A study of the frog lung fluke Haematoloechus (trematoda: haematoloechidae) collected from areas of Kentucky and Indiana.

ABSTRACT

One hundred seventy-six frogs and toads collected from areas of the Ohio River Valley region of Kentucky and Indiana were examined for the presence of lung flukes (Haematolechus spp.). Of the six recognized species of Haematolechus in North America, four were identified from the frogs examined in this study. The following species of lung flukes were recovered: 412 H. complexus, 63 H. longiplexus, 23 H. breviplexus and 8 H. varioplexus. Prevalence of infected frog species was 54% Rana utricularia, 42% R. clamitans, 38% R. catesbeiana. The mean number of lung flukes per infected frog species was as follows: R. utricularia, 13.6; R. clamitans, 8.9; R. catesbeiana, 6.3. The highest intensity was 71 mature H. complexus in one R. clamitans specimen. The overall prevalence and intensity of lung flukes were significantly higher in male frogs than in female frogs. There was no significant difference in prevalence or intensity of lung flukes with regard to right or left lung. Two of the four Haematoloechus sp ecies (H. complexus and H. varioplexus) have not been reported from Kentucky or Indiana. These records constitute a new geographical distribution for these two parasite species. Additionally, unusual variations in sucker sizes of H. longiplexus are reported. The surface topography of a representative specimen of H. complexus was studied by scanning electron microscopy. Almost the entire body of the fluke was covered by flattened conical-shaped spines. However, the spines around the periphery of the genital pore and the rim of the oral sucker were sharply-attenuated. No sensory papillae were noted, and the entire tegument had a porous spongy structure, which may function in secretion.

Keywords: Trematoda, Haematoloechus, frog, lung fluke

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Trematodes from the lungs of Anura were first reported over 200 years ago by Goeze (1782), who recognized a lung fluke in a frog and called it Planaria cylindrica. Rudolphi (1819) described a lung fluke from Rana esculenta and named it Distomum variegatum. This fluke was also studied and described as Monostoma bombynae by Zeder (1800) because he failed to see the ventral sucker. Looss (1894) described M. bombynae and later (1899) proposed a separate genus, Haematoloechus, for the frog lung fluke. Because of Still's hemipteran genus Haematoloecha established in 1874, Looss (1902) emended the genus Haematoloechus to Pneumonoeces. Harwood (1932) and Ingles (1932) independently pointed out that the name first chosen by Looss for this genus was not invalidated and should stand in accordance with International Code of Zoological Nomenclature. Although some authors have retained Pneumonoeces, Haematoloechus is widely accepted today.

A large number of amphibian helminth surveys have been published in North America. Early compilations included Stiles & Hassall (1894) and Pratt (1900). The first comprehensive systematic accounts were those of Stafford (1902a, b, 1905). Later studies on amphibian helminths from the United States include those by Cort (1915), Harwood (1932), Ingles (1936) and Manter (1938). Extensive work has also been done in Oklahoma (Kuntz & Self 1944), Iowa (Ulmer 1970) and Nebraska (Brooks 1976). More recent surveys have been conducted in Michigan (Muzzall 1991), Illinois (Andrews et al. 1992) and Arizona (Goldberg et al. 1998).

Our present knowledge of trematode parasites of amphibians from Indiana and Kentucky is scarce. Cort (1915) reported finding the lung flukes H. longiplexus and H. breviplexus from North Judson, Indiana. Krull (1931) also found H. longiplexus in a bullfrog from Indiana. Also, Lank (1971) found H. longiplexus and H. breviplexus in Indiana.

Several species of flukes have been studied by scanning electron microscopy (SEM) (Tandon & Maitra 1983; Srisawangwonk et al. 1989; Ferrer et al. 1996). Oliver et al. (1984) used SEM to study the surfaces of five Trematoda species from frogs, which included H. similes and H. variegates. Little additional work has been done to study the tegumental surfaces of Haematoloechus spp. The present study was undertaken to examine Haematoloechus spp. in frogs from the Ohio River Valley region of Indiana and Kentucky. Host specificity, geographical distribution, parasite prevalence and parasite intensities were among the parameters studied. Additionally, SEM was used to examine surface features, with special emphasis on the distribution and morphology of spines, of a representative H. complexus specimen.

METHODS

Frogs and toads were collected from five Kentucky counties (Jefferson, Meade, Henry, Oldham and Marion) and two counties in Indiana (Clark and Harrison). Frogs were collected at night by hand or with a frog gig, and immediately placed on ice. The frogs were taken to the laboratory, pithed and examined for parasites within 24 h after collection. They were catalogued by body weight, sex and locality. The lungs were removed and placed in individual containers of physiological saline. Each lung was teased apart under a binocular dissecting microscope.

All lung flukes were washed thoroughly in phosphate buffered saline (PBS) before fixing. Flukes were put in distilled water to allow discharge of eggs and fixed in hot acetic formyl alcohol. The flukes were stained in alcoholic carmine and dehydrated by passage through a graded series of ethyl alcohol. They were then cleared in Terpineol (J.T. Baker, Inc., Phillipsburg, New Jersey) and mounted in Euparal (ASCO Laboratories, Manchester, England).

A representative H. complexus specimen was chosen for analysis by SEM. This fluke was washed 3X in PBS and fixed in a cold 3% glutaraldehyde solution buffered to pH 7.3 with cacodylate. As much debris as possible was removed from the surface of the worm by using a camel's hair brush prior to fixing in glutaraldehyde. The specimen was rinsed in PBS, dehydrated through a graded series of acetone, and dried by a brief treatment with hexamethyldisilazane (Sigma Chemical Co., St. Louis, Missouri). It was coated with 50--60 nm of gold in a Polaron PS2 sputter coater, and examined with an ISI-40 scanning electron microscope.

Statistical analysis was performed with the aid of Minitab Statistical Software (Minitab, Inc., State College, Pennsylvania). The relationships between parasites and right and left lung and male and female hosts were examined by comparison of the binomial proportion to the z distribution (Ott 1988). Relationships between host size and lung fluke intensities were analyzed by simple linear regression. A value of P < 0.01 was accepted as statistically significant.

Line drawings of flukes were made by the author with the aid of a Bausch and Lomb microprojector.

RESULTS

Host and parasite species.--One hundred seventy-six frogs and toads were examined for the presence of lung flukes. Of these, 95 were Rana clamitans (green frog), 63 were Rana catesbelana (bullfrog), 13 were Rana utriculana (southern leopard frog), 3 were Bufo americanus (American toad), and one each was Bufo woodhousii (Fowler's Toad) and Hyla chrysocelis (southern gray treefrog). Of the R. clamitans examined, 40 (42%) were infected. Twenty-four (38%) of R. catesbeiana were infected. Seven (54%) of the R. utricularia were infected. No lung flukes were found in any of the Bufo species or the Hyla chrysocelis specimen, but samples were very small in these. Of the six recognized species of Haematoloechus in North America, four were recovered from the frogs examined in this study. These species included H. longiplexus, H. breviplexus, H. varioplexus, and H. complexus (Figs. 1-5). Haematoloechus longiplexus specimens from one female R. cates-beiana collected from Oldham County, Kentucky had significantly larger or al sucker and acetabulum sizes as compared to other H. longiplexus collected during this study (Fig. 2).

Analysis of parasite population parameters.--Of the 23 H. breviplexus, all (100%) occurred in R. catesbeiana. Of the 63 H. longiplexus, 61(97%) occurred in R. catesbejana and only 2 (3%) were found in R. clamitans.

All 8 (100%) H. varioplexus were found in a single R. clamitans specimen. Of the 412 H. complexus found, 316 (77%) occurred in R. clamitans, 95 (23%) occurred in R. utricularia, and one was found in R. catesbeiana. Table t compares the prevalence and mean intensities of flukes in their frog hosts. The highest intensity found was 71 mature H. complexus found in one R. clamitans specimen. Thirty-six were from the right lung and 45 from the left lung. Prevalence of infected frog species was 42% for R. clamitans, 38% for R. catesbeiana and 54% for R. utricularia (Table 1).

In R. catesbeiana the proportion of H. longiplexus was higher in the right lung as compared to the left lung. However, the proportion of H. breviplexus in the same frog species was higher in the left lung. The overall prevalence of lung flukes between the right and left lung of frog species showed no significant differences. There was also no significant difference in overall intensity of lung flukes with regard to right or left lung. The overall prevalence of lung flukes was significantly higher in male frogs as compared to female frogs (P < 0.01). Also there was a significantly higher intensity of lung flukes in male frogs as compared to female frogs (P < 0.001). There was no correlation between lung fluke intensity and size (weight) of any of the infected frog species.

Only three frogs had multiple infections of lung fluke species. One R. catesbeiana from Marion County, Kentucky was infected with 12 H. longiplexus and two H. complexus. Another R. catesbeiana also from Marion County, Kentucky was infected with seven H. longiplexus and four H. breviplexus. A R. clamitans specimen from Henry County, Kentucky was infected with one each of H. complexus and H. longiplexus. All R. utricularia were infected with only H. complexus. Fifteen frogs (21% of infected frogs) harbored immature lung flukes. Flukes were considered to be immature by either absence of eggs in the uterus or incomplete egg development.

To analyze the possibility of a crowding effect of the 71 H. complexus found in one frog, each of the measured characters was compared to the same character from the rest of the H. complexus. There was no decrease in these characters, as would be expected if there were a crowding effect; instead, all of the characters except for the ratio between the diameter of the oral sucker and that of the acetabulum (O/A ratio) and the ratio between the diameter of the oral sucker and that of the pharynx (O/P ratio) showed a significant increase in size as compared to the rest of the H. complexus population (P < 0.01).

Analysis of the H. longiplexus specimens with the larger than average sucker sizes revealed the following results. The oral sucker, pharynx and acetabulum sizes were all significantly larger than in the rest of the H. longiplexus found (P < 0.001). Although these structures were considerably larger than average, there was no significant difference in O/A ratio, O/P ratio or length and width of these flukes as compared to the rest of the H. longiplexus population.

Scanning electron microscopy.--The tegumental surface of a representative specimen of H. complexus was studied by SEM. Special emphasis was on the distribution and morphology of the spines. The elongate body of the fluke was covered with spines, except for the rim of the oral sucker. Two distinct spine types could be seen. These included flattened conical-shaped spines (Figs. 6, 7) covering the entire body (except the aspinose rim of the oral sucker) and sharply-attenuated spines seen primarily around the periphery of the genital pore (Figs. 6, 8). The sharply-attenuated spines could also been seen around the periphery of the rim of the oral sucker (Fig. 9). All spines were directed posteriorly except for those surrounding the genital pore (Fig. 6). The inner rim of the oral sucker is aspinose and has a spongy pitted appearance (Fig. 10). The tegument between spines can also be seen to have numerous pores or pits (Fig. 8). No sensory papillae could be identified.

DISCUSSION

Haematoloechus is one of the most common genera of trematodes found in frogs. The sample of frogs studied consisted of six species with 40% infected with Haematoloechus spp. A total of four Haematoloechus spp. was recovered from this sample, two of which (H. complexus and H. varioplexus) has not been reported from either Kentucky or Indiana and therefore constitutes a new geographical distribution for these two species.

Of the six species of frogs collected, only the three Rana spp. were infected. Neither of the Bufo spp. nor the Hyla chrysocelis was infected. Even though the numbers of Bufo and Hyla specimens examined were few, this lack of infection is consistent with other amphibian parasite surveys. These two genera are more terrestrial in their habitats and usually return to the water only to mate. This gives them only limited exposure to infected intermediate hosts of Haematoloechus spp. On the other hand, Rana spp. live in aquatic environments where there are ample opportunities to eat infected dragonflies or damselflies.

There was no correlation between size of frogs examined and presence of lung flukes in any of the three frog species collected. There was a wide range of sizes among infected frogs for all three Rana spp. The largest size range occurred with R. catesbeiana in which there were infected frogs from 15.6-203.5 g. Other studies (Dronen 1977) which have examined the relationship between frog weight and lung fluke infections have indicated that R. catesbeiana smaller than 20 g were rarely infected with Haemnatoloechus spp. The findings of this study were consistent with those of Dronen (1977) in that only one infected R. catesbeiana specimen was less than 20 g.

Although Haematoloechus spp. are capable of developing in more than one frog species, they are mainly associated with only one or two species of frogs and rarely other genera, e.g., Bufo spp. In the frog sample studied, R. clamitans harbored mainly H. complexus; R. utricularia harbored exclusively H. complexus; and R. catesbeiana harbored mainly H. longiplexus and H. breviplexus. One species of lung fluke, H. varioplexus, was found exclusively in one R. clamitans frog. There were only three cases of multiple infections. Haematoloechus longiplexus and H. complexus were found in the same R. clamitans; also, the same two species were found in a R. catesbeiana specimen. Haematoloechus longiplexus and H. breviplexus were found coinfecting the same R. catesbeiana specimen.

The number of mature flukes is usually not large in naturally-infected frogs, with 10 being the average number in this study. There are exceptional cases, however, in which the number far exceeds the average. A leopard frog examined by Cort (1915) had 42 mature lung flukes; and Fortner (1923) found 72 in one leopard frog, but unfortunately did not state whether these were mature or immature. Krull (1933) reported 74 mature lung flukes recovered from a single frog. In the current study, the largest number (71) of mature lung flukes was taken from a single R. clamitans collected from Henry County, Kentucky. There were no noticeable decreases in any of the measured characters of these flukes as might be expected due to the large number of them in a single host (crowding effect).

The level of parasitism was significantly higher in males than in females. Also, male frogs had a higher intensity of lung flukes than did female frogs. These results are consistent with other studies (Lees 1962; Hollis 1972). The difference in parasitism levels of male and female frogs, which is well-marked during the breeding season and less well-marked during the remainder of the year, may be explained by the fact that the female sex hormones depress the level of parasitism (Lees & Bass 1960).

Haematoloechus longiplexus specimens recovered from a particular frog from Oldham County, Kentucky had unusually large suckers (Fig. 2), the largest measuring 0.56 mm long by 0.53 mm wide with an acetabulum 0.33 mm wide. Cort (1915) reported the oral sucker size of one of his largest H. longiplexus being 0.36 mm long by 0.42 mm wide with an acetabulum 0.17 mm wide. More recently, Kennedy (1981) examined 203 H. longiplexus specimens and found the largest oral sucker size to be 0.49 mm long by 0.49 mm wide, and the largest acetabulum width was 0.24 mm. Therefore, these large sucker variants appear to be unique among the Haematoloechus spp. The significance of this variation in sucker sizes is unknown. Unfortunately, only one frog was collected from this particular site; and no other H. longiplexus specimens exhibited this variation. A more extensive survey of H. longiplexus could determine if these differences in sucker sizes are due to geographic or host-specific differences.

Spines are a common feature on the surface of many flukes. SEM was used to study the surface tegument of a representative specimen of H. complexus, and two distinct spine types were identified. Variations of surface spines have been reported by many other investigators (Tandon & Maitra 1983; Smales & Blankespoor 1984; Srisawangwonk et al. 1989). Variations seen in H. complexus included flattened conical-shaped spines covering the entire body and sharply-attenuated spines seen around the periphery of the genital pore and the oral sucker. The reasons for this variation are unclear. This may be a true morphological variation, or it may be due to the attenuated spines receiving less direct exposure to the host environment. A pitted tegumental surface has been reported for a number of flukes (Ogbe 1982; Probert & Awad 1987). The entire surface of the fluke, including the rim of the oral sucker, has a porous pitted appearance. The presence of pitting or pores suggests that the tegument may have a secretory function .

Table 1

Prevalence and intensities of Haematoloechus spp. in infected frogs.
* Percentage of infected frogs. + No. of lung flukes per infected frog.
NA, Not Appliable.

                                    No.       No.
Frog species      Fluke species   examined  infected  Prevalence *

Rana clamitans    H. longiplexus     95        40         42
                  H. complexus
Rana catesbeiana  H. breviplexus     63        24         38
                  H. longiplexus
                  H. complexus
Rana utricularia  H. complexus       13         7         54
Bufo americanus         --            3         0         --
Bufo woodhousii         --            1         0         --
Hyla chrysocelis        --            1         0         --
 Total                  --          176        71         40

                    Mean        Highest
Frog species      intensity +  intensity

Rana clamitans       8.9          71

Rana catesbeiana     6.3          56


Rana utricularia    13.6          33
Bufo americanus       --          --
Bufo woodhousii       --          --
Hyla chrysocelis      --          --
 Total                NA          NA

ACKNOWLEDGMENTS

The author thanks Dr. Bob Vogel for his assistance with statistical analysis and Dr. Ralph Lichtenfels for lending numerous slides of frog lung flukes from the U.S. National Helminthological Collection, Beltsville, Maryland. I wish to thank Dr. Fred Whittaker for his advice on this project and David Birch for his technical assistance with the electron microscope. I am indebted to Dr. Chris Mullins, Michael Elliott and Susanne Buckler for their valuable assistance in collecting frog specimens. Special thanks go to the many landowners who allowed me access to their property late at night to collect frogs. Without their cooperation, this study would not have been possible.

The opinions and assertions contained herein are those of the author and are not to be considered as reflecting the views of the U.S. Army or the Department of Defense.

Manuscript received 30 May 2001, revised 16 October 2001.

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Chris A. Whitehouse (1)

(1.) Current address: Virology Division, U.S. Army Medical Research Institute of Infectious Diseases, Ft. Detrick, Maryland 21702-5011