Hi Eduardo, I’ve injected many insects over the years. I’ve used Hamilton and Nanoject systems very successfully, depending on the application, but Nanoject is for much smaller volumes and very expensive equipment.

First a question: did you really mean 30-100 microlitres per pupa? This is a big volume for a 1cm insect.

Hamiltons are relatively inexpensive and the volume adjustment is sensitive enough for your goal. Bear in mind that injection will have a major impact on the health and stress levels of your insect. Be sure to sterilise the cuticle before injection and minimise handling stress. I would have additional controls (unhandled, uninjected) as well to check this impact.

It's good that you are using dye to test your injection methods. Some poor injection sites will impede dsRNA dispersal, e.g. if there is a lot of fat body in the locale, and might cause leakage by acting like a barrier. Injecting smaller quantities in several pulses usually helps for large volumes, as it gives time for the solution to disperse (inject, pause for a few seconds, inject, pause, etc - don’t take the needle out until the end). If you inject such a large volume all at once you will get leakage from the puncture wound. Also, of course, try to hold your insect as gently as possible so that you are not squeezing out haemolymph.

If you are breaking glass needles by pushing into the cuticle, then you need to find an injection site where you can avoid the cuticle and inject directly into e.g. arthrodial membrane. Even the best armoured insects will have chinks in their armour. The Hamilton syringes are quite good because the tip is strong and also isn’t angled. (With normal medical needles you have to insert it in quite a long way to account for the angle of the needle tip).

Nanoject (made by Drummond) is also a really nice system, but expensive. I used this for 3 years, working on mosquitoes. You can set the volume from about 2 to about 70nL per injection (so you can do multiple injections to get larger volumes). You hold it like a pen and you can make your own needles inexpensively by pulling glass capillaries. It’s very easy to see if your needle becomes blocked, and if it does you can sometimes snap off the end and continue injecting (depending on how skilled you are at making needles). There’s also a foot pedal to control injections. There's also a speed setting, so you can choose to inject very s-lo-w-l-y (possibly and important consideration since you report problems with leakage). In my opinion, for injecting insects you wouldn’t need the stand or micromanipulator - it’s fine holding it in your hand.

Often it helps if you can stop insects feeding for a few hours before injection (so the haemolymph volume is less). But, I am guessing that the pupae are not feeding anyway.

However you set up your equipment, try to modify your work area so that you can rest both your forearms and try to relax your shoulders while injecting. It will make a big difference to the pressure you use to make the puncture, and the subsequent health of the insect.

In order to be able to search the best answer, could you say what is the volume you want to inoculate and also the size of the fire ant pupae?

Best regards

Hello Cid, thanks for the response, a fire pupa is a bit over 1cm long, and the volume I would like to inject is 30-100ul.

Best,

I recommend you the Hamilton syringes, they have different needles, volume of syringe and chaney adapter to assures repetitive application.

http://www.hamiltoncompany.com/home.php

I agree with Hamilton syringes, they are very good. 30-100 ul is big enough to inject without problem (nanoliters insted are very difficult to make reproducible). I think it is very important to clean the needle very well between samples because the hemolymph of the larvae plug it and then the next sample is "misinjected"

Best regards

I used Hamilton syringes to inoculate saliva from sandflies (Diptera, Psychodidae) into ears of mice for histological studies. The needle I used was 30G, the thinnest one I found at that time. I had good results. Best regards.

Yes I am tempted to trying a Hamilton syringe, now they have these Neuros Syringes which seem to be pretty precise. Se below:

http://www.hamiltoncompany.com/products/syringes/c/1216/

Will try one out and let you know my impression.

Just one another thing, someone told me the metal needles could interact in DNA in an unfavourable way, thus affecting the injections. Anyone here had any issues with using metal needles for DNA/RNA injections?

Hi Eduardo, I’ve injected many insects over the years. I’ve used Hamilton and Nanoject systems very successfully, depending on the application, but Nanoject is for much smaller volumes and very expensive equipment.

First a question: did you really mean 30-100 microlitres per pupa? This is a big volume for a 1cm insect.

Hamiltons are relatively inexpensive and the volume adjustment is sensitive enough for your goal. Bear in mind that injection will have a major impact on the health and stress levels of your insect. Be sure to sterilise the cuticle before injection and minimise handling stress. I would have additional controls (unhandled, uninjected) as well to check this impact.

It's good that you are using dye to test your injection methods. Some poor injection sites will impede dsRNA dispersal, e.g. if there is a lot of fat body in the locale, and might cause leakage by acting like a barrier. Injecting smaller quantities in several pulses usually helps for large volumes, as it gives time for the solution to disperse (inject, pause for a few seconds, inject, pause, etc - don’t take the needle out until the end). If you inject such a large volume all at once you will get leakage from the puncture wound. Also, of course, try to hold your insect as gently as possible so that you are not squeezing out haemolymph.

If you are breaking glass needles by pushing into the cuticle, then you need to find an injection site where you can avoid the cuticle and inject directly into e.g. arthrodial membrane. Even the best armoured insects will have chinks in their armour. The Hamilton syringes are quite good because the tip is strong and also isn’t angled. (With normal medical needles you have to insert it in quite a long way to account for the angle of the needle tip).

Nanoject (made by Drummond) is also a really nice system, but expensive. I used this for 3 years, working on mosquitoes. You can set the volume from about 2 to about 70nL per injection (so you can do multiple injections to get larger volumes). You hold it like a pen and you can make your own needles inexpensively by pulling glass capillaries. It’s very easy to see if your needle becomes blocked, and if it does you can sometimes snap off the end and continue injecting (depending on how skilled you are at making needles). There’s also a foot pedal to control injections. There's also a speed setting, so you can choose to inject very s-lo-w-l-y (possibly and important consideration since you report problems with leakage). In my opinion, for injecting insects you wouldn’t need the stand or micromanipulator - it’s fine holding it in your hand.

Often it helps if you can stop insects feeding for a few hours before injection (so the haemolymph volume is less). But, I am guessing that the pupae are not feeding anyway.

However you set up your equipment, try to modify your work area so that you can rest both your forearms and try to relax your shoulders while injecting. It will make a big difference to the pressure you use to make the puncture, and the subsequent health of the insect.

Dear Miranda,

Many, many thanks for such a detailed response. Indeed on the volume I have friends that inject 200 nl into similar-sized insects, so I could consider readjusting the volume. My worry is that dsRNA could prove viscous at higher concentrations, and I have read about people injecting honeybee pupae (about 3x volume) with 90ul. I have done some preliminary tests and the fire ant male pupae seem to be remarkably tough, they survive injections >60% of times, even if I am not careful or too much dye goes in. My main issue is that with FemtoJet we cannot control for volume or use lower injection pressures. I am inclined to using Hamiltons (since the insects prove being so tough) and now I only worry about a possible negative interaction between metal needles and dsRNA.

Please on that specific point, please did you hear about any issues with using metal needles for injections of dsRNA? Can you please indicate your papers which employed Hamilton syringes with metal needles so I can reference to them in any case?

Many thanks for a clarifying answer.

just coming back to clarify than indeed I originally meant nanoliters, as I revisited the protocol and checked this there. Maybe I will have to make some volume adjustments for a Hamilton syringe then, let us see. 

Hi Eduardo,

I would also recommend Hamilton syringes. I used them to inject virus into adult stable flies, using a 33G needle. A note on the needles: I could only get them through a medical office (I used the veterinary school on campus), as sale was restricted (in the U.S., anyway). The syringe (50uL Luer tip 1700 Series Gastight Hamilton syringe) was attached to a repeating dispenser that injected the same volume of solution at each press of the button (i.e., in every specimen). I injected 6uL, but it could have been a smaller volume. The minimum size of the dispensed volume depends on the volume of the syringe the dispenser is fitted to.

I do not have information on the issues of injecting dsRNA with metal needles. Sorry.

I also recommend Hamilton Syringes.

You can blow your own glass syringe of microsize or can use a Luer tip 1700 Series Gastight Hamilton syringe for injection of pupae. 

Hi, I agree with use the Hamilton Syringes.

See this Reference1

https://jcs.biologists.org/content/joces/s3-91/14/217.full.pdf

2

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3523423/

3

https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=6&cad=rja&uact=8&ved=0ahUKEwjoqJf9jqfSAhVoP5oKHSPQA8gQFghZMAU&url=https%3A%2F%2Fwww.jove.com%2Fvideo%2F4295%2Frearing-injection-manduca-sexta-larvae-to-assess-bacterial&usg=AFQjCNG32SOWX46XfXVSCEx79HcuUsaZhQ&sig2=_VOT9dSajzbh9_Yl6Reduw

4

https://books.google.com/books?id=N2tQKmjKGFIC

I recommend Hamilton microsyringe with variable volumes. Care about injection to be parallel to the long axis of the insect body to avoid gut injury 

This thread has been very helpful for what we do in our lab (injecting dsRNA into 7th instar cricket larvae).  Our Nanoject has not been working well, so we are thinking of switching to a Hamilton Syringe.  My concern is cross-contamination, since we use several different dsRNAs.  I'd love to know if rinsing the Hamilton syringe with ethanol and then water is sufficient to ensure there will be no contamination of dsRNA samples?  Thanks!

Can any body recommend me good and economical "complete microinjection system" suitable for mosquitoes?