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.     

Importance of spores,crystals, and δ-endotoxins in the pathogenicity of different varieties of Bacillus thuringiensis in Galleria mellonella and Pieris brassicae

Physical methods were used to produce spores containing impurities of 0.02–0.05% crystals and crystals containing impurities of 0.001–0.01% spores from cultures of Bacillus thuringiensis. In Galleria mellonella larvae, these preparations from varieties galleriae, aizawai, and wuhanensis were only moderately active compared to 1:1 mixtures of spores and crystals. Spores of an acrystalliferous aizawai mutant were inactive and did not contain a polypeptide of the same size as the potent M_r 138000 δ-endotoxin present in spores and crystals of all three wild-type strains. Thus, this polypeptide probably contributed to the moderate activity of wild-type spores. Spore impurities in the crystal preparation were killed by γ irradiation without harming the crystals. The crystals without live spores were virtually inactive (LC₅₀s, ca. 10¹⁰ crystals/g insect food). Addition of 10³ spores to 10⁸ crystals/g food (0.001% spores) increased the mortality of larvae from 0 to 36%, and addition of 10⁴ spores (0.01% spores) killed 64% larvae. Thus, the addition of low levels of spores increased the potency of crystals in G. mellonella from virtually zero to moderate levels, suggesting that the live spore impurities in the crystal preparations were responsible for the observed moderate potency of crystals before γ irradiation, a view supported by a reduction of potency of crystal preparations following admixture of streptomycin to the insect food. In contrast to the results with G. mellonella, crystals were ca. 30 times as active as spores in Pieris brassicae larvae. Many authors have found crystals purified by physical methods to be highly active in a range of lepidopterous hosts. The present work indicates that the role of the spore impurities in these species may need further investigation. Absence of live spores of B. thuringiensis may impair the control of some insect species feeding on spore-free products and on microorganisms or plants into which endotoxins have been introduced by genetic manipulation.     

Infection of Tribolium castaneum with Bacillus thuringiensis: Quantification of Bacterial Replication within Cadavers,Transmission via Cannibalism,and Inhibition of Spore Germination

Reproduction within a host and transmission to the next host are crucial for the virulence and fitness of pathogens. Nevertheless, basic knowledge about such parameters is often missing from the literature, even for well-studied bacteria, such as Bacillus thuringiensis, an endospore-forming insect pathogen, which infects its hosts via the oral route. To characterize bacterial replication success, we made use of an experimental oral infection system for the red flour beetle Tribolium castaneum and developed a flow cytometric assay for the quantification of both spore ingestion by the individual beetle larvae and the resulting spore load after bacterial replication and resporulation within cadavers. On average, spore numbers increased 460-fold, showing that Bacillus thuringiensis grows and replicates successfully in insect cadavers. By inoculating cadaver-derived spores and spores from bacterial stock cultures into nutrient medium, we next investigated outgrowth characteristics of vegetative cells and found that cadaver-derived bacteria showed reduced growth compared to bacteria from the stock cultures. Interestingly, this reduced growth was a consequence of inhibited spore germination, probably originating from the host and resulting in reduced host mortality in subsequent infections by cadaver-derived spores. Nevertheless, we further showed that Bacillus thuringiensis transmission was possible via larval cannibalism when no other food was offered. These results contribute to our understanding of the ecology of Bacillus thuringiensis as an insect pathogen.     

Spore Load of Ascosphaera Species on Emerging Adults of the Alfalfa Leafcutting Bee, Megachile rotundata

The spore load of Ascosphaera species spores on larval chalkbrood cadavers and newly emergent adults of the alfalfa leafcutting bee, Megachile rotundata, was determined. The spore content of chalkbrood cadavers ranged from 3 × 10⁶ to 5 × 10⁸. Adults emerging through zero to nine cadavers carried spores on all body parts examined by scanning electron microscopy. Estimates of the total number of spores obtained from a series of adult washes ranged from 9 × 10⁴ to 8 × 10⁷. Some adult males which emerged through no cadavers carried 10⁴ to 10⁵ spores, indicating that nesting materials might also have been contaminated. However, the control of chalkbrood in commercial bee populations may not be accomplished simply by providing clean nesting materials as adults may still emerge through diseased larvae.     

The in vivo production of Bacillus popilliae var. rhopaea spores

The effect of various factors on the yield of Bacillus popilliae var. rhopaea spores formed in Rhopaea verreauxi larvae have been studied. Lack of adequate food, temperatures above and below 23°C, and infecting doses above 10⁶ spore larva, all significantly lowered spore yield per larva. Larval age had a pronounced effect; second-instar and young third-instar larvae produ ed about 1 × 10¹⁰ spores while old third-instar larvae produced about 4 × 10¹⁰ spores. Incubation of larvae for longer than 4 weeks did not increase spore yield per larva. Yields were similar whether larvae were infected by injection or per os. Three other host species could be used to mass-produce B. popilliae var. rhopaea spores but all were less efficient than R. verreauxi. Milky third-instar R. verreauxi larvae, which were field collected, yielded 1.57 × 10¹⁰ spores per larva.     

In vivo development and pathogenicity of Ascosphaera proliperda (Ascosphaeraceae) to the alfalfa leafcutting bee, Megachile rotundata

First instar larvae of the leafcutting bee, Megachile rotundata, were fed on either artificial or natural provisions containing spores of Ascosphaera proliperda. Two isolates were used as a source of inocula: one originated from in vitro isolates obtained while culturing what was thought to be pure spores of A. aggregata, the second originated from in vitro cultures from Denmark. Histological and scanning electron microscopy studies revealed that the spores germinated in the gut lumen and the developing hyphae invaded all tissues, after which they penetrated through larval integument and began the sexual phase of the life cycle aerially. Virtually all fungus-exposed larvae developed symptoms of disease regardless of source of inoculum, type of provision, and spore dose (1.5 × 10³ to 3 × 10⁶) per insect. It was concluded that the fungus was pathogenic to the alfalfa leafcutting bee under laboratory conditions and future studies should be conducted to determine its etiology, cross infectivity, and natural distribution in other bee taxa.     

A laboratory evaluation of the pathogenicity of Bacillus popilliae var. rhopaea, the agent of milky disease in Rhopaea verreauxi (Coleoptera: Scarabaeidae)

Two methods of infection, i.e., feeding known numbers of spores and rearing larvae in contaminated peat, were used to bioassay the susceptibility of Rhopaea verreauxi to Bacillus popilliae var. rhopaea at 23°C. The susceptibility of the three larval instars was similar as measured by the ID₅₀ and IC₅₀ values. However, within an instar, newly molted larvae were less susceptible than mature larvae when infected by the contaminated peat method. It is suggested that this was due to reduced food intake. The range of ID₅₀ values for all bioassays with R. verreauxi larvae were 1.1 × 10⁷ to 4.0 × 10⁷ spores per larva, and IC₅₀ values were 3.4 × 10⁶ to 5.0 × 10⁷ spores per g of contaminated peat. The slope of the probit line was always low (0.6 to 1.8) except for young first-instar larvae infected by contaminated peat when the slope was 4.0. Disease per se did not affect food intake, though intake was reduced at high doses of contaminated peat. Young larvae often died without developing symptoms but, with increasing age, infected larvae were more likely to develop symptoms. Bioassays with Othnonius batesi and Rhopaea morbillosa indicated a much lower susceptibility per os than for R. verreauxi. It is concluded that the potential for using B. popilliae var. rhopaea to control R. verreauxi is high, but the bacillus is unlikely to be of value in control of O. batesi or R. morbillosa.     

Pathogenicity of a nuclear polyhedrosis virus of the almond moth, Cadra cautella

Tests were conducted with neonate Cadra cautella larvae to determine the pathogenicity of a nuclear polyhedrosis virus. A bioassay on an agar base diet showed that concentrations of 0.25, 0.50, 2.00, and 4.00 polyhedra/mm² killed 27, 55, 87, and 92% of the test larvae, respectively. A study of the time of death showed that most larvae died on the 9th or 10th day after exposure to 4 polyhedra/mm² at 27°C. When larvae were exposed to 8, 16, 32, and 64 × 10³ polyhedra/g of bran diet, recorded mortalities were 18, 22, 48, and 80%, respectively. All the samples of virus in bran diet which were incubated at various temperatures for 7, 14, and 28 days remained stable at all test conditions except the sample incubated at 42°C for 14 days, and those held at 37° and 42° for 28 days. Larvae of C. cautella, Plodia interpunctella, Ephestia elutella, and Paramyelois transitella placed on a diet with 40 × 10³ polyhedra/g had mortalities of 75, 59, 16, and 4%, respectively. Light and electron microscopical examination of P. interpunctella cadavers showed that they were infected with a multiply occluded nuclear polyhedrosis virus.     

The susceptibility ofTribolium castaneum [Col.: Tenchrionidae] toFarinocystis tribolii [Protozoa: Schizogregarinida]

The LT₅₀ ofFarinocystis tribolii Weiser to larvae ofTribolium castaneum (Herbst) increased with the age of the insect indicating that older larvae were relatively more tolerant to the infection though there was 100 % mortality ultimately. The adults were less susceptible than larvae and between sexes, females were more susceptible than males. The number of spores produced increased with the stage of the larvae, but there was no variation in the size of spores in the different instars. The LC₅₀ on 20th and 40th day of inoculation were 1.4×10⁷ and 2.1×10⁶ respectively. Mortality-time due toF. tribolii was shorter at 35 °C than at 25 °C. Sporulation occurred earlier at 35 °C than at 25 °C.     

Comparative virulence of Nosema plodiae and Nosema heterosporum in the Indian meal moth, Plodia interpunctella

When larvae of the Indian meal moth, Plodia interpunctella, were fed diets containing spores of Nosema plodiae, the number that survived to the adult stage decreased and the rate of adult emergence was retarded as the concentration of spores was increased; all surviving adults were infected. Also, when larvae were reared on diets containing spores of Nosema heterosporum, the number that survived to the adult stage decreased as the concentration of spores was increased; however, no relationship was apparent between concentration of spores and the rate of adult emergence. The LC₅₀'s of N. plodiae and N. heterosporum were 8.09 × 10⁶ and 4.52 × 10³ spores/g diet, respectively, which confirmed preliminary observations regarding the relative virulence of the two species of Nosema to Indian meal moth larvae.     

A novel milky disease organism from Australian scarabaeids: Field occurrence,isolation, and infectivity

A novel milky disease organism has been found causing disease in Aphodius tasmaniae and other scarabaeid larvae in the field in Australia. The sporangium is exceptionally long, measuring 10.5 × 1.5 μm, with a small central spore, measuring 1.0 × 0.6 μm. The vegetative cell is about half the size of the sporangium. The disease was easily transmitted by injection of spores into the hemocoel, with typically milky symptoms developing in 2–4 weeks. Spores will form in vivo at temperatures down to 12°C. For A. tasmaniae third-instar larvae, the ID₅₀ by injection was 3 × 10² spores/larva, yet no infection resulted when larvae were reared in peat containing up to 10⁸ spores/g, i.e., the disease was not successfully transmitted per os. All 10 species of scarabaeids tested were susceptible to the disease when spores were injected; however, all attempts to infect larvae per os were unsuccessful. In vitro culture was also unsuccessful.     

Some characteristics of a new isolate of Helicosporidium and its effect upon mosquitoes

A Helicosporidium sp. was isolated by feeding spores concentrated by continuous flow centrifugation from ditch water to starved Heliothis zea larvae. This Helicosporidium sp was infectious to Anopheles quadrimaculatus, Culex salinarius, and Aedes aegypti with IC₅₀'s of 4.4 × 10², 2.6 × 10⁴, and 2.4 × 10⁴ spores/ml, respectively. Larval mortality was dosage dependent with LC₅₀ values 72 hr postexposure of 6.8 × 10⁴ for An. quadrimaculatus, 9.4 × 10³ for Cx. salinarius, and 1.5 × 10⁵ for Ae. aegypti. The spores of this Helicosporidium were also tolerant of freezing and desiccation. Because of these traits and the melanization response they provoked in host tissues, this is probably not naturally a mosquito pathogen and is most likely from a terrestrial insect.     

The reproduction potentials of four entomopathogenic nematode strains related to cost-effective production for biological control

Bioassays to evaluate the mortality, virulence and reproduction potentials of four indigenous EPN strains, S-PQ16, S-BM12, H-KT3987 and H-CB3452 on insect larvae of mealworm (Tenebrio molitor) and greater wax moth (Galleria mellonella) revealed the highest mortality rates of two insect larvae at the highest inoculation dose of 100 IJs to range from 89 to 100 percent and 94.3–100 percent at 48 h after inoculation, respectively. Virulence was high for all nematode strains, with LC₅₀ values between 29.6 and 47.3 IJs/insect host. The highest IJ yields were different between nematode strains and insect host, from 66.8 × 10³ IJs (S-PQ16) to 118.6 × 10³ IJs (H-KT3987) on T. molitor, and from 54.2 × 10³ IJs (S-BM12) to 163.3 × 10³ IJs (H-KT3987) on G. mellonella. The culturing cost in terms of food expenditure for rearing insect larvae varied between insect larvae and nematode strains, from 6.76 to 26.63 USD per billion IJs for nematode strains cultured on T. molitor larvae and from 3.54 to 7.81 USD per billion IJs for nematode strains cultured on G. mellonella larvae. The full cost for a nematode product of 2.5 × 10⁹ IJs per hectare, produced through in vivo mass culturing, of the most efficient nematode strain, H-KT3987, was 191.3 USD, slightly cheaper than 199.4 USD for the same nematode product produced through in vitro mass culturing.     

Mass production and storage of Vairimorpha necatrix (protozoa: Microsporida)

Mass production and storage methods were evaluated for maximization of spores of Vairimorpha necatrix, a promising protozoan for microbial control due to its virulence and prolificity in lepidopterous pests. In vivo spore production was at a maximum when 3rd instar Heliothis zea were exposed to 6.6 spores/mm² of artificial diet surface and reared for 15 days. Approximately 1.67 × 10¹⁰ spores/larva were produced, or ca. 1 × 10¹⁰ spores/larva after partial purification of the spores by homogenization of the larvae in water, filtration, and centrifugation. The spores were inactivated by relatively short exposures to several chemicals which were tested to counteract contamination of the diet surface by fungi in the spore inoculum. Spores of V. necatrix were stored at refrigerated and freezing temperatures for up to 2 years and bioassayed periodically with 2nd instar H. zea. Spores lost little infectivity after 23 months at 6°C if they were stored in a purified water suspension plus antibiotic, but they were noninfective after 18 months at 6°C if stored in host tissue. Storage at ?15°C caused little loss of infectivity whether the spores were stored in water and glycerine, in host tissue, or after lyophilization. The spores withstood lyophilization in host cadavers better than in purified water suspension. Samples of a dry V. necatrix-corn meal formulation, which was prepared for field efficacy tests and stored at ?15° and 6°C, were highly infective after 9 months. Large numbers of V. necatrix spores can thus be produced and later made available for microbial control field trials with little loss of infectivity.     

Persistence of spores of Pleistophora schubergi (Cnidospora: Microsporida) in the field,and their application in microbial control

Spores of Pleistophora schubergi, when applied to oak trees in the field at 2 × 10⁸ spores/ml with a uv protectant, “Shade,” infected 88% of Anisota senatoria larvae at 4 days after spray application. Spores without the uv protectant infected only 10% of the larvae at 4 days after application. When the spores were applied at the rate of 2 × 10⁸ and 2 × 10⁷ spores/ml in the field, 96 and 72% of the A. senatoria larvae and 100 and 100% of the Symmerista canicosta larvae were infected 14 days after spray application.     

Foliar application ofNosema pyrausta for suppression of populations of European corn borer

In 1974, an application of the microsporidan,Nosema pyrausta (Paillot, 1927)Kotlan, 1928, with a back-pack type sprayer (22.5×10⁷ spores/plant) to whorl stage maize infested with European corn borers,Ostrinia nubilalis (Hübner) reduced the number of larvae/plant by 48.1% and produced an infection of 15.3×10⁴ spores/mg of larval weight in 62.1% of the collected larvae. In 1975, applications of 24.3×10⁷ spores/plant to similar maize, in 2 separate tests, reduced the number of larvae/plant by 18.8 and 43.8% and caused an infection of 14.3 and 19.1×10⁴ spores/mg of larval weight in 65.9 and 63.3% of the collected larvae. Also, in 1975, applications of 24.3×10⁷ spores/plant to pollen shedding maize in 2 separate tests reduced the number of larvae/plant by 17.2 and 14.1% and caused an infection of 24.3 and 27.2×10⁴ spores/mg of larval weight in 99.2 and 95.2% of the collected larvae.     

Pathogenesis of infection by the hyphomycetous fungus,Tolypocladium cylindrosporum inAedes sierrensis andCulex tarsalis [Dip.: Culicidae]

The mode of infection and cycle of development ofTolypocladium cylindrosporum Gams was examined inAedes sierrensis andCulex tarsalis. Larvae were found to be infected through the external cuticle, the pharynx and the midgut. Blastospores and conidia were both infective although for equal numerical concentrations blastospores proved more virulent causing high mortality within the first 48 h after inoculation (80 % for L2 larvae exposed to 5×10⁵ spores/ml), while conidia generally took 7–10 days to produce the same results. Sporulation did not occur on submerged cadavers. Conidia were produced only on floating cadavers in contact with air. Conidial production on floating 4th instar larvae was found to average 1.8×10⁷ conidia/larva. Invasion of the haemocoele and fairly extensive growth of the fungus almost invariably occurred before larvae were killed. This was particularly true forAedes sierrensis larvae. Details are presented of growth within the host and post-mortem penetration of the fungus out of the cadaver. AdultA. sierrensis sprayed with a conidial suspension proved susceptible to infection with 100 % mortality being recorded at 10 days. Infections originated in the thorax, suggesting, the integument or possibly the thoracic spiracles to be the most probable site of infection.     

Microbial control of the fruit tree leafroller,Archips argyrospila [Lep.: Tortricidae] in California

Field populations of larvae of the fruit tree leafrollerArchips argyrospila (Wlk). were practically eliminated following spray application ofBacillus thuringiensis Berliner serotype III at 192 and 80×10⁶ I.U. per litre on the host trees,Cercis occidentalis. Spray applications of lower rates ofB. thurienginsis serotype III at 18.9 and 32.1×10⁶ I.U. per litre and mist application ofB. thuringiensis serotype 1 at 8.0 and 16.0×10⁶ I.U. per litre gave partial control of populations ofA. argyrospila larvae. A granulosis typeBaculovirus, applied by hand sprayer at 1.4×10⁹ granules/ml produced approximately 50% reduction of 5th instarA. argyrospila larvae onC. occidentalis trees. It was concluded thatB. thuringiensis and the granulosis typeBaculovirus are promising control agents forA. argyrospila larvae.     

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.     

Production of two entomopathogenic Aspergillus species and insecticidal activity against the mosquito Culex quinquefasciatus compared to Metarhizium anisopliae

The spore productivity and insecticidal activity of two opportunistic insect pathogenic Aspergillus species (namely: Aspergillus clavatus Desmazieres and Aspergillus flavus Link (Ascomycota: Eurotiales, Trichocomaceae)) were compared to Metarhizium anisopliae sensu lato (Metchnikoff) Sorokin (Ascomycota: Hypocreales, Clavicipitaceae) for mosquito (Diptera: Culicidae) control. The production of aerial spores on wheat bran and white rice was investigated in solid-, semi-solid-, and liquid-state media supplemented with a nutritive solution. Wheat bran-based media increased the spore yield in solid-state from three to sevenfold: A. clavatus produced 48.4?±?5.2 and 15.7?±?1.6?×?10⁸ spores/g, A. flavus produced 22.3?±?4.1 and 3.1?±?2.5?×?10⁸ spores/g, and M. anisopliae produced 39.6?±?6.5 and 13.1?±?2.6?×?10⁸ spores/g of wheat bran or white rice, respectively. A. clavatus, A. flavus and M. anisopliae spores harvested from wheat bran-based solid-state media showed lethal concentrations (LC₅₀) of 1.1, 1.8, and 1.3?×?10⁸ spores/ml against Culex quinquefasciatus Say larvae in 72?h. Because A. clavatus and M. anisopliae displayed similar features when cultured under these conditions, our results suggest that insect pathogenic Aspergillus species may be as productive and virulent against mosquito larvae as a well-recognised entomopathogenic fungus.