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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

The Effect of Glyphosate on the Reproduction of Estuarine Crabs: Neohelice granulata as a Study Model.

This review summarizes the bulk of evidence about the effect of glyphosate, both technical and formulated, on the ovarian maturation of Neohelice granulata female crabs, as well as the effects of glyphosate on sperm production in males of the same species. After long-term in vivo assays, made during the 3-month pre-reproductive period of this species, both formulated and technical glyphosate were able to produce a significant incidence of oocyte reabsorption in the ovary, together with a concomitant decreased of vitellogenin content, at concentrations ranging from 0.2 to 1 mg/L. Despite this, after 32-day in vivo assays, glyphosate stimulated oocyte growth, in terms of a higher percentage of vitellogenic oocytes, suggesting that glyphosate could be acting as an endocrine disruptor. In vitro assays made with isolated ovarian pieces showed a decrease of vitellogenin content, in correlation with lower protein synthesis, although some advance in maturation was observed in the histological analysis. In male crabs exposed in vivo to both technical and formulated glyphosate at 1 mg/L, several reproductive imbalances were noted, such as a significant decrease of the sperm count, abnormal spermatophores, and possible disrupting effects of glyphosate on the androgenic gland.

ß-1,3-Glucan from Euglena gracilis as an immunostimulant mediates the antiparasitic effect against Mesanophrys sp. on hemocytes in marine swimming crab (Portunus trituberculatus).

ß-1,3-glucans, natural polysaccharide groups, exert immunomodulatory effects to improve the innate response and disease resistance in aquatic species and mammals. However, this ß-glucan stimulant is yet to be assayed in swimming crab (Portunus trituberculatus) hemocytes. In this study, we explored the immunomodulatory effect of ß-1,3-glucans (derived from Euglena gracilis) via in vitro 24 h stimulation assays in swimming crab hemocytes. We found that this algal ß-1,3-glucans in crab hemocytes significantly elevated cellular enzymes related parameters, including phenoloxidase (PO), lysozyme, acid phosphatase (ACP) activities, and superoxide anion generation (O2-) rate both at intracellular (P < 0.05) and extracellular (P < 0.05) levels. Besides, alkaline phosphatase (AKP) in hemocytes exhibited no significant differences across the groups (P > 0.05). ß-glucan significantly influenced (P < 0.05) the activities of the antioxidant enzyme, superoxide dismutase (SOD) in hemocytes. Moreover, the relative mRNA expression of numerous immune-related genes, including proPO, TLR-2, Alf-1, NOX, Lysozyme, Crustin-1, and Cuznsod, was significantly higher stimulated hemocytes than in control (P < 0.05). We also reported the dose-dependent antiparasitic activity against Mesanophyrs sp., in stimulated hemocytes than in the control (P < 0.05). The present study collectively demonstrated that ß-glucan potentially stimulates innate immunity by elevating cellular enzyme responses and up-regulating the mRNA expression of genes associated with crab innate immunity. Thus, ß-glucan is a promising immunostimulant for swimming crab farming in crustaceans aquaculture.

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.

Effects of environmental antidepressants on colour change and locomotor behaviour in juvenile shore crabs, Carcinus maenas.

Juvenile crabs of Carcinus maenas thrive in coastal waters reputed to be the receptacle of continental pollution. Amongst the many pollutants encountered, antidepressants, such as fluoxetine (FLX) and venlafaxine (VEN), often detected at the ng•L-1 range, are particularly worrying because of their action on the levels of monoamines, such as serotonin, noradrenaline and dopamine. In crustaceans, those monoamines are involved in colour change through their action on neuropeptide hormones. In addition, they are known to have a role in different behaviours, such as locomotion. Both colour change and locomotion are strategies used by juvenile crabs to hide and escape from predators. To investigate if the presence of antidepressants may alter behaviours of ecological importance, juvenile crabs were exposed to environmentally realistic concentrations of either 5 ng•L-1 of FLX alone or in combination with VEN at 5 ng•L-1. The ability to change colour depending on the environment and the locomotor activity of juvenile crabs were monitored weekly over 25 days. Animals exposed to antidepressants displayed a different pattern of colour change than the controls, especially those exposed to the combination of FLX and VEN at 5 ng•L-1 each, and were less efficient to adapt to their environment, i.e., they were not as pale and not as dark as controls or crabs exposed to FLX at 5 ng•L-1. Moreover, juvenile crabs exposed to the combination of antidepressants exhibited an enhanced locomotor activity throughout the exposure period with a higher velocity and distance moved as well as more time spend moving. The alteration of cryptic behaviours, such as colour change and locomotion by antidepressants persistently present in marine environment at low concentrations may have an impact on the survival of juvenile of C. maenas on the long term.

Untargeted lipidomics reveals metabolic responses to different dietary n-3 PUFA in juvenile swimming crab (Portunus trituberculatus).

While tissue fatty acid compositions reflect that of the dietary lipid source, little information is available on how dietary oils modify lipid class and molecular species profiles in hepatopancreas of crustacean. Herein, an 8-week nutritional trial and untargeted lipidomic analysis were used to investigate the impacts of dietary n-3 PUFA lipid sources including fish oil, krill oil and linseed oil on the lipidomic characteristics of hepatopancreas of swimming crab (Portunus trituberculatus). Dietary krill oil significantly increased distribution of 20:5n-3 and 22:6n-3 at sn-2 in phosphatidylcholine and phosphatidylethanolamine compared to fish oil. Fish oil intake promoted the deposition of 20:5n-3 and 22:6n-3 at sn-1,2,3 in triglyceride compared to linseed oil, which significantly increased the specific accumulation of 18:3n-3 at sn-1,3 in triglyceride and sn-2 in phosphatidylcholine and phosphatidylethanolamine. The study revealed metabolic responses to different dietary n-3 PUFA in swimming crab, which provided novel insight into the lipid nutrition of crustacean.

Mass Spectrometric Profiling of Neuropeptides in Response to Copper Toxicity via Isobaric Tagging.

Copper is a necessary nutrient but quickly becomes toxic at elevated levels. To properly handle environmental copper influxes and maintain metal homeostasis, organisms utilize various methods to chelate, excrete, and metabolize heavy metals. These mechanisms are believed to involve complex signaling pathways mediated by neuropeptides. This study incorporates custom N,N-dimethyl leucine isobaric tags to characterize the neuropeptidomic changes after different time points (1, 2, and 4 h) of copper exposure in a model organism, blue crab, Callinectes sapidus. Using a modified simplex optimization strategy, the number of identifiable and quantifiable neuropeptides was increased 3-fold to facilitate a deeper understanding of the signaling pathways involved in responding to heavy metal exposure. The time course exposure showed many interesting findings, including upregulation of inhibitory allatostatin peptides in the pericardial organs. Additionally, there was evidence of transport of a pigment dispersing hormone from the sinus glands to the brain. Overall, this study improves the multiplexing capabilities of neuropeptidomic studies to understand the temporal changes associated with copper toxicity.

Influences of dietary vitamin D3 on growth, antioxidant capacity, immunity and molting of Chinese mitten crab (Eriocheir sinensis) larvae.

This study investigates the effects of vitamin D3 (VD3) on growth performance, antioxidant capacity, immunity and molting of larval Chinese mitten crab Eriocheir sinensis. A total of 6,000 larvae (7.52 ± 0.10 mg) were fed with six isonitrogenous and isolipidic experimental diets with different levels of dietary VD3 (0, 3000, 6000, 9000, 12000 and 36000 IU/kg) respectively for 23 days. The highest survival and molting frequency were found in crabs fed 6000 IU/kg VD3. Weight gain, specific growth rate, and carapace growth significantly increased in crabs fed 3000 and 6000 IU/kg VD3 compared to the control. Broken-line analysis of molting frequency, weight gain and specific growth rate against dietary VD3 levels indicates that the optimal VD3 requirement for larval crabs is 4825-5918 IU/kg. The highest whole-body VD3 content occurred in the 12000 IU/kg VD3 group, and the 25-dihydroxy VD3 content decreased with the increase of dietary VD3. The malonaldehyde content was lower than the control. Moreover, the superoxide dismutase activity, glutathione peroxidase and total antioxidant capacity of crab fed 6000 IU/kg VD3 were significantly higher than in control. Crabs fed 9000 IU/kg showed the highest survival after 120 h of salinity stress, and the relative mRNA expressions indicate vitamin D receptor (VDR) is the important regulatory element in molting and innate immunity. The molting-related gene expressions showed that the response of crab to salinity was self-protective. This study would contribute to a new understanding of the molecular basis underlying molting and innate immunity regulation by vitamin D3 in E. sinensis.

Effects of a bacteria-produced algicide on non-target marine invertebrate species.

Harmful algal blooms (HABs) affect both freshwater and marine systems. Laboratory experiments suggest an exudate produced by the bacterium Shewanella sp. IRI-160 could be used to prevent or mitigate dinoflagellate blooms; however, effects on non-target organisms are unknown. The algicide (IRI-160AA) was tested on various ontogenetic stages of the copepod Acartia tonsa (nauplii and adult copepodites), the blue crab Callinectes sapidus (zoea larvae and megalopa postlarvae), and the eastern oyster Crassostrea virginica (pediveliger larvae and adults). Mortality experiments with A. tonsa revealed that the 24-h LC50 was 13.4% v/v algicide for adult females and 5.96% for early-stage nauplii. For C. sapidus, the 24-h LC50 for first-stage zoeae was 16.8%; results were not significant for megalopae or oysters. Respiration rates for copepod nauplii increased in the 11% concentration, and in the 11% and 17% concentrations for crab zoeae; rates of later stages and oysters were unaffected. Activity level was affected for crab zoeae in the 1%, 11%, and 17% treatments, and for oyster pediveliger larvae at the 17% level. Activity of later stages and of adult copepods was unaffected. Smaller, non-target biota with higher surface to volume could be negatively impacted from IRI-160AA dosing, but overall the taxa and stages assayed were tolerant to the algicide at concentrations required for dinoflagellate mortality (EC50 = ~ 1%).

The effects of vesicle toxin from the sea anemone Bunodosoma cangicum on the behavior of a freshwater shrimp, Palaemon argentinus, and shore crab, Neohelice granulata.

Sea anemones of the genus Bunodosoma possess along their body column, longitudinally arranged brown-colored vesicles. We have shown that in B. cangicum, these warty structures contain a mixture of potent toxins. This work highlights the neuro-inhibitory effects exhibited by two decapod crustacean species exposed to the extracts from these vesicles. For this, we use the unrefined toxin in doses, exposure times, and different exposure pathways. The findings show that at least one neuro-inhibitory compound is present and remains active regardless of the exposure method or dose tested. This toxin affects neuro-motor pathways but not neuro-sensory pathways. Shrimp exposed to toxin could continue to perceive and track food pellets but could not secure and consume their ration. Of six anatomical reflexes tested under the toxin's influence, voluntary movements of the mouthparts were impacted most commonly. Interestingly, all subject animals recovered from the toxin exposure within 2 h. Finally, we propose Reflexive Action Analysis (RAMP) as a tool to evaluate the potency of other neurotoxic or neuro-inhibitory compounds in crustacea. This work is the first to show the neuro-inhibitory activity of extracts from these sea anemone columnar vesicle structures and the first to evaluate these effects using RAMP reflex analysis.