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.