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Transforming growth factor-β (Tgf-β) contributes to the development of liver diseases through its regulation of various cell types. While Tgf-β signaling to hepatic stellate cells (HSCs) and hepatocytes was shown to mediate hepatic damage, the effect of Tgf-β on other cells in liver is yet to be clearly defined. Herein we identified a regulatory function of macrophage Tgf-β signaling in liver injury. We found that transgenic mice expressing constitutively active Tgf-β receptor type I ( TβRI CA ) under the control of Fsp1-Cre ( TβRI CA / Fsp1-Cre mice) were less susceptible to concanavalin A (conA)-induced autoimmune hepatitis. Liver tissue examination showed a decrease of necrotic area in conA-treated TβRI CA /Fsp1-Cre liver compared to those of wild-type mice. Blood test revealed that serum aminotransferases were significantly reduced in conA-treated TβRI CA /Fsp1-Cre mice as compared to those of wild-type mice. Immunohistochemistry for CD3 and myeloperoxidase demonstrated that there was a decreased accumulation of T cells and neutrophils, respectively, whereas ELISA showed that IL-4, IL-5, IL-10, IL-12 and IFN-γ was increased in livers of conA-treated TβRI CA /Fsp1-Cre mice. Alternatively activated macrophage (M2) polarization was significantly elevated in livers of conA-treated TβRI CA /Fsp1-Cre mice as indicated by enhanced hepatic expression of CCR2 and CD206 as well as increased numbers of liver macrophages expressing M2 subtype marker, CD163. qPCR analysis indicated an increased expression of TβRI CA , Arg1, Ym1, CD206, Snail1, Foxo1 and IRF4 as well as a decreased expression of MHC class II and CD1d in liver macrophages that were isolated from TβRI CA / Fsp1-Cre mice. Moreover, flow cytometry analysis showed a lower number of NKT cells in livers of conA-treated TβRI CA / Fsp1-Cre mice when compared to those of wild-type mice. In conclusion, Fsp1-Cre-mediated expression of TβRI CA lead to a decreased conA-induced liver injury that was associated with enhanced M2 macrophage polarization and reduced NKT cell recruitment., Competing Interests: 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., (© 2025 The Authors.)

This study evaluated the growth and gut performance of shrimp fed three isonitrogenous diets (37% crude protein) with varying inclusions of fish meal (FM) and soybean meal (SBM): F1 (27.5% FM), F2 (10% FM + 23.5% SBM), and F3 (38% SBM). Over a 28-day period, feed intake, feed conversion ratio (FCR), and survival rates showed no significant differences among the groups. However, shrimp fed F2 and F3 exhibited significantly higher weight gain and average daily growth (ADG) compared to those fed F1 (P < 0.05). Gut performance analysis revealed that F3 consistently had the highest gut passage time (GPT), while F1 had the lowest. By day 28, shrimp fed F2 displayed elevated gut retention time (GRT). F1-fed shrimp showed a high gut passage rate (GPR), whereas F3-fed shrimp had a low GPR until day 21, with differences becoming negligible by day 28. Histological examination of the hepatopancreas revealed an increased R-cell population in shrimp fed F3. These findings highlight the adaptability of shrimp to different dietary compositions and underscore the importance of considering multiple factors when assessing the impacts of feed on growth and physiology., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Our previous studies revealed a mating attractant or possibly a pheromone released from molting reproductive mature female prawns, Macrobrachium rosenbergii, stimulates the expression of insulin-like androgenic gland hormones in a co-culture system. The released attractant is perceived by olfactory receptors with setae located on the short lateral antennules (slAn), which connect to the olfactory neuropil in the central nervous system (CNS) of male prawns. This neural signaling propagating through the CNS is mediated by at least four neuropeptides, namely neuropeptide F (NPF), short NPF (sNPF), tachykinin (TK), and allatostatin-A (ATS-A) whose transcripts have been detected in the present study. These deduced sequences, along with their conserved domains, serve as signatures of the identified neuropeptides, which were then compared with those found in other crustaceans and insects, whose nucleotide sequences were obtained from the nucleotide database. RT-PCR identified the expressions of the transcripts encoding these neuropeptides in the CNS. In situ hybridization specifically localized these transcripts in olfactory-associated neurons of cluster 9/11 of the deutocerebrum. Quantitative real-time PCR was used to quantify the expressions of the transcripts in response to the female attractants under different co-culture conditions: males with molting females (G1), males with intermolt females (G2), and slAn ablated males with molting females (G3). The transcripts were significantly increased on days 4-8 in the brain (Br) of males in G1 but not in G2 and G3. This suggests that expressions of the transcripts encoding the neuropeptides are associated with the perception of female mating pheromones through the slAn. This study is the first to show that female mating chemicals regulate the expressions and abundance of the olfactory neuropeptides, thus providing valuable insights for manipulation of mating of this species in aquaculture production., 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 © 2025 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., (© 2024. The Author(s).)

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., (© 2024. The Author(s).)

White spot syndrome virus (WSSV) presents a considerable peril to the aquaculture sector, leading to notable financial consequences on a global scale. Previous studies have identified hub proteins, including WSSV051 and WSSV517, as essential binding elements in the protein interaction network of WSSV. This work further investigates the functional structures and potential applications of WSSV hub complexes in managing WSSV infection. Using computational methodologies, we have successfully generated comprehensive three-dimensional (3D) representations of hub proteins along with their three mutual binding counterparts, elucidating crucial interaction locations. The results of our study indicate that the WSSV051 hub protein demonstrates higher binding energy than WSSV517. Moreover, a unique motif, denoted as "S-S-x(5)-S-x(2)-P," was discovered among the binding proteins. This pattern perhaps contributes to the detection of partners by the hub proteins of WSSV. An antiviral strategy targeting WSSV hub proteins was demonstrated through the oral administration of dual hub double-stranded RNAs to the black tiger shrimp, Penaeus monodon, followed by a challenge assay. The findings demonstrate a decrease in shrimp mortality and a cessation of WSSV multiplication. In conclusion, our research unveils the structural features and dynamic interactions of hub complexes, shedding light on their significance in the WSSV protein network. This highlights the potential of hub protein-based interventions to mitigate the impact of WSSV infection in aquaculture., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Panrat et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The presence of endogenous viral elements (EVE) in the penaeid shrimp genome has been recently reported and suggested to be involved in the host recognition of viral invaders. Our previous report of a search for EVE of infectious hypodermal and haematopoietic necrosis virus (IHHNV-EVE) in the Thai Penaeus monodon whole genome sequence project (GenBank accession no. JABERT000000000) confirmed the presence of three clusters of EVE derived from IHHNV in the shrimp genome. This study aimed to compare an immunohistochemistry method (IHC) and a PCR method to detect infectious IHHNV infection in shrimp. First, specimens collected from farms were checked for IHHNV using three PCR methods; two methods were recommended by WOAH (309 and 389 methods), and a newly established long-range PCR for IHHNV (IHHNV-LA PCR) targeting almost the whole genome (>90%) of IHHNV. Among 29 specimens tested, 24 specimens were positive for WOAH methods (at least one method). Among 24 WOAH-positive specimens (WOAH+), there were 18 specimens with positive IHHNV-LA PCR method (WOAH+/LA+), six specimens with negative IHHNV-LA PCR method (WOAH+/LA-). Six specimens were negative for all methods (WOAH-/LA-). The positive signals detected by IHC method were found only in the specimens with WOAH+/LA+. The results suggest that the WOAH+/LA- specimens were not infected with IHHNV, and the positive WOAH method might result from the EVE-IHHNV. The study recommends combining the IHHNV-LA PCR method and IHC with positive PCR results from WOAH's recommended methods to confirm IHHNV infection., (© 2023 The Authors. Journal of Fish Diseases published by John Wiley & Sons Ltd.)

Seawater (SW)-acclimated Nile tilapia, Oreochromis niloticus, can tolerate up to 30 g.L -1 SW but rarely produce offspring. The embryos of SW-acclimated O. niloticus survived equally well from 0- to 10-g.L -1 environment but not under 20-g. L -1 . However, when the embryos were incubated under 10 g.L -1 during days 0-3, and then the salinity was suddenly shifted to and maintained at 20 g.L -1 during days 4-6, their survival rate was comparable to those incubated under 0 and 10 g.L -1 . To elucidate a molecular adaptation of the embryos that survived different salinity environments, the proteomic profiles of the newly hatched embryos, or early larvae, hatched under 0 g.L -1 , 10 g.L -1 , and those being incubated at 10 g.L -1 during days 0-3 followed at 20 g.L -1 during days 4-6 were compared. Total proteins extracted from the samples were identified with a gel-free shot-gun proteomics approach using the Nile tilapia protein database. The early larvae from the three groups expressed 2295 proteins, and 279 proteins showed statistically different expressions among groups. Downregulation of the 182 proteins in the larvae hatched under 10 and 20 g.L -1 was found to include 22 proteins that are responsible for cellular responses to osmotic stress. This adaptation may be a crucial factor in reducing cellular metabolism and ion transport between the intra- and extra-cellular environment to stabilize cellular osmolality. In addition, some of these proteins suppress cellular damage from oxygen free radicals generated from the osmotic stress. Eighty-seven proteins significantly changed in the larvae hatched under 20 g.L -1 were clustered. Nineteen of the cellular stress response proteins, which were considered to be mortality induction, were described., 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.)

Microsporidia are obligate intracellular parasites that lost several enzymes required in energy production. The expansion of transporter families in these organisms enables them to hijack ATP from hosts. In this study, nucleotide transporters of the microsporidian Enterocytozoon hepatopenaei (EHP), which causes slow growth in economically valuable Penaeus shrimp, were characterized. Analysis of the EHP genome suggested the presence of four putative nucleotide transporter genes, namely EhNTT1, EhNTT2, EhNTT3, and EhNTT4. Sequence alignment revealed four charged amino acids that are conserved in previously characterized nucleotide transporters. Phylogenetic analysis suggested that EhNTT1, 3, and 4 were derived from one horizontal gene transfer event, which was independent from that of EhNTT2. Localization of EhNTT1 and EhNTT2 using immunofluorescence analysis revealed positive signals within the envelope of developing plasmodia and on mature spores. Knockdown of EhNTT2 by double administration of sequence specific double-stranded RNA resulted in a significant reduction in EHP copy numbers, suggesting that EhNTT2 is crucial for EHP replication in shrimp. Taken together, the insight into the roles of NTTs in microsporidian proliferation can provide the biological basis for the development of alternative control strategies for microsporidian infection in shrimp., (© 2023. The Author(s).)

Methyl farnesoate (MF), a crustacean equivalent of juvenile hormone (JH) of insects, is known to be produced from the mandibular organ (MO). This study reports transcriptome analysis of Penaeus monodon MO and identifies putative genes encoding enzymes in the sesquiterpenoid pathway. A total of 44,490,420 clean reads were obtained and utilized for subsequent analysis. De novo assembly created 31,201 transcripts and 31,167 unigenes. To archive the functional annotation, all unigenes were annotated with KOG, KEGG, and GO. Putative genes encoding enzymes and regulatory proteins involved in the sesquiterpenoid pathway were obtained from the MO transcriptome data based on the conserved domains and sequence homology. They included S-adenosylmethionine synthetase, farnesyl pyrophosphate synthase, short chain dependent dehydrogenase/reductase (SDR), NAD(P) + -dependent aldehyde dehydrogenase, S-adenosylmethionine-dependent methyltransferases or juvenile hormone acid-O-methyl transferase (JHAMT), farnesoic acid O-methyl transferase (FAMeT), juvenile hormone binding protein, cytochrome C/P-450 family 15 (CRYP15A1)/methylfarnesoate epoxidase (MFE), juvenile hormone epoxide hydrolase (JHEH), and juvenile hormone esterase (JHE). We first identified and characterized JHAMT orthologs inP. monodon(PmJHAMT). The complete cDNA sequence ofPmJHAMTconsisted of 1,221 nt encoded 271 amino acids with a conserved S-adenosyl methionine (SAM) binding domain. Phylogenetic analysis clusteredPmJHAMTinto the group JHAMT with the same clade of the crabPortunus trituberculausJHAMT. Moreover, the predicted three-dimensional structure of PmJHAMT showed remarkable similarity with the recent crystal structure ofthe Bombyx moriJHAMT homodimer. RT-PCR analysis revealed that PmJHAMT was exclusively expressed in MO and initially expressed at stage 3 postlarvae. In situ hybridization with a specific probe to PmJHAMT validated the specific expression of this gene in MO cells. Finally, we evaluated the regulation of MO by eyestalk inhibitory peptides. Diminishing MO inhibitory hormone through unilateral eyestalk ablation resulted in a significantly higher expression ofPmJHAMTin MO by quantitative PCR. This result indicated that the eyestalk inhibitory hormone inhibited MF synthesis byPmJHAMTgene suppression in the MO. This finding provides insight into the crustacean sesquiterpenoid pathway and improves our understanding of crustacean endocrinology., 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.)

Maternal stress (MS) has long-term effects on fetal brain development and consequently increases the risk of neuropsychiatric diseases in the offspring, however, the mechanism that links between early life stress and subsequent neuropsychiatric diseases is still not clear. It is well known that both neuroinflammation and autophagy dysfunction contributes to the pathology of psychiatric disorders. We hypothesized that MS might alter autophagy function and activate the neuroimmune response in the pup's brain. To test this hypothesis, we investigated the effects of MS on the expression of the autophagy biomarker and neuroimmune response in the hippocampus of rat pups. Results revealed that MS-induced a long-term decrease of LC3B-II throughout the postnatal periods, together with an increase of IL-6 and IL-10 in the hippocampus of rat pups during adolescence. These changes lasted at least until adulthood. Results from the In vitro studies showed that a partially toxic dose of corticosterone (CORT) induced a significant decrease of LC3B-II, together with an increase of IL-6 and IL-10, in the SH-SY5Y cells. Moreover, suppression of autophagy by mycophenolic acid (MPA) leads to an increased IL-6 and IL-10 expression in the CORT-treated SH-SY5Y cells. Findings suggested that CORT decreased autophagy dysfunction could activate neuroimmune response in the SH-SY5Y cells. Results from this study provides initial evidence for the relationship between stress hormone, autophagy dysfunction, and neuroimmune activation, which may be the linking mechanism between early-life stress and subsequent neuropsychiatric disorders., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Transformer 2 ( tra 2 ) and fruitless ( fru ) genes have been proven to play a key role in sex determination pathways in many Arthropods, including insects and crustaceans. In this study, a paralog of  P. monodon tra 2 (Pmtra 2), P. monodon ovarian associated transformer 2 ( PmOvtra 2) and 2 isoforms of  P. monodon fruitless -like gene ( Pmfru-1 and Pmfru-2 ) were identified and characterized . The full cDNA sequence of  PmOvtra 2 consisted of 1,774 bp with the longest open reading frame (ORF) of 744 bp encoding for 247 amino acids. The PmOvtra 2 exhibited a predicted RNA-recognition motif (RRM) domain and two arginine-serine (RS) regions, suggesting its function in RNA splicing. The full cDNA sequence of  Pmfru-1 consisted of 1,306 bp with 1,182 bp ORF encoding for 393 amino acids, whereas the full cDNA sequence of  Pmfru-2 consisted of 1,858 bp with 1,437 bp ORF encoding 478 amino acids. The deduced amino acid sequences of Pmfru-1 and Pmfru-2 exhibited highly conserved domains of Fru proteins, including Broad-complex, Tramtrack and Bric-a-brac (BTB), and zinc finger (ZF) domains. In addition, Pmfru-1 and Pmfru-2 were suggestively originated from the same single genomic locus by genomic sequence analysis . Specifically, Pmfru pre-mRNA was alternatively spliced for Pmfru-1 and Pmfru-2 to include mutually exclusive exon 7 and exon 6, respectively. Temporal and spatial expression of  PmOvtra 2 , Pmfru-1, and Pmfru-2 were also investigated by qPCR. The results showed that all were expressed in early developmental stages with undifferentiated gonads starting from nauplius until postlarvae . The expression of  PmOvtra 2 started at nauplius stage and gradually increased from mysis to postlarvae (PL) 1. However, the expression of  Pmfru-1 was low at the nauplii stage and slightly increased from protozoea to PL5, whereas the expression of  Pmfru-2 maintained a low level from nauplius to mysis and then gradually increased at the PL stages. Expressions of  PmOvtra 2, Pmfru-1, and Pmfru-2 were detected in various tissues including nervous tissue, gill, heart, hepatopancreas, gut, and gonads. Interestingly, the sexually dimorphic expression of  PmOvtra 2, Pmfru-1, and Pmfru-2 was demonstrated in fully developed gonads in which the ovary showed significantly higher expressions than the testis . The great difference in the expression pattern of  PmOvtra 2, Pmfru-1, and Pmfru-2 in the ovary and testis suggested their roles in the female sex determination in P. monodon ., Competing Interests: The authors declare there are no competing interests., (©2022 Thaijongrak et al.)

Enterocytozoon hepatopenaei (EHP) is an obligate intracellular parasite causing hepatopancreatic microsporidiosis (HPM) in cultivated shrimp in Asian countries. One strategy to control EHP is to identify and eliminate biological reservoir(s) in shrimp ponds. Several marine and brackish-water organisms, including false mussels (Mytilopsis) have been reported to test positive for EHP using the PCR method. Thus, we tested Thai false mussel Mytilopsis leucophaeata collected from the 6 ponds with EHP-infected shrimp for the presence of EHP using SWP-PCR. Results revealed the sampled mussels from all 6 ponds were PCR positive. Subsequent bioassays were carried out to study EHP transmission between mussels and shrimp. Firstly, the naïve mussels were cohabitated with EHP-infected shrimp and all mussels were SWP-PCR positive at day 20 post cohabitation. One batch of such PCR-positive mussels was transferred for cohabitation with naïve shrimp and 37.5% EHP-positive shrimp were observed within 10 days. Tissue analysis of the SWP-PCR-positive mussels using light microscopy, in situ hybridization technique and electron microscopy did not confirm EHP infection. In summary, there was no evidence demonstrating that Mytilopsis leucophaeata was itself infected with EHP. However, the false mussels were apparently capable of carrying infectious spores for some period after ingestion and serving as a mechanical or passive carrier. The results support previous reports warning of the danger of feeding living or fresh bivalves to broodstock shrimp in hatcheries or shrimp in rearing ponds without prior heating or freezing., (Copyright © 2021 Elsevier Inc. All rights reserved.)

In this study, a novel Crustacean Hyperglycemic Hormone-type II gene (CHH-type II) was identified and biologically characterized in a shrimp, Penaeus monodon . Based on its structure and function, this gene was named P. monodon vitellogenesis-inhibiting hormone ( PemVIH ). The complete cDNA sequence of PemVIH consisted of 1,022 nt with an open reading frame (ORF) of 339 nt encoding a polypeptide of 112 amino acids. It was classified as a member of the CHH-type II family based on conserved cysteine residues, a characteristically positioned glycine residue, and the absence of CHH precursor-related peptide (CPRP) domain. The deduced mature PemVIH shared the highest sequence similarities with giant river prawn sinus gland peptide A. Unlike P. monodon gonad-inhibiting hormone ( PemGIH ), PemVIH was expressed only in the brain and ventral nerve cord, but not the eyestalks. Whole mount immunofluorescence using a newly generated PemVIH antiserum detected positive signals in neuronal cluster 9/11 and 17 of the brain, commissural ganglion (CoG), and neuronal clusters of ventral nerve cord. The presence of PemVIH-positive neurons in CoG, a part of stomatogastric nervous system, suggested a potential mechanism for crosstalk between nutritional and reproductive signaling. The role of PemVIH in vitellogenesis was evaluated using RNA interference technique. Temporal knockdown of PemVIH in female subadults resulted in a 3-fold increase in ovarian vitellogenin expression, suggesting an inhibitory role of PemVIH in vitellogenesis. This study provided novel insight into the control of vitellogenesis and additional strategies for improving ovarian maturation in P. monodon without the current harmful practice of eyestalk ablation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Laphyai, Kruangkum, Chotwiwatthanakun, Semchuchot, Thaijongrak, Sobhon, Tsai and Vanichviriyakit.)

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)., Competing Interests: Conflict of interestThe authors declare that they have no conflict of interest to disclose., (© The Author(s), under exclusive licence to Springer Nature B.V. part of Springer Nature 2021.)

In crustacean, hemocytes are known as crucial components of crustaceans' innate immunity against pathogens. Drastic hemocytes reduction during infectious disease is apparently related to disease severity and calls for a health status evaluation and aquaculture management. The molecular pathogenesis of hemocytes loss during bacterial infection was elucidated with VP AHPND challenged in M. rosenbergii. We report herein a correlation between hemocyte loss and the pathogenicity and aggressive immune response in hematopoietic tissues of moribund M. rosenbergii. In this study, adult freshwater prawn was administered an LC 50 dose of VP AHPND ; bacterial clearance ensued, and success was reached within 24 h. Hemocytes increased in survival, yet drastically decreased in moribund prawn. Pathological analysis of hematopoietic tissue of moribund prawn showed apparent abnormal signs, including the presence of bacteria, a small number of mitotic cells, cellular swelling, loosening of connective tissue, and karyorrhectic nuclei cells. A significant upregulation of a core apoptotic machinery gene, caspase-3, was detected in hematopoietic tissue of moribund shrimp, but not in those of Escherichia coli DH5α (non-pathogenic bacteria) and VP AHPND survival prawn. The highest level was found in the moribund group, which confirms the occurrence of apoptosis in this hematopoietic tissue. Further, our results suggest that hematopoietic tissue damage may arise from inflammation triggered by an aggressive immune response. Immune activation was indicated by the comparison of immune-related gene expression between controls, E. coli (DH5α)-infected (non-pathogenic), and VP AHPND -infected survival groups with moribund prawn. RT-PCR revealed a significant upregulation of all genes in hematopoietic tissues and hemocytes within 6-12 h and declined by 24 h. This evident related to the almost VP AHPND are clearance in survival and E. coli (DH5α) challenged group in contrast with drastic high expression was determined in moribund group. We conclude that a reduction of renewing circulating hemocytes in fatally VP AHPND -infected prawn was caused by an acute self-destructive immune response by hematopoietic cells., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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.

Streptococcus agalactiae secrete virulence factors believed to be able of killing host tissues, especially under elevated water temperature. A direct effect of S. agalactiae secretory products on tilapia cells was tested on the tilapia kidney (TK-1) cell culture. The bacteria were cultured under four different temperature levels: 22, 29, 32 and 37°C; the cell-free portion was processed through SDS-PAGE; and distinct bands were identified by LC-MS/MS. At least, three virulence factors were identified, Bsp, PcsB and CAMP factor, with increasing levels as the cultured temperature rose. Expressions of bsp, pcsB and cfb were also up-regulated with the rising of the temperature in S. agalactiae culture. The supernatant from the bacteria cultured under specified temperatures was added into TK-1 cell-cultured wells. Morphological damage and mortality of the cultured cells, as determined by MTT method, were increased progressively from the supernatant treatment according to the rise of temperature in S. agalactiae culture. This study suggests that the production of the three virulence factors of S. agalactiae reported herein is temperature-dependent, and it is likely that CAMP factor directly kills the TK-1 cells since the other two types of protein are involved in S. agalactiae cell division and the bacterial adherence to host tissues., (© 2020 John Wiley & Sons Ltd.)

One of the clinical manifestations of streptococcosis is swimming errors of the infected fish, which is likely caused by lesions in the brain. As most studies described brain histopathology in streptococcosis as meningitis, with a limited description of lesions in the whole brain, the aim of this study was therefore to explore histopathology of the whole brain of red tilapia experimentally infected with Streptococcus agalactiae serotype III. Transcripts relating to motoneuron functions and inflammatory responses were also investigated. In the S. agalactiae-infected fish, the parenchyma of the whole brain and its associated meninx primitiva were found to be markedly infiltrated by mononuclear cells and Gram-positive cocci. Hemorrhage, neuronal necrosis, and localized spongiform histopathology were observed, especially within the midbrain and the cerebellum. The lesion was observed in the medial longitudinal fasciculus and its nucleus. Expressions of the transcripts CD166, GAP43, SMN, and SV2B of the infected fish did not change, while those of IL-1β and TNF-α were significantly upregulated. It is likely that S. agalactiae cause extensive damage to the fish brain, especially in areas that control swimming activities, through both direct invasion of the bacteria and acute inflammatory responses of the brain resident macrophages, or microglia., (© 2020 Wiley Periodicals, Inc.)

Insulin-like androgenic gland hormone (IAG) controls development of primary and secondary male sex-characteristics in decapod crustaceans. In male giant freshwater prawns, Macrobrachium rosenbergii, the IAG concentration correlates with male reproductive status and aggressiveness. When female prawns are co-cultured with males this can result in male size variations while this variation does not occur when males are cultured in monosex conditions. It was hypothesized that pheromone-like factors from female prawns may affect the abundance of IAG mRNA and protein in co-cultured males which would affect the pattern of sexual maturation of these males. In the present study, late premolt to postmolt females co-cultured with males for 7 days had a greater abundance of MrIAG mRNA transcript in all male phenotypes as well as for the gonad-somatic indexes (GSI). The abundance of MrIAG mRNA gradually increased from days 1 to 7 and using Western blot procedures MrIAG protein also increased in a similar pattern. Furthermore, with use of BrdU labeling, there was an increased cell proliferation in the spermatogenic zone of testicular tubules and in the spermatic duct epithelium during the 1 to 7 day co-culture period when there were increases in MrIAG mRNA and protein. In contrast, these effects were negated if short lateral antennules of males were ablated. Thus, results of the present study provide evidence that there might be female-molting factors which function as important regulators of androgenic gland function and gonadal maturation that were perceived by males via their short lateral antennules which are the olfactory organs., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Unlike that of vertebrates, the penaeid shrimp stomach is of ectodermic origin and is thus covered by a cuticle that is sloughed upon molting. It is composed of two chambers, here called the anterior and posterior stomach chambers, ASC and PSC, respectively. The PSC contains a filtration structure variously called a pyloric filter, filter press, gastric filter or gastric sieve (GS), and the last of these will be used here. The GS resembles an elongated, inverted-V, dome-like, chitinous structure with a midline ridge that is integral to the ventral base of the PSC. The dome surface is covered with a carpet-like layer of minute, comb-like setae bearing laterally branching setulae. This carpet serves as a selective filter that excludes large partially digested food particles but allows smaller particles and soluble materials to enter hepatopancreatic ducts that conduct them into the shrimp hepatopancreas (HP), where further digestion and absorption of nutrients takes place. Although the GS function is well known, its exclusion limit for particulate material has not been clearly defined. Using histological and ultra-structure analysis, we show that the GS sieve pore diameter is approximately 0.2-0.7 µm in size, indicating a size exclusion limit of substantially less than 1 µm. Using fluorescent microbeads, we show that particles of 1 µm diameter could not pass through the GS but that particles of 0.1 µm diameter did pass through to accumulate in longitudinal grooves and move on to the HP, where some were internalized by tubule epithelial cells. We found no significant difference in these sizes between the species Penaeus monodon and Penaeus vannamei or between juveniles and adults in P. vannamei This information will be of value for the design of particulate feed ingredients such as nutrients, therapeutic drugs and toxin-absorbing materials that may selectively target the stomach, intestine or HP of cultivated shrimp., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

The hematopoietic organ (HO) of the giant freshwater prawn Macrobrachium rosenbergii is a discrete, whitish mass located in the epigastric region of the cephalothorax, posterior to the brain. It is composed of hematopoietic cells arranged in a thick layer of numerous lobules that surround a central hemal sinus from which they are separated by a thin sheath. At the center of the sinus is the muscular cor frontale. The lobules extend radially outward from the sinus in three developmental zones. Basal Zone 1 nearest the sinus contains large hematopoietic stem cells with euchromatic nuclei that stain positive for proliferation cell nuclear antigen (PCNA). Zone 2 contains smaller, actively dividing cells as indicated by positive 5-bromo-20-deoxyuridine (BrdU) staining. Distal Zone 3 contains small, loosely packed cells with heterochromatic nuclei, many cytoplasmic granules and vesicles indicating that they will eventually differentiate into hemocytes and enter circulation. Three main arteries, namely the ophthalmic and the 2 branches of the antennary, connect the heart to the HO. Use of India ink and 0.1 μm fluorescent micro-beads injected into the heart revealed that the cor frontale could immediately remove foreign particles from hemolymph by filtration. Fluorescent beads were also detected in the hematopoietic tissue at 30 min after injection, indicating that it could be penetrated by foreign particles. However, the fluorescent signal completely disappeared from the whole HO after 4 h, indicating its role in removal of foreign particles. In conclusion, the present study demonstrated for the first time the detailed histological structures of the HO of M. rosenbergii and its relationship to hematopoiesis and removal of foreign particles from hemolymph., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

White tail disease caused by Macrobrachium rosenbergii nodavirus (MrNV) infection takes place only in nauplii, not adults, of M. rosenbergii prawn. Hemocyte homeostasis and immune-related functions derived from the hematopoietic tissue (Hpt) in adult prawn are presumed to play roles in resisting viral infection. To elucidate the role of the Hpt cell response to MrNV, a comparative transcriptome analysis was performed with MrNV-infected prawn at various time intervals. The results showed that there were 462 unigenes that were differentially expressed between mock and infected samples. BlastX sequence analysis revealed that two proteins, crustacean hematopoietic factor (CHF) and cell growth-regulating zinc finger protein (Lyar), are involved in hemocyte hematopoiesis and are up-regulated during MrNV infection. In fact, genes involved in cell growth regulation and immunity were highly expressed at 6 h and decreased within 24 h post-infection. Localization studies in the Hpt tissue revealed the presence of anti-lipopolysaccharide factor (ALF) and CHF mRNAs in Hpt cells. Considering these findings, we concluded that resistance to MrNV infection in adult prawn is due to an increase in humoral immune factors and the acceleration of hemocyte homeostasis by the dual roles of the Hpt organ in M. rosenbergii., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The giant freshwater prawn, Macrobrachium rosenbergii, is an important aquaculture species. A better understanding of the molecular components of reproduction in this species would help to advance the prawn production. In the present study, we demonstrated the presence of an egg laying hormone (ELH)-like peptide in the male reproductive system. First, an antibody to the abalone (a)ELH was generated, and by Western blot it was shown to specifically bound to a protein from the male M. rosenbergii reproductive tissues with a similar size to molluscan ELH. This aELH-like peptide was localized in spermatogonia in the testes of all three male morphotypes: blue claw, orange claw and small males. Moreover, the aELH-like peptide was detected in the epithelium of the spermatic duct and its associated smooth muscle cell layers and on the outer surface of spermatozoa. As well, the aELH-like peptide was detected in the spermatophore located in the female thelycum at 4-6 h post-mating, indicating that it was transferred to the female during copulation. Taken together, we suggest that this aELH-like peptide could be as a male inducing factor that helped to accelerate female spawning. Liquid chromatography of crude extracts and immunoblot analysis suggested that the aELH-like peptide could be further purified for ultimate characterization., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

In Southeast Asia, a new disease called scale drop disease (SDD) caused by a novel Megalocytivirus (SDDV) has emerged in farmed Asian sea bass (Lates calcarifer) in Singapore, Malaysia and Indonesia. We received samples from an Eastern Thai province that also showed gross signs of SDD (loss of scales). Clinical samples of 0.2-1.1 kg L. calcarifer collected between 2016 and 2018 were examined for evidence of SDDV infection. Histopathology was similar to that in the first report of SDDV from Singapore including necrosis, inflammation and nuclear pyknosis and karyorrhexis in the multiple organs. Intracytoplasmic inclusion bodies were also observed in the muscle tissue. In a density-gradient fraction from muscle extracts, TEM revealed enveloped, hexagonal megalocytiviral-like particles (~100-180 nm). By PCR using primers derived from the Singaporean SDDV genome sequence, four different genes were amplified and sequenced from the Thai isolate revealing 98.7%-99.9% identity between the two isolates. Since viral inclusions were rarely observed, clinical signs and histopathology could not be used to easily distinguish between SDD caused by bacteria or SDDV. We therefore recommend that PCR screening be used to monitor broodstock, fry and grow-out fish to estimate the current impact of SDDV in Southeast Asia and to prevent its spread., (© 2018 John Wiley & Sons Ltd.)

Background: The microsporidian Enterocytozoon hepatopenaei (EHP) is a spore-forming, intracellular parasite that causes an economically debilitating disease (hepatopancreatic microsporidiosis or HPM) in cultured shrimp. HPM is characterized by growth retardation and wide size variation that can result in economic loss for shrimp farmers. Currently, the infection mechanism of EHP in shrimp is poorly understood, especially at the level of host-parasite interaction. In other microsporidia, spore wall proteins have been reported to be involved in host cell recognition. For the host, heparin, a glycosaminoglycan (GAG) molecule found on cell surfaces, has been shown to be recognized by many parasites such as Plasmodium spp. and Leishmania spp., Results: We identified and characterized the first spore wall protein of EHP (EhSWP1). EhSWP1 contains three heparin binding motifs (HBMs) at its N-terminus and a Bin-amphiphysin-Rvs-2 (BAR2) domain at its C-terminus. A phylogenetic analysis revealed that EhSWP1 is similar to an uncharacterized spore wall protein from Enterospora canceri. In a cohabitation bioassay using EHP-infected shrimp with naïve shrimp, the expression of EhSWP1 was detected by RT-PCR in the naïve test shrimp at 20 days after the start of cohabitation. Immunofluorescence analysis confirmed that EhSWP1 was localized in the walls of purified, mature spores. Subcellular localization by an immunoelectron assay revealed that EhSWP1 was distributed in both the endospore and exospore layers. An in vitro binding assay, a competition assay and mutagenesis studies revealed that EhSWP1 is a bona fide heparin binding protein., Conclusions: Based on our results, we hypothesize that EhSWP1 is an important host-parasite interaction protein involved in tethering spores to host-cell-surface heparin during the process of infection.

In a number of marine animals, sperm serine proteases are important for fertilization. Penaeus monodon sperm require trypsin-like activity for a complete acrosome reaction, which exclusively occurs in sperm residing in the female thelycum. In this study, a complete cDNA sequence of reproductive tract-related Serine protease inhibitor (rrPmserpin) was identified. The longest open reading frame was composed of 1,366 nucleotides encoding 402 amino acids with a predicted pI of 6.86 and molecular mass of 44.88 kDa. The signal peptide cleavage site was identified as the 17th amino acid residue in the amino-terminus, and two potential N-glycosylation sites were predicted as post-translation modifications. A conserved reactive loop and fold similarities, identified through three-dimensional modeling, suggested that this gene is a member of the serpin family. The expression of rrPmserpin mRNA was prominent in the reproductive organs, including the testis, vas deferens, terminal ampoule containing the spermatophore, and the female thelycum. Inhibitory activity of recombinant rrPmSERPIN-6His was revealed from the negative correlation between the abundance of rrPmserpin mRNA and sperm trypsin-like activities, along with its inhibitory effects on chymotrypsin, trypsin, and thelycal proteases. Therefore, our results suggest that rrPmserpin participates in the regulation of the activity of a sperm protease and the decapacitation process., (© 2018 Wiley Periodicals, Inc.)

A 250-kDa protein was isolated from fluid in the middle spermatic duct (MSD) of the blue crab (Portunus pelagicus). N-terminal and partial amino acid sequences revealed that this MSD-specific protein is highly similar to the plasma-enriched protein Alpha-2 macroglobulin (α2M). The P. pelagicus ortholog (Ppα2M) is a large glycoprotein possessing mannose and N-acetylglucosamine residues. Ppa2m mRNA was detected in the spermatic duct, androgenic gland, and hematopoietic tissue, whereas the protein was primarily observed in the apical cytoplasm of MSD epithelium and in the matrix of the acrosome of MSD sperm; distally within spermatic duct, Ppα2M was lost from the sperm membrane but remained in the sperm acrosome. These results suggest that Ppα2M is expressed and glycosylated in the epithelium of spermatic ducts, secreted into MSD fluid, taken up by sperm in the MSD, and removed from the surface of sperm during its transit towards the female spermatheca. Given that Ppα2M also exhibits protease inhibitor activity, we hypothesize that acrosome localized Ppα2M may suppress premature acrosome reaction during post-testicular sperm maturation in this crab., (© 2017 Wiley Periodicals, Inc.)

White spot syndrome virus (WSSV) is a major causative agent in shrimp farming. Consequently, RNAi technology is an effective strategy to prevent WSSV infection in shrimp especially dsRNA targeting to rr2 of WSSV. In an effort to develop dsRNA expression in shrimp for control of WSSV infection, we developed a recombinant baculovirus expressing recombinant VP28 as the gene delivery system to carry a gene encoding dsRNA specific to rr2 for triggering the RNAi process in shrimp. The results showed that the recombinant baculovirus harboring VP28 was able to express VP28 indicated by Western blot with polyclonal antibody specific to VP28. VP28 transcript was detected in shrimp hemocytes after co-culture hemocytes with the recombinant baculovirus displaying VP28. In addition, we found that shrimp injected with the recombinant baculovirus displaying VP28 and encoding dsRNA synthetic gene specific to rr2 (Bac-VP28-dsrr2) showed the lowest cumulative mortality (33%) at 14days post infection (dpi) when compared to shrimp injected with baculovirus displaying VP28 (Bac-VP28) (64% cumulative mortality) (p<0.05). According to the results, shrimp injected with Bac-VP28-dsrr2 also showed significantly lower WSSV copies than shrimp injected with Bac-VP28 (p<0.05) along with the down-regulation of rr2 expression at 1, 3 and 7dpi. In conclusion, the Bac-VP28-dsrr2 was effective in prevention of WSSV infection. Therefore, the results obtained here can be applied to the prevention of WSSV infection by mixing the recombinant baculovirus with shrimp feed in the future., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Protein and lipid composition of sperm plasma membrane are modified as these gametes continue to mature during their transit along the spermatic tract. Our previous study revealed that during its journey through the spermatic duct of the black tiger prawn, Penaeus monodon, sperm cholesterol content decreases through the action of lipid-binding proteins within the luminal environment. In this study, the full cDNA sequence of epididymal secretory protein E1 (HE1), or Niemann-Pick C2 (NPC2), was cloned from P. monodon (termed Pmnpc2), and its conserved cholesterol/lipid-binding domain was characterized. The putative tertiary structure of PmNPC2 showed high similarity with the structure of Bos taurus NPC2. Pmnpc2 is expressed in many tissues, including the spermatic tract (i.e., testis, vas deferens, terminal ampoule) and the female thelycum. In situ hybridization revealed the presence of Pmnpc2 transcripts in the vas deferens, terminal ampoule, and thelycum epithelia, suggesting that PmNPC2 could be secreted into the lumen of the spermatic duct. A recombinant hexahistidine-tagged PmNPC2 (rPmNPC2-6His) was able to bind cholesterol and sperm lipid extracts, while co-incubation of sperm from the vas deferens with rPmNPC2-6His resulted in the depletion of cholesterol from these gametes. Together, these results suggest that PmNPC2 participates in sperm cholesterol efflux during the sperm maturation process in P. monodon. Mol. Reprod. Dev. 83: 259-270, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Schistosomiasis mekongi is one of the most important human parasitic diseases caused by Schistosoma mekongi in South-east Asia. The endemic area is the Mekong River sub-region from Laos to Cambodia. This parasite also infects dogs and pigs which are its alternative host species. Currently, the lack of reliable rapid diagnosis makes it difficult to monitor the infection and spreading of the disease. In this study, we screened the antigens of the parasite with sera of infected mice using Western blotting and identified proteins of interest with LC-MS/MS to obtain potential candidate proteins for diagnostic development. This assay yielded 2 immunoreactive bands at molecular masses of 31 and 22kDa. The 31kDa protein was the major band identified as cathepsin B, and its gene was cloned to obtain a full cDNA sequence (SmekCatB). The cDNA consisted of 1123bp and its longest reading frame encoded for 342 amino acids with some putative post translation modifications. The recombinant SmekCatB (rSmekCatB) with hexahistidine tag at the C-terminus was expressed in Escherichia coli and purified by Ni-NTA resin under denaturing conditions. The rSmekCatB reacted with sera of S. mekongi-infected mice. Indirect ELISA using rSmekCatB as the antigen to detect mouse antibodies, revealed a sensitivity of 91.67% for schistosomiasis mekongi and the specificity of 100%. Our data suggested that SmekCatB is one of the most promising parasitic antigens that could be used for the diagnosis of S. mekongi infection., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Scabraside D, a sulfated triterpene glycoside extract from sea cucumber Holothulia scabra, shows various biological activities, but effects on human cholangiocarcinoma cells have not previously been reported. In the present study, we investigated the activity of scabraside D against human cholangiocarcinoma (HuCCA) both in vitro and for tumor growth inhibition in vivo using a xenograft model in nude mice. Scabraside D (12.5-100 μg/mL) significantly decreased the viability and the migration of the HuCCA cells in a dose-dependent manner, with 50% inhibitory concentration (IC50) of 12.8 ± 0.05 μg/mL at 24 h. It induced signs of apoptotic cells, including shrinkage, pyknosis and karyorrhetic nuclei and DNA fragmentation on agarose gel electrophoresis. Moreover, by quantitative real-time PCR, scabraside D effectively decreased Bcl-2 while increasing Bax and Caspase-3 gene expression levels suggesting that the scabraside D could induce apoptosis in HuCCA cells. In vivo study demonstrated that scabraside D (1 mg/kg/day, i.p. for 21 days) significantly reduced growth of the HuCCA xenografts without adverse effects on the nude mice. Conclusively, scabraside D induced apoptosis in HuCCA cells and reduced the growth of HuCCA xenographs model. Therefore, scabraside D may have potential as a new therapeutic agent for cholangiocarcinoma.

In crustaceans, mating occurs during the ecdysis after female molting. During this period, a male transfers its spermatophore into a female which, in some species, stores the spermatophore for a long period prior to spawning and fertilization. However, in some species including the giant freshwater prawn, Macrobrachium rosenbergii, the male deposits its spermataphore onto the external surface of the thoracic segment of the female which affects the spawning time and maternal behavior. This study investigated the spawning behavior of the M. rosenbergii females, which was divided into pre-spawning, spawning, and post-spawning phases. It was revealed that mated female prawns with attached spermatophore exhibited an earlier spawning than unmated individuals, leading to assessment of the factors that may elicit this phenomenon. Four groups of female prawns were allocated to groups including mating females with spermatophore still attached, mating females with the spermatophore removed, artificially inseminated females with spermatophores, and an unmated control. There was a significant reduction in the time of egg-spawning in the presence of spermatophores, and the mating activity was also a contributing factor. Furthermore, over 90% of the mated and artificially inseminated females in which spermatophores were deposited carried the eggs in the abdominal brood chamber until completion of embryonic development while others discarded the eggs within 2 days post-spawning. This study implies that the spermatophore may contain ovulation-inducing factors which stimulate an earlier spawning and fostering of brooding behavior., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Coelomocytes are the first line of immune defense in marine animals. Their distributions are greatly variable even in the close animal species. In this study, we used lectin staining to aid in the classification and purification of these cells for further investigation of SOD distribution among coelomocytes of H. scraba. We classified coelomocytes into four types: type 1, lymphocytes; type 2, phagocytes; type 3, spherulocytes; and type 4, giant cells. Among four lectins used, Con A appeared to give a broad reactivity against most coelomocytes, except for giant cells. In addition, phagocytes usually engaged the highest fluorescent intensity with most lectins, with the exception of PNA, for which spherulocytes possessed the highest fluorescent intensity. Using FACS for fraction collection, it was found that F1 fraction contained the purest phagocyte population (> 95%), which was highly reactive with anti- superoxide dismutase (SOD) as revealed by immunoblotting and immunofluorescence staining, although some minor staining was also detected in spherulocytes. Our results thus provide a fundamental platform for comparing alterations that may happen to the population and SOD contents of coelomocytes when the sea cucumber is subjected to environmental changes that would activate their immune responses.

2-Cys peroxiredoxin (Prx) is the main antioxidant enzyme in Fasciola species for detoxifying hydrogen peroxide which is generated from the hosts' immune effector cells and the parasites' own metabolism. In this study, the recombinant Prx protein from Fasciola gigantica (rFgPrx-2) was expressed and purified in a prokaryotic expression system. This recombinant protein with molecular weight of 26 kDa was enzymatically active in reduction of hydrogen peroxide both in presence of thioredoxin and glutathione systems, and also protected the supercoiled plasmid DNA from oxidative damage in metal-catalyzed oxidation (MCO) system in a concentration-dependent manner. By immunoblotting, using antibody against rFgPrx-2 as probe, a native FgPrxs, whose MW at 25 kDa, was detected in all developmental stages of the parasite. Concentrations of native FgPrxs were increasing in all stages reaching highest level in adult stage. The antibody also showed cross reactivities with corresponding proteins in some cattle helminthes. Natural antibody to FgPrxs could be detected in the sera of mice at 3 and 4 weeks after infection with F. gigantica metacercariae. By immunofluorescence, FgPrxs was highly expressed in tegument and tegumental cells, parenchyma, moderately expressed in cecal epithelial cells in early, juvenile and adult worms. Furthermore, FgPrxs was also detected in the female reproductive organs, including eggs, ovary, vitelline cells, and testis, suggesting that FgPrxs might play an essential role in protecting parasite's tissues from free radical attack during their life cycle. Thus, FgPrxs is one potential candidate for drug therapy and vaccine development., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Sperm maturation in the epididymis involves multiple complex events, that include the adsorption of epididymal secretory proteins, re-organization and removal of sperm surface ligands. In this study, we investigated the existence and distribution of cathepsin D (CAT-D) transcripts and proteins in mouse reproductive tissues and proposed a transfer mechanism of CAT-D to the sperm surface. CAT-D transcripts were highly expressed in cultured Sertoli cells, but not in germ cells. The transcriptional level was relatively higher in the caput epididymis (CP) than in the cauda epididymis (CD). At the translational level, CAT-D was detected in testicular somatic cells and in the principal and basal cells in the CP. The expression of CAT-D was fairly specific to the clear cells in the CD. All forms of CAT-D were detected in ultracentrifuged epididymosomes. In conjunction with the expression levels in epididymal epithelium and epididymosomes, CAT-D expression level on the sperm surface was relatively high in CP sperm, but gradually declined toward the CD. Overall, our results indicated that CAT-D was not inherent to sperm themselves, but rather of epididymal origin and was presumably transported to the sperm surface via epididymosomes., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

In the giant male prawn, Macrobrachium rosenbergii, the olfactory system is thought to be the main pathway for modulating sexual behavior through pheromone perception. In this report, we first used gross anatomical, histological, and SEM methods to describe the structures of the olfactory receptors (sensilla setae), their neural pathways, and possible role in modulating mating behavior. On the surfaces of antennule and antenna filaments there are four types of sensory receptors, viz single spike-like setae, single flagellum-like setae, multiple flagella-like setae, and aesthetascs (ASs). The ASs, which had previously been proposed to be odor receptor setae, are found only on the short filament of lateral antennule (slAn). Each AS on the slAn connects with olfactory receptor neurons (ORNs), whose axons form an outer central antennule nerve (ocAnNv), which then connects with the olfactory neutrophil (ON) of the brain. Thus, the slAn is the major olfactory organ that conveys sensory inputs from each AS to the ON within the deutocerebrum. GABA immunoreactivity was present in ASs, neurons of ORNs, inner central antennular, lateral tegumentary nerve, ocAnNv and the ON, inferring that GABA is the likely neurotransmitter in modulating olfaction. Disruption of the slAn by ablation or covering with Vaseline, resulted in significant reduction of mating behavior, indicating that this organ is crucial for sex pheromone perception. Identification of the active pheromones and further bioassays are now being performed., (Copyright © 2013 Wiley Periodicals, Inc.)

We have recently shown that water-soluble materials from the egg extracellular cortical rods (wsCRs) exert the ability to induce the sperm acrosome reaction in Penaeus monodon. In this study, we further demonstrated that the thrombospondin protein family (TSP) existed in wsCRs, and that their mRNA transcripts were detected in developing oocytes as early as stage I. Full sequence analysis revealed that our pmTSP sequence was considerably different from the recently reported pmTSP in the 5' nonconserved region and in many TSP signature domains, hence, the name pmTSP-II was given to our variant. The transcripts of pmTSP-II were detected only in early developing oocytes (stage-I and -II) while TSP-like proteins were detected in all developing oocytes, particularly at the outer rim of cortical rods situated in the extracellular crypts of the mature, stage-IV oocytes. In addition, wsCRs contained anti-TSP-reactive proteins, suggesting that TSP-like proteins are dissolved in and are part of the egg water during spawning. The functional importance of TSP-like proteins was evident by the interference of a wsCR-induced acrosome reaction response with anti-TSP in a concentration-dependent manner. In summary, we found that pmTSP-II transcripts were present in the developing oocytes and pmTSP-II protein accumulated in cortical rods, which are partly secreted and thus solubilized to produce dissolved TSP-like proteins that participate in induction of the sperm acrosome reaction-a novel reproductive role for TSP protein family., (Copyright © 2013 Wiley Periodicals, Inc.)

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.

Proprotein convertase subtilisin/kexin 4 (PCSK4) is implicated for sperm fertilizing ability, based on studies using Pcsk4-null mice. Herein we demonstrated proprotein convertase (PC) activity in intact sperm and acrosomal vesicles. To determine whether this activity was important for sperm fertilizing ability, a peptide inhibitor was designed based on PCSK4 prodomain sequence (proPC4(75-90)), which contains its primary autocatalytic cleavage site. ProPC4(75-90) inhibited recombinant PCSK4's activity with a K(i) value of 5.4 µM, and at 500 µM, it inhibited sperm PC activity almost completely. Treatment of sperm with proPC4(75-90) inhibited their egg fertilizing ability in a dose dependent manner. Correlation between sperm PC activity and fertilizing ability showed a high co-efficient value (>0.9), indicating the importance of sperm PC activity in fertilization. In particular, sperm PC activity was important for capacitation and zona pellucida (ZP)-induced acrosome reaction, since proPC4(75-90) -treated sperm showed markedly decreased rates in these two events. These results were opposite to those observed in Pcsk4-null sperm, which contained higher PC activity than wild type sperm, possibly due to overcompensation by PCSK7, the other PCSK enzyme found in sperm. ADAM2 (45 kDa), a sperm plasma membrane protein, involved in sperm-egg plasma membrane interaction, was also processed into a smaller form (27 kDa) during capacitation at a much reduced level in proPC4(75-90) -treated sperm. This result suggested that ADAM2 may be a natural substrate of sperm PCSK4 and its cleavage by the enzyme during acrosome reaction may be relevant to the fertilization process., (Copyright © 2011 Wiley-Liss, Inc.)

We found that the androgenic gland (AG) of Macrobrachium rosenbergii possesses three cell types. Type I cells are small polygonal shaped-cells (13.4 μm in diameter), stain strongly with hematoxylin-eosin (H&E), have abundant multilayered rough endoplasmic reticulum (rER), and nuclei containing mostly heterochromatin. Type II cells are slightly larger (18.6 μm in diameter), stain lightly with H&E, have rER with dilated cisternae, and nuclei containing mostly euchromatin. Type III cells (previously undescribed) are similar in size and shape to type I cells, but the cytoplasm is unstained and they have a high amount of smooth endoplasmic reticulum (sER) and mitochondria with tubular cristae. Bilateral eyestalk-ablation resulted in AG hypertrophy with a proliferation and predominance of type I cells as determined by bromodeoxyuridine (BrdU) assays. Expression of insulin-like androgenic gland hormone (Mr-IAG), determined by immunohistochemistry, was weak in type I cells, strong in type II cells of both the intact and eyestalk-ablated, and negative in type III cells. It was also detected in spermatogonia, nurse cells, and epithelium lining of the spermatic duct. The function of Mr-IAG in these tissues is yet to be elucidated but the distribution implies a strong role in male reproduction., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Arylsulfatase A (AS-A) is a lysosomal enzyme, which catalyzes the desulfation of certain sulfogalactolipids, including sulfogalactosylglycerolipid (SGG), a molecule implicated in cell adhesion. In this report, immunocytochemistry revealed the selective presence of AS-A in the corpus luteum of mouse ovaries. Immunoblotting indicated that mouse corpus luteum AS-A had a molecular mass of 66 kDa, similar to AS-A of other tissues. Corpus luteum AS-A was active, capable of desulfating the artificial substrate, p-nitrocatechol sulfate, at the optimum pH of five. To understand further the role of AS-A in female reproduction, levels of AS-A were determined during corpus luteum development in pseudopregnant mice and during luteolysis after cessation of pseudopregnancy. Immunocytochemistry, immunoblotting and desulfation activity showed that AS-A expression was evident at the onset of pseudopregnancy in the newly formed corpora lutea, and its level increased steadily during gland development. The increase in the expression and activity of AS-A continued throughout luteolysis after the decrease in serum progesterone levels. We also observed the selective presence of SGG on the luteal cell surface in developed corpora lutea, as shown by immunofluorescence of mouse ovary sections as well as high-performance thin-layer chromatography of lipids isolated from mouse and pig corpora lutea. The identity of the "SGG" band on the thin layer silica plate was further validated by electrospray ionization mass spectrometry. Significantly, SGG disappeared in regressing corpora lutea. Therefore, lysosomal AS-A may be involved in cell-surface remodeling during luteolysis by desulfating SGG after its endocytosis and targeting to the lysosome.

Trypsin-like enzymes in egg water (EW), a natural acrosome reaction (AR) inducer, are known for their importance in shrimp AR. In this report, we describe a unique phenomenon of the AR of black tiger shrimp (Penaeus monodon) sperm. It was completed within 45-60 sec and comprised only the acrosomal exocytosis and depolymerization of the sperm head anterior spike. We used peptidyl fluorogenic substrates to show the presence of trypsin-like enzymes in P. monodon EW and sperm, but minimal activities of chymotrypsin-like enzymes. In sperm, these trypsin-like enzymes existed both on the sperm surface and in the acrosome. The acrosomal enzyme was revealed as a 45-kDa band by fluorogenic substrate in-gel zymography. Although EW possessed high trypsin-like enzyme activities, they were not essential for the AR induction; EW pretreated with an irreversible trypsin inhibitor, or heat-inactivated EW (HI-EW), to abolish the trypsin-like activities could still induce the AR. The HI-EW-induced AR was inhibited by the presence of a membrane impermeant soybean trypsin inhibitor (SBTI) in the sperm suspension, indicating the significance of sperm-borne trypsin-like enzymes (on the surface and/or in the acrosome) in this AR process. However, pretreatment of sperm with SBTI followed by its removal from the suspension still allowed the AR to occur within 5 min of sperm exposure to HI-EW. Since trypsin-like activity of the SBTI-pretreated sperm surface at 5 min after SBTI removal was at the minimal level, our results suggest the importance of the acrosomal trypsin-like enzyme in the AR process.