Tunicates (Phylum: Chordata, Subphylum: Tunicata/Urochordata) are a diverse group of more than 3000 filter feeding animals distributed in all of the world’s oceans, from shallow coastal waters to the deep sea (Smith 1989; Shenkar et al. 2019). They vary in size, body plan and life histories from sessile to free living, solitary to colonial (Brusca et al. 2016). They are among the most derived of all invertebrate phyla, exhibiting four key chordate features during development and retaining some of these features into mature life-stages (Ogura and Sasakura 2013). Recent phylogenetic studies suggest that Tunicata is the sister taxa to Vertebrata (Shenkar and Swalla 2011). Ascidians (Class: Ascidiacea, Oder: Enterogona, Suborder: Phlebobranchiata, commonly, sea squirts) lie within the Tunicata and are so named after their cellulose-containing epidermal test layer, called the tunic.

This report focuses on the temperate and tropically-distributed phlebobranch ascidian, Ascidia sydneiensis (Stimpson, 1855) and the curious ability of these animals to accumulate the transitional metal vanadium in their blood. The ascidian specimens upon which this report was based were collected at Manly boat harbour in Moreton Bay, Queensland by way of autonomous reef monitoring structures (ARMS).

NB: The identification of this species has been as accurate as possible. I have arrived at this identification based on key distinguishing morphological traits such as: colouration, number of lobes on atrial and buccal siphons, colour of ocelli and structure and positioning of the unpaired gonads (Enterogona) within the gut loop. Without having been able to acquire a clear scanning electron microscope image and count the number of stomata in the branchial basket, it is impossible to say conclusively that this identification is incontrovertible.
That being said, all of the information supplied in this page is to the best of my knowledge, true of this species. 

Tunic

The tunic of an ascidian is excreted by the ectoderm and surrounds the fragile body wall and internal organs (Kott 1985), as well as providing other biological functions (Hirose 2001). In the observed A sydneiensis specimens, the tunic was wrinkled and a translucent, light-brown colour and had visible test vessels (Figure 4).

There was light algal growth, as well as commensal, tube-building annelids encrusting the tunics of all of the studied specimens. The test (tunic) is a secreted ectodermal layer comprised of proteinaceous fibres, test cells, test vessels, blood cells and tunicin (Kott 2005; Satoh et al. 2014). Tunicin is the only known cellulose that is found in animal tissue, with all other examples seen in bacteria, plants and fungi (Passamaneck and Di Gregorio 2005). The acidity of the tunic, due to the tunic acid present in the cells, could serve the purpose of anti-biofouling in a sessile community (Hirose 2001). 

Internal body structure

The internal body structures of the ascidian are contained within a fragile body wall, enclosed within the vascularised tunic. The major observable structures are the large pharynx, pharyngeal basket, cylindrical heart, neural ganglion and a deeply curved gut loop (Figure 5). The gut loop (Figure 6) is comprised of an oesophagus, pyloric gland, folded stomach, intestine and anus. The intestinal tract surrounds the prominent ovary. Testis were not observed. Figure 7 shows the muscularisation and folding of the stomach walls. 

Also apparent in the histological slides examined was the neural ganglion (Figure 8). The purpose of this ganglion is disputed but likely plays a role in sensory input and neurosecretory function and has some influence over hormonal control of reproductive activity (Saad et al. 2014). 

Using a scanning electron microscope, the specimen's endostyle was visible (Figure 9). The endostyle is a ciliated groove that secretes the mucosal that lines the pharyngeal basket (Graham and Richardson 2012). This mucous that serves as fly-paper for food particles passing through the pharyngeal basket (Figure 10) is secreted by the endostyle (Graham and Richardson 2012)


The pharyngeal basket 
The vascularisation by longitudinal vessels of the unfolded pharyngeal basket is a defining feature of the phlebobranchs (Kott 1985; Kott 2005). The pharyngeal basket serves both a respiratory and feeding function. The basket captures organic matter as it passes through the inhalant oral siphon and across the mucous covered pharyngeal gill slits before filling the atrium, passing through the digestive system and being ‘exhaled’ out of the atrial siphon, along with digested organic matter. (Graham and Richardson 2012; Brusca et al. 2016). The branchial baskets of the A. sydneiensis specimens were highly vascularised, and blood cells were visible through a dissecting microscope.

The heart
The short and cylindrical heart is located in the proximal section of the body and is pumped by myocardium filaments that contract in series along the length of the heart (Brusca et al. 2016). Due to the sequential arrangement of organs in the ascidian body, whereby nutrients and oxygen etc. are moved along from the heart region through to the exhalant siphon, the oxygenative quality of blood declines progressively, as oxygen is depleted by the previous section (Brusca et al. 2016. To combat this, by action of automatic centres on either end of the cylinder, ascidian hearts undergo periodic reversal, causing blood to travel through the body in opposite the direction (Passamaneck and Di Gregorio 2005). Heart beat begins in juvenile ascidians following the body rotation in metamorphosis (Passamaneck and Di Gregorio 2005).  

Vanadium is found in oceans around the world at an average concentration of ~35nM (Ueki et al. 2019). Vanadium was first documented accumulating in ascidians by Dr. Friedrich Wolfgang Martin Henze in in 1911. This transitional metal accumulates highest in the blood cells of the phlebobranchs compared to all other ascidian orders, with Ascidia gemmata observed containing up to 10⁷ the concentration of vanadium occurring in seawater (Michibata 1996). Though the concentration of vanadium in A. sydneiensis is not quite as high as that figure, it is nonetheless classed as a vanadium-rich ascidian (Ueki 2003). Interestingly, vanadium is a highly toxic heavy metal in high concentrations and uptake in ascidian cells is most effective in acidic conditions, with a pH ranging between 1-3 (Treberg et al. 2012).  This vanadium accumulation phenomenon has not been observed at such magnitude in any other marine species (Michibata and Sakurai 1990). Consequently, there has been extensive study into ascidian biochemistry (particularly of the blood) in the century following this discovery (Chasteen 1990). 

Vanadium is currently used in commercial vanadium redox batteries (VRB) for grid-level energy storage for renewable energies (Prifti et al. 2012). When output is reduced, owing to a reduced availability factor, the stored energy in the VRB can be accessed for power supply. Due to the molecular structure of vanadium, it easily oxidises and reduces, releasing and receiving electrons (Rychcik and Skills-Kaszacos 1988; Clark and Marsden 2019). By creating a flow of electrolytes between anolyte and catholyte solutions, resulting in the reduction and oxidation of vanadium (between charged and discharged states) on both sides of the VRB, an electrical current is created (Prifti et al. 2012). On one side of the tank, V3+ reduces to become V2+ and VO²⁺ oxidises to become VO2+ on the other (Parasuraman et al. 2013). A VRB follows this cycle-discharge cycle, and doesn’t tend to lose recharge potential over time like a traditional battery, meaning that the battery can cycle nearly limitlessly with no emissions (Prifti et al. 2012; Rychcik and Skills-Kaszacos 1988; Parasuraman et al. 2013). Given the potential use of an emissions-free, near-limitlessly powered battery, the far-reaching importance of vanadium accumulation in a short lived species with high reproductive output becomes abundantly clear. With other heavy metals also being accumulated by these organisms, their potential roles as bioremediators is an application currently being explored (Romaidi and Ueki 2016; Kustin 1978). Romaidi and Ueki (2016) were able to isolate bacterial species occurring within the gut of A. sydneiensis var. samea which have some vanadium binding potential, which could be applied in systems with excessive heavy metal leaching, to reduce environmental degradation.  

Vanadium is found in the cells of the Auplousobranchia, Phlebobranchia, and Stolidobranchia suborders of the order Enterogona. The metal binds to vanadium binding proteins called Vanabins (Ueki et al. 2008). The phlebobranchs tend to have higher accumulation of the metal, with the highest known concentrations occurring in blood cells of Ascidia gemmata (Michibata 1996; Ueki et al. 2008). Vanadium binds to Vanabins found in ascidian blood cells known as vanadocytes (Ueki et al. 2003; Ueki et al. 2008). Vanadocytes may include: signet ring cells, bivacuolated cells and compartment cells, though there is debate as to whether these cells are in fact just progressive developmental stages of fewer cell types (Degnan, S. pers. comm.). Suggested roles of the accumulated metal include oxygen acquisition (Carlisle 1968), the formation of the tunic, chemical defence from predators and even prevention of excessive attachment of biofouling species upon the tunic (Michibata 1996). A study by Odate and Pawlik (2007) revealed that parrotfish found vanadium-containing salts unpalatable, supporting the chemical defence hypothesis. Smith (1989) suggested that vanadium uptake was a passive byproduct of uptake of another ion and that vanadocytes serve as proton and electron sink and strengthens the tunic. Perutz (1939) found that vanadium was highly concentrated in sandstone reef cores in Devonshire, suggesting that reef-living ascidians and indeed other reef-dwelling species, may be exposed to abnormally high amounts of the heavy metal throughout their lifetime, thus potentially shedding some light on this biological phenomenon.  

Live specimens of A. sydneiensis were retrieved from ARMS plates that had been deployed at Manly boat harbour. Using forceps and scissors, one specimen was dissected by cutting from the opening of the branchial (incurrent) siphon to the atrial (excurrent) siphon and continuing the incision along this dorsal line to the posterior end, opening the tunic to reveal the body wall and contained internal structures. The body wall was removed from the tunic and soaked in a saturated 2,2'-bipyridine solution (Figure 11). The dissected tunic was also bathed in the solution (Figure 12). After four minutes, some of the vanadocytes in the body wall of sample A began to stain purple, indicating the presence of vanadium (III) in Vanadocytes (Brand et al. 1990). The cells in the tunic did not appear to take up the 2,2'-bipyridine solution and the purple stain was not apparent until 24 minutes had passed (Figure 13). Purple stained vanadocytes were most numerous within the pharyngeal basket and oral siphon and in the muscles of the body wall. Figure 12 is an image of tunic tissue, taken through the microscope lens during the 2,2'-bipyridine accumulation phase, where only one vanadocyte has stained purple after 24 minutes (Figure 14). When observed under a higher powered microscope, the high density of vanadium-containing cells in a tissue sample of 2,2'-bipyridine treated pharyngeal tissue was apparent (Figure 15).