Rapid quantification of juvenile hormones and their metabolites in insect hemolymph by liquid chromatography-mass spectrometry (LC-MS)

Department of Animal Ecology I, University of Bayreuth, Universitätsstrasse 30, 95440 Bayreuth, Germany.
Analytical and Bioanalytical Chemistry (Impact Factor: 3.44). 07/2004; 379(3):540-3. DOI: 10.1007/s00216-004-2598-x
Source: PubMed


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.

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Available from: Klaus H Hoffmann
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    • "Concentrations of the juvenile hormone homologs (JH I, JH II, and JH III) in hemolymph and tissues were quantified by the LC– MS method as described previously (Westerlund and Hoffmann, 2004 "
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    ABSTRACT: In the polyandric moth, S. frugiperda, juvenile hormone (JH) is transferred from the male accessory reproductive glands (AG) to the female bursa copulatrix (BC) during copulation (see Hassanien et al., 2014). Here we used the RNA interference technique to study the role of allatoregulating neuropeptides in controlling the synthesis and transfer of JH during mating. Knockdown of S. frugiperda allatostatin C (Spofr-AS type C) in freshly emerged males leads to an accumulation of JH in the AG beyond that in the control and mating results in a higher transport of JH I and JH II into the female BC. Knockdown of S. frugiperda allatotropin 2 (Spofr-AT2) significantly reduces the amount of JH in the AG as well as its transfer into the female BC during copulation. Knockdown of S. frugiperda allatostatin A (Spofr-AS type A) and S. frugiperda allatotropin (Spofr-AT; Hassanien et al., 2014) only slightly affects the accumulation of JH in the AG and its transfer from the male to the female. We conclude that Spofr-AS type C and Spofr-AT2 act as true allatostatin and true allatotropin, respectively, on the synthesis of JH I and JH II in the male AG. Moreover, both peptides seem to control the synthesis of JH III in the corpora allata of adult males and its release into the hemolymph.
    Full-text · Article · Jul 2014 · Journal of Insect Physiology
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    • "Although classical studies suggested that the patterns of JH-titer transitions during intermolt determined the future caste differentiation, precise measurements of JH titer were not performed until the establishment of JH quantification methods (Zera, 2007). So far, three major methods for JH quantification have been reported: radioimmunoassay (Greenberg and Tobe, 1985; Goodman et al., 1990), gas chromatography-mass spectrometry (GC-MS) (Shu et al., 1997), liquid chromatography-mass spectrometry (LC-MS) (Westerlund and Hoffmann, 2004). The JH quantification by GC-MS was developed earlier than those using LC-MS. "
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    ABSTRACT: A colony of social insects is not only an aggregation of individuals but also a functional unit. To achieve adaptive social behavior in fluctuating environmental conditions, in addition to coordination of physiological status in each individual, the whole colony is coordinated by interactions among colony members. The study on the regulation of social-insect colonies is termed "social physiology." Termites, a major group of social insects, exhibit many interesting phenomena related to social physiology, such as mechanisms of caste regulation in a colony. In their colonies, there are different types of individuals, i.e., castes, which show distinctive phenotypes specialized in specific colony tasks. Termite castes comprise reproductives, soldiers and workers, and the caste composition can be altered depending on circumstances. For the regulation of caste compositions, interactions among individuals, i.e., social interactions, are thought to be important. In this article, we review previous studies on the adaptive meanings and those on the proximate mechanisms of the caste regulation in termites, and try to understand those comprehensively in terms of social physiology. Firstly, we summarize classical studies on the social interactions. Secondly, previous studies on the pheromone substances that mediate the caste regulatory mechanisms are overviewed. Then, we discuss the roles of a physiological factor, juvenile hormone (JH) in the regulation of caste differentiation. Finally, we introduce the achievements of molecular studies on the animal sociality (i.e., sociogenomics) in terms of social physiology. By comparing the proximate mechanisms of social physiology in termites with those in hymenopterans, we try to get insights into the general principles of social physiology in social animals.
    Full-text · Article · Apr 2014 · Frontiers in Physiology
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    • "None of the experimental animals died during the treatments. Titers of JH III, ecdysone, and 20-hydroxyecdysone 25 in the haemolymph of the females were determined by high-performance liquid chromatography/mass spectrometry as previously described (Alamer and Hoffmann 2013; Westerlund and Hoffmann 2004). "
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    ABSTRACT: Adult females of the ovoviviparous Argentinian cockroach, Blaptica dubia, were repeatedly treated with either 100 μg methoprene or 100 μg pyriproxyfen in 5 μL acetone either during the first vitellogenic cycle or during the period of gestation. Treatment during the first vitellogenic cycle (days 2–20 of adult life) did not inhibit vitellogenesis and oocyte growth, but prevented the formation of an ootheca. This was accompanied with a significant reduction of the titer of juvenile hormone (JH) III and an increased amount of ecdysone (E) and 20-hydroxyecdysone (20E) in the haemolymph of the animals. Treatment of adult females during the period of gestation (days 30–70) resulted in a complete degradation and resorption of the ootheca and induced another vitellogenic cycle. Again, this was associated with a decrease in haemolymph JH III titer, but an increase in the concentrations of free ecdysteroids.
    Full-text · Article · Jan 2014 · Invertebrate Reproduction and Development
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