Inhibitory modulation of action potentials in crayfish motor axons by fluoxetine.

The goal of this report is to explore how K2P channels modulate axonal excitability by using the crayfish ventral superficial flexor preparation. This preparation allows for simultaneous recording of motor nerve extracellular action potentials (eAP) and intracellular excitatory junctional potential (EJP) from a muscle fiber. Previous pharmacological studies have demonstrated the presence of K2P-like channels in crayfish. Fluoxetine (50 µM) was used to block K2P channels in this study. The blocker caused a gradual decline, and eventually complete block, of motor axon action potentials. At an intermediate stage of the block, when the peak-to-peak amplitude of eAP decreased to ∼60%-80% of the control value, the amplitude of the initial positive component of eAP declined at a faster rate than that of the negative peak representing sodium influx. Furthermore, the second positive peak following this sodium influx, which corresponds to the after-hyperpolarizing phase of intracellularly recorded action potentials (iAP), became larger during the intermediate stage of eAP block. Finally, EJP recorded simultaneously with eAP showed no change in amplitude, but did show a significant increase in synaptic delay. These changes in eAP shape and EJP delay are interpreted as the consequence of depolarized resting membrane potential after K2P channel block. In addition to providing insights to possible functions of K2P channels in unmyelinated axons, results presented here also serve as an example of how changes in eAP shape contain information that can be used to infer alterations in intracellular events. This type of eAP-iAP cross-inference is valuable for gaining mechanistic insights here and may also be applicable to other model systems.

EVALUATION OF IMMERSION IN EMULSIFIED ISOFLURANE OR PROPOFOL AS PART OF A TWO-STEP EUTHANASIA PROTOCOL IN MARBLED CRAYFISH (PROCAMBARUS VIRGINALIS).

The marbled crayfish (Procambarus virginalis) is a parthenogenetic invasive species across much of the world, and when found, euthanasia is often recommended to reduce spread to naïve ecosystems. Euthanasia recommendations in crustaceans includes a two-step method: first to produce nonresponsiveness and then to destroy central nervous tissue. Minimal data exist on adequate anesthetic or immobilization methods for crayfish. A population of 90 marbled crayfish was scheduled for euthanasia due to invasive species concerns. The population was divided into six treatment groups to evaluate whether immersion in emulsified isoflurane or propofol solutions could produce nonresponsiveness. Each group was exposed to one of six treatments for 1 h: isoflurane emulsified at 0.1%, 0.5%, 2%, 5%, and 15% or propofol at 10 mg/L and then increased to 100 mg/L. Crayfish from all treatment groups were moved to nonmedicated water after completion of 1 h and observed for an additional 4 h. All crayfish treated with isoflurane showed lack of a righting reflex at 5 min and loss of movement after 30 min. By 240 min (4 h), none of the crayfish from the isoflurane treatment groups regained movement. None of the crayfish in the propofol treatment achieved loss of reflexes or responsiveness, and all remained normal upon return to nonmedicated water. Isoflurane emulsified in water produces nonresponsiveness that is appropriate for the first step of euthanasia, while propofol was insufficient at these treatment doses.

Bisphenol S exposed changes in intestinal microflora and metabolomics of freshwater crayfish, Procambarus clarkii.

Bisphenol S (BPS), a typical endocrine-disrupting chemical (EDC), can cause hepatopancreas damage and intestinal flora disturbance. Comprehensive studies on the mechanisms of acute toxicity in crustaceans are lacking. In this study, 16S rRNA and liquid chromatography were used to investigate intestinal microbiota and metabolites of freshwater crayfish (Procambarus clarkii). In this study, freshwater crayfish were exposed to BPS (10 µg/L and 100 µg/L). The results showed a significant decrease in catalase (CAT) and superoxide dismutase (SOD) activities after exposure to BPS, which inhibited the Nrf2-Keap1 signaling pathway and induced oxidative stress toxicity in freshwater crayfish. In addition, BPS exposure induced the structural changes of intestinal microbial in the freshwater crayfish, showing different patterns of effects. The number of potentially pathogenic bacteria increased, such as Citrobacter, Hafnia-Obesumbacterium, and RsaHf231. A total of 128 different metabolites were analyzed by LC-MS/MS. The inositol and leukotriene (LT) contents in the hepatopancreas of freshwater crayfish were significantly decreased after 10 µg/L BPS exposure, which in turn led to the accumulation of lipids causing hepatopancreas damage. In conclusion, when the concentration of BPS in the water environment exceeded 10 µg/L, the freshwater crayfish intestinal microbiota was dysbiosis and the hepatopancreas metabolism was disturbed.

Toxic mechanisms of nanoplastics exposure at environmental concentrations on juvenile red swamp crayfish (Procambarus clarkii): From multiple perspectives.

Nanoplastics pollution has emerged as a global issue due to its widespread potential toxicity. This study delved in to toxic effects of nanoplastics on juvenile P. clarkii and molecular mechanisms from perspectives of growth, biochemical, histopathological analysis and transcriptome level for the first time. The findings of this study indicated that nanoplastics of different concentrations have varying influence mechanisms on juvenile P. clarkii. Nanoplastics have inhibitory effects on growth of juvenile P. clarkii, can induce oxidative stress. The biochemical analysis and transcriptome results indicated that 10 mg/L nanoplastics can activate the antioxidant defense system and non-specific immune system in juvenile P. clarkii, and affect energy metabolism and oxidative phosphorylation. While 20 mg/L and 40 mg/L have a destructive influence on the immune function in juvenile P. clarkii, leading to lipid peroxidation and oxidative damage, and induce apoptosis, can affect ion transport and osmotic pressure regulation. The findings of this study can offer foundational data for delving further into impacts of nanoplastics on crustaceans and toxicity mechanism.

Gradual effects of gradient concentrations of perfluorooctane sulfonate on the antioxidant ability and gut microbiota of red claw crayfish (Cherax quadricarinatus).

Perfluorooctane sulfonate (PFOS) is a typical persistent organic pollutant that is characterized by environmental persistence, bioaccumulation, and toxicity. In this study, we investigated the gut microbial response of the red claw crayfish Cherax quadricarinatus after 28 days of exposure to 0 ng/L, 1 ng/L, 10 µg/L, or 10 mg/L of PFOS as a stressor. We measured oxidative stress-related enzyme activities and expression of molecules related to detoxification mechanisms to evaluate the toxic effects of PFOS. We found that PFOS disturbed microbial homeostasis in the gut of C. quadricarinatus, resulting in increased abundance of the pathogen Shewanella and decreased abundance of the beneficial bacterium Lactobacillus. The latter especially disturbed amino acid transport and carbohydrate transport. We also found that the activities of glutathione S-transferase and glutathione peroxidase were positively correlated with the expression levels of cytochrome P450 genes (GST1-1, GSTP, GSTK1, HPGDS, UGT5), which are products of PFOS-induced oxidative stress and play an antioxidant role in the body. The results of this study provided valuable ecotoxicological data to better understand the biological fate and effects of PFOS in C. quadricarinatus.

Epigallocatechin-3-gallate attenuates the sulfamethoxazole-induced immunotoxicity and reduces SMZ residues in Procambarus clarkii.

Sulfamethoxazole (SMZ) is a commonly used antibiotic in aquaculture, and its residues in water bodies pose a significant threat to aquatic organisms in the water environment. In the present study, epigallocatechin-3-gallate (EGCG), a catecholamine, was used to mitigate the immunotoxicity caused by SMZ exposure in Procambarus clarkii. EGCG reduced the apoptosis rate, which was elevated by SMZ exposure, and increased the total hemocyte count. Simultaneously, EGCG enhanced the activities of enzymes related to antibacterial and antioxidant activities, such as superoxide dismutase (SOD), catalase (CAT), lysozyme (LZM), acid phosphatase (ACP), and GSH, which were decreased following SMZ exposure. Hepatopancreatic histology confirmed that EGCG ameliorated SMZ-induced tissue damage caused by SMZ exposure. In addition to EGCG attenuating SMZ-induced immunotoxicity in crayfish, we determined that EGCG can effectively reduce SMZ residues in crayfish exposed to SMZ. In addition, at the genetic level, the expression levels of genes related to the immune response in hemocytes were disrupted after SMZ exposure, and EGCG promoted their recovery and stimulated an increase in the expression levels of metabolism-related transcripts in hemocytes. The transcriptome analysis was conducted, and "phagosome" and "apoptosis" pathways were shown to be highlighted using Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. To the best of our knowledge, this is the first study to confirm that EGCG attenuates SMZ-induced immunotoxicity in aquatic animals and reduces SMZ residues in aquatic animals exposed to SMZ. Our study contributes to the understanding of the mechanisms by which EGCG reduces the immunotoxicity of antibiotic residues in aquatic animals.

Integration of transcriptome, gut microbiota, and physiology reveals toxic responses of the red claw crayfish (Cherax quadricarinatus) to imidacloprid.

Imidacloprid enters the water environment through rainfall and causes harm to aquatic crustaceans. However, the potential chronic toxicity mechanism of imidacloprid in crayfish has not been comprehensively studied. In this study, red claw crayfish (Cherax quadricarinatus) were exposed to 11.76, 35.27, or 88.17 µg/L imidacloprid for 30 days, and changes in the physiology and biochemistry, gut microbiota, and transcriptome of C. quadricarinatus and the interaction between imidacloprid, gut microbiota, and genes were studied. Imidacloprid induced oxidative stress and decreased growth performance in crayfish. Imidacloprid exposure caused hepatopancreas damage and decreased serum immune enzyme activity. Hepatopancreatic and plasma acetylcholine decreased significantly in the 88.17 µg/L group. Imidacloprid reduced the diversity of the intestinal flora, increased the abundance of harmful flora, and disrupted the microbiota function. Transcriptomic analysis showed that the number of up-and-down-regulated differentially expressed genes (DEGs) increased significantly with increasing concentrations of imidacloprid. DEG enrichment analyses indicated that imidacloprid inhibits neurotransmitter transduction and immune responses and disrupts energy metabolic processes. Crayfish could alleviate imidacloprid stress by regulating antioxidant and detoxification-related genes. A high correlation was revealed between GST, HSPA1s, and HSP90 and the composition of gut microorganisms in crayfish under imidacloprid stress. This study highlights the negative effects and provides detailed sequencing data from transcriptome and gut microbiota to enhance our understanding of the molecular toxicity of imidacloprid in crustaceans.

Early Changes in Crayfish Hemocyte Proteins after Injection with a ß-1,3-glucan, Compared to Saline Injected and Naive Animals.

Early changes in hemocyte proteins in freshwater crayfish Pacifastacus leniusculus, in response to an injection with the fungal pattern recognition protein ß-1,3-glucan (laminarin) were investigated, as well as changes after saline (vehicle) injection and in naïve animals. Injection of saline resulted in rapid recruitment of granular hemocytes from surrounding tissues, whereas laminarin injection on the other hand induced an initial dramatic drop of hemocytes. At six hours after injection, the hemocyte populations therefore were of different composition. The results show that mature granular hemocytes increase in number after saline injection as indicated by the high abundance of proteins present in granular cell vesicles, such as a vitelline membrane outer layer protein 1 homolog, mannose-binding lectin, masquerade, crustin 1 and serine protease homolog 1. After injection with the ß-1,3-glucan, only three proteins were enhanced in expression, in comparison with saline-injected animals and uninjected controls. All of them may be associated with immune responses, such as a new and previously undescribed Kazal proteinase inhibitor. One interesting observation was that the clotting protein was increased dramatically in most of the animals injected with laminarin. The number of significantly affected proteins was very few after a laminarin injection when compared to uninjected and saline-injected crayfish. This finding may demonstrate some problematic issues with gene and protein expression studies from other crustaceans receiving injections with pathogens or pattern recognition proteins. If no uninjected controls are included and no information about hemocyte count (total or differential) is given, expressions data for proteins or mRNAs are very difficult to properly interpret.

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.

Comparative transcriptome analysis of the gills of Procambarus clarkii provide novel insights into the response mechanism of ammonia stress tolerance.

Procambarus clarkii is an important model crustacean organism in many researches. Ammonia nitrogen is one of common contaminants in aquatic environment, influencing the health of aquatic organisms. The primary objective of this study was to investigate molecular mechanisms on ammonia stress in gills of P. clarkii to provide new insights into the strategies of aquatic animals in responding to high concentration of ammonia in the environment. Procambarus clarkii were randomly assigned into two groups (ammonia stress group, AG; control group, CG), and gill samples were dependently excised from AG and CG. Then response mechanisms on ammonia stress were investigated based on transcriptome data of P. clarkii. 9237 differentially expressed genes were identified in ammonia stress group. The genes of ion transport enzymes (NKA and SLC6A5S) were significantly up-regulated. Whereas the immune-related genes (e.g. MAP3K7, HSP70, HSP90A, CTSF, CTSL1, CHI and CTL4) and pathways were significantly up-regulated, which played an important role in reacting to ammonia stress. Procambarus clarkii may enhance immune defense to counteract ammonia toxicity by the up-regulation of immune-related genes and signaling pathways. The activities of ion transport enzymes are changed to mobilise signal transduction and ion channel regulation for adapting to ammonia environment. These previous key genes play an important role in resistance to ammonia stress to better prepare for survival in high concentration of ammonia.

Enhancement of synaptic responses in ascending interneurones following acquisition of social dominance in crayfish.

When crayfish have attained dominant status after agonistic bouts, their avoidance reaction to mechanical stimulation of the tailfan changes from a dart to a turn response. Ascending interneurones originating in the terminal ganglion receive sensory inputs from the tailfan and they affect spike activity of both uropod and abdominal postural motor neurones, which coordinates the uropod and abdominal postural movements. Despite the varying output effects of ascending interneurones, the synaptic responses of all interneurones to sensory stimulation were enhanced when they acquired a dominant state. The number of spikes increased as did a sustained membrane depolarizations. Regardless of social status, the output effects on the uropod motor neurones of all interneurones except VE-1 remained unchanged. VE-1 mainly inhibited the uropod opener motor neurones in naive animals, but tended to excite them in dominant animals. Synaptic enhancement of the sensory response of ascending interneurones was also observed in naive animals treated with bath-applied serotonin. However, subordinate animals or naive animals treated with octopamine had no noticeable effect on the synaptic response of their ascending interneurones to sensory stimulation. Thus, enhancement of the synaptic response is a specific neural event that occurs when crayfish attain social dominance and it is mediated by serotonin.

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.

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.

Evaluation of the acute toxic effect of azoxystrobin on non-target crayfish (Astacus leptodactylus Eschscholtz, 1823) by using oxidative stress enzymes, ATPases and cholinesterase as biomarkers.

Azoxystrobin is a broad-spectrum fungicide used worldwide. Since azoxystrobin spreads to large areas, its toxic effects on non-target organisms have aroused interest. In this study, the acute toxicity (96 h) of azoxystrobin on the crayfish (Astacus leptodactylus) was examined by using various biomarkers. The 96 h-LC50 dose (1656 mg L-) and its three sub-doses (828, 414, 207 mg L-1) were applied to crayfish. Superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities were increased significantly compared to the control in hepatopancreas, gill and muscle tissues. The activities of acetylcholinesterase (AChE) and glutathione S-transferase (GST) increased, and glutathione reductase (GR) activity decreased significantly in hepatopancreas. Level of reduced glutathione (GSH) decreased significantly. The content of malondialdehyde (MDA) increased in a dose-dependent manner in all azoxystrobin treatments with the exception of the lowest dose (207 mg L-1)treatment. ATPases (Na+/K+ -ATPase, Mg2+ -ATPase, Ca2+ -ATPase, total ATPase) were significantly inhibited in gill and muscle tissues. The results of the present study indicate that azoxystrobin induces oxidative stress, and has adverse effects on activities of AChE and ATPases in crayfish.

Roles of a small GTPase Sar1 in ecdysteroid signaling and immune response of red swamp crayfish Procambarus clarkii.

Secretion-associated and ras-related protein 1 (Sar1) is a small GTPase that plays an important role in the transport of protein coated with coat protein complex II vesicles. However, its alternative roles in the biological processes of Procambarus clarkii remain unclear. Here, a sar1 gene (named as Pc-sar1) with an open reading frame of 582 bp from P. clarkii was identified. Pc-sar1 was expressed in all examined tissues with highest expression levels in muscle, which was determined by real-time PCR and western blotting. After the induction of lipopolysaccharide (LPS) and polycytidylic acid (Poly I: C), the transcriptional levels of Pc-sar1 differed in hepatopancreas, gill, muscle and intestine. In contrast, the expression of Pc-sar1 was upregulated by 20-hydroxyecdysone in these four tissues. In addition, the RNA interference of Pc-sar1 significantly affected the expression levels of immune and hormone-related genes. These results indicate that Pc-sar1 is involved in the innate immune response and ecdysteroid signaling pathway.

In vitro effects of selected endocrine disruptors (DEHP, PCB118, BPA) on narrow-clawed crayfish (Astacus leptodactylus) primary cells.

Environmental pollutants with endocrine-disrupting effect are of global importance due to their contribution to the aethiologies of variety of complex diseases. These lipophilic pollutants are persistent in the environment and able to bioaccummulate in nontarget organisms. BPA, DEHP and PCB118 (dioxin-like PCB) are associated with endocrine disruption effects, while information on their effects on aquatic invertebrates are limited. In the current study, the effects of these compounds, which are ubiqutous and present at low concentrations in the environment, are studied in the primary hepatopancreas, muscle, gill, intestine and gonadal cultures of narrow-clawed crayfish (Astacus leptodactylus Eschscholtz, 1823), a widely distributed freshwater crayfish in Turkey with high economic importance. IC50 values following MTT assay ranged 0.27-12.61 nM; when compared with other tissues, the gonads were more affected with lower IC50 values. PCB118 induced higher cytotoxicity, while DEHP was the least toxic compound. This is the first study on the primary culture of A. leptodactylus¸ and the toxic effects of these compounds in this organism providing mechanistic insights on the responses and detoxification capacity of the organs. This study provides basis to unravel the mechanism of action of the tested EDCs in crayfish and improvement of cell culture conditions for ecotoxicity and screening assays.

Acute and sub-chronic effects of copper on survival, respiratory metabolism, and metal accumulation in Cambaroides dauricus.

Trace metal contamination in the aquatic ecosystem occurs worldwide: although copper is an essential trace metal, it is considered as a pollutant at certain levels in China. Freshwater crayfish Cambaroides dauricus is a commercially important wild species in northeastern China, in which is an important heavy industry area. The population of C. dauricus was decreasing sharply due to the environmental pollution and human intervention over the past 20 years. However, nothing is known regarding the responses of this species to trace metal toxicants. This study aimed to determine the acute and chronic toxicity of Cu and its toxicological effects on respiratory metabolism, as well as Cu accumulation in C. dauricus. The acute (96 h) median lethal concentration (LC50) value of 32.5 mg/L was detected in C. dauricus. Then, acute (96 h; 8.24, 16.48 mg/L) and sub-chronic (14 days; 2.06, 4.12 mg/L) exposure in Cu was investigated by estimating the oxygen consumption rate, ammonium excretion rate, and Cu accumulation. Both acute and sub-chronic Cu exposure induced an inhibition of the oxygen consumption rate and ammonium excretion rate, and thereby, an increased O:N ratio. The shift in O:N ratio indicated a metabolic substrate shift towards lipid and carbohydrate metabolism under Cu stress. Cu accumulation in the hepatopancreas and muscles throughout the study was found to be time-dependent and concentration-dependent. The maximum accumulation in the hepatopancreas and muscle were almost 31.6 folds of the control after 14 days' exposure to 4.12 mg/L concentration. Based on the present work, we suggest that crayfish be considered a potential bioindicator of environmental pollution in freshwater systems. The study provides basic information for further understanding of the toxicological responses of this species to trace metals.

The effects of tricaine mesylate on arthropods: crayfish, crab and Drosophila.

Tricaine mesylate, also known as MS-222, was investigated to characterize its effects on sensory neurons, synaptic transmission at the neuromuscular junction, and heart rate in invertebrates. Three species were examined: Drosophila melanogaster, blue crab (Callinectes sapidus), and red swamp crayfish (Procambarus clarkii). Intracellular measures of action potentials in motor neurons of the crayfish demonstrated that MS-222 dampened the amplitude, suggesting that voltage-gated Na + channels are blocked by MS-222. This is likely the mechanism behind the reduced activity measured in sensory neurons and depressed synaptic transmission in all three species as well as reduced cardiac function in the larval Drosophila. To address public access to data, a group effort was used for analysis of given data sets, blind to the experimental design, to gauge analytical accuracy. The determination of a threshold in analysis for measuring extracellular recorded sensory events is critical and is not easily performed with commercial software.

Effects of exposure to waterborne polystyrene microspheres on lipid metabolism in the hepatopancreas of juvenile redclaw crayfish, Cherax quadricarinatus.

Previous research has identified microplastics as new environmental pollutants that are widely distributed in a variety of environments, including aquaculture environments. However, the potential hazard of microplastics to aquaculture animals, especially toward lipid metabolism involved with the survival and growth of aquatic animal, has not yet been investigated. In the present study, redclaw crayfish (Cherax quadricarinatus) were exposed to different concentrations of 200 nm-sized polystyrene microspheres (0, 0.5, and 5 mg/L) for 21 days, to investigate the effects of microplastics on lipid metabolism. After ingestion, the microplastics were distributed in the intestines and hepatopancreas, and appeared to inhibit the growth of Cherax quadricarinatus. Subsequently, the lipid levels in the hepatopancreas and hemolymph was detected, and found that after 21 days of exposure, the lipid content and free fatty acids in the hepatopancreas and hemolymph decreased significantly, and total cholesterol and triglycerides levels increased significantly in the hemolymph. This might have been caused by insufficient intake of exogenous fat. A significant decrease in lipase activity also supported this view. The activity of lipoprotein lipase related to lipolysis in the hepatopancreas increased significantly, while the activity of fatty acid synthase related to fat synthesis increased, and the activity of acetyl-CoA carboxylase decreased. These results indicated disturbed lipid metabolism in the hepatopancreas. The significant increase of lipid transport-related low-density lipoprotein indicated that the lipolytic capacity was higher than the lipid synthesis capacity. The expression levels of fatty acid metabolism-related genes FAD6 and FABP decreased significantly, indicating that the fatty acid utilization ability of hepatopancreas cells was inhibited, which was consistent with the results of enzyme activities. Thus, microplastics represent a potential hazard to redclaw crayfish, at least on lipid metabolism. This study provided basic data on the ecotoxicological effects of microplastics on crustaceans.

Effects of chloridazon on early life stages of marbled crayfish.

The effects of chloridazon exposure at concentrations of 2.7 µg/L (maximal real environmental concentration in the Czech Republic), 27 µg/L, 135 µg/L and 270 µg/L on early life stages of marbled crayfish (Procambarus virginalis) were evaluated. Significantly higher glutathione S-transferase activity and reduced glutathione level was observed at all tested concentrations of chloridazon compared with the control. Chloridazon in concentrations 27, 135 and 270 µg/L caused delay ontogenetic development and slower growth. Histopathological changes in hepathopancreas were found in two highest tested concentrations (135 µg/L and 270 µg/L). Crayfish behaviour was not altered in control vs. exposed animals, while the activity parameters tend to decline with increasing chloridazon concentrations.