Effects of wortmannin, sodium nitroprusside, insulin, genistein, and guanosine triphosphate on chemotaxis and cell growth of Entodinium caudatum, Epidinium caudatum, and mixed ruminal protozoa

The mechanisms by which ruminal protozoa sense and migrate toward nutrients are not fully understood. Chemotaxis by many diverse eukaryotic cells is mediated by phosphatidylinositol-3-kinase, which is highly conserved in receptor tyrosine kinase (RTK) signaling pathways and consistently inhibited by wortmannin. In experiment 1a, increasing the concentration of wortmannin inhibited cell growth nonlinearly at 24 h of a culture of the rumen protozoan Entodinium caudatum, but high variability prevented growth inhibition of Epidinium caudatum from reaching significance. In experiment 1b, increasing the insulin concentration recovered 24-h cell counts for both cultures, depending on wortmannin concentration. In experiment 2, addition of sodium nitroprusside (Snp; activator of protein kinase G for cilial beat reversal in nonrumen ciliate models) at 500 µM or wortmannin at 200 µM in beakers containing rumen fluid decreased random swimming by mixed entodiniomorphids into capillary tubes (inserted into beakers) containing saline. Both Snp and wortmannin increased chemotaxis into tubes containing glucose compared with the beaker control. For isotrichids, beaker treatments had no response. Glucose increased chemotaxis, but peptides decreased chemotaxis even when combined with glucose. In experiment 3, we assessed preincubation of genistein (a purported RTK blocker in nonrumen ciliate models) at 40 or 400 µM in beakers and guanosine triphosphate (GTP; a universal chemorepellent in nonrumen ciliate models, perhaps mediated through an RTK) at 10 or 100 µM combined with glucose in capillary tubes. Neither genistein nor GTP affected chemotaxis toward glucose for entodiniomorphids. However, GTP at 100 µM reduced chemotaxis toward glucose for isotrichids. After the animal is fed, isotrichids that are depleted in glycogen migrate to the dorsal area of the rumen, and the rapid uptake of sugars is enhanced through strong chemotaxis but can be reversed by peptides or GTP. In contrast, entodiniomorphids are less intensely chemoattracted to glucose than isotrichids but are chemoattracted to peptides. Entodiniomorphids' chemoattraction appears to be integrated with slower but prolonged availability of energy from digesting starch and fiber.