Proteogenomic Analyses Revealed Favorable Metabolism Pattern

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Bioactive Constituents, Metabolites, and Functions

Proteogenomic analyses revealed favorable metabolism pattern alterations in rotifer Brachionus plicatilis fed with Selenium-rich Chlorella Xian Sun, Yizhi Cui, Qing Wang, Shengquan Tang, Xin Cao, Hongtian Luo, Zhili He, Xiaonong Hu, Xiangping Nie, Yufeng Yang, and Tong Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00139 • Publication Date (Web): 06 Jun 2018 Downloaded from http://pubs.acs.org on June 7, 2018

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Proteogenomic analyses revealed favorable metabolism pattern alterations in rotifer Brachionus plicatilis fed with Selenium-rich Chlorella Xian Sun,†1 Yizhi Cui,§1 Qing Wang,† Shengquan Tang,§ Xin Cao,§ Hongtian Luo,† Zhili He,¶ Xiaonong Hu, † Xiangping Nie,†* Yufeng Yang†* and Tong Wang §*

† Institute of Hydrobiology and Key Laboratory of Aquatic Eutrophication and Control of Harmful Algal Blooms, Guangdong Higher Education Institutes § Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes and Institute of Life and Health Engineering, Jinan University, Guangzhou 510632, China. ¶ School of Environmental Science and Engineering, Sun Yat-Sen University, Guangzhou 510275, China. 1 These authors contributed equally to this work.

Corresponding authors: Yufeng Yang, Fax:+86-20-85221397, Email: [email protected]; Tong Wang, Fax: +86-20-85222616, Email: [email protected]; Xiangping Nie, Fax: +86-20-85222616, Email: [email protected].

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Abstract

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Organoselenium have garnered attention because of their potential to be used as ingredients in new

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anti-aging and antioxidation medicines and food. Rotifers are frequently used as a model organism for

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ageing research. In this study, we used Se-enriched Chlorella (Se-Chlorella), a novel organoselenium

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compound, to feed Brachionus plicatilis to establish a rotifer model with a prolonged lifespan. The

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results showed that the antioxidative effect in Se-enriched rotifer was associated with an increase in

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guaiacol peroxidase (GPX) and catalase (CAT). Authors then performed the first proteogenomic

8

analysis of rotifers to understand their possible metabolic mechanisms. With the de novo assembly of

9

RNA-Seq reads as the reference, we mapped the proteomic output generated by iTRAQ-based mass

10

spectrometry. We found that the differentially expressed proteins were primarily involved in

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anti-reactive oxygen species (ROS) and anti-lipid peroxidation (LPO), selenocompound metabolism,

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glycolysis, and amino acid metabolisms. Furthermore, the ROS level of rotifers was diminished after

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Se-Chlorella feeding, indicating that Se-Chlorella could help rotifers to enhance their amino acid

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metabolism and shift energy generating metabolism from tricarboxylic acid cycle to glycolysis, which

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leads to reduce ROS production. This is the first report to demonstrate the anti-aging effect of

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Se-Chlorella on rotifers and to provide a possible mechanism for this activity. Thus, Se-Chlorella is a

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promising novel organoselenium compound with potential to prolong human lifespans.

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Key words: Rotifers, Ageing, Proteogenomics, Organoselenium, Anti-reactive oxygen species

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Introduction

Rotifers (e.g. Brachionus spp.) are a linker in the pelagic food chain among bacterioplankton,

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phytoplankton and higher trophic level organisms. While they are often used as the first-feed for the

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larvae of many commercially produced marine fish species, rotifers have been shown to improve the

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water quality in a biofloc system1, 2. Recently, rotifers have been deemed as a good model to study

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ageing, primarily owning to their short life cycles (a few days to several weeks), and are easy to grow1,

27

3

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In this field, rotifer ageing mechanisms have been associated with insulin-like growth factor signaling

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pathway4, 5, restricted calorie intake6, glycerol uses7 and mammalian target of rapamycin (TOR) and

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c-Jun N-terminal kinase (JNK) pathways8.

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. Such features make rotifers potentially useful organism-based models for anti-ageing drug testing.

It has recently been demonstrated that Se-enriched Chlorella vularis can effectively promote the

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reproduction of the rotifer B. plicatilis 9. For ageing studies, selenium (Se) is one of the most

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intensively investigated metals despite with contradictory roles. For example, a Se-enriched rotifer

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was shown to benefit the growth and development of red sea bream Pagrus major larvae, a bottom-up

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profit10; however, excessive Se in waters has also been associated with top-predator removal, which

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makes Se an ecotoxin11, 12. For humans, Se has been found to have anti-cancer, anti-ageing and

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anti-oxidation activities13. This conservative anti-aging effect of Se from rotifers to humans leads to

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an interesting question that whether rotifers share similar anti-ageing mechanisms with humans,

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which requires genome-wide analyses.

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However, only a few rotifer species have been sequenced with their whole genome and transcriptome information14, 15, and even for those sequenced rotifers, no proteomics analysis has been 3


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published to date, especially regarding the ageing model of rotifers. Since proteins are considered as

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the endpoint determinants of phenotypes, this makes transcriptomic and/or proteogenomic analysis of

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rotifers a fundamental requirement to move the field forward. As one of the most widely distributed

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rotifers in salt lakes around the world, B. plicatilis has been deemed as a model system in ecology and

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evolution studies. In addition, it is the only industrialized food for feeding fish larvae, while its whole

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genome has not been sequenced yet.

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In this study, we aimed to understand ageing mechanisms of B. plicatilis fed with

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selenium-enriched Chlorella (Se-Chlorella) using proteogenomic approaches. Our previous study has

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shown the green alga Chlorella is tolerant to Se with increased anti-oxidant enzyme production16. We

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hypothesized that Se-Chlorella would increase the life span of rotifers B. plicatilis with similar

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mechanisms to humans with the reactive oxygen species (ROS) level. To address this hypothesis, we

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report here the first proteogenomic evidence on the Se feeding-mediated energy metabolism pattern

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polarization toward glycolysis to avoid ROS injury, favorable to the rotifer’s survival and

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reproduction. This study is not only useful for understanding human ageing mechanisms, but also

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systematically justifies the potential of using B. plicatilis for screening anti-ageing drugs to prolong

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human lifespans.

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Materials and methods

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Rotifer culture. B. plicatilis was originally collected from the South China Sea and had been

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continuously propagated in the laboratory since 2012. The clonal culture was subjected to periodic

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resting egg production, collection, and storage. Rotifers used in this study were hatched from a resting

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egg batch produced on March 1, 2013. After hatching in 25 mL of EPA medium supplemented with

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10 g/L NaCl, incubated overnight (22–24 h) under constant fluorescent illumination (2000 lux) at

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25 °C, a clonal culture was established using the clonal culture of the green alga Chlorella

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pyrenoidosa as food and maintained in reconstituted hard water EPA medium17, plemented with 10

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g/L NaCl. For feeding, algae at log phase were harvested by centrifugation at 3,000 rpm for 10 min,

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and resuspended in distilled water, and the density of Chlorella was estimated with a haemocytometer

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as reported previously16.

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Se uptake and intracellular organic Se concentration determination in Chlorella. According

71

to our previous findings 16, the C. pyrenoidosa culture was treated by sodium selenite (Na2SeO3) at 75

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mg/L to reach optimum intracellular organic Se loading and conversion. The inductively coupled

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plasma – mass spectrometer (ICP-MS) method was used to determine the Se concentration as we

74

previously described 16. For a control, triplicated cultures were set up essentially the same manner as

75

the Se enrichment experiment, except that the algal prey was grown without Se.

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Spawning rate assay. From both the Se-Chlorella and no-Se Chlorella fed culture groups,

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rotifers were observed for every 8 h under a dissecting scope and eggs were collected and counted.

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The procedure was repeated until all rotifers deceased.

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Deep sequencing of mRNA and transcriptome assembly. For transcriptomic analysis, samples

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were collected. The RNA-Seq for transcriptome analyses was performed as described previously 18, 19.

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In brief, total RNA was isolated by using TRIzol reagent (ThermoFisher Scientific, Guangzhou,

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China), which were subjected to mRNA capture, libraries construction, next generation sequencing 5


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(NGS) and transcriptome de novo assembly. The RNA-seq on mRNAs was performed using a

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HiSeq-2000 sequencer (Illumina, San Diego, California). After quality filtering, high quality reads

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were de novo assembled using the short reads assembling program – Trinity (release-20130225) 20,

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with parameters --seqType fq --min_contig_length 100 --min_glue 3 --group_pairs_distance 250

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--path_reinforcement_distance 85 --min_kmer_cov 3. Fragments Per Kilobase of transcript per

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Million mapped reads (FPKM) were used for gene expression quantification. All sequencing datasets

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of B. plicatilis are publically available at Sequence Read Archive (accession number: SRP070933).

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Total protein extraction from rotifers. Rotifers were sieved with a 60 µm plankton net, washed

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with artificial sea water (ASW), and resuspended in 2 mL of ASW with 2 mL tubes. To remove the

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alga, tubes were centrifuged at 5,000 × g for 10 min, and supernatant (~1.8 mL) were transferred into

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new tubes. According to Denekamp et al. 21, ethanol was added to a final concentration 10% to

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demobilize rotifers, which allowed them to be pelleted by another brief centrifugation (5,000 × g for

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10 min). After removing the supernatant, rotifer pellets were resuspended with ~100 µL ASW and

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transferred to 300 kDa MWCO Nanosep ultracentrifuge tubes (Pall Life Sciences, Ann Arbor,

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Michigan). With an ultracentrifugation at 13,000 × g, rotifers were purified and the wet weight was

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measured. The rotifers were lysed on membrane by 100 µL T-PER tissue protein extraction reagent

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(ThermoFisher), followed by the liquid nitrogen grinding. The lysate was centrifuged for 15 min

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(17,000 × g, 4 °C) to pellet the debris, and protein in the supernatant was collected and quantified.

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Protein samples were prepared from three independently biological replicates.

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Isobaric tagging with iTRAQ and mass spectrometry. A) Experimental Design and Statistical Rationale – To determine the significantly and differentially expressed proteins (DEPs), three 6



104

independent experiments were performed. The iTRAQ 113, 117 and 119 were used to label the

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untreated group, while the iTRAQ 114, 116 and 121 were employed for the Se-Chlorella group

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(Supplementary Figure S1).

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B) The iTRAQ labeling and MS - Protein samples were digested in-solution with trypsin as we

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previously described22. The resulting peptides were labeled by using an 8-plex iTRAQ kit

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(ThermoFisher) following the manufacturer’s instructions. Labeled peptides were fractionated by

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strong cationic exchange chromatography using the Shimadzu LC-20AB HPLC Pump system. MS

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analyses were performed with a Triple TOF 5600 System (AB SCIEX, Guangzhou, China) as we

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previously described 22, 23 with minor modifications. MS parameters: ion spray voltage, 2.5 kV; curtain

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gas, 30 PSI; nebulizer gas, 15 PSI; interface heater temperature, 150 °C; resolution, >30 k fwhm;

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charge state, 2+ ~ 5+; total cycle time, 3.3 s; pulser frequency value, 11 kHz; detector monitoring

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frequency value, 40 GHz; sweeping collision energy, 35 ± 5 eV; dynamic exclusion, less than twice

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per 1/2 of peak width (~15 s).

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Database construction, search and protein annotation. We used the de novo assembly of

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transcriptome to construct the reference database for iTRAQ proteomic data searches. The

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transcriptome sequences were theoretically three-frame translated by using the nt2aa function in

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MATLAB R2014a (MathWorks, Natick, Massachusetts), which was integrated into reference database

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in fasta format for MS searches. Database searching and protein quantification were performed by

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employing the Mascot (version 2.5.0) software as we previously described22, 23 with minor

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modifications. Searching parameters were used as follows: enzyme, trypsin; fixed modification,

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carbamidomethyl (C); variable modifications, Gln → pyro-Glu (N-term Q), Oxidation (M); 7


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quantitation, iTRAQ 8plex; fragment ion mass tolerance, 0.05 Da; parent ion tolerance, 15 ppm.

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Finally, we had set the peptide level FDR < 1%; missed cleavage allowed, 2. All MS raw data are

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publicly available in the iProX database (accession number: IPX00073600). To obtain functional

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annotations of identified proteins, protein coding genes were blasted against the NCBI non-redundant

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(nr) protein database. Then GO and KEGG annotation and pathway analysis were performed. All

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annotation and analysis mentioned above were performed by Blast2GO24. All peptide and protein

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identification information could be found in Table S1.

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Reactive oxygen species assay. ROS was measured from both Se-enriched and no-Se cultures of

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rotifer. We used carboxylated H2DCFDA (CDCFDA, ThermoFisher) to determine the intracellular

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ROS concentrations as reported by Kim et al.25. with minor modifications. In brief, 18 rotifers were

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kept in fresh ASW for 60 min to evacuate their guts, followed by the 60 min incubation in 0.5 mL of

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ASW with CDCFDA (50µM) in a 24-well plate in dark. Rotifers were then rinsed with ASW,

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anesthetized with 0.5 mL of club soda, and fixed with 10 µL of 20% formalin. Fluorescence was

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determined by using a LS-5 spectrofluorometer (PerkinElmer, Shanghai, China) with the excitation

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wavelength of 485 nm and the emission wavelength of 535 nm. Fluorescence values were obtained by

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using a standard curve of CDCFDA.

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Antioxidant enzymes and lipid peroxidation assay. The B. plicatilis were resuspended in

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pre-chilled phosphate buffer (pH 7.0) and disrupted by ultrasonication. The homogenate was

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centrifuged at 10,000 g for 30 min and the supernatant was collected. The total protein content in the

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supernatant of homogenate extracts was determined according to the Bradford method, using bovine

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serum albumin as a standard. 8



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The activities of antioxidant enzymes and level of lipid peroxidation in the protein extract were

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examined using spectrophotometric diagnostic kit (NanJing JianCheng Bio Inst, Nanjing, China).

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Basically, superoxide dismutase (SOD) activity was determined using the xanthine oxidase method,

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based on its ability to inhibit the oxidation of hydroxylamine by the xanthine–xanthine oxidase system.

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Catalase (CAT) activity was measured according to the ammonium molybdate spectrophotometric

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method, based on the fact that ammonium molybdate could rapidly terminate the H2O2 degradation

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reaction catalysed by CAT and react with the residual H2O2 to generate a yellow complex which could

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be monitored by the absorbance at 405 nm. Guaiacol-dependent peroxidase (GPX) activity was

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measured by quantifying the rate of H2O2-induced oxidation of GSH to oxidised glutathione (GSSG),

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catalysed by GPX. The GPX content in the supernatant was measured by reaction with

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dithionitrobenzoic acid (DTNB) and monitored by absorbance at 412 nm.

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The level of lipid peroxidation (LPO) was estimated by malondialdehyde (MDA) levels, which

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were measured using the spectrophotometric diagnostic kits (Nanjing Jiancheng Biotechnology

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Institute, China).

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Statistics. Jarque-Bera test was employed for the data normality test. Cluster analysis, the

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statistics of Jarque-Bera test and data distribution were calculated by using MATLAB R2014a

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software package (MathWorks). In the survival analyses, the Mantel-Cox test and

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Gehan-Breslow-Wilcoxon test were employed for Kaplan-Meier curve comparison assisted by the

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Prism software (version 6.01, GraphPad Software, La Jolla, California). The PLGEM was used to

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detect differentially abundant proteins26, 27, with the Bioconductor package ‘plgem’ run in the R

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version 3.2.2 software environment. Data that passed the normality test were shown as mean ±

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standard deviation (SD). Statistical difference was accepted when p < 0.01.

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Results

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Se-enriched Chlorella increased B. plicatilis lifespan and fecundity. To establish the model of

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rotifers for lifespan studies, we fed the rotifers with Se-enriched Chlorella (Se-Chlorella) and

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documented the survival period of all individuals (12-18 rotifers at the starting time point). In

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comparison with the non-Se-enriched alga fed rotifers (the control group), the Se-Chlorella rotifer

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group exhibited a significantly longer lifespan (p < 1×10-4 and p = 0.0025, with Mantel-Cox and

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Gehan-Breslow-Wilcoxon tests, respectively) (Figure 1A). The median survival time of the

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Se-Chlorella group was 11 days compared to 8 days for the control group, accounting for a 37%

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increase. According to log-rank (Mantel-Cox) test, the hazard ratios (HRs) of the control group and

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the Se-Chlorella group were 2.11 (95% CI: 1.96 to 4.84) and 0.47 (95% CI: 0.21 to 0.51), respectively.

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In addition, the HRs calculated by Gehan-Breslow-Wilcoxon were 3.08 (95% CI: 1.81 to 5.22) for the

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control group and 0.33 (95% CI: 0.19 to 0.55) for the Se-Chlorella group. It has been reported that the

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fecundity of rotifers can be increased by exposure to certain combinations of antioxidant

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supplements28. The same phenomenon was observed in this study, showing the spawning rate was

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significantly (p < 1×10-4) increased in the Se-Chlorella group (Figure 1B). The above results

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demonstrated an increased lifespan and fecundity in B. plicatilis fed with Se-Chlorella.

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De novo assembly of B. plicatilis transcriptome. The transcriptome sequencing (RNA-Seq)

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information was summarized in Table S2. Approximately 227 million reads were generated by

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Illumina HiSeq-2000 with over 213 million reads passed the quality control and used for de novo

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assembly. Information of contigs and unigenes, output by Trinity, was summarized in Table S3. The 10



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assembly yielded 34 to 41 thousand contigs in a single sample with a total length of 17 to 21 Mb,

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resulting in 24,691 to 29,335 unigenes with a total length of over 17 to 24 Mb for each sample (Figure

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2A).

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Proteome of B. plicatilis. We next used the union set of unigenes from all four samples, known

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as AllUnigene, to construct the protein sequence database. In general, there were 25,757 unigenes,

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with 26,774,971 nt in length collectively, in such a union set. The mean length of AllUnigene was

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1040 nt and the N50 was 1550 nt. Products of RNAs shorter than 400 nt were regarded as

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oligopeptides instead of proteins. Such sequences were removed and the remains were theoretically

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three-frame translated in silicon to generate all possible protein sequences. As a result, we obtained a

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protein reference database in the FASTA format with 55,716 entries.

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To further investigate the biochemical mechanism of the lifespan extension of rotifers fed with

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Se-Chlorella, we employed the isobaric tagging quantification proteomic technology, iTRAQ, to

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quantify proteins from three replicates in a single tandem mass spectrometry (MS/MS) run. After

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searching for the database, we identified 2,628 proteins in B. plicatilis, and a total of 2037 proteins

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were quantified across all six samples (Figure 2B).

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We used the Blast2GO software to annotate proteins from published databases based on the

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sequence similarity24. To maximize the rate of annotations, we used the gene sequences of the

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quantified proteins for Blast2GO annotation. Most hits were from 13 species including B. plicatilis,

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with most hits in Crassostrea gigas (Figure 2C), and a total of more than 90% of 2027 proteins were

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annotated (Figure 2D).

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Determination of differentially expressed proteins of B. plicatilis. To quantitatively

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summarize proteomics data, the coefficient of variation (CV) for quantified proteins across all six

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samples was calculated. The results showed that the majority of quantified proteins had the CV under

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30% (Figure 3A), indicating that a high quality of proteomic dataset was obtained. Pavelka et al. has

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proposed that the identification of differentially expressed genes or proteins are affected by the

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variability of the data26, especially in data generated from biologically replicated samples. Here, we

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employed the power law global error model (PLGEM) to better identify the DEPs in rotifers fed with

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Se-Chlorella (Figure 3B and C). Per median fold change biases (1.02-1.05) observed in iTRAQ-based

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MS29, the proteins with fold change ≥ 1.2 and p-value < 0.05 were considered to be the statistically

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significant DEPs. We identified 235 such DEPs, 119 of which were up-regulated, and 116 were

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down-regulated. By employing the hierarchical clustering analysis, DEPs could reflect our

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experimental treatment perfectly in numeric, since all replicates in the same group were clustered in

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the same hierarchy (Figure 3D). Here, Euclidean distances and Ward linkage (inner squared distance)

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were used in calculating distances and linkages between rows and columns, respectively.

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Enhanced glycolysis and amino acid metabolism in Se-Chlorella fed rotifers. In order to

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uncover the mechanism of the lifespan extension of B. plicatilis, pathway and DEP analyses were

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performed according to Gene Ontology Biological Processes (GO_BP) and KEGG pathways. For the

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annotated up-regulated proteins in the Se-Chlorella group, by counting the GO_BP annotation, over

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half of these proteins were found to be involved in biological processes related to the anti-oxidation

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responses, such as the cellular nitrogen compound metabolic process (19 sequences), biosynthetic

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process (19 sequences), cellular amino acid metabolic process (7 sequences), responses to stress (6

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sequences), and protein folding (5 sequences) (Figure 4A).

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KEGG pathways indicated that DEPs were connected to a series of biochemical reactions. The

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thioredoxin reductase (EC:1.8.1.9) participating in selenocompound metabolism was significantly

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up-regulated (Figure 4B), which could serve as a positive control for our analyses. Both

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phosphoglycerate

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glycolysis/gluconeogenesis

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dephosphorylation of glycerate-2,3P2 (Figure 4C). The significant decrease of adenosine

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5‘-monophosphate (AMP)-activated protein kinase (AMPK) in the mTOR pathway was computationally

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detected (Supplemental Figure S2). Since AMPK is a key enzyme in cellular energy homeostasis,

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such an observation can be deemed as an energy metabolism response in rotifers fed with

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Se-Chlorella. Furthermore, the aldehyde dehydrogenase (EC:1.2.1.3), a detoxification enzyme with a

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variety of substrates, was detected to be up-regulated (Figure 4D). In addition, the transaminase

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(EC:2.6.1.52, Figure 4E), hydroxymethyltransferase (EC:2.1.2.1, Figure 4F) and adenosyltransferase

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(EC:2.5.1.6, Figure 4G) were significantly up-regulated in rotifers fed with Se-Chlorella. These

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enzymes were regulators of a variety of amino acid metabolism pathways, including the valine,

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leucine, isoleucine, and lysine degradation, as well as the metabolism of beta-alanine arginine, proline,

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glycine serine, threonine, histidine and tryptophan. Therefore, the results indicated that glycolysis and

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amino acid metabolism were enhanced in the rotifers fed with Se-Chlorella, which might alter their

247

energy supply patterns and protein (or amino acid) biosynthesis.

mutase

(EC:5.4.2.4) were

and

significantly

phosphatase up-regulated,

1 which

(EC:3.1.3.13) would

in

accelerate

the the

248

Relationships between transcriptomic/proteomic data and ROS levels in rotifers. The above

249

metabolic changes suggested that the gain of functions in rotifers fed with Se-Chlorella in terms of 13


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enhanced use of glycerol via glycolysis for energy production and improved recycling of amino acids

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would be beneficial for the prolonged lifespan in rotifers observed in his study. Especially, we noted

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that the thioredoxin reductase increased after Se-Chlorella feeding when TCA pathways were shifted

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to glycolysis. Therefore, we hypothesized that the rotifer’s tolerance to stresses, especially regarding

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the oxidative stress as a primary factor in ageing, should be improved by Se intake.

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We integrated the information acquired from transcriptome and proteome analyses to compare the

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consistency of quantification between mRNA and proteins. Among all six KEGG key enzymes

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detected above, such abundance consistency were observed in four of their counterpart genes, and the

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other two showed inconsistent fold changes between mRNA and protein (Figure 5A). Similar

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inconsistent results have been reported in human cells previously, showing that the linear correlation

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of mRNA and protein abundance in steady-state human cells was less than 0.418. This result also

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verified the importance of proteogenomic analyses to discern biological metabolism processes more

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accurately not just based on transcriptome alone.

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Animals have evolved defenses against ROS damage, and the balance between oxidative stress

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and concentration of available antioxidants has a major influence on lifespan. Important elements of

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antioxidant defenses include scavenging enzymes such as catalase (CAT), glutathione peroxidase

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(GPX), and superoxide dismutase (SOD)13. We further found that the total LPO and ROS level in

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rotifer had a longitudinal increase trend in both the control and the Se-Chlorella groups (Figure 5B).

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The antioxidative effect in Se-enriched rotifer was associated with an increase in GPX and CAT

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(Figure 6). These results suggest that regulating oxidative damage is an important feature of rotifer

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aging, and that Se treatments that enhance the antioxidant capacity of rotifers can extend lifespan. 14



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LPO and ROS level were found to be significantly lower in the Se-Chlorella group than that in the

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control group at all time points tested in this study (Figure 5B and Figure 6C), which supports our

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hypothesis.

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275 276

Discussion It has been a long-term goal for biologists to understand the ageing mechanisms and prolonging

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the lifespan of organisms, especially humans. For aging research, rotifers have several advantages.

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Most rotifers have very short lifespans (10–15 days), are easy to use in life table experiments, and

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exhibit survivorship curves similar in shape to many other animals1. Rotifer phenotypes associated

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with aging like changes in appearance and vitality are well characterized and easy to measure3.

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Rotifers also may be favored aging test animals because of their short generation time, ease of culture,

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and the commercial availability of their resting eggs. B. plicatilis is among the best studied rotifers,

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having been used as a model system for investigations of population dynamics speciation, the

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evolution of sex, and ecotoxicology.

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Here, we showed Se-Chlorella feeding could increase the lifespan and fecundity of rotifers largely

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due to systematic pattern alterations in energy supply from TCA to glycolysis and enhanced amino

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acid metabolism. The Se-mediated benefits can be explained by the conceptual model based on our

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major findings in this study as well as previous literatures (Figure 7). On the one hand, the increased

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level of glycolysis is expected to decrease the energy demands from TCA, which in turn leads to a

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reduced amount of TCA-dependent ROS production. On the other hand, Se-Chlorella feeding allows

291

rotifers to use amino acids more efficiently for protein/amino acid biogenesis and energy supply. The 15


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collective outcome of these effects can lead to an increased lifespan in rotifers fed with Se-Chlorella.

293

To consider whether Chlorella have toxic effects on human cells, hot water extracts of Chlorella have

294

been shown to independently induce apoptosis of human lung cancer cells30, 31 and liver cancer cells30,

295

while with no impact on normal cells.

296

In this study, we firstly showed the unbiased enrichment of thioredoxin reductase

297

(TrxR)-mediated selenocompound metabolism pathways from the proteomics data. As the protein

298

searches were solely based on the de novo assembly of the transcriptome, the Se metabolism

299

relevance of DEPs was a critical control for deducing valid conclusions in this study. In humans, as a

300

selenoprotein32, TrxR has been found to reduce methylseleninate for methylselenol production that

301

mediates cancer cell death33, without remarkably affecting normal cells34. Although it has differential

302

roles in ageing at various tissues and developmental stages35, TrxR discerned in this study may be

303

deemed as a response to the Se-enrichment. Favorable to this notion, Penglase et al. have found that

304

rotifers have much lower (> 30 folds) Se concentrations, and Se-enriched rotifers increase

305

Se-dependent enzyme mRNA expression36.

306

The increased TrxR abundance and shift of energy-producing metabolism from TCA toward

307

glycolysis observed in this study may explain the diminished ROS production in the Se-Chlorella fed

308

rotifers. The role of ROS in the ageing study may be one of the most debatable aspects ageing37-39.

309

The mitochondria are primary source of ROS production and they are also vulnerable to the oxidative

310

stress, which is one of main causes of ROS-mediated ageing. However, James et al. have found that

311

senescent human fibroblasts in vitro are featured by the upregulated glycolysis and virtually depleted

312

TCA bioprocess40. This is a very interesting finding as it is against the traditional perception for

16



313

considering enhanced glycolysis solely as a biomarker of aged cells41. It suggests that ageing human

314

cells are able to survive via acquiring energy from glycolysis when mitochondria are not sufficiently

315

functioning. As mitochondria are degenerating and glycolysis is boosting in ageing cells, these

316

findings imply that mitochondria consist of a primary source of senescence driving force. In this study,

317

our results suggest that Se-Chlorella could lead rotifers to use more glycolysis enzymes

318

physiologically to reduce the burden of mitochondria for energy production. Our theory of energy

319

supply with rotifers is directly supported by Yan et al. (2012), showing that Se could promote

320

intracellular ATP production via both glycolysis and TCA pathways in normal human chondrogenic

321

cells42.

322

Secondly, we demonstrated that amino acid metabolism in rotifers could be enhanced by

323

Se-Chlorella feeding. In humans, both anabolism and catabolism of proteins/amino acids are known

324

to be diminished in the elderly43, 44, and Se plays important roles in multiple processes of amino acid

325

metabolism32. Our findings tentatively move the field forward by not only demonstrating the

326

comparability of humans and rotifers as ageing models with Se enrichment, but also providing a

327

systematic and mechanistic overview of the key enzymes involved in the ageing process (Figure 7).

328

For example, the enhanced glycolysis in rotifers fed with Se enrichment algae may be attributed to the

329

better use of glycerol by phosphoglycerate mutase and phosphatase 1. Such a feature in rotifers

330

induced by feeding Se enrichment algae is comparable to the human dietary-dependent glycerol

331

acquisition.

332 333

In conclusion, we established a Se-Chlorella facilitated lifespan extension rotifer model that is suitable to be a mimic system of human ageing process. Using proteogenomic analysis for the first

17


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time on rotifers B. plicatilis, we provided the first systematic evidence that organoselenium could alter

335

the metabolic pattern of rotifers toward acquiring energy supplies with reduced ROS production, and

336

enhancing amino acid metabolism. With the current systems biology findings, we propose that rotifers

337

are a useful model for understanding human ROS-relevant ageing mechanisms, and Se-Chlorella is a

338

promising novel organoselenium source with potential to developing novel drugs to prolong human

339

lifespans.

340 341 342

Acknowledgements

343

Special thanks are due to Prof. Senjie Lin (University of Connecticut, USA) for very valuable

344

comments on the manuscript. This work was supported by the joint Project of NSFC-Guangdong,

345

China (U1301235 to Y. Y.) , the Project of NSFC (41173079 to Y.Y.), the National Basic Research

346

Program “973” of China (2014CBA02000 to T.W.) and the Guangdong Natural Science Foundation

347

(2014A030313369 to T.W.).

348 349

Author Contributions

350

Y.Y. and T.W. conceived the idea, designed the study. T.W. wrote the paper. X.S. performed the B.

351

plicatilis culture, Se-enrich treatment, RNA extraction and drafted the manuscript. Y.C. contributed to

352

the proteomic experiments, most of the data analyses and paper writing. Q. W., X. N., Z. H. and X. H.

353

contributed base ideas, carried out rotifer experiments and/or revised the manuscript. S.T. performed

354

the total protein extraction. Y.Z and H.L performed the B. plicatilis culture. X.C. performed the Trinity

18



355

assembly.

356 357

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Figure 1. Lifespan and spawning rate of B. plicatilis. (A) Kaplan-Meier curve of B. plicatilis: Control, fed with normal alga (red); Se-Chlorella, fed with Se-enriched alga (blue). (B) Statistical comparison of the spawning rate of B. plicatilis. The spawning rate was normalized to the median survival day of each group, respectively. *p < 1×10-4, n=4. Data are shown as mean ± SD.

25


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Figure 2. Quality of the de novo assembly of B. plicatilis transcriptome, and the Blast2GO annotation of identified proteins. (A) Contig length distribution (blue) and its cumulative curve (red) of de novo assembly. (B) Venn diagram of quantified proteins from different samples. The protein number with different iTRAQ tags were shown. (C,D) Blast2GO annotations. Distribution of top Blast hit species with more than 20 sequences (C) and proportions of protein annotations distribution (D) are shown.

26



Figure 3. Comparative proteomics of B. plicatilis. (A) The coefficient of variation (CV) distribution (blue) and its cumulative curve (red) of iTRAQ-based MS identifications and quantifications. (B, C) Residuals vs. rank graph (B) and normal Q-Q plot (C) of the 2037 quantified proteins by employing PLGEM algorithm. (D) Clustergram of significantly changed proteins. All iTRAQ ratios were normalized to the Control 1 group. Each column indicates a single independent experiment, and each row indicates a quantified protein.

27


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28



Figure 4. Pathway analysis of significantly up-regulated proteins induced in Se-Chlorella fed B. plicatilis. (A) The top 10 biological processes of significantly up-regulated proteins (blue), and cumulative curve of number of hit sequence (red). (B) Selenocompound metabolism pathway enriched by KEGG pathway analysis. Colored boxes with numbers indicated experimentally identified proteins and their enzyme codes. (C to G) Key enzymes with their first neighbors in the enriched KEGG pathways. The enzymes include, mutase and phosphatase (C), dehydrogenase (NAD+) (D), transaminase and hydroxymethyl-transferase (E), CoA-transferase (F) and adenosyltransferase (G). Blue circles with numbers depict experimental quantified enzymes by MS and their enzyme codes, while the white circles illustrate the curated enzymes.

29


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Figure 5. Quantitative verification of key enzymes and biological validations. (A) Quantitative comparison of KEGG key enzymes at mRNA and protein levels. Logarithm of fold changes were calculated based on mean of FPKM and iTRAQ ratio, regarding transcriptome (white) and iTRAQ-based proteome (black) data, respectively. (B) Statistical comparison of intracellular ROS. Longitudinal data of Day 3, 6 and 9 days post feeding are respectively shown. P-values of each comparison are shown respectively, n=3.

30



Figure 6. Changes in antioxidant enzyme activities and lipid peroxidation of B. plicatilis during cultivation time under Se treatments. (A) GPX, (B) CAT, (C) LPO. *p < 0.01, n=3. Data are shown as mean ± SD.

31


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Figure 7. Possible mechanisms of Se-enriched Chlorella feeding mediated rotifer lifespan extension.

32



Lifespan and spawning rate of B. plicatilis. (A) Kaplan-Meier curve of B. plicatilis: Control, fed with normal alga (red); Se-Chlorella, fed with Se-enriched alga (blue). (B) Statistical comparison of the spawning rate of B. plicatilis. The spawning rate was normalized to the median survival day of each group, respectively. *p < 1×10-4, n=4. Data are shown as mean ± SD. 75x88mm (300 x 300 DPI)


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Quality of the de novo assembly of B. plicatilis transcriptome, and the Blast2GO annotation of identified proteins. (A) Contig length distribution (blue) and its cumulative curve (red) of de novo assembly. (B) Venn diagram of quantified proteins from different samples. The protein number with different iTRAQ tags were shown. (C,D) Blast2GO annotations. Distribution of top Blast hit species with more than 20 sequences (C) and proportions of protein annotations distribution (D) are shown. 158x119mm (300 x 300 DPI)



Comparative proteomics of B. plicatilis. (A) The coefficient of variation (CV) distribution (blue) and its cumulative curve (red) of iTRAQ-based MS identifications and quantifications. (B, C) Residuals vs. rank graph (B) and normal Q-Q plot (C) of the 2037 quantified proteins by employing PLGEM algorithm. (D) Clustergram of significantly changed proteins. All iTRAQ ratios were normalized to the Control 1 group. Each column indicates a single independent experiment, and each row indicates a quantified protein. 160x129mm (300 x 300 DPI)


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Pathway analysis of significantly up-regulated proteins induced in Se-Chlorella fed B. plicatilis. (A) The top 10 biological processes of significantly up-regulated proteins (blue), and cumulative curve of number of hit sequence (red). (B) Selenocompound metabolism pathway enriched by KEGG pathway analysis. Colored boxes with numbers indicated experimentally identified proteins and their enzyme codes. (C to G) Key enzymes with their first neighbors in the enriched KEGG pathways. The enzymes include, mutase and phosphatase (C), dehydrogenase (NAD+) (D), transaminase and hydroxymethyl-transferase (E), CoAtransferase (F) and adenosyltransferase (G). Blue circles with numbers depict experimental quantified enzymes by MS and their enzyme codes, while the white circles illustrate the curated enzymes. 146x235mm (300 x 300 DPI)



Quantitative verification of key enzymes and biological validations. (A) Quantitative comparison of KEGG key enzymes at mRNA and protein levels. Logarithm of fold changes were calculated based on mean of FPKM and iTRAQ ratio, regarding transcriptome (white) and iTRAQ-based proteome (black) data, respectively. (B) Statistical comparison of intracellular ROS. Longitudinal data of Day 3, 6 and 9 days post feeding are respectively shown. P-values of each comparison are shown respectively, n=3. 105x110mm (300 x 300 DPI)


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Changes in antioxidant enzyme activities and lipid peroxidation of B. plicatilis during cultivation time under Se treatments. (A) GPX, (B) CAT, (C) LPO. *p < 0.01, n=3. Data are shown as mean ± SD. 63x153mm (300 x 300 DPI)



Possible mechanisms of Se-enriched Chlorella feeding mediated rotifer lifespan extension. 104x70mm (300 x 300 DPI)


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Proteogenomic Analyses Revealed Favorable Metabolism Pattern

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