Tuatara: Volume 5, Issue 1, January 1953
The Frog, Hyla aurea as a Source of Animal Parasites
The Frog, Hyla aurea as a Source of Animal Parasites
This paper describes a group of animals illustrating differing measures of parasitic habit, examples of commonly neglected major groups, but readily accessible to the biology student. Few laboratories fail to handle the frog in the course of the year, and the frog is an excellent host harbouring a variety of protozoa, trematodes, and at least one nematode. Our common frog is Hyla aurea Lesson, introduced from Australia, and with it came an unsuspected fauna, for the parasites we have found are those known in Australia through the pioneer studies of S. J. Johnston and Janet Raff. Hyla aurea fails in two respects. It apparently harbours no blood protozoa nor a cestode, but the fauna is nonetheless rich and worthy of study. The species dealt with here are fairly fully described to encourage study of them.
The small Entamoeba is an active species fully illustrating the major features of Amoeba and very readily cultured. The flagellates are minute but show the anatomy and behaviour of close relatives which are significant parasites of man and his domesticated animals. Protoopalina is a ciliate, slow in movement and beautifully demonstrates ciliary action, lacks any digestive apparatus, so is a complete saprozoite having no food vacuoles. When prepared as described, some examples show various stages of mitosis. Nyctotherus is a large, transparent, slow-moving ciliate clearly showing ciliate morphology and superior in this to Paramoecium. The trematode Gorgodera is very common, and in life is transparent enough for a general study of trematode anatomy, even flame cells can be seen. It yields brilliant preparations with acetic-alum-carmine, and is excellent for practice at ‘in toto’ staining. Diplodiscus is not common and too dense for study in life. The lung nematode, Rhabdias, has an interesting life-history since it is viviparous. The larvae occur free in the frog's rectum and can live in an organically rich medium away from the host. All the above parasites survive well on a slide at room temperatures in Ringer's solution.
In the course of our work one important point has arisen. Our specimens of Nyctotherus cordiformis are all very typical of that species. Miss Raff, however, records specimens twice the usual size from H. aurea in Australia. page 13 In all other respects our species is identical with that described by Miss Raff. It seems possible that Miss Raff has made some error in her measurements, because ours agree with other accounts of this species. Miss Raff has described Entamoeba morula from Australian frogs, including H. aurea. Here again our material is half the size of Miss Raff's. Possibly a similar error in measurement has been made.
It is clear that the frog offers more than just anatomy to a biology student, and it is still a fertile host for the parasitologist since we cannot feel confident we have fully listed its parasitic fauna.
The culture medium, fixatives and stains described below should be made up before the frog is killed.
Place a live frog in a jar with a wad of cotton-wool moist with chloroform or ether, and cover tightly until the frog is dead. Then place it on its back and pin down. Cut through the skin and muscles from the pelvis to the jaws and separate the flaps to show the internal organs. Remove the gut, bladder and lungs and put them in separate glass dishes in Ringer's solution. Slit open the rectum with fine-pointed scissors. Smear some of the nearly liquid contents thinly on a slide with a scalpel blade and fix as below. Living material for microscopical examination may be prepared by adding a drop or two of Ringer's solution to the smear and lowering a coverslip on to the drop. Diplodiscus may be found in the rectum and should be removed to a watch-glass of Ringer's. To obtain Gorgodera open the bladder and remove the trematodes with a mounted needle or paint brush. Some may hold firmly to the bladder-wall. Relax these with magnesium sulphate (Epsom salts) — a few crystals on the surface of the Ringer's solution. Later they can be removed with a paint brush. Flame cells in living specimens can be made more active by the addition of a few drops of 1% urea to the Ringer's solution. Tease out the tissue of the lung with a pair of needles to obtain the nematode Rhabdias. Movement of the worm makes it fairly easy to detect.
To make permanent preparations for study, the material has to be fixed, stained and mounted. Protozoa are fixed in Schaudinn's solution, the trematodes and the nematode in hot formalin-acetic-alcohol. Iron haematoxylin is a standard stain for the parasitic protozoa; acetic-alumcarmine, for the trematodes. (Nematodes are not stained as a rule.) After staining and before mounting the material must be dehydrated and cleared. Dehydration is accomplished by placing the material in successive changes of alcohol of increasing concentration, viz. 50%, 70%, 95% and 100% ethyl alcohol. Material should then be cleared in xylol and mounted in canada balsam. A few minutes (1 to 3) in each grade of alcohol and xylol is all that is required for smear preparations, but the thicker-bodied animals such as Gorgodera and Diplodiscus will take up to 15 minutes in each step. page 14 It is essential to remove all water from the material before clearing in xylol or a milky precipitate will result. Should this happen, return the material to 100% alcohol for a further period.
Formulae. (All chemicals should be C.P. standard.):—Ringer's Solution. (Culture medium and for examining live material.) Sodium chloride 6.5 grm.; potassium chloride 0.3 grm.; calcium chloride 0.25 grm.; distilled water 1000 cc. For the apparatus, etc., necessary for the culture of the parasites from the frog see Fig. 11. For cultures:—Grind up 2 or 3 grains of rice with pestle and mortar. Shake with Ringer's in a test-tube. Add 2 to 3 drops of cloudy suspension to 10 ccs. of Ringer's. Place a small quantity of rectal contents above the cotton wool in the glass tube and place the whole in a vial with culture solution. Remove the glass tube after 24 hours, by which time the protozoa should have migrated through the cotton wool into the vial.
Fixatives: Schaudinn's solution. (Poison). 100 cc. saturated solution mercuric chloride; 50 ccs. glacial acetic acid. Used cold. Leave smears in fixative for 15 mins. Keep Metal Instruments from Contact with this Solution. Formalin-acetic-alcohol (F.A.A.). (See Manter, Tuatara IV (1): 57.)
Stains: Iron haematoxylin (Heidenhain's formula). Solution 1. Stain. 95 ccs. distilled water; 1 grm. haematoxylin. Heat gently to dissolve haematoxylin. To ripen solution: (a) by natural means. Put solution in a wide-mouthed bottle loosely plugged with cotton wool. Place in the sun until the solution changes to dark purple. Or: (b) chemically. Add 5 cc. carbolic acid to dissolved haematoxylin. Solution is ‘ripe’ on cooling. (The chemically ripened solution has a short life, 3 to 5 months at most. Naturally ‘ripened’ lasts a year or more.) Solution 2. Mordant. 2 grm. ferric ammonium sulphate (iron alum, use violet crystals only); 100 cc. distilled water. Never Contaminate Stain with Mordant.
Staining Procedure for Iron Haematoxylin: Can be used following fixatives employing alcohol, formalin, picric acid or mercuric chloride, which must be removed by washing with water before staining; but mercuric chloride is removed from Schaudinn-fixed material by placing material in a mixture of 1 part Gram's Iodine (iodine 1 grm.; potassium iodide 2 grm.; and water 300 cc.) and 1 part 70% alcohol. After washing, place material to be stained in Solution 2 (the mordant) for 12 hours. Then Wash thoroughly by repeated changes of water. Then into a 50% concentration of Solution 1 in distilled water for 12 hours. Rinse. Destain in mordant till the cytoplasm is virtually unstained. (This process must be checked under the microscope, no exact time can be given. Nuclear chromatin should remain intensely black.) Wash. Dehydrate and clear. Mount in balsam. The slide can be hardened in an oven at 35°C. Acetic-alum-carmine:—Boil excess powdered carmine in a saturated solution of potash-alum for 15 minutes. Add 10 vols. of solution to 1 vol. glacial acetic acid. Stand for 1 week. Filter. Store in tightly-corked bottle. Dilute about 30 times with distilled water for long staining, or 1:10 for shorter. Differentiate with acid-alcohol (98 parts 70%, with 2 parts conc. hydrochloric acid).page 15
Procedure for Nematodes:— Place the nematodes in 5% aqueous solution of glycerine with thymol (1 crystal to 200 cc. of solution) to prevent growth of mould. Leave in this solution for approximately 10 days on top of an oven or in another suitably warm position. Cover against dust. After 10 days the glycerine should have concentrated by evaporation to pure glycerine. Note:— An ample volume of solution must be used so that when concentrated the specimen is still covered. The worms can now be examined in pure glycerine or if permanent mounts are required, mounted in glycerine jelly. (This technique is useful for trematodes after staining with acetic-alum-carmine.)
Entamoeba morula Raff 1912. (Fig. 7.) A small Entamoeba agreeing with the original account (Raff, 1912) but only half the size there given. Irregularly ovoid in outline, relatively flattened. Ectoplasm generally very thin, not readily recognisable except as a more or less broad posterior sheet, often the width of the animal. Ectoplasm also extended as one or more ventral, thin small blunt lobes close to or at the anterior end. These lobes show little change in form during locomotion and are not obviously pseudopodial. Endoplasm, minutely granular, highly vacuolated in life, the vacuoles generally refractile. In fixed and stained material the vacuoles are small and moderately dispersed. Food vacuoles, numerous, generally small. Inclusions, sparse, small. Crystals, not observed. Contractile vacuole variable in position, slightly smaller than the nucleus but not observed in all animals. Nucleus large, its greatest diameter almost one-third the width of the animal, clearly visible in life, always anterior. Nuclear membrane very thin, nucleoplasm almost hyaline, nucleolus large, its width nearly half that of the nucleus, essentially central but with chromatin bridges to the nuclear membrane. Minute chromatin granules on nuclear membrane regularly dispersed, not widely spaced out. Length:—0.04 to 0.07 mm.
Found in the rectum of two frogs where it was very common. Can be overlooked because of its small size. Moderately active. Survives well on slide in Ringer's solution and cultures easily with rice starch, reproducing rapidly and surviving a month in a corked vial. (E.C.B.) Reference: Raff, 1912.
Protoopalina australis Metcalf 1923 (Figs. 8 and 9). Cylindrical, elongate, bluntly rounded anteriorly, attenuated and tapering gradually to a sharp pointed tail. Spiralled in life. In stained preparations, the tail often bent in a hook. Ectoplasm, hyaline, uniformly narrow, distinct. Endoplasm with numerous granules, but in general rather clear, non-vacuolate. Cilia longer anteriorly. Nuclei large, ovoidal, longest diameter about half greatest width of the animal, and the nuclei separated by little more than their greatest diameter. A binucleate condition is commonest, but specimens may have one to four nuclei according to their stage in fission. No nucleolus. Length 0.085 to 0.130 mm.
Abundant in the rectum of most frogs examined. Not found with P. hylarum. Moves moderately slowly, spiralling and showing a clear and beautiful ciliary action. Nuclei (0.01 × 0.0075 mm.) visible in life. Did not survive longer than two days in culture with Ringer's solution and rich starch. (J.M.) Reference: Metcalf, 1923.page 16
Protoopalina hylarum Raff 1911 (Fig. 10). Very large, cylindrical, elongate, tapering, blunt at both ends. Our specimens green, but this not necessarily characteristic. Spiralled on the long axis in life. Pellicle, ciliation, ectoplasm and endoplasm as in P. australis. Nuclei spaced out from one another by about half the length of the animal, each being about one-third the greatest body width. Length (average), 0.42 mm.; width (average), 0.07 mm.
Occurred once in the present study, and the above account based largely on fixed and stained material. The large size of this species, about four times the length of P. australis, makes this a good species for study. (J.M.) Reference: Raff, 1911; Metcalf, 1923.
Nyctotherus cordiformis (Ehrenberg) 1838 (Fig. 12). None of our material reaches the size recorded by Raff (1911); but it agrees well with Wichterman's recent account of N. cordiformis (J. Morphol. 60 (2): 568).
Fig. 1: Gorgodera australiensis. Fig. 2: G. australiensis, detail of anterior part of reproductive system. Fig. 3: Rhabdias hylae. Fig. 4: R. hylae, detail of anterior portion. Fig. 5: R. hylae, detail of posterior portion. Fig. 6: Diplodiscus megalochrus. Fig. 7: Entamoeba morula. Fig. 8: Protoopalina australis, showing spiralling of body. Fig. 9: P. australis. Fig. 10: Protoopalina hylarum. Fig. 11: Apparatus for culturing protozoa.
Explanation of plate and text-figure: A, anus; AC, acetabulum; BW, body wall; C, cilia; CE, cuticular envelope; CK, cork; CP, cytopyge; CS, cirrus; CV, contractile vacuole; CW, cotton wool; D, diverticulum; E, eggs; EC, ectoplasm; EN, endoplasm; F, flagellum; FG, forked gut; G, gut; GA, genital aperture; GT, glass tubing; IP, intestinal pigment; L, lips; M, mouth; N, nucleus; O, ovary; OE, oesophagus; OS, oral sucker; P, pharynx; R, rectum; RS, Ringer's solution; T, testis; U, uterus; V, vacuole; VD, vas deferens; VS, vesicula seminalis; VU, vulva; YG, yolk glands.
Micronuclei not observed. Division apparently rare, not observed. Does not reproduce rapidly in culture. Length: 0.13 to 0.145 mm.; width: 0.09 mm. to 0.125 mm.
Occurred in the rectum of most frogs examined. A large transparent ciliate, slow-moving and from its form and transparency ideal for study, even showing the nucleus clearly in the living specimen. Movement of the cilia relatively slow and beautifully manifest. Survives well mounted in Ringer's solution on a slide. Cultures readily in Ringer's solution with rice starch in a corked vial for a month. Best studied alive, but stains attractively with iron haematoxylin. (P.A.M.) Reference: Kudo, 1947; Raff, 1911; Wichterman, 1936.
Copromonas subtilis Dobell 1907 (Fig. 13). A small pyriform flagellate pointed anteriorly, broadly rounded posteriorly, and apparently ovoidal in cross section. Length slightly more than twice greatest width. Pellicle appears plain, very thin. Ectoplasm, hyaline, very thin. Endoplasm, finely granular; in fixed and stained specimens richly but finely vacuolated. Numerous small non-vital inclusions in the endoplasm. Flagellum, as seen, about the length of the body. Nucleus poorly defined, granular, slightly anterior and the diameter no more than one-third the greatest width of the body. Cytostome not observed. Length: 0.011 mm.; width: 0.0045 mm.
Not common in the rectum of frog examined, but described by Raff (1911) as the commonest flagellate in the intestine. In life, this is a relatively slow-moving flagellate, but difficult to study owing to its small size. Was not cultured in rice starch with Ringer's solution. (B.R.C. and R.B.C.) Reference: Kudo, 1947; Raff, 1911.
Eutrichomastix batrachorum (Dobell) 1907 (Fig. 14). A small spindle-shaped flagellate, anterior end bluntly tapered, posteriorly attenuated as a tail about half the body length. Pellicle and ectoplasm very thin, endoplasm finely granular with two very large vacuoles obvious in stained specimens. Three long thin anterior flagella and one thin posteriorly directed flagellum approximately the same length as the anterior. Nucleus oval, thickly granular, located anteriorly, well-defined; but not visible in live specimens. A single kinetoplast situated at the anterior end. Axostyle not observed in stained material. Length: 0.015 mm.; width: 0.0045 mm.
In large numbers in Schaudinn-fixed smears of rectal contents stained with iron haematoxylin. When detected were abundant. Swims with an undulating jerky movement. In living specimens the tail appeared to have a swelling halfway along its length; but not in the stained specimen. Thrived for over three weeks in culture of Ringer's solution with rice starch in a corked vial. (B.R.C. and R.B.C.) Reference: Kudo, 1947; Raff, 1911.
Tritrichomonas batrachorum (Perty), (Fig. 15). Somewhat lemon-shaped, the body generally rounded but extended anteriorly and posteriorly, bluntly rounded at these extremities. Pellicle and ectoplasm very thin. Endoplasm minutely granular with a few small scattered vacuoles. A few minute granular inclusions. Axostyle not observed in fixed and stained specimens. In fixed and stained specimens, three short anterior flagella each less than half the body length, and page 19 a fourth originating with the others but directed posteriorly along an undulating membrane and extending beyond the posterior end of the body. Nucleus small, very anterior, spherical, vesicular, with a few chromatin granules on the nuclear membrane; an obvious but not large nuclcolus. Length: 0.0125 mm.; width: 0.0056 mm.
Text Figures: Fig. 12: Nyctotherus cordiformis. Fig. 13: Copromonas subtilis. Fig. 14: Eutrichomastix batrachorum. Fig. 15: Tritrichomonas batrachorum. For explanation of abbreviations see Plate I.
Diplodiscus megalochrus Johnston 1912 (Fig. 6). A thick-bodied, bluntly cenical trematode generally circular in section, tapering anteriorly from the posterior region. In life convex dorsally, venter broadly concave longitudinally. Skin, thick but without spines or spicules. Oral sucker, about one-tenth of body width, surrounded by a less obvious narrow ring of muscle as if an additional attachment organ. Acetabulum posterior, subterminal, about three times the width of the oral sucker and surrounded by a prominent ring of muscle, three-quarters of page 20 greatest body width and apparently a powerful accessory organ; but no specimens were attached to the wall of the rectum. Mouth opens into a length of oesophagus surrounded by long muscular diverticula from the oral sucker. Posterior to this a short length of oesophagus leading to a minute pharynx. The gut divides into two blind tubular short limbs which barely reach to the accessory muscular ring of the acetabulum. A single subspherical median testis a little smaller than the acetabulum located between the limbs of the gut. Vas deferens not seen but narrow cirrus leads to common genital atrium ventral to the fork of the gut. Ovary not distinguishable in size or texture from the many irregularly-shaped yolk glands which are located generally lateral to, and posterior to the ends of, the gut. Uterus a wide thin-walled tube apparently transversely coiled behind the ends of the gut and between the limbs extending so to the common genital atrium. Eggs large, about 0.1 mm. in length. Average length of body: 2.2 mm.; average width: 1.1 mm.
Found in rectum of three frogs. When present, as many as eight in one frog. A thick skin conceals detail in the live specimens but good preparations with acetic-alum-carmine can be made from large specimens flattened gently during fixation. Young specimens move with an extreme attenuation of the body, extending it to at least twice its resting length. (J.C.Y.) Reference: Johnston, 1912.
Gorgodera australiensis Johnston, 1912 (Fig. 1). A flat trematode, narrowly oval in cross section, tapering anteriorly in a highly mobile preacetabular region but wider and less mobile posteriorly. Oral sucker about two-thirds the width of the acetabulum which is about half the width of the posterior region and slightly more than the width of the anterior region of the body. Skin smooth, no spines. Mouth at base of oral sucker followed by a short oesophagus dividing just posterior to the oral sucker. Limbs of the intestine narrow, tubular, and extending almost to the posterior end of the body. Nine testes, each irregularly lobed, arranged in two longitudinal series of four and five respectively, connected longitudinally by delicate vasa deferentia which unite near the anterior margin of the acetabulum opening into a vesicula seminalis which passes into the cirrus. A single smoothly-lobed ovary level with the anterior testis. Two compact yolk glands are arranged as a transverse pair anterior to the gonads and immediately posterior to the acetabulum. Transverse vitelline duct occasionally visible. The convoluted uterus extends posteriorly as a median narrow tube between the gonads to the posterior end of the body, and anteriorly as two branches lateral to the intestine up to the level of the ovary. The median branch extends dorsal to the acetabulum to terminate at the female aperture in the common genital atrium. Common genital aperture immediately posterior to fork of the intestine. Length of body: 4.0 to 6.0 mm.; width: 0.75 to 1.5 mm.
A common trematode of the bladder, present in all frogs examined with 3 as a minimum and 24 as a maximum and averaging about 5 per frog. The living worm transparent, so that much internal anatomy is visible, and even flame cells can be seen. Specimens can be kept alive at least five to six hours in Ringer's solution. Stains brilliantly with acetic-alum-carmine. (B.G.McF. and J.B.N.) Reference: Johnston, 1912; Dawes, 1946.
Rhabdias hylae. Johnston and Simpson, 1942. (Figs. 3, 4, 5.) Body stout, tapering gradually anteriorly from the level of the base of the oesophagus, and posteriorly tapering rather abruptly from the level of the anus to a short conical tail. The dark-brown pigmented intestine is characteristic.
Cuticle thin, with faint longitudinal striations. All specimens examined showed the appearance of a ‘halted’ moult, each worm being enclosed in a wrinkled cuticular envelope. Mouth, surrounded by six very low, scarcely-discernible lips, opens into a short buccal capsule. Oesophagus, muscular throughout, with a slight posterior swelling; its length approximating one-seventh of total body length. Straight, thick-walled intestine constricted slightly posteriorly, expanding again before joining the short rectum opening at the sub-terminal anus. Vulva prominent, situated near the middle of the body, opening directly into the opposed uterine branches. Ovaries, reflexed; but much of the reproductive system is concealed by the pigmented intestine. Eggs containing advanced embryos can be seen in the distal portions of the uterine branches. Total length: 5.5 mm.; maximum diameter: 0.39 mm.; length of tail: 0.37 mm.
Barely 10% of the frogs examined were infected. An interesting fact is that all specimens were ‘female’ forms (protandrous hermaphrodite), this being the parasitic generation. Probably a free-living sexual generation occurs. The specimens were cleared in glycerine for examination and mounted in glycerine jelly. (J.M.McE. and R.J.S.) Reference: Baylis and Daubney, 1926; Johnston and Simpson, 1942.
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Dawes, B., 1946.—The Trematoda. Cambridge U.P. 1-644.
Johnston, S. J., 1912.—On some Trematode Parasites of Australian Frogs. Proc. Linn. Soc. N.S.W. XXVII (2): 285-362.
Johnston, T. H., and Simpson, E. R., 1942.—Some Nematodes of Australian Frogs. Trans. Roy. Soc. S. Aust. 66, (2): 172-179.
Kudo, R. R., 1947.—Protozoology. Thomas. 1-778.
Metcalf, M. M., 1923.—The Opalinad Ciliate Infusorians. Bull. 120, U.S. Nat. Mus.: 1-484.
Raff, J. W., 1911.—Protozoa Parasitic in the Large Intestine of Australian Frogs, Pt. I. Proc. Roy. Soc. Victoria 23 (2): 586-594.
Raff, J. W., 1912.—Protozoa Parasitic in the Large Intestine of Australian Frogs, Pt. II. Proc. Roy. Soc. Victoria 24 (2): 343-352.
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