Copper-Induced Chemosensory Impairment is Reversed by a Short Depuration Period in Northern Clearwater Crayfish (Faxonius propinquus).

Crayfish rely on their chemosensory system for many essential behaviours including finding food, finding mates, and to recognize individuals. Copper can impair chemosensation in crayfish at low concentrations; however, it is not clear if the effect is ameliorated once copper is removed. To better understand the effect of and recovery from copper exposure in crayfish, we exposed Northern clearwater crayfish (Faxonius propinquus) to 31.3 [Formula: see text] copper for 24 h and measured the response of the crayfish to a food cue. The crayfish were then placed into clean water to depurate for an 24 h. The results demonstrated that the crayfish did not respond to a food cue if they had been exposed to copper, but showed a full response after a 24 h recovery period without copper. Higher concentrations of copper have shown a much longer-term effect in rusty crayfish (Faxonius rustics), indicating there is a concentration where the copper is causing longer-term damage instead of just impairing chemosensation. These results highlight the fact that even though contaminants like copper can have profound effects at low concentrations, by removing the contaminants the effect can be ameliorated.

Effects of carbon source addition in rearing water on sediment characteristics, growth and health of cultured marron (Cherax cainii).

Carbon sources are considered as critical input for the health and immunity of aquatic animals. The present study investigated the impact of different carbon sources on water quality parameters, carbon to nitrogen (C/N) ratio and microbial community in sediments, and health responses of marron (Cherax cainii) under laboratory conditions. Following one week of acclimation, 120 marron were randomly assigned to 12 experimental tanks. There were four treatments including one untreated control and three groups with carbon addition to maintain a C/N ratio of 12 maintained in culture water. Carbon supplementation groups included corn flour (CBC12), molasses (MBC12) and wheat flour (WBC12). At the end of the 60-day trial, MBC12 resulted in the highest sediment C/N ratio, followed by CBC12. Weight gain and specific growth rate were higher in MBC12, compared to control. The protease activity in marron hepatopancreas, total haemocyte count and lysozyme activity in haemolymph were highest in MBC12. Analysis of 16S rRNA sequence data of tank sediments revealed increased bacterial alpha diversity in MBC12 and WBC12. Proteobacteria was the most abundant phylum in MBC12 (88.6%), followed by control (82.4%) and CBC12 (72.8%). Sphingobium and Novosphingobium were the most abundant genera in control and MBC12 groups, respectively. Higher Aeromonas abundance in CBC12 and Flavobacterium in WBC12 were observed. Overall results indicated that MBC12 led to improved water quality, retaining high C/N ratio and enriched the bacterial populations in sediments resulting in improved growth and immune performance of marron.

Temperature and hypoxia trigger developmental phenotypic plasticity of cardiorespiratory physiology and growth in the parthenogenetic marbled crayfish, Procambarus virginalis Lyko, 2017.

Attempting to differentiate phenotypic variation caused by environmentally-induced alterations in gene expression from that caused by actual allelic differences can be experimentally difficult. Environmental variables must be carefully controlled and then interindividual genetic differences ruled out as sources of phenotypic variation. We investigated phenotypic variability of cardiorespiratory physiology as well as biometric traits in the parthenogenetically-reproducing marbled crayfish Procambarus virginalis Lyko, 2017, all offspring being genetically identical clones. Populations of P. virginalis were reared from eggs tank-bred at four different temperatures (16, 19, 22 and 25 °C) or two different oxygen levels (9.5 and 20 kPa). Then, at Stage 3 and 4 juvenile stages, physiological (heart rate, oxygen consumption) and morphological (carapace length, body mass) variables were measured. Heart rate and oxygen consumption measured at 23 °C showed only small effects of rearing temperature in Stage 3 juveniles, with larger effects evident in older, Stage 4 juveniles. Additionally, coefficients of variation were calculated to compare our data to previously published data on P. virginalis as well as sexually-reproducing crayfish. Comparison revealed that carapace length, body mass and heart rate (but not oxygen consumption) indeed showed lower, yet notable coefficients of variation in clonal crayfish. Yet, despite being genetically identical, significant variation in their morphology and physiology in response to different rearing conditions nonetheless occurred in marbled crayfish. This suggests that epigenetically induced phenotypic variation might play a significant role in asexual but also sexually reproducing species.

Effect of psychoactive substances on cardiac and locomotory activity of juvenile marbled crayfish Procambarus virginalis.

Pharmaceutically active compounds are common and increasing in the aquatic environment. Evidence suggests they have adverse effects on non-target organisms, and they are classified as emerging pollutants for a variety of aquatic organisms. To determine the effects of environmentally relevant levels of psychoactive compounds on non-target organisms, we analyzed cardiac and locomotory activity in early developmental stages of marbled crayfish Procambarus virginalis. Responses to sertraline, methamphetamine, and a mixture of citalopram, oxazepam, sertraline, tramadol, venlafaxine, and methamphetamine at a concentration of 1 µg L-1 of each compound were assessed. On day four of exposure, cardiac activity was recorded for 5 min, and on day eight, locomotory activity was recorded for 15 min. There was a significant increase (p < 0.01) in heart rate in methamphetamine-exposed and Mix-exposed juveniles compared to the unexposed control and there was significant difference (p < 0.01) in proportion of time (activity %) was observed with sertraline-exposed, whereas velocity, and distance moved did not significantly differ (p > 0.05) in exposed and control animals. These findings revealed that low concentrations of chemicals and their mixtures can modify the physiological state of aquatic animals without outward manifestations (activity, distance moved, and velocity). Aquatic animals can be impacted earlier than is visible, but effects can potentially lead to substantial changes in populations and in ecosystem processes. Additional research to investigate chemical combinations, exposure systems, and organism physiological and molecular responses may provide evidence of broad impact of environmental pharmaceuticals.

Quantification of proteomic profile changes in the hemolymph of crayfish during in vitro coagulation.

Hemolymph is the circulatory fluid that fills the body cavity of crustaceans, analogous to blood in vertebrates. Hemolymph coagulation, similar to blood clotting in vertebrates, plays a crucial role in wound healing and innate immune responses. Despite extensive studies on the clotting process in crustaceans, no comparative quantitative analysis of the protein composition of non-clotted and clotted hemolymph in any decapod has been reported. In this study, we used label-free protein quantification with high-resolution mass spectrometry to identify the proteomic profile of hemolymph in crayfish and quantify significant changes in protein abundances between non-clotted and clotted hemolymph. Our analysis identified a total of two-hundred and nineteen proteins in both hemolymph groups. Furthermore, we discussed the potential functions of the top most high and low-abundant proteins in hemolymph proteomic profile. The quantity of most of the proteins was not significantly changed during coagulation between non-clotted and clotted hemolymph, which may indicate that clotting proteins are likely pre-synthesized, allowing for a swift coagulation response to injury. Four proteins still showed abundance differences (p < 0.05, fold change>2), including C-type lectin domain-containing proteins, Laminin A chain, Tropomyosin, and Reverse transcriptase domain-containing proteins. While the first three proteins were down-regulated, the last one was up-regulated. The down-regulation of structural and cytoskeletal proteins may affect the process of hemocyte degranulation needed for coagulation, while the up-regulation of an immune-related protein might be attributed to the phagocytosis ability of viable hemocytes during coagulation.

Antioxidation, immunity and hepatopancreatic histology of red swamp crayfish (Procambarus clarkii) subjected to low-temperature aerial exposure stress and re-immersion.

Desiccation is a stressful situation that decapods often experience during live transportation. This study investigated the effects of low-temperature aerial exposures (LTAEs) (dry exposure (DL) and moist exposure (ML) at 6 °C) and re-immersion on the antioxidative and immune responses and hepatopancreatic histopathology in P. clarkii. Compared to the control group (normally feeding at 24.0 °C water temperature), the crayfish under LTAEs showed overall severe hepatopancreatic oxidative damage, with significantly increased malondialdehyde (MDA) contents and significantly reduced total antioxidant capacity (T-AOC), and oxidant damage was not fully recovered even after 12 h of re-immersion; the expression of hsp70 was significantly increased within 24-48 h of stress and re-immersion. The activity of hemolymphatic acid phosphatase (ACP) was significantly increased during 24-48 h of the stress and at 12 h of re-immersion; the activity of aspartic aminotransferase (AST) and alanine aminotransferase (ALT) was significantly increased throughout the experiment; and the gene expression of proPO or TLR was significantly increased during 12-48 h of the stress. Severe histopathological changes (lumen dilatation, vacuolation of epithelial cells and reduced cell numbers) were observed in hepatopancreas at 48 h of stress and 12 h of re-immersion. These results indicated that 48 h of low-temperature aerial exposure stress stimulated the non-specific immunity but adversely affected the antioxidation and hepatopancreatic histomorphology of P. clarkii, whereas 12 h of re-immersion was not sufficient to restore crayfish from stress to a normal state.

Short-term effects of an environmentally relevant concentration of organic UV filters on signal crayfish Pacifastacus leniusculus.

Personal care products, including organic UV filters, are considered emerging contaminants, with their toxic effects being a concern in recent decades. UV filters continually enter surface waters via wastewater and human activity. Despite the presence of organic UV filters in the freshwater environment, little is known of their impact on aquatic biota. In this study, we evaluated the cardiac and locomotor responses of signal crayfish Pacifastacus leniusculus exposed to environmentally relevant concentrations of either 2-Phenylbenzimidazole-5-sulfonic acid (PBSA, 3 µg/L) or 5-Benzoyl-4-hydroxy-2-methoxybenzenesulfonic acid (BP4, 2.5 µg/L). Specimens exposed to the tested compounds for 30 min exhibited significantly greater changes in distance moved and time active than did unexposed controls. Significant differences of mean heart rate change compared to control were detected in both PBSA and BP4 experimental groups. Such behavior and physiological alterations demonstrate ecological effects of personal care products with the tested sunscreen compounds even with a short exposure. Evidence of the consequences of organic UV filters on aquatic organisms is scarce and is an important topic for future research.

Sub-chronic ammonia exposure induces hepatopancreatic damage, oxidative stress, and immune dysfunction in red swamp crayfish (Procambarus clarkii).

Ammonia, as one of the primary water pollutants in aquaculture, has been shown to induce a wide range of ecotoxicological effects on aquatic animals. In order to investigate the antioxidant and innate immune responses in crustaceans disrupted by ammonia, red swamp crayfish (Procambarus clarkii) were exposed to 0, 15, 30, and 50 mg/L total ammonia nitrogen for 30 d, the alterations of antioxidant responses as well as innate immunity were studied. The results showed that the severity of hepatopancreatic injury were aggravated by the increasing ammonia levels, which were mainly characterized by tubule lumen dilatation and vacuolization. The swollen mitochondria and disappeared mitochondria ridges suggested that oxidative stress induced by ammonia targets the mitochondria. Concurrently, enhanced MDA levels, and decreased GSH levels as well as the decreased transcription and activity of antioxidant enzymes, including SOD, CAT, and GPx were noticed, which suggested that high concentrations of ammonia exposure induce oxidative stress in P. clarkii. Furthermore, a significant decrease of the hemolymph ACP, AKP, and PO along with the significant downregulation of immune-related genes (ppo, hsp70, hsp90, alf1, ctl) jointly indicated that ammonia stress inhibited the innate immune function. Our findings demonstrated that sub-chronic ammonia stress induced hepatopancreatic injury and exert suppressive effects on the antioxidant capacity as well as innate immunity of P. clarkii. Our results provide a fundamental basis for the deleterious effects of ammonia stress on aquatic crustaceans.

Long-term macrophyte and snail community responses to population declines of invasive rusty crayfish (Faxonius rusticus).

A central focus of invasive species research has been on human efforts to eradicate invaders or reduce their abundance to mitigate the worst of their impacts. In some cases, however, populations of invasive species decline without human intervention, which may inform management responses to these invaders. Such is the case of the invasive rusty crayfish (Faxonius rusticus) in northern Wisconsin, USA, where systematic population monitoring since 1975 has revealed population declines in approximately half of the lakes surveyed. Population declines of invasive species without human intervention remain understudied, but there is even less research on how communities respond following such declines. Using 10 lakes in Vilas County, Wisconsin, we investigated community recovery of habitat (macrophytes) and prey (freshwater snails) of F. rusticus following up to 33 years of declines of this invader in some lakes using a dataset with a rare, long-term span over which consistent data were collected (1987, 2002, 2011, and 2020). We compared community responses in lakes where F. rusticus populations reached a peak and subsequently declined (boom-bust lakes) and lakes where our dataset only captured the decline of F. rusticus (bust lakes) to reference lakes with consistently high or low crayfish abundance over time. We found partial recovery of macrophytes and snails in the bust and boom-bust lakes where F. rusticus has declined, with recovery of macrophyte abundance and richness in the boom-bust lakes achieving levels observed in the low-crayfish reference lakes. Snail abundance and richness increased after declines of F. rusticus, though not to the level of the low-crayfish reference lakes, suggesting that snail recovery may lag macrophyte recovery because snails are dependent on macrophytes and associated periphyton for habitat. The recovery we document potentially represents long-term ecosystem resilience of lakes to biological invasions. Our results suggest that lake communities may recover without active restoration interventions after invasive crayfish population declines, although identifying which lakes experience these natural declines remains a priority for future research and management.

Factors influencing accumulation of Zn, Cu, and Ca in the tissues of spiny-cheek crayfish (Faxonius limosus, Rafinesque, 1817).

Both physicochemical and biological factors affect the degree of metal accumulation in crayfish tissues. The content of metals and correlations between the metal concentrations in different tissues and the total length of crayfish is suitable indicators of contamination of the aquatic environment. The aim of the study was to analyse the effect of age and sex of crayfish on the degree of accumulation of Ca, Cu, and Zn in the muscle and exoskeleton. A total of 100 individuals of the spiny-cheek crayfish (Faxonius limosus, Rafinesque, 1817) were caught from Glowinsk reservoir (Poland) in October 2019 using fyke nets. Metal concentrations were determined in freeze-dried samples of the abdominal muscle, exoskeleton, bottom sediment, and water using atomic absorption spectroscopy (AAS). Here, we show that the highest concentrations of Zn were found in the muscle of 4-year-old females, Cu in 3-year-old males, and Ca in 4-year-old males. Sex was a significant factor affecting the content of Ca in the muscle and Zn in the exoskeleton. Age was a significant factor affecting the content of Zn, Cu, and Ca in the muscle and Zn and Cu in the exoskeleton. The bioconcentration factor (BCF) of Zn and Cu in the muscle and exoskeleton of spiny-cheek crayfish was much higher from water than from sediments, unlike Ca. Furthermore, we found significant correlation for muscle between Zn and total length in 3-year-old females and 4-year-old males and between Cu and TL in 3-year-old males. Analysing the recommended daily intake (RDI) for the investigated minerals confirmed that the consumption of 100 g of spiny-cheek crayfish muscle could meet daily requirement for Zn up to 27.5%, for Ca in 12.4%, and over 100% for Cu. The conducted analyses confirmed that the consumption of crayfish meat was safe for the health of potential consumers in terms of the analysed metal content.

Activity-dependent decline and recovery of synaptic transmission in central parts of surviving primary afferents after their peripheral cut in crayfish.

Axons deprived of their nucleus degenerate within a few days in mammals but survive for several months in crustaceans. However, it is not known whether central synapses from sensory axons may preserve their molecular machinery in the absence of spiking activity. To assess this, we used peripheral axotomy, which removes their nuclei combined with electrophysiology techniques and electron microscopy imaging. We report the following. (1) Electron microscopy analysis confirms previous observations that glial cell nuclei present in the sensory nerve proliferate and migrate to axon tubes, where they form close contacts with surviving axons. (2) After peripheral axotomy performed in vivo on the coxo-basipodite chordotonal organ (CBCO), the sensory nerve does not convey any sensory message, but antidromic volleys are observed. (3) Central synaptic transmission from the CBCO to motoneurons (MNs) progressively declines over 200 days (90% of monosynaptic excitatory transmission is lost after 3 weeks, whereas 60% of disynaptic inhibitory transmission persists up to 6 months). After 200 days, no transmission is observed. (4) However, this total loss is apparent only because repetitive electrical stimulation of the sensory nerve in vitro progressively restores first inhibitory post-synaptic potentials and then excitatory post-synaptic potentials. (5) The loss of synaptic transmission can be prevented by in vivo chronic sensory nerve stimulation. (6) Using simulations based on the geometric arrangements of synapses of the monosynaptic excitatory transmission and disynaptic inhibitory pathways, we show that antidromic activity in the CBCO nerve could play a role in the maintenance of synaptic function of inhibitory pathways to MNs, but not monosynaptic excitatory transmission to MNs. Our study confirms the deep changes in glial nuclei observed in axons deprived of their nucleus. We further show that the machinery for spike conduction and synaptic release persists for several months, even if there is no longer any activity. Indeed, we were able to restore, with electrical activity, spike conduction and synaptic function after long silent periods (>6 months).

Distinct vulnerability to oxidative stress determines the ammonia sensitivity of crayfish (Procambarus clarkii) at different developmental stages.

Red swamp crayfish (Procambarus clarkii) has increasingly become a high-value freshwater product in China. During the intensive cultivation, excessive ammonia exposure is an important lethal factor of crayfish. We investigated the toxic effects and mechanisms of ammonia on crayfish at two different developmental stages. A preliminary ammonia stress test showed a 96-h LC50 of 135.10 mg/L and 299.61 mg/L for Stage_1 crayfish (8.47 ± 1.68 g) and Stage_2 crayfish (18.33 ± 2.41 g), respectively. During a prolonged ammonia exposure (up to 96 h), serum acid phosphatase and alkaline phosphatase showed a time-dependent decrease. Histological assessment indicated the degree of hepatopancreatic injury, which was mainly characterized as tubule lumen dilatation, degenerated tubule, vacuolization and dissolved hepatic epithelial cell, increased with exposure time. Enhanced malondialdehyde level and reduced antioxidant capacity of hepatopancreas were also observed. The mRNA expression and activity of catalase and superoxide dismutase showed an initial up-regulation within 24 h, and then gradually down-regulated with the exposure time. In the post-treatment recovery period, the Stage_2 crayfish exerted a stronger antioxidant and detoxification capacity than that of the Stage_1 crayfish, and thus quickly recovered from the ammonia exposure. Our findings provide a further understanding of the adverse effects of ammonia stress and suggest guidelines for water quality management during crayfish farming.

The Damage of the Crayfish (Procambarus Clarkii) Digestive Organs Caused by Citrobacter Freundii Is Associated With the Disturbance of Intestinal Microbiota and Disruption of Intestinal-Liver Axis Homeostasis.

As a common conditional pathogenic bacterium in nature, C. freundii has posed a threat to crayfish culture and may infect humans through consumption. However, the pathogenic mechanism of C. freundii in crayfish remains unknown, which poses difficulties for the prevention and control of the bacterium. In this study, the effects of C. freundii on the digestive organs, intestine and hepatopancreas, of crayfish were investigated by high-throughput sequencing technology combined with histological analysis and flow cytometry. The findings suggested that C. freundii caused disruption of the intestinal microbiota, leading to intestinal inflammation and disrupting intestinal integrity. Meanwhile, C. freundii infection stimulates bile acid biosynthesis in the intestinal microbiota. Transcriptomic results showed significant upregulation of hepatopancreatic lipid degradation pathway and cytochrome P450-related pathways. Follow-up experiments confirmed a decrease in intracellular lipids and an increase in ROS and apoptosis. All the results indicated the disruption of intestinal-liver axis homeostasis due to disturbed intestinal microbiota may as a potential basis for C. freundii pathopoiesis in crayfish. These results provide new insights into the pathogenic molecular mechanisms of C. freundii in the infection of crayfish.

The effects of salinities stress on histopathological changes, serum biochemical index, non-specific immune and transcriptome analysis in red swamp crayfish Procambarus clarkii.

This study aimed to investigate whether different salinity stresses (0, 2, 4, 6, or 8 ppt NaCl) affect the histoarchitecture, serum biochemical indices, antioxidant responses, and transcriptome analysis of the red swamp crayfish Procambarus clarkii. Transmission electron microscopy results showed that the degree of damage to the nuclei and mitochondria in the hepatopancreas increased with increasing salinity. Scanning electron microscopy results showed that the degree of gill wrinkles was enhanced under salinity stress. Serum biochemical indices demonstrated that the cholesterol significantly decreased while the triglyceride, aspartate transaminase, and alanine transaminase contents significantly increased with increasing salinity. The antioxidant parameters, including catalase, total antioxidant capacity, and glutathione peroxidase, significantly decreased, while the malondialdehyde content significantly increased under salinity stress. Transcriptome analysis revealed that the expression pattern of immune-related genes showed a downward trend. These findings enrich our knowledge about the salinity stress response of farmed organisms and provide a theoretical base for salinity domestication and saline soil cultivation of P. clarkii, which might contribute to income improvement, employment generation, food security, and environmental safety.

Anesthesia with Tricaine Methanesulfonate (MS222) and Propofol and Its Use for Computed Tomography of Red Swamp Crayfish (Procambarus clarkii).

Crayfish (Decapoda: Astacoidea and Parastacoidea) are among the few animals that have stem cells in hemolymph, with the capacity to continuously produce differentiated neuronal structures throughout life. As the use of crayfish and other invertebrates increases in biomedical research, we must develop laboratory standards and guidelines for performing clinical procedures. This manuscript presents introductory protocols for anesthesia in crayfish during diagnostic imaging. Five anesthetic protocols were evaluated: immersion in buffered tricaine methanesulfonate (MS222; 50 mg/L); immersion in buffered MS222 (150 mg/L); immersion in propofol (65 mg/L); injection of propofol (50 mg/kg); and injection of propofol (100 mg/kg) into the ventral surface of an abdominal somite. MS222 immersion (50 and 150 mg/L) had no observable effect on crayfish. After an extended period of time, immersion in propofol (65 mg/L) created a sedative effect suitable for short-term handling. Propofol injection (50 mg/kg) into the ventral surface of an abdominal somite created an effective plane of anesthesia without adverse effects during or after recovery. Propofol injection at 100 mg/kg had adverse effects and is not recommended for use in crayfish. CT imaging was performed successfully as proof of concept for handling anesthetized crayfish. These findings provide initial data for the anesthetization of crayfish used in research settings.

Functions of potassium channels blocked by low micromolar 4-aminopyridine in the crayfish nervous system.

4-aminopyridine (4-AP) is a potassium channel blocker that has been used to treat patients with multiple sclerosis and Lambert-Eaton disease. The concentration of this drug in the blood of patients was estimated to be in low or submicromolar range. Animal studies have shown that 4-AP at such low concentration selectively blocks a subset of channels in Kv1 or Kv3 families. The crayfish opener neuromuscular junction and ventral superficial flexor (VSF) preparations were used to examine functions of K+ channels blocked by low concentrations of 4-AP. At opener motor axons, intracellular recordings show that 4-AP could increase action potential (AP) amplitude, duration, and after-depolarization (ADP) at 10 µM. As 4-AP concentration was increased, in twofold steps, AP amplitude did not increase further up to 5 mM. AP duration and ADP increased significantly mainly in two concentration ranges, 10-50 µM and 1-5 mM. The effects of 50 µM 4-AP on the VSF were less consistent than that observed at the opener motor axons. 4-AP did not change AP amplitude of motor axons recorded with an extracellular electrode and change in AP repolarizing potential was observed in ∼25% of the axons. EPSP recorded simultaneously with AP showed an increase in amplitude with 4-AP treatment only in 30% of the axon-EPSP pairs. 4-AP also increased firing frequencies of ∼50% of axons. In four animals, 4-AP "awakened" the firing of APs from an axon that was silent before the drug. The mixture of positive and negative 4-AP effects summarized above was observed in the same VSF preparations in all cases (n = 8). We propose that there is a significant diversity in the density 4-AP-sensitive potassium channels among motor axons of the VSF. Functional significance in the differences of 4-AP sensitivity of the two motor systems is discussed.

Hemolymph Glycemia as an Environmental Stress Biomarker in the Invasive Red Swamp Crayfish (Procambarus clarkii).

AbstractSeveral freshwater crayfish species, including Procambarus clarkii, are both ecologically important and commercially important benthic macroinvertebrates, remarkable for their potential to adapt and reproduce but also for their unique abilities to face distinct abiotic and biotic environments and become successful invaders. While much work has been done to study crayfish introductions, less focus has been given to how crayfish cope with pollution and other environmental stressors, in terms of physiological responses, and whether crayfish responses can be used to assess the effective state of their living environment. Here, we used a mixed approach combining laboratory experiments with field data to validate the use of hemolymph glucose as a relevant biomarker of red swamp crayfish (Procambarus clarkii) stress response. Three meaningful sampling locations were chosen across southern Portugal that are representative of different environments where crayfish live and are frequently captured for human consumption but also correspond to different pollution levels. To reference field measurements of glucose levels, we performed two lab-based experiments: (a) crayfish were exposed to different levels of stress (stress challenge) and (b) crawfish were exposed to a maze dispersal test, with or without water. Crayfish glucose levels were responsive to induced stress but were not correlated with dispersal efforts. Wild crayfish's body condition and stress levels responded differently to environmental conditions, with more challenged individuals showing higher glycemia levels but similar body condition. The glucose levels of the more stressed wild crayfish were visually similar to lab-based crayfish subjected to the higher stress levels (electric shocks), while the levels of glucose of crayfish at the less polluted site corresponded to those measured before the start of the challenge (baseline). The maintenance of high levels of glycemia in crayfish inhabiting more challenging habitats is revealing of their higher energetic demand state. Since P. clarkii ia globally distributed and easily sampled invasive species, quantifying its hemolymph glucose levels can be a particularly useful proxy for assessing environmental quality.

Zeolite mediated processing of nitrogenous waste in the rearing environment influences gut and sediment microbial community in freshwater crayfish (Cherax cainii) culture.

Zeolite is known to uptake toxic metals and filter nitrogenous waste from aquaculture effluents. The present study aimed to investigate the impacts of zeolite in three different applications namely, dietary zeolite (DZ), suspended zeolite (SZ) in the water column, and a combination of both (DZSZ) relative to unexposed freshwater crayfish, marron (control). At the end of the 56-days trial, the impact was assessed in terms of characterization of microbial communities in the culture environment and the intestine of marron. Alongside the microbial communities, the innate immune response of marron was also evaluated. The 16S rRNA data showed that marrons exposed to the suspended zeolite had a significant increase of bacterial diversity in the gut, including the restoration of marron core operational taxonomic units (OTUs), relative to other forms of exposures (DZ, DZSZ) and the control. Suspended zeolite alone also increased the number of unshared OTUs and genera, and improved predicted metabolic functions for the biosynthesis and digestion of proteins, amino acids, fatty acids, and hormones. In the tank sediment, the shift of microbial communities was connected more strongly with the time of experiment than the type of zeolite exposure. In the second case, only control marron had a different microbial ordination in terms of rare taxa present in the community. Nevertheless, the modulation in the gut environment was found more prominent in DZ, relative to modulation in the tank sediments. The taxa-environment correlation identified Rhodoferax as the most potential bacteria in removing nitrogenous waste from the rearing environment. Further analysis showed that SZ resulted in the upregulation of genes associated with the innate immune response of marron. Overall results suggest that SZ can be used to enrich microbial communities in the gut and tank sediments and better immune performance of marron.

Impact of polyaluminum chloride on the bioaccumulation of selected elements in the tissues of invasive spiny-cheek crayfish (Faxonius limosus) - Potential risks to consumers.

The effect of coagulants used in lake reclamation on crayfish is poorly understood. Therefore, the aim of this study was to evaluate changes in the bioaccumulation of Al, Ca, Cu, Fe, K, Mg, Na and Zn in the gills, exoskeleton, muscle and hepatopancreas of spiny-cheek crayfish (Faxonius limosus) as a result of exposure to PAX®18 coagulant, containing polyaluminum chloride. The study also evaluated the risk to human health from the consumption of crayfish muscle. Metal levels, determined using atomic absorption spectrometry, differed between metals (the highest concentrations for Ca, K, Na, Mg) and the body part. Calcium was most abundant in the exoskeleton, K in the muscles, while Cu and Al in the hepatopancreas. The bioaccumulation of metals was affected by exposure to the coagulant, with a statistically significant (p < 0.05) increase in muscle concentration of Al and Na and a decrease in Ca and Fe. The concentrations of elements (in µg g-1) in the muscle of the control group crayfish and those in contact with the coagulant were, respectively: K (2150; 2090), Na (1540; 2020), Ca (749; 602), Mg (207; 174), Al (103; 164), Zn (21.1; 19.1), Fe (7.6; 3.8) and Cu (8.4; 7.6). Most elements were below 12% of the Dietary Reference Values (DRV). The Al concentration in the muscle exceeded the tolerable weekly intake (TWI) (maximum 164% TWI for muscle of crayfish exposed to polyaluminum chloride). In conclusion, the studied F. limosus had typical elemental bioaccumulation for a crayfish, but the contact with the coagulant increased Al concentration and decreased Fe, Ca, Mg, Zn and Cu concentrations. The muscle of crayfish can be used as a supplementary source of essential elements in the human diet, but it seems necessary to introduce obligatory control of Al levels due to the use of polyaluminum chloride in lake restoration.

Behavioral effects of ethanol in the Red Swamp Crayfish (Procambarus clarkii).

Alcohol abuse remains one of the primary preventable sources of mortality in the United States. Model species can be used to evaluate behavioral and other biological changes associated with alcohol and to identify novel treatments. This report describes methods for evaluating the behavioral effects of ethanol (EtOH) in crayfish. Crayfish (Procambarus clarkii) were immersed in ethanol concentrations ranging from 0.1 to 1.0 molar, for 10-30 min. Studies evaluated hemolymph alcohol concentration, locomotion in an open field and anxiety-like behavior using a Light/Dark transfer approach. EtOH immersion produced dose-dependent increases in hemolymph EtOH (up to 249 mg/dL) and reductions in open field locomotion that depended on EtOH concentration or exposure duration. Untreated crayfish exhibit avoidance of the open parts of the locomotor arena and a preference for a covered portion. Acute EtOH immersion decreased time spent in the covered portion of the Light/Dark arena, consistent with a decrease in anxiety-like behavior. Daily EtOH immersion for 5 days did not alter locomotor responses, however, activity was increased 3 days after the repeated EtOH regimen. Overall, this study shows that this inexpensive, easily maintained species can be used for behavioral pharmacological experiments designed to assess the acute and repeated effects of EtOH.