Gorgodera australiensis is a trematode parasite which commonly occurs in the urinary bladder of frogs. Because of its ubiquity, its typical trematode life history, and the comparative ease with which its intermediate hosts can be obtained and infected, it is a good subject for class study. It does not require much planning to ensure that live material of all stages of the life history is simultaneously available to students.

The adult of Gorgodera australiensis was first described by Johnston (1912), from Australian frogs. McFarlane and Northern (1953) described it from New Zealand frogs, and gave some notes on the collection and preparation of the trematode. For convenience of reference, a figure is given here, and some further information added. Most of the details of the body structure can be observed by flattening the live fluke under a coverslip, when the functioning excretory and reproductive systems can be watched under high power. Fixed specimens stained with Delafield's haematoxylin show good definition of the reproductive ducts. Several Gorgodera adults of various ages may be found in each frog bladder, and the most useful specimens are the smaller, pale worms. The larger, elderly yellow flukes are often so packed with mature egg capsules that little else can be seen.

Adult

The testes are prominent, and are arranged in two series of five and four. They are connected longitudinally by two vasa deferentia which unite near the anterior margin of the acetabulum and pass into the large vesicula seminalis. The latter curves about the genital atrium and passes into the cirrus. The vasa defferentia may contain sluggishly moving sperm, and are particularly prominent in younger individuals.

The ovary is compact, with smooth lobes, and has a central clear patch of formed ova which marks the beginning of the oviduct. This ducts begins as a narrow tube running slightly to one side, then widens into a fertilisation space which is frequently full of active page 90 sperms drawn up by the strong contractions of the oviduct. Occasionally, one or more ova may be seen in the duct, surrounded by frenzied sperm. Laurer's canal is a narrow tube opening just behind the vitelline lobe. It is contractile, and may contain a few sperms.

The vitelline glands are lobed structures posterior to the acetabulum, joined by transverse ducts to a median vitelline reservoir, and this in turn opens to the ootype. Vitelline cells may be seen in the ducts. The ducts and reservoir are contractile, and groups of cells are forced backwards and forwards until a few drop into the ootype. Mehlis' gland surrounds this chamber at the junction of vitelline duct and oviduct. It is a rounded clump of elongate cells usually showing in the space between and just anterior to the vitelline glands, and obscures further observation of the ducts at this point. When the duct becomes visible again, the first stage of egg shell development is apparent as a delicate elastic membrane surrounding the ovum and vitelline material. The shell is still soft, and may bend double in a turn of the uterus. Further down, the older shells are firm, and the vitelline cells eventually disappear.

The coils of the uterus extend through the posterior part of the body, forming loops between the testes, and between the testes and the edge of the body. The ascending coil makes several transverse turns in the region of the ovary and runs forward to the genital atrium. In old specimens these coils may obscure the ovary and vitelline area. The oldest capsules in the distal part of the uterus contain fully developed mobile miracidia.

A median longitudinal excretory bladder extends from a point just behind the oviduct to an opening at the posterior tip of the body. At the anterior end it is joined by two large collecting tubules which run forward to about the level of bifurcation of the gut. The ducts are extremely convoluted and difficult to follow at this point, but appear to reflex and run posteriorly for a short distance before dividing into a large posterior branch and a smaller anterior branch on each side. This agrees with the diagnosis for the genus given by Byrd, Venard and Reiber (1940). The anterior collecting tubule has a number of flame systems in the area from page 91

Plate 1:
Fig. 1: Adult. Fig. 2: Ovary and associated structures. Fig. 3: Miracidium, showing attitudes, a: in capsule, b: swimming, c: exploring, d: detail in capsule.
Fig. 4: Plan of main adult excretory ducts. Detail of accessory tubules shown on left in testes region to indicate type of branching.
Abbreviations: a, actabulum; ac, anterior collecting tubule; eb, excretory bladder; f, flame cell; g, gut; ga, genital atrium; L, Laurer's canal; m, Mehlis’ gland; mc, capsules with miracidia; o, ovary; od, oviduct; oe, oesophagus; os, oral sucker; ov, oviduct; pc, posterior collecting tubule; t, testis; u, uterus; ut, coil of uterus ascending to atrium; vas, vas deferens; vd, vitelline duct; vg, vitelline gland; vr, vitelline reservoir; vs, vesicula seminalis.

page 92 the base of the acetabulum forward, and the posterior branch seems to have four systems, each with possibly four or six flame cells. Byrd, Venard and Reiber give a pattern of 2 × 8 × 4 = 64 flame cells for the material they studied.

Miracidium

Eggs are laid in small groups from the genital atrium, and pass out from the frog with the urine. Miracidia can be recovered from water in which infected frogs have been kept, but it is more convenient to obtain them by breaking up an old Gorgodera adult to release mature capsules from the uterus. If kept in physiological solution the capsules do not hatch. When they are put in tap-water, however, the miracidial cilia begin to beat rapidly, the flame cells become noticeably active, the miracidium revolves energetically and bursts out of the capsule in 10-30 seconds. There is no operculum. The miracida move so quickly when released that is profitable to observe them first before they hatch. While in the capsule, the miracidium usually has its mobile anterior tip tucked in, but when swimming this portion is protruded slightly. There appears to be a sac-like inner cavity, and anteriorly there are two dense bodies, possibly glandular, which stain with Neutral Red. The two flame cells are placed posteriorly, and open by separate ducts. The cilia are borne on epidermal plate cells, which are difficult to observe. Silver nitrate preparations seem to indicate three rows of hexagonal plates.

The intermediate hosts which receive the miracidia are the freshwater pea shells, small (6mm), pale bivalves of the family Sphaeriidae, found in ponds and streams. The commonest is Sphaerium novaezelandiae, often found climbing in water weeds. So accomplished is it in the arboreal habit, that scoops from the floor of the pool may only yield dead shells, while weeds growing up to higher levels contain a flourishing population. Not all populations are infected; still, permanent ponds are most promising. Faster running streams, for instance, may support all three hosts but not the parasite, presumably because the miracidia and cercariae are swept away by the current. The largest molluscs are the most likely to be infected though sizes down to 3mm have been found to contain parasites. Such large, infected specimens are found to be not breeding, at times when most other uninfected shells contain young bivalves. Sphaeriids can be maintained for weeks in the laboratory under good aquarium conditions.

The miracidia do not seem to be attracted to the sphaeriids, and though the ciliary current of the mollusc can be seen to draw them in, many will bounce off and swim away. It seems that infection depends on the possibility of the miracidium becoming caught in page 93

Plate II:
Fig. 5: (to same scale) a: two sporocysts, one mature, one old; b: stages of cercaria development found in sporocyst; c: swimming cercaria. FIG. 6: Detail of cercarial chamber. FIG. 7: Cercaria. FIG. 8: Metacercaria. FIG. 9: Metacercaria in cyst.
Abbreviations: c, chamber; cb, cercarial body; cy, cystogenous cells; e, excretory tubules; eg, excretory globules; pg, penetration glands; s, stylet; w, wall of chambers. Others as in Plate I.

page 94 the gill, and this commonly happens. If a piece of the gill is dissected out into a drop of water, the cilia continue to beat, and the miracidia can be watched as they become trapped. Arrested miracida revolve slowly with the anterior tip pressed against the gill, sometimes producing drops of fluid, and this is presumed to be the attachment process, perhaps involving the anterior glands.

Sporocyst

Uninfected Sphaerium novaezelandiae exposed to miracidia in the laboratory were found after a week to contain a crop of very small sporocysts, each attached to the gill by a narrow neck-like portion. They move sluggishly, and the ciliated epidermal plates are lost. Mature sporocysts are simple thin walled sacs, up to 1/8in in length, attached by a slender stalk. Each contains about four large cercariae, as well as several less developed forms and a number of undifferentiated balls of cells. The long cercarial tails become inflated before emergence, and distend the sporocyst. Mature cercariae are very active, and can sometimes be seen to pierce the wall of the sporocyst with a sharp blow of their stylets. There is no birthpore, and cercariae are seen emerging from various points. This resembles the emergence of another gorgoderid, Cercaria duplicata Reuss, which is described as emerging through a rupture which closes behind it. The sporocyst collapses as the cercariae leave, and old ones are darkened and deflated.

Cercaria

The cercaria is 3-4mm long, the body a tenth the length of the tail. There is a chamber at the anterior end which encloses the body, and the latter is attached by a small papilla in the chamber floor which fits into the posterior excretory opening of the body. When extended, the anterior half of the body projects from the chamber, but during swimming it is held contracted and bent back on itself. When the cercaria leaves its chamber, the body moves convulsively and detaches itself from the papilla. The tail tapers at the tip, and a fin runs along the tail and up to the lips of the chamber. A stylet with a sharp point and broad base is set over the oral sucker. The penetration glands lie anterior to the acetabulum, and their ducts run a convoluted course forward to open half way along the stylet. The excretory system shows the adult pattern. The excretory bladder is bordered by a row of large oblong cells with dense granular contents. These cells occur in other gorgoderids, and are presumed to produce the cystogenous material. They are certainly absent or reduced in metacercariae.

The newly emerged cercaria has an inflated tail, but the chamber does not as yet surround the body. A collar-like fold pushes page 95 forward, and the cercarial body, previously extended, now doubles back and contracts. The folds of chamber and fin come forward and enclose the body. This process takes place before the cercariae leave the mollusc shell. The cercariae swim to the surface and remain there for a time, but may swim down again or laterally, so that they traverse a wide area. The chamber is held downwards, while the tail sweeps through an arc of 180 degrees at each stroke, driving the cercaria rapidly through the water. Swimming is continuous except for brief halts of a second or two, and is maintained strongly for twenty-four hours, with decreasing power till thirty-six hours, and the cercaria is moribund at the bottom in forty-eight hours. Cercariae were released in the laboratory at about twenty-four hour intervals, sometimes with great regularity, in groups of 1-7 per mollusc per day.

The movements of the cercariae attract the attention of nymphs of the damsel flies, Austrolestes colensonis and Xanthocnemis zelandica. The nymphs do not see the cercariae unless they are fairly close and moving energetically, but having sighted one, move up slowly on it. The labium is shot forward, and the cercaria stuffed into the mouth. The cercariae are completely unco-operative; they are not attracted to the numph, struggle when caught, and sometimes escape and swim off. A nymph which has suffered this experience may not attempt another for a time. Once within the jaws of the nymph, the cercaria is free of the chamber within a few seconds and penetrates through the wall of the anterior part of the oesophagus. Some nymphs are transparent enough to enable penetration to be watched. The cercaria wanders in the body space for a time, and has usually encysted within half an hour. Commonly the cyst is formed in the thorax, but may be in the anterior segments of the abdomen, or in the head. The tail and chamber are abandoned as soon as the nymph takes the cercaria. The chamber is assumed to be protective against the jaws of the nymph, but is not always effective and a small percentage of cercariae are found crushed in the nymph's gut.

If nymphs can be obtained from the same pond as infected molluscs, most large ones will contain at least one cyst. Uninfected nymphs put in with infected molluscs soon carry cysts, and one such in the laboratory was found to have collected twenty three cysts in a few days. If nymphs are starved for a day or so they will take cercariae immediately.

A cercaria freed from its chamber and introduced into a drop of fluid from a freshly killed nymph, or a drop of insect physiological solution, can occasionally be induced to form a cyst. The cercaria forms a thin, elastic membrane about itself, closely applied to the body at first, and moves actively inside this in a regular fashion, smoothing the cyst material as it is laid down. The cyst is often page 96 asymmetrical at first, with slight corners at the points successively occupied by the head and tail, and this small irregularity may sometimes be observed in older metacercaria. Generally the completed cyst is spherical.

Metacercaria

The cyst becomes elaborated with age, the walls thickened and eventually quite rigid. It can be opened with fine needles. The stylet is abandoned in the cyst and fragments of it may be seen floating inside. The metacercaria increases considerably in length, most of the increase being behind the acetabulum, and consequently the body becomes coiled within the cyst. The excretory bladder becomes filled with globules of greenish liquid, and in old specimens these become concretions, dark greenish in colour and irregularly shaped. These will squeeze out readily if the metacercaria is freed. The main pattern of the excretory tubules remains the same. Rudiments of the reproductive system are differentiated. In the cercaria, the ovary — vitelline mass and some clumps of cells roughly in the position of the testes are visible with difficulty, but in the metacercaria the nine testes are clear, and the ovary and vitelline mas differentiated. The uterus forms as a string of cells between the vitelline region and the position of the genital atrium, growing progressively longer and more looped. The gut lengthens and becomes plainly visible. The penetration ducts and glands remain, but are less obvious.

In the laboratory, young frogs were fed nymphs containing metacercariae over a period of four days. The frogs were then killed, twenty-four hours after the last feeding. One cyst was found in the stomach, and several in the rectum. The metacercariae in the latter were very active, grasping the cyst wall with the suckers and drawing it in strongly. Some excysted worms were found in the rectum, none were found in the cloaca, but two were seen migrating up the ureter, and a number were recovered from the macerated kidney. It is not known how they find their way, but it is possible that a chemical trail is followed. The worms were actively feeding in the substance of the kidney, and the gut was distended with yellowish granular material. One frog brought in from the field had a massive infection of 112 young worms in the kidneys.

When about lmm in length, the young Gorgodera move into the ureter again, and several dozen may be found, visibly distending the ureter. They migrate down to the bladder, and the youngest found there were 1-1.5mm, although the oldest in the ureter measured 2mm. When the fluke is 2mm in length, the ovary is page 97 functional, the uterus forms coils down the midline, and may contain new eggs. At 3-4mm the uterus may contain old capsules with miracidia.

All stages of the life cycle of Gorgodera australiensis can be found at most times of the year, though the percentage of infection varies a little with the season. Adults can be found in frogs whenever the latter are available, and frogs kept in artificial ponds through the winter yielded both young and old adults in July. Metacercariae overwinter in the nymph, and therefore are available to infect the frogs as long as the latter are feeding in autumn, and as soon as they become active in the spring. Some young adults may develop slowly through the winter in the frog. Fewer large nymphs are available in late summer and early autumn, when those from the previous year have become images, and the new generation are still very small. Cercariae are available through the year, although the percentage of infection varies. It is very low in late winter before the frogs appear, and is highest through summer and autumn.