Epigenetic-associated phenotypic plasticity of the ocean acidification-acclimated edible oyster in the mariculture environment.

For marine invertebrates with a pelagic-benthic life cycle, larval exposure to ocean acidification (OA) can affect adult performance in response to another environmental stressor. This carry-over effect has the potential to alter phenotypic traits. However, the molecular mechanisms that mediate "OA"-triggered carry-over effects have not been explored despite such information being key to improving species fitness and management strategies for aquafarming. This study integrated the genome-wide DNA methylome and transcriptome to examine epigenetic modification-mediated carry-over OA impacts on phenotypic traits of the ecologically and commercially important oyster species Crassostrea hongkongensis under field conditions. Larvae of C. hongkongensis were exposed to control pH 8.0 and low pH 7.4 conditions, mimicking near future OA scenario in their habitat, before being outplanted as post-metamorphic juveniles at two mariculture field sites with contrasting environmental stressors for 9 months. The larval carry-over OA effect was found to have persistent impacts on the growth and survival trade-off traits on the outplanted juveniles, although the beneficial or adverse effect depended on the environmental conditions at the outplanted sites. Site-specific plasticity was demonstrated with a diverse DNA methylation-associated gene expression profile, with signal transduction and the endocrine system being the most common and highly enriched functions. Highly methylated exons prevailed in the key genes related to general metabolic and endocytic responses and these genes are evolutionarily conserved in various marine invertebrates in response to OA. These results suggest that oysters with prior larval exposure history to OA had the ability to trigger rapid local adaptive responses via epigenetic modification to cope with multiple stressors in the field.

Quantitative analysis of oyster larval proteome provides new insights into the effects of multiple climate change stressors.

The metamorphosis of planktonic larvae of the Pacific oyster (Crassostrea gigas) underpins their complex life-history strategy by switching on the molecular machinery required for sessile life and building calcite shells. Metamorphosis becomes a survival bottleneck, which will be pressured by different anthropogenically induced climate change-related variables. Therefore, it is important to understand how metamorphosing larvae interact with emerging climate change stressors. To predict how larvae might be affected in a future ocean, we examined changes in the proteome of metamorphosing larvae under multiple stressors: decreased pH (pH 7.4), increased temperature (30 °C), and reduced salinity (15 psu). Quantitative protein expression profiling using iTRAQ-LC-MS/MS identified more than 1300 proteins. Decreased pH had a negative effect on metamorphosis by down-regulating several proteins involved in energy production, metabolism, and protein synthesis. However, warming switched on these down-regulated pathways at pH 7.4. Under multiple stressors, cell signaling, energy production, growth, and developmental pathways were up-regulated, although metamorphosis was still reduced. Despite the lack of lethal effects, significant physiological responses to both individual and interacting climate change related stressors were observed at proteome level. The metamorphosing larvae of the C. gigas population in the Yellow Sea appear to have adequate phenotypic plasticity at the proteome level to survive in future coastal oceans, but with developmental and physiological costs.

Comparative and quantitative proteomics reveal the adaptive strategies of oyster larvae to ocean acidification.

Decreasing pH due to anthropogenic CO2 inputs, called ocean acidification (OA), can make coastal environments unfavorable for oysters. This is a serious socioeconomical issue for China which supplies >70% of the world's edible oysters. Here, we present an iTRAQ-based protein profiling approach for the detection and quantification of proteome changes under OA in the early life stage of a commercially important oyster, Crassostrea hongkongensis. Availability of complete genome sequence for the pacific oyster (Crassostrea gigas) enabled us to confidently quantify over 1500 proteins in larval oysters. Over 7% of the proteome was altered in response to OA at pHNBS 7.6. Analysis of differentially expressed proteins and their associated functional pathways showed an upregulation of proteins involved in calcification, metabolic processes, and oxidative stress, each of which may be important in physiological adaptation of this species to OA. The downregulation of cytoskeletal and signal transduction proteins, on the other hand, might have impaired cellular dynamics and organelle development under OA. However, there were no significant detrimental effects in developmental processes such as metamorphic success. Implications of the differentially expressed proteins and metabolic pathways in the development of OA resistance in oyster larvae are discussed. The MS proteomics data have been deposited to the ProteomeXchange with identifiers PXD002138 (http://proteomecentral.proteomexchange.org/dataset/PXD002138).

Evidence of compositional and ultrastructural shifts during the development of calcareous tubes in the biofouling tubeworm, Hydroides elegans.

The serpulid tubeworm, Hydroides elegans, is an ecologically and economically important species whose biology has been fairly well studied, especially in the context of larval development and settlement on man-made objects (biofouling). Nevertheless, ontogenetic changes associated with calcareous tube composition and structures have not yet been studied. Here, the ultrastructure and composition of the calcareous tubes built by H. elegans was examined in the three early calcifying juvenile stages and in the adult using XRD, FTIR, ICP-OES, SEM and Raman spectroscopy. Ontogenetic shifts in carbonate mineralogy were observed, for example, juvenile tubes contained more amorphous calcium carbonate and were predominantly aragonitic whereas adult tubes were bimineralic with considerably more calcite. The mineral composition gradually shifted during the tube development as shown by a decrease in Sr/Ca and an increase of Mg/Ca ratios with the tubeworm's age. The inner tube layer contained calcite, whereas the outer layer contained aragonite. Similarly, the tube complexity in terms of ultrastructure was associated with development. The sequential appearance of unoriented ultrastructures followed by oriented ultrastructures may reflect the evolutionary history of serpulid tube biominerals. As aragonitic structures are more susceptible to dissolution under ocean acidification (OA) conditions but are more difficult to be removed by anti-fouling treatments, the early developmental stages of the tubeworms may be vulnerable to OA but act as the important target for biofouling control.

Ocean acidification through the lens of ecological theory.

Ocean acidification, chemical changes to the carbonate system of seawater, is emerging as a key environmental challenge accompanying global warming and other human-induced perturbations. Considerable research seeks to define the scope and character of potential outcomes from this phenomenon, but a crucial impediment persists. Ecological theory, despite its power and utility, has been only peripherally applied to the problem. Here we sketch in broad strokes several areas where fundamental principles of ecology have the capacity to generate insight into ocean acidification's consequences. We focus on conceptual models that, when considered in the context of acidification, yield explicit predictions regarding a spectrum of population- and community-level effects, from narrowing of species ranges and shifts in patterns of demographic connectivity, to modified consumer-resource relationships, to ascendance of weedy taxa and loss of species diversity. Although our coverage represents only a small fraction of the breadth of possible insights achievable from the application of theory, our hope is that this initial foray will spur expanded efforts to blend experiments with theoretical approaches. The result promises to be a deeper and more nuanced understanding of ocean acidification'and the ecological changes it portends.

Interactive effects of ocean acidification, elevated temperature, and reduced salinity on early-life stages of the pacific oyster.

Ocean acidification (OA) effects on larvae are partially attributed for the rapidly declining oyster production in the Pacific Northwest region of the United States. This OA effect is a serious concern in SE Asia, which produces >80% of the world's oysters. Because climate-related stressors rarely act alone, we need to consider OA effects on oysters in combination with warming and reduced salinity. Here, the interactive effects of these three climate-related stressors on the larval growth of the Pacific oyster, Crassostrea gigas, were examined. Larvae were cultured in combinations of temperature (24 and 30 °C), pH (8.1 and 7.4), and salinity (15 psu and 25 psu) for 58 days to the early juvenile stage. Decreased pH (pH 7.4), elevated temperature (30 °C), and reduced salinity (15 psu) significantly delayed pre- and post-settlement growth. Elevated temperature lowered the larval lipid index, a proxy for physiological quality, and negated the negative effects of decreased pH on attachment and metamorphosis only in a salinity of 25 psu. The negative effects of multiple stressors on larval metamorphosis were not due to reduced size or depleted lipid reserves at the time of metamorphosis. Our results supported the hypothesis that the C. gigas larvae are vulnerable to the interactions of OA with reduced salinity and warming in Yellow Sea coastal waters now and in the future.

Weakening mechanisms of the serpulid tube in a high-CO2 world.

Many benthic marine organisms produce calcium carbonate (CaCO3) structures for mechanical protection through a biologically controlled calcification process. However, the oceans are becoming unfavorable for calcification because of the stress associated with ocean acidification (OA) and associated chemical changes such as declining saturation state of CaCO3 and decreasing seawater pH. This work studies the impacts of OA-driven decreased pH on the calcareous tubes produced by the serpulid tubeworm Hydroides elegans. Tubes grown under control and OA experimental conditions were measured for structural and mechanical properties, and their mechanical properties were further interpreted using finite element analysis (FEA). The near-future predicted pH value of 7.8 altered tube ultrastructure, volume, and density and decreased the mean tube hardness and elasticity by ∼ 80 and ∼ 70%, respectively. The crushing force required for breaking the tube was reduced by 64%. The FEA results demonstrated how a simulated predator attack may affect the structure with different structural and mechanical properties and consequently shift the stress development and distribution in the tubes, causing a more concentrated stress distribution and therefore leading to a lower ability to withstand attacks.

Proteomic response of marine invertebrate larvae to ocean acidification and hypoxia during metamorphosis and calcification.

Calcifying marine invertebrates with complex life cycles are particularly at risk to climate changes as they undergo an abrupt ontogenetic shift during larval metamorphosis. Although our understanding of the larval response to climate changes is rapidly advancing, the proteome plasticity involved in a compensatory response to climate change is still unknown. In this study, we investigated the proteomic response of metamorphosing larvae of the tubeworm Hydroides elegans, challenged with two climate change stressors, ocean acidification (OA; pH 7.6) and hypoxia (HYP; 2.8 mg O2 l(-1)), and with both combined. Using a two-dimensional gel electrophoresis (2-DE)-based approach coupled with mass spectrometry, we found that climate change stressors did not affect metamorphosis except under OA, but altered the larval proteome and phosphorylation status. Metabolism and various stress and calcification-related proteins were downregulated in response to OA. In OA and HYP combined, HYP restored the expression of the calcification-related proteins to the control levels. We speculate that mild HYP stress could compensate for the negative effects of OA. This study also discusses the potential functions of selected proteins that might play important roles in larval acclimation and adaption to climate change.

Wild oyster population resistance to ocean acidification adversely affected by bacterial infection.

The carbon dioxide induced ocean acidification (OA) process is well known to have profound effects on physiology, survival and immune responses in marine organisms, and particularly calcifiers including edible oysters. At the same time, some wild populations could develop a complex and sophisticated immune system to cope with multiple biotic and abiotic stresses, such as bacterial infections and OA, over the long period of coevolution with the environment. However, it is unclear how immunological responses and the underlying mechanisms are altered under the combined effect of OA and bacterial infection, especially in the ecologically and economically important edible oysters. Here, we collected the wild population of oyster species Crassostrea hongkongensis (the Hong Kong oyster) from their native estuarine area and carried out a bacterial challenge with the worldwide pervasive pathogen of human foodborne disease, Vibrio parahaemolyticus, to investigate the host immune responses and molecular mechanisms under the high-CO2 and low pH-driven OA conditions. The wild population had a high immune resistance to OA, but the resistance is compromised under the combined effect of OA and bacterial infection both in vivo or in vitro. We classified all transcriptomic genes based on expression profiles and functional pathways and identified the specifically switched on and off genes and pathways under combined effect. These genes and pathways were mainly involved in multiple immunological processes including pathogen recognition, immune signal transduction and effectors. This work would help understand how the immunological function and mechanism response to bacterial infection in wild populations and predict the dynamic distribution of human health-related pathogens to reduce the risk of foodborne disease under the future climate change scenario.

Ocean acidification drives gut microbiome changes linked to species-specific immune defence.

Ocean acidification (OA) has important effects on the intrinsic phenotypic characteristics of many marine organisms. Concomitantly, OA can alter the extended phenotypes of these organisms by perturbing the structure and function of their associated microbiomes. It is unclear, however, the extent to which interactions between these levels of phenotypic change can modulate the capacity for resilience to OA. Here, we explored this theoretical framework assessing the influence of OA on intrinsic (immunological responses and energy reserve) and extrinsic (gut microbiome) phenotypic characteristics and the survival of important calcifiers, the edible oysters Crassostrea angulata and C. hongkongensis. After one-month exposure to experimental OA (pH 7.4) and control (pH 8.0) conditions, we found species-specific responses characterised by elevated stress (hemocyte apoptosis) and decreased survival in the coastal species (C. angulata) compared with the estuarine species (C. hongkongensis). Phagocytosis of hemocytes was not affected by OA but in vitro bacterial clearance capability decreased in both species. Gut microbial diversity decreased in C. angulata but not in C. hongkongensis. Overall, C. hongkongensis was capable of maintaining the homeostasis of the immune system and energy supply under OA. In contrast, C. angulata's immune function was suppressed, and the energy reserve was imbalanced, which might be attributed to the declined microbial diversity and the functional loss of essential bacteria in the guts. This study highlights a species-specific response to OA determined by genetic background and local adaptation, shedding light on the understanding of host-microbiota-environment interactions in future coastal acidification.

Transgenerational responses to seawater pH in the edible oyster, with implications for the mariculture of the species under future ocean acidification.

The majority of common edible oysters are projected to grow more slowly and have smaller impaired shells because of anthropogenic CO2-induced reductions in seawater carbonate ion concentration and pH, a process called ocean acidification (OA). Recent evidence has shown that OA has carryover effects, for example, larvae exposed to OA will also exhibit either positive or negative effects after metamorphosis. This study examined the hidden carryover effects of OA exposure during parental and larval stages on post-metamorphic traits of the commercially important oyster species Crassostrea hongkongensis. Adults of C. hongkongensis were exposed to control pH (pHNBS 8.0) and OA-induced low pH (pHNBS 7.4) conditions. Their larval offspring were then exposed to the same aquarium conditions before being out-planted as post-metamorphic juveniles at a mariculture site for 10 months. Initially, larval offspring were resilient to low pH with or without parental exposure. The larvae exposed to low pH had significantly faster development and higher percentage of settlement success compared to control groups. The out-planted juveniles with parental exposure had improved survival and growth compared to juveniles without parental exposure, regardless of the larval exposure history. This implies that transgenerational effects due to parental exposure not only persists but also have a greater influence than the within-generational effects of larval exposure. Our results shed light on the importance of linking the various life history stages when assessing the OA-induced carryover capacity of C. hongkongensis in the natural environment. Understanding these linked relationships helps us better predict the species rapid adaptation responses in the face of changing coastal conditions due to OA.

DNA methylation changes in response to ocean acidification at the time of larval metamorphosis in the edible oyster, Crassostrea hongkongensis.

Unprecedented rate of increased CO2 level in the ocean and the subsequent changes in carbonate system including decreased pH, known as ocean acidification (OA), is predicted to disrupt not only the calcification process but also several other physiological and developmental processes in a variety of marine organisms, including edible oysters. Nonetheless, not all species are vulnerable to those OA threats, e.g. some species may be able to cope with OA stress using environmentally induced modifications on gene and protein expressions. For example, external environmental stressors including OA can influence the addition and removal of methyl groups through epigenetic modification (e.g. DNA methylation) process to turn gene expression "on or off" as part of a rapid adaptive mechanism to cope with OA. In this study, we tested the above hypothesis through testing the effect of OA, using decreased pH 7.4 as proxy, on DNA methylation pattern of an endemic and a commercially important estuary oyster species, Crassostrea hongkongensis at the time of larval habitat selection and metamorphosis. Larval growth rate did not differ between control pH 8.1 and treatment pH 7.4. The metamorphosis rate of the pediveliger larvae was higher at pH 7.4 than those in control pH 8.1, however over one-third of the larvae raised at pH 7.4 failed to attach on optimal substrate as defined by biofilm presence. During larval development, a total of 130 genes were differentially methylated across the two treatments. The differential methylation in the larval genes may have partially accounted for the higher metamorphosis success rate under decreased pH 7.4 but with poor substratum selection ability. Differentially methylated loci were concentrated in the exon regions and appear to be associated with cytoskeletal and signal transduction, oxidative stress, metabolic processes, and larval metamorphosis, which implies the high potential of C. hongkongensis larvae to acclimate and adapt through non-genetic ways to OA threats within a single generation.

DNA methylation changes in response to ocean acidification at the time of larval metamorphosis in the edible oyster, Crassostrea hongkongensis.

Unprecedented rate of increased CO2 level in the ocean and the subsequent changes in carbonate system including decreased pH, known as ocean acidification (OA), is predicted to disrupt not only the calcification process but also several other physiological and developmental processes in a variety of marine organisms, including edible oysters. Nonetheless, not all species are vulnerable to those OA threats, e.g. some species may be able to cope with OA stress using environmentally induced modifications on gene and protein expressions. For example, external environmental stressors including OA can influence the addition and removal of methyl groups through epigenetic modification (e.g. DNA methylation) process to turn gene expression "on or off" as part of a rapid adaptive mechanism to cope with OA. In this study, we tested the above hypothesis through testing the effect of OA, using decreased pH 7.4 as proxy, on DNA methylation pattern of an endemic and a commercially important estuary oyster species, Crassostrea hongkongensis at the time of larval habitat selection and metamorphosis. Larval growth rate did not differ between control pH 8.1 and treatment pH 7.4. The metamorphosis rate of the pediveliger larvae was higher at pH 7.4 than those in control pH 8.1, however over one-third of the larvae raised at pH 7.4 failed to attach on optimal substrate as defined by biofilm presence. During larval development, a total of 130 genes were differentially methylated across the two treatments. The differential methylation in the larval genes may have partially accounted for the higher metamorphosis success rate under decreased pH 7.4 but with poor substratum selection ability. Differentially methylated loci were concentrated in the exon regions and appear to be associated with cytoskeletal and signal transduction, oxidative stress, metabolic processes, and larval metamorphosis, which implies the high potential of C. hongkongensis larvae to acclimate and adapt through non-genetic ways to OA threats within a single generation.

Protein expression during the embryonic development of a gastropod.

Despite the potential use of gastropod embryos in basic and applied research, little is known about their protein expression. We examined, for the first time, changes in proteomic profile during embryonic development of Pomacea canaliculata from an embryo without a shell (stage II) to an embryo with a fully formed shell (stage III) to understand the roles that proteins play in critical developmental events, such as the formation of shell, operculum and heart, and the differentiation of head and foot. To analyze protein expression during development, we used 2-DE to detect, MS to analyze, and de novo peptide sequencing followed by MS-BLAST to identify the proteins. The de novo cross-species protein identification method was adopted because of a lack of genomic and proteomic data in the whole class of Gastropoda. 2-DE detected approximately 700 protein spots. Among the 125 spots that were abundant, 52% were identified, a marked improvement over the conventional direct MS-BLAST method. These proteins function in perivitelline fluid utilization, shell formation, protein synthesis and folding, and cell cycle and cell fate determination, providing evidence to support that this embryonic period is a period of dynamic protein synthesis and metabolism. The data shall provide a basis for further studies of how gastropod embryos respond to natural and human-induced changes in the environment.

2D gel-based multiplexed proteomic analysis during larval development and metamorphosis of the biofouling polychaete tubeworm Hydroides elegans.

Larval settlement and metamorphosis of a common biofouling polychaete worm, Hydroides elegans, involve remarkable structural and physiological changes during this pelagic to sessile habitat shift. The endogenous protein molecules and post-translational modifications that drive this larval transition process are not only of interest to ecologists but also to the antifouling paint industry, which aims to control the settlement of this biofouling species on man-made structures (e.g., ship hulls). On the basis of our recent proteomic studies, we hypothesize that rapid larval settlement of H. elegans could be mediated through changes in phosphorylation status of proteins rather than extensive de novo synthesis of proteins. To test this hypothesis, 2D gel-based multiplexed proteomics technology was used to monitor the changes in protein expression and phosphorylation status during larval development and metamorphosis of H. elegans. The protein expression profiles of larvae before and after they reached competency to attach and metamorphose were similar in terms of major proteins, but the percentage of phosphorylated proteins increased from 41% to 49% after competency. Notably, both the protein and phosphoprotein profiles of the metamorphosed individuals (adult) were distinctly different from that of the larvae, with only 40% of the proteins phosphorylated in the adult stage. The intensity ratio of all phosphoprotein spots to all total protein spots was also the highest in the competent larval stage. Overall, our results indicated that the level of protein phosphorylation might play a crucial role in the initiation of larval settlement and metamorphosis.

Autophagy Dually Induced by AMP Surplus and Oxidative Stress Enhances Hemocyte Survival and Bactericidal Capacity via AMPK Pathway in Crassostrea hongkongensis.

Crassostrea hongkongensis (Hong Kong oyster) is an ecologically and economically valuable shellfish endemic to South/Southeast Asia. Due to ocean acidification and warming waters, they have become increasingly vulnerable to invading microbes including Vibrio parahaemolyticus, a significant foodborne human pathogen. In recent years, outbreaks of V. parahaemolyticus have emerged as a perennial phenomenon in parts of the world, necessitating to better understand the biology of host-pathogen interactions in this under-examined marine invertebrate. Although an immunologically relevant autophagy apparatus has been identified in Crassostrea gigas, an evolutionarily close mollusk cousin, the precise mechanistic details of C. hongkongensis autophagy during V. parahaemolyticus infection are still wanting. Here, we compellingly demonstrated that in vivo V. parahaemolyticus challenge robustly triggered autophagic signaling in C. hongkongensis hemocytes peaking at 6 h post-infection, which subsequently promoted bacterial clearance and dampened premature apoptosis. Simultaneously, a large surplus of adenosine monophosphate (AMP) and elevations in reactive oxygen species (ROS, specifically mitochondrial O2 - and cellular H2O2) formation were observed post-infection. Extrinsically applied AMP and ROS could synergistically induce AMP-activated protein kinase (AMPK) phosphorylation to stimulate downstream autophagic events. V. parahaemolyticus infection-induced autophagy was pharmacologically shown to be AMPK-dependent in vivo. Overall, our results establish autophagy as a crucial arm of host defense against Vibrio infections in mollusks, and provide new insights into the underappreciated roles of ROS and AMP as co-regulators of autophagy.

Proteomic analysis during larval development and metamorphosis of the spionid polychaete Pseudopolydora vexillosa.

BACKGROUND: While the larval-juvenile transition (metamorphosis) in the spionid polychaete Pseudopolydora vexillosa involves gradual morphological changes and does not require substantial development of juvenile organs, the opposite occurs in the barnacle Balanus amphitrite. We hypothesized that the proteome changes during metamorphosis in the spionids are less drastic than that in the barnacles. To test this, proteomes of pre-competent larvae, competent larvae (ready to metamorphose), and juveniles of P. vexillosa were compared using 2-dimensional gel electrophoresis (2-DE), and they were then compared to those of the barnacle. RESULTS: Unlike the significant changes found during barnacle metamorphosis, proteomes of competent P. vexillosa larvae were more similar to those of their juveniles. Pre-competent larvae had significantly fewer protein spots (384 spots), while both competent larvae and juveniles expressed about 660 protein spots each. Proteins up-regulated during competence identified by MALDI-TOF/TOF analysis included a molecular chaperon (calreticulin), a signal transduction regulator (tyrosin activation protein), and a tissue-remodeling enzyme (metallopeptidase). CONCLUSIONS: This was the first time to study the protein expression patterns during the metamorphosis of a marine polychaete and to compare the proteomes of marine invertebrates that have different levels of morphological changes during metamorphosis. The findings provide promising initial steps towards the development of a proteome database for marine invertebrate metamorphosis, thus deciphering the possible mechanisms underlying larval metamorphosis in non-model marine organisms.

Proteomic analysis of larvae during development, attachment, and metamorphosis in the fouling barnacle, Balanus amphitrite.

The barnacle, Balanus amphitrite, is one of the primary model organisms for rocky-shore ecology studies and biofouling research. This barnacle species has a complex life cycle during which the swimming nauplius molts six times and transforms into a cyprid stage. Cyprids must attach to a surface to metamorphose into a juvenile barnacle. To clarify the overall profile of protein expression during larval development and metamorphosis, 2-DE was used to compare the proteome of the nauplius, the swimming cyprid, the attached cyprid, and the metamorphosed cyprid. The proteome of the swimming cyprid was distinctly different from that of other life stages and had about 400 spots. The proteomes of the attached and metamorphosed cyprids were similar with respect to major proteins but had significantly lower numbers of spots compared to that of swimming larval stages. Obviously, synthesis of most proteins from swimming cyprids was switched off after attachment and metamorphosis. Our advanced MS analysis (MALDI-TOF/TOF MS/MS) allowed us to identify the proteins that were differentially and abundantly expressed in the swimming cyprid. These proteins included signal transduction proteins (adenylate cyclase and calmodulin) and juvenile hormone binding proteins. In summary, for the first time, we have analyzed the global protein expression pattern of fouling marine invertebrate larvae during metamorphosis. Our study provides new insights into the mechanisms of barnacle larval metamorphosis and also provides a foundation for exploring novel targets for antifouling treatments.

Evaluation of terminal-restriction fragment length polymorphism analysis in contrasting marine environments.

Terminal-restriction fragment length polymorphism (T-RFLP) analysis is widely used in microbial ecology studies. In the present study, T-RFLP analysis of PCR products digested by five restriction enzymes (AluI, HaeIII, MspI, Sau3AI and TaqI) was applied for 20 samples from three contrasting coastal environments to assess the biases associated with the choice of enzyme digestion and T-RF analysis. The five enzyme digestions produced highly variable species richness (in terms of number of T-RFs). Analysis of peak areas with a threshold of 0.5% of the total peak area, which recovered 92-96% of the total peak area, revealed different diversity indexes from the five enzyme digestions. Multidimensional scaling, based on matrices that were generated by scoring peak presence/absence and area, revealed similar bacterial community structure patterns among the 20 samples, regardless of the choice of restriction enzymes. Our results strongly argue that the choice of different digestion enzymes in the T-RFLP technique generated valid and consistent bacterial community structures but highly variable species richness and diversity indices. The biases associated with the choice of digestion enzymes needs to be evaluated carefully or at least to be addressed when using T-RFLP analysis.

Differential sensitivity of larvae to ocean acidification in two interacting mollusc species.

Anthropogenically-induced ocean acidification (OA) scenarios of decreased pH and altered carbonate chemistry are threatening the fitness of coastal species and hence near-shore ecosystems' biodiversity. Differential tolerances to OA between species at different trophic levels, for example, may alter species interactions and impact community stability. Here we evaluate the effect of OA on the larval stages of the rock oyster, Saccostrea cucullata, a dominant Indo-Pacific ecosystem engineer, and its key predator, the whelk, Reishia clavigera. pH as low as 7.4 had no significant effect on mortality, abnormality or growth of oyster larvae, whereas whelk larvae exposed to pH 7.4 experienced increased mortality (up to ∼30%), abnormalities (up to 60%) and ∼3 times higher metabolic rates compared to controls. Although these impacts' long-term consequences are yet to be investigated, greater vulnerability of whelk larvae to OA could impact predation rates on intertidal rocky shores, and have implications for subsequent community dynamics.