Common structural features facilitate the simultaneous identification and quantification of the five most common juvenile hormones by liquid chromatography-tandem mass spectrometry

https://doi.org/10.1016/j.ibmb.2019.103287Get rights and content

Highlights

  • This protocol allows the simultaneous analysis in a single LC-MS/MS run of JH I, JH II, JH III, JHB3 and JHSB3.

  • The JH homologs common structural features led to similar chromatographic behavior, as well as related fragmentation patterns.

  • Addition of deuterated JH III as an internal standard permits the absolute quantification of the different JHs.

  • The protocol detects JHs in the low femtomole range, allowing often the analysis of JH in individual insects.

  • We identified and quantified JHs from samples of different species of Diptera, Lepidoptera, Heteroptera and Hymenoptera.

Abstract

This study reports the development and application of a liquid chromatography method coupled to electrospray tandem mass spectrometry (LC-MS/MS) for the identification and quantification of the five most common juvenile hormone (JH) homologs and methyl farnesoate (MF). The protocol allows the simultaneous analysis in a single LC run of JH I, JH II, JH III, JH III bisepoxide (JHB3) and JH III skipped bisepoxide (JHSB3). The identification of JHs is based on multiple reaction monitoring (MRM), using two of the most abundant fragmentation transitions for each hormone. Addition of deuterated JH III as an internal standard permits the absolute quantification of the different JHs. The JH homologs common structural features led to similar chromatographic behavior, as well as related fragmentation patterns, which facilitated the simultaneous detection of all the homologs in a single LC-MS/MS run. The protocol detects JHs in the low femtomole range, allowing often the analysis of JH in individual insects. Fragmentation of each of the JH homologs generates unique diagnostic ions that permitted the identification and quantification of JHs from samples of different species of Diptera, Lepidoptera, Heteroptera and Hymenoptera. Having a simple protocol, which can undisputedly determine the identity of the homologs present in a particular species, provides us with the opportunity to identify and quantify JHs existing in insects that are pests, vector of diseases or important research models.

Introduction

Juvenile hormones (JHs) are synthesized by the corpora allata glands (CA). They play key roles in many processes in insect development and reproduction, including inhibition of metamorphosis, caste determination and differentiation, stimulation of flight and migration, regulation of reproduction, control of diapause, stress resistance, and aging (Goodman and Cusson, 2012; Zhu and Noriega, 2016). Consequently, JHs have been considered as targets for the development of novel insecticides (Cusson et al., 2013). Several JH homologs have been identified in insects. The first two JHs, JH I and II, were isolated from the moth Hyalophora cecropia (Röller et al., 1967; Meyer et al., 1968). JH III, the homolog found in most insects, was described from the moth Manduca sexta (Judy et al., 1973). In addition, two double-epoxidated compounds were later reported, JH III bisepoxide (JHB3) in Drosophila melanogaster (Richard et al., 1989), and JH III skipped bisepoxide (JHSB3) in the heteropteran Plautia stali (Kotaki et al., 2009, 2011). JH titers in insects are often in the femtomole to picomole range, which makes it challenging to measure them by most typical analytical techniques, such as radioimmunoassay and mass spectrometry (MS) coupled with gas or liquid chromatography and capillary electrophoresis (reviewed in Rivera-Perez et al., 2014). Previously, we described the detection and quantification of JH III using a liquid chromatography method coupled to electrospray tandem mass spectrometry analysis (LC-ESI-MS/MS) that increased sensitivity and reproducibility, while reducing the analysis time (Ramirez et al., 2016). In the present study, we optimized a LC-MS/MS method to identify and quantify simultaneously several different JH homologs. The protocol allows the concurrent analysis in a single LC run of the five most common JH homologs: JH I, JH II, JH III, JH III bisepoxide (JHB3) and JH III skipped bisepoxide (JHSB3), as well as methyl farneosate (MF). We utilized multiple reaction monitoring (MRM), selecting two of the most abundant fragmentation transitions for each hormone. Including a deuterated JH III as an internal standard permitted the absolute quantification of the different JHs. The protocol detects JHs in the low femtomole range (pg/ml), allowing often the analysis of JH in individual insects. Fragmentation of each of the JH homologs produced unique diagnostic ions that allowed the identification and quantification of JHs from samples of species of Diptera, Lepidoptera, Heteroptera and Hymenoptera. This simple protocol can unquestionably determine the identity of the JH homolog present in a particular species, and provides the opportunity to identify and quantify JHs existing in insects that are pests, vector of diseases or important research models.

Section snippets

Materials and reagents

Certified standard solutions for JH I, JH II, JH III skipped bisepoxide (JHSB3), JH III and its deuterated analog (JH III-D3) were obtained from Toronto Research Chemicals (Toronto, Canada). JH III bisepoxide (JHB3) and methyl farnesoate (MF) were from Echelon (Salt Lake City, Utah). Sodium chloride, potassium chloride, hydrochloric acid, sodium hydroxide, ammonium acetate, ammonium formate and ammonium hydroxide salts were analytical grade or better (Fisher Scientific, Pittsburgh, PA). Water,

The common structural features of the JH homologs facilitated their simultaneous analysis

The five JH homologs analysed are sesquiterpenes (16C) that have a methyl ester (α, β-unsaturated) at the C1 position and an epoxide ring at the C10–C11 position; with the different JH homologs displaying changes in the numbers and positions of carbons and epoxide groups (Fig. 1). These structural similarities, which include the 2E,6E geometry of the JH skeleton, dictated by two stereogenic double bonds, are critical for any JH to exert its agonist activity, through binding to the JH receptor (

Conclusions

We developed an analytical workflow for the fast, ultra-trace quantitation of the five most common JHs described in insect samples. The protocol was optimized for accurate quantitative analysis, with higher sensitivity and a reduced number of sample preparation steps. The protocol detects the hormones in the low femtomole range, allowing the analysis of JH in individual insects. The method is highly reproducible, with little variation among different individual experiments. The common

Acknowledgements

We thank Lacy Barton, Callum Kingwell, Salvador Hernandez-Martinez and Fabian Ramos for providing hemolymph from different insects. This work was supported by the National Institutes of Health (Grant No. 2R01AI045545-19 to FGN), and the Advanced Mass Spectrometry Facility of Florida International University.

References (27)

  • J. Zhu et al.

    The role of juvenile hormone in mosquito development and reproduction

    Adv. Insect Physiol. Prog. Mosquito Res.

    (2016)
  • J.-P. Charles et al.

    Ligand-binding properties of a juvenile hormone receptor, methoprene-tolerant

    Proc. Natl. Acad. Sci. U.S.A.

    (2011)
  • M. Cusson et al.

    Juvenile hormone biosynthetic enzymes as targets for insecticide discovery

  • Cited by (33)

    • Sexual dimorphism of diapause regulation in the hemipteran bug Pyrrhocoris apterus

      2022, Insect Biochemistry and Molecular Biology
      Citation Excerpt :

      The upper hexane phase was transferred to a new silanized vial and stored at −20 °C until further use. The JHSB3 amounts present in the hemolymph were quantified by liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) (Ramirez et al., 2020). Accessory glands were dissected in Ringer's solution for the analysis of JHAMT expression.

    • Juvenile hormone mediates lipid storage in the oocytes of Dipetalogaster maxima

      2021, Insect Biochemistry and Molecular Biology
      Citation Excerpt :

      The resulting material was centrifuged at 15,000×g for 30 min at 4 °C and the supernatants collected and used for ELISA. The JHSB3 amounts present in the hemolymph were quantified by liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) (Ramirez et al., 2020). Briefly, 60 μl of hemolymph were collected from females and placed on cold glass silanized vials (Thermofisher Scientific, Waltham, MA, USA) containing 60 μl of anticoagulant solution (PBS with 10 mM Na2EDTA, 26 mM sodium citrate, 26 mM citric acid and 100 mM glucose).

    • The involvement of insulin/ToR signaling pathway in reproductive performance of Rhodnius prolixus

      2021, Insect Biochemistry and Molecular Biology
      Citation Excerpt :

      In this context, the FoxO factor has been reported in B. germanica and T. castaneum to be a regulator, not only of JH biosynthesis, but also of Vg production (Süren-Castillo et al., 2012; Sheng et al., 2011). Interestingly, in the triatomines Dipetalogaster maxima and R. prolixus, it was recently reported that the circulating JH is the double-epoxidated JHSB3, a homolog also identified in other heteropteran species (Ramirez et al., 2020; Villalobos-Sambucaro et al., 2020). Knowing the precise JH in R. prolixus now allows examination of the interaction between JHSB3 and the insulin/ToR signaling pathways during reproduction in R. prolixus.

    View all citing articles on Scopus
    View full text