JEZ 797 THE JOURNAL OF EXPERIMENTAL ZOOLOGY 278:339–348 (1997) Isolation and Characterization of the High-Density Lipoproteins From the Hemolymph and Ovary of the Penaeid Shrimp Penaeus semisulcatus (de Haan): Apoproteins and Lipids E. LUBZENS,1* T. RAVID,1,2 M. KHAYAT,1,2 N. DAUBE,1 AND A. TIETZ2 1 Israel Oceanographic and Limnological Research, National Institute of Oceanography, Haifa 31080, Israel 2 Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 699878, Israel ABSTRACT The high-density lipoproteins (HDLs) found in the male and female hemolymph of Penaeus semisulcatus de Haan were isolated by NaBr (1.22 g/ml) followed by sucrose gradient (5–25%) ultracentrifugation. The male HDL contained one protein, lipoprotein 1 (LP1), composed of one 110-kDa peptide subunit. The female HDL contained two proteins: 1) the LP1 that was immunoidentical to the male LP1 and was similarly composed of one 110-kDa peptide subunit and 2) vitellogenin (Vg), reacting positively with the rabbit antiserum generated against vitellin (Vt) that was isolated from vitellogenic ovaries. Both Vg and Vt consisted mainly of three polypeptide subunits (200, 120, and 80 kDa) as revealed by denatured PAGE and Western blot. The LP1 from males or females did not react with the Vt rabbit antiserum. Similarly, Vg and Vt did not react with the rabbit antiserum prepared against LP1. Phospholipids (PL) constituted 71–76% of the total lipids in the hemolymph and HDLs of both male and female hemolymph. Cholesterol (Ch) amounted to 17–20% , and small amounts (5%) of diacylglycerols (DAG) were also carried by these HDLs. Both the PL and DAG contained highly unsaturated fatty acids (20:5 ω3 and 22:6 ω3) that are transported from the food or hepatopancreas to the tissues, including the vitellogenic ovaries in females. In the present study we show for the first time the separate lipid composition of female LP1 and Vg and compare them with the lipids attached to the Vt. Vg had a lower lipid content than LP1 (540 and 1089 mg/g protein, respectively). Differences were also found in the relative abundance of PL, Ch, and DAG classes in the LP1 in comparison with Vg. Furthermore, small amounts (~3.8%) of triacylglycerols (TAG) were found only in the hemolymph of vitellogenic females, and they were associated with the Vg. Although Vg and Vt were composed of similar polypeptides, their lipid composition was different. Vt, in contrast to Vg, carried considerable amounts of TAG (~22%) and only trace amounts of DAG. The significance of the TAG in the hemolymph of vitellogenic females is not known, and the functional relationship between Vg and Vt requires future extensive studies. Lipids were not detected in hemocyanin that was purified from clotted hemolymph. J. Exp. Zool. 278:339–348, 1997. © 1997 Wiley-Liss, Inc. Yolk is the nutritive material accumulating in substantial quantities in the ooplasm of developing oocytes and is intended to meet the basic requirements of embryonic development, independent of the maternal organism. It is accepted that yolk proteins provide the basic structural material needed for tissue buildup, whereas lipids, especially the neutral lipids, serve as the major fuel and a source of essential fatty acids. The main protein fraction of the crustacean egg proteins is a high-density lipoprotein/lipoglycoprotein frequently associated with carotenoid pigments and usually referred to © 1997 WILEY-LISS, INC. as lipovitellin. In addition to lipovitellin, lipid spheres or globuli have been observed within the developing oocytes (Adiyodi and Subramonian, ’83; Charniaux-Cotton and Payen, ’88). The ovary increases in weight during oocyte development, reaching 7–15% of the total fresh weight in females of Penaeus semisulcatus de *Correspondence to: E. Lubzens, Israel Oceanographic and Limnological Research, National Institute of Oceanography, P.O. Box 8030, Haifa 31080, Israel. Received 10 June 1996; accepted 30 January 1997 340 E. LUBZENS ET AL. Haan (Shlagman et al., ’86). This increase is associated with the accumulation of proteins and lipids. Protein content in the ovary of Penaeus semisulcatus increases from 1 to 12 mg/g fresh body weight (Shafir et al., ’92). Lipids constitute between 18–41% of the dry weight of the mature ovary in various crustaceans (Teshima and Kanazawa, ’83; Castille and Lawrence, ’88; Lautier and Lagarrigue, ’88; Harrison, ’90). In most species, phospholipids (PL), triacylglycerols (TAG), and sterols are the most abundant lipid classes in the ovaries. The fatty acid composition of PL and TAG showed an abundance of highly unsaturated fatty acids (HUFA) including 20:5 ω3 (EPA) and 22:6 ω3 (DHA), supporting the assumption that HUFA are necessary for vitellogenesis in penaeid shrimp (Middleditch et al., ’80; Teshima et al., ’89). Furthermore, the so-called “quality” of penaeid shrimp eggs is usually correlated with their ω3 HUFA content (Middleditch et al., ’80; Cahu et al., ’86, ’94; Lytle et al., ’90). The origin of the lipids reaching the crustacean ovary and the mode of their transport in the hemolymph are not fully known. The hepatopancreas (HEP) was suggested to operate as a metabolic juncture for lipids coming in from the gut (Teshima and Kanazawa, ’80a, b; Lee and Puppione, ’88). In several cases, it was shown that these lipids are stored in the HEP and transported to the ovary during egg development (Castille and Lawrence, ’88; and a review on this topic by Harrison, ’90). Teshima et al. (’88) showed that intensive labeling of the HEP lipids occurred after female shrimp were fed a diet supplemented with [9,10-3H]palmitatic acid and [1-14C]linolenic acid. These labeled lipids were transported to the ovaries in the group of females that were subjected to eyestalk ablation, which led to acceleration of ovarian development. Teshima et al. (’86a, b) also showed that female shrimp doubled their food consumption, further supporting that the lipids accumulating in the ovary originate in the food. Lipids, being insoluble in water, are transported in the hemolymph as lipoproteins (LPs). Three LPs were isolated from crustacean hemolymph; all belong to the high-density lipoprotein (HDL) and very high density lipoprotein (VHDL) classes (Lee and Puppione, ’78, ’88; Teshima and Kanazawa, ’80a, b; Puppione et al., ’86; Spaziani et al., ’86; Lee, ’90; Komatsu and Ando, ’92; Komatsu et al., ’93; Chen and Chen, ’94; Hall et al., ’95). One of them is the female-specific vitellogenin (Vg), the presence of which is correlated with ovarian development. The other LPs, lipoprotein 1 (LP1) and VHDL, are common to both sexes. Spaziani et al. (’86) found two LP1s, one belonging to the HDL2 and the other to the HDL3 density class. Both had similar subunit profiles on SDS-PAGE, suggesting that these two proteins serve as a reusable lipid shuttle transporting lipid from the HEP to other tissues (Lee and Puppione, ’88) with the HDL2 being loaded and HDL3 being the unloaded phase of the same LP, in a manner similar to that found in insects. Komatsu et al. (’93) suggested that VHDL was involved in lipid transfer reaction between lipoproteins in crustaceans. Hall et al. (’95) showed that HDL and VHDL were identical to two proteins previously studied for their role in immune recognition and clotting in the crayfish Pacifastacus leniusculus. Recently, Spaziani et al. (’95) argued that there are no femalespecific proteins in crustaceans. In the present work we isolated the high-density lipoprotein (LP1) found in both the male and the female hemolymph of adult Penaeus semisulcatus (de Haan) and the Vg specific to females. We also isolated the vitellin (Vt) from vitellogenic ovaries. We determined for the first time, for each of the lipoproteins, their apoprotein profiles, lipid classes, and fatty acid composition. The results presented here and in Ravid (’94) and Ravid et al. (in preparation) will permit the construction of a model explaining the transport mechanism of lipids into maturing ovaries in this commercially important species. Furthermore, this information will be relevant in future determinations of evolution and comparative trends with other invertebrate groups, including insects, which are also arthropods. MATERIALS AND METHODS Animals Marine shrimp (Penaeus semisulcatus de Haan) caught in Haifa Bay, Israel, were kept in 3-m3 tanks in running sea water (salinity 400/00; three exchanges per day) and fed each morning with food pellets (prepared by Dr. George Kissil, National Center for Mariculture, Israel Oceanographic and Limnological Research, Eilat, Israel) and in the afternoon with bait shrimp and squid. Water temperatures ranged between 19–21°C year-round. Under these conditions, females produced vitellogenic ovaries all year round. Determination of oocyte diameter The stage of oocyte maturation was determined in all females used. A piece of tissue was removed HDL APOPROTEINS, LIPIDS, SHRIMP from the ovary of each female and fixed in 4% formalin in seawater, and the average oocyte diameter (AOD) was determined (Shlagman et al., ’86). Previtellogenic females were those with oocytes of 100 µm or less in diameter. Isolation and purification of proteins Isolation of high-density lipoproteins (HDL) Hemolymph was collected, after cutting off the anterior part of the cephalothorax, near the base of the eyestalks, into a 10% sodium citrate solution or alternatively was allowed to clot at 4°C for 1–2 hr. Protease inhibitors were added immediately after collection of hemolymph to a final concentration of 1 mM phenylmethylsulphonylfluoride (PMSF) and 1 µg/ml aprotinin. Hemolymph samples from several individuals were pooled (separately for males or females) and centrifuged (10 min at 9,000 × g in a Sorval 5-CB, Dupont, USA) to remove cells and the clot. To isolate lipoproteins, NaBr was added to the supernatant at a final density of 1.22 g/ml and subjected to 48-hr density ultracentrifugation (125,000 × g and 12°C, in a T-875 fixed angle rotor, Sorval, Dupont). The upper yellow colored fraction containing the HDL was collected. The HDL samples were dialyzed for 24 hr at 4°C to remove the NaBr, in two changes of 1 L dialysis buffer (10 mM phosphate buffer solution containing 150 mM NaCl and 0.01% EDTA at pH 7.4 and protease inhibitors as described above). Samples were stored in sterile tubes in the dark at 4°C and used within 1–2 mo after isolation. Vg and LP1 from female hemolymph HDL and LP1 from male hemolymph HDL were obtained from hemolymph samples that were allowed to clot. The HDL (1–1.5 mg) was incubated at room temperature for 45 min in a final concentration of 0.1% acytelated Sudan black B (Ribero and McDonald, ’63) and loaded on a stepwise sucrose gradient of 5, 10, 15, 20, and 25% (w/w prepared in phosphate buffer saline). After a 24-hr centrifugation (swinging bucket T-641 rotor, at 180,000 × g and 6°C; Sorval, Dupont), one stained band was observed in the tube containing the male HDL samples. The female HDL showed two stained bands. One was running in the same position as the male LP1 and was presumed to be the female LP1. The second lower band was assumed to be Vg, because its mobility was similar to that of Vt that was run on an identical gradient. The purified bands were collected from the side of the tube by piercing and suction into a 5-ml syringe and 341 stored at 4°C in the dark until used in electrophoresis, Western blot, and lipid determinations. Apoprotein and lipid determinations of LP1 (from males and females) and Vg were performed on sucrose gradient purified proteins. Isolation of vitellin (Vt) Vt was isolated and purified from the ovaries of vitellogenic females by two methods giving similar results: 1) as described by Browdy et al. (’90) and Tom et al. (’92), except that 1 mM PMSF, 10 µg/ml leupeptine, and 1 µg/ml aprotinin were added during the first homogenation step and PMSF and aprotinin were included in the buffers used during the gel and ion exchange chromatography and 2) through high-density ultracentrifugation, using the same method described above for isolation of hemolymph HDL. Briefly, ovaries were homogenized in phosphate buffer containing protease inhibitors (as mentioned earlier) and centrifuged (10 min at 10,000 × g and 4°C; Sorval 5-CB, Dupont) to remove cell debris. The supernatant was subjected to NaBr and sucrose gradient ultracentrifugation as described above for HDL. After sucrose gradient ultracentrifugation, only one green band was observed and collected for further lipid and protein analysis. Protein determinations Protein concentrations were determined by a modification of the Lowry procedure (Markwell et al., ’78). Characterization of apoproteins Male and female HDL and sucrose isolated fractions were characterized by electrophoresis on native and denatured polyacrylamide gels (PAGE) as described previously (Khayat et al., ’94a). Protein bands were visualized after staining with Coomassie blue. Immunoidentity of proteins was established after the transfer of proteins to nitrocellulose in Western blot and was visualized as described by Khayat et al., ’94b. Preparation of rabbit-specific antisera Specific polyclonal antibodies against purified Vt were generated by Dr. M. Tom (Browdy et al., ’90; Tom et al., ’92). An LP1 polyclonal antibody was generated in rabbits that were injected with LP1 from males. The HDL prepared from male hemolymph was run on a native PAGE, and the Coomassie blue stained band was cut and injected into rabbits (Yeda, Weizmann Institute, Rehovot, Israel). Preimmunized and immunized samples 342 E. LUBZENS ET AL. were tested by dot blot and PAGE followed by Western blot, as described above. Extraction and lipid determinations Lipids from male and female HDL samples obtained after high-density NaBr ultracentrifugation and LP1 and Vg samples obtained by sucrose stepwise gradient centrifugation were extracted in methanol:chloroform (2:1 v/v) as described by Bligh and Dyer (’59). Lipids were separated by thin layer chromatography (TLC; silica gel 60, Merck, Darmstadt, Germany) employing benzene: ethylether; ethylacetate:acetic acid (80:10:10:0.2 by volume) for separation of neutral lipids and chloroform:methanol:acetic acid:water (100:20: 12:5 by volume) for the separation of PL. Lipid spots were detected after spraying with 2,7 dichlorofluorescine (0.2% in ethanol). Lipid spots were collected, saponified with 0.5 M methanolicKOH at 50°C, and the fatty acids were recovered after acidification by hexane extraction. The fatty acids were methylated with diazomethane (Schlenck and Gellerman, ’60) and separated by gas liquid chromatography on a 25-M fused silica column cross-linked with methylsilicone (0.52-m film thickness) at a temperature range of 185–230°C employing a Hewlett-Packard 5990A gas chromatograph equipped with a flame ionization detector. The relative composition of a fatty acid mixture was calculated from the areas under the peaks employing a Hewlett-Packard 3390A integrator. For quantitative analysis, heptadecanoic acid was added as the internal standard, assuming that the response of the detector for all methylfatty esters was identical. Ch was eluted from silica gel with chloroform-methanol (1:2, by volume) and quantitated by using Boehringer’s cholesterol-oxidase kit (Boehringer, Mannheim, Germany). The efficiency of the procedure was estimated by adding trace amounts of 14C-cholesterol prior to TLC, and the results were corrected for losses. RESULTS Male and female lipoproteins The hemolymph lipoproteins isolated from the top fraction after ultracentrifugation in NaBr at a density of 1.22 gm/ml fall in the class of HDL. No lipoproteins were found after ultracentrifugation in NaBr at a density of 1.063 gm/ml. Density sucrose ultracentrifugation revealed one Sudan black stained protein in the HDL of males and two in that of adult vitellogenic females, and one band in lipoproteins isolated from the ovary (Fig. 1). The differences in the staining intensity of the Fig. 1. Purification of high-density lipoproteins (HDL) of male and female hemolymph and vitellin (Vt) by density sucrose ultracentrifugation. The HDLs and Vt were obtained by NaBr density ultracentrifugation, stained with Sudan black B and loaded on a stepwise sucrose gradient as described in Materials and Methods. Vt is shown in lane 1, and female and male HDL separations are shown in lanes 2 and 3, respectively. The lower band in lane 2 (indicated by an arrow) correlates with Vt. two lipoproteins in female HDL after sucrose ultracentrifugation indicate the lower concentration of Vg (lower band) in comparison with LP1 (upper band). An excess stain of Sudan black precipitated after ultracentrifugation to the bottom of the tube holding the male HDL (Fig. 1., lane 3). One Coomassie blue stained band was revealed after gel electrophoresis under native conditions in each of the following preparations obtained after sucrose gradient ultracentrifugation of HDL: male LP1 (Fig. 2, lane 1) from the hemolymph of males, the upper band and lower bands of female HDL (Fig. 2; lanes 2 and 3, respectively) from the hemolymph of females, and Vt (Fig. 2, lane 4) from the ovarian homogenates. The upper Sudan black stained band in the female HDL subjected to sucrose gradient ultracentrifugation showed similar mobility on PAGE to that of LP1 of males and reacted positively with the polyclonal antibody prepared against male LP1 in Western blot analysis (Fig. 3, lanes 1 and 2). Vg and Vt run on the same gel did not react with the LP1 antibody. Furthermore, both the LP1 of males and the LP1 of females were found to be composed of one 110-kDa polypeptide, as revealed by gel electrophoresis under denaturing and reduced conditions (Fig. 4a) and HDL APOPROTEINS, LIPIDS, SHRIMP Fig. 2. Polyacrylamide gel (5%) electrophoretic analysis (PAGE) under native conditions of lipoproteins collected by sucrose gradient ultracentrifugation (shown in Fig. 1). The gel was stained with Coomassie blue. Lane 1: male lipoprotein (m-LP1; see Fig. 1, lane 3); lane 2: upper fraction of female lipoproteins (f-LP1; see Fig. 1, lane 2); lane 3: lower fraction of female lipoproteins (vitellogenin [Vg]; see Fig. 1, lane 2); lane 4: vitellin isolated from vitellogenic ovaries (Vt; see Fig. 1, lane 1). 343 Fig. 4. Electrophoretic and Western blot analysis of male and female LP1 proteins, under denatured and reduced conditions (7.5–17% gel). Coomassie blue stain of molecular weight standards (left lane), male (m-LP1), and female (fLP1) LP1 are shown in (a). Proteins were transferred to a nitrocellulose membrane and incubated with LP1 antiserum (b). The color was developed as described in Figure 3. by Western blot analysis (Fig. 4b). On several occasions, the female LP1 migrated slightly differently from the male LP1 on a native gel (not shown). The higher density protein (lower stained band in Fig. 1) found in sucrose gradient ultracentrifugation of the female HDL is Vg because it reacted positively with the polyclonal antibody prepared against purified Vt in Western blot analysis (Fig. 5). Coomassie blue staining and Western blot analysis revealed three stained bands at 200, 120, and 80 kDa in both Vg and Vt (Fig. 6a, b). The relative abundance of the polypeptide species of Vg was similar to that of Vt. Fatty acid and lipid composition Fig. 3. Western blot analysis of high-density lipoproteins (HDL) showing immunoidentity with LP1. Lipoproteins were collected after sucrose gradient ultracentrifugation (see Fig. 1) and were subjected to polyacrylamide gel electrophoresis (5%) under native conditions. Proteins were transferred to a nitrocellulose membrane and incubated with LP1 antiserum, followed by GAR-HRP. The color was developed after incubation with 3,3´-diaminobenzidine and hydrogen peroxide. Lane 1: male lipoprotein (m-LP1; see Fig. 1, lane 3); lane 2: the upper fraction in sucrose gradient (f-LP1; see Fig. 1, lane 2); lane 3: the lower fraction in sucrose gradient (vitellogenin [Vg]; see Fig. 1, lane 2); lane 4: vitellin (Vt; see Fig. 1, lane 1). Table 1 shows the lipid composition of male and female hemolymph and the HDLs isolated from them. Because the HDL of females contains two lipoproteins, LP1 and Vg, the lipid composition of each lipoprotein was determined separately (Table 2). Table 2 also presents the lipid composition of ovarian Vt. Considerable amounts of lipids were detected in the hemolymph of male and female shrimp, most of which were concentrated in the HDL upon centrifugation, after addition of NaBr. PL constituted 71–76% of the total lipid in the hemolymph and the HDL isolated from it. Ch amounted to 17–20%, yielding a PL:Ch ratio of 3.0:5.3. Only relatively small amounts of diacylglycerol (DAG) were detected. TAGs were detected only in the hemolymph and HDL of vitellogenic females and were carried by Vg (Table 2). The average lipid concentrations of 94 mg% (mg/100 ml), 344 E. LUBZENS ET AL. Fig. 5. Western blot analysis of high-density lipoproteins (HDLs) showing immunoidentity with vitellin (Vt). Lipoproteins were collected after sucrose gradient ultracentrifugation (see Fig. 1) and were subjected to polyacrylamide gel electrophoresis (5%) under native conditions. Proteins were transferred to a nitrocellulose membrane and incubated with Vt antiserum, followed by GAR-HRP. The color was developed after incubation with 3,3´-diaminobenzidine and hydrogen peroxide. Lane 1: male lipoproteins (m-LP1); lane 2: the upper fraction in sucrose gradient (f-LP1; see Fig. 1, lane 2); lane 3: the lower fraction in sucrose gradient (vitellogenin [Vg]; see Fig. 1, lane 1); lane 4: vitellin (Vt) isolated from vitellogenic ovaries. 136 mg%, and 113 mg% was found in males, vitellogenic, nonvitellogenic and young females, respectively. A ratio of 1.01, 0.78, and 0.94 lipids per 1 g protein was found for the HDL of males, vitellogenic, and nonvitellogenic females, respectively (Table 1). Table 2 shows the lipid composition of female LP1, Vg, and ovarian Vt. The lipid content of Vg is much smaller than that of LP1 (0.54 and 1.09 mg/g protein, respectively). This difference can explain the higher lipid content of male HDL compared with female HDL (Table 1). The lipid composition of female LP1 is very similar to that of male HDL. In contrast, Vg carries relatively less Ch (12%) than LP1, yielding a PL:Ch ratio of 7.1 DAG are carried by both Vg and LP1, but TAG are carried only by Vg. Vt, in contrast to Vg, carries considerable amounts of TAG (22.1 and 7.6%, respectively) and negligible amounts of DAG. Table 3 shows the fatty acid (FA) composition of LP1, Vg, and Vt. Because the FA composition of lipids isolated from male and female hemolymph and of male and female HDL are very similar to that of female LP1, these results are not described in detail in this table. All the lipoproteins carried EPA and DHA. Generally, the HUFA content of PL is higher than that of DAG and TAG (Table 3), and the ratio of ω3:ω6 fatty acids is also higher (1.71–1.72) in the PL than in the DAG (0.95–1.02) of the hemolymph lipoproteins. Vt showed a relatively high ω3:ω6 ratio in both the PL and TAG (2.00 and 2.30, respectively). Table 4 compares the fatty acid composition of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) isolated from female LP1, Vg, and Vt. The results shown for female LP1 are very similar to those determined for male and female hemolymph and HDL and are not presented in this table. PE constituted about 10% of the PL of all lipoproteins. As indicated by the SAT:HUFA fatty acid ratio, PE contained more HUFA than PC and less 16:0 and 18:1. The hemocyanin isolated from clotted hemolymph contained only trace amounts of lipids whereas that isolated from citrated (nonclotted) hemolymph contained 2.5–3.7 mg/g hemocyanin. DISCUSSION Fig. 6. Electrophoretic and Western blot analysis of vitellin (Vt) and vitellogenin (Vg) under denatured and reduced conditions (7.5–17% gel). Coomassie blue stain of molecular weight standards (left lane), Vt, and Vg are shown in (a). Proteins were transferred to a nitrocellulose membrane and incubated with Vt antiserum (b). The color was developed as described above in Figure 3. One lipoprotein was found in the hemolymph of Penaeus semisulcatus males that consisted of one 110-kDa peptide unit, supporting results reported previously (Khayat et al., ’94a; Lubzens et al., ’95). This lipoprotein, known as LP1, was found in several species of crustaceans, including HDL APOPROTEINS, LIPIDS, SHRIMP 345 TABLE 1. Lipid content of hemolymph (HL) and high-density lipoproteins (HDL) PL Male HL Female HL Non-vit female HL Male HDL Female HDL Non-vit female HDL DAG 68.1 ± 7.3 100.5 ± 17.5 81.3 ± 20.4 775 ± 361 559 ± 251 674 TAG 7.1 ± 1.7 11.9 ± 4.9 11.9 ± 4.9 50 ± 11 43 ± 15 40 Trace 4.5 Trace Trace 36 ± 24 Trace Ch PL/Ch Total 19.0 ± 0.9 19.0 ± 4.1 19.2 ± 3.9 185 ± 50 146 ± 41 225 3.6 5.3 4.2 4.2 3.8 3.0 94 136 113 1,012 784 939 Hemolymph was collected with citrate (10%), and HDL was prepared as described in Materials and Methods. The lipid content of hemolymph is expressed as mg100 ml hemolymph. The lipid content of HDL is expressed as mg/g protein. The results for HDL of nonvitellogenic (non-vit) females were performed only once. All other results represent averages and SD for three different preparations. other penaeid species (Lee and Puppione, ’88; Lee, ’90; Komatsu et al., ’93; Hall et al., ’95; see also Table 1 in Yepiz-Plascencia et al., ’95). A protein immunologically identical to male LP1 and similarly possessing one 110-kDa peptide was also found in the hemolymph of females. The site of synthesis of LP1 is not known. Although Lee and Puppione (’88) suggested that LP1 was synthesized in the HEP, no direct evidence has been presented. Direct evidence for cell-free translation of LP1 mRNA extracted from the male HEP failed, because of the relatively greater abundance of mRNA encoding hemocyanin in this organ (Khayat et al., ’95). The turnover rate of LP1 in the P. semisulcatus hemolymph was found to be significantly lower than that of Vg (Khayat et al., ’94a). This may be attributed to a relatively higher concentration of LP1 or higher removal rate of Vg from the hemolymph. Recently, Hall et al. (’95) have shown that major lipoproteins in crayfish hemolymph are also involved in immune recognition and clotting in addition to their role of lipid transport and suggested that the LP1 is identical to the β-1,3-glucan binding protein. The hemolymph of vitellogenic females contained a second lipoprotein, not found in males. It was found to react with the Vt antiserum, the main lipoprotein found in mature ovaries. By definition, this protein is termed Vg. In previous publications we showed that Vt is synthesized within the ovary of P. semisulcatus (Browdy et al., ’90; Khayat et al., ’94b). However, the HEP was also found to synthesize a protein that was immunologically identical to Vt and was therefore termed Vg (Fainzilber et al., ’92). The exact relationship between Vg and Vt in penaeid shrimp is not fully established. In vertebrates and most insects extraovarian synthesis of Vg has been shown. The Vg was found to enter the ovary through receptor mediated endocytosis and, following some modifications, forms the Vt (Byrne et al., ’89; Sappington et al., ’95; Schneider, ’95). The results reported here for P. semisulcatus show that all three Coomassie blue stained polypeptide bands of Vg were also found in the Vt, and the antiserum prepared against Vt reacted with all three bands of Vg. In P. monodon the isolated Vg consisted of 82- and 170-kDa subunits and was very different from the purified Vt that contained eight subunits, with molecular weights ranging from 35 to 91 kDa (Chang et al., ’94). The Vt of several penaeid species had molecular weights ranging between 283–540 kDa, with two to eight subunits in denatured gels (see comparisons in Lubzens et al., ’95). Because in most cases, Vt was isolated in the absence of protease inhibitors, the subunits reported in the various publications could represent products of enzymatic digestion. Surprisingly, Chen and Chen (’93), did not find cross-immunoreactivity between three of TABLE 2. Lipid content of LP1 and vitellogenin (Vg) isolated from feamle HDL, and vitellin (Vt) isolated from ovaries LP1 Vg Vt PL DAG TAG Ch PL/Ch Total 847 ± 119.9 (77.8) 382.9 ± 20.5 (70.9) 324.4 ± 114.4 (67.0) 54.0 ± 10.8 (5.0) 51.1 ± 17.4 (9.5) — — 220.9 ± 24.2 (20.3) 64.9 ± 37.1 (12.0) 38.8 ± 5.0 (8.6) 3.9 ± 0.95 1,088.9 ± 114.6 7.1 ± 3.2 539.8 ± 50.2 8.7 ± 4.3 320.0 ± 248.2 40.9 ± 10.2 (7.6) 99.0 ± 13.6 (22.1) HDL was obtained from clotted hemolymph. Vg and LP1 were isolated on a sucrose gradient as described in Materials and Methods. Results were expressed as mg lipid/g proteins. The results represent the average and SD for three different preparations. 346 E. LUBZENS ET AL. TABLE 3. The fatty acid composition (in % of total) of vitellogenin and LP1 isolated from vitellogenic female high-density lipoproteins (HDL) and vitellin that was isolated from the ovaries Phospholipids Fatty acids 16:0 16:1 ω7 18:0 18:1 18:2 ω6 20:1 ω9 20:4 ω6 20:5 ω3 22:6 ω3 ω3/ω6 1 2 Diacyglycerols Triacylglycerols LP1 Vg Vt LP1 Vg Vg Vt 18.5 ± 3.8 5.2 ± 1.3 7.8 ± 0.4 18.1 ± 2.0 8.0 ± 3.5 2.11 3.9 ± 0.3 10.7 ± 0.4 9.8 ± 2.9 1.72 18.9 ± 3.3 5.6 ± 1.4 8.1 ± 3.2 19.1 ± 2.7 8.6 ± 3.1 192 4.4 ± 0.4 11.9 ± 1.7 9.8 ± 2.2 1.71 12.9 ± 3.0 4.0 ± 1.2 7.7 ± 0.6 17.9 ± 2.1 6.0 ± 0.9 3.7 ± 1.4 2.9 ± 0.7 8.1 ± 1.4 9.8 ± 1.3 2.00 26.2 ± 2.1 5.7 ± 2.0 8.5 ± 3.6 20.0 ± 2.7 8.3 ± 3.4 4.21 4.9 ± 1.2 8.4 ± 0.8 4.2 ± 0.8 0.95 23.2 ± 5.9 5.2 ± 1.5 8.4 ± 2.6 19.2 ± 1.6 7.7 ± 3.6 2.01 5.8 ± 0.7 9.4 ± 0.8 4.4 ± 1.0 1.02 29.7 ± 2.7 4.6 ± 1.1 7.5 ± 2.9 22.7 ± 2.2 6.6 ± 2.6 4.51 2.1 ± 0.1 5.3 ± 1.8 4.2 ± 0.1 1.09 15.2 ± 1.9 3.5 ± 0.9 3.3 ± 0.6 11.0 ± 4.8 3.8 ± 0.7 4.3 ± 1.1 0.9 ± 0.1 4.0 ± 1.3 5.1 ± 1.5 2.30 Found only in two replicate samples. Found only in one replicate sample. the four polypeptides of Vt of P. monodon. On the other hand, immunocross-reactivity was reported between the antiserum prepared against one penaeid species with the Vt of other species. For example, the antiserum raised against Vt of P. monodon cross-reacted in immunodiffusion with the ovarian extracts from mature P. indicus, P. merguiensis, and P. semisulcatus (Quinitio et al., 90). Similarly, the antiserum raised against P. semisulcatus was found to react with the Vt of P. vannamei (Tom et al., ’92). Most of the lipids in the hemolymph of both males and females are carried by HDL:LP1 in males, LP1 and Vg in females. PL and Ch are the major lipid components (71–77% and 18–20%, respectively; Tables 1 and 2) of LP1. DAG constitute only approximately 5%. Vg in comparison with LP1 has a lower lipid content (540 and 1,089 mg/g protein, respectively); it carries much less Ch (12%) and slightly more DAG (9.5%). This may contribute to the differences observed between male and female HDL (Table 1) and to the lower intensity in the Vg stained with Sudan black and subjected to ultracentrifugation (Fig. 1). Vg also carries TAG, which are not found in male or female LP1 and in the hemolymph of nonvitellogenic females. Although Vg and Vt are composed of similar polypeptides, their lipid composition is different. Vt, in contrast to Vg, carries considerable amounts of TAG (22%) and only trace amounts of DAG. The relatively high amounts of TAG are reminiscent of the lipid composition of intact ovaries at the last stages of oocyte development in P. semisulcatus (Ravid et al., in preparation). All lipoproteins contain EPA and DHA. Generally, the HUFA content of PL is higher than that of DAG or TAG (Table 3), and the ratio ω3/ω6 fatty acids was higher in the PL than in the DAG of the hemolymph lipoproteins. The higher ω3/ω6 ratio in both the PL and TAG (2.0 and 2.3, respectively) TABLE 4. The fatty acid composition (in % of total) of the phosphatidylcholine (PC) and phosphoethanolamine (PE) from the phospholipids (PL) of the LP1, vitellogenin (Vg), and vitellin (Vt)1 Fatty acid Vg PL PC PE PL1 PL PC PE Vt PL PC PE 1 16:0 18:0 18:1 18:2 20:5 20:6 SAT/HUFA 23.5 24.5 18.9 8.5 10.0 13.2 15.5 16.6 6.8 12.7 13.2 6.8 9.7 10.0 16.3 12.9 13.9 15.3 1.4 1.4 1.0 23.5 23.4 8.2 7.3 7.2 11.5 15.3 16.3 8.2 12.8 14.3 6.6 14.1 10.2 24.6 10.2 14.2 21.3 1.3 1.3 0.4 17.5 18.6 10.0 8.2 7.6 8.8 19.9 19.3 9.6 8.8 9.0 5.6 12.2 10.4 20.6 16.0 13.9 20.7 0.9 1.1 0.5 The ratio of saturated to highly unsaturated fatty acids (SAT/HUFA) is shown in the last column. HDL APOPROTEINS, LIPIDS, SHRIMP was found in Vt. Although Vt and several of its lipids are synthesized within the ovary (Browdy et al., ’90; Fainzilber et al., ’92; Shenker et al., ’93), the HUFA originate from the food. Lipid fatty acid compositions were similar among the HDLs from male, female, and vitellogenic female blue crabs (Lee and Puppione, ’88), with phosphatidylcholine as the predominant lipid (80–85%). Phosphatidylethanolamine (3%), lysophosphatidylcholine (~1%), TGs (5–8%), cholesterol (Ch) (3–4%), and sphigomyelin (2–3%) were also carried by these HDLs. The functional relationship between Vg and Vt has not been established so far. One possibility, that Vg is associated with lipid transport to the ovary, as previously suggested by Quackenbush (’89), has yet to be verified. Lipids are carried from the HEP by the hemolymph lipoproteins, and we have not established the role of each lipoprotein in this process. The presence of an ovarian receptor for Vg has been demonstrated in a lobster (Lavedure and Soyez, ’88). It is not known whether this receptor is specific only to Vg or could serve for receptor-mediated transport of lipids also for LP1, as has been shown for the Vg/VLDL ovarian receptor in the chicken (Schneider, ’92). Recently, we have preliminary results showing the presence of a Vg/Vt receptor in ovarian membranes of P. semisulcatus (Tietz et al., ’96). These membranes also bind LP1, however, at a lower affinity. In recent years it has been claimed that hemocyanin functions as a lipid carrier (Hall et al., ’95). We did not detect any lipids on hemocyanin that was isolated from clotted hemolymph. However, small amounts of lipids, with a composition identical to that of HDL, were detected in hemocyanin that was isolated from citrated hemolymph (unpublished results). Whether these lipids are associated with the hemocyanin, the shrimp VHDL (Komatsu et al., ’93; Hall et al., ’95), or constitute a contamination of LP1 is currently being investigated. ACKNOWLEDGMENTS This work was supported by grants from the National Academy of Sciences and Humanities (231/91) and the Binational Science Foundation (93-00083). LITERATURE CITED Adiyodi, R.G., and T. Subramonian (1983) Oogenesis, oviposition and oosorption. In: Reproductive Biology of Invertebrates, Vol. 1. K.G. Adiyodi and R.G. Adiyodi, eds. Wiley Press, New York, pp. 443–495. 347 Bligh, E.G., and W.J. Dyer (1959) A rapid method for total lipid extraction and purification. Can. J. Biochem. Physiol., 37:911–917. Browdy, C.L., M. Fainzilber, M. Tom, Y. Loya, and E. Lubzens (1990) Vitellin synthesis in relation to oogenesis is in vitro incubated ovaries of Penaeus semisulcatus (Crustacea, Decapoda, Penaeidae). J. Exp. Zool., 255:205–215. Byrne, B.M., M. Gruber, and G. Ab (1989) The evolution of egg yolk proteins. Prog. Biophys. Mol. Biol., 53:33–69. Cahu, C., C. Fauvel, and Aquacop (1986) Effect of food acid composition of P. vannamei broodstock on egg quality. ICES, Mariculture Committee, 1986 F/28, 10 pp. Cahu, C., J.C. Guillaume, G. Stéphan, and L. Chim (1994) Influence of phospholipid and highly unsaturated fatty acids on spawning rate and egg and tissue composition in Penaeus vannamei fed semi-purified diets. Aquaculture, 126:159–170. Castille, F.L., and A.L. Lawrence (1988) Relationship between maturation and biochemical composition of the gonads and digestive glands of the shrimp, Penaeus aztecus (Ives) and Penaeus setiferus (L.). J. Crust. Biol., 9:202–211. Chang, C.F., F.Y. Lee, Y.S. Huang, and T.H. Hong (1994) Purification and characterization of the female-specific protein (vitellogenin) in mature female hemolymph of the prawn, Penaeus monodon. Invert. Reprod. Dev., 25:185–192. Charniaux-Cotton, H., and G. Payen (1988) Crustacean reproduction. In: Endocrinology of Selected Invertebrate Types, Vol. 2. H. Laufer and R.G.H. Downer, eds. Alan R. Liss, Inc., New York, pp. 279–303. Chen, C.C., and S.N. Chen (1993) Isolation and partial characterization of vitellin from the egg of giant tiger prawn, Penaeus monodon . Comp. Biochem. Physiol., 106B:141–146. Chen, C.C., and S.N. Chen (1994) Vitellogenesis in the giant tiger prawn, Penaeus monodon (Fabricius, 1789). Comp. Biochem. Physiol., 107B:453–460. Fainzilber, M., M. Tom, S. Shafir, S.W. Applebaum, and E. Lubzens (1992) Is there extraovarian synthesis of vitellogenin in penaeid shrimp? Biol. Bull., 183:233–241. Hall, M., M.C. van Heusden, and K. Söderhäll (1995) Identification of the major lipoproteins in crayfish hemolymph as proteins involved in immune recognition and clotting. Biochem. Biophys. Res. Commun., 216:939–946. Harrison, K.E. (1990) The role of nutrition in maturation, reproduction and embryonic development of decapod crustaceans: a review. J. Shellfish Res., 9:1–28. Khayat, M., O. Shenker, B. Funkenstein, M. Tom, E. Lubzens, and A. Tietz (1994a) Fat transport in the penaeid shrimp, Penaeus semisulcatus (de Haan). Isr. J. Aquacult. Bamidgeh 46:22–32. Khayat, M., E. Lubzens, A. Tietz, and B. Funkenstein (1994b) Cell-free translation of vitellin in the shrimp Penaeus semisulcatus (de Haan). Gen. Comp. Endoc., 93:205–213. Khayat, M., B. Funkenstein, A. Tietz, and E. Lubzens (1995) In vivo, in vitro and cell-free synthesis of hemocyanin in the shrimp Penaeus semisulcatus (de Haan). Comp. Biochem. Physiol., 112B:31–38. Komatsu, M., and S. Ando (1992) Isolation of crustacean egg yolk lipoproteins by differential density gradient ultracentrifugation. Comp. Biochem. Physiol., 103B:363–368. Komatsu, M., S. Ando, and S.I. Teshima (1993) Comparison of hemolymph lipoproteins from four species of Crustacea. J. Exp. Zool., 266:257–265. Lautier, J., and J.G. Lagarrigue (1988) Lipid metabolism of 348 E. LUBZENS ET AL. the crab Pachygrapsus marmoratus during vitellogenesis. Biochem. Syst. Ecol., 16:203–212. Laverdure, A.M., and D. Soyez (1988) Vitellogenin receptors from lobster oocyte membrane. Solubilization and characterization by a solid phase binding assay. Int. J. Inv. Reprod. Dev., 13:251–266. Lee, R.F. (1990) Lipoproteins from the hemolymph and ovaries of marine invertebrates. Adv. Comp. Env. Physiol., 7:187–207. Lee, R.F., and D.L. Puppione (1978) Serum lipoproteins in the spiny lobster, Panulirus interruptus. Comp. Biochem. Physiol., 59B:239–243. Lee, R.F., and D.L. Puppione (1988) Lipoproteins I and II from the hemolymph of the blue crab, Callinectes sapidus, lipoprotein II associated with vitellogenesis. J. Exp. Zool., 248:278–289. Lubzens, E., M. Khayat, T. Ravid, B. Funkenstein, and A. Tietz (1995) Lipoproteins and lipid accumulation within the ovaries of penaeid shrimp. Is. J. Aquacult. Bamidgeh 47:185–195. Lytle, J.S., R.F. Lytle, and L.T. Ogle (1990) Polyunsaturated fatty acid profiles as a comparative tool in assessing maturation diets of Penaeus vannamei. Aquaculture 89:287–299. Markwell, M.A.K., S.M. Haas, L.L. Bieber, and E. Tolbert (1978) A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal. Biochem., 87:206–210. Middleditch, B.S., S.R. Missler, H.H. Hines, J.P. McVey, A. Brown, D.G. Ward, and A.L. Lawrence (1980) Metabolic profiles of penaeid shrimp: dietary lipids and ovarian maturation. J. Chromatogr., 195:359–368. Puppione, D.L., D.F. Jensen, and J.D. O’Connor (1986) Physiochemical study of rock crab, Cancer antennarius lipoproteins. Biochem. Biophys. Acta, 875:536–568. Quackenbush, L.S. (1989) Yolk protein production in the marine shrimp Penaeus vannamei. J. Crust. Biol., 9:509–516. Quinitio, E.T., A. Hara, K. Yamauchi, and A. Fuji (1990) Isolation and characterization of vitellin from the ovary of Penaeus monodon. Invert. Reprod. Dev., 17:221–227. Ravid, T. (1994) Lipid transport and accumulation in ovaries of female shrimp, Penaeus semisulcatus (de Haan). M.Sc. thesis, Tel Aviv University, Tel Aviv, Israel. Ribero, L.P., and H.J. McDonald (1963) Electrophoresis of lipoproteins using pre-stained serum. J. Chromatogr., 10:443–449. Sappington, T.W., A.R. Hays, and A.S. Raikhel (1995) Mosquito vitellogenin receptor: purification, developmental and biochemical characterization. Insect Biochem. Mol. Biol., 25:807–817. Schlenck, H., and J.L. Gellerman (1960) Esterification of fatty acids with diazomethane on a small scale. Anal. Chem., 32:1412–1414. Schneider, W.J. (1992) Lipoprotein receptors in oocyte growth. Clin. Investig. 70:385–390. Schneider, W.J. (1995) Yolk precursor transport in the laying hen. Curr. Opin. Lipidol., 6:92–96. Shafir, S., M. Tom, M. Ovadia, and E. Lubzens (1992) Protein, vitellogenin and vitellin levels during ovarian devel- opment in Penaeus semisulcatus (de Haan). Biol. Bull., 183:394–400. Shenker, O., A. Tietz, M. Ovadia, and M. Tom (1993) Lipid synthesis from acetate by the in vitro incubated ovaries of the penaeid shrimp, Penaeus semisulcatus. Mar. Biol., 117:583–589. Shlagman, A., C. Lewinsohn, and M. Tom (1986) Aspects of reproductive activity of Penaeus semisulcatus (de Haan), along the southeastern coast of the Mediterranean. Mar. Ecol., 7:15–22. Spaziani, E., R.J. Havel, R.L. Hamilton, D.A. Hardman, J.B. Stoudemire, and R.D. Watson (1986) Properties of serum high density lipoproteins in the crab Cancer antennarius(Stimpson). Comp. Biochem. Physiol., 85B:307–314. Spaziani, E., W.L. Wang, and L.A. Novy (1995) Serum highdensity lipoproteins in the crab Cancer antennarius. IV. Electrophoretic and immunological analyses of apolipoproteins and a question of female-specific lipoproteins. Comp. Biochem. Physiol., 111B:265–276. Teshima, S., and A. Kanazawa (1980a) Lipid transport in crustaceans. Min. Rev. Data File Fish. Res., 1:1–25. Teshima, S., and A. Kanazawa (1980b) Lipid constituents of serum lipoproteins in the prawn. Bull. Jpn. Soc. Sci. Fish., 46:57–62. Teshima, S., and A. Kanazawa (1983) Variation in lipid composition during the ovarian maturation of the prawn. Bull. Jpn. Soc. Sci. Fish., 49:957–962. Teshima, S., A. Kanazawa, and Y. Kakuta (1986a) Role of dietary phospholipids in the transport of [14C] cholesterol in the prawn. Bull. Jpn. Soc. Sci. Fish., 52:719–723. Teshima, S., A. Kanazawa, and Y. Kakuta (1986b) Role of dietary phospholipids in the transport of [14C] tripalmitin in the prawn. Bull. Jpn. Soc. Sci. Fish., 52:519–524. Teshima, S., A. Kanazawa, K. Horinouchi, and S. Koshio (1988) Lipid metabolism in destalked prawn, Penaeus japonicus: induced maturation and transfer of lipid reserves to the ovaries. Nippon Suisan Gakkaishi, 54: 1123–1129. Teshima, S., A. Kanazawa, S. Koshio, and K. Horinouchi (1989) Lipid metabolism of the prawn, Penaeus japonicus during maturation: variation in lipid profiles of the ovary and hepatopancreas. Comp. Biochem. Physiol., 92B: 45–49. Tietz, A., T. Ravid, M. Khayat, N. Daube, and E. Lubzens (1996) The function of lipoproteins during vitellogenesis in a marine shrimp (Penaeus semisulcatus). In: 2nd European Crustacean Conference, Liège, Belgium, September 2–6, 1996, Abstract, p. 88. Tom, M., M. Fingerman, T.K. Hayes, V. Johnson, B. Kerner, and E. Lubzens (1992) A comparative study of the ovarian proteins from two penaeid shrimp, Penaeus semisulcatus (de Haan) and Penaeus vannamei (Boone). Comp. Biochem. Physiol., 102:483–490. Yepiz-Plascencia, G.M., R. Sotelo-Mundo, L. Vazques-Moreno, R. Ziegler, and I. Higuera-Ciapara (1995) A non-sex-specific hemolymph lipoprotein from the white shrimp Penaeus vannamei Boone. Isolation and partial characterization. Comp. Biochem. Physiol., 111B:181–187.