6.2.1. Method for collecting haemolymph

From Stephens (1962):

  1. Larvae can be field-collected (see Section 6.1.) or reared in vitro (Section 3).
  2. Anaesthetize the larvae with CO2 until visible movement ceases. This makes it easier to handle larvae since they are otherwise quite active.
  3. Surface sterilize the larvae per Section 6.6. or with a hypochlorite solution (24 ml Millendo bleach in 1 l distilled water) for 5 minutes.
  4. Rinse the larvae twice with distilled water.
  5. Dry the larvae on sterile blotters at 30ºC until normal movement resumes.
  6. Wax moth larvae can be bled by cutting a proleg from the body or puncturing the proleg with a sterile needle and collecting the haemolymph that pools at the wound.
  7. Collect haemolymph by capillary action into sterile capillary tubes. Larvae from which only a small amount of haemolymph is collected can survive, complete their development, and reproduce normally.

    Modifications

  • The haemolymph can be transferred to pre-cooled Eppendorf tubes containing a few crystals of phenylthiourea. This prevents melanization (Vilcinskas et al., 1997, Wedde et al., 1998).
  • 1 ml aliquots can be centrifuged twice at 10,000 g for 5 min to remove haemocytes (Wedde et al., 1998).