Biofouling ascidians such as Styela plicata pose a risk to the economy and the environment. Understanding their biology and ecology, is important for predicting and assisting management strategies to reduce their impact. This study tested whether ascidian size difference influenced the amount of epifauna that settled on it. Thirty ascidians (Styela plicata) were collected from Manly Boat Habour, Queensland, Australia. They were provided by teaching staff at the University of Queensland, St Lucia.  Their volumes were calculated and they were weighed and measured. It was predicted that the higher the density and surface area, the more epifauna would be present. Data showed both density and surface area had strong significant effects on the number of organisms. It was shown that high density and surface area led to high epifaunal presence. 

Keywords

Biofouling, Ascidian, Styela plicata, Epifauna, Organism, Density, Surface Area, Volume

Styela plicata (Lesueur, 1823) (pleated sea squirt) is a solitary ascidian, found inhabiting marinas and harbors in oceans around the world (Pineda et al., 2011). These ascidians are prominent components in epibenthic marine communities and are a major source of biofouling (Thiyagarajan & Qian, 2003). The importance of ascidians is well recognized in the study of marine introductions (Lambert & Lambert, 2003; Lambert, 2007). They represent one of the most common marine invaders in the world, largely due to transport via anthropogenic vectors (Lambert, 2007). Despite this, little is known about their biology, due to being of little economic value (Bullard & Carman, 2009). Studies such as this are therefore important to not only the scientific community, but to environmental management. It will assist those needing to make decisions towards limiting ascidian impact on the economy and environment.

Invasive introductions have increased over the century, threatening global biodiversity and altering many ecological communities (Pineda et al., 2011). A major increase in aquaculture facilities has provided new surfaces (oyster shells, ship hulls) for colonization (Lambert, 2007). This biofouling represents a significant loss to the economy in the form of prevention/control and poses a risk to biodiversity (Adams et al., 2011). Approximately 15% of total annual costs of shellfish aquaculture is used on the removal of biofouling organisms (Rosa et al., 2011). To reduce these costs and risks to the environment, further research needs to be undertaken.

Although previous studies have been carried out, causation for successful invasions of Styela plicata is not well understood. Pineda et al., 2011 carried out an experiment to determine if genetic diversity played a role on ascidian establishment in a new area. The study found that geographic distributions of the ascidian did not show any consistent pattern. Bullard & Carman, 2009, could only correlate their invasiveness to their competitiveness and environmental tolerance. The aim of this study is to determine if size difference of Styela plicata has any implication to the contributed biofouling caused by epifauna. This will be done by distinguishing surface area and density differences whilst counting the epifauna present on each ascidian. It is predicted that the larger surface areas and densities, the more epifauna present. 

Data collection

(Refer to Figure 1- Thirty Styela plicata) A total of 30 individual ascidians (Styela plicata) were provided by the teaching staff of BIOL3211, University of Queensland, St Lucia. These were collected from Manly Boat Harbor in Queensland, Australia. Data collection took approximately 8 hours over a course of two weeks. During these weeks, ascidians were kept alive in a controlled aquarium on the campus.  

Each ascidian was placed underneath a dissection microscope in a container with sea water (as seen in Figure 2- Counts using a Dissection Microscope). The outside material was rubbed and peeled off the ascidian and mixed into the sea water. Once the ascidian was stripped clean of any material, epifauna was counted. After this was completed, the length and weights of each ascidian was taken (as seen in Figure 3- Length Measurement & Figure 4- Weight Measurement). Lastly, using a graduated cylinder, the volume of each ascidian was obtained by noting its displacement within the water (as shown by Figure 5- Volume: Graduated Cylinder). 

Data analysis

Data analysis was carried out using the free software program R, as well as Microsoft Excel. Before determining a statistical model to be fitted, data was visualised using scatter plots for surface area and density. Outliers were removed using the 1.5*IQR rule and visualising data, removal resulted in consistent variation. Independence was fine considering each ascidian was only tested once and then separated. Linearity was demonstrated by the line in both plots as well as normality.

Normality was shown by QQ normal plots; normality was achieved after the removal of outliers. QQ plots were used instead of histograms, as they weren't useful for this sample size (less than 100). These assumptions being satisfied meant linear regression was utilised in the graphing. As the sample size was 15≤N≤40 and standard deviations were different, a two sided t-test was used. Density was calculated using D=Mass/Volume and surface area SA= 4πr2 assuming ascidian shape represents a circle. 

Analysis: Surface Area (cm²) 

Refer to Figure 6: Scatter Plot, Number of Organisms vs. Surface Area (cm²). This scatter plot was used to visualise the data, reasonably consistent variation is shown and line represents linearity.(Refer to Figure 7: Normal Q-Q Plot, Number of Organisms) The normality shown here is reasonably linear, although there seems to be a slight combination of left and right skewness. (Refer to Figure 8: Normal Q-Q Plot, Surface Area (cm²)) Normality seems to be a lot better here, with no obvious skewness occurring. 
 
Analysis: Density (g/cm³)

Surface Area

A two sided t-test showed that there was strong significant interaction between surface area and number of organisms (P<0.05) (t = 10.989, df = 43, p-value = P<0.0001) (Surface Area SD: +/- 1.48, Organism SD: +/- 4.14), (Figure 11). The higher the surface area the higher the number of organisms recorded as shown by the linear line (Figure 11). The Pearson Correlation Coefficient showed a strong relationship, with 83.92% of the variability being explained by surface area (Figure 11)

Density

A two-sided t-test showed that there was strong significant interaction between density and number of organisms (P<0.05) (t = 6.7048, df = 47, p-value = P<0.0001) (Density SD: +/- 0.214, Organism SD: +/- 7.46), (Figure 12). The higher the density the higher the number of organisms recorded as shown by the linear line (Figure 12). The Pearson Correlation Coefficient showed a moderate relationship, with 53.54% of the variability being explained by density (Figure 12).

Biofouling caused by ascidians can be devastating to the environment and the economy (Thiyagarajan & Qian, 2003). Economic costs associated with preventing and removing the damage done by these biofoulers can be enormous. Aquaculture companies can spend large amounts of money removing biofouling organisms (Rosa et al., 2011). Their invasiveness can also put the environment at risk, altering many community structures (Lambert, 2007). With little success, many studies have struggled to find causation of ascidian invasiveness. This highlights a major lack of biological and ecological knowledge of these ascidians.

This study aimed to contribute to the scientific community by helping further understand ascidian biology. It found that there was significant interaction between both density and surface area in relation to number of organisms present (P<0.05). After data was analysed a two-way t-test was performed for density (t = 6.7048, df = 47, p-value = P<0.0001) (Graph 7) and surface area (t = 10.989, df = 43, p-value = P<0.0001) (Graph 7). This result supported the hypothesis that, more organisms would be present on ascidians with higher densities and surface areas.

The results were expected as high surface areas correlate with more space for organisms to attach. It was noted that the sedentary tube worms and encrusting bryozoans were the most dominant organisms attached to the ascidians. This could be due to more surface area for species to live commensally. High densities meant higher mass and volumes, both which can result in higher surface areas. Higher volumes can also contribute to the amount of organisms that live within the siphons of the ascidians. As ascidians are filter feeders, the siphons provide space, food and protection from predation, a perfect environment.

A significant limitation of this experiment was the number of ascidians available. Although a reasonably good estimate, this sample size was quite small. More samples could have helped reduce slight skewness, prevent high numbers of outliers and solidify a stronger normality. The longer the ascidians were kept in the aquarium, the risk for epifauna to die or move through the filtration system. This was enhanced by the fact that data could only be collected in a 5 hour slot, once a week. There may have also been microscopic epifauna that was not observed with the use of a dissecting microscope. Given the size of the experiment and time constraints, sifting through water samples to find microscope epifauna did not seem feasible.

This experiment tested how ascidian size differences can contribute to higher epifauna numbers and thus increased biofouling. The study concluded that larger densities and surface areas had significant effects on epifauna numbers. With this knowledge, it has contributed to the overall understanding of ascidian biology. This will help support management decisions in terms of prevention and control of not only biofouling ascidians, but perhaps other biofouling species. To expand on knowledge attained from this experiment, future studies should investigate whether epifauna follow ascidian larvae and settle. Alternatively, if epifauna prefer an established ascidian to settle on. Secondly, a study on the number of organisms inhabiting the internal structure of ascidians would greatly contribute to this experiment. 

Here is a YouTube video to give an idea of the distribution and biofouling impact of Styela plicata 

Cilsick, R. (March 5, 2013). Pleated Sea Squirt [Video file]. Retrieved 27/5/2015 from https://www.youtube.com/watch?v=n8ARUKWPJAE

I'd like to thank the BIOL3211 tutors and teaching staff for not only providing the specimens, but providing guidance and assistance whenever it was needed. 

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