Although isotrichid protozoa have been studied for their migratory behavior followed by sedimentation to the ventral reticulum and rumen, it has not been determined whether the more predominant entodiniomorphid species exhibit chemotaxis following feeding and, if so, whether they also have a detrimental effect on the amount of dietary protein that becomes available to the cow. In chapter 3, I hypothesized that chemotaxis toward nutrients is responsible for the attraction of entodiniomorphid protozoa toward feed particles and therefore pass with the potentially digestible particulate phase (or phases if there is a buoyancy delay). In Chapter 3, objectives were to compare dose-responsive chemotaxis by isotrichids and entodiniomorphids to glucose (Experiment 1), glucose or xylose when protozoa were harvested from a fed cow (Experiment 2), peptides of bacterial, protozoal, and soy origin (Experiment 3), or toward glucose when mixed ruminal protozoa were previously incubated for 0, 3, or 6 h in the presence of emulsified polyunsaturated fatty acids (PUFA; Liposyn II). In experiment 1 isotrichid protozoa decreased chemotaxis toward increasing glucose concentration when the cow was fasted. However, entodiniomorphids exhibited chemotaxis to a lesser degree, regardless of feeding, but to similar glucose concentrations as isotrichids. In experiment 2, our results do not support xylose as a major chemotactic molecule that draws protozoa toward non-starch particulate matter in the rumen. Experiment 3 showed that entodiniomorphids are not selectively chemoattracted toward bacterial or protozoal peptides. In experiment 4, despite isotrichid populations being inhibited by fat, chemotaxis to glucose remained unchanged. In contrast, chemotaxis by entodiniomorphids was more complex and glucose tended to recover loss of chemotaxis from PUFA incubation.
In chapter 4, the objective of experiment 1 was to test the hypothesis that wortmannin (an inhibitor of phosphatidylinositol-3-kinase) would inhibit growth of single cultures of the rumen protozoa Entodinium caudatum and Epidinium caudatum unless cultures were subsequently rescued dose-responsively by increasing concentrations of insulin added to the culture medium. Results showed dose-responsive insulin recovery from wortmannin inhibition by both cultures. In Experiment 2 of chapter 4, I hypothesized that wortmannin should decrease chemotaxis, insulin should recover chemotaxis in wortmannin-incubated treatments, and sodium nitroprusside (SNP; activates guanylyl cyclase and resultant activation of protein kinase G) should increase chemotaxis toward glucose and peptides as in other ciliate models from aerobic ecosystems, but we expected interactions in insulin would overcome inhibition by WORT but not SNP. Sodium nitroprusside at 500 μM and wortmannin at 200 μM decreased (P < 0.05) random swimming to saline control but both increased (P < 0.05) chemotaxis toward glucose for entodiniomorphids. Sodium nitroprusside at 500 μM and wortmannin at 200 μM decreased chemotaxis toward glucose or peptides on isotrichids. Peptides also decrease chemotaxis on isotrichids. In Experiment 3, we assessed the role of receptor tyrosine kinases (RTK) on rumen protozoal chemotaxis toward glucose using genistein and guanosine triphophate (GTP; universal chemorepellent by ciliates). Neither genistein (reported RTK blocker in other ciliate models) nor GTP affected chemotaxis toward
glucose for entodiniomorphids. However, GTP at 100 μM reduced chemotaxis toward glucose for isotrichids. The repellence by GTP was reversed when cells were incubated in the presence of genistein at 40 or 400 μM.
In chapter 5, we screened and evaluated dose-responsiveness of various compounds used to study motility of non-rumen ciliates. The current objectives were to evaluate effects and potential interactions among wortmannin (blocking phosphoinositide signaling used in phagocytosis or chemotaxis), insulin (previously rescued wortmannin’s inhibition of cell growth, potentially through a RTK), genistein (RTK blocker, potentially inhibiting chemotaxis), U73122 (inhibitor of phospholipase C, PLC, potentially disrupting Ca++ gradients needed for swimming and turning), and sodium nitroprusside (SNP, protein kinase G activator to enhance turning toward a chemoattractant) preloaded for 3 h in flocculated ruminal fluid maintained anaerobically at 39°C. Chemoattractants were glucose, peptides, and their combination; peptides also were combined with GTP). Isotrichids increased chemotaxis to glucose, which was countered by wortmannin. Peptides were strongly chemorepellent to isotrichids, even in the presence of glucose and especially when preloaded with genistein or SNP. In this study, GTP did not reverse peptide repellence by isotrichids or chemoattraction by entodiniomorphids. For entodiniomorphids, U73122 increased random swimming into saline controls. As in a previous study, wortmannin decreased or did not affect random swimming into saline but enhanced chemotaxis by entodiniomorphids to glucose. Wortmannin’s opposite results for entodiniomorphids versus isotrichids appear to be mediated through differences in vacuolization or, more likely, through RTK signaling. For entodiniomorphids, motility toward chemoattractants appears to be sensitized by energy deprivation (as with
wortmannin inhibition of digestive vacuole formation and recycling). Turning toward gradients is mediated through PKG; however, we could not support a direct PLC role because results were opposite of results from other ciliate models.