5.4.1. Queen spermathecae
- Place the test queen in a freezer (-20ºC is sufficient) 4-6 mins or until immobilized.
- Remove the queen from freezer and weigh to the nearest 0.1 mg on digital scale. Measure the queen’s thorax and head using micro-calipers to the nearest 0.1 mm and record. *NOTE: Non-destructive morphometric measures such as these may be helpful and potentially important correlates of other measures of queen reproductive potential (see Delaney et al., 2011).
- Dissect out the spermatheca:
a. Euthanize the queen by removing her head and pin her body to a dissection tray.
b. Cut her abdomen along both sides.
c. Grasp stinger with forceps and gently pull out until the ovaries are exposed.
d. Gently push hindgut aside to reveal the spermatheca (off-white, semi-hard sphere; see Fig. 21).
- Carefully pull the spermatheca out and place on a watchglass. Remove the tracheal netting covering it if still attached.
- Set dissection microscope to maximum magnification, and use graduations on ocular to measure spermatheca diameter. Measure two diameters and record the average. This is an optional but potentially useful measure for spermatheca volume, which can be used to calculate the theoretical maximum storage capacity and therefore the percentage filled (see Tarpy et al., 2011).
- Place spermatheca in 0.5 ml sperm diluent (e.g. Table 4) in a small glass beaker, then use forceps to burst it to release sperm.
- Immediately place remainder of queen in a labelled 1.5 ml microcentrifuge tube and place in -80°C freezer for any further analyses (e.g. PCR, GC-MS, etc.).