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Ronny Larsson

Cytology and taxonomy
of the microsporidia

Project description

Spores from the microsporidium Amphiacantha longa. This species is a hyperparasite living inside the cells of a gregarine which, in turn, is a parasite of a polychaete worm. A part of a gregarine cell is seen above containing spherical spores inside elongated sporophorous vesicles. Spores are also seen outside the vesicles signifying that A. longa has two different sporulation pathways.

Nearly thirty years ago a protistology laboratory was established in Lund. From the beginning the activity was focused on parasites, especially microsporidia. Publications have treated, in addition to microsporidia, amoebae, coccidia, eugregarines, neogregarines, haplosporidia and myxosporidia, and there are also a few publications on non-protistological groups like viruses and rickettsiae. The laboratory has built a large reference collection of parasites, which includes donations from foreign colleagues.
 

Microsporidia

Living spores of the microsporidium Glugea anomala.

The microsporidia constitute a phylum of spore-forming unicellular parasites. The about 1 500 named species known today are only a minor fraction of the real number, probably exceeding one million, and new species are described in rapid succession. Microsporidia are restricted to animal hosts, and all major groups of animals host microsporidia. They are especially common and important parasites of insects, crustaceans and fish. Approximately 10 per cent of the species are parasites of vertebrates, including ten species in man.

A xenoma formed by the microsporidium Glugea anomala in a stickleback.

Many species of microsporidia appear to be host specific. They influence their hosts in various ways and all organs and tissues are invaded. Some species are lethal, and a few are used in biological control of insect pests. Parasitic castration, gigantism, change of host sex are effects of microsporidian parasitism. In the most advanced cases of parasitism the microsporidium rules the host cell completely and controls its metabolism and reproduction (forming a xenoma, see image above).
 

Characteristics of microsporidia

The spore is the only life cycle stage of a microsporidium with distinct characteristics. Spores of most species are oval or pyriform, but rod-shaped or spherical spores are not unusual, and a few genera produce spores of unique shape for the genus. 

Microsporidian spores, click here for a magnified image (168 K). A-B Alfvenia nuda; C-D Episeptum inversum; E-F Napamichum dispersus; G-H Bohuslavia asterias; I Amblyospora sp; K Amblyospora callosa; L Toxoglugea variabilis; M Helmichia aggregata; N-O Cougourdella polycentropi; P Bacillidium sp; Q Resiomeria odonatae.

Living spores of most species measure 1 to 5 micrometer, but a small number of species produce large rod-shaped spores, up to 40 micrometer long. The spore wall is a thick protecting cover, which enables the spore to survive for several years in nature. Three wall layers are distinct: an external electron-dense exospore, a median, wide and seemingly structureless endospore, containing chitin, and an internal plasma membrane. The structure of a spore is shown in the micrograph below. 

Episeptum inversum, longitudinally sectioned microsporidian spore, click here for a magnified image (211 K). A anchoring disc; EN endospore; EX exospore; MB membranes; N nucleus; P polar sac; PA anterior part of polaroplast; PF polar filament; PL plasma membrane; PP posterior part of polaroplast; R ribosomes; RU rough endoplasmic reticulum; S septum in the exospore; V posterior vacuole; * outer layer of the exospore. Transmission electron micrograph.

The spore is a single cell with a central nucleus or with two coupled nuclei (diplokaryon). It is equipped with a unique infection apparatus composed of three components. The anterior half of the spore is occupied by a system of lamellae or sacs delimited by unit membranes (the polaroplast). The polaroplast surrounds the anterior part of a long thread-like organelle (the polar filament) which is attached to an anchoring apparatus at the anterior pole of the spore. The anterior part of filament proceeds straight backwards through the centre of the spore, while the posterior part usually is coiled up in one, or sometimes more, layers of coils in the posterior half of the spore. Transverse sections through the coils reveal a telescopic construction with concentric layers of different electron density. Externally the filament is covered by a unit membrane continuous with the membrane component of the polaroplast. The width of the filament is either uniform (isofilar filament) or the anterior part of the filament is wider (anisofilar filament). The third component of the extrusion apparatus is a vacuole, a membrane-lined cavity, at the posterior pole of the spore.
 

Infection, presporal stages and life cycle

The host is normally infected by food or drink, but in addition vertical transmission occurs regularly in some genera. In the gut of the specific host, the spore is activated and the polar filament is ejected. The material of the filament is rearranged to form a tube which functions as a hypodermic needle and penetrates the gut epithelium. The nucleus (or nuclei) and the cytoplasm aquires a new plasma membrane from the membrane system of the polaroplast, and the cell formed in this way, the sporoplasm, is injected safely into the host cell. The plasma membrane of the spore remains as the internal wall layer of the empty spore. The sporoplasm either remains as a parasite of the gut epithelium, or proceeds to deeper tissues. See the two images below.

Nosema tractabile, microsporidian spores with ejected polar filaments. Photo micrograph.

Nosema tractabile, microsporidian spore with ejected polar filament transformed into a tube. The diplokaryotic sporoplasm is visible outside the tube. Scanning electron micrograph.

Depending on microsporidian species, the sporoplasm divides by binary or multiple fission. A phase of vegetative reproduction starts as soon as the parasite has entered the host cell. Normally the vegetative reproduction is directly followed by a sporogony by the same mode of division as in the earlier phase. Most diplokaryotic species conserve their diplokarya in all stages of the life cycle. However, there are also genera that are diplokaryotic in the vegetative reproduction, but shift to isolated nuclei in the sporogony. These microsporidia have a sexual process at the beginning of sporogony, with nuclear fusion followed by meiosis. Sexually reproducing microsporidia might have a life cycle with alternate hosts.
 

Cells of unusual cytology

The microsporidia express an unusual cytology and they develop in intimate association with the components of the host cell. For example Chytridiopsis and related genera sporulate in an invagination of the host nucleus, see image below. 

Chytridiopsis trichopterae, microsporidian sporogony inside an invagination of the host nucleus, click here for a magnified image (171 K). Transmission electron microscopy.

As all microsporidia lack mitochondria in all life cycle stages, they are completely dependent on the host cell for the supply of energy. The mitochondria of the host cell can aggregate around the periphery of the developing parasite, see image below. Microsporidia lack hydrogenosomes and peroxisomes, the Golgi apparatus is not of a typical construction for an eukaryotic cell, and the RNA is of the prokaryotic type. 

Buxtehudea scaniae, the microsporidian parasite is surrounded by the mitochondria of the host cell, click here for a magnified image (223 K). Transmission electron microscopy.

Phylogeny

The unusual cytology caused non-parasitologist for a period to consider microsporidia to be one of the most ancient groups of eukaryotes (subkingdom Archezoa). Parasitologists have all the time interpreted the lack of mitochondria as a reduction. This has now been verified by molecular results.

When the history of microsporidia started in 1857 with the description of Nosema bombycis, the famous agent of the pepper-spot disease in the larvae of the silk moth, the microsporidia were interpreted as a group of fungi. However, this classification was never accepted, and up to now microsporidia have been treated as protozoa (since 1977 as the phylum Microspora). In the most recent phylogenetic analyses microsporidia branch among the fungi. If this is a true picture of the phylogeny a considerable nomenclature problem has to be solved, as no microsporidium has been described in a way satisfactory to the botanical code of nomenclature.
 

Ongoing projects

1. Pollinator parasites. Taxonomic partner in a project financially supported by the European Community, coordinated from Uppsala, and with collaborators in Zürich, Belfast, Tilburg, Wageningen and Lund.
2. Microsporidia of aquatic hosts.
3. Taxonomic revision of the families Metchnikovellidae, Chytridiopsidae and Mrazekidae.

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Contact: Ronny Larsson, Professor
Address:	Department of Cell and Organism Biology,  Zoology Building, Helgonavägen 3,  SE-223 62 Lund, Sweden
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