Molecular Properties of the Theromyzon (Annelida: Hirudinea) Cocoon Daniel H. Shain
Cocoons provide a micro-environment necessary for development in several animal groups (e.g., arthropods, annelids, flatworms). Perhaps the most well known cocoon is spun by the silkworm, Bombyx mori, which provides the crude material for extracting silk. Lesser known are cocoons secreted by clitellate annelids (e.g., earthworms, leeches) which have several fascinating properties of their own. For example, aquatic species secrete specialized proteins that assemble underwater into a membranous sheath sealed at either end by an underwater "glue", requiring the synthesis of thousands of kilometers of "fiber" within a few hours. The goal of this proposal is to examine the molecular aspects of the cocoon produced by Theromyzon rude, an aquatic leech endemic to North America and sanguivorous to waterfowl. The T. rude cocoon is particularly resilient to denaturing chemicals and heat (>250C), yet is a thin, flexible and transparent membrane. A major protein component of the T. rude cocoon membrane has been isolated (called Theromyzon cocoon protein, or Tcp) and its amino acid sequence revealed 6 tandem repeats, each comprising 12 ordered Cys residues in a ~62 amino acid repeating unit. Tcp contains an unusually high Cys content (18%); other abundant amino acids include Glu (13%), Val (10%) and Pro (9%). The first half of each repeat (Cys residues 1-6) shares strong sequence similarity with antistasin, a leech anticoagulant with protease inhibitor properties, while the second half (Cys residues 7-12) shares similarity with notch/EGF-like protein domains which play roles in cell signaling. Tcp-like repeats (i.e., 12 ordered Cys residues) with unknown functions are present in disparate animal phyla including Arthropoda and Nematoda, suggesting that a pre-Cambrian Tcp-like ancestral protein was co-opted for a structural role in clitellate annelid cocoons. In this proposal, Tcp gene expression will be examined in situ to monitor events within the clitellum leading up to cocoon production, and the Tcp promoter will be characterized to understand the regulated expression of cocoon synthesis. The underlying architecture of the cocoon membrane will be investigated by classical electron microscopy, and with a Tcp-specific antibody aimed at determining the assembly, contribution and spatial arrangement of Tcp monomer in the cocoon membrane. Collectively, these research aims will explore the fundamental properties of an unusual biomaterial in the context of leech biology, while expanding our knowledge in a scientifically underrepresented phylum (Annelida). The resources to support these research objectives will impact a variety of academic activities on the Rutgers/Camden campus. Undergraduate students within the biology department will gain opportunities to address the proposed research aims through an independent study research program and will report their results as part of a weekly, departmental seminar series. Components of this study will be integrated into existing 'Electron Microscopy' and 'Computers in Biology' courses that will provide students an opportunity to become engaged in an active research area within the department. In addition, these funds will facilitate student participation in state and local symposia (e.g., Rutgers University Annual Undergraduate Symposium, New Jersey Academy of Sciences) and will help to provide the infrastructure for the PI's continued participation in research/education outreach programs.
