Sand Dollar Protocol
8/4/12- Bleaching and lesioned sand dollars from East Sound. Sand dollars were collected on August 4th at __ Beach in East Sound. They were kept separated in two buckets- healthy and bleached/lesioned for transport back. Overall, it was very difficult to find specimens with no signs of disease, prevalence seems very high. We tried to collect an assortment of sizes and pathologies.

I. Gross Dissection
Individuals were cracked in half by hand along the axis of symmetry if possible to access the organ cavity. Organs identified and examined were the digestive tract and the gonads.
Observations:
Healthy (SB-1-H)- coelomic fluid clear, organs dark.
Diseased (SB-3-B)- coelomic fluid milky, though could have been disruption of gonads (male), which were white. Interior overall looked pale. Digestive tract looked normal. Objectively, bleached specimens seemed easier to crack open, though differences in size were not accounted for.

External description:
-Normal: normal individuals have short fuzzy spines covering whole body surface which move actively when removed from water, which visually looks somewhat shimmery. They are dark purple across the whole individual and bury themselves within a few minutes when placed in water.
-Bleached: bleached individuals have lightened discoloration on first oral surface, then aboral surface on end that burrows, end associated with largest petalloid. Spines and epidermis turn pale and white quite gradually (temporally), and leading edge of infection cuts straight across individual. In advanced infection, the bleaching works its way across the entire individual, then spines are lost from bleached end and the individual dies. In small individuals, the radiating food grooves may turn pale first, or the underside may lose spines and color without any signs of bleaching on the aboral surface.
-Lesions: lesioned individuals (all also bleached) have green lesions associated with injuries and/or small (1-2 mm) spots. The calcium is exposed and turns green, potentially with algal fouling.

II. Histology
Organs from SB-1-H and SB-3-B were collected and put into cassettes, then Davidson's for 48 hours, then 70% ethanol but will not be sent for analysis.
Whole small (<3 cm) individuals were fixed in formalin for 48 hours then transferred to ethanol. They will be put into cassettes, wrapped in ethanol soaked paper towels, triple bagged, and sent to Cornell for histological prep, then sent to Drew's friend at the Bronx Zoo who specializes in echinoderm histology. Preserved 4 diseased and 3 healthy small individuals.

III. Cultures
Two sets of plates were prepared for each individual on TCBS and Marine Agar. Each plate was divided in half, then swabbed with interior coelomic fluid on one side and a scrape of the exterior epithelial/spine fauna from the same individual. We did not take an internal swab of the smallest individual, which was prepared whole for histology. For lesioned individuals, the lesion itself was swabbed for the exterior treatment, and the internal fluid was just taken from where it was. A total of 14 plates were prepared- 7 agar, 7 TCBS, two from one individual.

Accession #	Bleaching	Lesions	Histo?	Plates	Swab
SB-1-H (large)	no	no	organs	Marine Agar & TCBS	Interior and Exterior
SB-6-H (small)	no	no	whole individual	Marine Agar & TCBS	Exterior
SB-2-B	yes	no	no	Marine Agar & TCBS	Interior and Exterior
SB-3-B	yes	no	organs	Marine Agar & TCBS	Interior and Exterior
SB-4-BL	yes	yes	no	2x Marine Agar & TCBS lesion and bleaching	Bleaching Interior and Exterior, Lesion Interior and Exterior
SB-5-BL	yes	yes	no	Marine Agar & TCBS	Lesion Interior and Exterior

III. Transmission Experiment
To examine whether the disease was transmissible from diseased to healthy specimens, we set up a sea table to control exposure. We set up 6 mesh sided boxes in the same sea table and filled them with 2-3 cm of mud (with rocks removed) taken from Argylle Lagoon (a site with much fewer sand dollars that may have been struck with the same epizootic in the last few years). Tubes were set up in the sea table to approximate equal flow to each mesh sided box. To establish identity, we measured each sand dollar with calipers to the nearest mm along the axis of symmetry of the sand dollar. Some healthy individuals have signs of previous predation or injury, but we only selected these if it looked as though their injuries were completely healed (injured area covered with spines and purple epithelium again). Three boxes served as controls with three healthy individuals per box. We placed these individuals randomly with respect to size and previous predation. Three boxes are our transmission experiments, with one severely diseased individual sharing a box with two healthy individuals. In addition, we have held a small number of the smallest individuals in boxes to the side, three healthy and three bleached. All three healthy baby sand dollars have been preserved for histology. We checked on the health of the sand dollars every other day since the start of the experiment, taking notes on severity of bleaching and whether individuals were touching or not. In the field we observed that many individuals stacked within the sediment, and we hypothesize that stacking may increase contact and therefore may increase transmission.


-mesh sided boxes in common sea table (same water in all boxes)
-individuals placed touching in boxes at start
-new mud placed in boxes immediately prior to placement
-length is measured along the axis of symmetry of the sand dollar
-Will check on health of sand dollars daily.
-Some healthy sand dollars had evidence of previous injuries or predation, but all injuries were completely healed.
-All extra sand dollars (all are bleached or lesioned) are being kept in separate sea table.

Box 3 2 Healthy -51 mm -66 mm 1 Bleached -77 mm	Box 6- Control 3 Healthy -78 mm -75 mm -58 mm (previous predation)	Box 2- Healthy baby holding box -22 mm (has lightening to underside) -31 mm -30 mm
Box 8 - Control 3 Healthy -48 mm -71 mm (previous predation) -74 mm (previous predation)	Box 13 2 Healthy - 72 mm -67 mm 1 Bleached -66 mm	Box 4- Bleached baby box -29 mm -30 mm -26 mm
Box 16 2 Healthy -46 mm -68 mm 1 Bleached -89 mm	Box 14 3 Healthy -72 mm -49 mm -54 mm	empty

IV. Bacterial and Sand Dollar tissue preservation for future DNA extraction and PCR

We plan on isolating bacterial colonies from the plate cultures, culturing in a tub, and freezing them down for potential future research.