Bailey et al. 1 A comparison of the Mitotic Index of Zooxanthellae in two species of Anthopleura By Brooke Bailey, Maja Barlo, Susan Bonar, Jordan Bonnet, Riley Charlebois, Phillida Drummond, Carissa Graydon, Andrew McCurdy, Lindsey Ogston, Manon Picard, Bailey Rankine, Brittany Richardson, and Ashley Smith A REPORT SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR ECOLOGY OF MARINE SYMBIOSIS at BAMFIELD MARINE SCIENCES CENTER Instructor: Amanda Bates TA: Lisa Raeburn THE BAMFIELD MARINE SCIENCES CENTER Brooke Bailey, Maja Barlo, Susan Bonar, Jordan Bonnet, Riley Charlebois, Phillida Drummond, Carissa Graydon, Andrew McCurdy, Lindsey Ogston, Manon Picard, Bailey Rankine, Brittany Richardson, and Ashley Smith June 05, 2010
Bailey et al. 2 Abstract A comparison of the Mitotic Index of Zooxanthellae in two species of Anthopleura There are two common species of anemone found on Vancouver island, Anthopleura elegantissima and Anthopleura xanthogrammica. Both of these species have a symbiotic relationship with zooxanthellae and zoochorellae. This study analysed the mitotic index of the two species. Samples were obtained from 3 sites: Dixon island, Scott's bay and Aguilar point. 20 samples were collected from each species at each site. In the lab, the tentacles were mashed onto a slide and the number of dividing cells were counted using a dissecting microscope. The data was analysed using Levine's test, Kolmogorov-Smirnov analysis, two-way ANOVA and a regression test. A. elegantissima had a higher mitotic index than A. xanthogrammica. There was no difference between sites, anemone size or symbiont type. Keywords: mitotic index, Anthopleura, division, symbiosis Introduction Symbiotic relationships are common in marine invertebrates. Anthopleura provide an excellent example of a mutualistic partnership between a cnidarian and an algal symbiont. This relationship benefits both partners as the anemones are provided with an additional carbon source, while the algae are provided with nitrogen, phosphate and a constant environment in which to carry out their life processes. Anthopleura inhabit the intertidal regions of the Pacific Northwest from Alaska to Mexico. They reside in tide pools, crevices and nooks along rocky intertidal shores. In this study, we focused on two native species found in the intertidal zone on the west coast of Vancouver Island, Anthopleura elegantissima and Anthopleura xanthogrammica. Both species have a symbiotic relationship with zoochlorellae, a green algal symbiont, or zooxanthellae, a brown dinoflagellate; mixed populations also occur. A. elegantissima is frequently associated with zooxanthellae symbionts, which in regions north of Oregon has been identified as the species Symbiodinium muscatinei (LaJeunesse and Trench, 2000). A. xanthogrammica containing mixed
Bailey et al. 3 populations of symbionts or exclusively brown zooxanthellae are normally distributed from the intermediate to the high intertidal zone respectively, while individuals with only green symbionts are predominantly found in the low intertidal and deeper waters of tide pools (Bates, 2000). An individual zooxanthella cell has twice the mass of a zoochorella cell, and therefore zooxanthellae have significantly more chlorophyll and a greater respiration rate (Verde & McCloskey 1999). Zooxanthellae generally have a much lower mitotic index than zoochlorellae (Verde & McCloskey 1999). Theoretically, one might assume that an anemone with a mixed population of symbionts, such as A. xanthogrammica, might have a lower mitotic index than individuals which possess only zooxanthellae. Our research hypotheses are as follows: 1. There is a significant difference between the mitotic index of A. elegantissima and A. xanthogrammica. 2.. The mitotic index of A. elegantissima is higher than that of A. xanthogrammica. 3. There is a significant difference in mitotic index between A. xanthogrammica with only zooxanthellae than those with mixed symbiont populations. Methods We clipped tentacles of A. elegantissima and A. xanthogrammica from Dixon Island (48 51 09 N, 125 07 21 W), Scott s Bay (48 50 3 N, 125 08 50 W) and Aguilar Point (48 50 14 N, 125 08 35 W). We ran a transect parallel to the water line at 1 m elevation above low tide, which was determined using a laser level. We took one sample of each species every 3 m beginning at 0 m along the transect line. Samples were taken from a 1 m belt surrounding the transect line (0.5m on each side of the transect). If no suitable specimens were present at a given
Bailey et al. 4 interval, we sampled at the next possible interval; the transect line was extended if necessary. We collected 20 tentacle samples from each species. Specimen with accessible tentacles were measured prior to sampling and only individuals measuring 3-6 cm at the base were selected. We kept each clipping in an individually labelled vial, and stored them on ice during transport, until analysis. We crushed the tentacle clippings and homogenized them using seawater. Slides were prepared with blended samples and were viewed under the compound microscope at 400x magnification. We counted the number of dividing cells within a cluster of no less that 100 zooxanthellae cells to determine the mitotic index. Samples that contained exclusively zoochlorellae were recorded with a mitotic index of zero. For samples exhibiting mixed populations, only the mitotic index for zooxanthellae was recorded. We used Levine's test to determine the equality of variance and Kolmogorov-Smirnov analysis to verify the normality of the data. We ran a two-way ANOVA to assess variation in zooxanthellae mitotic index within the two anemone species between sites. Finally, regression was used to determine if the size of the anemone related to the zooxanthellae mitotic index. Results Mitotic indices of both A. elegantissima and A. xanthogrammica, at all three sites, followed a normal distribution and had equal variance. When comparing the mitotic indices of the two Anthopleura species between sites, no significant difference was observed (Fig. 1; P=0.751, Table 1). Therefore, the data from each site was pooled and the overall species mitotic indices were compared. A. xanthogrammica had mean mitotic index of 6.55 + 3.79 and A. elegantissima had a mean mitotic index of 9.44 + 5.50 (Fig. 2). These two means were
Bailey et al. 5 significantly different with A. elegantissima exhibiting a higher percent of dividing algal cells (P= 0.020, Table 1). Mitotic indices observed in specimens with mixed symbiont populations showed no significant difference (P=0.935, Table 2). Regression analysis showed no significant difference between anemone size and mitotic index (r 2 = 0.02, p > 0.05). Fig. 1. Box plot of mitotic index by sampling site. Mean values: Dixon Island = 7.45, Scott's Bay = 8.52, Aguilar = 8.05. Standard deviation (SD): Dixon Island = 5.27, Scott s Bay = 5.28, Aguilar = 4.77. Range: Dixon Island = 0-24%, Scott s Bay = 0-22%, Aguilar = 0-19%. N: Dixon = 30, Scott s Bay = 31, Aguilar = 39.
Bailey et al. 6 Effects of species and sample site Source Type III Sum of Squares df Mean Square F P-value Corrected Model 194.023 a 5 38.805 1.564 0.178 Intercept 5668.140 1 5668.140 228.385 0.000 Species 138.408 1 138.408 5.577 0.020 Site 14.234 2 7.117 0.287 0.751 Species * Site 19.125 2 9.563 0.385 0.681 Error 2332.929 94 24.818 Total 8952.256 100 Corrected Total 2526.951 99 a. R Squared =.077 (Adjusted R Squared =.028) Table 1. Two-way ANOVA testing the combined effects of species (A. elegantissima and A. xanthogrammica) and sample site (Dixon Island, Scott s Bay, and Aguilar), showing degrees of freedom (df), mean squares, F-values, and P values for each source. The P-value is significant only for species effect. Species*Site values show the interaction between species and site and is not significant. N(algal type) A. xanthogrammica = 41, A. elegantissima = 59. N(site) Dixon Island = 30, Scott s Bay = 31, Aguilar = 39.
Bailey et al. 7 Fig. 2. Box plot of mitotic index by species. Mean: A. xanthogrammica = 6.55%, A. elegantissima = 9.44%. Range: A. xanthogrammica = 0-19%, A. elegantissima = 0-24%, with dots showing outliers. SD: A. xanthogrammica = 4.09, A. elegantissima = 5.43. N: A. xanthogrammica = 41, A. elegantissima = 59. Effects of sites and algal type on the mitotic index of A. xanthogrammica Source Type III Sum of Squares df Mean Square F P-value Corrected Model 15.968 a 5 3.194 0.178 0.969 Intercept 1523.505 1 1523.505 85.059 0.000 Algal Type 0.119 1 0.119 0.007 0.935 Site 10.573 2 5.287 0.295 0.746 Algal Type * Site 6.881 2 3.441 0.192 0.826 Error 608.976 34 17.911 Total 2427.250 40 Corrected Total 624.944 39 a. R Squared =.026 (Adjusted R Squared = -.118) Table 2. Two way ANOVA showing the combined effects of sites and algal type on the mitotic index of A. xanthogrammica. showing degrees of freedom (df), mean squares, F-values, and P values for each test. There is no significant effect of site or algal type on mitotic index. N(algal type): zooxanthellae = 18, zooxanthellae/zoochorellae=23. N(site): Dixon Island = 10, Scott s Bay = 11, Aguilar = 19. Discussion We predicted that A. elegantissima would have a higher mitotic index than A. xanthogrammica, and that there would be no significant difference between mitotic indices of a host with only zooxanthellae versus a host with a mixture of zooxanthellae and zoochlorellae. The results show a higher mitotic index in A. elegantissima and no significant difference between
Bailey et al. 8 the mitotic index of anemones with zooxanthellae and anemones with a mixture of zooxanthellae and zoochlorellae. Figure 2 compared the difference of the mitotic index of zooxanthellae between the two species of anemones. The mitotic index was found to be significantly different between the two, confirming that the activity of cell division in the symbiont is different in each of the host species - the number of dividing symbiotic cells was greater in A. elegantissima. Site variability was irrelevant; the mitotic index for both species was not significantly different between the three sites. A. elegantissima had a consistently greater mitotic index at all three sites. Table 2 focuses solely on A. xanthogrammica. This species contained either zoochlorellae, zooxanthellae, or both. We conducted a two-way ANOVA on A. xanthogrammica since A. elegantissima did not contain zoochlorellae. This test was done to confirm that the presence of mixed algal symbionts did not affect the mitotic index. There was no significant difference between the mitotic index of the zooxanthellae or zooxanthellae/zoochlorellae mixed populations in A. xanthogrammica. The presence of zoochorellae in duel populations does not alter the division rate of zooxanthellae. The data set contained a narrow size range of anemones (3-6 cm), which is the estimated overlap size of adults in both species (Sebens, 1981). We preformed a regression analysis comparing the mitotic index and size of both species. The R 2 was 0.02, which indicates that the mitotic index was not correlated with the individual size. There were abnormally high mitotic indices in A. elegantissima, 20-24%, compared to 15% in Saunders and Muller-Parker (1997). Small sample sizes of 100 cells, used to determine mitotic index, may be responsible for these outlying values. On the collection day (June 1, 2010)
Bailey et al. 9 the ultraviolet index was 2. Zooxanthellae has been shown to increase in density at low light levels, given the conditions this presents another theory to explain the abnormally high mitotic indices (Saunders and Muller-Parker, 1997). Low light could have increased the number of cell divisions in the symbiont population, but to confirm these speculations a higher algae sample size should be used. To improve this study, the field methods would need to be better coordinated between groups. Field communications between all three teams were not possible. Due to this and multiple data collectors, there may have been a strong observer bias. To confirm findings in this study, fewer observers should be used and a more consistent field procedure should be decided upon prior to execution. Conclusion This study revealed a significant difference between the mitotic index of zooxanthellae in A. elegantissima and A. xanthogrammica. Zooxanthellae within A. elegantissima exhibited a greater rate of division than those within A. xanthogrammica. In A. xanthogrammica there was no decrease in the mitotic index of zooxanthellae, which shared its host with zoochlorellae. An interesting dynamic to reflect on may be preferential selection by the symbiont for the host. Zooxanthellae may opt for A. elegantissima as a host since the mitotic index is greater, suggesting that it is a more favorable environment for growth and reproduction.
Bailey et al. 10 References Bates, A. [Electronic version]. 2000. The intertidal distribution of two algal symbionts hosted by Anthopleura xanthogrammica. J. Exp. Mar. Bio.Eco. 249(2): 249-262. LaJeunesse, T.C., and R.K. Trench. [Electronic version]. 2000. Biogeography of two species of Symbiodinium inhabiting the intertidal sea anemone Antopleaura elgantissima. Biol. Bull. 199:126-134. Lamb, A. And B. Hanby. [Electronic version]. 2005. Marine Life of the Pacific Northwest. Harbour Publishing, Madeira Park. Saunders, B.K., Muller-Parker, G. [Electronic version]. 1997. The effects of temperature and light on two algal populations in the temperate sea anemone Anthopleura elegantissima. J. Exp. Mar. Bio. Eco. 211(2): 213-244. Sebens, K. P. 1981. Reproductive ecology of the intertidal sea anemones Anthopleura xanthogrammica (Brandt) and A. elegantissima (Brandt): body size, habitat, and sexual reproduction. J. Exp. Mar. Biol. Ecol. 54: 225 250. Verde, A. and L.R. McCloskey. [Electronic version]. 1999. Photosynthesis and respiration of two species of algal symbionts in the anemone Anthopleura elegantissima (Cnidaria; Anthozoa). J. Exp. Mar. Bio.Eco.195(2): 187-202.