The rhythmic defecation behavior in the nematode C. elegans represents a unique opportunity to study acid-base regulation and its intersection with oscillatory calcium signaling in a genetic model organism. The defecation period is ~50 seconds and behavior is initiated by a calcium wave that propagates through the twenty cells of the nematode intestine^1,2,3. We have shown using a combination of fluorescent biosensors and dynamic imaging that pH oscillates during defecation, as well⁴. These oscillations contribute to the behavioral output and help to maintain normal growth of the organism. Between behavioral episodes, the lumen of the intestine rests ~pH 4.1. As the behavior initiates, proton equivalents cross the apical membrane, causing robust cellular acidification coincident with luminal alkalinization. We hypothesize that resting luminal pH facilitates nutrient absorption through proton-coupled processes, and that protons which would otherwise be lost during expulsion of the luminal contents are instead conserved during defecation through entry into the cell. In support of this idea, deletion of VHA-6, an intestine-specific V-ATPase a subunit, or proton pump, that resides at the apical membrane, results in starvation and early larval arrest. To study VHA-6 function during defecation in adult worms we used single-generation RNA interference of vha-6. This treatment reduced vha-6 expression, prevented reacidification of the intestinal lumen following defecation, caused a loss of fat stores, and prevented absorption of dipeptides through the proton-dipeptide symporter OPT-2, confirming our hypothesis. An mCherry-tagged fusion construct was shown to rescue the vha-6 mutant phenotype, and we are exploring the use of this system to study calcium-regulated trafficking and assembly of V-ATPase subunits as well as a genetic model for treating acid-peptic disorders. A second rationale for rhythmic cellular acidification revolves around a unique method for rapid signaling between adjacent cells. The first motor step of the digestive program, the posterior body wall muscle contraction (pBoc), occurs without neuronal input. Instead, protons are extruded from the acidified intestine across the basolateral membrane. These protons act as fast transmitters to elicit a muscle response. Of particular novelty is the finding that a sodium-proton exchanger, NHX-7 (also called PBO-4), underlies intestinal proton signaling to muscles^4,5; this is the first example of this well-characterized, widespread, and evolutionarily conserved class of transporter being involved in cell communication. NHX-7 activity is precisely timed but the mechanism underlying its activation is unclear. Our data suggest that calcium is not only the pacemaker for timing defecation events, but is also required for triggering NHX-7 activity, and the cytoplasmic C-terminus of NHX-7 contains multiple consensus binding and phosphorylation motifs for potential regulation by calcium. In addition, mutation of a calcineurin homologous protein (chp-1/pbo-1), a co-factor for sodium-proton exchangers in mammals, results in a defect in pBoc as well as a starvation phenotype. Chp-1/pbo-1 may in fact coordinate the cyclic calcium, proton signaling, and nutrient uptake through interaction with multiple worm sodium-proton exchangers in the intestine.