Bilateral eyestalk ablation of the blue swimmer crab, Portunus pelagicus, produces hypertrophy of the androgenic gland and an increase of cells producing insulin-like androgenic gland hormone

The androgenic glands (AG) of male decapod crustaceans produce insulin-like androgenic gland (IAG) hormone that controls male sex differentiation, growth and behavior. Functions of the AG are inhibited by gonad-inhibiting hormone originating from X-organ-sinus gland complex in the eyestalk. The AG, and its interaction with the eyestalk, had not been studied in the blue swimmer crab, Portunus pelagicus, so we investigated the AG structure, and then changes of the AG and IAG-producing cells following eyestalk ablation. The AG of P. pelagicus is a small endrocrine organ ensheathed in a connective tissue and attached to the distal part of spermatic duct and ejaculatory bulb. The gland is composed of several lobules, each containing two major cell types. Type I cells are located near the periphery of each lobule, and distinguished as small globular cells of 5-7 μm in diameter, with nuclei containing mostly heterochromatin. Type II cells are 13-15 μm in diameter, with nuclei containing mostly euchromatin and prominent nucleoli. Both cell types were immunoreactive with anti-IAG. Following bilateral eyestalk ablation, the AG underwent hypertrophy, and at day 8 had increased approximately 3-fold in size. The percentage of type I cells had increased more than twice compared with controls, while type II cells showed a corresponding decrease.     

Chemical synthesis of N‐glycosylated insulin‐like androgenic gland factor from the freshwater prawn Macrobrachium rosenbergii

Crustacean insulin‐like androgenic gland factor (IAG) of Macrobrachium rosenbergii, a heterodimeric peptide having both four disulfide bonds and an N‐linked glycan, was synthesized by the combination of solid‐phase peptide synthesis and the regioselective disulfide formation reactions. The disulfide isomer of IAG could also be synthesized by the same manner. The conformational analysis of these peptides by circular dichroism (CD) spectral measurement indicated that the disulfide bond arrangement affected the peptide conformation in IAG. On the other hand, the N‐linked glycan attached at A chain showed no effect on CD spectra of IAG. This is the first report for the chemical synthesis of insulin‐like heterodimeric glycopeptide having three interchain disulfides, and the synthetic strategy shown here might be useful for the synthesis of other glycosylated four‐disulfide insulin‐like peptides.     

The identification and distribution of gonadotropin-releasing hormone-like peptides in the central nervous system and ovary of the giant freshwater prawn, Macrobrachium rosenbergii

In the present study, we demonstrated the existence of GnRH-like peptides in the central nervous system (CNS) and ovary of the giant freshwater prawn, Macrobrachium rosenbergii using immunocytochemistry. The immunoreactivity (ir) of lamprey (l) GnRH-III was detected in the soma of medium-sized neurons located in neuronal cluster number 11 in the middle part of supraesophageal ganglion (deutocerebrum), whereas ir-octopus (oct) GnRH was observed in the soma of both medium-sized and large-sized neurons in thoracic ganglia, as well as in the fibers innervating the other medium-sized and large-sized neuronal cell bodies in the thoracic ganglia. In addition, ir-lGnRH-I was observed in the cytoplasm of late previtellogenic oocyte and early vitellogenic oocyte. These data suggest that M. rosenbergii contain at least three isoforms of GnRH: two GnRH isoforms closely related to lGnRH-III and octGnRH in the CNS, whereas another isoform, closely related to lGnRH-I, was localized in the ovary. This finding provides supporting data that ir-GnRH-like peptide(s) may exist in this decapod crustacean.     

Effects of All-trans retinoic acid on germ cell development of embryos and larvae of the giant freshwater prawn, Macrobrachium rosenbergii

Embryos of the giant freshwater prawn, Macrobrachium rosenbergii, were treated with 1, 10 or 50 μg ml⁻¹ all-trans retinoic acid (AtRA) for 2 days. Survival and hatching rates were not affected. However, an increase in the number of primordial germ cells (PGCs), progenitors of gametes, and a slightly more advanced stage of the gonads were found in those treated with 10 or 50 μg ml⁻¹ AtRA. Newly hatched larvae were treated with 0.1, 0.5 or 1 μg ml⁻¹ AtRA for 2 days. Survival rates were lower in those treated with 0.5 or 1 μg ml⁻¹ AtRA; nevertheless, the gonads were slightly more developed. The results indicated that AtRA, an active metabolite of vitamin A, affected germ cell and gonad development of embryos and the larvae of giant freshwater prawn.     

Effect of hypoxia on the immune response of giant freshwater prawn Macrobrachium rosenbergii and its susceptibility to pathogen Enterococcus

Giant freshwater prawns Macrobrachium rosenbergii (14-19 g) were challenged with Enterococcus (3 x 10(5) cfu prawn(-1)) previously incubated in TSB medium for 24 h, then placed in water having concentrations of dissolved oxygen (DO) at 7.75, 4.75, 2.75 and 1.75 mg l(-1). Onset of mortality occurred after 6 h exposure to 1.75 mg l(-1) DO, and after 12 h exposure to 2.75 mg 1(-1) DO. Cumulative mortality of prawns at 1.75 mg l(-1) DO was significantly higher than that at 4.75 and 2.75 mg l(-1) DO, and cumulative mortality of prawns at 4.75 and 2.75 mg l(-1) DO was significantly higher than that at 7.75 mg l(-1) DO after 96 h. The prawns (20-30 g) which had been placed in water for 0 to 120 h at 7.75, 4.75, 2.75 and 1.75 mg l(-1) DO were examined for the THC (total haemocyte counts), DHC (differential haemocyte counts), phenoloxidase activity, respiratory burst, percentage phagocytosis and clearance efficiency. No significant difference in semi-granular cells and granular cells of prawns was observed among four treatments. The prawns following 120 h exposure to 2.75 mg l(-1) DO decreased significantly the hyaline cells and THC by 39% and 36%, respectively. Phenoloxidase activity and respiratory burst decreased significantly by 33% and 11% when the prawns were exposed to 2.75 mg l(-1) DO after 24 h, respectively. Percentage phagocytosis and clearance efficiency to Enterococcus decreased significantly by 44% and 54% for the prawns following 12 h exposure to 2.75 mg l(-1) DO, respectively. It is concluded that DO as low as 2.75 mg l(-1) and 4.75 mg l(-1) causes depression in immune system of M. rosenbergii, and increases its susceptibility to Enterococcus infection.     

Characteristics of NADH-dependent lipid peroxidation in sarcoplasmic reticulum of white shrimp,Litopenaeus vannamei,and freshwater prawn,Macrobrachium rosenbergii

The iron-catalyzed NADH-dependent lipid peroxidation system in sarcoplasmic reticulum (SR) of cultured white shrimp, Litopenaeus vannamei and freshwater prawn, Macrobrachium rosenbergii was characterized. Production of thiobarbituric acid reactive substances was used to measure the activity of lipid peroxidation. In both species, the system preferred NADH to NADPH as the reducing agent. Lipid peroxidation activities of SR from both species increased when reaction temperatures increased from 6 to 26 °C. At 66 °C, the reaction was no longer NADH-dependent. Acidic pH amplified the lipid peroxidation activity. Sarcoplasmic reticular lipid peroxidation activity in white shrimp was always greater than in freshwater prawn. Fatty acid composition of SR lipids could be a major factor for this outcome. The proportion of n–3 highly unsaturated fatty acids, such as C20:5 and C22:6, in sarcoplasmic reticular lipids of white shrimp was twice of that in freshwater prawn. The results of this study provide important tools required for anti-oxidative nutrient study at sub-cellular level.     

Immunomodulatory effect of Withania somnifera supplementation diet in the giant freshwater prawn Macrobrachium rosenbergii (de Man) against Aeromonas hydrophila

The effect of Withania somnifera extract supplementation diets on innate immune response in giant freshwater prawn Macrobrachium rosenbergii (de Man) against Aeromonas hydrophila was investigated. The bacterial clearance efficiency significantly increased in prawn fed with 0.1% and 1.0% doses of W. somnifera supplementation diet against pathogen from weeks 1-4 as compared to the control. The innate immune parameters such as, phenoloxidase activity, superoxide anion level, superoxide dismutase activity, nitrate, and nitrite concentrations were significantly enhanced in prawn fed with 0.1% and 1.0% doses of W. somnifera supplementation diet from weeks 1-4 against pathogen. The total hemocyte counts (THC) significantly increased in prawn fed with 0.1% and 1.0% doses diet from weeks 1-4 against pathogen as compared to the control. These results strongly suggested that administration of W. somnifera through supplementation diet positively enhances the innate immune system and enhanced survival rate in M. rosenbergii against A. hydrophila infection.     

Identification of lectin isoforms in juvenile freshwater prawns Macrobrachium rosenbergii (DeMan, 1879)

From the serum of juvenile freshwater prawns, we isolated by affinity chromatography on glutaraldehyde-fixed rat erythrocytes stroma, immobilized in Sephadex G-25, a sialic acid specific lectin of 9.6[emsp4 ]kDa per subunit. Comparative analysis against adult organisms purified lectin, by chromatofocusing, showed that the lectin from juvenile specimens is composed by four main isoforms with a pl of 4.2, 4.6, 5.1, and 5.6, whereas the lectin from adults is eluted at pH 4.2. The amino acid composition of the lectin obtained from adult and juvenile stages suggest identity, but the compositions are not identical since a higher content of carbohydrates was found in the lectin from younger organisms. The freshwater prawn lectin showed specificity toward N-acetylated amino sugar residues such as GlcNAc, GalNAc, Neu5Ac and Neu5,9Ac; but in juvenile organisms the lectin showed three times less hemagglutinating activity than the lectin from adults. Both lectins agglutinated rat, rabbit and chicken erythrocytes, indicating that Neu5,9Ac in specific O-glycosydically linked glycans seems to be relevant for the interaction of M. rosenbergii lectins with their specific cellular receptor. Our results suggest that the physicochemical characteristics of the lectin from the freshwater prawn are regulated through maturation.     

Viral diseases of the giant fresh water prawn Macrobrachium rosenbergii: a review

The giant freshwater prawn Macrobrachium rosenbergii is cultivated essentially in Southern and South-eastern Asian countries such as continental China, India, Thailand and Taiwan. To date, only two viral agents have been reported from this prawn. The first (HPV-type virus) was observed by chance 25 years ago in hypertrophied nuclei of hepatopancreatic epithelial cells and is closely related to members of the Parvoviridae family. The second, a nodavirus named MrNV, is always associated with a non-autonomous satellite-like virus (XSV), and is the origin of so-called white tail disease (WTD) responsible for mass mortalities and important economic losses in hatcheries and farms for over a decade. After isolation and purification of these two particles, they were physico-chemically characterized and their genome sequenced. The MrNV genome is formed with two single linear ss-RNA molecules, 3202 and 1250 nucleotides long, respectively. Each RNA segment contains only one ORF, ORF1 coding for the RNA-dependant RNA polymerase located on the long segment and ORF2 coding for the structural protein CP-43 located on the small one. The XSV genome (linear ss-RNA), 796 nucleotides long, contains a single ORF coding for the XSV coat protein CP-17. The XSV does not contain any RdRp gene and consequently needs the MrNV polymerase to replicate.     

Identification and characterization of a hyperglycemic hormone from freshwater giant prawn, Macrobrachium rosenbergii

Crustacean hyperglycemic hormone (CHH), a physiologically important neurohormone stored in the sinus gland of eyestalks, primarily regulates carbohydrate metabolism and also plays significant roles in reproduction, molting and other physiological processes. In the freshwater giant prawn, Macrobrachium rosenbergii, an injection of X-organ sinus gland (XOSG) extract evoked a hyperglycemic response, peaked in 1 h. The hyperglycemic effect of the eyestalk extract was maximal at the dose of 0.5 eyestalk equivalent. CHH fractionated by RP-HPLC, in M. rosenbergii was identified by its hyperglycemic activity and partial amino acid sequence, and the molecular weight of 8534 was determined by matrix-assisted laser desorption ionization mass spectrometry--time of flight analysis (MALDI-TOF). The amino acid sequence of the first 25 residues of CHH showed 72% homology with the first 25 residues of CHH A and CHH B of the American lobster Homarus americanus.     

Induction of Female Breeding Characteristics by Ovarian Tissue Implants in Androgenic Gland Ablated Male Freshwater Prawns Macrobrachium rosenbergii (de Man) (Decapoda,Palaemonidae)

Summary

Implantation of ovarian tissue into androgenic gland ablated male prawns, Macrobrachium rosenbergii (de Man) induced the development of ovigerous and ovipositing setae and brood chambers, suggesting that in females, these secondary sexual characteristics are induced by the ovary. This model system has potential for the study of morphological changes associated with breeding in the Decapoda.     

Distribution of dopamine and octopamine in the central nervous system and ovary during the ovarian maturation cycle of the giant freshwater prawn, Macrobrachium rosenbergii

Dopamine (DA), octopamine (OA) and serotonin (5-HT) are the key neurotransmitters that control gonadal development in decapod crustaceans. 5-HT stimulates, while DA and OA delay gonadal development in Macrobrachium rosenbergii. In the present study, we have further investigated the distribution patterns of DA and OA in the central nervous system (CNS) and ovary during various stages of the ovarian maturation cycle of this giant freshwater prawn. DA- and OA-immunoreactive neurons and fibers were distributed extensively in several regions of the brain, subesophageal ganglion (SEG), thoracic ganglia and abdominal ganglia. In the brain, the two neurotransmitters were present in neurons of clusters 6, 7, 11, 17, and nearby neuropil regions. In the SEG, thoracic ganglia and abdominal ganglia, immunoreactive neurons and fibers were found along the midline and in several neuronal clusters around each neuropil region. Staining for DA and OA was more intense in the thoracic ganglia than in other parts of the CNS. In the ovary, DA- and OA-immunoreactivities were present at high intensity in early oocytes. The presence of DA- and OA-immunoreactivities in neural ganglia as well as ovary suggests that DA and OA may also be involved in the reproductive process, particularly ovarian development and differentiation of oocytes in this species.     

Effect of infectious hypodermal and haematopoietic necrosis virus (IHHNV) infection on caspase 3c expression and activity in freshwater prawn Macrobrachium rosenbergii

Caspase 3c (MrCasp3c) was sequenced from the freshwater giant prawn Macrobrachium rosenbergii using Illumina Solexa Genome Analyzer Technique. MrCasp3c consisted of 2080 bp nucleotide encoded 521 polypeptide with an estimated molecular mass of 59 kDa. MrCasp3c sequence contains caspase family p20 domain profile and caspase family p10 domain profile at 236-367 and 378-468 respectively. The quantitative real time PCR analysis revealed a broad expression of MrCasp3c with the highest expression in haemocyte and the lowest in stomach. The expression of MrCasp3c after challenge with the infectious hypodermal and haematopoietic necrosis virus (IHHNV) was tested in haemocyte. In addition, MrCasp3c was expressed in Escherichia coli by prokaryotic expression plasmid pMAL-c2x. The enzyme activity of MrCasp3c was also found to be up-regulated by IHHNV in haemocyte and hepatopancreas tissues. This study suggested that MrCasp3c may be an effector caspase associated with the induction of apoptosis which is potentially involved in the immune defence of M. rosenbergii.     

Microsatellite loci in the eastern form of the giant freshwater prawn (Macrobrachium rosenbergii)

Six microsatellite loci were identified and characterized in the eastern form of the widespread and commercially important giant freshwater prawn (Macrobrachium rosenbergii). The loci were detected by randomly screening for dinucleotide and trinucleotide repeat units within a partial genomic library developed for the species. In a sample of 29 prawns, number of alleles and heterozygosity per locus ranged from 12 to 18 and from 0.66 to 0.90, respectively. These markers provide powerful tools for the conservation and management of wild stocks, the improvement of cultured stocks of M. rosenbergii, and for investigating evolutionary processes underlying genetic divergence among populations.     

Isolation and characterization of microsatellite loci in the Malaysian giant freshwater prawn, Macrobrachium rosenbergii

Eight single locus microsatellite markers were developed to characterize the Malaysian giant freshwater prawn, Macrobrachium rosenbergii. These microsatellites were isolated from an enriched genomic library contained by using a 5'-anchored polymerase chain reaction technique. Primers were designed to flank the repeat sequences and subsequently used to characterize 30 unrelated individuals of the giant freshwater prawn. The polymerase chain reaction amplification products of these eight microsatellite loci were polymorphic with the number of alleles ranging from two to 10 alleles per locus while the levels of heterozygosity ranged from 0.6333 to 0.8667.     

Hyperglycemic responses to cold shock in the freshwater giant prawn, Macrobrachium rosenbergii

Crustacean hyperglycemic hormone (CHH), a neurohormone synthesized and released from the x-organ sinus gland complex, is primarily involved in carbohydrate metabolism; biogenic amines and peptidergic neuroregulators are known to modulate the release of CHH. Marked elevations of hemolymph glucose titers, which peaked within 2 h, were observed in both intact and bilaterally eyestalk-ablated prawns, Macrobrachium rosenbergii, when they were transferred directly from their optimal temperature of 28 °C to lower temperatures close to their lethal limit. Hyperglycemia can therefore be considered a characteristic response in this species under cold shock. Involvement of biogenic amines in the hyperglycemic response was also demonstrated. Hyperglycemic effects of epinephrine, dopamine and serotonin were mediated through CHH at the eyestalk level, but the response under cold shock was not exclusively mediated through CHH. It is suggested that factor(s) other than CHH are involved in the hyperglycemic response, possibly norepinephrine or/and octopamine. Accepted: 24 October 1998     

Microsatellite DNA markers in the giant freshwater prawn, Macrobrachium rosenbergii: a tool for genetic analysis

Eight microsatellite loci were identified and characterized in the commercially important giant freshwater prawn, Macrobrachium rosenbergii. The microsatellite loci were detected by the random screening for dinucleotide repeat units within a partial genomic library developed for the species with biotinylated probes (CA)(15) , (AT)(15) and (GA)(15) . All the eight loci were found to be polymorphic. The number of alleles and observed heterozygosities per locus ranged between three to 16 and 0.22 to 0.71, respectively. These microsatellite markers will be useful for the conservation and management of wild and cultured stocks and population genetic studies of freshwater prawn.     

Enzymatic digestion--a safe and rapid technique for individual separation of Macrobrachium rosenbergii embryos for cryopreservation studies

A new, safe, and rapid technique for the individual separation of the embryos of giant freshwater prawn Macrobrachium rosenbergii de Man is described. Two protease enzymes, e.g., trypsin and collagenase were used. Embryos in the advanced stage of development (gray embryos with eyespot and heart beat) were selected for the study. Treatment with collagenase and trypsin at respective concentrations of 0.05 and 0.25% for 30 min resulted in 100% separation of 35-40 mg of embryonic mass (approximately 180 embryos). A chelating agent, EDTA (ethylenediaminetetraacetic acid disodium salt: dihydrate) at 400 mg l(-1) enhanced the activity of trypsin. Trypsin and collagenase, when used together, were found to act synergistically. The separated embryos revealed no morphological injury when observed under the microscope. Further, in vitro hatching of the separated embryos was successful indicating that the present technique is safe and effective in achieving individual separation of prawn embryos.     

Direct evidence for the function of crustacean insulin-like androgenic gland factor (IAG): Total chemical synthesis of IAG

Insulin-like androgenic gland factor (IAG) is presumed to be a sex differentiation factor so-called androgenic gland hormone (AGH) in decapod crustacean, although the function of IAG peptide has not yet been reported. In this study, we synthesized IAG from the prawn, Marsupenaeus japonicus, and its function was assessed by an in vitro bioassay. As a result, IAG with the insulin-type disulfide bond arrangement showed biological activity, whereas its disulfide isomer did not. These results strongly suggest that the native IAG peptide has an insulin-type disulfide, and it is the decapod AGH.