J. Euk Microbrol.. 42(2), 1995. pp. 158-165 0 I995 by the Society of Protozoologtsts

Vittaforma corneae N. Comb. for the Human Microsporidium Nosema corneum Shadduck, Meccoli, Davis & Font, 1990, Based on its Ultrastructure in the

Liver of Experimentally Infected Athymic Mice HENRIQUE SILVEIRA and ELIZABETH U. CANNING’

Department of Biology, Imperial College of Science Technology and Medicine3 London, S W7 2AZ, United Kingdom

ABSTRACT. A new genus, Vittuforma n. g. is proposed for the human microsporidium Nosema corneum Shadduck, Meccoli, Davis & Font, 1990, based on the ultrastructure ofdevelopmental stages in the liver ofexperimentally infected athymic mice. The diplokaryotic arrangement of the nuclei is the only character that conforms with the description of the genus Nosema. Sporogony is polysporoblastic, sporonts are ribbon-shaped, constricting to give rise to linear arrays of sporoblasts and each parasite is enveloped by a complete cisterna of host endoplasmic reticulum. Comparison of N. corneum, with established genera revealed that there were none with the same combination of characters. Consequently it is proposed that Nosema corneum be placed in a new genus as Vittaforma corneae n. comb.

Supplementary key words. Diplokarya, host endoplasmic reticulum, new genus, polysporoblastic sporogony.

microsporidium, first reported in the cornea of an immu- A nocompetent patient [8], was later propagated in vitro and named as a new species Nosema corneum [ 181. The species was placed in the genus Nosema on the basis of its presumed dis- poroblastic sporogony, and the diplokaryotic arrangement of the nuclei. The presence of a cistema of host endoplasmic re- ticulum (ER) surrounding each parasite was noted as being un- usual for the genus.

In the present study a culture of Nosema corneum was used to establish infections in athymic mice [ 191 and pieces of heavily infected liver were excised and processed for electron micros- copy. The ultrastructural observations suggested that the species could not be retained in the genus Nosema and, after comparison with other existing genera, it is proposed that a new genus be established to accommodate it. The characters of the genus are presented, as are further observations on the ultrastructure of the species.

MATERIALS AND METHODS Spores obtained from N . corneurvz-infected cultures of Madin

Derby Canine Kidney cells were used to infect 5-6 week old, BALB/c athymic mice (BALB/c-nu/nu/Ola/Hsd). Mice were in- oculated intraperitoneally with 1 0’ purified N . corneurn spores in PBS and were sacrificed fourteen days postinfection. Pieces of liver were excised, transferred into 2.5% v/v glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, and cut into small cubes of approximately 1 mm3. Fixation was for 10 min at room tem- perature and for 1 h at 4” C in fresh fixative. The specimens were washed twice in the cacodylate buffer. postfixed in 1% osmium tetroxide in the 0.1 M cacodylate buffer for 1 h, rinsed with 0.1 M sodium acetate, transferred to 2% v/v aqueous uranyl acetate for 1 h, rinsed in 0.1 M sodium acetate, passed through 35% v/v and 50% v/v aqueous acetone solutions into a solution of 1% v/v phosphotungstic acid and 1% w/v uranyl acetate in 70% v/v acetone overnight, dehydrated in an acetone series and embedded in Spurr resin. Sections were mounted on copper grids and stained in 2% w/v uranyl acetate and 2% w/v lead citrate. The sections were examined using a Phillips EM300 electron microscope. Spores were measured fresh from cultures to obtain length. Width was obtained from electron micro- graphs.

RESULTS Hepatocytes were heavily infected, with the parasites occu-

pying most of the cytoplasm (Fig. 1). Stages of merogony, spo- rogony and spore maturation were randomly distributed in the cell. Hepatocytes were frequently so heavily infected that little

I To whom correspondence should be addressed.

remained of normal cytoplasm but, where present, the organelles were intact. An abundance of host ribosomes, mitochondria and endoplasmic reticulum (ER) was distributed between the par- asites. Each parasite was enveloped by a complete cisterna of rough ER, with ribosomes attached mainly to the face in contact with host cell cytoplasm (Fig. 2,3). The inner face ofthe cisterna was usually so closely applied to the parasite that it followed the contours and appeared to be a second parasite membrane (Fig. 3).

Meronts were elongate cells, often highly irregular in shape, each containing I or 2 pairs of nuclei in diplokaryotic arrange- ment (Fig. 2 , 4). The paired nuclei were closely appressed, the four membranes of the two envelopes appearing at low power as a dense band between the two nuclei. The cytoplasm con- tained free ribosomes and some rough ER. During cytoplasmic division to separate daughter cells each with a diplokaryon, the inner membrane of the ER cisterna remained tightly attached to the parasite surface, dividing with the parasite. For a while the division products were enclosed in a common vacuole, until the outer membrane of the cistema invaginated and fused be- tween the daughter meronts so that each was again enveloped by a complete ER cisterna (Fig. 4).

Meronts were often observed with “clefts” resembling those of Enteroc-vtozoon bieneusi [3]. These were identified in some cases as enlarged spaces between the two membranes of the nuclear envelope and in others as expansions of ER in the cy- toplasm. Usually the clefts were bounded by membranes with a coat of electron dense material facing the lumen. Sometimes two such thickened membranes lay on one side of the cleft with no membrane, or at most a disrupted membrane, on the op- posite face (Fig. 5, 6), as if the two membranes had remained in contact during fixation with one tearing away from its ad- jacent cytoplasm. The electron dense material resembled that of the surface coat of sporonts.

Sporogony commenced with deposition of electron dense ma- terial on the plasma membrane, adjacent to the inner membrane of the cisterna of ER. Initially the electron dense surface coat was laid down in small patches, which appeared as curved, lenticular structures in sections (Fig. 7, 8). These extended to form a uniform coat surrounding the cell.

The external cistema of ER persisted around the sporont al- though the inner membrane lost its close contact with the par- asite’s surface. Sporonts were narrower than meronts and be- came greatly elongated during the deposition of the surface coat while the number of diplokarya increased. as did the amount of ER. Prior to division, sporonts became moniliform with con- stricted areas between bulges that contained the nuclei. The maximum number of diplokarya could not be determined, but, based on the bulges and constrictions the number could have been at least eight (Fig. 9-1 1) . Division of the sporont gave rise

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SILVEIRA & CANNING- VITTAFORMA CORNEAE N. COMB. FOR NOSEMA CORNEUM 159

to chains of sporoblasts. Again, the maximum number of spo- roblasts per sporont could not be determined but was at least four and probably more.

During sporont division, the cell membrane, together with the inner membrane of the enveloping ER cisterna, invaginated to form membrane whorls which were connected to the surface, at least at first, by a narrow channel, although they often ap- peared internalised (Fig. 12). These formations, which contained electron dense deposits as well as membranes, were positioned at the points where internal membranes would form to separate the sporont into sporoblasts (Fig. 13). They clearly provided the mechanism by which the sporoblasts were produced, still en- closed in host ER.

The parasite plasma membrane and inner ER membrane later extended across the entire width o f the sporont, apparently by invagination from the whorl, to meet the membranes on the other side. At the same time electron dense material was de- posited on the two new sections of plasma membrane (Fig. 13- 15). Final separation of the sporoblasts involved invagination of the external membrane of the ER as in the meront.

Sporoblast development culminated in spore formation. The sporoblasts gradually diminished in size, became more electron dense, and their initially irregular shape first became rounded, then ellipsoid. Several sections of the developing polar tube were observed as electron dense circles when seen in transverse sec- tion, with an outer membrane enclosing a core. Membranous stacks, mainly associated with the developing polar tube, were present especially in positions posterior to the diplokaryon (Fig.

The polar tube continued its development in immature spores (Fig. 19, 20). In transverse sections 8 layers were visible around a central core. The concentric layers alternated in electron den- sity, with the dark layers being narrower than the lucent layers. In immature spores, as judged by the poor development of the endospore, the posterior 2-3 coils of the polar tube were of smaller diameter and had only 4-5 alternating layers. Up to 6 vesicles with amorphous contents, which occupied the posterior end of the spore, appeared to function as the origin of the polar tube. At this stage the spore was still enveloped by the cisterna of host ER, with the inner membrane lying close to the exospore, while the outer membrane was loose, creating a large cisternal space. The electron dense exospore was separated from the plas- ma membrane by a narrow lucent endospore (Fig. 20).

16-18).

Mature spores were elongate, measuring 3.8 2 0.75 by 1.02 & 0.25 fim (n = 25), with 5 to 7 coils of the polar tube of similar diameter (Fig. 2 1). The nuclei, still closely appressed, lay along the longitudinal axis, surrounded by regularly-arranged ribo- somes (Fig. 22). At the anterior end of the spore there was a broad polar cap composed of alternating electron dense and lucent layers, surrounding the base of the polar tube and ex- tending back as a narrow sac over the lamellae of the polaroplast. The polaroplast consisted o f closely packed membranes running parallel to the straight part of the polar tube (Fig. 23). The spore wall was composed of a slightly corrugated electron dense exo- spore, and the endospore now considerably thicker than the exospore, abutting the plasma membrane (Fig. 24). Both were surrounded by the ER cisterna. In some spores the exospore and ER cisterna layers appeared more complex, with an addi- tional, very dense layer interpolated between the exospore and the ER (Fig. 25). It was not clear whether the layer originated as a component of the exospore or of the inner membrane of the ER. Its presence was not correlated with spore maturation as it was present in only a proportion of mature spores.

At all stages of development, membrane-bound vesicles con- taining spherical electron dense granules were present in host cell cytoplasm close to the ER cisterna enveloping the parasites (Fig. 2, 7, 10, 12 curved arrows). There was some evidence that the granules were first present in the enveloping ER cisterna (Fig. 2 curved arrows), suggesting that the free vesicles were subsequently pinched off from the ER cisterna. No function can be attributed to them on the available evidence.

DISCUSSION Relationship with host endoplasmic reticulum and host cell

nuclei. The envelopment of every stage of N. corneum by a complete cisterna of ER with attached ribosomes was a constant feature in all material observed by electron microscopy. This character is not common among microsporidia but some as- sociation with host cell ER has been observed in several genera.

The meronts of Glugea anomala parasitising sticklebacks are encircled by a cisterna of host ER and the cisterna divides as the meront divides [5 ] . However, in this case the cisterna dis- perses at the onset of sporogony and sporogenesis continues inside sporophorous vesicles. A similar arrangement occurs in Loma branchialis infecting haddock [ I 51. Two microsporidia utilise the lumen of host cell ER for part of their development.

+ Fig. 1-8. Ultrastructure of N. corneum. 1. Heavily infected hepatocyte with parasites occupying most of the cytoplasm. Note host cell nucleus.

Bar = 2 pm. 2. Meront with two diplokarya. At one point (curved arrow) the enveloping ER cisterna is dilated and contains electron dense granules. Small vesicles with similar granules lie free in the host cell cytoplasm. Bar = I pm. 3. Detail of the boxed area from 2 showing the ER cisterna around 2 meronts (a, b), with ribosomes (white arrowheads) attached to the outer ER membrane of both cisternae. The inner ER membranes (black arrowheads) follow closely the contours of the parasites. Bar = 0.1 pm. 4. Final stages of meront division: the outer membrane of the cisterna of ER invaginates between the daughter meronts to complete the separation (arrowheads). The inner ER membrane has already invaginated and fused around the separate meronts. Bar = 1 pm. 5,6. Meronts with “clefts,” which are seen as enlarged spaces between the two membranes of the nuclear envelopes or as expansions of ER in the cytoplasm. Bar on 5 = 1 pm. Bar on 6 = 0.4 pm. 7. Early sporont with two visible diplokarya, on which the deposition of extramembranous coat has started (arrowheads). Note vesicles with dense granules (curved arrow) close to the ER cistema. Bar = 2 pm. 8. Detail of boxed area of 7 showing the deposition of extramembranous coat, and the close relationship of the parasite surface with the inner membrane of the host ER cistema. Bar = 0.2 pm.

Fig. 9-18. Electron micrographs of sporogony of N. corneum. 9. Bead-like sporont with incomplete surface coat prior to division: note the bulges and constrictions signifying division which will give rise to sporoblasts. The micrograph suggests that the number of resulting sporoblasts may be eight, Bar = 2 pm. 10. Sporont with 4 visible diplokarya (arrows). Vesicles with dense granules (curved arrow) lie in host cell cytoplasm. Bar = 1 pm. 11. Division into irregularly-shaped sporoblasts. Bar = 1 pm. 12. Membrane whorl formed by invagination of the cell membrane, together with the inner membrane of the ER cisterna that envelops the cell: the whorl is connected to the surface by a narrow channel (arrowhead). Vesicles with dense granules (curved arrow) lie in the host cell cytoplasm. Bar = 1 pm. 13. Membrane whorl at the point where internal membranes are forming to separate the sporont into sporoblasts. 14. Dividing sporont: note membrane whorls in position close to the transverse division planes. Bar = 0.5 pm. 15. Detail of part of 14 showing connection of membrane whorls with the surface and division planes; note that the lamellar structure and electron dense material in the lower formation (arrow) is disappearing as the division plane is completed. Bar = 2 pm. 16. Sporoblast. Bar = 0.5 pm. 17, 18. Detail of 16 showing membranous stacks closely associated with the polar tube. Bars = 0.1 pm.

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Fig. 19-25. Electron micrographs of spore morphogenesis in N. corneum. 19. Immature spore: note the vesicles with amorphous content (v). Bar = 0.3 pm. 20. Detail of part of 19 showing transverse sections of the polar tube, these have 8 layers around a central core. Some of the sections of the polar tube have a different diameter, Bar = 0.05 pm. 21. Spore, with 5-6 coils of the polar tube. Bar = 1 pm. 22. Spore diplokaryon surrounded by regularly arranged ribosomes. Bar = 0.2 pm. 23. Detail of the spore polar cap which is composed of alternating dense and lucent layers, and of the polaroplast which is composed of closely packed membranes running parallel to the straight part of the polar tube. The spore wall has become detached from the cytoplasmic structures at the left of the picture. Bar = 0.1 pm. 24. Detail of the spore wall. Electron dense exospore (ex), and endospore (en) external to the plasma membrane, and envelopment of the spore by a cisterna of host ER. Bar = 0.1 pm. 25. Spore wall showing unusual electron dense layer (ed) as well as exospore (ex), endospore (en) and cisterna of host ER. Bar = 0.1 pm.

SILVEIRA & CANNING- VITTAFORMA CORNEAE N. COMB. FOR NOSEMA CORNEUM 163

Thus, in Microgemma hepaticus, infecting mullet, the meronts appear to lie within the lumen of an expanded ER cistern but the membrane is lost at the onset of sporogony [ 171. Tetramicra brevijilum in turbot also appears to develop transiently within the lumen of host ER [ 141.

The entire development of some species of microsporidia, such as Encephalitozoon cuniculi, occurs within a parasitopho- rous vacuole (PV). The vacuole, bounded by a membrane of host origin, encloses all stages of the parasite into one micro- environment. The origin of the vacuolar membrane has not been established with certainty but even if, as suggested by Issi [lo], PV are expansions of host cell ER, the relationship of the par- asites with the ER is different in N. corneum. In N. corneum each parasite is isolated and enveloped by both layers of a cis- terna of ER, while in E. cuniculi a single membrane, the vacuolar membrane, surrounds all the individuals which have resulted from one infective sporoplasm injected by the spore into the cell. Furthermore, E. cuniculi PV are organised structures, in which merogony is peripheral and sporogonic development is in the centre.

Among the microsporidia in invertebrates, several have a partial association with host cell ER. The close association of host ER cisternae with the surface of meronts was observed in Nosema wistmansi parasitising winter moths [4], but the cis- ternae appeared to be incomplete and became disassociated from the surface as the electron dense surface coat was laid down at the beginning of sporogony. A triple-layered pellicle was described around meronts of Nosema lepocreadii parasitis- ing a fish trematode [6]. The authors interpreted these layers as the parasite plasma membrane and a cistema of parasite ER lying directly underneath the plasma membrane. However, after re-examination of the electron micrographs and comparison with sporonts, which lack the two extra membrane layers, it is more likely that the ER is of host origin and lies external to the parasite. In places it is incomplete and, like the foregoing par- asites, the parasite-associated host ER becomes disassociated at the onset of sporogony.

In the genus Endoreticulatus all stages are enveloped by a complete, flattened cisterna of host ER, the inner membrane of which lies close to the surface of meronts, while the outer mem- brane is spatially separated and ribosome-studded [ 11. Meronts, whether uninucleate and spherical or multinucleate and ribbon- like are individually encased in the ER cistema. In contrast, sporogonic division occurs within the enlarging space bounded by the two membranes of host ER and culminates in groups of spores surrounded by ER. The genus Endoreticulatus was es- tablished, by Brooks et al. [ I ] , for E. fidelis from the Colorado potato beetle. Another species, formerly Pleistophora schubergi, from the spruce bud worm, was transferred to Endoreticulatus by Cali & El Garhy [2]. Their research showed that the ER cisterna invaginates at the constrictions during cytoplasmic di- vision and separates the daughter meronts each into their own cisterna, as in N. corneum. The delayed invagination of the outer ER membrane was also observed in E. schubergi. Most recently, the name Endoreticulatus durforti has been established for a species in brine shrimps [13]. In this species “vacuoles” con- taining individual meronts appeared to fuse with larger “vac- uoles” containing sporogonic stages.

The advantages of envelopment by host ER to the microspo- ridia are not clear. The possibility exists that the ER prevents direct contact between the parasite and cytoplasm containing lysosomes and thus, may help to disguise the parasite within the cell. The abundance of ribosomes, mitochondria and ER in the host cell indicates that parasitised cells have a high metabolic rate, possibly stimulated by the parasite. This suggests another

explanation of the close relationship of parasite and host ER, i.e. that the parasite takes control of the host’s synthetic appa- ratus, first causing the nucleus to increase production of ribo- somes, to enhance protein synthesis. In turn, the ER products may be made available to the parasite for continued parasite growth, or used to enlarge the cell. Numerous small vesicles, containing dense granules and distributed in the host cell cy- toplasm, appeared to be derived from the ER cisterna envel- oping the parasites but their role in parasite development or host cell survival could not be determined. A similar accumu- lation of granules in the ER cisterna around a sporont was present in one micrograph in Shadduck et al. [ 181 (their Fig. 1 at left of the dividing sporont) but was not mentioned by them. It was not possible to determine from our material whether the granules were formed in the parasites and passed through the membranes into the ER or were secreted by the ER. The cisterna surrounding N . corneum and Endoreticulatus spp. would appear to serve similar purposes.

The effect of N. corneum on host cell nuclei is demonstrated by the abnormal, multinucleate condition of the cells in culture, as seen by light and by electron microscopy when several nuclear profiles are visible in infected hepatocytes.

Nuclei and division. In N. corneum, merogony was generally by binary fission. The presence of two diplokarya was often observed. The initiation of sporogony was signalled by the de- position of an electron dense surface coat and considerable nar- rowing of the body. The diplokaryotic condition of the nuclei was maintained throughout sporogony and sporonts with at least four diplokarya were observed. The number may be as great as eight, making N. corneum tetra- or octosporoblastic. No syn- aptonemal complexes or other sings of meiosis were observed and it was concluded that N. corneum is in dihaplophase throughout its life cycle.

Cytoplasmic “clefts” and membrane whorls. Several meronts were observed with “clefts” bordered by electron dense material resembling those of Enterocytozoon bieneusi [3]. In both mi- crosporidia the clefts were either expansions of the nuclear en- velope or of cytoplasmic ER. Cali et al. [3] proposed that the clefts may contain storage material used in the formation of the polar tube and other spore structures. The appearance of these structures as clefts in E. bieneusi was later shown to be an artifact of fixation [9] since, when femosmium was used as fixative, the clefts appeared as multilamellar structures. These phospholipid structures were believed to contribute to division of the spo- rogonial plasmodia enabling rapid formation of sporoblasts [9]. The open appearance of the “clefts” found in N. corneum may also be artifactual. The membranes of the “clefts” with their coat of electron dense material, suggest that they may also be multilamellar structures.

Structures which appear to play a part in cell division and secretion of the sporont coat of N. corneum are the membrane whorls which appear in the sporonts and are involved in the invagination of the inner ER membrane. It is possible that these are derived in part from the “clefts” of the meronts. The whorls form in places where cell cleavage is initiated and the mem- branes and dense deposits within the whorls appear to contribute to the formation of the new cell membranes and their electron dense layers, as the sporont divides into sporoblasts. The in- vaginated ER membranes serve to maintain the complete en- velopment of each parasite within its own cisterna of ER after sporogonic division. Structures resembling the invaginated membrane whorls have been described previously and named scindosomes [2 11 a name later dropped in favour of “paramural bodies” (Vivra, J. 1976. The occurrence of paramural bodies in microsporidia. Abstract. J. Euk. Microbiol., 23:2 IA.). Par-

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amural bodies were formed as whorls of membranes or oftubuli, and were thought to be involved in formation of the extramem- branous coat. The structures observed in N. corneum, appeared to have the triple role of dividing the sporont. laying down the surface coat and ensuring that the sporoblasts were enveloped in ER.

Whorls of membranes in the cytoplasm have also been ob- served in Orthosomella operoptherae [4, 71. These were occa- sionally seen in areas of constriction in multinucleate sporonts and might also have been involved in division, although the authors suggested that arrays of ER stretching between the con- stricting surfaces in dividing sporonts could have provided the membrane for division.

Taxonomic aspects. The genus Noserna is currently classified in the family Nosematidae, superfamily Nosematoidea, order Dissociodihaplophasea and class Dihaplophasea of the phylum Microspora [20]. The principal character of the class IS that nuclei are paired as diplokarya. each nucleus being haploid so that the diplokaryon is the functional equivalent of a diploid nucleus. In the order Dissociodihaplophasea, haplosis (resto- ration of the haploid phase), if it occurs, is by dissociation of the two nuclei. In the genus Noserna this dissociation is rare, most stages of the life cycle being diplokaryotic. All stages of Nosema lie in direct contact with the host cell cytoplasm without an intervening membrane of host or parasite origin and spo- rogony is disporoblastic, i.e. producing two sporoblasts and ul- timately two spores from a sporont. In the type species, Noserna bombycis, early spores responsible for spread of infection within the host and late spores, with slightly different morphology, responsible for spread between hosts, have been described [ 1 1,

N. corneum was placed in the genus Nosema on the basis of its diplokaryotic nuclei and presumed disporoblastic sporogony. The envelopment of parasite by membranes, presumed to be host ER, was observed and it was noted that this envelopment was unusual for Nosema [18]. In the present study the envel- opment of all stages by host ER was confirmed and it has been shown that sporogony is at least tetrasporoblastic, if not octospo- roblastic. No evidence was obtained for the existence of early and late spore forms as in N. bombjris but their existence cannot be ruled out completely, as cultures were only fixed for electron microscopy when infections were heavy and early spores would have been outnumbered by late spores. However, the electron dense layer at the surface of some mature spores is not consid- ered as a sign of spore dimorphism. It may represent an excess of exospore protein deposited by the parasite.

Although the present study was carried out in athymic mice. there is no reason to suspect that the development was abnor- mal. The only character in common with the genus Noserna is the diplokaryotic arrangement of the nuclei and, consequently, the species should be transferred to another existing genus or a new genus. It does not fit into the two other genera assigned to the family Nosematidae, Hirsutosporos or Issia, both of which are disporoblastic [20]. Equally, N. corneum cannot be placed in any of the genera with diplokaryotic nuclei, which have been placed in the superfamily Nosematoidea. These genera are either polysporoblastic with spherical, multinucleate sporogonial plas- modia or are disporoblastic. None are enveloped by host ER.

The combination of characters exhibited by N. comeum. namely diplokaryotic arrangement of the nuclei, all stages in- dividually surrounded b y host ER, ribbon-shaped sporonts and polysporoblastic sporogony, is not found in any established ge- nus [20]. The genera with elongated moniliform sporonts. Ame- son, Perezia and Orthosomella all develop in direct contact with host cell cytoplasm and have unpaired nuclei in sporogony and

121.

uninucleate spores. Of the genera which develop in vacuoles, Cwtosporogenes. Baculea, Encephalitozoon and Septata all have unpaired nuclei throughout development and the vacuoles are bounded by a single membrane.

Microsporidia which develop within vacuoles bounded by two membranes are Buxtehudea, Merocinta and Endoreticula- tus. The early stages of development of Buxtehudea are un- known but sporogony is polysporoblastic, by division of a plas- modium into 50-100 sporoblasts. The genus Merocinta is dimorphic in its host, Mansonia africana. with a different cycle in adult and larval stages [ 161. In its larval host, multinucleate diplokaryotic meronts are individually surrounded by host ER but in sporogony, the nuclei separate and the division of the multinucleate sporogonial plasmodium into sporoblasts takes place in a common vacuole bounded by ER. Endoreticulatus is similar in its relationship with ER to the sporogonic cycle of Merocinta except that all stages have unpaired nuclei. Although Endorericulatus and N. corneuin do not appear to be related on morphological grounds because the former has unpaired nuclei and the latter is diplokaryotic, data from sequencing the 16s rRNA indicate that they have a closer relationship to each other than either has to Nosema (Baker, M. D.. Vossbrinck, C. R., Didier. E. S. & Shadduck, J. A., pers. commun.).

Since no established genus has the unique combination of characters exhibited by N. corneum, the species cannot be trans- ferred from Nosema to an existing genus and thus a new genus should be established for it. The name Vittaforma is proposed, derived from the Latin Vitta (a band, fillet) and forrna (shape), referring to the ribbon or band-like sporonts. Furthermore, as the specific name is derived from cornea, itself a feminine de- rivative of a Latin noun, the feminine form corneae, meaning “of the cornea” is more correct. The species which most closely resembles V. corneae is N. lepocreadii which is also diplokaryotic and produces chains of sporoblasts from a beaded sporont. How- ever, although the meronts of N. lepocreadii are probably sur- rounded by host ER, the cisterna of ER disappears in sporogony. It is likely that N. lepocreadii, parasitising a trematode in fish, would have diverged at an early stage from the line leading to N . corneum parasitising mammals. It is unlikely that they could be placed in the same genus.

Taxonomic summary. L’ittaforma n. g. Nuclei in diplokar- yotic arrangement throughout the life cycle. All stages individ- ually enveloped by a cisterna of host endoplasmic reticulum studded with ribosomes. Merogony by binary fission. Sporogony polysporoblastic utilising paramural bodies. gives rise to 4-8 linearly arranged sporoblasts.

Vittaforma corneae (Shadduck et al. [IS]) as in original de- scription, except that mean spore measurements 3.8 x 1.2 pm (present data) (versus 3.7 x 1.0 pm given by Shadduck et al.) and the number of coils of the polar tube 5-7 (present data) (versus 5-6 Shadduck et al.)

The authors refrain from suggesting a placement of Vittaforma into higher taxa because the data on sequences of the 16s RNA indicate that many changes may be necessary to the present taxonomic systems which are based on morphology.

ACKNOWLEDGMENTS The authors wish to thank Dr. E. S. Didier and Dr. J. A.

Shadduck for the supply of N. corneum culture. H. Silveira was supported by a scholarship from JNICT, Portugal and E. U. Canning by the Wellcome Trust. We are pleased also to ac- knowledge the Centro de Malaria e Outras Doenqas Tropicais, Universidade Nova de Lisboa for assistance with the photo- graphs.

SILVEIRA & CANNING- VITTAFORMA CORNEAE N. COMB. FOR NOSEMA CORNECJM 165

LITERATURE CITED 1. Brooks, W. M., Becnel, J. J. & Kennedy, G. G. 1988. Establish-

ment of Endoreticulatus n. g. for Pleistophora fidelis (Hostounsk? & Weiser, 1975) (Microsporida: Pleistophoridae) based on the ultrastruc- ture of a microsporidium in the Colorado potato beetle, Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae). J. Protozool., 3548 1- 488.

2. Cali, A. & El Garhy, M. 1991. Ultrastructural study of the de- velopment of Pleistophora schubergi Zwolfer, 1927 (Protozoa, Micro- sporida) in larvae of the spruce budworm, Choristoneura fumiferana and its subsequent taxonomic change to the genus Endoreticulatus. J. Protozool., 38: 21 1-27 8.

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Received 5-23-94; accepted 10-3-94