1. What exactly are Wolbachia and how do we know if an insect is infected with them?

2. Feminization, Parthenogenesis and C.I., oh my! Wolbachia-infected wasp embryo Feminization – Somehow, maybe by influencing hormone levels, Wolbachia cause developing male woodlice to be born as female. Parthenogenesis-inducing – In wasps and some other arthropods, females are diploid (result of fertilization) and males are haploid (develop from unfertilized eggs). Wol- bachia prevent chromosome division so eggs remain diploid, resulting in all female births. Wolbachia are intracellular parasites Cytoplasmic Incompatibility (CI) – Infected females can reproduce with any mate whereas uninfected females are restricted to breeding with fewer uninfected males. Q. How might Wolbachia affect gene flow in a population? How might this lead to sympatric speciation? Important!! Wolbachia are inherited via infected eggs.

3. What do we use to “hook” Wolbachia DNA? http://nitro.biosci.arizona.edu/c ourses/EEB105/lectures/DNA_ replication/DNA_rep.html http://nitro.biosci.arizona.edu/c ourses/EEB105/lectures/DNA_ replication/DNA_rep.html WSPEC-F (5’-CATACCTATTCGAAGGGATAG-3’) WSPEC-R (5’-AGCTTCGAGTGAAACCAATTC-3’) Primers – a strand of RNA that serves as the starting point for DNA replication. DNA copies Bacteria “fishing” DNA sequence DNA Replicatiton Use primers in PCR to help copy DNA (animation) - http://www.wiley.com/legacy/college/boyer/0470003790/animations/pcr/pcr.htm http://www.wiley.com/legacy/college/boyer/0470003790/animations/pcr/pcr.htm PCR + primer animation : What gene (DNA) is being copied using PCR? http://www.mcb.harvard.edu/Losick/images/TromboneFINALd.swf

4. http://en.wikipedia.org/ wiki/Ribosomal_RNA http://en.wikipedia.org/ wiki/Ribosomal_RNA Stems (sites that rarely mutate & are conserved) Loops (sites that are more free to mutate & evolve faster) Objective: Use primers to copy the 16S rDNA gene in Wolbachia bacteria Ribosome synthesizing a protein Bacterium with ribosomes Campbell & Reece, 6 th Ed. Atomic structure of the small subunit a ribosome. The rRNA, shown in orange, helps match the mRNA (codon) to the tRNA (anticodon). http://en.wikipedia. org/wiki/Image:10_ small_subunit.gif http://en.wikipedia. org/wiki/Image:10_ small_subunit.gif Small subunit ribosomal RNA 5’3’ Forward primerReverse primer Conserved region Variable regions 16S rDNA gene – codes for making SSU rRNA F R Copied DNA (using PCR)