Reproduction and lipids in the sub-Antarctic limpet Nacella (Patinigera) macquariensis Finlay, 1927

33

J. exp. mar. Biol. Ecol., 1982, Vol. 56, pp. 3348

Elsevier Biomedical Press

REPRODUCTION NACELLA

AND LIPIDS IN THE SUBANTARCTIC (PATiNIGERA)

MACQUARIENSLS

LIMPET

Finlay, 1927

R. D. SIMPSON Department of Zoology, University of New England, Armidale, N.S. W., 2351 Ausrrulia

Abstract: The reproductive cycle of the sub-Antarctic limpet, Nacella (Patinigera) macqwriensis Finlay, 1927 is described from monthly gonad indices and slide smears of gonadaf tissue. Two populations were sampled, from the eulittoral zone and at ~4 m depth into the sublittoral zone. Gametogenesis started 2 months earlier in the eulittoral zone population; the breeding period, however, was sufficiently long to overlap in the two populations. This phase difference in reproductive cycles is discussed with respect to possible genetic divergence and correlation between timing within the reproductive cycle and environmental factors. Lipid was a prominent component of both the digestive gland and the gonad, but not of the foot. Changes in lipid levels during the reproductive cycle indicated that the digestive gland stored lipids which were transferred to the gonad during gametogenesis.

INTRODUCTION

In latitudinal comparisons of the reproduction of littoral invertebrates, the subAntarctic region is an important link in the chain. There is a wide oceanic gap between temperate regions and the Antarctic continent and, apart from the southern tip of South America, sub-Antarctic shore biota are restricted to a few scattered islands (Fig. 1). Invertebrates were collected from the sub-Antarctic Macquarie Island (54”38’S : 158”53’E) to investigate their reproductive biology (Simpson, 1977, 1981). The patellid limpet ~acefZa (~atinigera) macq~riensi~ Finlay, 1927 formed part of these collections. N. (P.) macquariensis is endemic to Macquarie Island. It inhabits rocky surfaces and is abundant in the eulittoral zone and at depths down to 10 m in the sublittoral zone (Simpson, 1976a). It has also been dredged from a depth of 60 m (Tomlin, 1948). The wide vertical range of N. (P.) macq~ar~ens~s permitted a comparison of reproduction, particularly the timing of the reproductive cycle, between populations from two very different habitats - the eulittoral and shallow sublittoral zones. Unfortunately, there were no facilities for repeated collections from deeper waters. The lipid levels in tissues of N. (P.) macquariensis were determined to assess the importance of lipid as a storage material in relation to the reproductive cycle.

~22-O98~/82/~00-~0~$02.75

Q 1982 Elsevier Biomedical Press

34

R. D. SIMPSON

Fig. 1. Location of Macquarie and other islands in the sub-Antarctic and Antarctic regions.

MATERIALS AND METHODS REPRODUCTION

Many methods have been used to investigate the reproductive cycles of marine invertebrates (Giese, 1959a; Giese & Pearse, 1974). A number of criteria can be used to indicate reproductive activity such as spawning, number of larvae, gametogenesis, copulatory activity, appearance of eggs or broods, and relative sizes of gonads. The ratio of gonad size to body size has been used extensively and is applicable to animals where an immature or spent gonad is small and a ripe gonad large. This gonad index has been calculated in several ways, for example: volume of gonad to wet weight of the animal (Lasker & Giese, 1954; Farmanfarmaian et af., 1958; Giese et al., 1959); weight of gonad to weight of animal (Kowalski, 1955; Pear-se & Giese, 1966a); volume of gonad to volume of animal (Moore, 1934); gonad area to shell length of abalones (Boolootian et al., 1962). Whatever the method, plotting the gonad index against time gives a graphical representation of the average reproductive state of the population. Gonad indices together with microscopical examinations of slide smears of fresh gonad were used to determine the reproductive cycle of .N. (P.) ~?ae~ti~~~ei?,~~~.

REPRODUCTION

OF A SUB-ANTARCTIC

LIMPET

35

Collections were made monthly from two habitats: (1) the eulittoral zone (more specifically, the Lower Red Zone of the eulittoral zone - see Simpson, 1976b), and (2) from a depth of x4 m. This latter habitat is subsequently referred to as the diving station. The depth measurement was taken from the top of the sublittoral zone as set by Simpson (1976b). It was obviously important to determine whether limpets moved between these sampling areas during the course of the study. Marked individuals in the eulittoral zone tended to remain in a small area ( < 1 m2) and movements >3 m were rare, usually being horizontal movements within the same zone. No limpets from diving stations were marked for studies of movement, although the healthy and extensive encrustation of coralline algae over the shells of specimens indicated that they had been continuously submerged at some depth. Limpets from the top of the sublittoral zone had a sparser covering of coralline algae on their shells and limpets from rock surfaces in the eulittoral zone usually had no covering. All limpets collected from the eulittoral zone for this study had no coralline algal cover on the shells. Limpets from rock-pools in the eulittoral zone had some degree of coralline algal cover, depending on the depth of the pool, but limpets in rock-pools were very fixed in such locations. A similar relationship between coralline algal cover on the shell and habitat was noted for the Antarctic limpet N. (P.) concinna (= Patinigera polaris) by Walker (1972). Nacella (P.) macquariensis reaches a maximum length of 60-70 mm. Animals of 35-50 mm in length were used in this study. Each limpet was blotted dry to remove excess water and then dissected to obtain weights of whole body, shell, and gonad. Gonad index was calculated as: 100 x wet wt gonad/wet wt total soft parts. This avoided errors caused by differences in shell morphology and degree of encrustation. Gonad indices for the first ten males and the tirst ten females collected from each of the habitats were determined monthly for 1 yr except that limpets were not collected at the diving station for three of the months. Usually N. (P.) macquariensis cannot be sexed from external characters. At the peak of gonadal maturity, however, males had a pale, longitudinal streak down the centre of the foot, apparently the result of pressure from the enlarged testis. For limpets not in the regressed stage sex was easily determined from the colour of gonads: testes were light yellow while ovaries were brownish orange. Spent gonads of males and females in the regressed stage had a similar yellow colour but sex could still be determined by microscopical examination. Artificial fertilization was studied in N. (P.) macquariensis to establish the time taken to reach the trochophore stage and whether fertilization would occur between limpet populations from the two collection localities. For female limpets, a ripe ovary was removed and then broken up in a Petri dish containing filtered sea water at 7-9 “C. The eggs were then collected by filtering through 118 micron gauze and added to a large beaker containing 5 1 of filtered sea water constantly stirred at a moderate speed. For male limpets, the testis was removed and placed in a Petri

36

R. D. SIMPSON

dish containing a small amount of filtered sea water. An incision was made in the testis and a suspension was allowed to form for 15 min. Then 10 drops of the suspension were added to the eggs. Samples were removed at regular intervals and checked for fertilization and development. LIPIDS

Lipid measurements were undertaken monthly on limpets from only the eulittoral zone. Lipid concentrations as % dry wt were determined for gonad, digestive gland, and foot. For each sample the total lipid content for testis and ovary was calculated by standardizing the data to that for a limpet of 8 g soft body wt and with the mean gonad index for each month. Indices for the comparative size of the digestive gland (commonly a corollary of studies of biochemical levels) were not obtained. This gland was very soft and was wound around the intestine, making it difficult to measure the weight or volume of the gland accurately. A suitable section of digestive gland and small pieces from the middle of the foot muscle were removed for lipid determinations. Lipids were measured in a monthly sample of three males and three females. To offset the small sample size, the three specimens with gonad indices closest to the monthly average were taken from the set of limpets investigated for reproductive studies, Thus, the animals were standardized to the reproductive cycle. Other studies (Simpson, unpubl.) showed that the feeding activity of limpets in the eulittoral zone was determined by their state of submersion. The monthly collections were always made at low tide, ensuring that the limpets were at the same stage of their feeding cycle at each collection. The limpets were dissected within 24 h of collection. The tissue samples were weighed and then dried over sulphuric acid in a vacuum desiccator for = 24 h. Lipid was extracted from ground tissue by Soxhlet reflux with diethyl ether. The tissue samples were x 100 mg when possible and 10 to 15 ml of diethyl ether were used for extracting from such a weight over a Z-h period. As indicated by Giese (1966, 1967) and Morris & Cuikin (1976), a Soxhlet extraction using mild heat with a non-polar solvent (e.g. diethyl ether) fails to extract structural and more tightly bound lipids completely; such a method extracts mostly stored lipids occurring as free globuIes in cells whereas polar solvents (e.g. chloroform-methanol) are required for the complete extraction of lipids. The aim of the present study was to follow changes and possible re-distribution of loosely held stored lipids within the animal and, consequently, the Soxhlet method was regarded as adequate. Some molluscs, e.g. bivalves and scaphopods (Masumoto et al., 1934; Greenfield, 1953; Srinivasan, 1963; Ansell ef al., 1964; Giese, 1966; Ansell, 1975) store large amounts of glycogen while others e.g. chitons and limpets (Barry & h’lunday, 1959; Giese & Araki, 1962; Blackmore, 1969b) do not. Giese (1966) suggested that

REPRODU~ION

OF A SUB-ANTARCTIC

37

LIMPET

molluscs are probably of two kinds. Some have a prominent glycogen economy with a correspondingly lesser storage of lipid; others have a prominent lipid economy but store little glycogen. The studies by Barry & Munday (1959) and Blackmore (1969b) showed that Patella vulgara belonged to the second category. This suggests that the major food reserve in Nacella (P.) macquariensis (also a patellid limpet) would be lipid. Although it would have been interesting to compare the seasonal levels of glycogen with the lipid levels in tissues of N. (P.) macquariensis, investigation was confined to the expected major food reserve. RESULTS REPRODUCTION

The mean gonad indices of males and females from the two collecting locations over 12 months are outlined in Fig. 2. These graphs clearly show an annual reproductive cycle and a phase difference in the reproductive cycles of limpets from the two habitats. Coefficients of variation about the mean gonad indices were high 301

__ AMJJASONOJFM MONTHS Fig. 2. ~onth1~

mean

gonad indices for Ikeella n = females; diving station

(P.) ~~cq~~~~~~j~~ eulittoral - 0 = males, x = females.

zone

-

A = males,

during the period of gonadal regression in the cycle, i.e., 32-73”/, for the eulittoral sample and 888105% for the diving station sample. For the remainder of the cycle, coefficients of variation were lower - 13-32x for the eulittoral limpets and 14-3504) for the diving station limpets, the lowest variabilities being at, or just before, peak maturation of the gonads, Males had significantly larger gonad indices than females in all samples except for months when gonads had regressed, i.e., in May and July (eulittoral) and August and September (diving station) (P < 0.05, Student’s t-test). A marginal difference occurred in the January figures for the diving station sample (0.10 > P 2-0.05).

R. D. SIMPSON

38

The progressive

stages in the reproductive

cycles can be recognized

in Fig. 2.

Male and female cycles at each site were harmonious. Limpets in the eulittoral zone had regressed gonads from May to July. Development of the gonads commenced in August and peak maturation drop in gonad index indicated

was reached in November. The following sharp the first spawning. There was a second peak in

January, designating a second burst of gonadal development. The decline of the curve after the January peak indicated a second spawning. Limpets from diving stations had a regressed stage from about July to September. Development of the gonads commenced in October and peak maturation was reached in January. There was no sample from the diving station in March so there was no direct record of a second peak of gonadal development to show that the breeding season was of equivalent length to that for the eulittoral limpets. Indirectly, the gonad index values in April for the diving station limpets suggested that this would be the case; that is, if the cycle was in the same synchrony in the previous year, the April values appear to be too high for an uninterrupted drop from the February values. The interpretation of reproductive states from the gonad index cycles was supported by microscopical examinations of whole gonads and smears. Oocytes and spermatocytes in early developmental stages were noted in the gonads of limpets from the eulittoral zone in August-September when the gonads of limpets from the diving stations were in a regressed condition. In October-November, the gonads of the eulittoral zone limpets were approaching their peak maturation. In October, the eggs were individually visible to the naked eye but were not uniform in size and were still tightly packed and held by the germinal tissue. In November, the whole ovary was a mass of eggs of the same size, each egg enclosed by a chorion ; those on the surface were quite loose. In males, fully developed spermatozoa were present in October but there was still a high proportion of spermatids. In November, the testes were easily ruptured, releasing a milky fluid with a high proportion of spermatozoa. The same sequence of predominant gametogenic stages for limpets from the diving stations was two months behind. In December, eulittoral zone limpets had obviously spawned. Eggs on the surface of the ovary were extremely loose with noticeable gaps amongst them. Testes often had patches, darker in colour, and lacking in the tension within the tissue in other parts of the testis. January saw the second peak for the eulittoral zone limpets when gonads closely resembled those of November. Early stages of gametogenesis were present in November and January but formed a very small part of the gonadal tissue. The February sample showed that spawning had again recently occurred, using the criteria mentioned previously. Again, corresponding stages in the diving station limpets followed 2 months behind. In ovaries of eulittoral limpets in April and of diving station limpets in May-June, the eggs regressed to an irregular shape indicating degeneration and resorption. Gametes of limpets from both the eulittoral zone and the diving station were successfully cross-fertilized. Fertilization and development studies on limpets from

REPRODUCTION

OF A SUB-ANTARCTIC

LIMPET

39

the eulittoral zone showed that first cleavage stages occurred 6 h after introducing spermatozoa to the eggs, and fully developed trochophores after 50 h. Of a total of 504 limpets dissected, with a length of 35 mm or greater, 243 were females and 259 were males, giving a ratio not signi~cantly different from 50: 50. The remaining two were hermaphroditic with separate sections of both male and female gonadal tissue. LIPIDS

Figs. 3 and 4 show monthly lipid concentrations and contents for the gonad and concentrations for the digestive gland and foot muscle of male and female N. (P.) macquariensis, each point representing the mean value for three individuals, The

MONTHS Fig. 3. Monthly mean lipid levels in male Nucellu (P.) macquariensis from the eulittoral zone: lipid concentrations (% dry wt) - 0 = in digestive gland, m = in testis, A = in foot muscle; total lipid content (mg) - x = for testis of a “standard” limpet of 8 g soft body weight and with the mean gonad index at each month.

variability in lipid concentrations at each month was small and this was presumably due to the samples comprising those animals with gonad indices closest to the mean value. The analyses indicated that lipid is a prominent component in the gonad and the digestive gland. Over the year, mean monthly lipid concentrations ranged from 8.6-25.6% of the dry wt for ovaries, and 6.7-14.2x for testes. In the digestive gland, the range was from 7.2-19.2x of the dry wt in females, and 5.8-18.3x in males. In foot muscle the lipid concentration was very low, the range of averages being from 2.6-M% with no difference apparent between the sexes. During maturation of the gonads, the lipid concentration increased giving an additional factor of increase to the total lipid content of the gonads as they enlarged. At the peak of maturation, in the ripe condition, the lipid content of the testes was far below that of the ovaries. Further changes in the monthly lipid contents of the gonads were in accord with events in the reproductive cycle. That is, there was a decrease in lipid content in December, after spawning, and the lipid content again

R. D. SIMPSON

40

rose as the gonads built up to a ripe condition in January. spawning, the lipid content declined as the gonads regressed.

After the second

-12op -100; .80 FJ 6 -60 * F LO i .20 & 5 “AMJJASONDJFM” MONTHS

Fig. 4. Monthly mean lipid levels in female Nacella (P.) macquariensis from the eulittoral zone: lipid concentrations (% dry wt) - 0 = in digestive gland, n = in ovary, A = in foot muscle; total lipid content (mg) - x = for ovary of a “standard” limpet of 8 g soft body weight and with the mean gonad index at each month.

A reciprocal relationship between the lipid levels of the gonad and the lipid levels of the digestive gland of the female and, to a lesser extent, the male is evident in Figs. 3 and 4. There was no marked change or pattern in the lipid levels of the foot muscle. DISCUSSION REPRODUCTION

The reproduction of patellid limpets has now been studied in tropical, temperate, and Antarctic regions although the majority of studies have been on temperate species (e.g. Orton et al., 1956; Anderson, 1962; Blackmore, 1969a; Balaparameswara Rao, 1973; Branch, 1974; Picken, 1980). In all cases, these limpets release gametes for external fertilization and subsequent larval development. Most archaeogastropods reproduce this way, which is regarded as a primitive trait. Patellidae and Haliotidae are the only families that do not include species with a protected form of larval development (Webber, 1977). This is a curious phylogenetic restriction for the Patellidae. The patellid limpets are regarded as primitive molluscs and have similar features to another group of molluscs that are also regarded as primitive in structure - the Polyplacophora (chitons). Neither of these groups have secondary sexual anatomy for storage of gametes, glandular products or copulation. Both have a pallial groove, housing a row of gills, along each side of a broad foot. Some species of chitons

REPRODUCTION

OF A SUB-ANTARCTIC

LIMPET

41

have used this groove for brooding young (Simpson, 1977; Pearse, 1979) but the patellid limpets have not. This difference may be due to the lack of mechanisms in patellid limpets to keep the eggs clustered together. Such mechanisms (mucus and projections from the hull of the egg) have been noted in chitons and this has been suggested as an intermediate step in the development of brooding in chitons (Dell, 1962; Pearse, 1979). The developmental time to trochophore stage for N. (P.) macquariensis was longer than that reported for the northern temperate limpet Fate&z vulgata (Dodd, 1957) and far shorter than that for the Antarctic limpet ~aceZla (Pu~in~g~r~j continua (Shabica, 1976). As Dodd (1957) has stressed, temperature greatly affects devefopmental times. The temperature of the sea water used for the experimental rearing of trochophores of N. (P.) macquariensis was slightly above the sea temperature and, therefore, the developmental times recorded may be slightly shorter than in nature. It is possible that patellid limpets may have no temperature adaptation with respect to larval development - the colder the climate, the longer the larval development. As Picken (1980) suggested, the larvae in colder climates may, however, adopt demersal rather than planktonic development; this could serve as a means of reducing mortality. An unclear feature of patellid limpets is whether the veliger larvae feed while in the plankton. Anderson (1962) and Webber (1977) cite the veliger of Pate& vuigata as being planktotrophic, that is, actively feeding during planktonic life. Two of the sources for this citation (Smith, 1935; Crofts, 1955) simply, however, state that the larval stages of P. vulgata are planktonic. (Webber’s further reference to “Dodd (1956)” on this point presumably should have been “Dodd (1957)“.) Only Dodd (1957) claimed success in the feeding of P. vulgata veligers by noting that metamorphosis was obtained with a suspension of a particular species of Chrysophyceae algae and not when other foods were added; even then, the food was added when t!-%larva was adopting a sedentary habit, suggesting demersal feeding, When would the larvae of patellid limpets actually feed? The alimentary system does not appear to be developed enough for the assimilation of food until the post-torsional phase and by then the larvae are alternating between planktonic and sedentary habits before finally settling. Also, as in other archaeogastropods, the velum is a small inconspicuous organ, which suggests that it is not used for feeding. This would be in accord with the short larval life experienced by archaeogastropods relative to the longer larval life of meso- and neogastropods that have planktotrophic veligers with well-developed vela (Webber, 1977). Whether the larva of a particular species feeds in the plankton is obviously basic to attempts to assess the importance of the correlation between available phytoplankton food and larval occurrence in the sea, as has been recorded for feeding vehgers (Underwood, 1974). Phytop~ankton in the inshore waters at Macquarie Island, as indicated by chlorophyll, were at high levels in December and January

42

R. D. SIMPSON

(Simpson, 19’76b). This was a relatively short period of elevated phytoplankton levels; it is typical for high latitudes and has been put forward as a possible disadvantage for development via a feeding planktonic larva (Mileikovsky, 1971). The reproductive cycles of Nucella (P.) macquariensis show that both populations could have generated planktonic larvae during December and January. It is, however, unlikely that N. (P.) rnacq~~r~en~j~has a planktotrophic larva and hence the correlation most probably has no causal connection. Studies of reproductive cycles of marine invertebrates have shown that times for peak maturation, spawning, and resting stages can differ markedly in successive years, e.g. Farmanfarmaian et aE. (1958), Giese (1959b). Orton et al. (1956) showed this occurred in the limpet Patella vulgata; they also found that the time involved in maturation and spawning in the same year differed with the locality (within England and Scotland). Ballantine {cited by Morton & Mitler, 1968, pp. 324-325) found that low tidal populations of P. vulgata have earlier seasonal maturation of the gonads than high tidal populations. In contrast, Blackmore (1969a) found no difference in time of reaching peak maturation in P. v~~gata situated at different levels in the littoral zone. Such findings indicate that events within the reproductive cycle are susceptible to environmental influence. Giese (1959a) suggests that the precise pattern of a reproductive cycle depends upon the external factors which entrain or time the endogenous drive. Light, salinity, temperature, and food are possible mechanisms for controlling marine invertebrate reproductive cycles. The phase difference in the reproductive cycles of Nacella (P.) macquariensis from the two locations can be used to suggest possible mechanisms controlling the cycle by noting those factors that are not the same for the two populations. The diving station limpets were still subjected to good light penetration and, therefore, photoperiodicity could not have acted as a single determining factor on the reproductive cycle pattern. Diving station limpets encountered constant salinity values. Salinity was more variable for limpets in the eulittoral zone but the variation was small and sporadic and not likely to follow any seasonal trend under the influence of Macquarie Island’s equable climate (Simpson, 1976b). The diets of limpets in the two habitats were different. In the eulittoral zone, algal film and diatoms were grazed, while at the diving station the staple diet was coralline algae. There was, however, no obvious reason for these different diets to have an influence on the reproductive cycles. What is more likely to have an effect is availability of food. If there were insufficient food intake, there might not be enough energy for the animal to undertake gametogenesis successfully. Sutherland (1970) showed that the acmaeid limpet, Acmaea scabra had different reproductive patterns at different shore levels. Higher limpets had a distinct seasonal pattern while lower limpets had more constant reproductive activity. Sutherland attributed this to seasonal and constant availability of food in the higher and lower zones, respectively. Lawrence (1976), in reviewing relationships between patterns of storage of lipids in marine invertebrates and their reproductive cycles, suggested that there

REPRODUCTION

OF A SUB-ANTARCTIC

LIMPET

43

may be an obligation on the animal to store reserves for reproduction if there is a seasonality in food intake. In considering these hypotheses with respect to Nuceflu (P.) macquariensis in the two habitats, relevant findings were : (a) both populations had a seasonal reproductive cycle; a constant food supply for submerged limpets did not change their reproduction to one of constant output; (b) sublittoral limpets had more available time for feeding but the eulittoral limpets had much longer feeding periods when they were able to feed, that is during high tides (Simpson, unpubi.); and (c) the eulittoral limpets stored lipids in the digestive gland in the winter when it would be expected that there would be less food available on the rock surfaces. These findings lead to the hypothesis that N. (P.) ~ucq~arj~~~sis first has a commitment to a synchronized, seasonal reproductive cycle. It has a storage mechanism that is used in harmony with the reproductive cycle, and most likely for other energetic requirements as well. It then feeds for the required length of time to obtain the necessary food intake to support such an energy budget. In both habitats there was apparently enough food during the study period. At other times, shortage of food may not allow enough time for the required cropping, especially in the eulittoral zone. There were some correlations between temperature changes (Fig. 5) and the reproductive cycle of limpets in the eulittoral zone. The peak maturation of diving

AMJJASONDJFM 1968

Fig. 5. Monthly mean maximum.

MONTHS

1969

air and sea temperatures at Macquarie Island during the collecting period: air - 0 = x = mean minimum, solid vertical lines = range; sea - A = mean, vertical dotted lines = range.

station limpets coincided with peak sea temperature, but there was no temperature rise to correspond with the onset of development. The increase in temperature from insolation would affect limpets in the eulittoral zone and there was a sharp increase

R. D. SIMPSON

44

in average daily sunshine in August (Fig. 6). Air and sea temperatures both rose sharply in August and this rise, coupled with the sharp increase in sunshine, may

1968

Fig. 6. Mean daily sunshine

MONTHS

(h) at Macquarie

1969

Island for each month

during

the collecting

period.

have caused the earlier start to the development of gonads in the eulittoral population. The heavy December spawning for eulittoral zone limpets occurred in a month when there was a marked reduction in wave action in the area (east coast) from which the samples were taken (Simpson, 1976b). Correlations have been noted between the spawning of molluscs and both rough and calm sea conditions (Giese, 1959a). Calm seas on the shores of Macquarie Island would perhaps increase the possibility of the meeting of gametes and hence fertilization of eggs in the littoral zone. If the difference in timing of the reproductive cycle is accentuated to the point that spawning no longer coincides in two populations (perhaps deeper limpets may be even further out of phase), it is possible that speciation through reproductive isolation could result. Endler (1973) has proposed that speciation is theoretically possible even with some gene flow between populations along an environmental gradient. The limpet’s shedding of gametes into the sea, however. would facilitate gene flow. Consequently, for speciation to occur, there would need to be some restriction on the gene flow such as differences in spawning times (as above) or spawning

during

calm weather

resulting

in restricted

dispersal

of the gametes.

LIPIDS

Studies on the lipid levels in the body components of molluscs and other marine invertebrates have been reviewed by Giese (1966, 1969) and Lawrence (1976). Of the possible effects on the patterns of lipid storage in marine invertebrates noted by Lawrence (1976) three could apply to N. (P.) macquariensis: the physical environment, nutritional state of the animal, and the annual reproductive cycle.

REPRODUCTION

There have been suggestions

OF A SUB-ANTARCTIC

45

LIMPET

that colder water animals,

have higher lipid levels than those at higher temperatures has not been borne out for benthic invertebrates (Giese,

particularly

zooplankton,

(Lawrence, 1976); this 1966; Pearse & Giese,

1966b). For N. (P.) macquariensis, the lipid levels were within ranges of values obtained elsewhere for gastropods and other molluscs - most data coming from temperate regions (Giese, 1966, 1969; Blackmore, 1969b; Barnes & Blackstock, 1973). Any such comparisons are, however, still far too coarse. There may yet be subtle important differences for invertebrates from different latitudes when variations from different methods of lipid measurement, different lipid forms, and different physiological states of the animals are resolved. The lipid levels in the body components of marine invertebrates have been noted to vary considerably, and nutritional state is one of the most likely sources of variation in field studies (Giese, 1966; Lawrence, 1976). Consequently, it could be argued that any variation in the digestive gland of N. (P.) macquariensis is caused by a seasonal change in the amount of food ingested. As already discussed, lipid increased in the digestive gland in the winter when less food would be expected to be available on the rock surfaces. The guts of limpets also contained food at all seasons, although no quantitative comparisons between seasons were made. It would be hard to reconcile the 7-month period of development and spawning and the corresponding depressed lipid concentration of the digestive gland with a sustained period of decreased food intake. Changes in lipid levels in gonads and other organs and tissues of marine invertebrates have been studied to determine both (a) the role of lipids in the energy requirements of the reproductive cycle and (b) possible storage sites and subsequent transfer from these sites to the gonads. Reciprocal relationships of the size of an organ and/or its lipid level with those of the gonads constitute the bulk of the evidence for transfer of lipid from some somatic storage to the gonads during gonadal development (Giese, 1969; Lawrence, 1976). For N. (P.) macquuriensis, the reciprocal relationship between lipid levels of the gonad and of the digestive gland during the reproductive cycle suggested that the increase in lipid in the gonads during maturation occurred, in part, at the expense of that stored in the digestive gland. More direct evidence of such a transfer of lipid in other marine

invertebrates

has been obtained

(Lawrence,

1976).

During gonadal development, much more lipid accumulated in the ovary than in the testis (almost double the amount in the ripe condition). This difference is understandable on the premise that more lipid would be required for storage in the eggs, as shown in a chiton by Lawrence & Giese (1969). Yet, depletion in lipid concentration of the digestive gland of male N. (P.) macquarirnsis at this time was even greater than that of females. This appears to be contrary to an hypothesis that lipid reserves of the digestive gland are utilized for gonadal growth. Materials from the digestive gland, however, could still have been used in the manufacture of lipids which would not have been fully extracted by the technique employed, for instance phospholipids which have been shown to form a more prominent com-

46

R. D. SIMPSON

ponent

in spermatozoa

of marine

For the foot, both the lowness and the lack of seasonal

variation

invertebrates (indicative

(Lawrence

& Giese,

of only structural

in lipid concentrations,

1969).

lipids,

indicated

Giese,

1966)

that the foot

muscle was not a storage site for lipid reserves. This contrasted with findings for the chiton, Katharina tunicata, in which the foot had considerably higher lipid levels which fell during

gonadal

growth

and starvation

(Lawrence

& Giese,

1969).

ACKNOWLEDGEMENTS

The study was supported by the Antarctic Division, Department of Science and the Environment, Australian Government and later by internal research grant from the University of New England. I am grateful to Dr. R. Carrick for his advice and encouragement during the study. Mr. T. Gadd and Mr. S. Harris ably assisted in the field and the laboratory. Thanks are extended to Dr. D. Woodland for useful comments on the manuscript.

REFERENCES

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