A Novel In Situ Toxicity Identification Evaluation (iTIE) System for Determining which Chemicals Drive Impairments at Contaminated Sites.

Human-dominated waterways contain thousands of chemicals. Determining which chemical is the most important stressor is important, yet very challenging. The Toxicity Identification Evaluation (TIE) procedure from the US Environmental Protection Agency uses a series of chemical and physical manipulations to fractionate compounds within a matrix and systematically identify potential toxicants through laboratory bioassay testing. Although this may provide useful information, it lacks ecological realism because it is subject to laboratory-related artifacts and is resource intensive. The in situ Toxicity Identification Evaluation (iTIE) technology was developed to improve this approach and has undergone a number of modifications over the past several years. The novel prototype 3 consists of an array of iTIE ambient water fractionation units. Each unit is connected to a peristaltic pumping system with an organism exposure chamber that receives water from a resin chamber to chemically fractionate test site water. Test organisms included freshwater and marine standard toxicity test species. Postfractionation waters are collected for subsequent chemical analyses. Currently, the resins allow for separation of ammonia, metals, and nonpolar organics; the subsequent toxicity responses are compared between treatments and unfractionated, ambient exposures. The iTIE system was deployed to a depth of 3 m and evaluated in streams and marine harbors. Chemical analyses of water and iTIE chemical sorptive resins confirmed chemical groups causing lethal to sublethal responses. The system proved to be as sensitive or more so than the traditional phase 1 TIE test and required almost half of the resources to complete. This iTIE prototype provides a robust technology that improves stressor-causality linkages and thereby supports strong evidence for ecological risk weight-of-evidence assessments. Environ Toxicol Chem 2020;39:1746-1754. © 2020 SETAC.

Establishment of the novel bivalve body plan through modification of early developmental events in mollusks.

Mollusks have a wide variety of body plans, which develop through conserved early embryogenesis, namely spiral embryonic development and trochophore larvae. Although the comparative study of mollusks has attracted the interest of evolutionary developmental biology researchers, less attention has been paid to bivalves. In this review, we focused on the evolutionary process from single-shell ancestors to bivalves, which possess bilaterally separated shells. Our study tracing the lineage of shell field cells in bivalves did not support the old hypothesis that shell plate morphology is due to modification of the spiral cleavage pattern. Rather, we suggest that modification of the shell field induction process is the key to understanding the evolution of shell morphology. The novel body plan of bivalves cannot be established solely via separating shell plates, but rather requires the formation of additional organs, such as adductor muscles. The evolutionary biology of bivalves offers a unique view on how multiple organs evolve in a coordinated manner to establish a novel body plan.

Embryo and larval development of the yellow clam Mesodesma mactroides (Reeve, 1854) (Mesodesmatidae) in laboratory.

The yellow clam Mesodesma mactroides (Reeve, 1854) is a sand mollusc with historical and socioeconomic importance in Brazil, Uruguay and Argentina. A guaranteed form to access a successful reestablishment of the species in their natural environment is directly linked to their reproduction biology. Then, our report introduces the embryonic and larval development of the yellow clam reared in laboratory for such purposes. M. mactroides broodstock were selected as specimens who possess a mean total shell length and weight of 66 ± 3.82 mm and 27.15 ± 4.07 g for an afterwards spawn induction through stripping technique. Regarding the embryonic development, newly fertilized oocytes exhibited a mean diameter of 51.20 ± 6.64 µm. The first polar corpuscle, trochophores and D-veliger appeared at 20 min, 18 and 24 h after fertilization, respectively. Umbonate and pediveliger larvae were noticed on the 8th and 25th day, respectively, with complete metamorphosis occurring only at the 27th day, when all larvae were retained in a 200 µm nylon mesh. Therefore, with that basic understanding of the embryonic and larval development of M. mactroides in the laboratory, forwards studies will focus in establish a technological package for this species.

Molecular characterization of a novel sulfide:quinone oxidoreductase from the razor clam Sinonovacula constricta and its expression response to sulfide stress.

The razor clam Sinonovacula constricta is a commercial benthic bivalve, and burrows the deeper cave than the other buried benthic bivalves. Due to the little exchange of seawater and to anoxic conditions, S. constricta is exposed to considerable amounts of sulfide during low tide, but exhibits strong sulfide tolerance. Mitochondrial sulfide oxidation is a particular defense strategy against sulfide toxicity of sulfide-tolerant organisms, for which sulfide:quinone oxidoreductase (SQR) is the first key enzyme. In order to investigate the mechanism of sulfide tolerance in S. constricta, its SQR (designated as ScSQR), was cloned and characterized. The full-length cDNA of ScSQR was 3698 bp and encoded 443 amino acids. The deduced ScSQR protein contained conserved FAD-binding domains, two cysteine residues, two histidines, and one glutamic acid, which are the essential elements for the catalytic mechanism of SQR. Subcellular localization analysis by the TargetP 1.1 prediction and the Western blot confirmed that ScSQR was only located in the mitochondria. The response of ScSQR in the gill and liver of S. constricta were investigated during sulfide exposure (50, 150, and 300 µM sulfide) for 0, 3, 6, 12, 24, 48, 72, and 96 h by qRT-PCR. Moreover, the time-course expressions of ScSQR protein in the S. constricta gill were detected when exposed to 150 µM sulfide by Western blot. The expression level of ScSQR increased significantly and showed a time-dependent pattern. In addition, under sulfide stress, the expression level of the gill was higher than that of liver. Together, our results suggest that ScSQR may perform important roles in protecting cells from sulfide stress by participating in mitochondrial sulfide detoxification and providing high sulfide tolerance to S. constricta.

Impact of environmental hypercapnia on fertilization success rate and the early embryonic development of the clam Limecola balthica (Bivalvia, Tellinidae) from the southern Baltic Sea - A potential CO2 leakage case study.

Carbon capture and storage technology was developed as a tool to mitigate the increased emissions of carbon dioxide by capture, transportation, injection and storage of CO2 into subterranean reservoirs. There is, however, a risk of future CO2 leakage from sub-seabed storage sites to the sea-floor sediments and overlying water, causing a pH decrease. The aim of this study was to assess effects of CO2-induced seawater acidification on fertilization success and early embryonic development of the sediment-burrowing bivalve Limecola balthica L. from the Baltic Sea. Laboratory experiments using a CO2 enrichment system involved three different pH variants (pH 7.7 as control, pH 7.0 and pH 6.3, both representing environmental hypercapnia). The results showed significant fertilization success reduction under pH 7.0 and 6.3 and development delays at 4 and 9 h post gamete encounter. Several morphological aberrations (cell breakage, cytoplasm leakages, blastomere deformations) in the early embryos at different cleavage stages were observed.

Biogeographic vulnerability to ocean acidification and warming in a marine bivalve.

Anthropogenic CO2 emissions are rapidly changing seawater temperature, pH and carbonate chemistry. This study compares the embryonic development under high pCO2 conditions across the south-north distribution range of the marine clam Limecola balthica in NW Europe. The combined effects of elevated temperature and reduced pH on hatching success and size varied strongly between the three studied populations, with the Gulf of Finland population appearing most endangered under the conditions predicted to occur by 2100. These results demonstrate that the assessment of marine faunal population persistence to future climatic conditions needs to consider the interactive effects of co-occurring physico-chemical alterations in seawater within the local context that determines population fitness, adaptation potential and the system resilience to environmental change.

The byssus threads of Pinna nobilis: A histochemical and ultrastructural study.

The byssus of Pinna nobilis, the largest bivalve mollusc in the Mediterranean Sea, was investigated by histochemistry, immunohistochemistry, Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM). At low magnification, the byssus threads appeared distinctively elliptical in cross-section, with a typical size approaching 50 x 25 micron and a featureless glassy appearance. Histochemical and immunohistochemical techniques confirmed the presence of elastic domains but the absence of collagen, which is known to be the main component in other molluscs. Ultrastructural analysis by TEM revealed the presence of at least two components within the thread, and an inner arrangement of straight, tightly packed longitudinal streaks. SEM observations while confirming the inner packing of straight, parallel subfibrils, suggested in the fracture surfaces the presence of unidentified substance which cemented together the same subfibrils and which was removed by exposure to extreme pH values. AFM micrographs added further evidence for the tight packing of subfibrils and provided some evidence of orthogonal, barely visible connecting structures. Finally, HCl or NaOH treatment left the subfibrils clean and free from any other component.Â.

Lineage tracing of the bivalve shell field with special interest in the descendants of the 2d blastomere.

By evolving bilaterally separated shell plates, bivalves acquired a unique body plan in which their soft tissues are completely protected by hard shell plates. In this unique body plan, mobility between the separated shell plates is provided by novel structures such as a ligament and adductor muscles. As a first step towards understanding how the bivalve body plan was established, we investigated the development of the separated shell plates and ligament. Over 100 years ago, it was hypothesized that the development of separated shell plates is tightly linked with the unique cell cleavage (division) pattern of bivalves during development, wherein each bilateral daughter cell of the 2d descendant 2d(1121) develops into one of the bilateral shell fields. In this study, we tested this hypothesis by tracing the cell lineages of the Japanese purple mussel Septifer virgatus. Although the shell fields were found to be exclusively derived from the bilateral descendant cells of 2d: 2d(11211) and 2d(11212), the descendants of these cells were not restricted to shell fields alone, nor were they confined to the left or right side of the shell field based on their lineage. Our study demonstrated that ligament cells are also derived from 2d(11211) and 2d(11212), indicating that the ligament cells emerged as a subpopulation of shell field cells. This also suggests that the establishment of the novel developmental system for the ligament cells was critical for the evolution of the unique body plan of bivalves.

TOXICITY OF CRYOPROTECTIVE AGENTS AND SIGNALING OF INSULIN-LIKE GROWTH FACTOR IN HEN CLAM (MACTRA CHINENSIS) EMBRYOS.

BACKGROUND: Signaling of Insulin-like growth factor-I (IGF-I) is involved in development, growth, reproduction and aging of organisms. OBJECTIVE: The work investigated the toxicity of glycerol, dimethyl sulfoxide (DMSO), and ethylene glycol (EG) to hen clam (Mactra chinensis) embryos, as well as the possible role of the insulin-like growth factor-I (IGF-I) during the development and growth of embryos after freeze. MATERIALS AND METHODS: Effects of glycerol, DMSO and EG at different concentrations were tested. The relationship between larval viability and signaling of IGF-I receptor after cryoprotective treatment and/or freezing was examined using immuno-blot analysis. RESULTS: Glycerol had the highest toxicity, followed by DMSO or EG. No embryo survived freeze and thaw without CPAs. After freeze, the activation of the IGF-I signaling pathway, including the IGF-I receptor (IGF-IR) ß-subunit, could be detected in freeze-thawed embryos. The level of IGF-IR expression was very weak in freeze-thawed embryos. CONCLUSION: The survival and developmental rate of embryos was closely related to CPA concentration. IGF-IR was activated and regulated the downstream IGF-I signaling in embryos. The reduced activation of IGF-IR could be related to the death of hen clam embryos.

Paternally transmitted mitochondria express a new gene of potential viral origin.

Mitochondrial ORFans (open reading frames having no detectable homology and with unknown function) were discovered in bivalve molluscs with doubly uniparental inheritance (DUI) of mitochondria. In these animals, two mitochondrial lineages are present, one transmitted through eggs (F-type), the other through sperm (M-type), each showing a specific ORFan. In this study, we used in situ hybridization and immunocytochemistry to provide evidence for the expression of Ruditapes philippinarum male-specific ORFan (orf21): both the transcript and the protein (RPHM21) were localized in spermatogenic cells and mature spermatozoa; the protein was localized in sperm mitochondria and nuclei, and in early embryos. Also, in silico analyses of orf21 flanking region and RPHM21 structure supported its derivation from viral sequence endogenization. We propose that RPHM21 prevents the recognition of M-type mitochondria by the degradation machinery, allowing their survival in the zygote. The process might involve a mechanism similar to that of Modulators of Immune Recognition, viral proteins involved in the immune recognition pathway, to which RPHM21 showed structural similarities. A viral origin of RPHM21 may also support a developmental role, because some integrated viral elements are involved in development and sperm differentiation of their host. Mitochondrial ORFans could be responsible for or participate in the DUI mechanism and their viral origin could explain the acquired capability of M-type mitochondria to avoid degradation and invade the germ line, that is what viruses do best: to elude host immune system and proliferate.

Nuclear genes with sex bias in Ruditapes philippinarum (Bivalvia, veneridae): Mitochondrial inheritance and sex determination in DUI species.

Mitochondria are inherited maternally in most metazoans, but in bivalves with Doubly Uniparental Inheritance (DUI) a mitochondrial lineage is transmitted through eggs (F-type), and another through sperm (M-type). In DUI species, a sex-ratio distortion of the progeny was observed: some females produce a female-biased offspring (female-biased family), others a male-biased progeny (male-biased family), and others a 50:50 sex-ratio. A peculiar segregation pattern of M-type mitochondria in DUI organisms appears to be correlated with the sex bias of these families. According to a proposed model for the inheritance of M-type mitochondria in DUI, the transmission of sperm mitochondria is controlled by three nuclear genes, named W, X, and Z. An additional S gene with different dosage effect would be involved in sex determination. In this study, we analyzed structure and localization of three transcripts (psa, birc, and anubl1) with specific sex and family biases in the Manila clam Ruditapes philippinarum. In situ hybridization confirmed the localization of these transcripts in gametogenic cells. In other animals, homologs of these genes are involved in reproduction and ubiquitination. We hypothesized that these genes may have a role in sex determination and could also be responsible for the maintenance/degradation of spermatozoon mitochondria during embryo development of the DUI species R. philippinarum, so that we propose them as candidate factors of the W/X/Z/S system.

Sex-linked mitochondrial behavior during early embryo development in Ruditapes philippinarum (Bivalvia Veneridae) a species with the Doubly Uniparental Inheritance (DUI) of mitochondria.

In most metazoans mitochondria are inherited maternally. However, in some bivalve molluscs, two mitochondrial lineages are present: one transmitted through females (F-type), the other through males (M-type). This unique system is called Doubly Uniparental Inheritance (DUI) of mitochondria. In DUI species, M-type mitochondria have to invade the germ line of male embryos during development, otherwise sperm would transmit F-type mtDNA and DUI would fail. The mechanisms by which sperm mitochondria enter the germ line are still unknown. To address this question, we traced the movement of spermatozoon mitochondria (M-type) in embryos of the DUI species Ruditapes philippinarum by fertilizing eggs with sperm stained with the mitochondrial-specific vital dye MitoTracker Green. As in Mytilus DUI species, in R. philippinarum the distribution of sperm mitochondria follows two different patterns: an aggregated one in which these organelles locate near the first cleavage furrow, and a dispersed one in which sperm mitochondria are scattered. The presence of the two mitochondrial patterns in these taxa, together with their absence in species with Strictly Maternal Inheritance (SMI), confirms that their occurrence is related to DUI. Moreover, a Real-Time qPCR analysis showed that neither M-type nor F-type mitochondria undergo replication boosts in the earliest embryo development. This is the first study on sex-linked mtDNA copy number carried out by qPCR analysis on embryos of a DUI species and the first time the segregation patterns of sperm mitochondria are described in a DUI system other than Mytilus.

Embryonic development of Anodontites trapesialis (Lamarck, 1819) (Bivalvia: Mycetopodidae).

The phases of embryonic development of Anodontites trapesialis lasidia are described for the first time. Adult specimens were obtained from two fish farms located in Londrina, Paraná, Brazil. The internal demibranchs of 120 individuals were studied using a routine histological technique; 70 of these carried eggs and/or larvae in the marsupium and were utilized for the description of the phases of embryonic development. The demibranchs of five specimens were evaluated by scanning electron microscopy to detail the morphology of the larvae. Five phases of development were established: phase I, corresponding to the initial stage of cleavage with the formation of apical cells; phase II, including the stages of the morula and blastula; phase III, where the gastrula forms; phase IV, where the larva formed is still inside the egg envelope; and phase V, where the lasidium can still be identified immediately after eclosion.

Potential role of cathepsin B in the embryonic and larval development of clam Meretrix meretrix.

This study was designed to investigate the possible role of Meretrix meretrix cathepsin B (MmeCB) in embryonic and larval development. MmeCB mRNA expression profile was revealed by semi-quantitative RT-PCR. The level of MmeCB mRNA expression was low in trochophore stage but high in pedveliger stage. MmeCB protein expression was detected in the digestive gland, velum, and epidermis along the edges of the shell in D-larvae and pedveligers by immunocytochemistry. In post larvae, MmeCB protein expression was noticed abundant in the digestive gland, whereas a modest expression was identified in the gill filament. The average shell length of larvae hatched from embryos treated with 0.01, 1, and 10 µmol/L Ca074Me (a cathepsin B inhibitor) was significantly shorter than that of control groups. The metamorphosis rates of larvae treated with 0.01 and 1 µmol/L Ca074Me were significantly lower than that of control groups in 4-day larvae, but not in 5-day larvae. Taken together, these results indicated that MmeCB may have stimulatory effects on embryonic development, metamorphosis, and larval growth during M. meretrix larval development.

Early development and cleavage pattern of the Japanese purple mussel, Septifer virgatus.

Despite easy access to bivalves, few studies have examined the development of these animals, at least in part because most bivalve eggs are very small. In addition, annotating cells of the early bivalve embryo is difficult because few landmarks are present. We conducted detailed cell annotations of the Japanese purple mussel, Septifer virgatus, during early embryogenesis because of its relatively large eggs (ca. 130 microm in diameter). Septifer virgatus underwent the unique cell division profile reported for four other bivalve species, suggesting that the cleavage pattern itself is important for bivalve morphogenesis. The shell field invagination was led by 2d (X) lineage cells, supporting the hypothesis that lineage cells differentiate into cells excreting the shell matrix. The large egg size enabled us to trace cell movements in the early gastrulation phase, during which the invagination of the archenteron was initiated by 4d (M) lineage cells. These observations will serve as a basis for future analyses of S. virgatus embryogenesis and will contribute to understanding the evolution of the molluscan body plan, which is achieved by modification of early embryogenesis.

An in-situ study of the impacts of urban wastewater on the immune and reproductive systems of the freshwater mussel Elliptio complanata.

The goal of this study was to examine the disruptive effects of municipal effluents on the immune and reproductive systems of freshwater mussels. For 30 days, caged mussels were immersed in the Rivière des Mille Iles (Quebec, Canada), 150 m both upstream and downstream from two urban wastewater treatment plants: station F (Fabreville) and station A (Auteuil), which serve the city of Laval. Station F is 12 km upstream from station A. The immune and reproductive statuses of the mussels were thereafter determined. Though the weight/shell length ratio was not affected, the effluent induced mortality up to 60% at downstream sites. Total hemocyte counts increased, and phagocytosis and lysozyme activities were induced at station F, whereas these responses were suppressed at station A. Heterotrophic bacteria levels in mussels were negatively correlated with phagocytosis, showing the importance of this process in defending against infection. Inflammation biomarkers such as nitric oxide and cyclooxygenase activity were the same for all sites but were positively correlated with phagocytosis activity. The production of vitellogenin (Vtg)-like proteins was significantly induced at the site downstream from station A and was strongly associated with phagocytosis. This was further supported through analysis of covariance, of Vtg responses against phagocytosis, revealing that Vtg was no longer induced at the sites upstream and downstream from station A. The data support the contention that Vtg was involved, in part at least, in the immune system in mussels. Both Vtg and immune status are impacted by urban effluents and should be considered when using the Vtg biomarker to search for the presence of (xeno)estrogens in contaminated environments.

A novel role for dpp in the shaping of bivalve shells revealed in a conserved molluscan developmental program.

During the molluscan evolution leading to the bivalves, the single dorsal shell was doubled. To elucidate the molecular developmental basis underlying this prominent morphological transition, we described the cell cleavage and expression patterns of three genes, brachyury, engrailed, and dpp in the Japanese spiny oyster Saccostrea kegaki, and examined the function of dpp in this species. The cleavage pattern of the S. kegaki embryo was nearly the same as the previously described pattern of other bivalve species, suggesting that the pattern itself is highly important for the establishment or the maintenance of the bivalve body plan. The expression pattern of a brachyury homolog in S. kegaki (SkBra) was similar to the pattern in gastopods even at the single cell level despite the deep divergence of gastropods and bivalves. Engrailed and dpp were previously found to be expressed around the shell anlagen in gastropods. Like that of gastropods, an engrailed homolog in S. kegaki (SkEn) was found to be expressed around the shell anlagen. However, the dpp homologin S. kegaki (SkDpp) was expressed only in the cells along the dorsal midline. ZfBMP4 treatment experiments revealed the importance of dpp in establishing the characteristic shape of the bivalve shell anlagen.

Spawning, development, and the duration of larval life in a deep-sea cold-seep mussel.

We describe culturing techniques and development for the cold-seep mussel "Bathymodiolus" childressi, the only deep-sea bivalve for which development has been detailed. Spawning was induced in mature mussels by injection of 2 mmol l(-1) serotonin into the anterior adductor muscle. The mean egg diameter is 69.15 +/- 2.36 microm (+/-S.D.; n = 50) and eggs are negatively buoyant. Cleavages are spiral and at 7-8 degrees C occur at a rate of one per 3-9 h through hatching, with free-swimming blastulae hatching by 40 h and shells beginning to develop by day 12. When temperature was raised to 12-14 degrees C after hatching, larvae developed to D-shell veligers by day 8 without being fed. Egg size and larval shell morphology indicate that "B." childressi has a planktotrophic larva, but we did not observe feeding in culture. Wide distribution of this species throughout the Gulf of Mexico and amphi-Atlantic distributions of closely related congeners suggest that larvae may spend extended periods in the plankton. Duration of larval life was estimated for "B." childressi by comparing calculated settlement times to known spawning seasons. These estimates suggest variability in the larval duration, with individuals spending more than a year in the plankton.

Sequential toxicity identification evaluation (TIE) for characterizing toxicity of Venice Lagoon sediments: comparison of two different approaches.

A toxicity identification evaluation phase-I (TIE-1) procedure was carried out on five pore water samples extracted from sediments of the Venice Lagoon previously investigated to assess both chemical contamination and toxic effects on the biota. Two different sequential TIE procedures were tested. A first sequence (TIE-1) provided for adding Na2S2O3, adding Na-EDTA, filtering, elution through a C18-SPE column and removing ammonia using the macroalgae Ulva rigida Agardh 1823, while a second procedure (TIE-2) was set up using U. rigida treatment for ammonia removal as first step, keeping unchanged the sequence of the other manipulations. Two different exposure time to the macroalgae were tested (3-h and 15-h). Sperm-cell toxicity test with the echinoid Paracentrotus lividus and embryotoxicity tests with the bivalves Mytilus galloprovincialis and Crassostrea gigas were performed on pore-water samples to assess the effect of the sequential treatments on the overall toxicity. The results confirmed that ammonia contribution to toxicity is strong in most of the samples and that metals, specially Cu, are of concern at least in three sites. The TIE-2 procedure provided more reliable results for the samples characterized by high ammonia contribution to the overall toxicity, whereas the results of TIE-1 and TIE-2 were equivalent for the samples where ammonia contribution was not prevailing. Chemical analyses and test results showed that a 3-h U. rigida exposure is suitable to remove ammonia toxicity minimizing potential metal up-take.

Larval morphology of the brooding clam Lasaea adansonii (Gmelin, 1791) (Bivalvia, Heterodonta, Galeommatoidea).

The strongly modified mode of development of the small and brooding galeommatoid bivalve Lasaea adansonii (Gmelin, 1791) [syn. Lasaea rubra (Montagu, 1803)] has been studied by means of transmission and scanning electron microscopy and by fluorescent staining of the muscular system and of two neurotracers, FMRFamide and serotonin. In addition, two developmental stages were visualized using computer-aided 3D-reconstruction. All larval stages of L. adansonii lack ciliary rings. The apical organ appears invaginated: the base of the duct contacts the cerebral ganglia and opens on the preoral region. Larval protonephridia are lacking. The adult kidneys develop independently of the pericardial cavity and contain a protonephridial part that enables excretory function until the pericardium is formed. The larval muscular system is composed of smooth muscle fibers; striated fibers are lacking. Posteriorly and immediately below the ligament, a paired cell of unknown function is present that contains serotonin and FMRFamide. In summary, L. adansonii exhibits the direct mode of development. Only few truly larval structures (e.g., the modified apical organ) are elaborated.