Pharmaceutical induced behavioral changes in the American horseshoe crab, Limulus polyphemus
Pippins, Amber R.
The American Horseshoe Crab, Limulus polyphemus, is both economically and ecologically important and has been used in the laboratory for decades as a model organism for neurobiological studies. More recently, the locomotor activity and biological rhythms of this estuarine keystone species have been shown to be strong and robust, allowing for these behaviors to be used to assess the effects of environmental and physiological perturbations. An emerging threat for marine species is the appearance of hundreds of pharmaceuticals in their environment. Over the past several decades, pharmaceutical sales have increased dramatically worldwide and over four billion prescriptions were issued in 2017 in the United States alone. Unfortunately, a large percentage of these pharmaceuticals pass through humans and wastewater treatment plants virtually unchanged and enter marine and freshwater environments. Thus, pharmaceuticals have been characterized by the EPA as “contaminants of emerging concern” and dozens are readily detected in many of these environments globally. While numerous studies have documented the prevalence and concentration of these compounds in these environments, few studies have addressed the potential harm that these chemicals may have on the development or behavior of these aquatic organisms. Since horseshoe crabs develop and often live in estuaries, this species may be especially affected by pharmaceuticals in the aquatic environment. The first aim of this research was to determine the developmental and behavioral effects of three pharmaceuticals that are readily found in marine systems on juvenile horseshoe crabs, Limulus polyphemus. Fertilized horseshoe crab eggs were reared in environmentally relevant concentrations of carbamazepine (0-100?g/L), fluoxetine hydrochloride (0-1000 ng/L), and propranolol hydrochloride (0-100 ?g/L) for approximately two months and developmental observations were recorded. After metamorphosis into juveniles, a portion of these animals were further exposed to an environmentally relevant mixture of all three pharmaceuticals. After exposure to one, or all three of these chemicals, activity (linear velocity) was recorded in individuals over 10 days using a video recording system. When horseshoe crabs were exposed to a mixture of all three pharmaceuticals, linear velocity was significantly decreased. Exposure of any one of these pharmaceuticals alone did not significantly affect developmental measures, biological rhythms, or linear velocity. These findings are among the few to indicate behavioral impacts of environmental pharmaceuticals and may be especially important as Limulus is a keystone estuarine species. The second research aim was to determine if the molecular processes of transcriptional and/or post translational regulation control the circatidal rhythms in juvenile horseshoe crabs, Limulus polyphemus. Virtually all organisms, including horseshoe crabs, exhibit a variety of endogenous biological rhythms including circadian and circatidal rhythms. Protein synthesis and subsequent phosphorylation are important parts of the ubiquitous circadian clock mechanism and similar processes are likely involved in the mechanism controlling circatidal rhythms. In this study, kinase inhibitors caused significant lengthening in periodicity as the dose of casein kinase 1 inhibitors increased, suggesting protein kinases play an important role in the molecular mechanism of circatidal rhythms. Additionally, cycloheximide, a translational protein synthesis inhibitor, significantly affected the phase of circatidal rhythms in horseshoe crabs, while the transcriptional inhibitor, actinomycin D, did not. These results suggest that translational regulation plays a more important role than transcriptional regulation in horseshoe crab circatidal rhythms. Overall, the results of this thesis show that juvenile horseshoe crabs are an excellent model for investigating the mechanisms of circatidal rhythms as well as the effects of environmental toxins and pharmaceuticals. The recording system used in this investigation can also be used very broadly with other early developmental stage organisms for both toxicological and behavioral rhythm research.