Dear Kimberly,

Protostrongylid larvae should sink in 4% formalin. Just make sure you leave the flasks undisturbed for 10-15 minutes and gently use a syphon to bring the volume down, otherwise you can centrifuge and read only 1-2mL - using total counts or aliquots to estimate LPG). However, formalin-fixed larvae won't be ideal for any subsequent molecular species identification, and molecular information is lacking for many protostrongylid nematodes, especially those infecting wild ungulates and lagomorphs (and further info is always welcome in the field!).

Perhaps, fixing in 70% (or higher) ethanol is better, and DNA will be nicely preserved.

Also, most protostrongylid larvae survive just fine in water, refrigerated or frozen , for long periods (in my experience, Dictyocaulus sp. larvae wouldn't). But I understand that in case of Baermanns in the field preserving in ethanol should also be the ideal.

Hope this helps.

Best,

Gui Verocai

Thanks, Gui. This helps hugely. We have no plans to conduct any molecular analyses. While such analyses would be useful and interesting, it is outside of the scope of your research goals.

Dear Kimberly,

I do not know which type of Baermann technique you use but I can strongly recommend the flask recovery apparatus described by McKenna (1998) - see http://www.sciencedirect.com/science/article/pii/S0304401798002234. It is very easy, less laborious procedure and it is more sensitive in comparison to traditional funnel method.

I do not recommend formaldehyde as a fixative solution for preserving hair-like nematodes (like trichostrongylid and lungworm larvae). It is better to use Barbagall solution (20 g of NaCl and 30 ml of formalin in 1 000 ml of dd H20) which is not so destructive as "pure" formalin. Still prefer to use alcohol, for similar reasons as Guilherme.

However, it should be noted that larvae tend to curl up when they are in uncomfortable conditions or when they die (from natural causes or are killed). As you know, tail morphology is crucial for morphological identification of lungworm larvae. The identification is almost impossible when the larvae are curl up. However you can obtain relaxed larvae when you use an appropriate fixative solution and heat this suspension to 60 oC for about several minutes - see van Wyk & Mayhew, 2013. I use this procedure for preserving Muellerius larvae and they do not curl up.

Good luck!

Jarda

Collect the liquid from your Baermann funnels into a conical centrifuge tube and let it sediment 10-15 minutes (more if a 50 ml tube). Then you can aspirate most of the supernatant liquid off the top and add you formalin/formal saline, at any concentration you choose. JPCaruso, PhD, Veterinary Parasitology, 1983, University of Georgia, USA.