Reproduction and Development of Ferrissia Rivularis (Say) (Basommatophora: Ancylidae) and the Effects of Maleic Hydrazide on Its Development and Fecundity

Brigham Young University BYU ScholarsArchive

Theses and Dissertations

1973-04-01

Reproduction and development of Ferrissia rivularis (Say) (Basommatophora: Ancylidae) and the effects of maleic hydrazide on its development and fecundity

Yuan-Hsu Kang Brigham Young University - Provo

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BYU ScholarsArchive Citation Kang, Yuan-Hsu, "Reproduction and development of Ferrissia rivularis (Say) (Basommatophora: Ancylidae) and the effects of maleic hydrazide on its development and fecundity" (1973). Theses and Dissertations. 7797. https://scholarsarchive.byu.edu/etd/7797

This Dissertation is brought to you for free and open access by BYU ScholarsArchive. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of BYU ScholarsArchive. For more information, please contact [email protected], [email protected]. REPRODUCTION AND DEVELO?.MENT OF FERRISSIA RIVULARIS (SAY) (BAS0MMAT0PH0RA: ANCYLIDAE) AND THE EFPECTS OF MALEIC HYDRA.ZIDE ON ITS DEVELOPMENT AND FECUNDITY

A Dissertation

Presented to the

Department of Zoolcgy

Brigham Young Dniversity

In Partial FulfillmPnt

cf the Rc:!quirerr,ents for the Degree

Doctor of PhiloGophy

Yl' .a.~,---:.-Isu Ran1:,r

April 1973 iii

ACKNOWLEDGMENTS

Grateful acknowledgment is made for the valuable

suggestions, encouragement, and help given by the chairman

of my advisory committee, Dr. Lee F. Braithwaite, and other members of my committee, Dr. James R. Barnes, Dr. Wilmer VJ.

Tanner, and Dr. J. Keith Rigby. Special thanks are also due to Dr. Barnes for readinq the manuscript.

I am also indebted to Mr. James V. Allen for his valuable technical assistance in electron microscopy and to

Dr. Samuel R. Rushforth and Mr. Mou-Shen Cheng for their help in diatom identification. TABLE OF CONTENTS

Page

ACKNONLEDGMENTS •. . . . iii LIST OF TABLES . . vi LIST OF FIGURES ...... viii INTRODUCTION 1

REVIEW OF LITERATURE ...... 5 MATERIALS AND METHODS ...... 12 Generul Biology 12 Reproduction •.. 13

Fect:ndity Anatomy and histology of reproductive system, and gametogenqsis Electron microsc0p2 study

Developne~t .... 15

Embryonic and larval development Shell development Post:-embryoni~ df.::::veJ_cpm12ntof ovo-i.::esti.s ~ffccts of 1.5 and 0.5% maleic hydrazide on developmer:t Effect of maleic hydrozide on f e:cunc:U_ty RESUL'l'S , ...... 19

General Biology. • • • • s • 19 O·.:riposi t.ion 24 E

Embryonic and Larval Development. 112 Shell Development •..•••.. 161

Ovotestis post-embryonic development

Morphogenic Effects of Maleic Hydrazide on the Early Development . . . • . • 171 Maleic Hydrazide and Fecundity •••. 194

DISCUSSION • • 197

General Biology 197

Taxonomical problem

Reproduction ...•.. 198

Polyvitelline and empty eggs Anatomy and histology of the reproductive organs Fecundity Gametogenesis Electron microscope obser~ations

Development 223

Embryonic and larval development Egg capsule Self-fertilization Veliger Albumen fluid Hatching of gastrula Development of the shell

Effects of Maleic Hydrazide on Development and Fecundity ...... • 234

Malformations induc8d by maleic hydrazide treatwents Effect of Maleic hydrazide on fc➔ cundity

CONCLUSIONS AND srn:1MARY 243

Lrl'ERr'\TURE CI'J'ED . 248 LIST OF TABLES

Table Page

1. Number of oligochaeta worms on individual limpets during August and September, 19 7 ]...... 25

2. Percentage of various capsules observed during the summer of 1971 (June, July, August) 33

3. Egg production of 20 limpets in a four-day perjod from June 21 to June 24 .... 34

4. Egg production of Batch Bin a two-day period from June 28th to 29th ...... • . • • 35

5. Egg production of Batch C in 23 days from July 13 to August 4, 1971 36

6. Number of eggs deposited by Batch Din 18 days from July 16 to August 2, 1971 ...... 38

7. Egg production of Batch E in 14 days from July 22 to August 4, 1971 ....•• 39

8. Number of eggs deposited by Batch F .in 25 days from July 26 to August 19, 1971 40

9. Number of egos produced by Bat.ch G in 18 days front August 2-·19, 1971 ... . . 41 10. l\ compar:i.sor:. of slie).l ~~ize with -the sizes of ovote~:;t:Ls r common atrimrt 2u1d f,crninal

vesiclt:?: . . , . . . . .,. .., . 111 • • 42

11. Embryonic and larval arowth of v rivularis at the temper2ture of 21 ± 1~ C~--- (Studied in July, August, September, J.. 9 7 0 . . . ,...... 124

12. Changes of the volume of egg cell prior to the Il.:cGt clE::avage .....•••••• 126 vii

Table Page

13. Chronology of the development of F. rivularis at temperatures of 21 ± 1° C .- • • • 158

14. Relation between shell size (age) and the number and size of developing eggs 170

15. Cell volume changes of the egg through the second cleavage stage at 21 ± 1° C during and after treatment with 1.5% maleic hydrazide temperature ..•.. 172

16. Effects of 1.5% maleic hydrazide on the early development of r- rivularis •.••• 173 17. Cell volume changes during and afteY treatment with 1.5% maleic hydrazide at the two-cell stage . . • . • • . . • . ••. 177

18. Effects of 0.5% rnaleic hydrazide on the early de-;relopmen!c of F. ri vularis . . . 184

19. Effect of maleic hydrazide on the fecundity of F. rivularis at 21 ± 1° C ..••.• 196 LIST OF FIGURES

Figure Page

1. Egg collection apparatus • 20

2. Size distribution of Ferrissia rivularis over a five-month period o{ time . . • • . 20

3. External morphology of egg capsule . 28

4. Egg capsule showing a dwarf egg 28

5. Egg capsules •. 31

6 • Distal portion of the reproductive system of F. rivularis . . . •• 45

7. Ovotestis, longitudinal section 48

8 • Ovotestis and distal portion of reproductive duct, gross morphology. 48

9. Egg capsules . 49

10. Spermatheca, E.::xternal morphology . 50

11. Spermatheca, longitudinal section 50

12. Sagital section of an ovotestis illustrating amoeboid Sertoli cells a.nd sperma ti.ds . . . 53

13. A ruptured ser,:iinal vesicle shmJing mature S}Jer1n ., ., • ., 53

14. A. longi t.udina l section of the corrmon atrium showj_ng columnar enithelium, connective 54

15. Longitudinal s2ction of a seminal vesicle showing evaginations of the hermaphroditic duct and packed sperm 54 ix

Figure Page

16. Longitudinal section of the hermaphroditic duct leading from the common atrium to the seminal vesicle . . . • • • • • • • • • 56

17. Cross section of the ovotestis showing spermatogonia and developing germinal epithelium . • . . •..•.•• 56

18. Distal portion of the reproductive system, sagital section...... • • . • • • 57

19. Developing spermatogonium pulling away from the acinus wall ••.••••. 57

20. Cross section of an ovotestis showing four acini with an ovulated oocyte in the lumen of one ..•.. 58

21. Spermatogenesis of Ferrissia rivularis . 59

22. Seminal vesicle smear showing mature sperm 62

23. "Diakinesis" of primary spermatocytes 62

24. Ovotestis smear showing spermatogonia and a spermatid tail with a cytoplasmic globule . . . • . . • •. 63

25. Cross section of an ovotestis showing first oaturation division, secondary spermatocytes, and a developing Sertoli cell...... 63

26. Interphase of primary spermatocytes 64

27. Secondary spermatocytes surrounding a young Sertoli cell ...... 64

28. Young spermatids 66

29. Young spermatids showing post-nuclear apparatus ...... • . . . • 66

30. Advanced spermatids 68

31. Late spermatids showing cytoplasmic remnants ...... •. 68

32. A mature spermatozoon of F. ri v~~;ris 7 0

33. Longitudinal section of an ovotestis showirig oogenetic and spermatogcn2tic zones . . 70 X

Figure Page

34. Oogonia, oocytes, and follicle cells •. 73

35. Ovotestis cross section showing growing oocytes •..•.•• 73

36. Growing oocytes showing the vacuolated condition of the cytoplasm ...• 75

37. Types of nucleoli present during oogenesis 77

38. A fertilized egg in the acinus lumen ... 80

39. (A) Transverse section through the upper portionvcommon atrium. . •.... 83 oft (B) Transverse section of the ciliated epithelial cells of the lower oortion of the common atrium • • . . • • • • • 83

40. Transverse section of the wall of the seminal vesicle ....•. 85

41. Cross sections through the middle 7 piece of advanced spermatids . . ...•.. 87

42. Transverse sections through the middle- piece of young spermatids showing non- glycogen contained compartments .... 90

43. Cross sections through the middle-piece of advanced spermatids showing variable numbers of primary helices ... 90

44. Developing spermatid tail 93

45. Cross sections through middle-piece and end·-piece of mcJ.ture sperm tails . • . • 93

46. Longitudinal sections through the middle- piece of mature spermatozoR . . . • . . 95

47. Cross sections through the middle-piece of mature spermatozoa ...... 95

48. Longitudinal section through the end-piece of the tail of a mature spermatozoon . • . 98

49. Edge of the "mitochondrial. cloud" in the cytoplasm of a previtellogenctic oocyte o f F • r i vu la r i s • • • . • • • • . . 9 8 xi

Figure Page

50. Electron micrograph of a previtellogenetic oocyte showing "mitochondrial cloud", vitelline nucleus or Balbiani's body, endoplasmic reticulum, and connective tissue cell of Ancel's layer •.... 101

51. Electron micrograph of two previtellogenetic oocytes sharing a single follicle cell .•. 101

52. Electron micrograph of a previtellogenetic oocyte surface with its adjoining follicle cells • . . . • •.• 104

53. Electron micrograph of a part of a young oocyte showing two types of nucleoli and dispersed mitochondria .....•. 104

54. Electron micrograph of a vitellogenetic oocyte showing vitelline membrane, microvilli, and yolk granules • . . 107

55. Electron micrograph depicts the two types of yolk granules and unidentified bodies ... 107

56. High magnification of yolk granules containing electron-dense yolk platelets, and lysosomes containing virus-like pc1r·ticles ...... 110

57. Cross section of a vitelJ.ogenetic oocyte showing autophagic vacuoles and yolk granules ...... • . • . 110

58, Electron micrographs of a germinal vesicle and nucleolus of an oocyte during vitellogenesis . 113

59. Electron micrograph showing internal and external follicular layers . 113

60. Electron micrograph of a follicle cell showing the ER system and mitochondria . . ... 115

61. Electron micrograph of a vitellogenetic oocyte showing protion of vitelline nuclellS ...... 115

62. Growth of embryos and larvae of F. rivularis 120

63. Young trochophore, the fourth day of development ... 120 xii

Figure Page

64. Germinal vesicle breakdown in one primary oocyte, probably due to mechanical stimulation. . . . • • • . . • • . • • 121

65. Freshly laid egg showing "rounding off" and maturation division. . • • • • . • • . 122

66. First cleavage. 128

67. Early cleavage .. 130

68. Second cleavage, showing different cleavage patterns . . ••.••• 135

69. Third cleavage ... 138

70. Fourth cleavage and blastula. 141

71. Early developmental stages •. 145

72. Five-day trochophore, six-day veliger, and seven-day veliger •.•...••• 151

73. Post-veliger 153

74. Young limpet, 10-days old 156

75. Late trochophore and veligers 157

76. A hatched eqg capsule showing top lid, bottom floor, and operculate suture 160

77. Pour-day trochophore showing shell gland, larval liver, and stornodaeurn 160

78. Shell and associated shell gland .. 163

79. Shell from an eight··day :post--veliger showing its calcification pattern 163

80. Young oocytes and follicle cells .... 169

81. Longitudinal section of a young ovotestis lacking distinct zonation between oogenesis and sperm&togenesis . 169

82. Morphology of the normal gastrula and vari.ous types of exogastrulae and a hydropic trochophore ...•..• 179 xiii

Figure•· Page

83. Non-hydropic exogastrula . 187

84. Hydropic veliger ••.. 191

85. Hydropic post-veliger 191

86. Separation of blastomeres 192

87. Hydropic veliger with an everted radula 192

88. Deformed limpet larva showing shell malformation •...•.... 193

89. A Sertoli cell with attached spermatids 193

90. Metaphase of an unovulated oocyte 221

91. Yolk formation theories 222

92. Cross section of adult limpet shell showing periostracum ...... 239 93. An ovotestis and two ovulated oocytes in a coITut1.on atrium 239

94. Growing oocytes and follicle cells . 240

95. A full-grown oocyte 240

96. Hydropic ycung trochophore . 241

97. Hydropi~ veliger . 241

98. Bertoli ceJ.ls with their attached late sperrnatids a.nd fnll· ..grown oocyte in the r:cinus lurnen of oocrenetic zone ...... 242

99. Trjophthalrnic post-veligcr . 242 INTRODUCTION

In North America, the genus F'errissia is the most geographically widespread of the freshwater limpets, has the grestest variety of species, and occupies the most di- verse types of habitats (Basch, 1963). Ferrissia rivularis was the first American freshwater limpet to be described.

Thomas Say in 1317 named it Ancylus rivularis (Basch, 1963).

According to a modern mollusc classification (e.g., Purchon,

1968), the taxonomic position of this species is as follows:

Class Gzstropoda

Subclass Basom.rnatophora

Order Patelliformia

Family A.ncylidae

Genus Ferrissia

found in rivers or streams ·with gravel bottoms. This 2-s uhy it gained the name "river limpet." It has a fairly \\ride distribution in the United States, thus far being found from

Massachusetts west to Colorado, Wyoming, and Utah (Chamber- lin, 1929), and hns also been collected from Ohio northward to Wisconsin and Michigan (Baker, 1928). In the state of

Utah this limpet has only been n-~ported from Utah Lake

(Chamberlin, 1929). 2

Many aspects of the biology of Ferrissia have been

overlooked, in particular, reproductive and developmental

biology. This is probably because of their small size.

Ferrissia rivularis was selected for research be-

cause of its local accessibility and because essentially

nothing was known of its reproduction and development. Re-

productive and developmental biology is first described herein from the natural environment. This is then compared with the effects created by experimental exposure to a

selected herbicide, maleic hydrazide, which might be a

potential threat to certain invertebrates in the natural

environment.

Follicle cells of vertebrates and some invertebrates

have been reported to be responsible for nutrient transport,

from other body sites to growing oocytes (Raven, 1961;

Balinsky, 1970). In pulmonate snails, Raven (1963) believes

that follicle cells play an important role in the determina-

tion of cortical morphogenetic fields in Lymnaea eggs.

Follicle cells may serve as food for developing oocytes in

Helix (Balinsky, 1970). Joosse and Reitz (1969) have

stressGd the ir:1portcmce of Lym0a~....9..follicle cells in re-

lation to the nutritional aspect of growing oocytes.

Follicle cells may be involved in the yolk synthesis of developing oocytcs. Yet, no extensive study has clarified this issue. Therefore, the ultrastructure of oocytes,

follicle cells, and other cells of F. rivularis was ob-

served in an attempt to understand the reproductive biology of this freshwater limpet. 3 Since nothing is known concerning the fine structure of spermatid and mature sperm tails in ancylid snails, a comparative study of the two types of tails is elaborated in this paper.

Maleic hydrazide (1, 2-dihydropyridazine-3, 6- dione) was first synthesized by the Naugatuck Chemical

Division of the U. S. Rubber Company in 1947 and was subsequently discovered to be a synthetic plant growth regulant (Schoene, 1949). Since then, pure maleic hydrazide and its formulations have been widely used in agriculture as a weed killer .:i.nd sprouting inhibitor. Cytological and biochemical studies on the effect of maleic hydrazide on plant cells and tissues have revealed that maleic hydrazide causes chromosome breakage and aberration, and inhibits DNA synthesis (Carlson, 1954; Evans, et al., 1964). A warning on the use of this chemical was announced by Darlington

(1951): "Since nearly all chromosome-breaking agents have so far proved to be cancer-producing as well, we must hope that the agricultural use of this new agent will not be encouraged before suitable tests are rnRde. 11 The question as to whether maleic hydrazide is carcinogenic to animals is still uncertain.

If muleic hydrazide also causes chromosome breakage and aberration and inhibits DNA synth2sis in animal cells or tissues during mitosis or meiosis, as shown in plants; conspicuous abnormalities may occur when the dividing eggs, developing embryos, or reproducing anirnnls are s~bjected 4 to exposures of this chemical. Therefore, river limpet eggs, embryos, and adult animals were used in experiments to establish exact effects on a local freshwater organism.

This work is divided into three major parts. The first includes reproductive biology and gametogenesis, as revealed by light and electron microscopes. The second deals with embryonic and larval development and growth, and the third is concerned with the effects of maleic hydrazide on the early embryonic development and the fecundity of F. rivularis. REVIEW OF LITERATURE

Early investigations on the reproductive behavior of the European river limpet, Ancylus fluviatilis, were made by Bouchard-Chantereaux (1832) and Gassies (1851).

More recently, Nekrassow (1928) and Bondesen (1950a, 1950b) have studied egg capsules, and Hunter (1953) and Geldiary

(1956) have reported on post-embryonic growth and seasonal population cycles of this species. Comandon and Fonbrune

(1935) have studied the embryology of lmcylus lacustris by cinematography.

In North America, eggs of a limpet identified as

Ancylus rivularis were followed through the first few cleavages by Holmes (1899). Clapp (1921) studied the eggs and young of Ancylus fuscus, and Baker (1928) published illustrations of the egg capsule of an undetermined species of Ferrissia from Wisconsin. Basch (1959) reported on the development and reproduction of Ferrissia shimekii. Burky (1971) has reported recently on the reproduction and life cycle of Ferrissia rivularis from New

York state.

Wautier, et al. (1966) did a brief study of gametogenesis and fertiljzation of Gundlachia wautieri, and proved that seJf-fertilizQtion and parthenogenesis were 6

possible in this limpet species.

Many papers concerned with the anatomy and histology

of various species of freshwater limpets have been pub-

lished. Hoff (1940) reported on the anatomy and histology

of Ferrissia tarda. It was the first paper dealing with the

morphology of a North Ainerican freshwater limpet. Basch

(1959) did an extensive study on the anatomy of Laevapex

fuscus and also reported on the anatomy and histology of

. Rhodacrnea cahawbensis (1960). A comparative study on the

anatomy of South African species of Ferrissia was made by

Brown (1967). Burch (1965) did cytotaxonomic studies of

Japanese Ferrissia and Gundlachia species. Hubendick

described the anatomy of Acroloxus (1962) and published an

anatomical monograph on Ancylidae (1964). Wautier,

Hernandez, and Richardot (1966) studied the anatomy, his-

tology and life cycle of Gundlachia wautieri.

A large number of studies on the anatomy and his-

tology of other ~ulmonates has also been published. Joosse

and Reitz (1969) reported on the functional anatomy of the

ovotestis in Lymnaea ~.!=-agnalis. Holm ( 194 6) reported on

the histology and functional morphology of the genital

tract of Lymnaea stagnalis appressa, and the anatomy of the

genital organs of Heliosoma trivolvis and Biomphalaria

bot_~sy:i:_was described by l\bde1-·Ma1ek (1954) . A comparative

study on the genital systems of the freshwater Basomma-

tophora was reported by Duncan (1960).

Classical papers dealing with the garnetogenesis of 7 pulmonates include those of Gatenby on Helix and Arion.

Spermatogenesis of pulmonates was first elaborated on by

Gatenby (1917, 1918). He described in 1918 the role of mitochondria in spermiogenesis and called nurse cells

"yolk cells" because of the presence of yolk, instead of

Sertoli cell characteristics. Hickman (1931) and Watts

(1951, 1952) observed that mitochondria formed a spiral

sheath around the axial filament of the sperm in Succinea ovalis and slugs (Arion and Deroceras), and the cytoplasmic remnants sloughed off through the tail end. The differentiation of mitochondria during spermatogenesis in

Viviparus contectoides was studied by Kaye (1958). In the same year, Yasuzumi and Tanaka (1958) demonstrated typical and atypical spermatogenesis in the pond snail, Cipango- paludina malleata. Later, in 1960, they reported on the relation between nutritive cells (Sertoli cells) and the developing spermatids in the same species of pond snail, and described that a mantle, formed by rn.rnerous elongate pseudopodia of the nutritive cells around the head of spermatids, served as a conductor system for nutritional supply, from the nutritive cells to the developing typical spermatids.

Fahmy (1949) found that mitochondria formed a huge zone at one end of an oocyte during the period of active oocytic growth and were probably involved with chemical synthesis of yolk from raw m~terials in the ooplasm. He also concluded that yolk spheres appeared in the ·interior 8

of Golgi complexes. Bretschneider and Raven (1951)

reported that during oogenesis in Lymnaea stagnalis

protein yolk consisted of two kinds of granules of differ-

ent size and shape: ( S- and r-g-ranules) . Both were formed

by the Golgi bodies.

The relation between growing oocytes and the

follicle cells has been considered for a number of years.

Trujillo-Cenoz and Sotelo (1959) showed that in the young rabbit oocyte, the oocyte membrane and the follicle cell membrane faced each other over the entire oocyte surface, but were always separated by an intervening space, and

interdigitations between membranes occurred at many points.

Anderson and Beams (1960) proposed that follicle cells of guinea pigs may synthesize nutritive material, and this material may eventually reach the oocyte by diffusion and pinocytosis. In the ovarian eggs of the urochordate

Ciona, Reverberi and Mancuso (1960) observed that follicle cells developed eventually into a chorion layer after oogenesis. Recently Anderson and Spielman (1971) studied the permeability of the ovarian follicle of ~edes aegypti and found that the outermost layer of the follicular

sheath (basement lamina) was a coarse mechanical filter;

and the follicle epithelium of vitellogenic follicles was penetrated by narrow inter-cellular channels which may

serve as paths of nutrient transportation.

~oui_1 • J 1 on ( i~~, ,...r.· G) reportea , in. Cepe~ snai . 1 s th - a:t temperature controlled the direction of primary gonocyte 9 development. A low temperature was more favorable for development in the female direction and high temperature promoted development in the male direction. Ghose (1960) found both male and female germ cells in the same follicle of the ovotestis in Arion fulica. Also, no seasonal activity was found in the male germ cells, and spermatozoa were produced throughout the year; however, periodic activity was present in the female germ cells.

Effects of chemicals on the development of freshwater pulmonates have been reported by several authors.

Raven {1942) found that when eggs of Lymnaea stagnalis were treated at early stages of development with weak solutions of lithium chloride, exogastrµlae and cyclo- cephalic malformations were formed. When the eggs of

Ly_:r1maea stagnalis were exposed to dilute LiC1 2 during the period of development between oviposition and the 24-cell stage, various head malformations were formed, including cyclopic, synphthalmic, anophthalrnic and triophthalmic embryos (Raven, 1949). Geilenkirch and Nijenhuis {1962) reported that in Lymnae.~ stagnalis developmental progress was attended by a gradual decline of the general sensitivity of the embryo, as well as sensitivity of the endodcrm, when e:(posed to lithium chloride. Mancuso (1955) found that sodium azide caused exogastrulation and head malforrr.ations .i.n _?hysa rivularis, v:hen the eggs were treated with this chemical. norrill (1963) observed that cobaltous chloride also caused: (a) separation of 10

blastomeres, (b) vesicular and dumbbell-shaped exogas-

trulae, (c) arrested gastrulae, (d) veligers with a reduced larval liver, shell, and foot, and also with hydrop-

ic cavities, (e) shell-less snails, and (f) helmet-shaped

shells with abnormally wide apertures. Chloroacetophenone

also induced exogastrulation in Planorbis (Indoplanorbis) exustus (Mullherkar and Sherbet, 1963).

Since Darlington's published warning on the use of maleic hydrazide in 1951, research on the effects of maleic hydrazide on animals has intensified. Maleic hydrazide was claimed to be selectively toxic to plants, but not to bacteria, fungi, rodents, and dogs by Zukel

(1949, 1963). On the contrary, Nasrat (1965, 1967) found that it was mutagenic to the fruitfly Drosophila malano-

_sraster_. It produced cytotoxici ty, mitotic inhibition, and unbalanced growth in cultured mammalian cells (McCarthy,

1968). Timson (1968) found that maleic hydrazide caused a reduction in the mitotic index in phytohaemaglutinin stim- ulated human l:ymphocytes cultured in vitro. Davide (1968) reported that rnaleic hydrazide reduced the rate of development and affected the direction of sexual differentiation in nematode larvae. Experi~ents on Swiss mice proved that this herbicide was very carcinogenic and induced hepatomas in the liver (Epstein, 1967). The experi-· ments of Povolotaskaya (1967) indicated that maleic hydrazide in the fo:nn of Na or diehtanol amine salt did not have favorable effect on the weight and general 11 condition of white rats, and no accumulation of maleic hydrazide was found in organ muscles, livers, kidneys, nor brains. Greulach (1950) found that 2.0% was lethal to all treated Ambystoma punctatum larvae. MATERIALS AND METHODS

General Biology

Specimen collection and field observations were made at Lincoln Beach of Utah Lake. Most animals used in this study were collected from water about one·-half to one meter deep and about two to three meters from the shore.

Limpet populations were sampled in September, October and

November of 1971, and during April and May of 1972 in order to discover the period of breeding for the species.

No samples ·were made in December 1971 and January 1972 while the lake had ice cover. A few specimens were collected for histological study in February and March

1972, after the ice melted.

Stomach and intestine contents were examined by dissection and were observed in thin sections. Diatom shells were isolated by dissolving stomachs and intestines in concentrated sulfuric acid overnight, washed with distilled water and concentrated by centrifugation.

Cornm2n2,alistic olio;ochaete worms (Chaetogaster li~e~) v.-ere found on limpets and removed from the hosts with fine glass needles under a dissecting microscope.

Numbers of worms on each limpet were recorded. 13

Reproduction

Fecundity

Before collecting egg capsules on microscope slides, numbered slides were "pre-cultured" with diatoms by immersing them in lake water for a few days; mature limpets, with a minimal shell length of 2.5 mm were then placed on the slides for capsule deposition. Each slide was hung in lake water at 21 ± 1° C by a plastic clothes pin supported by a metal wire (Fig. 1). Aquariurn water was aerated with an airpump and water temperature was maintained at 21° C during all microscope observations.

Ninety-three mature limpets were used to study egg production during June, July, and August 1971. Limpets were kept on glass slides for periods of 2, 4, 14, 18, 23, and 25 days. Whenever egg capsules were found on a slide, the animal was removed and put on another slide for further observation. ~rhe diameter of the eg<-;;cap::mle and number of eggs were recorded daily.

Anatomy and Histology of Reproductive System, and Garnetogene;;is

Under a binocular dissecting microscope the ovotestls and associated ducts were dissected from mature limpets using extremely fine glass needles and forceps.

After removal they ·were transferred into a drop of filtered lake v:ater on a slide. 'l1he preparation was then covered 14 with a cover glass supported at the corners, about 1 mm high, with pieces of clay.

For histological study, both dissected ovotestes and whole limpets were fixed in Bouin's fluid for a minimum of

24 hours, dehydrated with a graded series of ethanol, and then embedded in Paraplast. Tissues were sectioned at six and eight microns and stained with alum hematoxylin and eosion.

Electron Microscope Study

The ovotestes and accessory reproductive organs were dissected out under a dissecting microscope and then fixed immediately in a 3% glutaraldehyde-acrol-ein mixture which was buffered with 0.2 M sodium cacodylate at a pH of 7.3 for two hours, or overnight, at room t.em.perature. Specimens ·were then washed in th.e same buffer solution six times in one hour and fixed in 1% OsO4 for two hours in an ice bath. Following this, specimens were rinsed several times in the buffer solution and then stained with 0.5% uranyl acetate for two days at room temperature. After fixc1tion, samples were dehydrated directJ.y, without washing in distilled water or buffer, in a graded series of ethyl alcohol. Specimens, embedded in Mollenhauer's plastic, were sectioned with glass knives using a Porter·· Bi.um !,11T-2 microtome. All sections were mounted on copper grids with or without a coating of

F'ormvar film, and were then post- stained 1-;ith lead citrate for 10 minutes before being examine~ under a Hitachi HS-7 15 electron microscope. Micrographs were taken with Kodak lantern slide plates at 2,500 to 25,000 magnifications.

Development

Embryonic and Larval Development

Egg capsules were collected using the same method described in the fecundity section. Drops of filtered lake water were placed on the egg capsules periodically to prevent dessication during microscopic observations.

Growth and morphological changes of embryos and larvae were recorded approximately every 24 hours. After the post-veliger stage, larval growth w~s indicated by shell growth (Crabb, 1929; Hunter, 1953).

Embryos or young larvae were dissected from egg capsules using fine glass needles and forceps. Diluted toluidj_ne blue and 1% erythrosin were used for vital staining and observations of large albumen cells and cilia.

Egg cell and blastomere volumes wsre claculated according t.o the formula V = 1/6 n ab 2 , in ,vhich "a" is the diameter in the direction of the egg axis and "b" is the diameter at right angles to it (Raven, Bezem, and Geelen,

1953).

Shell Development

In order to analyze shell development, veliger larvae of varying ages were dissected from egg capsules 16 with glass micro-needles and fine forceps, and were then placed on glass slides. The larvae were then carefully squashed with a cover glass. The dissociated shells were then separated from the tissues of the larvae and observed with a microscope to determine structural detail. A 1%

HCl solution was used for testing shell calcification.

Large, adult shells were analyzed by cleaning the shells with 70% alcohol, embedding them in resin, and finally grinding them to about 1/10 of a millimeter in thickness.

The structure and chemical composition of larval and adult shells were comparatively studied.

Post-embryonic Development of Ovotestis

Four groups of different sized limpets were fixed in Bouin's fixative overnight. Specimens were treated using the sarn.e procedure described in the section on garnetogcnesis. In Table 14, a "cell--index" of maximal length times maximal breadth (Bretschneider and Raven, 1951) has been 11sed as a measure of cell size, instead of actual cell volume. Since all measurements were made on fixed and sectioned material, the measured values were probably slightly smaller than the actual values, because of shrink- age during dehydration and embedding of the specimens. 17 Effects of Maleic Hydrazide on Development and Fecundity

Effects of 1.5 and 0.5% Maleic Hydrazide on Development

A total number of 132 eggs and embryos were treated with a 1.5% solution of maleic hydrazide 1 (diethanol amine salt of maleic hydrazide) at uncleaved, two-cell, four-cell, eight-cell, blastula, and gastrula stages for 60 minutes at

21° C water temperature. Developing eggs and embryos were checked and measured daily following the treatment.

All eggs and embryos exposed to a 0.5% maleic hydrazide treatment were produced by mature limpets collected in late October and November 1970. ~gg capsules were collected from limpets by temperature stimulation. Twenty- five eggs without treatment were used as experimental controls. Fifty-three eggs or embryos were separately treated at different stages with 0.5% maleic hydrazide solution for 60 minutes. Experiments and observations were carried out at a water ternpe::::-ature of 21 ± 1° C.

Effect of Maleic Hydrazide on Fecundity

One hundred fifty mature limpets were used to determine the effect of maleic hydrazide on fecundity.

Half of the number was divided into four groups and

1This chemical was donated by the Uniroyal Chemical Conpany, Division of Uniroyal, Inc., Naugatuck, Connecticut OG770. 18 separately treated with 1.5%, 0.5%, 0.25%, and 0.1% maleic hydrazide solution at 11° C for twelve hours. The purpose of treating the animals at a low temperature was to reduce or to prevent spawning during treatment. Half of the number was used for a control. All control animals were kept in lake water at 11° C for twelve hours. Observations of egg-deposition were carried out over a period of ten days. RESULTS

General Biology

In Utah Lake Ferrissia rivularis has only been

collected from Lincoln Beach, where the bottom is rocky and

silty. Animals are found on the undersurfaces of various

sizes of rocks and on the surfaces of dead twigs and submerged stems of Typh~ S£· on which diatoms are abundant.

Limpets are very numerous in the surrnner and fall but

are very scarce in the winter, at least after the ice melts.

The size-groups of populations in different months are shown

in Fig. 2. The ranges of shell size in each population

sampled during a five month period of time are as follows:

Date u}c-::>ec:r--e11 ,. ,.L,l . No. Rancres of shell size in mm

9--11-71 234 1.4 X 0.92 - 4.2 X 2.7

10·· 17--71 205 1.2 X 0.8 - 5.2 X 3.1

11--7•-·11 181 1.4 , .• 0.7 - 5.4 X 3.5

4--16-72 41 2.0 X 1.2 - 5.6 X 3.5

5-21--·72 38 3.9 X 2.3 - 6.5 X 4.1

Shell size of the river limpet ranged from 1.2 x 0.8 to G.5 x 4.1 mm.

Characteristics of the shell were originally de-

scribed by Say and recorded by Basch, 1963 as: "Shell 20

~Air

1-t------lr=t----F+---+-+--+--t----lr-:l::::::::::::::::::tt~- Slide

Water ----hi-• •4-1-----1-1--14--limpe t 0 0 Egg 0 0 capsule Q 0 0•

Fig. 1. Egg collection apparatus.

60 Septfrr.ber 1971 l,O so L 1'I~ 234 50 40 40 30 30 io 20 10 10 0 1-----=,,.___,___, _ _,__,____._,__-"=- 0-~ I 1.5 2 2.!; 3 .H ,;L~ +.5' 5 S-5' 6 H c > I 1-'i 2 2-5 3 3.5 4 4.S 5 SS 6 65

October 1971 GO 601 t1, 10s .!'? S-0 50 V ,- :, 40 "ti 40 > 30 30 "O C ,o 20 .,. 10 0 .. 0 '" 41 0-5 I 1-5' :; 2-!i 3 3.'i 4 4S-=t 5 H 6 6-5' 05 ~ -1i. E :, z NovcMll~r !971 ff., N: 1~2 so 40 30 10

o.r 1 , .., ✓, 2.,; 3 35' 40 ,is 5- s.r 6 6-5" 511~11 Lenglh (r.~m)

Fig. 2. Size distribution of Ferrissia rivularis over a five-month period of time. 21

corneous, opake [sic], conic-depressed, apex obtuse, nearer

to and leaning toward one .side and one end; aperture oval,

rather narrower at one end, entire; within milk-white.

Length 1/4 inch." Frequently the shells of the limpets are

decorated with green algae, a thin layer of silt, and

diatoms. Such decorations may provide the animal camouflage

and protection. The edge of the shell is not clacified so

that the flexibility of the periostracum enables the animal

to produce a strong adhesive power on the substrathum when

it was disturbed. Therefore, limpets can only be detached

from the substratum by sliding a sharp blade underneath the

shell.

Shells of several species of diatoms were found in

the stomach, intestine, and fecal pellets. Limpets grazing

on diatoms on the 0lass walls of aqua~ia were also observed

in the laboratory. Apparently this species of limpet feeds on diatoms which grow on the surfaces of substrata.

Numerous clacareous gastroli ths were found in tr1e stomach

(gizzard). Very likely the gastroliths are used to grind

food.

The stomach contents of limpets which were collected

in April, 1972 contained twenty-one genera incJ.uding thirty-

') five di ff ercnt species of diatorn.s ~ •· :=:ynedra rurnpens var.

') -'-Iclentific2tion of diatom ·was made by r1r. Mou·-Sheng Cheng and Dr. S. R. Rushforth, Department of Botany, Brigham Young University. 22 schizonemoidespatr, Nitzschia dissipata, Nitzschia fonticola,

Cocconeis placentula var. lineats, nitzschia ovalis, Steph- anodiscus niagarae, Gomphonema lanceolaturn, Gomphonema olivaceum, Achnanthes lanceolata, Nitzschia palea, Svnedra rumpens var. farnilians, Cyclotella bodanica var. michiganen- sis, Navicula. lanceolata var. lanceolata, Navicula crypto- cephala var. intermedia, Amphora ovalis, Cymbella prostrate,

Fragilaria vancheriae var. vancheria, Rhoicosphenia curvata,

Nitzschia amphibia, Nitzschia hrandersheimiensis, Cyrnbella tumids, Mastogloia elliptica var. edenseii, Diploneis pseudovalis, Cymbella. sp. Gorn.phonema sphaerophorum,

Surirella ova.ta, Bacillaria paradoxa, Denticula ~legans,

Epitherna turgida, Cymbella .:3p. Stephanodiscus invisitatus,

Cocconeis placentula var. euglypta, and Diatoma vulgare.

Limpets which were collected in the spring, summer and fall seasons were always found to be infested with

Chaetogaster lirn~asi Baer. One individual had as many as

27 oligochaete ,wrms attached to it in the summer of 1970.

A few worms (two or three) were often observed in some individuals in the winter of the same year. In August and

September of 1971. 1 twenty--r_;ix limpets, ranging from 3. 3 to

5. 2 rr~Ttin shell length, ,,-Jere examined. 11 he number of oligoc!1ae~e worms attached to the twenty-six limpets varied from one to twelve, totalled 12'1, and avera9ed 4.7 worms per lirnpet. Most ·worrns were found to inhabit the spaces between the nuchal region and the mantle and shell. A few were found in the palliQl grooves. The worm holds itself 23 to the mantle surface of the host by bundles of setae on the ventral side of the posterior tagma, and swings itself freely under the shell to ingest microorganisms. Chaetogaster limnaei Baer was first recognized as a commensal of freshwater gastropods feeding on microorganisms by Wagin (1931).

Backlund (1949) observed that cercariae of Fasciola hepatica were ingested by Chaetogaster. Wallace (1941) found that

C. limnaei lived in conunensal relationship - with Helisoma------sp. and fed on Cercariaeum mutable whose metacercariae encysted in the worms after penetrating the wall of the gut. Ruiz

(1957) later reported a similar observation with respect to

Schistosoma mansoni, but commented that the worm did not protect the snail from infection. Howev~r, Michelson (1964) found that Chaetogaster limnaei afforded protection to the snail Australorbi~ qlabratus when the snail was exposed to

Schistosorna mansoni miracidia; and to a lesser extent, the oligchaetes protected snails exposed to echinostome cercariae. Michelson also found Chaetoqaster limnaei with- --·---•--"·'------in the kidney of P11ys~ heterost:cophc}_. The presence of

Chaetoqaster l~nnaei in other species of snails has been reported. Krasnodcbski (1936) noted as many as 300 worms on a single specimen of Lymnaea staanalis, up to 60 worms on ---""----- Physa fontinalis, 0.nd a mean of l. 3 \Jorns on specimens of

Ancylus lacustris. An average of nine worms per snail from 105 Physa hetorostrouha was reported by Michelson

(1964). He also found the heaviest infestation appeared on old8r 2nd largc~r snnils, particularly those which have 24

overwintered. There is no strong evidence to indicate that

older snails are infested by more worms in Ferrissia

rivularis (Table 1).

Oviposition

In nature the first batch of egg capsules containing

gastrula embryos was found on the 16th of April, 1972. On

May 21, 1972 more egg capsules were found. On one rock more

than 15 capsules were observed. They were probably laid by

a single large limpet. It was interesting that no young

were found at that time, only large mature limpets ranging

from 3.9 x 2.3 to 6.5 x 4.1 mm. in shell size (Fig. 3).

The last sample of 1971 was collected on November 7, shell

sizes ranged from 1.4 x 0.7 to 5.4 x 3.3 mm. The dis-

tribution of shell sizes with the observations of egg

capsules in April may suggest that the oviposition of this

river limpet probably starts in the first half of April and

continues until late October.

In nature, egg capsules are found on the underside of rocks where dense algal growth is lacking or on the

surfaces of submerged stems of ~ypha .?P• and other plants.

Capsule color is very light brown. In the laboratory limpets were allowed to deposite egg capsules on glass slides only.

During the breeding season, after being brought into the

laboratory, most limpets laid eggs on slides the following day from 7 to 10 ZLM. and from 7 to 9 P.M. in the evening.

Only a few limpets laid eggs after 11 A.M. A given snail 25

Table 1. Number of oligochaeta worms on individual limpets during August and September, 1971.

Shell size (mm.) Number of ~-limnaei

August 1971 5.2 X 3.2 2 4.5 X 2.6 8 4.4 X 2.8 2 4.4 X 2.6 8 4.2 X 2.3 12 4.1 X 2.6 9 4.1 X 2.3 7 3.9 X 2.4 3 3.9 X 2.3 3 3.8 X 2.3 7 3.7 X 2.2 8 3.7 X 2.2 4 3.6 X 2.2 8 3.5 X 2.2 8

September 4.8 X 2.8 4 4.6 X 2.7 1 4.5 X 2.9 2 4.2 X 2.5 2 4.0 X 2.2 2 3.9 X 2.3 3 3.9 X 2.2 4 3.9 X 2.2 3 3.7 X 2.1 5 ? 0 3.6 X ~ . _, 4 3.3 X 2.1 1

may continue to lay eggs on slides for a few days. Unli]ce

A!:',cylus fluviatilis, which dies after spawning (Geldiay,

1956), all river limpets survive after spawning.

Spawning was not completely inhibited at 11° C during the breeding season. A few capsules were found on the glass walls of the container, but these eggs never developed nor hatched. Pre-chilled limpets generally spawned their first egg capsule about five hours and thirty 26 minutes after being transferred from 11° to 21° C water.

In the winter, artificial increase in water temperature is perhaps the only technique capable of inducing egg maturation and deposition. During a four- month period, from October 1970 to January 1971, 191 limpets were induced by different water temperatures (22°, 26°, 27°,

30°, and 35° C) to deposit 125 capsules containing 147 eggs.

It seems that 35° C is the most effective temperature to stimulate egg deposition. A high percentage (99.32%) of the eggs were fertile and developed normally in the early stages of development, only 0.68% (one egg) was sub-rectangular in shape and sterile. Among 125 capsules, only two were found to be empty.

The above observations suggest that temperature probably is the major factor controlling maturation of gametes and spawning beha.vior. Mature limpets of various sizes were examined in October and November 1971. All seminal vesicles of the animals were filled with spermatozoa, and grov,ing oocytes and sperm were observed in the ovotestes. Therefm:e, the function of higher temperatures is probably to initiate the final maturation and to stimulate the neurosecretory system ~1ich may elicit capsule formation and ovulation. There is no evidence which shows that light and food are important factors affecting spawning, although food is critical as far as enerqy sources are concerned.

If animals v1ere placed on clean slides where no food was available, the limpets were still able to lay normal eggs 27 for a few days.

Egg Capsule Morphology

Terms used for describing the egg capsules of this species are based on Bondesen's terminology (1950b).

Basically, the egg capsule is a low, dome-or-watch- glass-shaped structure, composed of a tough but transparent outer layer termed the capsula externa. This layer forms an upper lid which affords excellent protection against para- sites and foreign bodies, and a floor beneath the lid which prevents the contents of the capsule from coming into contact with the object upon which it is deposited. The capsula externa is secreted by a foot gland and can be distinguished from the internal envelope (capsula interna) by its granular composition and irregular, scalloped contour. Occasionally wri~zled folds appear at the terminal tail (exitus terminlais) siee on the top lid (Fig. 3).

There is a contact line between the top lid and the bottom floor of the capsule. It is termed the operculate suture or sutura opercula (Fig. 3). lt is assumed that the top licl splits off from the bottom floor along the opercu- latc suture when the young are ready to hatch. Details of the hatching mechanism will be described in a later section.

Eggs are located bet.ween the top lid and bottom floor of the ext.ernaJ. membrane. Egg cells and 21 lburnen fluids are surrounded by a thin-walled, very elastic membrane, known as the internal capsule membrane or tertiary 28

Fi g . 3 . Extern a l mor phol ogy of egg c apsu l e . CE , ex t er n a l c aps u le membrane , CI , i nterna l cap s ul e membrane ; Em, emb r yo ; OS , oper cul ate sutur e . 1 5 0 X.

DE - --.....,._'_Y}

Fig . 4 . Eg g capsule showinq a dwar f egg (DE). 1 50 x. 29 membrane. It collapses and breaks generally on the 9th day of development due to the consumption and enzymatic action of the developing larva (post-veliger). Markings or wrinkles on the internal membrane were not observed.

Live spermatozoa were frequently found in the albumen fluid of capsules at the time they were deposited.

Several sperm have been found in a single egg. Seven sperm, for example, were observed on one occasion in an egg. As many as twelve sperm have been found in a single egg. From the above observations, it may be theorized that ovulated egg cells or primary oocytes and mature spermatozoa appear simultaneously in the carrefour where the primary oocytes and the accompanying sperm are wrapped with albumen fluid by albumen gland secretion. They are finally coated with an internal membrane while they pass through the oviduct where the oothecal or nidamental gland empties. In several instances, live sperm were observed in the lumen of the ciliated carrefour. It appears that self-fertilization may take place in the carrefour where mature spermatozoa and primary oocytes meet for the second time.

Normally an egg contains one egg cell or egg proper.

Polyvitelline eggs have been found in Ferrissia rivularis on only three occasions in two years. In the first case, two blastula embryos were found in one egg in July 1970.

In the same year, another egg was found containing two egg cells, one:: norna.1 and one sub-·rectangular. 'l"hey were laid by one lirn;;Jct v.1)1ich had been previouf,ly subj cct to high 30

temperature. They died before the first cleavage. In the

third case, one polyvitelline egg, containing two twelve-

cell embryos (Figs. SC and D), was laid by a limpet which

was treated with 1.5% maleic hydrazide solution in the

summer of 1971. Double or triple-monster embryos have not

been observed in F. rivularis.

'l'he number of eggs contained in each capsule varies with the size (age) of the animal. There is a tendency for

larger limpets to produce larger egg capsules containing more eggs. The number of eggs in one capsule ranges from

one to six in F. ri vularis. !1ost young animals lay one to

two eggs in each capsule. Clapp (1921) observed that one

egg capsule of Ancvlus fuscus contained a maximum of nine

eggs. According to one of his illustrations, it appeared

that an egg contained five egg cells. Only one of them

developed L1to a trochophore, three became hydropic exogastrulae, and one died without advanced development.

One egg per capsule was reported by Basch (1959) in

Ferrissia shimekii. The size of the egg capsule ranges

from 1.20 to 1.95 rn.rn. in rctaxi1.c1,al di2n1eter.

In June, 1970, two capsules ~ere laid without egg cells by a rn.ature limpet of 4.2 x 2.8 mm. in size. The diameters of th-2 hm capsules ,('2re much smaller than that of a normal capsule zrnd measured O. 5 8 and O. 8 6 mr:1. (Fig. SA) .

Frequently this kind of egg ca~·'>Sule was laid by limpets which were treated ~ith maleic hydrazide or increased water temperature. In the summer of 197l (Juno, ,Tuly and August), 31

A ,.:

., • • ®

Fig . 5 . Egg capsules . A. Empty capsule (150 X), B. High magnification of A (300 X) , C . po l y- vitellinc egg (150 X), D. High magnifi c at i on of C (300 X) . 32 about 2.8% of the egg capsules lacked egg cells. Capsule diameters ranged from 0.58 to 1.2 mm. Geldiay (1956) reported in Ancylus flaviatilis that capsules without eggs were found more frequently towards the end of the breeding season. All empty capsules showed a common characteristic of having wrinkles on the top lid (Figs. SA, B), which is probably due to lack of albumen inside.

There is a diversity in egg shape. This is determined by the nrnnber of eggs present in a capsule. The egg is large and almost spherical in a one-egg capsule. Usually in two-egg capsules, the eggs are equal in size, and if one is much larger than the other, both may develop normally and hatch, or the smaller one (Fig. 4) may have arrested growth after the post-veliger stage (Fig. 9D) and never hatches.

The small egg was called a "dwarf egg" by Bon

Capsules with three eggs very frequently contain one larger egg and two smaller ones; the larger egg always occupies about two-thirds of the entire capsule, the smaller ones are almost equal in size. Four-egg capsules contain nearly triangular and equal-,sized eggs (Fi(]. 3). Five and six-egg capsules contain approximately triangular eggs which are always arranged in one row with some overlapping

(Figs. 9A, B).

Only a few five and six-egg capsules were collected in the sum:ner of 1970 and none were founu in the summer of

1971. 'I'lle frequency of capsule types is summarized in

Table 2. 33

Table 2. Percentage of various capsules observed during the summer of 1971 (June, July, August).

Type of capsule No. of capsules Percentage

0-egg 5 2.8 one-egg 86 48 two-egg 45 25 three-egg 29 16 four-egg 9 5 ·five-egg 0 0 six-egg 0 0

Fecundity

Animals of the first group (Batch A) were studied for four days, from the 21st to 24th of June. In the four day period of time, 27 capsules containing 35 eggs were deposited by 20 limpets, ranging from 2.9 x 1.8 to 4.2 x

2. 8 rmn. in shell size. Among 27 egg capsules, two capsules contained no egg cells. Table 3 shows the results obtained from Batch A.

Batch B consisted of 20 limpets, but only 12 animals laid eggs in two days from the 28th to 29th of June. Shell size in the batch ranged from 3.4 x 2.4 to 4.5 x 2.9 mm..

Seventeen capsules containing 31 eggs were observed. See

Table 4.

Egg production of Batch C was observed in a period of 23 days from July 13 to August 4, 1971. A total number of 24 capsules, containing 42 eggs, was produced by 12 limpets (Table 5). In Batch D, 36 egg capsules, containing

71 eggs, were laid by 15 lirrwets in 18 days. Two empty Table 3. Egg production of 20 limpets in a four-day period from June 21 to June 24.

:q_ange of Average Bat.ch Shsll capsule caDsule 0- one- two- three- four- Total Total A Size 6.2- crtnct. 12 r dia::o_-:eter egg egg egg egg egg capsule egg f•,;'11'1\• I ,J.., ~ ) (rrrn: • ) (mm.) l 2. 9xl. 8 1.1-1.8 1.4 - 2 - - - 2 2 3.lxl.9 1.1 1.1 1 1 2 - 1 - - - , 3 3.2x2.1 1.1 1.1 - 1 - - - .L 1 4 3.2x2.2 1.2 1.2 - 1 - - - 1 1 5 3.3x2.0 1.3 1.3 - - 1 - - 1 1 6 3.3x2.2 1.4 1.4 - 1 - 1 2 ..., - - I 3.3x2.2 1.4 1.4 - - 1 - - 1 2 8 3 .. 3)(2.3 1.1 1.1 - 1 - - - 1 1 9 3 . -~:: 2 . 2 1.2 1.2 - 1 - - - 1 l ", (iv') ? 10 ._;. :i: ...... ~- 1.1 1.1 l - - 1 1 "'"\ ~ - ,- ,.... - 11 .).:JXL..L 1.0-1.4 1.2 - 1 1 - - 2 3 12 3.5x2.2 1.7 1.7 -- - 1 - 1 3 13 3.5x2.3 1.5 1.5 - - 1 - - 1 2 14 3.5x2.l 1.2.-1.4 1.3 - 1 1 - - 2 3 " i:; 15 3.:Sx2.2 1.5 J.• ~ - 1 - - 1 2 16 3.5x2.3 1.1-1.5 1.3 - 1 1 - - 1 3 17 3.5x2.3 .9]_-1.2 1.05 - 2 - - - 2 2 18 3.5x2.3 .86 .86 - 1 - - - 1 1 1 19 4.2x2.6 1.1 1.1 - 1 - - - 1 .J. 20 4.2x2.8 .58xl.O .81 2 1 - - - 3 1

2 16 8 1 0 27 35

w ,t:,. Table 4. Egg production of Batch Bin a two-

Range of Average Batcl1 S~ell capsule capsule 0- one- two- three- four- Total Total B Size diameter di21.meter egg egg egg egg egg capsule egg (mm.) ( ;rim• ) (mm.)

1 3 . 11z2 . 4 1.4 1.4 - 1 - - - 1 1 2 3.5x2.2 1.3 1. 3 - - 1 - - 1 2 : ., 1 ~ . 1 3 3.7:x:2.5 .L. • L.. • L. - 1 -- - 1 -l. 4 3.8x2.5 1.7 l. 7 - - 1 - - 1 2 5 3 .. 9;-:2~5 1.5 1.5 - - 1 - - 1 2 6 3.9x2.5 1.5 1.5 - - 1 - - 1 2 7 3 .. 9:::2 ~ 7 l. 8 1.8 - - - 1 - 1 3 8 4.0x2.7 .96:.s:l.4 1.18 - 1 1 - - 2 3 9 4.1:~2.6 1.5 1.5 - - 1 - - 1 2 10 4.3x2.8 1.7 1. 7 - - - 1 - 1 3 11 4. 5:~2. 8 .96-1.4 1. 21 - 3 1 - - 4 5 12 4.Sx2.9 1.6-1~9 1. 75 -- 1 1 - 2 5

0 6 8 3 0 17 31

w u, ::aJ:-le 5. Egg production of Batch C in 23 days from July 13 to August 4, 1971.

:Range of Average Batcl1 Shell capsule capsule 0- one- two- three- four- Total Total " ~ C Size oia.1. 1-1ecer diameter egg egg egg egg egg capsule egg (r1·,n• ) ( ',Ylr.1•) (imn.) 1 2.8x2.0 1.5-1.6 1.55 - 1 1 - - 2 3 2 3.6x2.3 1. 4 1.4 2 - -, 1 - - 1 3 3,.7){2.J 1.2-1.7 1.45 - .L 1 - - 2 3 ~ Cv'1 1 4 - "_/,;::,...:,., . ._, 1.2-1.3 1.25 - 2 - - - 2 2 5 3.9:<2.5 1.2-1.6 -l . ,.,/, - 1 1 - - 2 3 6 3.9x2.7 1. 3 1.3 - 1 - - - 1 1 7 4.3x2.7 l. 3-1. 7 l. 5 - 1 1 - - 2 3 8 4.3x2.8 1.2-1.9 1.5 - 2 - 1 - 3 5 _,,0 4.2x2.7 1.4-1.8 1.56 - 2 - 1 - 3 5 10 4.4.x2.8 1. 9 1. 9 - - - 1 - 1 3 11 4.6x3111 1.8-2.1 1.95 - - 1 1 - 2 5 12 5.l:;-:3.2 1.1-2.3 1.86 - 1 - 2 - 3 7

0 12 6 6 0 24 42

w O"I 37 capsules were produced by two limpets, and five four-egg capsules were recorded (Table 6). Batch E consisted of seven limpets ranging from 3.1 x 2.2 to 5.0 x 3.2 m.~. in shell size. Fourteen eggs, contained in twelve capsules, were produced by this batch in 14 days from July 22 to

August 4 (Table 7). Fifteen limpets of Batch F were studied for 25 days from July 26 to August 19. Thirty- three capsules containing 66 eggs were produced by this batch (Table 8).

In 18 days, Batch G produced 65 eggs contained in 32 capsules (Table 9). One limpet 5.4 x 3.3 mm. in size laid eight egg capsules within 18 days. The highest number of eggs produced in 18 days was 12. These were laid by a limpet 4.2 x 2.5 rnm. in size.

The overall re □ uJ.ts of the observations show that the larger animals tend to produce larger capsules with more eggs than younger specimens. In other words, larger animals are more productive than smuller ones. A comparative study of the relationship of the ovotestis size with the animal size or age also indicates that larger J.impets possess a larger ovotestis, larger number of oocytes, and produce more sperm. Table 10 lists the data of ovotestis, con@on atrium, sern.inal vesicle, and spermatheca sizes obtained in August and September, 1971. T0.ble 6. Number of eggs deposited by Batch Din 18 days from July 16 to August 2, 1971.

Range of l-:1.verage Batch Shell ca~s~.;.le caMpsttie 0- one- tv:o- three- four- Total •:rotal D ;3ize c1ier-·te ...cer i:3.]_2'-:neter egg egg egg egg egg capsule egg (rr:m• ) (h'\~/,', ,it • ) (mm.)

1 3.2xl.7 1.3 1.3 - - 1 - - 1 2 ') L. 3.5x2.l .86-1.5 1.18 1 1 1 - - 3 3 3 3. 5z~2 ~ 4 l.1-L 6 1.35 - 1 - 1 - 2 4 4 3.Sx2.3 1.5 1.5 - - 1 - - 1 2 5 4.0x2.5 1.)-~.3 1.15 - 4 - - - 4 4 6 4.2x2.5 1.8 1. 8 1 1 3 ..., - - - - I 4 . 2::2 . 5 1.7 1. 7 - - - 1 - 1 3 3 4.2:-:::2.5 1.2-1.7 1.45 - 1 - 1 - 2 4 9 4o3;{2~8 1.2-1.7 l.45 1 -- 1 - 2 _,? 10 4.4x2.7 1. 5-1. 6 l. 55 - - 1 1 - 2 5 11 4.5x3.0 1. 3-l. 9 1. 6 - 1 1 - 2 4 11 12 4. 6;:3 .1 2.0 2.0 - - - - 1 1 4 13 4.6x3.6 1.1-1.7 1. 4 1 2 3 5 1 ,, - - - ~± 4.8x2.8 1.6-2.0 l. 72 - 2 1 - l 4 8 , ~ .L) 5.2J:3.l l..2x2.2 1.65 - 3 - l 1 5 10

2 14 8 7 5 36 71

w co Table 7. Egg production of Batch E in 14 days from July 22 to August 4, 1971

R2.nge Average Batch Sh_ell Cc".Dsule capsule 0- one- two- three- four- Total Total

E f,ize O.. ia_rne-'~er dia:-r1eter egg egg eao.., J egg egg capsule egg (r.rm. J (mm. J (mm • )

1 3 .. 1:;<2.2 1.2 1 • L...') - 1 - - - 1 1 2 3.5x2.3 1.2 1.2 - 1 - - - 1 1

3 3.Sx2.5 1.1-1.3 1 . ?~ 1 1 - - - 2 1 ., 4 3.9x2.3 ..79-1.G 1.2 l .l.. - 1 - 3 4 5 3.9x2.4 1.3 1.3 - - 1 -- 1 2 6 3.9x2.6 .96-1.5 1.3 1 1 1 - - 1 2 7 5.0x3.2 1.4 1.4 - - 1 - - 1 2

3 5 3 1 0 12 14

w I..O Table 8. Number of eggs deposited by Batch Fin 25 days from July 26 to August 19, 1971.

~a.r_ge of _Zl,.verage Batcl'-1 u.neJ-c· . ·1 ...... l C 2'..IJ s 1.1l (~ capsule 0- one- two- three- four- Total Total ,::, l. size dia1L1eter diar::ieter egg egg egg egg egg capsule egg (mm.) (r.m.) (rim.)

l •.L 3.2x2.2 1.1--1.3 1.2 - 1 1 -- 2 3 2 3.4x2.l 1. 2-1. 4 1. 3 - l 1 2 3 -, ") , r::. - - 3 3.6x2.3 J...L.-.L.O 1.4 - 1 1 -- 2 3 4 3.7x2.5 1.9 l. 9 - - - - l 1 4 5 3o9;,:2.3 L6 1.6 - - 1 - - 1 2 6 4.lx2.8 1. 7 1.7 - - - 1 - 1 3 7 4.2x2.7 1.8 1.8 -- - 1 - l 3 8 4. 2x2. 5 • 9G-l. 7 1.3 - 2 1 1 - 4 7 9 4.5x2.6 1. 4 1. 4 - - 1 - - 1 2 10 4.6~{2 .. 8 1.1-1. 9 1.4 - 3 1 1 - 5 8 7 7 .L .i.. 4.Gx3.0 1.2-1.9 1.7 - 3 2 1 - 6 10 12 4..8x2.8 1.96 1.96 -- 1 - - 1 2 13 4.Sx3.l 1.9 1. 9 - -- 1 - 1 3 14 5.0x3.l 1.7-1.8 1.8 -- 1 1 - 2 5 15 5.4x3.l 1.1-2.3 1.7 - 1 - 1 1 3 8

0 12 11 8 2 33 66

~ 0 Table 9. Number of eggs produced by Batch Gin 18 days from August 2-19, 1971.

Average Range ., of Batch Sl1sll capsuj_e capsu2-e 0- one- b,,;o- three- four- Total Total G size dia:rr;eter cl.ia,reter egg egg egg egg egg capsule egg \.111I "''Yr] • ) (mm • ) (mm. J

1 '? _ 1 7 J_ 3.5;,,:2.7 ..L ... ._:i .\.,.,, J_ .. 5 ·- .J..' - 1 - 2 4 ') 1 ;:; "-- 3.9x2.4 ..... 1.5 -- 1 - - 1 2 3 3.9x2.5 1.1-1.3 1.2 - 3 - - - 3 3 {, 4.0~{2 .. 5 " 1.6-1.8 1.7 -- 1 1 - 2 5 5 4.1x2.5 1.4-1.9 1.7 1 1 2 5 r - -- 0 4.2x2.5 .9-1.5 1.4 - 2 5 - - 7 12 7 4.4x2.8 1.4 1.4 - 1 - - - 1 1 8 4.5x2.6 1.3 1.3 - 1 - - - 1 1 9 4.5);.2.7 1.2-1.9 1.6 - 3 - - 1 4 7 10 4.9}·:3.2 1.7 1.7 - - 1 - - l 2 1 J_ 5.lx3,0 1.1-1.7 1.4 - 3 1 - - 4 5 " ') 1 .l..<'.: 5 (,4.x3. 3 l.l-1.8 1.5 - J.. 2 1 - 4 8

0 16 11 3 2 32 65

.r:,. I-' 42 Table 10. A comparison of shell size with the sizes of ovotestis, coTiu~on atrium, and seminal vesicle.

Shell size Ovotestis Common atrium Seminal vesicle

Length X width Height X width Height X width Length X width (mm.) (rnm. J tmm. J (mm.)

5.6 X 3.6 0.56 X 0.65 0.27 X 0.24 5.4 X 3.7 0.56 X 0.67 0.49 X 0.28 5.4 X 3.6 0.53 X 0.62 5.2 X 3.2 0.47 X 0.38 0.30 X 0.20 0.38 X 0.15 5.1 X 3.4 0.53 X 0.65 0.34 X 0.11 4. 9 X 3.3 0.56 X 0.53 0.33 X 0.17 0.23 X 0.11 4.9 X 2.8 0.48 X 0.53 4.8 X 2.8 0.49 X 0.51 0.30 X 0.13 0.26 X 0.11 4.8 X 2.8 0.47 X 0.34 0.38 X 0.19 0.23 X 0.15 4.6 X 3.1 0.49 X 0.59 0.39 ~·V 0.21 0.30 X 0.11 4.6 X 2.7 0.48 X 0.47 0.45 X 0.16 0.38 X 0.17 4.5 X 2.9 0.42 X 0.43 0.23 X 0.08 0.29 X 0.12 4.5 X 2. S 0.47 X 0.35 0. -11 X 0.16 4.5 X 2.5 0.43 X 0.38 0.38 X 0.15 0.26 X 0.17 4.4 X 2.8 0.45 X 0.38 0.41 X 0.17 0.23 X Oo08 4.4 X 2.6 0.32 X 0.26 0.23 X 0.11 4.3 X 2.8 0.46 X 0.27 4.3 X 2.3 0.25 X 0.23 4.2 X 2.5 0.57 X 0.49 4.2 X 2.5 0.45 X ().t;;l 0.34 X 0.21 0.23 X 0.08 4.2 X 2.3 0.38 X 0.40 0.41 X 0.17 0.23 X 0.11 4.2 X 2.3 0.38 X 0.34 0.23 X 0.12 <'.1.1 X 2.6 0.39 X 0.38 0.41 X 0.23 0.23 X 0.10 4.1 X 2.5 0.38 X 0.32 4.1 X 2.5 0.41 X 0.38 0.38 X 0.19 0.15 X 0.09 4.1 X 2 . ::)r' 0. ~15 X 0.38 0.49 X 0.2() 0.26 X 0.12 4.0 X 2.6 0.40 X 0.32 0.19 X 0.11 0.23 X 0.11 4.0 X 2.5 C.38 X 0.38 0.24 X 0.14 0.30 X 0.09 4.0 X 2.5 0.32 V 0.32 0.26 .,," 0.19 4.0 X 2.3 0.38 X 0.48 0.41 X 0. ].'/ 0.33 X 0.11 4.0 X 2.2 0.34 X 0.11 3.9 X 2.5 0.23 X 0,25 0.39 X 0.11 3.9 X 2 . ,1 0.37 X 0.32 0,20 ,,.V 0.12 0.26 X 0.08 3.9 X 2.3 0.35 X 0.36 0.33 x. 0.17 0.38 X 0.13 .J" ~ a-~ X 2.3 0. 3,1 X 0.32 0.23 X 0.13 3.9 X 2.3 0.38 X 0. 3 4. 0.38 X 0.19 0.30 X 0.13 ') ') 3.9 X L. • ..) ') 1 3.9 X ~ . --· 0.44 X 0.42 o. ,n X 0.21 0.32 X 0.09 3.9 X 2.3 0.38 X 0.3'1 0.38 X. 0. 19 0.30 X 0.13 3.9 X 2.3 0.39 X 0.34 0.32 ,i," 0.13 0.23 X 0.13 3.9 X 2.2 0.39 .,~V 0.32 0.30 X 0.17 0.50 X 0.11 3. 9 X 2.2 0.35 X 0.35 0.34 X 0.17 0.26 X 0.13 3.9 .,,V 2.2 0.11 X 0.34 0.38 ;~ 0.18 0.39 X 0.09 3.8 X 2.3 0.38 X 0.25 0.29 X 0.17 0.26 X 0 .1 '.1 43 Table 10. (continued)

Shell size Ovotestis Common atrium Seminal vesicle

Length X width Height x width Height X width Length x width (mm.J (mm. J (mm.) (mm.)

3.8 X 2.2 0.33 X 0.35 0.30 X 0.14 3.8 X 1.8 0.34 X 0.26 3.7 X 2.2 0.36 X 0.33 0.24 X 0.13 0.38 X 0.09 3.7 X 2.2 0.32 X 0.35 0.26 X 0.11 0.23 X 0.098 3.7 X 2.1 0.34 X 0.30 3.7 X 2.2 0.26 X 0.30 3.7 X 2.2 0.36 X 0.31 0.26 X 0.15 0.21 X 0.083 3.6 X 2.9 0.49 X 0.41 0.47 X 0.24 0.26 X 0.11 3.6 X 2.3 0.39 X 0.24 0.28 X 0.07 3.6 X 2.2 0.35 X 0.35 0.23 X 0.14 0.23 X 0.11 3.5 X 2.3 0.35 X 0.29 0.26 X 0.05 0.20 X 0.06 3.5 X 2.2 0.34 X 0.35 0.30 X 0.12 0.21 X 0.12 3.5 X .2.2 0.34 X 0.35 0.26 X 0.15 0.26 X 0.08 3.5 X 2 ? 0.34 X 0.35 0.30 X 0.12 0.21 X 0.12 3.3 X 2.1 0.34 X 0.34 0.23 X 0.08 0.23 X 0.15 44

Anatomy and Histology of Reproductive System

1. Reproductive system anatomy

Only the distal portion of the reproductive system was studied (Fig. 6). No attempt was made to study the entire reproductive system because the scope of this research-was focused on gametogenesis and fertilization.

Ferrissia rivularis (Say}, like that of other freshwater pulmonate snails, is hermaphroditic; the eggs and sperm being produced by a single reproductive gland, the ovotestis. This organ is nearly spherical or elliptical and distinctly lobed from top view, though slightly narrowing toward the joining common atrium. The ovotestis is placed beneath and sometimes slightly posterior to the ascending portion of the intestine, being surrounded by the right posterior portion of the liver or digestive gland which hides it from lateral view.

The ovotestis is pear-shaped. Externally it consists of four to six lobes. Each lobe represents one acinus (follicle) in \;rhich eggs and sperm are formed (Fig. 8).

The corn.man atrium is a thin-walled sac resoretbling a vase situated immecl.i.ately belm•J the ovotestis. This atrium gradually reduces its diameter towards the hermaphroditic duct. The hermaphroditic duct is modified to form a seminal vesicle half way along its length. The cownon atrium and the hermaphroditic duct, except the seminal vesicle portion, are transparent, thus ciliary movement inside is clearly 45

Fig. 6 . Distal portion of the reproductive system of F . rivularis . AG, a lbu men gla nd; CA, common-atrium; HD, hermaphroditic du ct; Ov , ovotc stis ; NG, nidamental gland ; SV, seminal vesicle. 300 X. 46 visible. Sometimes a small constriction appears between the ovotestis and the common atrium. It may function to control the movement of oocytes and sperm from the ovotestis.

The size of ovotestis varies considerably with the size (age) of an animal. Sixty-one limpets of 3.3 to 5.6 mm. in shell length were dissected and examined, and the sizes of the ovotestis and other accessory organs were measured during August and September of 1971 and April of 1972. All measurements are listed in Table 10. The size of the ovotestis varies between 0.56 x 0.65 and 0.34 x 0.34 mm.

Inside the living ovotestes, mature and growing oocytes, mature spermatozoa, and spermatids with Sertoli cells are visible. Occasionally small orange-brown bodies of unknown function are seen on top of the ovotestis. They, perhaps, have something to do with vitellogenesis (Joosse and Reitz, 1969).

'i'he si,:;e of the comrnon atrium range[~ from O. 49 x

0.2B to 0.23 x 0.08 m.'11. The proposed function of this atrium is given in the discussion section.

The seminal vesicle is very irregular in configuration, being formed by an evagination of the hermaphroditic duct. The vesicle retains and stores sperm at least until oocytes pass t}1rough. Sperm, few in number, are found in the cmn 1non atr:i_ uin and the hermo.phrodi tic duct leading to the seminal vec:;icle. The serclinal vesicle is ah·1ays completely filled with tiqht.ly packed sperm even in the \·!inter time; v1hile that portion of the hermaphroditic 47 duct leading away from the vesicle is empty or contains only a few sperm. There appears to be one constriction at each end of the seminal vesicle. The constrictions may control the movement of sperm through hermaphroditic duct.

The size of the seminal vesicle varies greatly with the number of sperm present and the size of the animal. The long axis of the vesicle is usually parallel to the long axis of the body and measures from 150 to 390 µro. The size of the vesicle (length x breadth) varies between 150 x 90 and 390 x 110 µm.

The hermaphroditic duct leads into the carrefour.

The width of this duct varies with limpet size, and the particular section of the duct. Generally it ranges from

45 to 27 µmin outer diameter, and 9 to 27 µmin inner diameter.

The spermat~eca (seminal receptacle) is pear-shaped and about 210-450 µm long by 120-210 µm wide. This organ was examined to show whether or not sperm were present.

The lumen cf the spermatheca in living specimens was filled with unidentified orange material (Figs. 10 and 11). No spenn were observed inside. Thin sections of the spennatheca also confirmed this observation. The wall of the spermathcca consists of two layers, a11 inner columnar secretory epithelium &nd an outer thin connective tissue

(Fiq. 11).

2. Ovotestis histology

Both longitudinal-- and cross·-sections of the ovotes-· 48

-.,,.------Oogonia _;:;.,~..,._----Full•grown Oogenetic oocyte zone H-+----Sertoli cell ~,'--;,/-~!----Follicle c e II ~1+----Sperm "----Growing oocyte

>'>'----Acinus lumen

.------,.Connective tiuue

epithelium

Fig. 7. Ovotestis, longitudinal section.

Oocytc Sperm Oocvte I 1·

Packed

Ovo!·es!is

Fig. 8. Ovotestis and distal portion of reproductive duct, gross morphology. 49

., ·~ .. .0 .

' - ,, •

C D

Fig . 9. Egg capsules . A. Six-egg capsule (200 X). B . Five-egg capsule (200 X). C. Egg capsule containing three juveniles (150 X) . D. Egg capsule containing one "dwarf" and one normal juvenileS(200 X) . 50

Fig. 10 . Spermatheca , external morphology . 200 X.

Fig. 11. Spermathcca , longitudinal section . 300 X. 51 tis were made. Fig. 7 shows a longitudinal section through the ovotestis. The entire ovotestis is covered by a thin connective tissue or "Ancel's layer" composed of two or more tiers of thin flat cells. The acini or follicles are separated from one another by at least two layers of this connective tissue.

All acini are lined internally with a single layer of cells, the germinal epithelium. This tissue differentiates into sperm, oocytes, and nurse cells (including follicle and Sertoli cells). There is a central lumen in each acinus, and it becomes more spaceous after ovulation (Fig. 20}. In mature limpets only the germinal epithelium divides into two distinct zones (Figs. 18 and

33), an upper oogenetic zone where eggs are produced and a lower spsrmatogenetic zone where sperm are formed.

Growing oogonia and primary oocytes with their follicle cells, as well as ovulated or fertilized oocytes without follicle cells, may be observed in the oogenetic zone (Fig. 33). Mature and growing oocytes are seen in the distal part of this zone. Young oocytes and oogonia are generaJ.ly located in the proximal portion. Frequently two or more bundles of late sperrnatids, attached to Sertoli cells, are seen in the distal part of the acinus lumen

(Figs. 18 2nd 90). Sperm and Sertoli cells develop from the germinal epithelium of the spermatogenetic zon2, however, their presence in the lumen of the oos:renetic zone may suggest that irregular-shaped Sertoli cells move from the 52 lower. zone by amoeboid movement (Fig. 12). Therefore, spermiation probably takes place in the vicinity of mature and growing oocytes. The occurrence of spermiation and sperm in this region may support the possibility of self- fertilization in the lumen of the oogenetic zone.

Spermatogonia, primary spermatocytes, secondary spermatocytes (very rare), various phases of spermatids, and developing Sertoli cells are present in the spermatogenetic zones of acini. Spermatid tails extend from this zone down to the upper portion of the common atrium. Prior to spermiation, the hsads of spermatids are always oriented toward the distal end of the ovotestis, and the tails extend downward, toward the other end of the ovotestis.

Later, after spermiation, the orientation is reversed.

Mature spermatozoa must make a 180° turn either in the lumen of the ovotestis or in the com.rrion atrium; since sperm are always oriented in this direction when the seminal vesicle or hermaphroditic duct is opened by dissection (Figs. 13 and 22). It is more likely that sperm rotate in the common atrium, since its large fluia-filled cavity could more easily facilitate turning.

3. Common atrium histology

The cornrnon dtrium' s wall conc::ists of two cellular layers and onE:' non-·cellular layer. The outer consists of thin connective tissue, which is the extension of Ancel's mc,mbrane. 'l'he insic'Je of the atrium is lined with a sing1G layer oE ciliated s:Lmple columnar epithelium. Between the 53

two layers is a thi~non-cellular basement membrane

(Fig. 14).

4. Seminal vesicle and hermaphroditic duct histology

The structure of seminal vesicle and hermaphroditic duct walls is similar to that of the common atrium, except

for the inner layer. The internal layer, as in the atrium,

is a ciliated epithelium; but unlike the atrium, the epithelium is thinner, non-glandular, and is composed of flat cells with relatively large nuclei (Figs. 15 and 16).

Gametogenesis

1. Spermatogenesis

(1) Germinal epithelium

The ovotestis acini of Ferrissia rivularis has the

same general appearance as that of Helix_ aspersa (Gatenby,

1971), Succinea ovalis (Hickman, 1931), Eremina desertorum

(Fahmy, 19~9), slugs (Pelluet and Watts, 1951), Arion

s~1bfuscus ("1"'7atts, 1952) and Lymnaea stagnalis (Joosse and

Reitz, 1969). Spermatogonia, Sertoli cells, oogonia, and follicle cells arise from the cells of the germinal epithelium throughout the life of the limpet. The germinal epithelimn con~,ists of small oblong cellf; which are greatly flattened until maturation progresses. Then the cells become pros_rressively cuboidal. GermL1al epithelial cells are arranged around the acinus wall in the spermatogenetic zone and occur as a lining one cell-·layer thick (Fig. 17). 53

Fig. 12 . Sagital section of an ovotestis illustratin g amoeboid Sertoli cells (Se C) a nd spermatids (Spt) . 130 0 x.

Fig . 13 . A ruptured semin al vesicle showing mature sperm . CA, common atrium·; Ov , ovotestis. 250 X. 54

Fig. 14. A lon g itudin a l s e ction of the co mmon atrium sh owing colu mna r ep i the lium (CE) , conn e c ti ve tis s ue (CT) , a nd ba se ment membr a ne (BL) . 1300 X.

Fig. 15 . Longitudinal section of a sem in al v es i cle showing ev a gin a tions of the h e rmaph r od i t i c du c t and packed sperm . 430 X. 55

(2) Spermatogonium

Spermatogonia are easily recognized as spherical cells of varying sizes. They contain a large nucleus surrounded by a thin layer of cytoplasm of almost uniform thickness (Figs. 17, 21B, and 24). The size of the spermatogonium ranges from 5 x 5 to 9 x 9 µm. Nuclei measured from 4 x 4 to 7 x 7 µm. An acidophilous nucleolus is always found in the peripheral region of the nucleus

(Fig. 21B). In most cases, nuclei are found at an interphase stage. In interphase, fine chromatic granules are clearly seen. Chromatin granules form a deeply stained, chromatin-reticular network, and nucleoli and Golgi bodies disappear at prophase. Often spermatogonia are loosely grouped at the site where they originate.

(3) Primary spermatocyte

The spermatogon:Lum is spherical up to the time of primary spernmtocyte formation; then it lengthens slightly.

The nucleus with its nucleolus moves to the broader end of the cell, where i-1.: comes to lie close to the cell membrane

(Figs. 21C and 26). Mitochondria and Golgi granules in the cytoplasm now become distinct and group in a mass on one side of the nucleus (Fig. 21C). All primary spermatocytes are characterized by elongated, large cells with large nuclei at on2 cnc'l of tl1e cell. The size of these cells

averacre lG x 7 11w.1 and the size of ·Lhe nucleus is about

7 X 6 pm.

Various phases of primary spermatocyte formation 56

Fig. 16. Longitudinal section of th e hermaphroditic duc t leading from the co~non atrium to the seminal vesicle. 1300 X.

Fig . 17. Cross section of the ovotestis sl1owing spermatogonia (Spg ) and developing germina l epithelium (GE) . 1300 X. 57

Fig. 18. Distal portion of the reproductive system , sagit a l section . OZ, oo ge netic zone; SZ , spermatogenetic zone; CA, common atrium ; HD, hermaphroditic duct; SV, seminal vesic]e . 300 X.

Fig . 19 . Developing spermatogonium (o ff the arrow ) pulling away from acinus wall. Sec , developing Sertolic cell ; Spq , spermatogonium . 1300 X. 58

Fig . 20. Cross section of an ovotestis showing four a ci n i with an ovu lat ed oocyte in the lumen o f one. 310 X. Fig. 21. Spermatogenesis of Ferrissia rivularis. x 1000. A. Undifferentiated germinal epithelium. B. Sper- matogonia and developing germinal epithelium. note the large nucleus, nucleolus and Golgi body. C. Primary spermatocytes. ~ote the eccentrically located nucleus and Golgi-mitochondria cloud. D. Metaphase of the first maturation division and developing Sertoli cell. E. Diakinesis of prophase of the primary spermatocytes. F. Secondary spermatocytes and developing Sertoli cell. G. Early spermatids and developing Sertoli cell. H. Spermatids \,1i th PNA and Sertoli cell. I. Inter- mediate spermatids and Sertoli cell. J. Late spermatids, a Sertoli cell. K. Spermatid showing head and tail.

CT, connective tiss~e of Ancel's layer; C~r, chromosome~ E Spt 1 early soonnatid; GE, germinal epitholium; GM, Golgi and mi tochonC!.ria cloud; P:'11\., post-nu:::lear apparatus; S Spc, secondary spermatocytei Se C, Sertoli cell; Spt, spermatid. 60

C 61 have been observed during the entire breeding season. The interphase stage shows a large nucleus with one nucleolus situated at the end of the nucleus where mitochondria and

Golgi granules are gathered in the cytoplasm (Fig. 26).

"Diakinesis" is often observed in some primary spermatocytes.

The cell at this stage becomes polygonal in shape due to crowding of adjacent cells. Paired chromatids appear as short rods scattered in and on the periphery of the nucleus

{Figs. 21E and 23). The ~ize of these cells at prophase is

8 x 8 um, and the nucleus is 7 x 7 um. Occasionally, primary spermatocytes arrange into a line next to a large young Sertoli cell, but they do not attach to it (Fig. 21D).

At metaphase the nuclear membrane disappears, and chromosomes are lined up at the equatorial plane (Fig. 25).

Telophase of the first maturation was not observed.

(4) Secondary spermatocyte

Secondary spermatocytes are distinguished from primary spermatocytes by their smaller si.ze, lesser con- centration of mitochondria, smaller size of nucleus, and by the absence of a nucleolus. Condensed chromatin granules are concentrated at the peripheral region of the nucleus.

Secondary spermatocyte cells are frequently found in regular groups arranged radially around a Sertoli cell (Figs. 21F and 27). The Sertoli cell in general is no different than the young pri.rnary oocyte in morphology, except the oocyte is always surrounded by three or four small flat follicle cells at this time. A large nucleus with a small amount of 62

Fig . 22 . Seminal vesicle smear showing matur e sp erm . 500 X.

Fig. 23. "Diakinesis" of primary spermatocytes . 1300 x. 63

Fig. 24. Ovotestis smear showing sbermatogonia and a spermaLid tail with a cytoplasmic globule . 700 X.

Fig. 25. Cross section of an ovotestis showing the first Maturation division, secondary spcrmatocytes {off the arrow), and a developing Scrtoli cell. 1300 X. 64

Fig . 26. Interphase of primary spermatocytes . 1300 x.

Fig . 27 . Secondary spermatocytes (off the arrow) surrounding a youn0 Sertoli cell (SeC) . 1300 X. 65 chromatin material is located in the center of each Sertoli cell. The second maturation division of the spermatocyte was rarely observed. s~all spermatids resulting from this division elongate distally.

(5) Spermiogenesis

Early spermatids: A young spermatid is 7 x 5 µmin size, and has a spherical nucleus situated at the end of the cell nearest an adjacent Sertoli cell (Fig. 21G). This side will eventually become the anterior end of the spermatozoon

(Fig. 28). A Golgi and mitochondria cloud is seen in the cytoplasm of the presumed anterior end of the spermatid

(Fig. 21G). The nucleus shows a dense rim at the peripheral area and a dark spot in the center, and is 2 x 2 µmin size.

Young spermatids are not physically attached to a Sertoli cell. A gap between the two cell types is seen at this stage..

Intermediate spermatids: Young spermatids attach, elongate, and radiate from the periphery of Sertoli cells.

The nucleus at this stage develops into a dark post-nuclear appa.ratus (PN.i":.) with a small black dot abo;1t 1 µm in diameter in the center (Fiq. 29). The size of the spermatid cell is 8 x 3 11m, and the I'NA is 2 µm in diameter and is concentrated at the end of the cell nearest an adjacent

Sertoli cell (Fig. 29). Sertoli cells 2nd their attached spermatids are always seen in the lumen of the spermatogenetic zone. 66

Fig. 28. Young spermatids (off th e arrow ). 130 0 x.

Fig. 29. Young spermatids showing post-nuclear apparatus (PNA). 1300 X. 67

In the early stage of spermiogenesis, young spermatids are only found on the lumen side of Sertoli cells. No

spermatids are attached to the side next to the acinus wall.

Sertoli cells move away from the acinus wall following the early spermatid stage, and hence intermediate spermatids are allowed to attach to the entire peripheral surface of the cell.

Late spermatids: Spermatids at this stage have bullet-shaped heads which remain attached to a Sertoli cell and bear one long belt-like tail (Fig. 30). As development progresses, the belt-like tail lengthens by becoming thinner. This is due to rapid growth of the central filament and decrease of volume in the tail by shedding cytoplasm through the free end of the tail. Small cytoplasmic beads are always seen along the length of the tail, particularly at the free tip where lar~e globules of cytoplasm accunmlate (Figs. 31 and 89). At this state, Sertoli cells are often amoeboid in shape and are seen with their attached late spermatids in the lumen of the upper oogenetic zone, particularly at the junction of oogenctic and spermatogenetic zones. RvidentJ.y Sertolj cells are able to move from the spermatogenetic zone up to the oogenetic zone, probably by amoeboid locomotion.

Fully developed sperm have short heads that are cork-screw-shaped and tails that are smooth and lack cytoplasmic globule remnants. These mature spermatids are now ready to spermiate. The mechanism of spcrmiation is 68

Fi g . 30 . Advanced sp er~a tids. H, head ; sec , Se rtoli cells ; T , t ail . 1300 X.

Fig. 31.. Late spermatids showing cytop l asmic rem nants . CR, cytoplasmic remnant ; sec, Sertoli cell; Spt , young spermati

Mature spermatozoa: Mature (spermiated) spermatozoa have thick cork-screw shaped heads that are four microns long, have pointed acrosomal tips, long tails which average 214 ~min length, ~nd spiral cytoplasmic keels which surround the flagellum or filament (Fig. 32). Details of the tail structure will be described in a later section.

( 6) Sertoli cell

In Ferrissia rivularis, Sertoli cells, as in other invertebrates, arise from the germinal epithelium. An early Sertoli ceJl is similar to a young oocyte in having a large nucleus and in being surrounded by a group of secondary spermatocytes on the surface exposed to the acinus lumen.

As sperrnatocytes divide, the Sertoli cell begins to pull away from the acinus wall, probably by amoeboid mover<1ent, to increase more area for spermatid attacl~ent. FinaJly, the

Sertoli cell loses its vesicular nucleus and becomes free in the lumen. It soon becomes radially surrounded by elongated sperrnatids. A Bertoli cell with its associated spermatids may move upward to the luuen of oosenetic zone during the met8.Inorphor;i,-~ of the spermatids. The cytoplasm of the

Sertoli cell dwindles before spermiation occurs; and the 70

•

Fig. 32 . A mature spermatozoon of F . rivularis . 1000 x.

Fig. 33. Longitudinal section of an ovotestis showing oogene tic and sperrnatogenet ic zones. 400 x. 71 nucleus becomes condensed and dark stained. The number of spermatids or sperm surrounding a single Sertoli cell has been observed to be from 22 to 28.

2. Oogenesis

(1) Germinal epithelium

The condition of the undifferentiated germinal epithelium in the oogenetic zone is the same as that described previously in the spermatogenetic zone. Both oogonia and follicle cells are derived from this epithelium.

Flat germinal epithelial cells are rareJ.y seen during the breeding season. The entire zone is almost completely lined internally ~ith growing and full-grown oocytes and a few oogonia (Fig. 33). In the young ovotestis, oocytes are seldom seen, only germinal epithelium and oogonia (Fig. 81).

( 2) Oosronia

Certain cells of the germinal epithelium grow and change into oogonia which contain a thin layer of cytoplasm and a large sphericaJ., oval, or irregular-shaped nuclei with large basophilous nucleoli (Fj_g, 34). A chromatin re::ticulcir netv1ork is faintly seen in the nucleus. The size of oogonia rnngos from 9 x 7 to 19 x 9 µm. Nucleus size is between 6 x 6 and 10 x 6 µm. The size of nucleolus is between 2 x 2 and 3 x 3 pm. Nucleoli are frequently eccentrically located in the nucleus. Two nucleoli have becc:n observod in some· oogonia, one-:; be.in9 small, ephemeral dcidophilous and the other larger, ring-like, and 72 basophilous. The same structures were also observed in an electron micrograph of a young previtellogenetic oocyte.

The basophilous nucleolus increases in size until about the middle of the oocytic growth phase. Growing oogonia are not surrounded by follicle cells until they begin to grow.

Developing follicle cells are, however, frequently seen adjacent to oogonia prior to this time (Fig. 34). The proximal end of an oogonium is attached to the acinus wall along about one quarter of the cell surface. This attachment is the presumed vegetal pole of the egg.

(3) Oocyte

An amoeboid phase (Bretschneider and Raven, 1951) is not observed in the oogenesis of this limpet. Oocytes grow characteristically by increasing greatly in size while elongating into the acinus lumen. The elongation of the growing oocyte is probably aided by rapid growth and crowding of adjacent oocytes (Fig. 34). A few cells may bulge into the acinus lumen, move away from the acinus wall, and may become Sertoli cells associated with spermatids or possibly degenerate (Figs. 34 and 35). The length of the cell is about twice that of the width at this time. As the oocyte grows, follicle cells move away from the acinus wall and bc9in to surronnd the apical and lateral regions of the oocytc (?igs. 34, 80, and 94). Only the basal part of an oocyte borders upon t11e connective tissue layer of the acinus wall. voJlicle cells have a relatively large nucleus ·wit:h dern;e basic chror1tatin granules, one small 73

Fi g. 3 4 . Oogonia , oocytes , and follicle c e l ls . Og , oo goniurn; Oo , oocyt e ; FC , fol li c l e c e ll . 900 X.

Fig . 35 . Ovotestis c ross sec ti on s howin g g rowing oocytes. Note the ce ll, off the a rr ow, mqvi ng away f rom aci nu s wal l. 1 3 00 X. 74 acidophil nucleolus, and a very small amount of cytoplasm.

The nucleus of a follicle cell is 7 x 11 µmin size, and the nuceolus is about 1.5 µmin diameter. Generally, one oocyte is surrounded by three or four follicle cells. In early stages of growth follicle cells and growing oocytes are physically attached to one another. As growth progresses, oocytes start to grow laterally and finally become rounded or oval. Follicle cells also become thinner.

The cytoplasm of an early oocyte is very dense and stains heavily with basic stain. As soon as an oocyte elongates and becomes enveloped by follicle cells, a mitochondrial cloud appears at the basal part of the cell which lies upon the connective tissue (Ancel's layer). This has been verified by electron microscopy. The basal region stains more deeply than cytoplasm of the apical region and has a more granular texture. The apical cytoplasm region stains light purple and is finely granular. A large nucleus or germinal vesicle is situated toward the apical regi.on. Mitochondria scatt~r over the cytoplasm prior to vitellogenesis. This dispersion of mitochondria is probably related to vitellogenesis. Details of yolk formation will be discussed in the electron microscope study section.

Cortical cytoplasm eventually becomes finely vascuolar in appei'lrc:,nce (I'ig. 36A) ; and the C!ntire cell is expanded, probably due to hydration. 'rhe volume is slightly larger in

this st2a_.,eC,l thc~tn -~n- a 111att1~e~J_ oo,•tnC _I - '-' (~i"· ~g • 36B)' : • 'J1he total volume of the egg is greatly incrca~;c~d from the oogonium to 75

Fig. 3G. Growing oocytes showing the vacuo l ated condition of the cytoplasm. 1300 X. 76 full-grown oocyte stages. It increases about 37 times, if volume is indicated by cell index measurement.

During oocyte growth all visible generative changes within the nucleus stop, chromosomes disappear and the vegatative function of the nucleus prevails, particularly the activity of nucleolus. The synthetic activity of the nucleus can be deduced from the parallel growth of the nucleus and cytoplasm. The nucleus of a young oogonium has 2 an index of about 24 µm, whereas the full-grown oocyte is 2 900 µm. Hence, the nucleus increases during growth up to

38 times its initial size. Chromatin granules and a reticular network are faintly seen in large germinal vesicles.

The nucleolus is the most distinct structure seen in the germinal vesicle. The size of the nucleolus also increases considerably during growth. Nucleolar index increases 36 times from the oogonium to full-irown oocyte stages. During th2 growth stage of an oocyte, the nucleolus shows a great variation in structure and chemical nature.

Three different types of nucleoli have been observed in growing oocytes, viz., basophilous, acidophilous, and arnph- inucleolar. Structural configurations of the nucleolus are shown in Fig. 37. Most nucleoli arc round or oval, others are irregular or. stellate in shape. Intranucl0olar vesicle;s and nucleolar bucls are often seen in the nucleoli of older oocytes. In younger oocy~es, the cortical portion of the nucleolus is a.cidoph:Llous, and the rn.::dulla is basophilous Fig. 37. Types of nucleoli present during oogenesis. A. .i\n ovulated egg. B-L. Developing oocytes. ltNcl, acidophilous nucleolus; Btlcl, baso;~hilous nucleolar bud; CT, connective tissue; GV, germinal vesicle. 78

•✓ • • •• 'i .• ·_.. __•

D 79

and has various shapes. The nucleolus of an ovulated egg

has a smaller, basophilous nucleolar bud and an acidophilous major portion (Fig. 38A). The nucleolar extrusion, as

described by Bretschneider and Raven (1951), was not ob-

served in the nucleus of the growing oocyte of Ferrissia

rivularis.

(4) Ovulation

Most full-grown oocytes are round in cross section

(Fig. 95). There is a narrow cleft between the oocyte and

the very thin follicle cells. Ovulation may take place by

autolysis of follicle cells. An ovulated oocyte is oval and

is 55 x 45 µmin size (Fig. 38). The germinal vesicle of an ovulated oocyte has an indentation as Reverberi (1967) described in ~ytilus egg.

(5) Insemination

Very late spermatids with their host Sertoli cell often appear in the lumen of the upper oogc~netic ,.:,one at the vicinity cf the full-grown oocytes (Fig. 98). If

spermiation takes place at the time when full-grown oocytes move into the lumEm through the autolyzed follicle cells, it

is plausible to concluae that self-fertilization takes place

in the lumen of the oogenetic zone. Only one instance was observed where an egg v1as fertilized iE the a.cinus lumen of

the ooqenetic zone (Fig. 38B). There are also two possibilities th~t eggs may be fertilized by the animal's own sperm in the coMnon atrium and in the carrefour. 80

Fi g . 38 . A ferti li zed egg i n the ac i nus l ume n. n. Gerrn i na. l ves i cle and amph.i.nuc l eolus . B. Same egg wi th two sperm (off the a r row ) i n the cytop l asm . 1300 X. 81

However, so far there is no direct evidence to support this assumption.

(6) Oocyte maturation

The maturation of the primary and secondary oocytes will be discussed in the section of embryonic development.

Electron Microscope Observations

1. Ovotestis and common atrium ultrastructure

Electron micrographs reveal Ancel's layer to be composed of three layers of flat connective tissue cells with oblong nuclei and small ar.-iounts of mitochondria. A very thin noncellular basement lamina separates Ancel's layer fror:t the gerrninal epithelium.

The structu~e of the atrial portion of the ovotestis is slightly different from that of the upper generative portion, rese:t: 1 bling the latter in all details oxecpt for the gerrninaJ. epithelial development. A thicker layer of secretory epithelium constitutes the inner portion of the wall of the cor:m10n atrium. This layer is composed of ciliated epithelial cells. 11icrovilli are seen on the inner surface of thes0 cells. 1'1itochondria, an endoplasmic reticulum, zyn~gen granules, and basal bodies of cilia are distinctly seen in the c7toplasm. A distinct intcrcellular space is seen between the cells. Desmosomes are also seen in the space close to the upper surface of the cells

(Fiq. 39B). 'i'he outer p0rtio~1 of the wall is a thin layer of connective tissue v1hich is a continuous membranous 82 extension of Ancel's layer from the generative portion of the ovotestis. There is also a thin basal lamina between

Ancel's layer and the inner ciliated epithelium.

2. Hermaphroditic duct and seminal vesicle ultrastructure

The hermaphroditic duct and the seminal vesicle are essentially the same structurally. The latter is formed by evaginations of the hermaphroditic duct. Electron micrographs show that the thin wall of the seminal vesicle is constructed of two cellular layers and one noncellular intermediate l.ayer, the basement lamina. The inner layer of the wall is a single layer of flat ciliated epithelial cells with comparatively large elongated nuclei and microvilli on the internal surface. The outer layer of the wall is relatively thi~ about one quarter of the thickness of the inner layer, and is made of a single layer of fibrous connective tissue cells. Collagen fibers are clearly visible (Figs. 40 and 49).

3. Ultrastructure of sperm tails

(1) Tajls of developing spermatids

In the light microscope, tails of developing spermatids appnE:r as bcl t·-like structures ,.,_,ith a few cytoplasmic

J-x~ads but td thout spiraJ cy toplas:mic keels, as seen in the mature spermatozoa. However, the e]ectron micrographs of the transverse sections through the middle sec-tion of these devclopinc; spcnnatids o.:-~hibit a strnr.turc resembiinq that of Fig. 39A. Transverse section through the upper portion common atrium. BB, Basal body; C, cilium; EP, cross section of tail end-piece; M, mitochondria; MP, cross section through middle-piece; Nv, microvillus; TT, cross section through tail tip. 20000 X.

Fig. 39B. Transverse section of the ciliated epithelial cells of the lm-1er portion of the cormnon atrium. BB, Basal body; C, ciJ. iu:ri.; D, desmosorae; Mv, rnicrovilli. 22000 X. 84 85

Fig. 40. Transverse section of the wall of the sem in al vesicle. N, nucleus ; C, cilia; Mv, microvillus; BL, basal la mina ; CT, connect ive tissue; CF , collagen fiber; Arrow indicates four primary helices . 6000 X. 86

Helix (Anderson and Personne, 1967; Favard and Andre, 1970).

The tail if surrounded by a double membrane, however, it is difficult to distinguish at this stage. A thin cytoplasmic layer with degenerated mitochondria is seen between the outermost plasma membrane and the developing mitochondrial complex. No endoplasmic reticulum and other common cell components, except degerating mitochondria, are observed in the cytoplasm (Fig. 41). Inside the cytoplasmic layer the metamorphosing mitochondria are externally wrapped by a microtubular manchette layer running vertically throughout the entire middle-piece of the tail (Fig. 41). The pattern of the manchette is similar to that of Helix (Anderson and

Personne, 1967). Spaces between microtubules are approximately the same as the diameter of the tubule. A metamorphosing mitochondrial complex and a coarse concentric lamellar structure, which envelopes the axoneme, are located immediately inside the manchette. In the developing mitochondrial complex there are three to eight intramitochondrial compartments which entirely lack glycogen granules in younger sperrnatids (Fig. 42), and contain only a few glycogen granules in older spermatids (Figs. 41 and 43).

Most intramitochondrial compartments are round in shape, only few larger ones are oblong or crescent-shaped. These compartm::mts are called "primary helix" (major helix), because they wind spirally along the axoneme. A "secondary helix'', found in mature sperm is absent in spermatid tails.

The central core of the flogellar tail consists of an Fig. 41. Cross sections through the middle-piece of advanced spermatids. Ax, axoneme; Cy, cytoplasm; DM, degenerating mitochondrion; f1S, developing mitochondrial sheath; Mt, microtubul8; PH, primary helix. 68000 X. 88 89 axoneme which is constructed of nine thick peripheral fibrils and two small central fibrils, as described by

Anderson and Personne (1970) in Lyrnnaea, Planorbis, and

Helix. The structure of the nine peripheral fibrils, rather than nine fibril pairs, contrasts with the condition in cilia and flagella. As cross sections of these fibrils are observed, each is found to be visibly divided into two parts: an electron-dense external half and a proximal, lighter inner half (Fig. 41). Glycogen granules are also sparsely distributed in the spaces between the central fibrils and the peripheral fibrils.

There are no intrarnitochondrial compartments in the mitochondriul sheath of the tail end-piece. A large amount of cytoplasm with degenerated mitochondria surrounds the mitochondrial sheath and the axoneme (Fig. 44).

Transverse sections of the free end of a spermatid tail disclose the presence of a sinall terminal cytoplasmic globule which lacks devel.oping mitochondria, axoneme, or ER.

This suggests that the cytoplasmic remnants may slough off through th2 tail (Fig. 44, CR), instead of head, as suggested by Joosse and Reitz (1969) during spermiogenesis

(2) Tails of mature spermatozoa

The structure of the mature sperm tail is slightly different f rorn that of developing spermatids. 'I'he tail shows spiral cytoplasmic keels winding along the central filament when observed with a light microscope. Cross- Fig. 42. Transverse sections through the middle-piece of young spermatids showing non-glycogen contained compartments (PH). Ax, axonerne; Cy, cytoplasm; Mt, microtubule; MS, mitochondrial sheath; Pl, plasma me~brane. 30000 X.

Fig. 43. Cross sections through the middle-piece of advanced sperm2_tids showing variable· numbers of primary helices. 34000 X. 91

Fig. 42

Fig. 43. ,/ 92 and longitudinal-sections through the middle section of the tail show that the tail is enclosed by a distinct double- membrane. A wide, electron transparent space is present between this outer double membrane and an inner mitochondrial sheath. The mitochondrial sheath consists of fine concentric paracystalline membranes derived from mitochondria. In contrast to the condition in spermatids, there are no microtubules on the peripheral surface of the sheath. With- in the sh2ath various numbers of glycogen compartments

(two to four), bounded by paracrystalline membranes

(mitochondrial derivatives), are visible in cross and longitudinal sections (Figs. 45 and 46). Each compartment contains dense fine glycogen granules. During spermiogenesis, glycogen is continuously synthesized and accumu1ated in the mitochondrial compartments. The secondary helix is not very distinct in this limpet, compared to the condition in Iymnaea (Favard and Andre, 1970). The secondary helices, shown in cross sections in Fig. 47, appear as a short chain consisting of four or more electron- dense vesicles united in the peripheral region of the mitochondrial sheath. No glycogen granules are seen in this small compartment. The small compartments contain a matrix consisting of Kreb' s cycle enzyrnes (Favard and Andre, 197 0) .

The axoneme of the mature sperm tail is slightly different from that of the spermatid, displaying in the former electron uniformity in the peripheral nine fibrils. Glycogen granules are distributed in the spaces between the Fig. 44. Developing spermatid tail. MC, developing mitochondrial complex; CR, cytoplasmic remnant; GC, Golgi complex. 18000 X.

Fig. 45. Cross sections through middle-piece (MP), and end-piece (EP) of mature sperm tails. PH, primary helixi Pl, plasmd membrane; SH, secondary helix. 40000 X. Fig . 44

Fig. 45 Fig. 46. Longitudinal sections through the middle-piece of mature spermatozoa. Pl, p~Lasma membrane; PM, paracry2talline me:::rtbranes of mi.tochond:cial derivatives. 37000 x.

Fig. 47. Cross sections through the middle-piece of mature spermatozoa. FIT, primary helix; SH, secondary helix; Ax, axoneme; MS, mitochondrial sheath; Pl, plasma membrane. 40000 X. 96

Fig. 4 6

Fig. 47 97 peripheral nine and central two fibrils (Fig. 46). as described in Lymnaea by Anderson and Personne (1970).

Transverse and longitudinal sections illustrate that the mature sperm tail end-piece is enveloped by a double plasma membrane outside, and has a small space between the plasma membrane and the thin mitochondrial sheath, but lacks glycogen compartments and secondary helices. A few glycogen granules are seen in axoneme spaces (Figs. 45 and 48).

The tail tips of mature sperm include only a thin layer of cytoplasm and axoneme without a mitochondrial sheath surrounding it (Fig. 39A).

4. Ultrastructure of oocytes and surrounding follicle cells

(1) Previtellogenetic oocyte

The young oocyte is characterized by having a large germinal vesicle with one or two nucleoli, a "mitochondrial cloud'' situated at the end where the cell lies against the

Ancel's layer (Fig. 49), and one membranous "vit.elline or yolk nucleus" (Rebhun, 1956) near the oocyte nucleus

(Fig. 50). The entire oocyte is surrounded by follicle cells, except at the basal end (Fig. 50). Two oocytes may share a single follicle cell between them (Fig. 51). The oocyte and follicle cells are bridged by processes arising from the oocyte and the follicle cells. The contact sur·- faces of the two processes arc considerably narrow and lack typiczil dosnosome features. Contact E:,urfnces may be termed

"electrica1 couplins-r," a:=; sugqest'-~d by lrnderson (pc,rsonal Fig. 48. Longitudinal section through the end-piece of the tail of a mature spermatozoon. Ax, axoneme; MS, mitochondrial sheath; PTT, primary helix; Pl, plasma membrane. 20000 X.

Fig. 49. Edge of the "mitochondrial cloud" in the cytoplasm of a previtelloge.:1etic oocyte of r. rivulari 13. Bl, basal lamina; CTC, connective tisi:me cell of Ancel I s layer; CF, collagene fiber; FC, follicle cell; M, mitochondria; N, nucleus, NM, nuclear membrane. 62000 X. 99 100 communication).

Numerous vacuole-like intercellular spaces, which vary in size and frequently show electron transparency and are located between the electrical couplings. These spaces may be designated "oocytic-follicular spaces." Neither pinocytes nor interdig~ations were observed in these spaces on oocyte and follicle cell surfaces (Fig. 52). There is no space between the oocyte and the connective tissue cells of the Ancel's layer at the basal end of the oocyte, except a thin basal (basement) lamina (Figs. 49 and 50).

In the previtellogenetic egg of the limpet, mitochondria are not evenly distributed in the cytoplasm, but are rather concentrated at the basal end of the cell as a

"mitochondrial cloud.'' Raven (1961) has suggested this end may be the future vegetal pole. Mitochondria in the oocyte can be distinguished from that in follicle cells by having small cisternae. However, the mitochondria seen in follicle cells always possess large cisternae. Later the

"mitochondrial cloud'' scatters into the cytoplasm, probably just before vitellogenesis.

The vitclline or yolk nucleus, mentioned in the previous paragraph, is situated close to the oocyte nucleus and "mitochondrial cloud" and is composed of a continuous concentric laminae which is continuous with a simple smooth endoplasmic reticulum (Fig-. 50) . The laminae, unlike those of Mytilu~ oocytes (Reverberi, 1967), are widely spaced.

In the vitelline nucleus, small mitochondria are sparsely Fig. 50. Electron micrograph of a previtellogenetic oocyte showing "r.iitochondrial cloud" (MC), vitelline nucleus or Ba.lbiani 1 s body (VN), endoplasmic reticulum (ER), and connective tissue cell of Ancel's layer (CT). BL, basal ~ascmentj lamina; EC, electric coupling; F, fold; FN, nucleus of follicle cell; M, mitochondria; N, nucleus of oocyte; Ncl, nucleolus; O, oocyte, OFS, oocytic- follicular space. 5500 X.

Fig. 51. Electron micrograph of two previtellogenetic oocytcs sharing a single follicle cell. FN, nucleus of follicle cell; M, mitochondria; N, nuclenE; Ncl, nucleolus, OFS, oocytic-follicular space. 14000 X. 102 103

scattered centrally and in the spaces between the laminae.

The mitochondria, except for smallness of size, is similar

to that found in follicle cells. The vitelline nucleus is

commonly seen in oocytes of invertebrates and has been

suggested to be responsible for yolk synthesis. Functional

details of the vitelline nucleus will be discussed in a

subsequent section.

A large round germinal vesicle is located in the

center of the oocyte and has slight electron transparency.

The nuclear membrane, as usual, is double; but a nuclear

pore is absent. In the young oocyte one electron opaque

nucleolus is four.d. Two nucleoli may be observed in older

previtellogenetic oocytes, one being massive and the other

being ring-shaped (Fig. 53).

A young oocyte may be surrounded by two or three

follicle cells, as described in the oogenesis section.

Follicle cells possess long irregular nuclei with dense,

fine granules, a11 undeveloped ER system, a few scattered mitochondria with wide distinct cisternae, and a vitelline

nucleus. Vesicular intercellular spaces and electric

couplings are also observed between the follicle cells.

Follicl8 cells are connected with oocytes by intercellular

bridges (electric couplings) which are the contacts of the

oocytic processes and follicular processes. The role of

follicle cells is unknown. A discussion of this will

follow. Fig. 52. Electrmmicrograph of a previtellogenetic oocyte surface with its adjoining follicle cells. EC, electric couplinq; ER, endoplasmic reticulum; FN, nucleus of follicle cell; Ncl, nucleolus; OFS, oocyLic-follicular space; PFC, process of follicle ceJ.l; PO, process of oocyte; \~J, vitelline nucleus. 10000 X.

Fig. 53. Electrmmicrograph of a part of a young oocyte showing two types of nucleoli and dispersed mitochondria. 6000 x. 105 106

(2) Vitellogenetic oocyte and follicle cells

Vitellogenesis occurs at the growth phase of oogenesis, therefore, a large amount of yolk is synthesized at this time. Abundant polymorphic mitochondria, Golgi complexes, large phagocytic vacuoles, unidentified bodies, and microvilli characterize the vitellogenetic oocyte

(Figs .. 54 and 55).

At this stage, vacuole-like, oocytic-follicular vesicles disappear, and a wide intercellular space appears.

In some cells very few contacts may be seen. Contacting surfaces are comparatively narrow and do not appear to have desmosome-like structures.

Numerous short microvilli are seen on the surface of the oocyte. A thin layer of perioocytic material, extending between the tips of microvilli and thought to be secreted by the egg, is termed the vitelline membrane

(Fig. 54).

Cell structure of the oocyte at the vitellogenetic stage is much more complicated t~an in the previtellogenetic oocyte. Polymorphic mitochondria and yolk granules occur in much larger amounts and have a more uniform distribution at the vitellogenetic staae. The form and size of mitochondria varies. Most mitochondria are elongate, peanut-like, and aumb-bell-shaped; a few are round, oval, or oblong, and have a large eccentrically situated vesicle.

Cistcrnae are distinct in all mitochondria and are very similar to those observed in follicle cells. A large Fig. 54. Electron micrograph of a vitellogenetic oocyte showing vitelline membrane (VM), microvilli (Mv), and yolk granules (YG). FC, follicle cell; ER, endoplasmic reticulum; GC, Golgi complex; M, mitochondria; Pl, plasma.lemma of oocyte; PVS, peri vi tel line space; arrow indicates large cisternal space in mitochondria. 12000 X.

Fig. 55. Electron nicrogrnph depicts the two types of yolk granules and unidentified bodies. The arrow points to a cross section of mature sperm, middle-piece. 7600 X. 108

Fig. 55 109 number of mitochondria is located in the cortical region of the oocyte close to the plasma membrane. Divisions of mitochondria by elongation at the two ends and constriction in the middle are observable in the cortical region of the oocyte (Fig. 54).

Golgi complexes, a few in number and composed of closely spaced membranes (Figs. 54 and 57), are seen in the peripheral cytoplasm of the oocyte. Yolk precursor granules, as described in the Limnaea oocyte by Recourt (Bluemink,

1969), are not seen in the Golgi system of the limpet.

Two types of yolk granules are found in the oocyte, one with smaller electron-dense granules without rod-like yolk platelets, and one with larger granules composed of rod-like yolk platelets (Fig. 56). Yolk platelets are electron opaque and are composed of a "band pattern" of yolk crystals (Wischnitzer, 1967). A type of lysosorne is present which resembles a yolk granule bounded by multiple membranes and contains electron-dense, ring-like structures similar to virus particles (Fig. 56). The origin and function of these virus-like particles are unknown. Another type of n:ultiple rnernbrane lysosome is also present in the cytoplasm of the oocyte. This lysosome contains membranous

V<'-~sicles of various sizes a.nd lamellae which may be the remnants of digested yolk (Anderson, personal conmmnication).

This typ~ of lysosome is called an autophagic vacuole

( 1.".1.g'L' • ....,t:;"7).

A large germinal vesicle is eccentrically located Fig. 56. High magnification of yolk granules containing electron-dense yolk platelets, and lysosomes containing virus-like particles. Ly, lysosome; YG, yolk granule; YP, yolk platelet. 36000 X.

Fig. 57. Cross section of a vitellogenetic oocyte showing autophagic vacuoles (AV) and yolk 9ranules (YG). GC, Golgi complex; M, mitochondria; VN, vitclline nucleus. 10000 ::. 111

F.. ig ' . 56

Fig. 57 112

in the oocyte and is enveloped by a double nuclear membrane

which lacks nuclear pores. One electron-dense nucleolus

is present in the germinal vesicle (Fig. 58).

One or two layers of comparatively thin follicle

cells surround the oocyte. The two layers are designated

the internal follicular layer and the external follicular

layer. The internal is distinguished from the external foll-

icular layer by having a complicated ER system and numerous

large mitochondria (Figs. 59 and 60). The external folli-

cular layer also appears to be less electron-dense and lacks

an endoplasmic reticulum and mitochondria.

The vitelline nucleus still persists in the cyto-

plasm of the oocyte. Small amounts of mitochondria are

visible in the spaces between the double membranes of this nucleus (Fig. 61).

Embryonic and Larval Development

Developmental stages of F. rivularis are arbitrarily defined by the author in t.erms of a comprehensive descriptive interpretation of the entire developmental process.

Successj.ve stages in development are not sharply defined, nor are detaLls of metamorphosis, for changes are much less discrete, as conpared to most marine gastropods.

Larval stages within the egg of freshwater or terrestrial gastropods have been termed trochophore and veliger by Lankestor (1874), Baily (1939), Raven (1946),

DeWitt (1954), Morrill (19G3), and IIess (1971), or trocho- Fig. 58. Electron micrograph of a germinal vesicle, and nucleolus (Ncl) of an oocyte during vitellogenesis. 5000 X.

Fig. 59. Electron ~icrograph showing internal and external follicular layers (IFL, EFL). 8500 X. 114

Fig. 59 Fig. 60. Electron micrograph of a follicle cell showing the ER system and mitochondria. M, mitochondria; Mv, microvilli; O, oocyte. 22000 X.

Fig. 61. Electron micrograph of a vitellogenetic oocyte showing protion of vitelline nucleus (VI1). 10000 X. 116

Fig. 61 117 phore and post-trochophore by Lowrance (1934). Regardless of the terminology, it is possible to recognize two stages within the eggs of land and freshwater snails corresponding to some extent to free-living larvae.

The following stages are recognized by this author during observations on the development of F. rivularis.

Stage I Early Segmentation

Stage II Blastula

Stage III Gastrula

Stage IV Trochophore (or trochosphere)

Stage V Veliger

Stage VI Post--veliger

Stage VII Miniature or juvenile limpet

I. EarJ.y Segmentation

1. Size of ovum

The size of ripe egg cells of freshwater gastropods has been reported by various authors as follows: Paludina,

18 µm; Limnae st2_g_nalis, 120 µm; 1,\chatin~ fulica, 300 11m

(Ghose, 1962), J::'.l.\t~9yrin~, 13·-150 Jjffi (DeWitt., 1954);

Stagnicola kinqi, 9 0--130 inn (Lmvrance ,- 19 3 4) ; Macrochlarnys indica, 100 µm (Chose, 1960); and ~.:L!-hynia :t:_entaculata_,

170 µm (he£,s, 1971).

Oocytes of Fcrrissi_~ :~'b_vu~~:ar_J~,in the ovotestis and the common atrium v1ere measured before germinc1l vesicle breakdown. The maximal diameter of an oocyte in the ovotesti s was 9 6 )lin. Oocytes in thE; comrnon atrium 'dere of 118

various sizes ranging from 83 to 128 µmin maximal diameter, with an average of 102 µm. The diameter of the germinal

vesicle of the atrial egg cell was about 42 µm. The

nucleolus of an oocyte was measured as 12 µm. Four oocytes without germinal vesicles were found in a carrefour. The

size of those egg cells was 72 x 69 µm. Spawned egg cells

are spherical after "rounding up" and the average size is

97 µmin diameter.

2. Primary oocyte maturation

Very likely the primary oocyte is fertilized in the

lumen of the ovotestis or in the carrefour by the animal's own sperm. Po1yspermy was observed in the lumen of an acinus (Fig. 38B). The germinal vesicle.probably does not break down until the fertilized egg reaches the carrefour,

since four oocytes without germinal vesicles were found in a carrefour chamber.

A great amount of time was spent on observations of oocyte change in the co1.11rr,onatrium to attempt to locate the

sites of fertilization and breakdown of germinal vesicles.

Most occytes re;11aj_n2cl unaltered .i.n ::he a triurn for quite a

few hours after ovulation (Fig. 93). In only one case did the oocyte divi.de into two extremely unegual cells, and

subseguently the smaller one disintegrated and disappeared.

Germinal vesicles of only a few oocytes were broken down after the oocytes had escaped from the ovotestis through the rupturing of the ovotestis or hermaphroditic duct ,... . ( 1:' J.q. 6 4) • Such brC'akdown may be due to mechanical 119 stimulation, rather than sperm stimulation.

The first maturation most likely does not occur until the egg cell has been laid. The egg cell is very irregular in shape immediately after being deposited. How- ever, the polarity of the egg cell is distinctly visible.

The animal pole is clear and less granular than the vegetal pole which is granular and dark because of the presence of large amounts of yolk. The first polar body, at the animal pole, is always already formed before egg cells "round off".

It takes about ten minutes for an egg cell to change from an irregular to a round shape. The second polar body is extruded during this process (Fig. 65B). The first polar body is completely separated from the egg, but remains attached to the egg cell by resting upon the second polar body. The second polar body is situated in a small space at the animal pole and is separated from the first polar body by the vitelline membrane (Fig. 66F). This space may be considered the 11perivitelline space" as seen in marine eggs. The first and second polar bodies never divide and re,main attached to the ovum until the time when the gastrula emerges from the vi.telline membrane. The discarded vitelline membrane with its attached polar bodies remains unchanged in the capsule fluid for a long period of time. The fj_rst and second polar bodies are almost equal in ~_;ize, however, the second is slightly larger than the first before the first cleavage takes pl.ace. They were neasured respectively as 15 and

12 µmin diameter. Later, at the eight c2ll stage, the size 120

Post- ES Bl Ga Troch Miniature lim et

1000

CJ .,.. 900 E 800 C:

0 700 >-. A.. ·e 60 I) 500 -0 I) M ., 400 300 0>"' ..C GI > 200 ct 100

2 3 4 5 6 7 8 9 10 \1 12 13 14 Age of embryo in days Fig. 62. Grm,1th of embryos and larvae of F. rivularis. ES, early segmentation; Bl, Elastula; Gi, Gastrula; Troch, trochophore.

Blastoporc

glc,nd

Fig. 63. Young trochophore, the fourth day of development. 121

Fig. 64. Germinal vesicle (GV) breakdown in one primary oocyte (arrow), probably due to mechanical stimulat ion. 400 X. Fig. 65. Freshly laid egg showing 11 rounding off" and maturation division. A. Irregular-shaped egg with extrusion of the first polar body (off the arrow), B. Beginning of "roundir.g off," C. Few minutes later, D. "Roundinq off'' completed, E. 25 minutes later, F. Two minutes later, G. 97 minutes later, H. The first cleavage starts. 300 X. 123

Fig . 6 5 124

Table 11. Embryonic and larval growth of F •. rivularis at the temperature of 21 ± 1°c. (Studied in July, August, September, 1970)

Age Average maximal No. of specimens Stage (days) diameter ( µm) measured

Early cleavage 1 97 40

Blastula 2 105 56

Gastrula 3 129 67

Trochophore 4 179 65 5 242 55

Veliger 6 346 54 7 459 53

Post-veliger 8 567 49 9 688 46

Miniature (juvenile) limpet 10 688 shell length 45 11 818 44 12 910 45 13 973 45 14 1044 27 125 difference becomes pronounced, when the second polar body turns more vacuolar and clear and becomes twice as large as the first polar body which condenses (Figs. 70A and 71F).

Chromatin granules and nuclear material are visible in the first polar body. After the completion of "rounding off" and formation of polar bodies, the diameter at the animal- vegetal axis of a mature egg cell is slightly shorter than that at the equator. The egg cell measured 93 x 96 µm.

3. Changes of egg cell volume before the first cleavage

Egg cell volume changed slightly prior to the first cleavage in F. rivularis (Table 12). In one case the egg cell volume showed an increase of 9.7 per cent in 80 minutes, from the time of "rounding off" until the first cleavage. Raven (1945) observed that in one case the egg cell volume in Limnaea increased 36 per cent in about four hours from the time of egg laying until one hour before the first cleavage. Clement (1938) found that an average increase in egg cell volume of Physa !1eterostropha was 29 to

38 per c2nt.

Apparently, the swelling of egg cell volume is brought about by an absorption of substances, especially water, from the surrounding fluid. Clement (1938) has connected it with the fo:rmc:ttion of vacuoles in the egg cytoplasm by a swelling of coarse unpigemented granules.

The physiology of such absorption is unknown. 126 Table 12. Changes of the volume of egg cell prior to the first cleavage.

Time Volume (PM) Size ( µm) ( ]Jffi 3) Events

6:47 Freshly laid, irregular; 1st polar already formed; 2nd polar body formed two minutes later; started "rounding off".

6:54 Animal pole slightly flat.

7:00 93 X 96 445686

7:10 93 X 96 445686

7:20 93 X 99 473976

7:30 95 X 98 474438

7:40 93 X 99 473976

7:50 96 "'_,., 99 489266

8:00 96 X 99 489266

8:10 96 X 99 489266

8:20 96 X 99 489266 Animal pole surface started flattening, cell elongated at equatorial plane.

8:30 87 X 7':i 254476 1st cleavage completed. One of 87 X 7'!. 226437 the b-vo blastomeres was slightly larger and may be considered the CD cell, aJ.though the polar lob0 is not fanned in this species. Polar bodies attached on the animal pole end of the larger CD cell.

V 8:35 81 ./> 78 2S6258 Blastorneres started expnnjing ~, Q 78 X I '--' 246768 at the equatorial plane; contact surface increased.

8: I'.'.0 ·18 X 78 246768 75 X 78 237276

------....-----·---~····~-_....--_.---- ·----- 127

4. Segmentation

As usual, the first and second cleavages are meridional and equal, and the third cleavage is equatorial and heteroblastic.

(1) The first cleavage

At approximately ninety-seven minutes following the completion of "rounding off," the first cleavage is initiated by a flattening of the egg surface at the animal pole. The flattening process occurs rapidly and is completed in a few seconds (Fig. 66A). Abruptly following this, a broad furrow or depression appears slightly beyond the polar bodies (Fig. 66B), encircling the egg in a plane of the animal-vegetal axis and finally pinches apart into two spherical and almost equal blastorneres. The vegetal pole starts to invaginate as soon as a distinct depression is formed at the animal pole (Fig. 66B). The first cleavage is completed in an average time of 9.5 minutes. The two daughter cells are oval irnrn2d.iate1y aft.er constriction is completed. Soon the cells become flattened on their contiguous sides by pulling toward each other, and the contact surface increases progressively at the same time (Figs. 66D,

E, F, and G). When first observed, the contact surface of the two blastornerer:: is 48 µFL. Eleven rrrinutcs later, it incrcaEJCS to 60 i.1rn, o.nd 29 minutes later, the contiguous side extends to 90 )lm (Fig. 6GJI). Finally, th2 two blastomeres become almost hemispherical in shape about 60 minutes after the constriction (Fig. 67A). During this Pig. 66. First cleavage. A. Cleavage furrow starts to form at animal pole, B. Vegetal pole starts to in_vaginate, C. T\>70 JJlastomeres are formed, D. T,~.ro blastomeres begin to round off and to pull against each other. The first and second polar bodies are attached to one of the two blastomeres (n, E, v). Vitelline membrane (off the arrow) is seen in F. 1 PB, first polar body; 2 PB, second polar body. 300 X 129

Fig. 6 6. Fig. 67. Early cleavage. Ai B, and C. Formation of cleavage cavity beti:1een tv10 blastomeres. D. Second cleavage starting at the animal pole of one of the two blastomeres. E. Seven minutes later. F. Five minutes later. G. Second cleavage completed. H. Large central cleavage cavity (CC). 300 X. 131

Fi g. 67 132

time, a temporary cleavage cavity appears between the two

blastomeres (Figs. 67B and C) and increases in size until

just before the next cleavage, when it suddenly disappears by contraction of the two blastomeres. The maximal breadth

of the lenticular cleavage cavity measured 18 µm (Fig. 67C).

Similar cavities have been reported at a corresponding stage by Holmes (1900) in Planorbis, Kofoid (1895) in Agriolimax,

Wierzejski (1905) in Physa, Lowrance (1934) in Stagnicola,

Comandon and Fonbrune (1935) in Ancylus lacustr_is. The phenomenon of formation and disappearance of the cleavage cavity has been extensively described in various species of

freshwater gastropods by Comandon and Fonbrune (1935).

(2) The second cleavage

Two different cleavage patterns have been observed in the second division of Ferrissia rivularis.

The size of the two blastomeres changes slightly before the second cleavage begins (Table 12). The two polar bodies may attach to either the larger or the smaller cell. The second cleavage begins about two hours and 33 minutes after completion of the first cleavage. Normally the t1,,o cells do not divid<~ &t the same l.ime. Division of one eel] always occurs a Ehort time before that of the other, but cleavage is norrnally never completed before division begins in the undivided cell. In only one case did one of the tv10 blastomeres remain nnchanged for a short period of t.imEo (about six rnj nutes). Uu1ike the usual manner previously described, its partner cell was completely 133

divided into new cells (Figs. 68D and E).

As the second division starts, one cell, probably

the AB blastomere, flattens first at the animal pole sur-

face (Fig. 67D) and then starts to invaginate. About seven minutes later, as the furrow reaches about 120°, the CD

cell starts to flatten (Fig. 67E). Five minutes later,

when the division of the AB cell is almost complete, the

cleavage furrow of CD orients to about 120° (Fig. 67F).

The entire second cleavage process takes approximately 19 minutes. The result of the second division is four cells

nearly equal in size, two of which, Band D, come in contact

bE!low, in the ventral cross furrow. 'l'he other two, A and C, meet in a cross furrow at the upper pole, which is nearly

at right angles to the lower one (Figs. 67G and 68H).

The four cells "round off" after division like the

cells produced by the first cleavage and later become almost

spherical. A small slit-like cleavage cavity is formed near

the center, bstwcen A and Band C and D blastomeres. Fin-

aJ_ly, the two srnnll cavi ti,:!S coalesce and form a large

central cleavage cavity filled with fluid secreted by the

secretory cones of the four blastomeres (Raven, 19~6). The

entire embryo is now very clo~,e to being spherical (Fig.

6 7JI) . 'l1 he diamei.~ers of t:he cavity and the embryo are m0asured respectively as 60 and 102 µm. Nineteen minutes

later, the embryo decreased in size to 96 x 88 µm, because of disappearance of the cleavage cavity. Fig. 68. Second cleavage, showing different cleavage patterns. A. Two almost equal blastomeres, B. One of the two blastomeres starts to flatten at animal pole, C. One minute later, D. Two minutes later, E. Six minutes later, F. 14 minutes later, G. Five minutes later, II. Second cleavage completed, cross furrow appears. 300 X. 135

G Fig . 68 136

(3) The third cleavage

The four-cell embryo with a large, almost round cleavage cavity is measured as 102 x 102 µm (Fig. 67H).

The cleavage cavity decreases to a hyaline slit before the next cleavage takes place, and the embryo consequently reduces its size,measuring 96 x 99 µm. Kofoid (1895) and

Raven (1946) claim that the cleavage cavity may serve as an excretory organ to gather and get rid of metabolic wastes by the contraction of the embryo. This phenomenon resembles the excretory function of the protozoan contractile vacuole.

The cross-furrow (6r polar-furrow) reappears before the commencement of the third cleavage.

The third cleavage in Ferrissia rivularis is dexio- tropic and starts about four hours after the completion of the second cleavage and about 50 minutes after the disappearance of the cleavage cavity. The cleavage plane is not quite equatorial, but is situated slightly nearer the animal pole, thus eight blastorneres of two different sizes are produced. The four cells at the animal pole have a smaller size and form the first quartette of micromeres.

The other four at the vegetal pole arc larger and constitute the macromeres. ~fuen cleavage starts, the four blastorncres do not divide ~d.multaneously. Instead, one

(probably A) of the two cells contacting the animal pole divides first, then the opposite one (C c0ll) (Fig. 69B).

About eight minutes after J\. cell starl·s cleaving, B cell comrnences i b, c1:tvision, and D cell st.arts to divide one Fig. 69. Third cleavage. A. Cleavage cavity disappears and cross furrow reappears, B. The third cleavage starts, C. Two minutes later, D. Two minutes later, E. Four minutes later, F. Third cleavage completed, G. 'l1 hree 1ninutes later, n. Five minutes later, large cleavage cabity appears. CC, cleavage cavity; Mi, micromere; Ma, macror:tere. 300 X. 138 rr -~-~

F i g . 69 139 minute later (Figs. 69D, E, and F). The entire cleavage time is about ten minutes. In the meantime, the cleavage cavity reappears and gradually increases in size, until cz1·;,,-. •'/i:111,./f~ large cavity is formed (Figs. 69G and 70A). The micromeres rotate slowly, ultimately taking their position on the furrows between and alternating in position with the macro- rneres. However, the micromeres do not lie exactly midway between the macromeres, but slightly nearer to the cells next to them in a clockwise direction. This is different from Holmes' observation on Ancylus rivularis (1899), which is synonomous with Ferrissia shimekii (Basch, 1959). The cleavage cavity does not disappear before the next cleavage.

The fourth cleavage starts with the division of the four macromeres. Observations on cleavage were only made up to the twelve-cell stage (Fig. 70). A large cleavage cavity also appears slightly nearer the animal pole in the twelve- cell embryo. The cross furrow is clearly seen from the animal pole at the sixteen-cell stage, but a cleavage cavity is not observed at this stage (Fig. 70F). The sixteen-cell embryo appeRrs about two hours and thirty minutes after the completion of the third cleavage. Further observations on subsequent stages of cleavage were not made, because of the yolk obscurity in the egg cell. Ghose (1962) observed -that the cleavage cavity disappeared before the fourth cleavage in Achatina fulica. Because of the high yolk content of the egg cell, no attempt was made to study cell lineage of the embryo. Fig. 70. Fourth cleavage and blastula. A. Lateral view of the eight-cell stage showing a lar~e cleavage cavity (CC), first and second polar bodies (PB), micromeres (Mi) and rnacromeres (Ma}, B. Fourth cleavage :beginning wi tb T'.1.acror.,ere di vision, pointed out with an ar=ow, C. Four minutes later, D. Nine minutes later, 12-cell stage, E. Lateral view of a 12- cell embryo, F. Polar view of 16-cell stage with distinct cross furrow, G. and H. Young blastula with large blastocoel (Bl). 141

Fig . 70 142

II. Blastula

The blastula is formed within 24 hours. The young blastula is nearly round in shape. The vegetal hemisphere is slightly wider than the animal hemisphere. A large blastocoel, smaller animal pole blastomeres, and large vegetal cells characterize the blastula. The blastula and blastocoel are measured respectively as 99 x 105 µm and 51 x

66 µm. Yolk granules are concentrated on the internal sides of the blastoderm, while the peripheral region of the blastoderm is less granular and more hyaline. Two large yolk-rich cells, stomoblasts, are seen at the vegetal end of the embryo. The internal halves of these two cells are heavily loaded with yolk, the other halves are less yolky and clear. At this time a vitelline membrane may be clearly seen between the two stomoblasts (Fig. 71A). The two cells complete their division in eleven minutes (Fig. 71B).

Approximately twenty-seven minutes later, the blastocoel disappears and the coeloblastula turns into a stereoblastula with a blastopore-like structure at the vegetal pole (now a depressed pit). About an hour and forty-five minutes later, the embryo rotates upwa:::-d about ninct.y de9re,-::s; consequently, the vegetal pole faces upward. Two stomoblasts and the blastopore primordiu.rn are f;c.c.:n at. this time from the vegetal poJ.e (Fig. 71C). The late blostula is ellipsoidal in shape.

Its equator is longer than the animal-vegetal (A-V) axis, ana measure::; 113 x 83 J1m. Tlwre arc numerous small vesicles on the periphery of the embryo. Prccedins:i gastrula 143

formation the cleavage cavity (blastocoel) disappears. In

its place a number of intercellular spaces appear between

the cells at the animal pole. Some of these may coalesce to become large vesicles. Occasionally the contents are extruded as a thick fluid in the surrounding albumen medium.

The vegetal half always appears very dark, possibly due to large amounts of yolk in the endodermal cells. Polar bodies, at this time, still remain attached to the animal pole. The second polar body is clearer and about twice as large as the first.

III. Gastrula

Gastrulae are formed on the third day of development.

The gastrulation process was not observed, however, it is probably accomplished first. by migration of J.arge albumen filled endoderm cells, called "albumen cells" by some authors, into the blastocoel from the veget.al end and by the invagination of smaller ectoderm cells. The embryo is equipped with cilia before hatching free of the vitelline membrane. 'I'he cilia cannot be seen using a light microscope. The hatching mechanism is unknown, but it is very likely due to physical rather than chemical rupturing since the discard.ed vi tel line membrane is complete. The gastrula discards its vitelline membrane and attached polar bodies and starU, rotating clocl:-,d_sc or anticlockwise after rnnerging. At this time, wide blastoporic depression and blastopore are visible at the broader vegetal pole. At the opposite anirnnl pole, there are always two large Fig. 71. Early developmental stages. A. Young blastula with stomoblasts (Sb), B. Young blastula, C. BlastuJa with stomoblasts (Sb) and blastopore prirnosdium {Bp), D. Gastrula outside of the vitelline membrane (VM) ,. E. Castrula showing blastoporic depression (20) and shell gland primordium (SGP), F. Discarded vitelline membrane, G. Young trochophore, note the larval. liver (LL). 300 X. 145

Fig. 71 146 intercellular vacuoles situated on either side of the shell gland primordium. Like protozoan contractile vacuoles, these vacuoles extrude their contents when they reach a certain size and then reappear later. Minute vesicles, which are seen in the ectoderm cells at the animal pole, may be albumen vacuoles formed by uptake of albumen fluid from the surrounding medium via pinocytosis, as suggested by

Raven (1946) and proved by Elbers and Bluemink (1960).

A pit-like depression appears eccentrically on the vegetal surface, and from this a broad shallow furrow extends across the center of the surface to the periphery.

Concomittantly, with the appearance cf the blastoporic depression, the embryo takes on a markedly bilateral symmetry. Cells constituting the ectodermal wall at the animal pole are uniform in size and are relatively small when compared with those of the vegetal pole. The late gastrula rotates vigorously. At this time, the larval liver primordium (''albumen cells") starts to appear

(Fig. 71D) in the interior portion of the vegetal region near the blastopore. These cells increase in size and become more vacuolar as the embryo grows older. The shell gland prirnodrium is situated on the side of the embryo opposite the side with the blastopore. The gland is formed at the contact site of ectodcrm with the archenteron (Raven,

1966; Hess, 1971). The full-grown gastru]a is about 117 x

75 µmin size. 147

IV. Trochophore

Young trochophores appear on the fourth day of the development. They measure about 167 µmin diameter. A young trochophore is characterized by, the presence of distinctly clear, and large endoderm cells (albumen cells) or larval liver cells, about thirteen in nunber, situated in the central portion of the larva (Figs. 71G and 77).

In general, the larva at this time is approximately spherical in shape. In one instance, the larval liver cell mass measured 128 µmin diameter. The peripheral region of the trochophore is covered by small ectodermal cells in which minute vesicles are frequently seen. In the young trochophore the blastoporic depression becomes shallow and broad and a dorsal blastoporic trough is barely seen

(Fig. 77). Cells of the shell gland primordium begin to assume a columnar form, and a ciliated prototroch forms at the periphery of the vegetal pole (Pig. 63).

On the fifth day, the trochophore starts to elongate antero-posteriorly (Figs. 72A and 75A). The larval liver becomes large, lobe-like, and translucent in appearance, apparently due to coalesc~nce of albumen cells and intensive uptake of albuminous material surrounding the larva. The p~~nordium of the buccal roass starts to appear in the middle of the anterior end, and the shell gland thickens, becoming more distinct at the posterior end of the trochophore. Ciliated velar lobes begin to appear on the right and left sides of the stomodeal oper1ing. Nuchal cells 148 are not seen at this time. One embryo measured 255 x 210 µm in size.

V. Veliger

The veliger of F. rivularis is well defined on the sixth day following egg deposition. The larva becomes more elongate antero-posteriorly, and the major body divisions become apparent. The anterior end becomes slightly wider and more blunt than the posterior end. The posterior end is rounded and smooth. At this time the stomodaeum differentiates into a mouth and buccal mass complete with a protrusile radula. The radula protrudes and withdraws slightly to suck in nutritive albumen from the surrounding medium (Morrill, 1963, 1964). A flat circular foot without developed muscles is well developed on the ventral-anterior half of the body. Two small ciliated velar lobes are now clearly visible on both sides of the mouth (Figs. 72B and

75B). There are two protuberances formed on the sides on the nuchal region slightly behind the mouth which later become the tentacles (Fig. 72B). The prototroch still persists. It is located behind the regions which develop tentacles. Numerous nuchal cells are visible in the neck region between the buccal bulb and the larval liver.

The ultimate fate of these cells is unknown; however, they disappear at or before the post-veliger stage. Secretion by the shell gland starts at this time, consequently a small almost transparent, conchiolin shell is formed on the top of Uw gl2rnd. Later the sh0ll gl,md migrates ~-rradually 149 upward to the dorsal surface of the embryo and is finally established on top of the visceral mass as the larva grows and develops. Muscles are somewhat developed at this stage, since the larva may undergo contorsions by contraction. Large albumen cells now are more concentrated between the head region and shell gland. The configuration of the larval liver now looks more like an actual digestive gland.

Up to this stage, the larva moves by ciliary rotation rather than muscular movement, even though a foot is fully formed. Apparently the foot, at this time, does not serve as a locomotory organ in the capsule fluid, and locomotion of the embryo is mainly by ciliary motion.

On the seventh day, the veliger becomes even more elongate and measures about 540 µmin length. The head region is now more differentiated than before. The buccal mass and radula are fully developed by this stage. Tentacle buds or fields become more prominent as two lobes on either side of the head. These lobes are located slightly behind the v0lar lobes (Figs. 72C and 75C). Eyes begin to appear at this time. As a larva enlarges, its ra

On top of the larval liver, a fairly distinct rounded. and partially calcified conchiolin shell is formed. 150

The shell is very elastic. This plasticity is obvious when the body strongly contracts. On the left side of the larva there appears a short rod-like kidney primordium of meso- dermal origin. According to Meisenheimer (1899), the protonephridium of ~ncylus originates from ecotoderm. All other authors (e.g. Rabl, 1879; Holmes, 1900; Wierzejski,

1905) agree that the basommatophoran protonephridium is derived from the mesoderm. This primordium develops into a curved, loop-like kidney at a later stage of development.

Anterior to the kidney primordium a heart primordium is visible. The heart primordium starts to exhibit faint contraction at this time. Two ciliated unidentified structures, resembling "flame cells" are present in the neck region on the sides of the esophagus. Strong beating action of the cilia is clearly visible, particularly in the hydropic veliger (Fig. 84A).

Osphradia have been described in marine primitive gastropods and in freshwater mussels and limpets. The function of osphradium in the latter animals is uncertain.

These ciliated structures could be osphradia or salivary duct,;. In spite of their resemblance to salivary ducts of ancylids, it is more likely true that the questionable structures are osphradia, due to their structure and position. 'rhe osphradj urn appears in larval stages and disappears in the 2.dult limpet. 'l'his has been reported in some t.erre::;trial pulmonates by t:c.ome authors. At this stage the internal mernbranc \,1hicll confines the c~Jq within the 151

F'ig. 72. 7'.• rive·--c1ay i..:rochophore. B. Six· ..day veliger. C. Scvcn--day voligQr. mr, buccal 1nass prirnordirnn; LL, larval liver; Pt, prototroch; SG, shell gland; TB, tentacle bud; Sh, shell; St, stomodaeurn; Mo, mouth; Ve, VQlun. 152 capsule still remains intact.

VI. Post-veliger

A distinct oval shell is formed on top of the post- veliger on the eighth day. The shell does not cover the entire animal but leaves the cephalic and nuchal regions uncovered. The larva in general outline resembles a turtle

(Fig. 73A). At this stage, the larva breaks through the internal membrane, probably by enzymatic action and escapes into the larger cavity of the egg capsule. In those egg capsules containing several eggs that develop normally, all of the larvae escape from their respective eggs on the eighth or ninth day and continue their development in the common chamber of the capsule (Fig. 9C). In some two-egg capsules, particularly those containing two unequal eggs, one of the two eggs, more frequently the smaller one, may not develop normally or is arrested ir. development at an early stage. In this event, the internal membrane of the arrested egg may remain intact for a period of time until the other larva grows to its full size and forces it to break. Clapp {1921) claimed that the internal membrane of

Ancylus_ !'..1:3E~'?uswas ru?tured by constant physical prodding of the larva and by rasping actions of the radula. This is not true j_n the case O .c"'j_ .... rivularis other than for the comparable rasping action of radul2. The rasping action in the latter is employed nhlinly, if not solely, to suck in albumen nutrients from the egg capsule.

The "turtle·-like larva" now po~;scsse:,, all the udult 153

------~------~---

Fig . 73. Post-vel i gcrs . A. Eight-dny post-vcJiger. B. Nine-day post - v eligcr . 300 X. 154

features, except for comparable size. There is a notch or shallow indentation between the two velar lobes.

Slightly behind the velar lobes short, broad tentacles

are developed. One red eye with a transparent lens in the

center appears at the base of the anterior end of each

tentacle. A brown, well-developed buccal bulb, in which a

radula and other associated structures are formed, is situated in the middle of the head. Nuchal cells are still present in the neck region. The digestive gland now develops into fewer but larger lobes and frequently develops a hyaline, bubbly texture. The kidney is identical to that

in adults. The heart primordium differentiates into one auricle and one ventricle. The heart starts to pulsate distinctly at an average of forty-seven times per minute.

The lowest rate observed was seventeen per minute. At the

same time, a large foot is present, and a short wide flap- like pseudobranch (Fig. 74B) is visible i11 the right mantle cavity. The post-veliger at this time may twist or turn by muscle contraction, or rotate by ciliary action.

On the ninth day, the post-veliger grows larger with little change in external morpholcgy (Fig. 73B). Tentacles become pointed distally and wider proximally. The head region remains uncovered by the shell. Radiating the shell striae are distinctly visible. The activity of the radula and buccal bulb continues. 155

VII. Miniature (juvenile) limpets

The miniature or juvenile limpet stage is well defined on the tenth day (Fig. 74). Normally this stage lasts in the capsule five days before hatching. Occasionally the young individuals may remain in the capsule for several days and finally hatch or die. This stage is characterized by having an elliptical shell covering the entire soft parts of the animal, including the head. Currents created by cilia in the mantle cavities are seen to flow from anterior to posterior. Fecal pellets, dispelled by the young, are always seen with the discarded internal membranes on the side of the capsule chamber. In a few cases, shed radular teeth were observed in the capsule chambers which were deposited by heat stimulation in the winter of 1970. The fluid which fills the capsule chamber at this time is probably just water and dissolved met&bolic wastes, since cilia operate more freely in this less viscous medium.

Young limpets continue to grow without any morphological changes for the next three or four days. Just before hatching, the growing limpets nearly fill the capsule.

Ordinarily they hatch on the fourteenth day from the time the egg is laid. Only in a few instances do the young remain inside the capsule for a fm1 more days. Some failed to hatch anct finally died inside the capsule for unknown reasons. This was observed more often in the eggs which were laid by artificial stinulation. Perhaps there is a correlation of mortality with available food reserves in the 156

Fig . 74. Young limpet , 1 0-days old . A. Dorsal vi ew ; B. ventra l view. B~ , bucc a l mass; DG, d i gestive g l and ; E , eye; EO, excretory organ; H, heart; Ps , pscudobranch ; Sh , shell; T, tentacle. 300 X. 157 ·

Fig. 75. Late trochophore and veligers. A. Five- d ay trochophorc , B. Six-day veliger showing shell gland (SG) , C. vel i ger , s ligh tly older than seven days . 250 x. 158 Table 13. Chronology of the development of F. rivularis at temperatures of 21 ± 1° C.

Time Developmental events

0 minute Freshly laid ovum, irregular in shape, with 1st polar body, without germinal vesicle; starts rounding off.

10 min "Rounding off" completes; 2nd polar body forms; polarity becomes distinct.

1 hr 37 min 1st cleavage starts.

1 hr 47 min Completion of 1st cleavage.

4 hr 36 min Completion of 2nd cleavage.

8 hr 30 min Completion of 3rd cleavage.

9 hr 50 min Formation of 12-cell embryo.

10 hr Formation of 16-cell embryo.

2 days Flastula; gastrulation barely starts.

3 days Gastrula hatches out; vitelline membrane is discarded; blastopore and archenteron are formed; formation of shell gland rudiment; rotation starts.

4 days Young trochophore, larg-e albumen cells or larval liver becoroe distinct; shell gland develops.

5 days Late trochophore; antero-posterj_or differentiation roughly starts.

6 days Young veliger, body elong2tes; larval liver, buccal mass rudi.ment, stomodaeum and shell gland become pronounced; conchiolin she:11 is secretccl; nuchal cclls appear.

7 days Late ve1iger, radula start:3 intaking capsule fluid; kidney and heart rudiment appear;: tE,ntacle bud,:; are f:ormr2d; conchjo1jn shell is partially calcified.

(etc.) 159

Table 13. (continued)

Time Developmental events

8 days Post-veliger, possesses of adult features except small shell and head and neck are exposed; heart starts pulsating.

9 days Late post-veliger; increase of size without important morphological alternation.

10 days Miniature limpet; soft part of body is fully covered by shell.

11 days Miniature limpet; soft part of body is fully covered by shell.

12 days Miniature limpet; soft part of body is fully covered by shell.

13 days Miniature limpet; soft part of body is fully covered by shell.

14 days Hatching from the egg capsule. 160

, •

Fig. 76 . A hatched egg capsul e showin g top lid , bottom floo r , and operculat e suture. 300 X.

Fi g . 77. Four - day trochophore show i ng shel l g l and (SG), larv al liver (LL) ., and stom odaeum (St). 500 x. 161 egg capsule. As the shell of a young limpet grows larger and higher, more pressure is put on the top lid of the capsule. Eventually the lid splits away from the floor along the operculate suture at a certain point. At this moment young limpets crawl into the new environment through the opening, abandoning the capsule. The breaking of the top li~ from the floor of the capsule is shown in Fig. 76.

Clapp (1921) mentioned that through constant prodding by the rasping action of the radula, an opening was made on the capsule through which the young escaped.

DeWitt (1954) observed that young snails of Physa gyrina escaped from the egg by first rasping through the internal and then through the external membrane. Per- foration of the membranes was aided by the snail periodically thrusting its foot against the wall. He also noted that the size of 1:_. _gyrina at hc1tchi:ng depended largely upon the supply of albuminous material available.

Abnormal development frequently occurred in very small eggs which were called "dwa:".'.'f eggs" by Bondesen (1950).

The°' function of the albumen fluid will be elaborated on in the discussion section.

Shell DGvelopment

The shell gland pr~nordiuill of r ■ rivularis first appears at th8 end (animal pole) opposite the blastopore of the gastrula on the third day of development. It is formed by a shallow invagination of ectoderm contacting the 162 distal end of the archenteron (Fig. 71E).

In the young trochophore, about four-day old, the shell gland appears as a shallow depressed columnar epithelial tissue at the posterior end of the embryo, opposite the stomodaeum (Fig. 77). There is no indication that the shell. gland is active in secretion at this time.

On the sixth day of development, an oblong conchiolin shell is secreted on the columnar epithelium (Fig. 78A) of the shell gland. The shell is transparent, shows no evidence of calcification, and has a few faint concentric lines around the center (Fig. 78B). The size of the shell is about 36 x 24 µmat this stage.

The overall body length of a seven-day old veliger was measured as 450 µm; its round shell was 248 x 248 µm.

The central apex and the peripheral rim of the shell did not show any calcification. Only the portion between the apex and the rim showed the appearance of finely dispersed cal- cium carbonate cr1stals. A drop of 0.5 per cent hydro- chloric acid solution dissolved all the calcareous spots, but not the cuticuJar periostracum. This suggests that the larval sholl may start to become calcified after the sixth day of development.. Jl"nothcr seven-day old veliger measured

435 pm in overall body len(~rth and 255 x 255 µm in shell size.

The structure of tl1is shell was the same as that previously described.

One veligcr which was slight,ly younger than seven

Fig. 78. Shell (Sh) and associ ated shell gland (SG) . A. Six-day veliger showing shell gland and conchiolin she ll. B. Conchiolin shell. 400 X.

Fig . 79. Shell from an eight-day post-velige r showing its calcifica~ion pattern. 400 X. 164 length. The shell, once separated from.the body and analyzed, showed no calcification. This confirmi the conclusion that calcification of the larval shell does not occur until the seventh day of the development.

As development progresses, the shell gland starts to shift over to a dorsal position and finally becomes situated on the dorsal surface of the larva on the eighth day. In the meantime, the mantle rudiment is formed by a thickened edge of the shell gland. Subsequently, shell growth is controlled by secretion of the mantle membrane. In other words, as the mantle grows, the shell increases in size, under normal conditions.

One eight-day post-veliger was 503 µmin overall body length, the maximum diameter of shell was 290 µm. The shell of this larva showed three distinct regions, the thickest being a calcified central portion. Next to this, a thinner, less calcified portion is probably formed mainly by the secretion of mantle; and the thinnest constitutes a non-calcified rim (Fig. 79).

Another eight-day veliger was studied which measured

450 µmin body length, and the shell measured 278 x 255 µm.

The shell of this veliger was uniformly calcified up to the periostracal rim. The shell begins to exhibit radiating lines on the eighth day of development.

One ninc-"day old post--veliger was decapsulated, and its overc 1 ll body length wa:.., measured as 563 pm. Its shell size measured 413 x 330 µm. Only the apex of the shell was 165 unevenly calcified, showing scattered thickened spots. The· periostracal rim was about 23 µmin width.

The shell continues to grow by the secretion of the mantle edge following the ninth day of development. Later in development, the shell starts to show distinct concentric growth lines. The average shell size of the animal immediately after hatching is 1044 µmin length. Clapp

(1921) claimed that in Ancylus fuscus no well-defined growth lines (concentric sculpture), nor radiating lines were observed in the 12 or 13 day old animals which were ready to leave the egg capsule.

Adult shells have three distinct layers. The top layer is a thin periostracum on which diatoms grow. The inner, calcareous layer is composed of two sub-layers, the

"ostracurn" and "hypostracum." An oblique line continues with the pcriostracum always growing obliquely across the ostracum forming what appears -to be grov,th lines on the shell (Fig. 92).

Ovotestis Post-embryonic Devclopm2nt

A comparative study on the ovotc,stis of different ages of limpets demonstrates that development and differentL,,t:i.on 0£ gou&cls is dircct:J.y related to tll8 age

(shell size) of the anirna.1. Diff0rcnti,1tion of the germinal epi tJwliurn probably ccmmences :=;hc)r tly a.fter hatching, particularly sperVi development. Spe~m~togenesis is very active at this tir:1e. Spcrmil.ticls and dividing SI>errnatognia ure 166

present in the young ovotestis; whereas, the egg cells still

remain in the young oogonium stage. No distinct oogenetic

zone can be seen until later. The previtellogenic oocytes

start to appear as the limpet grows to 2.3 mm in shell

length. As limpets grow older, more oocytes (vitellogenic

and mature) and less oogonia, are produced; and mature

spermatozoa start to appear in the lumen of the acini and

in the seminal vesicle. The relation between age and the

number and size of developing egg cells is shown in Table

14. Gonad development in each age group is described and

discussed below.

1. The youngest limpets studied ranged from 1.3 to

1.8 mm in shell length, averaging 1.5 mm. The ovotestis

measured 83 x 60 µm and consisted of three to four acini

(follicles). In each acinus various phases of gametogenesis were observed.

Spermatogenesis was observed with spermatogonia at

11 leptonema II and "bouquet II stages of prophase and ffiE';taphase

either free in the lurnen or attached tc ·che wall of ovotestis.

It ended with the intermediate spernwtid stage with short

cytoplasmic taiJ.s and da.rk PNA (post-nuclear apparatus).

Young spermatids lacking tails were also seen in the

acini. However, no sperrnatocytes and mature spermatozoa

were observed at this age.

Oogenesis was observed at the oogonium stage only.

A total number of six oogonia ·was recorded in one ovoter,tis. . 2 '11 hc cell :Lnclex of oosonia ran9ec1 from 49 to 135 µm 167 2 (106 µm average). One older oogoniurn with a large nucleus and acidophilous nucleolus was 15 x 10 µrn in size; the nucleus was 8 X 7 µrn and its nucleolus 3 X 3 µm. Some of the nucleoli were weakly basophilous.

2. The ovotestis of the limpets, ranging from

2.3 to 2.7 mm in shell length, were studied. The size of the gonad was 165 x 81 mm, and contained five acini. All the developmental stages of sperrnatogenesis were seen in the ovotestis at this stage. Free and rounded spermatogonia, a few at the leptonema stage, were seen in the lumen. A few younger spermatogonia still remained attached to the acinus wall. Both primary and secondary spermatocytes were present in the lumen close to the wall of the acini. A few of the secondary sperrnatocytes were at metaphase and anaphase. Young spermatids were short and gathered around a large Sertoli cell; they became elongated and finally developed into older spermatids with short cytoplasmic tails and PNA at the head end. Bundles of late spermatias ready to spermiate were seen in the lumen of a.cini and in the corn.ffton atrium.

As growth proceedPd, large amoeboid primary oocytes began to appear with follicle cells surrounding them. The primary oocytes had large germinal vesicles and distinct acidophilous or basophilous nucleoli. The cell index of 2 these oocytes ranged from 2GO to 420 vm , averaging 2 3 -75 JJrn . 'l'lie total nu;rber of primary oocytes was rdx, and oogonia were counted as three. The largest oocyte found 168 measured 29 x 18 µm, and its germinal vesicle was 15 x 9 µm.

The basophilous nucleolus measured 6 x 6 µm. No distinct zonation appeared between oogenesis and spermatogenesis

(Fig. 81) .

3. Limpet shells which were studied ranged from

2.6 to 2.8 mm (2.7 average). The size of one ovotestis was

110 x 90 mm. The development of the ovotestis and gametogenesis were very similar to group number 2 since the limpet age (reflected by shell size) was almost the same.

Various stages of spermatogenesis which were described above were present in the gonad of this group. But there were more bundles of spermatids than in group number

Three oogonia and eight primary oocytes were found in the upper portion of one ovotestis. Oogonial index 2 ranged from 63 to 171 µm. Oocytes varied from 20 x 13 to

35 x 16 µmin size. All oocytes possessed a large germinal vesicle a.nd a.n acidophilous or basophilous nucleolus which was 3 x 3 to 5 x 5 1tm in size.

4. This group of limpets contained the largest animals used in the present study. The average shell length was 4.1 mm. The ovotestis size of one specjmen was 338 x

263 µm and contained four acini. The ovotestis of this group was characterized by having more numerous mature oocytes, spermatozoa, and distinct zones of spermatogenesis and OO9en 0 r; is .

In one case 20 oocytes of various sizes and three 169

Fig . 80. Young oocytes and follicle cells. 1300 x.

Fig . 81. Longitudinal section of a young ovotestis lacking distinct zonatio n betwee n oogencsis and spermatogenesis . ,. , Ta:)J.e 14. Relation between shell size (age) and the number and size of developing eggs.

f,helJ.. A·?e~.:::-..sc No. of Oogonial Average No. of Oocyte Average le:1.gt.h s>:cll Ovotesti~-:; oogcnia inc.ex oogonial oocytes index oocyte Batch range le;;c,th s.:ize in ran;1e index in ran~e index nu::fber (m:n) (:cul'.) (µm) ovotestis (µm") (µm2) ovotestis (µm ) (µm2)

1 1. 3-1. 8 l.5 83 X 60 6 135 - 49 106 0 0 0

2 2.3-2.7 2.5 155 X 81 3 204 - 120 133 6 420-260 375

3 2.6-2.8 2.7 110 X 90 3 171 - 63 109 8 560-260 351

,1 3.9-4.2 4.1 338 X 263 3 120 - 99 122 20 3822-340 1282

I-' '-3 ·o 171 oogonia were observed; the cell index of oocytes ranging 2 from 340 to 3822 ~m , three times larger than that of the

oocytes in Batch 2 and 3.

Morphogenic Effects of Maleic Hydrazide on the Early Development

1. 1.5% Maleic Hydrazide Effect on Development

The morphological effects of 1.5% maleic hydrazide on Ferrissia eggs and larvae are given in Table 16. Maleic hydrazide at 1.5% dilution retards development and produces different abnormalities of the larva, but produces little

immediate mortality. Out of 132 treated eggs and larvae, only 15 developed normally and hatched. The residuum eventually expired at different stages. Overall developing time was also delayed by maleic hydrazide. In 15 hatched animals, the average hatching time was 19.2 days. In two cases, 26 and 36 days were recorded. Normally, the embryo takes about fourteen days to hatch from the time eggs are

shed. The morphological characteristics of the following malformations will be described in subsequent sections: hydropic and non-hydropic exogastrulae, hydropic trochophore, hydropic veliger, head, and shell. The effects of 1.5% maleic hydrazide at different periods of development is outlined below:

(1) Uncleaved-egg stage

Twenty-two uncleaved eggs at various phases were treat~d and studied. Maleic hydrazido solution (1.5%) caused strong dehydration of the u11cleaved eggs. The total 172 Table 15. Cell volume changes of the egg through the second cleavage stage at 21 ± 1° C during and after treatment with 1.5% maleic hydrazide temperature.

Accumulative time Size Volume (minutes) (µm) (µm 3)

0 93 X 93 418266 .µ 10 90 X 90 379080 i:: (l) 20 87 X 81 296820 b"E: 30 78 X 72 210263 i:: .µ ·r-l co 40 72 X 69 178252 H a, ::SH 50 72 X 69 178252 Q .µ 60 69 X 69 170825

61 69 X 69 170825 73 72 X 72 194089 95 75 X 75 219375 102 81 X 81 276349 133 84 X 81 286584 150 87 X 84 319213 170 87 X 87 342422 .µ 180 90 X 87 345229 s::: (l) 195 1st cleavage f:; .µ started rd 198 1st cleavage com- <11 ~~ pleted .µ 215 78 X 66 176679 H 2:50 75 X 75 219375 (j) .µ 75 X 75 219375 4-! 300 108 X 90j: 454896* ,< 306 105 X 90* 442260* 314 105 X 96* 503194* 336 105 X 96* 503194* 355 2nd clcav2.ge com-· pleted _, -----,~----· - ~,.- ~ -·- -·,.~., -~"'~ --- -:l·Mec"tsurernent of entire two·-cell embryo. T~ble 16. Eftects of l.59e maleic hydrazide on the early development of~- rivularis.

Normal dcv2lo;?~.cn.t ~otal no. to you:--1•; Sta~e of Total J_ir1pet st.:tge Abnormal develo:_:,ment treafed S?eci~ens mortali~y Arrested Exo- Hydropic Hydropic Head Hatch,~d Died blastula gastrula trochophore veliger :nalfor!:'!ation

1-cell 22 12 10 0 0 11 l 1 1

2-cell 22 17 5 3 0 14 0 0 0

4-C·3l.l 27 27 0 2 0 24 0 1 0

, 0 3-ccl.l 18 .Lu 0 0 0 17 0 l l

Blastul~ 19 19 0 0 13 5 0 1 0

Gastrulo. 24 24 0 0 0 24 0 0 0

f-J -..J w 174 volume of the egg cell was greatly reduced after a 60 minute treatment, but the original volume recovered gradually in two hours after being transferred into normal lake water. Egg cell volume changes during and after treatment are given in

Table 15.

Normally eggs start to divide about 97 minutes after being deposited. In treated eggs the first cleavage is delayed about three hours and 15 minutes. Dehydration is probably the main factor delaying the first cleavage. Since cytology of treated eggs was not investigated, cytological effects of maleic hydrazide on the dividing mechanism is unknovm. The cortical region of the two blastomeres resulting from the first cleavage became more hyaline, and the rest of the cell was dark and very granular.

Normal blastulae and gastrulae were developed respectively on the second and the third day, although a few gastrulae were slightly hydropic at the animal pole. Hydrop- ic exogastrulae hecame more apparent on the fourth day.

Various types of hydropic exogastrulae are shown in Fig. 82.

Most exogastrulae disintegrated and died within a few days.

The life span of the exooastrulae varied from eight to twenty-one days. Some of the exogastrulae appeared as a solid mass of cc:11s wi tllout any hyd:copic cavity but were ciliated.

After treatment, some eggs developed nonnally to the gastrula and young trochophore stages. In some of these trochophores a hydropic cavity began to appear abnormally 175 at the original animal end on the fifth day of the development. The resulting hydropic features of the trochophores became more distinct on the sixth day including: a large hydropic cavity filled with clear fluid, digestive tract with relatively small larval liver on the dorsal of the stomodaeum, few small mesoderm cells seen on the side of the hydropic cavity, and a distinct buccal mass rudiment

(Fig. 82D). The larval liver became less distinct and finally disappeared, probably due to metabolic consumption of yolk granules and albumen in the large endcderm cells.

The hydropic trochophore died twenty-three days after its appearance.

In one two-egg capsule, an egg developed into an exogastrula and the other developed to an extra large young embryo, measuring 1296 µmin shellJength. Apparently this embryo was nourished with additional amounts of nutrient from the neighboring egg which was arrested after the gastrula stage.

Cephalic malformation was observed in one embryo.

The radula protruded in one seven-day old veliger and the right tentac]a was slightly longer than the left one on the eleventh day of development.

(2) Two-cell stage

rrwenty-two two·-cell embryos v1ere subjected to exposure of 1.5% maJ.eic hydrazide solution for an hour. Two blastomeres were profoundly dehydrated after an hour exposure to the solution. Changes of volume in the two 176 blastomeres were recorded (Table 17).

Cell volumes were not completely recovered after being transferred in natural13.ke water. Prior to the commencement of the second cleavage, the volumes of the two blastomeres were still slightly smaller than the original volume before treatment. In this experiment the second cleavage was delayed for about 24 minutes. No cleavage cavity was observed between the two cells in the entire process. However, a serrate contact surface between the two cells was observed which might have been the vestigial cleavage cavity.

Among twenty-two treated embryos, nine developed normally up to the gastrula stage; eight of which developed up to post-veliger stage or young limpet, but only five hatched; three failed to hatch and died; one turned into a hydropic exogastrula and died afterwards. The time from egg deposition to hatching ranged from 16 to 26 days, an average of 19.8 days. The entire development time was very much delayed. I\ total of fourteen exogastrulae Cir,bryos were produced. Some of them passed through the normal gastrula stage and became hydropic exogastrulae on the fourth day. Only a few exogastrulae developed directly from normal blastulae on the third day. All exogastrulae died within a few days. One dumb-beJl shaped exogastrula

·wi t.h a distinct: vitelline membrane between the larger animal half and the ev2ginated vegetal half (Fig. 83) failed to hatch from tho vitelljne membran8 and finally died. 177 Table 17. Cell volune changes during and after treatment with 1.5% maleic hydrazide at the two-cell stage.

Accumulative time Size Volume (minutes) ( µm) ( µm 3) .. Events

0 87 X 75 254475 First cleava.ge completed 84 X 72 226437

. 9 81 X 78 256258 Treatment started 78 X 78 246767

39 66 X 60 123552 63 X 57 106437

51 60 X 51 81151 60 X 51 81151

69 57 X 51 77094 Treatment ended 57 X 51 77094

84 63 X 57 106437 15 minutes after being 63 X 57 106437 transferred in lake water

104 72 X 63 148599 69 L'>." 60 129168

114 72 X 66 163089 69 ".,, 62 142408

124 75 X 69 185679 72 X 66 163089

154 78 X 72 210263 78 X 69 193106

174 Second cleavage completed

·--· Fig. 82. Morphology of the normal gastrula and various types of exogastrulae and a hydropic trochophore. A. A normal gastrula. B, C, B, F, G. Exogastrulae. D. Hydropic trochophore. H. Abnormal embryo derived from a gastrula treated TTith MH. Ar, digestive tract. mw, buccal mass princrdium. Bp, mouth (stomodal opening). EC, endodennal cell. LL, larval liver. SGP, shell gland primordium. 179

SGP

D 180

(3} Four-cell stage

A batch of 27 four-cell embryos were treated with

1.5% maleic hydrazide for 60 minutes. Only two embryos developed normally up to the post-veliger stage, but they failed to hatch and died. Twenty-four exogastrulae were produced, and one resembled a ciliated morula embryo. One hydropic veliger was formed. Mortality of this group was

100%.

(4) Eight-cell stage

Only eighteen eight-cell embryos were studied. In this batch, 17 exogastrulae were produced and one hydropic veliger was observed. This malformed veliger was about

488 µmin length and was extremely hydropic. Its abnormal features included: everted radula, a comparatively small larval liver, an abnormally small shell, a stretched dorsal nerve ring across the esophagus, short and lobe-like tentacles, and a small kidney rudiment on the left postero- lateral side of the body (Figs. 84A and B). Mortality was

100%.

(5) Blastula stage

Among nineteen treated blastula embryos, two became hydropic blastulae, five became exogastrulae, and one developed into a hydropic veliger; eleven of the nineteen embryos disint8qrated and died after a one hour treatment

\vi tb. 1. 5% 1Tt2leic hydrazide solution. Hydropic exogastrulae produced hen2 were different from those of the other 181 experiments. After treatment, embryos started to break apart and the major fragment then gradually developed into an exogastrula, probably by re-organization and re- differentiation.

(6) Gastrula stage

Twenty-five gastrula embryos were treated in this experiment. Twenty-two gastrulae gave off small clusters of cells and then developed into 3mall exogastrulae by re- organization and re--differentiation following an hour treatment with 1.5% maleic hydrazide solution. Only two embryos died immediately after treatment, probably because of extreme dehydration. All exogastrulae derived from the fragment of the treated normal gastrulae·possessed a large central cavity filled with fluid and were ciliated. The central cavity disappeared before they died. lJone of them showed any further development beyond the gastrula stage.

2. 0.5% Maleic Hydrazide Effect on Development

The results of the effect of 0.5% maleic hydrazide on developrr1ent are surn:-iarized in Table 18. Data from the control group r,ho,:,1ed undesi.rablo results. Among a total of twenty-five eggs and embryos treated, only eight developed normally and natched; nine died before }1atching; four died in cxogastrulation; tHo died in the hyc 1.ropic trochophore state, and two expired as hydropic veligers. Results suggest that higher temperatures of 25-30° C are dc~trirnental to thE? dc.velopFtc?nt of eggs. Ovar:i.an eggs or freshly laid 182

eggs are very sensitive to heat injury.

The effects of 0.5% maleic hydrazide at different

developmental stages are described as follows:

(1) Uncleaved-egg stage

Twelve uncleaved eggs at different phases were

exposed to 0.5% maleic hydrazide for 60 minutes. One embryo

ceased to grow beyond the eight-cell condition. One embryo developed normally to a young-limpet stage, but failed to hatch and died. Four exogastrulae were produced, three of which were hydropic. The hydropic exogastrulae lived for

18 to 20 days. They died in the saffie manner as those treated with 1.5% maleic hydrazide. One non-hydropic exogastrula

died within eleven days. One egg developed into a normal young limpet, but became aborted on the twenty-·third day.

Two embryos died after becoming hydropic veligers and one after developing into a hydropic trochonhore.

(2) Two-cell stage

Only twelve two-cell embryos were treated with 0.5% maleic hydrazide. None of them developed normally. Eight hydropic exogastruJ.ae were formed and died on the average within 21 days. Three hydropic veljgers and one post- veliger were developed; none of which survived.

(3) Fm1r•acell stasre

Nineteen four-cell stages were treated with 0.5% maleic hydrazide for one hour. Fiftesn hydropic

exogastrulae developed and survived for six to twenty-six 183 days. One embryo's growth was arrested at the blastula stage and one at the six-cell stage. One hydropic veliger was produced. A great number of cells containing yolk granules were found in an egg containing a hydropic exogastrula. It eventually died after the number of cells greatly increased and filled the entire egg capsule on the thirteenth day. Three abnormal types of cells were observed: sickle, ball, and ring-like.

(4) Eight-cell stage

Only ten eight-cell embryos were treated. Seven hydropic exogastrulae and one hydropic post-veliger were developed. Two died during early cleavage stages. One of the latter two had eight blastomeres which lined up into two rows, four micromeres on top and four macromeres below.

A vitelline membrane was not observed. This indicated that

0.5% maleic hydrazide may have dissolved the vitelline membrane which protects and holds the blastomeres in place.

The other that died during early cleavage did so after the fourth cleavage when the sixtee~ blastomcres arranged into an abnormal pztttern (Fig. 86). All the blastorneres tended to be spherical in shupe and thence somewhat separated from one another.

(5) A few embryos wero treated prior to the blastula stage, mainly between the eight and thirty-two cell stagc!s. 'rl1e ::.;,,n1e pheno:rtcnon occurred in these embryos as described above. Blastomeres of the treated embryos , Table 18. Effects of 0.5% maleic hydrazide on the early development of r- rivularis.

Normal developr.,ent to young Total no. limpet stage Abnormal development* of Total Stages specimens mortulity Hydropic Hydropic Hydropic Hydropic post- Hatched Died exogastrula trochophore veligcr veliger Other**

'Cntrc°' ted S:ggs (control) 25 17 8 9 4 2 2 0 0 1-c,;ll (t;-eat:cd) 12 12 0 1 4 1 2 3 a-cell

2-ccll (t:".",2ated) 12 12 0 0 a 0 3 l 0 4-c'211 Blastula & (trea-:.ed) 19 19 0 1 15 0 l 0 6-cell 8-ce:!.l a-cell & (treat0.d) 10 10 0 0 7 0 0 1 16-cell

*l,Iead malfornati(,1n ·das not obse:!.:'ved i::1 this experiment.

**Non-specific malformation. Stages while the embryos were arrested.

I-' co ~ 185

scattered in a very irregular pattern. Half of the cells contained dense yolk granules and half were transparent.

3. Malformations

(1) Exogastrulation

Clapp (1921) was probably the first to describe the exogastrula embryos in freshwater limpets from the natural environment. He found in an egg containing four egg cells that only one developed normally [probably into a veliger], and the other three developed into very transparent embryos which were ciliated and able to rotate in a normal manner.

All transparent embryos broke down within 48 hours after ciliary motion had begun and cell fragments were scattered through the fluid of the capsule.

Exogastrulation seldom occurs in naturally deposited eggs of F. rivularis. However, exogastrula embryos are frequently observed in eggs which have been produced by temperature stimulation. Various exogastrula forms produced by maleic hydrazide treatments have been observed in this species naturally. In general, they may be grouped into two major types, hydropic and non-hydropic.

Embryos deformed at the gastrula stage frequently developed into vesicular exogastrulae with a supressed archenteron invagination (Raven, 1942). They often swelled into hydropic vesicles with a group of endodermal cells visible in the blastopore region (Morrill, 1963). Hydropic exogastrul2Le of the river limpet show a great variation in 186 appearance. Dumbbell, vesicular, and irregular forms are commonly seen in the hydropic type (Fig. 82). Vesicular hydropic exogastrulae are the most common in Ferrissia.

Such a malformed embryo possesses a large hyaline cavity at the animal pole which is sarrounded by a thin layer of ectodermal cells. Very often large or small endodermal cells with dense yolk granules are present in the cavity near the blastopore region of the embryo. Dissociated endodermal cells are always seen gathered at the blastoporic region. They are presumably formed by the everted endoderm cells, if the exogastrula is formed from a treated normal gastrula, or by the failure of invagination, if it is produced prior to the gastrula stage. Dumbbell-shaped exogastrulae occur less frequently. They are composed of two major parts approximately equal in size; the animal half is vesicular, as seen in the vesicular type; the vegetal half is solid and constructed of a large mass of yolk-laden endodermal cells. Sometimes a few small transparent vesicles are seen attached to the outer vegetal surface of the ernbryo .

In one case, a duml.)Lell--shaped exogastrula was formed inside the vitelline membrane (Fig. 83). Both the animal and vegetal halves were solid and spherical. A vitelline membrane was clearly visible between the two spheres. The two spheres ware unequal in size and measured fifty-seven and ei<;:rhty-one microns in diameter. Such an cxogastrula is of the :r.on··hydropic type and is infrequently seen in 1 8 7

Fig . 83. Non-hy

Ferrissia.

All exogastrulae died within a few days. Generally,

hydropic features disappeared, for embryos broke down and

turned into small solid pieces before dying. Frequently

solid fragments survived for a period of time and were able

to revolve by ciliary motion. No further development has

ever been observed in either type of exogastrula.

(2) Hydropic trochophore

A few embryos exposed at early stages to maleic

hydrazide treatment developed on the fifth day into hydropic

trochophores. Trochophores developing from maleic hydrazide-

treated embryos were smaller than the normal five-day

trochophore and measured 188 µm long. A small hydropic

cavity was seen at the animal end, opposite the stomodaeum.

Hydropic features appeared on the sixth day with

the appearance of a large transparent hydropic cavity

surrounding the entire digestive system (Fig. 96). The size of the larva was greatly increased due to the expansion of

the hydropic cavity. One six-day old trochophore was measured as 278 µmin length. It increased its size to a maximum of 390 µm on the tenth day, probably simpJ.y by

taking up water or fluid from the surrounding medium. The most conspicuous features of a six-day hydropic trocho-

phore included: a visible digestive tract, a few pro-

jections arisinq from the stomodeal region and attaching posteriorly to the body wall, a di.stinct buccal mass rudiment, an indented stomodeal opening or blastopore, and 189

a lack of shell and nuchal cells. Trochophores having

received maleic hydrazide treatment usually lived for about

18 days without undergoing further differentiation.

(3) Hydropic veliger

Early embryos treated with maleic hydrazide that

developed to the veliger or post-veliger stages often

showed various degrees of hydropia. Embryos which were

affected at early veliger stages frequently developed into

strongly hydropic veligers, and thos affected at late veliger stage conmtonly developed into moderately hydropic

embryos. A strongly hydropic veliger resembles a normal veliger in general appearance having an antero-posteriorly

elongated body with head differentiation and a digestive

tract includin9 a stomodeal opening, buccal mass or radular

sac, esophagus, intestine rudiment, larval liver and kidney

rudiments, and larval shell and shell gland. In addition

to the above described structures, a strongly hydropic

veliger has a large fluid-filled body cavity which distorts,

allowing some of the internal organs to be distinctly visible (Fig. 97). Concomitant with body cavity enlarge- ment, the larval liver is reduced to a small mass of

endodermal cells s:Ltuated 2t the posterior end of the body;

the shE,11 is small and limi teed to the area of the shell

gland prirnordium; a kidney pri:nordiurn is displciced at the

poste~ior side of the embryo; and nuchal cells are

scattered in the neck regi.on. The most importan~ feature

of tbis eir.bryo is the c~vertod radula. Later this structure 190 may entirely protrude out of the mouth (Fig. 87). Maleic hydrazide-treated embryos may develop further into hydropic post-veligers which have a very vesicular head and neck, poorly developed tentacles, a small rounded shell, a small larval liver, a distorted kidney, and a deformed heart on the left side.

· A moderately hydropic veliger frequently develops into a post-veliger stage. Such a larva shows hydropic features only in the cephalic and nuchal regions (Fig. 85).

All hydropic veligers and post-veligers lived for seventeen to twenty-one days without hatching. Frequently the shell of hydropic post-veligers became detached from the mantle before death (Fig. 88).

(4) Head malformations

Head malformations independent of hydropia may develop at the veliger stage. The most common and important head malformation is the protrusion of the radula. In young veligers the radular mass (odontophore) is everted as a small bud near the mouth. As the larva grows, the radula increases in size and completely protrudes from the mouth.

Radular teeth are often seen separated from the radula in subsequent development.

In one case, a triophthalmic post-veliger was developed (Fig. 99). Two eyes were developed at the base of the right tentacle, and one on the left. Finally the two ris:rht eyes fused into a large single ey(;. 'rhe right tentacle was slightly larqer tlEt.n the left and was short and 191

Fig. 84. Hydropic veliger. A. Dorsal view, B. Lateral view, BM, buccal mass; CG, cerebral ganglion; DG: digestive gland; EO,· excretory organ; F, foot; H. heart; In, intestine; NC, nuchal cells; Oe, esophagus; OG, optic ganglion; S, stomach; Sh, shell.

Fig. 85. Hydropic post-veJ.iger. T. tentacle. Other stnl_Ctt:re leqends refer to Fig. 84. 192

Fig . 86. Separation of blasto mer es . 550 X.

Fig. 87. Hydropic veliger with an everted radula. Ra , radula; RT, radular teeth. 300 X. 193

Fig. 88. Deformed limpet larva showing shell mal formation. Sh , shell . 150 X.

Fig. 89. A Sertoli cell with attached spermatids. sec , Sertoli cell; CR, cytoplasmic remnant . 400 x. 194 blunt at the tip, but wider than the normal left tentacle.

4. Direct mortality

Direct mortality immediately after maleic hydrazide treatment was only shown in embryos which were treated with

0.5% maleic hydrazide solution prior to the blastula stage or were treated with 1.5% maleic hydrazide at the blastula stage. In embryos treated with 0.5% maleic hydrazide before the blastula stage, especially at the eight-cell stage, blastom2res tended to be spherical and to separate from one 3 another (Fig. 86). Some macromeres showed that yolk granules were condensed at one end of the cells, and the other end was hyaline.

Maleic Hydrazide and Fecundity

Maleic hydrazide at 1.5% and 0.5% concentrations completely killed the animals aftGr a 12 hour treatment.

Non-fatal concentrations (< 0.25%) of maleic hydrazide produced inl1ibition of egg production in this species. Egg production tended to decrease after the middle of August in the natural environment. Results obt2ined from the treated groups indicated the same phf'~nomenon, that is, egg pro- auction decreased gradually after August. The results of the

-----··-- ..·-···---- 3 In two ca~-;C::s, a qreat mirc,ber of sntall yolky cells aonear in the egq a few d~vs after the dissociation of bj;storner0s. This conditi~n sugaests that the dissociated cc1ls nay be still alive ,rnd able to multiply rapidly by repeated di vision:::; in 0 short pt:-::riod of time. 195

experiments are summarized in Table 19.

Post-effect of maleic hydrazide on the development of the embryos of those treated animals was observed.

Hydropic trochophores and normal young developed. All hydropic trochophores died in a few days. Some young

limpets were aborted before hatching. Only a few of the young hatched among the treated groups. All embryos of the control groups developed normally and hatched within a normal perj.od of time. 196 Table 19. Effect of maleic hydrazide on the fecundity of F. rivularis at 21 ± 1° c.

Average No. of No. of shell Beginning capsules contained Treatment length (mm) date deposited eggs

Control 2.7 8/7/71 46 91

1.5% MH 2.5 0 0

Control 2.4 8/16/71 25 36

0.5% MH 2.4 0 0

Control* 3.4 8/19/71 19 21

0.25% 3.6 6 9

Control 3.5 8/31/71 14 19

0.25% 3.9 5 8

Control 2.2 9/12/71 4 8

0.1% 2.3 3 3

*Data from two-day period observation. DISCUSSION

General Biology

Taxonomical Problem

There is a taxonomic problem concerning Ferrissia rivularis and F. tarda, and this problem has never been clarified, as far as this author is concerned. Baker (1928) reported that r- rivularis was closely related to F. tarda and many specimens were difficult to classify. In Basch's

"Review of North America Freshwater Lirnpets, 11 (1963), he remarks, "Ferrissia rivularis has been the subject of a great deal of confusion for over a century. In particular, the distinction between rivularis and tarda has never been clear." I have examined s01,1e of the reproductive organs, such as the ovotestis, common atrium, hermaphroditic duct, seminal vesicle, and spermatheca of sixty-one specimens.

My observations show th3t there is a difference in the ovotestis between F. rivularis and t2rda. Hoff never mentioned there ·was a sac located immediately below the ovotcstis, however, there i~; a fairly large and distinct thin-walled sac, which freouently contains oocytes and numerous sperm, situated bc~b-Jeen the ovotestis and the hsrmaphrocUtic duct in I'. rivularis. This st.ruct:.ure has 198 been observed in fresh specimens and histological sections.

Nevertheless, Hoff described the atrium as merely a common chamber into which all the acini or follicles open. This does not correspond to the condition of the common atrium described by me in F. rivularis. In live specimens, ciliary movement was observed on the internal surface of the common atrium.

Paraffin sections of the common atrium failed to reveal the presence of cilia. Electron micrographs of this sac, however, clearly show cilia and microvilli on the internal surface of this sac (Fig. 39A, B). A similar condition is also found in Lymnaea stagnalis (,Joosse and

Reitz, 1969), in Vaginulus borellianus, and in Laevicaulis alte (Quattrini and Lanza, 1965).

If Hoff's observation is correct, a conclusion may be drawn that ------Ferrissia -·------rivularis and F. tarda are two different species, in spite of their s~nilarities in external morpholosy. Another evidence which may solve the identification problem in the future is the function of the sperwatheca. No spermatozoa have ever been observed by me in the spermatheca of F. rivularis. Although Eoff mentioned that copulation mi

Reproduction

Polyvitelline and Empty Eggs

The occurrence of polyvitel}ine and empty eggs in 199

freshwater and terrestrial pulmonates has been reported by a few authors. Crabb and Crabb (1927) found that the production of polyvitelline eggs in pond snails was not hereditary, and there was a tendency for polyvitelline eggs to be included in egg masses laid either near the beginning or end of the period of oviposition. The two authors also

found that egg masses with a small number of eggs usually were polyvitelline; mortality was high (80%) in these eggs.

Crabb (1931) found one egg of Physa gyrina which contained as many as 25 egg cells and there was a distinct tendency that snails producing the largest numbers of polyvitelline eggs also laid the largest number of eggs that lacked egg cells. Winsor and Winsor (1932) concluded that the production of polyvitelline in Lymnaea colurnella was influenced by heredity.

Carrick (1939) observed that in the gray field slug

(Agriolinax -~:9res tis) , that as many as thirty unfertilized egg cells result.2d from "budding" of a single egg cell, all were present in a single egg. Carrick also reported that mul ti.,·embryonated eggs with two to six egg cells per egg were Laid by a single slug, and multi-embryonated eggs took slightly longer to hatch than those of the same egg mass having fewer f~mbryos. Dc:~\1\iitt {19S4) reported for Ph 2.:_0a gryina that of the 51 individual snails studied, 10 pro-- duced polyvitelline eggs and there was no evidence that polyvitelline was controJled by hereditary factors.

Results show that polyvitclline and empty egg 200 production in the river limpet is extremely inconsistent.

In two years, only three polyvitelline and a few empty eggs have been observed. Each polyvitelline egg contained only two egg cells. It is most conceivable that production of polyvitelline and empty eggs in F. rivularis is controlled by environmental, rather than genetic factors, as suggested by Winsor and Winsor (1932).

Fecundity

This investigation was carried out in the laboratory.

Therefore, certain inherent artificial conclusions seem inevitable, since food supply, loss of limpets from slides, temperature fluctuation, and other environmental factors were unnatural. Water temperature was maintained at 21° C in the laboratory, but in nature water temperature fluctu- ated slightly daily. The data may nevertheless give a general pi.cture of the fecundity and its relationship with size of the ovotestis of Ferrissia limpet.

Ghose (1960) suggested that the number of eggs produced by an individual is dependent upon the amount of available albwnen, instead of upon the size of the ovotestis.

In Lymnc:wa stagnc1lif,. appressr1., the alburnen gland at various times seems to indicate that it may vary in appearance and structure with different physiological states of the animal; during sexual activity, the albumen gland is filled with a thin watery secretion so that the entire organ is quite turgid; at senility or starvati.on, the albumen regresses 201 in size and loses its turgidity (Holm, 1946). According to

Ghose, the snail will produce fewer eggs or none during starvation periods; more eggs will be produced in spite of the snail's size when sufficient food is provided. This does not agree with the results obtained from my limpet experiments. Most of the limpets used in this experiment were starved for a period of time before use. They laid normal egg capsules and eggs without showing abnormality, and egg deposition continued for a period of time. Joosse and his colleagues (1968) reported that Lymnaea snails stopped oviposition under the circumstance of starvation, but gametogenesis continued at a slower rate.

~Toosse and Reitz (1969) reported for Lymnaea stagna]is that the number of gametes produced by each acinus varies with the acinus size. It is likely that the ovotestis size is the main factor controlling the number of eggs produced, although albumen is important in the egg deposition process.

Anatomy and Histology of the Reproductive Organs

'l'hc morpholocJy of the ovotestis in fresh-water gastropods has been reported on by various authors. Draw- in9s of the ovotestir_~ of 1',ncylus f h1vi~ctilis by Lucaze-

Duthiers indicate there are about fifteen, rather distinct, and clearly sepdrated follicles (Basch, 1959). The ovotestis of Ferri~rnia !0£..<2.'2:.is nearly spherical and is drawn- out slightly to~ard the joining duct, and consists of five 202

to seven narrowing acini or follicles (Hoff, 1940). In

Laevapex fuscus the ovotestis is roughly hemispherical,

about one milimeter in diameter; consisting of twenty or

more elongated acini whose long axes lie in a plane perpen-

dicular to the flat surface of the organ (Basch, 1959).

Eight to ten acini in one ovotestis are found in the ovo-

testis of Rhodamea cahawbensis (Basch, 1960). The gonad of

Acroloxus lacustris consists of about ten diverticula or

acini (Hubendick, 1962). The maximum diameter of the ovo-

testis of 3.5 mm long Gundlachia wauterri (.Mirolli) lir.tpets

measured 450 to 500 µm, and the ovotestes consisted of four

to seven acini (Wauti.er, Hernandex, and Richardot, 1966).

Brown (1967) reported that the ovotestis of Ferrissia

burnupi consists of thre2 to five lobes connected to a

common atrium, each lobe being formed of a group of five to

seven acini. In Ferrissia rivularis (Say) four to six

acini have been observed in the ovotestis. I have never

found an ovotestis consisting of more than six acini.

Brown (1967) summarizing Eoff's 1940 pablicat.ion mentioned

the number of acini in the ovotestis of F. rivularis was

five to seven, and Hubendick (1964) reported five to many.

Apparently the number of acini is determined by two main

factors, species and age variations. In F. rivularis the

acinus number varintion is mainly dependent upon the size

or aae of the animal. Larger animals tend to have a larger

ovotestis and a larger number of acini, and they produce more eggs and spe~n1.. 'J'hese c01:1clusions are sum:.rnarized in 203

Tables 3-9 and 10.

Over sixty limpets were personally dissected and observed to gather anatomical data. The common atrium was consistently observed both in histological sections and live materials. This structure has been ignored in importance or neglected by most authors working on fresh-water limpets.

It was described in Ferrissia burnupi by Brown (1967); however, nothing was postulated for its function.

Regular histological sections failed to show the presence of cilia in the common atrium, hermaphroditic duct, and seminal vesicle; however, electron micrographs of these structures show clearly that cilia and microvilli are present on the internal surface. Hubendick is the only author who describ2s the epit~elium of the hermaphroditic duct of Acroloxus lacustris as being ciliated.

The atrium is always fiJ.led with fluid which is secreted by its glandular lining. Very often mature spermatozoa and ovulated oocytcs are ceen in this chamber. Four oocytes have been observed in one atrium. Frequently, late spermatids and spermatocytes are present in the atrium.

Therefore, the atcimn is assumed to serve as a reservoir for temporil.ry retention of sperm and oocyt.c~f3. 'rhis condition may assure the opportunity of self-fertilization. Ciliary motion if::; o'.:)servcd on the inner side of the atrium. Such ciliary movement may function to transport sperm and oocytes from the 21triu,11 to the :1.ermaphrodi tic duct.

The function of the spermatheca in ------Fe::rrissia 204 rivularis is vague. Hoff (1940) assumed it may be used for sperm storage after copulation of F. tarda. Basch (1959) never clarified its function in Laevapes fuscus, but he observed some orange substance in the cavity of spermatheca.

Burky (1971) reported that copulating pairs were observed one week before egg laying in F. rivularis. I have never observed actual copulation in tl1is species; although, behavior similar to copulation with one limpet lying over the other was observed, but the evertcd penis was never found while this behavior was proceeding. If Burky's observations are true, sperm should be seen in the spermatheca. Sperm are not present in the spermatheca (Fig. 11) ! Ghose (1960) stated, "Spermatozoa were found in the spermathecae of several Ma_crochlamys _indica, but only in one case in Achatina fulica. Autofertilization being common in them, the spermatheca has been thrown out function, but it was found well developed in all individuals." Self-fertil.:.zation has been proven and reported in Ferrissia shimekii by Basch (1959).

One fertilized oocyte was observed in the lumen of an acinus in F. rivularis. Evidently self-fertilization is fairly common in this sp,2cies, and it seems plausible that the spermatheca is merely a degenerated organ.

Comparing the oocyte size in the atrium with the diameter of the hen;iaph:coditic duct., the oocyte is about. four to eleven times larger than the inner diameter of the duct. The oocyte, therefore, must elongate and become very slender in order to pass through the narrow duct. This may 205 explain why freshly laid eggs always are very irregular in shape.

Mature limpets of various sizes were dissected or sectioned in August, September, October, and November of

1971, and February, March, and April of 1972. All seminal vesicles were found to be filled with sperm. This indicates that sperm may persist through the winter, and spermatogenesis may be carried out even at low temperatures. From this phenomenon the question arises as to how the oocyte can pass through the seminal vesicle in which sperm are heavily concentrated.

Gametogencsis

1. Germinal epithelium differentiation

Literature on developmental details of the germinal epithelium in pulmonates is conjectural. Ancel (1903, from

Raven's Morphogenesis, 1966) first developed the idea that the differentiation of the germinal epithelial cells into either male or female germ cells is dependent on the presence or abse11ce, respectively, of differentiated nurse cells. This idea was somewhat modified by Buresch (1911), who found that male c:rnd female germ cellc; develop in Helix side by side in the Lame acinus of the ovotestis, but the developing oocytes invariably lie near one or rnore nurse cells. Development in the male or female direction depends on the conditions of nutrition of the indifferent germ cells. 206

However, Bouillon (1956) advanced an idea that

temperature has an effect on sexual differentiation in the

Pulmonata. In the ovotestis of Cepea nemoralis L. Bouillon

found a low temperature is more favorable to development of primary gcnocytes in the female direction; and a high

temperature promotes development in the male direction.

Recently a hypothesis regarding the development of the

_germinal epithelium was established by Joosse and Reitz

(1969). 'rhey proposed that in Lymnaea stagnalis, all sex cells involved in gametogenesis originate from the germinal epithelial ring, and the arrangement of the sex cells in an acinus is determined primarily by the digestive gland; the

latter also determines the shape and size of the vitellogenetic area. The location of the spermatogenetic zone is in turn determined by the vitellogenetic area. Pelluet and

Lane (1961) suggest that in Arion a hormone produced in the tentacles rnay suppress egg production while stimulating

sperm production; and egg production is under the control of

a hormone prodl.-..ceo by the brain.

In the ovotestis of F. rivularis distinct oogenesis and spermato;ienesis zonation is distinguishable in mature limpets, and the entire ovotestis is embedded in the digestive gland. However, the oogenetic zone is more closely

associated with the digestive gland. No distinct oogenetic and spermatogenetic zones can be distinguished in the ova- testis of young limpets. However, late sperrnatids or mature

spermatozoa are developed in the lower portion of the acinus, 207 while the egg cells still remain at the young oogonium stage in the upper portion of the acinus. If the oogenetic zone is determined by the digestive gland, then the oogenetic zone controls the formation of spermatogenetic zone, as proposed by Joosse and Reitz. The formation of egg cells should be earlier than the development of sperm in

~- rivularis, since a functional digestive gland is already well developed after hatching. On the contrary, the development of sperm takes place much earlier than that of eggs in the limpet. It seems that low temperature does not completely inhibit spermatogenesis in the limpet.

Frequently, mature sperm and various phases of spermatogenesis were observed in the ovotestis of limpets which were kept at the water temperature of 11° C for a month or longer. At this temperature, egg cells remained inactive and there was no indication that viteJlogenesis was actively progressing. Therefore, it seems plausible to conclude that the differentiation of the germinal epithelium of F. rivularis is neither entirely dependent on the digestive gland nor on temperature differences. It mc:1y be controlled by age and ur~nown genetic factors. Concerning ru1trition, however, oocytic growth and vitellogenesis may be attributed to the digestive gland. The role of temperature is 1:1ainly one of garnetogcncsis promotion, rather than one of initio.tion of gE~rminal cpi thcliimt cell differentiation. At least it is true in this limpet. 208

2. Sertoli Cell

Hickman (1931) observed that.male germ cells

started to gather around Sertoli cells 'at the primary

spermatocyte stage in Succinea ovalis Say. Pelluet and

Watts (1951) and Watts (1952) also found the same phenomenon

in the ovotestis of slugs. In 1954, Abdel-Malek found that

in Helisoma trivulvis a "basal cell" or "Sertoli cell" was

surrounded by a number of spermatogonia and was able to move

in the acinus. Recently Joosse and Reitz (1969) reported that in Lymnaea stagnalis numerous spermatogonia were ob-

served surrounding a Sertoli cell. There is no indication

showing that the spermatogonia or the primary spermatocytes

surround the Sertoli cell in the ovotestis of F. rivularis.

Instead, the Sertoli cell is surrounded on its free side by a certain number of secondary spermatocytes at early

stages of development. The relationship between Sertoli cells and spermatjds has been studj_ed in Cipangopaludina malleata by Yaauzumi, et al. (1960) using an electron microscope. Electron micrographs reveal that a Sertoli cell gives rise to numerous slender, or broad, elongate pseudopodia whic!1 extend, surround, and finally coalesce to form a mantle around the sperm head. It is suggested that the mantle constitutes a conductor system for nutritional supply from tho Sertoli cell to developing spermatids. In the limpet, as spcrmatids develop, the cytoplasm of the

Sertoli cell dwindles and the cell membrane disappears~ consequently, only a nucleus is seen by the end of the 209 development. This may suggest that Sertoli cells really provide nutrients for spermatocyte development.

3. Vitellogenesis

At the beginning of the period of active oocytic growth, mitochondria granules are concentrated in the basal part of the oocyte where the cell lies against Ancel's layer. Later they increase in number and widely scatter in the ooplasm. It is thought that :mitochondria are probably concerned with chemical synthesis of yolk from raw material provided by the ooplasm. Electron micrographs of young and growing oocytes show clearly the significant relationship of mitochondria with vitellogenesis of the egg. Discussion regarding this will be explained in a later section. Fahmy

(1949) claimed that Golgi rods were related to yolk synthesis in Eremina des2rtorum eggs. Golgi bodies or rods were not encountered in the ooplasm of the limpet egg.

4. The role of follicle cells

Little attention has been paid to the functional morphology of follicle cells in pulmonate g2stropods.

Raven (1963) reportecl. that all larger oocytes of !.:.Y1'.1naea sta<:p1ali_~ llad six cells in the inner follicle layer. In the larger oocytes of ::_. tivul_ar.i~.1 only three or four follj_cle cells have been observed. Raven also found that follicle cells are responsible for determining the cortical structure of th2 egg. The corticaJ mosaic called ''blue- print information'' is responsible for the future 210 developmental pattern of the embryo. In addition to Raven's

idea, follicle cells may actively assist in the growth of oocytes by secreting substances which are taken up by oocytes through diffusion or pinocytosis. The morphological

relation between the oocyte and follicle cells will be discussed in detail in a later section.

5. Spermiation

Gatenhy (1918), Hickman (1931) and Watts (1952)

suggested that in pulmonate gastropods sperm become motile

and lose their contact with the Sertoli cells after the passage of cytoplasmic remnants along the tails of the

spermatids. Merton (1930) observed in Planorbis that during spermiation cytoplasmic globules originating from

Sertoli cells passed along the sperrnatid tails. However,

Barth and Jansen (1960) described the reverse in

Australorbis glab~atus where cytoplasmic globules in the

Sertoli cells were claimed to be cytoplarn~ic remnants of the spenr.atidf"~. Joosse and Reitz (19 6 9) also observed

the same~ phenomenon in Lymnaea staqrialis. I have never observed such structures in Sertoli cells of~- rivulari~,

in fresh spccirr:en~, nor in paraf f i:i sections (Fig, 9 8) . In

fresh specimens I did observe vesicular structures in the·

Scrtoli cells (Fig. 89). These vesicles are too

large to be the cytoplasmic glcbules which Joosse and

Reitz refer to. Cytoplasmic beads are visible along

the entire length of spermatid tails. A particularly 211 large globe of such material is found at the free tip of the tail. In F. rivularis it seems plausible that the classical spermiation idea applies, i.e., spermatids slough off cytoplasmic remnants through the tails instead of the heads.

6. Spermatozoa

The development of the filament in F. rivularis spermatids was not observed but cytoplasmic tails were clearly visible. Development of the filament has been described in spermatids of Helix aspersa (Gatenby, 1918),

Succinea ovalis (Hickman, 1931), Deroceras reticulatum

(Pelluet and Watts, 1951), and Arion ~ub!uscus (Watts, 1952).

Watts described the development of spermatids in Arion subfuscus as follows: "As the tail filament grows, it stretches the cell nembrane, pulling out the cytoplasm into a long, narrow strjp, the mitochondria are moved with the filament. Some becorrie closely associated with the filament, and these renain to form the spiral sheath. The others that are passed dovm the tail are sloughed off in the residual globules. Consequently, the entire tail filament becomes covered by a spiral mitochondria sheath." This spiral sheath is also observed in the middle piece of mature spermatozoa tails in the river liE,pet as a single spiral keel winding clo:~kwise around the cc,ntral filament. The end pic~ce of the tail is similar to a thin-·belt without shm,Jing a spiral feature, and seems to consist of two fine filaments which are connected by a thin cytoplnsm3c film (Fig. 32).

Or.1ing to such unusual configurations, an electron microscope 212 study was made to investigate the ultrastructure of sperm tails. The electron microscope study on the sperm tail is discussed in a separate section.

Two types of sperm have been mentioned in the same pulmonate by a few authors. Yasuzumi and Tanaka (1958) described two sperm types in Cipangopaludina malleata.

Typical sperm have a single flagellar tail, and atypical sperm possess a bundle of tail flagella (7 to 17), each with a centriole at its anterior end. Eupyrene and oligopyrene sperm were described in yiviparus viviparus by Hanson et al.

(1952). Eupyrene sperm contain seven haploid chromosomes; and oligopyrene sperm are infertile and have only one chromosome. In r ■ rivularis, only one type of_sperm has been observed in fixed specimens or in smears of live seminal vesicles.

Electron Microscope Observations

Nothing has been reported on the fine structure of the ovotestis anrl other reproductive organs of any freshwater limpet sp2cies. Electron microscope studies on the ovotestis of a slug (V_acrinulus borellia.nus) by QuatLrini and

Lanza (1965) and Lymn2ca staonalis by Joosse and Reitz

(1969) have been reported. The ultrastructure of the ovotestis, hermaphroditic duct, and seminal vesicle of the slug and the ovotestis of Lymn~ca is quite similar to that of F. rivularis. The ovotestis funnel (common atrium) of s].ugs resemble to a certain extent the same struciure in the 213 limpet in having an inner epithelium consisting of a single layer of ciliated epithelial cells with microvilli on the internal surface, one extremely thin non-cellular basal lamina, and one external connective tissue of Ancel's layer composed of flat cells. In Ferrissia the internal surfaces studied from the common atrium down to the carrefour, are equipped with cilia and microvilli. The presence of cilia leads one to conclude that oocytes and spermatozoa may be transported from the ovotestis to carrefour by ciliary motion, but the function of microvilli remains unknown.

Hickman (1931) described with light microscopy sperm tails of Succinea ovalis which had an inner column consisting of an axial filament surrounded by a sheath of mitochondria and an outer spiral cytoplasmic keel. Watts

(1952) reported that mitochondria form a spiral sheath around the axial filament of the Arion subfuscus sperm tails. In r- ri·Jul_?ri~, light microscopy of a mature sperm tail also showed that the axial filament is surrounded by cytoplasmic keels. Electron micrographs of longitudinal and transverse sections of middle· ..pieces and end-pieces of sperm tails, however, reveal that the axial filament or axoneme is surrounded by a mitochonclr.ial shE~ath and various numbers of intramitochondrial com2artments which produce the helical appearance of the middlc--piece. No cytoplasm was observed outside the mitochondrial sheath; merely a wide space was present between the sheath and double plasma membrane (Figs. 45 and 46). The sp~ce may be filled with a 214

fluid of unknown nature although it appears to be electron

transparent. It seems impossible that the wide space is

an artifact and empty, since it has been observed in sperm

of other species, such as Paludina (Personne and Anderson,

1970), Lymnaea (Favard and Andre, 1970), Helix (Anderson and

Personne, 1970), and Vaginulus borellianus (Quattrini and

Lanza, 19 6 5) .

A significant amount of glycogen is present in the

intramitochondrial compartments of the middle-piece in mature limpet sperm. A few glycogen granules also exist

in the spaces between the central fibrils and the peripheral

fibrils of the axoneme. The function of glycogen has been

reported for sperm of the pulmonates Planorbis, Lymnaea, and

Helisoma by Anderson and Personne (1970), who also show that

the sites of activity of some oxidative and respiratory

chain enzymes are localized in some invertebrate sperm in

both membranous and paracrystalline mitochondrial deriva-

tives. Glycogen in mature sperm may serve as an important

energy source ior the motility of sperm.

Hanson, et al. (1952) presumed that the tail sheath

of yivip2Fus sperm contain polysacchari.des. The actual movement of the F. rivularis sperm has never been observed,

because this limpet is assumed to perform self-fertj_lization within the reproductive tract. The existence of glycogen

in primary helices rr1ay serve primo.rily as the food source

for sperm in the semin,'il vesicle. or other reproductive organ sites since~ sperm are observc,d in the seminal vesicle 215 of limpets during or after winter.

Microtubules or manchette were observed enclosing the outside metamorphosing mitochondria of developing spermatids and disappeared after spermiogenesis in F. rivularis. The function of microtubules in the sperm tail has long been a subject of interest to cell biologists.

Studies of spermatogenesis in a lizard (Clark, 1967), in domestic fowl (McIntosh and Porter, 1967), and in insects

(Shoup, 1966; Kessel, 1970) have led several investigators to suggest that microtubules may be responsible for the elongation and shaping of the sperrnatid nucleus. However,

Facett, Anderson, and Phillips (1971) suggest that the shape of thf; sperm head may be largely dE;;termincd by genetic factors instead of microtubules. Since the ultrastructure of the limpet sperm head has not been studied, it remains 1..1.nknown whether n1icrot1.1bules c.re present in the head during spermiogenesis. Microtubules, however, may be responsible for shaping the middle-piece of lim?et sperm.

Kessel (1967) and others suggest that microtubules, especially in the middle-piece and tail of sperm, provide support for a cytoskeleton framework for such attenuated cells as mature sperm. Kessel also reported that microtubules develop in the middJ.e-picce and tail during spermiogencsis, persist in mature grasshopper spermatozoa, and may constitute a mechanism for giving direct motion to a cell cind its content. R0binson ( J 9 6 6) also claimed that the m:i crotubulc shc,~th in speormat:o ✓,oa of scale insect.s 216

probably plays a significant role in spermatozoan motility.

It does not seem probable that Kessel's and Robinson's

idea is applicable to limpet sperm, since microtubules

only appear in the developing spermatids during spermio-

genesis and do not persist in mature sperm. Moreover, since

self-fertilizdtion is believed to be the only means of

reproduction in F. rivu.laris, the motility of sperm seems to

be unimportant..

Anderson (1972, personal comrnunication) suggested

that the manchette may serve as a screen to keep cytoplasm

out of the developing mitochondrial complex. This idea

seems plausible to sperrniogenesis in the limpet, since rnicrotubules only appear in developing spermatids and dis-

appear with cytoplasm after spermiogenesis. The conclusion may be made that microtubules in spermatids play a role,

either in shaping the middle-piece er in keeping off

cytoplasm during spermiogenesis.

The function of the follicle cell in the development

of the oocyte of pulmonate gastropods has been an interesting

subject to developmental biologists. It has been mentioned

in a previous section that follicle cells play an important

role in the determination of the cortical morphogenetic

pattern in the (Raven, 1963). Unfortunately,

Raven's tl1eory is based on J.ight microscope observations,

usin9 spccia.1 stuining teclmiqees, but the procedure was not mentioned .. It remains uncertain whether follicle cells are

structurally associated with developing oocytcs in Limnnea. 217

Electron micrographs of previtellogenetic oocytes

with the surrounding follicle cells of Ferrissia show no

indications of coincidence of cortical egg pattern with the

arrangement of follicle cells. However, follicle cells and

the developing oocyte are intimately associated, as de-

scribed in a previous section by intercellular bridges

(electric couplings) and oocytic-follicular spaces. The

membranes are particularly thickened at the contacts of the

two processes. The oolemma and the plasmalewma of follicle

cells in the space area are fairly thin, but no pinocyte is

formed on the membrane. Anderson and Beams (1960) suggested

that the oocyte takes in fluid and dissolved substances from

the oocyte-folJ.icular space by means of pinocytosis.

Apparently electric couplings serve to hold the oocyte and

follicle cells together; thus, as the oocyte grows and

develops nutrients of low molecular size may be transported

into the oocyte through the fluid within the oocytic-

follicular spaces by diffusion, instead of pinocytosis.

Kemp (1956) estimated that microvilli of the oocyte of

Rana pip_iens increase,! the absorptive area of the surf ace

to about thirty-·five tirc,es that of a simple sphere.

Microvilli on the surface of the limpet oocyte may provide

a better abso:cption surface of nutrients from the

surrounding medium. Yolk precursors may be synthesized in

the digestive gland and passed over to the oocyte through

the oocytic-follicular spac2s, since these spaces reduce or

disappear probably before viteJ.logoncsis. One evi.dence 218 which may support the above assumption is that the ER system and mitochondria in follicle cells is well developed during vitellogenesis (Fig. 60). This indicates that the relationship between follicle cells and oocyte becomes more intimate and complicated.

A large lamellar structure arises near the nucleus and the "mitochondrial cloud'' in previtellogenesis and persists during vitellogenesis of the oocyte in the limpet.

This struct~ure has been termed the "yolk nucleus",

"vitelline nucleus","vitelline body", npallial substances", and "Balbiani's vitelline body" by different authors. The function of the vitelline nucleus has been an interesting subject investigated by cell biologists.

The number of vitelline nuclei varies with species, from one to several. Reverberi (1966) found five vitelline nuclei in the ~1yti_1U£ oocyte (1966), Vitelline nuclei are considered responsible for yolk synthesis. Rebhun (1956) mentioned the vitelline nucleus in the egg of the surf clam

(Spisula ~?-~~dissirna) cont2:'lined RNA and was responsible for yolk synthesis, since he found yolk plaLelets were present in the lamella.r struct1.1re. Based upon the obser-

11 n;:i.L.1'onsvc_L. ... of _ --:.~Iytil.... ' ...... b' oncvtE'S.1.._. '.J. -. 1 Reverberi (1966, 1967) con- eluded that if the~ function of the yolk nucleus is intended for the construction of yolk qranuJ2s, one can suppose that the substances which are used for yolk synthesis filter gradually tl1rough the different ordors of lamellar rings and herea.ft..er concentr,1te in U1e centred. area, from this 219 area the yolk granule emerges; but the reverse process is equally possible, if the yolk granule should emerge from the most peripheral ring. Reverberi (1966) claimed that the vitelline nucleus is responsible for yolk synthesis in invertebrates, although the exact mechanism is unknown.

On the contrary, Anderson and Beams (1960) claimed that the membranes of the "yolk nucleus" in the oocyte of guinea pigs are unlike the elements of the endoplasmic reticulum, i.e., it has no ribonucleoprotein granules associated with its membranous system. Also mitochondria are not present in this lamellar body, and are similar to

Golgi complexes in submicroscopic organization. They also suggested that it is probably best to drop the term "yolk nucleus" at least for guinea pig oogonia and instead refer to it as a Golgi complex.

In the case of the Ferrissia oocyte, only one vitelline nucleus is present in an oocyte. In is constructed of a few layers of a concentrjc double membrane resembling the endoplasmic reticulum; however, the overall structure is dj_fferent from that described in other molluscs by Rebhun (1956) and Reverberi (1966, 1967). A few small mi t.ochondria. are encJ osed :i.n the spaces betvzeen the>. lamellae and in tho central space. Evid2nce indicates that no yolk platelets nor granules are present in the vitellinc nucleus of the limpet oocyte. The only place which appears to have yolk plc1telets is the m0~rnbrane--bouncJ. yolL granules. It seems prob~ble to conclude at the present time that the 220 vitelline nucleus in the limpet oocyte is not responsible for actual yolk formation and its function is unknown.

At the previtellogenetic stage of the oocyte all mitochondria are aggregated into a "cloud" at the basal end of the cell. The cloud disperses through the cytoplasm during vitellogenesis. This suggests that mitochondria are possibly related directly and indirectly to yolk synthesis.

Mitochondria proliferate during vitellogenesis by binary division as described in the eight-cell stage of Arion ate2.:_ rufus by Sathananthan (1970). Metamophosis of mitochondria also occurs during this time. A number of mitochondria are apparently involved with yolk synthesis as described by

Lanzavecchia (in Wischnitzer, 1967). These mitochondria

(Pig. 54) possess a large vacuole-like cisterna of electron transparency which is eccentrically located. It is possible that yolk formation in F. rivularis may be attributed to mitochondria, as proposed by various authors in amphibian oocytes (Fig. 91).

According to Wischnitzer's review of theories on yolk formation, the autophagic vacuoles of the limpet oocyte show great similarity to the third step of the yolk formation process dE~scribed by Hartenberq in 1962

(Nischnitzer, 1967). The initial steps of the process were not observed in the vitellogenetic oocytes of the limpet,

F. rivuloris, therefore, final conclnsion~, cannot be drawn. 221

Fig. 90. Metaphase of an unovulated oocyte. As , aster ; Chr , chromosome; FC , follicle cell, 1300 X. 222

...... ' ...... ' 'a. . .

. . I!

C D 1:'i9. 91" Yolk fo1.T1ati.on thoocies. 1'. According to Lanzavccchia (1961); Il. Accmd:Lu:, ·::c-, Ward (l'.::S~>,); C. :\ccording to F'artcnbe:c·g (196;2); D. Accordinc'. to l3alinsky and .Devis (19G3). ('l'hc line loac'ling to this seric::o :l ,; hroJ·.en since the init.ial }.iocJy is pr0~.;urned to bo an at-ypicol rr1:i.tochondrion). (}\ftor: c;c,ul v.1ischnitzcr, 19(i"/) . 223

Development

Embryonic and Larval Development

Up to the present time only two complete reports on

freshwai.:er limpet development are encountered. The embryonic

growth and development of Ferrissia shimekii was described

by Basch (1959), and Clapp (1921) reported on the development of Ancylus Juscus. The development of r. rivularis, in general, is similar to that of the above two species.

Morphological features of each developmental stage, however,

are not extensively described, nor defined by the two authors.

There are distinct differences in the timing of the

various developmental stages, differences perhaps being

influenced by temperature and genetic factors. According

to Clapp, hatching in Ancylus fuscus takes place most often

in about eighteen days. The European river limpet, Ancylus

fluviatilis, emerges from the egg capsule after twenty-five

days (Hunter, 1953). F. shimekii embryos normally hatch

on the tenth day (Basch, 1959). The time of hatching

appears to b2 consistent for f2._rivula1~:~~, normally hatching

on the fourteenth day of development. Basch suggests that

en~ymatic action upon the capsule by bacteria, associated

\vith introd11ce.d foocl, m2c:y he significan-c. in inducing rL,pture

of thG capsule and hatching. Ba.sch I s intc~rpretation seems

very unlikely, for, j f hatching :Ls dependent on bacteria,

the young lirnriet r,;hould be able: to emerge from any point of the c~p~ule. Such a phenomenon was never observed in 224

F. rivularis. The opening of the capsule in this species

is fairly uniform with the dorsal lid always opening along the operculate suture named by Bondesen (1950). It is more

likely that hatching is mainly due to rapid body and shell growth and to the development of larval movements. Increase of shell height in addition to the action of the foot and mantle muscles allow the young to easily lift the dorsal lid along the weak contact line (operculate suture).

Rupturing does not take place at any other site. This evidence supports the proposed assumption that limpet hatching is mechanical rather than chemical.

A few small limpets did not hatch and finally died inside the egg capsules. These limpets developed very unusual features before dying. The mantle retracted and became separated from the shell and the digestive gland or liver became very dyst:r.ophic. Apparently they started to use the digestive gland and other tissues for energy sources after the capsule nutrients had been exhausted.

Embryonic and larval growth of F. rtvul~n-i_~ is depicted by a curve shown in Fig. 62. The size of the embryo sLlghtly increaf;es the first three days of development. Growth becomes pronounced following the third day, after gastrula hatching, and increases rapidly from the sixth to the tRnth day. According to the growth curve, apparently Uw grmvth of embryo during this period shows correlation with the uptake of albu~en fluid. Rapid growth ctarts2~ the time when tho embryosstart to absorb alLumen 225

after hatching from the vitelline membrane. Growth becomes

more rapid after the radula and accessory organs are devel-

oped. In embryos of Limnaea palustris, from the fourth to

the eighth day stage, protein levels of embryos continued

to rise, ·while albumen protein decreased (Morrill, 1964).

Although the protein content of the embryos or larvae and

albumen protein were not checked in F. rivularis, a decrease

of albumen was indicated by the change of viscosity of the

albumen fluid. As the embryos grew the fluid became more

watery and less particulate. Just prior to hatching, the

larvae grew slightly and the albumen was distinctly water-

like.

Growth of the river limpet is quite similar to that

of F. shiI".lekii (Basch, 1959). In F. shi2::.ie~~i, the first half-

day embryo is 75.7 µmin length, and the hatching size is

593.1 µmin length.

The following points in the embryogeny of F.

Ei ~~~aEi:E. ar2 particularly interesting.

Egg Capsule

The usefulness of an ancylid egg for experimental

or developmental biology was emphc:.siz0d by Bondesen

(1950a, b). For the studv of descriptive embryology ancylid

eggs are excellent because Lhey are easily handled and ohsorved, and not easily injured. However, for statistical analysis, ancylid eggs are not useful, since the number of eggs in each capsule is too small, and it is difficult to 226

gather a larg~ number of egg capsules. Ancylid eggs are

also unfavorable studying cell lineage and histogenesis

owing to the rich, opaque yolk contents of egg cells and

the surrounding medium. Therefore, the present study mainly focuses on morphogenesis.

Self-fertilization

Inseraination of pulmonates has been investigated by biologists since the early part of this century: particu-

larly, the prospect and discovery of self-fertilization

(autofecund.ation). The comrnonest method employed to

investigate self-fertilization has been to isolate young

animals from the time of hatching to the time of egg laying.

The possibility of laying fertile eggs in strict isolation

has been reported in a number of pulmonates, though the

degree of self-fertilization varies in different species.

In Lyrnnaea. (Colton, 1918; Crabb, 1927 f 1928), for instance,

individuals iL isolation deposit as rnany eggs as individuals

raised togei.:her in mass culture, and the percentage of egg

viability is equal in both groups. Ikeda and Emura (1934)

0 ~ ~c:,·i.c __ -i:i.~ ~v 1 :c,o>,·., .' "'Olll f ouno ~c:.• .Ll. 1.eJ.,,:,.,-·t'l'r;.,-· i .J.,.aL.lc.,n ·,, J·,.• l __. .Lao.., De"-:::.:::'. si.mJ_·1-· a1 -iC'___ ,-, st'm.J .. p., . ,

al though tl-)2 fecun:Jity v.1as low compared to that of the control group. Cain (1956) demonstrated self-fertilization

in Lym~ac:a stac::rna1:i s oyprer::;sa by gen2tic···cross techniques.

On]y two instances of self-fertilization have been

reported in frcshv.1c1ter limpets, Col ton (1918) discovered

self-·fertili zution in ~.nc?lus---•-~-~----•v~•v~-- (Ferd~-•--••-.-•-,-•- s,]i&)-~•- :f luv:L1tilis. 227

Basch (1959) subsequently isolated the young of Ferrissia shimekii immediately after hatching and reared them in solitude in small vials. He successfully obtained fertile eggs from these limpets. In respect to fertilization in

Ferrissia rivularis, as mentioned before, no copulation nor sperm in the spermatheca have ever been observed. Copu- lation has been reported by Burky (1971), but he may be mistaken, since feeding behavior, resembling copulation, is often seen in this species, as Bondesen (1950) described in Ancylus fluviatilis. Since sperm are never found in smears and serial sections of spermathecae during the breeding season, it seems very plausible to conclude that self-fertilization occurs in~- rivularis, and the animal reproduces essentially by this process, in spite of the evolutionary and genetic problems supposedly created by self-fertilization.

Concerning the exact location of self-fertilization,

Colton (1922) assu~ed that fertilization occurred in the acinus in !.::'/nn12ea ~olumella. Crabb (1927, 1928) observed that egqs of Lyrnnoea were fertjlized by its own sperrn in th-2 acim,s or in the hermaphordj tic duct. Polyspf-~rrny also

has been o}y3crved in. ·t'-1e1 E'CT''f"'.1_!':-_J0 of, __Iyrnn·,,·•,c, 1_.\•~·-G'--:~C~ l--Dv.I. {-J_,r:,,._...... ,. above t,,,,o\' authors and by Bretschneider (1948).

An egg of the river limpet w2s fertilized by two sperm in only one case in the aci.nus lumen and a few sperm were seen outside the egg at the site probably where the spc:.,rrn entered (Fig. 3 GB) . 'I'hc sperm L.1i 1 in the:~ oocyte 228 cross section (Fig. 55) confirms the conclusion that self-

fertilization takes place in the acinus lumen. It is also possible that self-fertilization may occur in the common atrium and in the carrefour, before the eggs are coated with albumen. Wautier, et al. (1966) reported that self- fertilization and parthenogenesis takes place in Gundal- achia wautieri. Metaphase of the first maturation was observed in an unovulated oocyte in one acinus (Fig. 90A,

B). This suggests that parthenogenesis may also occur in

F. rivularis. The problems of the locations of self- fertilization, function of the spermatheca, and parthenogenesis still remain unsolved.

Fretter and Graham (1968) made a significant statement: "A hermaphrodite is normally living in a habitat or ecological situation in which is confronted some difficulty in reproductive activity, nnd that this difficulty, to some extent at least, is eased by the adoption of aml)isexual state. Hermaphroditism has, therefore, been favored and perpetuated by natural selection.

Thus, hermaphrodites a.re commoner amons:r fresh-wa t.er than marine molluscs. A logical extensj.on of this is the increased efficiency introduced ·when self~·fe:ctilization becomes possible if an individual fails to find a partner with which it may copulate."

It has often b2en observed during the breeding season in Utah Lake that rocks with individuals of F. r:Lvu. ..} arJ_n. are isolated front one another by wide, soft, silty 229 substrata between rocks. Under such isolation situations, self-fertilization would be advantageous.

Colton and Pennypacker (1934) reported that self- fertilization in Lyrnnaea columella for 93 generations did not decrease the viability of the strain. This strongly suggests that self-fertilization plays a significant role in the evolution of hermaphroditic gastropods.

Veliger

Hess (1971) called all the larvae of freshwater gastropods which can swin freely by rotating within the egg capsule a "trochophore" larvae. He com:nented that the veliger stage is in most cases not typically developed by freshwater gastropods; in particular, the vela which are so prominent in larvae of many marine gastropods are not formed. Therefore, it is unsuitable to call the larva, which immediately follovrn the trochophore, a. veliger. The veliger of most marine gastropods is characterized by having ciliated velar lobes, a shell gland (or shell), foot, eyes, and a statocyst. However, these characterj_stics are possessed by larvae folJowing the trochophore in F. rivularis and some other freshwater gastropods, e.g.,

Ancylus fuscus. Obviously, there is no good reason why the terrn "trochophore larva" should be 9ivsn to all larval stages following the trochophore stage as Hess suggests in his description of the development of Bi thvnia t~~1:.?:_~.:_c_'.1_9Ulu.ta. 230

Albumen Fluid

Raven (1946) first noted that numerous vacuoles appear in the cytoplasm of forty-cell embryos. Contents were strongly eosinophilic. The color, with all stains used on the vacuole content, corresponded to that of the egg capsule fluid. Later Elbers and Bluemink (1960) verified Raven's observation by electron microscopy and also found that the capsule fluid was taken by the developing embryos by pinocytosis. Morrill (1963, 1964) found that tbe embryo started to absorb albumen fluid from the surrounding medium on the second day of development and ingested nutrients orally after the oesophageal invagination joined with the gut. Most of the albumen was con- sumed by the seventh day of developr:1ent in Lymn9-ea palustris. In the enbryos of F. rivul~):.~' large albumen cells make their first appearance at the late gastrula stage and b,?.come distinct on the fourth day of development

(Figs. 63 and 77). In the meantime, minute vesicles or vacuoles begin to appear in the peripheral region of the young trochophore. The larva starts to ingest albumen fluid by radular action at the late veligcr stage, as soon as the radula and tlle associate::d nusclcr:; are developed.

Albumen fluid becomes thinner as the ::=,rnbryo grows, and finally bec~~es watery before hatching. The above observations on the development of Ferri.ssia suggest the embryo sturts to take in albumen fluid from the surrounding medium at the gastrula stage, after it hatches from the 231

vitelline membrane. Intake of albumen continues until the

time of hatching. Before the gastrula stage, development of

the embryo apparently depends primarily upon consumption of

yolk contained in cells. The vitelline membrane may be a

barrier to restrict passage of albumen. This may explain

why large albumen cells and small vesicles do not appear

µntil after the gastrula stage. According to Raven,

albumen-contained vacuoles start to appear in the cytoplasm

of 40- and 120-cell embryos. It seems very difficult to believe that the cells of the embryo can engulf the albumen droplets through the thick, tough vitelline membrane by pinocytosis. The vitelline membrane was called a chorion

in Linmaea limosa by Comandon and de Fonorune (1935).

Usage of the term "chorion" implies that it is a thick membrane. Elbers and Bluemink (1960) also suggest that the

embryo can take in albumen by pinocytosis; of course, cells

of the embryo must be in direct contact with the fluid.

Therefore, they believe that pinocytosis probably takes place two days after the first cleavage, about the time the vitelline membrane comes off. Thus, Raven's observation remains questionable.

The composition of the capsule fluid of Limnaca

eggs h2:s been studied by Eon::;trnc1.rm (Elbers and Bluemink,

1960) ancl !'.iorrill (1963). It contains about 15 per cent dry matter, a 3·-6 per cent of which is galactogen. Protein content is G-8 per cent. Eighteen amino acids have been detcctecl in the egg albumen of Thus, the only way 232 the cells take in albumen substances is by pinocytosis.

Later the larva becomes equipped with a radula and mouth, then the nutrient of albumen may be taken in by ingension.

Therefore, albumen fluid, besides affording protection, serves as a food source for the growth and development of embryos and larvae.

Hatching of Gastrula

The gastrula of the river limpet sheds the vitelline membrane on the third day of development. The mechanism of hatching has never been reported. Due to rapid growth, the size of the embryo at the gastrula stage increases twenty-five per cent of its initial size. Two vacuolar structures are always seen at the animal end of the embryo.

They disappear at intervals, probably by contraction. Thick fluid is extruded into the surrounding medium when these vesicles disappear. It seems possible that rupture of the vitelline membrane is caused by rapid growth and by the contraction of the embryo. J\ftcr the membrane breaks, the embryo crawls out into the capsule fluid by ciliary motion.

This allows for uptake of albumen by pinocytosis. Electron micrographs of the vitellog8netic occyte verify that the v.ite11ine meirtbranc is a thin 1nernbrane along the periphery of microvi 1li of the oc>cyte. It is secreted by the oocyte.

This membrane perhaps corre~'.ponds to Raven's "zona radiata 11

'.rhe rnf;mbr anc iri the ovarian e9gs of Lvmnaea------·- --·-sta0nalis.---·-~--- was not observed in serial s~ctions of full-grown oocytes 233 of limpets stained with alum hematoxylin and eosion. No electron microscope study was made on the surface structure of the spawned egg cells of Ferrissia. However, the structure is assumed to be very similar to that of growing ovarian eggs. Therefore, Raven's «zona radiata" probably includes not only the vitelline membrane but also the microvilli on the peripheral surface of the egg cell.

Development of the Shell

The development of shell gland in pulmonates has been reported by a few authors. Hobnes (1900) reported that the shell gland of Physa fontialis made its appearance at a position opposite the stomodaeum some time after the closure of the blastopore. Lowrance (1934) reported that the shell gland of Stagnicola kingi became evident when the velum was

formed by the post·-trochophore (equal to veliger stage in this study), before either eyes or tentacles had made their appearance. Hess (1971) observed that in !?ith;{.£1-}-a tenta-

~-~a:t:~-' the shell gland appeared in the young trochophore larva at the dorsal side of the post-trochal ectoderm; later, a shell field wa~ formed in the center of the gland.

The gland was surrounded by a circular shell groove, and

started to i,ecretc:, a conchiolin shell primordium. During dE::veloprncnt. the~ ~;hell field (2Xtended more and more, and finally covered large parts of the visceral hump.

f;hell glan6 development in P. d;rularis shows some similarity to tliat of tbe c:d:_1ov·emcnt_ioned species. However, 234 the shell gland primordiumappears in gastrulae at the end opposite the blastopore where the invaginated ectoderm meets the endodermal cells of the archenteron. Raven (1952)

first proposed that the shell gland is formed by induction of endodermal cells at the tip of archenteron on the invaginated ectodermal cells; and if the gut invagination is prevented from developing or is retarded, the shell gland either does not develop or may develop in the abnormal area of contact. Evidence for an induction of the shell gland by endoderrnal material has also been found in the prosobranch snail Ilyanassa obsoleta (Cather, 1967). Cal- - ...... -·---~------cification of the conchiolin part of the limpet shell does not occur until the seventh day of development. 'The mechanism of calcification in the limpet is unknown. How- ever, one may assune that in the early stages, the calcification of the shell is blocked by the presence of unknown carbcnic anhydrase inhibitors (f'ree:rr"tan, 1960), or by the absence of carbonic anhydrase in the shell gland.

Ef fee ts of r1.aleic Hydrazide on Development and Fecundity

Malformation:::; Induced by Maleic Hydraz idc, '['rcatrnents

'J'he: ear: ly embryonic d2vcloprncnt of r. r i vu.laris was

altered by tr02tments with 1.5 and 0.5 per cent maleic hydrazide ::;o1utions at different stages. These amounts are actually far in excess of the levels that could be expected norm,1.lly from a~r.r:icul tural usaqc, bot :more con- 235 centrated dosages were used for this study in order to determine lethal levels and the exact malformation effect.

The overall effects of maleic hydrazide on early embryonic development of this limpet resembles greatly the effects of cobalt chloride on the embryos of larvae of

Lymnaea stagnalis and L. palustri~ (Morrill, 1963). Embryo malformations induced by CoC12 are: (a) separation of blastomeres, (b) vesicular and dumbbell-shaped hydropic exogastrulae, (c) arrested gastrulae, and (d) hydropic veligers. Most blastulae of Ferrissia were killed by 1.5 per cent maleic hydrazide solution.

'11 he most common type of abnormal ma.lformation induced by maleic hydrazide appeared to be-hydropic exogastrulae. The four-cell stage and young gastrulae seemed to be the most susceptible to the action of maleic hydrazide; and uncleaved eggs were the most resistant to it. Raven

(1952) and others report that the sensitivi.ty of Lymnaea

embryos to LiCl existed only during early c1eavag·e I with a 2 maximum s-2nsitivi ty at the four-cell ::;ta.go.

Exogastrulne which were induced by different agents have been reported in various species of pulmonates. Mancuso

(1955) found that 0.5 and 0.25 per cent sodium azide sol"L1tions produced vcr.,icnl..3r 2md dun:bbe11·-shaped hydropic exogastru.1.-2H~, o.nd Ui('.!!:e ""'· s 2. F\axi1(,1_·nt of sensit:.i vi ty to the action of the chemical at the four- an~ eight-cell stages of

I)hys;:~. riv1 3_layi_s_. Li thiurn c:::tloTidt~ and c'i.naerobiosis also induced forrna tion of e.xogastr\J 1a E,1T1bryos :i.n Lyn'.!1<1_~"'.1:_ 236 (Raven, 1959; Geilenkirchen and Nijenhuis, 1959;

Geilenkirchen, 1952). One -SH inhibitor, chloroacetophenone, produced exogastrulae in Planorbis (Indoplanorbis) exuxtus (Mulherkar and Sherbet, 1963).

The causes of the formation of exogastrulae are still obscure. Morrill (1963) suggested that exogastrulae, hydropic exogastrulae, and hydropic veliger larvae of

Limnaea are caused by an impairment in the development of mesomeres plus concomittant uptake of water by the blastocoel or body cavity. The effect of lithium on Limnaea eggs is two fold: {1) an inhibition of the animal gradient field leading to a suppression of animal differentiation, (2) an injurious action on the vegetative material of the egg which may lead to gastrulation disturbances or to an inhibition of differentiation of the endoderm (Raven and

Rijckevorsel, 1953).

The above proposed interpretations seemed logical;

• C' however, it l . .::, uncertain that maleic hydrazide has the same effect on eggs and ernb:cyos of Fe_~::_:i:-issi_aas CoC1 a:i1d LiC1 . 2 2 Nevertheless, rnaleic hydrazide does have an inhibitory effect on gast:rul3_tion and endoderrn cell differ·entiation.

The presence of cilia in the hydropic and non-hydropic cxogastrulae indic&tes that maleic hydrazide J1as no effect on the differentiation of ectodcrnal cells at the animal end.

An interesting question is raised as to how the normal qastrula is t:canr;foJ:-r,tcd into hyc1ropic exoqaE;trula. 237

This phenomenum has been described in a previous section.

All of the embryos in a group of twenty-four gastrulae treated withl.5 per cent maleic hydrazide solution for an hour, disintegrated at the vegetal ends and consequently developed into small hydropic exogastrulae. It seems probably that maleic hydrazide may also cause "de-gastrulation. 11

In a group of twenty-two treated eggs, ten developed normally and hatched and eleven became exogastrulae. The results suggest that the various cytological conditions of the egg, particularly its cortical structures, show various degrees of susceptibility to the action of maleic hydrazide.

It is possible tha.t maleic hydra:dde may· alter the cortical pattern of the egg, since it causes strong dehydration and shifting of the:: orqanization of yolk granules to a certain extent. At times, n.s per cent maleic hydrazide causes segregation of hlastomeres. Apparently malei.c hydrazide may change the surface structure of the blastomeres (Garber and Poscona, l!rOa, b) , dissolve the intercellular substance which holds the cells to9ether, or it. muy destroy the

"mernbraJ.18 knottin~T f.:ystem" between the blastomcres

(Wac1din9ton, Pe:o:y 1 and Okadc:,, 1.S 61) •

Up to the present tim2, there 1s no indication that rnalcic hydrctzid0 i::; carcinouc,ni.c to this fresh,·,ater lirnpet.

Electron microscope and auto:r ;::,d:Lc-qrapll techniques would. be recommended for further study of this aspect. 238

Effect of Maleic Hydrazide on Fecundity

According to the data shown in Table 19, it seems that the nonfatal concentrations of maleic hydrazide show to a certain extent inhibition of egg production in~. rivularis. Information on the effect of maleic hydrazide on the fecundity of other animals is extremely scanty. How- ever, Yule, Parups, and Hoffman (1966) showed that 1500 to

200 p.p.m. maleic hydrazide did not have any effect on the fecundity of Drosophila and the housefly nor on their development. '1,he inhibiting mechanism on egg production is unknown, since no histological studies have been made on the ovotestes of treated limpets. However, one may assume that maleic hydrazide causes disturbances in gametogenesis or reduces the secretion of the albumen gland. 239

Fig. 92. Cross section of adult limpet shell showing periostracum (Pe), ostracum (Os), and hypostracum ( Ho ) . 300 X.

Fig. 93. An ovotcstis and two ovulated oocytes in a common atrium. 150 X. 240

Fig. 94 . Growing oocytes and follicle cells. 1300 X.

Fig . 95. A full-grown oocytc . FC , follicle cell; GV, germinal vesicle ; Ncl , nucleolus. 1300 X. 241

Fig. 96. Hydropic young trochophore. Ar , archenteron; St, stom odaeum . 200 X.

Fig. 97. Hydropic veliger . E , eye ; In, intestine; NC, nuchal ce ll; NR, nerve ring ; Oe , esophagus ; OG, optic ganglion ; S , stomacl1; Sh , she ll. 200 X. 242

Fig. 98. Sertoli cells (SeC) with their attached late spermatids (L Spt) and full-grown oocyte in the acinus lumen of oogenetic zone. FC , follicle cells; Spt , young spermatid; ST , spermatid tail. 1 300 X.

Fig. 99. Triophth a lmic post-veliger. 300 X. CONCLUSIONS A.ND SUMMARY

Results on reproduction and development, and effects of maleic hydrazide (diethanol amine salt of maleic hydra-

zide) on the fecundity and early embryonic development of

Ferrissia rivularis (Say) indicates the following:

1. Tho ovotestis consists of oogenetic and sperm-

atogenetic zones. The cornmon atrium has an internal

columnar secretory epithelium which is ciliated and consists

of a thin external connective tissue. The hermaphroditic duct and seminal vesicle have the same structure as the

cormnon at:c ium, except the internal layer is not secretory.

2. Oviposition of this limpet starts in the middle of April in Utah Lake. Eggs are laid on the undersurfaces of rocks or on the surfaces of submerged sterns of aquatic pla11ts.

3. Deposition of eggs is probably mainly controlled by temperature in mature limpets, but not affected by light.

4. Larger animals possess larger ovotestes and lay more ecygs.

5. Copuldtion is not seen in this species.

Reproduction is carried out by self-fertilization. Eocrs_; _,

are proLably fertilized in the acirrns Jmnen, common atrium, or in the Cctr:n::four. No sperm lwve been observed in the 244 spermatheca; its exact function remains unknown and possibly may be vestigial.

6. Spermatogenesis commences earlier than oogenesis in young limpets. Sperm and eggs are present at the same time in older animals. Sperm start to surround a Sertoli cell at the secondary spermatocyte stage. Oocytes become surrounded by three to four follicle cells prior to vitellogenesis.

7. Ovulation takes place by autolysis of follicle cells, and eggs move down to a common atrium by amoeboid movement and by ciliary motion of cilia lining the common atrium.

8. Sertoli cells with attached spermatids may move up to the oogenetic zone where spermiation takes place.

9. Freshly laid eggs are amoeboid and irregular in shape. Polar bodies are extruded during a process of

"rounding off" of the egg. The average size of a mature egg is 97 pm; sperm size average is 4 pm in head length, and 214 vm in tail length.

10. Electro11 microg:r.aphs of spcrIT1.atid tail cross section:; shu1·1 thctt the tail is enveloped by an indistinct outer double mc~nbrane. A thin layer of cy·Loplasm vJithout

ER and mitochondrja is situated between the plasma membrane and a single layer of microtubule manchette. Inside the manchette is the developing mitochondrial complex which surrounds a11 axonc:me. 1·hth:i.n the ElitochoJ:c1ric1l complex, thE-:!re are various numbers of intramitochondrial cornpc,rtrnents 245 which contain a few glycogen granules in older spermatids.

Intramitochondrial compartments are not present in the end- piece of the tail. The microtubule manchette may play a role in shaping the middle-piece and in keeping off cytoplasm from the mitochondrial sheath. It does not persist in mature sperm. Cytoplasmic remnants of the sperm tail slough off through the tail instead of head during spermiogenesis.

11. The mature sperm tail shows some structural resemblances to that of spermatids. However, in the mitochondrial sheath of middle-piece, there are fewer glycogen compartments (Intramitochondrial compartments or primary helices) which contain dense glycogen granules; and there are three to four jointed vesicular secondary helices which contain only Kreb I s cycle enzyrnc~s. The primary helices are bounded by paracrystalline membranes of mitochondrial derivatives. No intra1,.1i tochondrial comp artments are present in the mitochondrial sheath of end-piece.

Mitochondrial sheath is not observed outside the axoneme in the tail tip.

12. Glycogen contained in the pri~ary helices is probably the main food source for ~ature sperm in the seminal ve3icles.

13. The previtelJogentic oocyte contai.ns one large germinal vesicle, on2 vit.elline nucleus, and one "mitoch- . . onc1rial cloud" at the basc1l cmd. T 111 ~; oocyt.e J_ s surrounded by three or four follicle cells. These two cell types are 246 connected by electric couplings and separated by oocytic- follicular spaces. At this stage, there is no evidence of membrane fusion or of continuity between their respective cytoplasm. Transportation of nutrients from follicle cells into the oocyte is probably through oocytic-follicular spaces by diffusion.

14. Two different types of yolk granules, lysosomes, autophagic vacuoles, large mitochondria, Golgi complexes,

ER, and a vitelline nucleus are present in the cytoplasm of the vitellogenetic oocyte. Perioocytic space appears between the oocyte and follicle cells at this stage.

Microvilli and vitelline membranes are present on the surface of oocytes. Mitochondria proliferate by binary fission. They may be responsible for yolk synthesis.

The entire developmental process is divided into seven stages: (a) early cleavage, (b) blastula,

(c) gastrula, (d) trochophore, (e) veliger, (f) post- veliger, and (g) juvenile. The total developing time on the average is fourteen days.

16. The shell gland primordium first appears at the aniroal end in th~ gastrula stage. The first non- calcified conchiolin shell is secreted by a shell gland and appears OJl the sixth day of devc::lopr:1ent. Shell calcif ic-· ation probably starts on the seventh dRy from the time of eqg depoE~ition.

17. Maleic hyJrazide ~;olution~; of 1. 5 and O. 5 per cc.mt kill adult lirnp(,,ts after hrnl ve honrs exposure. Non·- 247 fatal concentrations of maleic hydrazide seem to reduce egg production in F. rivularis.

18. Treatment with maleic hydrazide resulted in

(a) separation of blastomeres, (b) arrested blast.ulae,

(d) hydropic trochophores, (e) hydropic veligers, (f) head malformations, and (g) shell malformations.

19. Results of maleic hydrazide treatment experiments suggest that maleic hydrazide possibly dissolves intercellu].ar substances and vitelline membranes, alters the surface structure of blastorneres, and destroys the "membrane knotting'' system, thus allowing segregation of blastomeres in the embryos prior to blastulation.

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