Techn. Universiẗat, Diss., 2003--Braunschweig.

Intertidal habitats are characterized by dynamic, tidally-driven fluctuations in abiotic and biotic factors. Many of the environmental stressors that vary across the intertidal (e.g., temperature, oxygen, food availability, predation pressure) are strong drivers of metabolic rate in ectotherms. As such, we predicted that there may be pronounced differences in the metabolic and stress physiology of conspecific sessile invertebrates occupying at different relative tidal heights. The common acorn barnacle Balanus glandula represents an ideal model organism in which to investigate the possibility of tidal height-dependent physiological differences, owing to their wide distribution in the intertidal zone and their eurytolerant nature. In the first chapter of my thesis, we investigate the hypothesis that B. glandula anchored in the low intertidal have a greater capacity for anaerobic metabolism than conspecifics in the high intertidal, and that this is due to increased predation pressure during submersion. Further, we explore the temporal and spatial fidelity of certain tidal-height driven trends in lactate dehydrogenase activity previously observed in our lab (i.e., higher LDH activity in low intertidal barnacles; Horn et al., 2021), and attempt to identify environmental variables that drive plasticity in LDH activity. We found that, in general, there were higher densities of B. glandula and gastropod whelk predators in the low intertidal compared to the high intertidal, but follow-up studies in the lab revealed that opercular closure in B. glandula was induced by predator exposure (Acanthinucella spirata) for less than 24h. This time frame for shell closure is unlikely to result in internal hypoxia or enhance capacity for anaerobic metabolism. We were therefore not surprised to find that LDH activity in B. glandula was likewise not affected by predator exposures (48h) carried out in the lab. After failing to find an effect of predators on LDH activity in B. glandula, we attempted to replicate the previous finding that LDH activity was highest in low intertidal populations of B. glandula. We did this at the original location in San Luis Obispo Bay, CA as well as at three novel field sites and across seasons and years. While we did observe variation in LDH activity over time and between sites, we did not consistently observe the same trend in LDH activity whereby low intertidal barnacles had the highest activity. In response to these variable patterns, we attempted to identify what environmental parameters, other than predation, might be responsible for plasticity in LDH activity. Unfortunately, neither temperature nor emersion stress – the two variables we examined – had any significant an effect on LDH activity in B. glandula. These data suggest that there must be multiple, interacting stressors – including tidal position - that influence the anaerobic metabolic capacity of B. glandula. In the second chapter of my thesis, we went on to investigate how the response to thermal stress might differ between populations of B. glandula from different vertical heights in the intertidal zone. To this end, we assessed how aerial temperature stress affected oxygen consumption rates (MO2), superoxide dismutase (SOD) activity, and time to mortality in B. glandula collected from both low and high intertidal positions. We found that barnacles from the low intertidal showed a significant increase in MO2 with higher temperature, while MO2 was unaffected by temperature in B. glandula from the high intertidal. We also observed that SOD activity levels were higher in the high intertidal barnacles compared to the low intertidal barnacles, although neither group was increasing SOD activity under higher temperature. Finally, we observed significantly longer survival times during thermal stress in barnacles from the high intertidal zone (e.g., LT50 = 8.75 h vs 5 h at 33˚C for the high and low barnacles, respectively), although this advantage seemed to be lost with the addition of desiccation stress at these same temperatures. It is evident that life in highest reaches of the intertidal zones is physiologically challenging, and this has resulted in a population of B, glandula barnacles that are less sensitive to and better suited to tolerate temperature extremes than conspecifics in the lowest intertidal regions. Understanding how habitat variation may differentially impact the metabolic and thermal stress physiology of B. glandula is increasingly important as climate change progresses. This is particularly significant considering that organisms in the intertidal already reside within a relatively stressful environment and may be living closer to their thermal tolerance limits than animals from less extreme habitats.

Barnacles are dominant hard–fouling organisms in marine waters. They attach to substrates by secreting a complex proteinaceous adhesive. Understanding the chemical composition of this multi–protein underwater adhesive and how it is affected by environmental variables, such as oceanic temperatures, is critical for developing nontoxic solutions to control biofouling. Previous experiments in our lab revealed an inverse relationship between critical removal stress (CRS) and temperatures at which barnacles were reared. Further investigations showed that this correlation is not attributed to differences in physical properties such as barnacle size or short–term changes in the viscosity of adhesive. Therefore, the observed effects may be influenced by a physiological response to temperature during initial growth and development. We hypothesized that rearing temperature affects the expression of proteins found in the adhesive matrix. To elucidate the underlying mechanisms responsible for the temperature effect, we analyzed uncured barnacle adhesive using two-dimensional gel electrophoresis (2DGE) and matrix-assisted laser desorption/ionization-tandem time-of-flight (MALDI-TOF/TOF) mass spectrometry (MS). In our analysis, we 1) detected differences in protein expression at two experimental temperatures (15°C and 25°C) and 2) identified several proteins that may serve functional roles in the process of adhesion. Our data are also consistent with a model that the curing process of barnacle adhesive may be analogous to the process of wound healing in animals.

The intertidal zone is characterized by persistent, tidally-driven fluctuations in both abiotic (e.g., temperature, [O2], salinity) and biotic (e.g., food availability, predation) conditions, which makes this a very physiologically challenging habitat for resident organisms. The magnitude and degree of variability of these environmental stressors differs between intertidal zones, with the most extreme physiological stress often being experienced by organisms in the high intertidal. Given that many of the fluctuating conditions in this environment are primary drivers of metabolic rate (e.g., temperature, [O2], food availability), we hypothesized that sessile conspecifics residing in different tidal zones would exhibit distinct ‘metabolic phenotypes,’ a term we use to collectively describe the organisms’ baseline metabolic performance and capacity. To investigate this hypothesis, we collected acorn barnacles (Balanus glandula) from low, mid, and high intertidal positions in San Luis Obispo Bay, CA and measured a suite of biochemical (whole-animal citrate synthase (CS) and lactate dehydrogenase (LDH) activity, aerial [lactate]), physiological (O2 consumption rates), morphological (body size), and behavioral (e.g., cirri beat frequency, % time operculum open) indices of metabolism. We found tidal zone-dependent differences in B. glandula metabolism that primarily related to anaerobic capacity, feeding behaviors and body size. Barnacles from the low intertidal tended to have a greater capacity for anaerobic metabolism (i.e., increased LDH activity), feed less when submerged, and be smaller in size compared to conspecifics in the high intertidal. We did not, however, see differences between barnacles from different tidal heights in whole-animal [lactate] following 24h of air exposure, which indicates that the enhanced capacity of low intertidal barnacles for anaerobic metabolism may have evolved to support metabolism during more prolonged episodes of emersion (>>24h) or during events other than emersion (e.g., coastal hypoxia, predation). There were also no significant differences in CS activity or baseline oxygen consumption rates (in air or seawater at 14˚C) across tidal heights, which implies that aerobic metabolic capacity may not be as sensitive to tidal position as anaerobic processes. Understanding how individuals occupying different shore heights differ in their metabolic capacity becomes increasingly interesting in the context of global climate change, given that the intertidal zone is predicted to experience even greater extremes in abiotic stress.

The world’s oceans are becoming more acid in a process called ocean acidification. The pH of the ocean have already decreased by 0.1 units from pre-industrial time until today. Scientists predict that by the year of 2100 the pH will decrease by as much as 0.4 units. This is a big potential problem to many marine species, because they have developed in such a stable environment that has not changed for millions of years. It is difficult to predict how they might be affected by such a decrease in pH during a relatively short time period. Several studies have been made on marine species exposed to decreased pH-levels, the results showed changes in their physiology but it is hard to predict how these changes will affect the organism in a long-term scale and if this might change ecosystem dynamics. Our study measured the activity of Littorina littorea and Balanus improvisus when exposed to lower pH, the results of our study showed an increase in activity for the lower pH (pH 6.0-7.5) when compared to the control (~pH8). The area of ocean acidification is a field that requires further studies to fully understand its effects on the marine ecosystems and the species within it.

Crustacean muscle fibers are some of the largest cells in the animal kingdom, with fiber diameters in the giant acorn barnacle (Balanus nubilus) exceeding 3 mm. Sessile animals with extreme muscle sizes and that live in the hypoxia-inducing intertidal zone – like B. nubilus – represent ideal models for probing the effects of oxygen limitation on muscle cells. We investigated changes in metabolism and structure of B. nubilus muscle in response to: normoxic immersion, anoxic immersion, or air emersion, for acute (6h) or chronic (6h exposures twice daily for 2wks) time periods. Following exposure, we immediately measured hemolymph pO2, pCO2, pH, Na+, Cl-, K+, and Ca+ then excised tergal depressor (TD) and scutal adductor (SA) muscles to determine citrate synthase (CS) activity, lactate dehydrogenase (LDH) activity, and D-lactate levels. We also prepared a subset of SA and TD muscles from the chronic barnacles for histological analysis of fiber diameter (Feret’s), cross-sectional area (CSA), mitochondrial distribution and relative density, as well as nuclear distribution and myonuclear domain size. There was a significant decrease in hemolymph pO2 and pCO2 following acute and chronic anoxic immersion, whereas air emersion pO2 and pCO2 was comparable to normoxic levels. Fiber CSA and diameter did not change significantly in either tissue, while myonuclear domain size in SA muscle was significantly lower in the anoxic and emersion groups than the normoxic control. Neither CS, nor LDH activity, showed any significant treatment effect in either tissue, whereas both muscles had significantly higher D-lactate levels after air emersion following acute (though not chronic) exposure. Thus far, our findings indicate that B. nubilus experience a general reduction in aerobic metabolism under anoxia, emersion is only mildly oxygen-limiting, and that muscle plasticity is occurring during chronic emersion and anoxia.