Evaluation of Reference Genes and Expression Level of Genes Potentially Involved in the Mode of Action of Cry1Ac and Cry1F in a Susceptible Reference Strain of Chrysodeixis includens

Soybean looper (SBL), Chrysodeixis includens (Walker), is one of the major lepidopteran pests of soybean in the American continent. SBL control relies mostly on the use of insecticides and genetically modified crops expressing Bacillus thuringiensis (Bt) insecticidal Cry proteins. Due to the high selection pressure exerted by these control measures, resistance has developed to different insecticides and Bt proteins. Nevertheless, studies on the mechanistic background are still scarce. Here, the susceptibility of the laboratory SBL-Benzon strain to the Bt proteins Cry1Ac and Cry1F was determined in diet overlay assays and revealed a greater activity of Cry1Ac than Cry1F, thus confirming results obtained for other sensitive SBL strains. A reference gene study across larval stages with four candidate genes revealed that RPL10 and EF1 were the most stable genes for normalization of gene expression data obtained by RT-qPCR. Finally, the basal expression levels of eight potential Bt protein receptor genes in six larval instars were analyzed, including ATP-binding cassette (ABC) transporters, alkaline phosphatase, aminopeptidases, and cadherin. The results presented here provide fundamental knowledge to support future SBL resistance studies.

Can (We Make) Bacillus thuringiensis Crystallize More Than Its Toxins?

The development of finely tuned and reliable crystallization processes to obtain crystalline formulations of proteins has received growing interest from different scientific fields, including toxinology and structural biology, as well as from industry, notably for biotechnological and medical applications. As a natural crystal-making bacterium, Bacillus thuringiensis (Bt) has evolved through millions of years to produce hundreds of highly structurally diverse pesticidal proteins as micrometer-sized crystals. The long-term stability of Bt protein crystals in aqueous environments and their specific and controlled dissolution are characteristics that are particularly sought after. In this article, I explore whether the crystallization machinery of Bt can be hijacked as a means to produce (micro)crystalline formulations of proteins for three different applications: (i) to develop new bioinsecticidal formulations based on rationally improved crystalline toxins, (ii) to functionalize crystals with specific characteristics for biotechnological and medical applications, and (iii) to produce microcrystals of custom proteins for structural biology. By developing the needs of these different fields to figure out if and how Bt could meet each specific requirement, I discuss the already published and/or patented attempts and provide guidelines for future investigations in some underexplored yet promising domains.

Bacillus thuringiensis chimeric proteins Cry1A.2 and Cry1B.2 to control soybean lepidopteran pests: New domain combinations enhance insecticidal spectrum of activity and novel receptor contributions

Two new chimeric Bacillus thuringiensis (Bt) proteins, Cry1A.2 and Cry1B.2, were constructed using specific domains, which provide insecticidal activity against key lepidopteran soybean pests while minimizing receptor overlaps between themselves, current, and soon to be commercialized plant incorporated protectants (PIP’s) in soybean. Results from insect diet bioassays demonstrate that the recombinant Cry1A.2 and Cry1B.2 are toxic to soybean looper (SBL) Chrysodeixis includens Walker, velvetbean caterpillar (VBC) Anticarsia gemmatalis Hubner, southern armyworm (SAW) Spodoptera eridania, and black armyworm (BLAW) Spodoptera cosmioides with LC50 values < 3,448 ng/cm2. Cry1B.2 is of moderate activity with significant mortality and stunting at > 3,448 ng/cm2, while Cry1A.2 lacks toxicity against old-world bollworm (OWB) Helicoverpa armigera. Results from disabled insecticidal protein (DIP) bioassays suggest that receptor utilization of Cry1A.2 and Cry1B.2 proteins are distinct from each other and from current, and yet to be commercially available, Bt proteins in soy such as Cry1Ac, Cry1A.105, Cry1F.842, Cry2Ab2 and Vip3A. However, as Cry1A.2 contains a domain common to at least one commercial soybean Bt protein, resistance to this common domain in a current commercial soybean Bt protein could possibly confer at least partial cross resistance to Cry1A2. Therefore, Cry1A.2 and Cry1B.2 should provide two new tools for controlling many of the major soybean insect pests described above.

Effect of Urea Spray on Boll Shell Insecticidal Protein Content in Bt Cotton

Reproductive organs of Bacillus thuringiensis transgenic cotton, which contribute to cotton final yield, have low insect resistant efficacy, so it is important to improve their insect resistance. This study was conducted to find out the impact of different urea spray doses on the expression of Cry1A protein in boll shell of Bt cotton (Sikang 1 and Sikang 3), and nitrogen metabolism in this process was also studied to uncover the physiological mechanism. The experiment with six urea doses was organized during peak boll stage in 2017 and 2018. The results showed that urea spray could significantly increase boll shell insecticidal protein contents in both cultivars, with the highest Bt protein content observed at 28–32 kg ha−1 urea dose. In addition, urea spray increased the contents of soluble protein and free amino acid and the activities of GS, GOGAT, GOT, and GPT, but decreased the activities of peptidase and protease in boll shell. Correlation analysis showed that the amount of boll shell Bt protein was positively correlated with levels of soluble protein and amino acid, and activities of GS, GOGAT, GOT, and GPT, but negatively correlated with peptidase and protease activities. Thus, this study demonstrated that higher protein synthesis ability and lower proteolysis ability were related to increased Bt protein content in urea-sprayed boll shell.

Increasing plant density increases Bt toxin concentration of boll wall in cotton by decreasing boll setting speed

Background In order to uncover the mechanism of significantly reduced insect resistance at the late developmental stage in cotton (Gossypium hirsutum L.), the relationship between boll setting rate under different planting densities and Bacillus thuringiensis (Bt) insecticidal concentrations in the boll wall were investigated in the present study. Two studies were arranged at Yangzhou, China during the 2017–2018 cotton growth seasons. Five planting densities (15 000, 25 000, 45 000, 60 000 and 75 000 plants per hectare) and the flower-removal treatment were imposed separately on Bt cotton cultivar Sikang3 to arrange different boll setting rates, and the boll setting rates and Bt toxin content were compared. Results Higher boll setting rate together with lower Bt toxin contents in boll wall was observed under low planting density, whereas lower boll setting rate and higher Bt toxin contents were found under high planting density. Also, higher Bt protein concentration was associated with higher soluble protein content, glutamic-pyruvic transaminase (GPT), and glutamic oxaloacetate transaminase (GOT) activities, but lower amino acid content, and protease and peptidase activities. It was further confirmed that a higher boll setting rate with lower Bt protein content under flower-removal. Conclusions This study demonstrated that the insecticidal efficacy of boll walls was significantly impacted by boll formation. Reduced protein synthesis and enhanced protein degradation were related to the reduced Bt toxin concentration.

β-carotene and Bacillus thuringiensis insecticidal protein differentially modulate feeding behaviour, mortality and physiology of European corn borer (Ostrinia nubilalis)

Maize with enhanced β-carotene production was engineered to counteract pervasive vitamin A deficiency in developing countries. Second-generation biofortified crops are being developed with additional traits that confer pest resistance. These include crops that can produce Bacillus thuringiensis Berliner (Bt) insecticidal proteins. Currently, it is unknown whether β-carotene can confer fitness benefits through to insect pests, specifically through altering Ostrinia nubilalis foraging behaviour or development in the presence of Bt insecticidal toxin. Therefore the effects of dietary β-carotene plus Bt insecticidal protein on feeding behaviour, mortality, and physiology in early and late instars of O. nubilalis larvae were investigated. The results of two-choice experiments showed that irrespective of β-carotene presence, at day five 68%-90% of neonates and 69%-77% of fifth-instar larvae avoided diets with Cry1A protein. Over 65% of neonate larvae preferred to feed on diets with β-carotene alone compared to 39% of fifth-instar larvae. Higher mortality (65%-97%) in neonates fed diets supplemented with β-carotene alone and in combination with Bt protein was found, whereas <36% mortality was observed when fed diets without supplemented β-carotene or Bt protein. Diets with both β-carotene and Bt protein extended 25 days the larval developmental duration from neonate to fifth instar (compared to Bt diets) but did not impair larval or pupal weight. Juvenile hormone and 20-hydroxyecdysone regulate insect development and their levels were at least 3-fold higher in larvae fed diets with β-carotene for 3 days. Overall, these results suggest that the effects of β-carotene and Bt protein on O. nubilalis is dependent on larval developmental stage. This study is one of the first that provides insight on how the interaction of novel traits may modulate crop susceptibility to insect pests. This understanding will in turn inform the development of crop protection strategies with greater efficacy.

Effects of hydroxycinnamic acid esters on sweetpotato weevil feeding and oviposition and interactions with Bacillus thuringiensis proteins

Sweetpotato weevil (SPW) pest management is challenging because the pest target is sub-terranean, so the application of pesticides is impractical and usually ineffective. Host plant resistance and the genetic transformation of sweetpotatoes to produce entomotoxic Bt proteins offer potential for environmentally benign pest control. Resistance can be conferred by naturally occurring hydroxycinnamic acids which protect against oviposition by adults, but these compounds are restricted to the root surface so do not protect against the cortex bound larvae where the greatest damage occurs. Resistance could be enhanced if combined with expression of Bt proteins in transformed plants, but interactions between hydroxycinnamic acids and Bt proteins remain unknown. Here the bioactivity of Cry7Aa1 protein and hydroxycinnamic acid esters was evaluated individually and in combination against SPW larvae and mortality determined. Low and high concentrations of hydroxycinnamic acid esters alone caused significantly higher mortality of both weevil species in all experiments compared to the control. SPW larval mortality was greater when tested as a combination of hydroxycinnamic acid esters and Bt protein, but this effect was additive not synergistic. Although we report no evidence of antagonistic interactions, the antifeedant effects of the plant compounds conferring host plant resistance could have reduced consumption of the Bt protein in our assays leading to a lower efficacy when combined. Further work is required to determine whether the toxic effects of Bt proteins function alongside host plant resistance in sweetpotato under field conditions.

Toxicity of the Bt Protein Cry1Ac Expressed in Leaves of the Event of Transgenic Soybean Released in Paraguay Against Spodoptera cosmioides

Spodoptera cosmioides Walk (Lepidoptera: Noctuidae) is a polyphagous species, considered a secondary pest in soybean crop. The Intacta soybean (Bt soybean) event was released in Paraguay with Bt proteins which are supposed to be toxic for caterpillars. However, the effects of these proteins in the Spodoptera cosmioides biology still uncertainty. This study evaluated the biological activity of Bt soybean in S. cosmioides that were fed on Bt and non-Bt soybean leaves under laboratory conditions. The experiment was carried out at the Laboratory of Entomology; temperature 26&plusmn;2 &ordm;C; relative humidity: 60&plusmn;10%; photoperiod: 14:00 h where 150 neonate larvae were used per treatment. The period and viability of each stage, the mortality of larval, number of eggs and neonate per couple, and the period of larvae to adult were the assessed variables. Higher mortality was observed in larvae fed with intact soybeans, but without statistical differences with non-Bt soybeans. For larval survival, period, number and total period, no statistical differences were verified. Higher percentage of fertility was observed in soybeans non-Bt but no statistical differences.