Natural RNA interference directs a heritable response to the environment

RNA interference can induce heritable gene silencing, but it remains unexplored whether similar mechanisms play a general role in responses to cues that occur in the wild. We show that transient, mild heat stress in the nematode Caenorhabditis elegans results in changes in messenger RNA levels that last for more than one generation. The affected transcripts are enriched for genes targeted by germline siRNAs downstream of the piRNA pathway, and worms defective for germline RNAi are defective for these heritable effects. Our results demonstrate that a specific siRNA pathway transmits information about variable environmental conditions between generations.

similar inherited changes in gene expression. We also identify a specific RNAi pathway required for inheritance of environmental effects, show that extending the duration of stress increases the magnitude of the response and the number of affected generations, and determine that the effect is passed via the female germline.

Results
Our approach was to culture worms at 20uC, then split the culture into 20uC control and 25uC treatment for one generation (3 days at 25u), followed by return to 20uC, transferring to fresh culture plates each generation. Animals cultured at 25uC grow faster than animals cultured at 20uC but produce fewer progeny, evidence of mild stress. We chose early-stage embryos for comparisons between environmental histories, because transcript levels for most genes stay relatively constant over time through the 4-cell stage 34 . While this provides high sensitivity to detect differences in transcript levels, it is unknown whether the transcripts present at this stage are important for the response to mild heat stress, but they should be reliable endogenous reporters for the phenomenon.
We found 20 genes showing notable persistence of changes in transcript abundance for at least a generation after return to 20uC ( Figure 1 bottom left and top right corners, and Supplementary Table S1; for source data see Supplementary Table S2 and Gene Expression Omnibus accession GSE30666). Based on permutation tests, only one gene would be expected to show such changes by chance alone (Supplementary Figure S1; see Supplementary Notes for discussions of permutation tests and outlier genes). We selected two genes, one with elevated and one with reduced transcript levels, to measure by reverse transcription quantitative PCR (RT-QPCR) in multiple independent biological replicates ( Figure 1, right margin). This demonstrated the reproducibility of the observed transgenerational memory.
To investigate how long the effect persists, we measured transcript levels of the two genes over multiple 20uC generations following either one or two generations exposure to 25uC. We found that the effect can persist for at least four generations (Figure 2). Because a single C. elegans hermaphrodite worm produces hundreds of viable eggs and sperm, the dilution of material from any given developmental stage to the same stage in the next generation is expected to be at least a hundredfold. The reversibility of the heritable effects ( Figure 2) makes it unlikely that they are due to mutation of genomic DNA sequences. Therefore, we surmise that the heritable signal is either self-regenerating or amplifiable from a small number of molecules; otherwise, we would have expected the effect to disappear quickly due to dilution with each passing generation.
Heritable silencing of germline genes triggered by artificially introduced RNAs is similar in duration to what we see here (Figure 2), roughly two to four generations in the most detailed study so far 6 . To explore the possibility that an endogenous RNA interference-like phenomenon is involved in the heritable effect, we compared the mRNAs highlighted in Figure 1 with mRNAs targeted by short RNAs (sRNA) in oocytes 35 . We found that mRNAs showing transgenerational responses are highly enriched for genes targeted by endogenous antisense sRNA (Figure 3a and Supplementary Figure  S2, P of overlap with oocyte sRNA , 10 26 , by a cumulative hypergeometric distribution; see Supplementary Notes for discussion of those temperature-responsive genes that are not targeted by known sRNAs). Most of these sRNAs are 22 nucleotide residues long and start with a guanosine residue at the 59 end, and have therefore been dubbed ''22G'' RNAs 35 . Most 22G RNAs are thought to be secondary sRNAs made by RNA dependent RNA polymerases 20,35,36 .
The production or maintenance of endogenous 22G RNAs antisense to the temperature-responsive mRNAs identified here depends on several genes required for germline RNAi, including mut-2 (also known as rde-3), mut-7, mut-16 and multiple Argonaute-encoding genes 19,26,27,[35][36][37][38][39][40] (Figure 3b-c and Supplementary Figure S2). MUT-2, MUT-7, MUT-16 and the RNA dependent RNA polymerase RRF-1 concentrate together in mut-16 dependent structures adjacent to germline nuclei, consistent with a proposed role for these proteins in secondary sRNA production 41 . As previously reported, genes targeted by 22G secondary RNAs in oocytes are composed of two complementary classes (see Figure 4a): those targeted by 22G RNAs that co-immunoprecipitate with CSR-1, and those targeted by 22G RNAs that co-immunoprecipitate with WAGO-9 (also known as HRDE-1) 7,42 . CSR-1 is not known to be involved in gene silencing, and consistent with published results 42 , our microarray data shows that CSR-1 associated sRNAs target genes that tend to be highly expressed ( Figure 4b). These genes do not correspond to the temperature responsive genes identified here (Figure 4c). Growing evidence indicates that CSR-1 associated sRNAs activate or license transcription of  represent genes whose mRNA levels in each generation differ .23 between treatment and control and ANOVA/Student's T P , 0.01 (see Figure 1). All P values are from cumulative hypergeometric distributions. Unless noted otherwise, P values represent the probabilities of the observed or greater overlap between the genes highlighted in Figure 1 and the sRNA set, relative to all genes on the array that are also represented among the annotations used by the published studies (15,197 35   their target genes [43][44][45][46] . By contrast, our analysis of targets of WAGO-9, which is involved in RNAi-initiated heritable transcriptional silencing 7,14 , indicate that WAGO-9 binding 22G RNAs target the temperature-responsive transcripts (Figure 4d).
Heritable silencing can be triggered by PRG-1 and 21U RNAs [14][15][16] , which appear to generate WAGO-associated 22G secondary sRNA 23,24 . Therefore, we wondered whether the temperatureresponsive transcripts are also targeted by prg-1 dependent sRNAs.  Indeed, most of the temperature-responsive transcripts that are targets of oocyte 22G RNAs are also predicted 21U RNA targets where presence of 22G RNAs depends on prg-1 23,24 (see Supplementary  Table S1). Other primary sRNAs, specifically the 26G RNAs 21,47 , also direct production of 22G RNAs 32,48,49 . The Argonautes ALG-3 and ALG-4 act with 26G RNAs in the male germline 32 , while the Argonaute ERGO-1 acts with 26G RNAs found mostly in embryos and oocytes to silence genes in somatic tissues [48][49][50] . No known 26G RNA targets are represented among the highlighted genes in Figure 1. In conclusion, our analysis of published sRNA targets indicate that only PRG-1 dependent, WAGO bound 22G RNAs target transcripts associated with heritable effects of temperature.
Therefore, we sought to determine how well targeting by PRG-1 dependent germline sRNAs predicts heritable temperature effects. We first established a stringent statistical filter (less than one expected false positive based on permutation tests) to define a high confidence set of genes whose transcript levels vary in response to 25uC growth (G2 of Figure 1). We then defined, by consensus among published experiments, a high confidence set of transcripts that are targets of endogenous germline sRNAs. We found that among transcripts with strong evidence for an initial temperature response in G2, those that are likely targets of the PRG-1 pathway are more than ten times more likely than other transcripts to show a heritable effect upon return to 20uC (G3) ( Figure 5).
The presence of 22G RNAs antisense to the temperature-responsive transcripts raises the question of whether the 22G RNAs themselves respond to temperature as well. We used RT-QPCR to measure the abundance of one 22G RNA sequence antisense to each of B0286.1 and K10B3.5 before, during, and for two generations after return to 20uC. We found that the B0286.1 22G RNA levels increase when B0286.1 transcript decreases in response to 25uC, while K10B3.5 22G RNA decreases when K10B3.5 transcript increases in response to 25uC (Figure 6a; abundances vary with generation in N2 data, ANOVA P 5 2 3 10 25 for B0286.1 and 4 3 10 25 for K10B3.5). This would be expected if the changes in transcript levels are partly or wholly due to temperature-induced changes in 22G RNAs, the 22G RNAs acting as short interfering RNAs (siRNAs) to reduce transcript levels. Thus, endogenous silencing of K10B3.5 is stronger at 20uC than at 25u, while endogenous silencing of B0286.1 is stronger at 25u than at 20u.
If the endogenous sRNA silences the corresponding genes, one prediction is that inactivating germline RNAi would increase transcript levels. Among germline RNAi defective mutants, the mut-2(ne298) strain WM30 is unusual in that the worms remain sufficiently fertile at 25u to allow transgenerational experiments. As expected based on sequencing of sRNAs from mut-2(ne298) 40 , compared to wild type (N2), WM30 has reduced levels of the selected sRNAs that target B0286.1 and K10B3.5 (Figure 6a). Comparisons between wild-type and mutator strains are complicated by likely additional unknown mutational differences. Therefore, as a complementary approach, we used RNAi to disrupt germline RNAi in wild-type worms (N2). Specifically, we fed worms E. coli food that expresses mut-16 dsRNA 51 , or as a negative control, food that knocks down the reporter gene GFP and the muscle gene unc-22. In most cases, we saw a substantial increase in transcript levels when germline RNAi is impaired (Figure 6b). We conclude that B0286.1 and K10B3.5 are normally silenced by germline RNAi.
A second prediction is that inactivating germline RNAi would cause the loss of the temperature effect. At least in the case of B0286.1, the absence of germline RNAi nearly eliminates the heritable temperature response (Figure 6b, 2-way ANOVA tests for interaction, P 5 5 3 10 27 in the mut-2 experiment, and P 5 3 3 10 24 in the mut-16 experiment). We conclude that RNAi-defective worms do not respond to temperature in a wild-type manner. This result indicates that the sRNAs are not merely passive observers of transcript changes, but instead play an active role.
Recent work has suggested that siRNAs produced from artificially introduced dsRNA can be passed from parent to offspring in C. elegans 9 , and can persist for up to 3 or 4 generations after dsRNA exposure 7 , in which case siRNAs are presumably regenerated in each generation. In this light, it is interesting to note that B0286.1 22 nt RNA levels remain elevated for at least 1K generations after heat stress is removed -through the adult stage twice at 20u after exposure to 25u (Figure 6a, P 5 0.03, two-tailed Student's T-test for difference with 20u control). It is unknown whether any of this RNA is inherited, or alternately it is entirely regenerated in response to an unknown heritable molecule or mark. Nevertheless, the high levels of endogenous sRNA to specific genes in oocytes (Figure 3a) are suggestive of an inherited signal.
Finally, we note that spermatocytes contain a relatively high level of sRNAs antisense to B0286.1 52 (see Supplementary Table S1), leading us to wonder whether part of the heritable effect passes through the male line. To test this idea, we measured B0286.1 transcript levels in the progeny of 20uC raised hermaphrodites that were themselves the cross progeny of males and hermaphrodites raised at 20uC or 25uC (all four combinations tested; Figure 7). We found that the heritable effect passes almost entirely through the female line.

Discussion
Here we present a proof-of-principle finding that a common environmental stress triggers heritable changes in gene expression in animals. To identify genes that show a persistent change in gene expression in response to environmental change, we measured transcript levels in small populations of precisely staged animals that were transiently exposed to a mild heat stress. These data show that memory of the environmental stress persists as seen in altered transcript levels for two to three generations after return to the previous environmental condition. Extending the stress exposure to two   consecutive generations doubles the magnitude of the altered transcript levels and, for at least one gene, extends by at least a generation the duration of the memory. This indicates that the information persists for multiple generations, and also suggests that the inherited state is not a simple on-off switch (Supplementary Table S3).
We found that genes that show heritable changes in gene expression are normally silenced by endogenous RNAi. The affected transcripts are enriched for genes targeted by 22G germline antisense short RNAs, and at least for the two transcripts chosen for further study, the abundances of 22G RNAs change in the opposite direction of their target mRNAs ( Figure 6). These results imply that endogenous RNAi is important for initiating, maintaining, or expressing the heritable effects. One gene we investigated further is more efficiently silenced at 25uC than at 20uC, while the other is more efficiently silenced at 20u than at 25uC. Additionally, the effect of temperature on transcript levels is missing in RNAi defective worms. Although we analysed only two genes in detail, both categories of increasing and decreasing transcripts ( Figure 1) are targeted by siRNAs (Supplementary Figure S2 and Supplementary Table S1), indicating a specific response to temperature changes rather than a global change in gene silencing efficiency. Indeed, simple numerical models we constructed show that a single mechanism deployed at different temperatures is sufficient to explain inheritance of both increased and decreased transcript (Figure 8; Supplementary Table S3) Consistent with a single mechanism regulating temperature responsive genes that both increase and decrease in abundance, the set of genes is strongly enriched for WAGO-associated 22G sRNA targets. It is remarkable that none of the genes are targets of CSR-1associated sRNA. CSR-1 sRNAs predominantly target abundantly expressed maternal genes, protecting them from silencing by WAGO-dependent processes. We note that starvation induced transcripts do include CSR-1 sRNA targets 33 , although our study used different methods and looked at a different developmental stage and a different stress.
Analysis of our data combined with recent findings in other labs indicates that targets of PRG-1, an Argonaute protein in the Piwi family widely conserved among animal phyla, include the genes that show heritable temperature responses. Identifying the specific 21U RNAs (C. elegans piRNAs) involved is not straightforward because 21U RNAs do not need a perfect sequence match to act as primary siRNAs for secondary 22G RNA production, and individual 21U species are far less abundant than their secondary 22G RNA products. In the future, it will be interesting to see whether the production or maturation of specific 21U RNAs responds to ecologically relevant stimuli.
More generally, the identification of environment-sensitive siRNA expression may serve to identify transcripts sensitized for transgenerational epigenetic reprogramming. Indeed, recent studies have identified starvation-induced and dauer-induced siRNAs 33,53 with starvation being associated with transgenerational effects on targeted transcripts. Because sRNAs can target any arbitrary gene, a potentially large amount of information about environmental conditions can be encoded in sRNA profiles for transmission to future generations.
We also found that the heritable effect passes primarily through the female line. This contrasts with heritable silencing triggered by exogenous RNA in C. elegans 6 and heritable epigenetic effects in mammals 4,54,55 where heritable effects pass either through the male line or through both the female and male lines. However, our finding is consistent with inherited gene silencing triggered by a 21U RNA 16 . In an experimental system described in ref. 16, a heritably silenced locus can impose silencing on a second transgenic reporter, but only by way of the female line and not the male line. Since the two transgenes do not have to be simultaneously present for silencing to be transferred, the authors speculate that a diffusible factor in the oocyte cytoplasm is responsible for the heritable effect 16 .
Although many have been tempted to propose transgenerational inheritance of small RNAs, there is as yet no evidence that abundant secondary siRNAs, themselves the products of RNA directed RNA polymerase activity, can direct further amplification (see for example ref. 56) that can overcome the hundredfold or greater dilution that would occur with each generation. Thus, the precise marks or signals that mediate transgenerational inheritance remain unknown.
Variation in chromatin is a candidate for a heritable mark or signal that may act in concert with RNAi to preserve expression levels. Indeed, work on heterochromatin maintenance at centromeres in the fission yeast Schizosaccharomyces pombe and on heterochromatin-triggered gene silencing in the fruit fly Drosophila, suggests that RNAi-like mechanisms might work closely with chromatin modifications in a positive feedback loop 57,58 . In C. elegans effective RNAi requires heterochromatin-promoting activities, and RNAi triggers the accumulation of histone H3 lysine 9 methylation at silenced loci 5,51,[59][60][61] . Furthermore, manipulating histone modifications can trigger heritable changes in worm longevity 8 , although it is unknown whether this changes the abundances of siRNAs that target genes that affect aging.
Additionally, there is as yet no way to entirely rule out other signals such as transcription factors, prions, trace amounts of DNA methylation, or even hormones and nutrients deposited with the yolk as inherited temperature signals in C. elegans 3 . Thus, much additional work is needed to understand the mechanisms of transgenerational epigenetic inheritance.
Also of interest is whether the signal originates in somatic tissues, which then communicate with the germline 62 . RNAi in C. elegans is systemic; dsRNA introduced locally can spread to cause silencing in cells throughout the animals and in future generations. Thus, the involvement of an RNAi-related mechanism in the inherited response raises the possibility that gene-specific information about variable environmental conditions may be exchanged between somatic cells and the germline. In this regard we note that the effects we describe in this paper are independent of the SID-1 dsRNA channel that is responsible for systemic RNAi (see Methods), but we cannot rule out SID-1 dependent effects that may have gone undetected in this study. Additionally, there are many SID-1 independent ways that a signal could travel between somatic and germline tissues. Also  Table S3). The assumptions for this numerical model are that a new temperaturedependent silencing signal is generated at the silencing temperature and that this signal is maintained at the non-silencing temperature by limited, incomplete amplification resulting in dilution of the signal with each new generation.
www.nature.com/scientificreports SCIENTIFIC REPORTS | 4 : 7387 | DOI: 10.1038/srep07387 pertinent to this question is the observation that RNAi is more potent in the germline than the soma 63,64 , raising the question as to whether the heritable effects are restricted to the germline. In one study of heritable germline silencing of a GFP-expressing transgene in C. elegans, silencing did not escape the germline into somatic tissues 15 , but it is unclear whether the GFP experiment is representative of all piRNA-triggered heritable gene silencing.
Temperature change is a stress that C. elegans likely encounters frequently in the wild; therefore the ability to inherit responses should provide a selective advantage. In this regard it is noteworthy that the transcriptome of 4-cell embryos varies substantially in response to temperature among different geographical isolates of C. elegans 65 , and seven of the transcripts highlighted in Figure 1 show an interaction between environmental conditions and natural genotypic variation 65 . For example, both scrm-4 and K10B3.5 transcripts increase at 25uC in strains from the UK (N2), California (CB4857) Australia (AB2), and Germany (RC301), but in a divergent strain from Hawaii (CB4856), scrm-4 transcript is constitutively high and K10B3.5 transcript is constitutively low 65 . However, the physiological significance of any of the heritable gene expression changes remains unknown. The list of genes (Supplementary Table S1) provides few clues in this regard. If changes in multiple transcripts each contribute a small but significant amount to fitness, it may prove to difficult to demonstrate a selective advantage for expression changes for individual genes: A recent study showed that a majority of genes in C. elegans confer a significant but small fitness advantage in a single environmental condition 66 . It will be interesting to see whether different kinds of environmental stimuli produce distinctly different heritable patterns of gene expression, and whether the gene expression changes result in phenotypes relevant to surviving stresses.
Individuals show transcriptional responses to new environments within minutes, while populations show adaptive genetic change by natural selection over many generations. Mechanisms that enable adaptation to variable or recurring environmental conditions over intermediate timescales are poorly understood. The study of inheritance of acquired characteristics has long been plagued by controversy and irreproducible results (see for example ref. 67). Our work shows it is possible to identify specific, quantitative molecular markers of inheritance, which will facilitate replication. Studying transgenerational inheritance in C. elegans, with its wealth of molecular and genetic tools, will provide a unique perspective to this still largely unexplored territory.

Methods
Worm culture. We used standard methods to culture and handle C. elegans, except we used the nonpathogenic, sporulation-defective Bacillus subtilis strain RL1275 (spoIIAC::erm in a PY79 background, a kind gift from Richard Losick) as worm food for most experiments in order to see temperature effects independently of pathogen responses. B. subtilis is substantially less pathogenic to C. elegans than the standard C. elegans food E. coli strain OP50 at 25uC 68 .
The only experiment presented here that did not use RL1275 as food is the feeding RNAi experiment (right hand half of Figure 6B), where the RNase III defective E. coli strain HT115(DE3) bearing either the Ahringer library clone I-5E09 (L4440 with a mut-16 insert) or the plasmid pPD126.25 (GFP::unc-22 hairpin, lab of Andrew Fire) was used as worm food on NGM plates containing 25 mg/ml carbenicillin and 1 mM IPTG.
Before each of the experiments, we cultured the worms for at least three generations at 20uC without allowing the worms to deplete their food. Other than the 25uC treatments, done in a 25 6 0.5u incubator, all incubations and procedures involving live worms were done in a temperature controlled room at 20 6 0.3uC. We used HOBO miniature temperature loggers (Onset Computer Corporation, Bourne, MA, USA) to continuously monitor culture temperatures. To minimize temperature variation during handling, we used large (10 cm diameter) plates, wore insulated gloves while handling plates, and used an external light source for the dissecting microscope.
For the microarray experiment, we used a platinum wire loop to transfer several hundred embryos to a fresh plate for each and every generation, and used a platinum wire pick to remove any hatched worms that were carried along with the eggs. For the multigenerational follow-up experiments, we used a platinum wire pick to transfer 60 L4 larvae to a fresh plate for each generation. The purpose of transferring L4 larvae instead of embryos in the multigenerational experiment was to eliminate males from the cultures, thereby eliminating variation in the frequency of sex-specific imprinting (most C. elegans individuals are hermaphrodites, with a low rate of spontaneous males that increases with temperature).
All transfers were either to prewarmed 25u plates, or to precooled 20uC plates, as appropriate. For post-25uC steps of all experiments, we interleaved the constant 20uC (control) plates with post-25uC treatment plates in common stacks whenever possible, so that treatment and control plates experience the same microenvironment on average. In practice, the treatment plates were slightly staggered in time relative to the corresponding control plates of the same generation, because of time constraints and because development time from zygote to adult is ,17 hours faster at 25uC than at 20uC. The total time between transfers of eggs or L4 larvae to new plates -the total amount of time per generation -was 4 days for incubation at 20uC, or 3 days for incubation at 25uC.
Embryo collection. For each combination of condition and generation in the microarray experiment, we collected three replicates of the standard C. elegans laboratory strain N2 and three replicates of the strain HC445 (sid-1(qt9) in an N2 background). For all subsequent experiments (with the exception of the WM30 experiment), we used N2 alone.
We collected embryos and purified RNA as described 34 , except we increased the number of embryos per sample to 50. In brief, we picked young adults into water, chopped them in half, treated them 30 seconds with alkaline bleach (final concentration 80 mM KOH, 50 mM NaOCl) to destroy any RNA and protein not protected by the eggshell, stopped the bleach reaction with bovine serum albumin (final concentration 3%), collected and washed early embryos three times in nuclease-free water using a glass mouth pipette, flash-froze the embryos in liquid nitrogen in lowadhesion 600 ml microcentrifuge tubes, and transferred the tubes to a 280uC freezer for storage. For the microarray experiment in Figure 1, the total time from picking of the first worm to bleaching was not allowed to exceed 15 min. For all subsequent experiments, the time was not allowed to exceed 8 min. We added 100 ml Trizol (phenol/guanidine isothiocyanate; Invitrogen Corporation, now part of Life Technologies, Carlsbad, CA, USA), with added linear polyacrylamide (5 mg) and yeast tRNA (100 ng) as carriers, to the frozen embryos for RNA extraction and isopropanol precipitation. We used the RNA pellet directly for RT (reverse transcription).
Microarrays. We used a custom microarray (Agilent Technologies, Santa Clara, CA, USA) bearing 60-mer oligonucleotide spots corresponding to 15,208 C. elegans genes. Since this is fewer than the known number of genes in C. elegans, we limited the genes to predicted protein-coding genes, prioritizing genes with 151 best reciprocal similarity scores to C. briggsae. We selected oligonucleotide sequences corresponding to the 39-most predicted exon that allows design of a probe that minimizes crosshybridization with other genes. Gene positions on the array were randomized to minimize bias due to hybridization artifacts.
For microarray hybridizations, we performed two rounds of linear amplification 34,69 using the MessageAmp II kit (Ambion, now part of Life Technologies, Carlsbad, CA, USA) starting from a T7-oligo-dT RT primer. The following modifications were designed to increase the average lengths of the amplified RNA populations 34 : First-round cDNA construction from total RNA was carried out at 1/5 of the recommended quantities and total volume, while IVT was carried out at 1/2 of the recommended volume. 100 ng amplified RNA from the first round was used as input for the second round of amplification following the manufacturers protocol for amino-allyl modified nucleotides. We labelled 10 mg amplified RNA with Cy3 fluorescent dye using the Ambion MessageAmpII kit and used 1.65 mg labelled amplified RNA to hybridize with each array. To minimize post-amplification biases, we randomized the position and order of hybridizations. Hybridizations with obvious artefacts (a bubble, a fingerprint, or a notably skewed fluorescence profile) were redone. We scanned the arrays at 10% laser power to avoid signal saturation.
For microarray data, we used the Agilent Feature Extraction software GE1-v5_95_Feb07 to calculated fluorescence values. We log 2 transformed the fluorescence values and quantile-normalized 70 them among hybridizations. Normalization may introduce slight biases due to a shift in global transcript abundances at 25uC, but not enough to affect the paper's conclusions. With the exception of the 10% of genes with lowest fluorescence signal, the processed values closely fit a Gaussian distribution for each gene, allowing us to use standard parametric statistics. Supplementary Figure S1 summarizes randomization/permutation tests of the microarray data. Supplementary Figure S3 shows a test of linearity. Supplementary Figure S4 shows that the genes identified in Figure 1 have a random spatial distribution on the physical microarray, indicating that the identification of these genes is not due to common hybridization artefacts.
Initial analysis suggested the microarray data lack the statistical power to conclusively show sid-1 dependent heritable effects; therefore we set aside for the future description of such effects. A portion of the microarray data (three replicates of the first generation but none of the second) has been reported previously 65 .
Detailed microarray data and methods are available in Supplementary Table S2 and the Gene Expression Omnibus (GEO) GSE30666.
Reverse transcription-quantitative PCR. As reference genes for reverse transcription-quantitative PCR (RT-QPCR) we chose atf-6 and cpf-1, two wellcharacterized genes whose hybridization signal varies little in our microarray experiments. We performed Thermoscript RT (Invitrogen Corporation, now part of Life Technologies, Carlsbad, CA, USA) reactions using a mix of gene-specific RT www.nature.com/scientificreports SCIENTIFIC REPORTS | 4 : 7387 | DOI: 10.1038/srep07387 primers (''RT'' listed in Supplementary Table S4). A critical step to ensure proper mixing in setting up RT reactions was to mix by stirring with a pipette tip, monitored under a microscope. In order to get reliable amplification from 50-embryo samples, we needed to perform nested PCR. We amplified the cDNA in a multiplex PCR reaction containing ''RT'' and ''outer'' primers (listed in Supplementary Table S4) for 19 cycles in the presence of SYBR Green to monitor amplification. We diluted this first PCR product 1250-fold and divided it as template for separate QPCR reactions (Quantitect; Qiagen N.V., Hilden, Germany) for each gene (''QF'' and ''QR'' primers, Supplementary Table S4).
We calculated relative mRNA levels from cycle thresholds as . However, like with the microarray data, we calculated means and other statistics on logarithmic-scale instead of linear-scale data. Serial dilution experiments show that the RT-QPCR methods we used yield reasonably linear results (Supplementary Figure S5).
We focused primarily on collecting samples from the second generation after return to 20u and the corresponding generation of the constant 20u control. Plates with visible contamination or excessive mineral precipitate were discarded without sampling. Occasional QPCR reactions (,2%) failed to yield fluorescence within 25 or more cycles in the second PCR and were treated as missing data.
We used custom TaqMan (Applied Biosystems, now part of Life Technologies, Carlsbad, CA, USA) RT-QPCR assays to measure abundances of short RNAs in total nucleic acid extracted from 1.5 day old adult worms. Nucleic acid was prepared by proteinase K digestion for 10 minutes at 65u, followed by two rounds of phenolchloroform extraction, using silicone grease (vacuum grease; Dow Corning Corporation, Midland, MI, USA) to separate the aqueous phase from the organic phase, and then sodium acetate-isopropanol precipitation (based on a protocol suggested by Weifeng Gu). We performed the TaqMan reverse transcriptions at 1/3 the scale of the manufacturer's protocol, but used 13.36 ng nucleic acid (as measured by absorbance at 260 nm) as template for each reaction. Estimates are 2 2C T.