Date of Award

10-3-2014

Document Type

Restricted Access Thesis

Degree Name

Master of Science in Biology

Department

Department of Biological Sciences

Thesis Advisor

Christopher Chabot

Committee Member

Kerry Yurewicz

Committee Member

Winsor H. Watson, III

Abstract

Along its North American range, the American horseshoe crab, Limulus polyphemus, is ecologically and commercially valuable, supporting both a bait fishery and a biomedical fishery. While multiple stakeholders depend on this species, population declines in certain regions suggest the need for improved region-specific management strategies for the four L. polyphemus subpopulations. Development of such strategies requires knowledge of both the behavioral features of animals of these subpopulations and the impacts of harvest processes, specifically the relatively unassessed impacts of the growing biomedical fishery, on individual animals. The expression of circatidal (-12.4 h) activity patterns in L. polyphemus along much of the Atlantic coast provides a metric through which to examine the 1) the influences of harvest practices, 2) population-level behavioral differences in biological rhythm expression, and 3) the behavioral and physiological outputs of biological oscillators. Throughout New England and the Mid-Atlantic region, L. polyphemus is harvested by the bait industry and the biomedical industry, the latter of which extracts the hemolymph of these animals to produce Limulus Amebocyte Lysate (LAL), a bacterial endotoxin detection assay. Population declines in heavily-harvested regions suggest deleterious effects of harvest practices on female horseshoe crabs, the primary target of harvesters. While harvest processes have an immediate (1-17 day post-harvest) mortality rate of 10-30%, sublethal effects of harvest processes have received little attention. Sublethal behavioral and physiological alterations elicited by LAL harvest processes were investigated in 28 female horseshoe crabs. Linear and angular velocity, total distance moved, percent of time active, and expression of circatidal behavioral rhythms were significantly reduced after LAL harvest processes. Recovery of pre-harvest behaviors occurred by the third week after the process. Hemocyanin levels were significantly reduced in the week after the bleeding process, and remained suppressed for the duration of the study (6 weeks). These previously unrecognized behavioral and physiological effects of the LAL harvest suggest that harvest processes may decrease fitness of females returned to the wild post-harvest and may contribute to population declines in regions of heavy biomedical harvest. Along its geographic range, L. polyphemus experiences at least three different tidal regimes: two subpopulations experience semidiurnal (2 tides/day) tides, one subpopulation experiences mixed tides ( one high tide larger than the other), and one subpopulation experiences a microtidal environment (very small or nonexistent tides). Behavioral rhythms of animals from these three separate environments were compared in three different experimental tide conditions: constant water depth, two tides per day (every 12.4 hours), and one tide per day (every 24.8 hours). Across the three tide conditions, L. polyphemus from mixed tide environments (n = 21) consistently expressed unimodal rhythms, while those from two tide/day environments (n = 28) generally expressed bimodal rhythms. In contrast, animals from the microtidal site (n = 7), while they expressed unimodal activity patterns in constant depth, expressed both bimodal and unimodal rhythms in response to twice and once daily tides, respectively. Activity rhythms in L. polyphemus thus appear to reflect the tides of their source environment, but adults from the microtidal environments exhibited plasticity in behavioral rhythm expression. While it is unclear whether these behavioral differences are driven by genetic differentiation among the subpopulations or by the tidal environments experienced by individuals in early development, the ability of all animals to express a 24.8 hour tidal cycle suggests that circalunidian oscillators may control behavior in L. polyphemus. L. polyphemus from two tides/day environments express circatidal behavioral rhythms controlled by tidal clock(s). However, whether physiological functions, such as heart rate and respiration frequency, are similarly under control of tidal oscillator(s) is unknown. Rhythms of activity, heart rate, and respiration were recorded in freely moving (n = 12) and restrained L. polyphemus (n = 6) during periods of light/dark cycles, constant light, and tide cycles. Most (10/12) animals exhibited coordinated activity, heart rate, and respiration rhythms across the different conditions, though individual animals expressed tidal or daily rhythms in the three parameters. When restrained, most (4/6) animals continued to exhibit coordinated rhythms of heart rate and respiration, with daily rhythms (3/6) and tidal (1/6) rhythms of heart rate and respiration in individuals. In response to alterations in light and tide conditions, rhythms of activity, respiration, and heart rate responded in phase with one another. Cumulatively, these results suggest that the same tidal oscillators which govern activity rhythms also regulate rhythms of heart rate and respiration.

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