DNMT3.1 controls trade-offs between growth, reproduction, and life span under starved conditions in Daphnia magna

The cladoceran crustacean Daphnia has long been a model of energy allocation studies due to its important position in the trophic cascade of freshwater ecosystems. However, the loci for controlling energy allocation between life history traits still remain unknown. Here, we report CRISPR/Cas-mediated target mutagenesis of DNA methyltransferase 3.1 (DNMT3.1) that is upregulated in response to caloric restriction in Daphnia magna. The resulting biallelic mutant is viable and did not show any change in growth rate, reproduction, and longevity under nutrient rich conditions. In contrast, under starved conditions, the growth rate of this DNMT3.1 mutant was increased but its reproduction was reciprocally reduced compared to the wild type when the growth and reproduction activities competed during a period from instar 4 to 8. The life span of this mutant was significantly shorter than that of the wild type. We also compared transcriptomes between DNMT3.1 mutant and wild type under nutrient-rich and starved conditions. Consistent with the DNMT3.1 mutant phenotypes, the starved condition led to changes in the transcriptomes of the mutant including differential expression of vitellogenin genes. In addition, we found upregulation of the I am not dead yet (INDY) ortholog, which has been known to shorten the life span in Drosophila, explaining the shorter life span of the DNMT3.1 mutant. These results establish DNMT3.1 as a key regulator for life span and energy allocation between growth and reproduction during caloric restriction. Our findings reveal how energy allocation is implemented by selective expression of a DNMT3 ortholog that is widely distributed among animals. We also infer a previously unidentified adaptation of Daphnia that invests more energy for reproduction than growth under starved conditions.


Results
manga, we attempted to mutate this gene using the CRISPR/Cas system. As with the DNMT3L in mammals, DNMT3.1 lacks five out of six characteristic motifs of the MTase domain in catalytically active DNMT3 proteins except motif VI (Fig. 1a) 43 . gRNA was designed to bind upstream of the motif VI because the diverged MTase domain of DNMT3.1 may interact with another D. manga DNMT3 ortholog DNMT3.2 harboring all of the six motifs for de novo methylation as well as the interaction between DNMT3L and DNMT3A in mammals 44 . In addition, truncation of the C-terminus of DNMT3A/B led to a significant decrease of their protein levels in human embryonic stem cells 45 . Between this gRNA and the other D. magna DNMT genes, there are more than 6 base pair mismatches ( Supplementary Fig. S2). This specificity of the gRNA to DNMT3.1 could avoid off-target effects to the other DNMT genes because the DNA region with up to five base pair mismatches with gRNA is susceptible to editing by Cas9/gRNA complex 46,47 . We injected Cas9 ribonucleoproteins (RNPs) comprising the purified Cas9 proteins and targeting gRNA into 48 parthenogenetic female eggs, of which 15 survived until the adult stage. G2 progenies of these potential founder lines were used for genotyping. We cloned and sequenced genomic PCR products encompassing the gRNA-targeted site to find mutant lines. Of the 15, we found one founder animal that produced offspring harboring biallelic indel mutations around the targeted site (Fig. 1b). The biallelic indel mutations of this line led to frameshifts, resulting in premature STOP codons occurring in both alleles (Fig. 1b). We named this mutant line DNMT3.1 −/− and used it for phenotyping and transcriptional analysis.

DNMT3.1 mutation does not change growth and fecundity under nutrient rich conditions.
We investigated the phenotypes of the mutant line under nutrient rich conditions (5.12 × 10 7 Chlorella cells/daphnid). We cultured the wild type and DNMT3.1 −/− for 14 days and divided this culturing period into two phases, (1) growth phase (0-4 day) and (2) growth and reproduction phase (4-14 day) where growth and reproduction compete for energy from the food. The growth rate was measured in each phase and the number of offspring was counted in each clutch. In the growth phase, the growth rate of DNMT3.1 −/− was similar to that of wild type (P = 0.14) (Fig. 2a and Table 1). In the growth and reproduction phase, there was also no significant difference in the growth rate (P = 0.2) in addition to the number of offspring at each clutch or their cumulative clutch size (P = 0.7) between wild type and mutant lines (Fig. 2b, c and Table 1).

DNMT3.1 controls trade-off between growth and fecundity under starved conditions. Because
we previously had found upregulation of DNMT3.1 expression in response to caloric restriction 43 , we compared phenotypes between wild type and DNMT3.1 −/− mutant under starved conditions where the number of Chlorella was reduced eightfold compared to that in nutrient rich conditions. We first examined the effects of starvation on growth rate and reproduction in wild type daphnids. In the growth phase, the growth rate was reduced compared to nutrient rich condition (P = 7.575e−06) ( Supplementary Fig. S3a). In the growth and reproduction phase, the growth rate of starved daphnids was higher than that of well-fed daphnids (P = 1.167e−07). The starved daphnids produced a smaller number of eggs since the second clutch ( Supplementary Fig. S3b, c). This reduction of the food did not lead to a significant difference in the timing of molting between nutrient rich and starved conditions. We next investigated phenotypes of DNMT3.1 −/− mutants. There was no difference in growth (P = 0.12) between the wild type and the mutant in the growth phase ( Fig. 3a  www.nature.com/scientificreports/ reproduction phase, the growth rate of mutant daphnids (0.082 mm/day) was higher than that of wild type (0.051 mm/day) (P = 2.5E−10) ( Fig. 3a and Table 1). In contrast, mutants produced a smaller number of offspring at the second and third clutches compared to the wild type (P = 1.6E−06 and 8.7E−09, respectively) ( Fig. 3b and Table 1). These phenotypic differences in this phase motivated us to extend observation until 22 days-old for investigations of the phenotypes. We named this third period the reproduction phase because the growth rate in this phase becomes much lower than that in the earlier two phases. In the reproduction phase, we observed no differences in growth rate and clutch size between wild types and mutants ( Fig. 3 and Table 1). The timing of molting did not differ between wild type and DNMT3.1 −/− mutants.

DNMT3.1 mutation reduces life span under starved conditions.
We compared the life span of the DNMT3.1 mutant to that of the wild-type. In the nutrient-rich condition, wild type and DNMT3.1 −/− lines showed similar longevity (log-rank p = 0.9), with a median lifespan of 54 and 56 days, respectively (Fig. 4). In the  Table S1). Upregulation of DNMT3.1 expression in response to the starved condition was also confirmed both in wild type and DNMT3.1 −/− (Supplementary Fig. S1).
To examine how many genes were affected at mRNA level by the DNMT3.1 mutation, we performed RNA-Seq analysis to find differentially expressed genes (DEGs) between wild type and mutant lines in the second phase, growth and reproduction phase. In nutrient rich and starved conditions, numbers of differentially  Table 1. Growth rate and clutch size of D. magna under nutrient rich and starved condition. Values are mean ± SD; n = 15 in the nutrient rich and n = 20 in the starved. nr: not related. Significant differences between wild type versus DNMT3.1 −/− , were indicated by asterisks via pairwise t-test; ***P < 0.001.

Condition
Age ( www.nature.com/scientificreports/   www.nature.com/scientificreports/ expressed genes were 220 and 2770 respectively, demonstrating that, in the starved condition, disruption of DNMT3.1 showed a much larger effect on the transcriptome. Amongst the DEGs in starved animals, 1432 annotated genes were identified and we performed clustering of these by k-Means. 1364 genes passed the filter (as described in Materials and Methods) and were divided into five clusters (Fig. 5, Supplementary File S1). Furthermore, we analyzed the gene ontology (GO) for functional annotation of genes in each cluster by using Fisher's Exact Test (Supplementary File S2). Clusters A and B included genes showing higher expression in the mutant under starved conditions. The top enriched GOs in clusters A and B were mitotic cell cycle and transmembrane transport respectively (Table 2). In cluster A, we found a notable gene, Nuclear hormone receptor ftz-f1 (Dapma7bEVm011018t1) (ftz-f1), that showed an upregulation in the mutant under starved conditions (Table 2). Interestingly, in cluster B, we found a gene that codes for an ortholog of solute carrier family 13 member 5 (Dapma7bEVm010715t1), known as Slc13a5 or INDY (I'm not dead yet). The Daphnia INDY ortholog showed reduced expression in the wild type but upregulated expression in the mutant under starvation (Table 2 and Supplementary File S1). Genes in Cluster C showed down-regulation in both wild type and mutant under the starved condition and were more severely down-regulated in the mutant (Fig. 5). Importantly, vitellogenin genes, vtg1 (Dapma7bEVm024402t1), and 7 other genes related to lipid transport were included in this cluster (Supplementary File S1). Cluster D represented down-regulated genes in the mutant under starved conditions, many of which are involved in carbohydrate metabolic processes ( Fig. 5 and Table 2). We found a represented gene, target of brain insulin (Dapma7bEVm003111t1) known as tobi, an α-glucosidase. Finally, genes in cluster E were upregulated in the wild type under starved conditions, which included genes related to protein turnover (proteolysis) (Fig. 5 and Table 2). A represented gene of this GO was trypsin (Dapma7bEVm010425t1) known as epsilonTry. www.nature.com/scientificreports/

Discussion
Despite the long history of research on energy allocation, genes affecting energy allocation still remain largely unknown. In the cladoceran crustacean D. magna, energy allocation among competing life history traits under starved conditions has been extensively investigated. We have previously shown that Daphnia DNMT3.1 was upregulated in response to starvation, suggesting a potential function of this gene for energy allocation. In this study, we introduced a mutation into DNMT3.1 and analyzed phenotypes of its mutant under caloric restriction. We demonstrate that this gene is a key regulator for life span and energy allocation between growth and reproduction.

DNMT3.1 controls the trade-off between growth and reproduction under starved conditions.
The trade-off between growth and reproduction occurred in both wild-type and DNMT3.1 mutants under different food levels, which is in line with previous reports 25 . Interestingly, the DNMT3.1 mutant showed a higher growth rate and lower clutch size compared to wild type when both processes competed during a period from instar 4 to 8, indicating that DNMT3.1 allocates more energy to reproduction. These results suggest that DNMT3.1 controls the trade-off between growth and reproduction when growth and fecundity compete for energy from the food. Larger clutch size has a positive impact on expanding its population size, which in turn would lead to an increase in fitness. The correlation of DNMT3.1 gene activity with phenotypes in the natural population needs to be examined to further understanding of the functions of this gene in an ecological context.  52 In addition, catalytically inactive DNMT3B has been demonstrated to rescue the embryonic lethal phenotype of DNMT3B knockout mice 53 , suggesting that DNMT3B has a function independent of DNA methylation. www.nature.com/scientificreports/ Taken together, the DNMT3.1 mutant lacking a typical MTase domain and activity has lost the control of a large amount of genes differentially expressed under the starved conditions. Among the potential DNMT3.1 target genes under starved conditions, we listed five notable genes including INDY (Table 2). ftz-f1, a competence factor for juvenile hormone (JH) activation, has essential roles in developmental regulation and various aspects of insect adult life 54,55 . Vtg1 encodes vitellogenin, a precursor of a major yolk protein 56 , and has been used as an indicator of fecundity 42,57 . The downregulation of Vtg genes was consistent with the severe decrease of fecundity in mutants in response to the starved condition. trypsin encodes digestive protease in the gut of D. magna 58 and has been listed as involved in starvation resistance gene in flies 59 .

DNMT3.1 increases life span under starved conditions.
Our study demonstrates the physiological function of DNMT3.1 in extending longevity and energy allocation to reproduction under starved conditions. Although DNMT3.1 lacks an evolutionarily conserved sequence of the MTase domain, it changes the global transcriptome in response to starvation. In the future, the gene regulatory mechanisms can be investigated not only by investigating DNA methylation but also by chromatin immunoprecipitation for the identification of direct targets of this protein. We anticipate that this work will contribute to understanding the molecular mechanisms underlying energy allocation in the ecologically important Daphnia species. CRISPR/Cas9-mediated mutagenesis. Impairment of the DNMT3.1 gene was done with CRISPR/Cas9 technology by introducing a frameshift and premature STOP codon as described previously 28 . For the preparation of the DNMT3.1-targeting Cas9 RNPs, we purified Cas9 proteins as described elsewhere 61 . For the synthesis of the gRNA, DNA templates with the T7 promoter and target site (5′-GGA AGA GGT GTA CGA ACT CAAtgg-3′, protospacer adjacent motif shown in lowercase), was amplified by PCR 28 and purified by phenol/chloroform extraction. These DNA fragments were used as templates for in vitro transcription with mMessage mMachine T7 Kit (Life Technologies, California, USA), followed by column purification with miniQuick Spin RNA columns (Roche diagnostics GmbH, Mannheim, Germany), phenol/chloroform extraction, ethanol precipitation, and reconstitution in DNase/RNase-free water (Life Technologies, California, USA). We incubated 2 μM gRNA with 1 μM Cas9 protein to generate Cas9 RNPs and injected them into parthenogenetic female eggs according to established procedures 62 . Accordingly, the eggs were collected from 2-3 weeks daphnids after ovulation and placed in an ice-chilled M4 medium containing 80 mM sucrose (M4-sucrose). Approximately 0.2 nL volume of the generated Cas9 RNPs were injected by using N 2 gas pressure through a glass needle. After injection, to investigate the Cas9-induced mutations, G2 offspring were homogenized in 90 μL of 50 mM NaOH with zirconia beads. The lysate was heated at 95 °C for 10 min and then neutralized with 10 μl of 1 mM Tris-HCl (pH 7.5). This crude DNA extract was centrifuged at 12,000 rpm for 5 min and then used as a template in genomic PCR. The targeted genomic regions in the DNMT3.1 locus were amplified by PCR with Ex Taq Hot Start Version (Takara, Japan) and the following primers; DNMT3.1-U-gDNA 5′-TCC GGG TCG TGG TAC TCC -3′ and DNMT3.1-D-gDNA 5′-AGA CAA GAA ACG AGC AGG TGA ATA G-3′. The PCR products were analyzed by polyacrylamide gel electrophoresis and DNA sequencing.

Culture of D. magna under nutrient rich and starved conditions.
Before culturing under starved conditions, in order to control for maternal effects, 40 Daphnia of each line were cultured in 2.5 l ADaM medium and daily fed with 1.5 × 10 9 cells ml −1 Chlorella algae for at least 3 generations. Neonates from the third clutch were randomly assigned, transferred individually to a 50 ml conical tube containing 40 ml ADaM medium, and subjected to nutrient rich or starved treatments. Under the nutrient rich condition, 5.12 × 10 7 cells were given to each daphnid daily. For starvation, daphnids were fed with the eightfold lower amounts of the algae. During culture, the medium was changed every day in order to avoid carbon (detrital) accumulation that could differentially affect resource availability. For life-history traits observation, 15 and 20 neonates were randomly collected from culture under nutrient rich and starved conditions, respectively. Under the nutrient rich condition, the body length of daphnids was measured on days 0, 4, 7, and 14. The clutch size was recorded by counting the number of offspring produced from the 1st to 3rd clutch. During culturing, due to handling, one wild type and one mutant were killed. Therefore, we recorded the life span of 14 individuals of wild types and mutants. For starvation experiments, the body length of daphnids was measured at the two more time points, day 18 and day 22, in addition to day 0, 4, 7, and 14. Clutch size was recorded from 1st to 5th clutch. In this treatment, we could record the life span of 20 individuals both from wild types and mutants. For expression analysis by quantitative real-time PCR and RNA-seq, 5 daphnids were also cultured individually in each condition until producing 2nd clutch (day 12). Eggs were removed before homogenization for RNA extraction. Each treatment was repeated 3 times for triplicates of sampling.

Body length and growth rate analysis.
To examine the effect of starvation on D. magna growth, we measured body length from the center of the eye to the base of the tail 63  www.nature.com/scientificreports/ software (http:// rsb. info. nih. gov/ ij/). The growth rates of juvenile and adult daphnids were determined by using their body length on day 0-day 4 and day 4-day 14, respectively, and calculated as according to a previous study, where L 1 and L 0 are the final and the initial body lengths, respectively, and t is the time in days from the first to final observation 64 .
RNA extraction and cDNA synthesis.. To extract total RNA, 5 female adult daphnids were collected and briefly washed. Homogenization was performed with beads using a Micro Smash machine MS-100 (TOMY; Tokyo, Japan) in the presence of Sepasol-RNA I reagent (Nacalai Tesque Inc.; Kyoto, Japan). Total RNA was isolated according to the manufacturer's protocol, which was followed by phenol/chloroform extraction. One μg of purified total RNA was converted into the first-strand cDNA with PrimeScript II Reverse Transcriptase (Takara, Japan) and random primers (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's recommended protocol. RNA-seq data analysis. Sequencing data from 9 libraries of the above samples were analyzed using CLC Genomics Workbench 12 (Qiagen) (accession number: GSE158129). The other 3 libraries of the triplicates from wild type in nutrient rich condition (accession number: GSE150821) had been prepared and sequenced elsewhere by the same methods including Daphnia culturing condition as in this study 67 . The Daphnia magna genome that is available from wFleaBase.org 66 was used as a reference for mapping the reads. Mapping options were set at mismatch cost 2, insertion cost 3, depletion cost 3, length fraction 0.8, similarity fraction 0.8, expression value set to total counts. Differential expression analysis was performed with the 'Transcriptomics Analysis' toolbox, and comprised 'experimental set-up' , where treatments pairs were analyzed with the option ' All group pairs' . This setting uses the Wald test and reports the expression mean of each gene with a fold change between the treatment pair. Expression values were normalized using the options 'by totals' and 'state numbers in read 1,000,000′. The normalized values were transformed using the "Add a Constant" set at the value '1′. In order to identify the differential expressed genes (DEGs) between a pair of treatments, Anova and Likelihood ratio test were performed on the transformed values for each mapped gene, and DEGs were filtered based on false discovery rate (FDR) p-value cutoff FDR P < 0.05 and fold change cutoff FC ≥ |1.5|. k-Means clustering was performed by iDEP(v.9) 68 . A list of gene ID of DEGs merged counts was uploaded to iDEP and parameters for analysis were set following a previous study 69 . Accordingly, counts were filtered out by criteria of at least 0.5 counts per million in one of the samples and transformed by VST (variance stabilizing transform). Heatmap was visualized by blue-white-red color scheme and k-Means was clustered for all of the filtered genes by choosing 5 clusters. The gene ID from the Daphnia magna genome was annotated by NCBI homologs using Blast2GO features within OmicsBox 1.2.4 70 . The annotated genes in each cluster were examined for the enrichment of GO terms within the subsets of the assembled sequences via Fisher's Exact Test executed within OmicsBox 1.2.4 71 .
Statistics. Statistical analyses were conducted using statistical program R version 3.2.5 72 . Growth rate, clutch size, and median lifespan were analyzed by pairwise t-test and Student's t-test with Welch's correction. Fitted von Bertalanffy growth was performed by the "FSA" R package 73 . The boxplots were generated by "ggplot2" R package 74 . Kaplan Meier survival curves and log-rank test analysis were performed by the "survival" R package 75 . Two-way ANOVA and Fisher's LSD test were performed by "agricolae" R package 76 . The level of significance was P < 0.05.