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The spread of SARS-CoV-2 cannot be well monitored and understood in areas without capacity for effective disease surveillance. Countries with a young population will have disproportionately large numbers of asymptomatic or pauci-symptomatic infections, further hindering detection of infection in the population. Sero-surveillance on a country-wide scale by trained medical professionals may be limited in scope in resource limited setting such as Mali. Novel ways of broadly sampling the human population in a non-invasive method would allow for large-scale surveillance at a reduced cost. Here we evaluate the collection of naturally bloodfed mosquitoes to test for human anti-SARS-CoV-2 antibodies in the laboratory and at five field locations in Mali. Immunoglobulin-G antibodies were found to be readily detectable within the mosquito bloodmeals by a bead-based immunoassay at least through 10 hours post-feeding with high sensitivity (0.900 ± 0.059) and specificity (0.924 ± 0.080), respectively, indicating that most blood-fed mosquitoes collected indoors during early morning hours (and thus, have likely fed the previous night) are viable samples for analysis. We find that reactivity to four SARS-CoV-2 antigens rose during the pandemic from pre-pandemic levels. Consistent with other sero-surveillance studies in Mali, crude seropositivity of blood sampled via mosquitoes was 6.3% in October/November 2020 over all sites, and increased to 25.1% overall, with the town closest to Bamako reaching 46.7% in February of 2021. Mosquito bloodmeals a viable target for conventional immunoassays, and therefore country-wide sero-surveillance of human diseases (both vector-borne and non-vector-borne) is attainable in areas where human-biting mosquitoes are common, and is an informative, cost-effective, non-invasive sampling option.

These are the files necessary to reproduce the survival, oocyst, and feedingfigures in our mosquito marking manuscript, and the original gel images for other figures.&nbsp

Additional file 3: Table S1. Akaike’s information criterion values for distributions tested using accelerated failure time models.

Additional file 1: Figure S1. Flight sound file output (spectrogram view) in Audacity (a) and Raven Pro 1.5 software (b). Energy frequency (y-axis) and time of flight during the assay (x-axis) allowed extraction of flight durations during the assay. Panel a shows continuous flight bout of > 70 min (in white), followed by a > 40-min rest period (in red). Panel b (top) shows > 120-min long continuous flight (in black) ending in several shorter bouts, whereas the bottom panel shows multiple bouts spanning 2–20 s. Figure S2. Spearman and Pearson correlations between the key flight aptitude indices: total flight, longest flight and flight bouts. Upper row: scatter plots of flight aptitude indices pairs showing linear regression as trend lines (dark blue) across all data (N = 707 mosquitoes). Pearson and Spearman correlation coefficients are given with their significance level (*** and ns denotes P 0.05, in Pearson (p) and Spearman (s) coefficients respectively). Lower row: Scatter plots as above, showing the relationships for HFA mosquitoes (red; n = 261) and LFA mosquitoes (blue; n = 446). HFAs were defined based on any one significant flight aptitude index (or more). Figure S3. Wing length in Anopheles coluzzii by time of year (season). Mean length represented as diamond symbols (◊). Medians as horizontal lines inside boxes. Table S1. Results summary table for the three flight aptitude indices: total flight, longest flight and flight bouts per test groups (top section). Shared high flight activity (HFA) frequency in the three flight aptitude indices (bottom section).

Additional file 4: Figure S3. Raincloud plot indicating measured wing lengths as a proxy for body size for primed and unprimed mosquitoes. Boxplot, individual points, and density distribution are shown. Point shapes indicate which replicate the lengths are from.

Additional file 5: Figure S4. Correlations of wing areas and survival for replicates 2 and 3 of the experiment. No consistent correlations between wing area and lifespan were found between the two experiments.

Additional file 2: Figure S2. Boxplot and raincloud [61] plots showing the date of death ranges and distributions for each of the experimental groups.

Additional file 1: Figure S1. Temperature and Humidity profiles for Thierola, Mali (red, orange) and the priming incubator (green, blue) as recorded by a HOBO temperature and humidity logger.

The spread of SARS-CoV-2 cannot be well monitored and understood in areas without capacity for effective disease surveillance. Countries with a young population will have disproportionately large numbers of asymptomatic or pauci-symptomatic infections, further hindering detection of infection in the population. Sero-surveillance on a country-wide scale by trained medical professionals may be limited in scope in resource limited setting such as Mali. Novel ways of broadly sampling the human population in a non-invasive method would allow for large-scale surveillance at a reduced cost. Here we evaluate the collection of naturally bloodfed mosquitoes to test for human anti-SARS-CoV-2 antibodies in the laboratory and at five field locations in Mali. Immunoglobulin-G antibodies were found to be readily detectable within the mosquito bloodmeals by a bead-based immunoassay at least through 10 hours post-feeding with high sensitivity (0.900 ± 0.059) and specificity (0.924 ± 0.080), respectively, indicating that most blood-fed mosquitoes collected indoors during early morning hours (and thus, have likely fed the previous night) are viable samples for analysis. We find that reactivity to four SARS-CoV-2 antigens rose during the pandemic from pre-pandemic levels. Consistent with other sero-surveillance studies in Mali, crude seropositivity of blood sampled via mosquitoes was 6.3% in October/November 2020 over all sites, and increased to 25.1% overall, with the town closest to Bamako reaching 46.7% in February of 2021. Mosquito bloodmeals a viable target for conventional immunoassays, and therefore country-wide sero-surveillance of human diseases (both vector-borne and non-vector-borne) is attainable in areas where human-biting mosquitoes are common, and is an informative, cost-effective, non-invasive sampling option.