Biomphalaria glabrata: The Localization of PIWI in the Brain and Ovotestes of the NMRI (susceptible) and BS90 (resistant) Snail Stocks.

Undergraduate #59
Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology
Session: 1
Room: Exhibit Hall A

Kimberly Karolyi - University of the District of Columbia
Co-Author(s): Gissan Stewart, UDC, Washington, DC; Odain Stewart, UDC, Washington, DC; Trenesha Anderson, UDC, Washington, DC; Freddie Dixon, UDC, Washington, DC; Matty Knight, UDC, Washington, DC; Carolyn Cousin, UDC, Washington, DC

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The PIWI transcript encodes a protein with an evolutionarily conserved domain that is found in the argonaute family of proteins that are essential for maintaining the integrity of the genome of germ cells and somatic stem cells. By silencing the expression of mobile genetic elements, transposons and retrotransposons, by an RNA interference (RNAi) silencing mechanism, PIWI prevents aberrant mutations in the germ cells that can cause random insertions and thereby mutations in the genome of germ cells. By binding to small cognate piRNA, PIWI mediates suppression of expression of mobile elements, such as nimbus in the snail, Biomphalaria glabrata. This freshwater snail is the obligate intermediate host of Schistosoma mansoni, the parasitic flatworm that is the causative agent for the tropical disease known as schistosomiasis. Both susceptible (NMRI) and resistant (BS-90) snails are an invaluable resource in studying the molecular basis of the B. glabrata and S. mansoni interaction. NMRI is a susceptible snail that has been bred in the laboratory for its high susceptibility to the parasite and the BS90 snail is a wild-type snail that originally came from an endemic area in Brazil. By investigating the differential expression of the transcript encoding PIWI in these two snails with, and without, schistosome infection by real time qPCR, results showed that PIWI is downregulated in the susceptible NMRI snail but upregulated in the resistant BS90 snail. We hypothesized that the transcript encoding the PIWI protein will be differentially expressed in the brain and ovotestes of these snails depending on their susceptibility phenotypes to S. mansoni. Using a rabbit anti- PIWI antibody PIWI expression was localized and quantitated in these organs by using both light and electron microscopy (LM & EM) and also by Western Blot Analysis. For LM and EM, sections of the brain and ovotestes were fixed in 4% paraformaldehyde/ 0.5% glutaraldehyde in 2% sucrose in 100mM sodium cacodylate buffer, washed and postfixed in 0.05% Osmium Tetroxide buffered with 100mM sodium cacodylate. After treatment with antibodies, the sections were washed in inactivated 10% normal goat serum in 100mM Tris buffer, pH 7.6. For LM, the tissue was further processed by using goat anti-rabbit IgG and placed in phosphatase labeled streptavidin and the process continued until the sections were embedded in LR White (water soluble). For EM, immunogold labelled goat anti-rabbit IgG was used as the secondary antibody and further processed for EM using LR White (water soluble). Western Blot analysis was performed on protein samples extracted from the specific tissues of the aforementioned specific snail stocks, with and without schistosome infection, utilized in this study. Results showed that PIWI in BS90 snails was localized in various regions of the brain with a greater concentration in the cerebral lobe. Also, the ovotestes showed areas that reacted positively with the anti- PIWI antibodies. These results will be further confirmed and validated by using Western Blot analyses to examine the presence and expression of the PIWI encoding protein in these tissues. Funded by NSF-HBCU-UP Grants 1622811 and 1531014.

Funder Acknowledgement(s): Funded by NSF-HBCU-UP Grants 1622811 and 1531014.

Faculty Advisor: Carolyn Cousin, ccousin@udc.edu

Role: I removed and performed the immunocytochemistry for the brain and ovotestes for both EM and LM; the process used is described above in the abstract. I performed microtomy, as well as ultra microtomy.