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ie From BULLETIN OF THE BUREAU OF FISHERIES, Volume XXVIII, 1908

| Proceedings: of the Fourth International Fishery Congress: : Washington, 1908

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EXPERIMENTS IN THE ARTIFICIAL PROPAGATION
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From BULLETIN OF THE BUREAU OF FISHERIES, Volume XXVIII, 1908

Proceedings of the Fourth International Fishery Congress : : Washington, 1908

WASHINGTON : : : : : : GOVERNMENT PRINTING OFFICER : : : : : : 1910

1° 4

BUREAU OF FISHERIES, DOCUMENT NO. 671
Issued April, 1910 ‘ ;
- ne : ‘

EXPERIMENTS IN THE ARTIFICIAL PROPAGATION
OF FRESH-WATER MUSSELS

a
By George Lefevre and W. C. Curtis
Professors of Zoology, University of Missourt
&

Paper presented before the Fourth International Fishery Congress
held at Washington, U. S. A., September 22 to 26, 1908

CONTENTS.

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Method e's Se 5 Wea LS Wee ats Saar cack Gee ENV eee Pe) sti per es epee noe eee
Behavior of the fishes—Their susceptibility and resistance_____-_-___------------------
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‘Post-larvall growth! of musselsa2=— S06 = bao ete eee en Se ee
Tnfection:ofslargesntim bers yo fetis biases eee ee ee er en na

616

EXPERIMENTS IN THE ARTIFICIAL PROPAGATION
OF FRESH-WATER MUSSELS.

a

By GEORGE LEFEVRE and W. C. CURTIS,
Professors of Zoology, University of Missourt.

Bad

OBJECT AND SCOPE OF THE UNDERTAKING.

The threatened extinction in the Mississippi River and its more important
tributaries of those species of mussels whose shells have been taken in enormous
numbers during the past fifteen years for the manufacture of pearl buttons has led
the United States Bureau of Fisheries to undertake an extensive investigation
to determine the possibility of artificial propagation of these mollusks, and to
devise such means as may be practicable for restocking depleted waters which
present favorable conditions. The general direction of the investigation has
been placed in the hands of the writers, who for the past three years have
devoted to the work as much time as their professional duties would allow.
In certain phases of the work, however, many others have collaborated.

It was recognized at the outset that if the investigation was to have real
practical value it would have to be wide in scope and extend over a period of
at least several years. At that time little was known with accuracy of the
breeding habits and seasons of the commercial species, of the biological and
physical conditions under which they live, of their distribution, and of many
other essential matters, while it yet remained to be discovered whether artificial
propagation could be successfully accomplished. At the very inception of the
work, therefore, a comprehensive plan was outlined which was designed to
include every subject that might bear even remotely upon the central problem,
and, although many parts of this programme have not yet been touched, much
progress has been made in some of the more important lines.

The plan of work contemplated, besides a thorough investigation of artificial
propagation, a detailed study of the life history and ecology of the Unionide,
emphasizing especially the geographical distribution of the group throughout the
Mississippi Valley, the breeding habits and seasons, the physical conditions of
the waters in which the different species thrive, food, enemies, and diseases,

617

618 BULLETIN OF THE BUREAU OF FISHERIES.

rate of growth, post-embryonic development, and the mutual behavior of
glochidia, or larvee, and fishes as parasites and hosts.

The results that have already beén obtained, although far from being
complete, will serve as a basis for future investigations, while the lines of attack
in the main problems have been definitely indicated. We have proceeded far
enough to make it clear that the ultimate end of the investigation is assured,
and with proper facilities for the infection and care of large numbers of fish
the final success of the work is no longer in doubt. The essential facts in the
life history of the Unionide are known; the general conditions of infection have
been determined; the entire feasibility of artificially infecting large numbers of
fish has been established; while the requisite conditions for placing artificial
propagation on a practical basis are clearly understood. Furthermore, during
the summers of 1907 and 1908 several parties, working under the direction of
the Bureau of Fisheries, have made extensive observations on a number of
different rivers in the upper Mississippi Valley, with a view to determining the
distribution of all of the species of mussels occurring in these waters. The
rivers that have been explored in this manner are the upper Mississippi, Illinois,
Iowa, Minnesota, Wabash, Ohio, and Tippecanoe, as well as several smaller
streams in Indiana. When the results obtained by these parties have been
brought together, we should be in possession of detailed information regarding
the distribution and relative abundance of at least all of the important com-
mercial species throughout this region. In addition, the breeding seasons of
almost all of the important commercial species have been determined, and a
large mass of miscellaneous data relating to a great variety of subjects has
been accumulated.

Some of the more important results that have thus far been obtained in
the investigations are briefly presented here.

SOME FACTS IN THE REPRODUCTION AND LIFE HISTORY OF MUSSELS.

As is well known, the Unionide carry their young in the gills, which act
as a marsupium or brood chamber until the completion of the embryonic
development. The eggs are passed from the ovaries into the suprabranchial
chambers, where they are fertilized by spermatozoa brought in by the inhalent
current of water. In many species the eggs are bound together in masses by a
gelatinous or glutinous matrix which is secreted by the gills and which may be
very tenacious. This, however, in most cases gradually dissolves away as
development proceeds, until at the close of the embryonic period the larve or
glochidia are no longer agglutinated. These masses assume in different species
many forms of flat plates or cylindrical cords which fill the water tubes of
the gills. The latter may be greatly distended or modified in various ways
to receive the egg-masses. In some species, on the other hand, the condition

PROPAGATION OF FRESH-WATER MUSSELS. 619

just described is absent and the eggs are either not agglutinated at all or are
only loosely held together in a slightly viscid substance.

The extent to which the gills are modified as a marsupium varies greatly in
different genera. In many, as in Anodonta, Pleurobema, Strophitus, Symphynota,
and Unio, the entire outer gill of each side is thus utilized and when filled with
embryos or glochidia, presents a much swollen, padlike appearance. In others,
for example, Lampsilis, Obovaria, Plagiola, only the posterior portion, varying
from one-third to two-thirds of the entire outer gill, is used as a receptacle for
the eggs, a sharp demarcation existing between the marsupial region and the
anterior respiratory part. The former is highly modified in structure and may
be readily recognized, even when empty. In Obliquaria reflexa a varying
number of enlarged and distended water tubes contain the dense, glutinous
cords in which the embryos are embedded. Finally, there are at least two
genera, namely, Quadrula and Tritigonia, in which all four gills are utilized as
brood chambers. ‘This condition would seem to be the most primitive one, as
the gills show but little structural modification and when empty are indistin-
guishable from those of the male mussel.

In all of the species of the genus Lampsilis which we have examined the
posterior margin of the valves in the female is less pointed than in the male, and
by reason of this difference in the shape of the shells the sexes may be readily
distinguished externally. In most of the other genera that have come under
our observation, however, the shells of the two sexes are identical, and an inter-
nal examination is necessary in order to determine the sex of an individual.

The entire embryonic development takes place within the marsupium, and
at the end of this period the glochidium, which has become liberated from the
egg membrane within the gill, is fully formed. In those species which are
known as “summer breeders,’ the glochidia are discharged at once, while in
many others, the so-called “winter breeders,” they are carried in the gills for
several months before being set free in the water.

The glochidia, as has long been known, do not complete their development
unless they become attached to fishes, and the metamorphosis occurs while
they are living parasitically in a cyst formed by the epidermis of the host.
After discharge from the marsupium, the glochidia under natural conditions
lie quiescent on the bottom, their valves gaping widely apart, and unless chance
brings them in contact with a fish they will die. Once attached to the gills or
external parts of a fish, however, the rest of the development, if conditions are
favorable, is assured.

This unique peculiarity in the life history of fresh-water mussels has been
taken advantage of in artificial propagation, as it has been found that a single
fish, even though small, may successfully carry to the close of metamorphosis
several hundred glochidia. One fish may, therefore, be made to do the work

620 BULLETIN OF THE BUREAU OF FISHERIES,

of a large number in a state of nature, for under ordinary conditions it is
extremely improbable that more than a very few glochidia would succeed in
attaching themselves to a single fish.

After a parasitic life of variable length, during which the metamorphosis
is completed, the cyst breaks down, and the young mussel, thus liberated, falls
to the bottom and leads thereafter an independent life.

BREEDING SEASONS.

We have confirmed the conclusion, first reached by Sterki (Nautilus, vol.
IX, 1895), that the Unionide, with respect to their breeding seasons, fall into
two classes, the so-called ‘‘summer breeders’? and ‘‘ winter breeders.’’ ‘The
latter designation, however, is not strictly appropriate, for in the species which
belong to this group the eggs are fertilized during the latter part of the summer,
usually in August, and the glochidia, which are carried in the brood chambers
over the winter, are not discharged until the following spring and summer. In
fact, in some cases the close of one breeding period may overlap on the begin-
ning of the next, as one may still find in late July some females gravid with
glochidia, while in others the eggs are passing into the gills for the next season.
In the case of the summer breeders the eggs are fertilized during the late spring
and summer, and the glochidia are in all probability discharged before the end
of August, or certainly not later than the middle of September. In the species
of this class of which we have the completest records, ovulation begins in May
and early June and may last well into August, but after the end of August
gravid females have not been found.

In view of the above facts, it would seem in better accord with actual
conditions to separate the species with respect to the length of time that the
glochidia remain in the gills, designating them as those which have a “short
period” of gravidity and those having a “long period,” rather than to dis-
tinguish them as “summer breeders” and ‘‘ winter breeders,” respectively, for
in the case of the latter neither ovulation nor discharge of the glochidia takes
place in winter. ‘The breeding season is undoubtedly a generic character, for,
so far as our observations go, all species of a given genus belong to the same

?

class.
The following list shows the distribution, with respect to the period of
gravidity, of the genera we have had under observation:

Long period: Short period:
Alasmidonta. Obliquaria.
Anodonta. Pleurobema.
Lampsilis. Quadrula.
Obovaria. Tritigonia.
Plagiola. Unio.
Strophitus.

Symphynota.

PROPAGATION OF FRESH-WATER MUSSELS. 621

As there are important commercial species in each class, the futility of any
law which would prohibit the taking of mussels during their breeding season
becomes at once apparent, for there is no month in the year when some species
are not carrying either embryos or glochidia.

THE LARVA, OR GLOCHIDIUM.

Two well-marked types of glochidia occur in the Unionide, one provided
with stout hooks on the ventral margin of the valves and the other entirely
hookless. The former are characteristically parasitic on the fins and other
external parts of the fish, the latter on the gills. We have given special atten-
tion to a study of the physiological peculiarities correlated with these two types
of structure, including their differences in behavior in attachment.

The occurrence of the two types of glochidia in the genera which we have
examined is shown below.

Hookless glochidia: Hooked glochidia:
Lampsilis (except L. lavissimus). Anodonta.
Obliquaria. Lampsilis levissimus.
Obovaria. Strophitus.

Plagiola. Symphynota.
Pleurobema.

Quadrula.

Tritigonia.

Unio.

It is thus seen that the hookless type is by far the commoner one, and
furthermore, practically all of the commercial species of mussels belong to
the genera included in this class.

The type of glochidium is constant for the genus, so far as our observations
have gone, except in the genus Lampsilis, one species of which, L. levissimus,
produces glochidia having hooks, while all of the remaining species that we
have examined have the hookless forms. The glochidia of L. Jevissimus, more-
over, are of quite a different form from those of any other species we have
encountered, being shaped like an axe head when seen in side view.

The hookless glochidia are usually much smaller than those having hooks,
and also differ characteristically from the latter in shape.

We have repeatedly determined the fact that the hooked glochidia form
permanent attachment as a rule only to external parts of the fish, as the margins
of the fins, operculum, anus, and mouth, the barbules, or even to scales and
along the fin rays occasionally. Wherever a free border is presented that may
be grasped between the valves of the glochidium, attachment is possible, the
hooks holding securely as soon as the valves have closed. Hooked glochidia,
furthermore, are not infrequently found on the gill arches and rakers, as these
places furnish the requisite conditions for a firm grasp. Glochidia of this type,

622 BULLETIN OF THE BUREAU OF FISHERIES,

however, do not as a rule acquire a permanent lodgment on the delicate gill
filaments, as their hooks usually tear through these when grasping. Their
greater size would also seem to render it difficult for the hooked glochidia to
become properly embedded under the epidermis of the slender filament.
Hookless glochidia, on the other hand, are only rarely found on the external
parts of the fish, and although they become attached readily to such places, in
the absence of hooks their grasp is insecure, and the usual consequence is that
they are all soon brushed off. Being hookless and quite small, they are admi-
rably adapted to lodgment on the gill filaments, among which they may become
entangled in great numbers.
Interesting differences of a physiological nature also are correlated with
the two structural types, apparently in adaptation to their different parasitic

habits.
IMPLANTATION OF THE GLOCHIDIA.

Contact with any part of the fish affords the stimulus which causes the
adductor muscle of the glochidium to contract and close the valves. This stim-
ulus is purely a mechanical one, as the same result may be produced by touch-
ing with any object. In this way they may be caused to snap shut on a bristle
or hair, or on the edge of a piece of paper. Once attached to the fish, how-
ever, the glochidium exerts a stimulus upon the epidermal cells of the host
which causes them to undergo a rapid proliferation. The stimulus in this case
is undoubtedly a chemical one, and its immediate effect is to throw the cells
of the epidermis into active mitotic division. The epidermis now grows rapidly
over the glochidium, which may become completely embedded in three hours
after attachment in the case of the gill parasites. The proliferation of the epi-
dermis of the fins occurs more slowly and in this situation twelve to twenty-
four hours are usually required before the glochidia are covered.

After the process of implantation is completed, the glochidia are entirely
inclosed within the cellular cyst, where they are nourished by the host until
liberated by the rupture of the cyst at the end of the parasitic period. It is
during this stage of the development that the metamorphosis occurs and the
structure of the glochidium is replaced by that of the mussel.

EXPERIMENTAL INFECTIONS.
METHOD.

A very large number of experimental infections have been made, and the
conditions determining infection have been investigated as far as the material
at our disposal has allowed. ‘This study has included not only the behavior of
both types of glochidia, their manner and place of attachment, but the reac-
tions of the fish as well.

PROPAGATION OF FRESH-WATER MUSSELS. 623

Twenty different species of mussels, yielding both hooked and hookless
glochidia, have been used for the infections. The majority of these have been
species showing the long period of gravidity, although several of the summer
breeding species have also been employed. Eighteen species of fishes have
been used, and their reactions have been carefully observed, but, as all are
not equally favorable for receiving and carrying the parasites, the greater num-
ber of infections have been:made upon five or six species.

When only a few fishes are to be used at a time, the method which has been
employed is quite simple. The brood chambers are excised from the gravid
mussels, split open lengthwise, and the ripe glochidia washed and scraped out
into dishes of water. The fishes are then placed together with the glochidia in
small aquaria, and, if the infection is to be made with gill parasites, the water
is agitated by mechanical means, in order to keep the glochidia suspended and
thus insure their being taken into the mouth of the fish. When using hooked
glochidia for external attachment, however, it is better not to stir them up, as
the fishes usually settle down to the bottom of the vessel and, by rubbing their
fins among the glochidia, quickly become infected to any desired degree.

When making gill infections, the fishes must be examined at intervals of
one or two minutes for the purpose of observing the rapidity with which the
glochidia are attaching, and when the degree is reached which experience has
shown to be the optimum for the species of fish in question, the exposure is at
once discontinued. Usually the gill infections take place very quickly, and
in four or five minutes the fishes will have received as many glochidia as they
can safely carry, although of course the duration of exposure depends upon
the abundance of glochidia in the water. Other things being equal, the fewer
glochidia present the more prolonged the exposure must be. Experience has
shown that the best results are obtained when the glochidia are used in very
‘‘dilute mixtures,’ for it is difficult to avoid overinfection, if they are at all
concentrated. Some species of fishes, however, are less susceptible than others,
and a longer exposure is necessary to insure the attachment of an adequate
number of glochidia. In certain cases we have had to allow the fishes to
remain with the glochidia for from two to three hours, or even longer, in order
to obtain a satisfactory degree of infection.

In nearly all of our experiments we have used young fishes, from three to
six inches in length, as for many reasons they are more convenient than larger
ones.

BEHAVIOR OF FISHES—THEIR SUSCEPTIBILITY AND RESISTANCE.

The fishes when first placed in contact with glochidia give evidence of great
irritation, and as soon as the gills begin to be infected they exhibit violent and
rapid breathing movements, apparently in the attempt to expel the parasites.

624. BULLETIN OF THE BUREAU OF FISHERIES.

After a time, however, they become quiet, and when removed from the presence
of the glochidia behave normally, except that the more rapid respiratory move-
ments may continue even for days afterwards.

The fishes we have used in our experiments have shown marked differences
in their ability to retain the glochidia both on the fins and on the gills, more
especially on the latter. They vary even more strikingly with respect to their
capacity for withstanding the injurious effects of gill infection, and further-
more some fishes are undoubtedly much more susceptible than others, as the
glochidia become attached, especially to the gills, more readily.

Of the fishes which we have had under observation, the following species
have proved to be the most satisfactory: rock bass, large and small mouthed
black bass, green sunfish, and red-spotted sunfish. All of these species are very
susceptible and readily infected with gill parasites, large numbers of which may
be carried without apparent injury to the fish. A single individual of any of
the above-mentioned fishes, not more than 3 or 4 inches in length, will success-
fully carry at least 1,000 glochidia on its gills to the close of the parasitic period,
a number that would speedily kill many other species.

Yellow perch, blue-gill sunfish, and crappie, although quite susceptible, are
far less resistent to the injury and quickly succumb to overinfection. Young
bullheads are apparently very difficult to infect, and we have not succeeded
in causing glochidia to become attached to either their gills or fins in appre-
ciable numbers.

German carp are easily infected on external parts with hooked glochidia,
but thus far all efforts to infect this fish with gill parasites have been unsuccessful.
The glochidia will attach themselves to the gill filaments, but for some unaccount-
able reason implantation is imperfect and they soon drop off. Apparently the
epidermis of this fish does not react to the stimulus in such a way as to lead to
the formation of a permanent cyst.

In still other cases it has been found that failure is due to the fact
that, even after complete implantation on either the gills or fins, the walls of
the cyst slough away in a day or two and liberate the glochidia. Whether this
is caused by unfavorable physiological conditions in the fish or in the glochidia
we are unable to say, but there are certain facts that would seem to indicate
that such abortive implantations are the result of a physiological immaturity
of the glochidia, which therefore fail to call forth the necessary response on the
part of the fish’s tissues.

DURATION OF THE PARASITIC PERIOD.

The duration of the parasitic period is very variable. With respect to
the glochidia of those species of mussels which remain gravid over winter the
duration depends upon the time of year when the infection is made. Fishes

PROPAGATION OF FRESH-WATER MUSSELS. 625

infected in the late fall and winter will carry the glochidia for from two and a
half to nearly four months, while in spring, when the same kind of glochidia
are used, the metamorphosis is completed and the young clams liberated in a
little less than a month. ‘This great difference in the length of time that the
glochidia remain on the fish can not be due primarily to temperature, although
this may have some influence, for we have observed the same differences in the
laboratory, where the temperature of the water remains practically constant
throughout the winter and spring. As the glochidia of the mussels which have
the long period of gravidity are not normally discharged until spring and summer,
it is quite possible that the fall and winter glochidia are physiologically immature,
although capable of causing the formation of the cyst on the fish, and require
for this reason a much longer time for the post-embryonic development. Struc-
turally, however, they seem to be identical with those taken from the marsupium
in the spring. :

In the case of the spring infections the young clams all leave the fish
at about the same time, and within a very few days after the first are liberated
the last to go have come off. ‘This is not true, however, of the fall and winter
infections, for in these cases the setting free of the young mussels may be
extended over a period of three or four weeks, or possibly more.

. We have no observations on the duration of the parasitic period in the case
of the summer-breeding mussels. Although infections have been success-
fully made with several of these species and kept under observation for ten or
twelve days, we have not yet had an opportunity of following the metamorphosis
through to the end. The period is, however, undoubtedly short, probably not
more than four weeks. Harms (Zoologischer Anzeiger, bd. xxx, 1908) has
recently recorded a parasitic period for two European species of Unio, a genus
belonging to the group of summer breeders, of twenty-six to twenty-eight days.

POST-LARVAL GROWTH OF MUSSELS.

No data have been obtained on the rate of growth of fresh-water mussels
beyond a short period after the metamorphosis, as we have not succeeded in
keeping the young mussels alive in the laboratory for more than four weeks after
leaving the fish. The amount of growth during that time was only a fraction
of a millimeter. We have made, however, extensive plantings of young mus-
sels which have been allowed to drop off the fishes in ponds and streams where
the chances of reclaiming them in the future are very good, and it is hoped that
by another year or two some data on the rate of growth will be available. Care-
fully measured and weighed young mussels of different sizes have also been placed
in wire cages and set in ponds and rivers, and it is possible that additional data

will be obtained from these experiments.
B, B. F. 1908—40

626 BULLETIN OF THE BUREAU OF FISHERIES.

INFECTION OF LARGE NUMBERS OF FISH.

In order to test the feasibility of infecting large numbers of fish, which is
a prerequisite of artificial propagation, an extensive experiment, in which
over 25,000 fish were employed, was made at the substation of the Bureau of
Fisheries at La Crosse, Wis., in November, 1907. Seven species of fishes and
glochidia from four species of Lampsilis were used in the experiment. The
fishes, with the exception of the German carp and the bullheads, were success-
fully infected and retained in the tanks of the station for over two months, when
they were liberated in the west channel of the Mississippi River at that place.
The experiment completely demonstrated the possibility of making infections
successfully on a large scale, and of retaining the fish until near the close of
the parasitic period.

In November, 1908, a similar experiment was carried out at the station
of the Bureau at Manchester, Iowa, where about 12,000 large-mouthed black
bass and crappies were infected with the glochidia of several species of Lampszilis.
The fish were kept at the station for one month and then placed in the Maquoketa
River between two dams. As the species of mussels used in the experiment in
all probability do not occur in that stream, the results of the work may be
determined if we succeed in reclaiming any of them at a future time.

During the fall and winter of 1908-9 infected fishes were put into ponds in
the neighborhood of Columbia, Mo., where we are absolutely certain that the
species of mussels used do not occur, and where there is every reason for the
expectation of subsequent reclamation. ‘The latter experiments should yield
valuable data on the rate of growth.

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