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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: 3.58). 07/2004; 379(3):540-3. DOI: 10.1007/s00216-004-2598-x
Source: PubMed

ABSTRACT 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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    • "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.
    Journal of Insect Physiology 07/2014; 66. DOI:10.1016/j.jinsphys.2014.05.012 · 2.50 Impact Factor
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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.
    Invertebrate Reproduction and Development 01/2014; 58(1). DOI:10.1080/07924259.2013.810675 · 0.67 Impact Factor
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    • "However, it is still unclear whether JH titre is different among aphids producing different morphs. The recent development of the liquid chromatography-mass spectrometry (LC-MS) method to quantify JH titre in small insects (Westerlund & Hoffmann, 2004; Schwartzberg et al., 2008) enables us to now investigate the relationship between JH titre and reproductivemode change. In parallel, the recent sequencing and assembly of the pea aphid, Acyrthosiphon pisum, genome opens new routes to identifying the genes encoding key "
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    ABSTRACT: Most aphids show reproductive polyphenism, i.e. they alternate their reproductive modes from parthenogenesis to sexual reproduction in response to short photoperiods. Although juvenile hormone (JH) has been considered a likely candidate for regulating the transition from asexual to sexual reproduction after photoperiod sensing, there are few studies investigating the direct relationship between JH titres and the reproductive-mode change. In addition, the sequencing of the pea aphid genome has allowed identification of the genes involved in the JH pathway, which in turn allows us to examine their expression levels in relation to the reproductive-mode change. Using liquid chromatography-mass spectrometry in the pea aphid, JHIII titre was shown to be lower in aphids producing sexual morphs under short-day conditions than in aphids producing parthenogenetic morphs under long-day conditions. The expression levels of genes upstream and downstream of JH action were quantified by real-time quantitative reverse-transcription-PCR across the reproductive-mode change. The expression level of JH esterase, which is responsible for JH degradation, was significantly higher in aphids reared under short-day conditions. This suggests that the upregulation of the JH degradation pathway may be responsible for the lower JHIII titre in aphids exposed to short-days, leading to the production of sexual morphs.
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