Gene drives may be capable of addressing ecological problems by altering entire populations of wild organisms, but their use has remained largely theoretical due to technical constraints. Here we consider the potential for RNA-guided gene drives based on the CRISPR nuclease Cas9 to serve as a general method for spreading altered traits through wild populations over many generations. We detail likely capabilities, discuss limitations, and provide novel precautionary strategies to control the spread of gene drives and reverse genomic changes. The ability to edit populations of sexual species would offer substantial benefits to humanity and the environment. For example, RNA-guided gene drives could potentially prevent the spread of disease, support agriculture by reversing pesticide and herbicide resistance in insects and weeds, and control damaging invasive species. However, the possibility of unwanted ecological effects and near-certainty of spread across political borders demand careful assessment of each potential application. We call for thoughtful, inclusive, and well-informed public discussions to explore the responsible use of this currently theoretical technology.

/pdf/concerning-rna-guided-gene-drives-for-the-alteration-of-wild-2la0voj6g0.pdf

Concerning RNA-guided gene drives for the alteration of wild populations

Transgenic crops have revolutionized insect pest control, but their effectiveness has been reduced by evolution of resistance in pests. We analyzed global monitoring data reported during the first two decades of transgenic crops, with each case representing the responses of one pest species in one country to one insecticidal protein from Bacillus thuringiensis (Bt). The cases of pest resistance to Bt crystalline (Cry) proteins produced by transgenic crops increased from 3 in 2005 to 16 in 2016. By contrast, in 17 other cases there was no decrease in pest susceptibility to Bt crops, including the recently introduced transgenic corn that produces a Bt vegetative insecticidal protein (Vip). Recessive inheritance of pest resistance has favored sustained susceptibility, but even when inheritance is not recessive, abundant refuges of non-Bt host plants have substantially delayed resistance. These insights may inform resistance management strategies to increase the durability of current and future transgenic crops.

Surge in insect resistance to transgenic crops and prospects for sustainability.

Double-stranded RNAs (dsRNAs) targeted against essential genes can trigger a lethal RNA interference (RNAi) response in insect pests. The application of this concept in plant protection is hampered by the presence of an endogenous plant RNAi pathway that processes dsRNAs into short interfering RNAs. We found that long dsRNAs can be stably produced in chloroplasts, a cellular compartment that appears to lack an RNAi machinery. When expressed from the chloroplast genome, dsRNAs accumulated to as much as 0.4% of the total cellular RNA. Transplastomic potato plants producing dsRNAs targeted against the β-actin gene of the Colorado potato beetle, a notorious agricultural pest, were protected from herbivory and were lethal to its larvae. Thus, chloroplast expression of long dsRNAs can provide crop protection without chemical pesticides.

https://science.sciencemag.org/content/sci/347/6225/991.full.pdf

Full crop protection from an insect pest by expression of long double-stranded RNAs in plastids

Transgenic crop pyramids producing two or more Bacillus thuringiensis (Bt) toxins that kill the same insect pest have been widely used to delay evolution of pest resistance. To assess the potential of pyramids to achieve this goal, we analyze data from 38 studies that report effects of ten Bt toxins used in transgenic crops against 15 insect pests. We find that compared with optimal low levels of insect survival, survival on currently used pyramids is often higher for both susceptible insects and insects resistant to one of the toxins in the pyramid. Furthermore, we find that cross-resistance and antagonism between toxins used in pyramids are common, and that these problems are associated with the similarity of the amino acid sequences of domains II and III of the toxins, respectively. This analysis should assist in future pyramid design and the development of sustainable resistance management strategies.

Optimizing pyramided transgenic Bt crops for sustainable pest management

Can Pyramids and Seed Mixtures Delay Resistance to Bt Crops

The widespread planting of crops genetically engineered to produce insecticidal toxins derived from the bacterium Bacillus thuringiensis (Bt) places intense selective pressure on pest populations to evolve resistance. Western corn rootworm is a key pest of maize, and in continuous maize fields it is often managed through planting of Bt maize. During 2009 and 2010, fields were identified in Iowa in which western corn rootworm imposed severe injury to maize producing Bt toxin Cry3Bb1. Subsequent bioassays revealed Cry3Bb1 resistance in these populations. Here, we report that, during 2011, injury to Bt maize in the field expanded to include mCry3A maize in addition to Cry3Bb1 maize and that laboratory analysis of western corn rootworm from these fields found resistance to Cry3Bb1 and mCry3A and cross-resistance between these toxins. Resistance to Bt maize has persisted in Iowa, with both the number of Bt fields identified with severe root injury and the ability western corn rootworm populations to survive on Cry3Bb1 maize increasing between 2009 and 2011. Additionally, Bt maize targeting western corn rootworm does not produce a high dose of Bt toxin, and the magnitude of resistance associated with feeding injury was less than that seen in a high-dose Bt crop. These first cases of resistance by western corn rootworm highlight the vulnerability of Bt maize to further evolution of resistance from this pest and, more broadly, point to the potential of insects to develop resistance rapidly when Bt crops do not achieve a high dose of Bt toxin.

/pdf/field-evolved-resistance-by-western-corn-rootworm-to-39bllj40ja.pdf

Field-evolved resistance by western corn rootworm to multiple Bacillus thuringiensis toxins in transgenic maize

Crops producing toxins derived from the bacterium Bacillus thuringiensis (Bt) are widely planted to manage insect pests including western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), which is a significant pest of maize in the United States and Europe However, the widespread use of Bt maize places intense selection pressure on pest populations to evolve resistance, and field-evolved resistance to Bt maize by western corn rootworm has been documented in the United States In conjunction with non-Bt refuges, fitness costs of Bt resistance can delay resistance evolution Fitness costs arise in the absence of Bt toxin when individuals with resistance alleles have lower fitness than Bt-susceptible genotypes We quantified the level of resistance and fitness costs of resistance for a strain of western corn rootworm with laboratory-selected resistance to transgenic maize (Zea mays L) producing Bt toxin Cry3Bb1 Survival to adulthood on Cry3Bb1 maize was more than twofold higher for resistant insects vs susceptible insects, which is similar to the magnitude of resistance first observed in the field Fitness costs were measured in two experiments; the first used maize hybrids and the second used inbred lines The experiment with maize hybrids compared resistant and susceptible strains while the experiment with maize inbreds compared resistant, susceptible and heterozygous genotypes The only non-recessive fitness cost detected (ie cost affecting heterozygotes) was for adult size Recessive fitness costs (ie costs affecting the resistant strain) were observed for developmental rate, female survival and egg viability However, when reared on non-Bt maize, the resistant strain also displayed higher fecundity, higher survival for males and greater adult longevity compared to the susceptible strain These results suggest that resistance to Bt maize by western corn rootworm may not impose substantial fitness costs, and consequently, may evolve quickly and persist once present

/pdf/fitness-costs-of-resistance-to-cry3bb1-maize-by-western-corn-23sku72rip.pdf

Fitness costs of resistance to Cry3Bb1 maize by western corn rootworm

Fitness costs can delay pest resistance to crops that produce insecticidal toxins derived from the bacterium Bacillus thuringiensis (Bt), and past research has found that entomopathogens impose fitness costs of Bt resistance. In addition, entomopathogens can be used for integrated pest management by providing biological control of pests. The western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is a major pest of maize and is currently managed by planting of Bt maize. We tested whether entomopathogenic nematodes and fungi increased mortality of western corn rootworm and whether these entomopathogens increased fitness costs of resistance to Cry3Bb1 maize. We exposed western corn rootworm larvae to two species of nematodes, Heterorhabditis bacteriophora Poinar (Rhabditida: Heterorhabditidae) and Steinernema feltiae Filipjev (Rhabditida: Steinernematidae), and to two species of fungi, Beauveria bassiana (Balsamo) Vuillemin (Hypocreales: Cordycipitaceae) (strain GHA) and Metarhizium brunneum (Metschnikoff) Sorokin (Hypocreales: Clavicipitaceae) (strain F52) in two assay types, namely, seedling mat and small cup. Larval mortality increased with the concentration of H. bacteriophora and S. feltiae in the small cup assay, and with the exception of S. feltiae and B. bassiana in the seedling mat assay, mortality from entomopathogens was significantly greater than zero for the remaining entomopathogens in both assays. However, no fitness costs were observed in either assay type for any entomopathogen. Increased mortality of western corn rootworm larvae caused by these entomopathogens supports their potential use in biological control; however, the lack of fitness costs suggests that entomopathogens will not delay the evolution of Bt resistance in western corn rootworm.

Effects of entomopathogens on mortality of western corn rootworm (Coleoptera: Chrysomelidae) and fitness costs of resistance to Cry3Bb1 maize.

Background: Insecticide resistance monitoring is key for evidence-based control of Anopheles and Aedes disease vectors in particular, since the vast majority of insecticide-based public health adult vector control tools are reliant on pyrethroids. While widespread pyrethroid resistance in Anopheles species and Aedes aegypti has been described in many countries, data for Papua New Guinea are scarce. Available data indicate the local Anopheles populations remain pyrethroid-susceptible, making regular insecticide resistance monitoring even more important. Knowledge on Aedes insecticide resistance in PNG is very limited, however, high levels of Aedes aegypti resistance have been described. Here we present insecticide resistance monitoring data from across PNG generated between 2017 and 2022. Methods: Mosquito larvae were collected in larval habitat surveys and through ovitraps. Mosquitoes were reared to adults and subjected to insecticide treated filter papers in WHO insecticide susceptibility bioassays. Subsets of Aedes mosquitoes were subjected to sequencing of the voltage-sensitive sodium channel (Vssc) region to identify resistance mutations. Results: Overall, nearly 20,000 adult female mosquitoes from nine PNG provinces were used in the tests. We show that in general, Anopheline mosquitoes in PNG remain susceptible to pyrethroids but with worrying signs of reduced 24 h mortality in some areas. In addition, some Anopheles populations were indicated to be resistant against DDT. We show that Ae. aegypti in PNG are pyrethroid, DDT and likely bendiocarb resistant with a range of Vssc resistance mutations identified. We demonstrate that Ae. albopictus is DDT resistant and is likely developing pyrethroid resistance based on finding a low frequency of Vssc mutations. Conclusion: This study represents the largest overview of insecticide resistance in PNG. While Ae. aegypti is highly pyrethroid resistant, the Anopheline and Ae. albopictus populations exhibit low levels of resistance in some areas. It is important to continue to monitor insecticide resistance in PNG and prepare for the widespread emergence of pyrethroid resistance in major disease vectors.

Insecticide Resistance Surveillance of Malaria and Arbovirus Vectors in Papua New Guinea 2017-2022.

17 Introduction 18 Materials and Methods 20 Results 30 Discussion 33 Acknowledgments 37 Literature Cited 37 CHAPTER 3. EFFECTS OF ENTOMOPATHOGENS ON MORTALITY OF WESTERN CORN ROOTWORM AND FITNESS COSTS OF RESISTANCE TO CRY3BB1 MAIZE 50 Abstract 50 Introduction 51 Materials and Methods 54 Results 61 Discussion 62 Acknowledgments 66 Literature Cited 66 CHAPTER 4. GENERAL CONCLUSIONS 7750 Introduction 51 Materials and Methods 54 Results 61 Discussion 62 Acknowledgments 66 Literature Cited 66 CHAPTER 4. GENERAL CONCLUSIONS 77

https://lib.dr.iastate.edu/cgi/viewcontent.cgi?article=4257&context=etd

Amanda M. Hoffmann

Papers

Field-evolved resistance by western corn rootworm to multiple Bacillus thuringiensis toxins in transgenic maize

Fitness costs of resistance to Cry3Bb1 maize by western corn rootworm

Effects of entomopathogens on mortality of western corn rootworm (Coleoptera: Chrysomelidae) and fitness costs of resistance to Cry3Bb1 maize.

Insecticide Resistance Surveillance of Malaria and Arbovirus Vectors in Papua New Guinea 2017-2022.

Fitness costs associated with resistance to Cry3Bb1 corn in western corn rootworm