`Cloning of <Emphasis Type="Italic">vip1/vip2</Emphasis> genes and expression of Vip1Ca/Vip2Ac proteins in <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

Forty-one Bacillus thuringiensis (Bt) standard reference strains and 118 Bt local isolates were screened for vip1/vip2 genes by PCR amplification, with only three strains (HD201, HD109 and HD12) producing the desired bands. Southern blot showed that vip1/vip2 genes were located on a 10 Kb EcoRV fragment of their total DNAs. Furthermore, the vip1Ca/vip2Ac genes were cloned from a partial genomic library of HD201. Sequence homologous analysis revealed that vip2Ac gene was highly conserved and encoded a protein possibly having ADP-ribosyltransferase activity, and that vip1Ca gene was of low homology, especially at its 3-terminus. Western blot showed that Vip1Ca and Vip2Ac proteins could be detected from middle logarithmic phase to the stationary phase in Bt HD201. However, bioassays of HD201 supernatants exhibited no activity against Culex quinquefasciatus, Spodoptera exigua, S. litura, Helicoverpa amigera and Tenebrio molitor larvae. Whether Vip1Ca and Vip2Ac proteins have any toxicity to other susceptible targets still needs to be investigated.     

Cloning of acryIIIA endotoxin gene ofBacillus thuringiensis var.tenebrionis and its transient expression inindica rice

Damage caused to rice production by coleopteran insects like rice weevil (Sitophilus oryzae), a stored grain insect pest and rice hispa (Dicladispa armigera), a pest of the growing plant is quite high. In order to combat the damage, generation of insect resistant transgenic rice plant was considered desirable. CryIIIA endotoxin ofBacillus thuringiensis var.tenebrionis, a 65 kDa protein toxic to coleopteran insects, figured as the candidate gene product. Thus, the cryIIIA gene was isolated from a local isolate ofBacillus thuringiensis var.tenebrionis. The gene was tailored at the N-terminal end to its minimal size by using a synthetic ATG codon which replaced the first codon next to ATG of threonine to proline. This modification did not affect the functional property of the gene product. A chimeric construct of the modifiedcryIIIA gene was developed containing CaMV35S promoter andnos terminator for plant expression. The expressibility of thecryIIIA gene inindica rice was judged through test for transient expression in indica rice protoplasts.     

Effects of genetic and environmental host plant variation on the susceptibility of two noctuids toBacillus thuringiensis

TwoApium graveolens var.rapaceum (L.) cultivars that differ in their suitability for the survival and growth ofSpodoptera exigua (Hübner) andTrichoplusia ni (Hübner) were used to examine the effect of genetic and seasonal environmental variation in host plant suitability on the efficacy ofBacillus thuringiensis subsp.kurstaki (Berliner). The effects of host plant genotype andB. thuringiensis were generally independent, so thatB. thuringiensis efficacy was greatest on the resistant host plant cultivar. Host plant suitability varied within growing season for both insect species but, while host plant suitability decreased with increasing plant age forT. ni, the response ofS. exigua to plant age was not as clear. Within season variation in host plant suitability affectedB. thuringiensis efficacy and the interaction betweenB. thuringiensis and host plant cultivar forS. exigua but not forT. ni. Soluble protein and Folin-Denis phenolic concentrations of host plant tissue were not correlated with changes in host plant suitability to either insect species.     

Host Range of an Insecticidal Crystal Protein of Bacillus thuringiensis subsp. kurstaki Produced in Escherichia coli

A crylA(c)-like gene of Bacillus thuringiensis subsp. kurstaki strain HD1 was over-expressed in Escherichia coli from a multicopy plasmid. Biological toxicity tests conducted on the larvae of three lepidopteran insects showed that the host range of transgenic E. coli HB 101 (pRT 200) was a subset of the host range of B. thuringiensis kurstaki HD1. Both were toxic to the larvae of Helicoverpa armigera (Gram pod borer) and Bombyx mori (Silkworm). However. though the sporecrystal formulation of HDl was toxic to the larvae of Phthorimaea operculella (Potato tuber moth). the transgenic E. coli was not. Product of St toxin gene other than crylA(c) present In HD1 may be responsible for Its toxicity to the larvae of P. opercuiella.     

Cloning and expression inEscherichia coli of entomotoxic protein gene fromBacillus thuringiensis subspecieskurstaki

A plasmid borne larvicidal crystal protein gene from B.thuringiensis subspecieskurstaki was cloned inEscherichia coli using a specific 20-mer oligonucleotide probe. The gene expressed inE. coli at a high level. TransgenicE. coli cells produced large irregular bodies which looked bright under phase contrast microscopy. The phase bright bodies released by sonic disruption of cells could be pelleted by centrifugation. Toxicity trials on the larvae ofSpodoptera litura showed that the pellet was antifeedant and toxic to the larvae. The supernatant was only mildly antifeedant. Even short term feeding of larvae on the toxin delayed the onset of pupation.     

Kinetics of plasmid transfer among <Emphasis Type="Italic">Bacillus cereus </Emphasis>group strains within lepidopteran larvae

The cry toxin encoding plasmid pHT73 was transferred from Bacillus thuringiensis subspecies kurstaki KT0 to six B. cereus group strains in three lepidopteran (Spodoptera exigua, Plutella xyllostella and Helicoverpa armigera) larvae by conjugation. The conjugation kinetics of the plasmid was precisely studied during the larval infection using a new protocol. The infections were performed with both vegetative and sporulated strains. However, larval death only occurred when infections were made with spore and toxin preparations. Likewise, spore germinations of both donor and recipient strains were only observed in killed larvae, 44–56 h post-infection. Accordingly, kinetics showed that gene transfer between B. thuringiensis strain KT0 and other B. cereus strains only took place in dead larvae among vegetatively growing bacteria. The conjugational transfer ratios varied among different strain combinations and different larvae. The highest transfer ratio reached 5.83 × 10⁻⁶ CFU/donor between the KT0 and the AW05R recipient in Helicoverpa armigera, and all transconjugants gained the ability to produce the insecticidal crystal. These results indicated that horizontal gene transfer among B. cereus group strains might play a key role for the acquisition of extra plasmids and evolution of these strains in toxin susceptible insect larvae.     

Isolation of spore-forming bacilli from mosquitoes in natural breeding habitats in Iraq

New isolates of spore-forming bacilli from larvae and pupae of 3 species of mosquitoes are recorded in central Iraq.Bacillus sphaericus Meyer and Neide was isolated fromCulex pipiens (L.) larva.Bacillus carotarum Koch andBacillus cereus Frankland & Frankland were isolated fromTheobaldia longiareolata (Macquart) pupae.Bacillus laterosporus Laubach andBacillus thuringiensis (H 18) were isolated fromAedes caspius (Pallas) larvae. In addition, unidentifiedBacillus spp. were isolated fromCx. pipiens, T. longiareolata andAe. caspius larvae. Examination of soil samples collected from mosquito natural breeding habitats revealed isolates ofB. cereus, Bacillus thuringiensis H4a 4b; H 12 and H 16 and an unidentifiedBacillus sp.

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Résumé Des souches bactériennes sporogènes sont isolées de moustiques qui se trouvent dans la région centrale de l'Irak. Les résultats obtenus sont les suivants:Bacillus sphaericus Meyer & Neide [Culex pipiens (L.), larve],Bacillus carotarum Koch etBacillus cereus Frankland & Frankland [Theobaldia longiareolata (Macquart), nymphe],Bacillus laterosporus Laubach etBacillus thuringiensis (H 18) [Aedes caspius (Pallas), larvel]. L'examen des larves deCulex pipiens. T. longiareolata etAe. caspius, ainsi que l'analyse des échantillons du sol prélevés dans la région montrent la présence deBacillus cereus, Bacillus thuringiensis H4a 4b; H12 plus H16 et d'autresBacillus non identifiés.

     

Partial purification of Cry 1A toxin-binding proteins from Helicoverpa armigera larval midgut

Toxicity of insecticidal endotoxins produced by Bacillus thuringiensis correlates with the presence of specific proteins in the midgut of susceptible larvae. This study was aimed at identifying and purifying Cry 1A binding proteins from Helicoverpa armigera, an important crop pest of India. B. thuringiensis strain HD 73 which produces Cry 1Ac toxin, specific for H. armigera was used in this study. Toxin-binding proteins from insect larvae were detected by employing a toxin overlay assay using both radiolabelled as well as unlabelled toxin. Detergent-solubilized fractions of larval brush border membranes were subjected to soybean agglutinin (SBA) chromatography, from which N-acetylgalactosamine (NAG)-containing proteins were eluted. Analysis of the SBA-purified proteins indicated that four proteins of approximately 97, 120, 170 and 200 kDa could bind to Cry 1Ac toxin, and three proteins of 97, 170 and 200 kDa proteins could bind to Cry 1Ab. Furthermore, in the presence of excess Cry 1Ab toxin, the labelled Cry 1Ac toxin could bind only to 170 and 200 kDa proteins, implying that Cry 1Ab can also bind the 120 kDa protein. This study therefore demonstrates that in H. armigera, midgut proteins of 97, 120, 170 and 200 kDa have the ability to bind both Cry 1Ab and Cry 1Ac. Furthermore, while the 170 and 200 kDa proteins have higher affinity for Cry 1Ac, the 97 kDa has higher affinity for Cry1 Ab. This revised version was published online in July 2006 with corrections to the Cover Date.     

Expression and activity of a probable toxin from Photorhabdus luminescens

As an insect pathogen, Photorhabdus luminescens possesses an arsenal of toxins. Here we cloned and expressed a probable toxin from P. luminescens subsp. akhurstii YNd185, designated as Photorhabdus insecticidal toxin (Pit). The pit gene shares 94% nucleotide and 98% predicted amino acid sequence identity with plu1537, a predicted ORF from P. luminescens subsp. laumondii TT01 and 30% predicted amino acid sequence similarity to a fragment of a 13.6 kDa insecticidal crystal protein gene of Bacillus thuringiensis (Bt). The pit was expressed as a GST-Pit fusion protein in E. coli, most of which was insoluble and sequestered into inclusion bodies. The inclusion bodies were harvested and dissolved. The resultant protein was purified and the Pit was cleaved from the fusion protein by thrombin and purified from GST then used for bioassay. Pit killed Galleria mellonella (LD₅₀, 30 ng/larva) and Spodoptera litura (LD₅₀, 191 ng/larva) via hemocoel injection. Relative to a control that lacked toxin, Pit did not significantly increase mortality of S. litura and Helicoverpa armigera when introduced orally, but the treatment did inhibit growth of the insects. The present study demonstrated that Pit possessed insecticidal activity.     

Characterization of the cry1Ac17 Gene from an Indigenous Strain of Bacillus thuringiensis subsp. kenyae

An indigenously isolated strain of Bacillus thuringiensis subsp. kenyae exhibited toxicity against lepidopteran as well as dipteran insects. The lepidopteran active cry1Ac protoxin gene coding sequence of 3.5 kb from this strain was cloned into vector pET28a(+). However, it could not be expressed in commonly used Escherichia coli expression hosts, BL21(DE3) and BL21(DE3)pLysS. This gene is classified as cry1Ac17 in the B. thuringiensis toxic nomenclature database. The coding sequence of this gene revealed that it contains about 3% codons, which are not efficiently translated by these expression hosts. Hence, this gene was expressed in a modified expression host, Epicurian coli BL21-Codonplus (DE3)-RIL. The expression of gene yielded a 130-kDa Cry1Ac17 protein. The protein was purified and its toxicity was tested against economically important insect pests, viz., Helicoverpa armigera and Spodoptera litura. LC₅₀ values obtained against these insects were 0.1 ng/cm³ and 1231 ng/cm², respectively. The higher toxicity of Cry1Ac17 protein, compared to other Cry1Ac proteins, toward these pests demonstrates the potential of this isolate as an important candidate in the integrated resistance management program in India.     

Expression and characterization of inhA gene from Bacillus thuringiensis 8010

InhA, a zinc metalloprotease secreted by Bacillus thuringiensis, specifically hydrolyzes antibacterial peptides produced by insect hosts. In this study, the inhA gene was cloned from B. thuringiensis 8010 using a pair of degenerate primers and the deduced 796 amino acid sequence showed a high degree of similarity with other InhA proteins in the Bacillus cereus group. The deduced amino acid sequence contained the zinc-binding motif (HEXXH), which is characteristic of the zinc-metalloprotease family. Additionally, the inhA gene was expressed in Escherichia coli BL21 (DE3). The expressed InhA protein was shown to be toxic to the third larvae of Plutella xylostella, contrary to preliminary study concerning the effect of InhA on Bombyx mori. This study provided insights into the potential of InhA for the biological control of certain lepidopteran insects.     

Crystals of two mutants ofBacillus thuringiensis show increased toxicity against larvae ofSpodoptera littoralis

Crystals of two asporogenous mutants ofBacillus thuringiensis var.kurstaki strain HD-1 obtained following treatment with ozone and N-methyl-N′-nitro-N-nitrosoguanidine showed increased toxicity against larvae ofSpodoptera littoralis when compared to the wild-type crystal.      

Influence of laboratory hosts on the biological attributes ofChelonus blackburni [Hym.: Braconidae]

Among 6 laboratory hosts tested, the egg-larval parasitoidChelonus blackburni Cameron completed its development in only 3;Corcyra cephalonica Stainton,Phthorimaea operculella (Zeller) andAchroia grisella (Fabricius). The parasitoid failed to develop inSpodoptera litura (Fabricius),Galleria mellonella L. andSitotroga cerealella (Olivier). The development period of the parasitoids obtained from permissive hosts correspond to the development period of their hosts. Development period ofP. operculella, A grisella andC. cephalonica were 24.7±2.0, 39.5±2.1 and 50.5±4.1 days respectively while the development period of the parasitoid reared from these hosts were 25.8±1.6, 36.4±3.5 and 42.5±3.5 days respectively. The fecundity of the parasitoids reared onA. grisella, P. operculella andC. cephalonica were 365.2±52.8, 287.9±101.9 and 248.7±50.8 respectively. The size of the parasitoids reared from the above 3 hosts also followed the similar trend. However, no significant difference was observed on the percent parasitism and the longevity of the parasitoids reared on different hosts. This study was conducted at a temperature of 24±2°C and 60±5% RH. Contribution No 46001 of Biological Control Centre (NCIPM), Bangalore.     

Characterization of the genes encoding the haemolytic toxin and the mosquitocidal delta-endotoxin of Bacillus thuringiensis israelensis

Summary The crystalline parasporal inclusions (crystals) of Bacillus thuringiensis israelensis (Bti), which are specifically toxic to mosquito and black fly larvae, contain three main polypeptides of 28 kDa, 68 kDa and 130 kDa. The genes encoding the 28 kDa protein and the 130 kDa protein have been cloned from a large plasmid of Bti. Escherichiacoli recombinant clones containing the 130 kDa protein gene were highly active against larvae of Aedes aegypti and Culex pipiens, while B. subtilis recombinant cells containing the 28 kDa protein gene were haemolytic for sheep red blood cells. A fragment of the Bti plasmid which is partially homologous to the 130 kDa protein gene was also isolated; it probably corresponds to part of a second type of mosquitocidal toxin gene. Furthermore, restriction enzyme analysis suggested that the 130 kDa protein gene is located on the same Bti EcoRI fragment as another kind of Bti mosquitocidal protein gene cloned by Thorne et al. (1986). Hybridization experiments conducted with the 28 kDa protein gene and the 230 kDa protein gene showed that these two Bti genes are probably present in the plasmid DNA of B. thuringiensis subsp. morrisoni (PG14), which is also highly active against mosquito larvae.     

Reduction of resistance of Culex pipiens larvae to the binary toxin from Bacillus sphaericus by coexpression of cry4Ba from Bacillus thuringiensis subsp. israelensis with the binary toxin gene

The cry4Ba gene from Bacillus thuringiensis subsp. israelensis and the binary toxin gene from B. sphaericus C3-41 were cloned together into a shuttle vector and expressed in an acrystalliferous strain of B. thuringiensis subsp. israelensis 4Q7. Transformed strain Bt-BW611, expressing both Cry4Ba protein and binary toxin protein, was more than 40-fold more toxic to Culex pipiens larvae resistant to B. sphaericus than the transformed strains expressing Cry4Ba protein or binary toxin protein independently. This result showed that the coexpression of cry4Ba of B. thuringiensis subsp. israelensis with B. sphaericus binary toxin gene partly suppressed more than 10,000-fold resistance of C. pipiens larvae to the binary toxin. It was suggested that production of Cry4Ba protein and binary toxin protein interacted synergistically, thereby increasing their mosquito-larvicidal toxicity.     

Cloning and characterization of truncated <Emphasis Type="Italic">cry1Ab</Emphasis> gene from a new indigenous isolate of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

The insecticidal crystal protein(s) encoded by cry gene(s) of Bacillus thuringiensis (Bt) have been used for insect control both as biopesticides and in transgenic plants. A new 3′-truncated cry1Ab gene was cloned from an indigenous isolate of Bt, A19-31. Nucleotide sequencing and homology search revealed that the deduced amino acid sequence of Cry1Ab toxin of Bt strain A19-31 had a variation of two amino acid residues with the holotype sequence, Cry1Ab1. Expression of the 3′-truncated cry1Ab gene was studied in an acrystalliferous strain of Bt (4Q7). SDS-PAGE and immunostrip analysis of spore-crystal mixture revealed a low level expression of the 3′-truncated cry1Ab gene. Insecticidal activity assay showed that the recombinant 3′-truncated cry1Ab gene product was toxic to larvae of both Helicoverpa armigera and Spodoptera litura.     

High level expression of soybean trypsin inhibitor gene in transgenic tobacco plants failed to confer resistance against damage caused byHelicoverpa armigera

Helicoverpa armigera is a major pest of many tropical crop plants. Soybean trypsin inhibitor (SBTI) was highly effective against the proteolytic activity of gut extract of the insect. SBTI was also inhibitory to insect growth when present in artificial diet. The gene coding for SBTI was cloned from soybean (Glycine max, CVBirsa) and transferred to tobacco plants for constitutive expression. Young larvae ofH. armigera, fed on the leaves of the transgenic tobacco plants expressing high level of SBTI, however, maintained normal growth and development. The results suggest that in certain cases the trypsin inhibitor gene(s) may not be suitable candidates for developing insect resistant transgenic plants.     

The effects of temperature and host density on the development and survival of Copidosoma koehleri

Developmental rates for Copidosoma koehleri Blanchard (Hymenoptera: Encyrtidae) and its host, Phthorimaea operculella (Zeller), were determined at 10, 15, 23, 27, 29, 32, 34 and 35°C from host egg to adult. The developmental rates determined for both species showed good fit to mathematical models of insect development. At 15°C mean emergence of adult C. koehleri was 15 days after P. operculella adults emerged. At 29°C mean emergence of C. koehleri was only 5 days after that of P. operculella. P. operculella developed at 35°C, but parasitized larvae, and therefore C. koehleri, did not tolerate temperatures above 32°C. In the presence of C. koehleri, host survival was low (3%) at low host egg densities, but greater (20%) at higher host densities. Parasitized larvae of P. operculella were less able to compete for food resources, as measured by adult emergence.     

Insecticidal activity of a cryia(c) transgene in callus derived from regeneration-recalcitrant cotton (Gossypium hirsutum L.)

Summary The insecticidal effectiveness of a δ-endotoxin Cry protein from Bacillus thuringiensis in non-regenerable callus of a commercial Gossypium hirsutum L. variety was investigated. Two transgenic callus types were generated. The first callus type harbored the cry1A(c) gene and the hygromycin B phosphotransferase hpt selectable marker gene. The second callus type, the transgenic control, carried the marker genes β-glucuronidase (GUS) and hpt. Growth and survival rates of three major cotton moth species, Pectinophora gossypiella, Helicoverpa armigera, and Spodoptera littoralis, were examined with aseptic neonates reared on callus. Normal larval development occurred in all species supplied with non-transgenic callus, but insects died, or their growth was severely restricted, when reared on transgenic callus harvested from hygromycin B-supplemented medium. Development of larvae on transgenic control and on non-transgenic callus became very much alike after the transgenic control tissue had been subcultured on a hygromyein B-free medium for about 100 d prior to the insect-callus bioassay. Accordingly, for detection of Bt toxin activity without the interference of the influence of hygromycin B on insects, cry1A(c) callus was infested with insects after it had been propagated for more than 100 d on a medium free of the antibiotic. Under these experimental conditions all P. gossypiella and H. armigera, and most S. littoralis neonates died, and the growth (e.g., weight increment) of S. littoralis survivors was markedly impeded by cry1A(c) callus. Three new findings emerge from this study: first, P. gossypiella, a pest feeding in the field on bolls only, can be grown in vitro on cotton callus; second, in a host which is recalcitrant in terms of plant regeneration, the biological potency of an insectdetrimental transgene can nevertheless be evaluated by generating a transgenic host callus and conducting in vitro transgenic callus-insect assays; and third, our results suggest that hygromycin B is toxic to lepidopteran larvae.     

Sex determination in<Emphasis Type="Italic"> Drosophila melanogaster</Emphasis> and<Emphasis Type="Italic"> Musca domestica</Emphasis> converges at the level of the terminal regulator<Emphasis Type="Italic"> doublesex</Emphasis>

Sex-determining cascades are supposed to have evolved in a retrograde manner from bottom to top. Wilkins 1995 hypothesis finds support from our comparative studies in Drosophila melanogaster and Musca domestica, two dipteran species that separated some 120 million years ago. The sex-determining cascades in these flies differ at the level of the primary sex-determining signal and their targets, Sxl in Drosophila and F in Musca. Here we present evidence that they converge at the level of the terminal regulator, doublesex (dsx), which conveys the selected sexual fate to the differentiation genes. The dsx homologue in Musca, Md-dsx, encodes male-specific (MdDSX^M) and female-specific (MdDSX^F) protein variants which correspond in structure to those in Drosophila. Sex-specific regulation of Md-dsx is controlled by the switch gene F via a splicing mechanism that is similar but in some relevant aspects different from that in Drosophila. MdDSX^F expression can activate the vitellogenin genes in Drosophila and Musca males, and MdDSX^M expression in Drosophila females can cause male-like pigmentation of posterior tergites, suggesting that these Musca dsx variants are conserved not only in structure but also in function. Furthermore, downregulation of Md-dsx activity in Musca by injecting dsRNA into embryos leads to intersexual differentiation of the gonads. These results strongly support a role of Md-dsx as the final regulatory gene in the sex-determining hierarchy of the housefly.Edited by D. Tautz