Drosophila melanogaster was reared at 25°C with a 12 h:12 h light–dark cycle on autoclaved yeast-glucose medium [Y-G diet, comprising Brewer’s yeast and glucose (both at 83 g l−1, from MP Biomedicals), agar (10 g l−1, from Frutarom) and preservatives (0.04% phosphoric acid, 0.42% propionic acid, from Sigma)], and transferred to fresh medium weekly. Outbred populations of strains Canton-S and Oregon-R had been maintained on Y-G diet for at least 18 years. Strain Ithaca-83 is an isofemale line established from a single female collected at Littletree Orchard, New-field, New York in 2004, and maintained on Y-G diet since collection.
The experimental samples comprised: guts (from proventriculus to rectum, excluding Malpighian tubules) dissected from third-instar larvae and adults; whole first- to second-(‘early’) instar larvae (< 48 h after hatching: these insects were too small for gut dissections); pupae (which lack a gut); and eggs (< 20 h after deposition). All samples except the eggs were surface-sterilized in 10% sodium hypochlorite solution, followed by three rinses in sterile distilled water. Gut dissections were conducted in sterile Ringer’s solution on clean glass slides with sterilized forceps, using a dissecting microscope at × 7 magnification. This sampling design followed preliminary experiments that confirmed the presence of bacteria in all surface-sterilized samples except eggs (data not shown), consistent with published evidence that bacteria are borne within larvae, pupae and adults, but not internal to the eggshell (Bakula, 1969 (link)). All experiments used reagent-only controls comprising a drop of Ringer’s solution treated as for dissections (including swirling the dissection instruments in the solution), but without D. melanogaster materials.
The experimental samples comprised: guts (from proventriculus to rectum, excluding Malpighian tubules) dissected from third-instar larvae and adults; whole first- to second-(‘early’) instar larvae (< 48 h after hatching: these insects were too small for gut dissections); pupae (which lack a gut); and eggs (< 20 h after deposition). All samples except the eggs were surface-sterilized in 10% sodium hypochlorite solution, followed by three rinses in sterile distilled water. Gut dissections were conducted in sterile Ringer’s solution on clean glass slides with sterilized forceps, using a dissecting microscope at × 7 magnification. This sampling design followed preliminary experiments that confirmed the presence of bacteria in all surface-sterilized samples except eggs (data not shown), consistent with published evidence that bacteria are borne within larvae, pupae and adults, but not internal to the eggshell (Bakula, 1969 (link)). All experiments used reagent-only controls comprising a drop of Ringer’s solution treated as for dissections (including swirling the dissection instruments in the solution), but without D. melanogaster materials.
