Resistance to genetic insect control: modelling the effects of space

Genetic insect control, such as self-limiting RIDL(2) (Release of Insects Carrying a Dominant Lethal) technology, is a development of the sterile insect technique which is proposed to suppress wild populations of a number of major agricultural and public health insect pests. This is achieved by mass rearing and releasing male insects that are homozygous for a repressible dominant lethal genetic construct, which causes death in progeny when inherited. The released genetically engineered ('GE') insects compete for mates with wild individuals, resulting in population suppression. A previous study modelled the evolution of a hypothetical resistance to the lethal construct using a frequency-dependent population genetic and population dynamic approach. This found that proliferation of resistance is possible but can be diluted by the introgression of susceptible alleles from the released homozygous-susceptible GE males. We develop this approach within a spatial context by modelling the spread of a lethal construct and resistance trait, and the effect on population control, in a two deme metapopulation, with GE release in one deme. Results show that spatial effects can drive an increased or decreased evolution of resistance in both the target and non-target demes, depending on the effectiveness and associated costs of the resistant trait, and on the rate of dispersal. A recurrent theme is the potential for the non-target deme to act as a source of resistant or susceptible alleles for the target deme through dispersal. This can in turn have a major impact on the effectiveness of insect population control

High-resolution complex of papain with remnants of a cysteine protease inhibitor derived from Trypanosoma brucei

Attempts to crystallize a complex of papain (C. papaya) with a cysteine protease inhibitor from the parasitic pathogen T. brucei failed. However, over an extended period the mixture produced an ordered crystal of the protease carrying two peptide fragments in the active site. These correspond to dipeptides and tripeptides that are assigned as fragments of the inhibitor, which has presumably suffered proteolytic cleavage

The application of self-limiting transgenic insects in managing resistance in experimental metapopulations

This is the author accepted manuscript. The final version is available from Wiley via the DOI in this record.Data sharing: raw data for this study are available as supplementary data files.1. The mass release of transgenic insects carrying female lethal self-limiting genes can reduce pest insect populations. Theoretically, substantial releases can be a novel resistance management tool, since wild type alleles conferring susceptibility to pesticides can dilute resistance alleles in target populations. A potential barrier to the deployment of this technology is the need for large-scale area wide releases. Here we address whether localized releases of transgenic insects could provide an alternative, means of population suppression and resistance management, without serious loss of efficacy. 2. We used experimental mesocosms constituting insect metapopulations to explore the evolution of resistance to the Bacillus thuringiensis toxin Cry1Ac in a high-dose/refugia landscape in the insect Plutella xylostella. We ran two selection experiments, the first compared the efficacy of ‘everywhere’ releases and negative controls to a spatially density-dependent or ‘whack-a-mole’ strategy that concentrated release of transgenic insects in sub-populations with high levels of resistance. The second experiment tested the relative efficacy of whack-a-mole and everywhere releases under spatially homogenous and heterogeneous selection pressure. 3. The whack-a-mole releases were less effective than everywhere releases in terms of slowing the evolution of resistance, which in the first experiment, largely prevented the evolution of resistance. In contrast to predictions, heterogeneous whack-a-mole releases were not more effective under heterogeneous selection pressure. Heterogeneous selection pressure did, however, reduce total insect population sizes 4. Whack-a-mole releases provided early population suppression that was indistinguishable from homogeneous everywhere releases. However, insect population densities tracked the evolution of resistance in this system, as phenotypic resistance provides access to the 90% of experimental diet containing the toxin Cry1Ac. Thus, as resistance levels diverged between treatments, carrying capacities diverged and population sizes increased under the whack- a-mole approach. Synthesis and Applications Spatially density-dependent releases of transgenic insects, particularly those targeting source populations at landscape level, could suppress pest populations in the absence of blanket area-wide management. The resistance management benefits of self-limiting transgenic insects are, however, reduced in spatially localized releases, suggesting that they are not best suited for spatially restricted ‘spot’ treatment of problematic resistance. Nevertheless, area-wide and spatially heterogeneous releases could be used to support other resistance management interventions.This work was supported by the Biotechnology and Biological Sciences Research Council [grant numbers BB/L00948X/1 to MBB and NA, and BB/L00819X/1&2 to BR]

Combining the high-dose/refuge strategy and self-limiting transgenic insects in resistance management - a test in experimental mesocosms

This is the author accepted manuscript. The final version is available from Wiley via the DOI in this record.The high-dose/refuge strategy has been the primary approach for resistance management in transgenic crops engineered with Bacillus thuringiensis toxins. However, there are continuing pressures from growers to reduce the size of Bt toxin-free refugia, which typically suffer higher damage from pests. One complementary approach is to release male transgenic insects with a female-specific self-limiting gene. This technology can reduce population sizes and slow the evolution of resistance by introgressing susceptible genes through males. Theory predicts that it could be used to facilitate smaller refugia or reverse the evolution of resistance. In this study, we used experimental evolution with caged insect populations to investigate the compatibility of the self-limiting system and the high-dose/refuge strategy in mitigating the evolution of resistance in diamondback moth, Plutella xylostella. The benefits of the self-limiting system were clearer at smaller refuge size, particularly when refugia were inadequate to prevent the evolution of resistance. We found that transgenic males in caged mesocosms could suppress population size and delay resistance development with 10% refugia and 4% - 15% initial resistance allele frequency. Fitness costs in hemizygous transgenic insects are particularly important for introgressing susceptible alleles into target populations. Fitness costs of the self-limiting gene in this study (P. xylostella OX4139 line L) were incompletely dominant, and reduced fecundity and male mating competitiveness. The experimental evolution approach used here illustrates some of the benefits and pitfalls of combining mass-release of self-limiting insects and the high dose/refuge strategy, but does indicate that they can be complementary.This work was supported by the Biotechnology and Biological Sciences Research Council [grant numbers BB/L00948X/1 to MBB and NA, and BB/L00819X/1&2 to BR]

Intron-derived small RNAs for silencing viral RNAs in mosquito cells

Aedes aegypti and Ae. albopictus are the main vectors of mosquito-borne viruses of medical and veterinary significance. Many of these viruses have RNA genomes. Exogenously provided, e.g. transgene encoded, small RNAs could be used to inhibit virus replication, breaking the transmission cycle. We tested, in Ae. aegypti and Ae. albopictus cell lines, reporter-based strategies for assessing the ability of two types of small RNAs to inhibit a chikungunya virus (CHIKV) derived target. Both types of small RNAs use a Drosophila melanogaster premiRNA-1 based hairpin for their expression, either with perfect base-pairing in the stem region (shRNA-like) or containing two mismatches (miRNA-like). The pre-miRNA-1 stem loop structure was encoded within an intron; this allows co-expression of one or more proteins, e.g. a fluorescent protein marker tracking the temporal and spatial expression of the small RNAs in vivo. Three reporter-based systems were used to assess the relative silencing efficiency of ten shRNA-like siRNAs and corresponding miRNA-like designs. Two systems used a luciferase reporter RNA with CHIKV RNA inserted either in the coding sequence or within the 3’ UTR. A third reporter used a CHIKV derived split replication system. All three reporters demonstrated that while silencing could be achieved with both miRNA-like and shRNA-like designs, the latter were substantially more effective. Dcr-2 was required for the shRNA-like siRNAs as demonstrated by loss of inhibition of the reporters in Dcr-2 deficient cell lines. These positive results in cell culture are encouraging for the potential use of this pre-miRNA-1-based system in transgenic mosquitoes. </p

piggybac- and PhiC31-Mediated Genetic Transformation of the Asian Tiger Mosquito, Aedes albopictus (Skuse)

The Asian tiger mosquito, Aedes albopictus, is a highly invasive mosquito and has spread from South East Asia to Europe, the United States and northern areas of Asia in the past 30 years. Aedes mosquitoes transmit a range of viral diseases, including dengue and chikungunya. Aedes albopictus is generally considered to be somewhat less of a concern in this regard than Aedes aegypti. However a recent mutation in the chikungunya virus dramatically increased its transmission by Aedes albopictus, causing an important outbreak in the Indian Ocean in 2006 that eventually reached Italy in 2007. This highlights the potential importance of this mosquito, which can thrive much further from the Equator than can Aedes aegypti. This paper describes the first genetic engineering of the Asian tiger mosquito. This is an essential step towards the development of genetics-based control methods against this mosquito, and also an invaluable tool for basic research. We describe both transposon-based and site-specific integration methods

Comparison of Life History Characteristics of the Genetically Modified OX513A Line and a Wild Type Strain of Aedes aegypti

The idea of implementing genetics-based insect control strategies modelled on the traditional SIT (Sterile Insect Technique), such as RIDL (Release of Insects carrying a Dominant Lethal), is becoming increasingly popular. In this paper, we compare a genetically modified line of Aedes aegypti carrying a tetracycline repressible, lethal positive feedback system (OX513A) with a genetically similar, unmodified counterpart and their respective responses to increasing larval rearing density using a constant amount of food per larva. The parameters that we examined were larval mortality, developmental rate (i.e., time to pupation), adult size and longevity