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White spot syndrome virus (WSSV) is an exceptionally harmful virus that generally causes high levels of mortality in cultured shrimp. Attempts at viral suppression have been made to control the disease and have achieved limited efficiency. Recent advances in genome editing technology using CRISPR/Cas9 have led to potential innovations to prevent or treat many viral diseases. In this study, a CRISPR/Cas9 system was applied to WSSV genome cleavage to suppress WSSV infection in shrimp. The U6 promoter sequence was identified. A chimeric DNA vector consisting of the shrimp U6 promoter with gRNA expression sequences specific to two sites of the WSSV genome and the WSSV ribonucleotide reductase promoter with the Cas9 DNA sequence in pAC-sgRNA-Cas9 was constructed. The expression of gRNAs specific to the WSSV genome and Cas9 was determined in primary cultured hemocyte cells and in shrimp tissue via RT‒PCR. The efficacy of CRISPR/Cas9-WSSV for WSSV genome cleavage was determined in vitro and against WSSV-infected Penaeus vannamei. The reaction of synthetic gRNAs and recombinant Cas9 was able to cleave WSSV DNA amplicons, and shrimp that received CRISPR/Cas9-WSSV presented significantly lower WSSV DNA. In addition to interfering with viral DNA propagation, CRISPR/Cas9-WSSV encapsulated with IHHNV-VLP also stimulated an immune-related gene response. Treatment with CRISPR/Cas9-WSSV against WSSV challenge resulted in a significantly longer survival period. This finding has led to the development and application of a CRISPR/Cas9 system for WSSV infectious disease control, which could be used for managing shrimp aquaculture in the future. [ABSTRACT FROM AUTHOR]

In this study, the complex organization of the AnG in the giant freshwater prawn Macrobrachium rosenbergii was revealed using various techniques, including conventional histology, histochemistry, scanning electron microscopy, and X-ray tomography. The results showed the diversity of cells in the AnG and the detailed organization of the labyrinth's tubule into four radiated areas from the central to peripheral zones. The study also demonstrated the expression of some vertebrate kidney-associated homolog genes, aquaporin (AQP), solute carrier family 22 (SLC-22), nephrin, and uromodulin, in the AnG by qPCR. The result of in situ hybridization further showed the localization of SLC-22 and AQP transcript in the bladder and labyrinth's epithelium, specifically in regions 2, 3, and 4. Additionally, the study revealed neuropeptide expressions in the AnG by qPCR and in situ hybridization, i.e., crustacean hyperglycemic hormone (CHH) and molt inhibiting hormone (MIH), implying that the AnG may have a role in hormone production. Moreover, male and female prawns exhibited different levels of AQP, SLC-22, nephrin, and CHH expressions during the premolt and intermolt stages, suggesting a crucial role relevant to the molting stages. In conclusion, this study clarified the complex structure of the AnG in M. rosenbergii and demonstrated for the first time the expression of vertebrate kidney-associated genes and the possible endocrine role of the AnG. Further investigation is needed to clarify the role of these genes, particularly during ecdysis. The implications of these findings could significantly advance our understanding of the AnG in decapod crustaceans. [ABSTRACT FROM AUTHOR]

The giant freshwater prawn Macrobrachium rosenbergii is one of the most important aquaculture species in Southeast Asia. In this study, in vitro culture of its hematopoietic tissue cells was achieved and characterized for use as a tool to study its pathogens that cause major farm losses. By transmission electron microscopy, the ultrastructure of the primary culture cells was similar to that of cells lining intact hematopoietic tissue lobes. Proliferating cell nuclear antigen (PCNA) (a marker for hematopoietic stem cell proliferation) was detected in some of the cultured cells by polymerase chain reaction (PCR) testing and flow cytometry. Using a specific staining method to detect phenoloxidase activity and using PCR to detect expression markers for semigranular and granular hemocytes (e.g., prophenoloxidase activating enzyme and prophenoloxidase) revealed that some of the primary cells were able to differentiate into mature hemocytes within 24 h. These results showed that some cells in the cultures were hematopoietic stem cells that could be used to study other interesting research topics (e.g. host pathogen interactions and development of an immortal hematopoietic stem cell line). [ABSTRACT FROM AUTHOR]

Sequestering of cholesterol (CHO) is a hallmark molecular event that is known to be associated with sperm gaining their fertilizing ability in a broad array of animals. We have shown previously that the level of CHO declines in the Macrobrachium rosenbergii sperm membrane when they are migrating into the vas deferens, prompting us to search for CHO transporters, one of which is Niemann–Pick type 2C (NPC2), within the prawn male reproductive tract. Sequence comparison of MrNPC2 with other NPC2, from crustaceans to mammals, revealed its conserved features in the hydrophobic cavity with 3 amino acids forming a CHO lid that is identical in all species analyzed. Expressions of MrNPC2 transcript and protein were detected in testicular supporting and interstitial cells and along the epithelial cells of the vas deferens. As confirmed by live cell staining, the testicular sperm (Tsp) surface was devoid of MrNPC2 but it first existed on the vas deferens sperm, suggesting its acquisition from the luminal fluid, possibly through trafficking of multi-lamellar vesicles during sperm transit in the vas deferens. We further showed that recombinant MrNPC2 had a high affinity towards CHO in the lipid extracts, either from Tsp or from lipid vesicles in the vas deferens. Together, our results indicated the presence of MrNPC2 in the male reproductive tract, which may play an important role as a CHO modulator between the sperm membrane and vas deferens epithelial communication. [ABSTRACT FROM AUTHOR]

Phosphorylated sperm proteins are crucial for sperm maturation and capacitation as a priori to their fertilization with eggs. In the freshwater prawn, Macrobrachium rosenbergii, a male reproduction-related protein (Mar-Mrr) was known to be expressed only in the spermatic ducts as a protein with putative phosphorylation and may be involved in sperm capacitation in this species. We investigated further the temporal and spatial expression of the Mar-Mrr gene using RT-PCR and in situ hybridization and the characteristics and fate of the protein using immunblotting and immunocytochemistry. The Mar-Mrr gene was first expressed in 4-week-old post larvae and the protein was produced in epithelial cells lining the spermatic ducts, at the highest level in the proximal region and decreased in the middle and distal parts. The native protein had a MW of 17 kDa and a high degree of serine/threonine phosphorylation. It was transferred from the epithelial cells to become a major protein at the anterior region of the sperm. We suggest that it is involved in sperm capacitation and fertilization in this open thelycal species and this is being investigated. [ABSTRACT FROM AUTHOR]

We have investigated the cellular characteristics, especially chromatin condensation and the basic nuclear protein profile, during spermiogenesis in the common tree shrew, Tupaia glis. Spermatids could be classified into Golgi phase, cap phase, acrosome phase, and maturation phase. During the Golgi phase, chromatin was composed of 10-nm and 30-nm fibers with few 50-nm to 60-nm knobby fibers. The latter were then transformed into 70-nm knobby fibers during the cap phase. In the acrosome phase, all fibers were packed into the highest-order knobby fibers, each about 80–100 nm in width. These chromatin fibers became tightly packed in the maturation phase. In a mature spermatozoon, the discoid-shaped head was occupied by the acrosome and completely condensed chromatin. H3, the core histone, was detected by immunostaining in all nuclei of germ cell stages, except in spermatid steps 15–16 and spermatozoa. Protamine, the basic nuclear protein causing the tight packing of sperm chromatin, was detected by immunofluorescence in the nuclei of spermatids at steps 12–16 and spermatozoa. Cross-immunoreactivity of T. glis H3 and protamine to those of primates suggests the evolutionary resemblance of these nuclear basic proteins in primate germ cells. [ABSTRACT FROM AUTHOR]