DOI: 10.5061/dryad.4xgxd25j7
title: The transgenerational physiological and molecular data of Oryzias melastigma under seawater acidification stress

description: As shown in previous studies, anthropogenic CO2 emissions are increasing
 and cause lots of environmental changes, such as global warming; however,
 this change is also threatening the ocean. Several pieces of evidence
 pointed out that ocean acidification would impact marine life, especially
 in the early developmental stages. To understand the long-term and
 transgenerational effects of marine fish, we applied a marine model
 animal, Oryzias melastigma, to a CO2-induced acidified condition (pH 7.6).
 This study focused on the larvae's physiological acclimation and how
 they face acidified stress through gene expression changes. Furthermore,
 we checked the epigenetic modifications, like DNA methylation, to know if
 there were any parental effects under this stress.
description: <strong>Breeding
 conditions:</strong> We used the pH controller
 (E-M03, Macro, Taiwan) to monitor the pH value in each tank and pumped the
 extra CO<sub>2</sub> into the seawater to maintain a low pH
 condition (pH 7.6). The fertilized eggs from the control tank (WT, pH 8.1)
 were transferred immediately to acidified seawater until mature (F0), then
 swapped for the next generation (F1) and kept for the second generation
 (F2). The seawater system was kept in a similar
 condition (salinity 31–33, temperature 28±1 °C, 10 h dark/14 h light
 cycle) at Marine Research Station (the Institute of Cellular and
 Organismic Biology, Academia Sinica, Taiwan). During the exposure period,
 the water conditions were also recorded, like pH, temperature(T, °C),
 salinity (S, ‰), total alkalinity (TA,μM) and the total dissolved
 inorganic carbon (DIC,μM) and <em>p</em>CO<sub>2
 </sub>was calculated by CO2SYS module. The pH and
 temperature (°C) were measured using a pH meter (pH 3310, WTW, Germany)
 equipped with a pH electrode (SenTix® 41, WTW, Germany).
 TA (μM) was measured following a published method (Sarazin et al.
 1999). Ref: Sarazin, G., Michard, G., & Prevot,
 F. (1999). A rapid and accurate spectroscopic method for alkalinity
 measurements in sea water samples. <em>Water Research</em>,
 33(1), 290-294. doi: 10.1016/S0043-1354(98)00168-7 DIC
 (μM) was measured using an analyzer (AS-C3, Apollo SciTech, Newark, DE,
 USA) with quantification via a non-dispersive infrared CO2/H2O analyzer
 (LI-7000, LI-COR, Lincoln, NE, USA). 
 <strong>Larve growth patterns: </strong>
 We recorded the growth patterns from 2 days post-fertilization
 (dpf) to 7 dpf in each group. Measured the parameters (eye diameter, eye
 distance, body width, body height, and total length) under a microscope on
 a randomly selected individual every day. 
 <strong>Oxygen consumption and ammonium
 excretion: </strong> For the oxygen taking and
 ammonium excretion, 60 eggs were used for measurement as a simple point at
 5 dpf. The eggs were gently transferred to the airtight glass respiration
 chambers with filtered seawater, and then the oxygen concentrations were
 monitored in real-time by an OXY-4 mini multichannel fiber optic oxygen
 transmitter (PreSens) within one hour. After testing, water samples were
 collected from chambers and then measured the ammonium concentration
 following the published method (Holmes et.al., 1999).
 Ref: Holmes, R. M., Aminot, A., Kérouel, R., Hooker, B. A.,
 & Peterson, B. J. (1999). A simple and precise method for
 measuring ammonium in marine and freshwater ecosystems. <em>Canadian
 Journal of Fisheries and Aquatic Sciences</em>, 56(10), 1801-1808.
 doi: 10.1139/f99-128 
 <strong>Heartbeat frequency: </strong>
 A randomly chosen individual was gently placed under a
 microscope, and their heartbeat was recorded. Those videos were analyzed
 with Fiji to obtain a clear diagram. Further analyze the frequency of the
 heartbeat by changing the area of the heart. 
 <strong>RNA-seq
 profile: </strong> All RNA samples were extracted
 using the RNeasy Plus Universal Mini Kit (QIAGEN, Germany) in accordance
 with the manufacturer's instructions. Genomic (Taipei, Taiwan)
 performed the quality test using the BioAnalyzer2100 and Agilent RNA 6000
 Nano Kit. The passed samples were sequenced using the NovaSeq 6000
 (Illumina) 150 bp paired-end sequencing libraries for RNA-seq, giving 6 Gb
 data per sample, which was likewise processed by Genomics (Taipei,
 Taiwan). The reads were sufficiently trimmed and mapped to the
 <em>Oryzias melastigma</em> genome (ASM292280v2, NCBI) using
 the CLC genomic workbench (v. 20.0, CLC Bio-Qiagen, Denmark). The RNA-seq
 data were further analyzed by iDEP.96 (integrated Differential Expression
 Pathway analysis) to identify the significantly different Gene Ontology
 (GO) Molecular function pathway across groups. Based on the results, we
 selected some genes and then normalized reads count using Z-score to
 create a heatmap. Ref: Ge, S. X., Son, E. W., &
 Yao, R. (2018). iDEP: an integrated web application for differential
 expression and pathway analysis of RNA-Seq data.<em> BMC
 bioinformatics</em>, 19, 1-24. doi:
 10.1186/s12859-018-2486-6 
 <strong>Gene expression: </strong>
 To examine the gene expression of acid-base regulators, we
 synthesize the cDNA through SuperScript IV Reverse Transcriptase
 (Invitrogen, USA) and then measured the gene expression by a quantitative
 real-time PCR (qRT-PCR) with the Roche LightCycler 480 system (Roche
 Applied Science, Germany). The standard curves for each target gene were
 proven to be linear, with ribosomal protein L7 (<em>rpl7</em>)
 of <em>O. melastigma</em> serving as a reference
 gene. <strong>Epigenetic
 analysis:</strong>  The gDNA of 5 dpf larvae was
 purified using the Wizard® Genomic DNA Purification Kit (Promega, United
 States). Sonication was used to fragment the gDNA before proceeding with
 the MeDIP (Methylated DNA Immunoprecipitation) study, which followed the
 manufacturer's technique for the EpiQuikTM Tissue Methylated DNA
 Immunoprecipitation Kit (Epigentek, United States). After
 anti-5-methylcytosine immunoprecipitation, the immunoprecipitated (IP) DNA
 was collected and specific primers determined the promoter’s methylation
 levels. To confirm the dynamic DNA methylation pattern, we did the
 bisulfide conversion by EZ DNA MethylationTM Kit (ZYMO Research, United
 States) and amplified the AE1a’s promoter with bisulfite-treated
 DNA-specific primer by PCR. The PCR products were sequenced by Genomics
 (Taipei, Taiwan) to determine unmethylated and methylated CpG sites, which
 were predicted by MethPrimer.
description: # The transgenerational physiological and molecular data of Oryzias
 melastigma under seawater acidification stress
 [https://doi.org/10.5061/dryad.4xgxd25j7](https://doi.org/10.5061/dryad.4xgxd25j7) This data set provides the physiological and molecular results from the control group (WT) and three acidified acclimated generations (F0, F1, and F2). Due to our observations, the marine medaka was impacted by the acidified stress; however, they seemed to overcome this stress and acclimated to the low pH condition over generations. ## Description of the data and file structure **All raw data is separated, and the information for each file is as follows:** 1. **Om_Growth_parameters.csv**: Records growth parameters (in μm), including interocular distance, eye diameter, total body length, body height, and body width, measured from 2 dpf to 7 dpf. 2. **Om_metabolism.csv**: provide the oxygen consumption (μmol O2/h.g) and ammonium excretion (μmol NH4+ /h.g) at 5 dpf larvae. 3. **Om_Heartbeat_frequency_Hz.csv**: show the data from each individual. 4. **Om_RNA-seq_PCA.csv**: based on the RNA-seq results, we did the principal component analysis (PCA). 5. **Om_GO_term.csv**: the difference pathway among groups analysis by the Parametric Gene Set Enrichment Analysis (PGSEA) in iDEP.96. 6. **Om_Selected_genes.csv**: based on the pathway analysis, we selected the genes that were related to acidification stress among groups. All the values showed up in the Z-score. 7. **Om_Gene_expression.csv**: the relative gene expression of acid-base regulation genes in each group. 8. **Om_MeDIP.csv**: the DNA methylation levels (%) on the target gene’s promoter. 9. **Om_Methylation_position.csv**: showed the methylated CpG sites of the predicted AE1a’s promoter on each sample. ## Sharing/Access information The RNA-seq raw data were uploaded to NCBI (BioProject: PRJNA1086368).