Zinc oxide-Ulva lactuca nanocomposite is a robust dietary immunostimulant in the red swamp crayfish (Procambarus clarkii).

Aquaculture industry suffers significant limitations such as low resistance to diseases and expensive feed. This study investigated the antibacterial and immunostimulatory activities of ZnO-Ulva lactuca nanocomposite (ZnO-Ul NC) in the Procambarus clarkii. Zinc oxide nanoparticles (ZnO NPs) and ZnO-Ul NC were synthetized and characterized by electron microscopies as well as Fourier transform infrared spectroscopy. ZnO NPs and ZnO-Ul NC inhibited the growth of the isolated species Citrobacter freundii and Enterobacter hormaechei. For immunostimulatory evaluation, six crayfish groups (control, U. lactuca, ZnO L, ZnO H, ZnO-Ul L, and ZnO-Ul H) were fed on commercial diet, Ulva lactuca powder, and low or high dose of ZnO NPs or ZnO-Ul NCs, respectively for 90 days. The highest levels of total hemocyte count, granular cells%, phenoloxidase (PO) activity in hemolymph, and NO, superoxide dismutase (SOD), and GSH in hepatopancreas were all reported in the ZnO-Ul groups. The expression of proPO, SOD, and lysozyme exhibited the highest upregulation in the ZnO-Ul H group. Taken together, dietary ZnO-Ul NC significantly improved the non-specific immunity and antioxidant milieu of the crayfish at the genomic and proteomic levels. ZnO-Ul NC is cost effective, easily synthesized, and a promising immunostimulant for Procambarus clarkii that could be used in the aquaculture.

Expression of C/EBP and Kr-h1 transcription factors under immune stimulation in the noble crayfish.

Transcription factors (TFs) have an important role in the regulation of the gene expression network. The role of TFs in the immune response of freshwater crayfish is poorly understood, but leveraging the regulatory mechanisms of immune response could augment the resistance against the invasive oomycete pathogen, Aphanomyces astaci. Previous studies indicated that the TFs CCAAT/enhancer-binding protein (C/EBP) and putative Krüppel homolog-1 protein (Kr-h1) might play a role in immune and stress response of the noble crayfish (Astacus astacus). Here, we aimed to further characterise these two gene products to gain a better understanding of their evolutionary origin, domain organisation and expression patterns across different crayfish tissues. Furthermore, we conducted an immune stimulation experiment to observe the potential changes in the gene expression of C/EBP and Kr-h1 under immune challenge in different crayfish tissues. Our results showed that both C/EBP and Kr-h1 are closely related to other C/EBPs and Kr-h1s in Malacostraca. Gene expression analysis revealed that both TFs are present in all analysed tissues, with higher expression of C/EBP in the gills and Kr-h1 in the abdominal muscle. Immune stimulation with laminarin (mimicking ß-1-3-glucan in the oomycete cell wall) showed an activation of the crayfish immune system, with an overall increase in the total haemocyte count (THC) compared to untreated control and crayfish buffered saline (CBS) treatment. On the gene expression level, an up-regulation of the C/EBP gene was detected in the laminarin treated group in hepatopancreas and heart, while no changes were observed for the Kr-h1 gene. Our results indicate an early change in C/EBP expression in multiple tissues during immune stimulation and suggest its involvement in the immune response of the noble crayfish.

Ferritin Heavy-like subunit is involved in the innate immune defense of the red swamp crayfish Procambarus clarkii.

Iron-binding proteins, known as ferritins, play pivotal roles in immunological response, detoxification, and iron storage. Despite their significance to organisms, little is known about how they affect the immunological system of the red swamp crayfish (Procambarus clarkii). In our previous research, one ferritin subunit was completely discovered as an H-like subunit (PcFeH) from P. clarkii. The full-length cDNA of PcFerH is 1779 bp, including a 5'-UTR (untranslated region, UTR) of 89 bp, 3'-UTR (untranslated region, UTR) of 1180 bp and an ORF (open reading frame, ORF) of 510 bp encoding a polypeptide of 169 amino acids that contains a signal peptide and a Ferritin domain. The deduced PcFerH protein sequence has highly identity with other crayfish. PcFerH protein's estimated tertiary structure is quite comparable to animal structure. The PcFerH is close to Cherax quadricarinatus, according to phylogenetic analysis. All the organs examined showed widespread expression of PcFerH mRNA, with the ovary exhibiting the highest levels of expression. Additionally, in crayfish muscles, intestines, and gills, the mRNA transcript of PcFerH was noticeably up-regulated, after LPS and Poly I:C challenge. The expression of downstream genes in the immunological signaling system was suppressed when the PcFerH gene was knocked down. All of these findings suggested that PcFerH played a vital role in regulating the expression of downstream effectors in the immunological signaling pathway of crayfish.

Effects of glyphosate on neurotoxicity, oxidative stress and immune suppression in red swamp crayfish, Procambarus Clarkii.

Glyphosate, a prevalent herbicide, has raised concerns due to its potential ecological impact, especially on aquatic ecosystems. While it is crucial for managing agricultural productivity, its inadvertent effects on non-target aquatic species like the red swamp crayfish, Procambarus clarkii, are not fully understood. In the present study, the neurotoxicity, oxidative stress, and immune suppression of glyphosate on P. clarkii were investigated. Sublethal glyphosate exposure (5, 10 and 20 mg/L) for 96 h was found to significantly decrease AChE activity in both brain and hepatopancreas, correlating with reduced foraging efficiency and increased turnover time. Oxidative stress was evident through increased lipid peroxidation (LPO) and malondialdehyde (MDA) levels and altered antioxidant enzyme activities such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). In addition, the total antioxidative capacity (T-AOC) was inhibited at 10 and 20 mg/L of glyphosate exposure. Immune assays revealed a decrease in total hemocyte counts (THC) and suppression of key immune enzyme activities and transcriptional expressions at higher concentrations, suggesting compromised immune defenses. The findings demonstrate that glyphosate can induce considerable neurotoxic and immunotoxic effects in P. clarkii, disrupting essential physiological functions and behavior.

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.

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.

The identification, evolutionary analysis, and immune roles of Rab family members in red swamp crayfish, Procambarus clarkii.

The Rab GTPase constitutes the largest family of small GTPases that regulate intracellular trafficking. Different eukaryotes possess varying numbers of Rab paralogs. However, limited knowledge exists regarding the evolutionary pattern of Rab family in most major eukaryotic supergroups. This study cloned 24 Rab genes from transcriptome data of Procambarus clarkii haemocytes. The multiple sequence alignment and phylogenetic tree analysis revealed a relatively high degree of conservation for PcRab. Furthermore, PcRab exhibited similarities in motif composition with all members showing presence of G, PM, RabF, and RabSF motifs. The tertiary structure indicated that PcRab proteins mainly consisted of α-helices and ß-strands, and most PcRab proteins shared similar tertiary structures, and it was indicated that they have similar protein characteristics. Protein-protein interaction prediction identified a total of 20 interacting proteins involved in vesicle trafficking, phagocytosis, and signal transduction with 193 interactions. Expression analysis showed wide expression patterns for PcRab in P. clarkii organs. Upon infection by white spot syndrome virus and Aeromonas veronii, significant induction was observed for PcRab gene expression levels, indicating their involvement in pathogen response mechanisms. The present study represents the pioneering effort in comprehensively identifying and cloning the Rab family genes in crustacean, followed by a systematic investigation into their evolutionary patterns and immune response upon pathogen infection. The results provided valuable insights for further investigation into the molecular mechanism underlying the response of P. clarkii to pathogen infection.

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.

The molecular characterization of Rab11 and its immune roles in red swamp crayfish (Procambarus clarkii).

The Rab proteins primarily regulate vesicular transport between membrane-bound organelles and are important for innate immune. However, there is currently a lack of studies on crustaceans regarding Rab proteins, particularly core Rabs. We identified a Rab11 gene from Procambarus clarkii (PcRab11) and evaluated its potential involvement in immune response. The results showed PcRab11 was 1789 bp long, with an open reading frame of 645 bp encoding 211 amino acids and an estimated molecular weight of 23.8 kDa. Sequence analysis revealed its remarkable evolutionary conservation. The PcRab11 was widely expressed in various tissues, with highest levels in hepatopancreas, and localized within the cell cytoplasm. Upon infection with white spot syndrome virus (WSSV) or Aeromonas veronii, the expression of PcRab11 in immune organs was significantly induced. Furthermore, silencing PcRab11 reduced phagocytosis-related genes expression and haemocytes' phagocytic activity to FITC-labeled A. veronii, as well as decreased mortality and death time in WSSV or A. veronii infected P. clarkii. Additionally, the potential protein interaction between PcRab11 and 14-3-3ε was identified in haemocytes. Overall, our findings provided evidence for the involvement of Rab11 in P. clarkii's immune response, establishing a foundation to explore the immune role of core Rab proteins in crustaceans' innate immune system.

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

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

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.

Effects of Dietary Melatonin on Antioxidant Capacity, Immune Defense, and Intestinal Microbiota in Red Swamp Crayfish (Procambarus clarkii).

The aim of this study was to investigate the effects of melatonin (MT) feed supplementation on the antioxidant capacity, immune defense, and intestinal flora in Procambarus clarkii (P. clarkii). Six groups of P. clarkii were fed test feeds containing different levels of MT: 0 mg/kg (control), 22.5, 41.2, 82.7, 165.1, and 329.2 mg/kg for a duration of 2 months. The specific growth rate, hepatosomatic index, and condition factor were recorded highest in the test group of shrimp fed an MT concentration of 165.1 mg/kg. Compared to the control group, the rate of apoptosis was lower in hepatopancreas cells of P. clarkii supplemented with high concentrations of MT. Analyses of antioxidant capacity and immune-response-related enzymes in the hepatopancreas indicated that dietary supplementation of MT significantly augmented both the antioxidant system and immune responses. Dietary MT supplementation significantly increased the expression levels of antioxidant-immunity-related genes and decreased the expression levels of genes linked to apoptosis. Dietary MT was associated with an elevation in the abundance of the Firmicutes and a reduction in the abundance of the Proteobacteria in the intestines; besides, resulting in an increase in the abundance of beneficial bacteria, such as Lactobacilli. The broken-line model indicated that the suitable MT concentration was 154.09-157.09 mg/kg. MT supplementation enhanced the growth performance of P. clarkii, exerting a positive influence on the intestinal microbiota, and bolstered both immune response and disease resistance. Thus, this study offered novel perspectives regarding the application of dietary MT supplementation within the aquaculture field.

Induction and potential molecular mechanism of the enhanced immune response in Procambarus clarkii after secondary encountered with Aeromonas veronii.

For a long time, it was believed that invertebrates do not possess acquired immunity and mainly rely on innate immunity for protection against pathogens infection. However, an increasing number of studies have suggested that some form of "immune memory" can be initiated in invertebrates after primary exposure to the pathogen, which was defined as "specific immune priming". In the present study, two experiments were carried out to determine whether specific immune priming can be induced in crayfish (Procambarus clarkii) by Aeromonas veronii, if so, to identify the underlying mechanism. Once being "preimmunization" by formalin-killed A. veronii, the survival rate, in vitro antibacterial activity and haemocyte phagocytosis rate of crayfish were enhanced, which indicated that better immune protection was obtained. Furthermore, at some time points, the expression of antimicrobial peptide (AMP) and Down syndrome cell adhesion molecule (Dscam) genes was significantly higher in P. clarkii individuals that underwent stimulation twice than in those that were only stimulated once. Taken together, the results suggest that enhanced specific immune protection can be obtained in primed crayfish and that the Dscam molecule, haemocyte phagocytosis function, and AMPs may be involved in this immune priming. The present study provides a better understanding of the molecular mechanism of immune priming in invertebrates.

Effects of dietary quercetin on the innate immune response and resistance to white spot syndrome virus in Procambarusclarkii.

In recent years, the use of natural products with immune-stimulating and antimicrobial properties has attracted increasing attention in aquaculture researches. In our study, the effect of diet supplemented with quercetin, a flavonoid commonly found in some types of plants substance on the innate immune response and disease resistance in crayfish (Procambarus clarkii) against white spot syndrome virus (WSSV) is reported. It was found that dietary 40 mg/kg quercetin significantly reduced the mortality of crayfish and WSSV copy number after WSSV challenge. Dietary quercetin increased catalase (CAT), and lysozyme (LZM) activity in crayfish. Dietary quercetin increased the expression of NF-κB, anti-lipopolysaccharide factor (ALF) and toll-like receptor (TLR) genes in crayfish. The apoptosis rate of hemocyte was increased by quercetin supplement in crayfish. Our results suggest that dietary quercetin may affect the innate immunity of crayfish and protect crayfish from WSSV infection.

A Comprehensive Review on Crustaceans' Immune System With a Focus on Freshwater Crayfish in Relation to Crayfish Plague Disease.

Freshwater crayfish immunity has received great attention due to the need for urgent conservation. This concern has increased the understanding of the cellular and humoral defense systems, although the regulatory mechanisms involved in these processes need updating. There are, however, aspects of the immune response that require clarification and integration. The particular issues addressed in this review include an overall description of the oomycete Aphanomyces astaci, the causative agent of the pandemic plague disease, which affects freshwater crayfish, and an overview of crustaceans' immunity with a focus on freshwater crayfish. It includes a classification system of hemocyte sub-types, the molecular factors involved in hematopoiesis and the differential role of the hemocyte subpopulations in cell-mediated responses, including hemocyte infiltration, inflammation, encapsulation and the link with the extracellular trap cell death pathway (ETosis). In addition, other topics discussed include the identity and functions of hyaline cells, the generation of neoplasia, and the emerging topic of the role of sessile hemocytes in peripheral immunity. Finally, attention is paid to the molecular execution of the immune response, from recognition by the pattern recognition receptors (PRRs), the role of the signaling network in propagating and maintaining the immune signals, to the effector elements such as the putative function of the Down syndrome adhesion molecules (Dscam) in innate immune memory.

The identification of a nuclear factor Akirin with regulating the expression of antimicrobial peptides in red swamp crayfish (Procambarus clarkii).

Akirin is a highly conserved nuclear factor among different species. It is closely related to skeletal muscle development, innate immune response, and tumorigenesis in a variety of animals. In invertebrates, Akirin is mainly involved in gene transcription and NF-κB dependent natural immune response. In the present study, a nuclear factor Akirin was identified from Procambarus clarkii. The Akirin protein of crayfish consists of 204 amino acids and is conserved among its family members, especially the nuclear localization signal peptide motif (KRRR). PcAkirin was highly expressed in stomach, intestines, and hepatopancreas. After A. hydrophila challenge, the transcription level of Akirin significantly increased in hemocyte and hepatopancreas. In addition, the recombinant Akirin protein was produced successfully and helpful to resist WSSV infection by increasing the expression level of some immune related genes. On the contrary, after interfering with Akirin gene by dsRNA, the crayfish increased the sensitivity to A. hydrophila and WSSV infections. The results are more obvious in the accumulated mortality of P. clarkii infected with A. hydrophila and WSSV. All these results suggested that Akirin played a significant role in innate immune responses and protected it from WSSV and bacterial infection in crayfish.

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

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

A crayfish ALF inhibits the proliferation of microbiota by binding to RPS4 and MscL of E. coli.

Antimicrobial peptides (AMPs), most of which are small proteins, are necessary for innate immunity against pathogens. Anti-lipopolysaccharide factor (ALF) with a conserved lipopolysaccharide binding domain (LBD) can bind to lipopolysaccharide (LPS) and neutralize LPS activity. The antibacterial mechanism of ALF, especially its role in bacteria, needs to be further investigated. In this study, the antibacterial role of an anti-lipopolysaccharide factor (PcALF5) derived from Procambarus clarkii was analyzed. PcALF5 could inhibit the replication of the microbiota in vitro and enhance the bacterial clearance ability in crayfish in vivo. Far-western blot assay results indicated that PcALF5 bound to two proteins of E. coli (approximately 25 kDa and 15 kDa). Mass spectrometry (MS), far-western blot assay, and pull-down results showed that 30S ribosomal protein S4 (RPS4, 25 kD) interacted with PcALF5. Further studies revealed that another E. coli protein binding to PcALF5 could be the large mechanosensitive channel (MscL), which is reported to participate in the transport of peptides and antibiotics. Additional assays showed that PcALF5 inhibited protein synthesis and promoted the transcription of ribosomal component genes in E. coli. Overall, these results indicate that PcALF5 could transfer into E. coli by binding to MscL and inhibit protein synthesis by interacting with RPS4. This study reveals the mechanism underlying ALF involvement in the antibacterial immune response and provides a new reference for the research on antibacterial drugs.

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

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

CqPP2A inhibits white spot syndrome virus infection by up-regulating antimicrobial substances expression in red claw crayfish Cherax quadricarinatus.

Protein phosphatase 2A (PP2A) is an important serine/threonine phosphatase, a highly conserved enzyme widely expressed in eukaryotic cells, which accounts for a majority of the serine/threonine phosphatase activity in cells implicated in regulation of immune signaling pathways and antiviral response. However, most of studies about PP2A have been conducted in mammals but few in crustaceans. In this study, two subunits of PP2A (named as CqPP2Ab and CqPP2Ac) were characterized to be involved in white spot syndrome virus (WSSV) infection in the haematopoietic tissue (Hpt) cells from red claw crayfish Cherax quadricarinatus. The open reading frame (ORF) of CqPP2Ab was 1341 bp encoding 446 amino acids with seven WD40 domains, and the ORF of CqPP2Ac was 930 bp encoding 309 amino acids with a PP2Ac domain. Tissue distribution analysis showed that the mRNA transcript of CqPP2Ab and CqPP2Ac were both widely expressed in all the tested tissues with the highest expression in hemocyte, followed by high expression in Hpt. The gene expressions of CqPP2Ab and CqPP2Ac were both significantly down-regulated at 6 h post WSSV infection (6 hpi) in Hpt cells. Importantly, the expression of viral immediate early gene IE1 and late viral gene envelope protein VP28 were both significantly increased post WSSV infection after gene silencing of CqPP2Ab or CqPP2Ac in Hpt cells, suggesting that CqPP2Ab and CqPP2Ac could inhibit WSSV infection in Hpt cells, probably by increasing the antimicrobial substances expression in consideration to the significantly reduced expression of anti-lipopolysaccharide factor, crustin, and lysozyme after gene silencing of CqPP2Ab or CqPP2Ac, respectively. These findings provide a new light on the mechanism of WSSV infection and the antiviral response in crustaceans.