Characterizing the Xenoma of Vairimorpha necatrix Provides Insights Into the Most Efficient Mode of Microsporidian Proliferation

Abstract

Microsporidia are a group of obligated intracellular parasites that can infect nearly all vertebrates and invertebrates, including humans and economic animals. Microsporidian Vairimorpha necatrix is a natural pathogen of multiple insects and can massively proliferate by making tumor-like xenoma in host tissue. However, little is known about the subcellular structures of this xenoma and the proliferation features of the pathogens inside. Here, we characterized the V. necatrix xenoma produced in muscle cells of silkworm midgut. In result, the whitish xenoma was initially observed on the 12^th day post infection on the outer surface of the midgut and later became larger and numerous. The observation by scanning electronic microscopy showed that the xenoma is mostly elliptical and spindle with dense pathogen-containing protrusions and spores on the surface, which were likely shedding off the xenoma through exocytosis and could be an infection source of other tissues. Demonstrated with transmission electron microscopy and fluorescent staining, the xenoma was enveloped by a monolayer membrane, and full of vesicle structures, mitochondria, and endoplasmic reticulum around parasites in development, suggesting that high level of energy and nutrients were produced to support the massive proliferation of the parasites. Multiple hypertrophic nuclei were found in one single xenoma, indicating that the cyst was probably formed by fusion of multiple muscle cells. Observed by fluorescence in situ hybridization, pathogens in the xenoma were in merongony, sporogony, and octosporogony, and mature stages. And mature spores were pushed to the center while vegetative pathogens were in the surface layer of the xenoma. The V. necatrix meront usually contained two to three nuclei, and sporont contained two nuclei and was wrapped by a thick membrane with high electron density. The V. necatrix sporogony produces two types of spores, the ordinary dikaryotic spore and unicellular octospores, the latter of which were smaller in size and packed in a sporophorous vesicle. In summary, V. necatrix xenoma is a specialized cyst likely formed by fusion of multiple muscle cells and provides high concentration of energy and nutrients with increased number of mitochondria and endoplasmic reticulum for the massive proliferation of pathogens inside.

Keywords: Vairimorpha necatrix; microsporidia; proliferation; subcellular structure; xenoma.

Figure 1

The development of xenoma on the midgut of silkworm infected by V. necatrix BM. (A) The silkworm midgut from 5 to 16 dpi. The xenomas (arrowhead) could be observed after 12 dpi and were obvious after 13 dpi. The midgut was surround by massive xenomas after 16 dpi; (B) The infected silkworm larva was dissected in the 5^th instar; (C) Massive whitish xenomas (arrowhead) were shown on the outer surface of the infected midgut; (D, E) The V. necatrix BM in a xenoma produced a large number of meiospores (octospores) contained within a sporophorous vesicle (arrowhead). The bar indicates 10 µm.

Figure 2

FISH and DAPI staining of V. necatrix BM in xenoma. (A) The parasite purity was verified by FISH with a probe of V. necatrix ribosomal RNA (red) and IFA using an antibody against N. bombycis (green) in infected BmE cells, respectively. Bar, 10 µm. (B) The nucleus of V. necatrix BM in all stages was stained with DAPI (blue). The parasites in development were labeled using FISH with a probe of the ribosomal RNA (red). Red arrowhead, meront; Cyan arrowhead, sporont; Yellow arrowhead, mature spore; Green arrowhead, empty (germinated) spore; Bar, 5 µm.

Figure 3

The xenoma observed by SEM. (A, B) The elliptical and spindle intact xenoma. (C, D) The enlarged graph of the dotted box in panels (A, B, E) The mature spores (arrowhead) adhering to the surface of the xenoma, the bar is 50 µm. (F) The mature spores inlaid on the surface of the xenoma, the bar is 10 µm. (G, H) The mature spores embedded in a transverse xenoma. Bar, 10 µm.

Figure 4

The observation of xenoma nucleus. The nucleus (arrowhead) of xenoma and V. necatrix BM were stained with DAPI (blue). The hypertrophied nucleus was labeled with a dashed line. Bar, 10 µm.

Figure 5

The observation of xenoma mitochondria and endoplasmic reticulum (ER). (A) The xenoma was stained with Mito-Tracker Red for labeling mitochondria (red) and DAPI for dying nucleus (blue). (B) The xenoma was stained with ER-Tracker Red for labeling ER (red) and DAPI. The nucleus of meronts (red arrowhead) and spores (yellow arrowhead) were stained with DAPI (blue). Bar, 5 µm.

Figure 6

The xenoma observed by TEM. (A) The outer wall of xenoma (arrowhead) and V. necatrix BM inside. (B, C) The magnified outer wall of the xenoma. (D–F) The development of V. necatrix BM in xenoma. The arrowhead indicates the nuclei of the parasites.