Wolbachia density and distribution in the transient infected mosquitoes were compared 7 and 14 days post injection (dpi) to the wAlbB line using qPCR and fluorescence in situ hybridisation (FISH). DNA was extracted from stable and transiently Wolbachia infected mosquitoes using the DNeasy 96 Blood & Tissue kit (Qiagen) according to the manufacturer’s specifications. Quantitative PCR to determine the total relative Wolbachia densities of infected lines was performed as described by [30 (link)] using primers specific to the gene coding for the Wolbachia surface protein (wsp) (forward primer 5’-GCATTTGGTTAYAAAATGGACGA-3’, reverse primer 5’- GGAGTGATAGGCATATCTTCAAT-3’), as well as the Ae. aegypti actin gene (forward primer 5’- GACTACCTGATGAAGATCCTGAC-3’, reverse primer: 5’- GCACAGCTTCTCCTTAATGTCAC-3’) [24 (link)]. Statistical differences were determined using a Mann-Whitney (Graphpad Prism version 6.0f).
Wolbachia was localized in sections of paraffin-embedded 5–7 day old female mosquitoes by FISH, as described in [31 (link)], except that only one probe against 16S rRNA was used and its concentration was increased 10-fold to improve the signal. wAlbB was detected using AlbBW5: 5’-CTTAGGCTTGCGCACCTTGCAA-3’, labelled with Alexa 488 dye (green). DAPI was used to stain total DNA.
Wolbachia was localized in sections of paraffin-embedded 5–7 day old female mosquitoes by FISH, as described in [31 (link)], except that only one probe against 16S rRNA was used and its concentration was increased 10-fold to improve the signal. wAlbB was detected using AlbBW5: 5’-CTTAGGCTTGCGCACCTTGCAA-3’, labelled with Alexa 488 dye (green). DAPI was used to stain total DNA.
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