ISJ 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 S29 mailto:fiorenza.debernardi@unimi.it 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 Chemical classification EC50 (µM) Embryos 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 Organometallic anti-foulant 0.02 - 0.01 Bellas et al., 2005 Zinc pyrithione (Zpt) Zinc 1-oxidopyridin-1-ium-2- thiolate Organometallic bactericide, anti- foulant 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 Organo- phosphorus pesticide 15.70 - - ″ Diuron 3-(3,4-dichlorophenyl)-1,1- dimethylurea Urea derived herbicide 17.80 - - ″ Chlorothalonil 2,4,5,6-tetrachlorobenzene-1,3- dicarbonitrile Organochloride fungicide, anti- foulant 0.12 0.16 0.005 Bellas, 2006 Sea-Nine 211 (Kathon 930) 4,5-dichloro-2-octyl-1,2-thiazol-3- one Organochloride anti-foulant 0.37 0.15 0.013 ″ ″ Dichlofluanid N-(dichloro-fluoromethyl)sulfanyl- N-(dimethylsulfamoyl)aniline Organochloride anti-foulant 0.85 0.39 0.017 ″ ″ Tolylfluanid N-(dichloro-fluoromethyl)sulfanyl- N-(dimethylsulfamoyl)-4- methylaniline Organochloride anti-foulant 0.62 0.28 - ″ Irgarol 1051 N-tert-butyl-N'-cyclopropyl-6- methylsulfanyl-1,3,5-triazine-2,4- diamine Anti-foulant 8.34 >25.60 0.016 ″ ″ Imazalil 1-[2-(2,4-dichlorophenyl)-2-prop- 2-enoxyethyl]imidazole Organochloride triazole- imidazole 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 Organochloride triazole fungicide 29.56 - - ″ Fluconazole 2-(2,4-difluorophenyl)-1,3- bis(1,2,4-triazol-1-yl)propan-2-ol Organofluoride triazole fungicide 74.70 - - Groppelli et al., 2007 S30 http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=mesh&dopt=Full&list_uids=67080469 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 neurons with 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 and organic 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 concentration that determines larval malformation) values of Hg, Cu and Cd were very low indicating that these metals could effectively impair development and 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). 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 and disorganized blastomeres, lacking cytoskeletal elements. As a consequence, neurulation was blocked (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, Sea-Nine 211, 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 experiments. Triazole 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 S31 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 than their EC50 values on ascidian 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. S32 Table 2 Effects of different compounds on the development and/or morphology of larvae of three different ascidian species Effective concentrations (µM) Developmental stage Exposure time (h) Target organs Effects Styela plicata TBT 0.1/1 TPT 0.1 TCHT 0.1 2-4 cells, morula, gastrula 1 Mitochondria, cytoskeleton Block of cleavage Cima et al., 1996 Ciona intestinalis Pre-hatching larvae 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 5 Triadimefon 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). 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