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Effects of Conspecific Competition and Body Size on Pyura Stolonifera Filtration Rate


Cheyenne Mercy Moreau 2017

Abstract
Rocky shore dwelling marine invertebrates must battle the challenges of dynamic tides, harsh environment, and intense competition for space.  Many of these species have evolved colonial forms to outcompete solitary individuals, partition food, and increase resilience to predation.  Ascidians are well known for their colonial forms and fused chimera that help them thrive in these habitats.  However, one of the largest species has remained solitary and competes not only with other marine invertebrates but also with its own species. I investigated the effect of conspecific competition in feeding by observing changes in P. stolonifera filtration rate.  I also analyzed the influence of body size on filtration rate. The filtration rate of a group of six specimens and their respective individual rates were measured as a function of 664nm light ray absorption over time.  I found no significant difference between the group filtration rate and the sum of the individual rates.  However, I did see a slightly positive correlation, although insignificant, between body size and filtration rate.  Further studies with larger sample sizes could confirm these findings.

Introduction

Sessile marine invertebrates dominate the rocky shores of Queensland(Endean and Stephenson 1956).  These animals must endure dynamic environmental conditions on a daily basis.  During the high tide, they are exposed to marine predators such as fish and crabs (Quammen 1984) and cannot escape terrestrial predation by shorebirds at low tide(Zharikov 2003).  Low tide also brings dangers of desiccation and exposure to particularly high levels of UV radiation (Przeslawski 2005).  As the tide comes back in, these invertebrates must fight pounding waves to remain attached to their substrate and handle excess sedimentation(Lewis 1968, Gibbons 1988).  Aside from environmental challenges, sessile marine invertebrates must compete with well-adapted rivals for limited space on the substrate(Jackson and Buss 1975, Sebens 1982).  Many sessile marine invertebrate species have evolved colonial behaviors to cope with these harsh competitive conditions(Dayton 1971, Sebens 1979, Danchin and Wagner 1997).

Many studies have explored the advantages and drawbacks of colonization in marine invertebrates. Colonial animals frequently overgrow solitary animals giving them the edge over solitary forms in the absence of free space(Jackson 1977, Greene et al. 1983).  The ability for colonial animals to asexually bud also allows them to heal patches on the substrate created by predation whereas solitary organisms must recruit back to an area to replace an individual(Jackson 1977).  The colonial strategy dominates in cryptic environments within the sublittoral zone where wave action is high and populations dense with fierce competition; however, solitary forms flourish in the littoral zone which has abundant space and protection from wave action(Dayton 1971, Paine 1984).  Solitary forms of ascidians, in particular, tend to grow larger and are more fecund as they do not need to partition their food capture nor choose between asexual budding and sexual reproduction(Hughes 2005, Tarjuelo and Turon 2004, Lopez-Legentil et al. 2013). 

When ascidian larvae begin to settle, they use allorecognition to identify conspecifics(Rosengarten and Nicotra 2011, Allegretti 1978, Pawlik 1992). Once found, they decide whether to 1) fuse to the colony and compete for resources on a cellular level or 2) reject fusion and compete on a macro scale for space and food(Rosengarten and Nicotra 2011).  However, some ascidians are entirely solitary and are forced to compete with both other marine invertebrates and conspecifics.  Many consider animals of the same species to be functionally and ecologically equivalent, however, they are still individuals competing for resources in their solitary forms(Lomnicki 1988, Bolnick 2002).  Competition amongst individuals of the same species is referred to as conspecific competition in literature and has been documented for the solitary ascidian P. stolonifera as well as various other marine invertebrates(Dalby 1995, Bertness 1989, Buss 1990). 

This pilot study aims to investigate specifically the feeding aspect of conspecific competition in the ascidian P. stolonifera(Heller 1878).  Dalby (1995) conducted field experiments on P. stolonifera from Australia and found strong evidence for conspecific competition in this species.   In Australia, P. stolonifera are found on rocky shores of South Queensland and New South Wales in solitary or clumped distributions and up to 30cm in size(Monteiro et al. 2002).  If P. stolonifera, in fact, compete amongst themselves, we should see an increase in filtration rates when together in a confined space as each tries to outcompete the others for food.  Likewise, if kept separately, individual filtration rates should decrease once we remove the pressure of competition.  

Materials and Methods

Collection

I collected twelve Pyura stolonifera specimens from the rock groynes and jetty pilings of Amity Point, North Stradbroke Island, Queensland (Figures 1 & 2).  Collection took place during the low tide after Sunrise (6:19 am AEST) on May 15th, 2017.  I detached individuals from their respective substrates with the dull edge of a dive knife being careful not to damage the tunic of each P. stolonifera.  Once removed, I placed each specimen in an opaque bucket filled with enough seawater to submerge them.  Within five hours of collection, all specimens were relocated together into a flow-through tank at the University of Queensland Marine Aquaria.  They remained without food for five days prior to the filtration experiment.
1
Figure 1
2
Figure 2

Stage 1
I selected six P. stolonifera of similar size and measured their length (attachment end to tip of buccal siphon) and largest diameter (across the top) and calculated their body size as a cone. Then to the best of my ability, without damaging the specimens, I removed biofouling communities consisting of barnacles, mussels and tube worms to ensure that filtration rate measurements reflected the rate of P. stolonifera alone. Six individuals were then randomly placed adjacent to one another in a  23cmx3.5cmx18cm clear plastic container containing 1500ml filtered sea water (FSW). Once their siphons relaxed (Figure 3) and re-commenced pumping water, I added 5ml of Pavlova 1800 4-7μm liquid algae to the container(Figure 4). In order to keep the algae in suspension without causing the P. stolonifera to contract their siphons, I created turbulence by plunging a Gilson pipette repeatedly near the bottom of the container every 5 minutes. An equivalent container with identical conditions and lacking P. stolonifera acted as a control.

Experiment 1

To the best of my ability, without damaging the specimens, I removed biofouling communities consisting of barnacles, mussels and tube worms to ensure that filtration rate measurements reflected the rate of P. stolonifera alone. Six individuals were then randomly placed adjacent to one another in a A x B x C clear plastic container containing 1.5L filtered sea water (FSW). Once their siphons relaxed and recommenced pumping water (visible with the naked eye), I added 5ml of Pavlova 1800 4-7micron liquid algae to the container. In order to keep the algae in suspension without causing the P. stolonifera to contract their siphons, I created turbulence by plunging a Gilson pipette repeatedly near the bottom of the container every 5 minutes. An equivalent container with identical conditions and lacking P. stolonifera acted as a control.

Spectrophotometry

Pavlova algae has a high concentration of Chlorophyll-a with an absorption peak of red light at 664nm (SOURCE)(Figure 2). Thus, I measured the amount of red light absorption through my suspension using a Beckman Coulter General purpose UV/VIS spectrophotometer with fixed wavelength at 664nm. A decrease in absorption indicated a reduction in Chlorophyll-a via P. stolonifera filtration.

I set aside 1.5ml of FSW in a cuvette to use as a blank for spectrophotometer calibration. Every 30 minutes for 180 minutes from time 0, I pipetted 1.5ml of the algae/FSW suspension into three spectrophotometry cuvettes for each container. In using three cuvettes, I could average the absorption readings to achieve more precise measurements. After each measurement, I emptied the contents of the cuvettes back into their respective containers. Time and absorption of lambda (664nm) was recorded for both the experimental and control containers. Individuals were returned to separate tanks in the marine aquaria and kept without food for a week prior to the second stage of the experiment.

Experiment 2

After a week of separation and without food, I retrieved the same six P. stolonifera and allocated them into individual small circular containers of dimension BLAH. Each container held 0.5L FSW and 1.7ml of Pavlova to maintain the algal concentration used in the previous experiment. A seventh identical container sans P. stolonifera acted as a control. I used the exact spectrophotometer procedure from stage 1 for stage 2. Time and absorption of lambda (664nm) was recorded for the six experimental and single control containers.

Analysis

I calculated filtration rate as the change in 664 absorption over time as the slope delta(lambda)/t for the group of P. stolonifera in stage 1 and the individuals in stage 2.


Experiment 1

To the best of my ability, without damaging the specimens, I removed biofouling communities consisting of barnacles, mussels and tube worms to ensure that filtration rate measurements reflected the rate of P. stolonifera alone. Six individuals were then randomly placed adjacent to one another in a A x B x C clear plastic container containing 1.5L filtered sea water (FSW). Once their siphons relaxed and recommenced pumping water (visible with the naked eye), I added 5ml of Pavlova 1800 4-7micron liquid algae to the container. In order to keep the algae in suspension without causing the P. stolonifera to contract their siphons, I created turbulence by plunging a Gilson pipette repeatedly near the bottom of the container every 5 minutes. An equivalent container with identical conditions and lacking P. stolonifera acted as a control.

Spectrophotometry

Pavlova algae has a high concentration of Chlorophyll-a with an absorption peak of red light at 664nm (SOURCE)(Figure 2). Thus, I measured the amount of red light absorption through my suspension using a Beckman Coulter General purpose UV/VIS spectrophotometer with fixed wavelength at 664nm. A decrease in absorption indicated a reduction in Chlorophyll-a via P. stolonifera filtration.

I set aside 1.5ml of FSW in a cuvette to use as a blank for spectrophotometer calibration. Every 30 minutes for 180 minutes from time 0, I pipetted 1.5ml of the algae/FSW suspension into three spectrophotometry cuvettes for each container. In using three cuvettes, I could average the absorption readings to achieve more precise measurements. After each measurement, I emptied the contents of the cuvettes back into their respective containers. Time and absorption of lambda (664nm) was recorded for both the experimental and control containers. Individuals were returned to separate tanks in the marine aquaria and kept without food for a week prior to the second stage of the experiment.

Experiment 2

After a week of separation and without food, I retrieved the same six P. stolonifera and allocated them into individual small circular containers of dimension BLAH. Each container held 0.5L FSW and 1.7ml of Pavlova to maintain the algal concentration used in the previous experiment. A seventh identical container sans P. stolonifera acted as a control. I used the exact spectrophotometer procedure from stage 1 for stage 2. Time and absorption of lambda (664nm) was recorded for the six experimental and single control containers.

Analysis

I calculated filtration rate as the change in 664 absorption over time as the slope delta(lambda)/t for the group of P. stolonifera in stage 1 and the individuals in stage 2.
3
Figure 3
4
Figure 4

Spectrophotometry

Algae has a high concentration of Chlorophyll-α with an absorption peak of red light at 664nm(Figure 5). Thus, I measured the amount of red light absorption through my suspension using a Beckman Coulter General Purpose UV/VIS spectrophotometer with fixed wavelength at 664nm. A decrease in absorption indicated a reduction in Chlorophyll-α via P. stolonifera filtration.

I set aside 1.5ml of FSW in a cuvette to use as a blank for spectrophotometer calibration. Every 30 minutes for 180 minutes from time 0, I pipetted 1.5ml of the algae/FSW suspension into three spectrophotometry cuvettes for each container. In using three cuvettes, I could average the absorption readings to achieve more precise measurements. After each measurement, I emptied the contents of the cuvettes back into their respective containers. Time and absorption of λ664 were recorded for both the experimental and control containers. Individuals were returned to separate tanks in the marine aquaria and kept without food for a week prior to the second stage of the experiment. 
5
Figure 5

Stage 2

After a week of separation and without food, I retrieved the same six P. stolonifera and allocated them into individual small circular containers with a diameter of 11cm and height of 9.5cm. Each container held 0.5L FSW and 1.7ml of Pavlova 1800 4-7μm to maintain the algal concentration used in the previous experiment(Figure 6). A seventh identical container sans P. stolonifera acted as a control. I used the exact spectrophotometer procedure from stage 1 for stage 2. Time and absorption of λ664 was recorded for the six experimental and single control containers.
6
Figure 6

Analysis
I fit a linear regression to the absorption of λ664 over time. I related filtration rate to the slope (Δ λ664/t) of each regression line for the group of P. stolonifera data in stage 1 and the individuals data in stage 2. In the group experiment, absorption of λ664  stabilized after 90 minutes.  Points thereafter were removed from the regression to more accurately depict the steepness of the initial filtration rate.  Finally, the sum of the individual rates was compared to the group rate.  To see if body size affected filtration rate, I then graphed filtration rate of individuals against body size and ran a linear regression. I also investigated the relationship between body size and filtration rate using a linear regression. All calculations and graphs were done in the statistical computing software Rstudio®.

Results
Across all experiments,  λ664 absorption decreased over time indicating a decrease in chlorophyll-α concentration(Figure 7).  The control remained nearly constant over time with a slope of 0.00.  Figure 8 summarizes λ664 absorption over time for each experiment. While the adjusted R2 for each regression was high, p-values remained insignificant and weakly significant.  These statistical results require a much larger sample size to be considered conclusive as the power of this experiment was well under an acceptable 85%. Regardless, the decrease in chlorophyll concentration over time was quite clear to the naked eye(Figure 9).

In Figure 8 we also see that the combined filtration rate of the individuals is higher than that of the group by ~0.001.  Because there is only one sample, there is no comparative statistical tool to can confirm the significance of this difference.

Figure 10 illustrates a weak insignificant negative correlation between body size and change in λ664 absorption over time which correlates to a weakly positive relationship between body size and filtration rate (t4 = -1.55, p = 0.2, R2 = 0.32). 
7
Figure 7
8
Figure 8
9
Figure 9
10
Figure 10

Discussion

The group filtration rate stabilized after the 60-minute mark while individual rates seemed to drop across the entire time interval.  The fact that all six individuals filtered the same water at the same time explains the steeper rate seen in the group experiment.  P. stolonifera filter water extremely efficiently due to their large size and specialized pharynx for powerful pumping (Day 1974).  Once done feeding, they continue to pump water expelling food particles instead of capturing them (Klumpp 1984).  In addition, by the 60-minute mark, the water had noticeably lightened in color suggesting a much smaller concentration of algae available for filtering.  These factors combined likely explain the stabilization in chlorophyll a concentration towards the end of the experiment.

Though the sum of individual rates is greater than the group rate, a 0.01 difference is hardly significant. The sample size is simply too small to determine whether this small effect size is truly significant. Furthermore, I observed equivalent differences in concentration between time intervals for a single individual suggesting the 0.01 difference is fairly minute overall.  Perhaps because individuals remained without food for an additional two days they fed at a slightly faster rate.  Klumpp (1984) also found that within the first twelve hours of filtration, rates fluctuated due to handling disturbances and discomfort suggesting small differences may be a result of a short timescale. 

For the sum of individual rates to come so close to the group rate is remarkable in itself, however.  Biological and ecological studies typically anticipate large amounts of noise in their residuals(Fowler et al. 2013).  The fact that these ascidians appear to maintain their total filtration rate could suggest P. stolonifera filtration rates are not highly variable.  Klumpp (1984) investigated the effects of body size, food quality and food quantity on P. stolonifera filter feeding but no study has shown that their filtration rates remain constant over time.  A more precise experiment using fluorescent dye to repeatedly track filtration rate of individuals would provide better insight into this question. 

P. stolonifera are known to compete for resources amongst themselves(Dalby1995).  I expected these specimens to compete for algae as a food source by increasing their filtration rates to capture more food before their conspecifics.  The similarity between the group and sum of individual rates suggests this was not the case.  Future studies with a larger sample size could add significance to the small effect size of a .01 change in filtration rate.  In this study, the presence of conspecifics appears to cause P. stolonifera to decrease filtration rate.  This could indicate a partitioning of food resources, a behavior observed in P. stolonifera beds and various other filter feeding invertebrates(Seiderer and Newell 1988, Stuart and Klumpp 1984).

Increasing body size proved to have a weak and insignificant positive correlation with filtration rate in this study(sample size n = 6). However, a study by Klumpp (1984), found a very strong significant positive correlation between body size and filtration rate for P. stolonifera when n = 25.  Thus using a greater sample size would likely strengthen the results in this study.

Unfortunately, time constraints and resource limitations prevented a more rigorous analysis of changes to P. stolonifera filtration rate in the presence of conspecifics.  I only managed to collect twelve individuals, only seven of comparable size ideal for the experimental design.  I did not have enough time to complete a second collection of animals and other specimens in the aquaria were involved in other experiments. I also had to carry out stage 1 and 2 of my experiment a week apart to ensure the results were not conditional on hunger.  Without enough animals to run multiple experiments in parallel, I only achieved a sample size of one and no comparison could be made.  In addition, the experiment took place in a busy lab leaving the filtering ascidians prone to disturbances.  In general, ascidians are extremely sensitive to vibrations and changes in light and respond by retracting their siphons which prevents efficient filtration(Hoyle 1958). 

To conclude, this pilot study aimed to measure the effect of conspecific presence on P. stolonifera filtration rate. While I can draw no major conclusions given the statistical limitations brought on by a sample size of n = 1, the study shows that a filtration experiment for P. stolonifera could shine light on other aspects of filtration. Repeated experiments could demonstrate the degree of consistency in P. stolonifera filtration rates. Furthermore, P. stolonifera may not have the capacity to recognize the presence or absence of their conspecifics in such a short time span thus we saw no change in filtration rate.  A more natural field setting over a grander time scale would better reflect the real conditions under which conspecific competition occurs.  In-depth studies could more accurately identify feeding behaviors in various conditions to better understand intraspecific interaction within the species P. stolonifera.

Acknowledgements
I would like to thank first of all both my Professors Bernie and Sandie Degnan for encouragement and guidance throughout my project.  I also cannot thank enough all three of our class tutors for their time and commitment to help each individual student with our many questions and technical issues.  

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