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Three novel crayfish-infecting nudiviruses from crayfish in North America represent the first genomic confirmation of nudiviruses in crayfish: Faxonius propinquus nudivirus (FpNV), Faxonius rusticus nudivirus (FrNV), and Faxonius virilis nudivirus (FvNV). Histopathology and electron microscopy revealed nuclear infections, including nuclear hypertrophy in hepatopancreatic epithelial cells and the presence of membrane-bound bacilliform virions. Metagenomic sequencing resulted in complete circular genome assembly, and phylogenetic analyses (based on nudivirus core genes) placed these viruses within the unofficial Epsilonnudivirus genus. One of the nudiviruses was detected in the antennal gland of its host, and another is correlated with invasive crayfish decline in one infected lake ecosystem - suggesting a potential route for viral transmission through water, and possible population level impact. This study highlights the importance of genomic and ecological data in elucidating the diversity and evolutionary relationships of the Nudiviridae, while expanding their known diversity and range of host species., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Viruses have evolved various strategies to achieve early infection by initiating transcription of their own early genes via host transcription factors, such as NF-κb, STAT, and AP1. How the host copes with this immune escape has been a topic of interest. Tripartite motif (TRIM) family proteins with RING-type domains have E3 ubiquitin ligase activity and are known as host restriction factors. Trim has been reported to be associated with phagocytosis and is also believed to be involved in the activation of autophagy. Preventing the virus from entering the host cell may be the most economical way for the host to resist virus infection. The role of TRIM in the early stage of virus infection in host cells remains to be further interpreted. In the current study, a crayfish TRIM with a RING-type domain, designated as PcTrim, was significantly upregulated under white spot syndrome virus (WSSV) infection in the red swamp crayfish (Procambarus clarkii). Recombinant PcTrim significantly inhibited WSSV replication in crayfish. RNAi targeting PcTrim or blocking PcTrim with an antibody promoted WSSV replication in crayfish. Pulldown and co-IP assays showed that PcTrim can interact with the virus protein VP26. PcTrim restricts the expression level of dynamin, which is involved in the regulation of phagocytosis, by inhibiting AP1 entry into the nucleus. AP1-RNAi effectively reduced the expression levels of dynamin and inhibited host cell endocytosis of WSSV in vivo. Our study demonstrated that PcTrim might reduce early WSSV infection by binding to VP26 and then inhibiting AP1 activation, resulting in reduced endocytosis of WSSV in crayfish hemocytes. Video Abstract., (© 2023. The Author(s).)

The pathogenesis of white spot syndrome virus (WSSV) is largely unclear. In this study, we found that actin nucleation and clathrin-mediated endocytosis were recruited for internalization of WSSV into crayfish hematopoietic tissue (Hpt) cells. This internalization was followed by intracellular transport of the invading virions via endocytic vesicles and endosomes. After envelope fusion within endosomes, the penetrated nucleocapsids were transported along microtubules toward the periphery of the nuclear pores. Furthermore, the nuclear transporter Cq Importin α1/β1, via binding of ARM repeat domain within Cq Importin α1 to the nuclear localization sequences (NLSs) of viral cargoes and binding of Cq Importin β1 to the nucleoporins Cq Nup35/62 with the action of Cq Ran for docking to nuclear pores, was hijacked for both targeting of the incoming nucleocapsids toward the nuclear pores and import of the expressed viral structural proteins containing NLS into the cell nucleus. Intriguingly, dysfunction of Cq Importin α1/β1 resulted in significant accumulation of incoming nucleocapsids on the periphery of the Hpt cell nucleus, leading to substantially decreased introduction of the viral genome into the nucleus and remarkably reduced nuclear import of expressed viral structural proteins with NLS; both of these effects were accompanied by significantly inhibited viral propagation. Accordingly, the survival rate of crayfish post-WSSV challenge was significantly increased after dysfunction of Cq Importin α1/β1, also showing significantly reduced viral propagation, and was induced either by gene silencing or by pharmacological blockade via dietary administration of ivermectin per os . Collectively, our findings improve our understanding of WSSV pathogenesis and support future antiviral designing against WSSV. IMPORTANCE As one of the largest animal DNA viruses, white spot syndrome virus (WSSV) has been causing severe economical loss in aquaculture due to the limited knowledge on WSSV pathogenesis for an antiviral strategy. We demonstrate that the actin cytoskeleton, endocytic vesicles, endosomes, and microtubules are hijacked for WSSV invasion; importantly, the nuclear transporter Cq Importin α1/β1 together with Cq Ran were recruited, via binding of Cq Importin β1 to the nucleoporins Cq Nup35/62, for both the nuclear pore targeting of the incoming nucleocapsids and the nuclear import of expressed viral structural proteins containing the nuclear localization sequences (NLSs). This is the first report that NLSs from both viral structure proteins and host factor are elaborately recruited together to facilitate WSSV infection. Our findings provide a novel explanation for WSSV pathogenesis involving systemic hijacking of host factors, which can be used for antiviral targeting against WSSV disease, such as the blockade of Cq Importin α1/β1 with ivermectin.

The freshwater crayfish Procambarus clarkii is native to North America and Mexico, and it was introduced to China in 1929. The production and consumption of P. clarkii in China are the highest worldwide, reaching 208.96 million tons in 2020. The white spot syndrome virus (WSSV) is a major pathogen that affects shrimp, crayfish, crabs and lobsters, and it has caused widespread loss to the P. clarkii industry. Epigallocatechin-3-gallate (EGCG), a small-molecule compound, has a multitude of biological functions and the ability to bind to the 37 kDa/67 kDa laminin receptor (LamR). EGCG has potential antiviral effects against WSSV. In this study, we evaluated the potential anti-WSSV applications of EGCG in P. clarkii. We demonstrated that various concentrations (10 μg/g·bw, 20 μg/g·bw and 40 μg/g·bw) of EGCG can suppress WSSV infection in P. clarkii. Histopathological examination revealed no characteristic pathological changes due to EGCG administration in P. clarkii tissues. Furthermore, pharmacokinetics studies of EGCG in P. clarkii revealed its rapid absorption (T max  = 2 h), and the peak concentrations of EGCG were 73.78 µg/g in the liver and 24.87 µg/g in the muscle. Our results indicate the high potential applications of EGCG against WSSV in P. clarkii., (© 2021 John Wiley & Sons Ltd.)

Crayfish are a keystone species of freshwater ecosystems and a successful invasive species. However, their pathogens, including viruses, remain understudied. The aim of this study was to analyze the virome of the invasive signal crayfish ( Pacifastacus leniusculus ) and to elucidate the potential differences in viral composition and abundance along its invasion range in the Korana River, Croatia. By the high-throughput sequencing of ribosomal RNA, depleted total RNA isolated from the crayfish hepatopancreas, and subsequent sequence data analysis, we identified novel and divergent RNA viruses, including signal crayfish-associated reo-like, hepe-like, toti-like, and picorna-like viruses, phylogenetically related to viruses previously associated with crustacean hosts. The patterns of reads abundance and calculated nucleotide diversities of the detected viral sequences varied along the invasion range. This could indicate the possible influence of different factors and processes on signal crayfish virome composition: e.g., the differences in signal crayfish population density, the non-random dispersal of host individuals from the core to the invasion fronts, and the transfer of viruses from the native co-occurring and phylogenetically related crayfish species. The study reveals a high, previously undiscovered diversity of divergent RNA viruses associated with signal crayfish, and sets foundations for understanding the potential risk of virus transmissions as a result of this invader's dispersal.

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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

White spot syndrome virus (WSSV) is a serious pathogen threatening global crustacean aquaculture with no commercially available drugs. Herbal medicines widely used in antiviral research offer a rich reserve for drug discovery. Here, we investigated the inhibitory activity of 13 herbal medicines against WSSV in crayfish Procambarus clarkii and discovered that naringenin (NAR) has potent anti-WSSV activity. In the preliminary screening, the extracts of Typha angustifolia displayed the highest inhibitory activity on WSSV replication (84.62%, 100 mg/kg). Further, NAR, the main active compound of T. angustifolia, showed a much higher inhibition rate (92.85%, 50 mg/kg). NAR repressed WSSV proliferation followed a dose-dependent manner and significantly improved the survival of WSSV-challenged crayfish. Moreover, pre- or post-treatment of NAR displayed a comparable inhibition on the viral loads. NAR decreased the transcriptional levels of vital genes in viral life cycle, particularly for the immediately early-stage gene ie1. Further results showed that NAR could decrease the STAT gene expression to block ie1 transcription. Besides, NAR modulated immune-related gene Hsp70, antioxidant (cMnSOD, mMnSOD, CAT, GST), anti-inflammatory (COX-1, COX-2) and pro-apoptosis-related factors (Bax and BI-1) to inhibit WSSV replication. Overall, these results suggest that NAR may have the potential to be developed as preventive or therapeutic agent against WSSV., (© 2021 John Wiley & Sons Ltd.)

Invasive crayfish and the introduction of non-native diseases pose a significant risk for the conservation of endangered, white-clawed crayfish (Austropotamobius pallipes). Continued pollution of waterways is also of concern for native species and may be linked with crayfish disease dynamics. We explore whether crayfish species or environmental quality are predictors of infection presence and prevalence in native A. pallipes and invasive signal crayfish (Pacifastacus leniusculus). We use a seven-year dataset of histology records, and a field survey comparing the presence and prevalence of infectious agents in three isolated A. pallipes populations; three isolated P. leniusculus populations, and three populations where the two species had overlapped in the past. We note a lower diversity of parasites (Simpson's Index) in P. leniusculus ('Pacifastacus leniusculus Bacilliform Virus' - PlBV) (n = 1 parasite) relative to native A. pallipes (n = 4 parasites), which host Thelohania contejeani, 'Austropotamobius pallipes bacilliform virus' (ApBV), Psorospermium haeckeli and Branchiobdella astaci, at the sites studied. The infectious group present in both species was an intranuclear bacilliform virus of the hepatopancreas. The prevalence of A. astaci in A. pallipes populations was higher in more polluted water bodies, which may reflect an effect of water quality, or may be due to increased chance of transmission from nearby P. leniusculus, a species commonly found in poor quality habitats., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The peak period of morbidity and death in cultured Procambarus clarkii is around May each year and is called the "Black May" disease. The pathogen causing "Black May" disease is believed to be a white spot syndrome virus (WSSV). In 2018, a significant number of P. clarkii died in the pond culture of Xinglong Township, Xuyi County. Two sampling tests on the affected pond showed that, in addition to WSSV, a novel Dicistro-like virus (PcDV) was present. Genomic sequence analysis indicated that this new virus belongs to the Dicistroviridae family, Picornaviridaes order. A high number of spherical particles were detected in gill tissues of P. clarkii with "Black May" disease by electron microscopy, a finding consistent with viruses from the Picornaviridaes order. From October 2018 to September 2019, we took monthly samples from Hubei, Jiangsu and Anhui provinces, and tested for the presence of PcDV and WSSV in P. clarkii. The detection rates of PcDV in P. clarkii peaked from April to June, consistent with the onset of the "Black May" disease. In conclusion, we believe that the discovery of PcDV will provide new research directions for investigating the pathogens causing "Black May" disease in P. clarkii., (© 2020 John Wiley & Sons Ltd.)

Despite important ecological role and growing commercial value of freshwater crayfish, their diseases are underresearched and many studies examining potential crayfish pathogens do not thoroughly address their epizootiology, pathology or biology. This study reviews over 100 publications on potentially pathogenic viruses, bacteria, fungi and fungal-like microorganisms reported in crayfish and systematizes them based on whether pathogenicity has been observed in an analysed species. Conclusions on pathogenicity were based on successful execution of infectivity trials. For 40.6% of examined studies, microbes were successfully systematized, while for more than a half (59.4%) no conclusion on pathogenicity could be made. Fungi and fungal-like microorganisms were the most studied group of microbes with the highest number of analysed hosts, followed by bacteria and viruses. Our analysis demonstrated the need for: (a) inclusion of higher number of potential host species in the case of viruses, (b) research of bacterial effects in tissues other than haemolymph, and (c) more research into potential fungal and fungal-like pathogens other than Aphanomyces astaci. We highlight the encountered methodological challenges and biases and call for a broad but standardized framework for execution of infectivity trials that would enable systematic data acquisition on interactions between microbes and the host., (© 2020 John Wiley & Sons Ltd.)

White spot syndrome virus (WSSV) is currently the most severely viral pathogen for farmed crustaceans such as shrimp and crayfish, which has been causing huge economic losses for crustaceans farming worldwide every year. Unfortunately, study on the molecular mechanisms of WSSV has been restricted by the lack of crustacean cell lines for WSSV propagation as well as the incompletely annotated genomes for host species, resulting in limited elucidation for WSSV pathogenesis at present. In addition to the findings of anti-WSSV response in shrimp, some of novel cellular events involved in WSSV infection have been recently revealed in crayfish, including endocytosis and intracellular transport of WSSV, innate immune pathways in response to WSSV infection, and regulation of viral gene expression by host genes. Despite these advances, many fundamental gaps in WSSV pathogenesis are still remaining, for example, how WSSV genome enters into nucleus and how the progeny virions are fully assembled in the host cell nucleus. In this review, recent findings in WSSV infection mechanism and the antiviral immunity against WSSV in crayfish are summarized and discussed, which may provide us a better understanding of the WSSV pathogenesis as well as new ideas for the target design of antiviral drugs against WSSV in crustaceans farming., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

As the most severely lethal viral pathogen for crustaceans in both brackish water and freshwater, white spot syndrome virus (WSSV) has a mechanism of infection that remains largely unknown, which profoundly limits the control of WSSV disease. By using a hematopoietic tissue (Hpt) stem cell culture from the red claw crayfish Cherax quadricarinatus suitable for WSSV propagation in vitro , the intracellular trafficking of live WSSV, in which the acidic-pH-dependent endosomal environment was a prerequisite for WSSV fusion, was determined for the first time via live-cell imaging. When the acidic pH within the endosome was alkalized by chemicals, the intracellular WSSV virions were detained in dysfunctional endosomes, resulting in appreciable blocking of the viral infection. Furthermore, disrupted valosin-containing protein ( C. quadricarinatus VCP [ Cq VCP]) activity resulted in considerable aggregation of endocytic WSSV virions in the disordered endosomes, which subsequently recruited autophagosomes, likely by binding to Cq GABARAP via Cq VCP, to eliminate the aggregated virions within the dysfunctional endosomes. Importantly, both autophagic sorting and the degradation of intracellular WSSV virions were clearly enhanced in Hpt cells with increased autophagic activity, demonstrating that autophagy played a defensive role against WSSV infection. Intriguingly, most of the endocytic WSSV virions were directed to the endosomal delivery system facilitated by Cq VCP activity so that they avoided autophagy degradation and successfully delivered the viral genome into Hpt cell nuclei, which was followed by the propagation of progeny virions. These findings will benefit anti-WSSV target design against the most severe viral disease currently affecting farmed crustaceans. IMPORTANCE White spot disease is currently the most devastating viral disease in farmed crustaceans, such as shrimp and crayfish, and has resulted in a severe ecological problem for both brackish water and freshwater aquaculture areas worldwide. Efficient antiviral control of WSSV disease is still lacking due to our limited knowledge of its pathogenesis. Importantly, research on the WSSV infection mechanism is also quite meaningful for the elucidation of viral pathogenesis and virus-host coevolution, as WSSV is one of the largest animal viruses, in terms of genome size, that infects only crustaceans. Here, we found that most of the endocytic WSSV virions were directed to the endosomal delivery system, strongly facilitated by Cq VCP, so that they avoided autophagic degradation and successfully delivered the viral genome into the Hpt cell nucleus for propagation. Our data point to a virus-sorting model that might also explain the escape of other enveloped DNA viruses., (Copyright © 2020 American Society for Microbiology.)

Proteins and nucleic acids from ultrasonically ruptured white spot syndrome virus (WSSV) can infect crayfish and cause death as effectively as intact WSSV virions. In this study, ultrasound was used to rupture the virus and the resulting suspension was filtered through a 50 nm membrane. Analysis by PCR and SDS-PAGE showed that both viral genes (VP19, VP26, VP28 and DNA polymerase) and proteins (VP15, VP19, VP26 and VP28) were present in the filtered solution. Electron microscopy showed that there were no intact virions in the filtered solution. When crayfish were injected with the filtered solution or with intact WSSV, the mortality in each group was 100 %. The same result was seen when crayfish were challenged orally with the filtered solution and intact WSSV. The filtered solution of ultrasonically ruptured virus, which contains viral proteins and residual DNA genome, can thus infect the host as effectively as intact virions. When the solution of viral proteins and residual DNA genome was digested with DNase I and then injected into crayfish, the survival rate was 100 %. We also found that, although viral proteins (except VP15) in the solution of ruptured virus were destroyed by treatment with DNase I, DNase I did not destroy the structural proteins of intact virions. A remaining viral protein in the DNase I-treated solution protects the DNA genome from degradation and we concluded that this protein is VP15, which is a DNA-binding protein. Our study highlights the extreme danger in producing vaccines from proteins obtained by ultrasonic rupture of viruses sincethe viral DNA genome is difficult to degrade and, if present, will lead to viral infection., Competing Interests: Declaration of Competing Interest There are no patents, products in development or marketed products to declare. This does not alter our adherence to all the gene policies on sharing data and materials. The authors declare that they have no competing interests. Yongyong Lai declares that he has no conflict of interest. Fei Zhu declares that he has no conflict of interest. Yinglei Xu declares that she has no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The sea vegetable Hizikia fusiforme is not only a good source of dietary fiber but also enhances immunity. In this study, we investigated the effects of H. fusiforme on innate immunity in invertebrates, using white spot syndrome virus (WSSV) challenge in the crayfish, Procambarus clarkii. Supplementation with H. fusiforme significantly reduced mortality caused by WSSV infection and also reduced copy numbers of the WSSV protein VP28. Quantitative reverse transcription-polymerase chain reaction showed that supplementation of feed with H. fusiforme increased the expression of immune-related genes, including NF-κB and crustin 1. Further analysis showed that supplementation with H. fusiforme also affected three immune parameters, total hemocyte count, and phenoloxidase and superoxide dismutase activity. H. fusiforme treatment significantly increased hemocyte apoptosis rates in both WSSV-infected and uninfected crayfish. H. fusiforme thus regulates the innate immunity of crayfish, and both delays and reduces mortality after WSSV challenge. Our study demonstrates the potential for the commercial use of H. fusiforme, either therapeutically or prophylactically, to regulate the innate immunity and protect crayfish against WSSV infection., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The Janus kinase (JAK)/signal transducers and activators of transcription (STAT) signaling pathway is associated with the innate immune system and plays crucial roles in the mediation of immune response to viral infections. In this study, three STAT isoform cDNAs were cloned from the red swamp crayfish Procambarus clarkii, and they were designated as PcSTATa, PcSTATb, and PcSTATc. PcSTATa and PcSTATb were generated through the alternative splicing of the last exon, and PcSTATc was produced by intron retention. PcSTATa, PcSTATb, and PcSTATc contained 2382, 2337, and 2274 bp open reading frames encoding proteins with 793, 778, and 757 amino acid residues, respectively. Domain prediction analysis revealed that three isoforms of PcSTATs contain a STAT interaction domain, a STAT all-alpha domain, a STAT DNA binding domain, and a Src-homology 2 domain. The mRNA transcripts of three PcSTAT isoforms were detected in all examined tissues of male and female crayfish. The expression levels of the three PcSTAT isoforms in the hemocytes, gills, and intestines significantly changed after the white spot syndrome virus (WSSV) challenge. PcSTAT silencing by dsRNA interference could positively regulate the expression levels of three anti-lipopolysaccharide factors (PcALF1, PcALF2, and PcALF6) and two crustins (PcCrus1 and PcCrus2) and negatively regulate the expression levels of three ALFs (PcALF3, PcALF4, and PcALF5) and two crustins (PcCrus3 and PcCrus4). These results suggest that all three PcSTAT isoforms are involved in the host defense against WSSV infection., Competing Interests: Declaration of competing interest No potential conflicts of interest were disclosed., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Antibiotics used for humans and livestock are emerging as pollutants in aquatic environments. However, little is known about their effect on aquatic organisms, especially in crustaceans. In the present study, the freshwater crayfish Pacifastacus leniusculus was exposed during 21 days to environmental concentrations of sulfamethoxazole (SMX) (100 ng/L and 1 μg/L). Subsequently, the crayfish susceptibility to infection was evaluated by using White Spot Syndrome Virus (WSSV) challenge, a well-known crustacean pathogen. The median survival time of the infected crayfish exposed to 100 ng/L SMX was one day, whereas the control and the group exposed to 1 μg/L SMX survived for two and three days, respectively. In order to elucidate the effect of SMX upon the crayfish immune response, new sets of crayfish were exposed to the same SMX treatments to evaluate mRNA levels of immune-related genes which are expressed and present in hemocytes and intestine, and to perform total and differential hemocyte counts. These results show a significant down-regulation of the antimicrobial peptide (AMP) Crustin 3 in hemocytes from the 100 ng/L SMX group, as well as a significant up-regulation of the AMP Crustin 1 in intestines from the 1 μg/L SMX group. Semigranular and total hemocyte cell number were observed to be significantly lower after exposure to 100 ng/L SMX in comparison with the control group. The present study demonstrates that environmentally relevant SMX concentrations in the water at 100 ng/L led to an increased WSSV susceptibility, that may have been caused by a reduction of circulating hemocytes. Nevertheless, SMX concentrations of 1 μg/L could marginally and for a few days have an immunostimulatory effect., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Invertebrates rely solely on the innate immune system to protect against virus infection, while the viral infection must rely on the transcriptional system of the host cell to achieve the expression of viral genes, which is naturally regulated by the host's transcriptional system. However, the mechanism of the host against viral transcription in host cells is still poorly understood in crustaceans. Previously, we found that the partial transcript sequence of a negative elongation factor E (named as CqNELF-E) was up-regulated in a differentially expressed transcriptome library of the haematopietic tissue (Hpt) cells from red claw crayfish Cherax quadricarinatus upon white spot syndrome virus (WSSV) infection, suggesting a possible role of CqNELF-E in WSSV-host interaction. In the present study, we revealed the function of CqNELF-E. The full-length cDNA sequence of CqNELF-E was identified with 1726 bp from red claw crayfish, which contained an open reading frame of 816 bp, encoding 271 amino acids. Amino acid sequencing analysis revealed that the CqNELF-E had a conserved RNA recognition motif (RRM) and a leucine zipper motif (LZM). Tissue distribution analysis showed that CqNELF-E was widely expressed in various tissues with the highest expression in muscle, relatively abundant in Hpt and the lowest presence in heart. Interestingly, the gene expression of CqNELF-E was significantly up-regulated at both 6 and 12 hpi after WSSV infection in Hpt cell cultures in red claw crayfish. In addition, the expression of both the viral immediately early gene (IE) 1 (IE1) and a late gene envelope protein VP28 were significantly increased after gene silencing of CqNELF-E in Hpt cells, indicating the potential suppression role of CqNELF-E against the viral infection. Further study revealed that the CqNELF-E had an inhibitory effect on the promoter activity of WSSV IE genes WSV051, WSV069 (IE1) and WSV083 by a dual luciferase reporter gene assay. Taken together, these results suggest that CqNELF-E plays an antiviral role, probably via inhibition on the viral transcription activity in WSSV infection in a crustacean., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The cathepsin C, a lysosomal cysteine protease, involves the modulation of immune and inflammatory responses in living organisms. However, the knowledge on cathepsin C in red swamp crayfish (Procambarus clarkii), a freshwater crustacean with economic values, remained unclear. In the present study, we provide identification and molecular characterization of cathepsin C from P. clarkii. (Hereafter Pc-cathepsin C). The Pc-cathepsin C cDNA contained a 1356 bp open reading frame that encoded a protein of 451 amino acid residues. The deduced amino acid sequence comprised of cathepsin C exclusion domain and pept_C1 domain, and also catalytic residues (Cys 248 , His 395 and Asn 417 ). Analysis of the transcriptional patterns of the Pc-cathepsin C gene revealed that it was broadly distributed in various tissues of P. clarkii, and it was more abundant in the hepatopancreas and gut. Following a challenge with viral and bacterial pathogen-associated molecular patterns, the expression of Pc-cathepsin C was strongly enhanced at different time points. The knockdown of Pc-cathepsin C, altered the expression of immune-responsive genes, suggesting its immunoregulatory role in P. clarkii. This study has identified and provided the immunoregulatory function of Pc-cathepsin C, which will contribute to further investigation of the molecular mechanism of cathepsin C in crustaceans., Competing Interests: Declaration of competing interest The authors have declared that no competing interests exist., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Several investigations have recently assessed the ability of some aquatic invertebrates to act as tools for avian influenza A virus (IAV) surveillance as well as their potential role(s) in IAV ecology. Because of this, as well as the high IAV seroprevalence rates noted in select mesocarnivores that commonly inhabit aquatic and semi-aquatic habitats, we evaluated the effects that freshwater crayfish have on IAV in water at three dose levels and monitored for the presence of IAV in crayfish tissues (gill and green gland) and haemolymph at multiple time points. At relatively high, medium and low (approximately 10 4 , 10 3 and 10 2  EID 50 /ml, respectively) doses, mesocosms containing crayfish (Orconectes sp.) had less detectable IAV RNA present when final water samples were assayed (9 days post-contact [DPC]). In general, containers without crayfish present had nearly three-fold greater quantities of viral RNA at 9 DPC. A varying number of RNA positive samples were detected for the three crayfish sample types collected. Gill tissue produced the largest number of positive non-water samples (n = 26), with the highest quantities detected from crayfish sampled on 1 and 4 DPC (10 3.5  EID 50 equivalent/ml). On a few occasions, gill (n = 8) and haemolymph samples (n = 1) produced higher quantities of viral RNA than their respective water samples or water samples collected 1-2 DPC earlier, but these differences were typically minor. Based upon water samples, statistical models indicated that the interaction of dose and crayfish exposure days explained most of the variation in these data. Future efforts should address if crayfish exposed to IAV-laden water have the capacity to successfully transmit IAVs to mammals and birds which frequently prey upon them., (Published 2020. This article is a U.S. Government work and is in the public domain in the USA.)

Infectious hypodermal and haematopoietic necrosis virus (IHHNV) is a major viral pathogen in cultured penaeid shrimp. IHHNV has many hosts, mainly including crustaceans. It has recently been reported that Procambarus clarkii can be infected by IHHNV. In the present study, we studied the hepatopancreas of P. clarkii by transcriptome high-throughput sequencing to analyze the response of P. clarkii to IHHNV infection. After de novo assembly, there were 400,340,760 clean reads. A total of 237 differentially expressed genes (DEGs) were obtained, including 77 significantly up-regulated unigenes and 160 significantly down-regulated ones. The expression levels of 12 immune-related DEGs were validated by qRT-PCR, substantiating the reliability of RNA-Seq results. The enrichment analysis of DEGs showed that the immune-related pathways were closely related to apoptosis and phagocytosis. Moreover, a large number of pathways related to metabolic function were down-regulated, suggesting that IHHNV infection might affect the growth of P. clarkii., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Cherax quadricarinatus is a large-sized, highly fecund, and fast-growing species of freshwater crayfish, and has become one of the world's most intensely studied crustaceans. Decapod iridescent virus 1 (DIV1), a newly described species in the family Iridoviridae, is known to infect various crustaceans, including C. quadricarinatus, and may pose a new threat in the shrimp-farming industry. The present study performed de novo transcriptome sequencing of C. quadricarinatus hepatopancreas during DIV1 infection. A total of 114,784 transcripts and 56,418 genes were obtained; 1070 genes were upregulated and 775 genes were downregulated when compared with the uninfected samples (controls). Three pattern recognition receptor genes (fibrinogen-related protein, C-type lectin, and beta-1,3-glucan-binding protein) were upregulated during DIV1 infection. Among the top-30 upregulated unigenes, 9 unigenes were identified as vitellogenin (Vg) genes, and the top-3 upregulated unigenes were identified as involved in Vg lipid transport, lipid localization, and lipid transporter activity, which were all significantly over-representative GO terms in the GO enrichment analysis of total and upregulated differentially expressed genes (DEGs). Many genes associated with Jak-STAT signaling pathway, Endocytosis, Phagosome, MAPK signaling pathway, Apoptosis and Lysosome were positively modified after DIV1 infection. The predicted protein-protein interaction (PPI) analysis showed NF1 and TUBA, CRM1 and TUBB were involved in protein interactions. This research showed that DIV1 infection has a significant impact on the transcriptome profile of C. quadricarinatus hepatopancreas, and the results enhance our understanding of virus-host interactions. Furthermore, the high number of transcripts generated in the present study will provide information for identifying novel genes in the absence of a full C. quadricarinatus genome sequence., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

White spot syndrome virus (WSSV) is a serious epidemic pathogen of crustaceans and cause severe economic losses to aquaculture. However, no commercial drugs presently available to control WSSV infection. Genipin (GN) is a bioactive compound extracted from the fruit of Gardenia jasminoides and exhibits potential antiviral activity. In the study, the antiviral activity of GN against WSSV was investigated in crayfish Procambarus clarkii and in shrimp Litopenaeus vannamei. In vitro antiviral test showed that GN could inhibit WSSV replication in crayfish and in shrimp, and the highest inhibition on WSSV was over 99% when treatment with 50 mg/kg of GN for 24 h. In vivo antiviral test proved that GN could be used to treat and prevent WSSV infection. GN could also effectively protect crayfish from WSSV infection by reducing the mortality rate of WSSV-infected crayfish. Moreover, GN attenuated the WSSV-induced oxidative stress and inflammatory by upregulation the expression of antioxidant-related genes and downregulation the expression of inflammatory-related genes, respectively. Mechanically, GN inhibited WSSV replication at least via decreasing STAT (signal transducer and activator of transcription) gene expression to block WSSV immediate-early gene ie1 transcription. Additionally, the inhibition of BI-1 (Bax inhibitor-1) gene expression also played an important role in the suppression of WSSV infection. In conclusion, GN represented a potential therapeutic and preventive agent to block WSSV infection., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Hesperetin is a natural flavanone compound, which mainly exists in lemons and oranges, and has potential antiviral and anticancer activities. In this study, hesperetin was used in a crayfish pathogen challenge to discover its effects on the innate immune system of invertebrates. The crayfish Procambarus clarkii was used as an experimental model and challenged with white spot syndrome virus (WSSV). Pathogen challenge experiments showed that hesperetin treatment significantly reduced the mortality caused by WSSV infection, while the VP28 copies of WSSV were also reduced. Quantitative reverse transcriptase polymerase chain reaction revealed that hesperetin increased the expression of several innate immune-related genes, including NF-kappaB and C-type lectin. Further analysis showed that hesperetin treatment plays a positive effects on three immune parameters like total hemocyte count, phenoloxidase and superoxide dismutase activity. Nevertheless, whether or not infected with WSSV, hesperetin treatment would significantly increase the hemocyte apoptosis rates in crayfish. These results indicated that hesperetin could regulate the innate immunity of crayfish, and delaying and reducing the mortality after WSSV challenge. Therefore, the present study provided novel insights into the potential therapeutic or preventive functions associated with hesperetin to regulate crayfish immunity and protect crayfish against WSSV infection, provide certain theoretical basis for production practice., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

VP19 is a major envelope protein of white spot syndrome virus (WSSV), an important pathogen of farmed shrimp. However, the exact function of VP19 in WSSV assembly and infection is unknown. To understand the function of VP19, the gene was knocked down by RNA interference. We found that the dsRNA specific for vp19 gene dramatically reduced the replication of WSSV genomic DNA in infected animals. Further investigation by transmission electron microscopy showed that inhibition of VP19 prevented envelope coating of progeny virions, resulting in a high amount of immature virus particles without outer layer (envelope) in the host cells. This finding was further confirmed by SDS-PAGE analysis, which showed the loss of VP19 and other envelope proteins from the improperly assembled virions. These results suggest that VP19 is essential for WSSV envelope coating., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Arthropod hypervariable Dscam (Down syndrome cell adhesion molecule) may be involved in adaptive-like immune characteristics, namely immune priming, enabling the host to "learn" and "remember" pathogens previously encountered in arthropods. However, expression of Dscam in immune-primed arthropods after a second challenge has apparently not been confirmed. Herein, working with Dscam of Australian freshwater crayfish (Cherax quadricarinatus, i.e. CqDscam), we further investigated whether immune priming is mediated by "clouds" of appropriate (or "correct") CqDscam isoforms. In crayfish that survived a first WSSV challenge (immune priming), long-lasting CqDscam expression remained higher after a second WSSV challenge. Selective CqDscam isoforms were also induced after both challenges. Based on pathogen binding assays, these WSSV-induced CqDscam isoforms had a higher WSSV binding ability, perhaps mainly mediated by Ig3-spliced variants. We therefore hypothesized that in these crayfish survivors, an unknown selection process was generating a "correct cloud" of CqDscam against a previously encountered pathogen., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

We report the discovery of a new enveloped, spherical virus belonging to the Phenuiviridae family of negative ssRNA viruses associated with a massive outbreak in a French population of the endangered white-clawed crayfish Austropotamobius pallipes. We call this virus Bunya-like Brown Spot Virus (BBSV) and characterize it using transmission electronic microscopy, genome sequencing and clinical signs. Infected specimens show discolored brown spots on the cuticle. Using RNA-seq data we assembled a partial sequence for the L, M and S genome segments of BBSV. Phylogenetic analyses using all three segments show this virus is closely related to the Wenling crustacean virus 7 (China) and to a bunya-like virus found in feces of the European otter. Our survey of the mass mortality event indicates the virus is less virulent than the crayfish plague caused by Aphanomyces astaci. Overall, the discovery of BBSV provides a new important asset to monitor A. pallipes populations., (Copyright © 2019. Published by Elsevier Inc.)

The replication profile of white spot syndrome virus (WSSV) in crayfish, Procambarus clarkii, at different water temperature was investigated in this study. The WSSV detections were negative at 15 ± 1°C, and the natural infection ratio increased at 19 ± 1°C (24.2% ± 2.25%), reached 100% at 25 ± 1°C and decreased at 30 ± 1°C (93.2% ± 3.37%). The WSSV genome copies number was much higher at 25 ± 1°C (≥5 × 10 6.45 ± 0.35 /mg) than at 15 ± 1°C (≤5 × 10 1.13 ± 0.12 /mg), 19 ± 1°C (≤5 × 10 2.74 ± 0.48 /mg) and 32 ± 1°C (≤5 × 10 3.18 ± 0.27 /mg). Meanwhile, the significant transcription signals of immediate early gene ie1 and late gene vp28 and a large number of virus particles were detected in epitheliums of stomach, gut and gill, hepatopancreas, heart and muscle cells at 25 ± 1°C by using in situ hybridization (ISH) and transmission electron microscopy. The experimental infection of P. clarkii with WSSV infection showed reduced mortality and lower virus copies number at 19 ± 1°C (23.51% ± 0.84%, ≤5 × 10 3.41 ± 0.11 /mg) and 32 ± 1°C (38.42%  ±  1.21%, ≤5 × 10 3.72 ± 0.13 /mg) compared to 25 ± 1°C (100%, ≥5 × 10 4.99 ± 0.24 /mg). The water temperature regulated the transcription of immune-related genes (crustin2, prophenoloxidase (proPO) and heat shock protein70 (Hsp70)), with some differences between WSSV treatments and control treatments. These results demonstrate that water temperature has effect on WSSV proliferation, which may due to transcriptional response of immune-related genes to temperature., (© 2019 John Wiley & Sons Ltd.)

Caspase, an aspartate specific proteinase mediating apoptosis, plays a key role in immune response. In our previous study, the expression of a caspase gene was up-regulated in a transcriptome library from the haematopoietic tissue (Hpt) cells of red claw crayfish Cherax quadricarinatus post white spot syndrome virus (WSSV) infection. To further reveal the effect of caspase on WSSV infection, we cloned this caspase gene (denominated as CqCaspase) with an open reading frame of 1062 bp, which encoded 353 amino acids with a caspase domain (CASc) containing a p20 subunit and a p10 subunit. Tissue distribution analysis indicated that the mRNA transcript of CqCaspase was widely expressed in all tested tissues with the highest expression in Hpt, while the lowest expression in muscle. To further explore the effect of CqCaspase on WSSV replication, recombinant protein of CqCaspase (rCqCaspase) was delivered into Hpt cells followed by WSSV infection, which resulted in a significantly decreased expression of both an immediate early gene IE1 and a late envelope protein gene VP28 of WSSV, suggesting that CqCaspase, possibly by the enhanced apoptotic activity, had a strong negative effect on the WSSV replication. These data together indicated that CqCaspase was likely to play a vital role in immune defense against WSSV infection in a crustacean C. quadricarinatus, which shed a new light on the mechanism study of WSSV infection in crustaceans., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

MicroRNAs (miRNAs) as short noncoding RNAs play important regulatory roles in diverse biological processes by degrading the target mRNAs, and could be delivered by exosomes. WSSV is a highly pathogenic and prevalent virus, and has brought high mortality of P. clarkia. Till present, no studies focus on the miRNAs changes in exosomes during WSSV infection. To understand the different virulence of WSSV on miRNAs expression in P. clarkia hemolymph exosome, the deep sequencing was performed to compare the small RNA libraries from the hemolymph exosome of P. clarkia individuals with or without WSSV infections. From the TEM observations, NTA and Western Blot analysis, the extracted exosomes were well identified with classic characteristics. The 209 conserved miRNAs and 250 novel miRNAs were identified from the small RNA libraries. In response to WSSV infection, there were about 98 miRNAs significantly up-regulated and 59 miRNAs significantly down-regulated. The target genes prediction, GO and KEGG enrichment analysis revealed that some target genes of P. clarkia miRNAs were grouped mainly into the categories of biological regulation, immune system process, signal pathway and other more functions. This is the first report of comprehensive identification of P. clarkia hemolymph exosome miRNAs being differentially regulated in response to WSSV infection. These results will help to understand the hemolymph exosome miRNAs response to different virulence WSSV infection., (Copyright © 2018. Published by Elsevier Ltd.)

MicroRNAs (miRNAs) are important posttranscriptional regulators. They play an important role in the antiviral innate immunity of invertebrates. In the present study, high-throughput small RNAs Illumina sequencing systems were carried out to identify differentially expressed miRNAs (DEMs) in the gills of Procambarus clarkii, which was challenged with white spot syndrome virus (WSSV). Our results identified 11,617 known and 6 novel miRNAs in normal group (NG) and WSSV-challenged group (WG) small RNA libraries. Additionally, 27 DEMs were shown to participate in the antiviral innate immunity of P. clarkii and were significantly upregulated or downregulated. In addition, the results of the KEGG pathway prediction of the DEMs target genes showed that putative target genes of these 27 DEMs were related mainly to the RNA transport pathway, tight junction pathway, mRNA surveillance pathway, regulation actin cytoskeleton pathway, focal adhesion pathway, and MAPK signaling pathway. These results provide important information for future studies about the antiviral innate immunity of crustaceans., (Copyright © 2018. Published by Elsevier Ltd.)

The effects of temperature on the progression of White Spot Disease (WSD) have been studied in the freshwater crayfish Pacifastacus leniusculus. In this study, we aimed to understand the reason for previously observed low mortalities with white spot syndrome virus (WSSV) infected crayfish at low temperatures. The susceptibility of freshwater crayfish to WSSV was studied at different temperatures. The mortality rate at 6 °C was zero, meanwhile the animals kept at 22 °C developed WSD symptoms and died in a few days after WSSV injections, however upon transfer of animals from 6 °C to 22 °C the mortality reached 100% indicating that the virus is not cleared at 6 °C. Moreover, the VP28 expression at 6 °C was significantly lower compared to animals kept at 22 °C. We injected animals with demecolcine, an inhibitor that arrests the cell cycle in metaphase, and observed a delayed mortality. Furthermore, the VP28 expression was found to be lower in these animals receiving both injections with WSSV and demecolcine since cell proliferation was inhibited by demecolcine. We quantified WSSV copy numbers and found that virus entry was blocked at 6 °C, but not in demecolcine treatments. We supported this result by quantifying the expression of a clip domain serine protease (PlcSP) which plays an important role for WSSV binding, and we found that the PlcSP expression was inhibited at 6 °C. Therefore, our hypothesis is that the WSSV needs proliferating cells to replicate, and an optimum temperature to enter the host hematopoietic stem cells successfully., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Various known and unknown viral diseases can threaten crustacean aquaculture. To develop prophylactic and therapeutic strategies against viruses, crustacean cell lines are urgently needed for immunology and virology studies. However, there are currently no permanent crustacean cell lines available. In this study, we developed a new method for preparing crayfish plasma (CP) and found that CP enhanced the proliferative capacity of haematopoietic tissue (hpt) cells from Cherax quadricarinatus by an EdU (5-ethynyl-2'-deoxyuridine) assay. The optimal CP concentration for hpt cell culture and the optimal subculture method are discussed. To achieve efficient expression of a foreign gene in hpt cells cultured in vitro, different transfection methods and vectors were analysed. We found that Lipofectamine 2000 could be used to efficiently transfect a foreign vector into hpt cells and exhibited a lower level of cytotoxicity than the other methods tested, and transfection of pEGFP-N1/w249 and pDHsp70-EGFP-FLAG resulted in high EGFP expression. By transmission electron microscopy (TEM) and virus copy number analysis, we found that white spot syndrome virus (WSSV) could infect hpt cells and multiply efficiently. Our results implied that the crayfish hpt cell culture system we improved could be used as a replacement for immortal crustacean cell lines in viral infection studies. Our findings provide a solid foundation for future immortalization and gene function studies in crustacean cells., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Infectious hypodermal and haematopoietic necrosis virus (IHHNV) infects many crustacean hosts, including cultured penaeid shrimp. In the present study, we aimed to develop a novel sensitive SYBR Green-based real-time PCR method to specifically amplify DNA fragments of IHHNV. Our newly developed real-time PCR method with a 195-bp amplicon specifically detected IHHNV and showed no cross reaction with white spot syndrome virus (WSSV), hepatopancreatic parvovirus (HPV), Enterocytozoon hepatopenaei (EHP), infectious myonecrosis virus (IMNV) and yellow-head virus (YHV). This method could detect as low as one single copy of IHHNV plasmid DNA, more sensitive than other SYBR Green-based real-time PCR methods and less expensive and more convenient than the TaqMan probe-based real-time PCR. Moreover, our data using the newly designed method showed that 80% of IHHNV-fed Procambarus clarkii samples were IHHNV positive. Our findings further confirmed that P. clarkii can be infected by IHHNV., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The β-thymosins are a group of structurally related, highly conserved intracellular small peptides in vertebrates with various biological functions, including cytoskeletal remodeling, neuronal development, cell migration, cell survival, tissue repair and inhibition of inflammation. In contrast to vertebrates, the function of β-thymosin is not fully understood in crustaceans. Previously, we found that a thymosin-repeated protein1 (CqTRP1) gene was up-regulated after white spot syndrome virus (WSSV) challenge in hematopoietic tissue (Hpt) cells from the red claw crayfish Cherax quadricarinatus. To further identify the effect of CqTRP1 on WSSV infection, a full length cDNA sequence of β-thymosin homologue was cloned and analyzed from red claw crayfish followed by functional study. The CqTRP1 cDNA contains an open reading frame of 387 nucleotides encoding a protein of 129 amino acids with a putative molecular mass of 14.3 kDa. The amino acid sequence showed high identity with other β-thymosins and contained three characteristic thymosin β actin-binding motifs, suggesting that CqTRP1 was a member of the β-thymosin family. Tissue distribution analysis revealed a ubiquitous presence of CqTRP1 in all the examined tissues with the highest expression in hemocytes, Hpt and gonad at the transcriptional level. Interestingly, the gene silencing of endogenous CqTRP1 by RNAi enhanced the WSSV replication in Hpt cells. Meanwhile, the WSSV replication was significantly reduced in the Hpt cell cultures if overloaded with a recombinant CqTRP1. Taken together, these data clearly indicated that CqTRP1 was likely to be associated with the anti-WSSV response in a crustacean C. quadricarinatus, which provides new strategy against white spot disease in crustacean aquaculture., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Athtabvirus, a bunya-like virus and chequa iflavirus infect redclaw crayfish (Cherax quadricarinatus) and they may cause mortality reaching 20-40% after about three weeks following transportation stress. Lesions were seen in the muscles of broodstock and juveniles and nerve cords of craylings. Using NextGen sequencing, the whole transcriptomes of a farmed case crayfish and a tank-reared, unaffected crayfish were assembled producing over 500,000 contigs. The average depth of reads was 18 replicates with a range from 15 to 44. The near complete sequences of the large and middle genome segments of a bunya-like virus were detected along with chequa iflavirus. The internal bunya-like motifs; RNA-dependent RNA polymerase on the L segment, and glycoprotein n (Gn) on the M segment were easily identified. In the opposite, positive-sense direction on the M segment, another presumed glycoprotein (glycoprotein c) with a low-density lipoprotein receptor (cysteine-rich) motif was identified by position specific iterated (psi)-BLASTp. The athtabvirus was related to Whenzhou Shrimp Virus 2 (E = 0.0, 43% amino acid identity), an unassigned, -ve sense ssRNA virus, and to peribunyaviruses (E = 10 -50-20 ). In descending order of the number of RNA copies/0.2 mg of tissue, the organs most heavily infected were muscle (9.4 × 10 6 ), nerve cord (5.24 × 10 6 ), heart (4.07 × 10 6 ), gills (3.96 × 10 6 ), hepatopancreas (1.58 × 10 6 ) and antennal gland (6.6 × 10 5 ). Given the tissue tropism (muscle and nerves) of athtabvirus and the original lesions, this virus is implicated in being involved in the mortalities in crayfish after transportation., (Copyright © 2018 Elsevier B.V. All rights reserved.)

It is well known that iron is an essential element for all living organism. The intracellular iron availability is also important for the host's innate immune response to various pathogens, in which the iron homeostasis can be regulated by ferritin due to its iron storage property. In this study, a full-length cDNA sequence of ferritin (named as CqFerritin) was identified with 1410 bp from red claw crayfish Cherax quadricarinatus, which contained an open reading frame of 513 bp, encoding 170 amino acids with a conserved ferritin domain. Tissue distribution analysis demonstrated that CqFerritin was widely expressed in various tissues with high presence in haemocyte, haematopoietic tissue (Hpt) and heart, while lowest expression in hepatopancreas. In addition, loss-of-function of CqFerritin by gene silencing resulted in significantly higher expression of an envelope protein VP28 of white spot syndrome virus (WSSV) in red claw crayfish Hpt cell cultures, indicating the potential antiviral response of CqFerritin. To further explore the effect on WSSV replication by CqFerritin, recombinant CqFerritin protein (rCqFerritin) was transfected into Hpt cells followed by WSSV infection. Importantly, the replication of WSSV was obviously decreased in Hpt cells if transfected with rCqFerritin protein, suggesting that CqFerritin had clearly negative effect on WSSV infection. Furthermore, intracellular accumulation of iron ions was found to promote the WSSV replication in a dose-dependent manner, illustrating that the iron level regulated by CqFerritin was likely to be vital for WSSV infection in red claw crayfish. Taken together, these data suggest that CqFerritin plays an important role in immune defense against WSSV infection in a crustacean C. quadricarinatus., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The PI3K/AKT signaling pathway is commonly exploited to regulate viral replication and affect the fate of infected cells. In the present study, a PI3K-specific inhibitor (LY294002) was employed to pretreat crayfish to evaluate the effects of PI3K/AKT signaling pathway in WSSV replication. The results showed that the WSSV copy numbers in crayfish pretreated with LY294002 were significantly lower than those in Tris-HCl pretreatment crayfish on the sixth and tenth day after WSSV infection. In semigranular cells, the apoptosis rates were up-regulated on the third day post-WSSV infection, and a significantly lower proportion of apoptosis cells were observed in LY294002-pretreatment group. The expression level of Bax, Bax inhibitor-1 and lectin mRNA in haemocytes of crayfish were increased after WSSV infection. After the secondary stimulation with Tris-HCl, the Bax expression level in LY294002-pretreatment crayfish was significantly higher than that of crayfish pretreated with Tris-HCl on the third or sixth day, but the Toll and lectin mRNA expression decreased significantly on the third, sixth and tenth day. The Bax mRNA expression levels in LY294002-WSSV group were significantly higher than those in Tris-HCl-WSSV group on the third and tenth day. The Bax inhibitor-1 mRNA expression levels in LY294002-WSSV group were significantly lower than those in Tris-HCl-WSSV crayfish on the third day. These results together indicated that the hosts PI3K/AKT signaling pathway play positive roles in WSSV replication through the balance between host cell apoptois and innate immune responses. This information is helpful to further understand the role of PI3K/AKT signaling pathway on WSSV replication in Decapoda crustaceans., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Chequa iflavirus (+ve sense ssRNA virus) infects redclaw crayfish (Cherax quadricarinatus) and it may cause mortality reaching 20-40% after about three weeks following stress. The sequence of the RNA-dependent RNA polymerase at nucleotide position 8383-9873 was used for developing and comparing PCR-based detection protocols. The reverse transcription, quantitative, polymerase chain reaction (RT-qPCR) was specific against nine Picornavirales and crustacean viruses and its' measurement of uncertainty (0.07-1.37) was similar to PCRs for other crustacean viruses. In vitro, the reverse transcription loop-mediated isothermal amplification (RT-LAMP) read at 60min had poor repeatability for a linearized plasmid with an iflavirus insert when compared with RT-PCR visualised on an electrophoretic gel and RT-qPCR; both sensitive to 10 2 copies. In a limited, comparative sample of clinical crayfish haemolymph, the lowest, non-zero copies were 2.88×10 4 for RT-PCR and 4.60×10 1 for the RT-qPCR. In 68 further clinical crayfish haemolymph samples tested by RT-qPCR only, copy numbers ranged from 0 to 1.14×10 6 . For RT-qPCR, the amplification plots, melt curves and the C T values indicated that the C T above 34.0 is a potential negative result but examination of the melt curve is necessary for an accurate interpretation. A suggested program of testing for crayfish farmers would consist of non-destructive bleeding, labelling of crayfish and screening with RT-qPCR. Only those crayfish nominally negative (below detectable limits) would be used for broodstock or selective breeding., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Developing a rapid, accurate and quantitative method for detecting white spot syndrome virus (WSSV) is extremely urgent and critical for reducing the risk of white spot disease outbreaks. In the present work, an optimized double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was developed for quantitative detection of WSSV. The method employed rabbit polyclonal antibodies against WSSV as the capture antibody and previously produced anti-WSSV monoclonal antibodies as the detector antibody. A standard curve of the log concentration of WSSV versus OD value was established, which was linear in the concentration range of 120-7680ng/mL, and the linear regression equation was y=0.166x-0.151. Viral proteins in different tissues of crayfish (Procambarus clarkia) post artificial infection with WSSV were quantitatively measured using the DAS-ELISA. WSSV proliferated quickly within 60h post infection and gradually slowed down afterwards. According to the linear regression relationship, the viral proteins in hemolymph, gut and gonad were firstly able to be quantified at 24h post infection with the concentrations of 186, 158 and 128ng/mL, respectively. These three tissues also contained higher viral proteins than the gill, heart, hepatopancreas and muscle during the entire infection period. The viral protein concentration in gut reached the highest level of 6220ng/mL at 72h post infection. Real time quantitative PCR was also used to detect the dynamic change of viral copies in crayfish hemolymph post WSSV infection, with similar results for both assays. The developed DAS-ELISA could detect WSSV propagation from initial to moribund stage in infected crayfish and demonstrated potential application for diagnosis of WSSV., (Copyright © 2017 Elsevier B.V. All rights reserved.)

MicroRNAs (miRNAs) are small non-coding endogenous RNA molecules that play important roles in the innate immunity system of invertebrates, especially in the aspect of antivirus. In the present study, high-throughput small RNA Illumina sequencing systems were used to identify differentially expressed miRNAs (DEMs) from the intestines of Procambarus clarkii that were infected with white spot syndrome virus (WSSV). As a result, 39 known and 12 novel miRNAs were identified in both NG and WG small RNA libraries. Seven DEMs were determined to be involved in the antiviral innate immunity in the intestines of P. clarkii. The results of the target gene predictions of the DEMs showed that the putative target genes of these 7 DEMs are related to tight junctions, vascular smooth muscle contraction regulation of the actin cytoskeleton, focal adhesion, RNA transport, mRNA surveillance, viral carcinogenesis, and Salmonella infection. These results provide theoretical insights for future studies on the antiviral immunity of crustaceans.

Chequa iflavirus and a bunya-like virus infect redclaw crayfish (Cherax quadricarinatus) and they may cause mortality reaching 20-40% after about three weeks after a stress event. To complete River's postulates for viruses, virus-free animals are needed. Due to a lack of chequa iflavirus and bunya-like virus-free crayfish (testing shows>85% infection rate) coupled with the facts that iflavirus and bunyaviruses are found in insects and that crickets had been successful alternate hosts for crustacean viruses before, Acheta domesticus was trialled asa bioassay animal. There was no significant difference (P>0.05) in mortality rates between uninfected control crickets and infected crickets. Reverse transcriptase polymerase chain reaction for both viruses failed to find any trace of the RNA viruses in fed or inoculated crickets after 30days. The search for an alternative bioassay host will have to be widened., (Copyright © 2017 Elsevier Inc. All rights reserved.)

A novel iridovirus, Cherax quadricarinatus iridovirus (CQIV), was identified from diseased C. quadricarinatus in 2014. This virus is considered as a new threat to crustacean aquaculture because it is lethal to both peneaid shrimp and crayfish. Here, we determined the complete genome sequence of CQIV. The double-stranded DNA genome is 165 695 bp in length with a G+C content of 34.6 %. A total of 178 open reading frames (ORFs) have been predicted, encoding hypothetical proteins ranging from 50 to 1327 amino acids. Forty-seven of these exhibit similarities to proteins of known functions. Phylogenetic analysis based on multiple alignments of conserved proteins shows that CQIV clusters with the members of the family Iridoviridae, but is placed in a distinct clade from all the five known genera. It indicates that CQIV may represent a new genus in the family Iridoviridae, for which we propose the name Cheraxvirus based on the host organism.

MicroRNAs (miRNAs) were important post-transcriptional regulators and played vital roles in innate immunity system of invertebrates, especially in the aspect of antivirus. In this study, using high-throughput small RNAs Illumina sequencing system, differentially expressed miRNAs (DEMs) from lymph organs in red swamp crayfish, Procambarus clarkii, infected with white spot syndrome virus, were identified. As a result, 32 known miRNAs and 7 novel miRNAs were identified in crayfish lymph organ small RNAs library of NG and WG. Among them, 7 differentially expressed miRNAs (DEMs) were predicted to be involved in the lymph organ antiviral innate immunity of P. clarkii. Besides, the results showed that putative target genes of these DEMs were related with tight junction, RNA transport, regulation of actin cytoskeleton, focal adhesion, vascular smooth muscle contraction, mRNA surveillance pathway, NOD-like receptor signaling pathway, leukocyte transendothelial migration, and protein processing in endoplasmic reticulum. These results might provide the guiding theoretical foundation for future studies about crustaceans' antiviral innate immunity., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Serine proteases are involved in many critical physiological processes including virus spread and replication. In the present study, we identified a new clip-domain serine protease (PlcSP) in the crayfish Pacifastacus leniusculus hemocytes, which can interact with the White Spot Syndrome Virus (WSSV) envelope protein VP28. It was characterized by a classic clip domain with six strictly conserved Cys residues, and contained the conserved His-Asp-Ser (H-D-S) motif in the catalytic domain. Furthermore, signal peptide prediction revealed that it has a 16-residue secretion signal peptide. Tissue distribution showed that it was mainly located in P. leniusculus hemocytes, and its expression was increased in hemocytes upon WSSV challenge. In vitro knock down of PlcSP decreased both the expression of VP28 and the WSSV copy number in hematopoietic stem (HPT) cells. Accordingly, these data suggest that the new serine protease may be of importance for WSSV infection into hematopoietic cells., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

In 2014, northern Queensland crayfish from farms affected by particularly transportation and translocation stress, started to die with mortality reaching 20-40% after about three weeks and then mortalities subsided. Crayfish from 1 farm had 65% mortalities within 11 weeks. With histological examination of broodstock and juveniles, the muscle fibres were fractured with haemocytic infiltration reminiscent of viral infection or vitamin E/selenium deficiencies. Sequence dependent and independent PCRs failed to identify a viral aetiology. However, the whole transcriptomes of a case crayfish and an unaffected crayfish from a different population were assembled producing over 500,000 contigs. The complete sequence of a positive sense, single stranded RNA virus (+ve ssRNA virus; 9933bp) and the large and medium segments of a bunya-like virus were detected. Transcript back-mapping and newly developed PCRs indicated that the viruses were in the case crayfish but not the control crayfish. The +ve ssRNA virus is clearly in the order Picornavirales, marginally in the genus Iflavirus in a clade of Chinese and Northern American terrestrial arthropod viruses. The internal Picornavirales motifs; RNA-dependent RNA polymerase, helicase (P-loop) and 2 viral capsids genes were easily identified. This is the first iflavirus identified from crustacea and is named Chequa iflavirus. Whether these viruses are responsible for the stress-related mortalities is unproven but the Chequa virus' role seems limited as it appears it has been present in crayfish from at least the early 1990s; unless low-grade, chronic mortalities have been largely unnoticed., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Tolls or Toll-like receptors (TLRs) have an essential role in initiating innate immune responses against pathogens. In this study, a novel Toll gene, PcToll4, was first identified from the intestinal transcriptome of the freshwater crayfish, Procambarus clarkii. The PcToll4 cDNA is 4849bp long with a 3036bp open reading frame that encodes a 1011-amino acid protein. PcToll4 contains a signal peptide, 13 LRR domains, 3 LRR TYP domains, 2 LRR CT domains, an LRR NT domain, a transmembrane region, and a TIR domain. Quantitative RT-PCR analysis revealed that PcToll4 mRNA was detected in all tested tissues, and the expression of PcToll4 in the intestine was significantly upregulated after white spot syndrome virus (WSSV) challenge. Overexpression of PcToll4 in Drosophila Schneider 2 (S2) cells activates the antimicrobial peptides (AMPs) of Drosophila, including metchnikowin, drosomycin, attacin A, and shrimp Penaeidin-4. Results of RNA interference by siRNA also showed that PcToll4 regulates the expressions of 5 anti-lipopolysaccharide factors (ALFs) in the intestine of crayfish. Our findings suggest that PcToll4 is important for the innate immune responses of P. clarkii because this gene regulates the expressions of AMPs against WSSV., (Copyright © 2017. Published by Elsevier B.V.)