Some of the most important human diseases are borne by mosquitoes including malaria (Anopheles), filariasis (Culex, Mansonia and some Anopheles spp.), viral encephalitides (Culex), dengue and yellow fever (principally Aedes aegypti). An estimated 2 billion people live in areas where these diseases are endemic. The burden is heaviest in sub-Saharan Africa, where ~200 million cases of malaria are reported annually and many children succumb1. Emergence and spread of resistance to pyrethroids, organophosphates and carbamates is a particular threat, as most disease control programs rely heavily on these broad-spectrum chemical insecticides to reduce vector populations2,3,4,5. In recent years, anopheline malaria vectors in sub-Saharan Africa have gained sufficient resistance to render chemical insecticides largely ineffective5, 6.

The chemical insecticides used in mosquito control programs are directed at neuronal voltage-gated sodium (Na V ) channels, as are many insect-specific neurotoxins derived from arthropods, including the scorpion toxin AaIT that has been approved for release in several countries7. A recombinant strain of the entomopathogenic fungus Metarhizium anisopliae expressing AaIT killed Ae. aegypti mosquitoes faster and at lower spore doses than wild-type (WT) fungus8. Since AaIT and pyrethroids bind to different sites on insect Na V channels and channel mutations that confer resistance to pyrethroids actually increase binding of AaIT, the use of both pyrethroids and AaIT should mitigate against resistance acquisition9. Furthermore, combining toxins with different targets, such as voltage-gated sodium, potassium (K V ) and calcium (Ca V ) channels, could provide useful additive or synergistic effects and minimize the potential for cross-resistance. Ca V and K V channels are previously unexploited insecticide targets, reducing the likelihood of pre-existing resistance.

We compared the efficacy against Anopheles gambiae of recombinant Metarhizium pingshaense co-expressing green fluorescent protein (GFP) and one of several arthropod-derived toxins with different modes of action: AaIT, the Na V channel inhibitor μ-CUTX-As1a (As1a)10, the K Ca /Ca V blocker ω/κ-hexatoxin-Hv1a (Hybrid), the Ca V channel blocker ω-hexatoxin-Hv1a (Hv1a)11, and U 1 -AGTX-Ta1a (Ta1a), a weaponized insect hormone which has an unknown neuronal target12. With the exception of AaIT, all these toxins are derived from spider venom. To express the transgenes we used the Mcl1 promoter that targets expression to the hemocoel of infected insects8, 13, 14.

We previously determined that hyphal bodies appear in the hemolymph 2–3 days post infection and that Mcl1 expression commences within 20 min of contact with hemolymph15.

Transformants were selected by GFP expression and PCR/sequencing confirmation of the toxin gene, and initially bioassayed by spraying spore suspensions (~1 conidium/mosquito; ~3 conidia/mosquito; and ~6 conidia/mosquito) onto cold (4 °C) anesthetized adult female An. gambiae. Although they have different molecular targets (Na V , Ca V and K Ca channels), each toxin significantly (p < 0.05) improved the median lethal time (LT50) with infection loads of 3 or more spores (Fig. 1a,b, and Table 1) compared to WT, and therefore they constitute a potential arsenal that could be rotated and/or used in combination in a mosquito control program aimed at mitigating resistance. Hybrid, also known as VersitudeTM, was chosen for further study as the US EPA has already approved it for use as a stand-alone insect control agent. Since prevention of transmission of malaria is of primary importance in assessing a mosquito control technology, we took a holistic view of the life cycle of the parasite to determine if pre-lethal effects could contribute to Metarhizium preventing the spread of malaria.

Figure 1 (a) LT50 values for mosquitoes treated with 1 × 105, 1 × 106 and 1 × 107 M. pingshaense conidia/ml suspensions resulting in ~1, ~3 and ~6 conidia/mosquito, respectively, of M. pingshaense WT or M. pingshaense expressing Hybrid, AaIT or both AaIT and Hybrid. LC50 values are reported along the x-axis as the inverse of the estimated spore inoculum. Lettering represents statistical differences (p < 0.05) based on a log-rank test comparing the Kaplan-Meier survival curves. The LC50 dose for untreated mosquitoes was fixed at zero and reported for all spore concentrations for comparison. (b) Schematic representing infection timing (beige) and LT50s of mosquitoes treated with conidial suspensions and exposed to cloth impregnated with toxin expressing strains or WT. Lettering groups toxins by statistical significance (p < 0.05) based on a log-rank test comparing the Kaplan-Meier survival curves. Full size image

Table 1 L﻿T50s and LC50s (day 5) for Anopheles gambiae treated with Metarhizium pingshaense strains expressing arthropod-derived toxins alone and stacked (Hybrid/AaIT) compared with wild-type (WT) and a control (0.01% Tween). Full size table

We tested fungal efficacy against three anopheline vectors of human malaria: two wild-caught, insecticide-resistant species (An. coluzzii and An. gambiae s.s.) and one lab-reared, insecticide-susceptible species (An. gambiae Kisumu An. kisumu). We applied the fungus to sheets, as fungal-impregnated sheets hung in houses provide a resting area for mosquitoes that have taken blood meals16. Most malaria vectors prefer to blood feed and rest inside houses, thus maximizing the likelihood of fungal contact and infection17. We exposed mosquito populations for one hour to sheets impregnated with either: 1) Met-RFP, a strain with WT virulence expressing red fluorescent protein (RFP) as a marker, and 2) Hybrid toxin expressing fungus (Met-Hybrid) co-expressing GFP. We found mosquitoes exposed to the cloth for one hour in a WHO mosquito bioassay tube picked up an average of 129 ± 18 spores, a sufficient dose to kill all mosquitoes exposed to each fungal treatment. Mortality was monitored over 14 days and compared to uninfected controls.

Insecticide (Knockdown) resistance (kdr) in each mosquito population was quantified using PCR. The kdr mutation reduces sensitivity to DDT and pyrethroids and is the most prevalent form of insecticide resistance for West African mosquitoes6. The levels of kdr resistance in wild-caught An. coluzzii and An. gambiae s.s. were 98.3% and 92.9%, respectively (Supplementary Table 1). An. gambiae Kisumu mosquitoes are an established laboratory population of mosquitoes with stable susceptibility to insecticides, so their resistance was not tested.

Overall, insecticide resistance did not alter the susceptibility of the three mosquito species to Met-RFP and Met-Hybrid (Fig. 2). Within 2.5 days post-infection, mosquitoes exposed to Met-Hybrid were dying faster than those exposed to Met-RFP. LT50 (LT80) values for Met-Hybrid and Met-RFP were 4.14 ± 0.16 (5.47 ± 0.25) and 6.18 ± 0.14 (7.71 ± 0.16) days, respectively (mean ± standard error is reported). Fluorescent Metarhizium mycosis was observed on fungus-exposed cadavers, confirming mortality due to treatment. The number of mosquitoes surviving in the uninfected control group never dropped below 84.4% (Fig. 2).

Figure 2 Survival following WHO tube exposure: these three graphs represent survival curves for RFP expressing Metarhizium (Met-RFP) and Hybrid toxin-expressing Metarhizium (Met-Hybrid) against two wild-caught, insecticide-resistant (An. coluzzii and An. gambiae s.s.) and one lab-reared (An. gambiae Kisumu) human malaria vector mosquitoes. There are no significant differences in time to kill insecticide resistant and susceptible strains, but Met-Hybrid is more effective on all mosquito strains. Full size image

The wild-type M. pingshaense strain has a narrow host range. To investigate if expressing Hybrid toxin impacts host specificity, we assayed transformants against honeybees (Apis mellifera adansonii) as a representative local pollinator. Honeybees were infected with black cloth impregnated with ~2 × 106 conidia/cm2 in WHO tubes and spore dose was estimated using our selective media protocol. Met-RFP and Met-Hybrid did not kill any honeybees in the two weeks they were observed following infection (there was no difference in mortality rates between fungal exposed and unexposed bees, and no fungal emergence was observed on any bee cadaver). This is in agreement with previous studies indicating that the expression of insect toxins did not increase the host range of M. acridum 13. Interestingly, honeybees picked up significantly fewer spores (80 ± 3 spores, p < 0.05) than mosquitoes under the same conditions, despite the much smaller size of mosquitoes. Spraying bees with 1 × 108 spores/ml also failed to cause mortality.

Willingness and ability to blood feed were also tested, as even small changes in coordination could potentially interfere with behavior and disease transmission. Host-seeking (blood feeding) interest was quantified as the percentage of the mosquito population choosing the chamber closest to the host in a guinea pig choice chamber (Fig. 3a,b and Supplementary Figure 1a,b). At one day post-infection, 94.3% of untreated (controls) and treated (Met-RFP or Met-Hybrid) mosquitoes flew toward the blood source with no significant differences between treatments. The willingness of mosquitoes in the control group to blood feed did not change over the course of the experiment. In contrast, significantly (p < 0.05) fewer (56.9%) mosquitoes treated with Met-Hybrid flew to the blood source on day 3 as compared to Met-RFP (82.1%). By day 4 the number of Met-Hybrid infected mosquitoes in the guinea pig choice chamber (38.9%) was not significantly different than the 30% entering the chamber in the absence of a guinea pig. Met-RFP infected mosquitoes only became significantly (p < 0.01) less responsive than uninfected controls on day 4 (Fig. 3a). These results suggest a pre-lethal advantage to using transgenic fungi for mosquito control.

Figure 3 (a) Impact of fungal infection on blood-feeding at 1–5 days post-infection with either Met-Hybrid or Met-RFP. Mosquitoes were placed in a choice chamber with the guinea pig host outside of the chamber and just out of reach (Supplementary Figure 1a,b). Host-seeking (blood feeding) interest was quantified as the percentage of the mosquito population choosing the chamber closest to the host. The symbol “*” denotes no significant differences in mosquito choices with or without a host: 30 ± 3.05% of the mosquitoes chose the “host” chamber eve﻿n in the absence of a host. (b) Mortality and transmission of mosquitoes exposed to fungus, the light area represents the percent survival of mosquitoes for each treatment, while the dark area shows the proportion of mosquitoes in each treatment that are alive and would seek a host to blood feed (capable of malaria transmission). The upper dashed line represents the LT50 while the lower dashed line represents the 80% control threshold suggested by the World Health Organization (WHO) for a successful vector control agent18. Full size image

With this information, we projected the measured proportion of mosquitoes interested in blood feeding onto the mortality of mosquitoes to identify the fraction capable of malaria transmission (Fig. 3b). In contrast to Met-RFP, by day 5, Met-Hybrid infected mosquitoes passed the threshold in both metrics (8.35% malaria transmission and >80% mortality) for the 80% control threshold suggested by the World Health Organization (WHO) for a successful vector control agent18.

Like Hybrid, AaIT is US EPA approved. As AaIT targets Na V channels and the Hybrid toxin targets Ca V and K Ca channels, we examined whether their different sites of action produce synergistic effects. We found a clear benefit in terms of both effective spore doses and speed of kill in expressing both Hybrid and AaIT in a single strain (Met-Hybrid/AaIT) (Table 1). Mosquitoes sprayed with 1 × 105 spores/mL (~1 spore/mosquito) of Met-Hybrid/AaIT, Met-Hybrid or WT had LT50s of 8.90 ± 1.09, 9.08 ± 0.44, and 9.49 ± 0.60 days, respectively, and the differences between them fell short of significance (Fig. 1a and Table 1). At this low spore dose, Met-Hybrid/AaIT, Met-Hybrid and WT significantly (p < 0.05) reduced lifespan compared to untreated mosquitoes (LT50 of 11.4 ± 0.52 days), and high variation in Met-AaIT bioassays resulted in no statistical difference from untreated mosquitoes. However, increasing the spore doses reduced the LT50s of toxin expressing strains to a greater extent than WT, consistent with the toxins having threshold effects (Fig. 1a,b). Comparing toxins, mosquitoes sprayed with 1 × 106 Met-Hybrid/AaIT spores/ml (~3 spores/mosquito) achieved an LT50 of 5.30 ± 0.42 days that was significantly faster than the 6.39 ± 0.35 and 7.20 ± 0.84 days for Met-Hybrid and Met-AaIT alone. The Tabashnik synergism equation19 confirmed synergistic interactions as early as 4.5 days post-infection, and at 5 days it takes less than half the dosage of Met-Hybrid/AaIT to kill mosquitoes at the same rate as either Met-Hybrid or Met-AaIT applied alone (Supplementary Table 2). Such synergies suggest that optimizing the overall efficacy of the control strategy will require multiple transgenes, and a toxin arsenal could reduce effective conidial doses thereby reducing end-user costs (Fig. 1a and Table 1).

The spray infection method successfully probes efficacy differences between transgenes by delivering low spore doses, but contributes to variation in LT50 values because some mosquitoes escape infection at these low dosages. We estimated that inocula containing 1 × 107, 1 × 106 and 1 × 105 spores/ml deliver at least one spore to 95%, 75%, and 50% of the mosquitoes sprayed, respectively. As mosquito mortality increases with inoculum load, we plotted spore counts per mosquito versus the probability of death derived from our bioassays for the WT and transgenic strains (Met-AaIT, Met-Hybrid, and Met-Hybrid/AaIT) (Fig. 4). Together, these data revealed that the lethal dose per mosquito at which 100% of mosquitoes die (LD100) is 10 spores, 8 spores, 7 spores, and 6 spores for WT, Met-AaIT, Met-Hybrid and Met-Hybrid/AaIT, respectively. The results explain the incomplete mortality seen even with our highest spore concentration (1 × 107), and the complete mortality seen in mosquitoes treated through contact with oil-impregnated cloth, which delivers far more spores to every mosquito. This bodes well for oil-impregnated cloth as a delivery system for entomopathogenic fungal spores and for the translatability of spray method results to the field.

Figure 4 The number of spores infecting each mosquito after spraying with three different spore concentrations (1 × 105, 1 × 106, and 1 × 107 spores/mL in 0.01% Tween80) plotted against the probability of death. The mean number of spores delivered with each suspension is marked with a blue crossbar. Assuming that mosquitoes with a higher dose are more likely to die, we calculated the chance of death for each mosquito based on our survival data for each treatment at each concentration. Mosquitoes with an estimated 100% chance of death are colored in red, and 0–99% is represented with a green to orange gradient. The red dashed line indicates the estimated LD100 in each treatment (10 spores for WT, 8 spores for AaIT, 7 spores for Hybrid and 6 spores for Hybrid/AaIT). Full size image

In conclusion, fungi can be genetically modified to strategically maximize their success as biocontrol agents13, 20. When their impact on malaria transmission is considered, transgenic fungi applied on sheets meet WHO standards for effective control of malaria within 5 days post-exposure, indicating that the inclusion of transgenic Metarhizium in pre-existing control efforts would effectively decrease malaria transmission. Some female mosquitoes infected with transgenic fungi will lay eggs 3–4 days after a bloodmeal, so infected mosquitoes may still pass their genes onto the next generation, but infection will prevent further oviposition. Our study emphasizes the need to consider the effect of fungi on blood feeding for modeling of existing mosquito control techniques in conjunction with transgenic Metarhizium 21.