Internal biological clocks play important regulatory roles in virtually every living organism. The circadian clock in animals regulates daily activity rhythms and is composed of a molecular control system involving the protein PER. Recent preliminary evidence suggests that PER may also be an important part of the clock(s) controlling circatidal (~12.4 hour cycle) rhythms in the horseshoe crab, Limulus polyphemus. The first goal of the project is to use molecular techniques to investigate the molecular mechanisms that underlie both circadian and circatidal rhythms in Limulus and to localize the clocks that drive these rhythms to specific brain regions. A second goal will be to determine if circatidal rhythms are controlled by two 24 hr "circadian clocks" that each drive one of the high tide bouts of activity; an outstanding issue that has remained unanswered for decades. Third, for the first time in an aquatic invertebrate, the pattern of biological rhythms that are actually expressed by freely moving horseshoe crabs in their natural habitat will be recorded using a newly developed monitoring technique. The overall results will, for the first time in any species, localize a clock controlling circatidal behavior and will also identify the long sought after circadian clock in Limulus. Ultimately, the data obtained will allow for a full understanding of the mechanisms underlying the expression of rhythmic patterns of locomotion in this commercially and ecologically important species, from the molecular to the ecological level.
This project will significantly enhance the training of undergraduates in the sciences at an institution that educates many of the K-12 educators in the state. Over half of the student researchers are likely to be women and some will be from under-represented minorities. In addition, many of the students will be first generation university students from lower economic sectors.
Intellectual Merit: Internal biological clocks play important regulatory roles in virtually every living organism. The circadian clock in animals regulates a variety of daily rhythms and is composed of a molecular control system involving a number of core proteins including PERIOD and CLOCK. Many animals that inhabit the intertidal zone, like the American horseshoe crab (Limulus polyphemus) also exhibit tidal rhythms of activity that help them anticipate tidal cycles and synchronize the behaviors accordingly. The first goal of the project was to use molecular techniques to investigate the molecular mechanisms that underlie both circadian and circatidal rhythms in Limulus. We found that Limulus has virtually all of the circadian genes that are found in other species, the first time this has been demonstrated in any chelicerate. Interestingly, unlike most other species, the mRNA products from these genes do not appear to cycle by time of day nor do they appear to cycle by time of tide. These results suggest a potentially novel mechanism for the circadian and/or circatidal clock in this species. A second goal was to localize the clocks that control these rhythms. Using authentic antibodies we were also able to localize the product of one of these genes (CLOCK), to bilateral groups of cells in a small part of the brain, the cheliceral ganglia. Thus, the circadian clock controlling rhythms of eye sensitivity in this species may be located in this brain area. A third goal was to investigate the nature of the clocks controlling their behavior. We clearly demonstrated that the rhythms of eye sensitivity are controlled by a different clock system than the rhythms of locomotion. In addition, we also found clear evidence that the system controlling locomotion is controlled by at least two clocks that are tightly coupled in antiphase, each with a period of 24.8 hrs. This produces activity patterns with periods of 12.4 h or the average period of the tides and supports the "circalunidian hypothesis" of circatidal activity control. Our last goal was to record behavior in freely moving horseshoe crabs in their natural habitat. We found, for the first time that, like animals recorded in the lab, animals in their natural habitat express both daily and tidal rhythms, but only when water temperatures were above ~15oC. Furthermore, unlike several other populations of horseshoe crabs, this Great Bay NH population, found in a protected estuary (more than nine miles from the open ocean) overwinters in the bay. This suggests that other populations from other protected bays may not migrate to and from the open ocean but rather, may stay resident in those bays year-round. Overall, the data obtained help us to better understand the molecular and environmental factors that affect intertidal organisms. Broader Impacts: This project significantly enhanced the training of dozens of undergraduates in the sciences at institutions that educate many of the K-12 educators in the state. Several of these students were involved at every step of the scientific process, including as co-authors on publications. Over half of the student researchers were women and some were from under-represented minorities. In addition, many of the students were first generation university students from lower economic sectors. Finally, an additional study from the research demonstrated sub-lethal effects of biomedical bleeding on Limulus behavior. This study received much media attention and has helped to inform fisheries managers and the biomedical industry of the possible negative effects of the current practices.
