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Measuring food intake and nutrient absorption in CaenorhabditiselegansRafaelL.Gomez‐Amaro1,2,3†,ElizabethR.Valentine4†,MariaCarretero1,2,3,SarahE.LeBoeuf1,SunithaRangaraju1, 2, 3, CarolineD. Broaddus1,2,3, GregoryM. Solis1,2,3, JamesR.Williamson 4* andMichaelPetrascheck1,2,3*†Equalcontribution*Correspondingauthors1DepartmentofChemicalPhysiology,TheScrippsResearchInstitute,LaJolla,CA,USA2DepartmentofMolecularandExperimentalMedicine,TheScrippsResearchInstitute,LaJolla,CA,USA3DepartmentofMolecularandCellularNeuroscience,TheScrippsResearchInstitute,LaJolla,CA,USA4DepartmentofIntegrativeStructuralandComputationalBiology,TheScrippsResearchInstitute,LaJolla,CA,USARunningtitle:DissectingnutritioninC.elegansKeywords:Feeding,aging,nutrition,metabolism,denovoproteinsynthesisCorrespondingauthors:MichaelPetrascheckTheScrippsResearchInstituteMEM26810550NorthTorreyPinesRoadLaJolla,CA92037Phone:858‐784‐7923E‐mail:pscheck@scripps.eduJamesR.WilliamsonTheScrippsResearchInstituteMB‐3310550NorthTorreyPinesRoadLaJolla,CA92037Phone:858‐784‐8740E‐mail:jrwill@scripps.edu

Genetics: Early Online, published on April 21, 2015 as 10.1534/genetics.115.175851

Copyright 2015.

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ABSTRACTCaenorhabditis elegans has emerged as a powerfulmodel to study the genetics of

feeding,food‐relatedbehaviors,andmetabolism.DespitethemanyadvantagesofC.elegansasamodelorganism,directmeasurementofitsbacterialfoodintakeremainschallenging.Here,wedescribetwocomplementarymethodsthatmeasurethefoodintakeofC.elegans.The firstmethod is amicrotiter plate‐based bacterial clearing assay thatmeasures foodintakebyquantifying thechange in theopticaldensityofbacteriaover time.Thesecondmethod,termedpulse‐feeding,measurestheabsorptionoffoodbytrackingdenovoproteinsynthesis using a novel metabolic pulse‐labeling strategy. Using the bacterial clearanceassay,we compare the bacterial food intake of variousC. elegans strains and show thatlong‐livedeatmutantseatsubstantiallymorethanpreviousestimates.Todemonstratetheapplicability of the pulse‐feeding assay, we compare the assimilation of food for two C.elegansstrainsinresponsetoserotonin.Weshowthatserotonin‐increasedfeedingleadstoincreased protein synthesis in a SER‐7 dependentmanner, including proteins known topromote aging. Protein content in the food has recently emerged as critical factor indetermining how food composition affects aging and health. The pulse‐feeding assay, bymeasuring de novo protein synthesis, represents an ideal method to unequivocallyestablishhowthecompositionoffooddictatesproteinsynthesis.Incombination,thesetwoassaysprovidenewandpowerfultoolsforC.elegansresearchtoinvestigatefeeding,howfoodintakeaffectstheproteome,andthusthephysiologyandhealthofanorganism.

INTRODUCTION

Food intake has proven difficult tomeasure in smallmodel organisms. Yet, smallmodelorganismssuchasC.elegansoffersignificantadvantagesforthestudyofnutrition,appetite, andmetabolism.As amodel,C.elegans is amenable to genome‐wide screening,hasclearhumanorthologuesforgenesinvolvedinfeedingbehaviors(LUEDTKEetal.2010),andhasconservedmechanismsregulatingfeeding,fatstorage,andenergyexpenditure(DEBONOandBARGMANN1998;SZEetal.2000;ASHRAFIetal.2003;ASHRAFI2006;SRINIVASANetal.2008).

C. elegans captures food by pumping liquids containing bacteria into its mouth.Pumping is achieved by rhythmic contractions of a neuromuscular organ called thepharynx.Measurementsofpharyngealpumpinghavebecomethestandardforestimatingfoodintakeandinferringtheamountoffoodingested(AVERY1993;AVERYandYOU2012).Because C. elegans move freely, it is only possible to measure the rate of pharyngealpumping over short intervals of time (seconds). Thus, with pharyngeal pumping, short‐termmeasurementsmustbeextrapolatedtoestimateoverallfoodintake.

Ratherthan inferringthe ingestionof foodbymeasuringabehavioralchange(e.g.pumping), alternativemethods to estimate food intake quantify the ingestion of labeledbacteriaormicrobeads.Thesemethodsoffertheadvantageofdirectlymeasuringingestedmaterial,butrequireexpensiveandspecializedequipment,andagaincanonlymeasuretherateoffeedingovershortintervalsoftime.(YOUetal.2008;AVERYandYOU2012;PAEKetal.2012).While currently availablemethods allow the study of short‐term changes in foodintake, there is amajor need for simplermethods thatmeasure food intake over longerperiodsoftime.

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Here,we report two complementarymethods that quantifyC.elegans food intakeover several days. The first method, termed bacterial clearance, is a 96‐well microtiterplate‐based assay that measures the food intake of C. elegans in liquid medium byquantifying the change in optical density of bacteria over time. Using the bacterialclearanceassay,wecharacterizedthebacterialfoodintakeofC.elegansoverthecourseofitsdevelopmentand in response to serotonin.We find that smalleranimalseat less, andshowthatthelong‐livedeatmutantseatsubstantiallymorethanpreviousestimates.Whileothermethodsofbacterialclearancehavebeendescribed(VOISINEetal.2007;CABREIROetal.2013),ourassayuniquelyenablesthedirectmeasurementofbacterialfoodintakeperworm,issuitableforuseoverthelifespanoftheanimal,isnotinfluencedorlimitedbythehatching of larvae, and can measure food intake for hundreds of small populationssimultaneously, dramatically increasing its sensitivity. Importantly, the microtiter‐basedbacterial clearing assay can be expanded for medium‐throughput phenotype‐basedchemical and genetic (RNAi, candidate mutants) screens, and requires only standardlaboratoryequipment.

Thesecondmethod,termedpulse‐feeding,measurestheingestionofstableisotope‐labeledbacteria(15N),usingmassspectrometry(LC‐MS/MS) todetectnewlysynthesizedproteinsvia isotope incorporation into theproteome.Simplyput, it isametabolicpulse‐labelingstrategyinwhicha15Npulseisdeliveredbyfeedingworms15N‐labeledbacteria.Thus, any 15N in the proteomemust represent food intake within the pulse period.Weemploythemetabolicpulse‐labelingstrategytocomparetheassimilationof foodandthesubsequentdenovoproteinsynthesis for twoC.elegans strains inresponse toserotonin.We show that serotonin‐induced feeding in C. elegans leads to changes in the de novoproteome,includingincreasedtranslationandribosomebiogenesis.Toourknowledge,thisisthefirststudytomeasuretheabsorptionandutilizationofnutrients(i.e.aminoacids)bymeasuringdenovo protein synthesis, or change the isotopeenrichmentof anyC.elegansfoodsourcemid‐experiment,inamannersimilartopSILAC(SCHWANHAUSSERetal.2009),totimestamptheincorporationoftheisotopelabelintothewormproteome.Furthermore,asthemetabolicpulse‐labelingmethodcapturesdenovoproteinsynthesisinresponsetoanystimulus or condition, investigators can study the de novo proteome of an intactmulticellularorganisminbothnormalanddiseasedstates.

In combination, these two methods enable a comprehensive investigation of thegenesandmetabolites thataffect food intake,andwill facilitate the identificationofnewpharmacologicalinterventionsthataffectfeedingandmetabolism.

MATERIALSANDMETHODS

Strains and genetics: All strains were maintained at 20°C on NGM agar plates aspreviously described (Brenner, S. 1974 Genetics). Caenorhabditis elegans strains usedwere:Bristolstrain(N2),DA820eat‐18(ad820sd),DA1110eat‐18(ad1110),MT15434tph‐1(mg280) II, DA1814 ser‐1(0k345) X, OH313 ser‐2(pk1357) X, DA1774 ser‐3(ok2007) I,AQ866 ser‐4(ok512) III, FX02146 ser‐6(tm2146) IV, RB660 arr‐1(ok401) X, AZ30 sma‐1(ru18) V, CB502 sma‐2(e502) III, and CB491 sma‐3(e491) III. The strains DA2100 ser‐7(tm1325)X,RB2277ser‐5(ok3087)I,CB205unc‐26(e205)IV,andNM204snt‐1(md290)IIwere out‐crossed four times toBristolN2 and renamedVV122, VV130, VV78, andVV80respectively.

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FoodIntake(OD600)Assay:Foodintakewasassessedinliquidmedium(PETRASCHECKetal. 2007; SOLIS andPETRASCHECK2011) (S‐completemediumwith50µgml‐1 carbenicillinand 0.1 μg ml‐1 fungizone) in black, flat‐bottom, optically‐clear 96‐well plates (Costar)containing 150 μl total volume perwell. Plates contained 6‐12 nematodes perwell in 6mg/ml E. coli OP50 (1.5x 108 cfu/ml), freshly prepared four days in advance unlessotherwise specified.We used a carbenicillin resistant OP50 strain to exclude growth ofotherbacteria.Age‐synchronizednematodeswereseededasL1larvaeandgrownat20°C.Plates were covered with sealers (Nunc) to prevent evaporation. To prevent self‐fertilization, 5‐fluoro‐2’‐deoxyuridine (0.12mM final) (Sigma) was added 42‐45h afterseeding. Drugs were added 68h after seeding (day 1 of adult life) unless otherwisespecified.

Theabsorbanceat600nm(OD600)ofeachwellwasmeasuredusingamicroplatereader(TECAN).Measurementsweretakenevery24h,beginningonday1ofadulthood.BeforemeasuringOD600,eachplatewasplacedontoaplateshakerfor25min.ThefractionofanimalsaliveperwellwasscoredmicroscopicallyonthebasisofmovementonDay4ofadulthood.Eachplatewasplacedontoaplateshakerfor1‐2minpriortocounting.Strongmicroscopelightwasusedtoeffectivelystimulatemovementinyoungandoldanimals,toaidcountingofviableworms.Unlessstatedotherwise,foodintakeperwormwascalculatedasthebacterialclearancedividedbythenumberofwormsperwell.Forcomparisonsbetweenstrains,foodintakewasexpressedasapercentagerelativetocontrolanimals.Acompletestep‐by‐stepprotocolisavailableonline(NatureProtocolExchange,PetrascheckLab).

Severaltechnicalaspectsofouroriginalbacterialclearanceprotocolhaveprovenessentialtothesensitivityandrobustnessoftheassay,andmeritfurtherdiscussion.First,inliquidmedium,bacteriaclumptogetherandsettleonthebottomoftheassaywells.Simpleshakingoftheplatesfor25minonamicrotiterplateshakerpriortoeachOD600measurementwasfoundtobesufficienttodissolvebacterialaggregatesandre‐suspendthebacteriainsolution.Minimizingclumpsandsedimentationbyshakingsignificantlyimprovestheaccuracy,sensitivity,androbustnessofthebacterialclearanceassay.Second,bacteriaquicklysettletothebottomofthewellsaftershaking.Assuch,OD600measurementsbecomelessreliableastheintervaloftimebetweentheendofshakingandmeasurementincreases.Itisimportanttokeepthisintervalshort,preferablyunder10minutes,toensurerobustresults.Third,afterperformingseveralcontrolexperiments,weobservedthatdifferentbacterialpreparationshadanaffectonbasalfoodintake.Thus,itiscriticaltopreparethefeedingbacteriainexactlythesamemannerforeachexperiment,andtomeasurechangesinfoodintakerelativetoN2controlanimals.There are technical as well as biological explanations for the differences in basal foodintakebetweendifferentbacterialpreparations.OD600measuresabsorbance,andthusthenumberofparticles,irrespectiveoftheirsize.Bacteriaharvestedinthelogarithmicgrowthphasewillbemuchlargerthanbacteriainthestationaryphase,becausemanywillbeinadividingstate.OD600measurementsdonotdistinguishsize,andthussimpledifferencesinbacterial sizeor statemay lead to apparentbasal food intake changesbetweenbacterialpreparations. Of note, we identified two major determinants of basal feeding betweenbacterial preparations; i) the “freshness” of the bacteria, as inoculations from freshlystreakedbacteriaproducedgreater feeding thanbacteria fromprior inoculations, and ii)

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the“ageofthebacteria”orlengthoftimebacteriawerekeptinnutrient‐poorS‐completemedia after culture in the nutritionally rich TB medium. Bacteria aged four to six daysappearedoptimalforfeeding,assignificantlylowerbasalfoodintakewasobservedwhenwormswere fed bacteria aged for only one day (24 hrs.), independent of theC. elegansstrainorgeneticbackground. Imagingandbodylengthmeasurements:Animalswereimagedinindividualwellsofa96‐well optically clearmicrotiterplate, usingaMolecularDevices ImageXpressplatform.WormSizersoftwarewasusedtoextractthebodylengthofadult(Day4)nematodesfrombrightfieldimages(MOOREetal.2013).Statisticalanalysis:GraphPad Prism softwarewas used for data analysis. ComparisonsandP‐valuecalculationsweremadebetweenanimalsofthesameordifferentstrains,andtreated and untreated animals, using Student’s t‐test and ANOVA with corrections formultiple hypothesis testing.Wells containing less than 4 ormore than 15 animalswereexcludedfromstatisticalanalyses.Drugpreparation: Serotonin hydrochloride (Alfa Aesar) was freshly prepared for eachexperimentanddissolvedinwaterat50Xfinalconcentrationbeforeuse.Metabolic labeling of E. coli and C. elegans: Worms and bacteria were metabolicallylabeledwiththedesirednitrogenisotope(14Nor15N)aspreviouslydescribed(GOUWetal.2011; GEILLINGER et al. 2012). Prior to each experiment, worms were transferred tonitrogen‐freesolidagaroseplatesandseededwith14Nor15Nmetabolicallylabeledbacteriaas the sole source of nitrogen. After aminimum of three generations, age‐synchronizedlarvae were transferred to 10cm petri dishes and cultured in liquid medium plusmetabolicallylabeled14N(experimental)or15N(externalstandard)bacteria(6mg/ml).Forpulse‐labelingexperiments,cultureplateswerefirstclearedofall14Nlabeledbacteria,thenre‐seededwith50%14N/50%15N(M)labeledbacteriaandculturedforanadditionaltwodays.Unlessstatedotherwise, thestartdayofthepulse labelingwasDay5ofadulthood.Fortph‐1pulse‐labelingexperiments,approximately600wormswereharvestedperstrain.For serotoninpulse‐labeling experiments, 600‐800wormsper strainper conditionwereharvestedforanalysisbymassspectrometry.Tocontrolfordifferencesinwormnumbersbetween conditionswithin an experiment, each samplewas adjusted such that an equalnumberofwormswouldbeprocessed.Allpulse‐labelingexperimentswereperformedintriplicate,andindependentlyrepeatedthreetimes.Massspectrometryandproteomics:Lysedwormswerepreparedformassspectrometrybyprecipitationin13%trichloroaceticacid(TCA)overnightat4°C.Theproteinpelletwascollectedbycentrifugationatmaximumspeedfor20minutes.Thepelletwaswashedwith10%TCAandspunagainfor10minutesat4°C.Thepelletwasthenwashedwithicecoldacetoneandspunforanadditional10minutesat4°C.Theproteinpelletwasre‐suspendedin approximately 100‐200 μL of 100 mM ammonium bicarbonate with 5% (v/v)acetonitrile.10%(byvolume)of50mMDTTwasaddedandthesamplewasincubatedfor10minutesat65°C.This incubationwas followedbytheadditionof10%(byvolume)of1mMiodoaceticacid(IAA)anda30minuteincubationat30°Cinthedark.5μgoftrypsin

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wasaddedtoeachsampleandincubatedovernightat37°C.Trypsinizedsampleswerethencleaned up for mass spectrometry using PepClean columns (Pierce) following themanufacturer’s directions. Clean samples were dried in a speed vacuum and then re‐suspendedinapprox10μLof5%Acetonitrilewith0.1%(v/v)Formicacid.Sampleswerespunathighspeedtoremoveparticulatesbeforeplacing inmassspectrometry tubes foranalysis.LC‐MS: Samples were analyzed on an ABSCIEX 5600 Triple TOF mass spectrometercoupledtoanEksigentnano‐LCUltraequippedwithananoflexcHiPLCsystem.ConditionsontheABSCIEX5600wereasfollows.Thesourcegasconditionswereoptimizedforeachexperiment, andweregenerally set toGS1=8‐12,GS2=0, and curtaingas=25.The sourcetemperaturewassetto150C.IDAexperimentswererunwitha2secondcycletime,with0.5msaccumulation time in theMS1 scanandup to20 candidate ionsperMS/MS timeperiod.Them/zrangeintheMS1scanswas400‐1250Da,and100‐1800DafortheMS/MSscans.Target ionswereexcludedafter2occurrences for12seconds inorder to increasesequencingcoverage.Targetionswith+2to+5chargewereselectedforsequencingoncetheyreachedathresholdof100countspersecond.

ConditionsfortheEksigentnano‐HPLCwereasfollows.Gradientswererunwithatrap‐and‐elute setupwithwater plus 0.1% (v/v) formic acid as themobile phaseA, andacetonitrilewith0.1% (v/v) formic acid asmobilephaseB. Sampleswere loadedonto a200μm×0.5mmChromXPC18‐CL3μm120ÅTrapcolumnat2μL/minute.Gradientswererun from5%mobile phaseA to 40%mobile phase B over 2 hours on a 75 μm× 15cmChromXPC‐18‐CL3μm120Åanalyticalcolumn.Thiswasfollowedbyarapidjumpto80%B for10minutes to clean thecolumn,anda20minutere‐equilibrationat95%A.Watersample‐blanks were run between samples to rid the column of any residual interferingpeptides,includingashortgradient,followedbythe80%Band20minutere‐equilibrationwith5%A.LC‐MSdataanalysis:DatawasconvertedusingtheABSCIEXconversionsoftwaretomgfformatandMZMLformat.ThepeaklistwasgeneratedbysearchingaSwissProtdatabaseusing MASCOT, with the taxonomy set to C. elegans and E. coli simultaneously (MatrixScience).AMS/MSIonsearchwasperformedusingthe15Nquantification,withapeptidemass tolerance of ± 0.1 Da and a fragment mass tolerance of ± 0.1 Da. The maximumnumber ofmissed cleavageswas set to 2. MS1 scans for identified peptideswere fit tothree isotope distributions using ISODIST (SPERLING et al. 2008; CHEN et al. 2012). Thepercent labeling in the pulse was first fit using a floating variable to find the bestpercentage, and then fit with a fixed percentage value. This value depended on theexperiment,andvariedbetween42%and50%labeling.Thisvaluemostlikelyvariesdueto the amount of 14NE. coli left in the culture after switching the food to the 50% 15N‐labeledE.coli.Oneadvantageofourfittingmethodisthatwecaneasilyaccountforthesevariations,aswearefittingtheentireisotopicenvelope.Fractionlabeledwascalculatedas(50%15Nintensity)/(14Nintensity+50%15Nintensity).

RESULTS

ThebacterialclearingassaymeasuresfoodintakeofC.elegans

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C.eleganscanbegrownin96‐wellmicrotiterplatesinliquidmediumbyseeding4‐12developmentallysynchronizedL1 larvae into individualwellswithEscherichiacoli(E.coli)asafoodsource.YoungadultsraisedinthisformofliquidculturearemorphologicallyverysimilartoagematchedadultsraisedonNGM(Figure1A,B,SupplementaryFigure1).Theydonotshowtheelongatedandstarvedappearanceseeninothertypesof liquidculture.Incidentally,weobservedthatthewellsofmicrotiterplatescontainingwormsplusbacteria become optically clearer over the course of several days.We reasoned that, bymeasuringthechangeinbacterialconcentrationovertimeinawell,wecouldquantifytheamountofbacteriaeatenbytheworms.

Totestthis,weculturedwormsinmicrotiterplateswithanopticallyclearbottomtoallownon‐invasivemonitoringofbacterialconcentrationsbyopticaldensity(absorbance)at 600nm, herein referred to as OD600 (Figure 1C) (SOLIS and PETRASCHECK 2011). Tomaintainaconstantpopulation,viableoffspringwerepreventedfromhatchingbyadding5‐fluoro‐2’‐deoxyuridine (FUDR) at the L4 larval stage (RANGARAJU et al. 2015). For ourstudies,L4hermaphroditesweredefinedasday0adults.

To ensure that worms eating bacteria caused the observed changes in bacterialconcentrations, we conducted OD600 measurements in the presence and absence ofworms. OD600measurements were collected by a microtiter plate reader every 24 hrsover4days,beginningonday1ofadulthood. Inthepresenceofworms,bacteriaclearedovertimeresulting inaprogressivedrop inOD600values(Figure1D). Incontrast, littlebacterial clearance was observed in the absence of worms. Bacterial clearance wasobservedregardlessofwhetherthewormswerefedwithliveordead(gamma‐irradiated)E.coli (Figure1E), indicatingbacterialclearancewasduetotheconsumptionofbacteriabywormsratherthanthedeathorlysisofbacteria.

Afterfourdaysofadulthood,wormswillhavelaidhundredsofeggsineachwell.Todeterminetheinfluenceoflaideggsonopticaldensitymeasurements,wecomparedOD600valuesofindividualwellspriortoegglaying(day1)andagainaftereggshadbeenlaid(day4). To isolate the changes in OD600 caused by the eggs from those caused by bacterialclearance, we removed all bacteria, but not worms and eggs, prior to the OD600measurements.Despitethelargenumbersofeggsthatwerepresentonday4,theydidnotinfluenceOD600measurements(Figure1F,G,H).Thus,OD600measurementsspecificallydetectthepresenceofbacteriaanddonotdetectthepresenceofeggsorworms.Wormsareonlyindirectlydetectedbyactivelyeatingbacteria.

IfthedecreaseinOD600isduetowormseatingbacteria,themagnitudeofbacterialclearance (OD600)must depend on the number of worms per well. Operationally, wedefinedbacterialclearanceasthedifferenceinOD600betweentwotimepoints(OD600=OD600t1–OD600t2).Morewormsshould result ina largerdifference than fewerworms.Indeed,weobserved a strongpositive correlation (R2=0.77)betweenbacterial clearance(OD600)andthenumberofwormsineachwell(X0)(Figure1I).

Next,weaskedwhetherthefoodintakeofanindividualwormwasaffectedbythenumberofwormsinagivenwell.Toaccomplishthis,wenormalizedtheOD600changestowormnumberperwell(X0).Foodintakeperworm(OD600/X0)canthusbedefinedasthebacterialclearancedividedbythenumberofwormsinthewell(OD600/X0=(OD600t1–OD600t2)/X0).AsshowninFigure1J,thenumberofwormspresentineachwelldidnotaffectthefoodintakeperwormaslongasthenumberofwormsstayedbetween3and14.

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Importantly, by measuring the food intake per worm within the range above, we canperform meaningful comparisons of food intake between wells, populations, andexperimental conditions. Therefore, we used the food intake per worm as the unit ofmeasureforfoodintake.

Afterestablishingthebacterialclearanceassayasarobustmeasureoffoodintakeinadults,wenextmonitoredthefoodintakeofC.elegansthroughoutitsdevelopment.FoodintakepeakedasanimalstransitionedfromL4larvaetoyoungadults,coincidingwiththeonsetofeggproduction.Fromthere,foodintakedeclinedgraduallywithage,asexpected(HUANG et al. 2004) (Figure 1K). Food intake per day was greatest during the intervalbetween day 1 and day 4 of adulthood (D1:D4), making this period ideal for detectingdifferencesinfeedingovertime.Inthisreport,weusedtheD1:D4intervalasthestandardperiodtomeasurefoodintake.Comparingthefeedingratesofeatandsmallmutants

InC.elegans,mutationsintheeatgenesdisruptthefunctionofthepharynx,leadingto reduced pharyngeal pumping and a starved appearance (AVERY 1993; LAKOWSKI andHEKIMI 1998). However, the cumulative food intake of any eat mutant has not beendetermined.We compared the food intake ofwild‐type animalswith different eat‐2 andeat‐18alleles.Whilethefoodintakeofalleatmutantswasreducedcomparedtowild‐type(Figure2A),themagnitudeofthedecreasewasconsiderablysmallerthanpredicted,giventhat their pharyngeal pumping rates are approximately 10% of wild‐type (RAIZEN et al.1995;PETRASCHECKetal.2007).Thefoodintakeofeat‐2andeat‐18mutantswas80%and60%thatofwild‐typeanimals,respectively.Thiswasmuchhigherthanweexpected,butinthecaseofeat‐2(ad1116)correlateswellwithitsbroodsize(~60%ofwildtype)(HUGHESet al. 2007). Interestingly, the reduction in food intake we describe in long‐lived eatmutants is very close to the reduction in food intake necessary to extend lifespan inmammals(TAORMINAandMIRISOLA2014). Feedingdefects impair growth, causing the animals tobe small (MORCK andPILON2006).Weaskedifthereverseistrue,whethersmallanimalseatless.Comparingthefoodintakeofwild‐typeanimalstoseveralmutantswithreducedbodylength,weobservedthatsmalleranimalsdoeat less.Thiseffectwas independentof thegeneticpathwayreducingbody length (Figure 2B). Strains carrying mutations that disrupt TGFβ (sma‐2, sma‐3),embryonic constriction (sma‐1), global synaptic transmission (unc‐26, snt‐1), or reducepharyngeal pumping via disruption of neurotransmitter‐specific synaptic transmission(eat‐2,eat‐18,ser‐7,arr‐1)allatelessthanwild‐typeanimals(NONETetal.1993;MCKAYetal. 2004; BEAULIEU et al. 2005; STEGER et al. 2005; CH'NG et al. 2008). As expected, thereductioninfoodintakecorrelatedwithbodylength(MORCKandPILON2006)(Figure2C,D).Serotoninmodulatesfoodintakeviaser‐7

Thusfar,wehaveestablishedthatwecandeterminereductionsinfoodintake.Wenextsetouttomeasureincreasesinfoodintake.Theneurotransmitterserotonin(5‐HT)isa conserved regulator of energy balance (NOBLE etal. 2013).Treatmentwith exogenousserotonin increases the rate of pharyngeal pumping in a manner dependent on the G‐proteincoupledreceptors(GPCRs)ser‐1,ser‐5,orser‐7(SRINIVASANetal.2008;CUNNINGHAM

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etal.2012).Asexpected,treatingC.eleganswithserotoninincreasedbacterialclearanceinadose‐dependentmanner(Figure3A,B).

We next askedwhich of the known serotonin receptorsmediates increased foodintakebyserotonin.Serotonin‐inducedfeedingwascompletelyabolishedinser‐7(tm1325)mutants (Figure 3C). In contrast, ser‐1(ok345) mutants displayed a partial serotonin‐resistantphenotype.Inser‐1(ok345)mutants,boththerelativeandabsolutemagnitudeoffood intake in response to serotonin was reduced, indicating a reduction in theconsumption of total bacteria as compared to wild‐type N2 animals. The discrepancybetween pharyngeal pumping and bacterial clearance in the case of ser‐5(ok3087) wasunexpected,andwarrantsfurtherinvestigation(CUNNINGHAMetal.2012).

Having confirmed that exogenous serotonin increases food intake via SER‐7, weproceeded to investigate how endogenous serotoninmodulates basal food intake. In thecontext of thismanuscript,weuse the termbasal food intake todescribe the amountofbacteria the animals eat without stimulation by chemicals such as serotonin. The rate‐limiting enzyme for serotonin synthesis is tryptophan hydroxylase (tph‐1), andwell‐fedtph‐1mutants displaymultiplemetabolic phenotypes (SZE etal. 2000; CUNNINGHAM etal.2012). While mean food intake in N2 and tph‐1(mg280) mutant animals was similar(Figure 3D), food intake in tph‐1(mg280) mutants was more variable (Brown‐Forsythetest,P=1.5e‐06),suggestingdefectsintheregulationofbasalfoodintake.Thisisconsistentwithreportsofincreasedvariationinpharyngealpumpingratesoftph‐1(mg280)mutantsas compared towild‐type animals (HOBSON etal. 2006). Thus, themetabolic phenotypesobserved in tph‐1(mg280) mutants are unlikely to be the result of a reduction in foodintakeperse,butratherofdefectsinfoodsignaling.

Measuringnutrientincorporationbyquantitativemassspectrometry

Acompleteunderstandingoffeedingnotonlyrequiresustomeasuretheamountoffoodeaten,butalsotheamountincorporatedintotheanimal.Tomeasurefoodabsorption,wedevelopedthepulse‐feedingassay.Inthepulse‐feedingassay,a15NpulseisdeliveredtoC. elegans by feeding nitrogen‐isotope‐labeled (14N, 15N) bacteria (OP50). Nutrient (i.e.food)absorptionissubsequentlydeterminedbymeasuringisotopeincorporationintotheC.elegans proteome,which isproportional to thebacteria ingested (seeSupplementaryDiscussion).Toconductsuchanexperiment,C.elegansareraisedon14N‐labeledbacteria.Then,14N‐labeledbacteriaareexchangedwith50%14N/50%15N‐labeledOP50andfedtothewormthroughoutaperiodofinterest(pulse‐interval).Attheendofthepulse‐interval,proteins are extracted and the amountof incorporated 15N in theC.elegans proteome isdeterminedbymassspectrometry(Figure4A,B).

To quantitatively compare nutrient absorption between different samples, thepulse‐feedingmethod requires three different types of labeled bacteria (GEILLINGER etal.2012;GRUNetal. 2014). The first type is for growing theworms, the second for pulsingthem, and the third is to be added as an external standard. We used uniform 15Nenrichment of ammonium ions as the source of nitrogen. Toproduce the threedifferenttypes of feeding bacteria, OP50 were grown in M9 media containing three differentconcentrationsof15N‐enrichedammoniumsulfate(NH4SO4):only14NH4SO4,only15NH4SO4,ora50:50mixtureofthetwo.WerefertoOP50grownon14NH4SO4as”Light”or“L”OP50,toOP50grownona50:50mixtureas”Medium”or“M”OP50,andtoOP50grownon100%

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15NH4SO4 as “Heavy” or “H”OP50 (SYKESetal. 2010;GOUWetal. 2011; CHENetal. 2012;GEILLINGERetal.2012;CHENandWILLIAMSON2013).

Incomplete removal of the 14N‐labeled bacteria prior to the start of pulse‐feedingcanresult in the incomplete labelingofnewwormproteinswith50%15N labeledaminoacids.Wewereabletocorrectforthiseffectbyallowingthepercentageof15Ntovaryasanadjustableparameterwhenperformingtheleastsquaresfittingtotheisotopedistribution(SPERLING et al. 2008). Typically, we found that feeding the “Medium” OP50 (50% 15N)resultedinarelativeisotopeabundanceof~45%15N.Theincompletelabelingofproteinshad no effect on the feedingmeasurements,which only depend on the amplitude of theMediumisotopecomponent.

To understand how we identify individual peptides by mass spectrometry, it isnecessarytoaddressthecomplexityofthepeptidesamples.Foreachpeptide,threeisotopeenrichmentpatternsarepossible:100%14N‐enriched(L),ifitwassynthesizedbeforethepulse;50%15N‐enriched(M),ifitwassynthesizedduringthepulse;or100%15N‐enriched(H), if it was part of the external standard (Figure4A,B,C). Thus, each peptide in thesamplecanhavethreecomponentsinitsisotopedistributioninthemassspectra.Databasesearch engines cannot identify peptides from 50% 15N‐enriched parent ions and, as aconsequence,onlytheidentificationofthe100%14N‐and100%15N‐labeledpeptideswascarriedoutusingMASCOTwithsearches forbothE.coli andC.elegans(ENGetal.1994).Becausethelight,medium,andheavypeptidesco‐eluteduringliquidchromatography,theMS1scanfor them/zrangespanningall three labeledspeciescanbeextracted(L,M,H)(Figure5A).Misidentificationsarereadilyidentifiedbecausethespacingofthe14Nand15Npeaksdoesnotmatchthenumberofnitrogenatomsinthesequence.Thus,wecanensurecorrect identification of each peptide, despite the complexity of the isotope distributionwiththreecomponents.

In a typical experiment, 300‐400 proteins were identified based on one or morepeptides.Thisnumberislessthanwhatisachievedwithdeepproteomics,butismorethansufficient to determine the bulk metabolic labeling rate of the proteome. To determineisotope incorporation, and thus nutrient absorption, we calculated two values for eachpeptide: i) the fraction labeled, or fraction of partial 15N‐labeling, (M)/(L+M) and ii) theproteinlevelofthesample,(L+M)/(L+M+H).Thefractionlabeledindicatestheamountof15N incorporatedduringpulse‐feeding for eachprotein and, over the entireproteome, isindicative of the amount of bacteria eaten. Importantly, 15N incorporation representsdenovo protein synthesis occurring within the pulse‐feeding interval, and thus allowsidentificationofboththenewlysynthesizedandpre‐existingproteome.Theprotein levelindicatestherelativeproteincontentindifferentexperimentalsamples.

Wefirstaskedwhetherthepulse‐feedingassaywasabletoidentifyadifferenceinnutrient absorption between N2 and tph‐1(mg280) mutants. Since the bacterial clearingassayshowedthegreatestchangeinfoodintaketooccurbetweenL4andD1,wechosethisintervalforpulse‐labeling.N2ortph‐1(mg280)animalswereraisedonLightOP50,(100%14N)until theyreachedtheL4stage.AtL4,webeganthepulse‐labelingbyswitching thefeedingbacteriafromLighttoMediumOP50(50%14N;50%15N).Sixteenhours later,onday1of adulthood (D1), the animalswereharvested to extractprotein.To compare 15Nincorporation between different samples, we mixed each experimental sample with anexternalstandard.Theexternalstandardconsistedofproteinsextracted fromwormsfed100% 15N‐labeled OP50 bacteria over three generations and raised in parallel with the

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experimental samples. The samples were then processed for LC‐MS/MS massspectrometry.

PlottingthefractionlabeledforbothstrainsconfirmedthattherewasnodifferenceinfeedingornutrientabsorptionbetweenN2andtph‐1(mg280),asshownbythebacterialclearing assay. The results further show that,within 16 hours of pulse‐labeling, roughly80%ofthedetectedproteinsbecomelabeled(Figure5B).Wefoundthattheideallengthof the labelingperioddependson the food intakeof theanimals,with lower food intakerequiringlongerlabelingperiodsandhigherfoodintakerequiringshorterperiods.Wealsoobservedthategglayingrepresentsasubstantial“15Nleak”,aseggsarealmostcompletelydenovo synthesized from 15N. As it is difficult to ensure that all eggs are harvested,wedecided to start pulse‐feeding on day 5 in subsequent experiments, after the egg‐layingperiod was over. This approach prevents 15N loss through egg laying, and allowsmeasurementover longerperiodsof time, as both food intakeandprotein synthesis areloweratolderages.Serotoninincreasesproteinsynthesis

We next asked whether serotonin treatment also increased the absorption ofingested bacteria. On day 1 of adulthood,we added serotonin orwater towild‐type N2animals.Onday5,weexchangedLightOP50forMediumOP50(50%14N;50%15N)tostartpulse‐feeding. Inwild‐type animals treatedwith serotonin, the fraction labeled for eachpeptide increasedbyanaverageof (1.9±0.5)‐foldascomparedtowater‐treatedcontrolanimals(Figure5C).Hierarchicalclusteringoflabeledpeptidesfromwild‐typeN2water‐treatedvs.serotonin‐treatedsamples identifiedtwogroups:one inwhichthe increase infraction labeled was higher (2 ± 0.4 fold), consisting mainly of proteins involved intranslationandribosomalbiogenesis (SupplementalTableS1); andone inwhichthe increase in fraction labeled was smaller (1.5 ±0.3 fold), consisting of a variety ofdifferentproteins involved inmetabolism.Of the total153proteinswhosesynthesiswasincreasedinresponsetoserotonin‐inducedfoodintake,23arelikelytopromoteaging,asthey extend lifespanwhen suppressed by RNAi. Thus, the increased synthesis of theseproteinsprovidesapossiblelinktohowfoodintakepromotesaging.

Similartothebacterialclearingassay,theeffectofaddedserotoninonthefractionlabeledvaluewasabrogatedintheser‐7mutants(Figure5C,D,E).Proteinlevelsdidnotchangesignificantlybetweenwater‐treatedwild‐typeandser‐7mutantworms, indicatingthattheser‐7(tm1325)alleledidnothaveageneraleffectonthetotalamountofproteinintheworm(Figure5F).Thus,theser‐7receptorisspecificallyrequiredfortheincreaseinfoodintake,nutrientabsorption,andsubsequentdenovoproteinsynthesisandribosomalbiogenesisobservedinserotonin‐treatedwild‐typeN2worms.

DISCUSSIONThe present study outlines two independentmethods, based on entirely different

principles,whichwe used tomeasure food intake and nutrient absorption inC. elegans.These methods address two outstanding problems in the study of nutrition andmetabolism in C. elegans: the absence of a direct measurement for food intake overextendedperiodsoftime,andtheneedforinformationrelatingtohowwellnutrientsareabsorbedandassimilateduponingestion.

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Thebacterialclearingassayprovidesasimpleanddirectquantitativemeasurementof food intakeover timescales relevant to the studyof organismalhealth and lifespan. Itmeasuresfoodintakebythesameprinciplesasisusedinothermodelorganismssuchasflies and mice (MARTIN‐MONTALVO et al. 2013). The liquid culture format of the assayprovides a scalable framework for the testing of chemical, genetic, and environmentalperturbations inC.elegans. Importantly, thebacterialclearanceassay issuitable forbothsmall‐andlarge‐scalegenome‐widescreening,andrequiresequipmentreadilyavailableatmost institutions. The assay yields robust results over a range of temperatures andbacterialconcentrations,andinthepresenceofcommonchemicalsolvents(DMSO,aceticacid,etc.)(datanotshown).Undertheconditionsusedinthisstudy,thebacterialclearingassayconsistentlydetecteddifferencesinfoodintakeaslowas15%.

Studies of food intake inC. eleganshave revealed deep insights into food relatedbehaviors,suchasfoodchoiceandbehavioralresponsestofood(AVERYandYOU2012).Tofullyunderstandthemetabolicchainofeventsoffeeding,wemustalsoknowthenutrientlifecyclefromforagingbehaviortoassimilationoffoodintotheanimal.Toaddressthis,wedeveloped the pulse‐feeding assay that allows for the first time the determination ofnutrientabsorption intowormproteinsbymassspectrometry.Ourmethodestablishesadirectlinkbetweenafoodintakebehavior(pharyngealpumping)andproteinsynthesisbymeasuringingestion,absorption,andassimilationofnutrientsintotheproteome.Althoughthepulse‐feedingassayisnotthefirsttoaddressdenovoproteinsynthesisinC.elegansbymass spectrometry (LIANG et al. 2014), our metabolic pulse‐labeling approach offerssignificant advantages. First, metabolic labeling is non‐toxic, and therefore does notinfluenceanimalbehaviororphysiology.Second,metaboliclabelingwithheavynitrogenisnot subject to unwanted side reactions or chemical derivatization, nor does it requireadditionalgeneticmutationstosuppresstheseeffects(GOUWetal.2011).Third,thepulse‐feedingassaypresentedhereallowsforthequantizationofdenovoproteinsynthesisinaway that is not feasible by trace‐labeling with unnatural amino acids followed bymassspectrometry.Withtrace‐labeling,onlylabeledproteinscanbecomparedfromsampletosample. In contrast, thepulse‐feedingassayuniquely labelsproteinspresentbothbeforeand after the pulse, allowing comparison of protein levels and label incorporation ofproteinspriortoandafterpulse‐labeling.

Thegreateststrengthofthepulse‐feedingmethodisthat,whilemoreinvolvedthanbacterial clearing, it directly establishes nutrient utilization and is independent of theculture medium. Determining nutrient utilization directly is important, especially inanimals thatsuffer fromgutdefects,andcanprovidemeaningful informationevenwhenfoodintakeappearsnormal.Weproposethatthepulse‐labelingassayprovidesasuitablegoldstandardtomeasuretheamountoffoodutilizedbyananimal.Importantly,thepulse‐feedingassaycanbeadaptedforotherspecies, includingDrosophilamelanogaster,oranyotherorganismwhosefoodcanbegrowninstable‐isotope‐labeledmedia(PEREZandVANGILST2008;DESHPANDEetal.2014).

Asrecentlyshown,overabundanceofproteinintakeisassociatedwithpoorhealthand accelerated aging (SOLON‐BIET et al. 2014). How protein intake affects subsequentproteinsynthesis,theproteome,andthushealth,isanopenquestion.Asthepulse‐feedingassay is based onmeasuringdenovo protein synthesis, it provides an important tool tostudyhow foodcompositiondictatesprotein synthesis in theanimal, and thushow foodcompositionaffectstheproteome,physiology,andhealth.

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It revealed that serotonergic signals induce translation of ribosomes and other age‐promoting proteins, providing amechanistic link to how high food intakemay promoteaging. Thus, the two assays presented enable investigations into the complex biologicalinteractionbetween food and protein synthesis, and its subsequent effects on aging andhealth

ACKNOWLEDGEMENTS

Thisworkwasfundedbygrantsto:M.P.,fromtheNIH(DP2OD008398),agrantfromtheEllison Medical foundation (AG‐NS‐0928‐12), a grant to J.W. from the NIH (R37‐GM‐053757),anMDADevelopmentGrant forS.R., anAmericanheart fellowship toE.V (AHA10POST3500084)andaNSFGRFPFellowshipforG.M.S.StrainswereprovidedbyShigen‐JapanortheCGC,whichisfundedbyNIHOfficeofResearchInfrastructurePrograms(P40OD010440).

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Figure1.MeasurementofbacterialclearancewithC.elegans.(A)96‐wellliquidcultureformat.(B)Morphologyofday1(D1)adultN2wormsgrownonsolidNGMplates(top)orinliquidculture(bottom).(C)Thebacterialclearanceassay.Schematicofwormsplacedintowellswithanopticalbottomtomonitorbacterialconcentrationsbymeasuringtheopticaldensity(absorbance)at600nm(OD600).Sideandtopview.(D)Bacterialclearanceisonlyobservedinthepresenceofworms.Tukey‐styleboxplots.OD600depictingfourtimepoints,comparingwild‐typeN2vs.noworms,nwells=12biologicalreplicates(e.g.wells).Datarepresentfiveindependentexperiments.***P<0.001,Two‐wayANOVAwithBonferronipost‐hoctest.(E)Bacterialclearanceisobservedforbacteriakilledbyirradiation.Datarepresentthreeindependentexperiments.WellswithN2(nwells=6),wellswithnoworms(nwells=3).F‐H:OD600measurementsarenotinfluencedbythepresenceofeggs,anddependonlyonthepresenceofwormseatingbacteria.OD600onD1andD4forwellscontaining;(F)S‐completeonly(G)N2andeggsinS‐completewithOP50removed,or(H)N2plusbacteriainS‐complete.Datarepresentthreeindependentexperiments.(I)Bacterialclearancecorrelateswiththenumberofwormsperwell(X0).Valuesdepictbacterialclearanceover72hrs(D1:D4).Datadepict95%confidenceinterval(dashedlines),goodnessoffitstatistic(R2),andSpearman’scorrelation(P<0.0001).Datarepresentthreeindependentexperiments(nwells=84).(J)Datafrom(I)normalizedtowormsperwell.(K)Age‐relatedchangesinfoodintake.FoodintakeexpressedrelativetobacterialclearancewithintheL4:D1interval.Datarepresentthreeindependentexperiments(nwells=36).

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Figure2.Bodysizeinfluencesfoodintake.(A)Foodintakeoflong‐livedeatmutants.FoodintakeexpressedrelativetowildtypeN2(D1:D4).Datarepresentthreeindependentexperiments,nwells88.***P<0.01,One‐wayANOVAwithDunnett’smultiplecomparisonpost‐test.(B)Smallanimalseatless.Datarepresentthreeindependentexperiments,nwells43.***P<0.01,One‐wayANOVAwithDunnett’smultiplecomparisonpost‐test.(C)BodylengthofanimalsinB.BodylengthasmeasuredonDay4ofadulthood.Datarepresentthreeindependentexperiments,nwells28.***P<0.001,One‐wayANOVAwithDunnett’smultiplecomparisonpost‐test.(D)Interactionofanimalsizeandfoodintake.Foodintakeandbodylengthmeasurementsexpressedrelativetowild‐typeN2onDay4ofadulthood.Datarepresentthreeindependentexperiments.Linearregressionline(dashedredline)andthe95%confidenceinterval(dashedgreylines)areshown.Goodnessoffitstatistic,R2=0.753(P<0.002).

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Figure3.Modulationoffoodintakebyserotonergicsignaling.(A)Dose‐responsecurveforwild‐typeN2animalstreatedwithserotonin.Foodintakeexpressedrelativetocontroltreatment(water).Tukey‐styleboxplots,unlessotherwisestated,depictfoodintakeovertheD1:D4interval.Dataarerepresentativeofthreeindependentexperiments,nwells=20.***P<0.01,One‐wayANOVAwithDunnett’smultiplecomparisonpost‐test.(B)Foodintakeofpre‐andpost‐reproductivewild‐typeN2animalstreatedwithwaterorserotonin(2mM).FoodintakeexpressedrelativetotheD1:D4intervalofcontrolwater‐treatedN2animals.Dataarerepresentativeofthreeindependentexperiments,n=18.***P<0.0001,Student’st‐test.(C)Foodintakeinresponsetoserotonin(2mM)forwild‐typeN2animalsandserotoninreceptormutants.Dataforeachstrainarerepresentativeofaminimumofthreeindependentexperiments.Dataasdepictedingraphrepresenttwoindependentexperiments,nwells42.***P<0.001,Two‐wayANOVAwithBonferronipost‐testcomparingresponsetoserotoninforeachgenotype.###P<0.001,one‐wayANOVAwithDunnett’smultiplecorrectionpost‐testcomparingserotonin‐treatedanimalsofeachgenotypetowild‐typeserotonin‐treatedanimals.(D)Basalfoodintakeofserotonin‐synthesis‐deficienttph‐1mutants.Foodintakeexpressedrelativetowild‐typeN2.Datarepresentthreeindependentexperiments,nwells95.Student’st‐testusedtoestablishsignificance.Note:Foraversionofgraph3A,CshowingS.E.MandthusreproducibilitybetweenexperimentsseeSupplementaryFigure2.

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Figure4.MeasurementofnutrientabsorptioninC.elegansusingmetaboliclabelingcoupledwithquantitativemassspectrometry.Tometabolicallylabelworms,(A)wefirstgrewthreedifferentOP50“foods”byculturingOP50bacteriainminimalmediaenrichedwitheither100%(14NH4)2SO4,50%(14NH4)2SO4+50%(15NH4)2SO4,or100%(15NH4)2SO4.Thesefoodsweretermed“light”,“medium”and“heavy”respectively.Second,wegeneratedapopulationof“heavy”worms.Thesewormswerefed“heavy”OP50forthreegenerationstoensurefullyenriched100%(15NH4)2SO4wormproteins.(B)Third,lightandheavywormsweresynchronizedandculturedin“light”and“heavy”foodrespectively.Theheavywormswereusedasanexternalstandardtofacilitatethecomparisonofdifferentexperimentalsamples.14Nwormsweregiveneitherthedrugofchoiceorwateratday1.Atday5,orthestartdayforthepulselabeling,wormswerewashedandthefoodswitchedfrom“light”to“medium”fordrug‐orwater‐treatedworms,andfrom“heavy”to“heavy”forthemass‐spec‐standardworms.Thewormswereharvestedonday7afterpulselabelingandpreparedformassspectrometryanalysis.(C)Preparationofsamplesformassspectrometryanalysis.Foreachexperimentanexternalstandardwasgenerated,derivedfromheavywormsculturedinparallel.Eachsamplelysatewasspikedwiththeexternalstandard.ProteinswereextractedbyTCAprecipitation,digestedwithtrypsin,andanalyzedbyLC‐MS/MS.

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Figure5.Pulse‐feedingassay.(A)Wholewormlysateanalyzedonthemassspectrometer,withsampledatashownin(Figure4b,c).Eachpeptideinthespectrahasa“light”,“medium”and“heavy”component.Theintensityofthe“light”componentdependsontheamountofeachpeptidepresentbeforethepulse‐labelingperiod.Theintensityofthe“medium”componentdependsontheamountofingestedfoodafterthestartofthepulse‐labelingperiod.The“heavy”componentservesasastandardsample.(B)Correlationplotoffractionlabeledvaluesforwild‐typeN2(x‐axis)vstph‐1mutantworms(y‐axis)fromL4:D1.(C)Correlationplotoffractionlabeledvaluesbyproteinforwild‐typeN2treatedwithwater(x‐axis)orserotonin(y‐axis,5mM)fromD5:D7.(D)Correlationplotoffractionlabeledvaluesforser‐7mutantwormsgrownwithwater(x‐axis)ortreatedwithserotonin(y‐axis,5mM)fromD5:D7.(E)Correlationplotoffractionlabeledvaluesforwild‐typeN2(y‐axis)andser‐7mutants(x‐axis)treatedwithwaterfromD5:D7.(F)Histogramofproteinlevelvaluesforwild‐typeN2andser‐7mutantstreatedwithwaterorserotonin(5mM).