Anaesthetic Efficiency of Cymbopogon citratus Essential Oil and Clove Oil on Macrobrachium rosenbergii.

<b>Background and Objective:</b> Studies on plant herbs as alternatives to chemical anaesthetics in fish species are numerous, but little is known on crustaceans. A study was conducted to investigate the efficacy of <i>C. citratus</i> Essential Oil (EO) on the induction and recovery of <i>M. rosenbergii</i>. <b>Materials and Methods:</b> The <i>C. citratus</i> EO was obtained by hydrodistillation and analyzed using GC-MS. The prawns were exposed to <i>C. citratus</i> EO and clove oil in 100-1000 and 200-1000 µL L¹, respectively. Different stages of induction and recovery times were recorded. <b>Results:</b> In GC-MS, citral (78.47%) was detected as a major compound in <i>C. citratus</i> EO. Prawns reached loss equilibrium at 500-1000 µL L¹ <i>C. citratus</i> EO within 15.55-6.52 min. Exposure of prawn to <u><</u>500 µL L¹ <i>C. citratus</i> EO resulted in a high survival rate (100-94%). In clove oil, all tested concentrations caused significant induction in <i>M. rosenbergii</i> within 20.61-6.47 min. Recovery time and survival rate were significantly decreased with the increase of EO concentrations. The regression model showed the induction time in both anaesthetic agents was dependent on the concentration (R²= 0.86-0.96). The recovery time of <i>C. citratus</i> EO-exposed prawn was dependent on the concentrations (R²= 0.59). <b>Conclusion:</b> The study shows the potentiality of <i>C. citratus</i> EO as a natural anaesthetic in <i>M. rosenbergii</i>, although not as efficient as clove oil.

Identification and relative abundances of mRNA for a gene encoding the vWD domain and three Kazal-type domains in the ovary of giant freshwater prawns, Macrobrachium rosenbergii.

Understanding Macrobrachium rosenbergii ovarian maturation control at the genome level is an important aspect for increasing larvae production. In this study, an ovarian maturation related gene, M. rosenbergii vWD domain and three Kazal-type domains of a gene (MrvWD-Kazal) have been studied. The MrvWD-Kazal gene was isolated using a rapid amplification of cDNA end (RACE) method and the relative abundances of MrvWD-Kazal mRNA in the ovary, hepatopancreas, stomach, intestine and gill were determined by using the quantitative PCR technique. The MrvWD-Kazal gene is composed of 2194 bp with an open reading frame (ORF) of 1998 bp encoding 665 amino acids and has great similarity to the M. nipponense vWD-Kazal gene (91%). The qPCR analyses indicated the relative abundance of MrvWD-Kazal mRNA transcript varied among different stages of ovarian function (P < 0.05), but there were no differences abundance in hepatopancreas, stomach, intestine and gill (P> 0.05). In the ovary, relative abundance of MrvWD-Kazal mRNA transcript gradually increased with ovarian maturation from Stages 1 (Spent; 1.00-fold), to 2 (Proliferative; 3.47-fold) to 3 (Premature; 6.18-fold) and decreased at Stage 4 (Mature; 1.31-fold). Differential relative abundances of MrvWD-Kazal mRNA transcript in the ovary indicate the MrvWD-Kazal protein may have an important function in ovarian maturation of M. rosenbergii. The results of this study also indicate the MrvWD-Kazal is not involved in regulation of the reproductive related function of the hepatopancreas, digestive system (stomach and intestine) and respiratory system (gill).

Application MALDI TOF on protein identification of vitellogenin in giant grouper (Epinephelus lanceolatus).

A new proteomics technology has been implemented to study the protein repertoires of developing oocytes of giant grouper (Epinephelus lanceolatus). Knowledge of the chemical composition and physiochemical properties of vitellogenin (Vtg) is necessary to interpret the functional and biological properties attributed during ovulation. Vtg, as a biomarker indicator in sex determination, has been analyzed to determine the sex and maturational status of fish in the absence of the gonad tissue. A male giant grouper was induced by 2 mg/kg of 17ß-estradiol (E2), and blood was sampled at days 0, 1, 3, 5, and 10. SDS-PAGE 1D electrophoresis was used to analyze Vtg protein, and Vtg identification was done with 4800 Plus MALDI TOF/TOF™ mass spectrophotometer (Applied Biosystems/MDS SCIEX, USA). Meanwhile, MS/MS de novo sequencing identified the proteins by matching sequences of tryptic peptides to the known sequences of other species. Vtg was confirmed by MASCOT at 95% significant level, and molecular mass was 187 kDa. Protein resolved on SDS-PAGE as a double band of approximately the same mass as determined with MALDI-TOF. The N-terminal sequences and identification of Vtg were also determined. The potential of using MS methods to understand the structure and function of Vtg is discussed.

Metabolism of amino acids during hyposmotic adaptation in the whiteleg shrimp, Litopenaeus vannamei.

The penaeid prawn, Litopenaeus vannamei, was employed to investigate intracellular isosmotic regulation in situations where invertebrates encounter hyposmosis. Hemolymph osmolality was first analyzed to confirm osmoregulatory conditions in the experimental animals, followed by analysis of amino acids in muscle and hemolymph using high-performance liquid chromatography. Total muscle amino acid levels decreased when hemolymph osmolality was extremely low, whereas glycine and L-serine levels increased in the hemolymph. These results suggest that tissue amino acids were released into the hemolymph to lower the osmolality of the tissues for purposes of low-salinity adaptation. Next, oxygen consumption and ammonia excretion rates were examined, and the O/N ratio was determined. Oxygen consumption levels and ammonia excretion rates increased, and the O/N ratio decreased when the animals were exposed to low salinity. These results suggest that amino acids were abundantly consumed as an energy source when animals were exposed to low salinity. To confirm the consumption of particular amino acids, the specific activity of L-serine ammonia lyase was also examined. Specific activity was highest when L-serine levels in the hemolymph were highest. Thus, it appears that L-serine levels increased under hyposmotic conditions due to the consumption of L-serine as an energy source. It was concluded that particular amino acids as osmolytes are likely metabolized as energy sources and consumed for purposes of hyposmotic adaptation.

Characterization and expression of the putative ovarian lipoprotein receptor in the Kuruma prawn, Marsupenaeus japonicus.

Ovarian low density lipoproteins (LDL) such as vitellogenin (Vg) are the precursors of the major yolk protein vitellin, and constitute the major source of nutrients serving the developing embryo. The objective of this study was to gain a better understanding of crustacean egg development by focusing on the process of Vg internalization by its receptor (ovarian LDLR). First, an ovarian LDLR cDNA sequence in Marsupenaeus japonicus was determined. Ovarian LDLR mRNA expression was then examined, and was seen to be specific to the ovary, exhibiting highest levels during the previtellogenic stage. This pattern of ovarian LDLR expression is thought to signify preparation for yolk protein incorporation into the oocyte. Using immunoblotting techniques, an ovarian LDLR band was detected whose size was similar to that estimated from the deduced amino acid sequence. The ovarian LDLR protein was expressed only at the onset of vitellogenesis, and histological studies supported these observations. This is the first occasion that the ovarian LDLR and its expression dynamics during vitellogenesis have been fully characterized in a crustacean.

Molecular cloning and characterization of cortical rod protein in the giant freshwater prawn Macrobrachium rosenbergii, a species not forming cortical rod structures in the oocytes.

The formation of cortical rod structures is a characteristic of fully mature oocytes in penaeid prawns, but such structures are absent from oocytes of giant freshwater prawn Macrobrachium rosenbergii. In the present study, we first demonstrated the presence of a 30-kDa protein, which was immunologically related to kuruma prawn cortical rod protein (CRP), in the ovary of giant freshwater prawn, and subsequently purified this protein. Furthermore, a cDNA encoding the CRP-like protein was isolated. Based on the high homology (98%) in the amino acid sequence with kuruma prawn CRP, the 30-kDa protein has been identified as a CRP homologue of giant freshwater prawn, designated mrCRP. The RT-PCR analysis revealed that mrCRP mRNA was present in the ovary from a prawn with a gonadosomatic index (GSI) of 0.2. Western blot analysis revealed the presence of a CRP-immunoreactive band of 30kDa in the ovary with GSI of 1.6. By immunocytochemistry, CRP-immunopositive signals were detected in the ovary with GSI of 0.9, that had started to accumulate considerable amounts of vitellins and lipids in the peripheral cytoplasm. With progress of vitellogenesis, mrCRP was apparently accumulated in the mature oocytes, although it was not detectable, presumably because a relatively small amount of mrCRP was masked with large amounts of vitellin and lipids. In giant freshwater prawn without forming cortical rod structures, our findings indicate that the oocytes produce mrCRP, a homologue of CRP found in penaeid prawns.

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.

Isolation and cDNA cloning of ovarian cortical rod protein in kuruma prawn Marsupenaeus japonicus (Crustacea: Decapoda: Penaeidae).

Two cortical rod proteins having molecular weights of 28.6 kDa and 30.5 kDa were isolated from the mature ovary of Marsupenaeus japonicus using gel filtration and reversed-phase HPLC. Analysis of the N-terminal amino acid sequence of the 28.6 kDa molecule revealed that amino acid residues 1-21 corresponded to residues 9-29 of the 30.5 kDa molecule. Examination of homology using BLAST showed that 21 amino acids out of 29 residues of the 28.6 kDa molecule, and 14 out of 29 residues of the 30.5 kDa molecule were identical to that of the ovarian cortical rod proteins of Penaeus semisulcatus. Positive immunohistochemical reaction to antiserum raised against the 28.6 kDa protein was observed on cortical rods forming around the periphery of oocytes at the maturation stages. Western blotting analysis revealed that both the 28.6 kDa and 30.5 kDa molecules stained with the anti-28.6 kDa antiserum. Furthermore, the 28.6 kDa and 30.5 kDa proteins were both glycosylated, as evidenced by positive carbohydrate staining using Concanavalin A and production of positive PAS reaction. These results indicate that the cortical rods are comprised of the 28.6 kDa and 30.5 kDa molecules. We subsequently cloned two full-length cDNAs based on the N-terminal sequences of the 28.6 kDa and 30.5 kDa molecules. The open reading frame of 28.6 kDa and 30.5 kDa encoded 276 amino acid residues. Comparison analysis of the two cDNAs revealed that the location of the processing site and sequence of signal peptides differed, indicating that the two cDNAs are products of two separate genes and encode the 28.6 kDa molecule and 30.5 kDa molecule, respectively. Both proteins possessed one potential N-linked glycosylation site. It is considered that both molecules are components of the cortical rods, forming a jelly layer after fertilization.

Molecular characterization of a cDNA encoding vitellogenin in the coonstriped shrimp, Pandalus hypsinotus and site of vitellogenin mRNA expression.

In order to determine the primary structure of vitellogenin in a protandric species, the coonstriped shrimp Pandalus hypsinotus, we previously purified four vitellin components (designated as VnA, VnB, VnC, and VnD, respectively), and chemically analyzed their partial amino acid sequences. In this study, we subsequently cloned a cDNA encoding vitellogenin in this species based on the N-terminal and internal amino acid sequences of VnA, as well as the N-terminal sequence of VnC. The open reading frame of this cDNA encoded a pro-vitellogenin in which vitellins were arranged as follows: NH2-VnA-VnB-VnC/D-COOH. The deduced amino acid sequence possessed a single consensus cleavage sequence, R-X-K/R-R, along the lines of vitellogenins reported in other crustaceans and insects, and the N-terminal sequence of VnB was immediately preceded by this sequence. The comparison of primary structures revealed the existence of a basic and characteristic structure for the vitellogenin molecule in decapod crustacean species, and phylogenetic analysis reflected the current taxonomic classifications of Crustacea. An approximately 8 kb-long transcript of the vitellogenin gene was detected in the hepatopancreas of female shrimps having a gonadosomatic index higher than 1.0 by Northern blot analysis, but was not observed in the hepatopancreas and gonads of male shrimps and the hepatopancreas of female shrimps having a gonadosomatic index lower than 1.0. These results indicate that the hepatopancreas is responsible for vitellogenin synthesis.

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.

Deduced primary structure of vitellogenin in the giant freshwater prawn, Macrobrachium rosenbergii, and yolk processing during ovarian maturation.

A cDNA encoding vitellogenin (Vg) in the giant freshwater prawn, Macrobrachium rosenbergii, was cloned based on the cDNA sequence of vitellin (Vn) fragments A-N and B-42 determined previously, and its amino acid sequence deduced. The open reading frame (ORF) encoded 2,537 amino acid residues and its deduced amino acid sequence possessed three consensus cleavage sites, R-X-R-R, similar to those reported in Vgs of insects. The deduced primary structure of Vg in M. rosenbergii was seen to be similar to that of Penaeus japonicus, especially in the N-terminal region. It is therefore likely that Vgs in crustacean species including prawns and other related decapods exhibit a similar structural pattern. Based on the deduced primary structure of Vg and analysis of the various Vg and Vn subunits found in the hemolymph and ovary during ovarian maturation, we demonstrated the post-translational processing of Vg in M. rosenbergii. This is the first time that Vg processing has been clearly demonstrated in a crustacean species. Vg, after being synthesized in the hepatopancreas, is considered to be cleaved by a subtilisin-like endoprotease to form two subunits, A and proB, which are then released into the hemolymph. In the hemolymph, proB is possibly cleaved by a processing enzyme of unknown identity to give rise to subunits B and C/D. The three processed subunits A, B, and C/D are sequestered by the ovary to give rise to three yolk proteins, Macr-VnA, VnB, and VnC/D.