SUPPLEMENTARY MATERIALS AND METHODS:
Respirometry:
Flow-through respirometry was performed at 22-23 °C using two different
setups. A control setup was used to measure adult locusts that were not restrained or
implanted with optodes. These insects were measured in normoxia (n = 9) using
outside air scrubbed of CO2 and water vapour, as well as hypoxia (7.1 kPa, n = 4) and
hyperoxia (40.5 kPa, n = 4) using gas mixes produced by 0-1000 ml min-1 and 0-500
ml min-1 mass flow controllers (GFC-171, Aalborg, New York, USA) connected to
cylinders of compressed O2 and N2. Each locust was placed within an 8 ml
respirometry chamber constructed from a 10 ml syringe barrel and kept in semidarkness. Gas exchange was measured over an 8 h (n=5) or 2 h (n=4) period
following 1 h of acclimation. Briefly, the scrubbed air or gas mix was directed
through a mass flow controller (0–1000 ml min–1, Model 810C, Mass-Trak, Sierra
Instruments, Monterey, CA, USA; calibrated with a bubble flow meter, Gilibrator,
Sensidyne, Clearwater, FL, USA) at 350 ml min–1 standard temperature and pressure,
dry (STPD) then through the syringe barrel containing the resting locust. Excurrent air
from the respirometry chamber was directed through a small Drierite column to
remove water vapour before it entered a CO2 analyser (LI-820 LI-COR Biosciences,
Lincoln, NE, USA). The analogue outputs of the mass flow controllers and the CO2
analyser were recorded at 1 s intervals to a computer with a PowerLab data
acquisition system and LabChart software (ADInstruments, Bella Vista, NSW,
Australia). Immediately before and after each experiment the mass of each locust was
recorded to 0.1 mg on an analytical balance (AE163, Mettler, Greifensee,
Switzerland).
To measure intratracheal PO2 and CO2 release simultaneously, locusts (n = 3)
were restrained within a respirometry chamber constructed from a rectangular ABS
plastic enclosure (83 × 54 × 31 mm). The enclosure was mounted on an x/y rotational
stage (XYR1/M, Thor Labs, New Jersey, USA) beneath an Olympus VMZ dissecting
microscope (Olympus Australia, VIC, Australia). Adult locusts were cold knockedout by placing them in a freezer for 5-10 minutes. While immobilised, they were
quickly secured by their thorax to the lid of the respirometry chamber using melted
dental wax (Ainsworth Dental Company Pty. Ltd., Marrickville, NSW, Australia).
When the lid was screwed on to the respirometry chamber the locust was positioned
on a Perspex platform within the chamber with its abdomen directly above a reflective
IR detector (SFH 9202, Osram Opto Semiconductors, Regensburg, Germany). A 5
mm diameter hole in the lid of the chamber provided access to the locust’s thorax for
implantation of the oxygen optode. Flat-tipped, 140 µm diameter oxygen optodes
connected to an oxygen meter (TX3, PreSens GmbH, Germany) were calibrated using
a two-point calibration in pure N2 and a mix of O2 and N2 with a PO2 21.3 kPa. The
accuracy of the calibrated optodes were also checked in hyperoxia (PO2 = 40.5 kPa)
before implantation. A piece of cuticle less than 1 mm in diameter was cut from the
dorsal surface of the prothorax, either to the left- or right-hand side of the midline,
using a 25 gauge hypodermic needle. There was negligible bleeding during this
process as the haemolymph appeared quite viscous. The silvery surface of a thoracic
air sack was clearly visible through this incision. The air sack was then punctured
using the hypodermic needle and an X-Y-Z micromanipulator (World Precision
Instruments, USA) was used to carefully insert the oxygen optode through the hole in
the respirometer’s lid, and into the locust’s air sack (Figure S1). Once in place, a pin
was used to apply a small drop of polyvinylsiloxane polymer (President light body
dental impression material, Coltène Whaledent, Switzerland) to the optic fibre just
above where it penetrated the cuticle. The drop would run down the fibre and, upon
reaching the cuticle, would spread out, thereby forming an air-tight seal between the
cuticle and the optic fibre (figure S1). Once this had set, a small square of aluminium
foil, with a slit running from one edge to its centre to accommodate the optode, was
placed over the hole in the lid of the respirometry chamber. More polyvinylsiloxane
was applied over the top of the aluminium foil, where it would spread out over the lid
of the respirometry chamber, thereby sealing the optode into the lid of the
respirometry chamber (Figure S1). Within minutes the polymer had set completely
and a gas mixture of 21.3 kPa PO2 was passed from the mass flow controllers,
through a mass flow meter (0-1000 ml min-1, GFM-171S, Aalborg, New York, USA)
at 450 ml min-1, through the chamber and then into the Li-COR 820. A minimum of 1
h was then given before measurements began. Locusts were exposed to gas mixes
containing 5.1, 10.1, 15.2, 21.3, 30.4 and 40.5 kPa PO2. Voltage outputs from the
oxygen meter, mass flow meter, IR activity detector, and CO2 analyser were recorded
by a PowerLab data acquisition system and LabChart software running at 10 Hz.
Figure S1. Cutaway diagram showing the respirometry chamber used to measure CO2
release, intratracheal O2 level and abdominal pumping frequency simultaneously. The
inset shows location of the oxygen optode within the locust’s thoracic air sac. The
direction of air flow through the respirometry chamber is indicated by arrows.