2,4-Dinitrotoluene (2,4-DNT) exposure induces liver developmental damage and perturbs lipid metabolism and oxygen transport gene expression in zebrafish (Danio rerio).

2,4-Dinitrotoluene (2, 4-DNT) is a common environmental pollutant. The toxic effect on mammals of 2,4-DNT has been well studied, but its toxicity on aquatic organisms is little known. In this study, 126 healthy female zebrafish (Danio rerio) were exposed to different concentrations of 2,4-DNT (0, 2, 4, 8, 12 and 16 mg/L) to determine 96-h semi-lethal concentrations (LC50). And then, 90 female zebrafish were exposed to 0, 2, 4 and 8 mg/L 2,4-DNT for 5 days to study liver toxicity. Exposed zebrafish developed hypoxia features, such as floating head and breathing rapidly, and then died. 96-h LC50 of 2,4-DNT in zebrafish was 9.36 mg/L. Histological data revealed that 2,4-DNT severely damaged the liver tissues, following with the round nucleus, dense interstitial tissue, dense arranged hepatocyte cords and more inflammatory cells. Additionally, the further result showed that the lower levels of lipid transport and metabolism (apoα2, mtp, ppar-α and acox) were noticed. But, exposed to 2,4-DNT for 5 days significantly upregulated the expression levels of genes involved in respiration (hif1a, tfa and ho1, p < 0.05). These results indicated that 2,4-DNT exposure disturbed lipid transport and metabolism and oxygen supply in zebrafish, which could contribute to severe damage in liver and death.

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 10 µg/L for 28 days, and then transferred into clean water to pair spawn. The results showed that cadmium exposure at 5 or 10 µg/L for 28 days 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 5 µg/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 10 µg/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.

The Effect of Unpredictable Chronic Stress on Rare Minnow (Gobiocypris rarus): Growth, Behaviour and Physiology.

Fishes often adjust their behaviour patterns and physiological responses to cope with changing environments, and different life experiences affect them differently. Fishes might adapt to short-term stress, whereas long-term unpredictable stress may lead to various adverse effects. Although some studies have constructed unpredictable stress models of fish, the effect of unpredictable chronic stress (UCS) in the laboratory is poorly understood in fishes. In the current study, we exposed adult rare minnow to an unpredictable chronic stress protocol over 7 and 14 days and measured their response in terms of growth performance, cortisol, neurotransmitter levels (DA, 5-HT, and related metabolites), and behaviour patterns to comprehensively assess the effects of UCS on laboratory rare minnow. We discovered that specific growth rates were significantly decreased, and cortisol levels were lowered in both 7-days and 14-days stress groups. In the behaviour test, the activity level of the 14-days stress group increased, but there was no significant difference in the number of crossings to the center areas, time spent in the center areas, or the speed. In addition, the levels of DA and 5-HT did not change in the stress groups, but the DOPAC and 5-HIAA levels in the 14 days stress group were significantly higher than those in the control group. These results suggested that UCS influences rare minnow growth performance, behaviour patterns, and cortisol levels, and similar stress should be minimised in the laboratory.

Parental cadmium exposure during the spawning period reduces cadmium sensitivity through the antioxidant system in rare minnow (Gobiocypris rarus) larvae.

Cadmium (Cd) is a noxious heavy metal widely dispersed in aquatic systems. Parental Cd exposure of fish species at environmental concentrations has been shown to cause deformities and stunted growth in their offspring. However, the long-term effects and the mechanisms underlying parental Cd exposure in fish species on Cd sensitivity in their offspring remain unclear. To explore the impacts of parental Cd exposures on Cd sensitivity, rare minnow (Gobiocypris rarus) larvae whose parents were exposed to Cd at 0, 5 or 10 µg/L for 28 days were established. Results showed that parental Cd exposure in rare minnow increased the Cd content of its larvae. In terms of malformation rate, mortality rate and total length at 7 days of rare minnow larvae, parental Cd exposure at 5 or 10 µg/L reduced Cd sensitivity. Further mechanistic investigation demonstrated that parental Cd exposure significantly upregulated the expression of antioxidant gene regulated by nuclear factor erythroid 2-related factor 2 (Nrf2) and nuclear factor-kappa B (NF-кB) in rare minnow larvae. In addition, parental Cd exposure significantly elevated the level of reactive oxygen species (ROS) and malondialdehyde (MDA), but markedly decreased catalase (CAT), superoxide dismutase (SOD) and oxidized glutathione (GST) activity. The impact of parental Cd exposure to metallothionein (MT) content and the expression of MT mRNA, a detoxifying metallothionein, showed that parental Cd exposure of rare minnow induced oxidative stress in the larvae. Meanwhile, these results indicated that parental Cd exposure in rare minnow reduced the Cd sensitivity of the larvae via activating the Nrf2-mediated antioxidant system. This project helps us to further understand the toxicological mechanism of Cd in fish species and properly assess its potential ecological risk.

Long-term exposure to microplastics induces intestinal function dysbiosis in rare minnow (Gobiocypris rarus).

Microplastics are ubiquitous in the natural environment, especially in waters, and their potential impact is also a key issue of concern. In this study, we used 1 µm, 1000 µg/L, polystyrene (PS-MPs) particles to analyze the effects after exposure for 14 and 28 days in rare minnow (Gobiocypris rarus). Results indicated that PS-MPs induce structural alterations in the intestinal tissue, including epithelial damage, villi damage and the inflammatory cell infiltration, while the changes were severer after exposure for 28 days. Polystyrene microplastics also significantly increased the activities of catalase (CAT, increased 142 % and 385 % in 14d and 28d), superoxide dismutase (SOD, increased 17.76 % and 23.43 % in the 14d and 28d) and the content of malondialdehyde (MDA, increased 14.5 % and 442 % in the 14d and 28d), glutathione (GSH, increased 146 % and 298 % in the 14d and 28d). The results not only showed the characterization of gut microbial communities in rare minnow, but also indicated that microbial diversity and composition were altered in gut of fish exposed to PS-MPs. In the control groups, Proteobacteria (31.36-54.54 %), Actinobacteriota (39.99-52.54 %), Fusobacteriota (1.43-1.78 %), Bacteriadota (0.31-0.57 %) were the four dominant bacterial phyla in the intestinal of rare minnow. After exposure to microplastics, In the gut microbiota, the proportion of Proteobacteria increased 9.27 % and 30 % with exposure time, while Actinobacteria decreased 37.89 % and significantly different after 28 days. In addition, metabolomic analysis suggested that exposure to PS-MPs induced alterations of metabolic profiles in rare minnow and differential metabolites were involved in energy metabolism, inflammatory responsible secretion, oxidative stress, nucleotide and its metabolomics. In conclusion, our findings suggest that long-term exposure to microplastics could induce intestinal inflammation, oxidative stress, microbiota dysbiosis and metabolic disorder in rare minnow, and the alterations and severity were exacerbated by prolonged exposure. This study has extended our cognition of the toxicity of polystyrene, and enriched theoretical data for exploring the toxicological mechanism of microplastics.

The Effect of Environmental Enrichment on Laboratory Rare Minnows (Gobiocypris rarus): Growth, Physiology, and Behavior.

Environmental enrichment is a method to increase environmental heterogeneity, which may reduce stress and improve animal welfare. Previous studies have shown that environmental enrichment can increase the growth rate, decrease aggressive and anxiety-like behaviors, improve learning ability and agility, and reduce cortisol levels in animals. These effects usually differ between species. Unfortunately, habitat enrichment on laboratory fish is poorly studied and seldom adopted in care guidance. Rare minnows (Gobiocypris rarus) have been cultured as a native laboratory fish in China in barren banks without environmental enrichment since 1990; they have been widely used in studies on ecotoxicology, environmental science, and other topics. The purpose of this study was to investigate the effect of environment enrichment on the growth, physiological status, and anxiety-like behavior of laboratory rare minnows. We observed and analyzed SGR, cortisol levels, DA, DOPAC, 5-HT and 5-HIAA, and anxiety-like behavior indexes after one month of treatment in barren (control) and enrichment tanks. We found that there were no significant differences in SGR, anxiety-like behavior, DA, DOPAC, or 5-HIAA levels between the two treatments. However, higher cortisol and 5-HT levels were observed in the enrichment tanks. This study suggests that rare minnows might be influenced by their living environment, and future related studies should consider their environmental enrichment.

In Situ Assessment of Donghu Lake China Using Rare Minnow (Gobiocypris rarus).

In this work, rare minnow (Gobiocypris rarus) was applied as a sentinel organism and set in cages at control and test sampling sites in Donghu Lake for 4 weeks in March, June, September, and December 2016 to assess the biological toxicity of in situ water. Sampling for active biomonitoring and physicochemical variables was performed weekly. The control was obtained from the outdoor pool of the Institute of Hydrobiology, China. Superoxide dismutase, lipoperoxidation, metallothioneins, acetylcholinesterase activity, and Vtg mRNA expression were determined as biomarkers during the field exposure period. Survival and growth also were monitored to evaluate the overall physiological condition of the fish. The seasonal changes of organic pollutants and trace metals (As, Hg, Cr, Cu, Zn, Cd, Pb) in surface water were determined. The integrated biomarker response (IBR) index was applied to summarize biomarker responses and correlate stress levels with concentrations of organic pollutants and trace metals in the surface water. Results indicated that complex pollution by persistent organic pollutants and heavy metals was present in Donghu Lake and that the in situ exposed organisms were stressed. Moreover, the complex pollution of Donghu Lake in summer and autumn was more serious than that in spring and winter. Active biomonitoring combined with IBR analysis enabled good discrimination among different exposure seasons. The proposed protocol with caged rare minnow revealed marked biological effects caused by the investigated Lake and a useful approach that can easily be extended to monitor water pollution.

Chemicals affect color preference in rare minnow (Gobiocypris rarus).

Behavioral response of fish has been shown that was sensitive to chemicals in water. Herein, larval and adult rare minnows of Gobiocypris rarus were studied for their innate color preference and response to a concentration gradient of chemicals (cadmium ion [Cd2+], tricaine methanesulfonate [MS222], and p-chloroaniline). The results showed that both larval and adult rare minnows preferred blue and green over yellow and red in water with no chemicals added. Larval color preference changed significantly under concentrations of Cd2+ ≥ 0.4 mg/L, MS222 ≥ 3 mg/L, and p-chloroaniline ≥ 10 mg/L; for adults, color preference changed significantly when Cd2+ ≥ 3 mg/L, MS222 ≥ 34 mg/L, and p-chloroaniline ≥ 38 mg/L. In addition, the color preference priorities of both larvae and adults also changed at high concentrations of chemicals. The present study provides useful information on how changes in rare minnow behavior could be used as an early indicator of water pollution.

The Reproductive Strategy of the Rare Minnow (Gobiocypris rarus) in Response to Starvation Stress.

Food abundance plays an important role in the reproduction of fish, especially multiple spawners. Multiple spawners can exhibit various biological strategies when under starvation stress. However, the reproductive strategy used in these fish species remains unknown. To explore whether rare minnows (Gobiocypris rarus) prioritize survival over current reproduction under starvation conditions, paired adult rare minnows were starved for 0, 5, 10, 15 d and their spawning activities were recorded. Anatomical and histological characteristics of unpaired adult rare minnows were examined during starvation and following re-feeding. It was found that only 30-70% of paired rare minnows spawned within 5 d after deprivation of food. Though spawning activity was suppressed by starvation, rare minnows starved for 0, 5, 10, and 15 d waited 3.89 ± 0.78 d, 5.57 ± 3.36 d, 5.83 ± 5.15 d and 6.57 ± 4.50 d, respectively, after re-feeding to resume spawning. The average inter-spawning interval and length until egg production of those starved for 15 d was 4.60 ± 2.37 d and 139.1 ± 67.9 d, respectively, when they spawned with rhythm, which were significantly different from those starved for 0 d (P < 0.05). Anatomical and histological data further revealed that late maturing oocytes degraded after 8 d of starvation, but the gonadosomatic index recovered to initial levels after 8 d of re-feeding. Thus, reproduction of rare minnows was markedly affected by starvation, but rapidly returned to normal upon re-feeding. These observations demonstrated that paired rare minnows prioritize survival by channeling energy from the liver and absorbing late maturing oocytes instead of continuing to reproduce under food deprivation condition.

Susceptibility and immune responses after challenge with Flavobacterium columnare and Pseudomonas fluorescens in conventional and specific pathogen-free rare minnow (Gobiocypris rarus).

The susceptibility of fish from different culture environments to bacterial infection is not well known. The susceptibility and pathological changes of conventional (CV) and specific pathogen-free (SPF) rare minnow (Gobiocypris rarus) infected with two gram-negative bacteria, Flavobacterium columnare and Pseudomonas fluorescens are investigated. Rare minnows were intraperitoneally challenged with two bacterial species to first determine semi-lethal doses (LD50), and then with the LD50 dose, determine innate immune response. Infected rare minnows developed characteristic red bellies and then died. LD50 doses of F. columnare and P. fluorescens were 4.586 × 108 cfu/mL and 2.319 × 1010 cfu/mL for CV rare minnow, and 2.575 × 108 cfu/mL and 1.935 × 1010 cfu/mL, respectively, for SPF rare minnow. The results of RT-PCR showed that the highest levels of toll-like receptor 3 (TLR3), interleukin-6 (IL-6), interferon-2 (IFN-2) and rare minnow Z-DNA binding protein kinase (GrPKZ) mRNA were noticed at 6-48 h post-infection (hpi). In addition, TLR3, IL-6 and IFN-2 in F. columnare challenged rare minnow were more highly expressed than those in P. fluorescens challenged rare minnow, whereas as opposed in the expression of GrPKZ mRNA. Stimulation of innate immune responses is closely related to bacterial virulence. SPF rare minnow might be more susceptible to these bacteria than CV rare minnow, possibly due to their clean environment and lack of resistance. We speculate that clean environment renders rare minnow more susceptible to bacterial infections.

Bioconcentration and depuration of cadmium in the selected tissues of rare minnow (Gobiocypris rarus) and the effect of dietary mulberry leaf supplementation on depuration.

A 56-day trial was conducted to elucidate the bioconcentration and depuration of Cd in the liver and muscle of rare minnow (Gobiocypris rarus) and determine the effect of dietary mulberry leaf supplementation on depuration. Juvenile rare minnow were exposed to environmentally relevant doses of Cd (1 and 10 µg/L) for 28 days of uptake and then allowed 28 days of depuration. The bioaccumulation factors of the treated rare minnow in the liver and muscle were calculated to be between 4.13-4.675 and 1.76-1.94, respectively. This results suggested that Cd had high potential for bioconcentration in rare minnow. To investigate the effect of dietary mulberry leaf supplementation on depuration, the remaining fish of each group were allowed to depurate with different ratios (0%, 10%, and 30% dry weight) of dietary mulberry leaf supplementation for an additional 28 days. Fish weights did not differ significantly (p >  0.05) between the control and mulberry leaf treated groups. Mulberry leaf powder did not significantly affect Cd depuration in the 10 µg/L group or in the muscle of the 1 µg/L group, but caused a significant decrease in Cd content in the liver of the 1 µg/L group (p <  0.05). This work was the first to model the bioconcentration of Cd in rare minnow and showed that mulberry leaf supplementation decreased Cd residues in the liver of the 1µg/L group. Such a finding may promote the development of new approaches to mitigate the potential hazards of heavy metals to human health.

Effect of water temperature on reproductive performance and offspring quality of rare minnow, Gobiocypris rarus.

Water temperature plays a significant role in the reproductive processes of temperate fishes. In the present study, the effects of water temperature on the reproductive performance and offspring quality of rare minnow (Gobiocypris rarus) were evaluated by cultured parent fish at different temperature (18～30℃) in a 2-month trial. The results revealed that rare minnows could spawn continuously within the range from 18℃ to 30℃, and these at 24℃ and 27℃ spawned every 3-4 days. Batch size of rare minnow increased with increasing water temperature, while egg production increased with increasing water temperature and then decreased at 30℃. High water temperature (30℃) had significantly adverse effects on fertilization rate and hatching rate (P<0.05). It was found that the oocyte growth at 18℃, 21℃, and 30℃ were slower than those at 24℃ and 27℃. Histologic analysis further showed that low temperature (18℃ and 21℃) slowed down vitellogenesis and oocyte maturation, while high temperature (30℃) had suppressive effects on oocyte maturation and ovulation. Based on present results, it was concluded that 24-27℃ was optimal breeding temperature for rare minnows and water temperature higher than 30℃ resulting from climate change would pose a threat to its wild populations.