An Integrative Analysis of Transcriptomics and Proteomics Reveals Novel Insights into the Response in the Midgut of Spodoptera frugiperda Larvae to Vip3Aa

The insecticidal Vip3 proteins, secreted by Bacillus thuringiensis (Bt) during its vegetative growth phase, are currently used in Bt crops to control insect pests, and are genetically distinct from known insecticidal Cry proteins. Compared with Cry toxins, the mechanisms of Vip3 toxins are still poorly understood. Here, the responses of Spodoptera frugiperda larvae after Vip3Aa challenge are characterized. Using an integrative analysis of transcriptomics and proteomics, we found that Vip3Aa has enormous implications for various pathways. The downregulated genes and proteins were mainly enriched in metabolic pathways, including the insect hormone synthesis pathway, whereas the upregulated genes and proteins were mainly involved in the caspase-mediated apoptosis pathway, along with the MAPK signaling and endocytosis pathways. Moreover, we also identified some important candidate genes involved in apoptosis and MAPKs. The present study shows that exposure of S. frugiperda larvae to Vip3Aa activates apoptosis pathways, leading to cell death. The results will promote our understanding of the host response process to the Vip3Aa, and help us to better understand the mode of action of Vip3A toxins.

The economics of managing evolution

Humans are altering biological systems at unprecedented rates, and these alterations often have longer-term evolutionary impacts. Most obvious is the spread of resistance to pesticides and antibiotics. There are a wide variety of management strategies available to slow this evolution, and there are many reasons for using them. In this paper, we focus on the economic aspects of evolution management and ask: When is it economically beneficial for an individual decision-maker to invest in evolution management? We derive a simple dimensionless inequality showing that it is cost-effective to manage evolution when the percentage increase in the effective life span of the biological resource that management generates is larger than the percentage increase in annual profit that could be obtained by not managing evolution. We show how this inequality can be used to determine optimal investment choices for single decision-makers, to determine Nash equilibrium investment choices for multiple interacting decision-makers, and to examine how these equilibrium choices respond to regulatory interventions aimed at stimulating investment in evolution management. Our results are illustrated with examples involving Bacillus thuringiensis (Bt) crops and antibiotic use in fish farming.

Fitness Costs of Two Maize Lepidopteran Pests Fed on Bacillus thuringiensis (Bt) Diets Enriched with Vitamins A and C

Serious malnutrition problems occur in developing countries where people’s diets are mainly based on staple crops. To alleviate this, high-production crops are being developed that are better adapted to climate change, enriched in micronutrients and vitamins, or resistant to pests. In some cases, new varieties have been developed with several of the characteristics mentioned above, such as biofortified and pest-resistant crops. The development of biofortified Bacillus thuringiensis (Bt) crops raises the question of whether vitamin enrichment of Bt crops can in any way favor those pests that are not very susceptible to the Bt toxin that feed on these crops, such as Helicoverpa armigera (Hübner) or Mythimna unipuncta (Haworth) (Lepidoptera: Noctuidae). In this study, the response to a Bt diet enriched with vitamins A (β-carotene) and C (ascorbic acid) was somewhat different between the two species. M. unipuncta was less sensitive to the toxin than H. armigera, although the ingestion of the Bt diet resulted in oxidative stress (longer larval development and lower pupal weight) which was not mitigated by the vitamins. However, the two vitamins reduced the mortality of H. armigera larvae fed on a Bt-enriched diet; in addition, ß-carotene reduced the activity of the antioxidant glutathione S-transferase (GST) of both species, suggesting it has an antioxidant role. The results obtained here indicate that biofortified Bt crops will not favor the development of H. armigera very much and will not affect M. unipuncta’s development at all, although the effect of the increase in vitamins may be very variable and should be studied for each specific phytophagous.

Rapid spread of a densovirus in a major crop pest following wide-scale adoption of Bt-cotton in China

Bacillus thuringiensis (Bt) crops have been widely planted and the effects of Bt-crops on populations of the target and non-target insect pests have been well studied. However, the effects of Bt-crops exposure on microorganisms that interact with crop pests have not previously been quantified. Here, we use laboratory and field data to show that infection of Helicoverpa armigera with a densovirus (HaDV2) is associated with its enhanced growth and tolerance to Bt-cotton. Moreover, field monitoring showed a much higher incidence of cotton bollworm infection with HaDV2 in regions cultivated with Bt-cotton than in regions without it, with the rate of densovirus infection increasing with increasing use of Bt-cotton. RNA-seq suggested tolerance to both baculovirus and Cry1Ac were enhanced via the immune-related pathways. These findings suggest that exposure to Bt-crops has selected for beneficial interactions between the target pest and a mutualistic microorganism that enhances its performance on Bt-crops under field conditions.

Genetic Screening to Identify Candidate Resistance Alleles to Cry1F Corn in Fall Armyworm Using Targeted Sequencing

Evolution of practical resistance is the main threat to the sustainability of transgenic crops producing insecticidal proteins from Bacillus thuringiensis (Bt crops). Monitoring of resistance to Cry and Vip3A proteins produced by Bt crops is critical to mitigate the development of resistance. Currently, Cry/Vip3A resistance allele monitoring is based on bioassays with larvae from inbreeding field-collected moths. As an alternative, DNA-based monitoring tools should increase sensitivity and reduce overall costs compared to bioassay-based screening methods. Here, we evaluated targeted sequencing as a method allowing detection of known and novel candidate resistance alleles to Cry proteins. As a model, we sequenced a Cry1F receptor gene (SfABCC2) in fall armyworm (Spodoptera frugiperda) moths from Puerto Rico, a location reporting continued practical field resistance to Cry1F-producing corn. Targeted sequencing detected a previously reported Cry1F resistance allele (SfABCC2mut), in addition to a resistance allele originally described in S. frugiperda populations from Brazil. Moreover, targeted sequencing detected mutations in SfABCC2 as novel candidate resistance alleles. These results support further development of targeted sequencing for monitoring resistance to Bt crops and provide unexpected evidence for common resistance alleles in S. frugiperda from Brazil and Puerto Rico.

Hetero-oligomerization of Bacillus thuringiensis Cry1A proteins enhance binding to the ABCC2 transporter of Spodoptera exigua

The ABC transporters are membrane proteins that can act as putative receptors for Cry proteins from Bacillus thuringiensis (Bt) in the midgut of different insects. For the beet armyworm, Spodoptera exigua, ABCC2 and ABCC3 have been found to interact with Cry1A proteins, the main insecticidal proteins used in Bt-crops, as well as Bt-based pesticides. The ABCC2 has shown to have specific binding towards Cry1Ac and is involved in the toxic process of Cry1A proteins, but the role of this transporter and how it relates with the Cry1A proteins is still unknown. Here, we have characterized the interactions between the SeABCC2 and the main proteins that bind to the receptor. By labelling the Cry1Aa protein, we have found that virtually all of the binding is in an oligomeric state, a conformation that allowed higher levels of specific binding that could not be achieved by the monomeric protein on its own. Furthermore, we have observed that Cry1A proteins can hetero-oligomerize in the presence of the transporter, which is reflected in an increase in binding and toxicity to SeABCC2-expressing cells. This synergism can be one of the reasons why B. thuringiensis co-expresses different Cry1 proteins that can apparently have similar binding preferences. The results from in vitro competition and ex vivo competition showed that Cry1Aa, Cry1Ab and Cry1Ac share functional binding sites. By using Cry1Ab-Cry1Ac chimeras, the presence of domain I from Cry1A proteins was revealed to be critical for oligomer formation.

CRISPR-mediated mutations in the ABC transporter gene ABCA2 confer pink bollworm resistance to Bt toxin Cry2Ab

Crops genetically engineered to produce insecticidal proteins from Bacillus thuringiensis (Bt) have many benefits and are important globally for managing insect pests. However, the evolution of pest resistance to Bt crops reduces their benefits. Understanding the genetic basis of such resistance is needed to better monitor, manage, and counter pest resistance to Bt crops. Previous work shows that resistance to Bt toxin Cry2Ab is associated with mutations in the gene encoding the ATP-binding cassette protein ABCA2 in lab- and field-selected populations of the pink bollworm (Pectinophora gossypiella), one of the world’s most destructive pests of cotton. Here we used CRISPR/Cas9 gene editing to test the hypothesis that mutations in the pink bollworm gene encoding ABCA2 (PgABCA2) can cause resistance to Cry2Ab. Consistent with this hypothesis, introduction of disruptive mutations in PgABCA2 in a susceptible strain of pink bollworm increased the frequency of resistance to Cry2Ab and facilitated creation of a Cry2Ab-resistant strain. All Cry2Ab-resistant individuals tested in this study had disruptive mutations in PgABCA2. Overall, we found 17 different disruptive mutations in PgABCA2 gDNA and 26 in PgABCA2 cDNA, including novel mutations corresponding precisely to single-guide (sgRNA) sites used for CRISPR/Cas9. Together with previous results, these findings provide the first case of practical resistance to Cry2Ab where evidence identifies a specific gene in which disruptive mutations can cause resistance and are associated with resistance in field-selected populations.

Baseline Susceptibility and Laboratory Selection of Resistance to Bt Cry1Ab Protein of Chinese Populations of Yellow Peach Moth, Conogethes punctiferalis (Guenée)

Yellow Peach Moth (YPM), Conogethes punctiferalis (Guenée), is one of the most destructive maize pests in the Huang-Huai-Hai summer maize region of China. Transgenic Bacillus thuringiensis (Bt) maize provides an effective means to control this insect pest in field trials. However, the establishment of Bt resistance to target pests is endangering the continued success of Bt crops. To use Bt maize against YPM, the baseline susceptibility of the local populations in the targeted areas needs to be verified. Diet-overlay bioassay results showed that all the fourteen YPM populations in China are highly susceptible to Cry1Ab. The LC50 values ranged from 0.35 to 2.38 ng/cm2 over the two years of the collection, and the difference between the most susceptible and most tolerant populations was sevenfold. The upper limit of the LC99 estimates of six pooled populations produced >99% larval mortality for representative eight populations collected in 2020 and was designated as diagnostic concentrations for monitoring susceptibility in YPM populations in China. Hence, we evaluated the laboratory selection of resistance in YPM to Cry1Ab using the diet-overlay bioassay method. Although the resistant ratio was generally low, YPM potentially could evolve resistance to Cry1Ab. The potential developmentof resistance by target pests points out the necessity to implement resistance management strategies for delaying the establishment of pest resistance to Bt crops.

Compatibility of Bt biopesticides and adjuvants for Spodoptera frugiperda control

Spodoptera frugiperda is a pest of economic importance for several crops with resistance reports to Bt crops and pesticides. Eco-friendly Bt biopesticides may be an alternative to chemical insecticides due to their selectivity and specificity. However, the efficacy of Bt biopesticides may be influenced by the association with other chemicals, such as adjuvants. This study evaluated the compatibility and toxicity of Bt biopesticides mixed with adjuvants for the control of S. frugiperda. The treatments included the association of Dipel SC and Dipel PM with adjuvants. Compatibility tests were used to evaluate the Bt mixture. Bt suspensions obtained from mixtures of Bt and adjuvants at 106 and 3 × 108 spores/mL−1 were used to evaluate S. frugiperda mortality and distilled water was used as the control. The addition of the adjuvant LI increased growth and sporulation, indicating compatibility with Bt biopesticides. The other adjuvants were toxic to reducing Bt growth and sporulation. Only the mixture of Bt with LI and Bt alone was effective to S. frugiperda. The addition of adjuvants to Bt biopesticide affect the Bt sporulation, growth and mortality.