UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Relationships between seasonal biochemical changes and the reproductive cycle of the intertidal gastropod… Lambert, Philip 1970

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1970_A6_7 L34.pdf [ 4.18MB ]
Metadata
JSON: 831-1.0093364.json
JSON-LD: 831-1.0093364-ld.json
RDF/XML (Pretty): 831-1.0093364-rdf.xml
RDF/JSON: 831-1.0093364-rdf.json
Turtle: 831-1.0093364-turtle.txt
N-Triples: 831-1.0093364-rdf-ntriples.txt
Original Record: 831-1.0093364-source.json
Full Text
831-1.0093364-fulltext.txt
Citation
831-1.0093364.ris

Full Text

RELATIONSHIPS BETWEEN SEASONAL BIOCHEMICAL CHANGES AND THE REPRODUCTIVE CYCLE OF THE INTERTIDAL GASTROPOD THAIS  LAMELLOSA GMELIN (GASTROPODA, PROSOBRANCHIA) . by PHILIP LAMBERT B.Sc, University of V i c t o r i a , 1967 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Zoology We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA June, 1970 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study . I f u r t h e r agree t h a t permiss ion f o r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood that copying o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l ga in s h a l l not be a l lowed without my w r i t t e n p e r m i s s i o n . Department of The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada Date JULS 3L /<?70 i ABSTRACT The seasonal v a r i a t i o n i n the major biochemical constituents of T. lamellosa Gmelin have been studied i n r e l a t i o n to the reproductive cy c l e . Digestive gland, foot muscle and gonad were analysed f o r p r o t e i n , glycogen, l i p i d and ash over a period of one year. In addition to biochemical analyses, h i s t o l o g i c a l sections of digestive gland and gonad were made throughout the same period. H i s t o l o g i c a l data supplied information on feeding and gamete maturation. Two major periods of feeding a c t i v i t y occurred i n A p r i l and August. Garnetogenesis began i n l a t e summer and the peak spawning period was i n March. Glycogen i s at a maximum i n the digestive gland at times of maximum feeding, but food i s stored i n the digestive gland i n the form of l i p i d . Stored l i p i d i s u t i l i z e d by the animal during the winter. Glycogen i s at a low l e v e l i n a l l t i s s u e s and appears to be used p r i m a r i l y f o r l i p i d and yolk synthesis. The foot muscle does not store e i t h e r l i p i d or glycogen to any appreciable extent. Under normal f i e l d conditions during the winter, the d i g e s t i v e gland index decreases as reserves are u t i l i z e d , while the gonad siz e i s maintained u n t i l spawning. Animals which are maintained through the same period under a r t i f i c i a l summer conditions, show no l o s s i n the digestive gland index, but a decrease i n the s i z e of the gonad. None of the oogonia reach maturity and the mature oocytes are resorbed. The starved animals resorb more material from the gonad than fed animals. The possible r o l e of environmental factors i n c o n t r o l l i n g reproduction i s discussed. i i TABLE OF CONTENTS PAGE INTRODUCTION 1 MATERIAL AND METHODS 5 C o l l e c t i n g Area 5 C o l l e c t i o n of Samples 5 Histology 7 Chemical Analyses 9 L i p i d 9 Polysaccharide 10 Protein 12 Controlled Conditions 13 S t a t i s t i c a l Methods 15 RESULTS 16 F i e l d Observations 16 Environmental parameters 16 Reproductive a c t i v i t i e s 18 Internal Morphology 18 Gross anatomy 18 H i s t o l o g i c a l observations 21 Determination of reproductive cycle by oocyte diameter 21 Parasites 24 Histology of digestive gland 24 Per Cent Dry Weight 26 Gonad Index 26 Digestive Gland Index 29 Constituent Levels 29 Digestive gland 29 Protein l e v e l s 32 Glycogen and ash l e v e l s 32 Experimental Results 35 DISCUSSION 42 Reproduction and Development 42 Seasonal Biochemical Changes 46 Biochemical constituents of oocytes 46 Feeding 47 Explanation of terms 49 Constituents of digestive gland 49 Glycogen i n the digestive gland 51 Role of glycogen i n reproduction 52 Constituents of the ovary 54 Polysaccharide as a storage product 55 L i p i d l e v e l s i n T. lamellosa 56 Constituents of foot muscle 57 Conclusions about nut r i e n t t r a n s f e r 58 Conclusions from experimental observations 59 E f f e c t of Environmental Factors on Gonad Development 61 Lack of food 61 Temperature 62 i i i PAGE Day length 63 S a l i n i t y 64 Suggestions f o r Further Study " 64 SUMMARY 66 LITERATURE CITED 68 Iv < LIST OF TABLES TABLE PAGE 1 Subjective c l a s s i f i c a t i o n of digestive gland colour. 22 2 Subjective c l a s s i f i c a t i o n of numbers of granules i n the storage c e l l s of the dige s t i v e gland. 25 3 Summary of data f o r two experimental groups of T. lamellosa maintained i n summer conditions from August 31, 1968 to January 6, 1969. Data f o r females only. Numbers i n brackets are 1 S.E. c^ = f i e l d sample at s t a r t of experiment (Aug. 31). = f i e l d sample at end of experiment (Jan. 6). f = fed experimental group sampled at conclusion, s = starved experimental group sampled at conclusion. 38 V LIST OF FIGURES FIGURE PAGE 1 Map of research area showing main sources of run-off. 6 2 Mean monthly s a l i n i t y two feet below surface at Brockton Point. V e r t i c a l l i n e s indicate the range. The f i r s t and l a s t points do not represent complete months. 14 3 T o t a l p r e c i p i t a t i o n f o r each 15 day period at the Port Meteorological O f f i c e , Vancouver. Calculated from tables i n the Annual Meteorological Summary (Canada D.O.T. Meteorological Branch, 1969, 1970). 17 4 Mean monthly water temperature two feet below surface at Brockton Point. V e r t i c a l l i n e s indicate the range. 19 5 The points represent the average day-light hours per day f o r each month, calculated from tables i n North-c o t t (1968, 1969). 20 6 Oocyte diameter frequency polygons f o r each monthly c o l l e c t i o n of T. lamellosa. Each polygon represents a t o t a l of 125 oocytes and the points i n d i c a t e the per cent of the t o t a l i n each s i z e c l a s s . The abscissa i s i n per cent u n i t s . 23 7 Per cent of dry material i n a unit weight of f r e s h t i s s u e of T. lamellosa. The v e r t i c a l l i n e s i n d i c a t e + 1 S.E. Data f o r females only. 27 8 Gonad indices of male and female T. lamellosa as a function of month of year. Closed c i r c l e s ( • ) represent the mean ovary index and open c i r c l e s ( o ) represent i n d i v i d u a l males. V e r t i c a l l i n e s indicate + 1 S.E. 28 9 Digestive gland indices of male and female T. lamellosa as a function of month of year. Closed c i r c l e s ( • ) represent the mean of 8 females and the open c i r c l e s ( o ) represent i n d i v i d u a l males. V e r t i c a l l i n e s i n -dicate + 1 S.E. 30 10 Levels of protein, l i p i d and glycogen i n the digestive gland of T. lamellosa as a function of month of the year. Each point represents the per cent of a constituent i n a u n i t weight of dry t i s s u e . Closed symbols represent female means and open symbols represent i n d i v i d u a l males. V e r t i c a l l i n e s i n d i c a t e + !• S.E. 31 v i FIGURE PAGE 11 Levels of p r o t e i n , l i p i d and glycogen i n the foot muscle of T. lamellosa as a function of month of year. Each point represents the per cent of a constituent i n a u n i t weight of dry t i s s u e . Closed symbols represent female means and open symbols represent i n d i v i d u a l males. V e r t i c a l l i n e s i n d i c a t e + 1 S.E. 33 12 Levels of p r o t e i n , l i p i d and.glycogen i n the gonad of T. lamellosa as a function of month of year. Each point represents the per cent of a constituent i n a unit weight of dry t i s s u e . Closed symbols represent female means and open symbols represent i n d i v i d u a l males. V e r t i c a l l i n e s indicate + 1 S.E. 34 13 Per cent ash per unit weight of dry t i s s u e i n three t i s s u e s of the body. The data f o r digestive gland and foot muscle are means of pooled male and female t i s s u e . 36 14 Histograms represent the amount of dry t i s s u e accounted f o r by the protein, l i p i d and glycogen determinations. Data f o r ash i s a v a i l a b l e f o r only three of the c o l l e c t -ions. 37 15 Oocyte diameter frequency polygons f o r two experimental groups. Explanation as i n Figure 6. Animals kept under summer conditions from August 31, 1968 u n t i l January 6, 1969. 40 v i i LIST OF PLATES PLATE PAGE 1 Cross-section of digestive gland i l l u s t r a t i n g the subjective c l a s s i f i c a t i o n of granule content. 1 = lumen, t = tubule, g = storage granules. A. tubules f u l l of granules (2600 X) B. tubules h a l f f u l l (3400 X) C. tubules empty (3400 X) 25A ACKNOWLEDGMENTS I wish to express gratitude to my supervisor Dr. P.A. Dehnel f o r h i s assistance and constructive c r i t i c i s m during t h i s study. I would also l i k e to thank Dr. M.J. Smith f o r h i s assistance and encouragement during the i n i t i a l stages of the research, C. Rauchert who advised me on some of the biochemical techniques, D. Kramer f o r h i s advice and assistance with the photography, Dr. H. Nordan f o r making a v a i l a b l e f a c i l i t i e s f o r ash determination, and my fellow graduate students f o r a s s i s t i n g me i n ways too numerous to mention. 1 INTRODUCTION Reproductive cycles of various marine invertebrates based on h i s t o l o g i c a l i nvestigations or gonad indices are demonstrated i n the l i t e r a t u r e . Giese (1959) reviewed the work up to that time and since then fur t h e r species have been investigated: The chitons, Katherina tunicata and Mopalia h i n d s i i (Giese, Tucker, and Boolootian, 1959b) and Cryptochiton s t e l l e r i (Lawrence, Lawrence, and Giese, 1965) ; the abalone, H a l i o t i s c r a c h e r o d i i (Webber and Giese, 1969); the limpets, Acmaea mitra (Fritchman, 1961), Acmaea limatula (Seapy, 1966) , and F i s s u r e l l a barbadensis (Ward, 1966); the l i t t o r i n e s , L i t t o r i n a pintado, L. p i c t a , and L. scabra (Struhsaker, 1966); the bi v a l v e s , Crassostrea v i r g i n i c a (Sakuda, 1966), Aequipecten irradians (Sastry, 1966), Spisula solidissma (Ropes, 1968), Mytilus edulis  planulatus, Xenostrobus pulex, S e p t i f e r b i l o c u l a r i s , Brachidontes  v a r i a b i l i s , and Amygdalum glaberrimum (Wilson and Hodgkin, 1967); the asteroids, Odontaster validus (Pearse, 1965) and P i s a s t e r ochraceus (Mauzey, 1966); the ophiuroid, Gorgonocephalus c a r y i (Patent, 1969). Biochemical changes associated with the reproductive cycle have not been extensively studied (Giese et a l . 1959a; Giese and Araki, 1962; Barnes, Barnes and Finlayson, 1963; A n s e l l and Lander, 1967; Giese and Hart, 1967; Blackmore, 1969). For the most part, the reproductive cycles i n these studies were determined by gonad indices or spawning p o t e n t i a l , the l a t t e r being determined by standard spawn-ing s t i m u l i . Few studies u t i l i z e d the more precise method of measur-ing oocyte sizes i n h i s t o l o g i c a l sections to determine the stage of the reproductive cycle. The data obtained i n these studies provide 2 a more complete p i c t u r e of the i n t e r n a l changes, and allows the i n -ve s t i g a t o r to postulate the r e l a t i o n s h i p between n u t r i t i o n and the reproductive cycle. For example, Giese and Hart (1967) found seasonal changes i n the gonad and digestive gland indices of Katherina t u n i c a t a throughout the year. Analyses showed that most of the body components remained s i m i l a r i n t h e i r protein, l i p i d and t o t a l carbohydrate except f o r the ovary and t e s t i s . The l a t t e r organs showed marked changes i n protein and carbohydrate l e v e l , and the ovary showed a change i n l i p i d l e v e l which reached a maximum at the height of the breeding season. The carbohydrate l e v e l i n t e s t i s and ovary was maximal during the time of minimum gonad s i z e and minimal at the peak of breeding. From these data, they suggest that as the ovary develops, carbohydrate i s u t i l i z e d by the oocytes. They also suggest that carbohydrate i s converted to protein i n the t e s t i s , while l i p i d remains f a i r l y constant. I t i s suggested that carbohydrate i s converted to l i p i d and protein i n the ovary. Whether that i s i n f a c t what occurs would require more s p e c i f i c i n v e s t i g a t i o n , perhaps in v o l v i n g r a d i o a c t i v e t r a c e r s . However, b i o -chemical studies of t h i s type can at l e a s t provide a basis f o r explain-ing the mechanisms involved i n the timing and c o n t r o l of reproduction. Early work of t h i s type was done on commercially important bivalves such as the oysters, Ostrea gigas and 0. edulis ( M i t c h e l l , 1915-16; R u s s e l l , 1923; Masumoto, Masumoto, and Hibino, 1934; Hatanaka, 1940) and the mussel, Mytilus edulis (Daniel, 1921), while more recently, biochemical work has been performed on other groups such as echinoderms and chitons. Giese and h i s colleagues have contributed many papers to t h i s f i e l d of invertebrate physiology. There i s an obvious la c k of 3 biochemical information on marine prosobranch gastropods except f o r two recent papers on P a t e l l a vulgata (Blackmore, 1969) and on H a l i o t i s  c r a c h e r o d i i (Webber and Giese, 1969). Consequently, the marine i n t e r -t i d a l gastropod, Thais lamellosa Gmelin, a common species i n the area of Vancouver, B r i t i s h Columbia, was chosen f o r the present study. A study of t h i s type would provide basic information on the changes i n biochemical constituents and the reproductive cycle of Thais lamellosa, which might be important to furth e r e c o l o g i c a l or p h y s i o l o g i c a l i n -v e s t i g a t i o n . Some information has been published on the reproduction of T. lamellosa from f i e l d observations of breeding aggregations and egg-capsule deposition ( G r i f f i t h , 1967). Emlen (1966) did an e c o l o g i c a l study of T. lamellosa from the point of view of time, energy and r i s k and,, provided information on times of capsule l a y i n g , length of breed-ing season, and developmental time., i n a population from Port Townsend, Washington. Chapman and Banner (1949) report that the native d r i l l , T. lamellosa, lays eggs i n Puget Sound between March and June. They also performed some simple experiments on the s a l i n i t y tolerance of T. lamellosa. A f t e r 11 days at 15.2°/oo, 3 out of 10 died, while s a l i n i t i e s above t h i s had no observable e f f e c t . As the s a l i n i t y dropped below about 15°/°° "the s n a i l s tended to close up and remain i n a c t i v e ; hence, the s a l i n i t y i n d i r e c t l y affected feeding. The d i e t of T. lamellosa appears to be mussels and barnacles with a preference f o r the l a t t e r (Kincaid, 1957). D a l l (1915) reviewed the taxonomic h i s t o r y of Thais (Nucella) lamellosa Gmelin, and described f i v e v a r i e t i e s with type locations at 4 four places on the P a c i f i c coast. Kincaid (1957) presented many i l l u s t r a t i o n s of s h e l l v a r i a t i o n s from d i f f e r e n t locations on the P a c i f i c coast. For the purposes of t h i s study, animals were c o l l e c t e d from one l o c a t i o n and i d e n t i f i e d as Thais lamellosa from the de s c r i p t i o n of G r i f f i t h (1967), and no consideration was given to the d i f f e r e n t v a r i e t i e s . In t h i s paper, the biochemical constituents: l i p i d , carbohydrate, prote i n , and ash; i n gonad, digestive gland, and muscle t i s s u e , were determined f o r monthly samples and, the reproductive cycle was deter-mined from h i s t o l o g i c a l data and gonad indices. Environmental para-meters of temperature and s a l i n i t y were recorded concurrently with f i e l d c o l l e c t i o n s and, a l i m i t e d amount of experimentation was per-formed i n the laboratory under co n t r o l l e d conditions. This study was designed to provide some basic information which w i l l serve as the basis f o r furth e r experimentation on Thais lamellosa. MATERIAL AND METHODS 5 C o l l e c t i n g Area A sample of about 20 Thais lamellosa was c o l l e c t e d each month at Brockton Point, Stanley Park, Vancouver, B r i t i s h Columbia, ( F i g . 1) and t i s s u e was obtained from 8 females and 2 males. Animals were c o l l e c t e d between the 0 and 3 foot t i d e l e v e l s , the maximum t i d a l height at t h i s l o c a t i o n being about 15 feet . Only those animals longer than 4 cm. were chosen, as smaller animals did not y i e l d s u f f i c i e n t t i s s u e f o r i n d i v i d u a l chemical analyses. P r i o r to d i s -section, animals were kept i n aerated sea-water approximating f i e l d temperatures, 15°C i n summer and 8°C i n winter. These temperatures were c o n t r o l l e d by the cold tap water on a water table and were subject to the changes i n the water supply. The animals were main-tained i n t h i s way f o r at l e a s t 3 days to allow the gut to empty. Water temperatures were recorded at a depth of 2 feet with a Temp-scribe continuous temperature recorder (GBI S c i e n t i f i c ) o f f the Texaco O i l barge adjacent to the c o l l e c t i n g area. A water sample was c o l -l e c t e d once a week and s a l i n i t y was determined by standard t i t r a t i o n methods. C o l l e c t i o n of Samples The s o f t parts were removed by cracking the s h e l l open. External water on the whole animal was bl o t t e d with absorbant t i s s u e and the t o t a l s o f t parts, i n c l u d i n g the operculum, were weighed on a top-loading balance to + 0.01 grams. A portion of the gonad-digestive gland complex was removed and f i x e d i n Gilson's F l u i d (Galigher and Ko z l o f f , 1964) f o r l a t e r sectioning. The gonad, digestive gland and 6 FIGURE 1 Map of research area showing main sources of run-off. 7 foot muscle were dissected o f f , t r a n s f e r r e d to pieces of Parafilm and weighed to + 0.1 mg. The t i s s u e was dried i n a vacuum dessicator over D r i e r i t e f o r 48 hours which removed 98-99% of the water. From t h i s procedure, % dry weight, gonad index, and digestive gland index were obtained. The per cent dry weight i s the r a t i o of dry weight to wet weight and i s a measure of the t o t a l s o l i d s i n the wet t i s s u e . Since part of the complex had been removed f o r f i x a t i o n , a true com-ponent index could not be obtained; however, as near as possible, a proportional amount was removed from each animal. The component index obtained then, was a r e l a t i v e rather than an absolute value. There appears to be some confusion i n the l i t e r a t u r e regarding component index. Some authors c a l c u l a t e the index from the r a t i o of component volume to t o t a l wet weight times 100 (Giese, et a l . , 1959a; Giese, et a l . , 1959b; Giese and Araki, 1962), while i n more recent papers the r a t i o of component weight to t o t a l wet weight was used (Giese, 1967; Giese and Hart, 1967; Sastry, 1968; Webber and Giese, 1969). The l a t t e r method was used i n t h i s study. Histology Several f i x a t i v e s were tested, including Baker's, Carnoy's, Bouin's, and Gilson's f i x a t i v e s . The l a s t was found to give the most s a t i s f a c t o r y r e s u l t s and was employed as the f i x a t i v e f o r a l l the t i s s u e s . Later i n the course of the study, P i c r o - N i t r i c f i x a t i v e was found to give better f i x a t i o n of yolk but to maintain uniformity of shrinkage of oocytes G i l s o n T s F l u i d was used throughout the study. The method of oocyte measurement w i l l be described l a t e r . A segment from the d i s t a l end of the gonad-digestive gland complex 8 was f i x e d i n Gilson's F l u i d over night. Sectioning of the whole gonad indicated that development of the oocytes was synchronized throughout. The t i s s u e was then washed f o r several hours i n running tap-water to remove excess f i x a t i v e . The t i s s u e was dehydrated up to 70% EtOH where i t could be stored u n t i l processed. From t h i s point the t i s s u e was dehydrated with EtOH, cleared i n benzene and embedded i n Tissuemat (m.p. 56.7°C). The majority of the sections were cut at 8 microns but some ranged from 5-10 microns depending on factors such as s t a t i c e l e c t r i c i t y and sharpness of blade at time of sectioning, and f i x a t i o n of the specimen. Masson's Trichrome s t a i n (Pantin, 1964) was employed and gave black n u c l e i , green mucus and connective t i s s u e , pink e p i t -helium, and orange yolk granules. During the s t a i n i n g procedure the sections were exposed to Lugol's Iodine i n 70% EtOH which served to remove c r y s t a l s of mercuric chloride r e s u l t i n g from the f i x a t i v e . The gonad sections were analysed by the method of Pearse (1965). With an eyepiece scale c a l i b r a t e d i n microns, two measurements of oocyte diameter were made at r i g h t angles to each other, these u s u a l l y being the longest and shortest axes. From these two perpendicular measurements the mean diameter of each oocyte was calculated. These data were obtained f o r 25 randomly selected oocytes i n each of 5 females. Only those oocytes which were sectioned through the nucleus were mea-sured. Some of the large mature oocytes presented problems i n measure-ment since the nucleus was pyknotic and d i f f i c u l t to see among the large yolk granules; furthermore, the membranes of the mature oocytes were d i f f i c u l t to discern so the boundaries often had to be estimated 9 using clues from the shape of the surrounding t i s s u e and neighbouring oocytes. A l l the measurements were arranged into s i z e classes and a graph of siz e d i s t r i b u t i o n was drawn ( F i g . 6). The i n d i v i d u a l graphs or polygons ind i c a t e the per cent frequency i n each s i z e c l a s s f o r each monthly c o l l e c t i o n throughout the year. The sections of digestive gland were analysed f o r t h e i r storage granule content. The columnar c e l l s of the digestive gland tubules were s u b j e c t i v e l y c l a s s i f i e d as being f u l l of granules, h a l f f u l l , or empty (Table 2, Plate 1). Chemical Analyses Chemical analyses f o r l i p i d , p rotein, and polysaccharide were performed on the dry t i s s u e from i n d i v i d u a l animals rather than on pooled t i s s u e s from several animals. I n d i v i d u a l determinations pro-vided information on the amount of v a r i a t i o n i n the population and the differences between males and females, as well as Individual data which could be correlated with the h i s t o l o g i c a l r e s u l t s , observations of colour, and per cent water i n the t i s s u e . For p r a c t i c a l reasons, one determination of each component was performed i n each t i s s u e sample, as opposed t o pooling the t i s s u e s and doing several r e p l i c a t e s . L i p i d : L i p i d and polysaccharide determinations were performed on the same portion of t i s s u e . T o t a l l i p i d values were obtained by a method based on that of Sperry and Brand (1955). Approximately 20 mg. of dry t i s s u e were homogenized i n 2 ml. of a chloroform-methanol s o l -u t i o n (2:1) with a t i s s u e grinder (Pyrex Brand) and transferred to a centrifuge tube. The grinder was rinsed several times and the washings were added to the sample. The homogenate was centrifuged and the 10 supernatant containing the l i p i d was t r a n s f e r r e d to a screw-cap v i a l . The remaining p r e c i p i t a t e was retained f o r polysaccharide a n a l y s i s , which w i l l be described l a t e r . To the l i p i d extract, 5 ml. of a wash so l u t i o n of 1.6% C a C l 2 w a s added which, a f t e r shaking f o r 5 min., caused the separation of the l i p i d l ayer. A f t e r c e n t r i f u g a t i o n , the upper l a y e r was removed and discarded and about 2 ml. of a second wash s o l u t i o n , chloroform-methanol (2:1) plus 2% CaCl^ (Sperry and Brand, 1955), was layered c a r e f u l l y on the surface to remove any non-l i p i d impurities remaining at the surface. This wash l a y e r was removed with a Pasteur pipette a f t e r c e n t r i f u g a t i o n , and the remaining l i p i d was evaporated to dryness at 60°C with a stream of nitrogen. This l a t t e r procedure was to reduce the p o s s i b i l i t y of o x i d i z i n g the l i p i d . The l i p i d remaining was then dissolved with about 1 ml. of ether and tra n s f e r r e d to a pre-weighed piece of sponge on a planchet. The tube was rinsed a second time with 1 ml. of ether and was added to the planchet. The sponge allowed f o r f a s t e r evaporation and also prevented s p i l l a g e of the sample. When dry, the weight of the extracted l i p i d was determined and the t o t a l l i p i d / u n i t dry weight of t i s s u e was c a l -culated as a percentage. The percentage l i p i d present per u n i t dry weight w i l l be r e f e r r e d to as the l i p i d l e v e l i n t h i s paper, following the terminology introduced by Giese (1967). Polysaccharide: As mentioned i n the l i p i d analysis method, the residue from the l i p i d e xtraction was used f o r the polysaccharide analysis. This has two advantages: ( l ) both determinations can be performed on one small amount of t i s s u e , and (2) the p r i o r removal of l i p i d prevents foaming during the polysaccharide extraction (Giese, 11 1967). The t i s s u e was, f i r s t of a l l , heated with 4 ml. of 80% EtOH to p r e c i p i t a t e the polysaccharide and remove some soluble impurities. The s o l u t i o n was centrifuged and the supernatant discarded. The p r e c i p i t a t e was heated with 4 ml. of TCA at 100°C to extract the polysaccharide, the supernatant from the extraction being placed i n a 25 ml. volumetric f l a s k . This extraction was performed three times t o ensure complete removal of the polysaccharide. A 2 ml. aliquot was then analysed f o r polysaccharide by the anthrone method of S e i f t e r , et a l . (1950) with the modifications of Helbert and Brown (1955). The anthrone method depends on dehydration of the sugar to a f u r f u r a l d e r i v a t i v e by the s u l f u r i c acid component which combines with the anthrone to form a blue-coloured compound. A 0.16% so l u t i o n of anthrone i n 95% s u l f u r i c acid was made up fresh d a i l y . The anthrone s o l u t i o n when 4 hours old, was added to the 2 ml. sample while the l a t t e r was i n an ice-bath and being agitated with a magnetic s t i r r e r . The tubes were sealed with glass marbles and Parafilm and placed i n a b o i l i n g water-bath f o r 10 min. (+ 2 sees.). I t i s important to main-t a i n the bath at a constant temperature and t o time the incubation p r e c i s e l y f o r consistent r e s u l t s . A f t e r 10 min. at 100°C the tubes were again placed i n the ice-bath to stop the reaction. The tubes were then allowed to e q u i l i b r a t e with room temperature (about 20 min.) and read i n a Beckman DU spectrophotometer at a wavelength of 625 m i l l i m i c r o n s . An absorption spectrum was run under the same conditions and the point of maximum O.D. was v e r i f i e d as being 625 mu. The concentration of polysaccharide was calculated from a standard curve prepared from glucose standards. The r e s u l t s were expressed as per cent polysaccharide i n the dry t i s s u e and w i l l be referred to as 12 polysaccharide l e v e l . Protein; The Lowry co l o r i m e t r i c method was used f o r protein determinations, bovine albumin serving as the standard (Lowry, et a l . , 1951). Approximately 5 mg. of dry t i s s u e was dissolved i n 4 ml. of 1 N. NaOH f o r several hours and d i l u t e d to 25 ml. A 1 ml. aliquot was used f o r the protein determinations following the procedure of Lowry, et a l . (1951). In recent years the Lowry co l o r i m e t r i c method f o r determining proteins has come into widespread use (Giese, 1967). This method has several advantages." ( l ) I t i s as s e n s i t i v e as Nessler's reagent but requires no digestion. (2) I t i s 10 or 20 times more s e n s i t i v e than the measurement of the u l t r a v i o l e t absorption at 280 mu. and i s much more s p e c i f i c and much l e s s l i a b l e to disturbance by t u r -b i d i t i e s . (3) I t i s several f o l d more s e n s i t i v e than the ninhydrin rea c t i o n and i s somewhat simpler, as well as much easier to adapt to small scale analyses. (4) I t i s 100 times more s e n s i t i v e than the b i u r e t r e a c t i o n . There are two major disadvantages to the Lowry r e -action, ( l ) The amount of colour varies with d i f f e r e n t proteins; however, i n t h i s study the same type of t i s s u e was analysed through the year so only the r e l a t i v e changes were important. (2) The colour i s not s t r i c t l y p roportional to concentration. To overcome t h i s d i f -f i c u l t y , sample concentrations were kept i n the range of 0 to 100 |j.g./ml. where the standard curve was e s s e n t i a l l y l i n e a r . The 1 ml. sample aliqu o t was allowed to s i t with 5 ml. of a carbonate-copper s o l u t i o n f o r 10 min. and then to t h i s mixture was added 0.5 ml. of the F o l i n - C i o c a l t e a u phenol reagent while the tube was shaken. The colour was allowed to develop at room temperature f o r 30 min. and was measured i n a Beckman DU spectrophotometer at 750 mo-. 13 An absorption spectrum was obtained to v e r i f y 750 mu as the wavelength of maximum absorption. The concentration was expressed as the per cent protein per un i t dry weight of t i s s u e . As with l i p i d and glycogen, t h i s value w i l l be referred to as the protein l e v e l i n the t i s s u e . Ash weights were determined on pooled samples of dry t i s s u e f o r three periods i n the year using the method described i n O f f i c i a l Methods of Analysis of the A.O.A.C. (1960, page 419). The pooled sample was divided into two aliquots and the mean of the ash weights calculated. Ashing was done at a temperature of 525°C f o r about 12 hours t o constant weight. Controlled Conditions Two groups of Thais lamellosa, about 20 i n each, were maintained under c o n t r o l l e d "summer" conditions of s a l i n i t y (l8.9°/oo), temper-ature (15°C) and l i g h t (16 hours l i g h t ; 8 hours dark) i n p l a s t i c dishes supplied with aerated sea-water. The s a l i n i t y of 18.9°/op approximated the low values recorded i n May and June (Fig. 2). F u l l strength sea-water was d i l u t e d with glass d i s t i l l e d water to the desired value, and was changed weekly. The temperature of 15°C approximated the f i e l d water temperature i n the summer ( F i g . 4) and was regulated by a thermostatically c o n t r o l l e d water-bath (+ 1.0°C). The l i g h t regime represented the day length i n June ( F i g . 5) and was automatically c o n t r o l l e d by a timing mechanism. One group of animals was supplied with mussels and barnacles at a l l times, while the other was starved. Organisms such as barnacles growing on the s h e l l s of the starved group were removed and the walls of the dishes were kept clean of algae. At the end of 4 months t i s s u e s FIGURE 2 Mean monthly s a l i n i t y two f e e t below the surface at Brockton Point. V e r t i c a l l i n e s i n d i c a t e the range. The f i r s t and l a s t points do not represent complete months. 1968 MONTH OF YEAR 1969 of 8 females and 2 males were obtained and analysed f o r protein, l i p i d , polysaccharide, dry weight, and component index using the methods described previously. Also gonads and digestive glands were sectioned and analysed f o r oocyte s i z e d i s t r i b u t i o n and storage granule content. Data obtained from regular f i e l d samples at the beginning and end of the experimental period represented the normal changes during that time. S t a t i s t i c a l Methods Student's t - t e s t was used to determine s i g n i f i c a n t differences between mean values. P l e v e l s of 5% or l e s s were accepted as s i g n i f i -cant. Photographs were taken through a Zeiss Photo-Microscope with bright f i e l d optics on Kodak Panatomic-X f i l m . Sections were stained with Masson's Trichrome. 16 RESULTS F i e l d Observations Environmental parameters: Figure 2 shows the s a l i n i t y changes over a 14 month period from J u l y 1968 u n t i l September 1969. The maximum s a l i n i t y of 28.5°/oo, occurred i n the middle of March 1969; the minimum of 18°/oo, at the end of May and the beginning of Ju l y . This minimum corresponded to the maximum discharge of the Fraser River. During the winter the Fraser River discharge held steady at about 25,000 cubic feet/sec. and at maximum discharge reached 425,000 cubic feet/sec. ( P a c i f i c Oceanographic Group, 1951). The low s a l i n i t i e s occurring i n September and November can be explained by the r a i n f a l l data of Figure 3 (Canada D.O.T. Meteorological Branch, 1968). A peak of r a i n f a l l at the beginning of September res u l t e d i n a drop of s a l i n i t y i n the second h a l f of September. S i m i l a r l y , the dip i n s a l i n i t y i n November probably resulted from the r a i n f a l l peak i n early November. From December to March the s a l i n i t y increased slowly as most of the p r e c i p i t a t i o n f e l l as snow i n the surrounding mountains and run-off was consequently low. From A p r i l to J u l y the s a l i n i t y dropped as snows melted and run-off increased. The main sources of run-off i n the c o l l e c t i o n area are the Squamish River, Capilano River, Fraser River, Seymour Creek, and numerous smaller creeks draining the mountains around Burrard I n l e t ( F i g . l ) . Figure 4 shows the water temperature v a r i a t i o n over a period of 12 months from August 1968 to J u l y 1969. The minimum temperature of 2°C occurred i n l a t e December as a r e s u l t of unseasonally low a i r temperatures. The maximum of 15.5°C was i n J u l y 1969 at the end of FIGURE 3 T o t a l p r e c i p i t a t i o n f o r each 15 day period at the Port Meteoro-l o g i c a l O f f i c e , Vancouver. Calculated from tables i n the Annual Meteorological Summary (Canada D.O.T. Meteorological Branch, 1969, 1970). Vancouver S B J - i i S i § E s s I i § § i i i - s § I 3 i • i • j j _ J A S O N D J F M A M J 1968 MONTH OF YEAR 1969 J A the recording period. A p l o t of daylight hours ( F i g . 5) shows that the minimum water temperature corresponded to the shortest day length, and the maximum water temperature lagged behind the maximum day length by about a month. Reproductive a c t i v i t i e s : T. lamellosa was f i r s t observed to l a y egg-capsules at the beginning of January when the water temperature was near a minimum and the s a l i n i t y was about 25°/oo. No observations were made on the duration of capsule l a y i n g f o r the population but h i s t o l o g i c a l data, which w i l l be described l a t e r , indicate the l a y i n g continues u n t i l about A p r i l . The population of T. lamellosa appeared to move to lower t i d a l l e v e l s during the winter and e i t h e r formed aggregations f o r reproduction or buried themselves around the bases of rocks. They were found only i n the barnacle zone and seldom as high as the Mytilus zone, so i t appeared that T. lamellosa was feeding mostly on barnacles; however, these are only casual observations made during c o l l e c t i o n t r i p s and require more documentation. Internal Morphology Gross anatomy: The sexes are separate i n T. lamellosa; however, there are no external differences except f o r the presence of a penis i n the male which can sometimes be seen during copulation. The morphology of T. lamellosa i s v i r t u a l l y i d e n t i c a l to Nucella l a p i l l u s , an A t l a n t i c species which i s described i n d e t a i l i n F r e t t e r and Graham (1962, pages 332-338). The gonad l i e s on one side of the digestive gland i n the upper c o i l s of the v i s c e r a l mass. The ovary i s u s u a l l y yellow and granular i n appearance, while the t e s t i s i s more trans-lucent and homogeneous in texture. The digestive gland varies i n colour from dark brown to gray-green. Table 1 indicates the range FIGURE 4 Mean monthly water temperature two feet below surface at Brockton Point. V e r t i c a l l i n e s i n d i c a t e the range. FIGURE 5 The points represent the average day-light hours per day f o r each month, ca l c u l a t e d from t a b l e s - i n Northcott (1968, 1969). 21 of colour i n the digestive gland and the number of animals of a par-t i c u l a r colour i n each c o l l e c t i o n . A gray-green digestive gland predominated i n the February and May c o l l e c t i o n s , while i n August most animals had yellow-brown digestive glands. The gland was pre-dominantly brown t o dark brown i n the A p r i l c o l l e c t i o n . H i s t o l o g i c a l observations: H i s t o l o g i c a l sections showed the t e s t i s and ovary to consist of many tubules i n which the gametes appeared i n various stages of maturation. During reproductive periods, from January t o March, the tubules were large and c l o s e l y apposed to each other but i n non-reproductive periods the tubules were reduced i n s i z e and the connective t i s s u e spaces between tubules increased in s i z e . The h i s t o l o g i c a l analysis of the reproductive cycle was confined to females of T. lamellosa. An attempt was made to c l a s s i f y the ovary on a subjective scale ranging from r i p e t o spent; however, c l e a r cut changes did not occur i n the ovary of T. lamellosa. Large yolk granules were present at a l l times of the year, as were small immature oocytes. A f t e r the reproductive season, oocytes that remained i n the ovary appeared to dis i n t e g r a t e and the yolk granules became scattered throughout the tubule. Developing oocytes had prominent n u c l e o l i i n a large germinal v e s c i c l e and the cytoplasm contained small granules. In large mature oocytes the nucleus became dense and pyknotic and was often d i f f i c u l t t o locate among the large yolk granules which completely f i l l e d the oocyte. Determination of reproductive cycle by oocyte diameter: Figure 6 indicates the s i z e d i s t r i b u t i o n of oocytes i n each c o l l e c t i o n through-out the year. Each polygon represents 125 oocytes, 25 from each of 22 TABLE 1 Subjective c l a s s i f i c a t i o n of digestive gland colour. C o l l e c t i o n Date Sample Size Gray-Green Yellow-Brown Brown Dark Brown October 25, 1968 10 - 8 1 1 December 4 11 1 9 1 -January 17, 1969 10 4 4 2 -February 16 19 12 4 3 -A p r i l 4 20 5 1 9 5 May 15 20 11 4 4 1 J u l y 1 20 9 7 4 -August 25 20 — 17 3 _ FIGURE 6 Oocyte diameter frequency polygons f o r each monthly c o l l e c t i o n of T. lamellosa. Each polygon represents a t o t a l of 125 oocytes and the points ind i c a t e the per cent of the t o t a l i n each s i z e c l a s s . The abscissa i s i n per cent u n i t s . 800-900 r to I 700-800 600-700 500-600 400-500b 300-400 200-300 100-200 0-100 \ sept. 25 1968 o \ 0 25 PER CENT FREQUENCY f i v e females, and each point i s the percentage of the t o t a l , i n that s i z e c l a s s . For example, on September 25, 1968, 50% of the oocytes measured were i n the 0 to 100 u s i z e c l a s s , 22% were i n the 100 to 200 u s i z e c l a s s . The graphs indicate that oocytes i n the range of 500 u and greater, occur i n significant'numbers from September 1968 to February 1969 and again i n August 1969. In A p r i l the s i z e d i s t r i -bution, shows a lack of the la r g e r s i z e classes and probably indicates that spawning has taken place. In May the l a r g e r oocytes begin to appear but the small s i z e classes predominate. In J u l y the oocytes appear to be maturing and increasing i n s i z e as those i n the s i z e classes greater than 300 u begin to appear i n greater numbers. The data indicate that i n l a t e March or ea r l y A p r i l most of the population had spawned and, replenishment of large oocytes began immediately. From August to February mature oocytes, at l e a s t from the point of view of s i z e , were present i n the ovary. Parasites: In the A p r i l 4 c o l l e c t i o n , 6 out of 26 T. lamellosa examined were infected by the p a r a s i t i c trematode of the genus Renicola (Ching, pers. comm.). Infected T. lamellosa were not used f o r gonad indices or biochemical analyses. Histology of dige s t i v e gland: The digestive gland was examined microsc o p i c a l l y and the number of storage granules i n the c e l l s of the digestive gland tubules was c l a s s i f i e d s u b j e c t i v e l y , f u l l of granules, h a l f f u l l or empty (Table 2, Plate 1). I t i s assumed that the presence of storage granules indicates that the animals have been feeding. I f feeding stops, the granules w i l l gradually diminish i n numbers as the stored nutrients are mobilized f o r body maintenance. The number of storage granules was at a maximum i n August and remained f a i r l y high 25 TABLE 2 Subjective c l a s s i f i c a t i o n of number of granules i n the storage c e l l s of the digestive gland of Thais lamellosa. C o l l e c t i o n Date Sample Size F u l l H a l f - F u l l Empty August 6, 1968 11 10 1 -September 25 13 11 1 1 October 25 7 7 - -December 4 9 6 3 -January 17, 1969 9 6 3 -February 16 10 - 5 5 A p r i l 4 9 8 1 -May 15 9 6 1 2 J u l y 1 10 5 3 2 August 25 10 10 — Experimental Animals Aug. 31 to Jan. 6 (fed) 8 5 3 -Aug. 31 to Jan. 6 (starved) 10 3 5 2 PLATE 1 Cross-section of digestive gland i l l u s t r a t i n g the subjective c l a s s i f i c a t i o n of granule content. 1 = lumen, t = tubule, g = storage granule. A. tubules f u l l of granules (2600 X)" B. tubules h a l f f u l l (3400 X) C. tubules empty (3400 X) 26 u n t i l October 25. From December u n t i l February the number of granules i n the c e l l s dropped, probably i n d i c a t i n g that the animals were feed-ing l e s s and u t i l i z i n g stored nutrients. The number of granules increased i n A p r i l and i n May and J u l y the numbers dropped again. Per Cent Dry Weight The per cent dry weights of three body organs; gonad, digestive gland, and muscle, over a 12 month period are plotted i n Figure 7. The per cent dry weight of muscle remained e s s e n t i a l l y constant at about 27.5%. The per cent dry weight of ovary ranged from a maximum of 63% in December to a minimum of 40% i n July. The per cent dry weight of di g e s t i v e gland ranged from a maximum of 55% i n December to a minimum of 39% i n J u l y with a f u r t h e r low value of 40% i n February. The per cent dry weight indicates the amount of s o l i d material i n proportion to water content; consequently, when the gonads are f u l l of mature eggs and the inter-tubule spaces are smallest, the per cent dry weight i s at a maximum. S i m i l a r l y , when the animal has been feeding, a l l the c e l l s of the digestive gland are f u l l of stored material and no empty spaces are evident i n the h i s t o l o g i c a l sections, r e s u l t i n g i n a high per cent dry weight. Gonad Index Another method of representing the reproductive cycle i s by the gonad index. The gonad index cycle i s shown i n Figure 8. Only the females were sampled i n quantities which allowed c a l c u l a t i o n of mean and standard error. For t h i s population, the ovary index dropped from about 7 during the months of October to February, down t o 2 i n A p r i l , probably i n d i c a t i n g spawning. This correlates w ell with the data from egg si z e frequency ( F i g . 6) and per cent dry weight ( F i g . 7) which both FIGURE 7 Per cent of dry material i n a u n i t weight of fresh t i s s u e of T. lamellosa. The v e r t i c a l l i n e s indicate +1 S.E. Data f o r females only. 1968 MONTH OF YEAR 1969 FIGURE 8 Gonad indices of male and female T. lamellosa as a function of month of year. Closed c i r c l e s ( • ) represent the mean ovary index and open c i r c l e s ( O ) represent i n d i v i d u a l males V e r t i c a l l i n e s indicate + 1 S.E. 20 I o testis o • ovary 15 • o o uS o o I 10 • ° o 0' ' ' ' * ' * ' ' ' ' * ' 1 A S O N D J F M A M J J A 1968 MONTH OF YEAR 1969 dropped during t h i s same period. From A p r i l to May the ovary index rose again from 2 up to 5.5, and t h i s i s r e f l e c t e d i n the appearance of oocyte s i z e classes above 500 u i n the May 15 c o l l e c t i o n ( F i g . 6). The t e s t i s index appears to follow a s i m i l a r pattern to the ovaries but with higher absolute values during the winter months. Digestive Gland Index The changes i n the digestive gland indices are shown i n Figure 9. The maximum index of 22.5 f o r the females occurred i n l a t e August 1969 and the minimum of 7 i n February. The changes i n the d i g e s t i v e gland index were p a r a l l e l l e d by the per cent dry weight data i n Figure 7 and the storage granule data i n Table 2. The maximum index i n August corresponded to the greatest accumulation of storage granules, and the minimum index i n February corresponded to the lowest storage granule content. The male dige s t i v e gland index showed s i m i l a r changes to the female but due to the small sample s i z e minor differences could not be determined. I t should also be emphasized that due to the method of sampling as described previously, the indices arrived at are only r e l a t i v e values and they underestimate the true index. Constituent Levels Digestive gland: Figure 10 shows the l e v e l s of protein, l i p i d , and glycogen f o r the digestive gland; Figure 11 shows the l e v e l s i n the foot muscle; and Figure 12, the gonad. The changes i n the l i p i d w i l l be considered f i r s t . The greatest v a r i a t i o n i n l i p i d l e v e l occurred in the female digestive gland (Fig. 10), which went from 38.5% i n December to 20% i n A p r i l , followed by another increase to 35% i n J u l y . The l i p i d l e v e l i n the muscle remained e s s e n t i a l l y con-stant at 6 to 8%. The l i p i d l e v e l s i n the male digestive gland and FIGURE 9 Digestive gland indices of male and female T. lamellosa as a function of month of year. Closed c i r c l e s ( • ) represent the mean of 8 females and the open c i r c l e s ( o ) represent i n d i v i d u a l males. V e r t i c a l l i n e s i n d i c a t e + 1 S.E. FIGURE 10 Levels of protei n , l i p i d and glycogen i n the digestive gland of T. lamellosa as a function of month of the year. Each point represents the per cent of a constituent i n a un i t weight of dry t i s s u e . Closed symbols represent female means and open symbols represent i n d i v i d u a l males. V e r t i c a l l i n e s indicate + 1 S.E. Digestive gland 1968 MONTH OF YEAR 1969 32 muscle appeared to follow those of the females. The ovary l i p i d l e v e l f luctuated several times during the year with the most s i g n i f i c a n t change occurring between October and December from 26% down to 18% (p < .05). Fluctuations i n February, A p r i l and May were also s i g n i -f i c a n t (p < .05). The t e s t i s l i p i d appeared to drop down to about 10% in the winter with possibly a s l i g h t r i s e i n J u l y . Protein l e v e l s : In female t i s s u e the highest protein l e v e l s occurred i n muscle ( F i g . 11) with a high of 64% i n A p r i l and a low of 46.6% i n J u l y 1968. Ovary was s l i g h t l y lower with a maximum of 56% i n October and a general downward trend to A p r i l where the protein l e v e l was 46% ( F i g . 12). The digestive gland showed the greatest range in protein l e v e l from a maximum of 43.5% i n A p r i l to a minimum of 31% i n February. The protein l e v e l s i n male muscle and digestive glands were, as near as could be determined by the small sample s i z e , the same; but i n the t e s t i s , p rotein values were generally lower and averaged about 40%. Glycogen and ash l e v e l s : Emerson (1966) showed that the poly-saccharide of T. lamellosa i s 100% glycogen; hence, i n t h i s study the polysaccharide w i l l be r e f e r r e d to as glycogen. Glycogen was at very low l e v e l s i n a l l three t i s s u e s . In the ovary i t ranged from 2% to 4%; i n the muscle i t fluctuated from 2% to 4%; and i n the dig e s t i v e gland the range was s l i g h t l y greater, from 2% to 7.5%. Again, the glycogen l e v e l i n the male t i s s u e s was e s s e n t i a l l y the same. Ash weights f o r the three t i s s u e s at three times during the year are r e -presented i n Figure 13. Male and female t i s s u e s were combined i n a l l FIGURE 11 Levels of protei n , l i p i d and glycogen i n the foot muscle of T. lamellosa as a function of month of year. Each point represents the per cent of a constituent i n a un i t weight of dry t i s s u e . Closed symbols represent female means and open symbols represent i n d i v i d u a l males. Ver-t i c a l l i n e s indicate + 1 S.E. 80 ox 70 60 5 0 h to 6 40 Foot muscle 10 h o o A 1 lipid .2 glycogen i i A S O N D J F M 1968 MONTH OF YEAR M J J 1969 FIGURE 12 Levels of protein, l i p i d and glycogen i n the gonad of T. lamellosa as a function of month of year. Each point represents the per cent of a constituent i n a u n i t weight of dry t i s s u e . Closed symbols represent female means and open symbols represent i n d i v i d u a l males. V e r t i c a l l i n e s i n d i c a t e + 1 S.E. A S O N D J F M 1968 MONTH OF YEAR A M J J A 1969 35 cases except the gonad which represents only the females. Both muscle and gonad have s i m i l a r ash l e v e l s at the three c o l l e c t i o n dates; however, the ash l e v e l i n the digestive gland r i s e s from 3.4% i n August to 7% i n March. Figure 14 shows the amount of dry weight accounted f o r by l i p i d , glycogen, and protein analyses i n the digestive gland each month. In September, January and May, the ash weight values have been added. I t can be seen that these analyses account f o r only about 80% of the dry weight. Experimental Results As described i n the methods section, two groups of T. lamellosa were maintained i n summer conditions from August 31, 1968 u n t i l January 6, 1969} one group being fed, the other, starved. A regular c o l l e c t i o n , made at the beginning of the experiment from the same lo c a t i o n (Aug. 31) served to e s t a b l i s h the s t a r t i n g l e v e l s of protein, l i p i d , glycogen and dry weight. The experimental animals at the s t a r t were assumed to have the same biochemical l e v e l s as the f i e l d group since they were c o l l e c t e d from the same population. At the end of the experimental period another f i e l d c o l l e c t i o n was analysed; hence, the changes i n the normal animals during the experimental period were determined. At the end of the experiment, the fed and the starved groups were analysed f o r the various biochemical constituents. Table 3 summarizes the data, giving l e v e l s of l i p i d , glycogen, protein, dry weight and component indices f o r the f i e l d controls and the two experi-mental groups. The s t a r t i n g values f o r the dry weight l e v e l and com-ponent indices were not obtained on August 31 so the data from the September 25 c o l l e c t i o n were used as s t a r t i n g values. FIGURE 13 Per cent ash per u n i t weight of dry t i s s u e i n three t i s s u e s of the body. The data f o r digestive gland and foot muscle are means of pooled male and female t i s s u e . 10 i li JAN '69 digestive gland foot muscle ovary i i a | i I MAY '69 FIGURE 14 Histograms represent the amount of dry t i s s u e accounted f o r by the protein, l i p i d and glycogen determinations. Data f o r ash i s a v a i l a b l e f o r only three of the c o l l e c t i o n s . Digestive gland glycogen lipid H U M protein i — i ash 50 • s I § i fi § § I B S : • s I 11 I 1 I 5 I •I i • B s S 8 8 S LA i A S 1968 N D J F M MONTH OF YEAR A M J J 1969 38 TABLE 3 Summary of data f o r two experimental groups of T. lamellosa maintained i n summer conditions from August 31, 1968 to January 6, 1969. Data f o r females only. Numbers i n brackets are 1 S.E. c^ = f i e l d sample at s t a r t of experiment (August 31). c^ = f i e l d sample at end of experiment (January 6). f = experimental group fed, and sampled at conclusion. s = experimental group starved, and sampled at conclusion. Tissue L i p i d Glycogen Protein % Dry Weight Component Index C l 21.8(+1.2) 6.6(+0.9) 42.4(+0.7) 46.6(+3.0) (Sept. 25) 13.9(+1.0) (Sept. 25) Digestive C2 33.0(+2.9) 3.4(+0.4) 37.7(0.9) 47.9(2.6) 10.8(1.1) Gland f 32.9(+2.l) 7.0(0.6) 37.7(1.9) 46.2(2.2) 13.8(1.0) s 35.6(+1.9) 6.3(0.9) 33.8(1.9) 43.5(1.5) 12.4(1.1) C l 20.7(+1.0) 3.6(0.5) 48.8(1.4) 52.6(4.3) 5.5(0.6) Ovary C2 . 22.5(+1.0) 3.0(0.2) 49.9(0.7) 61.5(0.6) 6.6(0.6) f 31.4(+1.1) 4.2(0.5) 42.3(1.3) 54.4(3.4) 4.1(0.8) s 32.5(+1.9) 4.0(0.4) 42.2(2.1) 52.1(2.9) 2.5(0.4) C l 6.7(+0.2) 3.5(0.3) 55.7(1.3) 27.0(0.2) Foot C2 8.1(+0.1) 2 . 3 ( 0 . 3 ) 56.2(0.7) 26.9(0.5) Muscle f 6.7(+0.3) 4.2(0.6) 60.4(1.0) 25.6(0.7) s 6.6(+0.2) 3.4(0.2) 53.6(1.4) 24.0(0.6) The di g e s t i v e gland index of the f i e l d population dropped from 13.9 to 10.8 (p < .05) during the experimental period (Table 3). However, the dig e s t i v e gland indices of the fed and starved animals did not change. Table 2 shows the storage granule data f o r the two experimental groups. Most of the fed i n d i v i d u a l s were f u l l of storage granules, while the digestive glands of the majority of starved animals were h a l f - f u l l of granules. There was also a noticeable i n -crease i n the number of mucous c e l l s i n starved animals. The mean ovary index of the f i e l d population and the fed group did not change s i g n i f i c a n t l y during the experimental period. However, there was a s i g n i f i c a n t drop i n the ovary index of the starved group from 5.5 to 2.5 (p <. .05). Figure 15 shows the per cent frequency of oocytes i n each s i z e class f o r the two experimental groups. In both cases more than 75% of the oocytes measured are i n the range of 0 to 100 microns, and none occur in the classes greater than 500 u. In the digestive glands of the f i e l d controls the protein and glycogen l e v e l s dropped (p <~ .05), l i p i d increased (p .05), and the dry weight l e v e l remained constant. In the fed i n d i v i d u a l s the protein l e v e l dropped s i g n i f i c a n t l y (p <1 .05) and l i p i d increased s i g n i f i c a n t l y (p -<i .05), while glycogen and dry weight remained the same. As i n the fed i n d i v i d u a l s the di g e s t i v e glands of the starved animals showed a s i g n i f i c a n t drop i n protein (p <C .05), an increase i n l i p i d (p <: .05) and no change i n glycogen or dry weight. The ovaries of the f i e l d controls showed no s i g n i f i c a n t change i n the l i p i d , p r o t e i n , or glycogen l e v e l s . In both the fed and starved animals s i g n i f i c a n t changes occurred i n l i p i d and protein (p <L .05) FIGURE 15 Oocyte diameter frequency polygons f o r two experimental groups. Explanation as i n Figure 6. Animals kept under summer conditions from August 31, 1968 u n t i l January 6, 1969. g 600-700 o 1 500-600 400-500 300-400 fed < m 200-300 i — y 100-200 \ o 0-100 0 25 PER CENT FREQUENCY but glycogen and dry weight remained the same. During the experimental period the muscle t i s s u e of the f i e l d animals showed small changes i n the l i p i d and glycogen l e v e l s which were s i g n i f i c a n t at the 5% l e v e l . In the muscle t i s s u e of the fed i n d i v i d u a l s , l i p i d and glycogen l e v e l s remained the same but the protein l e v e l increased s i g n i f i c a n t l y (p < .05). The starved i n -div i d u a l s on the other hand, showed no change in protein, l i p i d , or glycogen l e v e l s . DISCUSSION 42 Reproduction and Development In a l l higher prosobranchs, f e r t i l i z a t i o n i s by copulation (Thorson, 1950). The f e r t i l i z e d eggs then proceed through develop-ment i n one of several ways. They may e i t h e r develop inside the female by v i v i p a r i t y or some form of i n t e r n a l brood protection; they may be shed s i n g l y into the water and proceed through development as pelagic larvae; they may develop i n a gelatinous mass or s t r i n g ; or, as i n Thais lamellosa, be deposited on the substrate within a capsule (Thorson, 1946). Larvae which develop in s i d e capsules leave a f t e r a c e r t a i n period and complete development as pelagic or non-pelagic young. The eggs of T. lamellosa, when l a i d , are i n the order of 500 microns i n diameter. This large s i z e i s apparently i n d i c a t i v e of non-pelagic development (Thorson, 1946). Some examples of egg sizes f o r species with non-pelagic development described by Thorson, are Trophon  hanleyi (500 u), Brachystomia r i s s o i d e s (380 u), Amauropsis i s l a n d i c a (1500 u), and Neptunea antiqua (300 u). Development within a capsule has several advantages.. Since every egg deposited i n the capsule i s f e r t i l i z e d , there i s l e s s "waste" than i n broadcast f e r t i l i z a t i o n . The large supply of yolk enables the larv a to develop to an advanced stage within the capsule, thereby reducing predation encountered by pelagic species. Thorson (1950) discusses to a greater extent the r e l a t i v e advantages of the d i f f e r e n t types of l a r v a l development i n r e l a t i o n t o the environmental conditions. The capsules of some species contain nurse eggs, which are oocytes that do not develop but become a food source f o r the other larvae ( F r e t t e r , 1941). The stage of development at hatching reached by the larvae which u t i l i z e nurse eggs, varies greatly, depending on the r a t i o of nurse eggs to larvae. Embryos w i l l leave the capsule when they have reached the stage of development that the nurse eggs w i l l support. Because of t h i s , embryos of a species i n one l o c a l i t y may hatch as non-pelagic young while i n another l o c a l i t y where fewer nurse eggs were produced, the embryos may hatch as pelagic larvae (Thorson, 1950). T. lamellosa does not produce nurse eggs (Ahmed and Sparks, 1970); hence the larvae always leave the capsule at the same stage of development. Emlen (1966) found that egg-capsules of T. lamellosa contained from 20 to 150 eggs with a mean of 81. He also estimated that a female can l a y about 200 capsules a year and assum-ing a spawning period of 5 months, 1.35 capsules per-day. The eggs of T. lamellosa are l a r g e r and more yolky than many other species, so oogenesis i s probably a longer process. I t i s reasonable to expect then, that T. lamellosa spawns only once a year, while some broadcast f e r t i l i z e r s which produce much smaller gametes, may spawn more often. The bi v a l v e , Spisula s o l i d i s s i m a (Ropes, 1968) and the h a r d - s h e l l clam, Venus mercenaria ( A n s e l l and Lander, 1967) have two spawning periods i n a year. According to Webber and Giese (1969), the abalone, H a l i o t i s c r a c h e r o d i i , which i s a broadcast f e r t i l i z e r , had two periods of gametogenesis i n the year although there was probably only one spawning period. The ovary showed an increase i n s i z e only during one gametogenic period. The second gametogenic period, which was said to have occurred a f t e r spawning, may have been an a r t i f a c t of the sampling technique, as explained below. In the present study, gametogenesis occurred l a t e r In the summer ( F i g . 6), and one spawning peak occurred i n March and A p r i l . The sudden increase i n the smaller s i z e classes i n A p r i l may not have n e c e s s a r i l y been due to an immediate increase i n t h e i r numbers but rather t o a lo s s of the large mature oocytes. The data are in percentages; hence i f no large oocytes are present the percentage of small oocytes appears to increase. This perhaps explains the con-cl u s i o n by Webber and Giese (1969) that there i s a second gametogenic period immediately a f t e r spawning i n H. c r a c h e r o d i i . Emlen (1966) determined the developmental time within the capsules of T. lamellosa to be 140 days. I f that i s true f o r the Brockton Point population the eggs l a i d i n March would hatch i n mid-summer. A subjective estimate, made on J u l y 1, 1969 indicated that about 30 to 40 per cent of the capsules checked were empty. Emlen (1966), study-ing a population at Port Townsend, Washington, reported the f i r s t appearance of capsules on November 20 i n 1964 and on December 10 i n 1965. Animals were s t i l l spawning on March 12, 1966 and had ceased by A p r i l 21, 1966. The breeding period f o r the population i n the present study agrees with that reported by Emlen, although the begin-ning of the capsule l a y i n g may have been s l i g h t l y l a t e r . The con-d i t i o n s at Port Townsend are probably more constant with respect to s a l i n i t y and temperature, since the area i s more d i r e c t l y Influenced by the P a c i f i c Ocean through the Juan de Fuca S t r a i t . The exact period and extent of spawning by T. lamellosa at Brockton Point was not determined by extensive f i e l d observations, but merely by obser-vation at the time of regular sampling. I t i s possible that capsules were l a i d at very low t i d a l l e v e l s (zero feet) before those f i r s t 45 observed i n January, 1969. The average s i z e of T. lamellosa from Port Townsend was 60 mm while those from Brockton Point averaged between 45 and 50 mm i n length. One i n d i v i d u a l of T. lamellosa c o l l e c t e d on October 25, 1968, and kept i n an aquarium, l a i d three egg-capsules on November 16, 1968. Whether t h i s incident can be extended to the f i e l d population i s d i f f i c u l t to say. A few i n d i v i d u a l s i n the f i e l d were observed cop-u l a t i n g on December 4, 1968 so conceivably, egg-laying may have taken place soon a f t e r . I t i s assumed that an oocyte requires one year to mature; however, there i s no d i r e c t evidence to support t h i s conclusion. Nimitz and Giese (1964) report that oogenesis i n Katherina tunicata i s a two year process, but they do not o f f e r evidence to support t h i s statement. The egg diameter frequency data ( F i g . 6) indicate that mature s i z e oocytes (500 a) are present i n the ovary from September to the time of the February c o l l e c t i o n . Furthermore, the gonad indices ( Fig. 8) during t h i s time are e s s e n t i a l l y the same. On the basis of oocyte s i z e and gonad index, i t appears that T. lamellosa has the p o t e n t i a l to spawn during the period from September to A p r i l . There are p o s s i b l y two periods of stimulation which may a f f e c t the timing of the breeding cycle. The f i r s t induces gametogenesis and oocyte development, and the second t r i g g e r s actual spawning or egg la y i n g . Thorson (1946) maintains that the inducement f o r gameto-genesis i s a temperature d i f f e r e n t from that required to induce spawn-ing; hence at the edge of a species' geographical range an animal could conceivably ripen i t s sexual products but not spawn them. Orton, Southward, and Dodd (1956) studying the limpet, P a t e l l a vulgata, con-46 eluded that the breeding cycle of a population i s r e l a t i v e l y constant from year to year; that i s , the population i s p o t e n t i a l l y ready to spawn during a s p e c i f i c time i n the year, e.g. September to A p r i l , but the spawning stimulus seems to over-ride the breeding c y c l e . Depending on the year or the l o c a l i t y , the time of the release of gametes may be quite v a r i a b l e . In the case of P. vulgata spawning appeared to correspond to rough seas rather than to temperature, t i d e s , or phases of the moon (Orton^ et a l . , 1956). Seasonal Biochemical Changes The r e s u l t s of the experimentation i n t h i s study suggest that temperature, l i g h t , or s a l i n i t y rather than food might induce gameto-genesis and egg l a y i n g i n T. lamellosa, but i t w i l l be necessary to perform c o n t r o l l e d experiments i n order to conclude which s t i m u l i are involved. The work presented here describes the biochemical changes which took place i n t h i s animal during the breeding cycle of 1968-69. From these data, conclusions w i l l be drawn about the t r a n s f e r of storage products within the various organs o f the s n a i l , and the re l a t i o n s h i p s among feeding, gametogenesis, spawning and the b i o -chemical l e v e l s . Biochemical analyses were performed on whole t i s s u e s ; hence any i n t e r n a l t r a n s f e r of constituents could not be measured but only suggested. Histochemical data from related species of mollusks and the morphological changes observed i n t h i s study w i l l be employed to explain the gross biochemical changes. Biochemical constituents of oocytes: Raven (1966) reviewed the types of yolk formed during v i t e l l o g e n e s i s i n mollusks. In the nudi-branch, Aplysia and the pulmonate s n a i l s , Limnaea, Helix, and Eremina, the f i r s t to be formed was protein yolk and i n l a t e r stages l i p i d was 47 added. Nudibranch eggs are 66% to 75% by volume protein yolk and only about 2% to 5% l i p i d yolk. However, some of the protein yolk may have l i p i d associated with i t . Crepidula eggs are reported to be 75% protein yolk (Raven, 1966). T. lamellosa eggs are probably s i m i l a r to those of nudibranchs and Crepidula, but since the eggs are l a r g e r and the larvae must l i v e o f f the y o l k content f o r a longer time than most other species, i t would seem economical to add more l i p i d . L i p i d provides more than twice as many c a l o r i e s per gram than e i t h e r carbohydrate or protein (White, Handler, and Smith, 1964). The l e v e l s of the biochemical constitutents i n T. lamellosa eggs were not deter-mined, but Bayne (1968) found that free l i p i d s were present i n the n u t r i e n t reserve of the eggs of Nucella l a p i l l u s , a c l o s e l y r e l a t e d species. For the maturation of oocytes, i t appears that protein and l i p i d are the e s s e n t i a l constituents. In order to supply the oocytes with the required biochemical constituents the animal may ingest food and t r a n s f e r i t d i r e c t l y to the gonad as i n Strongylocentrotus purpuratus (Giese et a l . , 1959a), or i t may store food i n another part of the body and t r a n s f e r the reserves to the gonad when required. P i s a s t e r ochraceus stores food reserves i n the p y l o r i c caeca and t r a n s f e r s them to the gonads during the breeding period (Farmanfarmaian et a l . , 1958). Balanus balanoides and B. balanus store material during the spring when food i s abundant and t r a n s f e r i t t o the gonads i n the winter (Barnes et a l . , 1963). Feeding: I t i s generally accepted that the c h i e f s i t e of diges-t i o n and storage of ingested food i n gastropods i s the digestive gland (Yonge, 1937; M i l l o t t , 1938; Graham, 1939; Howells, 1942; and Morton, 1951). In t h i s study, the presence of storage granules i n 48 the digestive gland d i v e r t i c u l a was used as an i n d i c a t i o n of feeding. This method was used by Mauzey (1966) as an i n d i c a t i o n of feeding i n P i s a s t e r ochraceus. Table 2 indicates that T. lamellosa was feeding i n August, September, and October, 1968 and to a l e s s e r extent i n December and January. In the February c o l l e c t i o n , the number of s t o r -age granules was l e a s t , suggesting the l e a s t amount of feeding. This period corresponded to the breeding time when most of the s n a i l s were at the zero t i d e l e v e l forming breeding aggregations, and were pre-sumably not a c t i v e l y feeding. Thorson (1958) documents "passive periods" i n several predator species of bottom-living invertebrates, e s p e c i a l l y during breeding. In the A p r i l c o l l e c t i o n , when most i n d i v i -duals had completed spawning, the increase i n storage granules suggest that feeding had resumed. The decrease of storage granules on May 15 and J u l y 1 imply that feeding was reduced. A reduction i n feeding might be explained by a decrease i n s a l i n i t y at the c o l l e c t i o n s i t e due to run-off ( F i g . 2). As mentioned i n the Introduction, Chapman and Banner (1949) found that reduced s a l i n i t y (l5°/oo) caused a reduction i n m o t i l i t y and even withdrawal into the s h e l l ; thereby i n d i r e c t l y reducing feeding. The e f f e c t of s a l i n i t y on the present population was not tested, although present work on t h i s aspect supports the suggestion (Johannsson, pers. comm.). The conclusions about feeding, based on the storage granule con-tent, are also supported by the digestive gland indices ( F i g . 9). The index drops from October u n t i l February then r i s e s i n A p r i l when feeding resumes a f t e r spawning. There was a drop i n the index during the period of low s a l i n i t y i n May and June, followed by a large increase i n the l a t t e r part of the summer. The index showed an increase cor-49 responding to the increase i n the number of storage granules. Explanation of terms; Before discussing the data on biochemical changes, several points should be made cl e a r . When presenting the l e v e l s of polysaccharide i n T. lamellosa, determined i n the present work, they w i l l be referred to as glycogen, since Emerson (1965) has shown by chromatography that the polysaccharide i n T. lamellosa i s 100 per cent glycogen. However, when reference i s made to other papers, the general term polysaccharide w i l l be used unless the author stated the s p e c i f i c polysaccharide which was measured. The constituent l e v e l s i n t h i s study are expressed as per cent of a unit weight of dry t i s s u e ; therefore care should be exercised i n i n t e r p r e t i n g the data. For example, i f one constituent i s u t i l i z e d from a t i s s u e , the remaining constituents, on a per cent b a s i s , may appear to increase. The change i n s i z e of an organ must be considered when i n t e r p r e t i n g the per cent data. Changes i n constituent l e v e l s i n conjunction with a decrease i n organ s i z e , may ind i c a t e that the constituents have a l l been u t i l i z e d but at d i f f e r e n t rates. Per cent data then, when considered with changes i n organ s i z e , can i n d i c a t e approximate rates of u t i l i z a t i o n . Constituents of digestive glandi I f ingested food i s f i r s t stored i n the digestive gland, there should be a p a r a l l e l increase i n one or more of the biochemical constituents and the digestive gland index. I f the constituent l e v e l s i n the gland ( F i g . 10) are compared to the digestive gland indices ( F i g . 9), i t w i l l be noted that the protein and glycogen l e v e l s peak at times corresponding to the index peaks, e s p e c i a l l y i n A p r i l and August, 1969. There was a protein and glycogen peak i n September, 1969, but no d i g e s t i v e gland index was obtained. 50 L i p i d l e v e l s peak about 3 months a f t e r the September and A p r i l peaks, on December 4, 1968 and J u l y 1, 1969, r e s p e c t i v e l y . S t a r t i n g on August 31, 1969, the l i p i d l e v e l began to increase and the glycogen l e v e l began to decrease. Since feeding was s t i l l occurring through August, September, and October, and the digestive gland was enlarging, the food was most l i k e l y being stored as l i p i d . The apparent drop in protein i s probably a passive r e s u l t of the increase i n l i p i d . Protein makes up the s t r u c t u r a l parts of t i s s u e s ; hence an increase i n stored material, such as l i p i d , w i l l r e s u l t i n a drop i n protein on a per cent basis. As a r u l e , there i s a net loss of protein only under s t a r v a t i o n conditions (White, Handler, and Smith, 1964). The fa c t that the protein curve i n Figure 10 i s the r e c i p r o c a l of the l i p i d curve probably indicates the passive nature of the protein changes i n r e l a t i o n to the stored l i p i d . The biochemical composition of barnacles, the food of T. lamellosa, based on the data from Barnes et a l . (1963) f o r two species of Balanus, show that the r a t i o of l i p i d to polysaccharide i s about 2:1. I f the barnacles eaten by T. lamellosa have a s i m i l a r composi-t i o n to those reported by Barnes et a l . (1963) then the s n a i l s w i l l ingest twice as much l i p i d as polysaccharide. The glycogen l e v e l i n the digestive gland does not reach more than 7%, while the l i p i d l e v e l does not f a l l below 20%. This suggests that ingested glycogen i s converted to l i p i d f a i r l y quickly, at l e a s t i n the f a l l when the animal i s preparing f o r reproduction. During the summer, when food i s p l e n t i f u l , the animal may u t i l i z e glycogen d i r e c t l y f o r energy rather than convert i t to l i p i d . In l a t e August, when feeding i s great, l i p i d i s at i t s lowest l e v e l and glycogen i s at a maximum, but 51 as f a l l and winter approach, reproductive demands increase and l i p i d becomes more prominent i n the s n a i l ' s economy. Emerson (1965) analysed several P a c i f i c west coast prosobranchs f o r t h e i r polysaccharide content and found a p o s i t i v e c o r r e l a t i o n between those with a carnivorous d i e t and the l a r g e s t polysaccharide content. He reported a glycogen l e v e l of 6.05 (+1.16, 95% confidence l i m i t ) per cent of the dry weight f o r the combined s o f t parts of T. lamellosa c o l l e c t e d i n July-August. This glycogen l e v e l reported by Emerson i s i n the same range as l e v e l s i n t h i s study, but a d i r e c t com-parison cannot be made, since h i s determinations were performed on the t o t a l 'soft parts rather than on i n d i v i d u a l t i s s u e s . The herbivorous prosobranchs had polysaccharide values i n the range of 1 to 3 per cent. Emerson (1965) also determined the t o t a l ether extractable material i n the herbivorous top s h e l l , Tegula f u n e b r a l i s , as being 12.1 (+ 4.7)% of the dry weight i n the female v i s c e r a l mass, and 2.3 (+ 0.9)% i n the male. The l i p i d i n the foot muscle was 4.3 (+ 2.4)% i n the female and 5.5 (+ 4.2)% i n the male. The difference i n the l i p i d l e v e l s of the v i s c e r a l mass between males and females was probably due to oocytes i n the ovary. The l i p i d l e v e l i n the v i s c e r a l mass of Tegula  funebralis i s much lower than that i n T. lamellosa, and i s perhaps a r e f l e c t i o n of the diff e r e n c e i n d i e t . Glycogen i n the digestive gland; The maximum l e v e l of glycogen i n T. lamellosa digestive gland i s twice as great as the maximum polysaccharide l e v e l of 3% i n the "gonad-free" t i s s u e of the limpet, P a t e l l a vulgata (Blackmore, 1969). The low polysaccharide l e v e l i n P. vulgata would be expected on the basis of the figures quoted by Emerson (1965) f o r the herbivorous limpets. Blackmore (1969) found 52 large amounts of ash i n P. vulgata, ranging from 15 to 37% of the t o t a l dry weight. He concluded that the ash content was highest when the a l g a l cover was at a minimum, and the limpet ingested r e l a t i v e l y more inorganic material along with the food. In T. lamellosa the highest ash weights were 8% i n the foot muscle and about 7% i n the d i g e s t i v e gland. Role of glycogen i n reproduction: Barry and Munday (1959) showed that i n P a t e l l a glycogen l e v e l s i n the digestive gland were c l o s e l y correlated with seasonal v a r i a t i o n s i n blood glucose, and they con-cluded that the increased glycogen l e v e l s were probably i n d i c a t i v e of feeding. There was a decrease i n glycogen l e v e l i n a l l t i s s u e s of P a t e l l a c o i n c i d i n g with the shedding of gametes i n January,, How-ever, a f t e r spawning, the resultant low l e v e l of glycogen remained constant throughout the winter. I t was concluded that P a t e l l a did not u t i l i z e glycogen to maintain metabolism during the winter. The gonad of P a t e l l a , during the year, never contained appreciable glycogen reserves as compared to the pulmonate, Helix, or the oyster, Ostrea gigas. Barry and Munday (1959) and Blackmore (1969) both concluded that i n Patella c,the major food reserve i s l i p i d , and that polysac-charides are not s i g n i f i c a n t as food reserves but vary with feeding. Giese and Hart (1967) found that polysaccharide l e v e l s i n Katherina  tunicata gonad were highest when the gonad was small and i n a c t i v e . Blackmore (1969) also found t h i s i n P a t e l l a vulgata, and came to the conclusion that polysaccharide occurred i n the i n t e r s t i t i a l t i s s u e rather than i n the oocytes. As stated previously, the amount of polysaccharide i n marine gastropod eggs i s low, most of the reserves being i n the form of protein yolk and l i p i d yolk. There i s an increase i n the glycogen l e v e l of the ovary of T. lamellosa a f t e r spawning, which l i k e P a t e l l a and Katherina, suggest that glycogen Is situated i n the i n t e r s t i t i a l t i s s u e of the ovary. What r o l e t h i s glycogen has i n the ovary i s not known, but Nimitz and Giese (1964), using histochemical techniques on the ovary of K. tu n i c a t a , found that glycogen i n the epithelium of the ovary gradually decreased to i t s lowest value i n May when spawning took place. Despite the decrease of glycogen i n the epithelium, no glycogen appeared i n the oocytes. There was a s i m i l a r decrease of l i p i d i n the epithelium, but unlike the glycogen, l i p i d began to appear i n the growing oocytes. Presum-ably, the glycogen was used i n the synthesis of l i p i d and protein yolk. A s i m i l a r s i t u a t i o n occurs i n the ovary of the oyster, Ostrea gigas. Glycogen reached a maximum i n A p r i l then began to decline as the l i p i d l e v e l increased. The l i p i d l e v e l peaked i n June a f t e r which spawning occurred. I t was postulated that glycogen i n the storage t i s s u e of the gonad was converted into l i p i d and transferred to the sexual products (Masumoto, Masumoto, and Hibino, 1934). Other h i s t o -chemical investigations have shown that the f o l l i c l e c e l l s around oocytes i n chitons can resorb nutrients from unshed oocytes, store them, and l a t e r pass them on to growing oocytes (Gabe and Prenant, 1949; Selwood, 1970). A histochemical study of T. lamellosa would re v e a l the r o l e of the various accessory c e l l s i n the movement and storage of nutrien t s . The glycogen l e v e l i n the ovary of T. lamellosa peaked i n September and A p r i l at the same time as i n the digestive gland. This suggests that glycogen from ingested food i s incorporated into the ovary as well as into the digestive gland, although not to the same extent. The glycogen l e v e l dropped i n both ovary and digestive gland following the September c o l l e c t i o n and at the same time protein and l i p i d l e v e l s i n the ovary Increased ( F i g . 12). This may have been due to the u t i l i z a t i o n of glycogen i n the synthesis of protein yolk and l i p i d yolk i n the oocytes. I f glycogen reserves were being used to synthesize yolk, i t would be expected that the glycogen l e v e l would drop and the protein and l i p i d l e v e l s In the ovary would increase. Unlike the digestive gland, the protein and l i p i d l e v e l s of the ovary change i n a p a r a l l e l manner, since both constituents are major con-t r i b u t o r s to yolk. Constituents of the ovary: At the time of spawning, the ovary index dropped, but the corresponding drop i n l i p i d and protein l e v e l i n the ovary was not dramatic ( F i g . 12) as found i n some other invertebrates (Giese and Hart, 1967; Giese, Hart, Smith and Cheung, 1967; Blackmore, 1969). The r e l a t i v e l y small biochemical changes i n the ovary of T. lamellosa might be explained by morphological evidence. Although the egg s i z e data showed loss of mature oocytes at spawning, i n f a c t some were s t i l l present i n the form of d i s i n t e g r a t i n g aggre-gations of yolk granules. They were not considered oocytes as the c r i t e r i o n required the presence of a nucleus. Unshed oocytes appeared to d i sintegrate and be resorbed. I t has been shown i n the chiton, Sypharochiton septentriones, that a l l the c e l l types i n the ovary, apart from the oocytes and l a t e f o l l i c l e c e l l s , possess lysosomes, and are capable of resorbing unshed, mature oocytes and l a t e r passing the nutrients to new oocytes (Selwood, 1968). Some yolk may be digested by the ingesting gland of T. lamellosa, situated between the albumen gland and the capsule gland. Sectioning of t h i s gland showed the presence of a few yolk granules. F r e t t e r (1941) also gave 55 evidence f o r the r o l e of the ingesting gland i n digesting unused yolk. The yolk granules i n a d i s i n t e g r a t i n g oocyte would probably y i e l d s i m i l a r r e s u l t s to the yolk i n a mature oocyte. The f a c t that the ovary i s never completely emptied of mature oocytes probably explains why the biochemical constituents did not show dramatic changes. Polysaccharide as a storage product: In animals which use polysaccharides as a storage product, the l e v e l s are much higher than the maximum glycogen l e v e l of 7% i n T. lamellosa. The clam, T i v e l a  stultorum, has from 10 to 25% glycogen i n the non-gonad t i s s u e , and 25 to 50% i n the gonads (Giese et a l . , 1967). The abalone, H a l i o t i s  c r a c h e r o d i i , has polysaccharide l e v e l s ranging from 5 to 25% i n the foot muscle (Webber and Giese, 1969). In the sea urchin,. Strongylo- centrotus purpuratus, the gonad appears to be the major s i t e of food storage. Here the maximum glycogen content was 10% at the time of highest gonad index, and the minimum was 1% (Giese et a l . , 1959a). Judging" from the r e l a t i v e l y low glycogen l e v e l s i n the gonad and foot muscle of T. lamellosa, and the low l e v e l s of glycogen i n the digestive gland, which f l u c t u a t e with feeding, glycogen i s not an important food reserve. A f t e r ingestion I t may be stored b r i e f l y then synthesized into e i t h e r l i p i d , . p r o t e i n , or yolk. Further evidence f o r the r o l e of glycogen i n T. lamellosa can be obtained from the experimental data i n Table 3. In the three t i s s u e s analysed, the difference i n glycogen l e v e l between fed and starved animals was almost n e g l i g i b l e . However, the glycogen l e v e l s i n digestive gland and foot muscle from the f i e l d population were lower on January 17, 1969, than at the beginning of the experiment on August 31, 1968. This drop i n glycogen may have been due to e i t h e r conversion to l i p i d , or u t i l i z a t i o n by the developing oocytes. In the fed and starved groups, the gonads did not develop. There was no requirement f o r glycogen i n the synthesis of protein or l i p i d yolk, so the glycogen l e v e l s did not decrease. I t Is possible that glycogen i s not r e a d i l y used during s t a r v a t i o n , but i s p r e f e r e n t i a l l y used f o r the developing oocytes. Martin (1961) described the work of May (1934) on the pulmonate, Helix pomatia. May found that glycogen was u t i l i z e d by the s n a i l during starvation, but galactogen was only drawn upon when glycogen was depleted. I t was speculated that the r o l e of galactogen was p r i m a r i l y as a reserve f o r reproduction, and because i t was not e a s i l y mobilized, the s n a i l was driven to exert i t s e l f i n f i n d i n g food and thus, c a r r i e d through the demanding reproductive process. This may be the case i n T. lamellosa except that protein and l i p i d i s used during s t a r v a t i o n , and that glycogen i s used f o r the develop-ing oocytes. However, more data are required to substantiate t h i s hypothesis. L i p i d l e v e l s i n T. lamellosa; The s i t e s of l i p i d storage are probably the digestive gland and the gonad. The l e v e l of l i p i d i n the foot muscle was about 7% throughout the year ( F i g - 11). Giese (1966) states that the l i p i d l e v e l i n crab muscle c e l l s , which h i s t o -chemically appeared to store no l i p i d , was 5.2%. Therefore, i t was concluded that a l e v e l of 5.2% represented only s t r u c t u r a l l i p i d , and anything above t h i s probably represents stored l i p i d . The l e v e l of l i p i d i n the foot muscle of T. lamellosa did not exceed 8%, so storage of l i p i d i n the foot muscle probably does not occur to any great extent. Species which histochemically have been shown to possess l i p i d vacuoles i n the foot muscle, have much l a r g e r l i p i d values. For example, the foot of the chiton, Mopalia h i n d s i i , has a l i p i d l e v e l of 21% and 17% in gravid and spent animals, r e s p e c t i v e l y (Giese and Arak i , 1962). The chiton, Cryptochiton s t e l l e r i , which spawns i n April-May, has a l i p i d l e v e l i n the foot muscle of 5% or l e s s , and i n the digestive gland, from 2% to 10% (Tucker and Giese, 1962). T. lamellosa, on the other hand, has a greater l e v e l of l i p i d i n the digestive gland, from 20% to S8%. These differences may be a r e f l e c t i o n of t h e i r d i e t s . C. s t e l l e r i i s mainly herbivorous and therefore, Ingests more polysaccharide, whereas T. lamellosa i s carnivorous and takes i n more l i p i d . Prolonged s t a r v a t i o n i n the sipunculid, Phascolosoma g o u l d i i , caused a drop i n a l l l i p i d except in the muscle. In f a c t , the l e v e l of l i p i d increased, suggesting that protein or some other constituent was u t i l i z e d at a greater rate (Wilber, 1947). Constituents of the foot muscle; The constituent of the foot muscle of T. lamellosa which did appear to change s i g n i f i c a n t l y was the protein. There was a high protein l e v e l i n the September c o l l e c t -ion ( F i g . 11) followed by a drop i n October. The high l e v e l i n September might have been due to the incorporation of ingested food into t i s s u e protein i n the foot muscle. S i m i l a r l y , the peak i n A p r i l was probably due to the resumption of feeding at that time. The protein l e v e l was probably not changing i n response to a l i p i d change, as i n the digestive gland, since the l i p i d l e v e l remained f a i r l y stable. The drop i n October corresponded to an increase of protein i n the ovary, which resulted from the formation of protein yolk. This decrease i n foot muscle protein also occurred during the time when feeding was reduced, which suggests that protein may be e i t h e r metabolized as a 58 source of energy, or u t i l i z e d by the oocytes. The experimental data i n Table 3 show that protein i n the muscle of the starved animals did not change but i n the fed group i t increased s i g n i f i c a n t l y (p <L .05). Further, i n the two experimental groups the ovaries did not increase i n s i z e and no mature oocytes developed. Hence, protein was probably not required f o r yolk production as i n the normal f i e l d population where the ovaries developed normally. The increase i n the protein l e v e l of the fed group may have been a r e s u l t of feeding. From t h i s evidence, i t i s suggested that the drop i n foot muscle protein a f t e r October and the peaks i n September and A p r i l were a r e s u l t of d i f f e r -ent rates of feeding by T. lamellosa, rather than as a r e s u l t of t r a n s f e r to growing oocytes. Conclusions about nut r i e n t t r a n s f e r : The c o l l e c t i o n on October 25, 1968, showed that the digestive gland index was at a peak. From t h i s data u n t i l February 16, 1969 c o l l e c t i o n , the digestive gland decreased i n s i z e , suggesting that reserves were being u t i l i z e d from that organ. Analyses of the animals from the c o l l e c t i o n following the digestive gland peak showed that the l i p i d had increased and the glycogen had decreased. I t i s speculated that the increase i n l i p i d was a r e s -ponse to a reduction of food a v a i l a b i l i t y . With the onset of a reduced food supply ingested nutrients and polysaccharide reserves may be directed into l i p i d synthesis f o r storage. The los s of weight from the dig e s t i v e gland was probably due to glycogen u t i l i z a t i o n f o r l i p i d or yolk synthesis, and l a t e r t o u t i l i z a t i o n of l i p i d reserves during winter. The s n a i l s presumably l i v e d o f f the accumulated l i p i d during the winter, and by A p r i l the l i p i d was at i t s lowest l e v e l . The glycogen l e v e l 59 had already begun to increase by A p r i l as feeding had resumed. When glycogen reaches a c e r t a i n l e v e l due to feeding, some of i t may then be converted into l i p i d f o r storage. This may explain the l a g i n the l i p i d peak following the onset of feeding i n A p r i l . Conclusions drawn from experimental observations: Data from the experimental work (Table 3) suggest what r o l e s the digestive gland and ovary play i n the metabolism of the s n a i l under normal reproductive demands and starvation. The mean index of the digestive glands of the f i e l d group decreased s i g n i f i c a n t l y during the experimental period, but the ovary index did not change. Thus, in the f i e l d population the s i z e of the ovary was maintained, but the digestive gland decreased. Bio-chemical analyses have shown that the weight l o s s was due to u t i l i z a t i o n of glycogen, l i p i d , and to some extent, protein. However, i f one looks at the two experimental groups, which due to the imposed conditions did not follow the normal reproductive pattern, i t was the digestive gland which remained at a constant s i z e , and rather the ovary s i z e which de-creased. I f the fed and starved groups are compared i t w i l l be noted that the digestive gland index of both did not decrease, but the ovary index of the starved animals did. Further, the ovary index of the starved group decreased more than the fed group. I t seems that under normal conditions, reserves i n the digestive gland are used, but during sta r v a t i o n nutrients are resorbed from the ovary. However, the starvation experiment was per-formed under abnormal conditions of l i g h t , temperature, and s a l i n i t y , so the e f f e c t of st a r v a t i o n under normal f i e l d conditions i s not known. In both the experimental groups the glycogen l e v e l did not decrease, not even i n the starved animals. This supports the suggestion;; that the primary 60 function of glycogen i s to supply the developing oocytes rather than to serve as a general energy reserve. Emerson and Duerr (1967) also came to the conclusion that l i t t l e glycogen i s used during s t a r v a t i o n i n L i t t o r i n a planaxis, and suggested that i t might be a reserve f o r anaerobiosis i f starvation was coupled with dessication. In summary, under normal conditions, the data suggest that the digestive gland supplies nutrients to the developing oocytes and f o r general body maintenance, as shown by the reduction of the s i z e of the gland and the decrease i n glycogen and l i p i d . Under abnormal conditions, when the ovary i s prevented from developing, i t appears that the animal draws on the reserves of the ovary to maintain i t s body functions. Von Brand, McMahon, and Nolan (1957) showed that s t a r v a t i o n i n the pulmonate s n a i l , A u s t r a l o r b i s glabratus, resulted i n a small l o s s of l i p i d and polysaccharide, but the majority of the weight loss was due to u t i l i z a t i o n of protein. They did not measure protein d i r e c t l y , but on the basis of oxygen consumption data, the amount of oxygen consumed did not account f o r the small loss i n l i p i d and polysaccharide. Data f o r a cephalopod indicated that protein as well as other reserves were metabolized, since the l i p i d l e v e l remained the same while the animal decreased i n s i z e (Giese, 1966). Emerson and Duerr (1967), on the other hand, found that a f t e r starving the prosobranch, L i t t o r i n a  planaxis, t o t a l l i p i d decreased s i g n i f i c a n t l y , while protein and poly-saccharide did not. The experimental data f o r T. lamellosa do not seem to suggest u t i l i z a t i o n of l i p i d during starvation. The conditions of the experiment, however, should be considered. Under the experi-mental conditions, the starved animals withdrew nutrients from the ovary rather than from the digestive gland. During the winter, when 61 food intake i s low, l i p i d i s u t i l i z e d from the digestive gland. In a review of l i p i d s i n marine invertebrates, Giese (1966) con-cluded that although l i p i d has been shown to be an important reserve mat e r i a l , protein i s generally present i n a l l t i s s u e s i n a quantity greater than e i t h e r polysaccharide or l i p i d , and may be used when the need a r i s e s . Giese mentions the following species as u t i l i z i n g protein during s t a r v a t i o n : the si p u n c u l i d , Phascolosoma a g a s s i z i , the chiton, Katherina t u n i c a t a , and the shore crab, Hemigrapsus nudus. As men-tioned previously, when an organ decreases i n s i z e , protein i s usually metabolized along with l i p i d and glycogen. The protein l e v e l i n the dig e s t i v e gland of the starved T. lamellosa showed more of a los s than the fed animals. E f f e c t of Environmental Factors on Gonad Development Lack of food: The ovary index of the starved animals decreased, p a r t l y because the experimental conditions did not allow maturation of the oocytes, and p a r t l y because of a lack of food. The ovary index of the fed animals also declined but not the same extent. Hence, a v a i l a b i l i t y of food allowed the ovary to maintain i t s s i z e , but the p h y s i c a l conditions prevented maturation of the oocytes. Mature oocytes which were present at the beginning were i n the process of being resorbed. The s i z e of the ovary of the starved animals was l e s s , presumably due to the greater demand on the reserves i n the ovary. Sastry (1966) found that starvation of scallops during the period of gonad growth resulted i n an absorption of oogonia and oocytes, but i f the animals had already accumulated gonad reserves, they released gametes, regardless. The pulmonate, Lymnaea s t a g n a l i s , i f starved f o r s i x weeks, maintained a l l stages of oogenesis and spermatogenesis i n the ovotestis; however the number of sex c e l l s and the s i z e of the ovotestis was reduced. A large number of mature oocytes degen-erated and were resorbed by the nurse c e l l s , but developing oocytes did not seem to be affected (Joosse, Boer, and Cornelisse, 1968). Temperature: In T. lamellosa, maintained under summer conditions of l i g h t , temperature, and s a l i n i t y , the sex c e l l s p r o l i f e r a t e d more i n fed than i n starved animals, but i n neither case did the oocytes mature. Thus, i t could be said that the condition f o r gonad matur-ation i s a f a c t o r other than food supply. Since only one combination of l i g h t , temperature, and s a l i n i t y was applied i n the experiment, i t was not possible to conclude which was the c o n t r o l l i n g f a c t o r . Sastry (1968) showed that there was a r e l a t i o n s h i p between food supply and temperature and the reproductive a c t i v i t y of the s c a l l o p , Aequipecten i r r a d i a n s . Optimum temperatures with food seemed to i n i t i a t e gonad development. When temperatures were below optimum, the reserves from the ingested food seemed to accumulate more In the digestive gland than i n the gonad. In the present study, experimental animals i n temperatures higher than the f i e l d , maintained the digestive gland s i z e , and u t i l i z e d reserves from the gonad. I f the temperature was f a r above the optimum, the reserves of A. irradians did not seem to accumulate i n the gonad e i t h e r because of increased metabolic u t i l i z a t i o n , f a i l u r e to regulate the synthetic processes of the grow-ing oocytes, or death of the s c a l l o p s . Furthermore, although oogonia may develop below the optimum temperature when food was supplied, oocyte development did not seem to occur. This may be the case i n T. lamellosa, but i n the opposite way. These s n a i l s breed at the lowest temperatures of the year; therefore an optimum low temperature may be required to allow the oocytes to mature. Two species of barnacles, Balanus balanoides and B. balanus, which breed i n November and February, r e s p e c t i v e l y , did not breed at temperatures of 14 to 18°C but did in the range of 3 to 10°C ( C r i s p , 1957). The dog whelk, Thais l a p i l l u s , under a r t i f i c i a l laboratory conditions, required 9°C to stimulate egg deposition. No information, however was given about the e f f e c t of temperature on gametogenesis or oocyte maturation (Largen, 1967). Day length: Day length has been implicated i n the c o n t r o l of breeding cycle s . Bo.olootian (1963) maintained male purple sea ' urchins at a constant temperature of 15°C and varied the day-length. On a 14-hour day, i n i t i a l development of gonial c e l l s began but no mature sperm resulted. On a 6-hour day, gonial c e l l s were reduced i n number as they developed into spermatocytes. The purple sea urchin used i n the study normally reproduces i n the winter. Barnes (1963) performed s i m i l a r experiments on winter-breeding B. balanus. Constant i l l u m i n a t i o n i n h i b i t e d breeding, e s p e c i a l l y the l a t e r stages of development. A period of 4 to 6 weeks at l e s s than 12 hours l i g h t per day was required f o r maturation of gametes. Webber and Giese (1969) analysed breeding data f o r H a l i o t i s  c r a c h e r o d i i and Katherina tunicata c o l l e c t e d over several years, and concluded that temperature did not act as an exogenous c o n t r o l of gonad growth. Photoperiod did not show a c o r r e l a t i o n with the increase i n gonad s i z e , but i t was suggested that gametogenesis was i n i t i a t e d by day lengths of greater than approximately 12 hours. I t was empha-sized that mere c o r r e l a t i o n of environmental changes cannot be viewed 64 as conclusive, and data from c o n t r o l l e d experiments should be obtained to e s t a b l i s h causal f a c t o r s . The r o l e of the endocrine system was also suggested as an important consideration f o r the future study of mollusk reproductive cycles. S a l i n i t y : S a l i n i t y has not generally been considered as a stimulus f o r reproduction, but i n s i t u a t i o n s such as Brockton Point, where r e l a t i v e l y large s a l i n i t y f l u c t u a t i o n s take place (17 to 29°/oo), i t should not be discounted. I t could be argued that i n regions on the open coast temperature and s a l i n i t y f l u c t u a t i o n s are very low, yet breeding occurs at regular times. I t may be that populations of a species i n d i f f e r e n t l o c a l i t i e s adapt to that p a r t i c u l a r environment, and u t i l i z e the most obvious regular changes i n the environment, to time the breeding cycle. For example, the population of H. c r a c h e r o d i i , studied by Webber and Giese (1969), encountered only small f l u c t u a -t i o n s i n temperature (range: 5°C) and s a l i n i t y , yet appeared to breed r e g u l a r l y . There was a c o r r e l a t i o n between day length and i n i t i a t i o n of gametogenesis, however. As mentioned in the previous paragraph, endogenous rhythms i n the endocrine system are probably important i n the regulation of* the breeding cycle. The r e s u l t s c i t e d in the previous few paragraphs f o r other inver-tebrates, give evidence f o r the r o l e of feeding, temperature, and l i g h t , i n the c o n t r o l of breeding cycles. U n t i l c o n t r o l l e d experi-ments, using various combinations of temperature, s a l i n i t y and l i g h t can be performed no conclusions can be drawn about the factors c o n t r o l -l i n g reproduction i n T h a i s l a m e l l o s a . Suggestions f o r Further Study The data c o l l e c t e d in t h i s study provide a basis f o r f u r t h e r 65 studies into T. lamellosa reproduction. For example, histochemical data would elucidate the d i s t r i b u t i o n of nutrients within the t i s s u e s , which so f a r can only be speculated. The techniques of rad i o a c t i v e l a b e l l i n g could be employed to show the s i t e s of synthetic a c t i v i t y and the sources of precursors i n r e l a t i o n to food. "Tracer experiments are needed to determine the movement of nutrient i n marine invertebrates" (Giese, 1966, p. 286). This method was also suggested by Barnes et a l . (1963) as a means of t r a c i n g the p a r t i t i o n of the various materials during growth and development of the gonads. A more accurate deter-mination of feeding times during the year could be obtained by measuring the blood glucose l e v e l s i n the manner described by Barry and Munday (1959). Also, biochemical analyses of the eggs would help to explain f u r t h e r the gross changes i n the gonad. Very l i t t l e work has been done on the endocrine system of molluscs and i t s r o l e i n the c o n t r o l of reproduction (Boer, Douma, and Koksma, 1968; Webber and Giese, 1969). A study of the e f f e c t s of environmental factors on reproduction, using c o n t r o l l e d conditions, would also provide a good opportunity to look at the r o l e of neurosecretion i n c o n t r o l l i n g the breeding cycles.-Several authors have pointed out the need f o r f u r t h e r i n v e s t i g a t i o n i n t o seasonal biochemical changes, and the r e l a t i o n s h i p s between nu-t r i t i o n and reproduction i n mollusks (Barnes et a l . , 1963; A n s e l l and Lander, 1967; Emerson, 1965, 1967; Giese, 1966). The present work has attempted to answer some of these questions with regard to the proso-branch, Thais lamellosa. The data show the general s i m i l a r i t i e s between T. lamellosa and other prosobranchs with regard to biochemical c o n s t i t -uents and reproduction. SUMMARY 66 1. A population of Thais lamellosa, from Brockton Point, Vancouver, B r i t i s h Columbia, was sampled approximately monthly from J u l y 28, 1968, u n t i l August 25, 1969. A study was made of the b i o -chemical changes associated with the reproductive cycle. 2. From the three t i s s u e s , d i g e s t i v e gland, foot muscle and gonad; per cent protein, glycogen, l i p i d , and ash were obtained. As well, gonad and digestive gland indices and h i s t o l o g i c a l sections of the gonad and digestive gland were obtained. 3. The reproductive habits of T. lamellosa and some of the properties of the eggs were discussed i n r e l a t i o n to length of breeding season, gametogenesis, developmental time within the cap-sule, and timing of the breeding c y c l e . 4. Estimates of storage granules i n the digestive gland d i v e r -t i c u l a e were used to determine periods of feeding a c t i v i t y . There was a feeding peak i n A p r i l and another i n August, During the winter months u n t i l spawning i n March, there was a decrease i n the amount of feeding. 5. In the f a l l ingested food i s accumulated i n the digestive gland as l i p i d , but in periods of active feeding, A p r i l and August, glycogen i s highest and l i p i d i s at a minimum. 6. I t was concluded that glycogen i s not used as an energy reserve, but i s e i t h e r converted to l i p i d i n the digestive gland or used by the growing oocytes f o r yolk production. 7. The foot muscle does not store l i p i d or glycogen to any appreciable extent, but the protein appears to fl u c t u a t e i n response to food intake. The protein l e v e l increased during maximum feeding and decreased when feeding was low. 8. In the f i e l d population, the digestive gland index decreased as reserves were used, but the s i z e of the ovary was maintained from about October u n t i l spawning i n March. 9. Under the experimental conditions, the digestive gland of both fed and starved animals maintained i t s s i z e , while the ovary decreased i n s i z e . The starved animals withdrew more material from the ovary than the fed animals. 10. Mature oocytes i n the ovary of the starved animals appeared to be resorbed, leaving the smaller oogonia, i n the range of 0 to 200 microns. Likewise, a f t e r spawning, unshed mature oocytes were resorbed. 11. The r o l e of temperature, l i g h t , and s a l i n i t y , i n timing reproduction was discussed, but conclusions could not be drawn based on c o r r e l a t i o n s with f i e l d data. 68 LITERATURE CITED Ahmed, M., and A. K. Sparks, 1970. A note on the chromosome number and i n t e r r e l a t i o n s h i p s i n the marine gastropod genus Thais of the United States P a c i f i c coast. V e l i g e r , 12:293-294. A n s e l l , A. D., and K. F. Lander, 1967. Studies on the ha r d - s h e l l clam, Venus mercenaria, i n B r i t i s h waters. I I I . Further observations on the seasonal biochemical cycle and on spawn-ing. J . Appl. E c o l . , 4:425-435. Association of O f f i c i a l A g r i c u l t u r a l Chemists, 1960. O f f i c i a l methods of analysis of the association of O f f i c i a l A g r i c u l t u r a l Chemists. Ninth ed., Assoc. O f f i c . Agr. Chem., Washington, D. C., 832 p. Barnes, H., 1963. Light, temperature, and breeding of Balanus  balanoides. J . Mar. B i o l . Assoc. U.K., 43:717-728. Barnes, H., M. Barnes, and D. M. Finlayson, 1963. The seasonal changes i n body weight, biochemical composition, and oxygen uptake of two common boreo-arctic c i r r i p e d e s , Balanus balanoides and B. balanus. J . Mar. B i o l . Assoc. U.K., 43:185-211. Barry, R.J.C., and K. A. Munday, 1959. Carbohydrate l e v e l s i n P a t e l l a . J . Mar. B i o l . Assoc. U.K., 38:81-95. Bayne, C. J . , 1968. Histochemical studies on the egg capsules of eight gastropod molluscs. Proc. Malacol. Soc. Lond. 38:199-212. Blackmore, D. T., 1969. Studies of P a t e l l a vulgata L. I I . Seasonal v a r i a t i o n i n biochemical composition. J . exp. mar. B i o l . E c o l . , 3:213-245. Boer, H. H., E. Douma, and J . M. A. Koksma, 1968. Electron microscope study of neurosecretory c e l l s and neurohaemal organs i n the pond s n a i l , Lymnaea s t a g n a l i s . Symp. zool. Soc. Lond., No. 22: 237-256. Boolootian, R. A., 1963. Responses of the testes of purple sea urchins to v a r i a t i o n s i n temperature and l i g h t . Nature, 197:403. Brand, T. von, P. McMahon, and M. 0. Nolan, 1957. P h y s i o l o g i c a l observations on starvation and dessica t i o n of the s n a i l , A u s t r alorbis glabratus. B i o l . B u l l . , 113:89-102. Canada Department of Transport. Meteorological Branch, 1969. 1968 Annual Meteorological Summary. Vancouver, B. C., 53 p. Canada Department of Transport. Meteorological Branch, 1970. 1969 Annual Meteorological Summary. Vancouver, B. C , 54 p. 69 Chapman, W. M., and A. H. Banner, 1949. Contributions to the l i f e h i s t o r y of the Japanese oyster d r i l l , T r i t o n a l i a japonica, with notes on other enemies of the Olympia oyster, Ostrea l u r i d a . B i o l . Rep. Dept. F i s h . , Wash. State, No. 49a:167-200. Cr i s p , D. J . , 1957. E f f e c t of low temperature on the breeding of marine animals. Nature, 179:1138-1139. D a l l , W. H., 1915. Notes on the molluscan subgenus Nucella (Thais) i n h a b i t i n g the northwest coast of America and adjacent regions. Proc. U.S. Nat. Mus., 49:557-572. Daniel, R. J . , 1921. Seasonal changes i n the composition of the mussel, Mytilus ed u l i s . Rep. Lanes. Sea-Fish. Labs, 1920:78-84. Emerson, D. N., 1965. Summer polysaccharide content i n seven species of west coast i n t e r t i d a l prosobranch s n a i l s . V e l i g e r , 8:62-66. Emerson, D. N., 1967. Carbohydrate oriented metabolism of Planorbis  corneus (Mollusca, Planorbidae) during starvation. Comp. Bio-chem. Ph y s i o l . , 22:571-579. Emerson, D. N., and F. G. Duerr, 1967. Some p h y s i o l o g i c a l e f f e c t s of s t a r v a t i o n i n the i n t e r t i d a l prosobranch, L i t t o r i n a planaxis ( P h i l i p p i , 1847). Comp. Biochem. Physiol., 20:45-53. Emlen, J.M., 1966. Time, energy and r i s k i n two species of carnivor-ous gastropods. Ph.D. Thesis. Univ. of Washington. Farmanfarmaian, A., A. C. Giese, R. A. Boolootian, and J . Bennett, 1958. Annual reproductive cycles i n four species of west coast s t a r f i s h e s . J . Exp. Zool., 138:355-367. F r e t t e r , V., 1941. The g e n i t a l ducts of some B r i t i s h stenoglossan prosobranchs. J . Mar. B i o l . Assoc. U.K., 25:173-211. F r e t t e r , V., and A. Graham, 1962. B r i t i s h prosobranch molluscs. Ray Society, London. 755 p. Fritchman, H. K., 1961. A study of the reproductive cycle i n the C a l i f o r n i a Acmaeidae (Gastropoda). V e l i g e r , 3:57-63. Gabe, M., and M. Prenant, 1949. Contribution a l ' h i s t o l o g i e de l'ovogenese chez l e s Polyplacophores. C e l l u l e 53:99-117. Galigher, A. E., and E. N. K o z l o f f , 1964. Ess e n t i a l s of p r a c t i c a l microtechnique. Lea and Febiger, Philadelphia, 484 p. Giese, A. C., 1959. Comparative physiology: Annual reproductive cycles of marine invertebrates. Ann. Rev. Phy s i o l . , 21:547-576. Giese, A. C., 1966. Li p i d s i n the economy of marine invertebrates. Physiol. Rev., 46:244-298. 70 Giese, A. C., 1967. Some methods f o r study of the biochemical con-s t i t u t i o n of marine invertebrates. Oceanogr. Mar. B i o l . Ann. Rev., 5:159-186. Giese, A. C., and G. Araki, 1962. Chemical changes with reproductive a c t i v i t y of the chitons, Katherina tunicata and Mopalia h i n d s i i . J . Exp. Zool., 151:259-267. Giese, A. C , L. Greenfield, A. Farmanfarmaian, H. Huang, R. Boolootian, and R. Lasker, 1959a. Organic p r o d u c t i v i t y i n the reproductive cycle of the purple sea urchin. B i o l . B u l l . , 116:49-58. Giese, A. C. , and M. A. Hart, 1967. Seasonal changes i n component indices and chemical composition i n Katherina tunicata. J . Exp. Mar. B i o l . E c o l . , 1:34-46. Giese, A. C., M. A. Hart, A. M. Smith, and M. A. Cheung, 1967. Seasonal changes i n body component indices and chemical com-p o s i t i o n i n the Pismo clam, T i v e l a stultorum. Comp. Biochem. Phy s i o l . , 22:549-561. Giese, A. C., J . S. Tucker, and R. A. Boolootian, 1959b. Annual reproductive cycles of the chitons, Katherina tunicata and Mopalia h i n d s i i . B i o l . B u l l . , 117:81-88. Graham, A., 1939. On the structure of the alimentary canal of the style-bearing prosobranchs. Proc. Zool. Soc. Lond., Ser. B., 109:75-112. G r i f f i t h , L. M., 1967. The i n t e r t i d a l univalves of B r i t i s h Columbia. B.C. P r o v i n c i a l Museum. Handbook No. 26, V i c t o r i a , B. C., 101 p. Hatanaka, M., 1940. Chemical composition of the oyster, Ostrea gigas. B u l l . Jap. Soc. Scient. F i s h . 9:21-26. Helbert, J . R., and K. D. Brown, 1955. Factors inf l u e n c i n g quantitative determination of methylpentoses and ketohexoses with anthrone. Anal. Chem. 27:1791-1796. Howells, H. H., 1942. The structure and function of the alimentary canal of Apl y s i a. Quart. J . Micr. S c i . , 83:357-397. Joose, J . , M. H. Boer, and C. J . Cornelisse, 1968. Gametogenesis and o v i p o s i t i o n i n Lymnaea stagnalis as influenced by y-radiation and hunger. Symp. Zool. Soc. Lond., No. 22:213-235. Kincaid, T., 1957. Local races and c l i n e s i n the marine gastropod, Thais lamellosa Gmelin - A population study. The Calliostoma Co., Seattle, Wash., 75 p. Largen, M. J . , 1967. The influence of water temperature upon the l i f e of the dog-whelk, Thais l a p i l l u s . J . Animal E c o l . , 36: 207-214. 71 Lawrence, A. L., J . M. Lawrence, and A. C. Giese, 1965. C y c l i c v a r i a t i o n i n the glandular oviduct of chitons. Science, 147: 508-510. Lowry, 0. H., N. J . Roseborough, A. L. Farr, and R. J . Randall, 1951. Protein measurement with the F o l i n phenol reagent. J . B i o l . Chem., 193:265-275. Martin, A. W., 1961. The carbohydrate metabolism of the mollusca. In Comparative physiology of carbohydrate metabolism i n hetero-thermic animals. Ed. A. W. Martin, pp. 35-64. Univ. of Washing-ton Press, Seattle. Masumoto, B., M. Masumoto, and M. Hibino, 1934. Biochemical studies of Magaki (Ostrea gigas Thunberg). I I . The seasonal v a r i a t i o n i n the chemical composition of Ostrea gigas Thunberg. Hiroshima Univ. J . S c i . , A4:47-56. Mauzey, K. P., 1966. Feeding behaviour and reproductive cycles i n Pi s a s t e r ochraceus. B i o l . B u l l . , 131:127-144. n May, F., 1934. Chemische und biologische Untersuchungen uber Galaktogen. Chem. T e i l . Z. B i o l . , 95:277-297. M i l l o t t , N., 1938. On the morphology of the alimentary canal, process of feeding and physiology of digestion of the nudibranch, Jorunna tomentosa. P h i l . Trans. Roy. Soc. B., 228:173-217. M i t c h e l l , P. H., 1915-16. N u t r i t i o n of oysters: glycogen formation and, storage. B u l l . U.S. Bur. F i s h . , 35:151-162. Morton, J . E., 1951. The ecology and digestive system of the S t r u t h i o l a r i i d a e (Gastropoda). Quart. J . Micr. S c i . , 92:1-25. Nimitz, M. A., and A. C. Giese, 1964. Histochemical changes correlated with reproductive a c t i v i t y and n u t r i t i o n i n the chiton, Katherina tunicata. Quart. J . Micr. S c i . , 105:481-495. Northcott, R. J . (ed.), 1968. The observer's handbook 1968. The Royal Astronomical Society of Canada, Toronto, Ont., 100 p. Northcott, R. J . (ed.), 1969. The observer's handbook 1969. The Royal Astronomical Society of Canada, Toronto, Ont., 100 p. Orton, J . H., A. J . Southward, and J . M. Dodd, 1956. Studies on biology of the limpets. I I . The breeding of P a t e l l a vulgata L. i n B r i t a i n . J . Mar. B i o l . Assoc. U.K., 35:149-176. P a c i f i c Oceanographic Group, 1951. Fraser River estuary project, 1950. J o i n t Committee on Oceanography, Nanaimo, B. C. 72 Pantin, C. F. A., 1964. Notes on microscopical technique f o r zoolo-g i s t s . Cambridge Uni v e r s i t y Press, Cambridge, 76 p. Patent, D. H., 1969. The reproductive cycle of Gorgonocephalus c a r y i (Echlnodermata, Ophiuroidea). B i o l . B u l l . , 136:241-252. Pearse, J . S., 1965. Reproduction p e r i o d i c i t i e s i n several con-t r a s t i n g populations of Odontaster validus Koehler, a common an t a r c t i c asteroid. In Biology of the a n t a r c t i c seas I I . Ed. G. A. Llano, A n t a r c t i c Res. Ser. Vol. 5, American Geo-ph y s i c a l Union. Raven, C. P., 1966. Morphogenesis: The analysis of molluscan development. 2nd ed. I n t e r n a t i o n a l Series of Monographs i n Pure and Applied Biology, Zoology Div., Vol. 2, Pergamon Press, London 365 p. Ropes, J . W., 1968. Reproductive cycle of the Surf Clam, Spisula s o l i d i s s i m a , i n offshore New Jersey. B i o l . B u l l . , 135:349-365. R u s s e l l , E. S., 1923. Report on seasonal v a r i a t i o n i n chemical composition of oysters. F i s h . Invest. Lond. Ser. 2, 6:No. 1. Sakuda, H. M., 1966. Reproductive cycle of American oyster Crassostrea v i r g i n i c a i n West Loch, Pearl Harbour, Hawaii. Trans. Amer. F i s h . S o c , 95:216-218. Sastry, A. N., 1966. Temperature e f f e c t s i n reproduction of the bay s c a l l o p , Aequipecten i r r a d i a n s Lamarck. B i o l . B u l l . , 130: 118-134. Sastry, A. N., 1968. The r e l a t i o n s h i p s among food, temperature and gonad development of the bay s c a l l o p , Aequipecten i r r a d i a n s Lamarck. Physiol. Zool., 41:44-53. Seapy, R. R., 1966. Reproduction and growth i n the f i l e limpet, Acmaea limat u l a Carpenter 1864. V e l i g e r , 8:300-310. S e i f t e r , S., S. Dayton, B. Novic, and E. Muntwyler, 1950. The estimation of glycogen with the anthrone reagent. Arch. Biochem., 25:191-200. Selwood, L., 1968. I n t e r r e l a t i o n s h i p s between developing oocytes and ovarian t i s s u e s i n the chiton, Sypharochiton septentriones (Ashby) (Mollusca, Polyplacophora). J . Morph., 125:71-104. Selwood, L., 1970. The r o l e of the f o l l i c l e c e l l s during oogenesis i n the chiton, Sypharochiton septentriones (Ashby) (Mollusca, Polyplacophora). Z. Z e l l f o r s c h , 104:178-192. Sperry, W. M., and F. C. Brand, 1955. The determination of t o t a l l i p i d e s i n blood serum. J . B i o l . Chem., 213:69-76. 73 Struhsaker, J . W., 1966. Breeding, spawning, spawning p e r i o d i c i t y and e a r l y development i n the Hawaiian L i t t o r i n a : L. pintado (Wood), L. p i c t a P h i l i p p i , and L. scabra (Linne). Proc. Male. Soc. Lond., 37:137-166. Thorson, G., 1946. Reproduction and l a r v a l development o f Danish marine bottom invertebrates with s p e c i a l reference to the planktonic larvae i n the Sound ( 0 r e s u n d ) . Medd. Komm. Havun-dersog., Kbh., Plankton, 4:1-523. Thorson, G., 1950. Reproduction and l a r v a l ecology of marine inver-tebrates. B i o l . Rev., 25:1-45. Thorson, G., 1958. P a r a l l e l level-bottom communities, t h e i r temper-ature adaption, and t h e i r "balance" between predators and food animals. In Perspectives i n marine biology. Ed. A. A. Buzzati-Traverso. pp. 67-86. Univ. of C a l i f . Press, Berkeley. Tucker, J . S., and A. C. Giese, 1962. Reproductive cycle of Crypto- chiton s t e l l e r i (Middledorf). J . Exp. Zool., 150:33-43. Ward, J . , 1966. The breeding cycle of the keyhole limpet, F i s s u r e l l a  barbadensis Gmelin. B u l l . Mar. S c i . , 16:685-695. Webber, H. H., and A. C. Giese, 1969. Reproductive cycle and gameto-genesis i n the black abalone, H a l i o t i s c r a c h e r o d i i (Gastropoda: Prosobranchiata). Mar. B i o l . , 4:152-159. White, A., P. Handler, and E. L. Smith, 1964. P r i n c i p l e s of b i o -chemistry. Third ed. McGraw-Hill Book Co., New York, 1106 p. Wilber, C. G., 1947. The e f f e c t of prolonged s t a r v a t i o n on'the l i p i d s i n Phascolosoma g o u l d i i . J . C e l l . Comp. Physiol., 29:179-183., Wilson, B. R., and E. P. Hodgkin, 1967. A comparative account of the reproductive cycles of f i v e species of marine mussels i n the v i c i n i t y of Fremantle, W. A u s t r a l i a . Aust. J . mar. Freshwat. Res., 18:175-203. Yonge, C. M., 1937. Evolution and adaption i n the digestive system of the metazoa. B i o l . Rev., 12:87-115. 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0093364/manifest

Comment

Related Items