The muscle LIM protein (MLP) is a member of the cysteine and glycine-rich protein (CSRP) family, composed of CSRP1, CSRP2 and CSRP3/MLP. MLP is involved in a multitude of functional roles, including cytoskeletal organization, transcriptional regulation, and signal transduction. However, the molecular mechanisms underlying its involvement in immune and stress responses remain to be elucidated. This study identified an MnMLP in the freshwater crustacean Macrobrachium nipponense. The isothermal titration calorimetry assay demonstrated that recombinant MnMLP was capable of coordinating with Zn2+. Upon challenge by Aeromonas veronii or WSSV, and exposure to CdCl2, up-regulation was recorded in the muscle and intestinal tissues, suggesting its involvement in immune and anti-stress responses. MnMLP protein was predominantly expressed in the cytoplasm of the transfected HEK-293T cells, but after treatment with LPS, Cd2+ or H2O2, the MnMLP was observed to be transferred into the nucleus. The comet assay demonstrated that the overexpression of MnMLP could mitigate the DNA damage induced by H2O2 in HEK-293T cells, suggesting the potential involvement of MnMLP in the DNA repair process. These findings suggest that DNA repair may represent a possible mechanism by which MnMLP may be involved in the host\'s defense against pathogens and stress.

Tropomyosin (TM) is the main allergen of Macrobrachium nipponense. Recombinant allergens have great prospects in the detection, diagnosis, and treatment of food allergens. The purpose of this study was to compare the differences in structure and allergenicity between natural TM and recombinant TM. Recombinant TM of M. nipponense with a molecular weight of 38 kDa was successfully expressed in the Escherichia coli system. The amino acid sequence as well as secondary structure between natural and recombinant TM were similar, which were verified by mass and CD spectrometry, respectively. Studies showed that both natural TM and recombinant TM had strong allergenicity, and recombinant TM was more allergenic, which could be used as a substitute for natural TM in the diagnosis and treatment of shrimp allergy. This study provided stable and reliable allergen components for the detection of crustacean allergens and the diagnosis and treatment of food allergies caused by crustacean allergens.

In this study, we used full-sib families to investigate the association between growth and gonad development during first sexual maturation of M. nipponense. We found that male GSI was significantly negatively correlated with growth traits (p < 0.01) and there were no significant correlations between female GSI (Gonadosomatic index) and growth traits (p > 0.05). HSI (Hepatopancreas index) in both males and females showed no significant correlations with growth traits (p > 0.05). We furthermore investigated the association between the specific allele of Mn-CTS L1 polymorphism and gonad development and growth traits. In total, 35 mutation loci were screened and 16 high-quality single-nucleotide polymorphisms (SNPs) loci were obtained after validation. Four and two SNPs proved to be strongly associated with all growth traits in female and male M. nipponense separately, among which A+118T might be a candidate SNP positively associated with large growth traits. Two and one SNPs were screened, respectively, in males and females to associate with GSI, while three SNPs were detected to associate with female HSI, among which A+1379C may be applied as a potential molecular marker for gene-assisted selection to improve both reproduction speed and growth traits in M. nipponense.

The ovary in mammals has developed specialized mechanisms for protection against pathogen infections; however, the understanding of the innate immune system in the ovary of crustaceans is still limited. To elucidate the ovary\'s defense mechanisms in response to viral challenges, we subjected oriental river prawns (Macrobrachium nipponense) to poly I:C, a double-stranded RNA analog that emulates viral dsRNA, and analyzed the ovary\'s transcriptome profiles. Concurrently, RNA-seq analysis was performed on the hepatopancreas, a well-recognized immune-related tissue, following poly I:C challenge to investigate the distinct response mechanisms of the ovary and hepatopancreas and to gain a comprehensive understanding of the immune responses in both tissues. The results indicate that 1368 genes are differentially expressed in the ovary, with 903 genes upregulated and 465 genes downregulated. Subsequent analysis reveals that these differentially expressed genes (DEGs) include numerous genes associated with innate immunity, such as members of the C-type lectin, fibrinogen-related protein (Frep), Toll-like receptor, and NOD-like receptor (NLR) gene families, as well as acid phosphatase, scavenger receptor, crustin, Down syndrome cell adhesion molecule (Dscam), hemocyanin, and lipopolysaccharide and beta-1,3-glucan binding protein (LGBP). Furthermore, the DEGs include several genes related to ovary development, such as sox8, vitellogenin, progranulin, cyclin-dependent kinase, ecdysone receptor, frizzled, and members of the Fox gene family. In the hepatopancreas, a total of 729 DEGs were identified. Comparison of the DEGs in both tissues indicates that only 91 genes are common to both groups, highlighting significant tissue-specific responses to poly I:C stimulation. This study aims to enhance our understanding of the immune protective mechanisms employed by the ovary in response to pathogen exposure and establishes a foundation for investigating ovarian reproductive immunity in crustaceans.

We identified a novel C-type lectin (CTL) from Macrobrachium nipponense, designated as Mn-clip-Lec. It consists of 1315 bp with an open reading frame of 1098 bp, encoding a polypeptide of 365 amino acids. Mn-clip-Lec contains 6 exons and 5 introns. Mn-clip-Lec possessed a CLIP domain at the N-terminal and two carbohydrate recognition domains at the C-terminal. Interaction between Mn-clip-Lec and MnLec was found by Yeast two-hybrid analysis. The expressions of Mn-clip-Lec, MnLec, prophenoloxidase (proPO)-activating system-associated genes (MnPPAF, MnPPAE, and MnPO), and antimicrobial peptides (AMPs) (MnALF and MnCRU) were up-regulated after the challenge with Staphylococcus aureus. RNA interference (RNAi)-mediated suppression of the Mn-clip-Lec and MnLec genes in S. aureus-challenged prawns reduced the transcripts of MnPPAF, MnPPAE, MnPO, MnALF and MnCRU. Knockdown of Mn-clip-Lec and MnLec resulted in decrease in PO activity in M. nipponense infected with S. aureus. The recombinant Mn-clip-Lec (rMn-clip-Lec) protein bound all tested bacteria and agglutinated S. aureus. A sugar-binding assay revealed that rMn-clip-Lec could bind to LPS or PGN. rMn-clip-Lec accelerated the clearance of S. aureus in vivo. Our findings suggest that Mn-clip-Lec and its interacting MnLec play important roles in the induction of the proPO system and AMPs expression in M. nipponense during bacterial infection.

NPC intracellular cholesterol transporter 1 (NPC1) plays an important role in sterol metabolism and transport processes and has been studied in many vertebrates and some insects, but rarely in crustaceans. In this study, we characterized NPC1 from Macrobrachium nipponense (Mn-NPC1) and evaluated its functions. Its total cDNA length was 4283 bp, encoding for 1344 amino acids. It contained three conserved domains typical of the NPC family (NPC1_N, SSD, and PTC). In contrast to its role in insects, Mn-NPC1 was mainly expressed in the adult female hepatopancreas, with moderate expression in the ovary and heart. No expression was found in the embryo (stages CS-ZS) and only weak expression in the larval stages from hatching to the post-larval stage (L1-PL15). Mn-NPC1 expression was positively correlated with ovarian maturation. In situ hybridization showed that it was mainly located in the cytoplasmic membrane and nucleus of oocytes. A 25-day RNA interference experiment was employed to illustrate the Mn-NPC1 function in ovary maturation. Experimental knockdown of Mn-NPC1 using dsRNA resulted in a marked reduction in the gonadosomatic index and ecdysone content of M. nipponense females. The experimental group showed a significant delay in ovarian maturation and a reduction in the frequency of molting. These results expand our understanding of NPC1 in crustaceans and of the regulatory mechanism of ovarian maturation in M. nipponense.

Macrophages are one of the important immune cells, which play important roles in innate and adaptive immune. However, the roles of macrophages in food allergy are not thoroughly understood. To investigate the roles of macrophages during food allergy, we focused on the relationship between macrophage polarization and allergic responses induced by tropomyosin (TM) in the present study. Arg 1 and CD206 expressions in the TM group were significantly higher than those of the PBS group, while iNOS and TNF-α expressions were no obvious difference, moreover, the morphology of macrophages stimulated by TM was similar to that of M2 macrophages. These results indicated macrophages were mainly polarized toward M2 phenotypes in vitro. The antibodies, mMCP-1, histamine and cytokines, revealed that macrophages could participate in food allergy, and macrophage polarization was associated with changes in allergic-related factors. The cytokine levels of M2 phenotypes were significantly higher than those of M1 phenotypes in peripheral blood. The mRNA expressions and protein levels of Arg1 and iNOS in the jejunum and peritoneal cells indicated that M2 phenotypes were the major macrophage in these tissues compared with M1 phenotypes. Hence, macrophage polarization plays an important role in food allergy.

C-type lectins (CTLs) execute critical functions in multiple immune responses of crustaceans as a member of pattern recognition receptors (PRRs) family. In this study, a novel CTL was identified from the exoskeleton of the oriental river prawn Macrobrachium nipponense (MnLec3). The full-length cDNA of MnLec3 was 1150 bp with an open reading frame of 723 bp, encoding 240 amino acids. MnLec3 protein contained a signal peptide and one single carbohydrate-recognition domain (CRD). MnLec3 transcripts were widely distributed at the exoskeleton all over the body. Significant up-regulation of MnLec3 in exoskeleton after Aeromonas hydrophila challenged suggested the involvement of MnLec3 as well as the possible function of the exoskeleton in immune response. In vitro tests with recombinant MnLec3 protein (rMnLec3) manifested that it had polysaccharide binding activity, a wide spectrum of bacterial binding activity and agglutination activity only for tested Gram-negative bacteria (Escherichia coli, Vibrio anguillarum and A. hydrophila). Moreover, rMnLec3 significantly promoted phagocytic ability of hemocytes against A. hydrophila in vivo. What\'s more, MnLec3 interference remarkably impaired the survivability of the prawns when infected with A. hydrophila. Collectively, these results ascertained that MnLec3 derived from exoskeleton took an essential part in immune defense of the prawns against invading bacteria as a PRR.

Environmental stresses play critical roles in the physiology of crustaceans. Food deprivation is an important environmental factor and a regular occurrence in both natural aquatic habitats and artificial ponds. However, the underlying physiological response mechanisms to starvation-caused stress in crustaceans are yet to be established. In the present study, the hepatopancreas tissue of Macrobrachium nipponense was transcriptome analyzed and examined for starvation effects on oxidative stress, DNA damage, autophagy, and apoptosis across four fasting stages (0 (control group), 7, 14, and 21 days). These results indicated that a ROS-mediated regulatory mechanism is critical to the entire fasting process. At the initial stage of starvation (fasting 0 d ~ 7 d), ROS concentration increased gradually, activating antioxidant enzymes to protect the cellular machinery from the detrimental effects of oxidative stress triggered by starvation-induced stress. ROS content production (hydrogen peroxide and superoxide anion) then rose continuously with prolonged starvation (fasting 7 d ~ 14 d), reaching peak levels and resulting in autophagy in hepatopancreas cells. During the final stages of starvation (fasting 14 d ~ 21 d), excessive ROS induced DNA damage and cell apoptosis. Furthermore, autophagolysosomes and apoptosis body were further identified with transmission electron microscopy. These findings lay a foundation for further scrutiny of the molecular mechanisms combating starvation-generated stress in M. nipponense and provide fishermen with the theoretical guidance for adopting fasting strategies in M. nipponense aquaculture.

The family of TIR domain-containing receptors includes numerous proteins involved in innate immunity. In this study, a member of this family was characterized from the ovary of the oriental river prawn Macrobrachium nipponense and identified as interleukin-1 receptor (MnIL-1R). Meanwhile, to elucidate the conservation of IL-1R, its orthologous were identified in several crustacean species as well. In addition, the expression pattern of MnIL-1R in various adult tissues and post different pathogen-associated molecular patterns (PAMPs) challenge in ovary was analyzed with qRT-PCR technology. Finally, the roles of MnIL-1R in the ovary were analyzed by RNAi technology. The main results are as follows: (1) MnIL-1R comprises a 1785 bp ORF encoding 594 amino acids and is structurally composed of five domains: a signal peptide, two immunoglobulin (IG) domains, a transmembrane region, and a TIR-2 domain; (2) the TIR domain showed a high conservation among analyzed crustacean species; (3) MnIL-1R is widely detected in all tested tissues including ovary; (4) MnIL-1R showed a positive response to challenges with LPS, PGN, and polyI:C in the ovary; (5) its IG domain showed strong binding ability to LPS and PGN, confirming its role as a pattern recognition receptor; (6) the expression patterns of several members of the Toll signaling pathway (Myd88, TRAF-6, Dorsal, and Relish) was similar to that of MnIL-1R after challenges with LPS, PGN, and polyI:C in the ovary; (7) the silencing of MnIL-1R resulted in down-regulation of theses gene\' (Myd88, TRAF-6, Dorsal, and Relish) expression level in the ovary. These results suggest that MnIL-1R can activate the Toll signaling pathway in the ovary by directly recognizing LPS and PGN through its IG domain, thereby contributing to the immune response in the ovary of M. nipponense.