Project description:BackgroundWhile it is established that vertebrate-like steroids, particularly estrogens (estradiol, estrone) and androgens (testosterone), are present in various tissues of molluscs, it is still unclear what role these play in reproductive endocrinology in such organisms. This is despite the significant commercial shellfishery interest in several bivalve species and their decline.Methodology/principal findingsUsing suppression subtraction hybridisation of mussel gonad samples at two stages (early and mature) of gametogenesis and (in parallel) following controlled laboratory estrogen exposure, we isolate several differentially regulated genes including testis-specific kinases, vitelline lysin and envelope sequences.ConclusionsThe differentially expressed mRNAs isolated provide evidence that mussels may be impacted by exogenous estrogen exposure.
Project description:Bivalves are widespread in coastal environments subjected to a wide range of environmental fluctuations: however, the rapidly occurring changes due to several anthropogenic factors can represent a significant threat to bivalve immunity. The mussel Mytilus spp. has extremely powerful immune defenses toward different potential pathogens and contaminant stressors. In particular, the mussel immune system represents a significant target for different types of nanoparticles (NPs), including amino-modified nanopolystyrene (PS-NH2) as a model of nanoplastics. In this work, the effects of repeated exposure to PS-NH2 on immune responses of Mytilus galloprovincialis were investigated after a first exposure (10 μg/L; 24 h), followed by a resting period (72-h depuration) and a second exposure (10 μg/L; 24 h). Functional parameters were measured in hemocytes, serum, and whole hemolymph samples. In hemocytes, transcription of selected genes involved in proliferation/apoptosis and immune response was evaluated by qPCR. First exposure to PS-NH2 significantly affected hemocyte mitochondrial and lysosomal parameters, serum lysozyme activity, and transcription of proliferation/apoptosis markers; significant upregulation of extrapallial protein precursor (EPp) and downregulation of lysozyme and mytilin B were observed. The results of functional hemocyte parameters indicate the occurrence of stress conditions that did not however result in changes in the overall bactericidal activity. After the second exposure, a shift in hemocyte subpopulations, together with reestablishment of basal functional parameters and of proliferation/apoptotic markers, was observed. Moreover, hemolymph bactericidal activity, as well as transcription of five out of six immune-related genes, all codifying for secreted proteins, was significantly increased. The results indicate an overall shift in immune parameters that may act as compensatory mechanisms to maintain immune homeostasis after a second encounter with PS-NH2.
Project description:Mitochondrial DNA (mtDNA) was thought to be inherited maternally in animals, although paternal leakage has been reported in mice and Drosophila. Recently, direct evidence of extensive paternal inheritance of mtDNA has been found in the marine mussel Mytilus. We give evidence that whereas female mussels are homoplasmic for a genome that is transmitted to eggs, male mussels are heteroplasmic for this genome and for a second genome that is transmitted preferentially to sperm. The results provide support for the existence of separate male and female routes of mtDNA inheritance in mussels. The two genomes show a base sequence divergence exceeding 20% at three protein coding genes, consistent with long term maintenance of the heteroplasmic state. We propose that the two genomes differ in fitness in males and females, possibly as a result of interaction with nuclear genes.
Project description:Various sequencing projects over the last several years have aided the discovery of previously uncharacterized invertebrate sequences, including new cytochrome P450 genes (CYPs). Here we present data on the identification and characterization of two CYP1-like and three CYP3-like genes from the bivalve mollusk Mytilus edulis, and assess their potential as biomarkers based on their responses to several known vertebrate aryl hydrocarbon receptor (AHR) agonists. Quantitative real-time PCR was used to measure CYP transcript levels in digestive gland, labial palps, adductor muscle, gill, foot, and different regions of the mantle. Levels of both CYP1-like genes were highest in digestive gland, whereas labial palps had the highest expression levels of the three CYP3-like genes followed by digestive gland and outer margin of the mantle. Mussels were exposed by injection to the AHR agonists, ?-naphthoflavone (BNF; 25 ?g g(-1)), 3,3',4,4',5-polychlorinated biphenyl (PCB126; 2 ?g g(-1)), or 6-formylindolo[3,2-b]carbazole (FICZ; 0.1 ?g g(-1)), or to Aroclor 1254 (a mixture of PCBs; 50 ?g g(-1)) for 24 h, followed by CYP expression analysis. There was no statistically significant change in expression of either of the CYP1-like genes after exposure to the various AHR agonists. The CYP3-like-1 gene was significantly up-regulated by BNF in gill tissues and the CYP3-like-2 gene was up-regulated in digestive gland by PCB126 and in gill tissue by BNF. These results suggest that distinct mechanisms of CYP gene activation could be present in M. edulis, although the importance of the CYP1-like and CYP3-like genes for xenobiotic and endogenous lipids biotransformation requires additional investigation.
Project description:Recent papers have suggested that epifaunal organisms use artificial structures as stepping-stones to spread to areas that are too distant to reach in a single generation. With thousands of artificial structures present in the North Sea, we test the hypothesis that these structures are connected by water currents and act as an interconnected reef. Population genetic structure of the blue mussel, Mytilus edulis, was expected to follow a pattern predicted by a particle tracking model (PTM). Correlation between population genetic differentiation, based on microsatellite markers, and particle exchange was tested. Specimens of M. edulis were found at each location, although the PTM indicated that locations >85 km offshore were isolated from coastal subpopulations. The fixation coefficient FST correlated with the number of arrivals in the PTM. However, the number of effective migrants per generation as inferred from coalescent simulations did not show a strong correlation with the arriving particles. Isolation by distance analysis showed no increase in isolation with increasing distance and we did not find clear structure among the populations. The marine stepping-stone effect is obviously important for the distribution of M. edulis in the North Sea and it may influence ecologically comparable species in a similar way. In the absence of artificial shallow hard substrates, M. edulis would be unlikely to survive in offshore North Sea waters.
Project description:The smooth-shelled blue mussel, Mytilus edulis is part of the Mytilus species complex, encompassing at least three putative species: M. edulis, Mytilus galloprovincialis, and Mytilus trossulus. These three species occur on both sides of the Atlantic and hybridize in nature, and both M. edulis and M. galloprovincialis are important aquaculture species. They are also invasive species in many parts of the world. Here, we present a chromosome-level assembly of M. edulis. We used a combination of PacBio sequencing and Dovetail's Omni-C technology to generate an assembly with 14 long scaffolds containing 94% of the predicted length of the M. edulis genome (1.6 out of 1.7 Gb). Assembly statistics were as follows: total length = 1.65 Gb, N50 = 116 Mb, L50 = 7, and L90 = 13. BUSCO analysis showed 92.55% eukaryote BUSCOs identified. AB-Initio annotation using RNA-seq from mantle, gills, muscle, and foot predicted 47,128 genes. These gene models were combined with IsoSeq validation resulting in 45,379 full CDS protein sequences and 129,708 isoforms. Using GBS and shotgun sequencing, we also sequenced several eastern Canadian populations of Mytilus to characterize single-nucleotide as well as structural variance. This high-quality genome for M. edulis provides a platform to develop tools that can be used in breeding, molecular ecology and evolution to address questions of both commercial and environmental perspectives.
Project description:Study of the genetic basis of gene expression variation is central to attempts to understand the causes of evolutionary change. Although there are many transcriptomics studies estimating genetic variance and heritability in model organisms such as humans there is a lack of equivalent proteomics studies. In the present study, the heritability underlying egg protein expression was estimated in the marine mussel Mytilus. We believe this to be the first such measurement of genetic variation for gene expression in eggs of any organism. The study of eggs is important in evolutionary theory and life history analysis because maternal effects might have profound effects on the rate of evolution of offspring traits. Evidence is presented that the egg proteome varies significantly between individual females and that heritability of protein expression in mussel eggs is moderate to high suggesting abundant genetic variation on which natural selection might act. The study of the mussel egg proteome is also important because of the unusual system of mitochondrial DNA inheritance in mussels whereby different mitochondrial genomes are transmitted independently through female and male lineages (doubly uniparental inheritance). It is likely that the mechanism underlying this system involves the interaction of specific egg factors with sperm mitochondria following fertilization, and its elucidation might be advanced by study of the proteome in females having different progeny sex ratios. Putative identifications are presented here for egg proteins using MS/MS in Mytilus lines differing in sex ratio. Ontology terms relating to stress response and protein folding occur more frequently for proteins showing large expression differences between the lines. The distribution of ontology terms in mussel eggs was compared with those for previous mussel proteomics studies (using other tissues) and with mammal eggs. Significant differences were observed between mussel eggs and mussel tissues but not between the two types of eggs.
Project description:The potential toxicity of engineered nanoparticles (NPs) for humans and the environment represents an emerging issue. Since the aquatic environment represents the ultimate sink for NP deposition, the development of suitable assays is needed to evaluate the potential impact of NPs on aquatic biota. The immune system is a sensitive target for NPs, and conservation of innate immunity represents an useful basis for studying common biological responses to NPs. Suspension-feeding invertebrates, such as bivalves, are particularly at risk to NP exposure, since they have extremely developed systems for uptake of nano and microscale particles integral to intracellular digestion and cellular immunity. Evaluation of the effects of NPs on functional parameters of bivalve immunocytes, the hemocytes, may help understanding the major toxic mechanisms and modes of actions that could be relevant for different NP types in aquatic organisms.In this work, a battery of assays was applied to the hemocytes of the marine bivalve Mytilus galloprovincialis to compare the in vitro effects of different n-oxides (n-TiO(2), n-SiO(2), n-ZnO, n-CeO(2)) chosen on the basis of their commercial and environmental relevance. Physico-chemical characterization of both primary particles and NP suspensions in artificial sea water-ASW was performed. Hemocyte lysosomal and mitochondrial parameters, oxyradical and nitric oxide production, phagocytic activity, as well as NP uptake, were evaluated. The results show that different n-oxides rapidly elicited differential responses hemocytes in relation to their chemical properties, concentration, behavior in sea water, and interactions with subcellular compartments. These represent the most extensive data so far available on the effects of NPs in the cells of aquatic organisms. The results indicate that Mytilus hemocytes can be utilized as a suitable model for screening the potential effects of NPs in the cells of aquatic invertebrates, and may provide a basis for future experimental work for designing environmentally safer nanomaterials.
Project description:Coastal monitoring in the 21st century faces challenges as measuring exposure using traditional toxicity approaches is not practical or financially feasible for the numerous contaminants entering oceans. Transcriptomics, high-throughput assays, and adverse outcome pathways (AOP) have been one approach to overcome these obstacles but development of these methods is challenging for non-model organisms and contaminants with unknown mechanisms. Endocrine Disrupting Compounds (EDCs) have been found to cause reproductive impairments such as intersex in bivalves, however, the mechanism linked to this adverse outcome (AO) is unknown as the estrogen receptor (ER) has been identified to be unresponsive to EDCs. To develop mechanism-based biomarkers that may be linked through an AOP we exposed Mytilus edulis to 17-alpha-ethinylestradiol (5 and 50ng/L) and 4-nonylphenol (1 and 100μg/L), and performed gene expression analysis on digestive gland tissue using a M. edulis microarray. Through network and targeted analyses, we identified the non-genomic estrogen signaling pathway and steroidogenesis pathway as the likely mechanism of action and identified biomarkers and potential key events (KE) for an AOP of endocrine disruption in marine mussels. The mechanism-based biomarkers were validated in the laboratory, tested in the field, and were responsive in areas known to be contaminated with EDCs.
Project description:Hypoxia is a characteristic feature of marine environments and a major stressor for marine organisms inhabiting benthic and intertidal zones. Several studies have explored the responses of these organisms to hypoxic stress at the whole organism level with a focus on energy metabolism and mitochondrial response, but the instrinsic mitochondrial responses that support the organelle’s function under hypoxia and reoxygenation (H/R) stress are not well understood. We studied the effects of acute H/R stress (10 min anoxia followed by 15 min reoxygenation) on mitochondrial respiration, production of reactive oxygen species (ROS) and posttranslational modifications (PTM) of the proteome in a marine facultative anaerobe, the blue mussel Mytilus edulis. The mussels’ mitochondria showed increased OXPHOS respiration and suppressed proton leak resulting in a higher coupling efficiency after H/R stress. ROS production decreased in both the resting (LEAK) and phosphorylating (OXPHOS) state indicating that M. edulis is able to prevent oxidative stress and mitochondrial damage during reoxygenation. Hypoxia did not stimulate the rearrangement of the mitochondrial supercomplexes but impacted the mitochondrial phosphoproteome including the proteins involved in OXPHOS, amino acid and fatty acid catabolism, and protein quality control. This study indicates that mussels’ mitochondria possess intrinsic mechanisms (including regulation via PTM mechanisms such as reversible protein phosphorylation) that ensure high respiratory flux and mitigate oxidative damage during H/R stress and contribute to the hypoxia-tolerant mitochondrial phenotype of this metabolically plastic species.