The Grigarinis of Cucumaria: Lithocystis minchinii Woodc. and Lithocystis cucumariae n.sp

This seems to be the most suitable name by which to distinguish the gregarine which goes through its whole life-history in the respiratory trees of C u c u m a r i a. It has appeared in only ten out of thirty-five specimens of C. saxicola examined from the Plymouth district since March 1927. A few of these holothurians were taken from the shallow tank in the old laboratory where they had probably been living for months. However, they seemed perfectly healthy and one individual had no infection at all, so they were not necessarily infecting each other. Another infected specimen came from Wembury Bay, and several others were taken in a trawl near the Mewstone and Stoke Point. Sometimes the infection was so heavy that the respiratory trees appeared to be more or less covered with the white cysts, while in other Cucumaria only two or three could be found.

Woodcock (1906) has described the vegetative stages lying inertly in the connective tissue-layer of the respiratory trees after their very early association, and his figs. 4, 18, 19, 31, and 32 represent this parasite. Many of mine have been at rather later stages than he showed in fig. 19. They were often larger—some being as much as 500 µ in diameter apart from the layers of flattened connective-tissue cells forming the host cyst. Many of the larger cysts were full of ripe spores. These cysts were 400 to 500 µ in diameter before compression and, as usual with gregarine cysts containing spores, they were rather more transparent than those containing earlier stages. They could thus be distinguished even macroscopically from the opaque cysts containing trophozoites.

The spores, crowded together indiscriminately within the cysts, were provided with long, flattened tails (Text-fig. 1) tapering to a point. They differed from most other Lithocystis spores in having a double funnel at the other end from the tail. The inner delicate episporal funnel was generally 2-3 µ deep. ² When seen in profile the four upstanding edges of the two funnels had often the appearance of refringent spines, but by examining a spore from its funnel end, the delicate circular-edges of inner and outer funnels could be clearly distinguished to be free from any spines or processes. While a sporozoite was forcing its way through the inner funnel it has been seen still surrounded by the outer one as usual—the use of it being quite obscure. The thick-walled endospore measured about l8µ, long and 7µ. at its widest part. The eight sporozoites were exceptionally short and broad. They were often arranged more or less in an upper and lower series round the central residuum of large refringent granules. They, however, appeared to be capable of rearranging themselves—sometimes most of them were lying obliquely across the spore. Each ripe sporozoite was somewhat fusiform and about 6µ long and 2 to ?µ wide—the nucleus, consisting of a small deeply staining caryosome in a clear vesicle, being towards one end. So transparent were the sporozoites that the most conspicuous part of the spore was the central spherical residuum. The delicate tails were a little wider than the endospore proximally and tapered to a whip-like extremity, being altogether about 5Oµ long. The episporal processes were already formed by the time that the four-nucleated stage of the sporocyst was reached, though they were at this stage even more delicate and easily pressed out of shape, and the funnels have shown clear evidence of being formed by the folding back on itself of the episporal wall. It should be noted that this gregarine, though having a spore with a long flattened tail very like the type Lithocystis (L.schneideri Giard), is very different from it in other ways, especially in having its active vegetative stage very much curtailed owing to neogamy. No doubt its precocious association is in correlation with its habitat, for it would be obviously impossible for unattached forms to remain in the lumen of the respiratory trees owing to the currents of water passing in and out.

Occasionally a small rounded trophozoite has been found embedded in the respiratory tree wall and on examination has been found to have a single nucleus only; one of these measured as much as l5Oµ. in diameter. In every one, however, the cytoplasm was found to be vacuolar and obviously necrotic, and there is no reason to think that solitary encystment is any more successful in this than in other gregarines.

Some early stages in nuclear division have been observed in the associates, but unfortunately no late stage and neither gametes nor zygotes have been obtained. This, no doubt, indicates that these sexual stages are passed through rapidly, as in most gregarines. A few empty cysts were found in the respiratory tree wall. There is no evidence that these cysts were burst during manipulation, so possibly the spores occasionally escape in this way and either cause auto-infection or pass to the exterior through the cloaca. We can at any rate now be certain that this gregarine passes through its whole life-history in the respiratory trees of C. saxicola—a habitat from which no other parasite has ever been recorded—and the following are its specific characteristics so far known.

Trophozoites neogamous and encyst in the wall of the respiratory tree.

Spores (approximately 18 µ long) provided with an epispore produced into a double funnel at one end and at the other into a long tapering tail.

Sporozoites, short and transparent, arranged round a conspicuous residuum of ref ringent granules.

Habitat: respiratory trees of C. saxicola.

Lithocystis minchinii Woodc.

Synonyms: Diplodina (Cystobia) minchinii Woodcock, 1916.

Gonospora minchinii Trégouboff, 1918.

This gregarine was found parasitic in fourteen out of thirty-five specimens of C. saxicola, but only four times simultaneously with L. cucumariae. In dealing with it I should like to refer first to the spores—these being the structures of most use among gregarines for determining species.

Each spore-containing cyst was held closely to or even embedded in the coelomic wall, mesentery or a retractor muscle, being surrounded by a thick connective tissue-layer forming a host cyst. The cysts themselves were generally from half to one millimeter in diameter and full of spores—no residuum being present. When compressed, the spores broke through the tough host cyst at a central spot on the free surface (Text-figs. 3 and 4) which will be referred to below.

The spores were examined immediately, while perfectly fresh, and even then considerable difficulty was experienced in making out their very peculiar structure (footnote, p. 277).

On the shoulder of each spore (Text-figs. 2? and b), round the delicate funnel through which the sporozoites escape, the epispore is raised up into a series of conical projections forming a more or less regular outer funnel; similar episporal projections occur at irregular intervals over the sides of the spore and at the posterior end there is a larger one forming a delicate flattened tail—to be seen whenever an exact profile view of a spore is obtained. Individual spores show much diversity in detail; the whole epispore has often, especially when fixed, the appearance of a loose thin-walled sack falling into folds about the refringent endospore.

The spores are small, the. endospore measuring generally 10 to I2µ by 7 to 8µ, and by far the most conspicuous part is the spherical mass of residual granules, which have a high refringency. Bound this mass the eight sporozoites are variously arranged; each is about 6µ long and almost transparent.

From their habitat, size, and general appearance, there can be no doubt that these are the spores that Woodcock ³ found and named after Professor Minchin. He said that their structure was very difficult to make out, as indeed it must have been in the preserved material that he used; some examined in alcohol he described as ‘crinkled and warty in outline ‘(1906, p. 57). Consequently, it is not surprising that the short tail was not observed and that this gregarine’s affinities with Lithocystis were overlooked. Even now these affinities arenot very close and it may be necessary to make for it a new genus when its complete lifehistory is known.

In addition to cysts containing spores, I have found in the same positions in the host exactly similar cysts still containing the associated gregarines with their two nuclei unchanged or occasionally in early stages of nuclear division: these cysts were also all surrounded by thick layers of connective tissue and embedded more or less in the host’s tissues, as shown in Woodcock’s fig. 16. Further, there can be little doubt, I think, that the bodies shown in Woodcock’s figs. 6 and 12 are, as he said, earlier stages of this same parasite. In Text-fig. 3 I have given an optical section of one of these bodies freshly detached from the body-wall of the host.

Here the associated trophozoites are enclosed in a stalked cup-shaped body, and some of the contractile fibres may be seen protruding from the proximal end of the long stalk where they have been torn away from the fibres in the connective tissue of the host’s body-wall. The lips of the cup enclosing the pair of gregarines have at this stage almost met at the distal end; they do not appear to fuse so long as the cyst projects at all into the coelom (Text-fig. 4), and when compressed under a coverglass, the cytoplasm followed by the nuclei of the gregarines could be made to shoot out with explosive suddenness through this central aperture in the host cyst. Even at the spore stage, as already mentioned, this is the only spot in the thick connective tissue cyst at which the spores could escape.

So definite were these cup-shaped bodies enclosing associated gregarines that one was tempted to suspect that they must be some special excretory organs of the host—such as the ciliated urns of Synapta. However, I have not been able to find them in Cucumaria uninfected with this gregarine, nor can I find any reference to similar structures in the literature of these Holothurians. The cup is lined with peritoneum, in places several layers thick (Text-fig. 3). The walls and stalk contain in addition to the contractile fibres many ordinary leucocytes and also a few of the large amoebocytes full of refringent globules which are of common occurrence in the general con-nective tissue of the body-wall. The globules of these amoebo-cytes stain mauve intra-vitam with cresyl blue, while the nuclei of leucocytes, peritoneal cells, and parasites stain blue.

The paired gregarines that I have measured in these cups have been from 300 µ to a millimetre long. Only once have I seen any division in the cytoplasm and that was possibly an abnormal specimen, but it had a trace of a partition at its proximal end, showing, as indeed the position of the nuclei also indicates, that association has been lateral. The cytoplasm could not be made to show any sexual dimorphism by means of intra-vitam stains. The nuclei were about 42 µ in diameter and appeared to contain a clear ñuid in which was suspended a vacuolated caryosome of 20 µ diameter. The stalks varied in length: the longest, as in Text-fig. 3, have been found on the actual body-wall where the connective tissue fibres have to pass through the circular muscles of the body-wall and then through the very thick coelomic epithelium—they were often to be found quite close to the longitudinal muscle-bands. Their relationships were confirmed by the study of serial sections through regions of the body-wall to which were attached cysts as well as the cup-shaped bodies. When once the full-grown pair of parasites has secreted its own thin and compressible cyst-wall, there can be little doubt that the stalk of the cup is shortened by the contraction of its fibres, for, although associated pairs of parasites are found in stalked cups or sessile (Text-figs. 3 and 4, and Woodcock’s figs. 12 and 16), no later stages have ever been found in the stalked forms. Cysts containing spores may be seen in sections either projecting partially into the coelom beyond the epithelium or completely embedded in the body-wall, having apparently been drawn down through the muscle-layers into the connective tissue-layers. Here presumably the spores, surrounded by their very tough cyst-walls, remain imprisoned during the life of the host and thus all chance of auto-infection is prevented.

The cysts embedded thus in the thick body-wall of the Cu cum aria are quite hidden from the outside of the animal as well as from the coelom, and were no doubt often entirelyoverlooked by me at first as well as by Woodcock. This would account for the fact that they seemed to be very rare. Several hosts appeared at first to have only a single cyst and that nearly always on the large retractor muscles of the buccal region—projecting only slightly from the surface. The thick connective tissue-wall was very tough to dissect, but when compressed the spores easily broke through the parasite cyst and emerged through the central aperture left in the host cyst or cup. Onone occasion a cyst with ripe spores was embedded in the mesentery supporting the alimentary canal, and in another Cucumaria I once found a cyst with the remains of a stalk free in the coelomic cavity. The contained parasites had reached an early stage of nuclear division and might possibly have developed further if they could have been removed and kept under aseptic conditions.

Strangely enough, no definite young stages of this gregarine have been seen—occasionally oval trophozoites have been found in the coelom, but they have been so limp and easily destroyed that it has often been impossible to pick them up with a pipette. They were probably specimens which had failed to pair and had become necrotic.

One may perhaps conjecture that sporozoites are liberated from spores drawn into the alimentary canal through the mouth and that they pair while finding their way through its wall into the coelom, where, if successful, they grow rapidly and stimulate the tissues of the host to grow out and envelop them, as described.

Even when they have been so enveloped they may sometimes fail to develop. Possibly if they fail to secrete a cyst the host’s phagocytes are able to attack them while in these cup-shaped bodies. Anyhow, I have found occasionally that the cups contained only limp pairs such as were sometimes free in the coelom and when touched the cytoplasm easily flowed out through the aperture. Similarly empty cup-shaped bodies were sometimes found, but only in specimens of Cucumaria infected with this gregarine.

It will thus be seen that in order to complete the life-history of this parasite a knowledge of the early vegetative stages is necessary as well as the gametes and zygotes. These stages would no doubt be most easily obtained by artificial infection of young Cucumaria. Possibly the pairs enclosed in their host cup could readily be induced to continue their development in vitro provided they were extracted under aseptic conditions and mounted in sterile coelomic fluid. I have not been able to carry out these experiments with Cucumaria, but we have already had some success in the development of gregarines from fresh-water animals ‘in vitro

Specific characteristics of L. minchinii Woodcock may be given at present as follows:

1. Association lateral and neogamous.

2. The host attaches the paired parasites to its coelomic wall throughout the greater part of their lives and tries to embed them in its connective tissue.

3. Spores with peculiar episporal projections including a short flattened tail.

Of two neogamous gregarines infecting C. saxicola and hitherto considered to be one and the same species, one—L. cucumariae n.sp.—was restricted to the respiratory trees and had spores with long flattened tails. The other—L.minchinii Woodc.—was enclosed throughout most of its life in a cup-like outgrowth of the host’s coelomic epithelium and connective tissue, and had spores with peculiar episporal processes including a short tail.

Department of Zoology and Comparative Anatomy, University Museum, Oxford.