Non-invasive monitoring of insect hemolymph pH and oxygenation by L-band EPR spectroscopy

Abstract Using L-band EPR spectroscopy, the acidity and oxygen concentration in the hemolymph of living Galleria mellonella larvae were determined in a non-invasive manner. As spin probes, we used a pH-sensitive nitroxyl radical with pK = 6.4 and a C-centered trityl spin probe (TAM OX063) as an oxygen probe. The pH values in the hemolymph of native larvae were 6.1–6.2. The oxygen concentration is about 150 µM, which is 30% less than the solubility of oxygen in water. Infection of larvae with the entomopathogenic fungus Metarhizium robertsii led to a significant decrease in the acidity of the hemolymph and depended on the degree of infection of the insects. Injection with immune activators, LPS and zymosan, did not lead to a significant change in pH in the hemolymph. However, zymosan led to a visible decrease in oxygen concentration, which is explained by the initiation of the melanization process in the hemolymph. In summary, the study demonstrates the capability of low-field EPR and spin probes for in vivo non-invasive monitoring of pH and oxygen content with good accuracy in investigations of various insects.