DOPA-containing proteins in the compound ascidian Botryllus schlosseri


 

ISJ 9: 1-6, 2012                                                                  ISSN 1824-307X 
 
 

MINIREVIEW 
 
Ascidian cytotoxic cells: state of the art and research perspectives 
 
L Ballarin 
 
Department of Biology, University of Padua, Padua, Italy 
 
 

   Accepted January 11, 2012 
 

Abstract 
Ascidian cytotoxic cells are multivacuolated cells, variable in morphology, abundantly represented 

in the circulation, playing important roles in ascidian immunosurveillance. Upon the recognition of 
foreign molecules, they are selectively recruited to the infection site where they release the content of 
their vacuoles. Their cytotoxic activity closely linked to the activity of the enzyme phenoloxidase (PO), 
a copper-containing enzyme widely distributed in invertebrates, contained inside their vacuoles 
together with its polyphenol substrata. Recent molecular data indicate that ascidian PO shares 
similarities with arthropod proPO but, unlike the latter, do not require enzymatic cleavage by 
extracellular serine proteinases for their activity. Possible ways of ascidian PO activation are 
discussed. 
 
Key Words: tunicates; ascidians; cytotoxic cells; phenoloxidase 

 
 
Introduction 

 
In recent years, the interest towards 

invertebrate immunity has considerably raised 
driven by comparative, evolutionary and ecological 
studies. Despite their relying only on innate 
immunity, invertebrates are capable of complex cell-
mediated and humoral responses able to guarantee 
the survival of individuals and, consequently, of the 
species. Two main immunocyte types are shared by 
invertebrates: professional phagocytes and 
cytotoxic cells which coordinate their efforts to cope 
with potentially pathogenic microbes having entered 
the organism. 

This paper will focus on ascidian cytotoxic cells, 
whose importance in immune surveillance, although 
still not completely clear, is progressively emerging 
thanks to the efforts of relatively few research 
groups focusing on a limited number of species. 
 
Ascidians: a renewed phylogenetic interest 

Since the publication of the paper by Delsuc et 
al. in 2006, changing their phylogenetic position 
within the phylum Chordata to the rank of vertebrate 
sister group, tunicates have experienced a period of 
scientific renown testified by the great increase in 
the interest of scientists towards this group of 
marine, filter-feeding organisms. Ascidians represent 
___________________________________________________________________________ 

 
Corresponding author: 
Loriano Ballarin 
Department of Biology, 
University of Padua, 
Via Ugo Bassi 58/B, 35100 Padua, Italy 
E-mail: loriano.ballarin@unipd.it

 

 
 
the best known and richest in species class of 
tunicates. Embryos give rise to free swimming 
tadpole-like larvae with a real notochord in their 
muscular tail, ventral to the neural tube which are 
replaced, at metamorphosis, by sessile, barrel-
shaped adults sharing, beyond the external tunic, a 
fissured pharynx, which occupies most of the 
volume of the organism, and a ventral endostyle 
able to secrete the mucous net useful in removing 
suspended particles from seawater passing through 
the pharyngeal slits (Ballarin and Burighel, 2002). 

Adult ascidians also have a well-defined tubular 
heart and an open circulatory system with 
hemolymph flowing within lacunae and sinuses 
inside internal tissues and, in most species, entering 
the tunic inside vessels derived from the epidermis. 
Hemocytes or blood cells represent an important 
constituent of the hemolymph, as they are involved 
in a variety of important biological processes such 
as wound repair, nutrient mobilization and transport, 
asexual reproduction and regeneration, waste 
accumulation, tunic synthesis, allorecognition and, 
last but not least, immune responses (Goodbody, 
1974; Wright, 1981). 
 
The ascidian professional killer cells and their poorly 
known history 

As invertebrates, ascidians rely only on innate 
immunity to cope with potentially pathogenic 
microbes or molecules having entered their 
organisms and their immune responses are mainly 
based on the activity of circulating immunocytes 
which include professional phagocytes, able to 

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mailto:loriano.ballarin@unipd.it


 

secrete a variety of immune-relevant molecules 
(Fuke and Fukumoto, 1993; Ballarin, 2008; Franchi 
et al., 2011), and a peculiar cytotoxic hemocyte-type 
which exert its biocidal activity through the 
enzymatic oxidation of polyphenol substrata and the 
induction of molecular damage consequent to the 
production of quinones and highly reactive oxygen 
species (ROS) (Cammarata et al., 1997; Ballarin 
and Cima, 2005). 

In most cases, these cells, which can 
collectively be called phenoloxidase (PO)-containing 
cells (POCC), assume a typical berry-like shape 
which justifies the name “morula cells” (MCs) used 
to indicate them and usually constitute one of the 
most abundant circulating haemocyte type 
(sometimes representing more than 50 % of the 
hemocytes). MCs are characterized by large 
diameters (10-15 μm) and cytoplasms filled by many 
vacuoles where the enzyme PO resides (Chaga, 
1980; Smith and Peddie, 1992; Arizza et al., 1995, 
Cammarata et al., 1997; Frizzo et al., 2000; Shirae 
and Saito, 2000; Shirae et al., 2002; Parrinello et al., 
2003; Cammarata et al., 2008). 

Until the end of the 1980s, POCCs were simply 
one of the numerous circulating hemocyte types 
reported in scientific papers, whose function was not 
well defined (Wright, 1981). They were considered 
too differentiated to be able to exert any important 
biological function and represented the research 
subject of relatively few investigators interested to 
the unusual acidic content of their vacuoles, which 
did not share any apparent relationship with the 
lysosomal machinery, and to their supposed ability 
to store metals such as vanadium (in most cases) 
and iron, hence the old names of vanadocytes and 
ferrocytes with which these cells were also known 
(Endean, 1960; Overton, 1966; Smith, 1970; Pirie 
and Bell, 1984; Milanesi and Burighel, 1978). This 
assumption resulted not true in the case of 
vanadium and the term vanadocytes today applies 
to cells different from MCs (Michibata et al., 1990; 
Nette et al., 1999; Yamaguchi et al., 2006). 

In 1980, in a paper written in Russian (which, 
unfortunately, limited its diffusion), Chaga reported, 
for the first time, that ascidian MCs have PO activity, 
a fundamental step towards the comprehension on 
the cytotoxic role of POCCs, but ten additional years 
were required for the first clear demonstration of the 
involvement of MCs in the induction of toxicity and 
the decisive advancement was made using botryllid 
ascidians as model organisms and one of the 
outcome of allorecognition, i.e., the rejection 
(nonfusion) reaction between contacting, genetically 
incompatible colonies of the same species resulting 
in the formation of a series of pigmented necrotic 
spots along the borders of the facing colonies, as 
the reference phenomenon (Hirose et al., 1990). 
Although known for many years, botryllid 
allorecognition was studied mainly in terms of 
morphology and formal genetics (Oka and 
Watanabe, 1957, 1960; Sabbadin, 1962; Mukai and 
Watanabe, 1974; Taneda et al., 1985) and papers 
dealing with the importance of hemocytes as 
mediators of the reaction appeared only from 1970s 
(Tanaka and Watanabe, 1973; Taneda et al., 
1982a, b); some observations suggesting a role of 
MCs in the rejection reaction were published in 

1980s (Taneda et al., 1982a; Scofield and 
Nagashima, 1983). The paper by Hirose et al. 
(1990) was the first histological analysis of the 
rejection reaction which unequivocally indicated 
MCs played a decisive role in the considered 
phenomenon. Today, we know that, during this 
reaction, MCs are selectively recruited from the 
general circulation to the lumen of the peripheral 
ampullae (the blind, sausage-like termini of marginal 
vascular vessels) facing the alien colony, then they 
leave the ampullar tips, cross the ampullar 
epithelium and enter the tunic where they 
degranulate and release their vacuolar content 
which, as reported below, is responsible of the 
induction of the observed cytotoxicity, a series of 
events sharing many features with vertebrate 
inflammation (Sabbadin et al., 1992). In B. 
schlosseri, where the nonfusion reaction has been 
particularly studied, MCs are activated by the 
recognition of soluble factors diffusing from the alien 
colony and express chemotactic molecules 
recognized by antibodies raised against mammalian 
proinflammatory cytokines, such as IL1α and TNFα, 
responsible of their selective recruitment inside the 
ampullar lumen and migration to the tunic (Cima et 
al., 2006; Ballarin, 2008). The selective recruitment 
of POCCs and their role in inflammatory and 
cytotoxic reactions consequent to graft 
transplantation or injection of nonself molecules was 
confirmed also in solitary ascidians (Cammarata et 
al., 1995, 1997, 2008; Parrinello, 1996; Parrinello et 
al., 1996). 

The cytotoxic role of POCCs, suggested by 
research on colonial and solitary ascidians, helped 
to insert in the same frame apparently dissimilar 
phenomena such as botryllid allorecognition, graft 
rejection and inflammatory reactions in solitary 
ascidians, contact reaction in mixed cultures of 
solitary species hemocytes, all induced by nonself 
recognition and involving POCCs as effector cells 
(Parrinello, 1996). Today, we can consider these 
cells as a sort of immunological sentinel able to 
recognize foreign molecules and quickly respond 
with the induction of cytotoxicity (Ballarin et al., 
2001, 2005). 
 
Phenoloxidase (PO): the ascidian molecular 
weapon against nonself 

POs are copper-containing enzymes, widely 
distributed in invertebrates, with 
orthodiphenoloxidase (catecholase) activity, able to 
convert phenolic substrata to quinones which, then, 
polymerize to form melanin. They exert a role in 
immune responses related to the induction of 
cytotoxicity, once released by specific hemocytes, 
through the production of ROS and semiquinones 
which can either induce oxidative stress or rapidly 
react with biomolecules altering their functionality. 
Quinones and melanin themselves are toxic: the 
former can undergo oxidation and generate reactive 
oxygen species (Nappi and Vass, 1993; Nappi and 
Ottaviani, 2000) or react with -SH groups on 
biomolecules (Kato et al., 1986), whereas the 
deposition of the latter in nodules of non-self 
material enveloped by host immunocytes 
contributes to the formation of the so-called brown 
bodies which prevent the survival of foreign 

 2



 

organisms in their interior (Nappi and Ottaviani, 
2000). 

The role of PO in ascidian cytotoxicity has been 
clearly demonstrated in both solitary and colonial 
species with the use of specific inhibitors in 
appropriate in vitro assays (Akita and Hoshi, 1995; 
Cammarata et al., 1997, 2008; Ballarin et al., 1998, 
2005; Hata et al., 1998; Parrinello et al., 2003). In B. 
schlosseri, during the rejection reaction, cytotoxicity 
is the consequence of a severe oxidative stress 
consequent to the depletion of reduced intracellular 
thiols (Ballarin et al., 2002). The involvement of the 
enzyme in the nonfusion reaction of other colonial 
species was also demonstrated (Shirae and Saito, 
2000; Shirae et al., 2002). Cytochemical analyses 
indicate that MC vacuoles also contain polyphenols, 
probably the PO natural substrata (Cammarata et 
al., 1997; Frizzo et al., 2000; Ballarin and Cima, 
2005). 

 
Ascidian PO: certainties and uncertainties 

The regulation of PO activity and its role in 
immune defense has been particularly studied in 
crustaceans, where the enzyme is usually stored, as 
inactive zymogen (proPO), inside the granules of 
PO-containing hemocytes, the morphology of which 
varies greatly among the different species (Aspán et 
al., 1995; Kawabata et al., 1995). The activation of 
proPO to PO requires the degranulation of PO-
containing cells, the release of the zymogen in the 
extracellular milieu and its conversion to active PO 
by extracellular serine proteases which are the last 
components of a finely regulated series of events, 
collectively called the proPO activating system, 
triggered by the recognition of foreign molecules on 
the surface of microbial cells (Cerenius and 
Söderhäll, 2004; Cerenius et al., 2008). 

Recent molecular investigations indicate that 
the primary, secondary and tertiary sequences of 
ascidian PO show many similarities with those of 
arthropod hemocyanins and proPOs, including the 
organization in three domains and the conservation 
of the histidines at both the copper-binding sites 
(Immesberger and Burmester, 2004). 

Similarly to arthropod POs, having a molecular 
weight ranging around 70 - 80 kDa (Decker et al., 
2007), the molecular weight of ascidian POs ranges 
between 60 and 90 kDa (Hata e et al., 1998; Frizzo 
et al., 1999; Parrinello et al. 2003; Cammarata et al., 
2008): these values agree molecular data predicting 
a molecular weight of 92 and 87 kDa for Ciona 
intestinalis POs (Immesberger and Burmester, 
1994). However, unlike arthropod POs, which 
assemble in vivo to form hexamers (Decker et al., 
2007), electrophoretic data indicate that ascidian 
PO monomers interact each other to form dimers 
(Frizzo et al., 1999; Parrinello et al., 2003; 
Cammarata et al., 2008).  

In addition, despite the absence of 
incontrovertible data, there is a general consensus 
that, like arthropods, a proPO activating system is 
present in all the invertebrates species in which PO 
activity has been demonstrated (Smith and 
Söderhäll, 1991; Cerenius and Söderhäll, 2004), 
ascidians included (Smith and Söderhäll, 1991; 
Jackson et al., 1993; Arizza et al., 1995; Ballarin et 
al., 1998; Shirae and Saito, 2000; Shirae et al., 

2002; Parrinello et al., 2003; Cammarata et al., 
2008). 

However, some doubts that this holds true also 
for ascidians come from the observation that the 
activation by exogenous serine proteases is never 
required for detecting the activity of ascidian PO in 
both hemocyte monolayers and hemolymph or 
hemocyte lysates (Smith and Söderhäll, 1991; 
Jackson et al., 1993; Arizza et al., 1995; Ballarin et 
al., 1994, 1998; Ballarin and Cima, 2005; Parrinello 
et al., 2003; Cammarata et al., 2008). Moreover, 
PO-positive MCs are easily observed in 
histoenzymatic assays on paraffin sections of 
colonial ascidians (Frizzo et al., 2000; Shirae et al., 
2002), implying that either the enzyme is 
constitutively active, at least in part, inside MC 
vacuoles or it can be easily activated during 
hemocyte collection or colony manipulation. The 
reported increase in enzyme activity observed after 
treatment with exogenous serine proteinases such 
as trypsin and chymotrypsin (Smith and Söderhäll, 
1991; Ballarin et al., 1994, 1998; Arizza et al., 1995; 
Cammarata et al., 2003, 2008; Parrinello et al., 
2003) which also leads to an increase in 
electrophoretic mobility of the enzyme (Parrinello et 
al., 2003), interpreted as evidences in favor of the 
presence of a proPO in ascidians, can be the result 
of unspecific effects of the exogenous proteinases 
which, acting on some cleavage sites and through 
the removal of enzyme fragment(s), decrease the 
protein molecular weight, with the consequent 
increase of its mobility during electrophoresis, and 
render the copper-binding sites more accessible to 
the substrate which results in the observed 
increment of enzyme activity. 

Since, as reported above, also the polyphenol 
substrata are contained inside the vacuoles of 
POCCs (Cammarata et al., 1997; Ballarin and Cima, 
2005), a control of the enzyme activity inside POCC 
vacuoles is required which can be achieved in at 
least two possible ways: i) the inhibition of the 
enzyme inside POCC vacuoles, and ii) the masking 
of the polyphenol substrata through chemical 
modifications. 
 
Conclusions and perspectives 

 
The two possibilities suggested above are 

supported by the fact that it is known that POCC 
vacuoles contain reducing compounds such as 
thiols (Ballarin et al., 1995) and tunichromes 
(Bruening et al., 1986; Oltz et al., 1988; Martoja et 
al., 1994) having inhibitory effects on PO (Ballarin et 
al., 1998; Hata et al., 1998). Tunichromes, in turn, 
are polyphenolic compounds (Bruening et al., 1986; 
Oltz et al., 1988) and, therefore, good candidates as 
natural substrata of PO. The modulation of PO 
activity by polyphenol substrata is an interesting 
point worth of deeper investigations. As for the 
second point, it is known that MCs contain sulfur in 
the form of bound sulfates inside their vacuoles (Bell 
et al., 1982; Scippa et al.,1985; Frank et al., 1987; 
Ballarin et al., 1995). The presence of the enzyme 
arylsulfatase inside Botryllus MC vacuoles (Ballarin 
et al., 1993) suggests that sulfates may be bound to 
the aromatic rings of polyphenols. Sulfates exert 
inhibitory effect on ascidian PO (Hata et al., 1998) 

 3



 

and the action of the enzyme may be important in 
detaching them thus rendering phenols available to 
PO. 

In conclusion, the importance of ascidian 
POCCs in immune responses is, today, definitively 
established; however, the regulation of the activity 
of PO, their main cytotoxic tool, and the 
relationships between polyphenols and PO remain 
controversial and require future investigations for a 
better clarification of these points. 
 
Acknowledgements 

Thanks are due to the Italian MIUR for 
supporting the research.  
 
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