Ex vivo physiological experiments using small insect models such as Drosophila larvae have become increasingly useful to address fundamental biological questions. To perform such experiments, various artificial saline solutions have been developed, but their osmolality varies significantly from one to the next. Such a variation of osmolality stems, in part, from the difficulty of determining the true value of haemolymph osmolality in Drosophila larvae. Thus, there is a pressing need to refine protocols for collecting and measuring the osmolality of the larval haemolymph. Two major obstacles are thought to impede the accurate analysis of haemolymph collected from small insects: melanin formation and gut-derived contamination. Here, we greatly refined existing haemolymph collection methods, evaluated the purity of the collected haemolymph under melanin-free conditions, and concluded that the true value of haemolymph osmolality is close to 306.0 mOsm kg–1 in Drosophila larvae.

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