Microsporidian infection in a free-living marine nematode

Abstract

Microsporidia are unicellular fungi that are obligate endoparasites. Although nematodes are one of the most abundant and diverse animal groups, the only confirmed report of microsporidian infection was that of the "nematode killer" (Nematocida parisii). N. parisii was isolated from a wild Caenorhabditis sp. and causes an acute and lethal intestinal infection in a lab strain of Caenorhabditis elegans. We set out to characterize a microsporidian infection in a wild nematode to determine whether the infection pattern of N. parisii in the lab is typical of microsporidian infections in nematodes. We describe a novel microsporidian species named Sporanauta perivermis (marine spore of roundworms) and characterize its infection in its natural host, the free-living marine nematode Odontophora rectangula. S. perivermis is not closely related to N. parisii and differs strikingly in all aspects of infection. Examination by transmission electron microscopy (TEM) revealed that the infection was localized in the hypodermal and muscle tissues only and did not involve the intestines. Fluorescent in situ hybridization (FISH) confirmed infection in the muscle and hypodermis, and surprisingly, it also revealed that the parasite infects O. rectangula eggs, suggesting a vertical mode of transmission. Our observations highlight the importance of studying parasites in their natural hosts and indicate that not all nematode-infecting microsporidia are "nematode killers"; instead, microsporidiosis can be more versatile and chronic in the wild.

Fig 1

Phylogenetic placement of the novel microsporidium, S. perivermis. The tree shown was obtained by Bayesian inference using microsporidian SSU rDNA sequences. The same tree topology was obtained by maximum likelihood. Support values from Bayesian inference, maximum likelihood bootstrapping, and the approximate likelihood ratio test Shimodaira-Hasegawa-like procedure (BI/MLB/aLRT-SH) are indicated in the figure. Support values below 50% are not shown. Other nematode-infecting microsporidia are denoted by an asterisk. Bar at the bottom indicates estimated number of changes per site.

Fig 2

Ultrastructure of S. perivermis spores by TEM. (A) Cross section of an infected O. rectangula nematode showing the localization of S. perivermis spores (S) in the hypodermal tissues (H). Note the absence of spores in the lumen (L) and intestinal (In) tissues. Localization of the spores in the muscle tissues (M) is also shown. Bar = 0.5 μm. (B) S. perivermis spores surrounded by muscle fibers (M). Bar = 2 μm. (C) S. perivermis spore showing the four coils of its polar filament (Pf), the exospore (Ex), the endospore (En), the polaroplast membranes (Pm), and a vesicle (V) enclosing the spore. Bar = 0.5 μm. (D) S. perivermis spores showing four coils of the polar filament (Pf), the anchoring disk (Ad), and the polaroplast membranes (Pm). Bar = 0.5 μm.

Fig 3

Localization of S. perivermis infecting the free-living marine nematode O. rectangula by fluorescent in situ hybridization (FISH). The columns show cross sections of nematode tissues at sequential depths, moving from interior to exterior (left to right) to show S. perivermis surrounding the intestine and localized to hypodermal and muscle tissues (long white arrows). Note the absence of S. perivermis in the intestine, whose position is clearly marked by the concentric rings of DAPI-stained nematode nuclei in the leftmost column. Each row shows a different light filter showing the nuclear stain DAPI (shown in blue), the S. perivermis fluorescent probe (Cy3 [shown in red]), a combination of the two dyes, and the view of the image under phase contrast. Bars = 10 μm.

Fig 4

S. perivermis infection in O. rectangula eggs by FISH. Each column shows a sequence of images taken on the same plane to show the localization of S. perivermis inside eggs. Each row shows a different light filter: the nuclear stain DAPI (shown in blue), the S. perivermis fluorescent probe (Cy3 [shown in red]), a combination of the two dyes, and the view of the image under phase contrast. Bars = 10 μm.