Development of a Method to Quantitate Nematode Pheromone for

Jan 24, 2013 - Pheromones produced by Caenorhabditis elegans are considered key regulators of development, mating, and social behaviors in this organi...
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Development of a Method to Quantitate Nematode Pheromone for Study of Small-Molecule Metabolism in Caenorhabditis elegans Kwang-Youl Kim,†,§ Hyoe-Jin Joo,†,§,⊥ Hye-Won Kwon,‡ Heekyeong Kim,† William S. Hancock,‡,¶ and Young-Ki Paik*,†,‡ †

Department of Biochemistry, College of Life Sciences and Biotechnology, Yonsei Proteome Research Center, Department of Biomedical Science, and ‡Department of Integrated Omics for Biomedical Research, World Class University Program, Yonsei University, Seoul, Korea ¶ Barnett Institute, Department of Chemistry, Northeastern University, Boston, Massachusetts, United States S Supporting Information *

ABSTRACT: Pheromones produced by Caenorhabditis elegans are considered key regulators of development, mating, and social behaviors in this organism. Here, we present a rapid mass spectrometry-based method (PheroQu) for absolute quantitation of nematode pheromones (e.g., daumone 1, 2, and 3) both in C. elegans worm bodies (as few as 20 worms) and in liquid culture medium. Pheromones were separated by ultra performance liquid chromatography and monitored by a positive electrospray ionization detector in the multiple-reaction monitoring mode. The daf-22 mutant worms were used as surrogate matrix for calibration, and stable deuterated isotope-containing pheromone was used as internal standard for measuring changes in pheromones in N2 wild-type and other strains under different growth conditions. The worm-body pheromones were extracted by acidified acetonitrile solvent, and the secreted pheromones were extracted from culture medium with solid-phase extraction cartridges. The run time was achieved in less than 2 min. The method was validated for specificity, linearity, accuracy, precision, recovery, and stability. The assay was linear over an amount range of 2−250 fmol, and the limit of quantitation was 2 fmol amounts for daumone 1, 2, and 3 in both worm bodies and culture medium. With the PheroQu method, we were able to identify the location of pheromone biosynthesis and determine the changes in different pheromone types synthesized, according to developmental stages and aging process. This method, which is simple, rapid, sensitive, and specific, will be useful for the study of small-molecule metabolism during developmental stages of C. elegans. with chemical analysis; this first pheromone to be characterized was named daumone (i.e., daumone 1).8 More than a dozen members of the pheromone family and their derivatives, collectively called ascaroside (ascr), have since been identified and characterized.9−12 These small molecules have been involved in development, mating, and social behaviors.8,13−16 Recently, more than 140 different structures of these small molecules have been identified in N2 wild-type and mutant strains of C. elegans.17 Thus, it is very important to accurately quantitate changes in these pheromone derivatives by a standard method when studying their relationship to different physiological states. From studies of many laboratories, the major pheromones (i.e., the most abundant in the worm body and secreted forms) found are daumones 1 (ascr#1, C7), 2 (ascr#2, C6), and 3 (ascr#3, C9).8,9,18,19 To maintain consistency in nomenclature

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ver the course of its life stage history (egg, L1−L4, young adult, adult), the free-living nematode Caenorhabditis elegans may be faced with several types of environmental stress (e.g., heat, food deprivation, or increased population density). If such stress occurs at the L1 larval stage, C. elegans can follow an alternative developmental pathway to form a morphologically and physically distinct type of L3 larva, termed dauer. In the dauer state, worms can remain alive for periods of 8−10 times longer than their average lifespan, even without taking in any food.1−4 The dauer state is regarded as nonaging because postdauer larvae (hermaphrodites) can usually live out the rest of the normal lifespan.5 Thus, dauer entry and the associated metabolic adjustments are critically important for survival of worms under adverse growth conditions.6 The key regulators of dauer entry are pheromones, first identified in crude extracts by the Riddle lab in 1982.7 Two decades after the seminal work by the Riddle group, the chemical identity and biological activity of a pheromone was determined for the first time in 2005 by the Paik group, through activity-based purification procedures in conjunction © 2013 American Chemical Society

Received: October 21, 2012 Accepted: January 24, 2013 Published: January 24, 2013 2681

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Figure 1. Chemical structures of three daumones (daumones 1−3) and stable deuterated isotope-containing daumones (D2-daumones 1−3) used as internal standards.

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EXPERIMENTAL SECTION Chemicals and Reagents. Analytical-grade ammonium acetate and formic acid were obtained from Sigma-Aldrich Chemical (St. Louis, MO, USA). Methanol, HPLC-grade water, and acetonitrile were supplied by Burdick & Jackson (Muskegon, MI, USA). Chemically synthesized daumones 1, 2, and 3, used as standards for generation of calibration curves and quality control (QC) samples, were prepared as previously described.20 Deuterated daumones 1, 2, and 3 (D2-daumone 1, D2-daumone 2, and D2-daumone 3, respectively), used as IS, were chemically synthesized in our laboratory (Figure 1). Oasis HLB μElution 96-well solid-phase extraction (SPE) plates (30μm particle size) were from Waters Corporation (Milford, MA, USA). Stock solutions of pheromones were prepared in methanol at a concentration of 1 mg/mL. Combined working solutions for daumones 1, 2, and 3 were further diluted appropriately to obtain a concentration of 1 pmol/μL and diluted serially in methanol to obtain final concentrations of 1, 2.5, 5, 10, 25, 75, and 125 fmol/μL. The IS mixture working solution (25 fmol/ μL for D2-daumone 1 and 3 and 12.5 fmol/μL for D2daumone 2) was prepared by diluting the stock solution in methanol. All stock solutions were stored at −70 °C until use. Preparation of Worm-Body and Liquid Culture Samples. Nematode strains were maintained on nematode growth media agar plates at 20 °C. The N2 Bristol strain, obtained from the Caenorhabditis Genetics Center (Minneapolis, MN, USA), was used for all experiments unless otherwise noted. Worm-body samples were obtained after worms were grown at 20 °C on nematode growth media agar plates containing Escherichia coli (OP50) and washed twice with distilled water. For each sample, 20 worms were collected, transferred into a 1.5 mL tube containing 20 μL of water and stored at −70 °C until use. Liquid culture medium was made by adding 1 mL of 1 M MgSO4, 1 mL of 1 M CaCl2, 1 mL of 5 mg/mL cholesterol, and 1 mL of 20 mg/mL streptomycin to 1 L of S basal liquid medium (5 g of NaCl, 25 mL of 1 M potassium phosphate) containing E. coli OP50 at an optical density of 2.5. Twenty synchronized L1 worms (N2 or daf-22) were transferred to a 2 mL conical tube containing 100 μL of liquid culture medium and incubated in a shaking incubator (180 rpm) at 20 °C.

and for convenience in our laboratory, we have referred to these pheromones as daumones 1−3 (ascr#1−3).8,18,20 Most C. elegans pheromones are composed of ascarylose and a methylated short-chain fatty acid. These pheromones exhibit multiple functions, depending on environmental concentrations (e.g., male mating attraction in the pM range; aggregation behavior in the nM−μM range; and chemoaversion above the μM range). Pheromones that are present in the worm body or secreted into the growth medium are distributed in distinct patterns.15,17 In most previously published methods for pheromone quantitation, including ours, pheromones are quantitated by analyzing m/z values and retention time (min) in the selected ion monitoring mode, using synthetic pheromone standards. These methods require large numbers of worms (>20 000 worms/assay)9,19 to quantitate either secreted or nonsecreted ascarosides and are therefore not suitable for accurate measurement of extremely low-level changes in studies of metabolic regulation and cellular localization of such small molecules. Sensitive methods are required for studies of pheromone metabolism and nematode development. Multiple-reaction monitoring (MRM) is a highly sensitive and selective method for detection and quantitation of small molecules in complex biological samples.21,24−26 Here, we describe a rapid and sensitive pheromone quantitation method, termed PheroQu, using ultra performance liquid chromatography (UPLC) coupled to MRM. The PheroQu method uses stable, deuterated, isotope-containing pheromone as internal standard (IS). It employs extracts of daf-22 mutant worms as surrogate matrix for calibration curve determination because the strain lacks any detectable endogenous pheromones (especially daumones 1−3), both of secreted and nonsecreted forms. Ammonium-adduct ions were chosen as the daumone precursors in positive-ion mode. This method enables rapid detection in the fmol amount of small molecules. It is suitable for use in localization studies of pheromone biosynthesis during different developmental stages and aging, and only small numbers of worms (approximately 20) are required for the assay. 2682

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Table 1. Optimized MS/MS Conditions of Daumones and Deuterated Internal Standardsa MRM transitions declustering potential (DP, V) collision energy (CE, V) collision cell potential energy (CXP, V) entrance potential (EP, V) a

daumone 1

D2-daumone 1

daumone 2

D2-daumone 2

daumone 3

D2-daumone 3

294.3/147.1 30 15 10 10

296.3/147.1 30 15 10 10

264.2/99.0 32 12 12 10

266.2/99.0 32 12 12 10

320.2/173.1 30 13 14 10

322.2/173.1 30 13 14 10

MRM, multiple reaction monitoring; D2, deuterium-containing form.

analytes were eluted at a flow rate of 700 μL/min. The sample manager was set to a temperature of 4 °C and programmed to deliver a 2 μL partial-loop needle overfill-mode injection. Construction of the Tissue-Specific DAF-22 Expression Vector. To make intestinal or hypodermal rescue worms of daf-22 (ok693) mutants, the 1.239 kb of plasmid DNA fragment covering full length of daf-22 cDNA (containing stop codon) was first ligated into the pPD 95.77 worm expression vector between the Xma1/Kpn1 restriction sites. The vha-6 promoter (1.248 kb in length), known as intestinal panpromoter, was inserted between Sal1/Xba1 upstream sites of the cloned daf-22 cDNA, creating the intestinal daf-22 rescue vector. For construction of the hypodermal rescue vector, the dpy-7 hypodermal pan promoter (868-bp length) was ligated between Pst1/Xba1 upstream sites of the cloned daf-22 cDNA. About 50 μg/mL of plasmid DNA containing each rescue vector was injected into daf-22 (ok693) mutants, coinjected with 50 μg/mL of the transformation marker pRF4 containing rol-6 (su1006).

Preparation of Calibration Standard and QC Samples. To measure the amounts of daumones in the worm body, calibration standards and QCs were prepared by spiking 20 daf22 worms in 20 μL of water with 40 μL of combined working solution. Quality control samples were also prepared at concentrations of 1.5 fmol/μL (low; QCL), 50 fmol/μL (medium; QCM), and 100 fmol/μL (high; QCH). To measure the amount of secreted pheromone, 10 μL of combined working solution was added to daf-22 worms in 90 μL of liquid culture medium to obtain a calibration standard and QCs, at the same concentrations described above. All samples were stored at −70 °C until use. Extraction of Pheromones from Worm-Body and Liquid Culture Samples. A frozen 20 μL sample containing 20 worms was thawed at ambient temperature, and 40 μL of methanol and 40 μL of IS working solution were added and mixed in a vortexer for several seconds. The sample was vortexmixed for 10 min with 100 μL of 0.1% (v/v) formic acid in acetonitrile using a multitube vortex mixer (DVX-2500, VWR Scientific, Atlanta, GA, USA). The acidic supernatant was dried in a Speed-Vac centrifuge (Savant SPD111 V-120, Thermo Scientific, USA). Before analysis, samples were reconstituted in 40 μL of 0.1% formic acid in 50% (v/v) acetonitrile and centrifuged at 13 000g for 5 min, and 2 μL was injected into the UPLC-tandem mass spectrometry (MS/MS) system. Liquid culture samples (100 μL) were spiked with 100 μL of IS in 1% formic acid in water and vortex-mixed for 30 s. Oasis HLB μElution 96-well SPE plates were preconditioned with 200 μL of methanol followed by 200 μL of water. Samples, including IS, were loaded onto an SPE plate. Next, the SPE wells were washed with 200 μL of water and eluted twice with 50 μL of methanol. The SPE eluates (2 μL) were injected into the UPLC-MS/MS system. Mass Spectrometry. Mass-spectrometric detection was performed on an API-4000 triple-quadrupole mass spectrometer (MDS SCIEX, Toronto, Canada) operated in MRM mode, using unit resolution on quadrupoles Q1 and Q3. Ionization of all analytes was carried out using electrospray ionization (ESI) in positive-ion mode. The turboion spray (TIS) source temperature was maintained at 650 °C, and the ionspray voltage was set at 5300 V. Both the nebulizer (GS1) and TIS (GS2) were set at 60 psi. The dwell time was set to 25 ms for each MRM transition. Mass parameters of analytes were optimized as shown in Table 1. Data processing was performed using Analyst 1.5.1 software (Applied Biosystems). Liquid Chromatography. Separation of analytes by UPLC was achieved on a C18 column (Zorbax Eclipse, Agilent, Santa Cruz, CA, USA; 1.7 μm, 2.0 × 100 mm) at 30 °C using a Waters Acquity UPLC system. Gradient conditions were as follows: mobile phase A, 0.1% (v/v) formic acid in 2 mM ammonium acetate buffer; mobile phase B, 0.1% formic acid in acetonitrile; 5−35% B for 0.7 min, followed by washing for 0.5 min at 90% B and re-equilibration at 10% B for 1 min. All

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RESULTS AND DISCUSSION Optimization of the UPLC-MS/MS Method. We first investigated the mass-spectrometric behavior of the three major C. elegans pheromones, daumones 1, 2, and 3, in positive- and negative-ion ESI modes by direct infusion. The infusion results revealed that both daumones 1 and 3 were efficiently deprotonated in negative-ion mode, because of their carboxylic acid groups. However, we did not observe deprotonation of daumone 2. In positive-ion mode, we observed formation of sodium and ammonium adducts of the daumones. After addition of ammonium acetate buffer, ammonium-adduct [M + NH4]+ ions for the daumones were detected with 3−5-fold higher sensitivity than ions corresponding to sodium adducts. Therefore, ammonium adduct ions were chosen as the daumone precursors in positive-ion mode and were efficiently fragmented at low collision energies (between 12−15 eV), with loss of ammonia yielding [M + H]+ ions as well as some product ions generated from the corresponding [M + H]+ ions. Among the product ions generated, the most abundant fragment for both daumones 1 and 3 was [M + NH4 − 147]+, generated by loss of ascarylose, whereas the most intense fragment of daumone 2 was [M + NH4 − 165]+, corresponding to the concomitant loss of ascarylose and water (Figure S-1, Supporting Information). Second, to achieve higher sensitivity and rapid separation, we optimized the UPLC-MS/MS conditions. We checked different ammonium acetate concentrations (1, 2, 5, 10, and 20 mM) to get the best adduct ion of pheromone precursor ions for the MS detection and found the following condition yielded the most satisfactory results: gradient elution with 2 mM ammonium acetate in water/acetonitrile (both water and acetonitrile containing 0.1% formic acid) and a 0.7 mL/min flow rate in a 1.7 μm particle C18 column (Figure 2). The 2683

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Figure 2. Extracted ion chromatogram of daumones and internal standards under optimized conditions by UPLC-MS/MS. Column, Zorbax Eclipse C18 (1.7 μm, 2.0 × 100 mm); flow rate, 0.7 mL/min; injection volume, 2 μL; mobile phase, 2 mM ammonium acetate in water/acetonitrile, both containing 0.1% (v/v) formic acid, under gradient conditions. D2 designates deuterium-containing forms.

at the same retention time position (Figure S-3, Supporting Information), indicating that the PheroQu method has very high chromatographic specificity with no carry over. The calibration curves were highly linear within the range of 2−250 fmol for pheromones from worm-body and liquid culture, and the correlation coefficients of all calibration curves were greater than 0.997. The current assay had a lower limit of quantitation of 2 fmol for both worm-body and culture samples, with a signal-to-noise ratio above 5, or within 15% of the nominal value of concentrations back-calculated from the calibration standards (Table 2). We established intra- and interday precision and accuracy by performing repeated analyses using

optimized MRM transitions for daumones and deuterated IS are listed in Table 1. Extraction of Pheromones from the Worm-Body and Liquid Culture Samples. To measure the pheromones in worm bodies, acetonitrile (ACN) containing 0.1% formic acid was employed, which produced the fastest run time and higher accuracy and reproducibility of pheromone analysis. This method was found to be equally efficient as other extraction methods (e.g., homogenization, sonication; Figure S-2, Supporting Information). To remove cellular debris and unwanted solutes, we used an SPE method for isolation of pheromones from liquid culture samples. We found that Oasis HLB μElution 96-well SPE plates, which allow efficient removal of salts and other contaminants from liquid media without additional enrichment, worked well (e.g., salt-out precipitation or column filtration). To improve the assessment of the recovery rate of pheromones, we added an IS dissolved in formic acid into the liquid media before filtering samples through the Oasis cartridge. Pheromones bound to Oasis cartridges were eluted with methanol. Method Validation. After optimizing extraction methods and separation conditions, we evaluated the method’s specificity and other important characteristics (carry over, linearity, precision, accuracy, recovery, and sample stability). For example, the specificity was verified by the fact that there were no differences in detection or retention time of either IS or pheromones that had been extracted from either worm-body or liquid-culture supernatant from the daf-22 mutant strain. To determine whether there was any carry-over material, blank samples were analyzed after high concentrations of pheromones were quantitated. We found that there were no detectable peaks

Table 2. Validation Data of Linearity and Limit of Quantitationa pheromone

matrix

regression equation

daumone 1

worm body liquid culture worm body liquid culture worm body liquid culture

y = 0.01674x + 0.001131 y = 0.01529x + 0.02435 y = 0.03370x + 3.02665 × 10−5 y = 0.03293x + 0.02468 y = 0.01905x + 0.00226 y = 0.01805x + 0.01103

daumone 2

daumone 3

R

b

0.99482

linear range (fmol)

LOQ (fmol)

2−250

2

2−250

2

2−250

2

0.9917 0.99383 0.99589 0.99422 0.99546

a

n = 5; LOQ, limit of quantitation. bShows the results with weighting: 1/x2. 2684

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Table 3. Summary of Intra- and Interassay Precision and Accuracy Results for the Quantitation of Quality Control (QC) Samples of Daumone 1, 2, and 3 in Both Worm-Bodies (W) and Liquid Culture Medium (L)a interassay actual amt. (fmol)

a

measured amt. (fmol)

intra-assay

precision (%, CV)

matrix

W

L

W

L

QCL_3 QCM_100 QCH_200

3.4 97.1 192.0

3.0 102.1 190.8

6.7 7.9 8.5

8.8 7.0 6.4

QCL_3 QCM_100 QCH_200

3.3 92.0 187.0

3.2 101.6 194.2

7.5 9.0 3.0

7.6 6.2 5.3

QCL_3 QCM_100 QCH_200

3.5 91.1 188.6

3.0 100.3 185.4

11.7 7.3 9.6

10.4 8.8 5.9

accuracy (%) W

L Daumone 1 112.1 98.8 97.1 102.1 96.0 95.4 Daumone 2 111.4 106.0 92.0 101.6 93.5 97.1 Daumone 3 115.4 99.3 91.1 100.3 94.3 92.7

measured amt. (fmol)

precision (%, CV)

accuracy (%)

W

L

W

L

W

L

2.9 98.7 203.4

2.9 105.5 206.6

13.7 7.1 7.6

11.9 7.4 2.9

97.6 98.7 101.7

94.0 103.2 102.8

3.4 90.5 185.2

3.0 100.4 189.8

7.4 7.3 6.5

5.7 5.9 8.2

114.4 90.5 92.6

101.3 96.4 98.2

3.2 100.5 180.3

3.1 99.0 188.2

11.8 3.9 12.9

12.8 4.7 12.7

105.5 100.5 90.2

103.0 99.0 94.1

Abbreviations used are QCL (low level, 3 fmol), QCM (medium level, 100 fmol), and QCH (high level, 200 fmol).

five sets of QC (quality control) samples over 5 days. From this experiment, we found that the intra- and interday precision for QCL (low), QCM (medium), and QCH (high) ranged from 3.0% to 11.7% and from 3.9% to 13.7% for worm-body and culture samples, respectively. The intra- and interday accuracies ranged from 91.1% to 115.4% and from 90.2% to 114.4%, respectively (Table 3). The mean extraction recoveries for daumone 1 ranged from 90.4% to 92.0% from worm bodies and from 91.4% to 93.2% from culture medium. For daumone 2, recoveries ranged from 90.1% to 92.0% from worm bodies and from 88.4% to 91.8% from medium. For daumone 3, recoveries ranged from 89.8% to 92.6% from worm bodies and from 90.1% to 93.2% from medium. All recoveries had relative standard deviations higher than 12.5% (Table 4). Given that

Localization of Pheromone Biosynthesis Site. As an example of application, our PheroQu method was employed to localize the pheromone biosynthesis site in the worm body. Having established the quantitative measurement of three major pheromones (daumones 1−3; Figure S-4, Supporting Information), their relative distribution was determined in various tissues. Although most pheromones are predicted to be synthesized in intestine or hypodermis, because three peroxisomal enzymes (ACOX-1, DHS-28, and DAF-22) are localized to the intestine and hypodermis (Figure 3A),18,20,22 the actual cellular location of pheromone biosynthesis site has not been investigated. To identify the biosynthetic site of three major pheromones (daumones 1−3), we prepared a construct expressing daf-22 cDNA under control of tissue-specific promoters, thus restricting DAF-22 protein to specific loci within the cell. For this experiment, we also took advantage of the daf-22 loss-of-function mutant, which cannot make pheromone but instead accumulates very long-chain fatty acids (VLCFA) in large lipid-droplet form (Figure 3B).18 The content of these large lipid droplets are mostly triglycerides from the diet.23 As shown in Figure 3B,C, when the mutation in daf-22 was rescued by the intestinal and hypodermal expression of daf-22 cDNA regulated by its own promoter, we observed that the large lipid droplets disappeared in the intestine and rescued the ability for daumone production. Whereas the large droplet phenotype and incapacity of daf-22 (ok693) for daumone biosynthesis were also rescued by the intestinal tissue-specific expression of DAF-22, they persisted in hypodermal DAF-22 expression. These results suggest that DAF-22 functions only in intestine, where the intestinal triglycerides or VLCFAs are metabolized into pheromone by DAF-22 and other pheromone biosynthetic enzymes. These results led us to conclude that the major pheromones are biosynthesized mainly in the intestine, where triglyceride or VLCFA accumulation is visible when intestinal DAF-22 is absent or defective. However, it remains to be determined what role is played by hypodermal DAF-22 in peroxisomal βoxidation. Changes in Pheromone Production during Development and Aging. The currently available pheromone quantitation methods generally have low sensitivity and, therefore, require much larger samples and a cumbersome pretreatment.16,19 To evaluate the detection sensitivity and

Table 4. Extraction Recovery of Daumones 1, 2, and 3 from Worm-Body and Liquid Culture Medium Samples matrix worm body

pheromones

nominal amt. (fmol)

recovery (%)

daumone 1

3 80 3 80 3 80 3 80 3 80 3 80

90.4 92 90.1 92 89.8 92.6 91.4 93.2 88.4 91.8 90.1 93.2

daumone 2 daumone 3 liquid culture medium

daumone 1 daumone 2 daumone 3

there are no reports in the literature regarding stability of pheromones during storage or analysis, we attempted to evaluate PheroQu using QCs for varying time periods and temperature conditions. Our results show that the pheromones tested were stable at least for 6 months when worm-body or liquid-culture samples were stored at −70 °C. Most of the stability values obtained from samples subjected to three freeze−thaw cycles stayed within 15% of theoretical values (Table S-1, Supporting Information). 2685

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Figure 3. Cellular localization of biosynthesis of daumones 1, 2, and 3. (A) Localization of the daumone biosynthetic enzyme, DAF-22. DAF22::green fluorescent protein (GFP) fluorescence was observed in N2 worms containing the daf-22p::GFP::daf-22 full-gene construct.18 Fluorescence of GFP was visible microscopically as foci both in hypodermis (left) and intestine (right). Scale bar, 50 μm. (B) Large lipid droplets in daf-22 rescue worms. Daf-22 rescue worms were transformed by injecting daf-22 (ok693) mutants with intestinal rescue vector, hypodermal rescue vector, or both (see Experimental Section for details of rescue vector construction). White arrow indicates large lipid droplets. Scale bar, 50 μm. (C) The amount of pheromones (in 2 μL injection volume) in daf-22 rescue worms. Quantitation was performed for three pheromones present in the worm-body of 20 young adult worms each, from the daf-22 (ok693) and the daf-22 rescue strains. Values represent means ± standard deviation from four independent sets. N.D., “not detected”; N.S., “not significant” relative to the amount of pheromones in the daf-22 rescue worms (intestinal and hypodermal).

Figure 4. The amount of worm body, secreted, and total pheromones (in 2 μL injection volume) during development and aging. Twenty N2 worms synchronized at L1 were incubated for 72 h at 20 °C in a shaking incubator. At the indicated developmental time, amount of worm body (A) and secreted (B) daumone and total pheromones containing both worm body and secreted daumones (C). Values represent means ± standard deviation from four independent sets of tubes. (D) Changes in the amounts of pheromones in worm body during adult aging. Synchronized adult N2 worms were grown at 20 °C for 20 days.

determine how the PheroQu method might be useful for detection of pheromones from the worms grown under various conditions, we attempted to quantitate pheromones from worms at different developmental and adult ages. For this comparison, we tested the PheroQu method using small numbers of animals (only 20 worms). Using the previous method, the authors for the previously described method reported that secretion of pheromones such as daumones 2 and

4 increase continuously as development proceeds, whereas daumone 3 increases until L4 but is not detected in adults.19 Using the PheroQu method, pheromone levels during development could be easily quantitated with high accuracy. Previously, in measurements of worm-body and secreted daumones from N2 worms grown in liquid culture for 10 days, we observed that daumone 2 is less abundant than daumones 1 and 3 in worm body, but it is the most abundant 2686

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secreted pheromone.18,20 Consequently, we anticipated that there would be differences in the amounts and distributions of the major pheromones (daumones 1−3). Therefore, we decided to separately quantitate pheromones from worm bodies and culture medium, using 20 worms that had been synchronized at the L1 stage. The worms were grown in liquid culture (100 μL); worm-body and liquid-media samples were prepared separately at 12 h intervals. Our results reveal that the levels of both secreted and nonsecreted daumone 1 and 2 increase as development proceeds (Figure 4A). However, in contrast to a previous report,19 the level of daumone 3 in both secreted and nonsecreted forms increase along with levels of other daumones (1 and 2; Figure 4B). This discrepancy in results may be attributed to differences in the methods of quantitation, reflecting sensitivity and specificity, in the different studies. Interestingly, although daumone 2 is the least abundant pheromone detected in worm-body samples of worms taken after 72 h of growth (1-day adults), daumone 2 was the most abundant secreted pheromone in samples taken from those worms after 24 h (Figure 4C). Thus, daumone 2 is very rapidly secreted after its biosynthesis, as compared to daumones 1 and 3, as previously reported.18 Furthermore, the level of daumone 2 decreases in worm body compared to daumones 1 and 3 only after 72 h, indicating that daumone 2 might saturate and become trapped in the worm body during that 72 h period and subsequently be excreted. When we compared the total quantities of secreted and nonsecreted forms of the three major pheromones, daumone 2 was the most abundant, followed by daumones 3 and 1, during the entire process of development (Figure 4C); this observation is consistent with previous reports.18,20,22 It is plausible that this difference in secretion rate might be caused by one of three factors. First, we initially believed that the different secretion rates might be due to differences in the stability of daumones 1−3, which might be greatly influenced by liquid-culture conditions and the pheromones’ interactions with the external environment. Alternatively, daumones 1 (C7) and 3 (C9) might be degraded into daumone 2 (C6) by an unknown enzymatic reaction in the media. However, we found that all three pheromones tested were quite stable and resistant to breakdown (Table S-1, Supporting Information). Second, differences in the cellular site of synthesis for the various pheromones might cause changes in the secretion rate. However, it was confirmed that daumones 1−3 were detected in the intestine at equal levels (Figure 3). Third, the pheromones may be exported by distinct transporters, each with its own diffusion rate or transport mechanism. This hypothesis can be tested after the pheromone transporters are identified. We observed changes in pheromone levels during the adult aging process. In these experiments, first, we determined the mean lifespan of N2 wild-type worms at 20 °C to be 21.4 ± 1 days. Therefore, we determined the changes in the quantity of pheromones in worm bodies over a period of 20 days, in N2 worms that had been synchronized at day 1. Following young adulthood, the amounts of both daumone 1 and 3 were found to increase for 10 days but decrease thereafter (Figure 4D). The level of daumone 1 in the adult worms was much higher than daumone 3, different from what is observed during development (Figure 4A). As anticipated, daumone 2 levels in the worm body remain constant throughout adulthood, possibly due to constant secretion from the body. In addition, none of the three pheromones was detected in bodies of dead

worms. Taken together, pheromone biosynthesis appears to influence the development and growth process throughout the life history.

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CONCLUSIONS In this study, we establish PheroQu as an improved method for daumone quantitation, which enables more accurate measurement of secreted and nonsecreted forms of the three major pheromones. The PheroQu method may also be applied to many similar pheromone derivatives. This method is simple, rapid (assay times of approximately 2 min), reproducible, highly sensitive, and specific. Thus, this method should be applicable to the study of pheromone metabolism with respect to cellular localization, monitoring of pheromone synthesis throughout the life history, and regulatory mechanisms.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Address: Room 423, Industry-University Research Building, Yonsei University, 50 Yonsei-ro, Sudaemoon-ku, Seoul, Korea, 120-749. E-mail: [email protected]. Tel: +82-2-2123-4242. Fax: +82-2-393-658. Present Address ⊥

Department of Genetics, University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA.

Author Contributions §

K.-Y.K. and H.-J.J. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Caenorhabditis Genetics Center for providing strains. This work was supported by grants from the National Research Foundation of Korea (2011-0028112), the World Class University Program (R31-2008-000-10086-0) funded by the Korean government (Ministry of Education, Science and Technology), and the National Project for Personalized Genomic Medicine (A111218-11-CP01 to Y.-K.P.).

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