In JoVE (1)

Other Publications (27)

Articles by Vengatesen Thiyagarajan in JoVE

 JoVE Biology

Characterization of Calcification Events Using Live Optical and Electron Microscopy Techniques in a Marine Tubeworm

1Department of Biological Sciences, Clemson University, 2Department of Marine Biodiversity Research (BioDive), Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 3Advanced Material Research Laboratory (AMRL), Clemson University, 4Swire Institute of Marine Sciences and School of Biological Sciences, The University of Hong Kong

JoVE 55164

Other articles by Vengatesen Thiyagarajan on PubMed

Development of a Marine Subtidal Epibiotic Community in Hong Kong: Implications for Deployment of Artificial Reefs

Biofouling. Feb, 2003  |  Pubmed ID: 14618687

A 2-year study was conducted in Hong Kong to examine the effects of substratum, season and length of submersion on the development of a subtidal epibiotic community using four types of settlement panels (concrete, steel, wood and tyre). The season and length of submersion had a strong influence on the total biomass and on community structure while the type of substratum had very little impact on the total biomass or the structure of the epibiotic community. The season of submersion determined the species composition of the newly submerged surfaces. In the spring and summer, tubeworms were the most abundant. In the autumn and winter, barnacles and tunicates dominated. Community succession was not obvious in the first year of submersion as it was intermingled with strong seasonal settlement, growth and death of barnacles and tunicates. In the second year of submersion, green mussels and tunicates settled and grew to occupy most of the panel surfaces, forming an assemblage that was characteristic of climax communities in the local subtidal waters. The results suggest that the type of construction material has limited impact on the development of epibiotic communities on artificial reefs deployed in Hong Kong; the season of submersion may affect community structure in the early successional stage, but not the characteristics of the climax communities. This study indicates that the type of substratum should not be of concern when deploying artificial reefs in the subtidal waters in this region. The design of artificial reefs should focus more on other physical and economical aspects such as durability, flow dynamics, stability, cost, and effects on the ambient environment.

Toxic Effects of Copper on Larval Development of the Barnacle Balanus Amphitrite

Marine Pollution Bulletin. 2005  |  Pubmed ID: 16291184

Experiments were conducted to assess the effects of copper on larval development in the barnacle Balanus amphitrite. In the first experiment, we compared the sensitivity of three naupliar stages to copper stress. Molting inhibition occurred at copper concentrations ranging from 32 microg l(-1) in nauplius II to 128 g l(-1) in nauplius VI. EC50 for molting ranged from 97 microg l(-1) in nauplius II to 129 g l(-1) in nauplius VI. Decreased survival occurred at 128 microg Cu l(-1) in all of the naupliar stages tested, with LC50 ranging from 145 in nauplius II to 213 microg l(-1) in nauplius VI. In the second experiment, we examined effects of copper on the development from nauplius II to cyprid. The larvae reached cypris stage only in treatments of 16 microg Cu l(-1). Our study therefore showed that molting was a more sensitive endpoint than survival, nauplius II was the most sensitive naupliar stage, and that whole larval development assay was more sensitive than assays using a particular larval stage. The results were discussed with respect to the use of this species in toxicity tests.

Cypris Habitat Selection Facilitated by Microbial Films Influences the Vertical Distribution of Subtidal Barnacle Balanus Trigonus

Microbial Ecology. May, 2006  |  Pubmed ID: 16645929

The potential driving force(s) of the vertical distribution of subtidal barnacle Balanus trigonus Darwin were investigated using both field and laboratory experiments. Early juveniles (approximately 24 h old) placed in intertidal [approximately 0.5 m above mean low water level (MLWL)] and subtidal (approximately 3 m below MLWL) habitats survived equally well, indicating that the intertidal absence of B. trigonus in Hong Kong waters was not determined by differential mortality. However, enhanced attachment of cyprids in subtidal habitats indicated the importance of differential larval choice in determining their vertical distribution. In the laboratory, cyprids preferred to attach in response to subtidal microbial films, which may implicate microbial films as a primary cue in driving the adult vertical distribution. Microbial films developed in these two habitats differed in their biomass (= total organic carbon), abundance of bacteria and diatoms (determined by fluorescence microscopy), and bacterial diversity (determined by DNA fingerprinting analysis). For example, 6-day films in subtidal habitat had a significantly higher biomass than in films from intertidal habitat (P < 0.05). There was no difference in the biomass of films from these two habitats in 9-day films (P > 0.05); however, bacterial abundance was greater in subtidal films than in intertidal films, irrespective of the age of the film, although there was no difference in diatom abundance in films from these two habitats. Neither the abundance of bacteria and diatoms nor the biomass correlated with the attachment preferences of cyprids. This study has not provided any data to prove the existence of inductive and inhibitive (to cyprid attachment) bacterial species in subtidal and intertidal films, respectively; however, results indicate that bacterial community provided qualitative information that might explain the preferential attachment of B. trigonus cyprids in subtidal habitat.

Evaluation of Terminal-restriction Fragment Length Polymorphism Analysis in Contrasting Marine Environments

FEMS Microbiology Ecology. Jul, 2008  |  Pubmed ID: 18503550

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.

Proteomic Analysis of Larvae During Development, Attachment, and Metamorphosis in the Fouling Barnacle, Balanus Amphitrite

Proteomics. Aug, 2008  |  Pubmed ID: 18654988

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.

2D Gel-based Proteome and Phosphoproteome Analysis During Larval Metamorphosis in Two Major Marine Biofouling Invertebrates

Journal of Proteome Research. Jun, 2009  |  Pubmed ID: 19341272

Larvae of some benthic invertebrates respond (metamorphose or not) to chemical cues within minutes or hours and often without excessive transcription or translation. Although protein phosphorylation is one of the most important molecular switching mechanisms that govern variety of rapid cellular responses in higher organisms, this is the first study to analyze the global protein expression and protein phosphorylation status during larval metamorphosis in two major marine biofouling invertebrates (a bryozoan Bugula neritina and a barnacle Balanus amphitrite). Results indicate that larval proteomic response to metamorphosis (inhibiton or induction) involves substantial change in the phosphorylation status of proteins rather than de novo protein synthesis. An abundantly expressed and an unnamed phosphoprotein that appears to play key regulatory role in larval metamorphosis was identified. When larvae of bryozoan and barnacle were challenged with a metamorphosis (and kinase) inhibitor, the genistein, the number of phosphoproteins in bryozoan were substantially reduced but drastically increased in barnacle. Taken together, this is the first time that the usefulness of employing 2DE-based proteomic and phosphoproteomic approaches was demonstrated for us to understand the molecular mechanisms of larval metamorphosis and to study the mode-of-action of chemical cues in marine organisms.

Response of Bacterioplankton Community Structures to Hydrological Conditions and Anthropogenic Pollution in Contrasting Subtropical Environments

FEMS Microbiology Ecology. Sep, 2009  |  Pubmed ID: 19619230

Bacterioplankton community structures under contrasting subtropical marine environments (Hong Kong waters) were analyzed using 16S rRNA gene denaturing gradient gel electrophoresis (DGGE) and subsequent sequencing of predominant bands for samples collected bimonthly from 2004 to 2006 at five stations. Generally, bacterial abundance was significantly higher in the summer than in the winter. The general seasonal variations of the bacterial community structure, as indicated by cluster analysis of the DGGE pattern, were best correlated with temperature at most stations, except for the station close to a sewage discharge outfall, which was best explained by pollution-indicating parameters (e.g. biochemical oxygen demand). Anthropogenic pollutions appear to have affected the presence and the intensity of DGGE bands at the stations receiving discharge of primarily treated sewage. The relative abundance of major bacterial species, calculated by the relative intensity of DGGE bands after PCR amplification, also indicated the effects of hydrological or seasonal variations and sewage discharges. For the first time, a systematic molecular fingerprinting analysis of the bacterioplankton community composition was carried out along the environmental and pollution gradient in a subtropical marine environment, and it suggests that hydrological conditions and anthropogenic pollutions altered the total bacterial community as well as the dominant bacterial groups.

Proteomic Analysis During Larval Development and Metamorphosis of the Spionid Polychaete Pseudopolydora Vexillosa

Proteome Science. Dec, 2009  |  Pubmed ID: 20003395

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.

Protein Expression During the Embryonic Development of a Gastropod

Proteomics. Jul, 2010  |  Pubmed ID: 20455212

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

Journal of Proteome Research. Sep, 2010  |  Pubmed ID: 20666481

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.

CO(2)-driven Ocean Acidification Alters and Weakens Integrity of the Calcareous Tubes Produced by the Serpulid Tubeworm, Hydroides Elegans

PloS One. 2012  |  Pubmed ID: 22912726

As a consequence of anthropogenic CO(2-)driven ocean acidification (OA), coastal waters are becoming increasingly challenging for calcifiers due to reductions in saturation states of calcium carbonate (CaCO(3)) minerals. The response of calcification rate is one of the most frequently investigated symptoms of OA. However, OA may also result in poor quality calcareous products through impaired calcification processes despite there being no observed change in calcification rate. The mineralogy and ultrastructure of the calcareous products under OA conditions may be altered, resulting in changes to the mechanical properties of calcified structures. Here, the warm water biofouling tubeworm, Hydroides elegans, was reared from larva to early juvenile stage at the aragonite saturation state (Ω(A)) for the current pCO(2) level (ambient) and those predicted for the years 2050, 2100 and 2300. Composition, ultrastructure and mechanical strength of the calcareous tubes produced by those early juvenile tubeworms were examined using X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM) and nanoindentation. Juvenile tubes were composed primarily of the highly soluble CaCO(3) mineral form, aragonite. Tubes produced in seawater with aragonite saturation states near or below one had significantly higher proportions of the crystalline precursor, amorphous calcium carbonate (ACC) and the calcite/aragonite ratio dramatically increased. These alterations in tube mineralogy resulted in a holistic deterioration of the tube hardness and elasticity. Thus, in conditions where Ω(A) is near or below one, the aragonite-producing juvenile tubeworms may no longer be able to maintain the integrity of their calcification products, and may result in reduced survivorship due to the weakened tube protection.

Analysis of Pacific Oyster Larval Proteome and Its Response to High-CO2

Marine Pollution Bulletin. Oct, 2012  |  Pubmed ID: 22921897

Most calcifying organisms show depressed metabolic, growth and calcification rates as symptoms to high-CO(2) due to ocean acidification (OA) process. Analysis of the global expression pattern of proteins (proteome analysis) represents a powerful tool to examine these physiological symptoms at molecular level, but its applications are inadequate. To address this knowledge gap, 2-DE coupled with mass spectrophotometer was used to compare the global protein expression pattern of oyster larvae exposed to ambient and to high-CO(2). Exposure to OA resulted in marked reduction of global protein expression with a decrease or loss of 71 proteins (18% of the expressed proteins in control), indicating a wide-spread depression of metabolic genes expression in larvae reared under OA. This is, to our knowledge, the first proteome analysis that provides insights into the link between physiological suppression and protein down-regulation under OA in oyster larvae.

Larval and Post-larval Stages of Pacific Oyster (Crassostrea Gigas) Are Resistant to Elevated CO2

PloS One. 2013  |  Pubmed ID: 23724027

The average pH of surface oceans has decreased by 0.1 unit since industrialization and is expected to decrease by another 0.3-0.7 units before the year 2300 due to the absorption of anthropogenic CO2. This human-caused pH change is posing serious threats and challenges to the Pacific oyster (Crassostrea gigas), especially to their larval stages. Our knowledge of the effect of reduced pH on C. gigas larvae presently relies presumptively on four short-term (<4 days) survival and growth studies. Using multiple physiological measurements and life stages, the effects of long-term (40 days) exposure to pH 8.1, 7.7 and 7.4 on larval shell growth, metamorphosis, respiration and filtration rates at the time of metamorphosis, along with the juvenile shell growth and structure of the C. gigas, were examined in this study. The mean survival and growth rates were not affected by pH. The metabolic, feeding and metamorphosis rates of pediveliger larvae were similar, between pH 8.1 and 7.7. The pediveligers at pH 7.4 showed reduced weight-specific metabolic and filtration rates, yet were able to sustain a more rapid post-settlement growth rate. However, no evidence suggested that low pH treatments resulted in alterations to the shell ultrastructures (SEM images) or elemental compositions (i.e., Mg/Ca and Sr/Ca ratios). Thus, larval and post-larval forms of the C. gigas in the Yellow Sea are probably resistant to elevated CO2 and decreased near-future pH scenarios. The pre-adapted ability to resist a wide range of decreased pH may provide C. gigas with the necessary tolerance to withstand rapid pH changes over the coming century.

Polybrominated Diphenyl Ethers Do Not Affect Metamorphosis but Alter the Proteome of the Invasive Slipper Limpet Crepidula Onyx

Marine Pollution Bulletin. Aug, 2013  |  Pubmed ID: 23743271

Man-made polybrominated diphenyl ethers (PBDEs) used as flame retardants in various consumer products may be harmful to marine organisms. Larvae of some marine invertebrates, especially invasive species, can develop resistance to PBDEs through altered protein expression patterns or proteome plasticity. This is the first report of a proteomics approach to study BDE-47 induced molecular changes in the invasive limpet Crepidula onyx. Larvae of C. onyx were cultured for 5 days (hatching to metamorphosis) in the presence of BDE-47 (1 μg L(-1)). Using a 2-DE proteomics approach with triple quadrupole and high-resolution TOF-MS, we showed that BDE-47 altered the proteome structure but not the growth or metamorphosis of C. onyx larvae. We found eight significant differentially expressed proteins in response to BDE-47, deemed the protein expression signature, consisting of cytoskeletal, stress tolerance, metabolism and energy production related proteins. Our data suggest C. onyx larvae have adequate proteome plasticity to tolerate BDE-47 toxicity.

Temperature Dependent Effects of Elevated CO2 on Shell Composition and Mechanical Properties of Hydroides Elegans: Insights from a Multiple Stressor Experiment

PloS One. 2013  |  Pubmed ID: 24265732

The majority of marine benthic invertebrates protect themselves from predators by producing calcareous tubes or shells that have remarkable mechanical strength. An elevation of CO2 or a decrease in pH in the environment can reduce intracellular pH at the site of calcification and thus interfere with animal's ability to accrete CaCO3. In nature, decreased pH in combination with stressors associated with climate change may result in the animal producing severely damaged and mechanically weak tubes. This study investigated how the interaction of environmental drivers affects production of calcareous tubes by the serpulid tubeworm, Hydroides elegans. In a factorial manipulative experiment, we analyzed the effects of pH (8.1 and 7.8), salinity (34 and 27‰), and temperature (23°C and 29°C) on the biomineral composition, ultrastructure and mechanical properties of the tubes. At an elevated temperature of 29°C, the tube calcite/aragonite ratio and Mg/Ca ratio were both increased, the Sr/Ca ratio was decreased, and the amorphous CaCO3 content was reduced. Notably, at elevated temperature with decreased pH and reduced salinity, the constructed tubes had a more compact ultrastructure with enhanced hardness and elasticity compared to decreased pH at ambient temperature. Thus, elevated temperature rescued the decreased pH-induced tube impairments. This indicates that tubeworms are likely to thrive in early subtropical summer climate. In the context of climate change, tubeworms could be resilient to the projected near-future decreased pH or salinity as long as surface seawater temperature rise at least by 4°C.

Proteomic Response of Marine Invertebrate Larvae to Ocean Acidification and Hypoxia During Metamorphosis and Calcification

The Journal of Experimental Biology. Dec, 2013  |  Pubmed ID: 24307710

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.

Decreased PH Does Not Alter Metamorphosis but Compromises Juvenile Calcification of the Tube Worm Hydroides Elegans

Marine Biology. 2013  |  Pubmed ID: 24391287

Using CO2 perturbation experiments, we examined the pre- and post-settlement growth responses of a dominant biofouling tubeworm (Hydroides elegans) to a range of pH. In three different experiments, embryos were reared to, or past, metamorphosis in seawater equilibrated to CO2 values of about 480 (control), 980, 1,480, and 2,300 μatm resulting in pH values of around 8.1 (control), 7.9, 7.7, and 7.5, respectively. These three decreased pH conditions did not affect either embryo or larval development, but both larval calcification at the time of metamorphosis and early juvenile growth were adversely affected. During the 24-h settlement assay experiment, half of the metamorphosed larvae were unable to calcify tubes at pH 7.9 while almost no tubes were calcified at pH 7.7. Decreased ability to calcify at decreased pH may indicate that these calcifying tubeworms may be one of the highly threatened species in the future ocean.

Ocean Acidification Impairs Vermetid Reef Recruitment

Scientific Reports. Feb, 2014  |  Pubmed ID: 24577050

Vermetids form reefs in sub-tropical and warm-temperate waters that protect coasts from erosion, regulate sediment transport and accumulation, serve as carbon sinks and provide habitat for other species. The gastropods that form these reefs brood encapsulated larvae; they are threatened by rapid environmental changes since their ability to disperse is very limited. We used transplant experiments along a natural CO2 gradient to assess ocean acidification effects on the reef-building gastropod Dendropoma petraeum. We found that although D. petraeum were able to reproduce and brood at elevated levels of CO2, recruitment success was adversely affected. Long-term exposure to acidified conditions predicted for the year 2100 and beyond caused shell dissolution and a significant increase in shell Mg content. Unless CO2 emissions are reduced and conservation measures taken, our results suggest these reefs are in danger of extinction within this century, with significant ecological and socioeconomic ramifications for coastal systems.

Interactive Effects of Ocean Acidification, Elevated Temperature, and Reduced Salinity on Early-life Stages of the Pacific Oyster

Environmental Science & Technology. Sep, 2014  |  Pubmed ID: 25014366

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.

The Proteome of Atlantic Herring (Clupea Harengus L.) Larvae is Resistant to Elevated PCO2

Marine Pollution Bulletin. Sep, 2014  |  Pubmed ID: 25110053

Elevated anthropogenic pCO2 can delay growth and impair otolith structure and function in the larvae of some fishes. These effects may concurrently alter the larva's proteome expression pattern. To test this hypothesis, Atlantic herring larvae were exposed to ambient (370 μatm) and elevated (1800 μatm) pCO2 for one-month. The proteome structure of the larvae was examined using a 2-DE and mass spectrometry. The length of herring larvae was marginally less in the elevated pCO2 treatment compared to the control. The proteome structure was also different between the control and treatment, but only slightly: the expression of a small number of proteins was altered by a factor of less than 2-fold at elevated pCO2. This comparative proteome analysis suggests that the proteome of herring larvae is resilient to elevated pCO2. These observations suggest that herring larvae can cope with levels of CO2 projected for near future without significant proteome-wide changes.

Weakening Mechanisms of the Serpulid Tube in a High-CO2 World

Environmental Science & Technology. Dec, 2014  |  Pubmed ID: 25415324

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.

Evidence of Compositional and Ultrastructural Shifts During the Development of Calcareous Tubes in the Biofouling Tubeworm, Hydroides Elegans

Journal of Structural Biology. Mar, 2015  |  Pubmed ID: 25600412

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.

Trans-generational Responses to Low PH Depend on Parental Gender in a Calcifying Tubeworm

Scientific Reports. Jun, 2015  |  Pubmed ID: 26039184

The uptake of anthropogenic CO2 emissions by oceans has started decreasing pH and carbonate ion concentrations of seawater, a process called ocean acidification (OA). Occurring over centuries and many generations, evolutionary adaptation and epigenetic transfer will change species responses to OA over time. Trans-generational responses, via genetic selection or trans-generational phenotypic plasticity, differ depending on species and exposure time as well as differences between individuals such as gender. Males and females differ in reproductive investment and egg producing females may have less energy available for OA stress responses. By crossing eggs and sperm from the calcareous tubeworm Hydroides elegans (Haswell, 1883) raised in ambient (8.1) and low (7.8) pH environments, we observed that paternal and maternal low pH experience had opposite and additive effects on offspring. For example, when compared to offspring with both parents from ambient pH, growth rates of offspring of fathers or mothers raised in low pH were higher or lower respectively, but there was no difference when both parents were from low pH. Gender differences may result in different selection pressures for each gender. This may result in overestimates of species tolerance and missed opportunities of potentially insightful comparisons between individuals of the same species.

Ocean Acidification Through the Lens of Ecological Theory

Ecology. Jan, 2015  |  Pubmed ID: 26236884

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.

Comparative and Quantitative Proteomics Reveal the Adaptive Strategies of Oyster Larvae to Ocean Acidification

Proteomics. Dec, 2015  |  Pubmed ID: 26507238

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 (

Quantitative Analysis of Oyster Larval Proteome Provides New Insights into the Effects of Multiple Climate Change Stressors

Global Change Biology. Jun, 2016  |  Pubmed ID: 26990129

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.

Assessment of Temperature Effects on Early Larval Development Survival of Hatchery-reared Tropical Oyster, Crassostrea Iredalei

Tropical Life Sciences Research. Nov, 2016  |  Pubmed ID: 27965748

The influence of the cool and warm temperatures on early life development and survival of tropical oyster, Crassostrea iredalei was studied. D-hinged larvae (day 1 larvae) were reared to three different temperatures (20°C, 27°C, and 34°C) for nine days. Oyster larvae reared in temperature 27°C, acted as control (ambient temperature). The highest survival rate occurred when the larvae were reared in 20°C and 27°C. Larvae reared at 34°C exhibited reduced survival but increase in the growth rate. The growth rate in larvae reared in high temperature (34°C) was significantly higher compared to larvae reared in 20°C and 27°C (p<0.05). The results from the present study indicated that tropical oyster larvae, C. iredalei had faster growth rate at the high temperature (34°C). However, the larval survival was decreasing according to days. There is no significant difference in the larval growth and survival in lower temperature (20°C) and control condition (27°C).

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