Instructions for milking Phasmids (Stick Insects)

 By Dr. Aaron T. Dossey


Approaching the Insect with a Vial
Simply getting the insect to spray into a vial is the simplest method to collect defensive spray substances from Phasmids. The vials I use are glass with lids with screw-cap lids that are lined on the interior with Teflon. This is useful in case one wishes to extract the substance with organic solvents later. I typically rinse new vials with deionized water and then with pure clean (HPLC grade) methanol, then let them dry for about 20 minutes. 
            To collect the defensive spray:
1)           Carefully approach the stick insect’s head with the open end of the vial. (Be sure to select an animal whose head is easily accessible and free from antennae or parts of other animals that may be nearby or mating with the target animal). Also, always wear safety glasses and gloves while milking these insects, as the defensive spray is dangerous if it gets in one’s eyes. Figure one shows a close-up of approaching the insect just before milking (It’s good to go slow even before you get to this point).
Figure 1: Approaching the insect with a vial. 
A blue circle is around the region of the insect from which the
glands actually shoot the defensive spray.
2)           Once you’re in position (maybe not even as close as in Figure 1) and ready to milk (and the insect has remained still), carefully but quickly apply the opening of the vial over the place on the insect where the defensive substance will come out of. Hold pressure on the insect with the vial opening (not so much that you damage the insect, just enough to keep it from escaping that position) until the insect has sprayed its defensive substance into the vial. Sometimes they don’t spray at all, so you shouldn’t hold the vial on the insect’s head for more than a few seconds (10 seconds max).  Figure 2 shows this process, as does the video. Be sure that there are no leaves, water droplets, insect feces, insect parts, leaves, twigs, or other debris in the area that might contaminate the sample by getting into the vial. This is important so that I do not end up chemically characterizing chemicals other than those that come from the defensive spray. 
Figure 2: Milking the insect.
Notice that the insect is pressed down and unable to move, yet not crushed. 
Also notice that there are no objects or other substances that make their way into the vial.  
1)            Repeat this until you have a “good sample” in one vial. You can milk several insects. If possible, please keep track of what milkings it tool to get a particular sample, ie: How many milkings, how many different insects, their life stages and their genders. Below in Figure 3 is shown a vial containing a small but useful sample of the substance (Figure 3A.), and another one with what I would call a good sized sample (not huge, but a good amount) (Figure 3B.) in the same type of vial that I have sent for you to collect in. If you have a “small” sample from one round of milkings, keep this in the freezer until your next round of milkings. When you get one vial that is a “good” sample or larger, go ahead and send it. For Anisomorpha, I can get a “good” sample in about 5-10 milkings within about 5-20 minutes (depending on if the insects are cooperating or not).
Figure 3:
2)            That’s all there is to it! Be sure to keep the cap on the vials as much as possible and when you’re storing the samples keep them in the freezer.