In the past few decades there has been an explosion of measurement techniques that have made finer and more intricate measurements on smaller invertebrates more commonplace. Developments in electronic and sensor instrumentation have allowed insect physiologists to piece together complex respiratory patterns in insects as small as Drosophila, to detect and quantify neurocontrol and muscular control in single neurons and muscle fibres, and to measure rapid changes in body temperature in individual micro-arthropods (mites and springtails — some the size of the period ending this sentence). Now Maxim Dokukin and colleagues, from Clarkson University, have added a new technique to the insect physiologist's tool kit, atomic force microscopy (AFM), allowing them to make finer, more intricate and higher frequency measurements of processes that were previously undetected (or even undetectable) in insects the size of ladybird beetles and smaller.
AFM employs a sharp, cantilevered probe that detects small variations in the sample surface, which necessitates very secure mounting to restrict animal motion. Most commonly the small cantilever deflections are detected optically with a laser reflected onto a photodiode. The reflections are then translated into force measurements with picometer precision. Recording the reflections with a high frequency (50,000 Hz) system, Dokunin's team measured the surface vibrations of adult ladybird beetles, Hippodamia convergens.
Once a sample is securely mounted, surface vibrations are recorded at high frequencies and very small amplitudes. To distinguish animal vibrations from background noise, the team made additional measurements on dead specimens and of room sounds. They then quantified patterns in these vibration recordings and interpreted them mathematically using Fourier spectral peak analyses — where peak height indicates amplitude and peak position frequency. Dokukin's team then used three approaches to identify physiological processes from spectral peaks: they placed the probe close to a target organ, induced a drinking stimulus, and measured the insects' responses to increasing air CO2 levels.
With the probe tip close to the heart the AFM physical approach showed previously documented heartbeat patterns at 0.6 Hz, and also a new high frequency peak at 293 Hz related to heart activity. Giving the beetles sugar water to drink the team identified a reduction in certain low frequency spectral peaks (15—17 Hz), shifts in higher frequency peaks (from 280 to 273 Hz), and new peaks at 18—20 and 350 Hz. The peak reductions and/or shifts may imply movement of fluids affecting tissue elasticity, while the new peaks may indicate muscle activities. Finally, when the team increased air CO2 levels they found that low frequency (<5 Hz) activities were suppressed and the 280 Hz peak decreased by 40% and was shifted to 273 Hz. These responses were similar to those recorded when the insects were drinking and may be a general response to an external stimulus. But there were also new peaks observed, strong at 133 Hz and weaker at 170, 350 and 480 Hz, which are likely associated with spiracular closer muscle activities.
These measurements are based only on minute picometer scale vibrations off the elytral surface and will require several extensive studies to understand the full physiological significance of the observations. Nevertheless, the authors' novel AFM approach has shown that selective positioning, application of external stimuli and mathematical interpretation of spectral patterns have identified several previously undetected physiological processes and this, as the authors said, ‘could lead to the emergence of “insect nanophysiology”’.
