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Aspects of the reproductive biology of Strombus gigas Egan, Brian Denis 1985

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ASPECTS OF THE REPRODUCTIVE BIOLOGY OF STROMBUS GIGAS by BRIAN DENIS EGAN BSc., U n i v e r s i t y of B r i t i s h Columbia, Vancouver, 1980 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Zoology) We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA July,1985 @ (c) B r i a n Denis Egan, fftfS In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available f o r reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by h i s or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of ^cxO^oC/  The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 i i ABSTRACT S h e l l .morphology changes r e l a t i n g to sexual m a t u r i t y and the r e p r o d u c t i v e c y c l e of Strombus gigas were s t u d i e d at a s i n g l e s i t e w i t h i n the B a r r i e r Reef lagoon of B e l i z e , C e n t r a l America. Monthly samples, from J u l y 1981 to June 1983, of tr a n s v e r s e t i s s u e s e c t i o n s , through the d i g e s t i v e gland and gonad, were f i x e d f o r examination by l i g h t microscopy. S e c t i o n s from a l l areas of the gonad were used t o develop a sexual m a t u r i t y s c a l e that would permit the maturity of a specimen to be determined from the c o n d i t i o n of a s i n g l e t i s s u e sample. The e f f e c t i v e n e s s of the technique d e s c r i b e d , for use in other meso- or neogastropods, i s dependent on simultaneous maturation throughout the gonad and on an even d i s t r i b u t i o n of r e p r o d u c t i v e s t r u c t u r e s w i t h i n . Sexual maturity i n S. gigas (as d e f i n e d by the development of mature gametes) f i r s t occurs when i n d i v i d u a l s have a f l a r i n g s h e l l l i p - t h i c k n e s s of approximately 4 mm. Sexual m a t u r i t y i s reached approximately 1 year a f t e r maximum s h e l l length i s reached and approximately 6 months a f t e r development of the f l a r i n g l i p , at an age of roughly 4 y e a r s . Ninety four percent of the p o p u l a t i o n s t u d i e d a t t a i n e d the l e g a l s h e l l l e n g t h , i n B e l i z e , of 175 mm. The time delay between a t t a i n i n g l e g a l l e n g t h and becoming s e x u a l l y mature means that there i s a p e r i o d , f o r 94% of the p o p u l a t i o n , when they may be l e g a l l y harvested before becoming s e x u a l l y mature.. Reproductive a c t i v i t y occurs year-round i n the p o p u l a t i o n s t u d i e d . An annual p a t t e r n of a c t i v i t y was not apparent through i n s p e c t i o n of the data alone. No s t a t i s t i c a l c o r r e l a t i o n was found between gonadal a c t i v i t y and average monthly a i r temperature or monthly r a i n f a l l . i v TABLE OF CONTENTS ABSTRACT i i LIST OF TABLES v i i LIST OF FIGURES v i i i ACKNOWLEDGEMENTS x Chapter 1: General I n t r o d u c t i o n to Research 1 General I n t r o d u c t i o n 1 P r o j e c t O b j e c t i v e s 2 The Need f o r Research 4 General Biology 9 Anatomy 12 General Methods and M a t e r i a l s 19 C o l l e c t i n g Specimens 24 Measurements 26 Ti s s u e Samples 26 Ti s s u e P r e p a r a t i o n 29 Ti s s u e A n a l y s i s 30 Chapter 2: H i s t o l o g y of the Gonad 31 I n t r o d u c t i o n 31 Spermiogenesis 33 Oogenesis 36 Methods and M a t e r i a l s 37 Re s u l t s and D i s c u s s i o n 38 The Male Gonad ..38 Sperm i n Strombus gigas 40 Spermiogenesis 41 Maturation Process of the Male Gonad 44 The Female Gonad 46 Oogenesis . . 4 7 Maturation Process of the Female Gonad 48 A M a t u r i t y Scale 50 U n d i f f e r e n t i a t e d "0" 51 Male C o n d i t i o n I "Beginning" 51 Male C o n d i t i o n II "Developing" 52 Male C o n d i t i o n III "Ripe" 53 Male C o n d i t i o n IV "Spent" 54 Female C o n d i t i o n I "Beginning" 55 Female C o n d i t i o n II "Developing" 55 Female C o n d i t i o n III "Ripe" 56 Female C o n d i t i o n IV "Spent" 56 Co n c l u s i o n s 57 Chapter 3: S h e l l Morphology at Sexual M a t u r i t y 80 I n t r o d u c t i o n 80 Methods and M a t e r i a l s 81 R e s u l t s 84 P r e c i s i o n of L i p Thickness Measurements 84 R e l a t i o n s h i p Between F l a r i n g L i p Thickness and Gonadal M a t u r i t y 84 R e l a t i o n s h i p Between Maximum S h e l l Length and Gonadal M a t u r i t y , 102 D i s c u s s i o n and Co n c l u s i o n s 106 Chapter 4: Season of Sexual M a t u r i t y 110 v i I n t r o d u c t i o n 110 Methods and M a t e r i a l s 111 R e s u l t s 113 S e r i a l S e c t i o n s 113 Weather Data 114 Gonadal A c t i v i t y versus Time and Weather C o n d i t i o n s .115 D i s c u s s i o n and Conclusions 131 T h i s Study 133 Concluding Remarks 136 References C i t e d 137 Appendix A - F i x a t i o n and P r e s e r v a t i o n 144 Appendix B - H i s t o l o g i c a l Technique 145 v i i LIST OF TABLES Table 1: Key to L e t t e r i n g In F i g u r e s 7 to 23 60 Table 2: The Extremes of S h e l l L i p Measures For Each Category of Sexual M a t u r i t y 90 Table 3: The Mean and Standard D e v i a t i o n s of S h e l l Length and L i p - T h i c k n e s s Measures of A d u l t s and J u v e n i l e s 100 Table 4: Table of Sampling Dates, Sample S i z e s and A c t i v i t y L e v e l s f o r A l l Samples 122 LIST OF FIGURES F i g u r e 1: Annual B e l i z e a n Conch Exports and T h e i r R e l a t e d Revenues, 1966 - 1982 6 F i g u r e 2: S h e l l C h a r a c t e r i s t i c s and R e l a t e d Measures 13 F i g u r e 3: General Anatomy of the Soft P a r t s 15 F i g u r e 4: Map of B e l i z e 20 F i g u r e 5: Map of Study s i t e 22 F i g u r e 6: Diagram I l l u s t r a t i n g T i s s u e P r e p a r a t i o n 27 F i g u r e 7: Closeup of The Dimorphic Sperm 62 F i g u r e 8: Closeup on Ovary Within Oviduct 63 F i g u r e 9: U n d i f f e r e n t i a t e d F o l l i c l e s ...64 F i g u r e 10: F i r s t D e t e c t a b l e Signs of S e x u a l i t y In Males ....65 F i g u r e 11: End of Male C o n d i t i o n 1 66 F i g u r e 12: Male C o n d i t i o n 11 - Low D e n s i t y F o l l i c l e s 67 F i g u r e 13: Sperm B u i l d - u p Within The Vas deferens of Young Males 68 F i g u r e 14: I n d i v i d u a l F o l l i c l e of Ripe Male 69 F i g u r e 15: Vas deferens Swollen With Stored Sperm 70 F i g u r e 16: Spent and/or R e s t i n g F o l l i c l e s of Male IV 71 F i g u r e 17: Spent and/ar R e s t i n g Vas deferens 72 F i g u r e 18: The F i r s t Signs of Oogonia 73 F i g u r e 19: End of Female C o n d i t i o n 1 74 F i g u r e 20: Female C o n d i t i o n 1 F o l l i c l e at Intermediate Stage of Development 75 F i g u r e 21: Female C o n d i t i o n 11 F o l l i c l e at Intermediate Stage of Development 76 F i g u r e 22: Overview of Ripe Female 77 F i g u r e 23: Spent and/or Resting F o l l i c l e s of Female IV 78 F i g u r e s 24A Through 24E: R e l a t i o n s h i p s Between C a t e g o r i e s of Sexual C o n d i t i o n and L i p Thicknesses i n One M i l l i m e t e r I n t e r v a l s 86 F i g u r e 25: S i z e D i s t r i b u t i o n of The E n t i r e Sample Set And of The Random Sample Set 92 F i g u r e 26A Through 26E: R e l a t i o n s h i p Between C a t e g o r i e s of Sexual C o n d i t i o n and S h e l l Length i n Ten M i l l i m e t e r I n t e r v a l s 96 F i g u r e 27: Graph of Average Monthly Weather Data From The B e l i z e Weather Bureau and Ambergris Caye 116 F i g u r e 28: Graph of Average D a i l y A i r And Water Temperature Readings f o r a Three Month P e r i o d 118 F i g u r e 29: Histogram of Gonadal A c t i v i t y vs. Time I n c l u d i n g a l l Data and Samples 120 F i g u r e 30: Histogram of the Percentage Frequency of Ripe Specimens vs. Time and the Re l a t e d 95% Confidence I n t e r v a l s 125 F i g u r e 31A and 31B: Histogram of Ripe Specimens vs. Time Along With A i r Temperature and R a i n f a l l f o r the Same P e r i o d 127 F i g u r e • 32: Histogram of Ripe Specimens vs. Time for Specimens with L i p Thicknesses Greater Than 10 mm Only .129 X ACKNOWLEDGEMENTS I would l i k e to thank my s u p e r v i s o r , Dr. T.R. Parsons, for h i s p a t i e n c e and support throughout the 4 years i t has taken to complete my MSc. I am g r a t e f u l to my e n t i r e s u p e r v i s o r y committee ( i n c l u d i n g Doctors CM. L a l l i , T.H. Car e f o o t and F.J.R. T a y l o r ) f o r t h e i r encouragement and the freedom they have a f f o r d e d me. Many people have c o n t r i b u t e d to t h i s t h e s i s e i t h e r d i r e c t l y or i n d i r e c t l y . The most notable i s S. S t r a s d i n e whose o r g a n i z a t i o n a l s k i l l s and d i l i g e n c e made t h i s t h e s i s p o s s i b l e . S p e c i a l thanks to W. M i l l e r and J . Gibson f o r t h e i r time and t r o u b l e , Dr. F.R. Bernard f o r h i s ideas, Dr. 'D.B. Quayle for ad v i c e , Kevin Gonzalez f o r h i s t e c h n i c a l a s s i s t a n c e and J . Bagshaw f o r h i s t o l o g i c a l p r e p a r a t i o n . L a s t , but not l e a s t , a s p e c i a l thanks to H.M. Dovey s u p e r s t a r , f o r t y p i n g the manuscript, e d i t i n g the t e x t , drawing the f i g u r e s and nagging me to get on with i t . I r e c e i v e d f i n a n c i a l support from the I n t e r n a t i o n a l Development Research Centre i n the form of a post p r o j e c t award - f o r which I am most g r a t e f u l . 1 CHAPTER U GENERAL INTRODUCTION TO RESEARCH GENERAL INTRODUCTION T h i s r e p o r t d e s c r i b e s the work and f i n d i n g s of s t u d i e s undertaken under the auspices of the Conch O p t i m i z a t i o n P r o j e c t (COP) i n B e l i z e , C e n t r a l America. The p r o j e c t was funded and d i r e c t e d by the I n t e r n a t i o n a l Development Research Centre (I.D.R.C.) i n Ottawa. I worked i n B e l i z e as a s t a f f member from August, 1980, u n t i l J u l y , 1982. T h i s report d e s c r i b e s only a small p o r t i o n of the many and v a r i e d s t u d i e s undertaken d u r i n g that p e r i o d . For s i m p l i c i t y and c l a r i t y , t h i s r e p ort has been d i v i d e d i n t o f i v e s e l f - c o n t a i n e d and r e l a t i v e l y independent c h a p t e r s . These a r e : 1. General I n t r o d u c t i o n 2. H i s t o l o g y of The Gonad; a Scale of Maturi ty 3. Morphology at F i r s t Sexual M a t u r i t y 4. Season of Sexual M a t u r i t y Chapters 2 through 4 are s e l f - c o n t a i n e d and i n c l u d e t h e i r own i n t r o d u c t i o n , methods and m a t e r i a l s , r e s u l t s , d i s c u s s i o n and. c o n c l u s i o n s . P r e l i m i n a r y to t h i s , however, Chapter 1 p r o v i d e s a d e s c r i p t i o n of the 2 o v e r a l l o b j e c t i v e s and methodology. •Project O b j e c t i v e s The u l t i m a t e g o a l s of the p r o j e c t were d e s c r i b e d by Dr. W.H.L. A l l s o p p , of I.D.R.C., w i t h i n the P r o j e c t Summary (I.D.R.C. document 3-P-79-0097, Nov. 1979) as f o l l o w s : "Object ives G e n e r a l l y : A) to study the l i f e h i s t o r y of the Caribbean conch Strombus gigas with a view to e s t a b l i s h i n g a f i r m b i o l o g i c a l b a s i s f o r the r a t i o n a l h a r v e s t i n g of the conch, i n c l u d i n g the c o n s i d e r a t i o n of e f f e c t i v e management measures s u i t a b l e f o r the improvement and cropping of t h i s r esource; and, B) to e x p l o r e the u t i l i z a t i o n of s h e l l s and other p r o d u c t s and the p o s s i b i l i t y of conch m a r i c u l t u r e . S p e c i f i c a l l y : A) to study the conch's l i f e h i s t o r y 3 ( i n c l u d i n g growth r a t e s , s i z e at m a t u r i t y , breeding h a b i t s , breeding seasons, food h a b i t s , e c o l o g i c a l p r e f e r e n c e s , s u r v i v a l and mi g r a t o r y h a b i t s ) ; B) to c o l l e c t s t a t i s t i c a l data f o r the assessment of the stocks a v a i l a b l e to the f i s h e r y ; C) to r e l a t e t h i s i n f o r m a t i o n i n e s t a b l i s h i n g the b a s i s f o r e f f e c t i v e r e g u l a t o r y and management measures i n regard to s i z e l i m i t s , c l o s e d seasons, breeding areas or p o s s i b l e aquaculture systems; and, D) to i n v e s t i g a t e the u t i l i z a t i o n of the s h e l l s for s h e l l c r a f t so as to a v o i d wastage and provide employment f o r f i s h i n g commun i t i e s . " To achieve these goals r e q u i r e d a great v a r i e t y of e d u c a t i o n a l , e c o l o g i c a l and s t a t i s t i c a l programs. Reproductive b i o l o g y , as an i n t e g r a l part of the o v e r a l l o b j e c t i v e s , was chosen as a t h e s i s t o p i c . 4 The Need f o r Research The Conch O p t i m i z a t i o n P r o j e c t was b e l i e v e d to be necessary, p a r t i c u l a r l y w i t h i n B e l i z e , f o r the f o l l o w i n g reasons. Strombus qigas , commonly r e f e r r e d to as the queen conch, (pronounced konk), i s the Caribbean r e g i o n ' s most commercially important mollusc (Brownell and S t e v e l y 1981). H i s t o r i c a l l y , i t s abundance in a c c e s s i b l e , shallow waters has f a c i l i t a t e d i t s use as a p r i n c i p a l p r o t e i n source f o r l o c a l i n h a b i t a n t s s i n c e settlement by the f i r s t American Indians (Brownell and S t e v e l y 1981). U n t i l as r e c e n t l y as the mid 1960's, the conch remained inexpensive and r e a d i l y a c c e s s i b l e . However, in the l a t e 1960's a l u c r a t i v e market for f r o z e n conch meat developed i n the United S t a t e s . Americans of Caribbean o r i g i n , l i v i n g p r i n c i p a l l y i n F l o r i d a and New York, began to provide a r a p i d l y growing export market. T h i s demand d r a m a t i c a l l y i n c r e a s e d the economic s i g n i f i c a n c e of S. gigas as a f i s h e r i e s resource throughout the Caribbean (Brownell and S t e v e l y 1981). Consequently, f i s h i n g pressure rose i n a number of c o u n t r i e s (e.g., B e l i z e , Turks and Caicos I s l a n d s ) . L u c r a t i v e f o r e i g n markets drove up l o c a l p r i c e s , thus reducing the a v a i l a b i l i t y of the meat to poorer Caribbean c i t i z e n s (Brownell and S t e v e l y 1981). Catch s t a t i s t i c s f o r the B e l i z e a n conch f i s h e r y provide a c l a s s i c example of stock d e p l e t i o n caused by commercial 5 o v e r f i s h i n g . Figure 1 d e s c r i b e s how conch exports r a p i d l y grew in the mid to l a t e 1960's, c u l m i n a t i n g at 500,000 kg i n 1972, then s u f f e r i n g c o n s i s t e n t d e c l i n e i n subsequent y e a r s . F i g u r e 1 a l s o i l l u s t r a t e s how the s t e a d i l y i n c r e a s i n g export p r i c e s for conch meat allowed revenues from the f i s h e r y to remain high d e s p i t e d e c l i n i n g c a t c h e s . P e r s i s t e n t f i s h i n g p r e s s u r e , d e s p i t e low c a t c h per u n i t e f f o r t , has c o n t r i b u t e d to the s e v e r i t y of the o v e r f i s h i n g problem. The poverty of B e l i z e a n and other Caribbean fishermen has promoted o v e r f i s h i n g to l e v e l s well beyond those which might be economically s u s t a i n e d i n more developed and w e a l t h i e r n a t i o n s . Export s t a t i s t i c s t h e r e f o r e do not adequately r e f l e c t the s e v e r i t y of stock d e p l e t i o n . A b r i e f assessment of conch s t o c k s performed by the COP s t a f f i n e a r l y 1981 c l e a r l y i n d i c a t e d that a l l known po p u l a t i o n s of S. g i g a s within B e l i z e were under heavy f i s h i n g pressure and many were e s s e n t i a l l y decimated. No u n e x p l o i t e d resources e x i s t e d . Stock d e p l e t i o n has f o r c e d most fishermen to abandon f i s h i n g e x c l u s i v e l y f o r conch. Instead they harvest the few that are i n c i d e n t a l l y encountered while f i s h i n g f o r other seafoods (e.g., spiny l o b s t e r or s c a l e f i s h ) . D e s p i t e the d r a s t i c r e d u c t i o n i n exports, conch remains the second l a r g e s t f i s h e r i e s export of B e l i z e . The s i g n i f i c a n c e of t h i s i s a p p r e c i a t e d most when one r e a l i z e s that f i s h e r i e s 6 FIGURE 1: Conch exports and r e l a t e d revenues ( i n kilograms and B e l i z e a n d o l l a r s ) 1965-1982. Source: B e l i z e F i s h e r i e s U n i t . Quantity of Revenues (Belizean Dollars) or Exports (Pounds) X 1000 8 products are B e l i z e ' s t h i r d l a r g e s t f o r e i g n exchange earner. The conch thus continues to be a r e l a t i v e l y s i g n i f i c a n t export, commodity ( S t r a s d i n e , M i l l e r and Gonzalez 1983). The COP was formulated to t r y to save what had become a n a t i o n a l l y v a l u a b l e f i s h e r y and what had always been an important l o c a l food source. At the request of the B e l i z e a n government, I.D.R.C., CUSO and the B e l i z e a n F i s h e r i e s Unit undertook the p r o j e c t . F o r e i g n a i d was necessary i n t h i s i n s t a n c e because, as a f i s h e r y w i t h i n predominantly t h i r d world c o u n t r i e s , p r i v a t e e n t e r p r i s e and/or wealthy c o u n t r i e s would not otherwise have funded such r e s e a r c h . The B e l i z e a n F i s h e r i e s U n i t (under the d i r e c t i o n of Mr. Winston M i l l e r ) has a l s o d i s p l a y e d c o n s i d e r a b l e i n t e r e s t i n management, but has been unable to f i n a n c e e x t e n s i v e r e s e a r c h on t h e i r own. P r e l i m i n a r y s t u d i e s of conch b i o l o g y w i t h i n B e l i z e began as e a r l y as 1976 ( B l a k e s l e y 1977). The COP was formulated i n order to modify e x i s t i n g B e l i z e a n management p r a c t i c e s and to help other Caribbean n a t i o n s do the same. As a member of the p r o j e c t s t a f f , I was pe r m i t t e d the independence to emphasize r e s e a r c h areas of my c h o i c e . A d e t a i l e d study of the r e p r o d u c t i v e b i o l o g y of the conch was co n s i d e r e d to be of fundamental importance to the p r o j e c t o b j e c t i v e s , while at the same time p r o v i d i n g a sub j e c t s u i t a b l e for a Masters T h e s i s . The comparatively small amount of f i e l d 9 time s p e c i f i c a l l y r e q u i r e d f o r gonad sampling allowed me to p a r t i c i p a t e i n a great v a r i e t y of other f i e l d s t u d i e s while accumulating the r e q u i s i t e M.Sc. data. T h i s was aided by the f a c t that h i s t o l o g i c a l p r e p a r a t i o n , a n a l y s i s and i n t e r p r e t a t i o n of gonadal t i s s u e was best l e f t f o r when I was i n Canada, where lack of equipment and e x p e r t i s e were not hindrances. GENERAL BIOLOGY Strombus gigas Linnaeus, i s a member of the marine mesogastropod f a m i l y Strombidae. Strombids are circumpolar i n o d i s t r i b u t i o n , but occur p r i n c i p a l l y w i t h i n the 20 C r e s t r i c t i v e isotherm i n shallow waters with hermatypic c o r a l ( H o r u i c h i and Lane 1965). S. gigas i s r e s t r i c t e d to shallow c o a s t a l waters of the Caribbean, north to Bermuda and south to B r a z i l (Brownell and S t e v e l y 1981). In g e n e r a l , S. Gigas are o p p o r t u n i s t i c h e r b i v o r e s . The dominant algae w i t h i n a p a r t i c u l a r h a b i t a t tends to be the p r i n c i p a l food (Brownell and S t e v e l y 1981). While on a sand s u b s t r a t e , they w i l l i n g e s t q u a n t i t i e s of sand from which they d e r i v e u n i c e l l u l a r algae and d e t r i t u s (Robertson 1961). When a v a i l a b l e , a wide v a r i e t y of macroscopic algae are grazed upon d i r e c t l y (Robertson 1961). Although S. gigas most commonly i n h a b i t s sandy bottoms with 10 abundant seagrass and a l g a l growth, i t i s are a l s o found on g r a v e l , c o r a l rubble and beach rock bottoms. T h e i r v e r t i c a l d i s t r i b u t i o n ranges from a depth of a few centimeters, down to 76 m but they are seldom below 30 m (Randall 1964). R e s t r i c t i o n to shallow waters i s g e n e r a l l y a t t r i b u t e d to l i m i t a t i o n s of l i g h t f o r p l a n t growth (Robertson 1961, Randall 1964). A unique mode of locomotion among the Strombidae d i f f e r e n t i a t e s them from other gastropods. They progress over the s u b s t r a t e by sudden leaps and not by the slow g l i d i n g movements of most s n a i l s (Parker 1922). The h o o k - l i k e operculum i s used i n locomotion and i n the r i g h t i n g of the s h e l l . The m o t i l i t y of Strombus i s an a i d i n escaping p r e d a t o r s and i s not r e l a t e d to predatory feeding h a b i t s (Robertson 1961). Reproduction i s by i n t e r n a l f e r t i l i z a t i o n . The penis i s extended under the female's f l a r i n g s h e l l l i p , through the si p h o n a l notch and up i n t o the g e n i t a l r e g i o n . C o p u l a t i o n occurs e i t h e r d u r i n g the day or night and can precede spawning by s e v e r a l weeks (Randall 1964, D'Asaro 1965). Spawning takes from 24 to 36 hours f o r completion (Randall 1964, D'Asaro 1965). In g e n e r a l , the eggs are l a i d i n areas with c l e a n c o r a l sand that i s low i n organic content, and where there i s open ocean water renewal (Brownell and S t e v e l y 1981). The eggs form a s i n g l e continuous tube which i s f o l d e d upon and s t i c k s t o i t s e l f and to sand g r a i n s , producing a compact, c r y p t i c mass. 11 The number of eggs per spawn has been estimated at between 300,000 and one h a l f m i l l i o n (Robertson 1959, R a n d a l l 1964, D'Asaro 1965). Up to e i g h t egg masses have been l a i d by a s i n g l e female w i t h i n a p e r i o d of s i x months (Davis and Hesse 1982). I n d i v i d u a l , encapsulated eggs hatch i n t o p l a n k t o t r o p h i c v e l i g e r l a r v a e a f t e r approximately f i v e days (D'Asaro 1965). Wi t h i n 20 to 30 days the l a r v a e have metamorphosed and s e t t l e d (Brownell and S t e v e l y 1981). While small (<80mm), the conch tends to be n o c t u r n a l , remaining b u r i e d d u r i n g the day and emerging at night to feed (Brownell and S t e v e l y 1981). D a y l i g h t a c t i v i t y i n c r e a s e s with age. Growth r a t e s have been estimated by a number of authors (Berg 1976, Brownell 1977, A l c o l a d a 1976, Hesse 1976, Wefer and K i l l i n g l e y 1980, Wood and Olsen 1982). Average mean lengths (taken from the t i p of the s p i r e to the d i s t a l end of the s i p h o n a l canal) a r e : 9.7 cm f o r the f i r s t year of growth, 15 cm a f t e r the second and 19 cm a f t e r the t h i r d . At approximately 2.5-3 years the conch i s r e p o r t e d to stop s p i r a l growth and to begin development of a f l a r i n g l i p (Berg 1976). I n i t i a l l y the f l a r i n g l i p i s t h i n and broad but, with age, the l i p erodes at i t s outer edge due to the a c t i v i t y of boring organisms and a b r a s i o n . T h i c k e n i n g of the l i p continues throughout the remainder of the l i f e c y c l e . S h e l l m a t e r i a l i s 1 2 added to the inner s u r f a c e s of the a p e r t u r e . The r e s u l t i s a t h i c k , narrow l i p and a d i m i n i s h e d a p e r t u r e (Brownell and S t e v e l y 1981). S h e l l weight i n c r e a s e s s i g n i f i c a n t l y a f t e r development of the f l a r i n g l i p , whereas v i s c e r a l weight shows l i t t l e change (Berg 1976, B l a k e s l e y 1977). Sexual m a t u r i t y i s b e l i e v e d to occur a f t e r the f l a r i n g l i p i s w e l l developed, at an age of 3-3.5 years. Longevity i s estimated at 6 years (Hesse 1975, Berg 1976). Predators of the conch i n c l u d e other gastropods, t u r t l e s , c r a b s , l o b s t e r , octopus and a great v a r i e t y of f i s h (Randall 1964). N a t u r a l p r e d a t i o n i s presumed to be the h e a v i e s t source of m o r t a l i t y of j u v e n i l e s while man i s the most s i g n i f i c a n t predator of conch longer than approximately 12 cm (Brownell and S t e v e l y 1 981 ) . Anatomy S h e l l c h a r a c t e r i s t i c s and r e l a t e d measurements are d e p i c t e d in F i g . 2. Clench and Abbott (1941) have d e s c r i b e d s h e l l f e a t u r e s more completely. The general anatomy of the s o f t p a r t s d e s c r i b e d below i s d e p i c t e d i n F i g . 3. Strombus gig a s has a long and extremely e x t e n s i b l e snout or p r o b o s c i s with a t e r m i n a l , v e r t i c a l , s l i t - s h a p e d mouth. Within 13 FIGURE 2: S h e l l c h a r a c t e r i s t i c s and r e l a t e d measures of Strombus g i g a s . 15 FIGURE 3: General e x t e r n a l anatomy of the s o f t p a r t s of Strombus g i g a s . LEGEND: cm - columella muscle es - eyestalk fg - female genital groove k - kidney mc - mantle collar oe - oesophogus op - operculum pd - posterior digestive gland r - rectum rad - right anterior digestive gland st - stomach stt - transparent area of stomach wall ut - uterus & uterine glands 1 7 the p r o b o s c i s i s a t a e n i o g l o s s a n radula and c h i t i n i z e d jaws ( L i t t l e 1965). The foot ( d e s t i n e d to be the f i l l e t ) i s l a r g e and powerful. The white muscle i s covered by a t h i c k s k i n . Only the a n t e r i o r p o r t i o n of the foot i s a p p l i e d to the s u b s t r a t e , the " s o l e " of the p o s t e r i o r two-thirds being rounded ( L i t t l e 1965). There i s a s i c k l e - s h a p e d operculum at the p o s t e r i o r end of the foot that i s only a t t a c h e d f o r o n e - t h i r d of i t s l e n g t h . The operculum i s strengthened by a c e n t r a l r i b and i s important i n the c h a r a c t e r i s t i c strombid l e a p i n g ( L i t t l e 1965). The white c o l u m e l l a muscle i s l a r g e and i s a t t a c h e d to the s h e l l c o l u m e l l a across the e n t i r e width of the body. ( I t i s t h i s connection that i s severed in the c l e a n i n g process.) The mantle i s g e n e r a l l y c o l o u r e d by a dark pigment a n t e r i o r l y and fades to a l i g h t grey more p o s t e r i o r l y . The kidney appears as a dark brown mass immediately behind the mantle c a v i t y . The n e p h r i d i a l gland and p e r i c a r d i u m are next to the kidney. The stomach i s p o s t e r i o r to these and c o n t a i n s a l a r g e c r y s t a l l i n e s t y l e . There are three d i g e s t i v e glands, one of which i s lar g e and p o s t e r i o r l y p l a c e d , running to the t i p of the v i s c e r a l hump where i t i s p a r t i a l l y covered by gonadal t i s s u e ( L i t t l e 1965). The g e n i t a l duct, a f t e r e n t e r i n g the mantle c a v i t y , runs 18 p a r a l l e l to the rectum ( L i t t l e 1965). In the female, the g e n i t a l groove runs across the foot and i n t o the pedal groove at the base of the a n t e r i o r end of the foot ( L i t t l e 1965). In the male the g e n i t a l groove runs to the base of the penis which protrudes from the r i g h t s i d e of the f o o t . A more complete d e s c r i p t i o n of conch anatomy i s p r o v i d e d by L i t t l e (1965). 19 GENERAL METHODS AND MATERIALS A l l sample animals were c o l l e c t e d w i t h i n B e l i z e a n waters i n an area r e f e r r e d to as Boca Chica ( F i g . 4 and 5). T h i s study s i t e was approximately 8 m i l e s south of my r e s i d e n t v i l l a g e of San Pedro on Ambergris Caye. A l l t i s s u e samples were taken from f r e s h specimens c o l l e c t e d by l o c a l fishermen who returned to us the f i r s t 50 t h i c k - l i p p e d conch from t h e i r day's c a t c h . For each animal, the fishermen were p a i d $2.50 Bz ($1.25 U.S.), twice what would normally be p a i d to the fishermen f o r the meat alone. I a c t i v e l y p a r t i c i p a t e d in the c o l l e c t i o n s during the f i r s t few months, as w e l l as during l a t e r sampling f o r the s i z e at maturity study. Fishermen were h i r e d to do the sampling f o r the f o l l o w i n g reasons: 1) we alone were unable to f i n d the necessary 50 animals w i t h i n a day; 2) the arrangement allowed p r o j e c t s t a f f to wait for the fishermen and be ready f o r immediate s e c t i o n i n g of the animals upon t h e i r r e t u r n ; 3) the fishermen gained a sense of p a r t i c i p a t i o n and t h i s was the c a t a l y s t f o r f u r t h e r i n t e r e s t ; and 4) the fishermen's s k i l l at l o c a t i n g the conch improved the q u a l i t y of the sample. A random sample from the e n t i r e a d u l t 20 FIGURE 4: Map of B e l i z e and i t s c o a s t a l waters i n c l u d i n g the study s i t e . 2) Scale: . 34 km 22 FIGURE 5: Diagram of the study s i t e showing thermograph l o c a t i o n and approximate depths. 24 p o p u l a t i o n was r e q u i r e d . Only experienced fishermen are capable of f i n d i n g the o l d , small and/or c r y p t i c a l l y overgrown s h e l l s . Sampling was g e n e r a l l y r e s t r i c t e d to the Boca Chica area i n order to l i m i t the study to a p o p u l a t i o n i n h a b i t i n g a r e l a t i v e l y homogenous environment. The Boca Chica r e g i o n was chosen because of i t s s i z e , i t s a c c e s s i b i l i t y (40 minutes by s k i f f ) , and i t s r e l i a b l e c a t c h p r o d u c t i o n as d e s c r i b e d by l o c a l f i shermen. There i s c o n s i d e r a b l e s u b s t r a t e v a r i a t i o n w i t h i n the region (dense to sparse grass, rock to rubble to sand bottoms), but t h i s i s a l s o true of s u b s t a n t i a l l y smaller a r e a s . In general the e n t i r e region b e n e f i t s from the input of open ocean water which comes in over the reef and e x i t s through reef breaks. Surrounding land masses, in c o n j u n c t i o n with the b a r r i e r r e e f , prevent severe bottom mixing dur i n g moderate winds. I t was f o r these reasons that the sample animals were a l l presumed to have been s u b j e c t to s i m i l a r p h y s i c a l and b i o l o g i c a l p r e s s u r e s . C o l l e c t i n g Specimens The sampling program r e q u i r e d that the fishermen leave San Pedro around 0600 h i n order to r e t u r n by approximately 1400 h. While f i s h i n g , the men use masks and f i n s . They drag t h e i r outboard-motored boat ( u s u a l l y a mahogany dugout) behind them on a long rope while c o n t i n u a l l y f r e e - d i v i n g to the bottom. The 25 c a t c h i s thrown i n t o the dory and l e f t u n t i l the fishermen's r e t u r n and t h e r e f o r e s u f f e r s some mechanical damage, as w e l l as exposure. M i s f o r t u n e and inclement weather sometimes made two days of c o l l e c t i n g necessary. During a n a l y s i s , these days have been combined where i n d i c a t e d . Back in San Pedro, p r o j e c t crew would r e c e i v e each day's sampling at a designated dock. A l l animals were immediately s e c t i o n e d while a l i v e and a c t i v e . The l i m i t e d exposure s u f f e r e d was not c o n s i d e r e d to be d e t r i m e n t a l . A l c o l a d o (1976) r e p o r t e d that 24 hours of continuous exposure during the Cuban summer was not f a t a l . Our s u b j e c t s were exposed f o r much l e s s time. When s e c t i o n i n g c o u l d not be completed w i t h i n the same day of sampling (a r a r e event), the conch were s t o r e d in a seawater bath and d e a l t with on the f o l l o w i n g day.' I s o l a t i o n of the f i e l d s i t e , a lack of s u p p l i e r s w i t h i n B e l i z e , v a g a r i e s of weather and o c c a s i o n a l poor catches f o r c e d sampling to be i n t e r r u p t e d or delayed on a number of o c c a s i o n s . These problems r e s u l t e d i n a sampling program which was s u b s t a n t i a l l y l e s s systematic than would be p o s s i b l e i n more developed areas, but which was s u c c e s s f u l when considered i n i t s t h i r d world c o n t e x t . 26 Measurements Three people took part in the s e c t i o n i n g process. One took weight measurements (using a beam bala n c e ) , another measured the s h e l l (using micrometer c a l i p e r s ) and cleaned the f i l l e t , and the t h i r d d i s s e c t e d out the t i s s u e samples and recorded data. T i s s u e Samples Tissue samples were cut using razor blades. Two 5 mm-wide tra n s v e r s e s e c t i o n s through the v i s c e r a l mass, j u s t p o s t e r i o r to the transparent stomach w a l l , were taken from a l l specimens. Each s e c t i o n i n c l u d e d a p o r t i o n of the gonad and the u n d e r l y i n g d i g e s t i v e gland. T i s s u e samples were immediately p l a c e d i n t i s s u e caps ( T i s s u e Tek II U n i c a s s e t t e s ) and f i x e d i n Davidson's s o l u t i o n with a c e t i c a c i d f o r 24 hours (see Appendix A). Long-term storage was i n Davidson's without a c e t i c a c i d . T i s s u e p r e p a r a t i o n i s i l l u s t r a t e d i n F i g . 6. The anterior-most s e c t i o n s were separated from the p o s t e r i o r - m o s t and held i n the l a b o r a t o r y i n B e l i z e . P o s t e r i o r s e c t i o n s were e i t h e r h a n d - c a r r i e d or commercially t r a n s p o r t e d to Nanaimo, B.C., i n s e t s c o v e r i n g 3-4 months as they became a v a i l a b l e . S p e c i a l s e r i a l samples were taken from 14 animals. In these samples the e n t i r e gonad was s e c t i o n e d from a n t e r i o r to 27 FIGURE 6: A schematic r e p r e s e n t a t i o n of the procedure used i n handling and p r e p a r a t i o n of gigas t i s s u e s e c t i o n s . 29 p o s t e r i o r i n 5 mm s l i c e s . P r e s e r v a t i o n was as d e s c r i b e d . In t h i s way 14 e n t i r e gonads were preserved and prepared f o r a n a l y s i s . T i s s u e P r e p a r a t i o n At the P a c i f i c B i o l o g i c a l S t a t i o n , Nanaimo, B.C., the t i s s u e samples were prepared f o r l i g h t microscopy a n a l y s i s by the r e s i d e n t h i s t o l o g i s t , Mr. John Bagshaw. The technique used was t h e i r standard h i s t o l o g i c a l s e c t i o n i n g and s t a i n i n g employed r o u t i n e l y on v e r t e b r a t e and i n v e r t e b r a t e t i s s u e , which i s summarized below. Appendix B d e s c r i b e s the technique i n d e t a i l . The t i s s u e caps, with enclosed t i s s u e samples were f i r s t taken through a 16 hour process of dehydration and p a r a f f i n impregnation, using i s o p r o p y l a l c o h o l as a dehydrator and x y l o l as a c l e a r i n g agent. The impregnated t i s s u e s were then bound to the o u t s i d e of the o r i g i n a l , l a b e l l e d t i s s u e caps w i t h i n a block of wax. The t i s s u e caps acted as mounting chucks during s e c t i o n i n g by a standard r o t a r y microtome. Two s t e e l wedge knives were employed, the f i r s t to roughly t r i m the block down to the l e v e l of the t i s s u e and another to f i n e l y s e c t i o n 6 pm s l i c e s . A p propriate s e c t i o n s were f l o a t e d onto a water and adhesive bath where they were taken up onto l a b e l l e d s l i d e s . Wet mounted s l i d e s were d r i e d f o r 15-45 min. In a f o r c e d hot a i r j e t and then s t a i n e d . 30 The s t a i n i n g process ( a l s o d e s c r i b e d i n Appendix B) r e q u i r e d that the p a r a f f i n be d i s s o l v e d i n x y l o l baths in order to allow f o r r e h y d r a t i o n in a l c o h o l and water. The f i r s t s t a i n was H a r r i s ' a c i d v a r i a n t hematoxylin ( b a s o p h i l i c ) , added f o r 4 minutes. A c i d a l c o h o l acted as the wash f o r d i f f e r e n t i a t i o n , and l i t h i u m carbonate was used f o r b l u e i n g . A c i d o p h i l i c c o u n t e r s t a i n i n g was done with e o s i n f o r 30 seconds. The s e c t i o n s were then dehydrated again i n baths of absolute a l c o h o l before f i n a l c l e a r i n g i n x y l o l . The l a s t step was the mounting of a c o v e r s l i p with n e u t r a l Canada Balsam in toluene (Permount). T i s s u e A n a l y s i s A n a l y s i s of the prepared s l i d e s was done at U.B.C, us i n g a Z e i s s compound l i g h t microscope with a v e r t i c a l l y mounted, 35 mm, magnetic r e l e a s e camera. A n a l y s i s began i n May of 1983 and was completed by September of 1983. A l l t i s s u e samples were a n a l y z e d f o r gross h i s t o l o g i c a l c h a r a c t e r i s t i c s a s s o c i a t e d with sexual m a t u r i t y as d e s c r i b e d i n Chapter I I . 31 CHAPTER 2: HISTOLOGY OF THE GONAD INTRODUCTION There has been a c o n s i d e r a b l e amount of r e s e a r c h done on the breeding c y c l e s of marine m o l l u s c s . Economically important s p e c i e s of b i v a l v e s and abalone have r e c e i v e d the g r e a t e s t amount of a t t e n t i o n because of the need fo r t h i s knowledge in resource management ( C h i p p e r f i e l d 1953, Quayle 1943, Leonard 1969, Loosanoff 1969, Young 1970, Berg 1969, Shepherd 1974). C o n s i d e r a b l y l e s s a t t e n t i o n has been given to higher gastropods as they are g e n e r a l l y of l e s s economic importance. Exceptions are nuisance species such as oyster d r i l l s (Manzi, Calabrese and Rawlins 1972) or s p e c i e s with an i n t e r e s t i n g l i f e h i s t o r y (e.g. C r e p i d u l a f o r n i c a t a , C h i p p e r f i e l d 1951). The bulk of gastropod l i f e h i s t o r y r e s e a r c h has emphasized common s p e c i e s where the subject was chosen because of i t s l o c a l abundance (Underwood 1974, Laws 1970, Mooers 1981, Rosenthal 1970). To the best of my knowledge, my study i s the f i r s t of a mesogastropod breeding c y c l e s p e c i f i c a l l y f o r f i s h e r i e s management reasons. As a consequence, there are no s u i t a b l e techniques a v a i l a b l e f o r determining the sexual maturity of mesogastropods from t i s s u e samples, as there are f o r b i v a l v e s and archaeogastropods. 32 Previous s t u d i e s on gastropod breeding c y c l e s have concentrated on temperate s p e c i e s and g e n e r a l l y report few d e t a i l s of a n a l y t i c a l technique (Feder et a_l. 1979, Rosenthal 1970, Mooers 1981, Laws 1970, Underwood 1974, Houston 1971). In order to study the now scarce t r o p i c a l mesogastropod, S. gigas , i t was necessary to develop a m a t u r i t y s c a l e that complemented i t s b i o l o g y , anatomy and my sampling program. Consequently, a l i g h t m i c r o s c o p i c a l study of the gonadal h i s t o l o g y of S. gigas was undertaken i n order to develop a system f o r determining the sexual m a t u r i t y of i n d i v i d u a l animals from s i n g l e , 6 /jm, gonadal c r o s s s e c t i o n s . No attempt i s made, w i t h i n t h i s chapter, to r e l a t e gonadal anatomy to the g e n e r a l p h y s i c a l c h a r a c t e r i s t i c s of the animal. In order to develop a system f o r determining sexual maturity from the s t a t e of gonadal development, the emphasis of the study f e l l upon changes in c h a r a c t e r i s t i c s that take p l a c e w i t h i n any and a l l p o r t i o n s of the gonad. C r o s s - s e c t i o n s from a l l areas of the gonad were t h e r e f o r e e x t e n s i v e l y s t u d i e d along with many s e c t i o n s taken from the same p l a c e i n each specimen. Although the r e s u l t s are not r e s t r i c t e d to gametic c y t o l o g y , emphasis was p l a c e d on gamete development. Post-spawning development has been r e p o r t e d by D'Asaro (1965) and i s not d e a l t with here. 33 Spermiogenesis Sperm dimorphism (the p r o d u c t i o n of two v a r i e t i e s of sperm w i t h i n the same t e s t i s ) i s t y p i c a l of many sp e c i e s of mesogastropods and neogastropods as w e l l as of the archaeogastropod Family N e r i t i d a e (Koike and Nishiwaki 1980). A l l gastropods produce normal, eupyrene sperm. In a d d i t i o n , many mesogastropods and neogastropods, i n c l u d i n g S. g i g a s , produce abnormal o l i g o p y r e n e or apyrene sperm (Webber 1977). Abnormal sperm g e n e r a l l y do not have a complete complement of chromatin ( o l i g o p y r e n e i m p l i e s a r e d u c t i o n while apyrene i m p l i e s that chromatin i s absent e n t i r e l y ) and do not p l a y a r o l e i n f e r t i l i z a t i o n (Webber 1977). The complicated polymorphic spermatozoa i n prosobranchs has s t i m u l a t e d c o n s i d e r a b l e r e s e a r c h on spermatogenesis i n t h i s group of gastropods. Important e a r l y reviews are provided by Tuzet (1930) and Franzen (1955, 1956). General r e f e r e n c e s have been t a b u l a t e d by Webber (1977) and f o r e l e c t r o n microscopy s t u d i e s by Koike and Nishiwaki (1980). Webber (1977), provides a d e t a i l e d account of gametogenesis in prosobranchs. Relevant d e t a i l s are summarized below. The c h a r c t e r i s t i c s of normal spermatogenesis to the beginning of spermiogenesis are s i m i l a r i n a l l prosobranchs. The three stages are commonly r e f e r r e d to as spermatogonia and 34 spermatocyte I and I I . Spermiogenesis ( d i f f e r e n t i a t i o n of spermatocytes) i s s i m i l a r among the mesogastropods and neogastropods but d i f f e r s s l i g h t l y i n the archaeogastropods. Spermatogonia i n prosobranchs are g e n e r a l l y small c e l l s of approximately 5 pm in diameter. Most spermatogonia c o n t a i n a s i n g l e l a r g e nucleus. A f t e r repeated m i t o t i c d i v i s i o n s the spermatogonia give r i s e to the primary spermatocytes. During the e a r l y spermatocyte I stage, there i s l i t t l e change i n nucleus or n u c l e o l u s but there i s an i n c r e a s e i n cytoplasmic volume. Before the f i r s t maturation (reduction) d i v i s i o n o c c u r s , c y t o p l a s m i c growth ceases, the nucleus c l e a r s , the n u c l e o l u s d i s a p p e a r s , the chromosomes take shape and the nuclear w a l l d i s a p p e a r s . In the spermatocyte II stage the nucleus reforms, chromatin becomes d i s p e r s e d and a r e s t p e r i o d ensues. T h i s p e r i o d v a r i e s with s p e c i e s and the number of mature spermatozoa w i t h i n the f o l l i c l e . The spermatocyte II stage ends with the second maturation d i v i s i o n a f t e r which the nucleus reforms, chromatin d i s p e r s e s and a second i n a c t i v e , or q u i e t stage i s entered. Spermiogenesis, the formation of spermatids and then spermatozoa, i n v o l v e s a v a r i e t y of cyt o p l a s m i c changes. For normal sperm, a head i s formed that i s elongate and compact. An elo n g a t e , uniform, m i t o c h o n d r i a l mass develops to i n t r u d e i n t o the middle s e c t i o n of the spermatozoa. The f i l a m e n t of the 35 f l a g e l l u m g e n e r a l l y a r i s e s from the a n t e r i o r c e n t r i o l e and passes through both the nuclear and m i t o c h o n d r i a l masses. Abnormal sperm are much l a r g e r and occur l e s s f r e q u e n t l y than normal sperm. The s i z e dimorphism i s apparent before the f i r s t maturation d i v i s i o n i n the spermatocyte I stage, with abnormal c e l l s a l r e a d y l a r g e r than the normal. T h i s s i z e d i f f e r e n t i a l i s maintained throughout development. Tuzet (1930) d e s c r i b e d three p a t t e r n s of abnormal spermatogenesis i n gastropods: without any maturation d i v i s i o n ; with one; or with both. Abnormal spermatozoa are m o r p h o l o g i c a l l y d i s t i n c t from normal sperm. They vary i n s i z e , shape and m o t i l i t y between s p e c i e s . The v a r i e t y ranges from small worm-like forms to gi a n t m u l t i - f l a g e l l a t e d ones (West 1978b). The r o l e of abnormal sperm i n re p r o d u c t i o n remains u n c l e a r : they may p l a y a r o l e i n the t r a n s p o r t of eupyrene sperm t o , and w i t h i n , females ( F r e t t e r 1953); t h e i r degeneration w i t h i n the female g e n i t a l t r a c t may r e l e a s e p o l y s a c c h a r i d e s and thereby provide nourishment f o r the eupyrene sperm (Hanson e t . a l . 1952); or they may pl a y a r o l e in the formation of nurse eggs found i n the egg ca p s u l e s of some neogastropods (Portmann 1927, 1931). There i s p r e s e n t l y no evidence to suggest that sperm dimorphism has occu r r e d i n the d i f f e r e n t genera f o r any s i n g l e reason. 36 With regard to s t u d i e s s p e c i f i c to spermiogenesis w i t h i n the Family Strombidae, works by Reinke (1912) and Kioke and Nishiwake (1980) stand out. Reinke p r o v i d e d a b r i e f , but d e t a i l e d , e a r l y account of abnormal spermiogenesis i n S. b i t u b e r c u l a t u s . Koike and Nishiwaki p r o v i d e d a d e t a i l e d d e s c r i p t i o n of normal and abnormal spermiogenesis i n two P a c i f i c strombid s p e c i e s , Conominex luhuanus and Lambis Iambis , by u s i n g t r a n s m i s s i o n e l e c t r o n microscopy. Relevant d e t a i l s from these s t u d i e s are i n c l u d e d where a p p r o p r i a t e w i t h i n the R e s u l t s and D i s c u s s i o n s e c t i o n of t h i s c h a p ter. Oogenesis Oogenesis i n prosobranchs has not been as e x t e n s i v e l y s t u d i e d as spermatogenesis. Reviews are given by Franc (1951), B a c c i (1954) and Raven (1961). S p e c i f i c r e f e r e n c e s have been t a b u l a t e d by Webber (1977). Terminology adopted i n t h i s r e p o r t i s taken from the general d e s c r i p t i o n of oogenesis i n mesogastropods, as r e p o r t e d by Webber (1977). Oogonia a r i s e from the germinal e p i t h e l i u m and have a l a r g e s p h e r i c a l , c l e a r nucleus that n e a r l y f i l l s the c e l l . They d i v i d e m i t o t i c a l l y up to the secondary oocyte stage. The important c h a r a c t e r i s t i c s of oocyte development i n c l u d e : p r e m e i o t i c nuclear changes (when the nucleus i n c r e a s e s i n s i z e ) ; c y t o p l a s m i c growth, which i s more r a p i d but accompanies n u c l e a r 37 growth; and f i n a l l y the maturation d i v i s i o n s . METHODS AND MATERIALS As p r e v i o u s l y d e s c r i b e d i n Chapter I, most t i s s u e samples were 5 mm t h i c k , s a g i t t a l s e c t i o n s through the v i s c e r a l mass j u s t p o s t e r i o r to the transparent stomach w a l l ( F i g . 6). In a d d i t i o n , the e n t i r e gonad of a number of specimens were s e c t i o n e d i n t o 5-mm s l i c e s i n order to study o v e r a l l gonadal changes d u r i n g maturation. A l l s e c t i o n s were embedded i n p a r a f f i n before being s e c t i o n e d i n t o 6 pm s l i c e s . The embedding procedure (Appendix B) s t a b i l i z e d the t i s s u e s u f f i c i e n t l y to minimize s e c t i o n i n g a r t i f a c t s . Mounted t i s s u e s e c t i o n s were s t a i n e d with hematoxylin and e o s i n . Two cr o s s s e c t i o n s were s p e c i f i c a l l y s t a i n e d to i d e n t i f y muscle and co n n e c t i v e t i s s u e u s i n g Gomori's Trichome s t a i n . A n a l y s i s was done using a Z e i s s compound, l i g h t microscope. 38 RESULTS AND DISCUSSION Note: F i g u r e s (photos) 7 to 23 appear together at the end of t h i s c h a p t e r . F i g u r e s 9 through 23 are arranged so as to i l l u s t r a t e the maturation process i n males and then females. The Male Gonad The gonad of male S. gigas v a r i e s in c o l o u r from white through orange and spreads over the d i g e s t i v e gland in the u l t i m a t e and i n part of the penultimate whorls of the s h e l l ( F i g . 3 ) . I t can develop to cover as much as h a l f of the d i g e s t i v e gland and shares with i t a s i n g l e e x t e r n a l membrane, the p a l l i a l e p i t h e l i u m . The r e l a t i v e c r o s s s e c t i o n a l area of the gonad, with regard to the d i g e s t i v e gland and/or other organs, i s g r e a t e s t at the a n t e r i o r end of the gonad, where i t meets with and surrounds the p o s t e r i o r p o r t i o n of the stomach. The volume of gonad w i t h i n an i n d i v i d u a l specimen v a r i e s with s i z e and sexual c o n d i t i o n , as i s d i s c u s s e d below. The gonad i s . e a s i l y d i s t i n g u i s h e d from the d i g e s t i v e gland and can be d i v i d e d i n t o somatic and gametic t i s s u e formations. Gametic t i s s u e l i e s w i t h i n s e m i n i f e r o u s tubules or f o l l i c l e s . These f o l l i c l e s are e a s i l y d i s t i n g u i s h e d from the surrounding somatic t i s s u e and c o n s i s t of branching tubes w i t h i n which 39 gametogenesis takes p l a c e . In c o n t r a s t , the i n t e r f o i l i c u l a r t i s s u e i s v a c u o l a t e d and s t a i n s weakly. In b i v a l v e s t u d i e s , i n t e r f o i l i c u l a r t i s s u e i s r e f e r r e d to by such names as f a t c e l l s (Brousseau 1981) or Leydig t i s s u e (Quayle per. Comm.). The i n t e r f o l l i c u l a r t i s s u e of S. gigas was i d e n t i f i e d f o r me by John Bagshaw (Dept. of F i s h e r i e s and Oceans, Nanaimo, B,C.) as c o n s i s t i n g of s i g n e t c e l l s , which are m o d i f i e d c o n n e c t i v e t i s s u e that f u n c t i o n i n energy s t o r a g e . Signet c e l l s surround the seminiferous f o l l i c l e s , presumably p r o v i d i n g mechanical support, and at the same time, n u t r i t i o n f o r a c t i v i t i e s w i t h i n the f o l l i c l e s . The network of gonadal f o l l i c l e s changes i n s i z e (volume) and complexity with age and m a t u r i t y . These developments are at the expense of the s i g n e t t i s s u e which decreases i n volume. Branches of the f o l l i c l e s appear to i n t e r c o n n e c t with and a r i s e from each other. They u l t i m a t e l y meet to form common tubes that are not themselves gametogenic. These tubes form the vas deferens at the p o s t e r i o r most s e c t i o n of the g e n i t a l duct. Although there i s a s i n g l e g e n i t a l duct l e a v i n g the t e s t e s ( L i t t l e 1965), many branches of the vas deferens are o f t e n v i s i b l e w i t h i n a s i n g l e gonadal c r o s s s e c t i o n . T h i s i s presumably the r e s u l t of c o n v o l u t i o n s . The vas deferens i s c l e a r l y v i s i b l e i n a d u l t s because i t a c t s as a seminal v e s i c l e 40 and t h e r e f o r e becomes swollen with mature sperm. Sperm s t o r e d w i t h i n the vas deferens are e a s i l y i d e n t i f i e d throughout the l e n g t h of a mature gonad. The vas deferens appears to u l t i m a t e l y form a s i n g l e tube which passes along the columnar s i d e of the gonad and i n t o the v i s c e r a l mass. From there i t passes through the region of the p r o s t r a t e gland, f i n a l l y a r r i v i n g at the penis ( L i t t l e 1965). Muscle c e l l s and c o l l a g e n occur w i t h i n the gonad. The volume of muscle t i s s u e i s r e l a t i v e l y small and does not appear to change with m a t u r i t y . A l a y e r of 5-6 c e l l s t h i c k n e s s occurs around the e n t i r e gonad and d i g e s t i v e gland ju s t beneath the p a l l i a l e p i t h e l i u m . C o l l a g e n i s mixed with and surrounds the muscle f i b r e s . C o l l a g e n f i b r e s are a l s o a p a r t of the p e r i p h e r y of i n d i v i d u a l f o l l i c l e s . The t h i c k , p e r i p h e r a l l a y e r of muscle c e l l s i s c l e a r l y o r g a n i z e d i n two d i r e c t i o n s . The primary r o l e of these muscle f i b r e s i s b e l i e v e d to be i n squeezing a l l , or p o r t i o n s , of the gonad d u r i n g c o p u l a t i o n . C o l l a g e n p r o v i d e s s t r e n g t h and support. Sperm i n Strombus gigas The most s t r i k i n g f e a t u r e of the male r e p r o d u c t i v e system i s the r a d i c a l l y dimorphic spermatozoa ( F i g s . 7 and 14). The s m a l l e r , normal sperm are roughly 500 times more abundant than 41 the abnormal (Reinke 1912). U l t r a s t r u c t u r a l l y , the normal sperm are nea r l y the same as in other f a m i l i e s of the meso- and neogastropods (Koike and Nishiwaki 1980). M o r p h o l o g i c a l l y they are not s i g n i f i c a n t l y d i f f e r e n t from those of other mesogastropod forms with the same sexual dimorphism (e.g., Paludina Reinke 1912). The t a i l p i e c e of the mature normal sperm i s of moderate length r e l a t i v e to that of others (e.g. L i t t o r i n a and F a s c i o l a r i a ;Reinke 1912). In c o n t r a s t to the normal sperm, the abnormal spermatozoa bear no resemblance to sperm of other genera (Reinke 1912). The mature abnormal, or apyrene, spermatozoa has a long, narrow and f l a t t e n e d c e l l body with membranes on e i t h e r s i d e ( F i g . 7 ) . A n t e r i o r l y , these membranes round out and meet, while at the p o s t e r i o r they j o i n to form a p o i n t e d " t a i l " p i e c e . The c e l l body i s f i l l e d with a number of polygonal bodies. Reinke (1912) suggested that these bodies were composed of albumen and played a n u t r i t i v e r o l e . Movement i s by way of c o o r d i n a t e d c o n t r a c t i v e waves moving along the membranes (Reinke 1912). Spermiogenesis Normal spermiogenesis begins at the p e r i p h e r y of the f o l l i c l e w a l l s ( F i g . 7 ) . Spermatogonia are small (approximately 4 pm i n diameter) with a s i n g l e nucleus. The primary and secondary spermatocyte stages are d i s t i n g u i s h e d from the 42 spermatogonia by t h e i r i n c r e a s e d s i z e . The " r e s t p e r i o d " before, and "quiet stage" a f t e r , the second maturation d i v i s i o n s , t h a t are reported f o r other mesogastropods (Webber 1977) were not evident w i t h i n t h i s study. The normal spermatocyte II stage c e l l s are c l e a r l y v i s i b l e as being r e l a t i v e l y l a r g e , round and d a r k l y s t a i n i n g . T h i s stage ends with the second maturation d i v i s i o n and produces the numerous spermatids. In t h i s phase, the c e l l s have been reduced to a r e l a t i v e l y small s i z e with dark oval bodies. The f i r s t of many ext e n s i v e changes that l e a d to the formation of spermatids i s the development of the t a i l f i l a m e n t . T h i s i s followed by the e l o n g a t i o n of the c e l l body and then f u r t h e r lengthening of the t a i l . U l t r a s t r u c t u r a l d e t a i l s of mature sperm and of the maturation process, of the P a c i f i c Strombids Conominex luhuanus and Lambis lambis are provided by Koike and Nis h i w a k i (1980). During the maturation process, the dev e l o p i n g sperm move from t h e i r p l a c e of o r i g i n (along the f o l l i c u l a r w a l l ) i n t o the lumen and then through the f o l l i c u l a r network to t h e i r p l a c e of storage i n the vas deferens. Abnormal spermatocytes are c o n s i d e r a b l y l a r g e r than those which w i l l form the normal sperm. They are pear-shaped with a round, d a r k l y s t a i n i n g nucleus. U n t i l l a t e i n the spermatocyte maturation process, they r e t a i n a s t a l k e d c o n n e c t i o n with the f o l l i c u l a r w a l l . A second maturation d i v i s i o n does not occur, 43 but i n s t e a d the nuclear w a l l breaks down and chromatin becomes d i s p e r s e d throughout the c e l l (Reinke 1912). Rapid growth leads d i r e c t l y i n t o abnormal spermatozoa from spermatocytes. The chromatin takes no f u r t h e r part i n the development process and g r a d u a l l y degenerates (Reinke 1912). The abnormal sperm of S. g i g a s i s t h e r e f o r e c o r r e c t l y ' r e f e r r e d to as being of the apyrene v a r i e t y . As with the normal sperm, the d e v e l o p i n g apyrene sperm move i n t o the f o l l i c u l a r lumen, g r a d u a l l y f i l l i n g i t and the lumen of the vas deferens as w e l l . My o b s e r v a t i o n s agree with those of Reinke (1912) and Hanson e_t a_l. (1952) i n that the apyrene sperm provide nourishment f o r the eupyrene. During storage w i t h i n the male, the eupyrene sperm do not adhere to the apyrene i n any way but they do occur together i n mixed clumps. The food r e s e r v e s of albumen (Reinke 1912) or g l y c o p r o t e i n (Koike and Nishiwaki 1980) that are s t o r e d w i t h i n the apyrene c e l l - bodies may be r e q u i r e d for m o t i l i t y of such l a r g e c e l l s but may a l s o be adequate to provide a n u t r i e n t - r i c h b r o t h w i t h i n the lumen of the f o l l i c l e s and vas d e f e r e n s . T h i s would allow the eupyrene sperm to remain v i a b l e f o r extended p e r i o d s of time while not themselves c a r r y i n g a l a r g e amount of food r e s e r v e s . A maintenance r o l e f o r apyrene sperm would become s t i l l more important dur i n g storage of semen w i t h i n females, as c o p u l a t i o n can precede spawning by s e v e r a l weeks (D'Asaro 1965). 44 Maturation Process of the Male Gonad Young specimens, c h a r a c t e r i z e d by t h e i r short l e n g t h and by the absence of a f l a r i n g l i p , have few f o l l i c l e s w i t h i n the gonad. The gonad i s c o r r e s p o n d i n g l y narrow in c r o s s s e c t i o n , and the f o l l i c l e s are u s u a l l y rounded and completely f i l l e d with f o l l i c u l a r t i s s u e . I n t e r f o l l i c u l a r space dominates, but s i g n e t c e l l t i s s u e i s not abundant as the volume of gonad i s s m a l l . As the gonad matures, the number of f o l l i c l e s v i s i b l e w i t h i n a l l areas of the gonad i n c r e a s e s . The e n t i r e gonad widens as the f o l l i c l e s expand and the s i g n e t c e l l s m u l t i p l y . With f u r t h e r m a t u r i t y , the gonad widens. T o t a l volume has inc r e a s e d but i n t e r f o i l i c u l a r d i s t a n c e s e v e n t u a l l y decrease as a r e s u l t of f o l l i c a l expansion. The f o l l i c l e s e v e n t u a l l y dominate the c e n t r a l areas of the gonad. P e r i p h e r a l areas, p a r t i c u l a r l y around the stomach, remain composed e n t i r e l y of s i g n e t t i s s u e . The vas deferens i s f i r s t v i s i b l e as r e l a t i v e l y small (5 10 u^m) round tubes which occur predominantly on the v e n t r a l s i d e of the v i s c e r a l mass. As mature sperm begin to accumulate, c o n v o l u t i o n s of the vas deferens become l a r g e r , more numerous and widely d i s t r i b u t e d . Sperm can p a r t i a l l y or e n t i r e l y f i l l t h e i r i n t e r i o r . Large, b u l g i n g c r o s s s e c t i o n s of the vas deferens are v i s i b l e to the eye i n prepared s e c t i o n s . They come to dominate l a r g e areas of c r o s s s e c t i o n s taken from anywhere i n 45 the gonad. Maturation of the gonad i s simultaneous throughout i t s e n t i r e l e n g t h . S i m i l a r i l y , i n c r e a s e s i n f o l l i c l e d e n s i t y are a l s o r e l a t i v e l y c o n s i s t e n t . T h i s i s not to say that the f o l l i c l e s are u n i f o r m l y d i s t r i b u t e d , but that they are clumped in the same numbers and i n s i m i l a r areas throughout the gonad. Absolute numbers of f o l l i c l e s v i s i b l e w i t h i n a c r o s s s e c t i o n are g r e a t e s t at the a n t e r i o r end where the gonad i s broadest. D e n s i t i e s are more c o n s i s t e n t i n regions p o s t e r i o r to the stomach. The vas deferens system i s a l s o v i s i b l e throughout the e n t i r e gonad. The p o s i t i o n and number of v i s i b l e branches shows some v a r i a t i o n but t h e i r c o n d i t i o n (immature, mature, spent) i s c o n s i s t e n t . The f o l l i c l e s of mature males do not g e n e r a l l y occupy any more than 50-80% of the gonad c r o s s s e c t i o n a l area. Sperm accumulation can be o c c u r r i n g when as l i t t l e as 20-30% of the gonad i s consumed by f o l l i c l e s . As accumulation c o n t i n u e s , the vas deferens system expands and so do the f o l l i c l e s . The r i p e gonad i s l a r g e , complex and crowded, with mature sperm f i l l i n g the f o l l i c l e s and vas deferens. The post-spawning gonad i s c h a r a c t e r i z e d by numerous f o l l i c u l a r remnants . These may be l a r g e and empty or long and 46 compressed. The vas deferens i s r e a d i l y apparent but empty. I t may be degenerate, or with the i n t e r n a l t i s s u e absent, or i n d i s a r r a y , or i t may be r e g e n e r a t i n g and have r e b u i l t the t h i c k inner l a y e r of columnar t i s s u e . A w e l l developed vas deferens makes i t easy to d i s t i n g u i s h the spent from the young specimens. The Female Gonad The female gonad i s s t r u c t u r a l l y very s i m i l a r to that of the male. For the sake of b r e v i t y , only s i g n i f i c a n t s i m i l a r i t i e s and d i f f e r e n c e s are d e s c r i b e d . The female gonad i s of s i m i l a r s i z e and l i e s i n the same p o s i t i o n r e l a t i v e to the s h e l l and d i g e s t i v e gland. The surrounding p a l l i a l e p i t h e l i u m , muscle and c o l l a g e n f i b r e s are present i n the same p o s i t i o n s , presumably f o r s i m i l a r reasons, as i n the male. Signet c e l l s separate the branching f o l l i c l e s which u l t i m a t e l y meet to form the o v i d u c t . The main stem of the ovi d u c t passes along the columnar si d e of the ovary, and from there i n t o the v i s c e r a l mass. U n l i k e the male vas d e f e r e n s , the o v i d u c t does not p l a y any apparent storage r o l e . Throughout the maturation process, c o n v o l u t i o n s of the oviduct remain as small round or o v a l tubes with a t h i c k i n n e r . l a y e r of columnar t i s s u e . They are i d e n t i c a l in appearance to the vas deferens of young males but do riot o b v i o u s l y expand with sexual m a t u r i t y ( F i g . 23). 47 Oogenesis Oogenesis i n S. gigas does not have unique f e a t u r e s . I t begins at the f o l l i c l e p e r i p h e r y , where the oogonia are f i r s t d e t e c t a b l e at a s i z e of 20 to 30 pm. They are i r r e g u l a r l y p l a c e d on the p e r i p h e r y and are surrounded by a t h i c k l a y e r of f o l l i c l e c e l l s . As oogenesis begins, the oogonia give r i s e to primary oocytes, o f t e n i n c l u s t e r s of 3-5. Oocytes are d i s t i n g u i s h e d by being l a r g e r than oogonia and having a l a r g e r nucleus. In the e a r l y oocyte, the nucleus occupies almost the e n t i r e c e l l volume. While s t i l l s m a l l , the oocytes remain a t t a c h e d to the f o l l i c u l a r w a l l s , c l o s e to other developing oocytes and f o l l i c a l c e l l s . As v i t e l l o g e n e s i s begins and the oocytes r a p i d l y i n c r e a s e i n volume, they become i n c r e a s i n g l y removed from the f o l l i c a l p e r i p h e r y and begin to f i l l the lumen. The v i t e l l o g e n i c oocyte i n i t i a l l y has a r e l a t i v e l y l a r g e nucleus. Toward the beginning of v i t e l l o g e n e s i s , the nucleus r a p i d l y i n c r e a s e s i n s i z e . As food r e s e r v e s accumulate the nucleus becomes p r o p o r t i o n a t e l y s m a l l e r reaching a maximum width of 1 5 u^m. As v i t e l l o g e n e s i s c o n t i n u e s , the c e l l s i n c r e a s e i n s i z e to 48 a maximum of 70 pm. Oocytes e v e n t u a l l y f i l l the f o l l i c u l a r lumen and cause f o l l i c u l a r expansion with a c o n s e q u e n t i a l decrease i n i n t e r f o l l i c u l a r d i s t a n c e s . T h i s process continues as more c e l l s reach m a t u r i t y . P e r i p h e r a l t i s s u e becomes compressed and i n t e r f o l l i c u l a r space d i m i n i s h e s . P o s t - v i t e l l o g e n i c oocytes are g e n e r a l l y b e l i e v e d to be r e l a t i v e l y q u i e scent u n t i l a c t i v a t i o n by spermatozoa (West 1981). Ripe oocytes are presumed to be h e l d w i t h i n the f o l l i c l e s u n t i l spawning, at which time the muscle f i b r e s may come i n t o p l ay to a i d i n d e l i v e r y of the oocytes along the g e n i t a l t r a c t . The exact s i z e of mature oocytes i s not known. N e i t h e r Robertson (1959), R a n d a l l (1964) nor D'Asaro (1965) reported the a c t u a l s i z e of spawned eggs when d e s c r i b i n g S. g i g a s egg masses in c o n s i d e r a b l e d e t a i l . During my a n a l y s i s , only one oocyte was observed w i t h i n an o v i d u c t ( F i g . 8 ) . T h i s oocyte was c e r t a i n l y approaching the s i z e at m a t u r i t y . I t s diameter was 60 pm which i s t h e r e f o r e presumed to'approximate the minimum dimension of an oocyte at the time of spawning, with the r e s e r v a t i o n that the widest p o r t i o n of the c e l l may not have been s e c t i o n e d . Maturation Process of the Female Gonad The maturation processes i n the male and female are s i m i l a r . U n d i f f e r e n t i a t e d male and female gonads are 49 i n d i s t i n g u i s h a b l e . There are few f o l l i c l e s in a r e l a t i v e l y s mall gonad. With the onset of oogenesis, as i n spermiogenesis, the f o l l i c u l a r branches i n c r e a s e i n number and s i z e . Oogonia and oocytes are v i s i b l e on the f o l l i c l e p e r i p h e r y as the lumen expands. Development i s r a p i d , with growing oocytes q u i c k l y crowding the p e r i p h e r y and then the lumen as food reserves accumulate. As the number and s i z e of f o l l i c l e s i n c r e a s e , the i n t e r f o l l i c u l a r d i s t a n c e s decrease, r e f l e c t i n g the volume of c o n f i n e d o o c y t e s . With m a t u r i t y , the i n t e r n a l complexity and o v e r a l l volume of the gonad i n c r e a s e s . Again, development i s synchronized throughout the gonad. U n l i k e the males, the f o l l i c l e s e v e n t u a l l y occupy the e n t i r e gonad l e a v i n g only a small band of s i g n e t c e l l s around the gonad p e r i p h e r y . Up to 95% of a g r e a t l y expanded gonad w i l l be f i l l e d by almost b u r s t i n g f o l l i c l e s but the oviduct does not p a r t i c i p a t e i n s t o r a g e . The spent female gonad i s c h a r a c t e r i z e d by l a r g e but empty f o l l i c l e s or by f l a t t e n e d remnants. I t i s o f t e n d i f f i c u l t to d i s t i n g u i s h the spent gonad from f i r s t development. The oviduct does not p r o v i d e c l u e s as in the males - only c r o s s s e c t i o n a l area and f o l l i c u l a r remnants may be used as i n d i c a t o r s . 50 A MATURITY SCALE If the e n t i r e gonad of each specimen had been a v a i l a b l e f o r study, i t would have been p o s s i b l e to develop a m a t u r i t y s c a l e with a l a r g e number of w e l l - d e f i n e d c a t e g o r i e s . However, u s u a l l y only a s i n g l e s e c t i o n from each animal was a v a i l a b l e and only a few broad, l o o s e l y d e f i n e d c a t e g o r i e s are p o s s i b l e . Had the gonads not matured simultaneously throughout, even broader c a t e g o r i e s might have been necessary, or c l a s s i f i c a t i o n i m p o s s i b l e . In the maturity s c a l e developed i n t h i s study, u n d i f f e r e n t i a t e d males and females were clumped together and d e s c r i b e d as c o n d i t i o n "0". The remaining c a t e g o r i e s are I, I I , I I I , and IV, with I being c o n s i d e r e d the beginning of the maturation p r o c e s s , III as r i p e and IV as p o s t - r i p e n e s s . D e f i n i t i v e c h a r a c t e r i s t i c s for each category vary between the males and females. Therefore, the c a t e g o r i e s do not n e c e s s a r i l y represent e q u i v a l e n t ages or s t a t e s of development between sexes. S i m i l a r i l y , since broad c a t e g o r i e s have been used, each represents a wide range of p o s s i b l e maturation s t a t e s with the boundaries between c a t e g o r i e s being somewhat a r b i t r a r y . 51 U n d i f f e r e n t i a t e d "0" In the u n d i f f e r e n t i a t e d s t a t e , sex i s not yet apparent c y t o l o g i c a l l y . I t i s m o r p h o l o g i c a l l y d i s c e r n a b l e by the presence or absence of a seminal groove. The gonad i s small i n c r o s s s e c t i o n with few f o l l i c l e s . F o l l i c u l a r t i s s u e commonly f i l l s the f o l l i c u l a r lumen ( F i g . 9). I t i s important to note that gametic d i f f e r e n t i a t i o n may have begun but i t i s too e a r l y fo r sex to be d i s t i n g u i s h e d . T h i s stage ends when males and females are d i s t i n c t . Male C o n d i t i o n I_ "Beginning" Of the two sperm v a r i e t i e s o c c u r r i n g w i t h i n the s e m i n i f e r o u s f o l l i c l e s , the apyrene are the f i r s t to be r e a d i l y i d e n t i f i e d at a s i z e of approximately 8 pm ( F i g . 10). Rapid growth of deeply s t a i n i n g cytoplasm and a l a r g e black nucleus d i s t i n g u i s h them from the small f o l l i c l e c e l l s . Round, black eupyrene spermatocytes are r e c o g n i z a b l e a f t e r they have become numerous w i t h i n the f o l l i c u l a r lumen. During c o n d i t i o n I, the gonad i s dominated by s i g n e t c e l l s . There are r e l a t i v e l y few f o l l i c l e s with l a r g e i n t e r f o l l i c u l a r spaces. In order to d i s t i n g u i s h animals which have j u s t become mature from r e g e n e r a t i n g or r e s t i n g i n d i v i d u a l s , s h e l l - l i p 52 t h i c k n e s s (which r e f l e c t s age by i t s presence and development) and signs of a w e l l developed vas deferens with remnant sperm were used as i n d i c a t i o n s of p r e v i o u s r e p r o d u c t i v e a c t i v i t y . Unless r e g e n e r a t i o n was o c c u r r i n g ( i . e . , f o l l i c l e s r e d e v e l o p i n g ) , t h i c k - l i p p e d animals with w e l l developed vas d e f e r e n s , which d i d not yet have accumulated sperm, were ass i g n e d to c o n d i t i o n IV and presumed to be i n a r e s t i n g phase. Male C o n d i t i o n 11 "Developing" The d e v e l o p i n g male gonad i s d i s t i n g u i s h e d from e a r l i e r stages by the presence of mature apyrene spermatozoa ( F i g s . 12 and 13). F o l l i c l e s are l a r g e r and more numerous than d u r i n g c o n d i t i o n I and the vas deferens i s c l e a r l y expanding, but i s not yet swollen with s t o r e d sperm. Young d e v e l o p i n g animals ( t h i n f l a r i n g l i p ) are e a s i l y d i s t i n g u i s h e d from o l d e r , r e g e n e r a t i n g i n d i v i d u a l s . The r e d e v e l o p i n g gonad i s g e n e r a l l y l a r g e r in c r o s s s e c t i o n , with many more f o l l i c l e s p r e s e n t . Convolutions of the vas deferens are c l e a r l y v i s i b l e i n o l d e r specimens, but they must not be s i g n i f i c a n t l y f i l l e d with sperm i f the specimen i s to be a s s i g n e d to c o n d i t i o n I I . Stage II ends when s i g n i f i c a n t amounts of sperm have accumulated and f o l l i c l e s are numerous. The d i s t i n c t i o n between c o n d i t i o n s II and III was s u b j e c t i v e . Here, more than at any 53 other p o i n t , i t i s p o s s i b l e that there have been i n c o n s i s t e n c i e s as a consequence of the l i m i t a t i o n a s s o c i a t e d with viewing s i n g l e c r o s s s e c t i o n s and not e n t i r e gonads. Study of s e r i a l s e c t i o n s showed that vas deferens development and sperm storage are not as c o n s i s t e n t throughout the gonad as are other f e a t u r e s . E a r l y c o n d i t i o n III may have been mistaken f o r l a t e II or the rev e r s e . Male C o n d i t i o n III "Ripe" The primary d i s t i n g u i s h i n g c h a r a c t e r i s t i c of r i p e males i s swollen, s p e r m - f i l l e d c o n v o l u t i o n s of the vas deferens F o l l i c l e s are g e n e r a l l y numerous, but t h e i r d e n s i t y and the r e s u l t i n g i n t e r f o l l i c u l a r d i s t a n c e s vary ( F i g s . 14 and 15). P a r t i a l l y spent i n d i v i d u a l s have been i n c l u d e d w i t h i n c o n d i t i o n I I I . In t h i s s t a t e , the f o l l i c l e s remain numerous and complex. At the same time, the vas deferens i s we l l developed but some p o r t i o n s appear completely or p a r t i a l l y empty, while others are bu l g i n g with s t o r e d sperm. I t i s presumed that previous accumulations of sperm promoted the o r i g i n a l development of the vas deferens and that c o p u l a t i o n or some other process has r e s u l t e d i n a l o s s of a p o r t i o n of the gametes. I f p r e s e r v a t i o n or s e c t i o n i n g produced the p a r t i a l l y spent appearance, then combining r i p e with p a r t i a l l y spent samples w i l l have helped to e l i m i n a t e e r r o r . 54 Male C o n d i t i o n IV "Spent" The spent male gonad occurs i n b a s i c a l l y two v a r i e t i e s . The f i r s t form i s what i s presumed to r e f l e c t c o n d i t i o n s soon a f t e r e j a c u l a t i o n . The second v a r i e t y i s b e l i e v e d to be the spent c o n d i t i o n p o s s i b l y a f t e r a r e s t i n g phase. Recently spent males have numerous f o l l i c l e s that are l a r g e and almost completely empty. The vas deferens i s w e l l developed but i s empty or almost so. R e s t i n g i n d i v i d u a l s have numerous but f l a t t e n e d f o l l i c l e s . The vas deferens i s w e l l developed but empty, with a regenerated inner l a y e r of columnar t i s s u e ( F i g s . 16 and 17). I t was presumed that a specimen was i n a r e s t i n g phase and not u n d i f f e r e n t i a t e d i f the s h e l l had a thickened f l a r i n g l i p (> 5 mm) and at the same time the gonad was wide, the vas deferens system was present and obvious, and f o l l i c u l a r remnants were numerous. These c h a r a c t e r i s t i c s imply that p r e v i o u s r e p r o d u c t i v e a c t i v i t y had o c c u r r e d . In order to minimize e r r o r , a s i n g l e c h a r a c t e r i s t i c alone was not accepted as adequate evidence. I t appears t h a t spent males r e t u r n to the maturation c y c l e at c o n d i t i o n I or I I . In order to do so, they must have gone through a phase s i m i l a r t o 0 but may pass through i t more 55 q u i c k l y the second time. Spent and, at the same time, u n d i f f e r e n t i a t e d samples were r a r e . Female C o n d i t i o n I_ "Beginning" Female c o n d i t i o n I begins when oogonia are f i r s t c l e a r l y d i s t i n g u i s h e d at a s i z e of approximately 20 to 30 pm. Small oogonia are r e c o g n i z e d by t h e i r l a r g e nucleus and r e l a t i v e l y small p r o p o r t i o n of cytoplasm ( F i g . 18). C o n d i t i o n I i n c l u d e s those samples with oocytes of a diameter l e s s than or equal to 50 u^m ( F i g . 19). The gonad widens as the f o l l i c u l a r network expands. Clumps of d e v e l o p i n g oocytes become apparent along the f o l l i c l e p e r i p h e r y as the f o l l i c u l a r lumen opens ( F i g s . 19 and 21). Signet c e l l s dominate throughout c o n d i t i o n I, occupying 90% or more of the t o t a l s u rface a r e a . Female C o n d i t i o n II "Developing" The number of oocytes i n c r e a s e s r a p i d l y , with f o l l i c l e p e r i p h e r i e s becoming crowded. There i s c o n s i d e r a b l e v a r i a t i o n i n oocyte diameter as c e l l s begin d e v e l o p i n g at d i f f e r e n t times ( F i g . 20). As the lumen f i l l s with growing oocytes, f o l l i c l e s expand and e v e n t u a l l y come i n t o c o n t a c t with each other, thus beginning c o n d i t i o n I I I . 56 During phase I I , i n t e r f o l l i c u l a r d i s t a n c e s n a t u r a l l y d i m i n i s h , e v e n t u a l l y r e a c h i n g zero. Signet c e l l s p e r s i s t but occupy l e s s than 50% of an expanded gonad by the end of stage II . The d i s t i n c t i o n between l a t e II and e a r l y III was often vague. When most f o l l i c l e s were in c o n t a c t with two or more other f o l l i c l e s , c o n d i t i o n III was a s s i g n e d . Female C o n d i t i o n III "Ripe" The r i p e female gonad i s dominated by b u l g i n g f o l l i c l e s that occupy most or a l l of the gonad su r f a c e area. Yolk g l o b u l e s are very prominent and o f t e n b l u r d i v i s i o n s between c e l l s ( F i g . 22). Convolutions of the o v i d u c t are apparent but they remain s m a l l . I n t e r f o l l i c u l a r t i s s u e may occupy as l i t t l e as 10% of the surface area of a c r o s s s e c t i o n . Female C o n d i t i o n IV "Spent" The spent female has a wide gonad with a l a r g e number of expanded but n e a r l y empty f o l l i c l e s . A few small oocytes may remain. Signet c e l l s occupy 50% of the s u r f a c e area of a c r o s s s e c t i o n ( F i g . 23). As with males, there appears to be a recovery or r e s t i n g 57 c o n d i t i o n . In t h i s s t a t e , there are numerous f o l l i c u l a r remnants in a r e l a t i v e l y l a r g e gonad. A specimen was not c o n s i d e r e d e l i g i b l e for the spent c o n d i t i o n u n l e s s i t s l i p t h i c k n e s s exceeded 5 mm. Specimens that seemed to have been p r e v i o u s l y mature (spent) but were at the same time u n d i f f e r e n t i a t e d were more common i n females than males. R e s t i n g females show no obvious sig n s of previous maturations other than numerous f o l l i c u l a r remnants. They were t h e r e f o r e presumed to reenter the c y c l e at e i t h e r c o n d i t i o n "0" or I r a t h e r than c o n d i t i o n I or II as i n the males. CONCLUSIONS T h i s study has d e s c r i b e d h i s t o l o g i c a l maturation of the gonad of S. gigas and p r o v i d e s a b a s i s for determining sexual m a t u r i t y of i n d i v i d u a l animals from s i n g l e c r o s s - s e c t i o n s of the gonad. The s c a l e developed f o r males uses f o l l i c l e s i z e , d e n s i t y and t h e i r i n t e r n a l complexity; gonad width; spermatocyte development; and accumulation of sperm w i t h i n c o n v o l u t i o n s of the vas deferens as c h a r a c t e r i s t i c s used to d e f i n e the maturity of specimens. Gonad width, i n t e r f o i l i c u l a r d i s t a n c e , f o l l i c l e s i z e and oocyte diameter were used as d e f i n i n g c h a r a c t e r i s t i c s 58 f o r female stages. F l a r i n g l i p t h i c k n e s s was used as an i n d i c a t o r of r e l a t i v e age du r i n g a n a l y s i s of both sexes. The gonad of S. gigas i s s t r u c t u r a l l y s i m i l a r to that of Colus stimpsoni as reported by West (1978a, 1979). Gametogenesis i s s i m i l a r to that d e s c r i b e d by Webber (1977) f o r other meso- and neogastropods. I t i s t h e r e f o r e probable that the maturity s c a l e developed f o r the study of S. gigas would be a p p r o p r i a t e f o r many other meso- and neogastropods. The s i m i l a r i t i e s between spermiogenesis i n S. gigas and t h a t reported f o r other strombids (Reinke 1912; Koike and Nishiwaki 1980) supports B a c c e t t i ' s (1970) view that sperm morphology may be used as a guide to p h y l o g e n e t i c a f f i n i t y w i t h i n v a r i o u s animal groups. The f u n c t i o n of the apyrene spermatozoa was not p o s i t i v e l y a s c e r t a i n e d , but i t i s thought that the long term v i a b i l i t y of the small eupyrene sperm w i t h i n the female seminal r e c e p t a c l e (D'Asaro 1965) may be r e l a t e d to an abundance of n u t r i e n t - r i c h apyrene spermatozoa w i t h i n f e r t i l i z e d females. Simultaneous maturation of the e n t i r e gonad i n both sexes allowed m a t u r a t i o n - c a t e g o r i e s to be d e s c r i b e d . L i m i t a t i o n s inherent i n the use of s i n g l e c r o s s s e c t i o n s p e r m i t t e d only broad c a t e g o r i e s to be used. A lack of samples of c o p u l a t i n g and/or spawning specimens has made i t imp o s s i b l e to d e s c r i b e 59 e x a c t l y what c o n s t i t u t e s m a t u r i t y and t h e r e f o r e to i n c l u d e t h i s as a w e l l d e f i n e d c a t e g o r y . I t was not p o s s i b l e to p i n p o i n t the p r e c i s e p o i n t , w i t h i n the maturation s c a l e , that sexual a c t i v i t y begins. 60 TABLE I - Key to F i g u r e s 7 to 23 AcS Accumulated Sperm ASc Abnormal Spermatocyte(s) ASz Abnormal Spermatozoon (apyrene) CT Columnar T i s s u e Cy Cytoplasm DG D i g e s t i v e Gland F F o l l i c l e ( s ) FL F o l l i c u l a r Lumen FT F o l l i c u l a r T i s s u e LT Leydig T i s s u e N Nucleus NSc Normal Spermatocyte(s) NSz Normal Spermatozoon (eupyrene) Oc Oocyte(s) Od Oviduct Og Oogonium Ov Ovum Sd Spermatids (eupyrene) Sg Spermatogonium VD Vas Deferens 62 FIGURE 7: C l o s e up of the two v a r i e t i e s of sperm of S. g i g a s . The abnormal (ASz) dominates the photograph and dwarfs the normal (NSz) sperm which appear as black c y l i n d e r s with an almost i m p e r c e p t i b l e t a i l p i e c e . FIGURE 8: Female o v i d u c t with ovum (Ov) en c l o s e d . The columnar t i s s u e (CT) of the o v i d u c t and nucleus (N) of the ovum are c l e a r l y v i s i b l e . F i g u r e T Mag. x W F i g u r e & Mag. x 7^9 64 FIGURE 9 : U n d i f f e r e n t i a t e d f o l l i c l e (F) with f o l l i c u l a r t i s s u e (FT) f i l l i n g i t s lumen. Leydig t i s s u e (LT) dominates the photograph. FIGURE 10: The f i r s t d e t e c t a b l e s i g n s of gamete development i n male §_;_ g i g a s . The l a r g e r abnormal spermatocytes (ASc) along with the dark black normal spermatocytes (NSc) accumulate i n the f o l l i c u l a r lumen (FL) and along the p e r i p h e r y . F i g u r e ^ Mag. x 3ffO Figure \0 Mag. x 930 66 FIGURE 1 1 : The presence of mature abnormal spermatocytes (ASc) separate c o n d i t i o n I from c o n d i t i o n 0 . Here, the f o l l i c l e has begun to open and developed sperm (ASz) are apparent. FIGURE 12 : A low m a g n i f i c a t i o n photograph i l l u s t r a t i n g how the gonad appears i n an i n t e r m e d i a t e c o n d i t i o n II male g i g a s . Leydig t i s s u e (LT) dominates the gonad but the f o l l i c l e s (F) are numerous and show c o n s i d e r a b l e i n t e r n a l complexity. F i g u r e 1/ Mag. x 3dO F i g u r e Mag. x % 68 FIGURE 13: A high m a g n i f i c a t i o n photograph showing the vas deferens of S_^  gigas while s t i l l s m a l l . Sperm have begun to accumulate. (Compare t h i s to the vas deferens of a r i p e specimen, F i g . 15.) FIGURE 14: The f o l l i c l e of a r i p e male. The f o l l i c l e s have grown r e l a t i v e l y wide and are packed with developed (ASz) and dev e l o p i n g (ASc) abnormal sperm. F i g u r e /3 A S : 4 ...;v * 9' It . LT * Mag. x 3&0 F i g u r e /f LT LT Mag. x 3S0 7 0 FIGURE 15: A low m a g n i f i c a t i o n photograph dominated by the g r e a t l y expanded vas deferens (VD) which, when f u l l y developed, are v i s i b l e on a t i s s u e s e c t i o n with the naked eye. FIGURE 16: The f o l l i c l e s of spent male gi g a s were t y p i c a l l y l a r g e , numerous and e s s e n t i a l l y empty. In t h i s photograph the f o l l i c u l a r remains of a spent male are v i s i b l e . A t h i n l a y e r of p e r i p h e r a l t i s s u e and a few spermatocytes (ASc, NSc) are a l l t h a t remain. r i g u r e 15 Mag. x °l5 F i g u r e 16 Mag. x 3%0 72 FIGURE 17: The vas deferens (VD) of spent males remain c l e a r l y v i s i b l e even while the gonad appears dormant. As i s i l l u s t r a t e d i n t h i s photograph, the r e s t i n g vas deferens develops a t h i c k inner l a y e r of columnar t i s s u e and continues to dominate l a r g e p o r t i o n s of the gonad. FIGURE 18: In female Gigas the f i r s t d e t e c t a b l e s i g n s of gamete development occurs along the f o l l i c u l a r p e r i p h e r y . In t h i s photograph an e a r l y d e veloping oocyte (Oc) with a l a r g e nucleus i s c l e a r l y v i s i b l e . F i g u r e Mag. x 95 F i g u r e Ifr Mag. x ^ 3 0 74 FIGURE 19: Female c o n d i t i o n I ends when oocytes (Oc) l a r g e r than 50 microns i n diameter are d e t e c t a b l e . T h i s photograph i l l u s t r a t e s f o l l i c u l a r c o n d i t i o n s at the end of female c o n d i t i o n I. FIGURE 20: T h i s photograph i s of a c o n d i t i o n I I female gigas f o l l i c l e at an in t e r m e d i a t e stage of development. The f o l l i c l e i s s t i l l r e l a t i v e l y narrow and the oocytes (Oc) small (compare t h i s to F i g . 22). 7S F i g u r e IS Mag. x 3S0 F i g u r e £0 Mag. x 380 76 FIGURE 2 1 : T h i s f i g u r e i s a low m a g n i f i c a t i o n overview of a female c o n d i t i o n II gonad. The f o l l i c l e s with e n c l o s e d oocytes (Oc) are both r e l a t i v e l y small and do not yet dominate the gonad. FIGURE 2 2 : T h i s photograph i s a low m a g n i f i c a t i o n overview of a r i p e female S gigas gonad. Almost the e n t i r e gonad i s dominated by f o l l i c l e s b u r s t i n g with r i p e ova. The boundaries of i n d i v i d u a l ova and f o l l i c l e s have become i n d i s t i n c t . Note the o v i d u c t (Od) i n the lower r i g h t c o r n e r . Figure Mag. x 95 Figure 3<3-Mag. x 78 FIGURE 23: The f o l l i c l e s of dormant gig a s females are g e n e r a l l y l a r g e , numerous and e s s e n t i a l l y empty. In t h i s photograph f o l l i c u l a r t i s s u e dominates the f o l l i c u l a r lumen and a few oocytes l i e around the p e r i p h e r y . 80 CHAPTER 3: SHELL MORPHOLOGY AT SEXUAL MATURITY INTRODUCTION In many s h e l l f i s h f i s h e r i e s (e.g., abalone, clams and o y s t e r s ) , management i s based on some measure of maximum l e n g t h as an i n d i c a t o r of adulthood or sexual m a t u r i t y . T h i s p r a c t i c e i s a p p r o p r i a t e only when l e n g t h remains i n d i c a t i v e of age throughout maturation. When growth p a t t e r n s are complex, some other measure i s r e q u i r e d to d e f i n e adulthood. Strombus gigas u n l i k e many other gastropods, does not continue to grow i n length throughout i t s l i f e h i s t o r y . Instead, s p i r a l s h e l l growth ceases at an age of 2.5-3 years and a f l a r i n g s h e l l l i p i s developed (Berg 1976, Brownell 1977, A l c o l a d o 1976). A f t e r t h i s time, s h e l l l e n g t h does not i n c r e a s e s i g n i f i c a n t l y but s h e l l weight does (Berg 1976, B l a k e s l e y 1977). At f i r s t , a t h i n broad f l a r i n g l i p i s formed which g r a d u a l l y grows t h i c k e r , but becomes l e s s f l a r i n g with age as ab r a s i o n and damage erode i t s edge (Brownell and Stevely 1981). Previous s t u d i e s of S. g i g a s have r e p o r t e d that maturation occurs a f t e r maximum len g t h has been reached and when the f l a r i n g l i p i s w e l l developed (Berg 1976, Brownell 1977 and B l a k e s l e y 1977). While these i n v e s t i g a t o r s were unable to d e s c r i b e e x a c t l y when f i r s t m a t u r i t y was a t t a i n e d , they d i d 81 report that l e n g t h alone was not a good i n d i c a t o r of sexual m a t u r i t y . Maximum s h e l l length r e s t r i c t i o n s are c u r r e n t l y used in the stock management p r a c t i c e s of B e l i z e on the b e l i e f that such r e s t r i c t i o n s w i l l permit most conch to reproduce before harvest and t h e r e f o r e a healthy breeding p o p u l a t i o n w i l l be maintained. In order to a p p r e c i a t e the p o t e n t i a l e f f e c t i v e n e s s of such management p r a c t i c e s , i t i s d e s i r e a b l e to have an understanding of s h e l l morphology at sexual m a t u r i t y . T h i s chapter i s devoted to an a n a l y s i s of the r e l a t i o n s h i p between the onset of sexual maturity and s h e l l s i z e , with emphasis on development of the f l a r i n g l i p . METHODS AND MATERIALS Over a 20 month p e r i o d , 1030 specimens of Strombus gigas were c o l l e c t e d from a 40 km area of s e a f l o o r w i t h i n the b a r r i e r reef of B e l i z e ( F i g s . 4 and 5). S h e l l s with developing or developed l i p s comprised the bulk of the specimens. T h i s i n c l u d e d a wide v a r i e t y of s h e l l l e n g t h s . The remainder of the monthly samples were made up of s h e l l s without f l a r i n g l i p s but that were c l o s e to l e g a l l e n g t h or g r e a t e r . On two occasions animals of a l l s i z e s were s p e c i f i c a l l y c o l l e c t e d i n order to guarantee that a l l ages would be represented w i t h i n the o v e r a l l sample. These s p e c i a l c o l l e c t i o n s i n c l u d e d 259 animals and were 82 taken i n May of 1982 and 1983, during the h o t t e s t time of year, when r e p r o d u c t i v e a c t i v i t y i s presumed to be at i t s peak (Brownell and S t e v e l y 1981). A v a r i e t y of s h e l l measurements were taken from each specimen. For the purpose of t h i s study, only maximum l i p -t h i c k n e s s and maximum t o t a l l e n g t h are important ( F i g . 2). L i p t h i c k n e s s measurements were taken, using c a l i p e r s , as c l o s e to the t h i c k e s t p a r t of the l i p as p o s s i b l e . I t should be noted that specimens without f l a r i n g l i p s ( i . e . , f l a r i n g l i p not yet formed), or with incompletely f l a r e d l i p s , were assig n e d a l i p -t h i c k n e s s of zero. In order to study the p r e c i s i o n of maximum l i p - t h i c k n e s s measurements, the f l a r i n g l i p s of 63 specimens were cleaned and se c t i o n e d l o n g i t u d i n a l l y u s i n g a diamond saw. T h i s made i t p o s s i b l e to f i n d , e x a c t l y , the t h i c k e s t p a r t of the s h e l l l i p . O r i g i n a l (estimated) maximum l i p - t h i c k n e s s measurements were then compared with the accurate measurements taken from d i s s e c t e d s h e l l s , on the b a s i s of percentage e r r o r where: percentage e r r o r = accurate measurement - estimated measurement x 100 accurate measurement T i s s u e samples were taken from each specimen while the animals were s t i l l a l i v e . These samples were 5 mm t r a n s v e r s e s e c t i o n s through the v i s c e r a l mass, from j u s t behind the 83 t r a n s p a r e n t stomach w a l l . T i s s u e samples were preserved as d e s c r i b e d i n Ch. 2. A r e l a t i v e s t a t e of sexual development was a s s i g n e d to each preserved s e c t i o n . The c a t e g o r i e s range from 0-4 i n both males and females, but of n e c e s s i t y d i f f e r i n t h e i r p r e c i s e d e f i n i t i o n s (see Chapter 2). In g e n e r a l , c o n d i t i o n 0 i m p l i e s that sex was not yet h i s t o l o g i c a l l y d i s c e r n i b l e , c o n d i t i o n I i n d i c a t e s that d i f f e r e n t i a t i o n had begun, c o n d i t i o n II i n d i c a t e s that m a t u r i t y had been reached and that the gonad was d e v e l o p i n g , and c o n d i t i o n III i m p l i e s r i p e n e s s . C o n d i t i o n IV i n d i c a t e s that the specimen was spent. The c a t e g o r i e s are broad in t h e i r d e f i n i t i o n s and act only as approximations. Sexual maturity i s d e f i n e d here as having been reached when c o n d i t i o n II i s f i r s t entered ( i n both the male and female ma t u r i t y s c a l e s ) . At t h i s p o i n t there are mature sperm v i s i b l e in males, and v i t e l l o g e n i s i s i s w e l l underway i n females. Although i n d i v i d u a l s are not n e c e s s a r i l y capable of mating at t h i s time i t i s presumed to be imminent. The s m a l l e s t l i p - t h i c k n e s s measurement that was observed fo r male and female c o n d i t i o n II was taken as the l i p - t h i c k n e s s at which sexual maturity i s f i r s t reached. 84 RESULTS P r e c i s i o n of L i p Thickness Measurements Of the 63 l i p measurements t e s t e d a g a i n s t those f o r s e c t i o n e d s h e l l s , 16 o v e r e s t i m a t i o n s occured with an average e r r o r of 17%. There were 33 u nderestimations, the average e r r o r of which was 20%. O v e r a l l , the l i p - t h i c k n e s s measurements e r r e d by an average of 16% (S.D. = 4.25). Although few s h e l l s with r e l a t i v e l y t h i n f l a r i n g l i p s (<8mm) were t e s t e d (10), t h e i r measurements suggested that p r e c i s i o n improved with i n c r e a s e d 1 i p - t h i c k n e s s . Measurements of s h e l l s with t h i n s h e l l l i p s were g e n e r a l l y underestimated (by a maximum of 60% and average of 40%). As a consequence of a r e l a t i v e l y high margin of e r r o r , e s p e c i a l l y i n t h i n - l i p p e d specimens, the p r e c i s i o n with which sexual m a t u r i ty and l i p - t h i c k n e s s can be r e l a t e d i s d i m i n i s h e d . R e l a t i o n s h i p Between F l a r i n g L i p Thickness and Gonadal M a t u r i t y F i g u r e s 24A through 24E r e l a t e c a t e g o r i e s of sexual c o n d i t i o n to l i p - t h i c k n e s s i n 1 mm i n t e r v a l s f o r both sexes combined. Study of these f i g u r e s i l l u s t r a t e s that only c o n d i t i o n s 0 and I occur in specimens with l i p - t h i c k n e s s below 3 85 mm. At l i p - t h i c k n e s s e s above 3 mm, c o n d i t i o n II begins to develop, while above 4 mm and 6 mm r e s p e c t i v e l y , c o n d i t i o n s III and IV begin to occur. Table II r e p o r t s the maximum and minimum l i p - t h i c k n e s s e s a s s o c i a t e d w i t h each category of sexual c o n d i t i o n f o r each sex. C o n d i t i o n II occurs at a minimum l i p - t h i c k n e s s of 3 mm i n males (the next t h r e e lowest val u e s were 3, 3 and 4 mm) and at 4 mm in females (the next three lowest values were 5, 5 and 5 mm). Each maturation category c o n t a i n s specimens with a wide range of l i p -t h i c k n e s s e s , which i m p l i e s that the maturation c y c l e i n v o l v e s a r e t u r n t o the i n i t i a l u n d i f f e r e n t i a t e d s t a t e . T h i s i n turn suggests t h a t there may be a r e s t i n g phase and that c o n t i n u a l gamete p r o d u c t i o n does not n e c e s s a r i l y occur i n a l l i n d i v i d u a l s w i t h i n a p o p u l a t i o n . The r e s u l t s suggest that maturity i s f i r s t a t t a i n e d when i n d i v i d u a l s have l i p - t h i c k n e s s e s of 3 to 4 mm. However, i t i s important t o note that t h i s a n a l y s i s assumes that a l l i n d i v i d u a l s mature at approximately the same time i n the development of t h e i r f l a r i n g l i p . Previous s t u d i e s suggest that t h i s i s a reasonable assumption (Berg 1976, Wefer and K i l l i n g l e y 1980). These s t u d i e s suggest that l i p - t h i c k n e s s i s i n d i c a t i v e of age a f t e r growth i n l e n g t h ceases and the f l a r i n g l i p forms. Nonetheless i t i s probable that there i s some minor v a r i a t i o n in l i p - t h i c k n e s s , at mat u r i t y , between i n d i v i d u a l s . 86 FIGURE 24; A through E: Histograms 24A through 24E show percentage f r e q u e n c i e s of animals having l i p t h i c k n e s s e s from 0 to 15 mm, with measurements grouped i n t o 1 mm i n t e r v a l s . The frequency of each l i p - t h i c k n e s s i n t e r v a l i s r e p o r t e d s e p a r a t e l y f o r each category of sexual c o n d i t i o n . C o n d i t i o n 0, I, I I , I I I and IV r e s u l t s are i n f i g u r e s 24A, B, C, D and E r e s p e c t i v e l y . Note: Animals with l i p - t h i c k n e s s e s exceeding 15 mm were not i n c l u d e d i n the a n a l y s i s . 100 n Condition 0 80-60-40-20-r~i i 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Lip thickness interval (mm) 100-1 8 0 -Condition I 60 -40 -20 -1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Lip thickness interval (mm) 100-i 80 Condition n 60 40 20--I 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Lip thickness interval (mm) 100-, 80 -6 0 -Condition DI 40-20-T 2 I 3 4 5 6 7 8 9 1 0 11 Lip thickness interval (mm) 12 13 14 15 100-i Condition DC 80-60-40-20-T 1 I I 2 3 4 5 6 7 8 Lip thickness interval Ho TABLE I I : T h i s t a b l e l i s t s the maximum and minimum l i p t h i c k n e s s measurements f o r each category of sexual c o n d i t i o n f o r each sex s e p a r a t e l y . Note the minimum l i p t h i c k n e s s e s encountered i n c r e a s e d i n going from c o n d i t i o n s 0 to IV. FEMALES | MALES L i p Thickness . (mm) max. (mm) Gonad C o n d i t i o n | L i p Thickness 1 min.(mm) max. 16 0 1 - 19 0 25 I I 0 22 4 27 II | 3 25 6 29 1 H I | 4 29 7 25 1 i v | 6 26 92 FIGURE 25; The s i z e d i s t r i b u t i o n s of gi g a s specimens c o l l e c t e d are i l l u s t r a t e d i n t h i s f i g u r e . The e n t i r e sample set i s p l o t t e d along with the d i s t r i b u t i o n of the May 1981 and 1982 samples (combined) which were c o l l e c t e d s p e c i f i c a l l y to pr o v i d e a random sample of s h e l l l e n g t h s . All samples Random samples only 170 190 210 230 250 SHELL LENGTH INTERVAL (mm) ^ r 290 96 FIGURE 26; A THROUGH E Histograms 24A through 24E show percentage f r e q u e n c i e s of gigas specimens having s h e l l lenghts i n i n t e r v a l s of 10 mm. The frequency of each s h e l l l e n g t h i n t e r v a l i s r e p o r t e d s e p a r a t e l y f o r each category of sexual c o n d i t i o n . C o n d i t i o n s 0, I, I I , I I I and IV r e s u l t s are re p o r t e d w i t h i n f i g u r e s 26A, B, C, D and E r e s p e c t i v e l y . N | 2 | 9 | 21 | 27 J 53 159 |90 |I43|I69 JI581146|I22| 47 | 21 | 4 | I | 0 | 100-1 5 804 o S 40 o K Ul 0. CONDITION 0 i > i i 270 290 130 150 170 190 210 230 250 SHELL LENGTH INTERVAL (mm) 100-CONDITION I >- 80-o z Ui => o UJ60-u. ui 40-o or ui o. 20-130 4-150 170 190 210 2 30 250 SHELL LENGTH INTERVAL (mm) 270 290 100 CONDITION II > 80 u z IfJ UJ 60 tr g 40 o r r UJ 20 • 130 150 170 190 210 230 250 SHELL LENGTH INTERVAL (mm) 270 290 100 n CONDITION III >- 60 o UJ UJ 60 or u. 5 40 or UJ o. 20 130 150 170 190 210 230 250 SHELL LENGTH INTERVAL (mm) 270 290 100 CONDITION IV _~h r - r > - r n - r - n - n 130 150 170 190 210 230 250 270 SHELL LENGTH INTERVAL (mm) 100 TABLE I I I : Table I I I pr o v i d e s the mean and standard d e v i a t i o n s of maximum l e n g t h and l i p - t h i c k n e s s f o r a d u l t and j u v e n i l e Strombus gigas (as d e f i n e d by gonadal c o n d i t i o n and l i p - t h i c k n e s s ) . NOTE: Some u n d i f f e r e n t i a t e d t h i c k - l i p p e d specimens are presumed to be i n c l u d e d i n the n o t - r i p e category and t h e r e f o r e are expected to i n c r e a s e a l l measures i n t h i s c a t e g ory. However, the mean of n o t - r i p e i s not s i g n i f i c a n t l y d i f f e r e n t , at the 95% conf i d e n c e l e v e l , from the j u v e n i l e s as d e f i n e d by a l i p - t h i c k n e s s of l e s s than 4 mm which suggests that the d i s t o r t i o n i s not m i s l e a d i n g . APPARENT-ADULTS (maturity c a t e g o r i e s I I , I I I and IV) SHELL LENGTH: mean 211.6 std.dev. 26.8 LIP-THICKNESS: mean 15.2 std.dev. 5.7 NOT-RIPE (maturity c a t e g o r i e s 0 and I) SHELL LENGTH: mean 208.7 std.dev. 24.4 LIP-THICKNESS: mean 6.8 std.dev. 6.3 LIP-ADULTS (g r e a t e r than 4 mm l i p - t h i c k n e s s ) SHELL LENGTH: mean 212.3 std.dev. 24.8 LIP-THICKNESS: mean 14.5 std.dev. 5.1 LIP-JUVENILES ( l e s s than 4 mm l i p - t h i c k n e s s ) SHELL LENGTH: mean 199.3 std.dev. 28.1 LIP-THICKNESS: mean 1.2 std.dev. 1.5 1 02 R e l a t i o n s h i p Between Maximum S h e l l Length and Gonadal M a t u r i t y U n l i k e l i p - t h i c k n e s s at m a t u r i t y , maximum s h e l l l e n g t h at mat u r i t y c l e a r l y v a r i e s between i n d i v i d u a l s from w i t h i n p o p u l a t i o n s and between p o p u l a t i o n s (Randall 1964). Of the specimens measured, the maximum l e n g t h of an a d u l t was 295 mm and the minimum 125 mm. This v a r i a t i o n in l e n g t h at adulthood, makes i t impossible to d e f i n e a maximum s h e l l l e n g t h at which a l l i n d i v i d u a l s i n a p o p u l a t i o n are s e x u a l l y mature. F i g u r e s 25 and 26A through 26E have been i n c l u d e d in order to i l l u s t r a t e the r e l a t i o n s h i p between s h e l l l e n g t h and sexual m a t u r i t y . F i g u r e 25 provides the d i s t r i b u t i o n of s h e l l l e ngths w i t h i n the e n t i r e sample set f o r t h i s study and w i t h i n the samples where random c o l l e c t i o n of a l l s i z e s was emphasized. If the d i s t r i b u t i o n of the o v e r a l l sample set and the random samples are s i m i l a r , i t i s p o s s i b l e to use the e n t i r e sample set to analyze s h e l l length at m a t u r i t y . I n s p e c t i o n of f i g u r e 25 r e v e a l s that the two d i s t r i b u t i o n s are s i m i l a r but that the standard d e v i a t i o n i n the random sample i s s l i g h t l y lower. The mean and standard d e v i a t i o n of the e n t i r e sample set are 210 mm and 25.9 r e s p e c t i v e l y and f o r the random sample set are 197 mm and 25.1 r e s p e c t i v e l y . The e n t i r e sample set i s t h e r e f o r e b i a s e d towards l a r g e r s h e l l s . By not having a g r e a t e r p r o p o r t i o n of smaller s h e l l s the p r e d i c t i v e value of s h e l l l e n g t h , as an i n d i c a t o r of sexual m a t u r i t y , i s d i m i n i s h e d f o r t h i s data s e t . However, the range of lengths covered by both 103 sample s e t s i s s i m i l a r and encompasses the lengths at which members of the p o p u l a t i o n become s e x u a l l y mature. T h e r e f o r e , the e n t i r e sample set may be used, with c a u t i o n , to i l l u s t r a t e the p r e d i c t i v e value of le n g t h versus l i p - t h i c k n e s s f o r s h e l l lengths s l i g h t l y below and above the l e g a l l ength in B e l i z e . F i g u r e s 26A through E have been prepared to permit comparison between the a b i l i t y of s h e l l l e n g t h and l i p - t h i c k n e s s to d e f i n e adulthood. F i g u r e 26A through 26E are histograms of percentage frequency of specimens with s h e l l l e ngths i n i n t e r v a l s of 10 mm. The frequency of each l i p - t h i c k n e s s i n t e r v a l i s re p o r t e d s e p a r a t e l y f o r each category of sexual c o n d i t i o n . I n s p e c t i o n of these f i g u r e s i l l u s t r a t e s t h a t there i s not a p r e c i s e r e l a t i o n s h i p between s h e l l l ength and sexual m a t u r i t y as has been shown to occur f o r l i p - t h i c k n e s s . There i s no s i n g l e s h e l l l e n g t h , w i t h i n the i n t e r v a l s s t u d i e d , i n which sexual m a t u r i t y can be i d e n t i f i e d as f i r s t o c c u r r i n g i n the specimens s t u d i e d . On a s t a t i s t i c a l b a s i s , l e n g t h can be demonstrated as being a poor i n d i c a t o r of adulthood i f i t can be shown that t h e r e i s no s i g n i f i c a n t d i f f e r e n c e in the average maximum s h e l l l e n g t h of a d u l t s versus j u v e n i l e s . I f , at the same time, average l i p -t h i c k n e s s measurements are s i g n i f i c a n t l y d i f f e r e n t between j u v e n i l e s and a d u l t s , then l i p - t h i c k n e s s would be a b e t t e r i n d i c a t o r of adulthood. 1 04 Table II r e p o r t s the mean and standard d e v i a t i o n s of maximum l e n g t h and l i p - t h i c k n e s s measures f o r appar e n t - a d u l t s (specimens from sexual m a t u r i t y c a t e g o r i e s I I , III and IV) , not- r i p e (specimens from sexual maturity c a t e g o r i e s 0 and I ) , l i p - a d u l t s (speciments with l i p - t h i c k n e s s e s g r e a t e r than or equal to 4 mm) and l i p - j u v e n i l e s (specimens with l i p - t h i c k n e s s e s l e s s than 4 mm). Two d e f i n i t i o n s of a d u l t s and j u v e n i l e s have been i n c l u d e d because of the p o s s i b i l i t y that there are a d u l t s , ( i n a r e s t i n g or r e g e n e r a t i v e phase), with underdeveloped gonads and t h e r e f o r e with maturity c o n d i t i o n s 0 or I ( n o t - r i p e ) . T h i s p o s s i b i l i t y c o mplicates the s e p a r a t i o n of j u v e n i l e s from a d u l t s based on m a t u r i t y c o n d i t i o n s alone. If a d u l t s do enter a re g e n e r a t i v e phase, and t h e r e f o r e occur w i t h i n the n o t - r i p e category, then the mean and standard d e v i a t i o n of the s h e l l l e n g t h and l i p - t h i c k n e s s measures would be higher f o r t h i s group than f o r true j u v e n i l e s a l o n e . The mean and standard d e v i a t i o n of a d u l t s and j u v e n i l e s , as d e f i n e d by l i p - t h i c k n e s s measures, are t h e r e f o r e i n c l u d e d to permit comparison. There i s no s i g n i f i c a n t d i f f e r e n c e (at the 95% conf i d e n c e l e v e l ) i n the mean lengths of a d u l t s or j u v e n i l e s based on e i t h e r of the d e f i n i t i o n s . I t i s t h e r e f o r e not p o s s i b l e to separate j u v e n i l e s from a d u l t s , i n the segment of the p o p u l a t i o n s t u d i e d , based on s h e l l l e n g t h measures alone. I t i s presumed, however, that i f a l l p o s s i b l e s h e l l lengths were i n c l u d e d i n t h i s a n a l y s i s that s h e l l l e n g t h of a d u l t s versus j u v e n i l e s would be s i g n i f i c a n t l y d i f f e r e n t because of the p o s s i b l e weighting of 105 very small s h e l l s . The l e n g t h / m a t u r i t y data a v a i l a b l e from t h i s study i s only s u f f i c i e n t to t e s t whether i t i s p o s s i b l e to separate j u v e n i l e s from a d u l t s that are w i t h i n the range of lengths that sexual m a t u r i t y does occur. Since i t i s i n t h i s range of lengths that the need to p r e d i c t m a t u r i t y a r i s e s , the a n a l y s i s i s meaningful. In c o n t r a s t to s h e l l l e n g t h measures, s h e l l l i p - t h i c k n e s s measures of a d u l t s do d i f f e r s i g n i f i c a n t l y (at the 95% confidence l e v e l ) , from those of j u v e n i l e s f o r the segment of the p o p u l a t i o n s t u d i e d , even when some a d u l t s are i n c l u d e d w i t h i n the n o t - r i p e or j u v e n i l e category and t h e r e f o r e r a i s e the average l i p - t h i c k n e s s and standard d e v i a t i o n . T h i s supports the a s s e r t i o n t h at l i p - t h i c k n e s s i s a more accurate i n d i c a t o r of age or sexual maturi t y a f t e r the onset of f l a r i n g l i p development. It i s , however, d i f f i c u l t to compare the p r e d i c t i v e value of the two measures because i t has not been p o s s i b l e to separate j u v e n i l e s from a d u l t s with c e r t a i n t y , based on the m a t u r i t y s c a l e employed and because the data i s b i a s e d towards l a r g e s h e l l s . 1 06 DISCUSSION AND CONCLUSIONS The r e s u l t s of the s h e l l l i p measures suggest that sexual m a t u r i t y i s a t t a i n e d i n males and i n females at approximately the same times. I t i s important to note however, that the c r i t e r i a used to d e f i n e the onset of sexual maturity (mature and/or maturing gametes) do not ensure that the animals are capable of e j a c u l a t i o n or spawning. While i t i s presumed to be imminent, i t i s p o s s i b l e that sexual a c t i v i t y does not occur f o r some time a f t e r the onset of c o n d i t i o n I I . The l e n g t h of development- time remaining may d i f f e r between the sexes. It i s t h e r e f o r e p o s s i b l e that the sexes mature at d i f f e r e n t l i p -t h i c k n e s s e s (ages) but t h i s study does not provide strong support f o r such an a s c e r t i o n . The a b i l i t y to reproduce f o r the f i r s t time i s b e l i e v e d to occur when the l i p i s approximately 4 mm t h i c k , in both sexes. At t h i s age, the l i p i s s l i g h t l y t h i c k e r than when the f i r s t mature gametes are v i s i b l e ( i . e . , the onset of c o n d i t i o n I I ) . Using t h i s l i p - t h i c k n e s s (4 mm) as the minimum f o r the onset of sexual m a t u r i t y a l l o w s time f o r complete maturation of the gametes and compensates both f o r the f a c t that l i p measurements tended to be underestimated and f o r the assumptions used to d e f i n e m a t u r i t y . The i m p l i c a t i o n s of v a r i a t i o n s i n maximum length at the ou t s e t of f l a r i n g l i p development i s that resource management 1 07 p r a c t i c e s , based on maximum s h e l l l e n g t h measures, are not very e f f e c t i v e at ensuring that a l l members of a p o p u l a t i o n have the o p p o r t u n i t y to reproduce before h a r v e s t . Any s h e l l l e n g t h that allows the average, or m a j o r i t y , of a d u l t s to enter the f i s h e r y before harvest w i l l n a t u r a l l y r e s u l t i n a c e r t a i n percentage becoming mature before reaching l e g a l l e n g t h . Of the p o p u l a t i o n s t u d i e d , only 6% stopped growth in length and began the development of the f l a r i n g l i p at lengths below the l e g a l length of 175 mm. T h i s i m p l i e s that the e x i s t i n g management r e g u l a t i o n s i n B e l i z e i n c l u d e the great m a j o r i t y of the conch p o p u l a t i o n w i t h i n the f i s h e r y but that t h i s same m a j o r i t y may be harvested before becoming s e x u a l l y mature. T h i s i s because of the time i t takes to reach maximum l e n g t h , a f t e r having reached l e g a l l e n g t h , then the time to develope a t h i c k e n e d f l a r i n g l i p and, f i n a l l y , to become s e x u a l l y mature. Wefer and K i l l i n g l e y (1980), working on specimens from Bermuda, estimated that i t takes s i x months f o r the f l a r i n g l i p to completely f l a r e and approximately another e i g h t months for i t to t h i c k e n to roughly 4 mm. T h e r e f o r e , a f t e r reaching maximum len g t h i t takes approximately 14 months to reach sexual m a t u r i t y . If one adds to t h i s the time i t takes to reach maximum length a f t e r exceeding the l e g a l l e n g t h , and allow f o r more f a s t e r growth in warmer waters than Bermuda, then there may be a delay of one to two years between e n t e r i n g the f i s h e r y i n B e l i z e and reaching sexual m a t u r i t y . The conch that tend to grow the l a r g e s t w i l l have the longest p e r i o d i n the f i s h e r y 108 before maturi t y and t h e r e f o r e w i l l have the g r e a t e s t chance of premature harvest and the l e a s t o p p o r t u n i t y f o r c o n t r i b u t i n g to the next g e n e r a t i o n . Only those conch that mature below the l e g a l l e n g t h of 175 mm are ensured of the o p p o r t u n i t y t o reproduce before h a r v e s t . The delay, f o r the bulk of the p o p u l a t i o n , between a t t a i n i n g l e g a l l e n g t h and becoming s e x u a l l y mature i s l i k e l y to have resource management i m p l i c a t i o n s o n l y when conch stocks are under heavy f i s h i n g p r e s s u r e . The more intense the f i s h i n g pressure, the more l i k e l y i t i s t h a t conch w i l l be h a r v e s t e d soon a f t e r e n t e r i n g the f i s h e r y but before having had a chance to reproduce. Under heavy f i s h i n g p r e s s u r e , the breeding stock w i l l tend to be the f a s t e s t growing and/or the s h o r t e s t i n d i v i d u a l s . If s h e l l l e n g t h i s to be used as a b a s i s f o r management of conch sto c k s , i t should be done with the understanding that length r e s t r i c t i o n s cannot be used to ensure that most members of a p o p u l a t i o n have the o p p o r t u n i t y to reproduce before h a r v e s t . Length r e s t r i c t i o n s may, however, be used to ensure that a c e r t a i n p o r t i o n of the p o p u l a t i o n (e.g. 6%) w i l l reproduce, and to prevent small animals from being h a r v e s t e d . Management p r a c t i c e s based on s h e l l l ength measurements should be combined with a knowledge of the r e l a t i o n s h i p between a d u l t p o p u l a t i o n s i z e and recruitment r a t e . T h i s r e l a t i o n s h i p i s c u r r e n t l y unknown. 109 Management of conch stoc k s based cn l i p - t h i c k n e s s c o u l d be used to ensure that a l l or most members of a p o p u l a t i o n have the o p p o r t u n i t y to breed before h a r v e s t . T h i s type of management would not n e c e s s a r i l y r e q u i r e an understanding of the r e l a t i o n s h i p between breeding p o p u l a t i o n s i z e and recruitment r a t e s . However, the p o s s i b i l i t y that there i s v a r i a t i o n i n l i p -t h i c k n e s s at adulthood, and the c o m p l i c a t i o n s a s s o c i a t e d with p r e c i s e l y measuring l i p - t h i c k n e s s , makes i t probable that the use of a minimum l e g a l l i p - t h i c k n e s s would a l s o r e s u l t i n the harvest of some j u v e n i l e s , u n l e s s the minimum were set very high (eg. 10 mm). Nonetheless, the advantage of l i p - t h i c k n e s s measures are that a c o n s e r v a t i v e minimum l e v e l c o u l d be set that would ensure that only a d u l t s were harvested (eg. 10 mm). T h i s i s not p o s s i b l e through the use of s h e l l l e n g t h measures. A p r a c t i c a l working d e f i n i t i o n of l i p - t h i c k n e s s at adulthood would be a w e l l developed and completely opaque l i p . At t h i s p o i n t the l i p i s approximately 4 mm t h i c k . T h i s d e f i n i t i o n may allow some conch to be prematurely harvested but in g eneral would provide a p r a c t i c a l , minimum d e f i n i t i o n of adulthood f o r use i n f i s h e r i e s management. 1 10 CHAPTER 4: SEASON OF SEXUAL MATURITY INTRODUCTION A knowledge of the breeding c y c l e of an animal i s fundamental i n a c q u i r i n g an understanding of the animal's b i o l o g y , and i s p a r t i c u l a r l y important i n managing economically important s p e c i e s . There i s a c o n s i d e r a b l e amount of l i t e r a t u r e on the r e p r o d u c t i v e b i o l o g y of s h e l l f i s h e x p l o i t e d by f i s h e r i e s (Berg 1969, Boolootian et a l . 1962, Calabrese 1970, Feder et a l . 1979, Kennedy 1983, Porter 1964, Shepherd and Laws 1974) but much l e s s has been done on other s h e l l f i s h s p e c i e s , p a r t i c u l a r l y on meso- or neogastropods. S t u d i e s of meso- and neogastropod breeding c y c l e s are g e n e r a l l y complicated by i n t e r n a l f e r t i l i z a t i o n and the r e s u l t i n g adaptive r a d i a t i o n in gamete form (Franzen 1955) and r e p r o d u c t i v e s t r a t e g i e s (West 1978b). At the same time, m o t i l e animals that copulate are presumed to be l e s s dependent on s e a s o n a l i t y than are s e s s i l e broadcast f e r t i l i z e r s which o f t e n r e q u i r e synchronized r e p r o d u c t i v e a c t i v i t y and r e l a t i v e l y l a r g e gametic outputs. The study of t r o p i c a l l i f e h i s t o r i e s i s f u r t h e r complicated by l e s s obvious s e a s o n a l i t y i n the f a c t o r s that would promote annual v a r i a t i o n s i n r e p r o d u c t i v e a c t i v i t y . For these reasons, the breeding c y c l e s of b i v a l v e s and archaeogastropods are more r e a d i l y s t u d i e d than are the meso- or 111 neogastropods, and temperate s p e c i e s more r e a d i l y than t r o p i c a l . In t h i s chapter, the gonadal a c t i v i t y of one l a r g e p o p u l a t i o n of Strombus gigas i s repor t e d f o r a 22 month p e r i o d . The observed p a t t e r n of a c t i v i t y i s d i s c u s s e d with respect to ( l i m i t e d ) p h y s i c a l and environmental data and to r e p o r t s on a c t i v i t y p a t t e r n s made by other authors. METHODS AND MATERIALS Si z e at mat u r i t y i s reported i n chapter III as o c c u r i n g at a l i p t h i c k n e s s of approximately 4 mm. An a n a l y s i s of season of maturity r e q u i r e s that a l l specimens i n c l u d e d w i t h i n the samples be at l e a s t p o t e n t i a l l y mature. The r e f o r e , a l l specimens below the s i z e of sexual maturity were e l i m i n a t e d from a n a l y s i s . The specimens i n c l u d e d i n the a n a l y s i s were t h e r e f o r e a d u l t s , or p o t e n t i a l l y so, and were s a i d to comprise 100% of each monthly sample. The percentage frequency of each sexual c o n d i t i o n f o r each sample date was determined with the formula: Percentage frequency = number with c o n d i t i o n Z X 100 t o t a l number i n months' sample (Where Z c o u l d be any one of 0, 1, 11, 111, or IV.) 1 1 2 95% confidence l i m i t s f o r percentages were estimated (where i n d i c a t e d ) by e x t r a p o l a t i o n from confidence l i m i t s t a b l e s (Rohlf and Sokal 1981). D i r e c t environmental monitoring at the sample s i t e was g e n e r a l l y not p o s s i b l e due to i t s i s o l a t i o n . A continuous thermograph was a v a i l a b l e f o r only 3 months. During the time t h a t i t was a v a i l a b l e , i t was anchored j u s t above the seabed, w i t h i n the sampling region (see F i g . 5 f o r s p e c i f i c l o c a t i o n ) . Weather data were a v a i l a b l e from two sources. U n t i l August 1980, a p r i v a t e s t a t i o n operated on Ambergris Caye only 6 km no r t h of the sampling s i t e ; measurements were taken of d a i l y a i r temperature (taken at 9 am) and of t o t a l d a i l y r a i n f a l l . The B e l i z e Weather Bureau (B.W.B.) i s l o c a t e d at the B e l i z e I n t e r n a t i o n a l A i r p o r t which i s on the mainland, approximately 10 km i n l a n d from the coast and 40 km from the sampling area. T h i s source s u p p l i e d maximum and minimum a i r temperatures (taken at 500 m) and t o t a l d a i l y r a i n f a l l d u r i n g 1979 through 1983 (500 m was suggested as being most r e p r e s e n t a t i v e of c o a s t a l c o n d i t i o n s of the a v a i l a b l e d a t a ) . Average d a i l y temperatures were estimated from the BWB data and converted to average monthly v a l u e s . The Ambergris Caye data were a l s o averaged monthly. Weather data f o r Ambergris Caye from 1979-1980 and from the 1 1 3 BWB from 1979-present have been graphed ( F i g . 25) i n order to allow e x t r a p o l a t i o n to weather c o n d i t i o n s i n the study region d u r i n g the sampling p e r i o d (1981-1983). Average, d a i l y , bottom-water temperature f o r 3 months (Nov. 10/82 to Feb. 11/83) i s p l o t t e d along with average d a i l y a i r temperature from the BWB ( F i g . 26). RESULTS S e r i a l S e c t i o n s I n d i v i d u a l s e c t i o n s from s e r i a l samples ( e n t i r e gonads) were analyzed on the same b a s i s as a l l other t i s s u e s e c t i o n s . T h i s was done i n order to t e s t whether the use of s i n g l e c r o s s -s e c t i o n s from t e s t s u b j e c t s would b i a s r e s u l t s by g i v i n g an i n c o r r e c t i n d i c a t i o n of the o v e r a l l s t a t e of a gonad. Fourteen animals were s e r i a l l y s e c t i o n e d f o r a t o t a l of 206 i n d i v i d u a l s e c t i o n s . As many as 21 and as few as 9 were taken from a s i n g l e animal. Only 7 s e c t i o n s (4%) were i n c o n s i s t e n t i n t h a t they d i d not provide the same r e s u l t as the m a j o r i t y of s e c t i o n s taken from the same animal. The i n c o n s i s t e n c i e s occurred p r i m a r i l y i n l a t e c o n d i t i o n II and e a r l y c o n d i t i o n III males. T h i s was a consequence of 1 1 4 v a r i a t i o n s i n the development of the vas deferens between i n d i v i d u a l s . Sperm accumulation w i t h i n the vas deferens was one c r i t e r i o n used to d i s t i n g u i s h males i n c o n d i t i o n II from those in c o n d i t i o n I I I . In the specimens with i n c o n s i s t e n t r e s u l t s , the volume of sperm accumulated w i t h i n v a r i o u s regions of the vas deferens d i f f e r e d s l i g h t l y and r e s u l t e d i n d i f f e r e n t s t a t e s of m a t u r i t y being assigned to v a r i o u s s e c t i o n s from the same spec imen. In g e n e r a l , maturation of the gonad was completely simultaneous throughout. V a r i a t i o n s i n development were minimal, causing minor i n c o n s i s t e n c i e s only when a n a l y z i n g specimens at the boundaries between m a t u r i t y c o n d i t i o n s . F o l l i c u l a r development and d e n s i t y were c o n s i s t e n t in both males and females. The use of a s i n g l e s e c t i o n i n f u r t h e r a n a l y s i s i s not b e l i e v e d to have bi a s e d r e s u l t s . Weather Data F i g u r e 27 r e l a t e s weather data recorded by the B e l i z e Weather Bureau (BWB) with that recorded at Ambergris Caye. While a i r temperature i s s l i g h t l y higher at Ambergris Caye, r a i n f a l l i s lower. In general the two areas have s i m i l a r weather c o n d i t i o n s . BWB data may t h e r e f o r e be used to approximate weather c o n d i t i o n s at the study s i t e . F i g u r e 28 r e l a t e s d a i l y BWB a i r temperature readings with 1 15 bottom water temperature dur i n g the three months that the continuous thermograph was a v a i l a b l e . In g e n e r a l , water temperature and a i r temperature f l u c t u a t e d t o g e t h e r . During the p e r i o d monitored, the maximum and minimum bottom water temperatures were 27.5 C and 23 C r e s p e c i v e l y . T h i s was d u r i n g a time when a i r temperature, at the BWB, f l u c t u a t e d between 28.2 CC and 20 °C. The c l o s e r e l a t i o n s h i p between BWB weather data and t h a t f o r the Boca Chica sampling region i s presumed to be a consequence of t h e i r p r o x i m i t y (approximately 40 km). The p r e v a i l i n g winds are from the east, passing over Boca Chica and then onto the mainland (towards the BWB). Gonadal A c t i v i t y versus Time and Weather C o n d i t i o n s F i g u r e 29 and Table 4 have been i n c l u d e d i n order to present sampling dates and percentage frequency data f o r a l l samples and c a t e g o r i e s of sexual m a t u r i t y . Only category I I I ( r i p e ) w i l l be i n c l u d e i n f u r t h e r a n a l y s i s s i n c e t h i s c a t e g o r y r e p r e s e n t s those specimens that are, or w i l l soon be, r e p r o d u c t i v e l y a c t i v e . The term 'gonadal a c t i v i t y ' w i l l t h e r e f o r e be used as being synonymous with 'percentage frequency of c o n d i t i o n I I I ' . F i g u r e s 30 i l l u s t r a t e s the percentage frequency d i s t r i b u t i o n of. gonadal a c t i v i t y ( c o n d i t i o n I I I ) f o r a l l a d u l t s , 116 FIGURE 27: Ambergris Caye and B e l i z e Weather Bureau data have been p l o t t e d together i n order to show the s i m i l a r i t y i n weather p a t t e r n s between the two r e g i o n s . The a i r temperatures used are d a i l y averages, averaged monthly and p l o t t e d i n the middle of each month. T o t a l monthly r a i n f a l l has a l s o been p l o t t e d i n the middle of each month. Note: T i s s u e sampling took p l a c e from J u l y , 1981 to May, 1983 when onl y B e l i z e Weather Bureau (BWB) data were a v a i l a b l e . DATE (MONTHS) 118 FIGURE 28: B e l i z e Weather Bureau a i r temperature data along with water temperature data taken from a continuous thermograph, anchored w i t h i n the sampling r e g i o n , have been p l o t t e d together f o r the three months t h a t the thermograph was a v a i l a b l e . Note how a i r and water temperature move together, showing s i m i l a r p a t t e r n s . DATE FIGURE 29: Percentage frequency data f o r a l l c a t e g o r i e s of sexual c o n d i t i o n used to d e s c r i b e S_^  g igas t i s s u e s e c t i o n s . A l l sample p e r i o d s have been i n c l u d e d . Sample s i z e i s l i s t e d at the top of each column. Spacing of columns i s meant to i n d i c a t e time e l a p s e d between samples. N = 46 48 49 100-1 42 37 48 32 45 27 21 29 29 20 23 23 39 41 38 32 25 0 n R 1 ! 2 ! I \ 7, 1 i 1 A S O N D I I I ' " " I ""I J F M A M J 1982 I , - ii , . . . . , , J A S O N D J F M A M 1983 SAMPLING DATE (MONTHS) H Condition 0 m Condition I CD Condition II I Condition III 0 Condition IV TABLE IV: Sampling dates, sample sizes and percent frequency occurrence of each of the categories of sexual condition are given. A l l data included. Date Category Sample July 2, Aug. 10 Sept. 5 Nov. 25 Jan. 12 Feb. 15 Mar. 23 Apr. 17 May 25/ June 18 July 18 Sept. 1 Oct. 16 Nov. 18 Dec. 20 Jan. 8-Feb. 23 Mar. 19 Apr. 21 May 30, 1981 , 1981 , 1981 , 1981 , 1982 , 1982 , 1982 , 1982 26, 1982 , 1982 , 1982 8, 1982 , 1982 , 1982 , 1982 10, 1983 , 1983 , 1983 , 1983 1983 0 1 I I II 1 III 1 IV | Size 0 1 207. | 477. 1 337. 1 0 | 46 0 1 157. | 297. 1 567. 1 0 | 48 0 1 167. | 167. 1 617. 1 n 1 49 57. I 437. | 217. I 27. 1 29% I 42 57. 1 437. | 307. 1 117. 1 11% 1 37 0 I 317. | 447. 1 217. 1 4% | 48 37. 1 417. | 287. I 227. 1 6% | 32 67. I 387. | 387. 1 97. 1 9% | 45 47. I 337. | 267. 1 377. 1 0 | 27 57. 1 337. | 147. 1 437. 1 5% 1 21 77. I 107. | 247. 1 527. 1 7% | 29 417. 1 247. | 0 1 217. 1 14% | 29 207. 1 157. | 107. 1 407. 1 15% | 20 97. I 397. | 227. 1 267. 1 4% | 23 317. 1 267. | 177. 1 137. 1 13% 1 23 57. 1 267. I 217. I 337. 1 15% 1 39 27. I 107. | 257. I 617. 1 2% | 41 87. I 187. | 377. I 327. 1 5% I 38 37. 1 287. | 317. 1 357. 1 3% | 32 0 I 40% 1 367. 1 167. 1 8% | 25 Pc /X3 /ntfSi C7 1 24 along with the 95% c o n f i d e n c e i n t e r v a l s f o r these data. Although small sample s i z e s produced l a r g e confidence i n t e r v a l s f o r a number of months, s i g n i f i c a n t d i f f e r e n c e s between d i f f e r e n t sampling dates do occur. F i g u r e 31A and 31B have average monthly a i r temperature and r a i n f a l l ( r e s p e c t i v e l y ) p l o t t e d over gonadal a c t i v i t y . Pearson Product moment and Spearman Rank C o r r e l a t i o n a n a l y s i s ( S e i g e l 1956, Sokal and Rolf 1973) were performed to t e s t f o r a c o r r e l a t i o n between gonadal a c t i v i t y and a i r temperature or r a i n f a l l . No c o r r e l a t i o n was found at the 90% c o n f i d e n c e l e v e l . F i g u r e 32 shows the percentage frequency d i s t r i b u t i o n and 95% confidence i n t e r v a l s f o r c o n d i t i o n III ( a l l a d u l t s ) with l i p - t h i c k n e s s e s of 10 mm or g r e a t e r . S i g n i f i c a n t d i f f e r e n c e s occur between v a r i o u s sampling months d e s p i t e some s m a l l sample s i z e s . T h i s p a r t i c u l a r a n a l y s i s was performed in order to t e s t whether o l d specimens would have a d i s t r i b u t i o n s i m i l a r to a l l a d u l t s ( a d u l t s being d e f i n e d as having l i p - t h i c k n e s s e s g r e a t e r than 4 mm). The f a c t that F i g u r e s 30 and 32 are very s i m i l a r suggests t h a t the working d e f i n i t i o n of a d u l t s (Chapter II) i s a p p r o p r i a t e . However, the p r o p o r t i o n of r i p e specimens i n the o l d e r samples i s higher at a l l times and f l u c t u a t i o n s are more gra d u a l . The i m p l i c a t i o n s are that o l d e r specimens may remain r i p e longer and/or undergo l e s s r a j i i d gonadal changes, r e l a t i v e to j u v e n i l e s . f a x L2-6 FIGURE 30: Percentage frequency d i s t r i b u t i o n of 5. gigas specimens i n c o n d i t i o n I I I ( r i p e ) o n l y . 95% c o n f i d e n c e i n t e r v a l s are i n c l u d e d . PERCENT FREQUENCY ±2-8 FIGURE 31A: Percentage frequency d i s t r i b u t i o n of c o n d i t i o n I I I ( r i p e ) gigas specimens, with average monthly a i r temperature from the B e l i z e Weather Bureau superimposed over top. FIGURE 31B: Percentage frequency d i s t r i b u t i o n of c o n d i t i o n I I I ( r i p e ) specimens, with t o t a l monthly r a i n f a l l from the BWB superimposed over top. •Aftnma O A T C ( M O N T H * ) >30 FIGURE 32: The percentage frequency d i s t r i b u t i o n of c o n d i t i o n I I I ( r i p e ) gigas specimens wit h l i p t h i c k n e s s e s equal to or g r e a t e r than 10 mm o n l y . 02) 131 DISCUSSION AND CONCLUSIONS In g e n e r a l , the r e s u l t s imply that 1) there i s c o n s i d e r a b l e v a r i a t i o n i n r e p r o d u c t i v e a c t i v i t y throughout the year, with some a c t i v i t y o c c u r r i n g year-round, 2) t h i s a c t i v i t y does not have an obvious annual p a t t e r n nor i s i t n e c e s s a r i l y r e l a t e d to a i r temperature or r a i n f a l l and, 3) that 4 mm i s an e f f e c t i v e d e f i n i t i o n of adulthood. Previous r e p o r t s on the r e p r o d u c t i v e a c t i v i t y of S. gigas have been based on o b s e r v a t i o n s of c o p u l a t i o n , spawning or appearance of the gonad ( B l a k e s l e y 1977; Brownell 1977; DAsaro 1965; Davis 1982 Hesse 1976; Ra n d a l l 1964). T h i s i s the f i r s t study to be based on d e t a i l e d gonadal a n a l y s i s and r e g u l a r sampling, from a l a r g e r e g i o n , f o r a continuous two year p e r i o d . Random sampling and gonadal a n a l y s i s were employed, r a t h e r than o b s e r v a t i o n s of spawning and/or c o p u l a t i o n , f o r p r a c t i c a l reasons. A q u a n t i t a t i v e measure of the change i n r e p r o d u c t i v e a c t i v i t y was d e s i r e d f o r use i n f i s h e r i e s management. O b s e r v a t i o n a l s t u d i e s of spawning and c o p u l a t i o n are time and labour i n t e n s i v e and do not lend themselves to q u a n t i t a t i v e a n a l y s i s when working with sparse p o p u l a t i o n s under heavy f i s h i n g p r e s s u r e . A l a r g e r sample of a d u l t s i s r e q u i r e d f o r spawning s t u d i e s than f o r gonadal a n a l y s i s because only a small f r a c t i o n of the a d u l t p o p u l a t i o n may be a c t i v e at any one time. 1 32 It would t h e r e f o r e be necessary to observe a very l a r g e number of a d u l t s (eg. 200) in order to have a r e p r e s e n t a t i v e sample. This would not have been p o s s i b l e because of the low p o p u l a t i o n d e n s i t y i n the a v a i l a b l e study s i t e . The time and equipment were not a v a i l a b l e f o r such a labour i n t e n s i v e program. Gonadal a n a l y s i s does not r e q u i r e as much labour because both sexes may be c o l l e c t e d and a c t u a l behavior p a t t e r n s need not be observed. Gonadal a n a l y s i s i s t h e r e f o r e more a p p r o p r i a t e f o r the study of a l a r g e , sparse p o p u l a t i o n when q u a n t i t a t i v e r e s u l t s are d e s i r e d . I t i s important to po i n t out that o b s e r v a t i o n s of spawning i s p r e f e r r a b l e to gonadal a n a l y s i s i f a p p l i c a b l e . There can be no doubt about r e p r o d u c t i v e a c t i v i t y when o b s e r v a t i o n s of spawning are made. In c o n t r a s t , gonadal a n a l y s i s i s a more coarse measure of r e p r o d u c t i v e a c t i v i t y and r e q u i r e s that the gonad can be p r o p e r l y analyzed. There i s however, c o n s i d e r a b l e precedent f o r such gonadal a n a l y s i s ( B o o l o o t i a n e_t. a_l. 1962; Brousseau 1981; Calabrese 1970; Caugant 1980; Penchaszadeh e t . a l . 1982) I t i s b e l i e v e d that random e r r o r s may have been int r o d u c e d i n t o the r e s u l t s as a consequence of two f a c t o r s . The f i r s t i s the p o s s i b i l i t y that conch i n a l l areas of the sample s i t e are not e q u a l l y r e p r o d u c t i v e l y a c t i v e at the same time. If t h i s i s the case, and a l l monthly samples do not i n c l u d e a c e r t a i n p o r t i o n from each subgroup, then there w i l l be v a r i a t i o n between 1 33 months that i s not a consequence of s e a s o n a l i t y . The low d e n s i t y of the p o p u l a t i o n s t u d i e d made i t probable that a l a r g e p r o p o r t i o n of the study s i t e would need to be v i s i t e d during c o l l e c t i o n s . However, t h e r e was c e r t a i n l y some d i f f e r e n c e s between months and t h e r e f o r e the p o t e n t i a l f o r t h i s e r r o r e x i s t s . The second source of e r r o r i s a consequence of using gonadal a n a l y s i s r a t h e r than a c t u a l o b s e r v a t i o n s . Although a specimen may appear to be r e p r o d u c t i v e l y a c t i v e on a h i s t o l o g i c a l b a s i s , i t i s not a c e r t a i n t y . Only specimens that had l a r g e accumulations of mature gametes were i n c l u d e d i n t h i s a n a l y s i s . I t t h e r e f o r e seems probable t h a t they would engage in r e p r o d u c t i v e a c t i v i t y around the time of sampling, even though the exact t i m i n g may be unknown. T h i s e r r o r i s not b e l i e v e d to have been l a r g e . The two sources of e r r o r are b e l i e v e d to have int r o d u c e d random e r r o r and not to have s t r o n g l y b i a s e d the r e s u l t s i n any p a r t i c u l a r d i r e c t i o n . A n a t u r a l p a t t e r n of a c t i v i t y i s t h e r e f o r e l e s s l i k e l y to be apparent but i f apparent i s not l i k e l y to be a r e s u l t of experimental d e s i g n . T h i s Study The r e s u l t s of t h i s study i n d i c a t e t h a t some r e p r o d u c t i v e a c t i v i t y occurs year-round. T h i s confirms B l a k e s l e y ' s (1977) 1 34 e a r l i e r r e p o r t . R a n d a l l ' s (1964) a s c e r t i o n that there i s a short i n a c t i v e p e r i o d may have been the r e s u l t of a c t i v i t y l e v e l s being too low to be e a s i l y observed and/or a c t i v i t y o c c u r r i n g i n i n a c c e s s i b l e areas. The a s c e r t i o n of others ( A l c o l a d o 1976; Berg 1976; Brownell 1977; DAsaro 1965; Davis 1982; Hesse 1976) t h a t there i s a long season of i n a c t i v i t y i s not s u b s t a n t i a t e d f o r the p o p u l a t i o n s t u d i e d . The lack of apparent c o r r e l a t i o n between sexual a c t i v i t y and water temperature i s i n c o n t r a s t to suggestions made by Brownell (1977), Hesse (1976) and Randall (1964). Brownell (1977) suggested that a r a p i d drop i n sea temperature of only o 1.1 C may be adequate to t r i g g e r a seasonal response. Hesse (1976) suggested that seasonal changes i n temperature are one of the f a c t o r s that t r i g g e r s spawning a c t i v i t y . During t h i s study, sea temperature f l u c t u a t e d by as much as 4.5 °C while gonadal a c t i v i t y remained high. The i m p l i c a t i o n s are that sea temperature does not act as an independent c o n t r o l of r e p r o d u c t i v e a c t i v i t y i n B e l i z e a n waters. The lac k of c o r r e l a t i o n between gonadal a c t i v i t y and the weather data that i s a v a i l a b l e may be a consequence of; the weather data being from a remote source, e r r o r s introduced through the use of gonadal a n a l y s i s rather than b e h a v i o u r a l o b s e r v a t i o n s , or because gonadal a c t i v i t y i s r e g u l a t e d by other environmental or p h y s i c a l f a c t o r s or combinations of f a c t o r s . I t has however been p o s s i b l e to show that r e p r o d u c t i v e a c t i v i t y 1 35 occurs year-round, at l e a s t at the s i t e s t u d i e d . I t i s p o s s i b l e that authors that reported short breeding seasons d i d not look hard enough f o r spawning i n d i v i d u a l s , d u r i n g the apparent o f f -season, or that seasonal weather p a t t e r n s are more extreme in other areas and t h e r e f o r e have more marked e f f e c t s on r e p r o d u c t i v e a c t i v i t y p a t t e r n s i n these areas. The c l o s e d f i s h i n g season i n B e l i z e i s meant to c o i n c i d e with peak breeding a c t i v i t y . The r e s u l t s of t h i s study are i n s u f f i c i e n t to p i n p o i n t the season(s) when a c t i v i t y peaks, and t h e r e f o r e no recommendations f o r change can be made. Since r e p r o d u c t i v e a c t i v i t y occurs year-round, the le n g t h of the c l o s e d season may be more c r i t i c a l than the t i m i n g . However the c l o s e d season would be most e f f i c i e n t at p e r m i t t i n g undisturbed spawning to occur, i f i t c o u l d be centered around the p e r i o d of peak breeding a c t i v i t y . 1 36 CONCLUDING REMARKS In c o n c l u d i n g t h i s t h e s i s I would l i k e to point out th a t , from a resource management p e r s p e c t i v e , the r e s u l t s of t h i s t h e s i s are l i t t l e or no value i n B e l i z e . Although i t has been p o s s i b l e to r e l a t e s h e l l l i p - t h i c k n e s s to sexual maturity and to re p o r t that some sexual a c t i v i t y appears to occur year round, n e i t h e r of these f i n d i n g s are of p a r t i c u l a r value to managers at t h i s time. The d e c l i n e i n conch stocks w i t h i n B e l i z e i s a consequence of o v e r f i s h i n g r e s u l t i n g from a lack of enforcement of e x i s t i n g laws; i t i s not because of a lack of b i o l o g i c a l knowledge. My r e s u l t s w i l l not permit managers to deal with the f a c t that the e x i s t i n g s i z e r e s t r i c t i o n and c l o s e d season are e s s e n t i a l l y ignored. The r e s u l t s may however be of some value i n other C a r r i b e a n c o u n t r i e s , to a q u a c u l t u r a l i s t s or to academics, and t h e r e f o r e a l l may not have been i n v a i n . 1 37 REFERENCES CITED A l c o l a d o , P.M. 1976. Growth, morphological v a r i a t i o n s of the s h e l l and some b i o l o g i c a l data of the conch ("cobo") Strombus gigas L. (Mollusca, Mesogastropoda). Oceanologica S e r i e s 34: 27pp. A l l s o p p , W.H.L. 1979. D e s c r i p t i o n and o b j e c t i v e s of p r o p o s a l : IDRC p r o j e c t 3-P-79-0097. I n t e r n a t i o n a l Development Research Centre, Ottawa. B a c c e t t i , B. (ed.) 1970. Comparative Spermatology. 573pp. Academic Press, New York. B a c c i , G. 1954. Rapporti t r a c e l l u l e n u t r i c i ed o v o c i t i n e l d i f f e r e n z i a m e n t o e n e l l a maturazione d e l l a gonadi. In "Problemi d i S v i l u p p a n " (C. Ambrosiana, ed.) pp. 123-145. Mi lano. Berg, C.J. 1969. Seasonal gonadal changes of a d u l t oviparous o y s t e r s i n Tomales Bay, C a l i f o r n i a . V e l i g e r Y2 (1 ): 27-36. Berg, C.J., J r . 1976. Growth of the queen conch Strombus gigas , with a d i s c u s s i o n of the p r a c t i c a l i t y of i t s m a r i c u l t u r e . Mar. B i o l . 34:191-199. B l a k e s l e y , H.L. 1977. A c o n t r i b u t i o n to the f i s h e r i e s and b i o l o g y of the queen conch, Strombus gigas L., i n B e l i z e . Presented at American F i s h e r i e s S o c i e t y 107th Annual Meeting, Vancouver, B.C., Sept. 15-17, 1977. B o o l o o t i a n , R.A., A. Farmanfarmaian and A.C. G i e s e . 1962. On the r e p r o d u c t i v e c y c l e and breeding h a b i t s of two western s p e c i e s of H a l i o t i s . B i o l . B u l l . 122:183-193. Brousseau, D.J. 1981a. Spawning c y c l e and f e c u n d i t y i n a p o p u l a t i o n of P e t r i c o l a p h o l a d i f o r m i s from M i l f o r d , C o n n e c t i c u t . V e l i g e r 24(1):56-60. Brousseau, D.J. 1981b. Gametogenesis and spawning i n a p o p u l a t i o n of Geukensia demissa from Westport, C o n n e c t i c u t . V e l i g e r 24(3):247~251. 138 Brownell, W.N. 1977. Reproduction, l a b o r a t o r y c u l t u r e , and growth of Strombus gigas , S. c o s t a t u s and S. p u g i l u s i n Los Roques, Venezuela. B u l l . Mar. S c i . 27:668-680. Brownell, W.N. and J.M. S t e v e l y . 1981. The b i o l o g y , f i s h e r i e s and management of the queen conch Strombus gigas . Marine F i s h e r i e s Review 43:1-12. Calabrese, A. 1970. Reproductive c y c l e of the coot clam Mulina  l a t e r a l i s (Say), i n Long I s l a n d Sound. V e l i g e r 12(3): 265-269. Caugant, D. and J . Bergerard. 1980. The sexual c y c l e and r e p r o d u c t i v e modality i n L i t t o r i n a s a x a t i l i s O l i v i . V e l i g e r 23(2):107-111. C h i p p e r f i e l d , P.N.J. 1951. The breeding of C r e p i d u l a f o r n i c a t a (L.) i n the R i v e r Blackwater, Essex. J . Mar. b i o l . Ass. U.K. 30:49-71. C h i p p e r f i e l d , P.N.J. 1953. Observations on the breeding and settlement of M y t i l u s e d u l i s (L.) i n B r i t i s h waters. J . Mar. b i o l . Ass. U.K. 32:449-476. Clench, W.J. and R.T. Abbott. 1941. The genus Strombus in the Western A t l a n t i c . Johnsonia 1:1-15. D'Asaro, C.N. 1965. Organogenesis, development, and metamorphosis in the queen conch, Strombus gigas with notes on the breeding h a b i t s . B u l l . Mar. S c i . 15:359-416. Davis, M. and C O . Hesse. 1982. T h i r d world l e v e l conch m a r i c u l t u r e i n the Turks and C a i c o s I s l a n d s . Presented at the 35th Annual Meeting G u l f and C a r i b . F i s h . I n s t i t . , Nov. 7-14, 1981, Nassau, Bahamas. Feder, H.M., J.C. Hendee, P. Holmes, G.J. M u e l l e r and A.J. P a u l . 1979. Examination of a r e p r o d u c t i v e c y c l e of Protothaca  staminea using h i s t o l o g y , wet weight-dry weight r a t i o s , and c o n d i t i o n i n d i c e s . V e l i g e r 22(2):182-187. 1 39 Franc, A. 1951. Ovogenese et e v o l u t i o n n u c l e o l a i r e chez l e s gasteropodes prosobranches. Ann. S c i . Nat. Zool. B i o l . Anim. 11:135-144. Franzen Ake. 1955. Comparative morphological i n v e s t i g a t i o n s i n t o the spermeogenesis among M o l l u s c a . Zoologiska Bidrag Fran Uppsala, Band 30:399-456. Franzen, A. 1956. On spermiogenesis, morphology of the spermatozoon, and bi o l o g y of f e r t i l i z a t i o n among i n v e r t e b r a t e s . Zool. B i o r . Uppsala 31:356-482. F r e t t e r , V. 1953. The t r a n s f e r e n c e of sperm from male to female prosobranch, with r e f e r e n c e , a l s o , to the p y r a m i d e l l i d s . Proc. L i n n . Soc. London 164:217-224. Giese, A.C. and H.H. Webber. 1969. Reproductive c y c l e and gametogenesis i n the black abalone H a l i o l i s c r a c h e r o d i c . J . Mar. B i o l . 4:152-159. Grave, B.H. 1922. An a n a l y s i s of the spawning h a b i t s and spawning s t i m u l i of Chaetopleura a p i c u l a t a (Say). B i o l . B u l l . 42:234-236. Hanson, J . , J.T. R a n d a l l , and S.T. Bayley. 1952. The m i c r o s t r u c t u r e of the spermatozoa of the s n a i l Vivipar'us . Exp. C e l l Res. 3_:65-78. Hesse, C. 1975. The conch i n d u s t r y i n the Turks and Carcos I s l a n d s . Unpubl. Manuscript., 18 p. PRIDE, Pine Cay, Turks and Carcos I s l a n d s , B.W.I. Hesse, K.O. 1976. An e c o l o g i c a l study of the queen conch, Strombus gigas . MSc. t h e s i s , Univ. of Conneticut, 107pp. Houston, R.S. 1971. Reproductive b i o l o g y of Thais emarginata (Deshayes, 1839) and Thais c a n a l i c u l a t a (Duclos , 1832). V e l i g e r J_3( 4 ) : 348-357 . H o r i u c h i , S., and C.E. Lane. 1965. D i g e s t i v e enzymes of the c r y s t a l l i n e s t y l e of Strombus gigas Linne I. C e l l u l a s e and some other carbohydrases. B i o l . B u l l . , Woods Hole 129:273-281. 140 Kennedy, V.S. 1983. Sex. r a t i o s i n o y s t e r s , emphasizing C r a s s o s t r e a v i r g i n i c a from Chesapeake Bay, Maryland. V e l i g e r 25:329-337. Koike, K. and S. N i s h i w a k i . 1980. The u l t r a s t r u c t u r e of dimorphic spermatozoa i n two s p e c i e s of Strombus. Venus (Jap. J . Malac) 38:25.9-274. Laws, H.M. 1970. Reproductive b i o l o g y and s h e l l s i t e p r e f e r ence in Hipponix conicus (Schumacher). The V e l i g e r 13(2):115-121. Leonard, V.K. J r . 1969. Seasonal gonadal changes i n two b i v a l v e mollusks i n Tomales Bay, C a l i f o r n i a . V e l i g e r 11:382-390. L i t t l e , C. 1965. Notes on the anatomy of the queen conch, Strombus gigas . B u l l . Mar. S c i . 15:338-358. Loosanoff, V.L. 1969. Maturation of gonads of o y s t e r s , C r a s s o s t r e a v i r g i n i c a , of d i f f e r e n t g e o g r a p h i c a l areas subjected to r e l a t i v e l y low temperatures. V e l i g e r 11:153-1 63. Manzi, J . J . , A. Calabrese and D.M. Rawlins. 1972. A note on gametogenesis in the Oyster D r i l l s , U r o s a l p i n x c i n e r e a (Say) and Eupleura caudata (Say). V e l i g e r 14:271-273. Mooers, M.G. 1981. D i e t and r e p r o d u c t i v e b i o l o g y of the rocky i n t e r t i d a l prosobranch gastropod T r i c o l i a p u l l o i d e s . V e l i g e r 24:103-108. Parker, G.H. 1922. The l e a p i n g of the stromb ( Strombus gigas L i n n . ) . J . Exp. Zool. 36(2):205-209. Penchaszadeh, P.E. and Juan J . S a l a y a . 1982. Reproduction and gonadal changes in Laevicardium laevigatum , of G o l f o T r i s t e , Venezuela. V e l i g e r 25:343-344. P o r t e r , H.J. 1964. Seasonal gonadal changes of a d u l t clams, Mercenaria mercenaria (L.) i n North C a r o l i n a . Proceedings N a t i o n a l S h e l l f i s h Assn. 55:35-52. 141 Portmann, A. 1927. Die N a h r e i e r b i l d u n g durch a t y p i s c h e Spermien b e i Buccinum undatum . Z. Z e l l f o r s c h . Mikrosk. Anat. 5: 240-243. Portmann, A. 1931. Die a t y p i s c h e spermatogenese b e i Bucc inum  undatum und Purpura l a p i l l u s . Z. Z e l l f o r s c h . Mikrosk. Anat. 1 2:307-326. Quayle, D.B. 1943. Sex, gonad development and seasonal gonad changes i n Paphia staminea Conrad. J . F i s h . Res. Bd. Canada. 6:140-151. Quayle, D.B. Personal communication, P a c i f i c B i o l o g i c a l S t a t i o n , Nanaimo, B r i t i s h Columbia. R a n d a l l , J.E. 1964. C o n t r i b u t i o n s to the b i o l o g y of the queen conch, Strombus gigas . B u l l . Mar. S c i . G u l f and C a r i b . J_4: 246-295. Raven, C P . 1961. "Oogenesis: The storage of developmental i n f o r m a t i o n . " Pergamon Press, Oxford. Reinke, E.E. 1912. A p r e l i m i n a r y account of the development of the apyrene spermatozoa i n Strombus and of the nurse c e l l s i n L i t t o r i n a . B i o l . B u l l . 22:319-327. Robertson, R. 1959. Observations on the spawn and v e l i g e r s of conchs ( Strombus ) i n the Bahamas. Proc. Malac. Soc. London 3_3: 164-171 . Robertson, R. 1961. The feeding of Strombus and r e l a t e d h e r b ivorous marine gastropods. Notulae Naturae 343:1-9. Rohlf, F . J . and R.R. Sokal. 1981. S t a t i s t i c a l T a b l e s : 2nd e d i t i o n , W.H. Freeman and Co., San F r a n c i s c o , pp 156-162. Rosenthal, R.J. 1970. Observations on the r e p r o d u c t i v e b i o l o g y of the R e l i e f s Whelk, K e l l e t i a k e l l e t i i . V e l i g e r J1_2:319-324. 1 42 S c h e i b l i n g , R.E. 1980. Abundance, s p a t i a l d i s t r i b u t i o n and s i z e s t r u c t u r e of p o p u l a t i o n s of Oreaster r e t i c u l a t u s (Echinodermata: A s t e r o i d e a ) on sand bottoms. Mar. B i o l . 57:107-119. S e i g e l , S. 1956 Nonparametric s t a t i s t i c s f o r the b e h a v i o r a l s c i e n c e s . McGraw H i l l : Toronto. 312pp. Shepherd, S.A. and H.M. Laws. 1974. S t u d i e s on Southern A u s t r a l i a n Abalone (Genus H a l i o t i s ) I I . Reproduction of f i v e s p e c i e s . Aust. J . mar Freshwat. Res. 25:49-62. S o k a l , R.R., and F . J . Rohlf. 1973. I n t r o d u c t i o n to b i o s t a t i s t i c s . W.H. Freeman and Company: San F r a n c i s c o . 368pp. S t r a s d i n e , S., W. M i l l e r and K. Gonzalez. 1983. Conch O p t i m i z a t i o n P r o j e c t , B e l i z e : F i n a l Report, Phase I. IDRC Reports, Ottawa. Tuzet, 0 . 1930. Recherches sur l a spermatogenese des prosobranches. Arch. Z o o l . Exp. Gen. 70:95-229. Underwood, A.J. 1974. The r e p r o d u c t i v e c y c l e s and ge o g r a p h i c a l d i s t r i b u t i o n of some common eastern A u s t r a l i a n Prosobranchs ( M o l l u s c a : Gastropoda). Aust. J . mar Freshwat. Res. 25:63-88. Webber, H.H. 1977. Reproduction of gastropoda : prosobranchia. In : Reproduction of Marine I n v e r t e b r a t e s V o l . IV A.C. Giese and J.S. Pearre (eds.) Academic Press, N.Y. 1-77pp. Wefer, G. and J.S. K i l l i n g l e y . 1980. Growth h i s t o r i e s of Strombid s n a i l s from Bermuda recorded i n t h e i r 0 - 1 8 and C -13 p r o f i l e s . Mar. B i o l . 60:129-135. West, D.L. 1978a. Reproductive b i o l o g y of Colus stimpsoni . 1) Male g e n i t a l system. V e l i g e r 20:266-273. West, D.L. 1978b. Reproductive b i o l o g y of Colus stimpsoni . 2) Spermiogenesis. V e l i g e r 21:1-9. 143 West, D.L. 1979. Reproductive b i o l o g y of Colus stimpsoni . 3) Female g e n i t a l system. V e l i g e r 21 : 432-438. West, D.L. 1981. Reproductive b i o l o g y of Colus stimpsoni (Prosobranchia : Buccinidae) IV. Oogenesis. V e l i g e r 24:28-38. Wood, R.S. and D.A. Olsen. 1982. A p p l i c a t i o n of b i o l o g i c a l knowledge to the management of the V i r g i n I s l a n d s conch f i s h e r y . Presented at the 35th Annual Meeting Gulf and C a r i b . F i s h . I n s t i t . , Nov. 7-14, 1982, Nasssau, Bahamas. Young, J.S. and DeMartini, J.D. 1970. The r e p r o d u c t i v e c y c l e , gonadal h i s t o l o g y , and gamatogenesis of the red abalone, H a l i o t i s rufescens (Swainson). C a l i f . F i s h and Game 56: 298-309. 1 44 APPENDIX A - FIXATION AND PRESERVATION F i x a t i o n : i n Davidson's s o l u t i o n with a c e t i c a c i d . 20 p a r t s : f o r m a l i n 10 p a r t s : g l y c e r i n 30 p a r t s : 95% e t h a n o l 10 p a r t s : g l a c i a l a c e t i c a c i d 30 p a r t s : seawater F i x a t i o n i n c h i l l e d Davidson's f o r 24 to 48 hours. P r e s e r v a t i o n : i n Davidson's s o l u t i o n without a c e t i c a c i d . [contents as above but without a c e t i c a c i d . ] T i s s u e caps, with e n c l o s e d t i s s u e samples, were i n d i v i d u a l l y packaged in Whirl Paks with s e v e r a l teaspoons of p r e s e r v a t i v e . 1 45 APPENDIX B - HISTOLOGICAL TECHNIQUE The M i l e s Labteck L a b o r a t o r i e s , T i s s u e Tek 2 u n i c a s s e t e system was employed. A f t e r a r r i v a l at the h i s t o l o g y l a b o r a t o r y t i s s u e samples, w i t h i n t i s s u e caps, underwent standard p a r a f f i n imbedding f o l l o w e d by hemotoxylin and eo s i n s t a i n i n g . Imbedding The p a r a f f i n imbedding procedure employed an Autotechnicon U l t r a Processor on a s i x t e e n hour c y c l e . The treatment c o n s i s t e d of ten stages o f : 50% i s o p r o p y l a l c o h o l 70% i s o p r o p y l a l c o h o l 80% i s o p r o p y l a l c o h o l 95% i s o p r o p y l a l c o h o l 95% i s o p r o p y l a l c o h o l a b s o l u t e i s o p r o p y l a l c o h o l a b s o l u t e i s o p r o p y l a l c o h o l with 50% x y l o l x y l o l x y l o l p a r a f f i n wax at 56.5 C* under vacuum A f t e r impregnation the blocks were mounted on the o u t s i d e of the t i s s u e caps w i t h i n a wax blo c k . F r e e z e r c h i l l e d b l o c k s 146 were trimmed with an American Op t i c a l / S p e n c e r Rotary Microtome f i t t e d with simple, s t e e l wedge knives . Rough trimming, down to t i s s u e l e v e l , was completed before f i n a l trimming, with a f r e s h k n i f e , at s i x microns. F i n a l s e c t i o n s were placed i n a 53 C* water bath that i n c l u d e d .05% g e l a t i n as adhesive. The s e c t i o n s were f l o a t e d onto s l i d e s and a i r d r i e d . S t a i n i n g The s t a i n i n g program c o n s i s t e d of : BATH CONTENTS EMMERSION TIME x y l o l 30 sec. x y l o l 30 sec. x y l o l 30 sec . abs o l u t e i s o p r o p y l a l c o h o l 30 sec. ab s o l u t e i s o p r o p y l 30 sec. ab s o l u t e i s o p r o p y l 30 sec. 80% i s o p r o p y l 30 sec. 50% i s o p r o p y l 30 sec. 30% i s o p r o p y l 30 sec. tap water 30 sec. hematoxylin 4 minutes ( H a r r i s ' a c i d v a r i a n t ) tap water 30 sec. a c i d a l c o h o l 30 sec. (70% ETOH and 0.5% HCL) 147 tap water 30 sec l i t h i u m carbonate s o l u t i o n 6 sec. 95% ETOH 15 sec . 95% ETOH 15 sec . eo s i n 30 sec. ( a c i d o p h i l i c c o u n t e r s t a i n , 0.5% i n 95% ETOH) abso l u t e i s o p r o p y l 30 sec. abs o l u t e i s o p r o p y l 30 sec. abso l u t e i s o p r o p y l 30 sec. abso l u t e i s o p r o p y l 30 sec. x y l o l 30 sec. x y l o l 30 sec. x y l o l 30 sec. The f i n a l s t e p was mounting of g l a s s c o v e r s l i p s with Permount, ( n e u t r a l Canada Balsam i n t o l u e n e ) . 

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