All developing embryos contain an extracellular matrix (ECM) consisting of proteins, glycoproteins, and proteoglycans. These components are important for morphogenetic processes such as cell migration, cell differentiation and cell death. The ECM of the starfish, Pisaster ochraceus, consists of three major components: A hyaline layer which coats the external surface of the embryo; a basal lamina which lines the basal surfaces of the epithelia; and a blastocoelic component which fills the embryonic cavity or blastocoel. Observations of chemically fixed asteroid embryos have revealed the hyaline layer to contain five sub-layers of fibrous strands encrusted with amorphous material. Strands of a similar nature form a meshwork within the fluid-filled blastocoel. Recent studies of the living embryo, however, have suggested that the ECM within the blastocoel of echinoderms, including the asteroid, is a gel-like substance and not a fluid with extracellular fibres. Since artefacts imposed by chemicals such as aldehydes and osmium are well documented, a method of preservation, which does not involve the use of these chemicals, may resolve the apparent conflict over the nature of the ECM of the asteroid embryo. Freeze substitution, an expensive cryofixation technique which has proven successful in fixing vertebrate tissue, does not require the use of aldehydes and osmium. The initial objective of this study was to devise an inexpensive, easily employable freeze substitution technique which would allow good preservation of cellular and extracellular elements of the embryonic starfish, Pisaster ochraceus. A plunge freezing apparatus was constructed which consisted of a Dewer flask filled with liquid nitrogen, a small cup was filled with cryogen and inserted into the nitrogen, and a motor which constantly stirred the cryogen. Embryos were isolated on copper freeze-fracture grids and plunged into the cryogen. After considering four different cryogens and four separate cryoprotectants, cryoprotecting asteroid embryos with propylene glycol and plunging them into supercooled propane was found to provide optimal preservation. Frozen embryos were freeze substituted in anhydrous ethanol at -90 °C, osmicated, and embedded for ultrastructural and histochemical analysis. Following freeze substitution, the blastocoel appears to contain a gel-like substance, rich in sulfated GAG's, with extracellular fibres and not a fluid with fibres. In addition, the hyaline layer was found to consist of at least six sub-layers of greater thickness than was seen in chemically fixed embryos. Histochemical studies demonstrated that both sulfated and unsulfated GAG's were present in these layers. The morphological differences among the sub-layers suggest that some sub-layers may have unique functions while others may have functions shared by other sub-layers. Freeze substitution also revealed the presence of microvillus associated bodies, structures which may represent major attachment points of the hyaline layer to the epithelium. Although the fixation of asteroid embryos by freeze substitution is a lengthy process, taking four to five days, the resulting preservation, particular!ly of the ECM components, justifies its use over chemical fixations. Material preserved by freeze substitution can be used for histochemical studies and, since aldehydes and heavy metals are not necessary for successful preservation, may also prove useful for immunocytochemical studies. / Medicine, Faculty of / Graduate

Most climate change predictions focus on the response of individual species to changing local conditions and ignore species interactions, largely due to the lack of a sound theoretical foundation for how interactions are expected to change with climate and how to incorporate them into climate change models. Much of the variability in species interaction strengths may be governed by fundamental constraints on physiological rates, possibly providing a framework for including species interactions into climate change models. Metabolic rates, ingestion rates and many other physiological rates are relatively predictable from body size and body temperature due to constraints imposed by the physical and chemical laws that govern fluid dynamics and the kinetics of biochemical reaction times. My dissertation assesses the usefulness of this framework by exploring the community-level consequences of physiological constraints. In Chapter 2, I incorporated temperature and body size scaling into the biological rate parameters of a series of realistically structured trophic network models. The relative magnitude of the temperature scaling parameters affecting consumer energetic costs (metabolic rates) and energetic gains (ingestion rates) determined how consumer energetic efficiency changed with temperature. I systematically changed consumer energetic efficiency and examined the sensitivity of network stability and species persistence to various temperatures. I found that a species' probability of extinction depended primarily on the effects of organismal physiology (body size and energetic efficiency with respect to temperature) and secondarily on the effects of local food web structure (trophic level and consumer generality). This suggests that physiology is highly influential on the structure and dynamics of ecological communities. If consumer energetic efficiency declined as temperature increased, that is, species did best at lower temperatures, then the simulated networks had greater stability at lower temperatures. The opposite scenario resulted in greater stability at higher temperatures. Thus, much of the community-level response depends on what species energetic efficiencies at the organismal-level really are, which formed the research question for Chapter 3: How does consumer energetic efficiency change with temperature? Existing evidence is scarce but suggestive of decreasing consumer energetic efficiency with increasing temperature. I tested this hypothesis on seven rocky intertidal invertebrate species by measuring the relative temperature scaling of their metabolic and ingestion rates as well as consumer interaction strength under lab conditions. Energetic efficiencies of these rocky intertidal invertebrates declined and species interaction strengths tended to increase with temperature. Thus, in the rocky intertidal, the mechanistic effect of temperature would be to lower community stability at higher temperatures. Chapter 4 tests if the mechanistic effects of temperature on ingestion rates and species interaction strengths seen in the lab are apparent under field conditions. Bruce Menge and I related bio-mimetic estimates of body temperatures to estimates of per capita mussel ingestion rates and species interaction strengths by the ochre sea star Pisaster ochraceus, a keystone predator of the rocky intertidal. We found a strong, positive effect of body temperature on both per capita ingestion rates and interaction strengths. However, the effects of season and the unique way in which P. ochraceus regulates body temperatures were also apparent, leaving room for adaptation and acclimation to partially compensate for the mechanistic constraint of body temperature. Community structure of the rocky intertidal is associated with environmental forcing due to upwelling, which delivers cold, nutrient rich water to the nearshore environment. As upwelling is driven by large-scale atmospheric pressure gradients, climate change has the potential to affect a wide range of significant ecological processes through changes in water temperature. In Chapter 5, my coauthors and I identified long-term trends in the phenology of upwelling events that are consistent with climate change predictions: upwelling events are becoming stronger and longer. As expected, longer upwelling events were related to lower average water temperatures in the rocky intertidal. Furthermore, recruitment rates of barnacles and mussels were associated with the phenology of upwelling events. Thus climate change is altering the mode and the tempo of environmental forcing in nearshore ecosystems, with ramifications for community structure and function. Ongoing, long-term changes in environmental forcing in rocky intertidal ecosystems provide an opportunity to understand how temperature shapes community structure and the ramifications of climate change. My dissertation research demonstrates that the effect of temperature on organismal performance is an important force structuring ecological communities and has potential as a tractable framework for predicting the community level effects of climate change. / Graduation date: 2013 / Access restricted to the OSU Community, at author's request, from Nov. 20, 2012 - Nov. 20, 2014