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The eyestalk of crustaceans, such as Macrobrachium nipponense, contains many neurosecretory hormones affecting the process of reproduction, molting, metabolism of glucose, and other functions. In this study, important metabolic pathways and candidate genes involved in male sexual development were selected from M. nipponense. The methodology involved performing long-read and next generation transcriptome sequencing of genes from the androgenic gland after eyestalk ablation. qPCR analysis revealed that the mRNA expression of Mn-IAG was significantly increased after ablation of both the single-side (SS) and double-side (DS) eyestalk, compared with the control group (CG). The long-read transcriptome generated 49,840 non-redundant transcripts. A total of 1319, 2092 and 4351 differentially expressed genes (DEGs) were identified between CG versus SS, SS versus DS and CG versus DS, respectively. These data indicated that ablation of the double-sided eyestalk played stronger regulatory roles than the single-side ablation on male sexual development in M. nipponense. This was consistent with the qPCR analysis. Cell Cycle, Cellular Senescence, Oxidative Phosphorylation, Glycolysis/Gluconeogenesis and Steroid Hormone Biosynthesis were the primary enriched metabolic pathways in all three comparisons, and the important genes from these metabolic pathways were also selected. qPCR permitted secondary confirmation of ten DEGs identified through RNA-seq. RNAi-mediated silencing analyses of Hydroxysteroid dehydrogenase like 1 (HSDL1) revealed that HSDL1 has a positive regulatory effect on testes development. This study provides valuable insight into male sexual development in M. nipponense, including metabolic pathways and genes, paving the way for advanced studies on male sexual development in this species and in other crustaceans.

Previous studies have revealed that SDHB has potential functions in the male sexual differentiation and development in M. nipponense through providing ATP. In this study, the functions of Mn-SDHB were further analyzed in depth using quantitative polymerase chain reaction (qPCR), in situ hybridization, western-blot, and RNA interference (RNAi), combined with the histological observations. The full-genome sequence of Mn-SDHB was 54,608 bp at Chromosome 34, including 7 introns and 6 exons. The full-length cDNA sequence of Mn-SDHB was 1,268 base pairs (bp) long with an open reading frame of 807 bp, encoding for 268 amino acids. The highest expression level of Mn-SDHB in different tissues was observed in the testis, and male prawns at post-larval developmental stage 25 during different developmental stages, indicating that SDHB was potentially involved in the male sexual development in M. nipponense. In situ hybridization and western-blot analysis indicated that SDHB plays essential roles in the testis development. The in situ hybridization analysis also implies the potential roles of Mn-SDHB in ovarian development. The expressions of Mn-IAG were decreased after Mn-SDHB dsRNA injection, indicating SDHB has the positive regulatory effects on IAG in M. nipponese. Thus, SDHB was involved in the mechanism of the male sexual development. The testis development was inhibited, and sperms were rarely observed after 10 days of Mn-SDHB dsRNA injection, indicating SDHB has positive effects on the male sexual development in M. nipponense. This study highlights the functions of SDHB in M. nipponense, which provide new insights for the future studies of the male sexual development in other crustacean species.

Parenteral vaccinations are not able to elicit effective systemic and gastrointestinal immune protection simultaneously because the lymphocytes are typically restricted to primed tissues. Although all-trans retinoic acid (atRA) was reported to trigger the gut-homing of immunocytes, the bioavailability and systemic immune responses remain limited for use in robust enteric vaccinations. Here, we show that co-delivery of atRA, CpG oligodeoxynucleotides (CpG), and antigens via engineered polymer/lipid nanoparticles (PLNPs) could exploit the amplifying function of draining lymph nodes (DLNs) for potent gut tropism and immune activations. After intramuscular injection, forming an immune-potentiated environment at the injection site, the PLNPs induced the designated transfer of primed dendritic cells (DCs) to the DLNs instead of the gastrointestinal tissues. Within the DLNs, the immune-potentiated environment markedly amplified the antigen presentation and homing receptor switch among immunocytes, which simultaneously stimulated the preferential dissipation of activated lymphocytes in the peripheral and gastrointestinal tissues, that is, exerted a DLN-amplifying effect. Compared with current atRA-containing formulations, the PLNPs not only boosted potent IgG secretions and T cell activations in the peripheral tissue but also provoked robust T cell homing and antigen-specific IgA levels in the gastrointestinal tracts in both ovalbumin and EV71 vaccinations. These data indicate that exploiting DLN amplification can stimulate potent systemic and gastrointestinal responses for more efficient enteric vaccinations.

The eyestalk of crustacean species secretes many hormones, affecting the process of reproduction, molting, metabolism of glucose, and other functions in crustaceans. In this study, important metabolic pathways and candidate genes involved in the male sexual development were identified through performing the transcriptome profiling analysis of the testis after the ablation of eyestalk from Macrobrachium nipponense. The histological observations revealed that the testis development became vigorous after eyestalk ablation, indicating that the hormones secreted by the eyestalk have negative effects on the testis development in M. nipponense. Transcriptome profiling analysis revealed that 1,039, 1,226, and 3,682 differentially expressed genes (DEGs) were identified between normal prawns (CG) vs single-side eyestalk ablation prawns (SS), SS vs double-side eyestalk ablation prawns (DS), and CG vs DS, respectively, indicating that the ablation of double-side eyestalk has more significant regulatory roles on male sexual development than that of single-side ablation, which was consistent with the histological observations. Lysosome, Apoptosis, Glycolysis/Gluconeogenesis, and Insulin signaling pathway were the main enriched metabolic pathways in all of these three comparisons, and the important genes from these metabolic pathways were also selected. The qPCR verifications of 10 DEGs from these metabolic pathways were the same as those of RNA-seq. The qPCR, in situ hybridization, and RNA interference analysis of Mn-NFkBα revealed that NFkBα has a positive regulatory effect on testis development. This study provided new insights on male sexual development in M. nipponense, promoting the studies on male sexual development in other crustaceans as well.

In the combat against pathogens, the immune systems were evolved with the immune recognitions against the various danger signals, which responded vigorously upon the pathogen invasions and elicited potent antibodies or T cell engagement against the re-infections. Envisage with the prevailing pandemics and increasing demands for cancer vaccines, bio-mimic particles were developed to imitate the natural traits of the pathogens, which conferred the optimal strategies to stimulate the immune engagement and let to the increased vaccine efficacy. Here, the recent development in bio-mimic particles, as well as the natural cues from the pathogens were discussed. As such, the designing principles that adapted from the physiochemical properties of the pathogens were unfolded as the surface characteristics (hydrophobic, nano-pattern, antigen display, charge), properties (size, shape, softness) and the delivered components (peptide, protein, nuclear acids, toll-like receptor (TLR) agonist, antibody). Additionally, the strategies for the efficient delivery, regarding the biodistribution, internalization and presentation of the antigens were also illustrated. Through reviewing the state-of-art in biomimetic particles, the lesson learnt from the natural traits and pathogenic invasion may shed light on the rational design for the enhanced vaccinations.

The eyestalk of crustacean species contained many neurosecretory structures, affected the process of reproduction, molting, metabolism of glucose and other function in crustaceans. In this study, we aimed to selected important metabolic pathways and candidate genes involved in the male sexual development through performing the long-reads and next generation transcriptome sequencing of androgenic gland after the ablation of eyestalk from Macrobrachium nipponense. qPCR analysis revealed that the mRNA expression of Mn-IAG was significantly increased after ablation both of the single-side (SS) and double-side (DS) eyestalk, compared with that of control group (CG). The long-reads transcriptome generated 49,480 non-redundant transcripts. A total of 1,319, 2,092 and 4,351 differentially expressed genes (DEGs) were identified between CG vs SS, SS vs DS and CG vs DS, respectively, indicating the ablation of double-side eyestalk has more important regulatory roles than that of single-side ablation on male sexual development, which was consistent with that of qPCR analysis. Cell cycle, Cellular senescence, Oxidative phosphorylation, Glycolysis/Gluconeogenesis and Steroid hormone biosynthesis were the main enriched metabolic pathways in all of these three comparisons, and the important genes from these metabolic pathways were also selected. The qPCR verifications of 10 GEDs were as the same as that of RNA-seq. The qPCR and RNAi analysis of Hydroxysteroid dehydrogenase like 1 (HSDL1) revealed that HSDL1 has the positive regulatory effected on testis development. This study provided valuable evidences on male sexual development in M. nipponense, promoting the studies on male sexual development in other crustacean as well.

A previous study identified slow-tonic S2 tropomyosin and slow tropomyosin isoform as sex-related genes in Macrobrachium nipponense. Their functions were analyzed using RNA interference. However, more than half of the specimens died approximately 8–12 h after injection of the respective double-stranded RNAs (dsRNAs), and HE staining indicated that the heart and gills were the most likely tissues responsible for the resultant deaths. In the current study, we conducted a comparative transcriptomic study of the gills and hearts of M. nipponense to identify potential target genes associated with acute death after dsRNA injection. A total of 68,772 annotated unigenes were generated. In the heart, differentially expressed genes (DEGs) were mainly enriched in glycolysis/gluconeogenesis and oxidative phosphorylation, while the most relevant pathways in the gills were lysosome, phagosome, and peroxisome. Ten DEGs were screened out and analyzed under lethal hypoxic stress. Among these, fructose 1, 6-biphosphate-aldolase (FBA), glyceraldehyde 3-phosphate dehydrogenase (GDPDH), alcohol dehydrogenase class-3 (ADC3), ATP-synthase subunit 9 (ATPS9), and acid ceramidase-like (ACL) were all differentially expressed under hypoxic conditions. This study shed light on the lethal mechanism caused by interference with tropomyosin genes in M. nipponense, and identifies the related pathways and key genes that could help to improve stress resistance and tolerance in M. nipponense.

Cancer vaccines have opened a new paradigm for safe and effective antitumor therapy, but they still suffer from shortcomings such as insufficient immunogenicity and immune tolerance, which seldom makes them the first choice in clinic. In fact, similar to providing a high-end product, a robust antitumor effect depends on the inherent supply chain, which attains, processes, and presents tumor-associated antigens via antigen presenting cells to T cells, which then leads to lysis of the cancer cells to release more antigens to complete the supply chain. Under these circumstances, the failure of cancer vaccines can be treated as a blockade or chain rupture. Thus, for effective tumor treatment, the key is to rationally design logistic systems to restore the supply chain.Under these circumstances, this Account summarizes our recent attempts to exploit the immunogenic trait of synthetic particles to enhance the distribution, presentation, and immune activations of the whole priming process in cancer vaccines: (1) Raw material (tumor antigen/signals) procurement: We illustrated the efforts to deliver antigens to antigen presenting cells (APCs) and draining lymph nodes for potent internalizations, and put more emphasis on the structural effect of sizes, charges, shapes, and assembly strategies for the antigen depot, lymph node transfer, and APC endocytosis. (2) Manufacture of cytotoxic T lymphocytes (CTLs) via APC recognition and presentation: We centered on exploiting the softness of two-dimensional graphene and Pickering emulsions to dynamically potentiate the immune recognition, and demonstrating the recent advances in lysosome escape strategies for enhanced antigen cross-presentations. (3) Marketing the accumulations of CTLs and the reversal of an immunosuppressive microenvironment within the tumor: We demonstrated the previous attempts to inherently cultivate the tumor tropism of the T cells via the multiantigenic repertoire and discussed the advances and challenges of combinatory cancer vaccines with an immune checkpoint blockade to reinforce the antitumor efficacy. Collectively, this Account aims to illustrate the potential of the particulate cancer vaccines to recapitalize the inherent host immune responses for the maximum antitumor effect. And by integrating the antitumor supply chain, optimized synthetic particles may shed light on the development of safe and effective particulate cancer vaccines.

In this study, a full-length cDNA sequence of SoxE (subgroup E within the Sox family of transcription factors) was cloned from Macrobrachium nipponense and named MnSoxE1. The full-length cDNA of MnSoxE1 is 1748 bp, consisting of a 110 bp 5′ UTR, a 105 bp 3′ UTR, and a 1533 bp ORF that encodes 510 amino acids. Conserved domains showed that MnSoxE1 has a high similarity to the SoxE gene of Penaeus vannamei. Phylogenetic tree analysis classified that MnSoxE1 with the SoxE gene of other arthropods into one clade. These results suggested that MnSoxE1 belongs to the SoxE subgroup. During embryonic development, MnSoxE1 was mainly expressed in the gastrula stage, implicating its involvement in tissue cell differentiation and formation. In the post-larval stages, the expression of MnSoxE1 continued to increase on days 1–10. The expression level in males was significantly higher than that in females. Males are clearly distinguishable from females on post-larval day 25, showing that MnSoxE1 may play a role in promoting early development and germ cell and gonadal differentiation, especially for males. qPCR analysis showed that MnSoxE1 may also be involved in oogonium proliferation during ovary development. Further in situ hybridization analysis revealed that MnSoxE1 was mainly located in oocytes and spermatocytes, especially in sertoli cells, and implies that it may be involved in the development of oocytes and spermatocytes, as well as the maintenance of testes in mature prawns. These results indicate that MnSoxE1 is involved in gonadal differentiation and development in M. nipponense, especially testis development.

Opsins are photoreceptors with important roles in reproductive regulation in birds and fishes. In the present study, we identified an opsin gene from the eyes of the oriental river prawn Macrobrachium nipponense using expressed sequence tag analysis and rapid amplification of cDNA ends. The full-length transcript contained 1382 base pairs, encoding 375 amino acids. It was classified into the long-wavelength opsin group by phylogenetic analysis, and designated Mn-LW. Mn-LW expression demonstrated significant seasonal variation in somatic tissues from both male and female prawns, with the highest expression in the eyes, and expression also shown in the ovary. The expression profiles of Mn-LW in eyes and ovary were positively related to ovarian development. In situ hybridization showed that Mn-LW was present in retinular cells in the eye and oocytes in the ovary. Injection of Mn-LW dsRNA in vivo effectively down-regulated Mn-LW expression levels compared with control levels. Mn-LW dsRNA injection also significantly reduced vitellogenin (Vg) expression, indicating a close relationship between Mn-LW and Vg in ovarian development. These results suggest that Mn-LW may play an important role in Vg synthesis and accumulation during ovarian maturation in M. nipponense.

As an important economical freshwater prawn, Macrobrachium nipponense has difficulty with adapting to hypoxia. In this study, comparative transcriptome analysis was used for the first time to explore the differences between different living states of Macrobrachium nipponense under hypoxia. A total of 94.22 Gb clean reads were obtained and assembled into 54,688 unigenes. A total of 224, 266, and 750 differently expressed genes were found in the comparison of the control and death groups, the control and moribund groups, and the control and survived groups, respectively. Three signal pathways closely related to hypoxia were found by enriching of the signal pathways in three comparison groups. In addition, much attention was focused on the differential genes in these pathways. Oxidative stress related genes, such as 70 kDa heat shock protein, phosphoenolpyruvate carboxykinase and cyclooxygenase were differentially expressed in different comparisons. After comparing with previous studies, cyclooxygenase was found to be an important hypoxia-related gene that is fully involved in the hypoxic response. Interestingly, two new genes with no Nr annotation were found in this manuscript. This manuscript will enrich our understanding of oxidative stress response to hypoxia and provide a theoretical basis for the subsequent solution of apoptosis caused by hypoxia.

The aim of this study was to explore the function of the Mn-Spook gene, which was found in the ovary transcriptome of the Oriental river prawn (Macrobrachium nipponense). The Spook gene, which is the precursor gene of 20-hydroxyecdysone (20E), plays an important role in the process of molting in many arthropods, but its function in M. nipponense is unclear. We cloned the full-length Mn-Spook gene from the ovary of M. nipponense and found that it had the same conserved domains as the P450 gene of the Halloween family of genes. The Mn-Spook gene was highly expressed in ovary and gill tissue during the breeding period. During ovarian development, Mn-spook gene expression was highest at the nearly-ripe stage, and it also was highly expressed in the zoea developmental stage. Cellular localization analysis showed that Mn-Spook signals accumulated in the cytoplasmic membrane and nucleus of oocytes. Finally, we used RNA interference to evaluate the function of the Mn-Spook gene. Compared with the control group, in vivo injection of Mn-Spook dsRNA effectively downregulated the expression of Mn-Spook and the content of 20E. The molting frequency of M. nipponense in the experimental group also was significantly inhibited. These results demonstrated that the Mn-Spook gene played an important role in the molting process of M. nipponense.

Macrobrachium nipponense has the characteristics of fast ovarian development cycle, which leads to the coexistence of multiple generations, the reduction of commodity specifications and the low economic benefit. Therefore, the study on the mechanism of ovarian development is of great significance to the development of industry. Cyclin A (CycA)is a key gene regulating ovarian development in vertebrates, but little information was available for its function in crustaceans. In this study, the full-length cDNA of Mn-CycA was obtained from the ovary. The full-length cDNA (2033 bp) with an open reading frame of 1368 bp, encoded a 456-amino acid protein. qRT-PCR revealed tissue-specific expression pattern of Mn-CycA, with abundant expression in the ovary. Results in different developmental stages of ovary indicated that Mn-CycA expression is positively correlated with ovarian maturation. qRT-PCR In different developmental stages, the expression of Mn-CycA mRNA gradually increased during the embryonic stage and decreased significantly on the first day of the hatching stage. At the 25th day of the metamorphosis stage, the expression level of Mn-CycAmRNA in female shrimp was 3.5 times higher than that in male shrimp, which may be related to the proliferation of oogonia and the formation of oocytes. In situ hybridization (ISH) of ovary showed Mn-CycA was examined in all stages and was mainly located in oogonia and oocytes. Compared with the control group, the obvious change of gonad somatic index (GSI) proved that injection of Mn-CycA dsRNA could delay the ovarian development cycle, which provided strong evidence for the involvement of Mn-CycA in ovarian maturation and oogenesis, and expanded a new perspective for studying the fast ovarian development cycle in M. nipponense.

The goal of this study was to analyze the functions of DNA polymerase zeta catalytic subunit (Rev3) in the oriental river prawn Macrobrachium nipponense (Mn-Rev3) with a focus on its potential roles in sex differentiation and development. The full length of Mn-Rev3 cDNA sequence was 6832 base pairs (bp) with an open reading frame of 6102 bp encoding 2033 amino acids. Mn-Rev3 showed the closest evolutionary relationship with Penaeus vannamei. The highest expression level of Mn-Rev3 occurred in the hepatopancreas and strong signals were observed in hepatopancreas cells, suggesting that Mn-Rev3 played a role in the immune system. Expression levels of Mn-Rev3 also were relatively high in the androgenic gland and testis, suggesting its potential roles in male sexual differentiation and development. During development, expression of Mn-Rev3 was highest on larval day 15 and relatively high from post-larval day 1 (PL1) to PL15, indicating that it played essential roles in promoting metamorphosis and gonad differentiation and development in M. nipponense. Strong Mn-Rev3 signals were detected in spermatids, spermatocytes, and sperm in the testes, and Mn-Rev3 expression was higher in the testes during the reproductive season than in the non-reproductive season. This result indicated that Rev3 promoted whole testis development, and especially sperm development, in M. nipponense. The expression level of Mn-Rev3 was high from ovary V to ovary II stages, indicating that Rev3 may be involved in yolk deposition. The expression level of Mn-insulin-like androgenic gland hormone (Mn-IAG) and the content of testosterone showed the same expression pattern as that of Mn-Rev3 after injection of double-stranded RNA of Mn-Rev3, which indicated that Rev3 had positive effects on male sexual differentiation and development in M. nipponense. The results of this study advance our understanding of male sexual development in M. nipponense and provide the basis for further studies of male sexual differentiation and development in crustaceans.

A better understanding of the mechanisms underlying the male sexual differentiation of Macrobrachium nipponense is urgently needed in order to maintain sustainable development of the M. nipponense industry. Environmental factors, especially temperature and illumination, have dramatic effects on gonadal development. The aim of the present study was to identify key genes and metabolites involved in the male sexual differentiation and development of M. nipponense through integrated metabolomics and transcriptome analyses of the testis in response to different temperatures and illumination times. A total of 268 differentially abundant metabolites and 11,832 differentially expressed genes (DEGs) were identified. According to integrated metabolomics and transcriptome analyses, glycerophospholipid and sphingolipid metabolism was predicted to have dramatic effects on the male sexual differentiation and development of M. nipponense. According to the KEGG enrichment analysis of DEGs, oxidative phosphorylation, glycolysis/gluconeogenesis, the HIF-1 signaling pathway, the citrate cycle, steroid hormone synthesis, and the spliceosome complex were predicted to promote male differentiation and development by providing adenosine triphosphate, promoting the synthesis of steroid hormones, and providing correct gene products. Quantitative polymerase chain reaction analysis and in situ hybridization showed that the SDHB, PDE1, HSDL1, CYP81F2, SRSF, and SNRNP40 genes were differentially expressed, suggesting roles in the male sexual differentiation and development of M. nipponense. Strong candidate sex-related metabolic pathways and genes in M. nipponense were identified by integrated metabolomics and transcriptome analyses of the testis in response to different temperatures and illumination times, as confirmed by PCR analysis and in situ hybridization.

Gem-associated protein 2-like isoform X1 (GEM) was previously predicted to be involved in the sexual development of male Macrobrachium nipponense. In this study, we analyze the GEM functions in depth using quantitative polymerase chain reaction (qPCR), in situ hybridization, and RNA interference (RNAi). The full-length Mn-GEM cDNA sequence was 1018 base pairs (bp) long with an open reading frame of 777 bp encoding 258 amino acids. qPCR analysis of Mn-GEM in different tissues and developmental stages showed that Mn-GEM was highly expressed in the gonad and from post-larval developmental stage day 5 (PL5) to PL15, which indicated that GEM has potential roles in gonad differentiation and development in M. nipponense. In situ hybridization and qPCR analysis of various stages of the reproductive cycle of the testis and ovary indicated that GEM may promote spermatid development and gametogenesis in M. nipponense. After injecting with double-stranded RNA (dsRNA) of Mn-GEM, mRNA expression of Mn-insulin-like androgenic gland hormone (Mn-IAG) and the content of testosterone increased with the decrease of Mn-GEM expression, indicating that GEM has negative effects on the male sexual differentiation and development in M. nipponense. Results of this study highlight the functions of GEM in M. nipponense, which can be applied to future studies of male sexual development in M. nipponense and other crustacean species.