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A simple, fast and sensitive method was developed for routine determination of juvenile hormone (JH), JH diols and JH acids in insect haemolymph, by liquid chromatography-mass spectrometry (LC-MS). Sample clean-up involves the precipitation of proteins by methanol/isooctane (1:1, v/v), centrifugation and partial evaporation of the organic solvents. Since JH is bound to a carrier protein in the haemolymph, a binding protein (BP) assay was performed to ensure JH is removed during precipitation. The JH compounds were separated on a C(18) column (ReproSil-Pur ODS-3) by gradient elution with water and methanol in less than 22 min and analysed by electrospray mass spectrometry. Due to the high abundance of Na(+) in insect haemolymph, [M+Na](+) is primarily formed. The limit of detection and quantification was 6 and 20 pg for JHs, and 8 and 25 pg for JH diols, respectively. To demonstrate the applicability of the method to different insect orders, haemolymph samples from the Mediterranean field cricket ( Gryllus bimaculatus), the fall armyworm ( Spodoptera frugiperda), the pea aphid ( Acyrthosiphon pisum) and an ant species ( Myrmicaria eumenoides) were analysed.

Despite their abundance and their enormous signiWcance for various ecological processes, endocrine systems of microcrustaceans have been poorly investigated. Here, we used a highly sensitive radioimmunoassay (RIA) to determine free and conjugated ecdysteroid levels in whole-body extracts of adult Daphnia magna during a complete molt cycle. Ecdysteroid levels were predominated by free ecdysteroids. Starting from basal levels in the postmolt stage the concentration of free ecdysteroids increased sharply in the early premolt stage, followed by a sharp decline back to basal levels just prior to ecdysis. Polar and apolar ecdysteroid conjugates were found in rather low amounts with little variation during the molt cycle. Only small amounts of ecdysteroids were found in newly deposited eggs of D. magna, which suggest a sparing investment of maternal ecdysteroids into the eggs for early embryogenesis. As in whole-body extracts, free ecdysteroids were the predominant ecdysteroids found in eggs of D. magna, together with small amounts of polar and intermediary amounts of apolar conjugates. Hence, the pathways leading to polar and apolar ecdysteroid conjugates appear to be of minor importance in D. magna. Analyses of the immunodetectable peak in free ecdysteroids by liquid chromatography-mass spectrometry (LC-MS) revealed that molting is induced most probably by an increased level of 20-hydroxyecdysone. Microcrustaceans of the genus Daphnia are key components in freshwater food webs. Examination of the functional role of ecdysteroids in controlling developmental processes might help to understand the performance of the herbivorous grazer in its environment, in particular with regard to the adverse eVects of environmental xenobiotics acting as endocrine disrupters.

We investigated the influence of juvenile hormones (JH) on the composition of cuticular hydrocarbons (CHCs) and the division of labor in colonies of the African ant Myrmicaria eumenoides. CHCs have long been implicated in nestmate recognition in social insect colonies. In M. eumenoides, the CHC profiles also vary with the task performed from brood-tender-type to forager type. The endocrine factors regulating the task allocation as well as the intracolonial recognition cues are not well understood, but JHs are prime candidates. Only JH III was identified in the hemolymph of M. eumenoides workers. Foragers had significantly higher JH III titers than brood tenders. The application of exogenous JH III and a JH analogue (methoprene) to M. eumenoides workers did not result in an observable acceleration of task change in our study. However, longevity of the focus workers, and thus the observational period, was reduced by the applications. Changes from a brood-tender-type to a forager-type CHC profile were accelerated by the application of JH III and methoprene, resulting in brood-tending workers that displayed forager-type CHC profiles. We present the first data supporting that recognition cues of an eusocial Hymenopteran are influenced by JH III, which could thus play a major role in the regulation of the dynamic nature of social insect colonies. JH III is connected to at least two key processes: the acceleration of CHC changes and the more long-term modulation of task shifting. Moreover, this indicates that changes in CHC recognition cues do not trigger task allocation in social insect colonies.

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