Hemolymph pH Research Articles

Effects of elevated CO2 on the critical oxygen tension (Pcrit) and aerobic metabolism of two oxygen minimum zone (OMZ) hypoxia tolerant squat lobster species.

Marine invertebrates living in oxygen minimum zones (OMZ), where low pO2 and high pCO2 conditions co-occur, display physiological mechanisms allowing them to deal with these coupled stressors. We measured aerobic metabolic rate (MRa) and the critical oxygen tension (Pcrit), and calculated the oxygen supply capacity (α) of both the red (Grimothea monodon) and yellow (Grimothea johni) squat lobsters, under two pCO2 scenarios (~414 and 1400 μatm). We also measured haemolymph pH, haemocyanin oxygen binding affinity (p50), and haemolymph lactate content in both species under normoxia, low pCO2 hypoxia and high pCO2 hypoxia. Our results revealed that both species show extremely low Pcrit and P50 values. The MRa increased under high pCO2 condition in both species but hypoxia tolerance was not negatively impacted by pCO2. Furthermore, hypoxia tolerance is enhanced at high pCO2 in the yellow squat lobster, and although not statically significant, α value follows the same trend. The red squat lobster has a better pHe regulation and lower reliance on anaerobic metabolism. While the yellow squat lobster had a poorer pHe regulation during high pCO2 hypoxia, relying more on anaerobic metabolism. Our research suggests that elevated pCO2 is crucial on respiratory processes in hypoxia tolerant organisms, ameliorating the effects of hypoxia alone. Learning from OMZ adapted species contribute to better predicting climate change consequences on these important ecosystems.

Rates of osmoconformation in triploid eastern oysters, and comparison to their diploid half-siblings

Triploid eastern oysters (Crassostrea virginica) suffer greater mortalities than diploids in the U.S. Gulf of Mexico estuaries when extreme low salinities (< 5) and elevated temperatures (≥ 28 °C) coincide. To investigate potential causes, changes in plasma osmolality, hemolymph pH, valve opening and mortality in diploid and triploid oyster half-siblings were compared during a step-down gradual acclimation from a salinity of 5 to 1 (5, 2.5, 2.0, 1.5, 1.0) at 23 °C (expt 1) and at 28 °C (expt 2). To further explore differences in diploid and triploid oyster responses to changing salinity, we compared their plasma osmolality after abrupt decreases in salinity from 20 to 10 and 5, followed by increases in salinity from 5 and 10 to 20 once oysters had osmoconformed to the lowered salinities (expt 3). Lastly, changes in wet weights of mantle tissue explants were compared between diploid and triploid oysters every 10 min for 40 min after being transferred from a salinity of 20 to 10 (expt 4). Oysters of both ploidies were able to osmoconform to water at salinities between 5 and 1.5. After a decrease in salinity, triploid oysters were slower to open their valves and osmoconform, were less efficient in maintaining acid-base status enduring longer periods of acidic hemolymph pH, and were less efficient in regulating tissue water content compared to half-sibling diploid oysters. At a salinity of 1.0, plasma of both diploid and triploid oysters remained hyperosmotic, their hemolymph acidic and their valves closed. Oysters osmoconformed faster at 28 °C than at 23 °C, but the combination of low salinity (≤ 1.5) and higher temperature caused rapid mortalities regardless of ploidies. Triploid oysters, however, started dying earlier and at greater percentages when salinity of 1.5 and temperature of 28 °C were combined. Triploids have been embraced as a means to support higher production, but results indicate superimposed stressors, such as low salinity and high temperature, may be more lethal to triploid than diploid oysters.

Examination of the potential of refrigerated seawater to improve live transport of the mussel Perna canaliculus: Physiological responses, meat quality and safety implications under different chilled storage conditions

Refrigerated seawater (RSW) based systems for post-harvest storage of green-lipped mussels (Perna canaliculus; also known as Greenshell™ mussels) were assessed. Two post-harvest storage trials were conducted in October 2020 and March 2021 (austral spring and summer respectively) on mussels recovered for 2.5 days after harvest in flow-through seawater. The mussels were randomly assigned to three post-harvest treatments, all conducted at 6.5 °C for 10 days: Chilled in air; immersion in recirculated RSW; and Periodic immersion in RSW (6 min h−1). A range of physiological, morphometric and quality measures were screened to assess their ability to indicate mussel performance. Further to this, mussels in duplicate systems were inoculated with Escherichia coli and Vibrio parahaemolyticus to inform on risks associated with pathogen proliferation. The mussels interacted with the seawater in both the RSW and Periodic RSW systems as seawater ammonia concentrations increased in both. Mussel mortalities counted across the 10-day trials were highest in the Chilled treatment (5.5%), followed by RSW (3.5%) and Periodic RSW (2.4%). Key measurements including haemolymph pH and osmolality, meat and intra-valvular water weight, and textural hardness, were all affected by the post-harvest treatments. For inoculated mussels, E. coli and V. parahaemolyticus most probable numbers (MPN) remained stable in both the Chilled and Periodic RSW treatments with reductions in both bacteria recorded in the RSW treatment. Higher variances in intra-valvular water retention and haemolymph haemocyte numbers and differences in volatile organic compound profiles were seen in the summer mussels, as compared with spring, highlighting mussel condition as a determining factor for post-harvest performance. RSW has therefore been demonstrated to be beneficial, but optimisation (e.g., through further reduction of metabolic activity) may further increase practical post-harvest transport distances and the quality of live/fresh mussel products.

Haemolymph pH of two important mollusc species is susceptible to seawater buffering capacity instead of pH or pCO2

The acid-base status and balance of molluscs are considered to be susceptible to environmental changes, especially in the context of ocean acidification (OA). Here, we studied the effects of manipulated seawater carbonate chemistry on the acid-base status of scallop Chlamys farreri and abalone Haliotis discus hannai. The haemolymph pH of the tested individuals showed a fast response to acidified seawater incubation, and the pH level was restored to a normal value within 1 h of recovery in control seawater. However, no significant correlation (P > 0.05) was found between haemolymph pH and seawater pCO2 or pH, while the squared Pearson correlation coefficient (R2) ranged from close to zero to 0.41. In addition, although the pCO2 level of total alkalinity (TA)-lowered seawater was lower than half of that in the control, molluscs eliminated less CO2 (less than 80%) to TA lowered waters than to the control waters. These findings seem to disagree with the crucial role of seawater pCO2 in influencing the acid-base balance of molluscs. CO2 elimination occurs in the microenvironment, and CO2 first diffuses to limited amounts of seawater that tightly surround the gills, causing dissolved inorganic carbon (DIC) accumulation in the ventilation sites, which leads to a sharp increase in the pCO2 of the surrounding seawater. Moreover, in this microenvironment, the pCO2 level increases much faster and more greatly if the environmental seawater is acidified or contains a lower level of TA. Therefore, mollusc acid-base status is influenced by rapidly varying pCO2 levels at the ventilation site, which is largely independent of that of the rest of the incubating seawater. In summary, CO2 elimination by molluscs relies heavily on the carbonate chemistry of environmental seawater, and seawater buffering capacity should be taken into consideration instead of considering only pCO2 or pH in studying the acid-base balance of marine molluscs.

Effect of high alkalinity on shrimp gills: Histopathological alternations and cell specific responses

High alkalinity stress was considered as a major risk factor for aquatic animals surviving in saline-alkaline water. However, few information exists on the effects of alkalinity stress in crustacean species. As the dominant role of gills in osmotic and ionic regulation, the present study firstly evaluated the effect of alkalinity stress in Exopalaemon carinicauda to determine changes in gill microstructure, and then explore the heterogeneity response of gill cells in alkalinity adaptation by single-cell RNA sequencing (scRNA-seq). Hemolymph osmolality and pH were increased remarkably, and gills showed pillar cells with more symmetrical arrangement and longer lateral flanges and nephrocytes with larger vacuoles in high alkalinity. ScRNA-seq results showed that alkalinity stress reduced the proportion of pillar cells and increased the proportion of nephrocytes significantly. The differentially expressed genes (DEGs) related to ion transport, especially acid-base regulation, such as V(H+)-ATPases and carbonic anhydrases, were down-regulated in pillar cells and up-regulated in nephrocytes. Furthermore, pseudotime analysis showed that some nephrocytes transformed to perform ion transport function in alkalinity adaption. Notedly, the positive signals of carbonic anhydrase were obviously observed in the nephrocytes after alkalinity stress. These results indicated that the alkalinity stress inhibited the ion transport function of pillar cells, but induced the active role of nephrocytes in alkalinity adaptation. Collectively, our results provided the new insight into the cellular and molecular mechanism behind the adverse effects of saline-alkaline water and the saline-alkaline adaption mechanism in crustaceans.

Hypoxia and Anoxia Tolerance in Diploid and Triploid Eastern Oysters at High Temperature

Increasing reliance on the use of triploid oysters to support aquaculture production relies on their generally superior growth rate and meat quality over that of diploid oysters. Reports of elevated triploid mortality have generated questions about potential trade-offs between growth and tolerance to environmental stressors. These questions are particularly relevant as climate change, coastal activities, and river management impact water salinity, temperature, nutrients, pH, and oxygen levels within key estuarine oyster growing areas. In particular, the co-occurrence of warm water temperatures and low dissolved oxygen concentration (DO) events are increasingly reported in estuaries, with potentially lethal impacts on sessile, oyster resources. To investigate potential differences in DO tolerance, diploid and triploid market-sized or seed oysters were exposed to continuous normoxia (DO > 5.0 mg L–1), hypoxia (DO < 2.0 mg L–1), and anoxia (DO < 0.5 mg L–1) at 28°C and their mortalities were monitored. The hemolymph of the market-sized oysters was collected to measure cellular and biochemical changes in response to hypoxia and anoxia, whereas their valve movements were also measured. In general, about half of market-sized oysters died within about 1 wk under anoxia (LT50: 5.7–8.9 days) and within about 2 wk under hypoxia (LT50: 11.9–19.4 days) with diploid oysters tending to die faster than triploid oysters. Seed oysters took longer to die than market-sized oysters under both anoxia (LT50: 9.5–12.1 days) and hypoxia (LT50: 21.8–25.0 days) with diploid oysters (LT50: 9.5–11.8 days) dying slightly faster than triploid oysters (LT50: 11.8–12.1 days) under anoxia. Hemolymph pH decreased and plasma calcium and glutathione concentrations increased with decreasing DO, with values under anoxia being different than those under normoxia. Hemocyte density was also lower under anoxia than under either normoxia or hypoxia. Overall, few differences in physiological responses to hypoxia and anoxia were found between diploid and triploid oysters suggesting that ploidy (2N versus 3N) had limited effect on the tolerance and response of eastern oysters to low DO.

Proteomic and Transcriptomic Responses Enable Clams to Correct the pH of Calcifying Fluids and Sustain Biomineralization in Acidified Environments.

Seawater pH and carbonate saturation are predicted to decrease dramatically by the end of the century. This process, designated ocean acidification (OA), threatens economically and ecologically important marine calcifiers, including the northern quahog (Mercenaria mercenaria). While many studies have demonstrated the adverse impacts of OA on bivalves, much less is known about mechanisms of resilience and adaptive strategies. Here, we examined clam responses to OA by evaluating cellular (hemocyte activities) and molecular (high-throughput proteomics, RNASeq) changes in hemolymph and extrapallial fluid (EPF-the site of biomineralization located between the mantle and the shell) in M. mercenaria continuously exposed to acidified (pH ~7.3; pCO2 ~2700 ppm) and normal conditions (pH ~8.1; pCO2 ~600 ppm) for one year. The extracellular pH of EPF and hemolymph (~7.5) was significantly higher than that of the external acidified seawater (~7.3). Under OA conditions, granulocytes (a sub-population of hemocytes important for biomineralization) were able to increase intracellular pH (by 54% in EPF and 79% in hemolymph) and calcium content (by 56% in hemolymph). The increased pH of EPF and hemolymph from clams exposed to high pCO2 was associated with the overexpression of genes (at both the mRNA and protein levels) related to biomineralization, acid-base balance, and calcium homeostasis, suggesting that clams can use corrective mechanisms to mitigate the negative impact of OA.

Effects of one-year exposure to ocean acidification on two species of abalone

Ocean acidification (OA) resulting from the absorption of excess atmospheric CO2 by the ocean threatens the survival of marine calcareous organisms, including mollusks. This study investigated the effects of OA on adults of two abalone species (Haliotis diversicolor, a subtropical species, and Haliotis discus hannai, a temperate species). Abalone were exposed to three pCO2 conditions for 1 year (ambient, ~ 880, and ~ 1600 μatm), and parameters, including mortality, physiology, immune system, biochemistry, and carry-over effects, were measured. Survival decreased significantly at ~ 800 μatm pCO2 for H. diversicolor, while H. discus hannai survival was negatively affected only at a higher OA level (~ 1600 μatm pCO2). H. diversicolor exhibited depressed metabolic and excretion rates and a higher O:N ratio under OA, indicating a shift to lipids as a metabolism substrate, while these physiological parameters in H. discus hannai were robust to OA. Both abalone failed to compensate for the pH decrease of their internal fluids because of the lowered hemolymph pH under OA. However, the reduced hemolymph pH did not affect total hemocyte counts or tested biomarkers. Additionally, H. discus hannai increased its hemolymph protein content under OA, which could indicate enhanced immunity. Larvae produced by adults exposed to the three pCO2 levels were cultured in the same pCO2 conditions and larval deformation and shell length were measured to observe carry-over effects. Enhanced OA tolerance was observed for H. discus hannai exposed under both of the OA treatments, while that was only observed following parental pCO2 ~ 880 μatm exposure for H. diversicolor. Following pCO2 ~ 1600 μatm parental exposure, H. diversicolor offspring exhibited higher deformation and lower shell growth in all pCO2 treatments. In general, H. diversicolor were more susceptible to OA compared with H. discus hannai, suggesting that H. diversicolor could be unable to adapt to acidified oceans in the future.

Meta-analysis suggests negative, but pCO2-specific, effects of ocean acidification on the structural and functional properties of crustacean biomaterials.

Crustaceans comprise an ecologically and morphologically diverse taxonomic group. They are typically considered resilient to many environmental perturbations found in marine and coastal environments, due to effective physiological regulation of ions and hemolymph pH, and a robust exoskeleton. Ocean acidification can affect the ability of marine calcifying organisms to build and maintain mineralized tissue and poses a threat for all marine calcifying taxa. Currently, there is no consensus on how ocean acidification will alter the ecologically relevant exoskeletal properties of crustaceans. Here, we present a systematic review and meta‐analysis on the effects of ocean acidification on the crustacean exoskeleton, assessing both exoskeletal ion content (calcium and magnesium) and functional properties (biomechanical resistance and cuticle thickness). Our results suggest that the effect of ocean acidification on crustacean exoskeletal properties varies based upon seawater pCO2 and species identity, with significant levels of heterogeneity for all analyses. Calcium and magnesium content was significantly lower in animals held at pCO2 levels of 1500–1999 µatm as compared with those under ambient pCO2. At lower pCO2 levels, however, statistically significant relationships between changes in calcium and magnesium content within the same experiment were observed as follows: a negative relationship between calcium and magnesium content at pCO2 of 500–999 µatm and a positive relationship at 1000–1499 µatm. Exoskeleton biomechanics, such as resistance to deformation (microhardness) and shell strength, also significantly decreased under pCO2 regimes of 500–999 µatm and 1500–1999 µatm, indicating functional exoskeletal change coincident with decreases in calcification. Overall, these results suggest that the crustacean exoskeleton can be susceptible to ocean acidification at the biomechanical level, potentially predicated by changes in ion content, when exposed to high influxes of CO2. Future studies need to accommodate the high variability of crustacean responses to ocean acidification, and ecologically relevant ranges of pCO2 conditions, when designing experiments with conservation‐level endpoints.

Effect of Air Exposure on the Acid–Base Balance of Hemolymph in Akoya Pearl Oyster Pinctada fucata martensii

The hemolymph acid–base status of Akoya pearl oyster Pinctada fucata martensii exposed to air at 20°C was investigated. Air-exposed Akoya pearl oyster showed a decrease in hemolymph pH from 7.568 to 6.825 after 24 h. The hemolymph total CO2 concentration increased from 2.25 mM/L to 4.50 mM/L during 24 h of air exposure. The hemolymph CO2 partial pressure (Pco2) was calculated by rearranging the Henderson–Hasselbalch equation. The hemolymph Pco2 increased from 1.0 torr to 14.8 torr, and bicarbonate ion concentration increased from 2.21 mM/L to 3.91 mM/L during 24 h of air exposure. The hemolymph calcium ion concentration ([Ca2+]) increased from 9.4 mM/L to 12.8 mM/L. These results indicated that Akoya pearl oysters showed hemolymph acidosis with partial metabolic compensation by mobilization of bicarbonate from the shell valve during prolonged air exposure. Immersion in seawater for between 4 h and 24 h decreased the effect of air exposure on hemolymph acid–base status, except for [Ca2+]. The hemolymph [Ca2+] of immersed Akoya pearl oysters was slightly higher than the initial level upon air exposure although hemolymph [Ca2+] had already decreased after immersion in seawater for 4 h and 24 h. The hemolymph acid–base balance of immersed Akoya pearl oysters recovered to the initial level after 4–24 h, even if the animals were exposed to the air for a prolonged time (24 h).

Alterations in Hemolymph Ion Concentrations and pH in Adult Daphnia magna in Response to Elevations in Major Ion Concentrations in Freshwater.

Increases in the concentrations of major ions (Na+ , K+ , Ca2+ , Mg2+ , Cl- ) in freshwater are a growing concern for ecosystem health. These increases may originate from anthropogenic activities such as road deicing, fracking spills, mining, and fertilizer application and have detrimental effects on freshwater organisms through disturbances in ionoregulation and acid-base balance. The cladoceran Daphnia magna is adapted for active ion uptake and reduction of ion loss to maintain osmotic balance, but alterations in ionic composition of the environmental water are associated with toxicity. In the present study, hemolymph ion concentrations were measured using ion-selective microelectrode techniques. Increases in the hemolymph concentrations of Na+ and K+ correspond to elevations in the concentrations of these ions in ambient water. Water concentrations associated with sustained increases in hemolymph ion concentrations correlate well with median lethal concentration values from previous toxicology studies, indicating that Na+ and K+ concentrations in hemolymph may predict toxicity. When water K+ concentration is increased, a simultaneous increase in water Na+ concentration mitigates the increase in hemolymph K+ concentration, a finding which is consistent with the reported mitigation of K+ toxicity by Na+ . When ambient concentrations of K+ , Na+ , and Cl- are increased, not only is there a rise in hemolymph ion concentration but hemolymph pH is altered and pH regulation appears to be prioritized over regulation of hemolymph Na+ , K+ , and Cl- in D. magna. Environ Toxicol Chem 2021;40:366-379. © 2020 SETAC.

Physiological response of the giant acorn barnacle, Balanus nubilus, to oxygen-limiting environments

Sessile invertebrates in the nearshore coastal and rocky intertidal habitats can experience oxygen limitation during low tide air exposure and environmental hypoxia events. For some organisms, unique morphological characteristics may make these events especially challenging. The giant acorn barnacle, Balanus nubilus, has the largest muscle fibers in the animal kingdom (diameters can exceed 3 mm in adults). At these extreme sizes, muscle cells may already be at the brink of insufficient oxygen delivery owing to low SA:V ratios and long intracellular diffusion distances. We are interested in characterizing the internal oxygen dynamics of B. nubilus during air exposure and seawater anoxia so that we can better understand how they maintain function of their giant muscle fibers during environmentally-induced oxygen limitation. To this end, we examined the following: 1) hemolymph pO2, pCO2, pH and ion (Na+, Cl−, K+, Ca2+) concentrations across 9 h exposure to air emersion, anoxic immersion and normoxic immersion (control), 2) oxygen consumption rates (MO2) of barnacles held in water and air for 6 h (at 10, 15 and 20 °C), and 3) respiratory behaviors (e.g., % time operculum open or cirri extended, cirral beat frequency) of barnacles during acute (6 h) exposure to emersion, anoxic immersion and normoxic immersion. Our data revealed that hemolymph pO2 declined significantly (by 3 h) in the anoxic barnacles, whereas the air-exposed barnacles maintained hemolymph oxygen levels that were intermediate to the control and anoxia barnacles. We also found that MO2 values for B. nubilus were very similar in seawater and air at a common temperature. These results suggest that B. nubilus can effectively acquire oxygen and support aerobic metabolism while in the air. This assumption was corroborated by our behavioral data, which revealed that air exposed (and anoxic) barnacles spent significantly more time engaged in cirral beating than control barnacles. Such a behavioral preference should enhance oxygen delivery to the internal respiratory surfaces inside the shell. Finally, we found significantly higher hemolymph [K+] in the emersed and anoxic barnacles, which - when coupled to the relatively stable pH values we observed in all treatments - may suggest involvement of K+ ions in an effective acid-base buffering system. In sum, we predict that muscle function would be preserved in B. nubilus during periods of low tide emersion, whereas environmental hypoxia events, which are increasing in frequency and duration as global climates change, have the potential to diminish functionality of their giant muscle fibers.

External pH modulation during the growth of Vibrio tapetis, the aetiological agent of brown ring disease.

Brown ring disease (BRD) is an infection of the Manila clam Ruditapes philippinarum due to the pathogen Vibrio tapetis. During BRD, clams are facing immunodepression and shell biomineralization alteration. In this paper, we studied the role of pH on the growth of the pathogen and formulated hypothesis on the establishment of BRD by V. tapetis. In this study, we monitored the evolution of pH during the growth of V. tapetis in a range of pH and temperatures. We also measured the pH of Manila clam haemolymph and extrapallial fluids (EPFs) during infection by V. tapetis. We highlighted that V. tapetis modulates the external pH during its growth, to a value of 7·70. During the development of BRD, V. tapetis also influences EPFs and haemolymph pH in vitro in the first hours of exposure and in vivo after 3days of infection. Our experiments have shown a close interaction between V. tapetis CECT4600, a pathogen of Manila clam that induces BRD, and the pH of different compartments of the animals during infection. These results indicate that the bacterium, through a direct mechanism or as a consequence of physiological changes encountered in the animal during infection, is able to interfere with the pH of Manila clam fluids. This pH modification might promote the infection process or at least create an imbalance within the animal that would favour its persistence. This last hypothesis should be tested in future experiment. This study is the first observation of pH modifications in the context of BRD and might orient future research on the fine mechanisms of pH modulation associated with BRD.

Hemolymph Changes Resulting from Injection of Escherichia coli Into the Larvae of the Wax Moth, Galleria mellonella (Lepidoptera: Pyralidae)

The present investigation used Galleria mellonella larvae as an infection model to describe the virulence of Escherichia coli, the most frequent causes of several common bacterial infections in humans and animals. Some hemolymph physical properties such as hemolymph volume - and its relation to body water content, hemolymph density, and pH, along with a quantitative estimation of hemolymph proteins, lipids and carbohydrates were recorded in G. mellonella larvae at different time intervals post-injection with a sub-lethal dose (LD20) of E. coli into the larval hemocoel.A decrease in fresh body weight and body water content with an increase in the hemolymph volume was observed at all time intervals post-larval treatment. This may be due to the loss of tissue water and gained it into the hemolymph. At the same time, bacterial injection decreased the hemolymph density and pH immediately following injection, while the viscosity and acidity of the hemolymph restored its original level with time. The bacterial injection also recorded an obvious decrease in the hemolymph proteins and lipids of the treated larvae at all time intervals post-treatment. This may be due to their elimination and/or their involvement in immune defence reactions or may be due to the intensive consumption and depletion of nutrition during infection. On the contrary, the levels of hemolymph carbohydrates increased at all-time intervals post bacterial injection into larvae. This increase may be due to the release of stored sugars (treehouse) which is responded strikingly due to bacterial infection causing an increase in the level of glucose and glycogen in the hemolymph. These results may lead to a better understanding of the regulatory events and the physiology of infected insects.

The effects of elevated temperature and PCO2 on the energetics and haemolymph pH homeostasis of juveniles of the European lobster, Homarus gammarus.

Regulation of extracellular acid-base balance, while maintaining energy metabolism, is recognised as an important aspect when defining an organism's sensitivity to environmental changes. This study investigated the haemolymph buffering capacity and energy metabolism (oxygen consumption, haemolymph [l-lactate] and [protein]) in early benthic juveniles (carapace length <40 mm) of the European lobster, Homarus gammarus, exposed to elevated temperature and PCO2 At 13°C, H. gammarus juveniles were able to fully compensate for acid-base disturbances caused by the exposure to elevated seawater PCO2 at levels associated with ocean acidification and carbon dioxide capture and storage (CCS) leakage scenarios, via haemolymph [HCO3-] regulation. However, metabolic rate remained constant and food consumption decreased under elevated PCO2 , indicating reduced energy availability. Juveniles at 17°C showed no ability to actively compensate haemolymph pH, resulting in decreased haemolymph pH particularly under CCS conditions. Early benthic juvenile lobsters at 17°C were not able to increase energy intake to offset increased energy demand and therefore appear to be unable to respond to acid-base disturbances due to increased PCO2at elevated temperature. Analysis of haemolymph metabolites suggests that, even under control conditions, juveniles were energetically limited. They exhibited high haemolymph [l-lactate], indicating recourse to anaerobic metabolism. Low haemolymph [protein] was linked to minimal non-bicarbonate buffering and reduced oxygen transport capacity. We discuss these results in the context of potential impacts of ongoing ocean change and CCS leakage scenarios on the development of juvenile H. gammarus and future lobster populations and stocks.

The Effects of Ocean Acidification in the California sea hare (Aplysia californica)

Fish and some invertebrates have demonstrated to be strong acid‐base regulators following exposure to ocean acidification‐relevant carbon dioxide (CO2) levels through the accumulation of bicarbonate (HCO3−) in extracellular and intracellular fluids. This allows for pH compensation, however, internal pCO2 and HCO3− remain elevated. This compensation has been hypothesized to cause negative behavioral impairments, by altering ion gradients (HCO3−/Cl−) along neuronal cell membranes that affect the function of the GABAA receptor, an important inhibitory receptor in the central nervous system of vertebrates and invertebrates. Aplysia californica, known for their simple and well‐mapped nervous system, have the potential to be strong model organisms to examine the relationship between CO2 compensation and impaired behavior in invertebrates. We hypothesized that CO2‐exposed animals would compensate for an acidosis by accumulating HCO3−. In the second portion of the study, we hypothesized that two important behaviors, the righting reflex and tail‐withdrawal reflex, would be impaired following CO2 exposure, since this has been noted in many other species. Aplysia were exposed for 4–11 days to either control (400 μatm), 1,200 μatm (close to end of century predictions), or 3,000 μatm CO2. Hemolymph pH was measured using a custom gas‐tight chamber with a fiber‐optic pH microsensor, and HCO3− and pCO2 were calculated using pH and measurements of total CO2 using the Henderson‐Hasselbach equation. The amount of time it took the animal to right following release from the water column (righting reflex), and the amount of time it took the animal to relax its tail to 50% of the original length (tail‐withdrawal reflex) following a mild tail depression were recorded. We observed that animals were able to fully compensate pH at 1,200 μatm CO2, but experienced a significant but mild acidosis at the 3,000 μatm level. Hemolymph HCO3− and pCO2 increased at both CO2 levels, and were statistically significant when compared to controls. Increased CO2 exposure did not significantly affect the righting reflex, but tail‐withdrawal reflex demonstrated a significant 36–37% decrease in relaxation time at both CO2 levels, suggesting increased boldness and altered behavior at projected future CO2 levels. Given these findings, Aplysia are promising models to study CO2‐induced behavioral impairments since they are capable of regulating HCO3− and have a CO2‐impaired behavior linked to simple neural networks amenable to electrophysiological testing. Further research using this model may help illuminate physiological mechanisms underlying CO2‐induced behavioral impairments noted in many marine species that could threaten ecosystems in future oceans.Support or Funding InformationThis research was supported by the National Institute of Health Bridge to Baccalaureate Program (Award R25GM050083).

An integrated investigation of the effects of ocean acidification on adult abalone (Haliotis tuberculata)

AbstractOcean acidification (OA) and its subsequent changes in seawater carbonate chemistry are threatening the survival of calcifying organisms. Due to their use of calcium carbonate to build their shells, marine molluscs are particularly vulnerable. This study investigated the effect of CO2-induced OA on adult European abalone (Haliotis tuberculata) using a multi-parameter approach. Biological (survival, growth), physiological (pHT of haemolymph, phagocytosis, metabolism, gene expression), and structural responses (shell strength, nano-indentation measurements, Scanning electron microscopy imaging of microstructure) were evaluated throughout a 5-month exposure to ambient (8.0) and low (7.7) pH conditions. During the first 2 months, the haemolymph pH was reduced, indicating that abalone do not compensate for the pH decrease of their internal fluid. Overall metabolism and immune status were not affected, suggesting that abalone maintain their vital functions when facing OA. However, after 4 months of exposure, adverse effects on shell growth, calcification, microstructure, and resistance were highlighted, whereas the haemolymph pH was compensated. Significant reduction in shell mechanical properties was revealed at pH 7.7, suggesting that OA altered the biomineral architecture leading to a more fragile shell. It is concluded that under lower pH, abalone metabolism is maintained at a cost to growth and shell integrity. This may impact both abalone ecology and aquaculture.

CO2-driven ocean acidification weakens mussel shell defense capacity and induces global molecular compensatory responses.

Oceanic uptake of atmospheric CO2 is reducing seawater pH and shifting carbonate chemistry within, a process termed as ocean acidification (OA). Marine mussels are a family of ecologically and economically significant bivalves that are widely distributed along coastal areas worldwide. Studies have demonstrated that OA greatly disrupts mussels' physiological functions. However, the underlying molecular responses (e.g., whether there were any molecular compensation mechanisms) and the extent to which OA affects mussel shell defense capacity remain largely unknown. In this study, the thick shell mussels Mytilus coruscus were exposed to the ambient pH (8.1) or one of two lowered pH levels (7.8 and 7.4) for 40 days. The results suggest that future OA will damage shell structure and weaken shell strength and shell closure strength, ultimately reducing mussel shell defense capacity. In addition, future OA will also disrupt haemolymph pH and Ca2+ homeostasis, leading to extracellular acidosis and Ca2+ deficiency. Mantle transcriptome analyses indicate that mussels will adopt a series of molecular compensatory responses to mitigate these adverse effects; nevertheless, weakened shell defense capacity will increase mussels' susceptibility to predators, parasites and pathogens, and thereby reduce their fitness. Overall, the findings of this study have significant ecological and economic implications, and will enhance our understanding of the future of the mussel aquaculture industry and coastal ecosystems.

Physiochemical changes mediated by \u201cCandidatus Liberibacter asiaticus\u201d in Asian citrus psyllids

Plant pathogenic bacteria interact with their insect host(s)/vector(s) at the cellular and molecular levels. This interaction may alter the physiology of their insect vector, which may also promote the growth and transmission of the bacterium. Here we studied the effect of “Candidatus Liberibacter asiaticus” (“Ca. L. asiaticus”) on physiochemical conditions within its insect vector, the Asian citrus psyllid (ACP), and whether these changes were beneficial for the pathogen. The local microenvironments inside ACPs were quantified using microelectrodes. The average hemolymph pH was significantly higher in infected ACPs (8.13 ± 0.21) than in “Ca. L. asiaticus”-free ACPs (7.29 ± 0.15). The average hemolymph oxygen tension was higher in “Ca. L. asiaticus”-free ACPs than in infected ACPs (67.13% ± 2.11% vs. 35.61% ± 1.26%). Oxygen tension reduction and pH increase were accompanied by “Ca. L. asiaticus” infection. Thus, oxygen tension of the hemolymph is an indicator of infection status, with pH affected by the severity of the infection.

Ocean acidification affects acid-base physiology and behaviour in a model invertebrate, the California sea hare (Aplysia californica).

Behavioural impairment following exposure to ocean acidification-relevant CO2 levels has been noted in a broad array of taxa. The underlying cause of these disruptions is thought to stem from alterations of ion gradients across neuronal cell membranes that occur as a consequence of maintaining pH homeostasis via the accumulation of . While behavioural impacts are widely documented, few studies have measured acid–base parameters in species showing behavioural disruptions. In addition, current studies examining mechanisms lack resolution in targeting specific neural pathways corresponding to a given behaviour. With these considerations in mind, acid–base parameters and behaviour were measured in a model organism used for decades as a research model to study learning, the California sea hare (Aplysia californica). Aplysia exposed to elevated CO2 increased haemolymph , achieving full and partial pH compensation at 1200 and 3000 µatm CO2, respectively. Increased CO2 did not affect self-righting behaviour. In contrast, both levels of elevated CO2 reduced the time of the tail-withdrawal reflex, suggesting a reduction in antipredator response. Overall, these results confirm that Aplysia are promising models to examine mechanisms underlying CO2-induced behavioural disruptions since they regulate and have behaviours linked to neural networks amenable to electrophysiological testing.