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The relationship between molting, reproduction, and a hemolymph female-specific protein in the lobster, Homarus americanus
Camp. Biochem Phvsiol. Vol. 77A, No. 4. pp. 749-757, 1984
Printed in Great Britain
f‘
0300-9629/84 $3.00+ 0.00 1984 Pergamon Press Ltd
THE RELATIONSHIP BETWEEN MOLTING, REPRODUCTION, AND A HEMOLYMPH FEMALE-SPECIFIC PROTEIN IN THE LOBSTER, HOMARUS AMERICANUS EDWARD
Department
H. BYARD
and
DAVID
E. AIKEN
of Biology, University of Winnipeg, Winnipeg, Canada, R3B 2E9. Department of Fisheries and Oceans, Biological Station, St. Andrews, New Brunswick, Canada, EOG 2X0 (Receired 2 I
July 1983)
Abstract-l. A female-specific protein (FSP), immunologically identical to oocyte lipovitellin, is present in the hemolymph of vitellogenic female lobsters (Homarus americanus). 2. FSP titers increase in the hemolymph during vitellogenesis, but the levels are maximum well prior to oviposition. 3. FSP does not appear in the hemolymph during the winter months, while the ovaries are quiescent, nor does it appear in females that are in late pre-molt stages, when vitellogenesis is suppressed prior to ecdysis. 4. It thus seems that the titer of hemolymph FSP is a good index of vitellogenesis, and that FSP is probably an extra-oocytic precursor of lipovitellin in transit to the oocyte.
INTRODUCTION
ods, in particular, pleopod sampling (Stevenson, 1972; Aiken, 1973), for “staging” incremental periods of proecdysis, whilst an equivalent method for monitoring reproductive events has not been practised to the same extent. Ovarian and testicular samples can be taken, but these invasive techniques do not leave the animal intact, and further, damage the very organ which is being studied. In our studies on molting and reproduction in the female lobster, we were able to quantitate the levels of a female-specific protein (FSP) in the hemolymph, which is immunologically identical to lipovitellin, the major egg yolk protein as described by Wallace et a/. (1967). This protein had been first reported to exist in Homarus americanus by Barlow and Ridgway (1969). We found that the levels of the FSP showed good correlation to the timing of vitellogenesis. This study describes, in detail, the occurrence and properties of the FSP in relation to molting and vitellogenesis in female lobsters. We feel that the FSP provides a rapid and reliable index of ovarian growth, and, coupled with the existing methods for monitoring proecdysis, will be a useful tool in experimental studies of lobster physiology.
Both molting and reproduction in crustaceans occur on a seasonal timetable and are, moreover, integrated since most crustaceans continue to molt and grow after reaching sexual maturity. In female crustaceans, ovarian maturation and molting must occur at appropriate times, since the newly laid egg mass is carried on the maternal abdomen, and an inopportune molt would lead to a loss of these eggs. The North American lobster, Homarus americanus, is a valuable resource, and one might expect that a good deal of information might exist on both the molting and reproductive physiology. In reality, a good deal of the available information on molting and reproduction in Homarus americanus is based largely on field studies (Squires, 1970; Ennis, 1971; Krouse, 1973) which, in general, have largely been unable to define, in detail, the relationship between molting and oviposition, since these field studies were conducted by making single observations on a large number of animals. In this study, we have made sequential observations on individual lobsters maintained in the laboratory over two full seasons, and, thus, have been better able to define the timing of molting and oviposition in individual animals. It is revealing to note that, although molting and reproduction in crustaceans are equally important and interesting events, reviews of crustacean physiology have historically not treated them as such (for example, see Carlisle and Knowles, 1959; Highnam and Hill, 1969; or Tombes, 1970). These reviews have extensive coverage of ecdysis and its control, but little as to the control or timing of reproductive events, although this imbalance has been redressed recently (Aiken and Waddy, 1976). It seems clear that this discrepancy has arisen since there exist several meth-
MATERIALS
AND METHODS
Experimental animals Lobsters (Homarus americanus, Milne-Edwards) were obtained from retailers in New Brunswick and Nova Scotia, Canada, The lobsters were originally caught in the Northumberland Strait region, south-west of Prince Edward Island, Canada. Lobsters were kept in community tanks, and were fed a mulch of shrimp, herring, sea urchin and cod. Stages of proecdysis were determined by examination of fresh, severed pleopod tips according to the criteria established by Aiken (I 973). Ovarian stages were determined by examining freshly dissected ovaries. 749
EDWAK~H. BYARDand DAVIDE. AIKEN
750 Hemolymph
samples
Lobster hemolymph samples (up to 3 ml per sample) were obtained by syringe from just under the ventral a~omina~ membrane of the first or second abdominal segment. The fresh hemoiymph sample was immediately placed into a centrifuge tube, and swirled gently until a visible clot formed. The tubes were left to stand undisturbed at 4’C overnight to ensure complete clotting. The clot was loosened with an applicator stick, and the clear serum was obtained by a IO-15 min centrifugation at 10,OOOg.Serum was either used fresh for protein dete~inations and e~ectrophoresis, or frozen at -20 C.
diffusion analysis as described by Ouchterlony (1958). Diffusion was allowed to proceed for up to 48 hr at either room temperature or at ISC. Alternatively, samples of either sera or ovarian fractions were first run on 79; acrylamide gels as previously described. and then pairs of gels were placed parallel to each other. about 4 cm apart, in the bottom of a IO cm square Petri dish. Warmed, buffered agar was poured over the gels so that it just covered them. After the agar had cooled. a l.Omm x 9.0cm slit was made in the agar so that it was equidistant and parallel to the two acrylamide gels. Antiserum was added to the trough, and diffusion was allowed to proceed for 24-48 hr at either room temperature or at
Isolation
I ST,
and purification
of lipocitellin
Fragments of ovarian tissue at various stages of vitellogenesis were dispersed in a Potter-Elvejehm homogenizer in 10 volumes of ice-cold 0.5 M NaCI containing 5 mM EDTA* (sodium salt). The homogenates were cent~fuged, in the cold, at 20,OOOgfor 20 min and the supernatants used for both electrophoresis or subsequent purification. The lipovitellin fraction was isolated from the 20,OOOg supernatants using the ammonium sulphate precipitation technique described by Wallace et al. (1967) except that centrifugations were reduced to 20,OOOgfor 30 min at O-5°C. The hulk of the ~ipovite~~inprecipitated at 67% saturation of ammonium sulphate at O’C. Purity of the resulting preparations was checked electrophoretically. Protein determinations
Total serum or ovarian protein levels were determined using the method of Lowry et ul. (1951) or the Biuret method of Henry et al. (1957). Lobster serum or ovarian proteins were separated electrophoretically on standard 7’4 acrylamide gels, poured into 90 x 5 mm glass tubes (Davis, 1964). All separations were carried out at 1o’C with a reservoir buffer of 0.06 M Tris-glycine at pH 8.8. The volume of the sample was always less than 5~1 and was adjusted so that between 100 and 180,ug of protein were present. Gels were stained by immersion in 0.X;, Amido black in 7’;,; aqueous acetic acid for l-8 hr. Gels were destained electrophoreticaily and stored for subsequent quantification in 7:; acetic acid. Some gels were stained with Sudan Black B (for lipid) or with the periodic acid-Schiff reagent (for carbohydrates) according to the methods described in Sargent (1969). Stained gels were scanned on a Photovolt Densicord densitometer that provided gel scans that were expanded I : 5 with respect io the original gel dimension. Areas under the curves were estimated by using the Integrator device attached to the densitometer. This device was shown to measure protein concentrations in a linear fashion as checked with a series of standard protein gels. The precipi~dted iipovite~lin fractions (in 67:/, ~monium sulfate) were resuspended in, and thoroughly dialyzed against 0.5 M NaCl at 5-IO’C. Anti-hpovitellin antibodies were raised against this fraction by injecting rabbits with 1ml each of a 5 mg/ml solution of protein and Freund’s complete adjuvant. Three injections were administered over a three-week period. About ten days after the last injection, the rabbits were bled via a peripheral ear vein, and the presence of anti-~ipovitelIin confirmed using the ring precipitin test as described by Campbell et al. (1970). Samples of rabbit sera were pooled for further use. Various samples were tested for the presence of antigens precipitable by anti-lipovitellin by loading them on to 1% agar gels (0.05 M barbital-HCI buffer, pH 7.2) for double .-~--
_ _ _ .-._
“EDTA: ~thy~en~iaminetetracetic
---._ acid.
Agar from either of the diffusion methods was finally washed in several changes of 0.9”; NaCI. stained by immersion in 0.5”,;, Amido black in 7’?,, acetic acid, and destained by washing in several changes of 7”,, acetic acid.
RESULTS
L$e history observations The mature female lobsters examined in this study alternated a molt with an oviposition, but on a variable schedule. Usually, molting occurred in one summer season with oviposition occurring in the next, but occasionally a molt was followed within a month or so with oviposition. A detailed description of the reproductive cycles for female Homarus umer icanus can be found in Aiken and Waddy (1976). and all the lobster reproductive patterns observed in the present study fit one of the patterns described in their review. In summary, females examined in this study were either undergoing their first round of vitellogenesis following a “puberty” molt (designated as pubertal and adult I) or were proceeding through their second vitellogenesis (thus adult II and adult III. depending on the timing of the molt). Adult 111 lobsters invariably followed the same seasonal moltoviposition pattern described above, that is, a molt followed within a month by an oviposition, whereas the adult II lobsters followed the more predominant pattern of molt and oviposition in successive seasons.
Hemolymph samples for ei~trophoretic analysis were taken from mature intermolt females during the summer months. An estimate of the vitellogenic state of the ovaries of these animals was made by calculating an ovarian factor (Table I). Densitometric scans of the electrophoretic profiles of the serum samples from females with ovarian stage I to V inclusive exhibited IO-1 1 detectable protein bands with measured R, values of 0.03-0.92. The method of electrophoretic analysis was reproducible; 40 randomly selected samples from the hundreds of samples analysed showed little variation in the relative migration of several stained components (Table 2). The e~ectrophoretic analysis of serum samples from females with vitellogenic ovaries (stages II to V inclusive) showed that a female specific protein (FSP) band (R, 0.107) was present at highest concentration in the hemolymph of females with ovaries in stage III. This band was absent from male hemolymph, and was not detectable in the hemolymph of either immature females or females with ovaries in stage I (Fig. I).
Female-specific protein in the lobster
a
I
Direction
of
miamtion
Fig. I. Representative photographs and densitometer present after separation on 7% acrylamide gels at pH as indicated (see Table 1 for description of stages). present, at an R,
tracings of female (a-e) and male (f) serum proteins 8.8-9.0. Females exhibited ovarian stages from I-V Arrows denote the female specific protein, where value of 0.107.
751
EDWARD H. BYARD and DAVIU E. AIKEN
152
Table I. Ovary stages and descriptions for Honxrrus Ovarian stage*
Description (in .SIfU) White, creamy Yellow, flaccid Light green Dark green Dark green
Y-120 80 160 127 178 200 320 320- 400
Physmlogical stage
I 11 III IV V
Immature Post-oviposltion Vitelloyenesis Vitellogenesis Mature, ovulation
umrricutm
Range of ovarian factors (0,) observedt
are similar to those used by Krause (1973) for Homarus and Bomirskl and Klek (1974) for Rhithrop~mopeu,~ hurr~.rii and Cranaon uunpon. +Ovarian factor (o ) = [ovary weight (mUI x 10
*Designations
americanus,
I
Table
2. Relative
electrophoretic mobihties proteins*
of
-iGijiaX*T some
lobster
Protein
Source
Observed mean R, it SE
Female specific protein “Complex” protein’r Hemocyanin (4-S bands)? Purified lioovitellin
Female serum
0.107 5 0.012 (N = 40)
Male and female serum Male and female serum Homogenate of mature ovarv
0.204 i_ 0.071 (N = 40) 0.809 * 0.039 to O.Y18+0.031 (N =40) 0.106 t 0.007 (N = 5)
*Gels used were 7% acrylamide at pH 8.8-9.0. run at S-10 C tIdentification after Fielder PI al. (1971).
Electrophoretic preparations of crude and partially purified lipovitellin (Fig. 2) exhibited a major band with an R, value of 0.106, identical to the R, value of the hemolymph FSP found in vitellogenic females. To confirm the identity of the hemolymph FSP and the lipovitellin fraction double immunodiffusion in agar was performed, using pooled preparations of rabbit anti-lipovitellin. The antibody formed single precipitin lines when tested against the original lipovitellin preparations, sera from females with ovaries in stages II to V inclusive, and crude ovarian homogenates (Fig. 3a). Precipitin arcs were confluent on
Fig. 2. Photographs and densitometer tracings of (a) a 20,OOOg supernatant of a 0.5 M NaCl ovarian homogenate, and (b) purified lipovitellin, after electrophoresis on 7”~ acrylamide gels at pH 8.8-9.0. Both samples are 50 pg protein. The major bands (arrows) have R, values of 0.106.
Female-specific protein in the lobster all plates tested, thus confirming the immunological identity of the serum FSP and lipovitellin. Male serum did not precipitate the anti-hpovitellin under the test conditions. When anti-lipovitellin was allowed to diffuse in agar toward electrophoretically separated samples of either female serum or ovarian homogenates, single precipitin arcs were seen adjacent to the I$ 0.107 position only (Fig. 3b). No other arcs were detectable, and, again. male serum did not
precipitate tions.
the anti-lipovitellin
753 under
these
condi-
Hemolymnph FSP and the molt cycle We were further interested in the relationship between FSP concentration, molt stage, and ovarian stage. Non-ovigerous females were sampled throughout the year, with particular attention paid to the months of July and August, the period when vitel-
a
Fig. 3. (a) Photograph and diagram of a stained immunodiffusion slide. The arcs are confluent (arrows), suggesting identity of the proteins in wells F, P, and 0. A = anti-lipovitellin; F = female serum previously shown to exhibit FSP on 7% acrylamide gels; M = male serum; 0 = crude ovarian homogenate supematant; P = purified lipovitellin. (b) Diffusion (in agar) of anti-hpovitellin (A) against electrophoretically separated samples (in polyacrylamide gels, origin at top of figure) of purified lipovitellin (P), female serum with FSP (F, i), and male serum (M, ii). A single, identical precipitin arc is seen with each of the purified lipovitellin and the female serum, but the male serum shows no visible precipitin reaction.
154
EDWARD
H. BYARU and
DAVID E. AIKEN
l
Intermolt
A Lote-premolt postmott
200 Ovary
factor
(0,)
Fig. 4. Concentrations of serum FSP as a function of ovarian factor (0,) in mature, non-ovigerous females during the July and August. Levels of FSP were determined from quantitation of serum samples separated on 7% acrylamide gels at pH 8.8-9.0. Each point represents a serum sample from a different lobster. The circles (a) represent samples taken from females in July-August in molt stages C to D, (intermolt to early premolt), whereas the open triangles (A) represent samples taken from females in molt stages D, (premolt) to stage B (postmolt).
logenesis is at its peak in the Northumberland Strait population of lobsters. The results of extensive sampling are summarized in Fig. 4. Females in molt stages Cz to D, (intermolt) showed variable concentrations of FSP, with a maximum occurring at ovarian stage III, that is, well before oviposition. Females in late premolt (stage D, or later) or postmolt (stage B) showed low to nil concentrations of FSP, although ovarian stages up to stage IV were observed. No mature (stage V) ovaries were observed in any of the late premolt group. In fact, an occasional premolt female was observed to be reabsorbing yolk from the oocytes, a condition which can be readily identified by the massive degeneration of most of the mature oocytes in the ovary, and the appearance of the released green yolk protein in the hemolymph. The levels of reabsorbed hemolymph protein is often so high as to give the lobster a dark-green or even black hemolymph. Seasonal variation in hemolymph FSP levels Sequential serum samples were obtained from several females throughout a complete calendar year in order to make observations on the seasonal fluctuations of circulating FSP. As expected, the FSP concentrations varied in accordance with the molting and oviposition patterns. Some representative patterns are shown in Fig. 5. Several trends emerged and can be summarized as follows: (a) FSP levels are always maximal prior to oviposition and are always low prior to molting (Fig. 5 a, b and c); (b) FSP levels are low (or even zero) in the winter months, when the water temperature is as low as 0°C (Fig. 5 a, b, c); (c) the levels of FSP show a good correlation to the vitellogenic condition of the oocytes; that is, if FSP is not present, there is no measurable oocyte growth
(Fig. 5~). Reabsorption of yolk from the oocytes (Fig. 5d) causes a major deviation in FSP measurements but causes no alteration in the timing of the next molt (compare Fig. 5 a, b, c with Fig. 5d).
DISCUSSION
Our findings indicate that the concentrations of the female-specific protein (FSP) in the hemolymph of gravid females is modulated by the molt cycle and by seasonal variations in the water temperature, and is generally a clear indicator of the vitellogenic state of the oocytes. Thus, these results are in agreement with many other studies that have reported the occurrence of female-specific hemolymph proteins in the crustacean orders Amphipoda, Isopoda, as well as in the Decapoda (as reviewed by Adiyodi and Adiyodi, 1970; Croisille et al., 1974). These investigations report that, as found in the present study, the concentrations of the FSP in the hemolymph is reported to increase as vitellogenesis in the oocytes becomes maximal. The FSP in the Crustacea appears completely analogous to the blood-borne vitellogenins that are synthesized in the livers of birds (Schjeide et (II., 1963; Heald and McLachlin, 1965), amphibians (Redshaw and Follett. 1971; Wallace and Bergink, 1974), and by the fat bodies of insects (Telfer, 1965; Engelmann, 1969, 1970). Vitellogenins are transported to the ovary where they become incorporated, by micropinocytosis, into the oocyte as yolk. In fact, in the vertebrates and the insects, virtually all the yolk protein in a mature oocyte is accumulated in this way; for example, Wallace et al. (1972) estimate that in the amphibian Xenopus luevis, 99’7” of the yolk plate-
Female-specific
10
protein
in the lobster
755
r (b)
A
s
ONDJFMAMJJAS Month
Fig. 5. Representative seasonal patterns of FSP levels in the serum of female lobsters sampled over an entire calendar year (August through the following September, inclusive). Each pattern graphed in (a) through (d) represents a single animal as followed throughout the year, and each point represents the measurement of the FSP concentration in a monthly serum sample. In (a) there was a molt (M) in one season, followed by a gradual increase in FSP levels and oviposition (0) the following summer. In (b) and (c) molt and oviposition both occurred in one summer, followed by only a molt the following summer. In (b), some vitellogenesis occurred (0, = 162), whereas in (c) no vitellogenesis occurred (0, = 91). Note that in (b), FSP was detectable in the spring of the second season, but this was not the case in (c). The typical pattern of reabsorption is shown in (d). Note that oviposition does not occur [compare with (b) and (c)l, and that high levels of FSP persist throughout the winter months, but that this has no effect on the timing of the molt in the second season-[compare (b) and (c) with (d)].
let proteins phosvitin and lipovitellin are heterologous in origin. The complete story with respect to the origin and fate of the crustacean FSP is less clear. Although FSP levels in the hemolymph of adult females seems to be a good index of vitellogenesis, with a maximum concentration occurring well prior to the maximum yolk accumulation in the oocytes, the central question as to the site of synthesis of FSP remains unclear. Two alternatives are proposed in the literature, and each alternative is supported by some, if incomplete, evidence. The first alternative is that the FSP is synthesized, as are vertebrate and insect vitellogenins, outside the oocyte, and is transported there via the hemolymph (Kerr, 1969; Wolin et al., 1973). The evidence for this point of view is considerable, although largely circumstantial, and is based on the consistent obser-
vations that the surface of the crustacean oocyte exhibits micropinocytotic pits and vesicles (Hinsch and Cone, 1969; Zerbib, 1973; Hinsch and Bennett, 1979; Schade and Shivers, 1980) and that the oocyte will take up exogenous proteins either from the hemolymph or from the external medium in vitro (Wolin et al., 1973: Zerbib, 1977; Schade and Shivers, 1980). It is not known if the micropinocytosis is a receptor-mediated process (Goldstein et a/., 1979), nor has the site of synthesis of FSP been satisfactorily established. The second alternative mechanism for the observed patterns of FSP in the hemolymph is that these proteins result only from reabsorption of yolk proteins that are synthesized in the oocyte. The evidence put forward for this alternative is both biochemical and ultrastructural. Lui et al. (1974) and Lui and O’Connor (1976) report that, in crayfish, each of the
756
EUWAKD H. BYAKU and DAVID E. AIKW
three subunits of hpovitelhn. the major yolk protein. are synthesized within the oocyte and conclude that extra-oocytic contributions to the total yolk protein seen in the mature oocyte are insignificant. Beams and Kessel (1963) and Carrion and Kessel (1972) concur, and describe the ultrastructural development of yolk granules in the extensive rough endoplasmic reticulum of the oocyte. Similarly, they conclude that but makes an micropinocytosis is present, insignificant contribution to the total yolk accumulated during vitellogenesis. Neither of these studies directly addresses the issue of the occurrence of female specific proteins present in the hemolymph. although Lui and O’Connor (1976) briefly state that these proteins could be accounted for by reabsorption of yolk, as has been described to occur in crayfish by Stephens (1952). Our observations are best explained by the first alternative, that is, that FSP is in transit to the oocyte from an external source. The levels of hemolymph FSP that we observed were always highest well prior to the maximum accumulation of yolk in the oocytes, and the levels dropped off markedly prior to oviposition. This pattern is consistent with the idea that the FSP represents an externally synthesized protein that logically is found at the highest level in hemolymph during the period when the oocytes are accumulating the maximum amounts of yolk. If reabsorption were accounting for hemolymph FSP, then one might expect the maximum levels when the oocytes are the maximum size, that is, just prior to oviposition. In any case, our observations show that reabsorption is an abrupt and isolated event in lobsters which occurs usually some time after an otherwise normal period of vitellogenesis. The reabsorbed yolk is present in the hemolymph at huge concentrations (e.g. greater than 20 mgiml) whereas FSP levels during vitellogenesis are considerably less (usually much less than 10 mgiml). In other words, reabsorption is an event distinct from normal vitellogenesis. Given the apparent contradiction in the evidence for yolk production in crustacean oocytes, it seems that the answer lies in a more complete assessment of the proportion of yolk that is synthesized in the oocyte. and the proportion that is imported from the hemolymph by micropinocytosis. There is clearly a component of the yolk that is synthesized inside the endoplasmic reticulum of the oocyte (Hinsch and Cone, 1969; Ganion and Kessel. 1972; Lui and O’Connor, 1976; Schade and Shivers, 1980), but it is equally clear that micropinocytosis is capable of taking up significant amounts of protein from the hemolymph (Schade and Shivers, 1980; Wolin et ul., 1973) and that micropinocytosis can be amplified by an appropriate hormonal signal (Hinsch and Bennett, 1979). Thus, that the total quantity of crustacean yolk protein is accumulated as a net result of both intraoocytic and extraoocytic syntheses seems certain; however, the relative contribution of each needs to be better defined. Our detailed observations on individual lobsters over two full reproductive seasons allowed us to establish unequivocally that molt and oviposition are alternated on a variable schedule. This alternation is not surprising in that both molt and oviposition
make major demands on metabolic reserves. But it is important to note that both molt and oviposition continue to occur in most reproductively mature crustaceans, thus separating the Crustacea from insects. to which they are often compared. Mature female insects do not molt, with the exception of the Thysanura (Rohdendorf and Watson. 1969). Thus the separation of molting and reproduction is temporally distinct. as are, presumably, whatever control mechanisms for the two processes that are at work. In the Crustacea, on the other hand, the control mechanisnls for molting and reproduction are more closely interwoven, and thus more difficult to analyze in isolation. That there is some kind of hierarchy between the two processes seems axiomatic, since an inopportune molt, that is. immediately after oviposition, would result in a loss of eggs on the cast exoskeleton. Our results indicate that in the period from late premolt (stages D, to D,) to postmolt (stage B), the hemolymph FSP levels are low to zero in non-ovigerous females, although the ovaries in these same animals are up to stage III in development. Thus, it appears that premolt events have suppressed vitellogenesis, as judged by the hemolymph titer of FSP. In addition, late premolt lobsters with mature ovaries will reabsorb yolk rather than oviposit. These two observations, taken together, lead to the conclusion that premolt events take precedence over, and may disrupt events leading to normal oviposition, A similar hierarchy exists in the Thysanuran, Lepisno&s inquilirzus, one of this order of exceptional insects that continues to molt and grow after reaching sexual maturity. In this species, the molting cycle overrides reproductive activity, and artificially induced molting will cause reabsorption of yolk from the oocytes of mature ovaries (Rhodendorf and Watson, 1969). It seems that premolt events and oviposition are incompatible. A similar conclusion can be derived from an experiment in which lobsters (Homarus mwricuntu) were subjected to 6 summer months in an artificially imposed B-month year (Aiken and Waddy, 1976). The mature ovary was reabsorbed only when the molt and reproductive cycles coincided such that molt would occur shortly after oviposition. In other words, it appears that the physiology of late premolt cannot include the final vitellogenesis (at least past stage III) and other preparations leading up to oviposition. In conclusion, we feel that the data on hemolymph FSP that are presented here provide a reliable index of vitellogenesis, and, could become a useful tool in further studies of lobster reproductive physiology. Acknor,led~emetlts-We thank Orysia Dawydiak for help in the preparation of figures. This investi~asion was supported by the Fisheries Research Board of Canada, and the National Canada.
Sciences
and
Engineering
Research
Council
of
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Female-specific
protein
Aiken D. E. and Waddy S. (1976) Controlling growth and reproduction in the American lobster. Proc. World Mariculture sot. 7, 415430. Barlow J. and Ridgway G. J. (1969) Changes in serum protein during the molt and reproductive cycles of the American lobster (Homarus americanus). J. Fish. Res. Bd Can. 26, 2101-2109. Beams H. W. and Kessel R. G. (1963) Electron microscope studies on developing crayfish oocytes with special reference to the origin of yolk. J. ceN Biol. 18, 621-649. Bomirski A. and Klek E. (1974) Action of eyestalks on the ovary in Rhithropanopeus harrisii and Crangon crangon (Crustacea: Decapoda). Mar. Biol. 24, 329-337. Campbell D. H., Garvev J. S.. Cremer N. E. and Sussdorf D.’ H. (1970) Methods in Immunology. W. A. Benjamin, New York. Carlisle D. B. and Knowles F. G. W. (1959) Endocrine Control in Crustaceans. Cambridge University Press, New York. Croisille Y., Junera H.. Meusy J. J. and Charniaux Cotton H. (1974) The female-specific protein (vitellogenic protein) in Crustacea with particular reference to Orchestia gammareila (Amphipoda). Am. 2001. 14, 1219-1228. Davis B. J. (1964) Disc electrophoresis-II. Method and application to human serum proteins. Ann. N.Y. Acad. Sci. 121, 404-427. Engelmann F. (1969) Female-specific protein: biosynthesis controlled by corpus allatum in Leucophaea maderae. Scienke 165, 407-409. Engelmann F. (1970) The Physiology oflnsect Reproduction, Pergamon Press, New York. Ennis G. P. (1971) Lobster (Homarus americanus) fishery and biology in Bonavista Bay, Newfoundland 196670. Fish. Res. Bd Can. Tech. Rep. 289, 291-297. Fielder D. R., Rangarao K. and Fingerman M. (1971) A female-limited lipoprotein and the diversity of hemocyanin components in the dimorphic variants of the fiddler crab,. Uca pugilator, as revealed by disc electronhoresis. Camp. Biochem. Phwiol. 29B. 291-297. Ganion C. R. and Kessel R. G. (1972) Intracellular synthesis, transport, and packaging of proteinaceous yolk of Orconectes immunis. J. cell Biol. 52, 420437. Goldstein J. L.. Anderson R. G. W. and Brown M. S. (1979) Coated pits, coated vesicles, and receptor-mediated endocytosis. Nolure, Lond. 279, 6799685. Heald P. J. and McLachlin P. M. (1965) The synthesis of phosvitin in vitro by slices of liver from the laying hen. Biochem. J. 94, 32-39. Henry R., Sobel C. and Berkman S. (1957) Interferences with the Biuret method for serum proteins: use of Benedict’s qualitative glucose reagent as a Biuret reagent. Analyt. Biochem. 29, 1491-1495. Highnam K. C. and Hill L. (1969) The Comparative Endocrinology of the Incertebrates. American Elsevier, New York. Hinsch G. W. and Bennett D. C. (1979) Vitellogenesis stimulated by thoracic ganglion implants into destalked immature spider crabs, Libinia emarginata. Tiss. Cell 11, 345-351. Hinsch G. W. and Cone M. V. (1969) Ultrastructural observations of vitellogenesis in the spider crab, Libinia emarginata (L). J. cell Biol. 40, 336-342. Kerr M. S. (1969) The hemolvmph nroteins of the blue crab, Callinectes sapidus--II.-A’lipdprotein serologically identical to oocyte lipovitellin. Deal Biol. 20, 1-17. Kraouse J. S. (1973) Maturity, sex ratio, and size composition of the natural population of American lobster, Homarus americanus along the Maine coast. Fishery Bull. 71, 165-I 73.
in the lobster
157
Lowry 0. H., Roseborough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin ohenol reagent. J. ‘biol. Chem. 193, 265-275. Lui C. W. and O’Connor J. D. (1976) Biosynthesis of lipovitellin by the crustacean ovary--II. Characterization of, and in vitro incorporation of amino acids into the purified subunits. J. exp. Zool. 195, 41-51. Lui C. W., Sage B. A. and O’Connor J. D. (1974) Biosynthesis of lipovitellin by the crustacean ovary. J. exp. Zool. 188, 289-296. Ouchterlony 0. (1958) Diffusion-in-gel methods for immunologicai analysis. Prog. Allergy 5, l-78. Redshaw M. R. and Follett B. K. (1971) The crvstalline yolk-platelet proteins and their soluble plasma precursor in an amphibian, Xenopus laevis. Biochem. J. 124, 759-766. Rohdendorf E. B. and Watson J. A. L. (1969) The control of reproductive cycles in the female firebrat, Lepismodes inguilinus. J. Insect Phvsiol. 15, 2085-2 101. Sargent J. R. (1969) Methods in Zone Electrophoresis. 2nd edn. BDH Chemicals, Poole. Schade M. L. and Shivers R. R. (1980) Structural modulation of the surface and cytoplasm of oocytes during vitellogenesis in the lobster, Homarus americanus. An electron microscope-protein tracer study. J. Morph. 163, 13-26. Schjeide 0. A., Wilkens M., McCandless R. G., Munn R., Peterson M. and Carlsen E. (1963) Liver synthesis, plasma transport, and structural alterations accompanying passage of yolk proteins. Am. Zool. 3, 167184. Squires H. J. (1970) Lobster (Homarus americanus) fishery and ecology in Port au Port Bay, Newfoundland, 196&65. Proc. natn. ShellJish. Ass. 60, 22-39. Stephens G. J. (1952) Mechanisms regulating the reproductive cycle in the crayfish, Cambarus. The female cycle. Physiol. Zodl. 25, 70-84. Stevenson J. R. (1972) Changing activities of the crustacean epidermis during the molting cycle. Amer. Zool. 12, 373-380. Telfer W. H. (1965) The mechanism and control of yolk formation. A. Rec. Enfomol. 10, 161-184. Tombes A. S. (1970) An Introduction to Invertebrate Endocrinology. Academic Press, New York. Wallace R. A. and Bergink E. W. (1974) Amphibian vitellogenin: Properties, hormonal regulation of hepatic synthesis and ovarian uptake, and conversion to yolk proteins. Am. Zool. 14, 1159-I 175. Wallace R. A., Walker S. L. and Hauschka P. V. (1967) Crustacean lipovitellin: isolation and characterization of the major high density lipoproteins from the eggs of decapods. Biochemistry 6, 1582-I 590. Wallace R. A., Nick01 J. M., Ho T. and Jared D. W. (I 972) Studies on amphibian yolk-X. The relative roles of autosynthetic and heterosynthetic processes during yolk protein assembly by isolated oocytes. Deul Biol. 29, 255-272. Wolin E. M., Laufer H. and Albertini D. F. (1973) Uptake of the yolk protein lipovitellin by developing crustacean oocytes. Deal Biol. 35, 160-170. Zerbib C. (1973) Contribution a l’etude ultrastructurale de l’ovocyte chez la Crustace Amphipode Orchestia gammarella Pallas. C.r. Acad. Sci., (Paris) Ser. D. Nat. Sci. 277, 1209-1212. Zerbib C. (1977) Endocytose ovocytaire chez le Crustace Amphipode Orchestia gammarellus (Pallas). Demonstration par la peroxydase. C.r. Acad. Sci. (Paris) Ser. D. Nat. Sci. 284, 757-760.
Printed in Great Britain
f‘
0300-9629/84 $3.00+ 0.00 1984 Pergamon Press Ltd
THE RELATIONSHIP BETWEEN MOLTING, REPRODUCTION, AND A HEMOLYMPH FEMALE-SPECIFIC PROTEIN IN THE LOBSTER, HOMARUS AMERICANUS EDWARD
Department
H. BYARD
and
DAVID
E. AIKEN
of Biology, University of Winnipeg, Winnipeg, Canada, R3B 2E9. Department of Fisheries and Oceans, Biological Station, St. Andrews, New Brunswick, Canada, EOG 2X0 (Receired 2 I
July 1983)
Abstract-l. A female-specific protein (FSP), immunologically identical to oocyte lipovitellin, is present in the hemolymph of vitellogenic female lobsters (Homarus americanus). 2. FSP titers increase in the hemolymph during vitellogenesis, but the levels are maximum well prior to oviposition. 3. FSP does not appear in the hemolymph during the winter months, while the ovaries are quiescent, nor does it appear in females that are in late pre-molt stages, when vitellogenesis is suppressed prior to ecdysis. 4. It thus seems that the titer of hemolymph FSP is a good index of vitellogenesis, and that FSP is probably an extra-oocytic precursor of lipovitellin in transit to the oocyte.
INTRODUCTION
ods, in particular, pleopod sampling (Stevenson, 1972; Aiken, 1973), for “staging” incremental periods of proecdysis, whilst an equivalent method for monitoring reproductive events has not been practised to the same extent. Ovarian and testicular samples can be taken, but these invasive techniques do not leave the animal intact, and further, damage the very organ which is being studied. In our studies on molting and reproduction in the female lobster, we were able to quantitate the levels of a female-specific protein (FSP) in the hemolymph, which is immunologically identical to lipovitellin, the major egg yolk protein as described by Wallace et a/. (1967). This protein had been first reported to exist in Homarus americanus by Barlow and Ridgway (1969). We found that the levels of the FSP showed good correlation to the timing of vitellogenesis. This study describes, in detail, the occurrence and properties of the FSP in relation to molting and vitellogenesis in female lobsters. We feel that the FSP provides a rapid and reliable index of ovarian growth, and, coupled with the existing methods for monitoring proecdysis, will be a useful tool in experimental studies of lobster physiology.
Both molting and reproduction in crustaceans occur on a seasonal timetable and are, moreover, integrated since most crustaceans continue to molt and grow after reaching sexual maturity. In female crustaceans, ovarian maturation and molting must occur at appropriate times, since the newly laid egg mass is carried on the maternal abdomen, and an inopportune molt would lead to a loss of these eggs. The North American lobster, Homarus americanus, is a valuable resource, and one might expect that a good deal of information might exist on both the molting and reproductive physiology. In reality, a good deal of the available information on molting and reproduction in Homarus americanus is based largely on field studies (Squires, 1970; Ennis, 1971; Krouse, 1973) which, in general, have largely been unable to define, in detail, the relationship between molting and oviposition, since these field studies were conducted by making single observations on a large number of animals. In this study, we have made sequential observations on individual lobsters maintained in the laboratory over two full seasons, and, thus, have been better able to define the timing of molting and oviposition in individual animals. It is revealing to note that, although molting and reproduction in crustaceans are equally important and interesting events, reviews of crustacean physiology have historically not treated them as such (for example, see Carlisle and Knowles, 1959; Highnam and Hill, 1969; or Tombes, 1970). These reviews have extensive coverage of ecdysis and its control, but little as to the control or timing of reproductive events, although this imbalance has been redressed recently (Aiken and Waddy, 1976). It seems clear that this discrepancy has arisen since there exist several meth-
MATERIALS
AND METHODS
Experimental animals Lobsters (Homarus americanus, Milne-Edwards) were obtained from retailers in New Brunswick and Nova Scotia, Canada, The lobsters were originally caught in the Northumberland Strait region, south-west of Prince Edward Island, Canada. Lobsters were kept in community tanks, and were fed a mulch of shrimp, herring, sea urchin and cod. Stages of proecdysis were determined by examination of fresh, severed pleopod tips according to the criteria established by Aiken (I 973). Ovarian stages were determined by examining freshly dissected ovaries. 749
EDWAK~H. BYARDand DAVIDE. AIKEN
750 Hemolymph
samples
Lobster hemolymph samples (up to 3 ml per sample) were obtained by syringe from just under the ventral a~omina~ membrane of the first or second abdominal segment. The fresh hemoiymph sample was immediately placed into a centrifuge tube, and swirled gently until a visible clot formed. The tubes were left to stand undisturbed at 4’C overnight to ensure complete clotting. The clot was loosened with an applicator stick, and the clear serum was obtained by a IO-15 min centrifugation at 10,OOOg.Serum was either used fresh for protein dete~inations and e~ectrophoresis, or frozen at -20 C.
diffusion analysis as described by Ouchterlony (1958). Diffusion was allowed to proceed for up to 48 hr at either room temperature or at ISC. Alternatively, samples of either sera or ovarian fractions were first run on 79; acrylamide gels as previously described. and then pairs of gels were placed parallel to each other. about 4 cm apart, in the bottom of a IO cm square Petri dish. Warmed, buffered agar was poured over the gels so that it just covered them. After the agar had cooled. a l.Omm x 9.0cm slit was made in the agar so that it was equidistant and parallel to the two acrylamide gels. Antiserum was added to the trough, and diffusion was allowed to proceed for 24-48 hr at either room temperature or at
Isolation
I ST,
and purification
of lipocitellin
Fragments of ovarian tissue at various stages of vitellogenesis were dispersed in a Potter-Elvejehm homogenizer in 10 volumes of ice-cold 0.5 M NaCI containing 5 mM EDTA* (sodium salt). The homogenates were cent~fuged, in the cold, at 20,OOOgfor 20 min and the supernatants used for both electrophoresis or subsequent purification. The lipovitellin fraction was isolated from the 20,OOOg supernatants using the ammonium sulphate precipitation technique described by Wallace et al. (1967) except that centrifugations were reduced to 20,OOOgfor 30 min at O-5°C. The hulk of the ~ipovite~~inprecipitated at 67% saturation of ammonium sulphate at O’C. Purity of the resulting preparations was checked electrophoretically. Protein determinations
Total serum or ovarian protein levels were determined using the method of Lowry et ul. (1951) or the Biuret method of Henry et al. (1957). Lobster serum or ovarian proteins were separated electrophoretically on standard 7’4 acrylamide gels, poured into 90 x 5 mm glass tubes (Davis, 1964). All separations were carried out at 1o’C with a reservoir buffer of 0.06 M Tris-glycine at pH 8.8. The volume of the sample was always less than 5~1 and was adjusted so that between 100 and 180,ug of protein were present. Gels were stained by immersion in 0.X;, Amido black in 7’;,; aqueous acetic acid for l-8 hr. Gels were destained electrophoreticaily and stored for subsequent quantification in 7:; acetic acid. Some gels were stained with Sudan Black B (for lipid) or with the periodic acid-Schiff reagent (for carbohydrates) according to the methods described in Sargent (1969). Stained gels were scanned on a Photovolt Densicord densitometer that provided gel scans that were expanded I : 5 with respect io the original gel dimension. Areas under the curves were estimated by using the Integrator device attached to the densitometer. This device was shown to measure protein concentrations in a linear fashion as checked with a series of standard protein gels. The precipi~dted iipovite~lin fractions (in 67:/, ~monium sulfate) were resuspended in, and thoroughly dialyzed against 0.5 M NaCl at 5-IO’C. Anti-hpovitellin antibodies were raised against this fraction by injecting rabbits with 1ml each of a 5 mg/ml solution of protein and Freund’s complete adjuvant. Three injections were administered over a three-week period. About ten days after the last injection, the rabbits were bled via a peripheral ear vein, and the presence of anti-~ipovitelIin confirmed using the ring precipitin test as described by Campbell et al. (1970). Samples of rabbit sera were pooled for further use. Various samples were tested for the presence of antigens precipitable by anti-lipovitellin by loading them on to 1% agar gels (0.05 M barbital-HCI buffer, pH 7.2) for double .-~--
_ _ _ .-._
“EDTA: ~thy~en~iaminetetracetic
---._ acid.
Agar from either of the diffusion methods was finally washed in several changes of 0.9”; NaCI. stained by immersion in 0.5”,;, Amido black in 7’?,, acetic acid, and destained by washing in several changes of 7”,, acetic acid.
RESULTS
L$e history observations The mature female lobsters examined in this study alternated a molt with an oviposition, but on a variable schedule. Usually, molting occurred in one summer season with oviposition occurring in the next, but occasionally a molt was followed within a month or so with oviposition. A detailed description of the reproductive cycles for female Homarus umer icanus can be found in Aiken and Waddy (1976). and all the lobster reproductive patterns observed in the present study fit one of the patterns described in their review. In summary, females examined in this study were either undergoing their first round of vitellogenesis following a “puberty” molt (designated as pubertal and adult I) or were proceeding through their second vitellogenesis (thus adult II and adult III. depending on the timing of the molt). Adult 111 lobsters invariably followed the same seasonal moltoviposition pattern described above, that is, a molt followed within a month by an oviposition, whereas the adult II lobsters followed the more predominant pattern of molt and oviposition in successive seasons.
Hemolymph samples for ei~trophoretic analysis were taken from mature intermolt females during the summer months. An estimate of the vitellogenic state of the ovaries of these animals was made by calculating an ovarian factor (Table I). Densitometric scans of the electrophoretic profiles of the serum samples from females with ovarian stage I to V inclusive exhibited IO-1 1 detectable protein bands with measured R, values of 0.03-0.92. The method of electrophoretic analysis was reproducible; 40 randomly selected samples from the hundreds of samples analysed showed little variation in the relative migration of several stained components (Table 2). The e~ectrophoretic analysis of serum samples from females with vitellogenic ovaries (stages II to V inclusive) showed that a female specific protein (FSP) band (R, 0.107) was present at highest concentration in the hemolymph of females with ovaries in stage III. This band was absent from male hemolymph, and was not detectable in the hemolymph of either immature females or females with ovaries in stage I (Fig. I).
Female-specific protein in the lobster
a
I
Direction
of
miamtion
Fig. I. Representative photographs and densitometer present after separation on 7% acrylamide gels at pH as indicated (see Table 1 for description of stages). present, at an R,
tracings of female (a-e) and male (f) serum proteins 8.8-9.0. Females exhibited ovarian stages from I-V Arrows denote the female specific protein, where value of 0.107.
751
EDWARD H. BYARD and DAVIU E. AIKEN
152
Table I. Ovary stages and descriptions for Honxrrus Ovarian stage*
Description (in .SIfU) White, creamy Yellow, flaccid Light green Dark green Dark green
Y-120 80 160 127 178 200 320 320- 400
Physmlogical stage
I 11 III IV V
Immature Post-oviposltion Vitelloyenesis Vitellogenesis Mature, ovulation
umrricutm
Range of ovarian factors (0,) observedt
are similar to those used by Krause (1973) for Homarus and Bomirskl and Klek (1974) for Rhithrop~mopeu,~ hurr~.rii and Cranaon uunpon. +Ovarian factor (o ) = [ovary weight (mUI x 10
*Designations
americanus,
I
Table
2. Relative
electrophoretic mobihties proteins*
of
-iGijiaX*T some
lobster
Protein
Source
Observed mean R, it SE
Female specific protein “Complex” protein’r Hemocyanin (4-S bands)? Purified lioovitellin
Female serum
0.107 5 0.012 (N = 40)
Male and female serum Male and female serum Homogenate of mature ovarv
0.204 i_ 0.071 (N = 40) 0.809 * 0.039 to O.Y18+0.031 (N =40) 0.106 t 0.007 (N = 5)
*Gels used were 7% acrylamide at pH 8.8-9.0. run at S-10 C tIdentification after Fielder PI al. (1971).
Electrophoretic preparations of crude and partially purified lipovitellin (Fig. 2) exhibited a major band with an R, value of 0.106, identical to the R, value of the hemolymph FSP found in vitellogenic females. To confirm the identity of the hemolymph FSP and the lipovitellin fraction double immunodiffusion in agar was performed, using pooled preparations of rabbit anti-lipovitellin. The antibody formed single precipitin lines when tested against the original lipovitellin preparations, sera from females with ovaries in stages II to V inclusive, and crude ovarian homogenates (Fig. 3a). Precipitin arcs were confluent on
Fig. 2. Photographs and densitometer tracings of (a) a 20,OOOg supernatant of a 0.5 M NaCl ovarian homogenate, and (b) purified lipovitellin, after electrophoresis on 7”~ acrylamide gels at pH 8.8-9.0. Both samples are 50 pg protein. The major bands (arrows) have R, values of 0.106.
Female-specific protein in the lobster all plates tested, thus confirming the immunological identity of the serum FSP and lipovitellin. Male serum did not precipitate the anti-hpovitellin under the test conditions. When anti-lipovitellin was allowed to diffuse in agar toward electrophoretically separated samples of either female serum or ovarian homogenates, single precipitin arcs were seen adjacent to the I$ 0.107 position only (Fig. 3b). No other arcs were detectable, and, again. male serum did not
precipitate tions.
the anti-lipovitellin
753 under
these
condi-
Hemolymnph FSP and the molt cycle We were further interested in the relationship between FSP concentration, molt stage, and ovarian stage. Non-ovigerous females were sampled throughout the year, with particular attention paid to the months of July and August, the period when vitel-
a
Fig. 3. (a) Photograph and diagram of a stained immunodiffusion slide. The arcs are confluent (arrows), suggesting identity of the proteins in wells F, P, and 0. A = anti-lipovitellin; F = female serum previously shown to exhibit FSP on 7% acrylamide gels; M = male serum; 0 = crude ovarian homogenate supematant; P = purified lipovitellin. (b) Diffusion (in agar) of anti-hpovitellin (A) against electrophoretically separated samples (in polyacrylamide gels, origin at top of figure) of purified lipovitellin (P), female serum with FSP (F, i), and male serum (M, ii). A single, identical precipitin arc is seen with each of the purified lipovitellin and the female serum, but the male serum shows no visible precipitin reaction.
154
EDWARD
H. BYARU and
DAVID E. AIKEN
l
Intermolt
A Lote-premolt postmott
200 Ovary
factor
(0,)
Fig. 4. Concentrations of serum FSP as a function of ovarian factor (0,) in mature, non-ovigerous females during the July and August. Levels of FSP were determined from quantitation of serum samples separated on 7% acrylamide gels at pH 8.8-9.0. Each point represents a serum sample from a different lobster. The circles (a) represent samples taken from females in July-August in molt stages C to D, (intermolt to early premolt), whereas the open triangles (A) represent samples taken from females in molt stages D, (premolt) to stage B (postmolt).
logenesis is at its peak in the Northumberland Strait population of lobsters. The results of extensive sampling are summarized in Fig. 4. Females in molt stages Cz to D, (intermolt) showed variable concentrations of FSP, with a maximum occurring at ovarian stage III, that is, well before oviposition. Females in late premolt (stage D, or later) or postmolt (stage B) showed low to nil concentrations of FSP, although ovarian stages up to stage IV were observed. No mature (stage V) ovaries were observed in any of the late premolt group. In fact, an occasional premolt female was observed to be reabsorbing yolk from the oocytes, a condition which can be readily identified by the massive degeneration of most of the mature oocytes in the ovary, and the appearance of the released green yolk protein in the hemolymph. The levels of reabsorbed hemolymph protein is often so high as to give the lobster a dark-green or even black hemolymph. Seasonal variation in hemolymph FSP levels Sequential serum samples were obtained from several females throughout a complete calendar year in order to make observations on the seasonal fluctuations of circulating FSP. As expected, the FSP concentrations varied in accordance with the molting and oviposition patterns. Some representative patterns are shown in Fig. 5. Several trends emerged and can be summarized as follows: (a) FSP levels are always maximal prior to oviposition and are always low prior to molting (Fig. 5 a, b and c); (b) FSP levels are low (or even zero) in the winter months, when the water temperature is as low as 0°C (Fig. 5 a, b, c); (c) the levels of FSP show a good correlation to the vitellogenic condition of the oocytes; that is, if FSP is not present, there is no measurable oocyte growth
(Fig. 5~). Reabsorption of yolk from the oocytes (Fig. 5d) causes a major deviation in FSP measurements but causes no alteration in the timing of the next molt (compare Fig. 5 a, b, c with Fig. 5d).
DISCUSSION
Our findings indicate that the concentrations of the female-specific protein (FSP) in the hemolymph of gravid females is modulated by the molt cycle and by seasonal variations in the water temperature, and is generally a clear indicator of the vitellogenic state of the oocytes. Thus, these results are in agreement with many other studies that have reported the occurrence of female-specific hemolymph proteins in the crustacean orders Amphipoda, Isopoda, as well as in the Decapoda (as reviewed by Adiyodi and Adiyodi, 1970; Croisille et al., 1974). These investigations report that, as found in the present study, the concentrations of the FSP in the hemolymph is reported to increase as vitellogenesis in the oocytes becomes maximal. The FSP in the Crustacea appears completely analogous to the blood-borne vitellogenins that are synthesized in the livers of birds (Schjeide et (II., 1963; Heald and McLachlin, 1965), amphibians (Redshaw and Follett. 1971; Wallace and Bergink, 1974), and by the fat bodies of insects (Telfer, 1965; Engelmann, 1969, 1970). Vitellogenins are transported to the ovary where they become incorporated, by micropinocytosis, into the oocyte as yolk. In fact, in the vertebrates and the insects, virtually all the yolk protein in a mature oocyte is accumulated in this way; for example, Wallace et al. (1972) estimate that in the amphibian Xenopus luevis, 99’7” of the yolk plate-
Female-specific
10
protein
in the lobster
755
r (b)
A
s
ONDJFMAMJJAS Month
Fig. 5. Representative seasonal patterns of FSP levels in the serum of female lobsters sampled over an entire calendar year (August through the following September, inclusive). Each pattern graphed in (a) through (d) represents a single animal as followed throughout the year, and each point represents the measurement of the FSP concentration in a monthly serum sample. In (a) there was a molt (M) in one season, followed by a gradual increase in FSP levels and oviposition (0) the following summer. In (b) and (c) molt and oviposition both occurred in one summer, followed by only a molt the following summer. In (b), some vitellogenesis occurred (0, = 162), whereas in (c) no vitellogenesis occurred (0, = 91). Note that in (b), FSP was detectable in the spring of the second season, but this was not the case in (c). The typical pattern of reabsorption is shown in (d). Note that oviposition does not occur [compare with (b) and (c)l, and that high levels of FSP persist throughout the winter months, but that this has no effect on the timing of the molt in the second season-[compare (b) and (c) with (d)].
let proteins phosvitin and lipovitellin are heterologous in origin. The complete story with respect to the origin and fate of the crustacean FSP is less clear. Although FSP levels in the hemolymph of adult females seems to be a good index of vitellogenesis, with a maximum concentration occurring well prior to the maximum yolk accumulation in the oocytes, the central question as to the site of synthesis of FSP remains unclear. Two alternatives are proposed in the literature, and each alternative is supported by some, if incomplete, evidence. The first alternative is that the FSP is synthesized, as are vertebrate and insect vitellogenins, outside the oocyte, and is transported there via the hemolymph (Kerr, 1969; Wolin et al., 1973). The evidence for this point of view is considerable, although largely circumstantial, and is based on the consistent obser-
vations that the surface of the crustacean oocyte exhibits micropinocytotic pits and vesicles (Hinsch and Cone, 1969; Zerbib, 1973; Hinsch and Bennett, 1979; Schade and Shivers, 1980) and that the oocyte will take up exogenous proteins either from the hemolymph or from the external medium in vitro (Wolin et al., 1973: Zerbib, 1977; Schade and Shivers, 1980). It is not known if the micropinocytosis is a receptor-mediated process (Goldstein et a/., 1979), nor has the site of synthesis of FSP been satisfactorily established. The second alternative mechanism for the observed patterns of FSP in the hemolymph is that these proteins result only from reabsorption of yolk proteins that are synthesized in the oocyte. The evidence put forward for this alternative is both biochemical and ultrastructural. Lui et al. (1974) and Lui and O’Connor (1976) report that, in crayfish, each of the
756
EUWAKD H. BYAKU and DAVID E. AIKW
three subunits of hpovitelhn. the major yolk protein. are synthesized within the oocyte and conclude that extra-oocytic contributions to the total yolk protein seen in the mature oocyte are insignificant. Beams and Kessel (1963) and Carrion and Kessel (1972) concur, and describe the ultrastructural development of yolk granules in the extensive rough endoplasmic reticulum of the oocyte. Similarly, they conclude that but makes an micropinocytosis is present, insignificant contribution to the total yolk accumulated during vitellogenesis. Neither of these studies directly addresses the issue of the occurrence of female specific proteins present in the hemolymph. although Lui and O’Connor (1976) briefly state that these proteins could be accounted for by reabsorption of yolk, as has been described to occur in crayfish by Stephens (1952). Our observations are best explained by the first alternative, that is, that FSP is in transit to the oocyte from an external source. The levels of hemolymph FSP that we observed were always highest well prior to the maximum accumulation of yolk in the oocytes, and the levels dropped off markedly prior to oviposition. This pattern is consistent with the idea that the FSP represents an externally synthesized protein that logically is found at the highest level in hemolymph during the period when the oocytes are accumulating the maximum amounts of yolk. If reabsorption were accounting for hemolymph FSP, then one might expect the maximum levels when the oocytes are the maximum size, that is, just prior to oviposition. In any case, our observations show that reabsorption is an abrupt and isolated event in lobsters which occurs usually some time after an otherwise normal period of vitellogenesis. The reabsorbed yolk is present in the hemolymph at huge concentrations (e.g. greater than 20 mgiml) whereas FSP levels during vitellogenesis are considerably less (usually much less than 10 mgiml). In other words, reabsorption is an event distinct from normal vitellogenesis. Given the apparent contradiction in the evidence for yolk production in crustacean oocytes, it seems that the answer lies in a more complete assessment of the proportion of yolk that is synthesized in the oocyte. and the proportion that is imported from the hemolymph by micropinocytosis. There is clearly a component of the yolk that is synthesized inside the endoplasmic reticulum of the oocyte (Hinsch and Cone, 1969; Ganion and Kessel. 1972; Lui and O’Connor, 1976; Schade and Shivers, 1980), but it is equally clear that micropinocytosis is capable of taking up significant amounts of protein from the hemolymph (Schade and Shivers, 1980; Wolin et ul., 1973) and that micropinocytosis can be amplified by an appropriate hormonal signal (Hinsch and Bennett, 1979). Thus, that the total quantity of crustacean yolk protein is accumulated as a net result of both intraoocytic and extraoocytic syntheses seems certain; however, the relative contribution of each needs to be better defined. Our detailed observations on individual lobsters over two full reproductive seasons allowed us to establish unequivocally that molt and oviposition are alternated on a variable schedule. This alternation is not surprising in that both molt and oviposition
make major demands on metabolic reserves. But it is important to note that both molt and oviposition continue to occur in most reproductively mature crustaceans, thus separating the Crustacea from insects. to which they are often compared. Mature female insects do not molt, with the exception of the Thysanura (Rohdendorf and Watson. 1969). Thus the separation of molting and reproduction is temporally distinct. as are, presumably, whatever control mechanisms for the two processes that are at work. In the Crustacea, on the other hand, the control mechanisnls for molting and reproduction are more closely interwoven, and thus more difficult to analyze in isolation. That there is some kind of hierarchy between the two processes seems axiomatic, since an inopportune molt, that is. immediately after oviposition, would result in a loss of eggs on the cast exoskeleton. Our results indicate that in the period from late premolt (stages D, to D,) to postmolt (stage B), the hemolymph FSP levels are low to zero in non-ovigerous females, although the ovaries in these same animals are up to stage III in development. Thus, it appears that premolt events have suppressed vitellogenesis, as judged by the hemolymph titer of FSP. In addition, late premolt lobsters with mature ovaries will reabsorb yolk rather than oviposit. These two observations, taken together, lead to the conclusion that premolt events take precedence over, and may disrupt events leading to normal oviposition, A similar hierarchy exists in the Thysanuran, Lepisno&s inquilirzus, one of this order of exceptional insects that continues to molt and grow after reaching sexual maturity. In this species, the molting cycle overrides reproductive activity, and artificially induced molting will cause reabsorption of yolk from the oocytes of mature ovaries (Rhodendorf and Watson, 1969). It seems that premolt events and oviposition are incompatible. A similar conclusion can be derived from an experiment in which lobsters (Homarus mwricuntu) were subjected to 6 summer months in an artificially imposed B-month year (Aiken and Waddy, 1976). The mature ovary was reabsorbed only when the molt and reproductive cycles coincided such that molt would occur shortly after oviposition. In other words, it appears that the physiology of late premolt cannot include the final vitellogenesis (at least past stage III) and other preparations leading up to oviposition. In conclusion, we feel that the data on hemolymph FSP that are presented here provide a reliable index of vitellogenesis, and, could become a useful tool in further studies of lobster reproductive physiology. Acknor,led~emetlts-We thank Orysia Dawydiak for help in the preparation of figures. This investi~asion was supported by the Fisheries Research Board of Canada, and the National Canada.
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