Protective effects of butyric acid during heat stress on the survival, immune response, histopathology, and gene expression in the hepatopancreas of juvenile pacific shrimp (L. Vannamei).

This study looked at the effects of adding butyric acid (BA) to the diets of juvenile Pacific shrimp and how it affected their response to survival, immunity, histopathological, and gene expression profiles under heat stress. The shrimp were divided into groups: a control group with no BA supplementation and groups with BA inclusion levels of 0.5 %, 1 %, 1.5 %, 2 %, and 2.5 %. Following the 8-week feeding trial period, the shrimp endured a heat stress test lasting 1 h at a temperature of 38 °C. The results showed that the control group had a lower survival rate than those given BA. Interestingly, no mortality was observed in the group receiving 1.5 % BA supplementation. Heat stress had a negative impact on the activities of alkaline phosphatase (AKP) and acid phosphatase (ACP) in the control group. Still, these activities were increased in shrimp fed the BA diet. Similar variations were observed in AST and ALT fluctuations among the different groups. The levels of triglycerides (TG) and cholesterol (CHO) increased with high temperatures but were reduced in shrimp-supplemented BA. The activity of an antioxidant enzyme superoxide dismutase (SOD) increased with higher BA levels (P < 0.05). Moreover, the groups supplemented with 1.5 % BA exhibited a significant reduction in malondialdehyde (MDA) content (P < 0.05), suggesting the potential antioxidant properties of BA. The histology of the shrimp's hepatopancreas showed improvements in the groups given BA. Conversely, the BA significantly down-regulated the HSPs and up-regulated MnSOD transcript level in response to heat stress. The measured parameters determine the essential dietary requirement of BA for shrimp. Based on the results, the optimal level of BA for survival, antioxidant function, and immunity for shrimp under heat stress is 1.5 %.

Efficacy of Annona glabra extract against acute hepatopancreatic necrosis disease in white-leg shrimp (Penaeus vannamei).

This study aims to investigate the use of pond apple (Annona glabra) compounds as a novel strategy to prevent and treat acute hepatopancreatic necrosis disease (AHPND) as well as to better understand the mechanism of health improvement in shrimp. The A. glabra leaf extracts were extracted using various solvents and examined for in vitro and in vivo activity against Vibrio parahaemolyticus strains. In comparison with ethanol and water extracts, methanol extract showed the strongest bactericidal effect (MBC/MIC ratio of 2.50 ± 1.00), with minimal inhibitory concentration (MIC) of 0.023 ± 0.012 mg ml-1 and minimum bactericidal concentration (MBC) of 0.065 ± 0.062 mg ml-1. White leg shrimp (P. vannamei, body weight 10.37 ± 0.27 g) fed A. glabra methanol extracts-containing diets (AMEDs) at 1 %, 1.5 %, and 2.0 % demonstrated no deleterious effects on survival and were significantly increased in length and weight after 30 days of feeding. The level of total haemocyte, hyaline haemocyte on day 15 and granulocyte on day 30 remarkably increased (p < 0.05) in shrimps fed AMEDs groups compared to those in the control group. The finding demonstrates that granulocyte was induced time dependently. In particular, the survival rate of V. parahaemolyticus challenged shrimps under medication with AMEDs at 1.5 % and 2.0 % was significantly higher (p < 0.05) than that of the control group. The decrease in bacterial load of Vibrio spp. and V. parahaemolyticus was obviously recorded in hepatopancreas shrimp given AMEDs 1.5 % and 2.0 % and may be linked to herb characteristics such as antibacterial activity, enhancing innate immunity, and its potential to maintain the integrity of hepatopancreatic tissue. Our findings suggest that A. glabra extract might be used as a health enhancer in commercial farmed shrimp.

Exploring the impact of nonylphenol exposure on Litopenaeus vannamei at the histological and molecular levels.

Nonylphenol (NP) is one of the common pollutants in the environment that have toxic effects on aquatic animals. Nevertheless, little is known about the possible toxicity mechanism of NP on the hepatopancreas of Litopenaeus vannamei. In the present study, the detrimental effects of NP on the hepatopancreas of the L. vannamei were explored at the histological and transcriptomic levels. The findings indicated that after NP exposed for 3, 12, and 48 h, the hepatopancreas histology was changed significantly. Transcriptomic analysis showed that a total of 4302, 3651, and 4830 differentially expressed genes (DEGs) were identified at 3, 12, and 48 h following NP exposure. All these DEGs were classified into 12 clusters according to the expression patterns at different time points. GO and KEGG enrichment analyses of DEGs were also performed, immunological, metabolic, and inflammatory related pathways, including arachidonic acid metabolism (ko00590), the PPAR signaling pathway (ko03320), and the regulation of TRP channels by inflammatory mediators (ko04750) were significantly enriched. Six DEGs were selected for validation by quantitative real-time PCR (qRT-PCR) and the results confirmed the reliability of transcriptome data. All results indicated that NP is toxic to L. vannamei by damaging the histopathological structure and disrupting the biological function. The findings would provide a theoretical framework for lowering or limiting the detrimental impacts of NP on aquaculture and help us to further study the molecular toxicity of NP in crustaceans.

Effects of glyphosate-based herbicide on gut microbes and hepatopancreatic metabolism in Pomacea canaliculata.

Roundup®, a prominent glyphosate-based herbicide (GBH), holds a significant position in the global market. However, studies of its effects on aquatic invertebrates, including molluscs are limited. Pomacea canaliculata, a large freshwater snail naturally thrives in agricultural environments where GBH is extensively employed. Our investigation involved assessing the impact of two concentrations of GBH (at concentrations of 19.98 mg/L and 59.94 mg/L, corresponding to 6 mg/L and 18 mg/L glyphosate) during a 96 h exposure experiment on the intestinal bacterial composition and metabolites of P. canaliculata. Analysis of the 16 S rRNA gene demonstrated a notable reduction in the alpha diversity of intestinal bacteria due to GBH exposure. Higher GBH concentration caused a significant shift in the relative abundance of dominant bacteria, such as Bacteroides and Paludibacter. We employed widely-targeted metabolomics analysis to analyze alterations in the hepatopancreatic metabolic profile as a consequence of GBH exposure. The shifts in metabolites primarily affected lipid, amino acid, and glucose metabolism, resulting in compromised immune and adaptive capacities in P. canaliculata. These results suggested that exposure to varying GBH concentrations perpetuates adverse effects on intestinal and hepatopancreatic health of P. canaliculata. This study provides an understanding of the negative effects of GBH on P. canaliculata and may sheds light on its potential implications for other molluscs.

Integrated histological, physiological, and transcriptome analysis reveals the post-exposure recovery mechanism of nitrite in Litopenaeus vannamei.

Nitrite is one of the most common toxic pollutants in intensive aquaculture and is harmful to aquatic animals. Recovery mechanisms post exposure to nitrite in shrimp have rarely been investigated. This study focuses on the effect of nitrite exposure and post-exposure recovery on the histological and physiological aspects of Litopenaeus vannamei and utilizes transcriptome sequencing to analyze the molecular mechanisms of adaptation to nitrite exposure. The results showed that histopathological damage to the hepatopancreas and gills caused by short-term nitrite exposure resolved with recovery. The total antioxidant capacity (T-AOC), superoxide dismutase (SOD), and catalase (CAT) of shrimp were significantly reduced during nitrite exposure and returned to the control level after recovery, malondialdehyde (MDA) levels were opposite to them. Restoration of the antioxidant system after exposure mitigated oxidative damage. Nitrite exposure results in reduced activity of the immuno-enzymes acid phosphatase (ACP) and alkaline phosphatase (AKP), which can be recovered to the control level. L. vannamei can adapt to nitrite exposure by regulating Na+/K+-ATPase (NKA) activity. Transcriptome analysis revealed that activation of glutathione metabolism and peroxisomal pathways facilitated the mitigation of oxidative damage in L. vannamei during the recovery period. Excessive oxidative damage activates the apoptosis and p53 pathways. Additionally, Sestrin2 and STEAP4 may have a positive effect on recovery in shrimp. These results provide evidence for the damage caused by nitrite exposure and the recovery ability of L. vannamei. This study can complement the knowledge of the mechanisms of adaptation and recovery of shrimp under nitrite exposure.

The impact of Roundup® Original DI on the hemolymph metabolism and gill and hepatopancreas oxidative balance of Parastacus promatensis (Crustacea, Decapoda, Parastacidae).

In Brazil, glyphosate is present in more than 130 commercial formulations, and its toxic effects have already been tested in different species to understand its impact on biota Decapod crustaceans are widely used as experimental models due to their biology, sensitivity to pollutants, ease of collection, and maintenance under laboratory conditions. We evaluated the changes in metabolism (hemolymph) and oxidative balance markers (gill and hepatopancreas) of a crayfish (Parastacus promatensis) after exposure to Roundup® (active ingredient: glyphosate). The crayfish were captured in the Garapiá stream within the Center for Research and Conservation of Nature Pró-Mata, Brazil. We collected adult animals outside (fall) and during (spring) the breeding season. The animals were transported in buckets with cooled and aerated water from the collection site to the aquatic animal maintenance room at the university. After acclimatization, the animals were exposed to different concentrations of glyphosate (0, 65, 260, 520, and 780 µg/L). The results showed a significant variation in the hemolymph glucose, lactate, and protein levels. We observed variations in the tissue antioxidant enzymatic activity after exposure to glyphosate. Finally, the increase in oxidative damage required a high energy demand from the animals to maintain their fitness, which makes them more vulnerable to stress factors added to the habitat.

Comparative Transcriptome Analysis of Hepatopancreas Reveals Sexual Dimorphic Response to Methyl Farnesoate Injection in Litopenaeus vannamei.

Sexually dimorphic traits such as growth and body size are often found in various crustaceans. Methyl farnesoate (MF), the main active form of sesquiterpenoid hormone in crustaceans, plays vital roles in the regulation of their molting and reproduction. However, understanding on the sex differences in their hormonal regulation is limited. Here, we carried out a comprehensive investigation on sexual dimorphic responses to MF in the hepatopancreas of the most dominant aquacultural crustacean-the white-leg shrimp (Litopenaeus vannamei). Through comparative transcriptomic analysis of the main MF target tissue (hepatopancreas) from both female and male L. vannamei, two sets of sex-specific and four sets of sex-dose-specific differentially expressed transcripts (DETs) were identified after different doses of MF injection. Functional analysis of DETs showed that the male-specific DETs were mainly related to sugar and lipid metabolism, of which multiple chitinases were significantly up-regulated. In contrast, the female-specific DETs were mainly related to miRNA processing and immune responses. Further co-expression network analysis revealed 8 sex-specific response modules and 55 key regulatory transcripts, of which several key transcripts of genes related to energy metabolism and immune responses were identified, such as arginine kinase, tropomyosin, elongation of very long chain fatty acids protein 6, thioredoxin reductase, cysteine dioxygenase, lysosomal acid lipase, estradiol 17-beta-dehydrogenase 8, and sodium/potassium-transporting ATPase subunit alpha. Altogether, our study demonstrates the sex differences in the hormonal regulatory networks of L. vannamei, providing new insights into the molecular basis of MF regulatory mechanisms and sex dimorphism in prawn aquaculture.

Efficacy of fennel (Foeniculum vulgare) and anise (Pimpinella anisum) essential oils as anaesthesics in common carp (Cyprinus carpio L. 1758).

In this study, the anaesthetic effects of fennel and anise essential oils were investigated on common carp. Fish (10 ± 0.45 g) were exposed to nine concentrations of essential oils (5, 10, 20, 50, 100, 200, 300, 400 and 500 mg L-1). Additionally, the histopathological effects on the fish tissues including gill, skin and hepatopancreas and physiological effects on some blood parameters (Na+, K+, Ca+2, Cl-, total plasma protein and glucose) of essential oils were investigated in carp. At the end of the experiment, fennel oil showed an anaesthetic effect at a concentration of 500 mg L-1 in carp (anaesthesia induction and recovery times were 308 and 472 s, respectively). Anise essential oil showed deep anaesthesia at a concentration of 100 mg L-1, but anaesthesia induction time was found to be very long (20 min). In addition, anise oil at concentrations above 100 mg L-1 caused 10% mortality in fish. Blood parameters except glucose level in both essential oils were unchanged during deep anaesthesia in carp. However, plasma glucose levels were found lower in fish anaesthetized with anise oil than control and fennel groups (P < 0.05). At the histopathological examination, no pathological findings were observed in any organ of fish in the fennel group. However, severe hyperemia and inflammatory cell infiltrations in gills, erosive lesions in the skin and slight inflammatory reactions in the skin were observed in the anise group. The present study demonstrated that fennel essential oil at 500 mg L-1 concentration can be used as an effective and safe anaesthetic in common carp, but anise essential oil is not suitable.

Histopathological Evaluation of the Curative Effects of EDTA on Lymnaea stagnalis Exposed to Subacute Cadmium.

We investigated the therapeutic effects of EDTA application for 14 and 28 days on cadmium (Cd) induced pond snail Lymnaea stagnalis (Linnaeus, 1758). The sublethal concentration of cadmium (63.4 mg/l Cd) caused tissue damages to the snail after an exposure for 28 days.In the groups treated with EDTA, the concentration of Cd in the foot, mantle and hepatopancreas tissues showed significantly decreased during the recovery period. The curative effects of EDTA on Cd-induced damage were assessed using a scoring system. Cadmium exposure led to histopathological changes including increased mucositis, pigment and protein cells, foot epithelium desquamation, muscle fibril damage, connective tissue cell atrophy, and increased lipid vacuoles in the mantle and hepatopancreas. However, these changes were less severe in snails treated with EDTA (2.00 mL/L for 28 day), indicating that EDTA reduces their susceptibility to heavy metal toxicity.

Effect of dietary Xiao-Chaihu-Decoction on growth performance, immune response, detoxification and intestinal microbiota of pacific white shrimp (Litopenaeus vannamei).

Xiao-Chaihu-Decoction (XCHD), a classical traditional Chinese medicine with diverse biological activities, is widely applied to prevent and treat many human diseases. Effects of dietary XCHD on growth performance, immune response, detoxification system, intestinal microbiota and resistance against aflatoxin B1(AFB1) of Litopenaeus vannamei was studied. Four isonitrogenous and isolipidic diets were formulated to contain 0, 1, 2, and 5 g/kg (control, XCHD1, XCHD2 and XCHD3) of XCHD, respectively. Seven hundred and eighty shrimp (1.16 ± 0.09 g) were assigned randomly to 12 tanks (400 L, three tanks each group, 65 shrimp in each tank) for 6 weeks. After sampling, 25 shrimp from each tank were selected for a 2-week AFB1 (2500 µg/kg) challenge experiment. The results indicated that the final weight, weight gain and specific growth rate in XCHD2 and XCHD3 groups were significantly increased compared to control. The protease, amylase, superoxide dismutase (SOD), glutathione s-transferase (GST), sulfotransferase (SULT) activities, total antioxidant capacity (T-AOC) and glutathione (GSH) contents in hepatopancreas were significantly increased in XCHD3 groups and the expressions of immune-related genes (Toll, Dorsal and Cru) in hepatopancreas were significantly up-regulated in XCHD2 and XCHD3 groups. High-throughput sequencing analysis revealed that the abundance of Proteobacteria decreased and the abundances of Bacteroidetes increased in XCHD2 and XCHD3 groups. Additionally, AFB1 challenge experiments showed that AFB1 caused histological damage to the hepatopancreas and significantly increased the levels of malondialdehyde (MDA) and protein carbonylation (PC) in hepatopancreas as well as the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Nevertheless, XCHD could effectively alleviated the growth toxicity, immunosuppression and macromolecular damage caused by AFB1 to shrimp by inhibiting the Phase I enzyme and enhancing Phase II enzyme and antioxidant system.

The characterization, expression and activity analysis of three superoxide dismutases in Eriocheir hepuensis under azadirachtin stress.

Superoxide dismutase (SOD) can effectively eliminate of excess ROS, which causes oxidative damage to lipids, proteins, and DNA. In this study, we cloned the CuZn-SOD, cMn-SOD1, and cMn-SOD2 genes in Eriocheir hepuensis, and found that the coding sequence (CDS) lengths were 627 bp, 861 bp and 1062 bp, which encoded 208, 286, and 353 amino acids, respectively. Phylogenetic analysis indicated that all SOD genes were evolutionarily conserved, while cMn-SOD2 had an extra gap (67 amino acids) in the conserved domain compared with cMn-SOD1 without huge changes in the tertiary structure of the conserved domain, suggesting that cMn-SOD2 may be a duplicate of cMn-SOD1. qRT-PCR showed that the three SOD genes were widely expressed in all the tested tissues, CuZn-SOD and cMn-SOD1 were mostly expressed in the hepatopancreas, while cMn-SOD2 was mostly expressed in thoracic ganglia. Under azadirachtin stress, the oxidation index of surviving individuals, including the T-AOC, SOD activity, and MDA contents increased in the early stage and then remained steady except for a decrease in MDA contents in the later stage. qRT-PCR showed that the three SOD genes displayed the same trends as SOD activity in surviving individuals, and the highest expressions of CuZn-SOD in the hepatopancreas, heart, and gill were 14.16, 1.41, and 30.87 times that of the corresponding control group, respectively. The changes were 1.35, 5.77 and 3.33 fold for cMn-SOD1 and 1.62, 1.71 and 1.79 fold for cMn-SOD2, respectively. However, the activity and expression of SOD genes in dead individuals were lower than that observed in surviving individuals. These results reveal that SOD plays a significant role in the defence against azadirachtin-induced oxidative stress.

Transcriptome analysis to elucidate the toxicity mechanisms of fenvalerate, sulfide gatifloxacin, and ridomil on the hepatopancreas of Procambarus clarkii.

Most antibiotics, insecticides, and other chemicals used in agricultural and fishery production tend to persist in the environment. Fenvalerate, sulfide gatifloxacin, and ridomil are widely used in aquaculture as antibacterial, antifungal, and antiparasitic drugs; however, their toxicity mechanism remains unclear. Thus, we herein analyzed the effects of these three drugs on the hepatopancreas of Procambarus clarkii at the transcriptome level. Twelve normalized cDNA libraries were constructed using RNA extracted from P. clarkii after treatment with fenvalerate, sulfide gatifloxacin, or ridomil and from an untreated control group, followed by Kyoto Encyclopedia of Genes and Genomes pathway analysis. In the control vs fenvalerate and control vs sulfide gatifloxacin groups, 14 and seven pathways were significantly enriched, respectively. Further, the effects of fenvalerate and sulfide gatifloxacin were similar on the hepatopancreas of P. clarkii. We also found that the expression level of genes encoding senescence marker protein-30 and arylsulfatase A was downregulated in the sulfide gatifloxacin group, indicating that sulfide gatifloxacin accelerated the apoptosis of hepatopancreatocytes. The expression level of major facilitator superfamily domain containing 10 was downregulated, implying that it interferes with the ability of the hepatopancreas to metabolize drugs. Interestingly, we found that Niemann pick type C1 and glucosylceramidase-ß potentially interact with each other, consequently decreasing the antioxidant capacity of P. clarkii hepatopancreas. In the fenvalerate group, the downregulation of the expression level of xanthine dehydrogenase indicated that fenvalerate affected the immune system of P. clarkii; moreover, the upregulation of the expression level of pancreatitis-associated protein-2 and cathepsin C indicated that fenvalerate caused possible inflammatory pathological injury to P. clarkii hepatopancreas. In the ridomil group, no pathway was significantly enriched. In total, 21 genes showed significant differences in all three groups. To conclude, although there appears to be some overlap in the toxicity mechanisms of fenvalerate, sulfide gatifloxacin, and ridomil, further studies are warranted.

Effect of maduramicin on crayfish (Procambius clarkii): Hematological parameters, oxidative stress, histopathological changes and stress response.

Maduramicin, an extensively used anticoccidial drug, has been introduced into environment due to poorly absorbed in the intestine of broiler chicken. To understand the potential ecological toxicity of maduramicin on aquatic organisms, acute and subacute toxicity, hemolymph biochemistry, histopathology and the expressions of drug metabolism and stress response genes of crayfish (Procambius clarkii) were investigated in this study. For the first time, the 96 h median lethal concentration (LC50) of maduramicin on crayfish was 67.03 mgL-1 with a 95% confidence interval (54.06-81.32 mgL-1). Then, the crayfish were exposed to 0.7 mgL-1 (1/100 LC50), 3.5 mgL-1 (1/20 LC50) and 7.0 mgL-1 (1/10 LC50) maduramicin for 28 days. Maduramicin significantly altered biochemical parameters including AST, ALT, CK, LDH and ALP of hemolymph in crayfish at several time points. The activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) of crayfish gills, hepatopancreas and abdominal muscle were significantly decreased or elevated by different concentrations of maduramicin treatment at varying time points. Furthermore, histopathological damage of crayfish gills, hepatopancreas and abdominal muscle were observed in a concentration-dependent manner. The expressions of metabolic and stress response genes (CYP450, GST, COX1, COX2, HSP70 and MT) in hepatopancreas of crayfish were significantly up-regulated by maduramicin (7.0 mgL-1) treatment for 8 h to 7 d, and returned to normal levels after the removal of maduramicin for 3-7 days. In conclusion, our findings demonstrated that environmental exposure of maduramicin threaten to the health of crayfish living in the areas nearby livestock farms or pharmaceutical factory. Crayfish exhibited resistance to the stress of maduramicin via activating drug metabolite and detoxification pathways.

Multi-biomarker assessment in the giant freshwater prawn Macrobrachium rosenbergii after deltamethrin exposure.

Deltamethrin (DM) is a synthetic pyrethroid used for agricultural purposes to control insects. However, its extensive use contaminates the aquatic environment and results in serious health problems in aquatic organisms. Knowledge about the toxic effect of DM in freshwater prawns is limited; therefore, this study aims to assess the toxicity of DM in Macrobrachium rosenbergii based on multiple biomarkers. Four-day acute toxicity tests showed that DM was highly toxic to M. rosenbergii with the 24 h, 48 h, 72 h and 96 h LC50 values to be 1.919, 0.603, 0.539, and 0.449 µg/L, respectively. According to 96 h LC50, prawns were exposed to DM at three concentrations (0.02, 0.08, and 0.32 µg/L) for 4 days, and then moved into fresh water for decontamination to investigate the toxic effect of DM in M. rosenbergii. At low concentration (0.02 µg/L and 0.08 µg/L), DM did not cause obvious histopathological damage to hepatopancreas and gill tissue, while at high concentration (0.32 µg/L), the histopathological harm was serious and the damage did not recover to the initial level after 7-day decontamination. 0.02 µg/L DM exposure did not induce significant changes in most of the biomarkers except the increased lactate dehydrogenase (LDH) activity, lactic acid (LD) level, and the first increased then decreased mRNA expression of immune-related genes, indicating the stimulation of DM on energy production and immunity. 0.08 µg/L and 0.32 µg/L DM exposure resulted in varying degrees of damage on prawns, but overall, their toxic effects showed similar trends based on the biomarkers. Increase in malonaldehyde (MDA) and hydrogen peroxide (H2O2) content and decrease in superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity after DM exposure demonstrated the oxidative stress caused by DM. The significantly increased acid phosphatase (ACP), alkaline phosphatase (AKP), LDH activity and LD level indicated hepatopancreatic dysfunction and respiration disruption. The first increased and then decreased expression pattern of immune-related genes indicated the immunosuppression caused by DM. After 7-day decontamination in freshwater, the activity/level of the biomarkers partly recovered. This study revealed the severe toxic effect of DM on Macrobrachium rosenbergii based on multiple biomarkers, providing fundamental knowledge for the establishment of DM toxicity assessment system with proper parameters in freshwater crustaceans.

The effect of chronic exposure to chloridazon and its degradation product chloridazon-desphenyl on signal crayfish Pacifastacus leniusculus.

The effects of chloridazon (Ch) and its metabolite chloridazon-desphenyl (Ch-D) at the environmentally relevant concentrations of 0.45 µg/L and 2.7 µg/L on signal crayfish Pacifastacus leniusculus were assessed in a 30-day exposure followed by a 15-day depuration period. Locomotion, biochemical haemolymph profile, oxidative and antioxidant parameters, and histopathology were evaluated. Crayfish exposed to Ch at 0.45 µg/L and 2.7 µg/L showed significantly (p < 0.01) higher CAT activity and GSH level in hepatopancreas and gill compared to controls. The concentration of Ch at 2.7 µg/L was associated with significantly (p < 0.01) higher levels of GLU, LACT, ALT, AST in haemolymph compared to controls. Chloridazon-desphenyl exposure at both tested concentrations caused significantly higher (p < 0.01) GLU, LACT, ALT, AST, NH3, and Ca in haemolymph; lipid peroxidation (TBARS) levels in hepatopancreas; and CAT activity and GSH level in hepatopancreas and gill. Alterations of structure including focal dilatation of tubules, increased number of fibrillar cells, and haemocyte infiltration in the interstitium were observed with 2.7 µg/L Ch and with both Ch-D exposures. Locomotion patterns did not vary significantly among groups. A 15-day recovery period was insufficient to restore normal physiological parameters in exposed groups. Chloridazon and its metabolite Ch-D exerts harmful effects on crayfish.

Co-exposure to nTiO2 impairs arsenic metabolism and affects antioxidant capacity in the marine shrimp Litopenaeus vannamei.

Aquatic animals are vulnerable to arsenic (As) toxicity. However, rarely does a contaminant occur alone in the aquatic environment. For this reason, this study was conducted to evaluate whether titanium dioxide nanoparticles (nTiO2) can interfere with the effects induced by As in Litopenaeus vannamei. Arsenic accumulation and metabolic capacity; expression and enzymatic activity of GSTΩ (glutathione-S-transferase omega isoform); antioxidant responses such as GSH, GR, and GST (reduced glutathione levels, glutathione reductase, and glutathione-S-transferase activity, respectively); and lipid peroxidation in the gills and hepatopancreas of shrimp were evaluated. The results are summarized as follows: (1) higher accumulation of As occurred in both tissues after exposure to As alone; (2) co-exposure to nTiO2 affected the capacity to metabolize As; (3) GSTΩ gene expression was not modified, but its activity was decreased by co-exposure to both contaminants; (4) As alone increased the GSH levels in the hepatopancreas, and co-exposure to nTiO2 reduced these levels in both tissues; (5) a decrease in the GST activity in the gills occurred with all treatments; (6) in the gills, GR activity was increased by As, and nTiO2 reversed this increase, whereas in the hepatopancreas co-exposure inhibited enzyme activity; (7) only in the hepatopancreas lipid damage was observed when animals were exposed to As or nTiO2 but not in co-exposure. The results showed that the As induces toxic effects in both tissues of shrimp and that co-exposure to nTiO2 can potentiate these effects and decrease the capacity to metabolize As, favoring the accumulation of more toxic compounds.

Roles of gonad-inhibiting hormone in the protandric simultaneous hermaphrodite peppermint shrimp†.

To date, the molecular mechanisms of the unique gonadal development mode known as protandric simultaneous hermaphroditism (PSH) are unclear in crustaceans. In this study, cDNA of a gonad-inhibiting hormone (Lv-GIH1) was isolated from the PSH peppermint shrimp Lysmata vittata, and its expression was exclusively found in the eyestalk ganglion. Real-time quantitative polymerase chain reaction (qRT-PCR) revealed that the expression of Lv-GIH1 increased during gonadal development of the functional male stages but decreased significantly at subsequent simultaneous hermaphroditism stage. Further in vitro experiment showed that recombinant GIH1 protein (rGIH1) effectively inhibited Vg expression in the cultured hepatopancreas tissues while the short-term injection of GIH1-dsRNA resulted in reduced expression of Lv-GIH1 and upregulated expression of Vg in the hepatopancreas. Moreover, long-term rGIH1 injection led to significantly reduced expression of Lv-Vg, Lv-VgR, and Lv-CFSH1, subdued growth of oocytes, and feathery setae as a secondary sexual characteristic in females. Interestingly, while germ cells in testicular part were suppressed by rGIH1 injection, the expression of Lv-IAGs showed no significant difference; and long-term GIH1-dsRNA injection results were contrary to those of rGIH1 injection. Taken together, the results of this study indicate that Lv-GIH1 is involved in gonadal development and might also participate in controlling secondary sexual characteristic development in L. vittata by inhibiting Lv-CFSH1 expression.

Effects of dietary lipid level on growth, fatty acid profiles, antioxidant capacity and expression of genes involved in lipid metabolism in juvenile swimming crab, Portunus trituberculatus.

The regulation of lipogenesis and lipolysis mechanisms related to consumption of lipid has not been studied in swimming crab. The aims of the present study were to evaluate the effects of dietary lipid levels on growth, enzymes activities and expression of genes of lipid metabolism in hepatopancreas of juvenile swimming crab. Three isonitrogenous diets were formulated to contain crude lipid levels at 5·8, 9·9 and 15·1 %. Crabs fed the diet containing 15·1 % lipid had significantly lower growth performance and feed utilisation than those fed the 5·8 and 9·9 % lipid diets. Crabs fed 5·8 % lipid had lower malondialdehyde concentrations in the haemolymph and hepatopancreas than those fed the other diets. Highest glutathione peroxidase in haemolymph and superoxide dismutase in hepatopancreas were observed in crabs fed 5·8 % lipid. The lowest fatty acid synthase and glucose 6-phosphate dehydrogenase activities in hepatopancreas were observed in crabs fed 15·1 % lipid, whereas crabs fed 5·8 % lipid had lower carnitine palmitoyltransferase-1 activity than those fed the other diets. Crabs fed 15·1 % lipid showed lower hepatopancreas expression of genes involved in long-chain-PUFA biosynthesis, lipoprotein clearance, fatty acid uptake, fatty acid oxidation, lipid anabolism and lipid catabolism than those fed the other diets, whereas expression of some genes of lipoprotein assembly and fatty acid oxidation was up-regulated compared with crabs fed 5·8 % lipid. Overall, high dietary lipid level can inhibit growth, reduce antioxidant enzyme activities and influence lipid metabolic pathways to regulate lipid deposition in crab.

Transcriptome analysis of Penaeus vannamei hepatopancreas reveals differences in toxicity mechanisms between phoxim and prometryne.

Due to overuse and terrestrial input, there are large quantities of phoxim and prometryne residues in some aquatic environments. In the present study, the effects of these compounds on Penaeus vannamei hepatopancreas were analysed at the transcriptome level to investigate toxicity in this nontarget aquaculture organism. Twelve normalised cDNA libraries were constructed using RNA from phoxim and prometryne treatment groups, and an untreated control group. A total of 667,750,902 clean reads were obtained. Analysis of differentially expressed genes (DEGs) identified 449 in control vs phoxim groups, 185 in control vs prometryne groups, and 183 in prometryne vs phoxim groups. In the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, arachidonic acid metabolism, pancreatic secretion, linoleic acid metabolism, and beta-alanine metabolism pathways were significantly enriched in control vs phoxim groups. In control vs prometryne groups, lysosome, pentose and glucuronate interconversion, antigen processing and presentation, and glycosaminoglycan degradation pathways were significantly enriched. In prometryne vs phoxim groups, protein digestion and absorption, extracellular matrix (ECM)-receptor interaction, PI3K-Akt signalling, cell adhesion molecule (CAM), AGE-RAGE signalling related to diabetic complications, focal adhesion, and renin secretion pathways were significantly enriched. In further detailed analysis, glutathione S-transferase (GST), glutathione peroxidase and basic phospholipase A2 were downregulated in the phoxim treatment group, indicating that phoxim damaged hepatopancreas. Upregulation of phospholipase A2 (secretory phospholipase A2-like) indicates possible inflammatory pathological injury to hepatopancreas caused by phoxim. Meanwhile, downregulation of CD63 indicates that prometryne affect the immune system.

Dietary glutathione supplementation enhances antioxidant activity and protects against lipopolysaccharide-induced acute hepatopancreatic injury and cell apoptosis in Chinese mitten crab, Eriocheir sinensis.

Eriocheir sinensis (E. sinensis) is an important aquaculture species in China. However, deteriorating water environments lead to oxidative stress in these crabs, which subsequently reduces their quality and yield. Glutathione (GSH) is an endogenous antioxidant that is used to mitigate oxidative stress. However, whether dietary GSH can enhance the resistance of E. sinensis to oxidative stress remains unclear. Herein, crabs were fed dietary GSH (the basal diet was supplemented with 0, 300, 600, 900, and 1200 mg/kg diet weight of GSH) for up to 3 weeks and, then, challenged with lipopolysaccharide (LPS; 400 µg/kg body weight). After 6 h, their hepatopancreas were sampled. Diet supplementation with 600 and 900 mg/kg diet weight GSH not only increased the content of GSH in the hepatopancreas, but also enhanced the activities and mRNA expressions of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and glutathione-S-transferase (GST) (P < 0.05), compared to that in control crabs challenged with LPS alone. Diet supplementation with 600 or 900 mg/kg GSH also significantly increased the enzyme activities of GSH reductase and γ-glutamyl cysteine synthetase (γ-GCS) in LPS-treated crabs. Haematoxylin-eosin (HE) staining, electron microscopy, and flow cytometry were used to examine the structure and subcellular structure of and apoptosis in the hepatopancreas. The histopathology and sub-microstructure analysis results also showed that diet supplementation with 600 or 900 mg/kg GSH significantly alleviated damage in crabs challenged with LPS and decreased reactive oxygen species (ROS) levels and cell apoptosis ratios in the hepatopancreas, compared to the LPS-treated crabs. To further understand the effect of dietary GSH on LPS-induced apoptosis, the activities and gene or protein expressions of apoptosis-related factors were evaluated. As a result, diet supplementation with 600 or 900 mg/kg GSH significantly decreased the activities of caspases-3, -8, and -9 and inhibited the relative expression of caspase-3 and -8 but increased the expression of B-cell lymphoma-2 (bcl-2) and B-cell lymphoma-2-associated X inhibitor (bax inhibitor) in crabs challenged with LPS. This treatment further significantly downregulated the relative protein levels of caspase-3, -8, -9 and Bax and upregulated those of Bcl-2 in crabs challenged with LPS. However, treatment with 1200 mg/kg GSH caused the opposite effects. Overall, our results reveal that appropriate diets supplemented with 600 or 900 mg/kg GSH could enhance the antioxidant capacity and anti-apoptotic mechanisms in crabs after LPS injection, thereby providing a theoretical basis for the application of dietary GSH in E. sinensis.