Let me ask you a few questions in order to help you. You understand that in any case you will need to recalculate the data obtained at low temperatures, for comparison with standard measurements at a certain temperature level, barometric pressure and humidity. I suggest that you fix all these parameters. Secondly, I know that the caterpillar lives at different temperatures. You need it for low temperature measurements. Still, let me tell the truth. Measurement capabilities depend on the quality of measuring instruments. I ask you to see the article. Perhaps the experience of the authors of this article will help you. Good luck in your work.

Hi,

What kind of difficulties are you experiencing? Are they technical or biological ones?  We are also performing flow-through respirometry using Sable Systems equipment.  Maybe I could help you.

Dear Claudio. I noticed that the young scientist was not satisfied with my answer. Please help Spiro understand the features of the methodology, which as you know has a number of important nuances. All the best.

Thank you both (Claudio and Vladimir) for your responses. Claudio, I think the difficulties may be a bit of both, technical and biological. The biological difficulty is dealing with very low MR at near freezing temperatures. The technical difficulty I believe is mostly due to my current setup. For example, for flow through measurements using the push method, the “plumbing” (i.e., tubing, scrubbing tubes, and the small respirometer chamber holding the animal) may be too extensive. The analyzers are on a bench with the incubator housing the animal just next to it. With the very low flow rate (~10ml/min) that I think is required, I am not detecting a drop in O2 or increase in CO2…I have run animals for as long as two hours. To give you a bit more info, I am using Sable systems UI-2 as the interface and their software but the analyzers are old AMETEK systems that are prone to drifting, especially the O2 analyzer.

I would focus on the change in carbon dioxide. It is present at a much lower concentration than oxygen in ambient air, thus its variation should be easier to detect and interpret. Also, consider adding multiple animals to the chamber at one time. If you know the masses of all individuals, you can calculate a mass-specific rate for the entire group. And, if masses of individuals are similar, then it would be reasonable to assume that this mass-specific rate is similar across individuals. If you do this, I suggest describing this experimental setup explicitly in your methods when you report results. Good luck.

Hi, I agree with John, you are probably at the limits of the sensitivity of your system.  CO2 measure is usually easier (more sensitive and stable instruments than for measuring O2).  I would also try measuring in stop-flow, to have an idea of the CO2 production rate of your animals. I do no think that low flow would be a problem; we also use reduced flow for insects with low metabolism.  However, if the volume of the chamber is to big, cleaning them may take long time and you miss the dynamics of the emission.  Do you know the respiration pattern of your insects?  If they perform discontinuous respiration and spiracles open at long intervals (in particular at low temperature), maybe that you are simply  missing the exchanges. All the best.

Claudio and John, Thank you for your responses and guidance. I have done some preliminary close-system CO2 measurements using a 60ml syringe barrel as the respirometry chamber. I drilled a hole at the far end that allowed me to flush the barrel with fresh air and take an initial air sample. Then I moved the plunger past the opening to the 40ml mark to seal the syringe for one hour. I used a syringe infusion pump to push the final sample through the CO2 analyzer at a constant rate (15ml/min)..this was done with the animal in the syringe. I think this method will work fine and have the benefit of running multiple animals at the same time. At these low temperatures, I don't worry much about animal movement, as you normally would with a closed-system setup. Thank you again. Spiro

Close system CO2 sensors and dip analyzers based on mass spectrometery can analyse respiration in caterpillars.