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.

Clones’ provenance and maintenance

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 Scenedesmus acutus. 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^rd clutch of offspring were born at the density of 1 individual per 20 ml of COMBO water with Scenedesmus food added daily to the concentration of 10⁵ cells/ml and water changed every four days. Offspring from their 2^nd and 3^rd 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.

Chronic mild intermittent hypoxia treatment

Daphnia 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 10⁵ Scenedesmus cells were added per mL per day. The water was changed, neonates removed, and a census of Daphnia cohorts conducted every three days. Chronic mild intermittent hypoxia treatment (CMIH thereafter) was achieved by bubbling N₂ 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 Daphnia (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 Daphnia 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.  

Lactate and pyruvate measurements

Lactate and pyruvate assays were conducted using CellBiolab kits on 15- to 20-day-old and 55- to 60-day-old Daphnia from two clones (GB and IL) sampled from the experimental tanks and stored frozen at -80˚C until assay time. Each Daphnia 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. 

Respiration and filtering rates measurements

Respiration rate was measured in individual Daphnia 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 O₂/L) or hypoxic (4 mg O₂/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 Daphnia into plastic 24-well plates with normoxic COMBO water containing the initial concentration of 2x10⁵ Scenedesmus 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, Daphnia were measured, weighed to determine their wet weight to the precision of 0.1 mg, and returned to the experimental tanks.

Mitochondrial membrane potential

Samples for mitochondrial membrane potential (ΔΨ_m) 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_m was measured by means of rhodamine-123 assays as described in Anderson et al (2022). Briefly, Daphnia 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 (> 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_m. We have previously shown that ΔΨ_m 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 Daphnia exposed to CMIH for a sufficient time.

Acute Severe Hypoxia experiments and sample collection for RNA-Seq

For acute severe hypoxia (ASH) tolerance measurement, 25-day-old Daphnia 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 Daphnia 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 O₂/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).

RNA Sequencing

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: Daphnia reared at normoxia; Daphnia reared at normoxia and exposed to acute hypoxia for 12 hours; Daphnia reared in CMIH; and Daphnia reared in CMIH and exposed to acute hypoxia for 12 hours. Daphnia 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 Daphnia 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.

RNAseq data analysis

Base-calling and reads filtering, demultiplexing, trimming, and mapping were accomplished using ONT Guppy software (ver. 3.6). Daphnia magna 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_adj < 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 < 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 D. magna genome annotation available at (http://arthropods.eugenes.org/genepage/daphniamagna/Dapma7bEVm000001t1; Gilbert 2002) and annotations obtained for the D. magna 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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