Localization of vitellogenin mRNA expression and vitellogenin uptake during ovarian maturation in the giant freshwater prawn Macrobrachium rosenbergii

In situ hybridization and immunohistochemical techniques were used to investigate the dynamics of vitellogenin (Vg) mRNA expression and Vg uptake during ovarian maturation in the hepatopancreas and ovary at differing stages of ovarian maturation in both intact and eyestalk ablated female Macrobrachium rosenbergii. In the hepatopancreas of intact animals, Vg mRNA expression was detected faintly two days after ecdysis, and signals showed a gradual increase as the molt cycle advanced to the premolt stages, but decreased at the late premolt stage. Vg mRNA was detected in the R-cells of the hepatopancreas, indicating that these cells are responsible for synthesizing Vg. No Vg mRNA expression was observed in the ovary. Immunohistochemistry results for the hepatopancreas showed a pattern of staining intensity similar to that of in situ hybridization. Increases in the accumulation of yolk protein in the oocytes occurred concomitantly with increasing Vg mRNA expression. In eyestalk ablated animals, Vg mRNA expression and Vg uptake showed similar but accelerated patterns to those of intact animals. This study has confirmed on the cellular level previous results that Vg synthesis is intrinsically correlated to ovarian maturation and the molt cycle in M. rosenbergii.     

Dynamics of vitellogenin mRNA expression and changes in hemolymph vitellogenin levels during ovarian maturation in the giant freshwater prawn Macrobrachium rosenbergii

The dynamics of vitellogenin mRNA expression during ovarian maturation in Macrobrachium rosenbergii were examined by measuring hemolymph vitellogenin (Vg) levels and Vg mRNA expression in the hepatopancreas and ovary at differing reproductive stages in both intact and eyestalk ablated animals. Vg mRNA was quantified using real-time RT-PCR and hemolymph Vg was measured by enzyme immunoassay. In intact animals, Vg mRNA levels in the hepatopancreas and hemolymph Vg levels showed a gradual increase during the molt cycle concomitant with increasing gonadosomatic index (GSI), with Vg levels decreasing prior to ecdysis although GSI continued to increase. Eyestalk ablation was seen to accelerate Vg synthesis as well as ovarian maturation, although it did not alter the overall pattern of Vg expression. Vg mRNA expression was negligible in the ovary of both intact and eyestalk ablated animals, confirming that the hepatopancreas is the principal site of Vg synthesis in M. rosenbergii with the ovary being only a minor contributor. This study has shown that Vg synthesis is correlated to ovarian maturation and the molt cycle in M. rosenbergii.     

Dynamics of vitellogenin synthesis in juvenile giant freshwater prawn Macrobrachium rosenbergii

The dynamics of vitellogenin (Vg) mRNA expression and patterns of Vg and vitellin distribution in the hepatopancreas and ovary of juvenile Macrobrachium rosenbergii were examined using real-time RT-PCR and immunohistochemical methods. Eyestalk ablation was seen to induce rapid development of the gonads and Vg synthesis in females. In the female hepatopancreas, Vg mRNA expression was observed several days following ablation, after which levels increased gradually with increasing gonadosomatic index (GSI). Vitellin accumulation in the oocytes also increased with increasing Vg mRNA synthesis; expression was however negligible in the ovary. Hemolymph Vg levels in females ranged from 0.04 to 2.2 mg/ml. SDS PAGE/Western blotting analysis of hemolymph samples revealed that juvenile Vg was composed of 199 and 90 kDa subunits; the 102 kDa subunit present in adult female Vg (Okuno et al., 2002. J Exp Zool 292:417-429) could not be detected at any stage of vitellogenesis in juveniles. Vg was not detectable in non-ablated juveniles. The results of this study confirmed that the mode of involvement of eyestalk factors in regulating vitellogenesis is intrinsic to both juveniles and adults, and that a basic pattern of Vg synthesis and processing is conserved. However, the fact that juveniles are not able to produce the same Vg levels observed in adult females, and do not reach high GSI levels culminating in spawning suggests that other factors and physiological conditions specific to adult females are necessary to demonstrate full reproductive ability.     

Site of vitellogenin synthesis determined from a cDNA encoding a vitellogenin fragment in the freshwater giant prawn, Macrobrachium rosenbergii.

Vitellogenesis is an important part of reproductive process in crustaceans, and the process is characterized by the synthesis and accumulation of yolk protein in the developing oocytes. The yolk proteins in crustaceans mainly consist of vitellogenin (Vg) and vitellin (Vn), which are respectively present in extra-oocyte tissues and intra-oocytes. The site and the process of yolk protein synthesis in crustaceans are still controversial. The synthesis site of Vg in a crustacean species, Macrobrachium rosenbergii, is determined by immunological and immunohistochemical techniques, and molecular cloning of a cDNA encoding the primary structure of Vn in this study. The hepatopancrease is clearly shown to be the synthesis site of Vg in this species. The length of Vg mRNA was estimated as about 6 kb from Northern blotting analysis. The partial primary structure of Vg gene is presented, and the post-translational processing are further discussed. For the first time, the partial primary structure of Vg gene and the synthesis site of Vg approached by molecular cloning in crustaceans are presented.     

Isolation and characterization of microsatellites in giant freshwater prawn Macrobrachium rosenbergii

Microsatellite loci were characterized in a freshwater prawn from enriched genomic library using six biotinylated probes: (AG)₁₀, (TG)₁₀, (CAA)₁₀, (CAG)₁₀, (GAT)₁₀ and (TAC)₁₀. Primers for DNA amplification were designed and synthesized for 20 loci. Ten loci were polymorphic with the number of alleles ranging from five to 17 alleles per locus and the observed heterozygosity ranging from 0.27 to 0.83 per locus. Developed microsatellite primers should prove useful for selective breeding programs and population genetic studies of freshwater prawn.     

Establishment of hematopoietic tissue primary cell cultures from the giant freshwater prawn Macrobrachium rosenbergii

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).     

Hepatopancreas is the likely organ of vitellogenin synthesis in the freshwater prawn, Macrobrachium rosenbergii

The objective of the present study was to investigate the source of vitellogenin in the freshwater prawn, Macrobrachium rosenbergii. Ovarian development of M. rosenbergii was classified into five stages (stage I-V). Vitellin/vitellogenin was detected in the ovary and the hepatopancreas in different stages by native-PAGE and Western blotting. Two and three subunits of vitellin were observed in the ovary at the early- (I-II), mid- and late- (III-V) stages, respectively. The subunit of vitellogenin was not detected in the hepatopancreas at different stages of prawns. Hepatopancreas had positive immunocytological staining (against vitellin antibody) in different ovarian stages of prawn. Only vitellogenic oocyte but not previtellogenic oocytes and follicle cells had a positive immunocytological staining. Hepatopancreas could synthesize radiolabeled immunoreactive proteins after incubation with radiolabeled glycine on the basis of immunoprecipitation (against vitellin antiserum). Therefore, it is concluded that hepatopancreas is the most likely organ to synthesize vitellogenin in the freshwater prawn, M. rosenbergii.     

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

This paper reports on isolation and characterization of 8 microsatellite loci from a partial genomic library of Macrobrachium rosenbergii using DIG labeled dinucleotides, (GT)₁₅ and (CT)₁₅ as probes. Primers were designed and PCR conditions optimized for repeat arrays. Polymorphism was studied using 24 individuals collected from the wild. All the loci were polymorphic with number of alleles ranging from 3 to 5 and observed heterozygosity 0.50–0.85. All the loci except one were in agreement with Hardy–Weinberg equilibrium. No significant pair wise linkage disequilibrium was found among the loci. These markers should prove useful for characterization of natural population as well as brood stock management of this species.     

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     

Dietary vitamin-E modulates antioxidant defence system in giant freshwater prawn, Macrobrachium rosenbergii

The objectives of the present study were to determine the effect of supplementary vitamin-E (200, 400 and 600 mg/kg feed) on lipid peroxidation (LPX) and antioxidant defence system in gills and hepatopancreas of the freshwater prawn, Macrobrachium rosenbergii. Results indicated that vitamin-E inhibited LPX in the hepatopancreas in a comparatively lower dose than gills. Superoxide dismutase (SOD) activity was decreased significantly in gills in response to all the three supplemented diet, but in hepatopancreas decrease was observed only in response to higher doses of vitamin-E (400 and 600 mg/kg feed). Catalase (CAT) activity was reduced significantly only in gills but not in hepatopancreas. While glutathione peroxidase (GPX) activity was significantly elevated in the hepatopancreas by vitamin-E, its activity remains unaltered in gills. On the contrary, glutathione reductase (GR) activity was decreased in gills but that of hepatopancreas was constant. Glutathione (GSH) content of both gills and hepatopancreas was substantially elevated in the vitamin-E supplemented prawns. Although the ascorbic acid (ASA) content of gills was unchanged by vitamin-E, its level elevated significantly in hepatopancreas. Thus the findings of the present investigation suggest that dietary vitamin-E is capable of reducing LPX level and can modulate antioxidant defence system in gills and hepatopancreas, nevertheless, the response is highly tissue specific. It is further observed that highest dose of vitamin-E (600 mg/kg feed) could not render much additional protection in both the tissues.     

Metschnikowia bicuspidata and Enterococcus faecium co-infection in the giant freshwater prawn Macrobrachium rosenbergii

In May 2001, an epizootic yeast and bacterial co-infection in the giant freshwater prawn Macrobrachium rosenbergii occurred in Taiwan causing a cumulative mortality of 25%. The diseased prawns had a yellowish-brown body color, milky hemolymph, opaque, whitish muscles, and were approximately 7 mo old with total lengths ranging from 8 to 10 cm. Histopathological examination showed marked edema, yeast infiltration, and necrotic lesions with inflammation in the muscles, hepatopancreas and other internal organs. We isolated 2 pathogens from the diseased prawns, one was a yeast (AOD081MB) and the other a gram-positive coccus (AOD081EF). The gram-positive coccus was identified as Enterococcus faecium by the API 20 Strepsystem, conventional biochemical tests, and it had 99% 16S rDNA sequence identity (GenBank Accession Number AJ276355) to E. faecium (GenBank Accession Number AF529204). The sequence of a PCR product from the D1/D2 domain of 26S rDNA (GenBank Accession Number AF529297) from the yeast gave 99% sequence identity to Metschnikowia bicuspidata (GenBank Accession Number U44822). Experimental infections with these isolates produced gross signs and histopathological changes similar to those observed in the naturally infected prawns. The lethal doses (LD50) for isolate E. faecium AOD081EF, M. bicuspidata AOD081MB and the co-infection were 4.7 x 10(4), 2.6 x 10(2), and 2.4 x 10(2) colony-forming units prawn(-1), respectively. This is the first report of a confirmed co-infection of M. bicuspidata and E. faecium in prawn aquaculture.     

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.     

Novel polymorphic microsatellite DNA markers from Malaysian giant freshwater prawn, Macrobrachium rosenbergii

Seven single locus microsatellite markers were characterized in Malaysian giant freshwater prawn, Macrobrachium rosenbergii from an enriched genomic library Primer pairs were designed to flank the repeat sequences and the loci characterized for this species. The bands resulting from the PCR amplifications of these eight microsatellite loci were polymorphic with the number of alleles ranging from 8 to 26 alleles per locus, whereas the observed heterozygosity ranged from 0.0641 to 0.6564. These newly developed microsatellite markers should prove to be useful for population studies and in the management of genetic variations in broodstocks of freshwater prawn, M. rosenbergii.     

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.     

Androgenic gland cell structure and spermatogenesis during the molt cycle and correlation to morphotypic differentiation in the giant freshwater prawn, Macrobrachium rosenbergii

The freshwater prawn Macrobrachium rosenbergii shows three male morphotypes: blue-claw males (final stage having high mating activity), orange-claw males (transitional stage showing rapid somatic growth), and small males (primary stage showing sneak copulation). This morphotypic differentiation is considered to be controlled by androgenic gland hormone, which is probably a peptide hormone. However, its physiological roles are not fully understood. In the present study, we examined the correlation of androgenic gland cell structure to spermatogenic activity and morphotypic differentiation histologically in M. rosenbergii. spermatogenic activity showed close correlation to the molt cycle in orange-claw males and small males. spermatogonia increased in number in the late premolt stage, becoming spermatocytes in the postmolt stage, and spermatocytes differentiated into spermatozoa in the intermolt and early premolt stages. Ultrastructure of the androgenic gland was additionally compared among the molt stages, but, distinct histological changes were not observed in relation to spermatogenesis during the molt cycle. On the other hand, among the three morphotypes, the androgenic gland was largest in the blue-claw males, containing developed rough endoplasmic reticulum in the cytoplasm. These results suggest that, during spermatogenesis which is related to the molt cycle, the androgenic gland hormone is at rather constant levels and plays a role in maintaining spermatogenesis rather than directly regulating the onset of a specific spermatogenesis stage and that, during the morphotypic differentiation, the androgenic gland is most active in the blue-claw males and plays a role in regulating the observed high mating activity in M. rosenbergii.     

Isolation and characterization of a female-specific DNA marker in the giant freshwater prawn Macrobrachium rosenbergii

In this study, a female-specific DNA marker in the freshwater prawn Macrobrachium rosenbergii was identified through amplified fragment length polymorphism (AFLP). The AFLP-derived sequence-characterized amplified region (SCAR) marker was tested in over 200 individuals, giving reproducible sex identification. Further molecular characterization of the sex-marker's genomic region (～ 3 kb long) revealed the presence of tandem and inverted repeats. The ～ 3-kb sequence was identified both in male and female prawns, but with subtle differences: a deletion of 3 bp (present in female prawn but absent in male prawn) identified upstream of the SCAR marker sequence and two female-specific single-nucleotide polymorphisms, both indicating that male prawns are homozygous, whereas female prawns are heterozygous in this locus. Fluorescent in situ hybridization showed the ～ 3-kb sequence to be unique: to the best of our knowledge, this is the first report of a unique sex-specific sequence observed in situ in crustaceans. The sex-specific marker identified in M. rosenbergii may have considerable applied merit for crustacean culture in that it will enable the determination of genetic sex at early developmental stages when phenotypic differences are not identifiable.