Cite this paper:
IBRAHIM G., ELGHAZALY M.. Molecular characterization of yolk proteins in the female crab Neptunus pelagicus (A. Milne-Edwards, 1861) from the Mediterranean Sea of Alexandria, Egypt[J]. HaiyangYuHuZhao, 2020, 38(2): 438-453

Molecular characterization of yolk proteins in the female crab Neptunus pelagicus (A. Milne-Edwards, 1861) from the Mediterranean Sea of Alexandria, Egypt

1 Department of Zoology, Faculty of Science, Alexandria University, Alexandria 21547, Egypt;
2 Department of Zoology, Faculty of Science, Damnhour University, El Behara 22516, Egypt
This study aimed to characterize the morphological changes in the ovary of the female crab Neptunus pelagicus and to identify specific fractions of vitelloginin and vitelline molecules during primary and secondary vitellogenesis. Samples of the blue crab were collected from the Mediterranean Sea of Alexandria monthly during 2017. Ovaries and oocytes in primary and secondary vitellogenesis were detached and treated for histological test. Native polyacrylamide gel electrophoresis (PAGE) Bis-Tris Gels was applied to identify vitelloginin (VN) and vitelline (VL) molecules. Protein Analyses were done by PAGE-SDS. The initial degenerate primers were built regarding the conserved amino acid domains of the yolk proteins. Primary and secondary vitellogeneses consisted of 8 phases. Lipoprotein fraction with molecular weight 550 kDa was identified in the hemolymph in secondary vitellogenesis. Two protein fractions (VLI & VLII) were identified in secondary vitellogenic oocytes. The electrophoresis performed with extract of stage I oocyte showed two protein fractions with molecular weights 550 kDa and 460 kDa. In stage II and III oocyte, 4 subunits were presented of 180, 195, 140 and 120 kDa in VLI and 2 subunits with molecular weight of 110 kDa and 95 kDa in VLII. Another two fractions in stage V oocyte presented with molecular weights of 380 kDa and 360 kDa. Western blot analysis proved that both fractions were of four major polypeptide subunits with molecular weight of 180, 125, 90 and 85 kDa in each of the two VLs. The hybridization signal obtained by the Northern blot was detected in the hepatopancreas during ovarian cycle and in the ovary during secondary vitellogenesis. The result of the reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that the mRNA that encodes the C-terminal region of the VN cDNA was found in the ovary in secondary vitellogenesis and in the hepatopancreas.
Key words:   
Received: 2019-05-24   Revised: 2019-07-08
PDF (2144 KB) Free
Print this page
Add to favorites
Email this article to others
Articles by IBRAHIM G.
Articles by ELGHAZALY M.
Abdu U, Davis C, Khalaila I, Sagi A. 2002. The vitellogenin cDNA of Cherax quadricarinatus encodes a lipoprotein with calcium binding ability, and its expression is induced following the removal of the androgenic gland in a sexually plastic system. General and Comparative Endocrinology, 127(3):263-272,
Ando H, Makioka T. 1999. Structure of the ovary and mode of oogenesis in a freshwater crab Potamon dehaani. Journal of Morphology, 239(1):107-114,<107::AID-JMOR8>3.0.CO;2-J.
Anger K. 1995. The conquest of freshwater and land by marine crabs:adaptations in life history patterns and larval bioenergetics. Journal of Experimental Marine Biology and Ecology, 193(1-2):119-145,
Antunes M, Zara FJ, López-Greco LS, Negreiros-Fransozo ML. 2016. Morphological analysis of the female reproductive system of Stenorhynchus seticornis(Brachyura:Inachoididae) and comparisons with other Majoidea. Invertebrate Biology, 135(2):75-86,
Avarre J C, Michelis R, Tietz A, Lubzens E. 2003. Relationship between vitellogenin and vitellin in a marine shrimp(Penaeus semisulcatus) and molecular characterization of vitellogenin complementary DNAs. Biology of Reproduction, 69(1):355-364,
Bambeck G S. 1996. Electrophoresis separation gel and method for preparing an electrophoresis separation gel.US Patent No. 5589104.
Becker C, Brandis D, Storch V. 2011. Morphology of the female reproductive system of European pea crabs(Crustacea, Decapoda, Brachyura, Pinnotheridae).Journal of Morphology, 272(1):12-26,
Bisen P S. 2014. Microbial staining. In:Bisen P S ed. Microbes in Practice. IK International, New Delhi, p.139-155.
Browdy C L, Fainzilber M, Tom M, Loya Y, Lubzens E. 1990.Vitellin synthesis in relation to oogenesis in in vitroincubated ovaries of Penaeus semisulcatus (Crustacea, Decapoda, Penaeidae). Journal of Experimental Zoology, 255(2):205-215,
Chang C F, Jeng S R. 1995. Isolation and characterization of the female-specific protein (vitellogenin) in mature female hemolymph of the prawn Penaeus chinensis. Comparative Biochemistry and Physiology Part B:Biochemistry and Molecular Biology, 112(2):257-263,
Choy S. 1988. Reproductive biology of Liocarcinus puber and L. holsatus (Decapoda, Brachyura, Portunidae) from the Gower Peninsula, South Wales. Marine Ecology, 9(3):227-241.
Cobo V J, Fransozo A. 2005. Physiological maturity and relationships of growth and reproduction in the red mangrove crab Goniopsis cruentata (Latreille)(Brachyura, Grapsidae) on the coast of São Paulo, Brazil.Revista Brasileira de Zoologia, 22(1):219-223,
de Souza L P, Silva J R F. 2009. Morphology of the female reproductive system of the red-clawed mangrove tree crab (Goniopsis cruentata Latreille, 1803). Scientia Marina, 73(3):527-539,
de Souza L P, Silva J R F, Araujo A M, Camargo-Mathias M I. 2013. Morphology of the female genital ducts of the blue land crab Cardisoma guanhumi (Crustacea:Brachyura:Gecarcinidae). Acta Zoologica, 94(3):300-307,
Demeusy N. 1962. Rôle de la glande de mue dans l'évolution ovarience du crabe Carcinus maenas Linné. Cahiers de Biologie Marine, 3:37-56.
Engelman F. 1979. Insect vitellogenin:identification, biosynthesis, and role in vitellogenesis. Advances in Insect Physiology, 14:49-108,
Ewers-Saucedo C, Hayer S, Brandis D. 2015. Functional morphology of the copulatory system of box crabs with long second gonopods (Calappidae, Eubrachyura, Decapoda, Crustacea). Journal of Morphology, 276(1):77-89,
Flávia S D, Tania M S C, Assunta M P. 2011. Morphology and histology of the female reproductive system of the mangrove land crab Ucides cordatus (Linnaeus, 1763)(Brachyura. Ocypodidae). Nauplius, 19:145-153,
Garfin D E. 1990. One-dimensional gel electrophoresis.Methods in Enzymology, 182:425-441,
Girish B P, Swetha C H, Reddy S P. 2014. Hepatopancreas but not ovary is the site of vitellogenin synthesis in female fresh water crab, Oziothelphusa senex senex. Biochemical and Biophysical Research Communications, 447(2):323-327,
Guan Z B, Yin J, Chen K, Shui Y, Cai Y J, Liao X R. 2016. The hepatopancreas and ovary are the sites of vitellogenin synthesis in female red swamp crayfish (Procambarus clarkii (Girard, 1852)) (Decapoda:Astacoidea:Cambaridae). Journal of Crustacean Biology, 36(5):637-641,
Hamasaki K, Fukunaga K, Kitada S. 2006. Batch fecundity of the swimming crab Portunus trituberculatus (Brachyura:Portunidae). Aquaculture, 253(3-4):359-365.
Hard W L. 1942. Ovarian Growth and Ovulation in the Mature Blue Crab, Callinectes sapidus Rathbun. Chesapeake Biological Laboratory, Solomons Island, MD. p.1-17.
Hotzel H, Müller W, Sachse K. 1999. Recovery and characterization of residual DNA from beer as a prerequisite for the detection of genetically modified ingredients.European Food Research and Technology, 209(3-4):192-196,
Ibrahim G A. 2015. Oogenesis in the cancer crab Parthenope longimanus. Journal of Advances in Biology, 6(3):1 036-1 065.
Ibrahim G A. 2018. Oogenesis of the carpet sea squirt Didemnum vestitum (Kott, 2004) (Ascidiacea-Aplousobranchia) in the Arabian Gulf-ultrastructural profile. International Journal of Oceans and Oceanography, 12(2):173-190.
Jeannot M A, Zheng J, Li L. 1999. Observation of sodium gelinduced protein modifications in dodecyl sulfate polyacrilamide gel electrophoresis and its implications for accurate molecular weight determination of gel-separated proteins by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry. Journal of the American Society for Mass Spectrometry, 10(6):512-520.
Komm B S, Hinsch G W. 1987. Oogenesis in the terrestrial hermit crab, Coenobita clypeatus (Decapoda, Anomura):II. Vitellogenesis. Journal of Morphology, 192(3):269-277.
Kung S Y, Chan S M, Hui J H L, Tsang W S, Mak A, He J G. 2004. Vitellogenesis in the sand shrimp, Metapenaeus ensis:the contribution from the hepatopancreas-specific vitellogenin gene (MeVg2). Biology of Reproduction, 71(3):863-870,
Lautenschlager A D, Brandis D, Storch V. 2010. Morphology and function of the reproductive system of representatives of the genus Uca. Journal of Morphology, 271(11):1 281-1 299,
Lee R F, Puppione D L. 1988. Lipoproteins I and II from the hemolymph of the blue crab Callinectes sapidus:lipoprotein II associated with vitellogenesis. Journal of Experimental Zoology, 248(3):278-289.
Liu H C, Li C W. 2000. Reproduction in the fresh-water crab Candidiopotamon rathbunae (Brachyura:Potamidae) in Taiwan. Journal of Crustacean Biology, 20(1):89-99.
Luo W, Zhao Y L, Zhou Z L, An C G, Ma Q. 2008. Digestive enzyme activity and mRNA level of trypsin in embryonic redclaw crayfish, Cherax quadricarnatus. Chinese Journal of Oceanology and Limnology, 26(1):62-68,
Mak A S C, Choi C L, Tiu S H K, Hui J H L, He J G, Tobe S S, Chan S M. 2005. Vitellogenesis in the red crab Charybdis feriatus:hepatopancreas-specific expression and farnesoic acid stimulation of vitellogenin gene expression.Molecular Reproduction and Development, 70(3):288-300.
Okuno A, Hasegawa Y, Ohira T, Katakura Y, Nagasawa H. 1999. Characterization and cDNA cloning of androgenic gland hormone of the terrestrial isopod Armadillidium vulgare. Biochemical and Biophysical Research Communications, 264(2):419-423.
Okuno A, Yang W J, Jayasanka V, Saido-Sakanaka H, Huong D T T, Jasmani S, Atmomarsono M, Subramoniam T, Tsutsui N, Ohira T, Kawazoe I, Aida K, Wilder M N. 2002. Deduced primary structure of vitellogenin in the giant freshwater prawn, Macrobrachium rosenbergii, and yolk processing during ovarian maturation. Journal of Experimental Zoology, 292(5):417-429,
Opplt J J. 1999. Discontinuous and nonsequential polymeric gel systems for separation of macromolecules. US Patent, No. 5968332.
Palacios E, Racotta I S, Vallalejo M. 2003. Assessment of ovarian development and its relation to mating in wild and pond-reared Litopenaeus vannamei shrimp in a commercial hatchery. Journal of the World Aquaculture Society, 34(4):466-477,
Poms R, Glössl J, Foissy H. 2001. Increased sensitivity for detection of specific target DNA in milk by concentration in milk fat. European Food Research Technology, 213(4-5):361-365,
Puengyam P, Tsukimura B, Utarabhand P. 2013. Molecular characterization of hepatopancreas vitellogenin and its expression during the ovarian development by in situ hybridization in the banana shrimp Fenneropenaeus merguiensis. Journal of Crustacean Biology, 33(2):265-274,
Qiu C. 1991. The cytochemistry of oocytes of Chinese shrimp Penaeus orientalis. Chinese Journal of Oceanology and Limnology, 9(2):106-114,
Rotllant G, González-Gurriarán E, Fernández L, Benhalima K, Ribes E. 2007. Ovarian maturation of the multi-spawning spider crab Maja brachydactyla (Decapoda:Majidae)with special reference to yolk formation., Marine Biology, 152(2):383-394,
Sal Moyano M P, Gavio M A, Cuartas E I. 2010. Morphology and function of the reproductive tract of the spider crab Libinia spinosa (Crustacea, Brachyura, Majoidea):pattern of sperm storage. Helgoland Marine Research, 64(3):213-221,
Sharifian S, Kamrani E, Safaie M, Sharifian S. 2015. Oogenesis and ovarian development in the freshwater Crab Sodhiana iranica (Decapoda:Gecarcinuaidae) from the south of Iran. Tissue and Cell, 47(2):213-220.
Sheer D G, Yamane D K, Hawke D H, Yuan P M. 1990. The use of micropreparative electrophoresis of protein/peptide isolations for primary structure determinations. Peptide Research, 3(2):97-104.
Supriya N T, Sudha K, Krishnakumar V, Anilkumar G. 2017. Molt and reproduction enhancement together with hemolymph ecdysteroid elevation under eyestalk ablation in the female fiddler crab, Uca triangularis (Brachyura:Decapoda).Chinese Journal of Oceanology and Limnology, 35(3):645-657,
Swiney K M, Shirley T C. 2001. Gonad development of southeastern Alaskan dungeness crab, Cancer magister, under laboratory conditions. Journal of Crustacean Biology, 21(4):897-904,
Tseng D Y, Chen Y N, Kou G H, Lo C F, Kuo C M. 2001.Hepatopancreas is the extraovarian site of vitellogenin synthesis in black tiger shrimp, Penaeus monodon.Comparative Biochemistry and Physiology Part A:Molecular & Integrative Physiology, 129(4):909-917.
Tsutsui N, Saido-Sakanaka H, Yang W J, Jayasankar V, Jasmani S, Okuno A, Ohira T, Okumura T, Aida K, Wilder M N. 2004. Molecular characterization of a cDNA encoding vitellogenin in the coonstriped shrimp, Pandalus hypsinotus and site of vitellogenin mRNA expression.Journal of Experimental Zoology Part A:Comparative Experimental Biology, 301(10):802-814,
Tufail M, Takeda M. 2002. Vitellogenin of the cockroach, Leucophaea maderae:nucleotide sequence, structure and analysis of processing in the fat body and oocytes. Insect Biochemistry and Molecular Biology, 32(11):1 469-1 476.
Vallina M, Sal Moyano M P, Cuartas E I, Gavio M A. 2014.Reproductive system and size maturity of the paddle crab Ovalipes trimaculatus (Brachyura:Portunidae) along the Argentine coast. Journal of Crustacean Biology, 34(3):357-366,
Vasquez-Boucard C, Ceccaldi H J, Benyamin Y, Roustan C. 1986. Identification, purification, et caractérisation de la lipovitelline chez un crustacé décapode Natantia Penaeus japonicus (Bate). Journal of Experimental Marine Biology and Ecology, 97(1):37-50,
Vehof J, Scholtz G, Becker C. 2017. Morphology of the female reproductive system of three dorippid crabs (Crustacea:Decapoda:Brachyura:Dorippidae) and the role of accessory cuticle structures associated with seminal receptacles. Invertebrate Biology, 136(3):271-289,
Wallace R A. 1985. Vitellogenesis and oocyte growth in nonmammalian vertebrates. In:Browder L W ed.Oogenesis. Developmental Biology (A Comprehensive Synthesis). Springer, Boston, MA, 1:127-177,
Wang Y Y, Sun H S, Wang Y J, Yan D C, Wang L. 2015.Cytochemical characterization of yolk granule acid phosphatase during early development of the oyster Crassostrea gigas (Thunberg). Chinese Journal of Oceanology and Limnology, 33(2):339-346,
Yang W J, Ohira T, Tsutsui N, Subramoniam T, Huong D T T, Aida K, Wilder M N. 2000. Determination of amino acid sequence and site of mRNA expression of four vitellins in the giant freshwater prawn, Macrobrachium rosenbergii.Journal of Experimental Zoology, 287(6):413-422.
Yin M C, Craik J C A. 1992. Biochemical changes during development of eggs and yolk-sac larvae of herring and plaice. Chinese Journal of Oceanology and Limnology, 10(4):347-358,
Yu Z Y, Wu X G, Chang G L, Cheng Y X, Liu Z J, Yang X Z. 2007. Changes in the main biochemical composition in ovaries and hepatopancreas of Chinese mitten crab, Eriocheir sinensis (H. Milne-Edwards) during the second ovarian development. Acta Hydrobiologica Sinica, 31(6):799-806. (in Chinese with English abstract)
Zinski S. 2006. The Blue Crab Archives. Available at
Zmora N, Trant J, Chan S M, Chung J S. 2007. Vitellogenin and its messenger RNA during ovarian development in the female blue crab, Callinectes sapidus:gene expression, synthesis, transport, and cleavage. Biology of Reproduction, 77(1):138-146,