George et al. Malaria Journal 2011, 10:219 http://www.malariajournal.com/content/10/1/219 RESEARCH Open Access Reduction in host-finding behaviour in fungus- infected mosquitoes is correlated with reduction in olfactory receptor neuron responsiveness Justin George1, Simon Blanford1,2, Michael J Domingue1, Matthew B Thomas1,2, Andrew F Read1,2 and Thomas C Baker1* Abstract Background: Chemical insecticides against mosquitoes are a major component of malaria control worldwide. Fungal entomopathogens formulated as biopesticides and applied as insecticide residual sprays could augment current control strategies and mitigate the evolution of resistance to chemical-based insecticides. Methods: Anopheles stephensi mosquitoes were exposed to Beauveria bassiana or Metarhizium acridum fungal spores and sub-lethal effects of exposure to fungal infection were studied, especially the potential for reductions in feeding and host location behaviours related to olfaction. Electrophysiological techniques, such as electroantennogram, electropalpogram and single sensillum recording techniques were then employed to investigate how fungal exposure affected the olfactory responses in mosquitoes. Results: Exposure to B. bassiana caused significant mortality and reduced the propensity of mosquitoes to respond and fly to a feeding stimulus. Exposure to M. acridum spores induced a similar decline in feeding propensity, albeit more slowly than B. bassiana exposure. Reduced host-seeking responses following fungal exposure corresponded to reduced olfactory neuron responsiveness in both antennal electroantennogram and maxillary palp electropalpogram recordings. Single cell recordings from neurons on the palps confirmed that fungal-exposed behavioural non-responders exhibited significantly impaired responsiveness of neurons tuned specifically to 1- octen-3-ol and to a lesser degree, to CO2. Conclusions: Fungal infection reduces the responsiveness of mosquitoes to host odour cues, both behaviourally and neuronally. These pre-lethal effects are likely to synergize with fungal-induced mortality to further reduce the capacity of mosquito populations exposed to fungal biopesticides to transmit malaria. Background alternative is fungal entomopathogens formulated as Chemical insecticides targeting adult female mosquito biopesticides. A number of studies have demonstrated vectors have been one of the most successful strategies that residual contact with substrates treated with fungal employed for malaria control [1]. However, the effective- sprays can lead to high levels of infection, reducing sur- ness and sustainability of insecticide-based interventions, vivalofarangeofmosquitovectorspecies,including such as indoor residual sprays (IRS) and insecticide-trea- insecticide resistant phenotypes [9-15]. Since it takes ted nets (ITNs), is being undermined by evolution of around two weeks for the malaria parasite to develop insecticide resistance [1-8]. Accordingly, there is now a within a mosquito following a blood feed, the life-short- pressing need for novel control tools including alterna- ening effects of fungal infection can dramatically reduce tive, non-chemical approaches [8]. One promising malaria transmission potential [14-17]. In addition, fun- gal pathogens cause a range of pre- or sub-lethal effects in other arthropods including reductions in feeding * Correspondence: [email protected] [18-21], fecundity [22-26], flight performance [27,28] 1Department of Entomology, Pennsylvania State University, University Park, PA 16802, USA and predator avoidance [29,30], together with elevation Full list of author information is available at the end of the article © 2011 George et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. George et al. Malaria Journal 2011, 10:219 Page 2 of 13 http://www.malariajournal.com/content/10/1/219 of metabolic rate [31] and alteration in development were fed Liquifry for five days and then on Tetrafin fish [32]. For malaria control, reductions in feeding propen- flakes. From approximately two weeks after egg hatch, sity could be particularly important since the parasite pupae were collected daily and placed in emergence can only be transmitted during a blood feed and if feed- cages. The adults that emerged were fed ad libitum on ing is reduced, it does not necessarily matter whether a10%glucosesolution.Allexperimentsusedthreeto the mosquito is alive or not. To date, fungal infection five day old adult female mosquitoes. has been shown to reduce feeding in Anopheles, Culex and Aedes mosquitoes [14,33,34]. The proximate Fungal isolates, formulation and application mechanisms associated with such changes, however, Two species of entomopathogenic fungi were used that remain unexplored. are known to vary in their impact on mosquitoes: Beau- Mosquitoes have a highly developed olfactory system veria bassiana isolate IMI39150, which has been investi- that uses specialized olfactory receptor neurons (ORNs) gated in a number of previous publications and shown to detect the odours emanating from their hosts [35]. to have a marked impact on mosquito survival and per- They also use olfactory information to locate other food formance [9,10,14,39], and Metarhizium acridum (for- sources, mates, and oviposition sites. The peripheral merly Metarhizium anisopliae var acridum [40] isolate olfactory organs that detect the olfactory cues are the IMI330189, which is a relatively specific pathogen of antennae and maxillary palps. In Aedes aegypti and Ano- grasshoppers [41] but has been shown to infect mosqui- pheles gambiae, maxillary palps harbour a single mor- toes although with only moderate virulence [39]. Both phological type of chemosensory sensillum, the capitate isolates were formulated separately in a mix of mineral peg, which is innervated by three ORNs. One of these oils (80% Isopar M:20% Ondina 22) and their concentra- 9 -1 threeORNsishighlyresponsivetoCO2.Thesecond tions adjusted to give 1 × 10 spores ml . ORN is most sensitive to 1-octen-3-ol, which is a major Application and mosquito exposure were as described component of human and other vertebrate volatiles by Bell et al [39]. In brief, spray applications of the [36,37]. In An. gambiae, this ORN expresses on its den- spore formulation were applied to the insides of waxed drites the odorant receptor (OR) called AgOR8, which is cardboard cups using a hand-held artist’sairbrushand activated by 1-octen-3-ol [38]. The third ORN is tuned allowed to dry. The spray method delivers an estimated to a broad panel of odorants and expresses the OR 2×1010 spores/m2 of which approximately 2 × 108 named AgOR28 on its dendrite. Furthermore, An. gam- spores/m2 are actually deposited on the cup surface biae responds with a significant dose-dependent electro- [39]. Mosquitoes were introduced to these cups for six palpogram (EPG) response profile to 1-octen-3-ol hours resulting in a spore acquisition in the region of 2 [37,38]. ×104 spores/mosquito [39]. After exposure, mosquitoes This study combines simple behavioural assays with were aspirated into mesh cages, provided with an ad electroantennogram (EAG) and electropalpogram (EPG) libitum supply of 10% glucose, and housed in a con- recordings to examine possible olfaction effects of fungal trolled environment incubatorat26°Cand80%RH infection at the peripheral level. Single sensillum record- where they remained for the duration of the experiment. ings (SSRs) were used to investigate individual capitate Control mosquitoes were handled in exactly the same peg neuronal responses after fungal infection. Results way but were exposed to untreated cups. Each treatment show that in addition to survival, feeding propensity, had four replicate exposure cups per treatment with upwind flight behaviour and the olfactory responses are between 50 and 60 mosquitoes per replicate. After their all affected by fungal infection and depend on the fungal time in the cups, mosquitoes were pooled into a single species examined. The results provide one of the first large cage according to their respective treatment, demonstrations of a pathogen having a functional allowed to mix together and then redistributed into four impact on insect olfaction. This could have important replicate cages (~ 30 insects per cage) for survival implications for the effectiveness of fungi for vector assessment. Mortality was monitored daily and the trial control. stopped when all treated insects were dead or the trial had run for 14 days, whichever was sooner. Methods Three trials were run for feeding propensity and elec- Mosquito rearing trophysiology recordings. The first had three treatments: Anopheles stephensi were reared under standard insec- mosquitoes held in untreated cups ("UnExp”), or tary conditions of 27°C, 80% humidity and 12:12 light: exposed to either B. bassiana-treated or M. acridum- dark photo-period. Eggs were placed in plastic trays (25 treated cups ("Exp”). The second had two treatments: cm×25cm×7cm)filledwith1.5lofdistilledwater. mosquitoes held in untreated cups or exposed to B. To reduce variation in adult size at emergence, larvae bassiana-treated cups. The third had un-exposed and B. were reared at a fixed density of 400 per tray. Larvae bassiana-exposed insects and these were used for single George et al. Malaria Journal 2011,