Photo credit: NOAA Health Assessment of Florida's Long-Spined Sea Urchin, Diadema antillarum: A Keystone Species on Florida s Coral reefs The Diadema Team!
The Diadema Team: University of Florida: Ruth Francis-Floyd Roy Yanong Shirley Baker Debbie Pouder Mark Flint Tom Waltzek Nicole Stacy Kayla Ripple Diadema Team Dec 2014 FWC: Jan Landsberg Yasu Kiryu John Hunt Bill Sharp Gabe Delgado Melissa Gilbert Morgan Beaton Guest Experts: Marin Moe Aquaculturist extraordinaire Greg Beck University of Massachusetts, Boston Roxanna Smolowitz Roger Williams University Thierry Works USGS Hawai i David Vaughan and Ken Leber Mote Marine Lab John Than Florida Aquarium
The Diadema Story Photo Credit: Wikipedia Diadema antillarum = the Long-Spined Sea Urchin The genus Diadema six species included D. antillarum Atlantic D. mexicanum Eastern Pacific D. savignyi East Africa D. setosum East Africa, Red Sea D. palmeri New Zealand, Australia D. paucispinum Hawaii, South Pacific Diadema Characteristics: Long spines Widespread, tropical Ecologically important Source: Muthiga, N. and McClanahan. 2013. Diadema. In: Sea Urchins: Biology and Ecology, Third Edition; J.M. Lawrence (ED). Elsevier.
Ecological Importance of Diadema: Photo Credit: Wikipedia Critically important herbivores Prevents overgrowth of macroalgae Many other herbivores over-fished Niche not filled in most reefs Human grooming required for successful reef restoration Not feasible or sustainable Powerful Bio-eroders Photo Credit: NOAA Recycle coral skeletons to sand Prevents accumulation of coral rubble Clean substrate required for recruitment Corals and Diadema itself Mechanical Protection of Reef No one wants to step on these!
The Diadema Die-Off of 1983-1984 Impacted area: Western range Caribbean Sea, Florida Keys Duration of event: 13 months Impact: >90% mortality Diadema populations have never recovered! Rare exceptions Coral reef habitat has continued to degrade Habitat destruction Over-Fishing Climate change, warmer temps Loss of Diadema Other??? Photo Credit: FWC
State Interests 1. Substantial progress with reproduction of some coral species Florida has 1.4M acres of coral reef habitat Most is in poor condition and declining 2. Overgrowth of algae, poor recruitment remain problematic 3. Interest in release of cultured Diadema, or translocation Genetic homogeneity already demonstrated Concern about release or spread of disease Aquaculture is a work in progress 4. Need to understand health status of current wild population 5. Need to understand normal flora 6. Need standardized diagnostic criteria
What do we know About Disease in These Organisms? Traditional Approach. Emphasis on Infectious Agents Parasitic Fungal Bacterial Viral Or.Emphasis on Events Caribbean 1983-84 Nova Scotia 1980s, 90s Norway 1980s Curacao New Zealand Canary Islands Or Described Diseases Bald Sea Urchin Disease Paramoeba invadens Neoparamoaba branchiphila Vibrio alginolyticus Chlostridium
What about Bald Sea Urchin Disease??? First description CA tide pools, 1970 Since then reported all over. Reflects symptoms of disease, not an etiology Loss of spines, pedicellariae, tube feet If test is perforated it is lethal If test not perforated, recovery reported Generically associated with bacterial disease Transmission reported via direct contact with necrotic material Abrasion seems required in many reports
Biscayne Bay, Sep 1983: Clostridial infections proposed in lab-reared D. antillarum Bauer and Agerter, 1987 D. Antillarum (n=2, died in aquarium) Florida waters (flow-through system) Clostridium perfringens Clostridium sordellii Photo from Wikipedia Associated with disease and mortality in tank-raised animals!! Effort to fulfill Koch s postulates effective. Very small test population. These are obligate anaerobes very difficult to find in aquatic animals Clostridium was detected using gram stains on material From anaerobic culture of extracts of digestive tract and gonadal tissue
D. Antillarum sub-species in Canary Islands Mortality Event, October 2009 - April 2010 Dykova et al, 2011; Clemente et al, 2014 Three factors may have contributed to disease. 1. V. alginolyticusae in coelomic fluid of diseased animals Positive cultures diseased and healthy animals (Were healthy animals getting ready to break?) Previous reports coelomic fluid should be sterile ) 2. N. branchiphilum? Separate study isolated amoeba from diseased urchins (Dykova et 2011) Previously isolated from healthy urchins (Dykova et al 2007) Organism found in coelomic fluid 3. Toxic epibenthic bloom of Ostreopsis sp. Co-occurred with this mortality event High numbers dinoflagellates overgrowing turf macroalgae Previous reports of sea urchin mortality associated with Ostreopsis (Shears and Ross 2009) (We know sea grasses magnify brevitoxin )
Our Approach: Diadema Workshop Dec 17-19, 2014 (Marathon, FL) Topics: 1. Clarify need for project, state interest 2. Review health certification protocols for corals 3. Diadema culture where are we? 4. Sea urchin anatomy and physiology 5. Review of sea urchin diseases/ diagnostic methodologies 6. Discussion of clinical techniques: Gross anatomy, necropsy Microantomy, histology Parasitology Cytology Microbiology Virology Immunology 7. Wet Labs Practice Sessions
Development of Standardized Protocols for Diadema Diagnostics Developed Diagnostic Flow Chart Initially based on flow chart developed for fish diagnostic work at UF Tested at Dec workshop Second workshop held in Jan at UF-TAL Final protocol developed prior to March collections Station 1: Immunology, Microbiology & Histology Station 2: Necropsy, Cytology, Parasitology, Virology, Tissue Archive
Field Collections Sampling protocol Spring and Fall Collections Three Regions: Upper, Middle and Lower Keys Numbers targeted: 25 adults/ region/ collection 25 juveniles/ region fall only Weather complications Small craft advisories Poor visibility in rough weather Field crews to record Collection date, time and location Water depth, temp, conditions Substrate type Animal behavior: Sheltered, Protective Posture: Adhesion to substrate, position of spines
Actual Samples Obtained: March 2015: Lower Keys - 25 Middle keys - 25 Upper Keys - 2 April 2015 * : Upper Keys - 22 September 2015: Lower Keys 46 Middle Keys 71 Upper Keys - 74 Holding time varied from a few hours to a full week. *Animals collected in April were transported to the UF Tropical Aquaculture Lab (Ruskin, FL) and held overnight in artificial sea water.
All Urchins: Behavioral Assessment, Physical Exam Urchins were assigned to one of two teams: Team 1: Immunology, Microbiology & Histology Team 2: Gross Necropsy and Cytology
Behavioral Assessment: Behaviors: 1. Adherence to substrate 2. Posture 3. Seeks shelter 4. Response to touch 5. Attempts to right itself Response to Touch Righting Itself Seeks Shelter
Physical Exam: 1. Weight 2. Physical Descriptions Gross Exam: Color Condition of spines Exam with Dissecting Scope: Condition of spines Damage to test Presence of ulcers Aristotle s Lantern 3. Test Diameter Height and Diameter Easiest and more accurate after hair cut
Necropsy 1. Gross exam Macroscopic Dissecting Scope 2. Cytology (amoeba exam) Fecal pellet preserved in Zn-PVA Coelomic fluid smear Fecal smear 3. Exam of Wet Mounts Primarily looking for protozoa Tissues examined: Coelomic fluid Gut contents Gonad Medial test Spine with attached test
Preliminary Results of Parasitology: External Parasites: Occasional amphipods Potential to cause damage in culture situation Internal Protozoa (GI tract primarily): Ciliates: Four seen with some regularity Conformation of ID in progress Presumed normal flora Unsure if Handling/ transport may affect load Amphipod Photo credit: P. Baker lab Flagellates Characterization pending Difficult to distinguish form motile blood cells Photo credit: H. Walden No amoeba seen (yet) in processed samples
Immunology Dr. Greg Beck, U. Mass - Boston 1. Collect 5-7 ml Coelomic Fluid 2. Mix with 1 ml cold modified Alsever s Solution 3. Refrigerate 4. March collections only (n=20) Fed Ex, on ice to Beck Lab!
Immunology Results (Beck Lab): D. antillarum from Keys very similar to D. antillarum from St, Croix Is this representative of all Caribbean D. antillarum? Humoral assay (LPS Stimulation) interesting for all D. antillarum tested Most D. antillarum do not respond to LPS stimulation Non-diadema urchins tested do respond to this test Coelomocytes are large and nongranular Collaboration with Dr. Nicole Stacy in progress to further characterize Cytometry data from Beck lab Source: Greg Beck
Microbiology Tissues Cultured: Spine (with test attached) Coelomic Fluid Lesions (rare) Method: Rinse with sterile sea water Heat sterilize instruments Modification from Klinger et. al. 2003 JZWM 34(2):206-207 Media: BHI broth Rock overnight Plate if cloudy Enriched blood agar
Preliminary Results on Microbiology: March Results: 71 isolates, most from spines Of 30 animals tested, CF + in 3* 26 cannot be identified using Biolog qpcr to be attempted Most are Vibrios V. harveyii most common Microbiome to be elucidated using 16S *Evidence of GI contamination demonstrated in some specimens
Histology 1. Spines removed ( hair cut ) 2. Animal cut it half 3. Placed in fixative Fixative: 1 part Formaldehyde: 9 parts sea water Processing in progress by FWC Aquatic Animal Health group
The Discovery of Intestinal Holes Photo by Y. Kiryu Needles holes in intestinal wall This could result in bacterial contamination of coelomic fluid! Photo by Y. Kiryu
Collections of Tissues for Virology/ Archive Virology: Tissues collected in RNA-later Frozen at -80 F Tissues selected: Spine/test, Gut, Gonad Tissue Archive: Tissues collected in Whirl-paks Frozen at -80 F Tissues Selected: Spine/ test, Gut, Gonad, Coelomic Fluid Archive to be maintained by FWC
Findings so far (Some Cool Stuff!) Potential for non-lethal examination: Behavioral Assessment Physical Examination Gut Cytology Coelomic Fluid Culture Need to characterize gut flora: Some descriptions in literature Affected by environmental change/ transport? Can it contribute to disease under some circumstances? Microbiology is very interesting: Coelomic fluid looks like it will be a very useful diagnostic Gut contamination of fluid a concern Hoping to ID some of the unknowns Very interested in presence/ absence of Clostridium
Questions?