Transcriptomic responses in the nervous system and correlated behavioural changes of a cephalopod exposed to ocean acidification.

BACKGROUND: The nervous system is central to coordinating behavioural responses to environmental change, likely including ocean acidification (OA). However, a clear understanding of neurobiological responses to OA is lacking, especially for marine invertebrates. RESULTS: We evaluated the transcriptomic response of the central nervous system (CNS) and eyes of the two-toned pygmy squid (Idiosepius pygmaeus) to OA conditions, using a de novo transcriptome assembly created with long read PacBio ISO-sequencing data. We then correlated patterns of gene expression with CO2 treatment levels and OA-affected behaviours in the same individuals. OA induced transcriptomic responses within the nervous system related to various different types of neurotransmission, neuroplasticity, immune function and oxidative stress. These molecular changes may contribute to OA-induced behavioural changes, as suggested by correlations among gene expression profiles, CO2 treatment and OA-affected behaviours. CONCLUSIONS: This study provides the first molecular insights into the neurobiological effects of OA on a cephalopod and correlates molecular changes with whole animal behavioural responses, helping to bridge the gaps in our knowledge between environmental change and animal responses.

Cross-generational response of a tropical sea urchin to global change and a selection event in a 43-month mesocosm study.

Long-term experimental investigations of transgenerational plasticity (TGP) and transgenerational acclimatization to global change are sparse in marine invertebrates. Here, we test the effect of ocean warming and acidification over a 25-month period of Echinometra sp. A sea urchins whose parents were acclimatized at ambient or one of two near-future (projected mid and end of the 21st century) climate scenarios for 18 months. Several parameters linked to performance exhibited strong effects of future ocean conditions at 9 months of age. The Ambient-Ambient group (A-A, both F0 and F1 at ambient conditions) was significantly larger (21%) and faster in righting response (31%) compared to other groups. A second set of contrasts revealed near-future scenarios caused significant negative parental carryover effects. Respiration at 9 months was depressed by 59% when parents were from near-future climate conditions, and righting response was slowed by 28%. At 10 months, a selective pathogenic mortality event led to significantly higher survival rates of A-A urchins. Differences in size and respiration measured prior to the mortality were absent after the event, while a negative parental effect on righting (29% reduction) remained. The capacity to spawn at the end of the experiment was higher in individuals with ambient parents (50%) compared to other groups (21%) suggesting persistent parental effects. Obtaining different results at different points in time illustrates the importance of longer term and multigeneration studies to investigate effects of climate change. Given some animals in all groups survived the pathogenic event and that effects on physiology (but not behavior) among groups were eliminated after the mortality, we suggest that similar events could constitute selective sweeps, allowing genetic adaptation. However, given the observed negative parental effects and reduced potential for population replenishment, it remains to be determined if selection would be sufficiently rapid to rescue this species from climate change effects.

The role of ligand-gated chloride channels in behavioural alterations at elevated CO2 in a cephalopod.

Projected future carbon dioxide (CO2) levels in the ocean can alter marine animal behaviours. Disrupted functioning of γ-aminobutyric acid type A (GABAA) receptors (ligand-gated chloride channels) is suggested to underlie CO2-induced behavioural changes in fish. However, the mechanisms underlying behavioural changes in marine invertebrates are poorly understood. We pharmacologically tested the role of GABA-, glutamate-, acetylcholine- and dopamine-gated chloride channels in CO2-induced behavioural changes in a cephalopod, the two-toned pygmy squid (Idiosepius pygmaeus). We exposed squid to ambient (∼450 µatm) or elevated (∼1000 µatm) CO2 for 7 days. Squid were treated with sham, the GABAA receptor antagonist gabazine or the non-specific GABAA receptor antagonist picrotoxin, before measurement of conspecific-directed behaviours and activity levels upon mirror exposure. Elevated CO2 increased conspecific-directed attraction and aggression, as well as activity levels. For some CO2-affected behaviours, both gabazine and picrotoxin had a different effect at elevated compared with ambient CO2, providing robust support for the GABA hypothesis within cephalopods. In another behavioural trait, picrotoxin but not gabazine had a different effect in elevated compared with ambient CO2, providing the first pharmacological evidence, in fish and marine invertebrates, for altered functioning of ligand-gated chloride channels, other than the GABAAR, underlying CO2-induced behavioural changes. For some other behaviours, both gabazine and picrotoxin had a similar effect in elevated and ambient CO2, suggesting altered function of ligand-gated chloride channels was not responsible for these CO2-induced changes. Multiple mechanisms may be involved, which could explain the variability in the CO2 and drug treatment effects across behaviours.

Effect of elevated CO2 and small boat noise on the kinematics of predator-prey interactions.

Oceans of the future are predicted to be more acidic and noisier, particularly along the productive coastal fringe. This study examined the independent and combined effects of short-term exposure to elevated CO2 and boat noise on the predator-prey interactions of a pair of common coral reef fishes (Pomacentrus wardi and its predator, Pseudochromis fuscus). Successful capture of prey by predators was the same regardless of whether the pairs had been exposed to ambient control conditions, the addition of either playback of boat noise, elevated CO2 (925 µatm) or both stressors simultaneously. The kinematics of the interaction were the same for all stressor combinations and differed from the controls. The effects of CO2 or boat noise were the same, suggesting that their effects were substitutive in this situation. Prey reduced their perception of threat under both stressors individually and when combined, and this coincided with reduced predator attack distances and attack speeds. The lack of an additive or multiplicative effect when both stressors co-occurred was notable given the different mechanisms involved in sensory disruptions and highlights the importance of determining the combined effects of key drivers to aid in predicting community dynamics under future environmental scenarios.

Predatory strategies and behaviours in cephalopods are altered by elevated CO2.

There is increasing evidence that projected near-future carbon dioxide (CO2 ) levels can alter predator avoidance behaviour in marine invertebrates, yet little is known about the possible effects on predatory behaviours. Here we tested the effects of elevated CO2 on the predatory behaviours of two ecologically distinct cephalopod species, the pygmy squid, Idiosepius pygmaeus, and the bigfin reef squid, Sepioteuthis lessoniana. Both species exhibited an increased latency to attack and altered body pattern choice during the attack sequence at elevated CO2 . I. pygmaeus also exhibited a 20% decrease in predation rate, an increased striking distance, and reduced preference for attacking the posterior end of prey at elevated CO2 . Elevated CO2 increased activity levels of S. lessoniana comparable to those previously shown in I. pygmaeus, which could adversely affect their energy budget and increase their potential to be preyed upon. The effects of elevated CO2 on predatory behaviours, predation strategies and activity levels of cephalopods reported here could have far-reaching consequences in marine ecosystems due to the ecological importance of cephalopods in the marine food web.

Ocean warming has a greater effect than acidification on the early life history development and swimming performance of a large circumglobal pelagic fish.

Ocean warming and acidification are serious threats to marine life; however, their individual and combined effects on large pelagic and predatory fishes are poorly understood. We determined the effects of projected future temperature and carbon dioxide (CO2 ) levels on survival, growth, morphological development and swimming performance on the early life stages of a large circumglobal pelagic fish, the yellowtail kingfish Seriola lalandi. Eggs, larvae and juveniles were reared in cross-factored treatments of temperature (21 and 25°C) and pCO2 (500 and 985 µatm) from fertilisation to 25 days post hatching (dph). Temperature had the greatest effect on survival, growth and development. Survivorship was lower, but growth and morphological development were faster at 25°C, with surviving fish larger and more developed at 1, 11 and 21 dph. Elevated pCO2 affected size at 1 dph, but not at 11 or 21 dph, and did not affect survival or morphological development. Elevated temperature and pCO2 had opposing effects on swimming performance at 21 dph. Critical swimming speed (Ucrit ) was increased by elevated temperature but reduced by elevated pCO2 . Additionally, elevated temperature increased the proportion of individuals that responded to a startle stimulus, reduced latency to respond and increased maximum escape speed, potentially due to the more advanced developmental stage of juveniles at 25°C. By contrast, elevated pCO2 reduced the distance moved and average speed in response to a startle stimulus. Our results show that higher temperature is likely to be the primary driver of global change impacts on kingfish early life history; however, elevated pCO2 could affect critical aspects of swimming performance in this pelagic species. Our findings will help parameterise and structure fisheries population dynamics models and improve projections of impacts to large pelagic fishes under climate change scenarios to better inform adaptation and mitigation responses.

Warming has a greater effect than elevated CO2 on predator-prey interactions in coral reef fish.

Ocean acidification and warming, driven by anthropogenic CO2 emissions, are considered to be among the greatest threats facing marine organisms. While each stressor in isolation has been studied extensively, there has been less focus on their combined effects, which could impact key ecological processes. We tested the independent and combined effects of short-term exposure to elevated CO2 and temperature on the predator-prey interactions of a common pair of coral reef fishes (Pomacentrus wardi and its predator, Pseudochromis fuscus). We found that predator success increased following independent exposure to high temperature and elevated CO2 Overall, high temperature had an overwhelming effect on the escape behaviour of the prey compared with the combined exposure to elevated CO2 and high temperature or the independent effect of elevated CO2 Exposure to high temperatures led to an increase in attack and predation rates. By contrast, we observed little influence of elevated CO2 on the behaviour of the predator, suggesting that the attack behaviour of P. fuscus was robust to this environmental change. This is the first study to address how the kinematics and swimming performance at the basis of predator-prey interactions may change in response to concurrent exposure to elevated CO2 and high temperatures and represents an important step to forecasting the responses of interacting species to climate change.

Ocean acidification alters predator behaviour and reduces predation rate.

Ocean acidification poses a range of threats to marine invertebrates; however, the emerging and likely widespread effects of rising carbon dioxide (CO2) levels on marine invertebrate behaviour are still little understood. Here, we show that ocean acidification alters and impairs key ecological behaviours of the predatory cone snail Conus marmoreus Projected near-future seawater CO2 levels (975 µatm) increased activity in this coral reef molluscivore more than threefold (from less than 4 to more than 12 mm min-1) and decreased the time spent buried to less than one-third when compared with the present-day control conditions (390 µatm). Despite increasing activity, elevated CO2 reduced predation rate during predator-prey interactions with control-treated humpbacked conch, Gibberulus gibberulus gibbosus; 60% of control predators successfully captured and consumed their prey, compared with only 10% of elevated CO2 predators. The alteration of key ecological behaviours of predatory invertebrates by near-future ocean acidification could have potentially far-reaching implications for predator-prey interactions and trophic dynamics in marine ecosystems. Combined evidence that the behaviours of both species in this predator-prey relationship are altered by elevated CO2 suggests food web interactions and ecosystem structure will become increasingly difficult to predict as ocean acidification advances over coming decades.

Near-future pH conditions severely impact calcification, metabolism and the nervous system in the pteropod Heliconoides inflatus.

Shelled pteropods play key roles in the global carbon cycle and food webs of various ecosystems. Their thin external shell is sensitive to small changes in pH, and shell dissolution has already been observed in areas where aragonite saturation state is ~1. A decline in pteropod abundance has the potential to disrupt trophic networks and directly impact commercial fisheries. Therefore, it is crucial to understand how pteropods will be affected by global environmental change, particularly ocean acidification. In this study, physiological and molecular approaches were used to investigate the response of the Mediterranean pteropod, Heliconoides inflatus, to pH values projected for 2100 under a moderate emissions trajectory (RCP6.0). Pteropods were subjected to pHT 7.9 for 3 days, and gene expression levels, calcification and respiration rates were measured relative to pHT 8.1 controls. Gross calcification decreased markedly under low pH conditions, while genes potentially involved in calcification were up-regulated, reflecting the inability of pteropods to maintain calcification rates. Gene expression data imply that under low pH conditions, both metabolic processes and protein synthesis may be compromised, while genes involved in acid-base regulation were up-regulated. A large number of genes related to nervous system structure and function were also up-regulated in the low pH treatment, including a GABAA receptor subunit. This observation is particularly interesting because GABAA receptor disturbances, leading to altered behavior, have been documented in several other marine animals after exposure to elevated CO2 . The up-regulation of many genes involved in nervous system function suggests that exposure to low pH could have major effects on pteropod behavior. This study illustrates the power of combining physiological and molecular approaches. It also reveals the importance of behavioral analyses in studies aimed at understanding the impacts of low pH on marine animals.

Ocean acidification boosts larval fish development but reduces the window of opportunity for successful settlement.

Locating appropriate settlement habitat is a crucial step in the life cycle of most benthic marine animals. In marine fish, this step involves the use of multiple senses, including audition, olfaction and vision. To date, most investigations of larval fish audition focus on the hearing thresholds to various frequencies of sounds without testing an ecological response to such sounds. Identifying responses to biologically relevant sounds at the development stage in which orientation is most relevant is fundamental. We tested for the existence of ontogenetic windows of reception to sounds that could act as orientation cues with a focus on vulnerability to alteration by human impacts. Here we show that larvae of a catadromous fish species (barramundi, Lates calcarifer) were attracted towards sounds from settlement habitat during a surprisingly short ontogenetic window of approximately 3 days. Yet, this auditory preference was reversed in larvae reared under end-of-century levels of elevated CO2, such that larvae are repelled from cues of settlement habitat. These future conditions also reduced the swimming speeds and heightened the anxiety levels of barramundi. Unexpectedly, an acceleration of development and onset of metamorphosis caused by elevated CO2 were not accompanied by the earlier onset of attraction towards habitat sounds. This mismatch between ontogenetic development and the timing of orientation behaviour may reduce the ability of larvae to locate habitat or lead to settlement in unsuitable habitats. The misinterpretation of key orientation cues can have implications for population replenishment, which are only exacerbated when ontogenetic development decouples from the specific behaviours required for location of settlement habitats.

Interactive effects of ocean acidification and rising sea temperatures alter predation rate and predator selectivity in reef fish communities.

Ocean warming and acidification are serious threats to marine life. While each stressor alone has been studied in detail, their combined effects on the outcome of ecological interactions are poorly understood. We measured predation rates and predator selectivity of two closely related species of damselfish exposed to a predatory dottyback. We found temperature and CO2 interacted synergistically on overall predation rate, but antagonistically on predator selectivity. Notably, elevated CO2 or temperature alone reversed predator selectivity, but the interaction between the two stressors cancelled selectivity. Routine metabolic rates of the two prey showed strong species differences in tolerance to CO2 and not temperature, but these differences did not correlate with recorded mortality. This highlights the difficulty of linking species-level physiological tolerance to resulting ecological outcomes. This study is the first to document both synergistic and antagonistic effects of elevated CO2 and temperature on a crucial ecological process like predator-prey dynamics.

Will jumping snails prevail? Influence of near-future CO2, temperature and hypoxia on respiratory performance in the tropical conch Gibberulus gibberulus gibbosus.

Tropical coral reef organisms are predicted to be especially sensitive to ocean warming because many already live close to their upper thermal limit, and the expected rise in ocean CO2 is proposed to further reduce thermal tolerance. Little, however, is known about the thermal sensitivity of a diverse and abundant group of reef animals, the gastropods. The humpbacked conch (Gibberulus gibberulus gibbosus), inhabiting subtidal zones of the Great Barrier Reef, was chosen as a model because vigorous jumping, causing increased oxygen uptake (MO2 ), can be induced by exposure to odour from a predatory cone snail (Conus marmoreus). We investigated the effect of present-day ambient (417-454 µatm) and projected-future (955-987 µatm) PCO2 on resting (MO2 , rest) and maximum (MO2 , max) MO2 , as well as MO2 during hypoxia and critical oxygen tension (PO2 , crit), in snails kept at present-day ambient (28°C) or projected-future temperature (33°C). MO2 , rest and MO2 , max were measured both at the acclimation temperature and during an acute 5°C increase. Jumping caused a 4- to 6-fold increase in MO2 , and MO2 , max increased with temperature so that absolute aerobic scope was maintained even at 38°C, although factorial scope was reduced. The humpbacked conch has a high hypoxia tolerance with a PO2 , crit of 2.5 kPa at 28°C and 3.5 kPa at 33°C. There was no effect of elevated CO2 on respiratory performance at any temperature. Long-term temperature records and our field measurements suggest that habitat temperature rarely exceeds 32.6°C during the summer, indicating that these snails have aerobic capacity in excess of current and future needs.

Marine mollusc predator-escape behaviour altered by near-future carbon dioxide levels.

Ocean acidification poses a range of threats to marine invertebrates; however, the potential effects of rising carbon dioxide (CO2) on marine invertebrate behaviour are largely unknown. Marine gastropod conch snails have a modified foot and operculum allowing them to leap backwards rapidly when faced with a predator, such as a venomous cone shell. Here, we show that projected near-future seawater CO2 levels (961 µatm) impair this escape behaviour during a predator-prey interaction. Elevated-CO2 halved the number of snails that jumped from the predator, increased their latency to jump and altered their escape trajectory. Physical ability to jump was not affected by elevated-CO2 indicating instead that decision-making was impaired. Antipredator behaviour was fully restored by treatment with gabazine, a GABA antagonist of some invertebrate nervous systems, indicating potential interference of neurotransmitter receptor function by elevated-CO2, as previously observed in marine fishes. Altered behaviour of marine invertebrates at projected future CO2 levels could have potentially far-reaching implications for marine ecosystems.

Impaired learning of predators and lower prey survival under elevated CO2 : a consequence of neurotransmitter interference.

Ocean acidification is one of the most pressing environmental concerns of our time, and not surprisingly, we have seen a recent explosion of research into the physiological impacts and ecological consequences of changes in ocean chemistry. We are gaining considerable insights from this work, but further advances require greater integration across disciplines. Here, we showed that projected near-future CO2 levels impaired the ability of damselfish to learn the identity of predators. These effects stem from impaired neurotransmitter function; impaired learning under elevated CO2 was reversed when fish were treated with gabazine, an antagonist of the GABA-A receptor - a major inhibitory neurotransmitter receptor in the brain of vertebrates. The effects of CO2 on learning and the link to neurotransmitter interference were manifested as major differences in survival for fish released into the wild. Lower survival under elevated CO2 , as a result of impaired learning, could have a major influence on population recruitment.

Ocean acidification slows retinal function in a damselfish through interference with GABAA receptors.

Vision is one of the most efficient senses used by animals to catch prey and avoid predators. Therefore, any deficiency in the visual system could have important consequences for individual performance. We examined the effect of CO2 levels projected to occur by the end of this century on retinal responses in a damselfish, by determining the threshold of its flicker electroretinogram (fERG). The maximal flicker frequency of the retina was reduced by continuous exposure to elevated CO2, potentially impairing the capacity of fish to react to fast events. This effect was rapidly counteracted by treatment with a GABA antagonist (gabazine), indicating that GABAA receptor function is disrupted by elevated CO2. In addition to demonstrating the effects of elevated CO2 on fast flicker fusion of marine fishes, our results show that the fish retina could be a model system to study the effects of high CO2 on neural processing.

Low global sensitivity of metabolic rate to temperature in calcified marine invertebrates.

Metabolic rate is a key component of energy budgets that scales with body size and varies with large-scale environmental geographical patterns. Here we conduct an analysis of standard metabolic rates (SMR) of marine ectotherms across a 70° latitudinal gradient in both hemispheres that spanned collection temperatures of 0-30 °C. To account for latitudinal differences in the size and skeletal composition between species, SMR was mass normalized to that of a standard-sized (223 mg) ash-free dry mass individual. SMR was measured for 17 species of calcified invertebrates (bivalves, gastropods, urchins and brachiopods), using a single consistent methodology, including 11 species whose SMR was described for the first time. SMR of 15 out of 17 species had a mass-scaling exponent between 2/3 and 1, with no greater support for a 3/4 rather than a 2/3 scaling exponent. After accounting for taxonomy and variability in parameter estimates among species using variance-weighted linear mixed effects modelling, temperature sensitivity of SMR had an activation energy (Ea) of 0.16 for both Northern and Southern Hemisphere species which was lower than predicted under the metabolic theory of ecology (Ea 0.2-1.2 eV). Northern Hemisphere species, however, had a higher SMR at each habitat temperature, but a lower mass-scaling exponent relative to SMR. Evolutionary trade-offs that may be driving differences in metabolic rate (such as metabolic cold adaptation of Northern Hemisphere species) will have important impacts on species abilities to respond to changing environments.

Increased CO2 stimulates reproduction in a coral reef fish.

Ocean acidification is predicted to negatively impact the reproduction of many marine species, either by reducing fertilization success or diverting energy from reproductive effort. While recent studies have demonstrated how ocean acidification will affect larval and juvenile fishes, little is known about how increasing partial pressure of carbon dioxide (pCO(2)) and decreasing pH might affect reproduction in adult fishes. We investigated the effects of near-future levels of pCO(2) on the reproductive performance of the cinnamon anemonefish, Amphiprion melanopus, from the Great Barrier Reef, Australia. Breeding pairs were held under three CO(2) treatments [Current-day Control (430 µatm), Moderate (584 µatm) and High (1032 µatm)] for a 9-month period that included the summer breeding season. Unexpectedly, increased CO(2) dramatically stimulated breeding activity in this species of fish. Over twice as many pairs bred in the Moderate (67% of pairs) and High (55%) compared to the Control (27%) CO(2) treatment. Pairs in the High CO(2) group produced double the number of clutches per pair and 67% more eggs per clutch compared to the Moderate and Control groups. As a result, reproductive output in the High group was 82% higher than that in the Control group and 50% higher than that in the Moderate group. Despite the increase in reproductive activity, there was no difference in adult body condition among the three treatment groups. There was no significant difference in hatchling length between the treatment groups, but larvae from the High CO(2) group had smaller yolks than Controls. This study provides the first evidence of the potential effects of ocean acidification on key reproductive attributes of marine fishes and, contrary to expectations, demonstrates an initially stimulatory (hormetic) effect in response to increased pCO(2). However, any long-term consequences of increased reproductive effort on individuals or populations remain to be determined.

Combined effects of climate change stressors and predators with contrasting feeding-digestion strategies on a mussel species.

We investigated the combined effects of Ocean Warming (OW), Acidification (OA) and predator cues (Non-Consumptive Effects; NCEs) of two predators with contrasting feeding-digestion strategies on the mussel Perumytilus purpuratus. We considered starfish-NCEs (partially external digestion) and snail-NCEs (internal digestion). Mussels were exposed for 13 weeks to cross-factored OA (~500 and ~1400 µatm, pCO2) and OW (~15 and ~20 °C) conditions, in the presence/absence of NCEs from one or both predators. Mussels exposed to both NCEs exhibited smaller length and buoyant weight growth than those under control or snail-NCEs conditions. Mussels exposed to starfish-NCEs exhibited smaller wet mass than control mussels. OW and starfish-NCEs in isolation or combined with snail-NCEs increased mussel oxygen consumption. Byssal biogenesis was affected by the three-factors interaction. Clearance rates were affected by the OW × OA interaction. We suggest that mainly starfish-NCEs, in isolation or interacting with OA or/and OW, can threat mussel traits and the associated community.

Marine invertebrate skeleton size varies with latitude, temperature and carbonate saturation: implications for global change and ocean acidification.

There is great concern over the future effects of ocean acidification on marine organisms, especially for skeletal calcification, yet little is known of natural variation in skeleton size and composition across the globe, and this is a prerequisite for identifying factors currently controlling skeleton mass and thickness. Here, taxonomically controlled latitudinal variations in shell morphology and composition were investigated in bivalve and gastropod molluscs, brachiopods, and echinoids. Total inorganic content, a proxy for skeletal CaCO3 , decreased with latitude, decreasing seawater temperature, and decreasing seawater carbonate saturation state (for CaCO3 as calcite (Ωcal )) in all taxa. Shell mass decreased with latitude in molluscs and shell inorganic content decreased with latitude in buccinid gastropods. Shell thickness decreased with latitude in buccinid gastropods (excepting the Australian temperate buccinid) and echinoids, but not brachiopods and laternulid clams. In the latter, the polar species had the thickest shell. There was no latitudinal trend in shell thickness within brachiopods. The variation in trends in shell thickness by taxon suggests that in some circumstances ecological factors may override latitudinal trends. Latitudinal gradients may produce effects similar to those of future CO2 -driven ocean acidification on CaCO3 saturation state. Responses to latitudinal trends in temperature and saturation state may therefore be useful in informing predictions of organism responses to ocean acidification over long-term adaptive timescales.