WILSON ET AL.: EFFECT OF SNAIL ON OYSTERS 



TREATMENTS: 4 WEEK EXPERIMENTAL PERIOD 



Unparasitized Controls 



Parasitized 



4 WEEK RECOVERY PERIOD 



Recovery Control 



Previously Parasitized 

 I 1 



Figure 1.— Position of plexiglass domes during 4-wk experimental period and during 4-wk 



recovery period. 



were frozen immediately on dry ice and kept at 

 -40°C until lyophilized. Prior to biochemical anal- 

 ysis, lyophilized tissues were weighed and homog- 

 enized on ice. Total lipids were isolated from a por- 

 tion of the homogenate using the water:methanol: 

 chloroform method of Folch et al. (1957) with a 

 modified ratio of 0.8:2.1. This isolation produced a 

 two phase system and an insoluble pellet after cen- 

 trifugation in the cold. The pellet was used for pro- 

 tein and glycogen assays. The water phase was used 

 for amino acid analysis. The organic phase was 

 divided and dried under Ng gas at 40°C. One half 

 was used to determine lipid phosphate concentra- 

 tion by the spectrophotometric method of White et 

 al. (1979). The other half was dissolved in chloro- 

 form to which nonadecanoic acid was added as an 

 internal standard. The redissolved lipids were frac- 

 tionated on a silicic acid (Unisil,^ 100-200 mesh) 

 column. Neutral lipids were recovered by eluting 

 with 10 times the column volume of chloroform. The 

 chloroform was removed under N2 at 40°C ((rehron 

 and White 1982). The resulting chloroform fraction, 

 containing fatty acid methyl esters, was analyzed 

 by gas chromatography using a capillary, nonpolar, 

 methyl silicone high performance column and flame 

 ionization detector. 



The amino acids were analyzed on a Dionex 3000 

 amino acid analyzer. Because residual chloroform 

 interfered with the analysis, a second extraction was 

 performed on the sample in the cold using a chloro- 

 form:water ratio of 1:3; a-amino-n-butyric acid (2.5 

 fimole • mL~^ sample) was used as an internal 

 standard. The amino acids accounting for the bulk 

 of the free amino acid (FAA) pool, taurine, hypo- 



'Reference to trade names does not imply endorsement by the 

 National Marine Fisheries Service, NOAA. 



taurine, aspartic acid, serine, threonine, glutamine, 

 glutamate, glycine, and alanine, were separated us- 

 ing a lithium citrate buffer and measured using 

 o-phthalaldehyde as the detecting compound. 



Total soluble protein was estimated by Peterson's 

 (1977) modification of the Lowry method. Protein 

 was precipitated using a final concentration of 10% 

 trichloroacetic acid in the cold. After centrifugation 

 at 4°C the resultant pellets were resuspended 

 in 50:50, 10% SDS:0.8N NaOH for 30 minutes. 

 Replicate samples were centrifuged and analyzed 

 spectrophotometrically for protein concentration. 

 Bovine serum albumin (Sigma) was used as the 

 standard. 



Glycogen was degraded to glucose enzymatically 

 with amyloglucosidase (Carr and Neff 1984). After 

 preincubation with amyloglucosidase, glucose was 

 measured using the glucose oxidase-peroxidase pro- 

 cedure (Sigma glucose kit) (Roehrig and Allred 

 1974). Rat liver glycogen (Sigma) was used as a 

 standard. 



Statistical Analysis 



To assess the effect of Boonea impressa on the 

 oysters during the 4-wk exposure period, the level 

 of snail parasitism for each oyster had to be deter- 

 mined. Because the number of snails on each oyster 

 was counted only every third day, the total number 

 of snails that parasitized each oyster was estimated 

 by assuming that the same number of snails were 

 present on days between counts as found on the 

 previous visit. The total number of snail-days per 

 oyster, the sum of the number of snails present on 

 each day during the 4-wk exposure, will be referred 

 to as the snail scale. For example, during the first 

 week of the exposure period, one oyster had 15 



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