Effects of chlorantraniliprole-based pesticide on transcriptional response and gut microbiota of the crucian carp, Carassius carassius.

Chlorantraniliprole (CAP) is a presentative diamide pesticide utilized in agricultural area and as well as rice-fish co-culture system for pest control. However, the understanding of toxic effects of CAP on fish species is still incomplete. In the present study, we performed an integrated study of the acute toxicity and bioaccumulation of CAP on the crucian carp, Carassius carassius, a fish species widely distributed in freshwater area in China and commonly farmed in the rice-fish co-culture systems. Besides, biochemical changes, transcriptional responses and gut microbiota of fish were investigated upon sub-chronic CAP exposure. The results showed that CAP is low toxic to crucian carp with a 96 h LC50 of 74.824 mg/L, but has considerable accumulation in the fish muscles when exposed to 3 mg/L of CAP for 14 d and still detectable after 18 d recovery in fresh water. For sub-chronic test, fish were exposed to CAP at 0, 0.3, 3 and 30 mg/L respectively for 14 d. CAP induced oxidative stress and detoxification inhibition in the liver of fish by decreasing antioxidative and detoxicated enzymes activities and downregulating relevant genes expression. In addition, disrupted gut flora composition was found in all experimental groups by the 16 S rRNA sequencing data, indicating the gut microbiota dysbiosis in crucian carp and potential adverse host effect. All the results suggest that CAP at sublethal concentrations has prominent toxic effect on crucian carp and more attentions should be paid especially using directly in an integrated aquaculture system.

First Isolation and Multilocus Sequence Typing of Brucella canis from a Subclinically Infected Pet Dog in China.

Canine brucellosis, a worldwide zoonotic disease, is mainly caused by Brucella canis. In the present study, we isolated a Brucella strain (CD3) from a subclinically infected pet dog in Sichuan Province, Southwestern China. Classical biotyping methods and molecular biological tests (BCSP31 and BcSS PCR) proved that the strain belonged to B. canis. Furthermore, B. canis CD3 and another two B. canis strains (WJ5 and YA4), which were all isolated from pet dogs in Sichuan, were genotyped using multilocus sequence typing (MLST). Our results showed that the three B. canis strains were identified as the same sequence type (ST21). The present study is the first to report B. canis strain from a subclinically infected pet dog in China, indicating a potential threat to public health posed by subclinical infections in pet dogs. We suggest that screening for B. canis should be incorporated into routine medical examination of pet dogs and other companion animals in areas with a history of animal or human brucellosis.

Characteristics of intestinal microbiota in C57BL/6 mice with non-alcoholic fatty liver induced by high-fat diet.

Introduction: As a representation of the gut microbiota, fecal and cecal samples are most often used in human and animal studies, including in non-alcoholic fatty liver disease (NAFLD) research. However, due to the regional structure and function of intestinal microbiota, whether it is representative to use cecal or fecal contents to study intestinal microbiota in the study of NAFLD remains to be shown. Methods: The NAFLD mouse model was established by high-fat diet induction, and the contents of the jejunum, ileum, cecum, and colon (formed fecal balls) were collected for 16S rRNA gene analysis. Results: Compared with normal mice, the diversity and the relative abundance of major bacteria and functional genes of the ileum, cecum and colon were significantly changed, but not in the jejunum. In NAFLD mice, the variation characteristics of microbiota in the cecum and colon (feces) were similar. However, the variation characteristics of intestinal microbiota in the ileum and large intestine segments (cecum and colon) were quite different. Discussion: Therefore, the study results of cecal and colonic (fecal) microbiota cannot completely represent the results of jejunal and ileal microbiota.

Imidacloprid induces locomotion impairment of the freshwater crayfish, Procambarus clarkii via neurotoxicity and oxidative stress in digestive system.

Imidacloprid (IMI) is used in integrated farming like the rice-crayfish co-culture system to prevent water weevil. However, the toxic effect of IMI on the freshwater crayfish Procambarus clarkii is unknown. In the current study, the effects of IMI on the locomotion, antioxidative status, digestion and intestinal microbiota of P. clarkii were investigated. The results showed that IMI caused locomotion impairment with reduced crawl velocity, and attenuated their dark preference, aggressiveness and reversal ability. Inhibited AChE in muscle and hepatopancreas indicates the neurotoxicity of IMI which may directly lead their locomotion dysfunction. The increase of antioxidative enzymes activity and MDA level were found after 25 µg/L and 250 µg/L exposure. Significant up-regulation of several antioxidative and immune-related genes, including CZ-SOD, CAT, GPx, GST, AFL, proPO, HSP27 and HSP70 confirmed that oxidative stress was induced in all treatments when exposed to IMI. In addition, there was significant increase of LDH, indicating the different energy allocation during the exposure. Meanwhile, results from DNA damage analysis showed elevated OTM value and 8-OHdG level in hepatopancretic cells. On the other hand, decreases of alpha-amylase, lipase and increase of trypsin in hepatopancreas was observed at 25 and 250 µg/L. In addition, significant changes of composition of intestinal microbiota at both phylum and genus levels were observed according to the 16S rRNA sequencing results. Increase of pathogenic genera and decrease of beneficial bacterial communities revealed the disequilibrium of intestinal flora of crayfish. In summary, results in the present study suggest that IMI at environmentally realistic concentration could induce AChE inhibition and oxidative stress, conjointly leading the locomotion impairment in crayfish. IMI also affected the digestive functions by enzymes inhibition and gut microbiota dysbiosis.

DNA damage, immunotoxicity, and neurotoxicity induced by deltamethrin on the freshwater crayfish, Procambarus clarkii.

Pyrethroid pesticides are applied to both agricultural and aquacultural industries for pest control. However, information of their impact on the commercial important freshwater crayfish, Procambarus clarkii is scarce. Therefore, the present study aimed to characterize to effects of a commonly used pyrethroid pesticide, deltamethrin on DNA damage, immune response, and neurotoxicity in P. clarkii. Animals were exposed to 7, 14, and 28 ng/L of deltamethrin, which correspond to 1/8, 1/4, and 1/2 of the LC50 (96 hours) of this pyrethroid to P. clarkii. Significant increase of olive tail moment (OTM) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) was found after deltamethrin exposure in a dose-dependent way. Total hemocyte counts (THC) and activities of immune-related enzymes including acid phosphatase (ACP), lysozyme (LZM), and phenoloxidase (PO) were all decreased and significantly lower than control at concentration of 28 ng/L after 96 hours exposure. Acetylcholinesterase (AChE) activity, an indicator of neurotoxic effect was investigated and it was decreased significantly in muscles at 14 and 28 ng/L after 24 hours exposure. The level of intracellular reactive oxygen species (ROS) in hemocytes was also measured and the significant increase of ROS was found at 14 and 28 ng/L concentrations. The results revealed that deltamethrin induced DNA damage, immunotoxicity, and neurotoxicity in P. clarkii by excessive generation of ROS. Because of the dose-dependent responses of all parameters under exposure of deltamethrin at environmentally realistic concentrations, these parameters could be used as sensitive biomarkers for risk assessment of deltamethrin in aquaculture area.

Antioxidative status, immunological responses, and heat shock protein expression in hepatopancreas of Chinese mitten crab, Eriocheir sinensis under the exposure of glyphosate.

As a broad-spectrum herbicide, glyphosate was extensively utilised in China for several decades. The contradiction between glyphosate spraying and crab breeding in the rice-crab co-culture system has become more obvious. In this study, the antioxidative status and immunological responses of Chinese mitten crab, Eriocheir sinensis, under sublethal exposure of glyphosate were investigated by detecting the antioxidative and immune-related enzyme activity, acetylcholinesterase (AChE) activity and relative mRNA expression of heat shock proteins (HSPs) in hepatopancreas. The results showed that high concentrations of glyphosate (44 and 98 mg/L) could induce significant alteration of superoxide dismutase (SOD), peroxidase (POD), acid phosphatase (ACP), alkaline phosphatase (AKP), and phenoloxidase (PO) activities by first rising then falling during the exposure. However, AChE activity in all treatments including 4.4 mg/L was inhibited markedly after 6 h of exposure. In addition, the relative mRNA expression of HSP 60, HSP 70, and HSP 90 was significantly upregulated at both 48 h and 96 h. These results revealed that glyphosate has a prominent toxic effect on E. sinensis based on antioxidative and immunological response inhibition and AChE activity reduction even at the lowest concentration of 4.4 mg/L, and a protective response by upregulation of HSPs was carried out by the species to ease the environmental stress.

Effects of deltamethrin on the antioxidant defense and heat shock protein expression in Chinese mitten crab, Eriocheir sinensis.

In the present study, the status of antioxidant response and molecular regulation in Chinese mitten crab, Eriocheir sinensis under the exposure of synthetic pyrethroid deltamethrin were investigated by means of measuring the antioxidative enzyme activity and relative mRNA expression of heat shock proteins (HSPs) in hepatopancreas. The results showed that activity of superoxide dismutase (SOD) and catalase (CAT) decreased remarkably in all treatments except the SOD activity at concentration of 0.073 µg/L. The oxidative stress products malondialdehyde (MDA) and hydrogen peroxide (H2O2) increased significantly at high concentrations while no significant difference was observed at concentrations of 0.073 and 0.146 µg/L throughout the experiment. Meanwhile, the relative mRNA expression of HSP 60, HSP 70 and HSP 90 was significantly up-regulated in all treatments at each time point. All resutls above indicated that deltamethrin has prominent toxic effect on E. sinensis based on antioxidative enzyme inhibition and oxidative products accumulation at environmental related concentrations, and a protective response by up-regulation of HSPs was carried out by animals to mitigate the oxidative stress. In addition, SOD, CAT, MDA, H2O2 and the expression of heat shock proteins, especially HSP 70 in hepatopancreas could be sensitive biomarkers in the assessment of toxic effect of deltamethrin on E.sinensis.

Assessment of the oxidative and genotoxic effects of the glyphosate-based herbicide roundup on the freshwater shrimp, Macrobrachium nipponensis.

In the present study, an acute toxic test was performed to assess the oxidative stress and genotoxic effects of the herbicide on the freshwater shrimp Macrobrachium nipponensis. The results showed that the 48-h and 96-h LC50 values of Roundup to M. nipponensis were 57.684 mg/L and 11.237 mg/L, respectively. For further investigation, the shrimps were exposed to sublethal concentrations of 0.35, 0.70, 1.40, 2.80 and 5.60 mg/L for 96 h. A significant decrease in total haemocytes count (THC) was observed at concentration of 5.60 mg/L throughout the experiment. The level of superoxide dismutase (SOD), catalase (CAT) and total antioxidant capacity (T-AOC) in all the treatments decreased in a dose- and time-dependent manner except for the concentration group of 0.35 mg/L. The malondialdehyde (MDA), hydrogen peroxide (H2O2) and protein carbonyl in serum increased significantly at concentrations of 2.80 mg/L and 5.60 mg/L. A significant decrease in acetylcholinesterase (AChE) activity was observed at each concentration (P＜0.05). In addition, the micronucleus (MN) frequency of haemocytes significantly increased (P＜0.05) at concentrations of 1.40, 2.80 and 5.60 mg/L, whereas the comet ratio and %DNA in the tails exhibited a clear time- and dose-dependent response during the exposure. The analysis of the integrated biomarker response (IBR) showed the induction of oxidative stress biomarkers and the inhibition of antioxidants, and this dose-dependent relation suggests the sensitivity and availability of all the biomarkers. These results revealed that Roundup had a prominent toxic effect on M. nipponensis based on the antioxidative response inhibition and genotoxicity.

Occurrence and genotyping of Giardia duodenalis and Cryptosporidium in pre-weaned dairy calves in central Sichuan province, China.

Giardia duodenalis and Cryptosporidium spp. are common human and animal pathogens. They have increasingly been reported in dairy calves in recent years; however, multilocus genotyping information for G. duodenalis and Cryptosporidium infecting pre-weaned dairy calves in southwestern China is limited. In the present study, the prevalence of G. duodenalis and Cryptosporidium spp. in pre-weaned dairy calves in central Sichuan province was determined and the pathogens were analyzed molecularly. Of 278 fecal samples from pre-weaned dairy calves, 26 (9.4%) were positive for G. duodenalis and 40 (14.4%) were positive for Cryptosporidium spp. Cryptosporidium bovis (n = 28), Cryptosporidium ryanae (n = 5) and Cryptosporidium parvum (n = 7) were detected. All seven C. parvum isolates were successfully subtyped based on the gp60 gene sequence, and only IIdA15G1 was detected. Multilocus sequence typing of G. duodenalis based on beta-giardin (bg), triose phosphate isomerase (tpi) and glutamate dehydrogenase (gdh) genes revealed 19 different assemblage E multilocus genotypes (two known and 17 unpublished genotypes). Based on eBURST analysis, a high degree of genetic diversity within assemblage E was observed in pre-weaned dairy calves in Sichuan province. To the best of our knowledge, this is the first study using multilocus sequence typing and eBURST analysis to characterize G. duodenalis in pre-weaned dairy calves in southwestern China.

Oxidative stress and genotoxic effect of deltamethrin exposure on the Chinese mitten crab, Eriocheir sinensis.

The oxidative stress and genotoxic effect of deltamethrin on the Chinese mitten crab Eriocheir sinensis were assessed using several commonly used biomarkers in this study. The results showed that the 48 h and 96 h LC50 values of deltamethrin to E. sinensis were 2.319 and 1.164 µg/L, respectively, and the safe concentration was 0.293 µg/L. According to these results, deltamethrin was applied at concentrations of 1/16, 1/8, 1/4, 1/2 and 1/1 96 h LC50 for 8 d in an exposure experiment. The activity of superoxide dismutase (SOD) increased remarkably at 1 d, but decreased at 4 d in concentration group of 1/4, 1/2 and 1/1 LC50, whereas catalase (CAT) activity decreased during the exposure. The total antioxidant capacity (T-AOC) at the concentrations of 1/4, 1/8 and 1/16 LC50 increased significantly at 1 d or 2 d respectively, whereas it decreased gradually until the end of the experiment under the concentrations above 1/4 LC50. The oxidative stress products malondialdehyde (MDA) and hydrogen peroxide (H2O2) in serum increased significantly at each concentration except H2O2 at concentration of 1/16 LC50. Additionally, the micronucleus (MN) frequency of haemocytes increased at the concentrations of 1/4, 1/2 and 1/1 LC50 throughout the exposure, similar trend was observed in the comet ratio and percentage of tail-DNA (%DNA) in haemocytes. These results revealed that deltamethrin had a prominent toxic effect on E. sinensis based on antioxidative response inhibition and genotoxicity that was possibly due to lipid peroxidation (LPO) induced by oxidative products, and the accumulation of peroxide, even under a sublethal concentration.

Occurrence and genetic characterization of Giardia duodenalis and Cryptosporidium spp. from adult goats in Sichuan Province, China.

Giardia duodenalis and Cryptosporidium spp. are common gastrointestinal protozoa in mammals. Many studies have been conducted on the distribution of G. duodenalis and Cryptosporidium spp. genotypes in sheep and cattle. However, in China, information about molecular characterization and genetic analysis of G. duodenalis and Cryptosporidium spp. in goats is limited. In this study, 342 fecal samples from adult goats were collected from 12 farms in Sichuan Province, China. The occurrence of G. duodenalis and Cryptosporidium spp. in adult goats was 14.9% (51/342) and 4.7% (16/342), respectively. All G. duodenalis were identified as assemblage E, with two novel genotypes (assemblages E17 and E18) being detected at the beta-giardin (bg) locus. Based on three loci-beta-giardin (bg), triose phosphate isomerase (tpi), and glutamate dehydrogenase (gdh)-multilocus sequence typing revealed three novel multilocus genotypes (MLGs) of assemblage E (MLG-E1, E2, E3 (sc)). Small Subunit (SSU) rRNA-based PCR identified two Cryptosporidium species, namely C. xiaoi (11/16) and C. suis (5/16). This study is not only the first to report C. suis infection in adult goats in China but is also the first to use the MLG approach to identify G. duodenalis in adult goats.

Effects of glyphosate on immune responses and haemocyte DNA damage of Chinese mitten crab, Eriocheir sinensis.

As a broad-spectrum organophosphorus herbicide, glyphosate is widely utilized around the world. The toxic effects of glyphosate on Chinese mitten crab, Eriocheir sinensis, were assessed using immunotoxicity and genotoxicity biomarkers in this study. The results showed that 24 h and 96 h LC50 values of glyphosate for E. sinensis were estimated as 461.54 and 97.89 mg/L, respectively, and the safe concentration was 4.4 mg/L. According to the results above, glyphosate was applied at concentrations of 0, 4.4, 9.8, 44 and 98 mg/L, for 96 h in the exposure experiment. Total haemocyte count (THC) and percentage of granulocytes decreased significantly following 6 h exposure to each concentration of glyphosate and tended to gradually stabilize after 12 h except in 4.4 mg/L, which rapidly recovered to a normal level in 12 h. Phagocytic activity in all treatments decreased dramatically at 6 h and maintained stability until the 96-h mark. Comet tail has been observed early at 24 h in each treatment, and the comet ratio and percentage of DNA (% DNA) in the tail increased as the exposure experiment progressed. Immune-related enzyme activity varied during the experiment. Acid phosphatase (ACP) and alkaline phosphatase (AKP) activities in 44 and 98 mg/L treatments decreased significantly after 48 h exposure, while AKP activities in all concentrations increased markedly at the beginning of exposure. The superoxide dismutase (SOD) and peroxidase (POD) activities increased significantly after 6 h exposure to 44 and 98 mg/L of glyphosates but decreased at 24 h. In addition, the ß-glucuronidase (ß-GD) activities in the 9.8, 44 and 98 mg/L groups, increased after 6-h exposure and were significantly higher than those in the control at 96 h. These results indicated that glyphosate has evident toxic effect on E. sinensis by immune inhibition that is possibly due to the haemocyte DNA damage and a sharp decline in haemocyte numbers, which subsequently induced changes in activities of immune-related enzymes and haemocyte phagocytosis.