Probing problems

Our probe synthesis for the sea star ISH doesn’t seem to be working. We tried to synthesize probes two different times, and neither the probe nor the plain dNTP control showed up in the gel. However, a band of DNA shows up for caecum of diseased sea star when we run normal PCR, indicating that something is going wrong between the PCR and the probe synthesis step. Ruth is looking into different reaction kits available to us, tweaking the synthesis reagents, and altering reaction conditions so that maybe we can get a working probe for the sea star slides like we have for Sarah’s coral slides. If this works, it will be really cool to drop our DNA probes onto tissue slides to detect the presence of the pathogen. This step takes lots of time to get right because if you need to tweak conditions, you can only get in about a maximum of 3 runs through the thermocycler in one day, since each probe synthesis attempt takes about 30 min of prep and 2.5 hours of temperature-controlled DNA replication. Theoretically, getting successful probes should be the major bottleneck (or maybe it’s one of many).

We still don’t know for sure whether the pathogen is absent in the tissues of healthy sea stars, since the PCR has shown mixed results and it’s hard to define if sea stars are truly uninfected or just asymptomatic until the right environmental conditions hit, especially now that most sites have infected stars. However, if healthy stars truly have no (or very low) wasting disease pathogen, then I can see ISH being a very interesting addition to diagnostic tools for this disease. A lot of times, ISH requires removing the organism from the field and sacrificing it to collect the tissues. However, since sea stars can regrow their arms, samples could be collected by cutting off a piece of the arm, taking a few slices for slides, and placing the animal back in its habitat (theoretically, if Fish and Wildlife would approve that). Or in the case of pycnopodia, you could just grab one arm firmly and they would probably let you have it. If healthy, the sea star would regrow its arm and continue serving its role as a predator in intertidal and subtidal ecosystems. If it was infected, the loss of an arm may hinder the immune system by producing additional stress. Convserely, according to informal laboratory observations from Drew and Morgan, loss of an arm in pycnopodia may help the immune system through removal of infected tissue.

In the past, taking a small number of animals like this for monitoring disease would not be an issue. However, since many sea star populations are now very low, scientists are hesitant to remove any more animals than necessary.

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