2.10 ∣ Determination of hormone titers
Juvenile hormone abundance in insects was determined using a high
performance liquid chromatography (HPLC)
(Feyereisen & Tobe, 1981). Authentic
JH-III (Sigma; 25, 50, 100, 200 and 400ng·µl-1) was
used for a standard curve (Supplementary Figure 20). Approximately 5mg
females or males were homogenized in 2ml methanol/diethyl ether (1:1)
for 3-5min on ice, and centrifuged for 10min at 4000rpm/min, and
supernatants were collected. Supernatants were dried under high purity
N2 and there then dissolved in methanol/water (75:25). The operating
conditions were a C18 column (ID 45µm, 4.6 mm x 150mm
in length); a LC200 HPLC pump with an ML-425 micro injection system
(with a 10 µL sampling loop); and a UV-monitor with peak monitoring at
218nm. Recording was made at a 1 mV full scale. Methanol /water mixture
(75: 25) was used as the developing solvent at a
0.6ml·min-1 flow rate and 6-7
kg.cm-2 pressure.
Ecdysones in males and females were quantified using an enzyme-linked
immunosorbent assay (ELISA) as previously described
(Kelly et al., 1992). Authentic 20E
(Sigma; 0.625, 1.25, 2.5, 5 and10 ng·ml-1) was used to
generate a standard curve (Supplementary Fig. 20). Approximately 5mg
insects in each assay was homogenized thoroughly using a motorized blue
pestle (10sec) in 200μl of methanol and centrifuged for 5 min (13.3K
rpm) at room temperature. The extracts were pooled and dried under
SpeedVac centrifuging conditions and were then dissolved into 50μl EIA
buffer for least 2 hours or at 4℃ overnight. The samples were then
analyzed via ELISA.