Table 3 Experimental design of S. titanus transmission trials. No. of individuals (donors + receivers) Transmission type Acquisition time Destination 20 (10 + 10) Co-feeding with Asaia 24 hours q-PCR 38 (19 + 19)   48 hours   28 (14 + 14)   72 hours   20 (10 + 10)   96 hours   8 (4 + 4)   48 hours FISH Tot. co-feeders: 114 (57 + 57)       10 (5 + 5) Asaia ABT-199 chemical structure Venereal transfer (male to female) 24 hours q-PCR 10 (5 + 5)   48 hours   10 (5 + 5)   72 hours   14 (7 + 7)  

96 hours   10 (5 + 5)   48 hours FISH 10 (5 + 5) Asaia Venereal transfer (female to male) 24 hours q-PCR 14 (7 + 7)   48 hours   10 (5 + 5)   72 hours   12 (6 + 6)   96 hours   8 (4 + 4)   48 hours FISH Tot. mated: 108 (54 + 54)       6 (3 + 3) Co-housing control trial (males with males) 24 hours   6 (3 + 3)   48 hours   6 (3 + 3)   72 hours   6 (3 + 3)   96 hours   10 (5 + 5) Co-housing control trial (females with females) 24 hours

q-PCR 6 (3 + 3)   48 hours   6 (3 + 3)   72 hours   6 (3 + 3)   96 hours   Tot. co-housed: 52 (26 + 26)       20 (10 + 10) Negative control for Co-feeding 24 hours q-PCR 22 (11 + 11)   48 hours   28 (14 + 14)   72 hours   32 (16 RG7204 order + 16)   96 hours   10 (5 + 5)   48 hours FISH Tot. co-feeders: 112 (56 + 56)       16 (8 + 8) Negative control for venereal transfer (male to female) 24 hours q-PCR 10 (5 + 5)   48 hours   8 (4 + 4)   72 hours   14 (7 + 7)   96 hours   10 (5 + 5)   48 hours FISH 8 (4 + 4) Negative control for venereal transfer (female to male) 24 hours q-PCR 14 (7 + 7)   48 hours   12 (6 + 6)   72 hours   10 (5 + 5)   96 hours   10 (5 + 5)   48 hours FISH Tot. mated: 112 (56 + 56)       Number of insect specimens used for each trial. The duration of the acquisition period, as well as the type of analysis carried out, are indicated both for samples submitted 5-Fluoracil cost to experiments performed with Gfp-tagged Asaia and for negative controls. Venereal transmission trials When Gfp-tagged Asaia-infected

males were mated with uninfected females, transfer of Gfp-tagged symbiotic cells was observed, although a longer period was required to reach infection rates similar to those of the co-feeding trials. After a 24 hour incubation time subsequent to mating, only 20% of females (1 out of 5 individuals) were gfp gene-positive, with 40% (2 out of 5) positive after 48 hour, 60% (3 out of 5 individuals) at 72 hours, with 4 out of 7 individuals infected at 96 hours (Figure 1B). The average concentration of the marked symbiont in the body of S. titanus also increased with longer incubation periods, even though it remained significantly lower than that of donor individuals (df= 18; F= 11.663; P<0.05) (Figure 1E).