Oviposition Cues as a Tool for Developing a New Malaria Control Strategy

Abstract

Anopheles gambiae sensu lato mosquitoes are among the dominant malaria vectors in sub-Saharan Africa. However, not much is known about the oviposition behaviour of these species. This knowledge is important for the development of malaria vector control strategies. New methods that can complement the currently used vector control methods: long lasting insecticidal nets (LLINs) and indoor residual spraying (IRS), are necessary due to the growing incidence of resistance to these methods. With the aim of investigating cues associated with selected oviposition sites, artificial oviposition sites- ponds were set-up in an open field at Mbita, Western Kenya in 2012 and 2013. These ponds were plastic basin buried into the ground for concealment which contained 2kg of soil mixed with 30 L of water. They were allowed to be colonized by wild An. gambiae s.l.. The numbers of Anopheles early instar larvae were counted and used as a proxy for oviposition preference. Water samples were then analysed for physicochemical, bacterial and chemical profiles. The bacterial profiles were analysed using denaturing gradient gel electrophoresis (DGGE) and the chemical profiles with gas chromatography-mass spectrometry (GC-MS). The detection of possible oviposition cues from oviposition substrates requires sensitive analytical methods. The detection of volatiles was improved seven times when 0.15 g/ml sodium chloride (NaCl) was added to the substrates and the volatiles trapped on Tenax traps for 20 h, were thermally desorbed compared to volatiles trapped on Porapak traps for 20 h that were eluted by liquid desorption with a solvent. Furthermore, to improve bacteria community profiling with denaturing gradient gel electrophoresis (DGGE), the detection of bacteria deoxyribonucleic acid (DNA) bands with DGGE was improved. This was achieved by pooling two replicate polymerase chain reactions and concentrating these to 10 μl which resulted in a minimum DNA concentration of 50 ng/μl. These two improved detection methods allowed for bacterial and chemical profiling of water samples taken from oviposition ponds. Results showed that ponds were colonized differently. Fresh ponds were preferred over slightly older ponds and contained two times more Anopheles early instar larvae. Bacterial analysis revealed that the bacterial load may play a role in oviposition site selection where ponds with a low number of bacteria colony forming units (CFU) was preferred for oviposition. Furthermore, diverse groups of chemicals were associated with the preferred ponds. Taken together for all the data analysed, there was no volatile detected that was common in all the rounds within an experiment. However some volatiles, including: 6,10-dimethyl-5,9-undecadien-2-one (geranylacetone) and 4-ethylbenzaldehyde, were associated with the oviposition preferred pond. In addition, physicochemical parameters such as low pH and high turbidity were associated with the ponds selected for oviposition. Finally, fungi isolated from the rhizomes of nut grass that was collected from the same natural oviposition site as the soil used in the open field experiment, yielded a promising array of volatiles of which one is known to attract oviposition site seeking malaria mosquitoes. This finding opens the door for a cost effective and environmental friendly method of using fungi in an “attract and kill” strategy targeting malaria vectors.