Ecology of the Trematode, Clinostomum Marginatum, and Its Hosts in Eastern Kansas ERWIN E

Home , Clinostomum marginatum, Largemouth bass

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Ecology of the Trematode, Clinostomum marginatum, and Its Hosts in Eastern Kansas ERWIN E. KLAAS Introduction Much of the fishing in eastern Kansas is done in farm ponds and small reservoirs; occasionally, the State Biological Survey of Kansas re- ceives inquiries from fisherman about "wormy" fish, usually with reference to farm ponds. The infections most often noted by anglers are metacercariae of Clinostomum marginatum (Rudolphi), a trematode (flatworm), commonly called the yellow grub. Although the parasite is harmless to man and there is little evidence that the yellow grub seriously affects the health of fishes, many fishermen discard infected fish because they are unattractive for human consumption. The life cycle of the yellow grub, as described by Hunter and Hunter (1934), is typical of trematodes for which fishes serve as intermediate hosts. A ciliated larva (miracidium) hatches from the egg in water, enters a snail of the genus Helisoma (first intermediate host) and, shedding its cilia, changes into a sporocyst. Rediae are produced parthen- ogenetically from germ cells within the sporocyst. Similarly, reproductive cells within the redia give rise either to larvae known as cercariae, or to daughter rediae which then produce cercariae. Cercariae leave the snail and, swimming by means of forked tails, come into contact with and penetrate fishes (second intermediate hosts). On entering the fish, cercariae shed their tails and secrete cysts, becoming metacercariae ("yellow grub" stage objectionable to anglers). These become adults on ingestion of the fish by the definitive host (a bird, usually a heron). Soon after reaching the stomach of the bird, the cysts surrounding the metacercariae are digested by enzymes in the stomach of the bird. The metacercariae migrate up the esophagus where the worms attach to the inner lining of the buccal cavity, upper esophagus, or trachea and become sexually mature adults in four to six hours. As the heron feeds, eggs are expelled into the water and the cycle is completed. In view of the possible significance of yellow grub infections in future fisheries management in Kansas, this investigation was undertaken to ascertain: (1) the extent of yellow grub infections in a series of farm ponds of unknown degree of infection, and (2) ecological factors associated with the occurrence and degree of infection in these ponds, and (3)

Transactions of the Kansas Academy of Science, Vol. 66, No. 3, 1963. [519] 520 Transactions Kansas Academy of Science degree of infection according to nearness of ponds to a nesting colony of Great Blue Herons, Ardea herodias, the definitive host of the yellow grub. Methods and Materials STUDY OF HERONS The foraging movements of Great Blue Herons were studied in Douglas County, Kansas, from the summer of 1960 until the fall of 1961. Ponds, lakes, and streams were visited; also, the movements of herons of one nesting colony were observed for approximately 70 hours at various hours of the day in March, April, and May, 1961. The nesting colony is on an intermittent tributary of Washington Creek in Douglas County, Section 17, Township 14 South, Range 19 East. In February, 1961, 46 old nests were present, approximately 60 to 80 feet from the ground, in nine large sycamore trees. A blind was con- structed about 50 feet above the ground in one of the trees, in the hope that nesting birds could be marked with dyes by means of a spray-gun operated at the blind. However, use of the blind so disturbed the herons that all nests in the blind-tree were deserted in the second week of incubation. Other observations were made from a hill about one-quarter mile from the heron colony, and from the top of a large silo about one- half mile away. From these vantage-points herons could be seen leaving and approaching the colony for a radius of about one mile. Ten herons were killed and examined for adult yellow grubs. All of the flukes that were found in the herons were fixed in 10 per cent formalin or alcohol-formol-acetic fixative in the field. The flukes were later stained with Semichon's carmine or Harris's hematoxylin and mounted on slides with Canada balsam. NUMBER AND DESCRIPTION OF PONDS SAMPLED Hastings and Cross (1962) reported the number of ponds. their distribution and general physical characteristics, and the predominant kinds of fish in Douglas County, Kansas, in 1954. They found in 1316 farm ponds of which 89.5 per cent were one-half acre or less in size. The largest of the ponds that I seined was about one and one-half acres, but most ponds were between one-tenth and one-fourth acre in size, and agreed with descriptions given by Hastings and Cross. All but eight of the ponds were used by livestock for wading. The average depth was about four feet and soft bottom-mud averaged 6 to 8 inches deep. Watersheds consisted mainly of permanent pasture, but all ponds received some drainage from cultivated fields. Samples of fish and snails were obtained from ponds in two transects, each about one mile wide and ten miles long; one extended north

Ecology of Trematode and its Hosts in Kansas 521

from the heron colony and the other east. Ponds in these transects were selected from aerial photographs that were taken in 1954, a period of severe drought. Therefore, it is reasonably certain that the ponds investi- gated are permanent, and all are several years old. Insofar as possible, samples were obtained from at least one pond in each section of each ten- mile transect. The ponds east of the colony will be referred to as Series E; the ponds north of the colony will be referred to as Series N. The ponds were numbered according to their relative distances from the heron colony as shown in Figure 1. All of the ponds are listed below with

KANSAS RIVER 100 97

0N-16 39 39 • N—I5 0 N—I4 38 38 O N—I3 • N—I2 g N—I 1 100 97 N—I0 ON-9 0 N-8 T. WAKARUSA RIVER 13 O N-7 /N-6 o0N-5 • N-4 MILES 0 1 2 o N-3 O N-2 T. o N—I 13 E-5 E-6 E-- I I E—I E_ 3 / E-7 o • • • - o 0E—I4 HERON / 0 E* 8 °0E-13 - 0 COLONY 0E-4 0 OE-10 E 150 •- E-2 —E-9 E—I2 E— IL R.I9 E R.20 E Figure 1. Localities of Ponds Seined in Relation to Location of Heron Colony. Solid Circles indicate ponds having infected fish. Ponds N-3, N-5, N-8, and N-14 did not have fish. Small dot on inset map of Kansas indicates general location of the study area. 522 Transactions Kansas Academy of Science the localities, surface areas, dates of seining, and the names of the landowners. SERIES E. E-1; Sec. 16, T. 14 S, R. 19 E; 1/4-acre; Aug. 1, 1960, Aug. 4, 1961; F. Deay. E-2; Sec. 21, T. 14 S, R. 19 E; 1/2-acre; Aug. 1, 1960, Aug. 1, 1961; E. L. Russell. E-3; Sec. 15, T. 14 S, R. 19 E; 1/4-acre; Aug. 2, 1960, Aug. 4, 1961; J. S. Beeghley. E-4; Sec. 15, T. 14 1 S, R. 19 E; /3-acre; Aug. 2, 1960, Aug. 4, 1961; D. F. Beeghly. E-5; 1 Sec. 15, T. 14 S, R. 19 E; /3-acre; Aug. 3, 1960, Aug. 1, 1961; R. L. Markeley. E-6; Sec. 14, T. 14 S, R. 19 E; 1/4-acre; Aug. 3, 1960, Aug. 17, 1961; P. R. Ulrich. E-7; Sec. 14, T. 14 S, R. 19 E; 1/4-acre; Aug. 2, 1960, Aug. 17, 1961; P. R. Ulrich. E-8; Sec. 13, T. 14 S, R. 19 E; 1/4-acre; Aug. 4, 1960, Aug. 1, 1961; R. P. Harrison. E-9; Sec. 13, T. 14 S, R. 19 E; 11/2-acres; Aug. 4, 1960, Aug. 1, 1961; R. P. Harrison. E-10; Sec. 18, T. 14 S, R. 20 E; 1/10-acre; Aug. 3, 1960; D. A. Wray. E-//; Sec. 16, T. 14 S, R. 20 E; 1/10-acre; Aug. 3, 1961; R. Hoskinson. E-12; Sec. 16, T. 14 S, R. 20 E; 1/4-acre; Aug. 3, 1960, Aug. 3, 1961; G. Hoskinson. E-13; Sec. 16, T. 14 S, R. 20 E; 1/4-acre; Aug. 3, 1960, Aug. 3, 1961; G. Hoskinson. E-14; Sec. 14, T. 14 S, R. 20 E; 1/10-acre; Aug. 5, 1960; W. E. Hoskinson. E-15; Sec. 13, T. 14 S, R. 20 E; 1/10- acre; Aug. 4, 1960, Aug. 3, 1961; E. F. Johnson. E-16; Sec. 13, T. 14 S, R. 20 E; 1/2-acre; Aug. 4, 1960, Aug. 3, 1961; R. 0. Johnson. SERIES N. N-1; Sec. 8, T. 14 S, R. 19 E; 1/10-acre; Aug. 8, 1960; D. Cleff. N-2; Sec. 5, T. 14 S, R. 19 E; 1/4-acre; Aug. 8, 1960. Aug. 18, 1961; R. Williams. N-3; Sec. 32, T. 13 S, R. 19 E; 1/4-acre; Aug. 8, 1960; H. Schellhorn. N-4; Sec. 29, T 13 S, R. 19 E; 1/2-acre; Aug. 8, 1960, Aug. 18, 1961; W. Thome. N-5; Sec. 29, T. 13 S, R. 19 E; 1/2-acre; Aug. 10, 1960; L. W. McNess. N-6; Sec. 29, T. 13 S, R. 19 E; 11/4-acre; Aug. 10, 1960; L. W. McNess. N-7; Sec. 20, T. 13 S, R. 19 E; 1/4-acre; Aug. 9, 1960, Aug. 19, 1961; G. A. Weeks. N-8; Sec. 8, T. 13 S, R. 19 E; 1/10-acre; Aug. 9, 1960; E. M. Hamming. N-9; Sec. 5, T. 12 S, R. 19 E; 1-acre; Aug. 9, 1961; W. 0. Mitchell. N-10; Sec. 6, T. 12 S, R. 19 E; 1/10-acre; Aug. 10, 1960, Aug. 10, 1961; R. Wulfkuhl. N-//; Sec. 6, T. 12 S, R. 19 E; 1/10-acre; Aug. 10, 1960; 1 R. Wulfkuhl. N-12; Sec. 32, T. 12 S, R. 19 E; /3-acre; Aug. 9, 1961; G. M. Schneck. N-13; Sec. 32, T. 12 S, R. 19 E; 1/10-acre; Aug. 10, 1960, Aug. 18, 1961; C. Hird. N-14; Sec. 29, T. 12 S, R. 19 E; 1/3-acre; Aug. 11, 1960; W. F. Elkin. N-15; Sec. 29, T. 12 S, R. 19 E; 1/4-acre; Aug. 10, 1960, Aug. 10, 1961; F. E. Emery. N-16; Sec. 20, T. 12 S, R. 19 E; 1/10-acre; Aug. 11, 1960, Aug. 10, 1961; B. F. Stegmaier. At the time the ponds were seined the following notes were recorded; (1) estimates of pond-area, average depth, and depth of bot- Ecology of Trematode and its Hosts in Kansas 523 torn ooze; (2) the presence or absence of submergent and emergent vegetation; (3) the condition of the banks (bare, sparsely vegetated, or grassy); (4) the amount of use by livestock; (5) the presence or absence of aquatic snails; (6) the year in which the pond was built (if known to the landowner). Measurements of temperature and turbidity were not made; however, all of the ponds were warm and turbid. These factors did not vary greatly from pond to pond except that where cattle were kept out of the pond-area and where most of the watershed was well vegetated the ponds were noticeably less muddy. COLLECTION AND EXAMINATION OF SNAILS In August, 1961, at the time the study-ponds were seined, snails were collected by hand-picking them from vegetation and debris in shallow water. Two persons collected at each pond for 10 minutes. The snails were brought into a laboratory and isolated in 50-ml. jars of tap-water to allow cercariae to emerge. After 24 hours the water in the jars was examined for cercariae of C. marginatum. All snails were then dissected under 200X magnification, in an effort to find rediae.

COLLECTION AND EXAMINATION OF FISH One seine-haul was made in each pond using a 50 ft. seine with

3/4 inch mesh. All fish taken were preserved in 10 per cent formalin and examined later for metacercariae. The following species were taken: Semotilus atromaculatus, creek chub; Notemigonus crysoleucas, golden shiner; Pimephales promelas, fat-headed minnow; Ictalurus melas, black bullhead; Ictalurus punctatus, channel catfish; Micro pterus salmoides, largemouth bass; Lepomis cyanellus, green sunfish; Lepomis macrochirus, bluegill; Lepomis humilis, orange-spotted sunfish; Pomoxis annularis, white crappie; Pomoxis nigromaculatus, black crappie. At the time the fish were examined for metacercariae, the total lengths of all fish in a sample were measured to 0.1 inch. When only part of a sample of fish was examined, at least 25 fish including a representative number from each size-class were examined for metacercariae. Catfish were examined by first cutting off each fin and then probing the muscle fibers at the base of the fin. Most metacercariae were exposed when the fin was removed. The head was then removed by a transverse section behind the gill covers. The head was split from the ventral side to expose the mouth cavity, and the gills, jaw musculature, and orbits were then probed. In each sample of catfish examined, the trunk and tail of several were sliced into thin cross-sections and examined for metacercariae in the body musculature. 524 Transactions Kansas Academy of Science

Scale-fish were examined by making a sagittal-section through the fish and removing each fin. The head and gills were probed and the viscera were removed. The right and left halves of the body musculature were held up to a strong light, which revealed metacercariae as dark spots in the translucent musculature. This method worked satisfactorily for all but largemouth bass more than 10 inches in length, and for bluegills which were heavily pigmented; those fish were examined by making thin slices through the musculature and probing. The number and location of metacercariae in each fish were recorded. The Definitive Host Although several species of wading birds may serve as definitive hosts of the yellow grub in North America (Hunter and Hunter, 1934: 275), the principal host is the Great Blue Heron, Ardea herodias. The species breeds throughout most of the eastern and southern United States in a variety of woodland and marshland habitats. Ardea herodias is a summer resident in Kansas, where more than 100 nesting colonies are known in the eastern and central parts of the State (Andrews and Stephens, mimeographed report, Kansas State College, Emporia, May, 1961). The herons most frequently nest in tall sycamores in riparian woodland along small streams. Observations at a nesting colony in Douglas County in March, April, and May, 1961, and observations of herons at ponds and streams throughout Douglas County, gave some information concerning seasonal activities. The herons began returning to the colony in early March; 45 to 50 birds were present by March 15. Cottrille and Cottrille (1958:5) reported that early in the season, the herons spent most of the time standing in the trees. This was also true at the colony I studied. Flights to and from the colony were infrequent; they usually occurred from dawn until 7 a.m. and in the hour before dark. Courtship started about March 15 and a heron was first seen sitting on a nest, apparently incubating, on March 20. Non-incubating herons were often seen carrying sticks used for nest-repair to their mates on the nests. The herons usually acquired nest-material in the immediate vicinity of the colony; I never observed them traveling more than one-fourth-mile to gather sticks. Young herons were first heard in the nests on April 23. The young were able to fly by early July and after July 15 herons were no longer seen at the colony. Initially an attempt was made to determine whether herons preferred to forage in some farm ponds more than others, and, if selective foraging occurred, whether it affected the incidence and degree of infection by yellow grubs. Ecology of Trematode and its Hosts in Kansas 525

Early in the breeding season, the herons remained near the colony; I never observed one leaving the valley. Herons occasionally made long flights from the colony after incubation had started, but many herons foraged in pastures, woodlands, and in a small stream less than one- fourth-mile from the colony. Presumably, food taken at the latter locations consisted mainly of insects, crayfish, and small rodents. The stream where the colony is located flowed until early May when it became pre- dominantly dry, with a few remnant pools. The stream was seined at several localities within a mile of the colony but no fish were obtained. Crayfish were not abundant but occurred in some deeper pools. Foraging near the colony was discontinued after young herons were heard in the nests, and flights to and from the colony became more frequent. Flights were most frequent in early morning but continued throughout the day. At the end of May the number of herons arriving at and leaving the colony averaged about four per hour. The number of flights to and from the colony was approximately equal in all directions. From my place of observation at the colony, it appeared that herons approaching the colony did so in a more or less straight line. Herons leaving the colony usually continued flight in the direction of departure until out of sight. Sometimes, however, a heron flew in one direction for a short time and then turned sharply to the right or left. Apparently the herons find places to forage by random wandering. Because of the large size of the colony the surrounding countryside probably is explored widely for food. I could not ascertain whether individual herons foraged at the same locations each day, but a few observations indicate that if a heron is successful at one locality, it will continue to visit that locality for several days thereafter. The owner of a pond four miles northeast of the colony reported that a heron visited his pond between 6:30 and 7:30 a. m. each day on six consecutive days (May 10 to May 15, 1961). The heron approached from the southwest (the direction of the colony), remained at the pond for 20 to 30 minutes, and then departed in a northerly direction. I visited pond N-4 on four successive days (August 20 to 23, 1961) between 1:00 and 3:00 p. m.; the first and third days a subadult heron was flushed from the pond, and on the second day, while four people were fishing from the banks of the pond, a subadult heron approached the pond but did not land. No heron was seen at the pond on the fourth day. On September 16, 1961, a subadult heron was accidently caught at pond N-4 in a No. 1 steel trap that had been set for muskrats. 526 Transactions Kansas Academy of Science

Little is known about the movements of young herons after they leave the nest. Recoveries of young European Common Herons, Ardea cinerea, banded as nestings (Owen, 1960:598), showed that for the first three months after leaving the nest they moved away in all directions. Great Blue Herons probably do the same. After fledging in early July, juveniles were often seen feeding in ponds and streams throughout the countryside, both singly and in groups. Herons were abundant throughout July, August, and September along the Kansas and Wakarusa rivers. Herons, heron-tracks and/or feathers were found at all but four of the ponds that were seined in August, 1960 and 1961. A Heron was seen feeding in pond N-8, which contained crayfish but not fish. The remains of partly digested fish, regurgitated by young herons in the nest when frightened or disturbed, were recovered and identified in the summer of 1960. The fish were mostly black bullheads and green sunfish, plus a few largemouth bass and bluegills. The presence of these species is consistent with their prevalence in my collections from farm ponds in Douglas County. Kirkpatrick (1940) reported that 92 per cent of the diet of Great Blue Heron nestlings consisted of fish. The other eight per cent consisted of crayfish, aquatic insect larvae, and small rodents. The stomach of one of the herons that I killed contained many crayfish, and numerous dragonfly larvae were found in the stomach of another. Fish made up the stomach contents of eight herons. All ten herons that were examined were infected by Clinostomum marginatum. Three had immature flukes in the posterior end of the esophagus, near the stomach. The immature flukes were smaller than full adults, and appeared white and opaque. Adult flukes were all found in the upper esophagus and buccal cavity, attached to the lining of the pharynx, tongue, internal nasal cavity, and in the folds of the glottis. A few were found inside the trachea. The number of flukes found in eight of the herons varied from one to eleven; 95 flukes were found in one heron and 48 in another. The sex and age of the herons and the location of the flukes in the body of the heron are given in Table 1. First Intermediate Host Cercariae of Clinostomunz marginatum, to my knowledge, have only been reported from snails of the genus Helisoma. Huter and Hunter (1934) experimentally infected Helisoma cam panulatum and H. antrosa while working out the life cycle of the fluke. Several authors have reported cercariae from H. trivolvis. The latter species is the most common representative of the genus in Kansas, and was found in 11 of 32 study ponds, usually together with Physa hawnii. Gyraulus parva was also collected in ponds N-12 and N-15. H. antrosa is rare in Kansas and

Ecology of Trematode and its Hosts in Kansas 527 is restricted to clean, flowing water (Leonard, 1959:60). H. trivolvis is generally found in quiet, shallow water, on ooze-covered bottoms, rocks covered by algae, and dead leaves and stems of plants. The abundance of snails in August was not correlated with the condition of the ponds. Snails were nearly as abundant in ponds E-11 and E-3, which were extremely turbid and heavily used by livestock, as in ponds N-15 and E-16, which were relatively clear, supported a dense growth of submergent vegetation, and were fenced from livestock. Table 2 shows the number and species of snails collected from each pond and examined for cercariae.

Table 1. List of herons examined showing age of heron, date and locality taken, number of C. marginahun found, and location of flukes in the heron. Asterisks designate sexually immature flukes. Number of Date Age & Sex Flukes Location in Heron July 14, 1960 Adult, a 79 Mouth and Upper Esophagus 16* Lower Esophagus July 14, 1960 Sub-adult, a 8 Mouth July 14, 1960 Sub-adult, 9 3 May 15, 1961 Adult, 9 3 fl May 24, 1961 Adult, 9 1 .. May 24, 1961 Adult, 6 2 11 July 11, 1961 Sub-adult, 8 8 2* Lower Esophagus July 18, 1961 Sub-adult, 9 40 Mouth and Upper Esophagus 6 Trachea 2* Lower Esophagus Aug. 22, 1961 Adult, 9 1 Trachea Sept. 16, 1961 Sub-adult, S 11 Mouth and Upper Esophagus

Table 2. Number of snails collected and examined for cercariae in August, 1961. Asterisks designate ponds in which infected fish were found. Pond Pond Number Helisoma Physa Number Helisoma Physa Gyraulus E-1 0 0 N-1 0 0 0 E-2* 35 78 N-2 0 0 0 E-3* 23 31 N-3 E-4* 68 22 N-4* 0 0 E-5* 100 22 N-5 .... E-6* 0 0 N-6 55 0 0 E-7 0 22 N-7 0 0 0 E-8 2 104 N-8 0 0 0 E-9* 35 92 N-9 0 12 0 E-10 0 0 N-10 0 0 0 E-11* 76 14 N-11 0 0 0 E-12 0 89 N-12* 78 0 12 E-13 0 106 N-13 0 0 0 E-14 0 0 N-14 E-15* 0 0 N-15* 130 0 15 E-16 135 0 N-16 0 10 0 528 Transactions Kansas Academy of Science

The furcocercus cercaria of C. marginatum has been described in detail by Krull (1934) and can be identified by its morphology and man- ner of swimming. Only five snails (one adult H. trivolvis from pond E-9 and four from pond N-12) of 735 examined (.68 per cent) were found to be infected by C/inostomum. H. trivolvis was found in 8 of 11 (73 per cent) of the ponds having infected fish-populations but the snail was found in only three of 17 (18 per cent) of the ponds in which the fish seemed free of infection even after an extended search. Ponds E-6, E-15, and N-4 all had infected fish but no H. trivolvis were found in those ponds, except for a few dead shells in N-4. Presumably Helisoma was present but extremely scarce in these ponds in August; possibly snails were more numerous at the time the yellow grub infection was acquired by the fish, but this was not evidenced by a large accumulation of dead shells. The above evidence indicates that neither an abundance of snails nor a high incidence of infected snails is necessary for infection of fishes This can be partly explained from the work of Edney (1950), who collected wild H. trivolvis and in one adult found 3500 rediae, several hun- dred of which contained mature cercariae and some of which contained both rediae and cercariae. In a six-hour period 8576 cercariae emerged from one snail. After experiments with laboratory-raised snails, Edney (1950) ascertained that snails (H. trivolvis) that are not more than two months old can be infected readily with C. marginatum. Snails two to 12 months of age were infected with difficulty, but snails older than 12 months could not be infected. Edney concluded that rediae in differ- ent stages of development are found in infected snails, from a few months to two or more years old, because of the continuous replacement of senile rediae by young productive rediae at the same time cercariae are produced Thus the infection is maintained in a snail for several years. According to Edney, the large yield of cercariae by old snails is explained by the presence of large numbers of productive rediae. My data show an absence or a low incidence of infection in snails in all ponds studied, an apparent absence of snails in some ponds having infected fish, and differences in rates and occurrences of metacercarial infections of fish in some ponds from year to year. Although conclusive evidence is lacking, some factors which apparently affect these conditions are: (1) natural fluctuations in the snail population in a pond from year to year, (2) a low incidence of infection in snails and (3) high mortality in some populations of snails as a result of redial infection by C. marginatum or other trematodes. In any event, one infected snail in a Ecology of Trematode and its Hosts in Kansas )29 pond, if it lives for an extended time, can produce enough cercariae to infect a large number of fish.

Second Intermediate Host The effect of the yellow grub on the health of fishes has been discussed in numerous reports, the most noteworthy of which are by Elliott and Russert (1949), Rabideau and Self (1953), and Woodbury (1940. A quantitative study of parasites of fishes with special reference to Clinostomum marginatum in the perch of Walsh Lake, Michigan. Un- published PhD thesis, University of Michigan). These authors found no direct correlation between the degree of parasitism and the condition of the fish. Occasionally an infection of epizootic proportions occurs, such as that reported by Forney (1955), in which the loss of fish can be attributed to a great number of cysts. Although the evidence indicates that infections are rarely so serious as to cause slow growth or mortality, infected fish conceivably are more susceptible to predation than uninfected fish. Until recent years it was believed that fish in temperate waters lost their infections of yellow grubs in winter. Fischthal (1949) has shown that the over-wintering loss of grubs in panfish is negligible. Species and numbers of fish caught and examined for yellow grubs, and the number of ponds in which each species occurred, are listed in Table 3. Eleven kinds of fish were taken from 28 ponds. The black bullhead (present in 21 ponds) and the green sunfish (present in 18 ponds) were most commonly taken. The fat-headed minnow was present in 11 ponds; all other species occurred in five or fewer ponds. Only three (E-4, N-6, and N-11) of the 28 ponds are used for the production of sport-fish.

Table 3. Species and numbers of fish caught and examined for yellow grubs from 28 ponds, Douglas County, Kansas, 1960 and 1961.

Number Number Number Number Species of Ponds Caught Examined Infected

Black Bullhead 21 4777 1715 163 Green Sunfish 18 2653 1087 19 Fat-headed Minnow 11 921 315 2 Bluegill 4 466 336 15 Golden Shiner 2 263 125 1 Orange-spotted Sunfish 1 126 126 103 White Crappie 2 96 70 1 Largemouth Bass 5 83 83 6 Channel Catfish 2 7 7 0 Northern Creek Chub 1 6 6 0 530 Transactions Kansas Academy of Science

Degree of Infection Table 4 lists ponds from which infected fish were seined in 1960 and 1961, together with the rate of infection in each species and pond. Ponds E-2, E-3, E-4, E-9 and N-4 had infected fish in both years. Only one yellow grub was found in one green sunfish in pond E-6 in 1960; in 1961 no infected fish were found in that pond. Ponds E-11 and N-12 were seined only in 1961. Ponds E-5, E-15, and N-15 were seined in both 1960 and 1961 but infected fish were found only in 1961. Tech- niques of sampling and examination were the same in both years; therefore, the rather large number of infected fish found in 1961 indicates

Table 4. List of fishes and their degree of infection in each pond, 1960 and 1961.

Number Number Per Cent Sample Size Examined Infected Infected Pond Fish Host 1960 1961 1960 1961 1960 1961 1960 1961 E-2 Black Bullhead 19 57 19 57 5 16 26.31 28.07 Green Sunfish 64 30 64 30 4 3 6.25 10.00 Golden Shiner 127 2 76 2 1 0 1.31 0.00 E-3 Black Bullhead 56 8 56 8 34 2 60.71 25.00 Largemouth Bass 3 25 3 25 0 2 0.00 8.00 Fat-headed Minnow 0 17 0 17 0 0.00 E-4 Channel Catfish 3 3 3 3 0 0 0.00 0.00 Bluegill 174 180 174 50 9 6 5.17 12.00 Green Sunfish 109 5 109 5 5 2 4.58 40.00 Largemouth Bass 1 9 1 9 0 4 0.00 44.44 E-5 Black Bullhead 105 138 40 44 0 6 0.00 13.63 White Crappie 70 42 41 42 0 1 0.00 2.38 E-6 Black Bullhead 682 58 118 58 0 0 0.00 0.00 Green Sunfish 286 34 67 34 1 0 1.49 0.00 Fat-headed Minnow 16 2 16 2 0 0 0.00 0.00 E-9 Black Bullhead 295 205 295 60 68 11 23.05 18.33 Green Sunfish 8 8 8 8 2 0 25.00 0.00 Fat-headed Minnow 15 1 15 1 1 1 6.60 100.00 E-11 Black Bullhead 101 101 26 25.74 E-15 Black Bullhead 267 64 65 64 0 10 0.00 15.62 N-4 Black Bullhead 108 57 58 57 0 0 0.00 0.00 Orange-spotted Sunfish 45 81 45 81 38 62 84.44 76.54 Green Sunfish 8 18 8 18 0 0 0.00 0.00 White Crappie 2 4 2 4 0 0 0.00 0.00 N-12 Black Bullhead 13 13 2 15.38 Bluegill 10 10 0 0.00 Green Sunfish - - -146 60 3 5.00 Fat-headed Minnow 3 3 0 0.00 Northern Creek Chub 6 6 0 0.00 N-15 Black Bullhead 256 35 75 35 0 10 0.00 28.57 Ecology of Trematode and its Hosts in Kansai 531 that infections in these three ponds were acquired between August 1960 and 1961, or the incidences of infections increased substantially in that interval. No changes were noted in the general features of the ponds from one year to the next. In ponds from which the sample of fish was large, the rate of infection ranged from 1.31 per cent to 84.44 per cent, but usually was less than 30 per cent. In pond E-9 in 1961, the fat-headed minnow shows 100 per cent infection but only one specimen was taken. Generally, the black bullhead had the highest rate of infection, varying from 13.63 per cent to 60.71 per cent (average 25.49 per cent) where infections occurred. Bullheads were present in ten of the eleven ponds in which infected fish were found. In five ponds (E-2, E-3, E-5, E-9 and N-12) the rate of infection in black bullheads exceeded the rate of infection in other species present in those ponds. In three ponds (E-11, E-15 and N-15) the black bullhead was the only species taken. The rate of infection in other species exceeded that in bullheads in only two ponds (E-6 and N-4). In E-6 only one fish was found with a minor infection; pond N-4 is a special situation and will be discussed below. In the one pond (E-4) in which black bullheads were not present, channel catfish were present but were not infected. With the exception of the orange-spotted sunfish in pond N-4, the rate of infection in the Centrarchidae (bluegill, green sunfish, white crappie, and largemouth bass) varied from 1.49 to 44.00 per cent with an average of 13.66 per cent. Minnows (golden shiner, fat-headed minnow and northern creek chub) occurred less frequently and were seldom infected. When an infection did occur its incidence was always low. In pond E-3, fifty-six bullheads showed a high rate of infection (60.71 per cent) in 1960, but only eight bullheads were taken in 1961. Soon after this pond was seined in 1961, all fish remaining were killed by rotenone and only one additional bullhead was recovered, together with several small bass. Pond N-4 had an unusual distribution of infection. Four species of fish, black bullhead, green sunfish, white crappie, and orange-spotted sunfish, were taken in 1960 and 1961. The orange-spotted sunfish was the only species infected by yellow grubs, and at a rate higher than that of any other species in any pond sampled. The owner of the pond stated that orange-spotted sunfish were stocked in pond N-4 in 1956, together with the other three species named. Subsequently, only black bullheads have been stocked. All fish taken were small and appeared stunted. Examination of scales of several specimens indicated that all 532 Transactions Kansas Academy of Science orange-spotted sunfish were less than three years old; therefore, it i.$ apparent that the infection was acquired in the pond. Pond N-4 is about one-half acre in area and was highly turbid in both 1960 and 1961, although the pond is fenced from livestock and the watershed is pre- dominantly in permanent pasture. The bottom-profile of pond N-4 differs from that in other ponds. Approximately one-half of the pond is shallow, having an average depth of about three feet, and is covered by American lotus ( Nelumbo lutea), a rooted plant with large floating leaves. The other half of the pond slopes abruptly to a depth of 15 feet and averages about 12 feet in depth. The banks of the entire pond are steep. No snails of the genus Helisoma were found; however, collecting was difficult because of the high turbidity and steep banks. The under- sides of leaves and the stems of vegetation were extensively examined for snails. The deep part of the pond could not be seined and all four species of fish were taken in the shallow part. It is not known why orange-spotted sunfish were heavily infected while other species in pond N-4 were not infected. Perhaps orange- spotted sunfish occupied part of the pond which placed that species in maximum contact with infected snails, whereas other species avoided that area. Most of the ponds I seined were "overpopulated" with fish, but high populations were not correlated with the occurrence or degree of infection. In many ponds that were overpopulated no infected fish were found; yet, pond E-4 had a relatively high degree of infection, although it has been properly managed for the production of sport-fish. The growth-rate of its fish was rapid. Number and Location of Cysts The mean number of cysts per fish varied from one to four, but in pond E-11 the mean was about 25 per fish. In some ponds, one or two fish had a much higher number of yellow grubs than any other individual of the same species and size. For example, one black bullhead, 5.1 inches long, from pond E-9 had 173 yellow grubs. Thirty-six other infected individuals, 5.0 to 5.9 inches long, had only 1.9 cysts per fish. A regression analysis showed no association between lengths of fish and number of yellow grubs per fish in any of the ponds. When the fish were examined for metacercariae the location of each cyst was recorded (Table 5). Cysts in catfish were most numerous beneath the skin at the base of the fins or in the head and gills. Cysts in the head were mostly beneath the skin on the inner or outer surfaces of the gill-covers, or beneath the epidermal lining of the dorsal surface of the mouth-cavity. Cysts were found in the trunk-and tail-musculature of Ecology of Trematode and its Hosts in Kansas 533 catfish only in fish that harbored a large number of metacercariae, such as the one in pond E-9 mentioned above. In scaled-fish, especially the orange-spotted sunfish, cysts were commonly found in the trunk- and tail-musculature as well as the region of the head, but fewer cysts were found at the bases of fins.

Discussion It is usually accepted that a predator will put most pressure on the food-source which is most abundant and/or most available. Food requirements in a large colony of herons are high; Kirkpatrick (1940) estimated that a young heron may consume, on the average, as much as two pounds of fish per day. The congregation of herons at a breeding colony and their foraging movements during the nesting season suggest that fish in ponds close to a nesting colony are most vulnerable to infections by yellow grubs. In Series E, more infections occurred in ponds less than five miles from the heron colony than in ponds more than five miles away (see Figure 1). The higher frequency of yellow grub infections in ponds of Series E nearest the colony may result from more frequent foraging by herons in these ponds. No correlation of infection with distance is found in the Series N transect. Proximity of a pond to the nesting colony may not importantly affect its use by herons; the number of ponds located within a given area may be more important. The first five miles of the transect east of the heron colony has an average of approximately 5.4 ponds per square mile (including ponds that were not seined) whereas the area of the east transect 6 to 10 miles from the colony has an average of only 1.8 ponds per square mile. Moreover, the transect north of the colony has an average of only 2.1 ponds per square mile, possibly accounting for the

Table 5. Site of metacercariae in body of fishes. Column-headings: M, muculature; CF, caudal fin; DF, dorsal fin; AF, anal fin; PI, pectoral fins; P2, pelvic fins; HG, head and gills.

No. Fish Species Infected M CF DF AF Pi P2 HG Golden Shiner 1 0 0 0 0 0 0 1 Fat-headed Minnow 2 2 1 0 0 2 0 1 Black Bullhead 163 8 33 255 137 204 57 388 Green Sunfish 19 0 2 3 1 8 1 10 Orange-spotted Sunfish 103 142 13 28 15 54 33 164 Bluegill 15 4 0 0 3 8 2 10 Largemouth Bass 6 4 24 18 13 9 4 25 White Crappie 1 0 0 0 0 0 0 1 534 Transactions Kansas Academy of Science low incidence of infection in ponds along that transect. The Kansas and Wakarusa rivers and their tributaries also provide good foraging in the area north of the colony and may reduce foraging in ponds. As previously mentioned, herons were seen flying northward from the colony as often as in other directions. Probably foraging is done at approximately the same degree in all directions from the colony because there are ample foraging sites in all directions. However, the most successful northerly foraging sites may be streams, whereas farm ponds receive more herons foraging east of the colony. Lone Star Lake, 198 surface-acres in size, approximately four miles west of the colony serves as a good foraging site (herons were often seen there), but there are no recent reports of fish caught at Lone Star Lake having yellow grubs. Infections occurred in some ponds located nearly ten miles from the colony. Infections may have been carried to those ponds by herons rang- ing widely from the breeding colony or by non-breeding, yearling herons; the latter are not strongly attached to nesting colony in early spring, but wander from place to place in search of food. Because of the high rate of infection in the herons, it is likely that fish in any pond in Douglas County, having snails of the genus Helisoma, are susceptible to infection. The abundance of ponds, presence of a particular genus of snails, availability of fish, and the size and proximity of a heron colony are only a few of the factors that may affect the frequency of yellow grub infections. If snails more than two months old are not readily infected, as Edney (1950) suggested, the chances of a snail population receiving an infection from herons may be limited to the time when snails of the appropriate age are present in the pond. If the breeding season of the snails in nature coincides with the breeding season of the herons, ponds subjected to heavy foraging by herons having young in the nest should be more susceptible to infection than ponds not heavily used by herons for foraging. Conversely, if young snails are present in late summer, the spread of the infection may be amplified by the radiation of newly-fledged herons. It is known that H. trivolvis breeds in Kansas in early spring, when the temperature of the water reaches 10° C., and again in the fall when temperatures begin to decline (A. B. Leonard, personal communica- tion). Unfortunately, little information is available concerning the length of the breeding season or general population ecology of Helisoma in Kansas. There is no apparent environmental factor in the farm ponds studied by me that can be correlated with the occurrence or degree of infection of any of the hosts, or which causes preferential foraging by the herons. Ecology of Trematode and its Hosts in Kansas 535

Control Three methods of possible control of yellow grubs of fish seem evident—elimination of herons, elimination of the snail-host, or the development of managerial practices that reduce the vulnerability of fishes. The elimination of herons is impractical because of their widespread distribution and nomadic wandering after the breeding season. Herons have aesthetic appeal to large numbers of citizens who would object if effort were made to eliminate the herons. Also, the herons may be beneficial in helping to delay "overpopulation" of fish in a pond (exces- sive numbers of slow-growing fish are a common cause of poor fishing in ponds of Douglas County). Control of the snail population seems, at first glance, to offer prac- tical protection from infestations of yellow grubs. Complete eradication of the snails may be necessary, because infections occur even in ponds where snails are so scarce that they escape detection. Batte, Murphy, and Swanson (1951) tested 33 compounds as molluscicides at various concentrations under laboratory conditions and found dinitro-o-cyclohexy- phenol and anhydrous cupric sulfate to be effective. These two compounds when tested on snails in aquaria gave 100 per cent mortality in concentrations of one part to one million parts of water. These compounds are said to be non-toxic to man, grass, and animals that might drink the treated water or eat the plants. They are relatively inexpensive and are not thought to be affected by the organic content of the water. The use of molluscicides under natural conditions presents several problems. Following a widespread snail-eradication program in Egypt, Van der Schalie (1960) reported that two years after treatment with copper sulfate snails were as abundant as before treatment. Even if a means of destroying snails is perfected, eradication would be undesirable because snails may serve as an important link in the food-chain in a pond. Moreover, the use of molluscicides may eliminate other organisms such as plankton and aquatic insects that are important food-sources for young fish. Molluscicides have not been tested in Kansas ponds and cannot be recommended until more information is available on their effects. The most desirable means of control is one which will permit a biological balance in the natural environment. Vegetation in ponds that I studied seemingly had little effect on the occurrence of snails or infected fish; but, when snails occurred in ponds that had a dense growth of submergent vegetation, the snails were abundant in the vegetation and occurred farther from shore than in ponds lacking vegetation. Conse- quently, more fish may contact infected snails in densely-vegetated ponds. Van Cleave and Mueller (1934) suggested that fish living in deep water 536 Transactions Kansas Academy of Science and weedless shallows where snails are absent are less affected by parasites than fish living in areas where snails are abundant. Thus, it can be postulated that the reduction of submergent vegetation might reduce the rate of infection in some ponds by minimizing snail-populations and con- fining them to shallow edges of the pond. Better natural controls may become evident when more information is obtained concerning the population ecology of H. trivolvis; we need especially to determine the time and length of the breeding season, reproductive potential, optimum and limiting environmental factors, and the effect of redial infections on snail populations. The economic loss of discarded fish can be lessened if fishermen will note that the great majority of cysts are just beneath the skin or at the base of the fins and can be easily removed in cleaning with no effect on the edible flesh. In any case, eating them involves no dangers to man.

Summary In recent years, fishermen have complained that fish in some ponds and lakes in Eastern Kansas are "wormy". The infections were mostly metacercariae of Clinostomum marginatum, a trematode (flatworm), commonly termed the yellow grub. Investigations in 1960 and 1961, to determine the extent of yellow grub infections and ecological factors associated with the occurrence and degree of infection, indicate that infections by yellow grubs in fish in farm ponds in Douglas County are frequent. Observations of the foraging movements of Great Blue Herons, the definitive host of the yellow grub, indicate that before young are in the nest, much of the foraging by breeding herons is done near the nesting colony. After the young hatch and food requirements increase, foraging activities are dispersed more widely, and in all directions from the nesting colony. Although the occurrence and degree of infections suggest that herons prefer to forage in an area where ponds are abundant, there was no indication of highly selective use of any particular pond. Only five of 735 Helisoma trivolvis examined for cercariae (.68 pet cent) were found to be infected by larvae of Clinostomum marginatum. A higher percentage of ponds where H. trivolvis was found had infected fish than did ponds where the snail was not found. Of thirty-two ponds seined in August of 1960 and/or 1961, 28 contained fish, and 11 had infections of yellow grub. Infected fish were found in five ponds in both years; one pond had infected fish only in 1960; and fish in three ponds were infected only in 1961. Two ponds that had infected fish were seined only in 1961. No apparent relation- Ecology of Trematode and its Hosts in Kansas 537 ships existed between yellow grub infections, size and abundance of fish, or physical features of the ponds (except possibly in pond N-4). The degree of infection in any one year ranged from 1.31 per cent to 84.44 per cent, but usually was less than 30 per cent. The degree of infection in black bullheads usually exceeded that recorded in other species. In catfish most cysts were found at the bases of the fins and in the head and gills. Scaled-fish were often infected in the trunk and tail. musculature, as well as on the fins and head. Cysts were located just beneath the skin except in fish having unusually heavy infections; thus most cysts can be removed by fisherman while cleaning the fish. A natural means of control is preferred over the impractical elimination of herons or snails. It is postulated that reduction of submergent vegetation in a pond in order to limit snails to a smaller area near the shoreline may be a partial control.

Acknowledgements I am especially grateful to Professors Frank B. Cross and Richard F. Johnston who guided me in my study and gave critical assistance with the manuscript. Additional suggestions were made by Professors E. Ray- mond Hall, A. Byron Leonard, Robert Sokal, William H. Coil, Mary E. Larson, and Mr. James Bee. Messrs. Hakim Al-Rawi, Donald Distler, Artie Metcalf, Gary Packard, Bill Simco, and William Stanley assisted me in the seining of ponds and collection of snails. Jon Barlow, Richard Morehouse, and especially the late Jack Knouse assisted with the study of the herons and Abbot Gaunt did the statistical analysis. I am grateful to Mr. M. A. Hartman, on whose farm the heron colony is situated, and to Mr. Virgil Flory, who allowed me to cross his farm when going to and from the colony. I am also indebted to the landowners who allowed me to take samples of fish from their ponds. Their names are listed with the localities of the ponds. My wife, Linda, drew Figure 1 and aided in typing of the manuscript. The project was financed by the State Bio- logical Survey of Kansas.

Literature Cited BATTE, E. G., J. B. MURPHY, and L. E. SWANSON. 1951. New molluscicides for the control of fresh water snails. Amer. Jour. Vet. Res., 12:158- 160. COTTRILLE, W. P. and B. D. COTTRILLE. 1958. Great Blue Heron: Behavior at the nest. Misc. Pub., Mus. Zool., Univ. of Michigan, No. 102, 15 pp. EDNEY, J. M. 1950. Productivity in Clinostomum marginatum. Trans. Amer. Micro. Soc., 69:186-188. 538 Transactions Kansas Academy of Science

ELLIOTT, A. M., and L. R. RUSSERT. 1949. Some conditions characteristic of a yellow perch population heavily parasitized by Clinostomum rnarginatum. Jour. Parasitol., 35:183-190. FISCHTHAL, J. H. 1949. The over-wintering of black and yellow grubs in fish. Jour. Parasitol., 35:191-192. FORNEY, J. L. 1955. Life history of the black bullhead, Ameiurus melas (Rafi- nesque), of Clear Lake, Iowa. Iowa State College, Jour. of Sci., 30: 145-162. HASTINGS, C. E., and F. B. CROSS. 1962. Farm ponds in Douglas County, Kan- sas. Univ. of Kansas, Mus. of Nat. Hist., Misc. Pub., No. 29, 21 pp. HUNTER, G. W., III, and W. S. HUNTER. 1934. Further studies on fish and bird parasites. Suppl. 24th Annual Rept., New York Conserv. Dept., No. IX, Rept. Biol. Surv. Mohawk-Hudson Watershed, pp. 267-283. KIRKPAnticK, C. M. 1940. Some food of the young Great Blue Herons. Amer. Midl. Nat., 24:594-601. ICRuLL, W. H. 1934. Some observations on the cercaria and redia of a species of Clinostomum. Proc. Helm. Soc. Wash., 1:34-35. LEONARD, A. B. 1959. Handbook of gastropods in Kansas. Univ. of Kabsas, Mus. Nat. Hist., Misc. Pub., No. 20, pp. 1-224, pl. 1-11, figs. 1-87. OWEN, D. F. 1960. The nesting success of the heron, Ardea cinerea, in relation to the availability of food. Proc. Zool. Soc. London, 133:597-617. RABIDEAU, T. R. and J. T. SELF. 1953. A study of the effects of black and yellow grubs on Lepomis cyanellus and L. humilis in a natural environment. Amer. Midl. Nat., 50:402-406. VAN CLEAVE, H. J. and J. F. MUELLER. 1934. Parasites of Oneida Lake Fishes. Part III. A biological and ecological survey of the worm parasites. Roosevelt Wildlife Annals, 3:161-334. VAN DER SCHALIE, HENRY. 1960. Egypt's new high dam—asset or liability. The Biologist, 42:63-70.

—State Biological Survey of Kansas, University of Kansas, Lawrence.