PhD thesis: Potential of the mosquito Aedes malayensis as an arbovirus vector in South East Asia Survey of the sylvatic mosquito fauna in a forested area of the Nakai Nam Theun National Protected Area Supervisors Dr. Paul Brey (IPL) Dr. Louis Lambrechts (Institut Pasteur / CNRS 2000) PhD Student Elliott Miot Background The Lao mosquito fauna is still not well characterized, especially in remote forested areas such as our study area. The work of Rueda et al. [1] began to establish a comprehensive checklist of mosquito species in the country, but it was mainly based on a literature review and only a few field collections. Methods In order to update the checklist of mosquitoes in our study area, we performed larval and adult mosquito collections in the NNT NPA along several rivers: Nam Theun (January 2012), Nam Mon (March 2012), Nam Noy (December 2011, March-April 2012, February, March, May, August, November, December 2017, and February-March 2018), and Nam On (May 2017) (Figure 1). Figure 1. Map showing sampling locations of mosquitoes in Lao PDR. KM = Khammuane Province; Black stars = sampling locations; Blue line = Mekong River and its tributaries. Mosquito larvae were collected in various types of breeding sites such as rock pools along the riverbanks, tree holes, temporary pools of water, fruit husks, and bamboo shoots. We also constructed bamboo traps, i.e. large bamboos cut in small pieces and filled with freshwater, that we deployed across the Nam Noy field site to create semi-natural breeding sites. Adult mosquitoes were captured using a combination of commercial traps (e.g. CDC light traps, BG sentinel traps) and active collections using vacuum-powered and mouth aspirators, and butterfly nets. Larvae were reared until adulthood and sorted morphologically back in the laboratory. Results During our field surveys, we identified 54 mosquito taxa belonging to 11 genera, of which 15 species were new records in Laos. We also described Ae. malayensis, a species found throughout South East Asia [2-4], for the first time in Laos based on morphological characters and molecular analyses. We produced photos of the morphological characters allowing proper identification of Ae. malayensis and distinction from closely related species that are found in sympatry, such as Ae. albopictus (Figure 2 & 3). We also provided information on the bionomics of Ae. malayensis. Figure 2: Morphological comparison of the thorax. (A)Ae. malayensis – a supraalar area of thorax with a patch of pale scales extended upward toward the scutellum. (B)Ae. albopictus – a supraalar area of thorax with a spot of pale scales not extended toward the scutellum. Figure 3: Morphological comparison of the abdominal terga IV- VI. (A) Ae. malayensis – dorsal white bands connected to lateral pale patches. (B) Ae. albopictus – dorsal white bands separated from lateral spots. Successful colonies of Ae. albopictus and Ae. malayensis were derived from the collections, which allowed subsequent laboratory studies on these sylvatic mosquito populations. Publications Motoki MT, Vongphayloth K, Rueda LM, Miot EF, Hiscox A, Hertz JC, and Brey PT. New records and updated checklist of mosquitoes (Diptera: Culicidae) from Lao People’s Democratic Republic, with special emphasis on adult and larval surveillance in Khammuane Province. J Vector Ecol. 2019;44: 76–88. doi:10.1111/jvec.12331 Motoki MT, Miot EF, Rueda LM, Vongphayloth K, Phommavanh N, Lakeomany K, Debboun M, Hertz JC, and Brey PT. First Record of Aedes (Stegomyia) malayensis Colless (Diptera: Culicidae) in the Lao People’s Democratic Republic, Based on Morphological Diagnosis and Molecular Analysis. US Army Med Dep J. 2018: 1–7. Identification of sylvatic mosquito species attracted to humans in a forested area of the Nakai Nam Theun National Protected Area Background Little is known about the vector status, ecology, and behavior of mosquito species in Laos. In addition to vector competence, vectorial capacity strongly depends on mosquito ecology and feeding behavior. Host preference, host availability, vector abundance, feeding frequency, encounter rate, and the temporal pattern of blood- feeding activity are some of the main ecological parameters that determine the effective contact between vectors and hosts [5]. Thus, characterizing the abundance, richness, diversity, and biting behaviors of mosquito species is an essential step in order to assess the risk of spillover of zoonotic, vector- borne pathogens to the human population [6]. The same is true for assessing the risk of spillback of human vector-borne pathogens establishing novel sylvatic transmission cycles in regions where they were previously absent [7,8]. Methods We conducted a total of four 1-week missions in August (rainy season), November-December (dry season) 2017 and twice in March 2018 (intermediate season) in the NNT NPA. Mosquito species attracted to humans were specifically targeted using human-baited double-net traps [9] (Figure 4) deployed in three habitats (riverside, low-cover forest, high-cover forest) along a trail going from the Nam Noy riverbank to deeper in the forest (Figure 5). Mosquitoes were captured around the clock, sacrificed, grouped per 1-hour interval, and identified morphologically back in the laboratory. Based on the captures, we evaluated the abundance, richness, diversity, and daily activity pattern of human-attracted mosquito species and compared these indices between habitats and seasons. Figure 4: Human- Figure 5: Map of baited double-net the Nam Noy field traps in situ. site with the location of the collection sites. Generated using ©Google Maps. Purple, red and yellow marks show the collection locations in the riverside habitat, low- cover forest habitat, and high-cover forest habitat, respectively. Results We collected a total of 1,018 females that were morphologically assigned to 9 genera and 26 mosquito taxa, of which 87.6% represented only 8 mosquito species (Figure 6). Some of them are known or putative arbovirus vectors such as Ae. albopictus, Cx. vishnui, Ar. subalbatus, and Ae. malayensis. Others have been poorly studied and little is known about their potential as pathogen vectors such as Heizmannia species, Ae. desmotes, and Ar. jugraensis. Figure 6: Species abundance of human-attracted mosquitoes stratified by season and habitat. Heat map of the log10- transformed a total number of mosquitoes caught in the human- baited traps shown by season (rainy, dry, intermediate) and habitat (riverside, low-cover forest, high-cover forest) for each species. Abundance ranges from dark blue (low) to red (high). Analyses of species richness and diversity revealed that they were significantly higher during the rainy season, especially in the low-cover forest habitat (Figure 7 & 8) Figure 7: Species richness of human-attracted mosquitoes stratified by season and habitat. Boxplots of the daily number of mosquitoes caught in the human-baited traps shown by season (rainy, dry, intermediate) and habitat (riverside, low-cover forest, high-cover forest). Each point in the boxplots represents the species richness of a 24-hour mosquito collection period. Letters above the boxplots indicate the statistical significance of the differences. Conditions sharing a letter are not significantly different. Figure 8: Diversity of human-attracted mosquitoes stratified by season and habitat. Boxplots of the daily Shannon diversity (H’) index of mosquitoes caught in the human-baited traps by season (rainy, dry, intermediate) and habitat (riverside, low- cover forest, high-cover forest). Each point in the boxplots represents the H’ index of a 24-hour mosquito collection period. Letters above the boxplots indicate the statistical significance of the differences. Conditions sharing a letter are not significantly different. Daily activity patterns showed that all human-attracted mosquito species collected were only active during daytime regardless of seasons or habitats (Figure 9). Host-seeking activity differed between the low-cover forest habitat, where mosquitoes were most active during the afternoon, and the high-cover forest habitat, where they were more active in the morning, regardless of the season. Figure 9: Daily pattern of human-seeking mosquito activity stratified by habitat and season. The mean hourly number of mosquitoes caught in the human-baited traps is plotted over time by habitat (riverside, low-cover forest, high-cover forest) and season (rainy, dry, intermediate). Hourly data were collected for 24-hour periods (rainy season: 6 consecutive days and nights; dry season: 6 consecutive days and nights; intermediate season: 2 x 6 consecutive days and nights). The results of this study improve our understanding of mosquito-human interactions in the NNT NPA by providing new information on the species abundance, richness, diversity, and daily activity pattern of mosquitoes caught in human-baited traps. We characterized several human-attracted mosquito species, including Ae. malayensis, in the primary forest of Laos, which paves to way for identification of potential bridge vectors in this area. Publication Miot EF, Motoki MT, Phommavanh N, Lakeomany K, Vongphayloth K, Hertz JC, Lambrechts L, and Brey PT. Diversity, abundance and daily activity pattern of human-seeking mosquitoes in a forested area of the Nakai district, Laos. Manuscript in preparation. Potential of the sylvatic mosquito Aedes malayensis to act as an arbovirus bridge vector in a forested area of the Nakai Nam Theun National Protected Area Background Following our initial field