Methods

           Mosquito Lagoon, which is the northernmost part of the Indian River Lagoon system, extends from Ponce Inlet to the north end of Merritt Island and covers 288.5 km2 of surface area (Dybas, 2002). Mosquito Lagoon is part of one of the most diverse estuaries in North America and serves as a spawning and nursery site for organisms from the lagoon and nearby ocean (Dybas, 2002). For our trials, approximately 90 green porcelain crabs (Petrolisthes armatus), 90 isopods (Sphaeroma quadridentata), and 90 eastern mud snails (Ilyanassa obsoleta) were collected from Mosquito Lagoon in February 2008. Once collected, all organisms from the lagoon were transported to Orlando in air-conditioned vehicles in five-gallon buckets filled with saltwater from collection sites. Trials started within two hours of collection. Over 1000 brine shrimp (A. salina) were purchased from two local aquarium stores. The first two trials, each from a different store, began on 9 February 2008. The third trial commenced a week later (16 February 2008), after the aquarium shop received a new shipment of brine shrimp.

            Green porcelain crabs, Petrolisthes armatus, are 1-1.5 cm in carapace width in the Atlantic and are somewhat larger in the Pacific. These crabs are filter feeders and live in rocky rubble, oyster reefs, and shallow inter-tidal and sub-tidal zones (King and Knott, 2008). The Eastern mud snail Ilyanassa obsoleta is commonly found in soft-sediment habitats. It is mainly a deposit feeder that focuses on diatoms, blue-green algae, and bacteria, but it will also actively feed on macroalgae when present (Kelaher, Levington, and Hoch, 2003). The isopod Sphaeroma quadridentata is an intertidal or shallow subtidal organism, and is a wood borer that frequently inhabits pilings and mangrove roots (Kensley, Nelson, and Schotte, 1995). Brine shrimp (Arthropoda) are approximately 10 mm in length as adults and feed on phytoplankton in the water column as well as benthic algae (USGS, 2005).

           To test the toxicity of S. terebinthifolius fruits on these invertebrates, 48-hour bioassays were performed. Materials needed for each assay included either Corning Inc. Costar 2.3 x 1.6 cm sterile 12-well, cell culture cluster, tissue culture treated, non-pyrogenic polystyrene well-plates or disposable 60 x 15 mm, sterile, polystyrene petri dishes (Falcon 1007). Each well or petri dish contained one organism. Each well in the Artemia salina trials included 2 mL of saltwater. S. quadridentata and I. obsoleta trials used 5 mL of saltwater in each well and P. armatus trials in petri dishes included 12 mL of saltwater per dish. The saltwater used in each trial matched the salinity of the collection site. The variation in water quantities was due to the size of the organisms; our goal was to minimize the amount of water needed per organism while still enabling the organisms to swim freely. Salinities were 35 ppt for all A. salina and I. obsoleta trials, 36 ppt for the S. quadridentata trial, and 40 ppt for the P. armatus trial. All of the assays were carried out at room temperature (22^o C).

           Immediately before commencing each trial, S. terebinthifolius treatments were randomly added to each of the petri dishes or wells. These treatments consisted of 0 fruits (control), 5 intact fruits, 5 crushed fruits, 10 intact fruits, or 10 crushed fruits. Fruits were crushed by placing them in the well or petri dish and crushing them with the bulbous side of a pipette. All pieces of the fruits were left in the container and then water and organism were added. Trials lasted 48 hours and observations of animal mortality were made at 0.5, 1, 2, 3, 6, 12, 24, and 48 hours. Organisms were recorded as dead if they were not moving and did not move upon agitation. Results were examined separately for each trial. The data were not normally distributed; therefore, non-parametric Kruskal-Wallis statistics followed by a posteriori comparisons of the ranks were used to determine if there were significant differences in survival among treatments at the end of 48 hours.

Results