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 pH 7.9 for 3 days, and gene expression levels, calcification and respiration rates were measured relative to pH 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 GABA receptor subunit. This observation is particularly interesting because GABA receptor disturbances, leading to altered behavior, have been documented in several other marine animals after exposure to elevated CO . 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.

Abstract:This study investigated the effects of seawater pH (i.e. 8.10, 7.85 and 7.60) and temperature (16 and 19°C) on (i) the abiotic conditions in the fluid surrounding the embryo (viz. the perivitelline fluid),(ii) growth, development and (iii) cuttlebone calcification of embryonic and juvenile stages of the cephalopod Sepia officinalis. Egg swelling increased in response to acidification or warming, leading to an increase in egg surface while the interactive effects suggested a limited plasticity of the swelling modulation. Embryos experienced elevated pCO 2 conditions in the perivitelline fluid (> 3-fold higher pCO 2 than that of ambient seawater), rendering the medium under-saturated even under ambient conditions. The growth of both embryos and juveniles was unaffected by pH, whereas 45 Ca incorporation in cuttlebone increased significantly with decreasing pH at both temperatures. This phenomenon of hypercalcification is limited to only a number of animals but does not guarantee functional performance and calls for better mechanistic understanding of calcification processes.

In recent decades, cephalopods have been shown to have very high capacities to accumulate most trace elements, regardless of whether they are essential (e.g., Cu and Zn) or non-essential (e.g., Ag and Cd). Among the different pathways of exposure to trace elements, the trophic pathway appears to be the major route of assimilation for numerous metals, including Cd, Co, Hg and Zn. Once assimilated, trace elements are distributed in the organism, accumulating in storage organs. The digestive gland is the main organ in which many trace elements accumulate, whichever of the exposure pathway. For example, this organ can present Cd concentrations reaching hundreds to thousands of ppm for some species, even though the digestive gland represents only a small proportion of the total mass of the animal. Such a specific organotropism towards the digestive gland of both essential and non-essential elements, regardless of the exposure pathway, poses the question of the detoxification processes evolved by cephalopods in order to sustain these high concentrations. This paper reviews the current knowledge on the bioaccumulation of trace elements in cephalopods, the differences in pharmaco-dynamics between organs and tissues, and the detoxification processes they use to counteract trace element toxicity. A peculiar focus has been done on the bioaccumulation within the digestive gland by investigating the subcellular locations of trace elements and their protein ligands.

Ag activity continued to increase in eggs, whereas 109 Cd kinetics displayed a significant decrease. Whatever the development stage, Cd was mainly associated with the eggshell all along the exposure experiment. In addition, both stable Cd concentrations and 109 Cd LCR remained low in the embryo all along the embryonic development, indicating that the eggshell acted as an efficient shield against the penetration of this metal. In contrast, 110mAg passed through the eggshell from day 30 onwards and was then accumulated in the embryo, which contained more than 40% of the whole egg metal burden at the end of the exposure period. In depuration conditions, it is noteworthy that Ag continued to accumulate in the embryo indicating translocation processes from the eggshell and a high affinity of the metal for the embryo tissues. Overall our results showed that at day 30 of the embryonic development the cuttlefish eggshell becomes permeable to Ag but not to Cd.Exposure to stable metals confirmed the saturation capacities of the eggshell for Cd and the Ag penetration properties.

The anthropogenic release of carbon dioxide (CO(2)) into the atmosphere leads to an increase in the CO(2) partial pressure (pCO(2)) in the ocean, which may reach 950 μatm by the end of the 21st century. The resulting hypercapnia (high pCO(2)) and decreasing pH ("ocean acidification") are expected to have appreciable effects on water-breathing organisms, especially on their early-life stages. For organisms like squid that lay their eggs in coastal areas where the embryo and then paralarva are also exposed to metal contamination, there is a need for information on how ocean acidification may influence trace element bioaccumulation during their development. In this study, we investigated the effects of enhanced levels of pCO(2) (380, 850 and 1500 μatm corresponding to pH(T) of 8.1, 7.85 and 7.60) on the accumulation of dissolved (110m)Ag, (109)Cd, (57)Co, (203)Hg, (54)Mn and (65)Zn radiotracers in the whole egg strand and in the different compartments of the egg of Loligo vulgaris during the embryonic development and also in hatchlings during their first days of paralarval life. Retention properties of the eggshell for (110m)Ag, (203)Hg and (65)Zn were affected by the pCO(2) treatments. In the embryo, increasing seawater pCO(2) enhanced the uptake of both (110m)Ag and (65)Zn while (203)Hg showed a minimum concentration factor (CF) at the intermediate pCO(2). (65)Zn incorporation in statoliths also increased with increasing pCO(2). Conversely, uptake of (109)Cd and (54)Mn in the embryo decreased as a function of increasing pCO(2). Only the accumulation of (57)Co in embryos was not affected by increasing pCO(2). In paralarvae, the CF of (110m)Ag increased with increasing pCO(2), whereas the (57)Co CF was reduced at the highest pCO(2) and (203)Hg showed a maximal uptake rate at the intermediate pCO(2). (54)Mn and (65)Zn accumulation in paralarvae were not significantly modified by hypercapnic conditions. Our results suggest a combined effect of pH on the adsorption and protective properties of the eggshell and of hypercapnia on the metabolism of embryo and paralarvae, both causing changes to the accumulation of metals in the tissues of L. vulgaris.

Abstract. Cephalopods play a key role in many marine trophic networks and constitute alternative fisheries resources, especially given the ongoing decline in finfish stocks. Along the European coast, the eggs of the cuttlefish Sepia officinalis are characterized by an increasing permeability of the eggshell during development, which leads to selective accumulation of essential and non-essential elements in the embryo. Temperature and pH are two critical factors that affect the metabolism of marine organisms in the coastal shallow waters. In this study, we investigated the effects of pH and temperature through a crossed (3 × 2; pH 8.1 (pCO 2 , 400 ppm), 7.85 (900 ppm) and 7.6 (1400 ppm) at 16 and 19 • C, respectively) laboratory experiment. Seawater pH showed a strong effect on the egg weight and non-significant impact on the weight of hatchlings at the end of development implying an egg swelling process and embryo growth disturbances. The lower the seawater pH, the more 110m Ag was accumulated in the tissues of hatchlings. The 109 Cd concentration factor (CF) decreased with decreasing pH and 65 Zn CF reached maximal values pH 7.85, independently of temperature. Our results suggest that pH and temperature affected both the permeability properties of the eggshell and embryonic metabolism. To the best of our knowledge, this is one of the first studies on the consequences of ocean acidification and ocean warming on metal uptake in marine organisms, Correspondence to: T. Lacoue-Labarthe (tlacouel@gmail.com) and our results indicate the need to further evaluate the likely ecotoxicological impact of the global change on the earlylife stages of the cuttlefish.

Uptake and depuration kinetics of inorganic mercury (Hg) were investigated in the juvenile common cuttlefish Sepia officinalis following exposures via seawater and food using a sensitive radiotracer technique ( Hg when exposed via seawater, with whole body concentration factors > 260 after only 10 d of exposure. The total Hg accumulated from seawater was depurated relatively fast with a radiotracer biological half-life (Tb ! ) of 17 d. During both exposure and depuration periods, accumulated Hg was mainly (> 70%) associated with the muscular parts of the cuttlefish. However, the proportion of the whole-body Hg content associated with the digestive gland increased during exposure and depuration phases, suggesting that the metal was transferred from the muscles towards this organ for detoxification. When fed with radiolabelled food, cuttlefish displayed high assimilation efficiency (> 90%) and the metal was found to be mainly located in the digestive gland (60% of the whole Hg content). Nevertheless, high depuration rates resulted in short Tb ! (i.e. 4 d), suggesting that this organ has a major role in Hg detoxification and depuration. Whatever the exposure pathway, a low proportion of Hg (< 2%) was found in the cuttlebone. Assessment of the relative contribution of the dietary and dissolved exposure pathways to inorganic Hg bioaccumulation in juvenile cuttlefish revealed that Hg was mainly accumulated from food, which contributed 77 ± 16% of the global metal bioaccumulation.

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