Gyrocotyle rugosa. Adults (Figs. 1, 2)

Body size and proportions vary considerably with contraction. The worms are strongly muscular and it is difficult to preserve them in their normal shape. Living specimens are soft and flabby, creamy-white in colour. Watson (1911) noted that in freshly killed hosts the worms were a pinkish, translucent flesh colour. Measurements of 22 specimens were: length 32 to 82 mm., greatest width 6.5 to 22 mm. A dead specimen, greatly extended, was 130 by 15 mm. Moderately contracted worms were 42 to 71 by 16 to 20 mm. Lateral undulations of the body are lacking. The lateral edges tend to curve slightly inward ventrally but the striking undulations present in other species do not occur. The rosette is highly frilled, fully as much so as in G. urna although usually less than is true for G. fimbriata. The diameter of the rosette is 33.1/3 to 50 per cent. of greatest body width, thus resembling G. urna.

The body surface is rugose even in extended or compressed specimens. The rugae are transverse except in the anterior half of the body ventrally where short longitudinal folds occur irregularly so that the surface is incompletely broken into irregular segments. Body spines such as occur in G. urna and G. fimbriata are lacking, but numerous exceedingly minute spines or points occur on the tips of small papilla-like elevations (Fig. 2). These papillae and spines are best observed by reflected light when the body surface is exposed to the air. They are invisible in cleared specimens. They are arranged more or less in transverse rows on the page 3 body ridges. Where the rugae or ridges are irregular the spines are also irregular. They occur on both the dorsal and ventral surfaces and on the rosette but appear to be lacking anterior to the vaginal pore.

The acetabular spines occupy a subtriangular area on each side opposite the posterior half of the acetabulum. In two specimens, the length of the spiny area was 71 per cent. and 74 per cent. of the acetabular length. The spines are lateral and ventral and perpendicular to the body surface. The area is longest on the edge of the body and tapers as it extends ventrally almost to the acetabulum. The largest spines are 0.357 by 0.065 mm. or 13 per cent. of the length of the acetabulum. The number of spines, estimated to he approximately 100 on each side, is greater than in either G. urna or G. fimbriata.

The genital notch is conspicuous on the right side of the body a little posterior to the acetabulum. The uterine pore is median about 1/6th body length from the anterior end. The male pore is at the tip of a pointed, cone-like elevation of the body surface, the genital papilla or genital cone. Fresh, living specimens may show this structure only slightly elevated, but after a few minutes outside the host and during killing and fixation it extends toward the right, usually to and even through the genital notch. Here it bends dorsally and may almost reach the vaginal pore. As has been noted by previous authors, it evidently can serve in self-copulation. It appears not to be a true cirrus or any specialized structure but merely an elevation of the body wall. When the cone is retracted, the male pore lies just median to the right longitudinal nerve and only slightly posterior to the genital notch. The vaginal pore is dorsal, well to the right of the longitudinal nerve, and near the genital notch. Excretory pores were not observed but the excretory tubes were seen to approach the edges of the body 14 mm. from the anterior end of a specimen the acetabulum of which was 2.5 mm. long. This distance of more than five times acetabular length is considerably more posterior than in the other species. In another specimen 44 mm. long and with acetabulum 2.2 mm. long, a diagonal excretory tube extended to each side of the body 16 mm. from the anterior end.

Testes are numerous, estimated 400 to 500 on each side, and close together. They extend anteriorly to near the base of the acetabulum and posteriorly well beyond the uterine sac, to about the tenth lateral coil of the uterus. Approximately half the longitudinal extent of the testes is posterior to the uterine sac. In a 32 mm. specimen with acetabulum 1.90 mm, long, the testicular field was 10 mm. or 31 per cent. of body length and more than five times acetabular length.

The ovary is approximately 26 per cent. of body length from the posterior end. The lateral coils of the uterus are much longer and much snore numerous than in other species. In 22 specimens measured, the width of the uterus was 40 to 81 per cent. of greatest body width. In only one specimen was it less than 50 per cent. and the average was 60 per cent. Since the gravid uterus is visible from the surface, this character alone easily distinguishes G. rugosa from G. urna and G. fimbriata which have short uterine coils. The vitelline follicles extend from about mid-acetabular level to a short distance posterior to the ovary. They are not limited to the lateral fields but extend across the body both ventral and dorsal to the acetabulum, the testes, and most of the uterus. Medianly they tend to be more sparse and are few or lacking opposite the ovary and the terminal eight or ten coils of the uterus. The distance between the posterior limit of the vitellaria and the posterior end of the body was 7.8 mm. in a 70 mm. specimen, and 7 mm. in a 40 mm. specimen, a greater distance than occurs in the other two species.

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Eggs and Larvae of Gyrocotyle rugosa. (Figs. 3, 4)

Eggs are fully embryonated in the rather short uterine sac and in the adjacent uterine coils. Such eggs, measured in alcohol mounts, are 122 to 135 microns (average 131 microns) long by 76 to 95 microns (average 80 microns) wide, thus being considerably larger than the eggs of G. urna or G. fimbriata. The egg shell is thin and colourless, the operculum inconspicuous. Most eggs show a small knob or boss at the anopercular end. No jelly was noted around the eggs and eggs did not tend to stick together.

Eggs removed by puncturing the terminal region of the uterus and placed in sea water immediately reveal very active embryos. The cilia beat vigorously and the embryo turns, twists, pushes, and struggles. Hatching occurs within a few minutes, apparently largely through the activity of the embryo. The lycophore larva swims very rapidly. It follows a spiral path but turns frequently. In a watch glass, the larvae swim to the top of the water every few seconds then turn and swim to the bottom. In addition, they speed in various directions as if searching for some host, as, indeed, they doubtless are. Since young larvae have been reported in the tissues of adult Gyrocotyle, a freshly collected adult was placed in a petri dish with large numbers of recently hatched larvae. No attraction toward the adult was noted and there was no evidence that any larvae adhered to or penetrated the worm. Reactions of the larvae to certain other objects will be discussed later.

Larvae were killed by pipetting into hot formal-alcohol-acetic solution. They were later stained with Delafield's hematoxlyn or with acetic alum carmine, and mounted in balsam.

Description of the lycophore larva of G. rugosa (Figs. 5–8)

Extended specimens, measured in temporary mounts in 70 per cent. alcohol, were 130 to 140 by 50 to 60 microns. The body, tapering from near the middle toward each end, is rounded anteriorly and somewhat truncated at the posterior end. Free swimming larvae as they rotate are seen to fee slightly bent or indented near the middle of one surface as noted in the case of G. fimbriata by Lynch (1945). The body is completely covered by long cilia of approximately equal length. No evidence of cuticular plates could be seen. Circular muscles lie just beneath the ciliated layer. Except for the posterior hooks, in general appearance and behaviour, this larva certainly resembles a miracidium.

Almost filling the anterior third of the body are three pairs of elongated, pyri-form, transparent sacs considered to be glands. The middle pair is almost twice as long as the others and in contracted specimens may be somewhat bent or folded. These structures do not stain but appear as transparent vesicles. The underlying short pair is usually not evident from a dorsal or a ventral view. Each gland opens separately at the anterior end of the body. There is evidence that one of the shorter pairs (the dorsal?) is physiologically different from the others. Among numerous larvae which had penetrated a mass of mucus from the spiral valve of the host, a considerable number, after being stained, showed this pair darkly stained.

Immediately posterior to the glands and just anterior to the midbody lies a large conspicuous bilobed mass of cells. It extends anteriorly lateral to the longer gland cells almost to the base of the shorter glands and posteriorly approximately to the midbody. Near one surface, considered as ventral. the organ narrows to form an isthmus-like band across the body. On each side the cells extend upward and page 5 near the other surface (dorsal) extend medianly but do not meet. This organ is probably a nervous centre. Radiating from the anterior end of the body are six narrow bands of slender nuclei or cells. A central non-cellular cord or tube extends backward between the large gland cells and appears to fork immediately anterior to the isthmus of the cellular mass. Although not granular it is more refractive than the glands. Two finely granular, inconspicuous spherical masses lie side by side immediately dorsal to the isthmus. They are probably not connected with the central cord or tube since they lie at a different level.

Posterior to the bilobed mass, nuclei are more scattered but a central collection and one or two small pairs of cell clusters can usually be observed. At least one pair of flame cells is present. These were not seen in entire specimens either living or stained but were clearly observed in an active state in a crushed, recently hatched specimen. They were located in the posterior half of the body. Each was-pointed diagonally backward and inward. Except for the beginning of the excretory tubule near the flame cell, excretory tubules or pores could not be seen. This record of flame cells is the first for lycophore larvae.

The most conspicuous feature of the lycophore larva is the presence of 10 large hooks withdrawn into the posterior end of the body. In G. rugosa these hooks are unusually large, measuring 34 to 36 microns in length or approximately 26 per cent. of body length. Eversion of the hooks was not observed. Several specimens possessing eight rather than ten hooks were observed. When the hooks are retracted an appearance of eight might result if certain hooks lie directly over others. Fig. 7 shows eight hooks as seen in a crushed specimen.

The lycophore of G. rugosa differs in several respects from the lycophores of G. urna and G. fimbriata. That of G. urna has ten pairs of unicellular glands extending nearly to the midbody, and relatively shorter hooks. The lycophore of G. fimbriata has hooks 19 to 23 microns long and only about l/16th body length. It has at least one pair of very long gland cells (Lynch, 1945). The anterior pair of clear vesicles mentioned by Lynch may correspond to what I have considered gland cells in the lycophore of G. rugose.

Postlarval Stages of G. rugosa. (Figs. 9, 10)

It was discovered more or less accidentally that numerous very small postlarval stages of G. rugosa occurred in the mucus of the anterior portions of the spiral valve of infected fishes. By scraping the wall of this organ with a scalpel living specimens can easily be found. They all possess a posterior, conspicuous, expanded, circular or subcircular, haptor-like organ bearing the larval hooks. The smallest specimens had a body length, not including the haptor, only slightly more than the length of the ciliated lycophore. AH were non-ciliated. The total body length, including the haptor, and the greatest width varied from 0.225 by 0.098 mm. to 0.957 by 0.280 mm. The body is uniformly filled with nuclei and shows less differentiation of cellular groups than does the ciliated lycophore. A depression is visible at the anterior end, and in the larger specimens a concentration of nuclei indistinctly outlines the rudiments of the acetabulum. In the 0.943 mm. specimen the acetabulum was 0.143 by 0.072 mm. The haptor contains more scattered nuclei. It was 0.108 mm. in diameter in a 0.686 mm. specimen. The hooks retain their original size but become more widely separated in the larger specimens. No evidence of the overgrowth of the haptor by the body as noted by Lynch for G. fimbriata was seen.

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Similar postlarval stages have been described embedded in the tissues of adult specimens of G. urna, G. fimbriata, and G. rugosa. None such were observed in the few specimens stained and cleared. In view of the common occurrence of these larvae in the spiral valve of the host, it seems probable that their penetration into an adult Gyrocotyle is accidental.

Fuhrmann (1930), commenting on the occurrence of young larvae in the tissues of adult specimens, stated; "Die 10-hakigen Larven bohren sich vielleicht normalerweise in die Mucosa des Darmes ein (anomalerweise ins Parenchym des Gyrocotyle), um sich dort zu entwickeln und dann wieder in den Darm zu gelangen, wo sie geschlechtsreif werden." He gave no evidence to support this possibility which he apparently proposed as a theory. It is supported by the above findings of numerous very young larvae in the mucosa. It will be shown below that there is a possibility that these larvae may have reached the mucosa by way of the blood stream rather than by way of the digestive tract.