Whole-Life-Stage Characterization in the Basic Biology of Daphnia

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Whole-Life-Stage Characterization in the Basic Biology of Daphnia magna and Effects of TDCIPP on Growth, Reproduction, Survival and Transcription of Genes Han Li, Siliang Yuan, Guanyong Su, Meng Li, Qiangwei Wang, Guonian Zhu, Robert J. Letcher, Yufei Li, Zhihua Han, and Chunsheng Liu Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b04569 • Publication Date (Web): 08 Nov 2017 Downloaded from http://pubs.acs.org on November 9, 2017

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Environmental Science & Technology

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Whole-Life-Stage Characterization in the Basic Biology of Daphnia

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magna and Effects of TDCIPP on Growth, Reproduction, Survival

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and Transcription of Genes

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Han Li¶, Siliang Yuan¶,ф, Guanyong Suξ, Meng Li§, Qiangwei Wang§, Guonian

6

Zhu§, Robert J. Letcher‡, Yufei LiЖ, Zhihua Han$, Chunsheng Liu¶,†,*

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¶

9

†

College of Fisheries, Huazhong Agricultural University, Wuhan 430070, China Collaborative Innovation Centre for Efficient and Health Production of Fisheries in

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Hunan Province, Changde 415000, China

11

ф

12

Control and Prevention, Wuhan 430070, China

13

ξ

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of Environmental and Biological Engineering, Nanjing University of Science and

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Technology, Nanjing 210094, China

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§

17

310058, China

18

‡

19

5B6, Canada

20

Ж

21

of PR China, Beijing 100045, China

22

$

Hubei Engineering Technology Research Center for Aquatic Animal Diseases

Jiangsu Key Laboratory of Chemical Pollution Control and Resources Reuse, School

Institute of Pesticide and Environmental Toxicology, Zhejiang University, Hangzhou

Departments of Chemistry and Biology, Carleton University, Ottawa, Ontario K1S

China Rural Technology Development Centre, Ministry of Science and Technology

Nanjing Institute of Environmental Science, MEP, Nanjing 210042, Jiangsu, China

23 24

*Author for correspondence:

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Tel: 86 27 87282113

26

Fax: 86 27 87282114.

27

Email: [email protected] or [email protected] (C. Liu) 1

ACS Paragon Plus Environment


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ABSTRACT

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Toxicity tests of chemicals have mainly focused on the partial life-cycle

30

evaluation of model animals. Limited information is available for the evaluation of

31

effects of chemicals from a whole-life-stage exposure perspective. The objective of

32

this study was to perform a whole-life-stage characterization in the basic biology of

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Daphnia magna (D. magna) and evaluate the effects of a known organophosphate

34

ester (OPE) contaminant, tris (1,3-dichloro-2-propyl) phosphate (TDCIPP), on growth,

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reproduction,

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characterization in growth, reproduction and survival of D. magna was conducted,

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and representative sampling time points for the three developmental stages were

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identified (day 6, day 32 and day 62). Transcriptomic profiles for these three stages

39

were compared and stage-specific PCR arrays of D. magna were developed. The

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whole-life-stage exposure to environmentally relevant or greater concentrations of

41

TDCIPP significantly inhibited growth and reproduction of D. magna and decreased

42

survival at the later stage of the exposure experiment (≥ 32 days). Such adverse

43

effects were not observed in the early stage of the exposure (< 32 days), suggesting

44

that short-term toxicity tests, such as the standard 21-day test, might underestimate the

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environmental risk of TDCIPP. Furthermore, expressions of genes selected at day 6,

46

day 32 and day 62 were significantly changed after TDCIPP exposure, and the

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changes in the expressions of partial genes were correlated to the inhibitory effects on

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growth, reproduction and survival.

survival

and

transcription

of

genes.

49 50 51

2


The

whole-life-stage

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INTRODUCTION

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Previous studies that have assessed the toxicity of exogenous chemicals, were

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mainly conducted based on partial life-cycle evaluations of selected model animals.1-3

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For example, experiments in 48-h acute and 21-day chronic exposure studies of

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flowback and produced wastewater (FPW) experiments were conducted using

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Daphnia magna (D. magna) to characterize toxicity of FPW.4 In another study,

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medaka fish (Oryzias latipes) were selected and a 28-day chronic exposure was

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performed to study cellular and molecular effects of Microcystin-LR on the liver.5

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Zebrafish were exposed to the organophosphate ester (OPE), triphenyl phosphate, for

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11-14 days to study the bioconcentration and tissue distribution of the chemical, thus

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evaluating its fate and potential toxicity.6 Collectively, these studies centralized the

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toxic effects to a certain life stage of a test organism although they usually had two

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typical shortcomings. Firstly, in these tests, the lowest observed effect concentrations

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of chemicals were relatively high and mostly not environmentally relevant due to the

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short exposure period. Therefore, it was difficult to accurately predict risks of

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chemicals at environmentally relevant concentrations. Secondly, in these standard

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toxicity tests, the evaluation of toxic effects of chemicals was performed in a partial

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life-cycle exposure period, and thus underestimated the environmental impacts of the

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chemical since in natural environments some wildlife might be exposed to some

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chemical pollutants throughout their life span and especially for those organisms with

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short life spans such as D. magna.

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To address this knowledge gap, in the present study a whole-life-stage toxicity 3



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test protocol with D. magna was developed and a exposure experiment was performed

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using the known OPE environmental contaminant, tris(1,3-dichloro-2-propyl)

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phosphate (TDCIPP) . D. magna is a model organism in ecotoxicology studies

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because it is an imperative animal link in the aquatic food chain.7,8 Furthermore, it has

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a short life span, and the whole-life-stage exposure of chemical pollutants in natural

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aquatic environment might be a frequent occurrence. Therefore, D. magna may be a

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suitable test animal for evaluating the toxic effects of chemicals after a

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whole-life-stage exposure. TDCIPP is a current-use flame retardant which has been

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added to a diverse array of commercial products.9,10 Being an additive flame retardant,

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TDCIPP can easily be released into the surrounding environment. Environmental

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monitoring has demonstrated that TDCIPP is extensively distributed in various

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environmental media,11-15 and the highest concentration reported in natural waters was

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377 ng/L.16 Previous studies reported that exposure to high or relatively low

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concentrations of TDCIPP could cause neurological, developmental and reproductive

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damage,17-27 however, to the best of our knowledge, all these studies were performed

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based on partial life stage tests with organisms. It remains unknown whether

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whole-life-stage exposure could cause greater environmental hazards compared with

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those of partial life stage tests.

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The objective of this study was to perform a whole-life-stage characterization in

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the basic biology of D. magna and evaluate the effects of a known organophosphate

94

ester (OPE) contaminant, TDCIPP, on growth, reproduction, survival and

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transcription of genes. Briefly, in this study, the whole-life-stage growth, reproduction 4


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and survival curves of D. magna were determined. Appropriate sampling time points

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for toxicity testing of the chemical TDCIPP were also determined. RNA-Seq

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technology was then used to obtain stage-specific genes of D. magna for PCR array

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development. Finally, TDCIPP was selected to perform a whole-life-stage exposure

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experiment and its effects at environmentally relevant or greater concentrations on

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growth, reproduction, survival and transcription of genes were evaluated.

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MATERIALS AND METHODS

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Chemicals and Reagents

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Tris (1,3-dichloro-2-propyl) phosphate (TDCIPP) was purchased from Sigma (St.

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Louis, MO, USA), and it was diluted with dimethyl sulfoxide (DMSO) using a serial

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dilution approach. TRIzol reagent, reverse transcription and SYBR Green kits were

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purchased from Takara (Dalian, Liaoning, China). d15-TDCIPP was purchased from

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Toronto Research Chemical (Toronto, Canada). All the other reagents used in this

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study were of analytical grade.

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Normal Culture of D. magna

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Animal maintenance and growth, reproduction and survival curves. D. magna

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was cultured as has been reported elsewhere.9 Culture medium was aerated tap-water,

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which was pretreated by an Angel water cleaner (J1205-ROB12, Shenzhen, China).

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The culture medium was changed completely every two days. To get a comprehensive

115

insight into its whole-life-stage characteristics, the growth, reproduction and survival

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curves of D. magna were determined. Here, two experiments were run in parallel. In

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the first experiment, a growth curve was produced. Briefly, three glass beakers 5



118

containing 1.5 L aerated tap-water were used, and each beaker contained 75 neonates

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(< 24 h) to ensure 20 mL per individual. The experiment was performed using a

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well-characterized single clone of D. magna, maintained indefinitely as pure

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parthenogenetic cultures as reported before.28 The culture media were renewed

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completely every two days and body length (from the apex of the helmet to the base

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of the tail spine) was measured every two or four days depending on the

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developmental stages of D. magna. This step was repeated until all the animals

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expired. Body length data were used to produce the growth curve. In the second

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experiment, reproductive and survival curves were obtained. Another three glass

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beakers containing 1 L culture medium and 50 neonates (< 24 h) were used to ensure

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20 mL per individual. During this experiment period, the number of offspring

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produced in each beaker was counted every day to generate the reproductive curve.

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Additionally, the number of dead D. magna in each beaker was recorded and a

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corresponding survival curve was generated. Similarly, this step was repeated until all

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the individuals expired.

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Transcriptomic sequencing. Based on growth, reproductive and survival curves,

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animals were sampled at day 0 (< 24 h), day 6, day 32 and day 62 for transcriptomic

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sequencing. The four time points (day 0, day 6, day 32 and day 62) were considered

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to be representatives of the stages of birth, growth, reproduction and death,

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respectively. Briefly, three glass beakers containing 2 L aerated tap-water were used,

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and each beaker contained 100 neonates (< 24 h) to ensure 20 mL per individual. D.

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magna were cultured as above and 50, 30, 10 and 10 individuals from each beaker 6


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were collected and pooled to generate one sample at day 0, day 6, day 32 and day 62,

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respectively, for transcriptomic sequencing. Transcriptomic sequencing was

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performed by Novogene Bioinformatics Technology Co., Ltd (Beijing, China). Briefly,

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total RNA was isolated by use of TRIzol reagent and the purity, concentration and

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integrity of RNA were determined. Once RNA samples were qualified, Oligo-dT

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beads were used for the isolation of mRNA. After that, the isolated mRNA was

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fragmented into short sections and used for the synthesis of first-strand cDNA. The

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synthesis of second-strand cDNA was conducted by use of dNTPs, DNA polymerase I

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and RNAase H. The purification of double-stranded cDNA was performed using

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AMPure XP beads, and then the purified double-stranded cDNA were used for end

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reparation, “A” base addition and ligation of sequencing adapters. After size selection

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with AMPure XP beads, PCR amplification was performed and the production was

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purified using AMPure XP beads for library construction. Finally, the library was

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sequenced as 2 × 150 bp paired-end reads on Illumina HiSeqTM P150 sequencer

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(Illumina, San Diego, CA, USA). Raw data were cleaned by removing reads with

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adaptors, low quality (> 50 %) or high-proportion unknown bases (> 10 %) and then

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were assembled by use of Trinity methodology.29,30 The annotation of transcriptome

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sequences were performed using the following seven databases: Nr (NCBI

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non-redundant protein sequences), Nt (NCBI nucleotide sequences), Pfam (Protein

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family), KOG/COG (eukaryotic Ortholog Groups and Clusters of Orthologous Groups

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of proteins), Swiss-Prot (A manually annotated and reviewed protein sequence

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database), KEGG (Kyoto Encyclopedia of Genes and Genomes) and GO (Gene 7



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Ontology). Gene expression was calculated using FPKM (expected number of

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fragments per kilobase of transcript sequence per millions base pairs sequenced), and

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differentially expressed genes between day 0 and day 6, day 6 and day 32, and day 32

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and day 62 were selected based on padj < 0.05. KEGG pathway analysis was

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conducted and corrected P value (FDR) cut-off was set at 0.05.

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Development of stage-specific PCR arrays. The life process of D. magna was

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artificially divided into four stages: birth stage, growth stage, reproduction stage and

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death stage. It was hypothesized that, compared with the last stage, when entering the

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next stage, expressions of some genes would be up-regulated to fulfill the biological

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function of the next stage. Therefore, in this study, up-regulated genes between two

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continuous life stages were screened for the development of stage-specific PCR arrays.

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Similar hypotheses were frequently used in previous studies to screen stage-specific

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functional genes.31,32 For the development of the present stage-specific PCR arrays,

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three steps were included. Firstly, differently expressed genes between day 0 and day

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6, day 6 and day 32, and day 32 and day 62 were obtained, and those up-regulated

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genes screened in each stage were used for KEGG pathway analysis; Secondly, those

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significantly enriched pathways obtained in KEGG pathway analysis were picked out

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and ranged based on the value of rich factor (RF); Thirdly, the top 5 up-regulated

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pathways in each stage were selected, and in each pathway the top 5 genes (readcount

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≥ 10) based on expression of fold change were used for the development of

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stage-specific PCR arrays. The gene list for each of the stage-specific arrays was

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provided in Table 1. 8


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TDCIPP Exposure Experiments

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TDCIPP exposure protocol. D. magna neonates (< 24 h) were exposed to 0, 300 or

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3000 ng/L TDCIPP in glass beakers until 90 days when survival rate of control group

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was < 50 %, and each beaker contained 1 L exposure solution and 50 animals and

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each concentration contained 3 replicate beakers. The exposure concentrations (300

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and 3000 ng/L) were selected based on a previous study.9 The 300 ng/L was an

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environmentally relevant concentration and 3000 ng/L was used a greater

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concentration. During the exposure period, the exposure media were renewed

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completely with the corresponding concentrations of solutions every two days and

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animals were fed everyday with the mixture of Chlorella pyrenoidosa and

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Scenedesmus obliquus at a concentration of 2.5 × 106 cells/mL. Fecundity and

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survival were monitored and recorded daily for calculating accumulated production of

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offspring and survival rates, respectively. At day 6, day 32 and day 62, animals were

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sampled for the measurement of body length and isolation of total RNA. For the

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isolation of total RNA, there were three replicates for each concentration, and each

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replicate included 15, 10 and 5 individuals at day 6, day 32 and day 62, respectively.

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Control and exposure groups received 0.01 % aqueous DMSO.

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Quantification of TDCIPP in exposure solutions. TDCIPP in the exposure solutions

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was extracted and quantified according to a previous study.33 Briefly, an aliquot of 3

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mL of exposure solution was transferred into a centrifuge tube, and then 4 mL hexane

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and 10 µL internal standard solution (10 µg/mL in methanol), d15-TDCIPP, were

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added. The mixture was blended and then ultrasonicated. After that, the supernatant 9



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was concentrated and then transferred into a clean glass tube. The extraction and

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supernatant collection was repeated one more time. Combined with the first

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supernatant and evaporated. Three hundred µL of methanol was added into the tube,

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and then vortexed. The concentration multiple of the water samples was tenfold in the

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present study. The resulting methanol solution was transferred into a liquid

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chromatography (LC) vial for TDCIPP analysis. Quantification of TDCIPP was

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performed using LC-tandem mass spectrometer system (LC-MS/MS) consisting of

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5500 QTRAP MS/MS system (AB SCIEX, Singapore) and Waters ACQUITY™

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system (Waters Corp., Milford, MA, USA). Chromatographic separation was

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conducted using ACQUITY UPLC ® HSS T3 (1.8 µm, 2.1 mm × 100 mm, Waters),

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and Water (A) and methanol (B) were used as mobile phases. The ESI source in

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positive mode was as follows: ion spray (IS) voltage: 5500 V; curtain gas (CUR): 20

218

psi; nitrogen collision gas (CAD): 8 psi; nebulizer gas (GS1): 40 psi; auxiliary gas

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(GS2): 40 psi; source temperature: 400 ℃. The instrumental and method detection

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limit of TDCIPP was 0.3 µg/L and 0.03 µg/L, respectively.

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Isolation of Total RNA and Quantitative Real-time PCR

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Total RNA was extracted with TRIzol reagent (Takara, Dalian, Liaoning, China)

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following the manufacturer’s instructions. After examination of the purity and

224

concentration of RNA, 500 ng of total RNA was used for reverse transcription using

225

the Prime ScriptTM RT reagent kits (Takara, Dalian, Liaoning, China). Real-time

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quantitative PCR were performed with SYBR Green Premix Ex TaqII kits (Takara,

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Dalian, Liaoning, China). In order to monitor the quantity assurance and quality 10


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control (QA & QC) of PCR reactions, melting curves and standard curves were

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utilized to check the purity of PCR productions and amplification efficiency of PCR,

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respectively.

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DAPPUDRAFT_311135) kept unchanged between different developmental stages or

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after exposure to different concentrations of TDCIPP, and thus this gene was used as

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an internal control or housekeeping reference gene. Primer sequences were designed

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with Primer Premier 6 software (Premier Company, Canada), and all the primers used

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in this study were tested for efficiency, and their efficiencies (95%-103%) were

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comparable. Real-time PCR thermal cycling was set as follow: 95 ℃ for 3 min, and

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then 40 cycles of 95 ℃ for 15 s and followed by 60 ℃ for 1 min. Expressions of genes

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were calculated by 2−∆∆Ct method, and were presented as fold change compared with

239

the control.

240

Statistical Analyses

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Statistical analyses were completed with Kyplot Demo 3.0 software (Tokyo, Japan).

242

Normality of variances for parameters was determined using Kolmogorov−Smirnow

243

and the homogeneity was checked using Levene’s test. One-way analysis of variance

244

and tukey’s multiple range test were used to determine significant differences of

245

parameters tested between control and exposure groups. The level of significance for

246

all statistical analyses was set at p < 0.05.

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RESULTS AND DISCUSSION

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Normal Culture of D. magna

249

Growth,

Expression

reproduction

of

and

gene

c9685_g1

survival

curves.

11


(hypothetical

The

protein

whole-life-stage


250

characterization in growth, reproduction and survival of D. magna was investigated in

251

this study, and the corresponding curves are shown in Figure 1. D. magna has been a

252

model species in the study of environmental toxicology;34-37 however, its basic

253

biological information remains limited. To the best of our knowledge, we report for

254

the first time on the characterization of growth, reproduction and survival of D.

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magna from birth to natural death. For D. magna, there was a rapid growing stage

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from day 0 to day 12 after birth, and during this period the body length of D. magna

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increased rapidly from 934.27 ± 44.98 µm at day 0 to 2733.47 ± 92.02 µm at day 12

258

(Figure 1a). After day 12, the body length of D. magna remained essentially constant

259

until death (Figure 1a). Therefore, the growth curve suggested that the days from 0 to

260

12 were a key period for the growth of D. magna, and thus the period might be a

261

sensitive window for the evaluation of effects of chemicals on growth. For

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reproduction, D. magna started to produce offspring at day 10, and since then the

263

individuals kept reproducing until death. The accumulated number of offspring was

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353.04 ± 7.85 at day 94 for each individual (Figure 1b). Previous studies reported that

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D. magna has two reproductive modes: cyclic parthenogenesis and sexual

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reproduction, and under favorable rearing conditions, cyclic parthenogenesis is the

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only reproductive mode.38,39 In this study, sexual reproduction was not observed since

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no resting eggs were produced throughout the whole-life-stage experiment, and thus

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all the offspring produced were considered to be from cyclic parthenogenesis.

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Additionally, our results demonstrated that the offspring of D. magna were initially

271

produced at day 10, and were consistent or comparable with results from previous 12


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studies.4,40 Furthermore, for the first time, we found that under favorable rearing

273

conditions, D. magna could maintain high reproductive capacity until the end of life.

274

This might be a unique reproductive strategy for the organisms with parthenogenesis.

275

For survival, from day 0 to day 53, no mortality was observed (Figure 1c). However,

276

survival rates of D. magna decreased from 100 % at day 0 to day 53 to 0 % at day 94

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(Figure 1c). The survival curve results suggested that D. magna had a relatively broad

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death period compared with its life span, and this phenomenon has also been reported

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for other organisms such as mice.41

280

Transcriptomic

281

Transcriptomic profiles between two continuous developmental stages were compared

282

and stage-specific PCR arrays were developed. More than 48,890 genes were detected

283

at day 0, day 6, day 32 and day 62 and the numbers of differently expressed genes

284

between day 0 and day 6, day 6 and day 32, and day 32 and day 62 were 7479, 2971,

285

and 886, respectively. In order to validate the results of transcriptomic sequencing,

286

seventeen genes were randomly selected for quantitative real-time PCR validation,

287

and the results demonstrated that expressions of genes selected were comparable in

288

the two parts: transcriptomic sequencing and quantitative real-time PCR (Supporting

289

Information, Figure S1).

290

Development of stage-specific PCR arrays. Compared with the birth stage (at day

291

0), twenty-one pathways were significantly up-regulated in the growth stage (at day 6)

292

(Figure 2; Table S1, see Supporting Information) and the top 5 up-regulated pathways

293

were all related to DNA replication and DNA repair. Previously, it has been reported

profiles

and

quantitative

real-time

13


PCR

validation.


294

that during the growth process of multi-cellular organisms, cell proliferation is the

295

most essential biological behavior, and the core part of cell proliferation is DNA

296

replication.42,43 Therefore, our results were consistent with the findings in these

297

studies. Furthermore, compared with growth stage, five pathways were up-regulated

298

in reproduction stage (Figure 2; Table S1, see Supporting Information), and three

299

pathways were related to DNA replication and RNA transcription. In this study,

300

parthenogenesis was the only reproductive mode for D. magna and where oocytes are

301

produced by stem cells in the ovary and undergo only the equational meiotic division

302

in accordance with previous study.44 During the process of parthenogenesis, DNA

303

replication and RNA transcription play important roles. Therefore, the up-regulation

304

of the three pathways was considered to be responsible for the reproduction of D.

305

magna. There were 6 up-regulated pathways in the death stage (at day 62) compared

306

with the reproduction stage (at day 32) (Figure 2; Table S1, see Supporting

307

Information). In the 6 up-regulated pathways, three were related to cardiopathy, and

308

thus we hypothesized that D. magna died from functional loss of the heart. For the

309

stage-specific PCR arrays, 15 pathways and 73 genes were included according to the

310

protocol of development of stage-specific PCR arrays above. The detailed information,

311

including names of pathways and genes involved in each pathway of the arrays, were

312

shown in Table 1. Previously, PCR arrays have been documented as a useful tool to

313

evaluate the effects of chemicals on expressions of genes involved in some certain

314

pathways and explore possible molecular mechanisms.45-47 Therefore, by the use of

315

the method of comparative transcriptome, stage-specific PCR arrays were developed 14


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for evaluation of the effects of TDCIPP on the expressions of genes which were

317

responsible for growth, reproduction and death in this study.

318

TDCIPP Exposure Experiments

319

Concentrations of TDCIPP in exposure solutions. Before water renewal, the actual

320

measured TDCIPP concentrations of the nominal 300 and 3000 ng/L exposure groups

321

were 191 ± 9.1 and 3138 ± 463 ng/L, respectively. After water renewal, the measured

322

TDCIPP concentrations were 238 ± 5.7 and 3930 ± 205 ng/L for the nominal 300 and

323

3000 ng/L exposure groups, respectively. Mean concentrations of TDCIPP for

324

samples taken before and after water renewing were 215 ± 7.2 and 3534 ± 298 ng

325

TDCIPP/L. No TDCIPP was detected in the control group samples. Therefore, these

326

data suggested that there were no significant depletion of TDCIPP in this D. magna

327

exposure system, and the results were consistent with a previous study.9

328

Exposure to TDCIPP inhibited growth and reproduction and decreased

329

survival rate. A whole-life-stage exposure protocol was developed using TDCIPP as

330

a case study chemical, and the effects on growth, reproduction and survival were

331

simultaneously evaluated within one life span of D. magna. In this study, exposure to

332

environmentally relevant or greater concentrations of TDCIPP significantly decreased

333

body length of D. magna at day 32 and day 62, while no significant effect was

334

observed at day 6 (Figure 3a). At day 32, the body length of control group was

335

3262.81 ± 57.03 µm, and after TDCIPP exposure the body length was significantly

336

decreased to 3081.29 ± 25.89 µm in 3534 ng/L group (Figure 3a). No significant

337

effect on body length was observed after exposure to 215 ng/L TDCIPP when 15



338

compared with the control. When the exposure time was prolonged to day 62, the

339

body length was significantly decreased in both the 215 and 3534 ng/L groups (Figure

340

3a). Previous studies demonstrated that treatment with environmentally relevant or

341

greater concentrations of TDCIPP caused developmental toxicity in various

342

organisms.21-27 For example, a recent study reported that environmentally relevant or

343

greater concentrations of TDCIPP significant inhibited body length of D. magna after

344

exposure for 21 and 28 days.9 Therefore, our results were consistent with this study.

345

Additionally, the rapid growth stage of D. magna was from day 0 to day 12, but the

346

decrease in body length was only observed at day 32 and day 62 after TDCIPP

347

exposure. No significant effect on body length was observed at day 6. The possible

348

reason was that the decrease in body length was actually caused from day 6 to day 12,

349

which resulted in the observed decrease at day 32.

350

Besides the inhibition of growth, reproduction of D. magna was also inhibited by

351

TDCIPP (Figure 3b). At day 90, accumulated number of offspring in the control group

352

was 316.62 ± 6.70, but exposure to 215 or 3534 ng/L significantly decreased the

353

accumulated number of offspring to 274.22 ± 6.85 and 263.45 ± 6.19, respectively

354

(Figure 3b). The effects of chemicals on reproduction are usually examined in the

355

evaluation of the environmental risk of chemicals.48,49 In the present study, exposure

356

to environmentally relevant or greater concentrations of TDCIPP for 21 days did not

357

change the number of accumulated offspring of D. magna, and this result was

358

comparable with a recent study.9 However, when the exposure time was prolonged to

359

90 days, a significant inhibitory effect on the production of accumulated offspring was 16


Page 16 of 34

Page 17 of 34


360

observed. The results demonstrated that a 21-day reproductive toxicity test might

361

underestimate environmental hazards of chemicals, especially for those organisms

362

with reproductive capacity throughout their lives. For survival, exposure to 215 or

363

3534 ng/L TDCIPP did not change the survival rates at day 6 and day 32, but when

364

the exposure time was prolonged to 62 days, the survival rate was significantly

365

decreased by 14.76 % compared with the control (Figure 3c). It should be noted that

366

the decrease in survival rate might be resulted from both natural death and TDCIPP

367

exposure. Previously, most relatively low concentrations in chemical exposure

368

experiments were used in a partial life stage, and thus lethal effects were infrequently

369

observed. However, in the present study, we found that the whole-life-stage exposure

370

of TDCIPP could shorten the life span of D. magna. Therefore, our results indicated a

371

potential risk of environmental chemical pollutants for survival of wildlife, especially

372

for those organisms that were continuously exposed to chemical pollutants during the

373

whole life stage.

374

Transcriptional responses to TDCIPP. Based on the developed PCR arrays,

375

transcriptional responses to TDCIPP were examined at day 6, day 32 and day 62. At

376

day 6, twenty-four of 25 genes involved in the 5 up-regulated pathways selected at the

377

growth stage were significantly down-regulated upon TDCIPP exposure, and among

378

them expressions of the 15 genes were down-regulated in both the 215 and 3534 ng/L

379

groups (Figure 4). The five enriched pathways, including homologous recombination,

380

DNA replication, mismatch repair, nucleotide excision repair and cytosolic

381

DNA-sensing pathway, were all related to DNA replication and DNA repair, which 17



382

was a core part for growth of multi-cellular organisms.44 Therefore, expressions of

383

genes involved in the five pathways should be up-regulated in fast growth stage

384

compared with the birth stage. The hypothesis was consistent with our findings in

385

comparative transcriptomic study in this study, where expressions of all the genes

386

involved in the five pathways were up-regulated. Therefore the down-regulation due

387

to TDCIPP exposure was considered to be responsible for the inhibition of growth

388

observed in this study. Here, it should be noted that no significant effect on body

389

length was observed after TDCIPP exposure at day 6, although the expressions of

390

those genes screened were down-regulated. The possible explanation was that there

391

might be a delayed effect on body length. At day 32, exposure to 215 or 3534 ng/L

392

TDCIPP significantly altered the expressions of 16 genes involved in the top 5

393

up-regulated pathways of reproduction stage, and expressions of the other 9 genes

394

involved in these pathways were not changed. Among the 16 genes, expressions of 13

395

genes were down-regulated and expressions of 3 genes were up-regulated compared

396

with the control (Figure 5). For the parthenogenesis of D. magna, oocytes are

397

produced by stem cells in the ovary and undergo only the equational meiotic

398

division.50 Therefore, changes in the expressions of genes involved in cell cycle,

399

splicesome and transcriptional misregulation in cancer pathways after TDCIPP

400

exposure were considered as a possible reason for inhibition of reproduction observed

401

in this study. At day 62, expressions of all the genes involved in dilated

402

cardiomyopathy and hypertrophic cardiomyopathy pathways were significantly

403

up-regulated after TDCIPP exposure compared with the control ((Figure 6). In the 18


Page 18 of 34

Page 19 of 34


404

comparative transcriptomic study, we found that the dilated cardiomyopathy and

405

hypertrophic cardiomyopathy pathways were up-regulated at day 62 compared with

406

day 32, and it was calculated that D. magna might die from functional loss of the

407

heart. Therefore, the up-regulation of genes involved in the two pathways due to

408

TDCIPP might promote functional loss of heart and accelerate death of D. magna. It

409

needs to be emphasized that the exact relationships between the up-regulation of

410

dilated cardiomyopathy and hypertrophic cardiomyopathy pathways, functional loss

411

of heart and decrease of survival rate were not firmly established in this study, and

412

further studies were needed to explore these relationships. Additionally, it should be

413

noted that the changes in the expressions of genes might have resulted from both

414

natural death and TDCIPP exposure. The expressions of genes involved in

415

GABAergic synapse, serotonergic synapse and neuroactive ligand-receptor interaction

416

pathways were also significantly up-regulated. However, the linkage between the

417

up-regulation of these genes and decreased survival rates needs to be explored further.

418

SUPPORTING INFORMATION

419

The Supporting Information is available free of change on the ACS Publications

420

websites. The significantly up-regulated pathways at the stage of growth, reproduction

421

and death for D. magna (Table S1); Sequences of primers for the genes tested in D.

422

magna (Table S2); Expressions of genes in D. magna randomly selected in

423

transcriptomic sequencing and real-time quantitative PCR (Figure S1).

424

ACKNOWLEDGEMENT

425

This work was supported by the Fundamental Research Funds for the Central 19



426

Universities (2662015PY036) and National Natural Science Foundation of China

427

(21622702). Dr. G. Su was supported by Natural Science Foundation of Jiangsu

428

Province (Grant No. 400 BK20170830), and “the Fundamental Research Funds for

429

the Central Universities” (Grant No. 30917011305).

430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 20


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638 639 640

641 642

Figure 1. The whole-life-stage growth (a), reproduction (b) and survival (c) curves of D. magna.

643

Values are the mean ± SD (n=3).

644 645 646 647 648 649 650 651

27



652 653

Figure 2. Numbers of significantly up-regulated gene pathways between different developmental

654

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655 656 657 658 659

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660 661 662

Figure 3. The body length (a), accumulated offspring (b) and survival rate (c) of D. magna after

663

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664

One-way analysis of variance and tukey’s multiple range test were used to determine

665

significant differences of parameters tested between control and exposure groups. * p

666

< 0.05.

667 668 669 670 671 672

29



673

674 675

Figure 4. Expression of genes involved in the top 5 up-regulated pathways in the growth stage of

676

D. magna were significantly down-regulated after exposure to 215 or 3534 ng/L TDCIPP for 6

677

days. Values are the mean ± SD (n=3). One-way analysis of variance and tukey’s multiple

678

range test were used to determine significant differences of parameters tested between

679

control and exposure groups. * p < 0.05.

680 681 682 683 684 685 686

30


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687 688 689

Figure 5. Expressions of genes involved in the top 5 up-regulated pathways of D. Magna in the

690

reproduction stage were significantly changed after exposure to 215 or 3534 ng/L TDCIPP for 32

691

days. Values are the mean ± SD (n=3). One-way analysis of variance and tukey’s multiple

692

range test were used to determine significant differences of parameters tested between

693

control and exposure groups. * p < 0.05.

694 695 696 697 698 699 700

31



701 702 703 704

Figure 6. Expressions of genes involved in the top 5 up-regulated pathways in the death stage D.

705

magna were significantly altered after exposure to 215 or 3534 ng/L TDCIPP for 62 days. Values

706

are the mean ± SD (n=3). One-way analysis of variance and tukey’s multiple range test

707

were used to determine significant differences of parameters tested between control.

708

and exposure groups. * p < 0.05.

709 710 711 712 713 714 715 716 32


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717

Table 1. A list of the top 5 genes and the corresponding pathways in the developed

718

stage-specific PCR arrays Life stage

Stage of growth

Stage of reproduction

Stage of death

Up-regulated pathway

Genes

Homologous recombination

rpa1, pold1, rad54l, rad52, mre11

DNA replication

rnha, rnaseh1, rfc3_5, pold1, mcm7

Mismatch repair

rpa1, pms2, podl1, msh6, rfc3_5

Nucleotide excision repair

pms2, pold1, pcna, msh6, rfc3_5

Cytosolic DNA-sensing pathway

rpc1_1, rpc1_2, rpc3, rpc1_3, rpc1_4

Chronic myeloid leukemia

runx1, shc1, abl1, cdk6, pik3c

Cell cycle

mcm2, ccnb, ccne, cdc25c, cdk6

Transcriptional misregulation in cancer

etv6_7, utx, flt1, prom1, yan

Spliceosome

hnrnpk, hspa1_8_1, hspa1_8_2, snrnp200, hspa1_8_3

Axon guidance

ablim, sgrap, plxna, smo, ptch1

GABAergic synapse

slc6a1_1, gnao, slc6a1_2, plcl1, slc32a

Dilated cardiomyopathy

sgcd, tpm3, actb_g1, adcy5_1

Neuroactive ligand-receptor interaction

grik2, sstr5, glra1, takr2, crhr1

Hypertrophic cardiomyopathy

sgcd, tpm3, actb_g1

Serotonergic synapse

ddc, gnao, cacna1b, slc18a1_2, adcy5_1

Internal control:

c9685_g1

719 720 721 722 723 33



724

TOC art

725

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Whole-Life-Stage Characterization in the Basic Biology of Daphnia

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