Study area and mosquito collection
Aedes spp. were collected at sentinel sites defined in urban, peri-urban, and rural landscapes across Bioko Island. Collections were performed using Human Landing Catch (HLC) and Centre for Disease Control Light Trap (CDC-LT) methods as a by-product of the routine entomological surveillance for malaria vectors between February 2020 and August 2021 [19]. Mosquitoes were collected throughout the night from seven PM to six AM the following morning in two houses randomly selected per sentinel monitoring site and per method. One volunteer each was placed indoors and outdoors of selected houses in a four person/night catch. Simultaneously, two CDC light traps each were installed indoors and outdoors of two other randomly picked houses. Resulting again in a four trap/night catch. Aedes spp. collected via HLC were preserved according to hour and place of collection in Eppendorf tubes, while Aedes spp. collected via CDC-LT were pooled in groups of ten per collection place. All mosquitoes were stored in DNA/RNA Shield (Zymo Research, USA) at room temperature until shipped to Swiss TPH in Allschwil, Switzerland, for further analysis.
Sample pooling and nucleic acid extraction
Out of 233 tubes received that contained between one to 14 individual mosquitoes, 30 tubes were extracted as individual samples derived from the same night, same site and same collection method. The remaining 194 tubes were grouped into 51 pools by collection district and whenever possible by sentinel collection site and collection year. The 30 vials extracted individually contained between one and seven Aedes spp. mosquitoes (median of two mosquitoes). The 51 pools contained between two and 29 mosquitoes (median of nine mosquitoes). For nucleic acid extraction, mosquitoes were homogenized for 12 minutes on a Disruptor Genie (Scientific Industries, USA) using stainless steel beads with a diameter of five mm (Qiagen, USA), followed by nucleic acid extraction using the QIAamp Viral RNA Mini Kit (Qiagen, USA) according to manufacturer’s protocol.
General qPCR settings
All qPCR reactions were set up as follows if not indicated otherwise: The qPCR and (RT-qPCR) assays were run on a Bio-Rad CFX96 Real-Time PCR System (Bio-Rad Laboratories, California, USA). Each qPCR reaction consisted of 1x Luna Universal Probe One-Step Reaction Mix (New England Biolabs), primer and probe mix and two μL of extracted nucleic acids in a total volume of 10 μL. The reverse transcription qPCR (RT-qPCR) runs were set up with the same master mix as the qPCR runs and complemented with 1x Luna WarmStart RT Enzyme Mix (New England Biolabs, USA). Samples were analysed in duplicate. In each qPCR run, non-template (molecular biology grade H2O) controls and controls positive for the target were included. The controls had to be non-template control, respectively positive to be encountered as valid. The runs were set up according to Table 1 whereas denaturation and annealing/ extension steps were repeated 45 times.
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Extraction control RT-qPCR
For quality control and quantification of the extracted RNA and DNA, a quantitative PCR assay (qPCR) targeting the 18S and 28S ribosomal RNA/DNA genes of Aedes spp. was used (see Additional file 1: Table S1). In addition, human RNaseP (HsRNaseP) was used to detect mosquito feeding on humans.
Aedes species identification
To distinguish A. albopictus and A. aegypti, a qPCR assay that targets a fragment of the A. aegypti gene AAEL017129, and a fragment of the A. albopictus gene AALF027084 was performed.
Identification of Aedes spp. transmitted human pathogens
To detect human pathogens, three different multiplex RT-qPCR assays were used. The first assay targets the NS5 gene of viruses belonging to the family Flaviviridae. The second assay was performed to detect and distinguish all four dengue virus serotypes (DENV-1, DENV-2, DENV-3, DENV-4). The last multiplex assay was used to screen for genetic material of CHIKV, Rickettsia spp., YFV and ZIKV. For all three assays, 1x Fast Virus 1-Step Master Mix (Applied Biosystems™, Thermo Fisher Scientific Inc) instead of Luna Universal Probe One-Step Reaction Mix was used.
Blood feeding of Aedes spp. identification
A RT-qPCR was used to test all mosquito samples for the source of the last blood meal. The mosquitoes were screened for presence of dog, cat and rat DNA targeting the cytochrome B gene. To screen for the presence of bird DNA, markers for 16S genes and an intergenic region specific for mice were used (Additional file 1: Table S1).
Sanger sequencing
The PCR product of samples positive for the avian primers were sent to Microsynth AG (Switzerland) for confirmatory Sanger sequencing. PCR was performed with the pan-avian qPCR primers to generate a 96 bp long amplicon of the 16S region.
Oxford Nanopore sequencing
Metagenomic sequencing of all nucleic acids was done for nine Aedes spp. pools according to a published protocol [20]. Reverse transcription of RNA molecules was accomplished using random hexamer primers with a known 20-nt tag sequence at the 5′-end and the RevertAid First Strand H minus cDNA synthesis kit (Thermo Fisher) according to the manufacturer’s manual. Second-strand synthesis using Klenow polymerase (Thermo Fisher) was performed, followed by sequence-independent single primer amplification and purification of amplified DNA (PureLink PCR micro kit; Invitrogen) and elution. Total DNA concentration was measured (Qubit fluorometer; Invitrogen) using the dsDNA High-Sensitivity assay. The sequencing library was prepared using the Native Barcoding Kit 96 (SQK-NBD114.96) according to manufacturer’s protocol before sequencing on a R10.4.1 flow cell (Oxford Nanopore Technologies).
For metagenomic analysis, the data was uploaded to the cloud platform BugSeq [21], for long-read taxonomic classification.
Screening for Wolbachia spp. DNA
Based on the sequences obtained in the metagenomic analysis, specific primer pairs for amplification of Wolbachia spp. were designed to facilitate screening for these bacteria by qPCR. Primers and probes were designed with Primer 3 (2.3.7) in Geneious Prime using an assembly of Wolbachia pipientis endosymbiont of Drosophila melanogaster (OX384529.1).
Primers amplify a 103 bp long fragment in the hscA gene (positions 858,261 to 858,363). Forward (GACGATTCAGCACGTAACGC) and reverse primer (TGGAGTAAGAAAGCGCTGCA) were used at a final concentration of 0.4 µM each. The probe (FAM-GCTGGCATAGAAGTTCTTCGC-BHQ1) was used at a final concentration of 0.2 µM.