August 1997                                          

SCSB# 387

Microsporidia (Protozoa):

A Handbook of Biology and Research Techniques

Biology

The spore

Figure 1. Electron micrograph of the ununucleate meiospore of Parathelohania anophelis (bar = 1 5m).

Figure 2. A diagram of a microsporidian spore showing, in cross section, showing the coiled polar filament, (PF), anchoring disc of the polar filament (A), the endospore (E) and exospore (Ex) of the spore wall, 2 nuclei (N), polaroplast (Pp), plasmalemma (Pl), posterior vacuole (PV), and ribosomes (R).

The structure of the spore is the most characteristic feature of microsporidia (Fig. 1 and 2). They are small, usually ranging between 1 - 15 µm in length and 0.5 - 5 µm in width and usually round, oblong, ovate or rod-shaped. Many have irregular shapes or ornamented spore coats that might obscure their identity. Microsporidian spores meet the environment with a resilient spore wall that consists of a proteinaceous exospore and chitinous endospore. It is backed by a plasmalemma that probably mediates the diffusion of ions and other small molecules between the cytoplasm of the spore and the external environment. The coils of the polar filament are frequently seen in electron micrographs as a series of cross sections lying in rows, just inside of the plasmalemma. The polar filament passes anteriorly through the polaroplast and attaches to an anchoring disc at the anterior end of the spore wall. The polaroplast, composed of vesicles, layers of tightly folded membranes, or a combination of the two, occupies the anterior third to half of the spore. Genetic material is carried in either a single nucleus or a diplokaryon (two nuclei in close association). The cytoplasm is rich in ribosomes and endoplasmic reticulum. At the end of the spore opposite the anchoring disc is a posterior vacuole containing a posteriosome, amorphous material, or a clear fluid.

Transmission

Microsporidian spores are unique in the way they invade a new host (Weidner, 1989). The spores sometimes leave the host with the feces but are usually released after the host dies. Another infection cycle commences after the spores are ingested by another host. A stimulus in the gut environment sets off a series of events initiating the process of germination. Within minutes of the initial stimulus, the polar filament ruptures through the spore wall in the anchoring disc region, everting to form a "polar tube." The tube elongates at its tip, penetrating the gut wall and other host tissues as it everts. After the polar tube is completely everted, the sporoplasm, which consists of one or two nuclei and some cytoplasm, passes quickly through the tube into the host cell. The expulsion phase, from initial rupture of the spore wall to the emergence of the sporoplasm, takes only about two seconds (Frixione et al., 1992). Although ingestion of spores is the most common means of horizontal transmission, infections have been passed from an infected host to an uninfected host on the ovipositors of parasitic insects (parasitoids). Microsporidioses are commonly transmitted vertically, through the infection or contamination of eggs within the infected female.

Germination and Infection

Figure 3. Scanning electron micrograph of a germinated Nosema algerae spore displaying the polar tube with the empty spore case (arrow) -- dents in the spore case are an artifact of drying -- at one end and the expelled sporoplasm at the other (bar =3 5m).

Figure 4. Edhazardia aedis spores germinating in the gut of Aedes aegypti showing the polar tubes penetrating through the peritrophic membrane (bar = 10 5m).

The spore wall is a tough, pressure-containment vessel. Within it lies the other elements of the discharge apparatus, the polar filament, polaroplast, and posterior vacuole (Vavra, 1976). After an appropriate set of stimuli, the polar filament is rapidly everted forming a polar tube, penetrating the gut barriers and into a host cell. Upon completion of polar tube formation, the sporoplasm, composed of the nuclei and cytoplasm, passes rapidly through the tube into the cell initiating a new infection. All of this activity occurs in only about two seconds. Figure 3 is a scanning electron micrograph showing the sporoplasm, polar tube and the empty spore case of a Nosema algerae spore that had been germinated in vitro (denting of the spore wall is an artifact of drying). By a technique combining in vivo and in vitro techniques, the polar tubes of Edhazardia aedis spores can be observed penetrating through the peritrophic membrane of its mosquito host, Aedes aegypti (Fig. 4). Under normal conditions, the polar tube is rapidly digested within the gut so that usually only the empty spore case can be seen. These rapid germination events can be observed in vitro at 400 x magnification using phase contrast. It can be slowed to provide for easier observation by increasing the osmolarity of the stimulating solution with sucrose or polyethylene glycol.

Some spores germinate soon after their formation to further disseminate the infection within the host (Avery and Anthony, 1983; Iwano and Ishihara, 1989; Iwano and Ishihara, 1991). Others germinate in situ to transmit the microsporidium to the eggs, infecting the next generation (Hazard and Weiser, 1968). Direct transfer of infection from the parent to offspring is called "vertical transmission" as opposed to "horizontal transmission" (transmission from individual to individual)which is accomplished by the ingestion of spores. The "internal spores" germinate within the host soon after their formation and are usually much less abundant than the more commonly found "external" or environmentally resistant spores. The internal spores have thinner walls than the external spores which are released into the environment to accomplish horizontal transmission. This class of spores need to withstand desiccation, freezing, or the osmotic stresses of immersion in water and remain capable of germination. Observations with N. algerae (Undeen, 1978) , Vavraia culicis, and others indicate that the application of the germination stimulus must be abrupt (Undeen and Avery, 1988a). Slow environmental changes inactivate the spores, leaving them temporarily incapable of germination, a condition that is readily reversed by a few hours in water. Probably the major importance of a germination stimulus is its absence in the environment in which the spore resides while waiting ingestion by a new host. Internal spores might germinate as soon as their development is complete, while external spores must avoid premature germination. Specificity of germination is only one factor, in some cases a minor one, in the mediation of host specificity. The lack of total specificity for a particular set of stimuli is revealed in the findings that stimuli effective in causing the germination of some spores in vitro are not the same as the stimuli in the gut of the host (Undeen and Epsky, 1990).

The force that propels the everting filament and drives the sporoplasm through the completed polar tube appears to be osmotic. Spores are permeable to water and contain high concentrate solutes, particularly the glucose disaccharide, trehalose . Thus, microsporidian spores in an aqueous environment have a high turgor pressure even before a stimulatory event (Undeen and Frixione, 1990). Although the propulsive force is probably osmotic in all species, the means by which the process of germination is initiated appears to be more variable and largely unknown. In some cases germination is initiated by a rapid increase in osmotic potential, apparently caused by an increased solute concentration from the conversion of trehalose to glucose (Undeen, et al., 1987; Undeen and Vander Meer, 1994; Undeen and Frixione, 1990). In other species, expulsion might be initiated by a weakening of the spore wall over the anchoring disc of the polar filament. The mobilization of calcium (Weidner and Byrd, 1982; Pleshinger and Weidner, 1985) seems to be a mediating mechanism in some instances. The stimuli that start the chain of events leading to discharge of the polar filament and sporoplasm are quite variable. Some of the stimuli that have been identified are ions, pH change, rehydration, direct pressure, and hydrogen peroxide. Temperature is an important variable (Undeen, 1978). A list of germination stimuli is found under "Spore Germination."

Developmental Cycles

Developmental stages can be seen on Giemsa-stained smears of specimens and with the electron microscope. The patterns of cell division are variable among microsporidia and aid us in their identification. Eventually, a new generation of spores is produced; and, depending upon the species, can be of the same type that initiated the infection or another, structurally different kind of spore with a different function. Below is a list--probably incomplete--of developmentally or functionally different types of spores.

1. External spores: Often called "environmental spores" these spores are produced in large numbers and released from the host to infect another host. They have a relatively thick spore wall and are resistant to a variety of environmental hazards. There are several developmentally different kinds of environmental spores.

a. Diplokaryotic (binucleate) spores. These are produced asexually from diplokaryotic sporonts and usually infect (per os) other members of the species in which they were formed.

b. Uninucleate spores formed by asexual reproduction from uninucleate sporonts. These spores, where known, infect other members of the species in which they were formed.

c. Dissociation spores. These spores arise from a diplokaryotic developmental sequence by dissociation of the diplokarya at the onset of sporogony. They are known only from mosquitoes and appear to transmit the microsporidium horizontally.

d. Meiospores. These spores formed from diplokarya by meiosis. In the case of mosquitoes, they only infect the copepod intermediate host. These spores are most often seen in packets of eight spores, frequently referred to as "octospores."

e. Uninucleate spores formed asexually in an intermediate host. These spores are incapable of infecting the host species in which the were formed. The only ones known so far are from copepods and infect mosquitoes.

2. Internal spores: This type of spore is usually produced in smaller numbers, has a thinner spores wall and germinates within the host in which it develops.

a. Early spores. The FC (few coil) spores of Ishihara (1993), more recently called "primary spores", are diplokaryotic spores that form within a few days of per os infection by environmental spores. They germinate immediately upon maturation, still within the host cell, to disseminate the infection in the host.

b. Binucleate spores that germinate in the ovary to infect the egg. In mosquitoes, this type of spore originates from a pathway starting with the union of gametes, with the nuclei remaining paired in the diplokaryotic state throughout all subsequent divisions. In some species, this spore occurs repeatedly in generation after generation of female hosts by vertical transmission.

Most microsporidia have been described on the basis of only one of their spores, usually an environmental spore. Hazard and Weiser (1968) discovered that a Nosema sp. and a Thelohania sp. from a mosquito host were but two parts of one species, demonstrating that a single microsporidium can have more than one type of spore. The "Nosema" was responsible for vertical transmission and the only thing known for sure about the "Thelohania" spores was that they did not infect mosquito larvae. Sweeney et al. (1985) discovered that this Thelohania-like spore infected a copepod intermediate host from which came yet a third type of spore that completed the cycle by infecting the mosquito larva. Since then, copepod intermediate hosts have been identified for several more microsporidia of mosquitoes. Thus, some microsporidia have been shown to have a life cycle that included two generations of a mosquito host and a copepod intermediate host, involving three different developmental pathways to three different spores. It is of special importance for taxonomists to note that these spores were once considered three distinct species--in three different genera.

Although the only microsporidia for which intermediate hosts have been identified are from mosquitoes (Amblyospora, Parathelohania), this situation might be common. Nosema necatrix and Thelohania diazoma were long considered to be dual infections in caterpillars. They were finally proven to be two spore types of one species, Vairimorpha necatrix, developing in their own way and their own time, in the same host (Fowler and Reeves 1974). The "Nosema type" spore seems to be the one usually responsible for transmission to the original host, leaving some doubt as to the role of the meiospores. So few complete life cycles have been thoroughly described that few generalities can be made. But, from what we already know, it seems as though many microsporidia are capable of producing more than one kind of spore and that each of these spores plays a specific role in the life cycle. Amblyospora californica (Fig. 5) in an example of complex developmental cycle in which two generations of mosquitoes and a copepod intermediate host are needed to compete it (Becnel, 1992). A different type of spore is formed in each host. Figure 6 outlines the relatively more simple life cycle of Nosema bombycis in which two types of spores are formed in the same host. In the genus Vairimorpha, an internal spore and two external spores--one a meiospore and the other a diplokaryotic external spore--are all produced in the same host. There is too much diversity in microsporidian developmental cycles for more than a cursory glance at them here.

Figure 5. Life cycle diagram of Amblyospora californica in its mosquito and copepod hosts.

Figure 6. Life cycle of Nosema bombycis, a parasite of some Lepidoptera.

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Document Prepared by:
Jeff D. Miller, Communications Specialist
Oklahoma State University