Effects of Aspen Phenolic Glycosides on Gypsy Moth (Lepidoptera: Lymantriidae) Susceptibility to \u3ci\u3eBacillus Thuringiensis\u3c/i\u3e

Performance of the gypsy moth, Lymantria dispar, on quaking aspen, Populus tremuloides, is strongly affected by foliar concentrations of phenolic glycosides. Because the microbial insecticide Bacillus thuringiensis is widely used against gypsy moths and has a mode of action similar to that of phenolic glycosIdes, we investigated the combined effects of the two toxins on gypsy moth larvae. The experimental design was a 2 x 2 factorial: two levels (0, +) of phenolicglycosides for each of two levels (0, +) of B. thuringiensis. The toxins were incorporated into artificial diets and bioassayed against first and fourth instars. Bacillus thuringiensis and phenolic glycosides ne~atively and addi· tively affected larval survival, growth and development tImes. Both agents slightly reduced consumption rates. In addition, B. thuringiensis reduced diet digestibility whereas phenolic glycosides decreased the efficiency with which food was converted to biomass. These results suggest that the efficacy of B. thuringiensis applications in aspen forests is likely to be affected by the allelo· chemical composition of foliage

Phylloplane Sterilization With Bleach Does Not Reduce Btk Toxicity for \u3ci\u3ePapilio Glaucus\u3c/i\u3e Larvae (Lepidoptera: Papilionidae)

Neonate tiger swallowtail larvae (Papilio glaucus) were used to bioassay the effects of Btk (Bacillus thuringiensis var. kurstaki) at 4 doses (0.268, 0.034, 0.008, and 0.004 BIU per cm leaf surface) with an untreated control. Larvae, obtained from females captured in Georgia and North Carolina, were fed leaves of either tulip tree (Liriodendron tulipfera) or black cherry (Prunus serotina) in experiments that either included a pre-treatment dip and rinse in 5% chlorox bleach or not, before the application of Btk (dipping leaves in serially diluted solutions of Foray 48B). The results show no difference between North Carolina and Georgia P. glaucus larval dosage sensitivities, but do illustrate a clear dosage effect for all 4 treatments (cherry with and without bleach pre-treatment; tulip tree with and without bleach pre-treatment). The larvae on the bleached leaves do not do better (for cherry or tulip tree host plant) as would be expected if microbial symbionts on the phylloplane synergize the Btk toxicity. These results show that Btk (at doses several thousand-fold less than aerial sprays across forests for gypsy moth control) will kill P. glaucus, with or without microbial synergism on leaf phylloplanes

Susceptibility of the Endangered Karner Blue Butterfly (Lepidoptera: Lycaenidae) to \u3ci\u3eBacillus Thuringiensis\u3c/i\u3e Var. \u3ci\u3eKurstaki\u3c/i\u3e Used for Gypsy Moth Suppression in Michigan

We investigated the phenological and physiological susceptibility of the endangered Karner blue butterfly (Lycaeides melissa samuelis) to Bacillus thuringiensis var. kurstaki (Bt), a product widely used for gypsy moth (Lymantria dispar) suppression in Michigan and other infested states. We monitored phenology of the bivoltine Karner blue in two regions of Michigan from 1993 to 1995 to determine if larval stages overlapped temporally with the period of Bt application for gypsy moth suppression. Karner blue larvae of the spring generation were found during the period that Bt was applied in nearby areas in 1993 only. However, spring-generation adults or newly laid eggs were observed up to 11 days before applications in 1994 and 1995. Since Karner blue eggs develop within one week, summer-generation larvae were most likely present during or shortly after 1994 and 1995 Bt application periods. These larvae would have been at risk, assuming Bt persistence of 4 to 6 days. Physiological susceptibility of Karner blue larvae to Bt was determined in a laboratory bioassay. Larvae were reared on wild lupine (Lupinus perennis) foliage that was untreated, or sprayed with Bt formulations at rates of 30-37 or 90 BIU/ha. A similar bioassay with second instar gypsy moth larvae on similarly treated white oak (Quercus alba) foliage was conducted concurrently. Karner blue survival was 100%, 27% and 14% on control, low and high Bt treatments, respectively. Early and late Karner blue instars were equally susceptible to Bt. Survival of gypsy moth was 80%, 33% and 5% on control, low and high Bt treatments, respectively, and did not differ significantly from Karner blue survival. We conclude that Karner blue is both phenologically and physiologically susceptible to Bt used for gypsy moth suppression, although the larval generation at risk and extent of phenological overlap may vary from year to year

Interaction between functional domains of Bacillus thuringiensis insecticidal crystal proteins

Interactions among the three structural domains of Bacillus 1huringiensis Cn.l toxins %~ere investigated by functional analysis of chinieric proteins. Hybrid genes were prepared by exchanging the regions coding for either domain 1 or domain III among CrylAb, Cn,lAc, CrylC, and CrylE. The activity of the purified trypsinactivated chimeric toxins was evaluiated by testing their effects on the viability and plasma membrane permeability of Sf9 cells. Among the parental toxins, only CrylC was active against these cells and only chimeras possessing domain II from CrylC were functional. Combination of domain 1 from CrylE Niith domains Il and III from CrylC, however, resulted in an inactive toxin, indicating that domain II from an active toxin is necessary, but not sufficient, for activity. Pores formed by chimeric toxins in which domain I was frorn Cr31M or CrylAc were slightly smaller than those formed by toxins in which domain I was from CrylC. The properties of the pores formed by the chimeras are therefore likely to result froin an interaction between domain I and domain II or 111. Domain III appears to modulate the activity of the chimeric toxins: combination of domain 111 from CrylAb with domains 1 and II of CrylC gave a protein which was more strongly active than CrylC. (Résumé d'auteur

Isolation of multiple subspecies of Bacillus thuringiensis from a population of the European sunflower moth, Homoeosoma nebulella

Five subspecies of #Bacillus thuringiensis# were isolated from dead and diseased larvae obtained from a laboratory colony of the European sunflower moth, #Homoeosoma nebulella#. The subspecies isolated were #B. thuringiensis# subspp. #thuringiensis# (H la), #kurstaki# (H 3a3b3c), #aizawai# (H 7), #morrisoni# (H 8a8b), and #thompsoni# (H 12). Most isolates produced typical bipyramidal crystals, but the #B. thuringiensis#. subsp. #thuringiensis# isolate produced spherical crystals and the #B. thuringiensis# subsp. #thompsoni# isolate produced a pyramidal crystal. Analysis of the parasporal crystals by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the crystals from the #B. thuringiensis# subsp. #kurstaki# and #aizawai# isolates contained a protein of 138 kDa whereas those from #B. thuringiensis# subsp. #morrisoni# contained a protein of 145 kDa. The crystals from #B. thuringiensis# subsp. #thuringiensis# contained proteins of 125, 128, and 138 kDa, whereas those from #B. thuringiensis# subsp. #thompsoni# were the most unusual, containing proteins of 37 and 42 kDa. Bioassays of purified crystals conducted against second-instar larvae of #H. nebulella# showed that the isolates of #B. thuringiensis# subspp.#aizawai#, #kurstaki#, and #thuringiensis# were the most toxic, with 50% lethal concentrations (LCS 50s) of 0.15, 0.17, and 0.26 ug/ml, respectively. The isolates of #B. thuringiensis# subspp. #morrisoni# and #thompsoni# had LC 50s of 2.62 and 37.5 ug/ml, respectively. These results show that a single insect species can simultaneously host and be affected by a variety of subspecies of #B. thuringiensis# producing different insecticidal proteins. (Résumé d'auteur

Development of Bacillus thuringiensis CryIC resistance by Spodoptera exigua (Huebner) (Lepidoptera : Noctuidae)

Selection of resistance in Spodoptera exigua (Hubner) to an HD-1 spore-crystal mixture, CryIC (HD-133) inclusion bodies, and trypsinized toxin from Bacillus thuringiensis' subsp, aizawai and B. thuringiensis subsp. entomocidus was attempted by using laboratory bioassays. No resistance to the HD-1 spore-crystal mixture could be achieved after 20 generations of selection. Significant levels of resistance (11-fold) to CryIC inclusion bodies expressed in Escherichia coli were observed after seven generations, Subsequent selection of the CryIC-resistant population with trypsinized CryIC toxin resulted, after 21 generations of CryIC selection, in a population of S. exigua that exhibited only 8% mortality at the highest toxin concentration tested (320 mu g/g), whereas the 50% lethal concentration was 4.30 mu g/g for the susceptible colony. Insects resistant to CryIC toxin from HD-133 also were resistant to trypsinized CryIA(b), CryIC from B. thuringiensis subsp. entomocidus, CryIE-CryIC fusion protein (G27), CryIH, and CryIIA. In vitro binding experiments with brush border membrane vesicles showed a twofold decrease in maximum CryIC binding, a fivefold difference in K-d, and no difference in the concentration of binding sites for the CryIC-resistant insects compared with those for the susceptible insects, Resistance to CryIC was significantly reduced by the addition of HD-1 spores, Resistance to the CryIC toxin was still observed 12 generations after CryIC selection was removed. These results suggest that, in S. exigua, resistance to a single protein is more likely to occur than resistance to spore crystal mixtures and that once resistance occurs, insects will be resistant to many other Cry proteins, These results have important implications for devising S. exigua resistance management strategies in the field

Histopathological effects of cypermethrin and Bacillus thuringiensis var. israelensis on midgut of Chironomus calligraphus larvae (Diptera: Chironomidae)

Pesticides are extensively used for the control of agricultural pests and disease vectors, but they also affect non-target organisms. Cypermethrin (CYP) is a synthetic pyrethroid used worldwide. Otherwise, bioinsecticides like Bacillus thuringiensis var. israelensis (Bti) have received great attention as an environmentally benign and desirable alternative. In order to evaluate the toxicity of those pesticides, Chironomus calligraphus was selected due to its high sensitivity to some toxicants. Third and fourth instars larvae were exposed to serial dilutions of CYP and Bti to determine LC50 values. In order to evaluate the potentially histopathological alterations as biomarkers, after 96-h of exposure, live larvae were fixed for histological analysis of the mid region of digestive tract. The 96-h LC50 values were 0.52 and 1.506 μg/L for CYP and Bti, respectively. Midgut histological structure of the control group showed a single layer of cubical cells with microvilli in their apical surface and a big central nucleus. The midgut epithelium of larvae exposed to a low concentration of CYP (0.037 μg/L) showed secretion activity and vacuolization while at high concentration (0.3 μg/L) cells showed a greater disorganization and a more developed fat body. On the other hand, Bti caused progressive histological damage in this tissue. Chironomus calligraphus is sensitive to Bti and CYP toxicity like other Chironomus species. The histopathological alterations could be a valuable tool to assess toxicity mechanism of different pesticides.Fil: Lavarias, Sabrina Maria Luisa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Limnología "Dr. Raúl A. Ringuelet". Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Limnología; ArgentinaFil: Arrighetti, Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Museo Argentino de Ciencias Naturales "Bernardino Rivadavia"; ArgentinaFil: Siri, Augusto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Limnología "Dr. Raúl A. Ringuelet". Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Limnología; Argentin

Bacteria as Ovipositional Attractants for \u3ci\u3eAedes Aegypti\u3c/i\u3e (Diptera: Culicidae)

The effectiveness of selected bacterial species as ovipositional attractants for Aedes aegypti was compared. Bacterial washes, in glass containers, were utilized as ovipositional substrates and subsequent egg counts determined the degree of ovipositional attractiveness. Among others, Bacillus cereus and Pseudomonas aeruginosa were noted as being effective attractants. It was concluded that A. aegypti displays discriminatory behavior in selecting individual bacterial species for oviposition

Dominant negative phenotype of Bacillus thuringiensis Cry1Ab, Cry11Aa and Cry4Ba mutants suggest hetero-oligomer formation among different Cry toxins.

Background - Bacillus thuringiensis Cry toxins are used worldwide in the control of different insect pests important in agriculture or in human health. The Cry proteins are pore-forming toxins that affect the midgut cell of target insects. It was shown that non-toxic Cry1Ab helix a-4 mutants had a dominant negative (DN) phenotype inhibiting the toxicity of wildtype Cry1Ab when used in equimolar or sub-stoichiometric ratios (1:1, 0.5:1, mutant:wt) indicating that oligomer formation is a key step in toxicity of Cry toxins. Methodology/Principal Findings - The DN Cry1Ab-D136N/T143D mutant that is able to block toxicity of Cry1Ab toxin, was used to analyze its capacity to block the activity against Manduca sexta larvae of other Cry1 toxins, such as Cry1Aa, Cry1Ac, Cry1Ca, Cry1Da, Cry1Ea and Cry1Fa. Cry1Ab-DN mutant inhibited toxicity of Cry1Aa, Cry1Ac and Cry1Fa. In addition, we isolated mutants in helix a-4 of Cry4Ba and Cry11Aa, and demonstrate that Cry4Ba-E159K and Cry11Aa-V142D are inactive and completely block the toxicity against Aedes aegypti of both wildtype toxins, when used at sub-stoichiometric ratios, confirming a DN phenotype. As controls we analyzed Cry1Ab-R99A or Cry11Aa-E97A mutants that are located in helix a-3 and are affected in toxin oligomerization. These mutants do not show a DN phenotype but were able to block toxicity when used in 10:1 or 100:1 ratios (mutant:wt) probably by competition of binding with toxin receptors. Conclusions/Significance - We show that DN phenotype can be observed among different Cry toxins suggesting that may interact in vivo forming hetero-oligomers. The DN phenotype cannot be observed in mutants affected in oligomerization, suggesting that this step is important to inhibit toxicity of other toxin

Perbedaan Daya Bunuh Bacillus thuringiensis Isolasi dari Habitat Tanah Sawah dan Tanah Bawah Pohon terhadap Larva Aedes aegypti

Bacillus thuringiensis telah banyak duigunakan dalam pengendalian vektor nyamuk karena efektif membunuh berbagai larva nyamuk, tidak menimbulkan resistensi vektor, bersifat spesifik target dan bersifat kosmopolit. Penelitian ini bertujuan untuk menilai perbedaan daya bunuh isolat Bacillus thuringiensis habitat tanah sawah dan tanah bawah pohon yang diambil di Kecamatan Pangkah, Kabupaten Tegal terhadap larva nyamuk Aedes aegypti. Metode penelitian ini adalah eksperimen dengan pre test-post test control group design. Penelitian ini menggunakan 25 ekor larva Aedes aegypti setiap perlakuan dengan 3 kali ulangan. Data dianalisis dengan menggunakan uji independent t test. Hasil penelitian ini menunjukkan bahwa tidak ada perbedaan daya bunuh isolat Bacillus thuringiensis terhadap larva Aedes aegypti antara habitat tanah sawah dan tanah bawah pohon dengan p value 0,606 (>0,05). Dari isolat-isolat yang berasal dari habitat tanah sawah dan tanah bawah pohon memiliki daya bunuh >70 % pada 24 jam dan bertahan hingga dalam waktu 48 jam perlakuan. Dari penelitian ini dapat disimpulkan bahwa isolat Bacillus thuringiensis yang berasal dari tanah sawah dan tanah bawah pohon di Kecamatan Pangkah, Kabupaten Tegal efektif membunuh larva Aedes aegypti dan tidak ada perbedaan daya bunuh Bacillus thuringiensis antara habitat tanah sawah dan tanah bawah pohon. Penelitian ini perlu dilanjutkan untuk mengetahui serotipe isolat Bacillus thuringiensis. Kata Kunci: Bacillus thuringiensis, habitat, Aedes aegypti, daya bunu