Sunday, April 2, 2017

Attempts at aphid identification

Since getting a focus stacking setup and starting to take on identification of some really obscure species I had some luck and some failure. It really did lower my productivity, as in a typical post is more than an hour work for sample preparation, photography, and stacking in Zerene Stacker.
If you have interest in trying to build a similar focus stacking system, I described it in detail in the amazon review for the Mitutoyo 5X microscope objective:

I was planning to do one big post of everything I learned since taking this on, but it is pretty clear than aphids deserve their own post.

 Aphids are a tough group to identify. In many cases my 5X magnification is not enough, and in extreme cases I was told a 200X phase contrast microscope is absolutely necessary. Still, there are many species which can be identified from a good photo and focus stacking aphids on a slide increases that number substantially.

The first step is collecting some adult aphids, and putting them on a slide. For this I use some combination of the technique described in the USDA video on slide mounting, and the instructions which came with a slide mounting kit I bought to mount the aphids in Euparal
  1. Put the aphids in a ~10% sodium hydroxide solution for 12-24 hours. I am not real scientific here, and just dump some crystals in until it looks like about 10%. Unfortunately this ruined a couple when I got the concentration a bit high but I am getting a good feel for it. For a supply of sodium hydroxide, I found that some drain cleaner works
  2. Squish the aphids as shown in the USDA video
  3. Put the aphids in specimen clearing solution, and heat them to 120-150 degrees F for a few hours until they look clear.
  4. Run them a few minutes each in 70% alcohol, 95% alcohol, and 99% isopropyl alcohol.
  5. Put a drop of Euparal on a slide and place 3-5 aphids on the slide bottom up. After some usually futile attempts to position such that all limbs are visible, put on a cover slip.
It would be best to heat the slides to dry them, but I just let them sit a day or two. It is clear it takes a couple weeks to completely dry them out. If I get really serious about this, I might switch to Canada Balsam, following this procedure. The advantage there is that the slides should last centuries rather than the years to decades Euparal is likely to survive. At the moment that advantage doesn't mean much to me, but if I end up with a big enough collection it might.

After drying the slides I use my microscope setup to photograph with as much magnification as my setup can do.
  1. Full body
  2. Third antenna segment
  1. Full body
  2. Third antenna segment
  3. Head, dorsal and ventral focus
  4. Apex of rostrum
  5.  Hind Tarsus
  6.  Abdomen, including cornicles and cauda, dorsal focus
  7.  Cornicle, especially apical 1/4 or so.
  8. Cauda, especially setae number and placement
Typically with the magnification my 5X objective can produce, I can get all those items into perhaps four to six photos.

Some examples of aphids rarely reported on iNaturalist I have been able to identify since taking this on:
Metopolophium dirhodum
Myzus persicae
Macrosiphum euphorbiae
Hysteroneura setariae
Neotoxoptera formosana
Aphis craccivora
Wahlgreniella nervata
Sitobion fragariae
Eulachnus rileyi

A couple of those are likely to get pushed back to genus when someone points out a look-alike, but most of them are correct.

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