Source: CLEMSON UNIVERSITY submitted to NRP
OYSTER BIOLOGY AND REAL TIME MICROSCOPY FOR MARINE BIOFOULING MANAGEMENT
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
0233320
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Jan 1, 2013
Project End Date
Sep 30, 2017
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
CLEMSON UNIVERSITY
(N/A)
CLEMSON,SC 29634
Performing Department
Biological Sciences
Non Technical Summary
Our mission at the Okeanos Research Laboratory is to understand biofouling and marine cellular biomineralization by advancing scientific knowledge of marine invertebrate systems at a cellular level. We accomplish this mission by using advanced optical and electron imaging techniques such as multi-photon microscopy . Over the next four years , we will further knowledge of marine invertebrate settling mechanisms and apply these results to the innovation of Clemsons patent pending anti- fouling deterrent technologies, in which the organisms own endocrine molecules when covalently bound to a polymeric coating act to deter settlement. In marine cellular biomineralization, we will continue our research on the cellular basis of molluscan bivalve shell formation, a field that was pioneered with Dr. Mounts 2004 publication in Science. In prior years, we reported that the oysters own blood cells or leukocytes nucleate and deliver calcium carbonate crystals to form the mineralized component of shell. In 2012, Dr. Mount was a co-author on the Nature article which reported on the first ever genome of the Pacific Oyster. The oyster genomic evidence further establishes the genetic mechanism for molluscan cellular biomineralization. The challenge now is to better understand the mechanistic basis of how intracellular nucleation occurs to form nanoscale crystals within living cells. With regard to marine biofouling, the negative impacts on marine life from the accumulation of heavy metals such as copper and tri-butyl-tins (TBT) in estuarine sediments (including Charleston Harbor, South Carolina) from metal-based antifouling paints on maritime vessels are well documented and have led to a complete worldwide ban of TBT based antifouling paints. To help address this deadly problem, we are developing the next generation of marine anti-fouling paints that are safe, non-toxic and environmentally benign. To further our research on cellular biomineralization, an important aspect of marine biofouling, we will fully characterize the intracellular mineralization compartment from where molluscan cellular biomineralization ensues. The knowledge gained will advance the science of marine cellular biomineralization and will enable future innovations in safe and effective and aquatic marine anti-fouling coatings and further scientific understanding from the growing threat of ocean acidification.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
13508111030100%
Knowledge Area
135 - Aquatic and Terrestrial Wildlife;

Subject Of Investigation
0811 - Shellfish;

Field Of Science
1030 - Cellular biology;
Goals / Objectives
OBJ-1.1- Perform comparative studies on the conservation of adrenergic receptors across diverse phyla and further characterization of oyster larval foot. OUT-1.1- FISH experiments will add to our prior results. OTC-1.1 -Comparative studies will improve fouling deterrence in tropical waters. Unification of signal transduction and neural pathways will provide valuable insight into the mechanisms of settlement by marine invertebrates. OBJ-1.2-Develop and evaluate synthetic analogs of noradrenaline and Sy-NA-polymer systems. OUT-1.2- Synthetic analogs of NA will have similar effects on cells and larvae as NA. Synthetics will have longer lasting bioactivity. OTC-1.2-The development of NA synthetic analogs will enable large scale manufacture of fouling deterrent coatings. OBJ-1.3- Characterize the toxicity of soluble NA, Sy-NA and Sy-NA-X-polymer leachate. OUT-1.3- NMR and mass spectrometry will identify chemical species released and their degradation products. OTC-1.3- Toxicity results will help further refine the design of synthetic NA. OBJ-2.1 Determine the cellular origin of lipids that make up the PMC at the mineralization front. OUT-2.1-Optical imaging of mineralizing living cells will confirm our prior SEM results. OTC-2.1- The determination that the oysters intracellular biomineralization compartment is vesicular will significantly advance the scientific understanding of biomineralization in the Mollusca. OBJ-2.2- Obtain and compare lattice orientation by TEM of prismatic calcite to that of foliated calcite plates. OUT- 2.2- The diffraction patterns will reveal crystalline structure and the a,b,c axes of orientation relative to the surface that the crystal was adhered to. OTC-2.2 -These results will help evaluate the "building block hypothesis" of shell layer formation. OBJ-2.3 -Ascertain if the crystallization of calcium carbonate proceeds through an amorphous intermediate phase within the refractive granulocytes of oysters OUT-2.3-Electron diffraction data and images of the hydrated cells will be collected OTC-2.3- the case of transient ACC, no diffraction pattern is expected whereas the oriented calcite will generate a pattern.
Project Methods
OBJ-1.1- FISH probes will be synthesized from consensus of barnacle and oyster adrenergic sequences. Once constructed and validated, these probes will be used to screen the bryozoans and polychaete tube worms. The oyster larval foot will be studied using high resolution electron and optical microscopes. Techniques such as immunofluorescence as well as immunogold labeling will be employed to identify key structures and signal transduction pathways from different types of cilia present on the pediveliger foot. OBJ-1.2- We will develop highly bioactive and persistent NA analogs that can be covalently linked and deployed as part of a polymeric coating system. OBJ-1.3-NA and Sy-NA analogs will be tested to determine both the effective and lethal concentrations using EPA approved bioassays and organisms. OBJ-2.1- We will analyze mineralizing refractive cells and mineralized implants by MPM. OBJ-2.2- Using a combination of SEM and high resolution TEM , we will obtain the electron diffraction patterns of the individual calcite plates. OBJ-2.3-We will observe the cellular samples by TEM to collect images and diffraction patterns. These data will be subsequently analyzed to ascertain the presence of either or possibly both transient ACC and oriented calcite phases in the refractive granulocytes.

Progress 01/01/13 to 09/30/17

Outputs
Target Audience:1. Marine biofouling: our ultimate target audience is any person or industry who has to eliminate the unwanted growth of aquatic and marine fouling organism on ships, water intake structures and nets and associated aquaculture gear. 2. Marine cellular biomineralization: The target audience is individuals/agencies/governments who need a better understanding of the potential biological impact of global climate change driven ocean acidification on marine food security and marine ecosystems. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Vera Bin San Chan, Postdoctoral scholar, Hong Kong University, Feb 2015- Feb 2018, continuing How have the results been disseminated to communities of interest?Chair, Special session on Biofouling, National Shellfisheries Association, Las Vegas, Nevada, February 22-26th, 2016 A.S. Mount and Vera Bin San Chan. The growing complexity of barnacle cyprid glue. National Shellfisheries Association, Las Vegas, Nevada, February 22-26th, 2016 A.S. Mount and Vera Bin San Chan. Oyster as a model of Cellular Biomineralization and its Implications for Ocean Acidification Research, Invited Speaker 2nd ISOACC Meeting, Hong Kong University, Dec 1-11th 2016, Hong Kong, SAR, China. Host, 20th anniversary of the Southeast Ultrafast Conference (SEUFC), Strom Thurmond Institute at Clemson University, Clemson, SC , January 18-19, 2017. A.S. Mount, Oyster shell formation and its implications for cellular biomineralization, 20th Southeast Ultrafast Conference, Clemson University, Clemson SC, January 18-19, 2017. Two papers presented at the International conference on Biomineralization 14, Tuksuka, Japan , October 2017 What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? OBJ-1.1- Perform comparative studies on the conservation of adrenergic receptors across diverse phyla and further characterization of the oyster larval foot. -UPDATE: In partnership with NEN-Biomedical we expressed our own unique chitin binding molecular probe which we conjugated to a far red fluorophore (red-CBP). This probe enabled us to detect the presence of chitin in the permanent adhesive plaque of attached cyprid larvae and in oyster hemocytes. The results of this research have been communicated in talks and posters to several federal agencies. Two oyster chitin papers are in preparation for publication in 2018. In collaboration with Nick Aldred at the University of Newcastle, we have discoveredchitin in the cyprid cement gland and as a component in its permanent adhesive. Research continues and a publication is planned. OBJ-1.2-Develop and evaluate synthetic analogs of noradrenaline and Sy-NA-polymer systems.UPDATE: We have synthesized and tested a novel antifouling peptide based on a conotoxin which interacts with the neuroendocrine transporter (NET). This peptide defeats larval biofouling in solution and is resistant to marine degradation. We engineered a new NET transporter inhibitorconopeptide system (Miata57) which had a glycine linker region and an azide group on the amino terminus. In July 2017, an NSF phase1 STTR grant was awarded to our commercialpartner, EngineeredMarine Coatings Inc. After six months of research, we now have a proof of concept miata57 based antifouling coating that will be scaled up as a commercial product, should an NSF phase 2 STTR be awarded. OBJ-2.1 Determine the cellular origin of lipids that make up the PMC at the mineralization front. UPDATE: We published an exosome based model of cellular mineralization in 2015. We are now exploring the role of chitin in PMC formation as noted above. OBJ-2.2- Obtain and compare lattice orientation by TEM of prismatic calcite to that of foliated calcite plates. Update: In progress. We are still developing TEM protocols to satisfy this objective. OBJ-2.3 Ascertain if the crystallization of calcium carbonate proceeds through an amorphous intermediate phase within the refractive granulocytes of oysters. A review article is currently in preparation and will be published in 2018. Update: In progress. We have completed the construction of a brood stock and a larval oyster holding facility. This facility will provide early stage oyster larvae that will be studied for carbonate uptake related to obj 2-2.

Publications

  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Price, Heather L; Gohad, Neeraj V; Mount, Andrew S; Wendt, Dean E; 2017. Investigation of larval settlement pathways in the marine bryozoan, Bugula neritina. Journal of Experimental Marine Biology and Ecology 486:69-76
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Essock-Burns, T. E. -, N. Gohad, B. O. Diaz, A. S. Mount, C. Spillmann, K. Wahl, D. Rittschof. 2017. Barnacle biology before, during and after settlement and metamorphosis: a study of the interface. Submitted to Journal of Experimental Biology. 220(2):194-207
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Chan, V.B.S., T. Toyofuku, G. Wetzel, L. Saraf, V. Thiaagarajan, A.S. Mount. 2017. Characterization of calcification events using live optical and electron microscopy techniques in the marine tubeworm. Journal of Visualized Experiments, 120 ,e55164-e55164


Progress 10/01/15 to 09/30/16

Outputs
Target Audience: Marine biofouling: our ultimate target audience is any person or industry who has to eliminate the unwanted growth of aquatic and marine fouling organism on ships, water intake structures and nets and associated aquaculture gear. Marine cellular biomineralization: The target audience is individuals/agencies/governments who need a better understanding of the potential biological impact of global climate change driven ocean acidification on marine food security and marine ecosystems. Changes/Problems:Our Coherent Chamelon VIS S laser failed in early 2015 and was replaced with a new unit in December 2015. The complete multiphoton system was brought online in April 2015 after an open pat refit. In November, 2016, Wolfgang Becker of Becker & Hickl successfully installed a two detector fluorescent lifetime imaging system as a demonstration project on the system. What opportunities for training and professional development has the project provided?1.Vera Bin San Chan, Postdoctoral scholar, Hong Kong University, Feb 2015- Feb 2017, continuing. 2. Zoe Rodgers, Naval Research Laboratory, August 2016 3.Andrew McQuiston, Research Technician Oct 215-May 2016 How have the results been disseminated to communities of interest?1.Wikfors, G, A.S. Mount. A. Croxton. Diverse Evidence for a Primary Role of Hemocytes in Oyster Shell Construction. International Oyster Symposium, Falmouth, MA. October 21-25, 2015 2.Zhang C. and Mount. A.S. (research mentor) Stuck at Sea: Using Imaris to visualize in 3D oyster and barnacle confocal data -poster. NSF REU Symposium 2015, October 29-30, 2015, NSF headquarters, Arlington, Virginia. 3.Lange, M, V.S. Chan, A. McQuiston, A.S. Mount. Inhibition of FAF1 reduces hemocyte aggregation and increase chitin production in the oyster hemocytes -poster, American Society for Cell Biology Annual Meeting, December 11-17, 2015, San Diego, California 4.Chan, V.B.S. and A.S. Mount. Chitin involvement at the biomineralization front on implant surfaces in the Eastern oysters, Crassostrea virginica -poster, American Society for Cell Biology Annual Meeting, December 11-17, 2015, San Diego, California 5.Mount. A.S. The Cellular Biology of Oyster Shell Formation and Barnacle Adhesion from a Materials Perspective - invited speaker. Southeast Ultrafast Conference, North Carolina State University, Raleigh, North Carolina. January 14-16, 2016 6.Lange, M, V.S. Chan, A. McQuiston, A.S. Mount. Inhibition of FAF1 reduces hemocyte aggregation and increase chitin production in the oyster hemocytes -poster, Southeast Ultrafast Conference, North Carolina State University, Raleigh, North Carolina. January 14-16, 2016 7.Chan, V.B.S. and A.S. Mount. Chitin involvement at the biomineralization front on implant surfaces in the Eastern oysters, Crassostrea virginica -poster, Southeast Ultrafast Conference, North Carolina State University, Raleigh, North Carolina. January 14-16, 2016 8.Mount, A.S. with C. Mangelson, patent examiner interview, re: Deposition of Nanocrystalline Calcite on Surfaces by a Tissue and Cellular Biomineralization, United States Patent and Trademark Office, Alexandria, Virginia, January 28, 2016. 9.McQusiton, A, V..Chan and A.S. Mount, Platinum blue staining mitigates charging and enhances cell membrane visualization in oyster hemocytes -poster. 13th 13th Advanced Imaging methods Workshop, UC Berkeley, February 8-12, 2016. 10.Chan, V.B.S. and A.S. Mount. Chitin involvement at the biomineralization front on implant surfaces in the Eastern oysters, Crassostrea virginica poster. 13th 13th Advanced Imaging methods Workshop, UC Berkeley, February 8-12, 2016. What do you plan to do during the next reporting period to accomplish the goals?We will host both NRL and international researchers in March 2017 to visualize chitin in cyprid and juvenile barnacles in March, 2017. We are preparing a special topic issue of Nature Frontiers in Marine Science that will focus on cellular biomineralizaton in marine invertebrates. We will continue to develop direct means to visualize metabolism by FLIM in unlabeled live specimens.

Impacts
What was accomplished under these goals? OBJ-1.1- Perform comparative studies on the conservation of adrenergic receptors across diverse phyla and further characterization of oyster larval foot. -UPDATE: In partnership with NEN-Biomedical we expressed our own unique chitin binding molecular probe which we conjugated to a far red flurophore (red-CBP). This probe enabled us to detect the presence of chitin in the permanent adhesive plaque of attached cyprid larvae and in oyster hemocytes. The results of this research havebeen communicated in talks and posters to several federal agencies, including ONR, NSF and NRL. Dr. Chan has continued to fully characterize red-CBP and has found it to be specific in oyster tissues and hemocytes.OBJ-1.2-Develop and evaluate synthetic analogs of noradrenaline and Sy-NA-polymer systems.UPDATE : We have synthesized and tested a novel antifouling peptide based on a conotoxin which interacts with the neuroendocrine transporter (NET) . This peptide defeats larval biofouling in solution and is resistant to marine degradation. We applied for patent protection for this new technology in May, 2015. OBJ-1.3- Characterize the toxicity of soluble NA, Sy-NA and Sy-NA-X-polymer leachate. UPDATE: The results were published in 2015. We are in the process of evaluating a second iteration of our NET conopeptide system for toxicity.OBJ-2.1 Determine the cellular origin of lipids that make up the PMC at the mineralization front.UPDATE: We published an exosome based model of cellular mineralization in 2015. Weare now exploring the role of chitin in PMC formation.OBJ-2.2- Obtain and compare lattice orientation by TEM of prismatic calcite to that of foliated calcite plates.Update: In progress. OBJ-2.3 Ascertain if the crystallization of calcium carbonate proceeds through an amorphous intermediate phase within the refractive granulocytes of oysters. We are still developing a methodology to accomplish this task. Update: In progress. We have completed the construction of a brood stock and larval oyster holding facility. This facility will provide early stage oyster larvae that will be studied for carbonate uptake related to obj 2-2.

Publications

  • Type: Journal Articles Status: Awaiting Publication Year Published: 2016 Citation: Burns, T. E. -, N. Gohad, B. O. Diaz, A. S. Mount, C. Spillmann, K. Wahl, D. Rittschof. 2016. Barnacle biology before, during and after settlement and metamorphosis: a study of the interface. Submitted to Journal of Experimental Biology
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Chan, V.B.S., T. Toyofuku, G. Wetzel, L. Saraf, V. Thiaagarajan, A.S. Mount. 2015. Direct Deposition of Crystalline Aragonite in the Controlled Biomineralization of the Calcareous Tubeworm. Front. Mar. Sci., 18 November 2015
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2016 Citation: Chan, V.B.S., T. Toyofuku, G. Wetzel, L. Saraf, V. Thiaagarajan, A.S. Mount. 2016. Characterization of calcification events using live optical and electron microscopy techniques in the marine tubeworm. Submitted to the Journal of Visulaized Experiments,


Progress 10/01/14 to 09/30/15

Outputs
Target Audience: Marine biofouling: our ultimate target audience is any person or industry who has to eliminate the unwanted growth of aquatic and marine fouling organism on ships, water intake structures and nets and associated aquaculture gear. Marine cellular biomineralization: The target audience is individuals/agencies/governments who need a better understanding of the potential biological impact of global climate change driven ocean acidification on marine food security and marine ecosystems. Changes/Problems:Our Coherent Chamelon VIS S laser failed in early 2015 and was replaced with a new unit in December2015. What opportunities for training and professional development has the project provided?Vera Bin San Chan, Postdoctoral scholar, Hong Kong University, Feb 2015- Feb 2016, continuing. Elizabeth Falwell, Graduated August 2015, PhD in Biological Sciences, Major advisor Paris Smith, NSF REU Computer Visualization Student, summer 2015 Caroline Zhang, Emory University, NSF REU visualization student, summer 2015 Tiffany Tang, UC-DAVIS, bioinformatics internship, Aug-September 2015 Felix Gravino, Clemson University, Undergraduate research experience, August 2015- May 2016 Morgan Lange, Clemson university, undergraduate research experience, August 2015- May 2016 Andrew McQuiston, Laboratory technician, September -2015 and continuing Taught Biosc 493, Spring 2015: Senior Seminar in Climate Change, at the Digital Research Lab, Cooper Library, Clemson University. How have the results been disseminated to communities of interest?Mount, A. S. The high frontier of materials science lives in the sea. Invited Speaker. Southeast Ultrafast Conference, Florida State University National High Magnetic Flux Laboratory, Tallahassee, Florida. January 15-16, 2015. NavalFuture Force Science & Technology EXPO 2015. Washington, D.C., February 4-5, 2015 Johnstone, M.B. N.V. Gohad, E. Falwell, D Hansen, A.S. Mount, K. Hansen. Cellular orchestrated biomineralization of crystalline composites on implant surfaces by the eastern oyster, Crassostrea virginica. National Shellfisheries Association Annual Meeting, Monterey Bay, California, March 22-26, 2015. Chan. B.S. and Mount, A.S. Observations of direct mineralization of aragonite in the serpulid tubeworm, Hydrodies elegans. National Shellfisheries Association Annual Meeting, Monterey Bay, California, March 22-26, 2015. Falwell, E. and Mount A.S. The role of folian and MMP on shell repair in the eastern oyster, Crassostrea virginica. National Shellfisheries Association Annual Meeting, Monterey Bay, California, March 22-26, 2015. Mount, A.S. Chair and session moderator on Calcification. National Shellfisheries Association Annual Meeting, Monterey Bay, California, March 22-26, 2015. Mount, A.S. New fouling deterrent strategies to defeat barnacle biofouling. ONR Biofouling & Coatings Program Review. Arlington, Virginia, 2-5 June, 2015. Mount, A.S. The complexity of barnacle glue. Naval Research Laboratory Guest Seminar, Arlington, Virginia, June 18, 2015. Mount, A.S. The cellular biology of oyster shell formation and barnacle adhesion from a materials perspective. National Science Foundation Invitational Seminar, Arlington, Virginia, June 19, 2015. Zhang, C and Mount, A.S. (research mentor) Stuck at Sea: Using Imaris to visualize in 3D oyster and barnacle confocal data. Research experience for undergraduates in collaborative data visualization applications at Clemson University NSF-REU, Clemson, South Carolina. June 1, 2015-July 24, 2015. Smith, P. and Mount A.S. (research mentor) The non-Io radio storms of Jupiter, a decametric catalog. Research experience for undergraduates in collaborative data visualization applications at Clemson University NSF-REU, Clemson, South Carolina. June 1, 2015-July 24, 2015. What do you plan to do during the next reporting period to accomplish the goals?Continuing to develop methods and experimental protocols that will be applied to objectives 2.1, 2.2 and 2.3

Impacts
What was accomplished under these goals? OBJ-1.1- Perform comparative studies on the conservation of adrenergic receptors across diverse phyla and further characterization of oyster larval foot. - UPDATE: In partnership with NEN-Biomedical we expressed our own unique chitin binding molecular probe which we conjugated to a far red flurophore. This probe enabled us to detect the presence of chitin in the permanent adhesive plaque of attached cyprid larvae and in oyster hemocytes. The results of this research has been communicated in talks and posters to several federal agencies, including ONR,NSF and NRL. OBJ-1.2-Develop and evaluate synthetic analogs of noradrenaline and Sy-NA-polymer systems. UPDATE : We have synthesized and tested a novel antifouling peptide which interacts with the neuroendocrine transporter (NET) . This peptide defeats larval biofouling in solution and currently we are developing a second cono-like peptide which can be covalently linked to a painted surface. We applied for patent protection for this new technology in May , 2015. OBJ-1.3- Characterize the toxicity of soluble NA, Sy-NA and Sy-NA-X-polymer leachate. UPDATE: The results were published in 2015. OBJ-2.1 Determine the cellular origin of lipids that make up the PMC at the mineralization front. UPDATE: we published an exosome based model of cellular mineralization in 2015. OBJ-2.2- Obtain and compare lattice orientation by TEM of prismatic calcite to that of foliated calcite plates. Update: In progress OBJ-2.3 -Ascertain if the crystallization of calcium carbonate proceeds through an amorphous intermediate phase within the refractive granulocytes of oysters. We are still developing a methodology to accomplish this task. Update: In progress

Publications

  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Chan, V.B.S., T. Toyofuku, G. Wetzel, L. Saraf, V. Thiaagarajan, A.S. Mount. 2015. Direct Deposition of Crystalline Aragonite in the Controlled Biomineralization of the Calcareous Tubeworm. Front. Mar. Sci., 18 November 2015
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Lee, E.H., A.S. Mount, B.W. Booth, W.Y. Chen. 2015. Prolactin and cisplatin combination treatment inhibits tumorsphere formation and delays breast tumor growth in mice. International Journal of Cancer and Oncology 2:1-7.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Overturf, C.L., A.M. Wormington, K.N. Blythe, N.V. Gohad, A.S. Mount, A.P. Roberts. 2015. Toxicity of noradrenaline, a novel anti-biofouling component, to two non-target zooplankton species, Daphnia magna and Ceriodaphnia dubia. Comp. Biochemistry and Physiology, Part C 171:49-54
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Johnstone M.B, N.V. Gohad, E.P. Falwell, D.C. Hansen, K.M. Hansen, A.S.Mount. 2015. Cellular orchestrated biomineralization of crystalline composites on implant surfaces by the eastern oyster, Crassostrea virginica . Journal of Experimental Marine Biology and Ecology 463:8-16


Progress 10/01/13 to 09/30/14

Outputs
Target Audience: 1. Marine biofouling : our ultimate target audience is any person or industry who has to eliminate the unwanted growth of aquatic and marine fouling organisms on ships, water intake structures and nets and associated aquaculture gear. 2. Marine cellular biomineralization: The target audience is individuals/agencies/governments who need a better understanding of the potential biological impact of global climate change driven ocean acidification on marine food security and marine ecosystems. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nature -Frontiers in Marine Science Journal, 2014 to present, Associate Editor Arctic Council, AMAP-AOA, Oslo, Norway 2013-present, permanent expert on Arctic Ocean acidification, USA representative Trained Vera Chan, PhD student , Hong Kong University, October-December, 2013 Participant, Edgar Brown Digital Resources Laboratory Training Workshop, 21 May 2014 Participant, Clemson University Critical Thinking2 Faculty Institute, Madren Center, Clemson SC June 16-20, 2014 and August 7, 2014 Taught Biosc 493, Spring 2014: Senior Seminar in Climate Change. This course is exceedingly popular as student enrollment increased 100% from 15 students in 2013 to 30 students in 2014. How have the results been disseminated to communities of interest? A.M. Wormington, C.L. Overturf, K.N. Blythe, N.V. Gohad, A.S. Mount, and A.P. Roberts. 2014. Toxicity of noradrenaline, a novel anti-biofouling component, to two non-target zooplankton species, Daphnia magna and Ceriodaphnia dubia. SETAC Regional Meeting. San Marcos, TX. A.M. Wormington, C.L. Overturf, K.N. Blythe, N.V. Gohad, A.S. Mount, and A.P. Roberts. 2014. Toxicity of noradrenaline, a novel anti-biofouling component, to two non-target zooplankton species, Daphnia magna and Ceriodaphnia dubia. SETAC North America Meeting. Vancouver, BC. N.V.Gohad, N. Aldred, C. Hartshorn, Y. J. Lee, B. Orihuela, M. Cicerone, A. Clare, D. Rittschof, and A. S. Mount. 2014. Synergistic Roles for Lipids and Proteins in the Permanent Adhesive of Barnacle Larvae 11th Advanced Imaging Methods Workshop, UC- Berkeley, Jan 29- 31 at UC Berkeley5.. N. V. Gohad and A.S. Mount . 2013. Development of marine coatings utilizing a fouling deterrence strategy. ONR Biofouling/Coatings Program Review, June 25-27, 2013, Arlington, Virginia. N.V.Gohad, N. Aldred, C. Hartshorn, Y. J. Lee, B. Orihuela, M. Cicerone, A. Clare, D. Rittschof, and A.S. Mount. 2014. Synergistic Roles for lipids and Proteins in the Permanent Adhesive of Barnacle Larvae 106th meeting of the National Shellfisheries Association Meeting, Mar 29 - Apr 2, 2014, Jacksonville, FL What do you plan to do during the next reporting period to accomplish the goals? Investigate conus peptides for biofouling, study chitin production in barnacles and oysters, study lattice ion regulation in barancles and oyster larvae.

Impacts
What was accomplished under these goals? New Award: Office of Naval Research - Award #N00014-14-1-0488 - $321,256.00 -Starts: 15 May 2014 Ends: 14 May 2016. - Title: New Fouling Deterrent Strategies to Defeat Barnacle Biofouling OBJ-1.1- Perform comparative studies on the conservation of adrenergic receptors across diverse phyla and further characterization of oyster larval foot. - UPDATE: Publications in preparation, Now studying chitin production in larvae. OBJ-1.2-Develop and evaluate synthetic analogs of noradrenaline and Sy-NA-polymer systems. UPDATE : Testing Xerogel coatings in 2015; now studying cone snail peptides. OBJ-1.3- Characterize the toxicity of soluble NA, Sy-NA and Sy-NA-X-polymer leachate. UPDATE: Publication submitted. OBJ-2.1 Determine the cellular origin of lipids that make up the PMC at the mineralization front. UPDATE: Published in Nature Communications, barnacle larval results. Working on oyster system. OBJ-2.2- Obtain and compare lattice orientation by TEM of prismatic calcite to that of foliated calcite plates. Update: In progress OBJ-2.3 -Ascertain if the crystallization of calcium carbonate proceeds through an amorphous intermediate phase within the refractive granulocytes of oysters Update: In progress

Publications

  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Neeraj Gohad, Nick Aldred, Christopher Hartshorn, Young Jong Lee, Beatriz Orihuela, Marcus Cicerone, Anthony Clare, Dan Rittschof, and Andrew Mount on Synergistic Roles for Lipids and Proteins in the Permanent Adhesive of Barnacle Larvae" published by Nature Communications on 11 July 2014
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Johnstone, M.B., N. Gohad, E. Falwell, D. Hansen, K.Hansen, A.S. Mount. 2015. Cellular orchestrated biomineralization of crystalline composites on implant surfaces by the eastern oyster, Crassotrea virgincia (Gmelin,1791). J. Exp. Mar. Biol. Ecol 46:8-16
  • Type: Other Status: Published Year Published: 2013 Citation: Mount et al. 2013. Deposition of nanocrystalline calcite on surfaces by a tissue and cellular biomineralization. United States Patent. US 8,541,031 , September 2013


Progress 01/01/13 to 09/30/13

Outputs
Target Audience: 1. Marine biofouling : our ultimate target audience is any person or industry who has to eliminate the unwanted growth of aquatic and marine fouling organism on ships, water intake structures and nets and associated aquaculture gear. 2. Marine cellular biomineralization: The target audience is individuals/agencies/governments who need a better understanding of the potential biological impact of global climate change driven by ocean acidification on marine food security and marine ecosystems. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? A doctoral student from the University of Hong Kong received training in two successive visits in the Spring and Fall of 2013. An undergraduate student was recruited to the lab in the Fall of 2013 and is currently receiving training. The lab is training a PhD candidate who is completing her research in 2014. A post doctoral scholar continued his lab training in 2013. How have the results been disseminated to communities of interest? The lab team presented its results in 2013 at four scientific meetings, the SPIE conference in San Francisco, Feb 2013; The National Shellfish Meeting in Nashville, TN in Mar 2013 (the labchaired a biofouling session). Dr. Mount presented cellular biomineralization results as it relates to ocean acidification in the Arctic in Bergen Norway , May 2013. Biofouling research progress was reviewed at the ONR coatings meeting in Arlington, Virginia , June 2013. What do you plan to do during the next reporting period to accomplish the goals? We have scheduled papers at the Advanced Imaging Methods 2014 meeting in Berkley, California. We are meeting with our microscopy and spectroscopy tool builders at SPIE in San Francisco in Feb 2014, We are planning to host another biofouling session and give papers at the upcoming National Shellfish meeting in Jacksonville Florida , spring 2014. Dr. Mount will attend a Gordon Research Confernce either on biomineralization or ocean acidification in July, 2014. A paper on biofouling has been submitted and is in peer review. Two more papers on marine cellular biomineralization are in preparation.

Impacts
What was accomplished under these goals? OBJ-1.1- Perform comparative studies on the conservation of adrenergic receptors across diverse phyla and further characterization of oyster larval foot. – UPDATE: In progress. OBJ-1.2-Develop and evaluate synthetic analogs of noradrenaline and Sy-NA-polymer systems. UPDATE: In collaboration with an academic lab, we have developed a covalently linked xerogel NA based coating and preliminary lab tests are encouraging. OBJ-1.3- Characterize the toxicity of soluble NA, Sy-NA and Sy-NA-X-polymer leachate. UPDATE: Additional soluble NA data on sub-lethal levels of NA have been collected. OBJ-2.1 Determine the cellular origin of lipids that make up the PMC at the mineralization front. UPDATE: We have a preliminary CARS result that the oyster's intracellular biomineralization compartment contains lipid. Research is ongoing. OBJ-2.2- Obtain and compare lattice orientation by TEM of prismatic calcite to that of foliated calcite plates. Update: In progress OBJ-2.3 -Ascertain if the crystallization of calcium carbonate proceeds through an amorphous intermediate phase within the refractive granulocytes of oysters Update: In progress

Publications

  • Type: Other Status: Published Year Published: 2013 Citation: Mount et al. 2013. Deposition of nanocrystalline calcite on surfaces by a tissue and cellular biomineralization. United States Patent. US 8,541,031
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Confocal microscopy-based goniometry of barnacle cyprid permanent adhesive N Aldred, NV Gohad, Mount, A.S. et al The Journal of experimental biology 216 (11), 1969-1972