Coastal marine ecosystems experience dynamic fluctuations in seawater carbonate chemistry. The importance of this variation in the context of ocean acidification requires knowing what aspect of variability biological processes respond to. We conducted four experiments (ranging from 3 to 22 days) with different variability regimes (pHT 7.4–8.1) assessing the impact of diel fluctuations in carbonate chemistry on the early development of the mussel Mytilus galloprovincialis. Larval shell growth was consistently correlated to mean exposures, regardless of variability regimes, indicating that calcification responds instantaneously to seawater chemistry. Larval development was impacted by timing of exposure, revealing sensitivity of two developmental processes: development of the shell field, and transition from the first to the second larval shell. Fluorescent staining revealed developmental delay of the shell field at low pH, and abnormal development thereof was correlated with hinge defects in D-veligers. This study shows, for the first time, that ocean acidification affects larval soft-tissue development, independent from calcification. Multiple developmental processes additively underpin the teratogenic effect of ocean acidification on bivalve larvae. These results explain why trochophores are the most sensitive life-history stage in marine bivalves and suggest that short-term variability in carbonate chemistry can impact early larval development.

Bivalve biomineralization is a highly complex and organized process, involving several molecular components identified in adults and larval stages. However, information is still scarce on the ontogeny of the organic matrix before calcification occurs. In this work, first shell formation was investigated in the mussel Mytilus galloprovincialis . The time course of organic matrix and CaCO 3 deposition were followed at close times post fertilization (24, 26, 29, 32, 48 h) by calcofluor and calcein staining, respectively. Both components showed an exponential trend in growth, with a delay between organic matrix and CaCO 3 deposition. mRNA levels of genes involved in matrix deposition (chitin synthase; tyrosinase- TYR) and calcification (carbonic anhydrase; extrapallial protein) were quantified by qPCR at 24 and 48 hours post fertilization (hpf) with respect to eggs. All transcripts were upregulated across early development, with TYR showing highest mRNA levels from 24 hpf. TYR transcripts were closely associated with matrix deposition as shown by in situ hybridization. The involvement of tyrosinase activity was supported by data obtained with the enzyme inhibitor N-phenylthiourea. Our results underline the pivotal role of shell matrix in driving first CaCO 3 deposition and the importance of tyrosinase in the formation of the first shell in M. galloprovincialis .

Nuclear Receptors (NRs) are a superfamily of transcription factors specific to metazoans that have the unique ability to directly translate the message of a signaling molecule into a transcriptional response. In vertebrates, NRs are pivotal players in countless processes of both embryonic and adult physiology, with embryonic development being one of the most dynamic periods of NR activity. Accumulating evidence suggests that NR signaling is also a major regulator of development in marine invertebrates, although ligands and transactivation dynamics are not necessarily conserved with respect to vertebrates. The explosion of genome sequencing projects and the interpretation of the resulting data in a phylogenetic context allowed significant progress toward an understanding of NR superfamily evolution, both in terms of molecular activities and developmental functions. In this context, marine invertebrates have been crucial for characterizing the ancestral states of NR-ligand interactions, further strengthening the importance of these organisms in the field of evolutionary developmental biology.

Bisphenol A-BPA, a widespread plastic additive, is an emerging contaminant of high concern and a potential endocrine disruptor in mammals. BPA also represents a potential threat for aquatic species, especially for larval stages. In the marine bivalve Mytilus galloprovincialis, BPA has been previously shown to affect early larval development and gene transcription. In this work, the effects of BPA (0.05-0.5-5 µM) were further investigated at different times post fertilization (24-28-32-48 hpf). BPA induced concentration-dependent alterations in deposition of the organic matrix and calcified shell at different larval stages, as shown by double calcofluor/calcein staining, resulting in altered phenotypes at 48hpf. Transcription of tTyrosinase-TYR, that plays a key role in remodelling of the shell organic matrix, and of HOX1, a member of homeobox genes involved in larval shell formation and neurogenesis, were evaluated by In Situ Hybrydization-ISH. BPA altered the spatial pattern of expression of both genes, with distinct effects depending on the concentration and developmental stage. Moreover, BPA affected the time course of mRNA levels for TYR from 24 to 48hpf. BPA impaired development of serotonin-5-HT-immunoreactive neurons at different times pf; at 48hpf, the reduction in the number of serotoninergic neurons was associated with developmental delay and downregulation of the 5-HT receptor-5-HTR. All the effects were observed from the lowest concentration tested, corresponding to detectable BPA levels in contaminated coastal waters. These data demonstrate that BPA interferes with key processes occurring during the first developmental stages of mussels, thus representing a potential threat for natural populations.

Tetrabromobisphenol A-TBBPA, a widely used brominated flame retardant detected in aquatic environments, is considered a potential endocrine disruptor-ED for its reproductive/developmental effects in vertebrates. In aquatic invertebrates, the modes of action of most EDs are largely unknown, due to partial knowledge of the mechanisms controlling neuroendocrine functions.In the marine bivalve Mytilus galloprovincialis, TBBPA has been previously shown to affect larval development in the 48 hours larval toxicity assay at environmental concentrations. In this work, the effects of TBBPA were further investigated at different times post-fertilization. TBBPA, from 1 µg/L, affected shell biogenesis at 48 hours post fertilization-hpf, as shown by phenotypic and SEM analysis.The mechanisms of action of TBBPA were investigated at concentrations of the same order of magnitude as those found in highly polluted coastal areas (10 µg/L). At 28-32 hpf, TBBPA significantly affected deposition of both the organic matrix and CaCO3 in the shell. TBBPA also altered expression of shell-related genes from 24 to 48 hpf, in particular of tyrosinase, a key enzyme in shell matrix remodeling. At earlier stages (24 hpf), TBBPA affected the development of dopaminergic, serotoninergic and GABAergic systems, as shown by in situ hybridization-ISH and immunocytochemistry. These data contribute draw adverse outcome pathways-AOPs, where TBBPA affects the synthesis of neutrotransmitters involved in key events (neurodevelopment and shell biogenesis), resulting in phenotypic changes on individuals (delayed or arrested development) that might lead to detrimental consequences on populations.

Contaminants of Emerging Concerns (CECs) are defined as chemicals not commonly monitored in aquatic ecosystems, but with the potential to cause adverse effects on biota. CECs include Endocrine Disrupting Chemicals (EDCs) and Neuro-Endocrine disruptors (NEDs) of vertebrates. However, most invertebrates only rely on neuroendocrine systems to maintain homeostatic processes. Although conserved neuroendocrine components have been characterized in ecologically relevant groups, limited knowledge on invertebrate neuroendocrinology makes it difficult to define EDCs and NEDs in most species. The monoamine serotonin (5-hydroxytryptamine, 5-HT) acts both as a neurotransmitter and as a peripheral hormone in mammals. In molluscs, 5-HT is involved in multiple physiological roles and molecular components of the serotonergic system have been identified. This review is focused on the effects of CECs on the serotonergic system of bivalve molluscs. Bivalves are widespread in all aquatic environments, estuarine and coastal areas in particular, where they are exposed to a variety of chemicals. In bivalves, 5-HT is involved in gametogenesis and spawning, oocyte maturation and sperm motility, regulates heart function, gill ciliary beating, mantle/siphon function, the ‘‘catch’’ state of smooth muscle and immune responses. Components of 5-HT transduction (receptors and signaling pathways) are being identified in several bivalve species. Different CECs have been shown to affect bivalve serotonergic system. This particularly applies to antidepressants, among the most commonly detected human pharmaceuticals in the aquatic environment. In particular, selective serotonin reuptake inhibitors (SSRIs) are frequently detected in seawater and in bivalve tissues. Information available on the effects and mechanisms of action of SSRIs on the serotonergic system of adult bivalves is summarized. Data are also reported on the effects of CECs on development of neuroendocrine pathways of early larval stages, in particular on the effects of model EDCs in the marine mussel Mytilus galloprovincialis. Overall, available data point at the serotonergic system as a sensitive target for neuroendocrine disruption in bivalves. The results contribute drawing Adverse Outcome Pathways (AOPs) for model EDCs and SSRIs in larvae and adults. However, basic research on neuroendocrine signaling is still needed to evaluate the potential impact of neuroendocrine disruptors in key invertebrate groups of aquatic ecosystems.

In the absence of standard methods for the detection/quantification of nanoplastics (NPs) in environmental samples, commercial nanopolymers are utilized as proxies for toxicity testing and environmental risk assessment. In marine species, a considerable amount of data are now available on the effects of nanopolystyrene (PS-NPs) of different size/surface characteristics. In this work, amino modified PS-NPs (PS-NH2) (50 and 100 nm), purchased from two different companies, were compared in terms of behavior in exposure media and of biological responses, from molecular to organism level, in the model marine bivalve Mytilus. Different PS-NH2 showed distinct agglomeration and surface charge in artificial sea water (ASW) and hemolymph serum (HS). Differences in behavior were largely reflected by the effects on immune function in vitro and in vivo and on early larval development. Stronger effects were generally observed with PS-NH2 of smaller size, showing less agglomeration and higher positive charge in exposure media. Specific molecular interactions with HS components were investigated by the isolation and characterization of the NP-corona proteins. Data obtained in larvae demonstrate interference with the molecular mechanisms of shell biogenesis. Overall, different PS-NH2 can affect the key physiological functions of mussels at environmental concentrations (10 µg/L). However, detailed information on the commercial NPs utilized is required to compare their biological effects among laboratory experiments.

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