Cross-infection of a nuclear polyhedrosis virus of the almond moth to the indian meal moth

A nuclear polyhedrosis virus isolated from the almond moth, Cadra cautella, was cross-infective to the Indian meal moth, Plodia interpunctella: Concentrations of polyhedra of 8, 16, 32, and 64 × 10³/g of bran diet killed 12, 19, 24, and 45% of the exposed neonate P. interpunctella larvae respectively. Viral replication in P. interpunctella occurred in cell nuclei of the hypodermis, tracheal matrix, fat, Malpighian tubules, in tissue associated with muscles, and in columnar cells of the midgut. However, infection in the alternate host developed more slowly, and the tissue tropisms were less apparent. Also, polyhedra in P. interpunctella were often cuboidal, although they always developed normally in C. cautella. The average number of virions per bundle and the average size of the occluded virions were about the same in both hosts.     

Synergism between Bacillus thuringiensis Spores and Toxins against Resistant and Susceptible Diamondback Moths (Plutella xylostella)

We studied the effects of combinations of Bacillus thuringiensis spores and toxins on the mortality of diamondback moth (Plutella xylostella) larvae in leaf residue bioassays. Spores of B. thuringiensis subsp. kurstaki increased the toxicity of crystals of B. thuringiensis subsp. kurstaki to both resistant and susceptible larvae. For B. thuringiensis subsp. kurstaki, resistance ratios were 1,200 for a spore-crystal mixture and 56,000 for crystals without spores. Treatment of a spore-crystal formulation of B. thuringiensis subsp. kurstaki with the antibiotic streptomycin to inhibit spore germination reduced toxicity to resistant larvae but not to susceptible larvae. In contrast, analogous experiments with B. thuringiensis subsp. aizawai revealed no significant effects of adding spores to crystals or of treating a spore-crystal formulation with streptomycin. Synergism occurred between Cry2A and B. thuringiensis subsp. kurstaki spores against susceptible larvae and between Cry1C and B. thuringiensis subsp. aizawai spores against resistant and susceptible larvae. The results show that B. thuringiensis toxins combined with spores can be toxic even though the toxins and spores have little or no independent toxicity. Results reported here and previously suggest that, for diamondback moth larvae, the extent of synergism between spores and toxins of B. thuringiensis depends on the strain of insect, the type of spore, the set of toxins, the presence of other materials such as formulation ingredients, and the concentrations of spores and toxins.     

Resistance to Bacillus thuringiensis by the Indian meal moth, Plodia interpunctella: comparison of midgut proteinases from susceptible and resistant larvae

Midgut homogenates from susceptible and resistant strains of the Indian meal moth, Plodia interpunctella, were compared for their ability to activate the entomocidal parasporal crystal protein from Bacillus thuringiensis. The properties of midgut proteinases from both types of larvae were also examined. Electrophoretic patterns of crystal protein from B. thuringiensis subspecies kurstaki (HD-1) and aizawai (HD-133 and HD-144) were virtually unchanged following digestion by either type of midgut homogenate. Changes in pH (9.5 to 11.5) or midgut homogenate concentration during digestion failed to substantially alter protein electrophoretic patterns of B. thuringiensis HD-1 crystal toxin. In vitro toxicity of crystal protein activated by either type of midgut preparation was equal toward cultured insect cells from either Manduca sexta or Choristoneura fumiferana. Electrophoresis of midgut extracts in polyacrylamide gels containing gelatin as substrate also yielded matching mobility patterns of proteinases from both types of midguts. Quantitation of midgut proteolytic activity using tritiated casein as a substrate revealed variation between midgut preparations, but no statistically significant differences between proteolytic activities from susceptible and resistant Indian meal moth larvae. Inhibition studies indicated that a trypsin-like proteinase with maximal activity at pH 10 is a major constituent of Indian meal moth midguts. The results demonstrated that midguts from susceptible and resistant strains of P. interpunctella are similar both in their ability to activate B. thuringiensis protoxin and in their proteolytic activity.     

Contribution of Bacillus thuringiensis Spores to Toxicity of Purified Cry Proteins Towards Indianmeal Moth Larvae

The influence of Bacillus thuringiensis subsp. kurstaki HD-1 spores upon the toxicity of purified Cry1Ab and Cry1C crystal proteins toward susceptible and BT-resistant Indianmeal moth (IMM, Plodia interpunctella) larvae was investigated. With susceptible larvae, HD-1 spores were toxic in the absence of crystal protein and highly synergistic (approximately 35- to 50-fold) with either Cry1Ab or Cry1C protein. With BT-resistant IMM larvae, HD-1 spores were synergistic with Cry1Ab and Cry1C protein in all three resistant strains examined. Synergism was highest (approximately 25- to 44-fold) in insects with primary resistance toward Cry1C (IMM larvae with resistance to B. thuringiensis subsp. aizawai or entomocidus). However, HD-1 spores also synergized either Cry1Ab or Cry1C toxicity toward larvae resistant to B. thuringiensis subsp. kurstaki at a lower level (approximately five- to sixfold). With susceptible larvae, the presence of spores reduced the time of death when combined with each of the purified Cry proteins. Without spores, the speed of intoxication and eventual death for larvae treated with Cry1C and Cry1Ab proteins was much slower than for the HD-1 preparation containing both spores and crystals together. Neither spores nor toxin dose affected the mean time of death of resistant larvae treated with either Cry1Ab or Cry1C toxins. Both Cry1Ab and Cry1C toxins appeared to reduce feeding and consequently toxin consumption. Received: 1 December 1995 / Accepted: 3 January 1996     

Spore Coat Protein Synergizes Bacillus thuringiensis Crystal Toxicity for the Indianmeal Moth (Plodia interpunctella)

Spores from Bacillus thuringiensis serovars kurstaki and entomocidus synergized crystal protein toxicity for larvae of the Indianmeal moth (Plodia interpunctella). Preparations of spore-crystal mixtures of either serovar were more toxic for the larvae than either purified spores or crystals alone (based on dry weight). Spores lost 53% of their toxicity for the Indianmeal moth after 2 h of UV-irradiation, but remained partially toxic (28%) even after 4 h of irradiation. Spore coat protein was toxic for the Indianmeal moth and was synergistic with B. thuringiensis serovar kurstaki HD-1 crystal protein. Enhanced toxicity of the combined spore-crystal preparation was attributed to a combination of crystal and spore coat protein, and included the effects of spore germination and resulting septicemia in the larval hemolymph. Ultraviolet irradiation of spores reduced the toxicity from septicemia but not the synergism caused by spore coat protein. The potencies of spore-crystal preparations must be carefully evaluated on the basis of contributions from all three factors. Received: 15 September 1997/Accepted: 21 October 1997     

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.     

Interaction of larval age and dietary formaldehyde on the susceptibility of tufted apple budmoth (Lepidoptera: Tortricidae)to Bacillus thuringiensis

Laboratory tests of Bacillus thuringiensis var. kurstaki (strain HD-1) against Platynota idaeusalis reared on a semisynthetic diet showed that the LC₅₀ values for neonates and 9-day-old larvae showed no significant difference in susceptibility; however, 14-day-old larvae were significantly more susceptible than neonates or 9-day-old larvae. There was no evidence of any interaction between 14-day-old larvae reared on the semisynthetic diets with and without formaldehyde and B. thuringiensis toxicity at any assessment reading. Likewise the toxicity of B. thuringiensis to 14-day-old larvae reared on apple leaves did not differ from the B. thuringiensis toxicity of 14-day-old larvae reared on semisynthetic diets with and without formaldehyde.     

Toxicity of Bacillus thuringiensis toward Aedes aegypti larvae.

Among six strains of Bacillus thuringiensis and five other species of Bacillus, only two strains of B. thuringiensis, strains HD-1 and BA-068, were toxic to Aedes aegypti larvae within 24 hr. The LC₅₀s were 5.6 × 10⁴ and 2.4 × 10⁵ spores/ml for strains HD-1 and BA-068, respectively. The toxic factor(s) was heat sensitive and γ ray resistant and preliminary evidences indicated that it was associated with the crystalline body of B. thuringiensis.     

Compatibility of Bacillus thuringiensis and granulosis virus treatments of stored grain with four grain fumigants

Wheat was fumigated with phosphine, methyl bromide, CCl₄-carbon bisulfide (80:20 by volume), or ethylene dichloride-CCl₄ (75:25 by volume) after it had been treated with either of two formulations of Bacillus thuringiensis or with a granulosis virus of the Indian meal moth, Plodia interpunctella. Only methyl bromide had an adverse effect: the granulosis virus was inactivated. Spores of B. thuringiensis washed from treated wheat after fumigation with methyl bromide did not produce colonies on nutrient agar plates, but the activity of the B. thuringiensis against Indian meal moths was not affected.     

Interaction of larval age and antibiotic on the susceptibility of three insect species to Bacillus thuringiensis

The addition of chlortetracycline hydrochloride to bioassay diets increased the LC₅₀S of the HD-1 isolate of Bacillus thuringiensis 2.4–67.1 times when bioassayed against 4-day-old larvae of Trichoplusia ni, Heliothis virescens, and Ostrinia nubilalis. There were no significant differences in LC₅₀s when bioassays were conducted with neonate larvae in the presence or absence of antibiotic, nor were there any highly significant differences between the LC₅₀s for HD-1 when neonate larvae were used in the presence or absence of antibiotic or when 4-day-old larvae were used in the absence of antibiotic. Viable counts of B. thuringiensis did not increase over time in any of three bioassay diets. There were no differences in activity of pure chlortetracycline hydrochloride and veterinary grade Aureomycin. There was a direct correlation between amount of antibiotic and elevation of HD-1 LC₅₀s when 4-day-old larvae were used. LC₅₀ elevation in presence of antibiotic first appeared in 2-day-old larvae.     

Toxic effects of spore/crystal ratios of Bacillus thuringiensis on European corn borer larvae

Bioassays to determine LC₅₀ values of spores and crystals of four varieties of Bacillus thuringiensis grown on nutrient agar plates were carried out against neonate and 6-day-old European corn borer, Ostrinia nubilalis, larvae. The four bacterial varieties were equally toxic against the neonates, but only B. thuringiensis var. kenyae, var. galleriae, and var. kurstaki were toxic to 6-day-old larvae. B. thuringiensis var. tolworthi was inactive against 6-day-old larvae. Different ratios of pure spores and crystals of the bacteria also were tested against neonate and 6-day-old larvae. Pure spores are not pathogenic to neonates or 6-day-old larvae. Pure crystals were toxic to both ages of the larvae, but a combination of spores and crystals was necessary for maximum larval mortality.     

Wolbachia sp. (Rickettsiales: Rickettsiaceae) a symbiont of the almond moth,Ephestia cautella: Ultrastructure and influence on host fertility

Wolbachia sp. is a maternally inherited symbiont of the almond moth, Ephestia cautella. It is transmitted through the cytoplasm of the egg and occurs normally in the gonads of all stages of the moth. The symbiont is responsible for reproductive cytoplasmic incompatibility between crosses of experimental laboratory strains of aposymbiotic female moths and symbiotic (normal) males. Although female moths were inseminated in laboratory tests, their eggs failed to hatch and exhibited no signs of embryonic development. The reciprocal cross, i.e., symbiotic female months × aposymbiotic males, produced normal progeny.The ultrastructure of Wolbachia was studied in sections of E. cautella larval testes. Symbionts, minute rod-shaped structures, were abundant in the cytoplasm of hypertrophied spermatids. There was no indication of deleterious influence of symbionts on sperm production or activity. Strains of Wolbachia occur in allopatric populations of insects where they may function as a genetic isolation mechanism. Microorganismal reproductive incompatibility has been suggested as a possible approach for insect control.     

Relationships between laboratory bioassay-derived potencies and field efficacies of Bacillus thuringiensis isolates with different spectral activities

Bacillus thuringiensis isolates with different spectral activities were not equally efficacious when applied to cabbage at the same number of IUs/ha for protection against larvae of the cabbage looper, Trichoplusia ni. Preparations of the isolates were standardized against T. ni larvae. Variety galleriae isolates (HD-196 and HD-153) were the most efficacious per applied IU, and a K-73 type variety kurstaki (HD-73) was the least efficacious per applied IU. A variety thuringiensis (HD-264) and a K-1 type variety kurstaki (HD-1) were intermediate in efficacy per applied IU. Speed of kill and, to some extent, differences in the amount of food consumed appear to be responsible for the differences in efficacy per applied IU. When more potent B. thuringiensis isolates are discovered and developed, the recommended field dosages for the new isolates must be determined by actual field experimentation rather than by extrapolation from existing HD-1 data.     

Field confirmation of a mechanism causing synergism between Bacillus thuringiensis and the gypsy moth parasitoid, Apanteles melanoscelus

In 16-ha plots aerially sprayed with single and double applications of Bacillus thuringiensis, percentage parasitism by A. melanoscelus and the number of A. melanoscelus cocoons under burlap strips were higher than in comparable untreated plots in the same area. Strong correlations occurred between percentage parasitism and caterpillar size, with plots having the smallest caterpillars being the most heavily parasitized. However, these parameters were also negatively correlated with number of caterpillars per plot. The increased numbers of parasitoid progeny, i.e., cocoons, found in treated plots showed that corresponding increases in percentage parasitism could not be due simply to improved parasitoid: host ratios. Evidence strongly suggests that the retarding effect of B. thuringiensis infection kept gypsy moth larvae small enough in the treated plots to permit A. melanoscelus females to parasitize relatively large numbers of caterpillars.     

Behavioral and physiological responses of susceptible and resistant diamondback moth larvae to Bacillus thuringiensis

To determine whether field-selected resistance of diamondback moth (Plutella xylostella L.) (Lepidoptera: Plutellidae) to Bacillus thuringiensis is based on behavioral or physiological adaptation, we measured mortality, consumption, and movement of larvae from a susceptible and a resistant colony when placed on untreated and B. thuringiensis treated cabbage. Colonies did not differ in mortality, consumption, or movement on untreated cabbage. However, for a given amount of consumption of treated cabbage, resistant larvae had lower mortality than susceptible larvae, demonstrating that resistance had a physiological basis. The movement patterns could not account for the differences between colonies in survival. Resistant larvae did not avoid B. thuringiensis more than did susceptible larvae. Thus, we found no evidence for behavioral resistance.     

Some Effects of Douglas Fir Terpenes on Certain Microorganisms

The Douglas fir terpene α-pinene was shown to inhibit the growth of a variety of bacteria and a yeast. Other terpenes of the Douglas fir, including limonene, camphene, and isobornyl acetate, were also inhibitory to Bacillus thuringiensis. All terpenes were inhibitory at concentrations normally present in the fir needle diet of Douglas fir tussock moth larvae. The presence of such terpenes in the diet of these insects was found to strongly influence the infectivity of B. thuringiensis spores for the Douglas fir tussock moth larvae. The terpene α-pinene destroyed the cellular integrity and modified mitochondrial activity in certain microorganisms.     

Nosema invadens sp. n. (Microsporida: Nosematidae), a pathogen causing inflammatory response in Lepidoptera

States in the life cycle of Nosema invadens sp. n. are described from the raisin moth, Cadra figulilella, and the almond moth, Cadra cautella. In laboratory tests, larvae of the following Lepidoptera were also susceptible: Ephestia elutella, Plodia interpunctella, Galleria mellonella, and Paramyelois transitella. The midgut wall and Malpighian tubules were invaded first, but subsequently most other tissues were also infected. Severe inflammatory response accompanied infection, including hemocytic encapsulation of infected areas and hemocytic infiltration of fat tissue.     

The comparative persistence of toxicity of Bacillus sphaericus strain 1593 and Bacillus thuringiensis serotype H-14 against mosquito larvae in different kinds of environments

Bacillus sphaericus strain 1593 and B. thuringiensis serotype H-14 were evaluated for persistence of toxicity against two species of mosquito larvae, Culex quinquefasciatus and Aedes aegypti, in a selected simulating plot in Bangkok. Both strains of bacteria demonstrated larvicidal activity towards both species of mosquito larvae. In tap water, the toxicity of B. sphaericus strain 1593 was found to be greater towards C. quinquefasciatus larvae than A. aegypti larvae, whereas the toxicity of B. thuringiensis serotype H-14 was found to be greater towards A. aegypti larvae than C. quinquefasciatus larvae. The persistence of toxicity of these two bacteria was found to be different. The lethal concentration of B. thuriengiensis H-14 against A. aegypti decreased from LC₉₀ to below LC₅₀ in about 15 weeks when tested in tap water. The decrease was faster in polluted water. The toxicity of B. sphaericus 1593 towards C. quinquefasciatus larvae persisted for at least 9 months in tap water and 6 months in polluted water. The multiplication of bacteria was indicated only in populations of B. sphaericus 1593 tested with C. quinquefasciatus larvae.     

Specificity of Activated CryIA Proteins from Bacillus thuringiensis subsp. kurstaki HD-1 for Defoliating Forest Lepidoptera

The insecticidal activity of the CryIA(a), CryIA(b), and CryIA(c) toxins from Bacillus thuringiensis subsp. kurstaki HD-1 was determined in force-feeding experiments with larvae of Choristoneura fumiferana, C. occidentalis, C. pinus, Lymantria dispar, Orgyia leucostigma, Malacosoma disstria, and Actebia fennica. The toxins were obtained from cloned protoxin genes expressed in Escherichia coli. The protoxins were activated with gut juice from Bombyx mori larvae. Biological activity of the individual gene products as well as the native HD-1 toxin was assessed as the dose which prevented 50% of the insects from producing frass within 3 days (frass failure dose [FFD₅₀]). The three toxins were about equally active against M. disstria. In the Choristoneura species, CryIA(a) and CryIA(b) were up to fivefold more toxic than CryIA(c). In the lymantriid species, CryIA(a) and CryIA(b) were up to 100-fold more toxic than CryIA(c). The toxicity of HD-1 was similar to that of the individual CryIA(a) or CryIA(b) toxins in all of these species. None of the CryIA toxins or HD-1 exhibited and toxicity towards A. fennica. Comparison of the observed FFD₅₀ of HD-1 with the FFD₅₀ expected on the basis of its crystal composition suggested a possible synergistic effect of the toxins in the two lymantriid species. Our results further illustrate the diversity of activity spectra of these highly related proteins and provide a data base for studies with forest insects to elucidate the molecular basis of toxin specificity.     

Interactions between Bacillus thuringiensis subsp. kurstaki HD-1 and midgut bacteria in larvae of gypsy moth and spruce budworm

We examined interaction between Bacillus thuringiensis subsp. kurstaki HD-1 (Foray 48B) and larval midgut bacteria in two lepidopteran hosts, Lymantria dispar and Choristoneura fumiferana. The pathogen multiplied in either moribund (C. fumiferana) or dead (L. dispar) larvae, regardless of the presence of midgut bacteria. Inoculation of L. dispar resulted in a pronounced proliferation of enteric bacteria, which did not contribute to larval death because B. thuringiensis was able to kill larvae in absence of midgut bacteria. Sterile, aureomycin- or ampicillin-treated larvae were killed in a dose-dependent manner but there was no mortality among larvae treated with the antibiotic cocktail used by [Broderick et al., 2006] and [Broderick et al., 2009]. These results do not support an obligate role of midgut bacteria in insecticidal activity of HD-1. The outcome of experiments on the role of midgut bacteria may be more dependent on which bacterial species are dominant at the time of experimentation than on host species per se. The L. dispar cohorts used in our study had a microflora, that was dominated by Enterococcus and Staphylococcus and lacked Enterobacter. Another factor that can confound experimental results is the disk-feeding method for inoculation, which biases mortality estimates towards the least susceptible portion of the test population.