Altered behavioral and olfactory receptor neuron response in the mosquito, Anopheles stephensi, due to infection with the fungus Beauveria bassiana, and the malaria parasite Plasmodium yoelii
Open Access
- Author:
- George, Justin
- Graduate Program:
- Entomology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- June 18, 2012
- Committee Members:
- Thomas Charles Baker, Dissertation Advisor/Co-Advisor
Thomas Charles Baker, Committee Chair/Co-Chair
Matthew Brian Thomas, Committee Member
James Lewis Frazier, Committee Member
James Harold Marden, Special Member - Keywords:
- Anopheles stephensi
Malaria
Fungal biopesticides
Electrophysiology
olfaction
Electropalpogram - Abstract:
- Malaria affects the lives of more than a quarter billion people, and still remains as one of the most important causes of human mortality. With resistance of malaria mosquitoes to insecticides growing in recent years, olfactory-based surveillance and attract- and-kill strategies are becoming more important for integrated vector management, which can be used in the fight against the malaria parasite, Plasmodium falciparum, and its principal vectors, mosquitoes in the genus Anopheles. My thesis research focused on using behavioral and neurophysiological techniques to study the olfactory responses of Anopheles stephensi adults, following fungal and malaria infection. I used electroantennogram (EAG), electropalpogram (EPG) and single sensillum recordings (SSR) to study the changes in the responsiveness to odor cues of neurons in the peripheral olfactory system of mosquitoes following fungal and malaria infections. I correlated these changes in neuronal responsiveness with alterations in the host-seeking behavior of adult females that I observed, using both Y-tube olfactometer and wind tunnel behavioral assays. I also investigated the attraction of mosquitoes to spores of entomopathogenic and non-entomopathogenic fungi using olfactometer bioassays. The attraction of female An. stephensi to caterpillars of two moth species that were infected or not infected with fungi, and the possible attraction-related role of CO2 emitted by these caterpillars, was also a part of my investigation into the attraction of An. stephensi to fungal spores. In chapter 1, I report the results of my studies on the effects of fungal infection on the responsiveness of the olfactory sensory neurons of An. stephensi females to mammalian host odors, using EAGs, EPGs and SSRs. I also report the results of behavioral bioassays that I conducted in the wind tunnel to study the effects of Beauveria bassiana fungal infection on females’ upwind flight host-seeking behavior. The results of these studies (George et al. 2011, Malaria Journal 10:219) showed that fungal infection reduces the responsiveness of female An. stephensi to host odor cues, both behaviorally and neuronally. Exposure to B. bassiana caused significant mortality and reduced the propensity of mosquitoes to respond and fly to a host-related stimulus. Infection of females with a different fungal pathogen, Metarhizium acridum spores induced a similar decline in feeding propensity, albeit more slowly than B. bassiana infection. Reduced upwind flight host-seeking responses following fungal infection was positively correlated with the reduced olfactory receptor neuron responsiveness, measured by both EAGs and EPGs. Single cell recordings from neurons in the capitate peg sensilla of the maxillary palps confirmed that fungal-infected, behavioral non-responders exhibited significantly reduced sensitivity of neurons tuned specifically to the known attraction-related mammalian host odor, 1-octen-3-ol. This was the first study that correlated fungal-induced reductions in mosquito host-seeking behavior with a decline in olfactory receptor neuron sensitivity. In chapter 2, I report the results of behavioral experiments that I performed using a Y-tube olfactometer, in which I investigated the attraction of An. stephensi females to the spores of the fungi, B. bassiana and M. anisopliae. These two entomopathogenic species are under development to be used in residual sprays applied to the interiors of human dwellings for control of Anopheline mosquito species for malaria vector control. My results show that 75-80 % of the females are attracted to fungal spores and they are more strongly attracted to the Beauveria and Metarhizium spores than they are to the spores of other species of fungi. Further experiments using B. bassiana-infected caterpillars of two moth species showed that infected caterpillars are more effective in attracting An. stephensi females than uninfected ones. My results also indicate that females landing on sporulating cadavers of caterpillars infected with B. bassiana become infected and die within 3-4 days after contact with the larvae covered with the spores. Volatile collections from dried fungal spores showed that they emit many volatile compounds at very low rates, but that some of these have been shown to be attractive to mosquitoes from other genera, including Aedes and Culex species. This is the first report of mosquito attraction to fungal spores, and it suggests that there is promise in developing the technique of applying fungal spores to surfaces as a biopesticide for malaria vector control. In chapter 3, I report the results of experiments I performed using olfactometer assays to investigate the attraction of female An. stephensi to fourth instar larvae of the moth species, Manduca sexta and Heliothis subflexa. As part of this effort to study the degree of attraction of mosquitoes towards uninfected caterpillars, and some of the possible stimuli involved, I measured the amount of CO2 produced by these larvae using a respirometer. Also, using large-scale cage assays I also measured the possible benefits of caterpillar feeding by female An. stephensi in terms of their increased longevity. Olfactometer assays using live and dead caterpillars of H. subflexa and M. sexta show that female An. stephensi are significantly attracted to caterpillars, feed on them, and survive significantly longer than females provided with no food whatsoever. The attraction of females to caterpillars is positively correlated with the emission levels of carbon dioxide from caterpillars. This part of my study suggests that caterpillar-feeding by An. stephensi, and perhaps other Anopheline species, might be occurring in nature, and the frequency of such feeding needs to be examined in the field. Such feeding could possibly be a mechanism by which fungal infection is spread in insects. In chapter 4, I report the results of my experiments in which I performed EPG recordings to investigate the degree to which olfactory neuron responsiveness of female An. stephensi changes during infection with the malaria parasite, Plasmodium yoelii, over the course of its life cycle within the mosquito. My results show that there are significant changes in olfactory receptor neuron sensitivity to odorants that are correlated with the different stages of malaria parasite development. The EPGs of infected mosquitoes are significantly lower than those of uninfected individuals during the oocyst (non-transmissible) stage of P. yoelii infection, whereas they are significantly higher during the sporozoite (transmissible) stage. This is the first report of shifts in mosquito peripheral olfactory neuron responsiveness caused by a malaria parasite. It suggests that Plasmodium yoelii, and perhaps other species of Plasmodium parasites, are able to “manipulate” the olfaction-related host-seeking system of their mosquito host to improve their transmission success. Such olfactory modulations during different stages of Plasmodium infection might play a significant role in the process of malaria transmission to humans, and understanding these shifts in olfaction as they relate to mosquito behavior may possibly contribute to better monitoring and control strategies against these pernicious disease vectors.