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

Factors affecting fungus-induced larval mortality in Anopheles gambiae and Anopheles stephensi

<p>Abstract</p> <p>Background</p> <p>Entomopathogenic fungi have shown great potential for the control of adult malaria vectors. However, their ability to control aquatic stages of anopheline vectors remains largely unexplored. Therefore, how larval characteristics (<it>Anopheles </it>species, age and larval density), fungus (species and concentration) and environmental effects (exposure duration and food availability) influence larval mortality caused by fungus, was studied.</p> <p>Methods</p> <p>Laboratory bioassays were performed on the larval stages of <it>Anopheles gambiae </it>and <it>Anopheles stephensi </it>with spores of two fungus species, <it>Metarhizium anisopliae </it>and <it>Beauveria bassiana</it>. For various larval and fungal characteristics and environmental effects the time to death was determined and survival curves established. These curves were compared by Kaplan Meier and Cox regression analyses.</p> <p>Results</p> <p><it>Beauveria bassiana </it>and <it>Metarhizium anisopliae </it>caused high mortality of <it>An. gambiae </it>and <it>An. stephensi </it>larvae. However, <it>Beauveria bassiana </it>was less effective (Hazard ratio (HR) <1) compared to <it>Metarhizium anisopliae. Anopheles stephensi </it>and <it>An. gambiae </it>were equally susceptible to each fungus. Older larvae were less likely to die than young larvae (HR < 1). The effect of increase in fungus concentration on larval mortality was influenced by spore clumping. One day exposure to fungal spores was found to be equally effective as seven days exposure. In different exposure time treatments 0 - 4.9% of the total larvae, exposed to fungus, showed infection at either the pupal or adult stage. Mortality rate increased with increasing larval density and amount of available food.</p> <p>Conclusions</p> <p>This study shows that both fungus species have potential to kill mosquitoes in the larval stage, and that mortality rate depends on fungus species itself, larval stage targeted, larval density and amount of nutrients available to the larvae. Increasing the concentration of fungal spores or reducing the exposure time to spores did not show a proportional increase and decrease in mortality rate, respectively, because the spores clumped together. As a result spores did not provide uniform coverage over space and time. It is, therefore, necessary to develop a formulation that allows the spores to spread over the water surface. Apart from formulation appropriate delivery methods are also necessary to avoid exposing non-target organisms to fungus.</p

Egg hatching, larval movement and larval survival of the malaria vector Anopheles gambiae in desiccating habitats

BACKGROUND: Although the effects of rainfall on the population dynamics of the malaria vector Anopheles gambiae have been studied in great detail, the effects of dry periods on its survival remain less clear. METHODS: The effects of drying conditions were simulated by creating desiccated habitats, which consisted of trays filled with damp soil. Experiments were performed in these trays to (i) test the ability of An. gambiae sensu stricto eggs to hatch on damp soil and for larvae to reach an artificial breeding site at different distances of the site of hatching and (ii) to record survival of the four larval stages of An. gambiae s.s. when placed on damp soil. RESULTS: Eggs of An. gambiae s.s. hatched on damp soil and emerging larvae were capable of covering a distance of up to 10 cm to reach surface water enabling further development. However, proportions of larvae reaching the site decreased rapidly with increasing distance. First, second and third-instar larvae survived on damp soil for an estimated period of 64, 65 and 69 hrs, respectively. Fourth-instar larvae survived significantly longer and we estimated that the maximum survival time was 113 hrs. CONCLUSION: Short-term survival of aquatic stages of An. gambiae on wet soil may be important and adaptive when considering the transient nature of breeding sites of this species in sub-Saharan Africa. In addition, the results suggest that, for larval vector control methods to be effective, habitats should remain drained for at least 5 days to kill all larvae (e.g. in rice fields) and habitats that recently dried up should be treated as well, if larvicidal agents are applied

The entomopathogenic fungus Beauveria bassiana reduces instantaneous blood feeding in wild multi-insecticide-resistant Culex quinquefasciatus mosquitoes in Benin, West Africa.

BACKGROUND: Mosquito-borne diseases are still a major health risk in many developing countries, and the emergence of multi-insecticide-resistant mosquitoes is threatening the future of vector control. Therefore, new tools that can manage resistant mosquitoes are required. Laboratory studies show that entomopathogenic fungi can kill insecticide-resistant malaria vectors but this needs to be verified in the field. METHODS: The present study investigated whether these fungi will be effective at infecting, killing and/or modifying the behaviour of wild multi-insecticide-resistant West African mosquitoes. The entomopathogenic fungi Metarhizium anisopliae and Beauveria bassiana were separately applied to white polyester window netting and used in combination with either a permethrin-treated or untreated bednet in an experimental hut trial. Untreated nets were used because we wanted to test the effect of fungus alone and in combination with an insecticide to examine any potential additive or synergistic effects. RESULTS: In total, 1125 female mosquitoes were collected during the hut trial, mainly Culex quinquefasciatus Say. Unfortunately, not enough wild Anopheles gambiae Giles were collected to allow the effect the fungi may have on this malaria vector to be analysed. None of the treatment combinations caused significantly increased mortality of Cx. quinquefasciatus when compared to the control hut. The only significant behaviour modification found was a reduction in blood feeding by Cx. quinquefasciatus, caused by the permethrin and B. bassiana treatments, although no additive effect was seen in the B. bassiana and permethrin combination treatment. Beauveria bassiana did not repel blood foraging mosquitoes either in the laboratory or field. CONCLUSIONS: This is the first time that an entomopathogenic fungus has been shown to reduce blood feeding of wild mosquitoes. This behaviour modification indicates that B. bassiana could potentially be a new mosquito control tool effective at reducing disease transmission, although further field work in areas with filariasis transmission should be carried out to verify this. In addition, work targeting malaria vector mosquitoes should be carried out to see if these mosquitoes manifest the same behaviour modification after infection with B. bassiana conidia

Anopheline and culicine mosquitoes are not repelled by surfaces treated with the entomopathogenic fungi Metarhizium anisopliae and Beauveria bassiana

ABSTRACT:\ud \ud BACKGROUND\ud \ud Entomopathogenic fungi, Metarhizium anisopliae and Beauveria bassiana, are promising bio-pesticides for application against adult malaria mosquito vectors. An understanding of the behavioural responses of mosquitoes towards these fungi is necessary to guide development of fungi beyond the 'proof of concept' stage and to design suitable intervention tools.\ud \ud METHODS\ud \ud Here we tested whether oil-formulations of the two fungi could be detected and avoided by adult Anopheles gambiae s.s., Anopheles arabiensis and Culex quinquefasciatus. The bioassays used a glass chamber divided into three compartments (each 250 × 250 × 250 mm): release, middle and stimulus compartments. Netting with or without fungus was fitted in front of the stimulus compartment. Mosquitoes were released and the proportion that entered the stimulus compartment was determined and compared between treatments. Treatments were untreated netting (control 1), netting with mineral oil (control 2) and fungal conidia formulated in mineral oil evaluated at three different dosages (2 × 1010, 4 × 1010 and 8 × 1010 conidia m-2).\ud \ud RESULTS\ud \ud Neither fungal strain was repellent as the mean proportion of mosquitoes collected in the stimulus compartment did not differ between experiments with surfaces treated with and without fungus regardless of the fungal isolate and mosquito species tested.\ud \ud CONCLUSION\ud \ud Our results indicate that mineral-oil formulations of M. anisopliae and B. bassiana were not repellent against the mosquito species tested. Therefore, both fungi are suitable candidates for the further development of tools that aim to control host-seeking or resting mosquitoes using entomopathogenic fungi

First report of the infection of insecticide-resistant malaria vector mosquitoes with an entomopathogenic fungus under field conditions

BACKGROUND: Insecticide-resistant mosquitoes are compromising the ability of current mosquito control tools to control malaria vectors. A proposed new approach for mosquito control is to use entomopathogenic fungi. These fungi have been shown to be lethal to both insecticide-susceptible and insecticide-resistant mosquitoes under laboratory conditions. The goal of this study was to see whether entomopathogenic fungi could be used to infect insecticide-resistant malaria vectors under field conditions, and to see whether the virulence and viability of the fungal conidia decreased after exposure to ambient African field conditions. METHODS: This study used the fungus Beauveria bassiana to infect the insecticide-resistant malaria vector Anopheles gambiae s.s (Diptera: Culicidae) VKPER laboratory colony strain. Fungal conidia were applied to polyester netting and kept under West African field conditions for varying periods of time. The virulence of the fungal-treated netting was tested 1, 3 and 5 days after net application by exposing An. gambiae s.s. VKPER mosquitoes in WHO cone bioassays carried out under field conditions. In addition, the viability of B. bassiana conidia was measured after up to 20 days exposure to field conditions. RESULTS: The results show that B. bassiana infection caused significantly increased mortality with the daily risk of dying being increased by 2.5 × for the fungus-exposed mosquitoes compared to the control mosquitoes. However, the virulence of the B. bassiana conidia decreased with increasing time spent exposed to the field conditions, the older the treatment on the net, the lower the fungus-induced mortality rate. This is likely to be due to the climate because laboratory trials found no such decline within the same trial time period. Conidial viability also decreased with increasing exposure to the net and natural abiotic environmental conditions. After 20 days field exposure the conidial viability was 30%, but the viability of control conidia not exposed to the net or field conditions was 79%. CONCLUSIONS: This work shows promise for the use of B. bassiana fungal conidia against insecticide-resistant mosquitoes in the field, but further work is required to examine the role of environmental conditions on fungal virulence and viability with a view to eventually making the fungal conidia delivery system more able to withstand the ambient African climate

Exploiting the behaviour of wild malaria vectors to achieve high infection with fungal biocontrol agents

BACKGROUND\ud \ud Control of mosquitoes that transmit malaria has been the mainstay in the fight against the disease, but alternative methods are required in view of emerging insecticide resistance. Entomopathogenic fungi are candidate alternatives, but to date, few trials have translated the use of these agents to field-based evaluations of their actual impact on mosquito survival and malaria risk. Mineral oil-formulations of the entomopathogenic fungi Metarhizium anisopliae and Beauveria bassiana were applied using five different techniques that each exploited the behaviour of malaria mosquitoes when entering, host-seeking or resting in experimental huts in a malaria endemic area of rural Tanzania.\ud \ud RESULTS\ud \ud Survival of mosquitoes was reduced by 39-57% relative to controls after forcing upward house-entry of mosquitoes through fungus treated baffles attached to the eaves or after application of fungus-treated surfaces around an occupied bed net (bed net strip design). Moreover, 68 to 76% of the treatment mosquitoes showed fungal growth and thus had sufficient contact with fungus treated surfaces. A population dynamic model of malaria-mosquito interactions shows that these infection rates reduce malaria transmission by 75-80% due to the effect of fungal infection on adult mortality alone. The model also demonstrated that even if a high proportion of the mosquitoes exhibits outdoor biting behaviour, malaria transmission was still significantly reduced.\ud \ud CONCLUSIONS\ud \ud Entomopathogenic fungi strongly affect mosquito survival and have a high predicted impact on malaria transmission. These entomopathogens represent a viable alternative for malaria control, especially if they are used as part of an integrated vector management strategy

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

Pyrethroid resistance in Anopheles gambiae leads to increased susceptibility to the entomopathogenic fungi Metarhizium anisopliae and Beauveria bassiana

<p>Abstract</p> <p>Background</p> <p>Entomopathogenic fungi are being investigated as a new mosquito control tool because insecticide resistance is preventing successful mosquito control in many countries, and new methods are required that can target insecticide-resistant malaria vectors. Although laboratory studies have previously examined the effects of entomopathogenic fungi against adult mosquitoes, most application methods used cannot be readily deployed in the field. Because the fungi are biological organisms it is important to test potential field application methods that will not adversely affect them. The two objectives of this study were to investigate any differences in fungal susceptibility between an insecticide-resistant and insecticide-susceptible strain of <it>Anopheles gambiae sensu stricto</it>, and to test a potential field application method with respect to the viability and virulence of two fungal species</p> <p>Methods</p> <p>Pieces of white polyester netting were dipped in <it>Metarhizium anisopliae </it>ICIPE-30 or <it>Beauveria bassiana </it>IMI391510 mineral oil suspensions. These were kept at 27 ± 1°C, 80 ± 10% RH and the viability of the fungal conidia was recorded at different time points. Tube bioassays were used to infect insecticide-resistant (VKPER) and insecticide-susceptible (SKK) strains of <it>An. gambiae s.s</it>., and survival analysis was used to determine effects of mosquito strain, fungus species or time since fungal treatment of the net.</p> <p>Results</p> <p>The resistant VKPER strain was significantly more susceptible to fungal infection than the insecticide-susceptible SKK strain. Furthermore, <it>B. bassiana </it>was significantly more virulent than <it>M. anisopliae </it>for both mosquito strains, although this may be linked to the different viabilities of these fungal species. The viability of both fungal species decreased significantly one day after application onto polyester netting when compared to the viability of conidia remaining in suspension.</p> <p>Conclusions</p> <p>The insecticide-resistant mosquito strain was susceptible to both species of fungus indicating that entomopathogenic fungi can be used in resistance management and integrated vector management programmes to target insecticide-resistant mosquitoes. Although fungal viability significantly decreased when applied to the netting, the effectiveness of the fungal treatment at killing mosquitoes did not significantly deteriorate. Field trials over a longer trial period need to be carried out to verify whether polyester netting is a good candidate for operational use, and to see if wild insecticide-resistant mosquitoes are as susceptible to fungal infection as the VKPER strain.</p