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).

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.