Local adaptation and species segregation in two mussel (Mytilus edulis X M. trossulus) hybrid zones

Few marine hybrid zones have been studied extensively, the major exception being the hybrid zone between the mussels Mytilus edulis and M. galloprovincialis in southwestern Europe. Here, we focus on two less studied hybrid zones that also involve Mytilus spp.; M. edulis and M. trossulus are sympatric and hybridize on both western and eastern coasts of the Atlantic Ocean. We review the dynamics of hybridization in these two hybrid zones and evaluate the role of local adaptation for maintaining species boundaries. In Scandinavia, hybridization and gene introgression is so extensive that no individuals with pure M. trossulus genotypes have been found. However, M. trossulus alleles are maintained at high frequencies in the extremely low salinity Baltic Sea for some allozyme genes. A synthesis of reciprocal transplantation experiments between different salinity regimes shows that unlinked Gpi and Pgm alleles change frequency following transplantation, such that post-transplantation allelic composition resembles native populations found in the same salinity. These experiments provide strong evidence for salinity adaptation at Gpi and Pgm (or genes linked to them). In the Canadian Maritimes, pure M. edulis and M. trossulus individuals are abundant, and limited data suggest that M. edulis predominates in low salinity and sheltered conditions, whereas M. trossulus are more abundant on the wave-exposed open coasts. We suggest that these conflicting patterns of species segregation are, in part, caused by local adaptation of Scandinavian M. trossulus to the extremely low salinity Baltic Sea environment.

Differential patterns of Male and Female mtDNA exchange across the Atlantic Ocean in the blue mussel, Mytilus edulis

Comparisons among loci with differing modes of inheritance can reveal unexpected aspects of population history. We employ a multilocus approach to ask whether two types of independently assorting mitochondrial DNAs (maternally and paternally inherited: F- and M-mtDNA) and a nuclear locus (ITS) yield concordant estimates of gene flow and population divergence. The blue mussel, Mytilus edulis, is distributed on both North American and European coastlines and these populations are separated by the waters of the Atlantic Ocean. Gene flow across the Atlantic Ocean differs among loci, with F-mtDNA and ITS showing an imprint of some genetic interchange and M-mtDNA showing no evidence for gene flow. Gene flow of F-mtDNA and ITS causes trans-Atlantic population divergence times to be greatly underestimated for these loci, although a single trans-Atlantic population divergence time (1.2 MYA) can be accommodated by considering all three loci in combination in a coalescent framework. The apparent lack of gene flow for M-mtDNA is not readily explained by different dispersal capacities of male and female mussels. A genetic barrier to M-mtDNA exchange between North American and European mussel populations is likely to explain the observed pattern, perhaps associated with the double uniparental system of mitochondrial DNA inheritance.

Positive selection on an acrosomal sperm protein, M7 lysin, in three species of the mussel genus Mytilus

Marine invertebrate sperm proteins are particularly interesting because they are characterized by positive selection and are likely to be involved in prezyogotic isolation and, thus, speciation. Here, we present the first survey of inter and intraspecific variation of a bivalve sperm protein among a group of species that regularly hybridize in nature. M7 lysin is found in sperm acrosomes of mussels and dissolves the egg vitelline coat, permitting fertilization. We sequenced multiple alleles of the mature protein-coding region of M7 lysin from allopatric populations of mussels in the Mytilus edulis species group (M. edulis, M. galloprovincialis, and M. trossulus). A significant McDonald-Kreitman test showed an excess of fixed amino acid replacing substitutions between species, consistent with positive selection. In addition, Kolmogorov-Smirnov tests showed significant heterogeneity in polymorphism to divergence ratios for both synonymous variation and combined synonymous and non-synonymous variation within M. galloprovincialis. These results indicate that there has been adaptive evolution at M7 lysin and, furthermore, shows that positive selection on sperm proteins can occur even when post-zygotic reproductive isolation is incomplete.

Analysis of marine bivalve shellfish from the fish market in Santos city, Sao Paulo state, Brazil, for Toxoplasma gondii

The aim of this study was to determine if Toxoplasma gondii are present in oysters (Crassostrea rhizophorae) and mussels (Mytella guyanensis) under natural conditions using a bioassay in mice and molecular detection methods. We first compared two standard protocols for DNA extraction, phenol-chloroform (PC) and guanidine-thiocyanate (GT), for both molluscs. A total of 300 oysters and 300 mussels were then acquired from the fish market in Santos city, Sao Paulo state, Brazil, between March and August of 2008 and divided into 60 groups of 5 oysters and 20 groups of 15 mussels. To isolate the parasite, five mice were orally inoculated with sieved tissue homogenates from each group of oysters or mussels. For molecular detection of T. gondii, DNA from mussels was extracted using the PC method and DNA from oysters was extracted using the GT method. A nested-PCR (Polymerase Chain Reaction) based on the amplification of a 155 bp fragment from the B1 gene of T. gondii was then performed. Eleven PCR-RFLP (Restriction Fragment Length Polymorphism) markers, SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, CS3 and Apico, were used to genotype positive samples. There was no isolation of the parasite by bioassay in mice. T. gondii was not detected in any of the groups of mussels by nested-PCR. DNA of T. gondii was apparently detected by nested-PCR in 2 groups of oysters (3.3%). Genotyping of these two positive samples was not successful. The results suggest that oysters of the species C. rhizophorae, the most common species from the coast of Sao Paulo, can filter and retain T. gondii oocysts from the marine environment. Ingestion of raw oysters as a potential transmission source of T. gondii to humans and marine mammals should be further investigated. (C) 2010 Elsevier B.V. All rights reserved.

Detection of the initial infective stages of the protozoan parasite Marteilia sydneyi in Saccostrea glomerata and their development through to sporogenesis

DNA probes were used in in situ hybridisation on histological sections of oysters exposed for defined intervals to Marteilia sydneyi infection to reveal the early development of the parasite in the oyster host, Saccostrea glomerata. The initial infective stages enter through the palps and gills whereupon extrasporogonic proliferation results in the liberation of cells into surrounding connective tissue and haemolymph spaces. Following systemic dissemination, the parasite infiltrates the digestive gland and becomes established as a nurse cell beneath the epithelial cells ill a digestive tubule. Here, cell-within-cell proliferation results in the eventual liberation of daughter cells from the nurse cell into spaces between adjacent epithelial cells. None of these stages had previously been described. Proliferation is associated with host responses, including haemocytic infiltration of the connective tissue and diapedesis across tubule epithelia. The responses cease as sporogenesis begins. (C) 2002 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.

The fan mussel Pinna rudis : occurrence and association with Pontonia pinnophylax in Terceira Island (Azores, NE Atlantic).

Conference: IMMR | International Meeting on Marine Research 2014. Peniche, Portugal, 10 Jul - 11 Jul, 2014. Presentation Type: Poster Presentation.

Immune responses in Bathymodiolus azoricus upon Vibrio challenges : approaches to characterize mussel survival strategies and physiological adaptations in deep sea extreme environments

Tese de Doutoramento, Ciências do Mar, especialidade de Biologia Marinha, 18 de Dezembro de 2015, Universidade dos Açores.

Host-symbiont interactions in the deep-sea vent mussel Bathymodiolus azoricus : a molecular approach

Tese de Doutoramento, Ciências do Mar, especialidade de Biologia Marinha, 19 de Dezembro de 2015, Universidade dos Açores.

Isolation and characterisation of metallothionein from the clam Ruditapes decussatus

Aquatic Toxicology 63 (2003) 307-318

Enzymatic characterization of Vibrio alginolyticus strains isolated from bivalves harvested at Venice Lagoon (Italy) and Guanabara Bay (Brazil)

The aquatic ecosystem is the natural habitat of microorganisms including Vibrio and Aeromonas genus which are pathogenic to human and animals. In the present investigation the frequency of these bacteria and the enzymatic characteristics of 34 Vibrio alginolyticus strains isolated from bivalves harvested in Venice Lagoon (Italy) and Guanabara Bay (Brazil) were carried out from November 2003 to February 2004. The mussels' samples were submitted to enrichment in Alkaline Peptone Water (APW) added with 1% of sodium chloride (NaCl) and APW plus 3% NaCl incubated at 37 ºC for 18-24h. Following the samples were streaked onto TCBS Agar (Thiossulfate Citrate Bile Sucrose Agar) and the suspected colonies were submitted to biochemical characterization. Also, the Vibrio alginolyticus strains were evaluated to collagenase, elastase and chondroitinase production. The results showed the isolation of 127 microorganisms distributed as follows: 105 Vibrio strains such as V. alginolyticus (32.4%), V. harveyi (19%) and V. parahaemolyticus (7.6%), 20 Aeromonas strains and two Plesiomonas shigelloides were the main pathogens isolated. We observed the production of the three enzymes from V. alginolyticus strains considered as the main virulence factors of the bacteria, especially in cases of human dermatological infection.

Gold nanoparticles for the detection of DNA adducts as biomarkers of exposure to acrylamide

The main objective of this thesis was the development of a gold nanoparticle-based methodology for detection of DNA adducts as biomarkers, to try and overcome existing drawbacks in currently employed techniques. For this objective to be achieved, the experimental work was divided in three components: sample preparation, method of detection and development of a model for exposure to acrylamide. Different techniques were employed and combined for de-complexation and purification of DNA samples (including ultrasonic energy, nuclease digestion and chromatography), resulting in a complete protocol for sample treatment, prior to detection. The detection of alkylated nucleotides using gold nanoparticles was performed by two distinct methodologies: mass spectrometry and colorimetric detection. In mass spectrometry, gold nanoparticles were employed for laser desorption/ionisation instead of the organic matrix. Identification of nucleotides was possible by fingerprint, however no specific mass signals were denoted when using gold nanoparticles to analyse biological samples. An alternate method using the colorimetric properties of gold nanoparticles was employed for detection. This method inspired in the non-cross-linking assay allowed the identification of glycidamide-guanine adducts and DNA adducts generated in vitro. For the development of a model of exposure, two different aquatic organisms were studies: a goldfish and a mussel. Organisms were exposed to waterborne acrylamide, after which mortality was recorded and effect concentrations were estimated. In goldfish, both genotoxicity and metabolic alterations were assessed and revealed dose-effect relationships of acrylamide. Histopathological alterations were verified primarily in pancreatic cells, but also in hepatocytes. Mussels showed higher effect concentrations than goldfish. Biomarkers of oxidative stress, biotransformation and neurotoxicity were analysed after prolonged exposure, showing mild oxidative stress in mussel cells, and induction of enzymes involved in detoxification of oxygen radicals. A qualitative histopathological screening revealed gonadotoxicity in female mussels, which may present some risk to population equilibrium.

Adhesive bioactive coatings inspired by sea life

Inspired by nature, in particular by the marine mussels adhesive proteins (MAPs) and by the tough brick-and-mortar nacre-like structure, novel multilayered films are prepared in the present work. Organic-inorganic multilayered films, with an architecture similar to nacre based on bioactive glass nanoparticles (BG), chitosan and hyaluronic acid modified with catechol groups, which are the main responsible for the outstanding adhesion in MAPs, are developed for the first time. The biomimetic conjugate is prepared by carbodiimide chemistry and analyzed by ultraviolet-visible spectrophotometry. The build-up of the multilayered films is monitored with a quartz crystal microbalance with dissipation monitoring and their topography is characterized by atomic force microscopy. The mechanical properties reveal that the films containing catechol groups and BG present an enhanced adhesion. Moreover, the bioactivity of the films upon immersion in a simulated body fluid solution for 7 days is evaluated by scanning electron microscopy coupled with energy dispersive X-ray spectroscopy, Fourier transform infrared spectroscopy and X-ray diffraction. It was found that the constructed films promote the formation of bone-like apatite in vitro. Such multifunctional mussel inspired LbL films, which combine enhanced adhesion and bioactivity, could be potentially used as coatings of a variety of implants for orthopedic applications.

Synchronous reproduction may facilitate introgression in a hybrid mussel (Mytilus) population

Mussel populations on the Irish Atlantic coast comprise an interbreeding mixture of the blue mussel, Mytilus edulis (L.) and the Mediterranean mussel, Mytilus galloprovincialis (Lmk.). The occurrence of hybrid genotypes varies between sites but can be as high 80%. This study compares the reproductive cycle of M. edulis, M. galloprovincialis and their hybrids to determine if the extensive hybridisation observed at Irish Atlantic coast sites is linked to spawning synchrony between the two taxa. Mussels (40-45 mm size class) were collected monthly from a sheltered shore in Galway Bay from January to December 2005. Two major spawning events (March- June and September-October) were observed and gametogenesis took place throughout the year. The spawning cycles of the three taxa were largely overlapping. Small differences were observed in the timing of peak spawning which occurred in March and October in M. galloprovincialis and in May-June and September in M.edulis. Spawning of hybrid individuals was intermediate between the parental genotypes. Fecundity was slightly higher in M. galloprovincialis females compared to the other taxa (up to 30% difference, p<0.05). This apparent advantage is not shared by the sexes and is likely being offset by high numbers of hybrid genotypes releasing gametes during peak spawning of M. galloprovincialis. There was no evidence for increased mortality in hybrid males; sex ratios did not deviate from the 1:1 ratio. The results show that in this region of the hybrid zone the timing of reproduction does not present a barrier to gene flow between M. edulis and M. galloprovincialis. Nonetheless, small differences in the timing of peak spawning may increase the likelihood of conspecific fertilisation at certain times of the year. Hybrids outnumber the parental genotypes, undergo complete gametogenesis and show no evidence of depressed fitness (i.e. hybrids are reproductively competent suggesting a high degree of introgression.

Sewage impact on the composition and distribution of Polychaeta associated to intertidal mussel beds of the Mar del Plata rocky shore, Argentina

The polychaete composition and distribution within mussel beds were studied in order to assess organic pollution due to domestic sewage in a rocky shore of Mar del Plata (Argentina) during 1997. Four stations and a control site were randomly sampled around the local effluent. Quantitative data on polychaetes, as well as sediment accumulated among mussels and its organic carbon content were measured. Polychaete distribution patterns are related to the organic matter gradient, being Capitella cf. capitata, Neanthes succinea (Frey & Leuckart, 1847) and Boccardia polybranchia (Haswell, 1885) the dominant indicator species close to the effluent. At medial distances, the cirratulids Caulleriella alata (Southern, 1914) and Cirratulus cirratus (Müller, 1776) are very important in abundance. The syllids Syllis prolixa Ehlers, 1901 and S. gracilis Grube, 1840 are distributed along the study area, but dominate at the medial stations and at the control site. The orbiniid Protoariciella uncinata Hartmann-Schröder, 1962 is subdominant at the control station.