The response of cancer cells to hypoxic conditions found within the interior of a tumor mass is mediated through the hypoxia inducible factor (HIF) cascade and is thought to promote metastasis. However, given their distant proximity from blood vessels as compared to normoxic cells at the vascularised tumor periphery, it is uncertain if these cells can migrate through the tumor mass to gain access. Hypoxia was simulated by exposure to cobalt chloride or deferoxamine in normal (MCF10A) and cancerous  cells. In this report, HIF1α expression and localization was measured using western blotting, ELISA, and immunofluorescence, cell proliferation by MTT assay, motility and invasion by wound healing, live cell imaging, matrigel and co-culture in chambered slides. We found that the expression and nuclear translocation of HIF1α was significantly elevated by hypoxia, which inhibited cell proliferation, but significantly increased motility of pII cells and their penetration into and through a dense layer of adjacent EII cells, as well as their selective emergence out of a co-culture. These data suggest that endocrine resistant pII cancer cells, having undergone epithelial to mesenchymal transition are able to penetrate through other cell layers, with possible enhancement in response to hypoxia. This condition is correlated with poor prognosis as well as poor outcome of anti-cancer therapies5.Hypoxia is a state of low oxygen level, and represents a pernicious effect of diseases such as cardiovascular diseases, diabetes, dementia and cancer. It is variably observed in most if not all solid tumors, depending on the type, size and stage6. Acute and chronic states of hypoxia can lead to variable outcomes in cancer cells7. Under conditions of acute hypoxia they adapt by decreasing reliance upon oxidative metabolism for energy generation, while increasing autophagy, reactive oxygen species (ROS) generation and metastasis, leading to tumor survival and progression12. Chronic hypoxia for a few hours to several weeks leads to high frequency of DNA breaks and accumulation of replication errors leading to genetic instability and mutagenesis15. Hypoxia also appears to be a driver of alternative splicing events in a large number of genes involved in multiple processes of tumorigenic development16.In the breast, hypoxia has been shown to induce genome-wide changes in DNA hydroxy-methylation that leads to the acquisition of breast tumor-initiating cell properties with up-regulation of tumor necrosis factor and activation of the p38-MAPK effector pathway17, is considered to be the major downstream transcription factor activated in both hypoxic and non-hypoxic conditions by stimuli such as nitric oxide (NO), ROS, cytokines, lipopolysaccharide, G-protein coupled receptors and toll-like receptors21. In the presence of oxygen, HIF-1α subunits are hydroxylated by oxygen-dependent enzymes such as prolulhydroxylases and factor inhibiting HIF-122 leading to poly-ubiquitination and proteasomal degradation of the α subunits23. Under hypoxic conditions, these oxygen-sensor enzymes lose their activity resulting in HIF-1α protein stabilization, accumulation and translocation to the nucleus to dimerize with the constitutively expressed HIF-1β subunits to bind to target hypoxia-responsive elements and induce expression of genes involved in cell anaerobic metabolism, survival, metastasis, angiogenesis and drug resistance. These include VEGF, SDF-1, Ang-II, MMPs 2 and 9, BNIP-3, p53, E-cadherin, CXCR4, LOX, CAIX, GLUT-1, and GSK30. A recent study indicated that the TRAF6-H2AX-γH2AX- axis mediates HIF1α enrichment in the nucleus of cancer cells leading to its stabilization and activation to enhance tumorigenesis, glycolysis and metastasis31. HIF-1-α was shown to be expressed in the majority of cancer biopsies at the leading edge of invading tumors32 and correlated with increased risk of metastasis and poor prognosis43.The hypoxia-inducible factor (HIF)in vitro by incubation in low oxygen environment using a specialized chamber, or by incubation with chemical agents. Exposure to cobalt chloride (CoCl2)  was shown to induce HIF-1α expression in PC-2 human pancreatic cancer cells46. Deferoxamine (DFO), a bacterial sidephore that chelates iron and inhibits iron-dependent prolyl hydroxylases thus preventing the degradation of HIF isoforms in normoxic conditions49 has also been used to induce a state of hypoxia in vitro. Kilic and colleagues recently showed increased release of extracellular vesicles from MCF-7 breast cancer cells, due to CoCl2 induced hypoxia50. These are increasingly implicated in cancer progression through inter-cellular communication and transfer of mediators.Hypoxia can be stimulated 53, and enhancement of cell proliferation45 and invasion54 which promotes tumour metastasis. This has led to the popular belief that metastasis is promoted by an acidic extracellular environment, which is expected to be found around anaerobically metabolizing cells producing and secreting excessive amounts of lactate , i.e. the hypoxic cells. It has also been suggested that there may be a ‘metabolic symbiosis’ between hypoxic cells producing lactate and oxygenated cells utilizing it as a substrate to feed into their oxidative cycle. However, the obvious but apparently un-addressed anomaly here is two-fold. Firstly, the hypoxic cells are buried within the core of the tumour and would have to penetrate through a dense meshwork of more oxygenated cancer and normal cells to reach the tumour periphery to contact blood vessels. Secondly, it is the cells at the neo-vascularized normoxic surface of the tumour mass that are actively proliferating, and therefore logically may be expected to have greater opportunity to metastasise. So we are faced with this intriguing question – which cells in the tumour mass actually metastasise? Clearly this issue will not be easy to resolve and longer term may be best addressed by video imaging of tumours in vivo when this becomes possible.It seems to be generally held that hypoxic environment modulates various effector functions of cancer cells such as induction of epithelial to mesenchymal transition (EMT)in vitro model that could provide some preliminary indications to address the questions posed above. Using the weakly-invasive estrogen receptor (ER) +ve MCF7 parental cells and the highly invasive ER silenced pII cells, we proposed to firstly examine their relative proliferative, motile and invasive abilities under normoxic/hypoxic conditions, comparing these also with normal MCF10A breast epithelial cells. Then, to try to simulate a tumor mass by mixing the different cells to determine whether they can penetrate through layers of each other before/after pre-treatment with HIF1α inducing agents to simulate the conditions of hypoxia.In the current report it is intended to look at this question in the limited setting of an 55. pII is ER-silenced while EII is a control line containing the shRNA carrying plasmid without ER down-regulation and constitutively expressing green fluorescent protein (GFP) as a marker. YS1.2 is MCF7 transfected with ER-directed shRNA but also failed to down-regulate ER and remained ER +ve56.MCF10A  was obtained from Dr E Saunderson St Bartholomews Hospital, London. MCF7 (estrogen receptor ER +ve breast cancer cells) were originally obtained from the ATCC . pII (ER −ve) and EII (ER +ve) are stable shRNA transfected cloned lines derived from the MCF7 line2 in Dulbecco’s modified eagle’s medium (Advanced DMEM), supplemented with 5% fetal bovine serum (FBS), 600 mg/mL L-glutamine, 100 U/Ml penicillin, 100 mg/mL streptomycin and 6 mL/500 mL 100 x non-essential amino acids. Cells were routinely grown in monolayer in 25 or 75 cm2 tissue culture flasks inside an incubator maintained at 37 °C with 5% CO2 atmosphere at 95% humidity.MCF10A cells were cultured in DMEM F12 medium supplemented with 5% horse serum, 1x Pen/Strep, 20 ng/mL EGF, 0.5 µg/mL hydrocortisone, 100 ng/mL cholera toxin and 10 µg/mL insulin. All other cell lines were routinely maintained at 37 °C in a humidified atmosphere of 5% COCell cultures were periodically treated with mycoplasma removal agent from Biorad (USA) and tested with detection kits from Invivogen (USA) and DAPI nuclear staining to ensure they remained free of mycoplasma.6 pII cells prior to incubation at 37 °C in 5% CO2 for 1 h. Following this incubation, the media was discarded and replaced with fresh media. The dye has an excitation of 405–585 nm and emission of 625 nm.Qtracker 625 cell labeling kit  was used to label pII cells (red dye) to monitor their motility. This was performed by mixing 1 µl each of solution A and B for 5 min followed by addition of 200 µl DMEM and mixing with 1 × 10Cells were cultured in 6 well plates with complete DMEM to 80–90% confluence, and the medium was subsequently aspirated off and cell monolayers harvested by scraping and re-suspension into 300 μl of lysis buffer containing 50 mM HEPES, 50 mM NaCl, 5 mM EDTA 1% Triton X, 100 µg/ml PMSF, 10 µg/ml aprotinin, and 10 µg/ml leupeptin. Protein concentration was determined by the Bradford assay using BSA as standard, and 8 μg protein lysate was mixed with an equal volume of 2 x SDS and heated at 90 °C for 10 min. Samples were loaded onto a 10% SDS-polyacrylamide gel and electrophoresed at 150 V for 1 h. Proteins were transferred to a nitrocellulose membrane and blocked with 2% BSA for 1 h before being incubated overnight at 4 °C with HIF1α or actin (loading control) antibody  (1:1000 dilution) prepared in 2% BSA. The membrane was washed and incubated with anti-HRP-conjugated secondary antibody  (1:1000 dilution) for 1 h, developed with Super Signal ECL and visualized with Kodak X-ray film.2 independent media) and allowed to settle for 48 h. The media was then removed and replaced with media containing either Cobalt (II) chloride (CoCl2) or Deferoxamine mesylate salt (DFO)  at 100 µM and left for 30 min, 1 h, 4 h, 24 h and 48 h at which times cells were fixed by the addition of 3.7% formaldehyde for 10 min and washed with ice-cold PBS. Cells were treated with 1% BSA and then incubated with HIF1α antibody (1:80 dilution) overnight at 4 °C. Unbound primary antibody was aspirated, cells washed with ice-cold PBS and incubated with anti-rabbit secondary antibody (1:250 dilution). Excess unbound secondary antibody was aspirated off, cells were washed with ice-cold PBS, fixed and stained with phalloidin  (1:125 dilution) to stain for F-actin and DAPI to stain the nucleus. After removal of the silicone scaffolding and mounting, cells were examined under a confocal microscope  at an excitation wavelength of 450 nm and images analyzed and processed using a ZEISS software package.Cells were seeded at approximately 5000 cells/well in an 8-chambered slide containing 200 µl/well DMEM media  or after 1 day and 4 days of growth. For the measurement, medium was removed and replaced with 500 µl of MTT reagent  (0.5 mg/ml) and left at 37 °C for 2 h; MTT solution was removed and 200 µl of acidic isopropanol added to dissolve the blue formazan crystals. Plates were scanned at 595 and 650 nm using a MULTISKAN SPECTRUM spectrophotometer.Cells were seeded into 24-well culture plates and allowed to grow to 30–35% confluency. The medium was removed and replaced with medium containing 100 µM CoCl2O), or various concentrations (25–100 µM) of CoCl2 or DFO. A fine scratch was made using a sterile yellow 200 µl Eppendorf pipette tip through the center of the YS1.2 cell monolayer and 1000 µl tip for pII and MCF10A cell monolayers. The width of the scratch was recorded under phase contrast light microscopy immediately (0 h) and after 24 h of incubation to determine the extent of wound closure.Cells were grown to 90% confluency in 12 well plates, and treated with vehicle (dH4 cells in 100 µl of DMEM media) at 37 °C, 5% CO2 overnight to attach. On the following day, the spacer between the cells was removed and 2 ml of CO2 independent culture media (to maintain pH at 7.4) was added and the slide placed inside an imaging chamber  heated to 37 °C with an airstream. Live cell microscopy with time-lapse photography, was used to continuously monitor the migration of cells (pII and EII) into their opposite compartment. For this, the plate was positioned to enable photography of both cells on the edges and an empty space in the middle and images (at 10 x magnification) were taken every 10 min. The AxioVision software (ZEISS) was used to combine all the pictures to generate a video.Relative movements of the different cell lines were studied using an Ibidi µ chamber plate  which allows segregated cultivation of cells. Non-invasive EII cells, which have intrinsic green fluorescence due to expression of GFP, and the highly invasive pII cells (previously labeled with the Qtracker red dye) were grown in adjacent chambers of an Ibidi plate (1 × 102 or DFO) were serum starved overnight at 37 °C and 5% CO2. On the following day, cells were harvested and re-suspended in serum free media and 100 µl containing 105 cells were added into the upper chamber. The lower chamber was loaded with 500 µl of serum free media (negative control) or media containing 40% FBS as a chemo-attractant. Cells were incubated at 37 °C, 5% CO2 and allowed to invade from the top to the bottom chamber. After 48 h, the media was aspirated from the upper chamber and the bottom washed with 1x cell washing buffer. Calcein-AM/cell dissociation solution was added to the bottom chamber and left for 1 h at 37 °C, 5% CO2. Cells present in the bottom chamber were quantitated by their ability to internalize Calcein-AM, with intracellular esterases cleaving the acetomethylester (AM) moiety generating free fluorescent calcein which could then be determined by recording the fluorescence emission using a microplate reader with a filter set of Excitation/Emission = 485/535 nm .The upper inset chambers of a Cultrex assay dish were coated with 100 µl of 1x basement membrane extract (BME) solution and incubated overnight at 37 °C. Meanwhile, cells  were added into the upper chamber and allowed to settle at 37 °C overnight. Then, 100 µl of pII cells (105) that had been 24 h serum starved either in the absence (control) or presence of 100 µM CoCl2 overnight at 37 °C and 5% CO2 and re-suspended in serum free media were added to form a layer on top of the EII cells. The plate was then left overnight at 37 °C in 5% CO2 and procedure followed as already described above.The Cultrex inset chamber was coated with 1x BME and left to set overnight as previously described. On the next day, serum starved EII cells were re-suspended in serum free media and 100 µl of cells  were created in the gel, 2.5 mm apart in a horizontal line, by insertion of a metallic mold as described previously57. Suspensions of pII and YS1.2 cells (4 × 104) in DMEM containing 5% FBS, that had previously been exposed for 24 h to CoCl2 or DFO (100 µM) or to vehicle only were loaded into the formed chambers. Plates were incubated at 37 °C in 5% CO2 humidified atmosphere. After 24 h, cells that had penetrated the agarose were manually counted by visual microscopic examination as previously described57.Ultra-pure agarose  (0.18 g) was dissolved by boiling in 10 ml dH4 cells suspended in 100 µl of DMEM were grown in separate compartments of an Ibidi chamber plate. The plate was incubated at 37 °C, 5% CO2 overnight. On the next day, the spacer between the cells was carefully removed and replaced with 2 ml of DMEM containing CoCl2 or DFO (100 µM) or vehicle (control). The number of pII cells migrating/invading through EII cells in all the conditions was recorded under phase contrast and fluorescence light microscopy immediately after removing the spacer and at various intervals thereafter (24–96 h).To monitor the migration/invasion of labeled pII cells towards EII cells, 1 × 104) suspended in 100 µl of DMEM were mixed together in different proportions  in an Ibidi chamber plate. The plate was incubated at 37 °C, 5% CO2 overnight. On the next day, the spacer was removed and medium replaced with 2 ml DMEM containing CoCl2 (100 µM) or vehicle. The number of pII cells moving/invading out of the mixture was recorded as previously described.To monitor the migration of labeled pII cells out of a co-culture with EII cells, the two cell cultures  cells and at various times (0.5–48 h) after addition of CoCl2 or DFO. As shown in Fig. 2 induced level of HIF1α observed in the ER−ve pII cells after 24 h was of four-fold greater magnitude than that seen in MCF10-A and YS1.2 cells. But interestingly the relative increase in HIF1α from the untreated control was approximately 26–30 fold in MCF10A cells and only 3–4 fold in the cancer cells. The effect of DFO was also tested on pII cells and was found to be similar to that of CoCl2  at 30 min to 48 h. Cells were fixed and labeled with HIF1α antibody, phalloidin  and DAPI (nuclear stain). Treatment of MCF10A cells with either CoCl2  or DFO (B) treatment at earlier times (0.5–4 h) while nuclear expression peaked at 24 h. In regard to pII cells  or DFO (B) treatment but at 24–48 h of stimulation HIF-1α was detected in the nucleus. Due to its poor immunoreactivity (possibly due to low expression) HIF1α staining in all the figures  treatment on cell proliferation was assessed using the MTT assay. As shown in Fig. 2 or DFO decreased proliferation of both normal (MCF10A) as well as the cancerous cells (YS1.2 and pII). This inhibition was seen at 24 h (A-C) and maintained at 96 h (D-F). The effect was more profound in the cancerous cells particularly at the shorter time and was also more pronounced with DFO. As this was somewhat unexpected, and to ensure that the results were not due to any impairment in mitochondrial function (at least in the enzymes involved in the MTT conversion) we also performed a proliferation assay with manual cell counting using a haemocytometer and found similar results (data not shown) to using the MTT assay.The effect of CoCl2 and DFO treatment significantly increased YS1.2 (A) and pII (B) cell motility with maximal effect at 100 µM. It should be noted that the scratch in YS1.2 was made with a yellow tip as compared to a blue tip for the pII scratch creating a much wider gap. Hence the actual movement (as opposed to % closure) of YS1.2 is much less than for pII. We also examined motility of MCF10A cells and surprisingly found that they were highly motile. As their culture medium contains stimulatory additives, we repeated the experiments omitting EGF in one case and in another, replacing the medium (24 h prior) with the DMEM medium used for the cancer cells. The data  assay was used to determine the effect of hypoxic simulation on the motility of ER +ve and ER −ve breast cancer cells. As illustrated in Fig. 2/DFO. As shown in Fig. 57, and CoCl2/DFO treatment did not significantly increase the number of cells migrating out of the well into the agarose. The low migratory capacity of YS1.2 cells was not influenced by either drug.The effect of hypoxia on random migration of ER +and ER− breast cancer cells was assessed using the agarose assay. Both YS1.2 and pII cells were pre-treated with either vehicle (control) or CoCl2/DFO for 24 h and then loaded into the top chamber separated from the bottom chamber containing medium supplemented with 40% serum as a chemo-attractant. Cells invading into the lower chamber were assessed after 24 h. Figure 2/DFO treatment did not significantly increase cell invasion of either through the BME layer.The Cultrex BME cell invasion assay was also used to assess the effect of hypoxic simulation on invasion of YS1.2 and pII cells. In this assay, both YS1.2 and pII cells were pre-treated with vehicle or CoClinto the EII cell monolayer since we could not distinguish between the two cell populations despite that the EII cells were fluorescently labelled with GFP.Live cell microscopy was also used to monitor the movement of ER −ve  and ER +ve  breast cancer cells co-cultured in an Ibidi chamber plate under normal conditions. As shown in Fig. 2 or DFO; snap shots were taken using phase contrast and fluorescence mode every 24 h. As shown in Fig. 2), pII cells started to move towards the EII monolayer and closed the space between them. On day 2, pII cells began to invade into the EII population, and at day 3 some pII cells penetrated through the EII monolayer. This was also observed with DFO treatment (data not shown). There was a 2-fold increase in the number of pII cells invading the EII monolayer under hypoxic conditions as compared to normoxia  and snap shots taken in phase contrast and fluorescence mode at 24 h intervals. In the first experiments we used lower confluency (about 60%) of EII cells (data not shown) and found that many pII cells were present inside the EII layer possibly because there was empty space so we increased EII cells to 80% confluency to test whether pII cells are able to penetrate a more solid barrier of another cell line. As shown in Fig. xia Fig. . Althougxia Fig. . There w2. Two experimental settings were performed; a), with an equal ratio of ER +ve and ER −ve cells (data not shown) and b), with ER +ve: ER −ve cells at a ratio of 4:1  were loaded into the top chamber of a transwell plate in which EII cells had previously been seeded on top of a BME layer. After 24 h incubation, only pII cells were found in the lower chamber but the number of these was not significantly different whether pre-exposed to vehicle or CoCl2. Moreover, the number of penetrating cells was far fewer than seen in a parallel set-up in which the upper chamber was seeded with a similar number of cells in the absence of EII cells, giving direct contact with the BME layer  or with 100 µM CoCl6. It has been reported that hypoxic cells are highly invasive59, so could these be the cells that metastasize from a tumor? This would entail migration into and possibly through the upper layers of cancer cells that remain adherent, in order to intravasate into physiologically functional blood vessels most likely located at the tumor periphery.An important feature of metastasis is that it appears to involve individual or small groups of cells detaching from the tumor mass and therefore the further question arises – which cells from within the tumor metastasize? Is this a random process or does it involve specific cells? Is it the cells at the tumor periphery or those further inside? Neo-vascularisation of a growing tumor mass is disorganized and incomplete, such that the microenvironment of cells nearer to the core is more hypoxic compared to the cells at the periphery, and this phenomenon may be a major factor in cancer progression and metastasis60. HIF1α protein and mRNA levels were elevated in human pancreatic cancer cells in response to (100–200 μM) CoCl2 treatment for 0–12 h in a time and dose dependent manner46. Also, Chan et al.13 demonstrated enhanced HIF-1α expression in human lung carcinoma cell lines grown in low O2 conditions. DFO treatment (30–300 µM for 24 h) of MDA-MB-231 cells enhanced HIF1α protein expression61. In this study, we showed that HIF1α expression in response to CoCl2 (100 µM) treatment was significantly increased particularly at 4–24 h of stimulation and declined at 48 h in all the cell lines  was seen in the ER −ve breast cancer cells (which have a higher proliferation and migration rate in comparison to ER + breast cancer cells), with the lowest expression in the normal epithelial breast cells. DFO treatment of pII cells also enhanced HIF-1α expression to similar levels  HIF1α was translocated to the nucleus in all the cell lines. This pattern was also observed following exposure to hypoxia (2.5% O2). In MCF-7 cells, induction of hypoxia using CoCl2 treatment (200 µM for 24 h) increased HIF1α nuclear localization and up-regulation of its expression63. In agreement with these studies, we too observed cytoplasmic HIF-1α only after hypoxic exposure, with subsequent translocation to the nucleus after 24–48 h.Under hypoxic conditions, HIF1α is accumulated into the nucleus2 for 36 h45 in MCF7 cells treated with CoCl2 showing a peak of proliferation at 150 µM with inhibition at higher doses64 and similarly in MDA-MB-231 cells, at 25 µM with higher concentrations decreasing proliferation rate64. Other reports showed decreased cell growth upon exposure to hypoxic environment. For example, Chan et al.13 reported that 72 h exposure of human lung carcinoma (H1299) cells to 0.2% O2 decreased their survival by 18%, with 0% O2 conditions leading to apoptosis. Pancreatic PC2 cancer cells were inhibited by CoCl2 (50–200 µM)46. Treatment of MDA-MB-231 cells with DFO  had no effect on their proliferation rate61. In this study, we observed that CoCl2 and DFO treatment  decreased both normal and breast cancer cell proliferation 65 as did treatment of MCF-7 with CoCl2 63 and MDA-MB-231 with DFO . We also demonstrated enhanced ER +ve and ER −ve cell motility (by approximately 2-fold) in response to both CoCl2 and DFO treatment  in non-invasive MCF7 cells enhanced their motility under hypoxic conditions  in association with EMT68. Vaapil and colleagues found no evidence for hypoxia-driven EMT69. Such reports have called into question the use of MCF10A as a representative of normal breast epithelia70. Or is this a cautionary indication that normal cells can exhibit some ‘cancer-like’ properties confronted with hypoxia or other stimulants? Either way this needs further investigation, but was not pursued here as it is not directly relevant to the objective of this study.A somewhat surprising observation was that MCF10A cells were found to be highly motile. However, this was found to be largely due to the presence of EGF in their special culture medium, as they showed little movement when this was omitted. Hypoxic conditions greatly stimulated motility irrespective of the medium used. Maeda and colleagues also observed significant motility (using same medium as ours) that was further enhanced by TGFβ2 (200 µM for 24 h) migrated faster than control63, MDA-MB-231 cell invasion increased by 24–129% by treatment with 30–300 µM of DFO for 24 h61 and hepatocellular carcinoma cells treated with 100 µM CoCl2 displayed approximately 10% increase in migration and invasion; this effect was reversed upon removal of the CoCl271.In agreement with previous observations57, our data using the Cultrex assay  we performed experiments in both normoxic and hypoxic conditions. Overall our data support the idea of cancer cells metastasizing as an escape from an unfavorable environment, in this case, hypoxia. Certainly, it appears to be a characteristic behavior of cells that have undergone EMT by whatever means Currently there is no data to indicate which cells within a tumor undergo this transformation or where they are located. Intuitively one might expect that it should be the ones at the tumor periphery in close proximity to functional blood vessels; while this may still be the case, our data suggests that EMT transformed cells can also migrate from further inside the tumor. Also, hypoxia significantly enhances endocrine resistant breast cancer cell migration/penetration into other cell monolayers but not through BME. Translating this into an in vivo scenario, one might envisage that hypoxia could drive O2 deprived cells to migrate towards the peripheral vasculature but not necessarily aid them to intravasate into it.In summary, the present study attempted to construct an The biggest limitation of our experimental approach is its two-dimensional nature and the use of pure cell populations. This could be improved with the use of three-dimensional organoid cultures with stromal elements and normal epithelial cells as well as live cell imaging of fluorescently labeled cells to track their movement in real time.Supplementary Figure legends.Supplementary information 1.Supplementary information 2.


Vibrio vulnificus. The TonB1 system contains three proteins: TonB1, ExbB1, and ExbD1. Both the TonB2 and TonB3 systems have been shown to also contain a fourth protein, TtpC2 and TtpC3, respectively. Here, we report and begin to characterize two additional proteins in the TonB2 and TonB3 systems: TtpB and TtpD. Both TtpB2 and TtpD2 are absolutely required for the function of the TonB2 system in V. vulnificus. However, although both TtpB3 and TtpD3 in the TonB3 system are related to the proteins in the TonB2 system, neither are active in iron transport. All six protein components of the TonB2 system—TonB2, ExbB2, ExbD2, TtpB2, TtpC2, and TtpD2—are essential for the uptake of both endogenously produced iron‐bound siderophores and exogenous siderophores produced from other organisms. Through complementation, we have shown that V. vulnificus is capable of using different TtpD2 proteins from other Vibrio species to bring in multiple siderophores. In contrast, we also demonstrate that TtpB2 must come from V. vulnificus, and not other species within the genus, to complement mutations in the TonB2 system.In gram‐negative bacteria, energy‐dependent active transport of iron‐bound substrates across the outer membrane is achieved through the TonB systems of proteins. Three TonB systems have been identified in the human pathogen  Vibrio vulnificus' TonB2 and TonB3 systems: TtpB and TtpD. Both TtpB2 and TtpD2 are absolutely required for the function of the TonB2 system in V. vulnificus for growth in iron‐limited conditions as well as the uptake of both endogenous and exogenous siderophores. Through complementation, we have shown that V. vulnificus is capable of using different TtpD2 proteins from other Vibrio species; while in contrast, we also demonstrate that TtpB2 must come from V. vulnificus, and not other species within the genus, to complement mutations in the TonB2 system.We report and begin to characterize two additional proteins in  These receptors bind nutrients, minerals, and iron‐bound siderophore complexes. The inner membrane (IM) is where the energy for the cell is derived. This can be in the form of adenosine triphosphate (ATP) or the proton motive force (PMF), which provides the energy needed to power the uptake of iron‐bound siderophores. The periplasmic space between these two membranes presents a challenge to gram‐negative bacteria. In order to breach that space, energy created by the PMF is transduced to the OM through a group of proteins known as the TonB system  or in minimal CM9 media  and 50 µg/ml kanamycin (Kmr) for E. coli, and 10 μg/ml Cm for V. vulnificus, when appropriate. Thiosulfate‐citrate‐bile salts‐sucrose agar  was used as the selective medium for V. vulnificus during conjugations. For iron‐limiting conditions, 2,2′‐dipyridyl was added at indicated concentrations. Iron‐rich conditions were obtained by the addition of ferric ammonium citrate (FAC) at indicated concentrations.The bacterial strains and plasmids used in this study are listed in Table 2.2V. vulnificus CMCP6 , V. vulnificus biotype 2 ATCC 33149 , V. cholerae 01 Biovar El Tor , V. parahaemolyticus RIMD , V. alginolyticus 12G01 , and V. anguillarum 775 .The percent identity matrices and protein alignments were generated through Clustal Omega  containing 15% sucrose. Colonies that grew on these plates were screened for Cm sensitivity, and deletions within the genes of interest were confirmed by PCR.In‐frame deletions of the entire coding sequences of genes were generated using splicing by overlap extension (SOE) PCR  was added to the solid and/or broth medium.Complementation of deleted genes was achieved by amplifying each gene of interest by PCR with primers containing restriction sites Table . The fra2.4600) of 0.02 into 25 ml of either TSBS or CM9 media and incubated at 37°C while shaking at 160 rpm. Readings were taken every 30 min for 300 min. For iron‐limiting conditions, 75 µM 2,2′‐dipyridyl was added to CM9 media. Complementation growth assays also contained 10 µg/ml chloramphenicol and 1 mM IPTG.Overnight cultures were diluted to an optical density at 600 nm (OD2.5600) of 0.02 into 25 ml of CM9 media and incubated at 37°C to an OD600 of 0.5 (∼3 hr). Cells were normalized to an OD600 of 0.5 in 1 ml of CM9 media. Cells were then centrifuged, and 0.5 ml of supernatant was used in the chrome azurol S (CAS) assay. A ratio of 1:1 of cell supernatant to CAS solution  was mixed and placed into 0.2‐cm cuvettes, and the OD630 was read using a spectrophotometer at 20 min after mixing. The assay was normalized to the wild type (WT), and the final values were inverted to show a positive change for ease of reading.Overnight cultures were diluted to an optical density at 600 nm (OD2.6V. vulnificus were as follows: 1 mg/ml iron‐free vibriobactin (EMC Microcollections), 1 mg/ml iron‐free aerobactin, 1 mg/ml iron‐free enterobactin, 1 mg/ml iron‐free ferrioxamine , and 500 μg/ml FAC (Sigma). Each bioassay plate was spotted with 2 μl of each indicated iron source. To test vibrioferrin, WT V. parahaemolyticus was streaked onto the plate. Vulnibactin and the hydroxamate siderophore, both from V. vulnificus, were tested by streaking either WT or the ΔvenB strain onto the bioassay plate. Halo growth around these streaks indicates that the strain within the plate can use the siderophore being produced by the streaked strain.Bacteria were grown overnight in TSBS with the appropriate antibiotics and diluted 1/500 into TSAS containing 100 µM 2′2‐dipyridyl. Complemented mutants also had appropriate antibiotics added as well as 1 mM IPTG. In these experiments, bacterial cells were included in the agar and, upon solidification, plates were spot inoculated with different iron sources. After incubation of the plates at 37°C for 24 hr, halos of bacterial growth surrounding the locations of the spots indicated positive results. In these experiments, ferric ammonium citrate (FAC), which does not require active transport to obtain, was included to confirm that the strain inoculated in the agar was viable. The purified compounds spotted on top of the bioassay plates to determine the functionality of the different TonB cluster of genes in 2.7V. vulnificus CMCP6 was grown overnight in TSBS and diluted 1/400 in CM9 with the addition of 250 µg/ml FAC for iron‐rich conditions or with the addition of 50 µM 2′2‐dipyridyl for iron‐limited conditions. Cultures were grown to an optical density at 600 nm (OD600) of 0.3. Samples were centrifuged, and RNA was harvested using Qiagen's RNeasy kit with Qiagen's RNAprotect following manufacturer's instructions. TurboDNase (Thermofisher) was used, and manufacturer's protocols were followed to ensure complete degradation of genomic DNA. At least three biological replicates were created for each condition.Wild‐type 2.8Purified RNA samples underwent reverse transcription to synthesize cDNA using the Qiagen QuantiTect Reverse Transcription kit following manufacturer's instructions. Control reactions without reverse transcriptase were also performed for each sample. Reverse transcriptase PCRs (RT‐PCRs) were then performed using 2 μl of each reverse transcription (RT) reaction mix using gene‐specific primers described in Table 33.1V. vulnificus contains three TonB systems spread across its two chromosomes  are present as the first and last ORFs in the tonB2 operon. Each appears to encode for hypothetical proteins of unknown function. We have designated the first ORF in this operon as ttpB2, and the last ORF as ttpD2 for, TonB2 complex‐associated transport protein B and D  PCR was performed. We saw a ~2.5‐fold and ~8‐fold elevated expression of ttpB2 and ttpD2, respectively, in iron‐depleted conditions compared to iron‐rich conditions  as a positive control. The housekeeping gene glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) was used for normalization. These results are very similar to what has been seen in another study  using microarray analysis . This family of proteins, DUF3450, appears in both eukaryotes and bacteria and always contains about 260 amino acids . When compared to other Vibrio species, the average similarity was about 50%  motifs throughout the protein  to other Vibrio species as compared to TtpB2 or any of the other four proteins in the TonB2 system  retained the ability to grow as well as the wild‐type strain and single mutants. This shows the importance and redundancy of the TonB1 and TonB2 systems when grown in these conditions and the inability of the TonB3 system to be used to overcome the loss of either of these systems. These data demonstrate the essentiality of TtpB2 and TtpD2 in the TonB2 system for growth in iron‐depleted conditions.Growth assays were used to determine the necessity and essentiality of TtpB2 and TtpD2 in the TonB2 system. An assortment of mutants was grown under iron‐rich and iron‐deplete conditions. When grown in iron‐rich media, TSBS, or minimal media, CM9, no differences in growth could be seen (data not shown). In contrast, when grown in CM9 plus the iron chelator dipyridyl (iron‐deplete conditions), a considerable difference in growth was noted in strains missing components of both the TonB1 and TonB2 systems Figure . Single V. vulnificus can produce two siderophores, vulnibactin, and the uncharacterized hydroxamate siderophore , or the TonB2 system was complemented (w/pttpB2 in a ttpB2 deletion or w/pttpD2 in a ttpD2 deletion), further confirming the importance of these proteins to the uptake of endogenous siderophores and the ability to use either  assays. Briefly, these colorimetric assays can be used to detect secreted iron‐bound complexes, like siderophores. When in iron‐depleted conditions, siderophores are secreted from tonB1 is deleted along with either tonB2, ttpB2, or ttpD2. This important result confirms the essentiality of both TtpB2 and TtpD2 in the TonB2 system.The results from these experiments can be seen in Figure venB, is the primary siderophore secreted under iron‐deplete conditions  or when a triple mutant, missing components of all three TonB systems, is used that the siderophores cannot be brought into the cell. These results provide additional evidence that both TtpB2 and TtpD2 are necessary components of the TonB2 system and must be functional for V. vulnificus to utilize exogenous siderophores.Each of the exogenous siderophores venB, were also unable to utilize exogenous siderophores listed above. Complementation of these quadruple mutants with either tonB1, ttpB2, or ttpD2 restored the uptake of all of the above exogenous siderophores . Complementing plasmids were moved into appropriate strains, and growth assays were performed.It has previously been demonstrated that the TtpC2 protein from different ttpC2, the ΔtonB1 ΔttpD2 ΔtonB3 mutant growth was complemented using the majority of other Vibrio species' homologous genes  was utilized. Results using the ΔtonB1 ΔttpB2 ΔtonB3 strains can be seen in Figure Vibrio species or TonB3 was not able to restore growth. These results demonstrate that the TonB2 system has a specificity for its own TtpB2 protein but not TtpD2.In contrast to these TtpD2 findings and previous findings with TtpC2, growth was only restored in a TtpB2 mutant when a complementing plasmid restoring TonB1 or a TtpB2 protein from a Vibrio strains show parallel results as growth assays—homologous strains did not restore siderophore utilization  as E. coli, the TonB2 and TonB3 systems of V. vulnificus appeared to have an additional gene, ttpC, as well as two ORFs found at the beginning and end of each operon  toward the N‐terminus, while TtpD2 contains a number of TPR motifs. Both of these conserved motifs suggest that TtpB2 and TtpD2 function as part of multiprotein complexes or through protein–protein interactions , cannot be complemented with the homolog from other species. Only the Biotype 2 strain with 97% homology to our wild‐type background was able to complement our ΔttpB2 mutation. Studies are underway to determine why this protein is unique in that other homologs of TtpB2 cannot complement its function within the V. vulnificus' TonB2 system. Studies are also underway to examine which portions of TtpD2, with its low level of conservation, are able to provide the conserved function that exists between species.Previously, both TonB2 and TtpC2 have been shown to be interchangeable among V. vulnificus' TonB systems and its contribution to growth and virulence (Alice et al., V. vulnificus growth and the uptake of endogenous and exogenous siderophores in iron‐limited conditions similar to those the organism experiences within a host. Both the specificity of TtpB2 and the ubiquitous nature of TtpD2 will be further analyzed in future studies.Previous studies have shown the importance of The authors have no conflicts of interest to disclose.R.J.K involved in conceptualization, funding acquisition, and project administration; provided the resources; and wrote the original draft of the article. A.D.B, H.J.P., B.R.Z., E.K.R., J.C.R., M.A.M., E.A.P., M.M.B., and R.J.K. curated the data; involved in formal analysis; investigated the study; designed the methodology; provided the software; supervised, validated, and visualized the study; wrote the review; and edited the manuscript.None required.


Access to asymmetrically functionalized polyoxometalates is a grand challenge as it could lead to new molecular nanomaterials with multiple or modular functionality. Now, a simple one‐pot synthetic approach to the isolation of an asymmetrically functionalized organic–inorganic hybrid Wells–Dawson polyoxometalate in good yield is presented. The cluster bears two organophosphonate moieties with contrasting physical properties: a chelating metal‐binding group, and a long aliphatic chain that facilitates solvent‐dependent self‐assembly into soft nanostructures. The orthogonal properties of the modular system are effectively demonstrated by controlled assembly of POM‐based redox‐active nanoparticles. This simple, high‐yielding synthetic method is a promising new approach to the preparation of multi‐functional hybrid metal oxide clusters, supermolecular systems, and soft‐nanomaterials. A one‐pot approach to the isolation of an asymmetrically functionalized organic–inorganic hybrid Wells–Dawson polyoxometalate is presented. The cluster bears two organophosphonate moieties with contrasting physical properties: a chelating metal‐binding group and a long aliphatic chain unit that facilitates solvent‐dependent self‐assembly into soft redox‐active nanostructures. Polyoxometalates (POMs) are anionic nanoscale metal oxide clusters comprising early transition metals in their highest oxidation states. They continue to attract significant interest owing to their vast structural diversity, excellent stability, and versatile chemical properties, making them ideal building blocks for functional materials.The covalent integration of organic moieties and POMs is an exciting and rapidly expanding area within the field.Herein, we present a simple, inexpensive, and high‐yielding synthetic approach to isolate the first reported example of an asymmetric bifunctional Wells–Dawson hybrid POM, obtained by exploiting the different solubilities of three hybrid POMs produced in the crude mixture. The cluster bears a chelating metal‐binding group, and a long aliphatic chain unit that allows solvent‐dependent self‐assembly into soft nanostructures Scheme . The sim3C21H16N3) (TPY), a terpyridine‐based ligand, and (PO4C24H43) (C18), an aliphatic chain group (see the Supporting Information). Hybridization was then conducted based on the previously reported method.18 were reacted with one molar equivalent of the potassium salt of the mono‐lacunary Dawson‐type anion, [P2W17O61]10− (see the Supporting Information). A 1:1 solvent mixture of N,N′‐dimethylformamide (DMF) and acetonitrile was used to adequately solubilize both ligands. After reaction completion, 31P NMR spectroscopy indicated the presence of three species 2[P2W17O57(PO3C21H14N3)(PO4C24H41)] (1) and the symmetric hybrids, (C2H8N)6[P2W17O57(PO3C21H14N3)2] (2), and K2(C2H8N)4[P2W17O57(PO4C24H41)2] (3). In the 31P NMR spectrum of 1 the signals for the covalently‐bound ligands, TPY and C18, appear at 13.31 and 16.77 ppm respectively, while the corresponding peaks appear at 14.10 and 15.96 ppm in the spectra of the respective symmetric structures, 2 and 3. Isolation of the desired product, 1, was achieved as follows. A large excess of ether was added to the reaction mixture upon which the solution turned cloudy. On centrifugation, a mixture of orange‐brown and green solid was collected and the yellow filtrate decanted. Removing the solvent from the filtrate leaves an orange‐brown crystalline solid that by 31P NMR is identified as 3. The remaining crude solid, a mixture of 2 and 1, was re‐dissolved in acetonitrile, in which 2 is sparingly soluble and can be separated by centrifugation. Finally, an excess of ether was used to re‐precipitate 1 while leaving any traces of 3 in solution. The process of dissolution in minimum acetonitrile to remove any insoluble 2, followed by re‐precipitating 1 from the filtrate with ether, can be repeated as necessary until no signals corresponding to 2 or 3 are visible by 31P or 1H NMR  and excellent purity.Functionalization of polyoxotungstates with aryl phosphonate groups enhances their photochemical properties and can be employed to direct their self‐assembly into micellar superstructures.1 was confirmed by 1H NMR, 31P NMR, ESI‐MS, elemental (CHN) analysis, thermogravimetric (TGA) analysis, and FTIR (see the Supporting Information). 1H NMR confirms the presence of both the aliphatic carbon chain and the aromatic TPY group in a 1:1 stoichiometric ratio. Interestingly, slight shielding of the TPY phosphorus and deshielding of the C18 phosphorus relative to their 31P chemical shifts in the respective symmetric hybrids suggests a degree of electronic communication via the POM core of the asymmetric structure.The composition and purity of 1, we explored the unique multifunctionality of the hybrid POM system, as derived from its asymmetric structure. First, we examined the self‐assembly of 1 enabled by the aliphatic chain ligand, C18, which imparts amphiphilic character to the hybrid POM (the POM core and TPY group acting as a polar head‐group). 1 was first dissolved in acetonitrile and then 9 equivalents of water were added to facilitate spontaneous self‐assembly. Dynamic light scattering (DLS) experiments on a 1.4 mm solution confirmed the formation of nanoscale assemblies of low dispersity with a hydrodynamic diameter (Dh) of approximately 6 nm . In agreement with this, Cryo‐TEM analysis of a solution of 1 in water–acetonitrile (9:1 v/v) deposited and frozen on a graphene‐oxide and holey carbon‐supported Cu grid showed spherical structures with diameters of 4–7 nm  mixture, indicative of reduced T2 transverse relaxation time arising from the slow tumbling of nanostructures .Following successful isolation of 1 in a 0.1 m DMF solution of TBA⋅PF6 revealed four distinct quasi‐reversible redox processes in the potential range of −0.5 to −2.25 V vs. Fc/Fc+ , all of which are positively shifted relative to the parent Wells–Dawson anion, [P2W18O62]6−, as is typical of organophosphonate hybrid POMs.E1/2 potentials of 1 were found to be intermediate to those of the respective symmetric hybrids, 2 and 3, with redox processes in 2 more positively shifted by an average of 5 mV and those of 3 more negative by an average of 20 mV, relative to the equivalent redox couples of 1 (see the Supporting Information for details).Cyclic voltammetry (CV) of 1 dissolved in a 0.1 m H2SO4:CH3CN  mixture, allowing for spontaneous assembly into micellar species as described above. Here, just two redox processes were observed between −0.5 and 0.5 V (vs. Ag/AgCl) in contrast to the three seen in pure DMF , as appears typical of micellar species of this type.3 .Recently, we showed that self‐assembled micellar nanostructures formed from hybrid POM species exhibit different redox properties to their constituent molecular species.1 was investigated by reacting the hybrid‐POM with 0.5 molar equivalents of FeCl2. After stirring the reactants at RT overnight in acetonitrile, the product, K7(C2H8N)3{Fe[P2W17O57(PO3C21H14N3)(PO4C24H41)]2} Fe‐1  in the 31P NMR spectrum of Fe‐1 demonstrate a degree of electronic conjugation across the whole asymmetric system. The dark purple color of the product is characteristic of a low spin d6 Fe2+ center, and UV/Vis absorption spectroscopy confirms the presence of a peak at 576 nm corresponding to a Fe2+‐TPY metal–ligand charge‐transfer (MLCT) band . CV studies in DMF reveal a reversible Fe2+/3+ redox couple (E1/2=0.543 V vs. Fc/Fc+) alongside the 4 quasi‐reversible POM‐centered processes .Post‐functionalization of 1 was then investigated by dissolving Fe‐1 in a water–acetonitrile (9:1 v/v) mixture at RT. DLS studies indicated the formation of nanoscale aggregates  with Dh(avg)=4 nm, and NMR studies again showed the disappearance of the peaks in D2O‐CD3CN (9:1 v/v) solution . CryoTEM analysis confirmed the presence of the assemblies .The combination of metal coordination and self‐assembly of In summary, we have isolated the first stable asymmetric Wells–Dawson hybrid POM using a simple and inexpensive procedure for the synthesis of novel multi‐functional hybrid materials based on molecular metal oxides. We have explored the multifunctionality of these asymmetric assemblies, driven by the nature of the appended organophosphoryl groups. The orthogonal and complementary functions of the aliphatic chain (facilitating solvent‐dependent self‐assembly) and the chelating group  allow us to tune the performance of the hybrid system through careful post‐functionalization and to obtain unique redox‐active soft nanostructures. This work provides a robust, modular, and easily accessible strategy for the creative design and development of new multi‐functional hybrid materials, with a range of potentially novel applications as redox‐, photo‐, and catalytically active soft nanomaterials.The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


Here we use atomistic simulation to predict the efficacy of C12A7 to encapsulate volatile fission products, in its stoichiometric and much more effective electride forms. In the stoichiometric form, while Xe, Kr and Cs are not captured, Br, I and Te exhibit strong encapsulation energies while Rb is only weakly encapsulated from atoms. The high electronegativities of Br, I and Te stabilize their encapsulation as anions. The electride form of C12A7 shows a significant enhancement in the encapsulation of Br, I and Te with all three stable as anions from their atom and dimer reference states. Successive encapsulation of multiple Br, I and Te as single anions in adjacent cages is also energetically favourable. Conversely, Xe, Kr, Rb and Cs are unbound. Encapsulation of homonuclear dimers  and heteronuclear dimers (CsBr and CsI) in a single cage is also unfavourable. Thus, C12A7 offers the desirable prospect of species selectivity.The nanoporous oxide 12CaO·7Al Among these, radioactive iodine is particularly challenging as it is highly mobile2, has high abundance in the spent fuel3 and can be concentrated by the body, increasing the radiotoxic impact. Caesium is also a particularly hazardous radio nucleotide but has a much longer half-life4. Therefore, separating iodine and caesium would be beneficial.Volatile radioactive species generated during the processing of spent nuclear fuel must be disposed of carefully to prevent contamination of the biosphere. The volatile species include chemically inert noble gases (Kr and Xe), alkali metals (Rb and Cs) and halogens (Br and I)5. Considerable effort has been devoted to modify and improve the effectiveness of filters using new materials. Porous materials including zeolites, silica, alumina and metal organic frameworks have been studied as these materials provide higher capacity to incorporate iodine9. The search for alternative filter materials exhibiting selectivity, thermodynamical stability, chemical activity, adequate mechanical properties and high capacity to incorporate guest nucleotides continues, while it is still a requirement they be manufactured at scale in a cost-effective manner.Impregnated carbon filters including charcoal filters are mostly used to remove iodine species in nuclear power plants2O3 (C12A7)12, and a constituent of aluminous cement13, is a candidate material for trapping volatile fission products because of its open crystal structure (consisting of 12 subnanometer-sized cages in a unit cell), but also its chemical and thermal stability12. The diameter of the cage inner free space is ~0.4 nm12. Neighboring cages are interconnected via ~0.1 nm wide openings. Furthermore, its constituent metal oxides (Al2O3 and CaO) are highly abundant, low cost and non-toxic. The framework of C12A7 is represented using the chemical formula [Ca24Al28O64]4+, in which each cage bears a charge of +1/3. The positive charge of the framework is compensated by negative ions occupying some of the cages; two O2− ions in stoichiometric C12A7 (C12A7:O2−)11, four OH− ions in fully hydrated C12A7: OH− 15, and four electrons in the electride form, C12A7 (C12A7:e−)17.Mayenite, a nanoporous complex inorganic oxide 12CaO·7Al− has the ability to accept and release electrons when guest species are present inside its cages. The extra-framework electrons can be replaced with other anions such as F− 18, Cl− 18, H− 15, S19, O2− 20 and OH− 15. Transition metals such as Au21 and Ru23 have also been incorporated in order to form catalysts (for example Ru in the synthesis of ammonia).Due to its extra-framework electrons, C12A7:eUsing density functional theory (DFT) together with dispersion corrections (DFT + D), the thermodynamic stability of gaseous volatile fission products  is investigated in both stoichiometric and electride forms of C12A7. DFT calculations, in addition to generating structural information, provides electronic structure and properties information. The present calculations consider fission products accommodated in the form of single atoms, homonuclear and heteronuclear dimers.11. The unit cell contains two 12CaO·7Al2O3 molecules (118 atoms) and twelve cages. The cages are approximately 6 Å wide and each cage wall is formed by 16 O ions, 8 Al ions and 6 Ca ions. The chemical composition of the unit cell is represented by [Ca24Al28O64]4+·(O2−)2, where [Ca24Al28O64]4+ is a positively charged framework 4 and the four electrons are localized in cages with the average occupation number of C12A7 exhibits a cubic crystal structure with space group of see Fig.  consistisee Fig. . The elesee Fig. .Figure 12−. The structure was first relaxed under constant pressure conditions allowing both lattice parameters and ion positions to relax. The calculated equilibrium lattice parameters  are in good agreement with experimental lattice parameters (a = b = c = 11.99 Å and α = β = γ = 90°)11 within a margin of ~0.45% error.The starting point of the study was to reproduce the experimentally observed crystal structure of C12A7:O2− ion  and the 2p states perpendicular to the bonding (non-bonded) respectively. Figure The relaxed structure of the cage containing the Oion Fig.  is slighsee Fig. . The smasee Fig. . The top− was relaxed under constant pressure. The calculated equilibrium lattice parameters were a = b = c = 12.06 Å and α = β = γ = 90°. Thus, the lattice volume of C12A7:e− is only slightly greater than that of C12A7:O2−. The calculated density of states (DOS) for C12A7:e− is shown in Fig. − means the system is metallic. The spatial distribution of the charge density associated with the extra-framework electrons localized in the cages is shown Fig. − to make four electrons in total) are distributed uniformly within the 12 cages. The electronic properties calculated for both C12A7:O2− and C12A7:e− are in good agreement with the previous theoretical study24.Next, the structure of C12A7:e2−, a single atom was incorporated into one of the ten empty cages in a supercell and encapsulation energies were calculated using a single atom reference state. Figure To establish the stability of volatile fission products inside C12A7:OBoth Xe and Kr demonstrate positive encapsulation energies with unmodified outer electronic configurations (refer to the Bader charge in Table 2+ ions. Br and I atoms assume charges of 0.75 and 0.68 |e| respectively (see Table The encapsulation energies for Br and I are negative (favourable) due to their strong electrostatic interaction with Ca2 molecules favourably occupy (separate) cage sites as Br− ions (i.e. with an energy gain) despite the molecular dissociation energy penalty. Conversely both I and Te are not stable inside cages from their dimers. The calculated dissociation energies (per atom) for Br2, I2 and Te2 in this study are 1.26 eV, 1.12 eV and 1.76 eV respectively. The stronger encapsulation energy of −1.47 eV for Br is thus due to the favourable balance of exothermic encapsulation energy (−2.73 eV) over the endothermic energy penalty for Br2 bond dissociation. In the case of I, a less negative but still favourable encapsulation energy (−0.44 eV) is observed. This is due to the slightly less exothermic encapsulation energy (−1.56 eV) for I, balance over its dissociation energy.The encapsulation energy calculated using the dimer as the reference indicates that even Br2+ ions in the cage wall  compared to its exothermic encapsulation energy of (−0.62 eV).The negative encapsulation energy for tellurium reveals that it is more stable inside the cage than as an isolated atom. This is due to the electron gain (0.49 |e|) by the Te and then the electrostatic attraction to CaRubidium exhibits a slightly negative (favourable) encapsulation energy, whereas for Cs the energy is slightly unfavorable. Both Rb and Cs transfer their single outer s-electrons to the lattice and exhibit a positive charge (see Table 2−) with respect to a single isolated species is negative but slightly smaller in magnitude compared to the first incorporation. Further, the encapsulation of a second Br is still strongly negative with respect to its dimer while the I:IC12A7:O2− energy is close to zero and the Te:TeC12A7:O2− energy is positive. The Bader analysis shows that the second Br or I gains the same charge as observed in the first encapsulation. Conversely, the second Te gains a smaller amount of charge.Next we considered up to 3 atoms  occupying separate cages . Calculations reveal that the encapsulation energy of a second atom  as single atoms occupying a cage in C12A7:e− with C12A7:O2−, the energies and distances are essentially the same.Xe exhibits a high positive encapsulation energy because of its large size and is non-bonding with the cage. This is supported by the almost zero Bader charge  as reference is strongly exothermic. This is because of the electron affinities of Br and I (3.36 eV and 3.06 eV respectively)25 which are gained when one of the extra-framework electrons complete the p-shells of these atoms.The encapsulation energies of Br and I in C12A7:e2− ion, close to completing its p-shell. Thus, unlike in C12A7:O2−, in C12A7:e− Te is stable in a cage with respect to dimer dissociation.Te is also predicted to exhibit a highly negative encapsulation energy. Indeed, its Bader charge is -1.23 electrons meaning that it is tending towards a Te2− ions . Bader charge analysis shows that both Rb and Cs donate ~one electron to form the +1 charge state. Once the positive ions are formed, they are attracted by the cage wall O−, 4BrC12A7:e−, 5BrC12A7:e− series. In 3BrC12A7:e− considerable charge resides in empty cages but this has localised onto Br− ions in 4BrC12A7:e−. The multiple anion encapsulation energies contrast strongly with the equivalent results in C12A7:O2− where even the encapsulation of a second halide came with a sharp fall in energy.Successive encapsulation was considered for up to 5 atoms of Br, I and Te inside separate unoccupied cages. Both Br and I show almost no change in the favourable encapsulation energies up to 4 atoms . Only the reactive fission products  were considered as homonuclear species.Calculations were performed to investigate the energetics associated with placing a single homonuclear or heteronuclear diatomic molecule within a single cage of C12A7:OThe relaxed structures of dimers (homo and hetero) within a cage are shown in Fig. 2 and I2 are 2.45 Å and 2.79 Å respectively, larger than their corresponding gas phase dimer values of 2.31 Å and 2.69 Å. This is partly because the overall Bader charges on Br2 and I2 are slightly negative meaning that if they were gas phase molecules there would be occupation of a more diffuse π* molecular orbital. However, these molecules also show strong induced polarization, which in I2 means the atom near the centre of the cage has a negative charge whereas the other atom has a positive Bader charge.The bond distances of Br2, there is an elongation of 0.16 Å in the bond length compared to the gas phase molecule. This elongation is similar to the values observed in Br2 and I2. However, the Te2 molecule is the most strongly polarized and has the smallest overall charge.In the case of TeCsBr and CsI molecules also experience strong distortion and their encapsulation energies are even more unfavourable that those of the homonuclear dimers. The bond distances of CsBr and CsI in the cage are ~0.30 Å shorter than their corresponding calculated gas phase dimers of 3.09 Å and 3.34 Å respectively, presumably due to the confinement induced by the cage. Nevertheless, the Bader charge distribution is roughly symmetric with absolute values as expected in a gas phase molecule.−. The relaxed structures of dimers introduced inside C12A7:e− are shown in Fig. Finally we considered both homo and heteronuclear dimers inside C12A7:e2 inside a cage is 2.68 Å, longer than its corresponding gas phase dimer, 2.31 Å . Nevertheless, the encapsulation energy for two Br atoms to occupy a cage with respect to an isolated dimer is exothermic . While the encapsulation energy of Br2 is exothermic, with respect to an isolated Br2 molecule, the reaction to split the molecule into two cages is strongly exothermic (−2.91 eV). Thus, if there is an unoccupied cage available, the molecule will dissociate into two Br− anions in separate cages. Furthermore, the energy to add ½ Br2 to a single existing Br− ion in a single cage is 1.92 eV. Thus, even if no cage remains unoccupied by a Br− anion, Br2 molecules within C12A7:e− will still not form.The bond distance for Br2, there is significant distortion of a cage occupied by two I atoms and the molecule orientation is asymmetric , thus it is really a Te2+ ions in the cage wall  or two Te atoms, due to the available electrons to be trapped by the additional atoms to form anions. Homonuclear  and heteronuclear dimers (CsBr and CsI) were allowed to occupy one of the cages in unit cells of C12A7:e− and C12A7:O2−. Calculations show (except for Br2) that both homo and hetero nuclear dimers are thermodynamically unfavourable in C12A7:O2− from their dimers since they introduce a significant distortion in the cage wall. While Br2 is stable as a molecular species, it is more stable as two anions occupying separate cages.More specifically, Xe, Kr, and Cs are unstable in both forms of C12A7. Rb shows a weak encapsulation in C12A7:O27. The exchange-correlation term was modelled using the generalized gradient approximation (GGA) parameterized by Perdew, Burke, and Ernzerhof (PBE)28. In all cases we have used a plane-wave basis set with a cut off value of 500 eV and a 2 × 2 × 2 Monkhorst-Pack29 k-point mesh, which yields 8 k points. Further increase in the k points resulted in a total energy difference of 0.6 meV per atom. Structural optimizations were performed using a conjugate gradient algorithm30 and the forces on the atoms were obtained from the Hellman-Feynman theorem including Pulay corrections. In all optimized structures, forces on the atoms were smaller than 0.001 eV/Å and the stress tensor was less than 0.002 GPa. We define the encapsulation energy for single atoms or dimers trapped inside the empty cages of the electride form of C12A7 through the following equation;−) is the total energy for bulk C12A7:e−, E (FP-C12A7:e−) is the total energy of the gaseous atom or atoms occupying the cages, E(FP) is the total energy of an isolated fission product (the reference state) and n is the number of fission product atoms considered in the process.The calculations were carried out using the spin-polarized mode of DFT as implemented in the VASP codeet al.31 (DFT-D3) in VASP.The inclusion of van der Waals (vdW) interactions is particularly important for the incorporation of highly polarizable noble gas and halogen atoms. Here, dispersion has been included by using the pair-wise force field as implemented by Grimme


This is rarely accounted for when assessing hypoxia tolerances of aquatic organisms. We investigated the impact of environmentally realistic increases in CO2 on responses to hypoxia in European sea bass (Dicentrarchus labrax). We conducted a critical oxygen (O2crit) test, a common measure of hypoxia tolerance, using two treatments in which O2 levels were reduced with constant ambient CO2 levels (~530 µatm), or with reciprocal increases in CO2 . We also assessed blood acid-base chemistry and haemoglobin-O2 binding affinity of sea bass in hypoxic conditions with ambient (~650 μatm) or raised CO2 (~1770 μatm) levels. Sea bass exhibited greater hypoxia tolerance (~20% reduced O2crit), associated with increased haemoglobin-O2 affinity  of red blood cells, when exposed to reciprocal changes in O2 and CO2. This indicates that rising CO2 which accompanies environmental hypoxia facilitates increased O2 uptake by the blood in low O2 conditions, enhancing hypoxia tolerance. We recommend that when impacts of hypoxia on aquatic organisms are assessed, due consideration is given to associated environmental increases in CO2.Global environmental change is increasing hypoxia in aquatic ecosystems. During hypoxic events, bacterial respiration causes an increase in carbon dioxide (CO In terrestrial ecosystems conditions of low O2 are rare. In contrast low O2, referred to as hypoxia, is much more common in freshwater and marine ecosystems4. Hypoxia occurs because high biological demand for O2 can exceed the rate of O2 supply to the ecosystem, leading to a reduction in environmental O2 levels6. However, the challenges of hypoxia are not solely a result of reduced O2. Organisms must also contend with simultaneous but reciprocal changes in the other respiratory gas, CO2.A lack of O2 decreases in aquatic systems there is a corresponding increase in CO28. This is a by-product of respiration, the same process that causes depletion of O2. As such high CO2 during hypoxia is ubiquitous and unavoidable. This coupling of O2 and CO2 has been highlighted numerous times in oceanographic sciences, most recently by Robinson5. Yet unaccountably, despite the known link between decreasing O2 and increasing CO2 during hypoxia9, the issue of increased environmental CO2 during periods of low O2 has been relatively overlooked by biologists.When O2 during hypoxia on aquatic organisms are particularly important to address in the face of human driven climate change. Hypoxic areas are predicted to become more common and more severe, particularly in marine systems, with the de-oxygenation of the world’s oceans recently highlighted as a major component of climate change12. In addition, there will be an increase in ambient CO2 as rising atmospheric CO2 is absorbed by the world’s oceans13. Non-linear interactive effects between higher atmospheric CO2 and CO2 accumulation during hypoxia will lead to increased CO2 levels during hypoxia in future oceans14. This means that effects of rising CO2 during hypoxia in marine systems will be amplified by climate change.Implications of rising CO2) and O2 (for examples see18). This creates low O2 conditions without the concurrent CO2 increase that would be expected in the environment. The lack of studies in which an environmentally realistic simultaneous decrease in O2 and increase of CO2 have been conducted may lead to mismeasurement of responses to hypoxia. Recently, several studies on marine fish and invertebrates have demonstrated interactive effects of low oxygen and increased CO220, with some species exhibiting loss of equilibrium (LoE) and death at higher O2 concentrations when CO2 is simultaneously elevated21. However, these experiments do not give insight into the physiological mechanisms underlying the influence of CO2 on hypoxic responses of fish.Typically, experiments which test responses to hypoxia or impacts from hypoxia on aquatic organisms create hypoxic conditions by off-gassing oxygen from water by gassing with pure nitrogen or a mix of nitrogen  by conducting a standard critical O2 level test (O2crit). Under normal O2 (normoxic) conditions fish maintain a minimum level of O2 consumption rate  in order to maintain and regulate this minimum level of 27. If environmental O2 continues to drop there comes a point at which fish are unable to regulate 2 level (O2crit). At O2 levels below O2crit fish become oxy-conformers (where 2 availability) and fish become increasingly reliant on anaerobic metabolism which is unsustainable in the medium to long term. In the past the measure of O2crit has been used as a proxy for overall hypoxia tolerance but recently this approach has been questioned29. Nevertheless O2crit does provide information related to the ability of fish to maintain O2 uptake and supply during hypoxia, and its prevalence in the literature allows comparison of responses between species30. Furthermore, we investigated whether any changes in O2crit could be linked to changes in blood acid-base chemistry and blood gas transport via alteration of Hb-O2 binding caused by rising environmental CO2. Our hypothesis was that the simultaneous increase in CO2 during a progressive decrease in O2 would decrease hypoxia tolerance (increase O2crit) and that this response may be a result of blood acid-base disturbance decreasing Hb-O2 affinity and O2 transport.Previously observed impacts of hypoxia-associated rises in CO2 conditions. This was indicated by measurements of O2crit in European sea bass being significantly different between fish exposed to either constant, or rising CO2 levels during O2crit tests when accounting for variation in SMR . A CO2 increase during O2crit tests resulted in a 20% reduction of O2crit  when compared to tests in which CO2 levels were maintained at ambient levels .There was evidence of enhanced tolerance to hypoxia in sea bass exposed to rising compared to constant CO2 during hypoxia within 5 hours (the period of exposure prior to blood sampling). Blood pH (pHe) was not different between fish exposed to constant ambient CO2 (7.87 ± 0.03) and fish exposed to progressively rising CO2 (7.88 ± 0.02) . The acidifying effect of the ~79% rise in blood pCO2 levels in the rising CO2 regime (0.272 ± 0.019 kPa CO2) compared to the constant ambient CO2 regime (0.152 ± 0.015 kPa CO2)  was fully compensated by elevating blood HCO3− . Plasma HCO3− was 88% higher under the rising CO2 regime (6.76 ± 0.29 mM) when compared to the constant ambient CO2 (3.60 ± 0.43 mM). There were no differences in haematocrit  or plasma lactate  between fish sampled under a constant ambient CO2 regime  or rising CO2 regime . Blood glucose levels were ~26% lower in fish exposed to a progressively rising CO2 regime (4.50 ± 0.53 mM) when compared to a constant ambient CO2 regime (6.09 ± 0.31 mM) .A comparison of blood chemistry parameters between the two treatment groups indicated that sea bass fully compensated for the rise in CO2 regime . Haemoglobin P50 was decreased by ~32.5% in fish under the progressively rising CO2 regime  compared to fish sampled with a constant ambient CO2 . There was no significant change in Hills number between treatments .Oxygen affinity of haemoglobin was increased in fish sampled under the progressively rising CO2 under conditions where O2 levels in water are depleted – a scenario that reflects the natural conditions during hypoxia which will be exacerbated by climate change – by demonstrating that ecologically relevant changes of CO2 impact physiological performance of a marine fish at both the molecular and whole organism level. We hypothesised that rising CO2 during progressive O2 decreases would lead to an increase in O2crit as a result of increased blood CO2, decreased blood pH and the associated Bohr/Root effect of fish haemoglobin (Hb), in which Hb-O2 affinity (Bohr effect) and the total capacity of Hb for O2 (Root effect) are reduced when pH falls. In contrast, we show that increasing CO2 as O2 declined led to enhanced hypoxia tolerance of sea bass with a 20% lower critical oxygen level . This provides a potential mechanistic basis to explain improved O2crit, enabling sea bass to enhance O2 uptake during hypoxia and thus maintain normal aerobic metabolism to lower environmental O2.Our results highlight the biological importance of simultaneously rising COvel Fig. . This chses Fig. . The cha2 affinity in sea bass exposed to concurrent O2 decline and CO2 rise is not clear from our results. Several allosteric factors that can modulate the affinity of haemoglobin for O2 could be involved, including pH, organic phosphates and inorganic ions. Fish haemoglobin is highly sensitive to pH, which modulates Hb-O2 affinity and carrying capacity via the Bohr and Root effects31, but we found no differences in blood pH of sea bass between treatment groups. In addition, the in-vivo increase in pCO2 in fish exposed to concurrent CO2 rises during hypoxia led to an opposite response of Hb-O2 affinity than would be expected by an in-vitro rise in pCO2 which would result in pH induced Bohr/Root effects. Increased Hb-O2 affinity could result from increased intracellular pH of erythrocytes32, as acute hypoxic exposure has been shown to stimulate a β-adrenergic stimulated increase in intracellular erythrocyte pH in rainbow trout33. Alternatively, increased Hb-O2 affinity could be due to decreased red cell nucleoside triphosphates (NTPs)34, a known hypoxia adaptation, but this can take more than 6 days to complete35. Sea bass may also have a particularly strong β-adrenergic response and/or fast NTP response, although there is little evidence to suggest this because P50 measurements from fish exposed to hypoxia at ambient CO2 levels do not differ from P50 measurements in normoxic fish from the same population . It is possible that rising CO2 during hypoxia may modulate the β-adrenergic response and/or the red cell NTP response within the time frame (~4–6 hours) of our treatments. However, direct measurements of red cell pHi and NTP content of sea bass in each treatment group would be needed to confirm this.The driver of increased Hb-O2 affinity in sea bass exposed to concurrent CO2 rise during hypoxia could be decreased erythrocyte chloride (Cl−)36. Although erythrocyte Cl− was not directly measured in our study, plasma HCO3− was approximately 3 mM higher in fish exposed to concurrent CO2 rises during hypoxia than fish which experienced constant ambient CO2 during hypoxia . This analysis identified two broad methods employed in O2crit measurements:2 is usually reduced by the O2 consumption of the fish (52 identified studies) or;Closed respirometry where O2 is usually reduced via gassing with pure N2 or combined N2 and O2 mixes (32 identified studies).Intermittent or flow-through respirometry in which OAnalysis of O2 rises as O2 is depleted by fish O2 consumption. The increase in ambient CO2 during closed respirometry is well known and often used as a criticism of this respirometry technique40. In contrast, use of intermittent-flow respirometry in O2crit trials normally necessitates the reduction of O2 in the water by aeration with N2 or a mix of N2 & O2. As a result CO2 would likely decrease during the time course of hypoxia induction . Such a change in CO2 during the O2crit trial would be the opposite of that seen in nature. Therefore it may be considered that closed respirometry provides conditions which give a more environmentally relevant measure of O2crit28.The use of closed respirometry in the majority of studies would result in concurrent CO2 during hypoxia directly affects the ability of sea bass to maintain O2 uptake, it could be expected that the use of closed respirometry methods would result in lower measurements of O2crit than intermittent-flow methods for the same species. This effect has not been documented for species in which a direct comparison has been made – with either there being no effect of respirometry method on O2crit41 or higher O2crit measurements when closed respirometry is used41. However, such comparisons are complicated by differences in the rate of hypoxia induction (RHI) by different studies, which in turn will influence how much time fish have to regulate blood pH when ambient CO2 is rising. For example, Regan and Richards41 have demonstrated that the faster rates of hypoxia induction (RHI) typical of closed respirometry O2crit trials lead to higher values of O2crit (i.e. lower hypoxia tolerance) when compared with longer trials using slower RHI’s typical of the intermittent-flow method. The effect of RHI on O2crit was proposed by Regan and Richards40 as a potential explanation of the results of Snyder et al. (i.e. higher O2crit in closed respirometry compared to intermittent-flow respirometry)42. The speed of RHI during closed respirometry will also effect the speed of CO2 rise. Almost all studies using closed respirometry to measure O2crit do not report changes in CO2 over the course of measurement period. When accumulation of CO2 during a closed respirometry O2crit trial was measured by Regan and Richards, CO2 levels were ~8,000 μatm after ~90 minutes41. However, we should note that this pCO2 level was measured after the O2crit point, when anaerobic metabolism continues to produce CO2 in the absence of O2 consumption, but also metabolic acid production and excretion further drives up water pCO2 in the respirometer due to excess H+ ions titrating ambient HCO3− to CO2. Regardless, at O2 levels above O2crit the rate of CO2 onset will be faster than those used in our current study as a result of the faster RHI. Increased speed of CO2 onset in closed respirometry trials may ‘outstrip’ the ability of fish to acid-base regulate, causing an uncompensated respiratory acidosis during the time of the trial, which in turn would decrease Hb-O2 affinity via the Bohr & Root effects and potentially increase O2crit. Similarly, fish species which have reduced ability to acid-base regulate may have an increased O2crit when rising CO2 is included in trials.As our results indicate that rising CO2 in European sea bass. This contrasts with previous research investigating interactive effects of CO2 and hypoxia on O2crit of fish. Woolly sculpin, Clinocottus analis , exposed to ~1100 μatm CO2 showed no impact on O2crit after 7 days acclimation but after 28 days had O2crit measurements ~34% higher than fish held in ambient (~400 μatm) conditions43. Higher O2crit after 28 days corresponded with higher RMR and Na+, K+, ATPase activity. This contrast in results could indicate that the beneficial effect of acute rises in CO2 associated with natural hypoxia documented in our study are potentially reversed when fish are exposed to long term constantly high CO2 associated with anthropogenic climate change. Moreover, acute changes in CO2 had no effect on O2crit of the estuarine fish species mummichog, Fundulus heteroclitus, and Norfolk spot, Leiostomus xanthurus when they were exposed to ~8,000–10,000 μatm CO2 immediately prior to an O2crit trial44. As such the effect of CO2 on O2crit will likely depend on differences in physiological responses to CO2 and O2 between species.Our results indicate improved hypoxia tolerance during rising CO2 also shows variable impacts on non-metabolic responses to hypoxia of several species. Cycling CO2 had no effect on aquatic surface respiration (ASR), the use of the thin surface layer of water for aquatic respiration45, or survival in juvenile Menidia menidia, Fundulus majalis, Fundulus heteroclitus or Morone saxatalis exposed to short term cycles of O246. In contrast, combined hypoxia and acidification resulted in an increase in the O2 level at which Menidia menidia and Menidia beryllina first performed ASR, consistently performed ASR, exhibited LoE, and finally died21. Additionally, combined high CO2  and hypoxia had no effect on survival of larval Cyprinodon variegatus, an additive negative effect on larval Menidia beryllina, and a synergistic negative effect on larval Menidia menidia47. This variation in effect of CO2 on hypoxia responses could be a result of methodological differences , the level of CO2 used in studies , differences in species and life stages used , or possibly variability in response as a result of differences in previous environmental experience49. The role of environmental variability in species sensitivities to CO2 has recently been outlined in the proposed Ocean Variability Hypothesis (OVH)50 and warrants testing on various species in the future.Simultaneously rising CO2 during a hypoxic event increased the hypoxia tolerance (i.e. reduces O2crit) of European sea bass which is at least partly explained by an enhanced ability of fish to uptake O2 via increased Hb-O2 affinity. Miller et al.21 also demonstrated impacts of concurrent CO2 rise on measurements of hypoxia tolerance, although in an opposite direction to that noted in our study. As concurrent CO2 rises during hypoxia are the norm in nature, evidence that this affects physiology of organisms exposed to hypoxia highlights an important shortcoming of research to predict tolerances to hypoxia of fish. More research on this issue is needed to clarify how common this modifying effect of CO2 on the response to hypoxia is and whether such measurements in the lab are ecologically relevant. A greater understanding of this issue may allow more accurate assessments of the impacts of hypoxic events on marine fish in nature, aiding management and conservation of fish species. With specific regard to measurements of O2crit we believe future studies should include concurrent rising CO2 in the following ways:2 relevant to hypoxic events that organisms may experience,Intermittent-flow respirometer studies should include increases of CO2 levels in the respirometer.Closed respirometry studies should report the start and end COOverall our results indicate that the environmentally realistic, simultaneous rises in CO2 increase for the species studied, and consistently report CO2 levels measured. By incorporating these recommendations we believe that future studies of O2crit will give more representative estimations of species hypoxia tolerance.In addition, both methods should aim to create an environmentally relevant rate of hypoxia induction/CO24). All experimental procedures were carried out under home office licence P88687E07 and approved by the University of Exeter’s Animal Welfare and Ethical Review Board.We collected juvenile sea bass from estuaries and coastal lagoons on the south Dorset coast and Isle of Wight in June 2017 . Prior to experimentation, these fish were held in the University of Exeter’s Aquatic Resource Centre in an aerated recirculating aquaculture system and fed a commercial pellet at a ration of ~1–2% body weight per day three times a week , and a measurement chamber into which a temperature-compensated fibre optic oxygen optode  was placed. Oxygen optodes were calibrated in water at the start of experiments at 100% air saturation and 0% air saturation according to manual instructions. Respirometry was conducted in a semi-closed system consisting of three 100 L experimental tanks fed by a 100 L sump, with overflowing water from the experimental tanks recirculating back to the sump. A second pump was used to periodically flush the respirometer system with water from the surrounding tank. This pump was controlled by an automated computer program  to intermittently flush the respirometer. Five respirometer chambers were distributed between the three experimental tanks . The sump was temperature controlled  using a heater/chiller unit  attached to a temperature exchange coil. Together these tanks formed a 400 L system with the same temperature, oxygen and water chemistry parameters for all respirometers.We determined oxygen consumption rates , chosen at random, were placed inside the respirometers and allowed an overnight recovery period, for a minimum of 13 hours, before O2crit test was conducted. Oxygen levels in the respirometer system were reduced from ~100% air saturation to ~15% air saturation over the course of 6 hours . Oxygen was regulated by gassing the sump and experimental tanks with a mix of N2 and O2  at a rate of 10 L min−1 following a pre-set automated protocol . Levels of CO2 within the system were controlled under one of two treatments (with 8 fish exposed to Treatment 1 and 8 separate fish exposed Treatment 2):2) - ambient levels of CO2 were maintained by including 0.04% CO2 as part of the gas mix delivered to the respirometer system.Treatment 1 (Constant CO2) - the proportion of CO2 in the gas mix was gradually increased as O2 was decreased. This increase in CO2 was designed to reflect environmentally realistic increases in CO2 predicted as a result of depletion of O2 by bacterial respiration (assumed respiratory quotient of 1)52, using the seawater carbonate chemistry calculator CO2sys .Treatment 2 /Dissolved Inorganic Carbon (DIC). Water chemistry of treatment 1 was monitored at the start and end of the treatment to ensure no change in water pCO2 occurred over the time course of the O2crit trial. Seawater DIC analysis was conducted using a custom built system described in detail by Lewis et al.53. These four parameters were then input into the seawater carbon calculator programme, CO2SYS to calculate pCO2 based on the NBS pH scale, equilibration constants from Mehrbach et al. refitted by Dickson and Millero, and KSO4 dissociation constants from Dickson. The reciprocal changes in O2 and CO2 during O2crit tests for each treatment are illustrated in Fig. 2crit tests were stopped once a minimum of 3 2crit trials experimental tanks were aerated with ambient air to swiftly restore O2 and CO2 levels.Water chemistry of treatment 2 was monitored once per hour by measuring pH2 levels reached ~21 kPa O2 (~100% air saturation). Fish were then removed from respirometers and background respiration was measured for a minimum of 1 hour (6 measurement cycles) for all respirometers immediately post trial.Sea bass were left to recover in respirometers, for a minimum of 1 hour post-trial, until O2 versus time data for each measurement period. The slope of this regression  was then used to calculate 2 kg−1 h−1) using the equation outlined by Svendsen et al.51:resp is the respirometer volume minus the volume of the fish (L), α is the solubility of O2 in water (mgO2 L−1 kPa−1) for the relevant salinity and temperature, and m is the mass of the fish (kg). Calculations of 2 of <0.98 were removed from subsequent analysis. For the purpose of establishing O2crit values from a plot of 2 level, the oxygen saturation of each measurement period was defined as the average of the dissolved O2 measurement over the measurement period. The average background respiration, over the 1 hour post-trial measuring period, for each respirometer  or quantile (q = 0.2) methods to calculate SMR we chose the mean of the 10 lowest values as it produced a value of SMR that most accurately matched the consistent low pO2 above the O2crit_point. Additionally, for each fish the coefficient of variation in the mean lowest normal distribution (MLND) was assessed using a ROUT test (Q = 1%) to check whether variation in low 2crit trials). The ROUT test removed one fish from the rising CO2 treatment which displayed a CoV of 34.6. Mean CoV of the remaining 15 fish was 11.85 ± 1.26 (±S.E.M).We calculated the standard metabolic rate (SMR) in R v3.5.32crit for each individual fish, using the estimated SMR of each individual, as detailed in the supplementary material of Claireaux & Chabot55. This function identifies the portion of the O2crit test where metabolic rate data follows an oxygen conforming relationship and fits a linear regression line through this data, O2crit is then calculated as the oxygen level at which this regression line crosses the calculated SMR of the individual fish. Plots of 2 during O2crit trials showing calculated O2crit for each individual can be seen in the supplementary material  and a maximum number of 7 2crit.We then used function calcO2crit from the package to calculate the Omentclass2pt{minim2crit trials sea bass were moved to individual 7 L chambers, which were aerated and supplied with seawater from the aquarium re-circulating system at a rate of about 4 L min−1. After an overnight acclimation period we then exposed the fish to a decrease in O2 levels to ~6.4 kPa O2 (30% air saturation) over a period of 4 hours . We chose to blood sample fish at an O2 level above O2crit to ensure that anaerobic metabolism did not influence blood chemistry. This was combined with the same CO2 regime each fish experienced during the O2crit test . Once an O2 level of ~6.4 kPa O2 (30% air saturation) was achieved the fish were allowed to acclimate for 1 hour before being individually anaesthetised in situ using a dose of 100 mgL−1 of benzocaine. Once fish were judged to be sufficiently anaesthetised  they were immediately transferred within 5 seconds to a gill irrigation table (with the same pO2 and pCO2 levels of the respective treatment), where anaesthesia was maintained with a dose of 37.5 mgL−1 of benzocaine. Gill ventilation was artificially maintained by a micro-pump, so that the operculum were just open and exhalant water flow could just be visualised. Once a stable gill water flow was established blood was sampled by caudal vessel puncture using a 1 ml heparinised syringe. This method has been demonstrated to obtain accurate measurements of blood chemistry parameters comparable to those achieved using cannulation . At the time of blood sampling water pCO2 was 656 ± 44 μatm (mean ± S.E.) for fish in the constant ambient CO2 regime and 1763 ± 43 μatm (mean ± S.E.) for fish in the progressively rising CO2 regime. Water pO2 was 8.1 ± 0.2 kPa  for fish in the constant ambient CO2 regime and 8.7 ± 0.2 kPa  for fish in the progressively rising CO2 regime. Following blood sampling fish were transferred to seawater isolation tanks containing ~20.8 kPa O2 (100% air-saturated) to recover from the anaesthetic. They were then monitored over a period of 24 hours before we returned them to their original holding tanks.Following OpO2 was measured at 18 °C in a temperature-controlled system . We measured extracellular pH on 30 µL of whole blood using an Accumet Micro pH electrode and Hanna HI8314 pH meter at 18 °C calibrated to pHNBS 7.04 and 9.21 specific buffers. Three 75 µL micro capillary tubes were then filled with whole blood and sealed with Critoseal capillary tube sealant (Fisher) and paraffin oil and centrifuged for 2 minutes at 10,000 rpm. Haematocrit was measured using a Hawksley micro-haematocrit reader. Plasma was extracted from these tubes for analysis of TCO2 using a Mettler Toledo 965D carbon dioxide analyser. Plasma pCO2 and HCO3− were then calculated from TCO2, temperature and blood pH using the Henderson-Hasselbalch equation with values for solubility and pK1app based on Boutilier et al.57. Haemoglobin content of the blood was also assessed by the cyanmethemoglobin method . Half the remaining whole blood was then centrifuged at 10,000 rpm for 2 minutes at 4 °C. The resulting plasma was separated and snap frozen in liquid nitrogen and stored at −80 °C before later being used to measure plasma glucose and lactate using a YSI 2900D Biochemistry Analyzer . All measurements or storage of blood occurred within 10 minutes of blood sampling.Immediately after sampling, whole blood 2 affinity using a Blood Oxygen Binding System , detailed in Oellermann et al.58. A sample of the same whole blood used for blood chemistry measurements was diluted at a ratio of 1:4 in its own plasma. 1 μL of this blood was then used for measurements. The BOBS exposed this blood sample to gas mixes with a progressive increase in O2 whilst measuring absorbance of light across a spectrum ranging from 200 to 800 nm. For each individual fish the gas mix that blood was exposed to matched the calculated pCO2 of the blood sample. The change in absorption of light at a wavelength of 435 nm was used to assess changes in oxygenation of Hb, as previously used by Verhille & Farrell59. Background changes in absorption of the blood sample were corrected using the isosbestic wavelength of 390 nm59. Following measurements the BOBS calculated the oxygen equilibrium curve of the sample using Hill’s formula before estimating P5058.We measured Hb-O54. There was no significant difference in mass of fish between the treatment groups  or of estimated SMR of fish between treatment groups . There was, however, a significant correlation between estimated SMR and calculated O2crit for all fish . As such the effect of CO2 treatment on O2crit was assessed using an ANCOVA with SMR as a covariate. All other analyses were conducted using general linear modelling (GLM). All values in the text are reported as mean ± standard error (S.E.).We conducted all statistical analysis in R v3.5.3Rising CO2 enhances hypoxia tolerance of a marine fish - supplementary materials