Metapenaeus Ensis Research Articles

Effects of high pressure freezing (HPF) on denaturation of natural actomyosin extracted from prawn (Metapenaeus ensis).

Effects of protein denaturation caused by high pressure freezing, involving Pressure-Factors (pressure, time) and Freezing-Factors (temperature, phase transition, recrystallization, ice crystal types), are complicated. In the current study, the conformation and functional changes of natural actomyosin (NAM) under pressure assisted freezing (PAF, 100,150,300,400,500MPaP-20°C/25min), pressure shift freezing (PSF, 200MPaP-20°C/25min), and immersion freezing (0.1MPaP-20°C/5min) after pressure was released to 0.1MPa, as compared to normal immersion freezing process (IF, 0.1MPaP-20°C/30min). Results indicated that PSF (200MPaP-20°C/30min) could reduce the denaturation of frozen NAM and a pressure of 300MPa was the critical point to induce such a denaturation. During the periods of B→D in PSF or B→C→D in PAF, the generation and growth of ice crystals played an important role on changing the secondary and tertiary structure of the treated NAM.

Effects of exposure to hypoxia and hydrogen sulfide on greasyback shrimp Metapenaeus ensis at the early life stages

Effects of exposure to hypoxia and hydrogen sulfide on greasyback shrimp <i>Metapenaeus ensis</i> at the early life stages

Inactivation Kinetics ofVibrio parahaemolyticuson Sand Shrimp(Metapenaeus ensis)by Cinnamaldehyde at 4°C

Sand shrimp(Metapenaeus ensis), shrimp shell, and shrimp meat were inoculated with a three-strain cocktail ofVibrio parahaemolyticuswith or without the natural antimicrobial cinnamaldehyde (2.5 mg/ml) and were, then, stored at 4°C for up to 25 days and 18 inactivation curves were obtained.V. parahaemolyticuswere inactivated down to the minimum level of detection (2.48 log CFU/g) on thiosulfate citrate bile salts sucrose agar (TCBS) plates within 7 and 10 days with low and high densities ofV. parahaemolyticusinoculation, 4.5 log CFU/g and 8.2 log CFU/g, respectively. With adding cinnamaldehyde, the inactivation process ofV. parahaemolyticuswith low populations, 4.5 log CFU/g, lasted for only 4 days. Therefore, cinnamaldehyde inactivated cells faster as expected. However, unexpectedly, in shrimp meat cases, cells have much more persistence of over even 25 days before entering the minimum level of detection both with and without cinnamaldehyde treatment. Therefore, a hypothesis was formed that when cells kept in cold environments (4°C) after several days recovered to up to 103–104CFU/g towards the end of the experiments and with starvation (shell and shrimp studies), cells might render a viable but nonculturable (VBNC) state.

Changes of structure and IgE binding capacity of shrimp (Metapenaeus ensis) tropomyosin followed by acrolein treatment.

Tropomyosin, a major allergen of shrimp, presents high stability to various conditions owing to the high content of α-helix. Chemical modification of tropomyosin can induce structural changes and affects IgE binding capacity. Since lipid peroxidant such as acrolein commonly exists during shrimp preservation, it is necessary to evaluate its influence on tropomyosin structure and IgE binding capacity. Western blot and inhibition enzyme-linked immunosorbent assay were used to illustrate IgE binding capacity, while structural changes were investigated by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and fluorescence spectroscopy. Results revealed that IgE binding capacity reduced and the secondary structure denatured following treatment with increasing concentration of acrolein. Furthermore, the identification of LC-MS/MS indicated that the free amines of lysine, tyrosine, and histidine residues were altered by acrolein. These amino acid modifications resulted in the collapse of epitopes, as well as structural changes responsible for the decreased IgE binding capacity of shrimp tropomyosin.

Effect of transglutaminase-catalyzed glycosylation on the allergenicity and conformational structure of shrimp (Metapenaeus ensis) tropomyosin.

Tropomyosin (TM), a myofibrillar protein, is a major allergen in shrimp. The aim of this study was to evaluate the effect of transglutaminase (TGase)-catalyzed glycosylation on the potential allergenicity and conformational structure of TM in Metapenaeus ensis. Results showed that glycosylation of TM induced unfolding of the primary protein structure followed by loss of the secondary structure. Cleavage of certain free amino groups was observed during TGase-catalyzed glycosylation. The glycosylation rate correlated with reaction temperature. Western blotting and indirect ELISA with TM-specific polyclonal antibodies from rabbit and sera from patients allergic to shrimp demonstrated that antigenicity and potential allergenicity of TM decreased, which correlated well with the conformational changes in its structure. Considering TGase is widely utilized in the food industry, these results indicate that TGase-catalyzed glycosylation has the potential to serve as a mild method for reducing the allergenicity of shrimp products.

Doctor, can I desensitize my food-allergic child using directly the allergenic molecules?

Food allergy has risen in prevalence worldwide and is one of the main causes of anaphylaxis, especially in children. The only possible therapeutic approach is specific immunotherapy. This review describes the recent approaches using allergenic molecules for specific immunotherapy. Hypoallergenic tropomyosins from Metapenaeus ensis have been cloned and constructed by direct mutagenesis or epitope deletion and have been successfully used in shrimp-sensitized mice. A modified carp parvalbumin is being used in phase I/IIa clinical trials in patients with fish allergy. Natural lipid transfer protein (Pru p 3) and profilin (Pho d 2) extracts have been used for the treatment of patients with plant food allergy. Treatments of egg-sensitized mice with glycated-ovalbumin and ovomucoid peptides led to a clinical and immunological improvement. A preventive treatment with synthetic β-lactoglobulin peptides was effective in reducing skin symptoms in mice sensitized to milk whey proteins but no dominant epitopes were found in α-lactalbumin. Finally peanut desensitization has been attempted using three modified recombinant peanuts but the protocol was interrupted and limited to a phase 1 study because of side-effects. Many molecules, including allergenic peptides or modified proteins are under consideration but clinical trials in food-allergic study participants are necessary to confirm tolerability and efficacy.

Effect of 4-hydroxy-2-nonenal treatment on the IgE binding capacity and structure of shrimp (Metapenaeus ensis) tropomyosin

Lipid peroxidation can react with free amines of proteins and induce modification of structural and functional properties. This study presents the IgE binding capacity and structural changes of shrimp tropomyosin (TM) under oxidative stress with 4-hydroxy-2-nonenal (HNE). IgE binding capacity was evaluated with the dot-blot assay and inhibition enzyme-linked immunosorbent assay. A decrease in IgE binding capacity of TM was found with 0.01mM HNE treatment, which was more significant when the HNE concentration was increased to 0.5mM. The conformational changes of TM, as characterized by fluorescence spectroscopy, circular dichroism spectroscopy and ultraviolet absorption spectroscopy, correlated well with IgE binding capacity changes. Further LC–ESI-MS/MS analyses showed that the side-chain groups of alanine, leucine, lysine and histidine had been modified by HNE. These results suggested that the HNE-induced conformational changes of TM significantly influenced its allergenicity and that these changes were caused by the modification of specific amino acids residues.

Development of pseudotyped retroviral system for effective gene transfer and expression in penaeid shrimp cells

The pantropic retroviral expression system has been widely used to stably introduce foreign genes into the genome of dividing cells from non-mammalian systems. However, we found that the current commercial retrovirus expression system did not work in inactively dividing primary shrimp cells. To overcome this, we successfully developed a triple-pseudotyped retroviral expression system that can be used for effective gene transfer and expression of reporter genes in both mammalian and penaeid shrimp cells by improving the commercial pantropic retrovirus system (VPK-305, Cell Biolabs) in two ways. First, the promoter regions of translationally controlled tumor protein (TCTP) genes were cloned from the shrimps Metapenaeus ensis and Marsupenaeus japonicus, named Pme-tctp and Pmj-tctp, respectively. We inserted them into the retroviral reporter vectors of pMCs-PLTR-GFP or eGFP, immediately after the retroviral promoter PLTR (5′LTR, long terminal repeat sequence). We then analyzed their expression efficiencies in mammalian and shrimp cells by the lipofection method. It was found that PLTR was a strong promoter in mammalian cells, but failed to effectively drive the reporter genes to express in the primary lymphoid cells (LCOO) derived from Oka organs of shrimp (M. ensis). However, inclusion of the shrimp promoters (Pme-tctp and Pmj-tctp) significantly improved the expression efficiency of the retroviral vectors in shrimp cells. Next, the revised retroviral vectors of pMCs-PLTR-Pme-tctp (or Pmj-tctp)-GFP or eGFP were pseudotyped with envelope protein VSV-G alone, or triply with VSV-G, VP19 and VP28 (two envelope proteins of shrimp white spot syndrome virus). And we then examined the corresponding infection and expression efficiencies in shrimp primary LCOO cells and embryo cells. We found that only the triple-pseudotyped retrovirus containing the shrimp-derived promoter could successfully mediate the delivery and fluorescent expression of GFP and eGFP genes, but VSV-G-pseudotyped retrovirus had poor tropism to shrimp LCOO cells. Confocal microscopic observation further confirmed the hypothesis that the improved tropism of the triple-pseudotyped retrovirus to shrimp cells might be attributed to the inclusion of VP19 and VP28 into the envelope of the packaged retrovirus. In spite of this, the expression efficiency and infection efficiency (20%–30%) of reporter genes in shrimp cells was still lower than those in mammalian cells. In infected shrimp primary embryo cells, strong green fluorescent signal was detected only in neuron-like cells, not in fibroblast-like cells. This triple-pseudotyped retroviral expression system ultimately may prove useful for immortalization of cultured shrimp cells and production of transgenic shrimps. Statement of relevance•We developed a novel triple-pseudotyped retroviral expression system that can be used in effective gene transfer and expression of foreign genes in shrimp cells.•This system may prove useful for the immortalization of cultured shrimp cells which are needed in the study and control of shrimp viral diseases.•This system may prove useful for the production of transgenic shrimps with new traits.

T cell epitope immunotherapy ameliorates allergic responses in a murine model of shrimp allergy.

Shellfish allergy is one of the most common food hypersensitivities worldwide but allergen-specific immunotherapy for shellfish allergy is not yet available. We believe that T cell peptide-based immunotherapy holds the potential for modulating allergic responses without IgE cross-linking. We sought to identify the immunodominant T cell epitopes of tropomyosin, the major shrimp allergen of Metapenaeus ensis (Met e 1), and to evaluate their therapeutic effects in a Balb/c mouse model of Met e 1 hypersensitivity. T cell epitopes of Met e 1 were first identified based on the proliferation and cytokine responses of splenocytes isolated from Met e 1-sensitized Balb/c mice upon stimulation by 18 synthetic peptides that span the full-length Met e 1. The immunodominant T cell peptides identified were then fed orally to Met e 1-sensitized Balb/c mice twice a week for four weeks. Allergic responses, serological antibody levels, intestinal histology and systemic and local cytokine profiles were compared between the treated and the untreated groups. Six major Met e 1 T cell epitopes were identified. Mice treated with the T cell epitope peptide mixture demonstrated an amelioration of systemic allergic symptoms and a significant reduction in Th2-associated antibody and cytokine responses. These benefits were accompanied by a shift to a balanced Th1/Th2 response, induction of IgG2a antibodies possessing in vitro and in vivo blocking activities and the induction of regulatory T cell responses. T cell epitope-based oral immunotherapy is effective in reducing allergic responses towards shrimp tropomyosin. This is a novel strategy for clinical management of shellfish allergy and is a model for mechanistic studies of oral immunotherapy.

Prediction of total volatile basic nitrogen contents using wavelet features from visible/near-infrared hyperspectral images of prawn (Metapenaeus ensis)

A visible/near-infrared hyperspectral imaging (HSI) system (400–1000nm) coupled with wavelet analysis was used to determine the total volatile basic nitrogen (TVB-N) contents of prawns during cold storage. Spectral information was denoised by conducting wavelet analysis and uninformative variable elimination (UVE) algorithm, and then three wavelet features (energy, entropy and modulus maxima) were extracted. Quantitative models were established between the wavelet features and the reference TVB-N contents by using three regression algorithms. As a result, the LS-SVM model with modulus maxima features was considered as the best model for determining the TVB-N contents of prawns, with an excellent RP2 of 0.9547, RMSEP=0.7213mgN/100g and RPD=4.799. Finally, an image processing algorithm was developed for generating a TVB-N distribution map. This study demonstrated the possibility of applying the HSI imaging system in combination with wavelet analysis to the monitoring of TVB-N values in prawns.

Shotgun assembly of the first mitochondrial genome of Metapenaeus (Metapenaeus ensis) with phylogenetic consideration

The first complete mitochondrial genome sequence of Metapenaeus was characterized from sand shrimp Metapenaeus ensis using shotgun assembly method. The complete mitochondrial DNA sequence is a 15 944 bp circular molecule, and contains 13 protein-coding genes, 22 transfer RNA genes, two rRNA genes, and a control region. The gene arrangements are consistent with the pancrustacean ground pattern. The phylogenetic trees were divided two clades besides outgroup, which belonged to Penaeus s.l. and Metapenaeus genus. The molecular analyses provided robust evidence for the monophyly of Fenneropenaeus, but Litopenaeus is not monophyletic.

Discrimination of shelled shrimp (Metapenaeus ensis) among fresh, frozen-thawed and cold-stored by hyperspectral imaging technique

The potential of a Vis-NIR (400–1000 nm) hyperspectral imaging system was investigated to discriminate fresh (F), cold-stored (C-S) and frozen-thawed (F-T) shelled shrimp (Metapenaeus ensis), to detect or prohibit illegally substituted and mislabeled products from the market. In this study, eight feature wavelengths (783, 689, 435, 416, 813, 639, 452 and 478 nm) were extracted by uninformative variable elimination (UVE) based on partial least squares (PLS) with successive projections algorithm (SPA) from the whole variables. Meanwhile, grey level co-occurrence matrix (GLCM) was applied to extract the first three principal components (PCs) images, which explained more than 99% of variances of all spectral bands. The spectral and textural data were combined for discrimination. Random forest (RF) and soft independent modeling of class analogy (SIMCA) classifiers were utilized to discriminate F, C-S and F-T shrimp based on: (1) whole spectral data, (2) optimal spectral data, (3) textural data, and (4) fusion of textural and optimal spectral data. Based on (4), satisfying results were obtained using RF and SIMCA with correct classification rates (CCRs) of 91.11% and 88.89% in the prediction sets, respectively, confirming the feasibility of hyperspectral imaging to classify fresh shrimps and those after cold storing or freezing.

In Silico Characterization and Comparative Analysis of Allergenicity of Allergic Proteins from Different Food Sources

Allergy is a steadily increasing health problem for all age groups. In general, it’s recommended that 10-35% of our daily calories come from protein. A complete protein source is one that provides all of the essential amino acids called high quality proteins. Although animal-based foods; for example, meat, poultry, fish, milk, eggs, are considered complete protein sources these are the common sources of causing allergenicity. In case of meat Bos taurus, prawn Penaeus monodon and egg Gallus gallus are found to be the most responsible for triggering allergenicity. The current study has disclosed the best alternative sources for meat, prawn and egg through in silico characterization and comparative analysis of allergic proteins (Myoglobin Ovomucoid, Lysozyme, Ovalbumin, Ovotransferrin, Tropomyosin) with other common sources of meat (Capra hircus, Ovis aries, Gallus gallus, Sus scrofa), prawn (Fenneropenaeus merguiensis, Macrobrachium rosenbergii, Metapenaeus ensis, Pandalus borealis) and egg (Anas platyrhynchos). Analyzing the results we found that Gallus gallus, Macrobrachium rosenbergii, Anas platyrhynchos would be the safe source for meat, prawn and egg respectively.

Problems with the use of liposome- and retrovirus-mediated gene transfer methods in the primary lymphoid cells of the Oka organs of the greasyback shrimp, Metapenaeus ensis (De Haan, 1844)

In this study, both liposome- and retrovirus-mediated gene transfer methods were examined for their potential to transfer and express two retroviral vectors containing the mouse c-Myc or the green fluorescent protein (GFP) gene into the primary lymphoid cell cultures (OKA) derived from “Oka” organs (= organs of the lymphoid system) of the greasyback shrimp Metapenaeus ensis (De Haan, 1844). It was found that the c-Myc gene could be delivered into OKA cells by the liposome-mediated method, but the introduced c-Myc gene could not be effectively transcribed into mRNA. In contrast, the pantropic retrovirus-mediated method failed to introduce the c-Myc gene into OKA cells, and GFP was not detected in the transformed cells, either. This work inferred two problems for the use of the two above-mentioned gene transfer methods in the non-dividing OKA cells: (1) the viral promoter of long terminal repeats (LTRs) had low activity in shrimp cells; (2) the pantropic retrovirus-mediated gene transfer system had a low tropism to shrimp lymphoid cells.

Nutrient Compositions and Properties of Antarctic Krill (Euphausia superba) Muscle and Processing By-Products

ABSTRACTThis study investigated the morphometrics, proximate chemical compositions, pH, total amino acid (TAA), fatty acid profile, and minerals of the processing by-products of Antarctic krill (Euphausia superba). The nutrient compositions and properties of the by-products were revealed by being compared to those of Antarctic krill muscle and the economically important species of freshwater prawns (Macrobrachium nipponense) and penaeid shrimps (Metapenaeus ensis). The by-products are worthy of utilization because of the high ratio to the total weight (65.7%). The crude protein contents in the muscle and by-products of krill are 17.4 and 11.7%, respectively. The krill proteins have higher contents of essential amino acids (EAAs). The EAAs constitute 42 and 37% of the TAAs in muscle and by-products, respectively. The krill processing by-products contain high levels of total lipid (3.3%), and polyunsaturated fatty acids constitute 34% of fatty acids with high levels of eicosapentaenoic acid (EPA; 19.08%) and docosahexaenoic acid (DHA; 10.02%). Krill meat provides considerable iron, zinc, calcium, selenium, and copper. It is imperative to lower the fluoride level (70.1 mg/kg, wet basis) in krill muscle. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) reveals that myosin heavy chain (MHC) and actin are the major proteins in muscle, and their contents vary between species.

Identification of oxidative modification of shrimp (Metapenaeus ensis) tropomyosin induced by malonaldehyde

In this study, shrimp tropomyosin was subjected to malonaldehyde (MDA)-induced oxidative stress in aqueous situation. The in vivo cross-linking of tropomyosin was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the structural changes were investigated by differential scanning calorimetry, circular dichroism, and Fourier transform infrared spectroscopy (FTIR). The location of the resulting protein carbonyls was determined by mass spectrometry. The SDS-PAGE of the cross-linked tropomyosin showed four new bands corresponding to two, three, four, and five-time molecular weights of tropomyosin. The conformational structures were partly destroyed because of the thermal denaturation at higher temperatures. The α-helix content increased, and new chemical bonds were formed by the MDA modification. These physiochemical properties were confirmed by the identification of amino acid side-chain modifications by using liquid chromatography–tandem mass spectrometry. Lysine (Lys), glutamine (Gln), and asparagine (Asn) residues in tropomyosin were modified at four lysine (Lys-76, 168, 189, and 233), five glutamine (Gln-61, 70, 118, 147, and 247), and four asparagine (Asn-17, 107, 203, and 215) sites. Apart from these MDA modification sites, oxidized methionine was observed by data search. These results indicate that some active food ingredients, such as MDA, can react with the side chains of amino acids resulting in structural changes in the protein.

The shrimp heat shock cognate 70 functions as a negative regulator in vitellogenin gene expression.

Within the 2.6-kb 5' flanking region of the shrimp (Metapenaeus ensis) vitellogenin gene (MeVg2), several clusters of putative heat shock factor (HSF) response elements were identified. Deletion of these response elements has caused significant increases in MeVg2 promoter activity, suggesting that the HSF and Hsc70 complex may regulate vitellogenin gene expression in a negative manner. To confirm the role of Hsc70 in the regulation of vitellogenin gene expression, the ovary cDNA for Hsc70 was cloned and characterized. The Hsc70 transcript level was high in the ovary and hepatopancreas of females at the early vitellogenic stage but dropped during ovarian maturation. In addition, Western blot analysis revealed the presence of Hsc70 in the nuclear but not in the cytoplasmic fraction during the early stage of ovary maturation. Electrophoretic mobility shift assay (EMSA) results showed that ovary nuclear extract contained a factor that binds to the HSF response element. Since the addition of ATP caused a decrease in the binding of Hsc70, Hsc70 may form a repressor complex with HSF to inhibit MeVg2 expression. An in vitro RNA interference technique was used to study the gene function of Hsc70. Hsc70 gene knockdown resulted in an increased MeVg2 mRNA level in the ovary (54%) and hepatopancreas (62%). In summary, this report describes the first study of vitellogenin gene regulation at the transcription level in crustaceans and provides strong evidence that Hsc70 acts as a molecular chaperone to negatively regulate MeVg2 gene expression in shrimp.

Potential of hyperspectral imaging for non-invasive determination of mechanical properties of prawn (Metapenaeus ensis)

The potential of using hyperspectral imaging in the wavelength region of visible and short-wave near infrared range (400–1000nm) was investigated for the rapid and non-invasive determination of mechanical properties in different parts of prawns (Metapenaeus ensis). Hyperspectral cubes were acquired at the second and fourth segments of prawn and their mean spectral data were extracted. The quantitative relationship between the spectral data and their corresponding reference mechanical properties (measured by Instron universal testing machine) were established by partial least square regression (PLSR) and least-squares support vector machines (LS-SVM) algorithms, respectively. Successive projections algorithm (SPA) was carried out to select the most important and effective wavelengths, which was an optimization process for improving the performance of established models. Then the optimized models were built using PLSR and LS-SVM based on the selected wavelengths and their performances were compared to find the best model for predicting mechanical properties of prawn. As a result, the SPA-LS-SVM model was considered as the best predictive model to determine the hardness, gumminess and chewiness values of prawn with correlation coefficient of prediction (RP) of 0.8489, 0.8096, and 0.8596 and root mean square error of prediction (RMSEP) of 0.1465, 0.1445, and 0.1258, respectively. Visualization of distribution maps for the mechanical properties within the region of interests (ROI) of prawns was also presented. The overall results revealed that hyperspectral imaging technique had a great ability for predicting the mechanical properties of prawn rapidly and non-invasively.

Characterization of the shrimp neuroparsin (MeNPLP): RNAi silencing resulted in inhibition of vitellogenesis

The full-length Metapenaeus ensis neuroparsin (MeNPLP) cDNA was cloned which encodes a shrimp protein homologous to the insect neuroparsin and vertebrate insulin-like growth factor binding protein (IGFBP). MeNPLP cDNA is 1389bp in length and the longest open reading frame is 303bp in length. The first 27 aa are predicted to be the signal peptide and aa 28–101 is the mature peptide with an estimated molecular weight of 7.83kDa and pI of 5. It shows high amino acid sequence similarity (42–68%) to the neuroparsin of insects and N-terminal end of the IGFBP of vertebrates. The cysteine residues in MeNPLP responsible for disulfide bond formation are conserved as in other neuroparsin-like proteins. The expression level of MeNPLP is the highest in the hepatopancreas, followed by the nerve cord, brain, heart, ovary, and muscle. However, it was not expressed in the testis. Using an insect neuroparsin antibody, MeNPLP could only be detected in the hepatopancreatic tubules, suggesting that MeNPLP may be a secretary product. Although MeNPLP expression was stimulated in the ovary, it was inhibited in the hepatopancreas after treatment with neurotransmitter serotonin (5-HT). In vivo gene silencing of MeNPLP could cause a significant decrease of vitellogenin transcript level in the hepatopancreas and ovary. As a result, a corresponding decrease in vitellogenin protein level was observed in the hemolymph and ovary. In conclusion, this study has provided the first evidence that MeNPLP is involved in the initial stage of ovary maturation in shrimp.

Comparative proteomic profiling during ovarian development of the shrimp Metapenaeus ensis

Two-dimensional electrophoresis and mass spectrometry were used to identify proteins that are differentially expressed during ovarian maturation in Metapenaeus ensis. 87 spots with consistently significant quantitative differences (≥ 1.5-fold for vol%) among stage I, III and V ovaries were chosen for MS/MS analysis. 45 spots were significantly matched to known proteins in the database (Mascot score >40). Half of them were down-regulated, in contrast to 9 out of 45 proteins that were up-regulated as ovarian maturation proceeded. Functionally, these identified proteins could be classified into five major groups, including cytoskeleton (11 %), metabolism (18 %), signal transduction (32 %), gene expression (14 %) and immune response (7 %). Among the differentially expressed reproduction-related proteins, the mRNA expression level of cellular retinoic acid/retinol binding protein in M. ensis (MeCRABP) during ovarian maturation was further characterized by quantitative real-time PCR. It was down-regulated during ovarian maturation. In situ hybridization further revealed that MeCRABP transcript was localized in ooplasm of previtellogenic oocytes but not in vitellogenic oocytes. These results demonstrate the application of proteomic analysis for identification of proteins involved in shrimp ovarian maturation and they provide new insights into ovarian development.