Apocrine secretion in the salivary glands of and other dipterans is evolutionarily conserved.

Apocrine secretion is a transport and secretory mechanism that remains only partially characterized, even though it is evolutionarily conserved among all metazoans, including humans. The excellent genetic model organism holds promise for elucidating the molecular mechanisms regulating this fundamental metazoan process. Two prerequisites for such investigations are to clearly define an experimental system to investigate apocrine secretion and to understand the evolutionarily and functional contexts in which apocrine secretion arose in that system. To this end, we recently demonstrated that, in , the prepupal salivary glands utilize apocrine secretion prior to pupation to deliver innate immune and defense components to the exuvial fluid that lies between the metamorphosing pupae and its chitinous case. This finding provided a unique opportunity to appraise how this novel non-canonical and non-vesicular transport and secretory mechanism is employed in different developmental and evolutionary contexts. Here we demonstrate that this apocrine secretion, which is mechanistically and temporarily separated from the exocytotic mechanism used to produce the massive salivary glue secretion (Sgs), is shared across and two unrelated dipteran species. Screening more than 30 species of from divergent habitats across the globe revealed that apocrine secretion is a widespread and evolutionarily conserved cellular mechanism used to produce exuvial fluid. Species with longer larval and prepupal development than . activate apocrine secretion later, while smaller and more rapidly developing species activate it earlier. In some species, apocrine secretion occurs after the secretory material is first concentrated in cytoplasmic structures of unknown origin that we name "collectors." Strikingly, in contrast to the widespread use of apocrine secretion to provide exuvial fluid, not all species use exocytosis to produce the viscid salivary glue secretion that is seen in . Thus, apocrine secretion is the conserved mechanism used to realize the major function of the salivary gland in fruitflies and related species: it produces the pupal exuvial fluid that provides an active defense against microbial invasion during pupal metamorphosis.