abstract / Biological Sciences / Doctoral / Doctor of Philosophy

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Populations of Crassostrea virginica within Mosquito Lagoon, Florida have recently undergone significant die-offs, which are a subject of major concern. Restoration efforts within Mosquito Lagoon are focusing on reconstructing the three-dimensional reef habitats. Before effective protocols can be established, however, important questions about the sources of juvenile and adult oyster mortality must be answered. Potential causes of Crassostrea virginica mortality in the Indian River Lagoon system include sediment loads, competition, predation, and disease. My research focused on the interactions between oysters and the competitors that may affect the settlement, growth, and survival of Crassostrea virginica. The four objectives of my thesis research were to: 1) identify potential oyster competitors in Mosquito Lagoon, 2) determine if the sessile species recruiting to oyster shells have changed over time, 3) determine how the dominant competitors, barnacles, affect oyster settlement, growth and survival, and 4) determine if oyster or barnacle larvae are better able to settle in increased sediment and flow conditions that are associated with high levels of recreational boating. Lift nets were deployed within Mosquito Lagoon to determine available competing species. I collected species inventory data at six sites to determine the sessile invertebrate species (competitors) present on oyster reefs. Nets were deployed intertidally, just above mean low water, on living oyster reefs. One and a half liters of live and dead oysters were placed within the nets upon deployment. The nets were picked up monthly and surveyed for all fauna. Upon retrieval, all oysters within each net were brought back to the lab where all sessile organisms were immediately identified and returned to the lagoon. This survey began June 2004 and continued for one year. Shells from historic shell middens (up to 15,000 years old) were examined to determine if the sessile species settling on oyster reefs have changed over time. Similar species were found on both shells of historic and extant reefs. One notable exception was the appearance of Balanus amphitrite, an invasive barnacle, on the extant reefs. Balanus amphitrite is thought to have invaded Mosquito Lagoon approximately 100 years ago. This has resulted in a five fold increase in barnacle abundance per oyster shell. Balanus spp. were identified as important potential competitors and thus my research focused on spatial competition between C. virginica and native versus invasive barnacles of the area. Over 300 barnacles, including a native species, Balanus eburneus, and an invasive, Balanus amphitrite, have been counted on a single oyster shell. To determine how Balanus spp. affected settlement, growth, and survivorship of C. virginica, laboratory and field experiments were conducted in which densities of Balanus amphitrite and Balanus eburneus were manipulated. Density treatments included: no barnacles (control), low, medium, and high coverage of barnacles. Laboratory settlement trials with cultured oyster larvae were run in still water and flow (recirculating flume) using all barnacle density treatments. Additionally, all treatments with 7-day oyster spat were deployed in the field to follow oyster spat growth and survivorship. Settlement was counted by microscopy, and growth and survivorship were measured every 3 days for 4 weeks. Settlement of oysters was affected by barnacle presence only in flowing water. Still water trials showed no oyster preference related to any barnacle density or species. The presence of barnacles affected the growth and survivorship of oyster spat. However, there were no species specific differences. Studies suggest that recreational boating activities, especially boat wakes that cause sediment resuspension, may decrease recruitment and this may then provide an advantage to sessile competitors less affected by flow and sediment loads. To address these issues, replicated laboratory trials were run in a laboratory flume to quantify the effects of water motion (0, 5, 10 cm/s) and sediment loads (0, 8, 16 g/ml) on oyster recruitment and the recruitment of an important, relatively new competitor in the system, the barnacle Balanus amphitrite. If B. amphitrite settles in a wider variety of flow rates and sediment conditions, it may have a competitive advantage over the native oyster in this space-limited habitat. I found that high flow and sediment loads reduced larval settlement of C. virginica. Alternatively, settlement of cyprids of B. amphitrite did not differ among treatments. Thus, continuous boat traffic during settlement times should favor recruitment of the invasive barnacle Balanus amphitrite over the native oyster Crassostrea virginica. Determination of the competitive interactions of Crassostrea virginica in Mosquito Lagoon gives us important insights into the ecological conditions necessary for reestablishment of these oyster populations. Crassostrea virginica in Mosquito Lagoon was significantly impacted by barnacles; settlement, growth, and survivorship were all reduced by Balanus spp. This information will help resource managers in planning restoration techniques to minimize oyster and barnacle competitive interactions and increase Crassostrea virgininca success.

The giant acorn barnacle, Balanus nubilus, is a resident of the subtidal and low intertidal rocky shoreline on the Pacific Coast of North America (Alaska to Baja California). B. nubilusis notable for having the largest muscle fibers in the animal kingdom; fiber diameters that can exceed 3mm in adults! At such extreme sizes these muscle cells may be at risk for insufficient oxygen delivery to mitochondria owing to low SA:V ratios and long intracellular diffusion distances. Oxygen limitation to these muscles may be further exacerbated during low tide air exposure (emersion) or environmental hypoxia events, which are increasing in frequency and duration along the world’s coastlines. We are interested in characterizing the internal oxygen conditions of B. nubilus during air emersion and anoxia so that we can ultimately investigate the physiologic mechanisms by which B. nubilus maintains function in their giant muscle fibers during environmental oxygen limitation. To this end, we examined the effects of air emersion and anoxia on 1) hemolymph gas, pH and ion levels, 2) oxygen consumption rates (MO2; emersion only), and 3) respiratory behaviors (e.g., cirri beating). In the first experiment, we measured hemolymph pO2, pCO2, pH and ion ([Na+], [Cl-], [K+], [Ca2+]) concentrations at 0, 3, 6 and 9h of exposure to air emersion, anoxic immersion and normoxic immersion (control). Next, we compared the average MO2 of barnacles held in water and air for 6h at three common temperatures (10, 15, or 20°C) using intermittent (aquatic) and closed-system (air) respirometry. Lastly, we investigated the respiratory behaviors (% time operculum open, %time testing, % time pumping, % time cirri beating, cirri beat frequency, opercular pulse frequency) of B. nubilusduring acute (6h) exposure to air emersion, anoxic immersion and normoxic immersion (control). Our data revealed that hemolymph pO­2 was significantly decreased in the anoxic barnacles by 3h and remained significantly depressed relative to the normoxic control for 9h. The air-exposed barnacles, however, maintained hemolymph oxygen levels that were intermediate to the control and anoxia barnacles for the entire experiment, achieving levels that were significantly lower than normoxic barnacles only by 9h. We also found that oxygen consumption rates for B. nubilus held at ecologically realistic temperatures were similar in air and water. From these data we conclude that B. nubilus is relatively adept at taking up oxygen from the environment while out of the water, which is common for certain barnacle species, and that air emersion represents a relatively mild environmental stress for this species (at least from a gas-exchange perspective). Efficient aerial gas-exchange by the giant acorn barnacle is likely facilitated by seawater pools stored in the mantle cavity, which can directly take up oxygen from the air and make it accessible to soft tissues and gill-like structures on the inside of the shell. This strategy, however, would require complementary behaviors aimed at oxygenating the mantle cavity fluid (e.g, aperture opening, cirri extensions to facilitate mixing), and this is exactly what we see. In our behavior experiment we found that air-exposed barnacles (and, more surprisingly, anoxic barnacles) spent significantly more time with their cirri extended than our control animals, who engaged more in an aperture pumping behavior with their cirri retracted. These behavioral preferences existed even though there were no significant differences in the total time spent with their aperture open (regardless of the behavior occurring while open) between any of the treatments. There were also interesting findings in the ion data. While there were no significant treatment effects on [Na+], [Cl-], or [Ca2+], we did observe significantly higher [K+] by 6h in both the emersion and anoxic groups relative to the normoxic group. We predict that this change in [K+] is likely attributable to its role in acid-base buffering. There was a strong correlation between pCO2 levels and pH across all treatments; however, decreases in pO2 levels in the hemolymph, which corresponded with increases in pCO2 levels, had only minimal effects on the hemolymph pH, indicating a well-buffered system. In conclusion, we found that air exposure does not inhibit aerobic metabolism in B. nubilus, owing largely to efficient aerial oxygen uptake and perhaps also effective acid base-buffering. We therefore predict that muscle function would be preserved during periods of low-tide emersion. Anoxia, on the other hand led to a significant decline in hemolymph oxygen content, which suggests that environmental hypoxia is likely to diminish functionality of their giant muscle fibers. In a parallel study, we intend to investigate the plastic response of B. nubilus muscle fibers to the same conditions (air emersion and anoxic immersion).

The rocky intertidal zone, experiencing fully marine and fully terrestrial conditions, has become increasingly investigated as a model ecosystem for studying the future implications of climate change. The barnacle, Balanus glandula, a common rocky intertidal inhabitant, plays an important role as a key prey item for many organisms. Low tide can be particularly challenging for barnacles as they are marine organisms subjected to the abiotic conditions of a terrestrial environment. The most stressful of these are increased temperature and decreased oxygen availability. This study aimed to investigate how low tide impacts the energy budget, specifically the digestion, of B. glandula. Barnacles are unable to feed at low tide however, if they were able to digest at low tide, they could maximize their energy intake by emptying their stomach to prepare to feed at the next high tide. However, digestion is a metabolically costly activity, which could make it less energetically favorable to digest when there’s less oxygen available. To test for an effect of low tide on digestion, barnacles were fed, and the time to first fecal production measured as a ‘baseline’. This was repeated, but barnacles were exposed to either a 16ºC or 35 ºC low tide immediately after being fed. The change in digestion time was calculated by comparing these two times for each barnacle. It was found that regardless of temperature, barnacles delayed their digestion by about 50-60 minutes after exposure to a one hour low tide. To determine the energetic cost of digestion, the rate of oxygen consumption was compared between starved and digesting barnacles. I was unable to detect any evidence of elevated metabolic activity during digestion. Additional testing is needed to confirm these results as the barnacles may have not fed during the trial, thus had no food to digest. While it appears that increasing temperatures associated with climate change will have little impact on the digestion of barnacles at low tide, if climate change alters the duration of low tide, there could be an energetic impact to barnacles due to the slowing of their metabolism as indicated by the delay in their digestion.

<p>The marine sponge <i>Geodia barretti</i> produces a range of secondary metabolites. Two of these compounds were isolated and elucidated guided by their ability to inhibit settlement of cypris larvae of the barnacle <i>Balanus improvisus</i>. The compounds barettin (cyclo-[(6-bromo-8-en-tryptophan)-arginine]) as E/Z mixture and 8,9-dihydrobarettin (cyclo-[6-bromo-tryptophan)-arginine]) were determined by using mass spectrometry, nuclear magnetic resonance and quantitative amino acid analysis.The bioactivity of these brominated dipeptides is in the range of antifouling substances used today: EC₅₀ values of 0.9 µM (barettin) and 7.9 µM (8,9-dihydrobarettin). The compounds were successfully synthesised and then tested in a field experiment to evaluate their antifouling properties. The compounds were incorporated in four different commerical, non-toxic marine coatings. The concentrations of the compounds were 0.1 and 0.01% (w/w) and coated panels were exposed to field conditions for eight weeks. The experiment evaluated the effect of barettin and 8,9-dihydrobarettin on recruitment of the barnacle <i>B. improvisus</i> and the blue mussel <i>Mytilus edulis</i> (major Swedish foulers). The most efficient paint was a SPC polymer, for which the reduction of recruitment of <i>B. improvisus</i> was 89% with barettin (0.1%) and 61% with 8,9-dihydrobarettin (0.1%). For <i>M. edulis</i> the reduction of recruitment was 81% with barettin (0.1%) and 72% with 8,9-dihydrobarettin (0.1%) with the same SPC paint. Furthermore, 14 analogs of barettin and dipodazine were synthesised and tested for their ability to inhibit larval settlement. Two of the analogs have a barettin scaffold and twelve have a dipodazine scaffold. Six of the analogs displayed significant settlement inhibition with the most potent inhibitor being benzo[g]dipodazine (EC₅₀ value 0.034 µM). The effect of benzo[g]dipodazine was also shown to be reversible. Finally, an investigation of the mode of action was performed on 5-HT receptors. Barettin demonstrated a specific affinity to 5-HT_2A, 5-HT_2C and 5-HT₄, while 8,9-dihydrobarettin interacted only with 5-HT_2C of the receptor subtypes tested (5-HT₁-5-HT₇).</p>

The marine sponge Geodia barretti produces a range of secondary metabolites. Two of these compounds were isolated and elucidated guided by their ability to inhibit settlement of cypris larvae of the barnacle Balanus improvisus. The compounds barettin (cyclo-[(6-bromo-8-en-tryptophan)-arginine]) as E/Z mixture and 8,9-dihydrobarettin (cyclo-[6-bromo-tryptophan)-arginine]) were determined by using mass spectrometry, nuclear magnetic resonance and quantitative amino acid analysis.The bioactivity of these brominated dipeptides is in the range of antifouling substances used today: EC50 values of 0.9 µM (barettin) and 7.9 µM (8,9-dihydrobarettin). The compounds were successfully synthesised and then tested in a field experiment to evaluate their antifouling properties. The compounds were incorporated in four different commerical, non-toxic marine coatings. The concentrations of the compounds were 0.1 and 0.01% (w/w) and coated panels were exposed to field conditions for eight weeks. The experiment evaluated the effect of barettin and 8,9-dihydrobarettin on recruitment of the barnacle B. improvisus and the blue mussel Mytilus edulis (major Swedish foulers). The most efficient paint was a SPC polymer, for which the reduction of recruitment of B. improvisus was 89% with barettin (0.1%) and 61% with 8,9-dihydrobarettin (0.1%). For M. edulis the reduction of recruitment was 81% with barettin (0.1%) and 72% with 8,9-dihydrobarettin (0.1%) with the same SPC paint. Furthermore, 14 analogs of barettin and dipodazine were synthesised and tested for their ability to inhibit larval settlement. Two of the analogs have a barettin scaffold and twelve have a dipodazine scaffold. Six of the analogs displayed significant settlement inhibition with the most potent inhibitor being benzo[g]dipodazine (EC50 value 0.034 µM). The effect of benzo[g]dipodazine was also shown to be reversible. Finally, an investigation of the mode of action was performed on 5-HT receptors. Barettin demonstrated a specific affinity to 5-HT2A, 5-HT2C and 5-HT4, while 8,9-dihydrobarettin interacted only with 5-HT2C of the receptor subtypes tested (5-HT1-5-HT7).

published_or_final_version / Biological Sciences / Master / Master of Philosophy

Like all intertidal species, the barnacle Balanus glandula must cope with temperature and desiccation stress during daily low tide exposure. The increase in temperature at low tide leads to both increased metabolic rate and the potential for increased ATP demand. With its additional inhibition of oxygen intake, low tide thus has an energetic cost that is often reflected in an increase in oxygen consumption following resubmersion. As anthropogenically induced global climate change increases air and water temperatures, its cost might increase. B. glandula individuals were exposed to 4‑hour low tides with maximal temperatures of 18, 30, 35, and 38°C, and their oxygen consumption rates and behaviors were recorded for 4 hours upon resubmersion. It was found that aerial respiration could be measured, though aerial rates were only a fraction of aquatic rates. It was further found that relative aquatic oxygen consumption rates were not elevated following low tide for any temperatures. However, B. glandula individuals exposed to 35 and 38°C low tides remained active a significantly greater portion of time through the first and second hours of recovery, respectively. This indicates that a low tide stress effect is evident in B. glandula, but that it manifests not as an increase in the respiration rate when active, but rather as an increase in the overall activity time. Thus, with increasing global temperatures B. glandula will likely have increased energy needs. This might lead to range relocations, a drive to find new energy sources, and/or reallocations of energy budgets.

As a unique habitat that encompasses steep environmental gradients, it is important to evaluate threats posed to the intertidal zone by rapid climate change. It is thought that intertidal ectotherms are living close to their physiological limit; therefore slight changes in temperature could result in high levels of mortality. Past studies on intertidal species measured thermal tolerance under constant temperatures, neglecting to consider the impacts of natural variation in field temperatures. I conducted a study on the barnacle, Balanus glandula, to assess if a variable thermal environment would alter thermal tolerance. Barnacles were acclimated in an intertidal mesocosm to either daily cold (maximum 20.4◦C), daily warm (maximum 26.5◦C), or variable (two days cold, two days warm) low-tide temperatures. I measured each barnacle’s critical thermal maximum (CTmax) by increasing air temperature 6◦C/hour and identifying the point at which the barnacle ceased to function. Barnacles exposed to any warm temperatures demonstrated an increased thermal tolerance, suggesting that this population of barnacles is capable of shifting their thermal maximum. Furthermore, acclimation to thermal heterogeneity raised thermal maximum, reinforcing the need for future thermal tolerance studies to incorporate biologically-relevant thermal regimes in laboratory experiments. These results demonstrate that B. glandula in the field are well-adapted for increasing air temperatures.