Effects of berberine hydrochloride on antioxidant response and gut microflora in the Charybdis japonica infected with Aeromonas hydrophila.

This study used berberine hydrochloride to treat the Asian paddle crab, Charybdis japonica infected with the Gram-negative bacterium Aeromonas hydrophila at concentrations of 0, 100, 200 and 300 mg/L. The effect of berberine hydrochloride on the survival rate and gut microbiota of C. japonica was investigated. Berberine hydrochloride improved the stability of the intestinal flora, with an increase in the abundance of probiotic species and a decrease in the abundance of both pathogenic bacteria after treatment with high concentrations of berberine hydrochloride. Berberine hydrochloride altered peroxidase activity (POD), malondialdehyde (MDA), and lipid peroxidation (LPO) in the intestinal tract compared to the control. Berberine hydrochloride could modulate the energy released from the enzyme activities of hexokinase (HK), phosphofructokinase (PFK), and pyruvate kinase (PK) in the intestinal tract of C. japonica infected with A. hydrophila. Zona occludens 1 (ZO-1), Zinc finger E-box binding homeobox 1 (ZEB1), occludin and signal transducer, and activator of transcription5b (STAT5b) expression were also increased, which improved intestinal barrier function. The results of this study provide new insights into the role of berberine hydrochloride in intestinal immune mechanisms and oxidative stress in crustaceans.

The impact of settleable atmospheric particulate on the energy metabolism, biochemical processes, and behavior of a sentinel mangrove crab.

We use the sentinel mangrove crab, Minuca rapax, as a model to investigate the effects of metallic settleable particulate matter (SePM) on wetland. Multiple levels of energetic responses, including (i) metabolic rate and energy budget, (ii) oxidative stress, and (iii) behavioral response by righting time, were assessed as well as the metal and metalloid content in crabs exposed to 0, 0.1 and 1 g.L-1 of SePM, under emerged and submerged conditions over five days, simulating the rigors of the intertidal habitat. Al, Fe, Mn, Cr, and Y exhibited a concentration-dependent increase. Metal concentrations were higher in submerged crabs due to the continuous ingestion of SePM and direct exposure through gills. Exposure concentration up to 1 g.L-1 decreased metabolic rate and enzymatic activities, reduced assimilation efficiency and energy for maintenance, and induces a slower response to righting time, probably by metal effects on nervous system and energy deficits. In conclusion, SePM exposure affects the redox status and physiology of M. rapax depending on he submersion regime and SePM concentration. The disruption to the energy budget and the lethargic behavior in M. rapax exposed to SePM implies potential ecological alterations in the mangrove ecosystem with unknown consequences for the local population.

Dietary lauric acid promoted antioxidant and immune capacity by improving intestinal structure and microbial population of swimming crab (Portunus trituberculatus).

Lauric acid (LA), a saturated fatty acid with 12 carbon atoms, is widely regarded as a healthy fatty acid that plays an important role in disease resistance and improving immune physiological function. The objective of this study was to determine the effects of dietary lauric acid on the growth performance, antioxidant capacity, non-specific immunity and intestinal microbiology, and evaluate the potential of lauric acids an environmentally friendly additive in swimming crab (Portunus trituberculatus) culture. A total of 192 swimming crabs with an initial body weight of 11.68 ± 0.02 g were fed six different dietary lauric acid levels, the analytical values of lauric acid were 0.09, 0.44, 0.80, 1.00, 1.53, 2.91 mg/g, respectively. There were four replicates per treatment and 8 juvenile swimming crabs per replicate. The results indicated that final weight, percent weight gain, specific growth rate, survival and feed intake were not significantly affected by dietary lauric acid levels; however, crabs fed diets with 0.80 and 1.00 mg/g lauric acid showed the lowest feed efficiency among all treatments. Proximate composition in hepatopancreas and muscle were not significantly affected by dietary lauric acid levels. The highest activities of amylase and lipase in hepatopancreas and intestine were found at crabs fed diet with 0.80 mg/g lauric acid (P < 0.05), the activity of carnitine palmityl transferase (CPT) in hepatopancreas and intestine significantly decreased with dietary lauric acid levels increasing from 0.09 to 2.91 mg/g (P < 0.05). The lowest concentration of glucose and total protein and the activity of alkaline phosphatase in hemolymph were observed at crabs fed diets with 0.80 and 1.00 mg/g lauric acid among all treatments. The activity of GSH-Px in hepatopancreas significantly increased with dietary lauric acid increasing from 0.09 to 1.53 mg/g, MDA in hepatopancreas and hemolymph was not significantly influenced by dietary lauric acid levels. The highest expression of cat and gpx in hepatopancreas were exhibited in crabs fed diet with 1.00 mg/g lauric acid, however, the expression of genes related to the inflammatory signaling pathway (relish, myd88, traf6, nf-κB) were up-regulated in the hepatopancreas with dietary lauric acid levels increasing from 0.09 to 1.00 mg/g, moreover, the expression of genes related to intestinal inflammatory, immune and antioxidant were significantly affected by dietary lauric acid levels (P < 0.05). Crabs fed diet without lauric acid supplementation exhibited higher lipid drop area in hepatopancreas than those fed the other diets (P < 0.05). The expression of genes related to lipid catabolism was up-regulated, however, and the expression of genes related to lipid synthesis was down-regulated in the hepatopancreas of crabs fed with 0.80 mg/g lauric acid. Lauric acid improved hepatic tubular integrity, and enhanced intestinal barrier function by increasing peritrophic membrane (PM) thickness and upregulating the expression of structural factors (per44, zo-1) and intestinal immunity-related genes. In addition, dietary 1.00 mg/g lauric acid significantly improved the microbiota composition of the intestinal, increased the abundance of Actinobacteria and Rhodobacteraceae, and decreased the abundance of Vibrio, thus maintaining the microbiota balance of the intestine. The correlation analysis showed that there was a relationship between intestinal microbiota and immune-antioxidant function. In conclusion, the dietary 1.00 mg/g lauric acid is beneficial to improve the antioxidant capacity and intestinal health of swimming crab.

Holistic assessment of dimethoate toxicity in Carcinus aestuarii's muscle tissues.

Dimethoate (DMT) is one of the most harmful and commonly used organophosphate pesticides in agricultural lands to control different groups of parasitic insects. However, this pesticide is considered a dangerous pollutant for aquatic organisms following its infiltration in coastal ecosystems through leaching. Yet, our investigation aimed to gain new insights into the toxicity mechanism of DMT in the muscles of the green crab Carcinus aestuarii, regarding oxidative stress, neurotransmission impairment, histological aspects, and changes in lipid composition, assessed for the first time on the green crab's muscle. Specimens of C. aestuarii were exposed to 50, 100, and 200 µg DMT L-1 for 24 h. Compared to the negative control group, the higher the DMT concentration, the lower the saturated fatty acids (SFA), and the higher the monounsaturated fatty acids (MUFA). The significant increase in polyunsaturated fatty acid n-6 (PUFA n-6) was related to the high release, mainly, of linoleic acid (LA, C18: 2n6) and arachidonic acid (ARA, C20: 4n6) levels. Biochemical biomarkers showed that DMT exposure promoted oxidative stress, highlighted by increased levels of hydrogen peroxide (H2O2), malondialdehyde (MDA), advanced oxidation protein product levels (AOPP), and protein carbonyl (PCO). Furthermore, the antioxidant defense system was activated, as demonstrated by the significant changes in the enzymatic activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and reduced glutathione (GSH) levels associated with an adaptation process of C. aestuarii to cope with the DMT exposure. This pesticide significantly impairs the neurotransmission process, as evidenced by the inhibition of acetylcholinesterase (AChE) activity. Finally, several histopathological changes were revealed in DMT-treated crabs, including vacuolation, and muscle bundle loss.This research offered new insights into the toxic mechanism of DMT, pointing to the usefulness of fatty acid (FA) composition as a sensitive biomarker in littoral crabs.

Effect of endocrine-disrupting chemicals on the expression of a calcium ion channel receptor (ryanodine receptor) in the mud crab (Macrophthalmus japonicus).

Endocrine-disrupting chemicals (EDCs) are toxic pollutants generated by artificial activities. Moreover, their hormone-like structure induces disturbances, such as mimicking or blocking metabolic activity. Previous studies on EDCs have focused on the adverse effect of the endocrine system in vertebrates, with limited investigations conducted on ion channels in invertebrates. Thus, in this study, we investigated the potential adverse effects of exposure to bisphenol-A (BPA) and di-(2-ethylhexyl) phthalate (DEHP) at the molecular level on the ryanodine receptor (RyR), a calcium ion channel receptor in Macrophthalmus japonicus. In the phylogenetic analysis, the RyR amino acid sequences in M. japonicus clustered with those in the Crustacean and formed separated branches for RyR in insects and mammals. When exposed to 1 µg L-1 BPA, a significant increase in RyR mRNA expression was observed in the gills on day 1, although a similar level to the control group was observed from day 4 to day 7. However, the RyR expression due to DEHP exposure decreased on days 1 and 4, although it increased on day 7 following exposure to 10 µg L-1. The RyR expression pattern in the hepatopancreas increased for up to 4 days, depending on the BPA concentration. However, there was a tendency for the expression to decrease gradually after the statistical significance increased during the early stage of DEHP exposure (D1). Hence, the transcriptional alterations in the M. japonicus RyR gene observed in the study suggest that exposure toxicities to EDCs, such as BPA and DEHP, have the potential to disrupt calcium ion channel signaling in the gills and hepatopancreas of M. japonicus crabs.

Abamectin-induced behavioral alterations link to energy metabolism disorder and ferroptosis via oxidative stress in Chinese mitten crab, Eriocheir sinensis.

The increasing application of abamectin (ABM) in agriculture has raised concerns regarding its environmental safety and potential adverse effects on aquatic environment safety. In the present study, the toxic effects of ABM exposure on the adult Chinese mitten crab, Eriocheir sinensis were investigated, with a focus on locomotion impairment, behavioral changes, oxidative stress, energy metabolism disruption, and ferroptosis. Crabs were exposed to sublethal concentrations of ABM at 2, 20 and 200 µg/L. After 21 d chronic exposure to 200 µg/L, residual ABM in hepatopancreas and muscles were detected as 12.24 ± 6.67 and 8.75 ± 5.42 µg/Kg, respectively. By using acute exposure experiments (96 h), we observed significant locomotion and behavioral alterations, alongside biochemical evidences of oxidative stress and energy metabolism impairment. The presence of ferroptosis, a form of cell death driven by iron-dependent lipid peroxidation, was notably identified in the hepatopancreas. Functional tests with N-acetylcysteine (NAC) supplementation showed restored behavioral responses and decrease of ferroptosis levels. It suggests that mitigating oxidative stress could counteract ABM-induced toxicity. Our findings highlight the critical roles of oxidative stress and ferroptosis in mediating the toxic effects of ABM on E. sinensis, underscoring the need for strategies to mitigate environmental exposure to pesticides.

Effects of glyphosate on sperm quality of the estuarine crab Neohelice granulata, under the "One Health" perspective.

This study was aimed at evaluating the effects of the commercial formulation Roundup Ultramax® on sperm mortality and viability, in terms of the capacity of spermatozoids (spz) to trigger the acrosome reaction (AR), using the estuarine crab Neohelice granulata as a model. To this, an in vivo assay comprising 100 days duration was carried out, on a control group and two groups exposed to the formulation (0.01 mg/L and 0.2 mg/L of glyphosate) under controlled conditions of photoperiod, feeding, and temperature. At the end of the assay, the right vas deferens (VD, proximal and middle portion) was dissected, and after homogenizing it in calcium-free saline solution, the acrosome reaction was induced in the phase containing the spz. In each treatment, the percentage of spz with total and partial AR was calculated, as well as that of dead spz. Compared to the control, crabs exposed to the herbicide showed a significant decrease in spz with full AR, together with an increase in the percentage of spz with partial AR. Furthermore, spz mortality was significantly higher in both glyphosate concentrations compared to the control, in a concentration-dependent manner. On the other hand, abnormal spermatophores, showing expanded walls and coalescence, were observed in a significant percentage in the left VD of the groups treated with the herbicide. The results obtained are compared with those from other studies on several invertebrate and vertebrate species that found inhibition of the AR and abnormal sperm, together with inhibition of spermatogenesis, endocrine disruption, and reduced sperm motility by effect of pure glyphosate and/or different glyphosate formulations. In summary, the available evidence highlights the possible impact of glyphosate on sperm quality, in a wide variety of species.

Two faces of one coin: Beneficial and deleterious effects of reactive oxygen species during short-term acclimation to hypo-osmotic stress in a decapod crab.

Exposure to environmental changes often results in the production of reactive oxygen species (ROS), which, if uncontrolled, leads to loss of cellular homeostasis and oxidative distress. However, at physiological levels these same ROS are known to be key players in cellular signaling and the regulation of key biological activities (oxidative eustress). While ROS are known to mediate salinity tolerance in plants, little is known for the animal kingdom. In this study, we use the Mediterranean crab Carcinus aestuarii, highly tolerant to salinity changes in its environment, as a model to test the healthy or pathological role of ROS due to exposure to diluted seawater (dSW). Crabs were injected either with an antioxidant [N-acetylcysteine (NAC), 150 mg·kg-1] or phosphate buffered saline (PBS). One hour after the first injection, animals were either maintained in seawater (SW) or transferred to dSW and injections were carried out at 12-h intervals. After ≈48 h of salinity change, all animals were sacrificed and gills dissected for analysis. NAC injections successfully inhibited ROS formation occurring due to dSW transfer. However, this induced 55% crab mortality, as well as an inhibition of the enhanced catalase defenses and mitochondrial biogenesis that occur with decreased salinity. Crab osmoregulatory capacity under dSW condition was not affected by NAC, although it induced in anterior (non-osmoregulatory) gills a 146-fold increase in Na+/K+/2Cl- expression levels, reaching values typically observed in osmoregulatory tissues. We discuss how ROS influences the physiology of anterior and posterior gills, which have two different physiological functions and strategies during hyper-osmoregulation in dSW.

Effects of Chronic Exposure to Low Doses of Rotenone on Dopaminergic and Cholinergic Neurons in the CNS of Hemigrapsus sanguineus.

Rotenone, as a common pesticide and insecticide frequently found in environmental samples, may be present in aquatic habitats worldwide. Exposure to low concentrations of this compound may cause alterations in the nervous system, thus contributing to Parkinsonian motor symptoms in both vertebrates and invertebrates. However, the effects of chronic exposure to low doses of rotenone on the activity of neurotransmitters that govern motor functions and on the specific molecular mechanisms leading to movement morbidity remain largely unknown for many aquatic invertebrates. In this study, we analyzed the effects that rotenone poisoning exerts on the activity of dopamine (DA) and acetylcholine (ACh) synthesis enzymes in the central nervous system (CNS) of Asian shore crab, Hemigrapsus sanguineus (de Haan, 1835), and elucidated the association of its locomotor behavior with Parkinson's-like symptoms. An immunocytochemistry analysis showed a reduction in tyrosine hydroxylase (TH) in the median brain and the ventral nerve cord (VNC), which correlated with the subsequent decrease in the locomotor activity of shore crabs. We also observed a variation in cholinergic neurons' activity, mostly in the ventral regions of the VNC. Moreover, the rotenone-treated crabs showed signs of damage to ChAT-lir neurons in the VNC. These data suggest that chronic treatment with low doses of rotenone decreases the DA level in the VNC and the ACh level in the brain and leads to progressive and irreversible reductions in the crab's locomotor activity, life span, and changes in behavior.

Ecotoxicological impact of heavy metals on wild mud crabs (Scylla olivacea) in Malaysia: An integrative approach of omics, molecular docking and human risk assessment.

Mud crab, one of the aquatic organisms found in estuary areas, has become a significant economic source of seafood for communities due to its delectable taste. However, they face the threat of heavy metal contamination, which may adversely affect their biological traits. This study explored the comparison of the mud crabs collected from Setiu Wetland as a reference site, while Kuala Sepetang is an area that contains a higher concentration of heavy metals than Setiu Wetlands. Heavy metal levels were quantified using inductively coupled plasma mass spectrometry (ICP-MS), while proteomes were assessed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and 1H nuclear magnetic resonance (NMR)-based metabolomics, respectively. Heavy metal contamination affects the proteome, metabolome, and putative molecular targets in mud crabs (Scylla olivacea), leading to oxidative stress. Mud crabs collected from the metal-polluted area of Kuala Sepetang in Perak had considerably elevated concentrations of nickel (Ni), copper (Cu), zinc (Zn), lead (Pb), chromium (Cr), and cadmium (Cd) in comparison to the reference site of Setiu Wetlands in Terengganu. The proteome analysis revealed an upregulation of the stress-response protein Hsp70, which triggered superoxide dismutase (SOD) and increased arginine kinase expression (5.47 fold) in the muscle tissue, results in the alteration of metabolite regulation in the mud crab from Kuala Sepetang. Additionally, in the muscle tissues of mud crabs obtained from Kuala Sepetang, uncharacterized myosin-tail 1 domain proteins and sarcoplasmic calcium-binding proteins were downregulated. The metabolomic investigation identified changes in metabolites associated with energy metabolism and osmoregulation. Exploration of docking analysis suggests potential connections between methylarsonic acid and essential proteins in mud crabs. These findings suggest that the presence of heavy metals disrupts physiological processes and highlights potential molecular targets that warrant further investigation.

Effects of high temperature and LPS injections on the hemocytes of the crab Neohelice granulata.

Temperature fluctuations, particularly elevated temperatures, can significantly affect immune responses. These fluctuations can influence the immune system and alter its response to infection signals, such as lipopolysaccharide (LPS). Therefore, this study was designed to investigate how high temperatures and LPS injections collectively influence the immune system of the crab Neohelice granulata. Two groups were exposed to 20 °C (control) or 33 °C for four days. Subsequently, half were injected with 10 µL of physiological crustacean (PS), while the rest received 10 µL of LPS [0.1 mg.kg-1]. After 30 min, the hemolymph samples were collected. Hemocytes were then isolated and assessed for various parameters using flow cytometry, including cell integrity, DNA fragmentation, total hemocyte count (THC), differential hemocyte count (DHC), reactive oxygen species (ROS) level, lipid peroxidation (LPO), and phagocytosis. Results showed lower cell viability at 20 °C, with more DNA damage in the same LPS-injected animals. There was no significant difference in THC, but DHC indicated a decrease in hyaline cells (HC) at 20 °C following LPS administration. In granular cells (GC), an increase was observed after both PS and LPS were injected at the same temperature. In semi-granular cells (SGC), there was a decrease at 20 °C with the injection of LPS, while at a temperature of 33 °C, the SGC there was a decrease only in SGC injected with LPS. Crabs injected with PS and LPS at 20 °C exhibited higher levels of ROS in GC and SGC, while at 33 °C, the increase was observed only in GC and SGC cells injected with LPS. A significant increase in LPO was observed only in SGC cells injected with PS and LPS at 20 °C and 33 °C. Phagocytosis decreased in animals at 20 °C with both injections and exposed to 33 °C only in those injected with LPS. These results suggest that elevated temperatures induce changes in immune system parameters and attenuate the immune responses triggered by LPS.

Systemic effects of settleable atmospheric particulate matter (SePM) on swamp ghost crab Ucides cordatus.

Metallurgical activities are a significant source of settleable atmospheric particulate matter (SePM). The material is exposed to wind action, leading to its deposition throughout terrestrial and aquatic ecosystems, thus promoting contamination by metals and metalloids. However, knowledge of the impacts on biota is scarce. In aquatic coastal zones, evaluating hemolymph in invertebrates makes it possible to have insights into the pre-pathogenic effects and health status of organisms. Our study aimed to evaluate bioaccumulation and the sublethal effects of SePM on the mangrove crab Ucides cordatus by assessing biomarkers of cito-genotoxicity in the hemolymph. Organisms underwent a 30-day experiment with four treatments: control; 0.01 g.L-1, 0.1 g.L-1, 1 g.L-1 of SePM, with hemolymph sampled at 2, 7, 15, and 30 days of exposure to assess lipid peroxidation (LPO), DNA damage (strand break), cholinesterase (ChE) and lysosomal membrane stability (LMS). The results revealed metals' bioaccumulation in soft tissues (Al, Fe+, Fe++, Cu, Zr, Nb) and dose-time-dependent responses for LPO, DNA strand break, ChE, and LMS. Significant correlation was found between LPO and Cu (tissue), reduced LMS and Al and Fe (tissue), and Cu, Zn, Ag, and Bi in water. Hemolymph was related to the toxicokinetic and toxicodynamic of metals and metalloids from SePM in Ucides cordatus. New toxicological evidence was obtained to shed light on the impacts of SePM on the ecological status of coastal zones.

The tricarboxylic acid cycle is inhibited under acute stress from carbonate alkalinity in the gills of Eriocheir sinensis.

Owing to population growth and environmental pollution, freshwater aquaculture has been rapidly shrinking in recent years. Aquaculture in saline-alkaline waters is a crucial strategy to meet the increasing demand for aquatic products. The Chinese mitten crab is an important economic food in China, but the molecular mechanism by which it tolerates carbonate alkalinity (CA) in water remains unclear. Here, we found that enzyme activities of the tricarboxylic acid (TCA) cycle in the gills, such as citrate synthase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, and malate dehydrogenase, were markedly reduced under CA stress induced by 40 mM NaHCO3. Secondly, the TCA cycle in the gills is inhibited under acute CA stress, according to proteomic and metabolomic analyses. The expressions of six enzymes, namely aconitate hydratase, isocitrate dehydrogenase, 2-oxoglutarate dehydrogenase, dihydrolipoyl dehydrogenase, succinate-CoA ligase, and malate dehydrogenase, were downregulated, resulting in the accumulation of phosphoenolpyruvic acid, citric acid, cis-aconitate, and α-ketoglutaric acid. Finally, we testified that if the TCA cycle is disturbed by malonate, the survival rate increases in CA water. To our knowledge, this is the first study to show that the TCA cycle in the gills is inhibited under CA stress. Overall, the results provide new insights into the molecular mechanism of tolerance to saline-alkaline water in crabs, which helped us expand the area for freshwater aquaculture and comprehensively understand the physiological characteristics of crab migration.

Mesocosm study of PAC-modified clay effects on Karenia brevis cells and toxins, chemical dynamics, and benthic invertebrate physiology.

Modified clay compounds are used globally as a method of controlling harmful algal blooms, and their use is currently under consideration to control Karenia brevis blooms in Florida, USA. In 1400 L mesocosm tanks, chemical dynamics and lethal and sublethal impacts of MC II, a polyaluminum chloride (PAC)-modified kaolinite clay, were evaluated over 72 h on a benthic community representative of Sarasota Bay, which included blue crab (Callinectes sapidus), sea urchin (Lytechinus variegatus), and hard clam (Mercenaria campechiensis). In this experiment, MC II was dosed at 0.2 g L-1 to treat bloom-level densities of K. brevis at 1 × 106 cells L-1. Cell removal in MC II-treated tanks was 57% after 8 h and 95% after 48 h. In the water column, brevetoxin analogs BTx-1 and BTx-2 were found to be significantly higher in untreated tanks at 24 and 48 h, while in MC II-treated tanks, BTx-3 was found to be higher at 48 h and BTx-B5 was found to be higher at 24 and 48 h. In MC II floc, we found no significant differences in BTx-1 or BTx-2 between treatments for any time point, while BTx-3 was found to be significantly higher in the MC II-treated tanks at 48 and 72 h, and BTx-B5 was higher in MC II-treated tanks at 24 and 72 h. Among various chemical dynamics observed, it was notable that dissolved phosphorus was consistently significantly lower in MC II tanks after 2 h, and that turbidity in MC II tanks returned to control levels 48 h after treatment. Dissolved inorganic carbon and total seawater alkalinity were significantly reduced in MC II tanks, and partial pressure of CO2 (pCO2) was significantly higher in the MC II-only treatment after 2 h. In MC II floc, particulate phosphorus was found to be significantly higher in MC II tanks after 24 h. In animals, lethal and sublethal responses to MC II-treated K. brevis did not differ from untreated K. brevis for either of our three species at any time point, suggesting MC II treatment at this dosage has negligible impacts to these species within 72 h of exposure. These results appear promising in terms of the environmental safety of MC II as a potential bloom control option, and we recommend scaling up MC II experiments to field trials in order to gain deeper understanding of MC II performance and dynamics in natural waters.

Differential effects of oxytetracycline on detoxification and antioxidant defense in the hepatopancreas and intestine of Chinese mitten crab under cadmium stress.

This study aims to evaluate the effects of oxytetracycline (OTC) on detoxification and oxidative defense in the hepatopancreas and intestine of Chinese mitten crab (Eriocheir sinensis) under cadmium (Cd) stress. The crab was exposed to 0.6 µM Cd, 0.6 µM OTC, and 0.6 µM Cd plus 0.6 µM OTC for 42 days. Our results showed that in the intestine, OTC alone enhanced protein carboxylation (PC) and malondialdehyde (MDA) contents, which was associated with the increased OTC accumulation. Compared to Cd alone, Cd plus OTC increased Cd and OTC contents, and reduced detoxification (i.e., glutathione (GSH) content, gene expressions of cytochrome P450 (CYP) isoforms, 7-ethoxyresorufin O-deethylase (EROD) activity, mRNA levels and activities of glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione-S-transferase (GST)), and antioxidant defense (i.e., gene expressions and activities of catalase (CAT) and superoxide dismutase (SOD)) in the intestine, leading to the increased in PC and MDA contents, suggesting that OTC had a synergistic effect on Cd-induced oxidative damage. In the hepatopancreas, although OTC alone increased OTC accumulation, it did not affect PC and MDA contents. Compared to Cd alone, Cd plus OTC reduced MDA content, which was closely related to the improvement of detoxification (i.e., GSH content, mRNA levels of CYP isoforms, EROD activity, gene expressions and activities of GPx, GR and GST), and antioxidant defense (gene expressions and activities of CAT and SOD, metallothionein content). Aryl hydrocarbon receptor (AhR) and nuclear factor E2-related factor 2 (Nrf2) transcriptional expressions were positively correlated with most detoxification- and antioxidant-related gene expressions, respectively, indicating that AhR and Nrf2 were involved in the regulation of these gene expressions. Our results unambiguously demonstrated that OTC had tissue-specific effects on Cd-induced toxicological effect in E. sinensis, which contributed to accurately evaluating Cd toxicity modulated by TCs in crab.

Untargeted LC-MS metabolomics reveals the metabolic responses in the Eriocheir sinensis gills exposed to salinity and alkalinity stress.

In recent years, saline-alkaline aquaculture development has become an important measure for China to expand its fishery development space to ensure food safety. Previous studies have verified that salinity and alkalinity positively influence the quality of Chinese mitten crabs (Eriocheir sinensis). However, the regulatory mechanism of E. sinensis endures saline-alkaline stress which remains obscure. This study investigated the metabolic changes in puberty-molting E. sinensis gills exposed to freshwater (FW), sodium chloride salinity of 5 ppt (SW), and carbonate alkalinity 10.00 mmol/L (AW) for 50 days using untargeted liquid chromatography-mass spectrometry metabolomics (LC-MS). A total of 5802 (positive-ion mode) and 6520 (negative-ion mode) peaks were extracted by LC-MS, respectively. A total of 188 (50 upregulated and 138 downregulated), 141 (94 upregulated and 47 downregulated), and 130 (87 upregulated and 43 downregulated) significantly regulated metabolites (SRMs) were observed in the FW-SW, FW-AW, and SW-AW treatments, respectively, wherein 42 generic SRMs were also found by Venn diagram analysis. Seven of the top 10 SRMs with the highest (variable importance in projection) VIP values were similarly identified in FW-SW and SW-AW. Integrated analysis of key metabolic pathways revealed glycerophospholipid, choline in cancer, phenylalanine, and butanoate metabolism. Overall, significant differences were observed in the metabolites and key metabolic pathways of E. sinensis gill exposed to salinity and alkalinity stress. These results will be helpful in understanding the environmental adaptability of aquatic crustaceans to saline-alkaline water.

Effect of size on concentrations and cadmium inducibility of metallothionein in the shore crab Carcinus maenas.

Metallothionein (MT) plays an important role in protecting organisms from the adverse effects of Cd, Hg, Zn and Cu. Investigations on mammals show variations in metallothionein concentrations and inducibility with age. This has never been investigated in invertebrates, and we determined the concentrations and inducibility of metallothionein in gills and midgut gland of different size classes of shore crabs from uncontaminated areas. Metallothionein concentrations in gills and midgut gland ranged between 30 and 40 µg g-1 dry weight with no differences among the different size classes. Concentrations of cadmium, copper and zinc in the midgut gland increased with increasing size of the crabs when the concentrations were expressed on a dry weight basis; water content in the midgut gland increased with the size and only the cadmium concentration increased with size when concentrations were expressed on wet weight basis. There was an inverse relationship between metallothionein and both copper and cadmium concentrations. Smaller crabs exposed to 1 mg Cd L-1 accumulated higher concentrations of cadmium in midgut gland and gills than larger ones and metallothionein concentrations in the midgut gland were higher in the smaller crabs. However, the increase in metallothionein concentration per accumulated unit of cadmium showed a linear increase with the size of the crabs. The ratio [Cd]midgut/[Cd]gills decreased with the size of the crabs. The overall conclusion is that baseline metallothionein concentrations do not change with age in shore crabs, but that the inducibility of metallothionein upon cadmium challenge does.

The effects of ammonia-N stress on immune parameters, antioxidant capacity, digestive function, and intestinal microflora of Chinese mitten crab, Eriocheir sinensis, and the protective effect of dietary supplement of melatonin.

Ammonia nitrogen pollution seriously affects the economic benefits of Chinese mitten crab (Eriocheir sinensis) farming. In this study, we first evaluated the protective effects of melatonin (MT) on immune parameters, antioxidant capacity, and digestive enzymes of E. sinensis under acute ammonia nitrogen stress. The results showed that ammonia-N stress significantly decreased the antibacterial ability of crabs, nevertheless MT could significantly improve it under ammonia-N stress (P < 0.05). Ammonia-N group hemolymph antioxidant capacity indicators (T-AOC, T-SOD, GSH-Px) were significantly decreased than control (p < 0.05), while the MT ammonia-N group hemolymph T-SOD activity significantly increased than ammonia-N group (p < 0.05). For hepatopancreas, ammonia-N group GSH-PX activity significantly decreased than control group, but MT ammonia-N group was significant increased than ammonia-N (p < 0.05). Ammonia-N stress has significantly increased the content of MDA in hemolymph and hepatopancreas (p < 0.05), but MT ammonia-N treatment significantly decreased than ammonia-N group (p < 0.05). Compared with the control group, ammonia-N significantly reduced the activities of Trypsin in the intestine and hepatopancreas (p < 0.05), while MT ammonia-N group can significantly improve the intestinal trypsin activity than ammonia-N (p < 0.05). The intestinal microbiota of E. sinensis results showed that ammonia-N stress significantly decreased the relative abundance of Bacteroidetes (p < 0.05). Ammonia-N stress significantly decreased the Dysgonomonas and Rubellimicrobium, and the Citrobacter significantly increased. In summary, melatonin has a protective effect on E. sinensis under ammonia-N stress. Acute ammonia-N stress may lead to the decrease of probiotics and the increase of pathogenic bacteria, which may be closely related to the impairment of digestive function and immune function.

Short communication: Effects of acute copper exposure on ionic regulation of the freshwater crab Aegla castro.

Aeglids are unique freshwater decapods whose habitats are being impacted by metallic compounds, such as copper (Cu). Thus, we investigated the effects of acute Cu exposure on ionic regulation of Aegla castro. For this, male specimens in intermolt were collected from a reference stream and acclimated for 5 days in laboratory. After which, crabs were exposed to 11 µg L-1 Cu (Cu11) or only to water (CTR) for 24 h. Hemolymph samples were withdrawn for the determination of Na+, K+, Ca2+, and Mg2+ concentrations and the posterior gills removed for the analysis of Na+/K+-ATPase, Ca2+-ATPase, H+-ATPase, and carbonic anhydrase (CA) activities. Increased Ca2+ and Mg2+ hemolymph concentrations were observed in animals from Cu11, when compared with CTR group. In addition, decreased activity of CA was observed in animals exposed to Cu. In the current study, alterations in Ca2+ and Mg2+concentrations probably indicate that animals activated exoskeleton reabsorption mechanisms, characteristic of the premolt. Therefore, increased Ca2+ and Mg2+ concentrations in hemolymph may indicate that a biochemical signal associated with the molting cycle was triggered by Cu exposure. Despite the known harmful effects of Cu on osmoregulatory enzymes, here we observed decreased activity only in CA. However, decreased activity of CA could trigger both acid-base imbalance and ionic disruption, since CA provides H+ and HCO3- for intracellular pH maintenance, and underpins Na+ and Cl- for ionic regulation. Therefore, understanding how aeglids respond to metal contamination in laboratory conditions is crucial to assess their potential as an alternative biological model for aquatic ecotoxicology.

A New C-Type Lectin Homolog SpCTL6 Exerting Immunoprotective Effect and Regulatory Role in Mud Crab Scylla paramamosain.

C-type lectin (CTL), a well-known immune-related molecule, has received more and more attention due to its diverse functions, especially its important role in development and host defense of vertebrate and invertebrate. Since the research on crab CTLs is still lack, we screened a new CTL homolog, named SpCTL6 from mud crab Scylla paramamosain. The full-length cDNA sequence of SpCTL6 was 738 bp with a 486 bp of ORF, and the deduced amino acids were 161 aa. SpCTL6 was predicted to have a 17 aa signal peptide and its mature peptide was 144 aa (MW 16.7 kDa) with pI value of 5.22. It had typical CTL structural characteristics, such as a single C-type lectin-like domain, 4 conserved cysteines, similar tertiary structure to that of vertebrate CTLs and a mutated Ca2+ binding motif Gln-Pro-Thr (QPT), clustering into the same branch as the crustacean CTLs. SpCTL6 was highly expressed in the entire zoeal larval stages and widely distributed in adult crab tissues with the highest transcription level in testis. During the molting process of juvenile crabs, the expression level of SpCTL6 was remarkably increased after molting. SpCTL6 could be significantly upregulated in two larval stages (Z1 and megalopa) and adult crab testis under immune challenges. Recombinant SpCTL6 (rSpCTL6) was successfully obtained from eukaryotic expression system. rSpCTL6 exhibited binding activity with PAMPs (LPS, lipoteichoic acid, peptidoglycan, and glucan) and had a broad spectrum bacterial agglutination activity in a Ca2+-dependent manner. In addition, rSpCTL6 could enhance the encapsulation activity of hemocytes and has no cytotoxic effect on hemocytes. Although rSpCTL6 had no bactericidal activity on Vibrio alginolyticus, rSpCTL6 treatment could significantly reduce the bacterial endotoxin level in vitro and greatly improved the survival of S. paramamosain under V. alginolyticus infection in vivo. The immunoprotective effect of rSpCTL6 might be due to the regulatory role of rSpCTL6 in immune-related genes and immunological parameters. Our study provides new information for understanding the immune defense of mud crabs and would facilitate the development of effective strategies for mud crab aquaculture disease control.