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Pyura stolonifera (Heller, 1878)

Candida Wong 2017



Phylum:     Chordata

Class:         Ascidiacea

Order:        Stolidobranchia

Family:      Pyuridae

Pyura stolonifera, also known as Cunjevoi, is a solitary, sessile ascidian that is found in low intertidal and subtidal zones in which most individuals form aggregates and adhere to rocky substrates (Day 1974; Kott 1985; Dalby 1995; Edgar 1997; Monteiro et al. 2002). They often form dense beds on rocky shores and play a significant role as ecosystem engineers in which a range of marine flora and fauna are dependent on them for survival (Kott 1985; Monteiro et al. 2002). The soft inner body of cunjevois are popular with fishermen who use them as fishing bait (Monteiro et al. 2002). They have a conspicuous brown, leathery and thick test which encloses the body wall; and two siphons (branchial and atrial) that are used for filter-feeding. Cunjevois are hermaphroditic and broadcast spawners (Day 1974; Marshall 2002). Populations of P. stolonifera are found across Australian, South African and South American coasts and are believed to be relics of the Gondwanaland species (Kott 1985; Davie & Queensland Museum 2011). Species complex between populations is controversial and most populations occurring in Australia were named Pyura praeputialis, although this remains unresolved (Day 1974; Kott 1985; Davie 2011). 

Figure 1

Physical Description

Individual cunjevois are either relatively cylindrical and upright (Fig. 2), or stubby with a base that is expanded (Kott 1985). P. stolonifera individuals can grow up to 30 cm high and have an upper base that is 10cm in diameter (Kott 1985). An individual adult that was collected from Caloundra Head, Caloundra, Queensland was approximately 11.5cm in length (Fig. 3). 

Figure 2 shows the external appearance of an adult P. stolonifera that was collected from the rocky intertidal zone of Caloundra head, Caloundra, Queensland. The upper surface of cunjevois are somewhat depressed and consists of atrial and branchial apertures, or siphons that are bordered with triangular-like lobes arranged close together (Kott 1985). These apertures appear rounded and can be seen protruding from the upper surface (Kott 1985). A curved ridge that borders the upper surface of the individual P. stolonifera can be seen surrounding the apertures (Kott 1985). The atrial siphon is pointed upwards whereas the branchial siphon is slightly pointed to the side (Kott 1985; Fig. 2).

A thick, solid and leathery brown test can be observed on an individual and is sometimes covered in embedded sand (Kott 1985). Populations found on the rocky intertidal zones of rocky shores are tightly aggregated individuals that are somewhat cylindrical, tall and have epiphytes growing around the apertures (Kott 1985; Fig. 4). The basal end of the animal is enclosed in a tough, leathery test which provides support to the top end of the tunic (Kott 1985; Fig. 2). On the other hand, individuals from sandy regions have long bearded, root-like basal ends of the test that are enmeshed into the sand (Kott 1985).

Populations in Tasmania and South Australia are known to be smaller than the ones that are found on the coast of New South Wales (Kott, 1985; Fig. 5). The ridge surrounding the apertures on the upper surface of the Tasmanian individuals is smooth-edged, bare and yellow (Kott 1985; Fig. 5). Furthermore, populations in Victoria, Australia are known to show dimorphism in which the yellow morph occurs on the inner shores and the brown morph lives primarily on the outer coasts (Jr. 1997).
Figure 2
Figure 3
Figure 4
Figure 5



In many regions of the Southern Hemisphere, aggregates of P. stolonifera individuals attached to rock substrates are known to form extensive, dense beds on exposed rocky shores in low intertidal as well as shallow subtidal zones (Kott 1985; Dalby 1995; Monteiro et al. 2002) (Fig. 6; Fig. 7). Populations that occur in habitats of low intertidal zones can extend into subtidal depths of 10 to 12 m in which rocky substrates are clear without any sediments (Day 1974; Kott 1985; Edgar 1997; Monteiro et al. 2002) (Fig. 8). Cunjevois have also been reported to occur and attach to artificial structures such as wharf piles in protected waters (Kott 1985). Additionally, solitary individuals, instead of aggregates can be found on sandy substrates (Kott 1985). 

Sessile filter-feeding P. stolonifera are conspicuous and frequently abundant in areas that are maximally exposed to strong waves and currents (Kott 1985; Otway 1989; Edgar 1997; Monteiro et al. 2002; Paine & Suchanek, 1983; Fielding et al. 1994) (Fig. 9). 

Video showing Pyura stolonifera individuals filter-feeding in subtidal rock pool with constant flow
of seawater due to wave action. Video was taken at Caloundra Head, Caloundra, Queensland.

In rocky shores where P. stolonifera are distributed within Australia, beds formed by these ascidians can provide clumped and sparse habitats (Monteiro et al. 2002). In clumped habitats, there is more than 50% cunjevoi bed cover which is dense and crowded with tightly packed individuals (Monteiro et al. 2002). Sparse habitats, on the other hand, have less than 50% cover in which cunjevois are mostly solitary or isolated from contact with conspecific individuals (Monteiro et al. 2002). Differences between both habitats are presumably due to varying dispersal, settlement, and post-settlement mortality rates; as well as environmental disturbances (Monteiro et al. 2002). 
Figure 6
Figure 7
Figure 8
Figure 9

Ecological Relationships

Pyura stolonifera are known as intertidal ecosystem engineers in which dense beds formed by these ascidians have crevices and interstices that provide an important ecosystem and habitat for a wide range of fauna (macroinvertebrates) and algae (Fielding et al. 1994; Jones et al. 1994; Monteiro et al. 2002). These organisms are able to settle on the extended surface area of P. stolonifera aggregates which support sheltered microhabitats of enriched environmental conditions (Fielding et al. 1994; Monteiro et al. 2002). Furthermore, this naturally built ecosystem gives access to a constant flow of food and oxygenated water while providing protection from strong wave action (Fielding et al. 1994; Monteiro et al. 2002).
Figure 10

Life History and Behaviour


Pyura stolonifera are hermaphrodites and synchronous spawners that broadcast their eggs and sperm when partially submerged in seawater during low tide (Day 1974; Marshall 2002; Marshall & Evans 2007). The fertilisation success in P. stolonifera populations is highly dependent on the concentrations of localised sperms (Marshall 2002; Marshall & Evans 2007). Additionally, the density of embryonic development is highly variable in which incoming waves can immediately wash some spawned eggs into the water, whereas others remain in a viscous matrix (Marshall 2002; Marshall & Evans 2007). 

Larval Development and Growth

The larval stage of P. stolonifera is quite short and displays a strong preference for settling on conspecific individuals (Marshall et al. 2002; Marshall & Evans 2007) (Fig. 11A).

The cell division of a fertilised egg is rapid, after which a trunk and tail region start to differentiate within 12 hours of gastrulation (Griffiths 1976). A thin layer of clear test can be seen developing within the trunk (Griffiths 1976). The newly emerged tadpole larva is covered by a thin test that gradually develops into a wide fin that terminates on the posterior end (Fig. 12). Tadpole larva generally only have brief periods of inactivity as they are active swimmers (Griffiths 1976). 

During the short planktonic stage (less than 1 day), the larva is induced by optimal conditions within its environment to swim vertically downward and settle permanently on a substrate using sensory and adhesive organs such as the apical papillae (Griffiths 1976; Pennati et al. 2015). Collocytes, or glue-forming cells are secreted from this organ to aid in substrate adhesion (Pennati et al. 2015). After attachment, the larvae starts metamorphosis in which the test thickens, resorption of the tail begins; and an ampullae develops that spreads around and permanently attaches the animal to the substrate (Griffiths 1976; Pennati et al. 2015). The branchial siphon of an early metamorphosing tadpole larva is located towards the anterior of the trunk and secured parallel to the substrate whereas the vertically held atrial siphon is in the posterior region (Griffiths 1976; Fig. 12). The secured larva continues metamorphosing until it transforms into a sessile young adult (Griffiths 1976; Pennati et al. 2015) (Fig. 11B). Sessile juveniles can be observed with bumps and projections on the uneven surface of the animal (Day 1974; Fig. 11B).
Figure 11
Figure 12


Pyura stolonifera are active sessile filter-feeders in which the branchial (inhalant) siphon is aimed horizontally towards oncoming waves and the higher atrial (exhalant) siphon is directed vertically, or upwards (Knott et al. 2004). Cunjevois that are facing away from the sea typically orient their siphons into the backwash (Knott et al. 2004). As water enters the branchial siphon, the ascidian actively pumps water through the branchial basket with its cilia (Knott et al. 2004). These siphons are normally extended when submerged but are closed at low tide on rocky shores (Day 1974; Knott et al. 2004). Furthermore, these ascidians have been studied to induce passive flow to increase suspension feeding of main phytoplankton as well as particulate detritus and silt (Knott et al. 2004; Seiderer & Newell 1998). Particles of approximately 0.5 to 20 µm are efficiently retained from most suspended natural matter (Klumpp 1984).

Pyura stolonifera actively filter-feeding on algal nutrient solution.
Video was taken at the Degnan Marine Aquarium in the University of Queensland.

Anatomy and Physiology


Two individual adult P. stolonifera were dissected showing the bisections (left and right) of the whole anatomy and the body that was removed from the test (Fig. 13 and Fig. 14).

Below are key anatomical features observed:-
  1. The external appearance of the test is thick and leathery and internal test is clear and gelatinous consisting of fiber networks (Day 1974; Kott 1985).
  2. Soft, fleshy and orange-red coloured body wall with longitudinal and circular muscle fibers (Day 1974; Kott 1985).
  3. A tubular and elongated heart that is enclosed in pericardium with twisted rope-like appearance (Goddard 1972; Day 1974; Kott 1985).
  4. Distinctly curved folds (about 6 to 7) in the branchial sac with internal longitudinal vessels (Day 1974; Kott 1985).
  5. Wide and curved intestinal loop (Day, 1974). Fully advanced and distinctive digestive glands (Day 1974). 
  6. Hermaphrodite gonads consisting of block-shaped polycarp sacs serially arranged (Day 1974; Kott 1985).
Figure 13
Figure 14

The Test and Body Wall

The test of the P. stolonifera is leathery, thick and tough and is used as a form of protection from predation and strong incoming waves (Day 1974; Kott 1985). The clear and gelatinous interior of the test consists of a network of fibres that follow the contours of the surface (Endean 1955b; Day 1974). Thin, solid and compact polysaccharide fibres, which are formed from iron-rich blood cells called ferrocytes, can be observed surrounding the cavity of the test (Endean 1955b; Day 1974). An extensive network of blood vessels (from anterior and posterior vessels of the test) intertwine and can be observed having prominent branches to all parts of the test (Endean 1955b; Day, 1974). However, these network of vessels (anterior and posterior vessels) are independent in supplying blood to the test (Endean 1955b; Day 1974).

The orange-red body wall of P. stolonifera is fleshy and soft with bands of longitudinal muscle fibres extending from the apertures towards the basal part of the body (Day 1974; Kott 1985). The body wall also has thin circular muscle fibres encircling the body, which start from the base of the siphons (strongly developed) and but don't extend very far ventrally (Day 1974; Kott 1985).  Both branchial and atrial siphons have thin red inner linings formed by inversions of the epidermis (Day 1974).

Respiratory and Circulatory System

The heart of P. stolonifera consists of a tubular and elongated structure that is enclosed in a pericardium entrenched in the right side of the body wall (Goddard 1972; Day 1974; Kott 1985). This muscular circulatory organ can be observed located to right of the endostyle, below the mantle and can be easily damaged due to a very delicate tissue wall (Goddard 1972; Day 1974). In ascidians, the direction of the heart-beat is reversed occasionally (Day, 1974). However, Day (1974) has described the circulatory system in a way that it is unidirectional in P. stolonifera, in which blood flows from the anterior end of the heart (Day 1974).

The main vessels found in the anterior end of the heart are the anterior and posterior subendostylar sinuses; as well as the subintestinal vessels (Day 1974). The hepatic, gastric and oesophageal vessels are found in the area of the oesophagus in which the endostyle and posterior end of the heart crosses (Day 1974). As blood flows from the anterior part of the main circulation organ, it reaches the branchial walls through the subendostylar sinuses and minor vessels; as well as the test through the anterior test vessel (Day 1974). Branches of tentacular vessels lead into the branchial and atrial siphons, body wall as well as the branchial tentacles (Day 1974). Additionally, blood flows into the dorsal vessel from the branchial siphon and oesophagal bulb passing the cerebral ganglion, thus leading into the dorsal sinus of the pharynx (Day 1974). At the posterior end of the heart, blood flows directly from the atrial siphon (Day 1974). 

The branchial sac consists of deeply curved folds in which internal longitudinal vessels are present (Day 1974; Kott 1985). Significant respiratory exchange takes place in the branchial tentacles as well as the branchial sac in which the vessels of the tentacles leading into the dorsal vessel are thin (Day 1974). Consequently, this provides efficient circulation of oxygenated blood from the dorsal vessel (Day 1974). 

Vacuolated Blood Cells and Vanadium Bioaccumulation

Bioaccumulation of vanadium in ascidians have been studied for over a century since Martin Henze, a German physiologist, discovered vanadium compounds in the blood cell of a sea squirt in 1911 (Michibata 1996; Michibata & Ueki 2012). The ascidian species, Ascidia gemmata has been recorded to have the highest concentration of vanadium (350 mM), which is 10 million times that of the concentration of vanadium in the sea (Michibata 1996; Michibata & Ueki 2012; Ueki et al. 2015). 

Vanadium is a type of multivalent, transition metal that is present in the earth’s crust and exhibits a wide range of oxidation states (from -1 to +5) (Assem & Levy 2011; Kanamori & Tsuge 2012). Low concentrations of vanadium are distributed in the seawater as this heavy metal is released into the environment from a succession of complex biogeochemical processes and anthropogenic activities (Collier 1984; Michibata 1996; Assem & Levy 2011). Biological and chemical applications of vanadium have been studied over time and was found to have bioremediation functions in which bacterial strains with metal-binding abilities from vanadium bioaccumulating ascidians can address issues pertaining to soil and water heavy metal decontamination (Ueki et al. 2003; Romaidi & Ueki 2016).

Vanadium ions are known to accumulate specifically in the vacuolated blood cells of ascidians in which vanadium ions sequestered from the seawater via the branchial sac are transported into the blood plasma and finally stored in blood cells (Ueki et al. 2015). Due to the vanadium storage functions of these vacuolated blood cells, they are also known as vanadocytes (Ueki et al. 2015). Vacuolated blood cells have fluid-filled vacuoles in the the cell cytoplasm that are enclosed in a lipid surface membrane and play essential roles in test development, wound repair and inhibit blood coagulation (Endean 1960; Taylor 1992). Vacuolated blood cells have been categorised into at least four types which are morula cells, signet ring cells, compartment cells, and small compartment cells (Michibata & Ueki 2012).

The blood cells of P. stolonifera were previously described and characterised by Endean (1955). In this recent mini study, a dissection on an individual P. stolonifera was performed in which the heart was sectioned and stained with hematoxylin and eosin to observe the vacuolated blood cells (Fig. 15). These blood cells were identified and characterised based on fixed sample observations (Table 1; Fig. 16).

There has been contention between various studies into the true vanadocytes. However, more studies have concluded that these are signet ring cells, bivacuolated cells, compartment cell Type II and vacuolated amoebocytes (Nette et al. 1998; Michibata et al. 2002; Ueki et al. 2015). According to Nette et al. (1998), bivacuolated cells are of the same cell lineage as signet rings cells. 

Ascidians that have been studied for vanadium uptake from the Stolidobranchia suborder are known to have lower concentrations of the transition metal compared to the suborder Phlebobranchia (Ueki et al. 2015). Furthermore, investigations into the blood cells of P. stolonifera have found that the blood cells do not store vanadium but are iron-rich instead (Endean 1955; Hawkins et al. 1980). These iron accumulating blood cells are termed ferrocytes which are morula-shaped (Hawkins et al. 1980). Perhaps more advanced studies involving sophisticated microscopy and biochemical techniques could be performed to further investigate the vacuolated blood cells of Pyura stolonifera in order to contribute to the growing research of vanadium uptake in bioaccumulating ascidians.
Figure 15
Figure 16

Digestive System

During feeding, mucous strings are produced by the endostyle in which they rapidly move out over prominent pharyngeal folds of a huge pharyngeal sac and are simultaneously passed into the narrow oesophagus that leads into the stomach of P. stolonifera (Day 1974). Undigested mucous strings have been found in the stomach of dead individuals of P. stolonifera within 30 minutes of the food particles entering the siphons according to a study conducted by Day (1974). P. stolonifera are known to have an indistinct stomach which is generally observed in large solitary ascidians (Day 1974).

The intestine is wide, curved and loops around the posterior and ventral part of the body (Day 1974). This intestinal loop forms part of the descending limb and the entire ascending limb of the gut and ends at the anus which is at the base of the atrial aperture (Day 1974).  The digestive gland, also known as the liver is very distinct and displays an advanced development from the diverticulum of some Stolidobranch ascidians (Day 1974).

Nervous System

The cerebral ganglion of P. stolonifera located above the dorsal tubercle has four primary nerve trunks and is elongated (Day 1974; Fig. 17). Two of the nerve trunks are connected to the base and tip of the branchial siphon and the others are similarly connected to the atrial siphon (Day 1974; Fig. 17).

The dark neural gland with a double spiral opening is located above the cerebral ganglion and is three times larger with two horns passing down on each side of the ganglion (Day 1974). This neural gland further connects between the dorsal and tentacular blood vessel leading to the oesophageal bulb (Day 1974; Kott 1985).
Figure 17

Reproductive System

A pair of block-shaped hermaphrodite gonads are embedded in the mantle wall of P. stolonifera. These gonads are split into a series of polycarp sacs that can be in paired (along the side of central ducts) or single rows (Day 1974; Kott 1985). The polycarp sacs of the left gonad are primarily located in the intestinal loop and the polycarp on the posterior end is normally above the gut or outside the intestinal loop (Day 1974). Ovary and testes can be found radially organised within each polycarp sac; and the gonoduct that links the polycarp sacs is subdivided by folds leading into the channels of ova and sperm (Day 1974). However, separate male and female gonopores that are positioned next to each other, or behind one another are observed at the base of the atrial siphon (Day 1974).

Biogeographic Distribution

Populations of P. stolonifera in Australia can be found on the western, eastern (Queensland and NSW), southern coasts (Victoria); and Tasmania (Day 1974; Kott 1985; Edgar 1997; Davie 2011). This species is also distributed along the coast of South Africa, northern coast of New Zealand and the Pacific coast of South America (Chile) (Day 1974; Kott 1985; Fielding et al. 1994; Davie & Queensland Museum 2011).

The distribution of P. stolonifera was hypothesized as evidence of relic populations from Gondwanaland when southern continents were joined together to form this super continent (Kott 1985; Davie & Queensland Museum 2011).

Evolution and Systematics

Phylogenetic Relationships

Colonial and solitary ascidians from the families Pyuridae, Styelidae and Molgulidae belong to the order Stoliobranchia (Pérez-Portela 2009). The family Mogulidae is sister to the families Styelidae and Pyuridae in which Styelidae is paraphyletic and Pyuridae is monophyletic (Pérez-Portela 2009; Figure 18). 

The species Pyura stolonifera belongs to the genus Pyura which is from the family Pyuridae (Pérez-Portela 2009). The relationship of the genus Pyura within the Pyuridae family is polyphyletic (Pérez-Portela 2009; Fig. 18). 
Figure 18

Species Complex

The relationships between populations from South Africa and Australia have been disputed and as such, the Australian species has been referred to as Pyura praeputalis (Heller, 1878) (Kott 1985; Davie & Queensland Museum 2011). Both populations were regarded as separate species based on the differences in morphological characteristics of the branching spicules and the coiling direction of the neural gland opening (Kott 1985). However, both characteristics were confirmed to be stable between populations; and therefore was not enough to justify separating African and Australian species (Kott 1985). 

Populations from the South American continent are also regarded as conspecific with the African and Australian populations (Kott 1985). There has been no considerable evidence that these populations have been isolated long enough to attain true biological isolation for the status of being separate species (Kott 1985). However, due to the geographic separation, it was suggested that each population could be regarded as a subspecies (Kott 1976a).

As shown in Figure 19, a recent study conducted by Rius and Teske (2013) using morphogenetic approaches revealed levels of cryptic diversity within the P. stolonifera species complex. At least five distinct cryptic species were sub-divided into lower-level evolutionary lineages (Rius & Teske 2011; Rius & Teske 2013):-
  1. Pyura stolonifera
  2. Pyura praeputalis
  3. Pyura doppelgangera n. sp.
  4. Pyura herdmani
  5. Pyura dalbyi

Results from this study showed that the P. stolonifera species complex requires further examination and scrutiny, especially in the evolutionary and ecological mechanisms that formed the current biogeographic distribution (Rius & Teske 2013). This means that the status of separating populations into distinct species still remains unclear (Davie & Queensland Museum 2011).

Figure 19

Conservation and Threats

Cunjevois have been frequently removed from rocky shores and used as bait by fishermen (Monteiro et al. 2002). As P. stolonifera populations are ecosystem engineers, their removal affects and changes the structure of the dense beds they have created and would cause disturbances in associated biota (Fielding et al. 1994; Monteiro et al. 2002; Rius & Teske 2013). In NSW, the maximum number of individual cunjevois that fishermen can collect at any one time is 20 (NSW Government 2017). However, P. stolonifera are generally common and have not been assessed by the IUCN Red List of Threatened Species, and are therefore not a threatened species (IUCN 2017).


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