A Genetic and Molecular Investigation of the Circadian and Circatidal Clocks in the Horseshoe Crab, Limulus polyphemus

Biological rhythms are cyclical behavioral and/or physiological changes that are driven by endogenous biological clocks. The most common type of rhythm, known as a circadian rhythm, oscillates with a period of about 24 hours. The endogenous clock that modulates circadian rhythms is composed of a set of “core” and accessory circadian genes. The core genes in animals are clock, cycle, period,nonphotoreceptive-cryptochrome2 and timeless and appear to be self-regulated by transcription-translation negative feedback loops thus allowing for synchronization and anticipation of the daily light and dark changes. Organisms that live in or visit the intertidal areas are also exposed to another type of environmental rhythm, which corresponds to the rise and fall of oceanic tides. Tidal rhythms are well documented in several intertidal animals, yet, unlike circadian rhythms, the molecular basis of the tidal clock system is largely unknown. One intertidal organism, the Atlantic horseshoe crab, Limulus polyphemus, is an important keystone species and has robust circadian and circatidal rhythms, which makes it a potential model system for the study of these rhythms and the mechanisms of the clocks that drive them. The aims of this thesis are:(1) to create Limulus genomic and transcriptomic datasets and (2) to investigate the presence of core clock gene homologs in the brain of Limulus as well as examine their potential modulation by time of day and time of tide. In these studies, the central nervous system of Limulus was used to develop a draft genome and transcriptome of this species. Four adult horseshoe crabs were selected and total RNA was extracted from their central nervous systems during the day and night and during high and low tides. Illumina library preparation, sequencing, subsequent assembly, and RNA-seq investigations indicated the presence of virtually all of the core clock gene homologs and accessory genes although with there was no clear rhythmic cycling of the transcription of any circadian core or accessory gene. Interestingly, total transcript expression showed more high versus low tidal phase differences than light versus dark photoperiodic differences. Additionally, the majority of the genes that seemed to be regulated according to these conditions did not have homologs in public bioinformatics databases, suggesting that these rhythmic genes are novel and currently unique to Limulus. Using data from the genome and transcriptome as a guide for primer design, QPCR was used to further explore the expression of the putative core circadian genes clock, npcryptochome2, cycle1 and timeless in the protocerebrum over conditions of photoperiod and tidal phase. To accomplish this aim, horseshoe crabs were placed into tide-simulating tanks and were subjected to light/dark and tidal cycles. QPCR was performed on the mRNA extracts from the central nervous system to quantify the expression of the circadian clock genes clock, cycle1, timeless and npcryptochrome2over photoperiodic and tidal conditions. While core circadian transcripts were found to be expressed in all investigated sections of the central nervous system, there were nostatistically significant rhythmic cycling of those genes within the protocerebrum with respect to either photoperiod or tidal phase. Overall, both the RNA-seq and QPC Rresults suggest a relatively constitutive expression of core circadian transcripts within the Limulus central nervous system. These results, when taken together, suggest at least five possible explanations; (1) These genes may not be involved with the circadian or tidal clocks; (2) Rhythmic mRNA expression of these genes may not be necessary for these clocks; (3) The circadian and tidal clocks share some genes and them RNA of those shared genes oscillates out of phase with each other, masking true rhythmic expression; (4) Non-clock cells in the tissues examined are also expressing these genes at a high enough level to “dilute” the rhythms of mRNA expression; (5)Finally, there may be a heavy reliance of Limulus clocks on post-transcriptional and post-translational modifications. In order to explore some of these potential explanations, future studies should aim to systematically investigate putative circadian genes in Limulus. This will help to determine whether or not these genes are involved with one or more of these clocks and whether or not their transcription is required for measurable rhythms