20 oh e

The ubiquitously occurring insect hormones 20-OH-E and JH are not only controlling molting processes of juvenile insects but, in many insects, influence also pheromone biosynthesis in the adult stage [14 , 17 (link), 57 ]. Here, using the acetone-application method, we investigated how 20-OH-E and JH III affect pheromone biosynthesis in N. giraulti males. To this end, we produced solutions of 20-OH-E (Sigma-Aldrich, Saint Louis, MO, USA) and JH III (Sigma-Aldrich) in acetone (5 μg/μl) and applied 207 nl of the solutions (representing a dosage of 1.04 μg or 1040 ng, referred to as JH 1000 and 20-OH-E-1000 in the results) to the abdomens of newly emerged males as described above. Control males were treated with pure acetone (n = 20 for each treatment). Given that we found a significant effect of JH III at this dosage (see Results), we tested this compound again with a tenfold diluted acetone solution of JH III (500 ng/μl, representing a dosage of 103.5 ng, referred to as JH 100 in the results, n = 20). Two days after the treatment, pheromone extracts of individual males were produced as described above and analyzed by GC/MS (see section Chemical Analyses).

All reagents and solvents used for the present work were of analytical and HPLC grade and purchased from several suppliers. Reference standard of 20‐OHE was from Sigma‐Aldrich, Chemie (Steinheim, Germany). The standard solution of 20‐OHE (stock and working solutions) was prepared in Methanol. The stock solution (1 mg/ml), was diluted to working solution of 100 and 1 μg/ml and stored at −20°C. The internal standard used for the LC MS analysis was 17a‐Methyltestosterone (50 μg/ml), whereas for the GCMS Analysis was a mixture of d3‐Testosterone (4 μg/ml), d3‐epitestosterone (4 μg/ml), d4‐androsterone glucuronide (43.7 μg/ml), d5‐ethiocholanolone (50 μg/ml), d5‐5B‐Androstane‐3a,17B‐diol (5 μg/ml) and 17a methyltestosterone (50 μg/ml).
Sixteen dietary supplements were openly sourced through the internet (amazon.com, USA). Table 1 shows names of the supplements and declaration on the label about the presence of ecdysterone. Methanol, sodium hydrogen carbonate, potassium carbonate, dithioerytritol (DTE) were obtained from Sigma Aldrich (Darmstadt, Germany). β‐glucuronidase from E. coli was from Roche Diagnostics GmbH (Mannheim, Germany), Diethyl ether from Merck (Darmstadt, Germany) while MSTFA was from Roche (Mennheim, Germany).