Early-life exposure to climate change impairs tropical shark survival

Sharks are one of the most threatened groups of marine animals worldwide, mostly owing to overfishing and habitat degradation/loss. Although these cartilaginous fish have evolved to fill many ecological niches across a wide range of habitats, they have limited capability to rapidly adapt to human-induced changes in their environments. Contrary to global warming, ocean acidification was not considered as a direct climate-related threat to sharks. Here we show, for the first time, that an early ontogenetic acclimation process of a tropical shark (Chiloscyllium punctatum) to the projected scenarios of ocean acidification (ΔpH = 0.5) and warming (+4°C; 30°C) for 2100 elicited significant impairments on juvenile shark condition and survival. The mortality of shark embryos at the present-day thermal scenarios was 0% both at normocapnic and hypercapnic conditions. Yet routine metabolic rates (RMRs) were significantly affected by temperature, pH and embryonic stage. Immediately after hatching, the Fulton condition of juvenile bamboo sharks was significantly different in individuals that experienced future warming and hypercapnia; 30 days after hatching, survival rapidly declined in individuals experiencing both ocean warming and acidification (up to 44%). The RMR of juvenile sharks was also significantly affected by temperature and pH. The impact of low pH on ventilation rates was significant only under the higher thermal scenario. This study highlights the need of experimental-based risk assessments of sharks to climate change. In other words, it is critical to directly assess risk and vulnerability of sharks to ocean acidification and warming, and such effort can ultimately help managers and policy-makers to take proactive measures targeting most endangered species.     

Additive impacts of deoxygenation and acidification threaten marine biota

Deoxygenation in coastal and open‐ocean ecosystems rarely exists in isolation but occurs concomitantly with acidification. Here, we first combine meta‐data of experimental assessments from across the globe to investigate the potential interactive impacts of deoxygenation and acidification on a broad range of marine taxa. We then characterize the differing degrees of deoxygenation and acidification tested in our dataset using a ratio between the partial pressure of oxygen and carbon dioxide (pO₂/pCO₂) to assess how biological processes change under an extensive, yet diverse range of pO₂ and pCO₂ conditions. The dataset comprised 375 experimental comparisons and revealed predominantly additive but variable effects (91.7%, additive; 6.0%, synergistic; and 2.3%, antagonistic) of the dual stressors, yielding negative impacts across almost all responses examined. Our data indicate that the pO₂/pCO₂‐ratio offers a simplified metric to characterize the extremity of the concurrent stressors and shows that more severe impacts occurred when ratios represented more extreme deoxygenation and acidification conditions. Importantly, our analysis highlights the need to assess the concurrent impacts of deoxygenation and acidification on marine taxa and that assessments considering the impact of O₂ depletion alone will likely underestimate the impacts of deoxygenation events and their ecosystem‐wide consequences.     

The reef-building coral Siderastrea siderea exhibits parabolic responses to ocean acidification and warming

Anthropogenic increases in atmospheric CO₂ over this century are predicted to cause global average surface ocean pH to decline by 0.1–0.3 pH units and sea surface temperature to increase by 1–4°C. We conducted controlled laboratory experiments to investigate the impacts of CO₂-induced ocean acidification (pCO₂ = 324, 477, 604, 2553 µatm) and warming (25, 28, 32°C) on the calcification rate of the zooxanthellate scleractinian coral Siderastrea siderea, a widespread, abundant and keystone reef-builder in the Caribbean Sea. We show that both acidification and warming cause a parabolic response in the calcification rate within this coral species. Moderate increases in pCO₂ and warming, relative to near-present-day values, enhanced coral calcification, with calcification rates declining under the highest pCO₂ and thermal conditions. Equivalent responses to acidification and warming were exhibited by colonies across reef zones and the parabolic nature of the corals'' response to these stressors was evident across all three of the experiment''s 30-day observational intervals. Furthermore, the warming projected by the Intergovernmental Panel on Climate Change for the end of the twenty-first century caused a fivefold decrease in the rate of coral calcification, while the acidification projected for the same interval had no statistically significant impact on the calcification rate—suggesting that ocean warming poses a more immediate threat than acidification for this important coral species.     

Diclidophora merlangi and Entobdella soleae: egg production and oxygen consumption at different oxygen partial pressures

The rates of respiration and of egg production of the monogenean trematodes Diclidophora merlangi and Entobdella soleae were compared at different oxygen partial pressures. In D. merlangi the respiratory rate declined sharply as the oxygen pressure in the water fell and egg production almost ceased at pO₂s little below air saturation. Diclidophora lives on the gills of a pelagic fish and the likelihood that the parasite normally has air saturated water passing over it is discussed. Both respiratory rate and egg production in E. soleae were almost independent of ambient pO₂. Entobdella lives on the undersurface of a flat fish, which spends part of its time buried in sandy mud.     

Flow velocity affects internal oxygen conditions in the seagrass Cymodocea nodosa

The internal oxygen status of seagrass tissues, which is believed to play an important role in events of seagrass die-off, is partly determined by the rates of gas exchange between leaves and water column. In this study, we examined whether water column flow velocity has an effect on gas exchange, and hence on internal oxygen partial pressures (pO₂) in the Mediterranean seagrass, Cymodocea nodosa. We measured the internal pO₂ in the horizontal rhizomes of C. nodosa in darkness at different mainstream flow velocities, combined with different levels of water column oxygen pO₂ using an experimental flume in the laboratory. Flow velocity clearly had an effect on the internal oxygen status. In stagnant, but fully aerated water the mean internal pO₂ was 6.9 kPa, corresponding to about 30% of air saturation. The internal pO₂ increased with increasing flow velocity reaching saturation of around 12.2 kPa (60% of air saturation) at flow velocities ≥7 cm s⁻¹. Flow had a relatively larger influence on internal pO₂ at lower water column oxygen concentrations. By extrapolating linear relationships between internal and water column pO₂ in this experimental setup, rhizomes would become anoxic at a water column oxygen pO₂ of 4–4.5 kPa (∼20% of air saturation) in flowing water, but already at 6.4 kPa (∼30% of air saturation) in stagnant water. Water flow may play an important role for seagrass performance and survival in areas with poor water column oxygen conditions and may, in general, be of importance for the distribution of submerged rooted plants.     

Comparison of two co-culture systems to assess the survival of in vitro produced bovine blastocysts after vitrification

The ability of bovine blastocysts to recover after cryopreservation and thawing procedures is often assessed by evaluating their re-expansion during in vitro co-culture. However, the influence of factors such as feeder cell type and gas atmosphere on blastocyst survival and evolution have never been considered. This study therefore compared two cell co-culture systems and two different gas atmospheres to assess survival of in vitro produced bovine blastocysts after vitrification. Day-7 blastocysts (n=181) were vitrified in a mixture of 25% glycerol/25% ethylene glycol. After warming and dilution, they were co-cultured either on Buffalo rat liver cells (BRL CC cell line) or on granulosa cells (GR CC primary culture) in TCM 199 supplemented with 10% FCS and under an atmosphere of 5% or 20% O₂. Surviving and hatching rates were recorded at 24 h intervals for 3 days. After 72 h of culture, surviving blastocysts were treated for differential counting of inner cell mass (ICM) and trophectoderm cells. Blastocyst survival rates were higher when BRL and granulosa co-culture were performed under 20% oxygen as compared to 5% oxygen (20% O₂: 62% vs. 5% O₂: 25%, P<0.0001). However, the quality of blastocysts surviving in the granulosa co-culture condition was lower under 20% O₂ than under 5% O₂ as indicated by lower total and trophectoderm cell numbers (respectively 79±6 and 56±6 at 20% O₂ vs. 100±10 and 74±10 at 5% O₂, P<0.05), by an altered ICM/trophectoderm ratio (20% O₂: 28% vs. 5% O₂: 23%, P<0.05), by a higher total nuclear fragmentation (20% O₂: 3.7% vs. 5% O₂: 1.5%, P<0.05) and a trend to decreased hatching (20% O₂: 32% vs. 5% O₂: 81%, P=0.07). Whereas, for BRL co-culture, 20% O₂ yielded higher quality blastocysts than 5% O₂ as evaluated by higher ICM and trophectoderm cell numbers (19±1 and 71±5 at 20% O₂ vs. 15±2 and 48±9 at 5% O₂, respectively, P<0.05), by lower nuclear fragmentation in the ICM (20% O₂: 2.2% vs. 5% O₂: 6.7%, P<0.05). In conclusion, co-culture conditions may influence blastocysts survival and quality after cryopreservation. In our conditions, co-culture with BRL cells under 20% O₂ seems to be the best combination to evaluate blastocyst survival and quality after vitrification.     

Complex and interactive effects of ocean acidification and warming on the life span of a marine trematode parasite

Human activities have caused an increase in atmospheric CO₂ over the last 250 years, leading to unprecedented rates of change in seawater pH and temperature. These global scale processes are now commonly referred to as ocean acidification and warming, and have the potential to substantially alter the physiological performance of many marine organisms. It is vital that the effects of ocean acidification and warming on marine organisms are explored so that we can predict how marine communities may change in future. In particular, the effect of ocean acidification and warming on host-parasite dynamics is poorly understood, despite the ecological importance of these relationships. Here, we explore the response of one himasthlid trematode, Himasthla sp., an abundant and broadly distributed species of marine parasite, to combinations of elevated temperature and pCO₂ that represent physiological extremes, pre-industrial conditions, and end of century predictions. Specifically, we quantified the life span of the free-living cercarial stage under elevated temperature and pCO₂, focussing our research on functional life span (the time cercariae spend actively swimming) and absolute life span (the period before death). We found that the effects of temperature and pCO₂ were complex and interactive. Overall, increased temperature negatively affected functional and absolute life span, e.g. across all pCO₂ treatments the average time to 50% cessation of active swimming was approximately 8 h at 5 °C, 6 h at 15 °C, 4 h at 25 °C, and 2 h at 40 °C. The effect of pCO₂, which significantly affected absolute life span, was highly variable across temperature treatments. These results strongly suggest that ocean acidification and warming may alter the transmission success of trematode cercariae, and potentially reduce the input of cercariae to marine zooplankton. Either outcome could substantially alter the community structure of coastal marine systems.     

INTERACTIONS BETWEEN OCEAN ACIDIFICATION AND WARMING ON THE MORTALITY AND DISSOLUTION OF CORALLINE ALGAE1

Coralline algae are among the most sensitive calcifying organisms to ocean acidification as a result of increased atmospheric carbon dioxide (pCO₂). Little is known, however, about the combined impacts of increased pCO₂, ocean acidification, and sea surface temperature on tissue mortality and skeletal dissolution of coralline algae. To address this issue, we conducted factorial manipulative experiments of elevated CO₂ and temperature and examined the consequences on tissue survival and skeletal dissolution of the crustose coralline alga (CCA) Porolithon (=Hydrolithon) onkodes (Heydr.) Foslie (Corallinaceae, Rhodophyta) on the southern Great Barrier Reef (GBR), Australia. We observed that warming amplified the negative effects of high pCO₂ on the health of the algae: rates of advanced partial mortality of CCA increased from <1% to 9% under high CO₂ (from 400 to 1,100 ppm) and exacerbated to 15% under warming conditions (from 26°C to 29°C). Furthermore, the effect of pCO₂ on skeletal dissolution strongly depended on temperature. Dissolution of P. onkodes only occurred in the high‐pCO₂ treatment and was greater in the warm treatment. Enhanced skeletal dissolution was also associated with a significant increase in the abundance of endolithic algae. Our results demonstrate that P. onkodes is particularly sensitive to ocean acidification under warm conditions, suggesting that previous experiments focused on ocean acidification alone have underestimated the impact of future conditions on coralline algae. Given the central role that coralline algae play within coral reefs, these conclusions have serious ramifications for the integrity of coral‐reef ecosystems.     

Oxygen poisoning in Drosophila

Fruit flies live longer at the partial pressure of oxygen found in air than at either larger or smaller partial pressures. Flies exposed to 1 atm of oxygen for 8 hr every day do not recover completely in the remaining 16 hr. In general, intermittent exposures to 1 atm of oxygen are better tolerated than continuous exposure to the same average oxygen concentration per day, but exposures to higher pressures of 2–5 atm of oxygen for as little as a half hour every two days markedly shorten the life-span. Older flies consume more oxygen per minute and are more sensitive to oxygen poisoning than young flies, and the rate of dying in 6 atm of O₂, or the reciprocal of the survival time, is a linear function of the age. The oxygen pressure-time curve can be well expressed by the general empirical equation (P_OO2)² x time = 120 where P is in atmosphere and survival time in hours. The progress of oxygen poisoning appears to be linear with time rather than exponential.     

Effects of ocean acidification increase embryonic sensitivity to thermal extremes in Atlantic cod,Gadus morhua

Thermal tolerance windows serve as a powerful tool for estimating the vulnerability of marine species and their life stages to increasing temperature means and extremes. However, it remains uncertain to which extent additional drivers, such as ocean acidification, modify organismal responses to temperature. This study investigated the effects of CO₂‐driven ocean acidification on embryonic thermal sensitivity and performance in Atlantic cod, Gadus morhua, from the Kattegat. Fertilized eggs were exposed to factorial combinations of two PCO₂ conditions (400 μatm vs. 1100 μatm) and five temperature treatments (0, 3, 6, 9 and 12 °C), which allow identifying both lower and upper thermal tolerance thresholds. We quantified hatching success, oxygen consumption (MO₂) and mitochondrial functioning of embryos as well as larval morphometrics at hatch and the abundance of acid–base‐relevant ionocytes on the yolk sac epithelium of newly hatched larvae. Hatching success was high under ambient spawning conditions (3–6 °C), but decreased towards both cold and warm temperature extremes. Elevated PCO₂ caused a significant decrease in hatching success, particularly at cold (3 and 0 °C) and warm (12 °C) temperatures. Warming imposed limitations to MO₂ and mitochondrial capacities. Elevated PCO₂ stimulated MO₂ at cold and intermediate temperatures, but exacerbated warming‐induced constraints on MO₂, indicating a synergistic interaction with temperature. Mitochondrial functioning was not affected by PCO₂. Increased MO₂ in response to elevated PCO₂ was paralleled by reduced larval size at hatch. Finally, ionocyte abundance decreased with increasing temperature, but did not differ between PCO₂ treatments. Our results demonstrate increased thermal sensitivity of cod embryos under future PCO₂ conditions and suggest that acclimation to elevated PCO₂ requires reallocation of limited resources at the expense of embryonic growth. We conclude that ocean acidification constrains the thermal performance window of embryos, which has important implication for the susceptibility of cod to projected climate change.     

Spatio-temporal relief from hypoxia and production of reactive oxygen species during bud burst in grapevine (Vitis vinifera)

Background and Aims Plants regulate cellular oxygen partial pressures (pO₂), together with reduction/oxidation (redox) state in order to manage rapid developmental transitions such as bud burst after a period of quiescence. However, our understanding of pO₂ regulation in complex meristematic organs such as buds is incomplete and, in particular, lacks spatial resolution.Methods The gradients in pO₂ from the outer scales to the primary meristem complex were measured in grapevine (Vitis vinifera) buds, together with respiratory CO₂ production rates and the accumulation of superoxide and hydrogen peroxide, from ecodormancy through the first 72 h preceding bud burst, triggered by the transition from low to ambient temperatures.Key Results Steep internal pO₂ gradients were measured in dormant buds with values as low as 2·5 kPa found in the core of the bud prior to bud burst. Respiratory CO₂ production rates increased soon after the transition from low to ambient temperatures and the bud tissues gradually became oxygenated in a patterned process. Within 3 h of the transition to ambient temperatures, superoxide accumulation was observed in the cambial meristem, co-localizing with lignified cellulose associated with pro-vascular tissues. Thereafter, superoxide accumulated in other areas subtending the apical meristem complex, in the absence of significant hydrogen peroxide accumulation, except in the cambial meristem. By 72 h, the internal pO₂ gradient showed a biphasic profile, where the minimum pO₂ was external to the core of the bud complex.Conclusions Spatial and temporal control of the tissue oxygen environment occurs within quiescent buds, and the transition from quiescence to bud burst is accompanied by a regulated relaxation of the hypoxic state and accumulation of reactive oxygen species within the developing cambium and vascular tissues of the heterotrophic grapevine buds.     

Use of models in understanding the mechanism of action of haemoglobin

Models with three, four and eight salt-bridges have been used to study the mechanism of action of haemoglobin. Both side chains forming a salt-bridge, i.e. the proton acceptor and the proton donor, are postulated to change pK on ligation of oxygen. The eight salt-bridge model is able to predict, as a unified theory, both the degree of oxygenation and the Bohr effect at any PH and p_O2 value; this has not been done by any other published model. The predicted pK values for the Borh groups corresponde well with those measured experiemntally. This model predicts the pK values of those side chains responsible for the acid Bohr effect, suggesting that these correspond to the proton acceptors of the salt-bridges. The model also fulfils the condition of linearity between the fractional degree of oxygenation and fractional number of protons released. It is postulated that there is a gradual change in structure on going from deoxy to oxyhaemoglobin, due to the rupture of salt-bridges. The path folowed during this process will be both pH and p_O2 dependent. A formula describing the number of intact or broken salt-bridges as a function of pH and p_O2 was developed. This formula shows that the fractional number of broken salt-bridges reaches a minimum value of 0.2 at around pH 6.3 in the absence of oxygen. However, if oxygen is added, this fractional number approaches 1.0 soon after the partial pressure of oxygen goes above 40 mm Hg.     

High Oxygen Condition Facilitates the Differentiation of Mouse and Human Pluripotent Stem Cells into Pancreatic Progenitors and Insulin-producing Cells

Pluripotent stem cells have potential applications in regenerative medicine for diabetes. Differentiation of stem cells into insulin-producing cells has been achieved using various protocols. However, both the efficiency of the method and potency of differentiated cells are insufficient. Oxygen tension, the partial pressure of oxygen, has been shown to regulate the embryonic development of several organs, including pancreatic β-cells. In this study, we tried to establish an effective method for the differentiation of induced pluripotent stem cells (iPSCs) into insulin-producing cells by culturing under high oxygen (O₂) conditions. Treatment with a high O₂ condition in the early stage of differentiation increased insulin-positive cells at the terminus of differentiation. We found that a high O₂ condition repressed Notch-dependent gene Hes1 expression and increased Ngn3 expression at the stage of pancreatic progenitors. This effect was caused by inhibition of hypoxia-inducible factor-1α protein level. Moreover, a high O₂ condition activated Wnt signaling. Optimal stage-specific treatment with a high O₂ condition resulted in a significant increase in insulin production in both mouse embryonic stem cells and human iPSCs and yielded populations containing up to 10% C-peptide-positive cells in human iPSCs. These results suggest that culturing in a high O₂ condition at a specific stage is useful for the efficient generation of insulin-producing cells.     

Partial pressure of oxygen in brain and peripheral nerve during damaging electrical stimulation

The studies were performed to elucidate the mechanism underlying the neural damage which may occur during prolonged electrical stimulation of either brain tissue or peripheral nerve. The partial pressure of oxygen (pO₂) was measured in the sciatic nerve and the cerebral cortex of adult cats before and during direct, local electrical stimulation of these neural tissues, using stimulus parameters capable of inducing neural injury. pO₂ was monitored by the polarographic method, employing a platinum microelectrode inserted into the tissue adjacent to or beneath the stimulating electrode. In the sciatic nerve there was no marked change in intrafascicular pO₂ in three cats upon initiation of the electrical stimulation. In a fourth animal intraneural pO₂ increased briefly upon intitiation of the stimulation. In no case did the intrafascicular compartment of nerves become significantly hypoxic. In the cerebral cortex, the start of stimulation was accompanied by a significant increase (approximately 12–15 Torr) in intracortical pO₂ beneath the stimulating electrode, and pO₂ remained at or above the pre-stimulus value for the duration of the stimulation. These results show that extracellular hypoxia is unlikely to be a significant factor in the neural injury induced in brain or peripheral nerve by prolonged electrical stimulation.     

Will krill fare well under Southern Ocean acidification?

Antarctic krill embryos and larvae were experimentally exposed to 380 (control), 1000 and 2000 µatm pCO₂ in order to assess the possible impact of ocean acidification on early development of krill. No significant effects were detected on embryonic development or larval behaviour at 1000 µatm pCO₂; however, at 2000 µatm pCO₂ development was disrupted before gastrulation in 90 per cent of embryos, and no larvae hatched successfully. Our model projections demonstrated that Southern Ocean sea water pCO₂ could rise up to 1400 µatm in krill''s depth range under the IPCC IS92a scenario by the year 2100 (atmospheric pCO₂ 788 µatm). These results point out the urgent need for understanding the pCO₂-response relationship for krill developmental and later stages, in order to predict the possible fate of this key species in the Southern Ocean.     

Root Growth and Oxygen Relations at Low Water Potentials. Impact of Oxygen Availability in Polyethylene Glycol Solutions

Polyethylene glycol (PEG), which is often used to impose low water potentials (ψ_w) in solution culture, decreases O₂ movement by increasing solution viscosity. We investigated whether this property causes O₂ deficiency that affects the elongation or metabolism of maize (Zea mays L.) primary roots. Seedlings grown in vigorously aerated PEG solutions at ambient solution O₂ partial pressure (pO₂) had decreased steady-state root elongation rates, increased root-tip alanine concentrations, and decreased root-tip proline concentrations relative to seedlings grown in PEG solutions of above-ambient pO₂ (alanine and proline accumulation are responses to hypoxia and low ψ_w, respectively). Measurements of root pO₂ were made using an O₂ microsensor to ensure that increased solution pO₂ did not increase root pO₂ above physiological levels. In oxygenated PEG solutions that gave maximal root elongation rates, root pO₂ was similar to or less than (depending on depth in the tissue) pO₂ of roots growing in vermiculite at the same ψ_w. Even without PEG, high solution pO₂ was necessary to raise root pO₂ to the levels found in vermiculite-grown roots. Vermiculite was used for comparison because it has large air spaces that allow free movement of O₂ to the root surface. The results show that supplemental oxygenation is required to avoid hypoxia in PEG solutions. Also, the data suggest that the O₂ demand of the root elongation zone may be greater at low relative to high ψ_w, compounding the effect of PEG on O₂ supply. Under O₂-sufficient conditions root elongation was substantially less sensitive to the low ψ_w imposed by PEG than that imposed by dry vermiculite.     

Influence of elevated CO2 concentrations on thermal tolerance of the edible crab Cancer pagurus

Current trends of global climate change affect marine ectothermal animals not only through the increase in ambient temperature. Synergistic effects of carbon dioxide and temperature changes as well as more frequent hypoxia events must also be considered. As a first attempt, the combined effects of warming and elevated CO₂ concentrations were investigated in the edible crab (Cancer pagurus). Arterial oxygen tension (PaO₂) in the haemolymph was recorded on-line during a progressive warming scenario from 10 to 22 °C and cooling back to 10 °C. Hypercapnia (1% CO₂) caused a significant reduction of oxygen partial pressure in the haemolymph as well as a large, 5 °C downward shift of upper thermal limits of aerobic scope. The present findings are the first to show that hypercapnia causes enhanced sensitivity to heat and thus, a narrowing of the thermal tolerance window of a marine ectotherm. Such interactions of ambient temperature and anthropogenic increases in ambient CO₂ concentrations will need to be considered during future investigations of the effects of climate change on ecosystems.     

Ocean Acidification Accelerates Reef Bioerosion

In the recent discussion how biotic systems may react to ocean acidification caused by the rapid rise in carbon dioxide partial pressure (pCO₂) in the marine realm, substantial research is devoted to calcifiers such as stony corals. The antagonistic process – biologically induced carbonate dissolution via bioerosion – has largely been neglected. Unlike skeletal growth, we expect bioerosion by chemical means to be facilitated in a high-CO₂ world. This study focuses on one of the most detrimental bioeroders, the sponge Cliona orientalis, which attacks and kills live corals on Australia’s Great Barrier Reef. Experimental exposure to lowered and elevated levels of pCO₂ confirms a significant enforcement of the sponges’ bioerosion capacity with increasing pCO₂ under more acidic conditions. Considering the substantial contribution of sponges to carbonate bioerosion, this finding implies that tropical reef ecosystems are facing the combined effects of weakened coral calcification and accelerated bioerosion, resulting in critical pressure on the dynamic balance between biogenic carbonate build-up and degradation.     

Some effects of low oxygen partial pressures on the development of Calliphora vomitoria;

The influence of wet conditions and low pO₂ on the survival and development of non-feeding final instar larvae and puparia of Calliphora vomitoria has been investigated. The larvae delay the formation of the puparium in wet conditions in air and in dry or wet conditions in 10 and 5% oxygen. This may be related to the susceptibility of the newly formed puparia to oxygen shortage. The pupal respiratory horns play an important part in maintaining O₂ uptake when the puparia are surrounded by particles covered with a film of water but are not involved in aiding survival in low pO₂. Zero age puparia are killed by a 2 day exposure to 10% O₂ but later stages can continue to develop in this gas. Fifty per cent of the 0, 1 and 9 day old puparia are killed by about a 12 hr exposure to 1% O₂ whereas 50 per cent of the 2 to 8 day old puparia can survive over 1·5 days exposure to this gas. Development, as measured by respiration rates and the timing of the emergence of the adults, is delayed by 1% O₂ by the amount of time that the insects spend in that gas. However, the first phase of elongation of the pharate adult longitudinal flight muscle, occurring between the third and fourth day of puparial life, is only slightly slowed down in 1% O₂. The variations in susceptibility to 1% O₂ and the growth of the muscles are discussed in relation to published accounts of protein synthesis in the puparium.     

Oxygen-Sensitive K+ Channels Modulate Human Chorionic Gonadotropin Secretion from Human Placental Trophoblast

Human chorionic gonadotropin (hCG) is a key autocrine/paracrine regulator of placental syncytiotrophoblast, the transport epithelium of the human placenta. Syncytiotrophoblast hCG secretion is modulated by the partial pressure of oxygen (pO₂), reactive oxygen species (ROS) and potassium (K⁺) channels. Here we test the hypothesis that K⁺ channels mediate the effects of pO₂ and ROS on hCG secretion. Placental villous explants from normal term pregnancies were cultured for 6 days at 6% (normoxia), 21% (hyperoxia) or 1% (hypoxia) pO₂. On days 3–5, explants were treated with 5mM 4-aminopyridine (4-AP) or tetraethylammonium (TEA), blockers of pO₂-sensitive voltage-gated K⁺ (K_V) channels, or ROS (10–1000μM H₂O₂). hCG secretion and lactate dehydrogenase (LDH) release, a marker of necrosis, were determined daily. At day 6, hCG and LDH were measured in tissue lysate and ⁸⁶Rb (K⁺) efflux assessed to estimate syncytiotrophoblast K⁺ permeability. hCG secretion and ⁸⁶Rb efflux were significantly greater in explants maintained in 21% pO₂ than normoxia. 4-AP/TEA inhibited hCG secretion to a greater extent at 21% than 6% and 1% pO₂, and reduced ⁸⁶Rb efflux at 21% but not 6% pO₂. LDH release and tissue LDH/hCG were similar in 6%, 21% and 1% pO₂ and unaffected by 4-AP/TEA. H₂O₂ stimulated ⁸⁶Rb efflux and hCG secretion at normoxia but decreased ⁸⁶Rb efflux, without affecting hCG secretion, at 21% pO₂. 4-AP/TEA-sensitive K⁺ channels participate in pO₂-sensitive hCG secretion from syncytiotrophoblast. ROS effects on both hCG secretion and ⁸⁶Rb efflux are pO₂-dependent but causal links between the two remain to be established.