Background Mosquito and human behaviour interaction is a key determinant of the maximum level of protection against malaria that can be provided by insecticide-treated nets (ITNs). Nevertheless, scant literature focuses on this interaction, overlooking a fundamental factor for efficient malaria control. This study aims to estimate malaria transmission risk in a Burkina Faso village by integrating vector biting rhythms with some key information about human habits. Methods Indoor/outdoor human landing catches were conducted for 16 h (16:00–08:00) during 8 nights (September 2020) in Goden village. A survey about net usage and sleeping patterns was submitted to half the households (October–December 2020). A subsample of collected specimens of Anopheles gambiae sensu lato was molecularly processed for species identification, Plasmodium detection from heads-thoraxes and L1014F pyrethroid-resistance allele genotyping. Hourly mosquito abundance was statistically assessed by GLM/GAM, and the entomological inoculation rate (EIR) was corrected for the actual ITN usage retrieved from the questionnaire. Results Malaria transmission was mainly driven by Anopheles coluzzii (68.7%) followed by A. arabiensis (26.2%). The overall sporozoite rate was 2% with L1014F estimated frequency of 0.68 (N = 1070 out of 15,201 A. gambiae s.l. collected). No major shift in mosquito biting rhythms in response to ITN or differences between indoor and outdoor catches were detected. Impressive high biting pressure (mean 30.3 mosquitoes/person/hour) was exerted from 20:00 to 06:00 with a peak at 4:00. Human survey revealed that nearly all inhabitants were awake before 20:00 and after 7:00 and at least 8.7% had no access to bednets. Adjusting for anthropological data, the EIR dropped from 6.7 to 1.2 infective bites/person/16 h. In a scenario of full net coverage and accounting only for the human sleeping patterns, the daily malaria transmission risk not targetable by ITNs was 0.69 infective bites. Conclusions The high mosquito densities and interplay between human/vector activities means that an estimated 10% of residual malaria transmission cannot be prevented by ITNs in the village. Locally tailored studies, like the current one, are essential to explore the heterogeneity of human exposure to infective bites and, consequently, to instruct the adoption of new vector control tools strengthening individual and community protection. Graphical Abstract

Background: Mosquito and human behaviour interaction is a key determinant of the maximum level of protection against malaria that can be provided by insecticide treated nets (ITNs). Nevertheless, scant literature focuses on this interaction, overlooking a fundamental factor for efficient malaria control. This study aims to estimate malaria transmission risk in a Burkina Faso village by integrating vector biting rhythms with some key information about human habits. Methods: Indoors/outdoors Human Landing Catches were conducted for 16 hours (16:00-08:00) during 8 nights (September 2020) in Goden village. A survey about net usage and sleeping patterns was submitted to half the households (October-December 2020). A subsample of A. gambiae s.l. collected specimens was molecularly processed for: species identification, Plasmodium detection from head-thoraxes, and L1014F pyrethroid-resistance allele genotyping. Hourly mosquito abundance was statistically assessed by GLM/GAM and the entomological inoculation rate (EIR) was corrected for the actual ITN usage retrieved from the questionnaire. Results: Malaria transmission was mainly driven by Anopheles coluzzii (68.7%), followed by Anopheles arabiensis (26.2%). The overall sporozoite rate was 2%, and L1014F estimated frequency of 0.68 (N=1,070 out of 15,201 A. gambiae s.l. collected). No major shift in mosquito biting rhythms in response to ITN, nor differences between indoor and outdoor, were detected. Impressive high biting pressure (mean 30.3 mosquitoes/person/hour) was exerted from 20:00 to 06:00 with a peak at 4:00. Human survey revealed that nearly all inhabitants were awake before 20:00 and after 7:00 and at least 8.7% had no access to bednets. Adjusting for anthropological data, the EIR dropped from 6.7 to 1.2 infective bites/person/16h. In a scenario of full net coverage and accounting only for the human sleeping patterns, the daily malaria transmission risk not targetable by ITNs was 0.69 infective bites. Conclusions: The high mosquito densities and the interplay between human/vector activities means that an estimated 10% of residual malaria transmission cannot be prevented by ITNs in the village. Locally-tailored studies, as the current one, are essential to explore the heterogeneity of human exposure to infective bites and, by consequence, to instruct the adoption of new vector control tools strengthening individual and community protection.

Background Some species of drosophilid flies belonging to the genus Phortica feed on ocular secretions of mammals, acting as biological vectors of the zoonotic eyeworm Thelazia callipaeda. This study describes an effective breeding protocol of Phortica variegata and Phortica oldenbergi in insectary conditions. Methods Alive gravid flies of P. oldenbergi, P. variegata and Phortica semivirgo were field collected in wooded areas of Lazio region (Italy) and allowed to oviposit singularly to obtain isofamilies. Flies were maintained in ovipots (200 ml) with a plaster-covered bottom to maintain high humidity level inside. Adult feeding was guaranteed by fresh apples and a liquid dietary supplement containing sodium chloride and mucin proteins, while larval development was obtained by Drosophila-like agar feeding medium. The breeding performances of two media were compared: a standard one based on cornmeal flour and an enriched medium based on chestnut flour. All conditions were kept in a climatic chamber with a photoperiod of 14:10 h light:dark, 26 ± 2 °C and 80 ± 10% RH. Results From a total of 130 field-collected Phortica spp., three generations (i.e. F1 = 783, F2 = 109, F3 = 6) were obtained. Phortica oldenbergi was the species with highest breeding performance, being the only species reaching F3. Chestnut-based feeding medium allowed higher adult production and survival probability in both P. oldenbergi and P. variegata. Adult production/female was promising in both species (P. oldenbergi: 13.5 F1/f; P. variegata: 4.5 F1/f). Conclusions This standardized breeding protocol, based on controlled climatic parameters and fly densities, together with the introduction of an enriched chestnut-based feeding medium, allowed to investigate aspects of life history traits of Phortica spp. involved in the transmission of T. callipaeda. Obtaining F3 generation of these species for the first time paved the road for the establishment of stable colonies, an essential requirement for future studies on these vectors in controlled conditions. Graphical abstract

Background - The establishment of arthropod colonies is an important tool to manipulate disease vectors, investigate their life history traits, and their vector competence, prevention of vector borne pathogen transmission, or to assess insecticidal/repellent activities. Some species of drosophilid flies belonging to the genus Phortica feed on ocular secretions of mammals, acting as biological vectors of the zoonotic eyeworm Thelazia callipaeda. This study describes an effective breeding protocol of Phortica variegata and Phortica oldenbergi in insectary conditions. Materials and methods - Gravid flies of P. oldenbergi, P. variegata and Phortica semivirgo were field collected in wooded areas of Lazio region (Italy) and allowed to oviposit singularly to obtain isofamilies. Flies were maintained in ovipots (200ml) with a plaster-covered bottom to maintain high humidity level inside. Adult feeding was guaranteed by freshly apple and a liquid dietary supplement containing NaCl and mucin proteins, while larval development was obtained by Drosophila-like agar feeding medium. The breeding performances of two media were compared: a standard one based on cornmeal flour and an enriched medium based on chestnut flour. All conditions were kept in a climatic chamber with a photoperiod of 14:10h light:dark, 26±2°C and 80±10% RH. Results - From a total of 130 field collected Phortica spp. three generations (i.e., 783 F1, 109 F2, 6 F3) were obtained. Phortica oldenbergi was the species with higher breeding performance, being the only species reaching F3. Chestnut-based feeding medium allowed higher adult production and survival probability in both P. oldenbergi and P. variegata. Adult production/female was promising in both species (P. oldenbergi: 13.5 F1/f; P. variegata: 4.5 F1/f), indicating the possibility to obtain stable colonies.Conclusions - This standardized breeding protocol, based on controlled climatic parameters and fly densities, together with the introduction of an enriched feeding medium, allowed to investigate aspects of life history traits of Phortica spp. involved in the transmission of T. callipaeda. Obtaining F3 generation of these species for the first time paved the road for the establishment of stable colonies, an essential requirement for future studies on these vectors in controlled conditions.