Pie Müller

Field-caught permethrin-resistant anopheles gambiae overexpress CYP6P3, a P450 that metabolises pyrethroids

Insects exposed to pesticides undergo strong natural selection and have developed various adaptive mechanisms to survive. Resistance to pyrethroid insecticides in the malaria vector Anopheles gambiae is receiving increasing attention because it threatens the sustainability of malaria vector control programs in sub-Saharan Africa. An understanding of the molecular mechanisms conferring pyrethroid resistance gives insight into the processes of evolution of adaptive traits and facilitates the development of simple monitoring tools and novel strategies to restore the efficacy of insecticides. For this purpose, it is essential to understand which mechanisms are important in wild mosquitoes. Here, our aim was to identify enzymes that may be important in metabolic resistance to pyrethroids by measuring gene expression for over 250 genes potentially involved in metabolic resistance in phenotyped individuals from a highly resistant, wild A. gambiae population from Ghana. A cytochrome P450, CYP6P3, was significantly overexpressed in the survivors, and we show that the translated enzyme metabolises both alpha-cyano and non–alpha-cyano pyrethroids. This is the first study to demonstrate the capacity of a P450 identified in wild A. gambiae to metabolise insecticides. The findings add to the understanding of the genetic basis of insecticide resistance in wild mosquito populations.

Identification and validation of a gene causing cross-resistance between insecticide classes in Anopheles gambiae from Ghana

In the last decade there have been marked reductions in malaria incidence in sub-Saharan Africa. Sustaining these reductions will rely upon insecticides to control the mosquito malaria vectors. We report that in the primary African malaria vector, Anopheles gambiae sensu stricto, a single enzyme, CYP6M2, confers resistance to two classes of insecticide. This is unique evidence in a disease vector of cross-resistance associated with a single metabolic gene that simultaneously reduces the efficacy of two of the four classes of insecticide routinely used for malaria control. The gene-expression profile of a highly DDT-resistant population of A. gambiae s.s. from Ghana was characterized using a unique whole-genome microarray. A number of genes were significantly overexpressed compared with two susceptible West African colonies, including genes from metabolic families previously linked to insecticide resistance. One of the most significantly overexpressed probe groups (false-discovery rate-adjusted P < 0.0001) belonged to the cytochrome P450 gene CYP6M2. This gene is associated with pyrethroid resistance in wild A. gambiae s.s. populations) and can metabolize both type I and type II pyrethroids in recombinant protein assays. Using in vitro assays we show that recombinant CYP6M2 is also capable of metabolizing the organochlorine insecticide DDT in the presence of solubilizing factor sodium cholate.

Transcription profiling of a recently colonised pyrethroid resistant Anopheles gambiae strain from Ghana.

BackgroundMosquito resistance to the pyrethroid insecticides used to treat bednets threatens the sustainability of malaria control in sub-Saharan Africa. While the impact of target site insensitivity alleles is being widely discussed the implications of insecticide detoxification – though equally important – remains elusive. The successful development of new tools for malaria intervention and management requires a comprehensive understanding of insecticide resistance, including metabolic resistance mechanisms. Although three enzyme families (cytochrome P450s, glutathione S-transferases and carboxylesterases) have been widely associated with insecticide detoxification the role of individual enzymes is largely unknown.ResultsHere, constitutive expression patterns of genes putatively involved in conferring pyrethroid resistance was investigated in a recently colonised pyrethroid resistant Anopheles gambiae strain from Odumasy, Southern Ghana. RNA from the resistant strain and a standard laboratory susceptible strain, of both sexes was extracted, reverse transcribed and labelled with either Cy3- or Cy5-dye. Labelled cDNA was co-hybridised to the detox chip, a custom-made microarray containing over 230 A. gambiae gene fragments predominantly from enzyme families associated with insecticide resistance. After hybridisation, Cy3- and Cy5-signal intensities were measured and compared gene by gene. In both females and males of the resistant strain the cytochrome P450s CYP6Z2 and CYP6M2 are highly over-expressed along with a member of the superoxide dismutase (SOD) gene family.ConclusionThese genes differ from those found up-regulated in East African strains of pyrethroid resistant A. gambiae and constitute a novel set of candidate genes implicated in insecticide detoxification. These data suggest that metabolic resistance may have multiple origins in A. gambiae, which has strong implications for the management of resistance.

Pyrethroid tolerance is associated with elevated expression of antioxidants and agricultural practice in Anopheles arabiensis sampled from an area of cotton fields in Northern Cameroon

Spraying of agricultural crops with insecticides can select for resistance in nontarget insects and this may compromise the use of insecticides for the control of vector‐borne diseases. The tolerance of the malaria vector, Anopheles arabiensis to deltamethrin was determined in a field population from a cotton‐growing region of Northern Cameroon both prior to and midway through the 4‐month period of insecticide application to the cotton crop. A 1.6‐fold increase in the median knockdown time was observed. To determine whether this increased tolerance was associated with constitutively elevated levels of genes commonly associated with insecticide resistance, RNA was extracted from F1 progeny from family lines of field‐caught mosquitoes and hybridized to the Anopheles gambiae detox chip. The experimental design avoided the confounding effects of colonization, and this study is the first to measure gene expression in the progeny of gravid, wild‐caught mosquitoes. Several genes with antioxidant roles, including superoxide dismutases, a glutathione S‐transferase and a thioredoxin‐dependent peroxidase, and a cytochrome P450 showed elevated expression in mosquito families collected during the insecticide‐spraying programme. These genes may constitute an important general defence mechanism against insecticides. Intriguingly, the levels of expression of these genes were strongly correlated suggesting a common regulatory mechanism.

Tracking of flying insects using pan-tilt cameras

Potent and affordable video and computer systems for automatic data acquisition are becoming increasingly important in behavioural neuroscience. It has remained challenging, however, to acquire data from small and fast-moving animals, such as insects in flight, due to the limited spatial and temporal resolution of the systems currently available. Our research on free-flying insects motivated the development of new methods in the context of two different experimental settings. First, the position and precise body axis direction of honey bees approaching a food source were automatically measured. Second, the flight trajectories of a phonotactic parasitoid fly homing in on its cricket host were recorded in 3D. We used pan-tilt cameras, i.e. cameras with moveable optics, to follow the animals path with a close up image. Novel methods were developed for image acquisition and position measurement using pan-tilt cameras, as well as calibration and data evaluation in 3D world coordinates. The innovations of this system comprise: (1) Acquisition of images in high spatial detail over large observation areas. (2) Image acquisition at a field rate of 50 Hz PAL. (3) Free positioning of the cameras for 3D acquisition. (4) Computation of the flight path in 3D world coordinates. We illustrate the capabilities of the system with data obtained from a calibration object as well as from the behaviour of unrestricted, free-flying flies and bees. Potential applications in behavioural neuroscience and the psychophysics of sensory perception are briefly discussed.

Power analysis for generalized linear mixed models in ecology and evolution.

‘Will my study answer my research question?’ is the most fundamental question a researcher can ask when designing a study, yet when phrased in statistical terms – ‘What is the power of my study?’ or ‘How precise will my parameter estimate be?’ – few researchers in ecology and evolution (EE) try to answer it, despite the detrimental consequences of performing under- or over-powered research. We suggest that this reluctance is due in large part to the unsuitability of simple methods of power analysis (broadly defined as any attempt to quantify prospectively the ‘informativeness’ of a study) for the complex models commonly used in EE research. With the aim of encouraging the use of power analysis, we present simulation from generalized linear mixed models (GLMMs) as a flexible and accessible approach to power analysis that can account for random effects, overdispersion and diverse response distributions. We illustrate the benefits of simulation-based power analysis in two research scenarios: estimating the precision of a survey to estimate tick burdens on grouse chicks and estimating the power of a trial to compare the efficacy of insecticide-treated nets in malaria mosquito control. We provide a freely available R function, sim.glmm, for simulating from GLMMs. Analysis of simulated data revealed that the effects of accounting for realistic levels of random effects and overdispersion on power and precision estimates were substantial, with correspondingly severe implications for study design in the form of up to fivefold increases in sampling effort. We also show the utility of simulations for identifying scenarios where GLMM-fitting methods can perform poorly. These results illustrate the inadequacy of standard analytical power analysis methods and the flexibility of simulation-based power analysis for GLMMs. The wider use of these methods should contribute to improving the quality of study design in EE.

Identification of Cryptic Anopheles Mosquito Species by Molecular Protein Profiling

Vector control is the mainstay of malaria control programmes. Successful vector control profoundly relies on accurate information on the target mosquito populations in order to choose the most appropriate intervention for a given mosquito species and to monitor its impact. An impediment to identify mosquito species is the existence of morphologically identical sibling species that play different roles in the transmission of pathogens and parasites. Currently PCR diagnostics are used to distinguish between sibling species. PCR based methods are, however, expensive, time-consuming and their development requires a priori DNA sequence information. Here, we evaluated an inexpensive molecular proteomics approach for Anopheles species: matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). MALDI-TOF MS is a well developed protein profiling tool for the identification of microorganisms but so far has received little attention as a diagnostic tool in entomology. We measured MS spectra from specimens of 32 laboratory colonies and 2 field populations representing 12 Anopheles species including the A. gambiae species complex. An important step in the study was the advancement and implementation of a bioinformatics approach improving the resolution over previously applied cluster analysis. Borrowing tools for linear discriminant analysis from genomics, MALDI-TOF MS accurately identified taxonomically closely related mosquito species, including the separation between the M and S molecular forms of A. gambiae sensu stricto. The approach also classifies specimens from different laboratory colonies; hence proving also very promising for its use in colony authentication as part of quality assurance in laboratory studies. While being exceptionally accurate and robust, MALDI-TOF MS has several advantages over other typing methods, including simple sample preparation and short processing time. As the method does not require DNA sequence information, data can also be reviewed at any later stage for diagnostic or functional patterns without the need for re-designing and re-processing biological material.

Using Drosophila melanogaster to validate metabolism-based insecticide resistance from insect pests

Identifying molecular mechanisms of insecticide resistance is important for preserving insecticide efficacy, developing new insecticides and implementing insect control. The metabolic detoxification of insecticides is a widespread resistance mechanism. Enzymes with the potential to detoxify insecticides are commonly encoded by members of the large cytochrome P450, glutathione S-transferase and carboxylesterase gene families, all rapidly evolving in insects. Here, we demonstrate that the model insect Drosophila melanogaster is useful for functionally validating the role of metabolic enzymes in conferring metabolism-based insecticide resistance. Alleles of three well-characterized genes from different pest insects were expressed in transgenic D. melanogaster : a carboxylesterase gene (αE7) from the Australian sheep blowfly Lucilia cuprina, a glutathione S-transferase gene (GstE2) from the mosquito Anopheles gambiae and a cytochrome P450 gene (Cyp6cm1) from the whitefly Bemisia tabaci. For all genes, expression in D. melanogaster resulted in insecticide resistance phenotypes mirroring those observed in resistant populations of the pest species. Using D. melanogaster to assess the potential for novel metabolic resistance mechanisms to evolve in pest species is discussed.

Plasmodium infection alters Anopheles gambiae detoxification gene expression.

BackgroundAnopheles gambiae has been shown to change its global gene expression patterns upon Plasmodium infection. While many alterations are directly related to the mosquitos innate immune response, parasite invasion is also expected to generate toxic by-products such as free radicals. The current study aimed at identifying which loci coding for detoxification enzymes are differentially expressed as a function of Plasmodium berghei infection in midgut and fat body tissues.ResultsUsing a custom-made DNA microarray, transcript levels of 254 loci primarily belonging to three major detoxification enzyme families (glutathione S-transferases, cytochrome P450 monooxygenases and esterases) were compared in infected and uninfected mosquitoes both during ookinete invasion and the release of sporozoites into the hemocoel. The greatest changes in gene expression were observed in the midgut in response to ookinete invasion. Interestingly, many detoxification genes including a large number of P450s were down-regulated at this stage. In the fat body, while less dramatic, gene expression alterations were also observed and occurred during the ookinete invasion and during the release of sporozoites into the hemocoel. While most gene expression changes were tissue-related, CYP6M2, a CYP previously associated with insecticide resistance, was over-expressed both in the midgut and fat body during ookinete invasion.ConclusionsMost toxicity-related reactions occur in the midgut shortly after the ingestion of an infected blood meal. Strong up-regulation of CYP6M2 in the midgut and the fat body as well as its previous association with insecticide resistance shows its broad role in metabolic detoxification.

Context-dependent stimulus presentation to freely moving animals in 3D

The presentation of controllable, dynamic sensory stimuli provides a powerful experimental paradigm, which has been extensively applied to explore sensory processing in walking and tethered flying insects. Recent advances in computer hardware and software technology provide the opportunity to track the 3D flight path of free-flying insects and process these data in real-time, opening up the possibility to present dynamic stimuli to free-flying animals. To accommodate for the increased complexity relating to 3D space, we partitioned experimental design, real-time data acquisition and stimulus control into multiple self-contained modules. 3D experimental scenarios were created in a stand-alone application by forging multiple 3D space-stimulus relationships. The use of dynamic cues is illustrated by an experiment, in which dynamic acoustic cues were presented to a free-flying parasitoid fly in a large 3D environment. The combination of loosely coupled modules provides robust and flexible solutions, allowing new paradigms to be readily implemented based on existing technologies. We demonstrate this with a test system that displayed a complex visual stimulus, controlled in real-time by the 2D position and orientation of a test object. The presented methods are applicable in a variety of novel experimental paradigms, including learning paradigms, for various sensory modalities in walking, swimming and flying animals.