A novel milky disease organism from Australian scarabaeids: Ultrastructure

The unusual ultrastructure of a milky disease bacterium isolated from Aphodius tasmaniae is described and compared with a similar bacterium from Tipula paludosa. Both organisms possess an elongate sporangium with a small central spore, a multilayered exosporium, and longitudinally arranged extrasporal fibrils which arise from plates attached to the ends of the spore. The morphogenesis of the sporangium is described and illustrated for the organism from A. tasmaniae. It is suggested this organism and that from T. paludosa are closely related. Their taxonomic position remains obscure.     

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.     

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.     

Microsporidium goeldichironomi n. sp. and Microsporidium chironomi n. sp. (Microsporida: Apansporoblastina): Two new microsporidia from Florida chironomids

Two new species of Microsporida belonging to the genus Microsporidium are described. Microsporidium goeldichironomi n. sp. parasitizes the fat body of Goeldichironomus holoprasinus and Microsporidium chironomi n. sp. infects Chironomus attenuatus. Both microsporidia form uninucleate spores from rosette-shaped sporonts. M. goeldichironomi sporonts form 4, 6, 8, 10, 12, 16, and possibly more spores. Two shapes of spores are produced, oval, or slightly pyriform spores measuring 3.70 ± 0.09 × 2.49 ± 0.13 μm and pyriform spores measuring 3.74 ± 0.44 × 2.04 ± 0.17 μm. Electron micrographs show that both types of spores are uninucleate, have 8 to 11 polar filament coils and a lamellate polaroplast showing several distinct regions. M. chironomi spores are pyriform and are often joined at the posterior end in groups of two or four. They measure 4.12 ± 0.37 × 2.45 ± 0.26 μm. The spores are uninucleate, have six to seven polar filament coils and a lamellate polaroplast showing two distinct regions. Neither species can be transmitted per os and thus are assumed to be transovarially transmitted. No pansporoblastic membrane is present in either species.     

The development of milky disease under laboratory and field temperature regimes

The effect of temperature on the development of milky disease agent, Bacillus popilliae var. rhopaea, in third-instar Rhopaea verreauxi larvae was studied in two experiments. In the first experiment, only small differences were found between the probit lines of final percentage infection against dose under five temperature regimes (constant 18, 23, and 28°C; alternating 13–23°C, and 18–28°C). The disease developed more slowly at the lower temperatures while alternating temperatures gave shorter ET₅₀ values than the equivalent constant temperatures. In the second experiment, a higher incidence of disease was obtained under field conditions than under any of the laboratory conditions tested. It was concluded that for up to 6 months/year temperature is not likely to limit the development of B. popilliae var. rhopaea in the field, and that, under field conditions, most larvae would achieve a Phase IV infection.     

Histopathology of virus-like particles in Heliothis spp

Larvae of Heliothis spp. collected from cotton, soybean, and peanut fields in South Carolina were found to be infected with virus-like particles (VLPs). Infected larvae became pale and swollen, stopped feeding, and remained alive for 2–3 weeks. Hemolymph from these larvae was milky and contained numerous spherical bodies ranging in diameter from 2 to 10 μm. The hemolymph also contained VLPs which were oval and measured 375 × 125 nm. Infectivity tests with crude saline extracts of infected larvae demonstrated that the pathogen could be transmitted by injection but not per os. The spherical bodies contained VLPs (387 × 149 nm) surrounded by two envelopes and packed together in clusters. These VLPs were also found in fat body cells, cuticular epithelial cells, tracheal cells, and connective tissue associated with the body wall and the gut. They were not found in muscle tissue or in midgut epithelial cells. Similar VLPs have been found in Heliothis zea from Mississippi and Trichoplusia ni from California, but a positive identification of the VLPs has not been made in any of these studies. Morphologically they appear to be distinct from any other previously described insect viruses.     

A method for isolating milky disease, Bacillus popilliae var. rhopaea, spores from the soil

A method based on the tyndallization procedure is described for isolation of Bacillus popilliae var. rhopaea spores from the soil. A soil suspension is diluted with a germinating medium, which promotes the germination of most spores except B. popilliae var. rhopaea, and is treated with a series of seven heat shocks (70°C for 20 min) at hourly intervals. This treatment reduced the number of contaminant spores by over 95%. The suspension is then plated out onto “J” medium which allows the germination and growth of all surviving spores including the milky disease spores. The plates are incubated anaerobically at 28°C for 7 days before the characteristic small transparent colonies of B. popilliae var. rhopaea are counted. In testing the method it was revealed that about 15% of the milky disease spores in the soil produced visible colonies, and that a spore concentration of over 1.2 × 10⁵ spores/g dry wt of soil could be quantified. This concentration of spores produces only 3% infection in Rhopaea verreauxi larvae. The method may be applicable to other varieties of B. popilliae which will grow on “J” medium.     

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.     

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.     

Impact of Bacillus popilliae,Rickettsiella popilliae and entomopathogenic nematodes on a population of the scarabaeid,Cyclocephala hirta

Larvae of the scarabaeid, Cyclocephala hirta, are major pests of turfgrass in California. A field test was conducted against third instars that included the following treatments: untreated control; chemical insecticide (bendiocarb); milky disease bacterium (Bacillus popilliae); and entomopathogenic nematodes (Steinernema feltiae and Heterorhabditis bacteriophora). There were no significant differences in population reduction among the treatments, but the larval population in all plots showed a dramatic decline. The C. hirta population had a natural occurrence of milky disease and blue disease caused by Rickettsiella popilliae. The prevalence of blue disease during the course of the study averaged < 10% but that of milky disease averaged about 20%. More significantly, the soil from all treatment plots when bioassayed for B. popilliae showed that 67–90% of the larvae became infected with this bacterium. None of the larvae became infected with the blue disease organism. We conclude that B. popilliae was occurring in epizootic proportions in our field tests and was a significant mortality factor in causing the decline of the C. hirta population.     

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.     

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.     

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.     

Ecology of Bacillus thuringiensis in storage moths

A disease-free stock of Plodia interpunctella was produced by a continuous rearing technique. In dense populations of this stock, 10⁴ or more spores of H serotype V Bacillus thuringiensis applied at one point on the surface of 200 g of food were required to cause epizootics, compared with 10⁷ or more when spread evenly over the surface. In infected populations, spores contaminated the surfaces of all stages of the insect. In diseased larval cadavers there were 5.6–42.2 × 10⁸ spores/g of dry insect (P. interpunctella, Ephestia cautella, Anagasta kuehniella, Ephestia elutella, and Galleria mellonella). Larvae did not cannibalize live larvae while food was present though they sometimes ate cadavers. This is the most potent means of natural spread of the disease. Occurring mainly in protected situations such as food stores, natural infections are usually light, but occasionally spectacular surface accumulations of dead larvae occur, possibly associated with stress, physiological condition of the larvae, serotype of the bacterium, or behavior pattern such as migration. Natural disease may curb infestations in debris, but it attacks too late to prevent excessive damage to stored food. A prophylactic, even admixture of 2 × 10⁹ spores/200 g of food is required for effective insect control.     

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.     

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.     

Heliothis zea larval mortality time from topical and per os dosages of Nomuraea rileyi conidia

Heliothis zea larval mortality time was established for topical applications and per os (feeding) with fungal conidia of Nomuraea rileyi. The 6-to 7-day-old larvae received per os dosages ranging from 6 × 10⁶ to 9 × 10⁹ and a topical dosage of ca. 1 × 10⁹. These spore loads initiated fungal infections in larvae resulting in 1.5–100% accumulated mortality during the 30-day testing periods. The data indicated that regardless of the larval treatment method (topical or per os), or conidial dosage rates, the larval mortality time was never shorter than 5 days post-treatment, nor longer than 22 days. The highest percentage of larval deaths occurred from 7 to 9 days with a maximum mortality at 8 days.     

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.     

The use of a microinjection procedure for large-scale production of the microsporidian Nosema eurytremae in Pieris brassicae

Nosema eurytremae, a microsporidian parasite of Malaysian trematodes, was injected at the rate of 1 × 10⁴ spores/larva into Pieris brassicae. The larvae, which subsequently pupated, were incubated at 25 to 26°C and on harvesting 19 days later yielded an average of 6 × 10⁸ spores/pupa. This was equivalent to 60,000 times the initial dose. Purity of filtered, washed spore suspensions ranged from 80 to 99% with up to 20% host debris.     

Effects of temperature and dosage on Vairimorpha sp. 696 spore morphometrics,spore yield,and tissue specificity in Heliothis virescens

The effects of temperature and dosage on a new microsporidian species, Vairimorpha sp. 696, were examined in H. virescens. The pathogen was evaluated for tissue specificity, spore size, cumulative percentage mortality, and spore production. All tissues examined bore infection at 32°C. Spore length was significantly longer at 19°C (5.9 μm) than at 32°C (4.7 μm). Spore widths at these two temperatures did not differ significantly. Octospores were not found at either temperature at 8 or 12 days postinoculation. One hundred percent mortality was attained in all dosages administered, but the initial rate of mortaily was more rapid in the higher dosages. Finally, spore yield was greater in larvae administered lower dosages. Maximum spore yield at 27°C was 4.87 × 10⁹ spores/larva.