Myxosporea-induced xenoma formation in pike (Esox lucius L.) renal corpuscles associated with Myxidium lieberkuehni infection

European Journal of

Europ.], Protisto!' 24, 271-280 (1989)

PROTISTOLOGY

Myxosporea-induced Xenoma Formation in Pike (Esox lucius L.) Renal Corpuscles Associated with Myxidium Iieberkuehni Infection Jiff Lorn and Iva Dykova Institute of Parasitology, Geske Budejovice, Branisovska, Czechoslovakia

Stephen Feist Ministry of Agriculture, Fisheries and Food, Directorate of Fisheries Research, Fish Diseases Laboratory, The Nothe, U.K.

SUMMARY Myxidium lieberkuehni infection of the urinary tract of the pike (Esox lucius L.) is in most cases associated with the presence of cystic structures in renal corpuscles. These structures are greatly hypertrophic host cells constituting xenomas with the cytoplasm replete with proliferating myxosporean cells. Their development results in primary cells each enclosing one secondary and two tertiary cells. Eventually the cell structure assumes an appearance which suggests that the stages are no longer viable and most xenomas are destroyed by host tissue reaction. Some cells, however, may be released into the tubule lumen, suggesting a possible further sporogonic development of M. lieberkuehni. Circumstantial evidence points at the identity of the xenoma stages (designated Nephrocystidium pickii by Weissenberg in 1921, referenced in Weissenberg, 1923 [26]) with M. lieberkuehni, and this applies also to intracellular stages found in the epithelium of the collecting renal ducts. The latter have the same cell-in-cell arrangement of primary, secondary and tertiary cells, but were not observed to show any signs of cell degradation.

Abbreviations: C1 EN HC N, Nz N3 P S T XC XN

= cytoplasmic inclusion endothelial cell nucleus host cell nucleus of the primary cell nucleus of the secondary cell nucleus of the tertiary cell primary cell secondary cell = tertiary cell = host cell xenoma cytoplasm = host cell xenoma nucleus = = = = = = =

Introduction A frequent occurrence within the renal glomeruli of pike

(Esox lucius L.) are hypertrophic cells with small intracellular parasitic organisms. The first to describe them was © 1989 by Gustav FischerVerlag,Stuttgart

Debaisieux in Belgium, who in a short note [4] and later in a full paper [5] described the "Parasitic cysts" in pike renal corpuscles. He identified these "cysts" as hypertrophic host cells full of parasitic bodies which he interpreted as special developmental stages of Myxidium lieberkuehni, which had by that time already been recognised as a common myxosporean parasite of the urinary tract of pike. He based his interpretation on observations of "cystic" stages released into the Bowman's space; he claimed that they then proceeded further into the tubules where they developed into sporogonic plasmodia of M. lieberkuehni. He also reported a single finding, within the cyst, of a small plasmodium and even described small Myxidium plasmodia in the renal corpuscles. He detected M. lieberkuehni in five out of six pike infected with the renal corpuscle parasite. One year later, Weissenberg (1921- referenced in Weissenberg, 1923 [26]), unaware of the work of Debaisieux, described these parasites as organisms of unknown tax0932-4739/89/0024-0271$3.50/0

272 . J. Lorn, 1. Dykova and S. Feist onomic position under the name Nephrocystidium pickii. Subsequently, in 1922 [25] and in a large paper [26] he presented a thorough analysis of the "cystic" organisms. Most of them were small bodies with one or two nuclei containing two small inner cells. When placed in 0,2% NaCI solution, the bodies developed thin pseudopodia using them for a rather fast forward locomotion. In addition to the small bodies, he recorded large structures in which several small bodies were contained within a larger plasmodium. He refuted Debaisieux's claim that these organisms were developmental stages of M. lieberkuehni, arguing that no solid evidence supported this assumption. He agreed, however, to consider them to be myxosporean parasites probably of a new species to be described, the first true intracellular parasite including cell hypertrophy. In his 1923 paper [26], Weissenberg does not seem to stick firmly to the idea of N. pickii as an organism of unknown position. Few of the subsequent workers paid attention to the renal corpuscle parasite. Kudo [11] found 13 pike specimens (Lucius reticulatus) which he examined in North America to be infected with M. lieberkuehni but failed to detect any renal corpuscle parasites. Zandt [28] briefly mentioned this parasite from pike (E. lucius) collected in Lake Constance (Bodensee). jirovec [8] found the parasite in 4 out of 8 pikes collected in Bohemia (Czechoslovakia). From stained preparations, he described the small parasite bodies which were identical to Weissenberg's [26] and mentioned the unsettled problem of their taxonomic position. Curiously enough, no one has tackled the problem since, although the parasite is quite common. The recently growing interest in extrasporogonic development in myxosporea, i.e., in stages that precede or run parallel to the stages instrumental in spore production [12], has greatly contributed to the understanding of myxosporean life cycles [1,3,9,13,14,15,17,18] and has also prompted our studies of the pike renal corpuscle parasites. This paper presents the results of light- and electron-microscope findings of these stages and on some little known developmental stages of M. lieberkuehni.

Material and Methods From 1984 to 1986, 87 pike (E. lucius) were collected in the southwestern region of Czechoslovakia and were examined together with 39 pike from southern England. Following the observation of fresh contents of the urinary bladder and fresh kidney squashes, the samples were fixed for light and electron microscopy and impression smears were prepared. For histologi-

cal observations, the material was fixed in 10% neutral buffered formalin and embedded in paraffin wax. 5 11m tissue sections were stained with haematoxylin and eosin and Giemsastains.For the electron microscopy, tissue samples were fixed in cold, 2% osmic acid in 0,1 M cacodylate buffer or in 4% glutaraldehyde in cacodylate buffer followed by 1% osmium tetroxide in the same buffer and embedded in Epon-araldite. Ultrathin sections, double-stainedwith uranyl acetate and lead citrate, were observedin Philips EM 420 and JEOL 1000CX electron microscopes. Some of the sections were treated by the Thiery method for carbohydrates [22].

Results Both light and electron microscopy clearly demonstrated that the hypertrophic cell, together with the mass of parasite cells filling its cytoplasm, represent a xenoparasitic complex or xenoma. These xenomas measuring up to 1 mm and appearing as whitish dots, were found in the renal corpuscles of 41 (47%) pike examined from Czech localities. As a rule, all stages of infection were encountered from initial stages to mature xenomas and those in the process of degeneration. Concurrent M. lieberkuehni infection in the urinary bladder was observed in 73 % of all pike examined. In many cases of M. lieberkuehni infection, small plasmodial stages were found in the lumen of the renal tubules as were intracellular developmental stages in the epithelium of some of these tubules. Detection of luminal and epithelial stages was difficult; therefore the prevalence rate found to be 24% is a low estimate. There was no seasonal variation in the incidence of renal corpuscle infection.

Light microscopy The earliest stages of the infection in the renal corpuscles that could be detected were cells measuring 35 x 24 urn in histological sections. The origin of the infected cell could not be precisely determined because of morphological transformation during the course of infection but a comparison of many findings of early infection stages indicates that the cell originally infected is an endothelial cell of the glomerular capillary (Figs. 1, 2). Occasionally the xenoma was seen growing within the thin wall of a dilated capillary (Fig. 1). There is no clear evidence thus far that podocytes or mesangial cells can be infected. The young xenoma tends to maintain a position at the periphery of the glomerulus (Fig. 1). There may be more than one xenoma per corpuscle. In the subsequent period

Figs. 1 and 3-7 are haematoxylin and eosin-stained paraffin-embedded tissuesections. Fig. 2 is a semi-thinsection of a resin-embedded sample stained with Toluidine blue. - Fig. 1. Early stage of the xenoma with a greatly hypertrophic nucleus and nucleolus in a distended capillary of the kidney of pike. Bar = 20 11m. - Fig. 2. Earlyxenoma in the glomeruluswith two host cellnuclei (arrows)and chromophil inclusionsin the cytoplasm. Bar = 20 11m. - Fig. 3. A grown xenoma with a mass of parasitic stages in the cytoplasm.Bar = 100 11m. - Fig. 4. Two small trophozoites in the kidney tubule, one of which contains two maturating sporoblasts. Bar = 20 I1m.Fig. 5. Large xenoma with lobed outlines. Bar = 50 11m. - Figs. 6 and 7. Mature xenomas surrounded by incipienthost tissuereaction; in Fig. 7, the host cell nucleus has split into two fragments, one containing the nucleolus. Bars = 100 11m.

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of growth (Figs. 3, 5, 6), the number of parasites increases enormously and eventually the cytoplasm is replete with them. The central hypertrophic nucleus has a much altered appearance with large, dense aggregations of chromatin present. Fragmentation of the nucleus into two or more parts is frequently observed (Figs. 6 and 7). The grown xenoma clogs the Bowman's space and inflicts pressure atrophy upon the glomerular capillary loops, eventually resulting in their disappearance from the corpuscle. The whole lesion, attaining about 1 mm, has a diameter approximately eight times that of the normal glomerulus. As long as the changes provoked by the infection take place within the renal corpuscle, with at least the parietal sheet of the Bowman's capsule intact, the surrounding tissue does not reveal signs of tissue reaction. This is so even if the glomerular capillary tuft has already been markedly altered or has disappeared completely. The large lesions then become surrounded by a fibrous capsule varying in thickness and elicit a severe granulomatous inflammatory reaction. In heavy infections, large volumes of renal parenchyma are replaced by granulomatous tissue and are thus rendered non-functional. Eventually, healing with reparative fibrosis occurs. In pike with infected renal corpuscles, the sporogonic stages of M. lieberkuehni might be found not only in the urinary bladder but all the way up to the proximal parts of renal tubules. In these tubules, small plasmodial stages were found (Fig. 4) which can be interpreted as early, still small, sporogonic plasmodia. They start producing spores in the tubules (Fig. 4) without attaining larger size. In addition, the epithelium of the renal collecting ducts harbour small intracellular stages, often in large numbers. The infected epithelium is greatly hyperplastic and produces papillary ingrowths into the lumen of the ducts.

Electron microscopy (a) The xenoma and stages in its cytoplasm The very early stages of the xenoma (Fig. 8) represent a host cell which to some extent still retains its original morphology. Its nucleus and cytoplasm appear close to normal, but huge electron dense inclusions may signal incipient changes in metabolism induced by the presence in the cytoplasm of several parasite stages. It is noteworthy that all parasitic cells encountered were primary cells with inner, secondary cells. In a grown xenoma morphological changes from the original host cell are enormous. The grown xenoma is covered by a simple cell membrane, either flat or crenated.

Invaginations of the cell membrane (Fig. 11) may serve for ingestion of the thin layer of amorphous electron-dense substance covering the surface. Occasionally, even host cells may be ingested (Fig. 9). Aggregations of amorphous electron-dense substance beneath the xenoma cell membrane sometimes give the false impression of a hemidesmosome (Fig. 12). This dense material is also distributed throughout the xenoma cytoplasm. The cytoplasm is replete with small vesicles, mostly with electron lucent contents (Figs. 10, 14). There are mitochondria, bundles of microfibrils, ribosomes, vacuoles, and large (up to 1 urn) inclusions filled with finely granular material, and somewhat smaller (about 0.5 um) dense inclusions. There is no special cortical layer in the xenoma. In the hypertrophic nucleus, which has a well preserved nuclear envelope, most of the volume is taken up by finely dispersed, granular chromatin (Fig. 15). The cytoplasm is filled with parasitic cells which may be loosely scattered but are usually densely packed and are consequently near to both the nuclear and cell membrane of the xenoma. They are not bound by a parasitophorous vacuole membrane. There is a whole array of cells from rarely-occurring simple cells, numerous primary cells containing one secondary cell and very numerous primary cells with one secondary and often two tertiary cells. Boundaries between the cells always consist of the two cell membranes of the respective cells. The few electron micrographs of cells, that appeared as simple cells without secondary cells (Fig. 14), may represent the beginning of the developmental series or, perhaps, just a section through the primary cell, not showing the secondary cell. At the stage of the primary cells enclosing one secondary cell, most of the volume of the former is occupied by the latter (Fig. 13). The cytoplasm of both cells contains a number of rounded or elongated mitochondria and sparse cisternae of endoplasmic reticulum. In the primary cell, Golgi bodies, various vesicles, vacuoles, fat inclusions and a few ribosomes occur. In addition, the cytoplasm may contain a strange, elongated or club-shaped, membranebound structure with an electron dense inner core (Fig. 16) which may also be present in later developmental stages when tertiary cells are present. Sometimes there is another layer of electron-dense material apposed to the structure (Fig. 17). The cytoplasm of the secondary cell has an electron-dense appearance due to a mass of free ribosomes. Close to the nucleus of the secondary, as well as of the tertiary cells (Fig. 23), there is the characteristic bundle of microtubules.

(In Figs. 8 to 14: Bar = 2 urn) - Figs. 8-26 are electron micrographs. - Fig. 8. Section through a very early xenoma with tightly ~ apposed endothelial cells (arrows), with an almost unchanged nucleus, two large and one small cytoplasmic inclusion and four parasitic cells. - Fig. 9. Part of the periphery of a grown xenoma engulfing a host cell (arrow). - Fig. 10. Appearance of the xenoma cytoplasm including a cross-section of a primary and secondary cell.- Fig. 11. Interface between the xenoma and the host cell, filledby an amorphous dense substance; arrows point at the invagination transporting this substance into the cytoplasm. - Fig. 12. Dense substance subtending the xenoma membrane at the host cell - xenoma interface (arrow). - Fig. 13. A primary cell containing a secondary cell in the xenoma cytoplasm. - Fig. 14. A simple uninucleate cell or a section passing only through the primary cell?

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In the next developmental stage, the primary cell, depleted of cell organelles and reduced to a thin envelope, encloses a large secondary cell with one, and eventually two, tertiary cells (Fig. 19). When the tertiary cell completes its binary fission, a conspicuous bundle of microtubules can be seen to extend across the partition arising between the two daughter cells (Fig. 20); at that time, the bundle does not seem to be associated with the nucleus. Flat cisternae of endoplasmic reticulum appear apposed to cell membranes of the tertiary cells (Figs. 19, 21). The outlines of nuclei of both secondary and tertiary cells become wavy and folded. At one point, a group of tightlypacked tubules can be seen to adhere to the outer surface of the nuclear envelope (Fig. 24), sometimes fitting within a depression of the nuclear envelope. It appears comparable to a primitive Golgi body and to a slight extent it is also reminiscent of microsporidian kinetic centers. In the following stage, the tertiary cells produce, at their surface, curious lobose or pseudopodia-like projections into the cytoplasm of the secondary cell (Fig. 22); the double-membrane boundary between the cells is always maintained. The projections grow longer and twisted and the tertiary cells become intertwined and interlocked (Fig. 23). At the same time, the cytoplasm of the secondary cell becomes rich in small, lucent vesicles (Fig. 23), which are almost of the same appearance as those in the xenoma cytoplasm (Fig. 10). Aggregations of ~-glycogen particles are prominent in the cytoplasm of the secondary cell (Fig. 23, asterisks) and are conspicuously visualized by the Thiery technique (Fig. 25). The xenoma parasites show no signs of further development. Features such as vacuolisation of the cytoplasm and excessive intertwining of tertiary cells, which become extremely electron dense, indicate stages of cellular degeneration and that these cells cease to be viable. Additional changes in these degenerated cells are formation of tiny vesicles pinched-off from one of the two membranes forming the boundary between the intertwining projections of the two tertiary cells (Fig. 18) and evaginations pinchedoff of the surface of the tertiary cell. While most of the xenoma parasites are eventually phagocytosed and destroyed by the host tissue reaction, some of them appear in the lumen of proximal renal tubules. Some are obviously degraded and non-viable; others seem to be capable of further development, possibly as sporogonic plasmodia. The latter are characterised by the presence of large bodies consisting of a compressed mass of lamellae.

(b) Stages in the collecting ducts of the renal tubules Some stretches of collecting ducts are heavily parasitized, all epithelial cells in a row being infected, and some of the cells harbouring two parasites (Fig. 26). The parasite stages observed are mostly primary cells infected with one secondary cell, but stages with tertiary cells are also detectable. The primary cells, irregularly lobose and filling the majority of the host cell cytoplasm, have a pale, rather lucent appearance, while numerous free ribosomes give the secondary and tertiary cells a dense appearance. There is no parasitophorous vacuole membrane surrounding the parasite. The stages in epithelial cells differ from those in the xenoma in that the inner cells (secondary and tertiary) lack the conspicuous lobose projections. The epithelial cells are heavily vacuolated and lack the well-organized appearance of uninfected cells.

Discussion The main problem which the xenoma parasites pose is whether they are direct precursors of the sporogonic plasmodia of Myxidium lieberkuehni [5] or unrelated myxospore an parasites (Nephrocystidium pickii), possibly requiring two hosts for completion of their life cycle [26]. The observation of certain features revealed in the present study, such as bundles of microtubules and numerous free ribosomes in the inner (secondary and tertiary) cells, presence of glycogen in the primary cells, the lack of centrioles and the cell-in-cell arrangement confirms their myxosporean nature. They may be considered to be equivalent to extrasporogonic stages, i.e., stages of myxosporean species which develop in a site different from that in which the spore-forming plasmodia are produced, and which allows for proliferation of the parasite in the body of the fish host and precedes or runs parallel to the development of sporogonic stages [12]. Similar extrasporogonic stages were first thoroughly described in the genus Sphaerospora, in the bloodstream of the host [2, 14] and in the tissue of the swimbladder [3]. The stages of Sphaerospora have the same characteristics as the stages found in pike, namely an uninucleated primary cell enclosing secondary cell(s), often containing tertiary cell(s). The cell-in-cell configuration or "enveloped condition" is typical for myxosporeans. Even if the "actinosporean theory" regarding the need for two hosts and the myxosporean/actinosporean transformation [27; El-Matbouli, personal communication] is

Fig. 15. A parasitic stage with primary (P) cell with one secondary (5) and two tertiary (T) cells close to the hypertrophic host cell nucleus (XN). Bar = 2 urn, - Fig. 16. Two club-shaped structures in the cytoplasm of the primary cell. Bar = 1 um, - Fig. 17. Transverse sectionthrough a club-shaped structureenclosed in an additional layerof electron-dense material.Bar = 1 urn,- Fig. 18. A microtubule bundlepassing through the partition formed between the tertiarycells whichhavealmostcompleted division. Bar = 1 urn. - Fig. 19. A spherical primary cell (P) with one secondary (5) and two tertiary (T) cells. Bar = 1 urn.- Fig. 20. A bundle of (spindle?) microtubules still connecting the two dividing tertiarycells with already intertwining celloutgrowths(asterisk). Bar = 1 urn, - Fig. 21. Pseudopodia-like cell projections (asterisk) of the tertiary cells with adjacent flat cisternae of the smooth endoplasmic reticulum (arrows). Bar = 2 urn.

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Myxidium Infection in Pike' 279

true and generally valid, there is no evidence that the extrasporogonic development of a myxospore an life cycle takes place in a different host to that of the sporogonic phase. Consequently, the sporogonic phase succeeding the renal corpuscle parasites should be looked for in the same host. The regular coincidence of the latter with the only myxosporean (i.e. M. lieberkuehni) infecting the same organ suggests the identity of these organisms. The few cases in which pike harboured renal corpuscle parasites without M. lieberkuehni being present can be explained either as the early infections before the sporogonic phase had begun or as infections persisting after the sporogonic plasmodia had vanished from the urinary tract. The significance of the renal corpuscle stages is not clear. Our observations indicate that most of the parasites having reached the stage of one primary cell with one secondary and two tertiary cells eventually become nonviable and that most of the xenomas are destroyed by the host tissue reaction. Our observations also show that a fraction of them may reach the tubular lumen and serve as a stem line for sporogonic development. Were it not for the apparently dubious viability of most of the "mature" stages observed, it would be a resonable presumption that the renal corpuscle stages pass into the renal tubules and start sporogonic development in the urinary tract. In fact, Debaisieux [5] claimed to have observed sporogonic plasmodia in the renal corpuscle and a single instance of a plasmodium in the xenoma. His assertion, however, that the renal corpuscle stages pass into the lumen of the tubules was by no means supported by sufficient evidence. In many ways, the xenoma stages are reminiscent of the intracellular stages from the hypertrophic epithelial cells of the renal tubules of the common carp (Cyprinus carpio) infected with Spbaerospora renicola. These stages were considered by Plehn [19], and still are considered by Molnar and Kovacs-Gayer [17, 18] to be vegetative stages of another carp renal tubule myxosporean, Hoferellus cyprini. Ultrastrucural observation [13] and epizootiological evidence [7] showed that these intracellular stages develop to produce a structure consisting of one primary cell with one secondary cell and several tertiary and quaternary cells. Eventually, however, this whole cell complex becomes non-viable and the parasite foci are eliminated by host tissue reaction. Thus, while the intracellular stages of S. renicola were deemed to represent an aberrant, curious developmental blind alley in the propagative cycle of the parasite, it is thought that at least a portion of the pike xenoma stages can transform into sporogonic stages of M.

lieberkuehni.

No direct evidence has been supplied that the intracellular stages found in the hyperplastic ductal epithelium pass into the lumen of the urinary tubules to initiate the sporogonic infection. This passage, however, seems probable since there were no signs of aberrant development or evidence of non-viability. Host tissue reaction associated with these stages was not observed. The identity of the intracellular stages with M. lieberkuehni seems most probable. Assuming the hematogenous spread of the initial stages of myxosporean infection [6, 20], the sporoplasms released from the spores in the intestine - or products of their proliferation - could reach the renal corpuscles and produce the xenomas there. They could also reach the interstitium and penetrate into the epithelium of renal ducts and tubules and start the extrasporogonic proliferation there and later pass into the lumen of the excretory tract. In rare cases, such as those observed by KovacsGayer and Ratz [10], they may produce sporogonic plasmodia directly in the interstitium. The ultrastructure of the extra sporogonic stages in the xenomas and ductal epithelium is, understandably, more simple than that of the sporogonic plasmodia where the cytoplasm includes various fibrils, vacuoles, dense inclusions composed of lamellae and crystal-like inclusions [16]. The different appearance of mitochondria, which in sporogonic plasmodia tend to have a very dense matrix, may reflect differences in metabolism. Concerning the cytology of the renal corpuscle stages, no direct relation between the microtubular bundle in the secondary cells and nuclear division could be seen in electron micrographs. The bundle and the nuclei in these cells do not appear to be directly associated. The groups of fine tubules associated with the surface of the secondary cell nuclei can perhaps be interpreted as a modified Golgi body. In protists, the Golgi body usually has a much simplified structure, e.g., a simple tubular network in microsporidia [21,23,24]. In some myxosporean cells, a typical Golgi body with stacks of cisternae has been seen in close association with the nucleus, as in primary cells of the intracellular stages of S. renicola [13]. A Golgi body with a stack of cisternae was not observed in secondary cells in the present investigation and it may well be represented by the simple vesicular structure. The greatly hypertrophic cells in renal corpuscles of pike are the first and only example of a myxosporean xenoma thus far reported, completely comparable with myxosporean xenomas produced by species of the genus Glugea, e.g., in the form of a viable, grossly-enlarged cell (with an

.... Fig. 22. A stage from the advanced xenoma. Tertiary cell bears conspicuous pseudopodia-like outgrowths; primary cell is reduced to a thin envelope (asterisk). Bar = 2 urn. - Fig. 23. Two tertiary cells with intertwining cell outgrowth (arrows); the cytoplasm of both primary and secondary cells is affected by vacuolisation (crosses). Asterisks mark the aggregates of glycogen particles. Bar = 2 Ilm.Fig. 24. A clump of vesicles (arrows) in a depression of the nuclear membrane of the tertiary cell. Bar = 1 urn. - Fig. 25. Thiery reaction demonstrates aggregates of ~-glycogen granules in the cytoplasm of the secondary cell, which are absent in the tertiary cell. Bar = 1 urn, - Fig. 26. Epithelial cells of the disorganized epithelium of the collecting duct which harbour one or two parasitic stages. Bar = 4 urn,

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equally enlarged nucleus) harbouring a mass of.prolifera.ting parasites. Reasons why the extrasporogom~ .stages 10 the epithelial cells in the renal duct do not elicit such a hypertrophy of the host cell cannot be elucidated at the present time.

14 Lorn J., Dykova I. and Pavlaskova M. (1983): "Unidentified"

15

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Key words: Myxosporea - Xenoma - Myxidium lieberkuehni - Esox lucius Jifi Lorn, Institute of Parasitology, 37005 Ceske Budejovice, Branisovska 31, Czechoslovakia