Assessment of the toxic effect exerted by fluorescent pseudomonads on embryos and larvae of the sea urchin Strongylocentrotus nudus

Ecotoxicology and Environmental Safety 115 (2015) 263–271

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Assessment of the toxic effect exerted by fluorescent pseudomonads on embryos and larvae of the sea urchin Strongylocentrotus nudus I.A. Beleneva a, E.V. Shamshurina a,b, M.G Eliseikina a,b,n a b

A.V. Zhirmunsky Institute of Marine Biology FEB RAS, Palchevsky Str. 17, Vladivostok 690041, Russia Far Eastern Federal University, Sukhanova Str. 8, 690950 Vladivostok, Russia

art ic l e i nf o

a b s t r a c t

Article history: Received 27 September 2014 Received in revised form 23 January 2015 Accepted 18 February 2015 Available online 27 February 2015

Strains of bacteria capable of growing on artificial culture media were isolated from the fouling of brass plates submerged in Nha Trang Bay, South China Sea, and from tissues of the seastar Distolasterias nipon, caught in Peter the Great Bay, Sea of Japan. According to the complex of data of genetic and physiological/ biochemical analyzes, two strains of cultivated bacteria were identified by us as the species Pseudomonas aeruginosa, two strains as Pseudomonas fluorescens, and one strain as Ruegeria sp. It was shown that the cultivated strains of P. aeruginosa released exotoxins, particularly phenazine pigments, into the environment. Production of the toxins did not depend on presence of a target organism in the system and was aimed at regulation of interactions in the microbial community. The toxicity of the studied natural isolates of fluorescent pseudomonads was analyzed by using embryos and larvae of the sea urchin Strongylocentrotus nudus, which are the sensitive and dynamic toxicological sea-urchin embryo test (SET) system. As was established, exotoxins produced by the strains of P. aeruginosa inhibit activity of cilia in sea urchin larvae, as well as disturb processes of cell differentiation in embryos and larvae. Their toxic influence is accompanied by disturbances of protein synthesis and the disruptions of cytoskeleton in the course of zygote cleavage and larval development. Unlike P. aeruginosa, the strains of P. fluorescens and Ruegeria sp. did not exert the toxic effect on SET. The obtained data allow considering objects of the environment as the natural reservoir of opportunistic microorganisms posing a potential threat to human, whereas the use of SET for determination of toxicity of isolated bacteria provides an opportunity to study the mechanisms of their interactions with organisms in marine ecosystems. & 2015 Elsevier Inc. All rights reserved.

Keywords: Marine heterotrophic bacteria Pseudomonas aeruginosa Pseudomonas fluorescens Ruegeria sp. Toxicity Bioassay SET

1. Introduction The genus Pseudomonas is a widespread and significant group of bacteria, which plays a major role in the cycle of biogenic matter in terrestrial and aquatic ecosystems. In the marine environment, pseudomonads are widely distributed, and strains of Pseudomonas aeruginosa, including those from oligotrophic waters of the open ocean, have been isolated relatively recently (Khan et al., 2007). Their ability to exist in a broad range of environmental conditions is determined by the great metabolic and genomic potential of these bacteria (Lessie and Phibbs, 1984; Jensen et al., 2004). Pseudomonads are a component of the normal bacterial flora in various marine animals (Olafsen, 2001; Avalos-Téllez et al., 2010); also they become an agent of diseases in many aquatic organisms, especially under changing environmental conditions (Dunn et al., n Corresponding author at: A.V. Zhirmunsky Institute of Marine Biology FEB RAS, Palchevsky Str. 17, Vladivostok 690041, Russia. Fax: þ7 423 2310 900. E-mail address: [email protected] (M. Eliseikina).

http://dx.doi.org/10.1016/j.ecoenv.2015.02.030 0147-6513/& 2015 Elsevier Inc. All rights reserved.

2001; Austin and Austin, 2007; Eissa et al., 2010). A disease emerges, as a rule, when the immune system gets weakened usually under disturbances of phagocytic activity of a blood cells (Speert, 1993; Garrity-Ryan et al., 2000; Rangel et al., 2014). While being a saprophytic microorganism, P. aeruginosa, a representative of fluorescent pseudomonads, is also an agent of diseases in a broad variety of organisms such as plants, invertebrates, and vertebrates including humans (Rahme et al., 1995). Another pseudomonad species, Pseudomonas fluorescens, also traditionally considered a saprophytic microorganism, is nevertheless able to cause bacteremia in humans (Hsueh et al., 1998) and possess the virulence factors (Picot et al., 2001; Sperandio et al., 2010). By the present time, many issues concerning pathogenicity, mechanisms of adhesion of fluorescent pseudomonads to cells of host organism, and also features of aquatic organism's immune response to invasion of a bacterial agent remain poorly studied, despite representatives of pseudomonads hold a leading position among cultivated halophilic bacteria. To study the pathogenic

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potential of bacteria, various model systems—cell cultures and organisms—are widely used (Mahajan-Miklos et al., 2000). Among vertebrates, these are mice (Zweerink et al., 1988); among amphibians, frogs (Brodkin et al., 1992); among bony fish, Danio rerio or zebrafish (Phennicie et al., 2010); among insects, Drosophila melanogaster (Corby-Harris et al., 2007), Galleria mellonella (Andrejko and Mizerska-Dudka, 2011); among crustacea, Artemia spp. (Marques et al., 2005); among nematodes, Caenorhabditis elegans (Mahajan-Miklos et al., 1999; Tan et al., 1999). Developing embryos and larvae are a highly sensitive and adequate test system for evaluating toxicity of various substances. Particularly, developing embryos and larvae of sea urchins—sea urchin embryo test (SET)— are a classical example of models like these (Zhuravel et al., 2006; Alvarez et al., 2010; Rock et al., 2011). A broad use of SET is determined by the availability of sea urchins as one of abundant representatives of benthic ecosystems, by the convenient way to obtain large quantities of sex cells, by external fertilization, and by the possibility to have a culture of synchronously developing embryos and larvae. The use of marine organisms for the study of pathogenic potential of marine saprophytic bacteria will provide an opportunity to find the probable mechanisms of their interactions in natural ecosystems. We isolated several strains of marine halophilic bacteria, which were able to grow on artificial culture media, possessed autofluorescence, and had manifested a high antimicrobial activity in earlier studies (Beleneva et al., 2013). The goal of this work is to identify isolated bacterial strains and to assess their pathogenic potential by using embryos and larvae of the sea urchin Strongylocentrotus nudus as embryonic toxicological test systems.

2. Materials and methods 2.1. Material The strains 1242 and 1248 were isolated from the surface of fouling on brass plates in Nha Trang Bay, South China Sea, Vietnam, in August 2008; the strain 1444, from the seawater of Nha Trang Bay, in 2009; the strains 1573 and 1574, from tissues of a seastar Distolasterias nipon, caught in Peter the Great Bay, Sea of Japan, Russia, in August 2010. Earlier, we described the procedure of sampling and isolation of bacteria from aquatic organisms and seawater (Beleneva, 2011), as well as from biofilm formed on the surface of metal plates submerged in the sea (Beleneva et al., 2013). Serial dilutions of homogenates, biofilm suspensions, and water samples (0.1 ml) were plated on solid Youschimizu–Kimura medium (Y–K) with the following composition (g/l): 5.0 peptone, 2.0 yeast extract, 1.0 glucose, 0.2 K2HPO4, 0.1 MgSO4  7H2O, 12.0 agar, 500 ml distilled water, and 500 ml seawater; the pH of the medium was 7.8– 8.0 (Youschimizu andKimura, 1976). Cetrimide agar (Serva), supplemented with glycerol in the proportion of 10 g/l, was used to isolate bacteria of the genus Pseudomonas. The inoculated cetrimide agar was incubated at room temperature for three days. We selected colonies with pronounced blue–green or fluorescent yellow–green pigmentation for the further study. Chromogenesis was studied on the King A and King B media (Bio-Merieux). The taxonomic status of the strains 1242, 1248, 1444 had been established through sequencing of the 16S rRNA gene in previous studies (Beleneva et al., 2013). Identification of the strains 1573 and 1574 belonging to genus Pseudomonas was based on morphological, cultural, and biochemical characteristics according to the earlier described methods (Beleneva et al., 2013). The investigated properties of the isolated strains included the presence arginine dihydrolase were determined according to Moeller (1955). Hydrolysis of gelatine and urease, glucose oxidation and

fermentation by Hugh and Leifson (1953), reduction of nitrates, the presence of cytochromeoxidase, the production of indole and the utilization of citrate were determined by the methods of Smibert and Krieg (1994). The ability to grow in various NaCl concentrations was determined in the medium containing 5.0 g peptone, 2.5 g yeast extract, 1.0 g glucose, 0.2 g K2HPO4, 0.05 g MgSO4, 1000 ml distilled water, 15.0 agar with 0–10% NaCl. Growth at different temperatures (5 and 42 °C) was tested using Y–K medium. DNA was isolated according to Marmur (1961). The DNA G þC content was determined from temperature denaturation curves (Owen et al., 1969). The bacterial strains were stored at  85 °C in cryotubes with seawater containing 1% peptone, 30% glycerol, and MgSO4 with a concentration of 3–5 g/l. The sensitivity of the strains to antibiotics was tested by using the method of diffusion in Mueller–Hinton agar (Difco), enriched in 2% NaCl, the results are interpreted according to CLSI (2012). Sensitivity to ampicillin (10 μg/disk), carbenicillin (100), oxacillin (5), tetracycline (30), oleandomycin (15), cefazolin (30), cefotaxime (30), ceftazidime (30), streptomycin (25), gentamicin (10), lincomycin (15), ciprofloxacin (5), chloramphenicol (30), polymyxin B (300 U) was determined by the diffusion method with Oxoid disks. For the experimental study the pathogenic potential, bacteria were cultivated in 5 ml of liquid Y–K medium in tubes under static conditions at room temperature for 4 days. The bacterial cultures were centrifuged at 9500 r.p.m. during 10 min (MiniSpin, Eppendorf, Germany), the cell sediment was washed in sterile seawater, then resuspended in sterile seawater, and the bacterial lysate (BL) was prepared through “freeze-thawing”. The BL solution in sterile seawater was made in such a way to reach the protein content of 1 mg/ml in the solution. This solution was used in the experiment as initial one (stock). The Y–K medium was also used in the experiment; bacteria were cultivated in this medium, containing their metabolites (CuM), during 4 days. The intact Y–K medium was used as the control (CoM). Sea urchins Strongylocentrotus nudus were caught in Vostok Bay from a depth of 4–4.5 m. The animals were kept in aquariums with aerated seawater. Spawning was stimulated by injecting 1 ml of 0.5 M KCl into the coelomic cavity. Sperm was collected by using the dry technique with subsequent dilution in sterile seawater 20000 times. Then fertilization was performed; the final dilution of sperm was 60000 times. For the experiment, we used both embryos (immediately after fertilization and formation of envelope) and larvae at the stage of hatched blastula (11 h of the development). All experiments were conducted at 18 °C. 2.2. Bioassay studies The embryos and larvae at the hatched blastula stage were placed in sterile seawater containing CuM (strains 1242, 1248, 1444, 1573, and 1574) diluted to the concentrations of 1/5, 1/10, 1/100, and 1/200. As a control, sea urchin embryos and larvae were placed in CoM with similar dilutions in the sterile seawater. Moreover, embryos were cultivated in the sterile seawater containing BL at the dilutions of 1/5 and 1/10; larvae, at the dilutions of 1/5, 1/10, 1/100, and 1/200. Development in the sterile seawater was studied in each of the experiments. Each variant of the experiment was performed in triplicate. We conducted the experiment in 24-well plates with 150–200 egg cells or larvae added to 2 ml of liquid in each well. The state of the material was evaluated under a binocular microscope 4 h after the beginning of the experiment. The culture of the embryos was fixed 8 h after the beginning of the experiment, when in the control wells they reached the stages of middle blastula (prior to hatching). The larvae were fixed after 24 h of incubation (35 h after fertilization), when ones in the control became the two-armed pluteus. The samples were fixed with 2.5% glutaraldehyde in seawater (рН 7.2) for 60 min

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and rinsed then some times in sterile seawater for 24 h. The material was stored in 70% ethanol. 2.3. Immunohistochemical analysis For the immunohistochemical analysis, the embryos after formation of the fertilization envelope and larvae at the hatched blastula stage were placed in sterile seawater containing CuM (strains 1242) diluted to the concentrations of 1/10 and 1/100. As a control, sea urchin embryos and larvae were placed in the sterile seawater. The sea urchin embryos (2 and 8 h of the cleavage) and larvae (20 h of incubation, 30 h after fertilization) were fixed in 4% paraformaldehyde (Sigma) in 0.1 M PBS, pH 7.5, for 60 min at 4 °C and rinsed three times with cold PBS. The material was stored in PBS with 0.03% NaN3 at 4 °C. For the WNT5 detection the rabbit polyclonal antibody on the conserved regions of WNT5 of holothurian Eupentacta fraudatrix were used (Girich et al., 2014). The antibody were kindly provided to us by I.Yu. Dolmatov. For the actin visualization the TRITC-labeled phalloidin (Molecular Probes) was used. To reduce non-specific binding, the samples were incubated overnight in a blocking solution containing 10% normal goat serum (Sigma), 0.25% BSA, 0.1% Triton X-100, and 0.03% NaN3 in PBS. For simultaneous detection of WNT5 and actin, fixed embryos and larvae were first incubated for 12 h at 10 °C in the blocking solution with the primary WNT5 Abs (diluted 1/100) and then after washing in PBS (3  10 min) were incubated with the secondary antibody (GAR Alexa Fluor 488 IgG, Molecular Probes, USA, diluted 1/1000) and TRITC-labeled phalloidin (diluted 1/100) 2 h at RT. The specimens were then washed and embedded in the Vectashield mounting medium (Vector Laboratories, Burlingame, USA) containing 0.1 μg/ml DAPI to reveal nuclei. For negative controls, primary Abs were omitted from the staining protocol. Specimens were analyzed with a confocal laser scanning microscope Zeiss LSM 780 equipped with a high sensitivity GaAsP detector (with the 25  oil-immersion objectives). From 25 to 64 thin optical sections of the specimens were scanned through along the z axis with a stepping size of 0.35–0.45 mm. The depth (z range) of the scanned areas varied between 10 and 40 mm. The resulting laser confocal stacks of optical sections were processed using the ZEN-2010 imaging software. For 3D reconstructions, the ImageJ (NIH) software was used.

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Table 1 Phenotypic characteristics of marine bacterial isolates. Test

Oxidation/fermentation oxidase Nitrate reduction Gelatinase Arginine dihydrolase Indole Urease Simmons citrate Growth at: 0% NaCl 3% NaCl 6% NaCl 8% NaCl 10% NaCl 5 °C 42 °C Pyocyanine Fluorescein G þ C content, mol%

Strain 1242

1248

1573

1574

1444

þ/ þ  þ þ   þ

þ/  þ  þ þ   þ

þ/ þ þ þ þ   þ

þ/ þ þ þ þ   þ

/ þ      no data

þ þ þ    þ þ þ 67.5

þ þ þ    þ þ þ no data

þ þ þ   þ   þ 63.6

þ þ þ   þ   þ 63.3

 þ þ    no data   58.0

3. Results and discussion The bacterial strains that grew well on the Y–K medium were isolated from the surface of brass plates submerged in Nha Trang Bay, South China Sea, Vietnam, and from tissues of the sea star D. nipon, caught in Peter the Great Bay, Sea of Japan, Russia. When cultivated on a dense medium at room temperature for 2–3 days, the strains 1242 and 1248 formed flat, whitish and glittering colonies with 0.4–0.6 cm in diameter, denser in the center and transparent in edges; the bright yellow–green pigment-fluorescein-diffused into the medium. When grown in the liquid medium, the culture liquid acquired a green–blue color (Fig. s1). The strains 1573 and 1574 on the dense medium formed slightly convex and semitransparent colonies with 0.2–0.3 cm in diameter and produced fluorescein; in the liquid medium, the color of the culture liquid did not change. The strain 1444 on the dense medium formed yellowish opaque, convex, and glittering colonies 0.3–0.4 cm in diameter, did not manifest autofluorescence, and was used in this work as the control. According to the results of earlier studies (Beleneva et al., 2013) and those conducted within this work we identified the strains 1242 and 1248 as the genus P. aeruginosa; the strain 1444, as Ruegeria sp.; the strains 1573 and 1574, as P. fluorescens (Table 1).

Fig. 1. The effect of bacterial culture media CuM on the development of Strongylocentrotus nudus embryos after 8 h of incubation. The dilutions of the media, in which embryos were incubated, are at the top (1/5, 1/10, 1/100, and 1/200); the strain numbers are given in the column on the left (1242, 1248, 1444, 1573, and 1574); contr-signifies sterile medium CoM in similar dilutions. Scale bar is 50 mm.

The species P. fluorescens and P. putida are very similar in their physiological and biochemical properties. However the ability of P. fluorescens to hydrolyze gelatin and grow at 4 °C, which is a differentiating character (Gilligan, 1995), enabled us to refer both

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Fig. 2. The effect of bacterial lysates BL on the development of Strongylocentrotus nudus embryos after 8 h of incubation. The dilutions of stock BL with a protein concentration of 1 mg/ml are at the top (1/5, 1/10); the strain numbers are in the column on the left (1242, 1248, 1444, 1573, and 1574); contr-signifies sterile seawater. Scale bar is 50 mm.

strains, 1573 and 1574, to this species. Clinical stains of P. fluorescens isolated in a hospital of Taiwan were differentiated from P. putida using tests of gelatin hydrolysis and growth at 4 °C (Hsueh et al., 1998). Colony and cell morphology, and biochemical characteristics of our isolates 1573 and 1574, such as the presence of gelatinase, NO3 – reduction, the production of indole and the utilization of citrate, corresponded to morphological and cultural properties of P. fluorescens from soil (Rekha et al., 2010). Biochemical properties of 1444 strain did not contradict those previously described (Lee et al., 2007). The antibiotic sensitivity testing showed a high degree of strains' resistance to most of tested anti-microbial drugs (Table s1). It should be noted that the strains of P. fluorescens showed a higher resistance than the P. aeruginosa strains did. Thus, the former ones proved to be sensitive only to drugs of the 2nd generation aminoglycoside group (gentamicin) and 3rd generation fluroquinolones (ciprofloxacin), whereas the P. aeruginosa strains were

sensitive to 2nd and 3rd generation aminoglycosides (streptomycin and gentamicin, respectively), ciprofloxacin, and polymyxin. The increase in the resistance of these two pseudomonad species to a broad spectrum of antibiotics, and, as a consequence, development of cross-resistance and multi-drug resistance are observed around the world (Chandrasekaran and Lalitha Kumari, 1998; Hancock and Speert, 2000; Maravić et al., 2012). All these facts are the evidence of a high pathogenic potential of these microorganisms. Thus, bacteria isolated from the marine environment may serve a reservoir for genes of resistance to clinically used antibiotics. The pathogenicity mechanisms in P. aeruginosa are studied enough widely by using various approaches such as genetic, proteomic, and cell biology methods. The studies of strains of various origins, both clinical and obtained from environmental objects, plants and animals, showed that all the studied bacteria have conservative genes responsible for toxin production (Bradbury et al., 2010). The issue of expression of these genes and its regulation under various conditions still remains open. As was mentioned earlier, during cultivation of the strains 1242 and 1248, the culture medium got a green–blue color that indicates the presence of phenazine pigments, which are typical toxins for this species of microorganisms (Mahajan-Miklos et al., 1999). Along with phenazines, P. aeruginosa produces a number of exotoxines, including exo A, which inhibits the protein synthesis in target cells and affects metabolism in multicellular organisms (Iglewski et al., 1977; Woods and Iglewski, 1983). The expression of genes that code toxicity factors in the studied pseudomonad strains, cultivated in the absence of target organism, was analyzed by using embryos and larvae of the sea urchin S. nudus. As embryonic test systems, particularly embryos and larvae of sea urchins (SET), are frequently used for study of the effect of various toxins on living organisms, SET may serve as an adequate and highly sensitive system to investigate the toxic impact of bacterial metabolic products on organisms of echinoderms. Moreover, embryonic and larval development is followed by processes of division, differentiation, and migration of cells. Hence, the use of SET provides an opportunity to judge about the influence of factors of bacterial origin on these manifestations of cellular activity. We used two variants of SET to study the effect exerted by metabolic products of the bacterial strains on living systems. The use of fertilized eggs of the sea urchin S. nudus enabled us to determine the influence of the studied components on the process of cell division; the use of S. nudus larvae since the hatched blastula stage allowed characterizing the influence of the studied factors on cell differentiation and migration processes. In the course of experiment, stages of embryogenesis and larval development were classified according to Buznikov and Podmarev (1990). Immediately after fertilization, embryos of the sea urchin S. nudus were placed in the sterile seawater containing either BL diluted to 1/5 and 1/10 or CuM of the strains 1242, 1248, 1444, 1573, and 1574 diluted to 1/5, 1/10, 1/100, and 1/200. At the same time, development was studied in the sterile seawater containing CoM at the similar dilutions (control 1) and in the sterile seawater (control 2). Four hours after fertilization, all the control embryos reached the stages of 4–8 blastomeres. No division was observed in the solutions containing BL and CuM of the strains 1242 and 1248 diluted to 1/5 and 1/10. When CuM was diluted to 1/100, over 50% of embryos were at the 2- and 4-blastomere stages. The retardation of cell division was followed by asynchronous cleavage and disturbance of cytokinesis, and, as a result, embryos were formed with uneven number of blastomeres of various sizes. When CuM was diluted to 1/200, the retardation of cell division and cleavage disturbances were observed in 8% of individuals. In the media with

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Fig.3. The effect of bacterial culture media CuM on the development of Strongylocentrotus nudus larvae after 24 h of incubation, 35 h after fertilization. The dilutions of media are at the top (1/5, 1/10, 1/100, and 1/200); the strain numbers are in the column on the left (1242, 1248, 1444, 1573, and 1574), contr 1-is sterile culture medium CoM in similar dilutions; contr 2-is sterile seawater with no additives. Scale bar is 50 mm.

BL and CuM of the strains 1444, 1573, and 1574, irrespective of dilution degree, no abnormalities were observed in development, and it did not differ from the development in the control (data are not provided in the figure). Eight hours after the beginning of the experiment, embryos in the control and in the medium with various concentrations of BL and CuM of the strains 1444 (Figs. 1I–L, 2E and F), 1573(Figs. 1M–P, 2G and H), and 1574 (Figs. 1O–T, 2I and J) reached the stages of either early or middle blastula. There was no cleavage in media with BL and CuM of the strains 1242 (Figs. 1A, 2A) and 1248 (Figs. 1E, 2C) diluted to 1/5, 100% of the embryos were fertilized eggs (Figs. s2A, s3A). At the dilution of 1/10, abnormal cleavage of embryos was observed only in case of the BL of the strain 1242 (Fig. 2B). There were 20.2% of fertilized eggs, 32.2% of blastula with 2–4 blastomers, 47.6% of embryos with different developmental abnormalities (Fig. s2A). In the other variants of media with this dilution, no cleavage took place and 100% of the embryos were fertilized eggs (Figs. 1B and F, s2A, s3B). Embryos, incubated in the medium containing CuM of the strains 1242 (Fig. 1C and D) and 1248 (Fig. 1G and H) at the dilutions of 1/100 and 1/200, developed with various deviations from the normal process such as

uneven cleavage of blastomeres. At the dilutions of 1/100 (Fig. s3C) the embryos on the stage of fertilized egg were 2.94% (1242) and 6.06% (1248), with the abnormalities of development were 97.06% (1242) and 93,94% (1248). At the dilutions of 1/200 (Fig. s3D) the embryos on the stage of blastula were 9.05% (1242) and 8.96% (1248), with the abnormalities of development were 90.95% (1242) and 91.04% (1248). For cultivation of larvae of the sea urchin S. nudus, we used the same composition of media as that for embryos. Immediately after rising to the surface (11 h after fertilization), larvae were placed in media containing BL, CuM, CoM, and sterile seawater without additives. Four hours after the beginning of incubation (15 h after fertilization), all the control larvae and those developing in the media containing BL and CuM of the strains 1573, 1574, and 1444 reached the stage of early gastrula. Larvae developing in the CuM and BL solutions of the strains 1242 and 1248 at all the dilutions (1/5, 1/ 10, 1/100, 1/200) manifested lower mobility than those in the controls and almost did not rise to the surface of wells; most larvae were able to move only around the body axis (data are not provided).

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Fig. 4. The effect of bacterial lysates BL on the development of Strongylocentrotus nudus larvae after 24 h of incubation, 35 h after fertilization. The dilutions of stock BL with a protein concentration of 1 mg/ml are in the top (1/5, 1/10, 1/100, and 1/200); the strain numbers are in the column on the left (1242, 1248, 1444, 1573, and 1574); contr-is sterile seawater. Scale bar is 50 mm.

Twenty four hours after the beginning of the experiment (35 h after fertilization), all the control larvae developing in sterile seawater reached the stage of two-armed pluteus (Figs. 3Y, 4U). In the media containing BL and CuM of the strains 1242 (Figs. 3A and B, 4A and B) and 1248 (Figs. 3E and F, 4E and F) at the dilutions of 1/5 and 1/10 development was abnormal: cells of larvae lost the differentiation signs, grew rounded, and the larvae died, all of 100% larvae had the developmental disorders (Figs. s2B, s4A and B). In the medium containing CuM of the strain 1242 at the dilutions of 1/100 and 1/200, larvae reached the late gastrula stage, i.e. development was found to have slowed down (Fig. 3C and D) In dilution 1/100 it was 98.04%, and in 1/200 it was 2.31% (Fig. s4C and D). A disturbance of migration of primary mesenchyme cells followed by irregular filling of the blastocoele by them was observed in larvae developing in the medium with CuM of the strain 1248 at the dilutions of 1/100 and 1/200 and all larvae (100%) had the developmental abnormalities (Fig. 3G and H, s4C and D). The BL of the strains 1242 and 1248 at the dilution of 1/100 slightly decelerated the development of larvae (Fig. 4C and G), which were at the stages of prism (78.2% and 90.5% respectively), whereas the dilution 1/200 did not have any significant influence on the

development, and the larvae were at the stage of two-armed pluteus (Figs. 4D and H, s2B). In the media containing dilutions of CoM (Fig. 4 U–X) and CuM of the strains 1444, 1573, and 1574 (Fig. 3I–T), development insignificantly slowed down in compare with seawater, obviously this is due to an increase in viscosity of the medium in which it was developing. In the media with the CuM and CoM diluted to 1/ 5–1/100, the overwhelming majority of larvae reached the late gastrula – early prism stage; at the dilution of 1/200, the early pluteus stage. The BL of the strains 1444, 1573, and 1574 exerted almost no effect on larval development at all the dilutions (Fig. 4I– T). Only solitary individuals with abnormal development were observed, while most animals, the same as the control ones (Fig. 4U), reached the pluteus stage. The toxicity of the bacterium P. aeruginosa is determined by a number of factors that exert effect on various targets (Kipnis et al., 2006). Particularly, one of the most studied toxins, exotoxin ExoA, influences protein synthesis, arresting the translation processes. Toxins also have effect on the actin cytoskeleton of eukaryotic cells that results in disturbances of its normal organization (GarrityRyan et al., 2000).

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Fig. 5. Сhanges in organization of the actin cytoskeleton and localization of WNT5 protein in the cells of the sea urchin Strongylocentrotus nudus embryos and larvae under the effect of metabolites produced by the strain 1242 of Pseudomonas aeruginosa. Embryos developing in sterile seawater: A-2 h of the cleavage, B -8 h of the cleavage, actin filaments form the reticulate structure. Embryos developing in sterile seawater with a bacterial culture medium CuM, 8 h of cleavage: C-CuM at the dilution of 1/100, actin filaments have normal structure; D-CuM at the dilution of 1/10, actin filaments forms of the globules (indicated by arrows). Larvae developing in a CuM and in sterile seawater (30 h after fertilization, 19 h of incubation): E-control, sterile seawater, actin filaments form the reticulate structure, the immunopositive in relation to WNT5 cells migrating to the cavity of larva, as well as sites labeled with antibodies within components of the matter that fills the blastocoele can be seen; F-CuM in the dilution of 1/100, the immunopositive by WNT5 cells are revealed only within components of the matter that fills the blastocoele, actin filaments form the normal structure; G-CuM in the dilution of 1/10, only trace amounts of WNT5 protein are detected, actin filaments forms of the globules (indicated by arrows). The cells are labeled with phalloidin for actin detection (red) and with WNT5 Abs (green). The nuclei are stained with DAPI (blue). Scale bar is 50 mm. The samples are analyzed with a confocal laser scanning microscope Zeiss LSM 780. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).

To identify molecular targets of metabolites, produced by the wild strains of P. aeruginosa found by us, we studied their effect on the actin cytoskeleton of sea urchin embryo and larva cells, as well as on WNT5, which is a protein participating in the process of differentiation of germ layers (McIntyre et al., 2013). Presence and features of localization of these molecules were studied for embryos and larvae cultivated in CuM of the strain 1242 diluted seawater to 1/10 and 1/100, whose metabolites had the effective influence on the development. Embryos and larvae that were growing in sterile seawater were used as the control. Labeling of actin filaments with phalloidin revealed a disturbance of organization of actin cytoskeleton in embryo and larva cells at a high (1/10) concentration of CuM. Instead of building the typical diffuse reticulate structure in the cell cytoplasm (Fig. 5A, B, C, E and F) actin filaments formed globules (Fig. 5D and G) that indicates a disturbance in the process of actin polymerization under the effect of toxins from P. aeruginosa. An immunochemical labeling did not reveal the presence of protein WNT5 in sea urchin embryos (Fig. 5A–D); this protein was detected only at larval stages of development, accompanied by differentiation of cells of germ layers (Fig. 5E–G). In control larvae, sites of antibody binding were localized in primary mesenchyme cells migrating to the blastocoele and localized in the base and upper part of archenteron in the course of gastrulation, as well as within the cytoplasm of mesenchymal cells at the pluteus stage (Fig. 5E). Moreover, immunopositive labeling was observed within the matter that fills the blastocoele. When larvae were cultivated in sea water containing CuM at 1/100, cells labeled for WNT5 were as a rule absent, and labeling remained only in the components of reticulate structure (Fig. 5F). In case larvae developed in sea water containing CuM at 1/10, when cells did not differentiate and their development was blocked, protein WNT5 either was absent or its content was minor (Fig. 5G). The use of immunochemical methods of studies, as well as such a sensitive and dynamic test system as SET, showed that the mechanism of action of toxins from the wild strain of P. aeruginosa,

isolated by us from objects of the marine environment, is accompanied by violations of the protein WNT5 synthesis and disorganization of the actin cytoskeleton that eventually results in blockage of development of sea urchin. Thus, development of embryos and larvae of S. nudus was disturbed in the solutions of supernatants and bacterial lysates of the strains 1242 and 1248 even at the lowest concentrations. High concentrations of the CuM and BL solutions of the strains 1444, 1573, and 1574 caused insignificant retardation of development, comparable with the pace of development of embryos in the CoM. Changes were reversible and caused by the higher viscosity of the cultural medium in compare with seawater. As a result of the conducted study, we established that representatives of two pseudomonad species—P. aeruginosa and P. fluorescens—occur in environmental objects (fouling or biofilms) and are a component of bacterial flora of marine invertebrates such as the seastar D. nipon. By using the adequate, dynamic, and sensitive test system, SET, we showed that natural isolates of P. aeruginosa, when cultivated under artificial conditions, release exotoxins into the medium. The green–blue color of the culture fluid indicates the presence of phenazine pigments in the medium. The results of our studies show that production of the pigments does not depend on the presence of a target organism in the system; their production by bacterial cells is aimed at regulation of interactions in the microbial community. The use of the sensitive and dynamic SET model enabled us to ascertain that the exotoxins, emitted by the strains of P. aeruginosa, inhibit activity of cilia in cells of sea urchin larvae, as well as disturb the processes of division and differentiation of embryo and larval cells. The obtained data do not contradict to the information available in the literature on mechanisms of the effect of toxins emitted by P. aeruginosa. Particularly, exotoxin ExoA is known to decelerate cell metabolism by inhibiting the protein synthesis (Iglewski et al., 1977; Woods and Iglewski, 1983). Toxins of P. aeruginosa suppress the locomotive activity of cilia (Wilson and Dowling, 1998). Hence, the data obtained by us allow considering the environmental objects as a

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natural reservoir of opportunistic microorganisms, which pose a potential threat to human. The use of SET for determination of toxicity of the isolated bacteria provides an opportunity to study probable mechanisms of their interaction with organisms in marine ecosystems.

Competing interests The authors have declared that no competing interests exist.

Acknowledgments Financial support was provided by the Russian Science Foundation, Grant no.14-50-00034 (M.G. Eliseikina, E.V. Shamshurina) and FEB RAS Grant no.12-I-П4-02 (I.A. Beleneva). The study was performed at the «Far Eastern center of electronic microscopy» (A. V. Zhirmunsky Institute of Marine Biology, FEB RAS, Vladivostok, Russia).

Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ecoenv.2015.02. 030.

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