: eaten J et < y vere a =~ - i lia é & ee es ee nner pac el ow el Rep Be dian STUDIES ON THE REPRODUCTION AND ARTIFICIAL PROPAGATION OF FRESH- Pee MUSSELS 4, 4. 2 From BULLETIN OF THE BUREAU OF FISHERIES, Volume XXX, 1910 IDUSTEA A DERE IN, GEOINT SS ee ae en eno ee Issued May ro, 1912 ——————— EEE WASHINGTON =; 3; =: : : ‘GOVERNMENT PRINTING OFFICE : : : : + ! : bo: 1912 D. OF 0. MAY 20 1912 Sided ¥¥ coon habe “4 eS STUDIES ON THE REPRODUCTION AND ARTIFICIAL PROPAGATION OF FRESH-WATER MUSSELS & peed — By George Lefevre and Winterton C. Curtis Professors of Zoology in the University of Missouri 105 CONTENTS. a Page. TEMG. SGN eh dooudy ge Oo dgUOId 40 0b TR GOGOM Aas Seen Oe SELIG O TAC AD ACEC arc Oe Cnn re rie 109 ESEA SLOGAN en ree eames Ste ieiates oe wore ete rae coyeieieys seis mb einin ik wisiwturs le Ps savers lafalale cero ore Givieletel na wielgoig eo III AP RENMOMICWMOMs es uit: iach oe MERCK te OR PENA A wile weer b elceie eis ielelelalelelcislelaid sitjsau(oridnneate ace 114 BURN EEL ANGEL foLer crass tee eotay ste eucteys pays ecaletes seclec alors s/aic sre et cte sfacatel alata caress lela lavate etaverele:sraleleioisialeterevske 116 Wse onthe: marsipitiman Classification, J=:\<\+)sei2.aa\<)as a REPRODUDTION AND ARTIFICIAL PROPAGATION OF FRESH-WATER MUSSELS. 163 In the case of the carp, while the fish is admirably suited to carrying the hooked glochidia of Anodonta and Symphynota, we have never been able to secure a successful infection of the gills with the hookless glochidia of the genus Lampsilis. The disappear- ance of the hooked glochidia of Anodonta and Symphynota from the gills of the carp may be due to the pulling away of these large and heavy glochidia from the delicate gill filaments, as suggested in our consideration of the survival of the two types of glochidia upon fins and gills, respectively. The disappearance of the hookless glochidia of Lampsilis from both gills and fins of the carp can not be explained in this manner; it suggests rather that there may be some reaction of the host’s tissues comparable to the processes which confer immunity against parasitic bacteria in higher vertebrates. With minnows (Notropis cayuga and N. lutrensis) 2 to 4 inches in length, we have not been able to secure any considerable infection with the glochidia of Symphynota complanata, for, although they will attach in large numbers during infection, they all drop from the fins and gills within a few days. ‘The fins of these minnows are much more delicate than those of the carp, and the explanation is perhaps that so large a glochidium is easily torn away; but the large-mouth black bass has hardly a delicate fin, and for this fish we have records of infections where no glochidia of S. complanata became attached during an exposure sufficient for the attachment of many to the gills. In this latter case, the extreme activity of the fish must be considered as a factor which might keep the hooked glochidia from attachment to the fins. Darters (Etheostoma ceruleum spectabile) 114 to 2 inches in length can not be infected successfully with the glochidia of Lampsilis, for although they may fasten so thickly to the fins that many fish die during the first day after their exposure, the surviving fish will slough off considerable portions of the fins and within a week show only the healed and regenerating parts as an indication of their recent experience. ‘The gill slits were so small in these fish that only an occasional glochidium was found upon them. Such cases as these are of great importance and should be followed up to determine whether the simple mechanical conditions like over-infection, delicacy of fin, or con- figuration of the mouth parts can give a satisfactory explanation; or whether the histo- logical changes of which the fish is capable, under stimulation by the glochidium, must be regarded as the cause of its immunity. We have not carried out a sufficient number of experiments to feel sure that the simpler explanations can be excluded. In any case, it is interesting that fish like the minnows and darters, which live close to the bottom, are not likely to become heavily infected by some of our most common glochidia. BEHAVIOR OF FISHES DURING INFECTION. The behavior of the fish during infection isa matter of some importance and has been already mentioned in an incidental manner. The rock bass, large-mouth black bass, and blue-gill sunfish, which are very active and which consequently exhibit powerful respiratory movements, are well adapted to artificial infection, and the proper suspen- sion of the glochidia in the water is secured by the movements of the fish alone. The crappie, which are sluggish and easily killed by handling, require some special device to 164 BULLETIN OF THE BUREAU OF FISHERIES. insure the optimum infection and are not well suited for work on a large scale because of their behavior during infection. Fish which rest upon the bottom are sometimes not so favorable as they might seem because they do not move about enough to keep the glochidia in motion. While other features may be of greater importance, the behavior of the fish as affecting the distribution of the glochidia in the water should always be considered in deciding how useful any fish may be for purposes of infection. INFECTION OF FISH IN LARGE NUMBERS. The infection of fish in large numbers has been attempted with a view to determining the feasibility of extending the methods described above to wholesale infections of fish ina hatchery. Asa result of two such attempts, we have no doubt that the successful development of the methods needed for infection in connection with the artificial propa- gation of mussels is only a matter of a little study in a properly equipped station. In December, 1907, about 25,000 small fish, under 6 inches in length, were placed at our disposal at the substation of the Bureau at La Crosse, Wis., and we were able on this occasion to infect by wholesale methods about 12,000 blue-gill sunfish, 3,700 yellow perch, 7,000 catfish, 2,000 crappie, 150 rock bass, 150 carp, and roo roach. ‘The greater number of these fish were infected with the glochidia of Lampsialis ligamentina, and, considering the fact that this was our first experience with so large a number of fish, the results were satisfactory. Smaller lots were infected with the glochidia of L. anodon- toides and L. recta, the results giving every indication that these two species are essen- tially like L. ligamentina in the conditions of their development. The most successful infections were obtained by placing from 100 to 200 fish in a common galvanized iron washtub about two-thirds full of water. It was found that by adding to this body of water the glochidia obtained from two or three specimens of Lampsilis, and, when it seemed necessary, stirring the water by hand, tolerably constant results could be secured. Our difficulties were with over- rather than with under-infection. It was also possible to use the same tub a number of times without changing the water or adding to the stock of glochidia. Infection was also attempted by lowering the water in the large retaining tanks of the station to a depth of 4 inches and confining the whole number of fish which had been held in the full tank to this much smaller body of water. This method was found, in the absence of any attempt to keep the glochidia properly distributed through the water, quite inadequate and it became necessary to reinfect these fish in the tubs. The mortality of the fish in these experiments was decidedly in excess of what one might expect for uninfected fish kept under similar conditions, a result clearly due to the over-infection which is the one thing most to be guarded against. At the end of six weeks some of the remaining fish were liberated in the west channel of the Mississippi River at La Crosse, a locality which we then believed might be suitable for this species of Lampsils. These infections were made under conditions of limited time and equipment and were wholly tentative, the aim being to make a test of our methods on a large scale. We revisited La Crosse a month after the infection, making careful examinations of the REPRODUCTION AND ARTIFICIAL PROPAGATION OF FRESH-WATER MUSSELS. 165 fish and by shipping several hundred to Columbia were able to follow the development of the glochidia under the conditions in our laboratory. The results were probably as favorable as could have been expected under the circumstances. In December 1908 a similar infection was attempted with about 6,200 large-mouth black bass and 3,800 crappie in the station of the Bureau at Manchester, Iowa. Upon this occasion the glochidia of Lampsilis ligamentina were again used in a majority of the infections, similar results being obtained with L. anodontoides, recta, and ventricosa, which were used for the minor infections. The black bass took the glochidia very readily and, having had only a limited experience with this species of fish, we gave them an amount of infection equal to that which had been carried successfully by the rock bass infected at La Crosse in the previous experiments. The infection was estimated at from 2,000 to 2,500 glochidia to a fish 4 or 5 inches in length. This proved entirely too heavy for the large-mouth black bass and the mortality among them amounted to about 55 per cent in the 30 days they were under observation. By the third day after the infection the hypertrophy of the gill tissue was so great as to be at once noticeable to the eye, and this was clearly the cause of death. An infection of not more than 1,000 glochidia per fish would have been more nearly the optimum load. The crappie did not take the infection well despite longer exposure, the reason for this being the size of their gill slits and their behavior as already discussed, and we do not consider small fish of this species favorable for infection with any of the glochidia from mussels which are of commercial importance. Thirty days after these infections the surviving fish were liberated in the Maquoketa River near Manchester, in a situation where the conditions were favorable for mussels and where the presence of a dam below the point of liberation, together with the absence of mussels of this species, made it seem possible that at some later period their appear- ance in this locality might be traced to this experiment. We have never made any sub- sequent examination of this stretch of the river with this in view, a thing which should be done by one of the parties engaged in the field work of the mussel investigation. These two experiments in the wholesale infection of fish, while disappointing in some respects, give no indication of any insurmountable difficulties. It is fair to con- clude that a little experimentation under hatchery conditions will make it as easy to carry the glochidia through their metamorphosis in large numbers as we have found it in small lots of fish kept in aquaria. The high mortality of the fish, being so clearly a matter of over-infection, is a thing which can be guarded against without reducing too greatly the load of glochidia which the fish may carry. It is then only a matter of dis- covering the most suitable species of fish and finding out how best to handle them in large numbers. One thing which seems necessary for the rapid and uniform infection of fish in large numbers is a device which will bring about a uniform distribution of the glochidia in the water during the whole period of the fishes’ exposure. Without something of the sort it will hardly be possible to handle large numbers of fish with constant and uni- form results. We have tried, though not very extensively, two means of effecting 166 BULLETIN OF THE BUREAU OF FISHERIES. this. The first consisted of a two-bladed propeller fastened in the middle of the bottom of a tub and rotated slowly, there being enough space in the water above the blades to allow the fish room to escape the stroke. This device was not very satisfactory, but as it was operated by hand and the blades roughly constructed, effective use might be made of a more carefully adjusted mechanism of this type. A second and more promising device consists of a branched system of iron pipes bored with many small holes (text fig. 3), through which fine jets of water are forced out at the bottom of a tank. The amount of pressure in these fine jets can be easily regulated from the main supply pipe, and the height to which the glochidia will be driven from the bottom is thus controlled. The tank may be allowed to overflow at the top and the glochidia Fic. 3.—Apparatus for keeping glochidia suspended in water while fish are being exposed to them for gill-infections. Tap water entering at S issues in fine jets through the very small holes placed along the top and sides of the pipes on the bottom of the aquarium, and an even distribution of glochidia throughout the water is thereby maintained. By regulating the force of the water entering the pipes at S the glochidia are prevented from rising to the top of the aquarium and escaping with the overflow. prevented from being carried off in the overflow by so adjusting the force of the jets that the glochidia will not rise quite to the surface. This device keeps the glochidia suspended in a very uniform way, and it may prove to be just what is needed for the uniform infection of large numbers of fish. . CONDITIONS NECESSARY FOR SUCCESSFUL INFECTION. Three factors should be considered in attempting the infection of any species of fish with glochidia, namely, the uniform suspension of the glochidia in the water, the reaction of the glochidia when stimulated by mechanical or chemical contact with the fish, and the reaction of the fish’s tissues after the glochidium has become attached. aD 6 OWN ~E age ws REPRODUCTION AND ARTIFICIAL PROPAGATION OF FRESH-WATER MUSSELS. 167 In any attempted infection of fish in large numbers, careful tests should first be made upon a few fish in small dishes, with microscopic examination of the infected parts from fish killed during the time of infection and for several days following, or until it is clear that the glochidia have become safely established in their host’s tissues. After even limited experience one learns approximately the number of glochidia needed and can determine roughly their suspension in the water by taking samples at random in a pipette, which when held against the light shows clearly the individual glochidia. Dur- ing infection it is possible to pick out individual specimens and by lifting up the oper- culum of the living fish, examine the gills with a hand lens. The glochidia are then seen individually and the progress of the infection can be watched. Fin-infecting glo- chidia may be seen individually if a fish is placed in a small dish against a black back- ground. It is not difficult to determine by these means the optimum time for the exposure. When 100 fish 5 to 6 inches in length are taken and the contents of a single marsupium of a large Lampsilis is placed in an ordinary washtub, infections may be obtained some- what as follows: Rock bass, exposed 30 to 40 minutes, 2,000 to 2,500 glochidia on gills of each fish; large-mouth black bass, exposed 15 to 20 minutes, 500 to 1,000 glochidia on gills; crappie, exposed 20 to 30 minutes, 200 to 400 glochidia on gills; yellow perch, exposed 20 minutes, 400 to 600 on gills; German carp (with Anodonta), exposed 30 to 40 min- utes, 200 to 500 on fins. ‘These figures are given as starting points for anyone attempt- ing artificial infections and can not be taken as representing the results of precise deter- minations of optimum infections for the fish in question, because the means for deter- mining the numbers and distribution of the glochidia have been only approximate. It will probably always be necessary, in the practice of artificial infection on a large scale, to have the fish examined microscopically by a properly trained observer, and this will be particularly true in the beginning of this work in hatching establishments, because the practical details of artificial infection on a large scale have yet to be solved. DURATION OF THE PARASITIC PERIOD. According to the experience of previous observers, the duration of the parasitic period varies inversely with the temperature of the water (Schierholz, 1888; Harms, 1907-1909). Although we have found this to be true in general, our experiments have not shown so definite a relation between temperature and parasitism as has been described by Harms, for example, and it is quite possible that other factors, which are obscure, exert a modifying influence upon the length of time the glochidia remain on the fish. Harms found that the glochidia of Anodonta completed the metamorphosis in 80 days at a temperature of 8° to 10° C; in 21 days at 16° to 18°; and in 12 days at 20°; while in the case of the hookless glochidia of Unio (which are gill parasites) the period was 26 to 28 days at a temperature of 16° to 17°. He is inclined to attribute the some- what longer time required for the metamorphosis of Unio to the fact that the glochidia in this genus when discharged are in a less advanced stage of development than are those of Anodonta—a difference that exists between all hookless and hooked glochidia. 18713°—12—5 168 BULLETIN OF THE BUREAU OF FISHERIES. A few typical cases, selected from our records of infections are given in the accom- panying table, which illustrates the far greater variability in the parasitic period than that observed by Harms. TABLE SHOWING INFECTIONS WITH GLOCHIDIA. = : B Young ani sxe temp, xperi- . xpos- ion o! uring ment. BS Mussel. Fish. ure. Firma Parasit- | parasit- ism. ism. HOOKED GLOCHIDIA. Min. Days. as OF ep aosenord Dec. 3,1909 | Symphynota compla- | Apomotis cyanellus..... Dec. 17-19..... 14-16 16.0 nata. 15 SF sceencat! Dec. 17,1909 |..... GOs os doncesraoted beat GOH tmpmaesonaacn too Dal fel fOr lab oer 15-18 16.3 Pomoxis annularis. So eccnnete Jan. 7,X9%0'|..... GO Sen ites cap ivictecie Apomotis cyanellus..... 12 | Jan. 18-21..... II-14 16.0 Pomoxis annularis. Betaatecrs Apr. 5,1910 }..... do: -eatacancy nares Apomotis cyanellus..... 30 | Apr. 14-18... 9-13 17 8 HOOKLESS GLOCHIDIA. .| Feb. 19,1910 | Lampsilis ligamentina..| Apomotis cyanellus..... 9 | Mar. s-12..... 14-21 17-8 .| Mar. 6,1909 |..... oC See aHne oooehevcc lose less. gan concen deoooad to-15 | Apr. 7-11..... 32-36 19.1 Micropterus salmoides. eink avalaieralat Apr. 8,1909 }..... i OG aemanecoogucaaty Apomotis cyanellus..... ro-15 | Apr. 27-May 1. 19-23 20.3 Micropterus salmoides. Bowen Apr. 13,1910 | Lampsilis subrostrata...}| Apomotis cyanellus..... 8-15 | May 2-8....... 19-25 18.1 CBanousacar May 2,1910 | Lampsilis ligamentina..|..... Gly Si@aeaccoennacman 7-10 | May 15-26..... 13-24 18.1 Micropterus salmoides. May 33,1910 | Lampsilis subrostrata. ..] Apomotis cyanellus. .... 50 | May 17-25..... 14-22 18.1 .| July 29.1909 | Unio complanatus......| Perca flavescens......... 7-14 | Aug. 12-14.... 14-16 23-0 Aug. 5,1908 | Quadrula plicata.. .....] Micropterus salmoides. . BO MANS nny. neni 12 24-4 In the case of Symphynota complanata, which has hooked glochidia essentially like those of Anodonta, the period varied from 9 to 18 days at average temperatures of 17.8° to 16° C., as compared with Harms’s 21 days at practically the same temperature. At lower temperatures, about 10°, we have recorded a period of 74 days for S. costata. The absence of a close correspondence between the temperature and the duration of the parasitism has been much more conspicuous in the case of hookless glochidia, which have shown not only a remarkable range in the period but a considerable irregularity in dif- ferent experiments made at about the same temperature. The shortest period recorded by us was seven days in an infection of black bass with the glochidia of Lampsilis sub- rostrata and L. recta in April when the average temperature during the parasitism was 20.5°, but this unusual time was only observed in this one instance. A still more remarkable case, but at the opposite extreme, was an infection of black bass and crappie with the glochidia of L. ligamentina and L. recta which remained on the fish for 13 to 16 weeks. The infection was made in November and the young mussels were liberated during a period of about three weeks in the following February and March; during the parasitism the temperature varied from about 16° to 18.° The cause of the extreme duration in this case is not known, for in no other experiment at the same temperature has the parasitism lasted for more than 25 days. REPRODUCTION AND ARTIFICIAL PROPAGATION OF FRESH-WATER MUSSELS. 169 As may be seen in the table, with hookless glochidia (aside from the extreme cases mentioned) the variation in the period has been from 12 to 36 days at average tempera- tures ranging from 24.4° to 17.8°; but even at practically the same temperature the difference may be quite marked, as in experiments no. 8 and no. 9. Experiment no. 6 should be noticed as being a case in which, contrary to expectation, quite a long period (32 to 36 days) was recorded at 19.1°, whereas in other experiments (no. 5 for example) the time was only 14 to 21 days at the lower temperature of 17.8°. It would seem clear that, although within certain wide limits the duration of the parasitism is dependent upon the temperature of the water, nevertheless other factors may enter into the case to either accelerate the metamorphosis or prolong it over a period which is much longer than the usual duration of the parasitism. These factors would seem to be associated with individual physiological differences in the interaction between the fish and the parasite and are probably nutritive in nature, for on one and the same fish some glochidia may remain several days longer than others. ° As may be seen from an examination of the table, in which the period of liberation is given in each experiment, not all of the young mussels leave the fish at the same time, but, on the contrary, the liberation may occupy a week or more. Harms found that it required from 5 to 6 days, the greater number leaving the fish during the middle of the period. Our experience has usually been in accord with these observations, but we have found the period to be somewhat more variable, from 2 to 11 days, or even much longer. IMPLANTATION AND CYST FORMATION. As has been described, the glochidium attaches itself to the fish by closing its shell firmly over some projecting region which can be grasped between the valves, like the free border of a fin or a gill filament. In so doing, a portion of the epithelium and underlying tisstie, including blood vessels and lymphatics and varying in amount with the extent of the ‘“‘bite,’’ becomes inclosed within the mantle space of the glochidium. This tissue early disintegrates into its cellular constituents, which are taken up by the pseudopodial processes of the larval mantle cells, and, as Faussek (1895) has described, are utilized as food during the early stages of metamorphosis. In figure 60, plate xv, drawn from a glochidium six: hours after attachment to a fin, the disintegrated tissue, consisting of loose epithelial cells, blood corpuscles, and fibers which lie scattered in the mantle cavity, is seen in the process of being ingested by the mantle cells. Figure 61, plate xv, shows a later stage, 24 hours after attachment, in which the detritus has been entirely taken up, and the mantle cells are now heavily charged with food material. Almost immediately after attachment proliferation of the epithelium begins as the initial step in the formation of the cyst which eventually incloses the entire glochidium. The overgrowth of the larva has been described by Faussek (1895) and Harms (1907-1909) as a healing process on the part of the fish’s tissues, resulting from the irritation caused by the wound. The proliferation starts around the line of constriction produced by the pressure of the edges of the valves on the epithelium, and, since the glochidium lies between and prevents the immediate closure of the lips of the wound, the extending 170 BULLETIN OF THE BUREAU OF FISHERIES. epithelium is forced to slide up over the surface of the shell on all sides, until the free margins meet and fuse over the back of the larva, as may be understood by reference to figures 59 to 61, plate xv, and 35 to 38, plate xr. So rapid is the overgrowth, especially in the case of implantation on the gills, that it would seem that something more than the mere mechanical irritation produced by the glochidium is concerned in causing the proliferation of the epithelium. We have, therefore, carried out a series of experiments with a view to determining whether or not a chemical stimulus is provided by the larva, and by using various methods have studied the action of glochidial extracts on the epithelium of both fins and gills. The results have been entirely negative, although the question has by no means been settled by the experiments which have been thus far attempted. By further improvements in the technique, some of the difficulties involved in the investigation, which is still in progress, may be overcome. The process of implantation and cyst formation may be readily observed on the fila- ments of an excised gill, which under favorable conditions will live long enough in a dish of water to enable one to see the glochidium completely covered by the proliferated epithelium. Figure 54, plate x1, drawn from the living excised gill, shows the distal end of a single filament bearing a glochidium of Unio complanatus which has become nearly covered by the walls of the cyst. In this case the gill was cut from the fish two hours after the infection and the drawing was made an hour later; immediately after the excision of the gill this particular glochidium was hardly half covered. The same glochidium was kept under observation, and two hours later (five hours after the infec- tion) the sketch was made which is reproduced in figure 55, plate xim. By this time the cyst, which is seen to have very thick walls, was completed, and formed a prominent mass near the end of the filament. Shortly afterwards the tissues of the gill began to disintegrate, but for at least three hours they remained alive and the proliferation of the epithelial cells proceeded rapidly, the entire process of cyst formation taking place in a perfectly normal manner. The histological changes which the epithelium undergoes in the formation of the cyst have been studied in this laboratory by Miss Daisy Young, and, as her results will soon be published in detail, only a brief reference will be made in this place to the essential points involved in the cellular changes occurring during implantation of the glochidium. Figure 59, plate xv, shows a very early stage, 15 minutes after attachment, in the formation of the cyst on the fin of a fish which had been infected with the glochidia of Symphynota complanata. The section is taken transversely through the glochidium and the free border of the fin on which the parasite has a firm grip. The mass of tissue, consisting of epithelial cells, connective tissue, and blood vessels in the mantle chamber of the glochidium, is the edge of the fin which was inclosed between the valves when attachment was effected. Already the proliferation of the epithelium is beginning in the neighborhood of the constriction, where two mitoses may be seen on the right in the figure. At the edges of the wound caused by the closure of the shell some of the bee. REPRODUCTION AND ARTIFICIAL PROPAGATION OF FRESH-WATER MUSSELS. I7I epithelial cells are undergoing degeneration, while on the left of the section quite a patch of these cells is sloughing off, a not infrequent occurrence. The region of most active growth and multiplication of cells is just below the line of constriction, and, as the cells at this level increase in number, they appear to push those lying above them up over the outside of the shell, so that the actual covering of the glochidium is due largely to this mechanical gliding of the epithelium over its surface. Sections give no conclusive evi- dence of amitotic division, while mitoses are generally abundant in the region of active proliferation. An intermediate step in the process of implantation is illustrated in figure 60, plate xv, less highly magnified than the last figure, which shows a glochidium about half covered in six hours after attachment. The free edges of the cyst wall even- tually meet over the dorsal side of the glochidium, where they then fuse. Figure 61, plate xv, shows a case of complete implantation on a fin at the end of 24 hours; now the epithelial covering is continuous and the glochidium entirely inclosed. The wall of the cyst is seen at this time to be quite thick, but it usually becomes thinner later on as the cells composing it flatten down. In the last two figures the mantle cells of the larva clearly show epithelial nuclei and cell detritus which have been ingested. In figures 62 and 63, plate xv, two stages are represented in the formation of the cyst on gill filaments, taken at one hour and three hours, respectively, after attachment. The glochidia are those of Lampsilis ligamentina. In figure 62, plate xv, the prolifera- tion has made some progress, especially on one side, and three or four mitotic figures are seen just below the glochidium and near the raw edge of the constricted epithelium. A large mass of the tissues of the filament is also shown in the figure inclosed within the mantle chamber of the glochidium. Figure 63, plate xv, represents a stage when the process is nearly completed and the edges of the epithelial covering have met but not yet quite fused. The cyst wall in this case is much thinner than that shown in figure 61, plate xv, but its thickness is quite variable. In about one week after attachment, as a rule, the wall of the cyst begins to assume a looser texture, the intercellular spaces becoming infiltrated with lymph, and from this time on to the end of the parasitic period there is little further change in its structure. Before liberation of the young mussel, the valves open from time to time and the foot is extended. By the movements of the latter the cyst is eventually ruptured, its walls gradually slough away, and the mussel thus freed falls to the bottom. Portions of the wall of the cyst often adhere to the shell after liberation, while, if the young mussel has hooks, it may hang for a time by shreds of the fin in which the hooks are embedded, as seen in figure 24, plate rx. METAMORPHOSIS WITHOUT PARASITISM IN STROPHITUS. In a brief paper (1911) we have recently announced the discovery that in the genus Strophitus Rafinesque the metamorphosis takes place in the entire absence of parasitism, and, since the life history of this form is without a parallel in the Unionide, so far as is known, reference may be made again to the interesting conditions which obtain in its development. 172 BULLETIN OF THE BUREAU OF FISHERIES. It has been known for a long time that in Strophitus the embryos and glochidia are embedded in short cylindrical cords which are composed of a semitranslucent, gelatinous substance, and that these cords, which are closely packed together, like chalk crayons in a box, lie transversely in the water tubes of the marsupium. ‘The blunt ends of the cords are seen through the thin lamella of the outer gill, which in this genus, as in Anodonta and others, constitutes the marsupium. The position of the masses of embryos, while contained within the gill, is so unusual that Simpson in his “Synopsis of the Naiades”’ established a special group, the Diagene, for Strophitus—the only genus of the family in which this peculiarity exists. In other genera the embryos are conglutinated more or less closely to form flat plates or cylindrical masses, each one of which is contained in a separate water tube and lies vertically in the marsupium. So far as we are aware, Isaac Lea (1838) was the first to observe this interesting arrangement which he described and figured, rather crudely to be sure, in Strophitus undulatus (Anodonta undulata). In several subsequent communications (1858, 1863) he added further details and illustrations, and also mentioned the occurrence of the transversely placed cords, or ‘“‘sacks,”’ as he called them, in S. edentulus. He recorded the former species as being gravid from September until March, and described the extrusion of the cords from the female, as well as the remarkable emergence of the glochidia from the interior of the cords after the latter have been discharged. The sacks were discharged into the water by the parent from day to day, for about a month in the middle of winter. Eight or ten young were generally in each sack, but some were so short as only to have room for one or two. Immediately when the sacks came out from between the valves of the parent, most of the young were seen to be attached by the dorsal margin to the outer portion of the sack, as if it were a placenta. The essential points in these observations have since been verified by other inves- tigators. Sterki (1898), following the suggestion of Lea, has called the cords, which differ strikingly from the conglutinated masses of Unio and other genera, ‘‘placentez,”’ thus indicating that he considered them to have a nutritive function. He also described the extrusion of the glochidia, when placed in water, and their attachment to the cord “by a short byssus thread whose proximal end is attached to the soft parts of the young.’ He further states that the glochidia are inclosed in the placente when the latter are first discharged, and that after their extrusion they remain attached for some time. Strophitus edentulus, which Ortmann (1909) regards as identical with wndulatus, is a rare species in all of the localities in which we have collected mussels, and, until recently, our only observations on this form were made upon a few gravid individuals which were taken in the Mississippi River near La Crosse, Wis., during the summer of 1908. Mention has already been made of our records with reference to the breeding season of Strophitus. After verifying the main observations of Lea and Sterki, so far as was possible at that season of the year, we examined the glochidia carefully with a view to determining whether their subsequent life history would exhibit any peculiarities, as might be sus- pected from their relation to the cords. At that time we did not observe the normal —_— REPRODUCTION AND ARTIFICIAL PROPAGATION OF FRESH-WATER:MUSSELS. 173 discharge of the cords by the female; but we removed them from the marsupium, placed them in water, and, after the glochidia had emerged (fig. 46, pl. xm), employed various means to bring about their attachment to fish. None of these attempts, however, was successful, although the fish were left in small dishes containing many cords for as long a time as 12 hours. In the light of these results, which indicated the inability of this glochidium to attach itself to fish, and in view of the fact that the cords so evidently seemed to be a nutritive device, we felt it to be highly probable that in this species the metamorphosis would be found to occur in the absence of parasitism—a prediction which has been recently verified. On February 6, 1911, a single female of Strophitus edentulus, which had been kept in the laboratory since the preceding November, was seen discharging its cords from the exhalent siphon. The discharge continued until March 25, and during that time the cords were thrown out in varying numbers from day to day. They measured from 2 to 10 mm, in length and about 1 mm. in diameter, although they became more or less swollen after lying in the water for a time. Each cord contained from ro to 24 glochidia arranged in an irregular row. In many cases the glochidia emerged from the cords in a few minutes after the latter were discharged, and then usually remained attached by the thread in essentially the same manner as has been described by Lea and Sterki (fig. 46, pl. x11). The thread, which is apparently a modified larval thread, is continuous at its distal end with the egg membrane, which generally remains embedded in the cord; so intimate, in fact, is the union between the two that at times the mem- brane, adhering to the thread, is dragged out of the cord when the glochidium is extruded, in which case, of course, the glochidium becomes entirely detached from the cord. All attempts to infect fish with these fully formed glochidia were again unsuccessful, even when the exposure was of long duration. Within a few days the extruded glochidia died in spite of every effort to provide the most favorable conditions for their maintenance. When the cords first began to be discharged, one of our students, Miss Daisy Young, happened to notice that not all of the larva were extruded, and that among those which remained in the cords some had lost the larval adductor muscle, possessed a protrusible foot, and showed other signs of having undergone the metamorphosis. Upon careful examination this was found to be true, and it was discovered that these young mussels— for such they undoubtedly are—are subsequently liberated by the disintegration of the cord after having passed through the metamorphosis in the entire absence of a parasitic period. We, therefore, have concluded that the emergence from the cords in the glo- chidial stage is premature, due possibly to some change which has taken place in the gelatinous substance surrounding them as a result of free contact with the water, or to release from the pressure to which they are subjected while in the marsupium. It is perfectly evident that these glochidia neither become attached to fish nor undergo any further development; they have simply come out too soon and are lost. The young mussels, on the other hand, which have developed inside the cords, when liberated by the disintegration of the latter or removed directly by teasing, are found to 174 BULLETIN OF THE BUREAU OF FISHERIES. have reached as advanced a stage of development as is attained by any unionid at the time it leaves the fish. They closely resemble the young of Anodonta at the close of the parasitic period, and upon examination have been found to possess the following struc- tures: The anteriorand posterioradductor muscles; the ciliated foot; two gill budsoneach side; a completely differentiated digestive tract, including mouth, esophagus, stomach intestine, and anus; liver; the cerebral, pedal, and visceral ganglia; otocysts; the rudiments of the kidneys, heart, and pericardium; while they also show a slight growth of the per- manent shell around the margin of the shell of the glochidium (fig. 45, pl. xir). The larval muscle has completely disappeared, although some of the mantle cells of the glochidium, as well as the hooks of the shell, are still present. They crawl slowly on the bottom of the dish by the characteristic jerking movements of the foot, after the manner of the young of other species at a corresponding stage, although the valves of the shell gape more widely apart and the foot is shorter and less extensible. We have not succeeded as yet in keep- ing them alive for more than 10 days, but it is difficult in the case of any species to main- tain young mussels of this age under laboratory conditions. One of these young mussels after removal from the cord is shown in figure 45, plate xu, in which many of the organs of the adult or their rudiments are clearly indicated. A comparison will show that it is essentially as advanced in its development as the young of Anodonta when it is liberated from the fish (cf. Harms’s figures, 1909, and also our fig. 47, pl. x11, of Symphynota costata). The conclusion is inevitable that we have here to do with a species which has no parasitism in its life history, although the presence of hooks and other typical glochidial structures would indicate that it has originated from ancestors which possessed the para- sitie stage like other fresh-water mussels. The cord is undoubtedly to be interpreted as a nutritive adaptation which arises in the marsupium during the early stages of gravidity, since the young embryos are at first contained in an unformed viscid matrix and the cords are a later product. The whole history of this exceptional species warrants a more detailed study, and Miss Young is now engaged in such an investigation. When her work is completed we hope that it may include the entire course of development, the method of formation of the cords, and the rearing of the young mussels during a much longer period than has thus far been possible. V. ATTEMPT TO REAR GLOCHIDIA IN CULTURE MEDIA. Since the relation of the glochidium to the fish is essentially a nutritive one, it seemed to us that it should be possible to rear the larve through the metamorphosis artificially, provided a suitable nutritive medium could be found, and accordingly a series of experiments, with this object in view, were undertaken at our suggestion by one of our students, Mr. L. E. Thatcher. Although the result has thus far been entirely negative, we have not despaired of ultimate success, and, since the experiments are to be continued, a brief mention of the methods employed may be made in this place. BPs yn — REPRODUCTION AND ARTIFICIAL PROPAGATION OF FRESH-WATER MUSSELS. 175 It was natural to suppose that the blood of the fish would offer the most favorable nutritive conditions for the development of the glochidia, and hence it has been used in most of the experiments, which, moreover, have been made in the spring, when the water in the laboratory was comparatively warm and the metamorphosis, if it had occurred, would have taken place as rapidly as possible. The glochidia of Lampsilis ligamentina and L. subrostrata were carefully removed from the marsupium with a sterilized pipette and then repeatedly washed in distilled water in order to obtain them as free as possible from bacteria and other organisms. A drop of blood was next taken from a fish’s heart and placed on a cover glass and a few glochidia immediately introduced into it. The cover glass was then inverted over a hollow slide containing a moist piece of filter paper, and the chamber sealed with vaseline. Every precaution was taken to avoid contamination by bacteria. As soon as the glochidia came into contact with the blood, of course they snapped shut in the manner already described and in doing so inclosed some of the corpuscles, which it was to be presumed would be ingested by the mantle cells. Although in some cases bacteria and infusoria, probably introduced with the glochidia, appeared, in a majority of the cases the cultures remained free from foreign organisms. In the latter event the glochidia lived for a few days, but finally died without showing any indication of further development. Experi- ments were tried with the blood of the frog and of Necturus, and also with extracts of fish’s tissues, bouillon and other nutritive media. In all, however, the results were negative. The failure may possibly have been due to insufficient aeration, and experi- ments are now being devised in which oxygen is to be introduced into the moist chambers, and it is hoped that we shall yet succeed in rearing the glochidia in nutritive media through the metamorphosis. VI. POST-LARVAL STAGES. BEGINNING OF THE GROWTH PERIOD AND LIFE ON THE BOTTOM. The changes occurring during the parasitism and by means of which the glochidium becomes transformed into the young mussel, ready for life on the bottom, are more prop- erly described by the term development than by the word growth. The latter process becomes the conspicuous feature only when the miniature mussel has left the fish. From this time onward there are very few changes to which the term development may be strictly applied; for, with the exception of the outer gill, all the important organs of the animal have been laid down and have assumed something of their definitive structure (fig. 47, pl. x1). As soon as they are liberated from the fish the young mussels become quite active and move about on the bottom of a dish by means of the foot (fig. 18, pl. vin, and fig. 48, pl. x1), securing a hold by flattening the ciliated distal end against the bottom, and then drawing up the body after the characteristic fashion of lamellibranchs. In these move- ments the cilia of the foot play an active part; they beat vigorously while the foot is being extended, and apparently are effective in part at least in causing the protrusion. When 176 BULLETIN OF THE BUREAU OF FISHERIES. the foot reaches its limit of extension, the cilia stop abruptly and remain quiet while the forward movement of the body is taking place, only to resume their activity when the extension begins again. Figure 18, plate vim, furnishes an excellent illustration of the various positions assumed as the young mussels crawl about in their twisting, jerking movements, and also shows the extent to which the shell has grown beyond the limits of the glochidial valves by the end of the first week of free life. In the great majority of forms, as appears from the work of other investigators and our own observations, the mussel leaves the fish with only a very narrow margin of adult shell protruding beyond the glochidial outline. The shape is still that of the glochidium, although all other resemblances to this larval stage have disappeared. In the larva of Symphynota costata this margin of the adult shell is so narrow, even after some days upon the bottom (fig. 47, pl. x11), as not to protrude beyond the glochidial outline when the young mussel is slightly contracted. Exceptions to this supposedly universal con- dition have been observed by Coker and Surber (1911) in the young of Plagiola dona- ciformis and Lampsilis (Proptera) levissima—forms in which there is a considerable growth of the definitive shell and presumably of the other organs during the parasitic period. These cases are unique so far as known, but in view of the small number of species which have been observed at all during this period of their existence other such exceptions may be looked for. No data bearing upon the duration or other conditions of the parasitic life are given in the paper in question, since the material studied was from the gills of a fish which had been preserved after its infection under natural conditions. These stages immediately following the parasitism and until the mussels are about 20 mm. in length are less known than any others. They have seldom been found by collectors, and the reasons for this are made clear by the work of Isely (1911), to which we shall presently refer. Pfeiffer first observed and figured in 1821 a small shell having the glochidial outline still visible at its umbo, and other cases have been recorded, notably by Schierholz (1888). Such specimens were taken from nature and not from mussels artificially reared. Indeed, no one has yet succeeded in following individual specimens for more than a few weeks beyond the beginning of life on the bottom. Recently Harms (1907, 1908, and 1909) has obtained these stages, by rearing, more extensively than his predecessors and has figured (19072, p. 811) the young of Anodonta with a very substantial increase in size at an age of six weeks after the parasitism, beyond which they could not be reared because of their destruction by small Crustacea. He concludes that the latter constitute a serious danger to the life of the young mussel. In our own work repeated attempts have been made to rear these stages to a size which can be more easily handled, but without success. Specimens of Symphynota costata (fig. 47, pl. x11) and of Anodonta cataracta have been kept alive in small dishes containing green plants for a period of from One to two weeks after they had left the fish, and Lampsilis ligamentina and subrostrata for a period of six weeks. Little or no growth was observed after the first week. The two species of Lampsilis formed a conspicuous border of new shell during the first few days of bottom life (fig. 18, pl. v1, and fig. 48, REPRODUCTION AND ARTIFICIAL PROPAGATION OF FRESH-WATER MUSSELS. 177 pl. x11) and then ceased growing although they continued to move actively about. This would indicate that the difficulty lies in the lack of a suitable food supply. Crus- tacea were not observed to play an important réle, though we do not doubt the cor- rectness of Harms’s observations in this respect. Figures 18, plate vim, 47 and 48, plate x11, will illustrate the appearance of the young mussels at this period and an examination of figure 47 will show how extensively the organs of the future adult have been laid down. Nothing remains to suggest the glochidium save the shell, and structure and habit alike indicate that the organism is now ready for a life on the bottom essentially like that of the adult. JUVENILE STAGES AND THE ORIGIN OF MUSSEL BEDS. For the sake of completeness, we shall discuss briefly at this point the present state of our knowledge regarding the stages between the one last mentioned and that repre- sented by the young mussels over 20 mm. in length, which are often found upon the natural beds. In common with the experience of other collectors, we have seldom found mussels under 20 mm. It would therefore seem clear that these early stages are not at all common in localities where the slightly later stages and the adults are found. Isely (1911) has published a preliminary note upon his study of this “juvenile” period. We shall refer to his results rather fully, since there are no other recorded observations which deal with these stages save in the way of incidental reference to single specimens. This author states the problem by saying (p. 77) that: ‘Much diffi- culty was experienced in finding young mussels for study and experimentation. I have collected many specimens from the size of a nickel (20 mm.) to a quarter (24 mm.), but mussels under the size of a dime (17 mm.) have been rare.’’ The latter he terms the “early juvenile” stages, including in this “the period following the time when the mussel completes the parasitic stage and leaves the fish to lead an independent life until it is about 15 mm. in length. This would cover, in most species, approximately the first year of independent existence. Other periods may be designated as later juvenile and adult life.’ He then reports the finding of 32 specimens in this early juvenile stage representing four genera and nine species, as follows: (1) Lampsilis luteola, two; (2) Lampsilis fallaciosa, one; (3) Lampsilis parva, four; (4) Lampsilis gracilis, three; (5) Plagiola elegans, one; (6) Plagiola donaciformis, sixteen; (7) Anodonta imbecillis, two; (8) Ptychobranchus phaseolus, two; (9) unnamed species, one. All these specimens were found in places where the water was fairly swift, from 1 to 2 feet in depth, and on a bottom of coarse gravel, the particles of which were 10 to 25 mm. in diameter. They were anchored by the threads of a byssus gland “strong enough to support the mussel in a rapid current”’ and capable of sustaining ‘“‘the weight of a number of small pebbles without breaking.” Here then, as Isely concludes, we have the clue to the habits and ecology of these so little-known stages. ‘The finding of representatives from so many genera and species, both heavy and light shelled, under identical environmental conditions and the presence of the functional byssus in all cases is pretty good evidence that this is the normal 178 BULLETIN OF THE BUREAU OF FISHERIES. condition for early juvenile life in a wide range of forms. It is, moreover, interesting to find in the Unionide, as in many other lamellibranchs (e. g., Mya and Pecten) a functional byssus in the early stages, though there is no such organ in the adult. As these results are very important and of convenience for reference in this paper we may here quote Isely’s conclusions in full. The facts noted above are closely related, not only to the ecology of the juvenile mussel, but also to the ecology of the adult. 1. They indicate the conditions essential for the most successful growth and early development of the Unionide. This kind of an environment gives a constant supply of oxygen and sufficient food; is frequented by suitable fish; is free from shifting sand and silt accumulation. ‘Those mussels that drop from the fish in these favorable situations develop in large numbers, while the less fortunate, that drop in shifting sand and silt, die early. 2. In the study of the ecological factors that are inimical to mussel life more attention should be given to the consideration of the juvenile habitat. Absence of gravel bars and stony situations may sometimes explain the scarcity of the Unionide in certain streams and lakes where frequently water content has been thought the chief unfavorable factor. 3. It is a well-known fact that in many streams certain stretches of mud bottom are found loaded with mussels, while other areas, in the same stream, equally favorable from the standpoint of the habitat of the adult mussels, have only scattering specimens. This distribution of the adults may be explained by the assumption (which is fairly well established by experimental study and will be discussed in a later paper) that the average mussel seldom travels far up or down the stream from the place where it begins successful development. Stretches favorable for juvenile development thus come to be the centers of dispersal in the streams where they occur. As a result, areas of mud bottom near these favorable habitats become loaded with mussels by migration. 4. In the study of the life history of the Unionidz we may consider the embryonic, the glochidial, the parasitic, the early juvenile, and the adult as distinct periods for separate and special study. These results of Isely’s are clearly of very great importance in the problem of arti- ficial propagation and it is to be hoped that his observations may be greatly extended in the near future. The number of different species which he has found is a most promising sign that he is on the right track, and we may hope that we shall soon reach a satisfactory understanding of this stage of the life cycle hitherto so little known. At this point a word regarding the formation of beds may be opportune. It is a familiar fact that many species are most likely to be found congregated in beds which in some of the larger streams must have contained, before the shells came into commer- cial use, numbers of mussels which are hardly conceivable. Elsewhere in the stream — the mussels are found scattered and wandering over the bottom. In the absence of any indication that the individuals of a species are in some manner attracted to one another, the simplest explanation of the formation of beds would be the same as that given in other cases of this sort. The conditions of food supply, current, character of bottom, etc., must differ considerably, and we may reasonably suppose that some places present the optimum conditions over an extended area and that in such a place a bed may be formed. As the mussels wander over the bottom they may by chance enter such an area of optimum conditions and will then move about less actively or come to rest, because in the absence of unfavorable conditions there is no stimulus to continued loco- motion. The result is that individuals which enter are likely to remain and more keep a arc REPRODUCTION AND ARTIFICIAL PROPAGATION OF FRESH-WATER MUSSELS. 179 coming in. This kind of an explanation has been offered, by the students of animal behavior in recent years, to account for the formation of aggregates in a great variety of the lower organisms; and it appears the most reasonable one in such cases as the one in hand, where there is no evidence that the gregariousness is due to a definite recognition of the presence of other individuals. RATE OF GROWTH. It has been quite generally believed, by those investigators who have given their attention to this matter, that the mussel shell grows during the warmer months of the year and that in winter there is no appreciable addition to its margin. When growth begins again in the spring, the winter’s rest has left a mark which appears as a dark line on light-colored shells or as a deeper groove in others where the color is not so con- spicuous. Finer lines may be found between these rings of growth, but the latter, like the rings of a tree, mark the years. It is certain that these more conspicuous lines or “rings,” as we may term them, indicate an alternation of growing and resting periods in the formation of the shell. It is not entirely certain that a single growth period must always correspond to a single year; for, when any lot of shells is carefully examined, some will be found in which the ‘“‘rings”’ are distinct and strongly suggestive of an annual increment, while others of the same size may not show these rings in any such distinct fashion, and one is forced to conclude either that the annual rings, if such they be, are not always clearly to be seen or that some mussels may grow at a very different rate from others. The examination of any considerable number of shells leads to the belief that even if the annual-ring theory can be proved conclusively the rings are often not sufficiently distinct from the intervening lines to give an unquestionable record of the age. Assuming that these rings, when clearly seen, do represent years, it would seem that the shell grows very rapidly during the first few years of the mussel’s life and after that much more slowly. To judge from the lines alone, we should say that many of the large Quadrula shells had reached one-half their size in ten or a dozen years and then taken forty or fifty for the remainder, so closely set are their later rings of growth; and that shells of these species can not reach the most desirable commercial size in a less period than twenty or thirty years. Since these are regarded as the best of all button shells, the outlook may seem discouraging, because, like hardwood timber, the best shells take too long to grow. The “ring theory” if proved would not, however, make the situation so discourag- ing as might seem from the species of Quadrula; for we have in some members of the genus Lampsilis shells which are almost if not equally desirable, and such evidence as we have from the rings indicates that shells like these may reach a commercial size in a very few years and that even forms like the quadrulas may become marketable within a period of four or five years. In a recent paper, Israél (1911) has reported his conclusion that there is no winter- rest period and that more than one ring may be formed ina single year. This statement 180 BULLETIN OF THE BUREAU OF FISHERIES. is based upon the examination of the shell margin in mussels collected at various seasons of the year and of mussels which had been placed in wire inclosures on the bottom of the stream after having been accurately measured. The results from these plantings were fragmentary because of the accidental destruction of most of the inclosures. In one case, however, he found specimens which ‘‘when placed in the inclosure in August, 1909, and measuring 18 mm. in length, had reached, at the time of their examination in June, 1910, a length of 26 mm.’’ He reports that other similar investigations are in progress, the results of which we shall await with interest. Since no accurate observations on the rate of growth of fresh-water mussels have ever been made, we have attempted to secure definite data bearing upon this problem. The data obtained are derived from two entirely different lines of observation, as indi- cated by the headings of the sections which follow, and although meager they show that with better facilities it should not be difficult to follow individual mussels from the juvenile to the adult stages, and thus to determine their rate of growth in an accurate manner. GROWTH OF MUSSELS IN WIRE CAGES. While engaged in mussel investigations at La Crosse, Wis., during the summer of 1908, we collected a number of young clams (fig. 68, pl. xvi1) belonging to 16 different species, and after weighing and measuring them accurately they were distributed in wire cages, which were then anchored by long wires in midstream to the piers of a bridge over the west channel of the Mississippi River opposite La Crosse. One hundred and sixty- three small mussels, belonging to the following genera and representing both thin and thick shelled forms, were planted out in this manner: Alasmidonta, Anodonta, Lampsilis, Obliquaria, Obovaria, Plagiola, Quadrula, and Unio. - Some of the cages contained only a single specimen of each species represented in it, in which case an absolute identification would be possible, should the cage be recovered later, while, if two or more individuals of a species were put in a cage together, only specimens of practically the same size were selected. In the latter case it would of course be impossible to subsequently distinguish an individual mussel, and only the average rate of growth could be determined for the individuals present. It was assumed that mussels of the same size and under the same conditions would grow at practically the same rate. These plantings were made at intervals from June 29 to August 10, 1908. An opportunity did not present itself to make an attempt to recover the cages for over two years, but in November, 1910, Dr. R. E. Coker, who knew of the experiment, made a search while on a visit to La Crosse and was fortunate enough to find 2 of the 11 cages planted by us in 1908. One of the cages was deeply buried in the mud and all of the mussels in it were dead; as they showed little or no growth, they were evidently killed shortly after the planting. In the other cage, however, 6 living mussels were found, as follows: 3 Lampsilis ventricosa, 1 Obovaria ellipsis, 1 Quadrula solida, 1 Anodonta imbecillis. ‘These 6 mussels, with the exception of the specimen of Obovaria ellipsis, were readily referred to definite individuals as recorded at the time the cage was set out. The comparative measurements and weights are given below. REPRODUCTION AND ARTIFICIAL PROPAGATION OF FRESH-WATER MUSSELS. I81 June 29, 1908. November 15, 1910. Lambsilis ventricosa: CES yO TI), LO STATIS cc sre'se erase veaierain s/aicts elas) «0 85 by 65 mm., 129.85 grams. (2) raveby 42 tiie, 5. SAMS. 6 ev sca ceescae ces ae see. s sO DY 57 MM.) 115.5 grams. (Ee TAy Eby: SOM Oss STARTS ea cei iclnc ae ve cnt’ lesa creel 96 by 67 mm., 145.2 grams. Obovaria ellipsis: (x) 52 by 52 mm., 59.1 grams.. ‘ sseeees--57 DY 55 mm., 74.6 grams. (The identification of this specimen is somew ee dieertain: ) Quadrula solida: (Ca) egy oyna G Winds ees OATS se ators cel taeieyaiole yercicrs)¥¥= eps laos. 45 by 46mm., 46.3 grams. Anodonta imbecillis: CONGO, Diyg Gores Or MELAINS 53-28 creo sie) «lc oie, cim'alace s eefaieiesas 61 by 28 mm., 13.3 grams. In each case, the first measurement is the greatest antero-posterior length of the shell, and the second the distance from the top of the umbo to the ventral margin taken approximately at right angles to the lines of growth. An interesting and important fea- ture of these specimens is the fact that the original margin is clearly indicated by a con- spicuous line on the shell of each, and as the measurements within this line correspond with the original measurements, the identification is made sure for each individual. We quote below an analysis of the results sent us by Dr. Coker,who made the second series of measurements after the recovery of the cages: Lampsilis ventricosa.—They have increased in length by 34 to 39 mm. and in height by 25 to 37 mm., and they now weigh approximately 7, 8 and 9 times as much, respectively, as when first put out. Furthermore, the added area of shell is divided by a conspicuous dark ring and a less distinct ring which, one is tempted to assume, represent the periods of cessation of growth during the two winters. If such an interpretation is made, the growth was accomplished chiefly during 1908 and 1909, while during the present year (1910), the mussel having reached adult size, the growth has been considerably less. Increase in size stated by percentage (present measurements compared with original measurements). Period, June 29, 1908, to November. 15, 1910, 2 years, 44 months: Length. Height. Weight. SPECLMEN INOS La ala sais ont ss ans anisesd Saye eh sual oi per cent.. 188 217 812 SHECINIERINO, 2 an. Moat se cietsinc Seine sav eee el nent doa... E72 178 77° Specimen no. 3. rsngto ea aatdle noose tie TOE RO mee 2OF 223 880 The proportion of increase is a slightly greater in aul than in length, and the coefficient of increase in weight is, as might be expected, something like the cube of the coefficient of increase in either dimension. Obovaria ellipsis.—The specimen has probably gained very little in length or height but materially in weight. It was nearer its adult size, is doubtless a slower growing species, and has probably gained in weight by increase of thickness of shell. But we are not so sure of the identity of this specimen. Quadrula solida.—Has gained nearly 30 per cent in length and height and 70 per cent in weight. Anodonta imbecillis—Has more than doubled in length, with negligible increase in height, while it has increased 66 per cent in weight. This is particularly interesting as showing a marked change in form from the young to the adult. Text figure 4, A and B, represents outline sketches of two of the three specimens of L. ventricosa described above, showing the exact size of each after the completion of the growth in the fall of 1910; the line marked a is the margin of the shell at the time the planting was made in 1908; while lines b and c are the two successive rings indicating cessation of growth. The two areas inclosed between these lines, representing the two chief periods of growth which have occurred, are not of equal extent in the three speci- 182 BULLETIN OF THE BUREAU OF FISHERIES. mens. In A they are of about equal width, while in B the second area is much greater than the first. The area between line c and the margin of the shell is in all three cases very narrow, showing that, as the mussel approaches the adult size, further increase in the shell must take place very slowly. The recovered specimen of Q. solida shows only one broad area of growth, and a very narrow one around the margin. This mussel was relatively much nearer adult size when put in the cage than the specimens of ventricosa. Dr. Coker comes to the following conclusion with respect to the age of the specimens of L. ventricosa: They are very significant, as they show clearly that growth is much more rapid than is generally suspected. Considering what the growth has been since the cages were put out, it is fair to assume that the specimens had only one year’s growth at that time. That is to say, they were glochidia in the spring of 1907, and, since they must have been carried in the gills of the mother over the preceding winter, their complete age at this time (Nov. 15, 1910) is a little over four years. Their age since the metamorphosis would therefore be about three years. Their probable history, on the above assumption, is as follows: 1. Eggs fertilized in August, 1906. 2. Glochidia discharged in spring or early summer, 1907. 3. Liberated from fish in summier, 1907. 4. Collected at age (since metamorphosis) of about one year and placed in cages June 29, 1908. 5. Recovered and remeasured, November 15, 1910. The rate of growth of these individuals is probably typical of the genus Lampsilts, and the experiment indicates at least that commercial mussels may reach a marketable size in three years from the time they leave the fish. With the heavier shelled species (those of Quadrula, for example) the rate of growth is probably slower and a longer time must elapse before they are large enough for commercial use. These experiments, meager as they are, are quite significant and furnish the first definite data, so far as we know, relating to the rate of growth of fresh-water mussels. With the proper facilities and the opportunity of examining the mussels at closer in- tervals, similar plantings could readily be made and exact information obtained on the growth of all the important species. To prevent the cages from being buried in the sand or mud would seem to be the chief precaution that should be taken in future experiments of this kind. AN ARTIFICIALLY REARED MUSSEL. Another experiment, although it does not throw light upon the question of the rate of growth in nature, might be mentioned in this connection on account of its significance for the problem of artificial propagation. Avlot of black bass which had been infected with the glochidia of Lampsilis ligamentina, ventricosa, and recta at Manchester, Iowa, on December 2, 1908, were brought to Columbia, Mo., and placed in a large tank con- taining sand. The fish were left in the tank, where the young clams were allowed to fall off in the hope that some would survive and be later recovered. The sand was examined at intervals thereafter but never thoroughly, as the chance seemed very slight that any of the young clams were still living. On December 26, 1910, however, a single REPRODUCTION AND ARTIFICIAL PROPAGATION OF FRESH-WATER MUSSELS. 183 small individual of Lampsilis ventricosa was found alive and active in the sand of the same tank. There can be no doubt that it was derived from the infection referred to, as no young clams of this species had ever been in the laboratory, and no subse- quent infections were made in that tank. The exact size of this young mussel was 41 by 30 mm. on December 26, 1910. It isstill alive, but as late as June, 1911, it was practically of the same size. Since it is over two years old, it is evident that it is quite a dwarf, and, had it been reared under favorable conditions, it undoubtedly would have been muchlarger by this time, The tank in which it has spent all of its life is supplied with tap water, which is obtained from deep wells and contains little that a mussel could utilize as food, and its small size is undoubtedly due to the fact that it has been underfed from the beginning. The shell shows no indication whatever of lines of inter- rupted growth, but this is only what might have been expected, as the mussel has never been exposed to low temperatures. It is evident, therefore, that it has been growing continuously, but very slowly, throughout its entire life. Thisindividual,however, is of no little interest, as it is Fic. 4.—Two individuals of Lampsilis ventricosa recovered on November 15, 1910, after having been confined in a wire cage in the Mississippi River for two years and four and a half months. The line a is the original margin of the shell at the time of planting, June 29, 1908, and the lines b and c represent the “rings’’ which are due to the periods of cessation of growth. Natural size. the first fresh-water mussel actually reared artificially from the glochidium, and in a sense 18713°—12——6 184 BULLETIN OF THE BUREAU OF FISHERIES. furnishes a demonstration of the feasibility of artificial propagation. Had the food supply in the tank been adequate, it would now be a mussel of about two-thirds the adult size. THE ORIGIN AND AGE OF MUSSELS IN ARTIFICIAL PONDS. A second line of evidence bearing upon the rate of growth has been obtained in connection with an examination of certain artificial ponds in the vicinity of Columbia, Mo. In this region it is customary for the farmers to construct, for the watering of cattle, ponds in which water is held the year round by the impervious clay soil. We have examined many of these small bodies of water and have records of the approximate, if not the exact, dates of their construction. In 12 of these ponds, the ages of which are from 5 to 40 years, we have found specimens of Lampsilis subrostrata and Unio tetralasmus in some numbers, and in two of the ponds the mussels are present in very great numbers. s The occurrence of the mussels in the different ponds has been considered, first, with a view to the question of their original introduction into a given pond, and, second, their rate of growth. ‘The first of these two considerations will be discussed here as a matter of convenience, although it should more properly be considered in a section dealing with the introduction of mussels into favorable localities. As to their origin in the ponds, we find the facts interesting because it is quite clear that a majority, if not all of the ponds, must have been stocked with mussels which were first introduced as parasites upon fish. The significant facts in this connection are: That we have never found a pond containing mussels but no fish, although there are a number of ponds containing fish in which we have thus far failed to discover any mussels, and that none of the ponds have outlets or other immediate connections with streams in which the mussels occur, but are situated, for the most part, on high ground far from the watercourses, making it impossible that the mussels could have worked their way into these bodies of water by any ordinary process of migration. Since it is very unlikely that persons have introduced adult mussels into so many places by intent or accident, the mussels must have appeared in these ponds by natural means and the most probable of these is their introduction while parasites upon the fish with which the ponds were stocked. The transportation of small individuals attached to the mud on the feet of birds or of terrestrial animals, so often suggested as a means of dispersal in a case like this, is a possible mode of origin, although it seems hardly a probable one in view of the excellent chance the mussels would have of being introduced while still parasites. One of the above ponds, which is about 40 by 60 feet in area and 1o feet in depth, is particularly interesting since it contains great numbers of Lampsilis subrostrata and also of the sunfishes (Lepomis humilis and A pomotis cyanellus), which we have found in our laboratory experiments to be very favorable hosts for the glochidia of this mussel. The mussels are of all sizes and the pond has existed for many years. We do not know its exact age nor how long ago fish were introduced. The mussels were first discovered in 1907 and have ever since been found in abundance. Their success is doubtless due, = PANG Hm mt : REPRODUCTION AND ARTIFICIAL PROPAGATION OF FRESH-WATER MUSSELS. 185 in large part, to the abundance of a fish favorable for their parasitism. Nothing in these specimens, nor in what we know of the history of this pond, gives a clue to the age of the mussels. Another pond has great numbers of Unio tetralasmus. This pond was constructed in rgo1 and during the first year was stocked with fish (the exact species unknown). In 1907 it contained a great many mussels as long as 4 inches, and since that year the largest individuals have slightly exceeded this size, which is near the maximum as we know it for this species. It is inconceivable that these unios were introduced as adults, for they are present in great numbers, and the farmer who owned the land was astonished to find them there four or five years after the pond was established, because it was near the entrance to his dooryard and he knew that no one had introduced mussels in any such numbers and that there was no watercourse connecting the pond with any creek in which mussels occurred. These mussels evidently came as parasites upon the fish with which this pond was stocked during the first year and they had reached a length of 4 inches in a period of five years. The abundance of the adults when the pond was six years old and the presence of some smaller specimens made it seem that more than one generation was represented, and hence some may have reached this size in a shorter time. The shell of Unio tetralasmus is light and is by no means a good button shell. Still it is not an impossibility, commercially speaking, for we have been assured by one of the leading button manufacturers, Mr. J. E. Krouse, of Davenport, Iowa, to whom we sent shells fram which buttons were cut, that a marketable button could be made from them and would be made if there were no other shells available. The appearance of Lampsilis subrostrata and Umo tetralasmus and no other species in all the ponds examined suggests the question, why have these two species and no others become established? If they were introduced as glochidia infecting fish, is it likely that the different lots of fish placed in so many ponds were infected solely with the glochidia of these two species? It seems much more probable that other mussels were introduced in the parasitic stages and that they were not able to survive long upon the bottom of these ponds. We have introduced large adult specimens of Quadrula metanevra and Symphynota complanaia into one of the ponds in question and found some of them still alive after two years. ‘This pond had a very soft mud bottom well covered with a layer of black muck filled with the soft coal soot from the smoke of a neighboring power-house chimney and seemed unsuitable for any variety of mussel. It had become, in spite of this, well stocked with Lampsilis subrostrata and is the pond referred to in detail in a previous paragraph. The survival here of these specimens of heavy shelled mussels for a period of two years shows that the adults are not at once killed even by unfavorable conditions, and we are therefore inclined to believe that when these species are introduced into the ponds on fish their destruction occurs in the early juvenile stages. If a small body of water can be so fully stocked by the scant infection of glochidia obtained by fish in nature, we should be able to introduce mussels like these into a pond far more effectively by the use of fish which had been artificially infected and to rear 186 BULLETIN OF THE BUREAU OF FISHERIES. them to adult size within a short term of years. Accordingly, we have attempted the introduction of Lampsilis ligamentina into one of the ponds where no mussels had ever been found by placing in the pond several hundred fish well infected with the glochidia of this species; but several examinations of the mud and silt from the bottom, made during the 18 months following, have failed to show anything as a result of the experiment. The conclusions drawn from these observations are encouraging because they indicate, first, that other species, like those of the genus Lampsilis, whose shells are of excellent quality for the best of buttons, may be reared to commercial size in about the same length of time, and, second, that restricted localities can be stocked with mussels by the introduction of fish infected with glochidia. The members of the genus Lampsilis have shells which are evidently not much heavier than the shell of Unio tetralasmus, a fact which better fits them for life upon soft bottoms where there is little current, and in such localities they often occur. They move about more actively than the heavier shelled species and this, doubtless, enables them readily to seek out the most favorable food conditions in any body of water, instead of remaining long in one place where the conditions are very stable, as do the heavier shelled species. The study of any mussel which can live in small ponds like those in question and from which button shells can be obtained should be followed up with care, since the extensive culture of mussels would be a far simpler matter in ponds than in any stream where high and low water and the shifting of the bottom might so largely interfere with the most carefully located beds. For this purpose the species of Lampsilis which give good button shells would seem the most desirable, because they are better adapted for the conditions and because our planting experiments indicate that they reach a market- able size in a shorter time than the quadrulas. We feel that there is nothing discouraging in what is at present known regarding the rate of growth under the average natural conditions. Moreover, it should be remembered that in most invertebrates where the growth rate has been studied this may be modified to an astonishing degree by the food supply and that the actual size of an individual furnishes no trustworthy clue to its age. It is not at all unlikely that proper study of the food and other conditions necessary for the maximum rate of growth will enable us to obtain shells of commercial size in even slow-growing varieties within a reasonable number of years. To judge from the supposed annual rings of specimens taken in nature, Quadrula ebena may take from 20 to 30 years to reach, under natural conditions, the size which is most desirable. The question whether this is a necessity, or only a result of the poverty of food conditions which most mussels meet in nature, is one which must wait upon the proper scientific analysis of the mussel’s food and rate of growth in this and other species, and there is no problem in connection with the attempted artificial propagation which has more pressing importance. REPRODUCTION AND ARTIFICIAL PROPAGATION OF FRESH-WATER MUSSELS. 187 VII. INVESTIGATIONS ON THE UPPER MISSISSIPPI RIVER. A brief reference may here be made to certain field studies which were carried on in connection with our mussel investigations during the months of June, July, and August, in 1908, on the upper Mississippi River. The Bureau of Fisheries put at our disposal for this purpose its substation, a small building provided with tanks and running water, at La Crosse, Wis., and also its steamboat, the Curlew, which not only furnished us with living quarters, but was of invaluable service for transportation from place to place on the river (fig. 65, pl. xvi). The boat, which is ordinarily used in the work of reclaiming young fish from the overflow of the river during the floods which occur in the spring and early summer, is equipped with aerated tanks, seines, and other apparatus and provided us with what was essentially a floating laboratory. With these facilities much was accomplished that would have otherwise been impossible. In addition to the usual crew of the Curlew, the party consisted, besides ourselves, of Messrs. W. E. Muns, Howard Welch, F. P. Johnson, and W. E. Dandy, students in the University of Missouri, who served as assistants. — The primary object of the expedition was a determination of the breeding seasons of the commercial species of musséls as far as possible at that time of the year and an examination of the depleted mussel beds in the upper Mississippi River, which have been all but destroyed as a result of the ravages of the mussel fisheries. With a clamming outfit of our own (fig. 69, pl. xv), consisting of a flat-bottomed skiff and “‘crow-foot’”’ dredges—the usual apparatus employed by the mussel fishermen— we were able to secure thousands of mussels, which were examined microscopically for the purpose of determining their sex and the stage of development of the embryos. The data thus obtained furnished a mass of detailed information, especially with respect to those species which breed in the summer, but as they are incorporated in the account already given of the breeding seasons, there is no need to refer to the subject again. The planting of young mussels in cages for a determination of the rate of growth was also made during this summer, with the result as described in a preceding section. Some attempts were made to infect fish with glochidia, but this phase of the work was greatly interfered with by the high water of the river, which remained at flood stage unusually late in the summer of 1908 and made the seining of fish very difficult. Some infections, however, were carried out with the glochidia of a few summer-breeding species, the fish being retained in the tanks at the La Crosse station throughout the parasitic period and the duration of the parasitism determined. A thorough survey of the mussel beds from Winona, Minn., to Lansing, Iowa, was made, and records taken at each locality where mussels were collected. No large beds at all were discovered, and in every instance where mussels were found indications of the ravages worked by the clammers were apparent. An account of the distribution of the species throughout this section of the Mississippi River and their relative abundance is not presented here, as the results of our observations in these respects will be incorpo- rated in the work of the several field parties which have been engaged in the study of 188 BULLETIN OF THE BUREAU OF FISHERIES. the geographical distribution of the Unionide throughout the Mississippi Valley under the direction of the Bureau of Fisheries during the past four or five years. While working in the neighborhood of La Crosse, we made a careful investigation of the west channel of the river at this locality, with a view to determining whether places of this nature presented favorable conditions for experimental rearing of young mussels. As is usually the case with the accessory channels of the river in this region, the west channel at La Crosse is dammed across its head for the purpose of confining the water in the main channel, and, although at high-water stages of the river the dam is sub- merged, during the greater part of the year the volume of water in the channel is greatly reduced and the current retarded. ‘These dams, however, are never tight, and a greater or less quantity of water constantly seeps through them. A thorough study of this channel showed that it contained very few mussels indeed, and of those species that were found living in small numbers under these conditions, the majority belonged to Lampsilis, ventricosa being by far the most abundant form. Whenever a channel of the river is dammed, the slackening of the current causes an enormous sedimentation to take place, and in these “‘sloughs,’’ as such obstructed channels are called, sand and mud bars and shoals have been formed to an extent varying with the length of time since the dam above them was built. The more sluggish species of mussels, like the quadrulas, are especially ill adapted to these conditions and are frequently buried and destroyed by the deposits of silt in the river, an occurrence of which we found abundant evidence. With the more actively moving and burrowing species, as those of Lampsilis, the case is different, for apparently they may adjust themselves more readily and by their far greater ability to move from place to place they may avoid the danger of being buried. We found little evidence that the quadrulas, for example, move abdut at all, while, on the contrary, the tracks of slowly wandering individuals belonging to the species of Lampsilis were everywhere conspicuous on the sandy bottoms of the shallow sloughs. An interesting case of the destruction of mussel beds in situ by sedimentation is shown in figure 70, plate xv11, which is a photograph taken on the bank of a slough, near Muscatine, Iowa, which was exposed by a gully washed out by rains and cut directly through an extinct mussel bed. The photograph shows the surface of the cut where the mussels are exposed as they lie embedded in the muddy bank. ‘The bed is buried under about a foot of mud, and it is interesting to note that the valves of the mussels are closed and lying together in pairs. The latter fact proves conclusively that this is not an old shell heap, for the valves of the shells would be found scattered and separated in that event, but a mussel bed which had once existed in the river near the bank. It was probably buried under the deposits of sand and mud which followed the building of the dam across the head of the slough. An investigation of the species represented in the bed showed that they all belonged to Quadrula, being chiefly ebena, pustulosa, and trigona, while not a single individual belonging to Lampsilis could be found init. It is probable, as already stated, that it is the sluggish species, like those of Quadrula, that are the prin- cipal sufferers in catastrophies of this nature, and are caught and smothered in the process of sedimentation, while the propensity to wander possessed by the more active species REPRODUCTION AND ARTIFICIAL PROPAGATION OF FRESH-WATER MUSSELS. 189 enables them to move out into deeper water when the deposit of silt becomes a menace. The result of our study of the conditions obtaining in sloughs like the west channel at La Crosse, which are closed by dams at their heads, proves conclusively that such waters afford a very unfavorable habitat for mussels, and that therefore they are not adapted to experimental uses. VIII. ECONOMIC APPLICATIONS. It may not be inadvisable to discuss briefly certain applications of the results obtained in the foregoing investigations to the practical work of artificially propagating fresh-water mussels on a commercial basis. It must be emphasized at the outset that the ultimate object of the investigations—the restocking of depleted waters with com- mercial species of mussels—is not dependent for its realization solely upon the success of rearing mussels artificially from the glochidia, but that other methods of attaining the same end may be employed which are of equal, if not greater, importance. PROTECTIVE LAWS. Much can undoubtedly be done by securing the passage of laws by State legislatures for the closing of certain streams or sections of streams against all clamming for a period of years of sufficient length to allow of a natural increase of the mussels; by laws pro- hibiting the use of the ordinary ‘“‘crow-foot”’ dredge, which takes immature and adult individuals indiscriminately,* and by laws prohibiting the discharge of sewage and factory refuse in the neighborhood of mussel beds. By these and other protective measures of a legal nature, a great deal might be accomplished in the way of conserving the supply of mussels in the more important waters, but, since in the case of many rivers the control isin the hands of two or more States, the passage of such laws would require, to be effective, similar action on the part of several legislatures, and such cooperation might not be obtained without the greatest difficulty. The utter futility of laws which would establish a closed season of the year against clamming is apparent in the light of our knowledge of the breeding seasons of the Unionidae. We have already seen that there is no month in the year when some species are not bearing embryos or glochidia, and as species of commercial value are found in both groups—those with the long and those with the short period of gravidity—a closed season at any time would be of little or no avail. Several species of Lampsilis, for example, which bear embryos or glochidia from August to July, furnish valuable shells for the pearl-button industry, while the species of Quadrula and other summer breeders, gravid from May to August, supply shells of the best quality. Any law then, designed to relieve the situation, which prohibits the taking of mussels during a sup- posed breeding season is based on ignorance of the facts, for the entire year is the breed- @ Mussels caught on a hook of the ‘‘ crow-foot’’ are generally so badly injured internally in the process that, even if they are afterwards thrown back into the river, the majority probably die. A special form of hook has been devised by Mr. J. F. Boepple which is so constructed that small mussels can not be caught by it. The use of some such selective apparatus should be required by law. 190 BULLETIN OF THE BUREAU OF FISHERIES. ing time of the Unionidae. A law, however, which would close a river or large section of a river for a period of five years or more would be most beneficial, for in that time much could be accomplished both by artificial and by natural means to restore normal conditions. Even artificial propagation, unaided by certain protective measures, could hardly be- come effective on however extensive a basis it might be carried on, for unless some means can be devised for saving the young mussels it is difficult to see how much head- way could be made against the destruction of the supply. It therefore becomes of vital importance not only to make illegal the use of any apparatus which will catch or injure young mussels, but to see that the law is rigidly enforced. Certain requisite conditions for the artificial culture of fresh-water mussels, based upon our knowledge of their life history and habits, may now be briefly referred to. SELECTION AND MAINTENANCE OF A FISH SUPPLY. Although only a comparatively few kinds of fishes have been thus far used in our experimental infections, and doubtless as our experience widens many more will be found to be favorable for the purpose, success has been attained chiefly with the black basses, rock bass, and the sunfishes. All of these fishes have proved to be extremely resistant to the injurious effects of gill infections (practically all of the commercial species of mussels have hookless glochidia, which are gill parasites); to be able to carry large numbers of glochidia through the parasitic period; and to be easily kept in confine- ment—three necessary conditions for the success of propagation. It is to be hoped that other fishes will be found to be equally useful, but at present those just mentioned afford the most promising material for the work. As has already been shown, some species of fishes are very easily killed even by light gill infections, while others, accord- ing to our experience, have resisted all attempts to bring about permanent implantation of glochidia on their gills. The latter is particularly true of German carp and catfishes. Fortunately, the basses and sunfishes can be obtained in large quantities without serious difficulty. In the reclamation work conducted by the Bureau of Fisheries along the upper Mississippi River, immense numbers of young bass are annually seined from the sloughs and ‘‘lakes”’ into which they are carried when the river rises over its banks during the flood stages of early summer. When the water recedes these young fish are caught outside the banks of the river, and only the small fraction of them which is reclaimed in the seining operations is saved from the wholesale destruction (fig. 67, pl. xvi). There is no limit to this supply of material for the work of mussel culture, and doubtless extensive use will be made of it at the Fairport station. Even more valuable for the purpose are the species of sunfishes which we have used (probably other species of the same group are equally good), for, besides being just as resistant and as readily infected as the black bass, they are more easily kept and are less subject to disease in confinement. An adequate number of breeding ponds, in which sunfishes could be left to multiply naturally, would insure a large and constant supply of these fish for artificial infections. REPRODUCTION AND ARTIFICIAL PROPAGATION OF FRESH-WATER MUSSELS. I9QI THE BEST SEASONS FOR INFECTIONS. It has already been stated that the duration of the parasitic period of the mussel is inversely proportional to the temperature of the water. This fact is obviously import- ant for mussel culture, since the longer the fish have to be kept while carrying the glo- chidia the greater is the loss from disease and other causes. The loss not only involves the fish but the potential mussels which they are nourishing as well. It therefore be- comes desirable to reduce, as far as possible, the length of time that the infected fish must be retained, and this we have seen depends upon the temperature. Late spring and summer, consequently, are the seasons when the maximum efficiency from arti- ficial infections should be obtained, for in the warmer water at that time the duration of the parasitism will be at the minimum—about two weeks or even less. The glo- chidia of Lampsilis are available all through the spring and as late as July, while those of Quadrula can be obtained during the summer months, and most of the commercial species of mussels fall in these two genera. Of course infections can successfully be made in the fall and winter and the duration of the parasitism reduced by keeping the water artificially warmed, but the difficulty of maintaining the fish alive under these con- ditions is greatly increased. THE MUSSEL SUPPLY. By far the greater number of species of commercial value, as has already been stated, belong to the genera Lampsilis and Quadrula, and, as both of these genera are widely distributed, practically all of the mussel-bearing streams of the Mississippi Valley may be drawn upon for a supply of material for cultural purposes. We have found that living mussels may be shipped even long distances with little or no mortality, especially in cool, weather, and it is therefore possible to obtain breeding material from places at quite a distance from the station where the infections are to be made, should the local supply be inadequate. We have had on several occasions large numbers of gravid mussels shipped from Terre Haute, Ind., to La Crosse, Wis., to Manchester, Iowa, and to Columbia, Mo., with scarcely the loss of an individual, and have successfully used the glochidia obtained from them in infecting thousands of fishes. According to our experience mussels thrive very well in confinement, in small ponds and laboratory tanks, and that without any special attention to a food supply. We have for years been keeping both pond and river forms alive in the laboratory for months at a time in tanks containing a few inches of sand on the bottom and supplied by tap water. Under such conditions mussels have frequently been retained in the laboratory from the fall to the following summer. It should therefore be an easy matter to keep mussels for breeding purposes in ponds with natural bottoms in any quantity desired, and, if the ponds are fed with river water, a natural food supply should be present in abundance. Since, as has been pointed out above, the species of Quadrula, Unio, and other sum- mer breeders abort their embryos and glochidia with astonishing ease when disturbed, it will be necessary, when making infections with the glochidia of forms exhibiting this peculiarity, to collect the material at a time prior to the fertilization of the eggs and to 192 BULLETIN OF THE BUREAU OF FISHERIES. allow them to enter upon the breeding season after being placed in the ponds of the station. We have had females of different species of Quadrula become gravid in the tanks of the laboratory after they had been held in confinement for weeks or even months, and therefore no difficulty should be encountered in obtaining a supply of glochidia from these forms under the conditions mentioned. REARING AND DISTRIBUTING YOUNG MUSSELS. After the fish have been infected, one of two things may be done in distributing the young mussels resulting therefrom: Either the fish, after having been retained in tanks or ponds until near the end of the parasitism, may be taken to the stream which is to be restocked and the clams allowed to drop off there, or the liberation may take place in ponds where the young mussels may be reared until they are of considerable size, say until they are a year old, and then distributed as desired. Both methods might be used successfully, but in the first case it is to be supposed that only a very small pro- portion of individuals thus liberated would succeed in reaching maturity, as they would be exposed to the same destructive agencies as are encountered under natural conditions. The difficulty and expense of transporting the infected fish, the mortality among the fish themselves resulting from shipment, and the subsequent loss of large numbers of the young mussels are considerations which lead one to regard this method as not an efficient one. It should be stated, however, that in using this method of distribution it would not be necessary to liberate the fish and thus lose them for subsequent infections, for they could be confined in wire-bottomed fish cars set out in the streams, and after the mussels had all fallen off and dropped through the bottoms of the cars the fish could be returned to the station. This would of course involve a very large amount of labor and much expense. It would, therefore, seem to be a far more effective practice to retain the young clams in ponds with natural bottoms until they could with safety be liberated in the streams. After infection, in this event, the fish could be set free in these ponds at once, and allowed to remain there throughout the parasitism of the glochidia, at the close of which they could be seined out and made to do service again. Supplied with river water, the ponds should furnish an adequate amount of food for a practically normal rate of growth of the young mussels, which at the end of a year at latest should be of sufficient size to be placed in favorable localities in the rivers. When ready for dis- tribution, the water in the ponds could be drawn off and the juvenile mussels raked carefully from the sand or mud. If properly packed, it should be possible to ship them in large numbers to considerable distances. It is only reasonable to suppose that a large proportion of the mussels thus reared would reach maturity after distribu- tion, and it is certain that the number coming through would be far greater than would be the case if the first method should be pursued. REPRODUCTION AND ARTIFICIAL PROPAGATION OF FRESH-WATER MUSSELS. 193 IX. CONCLUSION. Of course, many practical details essential to success will have to be worked out before the artificial propagation of fresh-water mussels will have passed beyond the experimental stage, for the efficiency of the work from an economic point of view will doubtless depend upon the satisfactory solution of certain problems in technique, which, although secondary in character, are nevertheless a prerequisite of success. However much is yet to be done—and it should be clear that the work is far from completion—the entire feasibility of artificial propagation has been demonstrated beyond the shadow of doubt. Besides filling in the gaps, some of them important ones, in the results already obtained, certain fundamental phases of the mussel investigations remain practically untouched. Chief among these is an exhaustive study of the physical conditions of the waters as affecting the growth of mussels: The relation between the mineral content of the water and shell formation; the relation between the character of the bottom, whether rocky, sandy, or muddy, to the habits of different species; and the relation between the rapidity of current to the life of the mussel and the kind of shell which it secretes. These and many other interesting problems of a similar nature await solution. The immense mass of data that have been collected by the Bureau of Fisheries with respect to geographical distribution of species and their relative abundance through- out the Mississippi Valley has not been digested, yet the results which will be derived from a careful analysis of this information will have a fundamental economic bearing upon mussel culture. It is essential to know the centers and limits of distribution of at least the more valuable commercial species for the purpose of effectively conducting the operations in restocking streams and of avoiding useless labor in attempting to establish a species where the chances of its survival would be slight. The whole problem of the food of mussels is as yet untouched. Not only are we ignorant of the specific food forms among the micro-organisms upon which mussels depend, but we do not know whether different species, or rather species living under different physical conditions and species possessing different habits, utilize different food forms. The possibility of artificially rearing cultures of the unicellular organisms used as food—when we know what these forms are—for enriching the water in which young mussels are retained before distribution should be determined, for it is undoubtedly true that results of the greatest practical importance and interest would be derived from such an investigation. Very little is known at present respecting the enemies and diseases of fresh-water mussels, yet the importance of information of this nature can not be overestimated. Especially should we know the relative susceptibility of different species to parasitic diseases, and whether certain species are immune against the invasion of parasites which in the case of other forms constitute serious enemies. A most fascinating and valuable field of investigation lies open in the study of the - causes of pearl formations, for since these concretions are due, in part at least, to the 194 BULLETIN OF THE BUREAU OF FISHERIES. presence of parasites, the possibility of producing them at will offers an interesting opportunity for experimental study. The Unionide, in short, are a group of animals which, for the great variety of problems, both scientific and economic, presented in their unique life history, their structure, functions, and habits, their many interesting adaptations, and in their economic relations, is scarcely excelled by any other invertebrates except the insects. At present we may be said to possess only an introduction to a knowledge of the family, and the writers of this paper will feel amply repaid for their labor if they have succeeded in exposing some of the problems which here lie open for investigation and at the same time in laying the foundation for the artificial culture of fresh-water mussels. REPRODUCTION AND ARTIFICIAL PROPAGATION OF FRESH-WATER MUSSELS. 195 BIBLIOGRAPHY. Bakr, C. E. von. 1830. Ueber den Weg den die Eier unserer Siisswassermuscheln nehmen um in die Kiemen zu gelangen. Archiv fiir Anatomie und Physiologie, bd. 7, p. 313-352. BLAINVILLE, DUCROTAY DE. 1828. Rapport fait 4 1’Académie des Sciences de Paris sur un mémoire de M. Jacobson. Annales des Sciences naturelles, t. 14, p. 22. Braun, M. 1878a. Ueber die postembryonale Entwicklung unserer Siisswassermuscheln. Berichte der physikalisch-medicinischen Gesellschaft zu Wiirzburg, Mai-Heft, p. 24-27; also in Jahrbuch der deutschen malakozoologischen Gesellschaft, jb. 5, p. 307-319. 1878b. Ueber die postembryonale Entwicklung unserer Siisswassermuscheln (Anodonta). Zoologischer Anzeiger, jg. I, p. 7-10. 1884. Ueber Entwicklung der Enten- oder Teichmuschel. Sitzungsberichte der Dorpater Naturforscher-Gesellschaft, bd. 6, p. 429-431. 1889. Die postembryonale Entwicklung der Najaden. Nachrichtsblatt der deutschen malako- zoologischen Gesellschaft, jg. 21, p. 14-19. Cau, R. E. 1887. Note on the ctenidium of Unio aberti Conrad. American Naturalist, vol. 21, p. 857-860. Carus, C. G. 1832. Neue Untersuchungen iiber die Entwickelungsgeschichte unserer Flussmuschel. Nova Acta Physico-medica Academie Cesaree Leopoldino-Caroline Nature Curiosorum, bd. 16, p. 1-87. Coker, R. E., and SurzBer, T. 1911. A note on the metamorphosis of the mussel Lampsilis levissimus. Biological Bulletin, vol. 20, p.. 179-182. Conner, C. H. 1907. The gravid periods of Unio. Nautilus, vol. 21, p. 87-89. 1909. Supplementary notes on the breeding seasons of the Unionidae. Nautilus, vol. 22, p. III, 112. FAausseK, V. 1893. Biologische Studien. I. Ueber Parasitismus und Viviparitat. Russkoje Bogatstwo, bd. r. 1895. Ueber den Parasitismus der Anodonta-Larven in der Fischhaut. Biologisches Central- blatt, bd. 15, p. 115-125. tgor. Ueber den Parasitismus der Anodonta-Larven. Verhandlungen des V. internationalen Zoologen-Congresses (Berlin), p. 761-766. 1903. Parasitismus der Amnodonta-Larven. Mémoires de 1’Académie des Sciences de St. Pétersbourg, vur sér., classe physico-mathématique, t. 13. 1904. Viviparitat und Parasitismus. Zoologischer Anzeiger, bd. 27, p. 761-767. FLEMMING, W. 1874. Ueber die ersten Entwicklungserscheinungen am Ei der Teichmuschel. Archiv fiir Mikroskopische Anatomie, bd. 10, p. 257-293. 1875. Studien in der Entwicklungsgeschichte der Najaden. Sitzungsberichte der kaiserlichen Akademie der Wissenschaften (Wien), ur. abth., bd. 71, p. 1-132. FRIERSON, L. S. 1904. Observations on the genus Quadrula. Nautilus, vol. 17, p. 111, 112. 196 BULLETIN OF THE BUREAU OF FISHERIES. Harms, W. 1go7a. Ueber die postembryonale Entwicklung von Anodonta piscinalis. Zoologischer Anzeiger, bd. 31, p. 801-814. 1907b. Zur Biologie und Entwicklungsgeschichte der Flussperlmuschel (Margaritana margari- tifera Dupuy). Ibid., bd. 31, p. 814-824. 1g907c. Die Entwicklungsgeschichte der Najaden und ihr Parasitismus. Sitzungsberichte der Gesellschaft zur Beférderung der gesammten Naturwissenschaften zu Marburg, p. 79-94. 1908. Die postembryonale Entwicklung von Unio pictorum und Unio tumidus. Zoologischer Anzeiger, bd. 32, p. 693-703. 1909. Postembryonale Entwicklungsgeschichte der Unioniden. Zoologische Jahrbiicher, Abteil- ung fiir Anatomie und Ontogenie, bd. 28, p. 325-386. IsELy, F. B. 1911. Preliminary note on the ecology of the early juvenile life of the Unionide. Biological Bulletin, vol. 20, p. 77-80. ISRAEL, W. 1911. Najadologische Miscellen. Nachrichtsblatt der deutschen malakozoologischen Gesell- schaft, p. 10-17. Jacogson, L. L. 1828. Undersdgelser til naermere Oplysning af den herskende Mening om Dammuslingernes Fremarling og Udvikling. Kongelige Danske Videnskabernes Selskabs Skrifter, ~ Naturvidenskabelig og Mathematisk Afdeling (Kjé6behavn), 1828, p. 251-297; reprinted in Bidrag til Bléddyrenes Anatomie og Physiologie, heft 1, Kjébenhavn, 1828, p. 249-362. Latter, O. H. 1891. Notes on Anodon and Unio. Proceedings of the Zoological Society of London, p. 52-59. 1904. The natural history of some common animals. Cambridge. Lea, Isaac. 1827. Descriptions of six new species of Unios, ete. Transactions of the American Philosophical Society, vol. 3, p. 259-273- 1838, 1858, 1863, 1874. Observations on the genus Unio, together with descriptions of new genera and species, vol. 2, 6, 10, 13. Philadelphia. (Originally printed in Transactions American Philosophical Society and Journal Academy of Natural Sciences, Philadelphia. ) LEEUWENHOEK, A. VAN. 1722. Arcana Nature Detecta, t. 2, epist. 83, and t. 3, epist. 95 and 96. Leyden. LEFEVRE, G., and Curtis, W. C. 1908. Experiments in the artificial propagation of fresh-water mussels. Proceedings of the Fourth International Fishery Congress (Washington), Bulletin of the Bureau of Fisheries, vol. xxv, p. 617-626. tg10a. The marsupium of the Unionide. Biological Bulletin, vol. 19, p. 31-34. 1g1ob. Reproduction and parasitism in the Unionide. Journal of Experimental Zoology, vol. 9) P- 79-115- 1911. Metamorphosis without parasitism in the Unionidae. Science, vol. 33, p. 863-865. Leyoic, F. 1866. Mittheilung iiber den Parasitismus junger Unioniden an Fischen in Noll. Tubingen, Inaugural-Dissertation. Frankfort a. M. Linus, F. R. 1895. The embryology of the Unionide. Journal of Morphology, vol. 10, p. 1-100. igor. The organization of the egg of Unio, etc. Ibid., vol. 17, p. 227-292. REPRODUCTION AND ARTIFICIAL PROPAGATION OF FRESH-WATER MUSSELS. 197 ORTMANN, A. E. 1909. The breeding season of Unionide in Pennsylvania. Nautilus, vol. 22, p. 91-95 and 99-103. 1gtoa. A new system of the Unionidz. Ibid., vol. 23, p. 114-120. 1910b. The discharge of the glochidia in the Unionidz. Ibid., vol. 24, p. 94, 95. ror. A monograph of the Najades of Pennsylvania. Memoirs of the Carnegie Museum (Pitts- burgh), vol. 4, p. 279-347. Peck, R. H. 1877. The minute structure of the gills of lamellibranch Mollusca. Quarterly Journal of Micro- scopical Science, vol. 17, p. 43-66. PFEIFFER, C. 1821. Naturgeschichte deutscher Land- und Siisswasser-Mollusken. Weimar. Poupart, F. 1706. Remarques sur les coquillages 4 deux coquilles, et premiérment sur les Moules (Anodontes). Mémoires de 1’Académie des Sciences de Paris, p. 51-61. QUATREFAGES, A. dE. 1835. Sur la vie intrabranchiale des petites Anodontes. Annales des Sciences naturelles, t. 4. 1836. Mémoire sur la vie intrabranchiale des petites Anodontes. Ibid., t. 5, p. 321-336. RATHEE, J. 1797. Om Dammuslingen. Naturhistorie Selskabets Skrifter (Kjébenhavn), t. 4, p. 139-179- ScHIERHOLZ, C. 1878. Zur Entwicklungsgeschichte der Teich- und Flussmuschel. Zeitschrift fiir wissenschaft- liche Zoologie, bd. 31, p. 482-484. 1888. Ueber Entwicklung der Unioniden. Denkschriften der kaiserlichen Akademie der Wissenschaften (Wien), Mathematisch-naturwissenschaftliche Classe, bd. 55, p. 183-214. ScumiptT, F. 188sa. Vorlaufiger Bericht iiber Untersuchungen der postembryonalen Entwicklung von Ano- donta. Sitzungsberichte der Dorpater Naturforscher-Gesellschaft, p. 303-307. 1885b. Beitrag zur Kenntniss der postembryonalen Entwicklung der Najaden. Archiv fur Naturgeschichte, jg. 51, p. 201-234. Simpson, C. T. 1900. Synopsis of the Naiades, or pearly fresh-water mussels. Proceedings of the United States National Museum, vol. 22, p. 501-1044. STERKI, V. 1895. Some notes on the genital organs of Unionide, etc. Nautilus, vol. 9, p. 91-94. 1898. Some observations on the genital organs of Unionide, ete. Ibid., vol. 12, p. 18-21 and 28-32. 1903. Notes on the Unionide and their classification. American Naturalist, vol. 37, p. 103-113. 1907. Note. Nautilus, vol. 21, p. 48. 198 BULLETIN OF THE BUREAU OF FISHERIES. EXPLANATION OF PLATES. (Drawings by G. T. Kline.] PLATE VI. Fic. 1. Gravid female of Ptychobranchus phaseolus. Actual length 96 mm. Fic. 2. Gravid female of Lampsilis subrostrata. Actual length 50 mm. Fic. 3. Gravid female of Symphynota complanata. Actual length 170 mm. PLATE VII. Fic. 4. Gravid female of Dromus dromus. Actual length 57 mm. Fic. 5. Gravid female of Quadrula ebena. Actual length 98 mm. Fic. 6. Gravid female of Lampsilis recta. Actual length 122 mm. Fic. 7. Gravid female of Obliquaria reflexa. Actual length 55 mm. Fic. 8. Gravid female of Cyprogenia irrorata. Actual length 38 mm. PLATE VIII. Fic. 9. Hooked glochidium of Symphynota costata, anterior end view. For measurements see text figure 1. Fic. 10. Hooked glochidium, as above. Lateral view of living specimen. Fic. 11. Axe-head glochidium of Lampsilis (Proptera) alata, anterior end view. For measure- ments see text figure 1. Fic. 12. Axe-head glochidium, as above. Lateral view. Fic. 13. Hookless glochidium of Lampsilis subrostrata, lateral view. For measurements see text figure 1. Fic. 14. Hookless glochidium, as above. Posterior end view. Fic. 15. Hookless glochidium, as above. Ventral view. Fic. 16. Detail of a conglutinate of Lampsilis ligamentina. The glochidia, still inclosed in the membranes, are less crowded together than those of figure 17, and are embedded in a mucilaginous matrix. Fic. 17. Detail of a conglutinate of Obliquaria reflexa, showing the membranes closely pressed and adhering together. Fic. 18. Young mussels (Lampsilis ligamentina) one week after liberation from the fish, showing various positions assumed in crawling, the ciliation of the foot, and the new growth of shell. PLATE IX. Fic. 19. Fin of a carp about 3 inches long, 7 days after infection with glochidia of Anodonta cata- racta, showing complete failure of the overgrowth of fin tissue in all places where the glochidia are greatly crowded. See explanation in the text, p. 159, of the conditions along the upper margin. Fic. 20. Tip of an over-infected fin, as above, 12 hours after infection, showing no appreciable over- growth because of the crowding. The shadows represent glochidia upon the under surface. Fic. 21. Pectoral fin of a carp, as above, 3% hours after infection; an optimum infection. Fic. 22. Ventral half of caudal fin of a carp, as above, 24 hours after infection; an optimum infection. Fic. 23. Tip of fin, as above, 32 days after infection. ‘The shadows represent glochidia upon the under surface. The infection is less than the optimum. The glochidia were well overgrown and all alive when the fish was killed. Fic. 24. Young Symphynota costata, attached by only a shred of tissue and about to drop from the fin after a parasitism of 74 days. ———— se er CS REPRODUCTION AND ARTIFICIAL PROPAGATION OF FRESH-WATER MUSSELS. 199 PLATE X. Fic. 25. Fin, as above, 36 hours after infection with glochidia of Anodonta cataracta, showing com- plete overgrowth of all glochidia which have become properly attached. Fic. 26. Glochidium of A. cataracta upon fin margin of carp, 34 hours after infection. Prolifera tion of cyst just beginning. Fic. 27. Glochidia, as above, upon fin margin of carp, showing different stages of cyst proliferation, even in neighboring glochidia. Fic. 28. Glochidia, as above, 24 hours after infection. Fic. 29. Hooked and hookless glochidia (A. grandis and L. recta) embedded and developing on a fin margin. Fic. 30. Glochidia of A. cataracta upon fin of carp, 3 days after infection, showing the cyst com- pletely formed. Fic. 31. Glochidium of A. cataracta upon fin of carp, developing normally after a shift of 90 degrees from the position first taken. Fic. 32. Two glochidia of A. cataracta, overgrown after 36 hours upon surface of a carp’s fin. Fic. 33. Glochidium of A. cataracta 35 days after infection. The metamorphosis is more advanced than in figure 30 and the rudiments of the foot and other organs have assumed greater size. PLATE XI. Fic. 34. Three gill filaments of the rock bass infected with glochidia of Lampsilis ligamentina. The metamorphosis of the glochidia has hardly begun, although they have been attached for 28 days. Fic. 35, 36, 37, and 38. Stages in the formation of the cyst surrounding a hookless glochidium (Lampsilis ligamentina) upon a gill filament of the black bass. Taken at 15 minutes, 30 minutes, 1 hour, and 3 hours, respectively, after infection. The transverse lines on the filaments indicate the lamelle. Fic. 39. Anterior gill of a black bass infected with glochidia of L. ligamentina, showing distribution - upon the gill as a whole and the appearance of the cysts. Fic. 40. Gill of yellow perch, as above. Fic. 41. Two conglutinates of Lampsilis ligamentina removed from the marsupium. One is shown from the flat surface, the other on edge. Actual length 17 mm. Fic. 42. Three conglutinates of Obliquaria reflexa removed from the marsupium. Actual length 17 mm. Fic. 43. Part of a gill of black bass infected with glochidia of L. ligamentina, showing the distribu- tion and orientation of the glochidia in an infection above the optimum for this fish. Only the row of filaments toward the observer is shown. PLATE XII. Fic. 44. Symphynota costata, dissected from fin of carp 47 days after infection. The anterior end istotheleft. Rudiments of foot, digestive tract, liver diverticula, and the first gill buds are recognizable; also the hooks and the degenerating adductor of the glochidium. Compare with figure 47. Actual size, 0.39 by 0.35 mm. Fic. 45. Strophitus edentulus, from a living specimen which had completed its metamorphosis without parasitism and which was actively crawling about on the bottom. Seen from the ventral side. The anterior and posterior adductors are well developed and within the foot the pedal ganglia and litho- cysts may be seen. Two gill buds are found on either side. Sections show that the internal organiza- tion is as far advanced as that of the young mussels shown in figures 47 and 48. 106. Fic. 46. A single cord discharged from the marsupium of Strophitus edentulus, showing glochidia extruded and others still within the cord. 13.5. Fic. 47. Symphynota costata, a young mussel which had been crawling upon the bottom for 6 days after a parasitism of 68 days. The very narrow margin of the adult shell has been drawn within the 18713°—12——7 200 BULLETIN OF THE BUREAU OF FISHERIES. valves. The glochidial shell and its hooks are still in evidence. In other respects the young mussel shows most of the features which are characteristic of the adult. The anterior end is to the right. Anterior and posterior abductors, anterior and posterior retractors, digestive tract divided into esopha- gus, intestine and stomach with its large diverticula, cerebral, pedal, and visceral ganglia, lithocysts, three gill buds, palp rudiments, the heart and pericardium will be recognized by their resemblance to the adult organs. Sections show the rudiments of the kidneys. From a stained and decalcified speci- men. Actual size, 0.39 by 0.35 mm. Fic. 48. Lampsilis ligamentina, a young mussel 1 week after the close of the parasitic period. The margin of the shell is extended well beyond the glochidial outline and shows the first lines of growth. More calcification has rendered the shell so opaque that the internal organs are no longer visible with- out decalcification. Stained specimens and sections show about the same degree of organization as in the Symphynota larva of figure 47. The foot with its cilia is shown extended and attached to the bottom preparatory to drawing the mussel forward. From a living specimen. Actual size, 23 by 20 mm. PLATE XIII. Fic. 49. Alasmidonta truncata. Horizontal section of a water tube of gravid marsupium, taken near ventral border of gill. The respiratory canals (r. c.) are small clefts, indistinctly shown under this magnification (cf. fig. 56); the marsupial space contains young embryos. Fic. 50. Quadrula ebena. Horizontal section of two adjacent water tubes (w. t.) of gravid mar-. supium containing young embryos. Fic. 51. Anodonta cataracta. Horizontal section of a water tube of gravid marsupium, showing respiratory canals (r. c.) and marsupial space (m. s_); the latter contains young embryos. Fic. 52. Symphynota complanata. Horizontal section of a water tube of gravid marsupium, show- ing respiratory canals and marsupial space; the latter contains glochidia. Note the thin, stretched interlamellar junctions. Fic. 53. Lampsilis ligamentina. Horizontal section of a water tube (w. t.) of gravid marsupium containing young embryos. Note the thin, stretched interlamellar junctions (i. j.). Fics. 54-55. Two stages showing process of implantation of a glochidium of Unio complanatus on a filament of a gill excised 2 hours after infection. Figure 54is taken 3 hours after attachment, while 55 is the same glochidium drawn 2 hours later. The greater part of the cyst was formed while the gill was in a watch glass. 3 PLATE XIV. Fic. 56. Alasmidonta truncata. Horizontal section through portion of lamella and water tube of gravid marsupium, showing respiratory canals (r. c.) near ventral border of gill; each canal is sep- arated from the marsupial space by a septum (s). The interlocking cells, forming the suture in the septum, are clearly seen. Fic. 57. Anodonta cataracta. Section similar to last, but taken before fusion of folds (s), which are seen not quite touching. The septum is formed by their fusion. Eggs contained in the marsupial space are in an early cleavage stage. : Fic. 58. Anodonta cataracta. Region marked X in last figure, highly magnified, showing glandular epitheliim of respiratory canals (r. c.), adjacent blood sinus (b. s.), and histological structure of sur- rounding tissues. Note the muscle fibers. PLATE XV. Fics. 59-61. Transverse sections of glochidia of Symphynota complanata, taken 15 minutes, 6 hours, and 24 hours, respectively, after attachment to edge of fish’s fin, showing three stages in formation of cyst. In 59 proliferation of epidermis is just beginning; in 60 glochidium is about half embedded; - while in 61 formation of cyst is completed. In 59, which is more highly magnified than the other two, and in 60 several mitoses are shown in region of proliferation. In 60 cellular detritus from enclosed edge of fin is being ingested by mantle cells of glochidium. REPRODUCTION AND ARTIFICIAL PROPAGATION OF FRESH-WATER MUSSELS. 20I Figs. 62-63. Transverse sections of glochidia of Lampsilis ligamentina, taken 30 minutes and 3 hours, respectively, after attachment to gill filament. In 62 the development of cyst has made con- siderable progress, while in 63 the cyst wall is practically completed. In 62 several mitotic figures are seen in the epidermis where multiplication of cells is taking place. Fic. 64. Highly magnified section of a portion of the glandular epithelium of an interlamellar junction in the gravid marsupium of Quadrula ebena, showing the large mucus cells and the nuclei of several leucocytes (1) with which the epithelium has become infiltrated. PLATE XVI. Fic. 65. Station of the Bureau of Fisheries at North La Crosse, Wis., and steamer Curlew, used in mussel investigations during summer of. 1908. Fic. 66. Interior of station at North La Crosse, equipped as a laboratory. Fic. 67. Seining young black bass near La Crosse in a ‘‘lake”’ which had been filled by the over- flow of the Mississippi River during the early summer. The fish thus obtained were artificially infected with glochidia. ; PLATE XVII. Fic. 68. Dredging for young mussels in a slough near La Crosse. Fic. 69. The clamming outfit used in the mussel work on the Upper Mississippi River. The two “crow-foot” dredges, with the mussels still clinging to the hooks just after a haul, are seen resting on the stanchions. Fic. 70. An old mussel bed near Muscatine, Iowa, buried under a foot or more of sand and mud but exposed in cross section by a gully washed out by rains. The mussels are seen in situ embedded in the wall of the gully. Bury. Uo. B. FS, L610. PLATE VI. Burr. Use. Ba F., ror]: PratH VIL. FIG. 4. iBiupeit, Wis Sis We Ihoq, Weoy PLATE VIII. | | FIG. 17. BuLE. U.S: Be E., 1910: PLATE IX. BULL AU. o> Bee, LOLS. lee Nanoy >< » Dole ere Aum BuLy. U.S. Be P., 1910: FIG, 39. 4o. Fic. a FIG. 4 FIG. 42. FIG. 41. Bur wUe ona: Pa. WOO. + Jeanie DOO —_—— FIG. 47. Fic. 48. A as Led But. U. S. B. F., 1910. PLATER XIII. ‘ oA AAR RG ao) 05 RC: FIG. 49. Fic. 54. FIG. 55- Biers User Bak., Lolo; PLATE XV. FIG, 56. 5.8 «PE On iba iS oe ae$ if wee va a. 2 beer) ise so ene