Dataset Information

A LAMP assay for the rapid and robust assessment of Wolbachia infection in Aedes aegypti under field and laboratory conditions.

ABSTRACT: With Wolbachia-based arbovirus control programs being scaled and operationalised around the world, cost effective and reliable detection of Wolbachia in field samples and laboratory stocks is essential for quality control. Here we validate a modified loop-mediated isothermal amplification (LAMP) assay for routine scoring of Wolbachia in mosquitoes from laboratory cultures and the field, applicable to any setting. We show that this assay is a rapid and robust method for highly sensitive and specific detection of wAlbB Wolbachia infection within Aedes aegypti under a variety of conditions. We test the quantitative nature of the assay by evaluating pooled mixtures of Wolbachia-infected and uninfected mosquitoes and show that it is capable of estimating infection frequencies, potentially circumventing the need to perform large-scale individual analysis for wAlbB infection status in the course of field monitoring. These results indicate that LAMP assays are useful for routine screening particularly under field conditions away from laboratory facilities.

SUBMITTER: Jasper ME

PROVIDER: S-EPMC6867633 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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A LAMP assay for the rapid and robust assessment of Wolbachia infection in Aedes aegypti under field and laboratory conditions.

Jasper Moshe E ME Yang Qiong Q Ross Perran A PA Endersby-Harshman Nancy N Bell Nicholas N Hoffmann Ary A AA

PloS one 20191120 11

With Wolbachia-based arbovirus control programs being scaled and operationalised around the world, cost effective and reliable detection of Wolbachia in field samples and laboratory stocks is essential for quality control. Here we validate a modified loop-mediated isothermal amplification (LAMP) assay for routine scoring of Wolbachia in mosquitoes from laboratory cultures and the field, applicable to any setting. We show that this assay is a rapid and robust method for highly sensitive and speci ...[more]

PMID: 31747432

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Project description:Wolbachia bacteria are now being introduced into Aedes aegypti mosquito populations for dengue control. When Wolbachia infections are at a high frequency, they influence the local transmission of dengue by direct virus blocking as well as deleterious effects on vector mosquito populations. However, the effectiveness of this strategy could be influenced by environmental temperatures that decrease Wolbachia density, thereby reducing the ability of Wolbachia to invade and persist in the population and block viruses. We reared wMel-infected Ae. aegypti larvae in the field during the wet season in Cairns, North Queensland. Containers placed in the shade produced mosquitoes with a high Wolbachia density and little impact on cytoplasmic incompatibility. However, in 50% shade where temperatures reached 39°C during the day, wMel-infected males partially lost their ability to induce cytoplasmic incompatibility and females had greatly reduced egg hatch when crossed to infected males. In a second experiment under somewhat hotter conditions (>40°C in 50% shade), field-reared wMel-infected females had their egg hatch reduced to 25% when crossed to field-reared wMel-infected males. Wolbachia density was reduced in 50% shade for both sexes in both experiments, with some mosquitoes cleared of their Wolbachia infections entirely. To investigate the critical temperature range for the loss of Wolbachia infections, we held Ae. aegypti eggs in thermocyclers for one week at a range of cyclical temperatures. Adult wMel density declined when eggs were held at 26-36°C or above with complete loss at 30-40°C, while the density of wAlbB remained high until temperatures were lethal. These findings suggest that high temperature effects on Wolbachia are potentially substantial when breeding containers are exposed to partial sunlight but not shade. Heat stress could reduce the ability of Wolbachia infections to invade mosquito populations in some locations and may compromise the ability of Wolbachia to block virus transmission in the field. Temperature effects may also have an ecological impact on mosquito populations given that a proportion of the population becomes self-incompatible.

Project description:BACKGROUND:Yellow fever, dengue, chikungunya and Zika viruses are responsible for considerable morbidity and mortality in humans. Aedes aegypti and Aedes albopictus are the most important mosquito vectors involved in their transmission. Accurate identification of these species is essential for the implementation of control programs to limit arbovirus transmission, during suspected detections at ports of first entry, to delimit incursions or during presence/absence surveillance programs in regions vulnerable to invasion. We developed and evaluated simple and rapid colorimetric isothermal tests to detect these two mosquito species based on loop-mediated isothermal amplification (LAMP) targeting the ribosomal RNA internal transcribed spacer 1 (ITS1). METHODOLOGY/PRINCIPAL FINDINGS:Samples were prepared by homogenizing and heating at 99 oC for 10 min before an aliquot was added to the LAMP reaction. After 40 min incubation at 65 oC, a colour change indicated a positive result. The tests were 100% sensitive and species-specific, and demonstrated a limit of detection comparable with PCR-based detection (TaqMan chemistry). The LAMP assays were able to detect target species for various life stages tested (adult, 1st instar larva, 4th instar larva and pupa), and body components, such as legs, wings and pupal exuviae. Importantly, the LAMP assays could detect Ae. aegypti DNA in mosquitoes stored in Biogents Sentinel traps deployed in the field for 14 d. A single 1st instar Ae. aegypti larva could also be detected in a pool of 1,000 non-target 1st instar Aedes notoscriptus, thus expediting processing of ovitrap collections obtained during presence/absence surveys. A simple syringe-sponge protocol facilitated the concentration and collection of larvae from the ovitrap water post-hatch. CONCLUSIONS/SIGNIFICANCE:We describe the development of LAMP assays for species identification and demonstrate their direct application for surveillance in different field contexts. The LAMP assays described herein are useful adjuncts to laboratory diagnostic testing or could be employed as standalone tests. Their speed, ease-of-use, low cost and need for minimal equipment and training make the LAMP assays ideal for adoption in low-resource settings without the need to access diagnostic laboratory services.

Project description:A novel strategy for controlling the spread of arboviral diseases such as dengue, Zika and chikungunya is to transform mosquito populations with virus-suppressing Wolbachia. In general, Wolbachia transinfected into mosquitoes induce fitness costs through lower viability or fecundity. These maternally inherited bacteria also produce a frequency-dependent advantage for infected females by inducing cytoplasmic incompatibility (CI), which kills the embryos produced by uninfected females mated to infected males. These competing effects, a frequency-dependent advantage and frequency-independent costs, produce bistable Wolbachia frequency dynamics. Above a threshold frequency, denoted pˆ, CI drives fitness-decreasing Wolbachia transinfections through local populations; but below pˆ, infection frequencies tend to decline to zero. If pˆ is not too high, CI also drives spatial spread once infections become established over sufficiently large areas. We illustrate how simple models provide testable predictions concerning the spatial and temporal dynamics of Wolbachia introductions, focusing on rate of spatial spread, the shape of spreading waves, and the conditions for initiating spread from local introductions. First, we consider the robustness of diffusion-based predictions to incorporating two important features of wMel-Aedes aegypti biology that may be inconsistent with the diffusion approximations, namely fast local dynamics induced by complete CI (i.e., all embryos produced from incompatible crosses die) and long-tailed, non-Gaussian dispersal. With complete CI, our numerical analyses show that long-tailed dispersal changes wave-width predictions only slightly; but it can significantly reduce wave speed relative to the diffusion prediction; it also allows smaller local introductions to initiate spatial spread. Second, we use approximations for pˆ and dispersal distances to predict the outcome of 2013 releases of wMel-infected Aedes aegypti in Cairns, Australia, Third, we describe new data from Ae. aegypti populations near Cairns, Australia that demonstrate long-distance dispersal and provide an approximate lower bound on pˆ for wMel in northeastern Australia. Finally, we apply our analyses to produce operational guidelines for efficient transformation of vector populations over large areas. We demonstrate that even very slow spatial spread, on the order of 10-20 m/month (as predicted), can produce area-wide population transformation within a few years following initial releases covering about 20-30% of the target area.

Project description:The survival of adult female Aedes mosquitoes is a critical component of their ability to transmit pathogens such as dengue viruses. One of the principal determinants of Aedes survival is temperature, which has been associated with seasonal changes in Aedes populations and limits their geographical distribution. The effects of temperature and other sources of mortality have been studied in the field, often via mark-release-recapture experiments, and under controlled conditions in the laboratory. Survival results differ and reconciling predictions between the two settings has been hindered by variable measurements from different experimental protocols, lack of precision in measuring survival of free-ranging mosquitoes, and uncertainty about the role of age-dependent mortality in the field.Here we apply generalised additive models to data from 351 published adult Ae. aegypti and Ae. albopictus survival experiments in the laboratory to create survival models for each species across their range of viable temperatures. These models are then adjusted to estimate survival at different temperatures in the field using data from 59 Ae. aegypti and Ae. albopictus field survivorship experiments. The uncertainty at each stage of the modelling process is propagated through to provide confidence intervals around our predictions.Our results indicate that adult Ae. albopictus has higher survival than Ae. aegypti in the laboratory and field, however, Ae. aegypti can tolerate a wider range of temperatures. A full breakdown of survival by age and temperature is given for both species. The differences between laboratory and field models also give insight into the relative contributions to mortality from temperature, other environmental factors, and senescence and over what ranges these factors can be important.Our results support the importance of producing site-specific mosquito survival estimates. By including fluctuating temperature regimes, our models provide insight into seasonal patterns of Ae. aegypti and Ae. albopictus population dynamics that may be relevant to seasonal changes in dengue virus transmission. Our models can be integrated with Aedes and dengue modelling efforts to guide and evaluate vector control, better map the distribution of disease and produce early warning systems for dengue epidemics.

Dataset Information

A LAMP assay for the rapid and robust assessment of Wolbachia infection in Aedes aegypti under field and laboratory conditions.

Publications

A LAMP assay for the rapid and robust assessment of Wolbachia infection in Aedes aegypti under field and laboratory conditions.

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