Jenny M. Lindh

Transstadial and horizontal transfer of bacteria within a colony of Anopheles gambiae (Diptera: Culicidae) and oviposition response to bacteria-containing water

In a paratransgenic approach, genetically modified bacteria are utilized to kill the parasite in the vector gut. A critical component for paratransgenics against malaria is how transgenic bacteria can be introduced and then kept in a mosquito population. Here, we investigated transstadial and horizontal transfer of bacteria within an Anopheles gambiae mosquito colony with the focus on spiked breeding sites as a possible means of introducing bacteria to mosquitoes. A Pantoea stewartii strain, previously isolated from An. gambiae, marked with a green fluorescent protein (GFP), was introduced to mosquitoes in different life stages. The following life stages or older mosquitoes in the case of adults were screened for bacteria in their guts. In addition to P. stewartii other bacteria were isolated from the guts: these were identified by 16S rRNA sequence analysis and temporal temperature gradient gel electrophoresis (TTGE). Bacteria were transferred from larvae to pupae but not from pupae to adults. The mosquitoes were able to take up bacteria from the water they emerged from and transfer the same bacteria to the water they laid eggs in. Elizabethkingia meningoseptica was more often isolated from adult mosquitoes than P. stewartii. A bioassay was used to examine An. gambiae oviposition responses towards bacteria-containing solutions. The volatiles emitted from the solutions were sampled by headspace-solid phase microextraction (SPME) and identified by gas chromatography and mass spectrometry (GC-MS) analysis. P. stewartii but not E. meningoseptica mediated a positive oviposition response. The volatiles emitted by P. stewartii include indole and 3-methyl-1-butanol, which previously have been shown to affect An. gambiae mosquito behaviour. E. meningoseptica emitted indole but not 3-methyl-1-butanol, when suspended in saline. Taken together, this indicates that it may be possible to create attractive breeding sites for distribution of genetically modified bacteria in the field in a paratransgenic approach against malaria. Further research is needed to determine if the bacteria are also transferred in the same way in nature.

Oviposition Responses of Anopheles gambiae s.s. (Diptera: Culicidae) and Identification of Volatiles from Bacteria-Containing Solutions

Abstract In this study, a dual-choice oviposition bioassay was used to screen responses of gravid An. gambiae toward 17 bacterial species, previously isolated from Anopheles gambiae s.l. (Diptera: Culicidae) midguts or oviposition sites. The 10 isolates from oviposition sites have been identified by phylogenetic analyses of their 16S rRNA genes. Eight of the 10 isolates were gram-positive, out of which six belonged to the Bacilli class. Solid phase microextraction and gas chromatography coupled to mass spectrometry (GC-MS) were used to identify the volatiles emitted from the bacterial isolates. Aromatic and aliphatic alcohols, aliphatic ketones, alkylpyrazines, dimethyl oligosulfides, and indole were among the chemical compounds identified from the headspace above bacteria-containing saline. The mosquitoes laid significantly more eggs in six of the bacteria-containing solutions compared with the sterile solution. These six bacteria did not emit any compounds in common that could explain the positive oviposition response. Instead, the bacteria were grouped according to principal component analysis (PCA) based on the relative amounts of volatiles emitted. The PCA-plots facilitated the identification of 13 putative oviposition attractants for An. gambiae mosquitoes.