Dear Julien,

This is an interesting question that many labs in the field currently come up against. I was at this year's ESA meeting, where during a symposium on RNAi both these studies were discussed. One feedback was that the rehydration method works well for Culex but will not work for Anopheles (not sure if people have tried it in Aedes). The chitosan feeding method has used successfully by other labs for Anopheles. So it depends which species you want to use the method. Maybe other people can comment on their experience with Aedes.

Good luck!

I work with Anopheles. Do you know why it did not work for this species?

Actually, I just tried to dehydrate my mosquitoes following the paper protocol and most of them died. I'll try to diminish the time left in the salt solution.

Cheers

I think Denlinger actually mentioned in the paper that they thought this would work for Culex given their larval habitat but would not likely be transferable to other mosquitoes. What you observed has been seen by other people - the high salt exposure kills Anopheles. I am not sure in how far people tried to modify the original protocol, but as far as I know this technique or any modification of the protocol has worked for Anopheles.

Completely agree with Jason's comment. Hans Michael Mueller tried larval injections years ago as well, and while the knockdown works, the survivorship is rather poor compared to injection of adults.

If you work with 4th stage larvae, then you must be aware that it may be difficult to see a strong gene knock down effect due to the important metabolic changes occuring during pupation and emergence (time for gene knock down is usually 48 to 72h with dsRNA).

Hi all,

We tried injecting dsRNA in larvae in our lab, but the survivorship is indeed really too poor to be used.

Concerning the efficiency of the Kd, I'll validate it with WB for sure. But as others have succeeded by injecting larvae, another way of transferring dsRNA into the organism should work too.

Kristin, you mentioned that this technique worked in Anopheles, do you recall the name of the lab?

Thank you all for your participation, it is great to be able to exchange on matters which cannot be found in publications

Julien, the work was performed by Hans-Michael Mueller in the Kafatos Lab when the lab was still at the EMBL. The work was never published, so you might want to get in touch with him by email hans-michael[dot]mueller[at]bzh[dot]uni-heidelberg[dot]de

Jason mentioned that Larry's lab has done this more recently?

Hi Julien,

May be you should use An. melas or An. merus, member of the Anopheles gambiae complex that are halophiles species for this experience.

Hi Julien,

Zwiebel's Lab injected dsRNA in 2nd instar larvae of An. gambiae to knockdown odorant receptors. However, no info was given on survival rate. See Liu et al 2010 in PLoS ONE (doi:10.1371/journal.pbio.1000467)

Hi Julien,

I was wondering what was your output about RNAi in larvae.

Would appreciate your answer!

Thanks,

Avital

Hi Avital,

I used the rehydration method and after optimizing for salt concentration and dehydration time with 4th instar larvae, I had a poor survival rate of 10-20% in Anopheles gambiae. But the knock down worked in the survivors.

Given the other options, it is still a good method for kd in larvae.

what are you going to try?

Best

Julien, thanks for your sharing, I wonder that have you noticed any knock down efficiency? is there any gene dependent? what method have you used for testing your knockdown efficiency?

It's an old thread. but here are my two cents:

Injection in mosquito larvae results in extremely low survival (my own experience). Dehydration/rehydration works well in culex (adapted for saline water I guess). We compared the dehydration/rehydration in Ae. aegypti with no salt (just adding dsRNA to water) and there was no difference in knockdown efficiency. For some genes, RNAi works by soaking Aedes larvae but not for all. Chitosan/lipid carriers ,etc. did not have any effect on knockdown efficiency.