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Dragonflies (Odonata, Anisoptera) are amphibiotic; the nymph is aquatic and breathes water using a rectal gill before metamorphosing into the winged adult, which breathes air through spiracles. While the evolutionary and developmental transition from water breathing to air breathing is known to be associated with a dramatic rise in internal CO 2 levels, the changes in blood-gas composition experienced by amphibiotic insects, which represent an ancestral air-to-water transition, are unknown. This study measured total CO 2 (TCO 2 ) in hemolymph collected from aquatic nymphs and air-breathing adults of Anax junius , Aeshna multicolor (Aeshnidae), Libellula quadrimaculata and Libellula  forensis (Libellulidae). Hemolymph P  CO 2   was also measured in vivo in both aeshnid nymphs and marbled crayfish ( Procambarus fallax. f. virginalis ) using a novel fiber-optic CO 2 sensor. The hemolymph TCO 2 of the pre- and early-final instar nymphs was found to be significantly lower than that of the air-breathing adults. However, the TCO 2 of the late-final instar aeshnid nymphs was not significantly different from that of the air-breathing adults, despite the late-final nymphs still breathing water. TCO 2 and P  CO 2   were also significantly higher in the hemolymph of early-final aeshnid nymphs compared with values for the water-breathing crayfish. Thus, while dragonfly nymphs show an increase in internal CO 2 as they transition from water to air, from an evolutionary standpoint, the nymph's ability to breathe water is associated with a comparatively minor decrease in hemolymph TCO 2 relative to that of the air-breathing adult.

Changes in hemolymph total CO2 content during the water-to-air respiratory transition of amphibiotic dragonflies