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FLORIDA’S WILDLIFE LEGACY INITATIVE FLORIDA’S STATE WILDLIFE GRANTS PROGRAM
FINAL REPORT
Project Title:
Coral Restoration in the Florida Keys Using Colonies Derived from Aquacultured Fragments
Project Director:
Ilze K. Berzins, PhD, DVM The Florida Aquarium 701 Channelside Drive Tampa, FL 33602
Coauthors:
Craig A.Watson, MAq; Roy P.E. Yanong, VMD; Kathy Heym Kilgore, VMD; and Scott Graves Tropical Aquaculture Laboratory, University of Florida/IFAS 1408 24th Street SE Ruskin, FL 33570
Casey Coy, DSO and Ryan Czaja The Florida Aquarium 701 Channelside Drive Tampa, FL 33602
Lauri MacLaughlin and Billy Causey, PhD Florida Keys National Marine Sanctuary, NOAA 33 East Quay Road Key West, FL 33040
Date Report Submitted:
August 15, 2007
Project Number:
SWG04-038 (Modification No. 1: FWC Agreement No. 05045)
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ABSTRACT
The Coral Reef Task Force estimates that 70% of the world’s coral reefs are threatened and 10% have been destroyed. Portions of Caribbean coral reefs have lost up to 80% of coral species and continue to be under increasingly destructive pressures from various sources including dredging, ship groundings, pollution, illegal collecting and harsh weather conditions. Florida coral reefs, the only shallow water reefs in the continental United States, have suffered considerable loss. Restoration of damaged coral sites is limited by the availability of coral colonies. Aquaculture is emerging as a viable method of large-scale production of coral colonies. Recent efforts have shown that many species of Atlantic Scleractinia can be fragmented and grown successfully in tanks and on underwater lease sites. Can these aquacultured fragments be utilized in reef restoration? Two primary questions emerge concerning the feasibility and direction of this effort: 1) will aquacultured corals become a vector for disease introduction when returned to a restoration site, and 2) are survival and growth success of reintroduced fragments affected by culture techniques? The Florida Aquarium, with partners from the University of Florida’s Tropical Aquaculture Laboratory (TAL) and the Florida Keys National Marine Sanctuary (FKNMS) began investigating these questions in 2005. The team cut 210 fragments from seven species (30 per species) of coral collected from the Truman Annex site in Key West Harbor. The aquacultured corals (Siderastrea radians, Solenastrea bournoni, Montastrea annularis, Montastrea cavernosa, Diploria clivosa, Dichocoenia stokesii, and Stephanocoenia michelinii) were distributed to two land-based culture locations (TAL and Mote Marine) and to the field restoration site (Miss Beholdin grounding site, Western Sambo Reef). The team made regular measurements of the growth and observations of the health of coral in both environments. In an attempt to support visual observations and to provide a library of normal vs. abnormal changes, histology and microbiological tests have been applied to many of the samples. The land-based study fragments were grown in culture for approximately six months prior to transplantation in the field. To ensure that cultured fragments do not become a vector for disease in the wild, The Florida Aquarium (FLAQ) and the TAL developed a protocol for a federal and state health certificate of coral fragments with guidance from the FKNMS and others. The certification process was approved through Florida Fish and Wildlife Conservation Commission (FWC) and a Special Activity License was issued permitting the transplantation of health fragments back into the wild. In December 2006, the team transplanted 88 fragments (out of 140) that had passed health certification or were not used for diagnostic sampling to the restoration site. Monitoring was to have continued at three month intervals but due to inclement weather, the team was unable to inspect the fragments until the first week of May 2007. The final evaluation period for this grant took place July 29th, 2007. Sampling included health assessments, photographs, and mucus sampling for microbial community analysis on select fragments. Of the seven species cultured, Siderastrea radians appeared to exhibit the best success both in culture and field conditions, and fragmented corals given time to ―heal‖ from the cutting process in land-based culture situations seem to be doing better than
The Florida Aquarium Florida’s Wildlife Legacy Initiative SWG04-038 3 fragments put immediately into the field. Use of aquacultured corals appears to be one viable solution to helping restore damaged reefs.
ACKNOWLEDGEMENTS
Abundant thanks are given to the numerous individuals from many different agencies assisting in this project. In particular we would like to thank Joanne Delaney and Brian Keller from the Florida Keys National Marine Sanctuary; Michael Terrell from The Florida Aquarium; Jeff Hill and Dan Bury from the Tropical Aquaculture Laboratory; Kim Ritchie, David Vaughn, Kevan Main, and Ken Leber from Mote Marine Laboratory; Mya Breitbart and Camille Daniels from the University of South Florida; Ken Nedimyer from SeaLife Inc., and Lisa Gregg from the Florida Fish and Wildlife Conservation Commission. We also thank Instant Ocean® for their contribution of sea salt to the Tropical Aquaculture Laboratory for use in this study. Portions of this study were funded by The Florida Wildlife Legacy Initiative grants program (grant #SWG04-038 (Modification No. 1: FWC Agreement No. 05045)).
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TABLE OF CONTENTS
Introduction………………………………………………………….…………5
Methods…………………………………………………………………………7
Fragmenting and Attachment Culturing Process Caribbean Coral Health Certification Process Diagnostics Histology and Microbiology Field Operation Training, Operational Procedures, Coral Planting, Site Mapping, Health Assessment, Mucus Sampling
Results…………………………………………………………………………18
Discussion……………………………………………………………….….…28
Management Recommendations………………………………………..…….30
Conclusions…………………………………………………………………...31
Literature Cited………………………………………………………………32
List of Appendices……………………………………………………………35
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INTRODUCTION
Florida is the only state in the continental United States that has extensive shallow coral reef formations near its coasts, from the Florida Keys starting south of Miami reaching west to the Dry Tortugas. Caribbean coral reefs are under increasingly destructive pressures from various sources, including dredging, ship groundings, pollution, and illegal collecting. The state’s Comprehensive Conservation Wildlife
Strategy of 2005 cited coral reefs as a priority habitat, labeled ―bad and in decline.‖ Over
200 species of birds, mammals, fish and invertebrates, including numerous coral species, were designated ―species of greatest conservation need.‖
The specific objectives of an overall coral initiative program at The Florida
Aquarium include: 1) construct a land-based propagation facility at FLAQ and use it to educate visitors, 2) produce fragments for exchange and research, 3) serve as a holding facility for damaged corals and orphaned corals (through the FKNMS Reef Medic and
Coral Nursery programs), 4) conduct cooperative research on captive coral health and propagation issues, and 5) develop restoration projects and health certification protocols.
In 2000-2001, through funding from the Association of Zoos and Aquariums’
(AZA) Conservation Endowment Fund (CEF) and NOAA’s Five-Star Restoration Grants
Program, The Florida Aquarium built a working propagation facility, the ―Coral Farm‖ on the exhibit pathway (Fig. 1, Appendix E). Educational brochures that describe the project are available to the public and additional programming tells the message through outreach programs, camps and teacher workshops (part of the Science Education at Sea
(SEAS) program). Research studies at The Florida Aquarium initially were focused on exploring propagation methods in culture situations, evaluating requirements such as
The Florida Aquarium Florida’s Wildlife Legacy Initiative SWG04-038 6 lighting, flow, attachment, size, and compatibility, among other factors. Over time, additional projects emerged including: delineating diseases in ―captivity‖, developing diagnostic tools and treatments for disease, and propagating sufficient ―lab rats‖ for cooperative research.
However, key questions remained unanswered. Could these fragments be successfully reintroduced to the wild and, if they could, was there a potential for larger scale production in aquaculture facilities to provide adequate numbers of fragments for restoration projects? There is information available on culture techniques and on transplantation techniques (Borneman and Lowrie, 2001; Clark and Edwards, 1995;
Custodio and Yap, 1997; Delbeek, 2001; Lindahl, 1998 and 2003; Precht, 2006; Smith and Hughes, 1999; Soong and Chen, 2003; Van Treech and Schumacher, 1997; Yap et al., 1992 and 1998) but this is the first study that has combined the use of aquacultured fragments with a health certification process to allow such fragments to be returned to the wild for restoration projects. With support from the FKNMS and a Florida Wildlife
Legacy Initiative grant (#SWG04-038) through the FWC, The Florida Aquarium and its key partner, the Tropical Aquaculture Laboratory, University of Florida, began addressing two primary questions concerning the use of aquacultured fragments for restoration: 1) would culture techniques affect survival and growth of reintroduced fragments and 2) could these fragments become a vector for disease when returned to a restoration site?
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METHODS
FKNMS made the corals for this study available. During construction activities at the
Truman Annex Mole Pier in Key West, Florida, over 3,500 corals and fragments from this one site were removed. Project corals were obtained from the Truman Annex site in
April of 2006. Seven species were utilized: Siderastrea radians, Solenastrea bournoni,
Montastrea annularis, Montastrea cavernosa, Diploria clivosa, Dichocoenia stokesii, and
Stephanocoenia michelinii. I can’t locate the reference….the list was in the initial stages when the strategy was being developed by the state…..will just delete… No branching species were available from this site. Coral colonies were selected for size and suitability for fragmentation. Genetically identical colonies were preferred but not available for all species due to size of parent colony stock. Where multiple parent colonies were used, each of the three experimental sites (two land-based culture and one field/open reef) received representative samples. Thirty fragments of each species were made to allow for 10 fragments per culture site, a total of 210 fragments in all.
The corals were fragmented using a tile saw with sea water as a coolant into approximately 2.5 cm x 2.5 cm pieces (Fig. 5, Appendix E). Using a two-part epoxy (Z-
Spar®), the team affixed each fragment to a standardized cement base, 8.5 cm in diameter and 1.5 cm thick (Fig. 6, Appendix E). Ten fragments of each species were distributed to each of the three sites (Fig. 7, Appendix E). The fragments were to be held in culture for a minimum of six months, during which periodic health assessments and disease diagnostics (a minimum of every three months) were performed. The field site was scheduled to be evaluated every three months (Figure 11, Appendix E). As part of the assessment process, digital images are taken of each fragment with a scale bar and a
The Florida Aquarium Florida’s Wildlife Legacy Initiative SWG04-038 8 visual health examination was recorded (including an assessment of condition and color), along with any mortality of test fragments.
Culturing Process
The culturing portion of the study compared growth and survival between two land- based aquaculture facilities and an open-water (field) site. The site is approximately 5 miles south of the Truman Pier. The depth of the transplant site is from 8 to 16 feet.
There is a very strong tidal current at the field site but actual comparison values are unknown at this point. In an attempt to design economically feasible culture techniques
(for which cost-benefit analyses would later be run), culture methods were relatively basic in design. The first land-based site was at the Tropical Aquaculture Laboratory
(TAL), University of Florida, in Ruskin, Florida (Fig. 2, Appendix E). Corals were maintained in a commercial-style greenhouse (30 x 72 foot (approximately 9.1 m x 22 m) with inflated double layer poly 30% shade cloth) with fan shutters and a propane heater.
Fragments were placed in two 350 gallon (1,325 liter) tanks, each 3 m x 0.75 m x 0.75 m
, and elevated on racks constructed of PCV pipe and plastic lighting grids (―egg crate‖).
Water was supplied to each tank utilizing a ―Carlson‖ surge generator constructed from plastic drums and PVC pipe. The system included a sump that measured 2 m x 1 m x
0.75 m, filled with approximately 20 cm of crushed coral, which served as a calcium source and assisted with biological filtration. The system was powered by a 1.0 hp centrifugal pump. The system also included a 0.5 ton water chiller. During the winter months, the greenhouse was heated. Temperatures were maintained at 78-80 ºF (25-
27ºC). This closed recirculating aquaculture system used artificial seawater made by combining reverse osmosis water with Instant Ocean® sea salt mixture to achieve 33 ppt.
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Water changes (at least 50%) were made once each month. The culture tanks were also covered with a PVC frame and 50% shade cloth. To help control algal growth and fouling by marine organisms such as Aptasia, herbivorous snails (Astraea sp.) and predatory shrimp (peppermint shrimp, Lysmata wurdemanni) were added to the culture systems. The exposed cement disc surfaces were also periodically cleaned by hand using a fine wire brush.
The second land-based site was at the Mote Marine Laboratory (MML) on
Summerland Key (Fig. 3, Appendix E). The tank set-up was similar the only thing being different was that the water was obtained directly from the inlet on open-flow system.
The field/open-water site (control site) was the Miss Beholden grounding site on Western
Sambo Reef, approximately four nautical miles south of Key West (Fig. 4 and 12,
Appendix E).
Caribbean Coral Health Certification Process
Prior to the reintroduction of aquacultured coral fragments, a health certification protocol needed to be established. Important considerations included: 1) developing working definitions for ―diseased‖ and ―healthy‖ cultured corals, 2) maximizing the potential for cultured corals to survive and thrive in reintroduction sites, and 3) minimizing the potential for release of cultured corals with ―exotic‖ disease pathogens or other pathogens of concern into the wild. The goal was to develop a reasonable and practical, health certification process for the State of Florida that could act as a template for other studies. But just what is reasonable and practical? There is no such thing as zero risk or 100% certainty therefore assessments should be based on present day science, economics, federal and state policies, and reality. All protocols should be part of a living
The Florida Aquarium Florida’s Wildlife Legacy Initiative SWG04-038 10 document, constantly under review and change.
The partners developed protocols leading to health certification in collaboration with NOAA, FKNMS, the United States Department of Agriculture/Animal Plant Health
Inspection Service (USDA/APHIS), and FWC. Upon approval, a Special Activity
License (SAL) from FWC was also required at the time of reintroduction. Parameters evaluated included visual inspection, water quality testing, biosecurity (for land-based facilities), and histology evaluation and microbial community analysis of aquaculture normal and diseased fragments.
Health assessments on parent colonies were done first at the time of collection
(Appendix A - Health Assessment Form), then repeated on the fragments during culture, one month prior to reintroduction for the issuance of the health certificate, and at periodic intervals after being introduced back into the field. Fragments are monitored for visual abnormalities based on the fact that known coral diseases are categorized by visual signs
(i.e. Black Band disease, White Spot disease, etc) (Breitbart et al., 2005: Cooney et al.,
2002; Ducklow and Mitchell, 1979; Frias-Lopez et al., 2004; Pantos et al. 2003;
Rosenberg and Falkovitz, 2004; Sekar et al., 2006).
Any coral colony in culture showing signs of disease undergoes an extensive work-up including a detailed visual description of the problem, photos, live microscopic and stereoscopic evaluation, microbiology testing and histological evaluation. To develop the list of diseases/syndromes reported in culture conditions further, the team is sending a Captive Coral Health Survey to other facilities propagating corals. An accurate and comprehensive list of pathogens for corals may prove difficult to compile, but evaluation of handling methods, prophylactic treatments, biosecurity protocols and
The Florida Aquarium Florida’s Wildlife Legacy Initiative SWG04-038 11 thorough evaluation of problems are being used to develop a method for screening the population for health (for specifics please refer to Appendix B and C). Considerable precedence already exists for similar aquatic animal health screening certifications, such as for interstate transport and/or stock enhancement releases of shrimp, bivalves and fish.
Based on the recommendations of the veterinarians involved in this study, standard best management practices have been collaboratively developed to quarantine corals in culture properly and to ensure that corals are healthy prior to reintroduction to restoration sites (Appendix B – Guidelines). These procedures and other diagnostic methods were used to develop criteria for issuance of a federal health certification
(Appendix C - Health Certificate) prior to reintroduction of corals to restoration sites or where otherwise required. The criteria outlined in the health assessment form are used to provide each fragment with a score. Based on the score, fragments either pass or fail inspection. Then, the entire group of fragments of a given species is also assessed. If one fragment out of ten failed, then only that failed fragment would not be transplanted.
However, if only one fragment out of ten passed, none of the fragments from that species would be transplanted. The delineation point of whether or not a species get transplanted is 50%: half of the inspected coral fragments from a given species need to pass inspection. Failed fragments are then returned to culture and evaluated on a monthly basis. At the time of field visits, the team will transplant those fragments passing inspection to the restoration site following the issuance of appropriate permits.
Diagnostics
Histology and Microbiology
In an attempt to support visual observations and to provide a library of normal vs.
The Florida Aquarium Florida’s Wildlife Legacy Initiative SWG04-038 12 abnormal changes, histology and microbiological tests have been applied to many of the samples. Samples are obtained for evaluation at the time of collection (from parent colony), in the presence of disease (includes abnormal and normal tissue), prior to reintroduction and the issuance of a health certificate, and during field sampling if necessary.
Fragments for histology are fixed in a four-part seawater to one-part buffered zinc formalin (Z-fix concentrate®) solution. They are then enrobed in SeaKem® agarose and exposed to vacuum pressure to pull agar into crevices. Afterward a window is cut into the stiffened agar to expose the skeleton, and the specimen is decalcified in neutral
EDTA. The remaining tissue, held by the agar in a normal position, is then processed by routine cutting and staining techniques (see Appendix D for more specific details).
Coral species secrete a surface mucopolysaccharide layer (SML) that provides a rich environment in which microbes reside. There is evidence to support that these surface microbes may be involved in disease protection as well as evidence that there may be a shift in community composition under stressful conditions which could result in increased susceptibility to disease (Brown and Bythell, 2005; Kline et al., 2006; Reshef et al. 2006; Ritchie, 2006; Ritchie and Smith 2004; Rohwer et al. 2001; Rohwer et al. 2002;
Wegley et al. 2004).
Identification of various coral diseases and syndromes has classically been limited to visual characterization, but by the time changes are evident the coral is often already compromised. Evaluating microbial communities may therefore help assess the overall health of the fragment. However, less than 1% of all bacteria present on coral is isolatable in laboratory culture (Ritchie, 2004), and without fulfilling Koch’s postulates
The Florida Aquarium Florida’s Wildlife Legacy Initiative SWG04-038 13 one cannot attribute disease to a particular isolate. How best then to address changes and potential risks? As part of our coral restoration project using colonies derived from aquacultured fragments, we have attempted to characterize the metabolic diversity of the surface microbiota of seven species of Atlantic Scleratinia using BiOLOG® EcoPlates™.
These are commercially available 96-well microplates that provide a selection of carbon- based substrates (31 carbon sources and a control with three replicates per plate) for use in determining the metabolic profile of a given bacterial community.
The fragment is sampled by gently drawing a sterile 20-ml syringe over a 2 cm2 area of the fragment surface collecting ~20 cc of mucus and seawater. The EcoPlatesTM are directly inoculated with the contents of the syringe. The culture plate is incubated with mucus from the coral fragment in question. The plate is incubated at 25-27°C and read at least every 24 hours for up to 192 hours. As the microbes grow, they use a variety of the available carbon sources available in the microwells, resulting in turbidity and color changes in those wells. The Biolog® automated reader then evaluates the plate at two different wavelengths to produce a profile of metabolic activity that provides information on community structure and function. While individual isolates are not identified, we are evaluating the results to see if the process can be developed into a cost-effective clinical tool similar to microbial community analyses used to assess health in other species groups, e.g., ratios of basic bacterial groups obtained from choanal (throat area) and cloacal (anal area) swabs in birds.
As one of the goals of our coral restoration project was to determine if survival and growth of reintroduced fragments was affected by various culture techniques, one question we attempted to address with the BiOLOG® EcoPlates™ was whether the
The Florida Aquarium Florida’s Wildlife Legacy Initiative SWG04-038 14 microbial populations in the SML would shift depending on their culture conditions (an artificial seawater, recirculating system in a greenhouse versus a land-based, natural seawater flow-through system). Using methods described by Gil-agudelo et al (2006), samples of the SML from two fragments of each coral species from the two land-based aquaculture sites were obtained after a six-month period in culture (November 2006)
(Table 2). At the time of sampling, a visual health assessment and health certification process was performed, and those fragments that passed inspection were then ―planted‖ on an open ocean site (December 2006) near the ―control‖ fragments that had been
―planted‖ in May 2006.
Both sets of fragments were then to be monitored approximately every three months weather-permitting and assessed for condition and color as well as photographically documented. Actual field assessments of these corals occurred only at 12 and 15 months
(May and July 2007 respectively) post transplantation due to difficult weather conditions
(in effect, the 9 month internal was not done). At the times when field assessments were possible, the SML of two fragments from each coral species was obtained for microbial community analysis.
Field Operations
Training
All working divers on the Coral Propagation Project (CPP) are under the auspices of The Florida Aquarium. FLAQ is an organizational member of the American Academy of Underwater Sciences (AAUS) and all divers working on this project have been certified as scientific divers as outlined in section 5.30 of the 2006 AAUS Standards for
Scientific Diving Manual.
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All divers received additional project-specific training under the direction of Lauri
MacLaughlin of NOAA. This training included a general project overview and on-site training of coral attachment methods.
Field Operational Procedures
The primary mode of diving utilized during the field investigations was open circuit scuba. This mode was chosen over other possible diving modes (surface supplied diving and semi-closed or closed circuit re-breather) because it allowed the greatest flexibility, logistical efficiency and effective site recording while allowing for a high level of safety and supervision.
The dive plan was created by the Florida Aquarium Dive Safety Officer and approved by The Florida Aquarium Diving Control Board. Each diving day the roster included one Captain responsible for all safety and topside supervision of diving operations, one lead diver, one lead research diver and up to four working divers. The
Florida Aquarium and the University of Florida’s Tropical Aquaculture Lab provided the equipment used to complete site surveys and coral attachment, which includes:
1. Diving Slates 2. Mylar and slate paper, Pencils 3. Transect tapes in meters 4. Plumb bob 5. Digital imaging camera’s (both still imagery and video imagery) 6. Two-part epoxy 7. Site prep tools – hammer, chisel, wire brush
Coral planting procedures
Sites were identified based on depth, available light, coral orientation and proximity to similar species. Each site was prepared for planting by removing all living
The Florida Aquarium Florida’s Wildlife Legacy Initiative SWG04-038 16 organisms (algae, boring worms, etc.) using handheld metal scrapers and wire brushes.
Each coral fragment was attached (using two part epoxy) to a small circular cement disc approximately 10 centimeters in diameter. These discs were attached to the prepared site using the two-part epoxy. The epoxy was carefully applied to ensure no gaps between the cement base and the reef structure to prevent boring organisms from compromising the attachment.
Corals were transported by wrapping them first individually with wet (with seawater) paper towels and bubble wrap. Wrapped pieces were then placed in 2 inch thick Styrofoam cooler and stacked just to two layers. Ice packs were placed in the coolers but not directly on any corals. From time of initial wrapping to transplantation in the field was about 30 hours.
Site mapping procedures
Each site was mapped using trilateration. The distances from each coral fragment and two adjoining fragments were measured, thus creating a triangle. By mapping all fragments to each other we were able to create a ―web‖ that tied all fragments together.
The reef structure was hand-drawn over the web-based diver observation in the field, video and photo documentation.
Health Assessment
Visual assessments were performed on the study corals to evaluate their overall health in terms of tissue condition and color. Each fragment was inspected for condition and color using an ordinal scale (see Appendix A). For condition, the scale ranged from
1 to 6 with 1 being a dead fragment and 6 being a fragment with no evidence of tissue loss. For color, the scale ranged from 1 to 4 with 1 again indicating a dead fragment (or
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100% bleached) and 4 indicating a fragment with good/normal coloration. Observations were recorded on slates and with digital photography.
Health assessments were performed on our coral fragments at the following timeframes:: 0 months – May 2006 (baseline – prior to placement in different culture conditions); 3 months – August 2006; 6/7 months – November/December 2006 (6 months for land-based facilities; 7 months for open ocean site); 12 months – May 2007; and 15 months – July/August 2007.
Mucus Sampling
Mucus samples were taken from coral surface using 20 ml syringes. Divers agitate the surface of the coral to express mucus to facilitate collection. Syringes were capped and kept on ice during transport to the laboratory. Samples were taken from the land-based fragments at the time of health certification and then from the same fragments after transplantation to the field at the 12th month (May 2007) and 15th month (July 2007) assessment intervals.
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RESULTS
Final Fragment Disposition
Table 1 summarizes the disposition of the 210 fragments cut from the seven species of coral used for this study. In review, 30 fragments were cut from each species.
Ten of each species went to three different sites: one set to an artificial seawater, recirculating system in a greenhouse (TAL); one set to a land-based, natural seawater flow-through system (MML); and one set (control set) was transplanted directly to the field site on Western Sambo Reef. Health assessments were done on all fragments prior to placement at the three sites to establish baseline information. After five months in culture and a month prior to transplantation of the cultured fragments, health assessments were done on the TAL and the MML corals. This is stipulated in the health certification process. One fragment from each species from each set was sacrificed for diagnostics
(histology and microbiology). Then, within 30 days of the inspection, the fragments passing the health assessment were transplanted to the field. Passing scores for condition were either a 5 (~95% of tissue alive) or a 6 (no apparent tissue loss), and all passing color scores were between a 3 (lighter than normal) and a 4 (good/normal color). In addition, if more than 50% of the fragments of any given species did not pass inspection, the entire group failed and was not transplanted. Therefore, a fragment could fail individually or it could fail because the majority of fragments in its species group failed.
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Table 1: Final disposition of 210 coral fragments from seven species.
These numbers only include the mortalities in the two groups of fragments that had been transplanted to the field. The original field fragment mortalities are represented by the ―Number Deceased at the 1st Planting.‖
Mortalities in the fragments that have remained in culture are as follows: Diploria – 1 fragment; Solenastrea – 1 fragment; Stephanocoenia – 2 fragments. These are not currently indicated on the table.
The number of mortalities at the 2nd planting site has been updated from what was submitted for the initial grant report –These are the correct numbers as of Aug 2007, and the totals have been updated as well on the revised version of the chart below.
Montastrea Dichocoeni Siderastrea Diploria Montastrea Solenastrea Stephanocoenia cavernosa a stokesii radians clivosa annularis bournoni michelinii Total Fragments at 30 30 30 30 30 30 30 Start Number Sacrificed for 2 2 2 2 2 2 2 Diagnostics Number Planted in May 10 10 10 10 10 10 10 2006 (1st planting) Number Passing Health 17 14 18 6 9 15 9 Cert (Planted in Dec 2006) (2nd planting) Number Failing Health Cert 1 4 0 12 9 3 9 (Species (Species (Species failed at failed at failed at Mote) Mote) Mote) Number Deceased (as of Aug 2007)
1st planting 5 (Planting 1) 5 1 1 3 1 1
2nd planting 2 (Planting 2) 2 2 0 0 1 0
Total Planted 27 24 28 16 19 25 19 Total Deceased 7 7 3 1 3 2 2
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Coral Health Assessment
It is often difficult to graphically represent non-parametric data such as the mean
ranks used to generate the stats on our health assessment scores. However, what I’ve
provided are the median, min, and max for the condition and color scores for each coral
species at each site at the baseline, 6-month, and 15-month time points.
Table 1. Median (min, max) condition and color scores for seven species of coral at the baseline assessment – May 2006. Asterisks indicate significantly different groupings discussed in the text.
M. cavernosa D. stokesii S. radians D. clivosa M. annularis S. bournoni S. mechelinii Cond Color Cond Color Cond Color Cond Color Cond Color Cond Color Cond Color Control 5 4 5 4 6 4 6* 4* 6 4 6 4 6 4 (5,6) (4,4) (5,5) (3,4) (6,6) (4,4) (5,6) (4,4) (6,6) (4,4) (6,6) (3,4) (6,6) (4,4) TAL 5* 4 5 4 6 4 5* 3* 6 4 6 4 6 4 (4,5) (4,4) (5,6) (4,4) (6,6) (4,4) (5,6) (3,4) (6,6) (4,4) (6,6) (4,4) (4,6) (3,4) MML 6* 4 5 4 6 4 6* 4* 6 4 6 4 6 4 (4,6) (4,5) (5,6) (3,4) (6,6) (4,4) (6,6) (4,4) (6,6) (4,4) (6,6) (3,4) (5,6) (4,4)
Table 2. Median (min, max) condition and color scores for seven species of coral at the time of health certification – November 2006. Asterisks indicate significantly different groupings discussed in the text.
M. cavernosa D. stokesii S. radians D. clivosa M. annularis S. bournoni S. mechelinii Cond Color Cond Color Cond Color Cond Color Cond Color Cond Color Cond Color Control 3* 3 4* 4 5* 4 4 4 4.5 4 4* 4 5* 4* (1,5) (1,4) (1,5) (1,4) (1,5) (1,4) (1,6) (1,4) (3,6) (4,4) (3,5) (3,4) (1,5) (1,4) TAL 5* 3 5* 4 6* 4 5 4 6* 4 6* 3 6* 4* (4,6) (3,3) (5,6) (4,4) (6,6) (4,4) (2,5) (4,4) (5,6) (4,4) (6,6) (3,3) (6,6) (4,4) MML 6* 3 4 4 5* 4 4 4 4* 4 5* 3 4* 3* (5,6) (3,3) (4,6) (3,4) (5,6) (3,4) (2,5) (3,4) (3,5) (4,4) (1,6) (1,4) (2,5) (3,4)
Table 3. Median (min, max) condition and color scores for seven species of coral at the 15-month assessment – August 2007. Asterisks indicate significantly different groupings discussed in the text. There is no data for the MML D. clivosa, M. annularis, and S. mechelinii as those species failed the 6-month health certification process and were not planted in December 2006.
M. cavernosa D. stokesii S. radians D. clivosa M. annularis S. bournoni S. mechelinii Cond Color Cond Color Cond Color Cond Color Cond Color Cond Color Cond Color Control 2.5 3 2.5 1.5 4.5 3 5 3 4* 4 3.75 3 5 4 (1,5) (1,4) (1,6) (1,4) (1,5) (1,4) (1,6) (1,4) (1,5) (1,4) (1,6) (1,4) (1,6) (1,4)
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TAL 5 4 4 4* 5 3 5 3 5* 4 5 3 5 4 (1,6) (1,4) (3,6) (3.5,4) (4,5) (3,4) (4,6) (3,4) (2,6) (2,4) (1,6) (3,3) (4,6) (3,4) MML 6 4 2 3* 4 4 N/A N/A N/A N/A 3.25 3.5 N/A N/A (1,6) (1,4) (1,5) (1,4) (1, 5.5) (1,4) (2,5) (2,4)
Because these data were based on ordinal scales, our statistical analysis involved
the use of a Kruskal-Wallis Test. This test was applied to the mean ranks of the scores
for both condition and color respectively in order to compare each coral species across
the three different culture conditions - an artificial seawater, recirculating system in a
greenhouse (TAL); a land-based, natural seawater flow-through system (MML); and an
open ocean (―control‖) site.
Baseline assessments of both condition and color revealed a difference in the
Diploria clivosa fragments (p = 0.005 and p < 0.001 respectively). In both cases, those
fragments that were bound for Mote and the open ocean site had better condition and
color scores than those bound for the TAL. There was also a difference noted in terms of
condition for the Montastrea cavernosa fragments (p = 0.0359). In this case, the
fragments bound for Mote had better condition scores than those bound for the TAL.
This difference in condition/color of our fragments at the start of the study is likely due to
the variability within the parent colonies used to create our fragments. Furthermore,
upon examination of the actual condition and color scores for these two species, all
condition scores were either a 5 or a 6, and all color scores were between a 3 and a 4
Therefore, while there was a difference detected, all the fragments were considered to be
in good condition and in good color.
Assessments at three months for condition revealed differences in all species
except the Diploria clivosa and Stephanocoenia mechelinii fragments. The Dichocoenia
stokesii fragments housed at MML scored better (p = 0.0330) than those on the open
The Florida Aquarium Florida’s Wildlife Legacy Initiative SWG04-038 22 ocean site. The Montastrea annularis fragments housed at the TAL and on the open ocean site scored better (p = 0.0003) than those housed at MML. The Montastrea cavernosa and Solenastrea bournoni fragments housed at both inland culture sites scored better (p = 0.0019 and p = 0.0010 respectively) than those on the open ocean site. The
Siderastrea radians fragments housed at the TAL scored better (p < 0.0001) than those housed at MML and on the open ocean site.
Assessments at three months for color revealed differences in the Diploria clivosa, Solenastrea bournoni, and Stephanocoenia mechelinii fragments (p = 0.0173, p =
0.0010, and p = 0.0014 respectively). In the case of the Diploria clivosa fragments, those fragments housed at the TAL scored better than those on the open ocean site. The
Solenastrea bournoni fragments housed at both inland culture sites scored better than those on the open ocean site. The Stephanocoenia mechelinii fragments housed at both
MML and on the open ocean site scored better than those housed at the TAL.
Assessments at six months (at the time of health certification) for condition again revealed differences in all species except for the Diploria clivosa fragments. The
Dichocoenia stokesii fragments housed at the TAL scored better (p = 0.0063) than those on the open ocean site. The Montastrea annularis fragments housed at the TAL scored better (p = 0.001) than those housed at Mote. The Montastrea cavernosa fragments housed at both the TAL and Mote scored better (p < 0.001) than those on the open ocean site. The Solenastrea bournoni, Stephanocoenia mechelinii, and Siderastrea radians fragments housed at the TAL scored better (p = 0.0003, p < 0.0001, and p< 0.0001 respectively) than those housed at MML and those on the open ocean site. In each case, one or both of the inland culture sites scored better than the open ocean site. The reasons
The Florida Aquarium Florida’s Wildlife Legacy Initiative SWG04-038 23 for this are not entirely certain, but the results do suggest that stabilization of corals in land-based culture facilities after fragmentation may allow for them to be more successful once transplanted. In those cases in which fragments from the TAL scored better than those at MML, the reason was attributed to an overgrowth of Aptasia present in the flow- through system at MML that compromised the health of the study fragments housed there.
Assessments at six months for color revealed a difference in the Stephanocoenia mechelinii fragments. Those fragments housed at both the TAL and on the open ocean site had better scores (p < 0.0001) than those housed at MML.
Assessments at 12 months for condition revealed differences in the Diploria clivosa, Montastrea annularis, and Montastrea cavernosa fragments (p = 0.0112, p =
0.0007, and p = 0.0134 respectively). In all cases, those fragments that had been housed at the TAL scored better than those on the open ocean site. For the Montastrea cavernosa fragments, those that had been housed at MML also scored better than those on the open ocean site; however, there were no Diploria clivosa or Montastrea annularis fragments from MML for comparison as those fragments did not pass the health certificate process to allow for them to be planted in December 2006. These results again suggest that stabilization of corals in land-based culture facilities after fragmentation may allow for better transplantation success. Other considerations include possible site- specific differences as well as the fact that the ―control‖ fragments were planted six months prior to those from TAL (which included the summer months).
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Assessments at 12 months for color revealed a difference in the Montastrea cavernosa fragments. Those fragments that had been housed at MML had better scores
(p = 0.0486) than those on the open ocean site.
Assessments at 15 months for condition revealed a difference in the Montastrea annularis fragments (p = 0.0060). Those fragments that had been housed at the TAL had better condition scores than those present on the open ocean site. As with previous assessments, these results suggest that stabilization of corals in land-based culture facilities after fragmentation may allow for them to be more successful once transplanted.
There were no Montastrea annularis fragments from MML for comparison as those fragments did not pass the health certification process to allow for them to be planted in
December 2006.
Assessments at 15 months for color revealed differences in the Dichocoenia stokesii fragments (p = 0.0188). Those fragments that had been housed at the TAL had better color scores than those that had been housed at MML. This is likely due to the fact that all of the fragments from the TAL had color scores between 3 (lighter than normal) and 4 (good/normal color) whereas of the five fragments from MML, two were dead which yields a score of 1. The Dichocoenia fragments from MML that were living had scores similar to those from TAL.
The fragments that had died in the field were covered with coralline algae overgrowth. It is unknown if the overgrowth was the cause or a consequence of the death of the fragment.
Histology
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Figures 8a, 8b, 9 and 10 in Appendix E provide examples of normal sections of a
Montastrea sp. fragment and an abnormal section. We are currently compiling an atlas of normals for each species and will incorporate abnormal examples as they are available. It was our experience that histology, although a good tool to evaluate tissue pathology, was not currently useful in the health certification process. Until more information can be compiled as to the delineation of disease identification at the microscopic level, visual examination of the coral tissue based on the scores of condition and color defined previously, will be more applicable for health assessment purposes. However, we strongly urge the continuation of compiling histological study sets as the lack of such information impedes understanding of coral health in general.
Microbiology
Microbial community results were analyzed using a Jaccard Index (J.I.) of
Similarity which is a statistical tool used for comparing the similarity and diversity of sample sets. For our research, the Jaccard Index was calculated by the following equation:
CJ = j / (a + b – j) in which j = the number of carbon sources utilized by both samples, a = the number of carbon sources used by sample one, and b = the number of carbon sources used by sample two. This gives a proportion of faunal similarity. A value of 1.0 indicates 100% similarity and 0.0 indicates 0% similarity.
At the time of health certification, comparison of microbial communities from the surface mucopolysaccaride layer (SML) of healthy coral fragments maintained in both
The Florida Aquarium Florida’s Wildlife Legacy Initiative SWG04-038 26 land-based culture conditions were similar (see Table 2). Bolded results indicate the comparison of the same species at each site.
Comparison of the microbial communities from the SML of the coral fragments sampled from the land-based culture facilities in November 2006 at the time of health certification to the coral fragments sampled from the open ocean site in May 2007 (12- month assessment) were also similar (see Table 3).
Microbial analyses comparing the fragments from May 2007 (12 month) to those of July 2007 (15 month) (see Table 4) also revealed little difference between the microbial communities present. See below
We do not have baseline microbial information from the start of the study (at the time of fragmentation) as we were still evaluating the best techniques to employ. This portion of the evaluation is part of the second grant project.
Table 2: Microbial analysis comparing the SML of coral fragments at the two land-based culture sites. A Jaccard Index of Similarity was used to compare samples obtained at each site, and analysis was performed after 72 hours of incubation. Bolded results indicate the comparison of the same species at each site. Abbreviations are as follows: MC, Montastrea cavernosa; DS, Dichocoenia stokesii; SR, Siderastrea radians; DC, Diploria clivosa; MA, Montastrea annularis; SB, Solenastrea bournoni; SM, Stephanocoenia mechelinii.
Recirculating, Artificial Seawater System 72 hr
MC DS SR DC MA SB SM
Flow
Ocean System MC 0.87 1.0 0.97 1.0 1.0 1.0 1.0
- through Open Open through DS 0.87 1.0 0.97 1.0 1.0 1.0 1.0
SR 0.84 0.97 0.94 0.97 0.97 0.97 0.97
DC 0.74 0.87 0.84 0.87 0.87 0.87 0.87
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MA 0.87 1.0 0.97 1.0 1.0 1.0 1.0
SB 0.87 1.0 0.97 1.0 1.0 1.0 1.0
SM 0.83 0.90 0.87 0.90 0.90 0.90 0.90
Table 3. Microbial analysis comparing the SML of coral fragments at the two inland culture sites in November 2006 versus those on the open ocean site in May 2007. Samples were obtained from two fragments of each species at each site. Analysis was performed after 72 hours of incubation.
Coral Species TAL vs. Open Ocean Mote vs. Open Ocean
Montastrea cavernosa 0.87 1.0
Dichocoenia stokesii 1.0 1.0
Siderastrea radians 0.97 0.97
Diploria clivosa 0.94 0.81
Montastrea annularis 1.0 1.0
Solenastrea bournoni 1.0 1.0
Stephanocoenia mechelinii 1.0 0.90
Table 4: Microbial analysis comparing the SML of coral fragments at the two offshore, open ocean sites in May 2007 versus July 2007. Samples were obtained from one fragment of each species at each site. Analysis was performed after 72 hours of incubation. Because we only sample the SML from one fragment of each species at each site (i.e., one fragment of each species from the 1st planting and one fragment of each species from the 2nd planting), data was combined from both sites to compare the SML from both fragments of each species in May versus both fragments of each species in June.
Coral Species Jaccard Index of Similarity
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Montastrea cavernosa 1.0
Dichocoenia stokesii 0.97
Siderastrea radians 1.0
Diploria clivosa 0.94
Montastrea annularis 1.0
Solenastrea bournoni 1.0
Stephanocoenia michelinii 1.0
We did not compare the SML of the open ocean fragments to those at TAL/Mote at the time of transplantation (Dec 2006) because that was error in sampling from the off- shore site. We do have data comparing the open ocean fragments in May2006 back to the samples obtained from the land-based sites in Dec 2006. That is the information provided in Table 3. We do have a comparison of one fragment from the first/control planting (Site 1) to one fragment from the second/previously cultured planting (Site 2) in both May and July as well – this was not included in the original grant report, but is now listed in Table 5.
Table No. 5 Microbial analysis comparing the SML of coral fragments at the two offshore sites in both May and July 2007. Samples were obtained from one fragment of each species at each site. Analysis was performed after 72 hours of incubation.
Coral Species May 2007 July 2007 M. cavernosa 0.81 0.90 D. stokesii 1.0 0.93 S. radians 0.94 0.87 D. clivosa 0.79 0.90 M. annularis 0.94 0.90 S. bournoni 0.77 0.97 S. mechelinii 0.87 0.90
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DISCUSSION
The initial question posed in this study was whether aquacultured fragments could be utilized in reef restoration. Two additional questions emerged concerning the feasibility and direction of this effort: 1) will aquacultured corals become a vector for disease introduction when returned to a restoration site; and 2) are survival and growth success of reintroduced fragments affected by culture techniques?
We hope to have minimized concerns about disease introduction from aquacultured corals with the development of a health certification process that has received initial approval both from state and federal officials (FWC, FKNMS and
NOAA),. The certification process has actually been implemented in a recent study, independent of this project. The veterinarians involved in developing the certification process were approved to assess the study corals for this separate study (being conducted at the University of Miami). While extremely useful in developing the certification process, the use of histology and microbiology in a reasonable and practical health certification assessment is uncertain. Histological slides are useful in helping define anatomy of different species and to delineate disease processes but are limited at this point in time in predicting the development of a clinically observed diseased condition.
This study has provided information that the culturable microbial community on health fragments appears to be relatively similar between types of culture facilities and in the wild. Microbial communities did not significantly change as a result of being held in different culture facilities. Additionally, future studies will examine differences in community structure at time of fragmentation as well. Histology is useful in providing a
The Florida Aquarium Florida’s Wildlife Legacy Initiative SWG04-038 30 more detailed evaluation of disease processes but until further developed is of unknown value to health certification at this time.
With regards to culture techniques affecting growth and survival, it appears that if properly maintained, fragments can be successfully grown in relatively simple land-based systems. The overgrowth of Aptasia noted at the one facility could possibly be managed by more frequent inspection of the system and/or employment of biological control mechanisms such as predatory shrimp. In the field it was noted that the cement bases used for fragment attachment were rapidly covered with coralline algal overgrowth. The fragments that had died in the field were also covered with coralline algal overgrowth and it is unknown if the overgrowth was the cause or consequence of the death of the fragment. Evaluation of different types of attachment processes is recommended.
Transplantation of coral fragments at different times during the year and degree of
―healing‖ (coral tissue growing over cut skeletal margins) after fragmentation needs to be further addressed. It was noted that there was a higher incidence of mortality among the fragments planted in May of 2006 when compared to fragments planted in December of
2006. But the fragments planted in May were also recently fragmented whereas those planted in December had been in culture for seven months and had been allowed to heal before transplantation to the field. The two effects need to be dissected apart for stronger transplantation recommendations.
The health certification methods developed are designed to be applicable to all coral species. Available species at the time of the study were restricted to boulder types of coral. Future studies should attempt to incorporate branching species. Study colonies were also selected for size and suitability for fragmentation, and although genetically
The Florida Aquarium Florida’s Wildlife Legacy Initiative SWG04-038 31 identical colonies were preferred, they were not available for all species due to size of parent colony stock. Further studies evaluating size of fragment cut, and genetic variability of coral colonies and its impact on growth and survival are recommended
(growth being defined as growth over cut margins. Growth beyond the initial fragment size is minimal and will be monitored through periodic field assessments over the next several years).
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MANAGEMENT RECOMMENDATIONS
As the study advanced, many additional questions arose that could impact the successful use of aquacultured corals to rehabilitate reefs. Which species should be collected and recovered? What were appropriate culture parameters? Was there a way to mitigate potential for introduction of disease? Are there geographical and genetic concerns? Over-riding environmental concerns such as climate change and water quality are also factors in the effectiveness of coral restoration. To help focus future studies, the initial partnership expanded to include additional coral restoration stakeholders from around the state. To help guide future studies, the working group, known as the Florida
Cultured Coral Conservation Consortium (The Florida C’s,) adapted a ten point list from
Blankenship and Leber (1995) on a reasonable approach to marine stock enhancement.
In sum, the ten points include: 1) prioritize species, 2) identify genetic objectives, 3) define quantitative measures of success, 4) avoid inbreeding, 5) include disease and health management, 6) consider ecological, biological life-history patterns, 7) assess stocking impact, 8) identify optimum release protocols, work with pilot releases to help define, 9) identify economic and policy guidelines, and 10) use adaptive management concepts (continually revisit and revise).
A recent NOAA workshop (August 22 and 23, 2007) aimed at updating a 1993 publication on the ―Guidelines and Recommendations for Coral Reef Restoration in the
Florida Keys National Marine Sanctuary‖ (Miller et al., 1993) will also include many of the recommendations presented in this study.
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CONCLUSIONS
In sum, it appears that aquacultured coral fragments provide a promising, valuable tool for coral reef restoration. A detailed history, including careful evaluation of adherence to best management practices, and visual inspection by qualified individuals currently will be most useful for health certification. Several of the veterinarians in this study have been approved by the various state and federal agencies to apply the health certification process to a select group of corals in Florida. These veterinarians are also
USDA accredited and can demonstrate experience with coral systems, health and disease.
Future studies will be focusing on modification of attachment techniques, genetic histories, and additional microbiological community analysis including assessment at time of fragmentation through transplantation.
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LITERATURE CITED:
Blankenship, H.L., and K. M. Leber. 1995. A Responsible Approach to marine stock enhancement. American Fisheries Society Symposium 15:167-175.
Borneman, E. H. and J. Lowrie. 2001. Advances in captive husbandry and propagation: an easily utilized reef replenishment means from the private sector? Bulletin of Marine Science 69 (2): 897-913.
Breitbart, M., Bhagooli, R., Griffin, S., Johnston, I., and F. Rowher. 2005. Microbial communities associated with skeletal tumors on Porites compressa. FEMS Microbiology Letters 243: 431-436.
Brown, B. E. and J. C. Bythell. 2005. Perspectives on mucus secretion in reef corals. Marine Ecology Progress Series 296: 291-306.
Clark, S., and A.J. Edwards. 1995. Coral transplantation as an aid to reef rehabilitation: evaluation of a case study in the Maldive Islands. Coral Reefs 14: 201-213
Cooney, R. P., Pantos, O., Le Tissier, M.D.A., Barer, M.R., O'Donnell, A.G., and J. C. Bythell. 2002. Characterization of the bacterial consortium associated with black band disease in coral using molecular microbiological techniques. Journal of Environmental Microbiology 4: 401-413.
Custodio, H. M , and H. T. Yap. 1997. Skeletal extension rates of Porites cylindrica and Porites (Synaraea) rus after transplantation to two depths. Coral Reefs 16: 267-268
Delbeek, J.C. 2001. Coral farming: Past, present and future trends. Aquarium Sciences and Conservation 3 (1-3): 171-181.
Ducklow, H.W. and R. Mitchell. 1979. Composition of mucus released by reef coelenterates. Limnology and Oceanography 24: 706-714.
Frias-Lopez, J., Klaus, J.S., Bonheyo, G.T., and B. W. Fouke. 2004. Bacterial community associated with black band disease in corals. Applied Environmental Microbiology 70(10): 5955-5962.
Gil-agudelo, D.L., L. Ali-hassan, K. Kim, and G.W. Smith. 2006. Characterization of coral surface microbiota using metabolic profiling. Proceedings of the 10th International Coral Reef Symposium, 1:149-152.
Kline, D. I., Kuntz, N.M., Breitbart, M., Knowlton, N. and F. Rohwer 2006. Role of elevated organic carbon levels and microbial activity in coral mortality. Marine Ecology Progress Series 314: 119-125.
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Lindahl, J. 2003. Coral reef rehabilitation through transplantation of staghorn corals: Effects of artificial stabilization and mechanical damages. Coral Reefs 22 (3): 217-223.
Lindahl U. 1998. Low-tech rehabilitation of degraded coral reefs through transplantation of staghorn corals. Ambio 27(8): 645-650
Miller, S.L, McFall, G.B. and A.W. Hulbert. 1993. Guidelines and recommendations for coral reef restoration in the Florida Keys National Marine Sanctuary. Workshop Report. A Publication of the National Undersea Research Cetner pursuant to NOAA Award # 40AANC300210.
Pantos, O. and J. C. Bythell. 2006. Bacterial community structure associated with white band disease in the elkhorn coral Acropora palmata determined using culture- independent 16S rRNA techniques. Diseases of Aquatic Organisms 69: 79-88.
Pantos, O., Cooney, R. P., Le Tissier, M., Barer, M.R., O'Donnell, A.G. and J. C. Bythell. 2003. The bacterial ecology of a plage-like disease affecting the Caribbean coral Montastrea annularis. Environmental Microbiology 5(5): 370-382.
Reshef, L., Koren, O., Loya, Y., Zilber-Rosenberg, I., and E. Rosenberg. 2006. The coral probiotic hypothesis. Environmental Microbiology. Online early.
Ritchie, K. B. 2006. Regulation of microbial populations by coral surface mucus and mucus-associated bacteria. Marine Ecology Progress Series 322: 1-14.
Ritchie, K. B. and G. W. Smith. 2004. Microbial communities of coral surface mucopolysaccharide layers. Coral Health and Disease. R. E. and Y. Loya. New York, Springer-Verlag: 259-264.
Rohwer, F., Breitbart, M., Jara, J., Azam, F., and N. Knowlton. 2001. Diversity of bacteria associated with the Caribbean coral Montastrea franksi. Coral Reefs 20: 85-91.
Rohwer, F., Seguritan, V., Azam, F. and N. Knowlton. 2002. Diversity and distribution of coral associated bacteria. Marine Ecology Progress Series 243: 1-10.
Rosenberg, E., and L. Falkovitz. 2004. The Vibrio shiloi/Oculina patagonica model system of coral bleaching. Annual Review of Microbiology 58: 143-159.
Sekar, R., Mills, D.K., Remily, E.R., Voss, J.D., and L. L. Richardson. 2006. Microbial communities in the surface mucopolysaccharide layer and the black band microbial mat of black band-diseased Siderastrea siderea. Applied and Environmental Microbiology 72: 5963-5973.
Smith, L.D. and T.P. Hughes. 1999. An experimental assessment of survival, reattachment and fecundity of coral fragments. J. of Experimental Microbiology 72: 5963-5973.
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Spong, K. and T. Chen. 2003. Coral transplantation: Regeneration and growth of Acropora fragments in a nursery. Restoration Ecology 11 (1):62-71.
Van Treeck, P., and H. Schumacher. 1997. Initial survival of coral nubbins transplanted by a new coral transplantation technology--options for reef rehabilitation. Marine Ecology Progress Series 150: 287-292.
Wegley, L., Y. Yu, Breitbart, M., Casas, V., Kline, D.I. and F. Rohwer. 2004. Coral- associated archaea. Marine Ecology Progress Series 273: 89-96.
Yap, H.T., Alino, P.M., and E.D. Gomez (1992) Trends in growth and mortality of three coral species (Anthozoa: Scleractinia), including effects of transplantation. Marine Ecology Progress Series 83: 91-101.
Yap, H.T., Alvarez, R.M., Custodio III, H.M., and R.M. Dizon. 1998. Physiological and ecological aspects of coral transplantation. J. Exp. Mar. Biol. Ecol. 229: 69-84.
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APPENDICES
A – Health Assessment Form ...... 36
B – Guidelines for Evaluating Corals for Health Certification ...... 37
C – Health Certificate Protocol ...... 40
D – Histology Protocol ...... 43
E – Photographs ...... 44
The Florida Aquarium Florida’s Wildlife Legacy Initiative SWG04-038 38 Appendix A
Coral Health Assessment Form Fragment Number Species Size Condition Color Comments
Condition: 1: dead, 2: <25% of tissue alive, 3: 25-50% of tissue alive, 4: 50-75% of tissue alive, 5: 75-95% of tissue alive, 6: no apparent tissue loss Color: 1: 100% bleached, 2: partial bleach, 3: lighter than normal, 4: good color Comment codes: TS: tissue severed, MD: metal deposits present, F: fouling organisms present, CS: cracked skeleton without severed tissue
The Florida Aquarium Florida’s Wildlife Legacy Initiative SWG04-038 39 Appendix B
Guidelines in Preparation for Coral Health Certification
Veterinarians involved with the coral health certification process must be USDA accredited, with coral health and disease training and/or experience, and familiarity with the aquaculturists and the culture facility (in veterinary medicine, this is known as establishing a good VCPR (Veterinary/client/patient relationship). The veterinarian should be working with the aquaculturists and facility well in advance of the health certification process.
The following is a list of subject areas with which the veterinarian should be familiar:
1. Collection History
a. Permitting process for corals b. Chain of custody: permit through FKNMS/NOAA c. Collection information i. Condition (1-6); (1-7 scale used for cultured fragments) ii. Color (1-4) iii. Disease iv. Growth anomaly v. Competing algae vi. Other organisms
2. Culture Information
a. System set up/design i. Recirculating system 1. Holding tank configuration structure (L x W x H); water depth; 2. Filtration: a. Biological type (if present) b. Mechanical type (if present) c. Chemical type (if present) 3. Water flow rates through holding tank (s) 4. Surge device configuration/rate ii. Flow through 1. Holding tank configuration structure (L x W x H); water depth; 2. Water flow rates through holding tank (s) 3. Surge device configuration/rate b. Coral fragment base/attachment configuration/materials
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c. Water quality information/management i. Parameters tested/periodicity of testing/acceptable ranges/records of testing: 1. Temperature 2. pH 3. Ammonia, nitrite, nitrate 4. Phosphate 5. Hardness 6. Salinity 7. Alkalinity 8. Calcium 9. Selenium and Mg (if possible) 10. Light reading 11. Other parameters/observations should be noted if unusual (e.g., turbidity, color) ii. Management for each parameter above (types of additions/water changes)/records of these d. History of disease problems i. Brief written description of disease events and resolution; include dates; number of fragments affected; species affected
3. Culture Information/History: Biosecurity/water supply
a. Artificial seawater i. Source of freshwater (potential for contamination) 1. ―Protected‖ source (deep well/spring?) a. If not protected what biosecurity/type of disinfection used prior to use (methods) 2. Record of source water testing i. Temperature ii. pH iii. Ammonia, nitrite, nitrate iv. Phosphate v. Salinity vi. Alkalinity vii. Calcium viii. Selenium if possible ix. Mg is possible x. Other parameters/observations should be noted if unusual (e.g., turbidity, color) ii. Seawater mix used: Instant Ocean; Reef Crystals; other? b. Caribbean/local (Keys) seawater—should be from same location as corals (―same location‖ to be defined by FKNMS); documentation c. Coral parent colonies will be collected from an area considered ―local‖ to region for stock enhancement (as defined by FKNMS); documentation
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d. Corals from ―different locations‖ in the Keys should not be mixed in culture (FKNMS definition for ―different locations‖) e. Documentation required verifying absence of Indo-Pacific (or from any other are) corals/organisms. Only SE Gulf/SW Atlantic or Keys organisms allowed in systems f. Presence of competing algae (non-zooxanthellae): filamentous/phytoplankton i. Management protocol (including use of chemicals?) g. Presence of other organisms i. Management protocol (including use of chemicals?)
4. Training and Experience with Corals (through sanctioned training programs/work with sanctioned veterinarians/disease course work and experience)
a. Familiarity with growth characteristics and normal acceptable variations (growth, color) for species in culture b. Familiarity with clinical signs of disease in corals i. Common diseases of concern (field and culture) ii. Assessment based on tissue condition; color iii. Disease diagnostics/sampling methods iv. Assessment
The Florida Aquarium Florida’s Wildlife Legacy Initiative SWG04-038 42 Appendix C
Coral Health Certification Protocol
Recommendations developed by The Florida Aquarium and the University of Florida, Tropical Aquaculture Laboratory
1. Collection History
a. Chain of custody: permit through FKNMS/NOAA b. Collection information i. Source of coral (e.g., rescue donation, grounding opportunity, permitted collection, or purchased from other source) ii. Condition (1-6); (1-7 scale is for fragments) iii. Color (1-4) iv. Growth anomaly v. Other potential disease issues
2. Culture Information/History: Biosecurity/water supply
a. Artificial seawater i. Type/brand ii. Source of freshwater (should be approved in advance) b. Caribbean/local (Florida Keys) seawater— seawater should be from within the same region of the Florida Keys from which corals were collected (e.g., Upper, Middle, Lower Keys, or Dry Tortugas) c. Mean salinity throughout culture period d. Photoperiod and type of lighting used throughout the culture period e. Documentation required verifying absence of any corals or organisms which are not from the Western Atlantic, Gulf of Mexico, or Florida Keys f. Only SE Gulf/SW Atlantic or Keys organisms allowed in systems g. Presence of visual diseases or abnormalities i. Disease 1. Description 2. Diagnostics done? ii. Growth anomaly 1. Description iii. Treatment protocol h. Presence/extent of competing algae (non-zooxanthellae): filamentous/phytoplankton. Treatments? i. Presence/extent of other organisms. Treatments?
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3. Visual inspection: Categorical Classification of Coral Health Parameters
Individual Fragments will be assessed, but status of species population as a whole will also be assessed.
If a group of fragments of one species is exhibiting variable health conditions, fragments rated 1-5 condition, 1-2 color, or have other disease conditions should be separated from the remaining colonies for purposes of evaluation; general health history from the aquaculturist of the population will be requested. These fragments will then be assigned a consideration score. This consideration score will be used for long term evaluation of species culturability and culture conditions and will also help avoid release of chronically diseased corals. All fragments that are assessed to be ―not acceptable, not releasable at this time‖ will be allowed to remain in culture for potential re-introduction and re-assessment if their consideration score is 2 or greater.
CRITERIA/SCORES (The 3 Cs)
a. Condition score 1- Dead 2- < 25% of tissue alive 3- 25-50% of tissue alive 4- 50-75% of tissue alive 5- 75-95% of tissue alive 6- No apparent tissue loss 7- Growth over formerly dead tissue/ Overgrowth over edges
b. Color score 1- 100% bleached 2- Partial bleach 3- Lighter than normal 4- Good color
c. Consideration score 1- Failed 10 assessments 2- Failed 7-9 assessments 3- Failed 4-6 assessments 4- Failed 1-3 assessments 5- First assessment
4. Overall Assessment of Corals: Population Assessment/Acceptable Parameters
a. Evidence of growth/thriving in culture/acceptable tissue condition: condition score 6 or 7 b. No significant evidence of bleaching: color score 3 or 4
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c. Consideration score: 2 or greater is acceptable; i. Consideration score of 1 will result in fragment designation of ―not acceptable, not releasable, permanent status‖ (see section 6 below) d. No evidence of significant other visual diseases
5. Verification of Regional Collection and Re-Introduction Compatibility
a. Verification required that coral will be re-introduced to the field within the same region of the Florida Keys from which they were collected (either Upper, Middle, and Lower Keys, or Dry Tortugas), unless compelling scientific information on genetic composition of any given coral species dictates otherwise
6. Disposition
a. Based upon history, adherence to biosecurity, origin of parent colonies, water supply requirements, visual inspection (with possible diagnostic and microbiological support) stocks will either be deemed: 1) acceptable and healthy for release; 2) not acceptable, not releasable at this time; or 3) not acceptable, not releasable, permanent status i. Acceptable and healthy for release 1. Fragments will have to be released within 30 days from evaluation; any changes in health status in this time period should be reported to FKNMS prior to release for consideration ii. Not acceptable, not releasable at this time 1. This will be based on assessment/scores, overall in culture species population status, and information on species culture growth characteristics 2. Description of disease problem (this can be categorical based on assessment sheet and can have percentage tissue affected for each fragment) and/or reason for rejection 3. FKNMS to provide guidance on disposition iii. Not acceptable, not releasable, permanent status 1. Evidence of major disease issue (especially presence of ―field disease type‖ clinical signs) may result in not releasable, permanent status 2. Description of disease problem (this can be categorical based on assessment sheet and can have percentage tissue affected for each fragment) and/or reason for rejection 3. FKNMS to determine disposition
The Florida Aquarium Florida’s Wildlife Legacy Initiative SWG04-038 45 Appendix D HISTOLOGY PROTOCOL FOR CORAL PROJECT
1. Filter sea water through 0.2um filter to be used for fixative solution 2. Prepare fixative solution using 4 parts Z-Fix® to 1 part filtered sea water 3. Using a tile saw, cut coral fragments measuring less than 2cm x 2cm x 1cm and including healthy and diseased tissue 4. Place each coral fragment in a container with fixative so that fixative to coral ratio is at least 10:1 5. Fix for at least 24 hours, gently agitate (swirl fixative in the container) every few hours 6. Empty fixative from container, and refill the container with clean seawater or tap water, change water 3-5 times 7. Samples to be agar enrobed which will help maintain tissue orientation a. Make 1.5% agarose solution (15g SeaKem® Agar/L water) using hot (90oC) distilled water with stirring b. Allow agarose to cool to 60oC or the point when the agar is almost solid c. Drain specimens well and blot dry with paper towels d. Place coral fragments in metal weigh boats and cover with fragments with agar e. Preheat vacuum oven to 40-56oC (gel temperature) f. Place in vacuum oven and pull pressure of 25mmHg twice g. Scrape away agar until there is ¼‖ agar on each sides, exposing the internal canals 8. Specimens will then be taken to the University of South Florida Pathology Lab for decalcification and processing of histology slides a. Raise sample above container bottom so that decal solution penetrates all surfaces b. Swirl decal solution in container several times a day to expose tissue to fresh solution c. Change decal solution twice a day d. Be careful to remove fragment from decal solution as soon as decalcified to avoid overexposure (leads to poor staining) e. Rinse fragment in deionized water f. Process as normal in Paraffin for histology g. Stain with routine stain (H & E) or special stains as needed
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Appendix E.
Figure 1. The Florida Aquarium’s coral propagation facility, ―Coral Farm”, on the exhibit pathway.
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Figure 2. The greenhouse culture system at the Tropical Aquaculture Laboratory, University of Florida.
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Figure 3. Open-flow system at the Mote Marine Laboratory on Summerland Key.
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Figure 4. – The Miss Beholden grounding site on Western Sambo reef, the current restoration site.
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Figure 5. Cutting coral colonies into fragments using a tile saw with sea water as a coolant. Measuring individual pieces.
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Figure 6. Attaching the coral fragment to a concrete base using a 2-part epoxy, Z-Spar®.
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Figure 7. Fragments in culture at the Tropical Aquaculture Laboratory, University of Florida.
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Figure 8a. Oral cross-section 40x (Montastraea sp).
Figure 8b. Oral cross-section 100x (Montastraea sp).
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Figure 9. Longitudinal section 100x ((Montastraea sp).
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Figure 10. Abnormal section 40x ((Montastraea sp).
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Figure 11. Fragment of Dichocoenia stokessi after 3 months in the field.
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Figure 12. Location of Western Sambo Reef
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