MicroRNAs (miRNAs) are endogenous, non-coding RNAs, which are functional in a variety of biological processes through post-transcriptional regulation of gene expression. However, the role of miRNAs in the interaction between Bacillus thuringiensis and insects remains unclear. In this study, small RNA libraries were constructed for B. thuringiensis-infected (Bt) and uninfected (CK) Spodoptera exigua larvae (treated with double-distilled water) using Illumina sequencing. Utilising the miRDeep2 and Randfold, a total of 233 known and 726 novel miRNAs were identified, among which 16 up-regulated and 34 down-regulated differentially expressed (DE) miRNAs were identified compared to the CK. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that potential target genes of DE miRNAs were associated with ABC transporters, fatty acid metabolism and MAPK signalling pathway which are related to the development, reproduction and immunity. Moreover, two miRNA core genes, SeDicer1 and SeAgo1 were identified. The phylogenetic tree showed that lepidopteran Dicer1 clustered into one branch, with SeDicer1 in the position closest to Spodoptera litura Dicer1. A similar phylogenetic relationship was observed in the Ago1 protein. Expression of SeDicer1 increased at 72 h post infection (hpi) with B. thuringiensis; however, expression of SeDicer1 and SeAgo1 decreased at 96 hpi. The RNAi results showed that the knockdown of SeDicer1 directly caused the down-regulation of miRNAs and promoted the mortality of S. exigua infected by B. thuringiensis GS57. In conclusion, our study is crucial to understand the relationship between miRNAs and various biological processes caused by B. thuringiensis infection, and develop an integrated pest management strategy for S. exigua via miRNAs.

Mythimna separata (Lepidoptera: Noctuidae) is an omnivorous pest that poses a great threat to food security. Insect antimicrobial peptides (AMPs) are small peptides that are important effector molecules of innate immunity. Here, we investigated the role of the AMP cecropin B in the growth, development, and immunity of M. separata. The gene encoding M. separata cecropin B (MscecropinB) was cloned. The expression of MscecropinB was determined in different developmental stages and tissues of M. separata. It was highest in the prepupal stage, followed by the pupal stage. Among larval stages, the highest expression was observed in the fourth instar. Tissue expression analysis of fourth instar larvae showed that MscecropinB was highly expressed in the fat body and haemolymph. An increase in population density led to upregulation of MscecropinB expression. MscecropinB expression was also upregulated by the infection of third and fourth instar M. separata with Beauveria bassiana or Bacillus thuringiensis (Bt). RNA interference (RNAi) targeting MscecropinB inhibited the emergence rate and fecundity of M. separata, and resulted in an increased sensitivity to B. bassiana and Bt. The mortality of M. separata larvae was significantly higher in pathogen plus RNAi-treated M. separata than in controls treated with pathogens only. Our findings indicate that MscecropinB functions in the eclosion and fecundity of M. separata and plays an important role in resistance to infection by B. bassiana and Bt.

The sugarcane giant borer, Telchin licus licus, is an insect pest that causes significant losses in sugarcane crops and in the sugar-alcohol sector. Chemical and manual control methods are not effective. As an alternative, in the current study, we have screened Bacillus thuringiensis (Bt) Cry toxins with high toxicity against this insect. Bioassays were conducted to determine the activity of four Cry toxins (Cry1A (a, b, and c) and Cry2Aa) against neonate T. licus licus larvae. Notably, the Cry1A family toxins had the lowest LC50 values, in which Cry1Ac presented 2.1-fold higher activity than Cry1Aa, 1.7-fold larger than Cry1Ab, and 9.7-fold larger than Cry2Aa toxins. In silico analyses were performed as a perspective to understand putative interactions between T. licus licus receptors and Cry1A toxins. The molecular dynamics and docking analyses for three putative aminopeptidase N (APN) receptors (TlAPN1, TlAPN3, and TlAPN4) revealed evidence for the amino acids that may be involved in the toxin–receptor interactions. Notably, the properties of Cry1Ac point to an interaction site that increases the toxin's affinity for the receptor and likely potentiate toxicity. The interacting amino acid residues predicted for Cry1Ac in this work are probably those shared by the other Cry1A toxins for the same region of APNs. Thus, the presented data extend the existing knowledge of the effects of Cry toxins on T. licus licus and should be considered in further development of transgenic sugarcane plants resistant to this major occurring insect pest in sugarcane fields.

Insect defensins, are cationic peptides that play an important role in immunity against microbial infection. In the present study, an anionic defensin from Plutella xylostella, (designated as PxDef) was first cloned and characterized. Amino acid sequence analysis showed that the mature peptide owned characteristic six-cysteine motifs with predicted isoelectric point of 5.57, indicating an anionic defensin. Quantitative real-time polymerase chain reaction analysis showed that PxDef was significantly induced in epidermis, fat body, midgut and hemocytes after injection of heat-inactivated Bacillus thuringiensis, while such an induction was delayed by the injection of live B. thuringiensis in the 4th instar larvae of P. xylostella. Knocking down the expression of nuclear transcription factor Dorsal in P. xylostella by RNA interference significantly decreased the mRNA level of PxDef, and increased the sensitivity of P. xylostella larvae to the infection by live B. thuringiensis. The purified recombinant mature peptide (PxDef) showed higher activity against Gram-positive bacteria, with the minimum inhibition concentrations of 1.6 and 2.6 µM against B. thuringiensis and Bacillus subtilis, respectively. To our knowledge, this is the first report about an anionic PxDef, which may play an important role in the immune system of P. xylostella against B. thuringiensis.

Maruca vitrata Fabricius (legume pod borer, LPB), the most serious economic pest of legume crops in the tropics, is primarily controlled by chemical pesticide application with serious consequences for the ecosystem and human health. In this study, various concentrations of three commercial biopesticides, NeemBaan®, Bactospeine® (Bacillusthuringiensis (Bt) subsp. kurstaki) and Florbac® (Btaizawai), were tested either in the field or laboratory or in both conditions. In the laboratory experiments, different concentrations of NeemBaan® exhibited significant effects on the mortality of all the tested larval instars and a mortality rate of over 80% was recorded at a dose of 3000 ppm. Bactospeine® was found to be more effective against M. vitrata than Florbac®. Bactospeine® applied at a lower dose of 500 ppm caused 100% mortality in the first-instar and second-instar larvae; however, at the same dose, Florbac® caused mortality of only 26.67% (first instar) and 20% (second instar). In the field experiments, a higher dose of NeemBaan® (6000 ppm) significantly reduced pod damage to approximately 20% in both the first and second cropping seasons. In conclusion, neem- and Bt-based biopesticide products have insecticidal potential to be used in an integrated pest management strategy for controlling M. vitrata in Thailand.

Bacterial endospores are resistant to many environmental factors from temperature extremes to ultraviolet irradiation and are generally more difficult to inactivate or kill than vegetative bacterial cells. It is often considered necessary to treat spores or samples containing spores with chemical fixative solutions for prolonged periods of time (e.g., 1–21 days) to achieve fixation/inactivation to enable electron microscopy (EM) examination outside of containment laboratories. Prolonged exposure to chemical fixatives, however, can alter the ultrastructure of spores for EM analyses. This study was undertaken to determine the minimum amount of time required to inactivate/sterilize and fix spore preparations from several bacterial species using a universal fixative solution for EM that maintains the ultrastructural integrity of the spores. We show that a solution of 4% paraformaldehyde with 1% glutaraldehyde inactivated spore preparations of Bacillus anthracis, Bacillus cereus, Bacillus megaterium, Bacillus thuringiensis, and Clostridium perfringens in 30 min, and Bacillus subtilis in 240 min. These results suggest that this fixative solution can be used to inactivate and fix spores from several major groups of bacterial spore formers after 240 min, enabling the fixed preparations to be removed from biocontainment and safely analyzed by EM outside of biocontainment.

Combinations of Bacillus thuringiensis subsp. kurstaki (Btk) and some plant extracts, namely Acacia arabica (Lam.) Willd., Annona squamosa L., Datura stramonium L., Eucalyptus globulus (Labille), Ipomoea carnea Jacq., Lantana camara L., Nicotiana tabacum L. and Pongamia pinnata (L.) Pierre, were studied for their efficacy and synergistic activity against Spodoptera litura (Fab.) larvae. D.stramonium seed extract fortified with Btk recorded highest mean larval mortality of 74.7%, followed by 61% with A. arabica seed extract, 54.4% with N. tabacum leaf extract and 53% with A. squamosa seed extract. Combination of A. arabica seed extract at 25% concentration and Btk resulted in considerable mortality within 24 h. All Btk fortified extracts caused higher larval mortality than with Btk alone, indicating synergistic action except for the A. arabica leaf extract and Btk combination, which showed antagonistic effect.

Traditional approaches to studying the effects of genetically modified (GM) crops on beneficial insects involve either field assays, comparing insect population levels between control and GM crops or tritrophic bioassays with contaminated insects – usually larvae or eggs of Lepidoptera – as preys. Here, we report the results of a bioassay using an artificial diet, suitable for predatory Coleoptera, to supply Bacillus thuringiensis (Bt) solubilized Cry1Ab and Cry3Aa as well as trypsin-activated Cry1Ab to Atheta coriaria and Cryptolaemus montrouzieri adults and young larvae of Adalia bipunctata. Water, solubilization buffer and trypsin-treated solubilization buffer were used as controls. In total, 1600 insects were assayed. Assays showed a relatively low mortality rate in the controls, ranging from as low as 7% after 15 days (C. montrouzieri) to about 15–20% after five days (A. bipunctata) or 15 days (A. coriaria). For all three predators, there were no statistical differences between the mortality recorded in any of the treatment groups and the corresponding controls. These results indicate a lack of short- (A. bipunctata) and long-term (A. coriaria and C. montrouzieri) mortality associated with oral ingestion of Cry1Ab and Cry3Aa at the high dose tested (50 μg ml−1). We discuss the relevance of these findings for the ecology of beneficial Coleoptera and compatibility with Bt and GM Bt crops.

Bacillus thuringiensis strain WZ-9, isolated from soil in Hebei province, China, was effective against Henosepilachna vigintioctomaculata larvae. The strain presented bipyramidal crystals with a protein band of 130 kDa in SDS–PAGE. The pH changes of the culture media showed important fluctuations during the 24 h growth cycle. The pH varied less in log and stationary phases than it did in the exponential phase. Bioassay results showed that the WZ-9 strain was only harmful to larvae of H. vigintioctomaculata and not to either adults of H. vigintioctomaculata or other several lepidopteran and coleopteran insects. LC50 to second-instar larvae of H. vigintioctomaculata was 2.95×107 cells/ml after 72 h. Genotypic investigations showed that this strain possessed the cry7 gene. Sequence analysis demonstrated that the encoding gene contained an open reading frame (ORF) of 3414 bp and encoded 1138 amino acid residues. The deduced amino acid sequence was 99.65% identical to that of the reported Cry7Ab2 sequences. This gene was designated by the Bt δ-endotoxin nomenclature committee as Cry7Ab3 with accession number BI 1015188 in the GenBank database.

A disrupted allele (r1) of a cadherin gene (Ha_BtR) is genetically associated with incompletely recessive resistance to Bacillus thuringiensis toxin Cry1Ac in a Cry1Ac-selected strain (GYBT) of Helicoverpa armigera. The r1 allele of Ha_BtR was introgressed into a susceptible SCD strain by crossing the GYBT strain to the SCD strain, followed by repeated backcrossing to the SCD strain and molecular marker assisted family selection. The introgressed strain (designated as SCD-r1, carrying homozygous r1 allele) obtained 438-fold resistance to Cry1Ac, >41-fold resistance to Cry1Aa and 31-fold resistance Cry1Ab compared with the SCD strain; however, there was no significant difference in susceptibility to Cry2Aa between the integrated and parent strains. It confirms that the loss of function mutation of Ha_BtR alone can confer medium to high levels of resistance to the three Cry1A toxins in H. armigera. Reciprocal crosses between the SCD and SCD-r1 strains showed that resistance to Cry1Ac in the SCD-r1 strain was completely recessive. Life tables of the SCD and SCD-r1 strains on artificial diet in the laboratory were constructed, and results showed that the net replacement rate (R0) did not differ between the strains. The toxicity of two chemical insecticides, fenvalerate and monocrotophos, against the SCD-r1 strain was not significantly different from that to the SCD strain. However, larval development time of the SCD-r1 strain was significantly longer than that of the SCD strain, indicating a fitness cost of slower larval growth is associated with Ha_BtR disruption in H. armigera.

The gene ap36 encoding a protein elicitor from Alternaria sp. was fused downstream of the slh (S-layer homology) motif of Bacillus thuringiensis S-layer protein gene ctc. The recombinant gene was then transferred into B. thuringiensis plasmid-free derivative strain BMB171. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the SLH–Ap36 fusion protein was expressed in B. thuringiensis BMB171. After tomato (Lycopersicum esculentum) leaves were treated for 90 min with the recombinant strain cultured at 28°C for 24 h, the activity of peroxidase and the amount of proline of tomato leaves were increased to 57.14% and 131.59%, respectively, compared to the control, and after the tomato leaves were treated with the cultured recombinant strain for 4 days, the activity of phenylalanine ammonia lyase was also higher than that in the control. Furthermore, tubers of treated potato (Solanum tuberosum) plants showed higher resistance to rot disease caused by Erwinia corotovora SCG1 compared to the control treatments.

A full-length cry1Ia gene fragment, obtained by polymerase chain reaction (PCR) amplification with a pair of primers designed according to cry1Ia-type gene sequences and DNA from Bacillus thuringiensis as template, was introduced into the expression vector pET-21b and transformed into Escherichia coli BL21(DE3). The molecular weight of the induced expression product was 81.2 kDa. The amino acid sequence of Cry1Ia was very different from sequences of 12 known Cry1Ia-type proteins. This gene was designated as cry1Ia8 by the International B. thuringiensis Insecticidal Protein Nomenclature Committee (accession number AF373207). The bioassay results indicated that Cry1Ia toxin protein showed distinct insecticidal activity against Ostrinia furnacalis and Plutella xylostella with LC50 of 0.268 μg/g and 2.227 μg/ml, respectively. It also had insecticidal activity against Leguminivora glycinivorella, but not against Pyrrhalta aenescens. The novel cry1Ia8 gene will be an important resource in constructing genetically engineered bacteria and transgenic plants for biocontrol of insect pests. It is also available for screening gene stacks to delay pest resistance.

A bioassay method was developed to use the parasporal crystal protein of Bacillus thuringiensis against plant-parasitic nematodes. Using this method, the parasporal crystal proteins of ten Bt strains showed activity against plant-parasitic nematodes. The toxicity of YBT-021 against Meloidogyne hapla, Pratylenchus scribneri, Tylenchorhynchus sp., Ditylenchus destructor and Aphelenchoides sp. was also assayed. The resulting LC50 values were 35.62 μg/ml, 75.65 μg/ml, 94.31 μg/ml, 215.21 μg/ml and 128.76 μg/ml, respectively.

Development of resistance to insecticides has generally been associated with fitness costs that may be magnified under challenging conditions. Lepidopterans which are resistant to the biopesticide Bacillus thuringiensis subsp. kurstaki (Btk) have been shown to have reduced fitness, such as lower survival when subjected to overwintering stress. Recently, resistance to Btk has been found in some populations of Trichoplusia ni Hübner in greenhouses in British Columbia. This situation provides an opportunity to investigate potential trade-offs between overwintering survival and insecticide resistance in a major pest species. Here, we assess the survival and eventual fecundity of Btk resistant and susceptible T. ni pupae exposed to cool temperatures. Contrary to our expectations, resistant T. ni had higher overwintering survival than susceptible individuals. This is the first account of a potential advantage associated with Btk resistance. Resistant and susceptible moths had reduced fecundity and smaller progeny after cold exposure compared to controls, and this may counteract the survival advantage. Nevertheless, it seems unlikely that this is sufficient to select out the resistant phenotype in the presence of strong selection for resistance and in the absence of immigration of susceptible moths. The appearance of resistance without evidence of a trade-off in overwintering survival presents a major challenge to management in production greenhouses.

The S-layer protein CTC surface display system of Bacillus thuringiensis was used to test the possibility of displaying H5N1 Avian influenza virus (AIV) haemagglutinin HA1 on the cell surface of B. thuringiensis. Two recombinant plasmids, pCTC-HA1P and pCSHA1P, were constructed by replacing the central part below the surface anchor sequence slh of S-layer protein gene ctc with part ha1 gene (ha1p). pCTC-HA1P harboured the fusion gene ctc-ha1p and pCSHA1P the fusion gene csa-ctc-ha1p, csa representing the csaAB operon (very important in anchoring S-layer protein on the bacterial cell surface). Two recombinant B. thuringiensis strains were constructed by electrotransferring recombinant plasmids to B. thuringiensis plasmid-free derivative strain BMB171. Strains obtained were CH (bearing pCSHA1P) and BCCH (bearing pCTC-HA1P as well as the csaAB operon-carrying plasmid pMIL-CSA). The vegetative cells of CH and BCCH were used as antigens in haemagglutination (HA) and haemagglutination inhibition (HI) assays. HA assay showed recombinant HA1 proteins successfully displayed on the cell surface of CH and BCCH. HI assay showed that these recombinant HA1 proteins were specific to standard positive HI (haemagglutination inhibition test) serum of subtype H5 AIV. After immunization of mice with vegetative cells, both CH and BCCH elicited a humoral response to HA1 and exhibited immunogenicity as indicated by enzyme-linked immunosorbent assay (ELISA). ELISA also showed that CH exhibited a higher immunogenicity than BCCH. The strategy developed in this study suggests the possibility of generating a heat-stable and oral veterinary vaccine against AIV with the B. thuringiensis S-layer protein CTC surface display system.

The area planted to genetically engineered crops has increased dramatically in the last ten years. This has generated many studies examining non-target effects of bioengineered plants expressing Bacillus thuringiensis endotoxins. To date, most have focused on population-level effects in the field or laboratory evaluation of specific plant-herbivore or plant-herbivore-predator trophic pathways. Using a post-mortem enzyme-linked immunosorbent assay, we examined the uptake of Cry1Ab-endotoxins by predatory coccinellids and the importance of anthesis to this trophic pathway. Adult Coleomegilla maculata, Harmonia axyridis, Cycloneda munda and Coccinella septempunctata contained low, but detectable, quantities of Bt-endotoxin when screened by ELISA. This was most evident in C. maculata, with 12.8% of 775 individuals testing positive for Cry1Ab-endotoxins. Interestingly, the presence of endotoxins in gut samples was not confined to periods around anthesis, but coccinellid adults tested positive two weeks before and up to ten weeks after pollen was shed, suggesting tri-trophic linkages in their food chain facilitates the transfer of endotoxins into higher-order predators. This contrasts with adult Coleomegilla maculata entering overwintering sites where Bt-endotoxins were not detected in gut samples, indicating low levels of persistence of Cry1Ab-endotoxins within coccinellid predators. This study enhances our understanding of complex interactions between transgenic crops and non-target food webs, but further research is required to quantify the significance of specific trophic linkages in the field.

Recombinant Bac-GV2 DNA was obtained by inserting a fused gfp gene with the Bacillus thuringiensis vip2A(c) gene encoding a possible enzymatic component under the control of the polyhedrin gene promoter of the baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). The Trichoplusia ni cell line TnHi5 was transfected with Bac-GFP and Bac-GV2 DNAs respectively. Fluorescent cells expressing the fusion protein GV2 were much fewer than those expressing green fluorescent protein (GFP) alone, and did not obviously increase in number from 2 to 5 days after transfection. This result showed that the Vip2A fusion protein might have an ADP-ribosylating activity on cell skeleton actin, exerting an influence on the production and diffusion of the budded virus from insect cells.

The intrinsic toxicity of lepidopteran-active Bt proteins necessitates assessment of non-target risks associated with environmental release of transgenic crops expressing these proteins. Principles of ecological risk assessment provide a means for assessing non-target risks when information regarding exposure to the toxin and species-specific effects are lacking. This is shown for the case of Bt Cry1F maize release in Japan, where off-field pollen dissemination and effect on butterfly species is of concern. The specific ecological entity of concern for the assessment of the non-target impact of Cry1F maize pollen was Yamato-shijimi (pale grass blue butterfly), Pseudozizeeria maha (Kollar), a commonly occurring, susceptible species. Yamato-shijimi is widely adapted in Japan where it occurs in both rural and metropolitan settings, corresponding to the distribution and habitat of katabami (Oxalis corniculata (L.)), the larval host plant. The northern extent of Yamato-shijimi habitat lies to the south of major maize production regions in Japan, but exposure may occur elsewhere where maize and Yamato-shijimi co-occur. Screening level assessment of potential adverse effects to Yamato-shijimi in the field environment considered the probability for spatial-temporal co-occurrence of the life stages of concern (1st and 2nd instars) and the stressor (Cry1F protein expressed in maize pollen) at environmentally relevant concentrations. In the event of exposure to maize pollen, early instars of Yamato-shijimi feed exclusively on the underside of katabami leaves, which further limits the portion of the butterfly population that would be exposed. Projected levels of exposure to Cry1F pollen are below the toxicity level of concern and, thus, indicate negligible risk. Most sensitive species characterization (intergenera sensitivity) similarly shows negligible risk to other Japanese butterfly species of concern when distributed beyond the maize field or field margin.