A pathogenic strain of Bacillus cereus isolated from the cigarette beetle, Lasioderma serricorne

A noncrystalliferous, aerobic, spore-forming bacterium (accession number DD-1019) isolated from the cigarette beetle, Lasioderma serricorne, was identified as a strain of Bacillus cereus based on morphological, biochemical, and cultural similarities. Pathogenicity was established by exposing hatching larvae of the cigarette beetle to doses of inocula ranging from 1.17 to 600 × 10⁶ spores per gram of rearing medium. The LD₅₀ and the LD₉₀ were calculated to be 4.29 × 10⁶ and 371 × 10⁶ spores per gram of medium, respectively. The cigarette beetle was effectively controlled by both the DD-1019 strain of B. cereus and the CM1-1 strain (originally isolated from and pathogenic to codling moth, Laspeyresia pomonella) but proved quite refractory to Bacillus thuringiensis var. thuringiensis.     

The effect of L-canavanine and L-canaline on the hemolymph amino acid composition of the tobacco hornworm,Manduca sexta (L.) (Sphingidae)

Tobacco hornworm larvae, Manduca sexta (L.) (Sphingidae), were administered L-canaline either by parenteral injection or by dietary consumption. The overt toxicity and the alteration of hemolymph amino acids caused by these nonprotein amino acids were evaluated. The LD₅₀ value for parenterally administered canavanine and canaline is 1.0 and 2.5 mg/g fresh body weight, respectively. A dietary concentration of 5.2 mM for canavanine and over 20 mM for canaline represent the respective LC₅₀ values. A large percentage of the larvae reared on diets supplemented with additional arginine, ornithine, or 2,4-diaminobutyric acid in addition to canavanine or canaline were unable to complete larval-pupal ecdysis. These toxic effects were associated with a decreased glutamic acid hemolymph titer and dramatically elevated ornithine. On the other hand, larvae administered canavanine or canaline alone, either by dietary consumption or parenteral injection, experienced less drastic developmental aberrations. These symptoms were in some cases correlated with increased ornithine and glutamic acid titers. Evidence is presented that even a canavanine- and canaline-sensitive insect such as M. sexta has a marked ability to eliminate these protective allelochemicals.     

Differential susceptibility of parasitized and nonparasitized larvae of Trichoplusia ni to a nuclear polyhedrosis virus

Nonparasitized second-instar larvae of Trichoplusia ni were twice as susceptible (at the LD₅₀ level) to the singly enveloped T. ni nuclear polyhedrosis virus as those parasitized by Hyposoter exiguae (Hymenoptera: Ichneumonidae). The LD₅₀ values for nonparasitized and parasitized larvae were 1.58 × 10³ and 3.16 × 10³ polyhedra/ml of diet, respectively. The LD₉₅ value for parasitized larvae was approximateely 5 times higher than that for nonparasitized larvae. The slopes (b values) were 1.2 for parasitized larvae and 1.7 for nonparasitized larvae. The LT₅₀ values for parasitized larvae also were significantly longer than those for nonparasitized larvae. No significant difference was found between the food consumption of parasitized and nonparasitized T. ni larvae.     

Dosage-mortality and time-mortality responses of the armyworm Spodoptera exempta to a nuclear polyhedrosis virus

Third-instar Spodoptera exempta larvae were fed on young maize leaves treated with 20 μl of polyhedral inclusion body (PIB) suspension of concentrations that varied from 1.6 × 10² to 1.6 × 10⁹ PIBs/ml. Daily observations were kept on mortality rates. A probit analysis on the results gave an LD₅₀ value of 48.4 PIBs/larva (lower and upper fiducial limits 39.2 and 59.4 PIBs/larva, respectively), and an LT₅₀ that varied from 146.2 to 221.3 hr, depending on the dosage. LD and LT values obtained show the high pathogenicity of S. exempta nuclear polyhedrosis virus to its host.     

Bioassay of a nuclear-polyhedrosis virus and Bacillus thuringiensis against the American bollworm, Heliothis armigera, in Botswana

Heliothis armigera is a serious pest in Botswana. Preparations of a local nuclear-polyhedrosis virus and Bacillus thuringiensis (Biotrol BTB-183) are being considered as treatments for control of this pest. Laboratory bioassays of these two preparations were carried out to provide a firm basis for comparison with past field trials and a standard for future field testing. The LC₅₀ of the virus and LD₅₀ of B. thuringiensis were found to compare favorably with those for similar materials against closely and distantly related lepidopterous pests. The majority of larvae were killed very rapidly by the B. thuringiensis, although some survived up to 14 days before death. The minimum time to mortality for the virus was 4 days. B. thuringiensis lowered the weight of both male and female surviving pupae, whereas the virus had no significant effect on its survivors. Concentrations of the two materials calculated from the LC₉₀ and LD₉₀ were considerably lower than those already found to give some or good control of H. armigera on sorghum. Improved spray coverage and protection of the materials against environmental degradation are required if spraying concentrations are to be reduced and yet remain effective.     

The comparative susceptibilities of Pieris brassicae and P. rapae to a granulosis virus from P. brassicae

A comparison was made of the dosage-mortality responses of larvae of Pieris brassicae and P. rapae to infection by P. brassicae granulosis virus (GV). Bioassays with first, second, third, and fourth-instar larvae of both species revealed a marked difference in susceptibility between instars and between species. Median lethal dosages (LD₅₀s) for P. rapae larvae ranged from five capsules for the first instar to 662 capsules for the fourth instar. With P. brassicae, this range extended from 66 capsules to 2.3 × 10⁷ capsules. The time-mortality responses of the two species were similar when fed virus dosages equivalent to an LD₉₀. Median lethal times (LT₅₀s) ranged from 5 days for first-instar larvae to 7–8 days for fourth-instar larvae. A comparison between a long-established laboratory stock of P. brassicae and a stock recently acquired from the field showed no significant difference in their susceptibility to GV. The implications of the pronounced species differences in susceptibility to GV infection are discussed in relation to the potential field control of P. rapae and P. brassicae.     

EFFECT OF TANNIC ACID ON THE EFFECTIVENESS OF BACILLUlS THURINGIENSIS VAR. KURSTAKI AGAINST HELICOVERPA ARMIGERA (HÜBNER)*

Abstract The role of tannic acid in increasing effectiveness of Bacillus thuringiensis var. kurstaki (HD-1) against Helicover pa armigera was examined in bioassays on semisynthetic diet. Concentrations of B. thuringiensis (0 %, 0.005 %, 0. 01 %, 0.015 %, 0.02 %, 0.025 % wet weight) were incorporated into the diet containing 0. 025% tannic acid and tannic acid-free diet. LD₅₀ of B. thuringiensis with tannic acid were 0.006% but that without tannic acid was 0.011%. Both B. thuringiensis and tannic acid retarded growth of H. armigera significantly, but there was no synergetic effect between them. Choice tests showed that B, thuringiensis deterred feeding of the fifth instar larvae of H. armigera but tannic acid had no such effect. Experiments on colony growth of B. thuringiensis on NBA media containing tannic acid (0, 1, 3, 6, 9, 12, 15, 18, 21 mg/100 ml) demonstrated that tannic acid reduced colony growth of B. thuringiensis, and inhibited sporulation above 15 mg/100 ml.     

Histopathological effects of Bacillus thuringiensis on the midgut of tobacco hornworm larvae (Manduca sexta): Low doses compared with fasting

The cytology and ultrastructure of the midgut cells of Manduca sexta larvae are described for untreated controls, larvae which fed on a spore preparation of Bacillus thuringiensis, and larvae which were fasted for either 24 or 48 hr. New observations on the ultrastructure of midgut cells in Manduca larvae included the finding of specialized Golgi vesicles in anteriormost columnar cells and of regular arrays of expanded rough endoplasmic reticulum in goblet cells of the posterior midgut region. The present observations reveal that the columnar cells of the midgut responded cytologically in the same way to fasting as they did to exposure to the toxic spores of B. thuringiensis. The goblet cells, however, appeared unaffected by fasting but became swollen in response to feeding of B. thuringiensis spore preparation.     

Effect of starvation upon baculovirus replication in larval Bombyxmori and Heliothisvirescens

The progression of baculovirus (BmNPV, BmCysPD, AcMNPV or AcAaIT) infection in larval Bombyx mori and Heliothis virescens (1st, 3rd or 5th instar) was investigated following various starvation regimes. When the larvae were starved for 12 or 24 h immediately following inoculation, the median lethal time to death (LT₅₀) was delayed by 9.5-19.2 h in comparison to non-starved controls. This corresponded to a delay of 10-23% depending upon the larval stage and virus that was used for inoculation. When a 24 h-long starvation period was initiated at 1 or 2 days post inoculation (p.i.), a statistically significant difference in LT₅₀ was not found indicating that the early stages of infection are more sensitive to the effects of starvation. Viral titers in the hemolymph of 5th instar B. mori that were starved for 24 h immediately following inoculation were 10-fold lower (p < 0.01) than that found in non-starved control larvae. Histochemical analyses indicated that virus transmission was reduced in 5th instar B. mori that were starved for 24 h immediately following inoculation in comparison to non-starved control larvae. In general, the mass of larvae that were starved immediately after inoculation was 30% lower than that of non-starved control insects. Our findings indicate that starvation of the larval host at the time of baculovirus exposure has a negative effect on the rate baculovirus transmission and pathogenesis.     

Influence of abiotic factors on bacterial proliferation and anoxic survival of the sea mussel Mytilus edulis L.

The effect of several abiotic factors (salinity, temperature and pH) on bacterial proliferation and survival time of the sea mussel Mytilus edulis L. were studied under anoxic incubations. In addition, the presence in the incubation media of ammonium and the volatile fatty acids propionate and acetate, both excreted fermentation products of the bivalve, was tested.Anoxic incubations with seawater diluted with demineralised water showed at the lowest salinity (50% seawater, SW) a significant increase in the capacity of M. edulis to survive anoxia as compared to both 75% SW and control [100% SW, corresponding to 32 practical salinity units (psu)]. Formation of biotic sulphide and ammonium occurred in all incubations. However, bacterial proliferation was postponed by 2-3 days at lowest salinity and accordingly, concentrations of both compounds were lower. Anoxic survival profiles of mussels collected from different habitats in the Dutch Scheldt area, characterised by differences in salinity (range from 17 to 31 psu), corresponded with the above salinity effect. Walsoorden mussels (17 psu) showed the longest (P<0.001) survival time under anoxia (LT₅₀=17.2 days) as compared with Paulina (27 psu) and Wemeldinge (31 psu) mussels (LT₅₀=12.8 and 9.8 days, respectively). Condition index (ratio of soft body weight to shell volume) was not correlated with anoxic survival time in untreated mussels, although this was clearly the case when the antibiotic chloramphenicol was added to the anoxic seawater.Acidification of the anoxic incubation medium had a positive effect on survival time. LT₅₀ values significantly (P<0.001) increased from 10.2 days at pH 8.1 to 11.6 and 11.5 days at pH 7.3 and 6.5, respectively. Biotic sulphide and ammonium accumulation as well as bacterial numbers were significantly lower at pH 7.3 and 6.5 as compared with pH 8.1. Anoxic incubations at 10 °C (LT₅₀=12.0 days) strongly increased survival time as compared to 18 °C (LT₅₀=5.9 days). The benefit of antibiotic addition was also stronger at lower temperature (10 °C).Addition of both propionate and acetate (0.5 mM) displayed no effect on mortality of mussels under anoxia, but ammonium (0.5 mM) caused a negative effect (P<0.001). Biotic sulphide and ammonium concentrations measured in both volatile fatty acid incubations were lower than the control situation, as well as total bacterial numbers.This study shows that environmental factors play a significant role in determining the course of bacterial infection and death of bivalves exposed to anoxia.     

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.     

Constituents of Musa balbisiana seeds and their activity against Cryptolestes pusillus

The chloroform and acetone extracts of the seeds of Musa balbisiana Colla (Musaceae) and four of its constituents were assayed in the diet for toxicity against the flat grain beetle (Cryptolestes pusillus Schöherr). A mixture of fatty esters of phytol and the flavan (+)-epiafzelechin showed a significant growth inhibition and toxicity against this economically important pest of stored cereals. The LD₅₀ was 6.3% for the most active compound, (+)-epiafzelechin.     

Development of Bacillus thuringiensis in Galleria mellonella larvae exposed to gamma radiation

A suspension of Bacillus thuringiensis was inoculated at 24 and 72 hr into the oral cavity of Galleria mellonella larvae following exposure to 20, 50, and 70 Kr of gamma radiation, respectively. The cytopathology was conducted after B. thuringiensis had developed for 3, 5, and 7 hr and after radiation damage had developed for 27, 29, 31, 75, 77, and 79 hr in the larvae exposed to 20, 50, and 70 Kr, respectively.B. thuringiensis spores appeared in the midgut lumen from 3 to 7 hr after inoculation of 20 Kr irradiated larvae. At 7 hr after B. thuringiensis infection, and 79 hr after 20 Kr irradiation, the following changes were seen: B. thuringiensis rods appeared adsorbed onto the walls of epithelial cells, a few spores appeared in hemolymph, epithelial cells developed vacuoles, and villi appeared detached from the basement membrane.Within a period ranging from 3 to 5 hr after infection, B. thuringiensis rods attacked vacuolated epithelial cells of most of the 50 and 70 Kr irradiated larvae. At 7 hr after infection and at 31 hr after 70 Kr irradiation, the spores reached the interior of some epithelial cells and were also seen concentrated near the basement membrane.In general, the midgut epithelial cells of the 70 Kr-irradiated groups of larvae appeared highly vacuolated, badly disrupted, and in most cases undistinguishable as a result of attack of B. thuringiensis.In short, B. thuringiensis did not show a characteristic pattern of pathology on 20 and 50 Kr-irradiated midgut cells. The problem of permeability of B. thuringiensis toxin into the irradiated cells needs further investigation.     

Antagonism between Cry1Ac1 and Cyt1A1 Toxins of Bacillus thuringiensis

Most strains of the insecticidal bacterium Bacillus thuringiensis have a combination of different protoxins in their parasporal crystals. Some of the combinations clearly interact synergistically, like the toxins present in B. thuringiensis subsp. israelensis. In this paper we describe a novel joint activity of toxins from different strains of B. thuringiensis. In vitro bioassays in which we used pure, trypsin-activated Cry1Ac1 proteins from B. thuringiensis subsp. kurstaki, Cyt1A1 from B. thuringiensis subsp. israelensis, and Trichoplusia ni BTI-Tn5B1-4 cells revealed contrasting susceptibility characteristics. The 50% lethal concentrations (LC₅₀s) were estimated to be 4,967 of Cry1Ac1 per ml of medium and 11.69 ng of Cyt1A1 per ml of medium. When mixtures of these toxins in different proportions were assayed, eight different LC₅₀s were obtained. All of these LC₅₀s were significantly higher than the expected LC₅₀s of the mixtures. In addition, a series of bioassays were performed with late first-instar larvae of the cabbage looper and pure Cry1Ac1 and Cyt1A1 crystals, as well as two different combinations of the two toxins. The estimated mean LC₅₀ of Cry1Ac1 was 2.46 ng/cm² of diet, while Cyt1A1 crystals exhibited no toxicity, even at very high concentrations. The estimated mean LC₅₀s of Cry1Ac1 crystals were 15.69 and 19.05 ng per cm² of diet when these crystals were mixed with 100 and 1,000 ng of Cyt1A1 crystals per cm² of diet, respectively. These results indicate that there is clear antagonism between the two toxins both in vitro and in vivo. Other joint-action analyses corroborated these results. Although this is the second report of antagonism between B. thuringiensis toxins, our evidence is the first evidence of antagonism between toxins from different subspecies of B. thuringiensis (B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. israelensis) detected both in vivo and in vitro. Some possible explanations for this relationship are discussed.     

The control of Monomorium pharaonis (Hymenoptera: Formicidae) with Bacillus thuringiensis

In this study, the effect of different preparations made from Bacillus thuringiensis var. thuringiensis (strains: CCEB 555 and CCEB 058) on ants, Monomorium pharaonis, under laboratory conditions is reported. The different preparations tested consisted of (1) a liquid culture of the strain B. thuringiensis CCEB 555 (containing spores and exotoxin), (2) the supernatant of the culture broth of strain CCEB 555 (containing exotoxin), and (3) the biological preparation “Bathurin” prepared from the strain B. thuringiensis CCEB 058 (containing spores and inclusions, without exotoxin). The preparations were used either pure or in alternation with borax, i.e., 1 wk borax, 3 wk the respective preparation for several months. All preparations were found to be toxic to M. pharaonis and their effect was characterized by a slow extinction of the ant colony. Administration of “Bathurin” (1.3%) yielded a 100% mortality after 20 wk. Using a liquid culture of B. thuringiensis var. thuringiensis, 100% mortality was recorded after 21 wk, a period of time which did not differ from that obtained with the supernatant of the culture containing exotoxin. The alternation with borax was found to accelerate ant mortality by 9–10 wk after administration. In all experiments, the worker ants died first, the queen ants surviving them by 1–3 wk.In experiments employing worker ants only, a 100 and 98% mortality, respectively, occurred within 3 wk after administration of a liquid culture of B. thuringiensis and “Bathurin” supplemented with borax.     

Inhibition of active K+ transport in the tobacco hornworm (Manduca sexta) midgut after ingestion of Bacillus thuringiensis endotoxin

The effect of Bacillus thuringiensis endotoxin after ingestion on the K⁺ transport of Manduca sexta midgut is described. Direct measurements of short circuit current and transepithelial potential demonstrate that the transport is inhibited at the end of 1 hr with many of the insects exhibiting both negative short circuit current and transepithelial potential by the end of a 4-hr ingestion period.     

Midgut juice components affect pore formation by the Bacillus thuringiensis insecticidal toxin Cry9Ca

The pore-forming ability of the Bacillus thuringiensis toxin Cry9Ca, its two single-site mutants R164A and R164K, and the 55-kDa fragment resulting from its proteolytic cleavage at R164 was evaluated under a variety of experimental conditions using an electrophysiological assay. All four toxin preparations depolarized the apical membrane of freshly isolated third-instar Manduca sexta midguts bathing in a solution containing 122 mM KCl at pH 10.5, but the 55-kDa fragment was considerably more active than Cry9Ca and its mutants. The activity of the latter toxins was greatly enhanced, however, when the experiments were conducted in the presence of fifth-instar M. sexta midgut juice. This effect was also observed after midgut juice proteins had been denatured by heating at 95 °C or after inorganic ions and small molecules had been removed from the midgut juice by extensive dialysis. A similar stimulation of toxin activity was also observed when the experiments were carried out in the presence of the lipids extracted from an equivalent volume of midgut juice. Depolarization of the cell membrane was also greatly enhanced, in the absence of midgut juice, by the addition of a cocktail of water-soluble protease inhibitors. These results indicate that, depending on the cleavage site and on the experimental conditions used, further proteolysis of the activated Cry9Ca toxin can either stimulate or be detrimental to its activity and that M. sexta midgut juice probably contains protease inhibitors that could play a major role in the activity of B. thuringiensis toxins in the insect midgut.     

Bioassay of nucleopolyhedrosis virus against larval instars of Malacosoma neustria

The relative susceptibility of third- and fourth-instar Malacosoma neustria larvae to a nucleopolyhedrosis virus was studied by bioassay in the laboratory. Larval mortality increased with increased dosage, whereas the dosage-incubation relationship was reversed. Larval age inversely affected mortality and incubation. The computed LD₅₀'s for third- and fourth-instar larvae were 1,405 and 12,320 polyhedral inclusion bodies (PIB's)/larva. The median lethal doses calculated as number of PIB's/mg body weight showed that third-instar larvae were only twice more susceptible to virus than fourths. The LT₅₀ values for 3 × 10⁵, 3 × 10⁴, and 3 × 10³ PIB's/larva were 5.9, 6.58, and 8.15 days, respectively, in third-instar assay; the corresponding figures for the two highest concentrations were 9.3 and 10.7 days in the older larvae. Lethally infected individuals died after one or, exceptionally, two molts. No correlation was found between pupal weight or adult emergence of survivors and the virus dose administered to the larvae.     

Insecticidal activity of Bacillus thuringiensis crystal proteins

Published data on insecticidal activity of crystal proteins from Bacillus thuringiensis are incorporated into the Bt toxin specificity relational database. To date, 125 of the 174 holotype known toxins have been tested in ∼1700 bioassays against 163 test species; 49 toxins have not been tested at all; 59 were tested against 71 Lepidoptera species in 1182 bioassays; 53 toxins were tested against 23 Diptera species in 233 bioassays; and 47 were tested against 39 Coleoptera species in 190 bioassays. Activity spectra of the tested toxins were summarized for each order. Comparisons of LC₅₀ values are confounded by high variability of the estimates, mostly due to within-species variation in susceptibility, and errors associated with estimation of toxin protein content. Limited analyses suggest that crystal protein toxicity is not affected by quarternary toxin rank or host used for gene expression, but that pre-ingestion treatment by solubilization or enzymatic processing has a large effect. There is an increasing number of toxin families with cross-order activity, as 15 of the 87 families (secondary rank) that are pesticidal are active against more than one order. Cross-order activity does not threaten environmental safety of B. thuringiensis-based pest control because toxins tend to be much less toxic to taxa outside the family’s primary specificity range.