Search Results (93)

Bradysia difformis is a notorious pest in mushroom production in China. Biological control using Bacillus thuringiensis (Bt) offers an environmentally friendly and effective strategy against this pest. Here, we show that the complete genome of strain JW-1 consists of one circular chromosome and seven circular plasmids. JW-1-Plasmid 4 comprises 127,921 bp with a GC content of 33.9%, and is predicted to contain 131 genes, including six insecticidal genes: cry4Aa, cry4Ba, cry10Ab, cry11Aa, cyt1Aa, and cyt2Ba. A 3542-bp fragment containing the cry4Aa gene was amplified from this strain. Phylogenetic analysis based on Cry4 toxin sequences showed that JW-1 Cry4 toxin belongs to the Cry4Aa toxin cluster. A Cry4Aa fusion protein was subsequently expressed in E. coli and purified using Ni-IDA affinity chromatography. A larval feeding assay showed that purified Cry4Aa was toxic to B. difformis larvae, with an LC₅₀ of 2.71 ng/mL. These results confirmed the identity and bioactivity of Cry4Aa from strain JW-1, offering a promising biological control agent against this major pest. Full article

A variety of pesticidal proteins derived from the bacterium Bacillus thuringiensis exhibit activity against the yellow fever mosquito Aedes aegypti and are used to control this insect vector. Several of these proteins, including Cry1Ca and Cry2Aa, additionally have activity against lepidopteran insects. Furthermore, the specificity of Cry2Aa has recently been shown to depend on domain I of the Cry protein, whereas it is generally recognized that domain II is the primary specificity-determining domain. This work has made use of disabled forms of three Cry proteins (Cry2Aa, Cry1Ca and Cry11Aa) and one naturally non-active protein (Cry2Ab) in an in vivo competition assay to investigate whether Cry2Aa and the dual-active Cry1Ca share a common receptor with the other pesticidal proteins. It was found that despite their differing specificities and potential modes of action, all of the Aedes-active proteins tested made use of a common receptor, although evidence is presented that Cry2Aa can use multiple receptors. When additional toxins (Cry41Aa, Cry1Aa, Cry1Ac) with no activity against this mosquito were tested, they too were found to share the same receptor, suggesting that Cry toxins may have evolved to utilize a common set of receptors in insects but that additional factors determine species specificity. Full article

Establishing refuges is a primary strategy for managing resistance in target pests against Bt maize. The larval feeding and dispersal, and adult oviposition behaviors of Spodoptera exigua (Hübner) on Bt and non-Bt maize plants are critical factors in determining optimal refuge configurations. This study employed laboratory and field experiments to evaluate the larval feeding and dispersal behaviors, as well as the oviposition preferences of S. exigua moths, on Bt (Cry1Ab + Vip3Aa19) and non-Bt maize plants. Results showed that as time of the choice test increased, the larval selection rate on Bt maize leaves declined progressively, with all instars (1st–5th) preferring to feed on non-Bt maize. After 48 h, the selection rates of larvae for non-Bt and Bt maize were 40.63–66.25% and 9.38–33.75%, respectively. Female moths exhibited no significant oviposition preference between Bt and non-Bt plants under undamaged conditions; however, when non-Bt maize was infested by the larvae, females preferentially oviposited on Bt maize plants (73.55%). Under the seed-mixture refuge pattern in field conditions, increasing the proportion of non-Bt maize significantly enhanced larval dispersal distances and facilitated larval transit damage between Bt and non-Bt plants. Our research clarifies the behavioral patterns of S. exigua on Bt and non-Bt maize, provides a scientific basis for optimizing refuge strategy to delay the development of resistance. Full article

Aedes aegypti, a crucial vector mosquito that transmits many diseases that cause millions of deaths worldwide, can be controlled with Bacillus thuringiensis subsp. israelensis (Bti). The larvicidal activity of Bti against Ae. aegypti is due primarily to Cry4Aa, Cry4Ba, and Cry11Aa, and Cyt1Aa, a protein that synergizes the activity of the Cry proteins. Interestingly, Galectins-6 and Galectins-14, members of a family of β-galactoside-binding proteins that play a role in immune responses insects, have been shown to decrease the activity of Bti toxins. The activity of other Galectins, particularly Galectin-8A, against the Cry proteins is not known. Toward this end, we cloned the gene coding for galactin-8A and expressed the recombinant protein and purified protein. The bioassay results indicated that Galectin-8A can also reduce the toxicity of Cry11Aa, but it was much stronger than Galectin-6. To investigate the interactions among Galectin-8A, Cry11Aa, and toxin receptors, Octet Red System analysis, Western blot, far-Western blot, and ELISA assay were also performed. The Octet Red System result showed that Galectin-8A could also bind to BBMVs of Ae. aegypti, with a lower kDa value than that of Galectin-6, indicating that Galectin-8A had a stronger binding affinity to BBMVs than Galectin-6. Western blot, far-Western blot, and ELISA assay analyses also demonstrated that Galectin-8A bound to Ae. aegypti receptor ALP1 and APN2, consistent with the protein docking simulation results. These findings support the conclusion that Galectin-8A blocks with ALP1 and APN2 more effectively than Galectin-6, which may subsequently reduce the toxicity of Cry11Aa in Ae. aegypti. Full article

The vegetative insecticidal protein Vip3Aa from Bacillus thuringiensis (Bt) has become a key plant-incorporated protectant (PIP) in transgenic crops targeting lepidopteran pests, particularly as resistance increasingly compromises the efficacy of Cry protein PIPs. More than a decade after its commercial deployment, Vip3Aa performance remains efficacious but increasingly vulnerable. Field screens have detected unexpectedly high baseline frequencies of Vip3Aa-resistant alleles and have produced highly resistant strains in several major pests, including Helicoverpa spp., Spodoptera spp., and Mythimna separata. Although structure–function experiments and studies on resistance to Vip3Aa have identified altered midgut processing and impaired receptor binding as candidate resistance mechanisms, the underlying genetic determinants remain poorly understood. Moreover, resistance to Vip3Aa appears to diverge from canonical Cry protein resistance pathways, underscoring the need for dedicated mechanistic studies. This review critically examines the available experimental evidence on Vip3Aa resistance mechanisms, highlighting major knowledge gaps and proposing research priorities to inform resistance monitoring and extend the durability of Vip3Aa-based pest control. Full article

We constructed a library of Mpp46Ab mutants, in which S¹⁵³ within the transmembrane β-hairpin was randomly replaced by other amino acids. Mutagenesis and subsequent primary screening yielded 10 different Mpp46Ab mutants in addition to the wild type. Remarkably, S¹⁵³ was replaced with a more hydrophobic amino acid in most of the mutants, and the S153I mutant in particular exhibited significantly increased toxicity. Electrophysiologic analysis using artificial lipid bilayers revealed that the single-channel conductance and P_K/P_Cl permeability ratio were significantly increased for S153I pores. This suggests that the formation of highly ion-permeable and highly cation-selective toxin pores increases the influx of cations and water into cells, thereby facilitating osmotic shock. In addition, the S153F, S153L, and S153I mutants exhibited significantly reduced synergistic toxicity with Cry4Aa. Electrophysiologic analysis showed that the S153F, S153L, and S153I mutants form toxin pores with a significantly reduced P_K/P_Na permeability ratio and a significantly increased P_K/P_Ca permeability ratio compared to wild-type pores. Thus, our results suggest that pore formation is central to the insecticidal activity of Mpp46Ab and that the ion permeability of toxin pores is a potential indicator correlated with both toxicity and synergistic toxicity with other toxins. Full article

The larvae of Hyphantria cunea feed on plant leaves, causing significant losses to forestry and agricultural production. At present, cry1 genes such as cry1Ac and cry1Ah are mainly used to control H. cunea. To delay the problem of pest resistance induced by a single insecticidal gene, it is crucial to discover and develop new insecticidal genes or gene combinations. This study found cry9Aa3 and cry9Aa4 showed insecticidal activity against H. cunea. The toxicity of 14 mutants of Cry9Aa3 was analyzed and the LC₅₀ of the triple-amino-acid substitution mutant 316LRG318AAA was 3.69 μg/g, which represents a 1.49-fold increase in insecticidal activity compared to Cry9Aa3. Additionally, enhanced stability of this mutant was detected in the midgut juice of H. cunea. Cry9Aa3 and 316LRG318AAA, in combination with Cry1Ah, demonstrated synergistic effects against H. cunea, with synergistic factors of 4.76 and 8.33, respectively. This study has identified the mutant 316LRG318AAA and its combination with Cry1Ah as exhibiting high toxicity against H. cunea, providing valuable genetic resources for the development of transgenic poplars and holding significant importance for delaying resistance in this pest. Full article

Bt maize is the main means to control many lepidopteran pests in the world, but its control efficacy against Agrotis ypsilon (Rottemberg), an important insect pest of maize seedlings, remains unclear until now. The interaction between the insect and Bt transgenic maize events (DBN9936 (expressing Cry1Ab), DBN9501 (expressing Vip3Aa), and DBN3601T (expressing Cry1Ab and Vip3Aa)) was investigated using bioassay and insect behavioral tests. The results show that the Cry1Ab contents in different tissues of DBN9936 were 47.78–82.60 μg·g⁻¹, and the Vip3Aa contents in DBN9501 were 15.29–27.78 μg·g⁻¹. The contents of Cry1Ab and Vip3Aa in DBN3601T were 32.08–79.08 and 10.16–17.52 μg·g⁻¹, respectively. There was no significant difference in total Bt protein content between the leaves and stems; however, that the content in both was significantly higher than that in the roots. The larvae were most sensitive to the Vip3Aa protein, and the corrected mortalities of larvae feeding on DBN9501 and DBN3601T were greater than 89.65% at the seedling stage, significantly higher than those feeding on DBN9936 (16.46–76.13%). The corrected mortalities of the third to the fifth instar larvae feeding on Bt maize root were as follows: DBN3601T (54.00–96.60%) > DBN9501 (24.67–70.88%) > DBN9936 (6.67–53.31%). The results of behavioral tests for Bt/non-Bt maize plant selection indicated that the larvae mainly fed on non-Bt maize while showing antifeedant behavior toward Bt maize, and the moth preferred to lay eggs on undamaged or slightly damaged Bt maize. It is concluded that DBN3601T maize has a strong control efficacy for A. ypsilon, which can play an important role in building an integrated pest management strategy for the insect. Full article

Public acceptance of genetically modified crops engineered with Bacillus thuringiensis (Bt) insecticidal protein genes (BT-GMCs), which confer resistance to various lepidopteran insect pests, is generally lacking. As a major concern over BT-GMCs is the allergenicity of insecticidal proteins, alleviating safety concerns should help increase public acceptance. In this study, three lepidopteran-specific Bt toxins, Cry1Aa, Cy1Ab, and Cry1Ac, were treated with simulated digestive fluids under various conditions. Western blotting using antiserum raised against individual segments (α-helices of domain I and β-sheets of domains II and III) of Cry1Aa showed that digestion produces a variety of polypeptides. In particular, the transmembrane α4–α5 of domain I, which may retain the ability to form pores, was the most resistant to digestion. Intact Cry1A toxins and these digests were then applied to RBL-2H3 cultured rat mast cells to determine whether the toxins directly induce histamine release. However, fluorescence microscopy revealed no specific binding of Cry1A toxins to RBL-2H3 cultured rat mast cells. In addition, neither the OPA method nor HPLC analysis detected significant histamine release from mast cells treated with Cry1A toxins and these digests. Our results provide important data supporting the safety of Cry1A toxins and potentially BT-GMCs. Full article

Developing simple and efficient multi-gene expression systems is crucial for multi-trait improvement or bioproduction in transgenic plants. In previous research, an IGG6-based bicistronic system from the nonpathogenic fungus Glarea lozoyensis efficiently expressed multiple enzyme proteins in yeast and maize, and the heterologous enzymes successfully performed their catalytic activity to reconstruct the biosynthetic pathway in the host organism. Unlike enzyme proteins, some heterologous functional proteins (such as insecticidal proteins) are dose-dependent and they need to express sufficient levels to perform their biological functions. It remains unclear whether the IGG6-based bicistronic system can achieve high expression of the functional proteins for practical applications in crops. In this study, two Bacillus thuringiensis (Bt) insecticidal genes, vip3Aa and cry1Ab, were linked via IGG6 to form a bicistron, while two glyphosate resistance genes, gr79epsps and gat, served as monocistronic selectable marker genes. Regenerated maize plants were produced through genetic transformation. RNA and immunoblot analyses revealed that the vip3Aa-IGG6-cry1Ab bicistron was transcribed as a single transcript, which was then translated into two separate proteins. Notably, the transcription and translation of cry1Ab were significantly positively correlated with those of vip3Aa. Through ELISA and leaf bioassay, we identified two transgenic maize lines, VICGG-15 and VICGG-20, that exhibited high insecticidal activity against fall armyworm (FAW; Spodoptera frugiperda) and Asian corn borer (ACB; Ostrinia furnacalis), both of which had high expression of Vip3Aa and Cry1Ab proteins. Subsequent evaluations, including silk, ear, and field bioassays, as well as glyphosate tolerance assessments, indicated that the VICGG-15 plants displayed high resistance to FAW and ACB, and could tolerate up to 3600 g acid equivalent (a.e.) glyphosate per hectare without adversely affecting phenotype or yield. Our finding established that the IGG6-based bicistronic system can achieve high expression of functional proteins in maize, and it is a potential candidate for multi-gene assembly and expression in plants. Full article

The corn earworm, Helicoverpa zea (Boddie), causes persistent ear damage to corn grown in the southeastern United States region. Increased levels of ear damage have been associated with mycotoxin contamination in addition to yield loss. Corn hybrids expressing proteins from the Bacillus thuringiensis (Bt) may provide corn earworm control. A selection of hybrids expressing various Bt traits were evaluated in field experiments across Georgia over two years to assess their efficacy for corn earworm control, grain yield and quality protection, and grain mycotoxin mitigation. Ear damage was significantly reduced only by Bt hybrids expressing the Vip3Aa20 protein. The remaining Bt hybrids expressing Cry proteins provided only marginal control. Ear damage had a variable effect on grain yield and was not correlated with grain aflatoxin contamination. In contrast, grain fumonisin contamination was positively associated with earworm damage. These results indicate Bt hybrids that effectively reduce corn earworm damage may also assist in reducing fumonisin contamination and possibly yield loss. Full article

The effect of caffeine on the behavior and sleep patterns of zebrafish larvae, as well as its underlying mechanisms, has been a topic of great interest. This study aimed to investigate the impact of caffeine on zebrafish larval sleep/wake behavior and the expression of key regulatory genes such as cAMP-response element binding protein (CREB) and adenosine (ADA) in the sleep pathway. To begin, the study determined the optimal dose and duration of caffeine exposure, with the optimal doses found to be 31.25 μM, 62.5 μM, and 120 μM. Similarly, the optimal exposure time was established as no more than 120 h, ensuring a mortality rate of less than 10%. The confirmation of these conditions was achieved through the assessment of angiogenesis and the inflammatory reaction. As a result, the treatment time point of 24 h post-fertilization (hpf) was selected to examine the effects of caffeine on zebrafish larval sleep rhythm (48 h, with a light cycle of 14:10). Furthermore, the study analyzed the expression of clock genes (bmal1a, per1b, per2, per3, cry2), adenosine receptor genes (adora1a, adora1b, adora2aa, adora2ab, adora2b), and key regulatory factors (CREB and ADA). The research confirmed that caffeine could induce sleep pattern disorders, significantly upregulate adenosine receptor genes (adora1a, adora1b, adora2a, adora2ab, adora2b) (p < 0.05), and markedly decrease the total sleep time and sleep efficiency of the larvae. Additionally, the activity of ADA significantly increased during the exposure (p < 0.001), and the tissue-specific expression of CREB was also significantly increased, as assessed by immunofluorescence. Caffeine may regulate circadian clock genes through the ADA/ADORA/CREB pathway. These findings not only enhance our understanding of the effects of caffeine on zebrafish larvae but also provide valuable insights into the potential impact of caffeine on human behavior and sleep. Full article

Dioryctria abietella Denis Schiffermuller (Lepidoptera: Pyralidae) is an oligophagous pest that mainly damages Pinaceae plants. Here, we investigated the effects of the Bacillus thuringiensis 2913 strain (Bt 2913), which carries the Cry1Ac, Cry2Ab, and Vip3Aa genes, on the D. abietella midgut transcriptome at 6, 12, and 24 h after infection. In total, 7497 differentially expressed genes (DEGs) were identified from the midgut transcriptome of D. abietella larvae infected with Bt 2913. Among these DEGs, we identified genes possibly involved in Bt 2913-induced perforation of the larval midgut. For example, the DEGs included 67 genes encoding midgut proteases involved in Cry/Vip toxin activation, 74 genes encoding potential receptor proteins that bind to insecticidal proteins, and 19 genes encoding receptor NADH dehydrogenases that may bind to Cry1Ac. Among the three transcriptomes, 88 genes related to metabolic detoxification and 98 genes related to immune defense against Bt 2913 infection were identified. Interestingly, 145 genes related to the 60S ribosomal protein were among the DEGs identified in the three transcriptomes. Furthermore, we performed bioinformatic analysis of zonadhesin, GST, CYP450, and CarE in the D. abietella midgut to determine their possible associations with Bt 2913. On the basis of the results of this analysis, we speculated that trypsin and other serine proteases in the D. abietella larval midgut began to activate Cry/Vip prototoxin at 6 h to 12 h after Bt 2913 ingestion. At 12 h after Bt 2913 ingestion, chymotrypsin was potentially involved in degrading the active core fragment of Vip3Aa toxin, and the detoxification enzymes in the larvae contributed to the metabolic detoxification of the Bt toxin. The ABC transporter and several other receptor-protein-related genes were also downregulated to increase resistance to Bt 2913. However, the upregulation of 60S ribosomal protein and heat shock protein expression weakened the resistance of larvae to Bt 2913, thereby enhancing the expression of NADH dehydrogenase and other receptor proteins that are highly expressed in the larval midgut and bind to activating toxins, including Cry1Ac. At 24 h after Bt 2913 ingestion, many activated toxins were bound to receptor proteins such as APN in the larval midgut, resulting in membrane perforation. Here, we clarified the mechanism of Bt 2913 infection in D. abietella larvae, as well as the larval immune defense response to Bt 2913, which provides a theoretical basis for the subsequent control of D. abietella using B. thuringiensis. Full article

Blended refuges for corn-expressing toxins from Bacillus thuringiensis (Bt) Berliner controls have been approved in the United States as an alternative resistance management approach to structured refuge. This study examined the impact of blended refuges up to 30% non-Bt seed on the corn earworm, Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae), larval numbers, and kernel injury, and its effect on corn grain yield and test weights. The percentage of infested ears, larval numbers, and kernel injury of the 20% blend were not significantly different from the non-Bt and nonblended Bt for the Cry1A.105 + Cry2Ab + Cry1Fa2 treatment, but infested ears, larval numbers, and kernel injury of the nonblended Cry1A.105 + Cry2Ab treatment was lower than the comparable non-Bt hybrids, with the 20% blend being intermediate. The nonblended Cry1A.105 + Cry2Ab + Vip3Aa20 had virtually no larvae in ears and no kernel injury. Ear infestation, larval numbers, and kernel injury of the non-Bt blends with Vip3Aa20 were proportional to the percentage of non-Bt seed in the blend, and all infested ears were from the non-Bt refuge plants. Grain yield and test weight were not significantly different among nonblended or blended treatments of any Bt product tested. Results indicate losses of grain yield and test weight by corn earworm in seed blends up to 30% non-Bt seed are unlikely with infestation levels observed in this study. Full article

Asian corn borer (ACB), Ostrinia furnacalis, is an important agricultural pest affecting maize production in southwestern China, but knowledge of the toxic effect of Bt maize on the pest has been insufficient until now. In this study, we determined the susceptibility of ACB to Cry1Ab, Vip3Aa, and their complex proteins and evaluated the efficacy of Chinese domestic Bt-(Cry1Ab+Vip3Aa) maize (“DBN3601T” event) against the pest in Yunnan Province of southwestern China. The susceptible bioassay indicated that the LC₅₀ values of the Cry1Ab and Cry1Ab+Vip3Aa proteins expressed by the Bt maize varieties against ACB larvae were 51.42 and 46.85 ng/g, respectively; however, the ACB larva was insensitive to the Vip3Aa protein. The Cry1Ab+Vip3Aa protein contents in V6–V8 leaves, VT tassels, R1 silks, R2 kernels, R3 stalks and R3 cobs of the Bt-(Cry1Ab+Vip3Aa) maize were 114.20, 30.69, 3.77, 8.92, 11.09 and 10.99 μg/g, respectively. The larval feeding test indicated that the Bt-(Cry1Ab+Vip3Aa) maize was more toxic to the early instar larvae, and the survival time of larvae fed on the leaves was the shortest, while it survived the longest on stalks. The identification of maize resistance levels in the field showed that both larval density and plant damage score of Bt-(Cry1Ab+Vip3Aa) maize were significantly lower than those in conventional maize. It is concluded that the Bt-(Cry1Ab+Vip3Aa) maize can be used for control of the ACB in southwestern China. Full article