A fluorescent biosensor enables high-sensitivity quantification of hemolymph trehalose in individual aphids.

OBJECTIVE: Trehalose is a major hemolymph sugar in aphids; however, its accurate quantification at the individual level has been technically challenging because of the small body size of aphids. To quantify hemolymph trehalose at the single-insect level in aphids using a GFP-based fluorescent biosensor (Tre‑C04), benchmark it against high-performance liquid chromatography (HPLC), and examine species- and wing-morph differences. RESULTS: HPLC of pooled adult Acyrthosiphon pisum hemolymph measured 218.9 ± 34.4 mM trehalose, while Tre‑C04 quantified 183.5-261.7 mM in individual adults (mean 227.3 ± 10.0 mM); the two methods did not differ significantly. Across nine species, single-insect measurements clustered near ~ 200 mM (191.7-249.2 mM), and we did not reproduce previously reported values > 600 mM in Aphis gossypii or Myzus persicae; pooling required by 1H-NMR likely increased concentrations via tissue contamination. Winged Sitobion ibarae had higher trehalose than wingless conspecifics, whereas Megoura crassicauda showed no morph‑dependent difference in trehalose levels under rainy or sunny conditions. These data validate Tre‑C04 for rapid, sensitive single‑insect sugar quantification, reveal a conserved trehalose set‑point of approximately 200 mM with modest species‑level variation, and indicate that morph effects are species-dependent.