GABAB receptors induce phasic release from medial habenula terminals through activity-dependent recruitment of release-ready vesicles.

GABAB receptor (GBR) activation inhibits neurotransmitter release in axon terminals in the brain, except in medial habenula (MHb) terminals, which show robust potentiation. However, mechanisms underlying this enigmatic potentiation remain elusive. Here, we report that GBR activation on MHb terminals induces an activity-dependent transition from a facilitating, tonic to a depressing, phasic neurotransmitter release mode. This transition is accompanied by a 4.1-fold increase in readily releasable vesicle pool (RRP) size and a 3.5-fold increase of docked synaptic vesicles (SVs) at the presynaptic active zone (AZ). Strikingly, the depressing phasic release exhibits looser coupling distance than the tonic release. Furthermore, the tonic and phasic release are selectively affected by deletion of synaptoporin (SPO) and Ca2+-dependent activator protein for secretion 2 (CAPS2), respectively. SPO modulates augmentation, the short-term plasticity associated with tonic release, and CAPS2 retains the increased RRP for initial responses in phasic response trains. The cytosolic protein CAPS2 showed a SV-associated distribution similar to the vesicular transmembrane protein SPO, and they were colocalized in the same terminals. We developed the "Flash and Freeze-fracture" method, and revealed the release of SPO-associated vesicles in both tonic and phasic modes and activity-dependent recruitment of CAPS2 to the AZ during phasic release, which lasted several minutes. Overall, these results indicate that GBR activation translocates CAPS2 to the AZ along with the fusion of CAPS2-associated SVs, contributing to persistency of the RRP increase. Thus, we identified structural and molecular mechanisms underlying tonic and phasic neurotransmitter release and their transition by GBR activation in MHb terminals.

The crustacean DNA virus tegument protein VP26 binds to SNAP29 to inhibit SNARE complex assembly and autophagic degradation.

Autophagy generally functions as a cellular surveillance mechanism to combat invading viruses, but viruses have evolved various strategies to block autophagic degradation and even subvert it to promote viral propagation. White spot syndrome virus (WSSV) is the most highly pathogenic crustacean virus, but little is currently known about whether crustacean viruses such as WSSV can subvert autophagic degradation for escape. Here, we show that even though WSSV proliferation triggers the accumulation of autophagosomes, autophagic degradation is blocked in the crustacean species red claw crayfish. Interestingly, the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex including CqSNAP29, CqVAMP7, and the novel autophagosome SNARE protein CqSyx12 is required for autophagic flux to restrict WSSV replication, as revealed by gene silencing experiments. Simultaneously, the expressed WSSV tegument protein VP26, which likely localizes on autophagic membrane mediated by its transmembrane region, binds the Qb-SNARE domain of CqSNAP29 to competitively inhibit the binding of CqSyx12-Qa-SNARE with CqSNAP29-Qb-SNARE; this in turn disrupts the assembly of the CqSyx12-SNAP29-VAMP7 SNARE complex, which is indispensable for the proposed fusion of autophagosomes and lysosomes. Consequently, the autophagic degradation of WSSV is likely suppressed by the expressed VP26 protein in vivo in crayfish, thus probably protecting WSSV components from degradation via the autophagosome-lysosome pathway, resulting in evasion by WSSV. Collectively, these findings highlight how a DNA virus can subvert autophagic degradation by impairing the assembly of the SNARE complex to achieve evasion, paving the way for understanding host-DNA virus interactions from an evolutionary point of view, from crustaceans to mammals.IMPORTANCEWhite spot syndrome virus (WSSV) is one of the largest animal DNA viruses in terms of its genome size and has caused huge economic losses in the farming of crustaceans such as shrimp and crayfish. Detailed knowledge of WSSV-host interactions is still lacking, particularly regarding viral escape from host immune clearance. Intriguingly, we found that the presence of WSSV-VP26 might inhibit the autophagic degradation of WSSV in vivo in the crustacean species red claw crayfish. Importantly, this study is the first to show that viral protein VP26 functions as a core factor to benefit WSSV escape by disrupting the assembly of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, which is necessary for the proposed fusion of autophagosomes with lysosomes for subsequent degradation. These findings highlight a novel mechanism of DNA virus evasion by blocking SNARE complex assembly and identify viral VP26 as a key candidate for anti-WSSV targeting.

A Crayfish-Inspired Sensor Fusion Platform for Super Additive Integration of Visual, Chemical, and Tactile Information.

The concept of cross-sensor modulation, wherein one sensor modality can influence another's response, is often overlooked in traditional sensor fusion architectures, leading to missed opportunities for enhancing data accuracy and robustness. In contrast, biological systems, such as aquatic animals like crayfish, demonstrate superior sensor fusion through multisensory integration. These organisms adeptly integrate visual, tactile, and chemical cues to perform tasks such as evading predators and locating prey. Drawing inspiration from this, we propose a neuromorphic platform that integrates graphene-based chemitransistors, monolayer molybdenum disulfide (MoS2) based photosensitive memtransistors, and triboelectric tactile sensors to achieve "Super-Additive" responses to weak chemical, visual, and tactile cues and demonstrate contextual response modulation, also referred to as the "Inverse Effectiveness Effect." We hold the view that integrating bio-inspired sensor fusion principles across various modalities holds promise for a wide range of applications.

Genome assembly of redclaw crayfish (Cherax quadricarinatus) provides insights into its immune adaptation and hypoxia tolerance.

BACKGROUND: The introduction of non-native species is a primary driver of biodiversity loss in freshwater ecosystems. The redclaw crayfish (Cherax quadricarinatus) is a freshwater species that exhibits tolerance to hypoxic stresses, fluctuating temperatures, high ammonia concentration. These hardy physiological characteristics make C. quadricarinatus a popular aquaculture species and a potential invasive species that can negatively impact tropical and subtropical ecosystems. Investigating the genomic basis of environmental tolerances and immune adaptation in C. quadricarinatus will facilitate the development of management strategies of this potential invasive species. RESULTS: We constructed a chromosome-level genome of C. quadricarinatus by integrating Nanopore and PacBio techniques. Comparative genomic analysis suggested that transposable elements and tandem repeats drove genome size evolution in decapod crustaceans. The expansion of nine immune-related gene families contributed to the disease resistance of C. quadricarinatus. Three hypoxia-related genes (KDM3A, KDM5A, HMOX2) were identified as being subjected to positive selection in C. quadricarinatus. Additionally, in vivo analysis revealed that upregulating KDM5A was crucial for hypoxic response in C. quadricarinatus. Knockdown of KDM5A impaired hypoxia tolerance in this species. CONCLUSIONS: Our results provide the genomic basis for hypoxic tolerance and immune adaptation in C. quadricarinatus, facilitating the management of this potential invasive species. Additionally, in vivo analysis in C. quadricarinatus suggests that the role of KDM5A in the hypoxic response of animals is complex.

Correlated evolution of conspicuous colouration and burrowing in crayfish.

Conspicuous colours have fascinated biologists for centuries, leading to research on the evolution and functional significance of colour traits. In many cases, research suggests that conspicuous colours are adaptive and serve a function in sexual or aposematic signalling. In other cases, a lack of evidence for the adaptive value of conspicuous colours garners interest from biologists, such as when organisms that live underground and are rarely exposed to the surface are nevertheless colourful. Here, we use phylogenetic comparative methods to investigate colour evolution throughout freshwater crayfishes that vary in burrowing ability. Within the taxa we analysed, conspicuous colours have evolved independently over 50 times, and these colours are more common in semi-terrestrial crayfishes that construct extensive burrows. The intuitive but not evolutionarily justified assumption when presented with these results is to assume that these colours are adaptive. But contrary to this intuition, we discuss the hypothesis that colouration in crayfish is neutral. Supporting these ideas, the small population sizes and reduced gene flow within semi-terrestrial burrowing crayfishes may lead to the fixation of colour-phenotype mutations. Overall, our work brings into question the traditional view of animal colouration as a perfectly adapted phenotype.

Insights from the past: Invasion trajectory and niche trends of a global freshwater invader.

Freshwater ecosystems are invaded by a non-random selection of taxa, among which crayfish stand out with successful examples worldwide. Species distribution models (SDMs) have been used to detect suitable areas for invasive species and predict their potential distributions. However, these prediction exercises assume the stability of realized environmental niches, which is uncertain during invasion. Worldwide evaluations involving cosmopolitan invaders may be particularly useful but have seldom been considered. Focusing on the successful invasion history of the red swamp crayfish, Procambarus clarkii, we assessed its geographic expansion and niche trends over time. Based on global occurrences from 1854 to 2022, multiple sequential SDMs have been implemented based on a set of bioclimatic variables. The environmental suitability for each period was projected through to the next period(s) using an ensemble procedure of commonly used SDM algorithms. As the records of the species are known, it was possible to check whether the modelling projections were concordant with the observed expansion of red swamp crayfish at a global scale. This also permitted analysis of its realized niche, and its dynamics, during different expansion phases. SDM maps based on past species records showed concordance with the known crayfish distributions and yielded similar spatial patterns with outputs overperforming random combinations of cells in term of suitability. The results also reflect the stability of the species niche, which despite some expansions during the invasion process, changed little in terms of main position in functional space over time. SDMs developed in the early stages of invasion provide useful insights but also tend to underpredict the potential range compared to models that were built for later stages. Our approach can be easily transferable to other well-documented taxa and represents valuable evidence for validating the use of SDMs, considering a highly dynamic world where biogeographical barriers are often bypassed.

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.

Salvia miltiorrhiza polysaccharide promotes the health of crayfish (Procambarus clarkii) by promoting hemocyte phagocytosis, protecting hepatopancreas and enhancing intestinal barrier function.

Plant polysaccharides as immunomodulators are considered one of the effective measures to reduce antibiotic therapy in aquaculture. The immunomodulatory function of Salvia miltiorrhiza polysaccharides (SMP) has been demonstrated and begun to be applied in vertebrates, but its potential effect on crustaceans is unclear. In this study, crayfish (Procambarus clarkii) was fed with 0 %, 0.3 %, 0.7 %, 1.1 %, and 1.5 % SMP for 4 weeks to investigate the effects of SMP on hemocytes phagocytosis, hepatopancreatic function, and intestinal barrier function. The results revealed that hemocyte phagocytic activity was increased in all SMP groups. During the process of hemocytes phagocytic recognition and formation of phagosomes and phagolysosomes, the mRNA expression levels of mas, hem, rab3, ctsb, and lamp-1 were up-regulated mainly in the 0.3 % SMP group. During the clearance phase of phagocytosis, respiratory burst activity, ROS level, T-SOD, CAT, GST, and LZM activities were mainly increased in the 1.5 % SMP group. Hepatopancreas AKP and GOT activity were no significant change in all SMP groups. ACP activity was significantly enhanced in the 1.1 % SMP group. The GPT activity of 0.3-0.7 % SMP group was significantly decreased. The 0.7 % SMP group had the highest intestinal fold height. The highest index values of OTUs, Ace, Chao, and Shannon were in the 0.3 % SMP group. The dietary addition of 0.3 % SMP led to a tendency of increased relative abundance of Firmicutes and Bacteroidota at the phylum level, while the relative abundance of Proteobacteria at the phylum level decreased. In conclusion, dietary SMP could promote crayfish health by enhancing phagocytosis, protecting hepatopancreas and enhancing intestinal barrier function. This study contributes to the theoretical foundation for exploring the potential application of plant polysaccharides in crustaceans.

PHB2 inhibits WSSV replication by promoting the nuclear translocation of STAT.

Prohibitins (PHBs) are ubiquitously expressed conserved proteins in eukaryotes that are associated with apoptosis, cancer formation, aging, stress responses and cell proliferation. However, the function of the PHBs in immune regulation has largely not been determined. In the present study, we identified PHB2 in the red swamp crayfish Procambarus clarkii. PHB2 was found to be widely distributed in several tissues, and its expression was significantly upregulated by white spot syndrome virus (WSSV) challenge. PHB2 significantly reduced the amount of WSSV in crayfish and the mortality of WSSV-infected crayfish. Here, we observed that PHB2 promotes the nuclear translocation of STAT by binding to STAT. After blocking PHB2 or STAT with antibodies or interfering with PHB2 or STAT, the expression levels of the antiviral genes ß-thymosin (PcThy-4) and crustin2 (Cru2) decreased. The gene sequence of PHB2 was analyzed and found to contain a nuclear introgression sequence (NIS). After in vivo injection of PHB2 with deletion of NIS (rΔNIS-PHB2), the nuclear translocation of STAT did not change significantly compared to that in the control group. These results suggest that PHB2 promoted the nuclear translocation of STAT through NIS and mediated the expression of antiviral proteins to inhibit WSSV infection.

An i-type lysozyme from a crustacean, Pacifastacus leniusculus, functions as a clot-destabilising enzyme.

Lysozymes are hydrolytic enzymes, and they are ubiquitous among all living organisms. They are mostly associated with antibacterial properties through their muramidase activity, while other properties such as iso-peptidase activity are also common. Invertebrate-type (i-type) lysozymes include the enzyme Destabilase, which is present in the salivary secretions of the medicinal leach Hirundo medicinalis. Destabilase has the ability to hydrolyse the ε-(γ-glutamyl)-lysine iso-peptide bonds formed by transglutaminase in fibrin of vertebrate blood, thereby destabilising blood clots. We have identified an i-type lysozyme from the hemocytes of the freshwater crayfish Pacifastacus leniusculus, which was found to be upregulated at the protein level in response to an injection of the ß-1,3-glucan laminarin. Based on its sequence we predicted that this lysozyme would lack muramidase activity, and therefore we decided to determine its putative immune function. The P. leniusculus i-type lysozyme (Pl-ilys), is a protein with 159 amino acid residues, including a 29 residue signal peptide, with a predicted molecular weight of 16 kDa and a predicted pI of 5.6. It is expressed primarily in the hemocytes and to a lesser extent in the hematopoietic tissue. A recombinant mature Pl-ilys using an E. coli expression system was produced, and we could ascertain that this enzyme was deficient of muramidase activity. Moreover, no iso-peptidase activity could be detected against the substrate l-γ-glutamine-p-nitroanilide. Analysis of the conserved domains in Pl-ilys showed a putative destabilase domain, and thus we tested the clot dissolving activity of this enzyme. We could show that the purified P. leniusculus clotting protein which had been coagulated and clotted with transglutaminase was dissolved by the addition of Pl-ilys. Taken together our results indicate that Pl-ilys has a clot dissolving or destabilising activity in crustacean blood.

Effects of antimicrobial peptides from dietary Hermetia illucens larvae on the growth, immunity, gene expression, intestinal microbiota and resistance to Aeromonas hydrophila of juvenile red claw crayfish (Cherax quadricarinatus).

Antimicrobial peptides (AMPs), which are widely present in animals and plants, have a broad distribution, strong broad-spectrum antibacterial activity, low likelihood of developing drug resistance, high thermal stability and antiviral properties. The present study investigated the effects of adding AMPs from Hermetia illucens larvae on the growth performance, muscle composition, antioxidant capacity, immune response, gene expression, antibacterial ability and intestinal microbiota of Cherax quadricarinatus (red claw crayfish). Five experimental diets were prepared by adding 50 (M1), 100 (M2), 150 (M3) and 200 (M4) mg/kg of crude AMP extract from H. illucens larvae to the basal diet feed, which was also used as the control (M0). After an eight-week feeding experiment, it was discovered that the addition of 100-150 mg/kg of H. illucens larvae AMPs to the feed significantly improved the weight gain rate and specific growth rate of C. quadricarinatus. Furthermore, the addition of H. illucens larvae AMPs to the feed had no significant effect on the moisture content, crude protein, crude fat and ash content of the C. quadricarinatus muscle. The addition of 100-150 mg/kg of H. illucens larvae AMPs in the feed also increased the antioxidant capacity, nonspecific immune enzyme activity and related gene expression levels in C. quadricarinatus, thereby enhancing their antioxidant capacity and immune function. The H. illucens larvae AMPs improved the structure and composition of the intestinal microbiota of C. quadricarinatus, increasing the microbial community diversity of the crayfish gut. Finally, the addition of 100-150 mg/kg of H. illucens larvae AMPs in the feed enhanced the resistance of C. quadricarinatus against Aeromonas hydrophila, improving the survival rate of the crayfish. Based on the aforementioned findings, it is recommended that H. illucens larvae AMPs be incorporated into the C. quadricarinatus feed at a concentration of 100-150 mg/kg.

Structural and functional analysis of transforming growth factor beta regulator 1 (TBRG1) in the red swamp crayfish Procambarus clarkii: The initial insight into TBRG1's role in invertebrate immunity.

The transforming growth factor beta regulator 1 (TBRG1) is a growth inhibitory protein that acts as a tumor suppressor in human cancers, gaining its name for the transcriptional regulation by TGF-ß. While extensive research has been conducted on the tumor-related function of TBRG1 in mammals, its significance in invertebrates remains largely unexplored. In this study, a homolog of TBRG1 was first structurally and functionally analyzed in the red swamp crayfish Procambarus clarkii. The full-length cDNA sequence was 2143 base pairs (bp) with a 1305 bp open reading frame (ORF) encoding a deduced protein of 434 amino acids (aa). The changes of PcTBRG1 transcripts upon immune challenges indicated its involvement in innate immunity. After knocking down PcTBRG1, the decline of bacteria clearance capacity revealed the participation of PcTBRG1 in the immune response. Furthermore, the downregulation of AMPs' expression after the cotreatment of RNAi and bacteria challenge suggested that PcTBRG1 might participate in innate immunity through regulating AMPs' expression. These results provided initial insight into the immune-related function of TBRG1 in invertebrates.

Differential expression of sNPF in male and female eyestalk leading to sex dimorphism of AMP expression in Procambarus clarkii intestine.

Antimicrobial peptide (AMP) is an important component of crustaceans' innate immune system. In this study, a short neuropeptide F (sNPF) gene (Pc-sNPF) and a Forkhead box O (FOXO) gene (PcFOXO) from Procambarus clarkii were identified. Analysis findings showed that the expression level of AMP genes differed between male and female P. clarkii. Furthermore, Pc-sNPF and PcFOXO were related to the sex dimorphism of AMP. Knockdown of Pc-sNPF in the eyestalk significantly upregulated the expression of PcFOXO and two anti-lipopolysaccharide factors (PcALF4 and PcALFL) in the intestine of P. clarkii. The expression of PcFOXO in the intestine of female P. clarkii was higher than in that of males. Results from RNA interference revealed that PcFOXO positively regulated the expression of PcALF4 and PcALFL in the intestine of male and female P. clarkii. In summary, our study showed that differences in Pc-sNPF expression in eyestalk of male and female P. clarkii leading to sex dimorphism of AMP expression in the intestine are mediated by the sNPF-FOXO-AMP signal pathway called the eyestalk-intestine axis.

Effects of replacing fishmeal with soybean meal on the immune and antioxidant capacity, and intestinal metabolic functions of red swamp crayfish Procambarus clarkii.

Excess utilization of plant protein sources in animal feed has been found to adversely affect the antioxidant properties and immunity of animals. While the role of gut microbes in plant protein-induced inflammation has been identified in various models, the specific mechanisms regulating gut microbes in crustaceans remain unclear. Accordingly, this study was designed to investigate the effects of replacing fishmeal with soybean meal (SM) on the hepatopancreas antioxidant and immune capacities, and gut microbial functions of crayfish, as well as the potential microbial regulatory mechanisms. 750 crayfish (4.00 g) were randomly divided into five groups: SS0, SS25, SS50, SS75, and SS100, and fed diets with different levels of soybean meal substituted for fishmeal for six weeks. High SM supplementation proved detrimental to maintaining hepatopancreas health, as indicated by an increase in hemolymph MDA content, GPT, and GOT activities, the observed rupture of hepatopancreas cell basement membranes, along with the decreased number of hepatopancreatic F cells. Moreover, crayfish subjected to high SM diets experienced obvious inflammation in hepatopancreas, together with up-regulated mRNA expression levels of nfkb, alf, and tlr (p<0.05), whereas the lzm mRNA expression level exhibited the highest value in the SS25 group. Furthermore, hepatopancreas antioxidant properties highly attenuated by the level of dietary SM substitution levels, as evidenced by the observed increase in MDA content (p<0.05), decrease in GSH content (p<0.05), and inhabitation of SOD, CAT, GPx, and GST activities (p<0.05), along with down-regulated hepatopancreas cat, gpx, gst, and mmnsod mRNA expression levels via inhibiting nrf2/keap1 pathway. Functional genes contributing to metabolism identified that high SM diets feeding significantly activated lipopolysaccharide biosynthesis, revealing gut dysfunction acted as the cause of inflammation. The global microbial co-occurrence network further indicated that the microbes contributing more to serum indicators and immunity were in module eigengene 17 (ME17). A structural equation model revealed that the genes related to alf directly drove the serum enzyme activities through microbes in ME17, with OTU399 and OTU533 identified as major biomarkers and classified into Proteobacteria that secrete endotoxins. To conclude, SM could replace 25 % of fishmeal in crayfish diets without negatively affecting immunity, and antioxidant capacity. Excessive SM levels contributed to gut dysfunction and weakened the innate immune system of crayfish.

Functional characterization of serine proteinase inhibitor Kazal-Type in the red claw crayfish Cherax quadricarinatus.

Serine protease inhibitors Kazal type (SPINKs) function in physiological and immunological processes across multicellular organisms. In the present study, we identified a SPINK gene, designated as CqSPINK, in the red claw crayfish Cherax quadricarinatus, which is the ortholog of human SPINK5. The deduced CqSPINK contains two Kazal domains consisting of 45 amino acid residues with a typical signature motif C-X3-C-X5-PVCG-X5-Y-X3-C-X6-C-X12-14-C. Each Kazal domain contains six conserved cysteine residues forming three pairs of disulfide bonds, segmenting the structure into three rings. Phylogenetic analysis revealed CqSPINK as a homolog of human SPINK5. CqSPINK expression was detected exclusively in hepatopancreas and epithelium, with rapid up-regulation in hepatopancreas upon Vibrio parahaemolyticus E1 challenge. Recombinant CqSPINK protein (rCqSPINK) was heterologously expressed in Escherichia coli and purified for further study. Proteinase inhibition assays demonstrated that rCqSPINK could potently inhibit proteinase K and subtilisin A, weakly inhibit α-chymotrypsin and elastase, but extremely weak inhibit trypsin. Furthermore, CqSPINK inhibited bacterial secretory proteinase activity from Bacillus subtilis, E. coli, and Staphylococcus aureus, and inhibited B. subtilis growth. These findings suggest CqSPINK's involvement in antibacterial immunity through direct inhibition of bacterial proteases, contributing to resistance against pathogen invasion.

Effects of dietary chitosan oligosaccharide on the growth, intestinal microbiota and immunity of juvenile red claw crayfish (Cherax quadricarinatus).

This study aimed to evaluate the potential benefits of chitosan oligosaccharide (COS) on red claw crayfish (Cherax quadricarinatus) and explore its underlying mechanisms. The crayfish were randomly divided into six groups, and the diets were supplemented with COS at levels of 0 (C0), 0.2 (C1), 0.4 (C2), 0.6 (C3), 0.8 (C4), and 1 (C5) g kg-1. Treatment with COS significantly improved the growth performance of the crayfish with a higher weight gain rate (WGR) and specific growth rate (SGR) in the C2 group compared to the C0 group. Additionally, the content of crude protein in the crayfish muscles in the C1 group was significantly higher than that of the C0 group. Regarding non-specific immunity, the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and alkaline phosphatase (AKP), and the levels of expression of the genes related to immunity (SOD; anti-lipopolysaccharide factor [ALF]; thioredoxin1 [Trx1]; C-type lysozyme, [C-LZM]; and GSH-Px) in the hepatopancreas and hemolymph increased significantly (P < 0.05) after supplementation with 0.4 g kg-1 of COS, while the content of malondialdehyde (MDA) decreased (P < 0.05). The survival rate of C. quadricarinatus increased (P < 0.05) in the C2, C3, C4, and C5 groups after the challenge with Aeromonas hydrophila. This study found that COS has the potential to modulate the composition of the intestinal microbiota and significantly reduce the abundance of species of the phylum Proteobacteria and the genera Aeromonas and Vibrio in the gut of C. quadricarinatus, while the abundance of bacteria in the phylum Firmicutes and the genus Candidatus_Hepatoplasma improved significantly. This study suggests that the inclusion of COS in the diet of C. quadricarinatus can enhance growth, boost immunity, and increase resistance to infection with A. hydrophila, especially when supplemented at 0.4-0.8 g kg-1.

Genetic prospective of a local invader: the strange pattern of Pontastacus leptodactylus population structure in Greece and Turkey based on microsatellite DNA.

BACKGROUND: The combination of the increasing demand of freshwater crayfish exports, the reduced population sizes due to overfishing, the crayfish plague epidemics and the habitat degradation, have led to unrecorded translocations of Pontastacus leptodactylus in Greek lakes. METHODS AND RESULTS: In the present study, the genetics of five narrow clawed crayfish (P. leptodactylus) populations were studied, namely three translocated populations inhabiting in Northern Greece, one native Greek population from Evros river and one potential progeny source population from Turkey. Nine microsatellite loci previously designed for the specific species were investigated, in order to assess the levels of genetic diversity and further to confirm the origin of these translocated populations some decades after the translocation events. Our results confirmed that the source population for the translocated Greek population is the Turkish lake Egirdir. Further, despite the low values of the number of alleles, heterozygosity, and FST the populations were generally diverse, providing evidence for local adaptation. CONCLUSIONS: The low values of FIS for the translocated populations in combination with the high values of gene flow, possibly indicate the existence of re-introducing events. Apart from the translocated populations, high levels of genetic diversity and heterozygosity were observed in Evros population, suggesting it as a possible unit for future conservation purposes both as a donor population for reintroduction purposes as well as a unique gene pool protection source. To the best of our knowledge this is the first study dealing with the genetic composition of Greek P. leptodactylus populations from Nothern Greece, operating as a first step towards the development of proper management practices for restocking events and monitoring of translocated populations.

Ensemble evaluation of potential distribution of Procambarus clarkii using multiple species distribution models.

Procambarus clarkii is a notorious invasive species that has led to ecological concerns owing to its high viability and rapid reproduction. South Korea, a country exposed to a high risk of introduction of invasive species due to active international trade, has suffered from recent massive invasions by invasive species, necessitating the evaluation of potential areas requiring intensive monitoring. In this study, we developed two different types of species distribution models, CLIMEX and random forest, for P. clarkii using occurrence records from the United States. The potential distribution in the United States was predicted along coastal lines and inland regions located below 40°N latitude The model was then applied to evaluate the potential distribution in South Korea, and an ensemble map was constructed to identify the most vulnerable domestic regions. According to both models, the domestic potential distribution was highest in most areas located at low altitudes. In the ensemble model, most of the low-altitude western regions, the eastern coast, and some southern inland regions were predicted to be suitable for the distribution of P. clarkii, and a similar distribution pattern was predicted when the model was projected into the future climate. Through this study, it is possible to secure basic data that can be used for the early monitoring of the introduction and subsequent distribution of P. clarkii.

Microplastic burden in native (Cambarus appalachiensis) and non-native (Faxonius cristavarius) crayfish along semi-rural and urban streams in southwest Virginia, USA.

Our comparative assessment is the first study to investigate microplastic body burden in native (Cambarus appalachiensis) and non-native (Faxonius cristavarius) crayfish along a semi-rural and urban stream across different seasons. Crayfish, sediment, and surface water were collected, processed, and characterized using µRaman spectroscopy to compare microplastic polymer types and shapes across compartments. Average surface water concentrations were significantly higher in our urban stream compared to our semi-rural stream (17.3 ± 2.4 particles/L and 9.9 ± 1.3 particles/L, respectively; P = 0.015). Average sediment concentrations were similar between urban and semi-rural streams (140 ± 14.5 particles/kg and 139 ± 22.5 particles/kg, respectively; P = 0.957). Our findings showed a significant interactive effect of season, site, and nativity (i.e., species) regarding microplastic body burden in crayfish (P = 0.004). The smaller, non-native crayfish amassed more microplastic particles than the native crayfish (0.4-2.0 particles/g versus 0.4-0.8 particles/g, respectively). Fibers and fragments were the most common polymer shapes across compartments, with white and black being the dominant particle colors. Our study identified 13 plastic polymer types in crayfish and three in surface water and sediment; polypropylene was the most common polymer across compartments. This study provides evidence that crayfish body burden of microplastics can differ across species, seasons, and locations, highlighting the need for future studies to consider that sublethal impacts associated with microplastic body burden may vary by region and species.

Microbiome analysis reveals the intestinal microbiota characteristics and potential impact of Procambarus clarkii.

The intestinal microbiota interacts with the host and plays an important role in the immune response, digestive physiology, and regulation of body functions. In addition, it is also well documented that the intestinal microbiota of aquatic animals are closely related to their growth rate. However, whether it resulted in different sizes of crayfish in the rice-crayfish coculture model remained vague. Here, we analyzed the intestinal microbiota characteristics of crayfish of three sizes in the same typical rice-crayfish coculture field by high-throughput sequencing technology combined with quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme activity, investigating the relationship between intestinal microbiota in crayfish and water and sediments. The results showed that the dominant intestinal microbiota of crayfish was significantly different between the large size group (BS), normal size group (NS), and small size group (SS), where Bacteroides and Candidatus_Bacilloplasma contributed to the growth of crayfish by facilitating food digestion through cellulolysis, which might be one of the potential factors affecting the difference in sizes. Follow-up experiments confirmed that the activity of lipase (LPS) and protease was higher in BS, and the relative expression of development-related genes, including alpha-amylase (α-AMY), myocyte-specific enhancer factor 2a (MEF2a), glutathione reductase (GR), chitinase (CHI), and ecdysone receptor (EcR), in BS was significantly higher than that in SS. These findings revealed the intestinal microbiota characteristics of crayfish of different sizes and their potential impact on growth, which is valuable for managing and manipulating the intestinal microbiota in crayfish to achieve high productivity in practice. KEY POINTS: • Significant differences in the dominant microflora of BS, NS, and SS in crayfish. • Cellulolysis might be a potential factor affecting different sizes in crayfish. • Adding Bacteroides and Candidatus_Bacilloplasma helped the growth of crayfish.