Comparative Observation and Analysis of Preference Behavior Based on Three Types of Taxes and Locomotor Activity in the Goldfish, Carassius auratus.

Psychophysiological studies in vertebrates have focused on taxes as indicators of behavioral change. Actually, a considerable number of studies about anxiety-like and anti-anxiety-like behaviors involving geotaxis, scototaxis, and thigmotaxis have been conducted on fish. However, few analyses considering these behaviors based on taxes in fish have been conducted. Here, using goldfish, we measured the time spent in the bright or dark area of a horizontally long rectangular tank (HLRT), in the upper or lower area of a vertically long rectangular tank (VLRT), and in the central or edge area of a circular tank (CT), respectively, for the first 30 min and the last 30 min in a 3-h period after fish had been introduced to tanks. Dark, lower, and edge preference behaviors were observed for the first 30 min in all tanks. While dark and edge preference behaviors were maintained even for the last 30 min, the lower preference was lost. Swimming distance and the number of area crossings in each tank were also compared between the first 30 min and the last 30 min. Both decreased significantly or tended to decrease in the last 30 min in the HLRT and the CT, but no change was observed in the VLRT. These results suggest that, in goldfish, preference behavior is stable for a short time, and that environmental habituation may depend on the shape of the tank and the elapsed time.

Initial assessment of the toxicologic effects of leachates from 3-dimensional (3-D) printed objects on sperm quality in two model fish species.

The use of 3-dimensional (3-D) printing is gaining popularity in life sciences and driving innovation in fields including aquatic sperm cryopreservation. Yet, little is known about the effects leachates from these objects may have on biological systems. In this study, we investigated if exposure to leachates from 3-D printed objects fabricated from different photo-curable resins could affect sperm quality in two model fish species, zebrafish (Danio rerio) and goldfish (Carassius auratus). Leachates were collected following contact periods of 10 min and 22 h with objects manufactured using a mask LCD resin printer and three different commercially available resins (i.e., standard, eco-friendly, and impact-resistant). Sperm cells were exposed to the leachates for 18 min, and parameters related to sperm motility, cell count, and membrane integrity were evaluated. All experiments were blinded. Leachate originating from contact with impact-resistant resin for 10 min significantly reduced the cell count of zebrafish sperm, while leachate originating from contact with standard resin for 22 h significantly increased the beat cross frequency of goldfish sperm. The changes were not observed across species and no adverse effects were recorded in percent motility, velocity, amplitude of lateral head movement, or membrane integrity of sperm. Our findings demonstrate that exposure to leachates from certain 3-D printed resins can affect sperm quality, while other resins may support sperm quality evaluation. Further investigations are warranted to assess other parameters, effects, and their biological relevance for a variety of aquatic species.

Low-Cost and Rapid Method of DNA Extraction from Scaled Fish Blood and Skin Mucus.

PCR-based DNA amplification has been one of the major methods in aquaculture research for decades, although its use outside the modern laboratory environment is limited due to the relatively complex methods and high costs. To this end, we investigated a swabbing and disc protocol for the collection of DNA samples from fish which could extract DNA from fish skin mucus by a non-invasion technique costing only $0.02 (USD) and requiring less than 30 seconds. The disc method that we chose could use the cheap filter paper to extract DNA from above 104 crucian carp blood cells, which is comparable to the commercial kit. By using skin mucus swabbing and the disc method, we can obtain amplification-ready DNA from mucus to distinguish different species from our smallest fish (medaka, ~2.5 cm and crucian carp, ~7 cm) to our biggest fish (tilapia, ~15 cm). Furthermore, the viral pathogen Carassius auratus herpesvirus (CaHV) of crucian carp was detected using our method, which would make performing molecular diagnostic assays achievable in limited-resource settings including aquafarms and aqua stores outside the laboratory environment.

Assessment bioaccumulation factor (BAF) of methyl siloxanes in crucian carp (Carassius auratus) around a siloxane production factory.

Methyl siloxanes are identified as emerging persistent toxic compounds and the ecological environment risks of these compounds have been caused of great concern worldwide. In this study, the concentrations of methyl siloxanes were reported in dissolved water and crucian carp around a methyl siloxane production factory located in Liaoning Province, Northeast China. D4, D5, D6, D7, L4, L5 and L6 were detectable both in dissolved water and crucian carp. The total concentrations of 7 methyl siloxanes (Σ7MS) were 14 ± 6.3 ng/L in dissolved water and 43 ± 22 ng/g ww in crucian carp, respectively. D5 has the highest concentration both in dissolved water (5.5 ± 3.5 ng/L) and crucian carp (17 ± 11 ng/g ww). Based on the monitoring values, bioaccumulation factor (BAF) of these compounds were calculated. Significant bioaccumulation potential was observed for D4 (BAF = 5900 ± 3500 L/kg) based on the bioaccumulation criteria suggested by USEPA and EU (BAF > 5000 L/kg). To our understanding, this is the first report of BAF values of methyl siloxane in field study, which will provide important support for further assessment of bioaccumulation of these compounds.

Volumetric analysis and morphological assessment of the ascending olfactory pathway in an elasmobranch and a teleost using diceCT.

The size (volume or mass) of the olfactory bulbs in relation to the whole brain has been used as a neuroanatomical proxy for olfactory capability in a range of vertebrates, including fishes. Here, we use diffusible iodine-based contrast-enhanced computed tomography (diceCT) to test the value of this novel bioimaging technique for generating accurate measurements of the relative volume of the main olfactory brain areas (olfactory bulbs, peduncles, and telencephalon) and to describe the morphological organisation of the ascending olfactory pathway in model fish species from two taxa, the brownbanded bamboo shark Chiloscyllium punctatum and the common goldfish Carassius auratus. We also describe the arrangement of primary projections to the olfactory bulb and secondary projections to the telencephalon in both species. Our results identified substantially larger olfactory bulbs and telencephalon in C. punctatum compared to C. auratus (comprising approximately 5.2% vs. 1.8%, and 51.8% vs. 11.8% of the total brain volume, respectively), reflecting differences between taxa, but also possibly in the role of olfaction in the sensory ecology of these species. We identified segregated primary projections to the bulbs, associated with a compartmentalised olfactory bulb in C. punctatum, which supports previous findings in elasmobranch fishes. DiceCT imaging has been crucial for visualising differences in the morphological organisation of the olfactory system of both model species. We consider comparative neuroanatomical studies between representative species of both elasmobranch and teleost fish groups are fundamental to further our understanding of the evolution of the olfactory system in early vertebrates and the neural basis of olfactory abilities.

Assessment of cadmium toxicity and its possible effects on goldfish (Carassius auratus), employing microscopy and biochemical techniques.

The present study aimed to evaluate and compare the toxicity level of dietary and waterborne Cadmium (Cd) source on the Goldfish Carassius auratus (C. auratus) using various biochemical and microscopic shreds of evidence. C. auratus were divided into two groups based on Cadmium supplementation methods subjected to the same concentrations in the diet and water; moreover, control (Ctrl) group was set up for comparison. Dietary supplemented group (DSG) group and (Aqueous supplemented groups) ASG were supplied with 0.5% of the Cadmium. Successful completion of the study resulted in the lucrative outcomes, and we noticed significantly high concentrations of Cadmium in the gills of the ASG fish while considerably low levels in the gills of DSG. Histopathological evidence showed clear and distinct changes in the liver cells, dilated sinusoids and unusual occurrence of Kupffer cells were noticed in the AS group fish. At the same time, there were also noticeable changes in the DS group compared to CTRL. Visible changes can be seen on the outer morphology of gills, lamella of ASG and DSG both were shrunk and broken, similar changes were noticed in the muscles myoepithelium. The stress parameters were differentially expressed in both the groups; waterborne Cadmium caused a pronounced release of AST, ALT, cortisol, and glucose concentrations. Comparative mortality showed the highest death rate in the ASG fish compared to DSG fish. The present study depicts the highest toxicity of Cadmium in the soluble Addendum form moreover, administered route and time length are crucial factors.

Toxicity assessment of municipal sewage treatment plant effluent by an integrated biomarker response in the liver of crucian carp (Carassius auratus).

In this study, crucian carp (Carassius auratus) was exposed to the increasing concentrations of municipal sewage treatment plant effluent (MSTPE) for 15 days, and the activities of antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and acetylcholinesterase (AChE), together with the contents of malondialdehyde (MDA) and glutathione (GSH) in the liver of C. auratus were investigated. Moreover, the integrated biomarker response (IBR) approach was applied to assess the adverse effects of MSTPE in freshwater. The aim of the study was to provide an effective biological indicator for evaluating the toxicity effects and ecological risks of MSTPE in the freshwater environment quantitatively. Results showed that MSTPE could cause oxidative damage to the liver of C. auratus, which reflected through the increasing MDA content over the exposure period. MSTPE also led to the biochemical responses of antioxidant defense in C. auratus liver, such as the enhancement of SOD, CAT, and GPx activities, as well as the inhibition of AChE activity and GSH content. It was found that MDA, SOD, GPx, and GSH could be used as the biomarkers for reflecting the adverse effects of MSTPE in the receiving freshwater on the 12th day of exposure. A significant increase of IBR values was observed as the increasing concentration of MSTPE, and the IBR values presented a significant positive correlation (r = 0.891, P < 0.05) with the increasing concentrations of MSTPE, indicating that IBR approach is a promising tool for assessing the toxicity effects of MSTPE in environmental freshwater.

Assessment of the Effects of Bisphenol A on Dopamine Synthesis and Blood Vessels in the Goldfish Brain.

Bisphenol A (BPA) is an abundant contaminant found in aquatic environments. While a large number of toxicological studies have investigated the effects of BPA, the potential effects of BPA exposure on fish brain have rarely been studied. To understand how BPA impacts goldfish brains, we performed a transcriptome analysis of goldfish brains that had been exposed to 50 µg L-1 and 0 µg L-1 BPA for 30 days. In the analysis of unigene expression profiles, 327 unigenes were found to be upregulated and 153 unigenes were found to be downregulated in the BPA exposure group compared to the control group. Dopaminergic signaling pathway-related genes were significantly downregulated in the BPA exposure group. Furthermore, we found that serum dopamine concentrations decreased and TUNEL (terminal deoxynucleotidyl transferase 2-deoxyuridine, 5-triphosphate nick end labeling) staining was present in dopamine neurons enriched regions in the brain after BPA exposure, suggesting that BPA may disrupt dopaminergic processes. A KEGG analysis revealed that genes involved in the fluid shear stress and atherosclerosis pathway were highly significantly enriched. In addition, the qRT-PCR results for fluid shear stress and atherosclerosis pathway-related genes and the vascular histology of the brain showed that BPA exposure could damage blood vessels and induce brain atherosclerosis. The results of this work provide insights into the biological effects of BPA on dopamine synthesis and blood vessels in goldfish brain and could lay a foundation for future BPA neurotoxicity studies.

Toxicological Assessment of Ammonia Exposure on Carassius auratus red var. Living in Landscape Waters.

To understand the toxic mechanism of ammonia and identify effective biomarkers on the oxidative stress for the fish Carassius auratus red var., acute and chronic toxicity tests were conducted. The 96-h LC50 of total ammonia nitrogen (TAN) for C. auratus was 135.4 mg L-1, the corresponding unionized ammonia (NH3) concentration was 1.5 mg L-1. The activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), glutathione-peroxidase (GSH-Px) and glutathione (GSH) showed an increase with a subsequent falling, while the malondialdehyde (MDA) increased during the chronic test. The SOD, MDA, and GSH could be effective biomarkers to evaluate the TAN oxidative stress, the maximum acceptable toxicant concentration (MATC) was 11.3 mg L-1 for TAN. To our knowledge, this is the first study to propose biomarkers to evaluate potential environmental risk and establish a risk threshold for TAN in C. auratus.

Bioaccumulation and health risk assessment of trace metals in fish from freshwater polyculture ponds in Chengdu, China.

The freshwater polyculture pond culturing occupied an important position in the aquaculture industry. Accumulation of trace metals was investigated in water, sediments, and fish (Carassius auratus, Cyprinus carpio, Ctenopharyngodon idellus) from typical polyculture ponds in Chengdu, China. The results showed most of the pond water in Chengdu were safe for fish cultivation. The Cd and Cr concentrations in sediment samples from sites S3, S4, and S9 which were near the industrial park and road with a high traffic volume were higher than those of the other sites. Cu, Cr, Fe, Zn, Mn, Ni, and Pb in sediments were unpolluted, while Cd was unpolluted to moderately polluted due to anthropogenic activities. Cu, Cd, and Pb in fish pond sediment of Chengdu had higher potential mobility under normal environmental circumstances. The trace metal concentrations in liver of three fish species were all higher than those in muscle tissues. The order of bioaccumulation factor (BAF) values for trace metals was Cr > Cu > Pb > Zn > Cd > Ni > 20. The concentrations of Cu, Cd, Pb, Zn, and Cr in the muscle of three fish species were all below the local and international maximum permissible levels. The target hazard quotient (THQ) and hazard index (HI) of trace metals in aquaculture fish ponds in Chengdu were lower than 1, which indicated that the consumption of grass, crucian, and common carp cultivated in the aquaculture ponds of Chengdu pose no health risk to the residents.

Binary patch assessment by goldfish under safe and dangerous conditions.

Food resources can occur heterogeneously in space or time and differ in their abundances. A forager should be able to determine the value of a patch and choose optimally how to exploit it. However, patch choice and exploitation may be influenced by predation risk. Using the Giving up Densities (GUDs) technique, we evaluated goldfish patch assessment and choice in a binary patch choice experiment. We offered a pair of unequal food patches containing high and low food quantity. We quantified goldfish foraging behavior in the presence and absence of a predator. Goldfish groups equalized the GUDs in the two patches in safe environments but left higher GUDs in the rich patch under predation risk. The results suggest that goldfish can use both "patch assessment rule" and "fixed time rule" to exploit resource patches and trade off food and danger.

Trace elements in four freshwater fish from a mine-impacted river: spatial distribution, species-specific accumulation, and risk assessment.

The concentrations of 16 elements (Mg, Al, Ca, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Sr, Cd, Ba, and Pb) were determined in four fish species (Carassius auratus, Squaliobarbus curriculus, Pelteobagrus fulvidraco, and Silurus asotus) collected in the Xiang River, a mine-impacted river in Southern China. The mean values of the elements analyzed in fish muscles were in the decreasing order of Mg > Ca > Zn > Fe > Sr > Al > Cu > Mn > Ba > As > Cr > Pb > Ni > V > Co > Cd. The concentrations of Mg, Ca, Sr, Ba, and Cu in omnivorous species were found to be significantly higher (p < 0.05) than those in carnivorous species. Negative correlations observed between most element concentrations and fish sizes indicated the younger individuals accumulated more elements than the older ones. Principle component analysis and orthogonal partial least squares discriminant analysis were employed to characterize the effects on element bioaccumulation using the element concentration matrix. The elemental profiles preferred to cluster according to differences in fish species rather than in sampling sites. The potential health risk evaluated through Monte Carlo simulation showed no appreciable adverse impact on human health from exposure to trace elements in fish muscles through consumption.

Ecological risk assessment of a contaminated stream using multi-level integrated biomarker response in Carassius auratus.

The goal of this study was to evaluate the adverse effects of wastewater effluents on freshwater crucian carp, Carassius auratus, inhabiting Sincheon stream using the integrated biomarker response (IBR) at the genotoxic (micronucleus [MN] test), oxidative stress (activity of catalase [CAT] and glutathione S-transferase [GST], and level of lipid peroxidation [LPO]), histopathological (degree of tissue changes [DTC]), and physiological (condition factor [CF] and liver somatic index [LSI]) levels. The CF and LSI were significantly (p < 0.05) enhanced in fish from downstream sites (DS1 and DS2) as compared to that of upstream (US) fish samples. Moreover, a significant increase in morphometric indices (DTC) was observed in C. auratus collected from downstream sites (p < 0.05) and histopathological responses showed the degree of pathogenicity in the order of liver > kidney > gills. The activities of CAT, GST, and LPO in fish from the DS1 and DS2 sites were notably increased in gills, liver, and kidney compared to that of fish from the US site. Additionally, the MN test level in C. auratus from the DS1 and DS2 were significantly increased (p < 0.05) when compared with that of the US site. Considering the higher bioaccumulation of Cd, Co, Cr, Mn, Ni, and Pb in gills, liver, and kidney of C. auratus collected from downstream sites compared to that of the upstream site (p < 0.05), the observed toxicity was likely attributable to metal accumulation. The multi-level IBR index was higher at the DS1 site (15.08) than at the DS2 (1.02) and the reference US (0.00) sites. Therefore, these findings demonstrated that wastewater effluent discharge induces significant DNA damage, oxidative stress, and tissue injuries in C. auratus and suggested that the multi-level IBR approach should be used to quantify these effects on fish in streams and rivers.

Comparative assessment of the adverse outcome of wastewater effluents by integrating oxidative stress and histopathological alterations in endemic fish.

This study evaluated the adverse effect of wastewater effluents on three fish species, Carassius auratus, Zacco platypus, and Zacco koreanus, collected in the Eungcheon, Mihocheon, and Busocheon streams, respectively. Fish gills, liver, and kidneys from the mixing zone (MZ) and sites upstream (US) and downstream (DS) of the MZ were analyzed for oxidative stress responses and histology. Catalase and glutathione S-transferase activity was significantly higher at MZ and DS than from US (p<0.05), indicating induction of antioxidant defense mechanisms. Additionally, degree of tissue changes (DTC) indicated highest histopathological alteration in MZ, followed by DS and US. Integrated biomarker response (IBR) for oxidative stress and histopathological alterations showed higher values consistently for Z. koreanus than other two species. Water temperature, EC, and TN levels seemed to be responsible for the observed biomarker responses. These findings indicate that thermal hot spring effluent discharged into Busocheon steam induced the most significant impact on the cool water species (Z. koreanus). Overall, this study suggests that the IBR index is a very useful tool for monitoring in situ adverse effects of wastewater effluents on fish, particularly for histopathological alterations representing prolonged impact.

Characterization and functional assessment of the NLRC3-like molecule of the goldfish (Carassius auratus L.).

The NLRC3-like (NLRC3L) molecule from the goldfish transcriptome database was identified and characterized. Quantitative gene expression analysis revealed the highest mRNA levels of NLRC3L were in the spleen and intestine, with lower mRNA levels observed in muscle and liver. Goldfish NLRC3L was differentially expressed in goldfish immune cell populations with highest mRNA levels measured in PBLs and macrophages. We generated a recombinant form of the molecule (rgfNLRC3L) and an anti-CT-NLRC3L IgG. Treatment of goldfish primary kidney macrophages in vitro with ATP, LPS and heat-killed Aeromonas salmonicida up-regulated the NLRC3L mRNA and protein. Confocal microscopy and co-immunoprecipitation assays indicated that goldfish rgfNLRC3L interacted with apoptosis-associated spec-like protein (ASC) in eukaryotic cells, indicating that NLRC3L may participate in the regulation of the inflammasome responses. The dual-luciferase reporter assay showed that NLRC3L over-expression did not cause the activation of NF-κB, but that it cooperated with RIP2 to down-regulate NF-κB activation. Our results indicate that the NLRC3L may function as a regulator of NLR pathways in teleosts.

Accumulation and health risk assessment of trace elements in Carassius auratus gibelio from subsidence pools in the Huainan coalfield in China.

Microelement (As, Cd, Cr, Cu, Ni, Pb, and Zn) concentrations were determined in the muscle, skin, gill, and liver tissues of Carassius auratus gibelio collected from subsidence pools at three different coal mines in the Huainan coalfield in China. The concentrations of elements in the water were within the allowable levels for raising fish. However, the higher levels of these metals in sediment may pose potential harm on fish. It was found that the concentrations of Cr, Ni, and Zn in all fish tissues were higher, while As, Cd, and Pb levels were relatively low. Microelement accumulation appeared to be more widespread in subsidence pools than that in natural water. Elements accumulated in fish tissues differently: the highest metal concentrations were generally found in the liver tissues of the fish analyzed, whereas the lowest were recorded in the muscles. The mean element concentrations in muscle tissue from C. auratus gibelio collected from subsidence pools (As, 0.16 mg/kg; Cd, 0.06 mg/kg; Cr, 6.21 mg/kg; Cu, 1.61 mg/kg; Ni, 3.88 mg/kg; Pb, 1.76 mg/kg; and Zn, 12.80 mg/kg dry weight) were far below the allowable limit of the hygienic standard in fish proposed by the Ministry of Health in China, suggesting that the fish were safe for human consumption. A health risk assessment also suggested there was no risk from the analyzed elements for inhabitants near the Huainan coalfield that consume fish.

Thyroid disruption in male goldfish (Carassius auratus) exposed to leachate from a municipal waste treatment plant: Assessment combining chemical analysis and in vivo bioassay.

Several classes of thyroid-disrupting chemicals (TDCs) have been found in refuse leachate, but the potential impacts of leachate on the thyroid cascade of aquatic organisms are yet not known. In this study, we chemically analyzed frequently reported TDCs, as well as conducted a bioassay, to evaluate the potential thyroid-disrupting effects of leachate. We used radioimmunoassay to determine the effects of leachate exposure on plasma 3,3',5-triiodo-l-thyronine (T3), 3,3',5,5'-l-thyroxine (T4), and thyroid-stimulating hormone (TSH) levels in adult male goldfish (Carassius auratus). We also investigated the impacts of leachate treatment on hepatic and gonadal deiodinases [types I (D1), II (D2), and III (D3)] and gonadal thyroid receptor (TRα-1 and TRß) mRNA expressions by using real-time polymerase chain reaction. The results indicated the presence of five TDCs (bisphenol A, 4-t-octylphenol, di-n-butyl phthalate, di-n-octyl phthalate, and diethylhexyl phthalate); their mean concentrations in the leachate were 18.11, 2.76, 4.86, 0.21, and 9.16 µg/L, respectively. Leachate exposure induced plasma T3 and TSH levels in male fish, without influencing the plasma T4 levels. The highly elevated D2 mRNA levels in the liver were speculated to be the primary reason for the induction of plasma T3 levels. Disruption of thyroid functions by leachate was also suggested by the up-regulation of D1 and D2 as well as TRα-1 mRNA levels in the gonads. Prominent thyroid disruptions despite the very low TDC concentrations in the exposure media used in the bioassay strongly indicated the existence of unidentified TDCs in the leachate. Our study indicated the necessity of conducting in vivo bioassays to detect thyroid dysfunctions caused by leachate.

Physiological and condition-related traits in the gynogenetic-sexual Carassius auratus complex: different investments promoting the coexistence of two reproductive forms?

BACKGROUND: Carassius auratus complex is an extraordinary species complex including the diploid and polyploid forms exhibiting asexual and sexual reproduction modes. The coexistence of both forms in the same habitats is currently reported. The stable coexistence of asexual and sexual forms assumes some disadvantages for asexuals that balance the costs of sex. In our study, we hypothesized and tested the differences in physiological (including heamatological and immunological), growth-related, condition-related, and fitness-related traits between gynogenetic females and sexuals. RESULTS: Our results revealed similar growth performance in gynogenetic females and sexuals measured by body size and weight, or expressed by condition factor. The energy allocation in reproduction measured by the relative size of gonads revealed no difference between gynogenetic and sexual females; in addition, both females in spawning expressed the same estradiol levels in blood plasma. We found a gender specific trade-off between investment in reproduction and immunocompetence (measured by the spleen-somatic index). Higher aerobic performance expressed by the heart index and higher oxygen-carrying capacity were found in sexual males, with increasing values before and during spawning. Our study evidenced significantly lower aerobic performance but higher oxygen-carrying capacity per erythrocyte in gynogenetic females when compared to sexuals. IgM production differed between gynogens and sexuals of C. auratus complex. CONCLUSIONS: Our study indicates that a similar amount of energy is invested by both gynogenetic and sexual females of C. auratus complex in reproductive behaviour. We suggest that lower aerobic performance in gynogens may represent their physiological disadvantage balancing the cost of sexual reproduction. A trade-off between the number of erythrocytes and the oxygen-carrying capacity per erythrocyte in sexual males and gynogenetic females may contribute to the coexistence of gynogenetic and sexual forms. In addition, the differences in specific immunity between gynogens and sexuals may also reduce the evolutionary disadvantage of sexual reproduction. In conclusion, we propose that several mechanisms contribute to the coexistence of the gynogenetic-sexual C. auratus complex.

In vitro and in vivo assessment of the effect of antiprotozoal compounds isolated from Psoralea corylifolia against Ichthyophthirius multifiliis in fish.

Ichthyophthirius multifiliis, an external fish parasite, often causes significant economic damage to the aquaculture industry. Since the use of malachite green was banned, the search of alternative substance to control I. multifiliis infections becomes stringent. In present study, in vitro and in vivo anti-ich efficacies of isopsoralen and psoralidin, two active compounds isolated from methanol extract of Psoralea corylifolia by bioassay-guided fractionation based on the efficacy of anti-ich encysted tomonts, were evaluated. In vitro antiprotozoal efficacy of psoralidin is much better than that of isopsoralen. Psoralidin can kill all theronts at concentrations of 0.8 mg/L or more during 4 h exposure; and terminate reproduction of I. multifiliis post 6 h exposure of protomonts to 0.9 mg/L and encysted tomonts to 1.2 mg/L. In vivo trials showed that 5 h exposure of infected fish to 2.5 mg/L of psoralidin significantly reduced the number of theronts released from tomonts. Furthermore, we observed that a part of protomonts, collected from infected fish post treatment, presented characteristic morphological changes of apoptosis after staining with Annexin V-EGFP/propidium iodide, indicating the possible mechanism of psoralidin against I. multifiliis trophont in situ. On the basis of these results, psoralidin can be used as a potential lead compound for the development of commercial drug against I. multifiliis.

Antagonistic joint toxicity assessment of two current-use phthalates with waterborne copper in liver of Carassius auratus using biochemical biomarkers.

Di-n-butyl phthalate (DBP) and di-2-ethylhexyl phthalate (DEHP) are two kinds of widely-used phthalates, whereas Cu (II) is a common valence state of copper. They have been ubiquitously detected in the aquatic environment, but information on their joint toxicity on aquatic organisms is scarce. In this study, we evaluated the combined effects of copper and these two phthalates to the goldfish (Carassius auratus) by detecting the antioxidant responses in liver after exposure for 7 and 21 days. The exposure concentrations were in a range relevant to their levels in the natural aquatic environment. The results indicated that DBP, DEHP and Cu (II) can affect the antioxidant status in fish liver, evidenced by the significant alterations of antioxidant defenses (superoxide dismutase, catalase, glutathione) and malondialdehyde. Antagonistic effects were found in the joint toxicity of Cu (II) and DBP or DEHP using the integrated biomarker response (IBR) index. These findings have important implications in the risk assessments of phthalates mixed with some heavy metals in the aquatic environment.