Biomineralization plasticity and environmental heterogeneity predict geographical resilience patterns of foundation species to future change

Although geographical patterns of species' sensitivity to environmental changes are defined by interacting multiple stressors, little is known about compensatory processes shaping regional differences in organismal vulnerability. Here, we examine large‐scale spatial variations in biomineralization under heterogeneous environmental gradients of temperature, salinity and food availability across a 30° latitudinal range (3,334 km), to test whether plasticity in calcareous shell production and composition, from juveniles to large adults, mediates geographical patterns of resilience to climate change in critical foundation species, the mussels Mytilus edulis and M. trossulus. We find shell calcification decreased towards high latitude, with mussels producing thinner shells with a higher organic content in polar than temperate regions. Salinity was the best predictor of within‐region differences in mussel shell deposition, mineral and organic composition. In polar, subpolar, and Baltic low‐salinity environments, mussels produced thin shells with a thicker external organic layer (periostracum), and an increased proportion of calcite (prismatic layer, as opposed to aragonite) and organic matrix, providing potentially higher resistance against dissolution in more corrosive waters. Conversely, in temperate, higher salinity regimes, thicker, more calcified shells with a higher aragonite (nacreous layer) proportion were deposited, which suggests enhanced protection under increased predation pressure. Interacting effects of salinity and food availability on mussel shell composition predict the deposition of a thicker periostracum and organic‐enriched prismatic layer under forecasted future environmental conditions, suggesting a capacity for increased protection of high‐latitude populations from ocean acidification. These findings support biomineralization plasticity as a potentially advantageous compensatory mechanism conferring Mytilus species a protective capacity for quantitative and qualitative trade‐offs in shell deposition as a response to regional alterations of abiotic and biotic conditions in future environments. Our work illustrates that compensatory mechanisms, driving plastic responses to the spatial structure of multiple stressors, can define geographical patterns of unanticipated species resilience to global environmental change.     

Maerl grounds provide both refuge and high growth potential for juvenile queen scallops (Aequipecten opercularis L.)

Human damage to biogenic substrata such as maerl has been receiving increasing attention recently. Maerl forms highly biodiverse and heterogeneous habitats composed of loose-lying coralline red algae, which fulfil nursery area prerequisites for queen scallops (Aequipecten opercularis) and other invertebrates. The benefits obtained by queen scallops utilising maerl were poorly understood, so we used both laboratory predation and field tethering experiments to investigate the refuge and growth potential provided by pristine live maerl (PLM) grounds over other common substrata. In aquaria, more juvenile queen scallops (<35 mm shell height) survived on PLM than on gravel substrata in the presence of the crab Carcinus maenas or the starfish Asterias rubens. Field tethering experiments indicated similar survivorship of juvenile queen scallops on PLM than gravel; additionally, their growth rates were similar on both substrata. PLM allows scallops to seek refuge from predators and position themselves to optimise their food supply. Other bivalve refugia have been shown to provide poor food supply as a consequence of their high heterogeneity, yet maerl grounds provide a ‘win-win’ scallop nursery area coupling refuge availability with high food supply.     

Ocean Acidification Has Multiple Modes of Action on Bivalve Larvae

Ocean acidification (OA) is altering the chemistry of the world’s oceans at rates unparalleled in the past roughly 1 million years. Understanding the impacts of this rapid change in baseline carbonate chemistry on marine organisms needs a precise, mechanistic understanding of physiological responses to carbonate chemistry. Recent experimental work has shown shell development and growth in some bivalve larvae, have direct sensitivities to calcium carbonate saturation state that is not modulated through organismal acid-base chemistry. To understand different modes of action of OA on bivalve larvae, we experimentally tested how pH, P_CO2, and saturation state independently affect shell growth and development, respiration rate, and initiation of feeding in Mytilus californianus embryos and larvae. We found, as documented in other bivalve larvae, that shell development and growth were affected by aragonite saturation state, and not by pH or P_CO2. Respiration rate was elevated under very low pH (~7.4) with no change between pH of ~ 8.3 to ~7.8. Initiation of feeding appeared to be most sensitive to P_CO2, and possibly minor response to pH under elevated P_CO2. Although different components of physiology responded to different carbonate system variables, the inability to normally develop a shell due to lower saturation state precludes pH or P_CO2 effects later in the life history. However, saturation state effects during early shell development will carry-over to later stages, where pH or P_CO2 effects can compound OA effects on bivalve larvae. Our findings suggest OA may be a multi-stressor unto itself. Shell development and growth of the native mussel, M. californianus, was indistinguishable from the Mediterranean mussel, Mytilus galloprovincialis, collected from the southern U.S. Pacific coast, an area not subjected to seasonal upwelling. The concordance in responses suggests a fundamental OA bottleneck during development of the first shell material affected only by saturation state.     

Metabolically induced pH fluctuations by some coastal calcifiers exceed projected 22nd century ocean acidification: a mechanism for differential susceptibility?

Anthropogenically mediated decreases in pH, termed ocean acidification (OA), may be a major threat to marine organisms and communities. Research has focussed mainly on tropical coral reefs, but temperate reefs play a no less important ecological role in colder waters, where OA effects may first be manifest. Herein, we report that trends in pH at the surface of three ecologically important cold‐water calcifiers (a primary producer and herbivores), under a range of fluid flows, differ substantially from one another, and for two of the three calcifiers, the pH, during darkness, is lower than the mean projected pH due to OA for the surface waters of the global ocean beyond the year 2100. Using micro‐electrodes, we show that each calcifier had a different pH gradient between its surface and mainstream seawater, i.e. within the diffusion boundary layer (DBL) that appears to act as an environmental buffer to mainstream pH. Abalone encountered only mainstream seawater pH, whereas pH at the sea urchins’ surface was reduced by ~0.35 units. For coralline algae, pH was ~0.5 units higher in the light and ~0.35 units lower under darkness than in ambient mainstream seawater. This wide range of pH within the DBL of some calcifiers will probably affect their performance under projected future reductions in pH due to OA. Differing exposure to a range of surface pH may result in differential susceptibility of calcifiers to OA. Such fluctuations are no doubt regulated by the interplay of water movement, morphology and metabolic rates (e.g. respiration, calcification and/or photosynthesis). Our study, by considering physics (flow regime), chemistry (pH gradients vs. OA future projections) and biology (trophic level, physiology and morphology), reveals that predicting species‐specific responses and subsequent ecosystem restructuring to OA is complex and requires a holistic, eco‐mechanical, approach.     

The structure of mollusc larval shells formed in the presence of the chitin synthase inhibitor Nikkomycin Z

Background  

Chitin self-assembly provides a dynamic extracellular biomineralization interface. The insoluble matrix of larval shells of the marine bivalve mollusc Mytilus galloprovincialis consists of chitinous material that is distributed and structured in relation to characteristic shell features. Mollusc shell chitin is synthesized via a complex transmembrane chitin synthase with an intracellular myosin motor domain.     

Slow‐flow habitats as refugia for coastal calcifiers from ocean acidification

The pH of the oceans’ surface water is dropping, termed ocean acidification (OA), and the 0.4 unit reduction in pH by 2100 is projected to negatively impact benthic coastal organisms that produce calcium carbonate “skeletons.” Research has focussed on identifying species that are susceptible to OA, but there is an urgent need to discover refuge habitats that will afford protection to vulnerable species. The susceptibility of calcium carbonate skeletons to dissolution by OA depends on the pH at their surface, and this is controlled by the interaction between seawater velocity and organismal metabolism. This perspective considers how seawater velocity modifies the responses of calcifying organisms (seaweed, shellfish, and tropical corals) to OA through its action on controlling diffusion boundary layer thickness and thereby the pH and calcium carbonate saturation state (Ω) at the organisms’ surface. Evidence is presented to support the idea that slow‐flow habitats, such as wave‐sheltered bays or the within canopies of seaweed/seagrass beds, might provide inexpensive refugia from OA for vulnerable coastal calcifiers.     

The O2, pH and Ca2+ Microenvironment of Benthic Foraminifera in a High CO2 World

Ocean acidification (OA) can have adverse effects on marine calcifiers. Yet, phototrophic marine calcifiers elevate their external oxygen and pH microenvironment in daylight, through the uptake of dissolved inorganic carbon (DIC) by photosynthesis. We studied to which extent pH elevation within their microenvironments in daylight can counteract ambient seawater pH reductions, i.e. OA conditions. We measured the O₂ and pH microenvironment of four photosymbiotic and two symbiont-free benthic tropical foraminiferal species at three different OA treatments (∼432, 1141 and 2151 µatm pCO₂). The O₂ concentration difference between the seawater and the test surface (ΔO₂) was taken as a measure for the photosynthetic rate. Our results showed that O₂ and pH levels were significantly higher on photosymbiotic foraminiferal surfaces in light than in dark conditions, and than on surfaces of symbiont-free foraminifera. Rates of photosynthesis at saturated light conditions did not change significantly between OA treatments (except in individuals that exhibited symbiont loss, i.e. bleaching, at elevated pCO₂). The pH at the cell surface decreased during incubations at elevated pCO₂, also during light incubations. Photosynthesis increased the surface pH but this increase was insufficient to compensate for ambient seawater pH decreases. We thus conclude that photosynthesis does only partly protect symbiont bearing foraminifera against OA.     

The ontogeny of shell secretion in Terebratalia transversa (Brachiopoda, Articulata). II. Formation of the protegulum and juvenile shell

The fine structure of the shell and underlying mantle in young juveniles of the articulate brachiopod Terebratalia transversa has been examined by electron microscopy. The first shell produced by the mantle consists of a nonhinged protegulum that lacks concentric growth lines. The protegulum is secreted within a day after larval metamorphosis and typically measures 140-150 micron long. A thin organic periostracum constitutes the outer layer of the protegulum, and finely granular shell material occurs beneath the periostracum. Protegula resist digestion in sodium hypochlorite and are refractory to sectioning, suggesting that the subperiostracal portion of the primordial shell is mineralized. The juvenile shell at 4 days postmetamorphosis possesses incomplete sockets and rudimentary teeth that consist of nonfibrous material. The secondary layer occuring in the inner part of the juvenile shell contains imbricated fibers, whereas the outer portion of the shell comprises a bipartite periostracum and an underlying primary layer of nonfibrous shell. Deposition of the periostracum takes place within a slot that is situated between the so-called lobate and vesicular cells of the outer mantle lobe. Vesicular cells deposit the basal layer of the periostracum, while lobate cells contribute materials to the overlying periostracal superstructure. Cells with numerous tonofibrils and hemidesmosomes differentiate in the outer mantle epithelium at sites of muscle attachments, and unbranched punctae that surround mantle caeca develop throughout the subperiostracal portion of the shell. Three weeks after metamorphosis, the juvenile shell averages about 320 micron in length and is similar in ultrastructure to the shells secreted by adult articulates.     

Diurnal fluctuations in seawater pH influence the response of a calcifying macroalga to ocean acidification

Coastal ecosystems that are characterized by kelp forests encounter daily pH fluctuations, driven by photosynthesis and respiration, which are larger than pH changes owing to ocean acidification (OA) projected for surface ocean waters by 2100. We investigated whether mimicry of biologically mediated diurnal shifts in pH—based for the first time on pH time-series measurements within a kelp forest—would offset or amplify the negative effects of OA on calcifiers. In a 40-day laboratory experiment, the calcifying coralline macroalga, Arthrocardia corymbosa, was exposed to two mean pH treatments (8.05 or 7.65). For each mean, two experimental pH manipulations were applied. In one treatment, pH was held constant. In the second treatment, pH was manipulated around the mean (as a step-function), 0.4 pH units higher during daylight and 0.4 units lower during darkness to approximate diurnal fluctuations in a kelp forest. In all cases, growth rates were lower at a reduced mean pH, and fluctuations in pH acted additively to further reduce growth. Photosynthesis, recruitment and elemental composition did not change with pH, but δ¹³C increased at lower mean pH. Including environmental heterogeneity in experimental design will assist with a more accurate assessment of the responses of calcifiers to OA.     

Chitin helicoids accompany protein helicoids in the periostracum of a whelk, Buccinum

The periostracum of the marine gastropod Buccinum has a helicoidal arrangement of its principal constituent which is a fibrous protein (Hunt and Oates, 1978). Chitin, chemically and physically identified, is present at a concentration of about 6% of the dry weight and can be seen in dispersates of whole periostracum as long fibrils and ribbons between 3 and 14 nm diameter. Deproteinization with hot alkali removes all protein leaving a chitinous 'ghost' of the periostracum. Dispersates, examined negatively stained, show only chitin fibrils and ribbons while sectioned material demonstrates a tenuous, part orthogonal, part helicoidal, architecture based on the chitin residue. The relative roles of the protein and polysaccharide components is speculated upon and comparisons with arthropod cuticle drawn.     

Carbonic anhydrase activity and biomineralization process in embryos, larvae and adult blue mussels Mytilus edulis L.

To decide whether a physiological role can be attributed to enzymatic activity with respect to crystal formation and biomineralization of the first larval shell, carbonic anhydrase (CA) activity was measured in embryos and larvae of the blue mussels Mytilus edulis L. Also, CA activity was determined in the mantle edge and gonads of adult mussels with different shell length and condition index. The intention was to find a possible correlation between CA activity and adult shell calcification, i.e. gonadal maturation. The comparison of CA activity in different developmental stages of mussels and the results of an X-ray diffraction study of biomineralization processes in embryonic and larval shells indicate that CA activity is maximal at the end of several developmental stages. Consequently, the increase in CA activity precedes some physiological changes, i.e. the somatoblast 2d formation and the occurrence of the first calcite and quartz crystals in embryos, shell field formation in the gastrula stage, shell gland and periostracum production in trochophores, and rapid aragonite deposition in larval prodissoconch I and prodissoconch II shells. Furthermore, it was found that in adult mussels CA activity was quite variable and that in the mantle edge it was frequently inversely related to the activity in the gonad. Received: 28 November 1998 / Received in revised form: 30 August 1999 / Accepted: 31 August 1999     

The interstitial crystal-nucleating sheet in molluscan Haliotis rufescens shell: A bio-polymeric composite

The interstitial green sheets in abalone shell nacre are shown to be bifacially differentiated trilaminate polymeric complexes, with glycoprotein layers sandwiching a central core containing chitin. They share some common feature with the organic matrix layers between the aragonite tablets in the nacre and the periostracum, and show similarities to the myostracum. Thus, although the green sheet is reported to be unique to the abalone shell, it represents an interesting model for the study of molluscan shell biomineralization processes. Indeed, during shell formation, prismatic and spherulitic aragonite precedes and follows the deposition of the interstitial green polymeric composite sheets, and there is evidence to suggest that these sheets demark the interruption of nacre synthesis and serve to nucleate the resumption of calcium carbonate crystal growth. The green polymeric interstitial sheet purified from the abalone shell was investigated by spectroscopic and imaging techniques: FTIR, confocal microscopy, scanning and transmission electron microscopy, and by pyrolysis combined with GC–MS. Structural and compositional differences are observed between the surfaces of the two sides of the interstitial polymeric composite sheets. Moreover, comparative crystallization experiments on the green sheet sides also reveal asymmetry with respect to the nucleation of calcium carbonate. These findings suggest that these bifacially differentiated interstitial composites may play an active role in the mineral assembly processes, with one of the surfaces acting as a crystal nucleator.     

Ocean acidification at high latitudes: potential effects on functioning of the Antarctic bivalve Laternula elliptica

Ocean acidification is a well recognised threat to marine ecosystems. High latitude regions are predicted to be particularly affected due to cold waters and naturally low carbonate saturation levels. This is of concern for organisms utilising calcium carbonate (CaCO₃) to generate shells or skeletons. Studies of potential effects of future levels of pCO₂ on high latitude calcifiers are at present limited, and there is little understanding of their potential to acclimate to these changes. We describe a laboratory experiment to compare physiological and metabolic responses of a key benthic bivalve, Laternula elliptica, at pCO₂ levels of their natural environment (430 µatm, pH 7.99; based on field measurements) with those predicted for 2100 (735 µatm, pH 7.78) and glacial levels (187 µatm, pH 8.32). Adult L. elliptica basal metabolism (oxygen consumption rates) and heat shock protein HSP70 gene expression levels increased in response both to lowering and elevation of pH. Expression of chitin synthase (CHS), a key enzyme involved in synthesis of bivalve shells, was significantly up-regulated in individuals at pH 7.78, indicating L. elliptica were working harder to calcify in seawater undersaturated in aragonite (Ω_Ar = 0.71), the CaCO₃ polymorph of which their shells are comprised. The different response variables were influenced by pH in differing ways, highlighting the importance of assessing a variety of factors to determine the likely impact of pH change. In combination, the results indicate a negative effect of ocean acidification on whole-organism functioning of L. elliptica over relatively short terms (weeks-months) that may be energetically difficult to maintain over longer time periods. Importantly, however, the observed changes in L. elliptica CHS gene expression provides evidence for biological control over the shell formation process, which may enable some degree of adaptation or acclimation to future ocean acidification scenarios.     

Scanning electron microscopy of glochidia and juveniles of the freshwater mussel,Hyriopsis myersiana

Summary

The ultrastructure of early stages of the mussel, Hyriopsis (Limnoscapha) myersiana (Lea, 1856), was observed by scanning electron microscopy from the glochidial period until the onset of the juvenile stage 10 days later. Further observations were performed for an additional 13 days to assess juvenile development. Glochidia extracted from the brood chambers have a hookless, semi-oval and equivalve calcareous shell with numerous pores in the internal surface, pits in the external surface and cuticular spines in the ventral region. Keratin fibers with a random arrangement in the cuticle of the glochidial shell were also detected. The appearance of the foot within 10 days of in vitro glochidial culture was considered the main feature of metamorphosis to the juvenile stage. Another change during the following 13 days was the formation of a new periostracum exhibiting growth lines under the old glochidial shell. This development occurs mainly in the anterior region and is followed by hardening of the periostracum matrix by calcium deposition. Periostracum growth gradually became apparent in the lateral and posterior regions at the end of this period. The retraction of spines and the alteration of the external surface of the old shell are also described. It is speculated that transcuticular filaments identified in the juvenile stage may have sensory or metabolic exchange functions. The prominent foot, gradually covered by long dense cilia, shows rhythmical movements which suggest a role in feeding. Similarly, cilia present in the mantle may also be involved in the capture of food, while microvilli may facilitate absorption of dissolved materials. Longer cilia, sparsely distributed in the mantle, may function as chemo- or tactile sensors.     

Molecular mechanisms underlying the physiological responses of the cold-water coral Desmophyllum dianthus to ocean acidification

Cold-water corals (CWCs) are thought to be particularly vulnerable to ocean acidification (OA) due to increased atmospheric pCO₂, because they inhabit deep and cold waters where the aragonite saturation state is naturally low. Several recent studies have evaluated the impact of OA on organism-level physiological processes such as calcification and respiration. However, no studies to date have looked at the impact at the molecular level of gene expression. Here, we report results of a long-term, 8-month experiment to compare the physiological responses of the CWC Desmophyllum dianthus to OA at both the organismal and gene expression levels under two pCO₂/pH treatments: ambient pCO₂ (460 μatm, pH_T = 8.01) and elevated pCO₂ (997 μatm, pH_T = 7.70). At the organismal level, no significant differences were detected in the calcification and respiration rates of D. dianthus. Conversely, significant differences were recorded in gene expression profiles, which showed an up-regulation of genes involved in cellular stress (HSP70) and immune defence (mannose-binding c-type lectin). Expression of alpha-carbonic anhydrase, a key enzyme involved in the synthesis of coral skeleton, was also significantly up-regulated in corals under elevated pCO₂, indicating that D. dianthus was under physiological reconditioning to calcify under these conditions. Thus, gene expression profiles revealed physiological impacts that were not evident at the organismal level. Consequently, understanding the molecular mechanisms behind the physiological processes involved in a coral’s response to elevated pCO₂ is critical to assess the ability of CWCs to acclimate or adapt to future OA conditions.     

Attachment of the juvenile queen scallop (Aequipecten opercularis (L.)) to maerl in mesocosm conditions; juvenile habitat selection

Substratum un-acclimated juvenile queen scallops (Aequipecten opercularis) (<18 mm and 18-30 mm shell height) were released in equal numbers onto pristine live maerl (PLM), impacted dead maerl (IDM), gravel and sand in choice chambers. Their habitat selection was monitored over a 4-day period in control and predator treatments (utilising Asterias rubens L. and Carcinus maenas (L.)). Microhabitat use of PLM by juvenile queen scallops and the presence of cues in live maerl were also investigated.In control and predator treatments juvenile queen scallops were observed to attach preferentially to PLM than IDM, gravel or sand. Juvenile queen scallops were observed to maintain a more exposed attachment site in the absence of predators but sought refuge within and between maerl nodules in the presence of both predators. Smaller queen scallops (<18 mm shell height) were more efficient at utilising maerl thalli as a refuge. Juvenile A. opercularis showed hierarchical cue responses mediated by predator presence, i.e. responding favourably to a factor associated with live maerl presence irrespective of heterogeneity in the absence of predators but favourably to higher maerl heterogeneity in their presence. If they also preferentially attach to PLM in the field, at some sites where PLM grounds cover large areas, they may thus be considered to constitute ‘nursery areas’. Habitat attachment preference appears to be predetermined and not a result of localised predator avoidance; however, habitat usage changes in the presence of predators. Maerl beds have been shown to be easily damaged by scallop dredging in Scotland and if such nursery areas are being destroyed extensively in the field, this could damage recruitment to localised adult populations.     

Relevance of mytilid shell microtopographies for fouling defence – a global comparison

Prevention of epibiosis is of vital importance for most aquatic organisms, which can have consequences for their ability to invade new areas. Surface microtopography of the shell periostracum has been shown to have antifouling properties for mytilid mussels, and the topography shows regional differences. This article examines whether an optimal shell design exists and evaluates the degree to which shell microstructure is matched with the properties of the local fouling community. Biomimics of four mytilid species from different regional provenances were exposed at eight different sites in both northern and southern hemispheres. Tendencies of the microtopography to both inhibit and facilitate fouling were detected after 3 and 6 weeks of immersion. However, on a global scale, all microtopographies failed to prevent fouling in a consistent manner when exposed to various fouling communities and when decoupled from other shell properties. It is therefore suggested that the recently discovered chemical anti-microfouling properties of the periostracum complement the anti-macrofouling defence offered by shell microtopography.     

Formation of the prismatic layer in the freshwater bivalve Hyriopsis cumingii: the feedback of crystal growth on organic matrix

The squeezing hypothesis and the organic frameworks preformation hypothesis propose two different mechanisms to explain the interaction between organic frameworks and crystals during biomineralization of the prismatic layer of the mollusk shell. In this study, we began to study Hyriopsis cumingii shell formation and discover that this species seemed to follow the squeezing hypothesis. During the formation of the aragonite prismatic layer in the freshwater bivalve H. cumingii, we found that crystal growth was involved in controlling initiation of formation of the interprismatic organic membranes. First, newly formed crystals were embedded in the periostracum. Next, the interprismatic organic membranes of the prismatic layer were produced via squeezing between neighboring crystals. The organic matrix secreted by the mantle continuously self‐assembled into the interprismatic organic membranes as the crystals grew. In the mature stage, the interprismatic organic membranes were shaped by crystal growth. These findings provide evidence to support the squeezing hypothesis and add to the existing knowledge about interactions that occur at the organic–inorganic interfaces during mollusk shell biomineralization.