Vol. 94: 211–224, 2011 DISEASES OF AQUATIC ORGANISMS Published May 9 doi: 10.3354/dao02331 Dis Aquat Org OPENPEN ACCESSCCESS Characterisation of two vitellogenins in the salmon louse Lepeophtheirus salmonis: molecular, functional and evolutional analysis Sussie Dalvin1, Petter Frost1, 4, Peter Loeffen2, Rasmus Skern-Mauritzen1, Jamil Baban1, 5, Ivar Rønnestad3, Frank Nilsen1, 6,* 1Institute of Marine Research, 5817 Bergen, Norway 2Intervet International bv, Wim de Körverstraat 35, PO Box 31, 5830 AA Boxmeer, The Netherlands 3Department of Biology, University of Bergen, PO Box 7803, 5020 Bergen, Norway 4Present address: Intervet Norbio AS, Thormøhlensgt. 55, 5008 Bergen, Norway 5Present address: Department of Chemistry, Biotechnology and Food Science, University of Life Sciences, PO Box 500, 1432 Aas, Norway 6Present address: Department of Biology, University of Bergen, PO Box 7803, 5020 Bergen Norway ABSTRACT: The salmon louse Lepeophtheirus salmonis Krøyer affects a variety of wild salmonoid hosts, but is also an important pest in aquaculture, which is a globally important and rapidly growing industry. Salmon lice have large reproductive outputs, and knowledge of reproductive processes may be crucial for the control of this parasite. Here, we report on the characterisation of 2 vitellogenins (LsVit1 and LsVit2), which are the precursors of salmon-louse egg-yolk glycoprotein. The structure of LsVit1 and LsVit2 was examined and compared to that in other oviparous animals. Phylogenetic analysis of LsVit1 and LsVit2 confirmed the view that crustaceans are a polyphyletic group. Tran- scriptional and translational analysis demonstrated production of LsVit1 and LsVit2 in the subcuticu- lar tissue of the adult female lice. LsVit1 and LsVit2 could also be found in maturing oocytes and developing embryos and early larval stages. LsVit2 was found to be processed into 2 smaller frag- ments, whereas LsVit1 was found to be full length when deposited into the oocytes. Degradation of LsVit1 and LsVit2 was characterised through embryogenesis and the early non-feeding larval stages. Finally, protein content and the level of free amino acids were analysed in embryos and larval stages and their role in nutrition and osmoregulation discussed. In conclusion, our results confirm the role of vitellogenins in reproduction as providers of embryonic and larval nutrition. KEY WORDS: Vitellogenin · Copepoda · Reproduction · Vitellin · Ectoparasite · Aquaculture · Sea lice · Nauplii Resale or republication not permitted without written consent of the publisher INTRODUCTION jor economic losses in aquaculture and also represents a threat to wild salmon populations (Bjorn et al. 2001). The The salmon louse Lepeophtheirus salmonis is an ec- reproductive system of salmon louse has been described toparasitic copepod with a lifestyle adapted to the low morphologically (Ritchie et al. 1996), but few molecular population density of their salmonid hosts. Female lice studies of reproduction in salmon louse and the associ- are long-lived, store sperm in spermatophores, have ated copepod group have been published (Todd et al. large reproductive output and produce larvae with con- 2005, Lee et al. 2008, Dalvin et al. 2009, Hwang et al. siderable dispersal capacity. Aquaculture in coastal ar- 2009). eas of Norway, Scotland, Ireland and Canada has dra- In oviparous animals, eggs are supplied with suffi- matically increased the number of available hosts. The cient nutrients to ensure proper development and re sulting increase in salmon lice abundance causes ma- growth until external food can be ingested and utilised *Corresponding author. Email: [email protected] © Inter-Research 2011 · www.int-res.com 212 Dis Aquat Org 94: 211–224, 2011 autonomously. In lecithotrophic embryos of some body proteins, other biomolecules and energy. No marine invertebrates, however, dissolved nutrients, studies have thus far investigated these issues in including amino acids (AA), may be absorbed from the salmon louse. surrounding water, thereby representing an additional A transcriptomic study of post-moulting growth in supply of organic compounds to the developing em - adult female salmon louse (Eichner et al. 2008) re - bryos (Manahan et al. 1982, Vavra & Manahan 1999). vealed several putative egg-yolk protein transcripts, AA are important substrates for synthesising body pro- including 2 female-specific vitellogenin transcripts. teins and act as precursors for a range of biomolecules, The corresponding genes were named LsVit1 and while they are also used as a source of energy by the LsVit2 (Lepeophtheirus salmonis vitellogenin 1 and 2) developing embryos. Vitellogenins are the major yolk and were expressed in adult females only. proteins in most invertebrate and vertebrate egg-lay- The main objective of the present study was to char- ing animals. Several different vitellogenins typically acterise these 2 vitellogenins. Sequencing and phylo- give rise to vitellin present in mature eggs (Sappington genetic analysis of LsVit1 and LsVit2 shows that cope- & Raikhel 1998); however, in some animals, such as pod and hexapod vitellogenins have the same domain insects, the role of multiple vitellogenin genes is un - structure and are closely related in the phylogenetic known (Tufail & Takeda 2008). Vitellogenins and pro- tree. By means of in situ hybridisation and immunohis- teins with domain structure similar to vitellogenins are tochemistry, the localisations of transcription and also involved in other developmental processes, such translation and vitellogenesis were identified. The as regulation of osmolarity in pelagic fish eggs (Kristof- issue of processing of LsVit1 and LsVit2 was also fersen et al. 2009), immunity (Liu et al. 2009) and clot- addressed when our data showed that they are differ- ting (Hall et al. 1999). entially processed. Finally, the function of LsVit1 and In vertebrates, vitellogenins are produced in the LsVit2 was addressed by investigating the degradation liver, and tissues with similar function have been of the proteins during embryonic and larval develop- reported to produce vitellogenins in invertebrates. In ment at both the protein and AA level. insects, vitellogenins are produced in the fat body, structurally modified and processed into smaller frag- ments (Tufail & Takeda 2008) and thereafter incorpo- MATERIALS AND METHODS rated into the developing eggs by receptor-mediated endocytosis (Sappington & Raikhel 1998). After incor- Animal culture. Salmon lice eggs were hatched in poration into the oocyte, vitellogenins are stored in a incubators with flowing seawater and used to infect crystalline form, named vitellin. salmon (Hamre et al. 2009). Salmon lice were kept in In crustaceans, the vitellogenins are primarily pro- culture on Atlantic salmon Salmo salar in tanks with duced in the hepatopancreas, but minor production seawater (salinity 34.5, 20 µM filtered). Fish were has also been observed in ovaries (Eastman-Reks & anaesthetised with a combination of metomidate and Fingerman 1985, Chen et al. 1999, Zmora et al. 2007, benzocaine. All experiments were conducted in accor- Tiu et al. 2009). As in insects, crustacean vitellogenins dance with Norwegian animal-welfare regulations. are commonly processed into smaller fragments before Peptide sequencing, purification of LsVit1 and incorporation into vitellins (Okuno et al. 2002). Despite LsVit2 and antibody production. Adult female lice the crucial role of vitellin degradation for nourishment were collected with forceps from anaesthetised fish. of the developing and growing embryo and also larval Un fertilised eggs were harvested by puncturing the survival, few molecular studies (Walker et al. 2006, genital segment. Water-soluble proteins from eggs Garcia et al. 2008) have investigated the degradation were extracted from 50 lice in 2.5 ml cold sonication of vitellins during ontogeney. buffer (50 mM Tris-HCl, pH 7.5, 50 mM NaCl and Similar to most other marine copepods, the adult 1 mM EDTA). Eggs were disrupted by sonication using salmon louse is an osmoconforming regulator in sea- a micro-ultrasonic cell disrupter. The sonicated extract water (Hahnenkamp & Fyhn 1985). In such osmo-con- was clarified by centrifugation (13 000 × g for 20 min at forming animals, free amino acids (FAA) serve impor- 4°C) and pellet and lipids were discarded. The total tant roles in cell volume regulation and are therefore protein content in the supernatant was analysed by present in high intracellular concentrations. During sodium dodecyl sulphate polyacrylamide gel elec- degradation of vitellins in developing lice, there is trophoresis (SDS-PAGE) and Coomassie staining. Pro- likely to be a high production of AA with large implica- tein bands were excised from the gel and internal AA tions for the turnover in the FAA pool. Due to the mul- sequence analysis performed by either EuroSequence tiple roles served by FAA in early development, it is (Groningen, Netherlands) or Probe (Proteomic Unit, important for the embryo to balance the strategy for University of Bergen, Norway). This involved in situ osmoregulation with the demand for substrates for tryptic digestion of the protein band, extraction of the Dalvin et al.: Vitellogenins in the salmon louse 213 peptides and reverse phase high performance liquid room temperature for 30 min followed by washing and chromatography (RP-HPLC) separation of the frag- developed with New Fuchsin Chromogen (DAKO) ments generated. The phenylthiohydantoin AAs with 1 mM levamisole (Sigma). Slides