Measuring juvenile hormone and ecdysteroid titers in insect haemolymph simultaneously by LC-MS: The basis for determining the effectiveness of plant-derived alkaloids as insect growth regulators
Aim of this thesis was to develop a liquid chromatography-mass spectrometry (LC-MS) method to monitor hormones and their metabolites in the haemolymph of insects simultaneously. Furthermore, some plant-derived alkaloids were structurally elucidated, which may be used as insect growth regulators thus affecting haemolymph hormone titers in putative pest species. Juvenile hormones (JHs), JH diols and ecdysteroids were easily separated by high performance-liquid chromatography (HPLC) in less than 20 min using a reversed-phase C18 column and a methanol-water gradient. Subjecting the JH-JHBP (juvenile hormone binding protein) complex to HPLC was sufficient in releasing JH from the JHBP. In order to prevent JH from binding to glass surfaces, it was necessary to include a carrier in the solvent. JHs have a high affinity to polypropylene vials and should therefore be avoided, if no carrier is being used, such as triton X-100. The darkening of the haemolymph due to eumelanin production by phenol oxidases served as a visual indicator of general enzyme activity in the haemolymph. Isooctane:MeOH (1:1, v/v) inactivated the phenol oxidase system when used at a solvent-haemolymph ratio of 10. An isooctane:MeOH extract of haemolymph was most efficient in keeping JH distributed evenly in solution and prevented JH from adhering to the glass vessel. JH concentration in standard solutions was reduced with increasing sonication time. Highest ionization of JH was achieved in MeOH for MS compared to ACN or by using formic acid as an additive. In the positive ESI (electrospray ionization) mode the most abundant ions formed in haemolymph extract of Gryllus bimaculatus was the sodium adduct for JHs, JH diols and JH acids. At higher JH concentrations, the potassium adduct was also observed. The sodium and the potassium adducts were present in ecdysteroid analysis. The same ionization pattern was observed in Spodoptera frugiperda, Myrmicaria eumenoides and Acyrthosiphon pisum haemolymph. Method validation of the LC-MS method confirmed reproducibility and repeatability. The LODs for JHs and JH diols were between 6 to 12 pg, and 93 pg for ecdysteroids. 72 haemolymph samples can be processed per day by the LC-ESI-MS method using an autosampler. JH and ecdysteroid titer measurements showed good agreement between haemolymph titers and developmental events in Spodoptera frugiperda and Gryllus bimaculatus larvae and Gryllus bimaculatus adults. In Gryllus bimaculatus female and male last instar larvae, the JH titers were low and steady until day 6. The ecdysone peak maximum shifted from day 3 to a 20-hydroxyecdysone maximum peak on day 5, coinciding with the onset of adult ecdysis. JH III titers increased on day 3 in paired Gryllus bimaculatus males, occurring simultaneously with spermatophore maturation and deposition. A similar response was seen with ecdysone titers. Mated female crickets experienced a JH III titer increase on day 4 which coincides with egg deposit on day 4. Ecdysone titers reach a maximum on day 3 and ovary weights on day 4. Besides JH III, JH I was found in 5 to 8-day old female adult crickets, and in 6 and 7-day old male adult crickets. 20-Hydroxyecdysone was found neither in female nor in male Gryllus bimaculatus mated adults. The “classical” interplay between JHs and ecdysteroids was observed in 5th instar Spodoptera frugiperda larvae. JH titers decreased towards the end of the larval stadium and 20-hydroxyecdysone gave a sharp peak on the last day of the 5th instar. Extremely low levels of JH were measured in 6th instar larvae. 20-Hydroxyecdysone and ecdysone titers increased simultaneously in prepupae. The already known alkaloids arborinine and arborine, and the for the first time isolated 4-methoxy-1-methyl-2(1H)-quinolinone from Glycosmis pentaphylla, were extracted from Glycosmis pentaphylla leaves and are discussed as possible insect growth regulators affecting hormone titers in the haemolymph of insect pest species.
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