Hepatopancreas but not ovary is the site of vitellogenin synthesis in female fresh water crab, Oziothelphusa senex senex B.P. Girish a,b,1 , CH. Swetha a,b,1 , P. Sreenivasula Reddy b,⇑ a Department of Biotechnology, Sri Venkateswara University, Tirupati 517502, India b Department of Zoology, Sri Venkateswara University, Tirupati 517502, India article info Article history: Received 23 March 2014 Available online 5 April 2014 Keywords: Oziothelphusa Vitellogenin Hepatopancreas and ovary abstract The objective of the present study was to explore the site of synthesis of vitellogenin (Vtg) in fresh water edible crab, Oziothelphusa senex senex. Vtg cDNA fragments were isolated from the hepatopancreas of female crabs using RT-PCR method, and the deduced amino acid sequence of O. senex senex showed more than 60% identity with other brachyuran Vtg sequences. RT-PCR analysis showed that Vtg mRNA can be detected only in hepatopancreas of female Oziothelphusa but not in other tissues including eyestalks, Y- organs, mandibular organs, thoracic ganglion, hypodermis and ovary. Antibodies were raised against vitellin purified from the ovary of O. senex senex. Immunoprecipitation analysis revealed the presence of Vtg in the hepatopancreas of vitellogenic stage I females and in the hemolymph, hepatopancreas and ovary extracts from vitellogenic stage II females but absent in hemolymph and hepatopancreas extract of males. These results suggest that Vtg is synthesized only in hepatopancreas but not in the ova- ries of O. senex senex. In addition, Vtg synthesized in hepatopancreas is transported to ovary through hemolymph. Ó 2014 Elsevier Inc. All rights reserved. 1. Introduction Vitellogenesis is hormone regulated phase in female reproduc- tive cycle of crustaceans, involving synthesis and accumulation of yolk proteins in the ovary. The major yolk protein, vitellin contains proteins, carbohydrates and phospholipids and serves as the nutri- tive material for embryogenesis and early larva [1–5]. In addition, carotenoid, thyroxine, retinol and riboflavin can be taken into oocytes during vitellogenesis by the carrier of vitellogenin (Vtg) [6–9]. The site of synthesis of Vtg (i.e. precursor of vitellin) is still controversial in crustaceans. Vtg is synthesized in the liver of vertebrates [10], fat bodies of insects [11] and intestine of nematodes [12], and then transported to ovary through circulation. In crustaceans, Vtg is postulated to be synthesized by extra-ovarian tissues and then transported through the hemolymph to the developing oocytes [13–15]. Detection of Vtg in hemolymph supported the above hypothesis [16]. Vtg recep- tor (VtgR) is detected on the oocytes and it is proposed that Vtg internalization is receptor mediated endocytotic process [16]. Sev- eral studies have demonstrated that Vtg is synthesized exclusively in hepatopancreas of Macrobrachium rosenbergii [17], Pandalus hypsinotus [18] and Charybdis feriatus [19]. While other studies have shown that Vtg is synthesized in the ovaries of Penaeus semi- sulcatus [20] and Callinectes sapdius [21] respectively. Recently, few studies also confirmed both ovary and hepatopancreas were iden- tified as sites of vitellogenesis but contribution of ovary is rela- tively less [22–25]. These variable results may reflect the existence of multiple sites of Vtg synthesis, species specificity, or may be a manifestation of differences in the methodologies adopted. Immunological detection, radiolabeling of proteins and expression of mRNA have been employed by several scientists to determine the site of vitellogenesis in crustaceans [16]. The pres- ent experiments were carried out to contribute to the discussion on this controversy using immunological and RT-PCR methodolo- gies in fresh water edible crab, O. senex senex as model organism. 2. Materials and methods 2.1. Collection and maintenance of animals Intact female crabs, O. senex senex (NCBI, Taxon ID: 324906) were collected from the rice fields and irrigation canals around Tirupati (13° 36 0 N, 79° 21 0 E), Andhra Pradesh, India. Crabs were housed 6–8 per glass aquaria (length:width:height = 60:30:30 cm) with sufficient ambient medium (salinity: 0.5 ppt) and water was http://dx.doi.org/10.1016/j.bbrc.2014.03.148 0006-291X/Ó 2014 Elsevier Inc. All rights reserved. ⇑ Corresponding author. Fax: +91 877 2249611. E-mail address: reddy_1955@yahoo.co.in (P.S. Reddy). 1 Joint first authors, contributed equally for this work. Biochemical and Biophysical Research Communications 447 (2014) 323–327 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc