Crustacean Proteins Research Articles

The molecular characterization of Rab11 and its immune roles in red swamp crayfish (Procambarus clarkii)

The Rab proteins primarily regulate vesicular transport between membrane-bound organelles and are important for innate immune. However, there is currently a lack of studies on crustaceans regarding Rab proteins, particularly core Rabs. We identified a Rab11 gene from Procambarus clarkii (PcRab11) and evaluated its potential involvement in immune response. The results showed PcRab11 was 1789 bp long, with an open reading frame of 645 bp encoding 211 amino acids and an estimated molecular weight of 23.8 kDa. Sequence analysis revealed its remarkable evolutionary conservation. The PcRab11 was widely expressed in various tissues, with highest levels in hepatopancreas, and localized within the cell cytoplasm. Upon infection with white spot syndrome virus (WSSV) or Aeromonas veronii, the expression of PcRab11 in immune organs was significantly induced. Furthermore, silencing PcRab11 reduced phagocytosis-related genes expression and haemocytes' phagocytic activity to FITC-labeled A. veronii, as well as decreased mortality and death time in WSSV or A. veronii infected P. clarkii. Additionally, the potential protein interaction between PcRab11 and 14-3-3ε was identified in haemocytes. Overall, our findings provided evidence for the involvement of Rab11 in P. clarkii's immune response, establishing a foundation to explore the immune role of core Rab proteins in crustaceans' innate immune system.

Rab proteins in fish and crustaceans: an overview

Rab proteins in fish and crustaceans: an overview

An ependymin-related blue carotenoprotein decorates marine blue sponge

Marine animals display diverse vibrant colors, but the mechanisms underlying their specific coloration remain to be clarified. Blue coloration is known to be achieved through a bathochromic shift of the orange carotenoid astaxanthin (AXT) by the crustacean protein crustacyanin, but other examples have not yet been well investigated. Here, we identified an ependymin (EPD)-related water-soluble blue carotenoprotein responsible for the specific coloration of the marine blue sponge Haliclona sp. EPD was originally identified in the fish brain as a protein involved in memory consolidation and neuronal regeneration. The purified blue protein, designated as EPD-related blue carotenoprotein-1, was identified as a secreted glycoprotein. We show that it consists of a heterodimer that binds orange AXT and mytiloxanthin and exhibits a bathochromic shift. Our crystal structure analysis of the natively purified EPD-related blue carotenoprotein-1 revealed that these two carotenoids are specifically bound to the heterodimer interface, where the polyene chains are aligned in parallel to each other like in β-crustacyanin, although the two proteins are evolutionary and structurally unrelated. Furthermore, using reconstitution assays, we found that incomplete bathochromic shifts occurred when the protein bound to only AXT or mytiloxanthin. Taken together, we identified an EPD in a basal metazoan as a blue protein that decorates the sponge body by binding specific structurally unrelated carotenoids.

Complement-related proteins in crustacean immunity

As an important part of innate immune system, complement system is widely involved in defense response and immune regulation, and plays an important biological role. The complement system has been deeply studied. More than 30 complement-related molecules and three major complement-activation pathways have been identified in vertebrates. Crustacean animals do not have complement system. There are only some complement-related proteins in crustaceans which are important for host defense. In this review, we summarize the current knowledge about complement-related proteins in crustaceans, and their functions in crustacean immunity. We also make a comparation of the crustacean pro-phenoloxidase activating system and the mammalian complement system. This review provides a better understanding of the evolution and function of complement-related proteins in crustaceans.

Liquid chromatography coupled to tandem mass spectrometry for comprehensive quantification of crustacean tropomyosin and arginine kinase in food matrix

Crustacean has been recognized as one of the most critical food allergens whose impact could develop or persist into adulthood. It's urgently needed to establish accurate and reliable methods to quantify tropomyosin (TM) and arginine kinase (AK), the primary allergenic proteins in crustaceans. Herein, we developed an ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) method based on stable isotope-labeled internal standard (SIIS) to quantify TM and AK simultaneously. The amino acid sequences of TM and AK from the mostly consumed crustaceans in the world were analyzed, and the signature peptides of the two allergenic proteins were selected given the specificity, stability, and mass spectrometry responsiveness. Under the optimal detection conditions, signature peptide FLAEEADR and VSSTLSSLEGELK were able to quantify TM and AK in a complex food matrix with high accuracy (recoveries: 94.11%–102.16%) and precision (intra- and inter-day RSD<10%). At the same time, a signature peptide of TM, ALSNAEGEVAALNR, was found to be absent in crab. The proposed method exhibited a broad linear range (0–1000 ng/mL) of the absolute quantification (AQUA) peptides with R2 > 0.99 and took approximately 11 h per analysis. Moreover, it was successfully applied for quantifying TM and AK in commercial food samples, indicating the great applicable potential. The proposed method can reveal the allergenic risk of crustacean-related food and thus help allergic individuals avoid such food intake.

PIWIs maintain testis apoptosis to remove abnormal germ cells in Eriocheir sinensis.

PIWI proteins play important roles in germline development in the mammals. However, the functions of PIWIs in crustaceans remain unknown. In the present study, we identified three Piwis from the testis of Eriocheir sinensis (E. sinensis). Three Piwi genes encoded proteins with typical features of PIWI subfamilies and were highly expressed in the testis. Three PIWIs could be detected in the cytoplasm of spermatocytes and spermatids, while in spermatozoa, we could only detect PIWI1 and PIWI3 in the nucleus. The knockdown of PIWIs by dsRNA significantly affected the formation of the nuclei in spermatozoa, which resulted in deviant and irregular nuclei. PIWI defects significantly inhibited the apoptosis of abnormal germ cells through the caspase-dependent apoptosis pathway and p53 pathway. Knockdown of PIWIs inhibited the expression of caspase (Casp) 3, 7, 8, and p53 without affecting Bcl2 (B-cell lymphoma gene 2), Bax (B-cell lymphoma-2-associated X), and BaxI (B-cell lymphoma-2-associated X inhibitor), which further significantly increased abnormal spermatozoa in the knockdown-group crabs. These results show a new role of PIWI proteins in crustaceans that is different from that in mammals. In summary, PIWIs play roles in the formation of the germline nucleus and can maintain apoptosis in abnormal germ cells to remove abnormal germ cells in E. sinensis.

Dietary methyl farnesoate, a potential growth inducer in male crab Oziothelphusa senex senex

Insect juvenile hormone-like compound methyl farnesoate (MF), identified through ‘reverse endocrinology’ in crustaceans is a sesqui-terpenoid and plays crucial role in growth well proved by direct administration into the animals at laboratory conditions. However, these studies are not reached to the cultural ponds. Moreover, dietary supplementation of MF and its effects on growth in crustaceans is still at infancy. The present study tested MF (concentration of 10−9, 10−8 and 10−7 moles/crab added to commercial shrimp pellet diet) in the growth of male crab Oziothelphusa senex senex (Oss) supplemented every alternative day for about 40 days. Along with experimental group control and eyestalk removed (ESX) groups are maintained. Dietary MF induced significant enhancement in the growth of male crab. The most effective group MF 10−8 moles/crab supplemented. The frequency of growth induction found in this study is MF 10−8>10−9>10−7 moles/crab ≤ ESX and molted percentage is 27.5%, 17.5%, 10%, 10% in each group, respectively. The dietary supplementation of MF effective in inducing growth in cultured crustaceans thereby increases the yield of crustacean protein.

Comparison of real-time PCR and ELISA for the detection of crustacean shellfish allergens

ABSTRACT Food allergies are a significant public health concern, and crustacean shellfish represent one of the major FDA regulated food allergens. Allergic individuals must avoid foods containing crustaceans, and this necessitates highly sensitive and accurate detection methods. Two of the major methods used are protein-based ELISA and DNA-based real-time PCR. In order to properly compare these very different methodologies, we used identical split samples for a side-by-side comparison and analysed them using four different real-time PCR methods and two different commercial ELISA kits. Three real-time PCR assays targeting the mitochondrial 12S genes of shrimp, crab, and lobster were compared to a commercial ELISA assay for total crustacean protein. A fourth real-time PCR assay targeting the tropomyosin gene of shrimp was compared to an ELISA assay for shrimp tropomyosin. All comparisons were carried out in two different food matrices: Manhattan clam chowder and fish sauce. PCR assays had a more broad dynamic range (0.1–106 mg/kg) as compared to ELISA (200–4000 mg/kg) and did not show matrix interference like ELISA. In cases where the ELISA assays did not have matrix interference, there was good qualitative agreement between PCR and ELISA.

Conception of active food packaging films based on crab chitosan and gelatin enriched with crustacean protein hydrolysates with improved functional and biological properties

This study aims to assess the effect of shrimp and crab protein hydrolysates (SPH and CPH) on structure, physicochemical, antioxidant and antimicrobial properties of composite films based on chitosan and fish gelatin. To this end, protein hydrolysates as bioactive additives were incorporated into edible films at different weight ratios 1.5, 3 and 6% (g/100 g polymer). SPH and CPH enriched films were colored and exhibited higher UV-barrier properties compared to the control chitosan-gelatin film. In addition, an improvement in the mechanical properties and thermal degradation temperatures was also observed with PHs incorporation. Furthermore, the hydrophobicity of the composite films was evaluated by determining the contact angle using different solvents (water, ethylene glycol and diiodomethane) during 120 s. With the PHs incorporation, a significant decrease, which was dependent on time and film composition, in the contact angle was found for the composite films. However, an increase in the surface wettability (especially polar component and surface tension) was observed for enriched films. Therefore, PHs addition increases the hydrophilicity of CS-gelatin based films. Interestingly, the composite films, evaluated by several in vitro assays, displayed higher antibacterial and antioxidant potential than control film. Consequently, composite films are promising alternative for active packaging to hinder food oxidation.

Characterization and functional analysis of fibrinogen-related protein (FreP) in the black tiger shrimp, Penaeus monodon

Ficolin is classified as an immune related protein containing collagen-like and fibrinogen-related domain (FreD). In invertebrates, the functions of fibrinogen-related proteins (FrePs) are of importance to innate immunity. In this study, a FreP in the black tiger shrimp Penaeus monodon was identified and characterized. The PmFreP cDNA is 1,007 bp long with a 921 bp-open reading frame that encodes for 306 amino acids. The deduced PmFreP sequence consists of a signal peptide, an unknown region and the FreD. Phylogenetic analysis showed that PmFreP was clustered with fibrinogen-like proteins in crustaceans which was separated from vertebrate ficolin-like proteins. The deduced fibrinogen-like domain contains four conserved cysteine residues (Cys96, Cys127, Cys249, and Cys262) that are responsible for the formation of disulfide bridges. Gene expression analysis shows that Pmfrep is mainly expressed in the intestine and the expression is significantly upregulated after Vibrio harveyi and white spot syndrome virus (WSSV) challenge. Recombinant PmFreP (rPmFreP) were successfully expressed and purified, and forms a trimeric structure as judged by native-PAGE. Bacterial binding assay showed that the rPmFreD can bind and agglutinate Gram-negative and Gram-positive bacteria in the presence of calcium (Ca2+) ions. Moreover, the rPmFreP facilitates the clearance of V. harveyi in vivo. Overall, our results suggested that the PmFreP may serve as pattern recognition receptors implicated in shrimp innate immunity.

Validation of Real-time PCR Reference Genes of Muscle Metabolism in Harvested Spiny-Cheek Crayfish (Faxonius limosus) Exposed to Seasonal Variation.

Simple SummaryIn the age of shrinking stocks and intensive animal production alternative raw materials are in the eye of the scientists. Here, we aimed to identify molecular tools to evaluate the quality of spiny-cheek crayfish meat. Results showed a set of genes that were steadily or deferentially expressed between seasons and sexes. Additionally, the authors revealed genes involved in molting cycle and muscle growth/atrophy of the crayfish. The suite of molecular tools developed in the study have potential for the profiling of crayfish raw material quality in the food sector.Real-time quantitative reverse transcription PCR (RT-qPCR) is a sensitive and broadly used technique of assessing gene activity. To obtain a reliable result, stably expressed reference genes are essential for normalization of transcripts in various samples. To our knowledge, this is the first systematic analysis of reference genes for normalization of RT-qPCR data in spiny-cheek crayfish (Faxonius limosus). In this study, expression of five candidate reference genes (actb, β-actin; gapdh, glyceraldehyde-3-phosphate dehydrogenase; eif, eukaryotic translation initiation factor 5a; ef-1α, elongation factor-1α; and tub, α-tubulin) in muscle samples from male and female F. limosus in spring and autumn was analyzed. Additionally, the most stable reference genes were used for accurate normalization of five target genes, i.e., tnnc, troponin c; ak, arginine kinase; fr, ferritin; ccbp-23, crustacean calcium-binding protein 23; and actinsk8, skeletal muscle actin 8. Results obtained using the geNorm and NormFinder algorithms showed high consistency, and differences in the activity of the selected actb with eif genes were successfully identified. The spring and autumn activities of the target genes (except ak) in the muscle tissue of males and females differed significantly, showing that both sexes are immensely involved in an array of breeding behaviors in spring, and females intensively recover in the autumn season. Characterization of first reference genes in spiny-cheek crayfish will facilitate more accurate and reliable expression studies in this key species.

Cyclin-dependent kinase 2 (Cdk-2) from the White shrimp Litopenaeus vannamei: Molecular characterization and tissue-specific expression during hypoxia and reoxygenation

The cell cycle comprises a series of steps necessary for cell growth until cell division. The participation of proteins responsible for cell cycle regulation, known as cyclin dependent kinases or Cdks, is necessary for cycle progression. Cyclin dependent kinase 2 (Cdk-2) is one of the most studied Cdks. This kinase regulates the passage through the G1/S phase and is involved in DNA replication in the S phase. Cdks have been extensively studied in mammals, but there is little information about these proteins in crustaceans. In the present work, the nucleotide and amino acid sequence of Cdk-2 from the white shrimp (Cdk-2) and its expression during hypoxia and reoxygenation are reported. Cdk-2 is a highly conserved protein and contains the serine/threonine catalytic domain, an ATP binding site and the PSTAIRE sequence. The predicted Cdk-2 structure showed the two-lobed structure characteristic of kinases. Expression of Cdk-2 was detected in hepatopancreas, gills and muscle, with hepatopancreas having the highest expression during normoxic conditions. Cdk-2 expression was significantly induced after hypoxia for 24 h in muscle cells, but in hypoxia exposure for 24 followed by 1 h of reoxygenation, the expression levels returned to the levels found in normoxic conditions, suggesting induction of cell cycle progression in muscular cells during hypoxia. No significant changes in expression of Cdk-2 were detected in these conditions in hepatopancreas and gills.

Insect (food) allergy and allergens

Insects represent an alternative for meat and fish in satisfying the increasing demand for sustainable sources of nutrition. Approximately two billion people globally consume insects. They are particularly popular in Asia, Latin America, and Africa. Most research on insect allergy has focussed on occupational or inhalation allergy. Research on insect food safety, including allergenicity, is therefore of great importance. The objective of this review is to provide an overview of cases reporting allergy following insect ingestion, studies on food allergy to insects, proteins involved in insect allergy including cross-reactive proteins, and the possibility to alter the allergenic potential of insects by food processing and digestion. Food allergy to insects has been described for silkworm, mealworm, caterpillars, Bruchus lentis, sago worm, locust, grasshopper, cicada, bee, Clanis bilineata, and the food additive carmine, which is derived from female Dactylopius coccus insects. For cockroaches, which are also edible insects, only studies on inhalation allergy have been described. Various insect allergens have been identified including tropomyosin and arginine kinase, which are both pan-allergens known for their cross-reactivity with homologous proteins in crustaceans and house dust mite. Cross-reactivity and/or co-sensitization of insect tropomyosin and arginine kinase has been demonstrated in house dust mite and seafood (e.g. prawn, shrimp) allergic patients. In addition, many other (allergenic) species (various non-edible insects, arachnids, mites, seafoods, mammals, nematoda, trematoda, plants, and fungi) have been identified with sequence alignment analysis to show potential cross-reactivity with allergens of edible insects. It was also shown that thermal processing and digestion did not eliminate insect protein allergenicity. Although purified natural allergens are scarce and yields are low, recombinant allergens from cockroach, silkworm, and Indian mealmoth are readily available, giving opportunities for future research on diagnostic allergy tests and vaccine candidates.

Development and Evaluation of an Enzyme-Linked Immunosorbent Assay Using a Nonpoisonous Extraction System for the Determination of Crustacean Protein in Processed Foods

Crustacean proteins are food allergens that cause severe allergic reactions in patients with food allergies; therefore, the identification of crustaceans such as shrimp, crab, and lobster as ingredients in processed food products is mandatory in Japan. We previously developed and validated an ELISA method coupled with an extraction process using the surfactant sodium dodecyl sulfate and the reductant 2-mercaptoethanol (2-ME) to quantify crustacean protein. However, 2-ME was designated as poisonous in Japan in 2008. Therefore, in this study, we developed and evaluated an ELISA method for detecting and quantifying crustacean protein that uses sodium sulfite (Na2SO3) in place of 2-ME for extraction. The proposed ELISA method showed high sensitivity, with an LOQ of 0.66 μg protein/g food sample. Furthermore, the proposed method showed high specificity for the Decapoda order within the subphylum Crustacea, with recoveries ranging from 83.8 to 100.8% for model processed foods, as well as high reproducibility (intra- and interassay CVs of ≤8.2%) and high correlation with our previously validated ELISA method for processed foods (correlation coefficient of 0.996). The proposed ELISA method does not require the use of poisonous reagents, provides acceptable accuracy, and is useful for the routine monitoring of food products.

Perturbation of the yeast mitochondrial lipidome and associated membrane proteins following heterologous expression of Artemia-ANT

Heterologous expression is a landmark technique for studying a protein itself or its effect on the expression host, in which membrane-embedded proteins are a common choice. Yet, the impact of inserting a foreign protein to the lipid environment of host membranes, has never been addressed. Here we demonstrated that heterologous expression of the Artemia franciscana adenine nucleotide translocase (ANT) in yeasts altered lipidomic composition of their inner mitochondrial membranes. Along with this, activities of complex II, IV and ATP synthase, all membrane-embedded components, were significantly decreased while their expression levels remained unaffected. Although the results represent an individual case of expressing a crustacean protein in yeast inner mitochondrial membranes, it cannot be excluded that host lipidome alterations is a more widespread epiphenomenon, potentially biasing heterologous expression experiments. Finally, our results raise the possibility that not only lipids modulate protein function, but also membrane-embedded proteins modulate lipid composition, thus revealing a reciprocal mode of regulation for these two biomolecular entities.

A study on β-glucan binding protein (β-GBP) and its involvement in phenoloxidase cascade in Indian white shrimp Fenneropenaeus indicus

The present study reports the purification of novel immune molecule β-1, 3 glucan binding protein from the heamolymph of the Indian white shrimp, Fenneropenaeus indicus (Fiβ-GBP). The purified Fiβ-GBP had 95kDa molecular weight in SDS-PAGE analysis. MALDI-TOF/TOF analysis revealed that the purified Fiβ-GBP showed similarity to various crustacean proteins; 48 and 46% similarity was observed for β-1, 3 glucan binding protein of Chinese white shrimp Fenneropenaeus chinensis and banana shrimp Fenneropenaeus merguiensis, with MOWSE score of 3.11e+12 and 2.05e+8, respectively. The phenoloxidase activity (PO) of Fiβ-GBP was evaluated and, in the presence of laminarin, PO activity increased significantly. Substrate specificity assay demonstrated that Fiβ-GBP had the specific binding site for soluble or insoluble β-glucan (laminarin), since the PO activity increased in the presence of laminarin when compared to other sugars. Enzymatic activities revealed that the optimum temperature and pH for Fiβ-GBP activating PO were 40°C and pH 7–8. Moreover, even at 100°C Fiβ-GBP enhanced PO activity highlighting that Fiβ-GBP was thermostable and thermophilic in nature. Among various divalent metallic ions, Fiβ-GBP significantly promoted the PO activity in presence of Mg2+ and Ca2+. The breakdown of para nitroanilide from Nα−Benzoyl-l-Arginine 4-Nitroanilide hydrochloride showed that serine protease activity was induced by Fiβ-GBP and also increased concentration of Fiβ-GBP evoked the activity. Furthermore, hemolytic activity tests revealed that PO reaction product induced RBC membrane damage and cell shrinkage. Lastly, Baclight bacterial viability assays showed maximum killing effect of PO reaction product on both Gram positive and Gram negative bacteria.

Sequence homology of the fly proteins tropomyosin, arginine kinase and myosin light chain with known allergens in invertebrates

Due to the rapid growth of the world’s population and the increasing demand for food, there is an urgent need for alternative, more sustainable sources of protein. Insects have an important role in the diet in some societies and current initiatives are exploring the potential that insects have to offer for the production of food and feed. In this context, the safety implications of both producing and consuming insects are an important aspect to investigate. Here we present a bioinformatics analysis of proteomics data obtained for larvae of four different species of fly to assess the homology of tropomyosin, arginine kinase and myosin light chain with the crustacean orthologous proteins and other known allergenic proteins. The results indicate that the three proteins share homology with known allergens and therefore it is likely that they are also potential allergens. The implications in relation to mass rearing of flies are discussed.

Molecular Mechanisms of Reception and Perireception in Crustacean Chemoreception: A Comparative Review

This review summarizes our present knowledge of chemoreceptor proteins in crustaceans, using a comparative perspective to review these molecules in crustaceans relative to other metazoan models of chemoreception including mammals, insects, nematodes, and molluscs. Evolution has resulted in unique expansions of specific gene families and repurposing of them for chemosensation in various clades, including crustaceans. A major class of chemoreceptor proteins across crustaceans is the Ionotropic Receptors, which diversified from ionotropic glutamate receptors in ancient protostomes but which are not present in deuterostomes. Representatives of another major class of chemoreceptor proteins-the Grl/GR/OR family of ionotropic 7-transmembrane receptors-are diversified in insects but to date have been reported in only one crustacean species, Daphnia pulex So far, canonic 7-transmembrane G-protein coupled receptors, the principal chemoreceptors in vertebrates and reported in a few protostome clades, have not been identified in crustaceans. More types of chemoreceptors are known throughout the metazoans and might well be expected to be discovered in crustaceans. Our review also provides a comparative coverage of perireceptor events in crustacean chemoreception, including molecules involved in stimulus acquisition, stimulus delivery, and stimulus removal, though much less is known about these events in crustaceans, particularly at the molecular level.

Three cDNAs encoding vitellogenin homologs from Antarctic copepod, Tigriopus kingsejongensis: Cloning and transcriptional analysis in different maturation stages, temperatures, and putative reproductive hormones.

Three full-length cDNAs encoding lipoprotein homologs were identified in Tigriopus kingsejongensis, a newly identified copepod from Antarctica. Structural and transcriptional analyses revealed homology with two vitellogenin-like proteins, Tik-Vg1 and Tik-Vg2, which were 1855 and 1795 amino acids in length, respectively, along with a third protein, Tik-MEP, which produced a 1517-residue protein with similarity to a melanin engaging protein (MEP) in insects Phylogenetic analysis showed that Vgs in Maxillopods including two Tik-Vgs belong to the arthropod vitellogenin-like clade, which includes clottable proteins (CPs) in decapod crustaceans and vitellogenins in insects. Tik-MEP clustered together with insect MEPs, which appear to have evolved before the apoB-like and arthropod Vg-like clades. Interestingly, no genes orthologous to those found in the apoB clade were identified in Maxillopoda, suggesting that functions of large lipid transfer proteins (LLTPs) in reproduction and lipid metabolism may be different from those in insect and decapod crustaceans. As suggested by phylogenetic analyses, the two Tik-Vgs belonging to the arthropod Vg-like clade appear to play major roles in oocyte maturation, while Vgs belonging to the apoB clade function primarily in the reproduction of decapod crustaceans. Transcriptional analysis of Tik-Vg expression revealed a 24-fold increase in mature and ovigerous females compared with immature female, whereas expression of Tik-MEP remained low through all reproductive stages. Acute temperature changes did not affect the transcription of Tik-Vg genes, whereas Tik-MEP appeared to be affected by temperature change. Among the three hormones thought to be involved in molting and reproduction in arthropods, only farnesoic acid (FA) induced transcription of the two Tik-Vg genes. Regardless of developmental stage and hormone treatment, Tik-Vg1 and Tik-Vg2 exhibited a strong positive correlation in expression, suggesting that expression of these genes may be regulated by the same transcriptional machinery.

In-Vitro Simulated Gastric Fluid Digestion and Immunogenicity of Different Crustacean Protein Extracts

To compare the immunogenicity and digestibility in-vitro simulated gastric fluid, the protein extracts of white-leg shrimp, Chinese shrimp (female and male shrimp were separated), Southern rough shrimp, Acetes chinensis, Mantis shrimp and crawfish were analyzed by SDS-PAGE, indirect ELISA and Western blotting. The results showed that except for Acetes chinensis, the larger proportion of molecular mass were near to 25 kDa (sarcoplasmic calcium-binding protein) and 38 kDa (tropomyosin). White-leg shrimp protein and Southern rough shrimp protein had a maximum similarity of 68.2%. White-leg shrimp protein had higher immunogenicity than others. There were much more IgE-binding bands in male Chinese shrimp protein, while Crawfish just had only one IgE-binding band (38 kDa, tropomyosin). In simulated gastric fluid system, the protein such as 25 kDa was rapidly degraded within 0.5 mins, while tropomyosin (38 kDa) was relatively resistant to pepsin digestion. The Western blotting results indicated that the allergenicity of those crustacean protein extracts still remained even they were digested with simulated gastric fluid for 60 min.