We keep noticing males showing up, and our aim is to experiment on isogenic N2 hermaphrodites. I don’t know why we are getting males so often. We are maintaining them properly (or I think) and new populations (by selfing) are started from single larvae frequently. If a population contains a small % of males, it is obviously no longer considered isogenic right? what are we doing wrong?
A “good” N2 stock should have <0.5% males. One expects an occasional (~1/1000) spontaneous males, since spontaneous loss of one X during meiosis is a rare but possible event. If you stress your worms (e.g., temperature shifts), you will increase the frequency of males due to an increased frequency of non-disjunction. Also, there are a number of mutations that increase the frequency of spontaneous males, sometimes only slightly. And some strains have a higher frequency of spontaneous males than N2 does.
If you are working with N2 and your frequency of males is higher than 1/200, I recommend using a ‘fresh’ N2 stock. That is, if you have some N2 that were frozen soon after receipt from the stock center, defrost some fresh worms and use those. If you do not have worms that were frozen soon after receipt from the stock center, you can request a fresh aliquot of N2 from the CGC. Just remember to freeze some aliquots soon after receipt.
The spontaneous males that arise in your stock are ~isogenic with the hermaphrodites, but having two sexes in your experiment will complicate the analysis. And a high frequency of spontaneous males (HIM phenotype) is an indication that your stock has drifted from N2.
Oh, I didn’t think it would have drifted from wild type:( But that sounds very logical.
so far, as an estimation, it is much less than 1 in every 200. But, then again, maybe we are not noticing them all. To be safe, I think it is a much better idea to get a fresh stock , freeze some and use the rest. as I think if left, it will be more problematic as you explained:)
Thank you very much for your reply, it was very helpful!