Environmental Persistence of Microsporidia
J.V. Maddox - Illinois Agricultural Experiment Station
Although many Protists other than microsporidia have pathogenic associations with insects, this review is restricted to microsporidia for two reasons. First, microsporidia, by far, are the most important group of Protists that parasitize insects. Second, there has been very little additional information on non-microsporidian Protists since Brook's (1988) review.
Microsporidia are obligate parasites of most animal phyla, including Arthropoda. This extremely diverse and parasitic group of organisms was once an order in the phylum Protozoa, but has been elevated to phylum status (Sprague et al 1992). All orders of insects contain representative species that are infected by microsporidia. The phylum Microsporida contains 160 genera, many with complex life cycles and/or dimorphic spore types and alternate hosts. Most, if not all, species of microsporidia have a common characteristic; they produce environmentally resistant spores that are responsible for horizontal transmission. Spores produced by infected hosts are present in the feces, the silk, or are liberated when an infected host dies. The environmentally resistant spores, when eaten by a susceptible host, germinate, send a special organelle, called the polar tube, into an insect's midgut epithelial cell, inject an infectious sporoplasm and the infection is initiated (Brooks, 1988). Microsporidia also may be transmitted from an infected female to her offspring either transovarially (inside the egg) or on the egg surface (transovum transmission). In addition, microsporidia may be transmitted from infected to healthy individuals via the oviposition of parasitic insects.
Unlike the other groups of insect pathogens (viruses, bacteria, fungi) and also the nematodes, the microsporidia have no serious contenders for extensive development and commercialization as microbial insecticides. Nosema locustae is registered as a microbial control agent of grasshoppers, and both Nosema algerae and Vairimorpha necatrix have been considered as possible candidates for development as microbial insecticides. Nevertheless, it is very unlikely that N. locustae will become a major product or that N. algerae or V. necatrix will ever be registered as microbial insecticides. For this reason, there has been relatively little interest in determining the environmental persistence of microsporidia relative to their performance as microbial insecticides.
Although microsporidia have little potential as microbial insecticides, they are very important natural control agents for many species of insects. To promote natural control systems, it is important to understand how microsporidia persist in nature and how different environmental factors affect the persistence and, ultimately, the horizontal transmission of microsporidia. Therefore, most of the research questions about the persistence of microsporidia in the environment have not been insecticidal questions such as the half-life of microsporidia on leaf surfaces or the formulation of more stable microsporidian insecticides, but rather how microsporidia are transmitted from infected hosts to susceptible hosts. During this transmission process the extracorporeal spores of microsporidia are exposed to many environmental conditions, and these conditions affect the efficiency of horizontal transmission.
The microsporidia are a very diverse group of organisms and, with the exception of the direct effects of sunlight, which can quickly kill most microorganisms, different groups of microsporidia do not respond uniformly to most other types of environmental stress. In addition, the effects of environmental stress on microsporidian spores can be determined only by bioassays against susceptible hosts. There are no vital stains that reliably can determine the infectivity of microsporidian spores, and, since microsporidia are obligate parasites, infectivity and/or viability cannot be determined by growing microsporidia on artificial media. Therefore, even though infectivity (can it infect a susceptible host?) and viability (are the spores alive?) may not technically be the same, since a spore can be alive and yet be unable to infect, infectivity and viability are indistinguishable as estimates of the survival of microsporidian spores in the environment.
Relatively few species of microsporidia have been examined for their reaction to environmental stresses, which means that the generalizations we make about how microsporidia respond to environmental stress maybe modified as we examine additional groups of microsporidia. I will attempt to emphasize that the persistence of microsporidia is based on their adaptations to the specific environment of their host and environmental conditions during the horizontal transmission process. In the following discussion, frequently I will refer to earlier reviews that address the persistence of Protozoa (Brooks, 1988; Maddox, 1977; Maddox, 1973).
Effect of Environmental Factors
Solar radiation / Sunlight
Direct sunlight kills the unprotected spores of all species of microsporidia within in a few hours (Brooks, 1988; Maddox, 1977; Kaya, 1977). The half-life of V. necatrix exposed to direct sunlight on glass microscope slides was about two hours (Maddox, 1977), and Ignoffo et al. (1977) reported a half-life of 2.1 hours when they exposed V. necatrix spores on Helocoverpa zea eggs to radiation from a germicidal lamp (peak radiation at 254 nm). Brooks (1988) summarized the research results of other workers on the effect of sunlight and ultraviolet radiation on the spores of nine species of microsporidia. Research methods were variable, but unprotected spores did not survive exposure for more than a few hours. There was more variability in the survival of spores between the substrates on which the spores were exposed than between the different species of microsporidia. It is likely that most species of microsporidia have similar responses to radiation.
Temperature
Most of the studies on the effects of high temperature on microsporidia were conducted in an attempt to eliminate microsporidian infections from insect colonies. Infected insect hosts as well as extracorporeal spores have been involved in these studies (Baribeau and Burkhardt, 1970; Benjakova and Verejskajs, 1958; Hartwig, 1970; Vandermeer and Gochnauer, 1969). Studies on extracorporeal spores have usually involved dry spores, and, since higher temperatures usually have a drying effect, it is difficult to distinguish between the drying effect of higher temperatures and the absolute effect of the higher temperature itself. For most species of microsporidia, time of survival is inversely proportional to the higher temperature. For example, Maddox (1977) found that dry V. necatrix spores survived for three weeks at 40°C but survived for only five hours at 50° and 30 minutes at 60°. Kaya (1977) found that V. necatrix spores survived for 144 hours at 35°C, suggesting that the moisture provided by the bean leaf on which the spores were placed reduced the effect of the higher temperature.
Different species of microsporidia respond very differently to low temperatures that are above freezing. Brooks (1988) and Maddox (1977) have reviewed this subject. Most microsporidia from terrestrial insects will survive for several years at 2 to 5°C in sterile water suspensions. In sterile water suspensions at 2 to 5°C, Oshima (1964) maintained viable spores of Nosema bombycis for 10 years and Revell (1960) stored viable spores of Nosema apis for seven years. If the water suspension contains organic debris and microbial growth occurs in the water suspension, the spores will not survive for more than a few days (Brooks, 1980; White, 1919). Some microsporidia from aquatic insects will not survive storage at lower temperatures (Undeen, Johnson and Becnel, 1993). Because microsporidia infect such a wide range of insects in many diverse habitats, it is likely that specific microsporidia have evolved characteristics that promote their survival in the habitat of their hosts.
A similar dichotomy exists between microsporidia from terrestrial insects and aquatic insects relative to their responses to subfreezing temperatures. Most microsporidia from terrestrial insects will survive the freezing process, while most species of microsporidia from aquatic hosts will not (Maddox and Solter, 1996). The length of time spores will remain viable over a range of temperatures while frozen has not been thoroughly investigated. Spores of many species of microsporidia from terrestrial insect hosts will survive in a water suspension in liquid nitrogen for more than 20 years and require no special freezing or thawing rates (Maddox and Solter, 1996), but the addition of cryoprotectants, such as glycerol, promotes survival of spores in liquid nitrogen. The length of time spores are reported to survive subfreezing temperatures from 0° to -35°C ranges from two to 24 months (Fuxa and Brooks, 1979; Maddox, 1973). In temperate climates spores often must overwinter in the habitat of their host to initiate infections in the host population in the spring. Therefore, it is likely that the spores of many species of microsporidia must withstand freezing for several months if they are to infect hosts successfully early in the spring. Conversely, microsporidia of aquatic hosts are much less likely to encounter freezing conditions. Ponds and rivers seldom freeze at the bottom, where the microsporidian spores occur.
Moisture/humidity
As with freezing, the microsporidia of aquatic insects generally are not able to survive completely dry conditions (Alger and Undeen, 1970; Brooks, 1988). Since freezing has a drying effect on organisms, the similar responses to these two environmental conditions are not unexpected. Microsporidia from terrestrial insects exhibit a range of longevity records when held in a dry conditions with limited exposure to ultraviolet light. Survival times for eight species of microsporidia held as dry spores over a range of conditions were from two weeks to more than a year (Maddox, 1973; Kramer, 1970). Spores of microsporidia such as Octosporea muscaedomesticae, transmitted in fecal deposits, survived six to 12 months.
While drying generally is harmful to most species of microsporidia, the addition of free water to the dry spores of some microsporidia is equally harmful. Spores of the microsporidium Nosema whitei, a pathogen of flower beetles, can survive for over a year as dry spores in flour, but when the spores are placed in water they germinate and extrude their polar filaments and thus lose their infectivity (Milner, 1972; Maddox, 1973). Kramer (1970) obtained similar results with the microsporidian O. muscaedomesticae. Dry spores survived on glass microscope slides for more than one year, but the addition of water to the slide greatly reduced infectivity, presumably because the water stimulated spore germination.
The Effect of pH
The pH of the medium encountered by microsporidian spores has a profound influence on the germination of spores (Frixione et al., 1992; Undeen, 1976). Nevertheless, because other factors are involved and germination usually requires more than simple exposure to a single pH medium, it is unlikely that the pH values of water encountered in nature would influence the spontaneous spore germination of many species of microsporidia. The long-term effects of pH as an additional variable on survival of microsporidian spores could be important, but has not thoroughly been examined.
Wind
The direct effect of wind on survival of microsporidian spores has not been investigated, but wind could have an indirect effect in many ways. Fecal pellets from microsporidian-infected insects often contain many spores, and wind may play an important role in the movement and redistribution of infected fecal pellets. Where the pellets are distributed will influence which of the above factors contribute to spore survival. Likewise, wind affects the distribution of infected insects which, in turn, affects the distribution and, ultimately, the survival of spores.
Interactions Between Above Factors
Although some of the above factors can be isolated in laboratory experiments, in nature they act simultaneously. In addition, each factor does not remain at a constant value. Ultraviolet radiation and temperature fluctuate greatly over a 24-hour period. The microclimate where microsporidian spores reside almost always is very different from ambient reported climatic data. While the survival times reported for spores of different microsporidian species for a specific factor (at a constant value) give us a "ballpark figure" of survival times, they may not accurately represent the survival times of spores in nature.
Some factors cannot be isolated, even in laboratory experiments. Solar or ultraviolet radiation produces heat, unavoidably making the effect of temperature and radiation related. Kaya (1977) wisely recognized this in his studies on radiation/temperature effects. Likewise, drying of spores is enhanced by both radiation and high temperatures. There have been few hypothesis-testing experiments involving the interactions of detrimental environmental factors on microsporidian spores.
Substrate Effects
It is probably restating the obvious to conclude that "any substrate which offers some protection from direct sunlight and provides a constant source of moisture extends spore longevity" (Maddox, 1977). This probably is true for all microsporidian species except those, such as Nosema whitei, that germinate in the presence of water. All studies involving substrate/radiation interactions concluded that spores on more complex substrates survive exposures to radiation for longer periods. For example, spores survived exposure to sunlight for three hours on a glass microscope slide, four and a half hours on a corn leaf, nine hours on an artificial diet surface, and more than 28 hours mixed with soil (Maddox, 1977).
Likewise, substrates that provide moisture for microsporidian species that are harmed by drying promote survival. Nosema algerae does not survive drying. If the water in which N. algerae spores reside dries completely, N. algerae spores die. Ephemeral pools of water are not desirable substrates for N. algerae.
Food
Host plant/insect interactions probably affect the ability of many species of microsporidia to infect their hosts. While host/plant interactions with entomopathogenic bacteria and viruses have been the subject of many studies, the effect of the plant on which the insect host is feeding on the infection of that insect host by microsporidia has been largely neglected. Smirnoff (1967) found that certain plants affected the susceptibility of the ugly-nest caterpillar, Archips cerasivoranus, to microsporidian infections. The mode of action of these interactions is unknown, and the extent of similar phenomena among the microsporidia remains unexplored.
Insect cadavers
It is documented that some species of microsporidia seasonally persist in the environment of their hosts in infected cadavers (White, 1919; Brooks, 1988; Fuxa and Brooks, 1979), and the cadaver unquestionably provides protection from ultraviolet radiation. It is not clear whether protection against any other environmental factors incur from overwintering in infected cadavers.
Infected Living Hosts
Many species of microsporidia persist during unfavorable environmental conditions in infected, living hosts. Nosema pyrausta may persist throughout the winter as free spores in corn stalks, but, relative to the development of epizootics in the spring, persistence in living, diapausing European corn borer larvae is most important (Siegel et al., 1988). This is true for many species of microsporidia, especially those such as Nosema pyrausta that are transovarially transmitted. Microsporidia are present in living, infected hosts (in diapause or aestivation) as spores or immature development forms (meronts, sporonts, sporoblasts). Different developmental stages of the host (egg, larva, pupa or adult) may survive through unfavorable environmental conditions as infected individuals. Eggs of the gypsy moth, Lymantria dispar, (McManus et al., 1989), pupae of the fall webworm, Hypantria cunea (Nordin and Maddox, 1974), and adults of the alfalfa weevil Hypera postica,(Solter et al., 1993), are examples of microsporidia that overwinter in infected, living hosts. This becomes much more complicated when microsporidian species involving alternate hosts are involved (Andreadis, 1993). Many microsporidia from aquatic insect hosts (especially mosquitoes) require development in an alternate host as part of the developmental cycle. Microsporidia may overwinter or survive drought conditions in living, alternate hosts such as copepods in areas where the mosquito host could not survive. The mosquito may distribute microsporidia from pool to pool (Dieterich et al., 1994; White et al., 1994).
It has been demonstrated that spores of some species of microsporidia can pass through the digestive tract of predators, both vertebrate and invertebrate (Kaya, 1979). This serves not only to distribute microsporidia to new locations, but also to protect microsporidia from unfavorable environmental conditions while they are in the gut of the predator. Parasitoids often have various associations with the microsporidia of their hosts (Brooks, 1993). Some associations may involve passive transport, while other associations may involve active infections in parasitoid larvae and adults. In either case, microsporidia may be transmitted from infected to healthy hosts. Microsporidia transmitted in this manner are protected from most unfavorable environmental factors such as ultraviolet radiation and drying.
Insecticides/adjuvants
Because microsporidia are not considered as having potential for development as microbial insecticides, few studies have been conducted on the direct effects of insecticides on extracorporeal microsporidian spores. The experiments that have been conducted have shown that technical preparations of organophospates and pyrethrins have no effect on viability of spores, but that solvents present in many insecticide formulations often are very harmful to spores (Maddox, 1977). Most disinfectants, such as sodium hypochlorite, killed spores at very low concentrations.
The spores of Vairimorpha necatrix and Endoreticulatus schubergi have been formulated with various ajuvants in an effort to protect spores from ultraviolet radiation (Brooks, 1980). The survival of V. necatrix spores exposed to sunlight greatly increased when UV protectants, such as Shade, were included in formulations in one study (Kaya, 1977), but V. necatrix persistence was affected more by spore dosage than by UV protectants in another (Fuxa and Brooks, 1978).
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Document Prepared by: M.E. Baur, Louisiana State University Agricultural Center