Frog Lung Fluke Research Articles

EVOLUTIONARY AVENUES FOR, AND CONSTRAINTS ON, THE TRANSMISSION OF FROG LUNG FLUKES (HAEMATOLOECHUS SPP.) IN DRAGONFLYSECOND INTERMEDIATE HOSTS

Metacercariae survival patterns and their distribution in second intermediate odonate hosts were examined for 4 species of frog lung flukes. Surveys of aquatic larvae and recently emerged teneral dragonflies and damselflies indicated that prevalence and mean abundance of Haematoloechus spp. metacercariae were significantly lower in teneral dragonflies than larval dragonflies, while there was no significant difference in prevalence or mean abundance of Haematoloechus spp. metacercariae among larval and teneral damselflies. Experimental infections of dragonflies indicated that metacercariae of Haematoloechus coloradensis and Haematoloechus complexus were located in the head, thorax, and branchial basket of dragonflies, whereas metacercariae of Haematoloechus longiplexus and Haematoloechus parviplexus were restricted to the branchial basket of these hosts. Metacercariae of H. coloradensis, H. complexus, and H. longiplexus infected the head, thorax, and abdomen of damselflies, but these insects were resistant to infection with H. parviplexus. Subsequent metamorphosis experiments on experimentally infected dragonflies indicated that most metacercariae of H. longiplexus were lost from the branchial basket during metamorphosis, but most metacercariae of H. coloradensis, H. complexus, and H. parviplexus survived dragonfly metamorphosis. These observations suggest that the observed ecological host specificity of H. longiplexus in semiterrestrial leopard frogs may be due to few metacercariae of H. longiplexus reaching these frogs in a terrestrial environment. Because of the uncertain validity of Haematoloechus varioplexus as a distinct species from its synonym H. parviplexus, their morphological characters were reevaluated. The morphological data on H. varioplexus and H. parviplexus indicate that they differ in their acetabulum length and width, ovary shape, testes length, and egg length and width. Experimental infections of plains leopard frogs, northern leopard frogs, and bullfrogs with worms from bullfrogs indicate that the synonymy of H. parviplexus with H. varioplexus is not warranted, and that these flukes are distinct species, i.e., H. parviplexus in bullfrogs and H. varioplexus in plains leopard frogs and northern leopard frogs.

SMALL FROGS GET THEIR WORMS FIRST: THE ROLE OF NONODONATE ARTHROPODS IN THE RECRUITMENT OF HAEMATOLOECHUS COLORADENSIS AND HAEMATOLOECHUS COMPLEXUS IN NEWLY METAMORPHOSED NORTHERN LEOPARD FROGS, RANA PIPIENS, AND WOODHOUSE'S TOADS, BUFO WOODHOUSII

Studies on the life cycles and epizootiology of North American frog lung flukes indicate that most species utilize odonates as second intermediate hosts; adult frogs become infected by ingesting odonate intermediate hosts. Newly metamorphosed frogs are rarely infected with these parasites, predominantly because they are gape-limited predators that cannot feed on large intermediate hosts such as dragonflies. We examined the role of the frog diet and potential intermediate hosts in the recruitment of the frog lung fluke, Haematoloechus coloradensis, to metamorphosed northern leopard frogs (Rana pipiens), Woodhouse's toads (Bufo woodhousii), and bullfrogs (Rana catesbeiana) from western Nebraska. Because of the uncertain validity of H. coloradensis as a distinct species from Haematoloechus complexus, morphological characters of both species were reevaluated and the life cycles of both species were completed in the laboratory. The morphological data on H. coloradensis and H. coimplexus indicate that they differ in their oral sucker to pharynx ratio, uterine loop distribution, and placement of vitelline follicles. However, in terms of their life cycles, both species are quite similar in their use of physid snails as first intermediate hosts, a wide range of nonodonate and odonate arthropods as second intermediate hosts, and leopard frogs and toads as definitive hosts. These results indicate that H. coloradensis and H. complexus are generalists at the second intermediate host level and might be able to infect newly metamorphosed leopard frogs and toads by using small nonodonate arthropods more commonly than other frog lung fluke species. Comparisons of population structure of adult flukes in newly metamorphosed leopard frogs indicate that the generalist nature of H. coloradensis metacercariae enables it to colonize young of the year leopard frogs more commonly than other Haematoloechus spp. that only use odonates as second intermediate hosts. In this respect, the generalist nature of H. coloradensis and H. complexus at the second intermediate host level is an avenue for the colonization of young of year frogs.

Phylogenetic and biogeographical relationships among some holarctic frog lung flukes (Digenea: Haematoloechidae).

A phylogenetic study of 8 North American and European species of frog lung flukes belonging to Haematoloechus was conducted using approximately 850 to 1,000 bases of the intemal transcribed spacer region (ITS 1 + 5.8S + ITS 2) and 1,250 bases of the large subunit (LSU) of the nuclear ribosomal DNA. Adequate phylogenetic resolution could not be obtained from 5.8S or ITS 2 data. Analysis of ITS 1 data produced 2 equally parsimonious trees that differed only in the position of Haematoloechus breviplexus relative to H. medioplexus and H. varioplexus. Single, identical trees were produced by analysis of both LSU sequence data and a data set comprised of all ITS and LSU data. All trees demonstrated 3 distinct evolutionary lineages within the Holarctic Haematoloechus examined. The results confirmed the taxonomic validity of H. abbreviatus and demonstrated that the presence or absence of extracecal uterine loops is not a character meaningful to the recognition of evolutionary lineages or differentiation of genera. Examination of ITS sequence data revealed almost no intraspecific variation within 5 species of Haematoloechus and demonstrated an approximately 150-base indel common to the North American H. longiplexus and the European H. asper. Two of 3 clades revealed by the phylogenetic analyses are comprised of both European and North American species, indicating that lineages of Haematoloechus arose before the breakup of Laurasia and radiated after Eurasia and North America split. Within each of 3 evolutionary lineages, members share similar patterns of arthropod host specificity distinct from patterns found in the other lineages. This suggests that second intermediate host specificity may be a trait that has been conserved through evolutionary time.

Behavioral Basis of Second Intermediate Host Specificity among Four Species of Haematoloechus (Digenea: Haematoloechidae)

Cercarial behavior patterns were examined in 4 species of frog lung flukes (Haematoloechus spp.). Cercariae of Haematoloechus complexus, Haematoloechus medioplexus, Haematoloechus longiplexus, and Haematoloechus varioplexus were exposed to 3 species of experimental arthropods and an inanimate control. The number of cercariae attached to an experimental host at 5 min postexposure was recorded. Haematoloechus longiplexus and H. complexus cercariae attached to experimental hosts at higher rates than cercariae of H. medioplexus and H. varioplexus. Cercariae of H. longiplexus attached to experimental hosts in approximately the same numbers as H. complexus, but H. longiplexus penetrated only damselfly naiads, and only at the base of the zygopteran caudal gills. Cercariae of H. complexus, a second intermediate host generalist, were able to penetrate and enter several arthropod species at the intersegmental membranes. Haematoloechus medioplexus and H. varioplexus are restricted to development in dragonfly naiads and cercariae rarely attached to and never penetrated experimental hosts. These behavioral patterns dictate the range of hosts suitable for metacercarial development of H. complexus, H. longiplexus, H. medioplexus, and H. varioplexus. The evolution of disparate patterns of behavior among the cercariae of these 4 congeners has directly affected subsequent patterns of transmission to the definitive host.

Second Intermediate Host-Specificity of Haematoloechus complexus and Haematoloechus medioplexus (Digenea: Haematoloechidae)

Second intermediate host-specificity was examined for 2 species of the frog lung fluke genus Haematolechus. Nine species of freshwater arthropods were exposed to cercariae of H. complexus and H. medioplexus. Metacercariae of H. complexus developed in all arthropod species used. Metacercariae of H. medioplexus developed only in anisopteran odonate naiads. This difference in host utilization may have epizootiological implications. The potential development of H. complexus in a greater number of arthropods than H. medioplexus may increase the chances of ingestion of H. complexus by an anuran host. The range of arthropods parasitized by H. complexus indicates that host-specificity of the metacercarial stage of this fluke is not restricted by the phylogeny of aquatic arthropods.

Occurrence and distribution of putative neurotransmitters in the frog-lung parasiteHaplometra cylindracea (Trematoda: Digenea)

The localisation and distribution of the cholinergic, serotoninergic and peptidergic components of the nervous system of the frog-lung fluke Haplometra cylindracea have been determined by the application of standard enzyme cytochemical and immunocytochemical techniques to cryostat sections and whole-mount preparations. Cholinesterase activity (ChE), as indicative of acetylcholine, has been demonstrated cytochemically in the CNS and PNS; however, the anterior ganglia were notably unreactive. The occurrence of serotonin was examined by an indirect immunofluorescence technique, and immunoreactivity (IR) was demonstrable in small, paired anterior ganglia and in fine nerve fibres associated with the somatic muscle, cirrus and gonopore. The peptidergic portion of the nervous system was investigated using antisera to 17 mammalian regulatory peptides and the invertebrate peptide FMRFamide, and was visualised by both indirect immunofluorescence and confocal scanning laser microscopy. Positive immunostaining occurred with antisera raised against pancreatic polypeptide (PP), peptide tyrosine tyrosine (PYY), substance P (SP), peptide histidine isoleucine (PHI) and FMRFamide. Immunoreactivity to PP, PYY and FMRFamide was widespread throughout the nervous system and was evident in large, paired anterior ganglia, the dorsal commissure, main nerve tracts and the extensive array of small fibres that constitute the PNS. In contrast, the distribution of nerves immunoreactive to SP and PHI was less apparent, with PHI-IR occurring exclusively within the fibrous neuropile of the ganglia and in fibres of the ventral nerve cord. Results are discussed with respect to the distribution of the various neurochemical elements and their roles as putative neurotransmitters and/or regulatory molecules.

Réinvestigation de l'ultrastructure du spermatozoïde d' Haematoloechus (Trematoda: Haematoloechidae)

en The threadlike spermatozoon of the frog lung fluke, Haematoloechus , shows two patterns of external ornamentation. The first type is anterior and consists of bristles on the membrane that coincide with certain peripheral microtubules. This already exists in the zone of differentiation (ZD) of the young spermatid. The ZD is pinched off from the cytoplasmic mass at the arching-membrane level. The centriolelike body and striated roots are depolymerized. The two centrioles, made up of triplets, and the bristles are retained in the anterior part of the spermatozoon. The second type is more posterior. At this level, the transverse sections of the spermatozoon are asymmetric; only the larger side has peripheral microtubules, arranged in a semicircle around the axoneme, and a thick external crenate layer on its membrane. After several micrometers, this zone ends in an open collar ( collerette ) enclosing the next region of the spermatozoon. Only the part anterior to the collar is motile; on spontaneously breaking off, this region is mobile, although the unbroken spermatozoon is not. After the collar there is a region without microtubules. Then comes the very long middle region with dorsally and ventrally placed microtubules; the nucleus is at the posterior end. This new spermatozoon pattern, with retained ZD and anterior centrioles, resembles what we described earlier in a didymozoid. It probably occurs often in Digenea and perhaps other Platyhelminthes as well.

Fine Structure of the Reproductive System of a Frog Lung Fluke. III. The Spermatozoon and Its Differentiation

Monopartite sperm of the frog lung fluke, Haematoloechus medioplexus, arise by fusion of three juxtaposed cytoplasmic processes that grow out from a spermatid. Paired centrioles serve as nucleation sites for the axial units that grow into the 2 lateral processes, while the nucleus migrates into the central process. Such basal bodies, and attached rootlets, may aid in stabilizing the threadlike processes so that fusion can efficiently occur; basal bodies or rootlets are not seen in a mature sperm. The sperm is about 400 uL long and less than 1 u in diameter; the shaft resists bending, except for a terminal portion (25 to 50 Au) that undulates rapidly in living material. Typical transverse sections through the middle piece, which represents most of the shaft, show paired axial units, one on each side, a central mitochondrion, and two sets of cortical microtubules, some of which may be attached to the plasma membrane. Gametogenesis in the frog lung fluke, Haematoloechus medioplexus, has been studied in some detail at the light microscopic level (Burton, 1960), and the electron microscope has provided some information on fertilization in this trematode (Burton, 1967a). Electron microscopic studies of the structure and differentiation of trematode spermatozoa are few in number and generally lacking in detail (Shapiro et al., 1961; Gresson and Perry, 1961; Hershenov et al., 1966; Tulloch and Hershenov, 1967; Sato et al., 1967), and this is also the case as regards such studies of spermatozoa of cestodes (Gresson, 1962; Rosario, 1964; Lumsden, 1965; von Bonsdorff and Telkka, 1965; Swiderski, 1968; Morseth, 1969). One factor contributing to the difficulty in studying differentiation of trematode spermatozoa is the complexity of the process. A single spermatogonial cell ultimately gives rise to a rosette of 32 spermatids. As a result of incomplete cytokinesis at each division, the 32 cells remain connected by cytoplasmic bridges at their basal ends. The spermatids undergo a complex process of differentiation typically inReceived for publication 27 May 1971. * Supported by U. S. Public Health Service Grant AI-06448 and a Career Development Award (1-K3-GM-8620) from the Institute of General Medical Sciences. volving the formation and fusion of three cytoplasmic filaments several hundred microns in length (Burton, 1960; Hendelberg, 1962). Testes are filled with thousands of these filaments in various stages of differentiation, as well as germinal and spermatogonial cells, spermatocytes, and early spermatids. An ultrathin section (500 to 900 A), as examined in the electron microscope, will contain sections at various angles through hundreds of the aforementioned elements, and it is difficult and time-consuming to reconstruct each step of the complex process. The present study will set forth in some detail the sequence of structural changes involved in the differentiation of the spermatid of H. medioplexus into a mature threadlike sperma-

Carboxylic Esterase Activity of Procyotrema marsupiformis Harkema and Miller, 1959 (Diplostomatidae)

The histochemical localization of carboxylic esterase activity was studied for Procyotrema marsupiformis, a parasite of the pancreatic duct of the raccoon. Appropriate substrates and inhibitors were utilized to differentiate simple esterases from cholinesterases. In all tests the pharynx demonstrated the most intense reaction, apparently due to a mixture of nonspecific (simple) esterases and acetylcholinesterase. A strong reaction for acetylcholinesterase activity was obtained in the oral sucker, acetabulum, pseudosuckers and their glandular cells, tegument and subtegumentary cells in the region of the oral sucker, nerve tissue, Laurer's canal, oviduct, descending uterus, crypts of the holdfast, bursa, genital papilla, and excretory pore. Nonspecific esterases were indicated in the subtegumentary cells of the holdfast, the outer tunics of the testes and seminal vesicle, vas deferens, prostatic cells, and within the crypts of the holdfast. Pseudocholinesterase was not demonstrated. Although numerous studies have been made on the histochemical localization of carboxylic esterase activity in trematodes, only a few species have been examined. Notable among these are studies employing Fasciola hepatica adults (Halton, 1967a, b; Krvavica et al., 1967; Sekardi and Ehrlich, 1962; Thorpe, 1967, 1968) and larvae (Panitz and Knapp, 1967) and Schistosoma spp. (Fripp, 1967; Lewert and Hopkins, 1965; Pepler, 1958). Considerable attention has been focused on esterases of strigeoid trematodes as well, particularly in the works of Bogitsh (1966), Erasmus and Ohman (1963), Ohman (1965, 1966a, b), and Lee (1962). Halton (1968) recently investigated esterase activity in the frog lung fluke Haplometra cylindracea and a variety of other trematodes. Halton (1967b) and Davis et al. (1969) conducted a similar study on the digestive tract of Haematoloechus medioplexus. Procyotrema marsupiformis is parasitic in the pancreatic duct of the raccoon. Its elongate holdfast extends from the acetabulum to the posterior end of the forebody and is completely enclosed by the ventral body wall. Host pancreatic duct tissue fills the holdfast cavity. This tissue is highly vascular hemorrhagic and shows no characteristic pancreatic duct tissue com-

Cytochemical and biochemical observations on the digestive tracts of digenetic trematodes: III. Nonspecific esterase in Haematoloechus medioplexus

The cytochemical localization of nonspecific esterase was investigated in the frog lung fluke, Haematoloechus medioplexus. Fixed or fresh material was incubated in bromoindoxyl acetate for light and electron microscope studies. Addition of sodium fluoride, silver nitrate, and β-phenyl propionate inhibited the reaction; low concentrations of E600 and eserine sulfate had no effect, and PCMB and phenyl mercuric chloride enhanced enzymatic activity. On the basis of the action of these inhibitors and activators, the enzyme was characterized as a cathepsin. At the light microscope level, the reaction product was found dispersed irregularly as droplets in the gastrodermal lining. At the electron microscope level, the enzyme was localized in V 2 vesicles which, after starvation, engulfed mitochondria and formed cytolysomes. Advanced cytolysomes showed a high degree of esterase activity. They may receive additional secretions directly from other V 2 vesicles. Residual bodies derived from the cytolysomes migrate to the edge of the gastrodermis where they are presumably expelled into the lumen. No enzymatic activity was found in the superficial digestive vacuoles or in the lumen of the gut.

Peroxidase activity in the trematode Haplometra cylindracea

A light and electron microscopic study has been made of peroxidase activity in a sanguinivorous frog-lung fluke, Haplometra cylindracea. The use of inhibitors has shown that a true peroxidase exists in the worm and that any pseudoperoxidase activity is confined to ingested host blood. Ultrastructural localization of enzyme activity has shown reaction product in relation to the membranes of mitochondria. All observed mitochondria showed reactivity, and little or no variation in staining was observed.

Light and Electron Microscope Studies of Carboxylic Esterase Activity in the Trematode Haplometra cylindracea

Histochemical studies, using the light and electron microscopes, have been made of carboxylic esterase activity in the tegumental cells and surface tegument of the frog lung fluke Haplometra cylindracea. Regional differences in reactivity for esterases were found. The tegumental cells and tegumental lining of the two suckers and tegumental cells connected to the tegument surrounding the oral sucker stained intensely for esterases, but the tegumental cells and associated tegument in the rest of the body stained only weakly or not at all. The use of specific substrates or eserine sulfate showed that most of the activity was due to acetylcholinesterase. With the electron microscope, enzyme reaction product was found at the nuclear membranes and endoplasmic reticulum of esterase-positive tegumental cells and, in large concentrations, on the membranes of numerous platelike inclusion bodies present in the cells and connecting tegument. No structural differences relating to enzyme activity were found between reactive and nonreactive tegumental cells and tegument. The acidophilic cells present in the oral region of the worm were at all times negative for esterase activity. The oral region of the digenetic frog lung fluke, Haplometra cylindracea, is known to be a site of fairly intense carboxylic esterase activity (Halton, 1967a). Nonspecific esterases have been demonstrated using light microscopy in the oral and ventral suckers and their tegumental lining, as well as in certain gland cells which lie immediately behind the oral sucker. Ductlike processes connect these cells to the tegument overlying the oral sucker, and their content and that of the associated tegument is esterase-positive. Electron microscope observations have shown that at least two cell types are in communication with the tegument in H. cylindracea. There are numerous tegumental cells with tubelike connections to the surface tegument or cuticle (Threadgold, 1968) and, in the anterior region, fewer, but somewhat larger, acidophilic cells which are drawn out into Received for publication 7 July 1968. ductlike structures that open to the exterior through pores in the tegument (Halton and Dermott, 1967). The present investigation was made in order to correlate these light and electron microscope observations and to determine the source of carboxylic esterase activity in this region of the worm. MATERIALS AND METHODS For light microscopy live specimens of H. cylindracea were removed from the lungs of locally caught frogs (Rana temporaria) and fixed for 3 hr at 4 C in 10% formalin, buffered to pH 7.2 with 0.2 M phosphate. The specimens were then washed in three 10-min changes of ice-cold phosphate buffer, frozen in 5% sucrose solution, and sectioned. Sections (10 ,u thick) were transferred to glass slides and allowed to dry in air (30 min) prior to incubation. Carboxylic esterase activity was demonstrated using the standard techniques described in Pearse (1960). The following incubation times and temperatures were used: 12 hr at 18 C for 5-bromoindoxyl acetate (5-BIA); 12 hr at 18 C for acetyl FIGURES 1-3. Light histochemical localization of carboxylic esterase activity in H. cylindracea. 1. Frontal section through oral region showing esterase activity in oral sucker and tegumental lining and in tegumental cells and connecting tegument overlying oral sucker. 5-BIA. X 140. 2. Sagittal section through part of oral sucker showing intense esterase activity in tegumental cells of the sucker, their connections to the tegument lining the sucker, and throughout the tegument itself. Note esterase-positive connections from cells behind oral sucker to tegument overlying the sucker. 5-BIA. X 210. 3. Section through esterase-positive tegumental cells located immediately behind oral sucker. These connect by tubelike processes to the tegument overlying oral sucker and shown in Figure 2. 5-BIA. X 350. Abbreviations: A.C., acidophilic cell; C., connection; G.E.R., granular endoplasmic reticulum; I.B., inclusion bodies; M., muscle; N., nucleus; Nu., nucleolus; O.S., oral sucker; Ph., pharynx; P.M., plasma membrane; S.T., sucker tegument; T., tegument; T.C., tegumental cell.

Cytochemical and biochemical observations on the digestive tracts of digenetic trematodes: I. Ultrastructure of Heamatoloechus medioplexus gut

As seen with the electron microscope, the gastrodermal lining of the frog lung fluke, Haematoloechus medioplexus, is a single layer of epithelial cells. These cells possess indefinite cell boundaries and rest on a noncellular basal membrane. Large nuclei are present, each bound by a nuclear membrane and possessing a large nucleolus. Elongated flattened cytoplasmic processes, often forming superficial vacuoles, are found extending from the luminal surface of the cells into the lumen of the caecum. Many large, irregularly shaped mitochondria are dispersed through the cytoplasm. An extensive system of rough endoplasmic reticulum is evident throughout the cells and is thought to be related to the production of enzymes released at the luminal surface. Two types of vesicles are present. One type, bound by rough endoplasmic epithelium, is considerably larger than the other and contains material of lesser density. No Golgi apparatus is present.

Fine Structure of the Reproductive System of a Frog Lung Fluke. II. Penetration of the Ovum by a Spermatozoon

In the trematode, Haematoloechus medioplexus, the plasma membrane of the spermatozoon fuses, along the entire length of the cell, with that of the ovum. An acrosome is not present in these spermatozoa. Upon fusion of gametes, the plasma membrane and peripheral microtubules of the spermatozoon remain associated with the surface of the ovum, although the surface area of the ovum is not increased in proportion to that of the spermatozoon with which it unites. After fusion, however, the volume of the ovum is increased by about 200 /3, which is the approximate volume of an entire spermatozoon. In connection with a recent study (Burton, 1967) of Mehlis' gland of a frog lung fluke (Haematoloechus medioplexus), it was noted that before the eggshell becomes sclerotized, it is possible to obtain ultrathin sections which show stages in the penetration of an ovum by a filamentous spermatozoon. The present electron microscopic observations clarify and extend knowledge of some of the early events in fertilization in this trematode, as described in a previous article based on studies with the light microscope (Burton, 1960). For brevity, the term ovum is used in this paper for the female cell after it leaves the ovary, although it is a primary oocyte until meiosis is completed following penetration by the spermato-

Fine Structure of the Reproductive System of a Frog Lung Fluke. I. Mehlis' Gland and Associated Ducts

Fine Structure of the Reproductive System of a Frog Lung Fluke. I. Mehlis' Gland and Associated Ducts

In vitro Uptake of Radioglucose by a Frog Lung-Fluke and Correlation with the Histochemical Identification of Glycogen

Haematoloechus medioplexus from Rana pipiens were tested histochemically for glycogen by using the Bauer-Feulgen method. Major concentrations of glycogen occur in the oral sucker, pharynx, anterior subcuticular parenchyma, and spermatozoa. Worms maintained in vitro in glucose-saline showed no depletion of tissue glycogen after 50 hr, while worms maintained in only saline showed a partial loss of glycogen from the oral sucker, pharynx, and subcuticular parenchyma. In other experiments worms were maintained in vitro in saline containing radioglucose. After 12, 24, and 48 hr worms were sectioned and autoradiographed and compared with sections treated histochemically. The distribution of radioactivity corresponded with that of glycogen, indicating that glucose was removed from the medium and synthesized into glycogen. Grain counts were made to determine the amount of radioactivity (as C14 glycogen). The most radioactive structures were the pharynx and the anterior subcuticular musculature and parenchyma. Considerable glycogenesis also occurs in the oral sucker and deep parenchyma, but relatively little glycogen synthesis occurs in the cuticle, Mehlis' gland, vitelline follicles, digestive ceca, and ovary. Spermatozoa synthesize glycogen only shortly after their formation. Older spermatozoa in seminal receptacles were not radioactive. In this research histochemical and autoradiographic techniques were utilized in studying the uptake and fate of exogenous glucose in a frog lung-fluke, Haematoloechus medioplexus Stafford (Trematoda: Plagiorchiidae). The objectives were to obtain a more detailed understanding of the localization of glycogen and to obtain data on relative rates of glucose uptake by various tissues. Two publications have dealt with the distribution of glycogen in H. medioplexus as shown by histochemical methods. Axmann (1947) used a key with subjective values from 0 (no glycogen) to (large number of glycogen granules) to describe the distribution and appearance of glycogen in various structures. Burton (1960), in a preliminary study, largely substantiated Axmann's findings and made certain supplementary observations. MATERIALS AND METHODS H. medioplexus was obtained from lungs of Rana pipiens purchased from commercial sources. Received for publication 25 July 1962. * This research was supported by grants from the Atomic Energy Commission [AT(11-1)-1016] and the National Science Foundation (G17657). Frogs were pithed and the lungs quickly removed and placed in Ringer's amphibian saline solution. Lungs were opened and, if infested, worms were removed and washed in Ringer's solution prior to further processing. Worms to be sectioned were slightly flattened under a cover glass and( fixed in absolute alcoholformalin (9:1) for 12 to 24 hr at 4 to 5 C. The Bauer-Feulgen method was used to determine the distribution of glycogen within tissues. Worms were sectioned at 10 to 12 ,u and serially mounted on slides. Sections were oxidized in 4% chromic acid for 1 hr and exposed to Feulgen reagent for 15 min; no counterstain was used. Control sections were treated with a 0.1% solution of malt diastase in buffered neutral saline for 1 hr at 37 C. The solution in which worms were maintained in vitro was made up by adding 20,000 units of Penicillin G and 1 img of streptomycin per 10 ml of Ringer's solution. In some experiments glucose was added to make 1 and 2% solutions. Worms to be isolated were washed in several changes of antibiotic saline after being selected by examination through a dissecting microscope. For specimens to be isolated in vitro and then studied histochemically, the following procedure was employed: after washing, no more than 5 worms were placed in 10 ml of the appropriate medium contained in a stender dish, the dish was then covered and kept at room temperature (22 to 25 C). In the autoradiographic studies two identical

Studies on the life history of a frog lung fluke,Haematoloechus complexus (seely, 1906) Krull, N. Comb

1. The snailPseudosuccinea columella has been determined as the first intermediate host ofHaematoloechus complexus. 2. Dragonflies,Sympetrum vicinum, Pachydiplax longipennis andHolotania (Libellula) insecta, the last named being laboratory-raised from eggs, have been infected experimentally withH. complexus. 3. Rana clamitans has been determined as a new final host forH. complexus; it was experimentally infected and also found infected in nature. 4. The cercaria and metacercaria ofH. complexus are described and the adult fluke redescribed. 5. Experiments to establish the life history ofH. complexus involved 2 snails, 27 dragonfly nymphs (4 of which were raised from eggs in the laboratory), 17Rana clamitans and 15R. catesbeiana. 6. The life history ofHaematoloechus complexus is very similar to those of other frog lung flukes. The presence of long sensory hairs on the body of the cercaria ofH. complexus is the most striking anatomical difference in the life history stages separating it from other frog lung flukes. The distribution of the metacercariae in the dragonfly nymph and the loss of cuticular spines during the growth of the fluke in the final host are the most important differences in the life history.

The Life History of Two North American Frog Lung Flukes

The Life History of Two North American Frog Lung Flukes

North American Frog Lung Flukes

North American Frog Lung Flukes