Ectoparasitic mites are hypothesized to horizontally transmit bacterial endosymbionts, especially male-killing Spiroplasma. In this study we test how Spiroplasma poulsonii MSRO affects fly physiology and behaviour, and potential mite interactions.

We measured the metabolic rates of age-matched MSRO+ and MSRO– female fly cohorts at 2, 6, 11, 15, 20, 26, or 34 days post-eclosion using flow-through respirometry. Flies were frozen post assay, and mass was weighed when possible. Metabolic rate was measured as the rate of carbon dioxide production (uL/min). A MAVEn-FT unit was used to set the flow rate at 30 mL/min flow rate and control which respirometer position was active (14 containing flies and 1 baseline). A Li-7000 CO2 sensor was used to measure CO2 in air that had flown through chambers that contained MSRO+ flies, MSRO– flies, or a baseline (empty) chamber. To improve sensitivity incoming air was purged of CO2 and water vapour and excurrent gas was purged of water vapour again using a magnesium perchlorate column.

We used negative geotaxis endurance assays to measure the resistance capacity of MSRO+ and MSRO– flies. The ability to maintain energetically demanding activity is a proxy measure of resistance that eliminates the effects of mite preferences. We marked empty, transparent vials at 5 cm above the base, then aspirated a single MSRO+ or MSRO– fly into a vial and allowed 15 minutes for acclimation. At the outset of the experiment (t=0) the experimenter tapped the vial causing the fly to fall to the bottom of the vial, inducing the fly to climb. This knock-down procedure was repeated whenever the fly reached the 5cm line. A fly was considered exhausted if it failed to ascend the vial after 10 seconds. We recorded two metrics of endurance: the number of knock-downs before the fly was exhausted (cycles, c), as well as the time until exhaustion (seconds, t); if a fly failed to climb following the initial knockdown both values were recorded as 0. The time it took for the fly to climb to the mark after the first knock-down was also recorded (first cycle time, f).

Necessary definitions are in the Meta worksheet.