DOI: 10.5061/dryad.p5hqbzkrt
title: The interplay between prior selection, mild intermittent exposure, and acute severe exposure in phenotypic and transcriptional response to hypoxia

description: Hypoxia has profound and diverse effects on aerobic organisms, disrupting
 oxidative phosphorylation and activating several protective pathways.
 Predictions have been made that exposure to mild intermittent hypoxia may
 be protective against more severe exposure and may extend lifespan. Both
 effects are likely to depend on prior selection on phenotypic and
 transcriptional plasticity in response to hypoxia, and may therefore show
 signs of local adaptation. Here we report the lifespan effects of chronic,
 mild, intermittent hypoxia (CMIH) and short-term survival in acute severe
 hypoxia (ASH) in four clones of Daphnia magna originating from either
 permanent or intermittent habitats, the latter regularly drying up with
 frequent hypoxic conditions. We show that CMIH extended the lifespan in
 the two clones originating from intermittent habitats but had the opposite
 effect in the two clones from permanent habitats, which also showed lower
 tolerance to ASH. Exposure to CMIH did not protect against ASH; to the
 contrary, Daphnia from the CMIH treatment had lower ASH tolerance than
 normoxic controls. Few transcripts changed their abundance in response to
 the CMIH treatment in any of the clones. After 12 hours of ASH treatment,
 the transcriptional response was more pronounced, with numerous
 protein-coding genes with functionality in mitochondrial and respiratory
 metabolism, oxygen transport, and, unexpectedly, gluconeogenesis showing
 up-regulation. While clones from intermittent habitats showed somewhat
 stronger differential expression in response to ASH than those from
 permanent habitats, there were no significant hypoxia-by-habitat of origin
 or CMIH-by-ASH interactions. GO enrichment analysis revealed a possible
 hypoxia tolerance role by accelerating the molting cycle and regulating
 neuron survival through up-regulation of cuticular proteins and
 neurotrophins, respectively.
description: <em>Clones’ provenance and maintenance</em>
 The geographic origins of the four clones used in this study are
 listed in Supplementary Table S1. Clones were obtained from Basel
 University (Switzerland) Daphnia Stock Collection (Dieter Ebert, personal
 communication) and propagated asexually in the lab in COMBO water (Kilham
 et al. 1998) at 20 °C under a 16:8 D:L light cycle and fed by a diet of
 <em>Scenedesmus acutus</em>. Prior to all experiments,
 experimental cohorts were established through the following procedure:
 Five randomly selected grandmother females from each clone were maintained
 from birth till their 3<sup>rd</sup> clutch of offspring were
 born at the density of 1 individual per 20 ml of COMBO water with
 <em>Scenedesmus</em> food added daily to the concentration of
 10<sup>5</sup> cells/ml and water changed every four days.
 Offspring from their 2<sup>nd</sup> and
 3<sup>rd</sup> clutches were used to establish the maternal
 generation, which was in turn maintained in the same conditions until
 enough offspring from the second or consecutive clutches could be
 collected to form the experimental cohorts. Neonates were maintained in
 groups of 20 in 200 mL jars with COMBO water for the first 6 days of their
 lives until transferred to the corresponding experimental tanks.
 <em>Chronic mild intermittent hypoxia
 treatment</em> <em>Daphnia
 </em>cohorts were maintained in 5-L tanks each containing eight
 plastic containers with 1 mm nylon mesh bottoms, which allowed free water
 exchange and the removal of neonates during water changes. Water volume
 and daily food ratios were adjusted every 4 days to maintain 20 ml of
 water per individual, and 10<sup>5</sup> <em>Scenedesmus
 </em>cells were added per mL per day. The water was changed,
 neonates removed, and a census of <em>Daphnia </em>cohorts
 conducted every three days. Chronic mild intermittent hypoxia treatment
 (CMIH thereafter) was achieved by bubbling N<sub>2</sub>
 through the experimental tanks with continuous monitoring of oxygen
 concentration by the Extech DO210 probe (Nashua, NH, USA) twice daily
 until the concentration was lowered to 4 mg/L. At the same time, the
 control tanks were aerated with ambient air until the oxygen concentration
 reached 8 mg/L. Between these procedures, the oxygen concentration in the
 hypoxia tanks typically raised to 6.5 mg/L by diffusion, whereas the
 control tanks typically experienced a drop in oxygen concentration of
 7–7.5 mg/L due to respiration of <em>Daphnia</em>
 (Supplementary Fig. 1new). At the age of 100 days (at about 20% survival)
 replicate tanks were combined in order to maintain constant volume per
 remaining individuals without dropping water level close to the
 containers’ mesh bottoms. Due to space and handling
 time limitations, the CMIH experiment was conducted in two blocks, with
 each block consisting of two CMIH and two control tanks. To investigate
 whether early life exposure to mild hypoxia provides the same protection
 as life-time exposure, an additional replicate tank was set up within the
 CMIH treatment which was switched to normoxic condition on day 30 of the
 experiment. Because, due to space and handling time constraints, this
 switch treatment included only one replicate tank, the results of this
 comparison will be interpreted as highly speculative and
 preliminary. At various time points, individuals from
 each cohort were sampled for body size, fecundity, feeding rate, and
 respiration rate measurements (with replacement), or for lactate and
 pyruvate concentration determination, acute hypoxia tolerance experiment,
 or RNAseq (without replacement). Additionally, hemoglobin concentration in
 tissues was measured in <em>Daphnia</em> from CMIH and
 normoxic control treatments by means of light microscopy (with
 replacement) and (without replacement) by light absorption in homogenates
 (Ekwudo 2021). These measurements showed the expected increase of
 hemoglobin concentration in the CMIH treatment (Ekwudo 2021) and are not
 reported here.   <em>Lactate and pyruvate
 measurements</em> Lactate and pyruvate assays
 were conducted using CellBiolab kits on 15- to 20-day-old and 55- to
 60-day-old <em>Daphnia</em> from two clones (GB and IL)
 sampled from the experimental tanks and stored frozen at -80˚C until assay
 time. Each <em>Daphnia</em> was homogenized in 100 µL ice-cold
 PBS with a pestle, and the homogenates were centrifuged at 4˚C. 25 µL of
 supernatant were pipetted into each of the lactate and pyruvate assay
 plates using the manufacturer’s protocol (CellBiolab catalog #s 101820174
 & 82320181). Additionally, two replicate aliquots of 15 µL of the
 supernatant each were used to quantify soluble proteins by Bradford assay,
 with 185 µl of Bradford colorimetric reagent added to each well. All assay
 well plates were analyzed using a BIOTEK plate reader (Agilent, Santa
 Clara, CA, USA). Lactate and pyruvate fluorescence assay sensitivity was
 set to 35, and the Bradford assay absorption was measured at 595
 nm.  <em>Respiration and filtering rates
 measurements</em> Respiration rate was measured
 in individual <em>Daphnia</em> sampled from the cohorts by
 placing them in either 200 μL or 1700 μL 24-well respirometry glass plates
 (Loligo ®, Denmark), sealing them with PCR sealing tape, and measuring
 oxygen concentration using SDR fluorescence sensors (PreSens, Germany).
 Either normoxic (8 mg O<sub>2</sub>/L) or hypoxic (4 mg
 O<sub>2</sub>/L) COMBO water was used in these measurements.
 Each measurement continued for 45–60 minutes, with oxygen concentration
 recorded every 15 seconds until the oxygen concentration dropped by at
 least 1 mg/L. The first 15 minutes after the transfer of Daphnia into the
 respirometry plates were discarded as the break-in period. The slope of
 the linear regression of oxygen concentration over time was used as the
 measure of oxygen consumption rate. Feeding (filtering)
 rate was measured by placing individual <em>Daphnia</em> into
 plastic 24-well plates with normoxic COMBO water containing the initial
 concentration of 2x10<sup>5</sup>
 <em>Scenedesmus</em> cells per mL and measuring chlorophyll
 fluorescence at the start and after 8, 12, and 24 hours of filtering
 activity. After both respiration and feeding rate
 measurements, <em>Daphnia</em> were measured, weighed to
 determine their wet weight to the precision of 0.1 mg, and returned to the
 experimental tanks. <em>Mitochondrial membrane
 potential</em> Samples for mitochondrial membrane
 potential (ΔΨ<sub>m</sub>) measurements were taken from the
 cohorts at the median lifespan of all cohorts, i.e., at the age of 80 days
 to maximize longevity-related effects. ΔΨ<sub>m</sub> was
 measured by means of rhodamine-123 assays as described in Anderson et al
 (2022). Briefly, <em>Daphnia</em> were exposed for 24 h to a 4
 μM solution of rhodamine-123 dye, washed 3 times, and photographed using a
 Leica DM3000 microscope with a 10x objective (0.22 aperture) equipped with
 a Leica DFc450C color camera, with a 488 nm excitation / broadband
 (&gt; 515 nm) emission filter. The fluorescence from the following
 tissues and organs was measured: antenna-driving muscle, epipodite, brain,
 and optical lobe, with the median intensity of fluorescence used as the
 measure of ΔΨ<sub>m</sub>. We have previously shown that
 ΔΨ<sub>m</sub> does not change much with age in most tissues
 (Anderson et al. 2022), so it is likely that these measurements are
 representative of any age of <em>Daphnia</em> exposed to CMIH
 for a sufficient time. <em>Acute Severe Hypoxia
 experiments and sample collection for RNA-Seq</em>
 For acute severe hypoxia (ASH) tolerance measurement, 25-day-old
 <em>Daphnia</em> were sampled from each of the four tanks in
 one of the two CMIH experiment blocks. They were moved into 70 mL cell
 culture flasks filled with COMBO water with the concentration of oxygen at
 or below 1 mg/L, sealed without air bubbles, 7
 <em>Daphnia</em> per flask, 5 replicate flasks per clone per
 treatment. Flasks were kept at 20˚C. The acute hypoxia experiment was set
 up at 9:00 p.m. and mortality was recorded 12 hours later and every hour
 thereafter. Individuals for RNA sequencing were frozen at the beginning of
 the experiment (controls) and after 12 hours of exposure (before any
 mortality occurred), sampling 2 individuals from each flask (ASH
 treatment). Flasks were then topped with 1 mg O<sub>2</sub>/L
 water and sealed again. Survival time analysis was performed by
 proportional hazards model with CMIH treatment and habitats as main
 effects with clones nested within habitats (all effects fixes) using the
 Survival platform of JMP statistical package (Ver. 16; SAS Institute
 2021), and by the same model with CMIH and habitats as fixed effects,
 clones as random effect nested within habitats and flasks as a random
 effect nested within clones using coxme R package (Therneau et al.
 2003). <em>RNA Sequencing</em>
 Two individuals from each of the four clones (IL, FI, GB, and HU)
 and from each of the hypoxia treatments were frozen during the acute
 hypoxia experiment (see above). The four treatments were in full factorial
 fashion with respect to CMIH and ASH: <em>Daphnia</em> reared
 at normoxia; <em>Daphnia</em> reared at normoxia and exposed
 to acute hypoxia for 12 hours; <em>Daphnia</em> reared in
 CMIH; and <em>Daphnia</em> reared in CMIH and exposed to acute
 hypoxia for 12 hours. <em>Daphnia</em> for RNAseq were taken
 directly from the CMIH and ASH experiments described above (with
 appropriate censoring of survival data). RNA was extracted using Qiagen
 RNAeasy kit (Cat ID: 74134) and quantified using a Qubit (ThermoFisher)
 fluorometer. Following extraction, RNAs were reverse
 transcribed, and sequencing libraries were constructed from the cDNAs as
 prescribed by the Oxford Nanopore Technology (Oxford, UK) PCR-cDNA
 Barcoding kit protocol (SQK-PCB109), with 3 biological replicates per
 clone per treatment, each replicate consisting of RNA extracted from two
 <em>Daphnia</em> individuals. Barcoded samples from the 4
 treatments within each clone were pooled together into 3 replicate
 libraries, purified separately, and pooled together immediately before
 adding the sequencing adapter. Libraries were then sequenced using Oxford
 Nanopore MinION for 24–48 hours per sequencing run, obtaining 2–4 Gb of
 reads in each run. <em>RNAseq data
 analysis</em> Base-calling and reads filtering,
 demultiplexing, trimming, and mapping were accomplished using ONT Guppy
 software (ver. 3.6). <em>Daphnia magna</em> reference
 transcriptome 3.0 (D.Ebert and P.Fields, NCBI BioProject ID: PRJNA624896)
 was used as a reference. For the purpose of this analysis, the reference
 transcriptome containing only the longest isoform for each gene was used,
 with a total of 33,957 transcripts, of which at least one read mapped to
 22,445 transcripts. Transcripts were then filtered to retain only those
 that contained at least 72 reads across all samples, resulting in a set of
 6050 transcripts retained for further analysis. As each
 library and each sequencing run consisted of 3 biological replicates of
 each of 4 combinations of CMIH and ASH treatments from a single clone,
 clones were fully confounded with replicate library preparation and
 sequencing runs. The advantage of this design is that each library
 preparation contains a balanced set of all 4 treatments with a
 clone-library replicate combination that can be used as a block effect in
 the Likelihood Ratio tests (see below). The disadvantage of this design is
 the lack of the ability to test for the difference among clones, since it
 does not allow untangling the variance among clones from random variance
 among library preparation and sequencing runs.
 Differential expression was analyzed using Likelihood Ratio tests
 (LRT) in DESeq2 (Love et al. 2014) with CMIH treatment, ASH treatment, and
 habitat of origin as orthogonal effects and clones as an effect nested
 within habitats. In DESeq2 analysis, log fold change noise was shrunk by
 the apeglm algorithm for the Wald test (Zhu et al. 2019). DESeq2 analysis
 was conducted separately for full data, for the two habitats of origin
 separately, and for the analysis of the CMIH factor for the subset of
 samples not exposed to ASH treatment (ASH=Control). For LRT analysis, the
 reduced model consisted of all factors except the factor being tested and
 all its interactions. Wald tests and LRTs yielded similar results; with
 only LRT results reported. An arbitrary adjusted p-value of
 p<sub>adj </sub>&lt; 0.1 was chosen as the cut-off for
 reporting differential gene expression in any given transcript. R scripts
 are available in the Supplementary materials.
 Enrichment analysis was conducted separately for lists of
 transcripts with a possible significant effect for each of the main
 effects (CMYH, ASH, habitat type) and interactions. Two sources of gene
 lists and two types of annotation data were used. Gene lists were obtained
 by selecting transcripts with an uncorrected p &lt; 0.01. The two
 approaches to the annotation data were: a closed, non-overlapping list of
 transcripts with functions a priori known to be hypoxia-related; and an
 open, overlapping list of GO categories. Firstly, we constructed a
 non-overlapping list of annotation terms that characterized pathways and
 functions a priori known to play a role in hypoxia response. The only
 deviations from the non-overlapping principle were the fused genes
 containing vitellogenin and superoxide dismutase domains (Kato et al.
 2004), which appeared both in the “vitellogenins” and “antioxidant
 pathway” gene lists. The source of annotation was a combination of
 descriptions obtained from the <em>D. magna</em> genome
 annotation available at (http://arthropods.eugenes.org/genepage/daphniamagna/Dapma7bEVm000001t1; Gilbert 2002) and annotations obtained for the <em>D. magna</em> 3.0 transcriptome by blast2GO (Götz et al. 2008) and PANTHER (Mi et al. 2013) software. Secondly, we used all GO annotations generated by blast2GO and PANTHER to construct an open list of all GOs; this list was filtered to include only GOs represented in the reference transcriptome by at least 10 transcripts. In both cases, the Fisher exact test was used to test for enrichment, testing the hypothesis that a given pathway, function, or GO had a higher than random representation in the list of candidate genes (left-handed FET).
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