Retrotransposon-mediated disruption of a chitin synthase gene confers insect resistance to Bacillus thuringiensis Vip3Aa toxin.

The vegetative insecticidal protein Vip3Aa from Bacillus thuringiensis (Bt) has been produced by transgenic crops to counter pest resistance to the widely used crystalline (Cry) insecticidal proteins from Bt. To proactively manage pest resistance, there is an urgent need to better understand the genetic basis of resistance to Vip3Aa, which has been largely unknown. We discovered that retrotransposon-mediated alternative splicing of a midgut-specific chitin synthase gene was associated with 5,560-fold resistance to Vip3Aa in a laboratory-selected strain of the fall armyworm, a globally important crop pest. The same mutation in this gene was also detected in a field population. Knockout of this gene via CRISPR/Cas9 caused high levels of resistance to Vip3Aa in fall armyworm and 2 other lepidopteran pests. The insights provided by these results could help to advance monitoring and management of pest resistance to Vip3Aa.

Critical Domains in the Specific Binding of Radiolabeled Vip3Af Insecticidal Protein to Brush Border Membrane Vesicles from Spodoptera spp. and Cultured Insect Cells.

Vegetative insecticidal proteins (Vip3) from Bacillus thuringiensis have been used, in combination with Cry proteins, to better control insect pests and as a strategy to delay the evolution of resistance to Cry proteins in Bt crops (crops protected from insect attack by the expression of proteins from B. thuringiensis). In this study, we have set up the conditions to analyze the specific binding of 125I-Vip3Af to Spodoptera frugiperda and Spodoptera exigua brush border membrane vesicles (BBMV). Heterologous competition binding experiments revealed that Vip3Aa shares the same binding sites with Vip3Af, but Vip3Ca does not recognize all of them. As expected, Cry1Ac and Cry1F did not compete for Vip3Af binding sites. By trypsin treatment of selected alanine mutants, we were able to generate truncated versions of Vip3Af. Their use as competitors with 125I-Vip3Af indicated that only those molecules containing domains I to III (DI-III and DI-IV) were able to compete with the trypsin-activated Vip3Af protein for binding and that molecules only containing either domain IV or domains IV and V (DIV and DIV-V) were unable to compete with Vip3Af. These results were further confirmed with competition binding experiments using 125I-DI-III. In addition, the truncated protein 125I-DI-III also bound specifically to Sf21 cells. Cell viability assays showed that the truncated proteins DI-III and DI-IV were as toxic to Sf21 cells as the activated Vip3Af, suggesting that domains IV and V are not necessary for the toxicity to Sf21 cells, in contrast to their requirement in vivo.IMPORTANCE This study shows that Vip3Af binding sites are fully shared with Vip3Aa, only partially shared with Vip3Ca, and not shared with Cry1Ac and Cry1F in two Spodoptera spp. Truncated versions of Vip3Af revealed that only domains I to III were necessary for the specific binding, most likely because they can form the functional tetrameric oligomer and because domain III is supposed to contain the binding epitopes. In contrast to results obtained in vivo (bioassays against larvae), domains IV and V are not necessary for ex vivo toxicity to Sf21 cells.

Effect of substitutions of key residues on the stability and the insecticidal activity of Vip3Af from Bacillus thuringiensis.

Modern agriculture demands for more sustainable agrochemicals to reduce the environmental and health impact. The whole process of the discovery and development of new active substances or control agents is sorely slow and expensive. Vegetative insecticidal proteins (Vip3) from Bacillus thuringiensis are specific toxins against caterpillars with a potential capacity to broaden the range of target pests. Site-directed mutagenesis is one of the most approaches used to test hypotheses on the role of different amino acids on the structure and function of proteins. To gain a better understanding of the role of key amino acid residues of Vip3A proteins, we have generated 12 mutants of the Vip3Af1 protein by site-directed mutagenesis, distributed along the five structural domains of the protein. Ten of these mutants were successfully expressed and tested for stability and toxicity against three insect pests (Spodoptera frugiperda, Spodoptera littoralis and Grapholita molesta). The results showed that, to render a wild type fragment pattern upon trypsin treatment, position 483 required an acidic residue, and position 552 an aromatic residue. Regarding toxicity, the change of Met34 to Lys34 significantly increased the toxicity of the protein for one of the three insect species tested (S. littoralis), whereas the other residue substitutions did not improve, or even decreased, insect toxicity, confirming their key role in the structure/function of the protein.

Antimalarial drugs and their metabolites are potent Zika virus inhibitors.

Studies aimed at repurposing existing drugs revealed that some antimalarial compounds possess anti-Zika virus (anti-ZIKV) activity. Here, we further tested 14 additional antimalarial drugs and their metabolites or analogs for anti-ZIKV activity using a phenotypic screening approach. We identified four compounds with varying anti-ZIKV activity, including a metabolite of amodiaquine termed desethylamodiaquine (DAQ) and N-desethylchloroquine (DECQ), a metabolite of chloroquine, which both exhibited low micromolar effective concentrations against three different ZIKV strains. Two other compounds termed dihydroartemisinin (DHA) and quinidine (QD) exhibited only partial inhibition of ZIKV replication. Characterization of the inhibitory mechanisms of DAQ and DECQ showed that both drugs target the entry step as well as postentry events of the viral replication cycle. These hits represent attractive starting points for future optimization of new anti-ZIKV drug candidates derived from antimalarial drugs and their analogs.

JAK-STAT Signaling Pathways and Inhibitors Affect Reversion of Envelope-Mutated HIV-1.

HIV can spread by both cell-free and cell-to-cell transmission. Here, we show that many of the amino acid changes in Env that are close to the CD4 binding pocket can affect HIV replication. We generated a number of mutant viruses that were unable to infect T cells as cell-free viruses but were nevertheless able to infect certain T cell lines as cell-associated viruses, which was followed by reversion to the wild type. However, the activation of JAK-STAT signaling pathways caused the inhibition of such cell-to-cell infection as well as the reversion of multiple HIV Env mutants that displayed differences in their abilities to bind to the CD4 receptor. Specifically, two T cell activators, interleukin-2 (IL-2) and phorbol 12-myristate 13-acetate (PMA), both capable of activation of JAK-STAT pathways, were able to inhibit cell-to-cell viral transmission. In contrast, but consistent with the above result, a number of JAK-STAT and mTOR inhibitors actually promoted HIV-1 transmission and reversion. Hence, JAK-STAT signaling pathways may differentially affect the replication of a variety of HIV Env mutants in ways that differ from the role that these pathways play in the replication of wild-type viruses.IMPORTANCE Specific alterations in HIV Env close to the CD4 binding site can differentially change the ability of HIV to mediate infection for cell-free and cell-associated viruses. However, such differences are dependent to some extent on the types of target cells used. JAK-STAT signaling pathways are able to play major roles in these processes. This work sheds new light on factors that can govern HIV infection of target cells.

The antimalarial drug amodiaquine possesses anti-ZIKA virus activities.

Zika virus (ZIKV) outbreak has emerged as a global health threat, particularly in tropical areas, over the past few years. No antiviral therapy or vaccine is available at present. For these reasons, repurposing clinically approved drugs against ZIKV infection may provide rapid and cost-effective global health benefits. Here, we explored this strategy and screened eight FDA-approved drugs for antiviral activity against ZIKV using a cell-based assay. Our results show that the antimalarial drug amodiaquine has anti-ZIKV activity with EC50 at low micromolar concentrations in cell culture. We further characterized amodiaquine antiviral activity against ZIKV and found that it targets early events of the viral replication cycle. Altogether, our results suggest that amodiaquine may be efficacious for the treatment of ZIKV infection.

Analysis of cross-resistance to Vip3 proteins in eight insect colonies, from four insect species, selected for resistance to Bacillus thuringiensis insecticidal proteins.

Bacillus thuringiensis Vip3 proteins are synthesized and secreted during the vegetative growth phase. They are activated by gut proteases, recognize and bind to midgut receptors, form pores and lyse cells. We tested the susceptibility to Vip3Aa and Vip3Ca of Cry1A-, Cry2A-, Dipel- and Vip3-resistant insect colonies from different species to determine whether resistance to other insecticidal proteins confers cross-resistance to Vip3 proteins. As expected, the colonies resistant to Cry1A proteins, Dipel (Helicoverpa armigera, Trichoplusia ni, Ostrinia furnacalis and Plodia interpunctella) or Cry2Ab (H. armigera and T. ni) were not cross-resistant to Vip3 proteins. In contrast, H. armigera colonies resistant to Vip3Aa or Vip3Aa/Cry2Ab showed cross-resistance to the Vip3Ca protein. Moreover, the Vip3Ca protein was highly toxic to O. furnacalis (LC50 not significantly different from that of Cry1Ab), whereas the Vip3Aa protein only showed moderate growth inhibition at the highest concentration tested (100 µg/g of diet). These results extend the cross-resistance studies between Vip3 and Cry proteins, show for the first time cross-resistance between proteins within the Vip3 subfamily, and points to O. furnacalis as a target for the Vip3Ca protein.

Purification of Zika virus RNA-dependent RNA polymerase and its use to identify small-molecule Zika inhibitors.

Background: The viral RNA-dependent RNA polymerase (RdRp) enzymes of the Flaviviridae family are essential for viral replication and are logically important targets for development of antiviral therapeutic agents. Zika virus (ZIKV) is a rapidly re-emerging human pathogen for which no vaccine or antiviral agent is currently available. Methods: To facilitate development of ZIKV RdRp inhibitors, we have established an RdRp assay using purified recombinant ZIKV NS5 polymerase. Results: We have shown that both the hepatitis C virus (HCV) nucleoside inhibitor sofosbuvir triphosphate and a pyridoxine-derived non-nucleoside small-molecule inhibitor, DMB213, can act against ZIKV RdRp activity at IC 50 s of 7.3 and 5.2 µM, respectively, in RNA synthesis reactions catalysed by recombinant ZIKV NS5 polymerase. Cell-based assays confirmed the anti-ZIKV activity of sofosbuvir and DMB213 with 50% effective concentrations (EC 50 s) of 8.3 and 4.6 µM, respectively. Control studies showed that DMB213 did not inhibit recombinant HIV-1 reverse transcriptase and showed only very weak inhibition of HIV-1 integrase strand-transfer activity. The S604T substitution in motif B of the ZIKV RdRp, which corresponds to the S282T substitution in motif B of HCV RdRp, which confers resistance to nucleotide inhibitors, also conferred resistance to sofosbuvir triphosphate, but not to DMB213. Enzyme assays showed that DMB213 appears to be competitive with natural nucleoside triphosphate (NTP) substrates. Conclusions: Recombinant ZIKV RdRp assays can be useful tools for the screening of both nucleos(t)ide compounds and non-nucleotide metal ion-chelating agents that interfere with ZIKV replication.

Identification of resveratrol analogs as potent anti-dengue agents using a cell-based assay.

Dengue virus (DENV) causes a variety of difficult-to-treat diseases that threaten almost half of the world's population. Currently, no effective vaccine or antiviral therapy is available. We have examined a series of synthetic resveratrol analogs to identify potential anti-DENV agents. Here, we demonstrate that two resveratrol analogs, PNR-4-44 and PNR-5-02, possess potent anti-DENV activity with EC50 values in the low nanomolar range. These two resveratrol analogs were shown to mainly target viral RNA translation and viral replication, but PNR-5-02 is also likely to target cellular factors inside host cells. Although the precise molecular mechanism(s) mediating anti-DENV activities have not been elucidated, further structure-guided design might lead to the development of newer improved resveratrol derivatives that might have therapeutic value. J. Med. Virol. 89:397-407, 2017. © 2016 Wiley Periodicals, Inc.

The connection domain mutation N348I in HIV-1 reverse transcriptase enhances resistance to etravirine and rilpivirine but restricts the emergence of the E138K resistance mutation by diminishing viral replication capacity.

Clinical resistance to rilpivirine (RPV), a novel nonnucleoside reverse transcriptase (RT) inhibitor (NNRTI), is associated an E-to-K mutation at position 138 (E138K) in RT together with an M184I/V mutation that confers resistance against emtricitabine (FTC), a nucleoside RT inhibitor (NRTI) that is given together with RPV in therapy. These two mutations can compensate for each other in regard to fitness deficits conferred by each mutation alone, raising the question of why E138K did not arise spontaneously in the clinic following lamivudine (3TC) use, which also selects for the M184I/V mutations. In this context, we have investigated the role of a N348I connection domain mutation that is prevalent in treatment-experienced patients. N348I confers resistance to both the NRTI zidovudine (ZDV) and the NNRTI nevirapine (NVP) and was also found to be associated with M184V and to compensate for deficits associated with the latter mutation. Now, we show that both N348I alone and N348I/M184V can prevent or delay the emergence of E138K under pressure with RPV or a related NNRTI, termed etravirine (ETR). N348I also enhanced levels of resistance conferred by E138K against RPV and ETR by 2.2- and 2.3-fold, respectively. The presence of the N348I or M184V/N348I mutation decreased the replication capacity of E138K virus, and biochemical assays confirmed that N348I, in a background of E138K, impaired RT catalytic efficiency and RNase H activity. These findings help to explain the low viral replication capacity of viruses containing the E138K/N348I mutations and how N348I delayed or prevented the emergence of E138K in patients with M184V-containing viruses.

A resveratrol analog termed 3,3',4,4',5,5'-hexahydroxy-trans-stilbene is a potent HIV-1 inhibitor.

HIV resistance to current anti-HIV drugs and drug toxicity have created a need for new anti-HIV agents. We have examined and characterized a synthetic resveratrol analog, termed 3,3',4,4',5,5'-hexahydroxy-trans-stilbene (M8), for potential anti-HIV activity. Here, we demonstrate that M8 possesses potent anti-HIV activity against several HIV variants with EC50 values in the low µM range. M8 was shown to act at a very early step of HIV entry prior to fusion to host cells. These results demonstrate that this novel resveratrol derivative possesses potent anti-HIV-1 activity and may have a mechanism of action that is different from current anti-HIV-1 drugs including entry inhibitors. Further structure-guided design might lead to the development of newer improved resveratrol derivatives that could have value either in therapy or as microbicides to prevent the sexual transmission of HIV-1.

Defective HIV-1 quasispecies in the form of multiply drug-resistant proviral DNA within cells can be rescued by superinfection with different subtype variants of HIV-1 and by HIV-2 and SIV.

OBJECTIVES: HIV-1 generates swarms of similar, but genetically distinct, variants termed quasispecies and many of these variants can be defective. A relevant question is whether such defective species can contribute to viral pathogenesis. Indeed, we previously reported that a presumed recombination of defective proviral DNA with other complementary defective proviral DNA or with wild-type viral DNA in the aftermath of superinfection could lead to the rescue of defective provirus and the production of replication-competent virus. We then wished to determine whether such rescue could be affected by viruses of different subtypes or even by other members of the retrovirus family. METHODS: Here, we have used drug resistance mutations within the HIV genome as markers of potential recombination. RESULTS: We show that a defective proviral DNA within cells can be rescued by the superinfection of MT2 cells by various subtypes of HIV-1, and by HIV-2 and simian immunodeficiency virus, but not by human T cell leukaemia virus type 1 or by human herpes virus-6. The drug-resistance phenotype of the rescued HIV was confirmed in a second round of infection. CONCLUSIONS: Defective proviral HIV-1 can be rescued by the infection by different variants of HIV-1 and by several other retroviruses as well.

Identification of an env-defective HIV-1 mutant capable of spontaneous reversion to a wild-type phenotype in certain T-cell lines.

BACKGROUND: Attempts to eradicate HIV from cellular reservoirs are vital but depend on a clear understanding of how viral variants are transmitted and survive in the different cell types that constitute such reservoirs. Mutations in the env gene of HIV may be able to exert a differential influence on viral transmission ability in regard to cell-free and cell-associated viral forms. METHODS: The ability of HIV containing an env G367R mutation in cell-free and cell-associated viruses to cause infection and to revert to wild-type was measured using several T cell lines. To determine factors that might potentially influence the reversion of G367R, we studied each of entry inhibitors, inhibitors of cellular endocytosis, and modulators of cell growth and activation. RESULTS: We demonstrate that an HIV-1 variant containing a G367R substitution within the CD4 binding site of gp120 was non-infectious as free virus in culture but was infectious when infected cells were co-cultured with certain T cell lines or when cells were transfected by a relevant proviral plasmid. Differences in viral infectivity by cell-associated G367R viruses were determined by the type of target cell employed, regardless which type of donor cell was used. Reversion was slowed or inhibited by entry inhibitors and by inhibitors of cellular endocytosis. Interleukin 2 was able to block G367R reversion in only one of the T cell lines studied but not in the other, while phorbol 12-myristate 13-acetate (PMA) inhibited G367R reversion in all the T cell lines. CONCLUSIONS: Env-defective HIV may have a different phenotype as cell-free versus cell-associated virus. The persistence of defective forms can potentially lead to the emergence of virulent forms. The heterogeneity of cell types that constitute the HIV reservoir can contribute to viral variability, even among similar types of cells. This is the first demonstration of a mutation in the HIV envelope, i.e. G367R, that can compromise infection by cell-free virus but less severely by cell-associated virus and that does so in a cell type-dependent manner.

Role of the K101E substitution in HIV-1 reverse transcriptase in resistance to rilpivirine and other nonnucleoside reverse transcriptase inhibitors.

Resistance to the recently approved nonnucleoside reverse transcriptase inhibitor (NNRTI) rilpivirine (RPV) commonly involves substitutions at positions E138K and K101E in HIV-1 reverse transcriptase (RT), together with an M184I substitution that is associated with resistance to coutilized emtricitabine (FTC). Previous biochemical and virological studies have shown that compensatory interactions between substitutions E138K and M184I can restore enzyme processivity and the viral replication capacity. Structural modeling studies have also shown that disruption of the salt bridge between K101 and E138 can affect RPV binding. The current study was designed to investigate the impact of K101E, alone or in combination with E138K and/or M184I, on drug susceptibility, viral replication capacity, and enzyme function. We show here that K101E can be selected in cell culture by the NNRTIs etravirine (ETR), efavirenz (EFV), and dapivirine (DPV) as well as by RPV. Recombinant RT enzymes and viruses containing K101E, but not E138K, were highly resistant to nevirapine (NVP) and delavirdine (DLV) as well as ETR and RPV, but not EFV. The addition of K101E to E138K slightly enhanced ETR and RPV resistance compared to that obtained with E138K alone but restored susceptibility to NVP and DLV. The K101E substitution can compensate for deficits in viral replication capacity and enzyme processivity associated with M184I, while M184I can compensate for the diminished efficiency of DNA polymerization associated with K101E. The coexistence of K101E and E138K does not impair either viral replication or enzyme fitness. We conclude that K101E can play a significant role in resistance to RPV.

Effect of mutations at position E138 in HIV-1 reverse transcriptase and their interactions with the M184I mutation on defining patterns of resistance to nonnucleoside reverse transcriptase inhibitors rilpivirine and etravirine.

Impacts of mutations at position E138 (A/G/K/Q/R/V) alone or in combination with M184I in HIV-1 reverse transcriptase (RT) were investigated. We also determined why E138K is the most prevalent nonnucleoside reverse transcriptase inhibitor mutation in patients failing rilpivirine (RPV) therapy. Recombinant RT enzymes and viruses containing each of the above-mentioned mutations were generated, and drug susceptibility was assayed. Each of the E138A/G/K/Q/R mutations, alone or in combination with M184I, resulted in decreased susceptibility to RPV and etravirine (ETR). The maximum decrease in susceptibility to RPV was observed for E138/R/Q/G by both recombinant RT assay and cell-based assays. E138Q/R-containing enzymes and viruses also showed the most marked decrease in susceptibility to ETR by both assays. The addition of M184I to the E138 mutations did not significantly change the levels of diminution in drug susceptibility. These findings indicate that E138R caused the highest level of loss of susceptibility to both RPV and ETR, and, accordingly, E138R should be recognized as an ETR resistance-associated mutation. The E138K/Q/R mutations can compensate for M184I in regard to both enzymatic fitness and viral replication capacity. The favored emergence of E138K over other mutations at position E138, together with M184I, is not due to an advantage in either the level of drug resistance or viral replication capacity but may reflect the fact that E138R and E138Q require two distinct mutations to occur, one of which is a disfavorable G-to-C mutation, whereas E138K requires only a single favorable G-to-A hypermutation. Of course, other factors may also affect the concept of barrier to resistance.

Subunit-selective mutational analysis and tissue culture evaluations of the interactions of the E138K and M184I mutations in HIV-1 reverse transcriptase.

The emergence of HIV-1 drug resistance remains a major obstacle in antiviral therapy. M184I/V and E138K are signature mutations of clinical relevance in HIV-1 reverse transcriptase (RT) for the nucleoside reverse transcriptase inhibitors (NRTIs) lamivudine (3TC) and emtricitabine (FTC) and the second-generation (new) nonnucleoside reverse transcriptase inhibitor (NNRTI) rilpivirine (RPV), respectively, and the E138K mutation has also been shown to be selected by etravirine in cell culture. The E138K mutation was recently shown to compensate for the low enzyme processivity and viral fitness associated with the M184I/V mutations through enhanced deoxynucleoside triphosphate (dNTP) usage, while the M184I/V mutations compensated for defects in polymerization rates associated with the E138K mutations under conditions of high dNTP concentrations. The M184I mutation was also shown to enhance resistance to RPV and ETR when present together with the E138K mutation. These mutual compensatory effects might also enhance transmission rates of viruses containing these two mutations. Therefore, we performed tissue culture studies to investigate the evolutionary dynamics of these viruses. Through experiments in which E138K-containing viruses were selected with 3TC-FTC and in which M184I/V viruses were selected with ETR, we demonstrated that ETR was able to select for the E138K mutation in viruses containing the M184I/V mutations and that the M184I/V mutations consistently emerged when E138K viruses were selected with 3TC-FTC. We also performed biochemical subunit-selective mutational analyses to investigate the impact of the E138K mutation on RT function and interactions with the M184I mutation. We now show that the E138K mutation decreased rates of polymerization, impaired RNase H activity, and conferred ETR resistance through the p51 subunit of RT, while an enhancement of dNTP usage as a result of the simultaneous presence of both mutations E138K and M184I occurred via both subunits.

Molecular mechanism of antagonism between the Y181C and E138K mutations in HIV-1 reverse transcriptase.

Etravirine (ETR) is an expanded-spectrum nonnucleoside reverse transcriptase inhibitor (NNRTI) approved for use as an antiretroviral agent in treatment-experienced patients. Y181C and E138K in HIV-1 RT are among 20 different drug resistance mutations associated with ETR. However, E138K can be consistently selected by ETR when wild-type viruses but not viruses containing Y181C are grown in tissue culture. This study was carried out to evaluate any possible mechanisms that might explain antagonism between the Y181C and E138K mutations. Accordingly, we performed tissue culture studies to investigate the evolutionary dynamics of E138K in both a wild-type (WT) and a Y181C background. We also generated recombinant enzymes containing Y181C and E138K alone or in combination in order to study enzyme processivity, rates of processive DNA synthesis, enzyme kinetics, and susceptibility to ETR. We now show that the presence of the Y181C mutation prevented the emergence of E138K in cell culture and that the simultaneous presence of E138K and Y181C impaired each of enzyme activity, processivity, rate of processive DNA synthesis, and deoxynucleoside triphosphate (dNTP) affinity. The addition of E138K to Y181C also decreased the level of resistance to ETR compared to that obtained with Y181C alone.

Managing Practical Resistance of Lepidopteran Pests to Bt Cotton in China.

China is one of the major cotton producers globally with small farmers. Lepidopteran pests have always been the main factor affecting cotton production. To reduce the occurrence of and damage caused by lepidopteran pests, China has employed a pest control method focused on planting Bt (Cry1Ac) cotton since 1997. Chinese resistance management tactics for the main target pests, the cotton bollworm and pink bollworm, were also implemented. For polyphagous (multiple hosts) and migratory pests such as the cotton bollworm (Helicoverpa armigera), the "natural refuge" strategy, consisting of non-Bt crops such as corn, soybean, vegetables, peanuts, and other host crops, was adopted in the Yellow River Region (YRR) and Northwest Region (NR). For a single host and weak migration ability pest, such as the pink bollworm (Pectinophora gossypiella), the seed mix refuge strategy yields a random mixture within fields of 25% non-Bt cotton by sowing second-generation (F2) seeds. According to field monitoring results for more than 20 years in China, practical resistance (Bt cotton failure) of target pests was avoided, and there were no cases of Bt (Cry1Ac) failure of pest control in cotton production. This indicated that this Chinese resistance management strategy was very successful. The Chinese government has decided to commercialize Bt corn, which will inevitably reduce the role of natural refuges; therefore, this paper also discusses adjustments and future directions of cotton pest resistance management strategies.

Rotation of Multiple Single-Gene Transgenic Crops Did Not Slow the Evolution of Resistance to Cry1F or Cry1Ie in Ostrinia furnacalis.

A common strategy for delaying the evolution of resistance to transgenic crops that produce insecticidal proteins from Bacillus thuringiensis is to ensure that insect pests are exposed to multiple toxins with different mechanisms of action (MoAs). This can take the form of planting crops in a rotation pattern when different crops expressing single toxins are available on the market. The efficacy of a rotation strategy is reliant on mathematical models based on biological assumptions. Here, we designed laboratory evolution experiments to test whether Bt-based insecticidal proteins with different MoAs used in rotation could delay resistance from developing in Asian corn borer (ACB), Ostrinia furnacalis. We investigated the proteins Cry1Ab, Cry1F, and Cry1Ie, which are widely utilized for commercial insect control. We found that rotation of multiple toxins did not slow the evolution of resistance to Cry1F or Cry1Ie. Furthermore, the evolution of ACB to the Cry1Ab toxin develops faster when Cry1F or Cry1Ie is present, as compared to Cry1Ab exposure only. Our results suggest that toxins used in a rotation fashion do not work as an effective strategy in delaying ACB resistance evolution to Cry toxins over one-toxin exposure. Our result highlights the need to better understand the biological factors leading to insecticidal protein resistance and to develop IRM strategies against target insects.

Seven Years of Monitoring Susceptibility to Cry1Ab and Cry1F in Asian Corn Borer.

Resistance monitoring in the Asian corn borer, Ostrinia furnacalis, is necessary to accommodate the commercial introduction and stewardship of Bt maize in China. The susceptibility of 56 O. furnacalis field populations, collected between 2015 and 2021 from the corn belt regions of China, to Cry1Ab and Cry1F toxins was determined. Neonate larvae (within 12 h after hatching) were placed on the surface of semi-artificial agar-free diet incorporating a series of concentrations of purified toxins, and mortality was evaluated after 7d. The median lethal concentration (LC50) values of Cry1Ab and Cry1F were 0.05 to 0.37 µg/g (protein/diet) and 0.10 to 1.22 µg/g, respectively. Although interpopulation variation in susceptibility to the toxins was observed, the magnitude of the differences was 5.8-fold and 8.3-fold for Cry1Ab and Cry1F, respectively. These results suggested that the observed susceptibility differences reflect natural geographical variation in response and not variation caused by prior exposure to selection pressures. Therefore, the O. furnacalis populations were apparently still susceptible to Cry1Ab and Cry1F across their range within China. The monitoring data established here will serve as a comparative reference for early warning signs of field-evolved resistance after the cultivation of Bt maize in China.