| In the horseshoe crab Limulus polyphemus, high tolerance to both environmental and toxin-induced stress is characteristic of late-stage embryos. Previous studies have identified a metal-tolerant population of horseshoe crabs in the Delaware Bay, and metalsusceptible population in Raritan Bay. Additionally, tagging and migration studies have proposed Raritan Bay horseshoe crabs as a resident population. In this study, the genetic structure of horseshoe crabs along their native range from Maine to the Gulf of Mexico was studied using mitochondrial gene markers to determine the existence of resident populations in the Chesapeake Bay, Raritan Bay, and Delaware Inland Bay system, within the larger Mid-Atlantic population structure. Additionally, overall tolerance and the effect of stress conditions on gene expression profiles were studied in late-stage embryos from three different populations of horseshoe crabs under short-term Zn- and Cu-stress. Finally, the overall mRNA expression and the changes to the miRNA population in response to short-term Zn and Cu stress were evaluated by deep-sequencing.;Composite mitochondrial haplotypes showed significant differentiation between Gulf and Atlantic horseshoe crabs. In addition, Maine, New Hampshire, and Massachusetts samples were genetically distinct from Mid-Atlantic samples as well as each other indicating significant structure in the North Atlantic. In the Mid-Atlantic, the Raritan Bay and Indian River Bay were not distinct from the Delaware Bay; however, the Chesapeake Bay was genetically distinct from other Mid-Atlantic populations, suggesting the presence of a resident population of horseshoe crabs there.;In the Raritan Bay, previously reported susceptibility to heavy metals was not observed. Of the two metals, Zn tolerance was highest in both Delaware Bay and Raritan Bay, with 48h LC50 ranging from 100-500mg/L, compared to approximately 10mg/L for Cu. Heavy-metal induced differential gene expression profiles were not observed after stress treatments, and may indicate a high constitutive expression of stress genes that precludes the need for an inducible stress response.;Thirty-nine Limulus miRNA candidates were identified by homology to known animal miRNA species, and one pre-miRNA sequence was identified by predicted stem-loop folding. Overall, in this study, the genetic structure of horseshoe crab from 11 different regions was determined, as well as the first deep-sequenced miRNA libraries, and the first deep-sequenced EST libraries in horseshoe crabs. Developing molecular biology resources for this species, including a sequenced genome, will allow further characterization of the less-conserved small RNA population, late-stage embryo mRNA expression, and gene expression in response to heavy metal stress. |
