Effects of inbreeding and genetic modification on Aedes aegypti larval competition and adult energy reserves

<p>Abstract</p> <p>Background</p> <p>Genetic modification of mosquitoes offers a promising strategy for the prevention and control of mosquito-borne diseases. For such a strategy to be effective, it is critically important that engineered strains are competitive enough to serve their intended function in population replacement or reduction of wild mosquitoes in nature. Thus far, fitness evaluations of genetically modified strains have not addressed the effects of competition among the aquatic stages and its consequences for adult fitness. We therefore tested the competitive success of combinations of wild, inbred and transgenic (created in the inbred background) immature stages of the dengue vector <it>Aedes aegypti </it>in the presence of optimal and sub-optimal larval diets.</p> <p>Results</p> <p>The wild strain of <it>Ae. aegypti </it>demonstrated greater performance (based on a composite index of survival, development rate and size) than the inbred strain, which in turn demonstrated greater performance than the genetically modified strain. Moreover, increasing competition through lowering the amount of diet available per larva affected fitness disproportionately: transgenic larvae had a reduced index of performance (95-119%) compared to inbred (50-88%) and wild type larvae (38-54%). In terms of teneral energy reserves (glycogen, lipid and sugar), adult wild type mosquitoes had more reserves directly available for flight, dispersal and basic metabolic functions than transgenic and inbred mosquitoes.</p> <p>Conclusions</p> <p>Our study provides a detailed assessment of inter- and intra-strain competition across aquatic stages of wild type, inbred, and transgenic mosquitoes and the impact of these conditions on adult energy reserves. Although it is not clear what competitive level is adequate for success of transgenic strains in nature, strong gene drive mechanisms are likely to be necessary in order to overcome competitive disadvantages in the larval stage that carryover to affect adult fitness.</p

Evaluation of a sticky trap for collecting Aedes (Stegomyia) adults in a dengue-endemic area in Thailand.

Development of new operational techniques for collection and monitoring of adult Stegomyia mosquitoes is considered a pressing need for surveillance and prevention of arboviruses. Here we report the results from a trial carried out in 2 dengue-endemic villages in Thailand to compare the ability to collect Aedes adults of a sticky trap versus a CDC backpack aspirator, which has been used routinely at the study area for entomological/epidemiological surveys.Our comparison was based on a comparable sampling effort required to carry out collections with 2 approaches. Over 19,000 specimens were collected, ∼90% of which were Culex spp. Sticky traps collected significantly more Aedes aegypti and Aedes albopictus females than did backpack aspirators when located outdoors. The percentage of positive sticky-trap catches was double for Ae. aegypti and almost 20 times higher for Ae. albopictus. Operational benefits of the sticky trap are discussed within the context of the results obtained

Spatial and temporal clustering of dengue virus transmission in Thai villages.

BackgroundTransmission of dengue viruses (DENV), the leading cause of arboviral disease worldwide, is known to vary through time and space, likely owing to a combination of factors related to the human host, virus, mosquito vector, and environment. An improved understanding of variation in transmission patterns is fundamental to conducting surveillance and implementing disease prevention strategies. To test the hypothesis that DENV transmission is spatially and temporally focal, we compared geographic and temporal characteristics within Thai villages where DENV are and are not being actively transmitted.Methods and findingsCluster investigations were conducted within 100 m of homes where febrile index children with (positive clusters) and without (negative clusters) acute dengue lived during two seasons of peak DENV transmission. Data on human infection and mosquito infection/density were examined to precisely (1) define the spatial and temporal dimensions of DENV transmission, (2) correlate these factors with variation in DENV transmission, and (3) determine the burden of inapparent and symptomatic infections. Among 556 village children enrolled as neighbors of 12 dengue-positive and 22 dengue-negative index cases, all 27 DENV infections (4.9% of enrollees) occurred in positive clusters (p &lt; 0.01; attributable risk [AR] = 10.4 per 100; 95% confidence interval 1-19.8 per 100]. In positive clusters, 12.4% of enrollees became infected in a 15-d period and DENV infections were aggregated centrally near homes of index cases. As only 1 of 217 pairs of serologic specimens tested in positive clusters revealed a recent DENV infection that occurred prior to cluster initiation, we attribute the observed DENV transmission subsequent to cluster investigation to recent DENV transmission activity. Of the 1,022 female adult Ae. aegypti collected, all eight (0.8%) dengue-infected mosquitoes came from houses in positive clusters; none from control clusters or schools. Distinguishing features between positive and negative clusters were greater availability of piped water in negative clusters (p &lt; 0.01) and greater number of Ae. aegypti pupae per person in positive clusters (p = 0.04). During primarily DENV-4 transmission seasons, the ratio of inapparent to symptomatic infections was nearly 1:1 among child enrollees. Study limitations included inability to sample all children and mosquitoes within each cluster and our reliance on serologic rather than virologic evidence of interval infections in enrollees given restrictions on the frequency of blood collections in children.ConclusionsOur data reveal the remarkably focal nature of DENV transmission within a hyperendemic rural area of Thailand. These data suggest that active school-based dengue case detection prompting local spraying could contain recent virus introductions and reduce the longitudinal risk of virus spread within rural areas. Our results should prompt future cluster studies to explore how host immune and behavioral aspects may impact DENV transmission and prevention strategies. Cluster methodology could serve as a useful research tool for investigation of other temporally and spatially clustered infectious diseases

Methods for microbial DNA extraction from soil for PCR amplification

Amplification of DNA from soil is often inhibited by co-purified contaminants. A rapid, inexpensive, large-scale DNA extraction method involving minimal purification has been developed that is applicable to various soil types (1). DNA is also suitable for PCR amplification using various DNA targets. DNA was extracted from 100g of soil using direct lysis with glass beads and SDS followed by potassium acetate precipitation, polyethylene glycol precipitation, phenol extraction and isopropanol precipitation. This method was compared to other DNA extraction methods with regard to DNA purity and size

Fine Scale Spatiotemporal Clustering of Dengue Virus Transmission in Children and Aedes aegypti in Rural Thai Villages

Background: Based on spatiotemporal clustering of human dengue virus (DENV) infections, transmission is thought to occur at fine spatiotemporal scales by horizontal transfer of virus between humans and mosquito vectors. To define the dimensions of local transmission and quantify the factors that support it, we examined relationships between infected humans and Aedes aegypti in Thai villages. Methodology/Principal Findings: Geographic cluster investigations of 100-meter radius were conducted around DENV-positive and DENV-negative febrile ‘‘index’’ cases (positive and negative clusters, respectively) from a longitudinal cohort study in rural Thailand. Child contacts and Ae. aegypti from cluster houses were assessed for DENV infection. Spatiotemporal, demographic, and entomological parameters were evaluated. In positive clusters, the DENV infection rate among child contacts was 35.3% in index houses, 29.9% in houses within 20 meters, and decreased with distance from the index house to 6.2% in houses 80–100 meters away (pAe. aegypti were DENV-infectious (i.e., DENV-positive in head/thorax) in positive clusters (23/1755; 1.3%) than negative clusters (1/1548; 0.1%). In positive clusters, 8.2% of mosquitoes were DENV-infectious in index houses, 4.2% in other houses with DENV-infected children, and 0.4% in houses without infected children (pAe. aegypti pupae and adult females were more numerous only in houses containing infectious mosquitoes. Conclusions/Significance: Human and mosquito infections are positively associated at the level of individual houses and neighboring residences. Certain houses with high transmission risk contribute disproportionately to DENV spread to neighboring houses. Small groups of houses with elevated transmission risk are consistent with over-dispersion of transmission (i.e., at a given point in time, people/mosquitoes from a small portion of houses are responsible for the majority of transmission)

Quantifying Rift Valley fever virus transmission efficiency in a lamb-mosquito-lamb model

Rift Valley fever virus (RVFV) is a (re)emerging mosquito-borne pathogen impacting human and animal health. How RVFV spreads through a population depends on population-level and individual-level interactions between vector, host and pathogen. Here, we estimated the probability for RVFV to transmit to naive animals by experimentally exposing lambs to a bite of an infectious mosquito, and assessed if and how RVFV infection subsequently developed in the exposed animal. Aedes aegypti mosquitoes, previously infected via feeding on a viremic lamb, were used to expose naive lambs to the virus. Aedes aegypti colony mosquitoes were used as they are easy to maintain and readily feed in captivity. Other mosquito spp. could be examined with similar methodology. Lambs were exposed to either 1-3 (low exposure) or 7-9 (high exposure) infectious mosquitoes. All lambs in the high exposure group became viremic and showed characteristic signs of Rift Valley fever within 2-4 days post exposure. In contrast, 3 out of 12 lambs in the low exposure group developed viremia and disease, with similar peak-levels of viremia as the high exposure group but with some heterogeneity in the onset of viremia. These results suggest that the likelihood for successful infection of a ruminant host is affected by the number of infectious mosquitoes biting, but also highlights that a single bite of an infectious mosquito can result in disease. The per bite mosquito-to-host transmission efficiency was estimated at 28% (95% confidence interval: 15 - 47%). We subsequently combined this transmission efficiency with estimates for life traits of Aedes aegypti or related mosquitoes into a Ross-McDonald mathematical model to illustrate scenarios under which major RVFV outbreaks could occur in naïve populations (i.e., R0 &gt;1). The model revealed that relatively high vector-to-host ratios as well as mosquitoes feeding preferably on competent hosts are required for R0 to exceed 1. Altogether, this study highlights the importance of experiments that mimic natural exposure to RVFV. The experiments facilitate a better understanding of the natural progression of disease and a direct way to obtain epidemiological parameters for mathematical models

CRISPR-Cas9 ribonucleoprotein-mediated co-editing and counterselection in the rice blast fungus

The rice blast fungus Magnaporthe oryzae is the most serious pathogen of cultivated rice and a significant threat to global food security. To accelerate targeted mutation and specific genome editing in this species, we have developed a rapid plasmid-free CRISPR-Cas9-based genome editing method. We show that stable expression of Cas9 is highly toxic to M. oryzae. However efficient gene editing can be achieved by transient introduction of purified Cas9 pre-complexed to RNA guides to form ribonucleoproteins (RNPs). When used in combination with oligonucleotide or PCR-generated donor DNAs, generation of strains with specific base pair edits, in-locus gene replacements, or multiple gene edits, is very rapid and straightforward. We demonstrate a co-editing strategy for the creation of single nucleotide changes at specific loci. Additionally, we report a novel counterselection strategy which allows creation of precisely edited fungal strains that contain no foreign DNA and are completely isogenic to the wild type. Together, these developments represent a scalable improvement in the precision and speed of genetic manipulation in M. oryzae and are likely to be broadly applicable to other fungal species

Community mobilization for malaria elimination: application of an open space methodology in Ruhuha sector, Rwanda

Background Despite the significant reduction of malaria transmission in Rwanda, Ruhuha sector is still a highly endemic area for malaria. The objective of this activity was to explore and brainstorm the potential roles of various community stakeholders in malaria elimination. Methods Horizontal participatory approaches such as ‘open space’ have been deployed to explore local priorities, stimulate community contribution to project planning, and to promote local capacity to manage programmes. Two open space meetings were conducted with 62 and 82 participants in years 1 and 2, respectively. Participants included purposively selected community and local organizations’ representatives. Results Malaria was perceived as a health concern by the respondents despite the reported reduction in prevalence from 60 to 20% for cases at the local health centre. Some misconceptions of the cause of malaria and misuse of preventive strategies were noted. Poverty was deemed to be a contributing factor to malaria transmission, with suggestions that improvement of living conditions for poor families might help malaria reduction. Participants expressed willingness to contribute to malaria elimination and underscored the need for constant education, sensitization and mobilization towards malaria control in general. Active diagnosis, preventative strategies and prompt treatment of malaria cases were all mentioned by participants as ways to reduce malaria. Participants suggested that partnership of stakeholders at various levels could speed up programme activities. A community rewards system was deemed important to motivate engaged participants, i.e., community health workers and households. Establishment of malaria clubs in schools settings was also suggested as crucial to speed up community awareness and increase skills towards further malaria reduction. Conclusions This bottom-up approach was found useful in engaging the local community, enabling them to explore issues related to malaria in the area and suggest solutions for sustainable malaria elimination gains

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