Single-Larva RNA Sequencing Identifies Markers of Copper Toxicity and Exposure in Early Mytilus californianus Larvae

One of the challenges facing efforts to generate molecular biomarkers for toxins is distinguishing between markers that are indicative of exposure and markers that provide evidence of the effects of toxicity. Phenotypic anchoring provides an approach to help segregate markers into these categories based on some phenotypic index of toxicity. Here we leveraged the mussel embryo-larval toxicity assay in which toxicity is estimated by the fraction of larvae that exhibit an abnormal morphology, to isolate subsets of larvae that were abnormal and thus showed evidence of copper-toxicity, versus others that while exposed to copper exhibited normal morphology. Mussel larvae reared under control conditions or in the presence of increasing levels of copper (3–15 μg/L Cu2+) were physically sorted according to whether their morphology was normal or abnormal, and then profiled using RNAseq. Supervised differential expression analysis identified sets of genes whose differential expression was specific to the pools of abnormal larvae versus normal larvae, providing putative markers of copper toxicity versus exposure. Markers of copper exposure and copper-induced abnormality were involved in many of the same pathways, including development, shell formation, cell adhesion, and oxidative stress, yet unique markers were detected in each gene set. Markers of effect appeared to be more resolving between phenotypes at the lower copper concentration, while markers of exposure were informative at both copper concentrations.

Bivalves rapidly repair shells damaged by fatigue and bolster strength

ABSTRACT Hard external armors have to defend against a lifetime of threats yet are traditionally understood by their ability to withstand a single attack. Survival of bivalve mollusks thus can depend on the ability to repair shell damage between encounters. We studied the capacity for repair in the intertidal mussel Mytilus californianus by compressing live mussels for 15 cycles at ∼79% of their predicted strength (critically fracturing 46% of shells), then allowing the survivors 0, 1, 2 or 4 weeks to repair. Immediately after fatigue loading, mussel shells were 20% weaker than control shells that had not experienced repetitive loading. However, mussels restored full shell strength within 1 week, and after 4 weeks shells that had experienced greater fatiguing forces were stronger than those repetitively loaded at lower forces. Microscopy supported the hypothesis that crack propagation is a mechanism of fatigue-caused weakening. However, the mechanism of repair was only partially explained, as epifluorescence microscopy of calcein staining for shell deposition showed that only half of the mussels that experienced repetitive loading had initiated direct repair via shell growth around fractures. Our findings document repair weeks to months faster than demonstrated in other mollusks. This rapid repair may be important for the mussels’ success contending with predatory and environmental threats in the harsh environment of wave-swept rocky coasts, allowing them to address non-critical but weakening damage and to initiate plastic changes to shell strength. We highlight the significant insight gained by studying biological armors not as static structures but, instead, as dynamic systems that accumulate, repair and respond to damage.

Investigating controls of shell growth features in a foundation bivalve species: seasonal trends and decadal changes in the California mussel

Marine bivalve mollusc shells can offer valuable insights into past oceanographic variability and seasonality. Given its ecological and archaeological significance, Mytilus californianus (California mussel) presents the opportunity to examine seasonal and decadal changes recorded in its shell over centuries to millennia. While dark–light growth bands in M. californianus shells could be advantageous for reconstructing past environments, uncertainties remain regarding shell structure, environmental controls of dark–light band formation, and the amount of time represented by a dark–light pair. By analyzing a suite of M. californianus shells collected in 2002, 2003, 2019, and 2020 from Bodega Bay, California, we describe the mineralogical composition, establish relationships among growth band pattern, micro-environment, and collection season, and compare shell structure and growth band expression between the archival (2002–2003) and modern (2019–2020) shells. We identified three mineralogical layers in M. californianus: an outer prismatic calcite layer, a middle aragonite layer, and a secondary inner prismatic calcite layer, which makes M. californianus the only Mytilus species to precipitate a secondary calcite layer. Within the inner calcite layer, light bands are strongly correlated with winter collection months and could be used to reconstruct periods with moderate, stable temperatures and minimal upwelling. Additionally, modern shells have significantly thinner inner calcite layers and more poorly expressed growth bands than the archival shells, although we also show that growth band contrast is strongly influenced by micro–environment. Mytilus californianus from northern California is calcifying differently, and apparently more slowly, than it was 20 years ago.

Self-assembling of Chimeric Mussel-inspired Bio-adhesives Originated from Mytilus Californianus and Anabaena Flos-aquae: A New Approach to Develop Underwater Adhesion

Bio-adhesives play a pivotal role in a wide range of medical applications. However, there are some problems about their application in varied pH values and low adhesion force under wet conditions. Here, we report new recombinant fusion protein achieved by mussel foot proteins (Mfps) of Mytilus Californianus and gas vesicle protein A (GvpA) of Anabaena flos-aquae by genetic engineering methods. These chimeric proteins self-assembled into ß-sheet rich fibres because of GvpA amyloid structure. Also, their adhesion forces were significantly increased especially in alkaline environment based on Mfp-3 and Mfp-5. This study illustrates that copolymer of Mfp-5-GvpA:GvpA-Mfp-3 can be used as an underwater sturdy adhesive with tolerance to auto-oxidation, especially at basic conditions.