key: cord-0010700-1sqdqrnm
authors: MOORE, CLYDE B.; BROOKS, WAYNE M.
title: An Ultrastructural Study of Vairimorpha necatrix (Microspora, Microsporida) with Particular Reference to Episporontal Inclusions During Octosporogony
date: 2007-05-01
journal: J Protozool
DOI: 10.1111/j.1550-7408.1992.tb01469.x
sha: f529e56e957f6697c991a7b4290295bd300a1f9e
doc_id: 10700
cord_uid: 1sqdqrnm

ABSTRACT. The life cycle of Vairimorpha necatrix was studied by electron microscopy. Disporous development has two distinct stages: 1) diplokaryotic meronts which are actively mitotic, and 2) diplokaryotic sporonts which are distinguished by reduced ribosome density and a thickened plasmalemma. After final division of the sporont, sporoblasts form spores which are ovocylindrical and measure 4.4 ± 0.08 × 2.3 ± 0.05 μm (mean ± SE). Octosporous development results in eight haploid spores being formed in a sporophorous vesicle. The uninucleate octospores were smaller than the binucleate dispores and the exospore was thicker but less crenulate in outline. Early in octosporogony, tubules are produced from the sporont plasmalemma and electron‐dense material accumulates in the episporontal space. The latter may be amorphous, vesiculated, or vacuolated in appearance and in later stages may take a stacked, lamellar form. At sporoblast formation, exospore material coats the plasmalemma and attached tubules; all inclusions in the episporontal space gradually disappear as spores are formed. These secretory products may have application to taxonomic distinction at the species level.

T the dimorphic microsporidium Vairimorpha necatrix (Kramer, 1965) as the type species. This microsporidium was first reported by Tanada and Chang [I61 as a mixture of two separate species which were subsequently described by Kramer [8] as Noserna necatrix and Thelohania diazoma. In his intensive study of these isolates, Maddox (Maddox, J. V. 1966 . Studies on Microsporidiosis of the Armyworm Pseudaletia unipuncta (Haworth) . Ph.D. Thesis, Univ. of Illinois, Urbana) indicated the possibility that the two species may only be physiological variants of the same species, a conclusion supported by subsequent studies [2, 1 I] .

In establishing the genus Vairirnorpha, Pilley [ 131 redescribed the developmental stages based on Giemsa-stained preparations. The genus was characterized primarily by a dominant developmental cycle involving binary fission and disporoblastic sporogony and an additional cycle involving multiple fission and octosporoblastic sporogony which occurs primarily at low temperatures. Since her work, several additional dimorphic species have been placed in this genus, including V. plodiae (Kellen & Lindegren, 1968 ), V. heterospomrn (Kellen & Lindegren, 1969) , V. ephestiae (Mattes, 1927) , V. hybomitrae (Levchenko & Issi, 1973) , V. invictae Jouvenaz & Ellis, 1986 , Vuirirnorpha antheraeae Yefimenko, 1987 , and an isolate referred to as Vairimorpha sp. originally discovered by J. J. Hamm from Bolivia [3] .

In addition, as noted by Pilley [13] , the occurrence of such dimorphic species exhibiting both apansporoblastic and pansporoblastic development undermines the separation of the order Microsporida into the two suborders Apansporoblastina and Pansporoblastina [ 171.

Larsson [9, I01 addressed the need to use ultrastructural cytology in defining microsporidian taxa and in developing systems for their classification. However, in previous comparative studies of various species of Vairimorpha, biochemical and immunological techniques have been used primarily to distinguish between species (see review in [6] ). Observations on ultrastructural cytology have been limited to V. plodiae [ 121, V. ephestiae [ 181, and V. invictae [7] . The lack of published observations on the ultrastructural features of the type species, V. necatrix, seriously limits our ability to make comparative Observations among species in the genus Vairimorpha. Such data would provide a more definitive generic characterization based on ultra-' To whom correspondence should be addressed. structural cytology. It is to these ends that the present study on the ultrastructural cytology of F' . necatrix was undertaken.

A sample of V. necatrix spores was received from Dr. J. V.

Maddox of the Illinois Natural History Survey. The spore suspension (50 fil of 1 x lo8 spores/ml) was given per 0s to 4-dayold larvae of Helicoverpa zea from a colony maintained at the North Carolina State University insectary. The larvae were maintained on a synthetic diet [ 11. Cohorts ofthe exposed larvae were kept in environmental chambers (14:lO L:D) at both 25" C and 20" C. Samples of primarily adipose tissue were taken from larvae at 1, 3, 7, 10, 18 and 24 days post exposure. Tissues were fixed in 2.5% cacodylate buffered glutaraldehyde (pH 7.4) overnight at 4" C. Samples were rinsed three times in 0.1 M cacodylate buffer (pH 7.4), and then post-fixed in 1% OsO, in 0.1 M cacodylate buffer for 2 h. They were rinsed three times, 15 min each, in distilled water and dehydrated through a graded ethanol series. Propylene oxide was used as the transition solvent to embedment in Spurr's low viscosity resin (Ladd Research Industries, Inc., Burlington, VT).

Thin sections, 60-90 nm, were cut on a Reichert OM-2 ultramicrotome. The sections were stained using 5% (w/v) uranyl acetate in 50% (v/v) methanol followed by lead citrate [14] . Sections were examined with a Philips 300 transmission electron microscope at 80 kV.

Meronts were relatively undifferentiated cells containing a single diplokaryon and lay directly in the host cell cytoplasm. They were roughly spherical to elliptical and ranged in size from 1.5 to 3.2 pm. The cytoplasm was evenly dense with ribosomes and contained a few cisternae of rough endoplasmic reticulum (RER) (Fig. 1) . Host cell mitochondria were often closely associated with the parasite's plasmalemma. Spindle plaques and microtubules were frequently observed, indicating active mitosis in this stage. In some cases rapid mitotic division resulted in chain-like forms where cytokinesis was delayed. These appeared to be late stage meronts or possibly presporonts as indicated by an increase in cisternae of RER and a slightly thickened plasmalemma (Fig. 2) .

Early stage sporonts were identified by their less dense cytoplasm. The plasmalemma also became thickened in areas as electron-dense material was secreted to the exterior eventually forming a continuous layer over the entire surface of the sporont (Fig. 3) . The nuclei replicated and at least one additional mitotic division appeared to take place (Fig. 4) . Cisternae of RER continued to increase and primordial structures of the mature spore began to appear in sporoblasts (Fig. 5) . Anteriorly, the anchoring disc of the polar filament formed and the isofilar polar tubule projected posterolaterally, coiling on the average 13 turns (range 12-14) around the centrically located diplokaryon (Fig. 6 ). The polar filament in cross-section was composed of six concentric layers, alternately electron lucent and dense, with an inner core ( Fig. 6 , inset). The diameter of the polar filament was 76 ? 0.2 nm (mean k SE). The polaroplast appeared as petal-like membranous folds surrounding the basal portion of the polar filament. Posteriorly, a vacuole was usually present and frequently a Golgi apparatus was apparent.

Fresh spores were ovocylindrical, slightly pointed anteriorly Polaroplast lamellae are evident anteriorly. x 18,000. Inset: enlargement of polar filament in cross section. x 70,000. and measured 4.4 k 0.08 x 2.3 ? 0.05 pm. The outermost layer of the spore coat, the exospore, was electron dense, 24 nm thick, and appeared crenulate (Fig. 6) . The electron-lucent endospore layer was uniformly 160 nm thick except anteriorly where the anchoring disc was attached.

While stages in octosporogony were occasionally seen in material which had been held at 25" C, these stages were most abundant in material held at 20" C. Early octosporonts were similar in appearance to the diplokaryotic sporonts where the cytoplasm was less dense and cisternae of RER became more abundant (Fig. 7) . The principal distinction was the formation of a membrane-like structure exterior to the plasmalemma that became the envelope of the sporophorous vesicle (SV). The envelope appeared as blebs at various places on the plasmalemma and electron-dense material was secreted into the episporontal space (ES) between the envelope and the sporont plasmalemma (Fig. 7) . As shown when examined in a favorable plane of section (Fig. 8, 9 ), these early octosporonts were binucleate. Separation of the two nuclei of the diplokaryon prior to meiosis is apparent in Fig. 8 and 9 , but synaptonemal complexes indicating meiotic activity were not observed. Following this stage, nuclear division and cytokinesis gave rise to tetraand octonucleate sporonts. Spindle plaques with microtubules radiating toward electron-dense chromatin masses, probably synapsed chromosomes, were observed in dividing nuclei (Fig. The cytoplasm of the early stage sporonts was observed containing material in vacuoles similar to the electron-dense material secreted into the ES (Fig. 9, I I) . This secretory material 10). ranged from being uniformly electron dense to more-or-less vacuolated in appearance (Fig. 8-11 ). In some sporonts the electron-dense material appeared to organize into lamellar structures (Fig. 12, 13 ) 21 to 34 nm (27 k 0.01) thick. In more advanced stages of octosporogony (Fig. 14) , the lamellae either disappeared or reorganized into the electron-dense amorphous masses of secretory material. Alternatively, this material may not always organize into lamellae and may persist in the ES during sporoblast formation. Tubular structures, 2 3 4 9 nm (36 f 0.8) in diameter, also occurred in the ES primarily in the space between the sporont and the secretory material ( Fig. 10-12) ; in some sections, they appeared to have been produced from or attached to the sporont plasmalemma (Fig. 15 ). The faintly staining tubules were usually closely associated with the secretory material but separate groups of tubules often occurred apart from this material. The tubules also occurred in sporonts exhibiting the stacked lamellae and usually persisted in the ES during sporoblast formation (Fig.  13) . Sporoblast development was marked by an increasingly electron-dense and thickened layer of exospore material on the sporont plasmalemma (Fig. 13-1 6) . As the exospore was deposited on the surface of the plasmalemma, it also coated the basal regions of the tubules still attached to the sporont plasmalemma (Fig. 16) . Tubules and secretory material gradually disappeared with sporoblast formation and spore maturation (Fig. 17, 18 ).

Relatively few of the mature spores formed during octosporogony were well fixed a n d o r infiltrated with embedment. Many of the octospores appeared to be aberrant and internal detail Fig. 7-10 . Electron micrographs of early stages in octosporogony of V. necutrix. 7. Initial octosporont within a sporophorous vesicle. Note presence of secretory material in the episporontal space between the sporont and the envelope of the sporophorous vesicle. x 15,000.8. Binucleate sporont. Note area of apposition between the paired nuclei of the diplokaryon. x 16,500. 9. A sporont whose nuclei are beginning to separate prior to meiosis. Note tubules (T) and amorphous masses of secretory material (arrow) in the episporontal space. Vacuoles (arrowheads) also are present in the cytoplasm which contain material similar to the electron-dense secretory masses that occur in the episporontal space. x 16,500. 10. Sporont undergoing division. Note spindle plaque (sp) with microtubules radiating toward chromatin masses. x 19,000.

was best preserved in immature spores. The polar filament was isofilar, exhibited 12 to 13 coils (Fig. 18) and in cross section was similar to that of the diplokaryotic spores, measuring 76 k 0.9 nm in diameter. An exospore layer overlaid the much thicker endospore. Fresh spores measured 3.8 ? 0.04 x 2.1 k 0.03 Ccm.

The life cycle of V. necatrix does not appear to differ significantly from that described by Pilley [ 131 or for V. plodiue [ 121. Meronts are typically undifferentiated as reported for many microsporidia and are mitotically active. A principal feature of Fig. 11-15 . Electron micrographs of sporogonic stages of V. necatrix. 11. Sporont with extensive mass of tubules (arrow) between the sporont plasmalemma and the masses of amorphous secretory material in the episporontal space. Note the secretory material is vesiculate in appearance. Vacuoles (arrowheads) containing apparent secretory material can also be seen in the sporont cytoplasm. x 19,800. 12,13. Appearance of stacked lamellae (L) in the episporontal space of sporont along with tubules (T). x 18,700. 14. Advanced sporont with six sporoblasts evident in the plane of section. Note greatly thickened plasmalemma of the sporoblasts, presence of polar filament primordia (PT) and Golgi apparatus (G), and reduced presence of tubules. x 16,500. 15. Attachment of tubules to sporont plasmalemma. ~62,000. meronts and disporogonial stages is the presence of diplokarya.

While cells often appear uninucleate in electron micrographs, this is most likely due to the plane of section. Likewise, daughter meronts are more often encountered with one in the plane of section and the second in transverse section. This and micrographs of early cleavage indicate that division is at an obtuse angle, although longitudinal division is seen in some cases. In both situations, mirror-image symmetry is the usual condition. Although no direct evidence was obtained, the early stages of octosporogony are believed to be meiotic. Synaptonemal com- plexes were found in early octosporonts of V. plodiae by Malone and Canning [ 121, and they have also been described in species of the dimorphic genera Amblyospora and Parathelohania [4, 51. In V. necatrix we did not observe karyogamy, but diplokaryotic sporonts and sporonts whose nuclei were undergoing disassociation prior to meiosis were observed (Fig. 8, 9 ). Better preservation of structures in such nuclei might have allowed resolution of the synaptonemal complexes.

Various types of inclusions occur within the ES during octosporogony ( Fig. 8-1 8) . Working with a Glugea sp., Takvorian and Cali [ 151 identified three types of tubular appendages continuous with the sporont's plasmalemma. Larsson [9] has reviewed other types of inclusions described in the ES of other species. In V. necatrix, Type I1 tubules of Takvorian and Cali's classification were observed. These clearly emanate from the plasmalemma of the sporont (Fig. 15 ) and are most abundant in early stages of octosporogony ( Fig. 9-13 ). Malone and Canning [ 121 described these as being parallel to the plasmalemma. In our study, tubules were most frequently seen in close association with the plasmalemma but not necessarily parallel. Frequently the tubules extended further into the ES with little apparent organization. Although these tubules have mostly disappeared in late octosporogonic stages, tubules still attached to the sporont plasmalemma when exospore material is deposited externally on developing sporoblasts are also coated basally (Fig. 16) .

The tubules are usually present and may be closely associated with the amorphous secretory material which accumulates early in the ES (Fig. 11-13) . The electron-dense material occurs ini-tially in small deposits between the sporont plasmalemma and the newly formed envelope of the sporophorous vesicle in early octosporonts (Fig. 9, 11 ). As the tubules appear subsequently, it seems unlikely that the tubules play a role in the formation of the amorphous secretory material. Rather, this material is probably formed within the sporont itself. Similar electron-dense material occumng within membrane-bound vacuoles is common in the cytoplasm of the sporonts (Fig. 8, 9, 11) and is probably released by the process of exocytosis.

The amorphous secretory material is usually electron dense and may become vacuolated as well as vesiculate in appearance (Fig. 11) . At some point during sporogony, this material may organize into lamellae with a stacked-layered appearance (Fig.  12, 13) . However, as deposits of the amorphous material occur frequently in late stage octosporonts (Fig. 14) , either the material does not always organize into lamellae or it changes back into its original nature before gradually disappearing along with the tubules during spore formation.

Takvorian and Cali [ 151 suggested that tubules (appendages) such as these be avoided as taxonomically significant features until more study has been completed. We feel that these may be useful in taxonomy, especially at the species level. For example, they are the one morphological difference which can be used to distinguish between V. necatrix and V. plodiae. species which are otherwise difficult to separate by ultrastructure. The SV inclusions, which are amorphous, and the tubules appear identical in each of these species, although the tubules of V. necatrix do not appear as tightly associated with the plasmalemma as do those of V. plodiae. However, the electron-dense lamellar structures are the most significantly different feature between these two species. In V. plodiae they make "intricate layered configuration(s)" which at the peak of development make fingerprint-like whorls in and around themselves. In V. necatrix, the lamellae are more parallel with a stacked membranous appearance (Fig. 13) . In Larsson's [9] work on ultrastructural cytology, they would require some expansion of the Type 5E in his description of inclusions in sporophorous vesicles.

The potential taxonomic value of inclusions in the ES is also shown when those occumng in V. necatrix are compared with those produced by V. ephestiae as described by Weiser and Pumni [ 181. In their study, the electron-dense secretory material also transforms into membranous layers similar in configuration to those of V. necatrix. The Type I1 tubules of Takvorian and Cali [I51 also are present with both inclusions disappearing during late stages of octosporogony. However, in their study each lamella was identified as having a well-organized central core and two identical surface layers, details not ascertained in V. necatrix. While Weiser and Pumni [ 181 made no attempt to differentiate their species from any of the other described species of Vairimorpha, the only significant difference other than that noted above was in the number of coils of the polar filament, apparently 16, of the binucleate spore, although they cite 14 coils in the summary of their paper for both types of spores. They also described stages involved in both disporogony and octosporogony as developing within "parasitophorous vacuoles" of host cell origin; however, this observation needs reinterpretation, especially since their figure of a diplokaryotic sporont (Fig. 37 in [ 181) in a "parasitophorous vesicle" complete with tubules and masses of electron-dense secretory material is more likely an early stage in octosporogony prior to meiosis similar to the stage shown in Fig. 8 and 9 in V. necatrix. Thus, while the two species appear to be similar ultrastructurally, further study will be required to determine if V. ephestiae might be conspecific with V. necatrix.

Laboratory techniques for rearing Heliothis species on artificial medium

Spore dimorphism in a m icrosporidian isolate

Interaction of the microsporidium Vairimorpha sp. with Microplitis croceipes (Cresson) and Cotesia marginiventris (Cresson) (Hymenoptera: Braconidae), two parasitoids of Heliothis zea (Boddie) (Lepidoptera: Noctuidae)

Meiosis and its implications in the life cycle of Amblyospora and Parathelohania (Microspora)

Karyogamy and meiosis in an Amblyospora sp. (Microsporida) in the mosquito Culex salinarius

Immunoblot analysis of exospore polypeptides from some entomophilic microsporidia

Vairimorpha invictae n. sp. (Microspora: Microsporida), a parasite of the red imported fire ant, Solenopsis invictae Buren (Hymenoptera: Formicidae)

Nosema necatrix sp. n. and Thelohania diazoma sp. n., microsporidians from the armyworm, Pseudaletia unipuncta (Haworth)

Larsson, R. 1988. Identification of microsporidian genera (Protozoa, Microspora). A guide with comments on the taxonomy

Vairimorpha (Protozoa: Microsporida) for Nosema necatrix Kramer 1965: pathogenicity and life cycle in Spodoptera exempta (Lepidoptera: Noctuidae)

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Light-and electron microscopic studies on the microsporidian, Vairimorpha ephestiae (Mattes) (Protozoa, Microsporida) in the meal moth, Ephestia kiihniella. Arch. Protistenk

bamer-housed), the rats tested at 10 wk PI were all reactive to the rat coronavirus (313 RCV) in addition to 3/3 for PVM. Therefore, the fourth column