3.1.1. Haemolymph collection

The most frequently analysed samples are haemolymph obtained from larvae or adult honey bees. Pupae typically have very low JH titres which are physiologically irrelevant. It is only the pharate adults (pupae undergoing pigmentation of the thorax and abdomen) that may be of interest, as in these stages JH becomes relevant for inducing vitellogenin expression.

1. For collecting haemolymph from feeding-stage larvae:

   1.1. Place the insect on a piece of Parafilm.
   1.2. Identify the position of the dorsal vessel, which is the transparent and easily visible vessel running all along the entire dorsal side of the larva.
   1.3. Puncture this vessel with a pair of forceps.
   1.4. Extruding haemolymph should be clear and can be collect with a microcapillary.

2. For collecting haemolymph from spinning stage larvae and prepupae:

   2.1. Puncture and collect extruding body fluid.
   As spinning stage larvae and prepupae are undergoing metamorphosis, the extruding fluid this is not clear haemolymph, but a whitish fluid that contains a lot of tissue debris.
   For obtaining the haemolymph fraction:
   2.2. Transfer to a centrifuge tube.
   2.3. Centrifuge at 10,000 x g for 5-10 min.
   A thin white sheet containing lipids will have now formed a top layer.
   2.4. Carefully insert a collection capillary tube through this sheet to collect the small volume of clear fluid directly below.
   Avoid aspirating the voluminous white bottom layer, which mainly contains cell debris.

3. For collecting haemolymph from pharate adults or adults:

   3.1. Immobilize the bee with two insect pins crosswise inserted into a wax-filled Petri dish and pressing the pins down over the waist.
   3.2. Haemolymph should preferably be collected with a microcapillary from an incision in the neck membrane of adult bees or from the thorax.
   This minimizes lipid content in the sample

When collecting haemolymph, a graduated microcapillary tube should be used to ensure accurate assessment of volume. For both GC-MS and RIA, 2 - 4 µl are normally sufficient for a sample. Depending on the stage being sampled, it may be necessary to pool haemolymph from multiple individuals to get readable results. After registering the exact volume of haemolymph, this is expelled into a glass collection vial already containing appropriate solvent.

The vials should be glass, fitted with a screw cap containing a Teflon-lined rubber septum. The Teflon cover should face the sample. These are low-cost glass vials customarily used for GC analysis. For JH analysis by RIA, vials of 1.25-2 ml volume containing 0.5 ml acetonitrile are recommended. For JH analysis by GC-MS, the samples should be collected into 8 ml vials containing 1.5 ml of 50% acetonitrile in water. Samples can then be stored for long periods of time at -20 oC. Special refrigeration on transport for short periods (up to two days) is not required.

Do not use plastic vials, such as Eppendorf tubes, to store samples, as JH binds to plastic. It is also not recommended to store haemolymph in microcapillaries in the freezer, as JH degrading enzymes may retain activity under such conditions.