Determinants of Insecticide Resistance Evolution: Comparative Analysis Among Heliothines.

It is increasingly clear that pest species vary widely in their propensities to develop insecticide resistance. This review uses a comparative approach to analyze the key pest management practices and ecological and biochemical or genetic characteristics of the target that contribute to this variation. We focus on six heliothine species, three of which, Helicoverpa armigera, Heliothis virescens, and Helicoverpa zea, have developed resistances to many pesticide classes. The three others, Helicoverpa punctigera, Helicoverpa assulta, and Helicoverpa gelotopoeon, also significant pests, have developed resistance to very few pesticide classes. We find that host range and movement between alternate hosts are key ecological traits that influence effective selection intensities for resistance. Operational issues are also critical; area-wide, cross-pesticide management practices that account for these ecological factors are key to reducing selection intensity. Without such management, treatment using broad-spectrum chemicals serves to multiply the effects of host plant preference, preadaptive detoxification ability, and high genetic diversity to create a pesticide treadmill for the three high-propensity species.Without rigorous ongoing management, such a treadmill could still develop for newer, more selective chemistries and insecticidal transgenic crops.

Drop-off behaviour of Bt-resistant and Bt-susceptible Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) larvae on Bt-cotton and non-Bt cotton plants.

The highest natural mortality rate of larval Lepidoptera in field populations occurs in the first instar, but it is highly variable. The pattern and degree of survival is not easily predicted but depends on their ability to establish on host plants. Lepidopteran larval dispersal behaviour, known as 'drop-off', happens when the host is unsuitable for larvae to settle and begin feeding. Understanding drop-off behaviour of Helicoverpa armigera (Hübner) with and without physiological resistance to Bt toxins on Bt and non-Bt cotton plants is an important component for resistance management strategies for this insect. We examined the drop-off behaviour of H. armigera to determine: (1) whether they move the same way or differently in response to Bt and non-Bt, and (2) could H. armigera larvae detect Bt toxin levels in cotton plants or did they move independently of toxin levels? In this study, we assessed the drop-off behaviour of Bt-resistant and Bt-susceptible H. armigera neonates on artificial diets and cotton plants with and without Bt toxin during the first 12 h after hatching. Bt-resistant and Bt-susceptible H. armigera neonates behaved differently on Bt and non-Bt substrates. The percentages of Bt-resistant larvae that dropped off Bt and non-Bt cotton plants were not significantly different. In contrast, significantly more Bt-susceptible larvae dropped off Bt cotton than non-Bt cotton plants over time. Although Bt-susceptible larvae could not detect Bt toxin, they showed preference on non-Bt toxin substrates and were more likely to drop off substrates with Bt toxin.

Genomic innovations, transcriptional plasticity and gene loss underlying the evolution and divergence of two highly polyphagous and invasive Helicoverpa pest species.

BACKGROUND: Helicoverpa armigera and Helicoverpa zea are major caterpillar pests of Old and New World agriculture, respectively. Both, particularly H. armigera, are extremely polyphagous, and H. armigera has developed resistance to many insecticides. Here we use comparative genomics, transcriptomics and resequencing to elucidate the genetic basis for their properties as pests. RESULTS: We find that, prior to their divergence about 1.5 Mya, the H. armigera/H. zea lineage had accumulated up to more than 100 more members of specific detoxification and digestion gene families and more than 100 extra gustatory receptor genes, compared to other lepidopterans with narrower host ranges. The two genomes remain very similar in gene content and order, but H. armigera is more polymorphic overall, and H. zea has lost several detoxification genes, as well as about 50 gustatory receptor genes. It also lacks certain genes and alleles conferring insecticide resistance found in H. armigera. Non-synonymous sites in the expanded gene families above are rapidly diverging, both between paralogues and between orthologues in the two species. Whole genome transcriptomic analyses of H. armigera larvae show widely divergent responses to different host plants, including responses among many of the duplicated detoxification and digestion genes. CONCLUSIONS: The extreme polyphagy of the two heliothines is associated with extensive amplification and neofunctionalisation of genes involved in host finding and use, coupled with versatile transcriptional responses on different hosts. H. armigera's invasion of the Americas in recent years means that hybridisation could generate populations that are both locally adapted and insecticide resistant.

Oviposition site selection and survival of susceptible and resistant larvae of Helicoverpa armigera (Lepidoptera: Noctuidae) on Bt and non-Bt cotton.

In Australia Bt cotton has been planted since 1996, and has greatly improved the control of its key target Helicoverpa armigera (Hübner). There is no strong evidence that genetically modified cotton has been selected for significant physiological resistance to Bt toxin in field populations. There are many possible explanations for the lack of apparent selection that range from high compliance with the resistance management strategy for this technology to a lack of behavioral preference in key traits such as oviposition that could favor survival. To date most experiments that test oviposition of H. armigera on Bt cotton vs. conventional cotton have been done with susceptible moths. We determine the oviposition preference of a field isolated Bt resistant line of H. armigera and a susceptible counterpart when given a choice of non-Bt cotton and Bt-cotton with the same genetic background, and test whether there is any relationship between oviposition site selection (different plant structures) and the survival of the first instar larvae. Within cotton plants, our experiments consistently showed that both resistant and susceptible moths did not choose plants or plant parts that were less toxic in terms of Bt toxin on which to lay eggs. There was one exception in that susceptible moths were more likely to lay eggs on squares of Bt cotton plants than squares of non-Bt cotton. As expected, the mortality of susceptible H. armigera neonates was significantly higher on structures of Bt cotton plants than on those structures of conventional cotton, and survival was greater on flowers than on other structures of Bt cotton. This confirms opportunities for selection for resistance, and demonstrates no advantage in this respect to carrying resistance genes that might overcome the Bt toxins.

High nucleotide diversity and limited linkage disequilibrium in Helicoverpa armigera facilitates the detection of a selective sweep.

Insecticides impose extreme selective pressures on populations of target pests and so insecticide resistance loci of these species may provide the footprints of 'selective sweeps'. To lay the foundation for future genome-wide scans for selective sweeps and inform genome-wide association study designs, we set out to characterize some of the baseline population genomic parameters of one of the most damaging insect pests in agriculture worldwide, Helicoverpa armigera. To this end, we surveyed nine Z-linked loci in three Australian H. armigera populations. We find that estimates of π are in the higher range among other insects and linkage disequilibrium decays over short distances. One of the surveyed loci, a cytochrome P450, shows an unusual haplotype configuration with a divergent allele at high frequency that led us to investigate the possibility of an adaptive introgression around this locus.

Dual Cry2Ab and Vip3A resistant strains of Helicoverpa armigera and Helicoverpa punctigera (Lepidoptera: Noctuidae); testing linkage between loci and monitoring of allele frequencies.

Considerable attention has been given to delaying the evolution of insect resistance to toxins produced by transgenic crops. The major pests of cotton in Australia are the Lepidoptera Helicoverpa armigera (Hubner, 1805) and Helicoverpa punctigera (Wallengren), and the toxins deployed in current and imminent transgenic cotton varieties are Cry1Ac, Cry2Ab and Vip3A from Bacillus thuringiensis. In this study, lines that carry alleles conferring resistance to Cry2Ab and Vip3A were isolated using F2 tests. Extensive work on the Cry2Ab resistant lines, and preliminary work on the Vip3A resistant lines, suggested a single common resistance to each toxin in both species thereby justifying the use of more efficient F1 tests as the primary means for monitoring changes over time. A potential further efficiency could be gained by developing a single resistant line that carries both types of Bt resistance. Herein we report on work with both H. armigera and H. punctigera that tests whether dual Cry2Ab-Vip3A resistant lines can be developed and, if so, whether they can be used to effectively monitor resistance frequencies. Furthermore, the creation of dual resistant lines allowed linkage between the Cry2Ab and Vip3A resistances to be investigated for H. punctigera. We show that dual resistant lines can be used to increase the efficiency of the F1 screen for recessive alleles, and that in H. punctigera there is no linkage between Cry2Ab and Vip3A resistance.

Micro-structural geometry of thin films intended for the inner lumen of nerve conduits affects nerve repair.

Damage to peripheral nerves can cause significant motor or sensory injuries. In serious cases, a nerve is sacrificed from another part of the body to repair a damaged nerve (autograft). The development of biodegradable polymer conduits may offer an alternative to autografts. This study investigated the surface topography and mechanical properties of smooth, pitted and grooved structures of ultra-thin poly (ε-caprolactone)/poly lactic acid blended, solvent-cast films. We have investigated the effect of the groove shape on cell morphology and alignment. Photolithography and dry/wet etching was used to develop patterned silicon substrates with grooves with accurate geometries (V shaped, sloped walls and square shaped). Using a neural cell line (NG108-15), in vitro experiments confirmed good cell attachment and proliferation on all the polymer scaffolds. Imaging techniques demonstrated that there was different cellular responses and morphology according to the shape of the groove. Studies showed that the geometry, particularly the angle of the slope and the space between grooves, affected cellular responses. In addition, biomechanical studies showed that the patterned films had excellent mechanical properties and were stronger than the natural nerve. The conduit tubes were made by rolling the films around a mandrel and using a thermal welding technique to join the edges. The promising biomechanical and in vitro results demonstrate that nerve cell responses are affected by the shape of longitudinal grooves, and particularly by the angle of the slope of the groove walls.

Investigation of 2D and 3D electrospun scaffolds intended for tendon repair.

Two-dimensional (2D) electrospun fibre mats have been investigated as fibrous sheets intended as biomaterials scaffolds for tissue repair. It is recognised that tissues are three-dimensional (3D) structures and that optimisation of the fabrication process should include both 2D and 3D scaffolds. Understanding the relative merits of the architecture of 2D and 3D scaffolds for tendon repair is required. This study investigated three different electrospun scaffolds based on poly(ε-caprolactone) fibres intended for repair of injured tendons, referred to as; 2D random sheet, 2D aligned sheet and 3D bundles. 2D aligned fibres and 3D bundles mimicked the parallel arrangement of collagen fibres in natural tendon and 3D bundles further replicated the tertiary layer of a tendon's hierarchical configuration. 3D bundles demonstrated greatest tensile properties, being significantly stronger and stiffer than 2D aligned and 2D random fibres. All scaffolds supported adhesion and proliferation of tendon fibroblasts. Furthermore, 2D aligned sheets and 3D bundles allowed guidance of the cells into a parallel, longitudinal arrangement, which is similar to tendon cells in the native tissue. With their superior physical properties and ability to better replicate tendon tissue, the 3D electrospun scaffolds warrant greater investigation as synthetic grafts in tendon repair.

Medical grade sterilization affects synthetic polymer film properties intended for peripheral nerve repair: an in vitro study.

In vitro characterization of synthetic scaffolds in the laboratory commonly employs sterilization techniques, such as, ultraviolet light or ethanol (EtOH) soaking. These sterilization methods are not sufficient, however, to gain Regulatory approval for therapeutic use. Neglecting the effects medical grade sterilization may have on material properties could lead to years of research never translating to the clinic. The objective of this study was to ascertain whether medical grade gamma irradiation affected the properties of solvent-cast poly(ε-caprolactone)/poly(D, L-lactic acid) blend films for peripheral nerve repair. Scaffolds were sterilized at eight incremental doses of Gamma radiation (0-45 kGy). With increasing radiation dose, tensile testing identified significant reductions in both maximum tensile strength (>40 %) and strain (>90 %); gel permeation chromatography showed a dose-dependent reduction in polymer molecular weight (>46 %) and differential scanning calorimetry highlighted an increase in crystallinity. NG108-15 nerve cells were cultured up to 7 days on gamma irradiated and EtOH soaked films (control). Scanning electron microscopy showed cells proliferated on all films during this time and confirmed cell morphology was unaffected by sterilization method. However, cellular proliferation and number were greater for gamma irradiated films compared to EtOH soaked. Despite material properties being significantly altered, the in vitro response was encouraging and gamma irradiation may prove effective for medical grade sterilization of films intended for peripheral nerve repair.

No evidence for change in oviposition behaviour of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) after widespread adoption of transgenic insecticidal cotton.

Cotton growing landscapes in Australia have been dominated by dual-toxin transgenic Bt varieties since 2004. The cotton crop has thus effectively become a sink for the main target pest, Helicoverpa armigera. Theory predicts that there should be strong selection on female moths to avoid laying on such plants. We assessed oviposition, collected from two cotton-growing regions, by female moths when given a choice of tobacco, cotton and cabbage. Earlier work in the 1980s and 1990s on populations from the same geographic locations indicated these hosts were on average ranked as high, mid and low preference plants, respectively, and that host rankings had a heritable component. In the present study, we found no change in the relative ranking of hosts by females, with most eggs being laid on tobacco, then cotton and least on cabbage. As in earlier work, some females laid most eggs on cotton and aspects of oviposition behaviour had a heritable component. Certainly, cotton is not avoided as a host, and the implications of these finding for managing resistance to Bt cotton are discussed.

Resistance Bioassays and Allele Characterization Inform Analysis of Spodoptera frugiperda (Lepidoptera: Noctuidae) Introduction Pathways in Asia and Australia.

The fall armyworm (FAW) Spodoptera frugiperda (Smith; Lepidoptera: Noctuidae) is present in over 70 countries in Africa, Asia, and Oceania. Its rapid dispersal since 2016 when it was first reported in western Africa, and associated devastation to agricultural productivity, highlight the challenges posed by this pest. Currently, its management largely relies on insecticide sprays and transgenic Bacillus thuringiensis toxins, therefore understanding their responses to these agents and characteristics of any resistance genes enables adaptive strategies. In Australia, S. frugiperda was reported at the end of January 2020 in northern Queensland and by March 2020, also in northern Western Australia. As an urgent first response we undertook bioassays on two Australian populations, one each from these initial points of establishment. To assist with preliminary sensitivity assessment, two endemic noctuid pest species, Helicoverpa armigera (Hübner; Lepidoptera, Noctuidae) and Spodoptera litura (Fabricius; Lepidoptera, Noctuidae), were concurrently screened to obtain larval LC50 estimates against various insecticides. We characterized known resistance alleles from the VGSC, ACE-1, RyR, and ABCC2 genes to compare with published allele frequencies and bioassay responses from native and invasive S. frugiperda populations. An approximately 10× LC50 difference for indoxacarb was detected between Australian populations, which was approximately 28× higher than that reported from an Indian population. Characterization of ACE-1 and VGSC alleles provided further evidence of multiple introductions in Asia, and multiple pathways involving genetically distinct individuals in Australia. The preliminary bioassay results and resistance allele patterns from invasive S. frugiperda populations suggest multiple introductions have contributed to the pest's spread and challenge the axiom of its rapid 'west-to-east' spread.

The use of a silicon strip detector dose magnifying glass in stereotactic radiotherapy QA and dosimetry.

PURPOSE: Stereotactic radiosurgery/therapy (SRS/SRT) is the use of radiation ablation in place of conventional surgical excision to remove or create fibrous tissue in small target volumes. The target of the SRT/SRS treatment is often located in close proximity to critical organs, hence the requirement of high geometric precision including a tight margin on the planning target volume and a sharp dose fall off. One of the major problems with quality assurance (QA) of SRT/SRS is the availability of suitable detectors with the required spatial resolution. The authors present a novel detector that they refer to as the dose magnifying glass (DMG), which has a high spatial resolution (0.2 mm) and is capable of meeting the stringent requirements of QA and dosimetry in SRS/SRT therapy. METHODS: The DMG is an array of 128 phosphor implanted n+ strips on a p-type Si wafer. The sensitive area defined by a single n+ strip is 20 x 2000 microm2. The Si wafer is 375 microm thick. It is mounted on a 0.12 mm thick Kapton substrate. The authors studied the dose per pulse (dpp) and angular response of the detector in a custom-made SRS phantom. The DMG was used to determine the centers of rotation and positioning errors for the linear accelerator's gantry, couch, and collimator rotations. They also used the DMG to measure the profiles and the total scatter factor (S(cp)) of the SRS cones. Comparisons were made with the EBT2 film and standard S(cp) values. The DMG was also used for dosimetric verification of a typical SRS treatment with various noncoplanar fields and arc treatments when applied to the phantom. RESULTS: The dose per pulse dependency of the DMG was found to be < 5% for a dpp change of 7.5 times. The angular response of the detector was investigated in the azimuthal and polar directions. The maximum polar angular response was 13.8% at the gantry angle of 320 degrees, which may be partly due to the phantom geometry. The maximum azimuthal angular response was 15.3% at gantry angles of 90 degrees and 270 degrees. The angular response at the gantry angle of 180 degrees was 6.3%. A correction function was derived to correct for the angular dependence of the detector, which takes into account the contribution of the azimuthal and polar angular response at different treatment couch positions. The maximum positioning errors due to collimator, gantry, and couch rotation were 0.2 +/- 0.1, 0.4 +/- 0.1, and 0.4 +/- 0.2 mm, respectively. The SRS cone S(cp) agrees very well with the standard data with an average difference of 1.2 +/- 1.1%. Comparison of the relative intensity profiles of the DMG and EBT2 measurements for a simulated SRS treatment shows a maximum difference of 2.5%. CONCLUSIONS: The DMG was investigated for dose per pulse and angular dependency. Its application to SRS/SRT delivery verification was demonstrated. The DMG with its high spatial resolution and real time capability allows measurement of dose profiles for cone applicators down to 5 mm in diameter, both accurately and rapidly as required in typical SRS/SRT deliveries.

Aligned electrospun polymer fibres for skeletal muscle regeneration.

Skeletal muscle repair is often overlooked in surgical procedures and in serious burn victims. Creating a tissue-engineered skeletal muscle would not only provide a grafting material for these clinical situations, but could also be used as a valuable true-to-life research tool into diseases affecting muscle tissue. Electrospinning of the elastomer PLGA produced aligned fibres that had the correct topology to provide contact guidance for myoblast elongation and alignment. In addition, the electrospun scaffold required no surface modifications or incorporation of biologic material for adhesion, elongation, and differentiation of C2C12 murine myoblasts.

Characteristics of resistance to Bacillus thuringiensis toxin Cry2Ab in a strain of Helicoverpa punctigera (Lepidoptera: Noctuidae) isolated from a field population.

In 1996, the Australian cotton industry adopted Ingard that expresses the Bacillus thuringiensis (Bt) toxin gene cry1Ac and was planted at a cap of 30%. In 2004-2005, Bollgard II, which expresses cry1Ac and cry2Ab, replaced Ingard in Australia, and subsequently has made up >80% of the area planted to cotton, Gossypium hirsutum L. The Australian target species Helicoverpa armigera (Hübner) and Helicoverpa punctigera (Wallengren) are innately moderately tolerant to Bt toxins, but the absence of a history of insecticide resistance indicates that the latter species is less likely to develop resistance to Bt cotton. From 2002-2003 to 2006-2007, F2 screens were deployed to detect resistance to CrylAc or Cry2Ab in natural populations of H. punctigera. Alleles that conferred an advantage against CrylAc were not detected, but those that conferred resistance to Cry2Ab were present at a frequency of 0.0018 (n = 2,192 alleles). Importantly, the first isolation of Cry2Ab resistance in H. punctigera occurred before significant opportunities to develop resistance in response to Bollgard II. We established a colony (designated Hp4-13) consisting of homozygous resistant individuals and examined their characteristics through comparison with individuals from a Bt-susceptible laboratory colony. Through specific crosses and bioassays, we established that the resistance present in Hp4-13 is due to a single autosomal gene. The resistance is fully recessive. Homozygotes are able to survive a dose of Cry2Ab toxin that is 15 times the reported concentration in field grown Bollgard II in Australia (500 microg/ml) and are fully susceptible to Cry1Ac and to the Bt product DiPel. These characteristics are the same as those described for the first Cry2Ab resistant strain of H. armigera isolated from a field population in Australia.

Why do F1 screens estimate higher frequencies of Cry2Ab resistance in Helicoverpa armigera (Lepidoptera: Noctuidae) than do F2 screens?

F2 and F1 tests to detect resistance to Cry2Ab in Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) were performed during the 2007-2008 summer. F2 tests indicated a resistance frequency of 0.006, which is similar to the published resistance frequencies for this species during the summers spanning 2002-2006. In contrast F1 tests indicated a resistance frequency of 0.033. Thus, F1 tests isolated Cry2Ab resistance alleles almost six-fold more frequently than the F2 method. A discrepancy might be expected if the F2 tests detected resistance conferred by more than one locus because F1 tests identify only the form of resistance present in the tester resistant colony. However, if so, F2 tests would detect more, not fewer, cases of resistance. In addition, complementation tests on 10 separate isolates indicate that there is only one common form of resistance. We hypothesized that some "resistance alleles" are homozygous lethal if autozygous (as generated in F2 tests) but not as allozygous homozygotes (as generated in F2 tests). The hypothesis was extended to accommodate the possibility that alleles at linked loci may be homozygous lethal. Neither of two tests of the hypothesis provided evidence that any alleles that confer resistance are associated with severe fitness costs. Thus we are presently unable to explain the basis of the difference in frequencies between the methods. Because of the simplicity of the F1 tests, it is difficult to imagine that it overestimates the frequency of resistance and we therefore accept that this test should provide a more robust method to estimate the frequency of Cry2Ab resistance in H. armigera.

Frequency of alleles conferring resistance to the Bacillus thuringiensis toxins Cry1Ac and Cry2Ab in Australian populations of Helicoverpa punctigera (Lepidoptera: Noctuidae) from 2002 to 2006.

Helicoverpa punctigera and Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) are important pests of field and horticultural crops in Australia. The former is endemic to the continent, whereas the latter is also distributed in Africa and Asia. Although H. armigera rapidly developed resistance to virtually every group of insecticide used against it, there is only one report of resistance to an insecticide in H. punctigera. In 1996 the Australian cotton industry adopted Ingard, which expresses the Bacillus thuringiensis (Bt) toxin gene cry1Ac. In 2004/2005, Bollgard II (which expresses Cry1Ac and Cry2Ab) replaced Ingard and has subsequently been grown on 80% of the area planted to cotton, Gossypium hirsutum L. From 2002/2003 to 2006/2007, F2 screens were used to detect resistance to Cry1Ac or Cry2Ab. We detected no alleles conferring resistance to Cry1Ac; the frequency was < 0.0005 (n = 2,180 alleles), with a 95% credibility interval between 0 and 0.0014. However, during the same period, we detected alleles that confer resistance to Cry2Ab at a frequency of 0.0018 (n = 2,192 alleles), with a 95% credibility interval between 0.0005 and 0.0040. For both toxins, the experiment-wise detection probability was 94%, i.e., if there actually was a resistance allele in any tested lines, we would have detected it 94% of the time. The first isolation of Cry2Ab resistance in H. punctigera was before the widespread deployment of Bollgard II. This finding supports our published notion for H. armigera that alleles conferring resistance to Cry2Ab may be present at detectable frequencies in populations before selection by transgenic crops.

Isolations of Cry2Ab resistance in Australian populations of Helicoverpa armigera (Lepidoptera: Noctuidae) are allelic.

Alleles conferring resistance to Cry2Ab toxin occur at a frequency of 0.0033 in Australian populations of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae), and it is evident that detectable levels of resistance predated the introduction of transgenic cotton expressing this toxin. From 2002 until 2006, 10 such resistant alleles were scored. Here, we examine colonies established from five of the 10 isolates by using complementation tests to determine their genetic relationships. The results demonstrate that the resistance in each colony is due to alleles at the same locus and that for each allele the resistance is recessive. This latter finding is in conflict with the frequency of apparently resistant individuals occurring in the initial F2 tests that were used to identify alleles that confer resistance. These frequencies were variable (range 6.7-35.6%, mean 16.2%), but they generally indicated a measure of dominance (i.e., were >6.25% expected for recessive resistance). We hypothesize that this conflict is the result of differences in the genetic background of the laboratory adapted resistant colonies and the initial field isolations.

Frequency of alleles conferring resistance to the Bt toxins Cry1Ac and Cry2Ab in Australian populations of Helicoverpa armigera (Lepidoptera: Noctuidae).

Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) is an important lepidopteran pest of cotton (Gossypium spp.) in Australia and the Old World. From 2002, F2 screens were used to examine the frequency of resistance alleles in Australian populations of H. armigera to Bacillus thuringiensis (Bt) CrylAc and Cry2Ab, the two insecticidal proteins present in the transgenic cotton Bollgard II. At that time, Ingard (expressing Cry1Ac) cotton had been grown in Australia for seven seasons, and Bollgard II was about to be commercially released. The principal objective of our study was to determine whether sustained exposure caused an elevated frequency of alleles conferring resistance to Cry1Ac in a species with a track record of evolving resistance to conventional insecticides. No major alleles conferring resistance to Cry1Ac were found. The frequency of resistance alleles for Cry1Ac was <0.0003, with a 95% credibility interval between 0 and 0.0009. In contrast, alleles conferring resistance to Cry2Ab were found at a frequency of 0.0033 (0.0017, 0.0055). The first isolation of this allele was found before the widespread deployment of Bollgard II. For both toxins the experiment-wise detection probability was 94.4%. Our results suggest that alleles conferring resistance to Cry1Ac are rare and that a relatively high baseline frequency of alleles conferring resistance to Cry2Ab existed before the introduction of Bt cotton containing this toxin.

Hydroxychloroquine retinopathy.

Hydroxychloroquine (HCQ; Plaquenil) is used increasingly in the management of a variety of autoimmune disorders, with well established roles in dermatology and rheumatology and emerging roles in oncology. Hydroxychloroquine has demonstrated a survival benefit in patients with systemic lupus erythematosus; some clinicians advocate its use in all such patients. However, Hydroxychloroquine and chloroquine (CQ) have been associated with irreversible visual loss due to retinal toxicity. Hydroxychloroquine retinal toxicity is far more common than previously considered; an overall prevalence of 7.5% was identified in patients taking HCQ for greater than 5 years, rising to almost 20% after 20 years of treatment. This review aims to provide an update on HCQ/CQ retinopathy. We summarise emerging treatment indications and evidence of efficacy in systemic disease, risk factors for retinopathy, prevalence among HCQ users, diagnostic tests, and management of HCQ retinopathy. We highlight emerging risk factors such as tamoxifen use, and new guidance on safe dosing, reversing the previous recommendation to use ideal body weight, rather than actual body weight. We summarise uncertainties and the recommendations made by existing HCQ screening programmes. Asian patients with HCQ retinopathy may demonstrate an extramacular or pericentral pattern of disease; visual field testing and retinal imaging should include a wider field for screening in this group. HCQ is generally safe and effective for the treatment of systemic disease but because of the risk of HCQ retinal toxicity, modern screening methods and ideal dosing should be implemented. Guidelines regarding optimal dosing and screening regarding HCQ need to be more widely disseminated.