Evolutionary insights into gut acidification: Invertebrate-like mechanisms in the basal vertebrate hagfish.

Acidification is a key component of digestion throughout metazoans. The gut digestive fluid of many invertebrates is acidified by the vesicular-type H+-ATPase (VHA). In contrast, vertebrates generate acidic gut fluids using the gastric H+/K+-ATPase (HKA); an evolutionary innovation linked with the appearance of a true stomach that greatly improves digestion, absorption, and immune function. Hagfishes are the most basal extant vertebrates, and their mechanism of digestive acidification remains unclear. Herein we report that the stomachless Pacific hagfish (Eptatretus stoutii) acidify their gut using the VHA, while searches of E. stoutii gut transcriptomes and the genome of a closely related hagfish species (E. burgerii) indicate they lack HKA, consistent with its emergence following the 2R whole genome duplication. Immunostaining revealed prominent VHA presence in the apical membrane of enterocytes and sub-apical expression of both VHA and sAC. Interestingly, akin to vertebrates, VHA was also observed in immature pancreatic-like zymogen granules and was noticeably absent from the mature granules. Furthermore, isolated gut sacs from fed hagfish demonstrate increased VHA-dependent luminal H+ secretion that is stimulated by the cAMP pathway. Overall, these results suggest that the hagfish gut shares the trait of VHA-dependent acidification with invertebrates, while simultaneously performing some roles of the pancreas and intestine of gnathostomes.