Solitary ascidians embryos (Chordata, Tunicata) as model organisms for testing coastal pollutant toxicity

Abstract

Marine coastal communities are daily exposed to several chemical compounds commonly used in agriculture and industrial activities. Therefore, toxicological studies evaluating the effects of these compounds on marine organisms are of primary importance for marine environment preservation. Different model organisms are used to perform toxicity tests with potential pollutants, under laboratory conditions. In last decades, solitary ascidians have been selected as valuable model organisms to run bioassays with embryos and larvae. In fact, by in vitro fertilization, it is easy to obtain thousands of embryos, rapidly developing and therefore allowing a fast screen of pollutant toxicity.The aim of this review was to summarize results from toxicity tests, run with heavy metals, organo-metal and organic compounds, on solitary ascidian development and settlement to evidence that these animals offer several advantages as models to perform these kind of studies. First of all, they have a sensitiveness directly comparable to that of other marine model organisms. Moreover, the effects of toxicants on exposed embryos and larvae could be studied using different approaches, from ultrastructure to genetic analysis. Finally, since ascidians are chordates morphological and gene expression analyses could provide data for comparative studies with vertebrates.

Full text

Page 1

ISJ 6: S29-S34, 2009


ISSN 1824-307X
REVIEW

Solitary ascidians embryos (Chordata, Tunicata) as model organisms for testing
coastal pollutant toxicity

G Zega, R Pennati, S Candiani, M Pestarino, F De Bernardi

Department of Biology, University of Milan, Milan, Italy


Accepted March 13, 2009

Abstract
Marine coastal communities are daily exposed to several chemical compounds commonly used in
agriculture and industrial activities. Therefore, toxicological studies evaluating the effects of these
compounds on marine organisms are of primary importance for marine environment preservation.
Different model organisms are used to perform toxicity tests with potential pollutants, under laboratory
conditions. In last decades, solitary ascidians have been selected as valuable model organisms to run
bioassays with embryos and larvae. In fact, by in vitro fertilization, it is easy to obtain thousands of
embryos, rapidly developing and therefore allowing a fast screen of pollutant toxicity.
The aim of this review was to summarize results from toxicity tests, run with heavy metals, organo-
metal and organic compounds, on solitary ascidian development and settlement to evidence that
these animals offer several advantages as models to perform these kind of studies. First of all, they
have a sensitiveness directly comparable to that of other marine model organisms. Moreover, the
effects of toxicants on exposed embryos and larvae could be studied using different approaches, from
ultrastructure to genetic analysis. Finally, since ascidians are chordates morphological and gene
expression analyses could provide data for comparative studies with vertebrates.

Key words: heavy-metals; antifoulants; pesticides; development; Tunicates; ascidians


Introduction

Marine environment pollution is a concrete risk
along densely populated coastal regions, where
urban and industrial development could facilitate the
dispersal of several chemical agents. Therefore,
marine coastal ecosystems could be endangered by
pollutants, such as heavy metals, pesticides and
antifoulants that could be easily detected in the
water column or in the sediment of harbours and
estuaries (Castillo et al., 2006; Antizar-Ladislao,
2008; Bellas et al., 2008). These areas, often very
rich in nutrients, host filter-feeders communities
encompassing bivalves, serpulids and ascidians.
Marine mussels have been selected early for the
study of coastal pollution impact on marine life.
More recently, ascidians have been selected as
potential model organisms for testing pollutants
toxicity as they offer several advantages for these
studies (Mansueto et al., 1993; Cooper et al., 1995;
Cima et al., 1996, 2008; Bellas et al., 2003).
___________________________________________________________________________

Correspondig author:
Fiorenza De Bernardi
Department of Biology
University of Milan
via Celoria 26, 20133 Milan, Italy
E-mail: fiorenza.debernardi@unimi.it


Solitary ascidians (Chordata, Tunicata) are
marine benthic filter-feeders that occur in dense
populations along eutrophic coastal habitats, and
therefore they could be easily sampled. They are
hermaphrodite organisms that reproduce sexually
by the simultaneous emission of eggs and sperm.
Fertilized eggs develop in the water column in about
a day into a planktonic tadpole larva that shows
some chordate characters, a dorsal hollow neural
tube and a notochord flanked by muscle cells. Adult
solitary ascidians of Ciona, Phallusia and Styela
genus are world wide distributed and fertile almost
all year round. Gametes can be easily obtained by
gonoduct dissection and, from in vitro fertilization, it
is possible to obtain thousands of synchronously
dividing embryos . Under laboratory conditions,
development is completed in about 16-24 hours in a
range of decreasing temperature from 22 to 16°C.
For these characteristics, solitary ascidians are
valuable and reliable organisms to run toxicity tests
on gametes and embryos, for the high number of
specimen easy available every time and the rapid
development.
In last decades, several studies have been made
to test the effect of different pollutants on ascidian
development that is evaluating the percentage of

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Table 1 List of compounds whose toxicity has been tested on solitary ascidians embryos to determine median
effective (EC50) concentration on development and settlement. When available the environmental concentration is
also listed in bold, together with its reference


Compounds
classification
Embryos
Chemical
EC50
(µM)
EC50 (µM)
Larval
settlement
Environmental
concentration
(µM)

Hg
Heavy metal 0.22 0.39
0.002
Bellas et
al., 2004;
OSPAR
Commission,
2000
″
Cu
Heavy metal 0.58 1.61
5.67
Cd
Heavy metal 6.42 6.7
0.23
″
Cr
Heavy metal 226 289
-
″
TBT
Tributyltin
Zinc pyrithione (Zpt)
Zinc 1-oxidopyridin-1-ium-2-
thiolate
Organometallic
anti-foulant
Organometallic
bactericide, anti-
foulant
0.02
-
0.01
Bellas et
al., 2005
0.23 0.11 -
Bellas,
2005
Lindane
1,2,3,4,5,6-
hexachlorocyclohexane
Organochloride
insecticide
15.20
-
0.004
Bellas et
al., 2005;
OSPAR
Commission,
2000
Chlorpyrifos
Diethoxy-sulfanylidene-(3,5,6-
trichloropyridin-2-
yl)oxyphosphorane
Diuron
3-(3,4-dichlorophenyl)-1,1-
dimethylurea
Chlorothalonil
2,4,5,6-tetrachlorobenzene-1,3-
dicarbonitrile
Sea-Nine 211 (Kathon 930)
4,5-dichloro-2-octyl-1,2-thiazol-3-
one
Dichlofluanid
N-(dichloro-fluoromethyl)sulfanyl-
N-(dimethylsulfamoyl)aniline
Tolylfluanid
N-(dichloro-fluoromethyl)sulfanyl-
N-(dimethylsulfamoyl)-4-
methylaniline
Irgarol 1051
N-tert-butyl-N'-cyclopropyl-6-
methylsulfanyl-1,3,5-triazine-2,4-
diamine
Organo-
phosphorus
pesticide
15.70 - -
″
Urea derived
herbicide
17.80 - -
″
Organochloride
fungicide, anti-
foulant
0.12 0.16
0.005
Bellas,
2006

Organochloride
anti-foulant
0.37 0.15
0.013
″
″ ″
Organochloride
anti-foulant
0.85 0.39
0.017
″
″ ″
Organochloride
anti-foulant
0.62 0.28 -
″
Anti-foulant 8.34 >25.60
0.016
″
″ ″
Imazalil
1-[2-(2,4-dichlorophenyl)-2-prop-
2-enoxyethyl]imidazole
Organochloride
triazole-
imidazole
fungicide
Organochloride
triazole
fungicide
Organofluoride
triazole
fungicide
0.67
-
0.47
Pennati et
al., 2006;
FAO, 2001
Triadimefon
1-(4-chlorophenoxy)-3,3-dimethyl-
1-(1,2,4-triazol-1-yl)butan-2-one
Fluconazole
2-(2,4-difluorophenyl)-1,3-
bis(1,2,4-triazol-1-yl)propan-2-ol




29.56 - -
″
74.70
-
-
Groppelli et
al., 2007

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normal larvae hatching from different treatments. In
fact, larvae have a simple body plan that allows the
rapid screening of malformed specimen. The
tadpole larva body is formed by a trunk and a tail.
The trunk bears main sensory organs: the three
adhesive papillae or palps, situated at its anterior
end, and one or two pigmented organs, situated in
the sensory vesicle. The palps contain sensory
neurons, through which the larva is able to choose a
substratum where to settle, and mucus secreting
cells to perform permanent attachment (Groppelli et
al., 2003, Pennati et al., 2007). Commonly, larvae
possess two pigmented organs, the ocellus and the
otolith respectively a photo- and gravity-receptor,
but some larvae could have only one, usually called
the photolith, as it perceives both kind of stimuli.
The sensory vesicle is the anterior portion of
the central nervous system that continues towards
the posterior end as a dorsal hollow tube, divided in
three portions, the neck, the visceral ganglion and
the tail nerve cord. The neck, devoid of neurons,
connects the sensory vesicle to the visceral
ganglion. This latter contains
ascending projections and motor neurons with
descending projections to tail muscle cells (Imai and
Meinertzhagen, 2007).
Interestingly, tunicates are considered the
vertebrate sister group (Delsuc et al., 2006) and
their embryos and larvae share basic homologies
with vertebrates also at the level of the expression
of developmental regulatory genes (Meinertzaghen
et al., 2004). In particular, the availability of Ciona
intestinalis genome sequences (Dehal et al., 2002)
could favour the study of toxicant effects on gene
expression. For example, a chip for cDNA
microarray analysis has been developed to
investigate gene expression profiles in TBT (Table
1) exposed ascidians (Azumi et al., 2004).
In this review, results from toxicity tests run with
heavy metals, organometals
compounds, such as pesticides and anti-foulants,
on ascidian development will be reported. This
overview has the aim of evidencing how it is
possible to take advantage of solitary ascidians to
perform toxicity studies, with several approaches.

Effects of heavy metals, organometallic and
organic compounds on ascidian development
and settlement

Among pollutants, heavy metals and organo-
metallic compounds showed the highest toxic
effects on ascidian development and settlement.
Exposure to Hg, Cu, Cd and Cr of C. intestinalis
embryos for 20 h severely reduced percentage of
hatching of normal larvae and of settlement. The
EC50 (median effective
determines larval malformation) values of Hg, Cu
and Cd were very low indicating that these metals
could effectively impair
consequently larval attachment (Table 1). Moreover,
C. intestinalis sensitiveness to such pollutants
resulted comparable to what previously reported for
other marine organisms commonly used in toxicity
test, such as the bivalve Mytilus galloprovincialis
and the sea-urchin Paracentrotus lividus (Bellas et
al., 2004).
neurons with
and organic
concentration that
development and
In the group of tested organic compounds,
organo-metallic ones resulted the most toxic for
ascidians such as Styela plicata and C. intestinalis.
Micromolar doses of organotin compounds (TBT,
TPT, TCHT), blocked development of S. plicata
embryos in a stage-dependent manner. In fact,
earliest developmental stages, 2-4 cells to gastrula,
were more sensitive (Cima et al., 1996) (Table 2).
The ultrastructural analysis of 1h exposed embryos
of different stages revealed the presence of
electron-dense precipitates in mithocondria, whose
membrane were severely damaged. Moreover,
blastomere shape and adhesion were also affected
most probably because organotin compounds could
interfere with cytoskeletal proteins. Similarly, C.
intestinalis embryos exposed from neurula stage for
1 h showed malformed
blastomeres, lacking cytoskeletal elements. As a
consequence, neurulation
(Dolcemascolo et al., 2005) (Table 2). The effect of
TBT was studied also on late developmental stage
of C. intestinalis. Pre-hatching and swimming larvae
exposed for 1 h to 0.1µm TBT showed severe tail
malformations. Muscle cells had an abnormal
distribution along the tail and irregularly shaped
nuclei. Moreover, the ultrastructure of sarcomeres
and muscle mitochondria appeared completely
compromised (Gianguzza et al., 1996) (Table 2).
When C. intestinalis embryos were exposed to TBT
throughout development (about 20h) development
was blocked and EC50 was 0.022µM (Bellas et al.,
2005). Another potent organometallic anti-foulant,
zinc pyritione (Zpt) showed similar effect on C.
intestinalis development and settlement (Table 1)
(Bellas, 2005).
Pesticides and anti-foulants are the last group
of compounds whose toxicity was investigated on
ascidian development (Table 1). These substances
have a broad-spectrum activity and their action on
ascidian embryos were studied mainly evaluating
dose-depending effects on development. For each
compound the EC50 value was calculated (Table 1).
For some organic pesticides and anti-foulants, such
as Lindane, Chlorpyrifos, Diuron, Irgarol 1051,
Triadimenfon and Fluconazole, EC50 values were
quite high in terms of toxicity, corresponding to
micro-molar concentrations. Organochloride anti-
foulants (Chlorothalonil,
Dichlofluanid, Tolylfluanid) instead resulted the most
toxic substances, for their very low EC50 values.
Moreover, among fungicides, Imazalil, that contains
two chlorine atoms, showed a similar toxicity for
ascidian embryos (Bellas et al., 2005; Bellas, 2006;
Pennati et al., 2006; Groppelli et al., 2007). Effects
of the triazole fungicide was also evaluated in terms
of teratogenicity as these substances induced
specific malformations whose severity was dose-
dependent. Therefore, larval phenotypes obtained
after triazole exposure throughout development
were classified using a dissection microscope and
further characterized by means of histology and
immunohistochemistry
exposed larvae showed typical malformation: the
trunk appeared shortened, the palps were fused or
not completely differentiated, and the sensory
vesicle was reduced with displaced pigmented
organs (Fig. 1A, B). Moreover, the anterior nervous
and disorganized
was blocked
Sea-Nine 211,
experiments. Triazole

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Fig. 1 Control larvae (A, C, E) and larvae developed from embryos exposed to 5 µm Imazalil (B, D, F) of the
solitary ascidian Ciona intestinalis. Control (A) and malformed larva (B) showing the typical Imazalil induced
phenotype. Immunohistochemical localization of β-tubulin in control (C, E) and malformed (D, F) larvae, whose
anterior nervous network appeared disorganized. Bars = 100 µm.




network was compromised, as evidenced by
immunolocalization of β-tubulin (Fig. 1C-F). In
these studies, the teratogenic action of triazoles
on ascidian development was directly compared
with what known on vertebrate embryos, where
these fungicides typically affect differentiation of
the anterior structures, interfering with retinoic
acid catabolism. The authors found evidences
that also in ascidians the observed phenotypes
could be due to an alteration of retinoic acid
signalling (Pennati et al., 2006; Groppelli et al.,
2007).

Conclusions

Coastal pollution could stress marine
communities determining a decrease in biodiversity
for the disappearing of more sensitive species
(Castillo et al., 2006; Bellas et al., 2008).
Marine benthic invertebrates are easily exposed
to toxic compounds commonly used in agriculture,
industrial and harbour activities, and have been
selected as model organisms to evaluate effects of
these substances on life processes.
Some of the listed inorganic and organic
compounds were proved to impair ascidian
development at very low doses, ranging from nano-
to micro-molar concentrations. Even if the average
environmental concentration of these compounds is
lower
development (Table 1), we believe that the chance
of endangering coastal populations of sessile
tunicates is a realistic risk. In fact, given the wide
production of pesticides (Tilman et al., 2001), the
possibility of local accumulation by accidental spills
must be also considered. Similarly, the extensive
uses of anti-foulants favour their accumulation in
harbours shallow waters (Bellas, 2006). Moreover,
among the substances considered in this review,
Cu, TBT and Imazalil were detected in water or soils
in concentrations directly comparable to their EC50
values on ascidian development (Table 1). From the
conspicuous studies reviewed here, it is clear that,
among benthic coastal invertebrates, solitary
ascidians are valuable organisms to be considered
among models to test toxicity of potential or known
pollutants on their development and settlement
ability. In fact, it is possible to run toxicity tests on a
high number of embryos and to rapidly (1 day)
screen the effects. Solitary ascidians embryos and
larvae can be used in these laboratory studies with
different approaches, from exposure bio-assays of
different developmental stages to morphological
analysis at different levels (ultrastructure, histology,
immunohistochemistry). Results summarized here
evidenced that several common pollutants strongly
impaired ascidians development and consequently
their dispersal and recruitment phases.
than their EC50 values on ascidian

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Table 2 Effects of different compounds on the development and/or morphology of larvae of three different
ascidian species

Effective
concentrations
(µM)
Styela plicata

TBT 0.1/1
TPT 0.1
TCHT 0.1
gastrula


Ciona intestinalis

Pre-hatching
larvae

Developmental
stage
Exposure
time (h)
Target organs
Effects


2-4 cells,
morula,
1
Mitochondria,
cytoskeleton
Block of cleavage

Cima et al.,
1996




TBT 0.1
Swimming
larvae
1
Tail muscle cells,
mitochondria,
cytoskeleton
Block of
hatching/swimming
Gianguzza
et al., 1996
TBT 0.1/10 Neurula 1
Mitochondria,
cytoskeleton
Block of neurulation
Dolcemascolo
et al., 2005
Phallusia mammillata
Imazalil
Triadimefon

5
125
2 cells 10
Palps, central
nervous system
Pennati et
al., 2006
Fluconazole 125 2 cells 18
Palps, central
nervous system
Groppelli et
al., 2007




Recently, considering among other advantages,
that ascidian have a sensitiveness, in terms of EC50,
directly comparable to that of other model
organisms, such as bivalves or sea-urchins, a
standardized protocol for ascidian embryo-larval bio-
assays has been formulated (Bellas et al., 2003).
Finally, as C. intestinalis genome has been
released (Dehal et al., 2002), it is also possible to
investigate how expression of target genes could be
altered by toxicants. For instance, Azumi and
colleagues (2004) found, through microarray
analysis of gene expression in C. intestinalis
exposed to TBT, strong differential expression of
more than 200 genes concerned with stress
response, detoxification, oxidoreduction reaction,
biosynthesis and catabolism. Considering basic
vertebrate homologies of ascidians (Meinertzaghen
et al., 2004; Delsuc et al., 2006), these genetic
analysis could be powerful tools to forecast possible
toxic effects on vertebrate organisms, including
humans.

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