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Fungal Pathogen Reduces Potential for Malaria Transmission

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Science  10 Jun 2005:
Vol. 308, Issue 5728, pp. 1638-1641
DOI: 10.1126/science.1108423

Abstract

Using a rodent malaria model, we found that exposure to surfaces treated with fungal entomopathogens following an infectious blood meal reduced the number of mosquitoes able to transmit malaria by a factor of about 80. Fungal infection, achieved through contact with both solid surfaces and netting for durations well within the typical post-feed resting periods, was sufficient to cause >90% mortality. Daily mortality rates escalated dramatically around the time of sporozoite maturation, and infected mosquitoes showed reduced propensity to blood feed. Residual sprays of fungal biopesticides might replace or supplement chemical insecticides for malaria control, particularly in areas of high insecticide resistance.

The use of pyrethroid insecticides on bednets or on walls and ceilings is the mainstay of malaria vector control. However, some forms of resistance are a threat to the long-term effectiveness of such measures (1, 2). With practical implementation of novel molecular interventions still years off (36), there is a pressing need for practical alternatives for malaria control (7).

Several studies have investigated the use of microbial agents for biological control of mosquitoes (see 810 for reviews). The most successful approach has been the use of microbial biopesticides, such as Bacillus thuringiensis, for control of the larval stages (8, 9). Here, we report the potential of fungal entomopathogens for indoor use against adult mosquitoes to reduce malaria transmission. Oil-based formulations of fungal entomopathogens are a relatively recent innovation that enables economically viable spore application in ultralow-volume sprays under a wide range of environmental conditions (11, 12). These formulations create an opportunity to apply fungal pathogens for use on indoor surfaces of houses or curtains where some malaria vector species rest after a blood meal or on bednets to which mosquitoes are attracted by the odor of the occupant. Fungal entomopathogens infect through external contact, with spores germinating and penetrating through the cuticle before proliferating in the hemocoel. Natural mosquito-mosquito transmission is unlikely, because this would require contact between an uninfected adult and a sporulating cadaver. In other pest-control contexts, contact with fungal spores in a spray residue has proved to be a highly efficient means of infecting insects (13, 14) because it does not rely on direct hit of the target and enables accumulation of high doses of pathogen over time through continued or repeat exposure.

Our experimental system comprised Anopheles stephensi and the rodent malaria Plasmodium chabaudi. Our first experiment was a basic mortality screen of eight Hyphomycetes fungal isolates from two common species, Beauveria bassiana and Metarhizium anisopliae. The specific isolates were selected based on their known biological activity, that is, either known generalists or those originally isolated from dipteran hosts (15). The basic assay technique exposed blood-fed adult female mosquitoes to oil-based spray residues inside replicated cardboard pots (16). Mosquitoes were introduced 24 hours after the pots were sprayed and, for this initial screen, remained in the sprayed pots for 14 days. The eight fungal isolates varied in virulence to A. stephensi (Fig. 1 and table S2), six of which produced >80% mortality by day 14, with >70% of the cadavers bearing sporulated fungi. High mortality by day 14 is encouraging because that is about the time taken for Plasmodium to develop from ingested gametocytes to infective sporozoites.

Fig. 1.

Cumulative proportional survival of adult A. stephensi after exposure to oil-based spray residues containing spores of different fungal entomopathogens. Data represent means ± SEM from four replicates, each containing 40 female A. stephensi. Details on the fungal isolates are provided in (16).

After initial screening, we selected the B. bassiana isolate IMI 391510 for further evaluation. Although it was not the most virulent isolate of those tested, this isolate is used in an existing agricultural biopesticide product, and the provision of a registration dossier would accelerate progress to field-scale evaluation.

We assessed the impact of IMI 391510 by direct topical application against A. stephensi and as a spray residue on cage mesh, partially simulating treatment of bednets (but with a longer exposure than would occur with free-flying mosquitoes approaching an occupied bednet). The treatments killed 91% and 93% of mosquitoes, respectively, by day 14. This compared with 38% mortality in the control mosquitoes sprayed with oil only. Significant differences in median survival times were seen between treatments: controls, >14 days; topical application, 8.0 days; treated netting, 7.0 days (log-rank statistic = 5.43, P = 0.02).

Anopheline mosquitoes tend to rest on structures such as walls and ceilings for less than 24 hours after a blood meal (17, 18). Thus, we simulated short-term exposures after repeat blood feeds by exposing adult A. stephensi to the fungal spray residue for various combinations of 6 hours at 3-day intervals [3 days representing the approximate length of the feeding cycle (19)]. No difference was detected between any of the various control batches at the end of the assessment period (table S3, F4,10 = 0.24, P = 0.91). For the fungal treatments, there was a significant difference in survival between exposure regimes 14 days after their first exposure (F4,10 = 6.0, P = 0.01), although this was entirely due to the more rapid mortality of the day 0 regime compared with the day 12 regime (Tukey HSD, P = 0.005). No other comparisons revealed significant differences. Thus, limiting the time that mosquitoes were exposed to the spray residue did not alter overall survival compared with lifetime exposure shown in Fig. 1. For example, the day 12 treatment, with just one 6-hour exposure to a 12-day-old spray residue, showed 89% mortality 14 days after exposure (Fig. 2).

Fig. 2.

Cumulative proportional survival of adult A. stephensi after exposure to oil-based spray residues containing the fungal pathogen B. bassiana, isolate IMI 391510. The lines indicate temporal exposure treatments. For the day 0 treatment, blood-fed A. stephensi were introduced to pots sprayed with B. bassiana for 6 hours and then removed to an unsprayed pot. On day 3, these mosquitoes were returned to the original sprayed pot for a further 6 hours and then removed again. This continued every three days until day 12. The remaining treatments were staggered such that the day 3 treatments were not introduced to a sprayed pot until day 3 and were subsequently exposed every 3 days for 6 hours until day 12. Days 6, 9, and 12 treatments were first introduced to sprayed pots on those days, and subsequently every 3 days as above. The day 12 treatment was, therefore, exposed only once, for 6 hours, to a pot sprayed 12 days previously. Matching controls for these treatments were moved between blank oil-sprayed pots and unsprayed pots at equivalent frequencies and exposure periods. Data for the respective treatments represent means ± SEM from four replicates, each containing ∼40 female A. stephensi. The control line shows the control data for the day 0 exposure treatment (other control lines have been omitted for clarity because there were no significant differences between control treatments).

To assess the potential of this biopesticide to reduce malaria transmission, we tested A. stephensi infected with the CW clone of the rodent malaria Plasmodium chabaudi (16) (Malaria), compared with uninfected controls (Control), a B. bassiana only treatment (Beauveria), and a treatment combining P. chabaudi and B. bassiana (Beauveria+Malaria). Sub-samples of mosquitoes were dissected to estimate Plasmodium prevalence at the oocyst (day 7) and sporozoite (day 14) stages.

Median survival times for the Control and Malaria treatments were not significantly different from one another (both treatments >14 days; log-rank statistic = 0.06, P = 0.81), nor was there a difference between the two fungal treatments (Beauveria and Beauveria+Malaria both 9.0 days; log-rank statistic = 0.34, P = 0.56). By day 14, 90% of the mosquitoes in the Beauveria treatment had died, with 95% mortality in the Beauveria+Malaria treatment (Fig. 3A). From day 11, there was a marked escalation in the mortality rate in the Beauveria+Malaria treatment, such that by day 14, daily mortality rate was about 65 times as high as that for the malaria-only treatment (Fig. 3B).

Fig. 3.

Effect of exposure to a biopesticide spray residue on the capacity of A. stephensi to transmit malaria. Mosquitoes were given a malaria-infected or uninfected blood meal and then, for B. bassiana treatments, the mosquitoes were immediately exposed to fungal sprayed pots for 6 hours and then subsequently exposed for 6 more hours to fungi every 3 days up to day 12. Control and Malaria treatments were exposed in the same manner to pots sprayed with oil alone. (A) Cumulative proportional survival (means ± SEM) of adult A. stephensi in the four experimental treatments. Each treatment was replicated six times with ∼50 A. stephensi per replicate. (B) Mean daily percent mortality rate from (A) above. Daily mortality was calculated from the number dead on a particular day divided by the number alive at the start of that day. Standard errors represent the variation in mean mortality rate between replicates in each treatment. (C) Estimated mean proportion (± SEM) of the starting population of mosquitoes in the Malaria and Malaria+Beauveria treatments positive for oocysts at day 7 and sporozoites at day 14 after an infectious blood meal.

There were no differences in the concentrations of gametocytes in mice fed the two malaria treatments (F1,11 = 0.19, P = 0.67). Similarly, oocyst prevalence (Fig. 3C) and mean number of oocysts per midgut did not differ (F1,11 = 0.55, P = 0.46, and F1,11 = 2.35, P = 0.16, respectively). However, on day 14, 35% (109/310) of the mosquitoes remaining in the Malaria treatment were found to be sporozoite positive, compared with only 8% (1/12) in the Beauveria+Malaria treatment. Taking into account the difference in survival to day 14 of these two groups (Fig. 3A), this equates to 31% (109/352) of the initial mosquitoes in the Malaria treatment group being alive and potentially able to transmit at day 14, compared with only 0.4% (1/256) in the Beauveria+Malaria treatment (Fishers exact test, P < 0.001). Thus, fungal exposure led to a reduction of transmission risk by a factor of about 80. This result is supported by a further experiment with a different P. chabaudi clone (20).

Our results demonstrate the potential of oil-based formulations of fungal entomopathogens to reduce malaria transmission by reducing adult mosquito survival and altering Plasmodium survival/maturation in the mosquitoes. In addition, we have found that between days 8 and 14, fungal infection interferes with the ability of A. stephensi to take a blood meal, likely reducing transmission potential still further (20, 21). Mosquitoes can be infected by direct contact with spray droplets or by contact with spray residues on treated surfaces and netting, with 6 hours exposure sufficient to cause high levels of infection; this is well within the post–blood feed resting period for the majority of Anopheles malaria vector species (17, 18). We identified a range of isolates causing ≥ 90% mortality within 12 to 15 days (2224). Further screening is likely to reveal more virulent isolates, but it is also likely that vector survival in the wild is less than under laboratory conditions (22). Furthermore, mosquitoes cannot transmit sporozoites until about 2 weeks after an infectious blood feed, and rapid killing of the mosquito is not necessary for reducing malaria transmission. As emphasized by MacDonald (25) in justifying the possibility of malaria eradication by indoor residual spraying of insecticide, even limited changes in daily probability of survival of mosquitoes can have a large impact on the prospects of survival through the time required for Plasmodium maturation to the infective stage; hence, the substantial reduction in sporozoite-positive mosquitoes and the escalating daily mortality rate that we observe due to fungal infection are highly significant in terms of malaria control.

Fungal biopesticides are already registered for agricultural use alongside chemical insecticides in a number of African countries (12, 26). Transferring use to mosquito targets could (subject to further safety testing and appropriate registration) be rapid, with biopesticide products slotting readily into existing chemical application methodologies and strategies (22). As part of an integrated strategy, they could be used to respond to, or avert, the emergence of serious levels of insecticide resistance. Development of resistance against fungal pathogens has not been reported for insects, but even if resistance does occur, cross-resistance with chemical insecticides seems extremely unlikely. In the longer term, there would seem to be additional promise for using residual sprays of fungal pathogens for novel paratransgenic approaches to deliver toxins or effector molecules that block sporogony within the vector (e.g., see 27, 28). Unlike malaria control by genetic modification of mosquitoes, the fitness of biopesticide transgenes could be quite low and, because secondary transfer of fungi from mosquitoes is very unlikely, fungal transgenes would be much easier to control than mosquito transgenes.

Supporting Online Material

www.sciencemag.org/cgi/content/full/308/5728/1638/DC1

Materials and Methods

Tables S1 to S3

References and Notes

View Abstract

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