This dissertation has been microfilmed exactly as received 69-11,725

YOKLEY, Jr., Paul, 1923- A STUDY OF THE ANATOMY OF THE NAIAD CORDATUM (RAFINESQUE, 1820) (: : UNIONOIDA).

The Ohio State University, Ph.D., 1968 Zoology

University Microfilms, Inc., Ann Arbor, Michigan A STUDY OF THE ANATOMY OF THE NAIAD

PLEUROBMA CORDATUM (RAFINESQUE, 1820)

(MOLUJSCA: BIVALVIA: UNIONOIDA)

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of the Ohio State University

By

Paul Yokley, Jr., B.S., M.A

******

The Ohio State University 1968

Approved by

jr.s. Adviser Department of Zoology and Entomology ACKNOWLEDGMENTS

This study has been made under the supervision of Dr. L.

S. Putnam who has been patient when patience counted and prompt when it benefited me most. His help and advice have been most appreciated.

To Dr. David H. Stansbery who is a very special friend, thanks is expressed, for the careful way in which he advised, made suggestions, and constructively criticized my work at regular intervals.

Dr. John L. Crites has been most helpful in reading the manuscript and providing constructive criticism. His efforts have been greatly appreciated.

The cooperative spirit and help provided by Mr. Billy

Carroll and Mr. Billy Ison have been greatly appreciated.

The effort and skill required in collecting specimens weekly throughout the year would have been inadequate without the help of Mr. Charles Gooch. His diving skill and experience as well as his enthusiasm kept my collecting regular over a two year period.

ii Special words of thanks are owed my mother and father who instilled in me the desire to get a good education. Their constant interest and enthusiasm in my work were a continuing help to me.

Finally, to my wife, Betty Yokley, much credit is due because she never lost sight of my goal and sacrificed her time to listen and encourage me. In addition, her skill in typing was most important in completing this study.

To these and many others who cooperated in so many ways,

I shall always be indebted.

iii VITA

August 3, 1923 Born - Mitchellville, Tennessee

1949 B.S., George Peabody College, Nashville, Tennessee

1950 M.A., George Peabody College, Nashville, Tennessee 1950 Instructor, Florence State College, Florence, Alabama

1960 National Science Foundation Fellow, The Ohio State University, Columbus, Ohio

1961 ..... Assistant Professor, Florence State College, Florence, Alabama

PUBLICATIONS

"Mussels of the Bear Creek Watershed, Alabama and Mississippi, with a Discussion of the Area Geology." The American Midland

Naturalist. 79 (1): 189-196, 1968.

"The Mussel Fauna of Duck River in Tennessee, 1965." The

American Midland Naturalist. 80 (1): 34-42, 1968.

FIELDS OF STUDY

Major Field: Zoology

Studies in Ornithology. Professor L. S. Putnam

Studies in Malacology. Professor D. H. Stansbery

iv Major Field: Zoology (continued)

Studies in Vertebrate Zoology. Professor J. W. Price

Studies in Entomology. Professor D. J. Borror

Studies in Botany. Professor C. E. Taft

v TABLE OF CONTENTS

ACKNOWLEDGMENTS

I. INTRODUCTION 1

II. SYSTEMATICS 4

III. DISTRIBUTION 7

1. Range 7

2. Habitat . 8

IV. MATERIALS AND TECHNIQUES 11

V. MORPHOLOGY 17

1. Methods ...... 17 2. Shell 18 3. ; 24 4. Foot » ...... • 29 5„ Muscular System 31 6. Digestive System 35 7. Circulatory System 56 8. Ctenidial System ...... 67 9. Nervous System 79 10. Reproductive System .93 11. Excretory System 115

SUMMARY 122

APPENDIX 126

BIBLIOGRAPHY 133

vi LIST OF TABLES

Table Page 1. Wilson Dam outlet water temperatures at weekly intervals for 1964 16

2. Wilson Dam outlet water temperatures at weekly- intervals for 1965 126

3. Wilson Dam outlet water temperatures at weekly intervals for 1966 ...... 127

4. Wilson Dam outlet water temperatures at weekly intervals for 1967 128

5. Wilson Dam outlet water temperatures at weekly intervals for 1968 129

vii LIST OF ILLUSTRATIONS

Figure Page 1. Pleurobema cordatum . . 5

2. Three of the four closely related forms ... 6

3. Caliper constructed of hardwood . . . . .13

4. Ohio Pigtoe in position to measure length ... 13

5. Ohio Pigtoe in position to measure height ... 14

6. Ohio Pigtoe in position to measure width ... 14

7. Section of mantle margin .23

8. Periostracal gland on margin of mantle.... 23

9. Cardiac muscle viewed longitudinally .... 32

10. Cardiac muscle .32

11. Lateral section of mouth, esophagus and stomach. . 37

12. Stomach, lateral section with folds ... .37

13. Median sagittal section of the esophagus and stomach 40

14. Style sac and intestine 41

15. Prointestine and style sac 41

16. Lateral section of the intestinal coils ... 46

17. Transverse section of the mid-intestine ... 46

18. Rectum as it passes through the pericardium . . 48

19. Chemosensory tissue on the posterior margin . . 48

20. Digestive diverticula .51

21. Transverse section of the secreting acini ... 51

22. Lobule of the digestive diverticulum .... 54 viii Figure Page 23. Transverse section of the ventricle and rectvim . . 58

24. Dorsal aorta with lateral branches . . . . . 58

25. Anterior aorta with main division 62

26. Pedal sinus in the median section of the foot . . 62

27. Transverse section of the dorsal half of a Pigtoe Mussel 64

28. Amoebocytes in the ventricle of the heart ... 64

29. Transverse section of the gill 73

30. Section of a gill through a water tube.... 73

31. Dorsal transverse section of inner laminae... 74

32. Dorsal transverse section where inner gills are connected 74

33. Dorsal transverse section through cloacal chamber . 76

34. Transverse section of outer female gill ... 76

35. Ventral edge of inner gill • 77

36. Transverse section through cerebro-pleural ganglia . 82

37. Multipolar neurons 82

38. Dorsal section of naiad 85

39. Pedal ganglia 85

40. Visceral ganglion 87

41. Sensory tissue . 87

42. Statocyst 92

43* Statocyst 92

44. Median sensory organ of the foot 94

45. Sensory organ of foot 94

46. Testes of Male Pigtoe ...... 96 ix Figure Page 47. Transverse section of mussel through testes. . . 96

48. Acini of the testes of a Pigtoe male .... 99

49. Sperm pore and sperm duct . .99

50. Acinus of testes in the spring 103

51. Mature spermatozoa 103

52. Male acinus in summer 105

53. Male acinus in summer 105

54. Male acinus in the fall 106

55. Male acinus in the fall 106

56. Sagittal section through ovaries 108

57. Acini (follicles) of the ovary 108

58. Acini of the ovary of female 112

59. Acini of the ovary 112

60. Acini of ovary from Pigtoe . 113

61. Acinus of ovary 113

62. Several acini of the ovary 114

63. Acinus of ovary 114

64. Renopericardial opening 117

65. Renopericardial opening . 117

66. Afferent arm of the kidney 119

67. External renal orifice 119

68. Profile of Tennessee River showing Dams. . . .130

69. Depth Profile of Pickwick Reservoir. .... 131

• 70. Diagram of the Digestive System of a Pigtoe. . . 132

x I. INTRODUCTION

The Ohio Pigtoe Mussel, Pleurobema cordatum. in the

Family , Subfamily Ambleminae, i8 a common ovoviviparous

naiad in the Tennessee River. It has had a significant economic

history. The Tennessee River was recognized as a source of raw

material for buttons as long ago as 1883 when a short-lived

button plant was established at Knoxville, Tennessee. In 1914

the Tennessee River furnished 650 tons of mussel shells to the

pearl button industry. After the mainstream dams were built,

mussel fishermen and many biologists predicted mussels would die

out in the river. But in 1945, many were still alive

and the shell harvest was resumed after a lapse of 9 years,

rising from 3,700 tons in 1945 to near 10,000 ton3 in 1947- The

mainstream reservoirs of the Tennessee River became the most

important source of freshwater mussel shells in the United

States (T.V.A., 1966). Pearl buttons were gradually replaced by

synthetics but the cultured pearl industry created a new demand

for the freshwater mussel shells. Again, the Tennessee River

naiad shells possessed the most desired characteristics

including color, luster and toughness required by this industry. 2

The shell harvest rose annually during the decade following 1945 but in 1956 the harvest was much reduced. After this reduction, the U. S. Fish and Wildlife Service (Scruggs, i960) made a survey of the Pigtoe Mussel which was one of the most desirable commercial species in the Tennessee River. This survey revealed that mussels were being harvested 23 times faster than they were being replaced by younger ones. Also, it showed that the majority of the mussels were older than 12 years.

The exact cause for this could not be determined since the life histories of the species of this complex were largely unknown.

The mussel harvest has continued to drop annually even though more effort has been expended and more boats and equipment used.

Because natural populations of Ohio Pigtoe Mussels have been declining for several years, a renewed study has been made of this species. The emphasis of this study has been on morphology including histological detail and seasonal changes.

The study began in the spring of 1964 and continued through the summer of 1968.

Included in this study are the seasonal gonadal changes related to oogenesis and spermatogenesis.

J

The aim of this inquiry was to study as many aspects of the life history, morphology, and ecological requirements as is 3

practicable from samples collected at weekly intervals through­

out several seasonal cycles.

A thorough histological study seems to be lacking in this or any closely related species. For this reason, the present work is intended to reveal as much detail of each organ system as possible to provide a foundation for future investigations of this and related species.

This study was conducted in the Department of Science,

Florence State University, Florence, Alabama. II. SYSTEMATICS

According to Ortmann (1919) and Simpson (1914) Pleurobema

cordatum (Rafinesque, 1820) has a wide distribution arid has

several closely related forms (species or varieties) but is uniquely characterized by having a shell shape and color unlike

the related forms. Stansbery (1967) lists this species as one of

a complex of four very similar forms but characterizes the shell

of P. cordatum as having an equilaterally triangular outline, a yellowish brown smooth , a well-formed sulcus, white

, and moderately high, anteriorly directed umbones (Fig. 1).

The common name for this species is "pigtoe" evidently

because of its obvious resemblance in outline to a pig1s toe.

Stansbery (1967) recommends calling it the Ohio Pigtoe since

Fusconaia flava (Rafinesque, 1820) is called the Wabash Pigtoe

(Baker, 1928: 53) or Common Pigtoe.

The species in this complex closely resembling the Ohio

Pigtoe are, according to Stansbery (1967), P. coccineum (Conrad,

1836), P. pyramidatum (Lea, 1834), and another undescribed form

confined to the Ohio River drainage system (Fig. 2).

Care was taken to exclude all individuals of these latter

three species from the material upon which this study is based. Fig, l,—Pleurobema cordatum, photograph of right

5 Fig. 2.—Three of the four closely related forms (species or varieties) of the Genus Pleurobema which occur in the Muscle Shoals Area of the Tennessee River. (1) Plenrobaaa cordatum (2) Pleurobma coccineom (3) Pleurobema (undescribed)

6 III. DISTRIBUTION

1. Range (Distribution)

The origin of this particular species is unknown.

Ortmann (1919) states that it is difficult to make out the limits

of its distribution but it is certain that it chiefly inhabits

the systems of the Ohio, Cumberland and Tennessee Rivers.

Stansbery (1968, personal communication) states that it is widely

distributed as Ortmann indicated in the largest rivers of the

Ohio River drainage system only. Scruggs (i960) states that this

naiad is the most abundant commercial species taken by the mussel

fishery on all parts of the Tennessee River. In an estimate of the total population of mussels in Wheeler Reservoir (mile 308-

316) of the Tennessee River, Scruggs (i960: 20) found the pigtoe

mussels making up 52 percent of the population averaging nearly

3 mussels per square yard with more than twenty and one half

million individuals. Fourteen of eighteen collecting sites on the Tennessee River recorded by van der Shalie (1939, Table I,

454) from Paducah, Kentucky, to Hiwassee River, Tennessee, re­

vealed Pigtoe Mussels. The Tennessee Valley Authority began an

investigation to determine the distribution and density of mussel

populations in July 1963, and the Tennessee River was examined 8

frcoi its mouth to Watts Bar Dam, a distance of 529 miles.

Habitat suitable for most freshwater naiads was found to be about

175 miles of river channel below the dams. The 1966 mussel

population of the suitable habitat was roughly estimated at 26

thousand tons with P. cordatum and Fusconaia ebena (Lea, 1831)

accounting for about one-third of this weight. The Pigtoe Mussel

was found at nearly every point sampled along the length of the

Tennessee River (T.V.A., 1966) from Paducah to Knoxville.

2. Habitat (Distribution)

This form, having many of the characteristics of other

species, is usually found confined to "beds" or concentrations

of a few hundred to many thousand individuals. Several miles of

river bottom may be completely devoid of mussels followed by

areas rich in this and other species. t-

Relatively strong current and relatively deep (20-30 feet)

water afford a suitable environment for the Pigtoe Mussel. The

current prevents accumulations of silt and probably provides a

high oxygen content.

Close observations while diving and searching for pigtoes

indicate the bottom surface to be composed of sand, gravel, or

'cobbles. However, under the coarse rocks, the composition is a

finer gravel, sand or mud bottom. The coarse rocks seem to 9 maintain the firmness and stability of the bottom in strong current. Frequently very large flat rooks occur in the mussel populated zones. Under and around the margins of these large rocks may be one to several mussels. Possibly some protection is provided by these rocks (Fig. 68, 69).

The beds of pigtoes in the Muscle Shoals Area of the

Tennessee River below Wilson Dam are at depths of from 5 to 30 feet. The plant growth that is closely associated with these mussels is attached filamentous algae and suspended (planktonic) material.

There are several other mussel species occupying the same general environment as the pigtoe at the collecting sites at

Mussel Shoals. Among these forms most often seen are Fusconaia ebena, Elliptio crassidens. E. dilatatus. Quadrula pustulosa,

Cyclonaias tuberculata. Tritogonia verrucosa. Amblema plicata,

Megalonaias gigantea. Obliquaria reflexa, and Lampsilis ovata.

Other mussel species are infrequently found.

Occasionally, gastropods are seen in the vicinity of the pigtoe mussels and quite frequently leeches are observed attached to the shells.

Fish species that have been seen are darters which as yet have not been caught and definitely identified but appear to be 1

10 the Logperch, Percina caprodes. The Longear Sunfish, Lepomis

megalotis. is usually close by and the Smallmouth Bass,

Micropterus dolomieui often followed our movements closely. The

Bluegill Sunfish, Lepomis macrochirus. frequents the general area.

The above fish species are near the mussel beds nearly every time they are visited. Other fish species are seen on rare occasions at these collecting sites.

Many of the Longear Sunfish were observed in June, 1968

spawning within a rather concentrated bed of Pigtoe Mussels.

This bed of mussels was located along the north bank of the old river bed in water about 15 feet deep and several (6 to 8)

Longear "nests" were seen among these naiads. IV. MATERIALS AND TECHNIQUES

Specimens used in the present study were collected over a five year period from beds in the Tennessee River. Live mussels were fixed directly in a 10 percent formalin solution containing a buffering salt, disodium phosphate, or in AGW

(75% ethyl alcohol, 5% glycerine and 20$ water) without previous anaesthetisation. Cellular affinity for stains seemed better when tissues were fixed in formalin solution, however, good results were obtained in both fixatives. The mussels were pegged and immersed in the fixative immediately. Later the shells were separated from the soft parts and placed in plastic bags with proper labels to maintain their specific identities. Laundry tags were attached to the edge of the foot of the soft parts with an identifying number for each specimen.

Soft parts were sectioned through the gonads and two slides of each specimen were made to determine the sex and condition of the gonad at the specific season each specimen was collected. These tissues were dehydrated in an ethyl alcohol series and cleared in toluene.

The assigned number was applied to the shell, the soft parts, and to the glass slides when specimens were sectioned

11 histologically. This number, in a record book, referred to a specimen collected at a specific place, on a specific date and, when sex was determined this information was added. Shell dimensions including height, width and length were recorded

(Stansbery, 196l).

Shell dimensions were measured with a special caliper constructed of hardwood having a plastic millimeter ruler attached to the side (Fig. 3). Measurements were made in three planes to the nearest millimeter. Length is the maximum antero­ posterior dimension of the shell (Fig. 4). Height is the mavirmim dorso-ventral dimension of the shell but does not include the (Fig. 5). Width is the maximum transverse dimension of the shell with both valves in normal position

(Fig. 6).

The tissues were embedded in paraffin wax. Sections were from 6-10 microns thick and stained with eosin Y and Harris1 hematoxylin. A few special stains were used to distinguish specific types of tissue or to demonstrate certain constituents

of fixed cells which were not made visible by hematoxylin and

eosin. These will be identified with the organ or tissue with which they have been used.

Drawings were made with the aid of a camera lucida. No absolute measurements are given in the text as reference can be Fig. 3.'—Caliper constructed of hardwood with millimeter ruler attached to the side and used in measuring the naiads in this study.

nuni-inoONIA11 4|tlWl tThtiiTnfmhtTliiiliiifiiilililLtit1

Fig. 4.—Ohio Pigtoe in position to measure length. 13 1

Fig. 5.—Ohio Pigtoe in position to measure height.

Fig. 6.—Ohio Pigtoe in position to measure width.

04 15 made to the scales given with the drawings and photomicrographs.

Serial sections were made of the smallest specimens because these were best for mounting on the relatively small glass slides and could be fixed whole. Even the smallest specimens were more than 25 millimeters in diameter.

At each visit to the collecting site, water temperatures, depths and atmospheric conditions were noted (Table l). Also, the bottom conditions and fish seen in the vicinity of the mussel beds were recorded.

Since the youngest forms are very difficult to find in a gravel substrate and because they are believed to be somewhat buried below the surface, many bottom samples from several habitats were screened through a series of sieve sizes in an effort to find small and very young individuals. Juveniles evidently comprise a very small percentage of the bottom contents. This method yielded nearly negative results despite many hours of labor. TABLE 1.—Wilson Dam outlet water temperatures at weekly intervals for 1964• The discharge of water in thousands of cubic feet per second reflects the rate of flow or current below a dam. For data of other years, see Appendix.

Temperature in degrees Monthly average of Centigrade for each week discharge in thousands of cubic feet water Month 1 2 3 4 5 per second

January 4 5 5 7 64.9

February 8 8 61.2

March 8 11 13 12 13 123.3

April 14 15 18 19 107.5

May 19 19 20 22 59.2

June 22 25 27 27 36.2

July 27 27 27 28 33.0

August 28 28 27 27 26 33.0

September 27 26 25 23 30.7

October 19 18 17 43.2

November 16 17 16 13 10 54.8

December 10 9 7 9 74.3

16 V. MORPHOLOGY

1. Methods

A. Gross Dissection

The Pigtoe Mussel is sufficiently large to dissect and locate systems macroscopically but details of the structural make-up cannot be discerned except by the use of histological sections. The soft parts of 350 preserved specimens were care­ fully removed from their shells by using the blunt end of a scalpel handle to work the anterior and posterior adductor muscles free on one side of the shell. Then the mantle and other parts were rather easily freed from the entire shell. These formalin or alcohol hardened specimens were returned to the original preservative after being properly tagged and numbered.

The anterior two-thirds of the soft tissue was cut off and discarded, leaving enough of the remaining to be pre­

pared and embedded in paraffin. This embedded part-contained gonadal tissue which revealed the sex of each animal. The shell was placed in a plastic bag and properly labeled. Measurements

of these shells were later made.

B. Histological

17 18

More than 300 specimens were collected at regular inter­ vals throughout 1964. These specimens, serving as the basis of this study, were embedded in paraffin and sectioned through the posterior one-third of the animal's soft parts so as to include the gonads. These sections were in the transverse plane.

Harris1 hematoxylin and eosin were used for staining. The sections were cut at 7 microns.

Small specimens were prepared and embedded whole in paraffin. These specimens were used for making serial sections.

Serial sections of the entire animal were made in the transverse, sagittal and frontal planes. Harris' hematoxylin and eosin were used for staining most of the sections. Several other stains were used to differentiate special tissues. Among those special stains used were iron hematoxylin for chromosomes, periodic acid-Schiff stain for components of blood, Van Gieson's stain for connective tissue fibers, Pianese III B stain for certain cytoplasmic material, and silver impregnations for nerve4elements.

2. Shell

A. General Description

The shell is, relative to other naiads, large and heavy.

Its outline is subtriangular (equilateral), rounded anteriorly, and somewhat flattened and produced posteriorly, especially in older specimens. The lower margin is gently convex in the 19 anterior part and slightly concave (emarginate) in the posterior part. The supero-posterior (dorsal) margin is gently curved.

The beaks are at or very near the anterior end of the shell, incurved, and pointed forward over a well-developed .

Beak is readily eroded away in Tennessee River specimens, especially in those over four years old. A coarse sculpture does exist however (Ortmann, 1919s 70). The shell is much wider anteriorly. The width is usually over fifty percent of the length.

In the samples which I measured from the Tennessee River, the width averaged 55 percent of the length in males while 56.5 percent in the females. This width averaged actually very ' nearly 1.5 millimeters more per individual female shell. The average length was only 0.3 millimeter more in the females than the males. While the width difference may appear to be so slight as to go unnoticed, a person familiar with this species of the Muscle Shoals Area may be able to detect shell differences, particularly in the older specimens. Of the 173 males measured, only 11 of them were more than 50 millimeters wide or just over

6 percent of the 1964 year sample. There were 25 females over

50 millimeters wide representing 17 percent of the samples.

While 12 of the males were under 40 millimeters wide, only 2 females were under this width and they were quite young specimens being only 51 or 52 millimeters long. These data support the 20 inference that very slight morphological differences may be readily discernable to the experienced eye.

In addition, the average height of the males wa3 only

1 millimeter less than the females. However, 38 percent of the females measured in the 19&4 year sample were more than 70 millimeters high while only 21 percent of the males reached this height. There were 18 specimens in the 1964 year collection which were indeterminate in sex on the basis of histological examinations. Either the gonads were so atrophied that there was no definite method of sexing the specimens or they had been cut away in preparing the tissue. If thrown away in the anterior part of the tissue, then these organs were probably greatly- reduced in size and not functional. These specimens were considered to be sterile. The "neuters" average width was 3.2 millimeters less than the males. They also were not as long nor as high as the known males and females. This became significant, however, when average percentage width to height was calculated and compared to the two sex groups. The neuter groups average width was 54-8 percent of the length, compared to 55 percent in males and 56.5 percent in females.

These data indicate that gonads (when functioning properly) cause a proportional increase in the shell width with the ovaries having a slightly greater effect than the testes. 21

Obvious sexual dimorphism in the shells occurs in many of the lampsiline (Subfamily Lampsilinae) naiad species as a result of eggs and embryos occupying the gills during a longer period of the growing season. In the Pigtoe Mussel, enlarged gills occur only a short time and would have less effect during low temperatures on shaping the shell of this species. But, enlarged gonads throughout a considerably longer period of the growing season could affect shell shape. This would be especially noticeable in the width but also in height and in . length.

The shell is compressed posteriorly, with a broad, rather distinct radial furrow or sulcus running from the beaks toward the post ventral margin. The shell surface is relatively smooth and unsculptured.

The epidermis is usually rather dark brownish becoming blackish especially in older specimens. Green to black capillary rays may radiate indistinctly down from the umbones for a short distance but disappear as the shell gets older. The growth rests are much darker than the remainder of the shell.

The are rather heavy and the nacre is clearly white. This very tough white pearly nacre has made the Tennessee

River Pigtoe a very desirable shell for cultured pearl nuclei. 22

It is tough, clear-white, and lustrous. This species is very

rarely tinted pinkish.

B. Ligament

The ligament of Pleurobema is external and situated

posterior to the beaks (opisthodetic). It is cylindrical on the

upper side (parivincular) with one edge attached to each valve

(Baker, 1928: 458). This structure becomes somewhat thickened

as the specimen gets older and develops a significant tension

opposing the action of adductor muscles. A single columnar

epithelial layer of the mantle adds layers of tissue to the

inside of the ligament. In gross examination of the ligament it

was noted to be layered toward the inside. No histological study

has been made of the ligament in this investigation.

C. Periostracum

The periostracum is a thin horny, pigmented layer of the

shell which is secreted by glands in a groove between the mantle

margins. Transverse sections of the mantle have revealed the

periostracum issues from folds in its margin in thin strands

(Fig. 7, 8). The fold of the mantle consists of tall compact

epithelial cells on one side pressed close to much lower

.epithelial cells on the other side. Within this folded tissue

issues forth the sheet of non-cellular substance called the Fig. 7.—Section of mantle margin showing periostracum layer as it is formed. 1Q0X

8.—Periostracal gland on margin of mantle with periostracum issuing from between the layers. 430X. 24 periostracum. This fold could very logically be called the periostracal gland as suggested by Sellmer (1967 •' 147). This secretion possibly becomes interrupted when the mussel is re­ moved from its environment and renewed growth of this is illustrated by a false growth rest. When undisturbed the strand of periostracum remains intact even when opening and closing the two valves of the shell.

3. Mantle "

The mantle is thin and uniformly two layers of epithelial cells with scattered connective tissue fibers forming a weblike mass of sinuses between the cell layers except at the free margins (Fig. 7) and along the dorsal side where the two lobes join and form the isthmus. The margins are thickened forming longitudinal folds and, posteriorly, the mantle lobes form the incurrent and excurrent apertures (so-called siphons).

The outer lateral epithelial layer is a single layer of low columnar epithelial cells resting on a rather thick basement membrane. Near the free margins, however, thi3 single epithelial layer changes to rather tall slender columnar cells with a different staining affinity than the low columnar cells. The cytoplasm of these low cells is agranular and slightly acid­ ophilic with numerous vacuolated spaces. Quite frequently 25 vacant clear spaces between adjacent cells indicate significant secretory activity. The nuclei form a relatively constant row toward the outside of the cells away from the basement membrane.

They vary in shape as a result of pressure from adjacent cells and the accumulation of secretions in the cytoplasm, but are oval to oblong in shape with one nucleus per cell. These nuclei are basophilic and evenly chromatic. The epithelial cells are taller dorsal to the point of attachment of the mantle to the shell at the and the cytoplasm stains more deeply.

Then, toward the free ventral margin these cells are replaced by a muscle layer which is attached directly to the valves obliquely at the pallial line. These muscle fibers extend as a bundle or fascicle down through the interior of the mantle between the epithelial layers terminating on the inner ventral margin of the mantle. The contraction of these muscle fibers withdraws the extended mantle margin as the valves are closed and aids in holding the opposite lobes of the mantle together.

Scattered muscle cells occur throughout the entire mantle and a very thin layer of muscle occurs just below the basement membrane of the outer mantle layer. This layer disappears dorsally toward the isthmus. This is understandable since the ventral half of the mantle is more motile than the upper half.

The inner layer of epithelium of the mantle is a single low columnar epithelial layer in the ventral half with very 26 numerous vacuoles and nuclei located toward the basal part of the cells. Cilia appear in patches along the free surface of these cells forming an irregular outline. These are probably arranged regularly in rows and thus particles entering the mantle cavity with the incoming water may be moved in a definite direction by action of these cilia. The dorsal half of the inner epithelial layer consists of low simple epithelium which is squamous and not apparently ciliated nor does it appear to have secretory characteristics.

The loose connective tissue between the epithelial layers is composed of scattered interlacing fibers similar to loose areolar connective tissue. The fibers are of both collagenous and elastic types providing numerous spaces or sinuses which are occupied by blood cells and connective tissue cells of many types. The blood cells will be described later.

The margin of the mantle is bordered by a single columnar epithelial cell layer. Beginning near the outside surface of the mantle border, the epithelial cells increase in height and closeness to each other. The nuclei of these cells are very elongated and deeply basophilic. These cells evidently produce the prismatic layer of the shell as indicated by their structure

.and location. The outer longitudinal folds of the outer margin of the mantle are bordered with cells as described above and 27 they produce the periostracum's outer layer (Fig. 7). The lower margin of the outer fold of the mantle consists of columnar cells with an abundance of pigment present in the cytoplasm. These cells apparently secrete a layer of the

periostracum containing pigments which color this layer of the shell. The pigmented cells closely resemble the pigmented epithelial cells of vertebrate which are color producing.

Between the epithelial cell layers are muscle bundles arranged in a variety of planes which are responsible for movement of the mantle margin.

The mantle is quite sensory containing scattered nerve fibers throughout, but more, near the free margin. Several features indicate possible light sensitive areas of the mantle margin. Special light receptors however, were not recognized in the mantle. The diffuse sensitivity to light in the clam, Mya arenaria, is confined to the . Under constant conditions of illumination, the siphon is extended but when illumination increases the siphon is withdrawn. The photoreceptors of the siphon of Mya are single cell photoreceptors each with a refractile body connected to neurofibrilHae (Light, 1930).

Concentrated amounts of black pigment granules occur in groups

of cells under the epithelial layer on the inner border of the mantle in the regions of the Pigtoe. The author ha3 28 noticed the response of naiads when a shadow occurred over the external apertures. The reduction in light intensity caused the valves to close or at least to contract the mantle margin temporarily, while under constant conditions of illumination, the mantle remains extended.

Dark adaptation in the clam has been studied rather carefully elsewhere. Sensitivity to light after a period of darkness is common to all photoreceptor systems. Hecht (1919) determined the course of dark adaptation in the clam, Mya. The animal was illuminated with a constant intensity of light for a prolonged period. This illumination was terminated suddenly and the animal was in total darkness. Immediately, the animal was stimulated with a flash of light and the reaction time was measured. This was repeated several times with the reaction time decreasing with increasing time in the dark.

In transverse section, the mantle lobes proceed dorsally and medially but as they approach the medial sagittal plane, they descend suddenly, then ascend to meet at the midline and form the ridge of columnar epithelial cells which secrete the inner layers of the ligament. These cells are tall, narrow, simple columnar and the cytoplasm stains rather deeply above the nucleus, while below the nucleus the cytoplasm is nearly colorless. The nuclei are large, very elongated, centrally 29 located and basophilic.

The outer epithelial layer of the mantle adheres closely to the inside of the shell but is most firmly attached to the shell at the pallial line.

Below this line the mantle moves more freely and has been designated the pallial curtain. There are no special papillae, membranes or folds on the inside of the inner margin of the mantle as described in several other species.

4. Foot

The foot of the Ohio Pigtoe is a well-developed, muscular organ occupying a large ventral area of the mantle cavity. It is wedge-shaped and attached below the visceral mass.

The foot can be extended to a distance which equals the naiad's length. When removed from the river bottom, the foot is often fully extended having anchored the animal firmly in the bottom.

The epithelium of the foot is thrown into folds or ridges and is a single columnar layer. The entire free surface of these cells is ciliated in the apical half of the foot but less cilia occur in the basal half. The columnar cells at the apical tip of the foot gradually decrease in height as they 30 approach the basal part and this same epithelial layer changes to a cuboidal form around the visceral mass. Beneath the

epithelial layers of the foot, numerous glands are present which

open laterally at the surface between adjacent epithelial cells

and these glands secrete a viscous material which is probably mucus. These cells are arranged in irregular masses and the cytoplasm stains deeply with hematoxylin. The nucleus of each cell is practically blotted out by the basophilic cytoplasm indicating a secretory function. The basic affinity of the numerous cytoplasmic granules results in the deep color. Many

of these cells appear to be stellate in shape. This, however, may partially be a result of shrinkage of these cells when fixed.

Toward the base of the foot, very faintly stained cells forming acini just beneath the epithelial layers make their appearance.

These cells evidently are mucus secreting cells as revealed by their arrangement and location, but possibly a slightly different form of mucus to the ones described earlier.

Much of the remaining tissue of the foot is composed of muscle fibers and muscle bundles running in various directions through its lower portion. These muscle bundles unite the two lateral faces of the foot. Dorsally and away from the

of the foot, these muscles are organized into more definite

layers. These layers run longitudinally, vertically, and diagonally through the foot making a variety of movements possible.

The center of the foot consists of loose connective tissue and numerous nerve fibers extend peripherally from the pedal ganglia to all of the muscle cells. Large blood vessels and sinuses occur in the loose connective tissue occupying the center of the foot.

Approaching the base of the foot and just below the epithelial layers the glands described earlier are replaced by sinuses through which the blood flows ventrally and posteriorly as it collects from other dorsal tissues.

5. Muscular System

A. Heart Musculature

In vertebrate animals three types of muscle fibers are recognized. Of the three, cardiac muscle is confined to the heart, while smooth and striated are more widely distributed.

The ventricle of the heart of the Pigtoe Mussel consists

of cardiac muscle fibers which branch and anastomose freely.

The fibers are multinucleated with the nuclei located in the

.center of the fibers (Fig. 9> 10). Between the branching and anastomosing fibers large sinuses are observed lined with Fig. 9.—Cardiac muscle viewed longitudinally. Note the anastomosing fibers typical of heart muscle. 430X

Fig. 10.—Cardiac muscle. Note the centrally located, chromatic nuclei. 970X

32 endothelial cells with nuclei bulging out along the surface of the muscle fibers. The nuclei of these endothelial cells are flatter and more chromatic and are not easily confused with the nuclei of the cardiac muscle cells. Intercalary discs, characteristic of cardiac muscle, were not observed since the ~ heart muscle was not prepared to demonstrate these units.

B. Pedal Musculature

The muscles which control the movements of the foot are more nearly like the "striated" type. The cells are very elongated, cylindrical and end abruptly. The nuclei of these V fibers are peripheral and elongated. Faint striations are observed in some of the longitudinal fibers but these are less prominent than in vertebrate animals. These cells are held together by delicate strands of connective tissue, the endoymsium. Perimysium encloses several such cells forming a bundle or fascicle. In cross sections of these bundles the endomysium can be seen as tiny threads between the adjacent muscle fibers whereas the perimysium surrounds the bundle.

Besides the scattered fibers and layers of muscle described earlier as part of the foot, there are the following better known ones. The anterior retractor muscles originate just posterior to the anterior adductor muscle scars on each valve. They extend posteriorly and ventrally to a point just 34

behind the esophagus where they become part of a muscle bundle

oriented perpendicularly to the retractor fibers. As the name

implies, these muscles withdraw the foot in contraction. The

posterior retractor muscles originate on each valve just dorsal

to the posterior adductor muscle scars. These muscles extend

anteriorly and ventrally to converge with each other forming a

muscle bundle. Thi3 bundle is joined by muscle fibers which

continue laterally down each side of the foot. There is a

circularly arranged muscle mass which originates dorsal to the

foot and as it approaches the foot the space enclosed by this

ring of muscle enlarges and contains a loose connective tissue.

This space receives blood from the anterior aorta and upon con­

traction of its muscular wall aids in extending the foot. The

foot expands with the volume of blood forced into it. This

muscular cone is closed dorsally but becomes a part of the lateral

muscle layers of the foot as it extends ventrally. This

structure plays a significant role in creating the pressure

that moves blood through the anterior organs.

C. Pallial Musculature

Pelseneer (1906) includes the adductor muscles with the

muscles of the mantle, designating them as transverse pallial

•muscles. These are the largest muscles in the Pigtoe. They

consist of parallel arranged bundles of muscle fibers which are 35

anchored firmly to the two valves. At the junction of the

adductor muscles to the shell valve the mantle cells are inter­

rupted and there is a concentration of nuclei, of the muscle cells

at this point. The adductor muscles respond rapidly to a

stimulus and can maintain a contracted state for long periods.

The adductors thus have striated muscle fiber bundles which respond by sudden contraction and smooth muscle fiber bundles that maintain sustained contraction. There is no definite arrangement of these two kinds of fibers, however, in this Ohio

Pigtoe. In the scallop, the oyster, and some other bivalves the striated bundles are designated as the quick muscles and the

smooth fibers as the catch muscles. In the clam, Mya arenaria. the adductor muscles and mantle retractor muscles receive 2 types of nerve fibers. One type discharges reflexly at high

frequency and causes rapid contraction; the other discharges

slowly and causes a prolonged tonic type of contraction with

low potential electrical response. Such a system is efficient

when a low level of contraction must be maintained and

occasional quick contractions superimposed (Pumphrey, 1938).

6. Digestive System

A. Alimentary Canal

The mouth opens ventrally just posterior to the anterior

adductor muscle and between the two pairs of labial palps. The 36 borders of the mouth are simple and continued as parts of the labial palps. These labial palps form a continuous membranous shelf overhanging the anterior aspect of the mouth (Fig. 11).

The mouth entrance is lined with simple columnar epithelium with a cuticular border for a very short distance before joining the esophagus.

The esophagus which is flattened to oval in cross section, has a lumen which has microscopic folds forming ridges in and grooves between the folds of epithelial lining. This organ is lined with simple ciliated columnar epithelium resting on a fairly distinct basement membrane. Some of these epithelial cells are noticeably shorter but have longer cilia and occur in groups along the length of the esophagus. These possibly serve efficiently as food grooves through which the small particles travel toward the stomach. These channels seem to spiral upward in the direction of the stomach rather than run length­ wise in the esophagus. The nuclei are mostly located in the center of the cells, are quite elongated, and basophilic. The columnar cells are progressively taller and more narrow from the mouth to the junction of the stomach. The cilia are longer on the taller cells and the gland cells decrease in number. The esophagus is abundantly supplied with mucus glands of the unicellular type. These are more plentiful in the lower esophagus but occur along the entire length of the tube. The Fig. 11.—Lateral section of mouth, esophagus and stomach. Note the post intestine posterior to the stomach. 5X

Fig. 12. Stomach, lateral section with folds. Note the dorsal caecum and digestive diverticula. 6X

37 38 cytoplasm in most of the epithelial cells is acidophilic and stains pink with hematoxylin-eosin (H-E) preparation. Just distal to the nucleus in the cytoplasm are scattered brownish pigment granules in many of the cells. Wedged between the bases of the columnar epithelial cells are granulocytes from the connective tissue below. These cells have eosinophilic granules and small eccentric nuclei that are very chromatic. Many of these cells appear to break up into smaller eosinophilic spherules and enter the lumen of the esophagus as a secretion while the others increase in refractile bodies within their cytoplasm. At the oral end of the esophagus below the epithelial layer a few scattered longitudinally arranged smooth muscle fibers occur which increase to a definite muscle layer toward the stomach. Outside the muscle layer is a loose connective tissue layer.

At the junction of the esophagus with the stomach the epithelial wall is evaginated toward the lumen of the esophagus and in the direction of the stomach forming a valve between these two organs. Simple ciliated columnar epithelium lines the lumen of the stomach and the variations in height, ciliation, and folding of this epithelial wall are variable and difficult to describe. There are numerous caeca especially a dorsal one and another on the right lateral portion of the stomach besides the small microscopic folds in several areas of the stomach. The 39

entrance to the stomach begins with a series of microscopic

folds of the epithelial lining (Fig. 13). Following the narrow

entrance to the stomach the dorsal wall straightens out and

extends posteriorly and dorsally becoming the anterior wall of

the dorsal caecum of the stomach. The epithelium along this

wall is moderately tall columnar with long functional cilia.

Approaching the dorsal caecum the epithelium becomes folded for

a short distance before forming the caecal wall where the

epithelial cells become low columnar to cuboidal in shape and

the cilia, shorter and more numerous. The caecum narrows at

the closed upper end and overall in longitudinal section, this

organ is shaped much like a cone. The epithelium, forming the

dorsal posterior wall of the closed end of the caecum, consists

of very tall narrow compactly placed simple epithelial cells

with eosinophilic cytoplasm and basophilic nuclei located nearer

the proximal end. The end of the crystalline style joins this

wall and according to Yonge (1932) this wall is the gastric

shield which erodes away the crystalline style as it is rotated

against it. The crystalline style is an acidophilic gelatinous

rod which originates in a style sac located in an evagination

of the gut parallel to the intestine, and separated from it and

from a shallow caecum on the floor of the stomach by a

• longitudinal fold (Fig. 14). The remaining epithelium which

forms the posterior wall of the dorsal caecum consists of Fig. 13 .—Median sagittal section of the esophagus and stomach with cellular detail of 3 regions as indicated. gastric SHIELD.

DORSAL CAECUM

VAlVE

STOM AC H LUMEN

INTESTINE ESOPHAGUS ."'VlVlWcuiUM 1 DUCT ^.DETRITUS Fig. 14.—Style sac and intestine in the center of the visceral mass. Note the digestive diverticulum branch and pedal ganglia. 5X

Fig. 15.—Prointestine and style sac. Note the ventral ridge between the closely associated organs. 100X 41 42 shorter, less eosinophilic, and actively secretory cells with mucus strands along the free surface distal to the cilia

(Fig. 15).

At the apex of each projecting fold of the stcmach into its cavity, the epithelial cells are somewhat taller; and more eosinophilic than elsewhere (Fig. 12). A shallow posterior caecum is located just dorsal to the intestine and may be important in secreting the crystalline style. Large quantities

of mucus can be seen on the surface of these cells combining

with strands from shallow folds in the stomach floor located anterior to the intestine's connection to the stomach.

The crystalline style according to Yonge (1932) is chemically composed of a protein matrix but absorbs amylase and is significant in digestion of starches extracellularly. Since the style is within the stomach cavity all the time, protein digestion apparently occurs elsewhere. It may occur in the digestive glands which join the stomach from the ventral side and are described under "Digestive Diverticula".

The caecum mentioned earlier located on the right lateral

border of the stcmach is best seen in a transverse section of

the stomach. It, too, is lined with a much folded wall of

columnar epithelium with cilia at least on the folded side. This

could be a food sorting organ because special cells at the closed 43 end take up food bits. These cells migrate into the connective tissue below. These are amoeboid cells occurring between and below the epithelial cells. These amoeboid cells have very small nuclei and are a greenish brown color in H-E preparations.

Similar cells are present in the larger ducts of the digestive gland and below the epithelial layers of the intestine. Their unusual color could be a result of numerous food bits in the cytoplasm which disappear as they are intracellularly digested or dissolved. These amoebocytes have been observed in each area representing the progressive changes described earlier.

The entire stomach is surrounded by a relatively thin smooth muscle layer which has circularly and longitudinally , arranged fibers. Outside this layer is the loose connective tissue stroma surrounding all of the visceral organs.

The intestine joins the stomach at its ventral posterior aspect and is separated from the style sac by a shallow longitudinal ridge. The style sac is on the left side of the intestine and lined with simple ciliated columnar epithelium which stains eosinophilic with' H-E preparations. The cilia are very tall and heavy with the acidophilic secretion from these cells quite abundant on their free surfaces. The nuclei of the cells are located naar the proximal end and are rather small, oval and chromatic. The lumen of the style sac contains copious 44 amounts of the secretion produced by the above cells.

The longitudinal fold that separates the style sac and the intestine is ventrally placed and includes the epithelial lining plus connective tissue fibers from the underlying area.

Simple ciliated columnar cells which line the right portion or true intestine are tall and narrow with the nuclei more centrally placed. The nuclei are very elongated and deeply chromatic.

Cytoplasm distal to these nuclei is finely granular and stains lavender to blue in H-E preparations in contrast to the eosinophilic affinity of the style sac cells. In transverse sections of the style sac-intestinal tube, the color contrasts of the two areas are quite noticeable. Below the nucleus of the intestinal lining cells, the cytoplasm is not as granular nor as basophilic. Empty spaces between the cells described above indicate significant amounts of secretion. Solvents, in preparation of these cells, may have dissolved the secretion from them leaving the vacant spaces.

Surrounding the epithelial layer of the style sac and intestine is a thin circular muscle layer followed by loose connective tissue. The intestine extends posteriorly, then, dorsally, then anteriorly following the curvature of the foot.

This tube then folds abruptly dorsally and posteriorly retracing the periphery of the foot until it reaches a ventral median 45 position in the visceral mass. It again folds back above itself running posteriorly, then turns above itself again, manning anteriorly, then arches dorsally and is directed posteriorly, extending through the pericardium and heart beyond the posterior adductor muscle before turning ventrally to terminate just dorsal to the excurrent aperture (Fig. 70).

The intestine is approximately k to 5 times as long as the foot but coiled back and forth upon itself as described above within the visceral mass (Fig. 16). The tissues forming the intestinal wall remain much the same as previously described up to that first point where it folds abruptly above itself. At this point the style sac lining disappears and only intestinal epithelium remains. The tube also changes its overall shape to a dorso-ventrally flattened organ (Fig. 17). The first length of the intestine is, for descriptive purposes, designated the prointestine. The next length is called the midintestine followed by the postintestine which arches dorsally and poster­ iorly to join the rectum.

The midintestine is lined with simple ciliated columnar

epithelial cells which are very tall and narrow and have

centrally located nuclei. These nuclei are extremely elongated and basophilic. The cytoplasm of these cells is lavender to

blue with H-E, Numerous mucous cells of the goblet type occur Fig. 16.—Lateral section of the intestinal coils. Note where style sac ends and mid-intestine begins. 5X

Fig. 17.—Transverse section of the midr-intestine. Note the shape changes to a flattened tube. 5X 46 47 between the cells described above. A thin circular muscle layer surrounds the epithelial layer followed by a layer of loose connective tissue.

The postintestine begins in the central area of the visceral mass just beyond the last 180 degree fold and includes this anterior extension plus the dorsal arch which joins the rectum as it passes through the pericardium (Fig. 18). This part of the intestine is characterized by having a dorsal infolding of epithelium and connective tissue forming a typhlosole.

The cells and tissues lining this section are so nearly like those of the midintestine that a separate description is omitted.

The rectum continues as a straight tube posteriorly and contains the typhlosole which originated in the postintestine.

The main differences in the postintestine and rectum are in the position of the typhlosole, and the cells which line this fold.

The typhlosole in the rectum is a ventral fold into the lumen of this organ, and the simple columnar epithelial cells bordering it are taller with granular eosinophilic cytoplasm. There are fewer mucous cells of the goblet type than in the same parts of the postintestine. The epithelial layer of the rectum is resting on a basement membrane outside of which is a connective tissue layer, surrounded by a smooth muscle layer. The circular muscles are covered by the pericardial membrane. This is a very .

Fig. IB.—Rectum as it passes through the pericardium. Note the ventral nuclei in the columnar cells of the typhlosole. Mallozy-Azan stain. IJOOX

Fig. 19.—Chemosensory tissue on the posterior margin of the rectum near the anus. 100X

48 49 delicate simple squamous epithelial layer.

Near the end of the rectum the circular muscle layer is somewhat thicker and the typhlosole disappears. At the anus the wall of the rectum consists of simple ciliated columnar epithelium. Peripheral to this epithelium a circular muscle .• layer occupies the anterior side •while on the posterior side this same area has numerous neurons which communicate with a small mass of sensory tissue. The posterior side of the rectum bulges into the cloacal chamber and is covered with low cuboidal epithelium containing pigment granules except at one point of enlargement which has the sensory tissue above composed of ciliated columnar epithelium (Fig. 19). This is believed to be an which is discussed under "Nervous System". The • cuboidal epithelium mentioned above which covers the posterior side of the rectum is continued as the cloacal lining. The anterior side of the rectum has loose connective tissue which is known as adventitia in vertebrates rather than the cuboidal epithelium. This adventitia blends in with similar tissue from surrounding organs and anchors the rectum in position. Yonge

(1941) states that the infolded wall of the postintestine and rectum is not a true typhlosole in Malletia. a protobranch, because it plays no role in absorption. He states that the intestine functions exclusively to form feces and the intestinal 50 fold is probably involved in molding said conveyance of the feces.

However, in the Pigtoe Mussel the fold is more than just an increase in the height of the epithelial cells as in proto- branchs. It possesses connective tissue, a few muscle fibers, and it is highly vascularized. Structurally, it fits the description of a typhlosole.

B. Digestive Diverticula

Digestive diverticula, "hepatic caeca" or "liver" are names which have been applied to the compound tubular gland which opens into the stomach on its ventral aspect. This organ has two main lobes. The right lobe occupies the right and ventral side of the visceral mass below the stomach and connects to the stomach floor just posterior to the esophagus by a duct on the right side. This duct branches dichotomously into many smaller ducts which eventually end in smaller tubules in the visceral mass (Fig. 20). The left lobe occupies the ventral, lateral and dorsal sides of the stomach joining the stomach by a duct from the lower left side. The left lobe is somewhat larger and more expansive than the right lobe possibly because a caecum of the stomach, on the right, occupies space that limits growth of the right lobe.

Thisccmpound gland has at least three morphologically different ducts. The duct system is constructed in much the Fig. 20.—Digestive diverticula. Note the irregular height of the columnar cells in the larger duct. 100X

Fig. 21.—Transverse section of the secreting acini of the digestive diverticula. Note the pale staining mucous cells and darker staining serous cells. 430X

51 52 same way as the ducts in similar organs (salivary glands, liver) of vertebrates and their lining cells have a similar affinity for stains. These ducts are designated the excretory, secretory, and intercalary tubules because of this similarity. The excretory ducts are those that join the stomach. These are lined vrith simple ciliated columnar epithelium and the epithelial cells vary in height creating a highly irregular surface

(Fig. 13 ). Occasionally, the connective tissue below the epithelium is included in the folds. The tallest cells are very narrow with basophilic and slightly granular cytoplasm. The shortest cells are more chromophobic. Both types have basophilic oval nuclei located near the center of the cells. The secretory ducts join the excretory ducts and are more numerous. They lead to each lobule of the digestive gland (Fig. 22). The epithelium of these tubes is composed of simple columnar epithelial cells which vary in height creating an irregularly shaped lumen when viewed in transverse section. Most of the cells have a rather basophilic cytoplasm and nucleus. The nuclei are oval and near the bases of the cells. Mucus secreting cells are interspersed throughout the wall and appear chromophobic in H-E stained slides.

Cilia are sparce or non-existent on the free surfaces of these cells. Copious amounts of secretion from these cells may 53 occupy the lumen and surround the food particles swept into this organ.

The intercalary tubules are very short but represent the bulk of the digestive organ. These tubules are branches of the secretory ducts and connect to it but are closed on the opposite end. They are lined with simple columnar epithelium of a rather low form. Some of these are cuboidal and sometimes they may be nearly squamous. Their shapes may be related to seasonal secretory activity. Two kinds of cells make up the wall. Mucous cells are the most numerous and these cells have very weakly acidophilic cytoplasm with basally located nuclei which are oval and basophilic (Fig. 21). Serous cells make up the second kind and these are wedge-shaped cells or they form crescents around the bases of the mucous cells. These serous cells' cytoplasmic granules stain deeply basic and have round to oval nuclei centrally placed. Only about 20 percent or less of the wall consists of serous cells scattered among the chrcmophobic mucous cells. Both kinds of cells contribute to the secretion that collects in the lumen. Peripheral to the epithelium of the excretory and secretory ducts is a circularly arranged muscle layer. Muscle fibers are observed among the smallest tubes but not in the form of a layer.

Ciliary action plus muscular action apparently provide Fig.22.—Lobule of the digestive diverticulum showing cellular detail of a secretory duct (1) and an acinus (2).

SECRETORY DUCT

X.S. of ACINUS

L.S. OR ACINUS

.GROOVE

• dm m MUCUS

.VACUOLE

MUSCLE CELL

MUCOUS CELL

ACINUS BASAL NUCLEUS .SEROUS CELL

SEROUS CRESCENT 55 the motility essential in moving food particles from the stomach into the most distantly located tubules of the digestive organ.

These food particles may be extracellularly digested, at least in part, in the lumina of the tubules of the digestive gland.

The molluscs provide the most varied array in combinations of intracellular and extracellular processes. Two specializations are of particular interest. One of these is the digestive gland, made up of branched glandular follicles which communicate with the stomach by a system of ciliated ducts. The curious process in connection with the intracellular digestive activities of the epithelial cells is a fragmentation of the.outer border of the cell to form spheres containing the food vacuoles together with waste products and some enzymes. These then pass into the stomach and may be the source of some of the enzymes there. The other special feature is the crystalline style which is a thick gelatinous rod concentrated with enzymes and rotated by strong cilia which force it gradually into the stomach where it is rubbed against a gastric shield resulting in a release of its enzymes and a mixing of the stomach contents (Owen, 1956).

Many of the epithelial cells have been observed with food bits occupying their cytoplasm. These particles are apparently digested intracellularly.

Much of the digestive process is accomplished by the 56 stomach and digestive diverticula but with the long rather complex intestine it seems some digestion and absorption must surely occur along its length. Amoeboid cells are often seen among the epithelial cells of the intestine wall which resemble those in the stomach caeca walls and hepatic glands. These cells are phagocytic but just exactly how they function in the intestine of Pleurobema is unknown. Lamellibranchs digest protein, fat, and disaccharides inside the cells of the digestive diverticula and in the wandering amoebocytes which are abundant throughout the gut. The amoebocytes migrate into the wall and lumen of the gut, charge their bodies with food particles, digest them, and make their way again into the tissue spaces of the animal (Yonge, 1923). Churchill (1924) found that juvenile mussels will pass particles through the rectum in from one to five hours after ingestion. This varies roughly with the size of the mussel. One reason why food could be processed this rapidly is the fact that the consumed bits are very small when they enter the mouth including protozoa, diatoms, and minute detritus or decayed debris. A given .mass in the form of a number of small particles has a greater surface in contact with enzymes, hence it is digested faster.

7. Circulatory System

A. Heart 57

The heart occupies a dorsal, posterior position in the pericardial cavity. It surrounds the rectum and consists of the muscular ventricle and two thin-walled atria. Each conical atrium joins the ventricle laterally at a valve (Fig. 23) which is open as blood enters from each atrium but closed at contraction of the ventricle. The atria are very thin-walled organs having only simple squamous endothelium and an extremely thin circular muscle layer. The muscle cells are not consistently present in the walls leaving only a single thin cell layer making up its thickness. The ventricle when viewed in transverse section through the valves is somewhat shield shaped (Fig. 23). A longitudinal section of the ventricle is observed to be long and tubular. In a frontal section through the heart of a

Pigtoe 4 centimeters long, the ventricle's posterior limits were approximately 1 centimeter from the posterior end while the anterior limits were about two and one-fourth centimeters from the posterior end. The ventricle was about one and one-fourth centimeters long with the thickest part of the wall posterior to the atrio-ventricular valves. The valves are located about half way between the ends. Most of the cardiac muscle fibers which make up the bulk of the heart wall are oriented longitudinally and run parallel to the rectum. However, other fibers or fiber branches extend in several directions so that the lumen of the heart is a very irregular space. These muscle 23.—Transverse section of the ventricle and rectum. Note the thin walled atrium on the left and the heart valves. 35X

24. Dorsal aorta with lateral branches which convey blood to the inner laminae of inner gills. 35X

58 59 fibers are arranged in a way that their contraction not only creates the pressure to move the blood but accomplishes opening and closing of the valves. Endothelium appears to cover all of the muscle fibers lining this space but it is difficult to trace from a cross section.

Blood collects in the two atria from the excretory organs, gills, and the mantle. Blood passes through the narrow ends of the atria into the ventricle by way of the atrio­ ventricular valves or ostia. Ventricular contraction forces the blood anteriorly and posteriorly through arteries. The electrocardiogram of freshwater mussels consists normally of a diphasic component near the beginning of contraction and a slow wave associated with contraction. The interpretation of the rapid and slow components in the molluscan electrocardiogram is uncertain, but the fast wave probably represents spread of excitation. There is some variability in shape, according to electrode placement (Taylor, 1941). The heart rate is 3low when compared with homeothermic animals and the rate may be altered by varying temperatures and certain chemicals. No attempt has been made in this study to determine the average adult heart rate. Two mussels from the same tank may have a significantly different heart rate. When a shell valve is removed to expose the heart, many new factors undoubtedly influence the normal 60 heart rate. Ellis (1931s 511) describes a method of viewing the heart without altering the physiology of the animal by re­ moving its shell. He ground the shell down adjacent to the heart providing a "window" through the shell.

B. Arteries

An anteriorly directed blood vessel above the rectum known as the anterior aorta conveys the blood to the viscera, foot, anterior adductor and other medially placed parts anterior to the heart. This artery originates at the point where the most anterior extent of the ventricle is attached to the ventral side of the rectum. At first this connection is narrow but increases until only a dorsal space remains which is the anterior aorta. This vessel is lined with endothelium surrounded by a thin circular muscle layer which is thicker on its ventral aspect blending in with muscle and connective tissue fibers of the rectum. The endothelium on the dorsal surface of the anterior aorta is continuous with the pericardial lining. This artery first gives off laterally a small right and left branch

(Fig. 24) through which blood flows into a network of sinuses at the periphery of the foot. In addition, these branches provide blood to the reflected folds of the inner gills, anterior to the point of attachment of these folds to the median visceral mass. The next branch of the anterior aorta is one directly 61 ventral to itself (Fig. 25) which also divides almost immediately into another median branch just beneath itself. Both of these branches extend anteriorly but ventrally just posterior to the stomach and disappear in a mass of loose sinuses. The original anterior aorta continues anteriorly to a loose sinus network dorsal to the digestive glands and esophagus where it loses its identity. The blood gets an extra measure of pressure from the contraction of the ring of muscles described earlier surrounding most of the blood spaces dorsal to the foot and anterior to the aorta. The blood eventually collects from all the median anterior sinuses into the large median pedal sinus. From this sinus blood is directed dorsally through veins which cannot be traced any great distance. The blood enters the narrow isthmus medial to the base of the gills and flows into the vena cava lying medially and ventral to the pericardium. This vein is closely associated with the folded walls of the kidneys. Blood may be filtered by the kidney tissues and much of it passes through the pericardial glands to the gills. From the gills the blood flows into a sinus network dorsal and posterior to the visceral mass and base of the gills. This blood then collects in the large right and left atria of the heart.

Blood flows posteriorly from the heart through a vessel ventral to the rectum called the posterior aorta. This vessel Fig. 25.—Anterior aorta with main division directly beneath it. 35X

IntMtin* -5' kjj&ai ifc' ' ft

Fig. 26. Pedal sinus in median section of the foot. Note the numerous intestinal sections in the visceral mass. 5X

62 63 almost immediately divides near the posterior retractor muscles into two longitudinal branches that are slightly below and lateral to the last length of the rectum before it turns abruptly beyond the posterior adductor to enter the cloacal region. These lateral branches divide again near the posterior adductor to supply blood to the mantle, posterior adductors and the terminal end of the alimentary tract. Blood flow is through loose sinuses of the above organs from the posterior vessels in an anterior direction then dorsally to the numerous sinus spaces leading to the atria. The posterior aorta is lined with endothelium and a thin layer of circular muscles.

C. Sinuses and Blood

The largest sinus is the one located medially in the ventral part of the foot, the pedal sinus (Fig. 26). This space collects the blood from nearly all the organs and parts supplied by the anterior aorta. There are other sinuses which are directed dorsally from the pedal sinus and these connect to the vena cava described earlier.

Two lateral sinuses ventral to the pericardial glands and close to the bases of the inner gills collect blood which has passed very closely to the kidneys (Fig. 27). Rather capacious sinuses posterior and dorsal to the atria collect Fig. 27.—Transverse section of the dorsal half of a Pigtoe Mussel approximately mid-way between the anterior and posterior end. Note the large sinuses which serve as blood reservoirs. 6X

Fig. 28.—Amoebocytes in-the ventricle of the heart. Note the small chromatic nucleus of each. 970X 64 65

blood enroute to the heart from the mantle and gills. The small

sinuses consist of a latticework of white and elastic fibers

and a scattering of smooth muscle fibers, while the large ones

have very large open spaces with little connective tissue.

Amoebocytes are very numerous in the heart. These cells

are less numerous in vessels and sinuses. They may also be seen

in extravascular or intercellular spaces. Their role in intra­

cellular digestion has been described by Yonge (1926) and others,

and several workers have described the phagocytic activities of

these cells in bivalves. Sellmer (1967) quoting Tripp (1958)

states that intravascularly-injected bacteria, erythrocytes and

yeast cells are digested by amoebocytes of the oyster.

These amoebocytes are nucleated and have no pigment but

the cytoplasm stains acidophilic with H-E preparations. At

least some of these cells are apparently generated in the heart

since mitotic figures have been observed in clusters of these

cells close to the wall of the heart (Fig. 28). In the heart

these cells are large, stellate, and have vesicular nuclei.

Their cytoplasm is acidophilic but not very granular. In the

active circulation the amoebocytes are smaller, more oval in

shape, with definitely more granular cytoplasm. These

• granules are large and acidophilic while the nucleus is very

much smaller, eccentrically located, and deeply chromatic. 66

There are two or three sizes of amoebocytes all of which have approximately the same staining affinity. The amoebocytes which appear more often in a phagocytic role have been the largest ones which are much like eosinophils in human blood.

The mantle appears to contain a high percentage of the amoebocytes and the pericardial gland contains a noticeably large number.

The blood is associated not only with nutrition, respiration, excretion, and the general well-being of the individual as in the vertebrate animals, but the blood also has a special mechanical function in connection with the peculiar locomotion of freshwater mussels (Ellis, 1931)• The foot, described earlier, may be expanded during activity to many times its contracted size by an inflowing of blood. A signi­ ficant volume of blood is required for this action and a storage area for this blood when the foot is retracted is essential. The volume of blood in proportion to the size of the animal is large and the numerous sinuses described earlier apparently serve as reservoirs for this blood.

Although no data are available for the exact total volume of the blood of freshwater mussels, suggestions have been made on how to obtain this information in a mussel species at a given time. Weinland (1919) describes the fluid as that which could be 67 drained or easily pressed from the soft parts of an animal.

This would not be pure blood nor would it represent all of the blood, but it would provide a rough estimate of the amount. He found this body fluid as described above, to constitute from 46 to 57 percent of the total weight of the animal, including the shell, or nearly two and one-half times the weight of the drained soft parts.

The blood volume is largely composed of the clear viscid fluid plasma while only a very small proportion of the blood is the amoebocytes. Drawn blood does not clot into a solid mass, but in from one to five minutes after removal from the body of the animal, particles of a whitish, opaque coagulum appear, suspended like bits of curd in the more watery, uncoagulated fluid. Dried mussel blood has a very faint, yellowish brown color (Ellis, 1931).

The specific gravity of the blood of fresh-water mussels is approximately 1.002 while the pH is definitely alkaline and averages about pH 7.9. The total solids, organic materials, blood sugars, blood gases and inorganic salts are constituents of the blood which have been analyzed and the values of each recorded by Ellis (1931).

8. Ctenidial System 68

A. Labial palps

The palps consist of two pairs of appendages which are evaginations from the inside wall of the mantle and lateral to the base of the foot. Each pair begins anterior to the mouth as short thick folds surrounded by epithelium and filled with loose connective tissue with scattered amounts of muscle fibers.

The epithelium is smooth on the unapposed surfaces and ridged on the apposed surfaces of each pair. They hang suspended in the mantle chamber. The junction between the two external palps of each pair occurs anterior and dorsal to the mouth and forms the upper Up. The internal palps of each pair fuse medially forming the lower of the mouth. The pairs extend post-, eriorly seme distance behind the mouth. They are, in overall appearance, somewhat leaf-shaped.

The unapposed epithelium of the external palps is simple cuboidal and continuous with the inside epithelium of the mantle.

These cells increase in height as they approach the distal end of the palp and contain numerous mucous cells. The unapposed epithelium of the internal palps is simple cuboidal and similar to the upper epithelium of the foot. Numerous mucus secreting cells are among the cells forming this layer and patches of cilia appear on their free surface. These cells stain rather eosinophilic in H-E prepared slides. These cells get 69

progressively taller as they reach the apex of the palp.

The epithelium lining the apposed sides of both internal

and external palps is simple ciliated columnar epithelium. The

cells are very tall and narrow with cytoplasm slightly basophilic

in most of the cells, while the remaining cells are chromophobic and represent mucus forming types. The nuclei are basophilic and very elongated. These nuclei are centrally placed in the

cells. The cilia are rather dense and longer in some areas than others. The folding of the apposed walls of each palp is in the form of vertical ridges with grooves along the sides of the ridges. The number of folds is not uniform from specimen to specimen but uniformity of the folds does seem to occur in

each pair of palps. These folds include the epithelium and underlying connective tissues. The grooves are composed of

shallow or very low cells surrounded by tall columnar cells.

Numerous acidophilic granular amoeboid cells occur between the

bases of the columnar cells and in the sinuses of the connective

tissues below. These are probably phagocytic and play a role in

intracellular digestion as well as being a part of the

defensive mechanism of the animal. Movement of the whole palp

is accomplished by the muscle fibers in each palp but

especially by muscular tissue in the mantle and at the base of

the pairs of palps. 70

The movement of food particles along the palps is in an anterior direction over the apical ends of the ridges to the mouth while other particles have been observed by Churchill

(1924) to pass down the furrows between the ridges to the ventral edges of the palps. He also described a longitudinal groove on the apposing face of each palp immediately ventral to the line of union of the two palps which functions especially when the mussel is feeding in heavy suspensions, This groove was located in the Pigtoe in the area described.

The vertical groove described earlier as located along the posterior side of each ridge near its base, and the more shallow anterior groove near the apex of each ridge are said to mark the boundaries between the forward-beating cilia on the ridge and the downward-beating cilia in the groove. There is never a reversal in the direction of the beating of the cilia according to Churchill (1924).

6. Ctenidia

The ctenidia or gills have been rather carefully des­ cribed by a number of workers. Among these are the anatomical descriptions made by Ortmann (1911). Others have dealt specifically with the physiology of the gills related to respiration and feeding, while some have been concerned with the function of the g-t n» as brood chambers. Only certain aspects 71 of the ctenidia will be discussed in detail here, but a summary of several aspects seems to deserve a place in this discussion.

In most lamellibranchs, a has an outer and inner demibranch or lamina. The main elements of the gills are the gill-filaments which are cylindrical or compressed rods.

These filaments arise from a common base between the foot and the mantle, and hang down into the branchial chamber. These filaments are doubled back upon themselves, the outer row outwardly, the inner row inwardly. Thus each gill is composed of two layers or lamellae and there are two gills on each side of the foot, an inner and an outer one. The tissue between the two layers of each gill develops and connects the lamellae • together but between these connections are numerous regular interlamellar spaces which communicate directly with the out­ side surface by way of tiny pores. In most naiads the inter­ lamellar cavity of each gill is thus divided by the connective tissue between the two lamellae into a number of smaller spaces called water-tubes which are generally oriented dorsoventrally between the filaments. The secondary lamina of each gill which is reflected dorsally connects with tissue along the base of the

original lamina but is somewhat separated from it in a way that an open space occurs between the two layers of each gill. These spaces open into the suprabranchial spaces and are very 72 important in a number of functional activities to be described later.

The gill filaments are deeply basophilic and homogeneous rods that resemble chondrin of vertebrate animals. Some of the cells which evidently secrete the filaments occupy lacunae within the filament. The cells covering the outside of the lamellae are columnar with an abundance of cilia. These cilia are of varying lengths related to the specific location of each cell along the surface. The cilia facing the surface are short while those along the entrance pores and folds created by the filaments are longer (Fig. 29-30). These ciliary tracts not only aid in sweeping food and water along but they are important

"sorting devices" and are divided into two oppositely moving tracts (Atkins, 1936).

Ortmann (1911) states that in the genus Pleurobema the inner lamina of the inner gill is only attached to the abdominal

sac at the anterior end, leaving a slit-like communication open

between the suprabranchial canal and the branchial chamber.

This was found to be true in all Pigtoes which were sectioned in

this study (Fig. 31). The posterior point of connection of the

inner gill layer is just anterior to the exit openings of the

sperm ducts, nephridiopores and oviduct openings. The

significance of this is not known, but, it would seem that a Fig. 29.—Transverse section of the gill. Van GiesQn*s stain. 120X

Fig. 30.—Section of gill through a water tube. Note the ostia and long cilia at the entrance of the ostium. Van Gieson's stain. 430X

73 31.—Dorsal transverse section of the inner laminae of the inner gills. Note the gills are not attached at this point. 35X

32.—Dorsal transverse section where inner gills are connected. 15X 74 75 stronger current of water in the closed part of the inner suprabranchial chamber would aid in moving those products posteriorly and throughout the branchial chamber.

Posterior to the abdominal sac and foot the inner laminae of the two inner gills unite in the median line of the body (Fig. 32). This again separates the suprabranchial cavities from the branchial cavity. Just posterior to the above connection the dorsal ctenidial connections of the primary laminae are lost. This provides a single dorsal space separated from the branchial chamber. This dorsal space has been designated the cloacal chamber and it connects to the exterior by the dorsal or excurrent aperture. The cloacal • chamber is bounded on its lateral margins by the reflected or secondary laminae of the outer gills which remain firmly attached to the mantle tissue. The dorsal boundary of this chamber is smooth and composed of simple cuboidal epithelium similar to that lining the interior wall of the mantle. The ventral boundary is irregular being formed by the connected edges of the four gills (Fig. 33). Ortmann (1911) states that in a posterior view of the cloacal chamber, its floor is formed by a short, simple, horizontal bridge, from which the gills hang down. He calls this bridge the diaphragm. Fig. 33.—Dorsal transverse section through cloacal chamber. Note the floor of this chamber is formed by the U connected gills. 10X

Fig. 34•—Transverse section of female cuter gill. Note the square shape of water tube between the septa which is typical of marsupial gills. 100X 76 Fig. 35.—Ventral edge of inner gill. Arrows indicate the direction food particles are carried by the cilia.

.endothsli*1 cell

CMTATED'Y : EPI TH E L I O M capillary

WATER TUBE

CONNECTIVE TISSUE.

C ILIATED GROOVE

77 78

The interlamellar tissue extends from near the base of the gill to its edge in the form of septa or continuous partitions and this tissue is vascular. Between the septa in

Pleurobema there are from 15 to 20 filaments. The above characteristics describe the gills of male mussels and the inner gills of female mussels. The outer gills of females differ from the other gills in having more septa per unit of gill length and fewer filaments from one septum to the next with an average of 10 - 15 in those counted (Fig. 34). It is possible to distinguish the sexes by macroscopic examination of the outer gills when held up between a bright light and the eyes.

The more crowded septa are present in the outer gills of all females whether or not the gill has developing embryos.

The shapes of the water tubes in males and non-marsupial inner female gills, in cross section, are elongated in the longitudinal direction of the gills because of the widely separated septa (Fig. 29). Females however, have more nearly square water tubes in cross section as a result of more crowded

septa. These septa in marsupial gills are folded and wrinkled but in males the septa are simple epithelium and not folded.

The edges of the marsupial gills may vary in different mussel species but in the Pigtoe there is no modification from the

condition in males. 79

Allen (1914) has made a very thorough study of the feeding process in freshwater mussels. He states that the food

particles are carried into the mantle chamber by the currents induced by cilia on the gills. These particles are covered with mucus and aggregate into clumps. Material falling on the

outer surface of the outer gill is carried upward by ciliary action to the dorsal edge of the gill and then forward to the

palps in the groove formed by the juxtaposition of the gill and mantle. Food striking the inner surface of the outer gill moves upward to the dorsal edge, where it passes to the outer face of the inner gill. The cilia of this gill beat downward, and all material reaching it by any means is carried downward to the ciliated groove on the ventral edge (Fig. 35). In this groove it is swept forward to the palp. The ventral

edge of the outer gill is rounded off, while the inner gill

possesses a longitudinal furrow which functions to move the

particles forward as stated above.

9. Nervous System

The description of the nervous system is based on a

study of serial sections. Special stains used were Bielschowsky-

Type stains containing silver nitrate. Certain random slides

were stained with these metallic stains in an attempt to locate

nervous tissue. Only major structures can be considered as eo definitely located and described.

The parts which were located and identified are arranged in the categories below.

(1) Cerebro - pleural ganglia:

- anterior adductor nerves

- anterior pallial nerves

- cerebral commissure

- cerebro-pedal connectives

- cerebro-visceral connectives

- renal nerves

(2) Pedal ganglia:

- pedal nerves

(3) Visceral ganglia:

- posterior adductor nerves

- branchial nerves

- posterior pallial nerves

- siphonal nerves

A. Ganglia and their branches

In primitive clams as Nucula and Solenomya the cerebral 81 and pleural ganglia are distinct and separate (Pelseneer, 1906) but in the more advanced species, these ganglia are fused into a single unit on each side as in the naiad, Pleurobema.

These cerebro-pleural ganglia are located somewhat lateral to the esophagus just dorsal to the base of the labial palps and the mantle (Fig. 36). They are well separated from each other but connected by a stout commissure which arches anteriorly and dorsally in front of the esophagus. Several branches have been observed which leave these ganglia. The medulla of the cerebro-pleural ganglia is faintly stained and consists of numerous fibers and a few cell bodies having nuclei.

The periphery of the ganglia is a thin cellular layer with nuclei that are chromatic and with prominent nucleoli in Mallory-Azan preparations. The cytoplasmic processes of many of these cells can be followed into the medulla while very few processes are directed toward the surface of the ganglion. The most common cell type is a typical multipolar neuron (Fig. 37). At the points where nerves emerge from ganglia the fibers of the medulla are directed out through the interrupted peripheral shell or cortex. This arrangement seems to be a pattern common in the ganglia and nerve branches in most vertebrate forms of animal life. Fig. 36.—Transverse section through cerebro-pleural ganglia. Note the labial palps and esophagus. 1QX

Fig. 37.—Multipolar neurons. The typical cells of the ganglia of the Pigtoe Mussel. 600X

82 83

The anterior pallial nerves are rather large and directed anteriorly and ventrally into the mantle lobes where they branch profusely and are lost. The anterior adductor pair of nerves is directed to the anterior adductor muscles where they branch and are no longer visible. Small fiber tracts are observed connecting to cells at the surface dorsal to the base of the anterior mantle.

The cerebro-pedal connectives are directed posteriorly and lateral to the esophagus and stomach, then ventrally into the floor of the visceral mass and base of the foot. These connectives join the pedal ganglia in the medial area of the foot ventral to the gonads. Not far dorsally and somewhat lateral to the pedal ganglia, are the statocysts, but, it is generally agreed (Woortman, 1926) that these sensory structures are innervated by branches from the cerebro-pleural ganglia which leave the cerebro-pedal connectives as these balancing organs are approached. These statocyst nerves could not be traced to their points of origin in this study. They do extend from the statocysts toward the median line and somewhat dorsal to the pedal ganglia, however.

The cerebro-visceral connectives leave the cerebro- pleural ganglia with the cerebro-pedal connectives but diverge very soon from the latter to extend posteriorly to the visceral 84 ganglia. These connectives follow a rather straight course never far from the dorsal side of the visceral mass and close to the isthmus or narrow connection medial to the base of the gills.

These connectives stain faintly and are rather small oval-shaped masses (Fig. 38). They pass laterally to the digestive diverticula and stomach, above the gonads, and under or medial to the kidneys before terminating at the visceral ganglia. The closeness of the convoluted walls of the kidney to these branches has led to the idea that the connectives actually pass through the kidneys, but these cords pass medially between the two kidneys. Branches of these two nerve elements to the kidneys are the excretory nerves. Before joining the visceral ganglia, the connectives pass laterally to the posterior retractor muscles below the point where these muscles have converged into a single bundle. The cerebro-visceral connectives must actually ascend from the base of the foot to the dorsal side of the visceral mass, then continue posteriorly and slightly dorsally to finally join the visceral ganglia just anterior and ventral to the posterior adductor muscles.

The pedal ganglia are not separated as are the cerebro- pleural ganglia but are actually joined together. The pedal ganglia are situated in the median plane on the ventral side of Fig. 38.—Dorsal section of naiad through the cerebro-visceral con­ nectives. Note the kidneys lateral to the connectives. Mallory-Azan stain. 35X

Fig. 39.—Pedal ganglia. Note the medullary bridge connecting the two ganglia. 100X 86 the visceral mass and just posterior to the stomach. They may be described as in the base of the foot at the juxtaposition of the foot and visceral mass. As these two ganglia converge and make contact the nucleated corticular covering of each ganglion is lost at the point of fusion, and a medullary bridge of fibers interconnects the two ganglia (Fig. 39).

These ganglia have mainly a medulla of fibers while on the surface there is a thin layer of nucleated cells whose cytoplasmic processes are directed toward the medulla or center of the ganglia.

Paired branches originate in the medulla and extend ventrally and laterally to the abundant muscle bundles of the foot. These branches are called the pedal nerves and can be seen medially for same distance posterior to the pedal ganglia with secondary branches extending laterally to each muscle fascicle in the wall of the foot.

The visceral ganglia are connected or fused together and are located medially just dorsal to the roof of the cloacal

cavity and slightly anterior and ventral to the posterior adductor muscles. These ganglia are somewhat elongated laterally lying in an arched position dorsal to the posterior ends of the

suprabranchial chambers (Fig. 40). Pavlov in 1885 experimented 40.—Visceral ganglion dorsal to the cloacal cavity. Note the cheoiosensory tissue in the lateral mall of the cloaca. 35X

41.—Sensory tissue (osphradium). Note the non-motile ciliated epithelium extending into cloacal cavity. 430X

87 88

with the mussel Anodonta. Spontaneous activity of the foot

stopped when the visceral ganglion was removed, but tonus could

still be maintained in the adductor muscles after they were

denervated. The posterior adductor muscles received motor

impulses from the cerebral ganglia. In Mytilus. if the visceral

ganglia are removed opening and closing reactions to changes in

freshness of water are lost. These and other observations seem to indicate fairly restricted control of local areas by specific

ganglia (Woortmann, 1926). Three pairs of branches leave these

ganglia.

The branchial nerves leave the visceral ganglia ventrally and just anterior to that point where the base of the gills lose their dorsal posterior connection to become the floor of the

cloaca. These nerves are large and as they enter the gill tissue the osphradial branches are separated and connect to the sensory

cells of this organ.

The posterior adductor nerves are smaller and emerge

from the visceral ganglia posterior to the branchial nerves where they soon branch throughout the muscle bundles and are

lost.

The posterior pallial nerves are large and are observed

just above the roof of the cloaca passing laterally into the base 89

of the mantle tissue. Branches of these extend to the margin of

the mantle and innervate the muscles controlling the entrance

of water. These branches are called the siphonal nerves.

B. Special sense organs

The osphradia, believed to be chemoreceptor organs,

(Allen, 1923) are located in the dorsal anterior region of the

cloacal chamber slightly anterior and ventral to the visceral

ganglia. These chemoreceptors form an evaginated fold of

epithelium at the posterior end of the base of the inner gills

that extends into the posterior suprabranchial chamber and

anterior dorsal cloacal chamber. The sensory cells of these

receptors are simple columnar cells with very chromatic, thin,

elongated nuclei. Cilia and mucus secreting cells are

interspersed throughout the osphradial epithelium. Proximal

to the base of these cells are multipolar neurons in the form

of small oval ganglia. There is 3ome controversy as to the

source of innervation of these receptors. It is stated by

Wilmoth (1967) that innervation arises from the cerebro-pleural

complex. However, because of the close association of the

visceral ganglia and the branchial branches, it seems that the

nerves to this tissue arise from the visceral ganglia. Further

physiological investigations must be done to clearly understand

the source of innervation to these organs. Because of their 90

location and similarity to osphradia of gastropods, earlier

workers readily concluded that they test the respiratory fluid

(Pelseneer, 1906) and initiate reflexes to regulate water

flowing past them. Allen (1923) concluded that they are sensitive

to a number of chemicals and that the animal responds in such a

way as to expell noxious materials and prevent their subsequent

entry.

Sel"Inter (19.67) states that, at first, the position of

the osphradia in the excurrent stream seems remote since the

chemicals in the water would not reach the osphradia until just

before leaving the animal. However, if particles that enter

with the water were not removed as the water circulates over the

gills and through the circuit in the mantle chamber, the sensory

function of the chemoreceptor organs could be impaired. The

water evidently retains dissolved substances which circulate by

the osphradia in the excurrent stream.

Chemo-sensory tissue was also located at the posterior

ventral margin of the rectum in the form of a thickened wall

(Fig. 41)• The cells of this organ are very tall and ciliated

and may function as an additional chemo-receptor osphradium. It

does appear to be nervous tissue with underlying nerve elements

also present, extending from the surface cells. A description

of this latter tissue has not been previously made. However, the 91 varied locations of the osphradium in different species seems to indicate that chemo-sensory tissue may be in many locations in the cloacal area.

The paired statocysts are situated just dorsal and lateral to the pedal ganglia in the ventral part of the visceral mass and just above the base of the foot. These organs consist of an oval capsule of simple cuboidal cells containing a smaller capsule attached to the medial wall of the original capsule.

Within the smaller capsule is a statolith (Fig. 42). The statolith and its capsule stain deeply basophilic. These appear to be hyaline or somewhat transparent, and cellular detail cannot be accurately described (Fig. 43). Cilia were not observed to be a part of the organ as has been experienced by several other investigators. Field (1922) dissected out the statocysts of living Mytilus edulis and found them to contain

"vibratile cilia". Drew (1906) found cilia in the histological preparations of statocysts of Pecten tenuicostatus. Other investigators including Dakin (1909) and Drew (1895) were unable to find cilia in bivalves they were studying. The statocyst of the Pigtoe Mussel may be composed of a boundary of sensory cells as found by Dakin (1909) in Pecten opercularis and Pecten maximus. Shrinkage of the tissue in making the preparation is probably responsible for the inability to Fig. 42.—Statocyst. Note this organ is just ventral to the testes (upper right corner). 100X

Fig. 43.—Statocyst. Note the statolith at right. 430X

92 describe the cellular detail. Buddenbrock (1937) shows a generalized statocyst which includes a nerve connection, a ciliated cell layer, and the statolith or concretion. The statocyst of Pleurobema is similar to this illustration except for the details of cilia. One statolith appears to be present in each statocyst in the Pigtoe Mussel.

Posterior to the pedal ganglia, a medially situated hollow tubular organ was located in the foot. This tube descends as it runs toward the posterior end of the foot and consists of low columnar ciliated cells bordering a small lumen or cavity, except near the anterior end and posterior end of this organ (Fig. 44). At these points, the tube is enlarged and contains a basophilic stained oval body at each terminus which is attached to the wall of the tube in at least one or two points (Fig. 45)- Previous reference to this organ has not been found and it would only be speculation to suggest its function. It does appear to be a part of the sensory nervous system of the mussel. Its structure and location hints pressure reception.

10. Reproductive System

The reproductive and excretory systems of mollusks are closely associated anatomically. During their evolutionary Fig. 44.—Median sensory organ of the foot. Note the ciliated lumen. 1000X

•'yJ

Fig. 45.—Sensory organ of the foot. Note the attachment of the centrally located spherical body to squamcus epithelial cells bordering the cavity. 430X

94 95 history, however, there has been a tendency toward separation of these systems until, in eulamellibranchs, they are completely distinct. The relationships of the past exist only in the closeness of their ducts opening to the exterior. The closeness of the exit openings of these ducts in Pleurobema cordatum sometimes is confusing in histological sections.

A. Male

Literature (Sellmer, 1967) indicates that the molluscan gonad is a paired structure but this is not always easy to recognize with closely packed follicles (Fig. 46).

Lobes of the testes extend anteriorly to the digestive diverticula and sometimes between them to a point below the stomach. From the anterior limits to the posterior end of the visceral mass, the gonads occupy most of the available space, except for the area in which the alimentary tract is located.

Dorsally, the gonads occupy all the available space up to the narrow isthmus ventral to the kidneys and nerve cords (Fig. 47).

The ventral limits of the gonads are just above the juxtaposition of the visceral mass and the foot.

Hermaphroditism is a rare occurrence among freshwater mussel species of commercial importance (van der Schalie, un­ published records). This condition was not found in any 46.—Testes of male Pigtoe. Note the crowded condition of the acini and their variable shapes. 35X

. 47.—Transverse section of mussel through testes. Note the entire visceral mass dorsal to the foot is filled with gonadal tissue. a 96 97 specimen which was studied by the author. However, Bates (1967) has found examples of two species with evidence of hermaphroditism. These were Pleurobema cordatum and Quadrula quadrula. The hermaphrodite condition accounted for only one half of one percent of the total sample population made by

Bates. Even though these forms are rare, a few salient points were discovered by a study of these hermaphrodites. The gonad of Pleurobema cordatum revealed that nearly 75 percent of the tissue was female while the remaining 25 percent was male tissue, but the male tissue alone was producing functional gametes. The female acini did account for 75 percent of the gonadal mass, but normal oocytes did not appear to be developing. The individual described by Bates was functioning only as a male, at least, at the time the specimen was collected.

In the instance of Quadrula quadrula. the male tissue accounted for nearly 90 percent of the total gonadal mass and, again, as in the pigtoe, the individual appeared to be functionally a male. Bates (1967) states that the relative proportion of gonadal tissue that is either male or female does not seem to be the determining factor in functional development.

Bates stated further that in all instances of hermaphroditism among the commercial species considered, the only functional tissue was the male. Female tissue, in all instances, appeared 98 to be non-functional. Mature oocytes were not developing in the female tissue. Further studies are currently being pursued in this field.

The pattern of structure in the testes appears to be

much like a compound alveolar gland. The two lobes of the testes are further divided into lobules and each lobule into several

oval or somewhat elongated acini (Fig. 48). As these acini

enlarge they become more crowded and pressure about them alters their shapes and arrangements. Therefore, it is difficult to

observe any common pattern of organization. Each acinus is

lined with an epithelial wall frequently designated as germinal

epithelium. This tissue is relatively thin throughout the year

but seasonal changes in the thickness may be noted upon careful

observation. The lumen of the acinus is occupied by the products

of spermatogenesis with the mature spermatozoa usually in the

center of the lumen. The concentration of dividing cells, the

wall thickness, and the stages of development represented in the

acini, are seasonal differences which will be discussed later in

this study.

From two or three to several acini are arranged around a

small tubule (vas efferens) which has thin walls that stain

faintly acidophilic (Fig. 48). The cells in this tubular wall

are at first simple flattened epithelium or squamous in shape 48.—Acini of the testes of a Pigtoe male. Note the vas efferens through which spermatozoa pass. 43QX

49.—Sperm pore and sperm duct. Note the ciliated cells Hn-tng the sperm duct. 100X

99 100 but very soon change to cuboidal cells. Cilia also appear and, at first, do not form a continuous layer but as the tubule

enlarges the concentration of cilia increases. This larger tubule consists of low ciliated coluoinar cells which stain

very faintly and have a small amount of muscle forming their thickness peripheral to the epithelium.

These tubules (vasa efferentia) converge to form the sperm ducts (vasa deferentia). The sperm ducts are directed dorsally from the testes along the lateral and upper margins of the visceral mass (Fig. 49). These tubes are lined with low, weakly staining, ciliated columnar epithelium and a rather thick longitudinal muscle layer occurs along the distal length of- these tubes. The lumen of each tube is rather wide and has a smooth wall up to a point just below the kidneys where the tube arches laterally and becomes somewhat convoluted as a result of

the folded epithelial wall. The cilia are almost as tall as the

cells near the exit. The sperm pores are located just ventral to the anterior ends of the kidneys in the medial suprabranchial T space formed by the inner gill and just posterior to the point

where the reflected inner gill attachment to the dorsal wall

ends (Fig. 49).

Pigtoe Mussels less than 4 years in age were not found.

. It is not known exactly how early in life the gonads develop 101 and become functional. Most of the specimens examined, histologically, from the youngest to the oldest, appeared to have functional gonads. Sections were made of individuals which were beyond 25 years in age, while the average age range was between 12 and 16 years. None of the oldest individuals showed signs of gonadal atrophy. Some of the individuals sectioned in all age groups showed less gonad tissue, but a partial explanation for this could be individual variation in the location of this type tissue, as well as, seasonal differences which will be discussed.

The gonadal tissue presents seasonal changes, and these, at first, appear rather slight, but upon careful examination these changes are observed to be distinct. For convenience of description these seasonal changes will be divided into three groups; fall, spring, and summer. The winter condition is actually developed during the fall months when the water temperature (Table 2-5) reduces metabolism to a minimum level.

The acini of the testes are the sites of spermatogenesis.

This process evidently occurs in the Pigtoe during all the warm months since spermatogenic stages are found at any season.

However, mature spermatozoa are much more plentiful in the lumina in early spring when spawning and fertilization occur. 102

Each acinus may differ slightly from another in spermatogenic progress but typically the following parts are observed. The epithelium is a single layer of spermatogonia recognized by their large but vesicular nuclei. In the lumina are many primary spermatocytes with round, more chromatic nuclei and a little smaller than the spermatogonia. Among the primary spermatocytes which fill much of the acini may be seen small clumps of secondary spermatocytes, which give rise to spermatids by meiosis. Large numbers of dividing cells showing the various chromosome figures are characteristic of this area in the fall months. Sertoli cells appear either yellow or pinkish in the lumina of the acini when H-E are used. Spermatids are arranged in clumps around the slightly stained Sertoli cells. From 2 to

20 or more may occur in one clump. Spermatids develop into spermatozoa which remain in the lumina attached to Sertoli cells until they pass outside. Each acinus, overall, stains quite basic with H-E preparations because of the concentration of chromatic material. Spermatozoa are elongated rods rounded on one end and slightly concave on the other. A very long flagellum has been observed at the concave end of each spermatozoan in material from fresh testes (Fig. 51)•

The condition of the testes in the spring months reveals the following characteristics (Fig. 50, 51). Fig. 50.—Acinus of testes in the spring 1964. Note the crowded spermatozoa in the lumen. 970X

Fig. 51-—Mature spermatozoa. A flagellum occurs on the concave end of each spermatozoan but the flagella are not revealed in this photograph. 1600X

103 104

1. Acini enlarged and close to each other with little

tissue separating them.

2. Lumina of acini filled with nature spermatozoa.

3. Periphery of acini with many secondary spermatocytes

undergoing meiosis.

4. Spermatozoa migrating through the externally directed

tubules.

The summer condition of the testes appears to be a recovery period with the following characteristics (Fig. 52, 53).

1. Acini reduced in size leaving a good deal of space

between each acinus occupied by connective tissue.

2. Very few mature spermatozoa in the lumina.

3. Acinus wall build-up begins with many primary

spermatocytes and fewer later stages.

4. Accumulations of nutrient matter in the Sertoli cells

and division of these cells.

The fall condition of the testes is characterized by the following observations (Fig. 54, 55).

1. The acini are enlarged but not as large as spring

conditions.

2. Lumina of acini with few mature spermatozoa. %

Fig. 52.—Male acinus in summer 1964. Note the small number of mature spermatozoa in lumen. 970X

Fig. 53. Male acinus in summer 1964. Note the chromosomes in the cells undergoing meiosis. 1600X

105 Fig. 54.—Male acinus in the fall 1964. Note the increase in spermatozoa. 970X

Fig. 55.—Male acinus in the fall 1964. Note the spermatids clustered around Sertoli cells. 1400X

106 107

3. Meiosis in primary spermatocytes to secondary

spermatocytes prevalent.

4. Clusters of these spermatocytes in Sertoli cells

throughout the acini.

5. Spermatids occupy the lumina of the acini.

There is some overlap in the above seasonal differences but these above conditions prevail in the majority of specimens examined from the Muscle Shoals Area of the Tennessee River.

B. Female

The main bulk of the ovaries lies posterior to the stomach. However, follicles do extend forward, ventral, and lateral to the stomach and below the digestive gland. The ovaries extend farther anteriorly -than do the testes. Some follicles of the ovary may extend anteriorly to a point behind the esophagus

(Fig. 56) in the floor of the visceral mass. In numerous specimens which were grossly divided posterior to the digestive glands and embedded in paraffin, the first histological sections were frequently anterior to the male gonads, but in practically none of the females were the sections anterior to the gonads.

The ovaries extend posteriorly to the muscle layers in the foot, dorsally to the narrow isthmus below the rectum occupying much of the loose tissue of the foot and equally- distributed Fig. 56.—Sagittal section through the ovaries. Note that the ovaries occupy much of the visceral mass. 4X

Fig. 57.—Acini (follicles) of the ovary of a Pigtoe female. Note that several acini converge and empty their oocytes into a small oviduct leading-toward the exterior. 100X

108 109 throughout.

The ovary, as the testis, is much like a compound alveolar gland with the oviduct or exit duct leading to smaller secondary ducts which in turn join the follicles, acini, or alveoli (Fig. 57). These alveoli are deeply placed and lined with germinal epithelium or primordial tissue. This tissue may form a very thin wall at one season or build up a significant thickness at another season. These seasonal differences will be described later in this paper. The lumen of each follicle contains a few to several cells in the process of becoming gametes. Variations in these cells occupying the lumina from spring to summer to fall provide the basis for describing seasonal differences in the ovaries.

The follicles are directly connected to the exterior by a short thin-walled secondary duct. This secondary duct is

lined with simple low cuboidal epithelium with cilia which in

turn joins a larger duct with a wider lumen, and with ciliated

epithelium slightly taller. Several of these larger ducts

converge to form the oviduct which is lined with tall columnar

epithelium that gets taller as the tube progresses toward the

external opening. This oviduct is also provided with an

irregularly shaped lumen created by the variation in height of

the columnar cells and to some extent by the contraction of a 110

thin circular muscle layer peripheral to the epithelial layer

which causes additional folding. These epithelial cells stain

very slightly and all of them are provided with motile cilia.

The two oviducts are directed dorsally and each one runs parallel

to the lateral surface of the foot just medial to the peripheral

muscle layer.

The oviduct openings are located just posterior to the

nephridiopores and ventral to the anterior end of the kidneys.

The gametes enter the dorsal mantle cavity just posterior to the

point where the inner reflected gill laminae lose the connection

to the dorsal wall. Many of these gametes are evidently carried

in the current of water in the suprabranchial canals flowing

posteriorly. Exactly how these sex cells pass from the ovaries

to the outer gills is not known by the author. These cells may

be caught up in the incurrent flow of water through the outer

gill ostia as are the spermatozoa where they settle in the

water tubes. A definite limiting factor in the successful pro­

pagation of this mussel would seem to be the synchronous

requirements of sperm and egg meeting in only a fractional part

of the very large environment of water surrounding them.

The alveoli of the ovaries are found to be sufficiently

•different in the spring, summer, and fall to be described in

.each season in a slightly different way. Ill

In the spring months the following characteristics of the ovaries occur (Fig. 58, 59).

1. The alveolar walls are very thin.

2. The lumina of the alveoli are crowded with large

oocytes.

3. The oocyte nuclei possess one or more nucleoli.

4. The alveoli are crowded close to each other in the

limited space of the visceral mass.

The summer characteristics of the ovaries resemble the above conditions in an overlapping way but the following characteristics generally apply (Fig. 60, 61).

1. The alveolar walls are beginning to rebuild.

2. The lumina are almost empty of large oocytes.

3. The oocytes remaining in the cavities are usually

smaller and devoid of nucleoli.

4. More space now exists between the smaller alveoli.

Conditions which occur in the fall are characterized by the following features (Fig. 62, 63).

1. The alveolar walls are restored in thickness aB a

result of the accumulation ,of divided cells.

2. The lumina of the alveoli are crowded with early Fig. 58.—Acini of the ovary of female Pigtoe collected June 28, 1964. Note the crowded acini filled with large oocytes and the thin walls of each acinus. 35X

Fig. 59.—Acinus of the ovary collected June 28, 1964. Note the very large oocytes and their nucleoli. 100X

112 Fig. 60.—Acini of ovary from Pigtoe collected August 6, 1964. Note the reduced size of each acinus with more space between each one. 10QX

Fig. 61.—Acinus of ovary collected August 6, 1964. Note the atretic material in lumen representing breakdown of old oocytes. 430X

113 Fig. 62.—Several acini of the ovary of a specimen collected November 2, 1964. Note the developing oocytes are increasing in size. 100X

Fig. 63.—Acinus of ovary collected November 2, 1964. Note the thick wall of the acinus and recently divided oocytes occupying the lumen. 430X

314 115

oocytes and meiotic products.

3. Most of these newly divided cells are uniformly

small at this season.

4. Atretic material representing older oocytes occupy

the center of many lumina. These materials are in

the process of break-down.

5. The alveoli again are enlarging in the visceral mass

thus occupying proportionately more of the available

space.

11. Excretory System

The organs of excretion in lamellibranchs have been variously called nephridia, renal tubes, organs of Bojanus, urocoels, and kidneys. These organs in Pleurobema are open, tubular structures consisting of a pair of modified coelomoducts leading from the pericardial cavity to the exterior. The lumina of these ducts, those of the genital organs and the pericardial cavity represent the coelom in molluscs (Sellmer, 1967). The excretory organ of the Pigtoe Mussel has the characteristics of a true , being a tube open at both ends, one opening leading into the coelomic cavity and the other to the outside world. Actually, the organ is wholly of coelomic origin in the true clam (i.e. Mercernaria). The term kidney is non-specific 116 meaning simply excreting tubules that may occur singly or segmentary in pairs, or, more specifically, excretory units collected together in compact units. Their function is to move water and dissolved metabolic wastes from a tissue space, body cavity or vascular fluid to the exterior. Thus, the name kidney for the excretory organ could be substituted for nephridium.

Some zoologists use the tern kidney when describing the naiad's excretory organ.

The nephridia of Pleurobema are U-shaped tubes which lie beneath the pericardium beginning below the anterior end of the ventricle and extending posteriorly to the region of the posterior adductor muscle. Blood is filtered through the wall of the ventricle into the pericardial chamber (Picken, 1937).

The nephrostomes, which are the 2 renopericardial openings of the nephridia, receive the pericardial filtrate and convey it to their lumina. These nephrostomes originate along the lateral wall of the pericardium (Fig. 6k), just anterior to the origin of the anterior aorta. These openings are recognized by the sudden increase in the height of the epithelial wall which in the pericardium is extremely thin squamous epithelium. The cells are quite irregular in height creating a rather uneven surface ;Ln the lumen of this tube. Some of the cells are tall ciliated columnar cells while some are very low. Peripheral to Fig. 64.—Renopericardial opening. Note the pericardial cavity and the bladder. 35X

r V*V s" i* Jterr '• '**" Wu£'. • v-

Fig. 65.—Renopericardial opening. Note the smooth muscle layer peri­ pheral to the ciliated columnar epithelium. 100X 117 118 the epithelial layer is a circular muscle layer (Fig. 65). The ciliary beat is described as being in the direction of the external openings (Scheer, 1957). The tube extends posteriorly joining the main cavity of the nephridium. This part of the excretory organ is an elongated tube and its lumen is quite irregular in size and shape because of the numerous folds in the wall. The wall consists of simple low columnar cells with a ragged free border. The surface of these cells does not appear to be ciliated but rather to be of a stringy or fibrous condition at or near their surface (Fig. 66). Dakin (1909) suggested this could be a form of excretory matter. Numerous vacuoles are present in these cells lining the epithelial wall. The nuclei are mainly near the basal ends of the cells. Possibly the free ends of these cells secrete by breaking off and thus become a part of the contents in the lumen, which is typical of apocrine glands. Each cell then regenerates to secrete again within a short time. The lumina of the various parts of the nephridia may have varying sized bodies and a coagulum representing solid wastes of cellular nature. Scheer (1948) indicates that materials are taken up from the blood by the cells of the nephridial tubule and passed into the lumen. He also thinks that secretion is important in the formation of the urine.

The afferent arm of the kidney changes in cellular nature Fig. 66.—Afferent arm of the kidney. Note the ragged free border of the cells and the coagulum in the lumen. 120X Mallory-Azan stain.

nephrostome

renal •xi t

Fig. 67.—External renal orifice in longitudinal view. Note the point of exit is very near the genital exit. 100X

119 120 gradually as it extends posteriorly and at the bend of the "U", its histological character has become thinner like that of the anteriorly directed arm. This efferent arm is an elongated sac which connects transversely with its partner at the posterior level of the ventricle. The lumen of this arm is at times large and at other times almost occluded by the variable crowding and pressure of other organs around it. The wall is made up of low columnar or cuboidal cells which stain slightly because of the vacuolated spaces which they contain. The free surfaces of these cells are often ragged and present an irregular surface.

This arm lies dorsal and to some extent lateral to the afferent arm. It actually seems to drape over the afferent arms at the point where the two efferent arms are connected. The efferent arms are often called bladders. The ventral part or afferent arm is thicker walled and glandular, whereas the dorsal part is thinner walled and nonglandular, forming a urinary bladder whose anterior end opens to the outside by a nephridiopore located between the inner gill laminae slightly posterior to the genital orifices. The epithelium increases in height at this external renal orifice and these cells are again ciliated. The lumen of the renal exit is irregular in shape as a result of the varying cell heights (Fig. 67).

The pericardial gland is not well understood but it is included in the organs of excretion. It stains deeply basic in 121

H-E preparations. It is a diffuse organ which is paired, with a right and left branch located dorsal to the base of the gills and latero-ventral to the heart and dorsal arteries. The cells of this organ form anastomosing cords with sinuses between these cords of cells. The cells are filled with small granules.

The location and structure of the pericardial gland suggests that it functions as an organ of filtration of the blood. It resembles lymph nodes in vertebrate animals which screen out materials. Blood cells may be generated in this

organ. Many amoebocytes are seen throughout the pericardial tissue. SUMMARY

1. The Pigtoe Mussel is concentrated in specific locations known as "beds" in the Tennessee River.

2. Pleurobema cordatum is accompanied by at least two other forms of the complex in the Muscle Shoals Area.

3. SCUBA and skin diving equipment enhance the collecting of

Pigtoes in the Muscle Shoals Area since Pigtoe beds occur at depths of 5 to 30 feet. Collector may select a variety of sizes and obtain many more specimens by this method.

4. Neutral formalin is not only a good naiad fixative but it also hardens the soft parts.

5. Soft tissues of naiads fixed directly in formalin without relaxation make excellent histological tissues.

6. Harris' hematoxylin and eosin Y stains reveal clearly the different systems, organs and tissues of naiads.

7. Morphological differences between male and female Pigtoe shells, even though slight, are noticeable, especially in older specimens.

122 123

8. Female Pigtoe shells are usually slightly larger in each dimension than males in the same age group.

9. Functional gonads may influence the growth rate of the shell.

10. Naiads with inactive gonads are usually smaller than those with active functional gonads.

11. The periostracum is a thin, pigmented, noncellular secretion from between the folds in the mantle margin.

12. Striated, smooth, and cardiac muscle cells are included in the muscular system of the Pigtoe.

13. Cardiac muscle cells are branching fibers found only in the ventricle wall of the heart.

14. The blood of the Pigtoe is a clear fluid plasma containing dispersed cells (amoebocytes).

15. Blood vessels are thin-walled tubes extending anteriorly and posteriorly from the heart. The blood vessels join numerous sinuses located throughout the soft tissues.

16. The heart is an elongated tubular organ with valvular openings through which blood flows in one direction. 124

17. Usually the entire outer gills of the female are the marsupial pouches, but the inner gills infrequently may serve as brood chambers in the Pigtoe.

18. Marsupial gills are constructed with more filaments per unit length of gill than non-marsupial gills. Thus the sexes may be distinguished by the macroscopic appearance of their outer gills.

19. Gonadal changes are seasonal in the Pigtoe. Gonadal tissue (ovary or testis) is sufficiently different in spring, summer and fall to be distinguished histologically, large numbers of spermatozoa occur in early spring in the testes.

Many large oocytes occupy each ovary in the spring.

20. Sexual maturity is reached at least by the fourth year and possibly earlier. Gonads remain functional throughout the life of each individual.

21. The nervous system consists of three paired ganglia with their connectives and numerous multipolar neurons.

22. Chemoreceptors, photoreceptors and, equilibrium receptors are special sensory structures that are located in specific tissue areas of the Pigtoe.

23. The two nephridia are U-shaped tubes which collect dissolved 125 materials from the pericardial cavity and convey the wastes throughout their length to the exterior. The nephrostomes open into the pericardial cavity while the nephridiopores open into the dorsal mantle cavity. TABLE 2.—Wilson Dam outlet water temperatures at weekly intervals for 1965. The discharge of water in thousands of cubic feet per second is shown.

Temperature in degrees Monthly average of Centigrade for each week discharge in thousands of cubic feet water Month 1 2 3 4 5 per second

January 10 10 7 70.8

February 5 6 8 9 76.0

March 7 7 8 9 11 • 98.3

April 13 16 17 18 73.4

May : 20 20 22 24 33.8

June 24 25 25 25 25 32.7

July 26 28 28 29 34.3

August 28 28 28 34.2

September 26 20 21 24 30.0

October 23 20 21 19 31.6

November 17 17 16 14 13 35.3

December 12 10 10 9 26.0

126 TABLE 3.—Wilson Dam outlet water temperatures at weekly intervals for 1966. The discharge of water in thousands of cubic feet per second is shown.

Temperature in degrees Monthly average of Centigrade for each week discharge in thousands of cubic feet water Month 12 3 4 5 per second

January 9 9 8 6 27.6

February 4 9 9 8 68.6

March 7 8 10 12 iu • 40.2

April 11 23 14 15 13.4

May 18 !9 19 21 21 48.1

June 22 23 24 25 22.4

July 26 28 29 30 23.2

August 29 28 27 28 26 40.1

September 27 25 24 23 31.3

October 22 21 19 18 33.1

November 14 14 14 Ur 33 46.1

December 10 9 9 8 60.9

127 TABLE 4.—Wilson Dam outlet water temperatures at weekly intervals for 1967. The discharge of water in thousands of cubic feet per second is shown.

Temperature in degrees Monthly average of Centigrade for each week discharge in thousands of cubic feet water Month 1 2 3 4 5 per second

January 7 6 6 5 7 50.3

February 7 7 7 7 58.4

March 7 12 12 14 64-3

April 15 16 17 18 13.8

May 18 18 20 20 49.2

June 22 24 25 27 36.8

July 26 26 26 25 26 69.8

August 24 24 57.7

September 23 23 23 23 50.6

October 21 20 20 18 16 47.4

November 14 13 12 11 63.5

December 10 10 10 9 136.1

128 TABLE 5 • —Wilson Dam outlet water temperatures at weekly intervals for 1968. The discharge of water in thousands of cubic feet per second is shown.

Temperature in degrees Monthly average of Centigrade for each week discharge in thousands of cubic feet water Month 1 2 3 4 5 per second

January 7 5 4 5 5 127.8

February 7 5 40.9

March 4 7 9 10 47.3

April 23 16 17 29.5

May 19 19 21 34.6

June 21 24 25 25 32.4

July 26 20 25 27 27 35.6

August 27 28 28 29

September 26 25 24 23

October

November

December

129 i 1

I

>

Fort Loudoun Watts Bar ^ 74!

Pickwick

Fig,68.--Profile of the Tennessee River showing dasas and elevations of lakes in feet above s&a level. Less depth and strong current immediately below daias provide better conditions for naiad growth. 220 Mi. 250 Mi.* TOP OF GATES 418 Feet Above Sea Level

410 Feet

PIGTOE BEDS 400 Feet

390 Feet

HEIGHT OF SUBSTRATE 380 Feet

*MILES UPSTREAM FROM MOUTH OF THE TENNESSEE RIVER 370 Feet

Fig. 69.—Depth profile of Pickwick Reservoir. Ordinary minimum depth is 408 feet above sea level. Full pool is 414 feet. ESOPHAGUS^ ANUS

MOUTH BIBLIOGRAPHY

Allen, William Ray (1914). The Food and Feeding Habits of Fresh- Water Mussels. Biological Bulletin, Marine Biological Laboratory, Woods Hole, Mass. 27 (3): 127-146, 3 pis.

, (1923), Studies of the Biology of Freshwater Mussels. II The Nature and Degree of Response to Certain Physical and Chemical Stimuli. Ohio Journal Sc. 23 (2): 57-82.

Atkins, D. (1936). On the Ciliary Mechanisms and Interrelation­ ships of Lamellibranchs. Parts I, II, and III. Quart. J. Micr. Sci. 314: IB1-308; 315: 339-373 J 315: 375-421.

Baker, Frank Collins (1921). Preparing Collections of the • Mollusca for Exhibition and Study. Transactions of the American Microscopical Society. 40: 31-46.

, (1928). Freshwater Mollusca of Wisconsin. Part II. Pelecypoda. Wisconsin Geological and Natural History Survey Bulletin. 70: 1-495, 76 pis., 96 figs.

Baldwin, Ernest (1964). An Introduction to Comparative Bio-1 chemistry. London. Cambridge at the University Press.

Balinsky, B. I. (i96i). An Introduction to Embryology. Philadelphia. W. B. Saunders Co.

Barnes, Robert D. (1965). Invertebrate Zoology, pp. 278-300. Philadelphia: W. B. Saunders Co.

Bates, John M. (1962). The Impact of Impoundment on the Mussel Fauna of Kentucky Reservoir, Tennessee River. American Midland Naturalist. 68 (l): 232-236.

, (1967). Unpublished report on commercial mussels in Kentucky Reservoir of Tennessee River mile 89-105. University of Michigan.

Braungart, Dale C. and Rita Buddeke (1964). An Introduction to Animal Biology, ed. 6, pp. 218-228. Saint Louis: The C. V. Mosby Company.

133 134 Brown, C. J. D., Clarence Clark and Bruce Gleissner (1938). The Size of Certain Naiades from Western Lake Erie in Relation to Shoal Exposure. The American Midland Naturalist. 19 (3): 682- 701.

Buddenbrock, W. v., (1937). Grundriss der vergleichenden Physiologie. Vol. II, Berlin, Bomtraeger.

Call, Richard Ellsworth (1900). Mollusca of Indiana. Annual Report Indiana Dept. Geological and Natural Resources 24: 335- 535, 1013-1017, 78 pis.

Churchill, E. P., Jr. and Sara I. Lewis (1924). Food and Feeding in Fresh-Water Mussels. Bulletin of the Bureau of Fisheries. 39 (963): 440-471.

Coker, Robert E. (1915). The Common and Scientific Names of Fresh-Water Mussels. Bureau of Fisheries. No. 15: 1-4.

, (1919)i Fresh-Water Mussels and Mussel Industries of the United States. Bulletin of the Bureau of Fisheries. 36: 11- 89, 46 pis.

Coker, R. E., A. F. Shira, H. W. Clark and A. D. Howard (1921). Natural History and Propagation of Fresh-Water Mussels. Bulletin of the Bureau of Fisheries. 37 (893): 77-181> 17 pis.

Coker, Robert E. and John B. Southall (1915). Mussel Resources in Tributaries of the Upper Missouri River. Bureau of Fisheries Document Number 812: 3-17.

Dakin, W. J. (1909). Pecten. Liverpool Mar. biol. Comm. Mem., No. 17: 1-136.

Danglade, Ernest (1922). The Kentucky River and Its Mussel Resources. Bureau of Fisheries Document No. 934: 1-8.

Dinamani, P. (1967). Variation in the Stomach Structure of the Bivalvia. Malacologia 5 (2): 225-268.

Drew, G. A. (1895). The Anatomy of Sphaerium sulcatum. Proc. Iowa Acad. Sci., 3: 173-182.

, (1906). The Habits, Anatomy, and Qribryology of the Giant Scallop, (Pecten tenuicostatus Meghels). Univ. Maine Studies, No. 6: 1-171. w s

R . I,-* , oVQ, ^ „<> * » „0> 0 '-'^ Lb " *5 ^ •=» o < ... r' "y ° S> (tj 135. ti f J "' ' '•• .. . ;« ^ ,/ ''" Si 0 f> Duwe, A. E. (1958). The MicroscopicStructure of the Kidney of Fresh-Water Clams in the Genus Unio. Turtox News 36 (7): 161- 162.

Ellis, M. M., Amanda D. Merrick, and Marion D. Ellis (1931). The Blood of North American Fresh-Water Mussels Under Normal and Adverse Conditions. Bureau of Fisheries Document No. 1097: 509- 542. Fenchel, Tom (1966). On the Ciliated Protozoa Inhabiting the Mantle Cavity of Lamellibranchs. Malacologia 5 (1): 35-36.

Field, I. A. (1922). Biology and Economic Value of the Sea Mussel, Mytilus edulis. Doc. No. 922. Bull. U. S. Bureau Fisheries, 38: 127-259.

Hecht, S. (1919). Dark Adaptation: Mya. J. Gen. Physiol. 1: 545-558.

Hoar, William S. (1966). General and Comparative Physiology. Englewood Cliffs. Prentice-Hall, Inc.

Howard, Arthur Day (1914). Experiments in Propagation of Fresh- Water Mussels of the Quadrula Group. Bureau of Fisheries Document No. 801: 36-47.

, (1917). A Second Generation of Artificially Reared Fresh-Water Mussels. American Fisheries Society, Trans. 46: 89- 92.

Isom, Billy G. and Paul Yokley, Jr., (1968). Mussels of Bear Creek Watershed, Alabama and Mississippi, With a Discussion of the Area Geology. The American Midland Naturalist. 79 (1): 189- 196.

, (1968). The Mussel Fauna of Duck River in Tennessee, 1965. The American Midland Naturalist. 80 (l): 34-42.

Jones, Richard 0. (1950). Propagation of Fresh-Water Mussels. U. S. Fish and Wildlife Service, pp. 13-25.

Lefevre, George, and Winterton C. Curtis (1912). Studies on the Reproduction and Artificial Propagation of Fresh-Water Mussels. Bulletin of the Bureau of Fisheries No. 756: 105-201, 17 pis., 70 figs.

Light, V. E. (1930). Morphology and Distribution of Photo­ receptors in Mya arenaria. J. Morph. Physiol. 49: 1-42. 236

Lufty, Ramsis G., and Emile S. Demian (1967). The Histology of the Alimentary System of Marisa comuarietis (Mesogastropoda: Ampullariidae). Malacologia 5 (3): 375-429.

Matteson, Max R. (1948). Life History of Elliptio complanatus (Dillwyn, 1817). The American Midland Naturalist. 40: (3): 690-723.

, (1955). Studies on the Natural History of the Unionidae. The American Midland Naturalist. 53 (l): 126-145.

Morrison, J. P. E. (1942). Preliminary Report on Mollusks Found in the Shell Mounds of the Pickwick Landing Basin in the Tennessee River Valley. Bureau of American Ethnology Bulletin 129, pp. 339-392,

Murray, Harold D. and A. Byron Leonard (1962). Handbook of Unionid Mussels. Lawrence, Kansas. University of Kansas.

Needham, J. (1950). Biochemistry and Morphogenesis. Cambridge University Press, London, 787p.

Ortmann, Arnold E. (1911). A Monograph of the Najades of Pennsylvania. Memoirs of the Carnegie Museum. 4 (6): 279-347. 4 pis., 8 figs.

, (1912). Notes Upon the Families and Genera of the Najades. Annals of the Carnegie Museum, 8 (2): 222-365.

, (1918). The Nayades (Freshwater Mussels) of the Upper Tennessee Drainage. With Notes on Synonymy and Distribution. Proceedings American Philosophical Society. 57: 521-626.

, (1919). A Monograph of the Naiades of Pennsylvania. Part III. Systematic Account of the Genera and Species. Memoirs of the Carnegie Museum. 8 (l): 1-344, 21 pis., 34 figs.

, (1920). Correlation of Shape and Station in Fresh- Water Mussels (Naiades). Proceedings of the American Philosophical Society. 19 (4)* 268-312.

, (1924). The Naiad-Fauna of Duck River in Tennessee. The American Midland Naturalist 9: 3-47.

, (1925). The Naiad-Fauna of the Tennessee River System below Walden Gorge. The American Midland Naturalist. 9 (8): 321-371. 137

Ortmann, Arnold E. (1926). The Naiades of the Green River Drainage in Kentucky. Annals of the Carnegie Museum, 17 (1): 167-188.

Ortmann, A. E., Bryant Walker (1922). On the Nomenclature of Certain North American Naiades. Occasional Papers of the Museum of Zoology, Univ., of Michigan. Publication 112: 1-75.

Owen, G. (1955-1956). Observations on the.Stomach and Digestive Diverticula of the Lamellibranchia. Quart. J. Microscop. Sci. 96: 517-537 and 97: 541-567.

Pelseneer, P. (1906). Part V. Mollusca. In: E. Ray Lankester (ed), A Treatise on Zoology. Adam and Charles Black, London. 355p. Pennak, R. W. (1953). Fresh-Water Invertebrates of the U. S. Ronald Press, New York. 769p.

Picken, L. E. R. (1937). The Excretory Mechanism in Certain Mollusca. J. exptl. Biology, 14: 20-34.

Pietsch, Helen G. and Reuben M. Cares (1964). Histologic Techniques for Larvae of Bivalve Molluscs. Turtox News 42 (9): 234-235. Prosser, C. Ladd, ed. (1950). Comparative Animal Physiology. Philadelphia. W. B. Saunders Co.

Pumphrey, R. J. (1938). Double Innervation of Clam Muscle. J. Exper. Biol. 15: 500-505.

Reuling, F. H. (1920). Experiments in the Artificial Rearing of Fresh-Water Mussels in Troughs Under Conditions of Control. American Fisheries Society, Trans. 49: 153-155.

Rogers, Charles Gardner (1938). Textbook of Comparative Physiology p. 265. New York. McGraw-Hill Book Co., Inc.

Scheer, Bradley T. (1948). Comparative Physiology. New York. John Wiley and Sons, Inc.

, Ed., (1957). Recent Advances in Invertebrate Physiology. University of Oregon Publ., Eugene, Oregon. 304p.

Scruggs, George D. Jr., (i960). Status of Fresh-Water Mussel Stocks in the Tennessee River. U. S. Fish and Wildlife Service Special Scientific Report - Fisheries. Publication 370: 1-41, 9 figs., 17 tables. 138

Sellmer, George P. (1967). Functional Morphology and Ecological Life History of the Gem Clam, Gemma gemma (Eulamelli.branchia: Veneridae). Malacologia. 5 (2): 337-224.

Sinclair, Ralph M. (I964). Clam Pests in Tennessee Water Supplies. Journal American Water Works Association. 56 (5): 592-599. Stansbery. David H. (1961). The Naiades (Mollusca, Pelecypoda, Unionacea) of Fishery Bay, South Bass Island, Lake Erie. Sterkiana Number 5: 1-27, 5 pis., 18 figs, 9 tables.

, (1964). The Mussel (Muscle) Shoals of the Tennessee River Revisited. Annual Reports for 1964 of the American Malacological Union. 25-28.

, (1965). The Naiad Fauna of the Green River at Munfordville, Kentucky. The American Malacological Union Reports for 1965s 33-14.

, (1965). The Molluscan Fauna. The Cleveland Museum of Natural History. 4 (l): 120-124.

, (1967). A Provisional Classification of the Pleurobema cordatum. (Rafinesque, 1820) Complex in the Mississippi River Drainage Basin of North America (Mollusca: Pelecypoda: Unionacea). The Ohio State Museum.

Stasek, Charles R. (1966). Views of the Comparative Anatomy of the Bivalved Mollusca. Malacologia 5 (l): 67-68.

Surber, Thaddeus (1912). Identification of the Glochidia of Freshwater Mussels. Bureau of Fisheries Document No. 771: 1-10, 3 pis.

Taylor, I. R., and E. M. Walzl (1941). Electrocardiogram of Qyster. J. of Cell and Comp. Physiol. 8: 43-84.

Tennessee Valley Authority (1966). The Mussel Resource of the Tennessee River. Fish and Wildlife Branch, 1-32.

Tripp, M. R. (1958). Studies on the Defense Mechanism of the Qyster, Crassostrea virginica. Unpublished PhD. thesis. Rutgers University, New Brunswick, N. J. 139 van der Schalie, Henry (1928). The Naiades (Fresh-Water Mussels) of the Cahaba River in Northern Alabama. Occasional Papers-of the Museum of Zoology, University of Michigan. Publication 392: 1-29.

. (1939). Additional Notes on the Naiades (Fresh- Water Mussels) of the lower Tennessee River. The American Midland Naturalist 22 (2): 452-457.

, (1945). The Value of Mussel Distribution in Tracing Stream Confluence. Papers of the Michigan Academy of Science, Arts, and Letters. 30: 351-373. van der Schalie, Henry and Annette (1950). The Mussels of the Mississippi River. American Midland Naturalist 44: 448-466.

Walker, Bryant (1917). The Method of Evolution in the Unionidae. Occasional Papers of the Museum of Zoology, University of Michigan, No. 45: 1-10.

Weinland, Ernst (1919). Beobachtungen Uber den Geswechsel von Anodonta cygnea L. Zeitschrift, Bd. 19: 384-422.

Wilmoth, James H. (1967). Biology of the Invertebrate, pp. 205- 212. Englewood Cliffs, New Jersey: Prentice-Hall, Inc.

Woortman, K. (1926). Function of Ganglia of Mytilus. Ztschr. vergl. Physiol. 4: 488-527.

Yonge, C. M. (1923). Feeding, Digestion and Assimilation in the Clam Mya. J. Exper. Biol. 1: 15-63.

. (1926). Structure and Physiology of the Organs of Feeding and Digestion in Ostrea edulis. Journal Mar. Bio. Ass. U. K., 14: 295-385.

, (1932). The Crystalline Style of the Mollusca. Sci. Progr., 26: 643-653.

, (1937). Review of Digestion in Metazoa, Biol. Rev. 12: 87-115.

Yonge, (1941). The Protobranchiate Mollusca: a Functional Interpretation of their Structure and Evolution. Phil, Trans. Roy. Soc. London, Series B, 230: 79-147.