Identification of Methyl Farnesoate from the Hemolymph of Insects

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Identification of Methyl Farnesoate from the Hemolymph of Insects Peter E. A. Teal,*,† Davy Jones,‡ Grace Jones,§ Baldwyn Torto,⊥ Vincent Nyasembe,⊥ Christian Borgemeister,⊥ Hans T. Alborn,† Fatma Kaplan,† Drion Boucias,∥ and Verena U. Lietze∥ †

Center for Medical, Agricultural and Veterinary Entomology, USDA-ARS, 1700 SW 23 Drive, Gainesville, Florida 32604, United States ‡ Graduate Center for Toxicology, University of Kentucky, Lexington, Kentucky 40506, United States § Department of Biology, University of Kentucky, Lexington, Kentucky 40536, United States ⊥ International Center of Insect Physiology and Ecology, P.O. Box 30772, Nairobi, Kenya ∥ Department of Entomology and Nematology, University of Florida, Gainesville, Florida 32608, United States S Supporting Information *

ABSTRACT: Methyl farnesoate, [methyl (2E,6E)-3,7,11-trimethyldodeca-2,6,10-trienoate (1)] has not been thought be present in the hemolymph of insects, although it is the immediate biosynthetic precursor of the circulating insect hormone juvenile hormone III (methyl (2E,6E)10,11-epoxy-3,7,11-trimethyl-2,6-dodecadienoate) (2). Compound 1 was identified from the hemolymph obtained from five orders of insects. Identification of 1 from the American bird grasshopper was facilitated using both electron impact and chemical-ionization GC-MS, GC-FTIR, and 2D NMR techniques. The identifications from other insects were made using GC-MS, and the amounts of all were quantified using LIM-CI-GC-MS. The ratios of 1 and 2 varied in these insects during different developmental stages. The present results underscore the need for further studies on methyl farnesoate (1) as a circulating hormone in insects. the fruit fly, Drosophila melanogaster (Meigen) (order Diptera),12 where it was found to be present at up to 50 times the concentration of 2. Additionally, it was shown that, for D. melanogaster, 1 has a much higher affinity for the insect RXR (ultraspiracle receptor) than either 2 or its bisepoxide analogue (methyl (2E,6E)-6,7;10,11-bisepoxy-3,7,11-trimethyl2-dodecenoate) (3) and that 1 alone is necessary to induce larvae to pupariate. Given these findings, it was hypothesized that 1 may be present in the hemolymph of other insect groups. To test this hypothesis, spectroscopic techniques were used to identify and quantify titers of 1 in the hemolymph of species covering five orders of insects. Initially, hemolymph was obtained 2 h before dark from the American bird grasshopper [Schistocerca americana (Drury), order Orthoptera], a hemimetabolous insect (egg−nymph− adult developmental stages). Analysis of samples of the hemolymph from sexually mature females using GC-CIMS (isobutane reagent gas) showed the presence of a prominent peak having a base peak at m/z 251 (M + 1) (Figure S1, Supporting Information). Significant fragment ions representing losses of CH3OH (m/z 219) followed by loss of CO (m/z 191) were indicative of a methyl ester. Subsequent analysis (Figure 1) using GC-EIMS showed a molecular ion at m/z 250

I

n addition to morphogenic effects, the presence or absence of insect juvenile hormones, most commonly, juvenile hormone III (methyl (2E,6E)-10,11-epoxy-3,7,11-trimethyl2,6-dodecadienoate) (2), has been implicated in regulation of a wide range of biological functions and events including reproductive competence, diapause, and even division of labor in insects.1 As such, these sesquiterpene epoxides are critically important to insect life. Although 2 has not been found in crustaceans, the structurally related homologue methyl farnesoate [methyl (2E,6E)-3,7,11-trimethyldodeca-2,6,10-trienoate] (1) is produced and released from the mandibular organ, a gland homologous to the corpora allata of insects that secretes 2.2 Methyl farnesoate (1) is well documented to have hormonal effects on larval development, molting, reproduction, morphogenesis, and behavior in crustaceans, thus directly paralleling the action of the epoxidized juvenile hormones of insects.2−4 Indeed, the only structural difference between 1 and the juvenile hormone homologue common to all insect species, 2, is the presence in the latter compound of an epoxide, rather than a double bond at carbons C10 and C11. Juvenile hormone III (2) is biosynthesized by insects from 1 via the action of an epoxidase in the corpora allata.5,6 Interestingly, embryos of primitive insects produce and physiologically respond to the administration of 1, and the cultured corpora allata of all insects synthesize 1.7−12 However, until recently there has been no demonstration that 1 exists as a circulatory hormone in any insect. Our group identified 1 recently from the hemolymph of © 2014 American Chemical Society and American Society of Pharmacognosy

Received: September 30, 2013 Published: January 27, 2014 402

dx.doi.org/10.1021/np400807v | J. Nat. Prod. 2014, 77, 402−405

Journal of Natural Products

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Table 1. NMR Spectroscopic Data for Natural and Synthetic Methyl Farnesoate (1) natural position

δ 13Ca [ppm]

2 4 5 6 and 10 8 9 12 14 13 15 OCH3

115.0 40.8 25.7 122.9 39.6 26.5c 25.5 15.7 17.4 18.7 50.6

synthetic

δ 1H [ppm]b

δ 13Ca [ppm]

δ 1H [ppm]b

1H 2H 2H 2H 2H 2H 3H 3H 3H 3H 3H

115.0 40.8 25.7 123.0 39.6 26.5 25.5 15.7 17.4 18.7 50.6

1H 2H 2H 2H 2H 2H 3H 3H 3H 3H 3H

5.67, 2.17, 2.17, 5.08, 1.98 2.05 1.68, 1.60, 1.60, 2.16, 3.68,

s s s m

s s s br s s

5.67, 2.17, 2.17, 5.08, 1.98 2.05 1.68, 1.60, 1.60, 2.16, 3.68,

s s s m

s s s br s s

a 151 MHz. b600 MHz. COSY and HSQC NMR spectroscopic data in CDCl3. Chemical shifts referenced to δ(CHCl3) = 7.26 ppm for 1H and δ(CHCl3) = 77.36 ppm for 13C. Coupling constants are given in hertz [Hz]. cC-9 signal is at the noise level in the natural sample due to the small amount of natural 1. 1H and 13C NMR chemical shifts were deduced from the 1D 1H and 2D HSQC spectra.

Figure 1. Effect of time of day on the amounts of 1 and 2 present in the hemolymph of mated females of S. americana (n = 5 samples/time ± SEM). Note that the y-axis is given in a log scale.

found, although the maximum amount of 1 2 h before the lights went off was only 0.08 ng/μL (±0.003 SE, n = 5). Thinking that 1 might be released as a pheromone for communication, volatiles released by both males and females, individually and in groups of three, were collected both during the day and overnight using standard techniques.14 Analysis of the volatiles using limited-ion-monitoring GC-CIMS did not reveal the presence of any 1 despite a detection limit of 0.25 pg. Thus, there is no evidence to support 1 being released as a pheromone. These results showed that, in this species, there is a daily rhythm associated with amounts of 1 in the hemolymph and that at certain times 1 is the dominant farnesoid present in the hemolymph. To determine if 1 was present in other hemimetabolous insects, we obtained extracts of hemolymph from adult females of three species of true bugs (order Hemiptera) including laboratory-reared females of the coconut bug (Pseudotheraptus wayi, Brown) (7 days old) (Figure S4, Supporting Information) and field-collected adult females of the Southern green stink bugs, Nezara viridula (Linnaeus) (Figure S6, Supporting Information), and leaf-footed bugs [Leptoglossus phyllopus (Linnaeus)]. All species contained greater quantities of 1 than 2. Extending the study to holometabolous insects (egg−larva− pupa−adult life stages), hemolymph was sampled from females of the housefly, Musca domestica Linnaeus, a member of the order Diptera. Methyl farnesoate (1) (Figure S7, Supporting Information) increased dramatically during the first four days of adult life from 1.7 pg/μL (SE = 1.1, n = 5) to 40.5 pg/μL (SE = 16, n = 5), while that of 2 increased only from a low of 0.3 pg/ μL (SE = 0.2) on day 1 to 3.6 pg/μL (SE = 1.3) on day 4 (Figure 2). JH III bisepoxide (3) did not change substantially during the first three days but was somewhat higher on day 4. Extracts of hemolymph of the third instar of worker honeybees (Apis melifera Linnaeus, order Hymenoptera) were also analyzed, and an average of 104.3 pg/μL (±23.4, SE, n = 8) of 1 (Figure S8, Supporting Information) and only 7.5 pg/μL (±1.5, SE, n = 8) of 2 were found, indicating that at this stage 1 was the predominant circulating farnesoid. However, the

coupled with loss of m/z 43 (m = 207) and ions at m/z 69, 91, and 121 all indicative of a sesquiterpene methyl ester having the formula C16H26O2, supporting its assignment as 1. Compound 1 was synthesized from (2E,6E)-3,7,11-trimethyldodeca-2,6,10trien-1-ol using common techniques13 and was purified by GC prior to GC-MS analysis (Figures S2 and S3, Supporting Information). Both CI-MS and EI-MS spectra as well as the retention indices on both polar (DB-35) and apolar (DB-1) GC columns of the synthetic 1 were identical to those of the natural product. Further structural confirmation that the natural compound was indeed 1 was obtained from GC-FTIR comparison of the natural compound with synthetic 1 (Figure S4, Supporting Information). Additionally, the natural compound, after GC purification, was compared to synthetic 1 using 1H and 13C COSY and HSQC NMR analysis. As shown in Table 1, all assignments for the natural compound, with the exception of the 13C NMR signal for C-9 of the natural material, were identical with those of synthetic 1. The amount of natural 1 obtained was too small to obtain a 13C NMR signal above noise level for C-9. Combining all spectroscopic and chromatographic information, it may be concluded that 1 is present in the hemolymph of the American bird grasshopper. In fact, when the amount of 1 was quantified from these samples using limited-ion-monitoring GC-CIMS, an average of 2.15 ng/ μL (±0.24 SE, n = 6) of 1 but only 0.03 ng/μL (±0.005 SE, n = 6) of 2 were recovered. The recovery of such large amounts of 1 relative to that of 2 from the grasshoppers was questioned, so the sampling was repeated using similarly aged females and males at different periods of the photophase. The amounts of 1 were high for both males and females 2 h before dark but were reduced 2 h after illumination and were lowest at the midpoint of the photophase (Figure 1). Thus, over the course of the photoperiod, the amounts of 1 showed a 200-fold variation. Interestingly, although also the amount of 2 was variable, it only changed by 3-fold in the hemolymph from females and 8-fold in males during the day. The same study was performed using third instar nymphs of unknown sex, and the same trend was 403

dx.doi.org/10.1021/np400807v | J. Nat. Prod. 2014, 77, 402−405

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Extraction and Analysis from Hemolymph. Sesquiterpenes were extracted from methanol extracts of hemolymph by injecting 250 μL of pentane, containing 1 pg/μL each of the internal standards Z-9tetradecenoic acid methyl ester (Z-9-14:OMe) and farnesyl acetate (FA), through the cap of the vial and then vortexing the mixture for 2 min.14 The emulsion was broken by centrifugation at 8000g for 5 min, and the pentane layer transferred to a new vial. The methanol extract was re-extracted using only pentane (200 μL) two additional times, and the three pentane extracts were combined and centrifuged as above to separate any water. The pentane extracts were transferred to new autosampler vials and concentrated to ca. 25 μL under a fine stream of N2 prior to chemical analysis. Routine chemical analyses were conducted using chemicalionization mass spectroscopy (CIMS, isobutane reagent gas) with Agilent 5975B and 5975C mass spectrometers interfaced to Agilent 6890N and 7890A gas chromatographs (GC), respectively. Both GCs were equipped with cool-on-column injectors fitted with 10 cm lengths of 0.5 mm (i.d.) deactivated fused silica tubing connected to a 1 m × 0.25 mm (i.d.) deactivated fused silica retention gap and connected either to a 30 m × 0.25 mm (i.d., 0.25 μm coating thickness) DB5MS analytical column in the 5875 GC or to a 30m × 0.25 mm (i.d., 0.25 μm coating thickness) DB35MS analytical column in the 7890 GC. The chromatographic conditions for both instruments were as follows: initial oven and injector temperatures, 30 °C, 5 min; oven and injector temperatures increased at 10 °C/min; final temperature, 225 °C; helium carrier gas, LVF 21 cm/min. For quantitative analysis the mass spectrometers were operated in the chemical-ionization selective-ion mode using m/z 241 for Z-9-14:OME; m/z 251, 219, and 191 for 1; m/z 267, 249, and 235 for 2; m/z 221 and 203 for FA; m/z 283, 265, and 251 for 3.16 Total ion spectra (60−300 amu) were obtained on both instruments. Electron-impact spectra (60−300 amu) were obtained using a third Agilent 5975B instrument interfaced to a 7890 GC equipped as above except that a 30 m × 0.25 mm (i.d., 0.25 μm coating thickness) DB1MS analytical column was used. For all analyses fragmentation patterns and retention times were compared with those of authentic standards. Fourier transformed infrared (FTIR) spectra were obtained using a Nicolet 6700 FTIR interfaced to an Agilent 6890 GC equipped with a cool-on-column injector fitted with a 10 cm length of 0.5 mm (i.d.) deactivated fused silica tubing connected to a 1 m × 0.25 mm (i.d.) deactivated fused silica retention gap, connected to a 30 m × 0.25 mm (i.d. 0.25 μm coating thickness) DB1MS analytical column. The column was maintained at 30 °C for 1 min, then increased at 15 °C to a final temperature of 260 °C. The analytical column was interfaced directly to the FTIR light pipe (240 °C) through a transfer line (240 °C). The light pipe was flushed with additional He makeup gas, and the exit was connected in series to the FID using 0.53 mm deactivated fused silica tubing. FTIR data were acquired from 4000 to 600 cm−1 using a resolution of four wave numbers and eight scan averaging. Samples of 1 were synthesized, and natural 1 from locust hemolymph were purified by preparative wide bore capillary GC using an Agilent 6890 GC with a cool-on-column injector and fitted with a 1 m length of 0.53 mm i.d. deactivated fused silica retention gap, which was connected to 30 m × 0.53 mm (i.d.) (0.5 μm film thickness) DB1 or DB35 column. The analytical column effluent was split using a Y capillary connector between equal lengths of 0.1 mm (i.d.) and 0.25 mm (i.d.) lengths of deactivated fused silica column. The effluent from the 0.1 mm (i.d.) column (ca. 25%) went to the GC FID, while the 0.25 mm (i.d.) column (ca. 75%) exited the wall of the GC and through the heated block (225 °C) of a Brownlee-Silverstein collector.17 Samples of 5 μL were injected onto the column at an initial temperature of 30 °C. After 2 min, the oven temperature was increased to a final temperature of 250 °C at 10 °C/min. The fraction having the same retention time as that of synthetic 1 was collected in a 30 cm cooled glass capillary.17 After collection, samples were recovered by washing the capillaries with three aliquots of either methylene chloride or 25 μL of deuterated chloroform (CDCl3, Cambridge Isotopes, 99.96%). Samples from multiple runs were combined and analyzed by GC-MS for purity and structural properties. Initially samples were fractionated using the DB1 column and then refractionated using the

Figure 2. Comparison of changes in concentrations of 1−3 with increasing age in the hemolymph from adult females of the housefly.

prepupal larvae of worker honeybees had approximately equal amounts of 1 and 2 (1 = 32.2 ± 9.3 pg/μL; 2 = 45.5 ± 9.4 pg/ μL, n = 8). Extracts from adult worker honeybees (15 days old) contained averages of 118.7 pg/μL (±21, n = 10) of 1 and 21.0 pg/μL (±6.1, n = 10) of 2. Hemolymph was sampled from field-collected female June beetles [Phyllophaga crinita (Burmeister)] (Figure S9, Supporting Information) and tilehorned Prionus beetles [Prionus imbricornis (Linnaeus)] and from laboratory-reared larvae of the small hive beetle [Aethina tumida (Murray)], all order Coleoptera. Full electron-impact and isobutane chemical-ionization mass spectra of 1 (60−300 amu) were obtained from all of these species. Thus, chemical data obtained for insects representing five different orders have shown conclusively that 1 is a circulating sesquiterpene in insect hemolymph (Table S1, Supporting Information) and, as would be expected by a hormone, the hemolymph titers were variable depending upon stage of development and/or time of day. Coupled with the facts already shown that 1 has metamorphosis-disrupting effects greater than 2 in larval Drosophila12,16 and has a higher affinity for the nuclear receptor USP than either 2 or 3 and that an analogue of 1 inhibits both adult transformation and embryonic development in a hemipteran,15 the present data provide compelling support for 1 as an important addition to 2 as a circulatory hormone in insects and thus should be included in studies of insect hormonal regulations.

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EXPERIMENTAL SECTION

Insects and Collection of Hemolymph. Schistocerca americana (American bird grasshopper), Aethina tumida (small hive beetle), and Apis melifera (honeybee) were from colonies maintained at CMAVE (Gainesville, FL, USA). Honeybees were collected from hives in early May. Musca domestica (houseflies) were from a colony maintained at the University of Florida (Gainesville, FL, USA). Pseudotheraptus wayi (coconut bug) were from colonies maintained at the International Centre of Insect Physiology and Ecology (ICIPE, Nairobi, Kenya). Females of Nezara viridula (southern green stinkbug), Leptoglossus phyllopus (leaf-footed bug), Phyllophaga crinita (June beetle), and Prionus imbricornis (tile-horned Prionus beetle) were selected from insects collected in a field under artificial lights in LaCross, Florida (longitude N 29.844171, latitude W 82.403993), USA, and identified by P.E.A.T. Information on retention of voucher specimens is provided in the Supporting Information. Hemolymph from houseflies was collected as described by Jones et al.16 For collection of hemolymph from other insects 100 μL capillary pipets drawn to a fine point were used. The point of the pipet was inserted into the dorsal vessel at the sixth abdominal intersegmental membrane. Hemolymph that flowed into the pipet was collected, placed into a Chromacol 750 μL conical autosampler vial held in ice, and then measured using a Hamilton 50 μL syringe. Samples were diluted 10-fold by addition of methanol, capped with Teflon-lined crimp caps, and vortexed for 2 min. Capped samples were stored at −80 °C until processed. 404

dx.doi.org/10.1021/np400807v | J. Nat. Prod. 2014, 77, 402−405

Journal of Natural Products

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DB35 column. Samples, both synthetic and natural, were concentrated to ca. 150 μL and placed in 2.5 mm NMR tubes (Norell). One- and two-dimensional NMR spectroscopy, including double-quantum filtered correlation spectroscopy and heteronuclear single-quantum coherence, was used for verification of methyl farnesoate. All NMR spectra were acquired at 22 °C using a 5 mm TXI cryoprobe and a Bruker Avance II 600 console (600 MHz for 1H, 151 MHz for 13C) (AMRIS-UF facilities, Gainesville, FL, USA). Residual CHCl3 was used to reference chemical shifts to δ(CHCl3) = 7.26 ppm for 1H and δ(CHCl3) = 77.36 ppm for 13C NMR spectra, processed using Bruker Topspin 2.0 and MestReNova (Mestrelab Research) software packages.

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ASSOCIATED CONTENT

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AUTHOR INFORMATION

S Supporting Information *

This material includes figures of electron-impact and chemicalionization mass spectra obtained from both synthetic and natural 1 obtained from different insect species studied representing five insect orders. This material is available free of charge via the Internet at http://pubs.acs.org. Corresponding Author

*Phone: 352-376-6259. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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REFERENCES

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