The Central Paratethys Sea-rise and demise of a Miocene European marine biodiversity hotspot.

The Miocene Climate Optimum (MCO, ~ 17-14 Ma) was a time of extraordinary marine biodiversity in the Circum-Mediterranean Region. This boom is best recorded in the deposits of the vanished Central Paratethys Sea, which covered large parts of central to southeastern Europe. This sea harbored an extraordinary tropical to subtropical biotic diversity. Here, we present a georeferenced dataset of 859 gastropod species and discuss geodynamics and climate as the main drivers to explain the changes in diversity. The tectonic reorganization around the Early/Middle Miocene boundary resulted in the formation of an archipelago-like landscape and favorable conditions of the MCO allowed the establishment of coral reefs. Both factors increased habitat heterogeneity, which boosted species richness. The subsequent cooling during the Middle Miocene Climate Transition (~ 14-13 Ma) caused a drastic decline in biodiversity of about 67%. Among the most severely hit groups were corallivorous gastropods, reflecting the loss of coral reefs. Deep-water faunas experienced a loss by 57% of the species due to changing patterns in circulation. The low sea level led to a biogeographic fragmentation reflected in higher turnover rates. The largest turnover occurred with the onset of the Sarmatian when bottom water dysoxia eradicated the deep-water fauna whilst surface waters-dwelling planktotrophic species underwent a crisis.

Chemical defences indicate bold colour patterns with reduced variability in aposematic nudibranchs.

The selective factors that shape phenotypic diversity in prey communities with aposematic animals are diverse and coincide with similar diversity in the strength of underlying secondary defences. However, quantitative assessments of colour pattern variation and the strength of chemical defences in assemblages of aposematic species are lacking. We quantified colour pattern diversity using quantitative colour pattern analysis (QCPA) in 13 dorid nudibranch species (Infraorder: Doridoidei) that varied in the strength of their chemical defences. We accounted for the physiological properties of a potential predator's visual system (a triggerfish, Rhinecanthus aculeatus) and modelled the appearance of nudibranchs from multiple viewing distances (2 and 10 cm). We identified distinct colour pattern properties associated with the presence and strength of chemical defences. Specifically, increases in chemical defences indicated increases in colour pattern boldness (i.e. visual contrast elicited via either or potentially coinciding chromatic, achromatic and/or spatial contrast). Colour patterns were also less variable among species with chemical defences when compared to undefended species. Our results indicate correlations between secondary defences and diverse, bold colouration while showing that chemical defences coincide with decreased colour pattern variability among species. Our study suggests that complex spatiochromatic properties of colour patterns perceived by potential predators can be used to make inferences on the presence and strength of chemical defences.

The impact of aragonite saturation variability on shelled pteropods: An attribution study in the California Current System.

Observations from the California Current System (CalCS) indicate that the long-term trend in ocean acidification (OA) and the naturally occurring corrosive conditions for the CaCO3 mineral aragonite (saturation state Ω < 1) have a damaging effect on shelled pteropods, a keystone group of calcifying organisms in the CalCS. Concern is heightened by recent findings suggesting that shell formation and developmental progress are already impacted when Ω falls below 1.5. Here, we quantify the impact of low Ω conditions on pteropods using an individual-based model (IBM) with life-stage-specific mortality, growth, and behavior in a high-resolution regional hindcast simulation of the CalCS between 1984 and 2019. Special attention is paid to attributing this impact to different processes that lead to such low Ω conditions, namely natural variability, long-term trend, and extreme events. We find that much of the observed damage in the CalCS, and specifically >70% of the shell CaCO3 loss, is due to the pteropods' exposure to naturally occurring low Ω conditions as a result of their diel vertical migration (DVM). Over the hindcast period, their exposure to damaging waters (Ω < 1.5) increases from 9% to 49%, doubling their shell CaCO3 loss, and increasing their mortality by ~40%. Most of this increased exposure is due to the shoaling of low Ω waters driven by the long-term trend in OA. Extreme OA events amplify this increase by ~40%. Our approach can quantify the health of pteropod populations under shifting environmental conditions, and attribute changes in fitness or population structure to changes in the stressor landscape across hierarchical time scales.

Compensatory shell thickening in corrosive environments varies between related rocky-shore and estuarine gastropods.

Few studies have considered the capabilities of gastropods living in minerally-deficient acidified coastal waters to compensate for outer shell corrosion or compromised growing edge shell production. We compared inner shell thickening between pristine shells (control) and corroded shells (experiment) of two related intertidal neritid gastropod species from reduced salinity and acidified environments. We predicted that the rocky-shore, Nerita chamaeleon, which has greater access to shell building biomineralization substrates, should better control shell thickness than the estuarine, Neripteron violaceum. Accordingly, N. chameleon was found to compensate perfectly for variation in the thickness of the outer calcitic blocky layer (BL). Optimal shell thickness (OST) was maintained by selective reabsorption of the aperture ridge of the distal shell (aragonitic crossed-lamellar layer, CL) and by increased internal deposition of proximal (older) shell (aragonitic protocrossed lamellar, PCL). Despite greater exposure to acidification and hyposalinity, N. violaceum showed no significant compensatory shell thickening. These findings reveal that shell thickening capability may vary greatly among intertidal gastropods and that this may be constrained by environmental biomineralization substrate availability. Such environmentally-related responses carry implications for predicted future reductions in coastal water pH and salinity.

Assessing phenotypic variation and plasticity of endemic gastropods from thermal water refugia using complex morphometric techniques: A case study of Lake Pețea melanopsids.

Understanding the underlying reasons for phenotypic plasticity and resulting morphological disparity is one of the key topics of evolutionary research. The phenotypic plasticity of extant and fossil melanopsids has been widely documented. Yet millennial-resolution, well-dated records from small aquatic habitats harboring endemics are scarce. The thermal spring-fed Lake Pețea is an ice age refugia harboring a unique endemic warm-water fauna. Subfossil melanopsids display incredible morphological variability from smooth to keeled, elongated to ribbed, shouldered forms. Numerous morphotypes have been considered as individual taxa with a fluent succession from the smooth elongated to the ribbed, shouldered types. This study presents an extensive morphometric analysis of subfossil melanopsids (ca. 3500 specimens) derived from stratified samples with an independent chronology. The aim was to separate morphotypes for investigations of temporal morphological disparity. Our results challenge the widely accepted hypothesis that proposes the evolution of shouldered, compressed, ribbed shells through a two-step process from smooth elongated spindle-shaped shells. Instead, it suggests that the subfossil shells belong to two distinct taxa present throughout the available stratigraphic data. The main components of shape variation, shape globularity, and shell coiling seem allometry-related. Ribs, striation, and keels appear randomly. High-spired spindle-shaped forms were considered to represent specimens of Microcolpia daudebartii hazayi. Bulkier low-spired and shouldered specimens represent phenotypes of Mi. parreyssii parreyssii. The collective and random distribution of morphotypes from the early stages of the lake's history also refutes the idea of a continuous transformation of the elongated forms into compressed, shouldered ones. Rather points to multiple events and environmental stimuli triggering development. Melanopsids appear in Late Glacial horizons, with Theodoxus prevostianus preferring temperatures above 23°C which may indicate the subordinate presence of hot water microhabitats in cooler waters.

Characterization of myoinhibitory peptide signaling system and its implication in larval metamorphosis and spawning behavior in Pacific abalone.

Myoinhibitory peptides (MIPs) affect various physiological functions, including juvenile hormone signaling, muscle contraction, larval development, and reproduction in invertebrates. Although MIPs are ligands for MIP and/or sex peptide receptors (MIP/SPRs) in diverse arthropods and model organisms belonging to Lophotrochozoa, the MIP signaling system has not yet been fully investigated in mollusks. In this study, we identified the MIP signaling system in the Pacific abalone Haliotis discus hannai (Hdh). Similar to the invertebrate MIPs, a total of eight paracopies of MIPs (named Hdh-MIP1 to Hdh-MIP8), harboring a WX5-7Wamide motif, except for Hdh-MIP2, were found in the Hdh-MIP precursor. Furthermore, we characterized a functional Hdh-MIPR, which responded to the Hdh-MIPs, except for Hdh-MIP2, possibly linked with the PKC/Ca2+ and PKA/cAMP signaling pathways. Hdh-MIPs delayed larval metamorphosis but increased the spawning behavior. These results suggest that the Hdh-MIP signaling system provides insights into the unique function of MIP in invertebrates.

Critical thermal maxima and oxygen uptake in Elysia viridis, a sea slug that steals chloroplasts to photosynthesize.

Photosynthetic animals produce oxygen, providing an ideal lens for studying how oxygen dynamics influence thermal sensitivity. The algivorous sea slug Elysia viridis can steal and retain chloroplasts from the marine alga Bryopsis sp. for months when starved, but chloroplast retention is mere weeks when they are fed another green alga, Chaetomorpha sp. To examine plasticity in thermal tolerance and changes in net oxygen exchange when fed and starving, slugs fed each alga were acclimated to 17°C (the current maximum temperature to which they are exposed in nature) and 22°C (the increase predicted for 2100) and measured at different points during starvation. We also examined increased illumination to evaluate a potential tradeoff between increased oxygen production but faster chloroplast degradation. Following acclimation, we subjected slugs to acute thermal stress to determine their thermal tolerance. We also measured net oxygen exchange before and after acute thermal stress. Thermal tolerance improved in slugs acclimated to 22°C, indicating they can acclimate to temperatures higher than they naturally experience. All slugs exhibited net oxygen uptake, and rates were highest in recently fed slugs before exposure to acute thermal stress. Oxygen uptake was suppressed following acute thermal stress. Under brighter light, slugs exhibited improved thermal tolerance, possibly because photosynthetic oxygen production alleviated oxygen limitation. Accordingly, this advantage disappeared later in starvation when photosynthesis ceased. Thus, E. viridis can cope with heatwaves by suppressing metabolism and plastically adjusting heat tolerance; however, starvation influences a slug's thermal tolerance and oxygen uptake such that continuous access to algal food for its potential nutritive and oxygenic benefits is critical when facing thermal stress.

Preliminary analysis of pathways and their implications during salinity stress in abalone.

Transcriptome sequencing has offered immense opportunities to study non-model organisms. Abalone is an important marine mollusk that encounters harsh environmental conditions in its natural habitat and under aquaculture conditions; hence, research that increases molecular information to understand abalone physiology and stress response is noteworthy. Accordingly, the study used transcriptome sequencing of the gill tissues of abalone exposed to low salinity stress. The aim is to explore some enriched pathways during salinity stress and the crosstalk and functions of the genes involved in the candidate biological processes for future further analysis of their expression patterns. The data suggest that abalone genes such as YAP/TAZ, Myc, Nkd, and Axin (involved in the Hippo signaling pathway) and PI3K/Akt, SHC, and RTK (involved in the Ras signaling pathways) might mediate growth and development. Thus, deregulation of the Hippo and Ras pathways by salinity stress could be a possible mechanism by which unfavorable salinities influence growth in abalone. Furthermore, PEPCK, GYS, and PLC genes (mediating the Glucagon signaling pathway) might be necessary for glucose homeostasis, reproduction, and abalone meat sensory qualities; hence, a need to investigate how they might be influenced by environmental stress. Genes such as MYD88, IRAK1/4, JNK, AP-1, and TRAF6 (mediating the MAPK signaling pathway) could be useful in understanding abalone's innate immune response to environmental stresses. Finally, the aminoacyl-tRNA biosynthesis pathway hints at the mechanism by which new raw materials for protein biosynthesis are mobilized for physiological processes and how abalone might respond to this process during salinity stress. Low salinity clearly regulated genes in these pathways in a time-dependent manner, as hinted by the heat maps. In the future, qRT-PCR verification and in-depth study of the various genes and proteins discussed would provide enormous molecular information resources for the abalone biology.

Within- and transgenerational stress legacy effects of ocean acidification on red abalone (Haliotis rufescens) growth and survival.

Understanding the mechanisms by which individual organisms respond and populations adapt to global climate change is a critical challenge. The role of plasticity and acclimation, within and across generations, may be essential given the pace of change. We investigated plasticity across generations and life stages in response to ocean acidification (OA), which poses a growing threat to both wild populations and the sustainable aquaculture of shellfish. Most studies of OA on shellfish focus on acute effects, and less is known regarding the longer term carryover effects that may manifest within or across generations. We assessed these longer term effects in red abalone (Haliotis rufescens) using a multi-generational split-brood experiment. We spawned adults raised in ambient conditions to create offspring that we then exposed to high pCO2 (1180 µatm; simulating OA) or low pCO2 (450 µatm; control or ambient conditions) during the first 3 months of life. We then allowed these animals to reach maturity in ambient common garden conditions for 4 years before returning the adults into high or low pCO2 treatments for 11 months and measuring growth and reproductive potential. Early-life exposure to OA in the F1 generation decreased adult growth rate even after 5 years especially when abalone were re-exposed to OA as adults. Adult but not early-life exposure to OA negatively impacted fecundity. We then exposed the F2 offspring to high or low pCO2 treatments for the first 3 months of life in a fully factorial, split-brood design. We found negative transgenerational effects of parental OA exposure on survival and growth of F2 offspring, in addition to significant direct effects of OA on F2 survival. These results show that the negative impacts of OA can last within and across generations, but that buffering against OA conditions at critical life-history windows can mitigate these effects.

The effect of total alkalinity on growth performance and calcification in juvenile Pacific abalone Haliotis discus hannai.

A 45-day trial was conducted to study the effect of seawater total alkalinity (TA) level up- and downregulation on the growth performance and calcification of Haliotis discus hannai Ino, while seawater pH was maintained at pHNBS = 8.1. Although seawater was not acidified, the results showed that TA downregulation caused a significant reduction (P < 0.05) in the somatic tissue growth of juvenile abalone, while TA upregulation significantly increased growth performance (P < 0.05). Similar to the impacts of pH reduction, TA downregulation also induces a decline in CO2 buffering capacity, which may be the reason why somatic tissue growth was reduced, as lowered CO2 buffering capacity was reported to shift the acid-base balancing of abalone. Parts of the periostracum layer weremissing and exposed the inner shell layers of the individuals from the TA-downregulated group. Scanning electron microscopy (SEM) results showed calcium carbonate densely deposited onto the inner shell in the control and TA-upregulated groups, while sparsely deposited calcium carbonate was observed in the TA-downregulated group. The C: N ratio in the shell of individuals from the TA-downregulated group was significantly lower than that of the other two groups, indicating that less inorganic carbon was added to the shell. As a result, abalone grew lighter and thinner shells in TA-downregulated seawater. Although seawater was not acidified, TA downregulation also caused a reduction in the calcium carbonate saturation state (Ω), which induced the erosion of the surface shell and the interruption of calcium carbonate generation. In conclusion, although seawater pH remained at ambient levels, the lowered CO2 buffering capacity and Ω induced by seawater TA downregulation also showed a detrimental effect on the growth and calcification of Pacific abalone. The impact of ocean acidification on the growth of abalone should not be assessed using only seawater pH and/or pCO2 but rather taking into account all of carbonate chemistry, particularly the CO2 buffering capacity. Abalone cultivation is suggested to be carried out in seawater with a higher level of CO2 buffering capacity and Ω, which can be achieved through integrated culture with seaweed or increasing the seawater TA level.

Effects of heating rate and shell colour on the cardiac thermal performance in a polymorphic gastropod Batillaria attramentaria.

Heating rate has gained extensive attention in mechanistic understanding of physiological responses to changing thermal conditions in the context of climate change. In polymorphic gastropods, differences in the absorption of solar energy between dark- and light-coloured individuals lead to supposable differences in their heating rates and body temperatures in sunshine. In the present study, we examined the effect of heating rate on heart rate (HR) in a polymorphic gastropod Batillaria attramentaria. By using biomimetic models, we found that daily maximum temperature of snails with a dark unbanded shell (D-type morph) was higher than snails with a white line on the upper side of each whorl (UL-type morph) by 0.6 °C when exposed to sunlight, but there was no apparent difference in heating rates between D- and UL-type models. We measured HR of snails at various heating rates from 3.0 to 9.0 °C h-1. Faster heating rates significantly increased maximum thermal tolerance in both D- and UL-type snails, highlighting the importance to have thorough knowledge on the heating rate in the field to obtain accurate maximum thermal limit of gastropods. Critical temperature at which HR precipitously declines was higher in D-type snails than UL-type snails. Our results suggested that the impacts of heating rate as well as the shell colour should be considered to gain a mechanistic understanding of the population dynamics of polymorphic gastropods.

Seawater carbonate parameters function differently in affecting embryonic development and calcification in Pacific abalone (Haliotis discus hannai).

pH or pCO2 are usually taken to study the impact of ocean acidification on molluscs. Here we studied the different impact of seawater carbonate parameters on embryonic development and calcification of the Pacific abalone (Haliotis discus hannai). Early embryonic development was susceptible to elevated pCO2 level. Larvae hatching duration was positively and hatching rate was negatively correlated with the pCO2 level, respectively. Calcium carbonate (CaCO3) deposition of larval shell was found to be susceptible to calcium carbonate saturation state (Ω) rather than pCO2 or pH. Most larvae incubated in seawater with Ωarag = 1.5 succeeded in shell formation, even when seawater pCO2 level was higher than 3700 µatm and pHT was close to 7.4. Nevertheless, larvae failed to generate CaCO3 in seawater with Ωarag ≤ 0.52 and control level of pCO2, while seawater DIC level was lowered (≤ 852 µmol/kg). Surprisingly, some larvae completed CaCO3 deposition in seawater with Ωarag = 0.6 and slightly elevated DIC (2266 µmol/kg), while seawater pCO2 level was higher than 2700 µatm and pHT was lower than 7.3. This indicates that abalone may be capable of regulating carbonate chemistry to support shell formation, however, the capability was limited as surging pCO2 level lowered growth rate and jeopardized the integrity of larval shells. Larvae generated thicker shell in seawater with Ωarag = 5.6, while adult abalone could not deposit CaCO3 in seawater with Ωarag = 0.29 and DIC = 321 µmol/kg. This indicates that abalone may lack the ability to directly remove or add inorganic carbon at the calcifying sites. In conclusion, different seawater carbonate parameters play different roles in affecting early embryonic development and shell formation of the Pacific abalone, which may exhibit limited capacity to regulate carbonate chemistry.

Metabolic and transcriptional responses demonstrating enhanced thermal tolerance in domesticated abalone.

Global warming threatens aquatic systems and organisms. Many studies have focused on the vulnerability and stress responses of aquaculture organisms to future thermal conditions. However, it may be of more practical significance to reveal their acclimation potential and mechanisms. In this study, the physiological, metabolic, and transcriptional responses to long-term temperature acclimation of northern and southern populations of Pacific abalone Haliotis discus hannai, a commercially important gastropod sensitive to environmental changes, were compared. This study conducted two common-garden experiments, including a thermostatic experiment in the lab and an aquaculture experiment on the farm. The abalone population cultured in warmer southern waters was tolerant of ongoing high temperatures, whereas the abalone population originally cultured in cooler northern waters exhibited vulnerability to high temperatures but could enhance its thermal tolerance through the process of natural selection in warmer southern waters. This difference was linked to divergence in the metabolic and transcriptional processes of the two populations. The tolerant population exhibited a greater capacity for carbohydrate and amino acid metabolism regulation and energy redistribution to cope with heat stress. This capacity may have been selected for, and accumulated, over many generations because the tolerant population originated from the intolerant population over two decades ago. This work provides insight into the vulnerability and acclimation potential of abalone to heat stress and discloses the molecular and metabolic traits underlying this phenomenon. Future research on the ability of abalone and other commercial shellfish species to acclimate to global warming should take this potential into account.

Differential effects of ocean acidification and warming on biological functioning of a predator and prey species may alter future trophic interactions.

Independently, ocean warming (OW) and acidification (OA) from increased anthropogenic atmospheric carbon dioxide are argued to be two of the greatest threats to marine organisms. Increasingly, their interaction (ocean acidification and warming, OAW) is shown to have wide-ranging consequences to biological functioning, population and community structure, species interactions and ecosystem service provision. Here, using a multi-trophic experiment, we tested the effects of future OAW scenarios on two widespread intertidal species, the blue mussel Mytilus edulis and its predator Nucella lapillus. Results indicate negative consequences of OAW on the growth, feeding and metabolic rate of M. edulis and heightened predation risk. In contrast, Nucella growth and metabolism was unaffected and feeding increased under OAW but declined under OW suggesting OA may offset warming consequences. Should this differential response between the two species to OAW, and specifically greater physiological costs to the prey than its predator come to fruition in the nature, fundamental change in ecosystem structure and functioning could be expected as trophic interactions become disrupted.

Playing it safe; risk-induced trait responses increase survival in the face of predation.

Predation risk effects are impacts on prey caused by predators that do not include consumption. These can include changes in prey behaviour, physiology, and morphology (i.e. risk-induced trait responses), which can have consequences to individual fitness and population dynamics (i.e. non-consumptive effects). While these risk-induced trait responses (RITRs) can lower individual fitness as compared to prey not exposed to risk, they are assumed to increase fitness in the presence of predators. While much work has been built upon this assumption, most evidence occurs in consumptive experiments where the trait values of consumed prey are unknown. We have little evidence showing individuals with a greater magnitude of RITR have greater survival. Here, we tested the hypothesis that RITRs increase survival in the presence of predators, but come at a cost to growth. We tested this hypothesis using Nucella lapillus as prey and Carcinus maenas as a predator and including mussels as a basal resource in a two-phase mesocosm experimental set-up. In phase 1, Nucella were placed into either a control or risk treatment (exposure to non-lethal Carcinus) for 28 days and their behaviour and growth measured. In phase 2, a lethal Carcinus was added to all mesocosms (non-lethal crabs were removed), and survival was recorded for 15 days. At the treatment (group) level, we found that Nucella exposed to predation risk in phase 1 had significantly greater risk aversion behaviour (summed score of risky vs. safe behaviour) and significantly lower growth. In phase 2, we found that Nucella exposed to predation risk had greater survival. At the individual level (regardless of treatment), we found that Nucella with greater risk aversion scores in phase 1 had significantly higher survival in phase 2 when exposed to a lethal predator, but this came at a cost to their growth. This study provides some of the first empirical evidence, at both the group and individual level, testing a long-held assumption that predation risk-induced behavioural responses increase survival in the face of direct predation, but that these responses come at a cost to the prey. These results add to our growing understanding of the benefits of RITRs to individual fitness and non-consumptive effects generally.

The paralytic shellfish toxin effect on bioenergetic constituents of the fishery resource Chorus giganteus (Gastropoda: Muricidae).

Alexandrium catenella, one of the most common harmful microalgae observed in southern Chile, produces paralytic shellfish toxins, which can affect many organisms throughout the trophic chain. This research evaluated how paralytic shellfish toxins affected the principal bioenergetic constituents and fatty acids composition of the carnivorous snail Chorus giganteus. Snails were separated into a "toxic" group that was fed the toxic clam Mulinia edulis (which was previously fed A. catenella), and a "non-toxic" group, fed non-toxic clams. Both groups were kept under these conditions for 63 days. Our results indicated no difference in the ingestion rate of toxic versus non-toxic snails; however, a higher protein level was identified in toxic snails. The total lipid content proved to be no different in toxic versus non-toxic snails; although, an effect of the toxic diet on the fatty acid profile of C. giganteus was observed. High levels of essential polyunsaturated fatty acids, especially docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) in toxic snails, were identified. Our results suggest that exposure to paralytic shellfish toxins, through diet, may cause changes in the biochemical composition of C. giganteus, which may have a subsequent impact on its energetic physiology.

Responses of early life stages of European abalone (Haliotis tuberculata) to ocean acidification after parental conditioning: Insights from a transgenerational experiment.

CO2 absorption is leading to ocean acidification (OA), which is a matter of major concern for marine calcifying species. This study investigated the effects of simulated OA on the reproduction of European abalone Haliotis tuberculata and the survival of its offspring. Four-year-old abalone were exposed during reproductive season to two relevant OA scenarios, ambient pH (8.0) and low pH (7.7). After five months of exposure, abalone were induced to spawn. The gametes, larvae and juveniles were then exposed for five months to the same pH conditions as their parents. Several biological parameters involved in adult reproduction as well as in larval, post-larval and juvenile fitness were measured. No effects on gametes, fertilisation or larval oxidative stress response were detected. However, developmental abnormalities and significant decreases in shell length and calcification were observed at veliger stages. The expression profile of a GABA A receptor-like gene appeared to be regulated by pH, depending on larval stage. Larval and post-larval survival was not affected by low pH. However, a lower survival and a reduction of growth were recorded in juveniles at pH 7.7. Our results confirm that OA negatively impacts larval and juvenile fitness and suggest the absence of carry-over effects on abalone offspring. This may compromise the survival of abalone populations in the near future.

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.

Functional analysis of LFRFamide signaling in Pacific abalone, Haliotis discus hannai.

The invertebrate LFRFamide (LFRFa) and short neuropeptide F (sNPF), consisting of 6 to 10 amino acids, are orthologs for bilaterian NPF/Y, which consist of 36 to 40 amino acids. Recently, a molluscan G protein-coupled receptor (GPCR) for NPF was characterized in Pacific abalone (Haliotis discus hannai). To address the functional evolutionary route of the invertebrate LFRFa and NPF signaling system, in this study, we identified cDNAs encoding LFRFa precursors and the sNPF receptor (Hdh-sNPFR) in Pacific abalone. Four LFRFa mature peptides with 6 or 7 amino acids were predicted: GSLFRFa, GGLFRFa, GTLFRFa, and GSTLFRFa. Hdh-sNPFR was identified as a classical rhodopsin-like GPCR and classified into a molluscan sNPFR group. In HEK293 cells, Hdh-sNPFR was mainly localized in the cell membranes and internalized in the cytoplasm following treatment with LFRFa peptides. Reporter assays demonstrated that LFRFa peptides inhibit forskolin-stimulated cAMP accumulation in Hdh-sNPFR-expressing HEK293 cells. LFRFa precursor and Hdh-sNPFR transcripts were more strongly expressed in the cerebral and pleural-pedal ganglia of Pacific abalone than in the peripheral tissues such as the ovary, gills, intestine, and hepatopancreas. The levels of LFRFa transcripts in the ovary, intestine, and hepatopancreas were significantly higher in mature female abalone than in immature females. Injection of LFRFa induced the egg release and spawning behavior of mature abalone, but suppressed food intake. These results suggest that LFRFa peptides are endogenous ligands for Hdh-sNPFR involved in food intake and reproduction through a Gαi-protein dependent signaling pathway.

Mollusc shell shape as pollution biomarkers: Which is the best biological model?

Alterations in mollusc shells have been proposed contamination biomarkers. We used geometric morphometrics analyses associated with analytical determinations of contaminants to select suitable biological models among species widely distributed on coastal zones. The study was carried out using Lottia subrugosa (herbivore limpet), Crassostrea brasiliana (filter-feeder bivalve), and Stramonita brasiliensis (carnivore gastropod) obtained along a marked contamination gradient at Santos Estuarine System (Brazil). L. subrugosa and S. brasiliensis presented distinct shapes along the gradient, while no significant differences in shell form were seen for C. brasiliana. Indeed, limpets and snails presented morphometric parameters consistent with measured contamination levels hazardous substances. Based on cross-validation models, the reliability of morphometric responses was over 75% for the herbivore and carnivore species. In addition, for S. brasiliensis, a 95.2% confidence was detected in most contaminated sites. Therefore, shell alterations on carnivorous gastropods should be further investigated, seeking to be effectively employed as pollution biomarkers.