Organogen.Sis of the Reproductive System of Helisoma Duryi Eudiacus (Pilsbry) (Pulmonata, Gastropoda), with Notes on Breeding Habits

Organogen.sis of the Reproductive System of Helisoma duryi eudiacus (Pilsbry) (Pulmonata, Gastropoda), with Notes on Breeding Habits

U~eIM~ Department of Zoology Master of Science

ABSTRACT

The development of the reproductive system of the frvShwater pulmonate, Helisoma duryi eudiscus (Pilsbry), was studied by means of seriaI sections. Development yas traced from embryos to mature speci mens. Chronological and histologic&l changes in the reproductive organs yere studied. The tyO priaordia which gave ris. to the entire reproductive tract yere clarified. Br.eding habits such as mode of hatching, type of fertilization, quantity of eggs laid, effects of o~gen and temperature on hatching and the resist &Dca of egg membranes to chemicals were additional aspects dealt Yith in this stuay.

• Organogenesis of the Reproductive System of He1isoma duryi eudiscus (Pi1sbry) (Pu1monata, Gastropoda), with Notes on Breeding Habits

Mabe1 Mai, B.Sc.

A thesis submitted to the Facu1ty of Graduate Studies and Research in partial fu1fi11ment of the requirements for t.he degree of Master of Science.

Department of Zoology YcGil1 University, Montreal Ju1y 1969 .'

l' r'::\. Mabe1 Mai 19'70 ;1 .i

ACKNOWLE1XHŒNTS

The wri~er Yi.h•• to thank Dr. Carol M. Lalli

for her close supervision over the major part of ~hi. study and for her patience and assistance in rea4ing

and correcting every detail of the manuscrip~. A special note of thanks is due to Dr. Tyrell Smith for suggesting

the ~opic and supervising part of the experiment. Ur.

Ronald Chalk has been Most helptul in providing ~echnical assistance in the preparation of the photomicrographs. The writer Tould like to thank many other staff members

of the Departmen~ of Zoology for ~heir advice and assist

ance in supplying various equipmen~s and materials. This study Tas supported partially by a

Scholarship from the Na~ional Research Council, and

by & research assistan~ship under Dr. Tyrell Smi~h,

Account No. 283-53 of the Na~ional Research Council. ii

TABLE OF CONTENTS

Page LIST OF FIGURES ------.------._.------iii LIST OP PLATES iv

INTRODUCTION ------.------1 MATERIALS AND METHODS ------7 GENERAL ------.------7 000 ANOG ENESIS ---_._------_ .. 8 BREEDING HABITS .lN]) HATCHING 9 RESULTS ------_. ------13 ORGANOGENESIS ------14

BREEDING HABITS AND HATCHING ------27

DISCUSSION ------31 ORGANOGENESIS ------31

BREEDING HABITS AND HATCHING ---- 35 SUMMARY ------39 LITERATURE CITED ------41 APPENDICES

1. FIGURES ------47 II. PLATES 52 iii

LIST OP PIGURES

Figure Page 1. Helisoma àuryi eudiscus, juvenile; shoYing the scheme used in taking maximum shell diameter.-- 48

2. Genital system of adult R. duryj eudiscus ----- 50

3. An egg capsule of R. duryi eudiscua.------51. iv

LIST OP PLATES

Plate Page 1. Cross section of preputial primordium of

Helisoma dUEYi eudiscus at 12 days alter

hatching --- . ---- 53 2. Cross section of cord of cel1s migrating from male tract of H. duryi eudiscus at 19 days after hatching - 53 3. Cross section of posterior digestive gland of li. durvi eudiscus at 19 days after hatching, showing the aggregation of germ cells 54

4. Oblique section of preputial complex of ~.

duryi eudiscus at 26 days after hatching 54 5. Longitudinal section of anterior female tract of H. duryi eudiscus at 26 days after hatching- 55 6. Longitudinal section of an ovotestis acinus to show hermapnroditic duct of li. durri

eudiscus at 26 days alter hatch~g 55 7. Cross section of vagina and seminal receptacle duct of li. duryi eudiscus at 3.5 mm stage 56 8. Oblique section of female gonopore area of ff. duryi eudiscus at 3.5 mm stage, showing gonopore plug 56 e 9. Cross section of uterus and prostate gland of v

li. duryi eudiscus at 4 mm stage ------57 10. Longitudinal section of preputium of g. duryi eudiscus at 4.5 mm stage, shoYing preputial

organ duct ------57 Il. Cross section of prostate gland of g. duryi

eudiscus at 4.5 mm stage ------58 12. Cross sections of hermaphroditic duct of g. duryi eudiscu! at 4.5 mm stage; a) 4 sections ll!l17erior to junction b); b) junction of hermaphroditic duct; c) 4 sections posterior to b) 58 13. Longitudinal section of hermaphroditic duct

of H.duryi eudiscus ·at 4.5 mm .stage ------59 14. Cross section of preputial organ of g. duryi

eudiscus at 6.0 mm stage ------59 15. Oblique section of penis of li. duryi eudiscus

at 6.0 mm stage ------60 16. Oblique section of oviduct and sperm duct of

g. dUrYi eudiscus at 6.0 mm stage ------60 17. Cross section of vagina and seminal receptacle duct of g. dUEri eudiscus at 6.0 mm stage ---- 61 18. Longitudinal section of reproductive tract of H. duryi eudiscus at 6.0 mm stage,------61 19. Longitudinal section of the oviduct at the level of the carrefour of g. duryi eudiscus vi.

at 6.0 mm stage ------.------62 20. LongitudinaL section of seminal vesicle of R. duryi eudiscus at 7.0 mm stage -- 62 1

INTRODUCTION There have been few thorough studies of the development of the basommatophoran reproductive system.

Here, Helisoma du~i eudiscus (Pilsbry) has been chosen for this particular field of research. Helisoma is usually classified as one of the subgenera of Planorbis, of the family Planorbidae a family of freshyater snails. Hyman (1967) recently has reviewed the taxonomy of this group. Helisoma is characterized by its discoi dal or planispiral shell with the apex flattened and depressed below the body whorl. The animal is sinistral and has an overall red colour due to the presence of red blood.

The genital system ~f common freshwater gas tropods has been the subject of much research. Boyever, most investigators limited their york to certain organs, mainly the penial complex and the ovotestis, rather than the.entire system. Büchner (1891) compared the penis of members of the genera Planorbarius, Segmentina, Planorbis, Anisus, Bathyomphalus and Gyraulus. Baker studied the systematics of Helisoma species in Wisconsin (1928) and the large planorbid snails of Europe and America (lQ31), based on the preputial complex and radu

lae •. He, as well. as most workers, emphasized the signi~i- 2

canee of the penial complex in taxonomie studies. The "penia! gland" reported by Baker Yas later re investigated by Abdel-Malek (1952), who determined its function in mating as a "hold-fast organ" and suggested that it should be named as such. The hi5tology and development of th. ovo testis of Vallonia pulchella Müll. yere studied by Whitney (1941). Hickman (1931) 1rOrked on the same organ in Succinea ovali. Say, but he yas concerned primarily Yith spermiogenesis. Watson (1919) worked on the affinities of Pyramidula, Patulastra, Acanthinu1a and Val10nia

s~ci.s, using the genital system as one of the bases

for comparison. The anato~ of the entire ad*Jt geni tal tract of Indop1anorbis exustus (Desh.) Yas investi gated by Rao (1923) and brief1y by Baker (1945). Crabb (1927) and Holm (1946) studied Lymnaea stagna1is appressa Say and attempted to correlate anatomy and tunction of the reproductive organs. Baker (1928, 1945) deàlt Yiih the reproductive systems of gastropods systematiea11Y. Morton (1955a) studied the evolution of the E1lobiidae, using their genital tract as one of the bases of refer ence. He also studied the functiona1 morpho10gy of the reproductive systems of the British El10biidae (1955b). 3

Dunc~ (1960) compared the reproductive systems ot Physa, Lymnaea, Planorbarius and Ancylus, and con cluded from similarity ot the reproductive tracts that the tamily Planorbidae yas ev01ved trom Physidae. The gross morphology of the &dult genital system of Helisoma trivolvis (Say) yas studied by Abdel-Malek (1954). In 1854, Hogg made the tirst observations of the develmpment of Lymnaea stagna1is, but he did not mention the development of the reproductive tract. H01mes (1900) studied the early development of Planorbis, from cleavage up to gastrulation. Fraser (1946) dealt with the embryology of the reproduc~ive ~ract of Lymnaea stagnalis appress~. Maturation of the reproductive system of Arion ~ Linnaeus yas treated by Smith

(1966), and that of Agriolimax re~iculatus (MÜller) by Runham and Laryea (1968). The mode of hatching of Most pulmonates has aroused the interest ot Many research yorkers, and the presence of an en~e to partially dissolve the inner egg membrane at the time of hatching has been a question open to dispute. Ankel (1937) investigated this in Nucella lapillus (L.) (prosobr~ch) and tound that a larval en~e yas involved (in Davis, 1961). Noland. 4

and Carriker (1946) .tudied the breeding habits of Lymnaea stagnaIi, appressa; their york included copu lation, fertilization, oviposition and hatching. Bondesen (1950) made a comparative morphological biological .analysis of the egg capsules of freshyater pulmonates an~ recorded the mechanism of hatching in many cases. Davis (1961) studied the emergence of AmQicola limosa Say (prosobranch). D'Asaro (1965) mentioned the dissolution of the apertural plug on egg capsules, probably through the production of en zymes, at the hatching of Strombus gigas (prosobranch) veligers. The same author (1966) reported similar observations for Thais haemastoma floridana (prosobranch). ------~ -----~~ Davis (1967) studied the emergence of veliger larvae from eggs laid by 6erithium algicola Adams (prosohranch) and by four.opisthobranchs: Phidiana. lyncaeus Bergh, Haminea antillarum d'Orbigny, Bertti.elinia caribbea Edmunds and Bursatella pleii Rang. In each case, he found that the egg membranes yere softened before the animaIs crawied O"At, and he concluded :t;hat this softening yas caused by the action of "an enzyme or enzymes;.' '.' Bayne (1966) studied the biochemical composi tion of the layers of the egg of Agriolimax reticulatus by means vf thin-layer chromatography, gel-filtration 5

and e1ectrophoresis, and 1ater (1968) emp10yed histo chemica1 techniques to study the biochemica1 components of the egg capsules of six pu1monates, one opistho branch and one prosobranch. Mi1kman (1954) carried out contro11ed experi ments on the hatching of Pundulus heteroc1itus eggs.

It was found that ex~ra oxygen has an inhdbitor,y effect on their hatching. Vaughn (1953) studied the effect of temperature on the hatching and growth of Lymnaea stagna1is appressa. Stephens (1966) ".orked on the effect of photoperiodic stimulation on oviposition of Helix aspersa. The possibility of self-ferti1ization ".as discussed by Crabb (1927~) and No1and &Carri~er (1946) for Lymnaea stagnalis appressa; by Whitney (1941) for - Va110nia pulchel1a; and by DeYitt (1967) for He1isoma duryi Yeatherby, Physa gyrina Say and Pseudosuccinea co1ume11a (Say). The mating habits of He1isoma trivo1vi, were described by Abde1-Ma1ek (1952). There has been considerable controversy as to the time and location of appearance of thé various reproductive organs in pu1monates. The purpose of the present stu~ Aas been to trace the deve10pment of the reproductive organs from embryos to mature specimens 6

of Helisoma duryi eudiscus. The chronological appear ance of the various organs has been noted, as weil as his~ological changes ûceurring during developmen~.

Attempts have been made to clear up certain aspects involved in and affecting the hatching of thi. species.

Yoreover, the type of fertiliza~ion &lso has been investigated. 7

MATERIALS AND METHODS

GENERAL The identification of the species of the snail, Helisoma duryi eudiscus, was confirmed by Dr. Morrison of the Smith.onian Institute, Waahington, D.C. (Haley, 1968). It was chosen for this study mainly because it is easily accessible and is prolific aIl through the year under laboratory conditions. Adult snails originally were obtained from a commercial aquarium shop. About 20 sexually mature snails were transferred to a glass aquarium containing coarse sand. The water temperature was maintained at 22-24o C by an electric aquarium-warmer, and the water was constantly aerated. The snails were fed occasion ally with commercial fish food, TetraMin. As the snails shoyed a tendency to deposit egg capsules on dark surfaces, microscopic slides Yere placed at the corners of the aquarium. Egg capsules deposited on the slides were collected every morning and transferred to individual coplin jars on which the dates of ovi position and hatching were marked, so that the age of the animaIs could be determined when they were killed. Aeration of young snails in coplia jars was accomp'lished by pumping & jet of air through the water every day 8

using a medicine dropper.

000ANOG ENESIS AlI measurements yere taken at maximum àhell diameter, i.e., from the tip of the anterior margin of the aperture to the back of the snail at the most distal point, as shown in Fig. 1. Embryos were fixed as yell as newly-hatched snails (about 300)1 in diameter).: Young snails were fixed at the ages of 7 days after hatching

(400~), 12 days (600p), 15 days (800p), 19 days (l,OOOp), 21 days (1,200p.), 26 days (1,600p), 30 days- (1,900Jl) and 40 days (2,050p). Oying to limited space, snails became difficult to raise in coplin jars after the age of about 40 days. Larger animaIs were selected from the aquarium according to body size. Snails Yith maximum diameters of 3.5 mm, 4.0 mm, 4.5 mm, 6.0 mm and 7.0 mm were sectioned. Their respective measurements after clearing in toluene became 2.8 mm, 3.6 mm, 3.7 mm, 4.5 mm and 6.0 mm, making an average of about 20% shrinkage. Adul t snails yere sectioned for comparison. The diagram of the entire reproductive tract (Fig. 2) was drawn from adult speci mens fixed in Bouinls fixative after being relaxed in 1% aqueous chloretone (Galigher, 1964). Unanasthetized snails were fixed in either hot Bouinls or Zenker's fixative. The acids in these 9

fixatives rapidly dissolved the shell, and the problem of removing the hard shells from live animaIs was elia ihated. Specimens .ere cleared in toluene and embedded in soft par affin (m.p. 560 C). Sections were cut at 8p, mounted serially and stained with Harris' Haematoxylin and Eosin, or Heidenhain's Iron Haematoxylin and O~ange G. BREEDING HABITS AND HATCHING ~ The number of days the embryos took to hatch was noted. At hatching, the embr,yos in the egg capsules were observed·in .ater under a stereomicroscope. Te investigate the problem of the hatching en~e, the follewing experiments .ere performed. A. Daoustfs substrate film method - modified (Thompson, 1966)

Since the hatching en~e supposedly acts on the egg membranes, frozen sections of hatching embryos were incubated with crushed membranes to see if any digestion would take place. Embryos at the stage of hatching were frozen and sectioned at 15~ in a cry.stat (_200 C). The tissue sections were transferred directly to gelatin-glycerol coated slides (gelatin:glycerol:water - 7:40:53; premelted on water bath). These slides were prepared by spreading 0.3 ml of this mixture over an 10

area of approximate1y 25 x 40 mm rith i;he tip of a pipette; i;he slides then yere laid horizonta11y to gel at room temperai;ure, 240C. The substrate yas prepared by mixing equal amounts of O. ~ crushed egg membranes and 5.~ aqueous ge1atin. Egg membranes yere eo11ected by removing the embryos at an ear1y deve10p mental stage (1-2 days). The membranes yere c1eaned in disti11ed yater, then subjected to 95% and abso1ute ethano1, and fina11y ether to remove unbound lipids. The dry yeight yas taken alter desiccation. 0.05 ml of this substrate mixture was spread over an area of approximate1y 25 x 40 mm on a slide, again using the tip of a pipette. The film Yas dried on a horizontal position and then fixed overnight in neutra1 1~ form a1dehyde. This preparation Yas yashed in disti11ed

Yate~ and al10yed to dry. The gelated tissue-bearing slides yere p1aced against the dry substrai;e film slides, and each pair Yas pressed firmly i;ogether. Incubation Yas carried out at room ~emperature, 240C, for 40 min. The slides yere separated and the tissue sections yere fixed by imme1sion in a horizontal posi tion in neutra1 1~ forma1in for 18 hours. A11 slides yere yashed in tyO changes of disti11ed yater and a110yed to dry. They Yere stained by the PAS method and Il

mounted in permount. B. Presence of hatching embryos Yith developing embryos It yas postulated that the hatching enzyme of newly-hatched snails, if present, ahould aid in the hatching of other snails. To demonstrate this, egg capsules containing young embryos st 2 to 5 d~s of development yere placed in coplLn jars where the embryos yere at the stage of hatching. Keeping aIl physical conditions normal, the number of days the young embryos took to hatch yas noted. The possible inhibitory effect of oxygen on hatching yas investigated by bubbling a continuous stream of pure oxygen into individual coplin jars containing egg capsules with embryos at the hatching stage. The oxygen stream yas stopped after 30 min, 60 min, 80 min, 90 min, 2 hours and 4 hours,respectively, and the time taken for the embryos to hatch y&S observed. The effect of temperature on h&tching Tas studied by subjecting neyly-laid capsules to various temperatures: -5 oC, 100 C, 20 0 C, 300 C and 40 0 C. Those subjected to freezing temperature yere taken out of the freezer after 3 or 4 days and yere alloyed to 'develop at optimal temperature. The number of days taken for the embryos to develop and hatch y&s recorded. 12

Solubility of the outer membranes of egg capsules of Helisoma duryi eudiscus was investigated by placing capsules in separate vials containing lN sodium hydroxide solution, lN hydrochloric scid, saturated urea, aqueous sodium borohydride and oxalic &cid, respectively. The results were noted after 24 hours. Egg counts were made on September 17, 1968; Oct.ber 8, 1968; April 25, 1969; and May 15, 1969. Twenty capsules containing a variable number of eggs were counted at a time. The total and average numbers of eggs per capsule were calculated. The possi.ility of self-fertilization was investigated by isolating individual sexually mature snails in beakers or coplin jars. 13

RESULTS

Helisoma duryi eudiscus is a hermaphroditic planorbid. The adult reproductive system (Pig. 2) is very similar to that described in Helisoma trivolvi~ by Abdel-Malek (1954). The system consists ot three major divisions: the hermaphroditic, female and male portions. The hermaphroditic portion includes the ovotestis (hermaphroditic gland), seminal vesicles and the hermaphroditic duct. The female portion con sists of the oviduct branching off from the hermaphro ditic duct, the albumen gland, mucus gland, uterus, oothecal gland and the vagina. A seminal receptacle duct leads trom the vagina into the seminal receptacle sac. The female genital pore is common to both the vagina and the seminal receptacle auct, and opens to the left of the male genital pore. The male portion of .the system consists of the sperm duct branc);lÎng ·off the hermaphroditic duct, the prostate gland, and the vas deferens which empties into the penis, or verge, in the proximal part of the penial çomplex. The distal part of the penial complex co•• ists of the preputium housing the preputial gland, or "hold-fast organ" of

Abdel-Malek (1952). This organ has ~ duct leading to the di aphr agm, which is a ring of muscle with a dilat- 14

able opening in the centre for the passage of the penis. The male genital pore is located at the base of the left tentaele. The Sp9rmatozoa pass from the ovotestis into the hermaphroditic duct, then through the secreting prostate gland into the vas deferens. The vas deferens is provided with long cilia, and it passes the sperm on to the seminal canal in the centre of the penis. In mating, the spermatozoa are discharged into the vagina of the partner. From the vagina, they may pass up the seminal receptaele duct to the seminal receptaele sac for temporary stor~e. More often, the spermatozoa travel up the uterus to the carrefour, where fertiliza tion occurs in the fertilization chamber. The albumen gland duct opens into the fertilization chamber. Fer tilized eggs go down the oviduct, through the muciyarous gland tp tie uteràa, wijere the ~othec~ g~d.8ecretes Oapaulea for the eggs. Then they are passed through the muscular vagina and oviposition follows.

ORGANOGENESIS The following description is made aecording to the sequence of organ dévelopment; in other words, those organs that developed before others were given priority. Bowever, this scheme contr~icts the direction 15

of the passage of gametes in most cases. Therefore, the reaaer is requested to bear in mind that the repro ductive products pass from the ovotestis down the length of the tract and are discharged through the appropriate gonopores, as described previously.

Embryos and newly-h&tched snails (30~ in diameter) Sllow' no trace of any reproductive organ,. nor is any primordium visible among the loose mesen chyme cells. At 7 days after hatching (40~), there is still no sign of development of the reproductive system. 12 days (60Op) -- Hear the base of the left tentacle, at the site of the future male gonopore, a group of cells near the epidermis cluster together.

These form a solid cord, about l5-~ in diameter, leading dorsally inwards (Plate 1). The cells are very small (5-6p) and tightly packed together. The length of this

cord is about 60-7~ at this stage of development. This cord of cells corresponds to the preputium in the adul t and will give rise to the preputial organ and the penis. 15 days (80Op) -- The cord of cells which appeared at 12 days has not differentiated much further. Meanwhile, groups of cells, very darkly-staiaing with 16

haematoxylin, hav~ appeared behind the digestive gland. The cells are small (5-6p) and contain very littl. ' cytoplasm. This is the first appearance of undiffer entiated germ cells which will give rise to the ovotestis.

19 days (l,O~) -- The cord of cells inYag ;i;il..at;i;ng from the future male gonopore has reached a length of l30-14Op. Though the cells are still small, a lumen is now clearly visible. This cord can be traced as far as the top of the odontophore on the left of the animal. About midway down this cord, a string of cells (about 20p in diameter) is seen branching off at an angle in the direction of the future female gonopore; i.e., lateral and to the left of the male gonopore (Plate 2). This string of cells corresponds to that portion of the vas deferens leading from the preputial complex to the side of the female tract (see Fig. 2). Posteriorly, the germ cella are confined to a small area behind the digestive gland. They are still undifferentiated and appear as nothing more than a group of darkly-stained cells (Plate 3) ~

21 days (1,20~) -- The undifferentiated male genital tract has reached a length of 200-250F. The cells lining the lumen become more tightly packed as the invagination goes deeper. The tract ends on the 17

1eft of the odontophore 8S a blind sac of ce11s about 30p in diameter. By this time, the female tract has appeared 1ateral and to the left of the male tract. It is a tube, blind at both ends, looping beneath the pharyngeal bulb and ending to the right of this bulb. This corresponds to the part of the female adult tract up to the uterus and the seminal receptacle sac (see Fig. 2). Although a lumen can be traced along the entire 1ength of this tract, the gonopore is not open, and the division between the vagina and the seminal receptacle duct is not yet distinguishable. Meanwhile, the germ cells are undergoing mit.tic divisions. Chroma tin material is more scattered and the cells stain much lighter and are larger (7-~)e The size of the ovo testis at this stage is about 60p in diameter and ISOp in length. Thi~ organ is not subdivided into acini.

26 days (1,60~) -- At a distance of about 200p from the male gonopore, the cells have begun to differentiate into organs of the preputia1 complexe There is a slight demarkation between the penis and the preputial organ. The cells are darkly-stained and grouped tightly together. There is no duct in the penis or in the preputial organ. The dimension. of the pre putial complex are about IOOp in diameter and 135p in 18

length (Plate 4). About l70p from the future female gonopore, there is a slight syelling of the femàle tract. The cells are dense and the lumen is about

7-~ in diameter. This swelling marks the position of the separation of the vagina from the seminal recep tacle duct. As the tract loops under the pharyngeal bulb, tyO distinct ducts can be seen coming off from the above-mentioned swelling (Plate 5). One of these, the seminal receptacle duct, leads to the blind s.minâl receptacle Rac; the other develops into the uterus. At this time, ~he ovotestis (120p in diameter, 400p in length) i. divided into several acini (.op in diameter, lO~ in length). The germ cells are easily located at the periphery of these acini and meiosis is taking Vlace. It is still very difficult to distinguish between the male and female germ cells since they aIl look similar in size and cellular components. A duct is seen leading from the acini; this is the hermaphroditic duct (Plate 6). 30 days (1, 90Owr1) - The preputial organ and the penis are yell-defined. The vaa deferens is seen coming off the base of the penis. The female genital tract is distinguishable up to the lev.l of the oviduct. Anteriorly, the tract ends blindly as a plug of cells 19

(35~ thick) b10cking the opening. The seminal recep tac1e sac at this stage appea.rs as an empty sac about

5~ in diameter and lOO~ in 1eDgth, ve.tra1 and slight1y to the right of the oesophagea1 bulb. Beside the seminal receptac1e duct roDS the undeve10ped ootheca1 gland region of the uterus. The uterus continues its way beyond the 1eve1 of the seminal receptac1e sac and terminates in the mid-ventral body 1ine. Since aIl the reproductive glands (i.e., ootheca1, mucus, albumen and prostate) are not yet developed, and the ce11s are undifferentiated histo10gica11y, it is very hard to pin point the specifie part of the system deve10ped at this time. However, the writer fee1s justified to assume the termination of this tract at the 1eve1 of the ovi duct, becQUse the tract goés on for a distance of about 400}l behind the seminal receptac1e sac. The acini of the ovotestis now are much more prominent. There are severa1 maturing ova with big nuc1ei st the periphery of each acinus; they have a maximum diameter of about

20-3ûJ1.~. Towards the centre of the acini, some sperme. tids and spermatozoa are found with carefu1 examination. The hermaphroditic duct has not deve10ped beyond the ovotestis. 40 days (2,05Op) -- The ce11s 1ining the preputial passage are noy columnar Yith ~ig nuclei. The preputial organ is a compact structure and is separated from the penis by a slight constriction on the preputial yall. The penis measures 130p in diameter and 16~ in length. The vas deferens leaves the base of the penis, loops foryard, and can be traced alongside the female traet up to the lev~l of the future mucus gland, where it will give rise to the prostate gland. The female tract, though larger in size, is not much different from that of animaIs at 30 days after hatching. The gonopore plug noy measures 21p. The cord of cells leading from the uterus continues in the mid-yentral body line. The lumen of the female taact gradually di:appears; it only can be spotted as a group of cells denser than the surrounding areas and it disappears completeit at the level of the gizzar4. In the ovotestis, maturation of gametes is taking p~ace. There is a large number of mature ova at the periphery of each acinus and numerous spermatids and spermatozoa in the central cavity. The acini are about 7~ in diameter and 250p

in length. A~, this stage, no seminal vesicles can be found. 3.5 mm -- Invaginations on the preputial organ become apparent, although the cells are still undiffer- 21

entiated. The seminal canal in the penis is not yet lined Yith ciliated columnar cells as in the adult, but is merely represented by a slit among undifferen tiated cells. Both the vagina and the oothecal gland region are now lined with tall columnar cells with elongated nuclei (Plate', 7). The uterus is lined by closely packed cuboitlal cells. The gODopDre is covered

only by a thin layer (~) of cells (Plate 8). 4 mm -- Short columnar or euboidal cells are found lining the inside of the preputial wall. The preputial organ is surrounded by an epithelium of the same type of cells. Tubules are found leading out from the centre of the organ and these are contin

uous Yith the epitbelial invagina~ions. These were also ebserved by Abdel-Yalek (1952) in the adult tract of Helisoma trivol vis. Since the preputi&l organ is not functional at this stage, the tubules are not as extensive as in the adult where they function in secretion. The penis is covered by an epithelium of long columnar cells with elongated basal nuclei. The vas deferens enters at the base of the ,penis, then beuds laterally and opens on the side about half the length up the peni •• The seminal canal is lined by ciliated columnar cells with basal nuclei. A few blood spaces and crystalline

1 22

concretions can be seen among the cells in the penis. Ciliation of the female tract can be seen quite dis tinctly from the gonopore area to the an~erior portion of the vagina and seminal receptacle duct. The oothe cal gland noy can be diatinguished easily ~rom the seminal receptacle duct because of its wide and folded lumen (30p in diameter), whereas the lumen of the latter is hardly recognizable. The uterus is lined Yith tall ciliated columnar cells, some of which are glandular. The uterus lumen is a long curved slit in cross-section. The prostate gland develops beside the uterus and is represented by conglomerations of darkly stained glandular cells which are not yet differentiated into tubules (Plate 9). A distinct hermaphroditic duct is seen collecting reproductive products from the ovo testis acini. Hoyever, the connec~ion of this duct to the uterus cannot be traced yet.

4.5 mm -- Â preputial organ duct leads from the centre of the organ to the outside of the preputium and re-entera at the diaphragme The duct is lined with tall columnar cella with elongated basal nuclei (Plate 10). The preputial organ itself becomes more convoluted on the surface and has more branching tubules. A~ the point where the seminal receptacle sac ends, the 23

prostate gland develops by out-pocketing from the male tract. The prostate glandular cells are arranged in acini Yith small lumens (Plate Il). The female gono pore ~ breaking through, since only a one-cell thick layer is seen covering the future pore. Beyond the level of the prostate gland, the uterus becomes glandQ aa.-r..' and the lumen constricted. This corresponds to the future position of the muciparous gland. The tract continues for a short distance and the lumen Yiden~ and branches out, where it dev610ps into the oviduct.

The .perm duct leading from the prostate gland il no~ yet completely separated from the oviduct. They inter trine and become continuous at several locations. Th'e albumen gland is not developed. The hermaphroditic duct begins at the junction of the oviduct and the sperm duct. It proceeds on the mid-ventral line of the body and meets the other portion of the duct leading

from the ovotestis. The junction (Plate l2b) is repre~ sented by a group of undifferentiated cells Yithout any lumen, whereas at either end of this portion (Plates 12a, 12c), the duct is well-defined. Within the ovotestis, the hermaphroditic duct is a distinc. structure with a cuboidal ciliated epithelium underlain by a thin connective tissue layer containing severai 24

muscle fibres (Plate 13). This duct is circular in cross-section. At this stage, the entire tract cao be traced without any disruption. Nevertheless, the albumen gland and the seminal vesicles are not as yet apparent. 6 mm -- The connective tissue lining the preputial wall now contains a large number of muecle fibres arranged both circularly and longitudinally. Thd muscle fibre arrangement also is found in the verge sac and to a lesser extent in the vas deferens. The

preputial organ (40~ in diamet~r) has Many tubular invaginations lined by columnar cells with hasal nuclei. These cells May be secreting, but the secretion is lost during fi.xation. The invaginations &;re continuous Yi th the central glandular canal lined Yith the same type of cells which are secreting profusely (Plate 14). The verge sac is highly muscular, Yith an internaI epi thelium of .hort columnar cells. The verge itselt

(36~ in diameter) has a surface epithelium of tall columnar cells Yith basal nuclei, overlying a layer of circular muscle fibres. The bulk of the verge is made up of connective tissue among which are numerous

blood spece~, thick muscle fibres arranged in .arious p'lanes, and a large number of crystalline concretions 25

(Plate 15), (see also Abdel-Malek, 1954). The vas deferens coming out from the base of the penis loops forward and runs parallel to the female tract. It is about 30p in diameter at this stage and has a tiny lumen lined with ciliated cuboidal cells. The wall of the vas deferens contains a relatively thick layer of circular muscle fibres. The vas deferens is folloyed by the prostate gland, which nOT is weIl developed and co~tains numerous tubules with cells filled with large globular secretory elements. These tubules unite and enter the sperm duct which is lined with short columnar cells and is very similar in a~arance to the tubules. This duct leads into the hermaphroditic duct (Plate 16). The female gonopDre has broken through the epidermis. The anterior part of the female tract is strongly mus cular, with a thick muscular wall of circularly arranged

fibres about 33p in thickness. The vagina and seminal receptavle duct now are histologically distinguishable

since the former has a more m~scular wall and a larger lumen (Plate 17). The seminal recep$acle duct leads into the seminal receptacle sac lined by cuboidal. cells with central nuclei. The vagina leads to the oothecal gland, characterized by its folded wall (Plate 18). This gland is continuous with the uterus, which is lined 26

with tall columnar cells, most of them glandular and mucus-secreting. The uterus joins the oviduct after p,assing through a small region kn0101 as the muciparous gland. This gland is an outgrowth of the female tract specialized for mucus secretion. The oviduct has a wide lumen of 26p in diameter. Along the wall there are a number of invaginations (Plate 19). These invagin ations, as weIl as the oviduct proper, are lined Yith tall ciliated columnar cells with central nuclei. The oviduct is convoluted and leads to a sac called the carrefour (Baker, 1945), yhich is essentially a swollen portion of the oviduct with a side pocket called the fertilization sac. Opening into the female tract at this region is the albumen gland consisting of a fey acini of cuboidal glandular cells. Where the hermaphro ditic duct passes into the ovotestis under the posterio~ region of the digestive gland, there are a number of protrusions on the duct followed by a large numbe~ of globular evaginations. These are the seminal vesicles, presently lined by ciliated cuboidal cells as in the hermaphroditic duct. Their lumens are filled with mature sperme The ovotestis con tains more mature gametes than before, and the acini are more elongat~d in longitudinal sections. 27

7 mm -- Purther development of the reproductive system proceeds simply by growth of the organs previously deseribed. The only significan":change is in the epi thelial lining of the seminal vesicles. The interiors of these vesicles are now lined by a thin syncytium with elongated nuclei (Plate 20). Furthermore, by this stage the albumen gland has become more tubu1ar and larger in size.

BREEDING HABITS AND HJ.TCHING At optimal temperature and with proper aeratiou, Helisoma duryi eudiscus hatches in 6-7 days. When the snail is ready to hatch, it crawls out of the inner membrane which has been ruptured by its radula (Noland & Carriker, 1946; Bondesen, 1950; Vaughn, 1953). The emergence from the capsule takes place through an opening formed by the bursting of the operculate suture (i.e., the margin of the outer membrane which .as pressed down firm1y onto the substratum by the action of the foot during oviposition). On hatching, this membrane is

lifted like a lid; this a~.e:·- Y&S observed by Bondesen (1950). Egg capsules are roundish-oval Yith -,a sha1loy curvature. Eggs are laid close to one another in a clockwise spiral (Pig. 3). It is believed that the 28

foot participates in the formation of the capsule, probably with a secretion to harden the capsule. The capsules are thin-walled with a firm surface membrane. Eggs are laid in one layer only. They measure 0.8 x 0.7 mm in size. Capsules are up to 8.0 mm in length.

Presence of Hatching En~e: A. Daoust's substrate film method - (modified) The substrate film slides were examined under the microscope. There was no sign of any en~atic digestion of membranes, aince there was no lighter

area .. the slides to indicate the action of aD en~e during incubation. B. Presence of hatching embryos with developing embryos Young embryos, in the presence of ha.tching sBails, took 6-7 days to hatch, as under normal condi- tion •• The results are presented as tollo'tfs:

DATE DATE PLACED DAYS OF DATE NO. OP DUS LAID WITBIUTCHING DEVELOPYENT H.ATCHED Im2UIRED FOR SIlAILS BEFORE TRANSFER IlA'lCHIHG Apr. 1 Apr. 3 2 Apr. 8 7 Apr. 15 Apr. 18 3 Apr. 21 6 Apr. 20 Apr. 24 4 Apr. 27 7 Apr. 21 Apr. 26 5 Apr. 28 7 29

Effect of Oxygen on Hatching: There was no evidence for an inhibitory effect of oxygen on hatching. The results are shoY.n as folloys:

DATE DATE OF DURATION OF DATE NO. OP DAYS LAID OXYGENATION CONTINUOUS HATCHED REgUIRED FOIl OXYGENATION HATCHIm

Mar. 19 Mar. 26 30 min Mar. 26 7

Mar. 20 Mar. 26 60 min Mar. 26 6

Mar. 22 Mar. 28 80 min Mar. 28 6

Mar. 22 Mar. 28 90 min Mar. 28 6

Mar. 27 Apr~ 2 2 hrs Apr. 3 7

Apr. 1 Apr. 5 4 hrs Apr. 8 7

Effect of ~emperature on Hatching: The eggs kept at _50 C did not develop. After four days, they were taken from the freezer and sub jected to optimal temperature, but there was no sign of revival of the embryos. The eggs kept at 100C for three d&ys did not show any development. Yhen optimal temperature was attsin.,

.4, they resumed developme~t and hatched in another 7 days. At 20°C, the embryos took 7 days to hatch. At 30°C, the embryos took 6-7 days to hatch. At 400 C, there was no development. After o four days, the eggs were restored to 24 C, but the embryos were dead. 30

Solubi1ity of Capsule Membranes: The results .ere recorded and are shown as folloTs: SOLUTION RESULTS AFl'ER 24. ROURS lN NaOH slightly softened lN HCl no change Saturated Urea no change Aq. Sodium borohydride no change Oxalic acid no change

Egg Counts: The results of egg counts are shown as folloTs:

~ NO. OP mGS/20 CAPS. NO./CAP. AVERAGE NO./CAP. sept.17, 379 4 - 28 19.0 1968 Oct.8, 361 2 30 18.1 1968 Apxil 25, 449 16 - 80 22.5 1969

May 15, 446 14 - 26 22~3 1969 Self-Pertilization: Isolated sexually mature snails failed to

lay eggs. This result also Tas observed in those indiY~a ••is; Thich had previous contact with other snails where copulation had taken place. 31

DISCUSSION OHGANOGENESIS The results of this study permit certain con clusions regarding the origin and development of repro ductive organprimordia in Helisoma duryi eudiscus. The genital system of this pulmonate does not start developing until the second yeek after hatchin.g. As pointed ou~ by Hyman (1967), most pulmoDates hatch as young snails with completely developed systems except for the reproductive one. In Helisoma duryi eudiscus, the entire reproductive system starts as 2 primordia, . one of which is an invagination of the skin ectoderm, whereas the other derives from mesenchyme cells at the posterior of the body. The former ectodermal invagina tion, which eventually becomes the male gonopore, is definitely the first to appear. This is folloyed closely by the mesenchymal ovotestis primordium at the pos~erior extremity of the body. The male tract appears around the lOth day after hatching as an eetodermal invagination at the base of the left tentacle. The anterior portion of the vas deferens appears between the second and the third week. The preputial complex begins to devèlop at the age of three yeeks, and in the fourth yeek, the 32

preputial organ and the penis can be seen. The rest of the vas deferens appears after the fourth week, and the prostate gland does not develop until the young snail reaches the size of 4 - 4.5 mm in maximum shell diameter. The sperm duct also develops at this time. The female tract .tarte from cells migrating from the original ectodermal invagination which forms the male gonopore. Th~se cells become apparent to the left of the male gonopore between the second and the third week after hatching. The female gonopore is formed by the extension of the tract anteriorly, breaking through the bo~ wall at about the 4.5 mm stage. The vagina and the seminal receptacle duct appear during the fourth week. The seminal receptacle sac is formed in the following few days, and the uterus is well represented at this time. The oothecal gland begins to develop from the uterus when the animal is about 3.5 mm. The oviduct appears early in the second month, but is not completely developed until the animal is about 4.5 mm in shell diameter. The oviduct becomes ,confluent with the sperm duct of the male tract and continues as the hermaphroditic duct, which joins the upper portion of the hermaphroditic duct coming from

the ovotestis. The muciparous gland and the al~umen 33

gland start developing at approximately the 6 mm stage. The ovotestis starts as a group of cel,ls among the 'tail' Mesenchyme, a few days after the first in vagination has appeared. These cells are mesodermal in origin, since there is no basement membrane between this group of cells and the loose Mesenchyme under the digestive gland. The ovotestis primordial cells of

Lymnaea stagnalis appressa are ~lso conaidered meso dermal by Fraser (1946). This organ is not divided into aeini until the fourth week. Maturation of germ cells t!kes place before the end of the first month, when the hermaphroditic duct starts developing and linking up the acini. This duct joins the lower portion around the 4.5 mm stage and the system is therefore complete except for the glands. The seminal vesicles develop by the time the animal measures 6 mm. Since large ova are more conspicuous and spermatogonia and spermatocytes are too small to be sorted out from the germinal epi thelium, it was very difticult to judge which sex gametes mature first. However, fully mature ova and apermatozoa

are found more or l8SS at the same time. 11his can be regarded as simultaneous hermaphroditism, a common fea ture in Most pulmonates (Hyman, 1967), although a slight protandry was claimed by Morton (1958) tor Otina, Physa 34

and Limax; and for Siphonaria hispida by Marcus and Marcus (1960) (in Hyman, 1967). Hoffmann (1922) believed that the entire reproductive system of Limax Maximus L. arises froD a single ectodermal invagination in the inner part of the mantle cavity. Brock (1886) reported similar findings for Agrio1imax agrestis (L.) (in Hyman, 1967).

Fraser (1946) reported three prim~ an1agen for Lymnaea stagna1is appressa: (1) a mesodermal rudiment yhich becomes th. oTotestis, (2) an ectoderma1 invagina tion which gives rise to the entire fema1e tract and

~ost of the male tract, and (3) another ectodermal invagination which gives rise to the preputium, penis and lower vas deferens. His observations differed from those of the present study in 3 major points. He c1aimed that a) the preputia1 comp1ex and the anterior portion of the vas deferens are deve10ped from a second ectoderma1 invagination independent of the primary invagination; b) this anterior portion of the vas deferens 1ater joins the upper portion which derives from the primary invagination; and c) the mouth of the

primar,v invagination later disappears yhil. tha~ of the second ecto4erma1 invagination persists as the male gonopore. The results of the present study shoy that 35

the male and female tracts are deve10ped from a single primordium in Helisoma duryi eudiscus. Evidences are:

(1) A cord of cel1s is seen migrating from the or~. iMwal invagination in the direction of the future femaie gonopore at an ear1y stage of development. (2) It has been observed that the preputia1 complex deve10ps by proliferation of ce1ls at the anterior portion of the male tract. Fu.rthermore, there ,,&S no trace of a:tly second invagination at this area throughout the entire c1ev.10pmental periode Hoyeve.r, the resul ts of this study are in agreemmt rith Fraser's Yith regard to the formation of the female gonopore in both pulmonates.

BREEDING HABITS AND HATCBING

There yas no evidence that a:tl en~e is in volved in the hatching of Helisoma duryi eudiscus. Howwver, Daoust's substrate film method is not a deci

sive one, sinee the egg ••mbrane is a very comp~ex structure (Bayne, 1968). Refined techniques and thorough kno"ledge of the membrane compoaents and their structural configuration are raquired before further histochemical analysis can be performed. Such york has not been done for the hatching of pu1monates. Although Anke1 (1937) found a larval enz.yme Yhich dissolved the capsule paug

for Nucella lapillus (~osobranch) (in Bayne, 1968), 36

and other YOrkers c1aimed that an enz.yme aids in hatch ing (D'Asaro, 1965, 1966; Davis, 1967), yet this has not been conc1usive1y demonstrated in· any other gastro pod. Bondesen (1950) was skeptica1 about the presence of such an en~e and be1ieved that Most pu1monate embr,yos hatch by guawing their way out, especia11y those in the fami1ies Physidae, Lymnaeidae and P1anorbidae. No1and

& Carriker (1946) observed that Lymnaca stagna1is appr~ssa hatches by continuous rasping off. of the inne:s: surface of the egg case. V~ghn (1953).a1so described the hatch t~g of the same species by rupture of the egg membrane. Davis (1961) determined the hatching process in Amnico1a liwlO-s & (prosobranch) 1;0 be non-enzymatic. The eggs of this pu1monate can survive a fœirly ride range of temperature&. Al thoUlgh the limi ting temperatures are yet to be found, they will be betyeen 10o -20 0 C for the loyer 1imit and bet.een aoo_40oC for the upper 1imit. Vaughn (1953) reported an effective range of Il o -320 C for growth and development of Lymnaea stagnali. appressa. Anot~er point to note is that the eggs of He1isoma duryi eudiscus can be kept at a coc!)! temperature (e.g., 100C) to temporari1y stop· the deve10pmeni., yhich yi11 resume as soon as optimal temper&ture is at$ained. The capsule membranes .ere found to be high1y resistant to several strong oxidizing and reducing agents. They presumably afford good protection against the attack of parasites and the adherence of foreign bodies. The extreme reisistance of the capsule is probably due to the strong linkage, possibly co-valent, between poly saccharide and protein (Bayne, 1968). The results of egg-counts show an increase of aumber in Spring. The difference is not significant, because 1) the snails had been kept in the laboratory for several generations and might have adapted to the lahoratory environment; 2) the frequency of oviposition increases with food and changa of water. Stephens (1966) reported that Helix aspersa reaches the peak of egg laying in late June and early July.

There was no evidence of self-fer~ilization in Helisoma duryi eudiscus. This is not surprising since the reproduotive tract is well-equipped for cross fertilization, with a hold-fast organ as weIl as a penis. This point of view a180 is held by Abdel-Ualek (1952) for Helisoma trivolvis, and by DeYitt (1967) for Helisoma duryi. Conflicting opinions are held by Crabb (1927a, 1927b) and Noland & Carriker (1946) for LYmnaea stagnalis appressa, and by DeYit~ (1967) for Physa gyrina and Pseudosuccinea columella. Whitney (1941) claimed that 38

self-fertilization l.S obligatory in Va,llonia pulchella. Though a fey breeding aspects noy have been investigated for this pulmonate species, extensive study still has to be done on the biochemical structure of the egg membranes and on the proof of the presence of a, hatching enz.yme. 39

SUMMARY

1. The reproductive tract of the pulmonate snail, Helisoma dutyi eudiscus, arises from two primordia. 2. The first primordium appears as an ectodermal invagination at the base of the left tentacle during the second week after hatching, when the

snail attains a size of 60~ in diameter. It gives rise to the duct system of both the male and female tracts. 3. The second primordium also appears in the second week, at the posterior end of the body behind and ventral to the digestive gland. It is meso dermal in origin and develop. into the ovotestis and upper part of the hermaphroditic duct. 4. The loyer part of the hermaphroditic duct is formed from the first primordium. It joins the upper

por~ion of the duct when the s~ail is about 4.5 mm. 5. The original invagination of the primary primordium persists as the male gonopore, whereas the anterior female tract breaks through the epidermis to form the female gonopore lateral and to the left of the ..1 •. gOllopore.

6. The entire system is complete at 4.5 mm stage exce~ for the seminal vesicles, the muciparous gland and 40

the albumen gland. The glands develop as évagina tions along the temaie tract. 7. The seminal vesicles also develop from the hermaph roditic duct by evaginations and become apparent at the 6 mm stage. 8. The ciliated heraaphroditic duct collects mature spermatozoa and ova trom the ovotestis acini and brings them to the sperm duct and oviduct, respectively, as it biturcates at the region of th. albumen gland. 9. This pulmonate takes 6-7 days to hatch under favor- able conditions.

10. An en~e involved in the mechanism of hatching

was not detected; o~gen has no effect on the period of hatching.

Il. Eggs are laid in roundish-oval capsules measuring up to 8.0 mm in length. Each capsule contains about 20 eggs, each of which measures 0.8 x 0.7 mm.

12. The eggs of ~is species survive a fairly wide o 0 0 0 range of temperature between 10 -20 C and 30 -40 C. The capsule membranes are extremely resistant to strong oxidizing and reducing agents. 13. There is no evidence of self-fertilization in this species. 41

LITERATURE CITED

Abde1-Ma1ek, E. T. 1952. The preputial organ of snails in the genus Helisoma. (Gastropoda: Pulmonata).

Amer. MidI. Nat., ~: 94-102. 1954. Morpho10gical studies on the fami1y Planorbidae (Yol1usca: Pu1monata).

Trans. Amer. Micro. Soc., ~(2): 103-124. Baker, F. C. 1928. The fresh water Mo11usca of Wisconsin. Wisconsin Acad. Sci. Arts & Lett. Monograph -

Bull. No. 70,. pt. 1. ______• 1931. The. classification of the large

planorbid snai1s of Europe and America. Proc. Zool. Soc. Lond., 1931: 575-592. ______• 1936. The freshwater mo11usc He1isoma

corpulentum and its relatives in Canada.

ottawa, J. O. _~atenaude, printer. ______• 1945. The mol1uscan fami1y P1anorbidae.

Ill. Biol. Monographs. Univ. of Ill. Press. Collation, revision and additions by H. J. van C1eave. 530 pp. Bayne, C. J. 1966. Observations on the composition of the layera of the egg of Agrio1imax reticu1atus. Comp. Biochem. Physio1., 1966, 19: 317-388. ______• 1968. Histochemica1 studies on the egg 42

capsules of eight gastropod molluscs.

Proc. malac. Soc. Lond., (1968) ~: 199-212. Bondesen, Poul. 1950. Comparative analysis of egg

capsules. Natura Jutlandica, ~: 1-209. Büchner, O. 1891. Beitrëge zur Kenntnis des Baues der

einheimischen Planorbiden. Jahresh. Ver. f. vaterlind. Naturk. Wurtemberg, !1: 35-118. Crabb, E.D. 1927a. Anatomy and function of the repro ductive system in the snail Lymnaea stagnalis appressa Say. Biol. Bull., §A: 55-66. ------_. 1927b. The fertilization process in the snail Lymnaea stagnalis appressa Say.

Biol. Bull., ~: 67-108. D'Asaro, C. N. 1965. Organogenesis, development, and metamorphosis in the queen conch, Strombus gigas, with notes on breeding habits.

Bull. Mar. Sci., 12(2): 359-416. ______• 1966. The egg capsules, embryogenesis,

and early organogenesis of a common oyster

predator, Thais haemastoma floridana (Gastropoda: Prosobranchia). Bull. Mar. Sei., 12 (4): 884-914. Davis, C. C. 1961. The hatching process in Amnicola limosa (Gastropoda, Prosobranchia). 43

Trans. Amer. Microsc. Soc., §Q: 227-230. Davis, C."C. 1967. Emergence of ve1iger 1arvae from eggs inige1at1nous masseS" laid by some Jamaican marine gastropods. Ma1aco10gia, 1967, 2(2): 299-309. DeWitt, R. M. 1967. Stimulation of egg production in a l.'hysid and a Lymnaeid. Ma1aco10gia, 1967, 2(3): 445-453. Duncan, C. J. 1960a. The evolution of the pu1monate genital system. Proc. Zool. Soc. Lond., !M: 601-609. 1960b. The genital systems of the fresh water b'asommatophora.

Pro~ Zool. Soc. Lond., ~: 339-356. Fraser, L. A. 1946. The embryology of the reproductive tract of Lymnaea stagnalis appressa Say. Irans. Amer. Microsc. Soc., 22(4): 279-298. Fretter & Graham, 1962. British proaobranch molluscs. Ray Society.

Galigher, A. E. & Kozloff, E. N. 1964. Easenti~s of practical microtechâique. Lea & Pebiger. 484 pp. Haley, G. P. 1968. Caloium storage in a freBhwater snail. Unpuhlished thesis, McGill Univèrsity. 44

Hickman, C. P. 1931. The spermiogenesis of Succinea ova1is Say with special reference to the components of the sperme Jour. Morph., 21: 243-289.

Hogg, J. 1854. Observations on deve10pmeD~ and growth of Lymnaea staana1is.

Trans. Microsc. Soc. ~ond., n.s., ~: 91-103. Ho1m, L. W. 1946. Histo10gica1 and functiona1 studies

on the genita1 tract of LY[~aea stagna1is appressa Say. Trans. Jmer. Micros. Soc., §§: 45-68. Ho1mes, S. J. 1900. The early deve10pment of P1anorbis. Jour. Morph., 12: 369-458.

Hyman, L. H. 1967. The Invertebra~es: Mo11usca I.

Vol. 4. McGraw Hi~l. 792 pp. Mi1kman, R. 1954. Contro11ed observation of hatching in PUndu1us heteroc1itus. Biol. Bull., !Q1:300. Morton, J. E. 1955a. The,evolution of the E110biidae

with a discussion o~ the origin of the Pu1monata.

Proc. Zo01. Soc. Lond., ~: 127-167. ______• 1955b. The functiona1 morpho1ogy of the

British E110biidae (Gastropod~Pu1monata) with special reference to the digestive an" reproductive systems. 45

Philos. Trans. Roy. Soc. Lond., ~: 89-160. No1and, L. E. & Carriker, M. R. 1946. Observations on the bio1ogy of the sn&i1 Lymnaea stagnalis appressa during twenty generations in 1abora

tory culture. Amer. MidI. Na~., !2:467-493. Rao, H.S.1923. On the anatomy of Indop1anorbis exustus, (Mo11usca, Pu1monata).

Rec. Indian Mus., ~: 199-219. Raven, C. P. 1958. Morphogenesis: the ana1ysis of Mo11uscan deve1opment. Pergamon Press, N.Y. Runham, N. W. & Laryea, A. A. 1968. Studies on the maturation' of the reproductive system of Agrio1imax reticu1atus (Pu1monata: Limacidae). Ma1aco1ogia, 1(1): 93-108. Smith, B. J. 1966. Maturation of the reproductive tract of Arion .!i!!: (Pu1monata: Arionidae).

Ma1acologia, ~(2): 325-349. Stephens, C. K. & Stephens, G. C. 1966. Photoperiodic stimulation of egg laying in the land snai1,

Helix asperaa. Nature, ~(5070): 1582. Thompson, S. W. 1966. S.e1ected histochemical and histo patho1ogical methods. C. Thomas publiaher. Vaughn, C. M. 1953. Effects of temperature on hatching

and. grorlh of LY!l!Daea stagnalis appress~ Say.

Amer. MidI. Nat., ~: 214-228. Watson, H. 1919. The affinities of Pyr.. idula, P~ulastra, Acanthinula, and Vallonia.

Proc. Yalacol. Soc. Lond., ~: 6-11. Whitney, M. E. 1941. The hermaphroditic gland and germ cells of Val10nis pu1che11a Yu11.

Pape Mich. Ac ad. Sei. Arts & Lett., ~J 311-338. Wilbur, K. M. & Yonge, C. M. 1964-1966. Physiology of Mollusca. Vols. 1 8: II. Academie Press', N.Y. APPENDIX I

FIGURES maximum she Il diameter

Pige 1. He1isoma durxi eudiscus, juv.eni1e; showing the scheme used in taking maximum she11 diameter.

Pige 2. Genital ~s~em of adult ~. duryi eudiscus.

ABBREVIATIONS al.g., albumen gland; f.g., female gono pore; h.d., hermaphroditic duct; m.g., male gono pore; .u.g., muciparous gland; oo.g., oothecal gland; ovd., oviduct; oTt., ovotestis; pre., pre putium; pre.o.d., preputial organ duct; pro.g., prostate gland; prs.h., protrusions on hermaphro ditic duct; s.r.d., seminal receptacle duct; s.r.s., seminal receptacle sac; s.v., seminal vesicles; sp.d., sperm duct; v.d.; vas deferens; v ••• ; verge sac; va, vagina; ut, uterus. 50 .- ...... #-' .' ..'" .. ' ...... '" al. g.---...... ,:,-,,'-' ~.. ~~~~~~I§i':;:=:--- pr5. h . .' . /,.' /YF---n.d • ,. /. ..' ~1'H_~--5p. d • .. :'" .,- ...... -+---i---ovd. mu.9._:"-··"2&~-_ pro. --+---ut.

OV Lo-----'~~·

~---5.r.5.

OO.g.

-~..---'''. 50 -+---5. r.d ...... ;..... -+---va. v.d.

-.fH--pre. 1 1 mm· 1 -·..-".---f.g. \z.:s:p::a:as::az:=~--'

Pige 2. Geni~al s,ystem of adul~ ~. duryi eudiscas. 51

.1mm.

Fig. 3. An egg capsule of ~. duryi eudiscus. APPENDIX II

PLA.!ES 53

Plate 1. Cross section of preputial primordium of Helisoma dUryi eudiscus at 12 d~s after hatching. AlI sections shown in Plates 1-20 were stained with Harris' haematoxylin and eosin. L, left side of snail; PH, ph~; PP, primordium of preputial complexe

Plate 2. Cross section of cord of cells migrat ing from male tract of H. duryi eudiscus at 19 days after hatching. White arroy shows direc~ion of migrat ion. Ct cells migrating f»om original primordium; PP, future preputium; L, left side of snail; 0, odontophore. "\

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( 54

Plate 3. Cross section of posterior digestive gland of B. ,du"i eudiscul. at 19 da~. after hatching, Iho..... îng the aggregation of germ cells. DG, digestive gland; GC, germ cells.

Plate 4. Oblique section of preputial complex of 1_ dUrIi eudlscus at 26 days ,after hatching. ·L, left side of snaii; 0, odontophore; PB, devèloping penis; PO, developing preputial organ. 33 .,u. ....,_.

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Pla~e 5. Longitudinal section of anterior female trac~ ofy. dUryi eudiscus a~ 26 days >af~~r ha~c~ing. L, left side of sn&il; CG, oo~hecal gland region; PSBD, position of seminal.recep~acle duct; PVA, posi~ion of vagina.

Pla~e 6. Longi~udinal sec~ion of an ovo~es~is acinus to show herma~rodi~ic duc~. of g.. dUlYi eudiscus at 26 days af~er ha~ching. DG, diges~ive gland; GO, germ cells; RD, hermaphrodi~ic

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Plate 7. Cross section of vagin a and seminal

re~eptacle duct of ~. duryi eudiscus at 3.5 mm st'age. L, left sid,e of snail; PRE, preputium; SRD, seminal receptacle duct; VA, vagina; VD, vas deferens.

Plate 8. Oblique section of lemale gonopore area of ~. dUryi eudiscus at 3.5 mm stage, showing gonopore plug. PGP, female gonopore plug; L, left side of snail; LU, lumen of anterior female tract; VD, vas deferens. \.

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Plate 9. Cross section of uterus and prostate gland of ~. duryi eudiscus at 4 mm stage. PG, prostate gland; UT, uterus.

Plate 10. Longitudinal section of preputium of ~. duryi eudiscus at 4.5 mm stage, showing pr~utia1 organ duct. L, 1eft side of snai1; PE, penis; PH, pharynx; PO, preputia1 organ; POD, preputial organ duct; SRD, seminal receptac1e duct; VA, vagina; VD, vas deferens. '~: ( / ,

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Plate Il. Cross section of prostate gland of ~. dutyi eudiscus at 4.5 mm stage. PG, prostate gland; SRS, se.inal receptacle S&4; UT, uterus.

Plate 12. Cross sections of hermaphroditic duct of !. durxi eudiscus at 4.5 mm stage; a) 4 sections anterior to junction b); ~) junction of hermaphroditic duct; c} 4 sections poste~or to b)~ BD, hermaphroditic duct; JHD, junction of hermaphroditic duct. o

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Plate 13. Longituainal section of hermaphroditic duct of !l. duryi eudiscus at 4.5 mm stage. AC, acinus of ovotestis; BD, hermaph~oditic duct.

Plate 14. Cross section of preputial organ of !l. duryi eudi'sc1,!§. at 6.0 mm'stage. CC, cent:tal canal rlth secretion; PO, preputial organ; TI, tubular invaginations; VD, vas deferens. \ ") "-. ~., / ---- 60

Plate 15. Oblique section of penis of R. duryi eudiscus at 6.0 mm s$.,e. BS, blood space; PE, penis; SC, seminal canal; SRD,. seminal receptacle duct; VA, vagina.

Plate 16. Oblique section of oviduct and sperm duct of n. durri eudiscus at 6.0 mm stage. BD, bermaphrodi tic duct; OVD, oviduct; SPD, sperm duct. 100 )..l 1 1

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Plate 17. Cross section of vagina and seminal receptacle duct of.g. duryi eudiscus at 6.0 mm stage. SRD, seminal recep tacle duct; VA, vagina; VD, vas . deferens.

Plate 18. Longitudinal section of reproductive tract of g. duryi eudiscus at 6.0 mm stage. OG, oothecal gland; aVD, ovi duct; PG, prostate gland; SR», seminal receptacle duct; SRS, seminal recep tacle sac; UT, uterus. () ..

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Pla~e 19. Longitudinal sec~ion of ~he oviduc~ a~, the level of the carrefour of R. du~i eudiscus a~ 6.0 mm stage. PC, fertilization chamber; OVD, ovi duc~; PG, pros~ate gland; SPD, sperm duct.

Plate 20. Longitudinal section of seminal vesicle of ~. duryi eudiscus a~ 7.0 mm stage. AC, acinus of ovo ~es~i8; SP, sperm; SV, seminal vesicle. ~~. ~" -' "-

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