Quantitative studies on the pathogenicity of Nosema carpocapsae,a microsporidian pathogen of the codling moth,Cydia pomonella,in New Zealand

The pathogenicity of Nosema carpocapsae for codling moth was studied using dose-infectivity experiments. The IC₅₀ (median infective concentration) was similar for the five larval instars (range 4.0 × 10³ to 6.7 × 10⁴ spores/ml). Spore loads in moths ranged from 6.0 × 10⁶ to 7.1 × 10⁷ spores per moth and varied with dose and with larval age at infection. The infection does not cause mortality but does reduce the fecundity and fertility of infected moths. Nosema carpocapsae is transmitted transovarially as well as horizontally. Infected eggs were not produced by healthy females mated with infected males, although such pairs generally produced fewer eggs than healthy pairs.     

Different course of proteolytic inhibitory activity and proteolytic activity in Galleria mellonella larvae infected by Nosema algerae and Vairimorpha heterosporum

The activity of protease inhibitors and proteases was studied in the hemolymph, gut, and fat body of 7th-instar larvae of Galleria mellonella infected by two microsporidia, Nosema algerae and Vairimorpha heterosporum. The increase in inhibitory activity in the hemolymph was substantial, and coincided with the development of the disease. The increase in inhibitory activity in the gut was almost doubled by N. algerae as compared with V. heterosporum, whereas the increase in inhibitory activity in fat body was found only in V. heterosporum-infected larvae. The course of proteolytic activity followed an inverse pattern to the elevated activity of inhibitors in the gut and the fat body, and rose only in moribund larvae at the end of the course of V. heterosporum infection. The differences in the pattern of proteases and inhibitors reflect the organ specificity of each of the microsporidia.     

Influence ofNosema bordati (Microsporida: Nosematidae) on the biological cycle ofChilo partellus (Lep: Pyralidae), a stem borer of cultivated graminae

The biological cycle ofChilo partellus (Swinhoe) was described on artificial diet. From egg to adult, it lasted 32 to 49 days with an average of 36.6 days. About 2,000 larvae from the 2^nd to the 5^th instars were artificially infected by ingestion with doses ofNosema bordati Goudegnon, varying from 2×10² to 2×10⁷ spores per ml. Only 72 survived (7.66 %) of these infected larvae.N. bordati, when present in the larvae, continued to multiply in the resulting pupae. The parasite affected the adults of this Pyralid reducing in a proportion of 5 the productivity of infected females and increasing the production of sterile eggs in the proportion of 8.     

Benomyl: Effectiveness against the microsporidian Nosema heliothidis in the corn earworm,Heliothis zea

The fungicide benomyl was studied as a possible antimicrobial agent for obtaining Nosema heliothidis-free laboratory colonies of Heliothis zea. Newly hatched, transovarially infected larvae were placed on artificial diets containing 250, 500, or 1000 ppm benomyl. While late-stage larvae were found to be free of Nosema spores, low-level infections were found in pupae and newly emerged adults. The reduced intensity of infection in adults reared as larvae on treated diets was not correlated with a significant reduction in the efficiency of transovarian transmission. The chemical effect of benomyl was manifested by aberrant spores and vegetative stages and a rapid reduction in the number of microsporidian stages. However, small, isolated centers of infection in various host tissues resulted in a rapid resurgence of the microsporidiosis in pupae and adults. Thus, at the concentrations tested, benomyl was not effective in eliminating infection by N. heliothidis in H. zea. A discussion of the necessity for careful evaluation of the apparent suppression of microsporidioses by antimicrobial agents is also presented.     

Insecticidal activity of spore-free mutants of Bacillus thuringiensis against the Indian meal moth and almond moth

Three oligosporogenic mutants of Bacillus thuringiensis were assayed for toxicity against larvae of the Indian meal moth, Plodia interpunctella, and the almond moth, Ephestia cautella. The results were compared with insecticidal activity obtained from the parent strain (HD-1) and two standard B. thuringiensis formulations (HD-1-S-1971 and HD-1-S-1980) against the same insect species. The toxicity of the sporeless mutant preparations was significantly diminished against the Indian meal moth (10- to 26-fold increase in LC₅₀) but exceeded the toxicity of the standards against the almond moth. The toxicities of the B. thuringiensis preparations toward the Indian meal moth were consistent with the number of spores in the test samples, but spores did not contribute to toxicity to E. cautella larvae. A rationale for basing dosage on soluble protein was demonstrated for use in situations where spores are not a contributing factor in toxicity.     

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.     

Infection of the corn earworm, Heliothis zea, with Nosema acridophagus and Nosema cuneatum from grasshoppers: Relative virulence and production of spores

Per os inoculations of 4- to 6-day-old larvae of the corn earworm, Heliothis zea, with suspensions containing 10⁶ spores of Nosema acridophagus or 10⁴, 10⁵, and 10⁶ spores of Nosema cuneatum retarded the growth and development of the larvae. Migratory grasshoppers, Melanoplus sanguinipes, inoculated with N. acridophagus produced fewer spores than similarly inoculated corn earworms, but spore production was similar in these insects when they were inoculated with N. cuneatum. Standard bioassay procedures showed that spores of both microsporidians were some-what more virulent when they were produced in corn earworms than when they were produced in grasshoppers. Spores of these microsporidians might be produced more efficiently in corn earworm larvae than in grasshoppers.     

Nosema whitei,a microsporidan pathogen of some species ofTribolium

The pathogenicity ofNosema whitei was studied using a dose-mortality technique; larvae ofTribolium castaneum were reared for the duration of each experiment in flour mixed with known numbers of spores. The susceptibility of each of the first 5 larval instars was compared. The LD₅₀ (for mortality after 20 days) increased consistently from the first instar (1.8×10⁶ spores/g) to the fifth instar (1.0×10¹⁰ spores/g). The slopes of the probit lines increased consistently as age increased (from b=1.1 to b=3.9). Two factors which reduce the development time ofT. castaneum, high temperature and high humidity, both reduced the pathogenicity ofN. whitei. Thus pathogenicity decreased as the temperature was increased fram 25°C (LD₅₀=4.2×10⁶) through 30°C (LD₅₀=1.3×10⁷) to 35°C (LD₅₀=3.2×10⁶), also pathogenicity decreased consistently as humidity was increased fram 10%, through 30, 50, 70% to 90% R.H. Adults, emerging fromNosema free larvae, became infected only when exposed to a very high dose (2×10¹⁰ spores/g for 14 days from the day of emergence). Infected larvae were treated for 1 hr. at 45°C in an attempt to cure the infection. The infected larvae were not cured, rather the treatment had an adverse alfect on their survival.

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Résumé La pathogénicité deNosema whitei a été étudiée en élevant des larves deT. castaneum dans de la farine mélangée à des quantités connues de spores. La sensibilité des larves diminue uniformément en fonction de l'age; La DL₅₀ varie de 1,8×10⁶/g (1^er stade) à 1,0×10¹⁰ spores/g (5^e stade). Deux facteurs, qui accélèrent le développement deT. castaneum, des températures et des humidités élevées, réduisent tous les deux la pathogénicité deN. whitei. Les adultes ne peuvent être infectés qu'en les exposant à la dose extrêmement élevée de 2×10¹⁰ spores/g. Un traitement par la chaleur (45°C pendant une heure) n'a pas réussi à guérir les larves.

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This work financed by a Science Research Council (U.K.) studentship is based on a thesis submitted for a degree of Ph. D. at the University of Newcastle-upon-Tyne.     

Nosema ceranae Can Infect Honey Bee Larvae and Reduces Subsequent Adult Longevity

Nosema ceranae causes a widespread disease that reduces honey bee health but is only thought to infect adult honey bees, not larvae, a critical life stage. We reared honey bee (Apis mellifera) larvae in vitro and provide the first demonstration that N. ceranae can infect larvae and decrease subsequent adult longevity. We exposed three-day-old larvae to a single dose of 40,000 (40K), 10,000 (10K), zero (control), or 40K autoclaved (control) N. ceranae spores in larval food. Spores developed intracellularly in midgut cells at the pre-pupal stage (8 days after egg hatching) of 41% of bees exposed as larvae. We counted the number of N. ceranae spores in dissected bee midguts of pre-pupae and, in a separate group, upon adult death. Pre-pupae exposed to the 10K or 40K spore treatments as larvae had significantly elevated spore counts as compared to controls. Adults exposed as larvae had significantly elevated spore counts as compared to controls. Larval spore exposure decreased longevity: a 40K treatment decreased the age by which 75% of adult bees died by 28%. Unexpectedly, the low dose (10K) led to significantly greater infection (1.3 fold more spores and 1.5 fold more infected bees) than the high dose (40K) upon adult death. Differential immune activation may be involved if the higher dose triggered a stronger larval immune response that resulted in fewer adult spores but imposed a cost, reducing lifespan. The impact of N. ceranae on honey bee larval development and the larvae of naturally infected colonies therefore deserve further study.     

Transovarian transmission of Nosema plodiae in the Indian-meal moth, Plodia interpunctella

Twenty-five adult female Plodia interpunctella infected with Nosema plodiae laid 856 eggs in laboratory tests; 12.5% of the eggs were infected transovarially. The highest level of transmission by an individual female was 14 infected eggs of 27 laid (51.8%); the lowest level of transmission observed was 1 of 43 eggs (2.3%). All stages of N. plodiae were not transmitted with equal frequency; moreover, most eggs harbored predominently only the trophozoite stage of the pathogen. Approximately 80% of the infected eggs contained trophozoites almost exclusively; about 13.1% contained about an equal number of spores and trophozoites, and about 6.5% contained mainly spores. Histological observations indicated that infections may be initiated in nurse cells and subsequently transferred to associated oocytes.     

Interactions between Nosema pyrausta (Microsporidia: Nosematidae) and Bacillus thuringiensis subsp. kurstaki in the European corn borer (Lepidoptera: Pyralidae).

Larval susceptibility to Bacillus thuringiensis was determined for Nosema pyrausta-infected and uninfected European corn borers, Ostrinia nubilalis (Hübner), in bioassays using a commercial formulation of B. thuringiensis subsp. kurstaki, Dipel ES, incorporated into diet. LC50 values for N. pyrausta-infected larvae were significantly lower (P<0.0001) than for uninfected larvae and declined with increasing levels of infection. LC50 values for a 15-d bioassay using field-colony first instars were 0.006 and 0.027 mg of Dipel ES/kg of diet for larvae moderately infected by N. pyrausta and uninfected larvae, respectively. Nosema pyrausta-infected larvae reared on Dipel ES-amended diets produced 70-fold fewer spores (P<0.0001) than larvae reared on standard diet. For example, 15 d after placement as first instars on standard diet, infected field-colony larvae produced 7.6-8.7 million N. pyrausta spores per larva; similar larvae placed on diet containing 0.09 mg of Dipel ES/kg of diet produced 85-103 thousand spores per larva. Infected larvae also weighed less and failed to mature on Dipel ES-amended diets. Increased susceptibility of N. pyrausta-infected larvae to Dipel ES and reduced N. pyrausta spore production in larvae feeding on diet containing Dipel ES suggest that Bt corn will have a direct adverse effect on the survival and continual impact of N, pyrausta as a regulating factor on European corn borer populations.     

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.     

The morphology and development of Nosema carpocapsae,a microsporidian pathogen of the codling moth,Cydia pomonella (Lepidoptera: Tortricidae) in New Zealand

The morphology of Nosema carpocapsae and its development in experimentally infected codling moth larvae are described. Spherical uninucleate meronts were the first stages. Nuclear division produced binucleate meronts which were the most abundant vegetative stage, although additional uninucleate and a few tetranucleate meronts were also observed at this time. All meronts were spherical and ranged from 2.8 to 5.8 μm in diameter. Uninucleate and binucleate fusiform sporonts then appeared followed by some tetranucleate and dividing forms. Oval sporoblasts developed after these and did not divide before maturing into spores. Sporonts were approximately 5.0 to 7.9 × 2.4 to 3.0 μm. Spores developed in all host tissues except the nervous tissue. The binucleate spores showed considerable variation in spore size, 2.4 to 3.9 × 1.3 to 3.1 μm (alcohol fixed, Giemsa stained). The polar filament was usually coiled 11 times (range 9 to 13) at an angle of 53° to the long axis of the spore. Its maximum observed length was 75 μm.     

Toxicity of parasporal crystals of Bacillus thuringiensis to the Indian meal moth, Plodia interpunctella.

Toxicity of Bacillus thuringiensis parasporal crystals to the Indian meal moth, Plodia interpunctella, is described. The numbers of insects killed were in relation to crystal dry weight. Mortality was determined by comparing adult emergence in diets treated with crystals to emergence in untreated diets. There was only a 30% survival at an application of 0.414 microgram/cm2, and the mean 50% lethal concentration value was found to be 0.299 microgram/cm2. The use of emergence data has provided a reliable and reproducible bioassay for comparing relative toxicities of crystals, spores, and other cellular components to this economically important insect.     

Toxicity of parasporal crystals of Bacillus thuringiensis to the Indian meal moth, Plodia interpunctella.

Toxicity of Bacillus thuringiensis parasporal crystals to the Indian meal moth, Plodia interpunctella, is described. The numbers of insects killed were in relation to crystal dry weight. Mortality was determined by comparing adult emergence in diets treated with crystals to emergence in untreated diets. There was only a 30% survival at an application of 0.414 microgram/cm2, and the mean 50% lethal concentration value was found to be 0.299 microgram/cm2. The use of emergence data has provided a reliable and reproducible bioassay for comparing relative toxicities of crystals, spores, and other cellular components to this economically important insect.     

Effect of timing of application of Nosema marucae on the control of the spotted stalk borer Chilo partellus infesting sorghum

A local variety of sorghum (Serena) was planted in a screenhouse and infested with first‐instar Chilo partellus larvae. Sorghum rows were then inoculated at random with an aqueous suspension of Nosema marucae containing 1.6 ×10⁸ spores ml^‐1 at 2‐day intervals from 1 to 16 days after infestation (DAI). Foliar damage was low in plants inoculated with the pathogen at 1 and 3 DAI, where the highest score was 0.2 on a scale of 0–9. However, leaf damage increased rapidly as the interval between infestation and spraying of N. marucae became longer. At 19 DAI the foliar damage score on plants that had been inoculated at day 16 was 8.4. Similarly, the proportion of plants with either fully or partially formed heads decreased from 97.9% in plants sprayed at 3 DAI, to 10.8% at 11 DAI and 0% at 16 DAI. The timing of application of N. marucae is therefore important in the utilization of the microsporidian for the biological control of stem‐boring lepidopterans. This study suggests that a single, well timed application of Nosema spores can effectively control C. partellus for up to 19 days following oviposition.     

Production of microsporidia pathogenic to the Colorado potato beetle (Leptinotarsa decemlineata) in alternate hosts

The microsporida Nosema gastroideae and N. equestris, which are highly pathogenic for Leptinotarsa, have been successfully produced in some other chrysomelid species, Gastrophysa polygoni and G. viridula. As the principal target host, Leptinotarsa is very susceptible to these pathogens, and death occurs before massive sporulation by the microsporidia. By contrast, the infected larvae of G. polygoni or G. viridula are able to develop until the adult stage when most of the tissues become filled with spores. In addition, the larvae and adults of these species can be reared in the laboratory on Polygonum aviculare and Rumex obtusifolius. These plants have longer vegetative periods and are better sources of food than potato leaves. In both species of Gastrophysa the yields of spores related to unit weight were about five times higher than in Leptinotarsa. In the adults of G. viridula there was up to 4.8 × 10⁶ spores mg^?1 body weight of N. gastroideae, or 9.1 × 10⁶ spores mg^?1 of N. equestris. The higher content of microsporidian spores facilitates their purification and isolation.     

Nosema tyriae n.sp. and Nosema sp., microsporidian parasites of Cinnabar moth Tyria jacobaeae.

Nosema tyriae n.sp. was found in 63% of a population of Cinnabar moth larvae (Tyria jacobaeae). The infection was found in the gut wall, silk glands, and fat body and was probably generalized but appeared to be of low pathogenicity. Merogony and sporogony were by binary fission of diplokaryotic stages. Fresh spores were elongate, slightly pointed at the anterior end, and measured 4.7 x 2.0 microm. Ultrastructural features of special interest were 20-nm tubules connecting the surface of sporonts with host cell cytoplasm and, in the spores, a deeply domed polar sac, polaroplast consisting of closely packed longitudinally arranged membranes and loosely packed horizontally arranged membranes, and 10.5-14 coils of the polar tube in a single rank. The 16S rRNA genes of N. tyriae and Nosema bombycis from silkworms, Bombyx mori, differed by only six nucleotides and N. tyriae spores gave a moderately positive reaction with a monoclonal antibody raised to N. bombycis. N. tyriae was infective to B. mori but was less virulent than N. bombycis. However, no amplification product was obtained by PCR using N. tyriae DNA and primers considered to be specific for N. bombycis. Also, the spores of the two species are of entirely different shapes. A second diplokaryotic microsporidium, Nosema sp., found as a light infection in only one of the larvae had much smaller developmental stages and spores measuring 3.8 x 2.0 microm (fixed). Ultrastructurally it was distinguished by an abundance of dense membranes in cytoplasmic vesicles in both meronts and sporonts. Spores with up to 15 coils of the polar tube in irregular clusters or with about 12 coils in a single rank were observed in the tissues fixed from the one larva infected with this parasite. As this larva had been kept with N. tyriae-infected larvae for a few days before examination, it is possible that the two types of spores resulted from a double infection.     

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.     

Susceptibility of spores of the microsporidian Nosema algerae to sunlight and germicidal ultraviolet radiation

Spores of the microsporidian Nosema algerae exposed to 1, 2, and 4 hr of sunlight and 121 μW/cm² of germicidal ultraviolet light were fed to first-instar Anopheles albimanus. Twenty days after feeding, the incidence and intensity of infection (spores/mosquito) were recorded from adult mosquitoes. While sunlight-treated spores showed no significant decline in incidence of infection after 1-, 2-, and 4-hr exposures, intensity of infection decreased significantly after the 2- and 4-hr exposures. Incidence of infection of mosquitoes fed bactericidal ultraviolet light-treated spores declined 48.2, 76.2, and 99.9% after 1-, 2-, and 8-min exposures, respectively. Measured by intensity of infection, activity of bactericidal light-treated spores decreased 87.2% after 1 min, 91.7% after 2 min, and 99.9% after 8 min. Levels of radiation required to inactivate spores of N. algerae fell within the range reported for other Microsporidia.