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This experiment was conducted to explore the effects of dietary vitamin C supplementation on non-specific immune defense, antioxidant capacity and resistance to low-temperature stress of juvenile mud crab (Scylla paramamosain). Mud crabs with an initial weight of 14.67 ± 0.13 g were randomly divided into 6 treatments and fed diets with 0.86 (control), 44.79, 98.45, 133.94, 186.36 and 364.28 mg/kg vitamin C, respectively. The experiment consisted of 6 treatments, each treatment was designed with 4 replicates and each replicate was stocked with 8 crabs. After 42 days of feeding experiment, 2 crabs were randomly selected from each replicate, and a total of 8 crabs in each treatment were carried out 72 h low-temperature challenge experiment. The results showed that crabs fed diets with 186.36 and 364.28 mg/kg vitamin C significantly improved the activities of alkaline phosphatase (AKP) and acid phosphatase (ACP) in hemolymph and hepatopancreas (P < 0.05). Crabs fed diet with 133.94 mg/kg vitamin C significantly decreased the concentration of nitric oxide (NO) and the activity of nitric oxide synthase (NOS) in hemolymph (P < 0.05). Diet with 133.94 mg/kg vitamin C was improved the activity of polyphenol oxidase (PPO) and the concentration of albumin (ALB) in hemolymph. Crabs fed diet with 133.94 mg/kg vitamin C showed lower concentration of malondialdehyde (MDA) in hemolymph and hepatopancreas than those fed the other diets. Meanwhile, crabs fed diet with 98.45 mg/kg vitamin C showed higher activity of total superoxide dismutase (T-SOD) in hemolymph, and crabs fed diet with 133.94 mg/kg vitamin C showed higher activity of T-SOD in hepatopancreas. Crabs fed diet with 186.36 mg/kg vitamin C significantly decreased the concentration of reduced glutathione (GSH) and the activity of glutathione peroxidase (GSH-PX) in hepatopancreas (P < 0.05). In normal temperature, crabs fed diets with 133.94 mg/kg vitamin C significantly up-regulated the expression levels of gpx (glutathione peroxidase) and trx (thioredoxin) in hepatopancreas compared with the control treatment (P < 0.05). The highest expression levels of relish, il16 (interleukin 16), caspase 2 (caspase 2), p38 mapk (p38 mitogen-activated protein kinases) and bax (bcl-2 associated x protein) in hepatopancreas were found at crabs fed control diet (P < 0.05). Moreover, crabs fed diet with 133.94 mg/kg vitamin C showed higher expression levels of alf-3 (anti-lipopolysaccharide factor 3) and bcl-2 (B-cell lymphoma 2) in hepatopancreas than those fed the other diets (P < 0.05). Under low-temperature stress, crabs fed diet with 133.94 mg/kg vitamin C significantly improved the expression levels of hsp90 (heat shock protein 90), cat (catalase), gpx, prx (thioredoxin peroxidase) and trx in hepatopancreas (P < 0.05). In addition, dietary with 133.94 vitamin C significantly up-regulated the expression levels of alf-3 and bcl-2 (P < 0.05). Based on two slope broken-line regression analysis of activity of PPO against the dietary vitamin C level, the optimal dietary vitamin C requirement was estimated to be 144.81 mg/kg for juvenile mud crab. In conclusion, dietary 133.94-144.81 mg/kg vitamin C significantly improved the non-specific immune defense, antioxidant capacity and resistance to low-temperature stress of juvenile mud crab., Competing Interests: Declaration of competing interest The authors confirm that they have no known financial conflicts of interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Ovarian development in animals is a complicated biological process, requiring the simultaneous coordination among various genes and pathways. To understand the dynamic changes and molecular regulatory mechanisms of ovarian development in mud crab (Scylla paramamosain), both histological observation and whole transcriptome sequencing of ovarian tissues at different mating stages were implemented in this study. The histological results revealed that ovarian development was delayed in unmated females (60 days after courtship behavior but not mating), who exhibited an oocyte diameter of 56.38 ± 15.17 μm. Conversely, mated females exhibited accelerated the ovarian maturation process, with females reaching ovarian stage III (proliferative stage) 23 days after mating and attained an average oocyte diameter of 132.19 ± 15.07 μm. Thus, mating process is essential in promoting the rapid ovarian development in mud crab. Based on the whole transcriptome sequencing analysis, a total of 518 mRNAs, 1502 lncRNAs, 18 circRNAs and 151 miRNAs were identified to be differentially expressed between ovarian tissues at different mating stages. Notably, six differentially expressed genes (DEGs) associated with ovarian development were identified, including ovary development-related protein, red pigment concentrating hormone receptor, G2/mitotic-specific cyclin-B3-like, lutropin-chorio gonadotropic hormone receptor, renin receptor, and SoxB2. More importantly, both DEGs and targets of differentially expressed non-coding RNAs (DEncRNAs) were enriched in renin-angiotensin system, TGF-β signaling, cell adhesion molecules, MAPK signaling pathway, and ECM-receptor interaction, suggesting that these pathways may play significant roles in the ovarian development of mud crabs. Moreover, competition endogenous RNA (ceRNA) networks were constructed while mRNAs were differentially expressed between mating stages were involved in Gene Ontology (GO) biological processes such as developmental process, reproduction, and growth. These findings could provide solid foundations for the future development of female mud crab maturation enhancement strategy, and improve the understanding of the ovarian maturation process in crustaceans., 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 Elsevier Inc. All rights reserved.)

As a prerequisite for the success of embryo development, embryonic genome activation (EGA) is an important biological event in which zygotic gene products in the embryo are activated to replace maternal-derived transcripts. Although EGA has been extensively studied in a large number of vertebrates and invertebrates, there is a lack of information regarding this event in crustacean crab. In this study, the timing of EGA was confirmed by examining a transcriptomic dataset of early embryonic development, including mature oocytes and embryos through six early developmental stages, and signaling pathways associated with EGA were identified in the mud crab, S. paramamosain. The comprehensive transcriptomic data identified a total of 53,915 transcripts from these sequencing samples. Notable transcriptomic change was evident at the 1-cell stage, indicated by a 36% transcript number shift and a reduction in transcript fragment length, compared to those present in the mature oocytes. Concurrently, a substantial increase in the expression of newly transcribed transcripts was observed, with gene counts reaching 3485 at the 1-cell stage, indicative of the onset of EGA. GO functional enrichment revealed key biological processes initiated at the 1-cell stage, such as protein complex formation, protein metabolism, and various biosynthetic processes. KEGG analysis identified several critical signaling pathways activated during EGA, including the "cell cycle," "spliceosome," "RNA degradation", and "RNA polymerase", pathways. Furthermore, transcription factor families, including zinc finger, T-box, Nrf1, and Tub were predominantly enriched at the 1-cell stage, suggesting their pivotal roles in regulating embryonic development through the targeting of specific DNA sequences during the EGA process. This groundbreaking study not only addresses a significant knowledge gap regarding the developmental biology of S. paramamosain, especially for the understanding of the mechanism underlying EGA, but also provides scientific data crucial for the research on the individual synchronization of seed breeding within S. paramamosain aquaculture. Additionally, it serves as a reference basis for the study of early embryonic development in other crustacean species., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Mud crab (Scylla paramamosain) has become an important mariculture crab along the southeast coast of China due to its strong adaptability, delicious taste, and rich nutrition. Several vertebrate steroid hormones and their synthesis-related genes and receptors have been found in crustaceans, but there are few reports on their synthesis process and mechanism. 3-beta-hydroxysteroid dehydrogenase (HSD3B) is a member of the Short-chain Dehydrogenase/Reductase (SDR) family, and an indispensable protein in vertebrates' steroid hormone synthesis pathway. In this study, the SpHsd3b gene sequence was obtained from the transcriptome data of S. paramamosain, and its full-length open reading frame (ORF) was cloned. The spatial and temporal expression pattern of SpHsd3b was performed by quantitative real-time PCR (qRT-PCR). SpHsd3b dsRNA interference (RNAi) and HSD3B inhibitor (trilostane) were used to analyze the function of SpHSD3B. The results showed that the SpHsd3b gene has an 1113 bp ORF encoding 370 amino acids with a 3β-HSD domain. SpHSD3B has lower homology with HSD3B of vertebrates and higher homology with HSD3B of crustaceans. SpHsd3b was expressed in all examined tissues in mature crabs, and its expression was significantly higher in the testes than in the ovaries. SpHsd3b expression level was highest in the middle stage of testicular development, while its expression was higher in the early and middle stages of ovarian development. RNAi experiment and trilostane injection results showed that SpHSD3B had regulatory effects on several genes related to gonadal development and steroid hormone synthesis. 15-day trilostane suppression could also inhibit ovarian development and progesterone level of hemolymph. According to the above results, crustaceans may have steroid hormone synthesis pathways like vertebrates, and the Hsd3b gene may be involved in the gonadal development of crabs. This study provides further insight into the function of genes involved in steroid hormone synthesis in crustaceans., Competing Interests: Declaration of Competing Interest The authors declare that there is no conflict of competing interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Laminin receptor (LR), which mediating cell adhesion to the extracellular matrix, plays a crucial role in cell signaling and regulatory functions. In the present study, a laminin receptor gene (SpLR) was cloned and characterized from the mud crab (Scylla paramamosain). The full length of SpLR contained an open reading frame (ORF) of 960 bp encoding 319 amino acids, a 5' untranslated region (UTR) of 66 bp and a 3' UTR of 49 bp. The predicted protein comprised two Ribosomal-S2 domains and a 40S-SA-C domain. The mRNA of SpLR was highly expressed in the gill, followed by the hepatopancreas. The expression of SpLR was up-regulated after mud crab dicistrovirus-1(MCDV-1) infection. Knocking down SpLR in vivo by RNA interference significantly down-regulated the expression of the immune genes SpJAK, SpSTAT, SpToll1, SpALF1 and SpALF5. This study shown that the expression level of SpToll1 and SpCAM in SpLR-interfered group significantly increased after MCDV-1 infection. Moreover, silencing of SpLR in vivo decreased the MCDV-1 replication and increased the survival rate of mud crabs after MCDV-1 infection. These findings collectively suggest a pivotal role for SpLR in the mud crab's response to MCDV-1 infection. By influencing the expression of critical innate immune factors and impacting viral replication dynamics, SpLR emerges as a key player in the intricate host-pathogen interaction, providing valuable insights into the molecular mechanisms underlying MCDV-1 pathogenesis in mud crabs., Competing Interests: Declaration of competing interest The authors declared that they have no conflicts of interest to this work. We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

As a crucial member of pattern-recognition receptors (PRRs), the Tolls/Toll-like receptors (TLRs) gene family has been proven to be involved in innate immunity in crustaceans. In this study, nine members of TLR gene family were identified from the mud crab (Scylla paramamosain) transcriptome, and the structure and phylogeny of different SpTLRs were analyzed. It was found that different SpTLRs possessed three conserved structures in the TIR domain. Meanwhile, the expression patterns of different Sptlr genes in examined tissues detected by qRT-PCR had wide differences. Compared with other Sptlr genes, Sptlr-6 gene was significantly highly expressed in the hepatopancreas and less expressed in other tissues. Therefore, the function of Sptlr-6 was further investigated. The expression of the Sptlr-6 gene was up-regulated by Poly I: C, PGN stimulation and Vibrio parahaemolyticus infection. In addition, the silencing of Sptlr-6 in hepatopancreas mediated by RNAi technology resulted in the significant decrease of several conserved genes involved in innate immunity in mud crab after V. parahaemolyticus infection, including relish, myd88, dorsal, anti-lipopolysaccharide factor (ALF), anti-lipopolysaccharide factor 2 (ALF-2) and glycine-rich antimicrobial peptide (glyamp). This study provided new knowledge for the role of the Sptlr-6 gene in defense against V. parahaemolyticus infection in S. paramamosain., Competing Interests: Declaration of competing interest We declare that we have no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Aiming to assess the effects of lard oil (LO) and fish oil (FO) on the nutritional value of mud crabs (Scylla paramamosain), non-targeted lipidomics analysis was performed on the muscle of crabs after eight weeks of feeding trail. Compared to FO, dietary LO reduced the content of phosphatidylethanolamine (PE) and phosphatidylserine (PS) with 18:0 bound at sn-1/3 site, the content of ether phospholipids containing 20:5n-3 (EPA) and 22:6n-3 (DHA) combined at sn-2 site, and increased the content of ether PE containing 18:0 and 18:1n-9. Furthermore, the deposition of 16:0, 16:1n-7, 18:2n-6, 18:3n-3, 20:4n-6, EPA and DHA at each site of PE, PS, phosphatidylcholine and/or triacylglycerols were reduced by dietary LO, while the DHA content at the sn-2 position of PE was increased. In conclusion, the nutritional value of mud crabs was reduced by dietary LO with the manifestation of variation in FA composition and positional distribution on phospholipids., 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 © 2023. Published by Elsevier Ltd.)

Oviposition is induced upon mating in most insects. Spawning is a physiological process that is fundamental for the reproduction of Scylla paramamosain. However, the molecular mechanisms underlying the spawning process in this species are poorly understood. Herein, comprehensive ovary transcriptomic analysis was conducted at the germinal vesicle breakdown stage (GVBD), spawning stage, 0.5 h post-spawning stage, and 24 h post-spawning stage of S. paramamosain for gene discovery. A total of 67,230 unigenes were generated, and 27,975 (41.61%) unigenes were annotated. Meanwhile, the differentially expressed genes (DEGs) between the different groups were identified, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was subsequently conducted. These results suggested that octopamine (OA) and tyramine (TA) could induce oviposition, while dopamine (DA) and serotonin (5-hydroxytryptamine [5-HT]) inhibit oviposition. The 20-hydroxyecdysone (20E) and methyl farnesoate (MF) signal pathways might be positively associated with oviposition. Furthermore, numerous transcripts that encode neuropeptides and their G-protein-coupled receptors (GPCRs), such as CNMamide, RYamide, ecdysis-triggering hormone (ETH), GPA2/GPB5 receptor, and Moody receptor, appear to be differentially expressed during the spawning process. Eleven unigenes were selected for qRT-PCR and the pattern was found to be consistent with the transcriptome expression pattern. Our work is the first spawning-related investigation of S. paramamosain focusing on the ovary at the whole transcriptome level. These findings assist in improving our understanding of spawning regulation in S. paramamosain and provide information for oviposition studies in other crustaceans., (© 2024 Japanese Society of Developmental Biologists.)

Hypoxia-inducible factors -1 (HIF-1) is a crucial transcription factor that regulates the expression of glycolytic genes. Our previous study proved that the Mud crab dicistrovirus-1 (MCDV-1) can induce aerobic glycolysis that favors viral replication in mud crab Scylla paramamosain. However, the role of HIF-1 on key glycolytic genes during the MCDV-1 infection has not been examined. In this study, the intricate interplay between HIF-1 and the key glycolysis enzyme, lactate dehydrogenase (LDH), was investigated after MCDV-1 infection. The expression of LDH was significant increased after MCDV-1 infection. Additionally, the expression of HIF-1α was upregulated following MCDV-1 infection, potentially attributed to the downregulation of prolyl hydroxylase domains 2 expression. Subsequent examination of the SpLDH promoter identified the presence of hypoxia response elements (HREs), serving as binding sites for HIF-1α. Intriguingly, experimental evidence demonstrated that SpHIF-1α actively promotes SpLDH transcription through these HREs. To further elucidate the functional significance of SpHIF-1α, targeted silencing was employed, resulting in a substantial reduction in SpLDH expression, activity, and lactate concentrations in MCDV-1-infected mud crabs. Notably, SpHIF-1α-silenced mud crabs exhibited higher survival rates and lower viral loads in hepatopancreas tissues following MCDV-1 infection. These results highlight the critical role of SpHIF-1α in MCDV-1 pathogenesis by regulating LDH gene dynamics, providing valuable insights into the molecular mechanisms underlying the virus-host interaction., Competing Interests: Declaration of competing interest The authors declared that they have no conflicts of interest in this work. We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Light is an essential ecological factor that has been demonstrated to affect aquatic animals' behavior, growth performance, and energy metabolism. Our previous study found that the full-spectrum light and cyan light could promote growth performance and molting frequency of Scylla paramamosain while it was suppressed by violet light. Hence, the purpose of this study is to investigate the underlying molecular mechanism that influences light spectral composition on the growth performance and molting of S. paramamosain. RNA-seq analysis and qPCR were employed to assess the differentially expressed genes (DEGs) of eyestalks from S. paramamosain reared under full-spectrum light (FL), violet light (VL), and cyan light (CL) conditions after 8 weeks trial. The results showed that there are 5024 DEGs in FL vs. VL, 3398 DEGs in FL vs. CL, and 3559 DEGs in VL vs. CL observed. GO analysis showed that the DEGs enriched in the molecular function category involved in chitin binding, structural molecular activity, and structural constituent of cuticle. In addition, the DEGs in FL vs. VL were mainly enriched in the ribosome, amino sugar and nucleotide sugar metabolism, lysosome, apoptosis, and antigen processing and presentation pathways by KEGG pathway analysis. Similarly, ribosome, lysosome, and antigen processing and presentation pathways were major terms that enriched in FL vs. CL group. However, only the ribosome pathway was significantly enriched in up-regulated DEGs in VL vs. CL group. Furthermore, five genes were randomly selected from DEGs for qPCR analysis to validate the RNA-seq data, and the result showed that there was high consistency between the RNA-seq and qPCR. Taken together, violet light exposure may affect the growth performance of S. paramamosain by reducing the ability of immunity and protein biosynthesis, and chitin metabolism., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The structure and function of the water microbial community can change dramatically between different rearing modes. Yet investigations into the relationships between microbial community and water quality remain obscure. We provide the first evidence that rearing modes alter bacterial community and water quality in the rearing water of the mud crab (Scylla paramamosain) larvae. The juveniles in the recirculating aquaculture system (RAS) had a higher viability than those in the water exchange system (WES). RAS had the significantly lower levels of total ammonia nitrogen (TAN), NH 3 , NO 2 - -N, total nitrogen (TN), total dissolved solids (TDS), and chemical oxygen demand than those of WES. The number of significantly different amplicon sequence variants between rearing modes increased as the larvae developed. NH 3 , TAN, TDS, NO 2 - -N, and TN were closely related to the late alterations in water bacterial community. Both the FAPROTAX tool and quantitative PCR analysis showed enhanced nitrogen cycling functional potential of water bacterial community of RAS. Random forest analysis identified the enriched water bacteria especially heterotrophic bacteria such as Phaeodactylibacter, Tenacibaculum, and Hydrogenophaga, which were vital in removing nitrogenous compounds via simultaneous nitrification and denitrification. Notably, RAS could save 18.5 m 3 of seawater relative to WES in larviculture on the scale of 2.5 m 3 . Together, these data indicate that RAS could function as microbial community and water quality management strategy in the larviculture of crab., 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 Elsevier Ltd. All rights reserved.)

The mud crab (Scylla paramamosain) possesses extensive regenerative abilities, enabling it to replace missing body parts, including claws, legs, and even eyes. Studying the genetic and molecular mechanisms underlying regenerative ability in diverse animal phyla has the potential to provide new insights into regenerative medicine in humans. In the present study, we performed mRNA sequencing to reveal the genetic mechanisms underlying the claw regeneration in mud crab. Several differentially expressed genes (DEGs) were expressed in biological pathways associated with cuticle synthase, collagen synthase, tissue regeneration, blastema formation, wound healing, cell cycle, cell division, and cell migration. The top GO enrichment terms were microtubule-based process, collagen trimer, cell cycle process, and extracellular matrix structural constituent. The most enriched KEGG pathways were ECM-receptor interaction and focal adhesion. The genes encoding key functional proteins, such as collagen alpha, cuticle protein, early cuticle protein, arthrodial cuticle protein, dentin sialophosphoprotein (DSPP), epidermal growth factor receptor (EGFR), kinesin family member C1 (KIFC1), and DNA replication licensing factor mcm2-like (MCM2) were the most significant and important DEGs suspected to participate in claw regeneration. The findings of this research offer a comprehensive and insightful understanding of the genetic and molecular mechanisms underlying claw regeneration in S. paramamosain. By elucidating the specific genes and molecular pathways implicated in this process, our study contributes significantly to the broader field of regenerative biology and offers potential avenues for further exploration in crustacean limb regeneration., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 Elsevier Inc. All rights reserved.)

In the present study, BGISEQ-500 RNA-Seq technology was adopted to investigate how Scylla paramamosain adapts to salinity tolerance at the molecular level and explores changes in gene expression linked to salinity adaptation following exposure to both low salinity (5 ‰) and standard salinity (23 ‰) conditions. A total of 1100 and 520 differentially expressed genes (DEGs) were identified in the anterior and posterior gills, respectively, and their corresponding expression patterns were visualized in volcano plots and a heatmap. Further analysis highlighted significant enrichment of well-established gene functional categories and signaling pathways, including those what associated with cellular stress response, ion transport, energy metabolism, amino acid metabolism, H 2 O transport, and physiological stress compensation. We also selected key DEGs within the anterior and posterior gills that encode pivotal stress adaptation and tolerance modulators, including AQP, ABCA1, HSP 10, A35, CAg, NKA, VPA, CAc, and SPS. Interestingly, A35 in the gills might regulate osmolality by binding CHH in response to low salinity stress or serve as a mechanism for energy compensation. Taken together, our findings elucidated the intricate molecular mechanism employed by S. paramamosain for salinity adaptation, which involved distinct gene expression patterns in the anterior and posterior gills. These findings provide the foothold for subsequent investigations into salinity-responsive candidate genes and contribute to a deeper understanding of S. paramamosain's adaptation mechanisms in low-salinity surroundings, which is crucial for the development of low-salinity species cultivation and the establishment of a robust culture model., 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 © 2023 Elsevier Inc. All rights reserved.)

Sulfamethoxazole (SMZ) is commonly used in aquaculture to treat bacterial infections, but its long-term residual properties in natural water can pose a direct threat to aquatic animals. This study is to investigate the effects of continuous exposure to SMZ on mud crabs (Scylla paramamosain) at four different concentrations (0, 10, 100, and 1000 ng/L) that reflect the range found in natural aquatic environments. The results confirmed that SMZ exposure reduced the expression levels of genes related to the innate immunity in mud crabs, including JAK, Astakine, TLR, and Crustin. It also stimulated oxidative stress, caused the production of reactive oxygen species and lower activities of antioxidant enzymes such as peroxidase, superoxide dismutase, catalase, and glutathione. SMZ exposure damaged the DNA of crab hemocytes and hepatopancreas tissue, and reduced the phagocytosis, ultimately leading to a decreased survival rates of mud crabs infected with Vibrio alginolyticus. These findings demonstrate that SMZ exposure has immunotoxic effects on mud crabs' innate immunity and reduces the ability to resist pathogen infections., Competing Interests: Declaration of competing interest The authors declared that they have no conflicts of interest to this work. We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

In this study, we examined the impact of geniposide on the innate immunity of the mud crab Scylla paramamosain, specifically in relation to WSSV infection. Through the use of in vitro cell culture experiments, we assessed the effects of geniposide on various parameters of hemocyte activity in S. paramamosain. Our findings revealed that high doses of geniposide inhibited hemocyte growth, with an optimal dose of 100 mg/kg determined. Additionally, we observed that geniposide increased the total hemocyte counts in S. paramamosain following WSSV infection. Geniposide also enhanced the enzymatic activities in hemolymph following treatment. The enzymes affected by geniposide encompassed ACP (acid phosphatase), POD (phenol oxidase catalase), PO (phenoloxidase), SOD (superoxide dismutase), CAT (catalase), and LZM (lysozyme). Furthermore, the activities of ACP, POD, PO, and LZM were also observed to increase subsequent to infection with WSSV. Notably, geniposide was found to enhance the phagocytosis of V. alginolyticus within the hemocytes. Geniposide can reduce hemocyte apoptosis rates after treatment, as well as hemocytes infected with WSSV. Furthermore, geniposide treatment significantly up-regulated the expression level of Myosin, but expression levels of Astakine, C-type lectin (CTL), STAT, JAK, proPO, minichromosome maintenance protein (MCM7), caspase-3 and crustin were down-regulated in the hemocytes. Additionally, geniposide treatment inhibited WSSV replication in hemocytes of S. paramamosain, and enhanced the survival rates of mud crabs following WSSV infection. These experimental results provide evidence that geniposide can improve the immune response by regulating humoral immunity and cellular immunity, and enhance pathogen resistance in S. paramamosain., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Variability in food availability leads to condition-dependent investments in reproduction. This study is aimed at understanding the metabolic response and regulatory mechanism of female Scylla paramamosain in response to starvation in a temporal- and tissue-specific manner. The mud crabs were starved for 7 (control), 14, 28, and 40 days for histological and biochemical analysis in the hepatopancreas, ovary, and serum, as well as for RNA sequencing on the hepatopancreas and ovary. We further highlighted candidate gene modules highly linked to physiological traits. Collectively, our observations suggested that starvation triggered endogenous ovarian maturation at the expense of hepatopancreas mass, with both metabolic adjustments to optimize energy and fatty acid supply from hepatopancreas to ovary in the early phase, followed by the activation of autophagy-related pathways in both organs over prolonged starvation. These specific adaptive responses might be considered efficient strategies to stimulate ovarian maturation of Scylla paramamosain under fasting stress, which improves the nutritional value of female mud crabs and other economically important crustaceans.

Scylla paramamosain is an important cultured crab species on the southeast coast of China. However, the molecular regulation mechanism of its gonadal development still has not been thoroughly studied. Dsx (doublesex) and foxl-2 (forkhead transcription factor gene 2) are important transcription factors involved in gonadal development. So far, studies on the functions of dsx and foxl-2 in crustaceans are very limited. Insulin-like androgenic gland hormone (IAG) is an effector molecule that regulates the differentiation, development and sex maintenance of testes in crustaceans. In this study, the promoter region of Sp-IAG was predicted, and several potential binding sites of dsx and foxl-2 were found. Site-directed mutagenesis was performed on the predicted potential binding sites, and their promoter activity was analyzed. The results showed that there was a dsx and a foxl-2 binding site, respectively, that could regulate the expression of Sp-IAG. In order to verify the regulatory effect of these two transcription factors on Sp-IAG, we constructed the expression plasmids of dsx and foxl-2 and co-transfected them into HEK293T cell lines with the promoter of Sp-IAG, respectively. The results showed that dsx could significantly promote the expression of Sp-IAG, while foxl-2 could inhibit its expression substantially. Then we carried out in vivo RNA interference experiment on mud crabs. The expression of dsx and foxl-2 in crabs was interfered respectively. The results of qRT-PCR showed that the expression of Sp-IAG was significantly inhibited after interfering with dsx, while significantly increased after interfering with foxl-2, which was consistent with the cell experiment. In conclusion, dsx and foxl-2 transcription factors play opposite roles in regulating the expression of Sp-IAG., 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 © 2023 Elsevier Inc. All rights reserved.)

Phenanthrene (PHE), a representative polycyclic aromatic hydrocarbons (PAHs), is widely found in aquatic environments, which exhibits high toxicity to aquatic organisms and has a substantial impact on overall health. In order to investigate the immunosuppressive effects of PHE exposure on marine crustacean species, the Scylla paramamosain was exposure to different concentrations of PHE, which was 0 μg/L (control group), 0.7 μg/L, 7 μg/L, or 70 μg/L PHE with 35 individuals in every group, respectively. The results showed that the color of hemocytes gradually deepened with increasing PHE concentration, and the total hemocyte count (THC) was activated and increased after PHE exposure analyzed by Flow cytometry. Meanwhile, compared with the control group, cryostat sections of hepatopancreas showed cell infiltration, cell steatosis, eosinophilic masses and vacuolization in PHE groups. The superoxide dismutase (SOD) activity was decreased immensely in PHE exposure groups, meanwhile, the acid phosphatase (ACP) activity and glutathione (GSH) activities were increased after PHE exposure compared with control group. Moreover, the expression profile of Crustin, TLR, MCM7, JAK, caspase, Moyosin and P53 were up-regulated significantly after 7th day PHE exposure in all treatment groups by Q-PCR. Those data illustrated that PHE exposure could inhibit the immune function of mud crab by causing hepatopancreas damage, induce the activity of antioxidant enzymes and the expression of immune genes. These data provide a scientific basis for evaluating the impact of PAH pollution on marine organisms., Competing Interests: Declaration of competing interest The authors declare that they don't have any competing financial interests or personal relationships that might have influenced this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Benzo[α]pyrene (BaP), a polycyclic aromatic hydrocarbon, is present in the aquatic environment and may be harmful to aquatic animals. We exposed the mud crab Scylla paramamosain to BaP for 7 days, the of superoxide dismutase (SOD), catalase (CAT), phenoloxidase (PO), lysozyme (LZM), glutathione (GSH), glutathione-S-transferase (GST), and acid phosphatase (ACP) activities in the hemolymph of mud crab were reduced. Additionally, the reactive oxygen species content was increased in mud crabs after exposed to BaP. When BaP concentration was increased, the total hemocyte count (THC), the survival rate of hemocytes and their proliferation were decreased. Histopathology analysis revealed damaged hepatopancreas cells, which indicating that BaP exposure is cytotoxic to crab hemocytes. However, the degree of DNA damage did not worsen with increasing BaP concentration. The expression levels of p53, MCM7, Caspase-3, and Myosin were changed with increasing concentration of BaP, which indicated that BaP exposure may affect apoptosis and phagocytosis in mud crabs. As BaP concentration was increased, the apoptosis rate of hemocytes was increased and the phagocytosis was decreased. These results confirmed that BaP exposure inhibited the innate immune response of mud crabs. A possible explanation for this effect is that BaP reduces the antioxidant enzyme activity and increases the reactive oxygen species content in mud crabs, thereby oxidizing and damaging hemocytes, which stimulates phagocytosis and apoptosis and negatively affects the innate immunity of S. paramamosain., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Smad，a member of the TGF-β superfamily，controls cell proliferation，growth and guiding cell differentiation, thus playing a crucial role in diseases. However, the presence as well as specific function of Smad in crabs is still unknown. In this study, two Smads (Smad1 and Smad2/3) were identified for the first time from the mud crab Scylla paramamosain. The complete open reading frames of SpSmad1 and SpSmad2/3 were 1,497bp and 1,338bp, encoding deduced proteins of 498 and 445 amino acids respectively. Moreover, under the administration of Vibrio alginolyticus and WSSV, the relative expression levels of SpSmad1 and SpSmad2/3 were significantly increased, indicating their involvement in the innate immune response of mud crabs. Knockdown of SpSmad1 and SpSmad2/3 in vivo not only led to the increasement of the expressions of NF-κB signaling genes and antimicrobial peptides genes, but also significantly affected the bacterial clearance process of mud crabs. Additionally, overexpression of SpSmad1 and SpSmad2/3 in HEK293T cells could markedly activate NF-κB signaling. These results indicated that Smad1 and Smad2/3 participated in the innate immunity of Scylla paramamosain, and might provide a better understanding of the presence and immune regulatory functions of Smad1 and Smad2/3 in crabs and even invertebrates., (Copyright © 2023. Published by Elsevier Ltd.)

Benzophenones (BPs) are commonly used in personal care products like sunscreens and are increasingly being released into the environment, raising concerns about their potential ecotoxic effects. BPs as emerging environmental contaminants, little is known about their toxic effects on estuarine organisms. This study firstly investigated the toxic effects of five commonly used BPs on mud crabs (Scylla paramamosain). The crabs were exposed to varying concentrations of BPs for 14 days. The results showed that BPs caused damage to antioxidant systems in crabs. Transcriptome sequencing revealed that BP-3 and BP-1 had a greater impact on the crabs compared to the other BPs. Specifically, BP-1 and BP-3 caused severe damage to organelles and ribosomes. BP affected catalytic activity and hydrolase activity, BP-2 affected phosphoenolpyruate carboxykinase activity, and BP-4 affected tRNA aminoacylation and hydrolase activity. These findings can enhance our understanding of the ecotoxicity of BPs and may help to protect estuarine ecosystems., 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 © 2023. Published by Elsevier Ltd.)

Pattern recognition receptors (PRRs) play a crucial role in the recognition and activation of innate immune responses against invading microorganisms. This study characterizes a novel C-type lectin (CTL), SpccCTL. The cDNA sequence of SpccCTL has a full length of 1744 bp encoding a 338-amino acid protein. The predicted protein contains a signal peptide, a coiled-coil (CC) domain, and a CLECT domain. It shares more than 50 % similarity with a few CTLs with a CC domain in crustaceans. SpccCTL is highly expressed in gills and hemocytes and upregulated after MCRV challenge, suggesting that it may be involved in antiviral immunity. Recombinant SpccCTL (rSpccCTL) as well as two capsid proteins of MCRV (VP11 and VP12) were prepared. Pre-incubating MCRV virions with rSpccCTL significantly suppresses the proliferation of MCRV in mud crabs, compared with the control (treatment with GST protein), and the survival rate of mud crabs is also significantly decreased. Knockdown of SpccCTL significantly facilitates the proliferation of MCRV in mud crabs. These results reveal that SpccCTL plays an important role in antiviral immune response. GST pull-down assay result shows that rSpccCTL interacts specifically with VP11, but not to VP12. This result is further confirmed by a Co-IP assay. In addition, we found that silencing SpccCTL significantly inhibits the expression of four antimicrobial peptides (AMPs). Considering that these AMPs are members of anti-lipopolysaccharide factor family with potential antiviral activity, they are likely involved in immune defense against MCRV. Taken together, these findings clearly demonstrate that SpccCTL can recognize MCRV by binding viral capsid protein VP11 and regulate the expression of certain AMPs, suggesting that SpccCTL may function as a potential PRR playing an essential role in anti-MCRV immunity of mud crab. This study provides new insights into the antiviral immunity of crustaceans and the multifunctional characteristics of CTLs., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Lysine crotonylation (Kcr) is a newly discovered type of post-translational modification. Although Kcr has been reported in several species, its role in crustaceans remains largely unknown. In this study, Kcr in hemocytes of mud crab (Scylla paramamosain) was characterized using pan anti-crotonyllysine antibody enrichment and high-resolution liquid chromatogram-mass spectrometry analysis after SpTRAF6 or SpEcsit silencing. Altogether, 1,800 Kcr sites with six conserved motifs were identified from 512 proteins. Subcellular localization analysis showed that the identified Kcr proteins were mainly localized to the cytoplasm, nucleus, and mitochondria. The cellular components analysis showed that the 'chromosomal region' was enriched in the hemocytes of SpTRAF6-or SpEcsit-silenced mud crabs. The KEGG and PPI analyses showed that the identified Kcr proteins in the hemocytes SpTRAF6-or SpEcsit-silenced mud crabs were related to the 'protein processing in endoplasmic reticulum'; of which the marker of endoplasmic reticulum stress (Bip) was identified to be crotonylated. These datasets present the first comprehensive analysis of the crotonylome in mud crab hemocytes, providing invaluable insights into the regulatory functions of SpTRAF6 and SpEcsit in Kcr. Additionally, our findings shed light on the potential role of these proteins in activating marker proteins during endoplasmic reticulum stress in invertebrates., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Potassium channel modulatory factor 1 (KCMF1), an E3 ubiquitin ligase, plays a vital role in renal tubulogenesis, preeclampsia, and tumor development in mammals. Nevertheless, the function of KCMF1 in invertebrates remains to be investigated. Here, we identified KCMF1-like from Scylla paramamosian, encoding 242 amino acids with two zinc finger domains at the N-terminal. Real-time quantitative PCR analysis revealed that KCMF1-like was expressed in all tested tissues, including hemocytes, brain, mid-intestine, subcuticular epidermis, gills, muscle, heart, and stomach, with higher levels in muscle and mid-intestine. KCMF1-like was up-regulated in the hemocytes of mud crabs challenged with white spot syndrome virus (WSSV). RNA interference (RNAi) was performed to investigate the impact of KCMF1-like on the proliferation of WSSV in mud crabs. Knock-down of KCMF1-like resulted in an increase of the WSSV copy number and an impairment of the hemocytes apoptosis rate in vivo. In addition, KCMF1-like could also affect the mitochondrial membrane potential. Collectively, these results revealed that KCMF1-like might play a crucial role in the defense against virus infection in mud crab. This study contributes a novel insight into the role of KCMF1-like in the antiviral immune defense mechanism in crustaceans., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

In this study, the best domestication time for mud crabs ( Scylla paramamosain ) that were domesticated in the same region was explored. MC crabs (Mang Cai City, Vietnam), TS crabs (Taishan City, Guangdong Province, China), and NB crabs (Ningbo City, Zhejiang Province, China) were used as experiment groups, which were all domesticated in the Sanmen area for 28 days. Then, free amino acids and flavor nucleotides in muscles and hepatopancreas were determined. In MC and NB crabs, flavor amino acids in hepatopancreas and equivalent umami concentration (EUC) of flavor nucleotides in the hepatopancreas and muscles were lower than those of CK1 and CK3 crabs before domestication (0 day). There was no significant difference between MC and CK1 and between NB and CK3 crabs after 28 days of domestication. The content of umami amino acids, the total content of flavor nucleotides, and EUC in the hepatopancreas in TS crabs were lower than those in CK2 crabs and were still lower than those in CK2 crabs after 28 days of domestication. Therefore, it takes at least a month to domesticate mud crabs. The theoretical basis and technical reference for the directional cultivation of the flavor quality of mud crabs were provided in this study..

Reactive oxygen species (ROS) are typically regarded as being generated by the cellular respiratory chain or by cells under pathological damage, which play a crucial role as signaling molecules in promoting hemocytes circulation and normal cellular physiological functions. In this study, the antioxidant N-acetylcysteine (NAC) was used to reduce ROS in vivo and in vitro, which to analyze the effect of ROS on innate immunity and viral infection of mud crab. The total hemocyte count (THC), phenoloxidase (PO), superoxide dismutase (SOD) activity, immune-relative genes were analyzed, respectively. Moreover, the effect of ROS on WSSV infection was analyzed by THC and hemocytes apoptosis. The data showed that NAC could effectively remove and inhibit intracellular ROS. The THC of NAC group was reduced at 12 h and 24 h compared with that of control. And the inhibition of ROS by NAC could increase the SOD activity with control group, while increased the PO activity caused by early WSSV infection. And NAC could up-regulate the expression of MCM7, JAK, TLR and proPO significantly, while down-regulate the expression of Astakine, proPO, caspase and p53. Similarly, NAC could inhibit WSSV-induced apoptosis of S. paramamosain hemocytes. The data illustrated that ROS participates in the interaction between hemocytes and virus infection by regulating innate immunity. Especially, after NAC inhibited ROS, the expression of hemocytes proliferation gene Astakine was also inhibited, which may indicate that ROS is related to the process of hemocytes proliferation. The data will show a preliminary exploration on the regulatory role of ROS in crustacean immune system., Competing Interests: Declaration of competing interest The authors declare that they don't have any competing financial interests or personal relationships that might have influenced this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Heat shock proteins play an important role in host defense, and modulate immune responses against pathogen infection. In this study, a novel HSC70 from the mud crab (designated as SpHSC70) was cloned and characterized. The full length of SpHSC70 contained a 58 bp 5'untranslated region (UTR), an open reading frame (ORF) of 2,046 bp and a 3'UTR of 341 bp. The SpHSC70 protein included the conserved DnaK motif. The mRNA of SpHSC70 was highly expressed in the hemocytes, heart and hepatopancreas, and lowly expressed in the intestine. The subcellular localization results indicated that SpHSC70 was localized in both the cytoplasm and the nucleus. Moreover, SpHSC70 was significantly responsive to bacterial challenge. RNA interference experiment was designed to investigate the roles of SpHSC70 in response to bacterial challenge. V. parahaemolyticus infection induced the expression levels of SpPO, SpHSP70, SpSOD and SpCAT. Knocking down SpHSC70 in vivo can decrease the expression of these genes after V. parahaemolyticus infection. These results suggested that SpHSC70 could play a vital role in defense against V. parahaemolyticus infection via activating the immune response and antioxidant defense signaling pathways in the mud crab., Competing Interests: Declaration of competing interest The authors declared that they have no conflicts of interest to this work. We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Mitogen-activated protein kinase kinase 4 (MKK4), serves as a critical component of the mitogen-activated protein kinase signaling pathway, facilitating the direct phosphorylation and activation of the c-Jun N-terminal kinase (JNK) and p38 families of MAP kinases in response to environmental stresses. In the current research, we identified two MKK4 subtypes, namely SpMKK4-1 and SpMKK4-2, from Scylla paramamosain, followed by the analysis of their molecular characteristics and tissue distributions. The expression of SpMKK4s was induced upon WSSV and Vibrio alginolyticus challenges, and the bacteria clearance capacity and antimicrobial peptide (AMP) genes' expression upon bacterial infection were significantly decreased after knocking down SpMKK4s. Additionally, the overexpression of both SpMKK4s remarkably activated NF-κB reporter plasmid in HEK293T cells, suggesting the activation of the NF-κB signaling pathway. These results indicated the participation of SpMKK4s in the innate immunity of crabs, which shed light on a better understanding of the mechanisms through which MKK4s regulate innate immunity., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

A member of the immunoglobulin superfamily designated leucine-rich repeats and immunoglobulin-like domains protein-1 (lrig-1) encoding a protein with 1109 amino acids with a characteristic IGc2 domain was identified from the transcriptome data of mud crab Scylla paramamosain. Lrig-1 contained: one signaling peptide; one LRR&#95;NT domain; nine LRR domains; three LRR&#95;TYP domains; one LRR&#95;CT domain; three IGc2 regions; one transmembrane region; C-terminal cytoplasmic tail. lrig-1 was widely expressed in all tissues of mud crab and was responsive in hemocytes to first and second Vibrio parahaemolyticus infections. lrig-1 knockdown mediated by RNAi repressed expression of several antimicrobial peptides significantly. Its orthologs in 19 crustacean species were identified and showed high conservation. These results suggest that lrig-1 have a vital role in mud crabs against V. parahaemolyticus infection through expression of multiple antimicrobial peptides. The results obtained in the present study imply the potential roles the lrig-1 played in immune priming in crabs., Competing Interests: Declaration of competing interest We declare that we have no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Yellow oil mud crab (YOC) is a new variant of mud crab (Scylla paramamosain), which was attracted much attention in recent years due to its high level of nutrition. However, the nutritive values and the physiological changes in YOC have not been clearly understood. In this study, we aimed to identify the nutrient compositions (including total carotenoid content (TCC), total lipid content (TLC), total antioxidant capacity (TAC), and fatty acids) and differences in genes related to the biosynthesis of fatty acids using transcriptome analysis in YOC in comparison with those of normal mud crabs. As a result, observations on the morphological characteristics showed that the YOC exhibits a difference in the color of the muscle, gills (orange-yellow), and hemolymph (yellow) compared with the normal female crabs (NFC) (blue or nattier blue). The TCC and TLC (84.96 ± 9.65 μg/g in muscle and 1.39 ± 0.10 μg/mL in hemolymph) or TAC (1.52 ± 0.17 mM in hemolymph) of YOC were higher than that of NFC and normal male crab (NMC). YOC had lower saturated fatty acids, but higher unsaturated fatty acids, as well as the ratio of n-3/n-6 of fatty acids in muscle and hemolymph, compared with those of NFC and NMC. Furthermore, the transcriptome profile revealed that the unigenes in YOC were enriched in the synthesis of n-3 fatty acids. Furthermore, more unigenes related to 'Biosynthesis of unsaturated fatty acids' were identified in muscle and hemocytes, while fewer were in the gonads of YOC. Additionally, the positive (in muscle and hemocytes) and a negative correlation (in gonads) between expressions of unigenes and contents of TLC, TCC, and UFA were found, indicating a better synthesis ability of fatty acids in the muscle and hemocytes of YOC. Overall, compared to NFC and NMC, YOC has higher nutrients and is a better food nutrient source for humans., 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 © 2023. Published by Elsevier Ltd.)

During pond culture or intensive culture system of crabs (mainly Eriocheir sinensis, Portunus trituberculatus and Scylla paramamosain), high-density farming has typically contributed to a higher limb autotomy level in juvenile animals, especially in S. paramamosain which has a high level of cannibalism. Due to the high limb autotomy level, the survival and growth rates in S. paramamosain farming are restricted, which limit the growth of the mud crab farming industry. MicroRNAs (miRNAs) are small noncoding RNAs that regulate a series of biological processes including innate immune responses by post-transcriptional suppression of their target genes. MiRNAs are believed to be crucial for innate immune process of host wound healing. Many miRNAs have been verified to be required in host immune responses to repair wound and to defense pathogen after tissue damage. However, to our best knowledge, the miRNAs functions of crustacean innate immune reactions against injury induced by limb autotomy have not been studied yet. Here in this study, for the first time, miRNAs involved in the S. paramamosain immune reactions against injury induced by cheliped autotomy were obtained by high-throughput sequencing. A total of 575 miRNAs (518 known miRNAs and 57 novel predicted miRNAs) were obtained, of which 141 differentially expressed microRNAs (93 up-regulated microRNAs and 48 down-regulated microRNAs) were revealed to be modified against cheliped autotomy, and the qPCR results of randomly selected miRNAs confirmed the expression patterns in the miRNAs sequencing data. Numerous immune-related target genes associated with innate immune system were mediated by miRNAs to induce host humoral immune and cellular immune defense to minimize acute physical damage. Furthermore, the genes expression in hemolymph coagulation and melanization pathways, as well as Toll and Imd signaling pathways were mediated by miRNAs to activate host immune responses including melanization and antimicrobial peptides for rapid wound healing and killing invaded pathogens. These results will help to understand injury-induced immune responses in crabs and to develop an effective control strategy of autotomy rate in crabs farming., 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 © 2023 Elsevier Ltd. All rights reserved.)

Endoplasmic reticulum oxidoreductase 1 (ERO1) is an important mediator in regulating disulfide bond formation and maintaining endoplasmic reticulum homeostasis. Its activity is transcriptionally regulated by the unfolded protein response (UPR) in the endoplasmic reticulum, which is known to be essential in immunity. However, whether ERO1 is involved in innate immunity in invertebrates remains unclear. In the present study, two subtypes of ERO1 from Scylla paramamosain were first identified and characterized. Sequence analysis revealed the conserved ERO1 domain and the oxidative capacity assay verified the oxidative capacity of SpERO1 recombinant protein. Moreover, SpERO1s were found to be ubiquitously expressed in all the tested tissues, with the highest expression observed in hemocytes. Two SpERO1s exhibited distinct expression patterns in response to Vibrio alginolyticus and White Spot Syndrome Virus (WSSV). Importantly, the downregulation of the expression of immune factors upon bacterial challenge in SpERO1-silenced crabs was observed. These results provided an initial foundation for further investigations into the role of ERO1 in the innate immunity of invertebrates., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Portunion is a rare endoparasitic isopod genus, recently observed inhabiting the hemocoel of the commercially important mud crab, Scylla paramamosain. For better understanding of the host-parasite interaction between S. paramamosain and Portunion sp., the metabolomic and transcriptomic changes in the hemolymph of the S. paramamosain were analyzed. We detected a total of 143 and 126 differentially accumulated metabolites in the positive and negative modes, respectively. Pathways related to amino acids and vitamin synthesis, such as Aminoacyl-tRNA biosynthesis, Tyrosine metabolism, Cysteine and methionine metabolism, Vitamin B6 metabolism, and Biotin metabolism were significantly enriched. Based on the transcriptomic data, a total of 942 differentially expressed genes were identified, of which 25 and 36 were significantly related to the immune system and metabolic pathways, respectively. Based on the metabolomic and transcriptomic data, 90 correlated metabolite-gene pairs were selected to build a regulatory network. Common significantly enriched pathways, including Starch and sucrose metabolism, Metabolism of xenobiotics by cytochrome P450, Aminoacyl-tRNA biosynthesis, Nitrogen metabolism, and Galactose metabolism were detected. On the basis of our analysis, the endoparasite Portunion sp. places a heavy metabolic burden on the host, particularly with respect to fundamental resources, such as amino acids, vitamins, carbohydrates, and lipids. In summary, these data provide an overview of the global metabolic and transcriptomic changes of the S. paramamosain resulting from Portunion sp. infection., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Tumor necrosis factor (TNF) is an inflammatory cytokine that is important in cell survival, proliferation, differentiation, and death. However, the functions of TNF in the innate immune responses of invertebrates have been less studied. In this study, SpTNF was cloned and characterized from mud crab (Scylla paramamosain) for the first time. SpTNF contains an open reading frame of 354 bp encoding 117 deduced amino acids, with a conserved C-terminal TNF homology domain (THD) domain. RNAi knockdown of SpTNF reduced hemocyte apoptosis and antimicrobial peptide (AMP) synthesis. Expression of SpTNF was initially down-regulated but subsequently up-regulated after 48 h in hemocytes of mud crabs after WSSV infection. Results of RNAi knockdown and overexpression showed that SpTNF inhibits the WSSV infection through activating apoptosis, NF-κB pathway, and AMP synthesis. Furthermore, the lipopolysaccharide-induced TNF-α factor (SpLITAF) can regulate the expression of SpTNF, induction of apoptosis, and activation of the NF-κB pathway and AMP synthesis. The expression and nuclear translocation of SpLITAF were found to be regulated by WSSV infection. Knocking down of SpLITAF increased the WSSV copy number and expression of VP28 gene. Taken together, these results proved the protective function of SpTNF, which is regulated by SpLITAF, in the immune response of mud crabs against WSSV through the regulation of apoptosis and activation of AMP synthesis., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Background: For more than a century, the Koch's postulates have been the golden rule for determining the causative agents in diseases. However, in cases of multiple pathogens-one disease, in which different pathogens can cause the same disease, the selection of microorganisms that regress infection is hard when Koch's postulates are applied. Microbiome approaches can obtain relatively complete information about disease-related microorganisms and can guide the selection of target microorganisms for regression infection. In the present study, whitish muscle syndrome (WMS) of Scylla paramamosain, which has typical symptoms with whitish muscle and blackened hemolymph was used as an example to establish a new research strategy that integrates microbiome approaches and Koch's postulates to determinate causative agents of multiple pathogens-one disease., Results: Microbiome results revealed that Aeromonas, Acinetobacter, Shewanella, Chryseomicrobium, Exiguobacterium, Vibrio and Flavobacterium, and Kurtzmaniella in hemolymph were bacterial and fungal indicators for WMS. A total of 23 bacteria and 14 fungi were isolated from hemolymph and muscle tissues, and among the bacteria, Shewanella chilikensis, S. xiamenensis, Vibrio alginolyticus, S. putrefaciens, V. fluvialis, and V. parahaemolyticus were present in hemolymph and/or muscle tissues in each WMS crab, and the last three species were also present in three Healthy crabs. The target bacteria and fungi were further screened to regression infections based on two criteria: whether they belonged to the indicator genera for WMS, whether they were isolated from both hemolymph and muscle tissues in most WMS crabs. Only S. chilikensis, S. putrefaciens, S. xiamenensis, V. alginolyticus, V. fluvialis, and V. parahaemolyticus met both two criteria. The six bacteria that met both two criteria and six fungi and another bacterium that unmatched any of two criteria were used to perform regression infection experiments based on Koch's postulates. S. chilikensis, S. putrefaciens, S. xiamenensis, V. alginolyticus, V. fluvialis, and V. parahaemolyticus met both two criteria, and the results indicate that they cause WMS in crabs independently., Conclusions: This study fully demonstrated that our research strategy that integrates the microbiome and Koch's postulates can maximize the ability to catch pathogens in one net for the situation of multiple pathogens-one disease. Video Abstract., (© 2023. The Author(s).)

Prolyl hydroxylase 2 (PHD2) is the key oxygen sensor that regulates the stability of the hypoxia-inducible factor -1α (HIF-1α). In this study, a novel PHD2 gene from the mud crab Scylla paramamosain, named SpPHD2, was cloned and identified. The full-length transcript of SpPHD2 was found to be 1926 bp, consisting of a 333 bp 5' untranslated region, a 1239 bp open reading frame, and a 354 bp 3' untranslated region. The putative SpPHD2 protein contained a Prolyl 4-hydroxylase alpha subunit homologues (P4Hc) domain in the C-terminal and a Myeloid translocation protein 8, Nervy, and DEAF-1 (MYND)-type zinc finger (zf-MYND) domain in the N-terminal. The mRNA expression of SpPHD2 was found to be widely distributed across all examined tissues. Additionally, the subcellular localization results indicated that the SpPHD2 protein was mainly localized in the cytoplasm. The in vivo silencing of SpPHD2 resulted in the upregulation of SpHIF-1α and a series of downstream genes involved in the HIF-1 pathway, while SpPHD2 overexpression in vitro dose-dependently reduced SpHIF-1α transcriptional activity, indicating that SpPHD2 plays a crucial role in SpHIF-1α regulation. Interestingly, the expression of SpPHD2 increased under hypoxic conditions, which was further inhibited by SpHIF-1α interference. Moreover, four hypoxia response elements were identified in the SpPHD2 promoter, suggesting that a feedback loop exists between SpPHD2 and SpHIF-1α under hypoxia. Taken together, these results provided new insights into the regulation of SpPHD2 in response to hypoxia in S. paramamosain., Competing Interests: Declaration of competing interest The authors declared that they have no conflicts of interest in this work. We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted., (Copyright © 2023 Elsevier Inc. All rights reserved.)

In our previous study, we found that the Spfoxl-2 transcript was highly expressed in gonads and explored its potential target genes in the ovary of Scylla paramamosain. In the current study, we primally analyzed its potential target genes in the testis through RNAi and RNA-Seq technology and compared with that in the ovary. The results showed that a total of 7892 unigenes were differentially expressed after Spfoxl-2 silencing in the testis, including plenty of conserved genes involved in testicular development, such as Dmrt family genes, Sox family genes, Caspase family genes, Cdk family genes, Kinesin family genes, Fox family genes and other genes. Further analysis revealed that these differentially expressed genes (DEGs) were enriched in crucial pathways involved in spermatogenesis, such as DNA replication, Cell cycle, Spliceosome, Homologous recombination, Meiosis and Apoptosis. The comparison results of potential target genes in the ovary and testis reveal 135 common potential target genes, including some genes involved in the immune response. According to our knowledge, the present work was the first to disclose the functions of foxl-2 in the testis of crustacean species using transcriptome analysis. It not only identifies key genes and pathways involved in the testicular development of S. paramamosain, but also reveals a new molecular-level understanding of the function of foxl-2 in testicular development., 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 © 2023 Elsevier Inc. All rights reserved.)

Cadmium is one of hazardous pollutants that has a great threat to aquatic organisms and ecosystems. The intestine plays important roles in barrier function and immunity to defend against environmental stress. However, whether cadmium exposure caused the intestine injury is not well studied. Thus, the aim of this study was to explore the potential mechanisms of cadmium toxicity in the intestine of mud crab (Scylla paramamosain) via physiological, histological, microbial community, and transcriptional analyses. Mud crabs were exposed to 0, 0.01, and 0.125 mg/L cadmium. After a 21-day of cadmium exposure, 0.125 mg/L cadmium caused intestine damaged by decreasing superoxide dismutase and catalase activities, and increasing hydrogen peroxide and malondialdehyde levels. Integrated biological index analysis confirmed that the toxicity of cadmium exhibited a concentration-dependent manner. Comparative transcriptional analyses showed that the up-regulations of several genes associated with heat shock proteins, detoxification and anti-oxidant defense, and two key signaling pathways (PI3k-Akt and apoptosis) revealed an adaptive response mechanism against cadmium exposure. Transcriptomic analysis also suggested that cadmium exposure disturbed the expression of ion transport and immune-related genes, indicating that it has negative effects on the immune functions of the mud crab. Furthermore, the intestinal microbial diversity and composition were significantly influenced by cadmium exposure. The abundance of the dominant phyla Fusobacteria and Bacteroidetes significantly changed after cadmium exposure. KEGG pathway analysis demonstrated that cadmium exposure could change energy metabolism and environmental information processing. Overall, we concluded that excessive cadmium exposure could be potentially exerted adverse effects to the mud crab health by inducing oxidative damage, decreasing immune system, disrupting metabolic function, and altering intestinal microbial composition. These results provided a novel insight into the mechanism of cadmium toxicity on crustaceans., 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 © 2023 Elsevier Ltd. All rights reserved.)

Glutaredoxin (Grx) is a glutathione-dependent oxidoreductase that plays a key role in antioxidant defense. In this study, a novel Grx2 gene (SpGrx2) was identified from the mud crab Scylla paramamosain, which consists of a 196 bp 5' untranslated region, a 357 bp open reading frame, and a 964 bp 3' untranslated region. The putative SpGrx2 protein has a typical single Grx domain with the active center sequence C-P-Y-C. The expression analysis revealed that the SpGrx2 mRNA was most abundant in the gill, followed by the stomach and hemocytes. Both mud crab dicistrovirus-1 and Vibrioparahaemolyticus infection as well as hypoxia could differentially induce the expression of SpGrx2. Furthermore, silencing SpGrx2 in vivo affected the expression of a series of antioxidant-related genes after hypoxia treatment. Additionally, SpGrx2 overexpression significantly increased the total antioxidant capacity of Drosophila Schneider 2 cells after hypoxia, resulting in a reduction of reactive oxygen species and malondialdehyde content. The subcellular localization results indicated that SpGrx2 was localized in both the cytoplasm and the nucleus of Drosophila Schneider 2 cells. These results indicate that SpGrx2 plays a crucial role as an antioxidant enzyme in the defense system of mud crabs against hypoxia and pathogen challenge., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Cytochrome P450 (CYPs) enzymes are one of the critical detoxification enzymes, playing a key role in antioxidant defense. However, the information of CYPs cDNA sequences and their functions are lacked in crustaceans. In this study, a novel full-length of CYP2 from the mud crab (designated as Sp-CYP2) was cloned and characterized. The coding sequence of Sp-CYP2 was 1479 bp in length and encoded a protein containing 492 amino acids. The amino acid sequence of Sp-CYP2 comprised a conserved heme binding site and chemical substrate binding site. Quantitative real-time PCR analysis revealed that Sp-CYP2 was ubiquitously expressed in various tissues, and it was highest in the heart followed by the hepatopancreas. Subcellular localization showed that Sp-CYP2 was prominently located in the cytoplasm and nucleus. The expression of Sp-CYP2 was induced by Vibrio parahaemolyticus infection and ammonia exposure. During ammonia exposure, ammonia exposure can induce oxidative stress and cause severely tissue damage. Knocking down Sp-CYP2 in vivo can increase malondialdehyde content and the mortality of mud crabs after ammonia exposure. All these results suggested that Sp-CYP2 played a crucial role in the defense against environmental stress and pathogen infection in crustaceans., Competing Interests: Declaration of competing interest The authors declared that they have no conflicts of interest to this work. We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

In this study, we extracted the polysaccharides from Hizikia fusiforme (HFPs) and evaluated their effects on the immune response of the mud crab Scylla paramamosain. Compositional analysis revealed that HFPs were composed mainly of mannuronic acid (49.05%) and fucose (22.29%) as sulfated polysaccharides, and the sugar chain structure was β-type. These results indicated that HFPs have potential antioxidant and immunostimulation activity in vivo or in vitro assays. Through this research, we found that HFPs inhibited viral replication in white spot syndrome virus (WSSV)-infected crabs and promoted phagocytosis of Vibrio alginolyticus by hemocytes. Quantitative PCR results showed that HFPs up-regulated the expression levels of astakine, crustin, myosin, MCM7, STAT, TLR, JAK, CAP, and p53 in crab hemocytes. HFPs also promoted the activities of superoxide dismutase and acid phosphatase and the hemolymph antioxidant activities of crabs. HFPs maintained peroxidase activity after WSSV challenge, thereby providing protection against oxidative damage caused by the virus. HFPs also promoted apoptosis of hemocytes after WSSV infection. In addition, HFPs significantly enhanced the survival rate of WSSV-infected crabs. All results confirmed that HFPs improved the innate immunity of S. paramamosain by enhancing the expression of antimicrobial peptides, antioxidant enzyme activity, phagocytosis, and apoptosis. Therefore, HFPs have potential for use as therapeutic or preventive agents to regulate the innate immunity of mud crabs and protect them against microbial infection., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

In the study, a new gene homologous to the known antimicrobial peptide Scygonadin was identified in mud crab Scylla paramamosain and named SCY3 . The full-length sequences of cDNA and genomic DNA were determined. Similar to Scygonadin , SCY3 was dominantly expressed in the ejaculatory ducts of male crab and the spermatheca of post-mating females at mating. The mRNA expression was significantly up-regulated after stimulation by Vibrio alginolyticus , but not by Staphylococcus aureus . The recombinant protein rSCY3 had a killing effect on Micrococcus luteus and could improve the survival rate of mud crabs infected with V. alginolyticus . Further analysis showed that rSCY3 interacted with rSCY1 or rSCY2 using Surface Plasmon Resonance (SPR, a technology for detecting interactions between biomolecules using biosensor chips) and Mammalian Two-Hybrid (M2H, a way of detecting interactions between proteins in vivo). Moreover, the rSCY3 could significantly improve the sperm acrosome reaction (AR) of S. paramamosain and the results demonstrated that the binding of rSCY3, rSCY4, and rSCY5 to progesterone was a potential factor affecting the sperm AR by SCYs on. This study lays the foundation for further investigation on the molecular mechanism of SCYs involved in both immunity and physiological effects of S. paramamosain .

Biogenic amines (BAs) play an important role in the aggressive behavior of crustaceans. In mammals and birds, 5-HT and its receptor genes (5-HTRs) are characterized as essential regulators involved in neural signaling pathways during aggressive behavior. However, only one 5-HTR transcript has been reported in crabs. In this study, the full-length cDNA of the 5-HTR1 gene, named Sp5-HTR1 , was first isolated from the muscle of the mud crab Scylla paramamosain using the reverse-transcription polymerase chain reaction (RT-PCR) and rapid-amplification of cDNA ends (RACE) methods. The transcript encoded a peptide of 587 amino acid residues with a molecular mass of 63.36 kDa. Western blot results indicate that the 5-HTR1 protein was expressed at the highest level in the thoracic ganglion. Furthermore, the results of quantitative real-time PCR show that the expression levels of Sp5-HTR1 in the ganglion at 0.5, 1, 2, and 4 h after 5-HT injection were significantly upregulated compared with the control group ( p < 0.05). Meanwhile, the behavioral changes in 5-HT-injected crabs were analyzed with EthoVision. After 0.5 h of injection, the speed and movement distance of the crab, the duration of aggressive behavior, and the intensity of aggressiveness in the low-5-HT-concentration injection group were significantly higher than those in the saline-injection and control groups ( p < 0.05). In this study, we found that the Sp5-HTR1 gene plays a role in the regulation of aggressive behavior by BAs, including 5-HT in the mud crab. The results provide reference data for the analysis of the genetic mechanism of aggressive behaviors in crabs.

The Dmrt (double-sex and mab-3 related transcription factor) gene family is considered to be a highly conserved gene family related to sex determination and sexual differentiation across species. In order to better understand the role of the idmrt-2 gene in gonad development in Scylla paramamosain, the idmrt-2 gene was cloned and analyzed. The cDNA contains a 1659 bp ORF region encoding 552 amino acids. The qRT-PCR results showed that idmrt-2 was significantly more expressed in the testis than in other tissues (p < 0.05). The expression of idmrt-2 was highest in the spermatids stage (T2 stage), followed by the mature sperms stage (T3 stage) and significantly higher than in the spermatocytes stage (T1 stage) (p < 0.05) during testicular development and the expression difference was not significant in different stages of ovarian development. RNAi studies revealed that after idmrt-2 was knocked down, the expression of Dmrt-like and foxl-2 genes in the testis decreased, as well as IAG expression in the androgenic gland. The findings suggest that idmrt-2 may be an IAG regulator and involved in testicular development., 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 © 2022 Elsevier B.V. All rights reserved.)

The structure of allergenic proteins provides important information about the binding of allergens to antibodies. In this study, the crystal structure of Scy p 4 with a resolution of 1.60 Å was obtained by X-ray diffraction. Epitope mapping of Scy p 4 revealed that linear epitopes are located on the surface of Scy p 4. Also, conformational epitopes are mostly located in the structural conservative region. Further structural comparison, surface electrostatic potential, and hydrogen bond force analysis showed that mutation of Asp 70 and Asp 18/20/70 would lead to calcium-binding capacity being lost and destruction of allergenicity. Furthermore, a comparative analysis of structure showed that sarcoplasmic-calcium-binding protein (SCP) had high sequence, secondary, and spatial structural identity in crustaceans, which may be an important factor leading to cross-reactivity among crustaceans. The structure of Scy p 4 provides a template for epitope evaluation and localization of SCPs, which will help to reveal cross-reactivity among species.

Cadmium (Cd) is a heavy metal contaminant and can be toxic to environment. What's more, Selenium (Se) protects organism as heavy metal antagonist. The present study aimed to investigate whether inorganic (Na 2 SeO 3 ) or organic (L-SeMc) Se have an effect on the Cd bioaccumulation, antioxidant and immunity of the mud crab (Scylla paramamosain) under Cd exposure. The study showed that the concentration of Cd in hepatopancreas under Cd exposure was higher than the inorganic or organic Se group (P < 0.05), notably, Cd concentration of hepatopancreas in organic Se treatment is less than that in inorganic Se treatment (P < 0.05). Furthermore, this study analyzed 28 gene expression about antioxidant and immune from transcriptome, the result indicated that L-SeMc (organic Se) can reduced intracellular ROS production and oxidative damage. Furthermore, apoptosis was enhanced after Cd exposure, but Se could protect against apoptosis via expression of cathepsin B. Consequently, Organic Se may have a better effect than inorganic Se on reducing Cd toxicity. This study could provide the molecular basis that Se might alleviate Cd toxicity and increases the understanding of the environmental contaminant on crustaceans., 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 © 2022. Published by Elsevier Inc.)

The fibroblast growth factor receptor (FGFR) belongs to the tyrosine kinase family consisting of four members (FGFR1-4). This study involved identification and characterization of FGFR1 and FGFR3 from mud crab Scylla paramamosain for the first time. The obtained cDNAs of SpFGFR1 and SpFGFR3 were 2,380 bp and 2,982 bp in length with a 1,503 bp and 2,310 bp open reading frame, respectively. The predicted SpFGFR1 protein included three immunoglobulin domains and a transmembrane region, while SpFGFR3 protein possessed a typical TyrKc (Tyrosine kinase, catalytic) domain. Real-time PCR analysis showed that SpFGFR1 and SpFGFR3 were highly expressed in the hepatopancreas. Furthermore, the expression levels of SpFGFR1 and SpFGFR3 in the hepatopancreas were enhanced following challenges with Vibro alginolyticus, Staphylococcus aureus, Poly (I:C) and White spot syndrome virus, which shows the involvement of SpFGFR1 and SpFGFR3 in innate immune response to infections from bacteria and virus. There was significant suppression of six antimicrobial peptide genes (SpALF1-5 and SpCrustin) and three NF-κB members (SpDorsal, SpIKK and SpRelish) when SpFGFR1 and SpFGFR3 was interfered in vivo. Also, treatment of the hemocytes with specific inhibitor of SpFGFR for 24 h consistently down-regulated SpDorsal, SpRelish and AMPs. These results suggested that SpFGFR1 and SpFGFR3 played important roles in regulating the Toll signaling pathway and immune deficiency (IMD) pathway through NF-κB signaling pathway. These findings may provide new insights into the role of FGFRs in the innate immune function of crustaceans., (Copyright © 2022. Published by Elsevier Ltd.)

Vitellogenesis in crustaceans is controlled by several steroid hormones. In humans, the expression of SF-1, a gene that regulates gonadal development and the synthesis of steroid hormones, is affected by DDX20. However, how the homologous gene FTZ-F1 is regulated by DDX20 and its association with vitellogenesis remains unknown in the mud crab Scylla paramamosain. In this study, SpDDX20 and SpFTZ-F1 were identified in the transcriptome of mature ovarian tissue from the mud crab. qRT-PCR results revealed that the expression levels of SpFTZ-F1 and SpVTG in the ovaries of crab in the experimental group injected with dsDDX20 (EO) were significantly higher (P < 0.05) than those in the negative control group injected with dsEGFP (NO) and the blank control group injected with SPSS (BO). The differentially expressed genes (DEGs) identified by comparative transcriptome analysis of the EO group and NO group were enriched into five pathways related to ovarian steroidogenesis. The expression of CYP17, CYP3A4, CYP1A1 and 3β-HSD were up-regulated in pathways related to steroid hormone production and biosynthesis. The expression of the INSR, IRS and PI3K genes in the insulin signaling pathway were significantly increased (P < 0.05). The expression level of the TGF-β gene was up-regulated (P < 0.05) in the transforming growth factor pathway, whereas the expression level of the Smad2 gene was down-regulated (P < 0.05). The expression of GnRHR, GS, AC and PKA genes in the gonadotropin-releasing hormone signaling pathway were up-regulated. Our data provide a foundation for investigating the relationship between DDX20 and FTZ-F1 in the regulation of vitellogenin expression in S. paramamosain., 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 © 2022 Elsevier Inc. All rights reserved.)

In previous study, we reported the identification, tissue distribution, and the roles of Spdsx played in the testis, androgenic gland, and ovary in Scylla paramamosain. Here, we primally identify its potential target genes in the ovary with RNAi and RNA-Seq technology. By comparing the transcriptome data of two groups (ovaries that injected with dsRNA for EGFP and Dsx), we found that 6520 Unigenes were differentially expressed, including a plenty of conserved crucial genes involved in ovarian development, such as vitellogenin (vtg), vtg receptor (vtgR), apolipoprotein D, adenylate cyclase 3, adenylate cyclase 5, cyclin A, cyclin B, and cell division cycle 2 (cdc2). In addition, these DEGs were also enriched in pathways related to ovary development, including PI3K-Akt signaling pathway, MAPK signaling pathway, insulin signaling pathway, Wnt signaling pathway, relaxin signaling pathway, estrogen signaling pathway, progesterone-mediated oocyte maturation, ovarian steroidogenesis, and oocyte meiosis. Moreover, several genes were selected for qRT-PCR to validate the accuracy of the bioinformatic result. According to current transcriptome result, we speculate that the Spdsx is a crucial regulator of ovary development in S. paramamosain. To the best of our knowledge, the current study was the first report about dsx function through comparative transcriptome analysis in crustacean species, which not only identified relevant genes and pathways involved in ovarian development of S. paramamosain, but also shed light on the regulatory mechanisms of dsx at the molecular level in crustacean., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The epidermal growth factor receptor (EGFR) is a pleiotropic glycoprotein which plays a role in regulating cell proliferation, migration and differentiation. However, the genetic diversity of EGFR in crustaceans as well as its function, such as whether it is involved in immune regulation, remains obscure. In this study, two EGFR genes, including EGFR1 and EGFR2, and three transcripts were identified and characterized in Scylla Paramamosain for the first time. To our knowledge, this is the first time that more than one EGFR gene was identified in a single species. The complete open reading frames (ORFs) of SpEGFR1, SpEGFR2a and SpEGFR2b were 4377 bp, 4404 bp and 4341 bp encoding deduced proteins of 1458 amino acids (aa), 1467 aa and 1446 aa, respectively. All EGFR had a signal peptide region and two Recep&#95;L&#95;domain region, followed by a transmembrane region and a conserved tyrosine kinase domain (TyrKc), and phylogenetic analysis demonstrated three SpEGFRs clustered together with invertebrate EGFR branch. Tissue specific expression analysis depicted that all SpEGFRs presented similar transcription patterns. The expression levels of SpEGFR1 and SpEGFR2s in hepatopancreas and gills were significantly altered after the stimulation of bacterial and viral pathogens including Staphylococcus aureus, Vibrio alginolyticus, White spot syndromre virus and Polycytidylinic acid. The in vivo RNA interference assays demonstrated that expression levels of SpIKK, two members of NF-κB (SpRelish and SpDorsal) and six antimicrobial peptide (AMP) genes (SpCrustin and SpALF1-5) were significantly reduced when SpEGFR1 or SpEGFR2 was silenced, respectively. The transcription patterns of SpIKK, SpRelish, SpDorsal and AMPs exhibited similar down- or up-regulation trend when the primary cultured hemocytes were treated with EGFR antagonist or agonist for 24 h. These results suggested that SpEGFR might play an important role in innate immune responses to bacterial and viral infections by regulating the NF-κB pathway. It also provided a better understanding of the origin or evolution of EGFR in crustaceans and even invertebrates., (Copyright © 2022 Elsevier Ltd. All rights reserved.)