THE JOURNAL OF EXPERIMENTAL ZOOLOGY 276:87-94 (1996) Larval Storage Protein of the Barnacle, Balanus amphitrite: Biochemical and Immunological Similarities to Vitellin KATSUHIKO SHIMIZU, CYRIL G. SATUITO, WAKANA SAIKAWA, NOBUHIRO FUSETANI Fusetani Biofouling Project, ERATO, JRDC, c l o Yokohama R&D Center of AND Niigata Engineering Co., Ltd., Isogo-ku, Yokohama 235, Japan ABSTRACT Biochemical and immunological characterization of cyprid major protein (CMP), the principal protein constituent of cypris larvae of the barnacle Balanus amphitrite (Crustacea: Cirripedia), revealed similarities to egg-yolk protein, vitellin, as follows: CMP and vitellin heavy chain both have a molecular weight of 170 kDa by polyacrylamide gel electrophoresis containing sodium dodecylsulfate; CMP was crossreactive with antiserum against vitellin heavy chain in immunoblot analysis. The sequence of 11 amino acids in the amino-terminus of CMP, however, is not perfectly homologous to that of vitellin heavy chain. Thus, it was deduced that CMP was an isoform of vitellin. Concentration of CMP abruptly increased during the latter naupliar stages, reaching a peak just after metamorphosis to the non-feeding cypris stage, and decreased thereafter with aging of cyprids or during the early juvenile period. Specifically, the concentration of CMP in newly metamorphosed juveniles within one day decreased to 20% that of cyprids. CMP, therefore, appears to function as a storage protein during settlement of cyprids as well as metamorphosis to juveniles. Immunohistochemical analysis using antiserum against vitellin heavy chain on sectioned cyprids suggested that CMP is accumulated in the haemocoel. o 1996 Wiley-Liss, Inc. Barnacles are crustaceans which have successfully evolved a sessile form of life style. In their life cycle, they undergo a series of larval developments in the planktonic stage, consisting of 6 feeding naupliar stages, followed by a metamorphosis to a non-feeding cypris stage. Cypris larva is a unique larval form highly adapted for settlement and subsequent metamorphosis, while barnacle nauplii are similar to those of other crustaceans (Anderson, '94). Cypris larvae do not feed and resemble the pupal stage in insects, but they actively swim and search for suitable substrata to settle, then metamorphose into juveniles. Since cyprids contain many conspicuous oil cells, lipid is believed t o be their main energy resource. In Semibalanus balanoides, the number of oil cells increases through nauplius stages and then decreases after metamorphosis t o the cyprid (Walley, '69). Lucas et al. ('79) suggested that protein, in addition t o lipid, was utilized during the cypris stage of S. balanoides. In insects, storage proteins which are the principal constituents of the haemolymph in larvae of the final instar play important roles as amino acid pools, the transporter of lipids or sugars in the pupae, and as components of some tis0 1996 WILEY-LISS, INC, sues in the adult (Kanost et al., '90). However, storage proteins have not yet been identified or observed in larvae of marine invertebrates, including barnacles. In the course of our studies on the mechanism of larval settlement and metamorphosis of barnacles, we have encountered a conspicuous protein present abundantly in cyprids. We refer to this protein as CMP, the cyprid major protein. In this paper, we describe the biochemical and immunological characteristics of CMP, the principal protein constituent in the cypris larvae of the barnacle Balanus amphitrite. We also determine its occurrence in the other stages in the life cycle of the barnacle and discuss its function as a storage protein. MATERIALS AND METHODS Animals Adult barnacles, Balanus amphitrite, attached on bamboo poles in oyster farms were collected Received December 19, 1995; revision accepted May 10,1996. Address reprint requests to Katsuhiko Shimizu, Fusetani Biofouling Project, ERATO, JRDC, d o Yokohama R&D Center of Niigata Engineering Co., Ltd., 27 Shin-isogo-cho, Isogo-ku, Yokohama 235,Japan. 88 K.SHIMIZU ET AL. from Lake Hamana in Shizuoka, Japan (137" 36'E, 34"45'N) and maintained in an aquarium at 25°C by feeding a1 diet of brine shrimp (Artemia salina) nauplii (cultured on marine chlorella Nannochloropsis oculata) and the diatom, Chaetoceros graci,!is. Every other day the barnacles were removed from the aquarium t o dry, thereby preventing diatoms from growing on the shell surfaces. The barnacles released 1-11 stage nauplii upon immersion in seawater after these were dried for 2 days. Nauplii were immediately collected and washed with filtered seawater. These were cultured in 31 glass beakers at an initial density of 3 larvae/ml by feeding with the diatom C. gracilis at a concentration of 2.0 x lo5 cells/ml. Cultures were maintained at 25°C with weak aeration, Seawater used for the cultures was GF/C (Whatman, Clifton, NJ) filtered and the salinity was adjusted to 27%0. Each day, larvae were collected on 100 pm nylon plankton nets, washed with seawater, and transferred to newly prepared algal diet suspensions. Seawater used in this study was purchased from Tokai Kisen Co., Ltd. (Tokyo, Japan) which had been collected off Hachijo Island (139"50'E, 33'10"). Larvae were sampled every day from the culture batch for analysefi after confirmation of the stage of development. Larvae reached the cypris stage in 5 days from start of the culture. The day of moulting to the cyprid is referred to as Day 0. Day 0 cyprids were allowed to attach to a nitrocellulose sheet to obtain juveniles. First, a nitrocellulose sheet was coated with the adult barnacle extract, promoter of attachment for cyprids, which had been prepared by the method of Crisp and Meadows ('621, before being glued to the bottom of a polypropylene container. The container was then filled with 100 ml of C. gracilis suspension at a concentration of 2.0 x lo5 celldml, to which aplproximately 1,000 Day-Ocyprids were added. About 300 cyprids attached to the nitrocellulose sheet and metamorphosed intojuveniles over a 24 h period. Swimming cyprids were then transferred to anew polypropylene container containing seawater, but were not provided with a nitrocellulose sheet for attachment. On the other hand, cyprids which attached but,have not completed metamorphosis were removed, leaving only juveniles on the sheet. Juveniles were fed with fresh algal suspension, while seawater of the swimming cyprids was renewed daily. various stages in the life cycle were extracted with the sample buffer containing 62.5 mM TrisHCl (pH 6.8), 2% (w/v>SDS, 2% (v/v) 2-mercaptoethanol, 0.005% (w/v) bromophenol blue, 10% (wh) glycerol, 2 mM N-ethylmaleimide (all chemicals above were purchased from Wako Pure Chemical Industries Ltd., Osaka, Japan), and 1 mM phenylmethanesulfonylfluoride (Sigma Chemical Co., St. Louis, MO). Samples of either 25 larvae or juveniles were homogenized with 10 pl of the sample buffer. Each homogenate was boiled for 3 min and then centrifuged at 15,000 rpm for 20 min at 4°C t o remove insoluble materials. Aliquots of samples containing either 40 larvae or juveniles were electrophoresed at 20 mA for 1.5 h at room temperature. The gels used consisted of separation gel (8 cm width x 5 cm length x 1 mm thickness) containing 0.325 M TrisHCl (pH 8.8), 0.1% (w/v) SDS, 7.5% (w/v) acrylamide (Wako Pure Chemical Industries Ltd.), and 0.2% (w/v) N,N'-methylenebisacrylamide (Wako Pure Chemical Industries Ltd.) with stacking gel (8 cm x 1.5 cm x 1 mm) containing 0.125 M TrisHCl (pH 6.81, 0.1% (wh) SDS, 3% (w/v) acrylamide, and 0.1% (w/v> N,N'-methylenebisacrylamide. Proteins in the gel were visualized by staining with Coomassie Brilliant Blue R-250 (CBB, Nakarai Tesque Inc., Kyoto, Japan). For quantitative determination of CMP, gels dried between cellophane membranes were analyzed with the densitometer (The Flying-Spot Scanner CS-9000, Shimadzu Corp., Kyoto, Japan). Calibration was carried out using bovine serum albumin (BSA, Sigma Chemical Co.) as standard. Concentration of total protein in extracts from larvae or juveniles were determined by the method of Lowry et al. ('51) with BSA as standard. Statistic analyses for amounts of CMP or total protein were carried out with Student's t-test. Immunoblot analysis Sample preparation from cypris larvae were as described in the section on SDS-PAGE. Ovary extract was prepared as described in the previous report (Shimizu et al., '96). Ovary was homogenized on ice with extraction buffer (EB) containing 100 mM NaC1, 50 mM TrisHCl (pH 7 . 9 , 5 mM ethylenediaminetetraacetic acid, and 1 mM diisopropyl fluorophosphate (Sigma Chemical Co.) t o a ratio of 1 : l O (weight of ovary/volume of EB). Sodium dodecylsulfate-polyacrylamidegel After centrifugation at 15,000 rpm for 15 min at electrophoresis (SDS-PAGE) 4°C (Hitachi CR20B2, RPR20-2 rotor), the superSDS-PAGE was carried out according t o the natant and lipid layer was dialyzed against 100 method of Laemmli ('70). Specimens obtained from volumes of EB at 4°C overnight. Ovary extract 89 MAJOR PROTEIN IN BARNACLE CYF'RIDS was then obtained after removing precipitate by centrifugation followed by filtration through Millex-GV 0.22 pm filter (Millipore, Bedford, MA). Concentration of proteins in the ovary extract was determined by method of Bradford ('76) using Protein Assay Kit (Bio-Rad, Richmond, CA) with immunoglobulin as standard. For immunoblot, the ovary extract was mixed 1:1 with 2x-concentrated sample buffer and then boiled for 3 min. Samples were electrophoretically transferred onto polyvinylidene-difluoride (PVDF) sheets (8 cm x 5 cm, Atto Co., Tokyo, Japan) at 80 mA for 2 h at room temperature after separation by SDSPAGE (Towbin et al., '79). The sheets were immunostained as follows. The PVDF sheets were first incubated in the blocking solution of Tris buffered saline (TBS, 150 mM NaC1,25 mM TrisHC1, pH 7.5) containing 1%(w/v) low fat milk and then in antiserum against vitellin heavy chain (Shimizu et al., '96) diluted to 1:1,000 with the blocking solution for 30 min at each step. It had been previously confirmed t h a t at this concentration, antiserum recognized only vitellin heavy chain among proteins in ovary extract by immunoblot analysis (Shimizu et al., '96). Antigen was detected with a Vectastain ABC kit (Vector Lab. Inc., Burlingame, CA), composed of biotinylated goat I&-anti rabbit IgG and avidin biotinylated horseradish peroxidase complex, according to the manufacturer's protocol. The antigen-antibody complex was visualized with 0.5 mg/ml 4-chloro-1-naphthol (Wako Pure Chemical Industries Ltd.) and 0.005% (w/v) H202(Santoku Chemical Industries Co., Ltd., Tokyo, Japan) in TBS. 1%(v/v> normal goat serum and then with antivitellin antiserum diluted to 1:1,000with the blocking solution. Following incubation with the primary antibody, antigen was detected with Vectastain ABC kit as described previously. For the negative control experiment, pre-immune serum was used instead of the antiserum. Antiserum against vitellin heavy chain-immunoreactive substances was stained brown with 0.1% (w/v) diaminobentizine tetrahydrochloride (Nakarai Tesque Inc., Kyoto, Japan) and 0.005% (w/v) Hz02in TBS. Specimens were counterstained with 1%(w/v> methyl green (E. Merck, Darmstadt, Germany) solution. RESULTS SDS-PAGE analysis for 170 kDa proteins in B. amphitrite A summary of larval t o post-larval stages of development and days elapsed from the start of cul- ture is shown in Table l. SDS-PAGE analysis showed the existence of a 170 kDa protein in various larval stages of B. amphitrite, most abundant in the cyprid (Fig. 1). We referred t o this protein as the Cyprid Major Protein (CMP). Nauplius larvae expressed CMP from day 1 of the culture. CMP specifically increased in amount with development of nauplius larvae, reaching a maximum in day 0 cyprids (CO), and decreased thereafter. Amounts of total protein or CMP in each lane were determined and summarized in Figure 2. Statistical analysis of results in Figure 2 is presented in Table 2. Amount of total protein exponentially increased from NO nauplius to CO cyprids, was constant from CO to C3 cyprids and then decreased. Amino acid sequencing Newly metamorphosed juveniles (JO) lost 60% of Proteins in cypris larvae or the ovary were sepatotal protein in CO cyprids and maintained such rated by SDS-PAGE, electrophoretically transamount of total protein for one week. ferred from gels onto PVDF sheets, and stained with 1%(w/v) amide black solution as described TABLE 1. Timint. of dewelowmental stages during culture above. Bands corresponding to CMP or vitellin heavy chain were sliced off the sheets, rinsed with Days Abbreviation of deionized water, then with methanol, and finally elapsed from larval and Stage juvenile stages dried. The sliced sheets were applied to a gas-phase start of culture sequencer (model 476A, Applied Biosystems, Fos- 0 1-11 nauplius NO ter City, CA). 1 11-111 nauplius N1 Immunohistochemistry Larvae were fixed with 4% (w/vj paraformaldehyde in seawater overnight, dehydrated, embedded in Paraplast (Sherwood Medical, St. Louis, MO), and sectioned to 5 pm thickness. After rehydration, sectioned specimens were incubated first with the blocking solution of TBS containing 2 3 4 5 6 8 12 111-lV nauplius V-VI naupliusl VI nauplius' Cyprid (Day 0 ) (Day l)/juvenile (Day 0) (Day 3Y (Day 2 ) (Day 711 (Day 6) 'Without the compound eyes. 2With the compound eyes. N2 N3 N4 co Cl/JO C3/J2 C7/J6 K. SHIMIZU ET AL. 90 A 200 K - -170 K 116 K66 K - 42 K- N1 N2 N3 N4 CO B 200 K - -170 K 116 K66 K- 42 K- co c1 c3 c7 JO J2 J6 Fig. 1. SDS-PAGE analysis of proteins in larvae and juveniles of B. amphitrite. A Lanes N1 to N4 correspond to Day 1 to Day 4 nauplius larme; lane CO, 0-day-old cyprids. B: Lanes CO, C1, C3, and C7 indicate 0-, 1-,3-, and 7-dayold cyprids. Lanes JO, 52, and J6 indicate 0-, 2-, and 6-day- old juveniles. Each lane contains the extract from 40 larvae or 40 juveniles. CMP is indicated by the arrow on the right margin. The position of marker proteins is shown by bars with their molecular weight in the left margin. By contrast, change of CMP amounts was different from that of total praltein. Amount of CMP was under 0.25 pg until N3 nauplius and increased abruptly during the late nauplius stages (N4) and during metamorphosis t o cyprids (CO). CMP con- tent formed 6% of total protein in CO cyprids. CMP contents decreased with aging of cyprids. CMP contents in newly metamorphosed juveniles reduced to 20% those in CO cyprids. A linear decrease in CMP content was observed as juveniles grew. A T 120 100 80 60 40 20 0 0 1 2 3 4 5 6 8 12 days froiii start of culture B T I 0 1 2.5 0.5 5 I0 0.25 Amount of BSA (pg) 0 1 2 3 4 5 6 8 days from start of culture Fig. 2. Change in amounts of total protein (A) and CMP (B)during larval or post-larval development of B. amphitrite. Amounts of total protein were estimated by colorimetric method. CMP amounts were estimated from the SDS-PAGE gel by means of the densitometer and then calibrated wtih BSA as standard (inset).Boxes filled with oblique lines rep- LL 12 resent mean values of total protein or CMP per larvae obtained from three independent experiments with nauplii; closed boxes, cyprids; dotted boxes, juveniles. Vertical bars indicate standard deviation. Significance of differences between two values was evaluated with Student's t-test and summarized in Table 2. K. SHIMIZU ET AL. 92 TABLE 2. Comparison of each pair using Student's &test* 6 ((21) 5 (CO) Day from start of culture Amount of total protein + 4 "4) 5 (CO) 6 (C1) - 8 ((23) 8 (52) 12 ((27) 12 (J6) - - - - - ++ ++ X 6 (JO) 8 (C3) 8 (52) 12 (C7) Amount of CMP 6 (JO) X - X ++ ++ - ++ X ++ 4 (N4) 5 (CO) 6 (C1) - - - ++ ++ ++ - 6 (JO) 8 ((33) 8 (52) 12 (C7j + - X + - + - - ++ ++ *Mean values for amounts of total protein and of CMP each day were compared + and ++ show pairs that were significantly different at a < 0.05, and <0.01, respectively; x, not significant a t u = 0.05; -, not determined. Abbreviated stages defined in Figure 1 are described in parentheses Comparison of CMP with vitellin Immunological and bi'ochemical characterization of CMP and vitellin were carried out in order to examine the homology between the two proteins. As shown in Figure 3 4 CMP and vitellin heavy chain both had a molecular weight of 170 kDa. Immunoblot analysis (Fig. 3B) indicated that the antiserum against vitellin heavy chain reacted t o vitellin heavy chain among ovary proteins and to CMP among cyprid proteins. CMP and vitellin were biochemically characterized by means of amino acid sequencing for N-terminal regions of these proteins (Table 3). Amino acid sequence of CMP was "YGFKPNNQY, while vitellin heavy chain had the sequence of ELGFQ- B A 200 K- K (CMP or Vitellin Heavy Chain) -170 116K66 K42 K1 2 1 2 Fig. 3. Immunoblot analysis of proteins in the ovary of adults and in cypris larvae with antiserum against vitellin heavy chain. Samples containing proteins from 40 cypris larvae (lane 1) or 10 pg ovary proteins (lane 2) were separated with SDS-PAGE and then stained with CBB (A). Otherwise, after separation by SDS-PAGE, followed by electroblotting onto PVDF sheets, proteins were reacted with the antiserum (B).The antiserum crossreacted with both vitellin heavy chain and CMP indicated by the arrow with their molecular weight in the right margin. The position of marker proteins is shown by bars with their molecular weight in the left margin. MAJOR PROTEIN IN BARNACLE CYPRIDS TABLE 3. Comparison of amino-terminal amino acid sequences between CMP and vitellin' Vitellin heavy chain CMP Consensus 'Sequences were aligned for maximal homology, and homologous residues were boxed. PKFtQY. The two sequences were not identical but aligned sequences showed a homology of 45%. Immunohistochemistry of CMP Distribution of CMP in the larvae was immunohistochemically studied with the antiserum against vitellin heavy chain. Positive immunoreaction was localized in the haemocoel of the cypris larva (Fig. 4A). Non-specific reaction was not observed in the negative control experiment using pre-immune serum instead of antiserum (Fig. 4B). Fig. 4. Localization of immunoreactivity in a cyprid with antiserum against vitellin heavy chain. Larval sections of 5 pm thickness were reacted with the antiserum. Positive reaction was stained brown and counter-staining with 1%methyl green took place. A Section of a cypris larva was reacted with anti-vitellin serum. Immunoreactivity was localized in the haemocoel. a, antennule; g , gut; h, haemocoel; 0,oil cell; t, thoracic appendages. B: Cypris larva reacted with pre-immune serum. Scale bars = 100 pm in A and B. 93 DISCUSSION In this paper, we have shown the presence of a storage protein, named CMP, cyprid major protein, in the cypris larva of the barnacle Balanus amphitrite. CMP is a 170 kDa protein as determined by SDS-PAGE. Previously we have also examined vitellin in B. amphitrite and have found that it consisted of two subunits, heavy chain and light chain with molecular weights of 170 and 85 kDa in SDS-PAGE, respectively (Shimizu et al., '96). CMP and vitellin heavy chain both had the same molecular weight. Immunoblot analysis also revealed that CMP was crossreactive with antiserum against vitellin heavy chain. Amino acid sequencing of the N-terminal regions of CMP and vitellin heavy chain, disclosed that CMP had the sequence of NNYGFKPNNQY, while that of vitellin heavy chain was ELGFQPKRQY, thereby indicating that CMP was not identical to vitellin heavy chain. Thus, CMP is a homologue or an isoform of vitellin, but gene of CMP is probably different from that of vitellin. Vitellin, a protein widely distributed in oviparous animals, is strictly limited in its existence to the ovary of adults under normal conditions. Vitellin is one of the storage proteins and is consumed during embryonic development. Although vitellin was found in larvae which could feed (de Chaffoy de Courcelles and Kondo, '801, this was likely remnants of egg yolk vitellin and was not newly synthesized. Since egg-yolk vitellin was consumed during embryogenesis and almost disappeared in stage I nauplius in the barnacle (Shimizu et al., '96), CMP from the latter nauplius to the cypris stage can be accounted to de novo synthesis. CMP is unique as compared to other constituent proteins, because of its unusual abundance in the cyprid and variation in its concentration during larval development. CMP content increased with growth during the feeding nauplius stages, and then reached a peak upon metamorphosis t o the non-feeding cyprid. CMP accumulated until the cypris stage reduced thereafter. The increase and decrease of CMP content were sharper than those of total protein content. This observation suggests that CMP may be a storage protein to be used for larval settlement and metamorphosis. Amounts of CMP decreased with time when cyprids were prevented from settlement, strongly indicating that CMP is utilized as an energy resource. Metamorphosing cyprids consumed CMP 94 K.SHIMIZU ET AL. far greater than swimming ones; two-thirds of CMP in 0-day-old cyprids were consumed during the metamorphosis to juveniles. This excessive consumption suggests more than just an energy resource; new tissues may also be generated from CMP during metamorphosis. If such amounts of CMP are necessary for metamorphosis, cyprids older than a few days are likely to fail to settle and metamorphose. In fact, settlement ability of cyprids reached a maximum at day 3 and decreased thereafter (Satuito et al., '96). Larval energy resources in several species of marine invertebrate, including barnacles, have been thought t o be lipids. Lucas et al. ('79), however, have shown that S. balanoides metabolized protein as well as lipid during aging or metamorphosis of cyprids. Proteins reported by Lucas et al. ('79) probably included CMP. Cyprids of B. amphitrite also possess a large number of oil cells which appear to reduce in both size and number with time. Thus, it is likely that both a protein such as CMP and lipidls are generally consumed as energy resources by cypris larvae. Finally, we discuss CMP and barnacle vitellin from a comparative biochemical view point. Nonfeeding larvae carrying abundant storage proteins have not been found among marine invertebrates. Insect pupae or nymphs possess storage proteins for metamorphosis to the adult (Kanost et al., '90). Arylphorin, one of insect storage proteins, is a main constituent in pupae or nymphs and is widely distributed among both hemi- and holometabolous insects (Telfer and Kunkel, '91), but amino acid compositions of arylphorins are different from those of vitellogenins (Kanost et al., '90). Primary structures, of both an arylphorin-type storage protein and vitellogenin of the silkworm Bombyx mori have already been determined, but revealed no significani, similarities between the two proteins (Fujii et al., '89; Yano et al., '94). Accumulation of vitellin-like protein such as CMP in barnacle larvae is, therefore, a novel finding in developmental biology. ACKNOWLEDGMENTS The authors are grateful to Dr. K. Okamoto (Univ. of Tokyo) for his assistance in collecting adult barnacles, Drs. G. Walker (Univ. of Wales) and E. Hunter (Fusetani Biofouling Project) for critical reading of the manuscript, and Ms. K. Natoyama and M. Yamazaki (Fusetani Biofouling Project) for their assistance in culturing the barnacle larvae. LITERATURE CITED Anderson, D.T. (1994) Barnacles. Chapman & Hall, London. Bradford, M.M. (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72:248-254. 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