The infection of nonmosquito hosts by injection with spores of the microsporidan Nosema algerae

Nosema algerae normally infects only mosquitoes by the per os route but developed in a number of different arthropods when spores were injected into the hemocoels. Representatives of other phyla were not infected when injected with N. algerae spores. Spores produced in these injected hosts were of normal size and were infectious when fed to mosquito larvae. Many more spores were produced in some of the injected hosts than were produced in the infected mosquitoes. One corn earworm larva produced as many spores as 2,000 mosquito larvae.     

Growth of Nosema algerae in Pig Kidney Cell Cultures*

SYNOPSIS. Nosema algerae, a microsporidan parasite of mosquitoes, can infect pig kidney cell cultures. Spores germinated in the culture medium, infected the cells within 30 min of germination, multiplied, and produced spores. The early developmental stages in the N. algerae life cycle are described.     

Nosema algerae: Infection of the white mouse by a mosquito parasite

Spores of Nosema algerae, a microsporidan parasite of mosquitoes, were subcutaneously injected into the ears, tail, and feet of white mice. Infections were transient and localized at the injection sites. Spore germination tests in blood plasma indicated that it is unlikely that spores injected by an infected mosquito bite would result in an infection.     

Infectivity of a microsporidium of mosquitoes (Nosema algerae) to larval stages of Schistosoma mansoni in Biomphalaria glabrata

Lai P. F. and Canning E. U. 1980. Infectivity of a microsporidium of mosquitoes (Nosema algerae) to larval stages of Schistosoma mansoni in Biomphalaria glabrata. International Journal for Parasitology10: 293–301. Nosema algerae derived from a closed colony of Anopheles stephensi was fed to Biomphalaria glabrata infected with Schistosoma mansoni. Mother and daughter sporocysts became hyperinfected but the snail tissues remained free of the microsporidia except for rare small aggregates of spores. These lay close to the sites occupied by mother or daughter sporocysts and were probably liberated from them. Irrespective of dose, fewer snails contained infected sporocysts when spores were given at 7 days post-miracidial infection than when given at 14 days. These periods corresponded respectively to stages when mother sporocysts only or daughter sporocysts as well were present in the snails. Infection of the sporocysts began in the tegumental cells, spread to the brood chamber and ultimately to the cercariae themselves. Heavily infected sporocysts contained fewer developing embryos. Doses of 10⁶ and 10⁷ spores/snail caused significant depression of cercaria output when given at 14 days but not at 7 days.     

A laboratory evaluation of the microsporidian Vavraia culicis as an agent for mosquito control

The susceptibility of the mosquitoes Aedes aegypti, Aedes taeniorhynchus, Anopheles albimanus, Culex pipiens quinquefasciatus, Culex salinarius, and Culex tarsalis to infection by the microsporidian Vavraia culicis was determined. Using 18-hr exposures to 5 × 10³, 1 × 10⁴, 5 × 10⁴, and 1 × 10⁵ spores/ml, C. salinarius, C. tarsalis, and A. albimanus were found to be significantly more susceptible than A. aegypti. The most severe infections were observed in C. salinarius and C. tarsalis, although heavy infections of approximately 1 million spores per adult were recorded at the higher dosages in all species tested except A. aegypti. Production trials indicated that up to 5.4 × 10⁸ spores could be routinely produced in individual corn earworms, Heliothis zea. Inactivation of the spores by sunlight was measured by observing the subsequent incidence of infection and spore production in A. albimanus. These two measurements provided an LT₉₀ of 5.5 and 3.3 hr, respectively.     

Effect of ultraviolet radiation on the microsporidian Octosporea muscaedomesticae with reference to protectants provided by the host Phormia regina

The effect of ultraviolet light on the microsporidian Octosporea muscaedomesticae in relation to infection in the adult black blowfly, Phormia regina, was investigated. A 30-Watt germicidal lamp, 253.7-nm wavelength, was used as source of uv light in five investigations. In addition, sunlight served as a uv source in two studies. Viable naked dried spores exposed to the uv lamp at a distance of 10 cm were killed after 15 min. Viable naked spores in an aqueous suspension were killed after 30 min of exposure to the uv lamp and after 3 hr of exposure to bright sunlight, respectively. Daily 30-min uv lamp exposures on living hosts harboring all life phases of the parasite did not interfere with the ensuing infection in the blowfly's midgut and the pathogen's developmental cycle. Spores harvested from uv-treated infected hosts were found to be as infective as spores retrieved from hosts not treated with uv. Spores contained in dried fecal droplets and exposed up to 3 hr to the uv lamp, or 12 hr to bright sunlight, respectively, remained infective. Addition of uric acid to a preparation of naked spores prior to 15- and 30-min uv irradiations yielded 100% infection in both host groups. A uv-protective function is ascribed to components provided by the host's tissues and feces.     

The preservation of infective spores of Octosporea muscaedomesticae in Phormia regina,of Nosema algerae in Anopheles stephensi,and of Nosema whitei in Tribolium castaneum by lyophilization

Infective spores of three species of microsporidia were subjected to the lyophilization process by employing varying media as cryoprotectants. The infectivity of the lyophilized spores was then tested against a standard fresh spore preparation in the appropriate host insect. Spores of Octosporea muscaedomesticae served as an experimental model and were rendered noninfective in host Phormia regina (Calliphoridae: Diptera) after lyophilization with the following cryoprotective agents: skim milk (12%), ascorbic acid (5%) combined with thiourea (5%), glycerol (10%), mesoinositol (5%), and equine serum. Spores of O. muscaedomesticae lyophilized or vacuum-dried in 50% sucrose as well as in the hosts' tissues remained highly infective for as long as 2 years at a dose of 10⁶ spores/fly and a trial length of 12 days. At a dose of 5 × 10⁴ spores/fly there was a slight decrease in infectivity of the spores which had been lyophilized in the host's abdomen after a 2-year storage period compared with that of fresh, nonlyophilized spores. Naked spores of Nosema algerae suspended in 50% sucrose and lyophilized produced infection in 50% of the host population of Anopheles stephensi (Culicidae: Diptera) compared with 70% infection produced by fresh non-lyophilized spores. Spores of Nosema whitei lyophilized within its host larva Tribolium castaneum (Tenebrionidae: Coleoptera) remained 100% infective at a dose of 5 × 10⁵ spores/gram diet. It is concluded that an aqueous solution of 50% sucrose and/or the host's tissues are excellent protectants for the cryogenic or vacuum-drying process of the above-named spores, and their protective function may apply also to other microsporidian species.     

Hyperparasitism of intrasnail stages of Fasciola hepatica by a mosquito microsporidian parasite

Laboratory studies were conducted to investigate the infective behavior of Nosema algerae spores when ingested by Fasciola hepatica free of F. hepatica-infected snails (Lymnaea cubensis). Among the F. hepatica-infected snails exposed to N. algerae, 38.09% harbored microsporidia-infected F. hepatica rediae. The F. hepatica-free snails exposed to N. algerae as well as the controls did not become infected. Light microscopical studies of Giemsa-stained microsporidia distinguished this organism from microsporidia previously described in trematode larvae. Based on the infectivity studies and morphological data, it was concluded that N. algerae, a mosquito pathogen, was transmitted to intrasnail stages of F. hepatica.     

The effect of the microsporidan, Nosema algerae, on Anopheles stephensi

A bioassay method was established to examine infectivity differences between different batches of Nosema algerae spores. The IC₅₀ of N. algerae spores produced in one unusual host, Heliothis zea, was the same as for spores from the normal mosquito host, Anopheles stephensi. Soil and sand bottoms caused an approximate 200–400 fold increase in the IC₅₀. Nosematosis had little effect on the survival of larvae and pupae but the adult life span was reduced to the extent that malaria transmission would be doubtful.     

Ultrastructural study of early development of Nosema algerae in Anopheles albimanus

Early development of Nosema algerae in Anopheles albimanus was studied by electron microscopy. Sporoplasms were observed in the thorax and first three abdominal segments at 1 hr after initial exposure. The first division occurred between 30 and 36 hr. Immature spores were present at 48 hr with mature spores observed at 54 hr. By 60 hr from initial exposure, some of the mature spores had extruded and sporoplasms were seen. By 96 hr, all stages were present and development was no longer synchronous.     

Monitoring of Commitment,Blocking, and Continuation of Nutrient Germination of Individual Bacillus subtilis Spores

Short exposures of Bacillus spores to nutrient germinants can commit spores to germinate when germinants are removed or their binding to the spores'' nutrient germinant receptors (GRs) is inhibited. Bacillus subtilis spores were exposed to germinants for various periods, followed by germinant removal to prevent further commitment. Release of spore dipicolinic acid (DPA) was then measured by differential interference contrast microscopy to monitor germination of multiple individual spores, and spores did not release DPA after 1 to 2 min of germinant exposure until ∼7 min after germinant removal. With longer germinant exposures, percentages of committed spores with times for completion of DPA release (T_release) greater than the time of germinant removal (T_b) increased, while the time T_lag − T_b, where T_lag represents the time when rapid DPA release began, was decreased but rapid DPA release times (ΔT_release = T_release − T_lag) were increased; Factors affecting average T_release values and the percentages of committed spores were germinant exposure time, germinant concentration, sporulation conditions, and spore heat activation, as previously shown for commitment of spore populations. Surprisingly, germination of spores given a 2nd short germinant exposure 30 to 45 min after a 1st exposure of the same duration was significantly higher than after the 1st exposure, but the number of spores that germinated in the 2nd germinant exposure decreased as the interval between germinant exposures increased up to 12 h. The latter results indicate that spores have some memory, albeit transient, of their previous exposure to nutrient germinants.     

Ultraviolet light sensitivity,unscheduled DNA synthesis and DNA repair in C7-10 Aedes albopictus mosquito cells

We have examined the relative sensitivity of Aedes albopictus C7-10 mosquito cells to irradiation with ultraviolet light from a germicidal lamp. On the basis of plating efficiency, C7-10 cells were approximately two times more resistant to UV light than human 293 leukemia cells. Recovery after UV irradiation was accompanied by an increase in unscheduled DNA synthesis (UDS), which was measured by incorporation of ³H-thymidine into acid-precipitable DNA in the presence of hydroxyurea. Under standardized conditions, UDS was maximal after a 10 min exposure (120 J/m²), and declined after longer exposures. In addition, UV treatment is associated with a small but reproducible increase in repair of plasmid DNA in transiently transfected cells. We anticipate that analysis of DNA repair activities in mosquito cells will identify molecular targets that might control longevity in transgenic mosquitoes.     

The Effect of Ultraviolet Radiation on the Germination of Nosema algerae Vávra and Undeen (Microsporida: Nosematidae) Spores1

Spores of Nosema algerae Vávra and Undeen were subjected to various dosages of 254 nm ultraviolet radiation (UV). Very high dosages of UV were required to block germination. Germination was normal immediately after UV dosages of 0.2 to 1.0 J/cm², followed by a delayed effect in which both percentage germination and the intrasporal concentration of trehalose decreased with time after UV exposure. Although a few spores were germinated, most of them were inactivated (rendered temporarily unable to germinate) by exposure to UV of 1.1 J/cm². Ultraviolet radiation between 1.1 and 3.4 J/cm² stimulated spores to germinate. However, spores were completely unable to germinate immediately after exposure to dosages above 3.8 J/cm². Ammonia had little effect on stimulation by UV but was inhibitory to germination after stimulation had occurred. These results demonstrate that UV behaves like a germination stimulus and are discussed in terms of the hypothesis that germination is initiated by the breakdown of barriers between trehalose and trehalase.     

Structural Alteration of the Plasma Membrane in Spores of the Microsporidium Nosema algerae on Germination1

The fine structure of the plasma membrane in spores of the microsporidium Nosema algerae, a pathogen of mosquitoes, was examined in the resting condition and after the spores were stimulated to germinate in vitro. Slow penetration of resin caused collapse of the germinated spores. Thin sections of germinated spores showed peculiar membrane infoldings that were never found in ungerminated samples. Analogous germination-dependent configurations of the plasma membrane were observed in freeze-fractured preparations of spores either fixed and impregnated with glycerol prior to freezing, or rapidly frozen with liquid propane while in the process of germination. In every case, the replicas presented germinated spores with indentations in the protoplasmic face of the plasma membrane, and apparently complementary blunt spines on the external face, that were absent in ungerminated spores. It suggests that these alterations of the plasma membrane result from a structural adjustment to a spontaneous contraction of the spore case after germination. We discuss this interpretation with regard to conflicting views on the nature of such morphological features.     

Production of Aseptic Spores of Vesicular-Arbuscular Endophytes and Their Viability After Chemical and Physical Stress

The survival of germinating spores of vesicular-arbuscular endophytes after treatments with oxidizing agents, antibiotics, moist heat, ultrasonic radiation, and ultraviolet radiation was compared with that of their contaminating microbes. Spores of three species were rapidly decontaminated by treatment with 0.42% (wt/vol) chlorine available from 5.0% (wt/vol) chloramine-T at 30°C for 20 to 40 min depending on the species and the soil from which they were extracted. This treatment did not change spore viability. The survival of spores was reduced by exposure for 20 min to 1.11% chlorine at 30°C for Glomus caledonius or at 35°C for Acaulospora laevis. Growth of any bacteria surviving treatment with oxidizing agents was inhibited by 100 μg of chloramphenicol per ml in agar; however, spore germination and germ tube growth were reduced only by concentrations greater than 200 μg/ml in agar. Spore germination was decreased by concentration of pimaracin, which controlled fungal growth. The spores survived moist heat at 40°C for 80 min, 55°C for 10 min, and 60°C for less than 1 min. The viability of spores was unaffected by ultrasonic irradiation for up to 4 min. Spores of G. caledonius and A. laevis were extremely resistant to ultraviolet radiation. Their viability was unaffected by exposure to 5 × 10⁸ ergs cm⁻² from an ultraviolet source of 253.7nm. The spores had very thick, pigmented walls, and the possibility that these provided some protection against the physical and chemical treatments is discussed. The degree of physiological damage to the spores caused by the treatments demonstrated some adverse effects of basic laboratory procedures. This information, together with that on the comparative sensitivity of contaminating microbes to the treatments, was used in the development of protocol for producing large numbers of uncontaminated spores.     

Ricin B lectin-like proteins of the microsporidian Encephalitozoon cuniculi and Anncaliia algerae are involved in host-cell invasion

Microsporidia are obligate intracellular pathogens capable of infecting a wide variety of hosts ranging from invertebrates to vertebrates. The infection process requires a step of prior adherence of Microsporidia to the surface of host cells. A few studies demonstrated the involvement of proteins containing a ricin-B lectin (RBL) domain in parasite infection. In this study Anncalia algerae and Encephalitozoon cuniculi genomes were screened by bioinformatic analysis to identify proteins with an extracellular prediction and possessing RBL-type carbohydrate-binding domains, being both potentially relevant factors contributing to host cell adherence. Three proteins named AaRBLL-1 and AaRBLL-2 from A. algerae and EcRBLL-1 from E. cuniculi, were selected and comparative analysis of sequences suggested their belonging to a multigenic family, with a conserved structural RBL domain despite a significant amino acid sequence divergence. The production of recombinant proteins and antibodies against the three proteins allowed their subcellular localization on the spore wall and/or the polar tube. Adherence inhibition assays based on pre-treatments with recombinant proteins or antibodies highlighted the significant decrease of the proliferation of both E. cuniculi and A. algerae, strongly suggesting that these proteins are involved in the infection process.     

Brugia pahangi: effects upon the flight capability of Aedes aegypti.

Aedes aegypti mosquitoes were adversely affected by infections of the filarial worm Brugia pahangi. Infected mosquitoes flew significantly shorter distances and showed marked reductions in total flight time during 24-hr flight mill tests compared to uninfected controls. Total flight range and duration flown by infected mosquitoes remained relatively constant throughout the infection process, while control mosquitoes flew further and longer with increasing time after their blood meal. Furthermore, a significantly greater number of infected mosquitoes either died or were rendered incapable of flight. Of flying and nonflying mosquitoes with 6-day-old or older infections dissected for parasite burdens, the nonflying group contained significantly more worms. Results of this study indicate that developing filarial larvae within this mosquito vector reduce its ability to survive and to transmit its infection by reducing its flight capabilities. Conclusions from this study relate only to A. aegypti homozygous for the gene fm which is fully susceptible to this filarial parasite.     

A serological comparison of some species of microsporida.

Double immunodiffusion techniques were used to investigate the taxonomic relationships between six different microsporidian isolates. Microsporidia used included Nosema bombycis, N. algerae, N. plodiae, and three organisms morphologically similar to N. necatrix. Antigens were extracted from spores after disruption in an MSK Braun cell homogenizer. Cross-reactions were seen between N. plodiae and two of the N. necatrix isolates, while the third N. necatrix, N. bombycis, and N. algerae were antigenically unrelated. One of the N. necatrix isolates revealed temperature-related antigenic differences, but no antigenic differences resulted from aging spores for 5 months before disruption.     

Gametocytocidal and sporontocidal effects of antimalarial drugs on malaria parasites. II. Action of the folic reductase inhibitors, chlorguanide, and pyrimethamine against Plasmodium cynomolgi

The antimalarial activity of the dihydrofolate reductase inhibitors, chlorguanide and pyrimethamine, against gametocytes and sporogony of the simian parasite, Plasmodium cynomolgi was tested. Plasmodium cynomolgi infected rhesus monkeys (Macaca mulatta) were treated orally with multiple doses of chlorguanide (5.6 or 11.3 mg base/kg/Day X5) or a single dose of pyrimethamine (3 mg base/kg). Batches of mosquitoes (Anopheles maculatus) were allowed to feed immediately prior to and at appropriate intervals after drug administration. The effects of the drugs on the developmental stages of the parasite were assessed within the mosquito host. The results indicated that sporogony was interrupted in mosquitoes fed 1 hr after initial dosing with chlorguanide. With pyrimethamine, a clear-cut sporontocidal action was shown as early as 15 min after the drug was given. Both compounds inhibited further development of young oocysts and successive batches of mosquitoes fed 1 hr or more after medication ware consistently negative for sporozoites. Administration of either compound in the indicated doses did not prevent exflagellation, zygote or ookinete formation. However, the behavior of the latter forms was apparently altered by the action of the drug. There was a marked retarding effect in the formation of ookinetes in mosquitoes fed 24 hr after initial medication and thereafter. The schizontocidal and gametocytocidal action of chlorguanide was more evident than it was in pyrimethamine.     

Stored Bacillus popilliae spores and their infectivity against Popillia japonica larvae

Bacillus popilliae spores were stored for about 7 years under three separate conditions: frozen in sterile distilled water, smeared on glass microscope slides, and stored in loam soil at room temperature. In separate experiments, each of the 7-year-old preparations was fed to Popilla japonica larvae at concentrations of 10³, 10⁵, 10⁷, and 10⁹ spores/g of soil. A significant decrease in the percentage of larvae infected occurred in all of the aged spore tests. B. popilliae spores stored in soil, for the extended period, produced 3% larval infection only at the 10⁹ spores concentration; similar results were obtained from frozen spores. When P. japonica larvae were fed spores stored dried on slides, about 20% of the larvae developed milky disease. When aged frozen spores were artificially injected into larvae, 12% became infected at concentrations of 1 × 10⁶ spores/larvae; dried spores at the same concentration infected about 38% of the insect larvae. We conclude from these data that aged B. popilliae spores are significantly less infective against P. japonica larvae than young spores.