Impacts of a cyanobacterium contaminating large-scale aquaculture feed cultures of Tetraselmis chui on survival and growth of bay scallops, Argopecten irradians irradians.
KEY WORDS: bay scallop, feeding experiment, cyanobacteria, Tetraselmis chui
The bay scallop (Argopecten irradians irradians), is a native species of the North Atlantic coast of the United States for which there once was an active fishery. However, destruction of habitat (Stewart et al. 1981) and short life history have reduced wild supplies for commercial fisheries. Rapid growth, available markets, and ease of seed production make the northern bay scallop an attractive candidate for aquaculture production. Estimates from Rhodes & Widman (1980) indicated that the costs of growing a scallop from 5 mm to 20 mm seed are about 0.05 cents per scallop in raceways with flowing seawater. However, some problems with using raceways to grow scallops include seasonal changes in temperature, predators, diseases, and food availability (Wikfors et al. 2000). Recirculating aquaculture systems are currently being used to grow fish (e.g., Rachycentron canadum Resley et al. 2006; red snapper Ogle & Lotz 2006), but they are not being used widely to grow bivalves to marketable size because of high costs associated with the production of microalgal diets (100-400 US dollars per dry kilogram of algal biomass; Wikfors et al. 2000). However, research by Epifanio et al. (1976) found that, for another commercial bivalve species, the northern quahog Mercenaria mercenaria, a marketable size clam could be achieved in 1 y in a recirculating system, versus the 3-5 y it takes in nature, thus indicating that reductions in costs associated with producing microalgal diets could make recirculating systems a feasible way to grow bay scallops.
Approximately 99% of the costs associated with producing live microalgal feeds for any bivalves are from the cost of electricity for the lights (Wikfors et al. 2000). The Greenhouse for research on algal mass production systems (GRAMPS) at the NMFS Laboratory in Milford, CT is a component of an integrated, recirculating seawater system that consists of a greenhouse for microalgal culture, a shellfish-rearing unit, and a seawater discharge treatment (Wikfors et al. 2004). The microalgal production unit, consisting of two 18,000-L oval tanks, can produce approximately 9,000 L per day of Tetraselmis chui (PLY429), an algal strain used widely in aquaculture. Meseck et al. (2005) found that using a greenhouse to grow large volumes of algal feed could potentially reduce lighting costs by 84%, thus making it more economical to use with recirculating systems to grow bivalves.
Growing large, open algal cultures can be problematic, because it is more difficult to control many of the factors affecting phytoplankton growth (e.g., light duration and intensity, temperature, and contaminating microorganisms). Numerous cultures of Tetraselmis chui have been grown in the GRAMPS system; however, these cultures periodically become contaminated with a cyanobacterium. How cyanobacterium are introduced into PLY429 cultures at this rime is unknown, but these are open cultures with many vectors for contaminate introduction. The effects of feeding bay scallops, Argopecten irradians irradians, a diet of PLY429 with this cyanobacterium are uncertain, but preliminary observations indicate poorer performance associated with the contaminant. This experiment, therefore, examined the survival and growth rates of bay scallops fed a diet of a cyanobacterial contaminant isolated from a GRAMPS tank culture of PLY429 and a mixed suspension of the two algal strains, comparing performance with a pure culture of PLY429.
Juvenile bay scallops (5.13-5.93 mm), Argopecten irradians irradians, for this study were laboratory-reared at the National Marine Fisheries Services in Milford, CT. The scallops were spawned on March 7, 2006 and were 75 days old at the start of the experiment. A slight modification of the automated rearing chambers, as described by Smith & Wikfors (1998), was used in this experiment. Instead of filling the chambers completely to 6 L, they were run at 3 L volume, and 0.1 [micro]m-filtered seawater was used. Seawater was gravity fed to the chambers at a flow rate of 100 mL [min.sup.-1]. There were 50 scallops in each chamber with initial live weights of 1.32-1.83 g.
The microalgal contaminate isolate from the greenhouse, GH-05-01, has morphological and pigment characteristics consistent with the genus Synechococcus. Cells are roughly spherical with a size of 1-2 [micro]m and contain no distinct nucleus, as determined by epifluorescence microscopy using the nucleicacid fluorochrome acridine orange. The presence of the phycobiliprotein phycocyanin was confirmed by the fluorescence method of Stewart & Farmer (1984), thereby placing the isolate unequivocally in the cyanobacteria; this fluorescence spectrum was identical to that of Synechococcus sp. (SYN strain from the Milford microalgal culture collection). The cells reproduce by binary fission, divide in a single plane, and there is no capsule: these characteristics together are diagnostic for the genus Synechococcus (Rippka et al. 1979, Table 3).
The algae, Tetraselmis chui (PLY429) and the cyanobacterium (GH-05-01), for this experiment were grown semicontinuously in E medium at a temperature of 19.5[degrees]C, a light intensity of 110 [micro]Einst, [m.sup.-2] [s.sup.-1], and 24 h light. The PLY429 culture was an axenic culture, whereas the cyanobacterium for this experiment was isolated from a greenhouse culture of PLY429, where it had appeared periodically as a contaminant. The scallops were given a diet of either 100% PLY429, a mixed diet (50% PLY429 with 50% cyanobacteria, by dry weight), 100% cyanobacteria, or starved (n = 3 for each). Based on a previous nutritional study (Wikfors et al. 1998), scallops were fed 2% of their live weight daily, with 16 feedings per day to maximize conversion efficiency.
Each week, filters and chambers were cleaned with filters being replaced biweekly. Weekly, scallops were analyzed for live/dead counts, live weights, biovolume, and shell height. Live weights were done after blotting with a paper towel to remove moisture from the outside of the shell.
At week 6, the scallops that had been fed 100% cyanobacterium were switched to a diet of 100% PLY429. The switching of diet was done to simulate the time it may take a hatchery to start a new culture of PLY429 to feed scallops, once the cyanobacteria are detected in a culture, to determine if the scallops could recover from being fed a contaminant for 6 wk.
For each week, an ANOVA was used to determine the effects of diet on the survival and growth of scallops. After 3 wk, the ANOVA test was run only on those scallops that received food so that the results would not be influenced by starved scallops that had died by this time. ANOVA was also used to determine if switching the diet from the cyanobacterium to PLY429 had an effect. STATGRAPHICS Plus (Manugistics, Rockville, MD) software was used for all statistical analysis.
Starved juvenile bay scallops had significantly (P < 0.01) lower survival than fed scallops by week 2, and there were no surviving starved scallops by week 3 (Fig. 1). A multiple range test, by Fisher's least square difference, found that all fed scallops were in the same homogenous group after two weeks. Scallops fed the cyanobacterial culture alone began to die (Fig. 1) by week 3; there was a significant difference (P = 0.01) at week 3 in survival on the different diets. Survival of scallops fed only cyanobacteria 77 [+ or -] 9%, was significantly lower than survival of scallops fed either a mixed diet or only PLY429, 97 [+ or -] 3% and 98 [+ or -] 1%, respectively at week 3 (Table 1). There was a significant difference in survival between all the diets for week 4 (P < 0.01 ), week 5 (P < 0.01 ), week 6 (P < 0.01), week 7 (P < 0.01), and week 8 (P < 0.01), however for all these weeks scallop survival on the cyanobacterium was significantly lower than that of scallops fed the mixed and only PLY429 diets (Table 1). At week 6, the scallops given cyanobacteria only were changed to a diet of PLY429 and ANOVA revealed a significant difference in survival over time (P < 0.01) for these scallops. A multiple range test found that there were three homogenous groups with week 1 and 2 being one group, weeks 3, 4, and 5 being a second group, and weeks 7 and 8 being the third group, with week 6 being in both the second and third group, thus suggesting that switching the diet had no negative effect on survival.
[FIGURE 1 OMITTED]
The highest increase in shell size of juvenile bay scallops was on the diet of 100% PLY429, with the 100% diet of cyanobacteria having the lowest increase in mean shell height of the three diets fed (Fig. 2). By week 2, there was a difference (P = 0.04) in shell height between the scallops being given food and the starved scallops (Table 1). There was no significant difference in shell height between the three fed treatments until week 4 (P = 0.01), and differences in shell height on the three diets remained significant for week 5 (P = 0.02), 6 (P < 0.01 ), 7 (P < 0.01), and 8 (P < 0.01; Table 1). At week 4, shell heights of scallops fed PLY429 and the mixed diet were statistically identical (7.5 [+ or -] 0.2 cm and 7.0 [+ or -] 0.2 cm, respectively), but scallops fed the cyanobacterium were smaller (6.4 [+ or -] 0.1 cm; Table 1). By week 6, shell heights of scallops fed the three diets were all significantly different, with PLY429 being 8.6 [+ or -] 0.3 cm, the mixed diet at 7.6 [+ or -] 0.5 cm, and the cyanobacterium at 6.5 [+ or -] 0.1 cm. (Table 1). The cyanobacteria-fed scallops were switched to PLY429 at week 6 and by week 8 a multiple range test found that the mixed diet and the cyanobacterial diet that was switched to PLY429 statistically identical (Table 1). An ANOVA testing shell height of scallops fed the cyanobacterial diet with respect to rime found that there was a significant change over time (P < 0.01), with three homogenous groups with week 1 and 2 being one, weeks 3, 4, 5, 6, and 7 being another, and week 8 being the third, significantly larger in shell size (6.9 [+ or -] 0.1) than all other weeks. Therefore, the change in diet at week 6 did have an effect on shell growth of the scallops. A linear regression of the shell height in Figure 2 found that the scallops fed PLY429 grew 0.5 cm week J (r = 0.99), those fed the mixed diet grew 0.3 cm (r = 0.99), and scallops fed the cyanobacterium alone grew 0.1 cm week l (r = 0.93). The growth rate per scallop was calculated by dividing the slope of the regression by the number of scallops alive at the end of week 8; for PLY429, shell growth was 198-[micro]m [scallop.sup.-l] [d.sup.- 1], the mixed diet supported shell growth of 82-[micro]m [scallop.sup.-1] [d.sup.- 1], and the cyanobacterium yielded only 65-[micro]m [scallop.sup.-1] [d.sup.-1].
As with shell size, the scallops fed a diet of PLY429 had the greatest increase in live weight, relative to the mixed and cyanobacterial diets (Fig. 3). Exponential relationships between live weight and week for the PLY429 and mixed diets were observed, with scallops fed the PLY429 diet increasing in weight more rapidly than those given the mixed diet (Fig. 3). No temporal relationship in live weight of scallops fed the cyanobacterium over rime could be found, which may be attributable to mortalities that occurred during the experiment (Fig. 1 for survival). Bay scallops did have a significant difference (P < 0.01) in live weight by week 2, with all fed scallops being larger than the unfed, which had lost weight as a consequence of mortalities. Live weights of scallops fed the three diets were significantly different (P = 0.03) at week three, with two homogenous groups (Table l); the scallops receiving cyanobacteria weighed approximately 1.0 g less than the scallops fed PLY429. The diet effect on live-weight growth was significant for weeks 4, (P = 0.02); 5, (P - 0.02); 6, (P < 0.01); 7, (P < 0.01); and 8, (P < 0.01) with PLY429 and the mixed diet being one homogenous group for weeks 4, 5, and 6 (Table 1). However, by week 7, live weights of scallops fed PLY429 were significantly greater than those of scallops fed the mixed diet (Table 1). For scallops fed the cyanobacterial diet and then switched to PLY429 at week 6, ANOVA found significantly increased growth (P < 0.01) after the change in diet.
[FIGURE 2 OMITTED]
[FIGURE 3 OMITTED]
This study shows that survival and growth of scallops were diminished by diets that included the cyanobacterial contaminant isolated from the GRAMPS Tetraselmis chui culture. A diet of cyanobacteria appeared to not be acutely toxic to bay scallops, indicating that a "zero-tolerance" approach is not warranted; however, significantly lower growth rates (65-[micro]m [scallop.sup.-1] [d.sup.-1]) were observed when scallops were fed this diet, compared with T. chui. From week 3 to week 6, there was very little growth of the scallops fed the cyanobacterium alone (Fig. 2). For growth to occur, energy assimilated by the scallop needs to exceed the maintenance need of the animal. The lack of growth between weeks 3 and 6 on the 100% cyanobacterial diet suggests that the cyanobacterium provides just enough nutrition to maintain basic maintenance needs. The decrease in survival during weeks 3-6 (Fig. 1) further suggests that feeding a diet of only cyanobacteria does not provide enough nutrition to support maintenance of a scallop population. Further biochemical analysis of the cyanobacterium could help to determine if digestibility or biochemical characteristics are responsible for the poor performance of juvenile bay scallops feeding on this cyanobacterial contaminant. As a prokaryote, the cyanobacterium is likely to be deficient in sterols and essential fatty acids that have been demonstrated to support molluscan shellfish growth (Wikfors et al. 1991, Milke et al. 2006).
Scallops fed 100% cyanobacterial diet were changed to 100% PLY429 at week 6 with no significant change in survival between week 6 and week 8, but by week 8 a significant increase in shell height was obtained (Table 1). This suggests that, if the cyanobacterial contaminant is detected, there is enough time to start a new culture of PLY429 before scallop mortalities occur. Once switched back to a diet of PLY429 the scallops should begin to grow again, according to these results.
Growth rates of bay scallops in natural environments range from 89-345-[micro]m [scallop.sup.-1] [d.sup.-1] (Rhodes & Widman 1980, Sisson 1981). The growth rates of scallops fed the cyanobacterial diet, mixed diet, and the PLY429 diet were all within the ranges found in the natural environment. Milke et al. (2006) fed juvenile bay scallops (10 mm starting size) varying diets of algae and had growth rates of 190-392-[micro]m [scallop.sup.-1] [d.sup.-1], which was dependant on the algal food source. The 100% PLY429 diet was the only diet that had growth rates within the range reported by Milke et al. (2006). In conclusion, for higher growth rates and survival it is important to minimize the presence of the cyanobacterial contaminants in Tetraselmis chui cultures, but lack of acute toxicity of the cyanobacterium, and a limited capacity for this organism to maintain scallop survival above starvation levels indicate that it is better to continue feeding a contaminated culture than to cease feeding while a replacement culture is prepared.
The authors thank David Veilleux for providing hatchery-reared scallops for this experiment and Yaqin Li for pigment analyses contributing to identification of the cyanobacterium.
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SHANNON L. MESECK, * GARY H. WIKFORS, JENNIFER H. ALIX, BARRY C. SMITH AND MARK S. DIXON
U.S. Department of Commerce, NOAA, National Marine Fisheries Service, 212 Rogers Ave., Milford, CT 06460
* Corresponding author: E-mail: Shannon.firstname.lastname@example.org
TABLE 1. ANOVA on the survival, height and live weight of bay scallops (Argopecten irradians irradians) feed a diet of either 100% Tetraselmis chui (PLY429), a mixed diet (50% PLY429:50% cyanobacterium),or 100% cyanobacteria for each week. Week Treatment 1 2 % Survival P Value 0.12 <0.01 Mean Survival [+ or -] SD (%) PLY429 98 [+ or -] 2 99 [+ or -] 1 (1) Mixed diet 99 [+ or -] 1 (1) Cyanobacterium 95 [+ or -] 4 (1) Starved 48 [+ or -] 2 (2) Height P value 0.21 0.04 Mean Height [+ or -] SD (cm) PLY429 6.2 [+ or -] 0.4 6.5 [+ or -] 0.4 (1) Mixed Diet 6.4 [+ or -] 0.2 (1) Cyanobacterium 6.2 [+ or -] 0.2 (1) Starved 5.8 [+ or -] 0.2 (2) Live Weight P value 0.06 <0.01 Mean Live Weight [+ or -] SD (g) PLY429 1.64 [+ or -] 0.21 2.15 [+ or -] 0.30 (1) Mixed Diet 1.97 [+ or -] O.19 (1) Cyanobacterium 1.66 [+ or -] 0.12 (1) Starved 0.74 [+ or -] 0.16 (2) Week Treatment 3 4 % Survival P Value <0.01 <0.01 Mean Survival [+ or -] SD (%) PLY429 99 [+ or -] 1 (1) 99 [+ or -] 1 (1) Mixed diet 97 [+ or -] 3 (1) 95 [+ or -] 5 (1) Cyanobacterium 77 [+ or -] 9 (2) 72 [+ or -] 10 (2) Starved Height P value 0.09 0.01 Mean Height [+ or -] SD (cm) PLY429 6.8 [+ or -] 0.3 7.5 [+ or -] 0.2 (1) Mixed Diet 7.0 [+ or -] 0.2 (1) Cyanobacterium 6.4 [+ or -] 0.1 (2) Starved Live Weight P value 0.03 0.02 Mean Live Weight [+ or -] SD (g) PLY429 2.36 [+ or -] 0.47 (1) 2.74 [+ or -] 0.66 (1) Mixed Diet 2.03 [+ or -] 0.23 (1) 2.24 [+ or -] 0.33 (1) Cyanobacterium 1.38 [+ or -] 0.21 (2) 1.27 [+ or -] 0.20 (2) Starved Week Treatment 5 6 % Survival P Value <0.01 <0.01 Mean Survival [+ or -] SD (%) PLY429 97 [+ or -] 3 (1) 97 [+ or -] 3 (1) Mixed diet 94 [+ or -] 6 (1) 93 [+ or -] 7 (1) Cyanobacterium 70 [+ or -] 8 (2) 63 [+ or -] 8 (2) Starved Height P value 0.02 <0.01 Mean Height [+ or -] SD (cm) PLY429 8.0 [+ or -] 0.4 (1) 8.6 [+ or -] 0.3 (1) Mixed Diet 7.5 [+ or -] 0.3 (1,2) 7.6 [+ or -] 0.3 (2) Cyanobacterium 6.5 [+ or -] 0.1 (2) 6.5 [+ or -] 0.1 (3) Starved Live Weight P value 0.02 <0.01 Mean Live Weigh [+ or -] SD (g) PLY429 3.22 [+ or -] 0.81 (1) 4.05 [+ or -] 0.91 (1) Mixed Diet 2.49 [+ or -] 0.555 (1) 2.86 [+ or -] 0.75 (1) Cyanobacterium 1.24 [+ or -] 0.18 (2) 1.14 [+ or -] 0.15 (2) Starved Week Treatment 7 8 % Survival P Value <0.01 <0.01 Mean Survival [+ or -] SD (%) PLY429 97 [+ or -] 2 (1) 93 [+ or -] 8 (1) Mixed diet 93 [+ or -] 7 (1) 96 [+ or -] 2 (1) Cyanobacterium 43 [+ or -] 21 (2) 43 [+ or -] 21 (2) Starved Height P value <0.01 <0.01 Mean Height [+ or -] SD (cm) PLY429 9.0 [+ or -] 0.2 (1) 9.4 [+ or -] 0.2 (1) Mixed Diet 7.6 [+ or -] 0.5 (2) 7.8 [+ or -] 0.5 (2) Cyanobacterium 6.6 [+ or -] 0.1 (3) 6.9 [+ or -] 0.1 (2) Starved Live Weight P value <0.01 <0.01 Mean Live Weight [+ or -] SD (g) PLY429 4.84 [+ or -] 1.02 (1) 5.14 [+ or -] 0.85 (1) Mixed Diet 3.03 [+ or -] 0.89 (2) 3.06 [+ or -] 0.85 (2) Cyanobacterium 0.86 [+ or -] 0.41 (3) 0.89 [+ or -] 0.45 (3) Starved
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|Author:||Meseck, Shannon L.; Wikfors, Gary H.; Alix, Jennifer H.; Smith, Barry C.; Dixon, Mark S.|
|Publication:||Journal of Shellfish Research|
|Date:||Dec 1, 2007|
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