One of the problems we have to deal with when approaching some of the parasite work we do is figuring out how to access small patches of tissue. With the red algal parasites, this is particularly true, because of the way they grow. Many of the cells that contain parasite DNA also harbor host DNA, because they are heterokaryon cells (Top of Figure 1).
Figure 1. Comparison of parasite life history and the carposporophyte stage of a typical red algal life history. Parasite cells devoid of host DNA occur only in a portion of the “erumpent pustule” that extends from the host. Image from Blouin & Lane 2012 (DOI:10.1002/bies201100139)
If one wants to work with the genomes of these organisms, it is essential to be able to identify and extract tissue that contains only parasite DNA, which proves to be a challenge. We have played with several methods without success… until now. Postdoc Nic Blouin has been work with the Rhode Island Children’s Hospital, using their laser capture microdissection (LCM) system to isolate red algal cells of interest.
With our parasites living on the West Coast, we had to devise a proof of concept with a local species. We picked the carposporophytes (bottom of Figure 1) of Chodrus crispus as a test case, based on their size similarity to many red algal parasites. Nic isolated the carposporophytes, dried and sectioned them in prep for LCM (Figure 2A).
Figure 2. Carposporopyte tissues was dried and sectioned using a freezing microtome. A) Thin sections were mounted for LCM. B) After cutting, the C) cystocarp material was excised from the carposporophyte.
Nic was able to use the UV laser to cut out the cystocarps (Fig. 2B) and then, using transfer film, was able to remove the excised cells (Fig 2C). An explanatory video of the capture process can be found here. Removal of the cystocarp cells is roughly equivalent to the size and nature of the parasite material we will be isolating in the near future. From here we plan to use multiple isolates (to avoid reaction biases) and whole genome amplification to produce enough DNA to create a library for Illumina sequencing. We should have a feel for how much DNA we can produce in a week or two.
References:
Blouin NA, & Lane CE (2012). Red algal parasites: models for a life history evolution that leaves photosynthesis behind again and again. BioEssays : news and reviews in molecular, cellular and developmental biology, 34 (3), 226-35 PMID: 22247039
Lane Lab @ URI 