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1.
Gen Comp Endocrinol ; 331: 114176, 2023 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-36410448

RESUMO

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.


Assuntos
Penaeidae , Sesquiterpenos , Animais , Penaeidae/metabolismo , Filogenia , S-Adenosilmetionina , Hormônios Juvenis/metabolismo , Metiltransferases/metabolismo , Clonagem Molecular
2.
Front Endocrinol (Lausanne) ; 12: 760538, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34867802

RESUMO

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.


Assuntos
Hormônios de Invertebrado/metabolismo , Ovário/metabolismo , Penaeidae/metabolismo , Vitelogênese/fisiologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/metabolismo , Clonagem Molecular/métodos , Feminino , Proteínas do Tecido Nervoso/metabolismo , Vitelogeninas/metabolismo
3.
Artigo em Inglês | MEDLINE | ID: mdl-34004320

RESUMO

The authors recently reported the presence and distribution of oxytocin/vasopressin-like peptide in Portunus pelagicus as well as demonstrated its function to inhibit ovarian steroid release (Saetan et al., 2018). Here, the full-length receptor of this peptide, namely oxytocin/vasopressin-like peptide receptor (PpelOT/VP-like peptide receptor) is reported. The coding region of the PpelOT/VP-like peptide receptor contained 1497 bp which translationally corresponded to 499 amino acids. Sequence analysis revealed its seven transmembrane characteristics, with -two N-linked glycosylation residues located before the first transmembrane domain (TM I). The phylogenetic tree revealed that the PpelOT/VP-like peptide receptor was placed in the group of invertebrate OT/VP-like receptors, and was clearly distinguishable from the V1R, V2R and OTR of vertebrates. Also, this receptor gene transcript was detected in several organs of the blue swimming crab with highest abundance found in brain tissue. In situ hybridization exhibited its distribution in all neuronal clusters of the eyestalk, brain, ventral nerve cord (VNC), as well as in the ovary. Comparative gene expressions between this receptor and its corresponding peptide in immature and mature female crabs revealed no significant difference of the PpelOT/VP-like peptide receptor gene expression in the central nervous system (CNS) and ovary. In contrast, the PpelOT/VP-like peptide gene was shown to significantly express higher in the VNC of immature crabs and in the ovary of mature crabs. Changes in expression of this peptide gene, but not its receptor, might result in ovarian steroid release inhibition. However, the detailed mechanism of this peptide in reproduction regulation will be included in our further studies.


Assuntos
Braquiúros/fisiologia , Ocitocina/metabolismo , Receptores de Peptídeos/metabolismo , Receptores de Vasopressinas/fisiologia , Vasopressinas/metabolismo , Animais , Sistema Nervoso Central/metabolismo , Feminino , Perfilação da Expressão Gênica , Ovário/metabolismo , Peptídeos/química , Filogenia , RNA Mensageiro/metabolismo , Receptores de Peptídeos/genética , Receptores de Vasopressinas/metabolismo
4.
Cytotechnology ; 73(2): 141-157, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33927472

RESUMO

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).

5.
Fish Shellfish Immunol ; 110: 10-22, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33383176

RESUMO

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 VPAHPND 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 LC50 dose of VPAHPND; 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 VPAHPND 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 VPAHPND-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 VPAHPND 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 VPAHPND-infected prawn was caused by an acute self-destructive immune response by hematopoietic cells.


Assuntos
Bactérias/patogenicidade , Expressão Gênica/imunologia , Sistema Hematopoético/imunologia , Imunidade Inata/genética , Palaemonidae/imunologia , Vibrio parahaemolyticus/fisiologia , Animais , Sistema Hematopoético/microbiologia , Sistema Hematopoético/patologia , Hemócitos/imunologia , Homeostase , Palaemonidae/microbiologia , Virulência
6.
Fish Shellfish Immunol ; 88: 415-423, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30872029

RESUMO

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.


Assuntos
Sistema Hematopoético/citologia , Sistema Hematopoético/imunologia , Palaemonidae/imunologia , Animais , Proteínas de Artrópodes/química , Células-Tronco Hematopoéticas , Hemócitos/imunologia , Hemolinfa , Palaemonidae/anatomia & histologia , Fagocitose , Antígeno Nuclear de Célula em Proliferação/química
7.
Acta Histochem ; 121(2): 156-163, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30558912

RESUMO

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.


Assuntos
Genitália Masculina/metabolismo , Palaemonidae/metabolismo , Hormônios Peptídicos/metabolismo , Espermatozoides/metabolismo , Animais , Água Doce , Masculino , Testículo
8.
Acta Histochem ; 121(2): 143-150, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30497687

RESUMO

The mud crab, Scylla olivacea, is a high value economic marine animal in Thailand. However, collection of these crabs from natural habitat for local consumption and export has caused rapid population decline. Hence, aquaculture of this species is required and to this measure understanding of endocrine control of their reproduction must be understood. Egg laying hormone (ELH) is a neuropeptide synthesized by the bag cells (neurons) in the abdominal ganglia of Aplysia gastropods. It plays a critical role in controlling egg production and laying in gastropods, and its possible homolog (ELH-like peptide) was reported in the neural and ovarian tissues of prawns and recently in female reproductive tract of the blue swimming crab, Portunus pelagicus. In this study, we have studied the histology of the male reproductive tract in Scylla olivacea which are comprised of anterior testis, posterior testis, early proximal spermatic duct (ePSD), proximal spermatic duct (PSD), middle spermatic duct (MSD) and distal spermatic duct (DSD), by immunohistochemistry, detected an abalone ELH- immunoreactivity (aELH-ir) in epithelium of ducts in posterior testis and epithelium of all parts of spermatic duct. Furthermore, we could detect aELH-ir in neurons of cluster 9, 11, olfactory neuropil (ON) in the brain and in the small neurons located between the third and the fourth thoracic neuropils (T3-T4) and between the fourth and the fifth thoracic neuropils (T4-T5) of thoracic ganglia. Thus, the presence of aELH in male S. olivacea was designated the role of female egg laying behavior in the male mud crab.


Assuntos
Braquiúros/metabolismo , Sistema Nervoso Central/metabolismo , Hormônios de Invertebrado/metabolismo , Hormônios Peptídicos/metabolismo , Reprodução/fisiologia , Testículo/metabolismo , Animais , Gastrópodes/metabolismo , Imuno-Histoquímica/métodos , Masculino , Neurônios/metabolismo
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