Effects of diphenhydramine on crayfish cytochrome P450 activity and antioxidant defence mechanisms: First evidence of CYP2C- and CYP3A-like activity in marbled crayfish.

Growing evidence has reported that diphenhydramine (DPH), an ionisable antihistamine, is widely present in surface waters across the world. Relative to vertebrates studied, its impact on invertebrates, particularly concerning cytochrome P450 (CYP) metabolism and oxidative stress, remains poorly understood. In this study, we aimed to investigate the effects of 2, 20, and 200 µg/L DPH on marbled crayfish (Procambarus virginalis) after 96-h exposure. Specifically, we assessed CYP activity, antioxidant enzyme responses, and acetylcholinesterase (AChE) activity in gills, muscle, and hepatopancreas. The crayfish CYP metabolised fluorogenic CYP-metabolic substrates of 7-benzyloxy-4-trifluoromethylcoumarin (BFC) and dibenzylfluorescein (DBF), which evidenced the activity of CYP2C and CYP3A isoforms, well known in mammalian detoxification metabolism. Both BFC and DBF dealkylations showed a positive correlation with each other but were negatively correlated to water and haemolymph DPH concentrations. Exposure to 200 µg/L DPH elicited an apparent inhibition trend, albeit not significant, in BFC- and DBF-transformation activities in crayfish. Other tested 7-benzyloxyresorufin and 7-pentoxyresorufin substrates were poorly metabolised, suggesting their relatively low activity or the lack of mammalian-like CYP1A and CYP2B isoforms in marbled crayfish. The significant modulation of antioxidant enzymes was demonstrated in gills and hepatopancreas. The exposure to DPH did not alter the activity of AChE. Integrated biomarker response version 2 showed the highest cumulative effect of DPH exposure on gills, implying that gill tissue is the most reliable matrix for evaluating DPH toxicity. Activities of glutathione peroxidase and glutathione-S-transferase were the most deviated determinants among the investigated biomarkers, providing insights into the DPH toxicity in crayfish. This study brought the first insight into utilising the fluorogenically active substrates BFC and DBF to demonstrate the CYP involvement in the detoxification metabolism in marbled crayfish. Further, our results provided information on valuable antioxidant defence mechanisms and biomarker responses for a future DPH toxicity assessment in aquatic organisms.

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.

Host-pathogen coevolution drives innate immune response to Aphanomyces astaci infection in freshwater crayfish: transcriptomic evidence.

BACKGROUND: For over a century, scientists have studied host-pathogen interactions between the crayfish plague disease agent Aphanomyces astaci and freshwater crayfish. It has been hypothesised that North American crayfish hosts are disease-resistant due to the long-lasting coevolution with the pathogen. Similarly, the increasing number of latent infections reported in the historically sensitive European crayfish hosts seems to indicate that similar coevolutionary processes are occurring between European crayfish and A. astaci. Our current understanding of these host-pathogen interactions is largely focused on the innate immunity processes in the crayfish haemolymph and cuticle, but the molecular basis of the observed disease-resistance and susceptibility remain unclear. To understand how coevolution is shaping the host's molecular response to the pathogen, susceptible native European noble crayfish and invasive disease-resistant marbled crayfish were challenged with two A. astaci strains of different origin: a haplogroup A strain (introduced to Europe at least 50 years ago, low virulence) and a haplogroup B strain (signal crayfish in lake Tahoe, USA, high virulence). Here, we compare the gene expression profiles of the hepatopancreas, an integrated organ of crayfish immunity and metabolism. RESULTS: We characterised several novel innate immune-related gene groups in both crayfish species. Across all challenge groups, we detected 412 differentially expressed genes (DEGs) in the noble crayfish, and 257 DEGs in the marbled crayfish. In the noble crayfish, a clear immune response was detected to the haplogroup B strain, but not to the haplogroup A strain. In contrast, in the marbled crayfish we detected an immune response to the haplogroup A strain, but not to the haplogroup B strain. CONCLUSIONS: We highlight the hepatopancreas as an important hub for the synthesis of immune molecules in the response to A. astaci. A clear distinction between the innate immune response in the marbled crayfish and the noble crayfish is the capability of the marbled crayfish to mobilise a higher variety of innate immune response effectors. With this study we outline that the type and strength of the host immune response to the pathogen is strongly influenced by the coevolutionary history of the crayfish with specific A. astaci strains.

Chemical synthesis and functional evaluation of the crayfish insulin-like androgenic gland factor.

Insulin-like androgenic gland factor (IAG) from the marbled crayfish Procambarus virginalis is an insulin-like heterodimeric peptide composed of A and B chains and has an Asn-linked glycan at the B chain. IAG is considered to be a male sex hormone inducing the sex differentiation to male in decapod crustacean, although there is no report on the function of IAG peptide in vivo. In order to characterize P. virginalis IAG, we chemically synthesized it and evaluated its biological function in vivo. A and B chains were prepared by the ordinary solid-phase peptide synthesis, and three disulfide bonds were formed regioselectively by dimethyl sulfoxide oxidation, pyridylsulfenyl-directed thiolysis and iodine oxidation reactions. An IAG disulfide isomer was also prepared by the same manner. Circular dichroism spectral analysis revealed that the disulfide bond arrangement affected the peptide conformation, which was similar to the other insulin-family peptides analyzed so far. On the other hand, the glycan moiety attached at the B chain had no effect on the peptide secondary structure. Injection of the synthetic IAG and its disulfide isomer to female crayfish did not induce male characteristics on the external morphology, but both peptides suppressed the oocyte maturation in vivo. These results suggest that IAG has a pivotal role on the suppression of female secondary sex characteristics.

Effects of s-metolachlor on early life stages of marbled crayfish.

The effects of s-metolachlor chronic exposure at concentrations of 1.1 µg/L (maximal real environmental concentration in the Czech Republic), 11 µg/L (environmental relevant concentration) and 110 µg/L on early life stages of marbled crayfish (Procambarus virginalis) was evaluated under laboratory conditions. All s-metolachlor exposures resulted in higher mortality, delay ontogenetic development with accompanied slower growth and excited behaviour (increase of total distance moved and walking speed). Significantly lower superoxide dismutase, catalase, glutathione S-transferase activity and reduced glutathione level was observed at two higher tested concentrations (11 and 110 µg/L) of s-metolachlor compared with the control. S-metolachlor in concentrations 110 µg/L showed alteration of the tubular system of hepatopancreas including focal disintegration of tubular epithelium and notable reduction in epithelial cells number, especially B-cells. In conclusion, potential risk associated with using of s-metolachlor in agriculture, due to effects on non-target aquatic organisms as documented on early life stages of marbled crayfish in this study, should be taken into account.

Protein from whole-body crayfish homogenate may be a high supplier of leucine or branched-chain amino acids - A call for validation on genus Procambarus sp.

Essential proteinogenic branched-chain amino acids (BCAA), particularly leucine (Leu) have been investigated for their role in enhancing human myofibrillar protein synthesis and biomedical research on tumor models. However, only a few protein sources in our current food system have high enough BCAA or Leu coefficients (% of total amino acids) to be considered as supplements for food, sport, or biomedical research. Mostly dairy-sourced proteins such as casein and whey or rarely plant source such as maize gluten are typically regarded as the gold standards. This study hypothesized that protein isolates derived from the whole-body homogenate (including the chitinous exoskeleton) of procambarid crayfish might exhibit unusually high BCAA and Leu content. The study provides open-access data on the amino acid compositions of two procambarid crayfish (Procambarus virginalis and P. clarkii), as well as a comparison with casein. The mentioned crayfish species could offer 6.36-7.39 g Leu 100 g-1 dry matter (at 43-48% protein only). Crayfish whole-body protein isolates exhibit a Leu coefficient (18.41±2.51% of total amino acids) and a BCAA coefficient (28.76±2.39% of total amino acids), which is comparable to or higher than of casein (Leu coefficient 8.65±0.08%; BCAA coefficient 20.03±0.73%). However, it is important to interpret these results with caution, due to the challenges associated with leucine and isoleucine separation, as well as potential interactions within the sample matrices. Hence, international validation of these findings is recommended. NOVELTY STATEMENT: Protein isolates from whole-body homogenate (including chitinous exoskeleton) of P. virginalis and/or P. clarkii are hypothesized to be dense in BCAA and Leu. For potential use in biomedical research or as additives in supplements for BCAA and Leu.

Dataset of the de novo assembly and annotation of the marbled crayfish and the noble crayfish hepatopancreas transcriptomes.

OBJECTIVES: Crayfish plague disease, caused by the oomycete pathogen Aphanomyces astaci represents one of the greatest risks for the biodiversity of the freshwater crayfish. This data article covers the de novo transcriptome assembly and annotation data of the noble crayfish and the marbled crayfish challenged with Ap. astaci. Following the controlled infection experiment (Francesconi et al. in Front Ecol Evol, 2021, https://doi.org/10.3389/fevo.2021.647037 ), we conducted a differential gene expression analysis described in (Bostjancic et al. in BMC Genom, 2022, https://doi.org/10.1186/s12864-022-08571-z ) DATA DESCRIPTION: In total, 25 noble crayfish and 30 marbled crayfish were selected. Hepatopancreas tissue was isolated, followed by RNA sequencing using the Illumina NovaSeq 6000 platform. Raw data was checked for quality with FastQC, adapter and quality trimming were conducted using Trimmomatic followed by de novo assembly with Trinity. Assembly quality was assessed with BUSCO, at 93.30% and 93.98% completeness for the noble crayfish and the marbled crayfish, respectively. Transcripts were annotated using the Dammit! pipeline and assigned to KEGG pathways. Respective transcriptome and raw datasets may be reused as the reference transcriptome assemblies for future expression studies.

A combination of six psychoactive pharmaceuticals at environmental concentrations alter the locomotory behavior of clonal marbled crayfish.

Pharmaceutically active compounds (PhACs) are ubiquitous in the aquatic environment worldwide and considered emerging contaminants. Their effects on growth, behavior, and physiological processes of aquatic organisms have been identified even at very low concentrations. Ecotoxicological investigations have primarily focused on single compound exposure, generally at a range of concentrations. In the natural environment, pollutants seldom occur in isolation, but little is known about the effects and risks of combinations of chemicals. This study aimed to investigate the effects of concurrent exposure to six psychoactive PhACs on locomotory behavior and life history traits of clonal marbled crayfish Procambarus virginalis. Crayfish were exposed to ~1 µg L-1 of the antidepressants sertraline, citalopram, and venlafaxine; the anxiolytic oxazepam; the opioid tramadol; and the widely abused psychostimulant methamphetamine. In the absence of shelter, exposed crayfish moved significantly shorter distances and at lower velocity and showed significantly less activity than controls. With available shelter, exposed crayfish moved significantly more distance, showed higher activity, and spent a significantly more time outside the shelter than controls. Molting, mortality, and spawning frequency did not vary significantly between the groups. Hemolymph glucose level did not vary among groups and was not correlated with observed behaviors. Results suggest that environmental concentrations of the tested compounds in combination can alter the behavior of non-target aquatic organisms as individual exposure of these compounds, which may lead to disruption of ecosystem processes due to their reduced caution in polluted conditions. Further research is needed using varied chemical mixtures, exposure systems, and habitats, considering molecular and physiological processes connected to behavior alterations.

The Invasive Round Goby Neogobius melanostomus as a Potential Threat to Native Crayfish Populations.

Despite the spread of round goby Neogobius melanostomus into freshwater streams, there is a lack of information with respect to its effect on macroinvertebrate communities, especially crustaceans. We studied foraging efficiency of N. melanostomus on Procambarus virginalis and Asellus aquaticus, using a functional response (FR) approach. Stocking density of the prey species was manipulated to determine its effect on consumer utilization, with prey offered separately or combined at 1:1, 3:1, and 1:3 at each tested density. For both prey species, N. melanostomus exhibited type II FR, occasionally with a high proportion of non-consumptive mortality. Procambarus virginalis suffered a significantly higher attack rate compared to A. aquaticus. Neogobius melanostomus killed significantly more of the most prevalent prey, regardless of species. In trials with prey species of equal proportions, a difference in the number of each species killed was observed only at the highest density, at which P. virginalis was preferred. Neogobius melanostomus may be an important driver of population dynamics of prey species in the wild. The non-selective prey consumption makes N. melanostomus a potential threat to macrozoobenthic communities of river tributaries.

Environmentally relevant concentrations of methamphetamine and sertraline modify the behavior and life history traits of an aquatic invertebrate.

Pharmaceutically active compounds are major contaminants of aquatic environments that show direct and indirect effects on aquatic organisms even at low concentrations. The aim of this study was to assess the effects of the illicit drug methamphetamine and the antidepressant sertraline on clonal marbled crayfish Procambarus virginalis. Crayfish exposed to the environmentally relevant concentrations of methamphetamine of ∼1 µg L-1 did not exhibit significant differences from unexposed controls in distance moved, velocity, and activity level with or without available shelter. Sertraline-exposed (∼1 µg L-1) crayfish were significantly more active, regardless of available shelter, and moved greater distances when shelter was available, compared to control crayfish. Crayfish exposed to methamphetamine and sertraline spent significantly more time outside the shelters compared to controls. Sertraline-exposed crayfish spawned more frequently and showed higher mortality than controls. The results suggest that the low environmental concentrations of the tested compounds could alter the behavior and life history traits of crayfish, resulting in higher reproductive effort and mortality.