grass carp and rare minnow infected by the GCRV-GZ1208 have no obvious hemorrhagic symptoms, and the final mortality rate was ≤10%, indicating that it may be a low virulent isolate. GZ1208 possessed highest genomic homology to 873/GCHV (GCRV-I) and golden shiner reovirus (GSRV). Additionally, it was found a 90.7–98.3% nucleotide identity, a 96.4–100% amino acid identity, and <50% identity with GCRV-II and III genotypes. Interestingly, the sequences of some segments of GZ1208 were similar to GCRV-8733/GCHV, whereas the remaining segments were more closely related to GSRV, suggesting that a recombination event had occurred. Bootscan analysis of the complete genomic sequence confirmed this hypothesis, and recombination events between 873/GCHV and other GSRV-like viruses were also accompanied by gene mutations.

Toxic effects of ZnSe/ZnS quantum dots on the reproduction and genotoxiticy of rare minnow (Gobiocypris rarus)

ZnSe/ZnS quantum dots (QDs) have excellent optical properties, but researchers have not clearly determined whether they cause harm to organisms. In the present study, the effect of ZnSe/ZnS QDs on the parents and offspring of rare minnow were evaluated for the first time. Exposure to ZnSe/ZnS QDs altered the testicular structure, caused sperm DNA damage and decreased sperm motility in males. They also suppressed the expression of reproduction-related genes, such as androgen receptor (Ar), DM-related transcription factor 1 (Dmrt1), estrogen receptor (Er), and X-ray repair cross complementing gene 1 (Xrcc1). Continued monitoring of the F1 generation revealed that the embryonic development of the F1 generation was abnormal and the growth index of the F1 generation of adult fish showed hormesis. A comet assay showed that the F1 generation still had DNA damage in the 400 and 800 nmol/L groups at 96 h post-fertilization (hpf). Thus, ZnSe/ZnS QDs damaged the reproductive system of the rare minnow, and this effect continued to the F1 generation.

Bisphenol A induces hepatic triglyceride level in adult male rare minnow Gobiocypris rarus

Bisphenol A (BPA), an endocrine disruptor, exist in almost all waters. In the present study, we expose adult male Gobiocypris rarus rare minnow to 15 μg/L BPA to study the effect BPA on fish hepatic lipid metabolism. Following 1, 3 and 5 weeks exposure, the liver tissue of rare minnow was separated. The change of the hepatic morphology, hepatosomatic index, lipid composition and expression of lipid metabolism related genes were analyzed through paraffin section, oil red O staining, lipidomic analysis, and quantitative real-time PCR. BPA can cause significant hepatic lipid deposition in male rare minnow, leading to an increase in triglyceride (TG) level (1.84–22.87-fold), but it is also accompanied by a decrease in diglyceride level (1.67–4.78-fold). The expression of lipid metabolism related genes showed that BPA exposure can up-regulate TG synthesis related genes expression, and down-regulate TG degradation genes expression. Expression of TG transport related genes were also disrupted by BPA. It suggests that BPA can up-regulate rare minnow hepatic TG level through multi-path, and ultimately lead to lipid accumulation in the liver. The results of the present study enrich the mechanisms of environmental endocrine disruptors affecting lipid accumulation in fish.

Development of Novel SNP Assays for Genetic Analysis of Rare Minnow (Gobiocypris rarus) in a Successive Generation Closed Colony

The complex genetic architecture of closed colonies during successive passages poses a significant challenge in the understanding of the genetic background. Research on the dynamic changes in genetic structure for the establishment of a new closed colony is limited. In this study, we developed 51 single nucleotide polymorphism (SNP) markers for the rare minnow (Gobiocypris rarus) and conducted genetic diversity and structure analyses in five successive generations of a closed colony using 20 SNPs. The range of mean Ho and He in five generations was 0.4547–0.4983 and 0.4445–0.4644, respectively. No significant differences were observed in the Ne, Ho, and He (p &gt; 0.05) between the five closed colony generations, indicating well-maintained heterozygosity. The F-statistics analysis revealed a relatively stable genetic structure of the closed colony. Furthermore, the genetic distance between the newer and older generations increased with the breeding generations in closed colonies. Our results confirmed previous findings in the same samples using microsatellite markers. The results will be beneficial for establishing genetic variability monitoring criteria and restoration of the wild population of the rare minnow and other laboratory fish.

Carbamazepine at environmentally relevant concentrations caused DNA damage and apoptosis in the liver of Chinese rare minnows (Gobiocypris rarus) by the Ras/Raf/ERK/p53 signaling pathway.

To assess genetoxicity and the underlying mechanisms of carbamazepine (CBZ) toxicity in fish, adult Chinese rare minnows (Gobiocypris rarus) were exposed to 1, 10, and 100μg/L CBZ for 28d. Comet assays indicated that hepatic DNA damage was significantly increased in groups of minnows exposed to CBZ at all concentrations in a dose-dependent manner compared to those of the control groups (p<0.05). Liver levels of 8-hydroxydeoxyguanosine (8-OHdG) were significantly increased at 10 and 100μg/L CBZ (p<0.05). TUNEL assays indicated that the average apoptotic rates of the livers of female and male minnows were significantly increased following exposure to CBZ at all concentrations for 28d (p<0.05). Significant increases in caspase 3 and 9 activities after CBZ exposure at all concentrations and caspase 8at 10 and 100μg/L CBZ exposure reflected the presence of mitochondrial apoptosis (p<0.05). The mRNA levels of gadd45a, mdm2, casp3 and casp9 in female and male minnows exposed to CBZ at all concentrations were significantly increased compared with those in the control groups (p<0.05). Significant increases in the levels of p21 in female minnows exposed to 1 and 100μg/L CBZ, p53 in female minnows at all CBZ treatments and bcl2 in male minnows exposed to 1 and 100μg/L CBZ were observed, indicating p53 pathway activation. The inhibition of ras levels in females and males exposed to CBZ at all concentrations and increased levels of raf1 in males exposed to CBZ at all concentrations indicated Ras/Raf1/MAPK (ERK) activation. Therefore, the present study demonstrates that CBZ at environmentally relevant levels induces DNA damage and apoptosis in Chinese rare minnows by the Ras/Raf/ERK/p53 signaling pathway.

Environmentally relevant concentrations of carbamazepine induced lipid metabolism disorder of Chinese rare minnow (Gobiocypris rarus) in a gender-specific pattern

Carbamazepine (CBZ), an anticonvulsant and mood stabilizer, is ubiquitous distributed in aquatic environment. Though the toxicity and endocrine disrupting effect of CBZ on non-target organisms have been studied, its lipotoxity are scarcely known. To assess the lipotoxicity of CBZ, 2-month-old Chinese rare minnow were exposed to 0, 1, 10, and 100 μg/L CBZ for 90 d. Obvious dyslipidemia was observed after 30 d and 90 d exposure, whereas overt hyperlipidemia was observed in males at 100 μg/L treatments. Severe lipid droplet accumulation in livers was observed at 10 and 100 μg/L treatments for 30 d and in females, whereas those was observed at all treatments in males. In addition, serious mitochondria damage was observed in males at 100 μg/L treatments. After 90 d exposure, the enzyme activities of FAS and ACCα were significantly increased at 10 and 100 μg/L treatments, whereas HMGCR were markedly increased at 100 μg/L treatments (p < 0.05). However, ACCβ were markedly decreased in females at 10 and 100 μg/L treatments and in males at all treatments (p < 0.05). The transcription levels of fasn, accα, hmgcrα, fdft1, idi1, plin1, plin2, caveolin1, and caveolin2 were significantly increased at 100 μg/L treatments (p < 0.05). Moreover, the body weight was obviously increased at 10 and 100 μg/L treatments in males (p < 0.05). Our results confirmed that environmental relevant concentrations CBZ induced lipid metabolism disorder and mitochondria damage of Chinese rare minnow in a gender-specific pattern, which provided a new insight into the lipotoxicity mechanism of CBZ.

A Novel Subunit Vaccine Based on Outer Capsid Proteins of Grass Carp Reovirus (GCRV) Provides Protective Immunity against GCRV Infection in Rare Minnow (Gobiocypris rarus).

The grass carp hemorrhagic disease, caused by the grass carp reovirus (GCRV), has resulted in severe economic losses in the aquaculture industry in China. VP4 and VP35 are outer capsid proteins of GCRV and can induce an immune response in the host. Here, three recombinant baculoviruses, AcMNPV-VP35, AcMNPV-VP4, and AcMNPV-VP35-VP4, were generated to express recombinant VP4 and VP35 proteins from GCRV type II in insect cells by using the Bac-to-Bac baculovirus expression system to create a novel subunit vaccine. The expression of recombinant VP35, VP4, and VP35-VP4 proteins in Sf-9 cells were confirmed by Western blotting and immunofluorescence. Recombinant VP35, VP4, and VP35-VP4 were purified from baculovirus-infected cell lysates and injected intraperitoneally (3 μg/fish) into the model rare minnow, Gobiocypris rarus. After 21 days, the immunized fish were challenged with virulent GCRV. Liver, spleen, and kidney samples were collected at different time intervals to evaluate the protective efficacy of the subunit vaccines. The mRNA expression levels of some immune-related genes detected by using quantitative real-time PCR (qRT-PCR) were significantly upregulated in the liver, spleen, and kidney, with higher expression levels in the VP35-VP4 group. The nonvaccinated fish group showed 100% mortality, whereas the VP35-VP4, VP4, and VP35 groups exhibited 67%, 60%, and 33% survival, respectively. In conclusion, our results revealed that recombinant VP35 and VP4 can induce immunity and protect against GCRV infection, with their combined use providing the best effect. Therefore, VP35 and VP4 proteins can be used as a novel subunit vaccine against GCRV infection.

Establishment of a rare minnow (Gobiocypris rarus) disease model for grass carp reovirus genotype II

Grass carp haemorrhagic disease (GCHD) is one of the most important diseases affecting grass carp aquaculture. The rare minnow (Gobiocypris rarus) is a small experimental fish that has been proven to be sensitive to GCRV infection. However, few studies have definitively shown that rare minnow can be used as a disease model for GCRV-II, a recently emerging dominant genotype. In the present study, we investigated clinical characteristics, tissue tropism, histopathology, and relative expression of immune-related genes in rare minnow infected with GCRV-II virulent strain HuNan1307. Hemorrhage of rare minnow was induced by the GCRV-II, which could proliferate in the fishes, and the viral load peaked on the day 7. The histopathologic changes in rare minnow were primarily vasodilation and hyperaemia in multiple organs, cell degeneration and necrosis, as well as loose intercellular in spleen, kidney, brain and heart. Electron microscopic observation in the kidney revealed virus similar in shape and size to those GCRV-II infections in grass carp. Genes encoding IL-8, Mx, TLR5, IgM and IFNα were relatively high up-regulated in different tissues, while multiple genes in liver and spleen were significantly up-regulated, which was consistent with GCRV-II infection in grass carp (P < 0.05). Our study suggested that rare minnow can be used as a disease model of GCRV-II as a tool to study pathogenesis and develop vaccine against GCRV-II.

BPA's transgenerational disturbance to transcription of ovarian steroidogenic genes in rare minnow Gobiocypris rarus via DNA and histone methylation

As a well-known estrogenic endocrine disruptor, bisphenol A (BPA) is of utmost concern since it is reported with harmful effects on animal reproduction. However, the adverse effects on progeny after parental BPA exposure are largely unknown in fishes. To investigate the epigenetic effects of BPA on progeny gonadal development, parental rare minnow (Gobiocypris rarus) were exposed to BPA (15 μg L−1) for two months, then were purged in clean water for one, two or three months, respectively. From the second month, parents were mated once a month and the offspring were reared to 5 months old. Results showed that parental BPA exposure inhibited the ovary development of the offspring by reducing the number of mature oocytes while the transcripts of steroidogenic genes (cyp11a1, cyp17a1, cyp19a1a and star) were significantly affected. And the negative effects of parental BPA exposure on the offspring were reversible. The DNA methylation and histone trimethylation levels (H3K9me3 and H3K27me3) together with the expression of dnmts (dnmt1, dnmt5 and dnmt7) and histone methyltransferase genes (setdb1, setdb2 and ezh2) were significantly altered in the ovaries of the 5-month old offsprings. BPA interfered the expression of steroidogenic genes by altering histone recruitment in star (H3K4me3 and H3K9me3), in cyp11a1 and cyp17a1 (H3K9me3 and H3K27me3), as well as in cyp19a1a (H3K4me3, H3K9me3 and H3K27me3). In addition, altering of DNA methylation at CpG site caused by BPA exposure involved in the regulation of star, cyp17a1 and cyp19a1a expression. These results suggest that BPA transgenerationally imposes detriment to reproduction and the epigenetic changes in DNA methylation and histone trimethylation might account for steroidogenic genes expression.

TBK1 regulates the induction of innate immune response against GCRV by phosphorylating IRF3 in rare minnow (Gobiocypris rarus)

Rare minnow (Gobiocypris rarus), a small cyprinid species that is highly sensitive to the grass carp reovirus (GCRV), is regarded as an ideal model to study the mechanisms of innate immunity in fish. In the present study, a TBK1 homologue from rare minnow (GrTBK1) was identified and its roles in defence against viral infection were investigated. Sequence analysis showed that GrTBK1 encoded a 727-amino acid peptide which shared 98% and 72% identity to the black carp (Mylopharyngodon piceus) and human (Homo sapiens) orthologues, respectively. The amino acid sequence analysis demonstrated that GrTBK1 contains a conserved Serine/Threonine protein kinases catalytic domain (S_TKc) at the N-terminus. Furthermore, cellular distribution proved that GrTBK1 was located in the cytoplasm region. Quantitative real-time PCR analysis revealed that GrTBK1 was ubiquitously expressed in all examined organs, but especially highly in liver. Temporal expression analysis in vivo showed that the expression levels of GrTBK1 were obviously up-regulated in response to GCRV infection. Meanwhile, qRT-PCR assay revealed that the levels of S7 RNA, an important segment of GCRV genome, were higher in the liver than in other tissues. This indicates that GrTBK1 might play a crucial role in responses to GCRV infection in fish. In addition, GrTBK1 activated several type I interferon (IFN) promoters and induced the expression of downstream type I IFN-stimulated genes (ISGs). Furthermore, GrTBK1 obviously phosphorylated the interferon regulatory factor 3 (IRF3). Furthermore, overexpression of GrTBK1 remarkably decreased the GCRV proliferation. In summary, we systematically characterized GrTBK1 and illustrated its role in the innate immune response to GCRV infections.

Combined toxic impacts of thiamethoxam and four pesticides on the rare minnow (Gobiocypris rarus).

To examine pesticide mixture toxicity to aqueous organisms, we assessed the single and combined toxicities of thiamethoxam and other four pesticides (chlorpyrifos, beta-cypermethrin, tetraconazole, and azoxystrobin) to the rare minnow (Gobiocypris rarus). Data from 96-h semi-static toxicity assays of various developmental phases (embryonic, larval, juvenile, and adult phases) showed that beta-cypermethrin, chlorpyrifos, and azoxystrobin had the highest toxicities to G. rarus, and their LC50 values ranged from 0.0031 to 0.86 mg a.i. L-1, from 0.016 to 6.38 mg a.i. L-1, and from 0.39 to 1.08 mg a.i. L-1, respectively. Tetraconazole displayed a comparatively high toxicity, and its LC50 values ranged from 3.48 to 16.73 mg a.i. L-1. By contrast, thiamethoxam exhibited the lowest toxic effect with LC50 values ranging from 37.85 to 351.9 mg a.i. L-1. Rare minnow larvae were more sensitive than embryos to all the pesticides tested. Our data showed that a pesticide mixture of thiamethoxam-tetraconazole elicited synergetic toxicity to G. rarus. Moreover, pesticide mixtures containing beta-cypermethrin in combination with chlorpyrifos or tetraconazole also had synergetic toxicities to fish. The majority of pesticides are presumed to have additive toxicity, while our data emphasized that the concurrent existence of some chemicals in the aqueous circumstance could cause synergetic toxic effect, leading to severe loss to the aqueous environments in comparison with their single toxicities. Thence, the synergetic impacts of chemical mixtures should be considered when assessing the ecological risk of chemicals.

Chemicals Weaken Shoal Preference in the Rare Minnow Gobiocypris rarus

Fish behavioral responses are sensitive to chemicals in the water. We tested rare minnow tested for their shoal preference, and the shoal (school) factors including nutritional status, body size, and shoal (school) size that can make their preference most stable were measured. Then shoal preference was measured again while fish and shoal were subjected to a concentration gradient of chemicals (cadmium ion [Cd2+ ], tricaine methanesulfonate [MS222], and p-chloroaniline). The results showed that single rare minnow preferred shoals over blank control tanks. In addition, this preference was most stable when the shoal was well fed and contained 20 individuals 2 cm long. Although there was no significant response after exposure to p-chloroaniline, the time spent from entering the tank to start moving decreased greatly at concentrations of Cd2+ >3 mg/L and MS222 >11 mg/L. The time the test fish spent close to the shoal significantly decreased at Cd2+ >3 mg/L, MS222 >11 mg/L, and p-chloroaniline >10 mg/L, and the frequency of boundary line crossing increased at the same concentrations. The behavioral parameters changed by 20, 5, and 8 min once the lowest-observed-effect concentrations of Cd2+ , MS222, and p-chloroaniline, respectively, were added. Our study provides useful information on rare minnow shoal preference that may be used for a biological early warning system. Environ Toxicol Chem 2020;39:2018-2027. © 2020 SETAC.

Toxicological interactions of cadmium and four pesticides on early life stage of rare minnow (Gobiocypris rarus).

Although chemicals have been traditionally regulated on an individual basis in aquatic ecosystems, they often co-exist as different types of complex mixtures. Laboratory assays were conducted for assessing the responses of rare minnow (Gobiocypris rarus) to individual and mixture chemicals [trace element cadmium (Cd), thiamethoxam, deltamethrin, malathion and prochloraz]. Data obtained from 96 h semi-static toxicity assays implied that deltamethrin elicited the highest toxic effect on the various developmental phases (larval, juvenile and adult phases) of G. rarus with LC50 values ranging from 0.00061 to 0.25 mg a.i. L-1, followed by prochloraz, malathion and Cd with 96-h LC50 values ranging from 0.49 to 1.1, from 7.1 to 26, and from 7.6 to 15 mg a.i. L-1, respectively. Thiamethoxam elicited the lowest toxic effect on the organisms with 96-h LC50 values ranging from 38 to 202 mg a.i. L-1. Larval phase was not always the most sensitive period in the three detected phases to most of chemicals. Chemical combinations containing deltamethrin and malathion displayed synergetic responses to the larvae of G. rarus. Besides, the binary mixtures of Cd-deltamethrin and Cd-prochloraz also exhibited synergetic response to rare minnows. Our results indicate that extra information is necessary to develop practical criteria for selecting chemical combinations that require legislative attention according to their likelihood to exert synergetic responses. Thence, more investigations on mixture toxicities of various chemicals should be taken as a priority for producing synergetic interaction to improve the environmental risk assessment of chemicals.

Sequence analysis of hepcidin in barbel steed (Hemibarbus labeo): QSHLS motif confers hepcidin iron-regulatory activity but limits its antibacterial activity

Fish hepcidin genes are generally classified into two groups: hamp1-and hamp2-type isoforms. Hamp1-type hepcidin exhibits iron regulatory and antimicrobial activity, while hamp2-type shows a unique role in the immune response against various pathogens. An iron-regulatory motif exists at the N-terminus of hamp1-type hepcidin; however, the functional effect of this motif in fish is not well understood. Here, cDNA of the barbel steed (Hemibarbus labeo) hepcidin gene was cloned and sequenced. The predicted amino acid sequence comprised a signal peptide, a prodomain, and a mature peptide. Phylogenetic tree analysis revealed that barbel steed hepcidin belongs to the fish HAMP1 cluster and is closely related to Chinese rare minnow (Gobiocypris rarus) hepcidin. Barbel steed hepcidin is constitutively expressed in healthy fish tissues, predominantly in the liver. Following iron dextran treatment or Aeromonas hydrophila infection, expression of barbel steed hepcidin increased significantly in tested tissues. In vivo administration of intact hepcidin mature peptide (hep25) significantly and dose-dependently reduced ferroportin 1 expression, while truncated hepcidin mature peptide (hep20) lacking a QSHLS motif had no such effect. In vitro treatment of barbel steed monocytes/macrophages with hep25, but not hep20, increased the labile iron pool levels. Hep25 and hep20 conferred antibacterial activity only against A. hydrophila and Vibrio vulnificus, with greater activity of the latter at low concentrations. Neither hep25 nor hep20 impaired the cell membrane integrity of A. hydrophila, but could hydrolyze its genomic DNA; lack of a QSHLS motif enables hep20 to have a better hydrolytic effect. In summary, we identified an iron-regulatory motif in a fish species and demonstrated that this motif confers hamp1-type hepcidin iron-regulatory activity, but attenuates its antibacterial activity.

Molecular cloning, functional characterization and expression analysis of P65 subunit in response to GCRV infection in rare minnow (Gobiocypris rarus)

P65, the all-important subunit of the transcription factor NF-κB, plays an important role in the regulation of immune response. In this study, the cDNA of P65 subunit of rare minnow Gobiocypris rarus (GrP65) was cloned, and its expression patterns and functional role in rare minnow were investigated. The GrP65cDNA encodes a polypeptide of 573 amino acids, containing a well-conserved Rel-homology domain (RHD). The amino acid sequence analysis showed that GrP65 shared 81% and 69% identity to the grass carp (Ctenopharyngodon idella) and human (Homo sapiens) orthologous, respectively. Phylogenetic analysis revealed that GrP65 clustered with homologues from other teleosts. Cellular distribution anallysis demonstrated that GrP65 was located in the cytoplasm and nucleus. Quantitative real-time PCR analysis showed that GrP65 was ubiquitously expressed in all examined tissues, but especially highly in liver. Temporal expression analysis in vivo showed that the expression levels of GrP65 were significantly up-regulated in liver in response to GCRV infection, which suggested that GrP65 might play a crucial role in recognition and responses to GCRV infection in fish. In addition, GrP65 activated several interferon (IFN) promoters and induced the expression of downstream IFN-stimulated genes (ISGs). Furthermore, overexpression of P65 remarkably decreased the GCRV proliferation, while knockdown of P65 obtained opposite effects. In summary, we systematically characterized GrP65 and demonstrated its role in the innate immune response to GCRV infections.