Fig. 1

Overview of methodology used to perform inter- and intra-species microbiota transplantations in mosquitoes. (Left) Preparation of Ae. aegypti and Cx. quinquefasciatus donor pools. Individual 3–4-day-old sugar-fed adult females from our standard laboratory colonies were collected, surface-sterilized, and homogenized (1). Individual homogenates were then pooled and centrifuged to collect debris prior to filtering of the resulting supernatant to produce a final filtrate (2). Filtrates, which ranged in volume from 500 μl (pool 10) to 4 ml (pool 80), were then adjusted to a total volume of 50 ml using sterile water prior to transplantation (2). (Right) Transplantation of donor pools into a focal host species (Ae. aegypti). Eggs laid by blood-fed adult females from the standard laboratory colony were surface-sterilized and hatched in sterile water to produce axenic larvae (3). Larvae were then transferred to replicate flasks (n = 5) containing a 50 ml suspension of a given donor pool and provided sterilized diet every other day until pupation (4). Pupae produced from replicate flasks containing the same donor pool were finally pooled in water from the larval rearing flasks and transferred to a sterile plastic chamber for adult emergence (5). Donor and recipient samples collected for sequencing are indicated in bold. See “Methods” for more information. Created with BioRender.com