Vitamin B12 induces memory of predation through vitellogenin provisioning

Domain: chemistry
Published: 2026-04-09
Editorial outcome: major_revision

Abstract
Nutritionaleffectsontraitinheritancearewidespreadinanimalsincludinghumanfamines,representingsomeofthemostsevereenvironmentalinfluencesonorganismalphenotypesandcanbetransmittedovergenerations.However,thechemicalnatureofthestimuliinducingsuchmemoryremainselusive.ThenematodePristionchuspacificusexhibitsmouth-formplasticityincludingpredationandrespondstoamultigenerationalNovosphingobiumdietwiththeinductionandtransgenerationalinheritanceofthepredatorymorph.Weshowthatbacteria-derivedvitaminB12isnecessaryandsufficientfortransgenerationalmemory.NovosphingobiummutantsdefectiveinvitaminB12productiondonotinducetransgenerationalinheritance,butvitaminB12supplementationcanrescuethememoryphenotype.DifferentvitaminB12concentrationsarerequiredfortheoriginalinductionandsubsequenttransgenerationalinheritanceofthepredatorymorph.Thisinheritedeffectactsthroughincreasedmultigenerationalvitellogenintranscriptionsuggestingelevatednutrientprovisioning.Consistently,mutantsinthevitellogeninreceptorPpa-rme-2arememory-defectiveindicatingthatavitaminacts通过maternalprovisioningtoprogeny.Thus,vitaminB12inducesvitellogeninprovisioningtocontrolorganismalphysiologyandbehavior.

Introduction
Dietaryandnutritionaleffectscanalterorganismalphenotypesandinfluencetraitinheritanceovergenerations,so-calledtransgenerationalepigeneticinheritance(TEI)1,2.Alargebodyofevidenceindicatesthatdietaryfactorscaninducevariousmorphological,physiological,andbehavioralchangeswhichareoftentransmittedforthreegenerationsormore.However,thechemicalnatureoftheinducingagentsanddownstreameventsfollowingtheoriginaldietarystimuliarelargelyunknown.OnemajorobstacleinidentifyinginducingstimulifromthedietandthegeneticmachineryunderlyingTEIinthereceivingorganismisthelackofmodelsystemswithrobustphenotypestobeanalyzedunderlaboratoryconditions.However,nematodessuchasCaenorhabditis elegans and Pristionchus pacificus with their short generation time, isogenic propagation, and simple husbandry, have startedtoprovidemechanisticinsight3–7.

Forinstance,P.pacificusexhibitsamouth-formdimorphismthatisanexampleofdevelopmentalplasticitywithmorphologicalandbehavioralimplicationsandtherefore,providesarobustsystemtoanalyzedietaryeffectsandTEI8–10. During postembryonic development,geneticallyidenticalP.pacificuswormsadopteitherthenarrow-mouthed“stenostomatous”(St)morphwithasingledorsaltooth,orthewide-mouthed“eurystomatous”(Eu)formwithaclaw-likedorsalandanopposingsub-ventraltooth(Fig.1a).Importantly,onlytheEu morph allows omnivorous feeding, including predation on other nematodes (Fig. 1a). Mouth-form plasticity is characterized as a bistabledevelopmentalswitchallowingtheunderlyinggeneregulatorynetwork(GRN)tobeelucidatedindetail10–13. Additionally, multiple environmental cues influence mouth-form development, including changesindiet,establishing aneasystudysystem forexperimentalmanipulation14.

Arecentstudyintroducedalong-termenvironmentalinductionexperimentwithdistinctdietsbypropagating110P.pacificusisogeniclines for 101 generations with associated food-reversal experiments (Fig. 1b)3. In addition to the standard nematode laboratory food EscherichiacoliOP50,thisstudyusedthePristionchusenvironment-derivedbacteriumNovosphingobiumL76thatwasoriginallyshowntoincreasekillingefficiencyofP.pacificus15.Thelong-termenvironmentalinductionexperimentstudyrevealeddiet-derivedinductionoftheEu mouth form during Novosphingobium L76 exposure and subsequent TEI of this predatory phenotype when wormswererevertedtoanE.colidiet(Fig.1b).Interestingly,TEIoftheEumorphrequiresaminimalexposure offive generations on the inducing NovosphingobiumL76 diet,whichisphenomenologicallydifferentfrommostotherexamples of TEI that require only a short-term stimulus. Subsequent genetic studiesidentifiedapreviouslyunknownmechanismintheregulation of TEI3. Forward genetic screens for mutants defective in transgenerationalinheritanceindicatedanessentialroleoftheubiquitinligase EBAX-1/ZSWIM8andthedestabilizationofclusteredmicroRNAsofthe miR-2235/miR-35 family for transgenerational memory. Specifically, Ppa-ebax-1mutantsshownomemory,whereasdeletionsofthemiR-35 clusterresultinprecociousandextendedTEIofthepredatorymouth form (Fig. 1b). While these studies start to provide molecular mechanismsunderlyingTEI,nothingisknownaboutthefactorsfrom NovosphingobiumthatinducethepredatorymouthformanditssubsequentTEI.

In this work, we studied bacterial metabolites and found that vitaminB12isbothnecessaryandsufficienttoinducethepredatorymouthformanditssubsequentmemoryinaconcentration-dependentmanner.Thiseffectisinheritedthroughincreasedvitellogenintranscription and consistent with this finding, mutations in the single vitellogenin germline uptake receptor Ppa-rme-2 are memory-defective. Thus, vitamin B12 induces vitellogenin provisioning in a multigenerational manner to control organismal physiology and behavior.

Results
VitaminB12inducesthepredatorymorphandtransgenerationalmemory
Long-termenvironmentalinductionisperformedbyswitchingnaïveP.pacificus RSC011 cultures that were always grown on the standard laboratoryfoodsourceE.coliOP50toNovosphingobiumL76(Fig.1b). ThisP.pacificusstrainfromLaRéunionIslandispreferentiallynon-predatory (30%Eu:70%St) when grown on E. coli. The original long-term environmental induction experiment with 110 isogenic lines derived fromthe same ancestralRSC011 individualrevealed dietary inductionofthepredatorymouthform(Fig.1b).Thisdietaryeffectis (i) immediate (in generation 1 on Novosphingobium), (ii) complete (100%Euprogeny),(iii)systemic(inall110lines),and(iv)permanent (throughouttheentireexperimentof101generations).Followingthe reversalfromNovosphingobiumbacktothestandardE.coliOP50diet, weobservedTEIofthepredatorymouthform(Fig.1b).Importantly, suchTEIisonlyseenafteraminimalexposuretoNovosphingobiumfor fivegenerations,whereasashorterexposurewillnotresultinmemory ofthepredatorymorph.

Wewantedtodeterminethemolecularnatureofthedietarystimulusinducingthepredatorymouthformanditstransgenerational inheritance in P. pacificus RSC011. The Novosphingobium L76 strain used in this experimental setup was originally isolated from a Pristionchus-associated environment and was shown to enhance killing efficiencyoftheP.pacificuswildtypestrainPS312,whichisnaturally 100%Eu15.ThisstudyalsoidentifiedbacterialvitaminB12production tocauseenhancedpredation,toacceleratewormdevelopmentand increase brood size15. Therefore, we wanted to know if vitamin B12 would alsoplay a role in dietary induction of the predatory mouth formandTEIinP.pacificusRSC011animals.Forthat,wetransferred singlenaïveJ4animalstovitaminB12-supplementedagarplateswithE. coliOP50asfoodsource(Fig.2a).ItisimportanttonotethatE.coli OP50doesnotproducevitaminB12butratherobtainsitsvitaminB12 from the tryptone in the agar15–17. We originally used final concentrations of 500,1000,and1500nMofoneofthetwoactive formsofvitaminB12,methyl-Cobalamin(Me-Cbl),forsupplementation,similartopreviousstudiesinP.pacificus15.Notethattheseconcentrationsaresubstantiallyhigherthanthoseusedinpreviouswork inC.elegans17. Strikingly,weobservedanimmediateinductionofthe Eu mouth form after vitamin B12 supplementation at all three concentrationsused(Fig.2b–dandSupplementaryFig.1b–d).However, vitaminB12supplementationresultsinastrongbutnotacomplete induction of the Eu mouth form. Specifically, in three independent biologicalreplicateswithatotalof60assayplates,wefoundhighbut incomplete induction in the first generation after vitamin B12supplementation(0.878≤HDI(θ)≤0.994,whereθistheinferred probabilityofdevelopingthepredatorymouthform).Thiseffectwas seenoveraperiodof5and10generationsandwasobservedinalllines (Fig. 2b–d and Supplementary Fig. 1b–d). Thus,vitamin B12 mimics Novosphingobium-basedinductionofthepredatorymouthforminP. pacificus.

To determine if vitamin B12 would also induce TEI of the predatorymouthform,weestablishedamini-assaywitha5-generationor 10-generationexposureofwormstoa vitamin B12-supplementedE. coli diet, followed by 5 generations on an un-supplemented diet (Fig.2a–dandSupplementaryFig.1a–d).Indeed,weobservedTEIof thepredatorymouthform(Fig.2b–dandSupplementaryFig.1b–d). Animalssupplementedwith1000and1500nMvitaminB12showed TEI of the Eu mouth form similar to animals that were exposed to Novosphingobiumfor5or10generations(Fig.2b,candSupplementaryFig.1b,c).Incontrast,asupplementationwith500nMofvitamin B12onlycausedtheinductionofthepredatorymouthformbutnofull TEIoftheEumorph(Fig.2d).Specifically,theresponseduringreversal onlylastedfortwogenerations,withallfollowinggenerationsbeing down to baseline Eu frequencies (0.841≤HD1(θ )≤0.987, 0.52≤HD1(θ )≤0.842, and 0.305≤HD1(θ )≤0.637) (Fig. 2d and Supplementary Fig. 1d). Such a response is referred to as “intergenerational”, but not transgenerational inheritance4. These results allow two major conclusions. First, vitamin B12 supplementation mimics diet-derived induction ofthe predatorymouth form and its subsequenttransgenerationalinheritance.Second,theeffectofvitaminB12isconcentration-dependentwithonly1500and1000nMbut not500nMofvitaminB12causingTEI.

VitaminB12inducesadistincttranscriptomicresponse
Next,wewantedtoknowwhethervitaminB12supplementationcan elicitsimilareffectsongeneexpressionasthosecausedbytheNovosphingobiumdiet.Inprinciple,theNovosphingobiumdietandvitamin B12supplementationmightinducesimilartranscriptomicresponses or alternatively, have limited overlap in individual genes only. We examined the overlap of differential gene expression (DGE) found between vitamin B12 supplementation on E. coli OP50 with Novosphingobium-inducedgeneexpressionchangesinRSC011animalsby usingsinglewormtranscriptomicsofday1adultindividuals(Fig.2g). After three generations of exposure, Novosphingobium-upregulated genes were in parts also upregulated upon vitamin B12supplementation(Fig.2gandSupplementaryData1).Specifically, of the 530 upregulated genes after a three-generation vitamin B12 supplementation, 103 (12.2% of all uniquely upregulated genes) were also upregulated on Novosphingobium. Notably, these shared genescorrespondto19.4%ofthevitaminB12-inducedand24.6%ofthe Novosphingobium-induced transcriptional responses. Analysis of PFAM enrichment among these shared genes revealed that Lipase GDSL(PF00657.26)andLectin_C(PF00059.25)wereenrichedinboth conditions,suggestingthatlipidmetabolismandcarbohydrate-binding/cell-surfacesignalingformacoretranscriptionalresponse.Beyond this overlap, vitamin B12-specific genes were uniquely enriched for CUB(PF00431.24),associatedwithextracellularproteininteractions, whereas Novosphingobium-specific genes were enriched for Yae1_N (PF09811.13) and EryCIII-like_C (PF06722.16), reflecting ribosome-associatedfunctionsandsecondarymetabolitebiosynthesis,respectively.Theseresultshighlightbothconservedandcondition-specific molecular programs activated in response to vitamin B12 supplementation and Novosphingobium exposure. (Fig. 2g and SupplementaryData1).Thus,vitaminB12supplementationtotheE. coliOP50dietexhibitslimitedoverlapwithNovosphingobium-induced geneexpressionchanges.

VitaminB12isnecessaryandsufficienttoinducememory
PreviousstudiesthatestablishedaroleofdietaryvitaminB12onlife historytraitsinP.pacificusandC.eleganswerebasedonscreeningTn5-based bacterial mutant libraries15,17. We used a Novosphingobium vitaminB12-deficientmutanttostudytheeffectofNovosphingobium-derivedvitaminB12onmouth-formplasticityandTEIofthepredatorymorph.Usingthe5-generationmini-assaydescribedabove,wefound thataNovosphingobiumvitaminB12-deficientdietwouldstillinduce the Eu mouth form (Fig. 2e). However, in contrast to a wild-type Novosphingobium diet, the induction is not complete and plateaus ~0.95% Eu (0.818≤HDI (θ )≤0.98 and 0.883≤HDI(θ )≤0.999). Theseresultsindicatethatadditionalfactor(s)besidesvitaminB12are abletoinducethepredatorymouthform.Thus,thecomplete(100%) inductionoftheEumorphofP.pacificusRSC011onNovosphingobium isduetomultiplefactors.

In contrary, we found that the Novosphingobium vitamin B12-deficientdietwouldnotcauseTEIofthepredatorymouthform.When werevertedwormlinesthatwereexposedtotheNovosphingobium vitaminB12-deficientdietfor5generationsbacktoE.coli,weobserved an immediate return to the base line response level (0.135≤HDI(θ )≤0.47)(Fig.2e).Similarly,whenwerevertedRSC011 linesthathadbeenexposedtothisdietfor10generations,wealsodid notobserveTEIofthepredatorymouthform(SupplementaryFig.1e). Incontrast,vitaminB12supplementationfor5or10generationstothe NovosphingobiumvitaminB12-deficientdietrestoredthenormalTEI response after reversal to E. coli OP50 (0.772≤HDI(θ )≤0.971, 0.65≤HDI(θ )≤0.919,and0.47≤HDI(θ )≤0.832)(Fig.2fandSupplementary Fig. 1f). These results indicate that vitamin B12 is both necessary and sufficient to induce the TEI of the predatory mouthform.

VitaminB12-inducedTEIofthepredatorymorphrequiresmetr-1
Inanimals,includinghumans,vitaminB12actsasacofactoroftwo enzymes,methionine-synthasein the cytoplasm and methylmalonyl coenzymeA(CoA)mutaseinmitochondria(Fig.3a)18.InP.pacificusas inC.elegans,themethionine-synthaseisencodedbyPpa-metr-1.To manipulatethemethylmalonylcoenzymeA(CoA)mutasepathway,we targeted the upstream factor Ppa-mce-1, that is one-to-one orthologoustoCel-mce-1.WetargetedPpa-metr-1andPpa-mce-1inP.pacificus RSC011byCRISPRtostudyapotentialroleindietaryinductionandTEI ofthepredatorymouthform(Fig.3b).Weobtainedtwoframeshift allelesofPpa-metr-1andPpa-mce-1each,andtestedalloftheminour standardassay(Fig.3b).First,westudiedtheresponseofPpa-metr-1 mutantstoanE.colidietsupplementedwith500,1000and1500nM vitaminB12.Ppa-metr-1mutantanimalsfailedtorespondtovitamin B12indicatingthattheroleofvitaminB12ininducingtheEumouth form requires Ppa-metr-1 (Fig. 3c). Second, we grew Ppa-metr-1 mutantsonNovosphingobiumandobservedameaninductionofθ = 0.96throughouttheexposureof5–10generations(Fig.3dandSupplementary Fig. 2a, b). This result is likely due to the other Novosphingobiumfactor(s)thatcaninducetheEumouthformasindicated intheexperimentsusingtheNovosphingobiumvitaminB12-deficient diet (Fig. 2e and Supplementary Fig. 2e). Importantly, when we revertedPpa-metr-1mutantsfromNovosphingobiumbacktoE.coli,we observed no TEI of the predatory mouth form (Fig. 3d and SupplementaryFig.2a,b).ThesefindingsindicatethatvitaminB12actsasa cofactorofmethionine-synthaseinthetransgenerationalinheritance oftheEu mouth form. Third, we tested Ppa-mce-1 mutants and observed an induction of the Eu mouth form on Novosphingobium similar to wild-type animals (Fig. 3e and Supplementary Fig. 2d, e). After reversal, Ppa-mce-1 mutant animals responded similar to wild typecontrols,indicatingthatPpa-MCE-1isnotinvolvedintheTEIof thepredatorymouthform(Fig.3eandSupplementaryFig.2d,e).Note however, that the response of Ppa-mce-1 mutants to vitamin B12-supplemented E. coli diet only resulted in a partial Eu induction (0.256≤HDI (θ)≤0.616 for the highest vitamin B12 concentration) (Supplementary Fig. 2c). Together, our mutant analysis identifies a requirementofPpa-metr-1forthevitaminB12-mediatedinductionof thepredatorymouthformanditssubsequentTEI.

TheEumorphisalreadyinducedby0.1nMvitaminB12supplementation
GiventhattheinductionoftheEumorphandthetransgenerational memoryofthepredatorymouthformrequiredifferentconcentrationsofvitaminB12,wenextwantedtoknowtheminimalamountof vitamin B12 necessary to induce the Eu morph. While our original experiments used a final concentration between 500 and 1500nM Me-Cbl(Fig.2),wenowreducedtheconcentrationofvitaminB12for severalordersofmagnitude(Fig.4).Supplementationexperiments using250nMand125nMvitaminB12,resultedinanimmediateEu mouth-form induction,butreversalexperiments after5 or10generationsresultedinanevenshortermemorythansupplementation with500nMvitaminB12.Specifically,theresponseseenafter250, 125,and50nMvitaminB12lastedforonlyonegeneration(Fig.4a–c andSupplementaryFig.3a,b).Whenwereducedtheconcentration of vitamin B12 even further and supplemented with 10, 5, 2, and 0.1nMvitaminB12,allfouroftheseconcentrationsstillresultedina stronginductionoftheEumouth-form(SupplementaryFig.3c–f).In contrast,wormshadlosttheirmemoryalreadyintheF5R2generation (for 50nM, 0.276≤HDI(θ )≤0.655, for 10nM, 0.238≤HDI(θ )≤0.612, and for 5nM, 0.27≤HDI(θ )≤0.645) (SupplementaryFig.3c–f).Thus,thereductionofvitaminB12concentrationinsupplementationexperimentsresultsinthelossofTEI ofthepredatorymouthform,whiletheinductionoftheEumorph remainshigh.WhenwereducedthevitaminB12concentrationeven further, we found no induction of the predatory mouth form at a concentration of 0.01nM vitamin B12 (Supplementary Fig. 3g). Instead,wormstreatedwiththisamountofvitaminB12stayedatthe baselinelevelofEumouth-form,similartoun-supplementedgrowth conditions.Therefore,TEIofthepredatorymouthformrequiresa substantially higher vitamin B12 concentration than the initial inductionoftheEumouthform.

Memoryofthepredatorymorphrequiresmethioninebutnotfolate
Next, we used supplementation experiments with two major metabolites in the one-carbon cycle (Fig. 3a). First, we used 10, 50, and 100μMoffolate(vitaminB9)forsupplementationofstandardE.coli platesusingsimilarrangeofconcentrationsasapreviousstudyinC.elegans19.However,wefoundnocredibleinductionofthepredatory mouthform(e.g.,for100μM,0.384≤HDI(θ )≤0.755,Fig.4d–f).Note thatthismightbeduetothemicromolardosesoffolatethathadtobe usedintheseexperimentsduetosolubility.Incontrast,methionine supplementation resulted in the induction of the predatory mouth formandsubsequentTEI;however,withdifferentdynamicsthanwhat is observed after vitamin B12 supplementation. Specifically, supplementationwith5,10,and20mMmethionineresultedintheinduction oftheEumouthformafterseveralgenerationsofexposure(Fig.4g–i). Note thatthis supplementation does not resultin 100% Eu animals similartosupplementationwithlowerconcentrationsofvitaminB12 (Fig. 4). However, reversal after five generations of exposure to methionineindicatesaTEIofthepredatorymouthformthatlastsfor threegenerations(Fig.4g–i).Giventhatmethioninesignificantlyalters osmolarity, we controlled for this effect using sorbitol20. However, sorbitolsupplementationdidnotinducetheEumorph,indicatingthat theobservedresponseisspecifictomethionineratherthanosmotic changes(SupplementaryFig.3h).Overall,theseresultssuggestthat vitaminB12mightexertitsfunctionthroughmethionineintheone-carboncycle.

Given that vitamin B12 and methionine supplementations can bothcauseTEIofthepredatorymorph,weperformedsingleworm transcriptomicanalysisofmethionine-supplementedwormsofday1 adultindividuals.Theoverlapofupregulatedgenesbetweenmethionine supplementation and Novosphingobium exposure was limited (6.8%),whereasvitaminB12supplementationshowedalargeroverlap with Novosphingobium (12.2%), indicating thatvitamin B12 recapitulatesalargersubsetofthebacterial-inducedtranscriptionalprogram (Fig.4j).Notably,theoverlapbetweensignificantlyupregulatedgenes after vitamin B12 and methionine supplementation represents the largestfractionof19%(Fig.4j).Together,thesepatternssuggestthat vitamin B12 accounts for a larger fraction of the Novosphingobium-inducedtranscriptionalresponses.

VitaminB12causeselevatednutrientprovisioning
Itisimportanttodeterminewhattypeofchangesactdownstreamof theoriginalstimulustocausetheinheritedeffect.Thisisofparticular importancegiventhedifferentvitaminB12concentrationsnecessary toinducethepredatorymouthformanditsTEI.Onepossibilitywould be that vitamin B12 is directly transmitted from the mother to the offspring.Alternatively,TEImightbeaconsequenceoffactorsacting downstream of vitamin B12. To test for the former possibility, we generatedmutantsinmrp-5,agenethatwasshowninC.eleganstobe responsible for the transport of vitamin B12 from mother to the offspring21.Indeed,P.pacificushasa1:1orthologofmrp-5andwewere abletocreatetwomutationsinPpa-mrp-5thatresultinframeshifts (Fig.5a).However,Ppa-mrp-5mutantsshowanormalTEIofthepredatory mouth form (Fig. 5b, c). These findings suggest that TEI is unlikelytobedrivenbydirectvitaminB12transmissionandinstead dependsondownstreamfactorsoralternativetransportmechanisms.

In principle, a multigenerational vitamin B12-rich diet could induceaplethoraoftranscriptionalchangesintheworm.Therefore, we extended ourgene expression analysistoculturesafter reversal andcomparedtranscriptionalprofilesofsinglenaïve,F3,andF5generationwormsonNovosphingobiumwithF3R2,F4R2,F5R2,andF10R2 generationsafterreversaltoE.coli(Fig.5d).WeusedFnR2ratherthan FnR1generationstoruleoutanyeffectofthekanamycintreatmentsin the FnR1 generation. Note that we could notuse single worm transcriptomicanalysesforthislargesetofconditionsandinstead,performedtheseexperimentsbyharvestingcultureswithpreferentially adult individuals. We found a consistently strong increase in the expression of vitellogenin genes, encoding the nematode yolk proteins, after Novosphingobium exposure with multiple vitellogenin genes remaining athigher expression levels in comparison tonaïve animals(FDR-correctedpvalue<0.01)(Fig.5e).Notethatvitellogenin geneswerealsoupregulatedinoursinglewormtranscriptomicdataset describedabove(Fig.2g),butseveralofthemwerenotsignificant.As vitellogeninsaretransmittedfrommotherstooffspring,theyrepresent potential candidates for a function in TEI of the predatory mouthform.

P.pacificusshowsamorecomplexvitellogenincompositionwhen comparedwithC.elegans.WhiletheC.elegansN2genomecontainssix vitellogeningenes, P.pacificusRSC011has 9genes,allofwhich are mostcloselyrelatedtoCel-vit-6(Fig.5f,g).Wethereforenamedthese genes Ppa-vit-6-A to Ppa-vit-6-I based on their location along the chromosome (Fig. 5f, g). While more similar to one another in sequencethantheCel-vitgenes,thedivergencebetweenthePpa-vit genesstillallowsthedistinctionofindividualgenesingeneexpression analysis (Fig. 5e). We found that 8 of the 9 vitellogenin genes are upregulateduponNovosphingobiumexposure,fourofwhichshowan increasedexpressionofatleasteightfoldintheF10generation(Fig.5e andSupplementaryData2).Asimilarincreaseinexpressionwasseen inallothertestedgenerationsonNovosphingobium.AfterreversaltoE. coli the majority of vitellogenin genes were less expressed than on Novosphingobium;however,expressionremainedsubstantiallyhigher than in naïve animals that had never been exposed to Novosphingobium (Fig. 5e). Thus, exposure to a multigenerational Novosphingobium diet and subsequent reversal to E. coli cause elevated vitellogenin transcription resulting in increased nutritional provisioning.

Receptor-mediatedendocytosis(rme-2)mutantshavenomemory
The correlation between elevated vitellogenin expression and the formation of the predatory mouth-form during and after Novosphingobiumexposuresuggestsa role ofnutritionalprovisioningin transgenerationalmemory.Toprovidemechanisticevidenceforthis hypothesis, we investigated the receptor involved in vitellogenin uptakeintothegermline.Innematodes,vitellogeninisproducedinthe intestine,andthelow-densitylipoproteinreceptor(LDLR)encodedby rme-2 (receptor-mediated endocytosis protein) wasshown to represent the single protein involved in vitellogenin uptake into the germline22,23.InC.elegans,rme-2mutantembryoscontainnodetectableyolkandthebroodsizeofhomozygousmutantanimalsisstrongly reduced22.InP.pacificus,thereisa1:1orthologPpa-RME-2witha55% amino acid sequencesimilarity (Fig. 6a).Strikingly, we hadisolated threemutantallelesofPpa-rme-2inourEMSmutagenesisscreenfor predatory mouth-form Transgenerational-inheritance-defective (Tid) mutants reported previously (Fig. 6a and Supplementary Table 2)3. Thisfindingwouldbeconsistentwitharoleofvitellogenininmemory formation.

ToruleoutthepossibilitythatEMS-inducedmutationsingenes otherthanPpa-rme-2haveresultedintheobservedTidphenotype,we generatedanadditionalknock-outmutationbyCRISPRengineering. ThePpa-rme-2(tu2155)allelehasa16bpinsertionthatresultsinapremature stop codon (Fig. 6a). When we tested the Ppa-rme-2(tu2155) allele in a 5-generation Novosphingobium assay, we observed an incomplete induction of the Eu morph for all five generations of Novosphingobium exposures (0.64≤HDI(θ )≤0.928, 0.664≤HDI(θ )≤0.94, and 0.628≤HDI(θ )≤0.921) (Fig. 6b). This result indicates thatintheabsenceofRME-2andvitellogeninuptake into the germline, the predatory mouth-form can still be induced, whichislikelyduetootherNovosphingobium-derivedfactorsaspreviouslyindicated(Fig.2c).Incontrast,afterthereversaltoE.coli,Ppa-rme-2(tu2155)didnotshowtransgenerationalmemoryofthepredatory morph (0.12≤HDI(θ )≤0.431, 0.147≤HDI(θ )≤0.484) (Fig. 6b).Thus, vitellogeninis involved in the formation of transgenerationalmemoryofthepredatorymouth-forminP.pacificusindicatingarolefornutrientprovisioninginthisprocess.

Methods
Experimentalmodelandsubjectdetails
TheancestralP.pacificusRSC011isolateusedinthisstudywasfrozen withinthefirst5–10generationsafteroriginalisolationfromCoteau Kerveguen on La Réunion to minimize domestication and thereby, facilitate the investigation of diet-induced plasticity and TEI. Nematodes were grown under standard nematode growth conditions on NGMplatesseededwitheitherE.coliOP50orNovosphingobiumL76 and maintained at 20°C. The RSC011 stock was not subjected to bleaching, starvation, extreme temperature fluctuations as they are known to influence mouth-form ratios. Nematode mutant strains described in this study (Ppa-metr-1, Ppa-mce-1, Ppa-mrp-5, and Ppa-rme-2)weregeneratedforthisstudyandareavailableuponrequest fromtheleadauthor.

Bacterialstrainsconditions
AllbacterialstrainsandmutantsweregrownovernightinLB(Lysogeny broth) supplemented with 50μg/ml kanamycin, where required to initiate food-reversal. Bacteria were grown at 30°C or 37°C, dependingonthespeciesand6cmnematodegrowthmedium(NGM) plateswereseededwith300μlbacterialovernightculturesandwere incubatedfor2days.TheNovosphingobiumL76strainandthevitamin B12-deficientmutantwereisolatedandgeneratedbyourgroupandare alsoavailableuponrequest.

Nematodecultureanddietaryreversalexperiments
Overnight cultures of E. coli OP50 and Novosphingobium L76 were spreadtoNGMplatesandincubatedatroomtemperaturefor2days. The P.pacificus RSC011 strain is preferentially St (20–40% Eu) on a standardE.coliOP50diet.Novosphingobiumexposureisperformedby pickingsingleJ4larvaeontoaNovosphingobiumL76diet.J4worms wereinitiallyleftforatleastanhouronaninitialNovosphingobiumdiet toreducetracesofE.coliOP50beforetransferringtofinalF1Novosphingobiumplates.ReversalexperimentstoanE.coliOP50dietwere performed afterexposing ofRSC011 wormsforvarious numbersof generations,i.e.,1,3,5,10,or15generationsonNovosphingobiumL76. WormswereinitiallytransferredfromNovosphingobiumL76toNGM platessupplementedwith50μg/mlofkanamycinforonegeneration, then back to E. coli OP50-seeded NGM plates for subsequent generations.Exposuretokanamycinforasinglegenerationtoeliminate tracesofNovosphingobiumdidnotaffectmouth-formresponse3.

Mouth-formphenotyping
Mouth-from phenotyping was performed using Zeiss Discovery V.20stereomicroscope(X150magnification)byobservingthenematodebuccalcavitybasedonmouth-formidentities9.Finalmouth-form frequenciesarethemeanofatleast10replicates,eachassaying20 animalsfromasingleplate.

Statisticsandreproducibility
ToestimatetheprobabilityofdevelopingtheprobabilityofdevelopingtheEumouthforminP.pacificus(θ),weconstructedahierarchical Bayesianmodelaspreviouslydescribed3

y =Bernoulli(θ)
θ =Beta(ω(κ-2)+1,(1-ω)(κ-2)+1)
ω =Beta(α,β)
κ =Gamma(3,1)

whereθiscalculatedforeachreplicateandhyperparametersωandκ link the biological replicates for a given generation under a given experimental condition37. Each experimental condition included independentbiologicalreplicatesconsistingof5–10plates,withupto 60 plates assayed depending on the condition, and 20 animals per plate. The hierarchical model accounts for replicate-level variation acrossplates.Themodelwasfittedtothelaboratorymeasurements using PyMC38 in Python 3.11with Numpy 1.25.239. The mean highest densityinterval(HDI)forθforagroupofobservationswasusedto visualizetheinferredprobabilityofdevelopingtheEumouthform.We ensured that the estimated θ values were stable using common convergencediagnosticswitheffectivesamplesize(ESS)≥1000040.
All experiments were performed with independent biological replicates,andresultswereconsistentacrossreplicates.Nostatistical methodwasusedtopredeterminesamplesize.Nodatawereexcluded from the analyses. The experiments were not randomized because experimentalgroupsweredefinedbypredefinedtreatmentconditions and generations. The investigators were not blinded to allocation during experiments and outcome assessment because the experimentalconditionsandmouth-formphenotypeswerereadilyidentifiableduringscoring.

CRISPR/Cas9-inducedmutations
Mutations in candidate genes were generated using CRISPR/Cas9 methodologies41. Specifically, gene-specific crRNAs and universal trans-activating CRISPR RNA were obtained from Integrated DNA Technologies. Equal volumes of (5μl) of each 100μM stock were mixed and denatured at 95°C for 5min, then allowed to anneal at room temperature. Cas9 endonuclease (New England Biolab) was addedtothehybridizedproductandincubatedatroomtemperature for 5min. TE buffer was used to adjust to final concentrations of 18.1μMforthesgRNAand2.5μMforCas9.Theresultingmixturewas microinjectedintothegermlineof40–50P.pacificusRSC011young adults.EggsfrominjectedP0swerecollectedupto16hpost-injection. Afterhatchingandtwodaysofgrowth,theF1progenywassegregated onto individual plates until they had fully-developed and laid eggs sufficiently.F1genotypeswerescreenedusingSangersequencingand mutationswereidentifiedpriortoisolationofhomozygousmutants. DetailsofsgRNAsandprimersusedinthisstudyareprovidedinthe supplementarytable.Evidencefornullalleleswasbasedonframeshift mutations causing premature stop codons in protein-coding sequences.

Metabolitesupplementationassays
Methylcobalamin (Vitamin B12 CAS No. 13422–55–4), L-methionine (CASNo.63–68–3),andfolate(CASNo.59-30-3)werepurchasedfrom Sigma and dissolved in water at the highest possible soluble concentrations to prepare stock solution. To quantify real phenotypic effectsandcontrolforalteredNGMosmolarity,D-sorbitol(CASNo.) wasadded20atafinalworkingconcentrationof5,10,and20mM.All stockswerepreparedfreshbeforeuseineachexperiment.Metabolite solutionsweremixedwithNGMagarattherequiredconcentrationjust before pouring to 6cm plates. Plates were allowed to dry at room temperaturefortwodaysandthenspottedwithE.coliOP50.Wefirst testeddifferentconcentrationsofvitaminB12andfoundthestrongest andmostreliabletransgenerationalepigeneticmemoryeffectwitha concentration of 1000 and 1500nM, which is most likely unphysiological. Similarly, in C. elegans, dose-dependent effects have beenseenforvitaminB12 17.Differentconcentrationsformethionine andfolatesupplementationwerebasedonpreviousstudies15,19.

RNAsequencinganddataanalysis
We used single worm transcriptomics (SWT) of naïve and RSC011 worms reared on Novosphingobium for three generations using five independentreplicates42.E.coliOP50supplementedwithvitaminB12 andmethioninewerealsoprocessedwiththeSWTprotocol.Incontrast,weusedmixedculturesforassayingindependentgenerational datapoints during Novosphingobium induction and reversal experimentsinF3R2,F4R2,F5R2,andF10R2generations.Forthat,worms wereharvestedfromthreeNGMplates.Ingeneral,wormswerepelletedforRNAextractionandtotalRNAwasisolatedusingDirect-Zol RNA Mini prep kit (Zymo Research) following the manufacturer’s instructions. The RNA-seq library preparation and sequencing were performedbythecompanyNovogene.Rawreadswerealignedtothe P. pacificus RSC011 reference genome using Hisat243 (version 2.1.0), and read counts were quantifiedwith featureCounts44basedonthe RSC011geneannotations.Fortheanalysisofvitellogeninexpression, wecombinedrawcountsofthethreetranscripts(RSC011000007769, RSC011000004509, and RSC011000017258) into a single vit-6-B count.DifferentialgeneexpressionanalysiswasperformedinR(version4.0.3)usingDESeq2(version1.18.1)45.Forsingle-wormRNA-seq samples, genes with padj<0.05 were considered differentially expressedtoaccountforhigherinter-individualvariability.Formixed-worm RNA-seq samples, a more stringent cutoff of padj<0.01 was applied, reflecting the lower variability and increased confidence in expression differences. Tests for overrepresentation of protein domains in sets of differentially expressed genes were performed using aFisher’s exacttestinR. WeusedtheFDRmethod as implementedinthep.adjustfunctioninRtocorrectformultipletestingand onlyretainedresultswithadjustedP-value<0.01.

Phylogeneticanalysis
One-to-oneorthologousgenesbetweenP.pacificusandC.eleganswas identifiedfromBLASTPsearchesandwerevalidatedbyphylogenetic analysis.ProteinswerealignedusingClustalOmega46andtheoutput FASTAfile wasuploaded to the IQ- TREE tool47,48. Analysis wasperformedunderdefaultsettingsusingtheautosubstitutionmodel,and 1000 bootstrap alignments were calculated with ultrafast setting. FigTreewasusedtovisualizedtheresultingphylogenetictree(http:// tree.bio.ed.ac.uk/software/figtree/).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Previous work from The Sommer lab identified vitamin B12 as an important factor in increasing predatory behavior in P. pacificus. Adding to that work, Quiobe et al., found that the function of vitamin B12 in the met/SAM cycle was needed for the transgenerational epigenetic inheritance (TEI) of the predatory mouth form. The authors identified upregulation of vitellogenin genes caused by a vitamin B12 rich diet and concluded that the change in nutritional provisioning to embryos was responsible for TEI of the predatory mouth form. This study uncovers an unexpected role of vitamin B12 in transgenerational memory of an animal’s morphology and behavior.\nOverall, the experimental design and results are clear and thorough. However, an alternative logical hypothesis has not been explored and there are several major and minor issues that need to be addressed to improve the paper.\nMajor comments:\nMuch of figure 1 presents experiments that were previously published and is discussed in the introduction. This is highly unusual, and we wonder whether it could just be presented in a cartoon, rather than re-showing data.\nThe first experiment of this paper shows that both female and male gametes can cause TEI of the predatory mouth form. There is no follow up from this experiment and the results don’t contribute further to the paper. This experiment could be removed entirely, or they should discuss more about what this could mean in terms of vitamin B12 and/or methionine as a memory.\nThe authors use a very high dose of 1,000 nM vitamin B12 and state that this is equivalent to that used in our Watson et al Cell 2013 paper, and they say this is common in C. elegans studies. This is not correct, the maximum dose used in the Watson paper (and most of our other papers, e.g., see also Bulcha et al, for a titration) Cell was 64 nM. This is a critical point because a high dose of vitamin B12 is required to observe a transgenerational effect and reducing this dose decreases the number of generations that the predatory mouth form is observed. This therefore suggests that the ‘memory’ phenotype is caused simply by passing vitamin B12 from mother (or father) to offspring. If this is true, the use of the term “transgenerational epigenetic inheritance” is not correct. The authors could test this hypothesis by using an mrp-5 mutant (assuming there is an ortholog in P. pacificus) because in C. elegans MRP-5 transports vitamin B12 from the mother to the offspring (Na et al, Cell Reports 2018).\nThe Walker lab has shown that SAMS-1 converts methionine into S-adenosylmethionine which is required to make phosphatidylcholine, an important component of membranes. The authors should examine whether this plays a role in the phenotypes they observe by testing a sams-1 mutant.\nHow and why are vitellogenin genes upregulated by vitamin B12? Does methionine activate the same genes? And is this related to any (histone) methylation, which is the function of the Met/SAM cycle and an epigenetic mark, or only methionine?\nWhat could be the ecological relevance of vitamin B12 activating the predatory form and having a transgenerational effect? And would vitamin B12 concentrations be sufficiently high in the wild?\nMinor comments:\nDid the authors control their gene expression data for developmental effects caused by vitamin B12? Also, the gene expression tables are difficult to navigate.\nReplace “vitamin B9” with “folate” in the abstract (or both) as readers will be more familiar with that.\nIn figure 2 the authors describe some panels out of order. (e.g. figure 2e before figure 2c).\nFigures are inconsistently indicated in the text (e.g. Fig. 2e vs. Figure 2E).\nAuthors did not mention if NGM concentration was adjusted to account for discrepancy in osmolarity and nutrient amount when adding supplements.\nmce-1 is indicated at the wrong reaction in Figure 3.\nPage 11 – it would be helpful to mention what vitellogenins are in this section.\nPage 13 – vitamin B12 can also be made by certain archaea, not only by bacteria.\nGabby Giese & Marian Walhout", "reviewer_2": "Quiobe et al. investigate the mechanisms of the transgenerational epigenetic inheritance (TEI) of the memory of ancestral environment. To this end, they use as a model system the mouth dimorphism of the Pristionchus pacificus nematode, which they previously found to be modulated by diet (the presence of a Novosphingobium bacterium) in a transgenerational manner. Through a series of well-conceived and well-performed experiments the authors demonstrated that bacteria-derived vitamin B12 is both necessary and sufficient for transgenerational memory; found a dose response of B12 with increasing concentration required for a transgenerational inheritance of the predatory morph during an increasing number of generations. They also found that the TEI requires methionine and methionine-synthase metr-1 but not folate. Finally, they found that vit B12 (likely) acts through increased provisioning of vitellogenin.\nOverall, the results are interesting and the conclusions are generally well supported.\nHowever, I have several major and minor points to address before publication.\nThe main problem I have is with this manuscript is that figure 1 which is almost entirely recycled from their recent paper (specifically panels b, c which are taken from Fig 1 c,d and fig 2 B-F of Quiobe et al 2025 Sci adv). While it is important to put the new finding in context with their previous findings, I think there is no need (and it is inappropriate) to include figures with data already published as it could mislead reader into thinking they are new results, unless clearly stated and an attribution to the original source next to the reproduced figure is clearly specified and properly referred to. Nevertheless, I think a scheme indicating the previous finding is enough to put the novel findings in context.\nA second major point is how do the authors reconcile that both female and male gametes transmit TEI of the predatory mouth form with the proposed mechanism based on increased vitellogenin provisioning which would implicate only oocytes?\nWhat do the authors make of the collagen expression changes with collagen being the most significantly overrepresented category among differentially expressed genes? It is usually a red flag for uncontrolled variation in developmental stage between transcriptomic samples.\nDoes increased vit transcription also translate into increased vit provisioning to the embryo?\nLikely yes but it would be nice to check using protein tags or even by using autofluorescence (see Perez et al 2017 Nature)\nMoreover, given that vitellogenins are constituent of a lipoprotein complex (similar to LDL) that carries lipids, the actual signal could be a lipid or even broad change in the composition of the lipids carried by the complex (and dependent on rme-2) rather than an increased in amount, as it has been shown already in at least two cases of intergenerational transmission in C. elegans (Wang et al 2023 Nature cell biology, and Wilhelm et al. 2025 biorxiv doi: https://doi.org/10.1101/2025.06.03.657568). Considering that vitamin b12 and methionine are major regulators of lipid metabolism in nematodes I think the authors should at least discuss this possibility in the discussion section."}, "author_response": "Response: We thank the reviewer for the overall judgement of our work.\nMajor comments:\nMuch of figure 1 presents experiments that were previously published and is discussed in the introduction. This is highly unusual, and we wonder whether it could just be presented in a cartoon, rather than re-showing data.\nResponse: We have followed this recommendation and have replaced Figure 1 with a cartoon. The reason for having Figure 1 in the first place was that the experimental design used in our study system is relatively novel and different from that of other studies. We just wanted to make sure that readers are not lost early on. But given that also reviewer 2 had a similar concern, we are happy to change figure 1 into a cartoon like summary.\nThe first experiment of this paper shows that both female and male gametes can cause TEI of the predatory mouth form. There is no follow up from this experiment and the results don’t contribute further to the paper. This experiment could be removed entirely, or they should discuss more about what this could mean in terms of vitamin B12 and/or methionine as a memory.\nResponse: We thank the reviewer for this suggestion. We had similar concerns that this piece of data is unconnected to the rest of the story. Therefore, we have removed this paragraph all together.\nThe authors use a very high dose of 1,000 nM vitamin B12 and state that this is equivalent to that used in our Watson et al Cell 2013 paper, and they say this is common in C. elegans studies. This is not correct, the maximum dose used in the Watson paper (and most of our other papers, e.g., see also Bulcha et al, for a titration) Cell was 64 nM. This is a critical point because a high dose of vitamin B12 is required to observe a transgenerational effect and reducing this dose decreases the number of generations that the predatory mouth form is observed. This therefore suggests that the ‘memory’ phenotype is caused simply by passing vitamin B12 from mother (or father) to offspring. If this is true, the use of the term “transgenerational epigenetic inheritance” is not correct. The authors could test this hypothesis by using an mrp-5 mutant (assuming there is an ortholog in P. pacificus) because in C. elegans MRP-5 transports vitamin B12 from the mother to the offspring (Na et al, Cell Reports 2018).\nResponse: We thank the reviewer for these comments. We have now clarified our wording and have addressed all these issues:\n1.) We had mistaken the reference of our work (Akduman, ref #15) with the C. elegans work by Watson et al (reference #17) when introducing the high vitamin B12 concentrations. We have now corrected this by saying that we started with vitamin B12 concentrations similar to previous Pristionchus work.\nSpecifically, work in our lab had used 500 nM in the context of killing efficiency (Akduman et al, 2020).\nWhen starting to work with vitamin B12 in the context of mouth-form plasticity, we started to also increase the concentration to 1,000 nM and 1,500 nM vitamin B12 and observed the strongest and most consistent effect with these concentrations. We have now clarified these issues in the text.\n2.) Lower concentrations, such as those used in C. elegans, cause strong induction but no transgenerational inheritance, indicating that induction and memory require different concentrations of vitamin B12. This is now properly described on pages 5/6 and 9 and in Figures 2 & 4.\n3.) The effect with 1,000 nM and 1,500 nM vitamin B12 are still a transgenerational inheritance as it lasts for three generations after stopping the supplementation with vitamin B12. In line with this, 500 nM gives intergenerational inheritance, whereas smaller concentrations have no effect. In our discussion, we point out – along the same lines as the reviewer – that these are very high doses of vitamin B12 and we discuss various boundary conditions.\n4.) We added two more concentrations of vitamin B12 in our supplementation experiments. At 0.1 nM vitamin B12, we still see induction of the predatory morph. In contrast, 0.01 nM vitamin B12 does not cause any response. We have now added these data to our analysis.\n5.) We had indeed generated and used mrp-5 mutants in P. pacificus. However, this mutant shows a wild type pattern of transgenerational inheritance (now presented as Fig. 5a,b), indicating that factors other than the direct passing of vitamin B12 from parents to offspring are ultimately responsible for the observed memory. We now introduce these findings in Figure 5 and on pages 11 and 12.\nThe Walker lab has shown that SAMS-1 converts methionine into S-adenosylmethionine which is required to make phosphatidylcholine, an important component of membranes. The authors should examine whether this plays a role in the phenotypes they observe by testing a sams-1 mutant.\nResponse: This is a great suggestion that also came to our mind. Indeed, we had planned to use mutations in the sams-1 gene. Unfortunately, P. pacificus has a massive expansion of the sams-1 gene with additional copy number variations between strains. Therefore, we cannot target this gene in CRISPR experiments.\nHow and why are vitellogenin genes upregulated by vitamin B12? Does methionine activate the same genes? And is this related to any (histone) methylation, which is the function of the Met/SAM cycle and an epigenetic mark, or only methionine?\nResponse: We thank the reviewer for this suggestion. We had not looked at gene expression after methionine supplementation. We have now added these experiments and have – also in response to reviewer 2 – moved for some experiments from mixed stage RNAseq to single worm RNAseq of day 1 adult animals. This protocol had been previously established in our lab. As a result, parts of Figure 2 have been changed accordingly, and the methionine data have been added to Fig. 4. This also addresses a major concern of reviewer 2.\nWhat could be the ecological relevance of vitamin B12 activating the predatory form and having a transgenerational effect? And would vitamin B12 concentrations be sufficiently high in the wild?\nResponse: We thank the reviewer for bringing up this question. The induction of the predatory form by vitamin B12 is likely of importance in the competitive environment of the decaying beetle ecosystems in which Pristionchus nematodes are found. There are multiple competing nematodes on beetle carcasses and Pristionchus has an advatnge through intraguild predation. Also, previous work from our lab has shown that Pristionchus selectively feeds on vitamin B12 producing bacteria when exposed to a mixture of diverse bacteria (Lo et al., Nature Communications, 2023), a striking finding that is currently further studied by my previous postdocs. We have now added a novel paragraph to the discussion section to address these issues.\nMinor comments:\nDid the authors control their gene expression data for developmental effects caused by vitamin B12? Also, the gene expression tables are difficult to navigate.\nResponse: As indicated above, we have redone gene expression analysis with single worm RNAseq. Also, we changed the representation of the data.\nReplace “vitamin B9” with “folate” in the abstract (or both) as readers will be more familiar with that.\nResponse: Done.\nIn figure 2 the authors describe some panels out of order. (e.g. figure 2e before figure 2c).\nResponse: Corrected.\nFigures are inconsistently indicated in the text (e.g. Fig. 2e vs. Figure 2E).\nResponse: Corrected.\nAuthors did not mention if NGM concentration was adjusted to account for discrepancy in osmolarity and nutrient amount when adding supplements.\nResponse: Corrected.\nmce-1 is indicated at the wrong reaction in Figure 3.\nResponse: Corrected.\nPage 11 – it would be helpful to mention what vitellogenins are in this section.\nResponse: We have added an explanatory sentence.\nPage 13 – vitamin B12 can also be made by certain archaea, not only by bacteria.\nResponse: Corrected.\nGabby Giese & Marian Walhout\nReviewer #2 (Remarks to the Author):\nQuiobe et al. investigate the mechanisms of the transgenerational epigenetic inheritance (TEI) of the memory of ancestral environment. To this end, they use as a model system the mouth dimorphism of the Pristionchus pacificus nematode, which they previously found to be modulated by diet (the presence of a Novosphingobium bacterium) in a transgenerational manner. Through a series of well-conceived and well-performed experiments the authors demonstrated that bacteria-derived vitamin B12 is both necessary and sufficient for transgenerational memory; found a dose response of B12 with increasing concentration required for a transgenerational inheritance of the predatory morph during an increasing number of generations. They also found that the TEI requires methionine and methionine-synthase metr-1 but not folate. Finally, they found that vit B12 (likely) acts through increased provisioning of vitellogenin.\nOverall, the results are interesting and the conclusions are generally well supported.\nResponse: We thank the reviewer for the overall judgement of our work.\nHowever, I have several major and minor points to address before publication.\nThe main problem I have is with this manuscript is that figure 1 which is almost entirely recycled from their recent paper (specifically panels b, c which are taken from Fig 1 c,d and fig 2 B-F of Quiobe et al 2025 Sci adv). While it is important to put the new finding in context with their previous findings, I think there is no need (and it is inappropriate) to include figures with data already published as it could mislead reader into thinking they are new results, unless clearly stated and an attribution to the original source next to the reproduced figure is clearly specified and properly referred to. Nevertheless, I think a scheme indicating the previous finding is enough to put the novel findings in context.\nResponse: We apologize if the reviewer got the wrong impression. It was never our intention to mislead the reader. As indicated above, we just wanted to make sure that readers understand our novel and complex study design.\nWe have now replaced Figure 1 by a graphical representation summarizing our previous findings.\nA second major point is how do the authors reconcile that both female and male gametes transmit TEI of the predatory mouth form with the proposed mechanism based on increased vitellogenin provisioning which would implicate only oocytes?\nResponse: As suggested by reviewer 1, we have removed this entire paragraph, also because it was not connected to the rest of our study.\nWhat do the authors make of the collagen expression changes with collagen being the most significantly overrepresented category among differentially expressed genes? It is usually a red flag for uncontrolled variation in developmental stage between transcriptomic samples.\nResponse: We thank the reviewer for bringing up this question. Previous work from our lab has shown that the change in mouth form (Eu vs ST) results in substantial gene expression changes, including several collagens (Sieriebriennikov et al., PLoS Genetics, 2020, Sun et al., Genome Research, 2022; Theska et al., Evolution&Development, 2024). We are currently working on some of these genes as interestings markers and protein constituents of the teeth.\nAs already indicated in response to reviewer 1, we have redone this part of the differential gene expression analysis and used single worm transcriptomics rather than mixed stage cultures. As indicated above, parts of Figure 2 have been replaced, and additional analysis has been added to Figure 4.\nDoes increased vit transcription also translate into increased vit provisioning to the embryo?\nLikely yes but it would be nice to check using protein tags or even by using autofluorescence (see Perez et al 2017 Nature)\nResponse: Unfortunately, autofluorescnce does not work in P. pacificus due to differences in lipid composition.\nWe had aimed for quite some time in tagging individual vit genes, which turned out to be inheritantly more difficult in P. pacificus than C. elegans, likely due to the nine independent copies of vit-6, that are all very similar to each other in sequence. We finally managed to obtain one line with a single (rather than two ) ALFA tag at the C-terminus of the protein. Unfortunately, it has a phenotype on its own. While we keep trying to generate a flagged protein, for the time being this experiment is unfeasible in P. pacificus.\nMoreover, given that vitellogenins are constituent of a lipoprotein complex (similar to LDL) that carries lipids, the actual signal could be a lipid or even broad change in the composition of the lipids carried by the complex (and dependent on rme-2) rather than an increased in amount, as it has been shown already in at least two cases of intergenerational transmission in C. elegans (Wang et al 2023 Nature cell biology, and Wilhelm et al. 2025 biorxiv doi: https://doi.org/10.1101/2025.06.03.657568). Considering that vitamin b12 and methionine are major regulators of lipid metabolism in nematodes I think the authors should at least discuss this possibility in the discussion section.\nResponse: We thank the reviewer for bringing up this point and we have added this to our Discussion.\nIn summary, we feel that we have been able to successfully respond to the majority of concerns (except for those that were technically not feasible. We feel that this has increased the quality of the data. Therfeore, we want to thank the reviewers once again for their input.\nThank you very much for consiedring this revised version.\nRalf J. Sommer, on behalf of all co-authors"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have done an excellent job revising the paper based on our and the other reviewer’s comments. We only have a few remaining comments to further improve the paper:\nMost importantly, the notion that direct transfer of vitamin B12 from mother to offspring can contribute to the transgenerational effects is not sufficiently disproved, especially since transgenerational effects correlate with supplemented B12 concentrations. The experiment with mrp-5 mutants shows that there is a bit of a decrease in the transgenerational effect (not dramatic). It would be possible that there are multiple transporters that deposit vitamin B12 in the offspring as well. We suggest a short paragraph expanding on this in the Discussion.\nThe authors use only micromolar doses of folate (presumably because folate has low solubility in water). It is possible this is simply not enough folate to induce the EU mouth-form. The authors should add a sentence addressing this, and mention whether they have some evidence to suggest micromolar doses of folate is sufficient to be uptaken by P. pacificus.\nThe use of both TEI and LTEI abbreviations can be confusing. Maybe one can be spelled out throughout? (very minor)\nLine 144: the sentence and symbol for the quantification are hard to understand, consider explaining/rephrasing?\nHow many animals were used for single animal RNA-seq? And how similar were the profiles among animals from the same experiment?\nLine 184: change “12,2” to “12.2”\nLine 234/235: methyl malonylcoA mutase is encoded by mmcm-1, not mce-1! Thus, the enzyme that uses vitamin B12 as a cofactor in propionate breakdown has not been directly studied here.\nFigure 3C needs no vitamin B12 in the plot.\nLine 254: change “…indicating the Ppa…” to “…indicating that Ppa…”\nWe suggest a three-way Venn diagram to convey the differentially expressed genes for diet, vitamin b12, and methionine.\nLine 344/345 “…harvesting worm plates with preferentially adult individuals.” – rephrase, worms were harvested not plates and preferentially adult is weird.\nGabby Giese & Marian Walhout", "reviewer_2": "The authors have addressed my main concerns and I am happy with the current version of the manuscript"}, "author_response": "Response: We thank the reviewer for this overall statement.\nMost importantly, the notion that direct transfer of vitamin B12 from mother to offspring can contribute to the transgenerational effects is not sufficiently disproved, especially since transgenerational effects correlate with supplemented B12 concentrations. The experiment with mrp-5 mutants shows that there is a bit of a decrease in the transgenerational effect (not dramatic). It would be possible that there are multiple transporters that deposit vitamin B12 in the offspring as well. We suggest a short paragraph expanding on this in the Discussion.\nResponse: We have added a pargraph to the discussion as suggested (page 15), writing the following:\nThe experiments with Ppa-mrp-5 mutants show a normal TEI suggesting that it is not the direct transmission of vitamin B12 but a downstream factor causing the observed memory. However, the transgenerational effect is slightly decreased compared to wild type and one possibility that cannot be completely ruled out is that multiple transporters in addition to Ppa-mrp-5 are involved in vitamin B12 transmission. Nonetheless, Ppa-mrp-5 is one-to-one orthologous to Cel-mrp-5; thus, there are no additional copies of this transporter in the P. pacificus genome.\nThe authors use only micromolar doses of folate (presumably because folate has low solubility in water). It is possible this is simply not enough folate to induce the EU mouth-form. The authors should add a sentence addressing this, and mention whether they have some evidence to suggest micromolar doses of folate is sufficient to be uptaken by P. pacificus.\nRespone: We thank the reviewers for bringing up this point and have now rephrased as follows:\nFirst, we used 10 μM, 50 μM and 100 μM of folate (vitamin B9) for supplementation of standard E. coli plates using similar concentrations as a previous study in C. elegans.41 However, we found no credible induction of the predatory mouth form (e.g., for 100 μM, 0.384 ≤HDI(θ )≤ 0.755, Fig. 4d-f). Note that this might be due to the micromolar doses of folate that had to be used in these experiments due to solubility.\nThe use of both TEI and LTEI abbreviations can be confusing. Maybe one can be spelled out throughout? (very minor)\nResponse: We have spelled out long-term environmental induction experiment throughout the manuscript.\nLine 144: the sentence and symbol for the quantification are hard to understand, consider explaining/rephrasing?\nResponse: We have changed this sentence accordingly, writing the following:\nSpecifically, in three independent biological replicates with a total of 60 assay plates we found high but incomplete induction in the first generation after vitamin B12 supplementation (0.878 ≤HDI(θ)≤ 0.994, where θ is the inferred probability of developing the predatory mouth form).\nHow many animals were used for single animal RNA-seq? And how similar were the profiles among animals from the same experiment?\nResponse: We used five replicates for all single animal RNA-seq experiments. The results were highly overlapping as already seen in the original paper where we established this method by using three replicates. This information is provided in the Materials and Methods section.\nLine 184: change “12,2” to “12.2”\nChanged.\nLine 234/235: methyl malonylcoA mutase is encoded by mmcm-1, not mce-1! Thus, the enzyme that uses vitamin B12 as a cofactor in propionate breakdown has not been directly studied here.\nWe are sorry for this confusion and have now changed accordingly:\nIn P. pacificus as in C. elegans, the methionine-synthase is encoded by Ppa-metr-1. To manipulate the methylmalonyl coenzyme A (CoA) mutase pathway, we targeted the upstream factor Ppa-mce-1, that is one-to-one orthologous to Cel-mce-1. We targeted Ppa-metr-1 and Ppa-mce-1 in P. pacificus RSC011 by CRISPR to study a potential role in dietary induction and TEI of the predatory mouth form (Fig. 3b).\nFigure 3C needs no vitamin B12 in the plot.\nResponse: Thanks for pointing this confusion out. Figure 3C shows the response of wild type and mutant animals to different concentrations of vitamin B12 supplementation. This is the only graph that has concentrations on the X-axis, rather than generations. We changed the outline to make that clear.\nLine 254: change “…indicating the Ppa…” to “…indicating that Ppa…”\nChanged accordingly.\nWe suggest a three-way Venn diagram to convey the differentially expressed genes for diet, vitamin b12, and methionine.\nResponse: We changed to a three-way Venn diagram.\nLine 344/345 “…harvesting worm plates with preferentially adult individuals.” – rephrase, worms were harvested not plates and preferentially adult is weird.\nChanged accordingly:\n…..by harvesting cultures with preferentially adult individuals.\nGabby Giese & Marian Walhout\nResponse: We thank the reviewer for this overall statement."}, {"round": 3, "reviewer_comments": {"reviewer_1": "We are happy with the revision."}, "author_response": ""}]

Decision letter