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The submergence experiments suggested that the adult ticks are able to close their spiracles for extended periods. Based on information from other ixodid ticks (Lighton, Fielden & Rechav, 1993; Fielden & Lighton, 1996) this should be the case. To verify spiracle closure for the species examined here, two approaches were used. First, gas exchange patterns were examined using flow-through respirometry at a range of

temperatures because previous work on a range of groups has shown that temperature has a profound influence on gas exchange patterns (Chown & Nicolson, 2004; Contreras & Bradley, 2010; Heinrich & Bradley, 2014). Carbon dioxide production (VCO2) was measured using a Li-Cor 7000 CO2/H2O infrared differential gas analyser (LICOR, Lincon, USA), sampling at a rate of 0.5 Hz, attached to a Sable Systems International (SSI) flow through respirometry system (SSI, www.sablesys.com, Las Vegas, USA)

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(Supplementary Fig. S2). Air was pumped through three scrubber columns (1 soda lime, 1 silica gel, 1 1/3 Drierite, 2/3 Soda Lime) creating a dry, CO2-free airstream. The flow rate was then set to 100 or 300 ml/min (depending on the volume of the animal chamber) using a Sierra SideTrack 840 series mass flow valve (Sierra Instruments, Monterey, USA) controlled by a MFC2 mass flow controller (SSI). The airstream was directed to the A-cell of the Li-Cor 7000, and then into a MUX-2 intelligent multiplexer (SSI) housing eight chambers (either 10 or 30 ml depending on the size of the animal). The MUX-2 was programmed to sequentially measure each chamber using Expedata (SSI). Once passing through the MUX-2, the air-stream was directed to the Li-Cor 7000 B-cell. Data

acquisition was via Expedata. To reduce potential detrimental effects of desiccation, animals were flushed with a humidified air flow (approximately 82% relative humidity) between measurements. This was achieved using a second compressed gas flow, which was again scrubbed of CO2 and H2O vapour and directed to a Sierra mass flow valve with a flow rate of 35 ml/min. This air flow was then bubbled through a saturated potassium chloride (KCl) solution and directed to the flush input of the MUX-2. Four identical versions of the above respirometry set-up were constructed inside a Panasonic MLR- 352H-PE Climate Chamber (Panasonic Healthcare Co., Ltd, Sakata, Japan) which provided temperature control for the assays. VCO2 was measured at up to 10 set point temperatures randomised in the order 20°C, 10°C, 15°C, 30°C, 25°C, 35°C, 40°C, 42.5°C, 45°C and 47.5°C. Temperature was recorded using a type-T thermocouple (Omega

Engineering Inc., Stamford, USA) and a TC-2000 (SSI), and data was recorded using Expedata. All trials were completed under dark conditions to reduce the activity of the animals. Activity of the ticks was measured using AD-2 activity detectors (SSI). For the

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experiment, 34 fed adult females, 27 unfed adult females, 34 fed nymphs, 34 unfed adult males, and 25 unfed nymphs and were examined (Supplementary Table S2). Each animal was weighed before and after each temperature measurement using a Metter Toledo XP2U microbalance (Metter Toledo, Greifensee, Switzerland).

Data correction and extraction were performed using Expedata. Data were initially

corrected for flow rate in a push system (Lighton, 2008), nearest neighbour smoothed and drift corrected using baseline data collected every 30 minutes for a period of 5 minutes. Gas exchange patterns were then identified for each individual following Marais et al., (2005). For animals showing DGE (typically only unfed adults and nymphs at 15°C and 20°C), for each DGE cycle the mean duration and mean VCO2 of a total cycle, and the mean duration, emission volume and VCO2 for each of the open phase (O) and joint closed (C) and flutter (F) phases (CF phases), were extracted. The flutter and closed phases were jointly considered because the F-phase may commence before CO2 release is detected (Wobschall & Hetz, 2004; Groenewald, Chown & Terblanche, 2014). Typically, two cycles were analysed per individual to obtain a mean value for each trait at each temperature. Individuals were then used as independent data points per temperature. For continuous gas exchange (CGE), only mean VCO2 was calculated across the full period of recording (fed ticks and ticks measured from 25°C onwards, though some DGE was observed in ticks at 25°C). Mean VCO2 was estimated to determine variation with temperature, life cycle stage and starvation level (see below for rationale).

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The second approach used to verify spiracle closure was flow-through normoxic-anoxic- normoxic (NAN) respirometry (Lighton & Fielden, 1996). If the spiracles are closed, the introduction of anoxic air during the CF phase should have no effect because oxygen will not leave the animal by diffusion either through closed spiracles or once the spiracles are partially opened for the F phase. By contrast, rapid outward oxygen diffusion during anoxia would be expected if spiracles were not closed, resulting in a large CO2 burst (Lighton & Fielden, 1996). Ten unfed adult female ticks were each placed within a single 12 ml animal chamber housed inside an AD-2 activity detector (SSI) (see Supplementary Table S2). A Li-Cor 7000 was used to measure VCO2 at a sampling rate of 0.5 Hz. Flow rate was set at 100 ml/min using a Sierra mass flow valve and the airstream was scrubbed of residual CO2 and H2O vapour using the same scrubber system used in the metabolic rate assays. Animals were held in normoxic air at 20°C until they exhibited DGE, at which time data recording commenced. Once the animal had entered into a closed phase

(indicated by low VCO2) for a period of 5 minutes, the airstream was switched to anoxic air for a period of 5 minutes, and then back to normoxic air for a further 15 minutes. Data recording and extraction were as above. Gas exchange traces were then adjudicated visually for the effects of anoxic air (Lighton & Fielden, 1996; Klok, Mercer & Chown, 2002).