Tetrabromobisphenol A acts a neurodevelopmental disruptor in early larval stages of Mytilus galloprovincialis.

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 h 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.

Bisphenol A interferes with first shell formation and development of the serotoninergic system in early larval stages of Mytilus galloprovincialis.

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 Tyrosinase-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.

Characterization of the main steps in first shell formation in Mytilus galloprovincialis: possible role of tyrosinase.

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 CaCO3 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 CaCO3 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 CaCO3 deposition and the importance of tyrosinase in the formation of the first shell in M. galloprovincialis.

Ocean pH fluctuations affect mussel larvae at key developmental transitions.

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.

Cytomegalovirus Identification in Blood and Urine of Newborns by Nested Polymerase Chain Reaction.

AIM: To study the frequency of congenital cytomegalovirus (CMV) infection in newborns admitted to the Division of Neonatology, using nested polymerase chain reaction (PCR) and DNA to detect differences in blood and urine specimens. METHODS: The study was carried out for eight months. Newborns (n = 520) hospitalized in five hospitals in Campo Grande, Mato Grosso do Sul, Brazil, were checked for CMV by analysing blood and urine samples. RESULTS: Cytomegalovirus was PCR positive in 13 urine and 10 blood samples. Of the 13 positive urine patients, three (23%) had no clinical signs suggestive of CMV, and another three (23%) patients admitted to the neonatal intensive care unit (NICU) had no definite findings of bacterial infection, with negative blood culture and some clinical signs consistent with CMV as cholestasis, hepatomegaly and eosinophilia. Three patients were on mechanical ventilation and showed improvement after prescription of ganciclovir. One CMV positive child progressed to death. CONCLUSION: Cytomegalovirus detection in urine was slightly more efficient than in blood, and showed better sensitivity than in serological analysis (p < 0.01) therefore, boiled urine may be a better and easier specimen tool for CMV diagnosis in neonatal infection. The findings of the present research suggest that patients admitted to the NICU, especially premature infants, whose laboratory results are not compatible with bacterial infection, and exhibiting signs suggestive of CMV infection should have PCR done on urine for confirmation.