Ascidians, more commonly known as Sea Squirts, are members of Urochordata, a subphylum of Chordata. Ascidians are benthic, generally colonial and are surrounded by a tunic, also known as a test (Brusca et al., 2016). A colonial ascidian is generally characterised by morphologically identical polyps (or zooids) embedded together in a tunic with a shared circulatory system (Epelbaum et al., 2009). Their body plan is built around upward facing incurrent and excurrent siphons that draw water through the body to feed (Brusca et al., 2016). Many species within Ascidiacea are biofouling and have spread over large geographic areas due to anthropogenic factors, such as being carried in the ballast or on the hull of ships (Lambert, 2007).

Like many animals, ascidians make use of a system of cellular transport, that allows for blood containing nutrients and waste to be transported around the body. However, some small colonial ascidians, known as Botryllids, also require cellular transport between polyps, sometimes directly next to or further away. They also make use of blood vessels that lead both through and between polyps, as well as through the test (tunic vessels), to facilitate this transport (Monniot et al., 1991).

Botrylloides leachii is a species of colonial ascidian first described in 1816 by J.C. Savigny in Mémoires sur les animauxsans vertèbres (originally Botryllus leachi). This species has spread around the world and has been found from the Red Sea and the north-east Atlantic through to the Mediterranean, as well as around northern Australia (Kott, 1985). It is also known to vary wildly in colony colour between colonies from purple to red to mustard (Page et al., 2014), but there is no limit to the colour combinations (Kott, 1985). In the species B. leachii, the previously mentioned tunic vessel networks can be observed under a microscope, as can be seen in Figure 3. In some cases, these networks travel relatively long distances through the test. These channels can lead between different clusters of polyps, or branch off towards pear shaped terminal ampullae (Page et al., 2014) (Figure 3) where the contents are stored.

Travelling through these channels is the blood cells and colourless plasma (Monniot et al., 1991). In ascidians, there are several different types of blood cells, serving different functions such as nutrient storage, immunity, removal of heavy metals and waste as well as tunic formation (Schlumpberger et al., 1984). The movement of these cells and plasma throughout the polyp and through the test is facilitated by the heart.

The internal transport system in an ascidian is powered by the small cylindrical heart, surrounded by a pericardium (Kriebel, 1968; Ruppert et al., 2004) (Figure 2). In colonial species, such as B. leachii, the heart connects to tunic vessels which act as part of the circulatory system, allowing blood to travel through channels (also referred to as sinuses or blood vessels) in and through the tunic (Ruppert et al., 2004). This heart has pacemakers at either end that control the rate of beating (Ruppert et al., 2004).

Ascidian hearts are unique due to their ability to change the direction of flow (Kriebel, 1968). This can be observed in a colony and is shown in the video below. Sometimes this change in direction is sudden, but other times it slows to a pause, known as a reversal-pause, before pumping in the opposite direction (Kriebel, 1968). This change in direction is facilitated by the competition between the pacemakers at either end of the heart, alternating in dominance (Ruppert et al., 2004). The subsequent beating of the heart is referred to as a pulsation-series (Kriebel, 1968), and this is what generates the current that carries blood through the organism.

Colony 4 at 22 degrees Celcius

The optimal temperature range of B. leachii has been previously researched in an experiment by Brunetti, L. Beghi, M. Bressan and M. G. Marin, who observed that an adult B. leachii colony was best suited to water temperatures between 17 and 25 degrees Celsius. They also noted that at times when temperature or salinity levels cause stress to the organism, it is able to respond by either entering what they refer to as hibernation or undergoing colonial regression.

This experiment aimed to determine the effect of decreased temperature on;

• The rate of flow of blood through the tunic
• The amount of time blood spent travelling towards and away from the polyp within the tunic

These may allow for a better understanding of the morphological limitations on the distribution of this species based on their response to the stimuli.

The tunic blood flow in these colonies appears to be affected by the decrease in temperature. This can be seen in Figure 6, which displays a trend of decreasing amount of time spent pumping the blood (both towards and away from the polyp) as the temperature decreases, and Figure 7, which displays a trend of decreasing speed of the blood flow (both towards and away from the polyp) as the temperature decreases. Although there were no significant differences between the colonies for any of the variables, there persists a large variance. This variance may have contributed to the lack of statistical significance of any of the results.

Some of the variation in these results may have appeared due to several limitations on the experiment. Firstly, the precise temperature was not stable, often at times increasing or decreasing from the recorded temperature over the course of the filming.

Statistically, the low number of replicates may have contributed to the lack of significance, however, due to availability restraints, further testing was not possible. Also, due to the variation in the colonies, although not significant but morphologically obvious, there can be difficulty directly comparing them and this may have also contributed to the high levels of variance observed in Figure 6 and Figure 7 and thus the low significance level.

Variation may have also resulted from the length of the time period that was recorded for each colony. As can be seen in Figure 9, there was often large amounts of variation in speed within each colony at each temperature. This may have been alleviated by increasing the amount of time recorded giving better averages. Increasing recorded period would likely benefit all data.

The variable nature of the structure of tunic vessels may have also played a part in experimental error. Due to the visible joining and splitting of channels, it is possible to see different channels affecting each other. If these multiple joining tunic vessels are flowing at different rates, as can be interpreted from Figure 10, it can result in the channel that leads away from the polyp flowing in a different direction or stalling due to the additional channels leading back to the polyp cancelling each other out. This effect has been referred to as pacemaker competition (Kriebel, 1968).

It is also unclear as to whether the colonies are genetically differentiated from each other. The majority of the samples originated from the same tank and may have budded (grown from) a single ancestor (neighbour). Some of them were also located in close proximity and thus make it possible that they were at some point fused by a single test that has since split.

For many animals, the ability to pump blood around the body is essential. As can be seen in the results, there is a trend, however not a significant one, that suggests that as temperature declines, the ability for B. leachii to move blood through its test decreases. This could imply that this species experiences decreased fitness, as what had been previously suggested by Brunetti, L. Beghi, M. Bressan and M. G. Marin, and would be therefore ineffective at populating regions where the water temperature is below their optimum range. However, a specific range was not determined in this experiment, and more study on this morphological trait would provide further insight into the importance of this feature and whether this would affect the theoretical range of the species.