Massive Occurrence of the Harmful Benthic Dinoflagellate Ostreopsis cf. ovata in the Eastern Adriatic Sea

In September 2015, a massive occurrence of the Ostreopsis species was recorded in central Adriatic Kaštela Bay. In order to taxonomically identify the Ostreopsis species responsible for this event and determine their toxin profile, cells collected in seawater and from benthic macroalgae were analyzed. Conservative taxonomic methods (light microscopy and SEM) and molecular methods (PCR-based assay) allowed the identification of the species Ostreopsis cf. ovata associated with Coolia monotis. The abundance of O. cf. ovata reached 2.9 × 104 cells L−1 in seawater, while on macroalgae, it was estimated to be up to 2.67 × 106 cells g−1 of macroalgae fresh weight and 14.4 × 106 cells g−1 of macroalgae dry weight. An indirect sandwich immunoenzymatic assay (ELISA) and liquid chromatography–high-resolution mass spectrometry (LC-HRMS) were used to determine the toxin profile. The ELISA assay revealed the presence of 5.6 pg palytoxin (PLTX) equivalents per O. cf. ovata cell. LC-HRMS was used for further characterization of the toxin profile, which showed that there were 6.3 pg of the sum of ovatoxins (OVTXs) and isobaric PLTX per O. cf. ovata cell, with a prevalence of OVTXs (6.2 pg cell−1), while the isobaric PLTX concentration was very low (0.1 pg cell−1). Among OVTXs, the highest concentration was recorded for OVTX-a (3.6 pg cell−1), followed by OVTX-b (1.3 pg cell−1), OVTX-d (1.1 pg cell−1), and OVTX-c (0.2 pg cell−1).

Ostreopsis blooms in the Mediterranean Sea are commonly accompanied by respiratory problems and skin irritation in humans exposed to marine aerosol containing algal toxins and/or cells debris and seawater [9][10][11][12] due to ability of some Ostreopsis species to produce toxins. Most of these toxins belong to the palytoxin (PLTX) group. PLTX and its analogs may affect human health by ingestion of contaminated seafood, skin contact with seawater, and inhalation of marine aerosols containing Ostreopsis cell debris and/or their toxins [12][13][14]. So far, human poisonings ascribed to ingestion of PLTX-contaminated seafood have been recorded in the Pacific and Indian Oceans following consumption of fish [13,[15][16][17][18][19] and crabs [20,21], while in the Mediterranean Sea, no cases have been reported so far. Chemical studies of the Mediterranean strains of O. cf. ovata showed the presence of small quantities of an isobaric PLTX [22,23] and larger amounts of structural PLTX congeners called ovatoxins (OVTXs), with a high prevalence of OVTX-a ( Figure 1) [24][25][26][27]. According to Funari et al. [28], the lack of toxicity in humans via food chain transfer during O. cf. ovata blooms in the Mediterranean area could be explained by the lower oral toxicity of OVTXs in comparison with that of PLTXs. This hypothesis has been supported by the in vitro cytotoxicity characterization of OVTX-a in relation to the reference compound PLTX, which revealed less toxic effect compared with PLTX, displaying lower cytotoxicity as well as lower hemolytic activity on human erythrocytes [29].
Ostreopsis blooms in the Mediterranean Sea are commonly accompanied by respiratory problems and skin irritation in humans exposed to marine aerosol containing algal toxins and/or cells debris and seawater [9][10][11][12] due to ability of some Ostreopsis species to produce toxins. Most of these toxins belong to the palytoxin (PLTX) group. PLTX and its analogs may affect human health by ingestion of contaminated seafood, skin contact with seawater, and inhalation of marine aerosols containing Ostreopsis cell debris and/or their toxins [12][13][14]. So far, human poisonings ascribed to ingestion of PLTX-contaminated seafood have been recorded in the Pacific and Indian Oceans following consumption of fish [13,[15][16][17][18][19] and crabs [20,21], while in the Mediterranean Sea, no cases have been reported so far. Chemical studies of the Mediterranean strains of O. cf. ovata showed the presence of small quantities of an isobaric PLTX [22,23] and larger amounts of structural PLTX congeners called ovatoxins (OVTXs), with a high prevalence of OVTX-a ( Figure 1) [24][25][26][27]. According to Funari et al. [28], the lack of toxicity in humans via food chain transfer during O. cf. ovata blooms in the Mediterranean area could be explained by the lower oral toxicity of OVTXs in comparison with that of PLTXs. This hypothesis has been supported by the in vitro cytotoxicity characterization of OVTXa in relation to the reference compound PLTX, which revealed less toxic effect compared with PLTX, displaying lower cytotoxicity as well as lower hemolytic activity on human erythrocytes [29]. According to literature (Table 1) the most abundant and widely distributed Ostreopsis species in the Adriatic Sea is the Atlantic/Mediterranean ribotype O. cf. ovata [7]. The first identification of O. cf. ovata in Croatian waters was from the central Adriatic Kaštela Bay in 1984 [30]. Thereafter, this species was not reported in Kaštela Bay, but it is possible that it remained undetected due to the absence of visible blooms and the lack of noted negative impacts on human health. Ostreopsis cf. ovata has been reported along the Italian coasts since the late 1990s and, with a few exceptions, almost all Italian regions are seasonally affected by O. cf. ovata blooms [28]. Cases of respiratory problems and skin irritations in humans associated with massive blooms of O. cf. ovata in Croatian waters were reported for the first time in the northern Adriatic Sea in 2010 [31]. In 2015, complaints of similar signs and symptoms came from the beaches along the Kaštela Bay coasts, indicating the development of another O. cf. ovata bloom, this time in the central part of the Adriatic Sea. According to literature (Table 1) the most abundant and widely distributed Ostreopsis species in the Adriatic Sea is the Atlantic/Mediterranean ribotype O. cf. ovata [7]. The first identification of O. cf. ovata in Croatian waters was from the central Adriatic Kaštela Bay in 1984 [30]. Thereafter, this species was not reported in Kaštela Bay, but it is possible that it remained undetected due to the absence of visible blooms and the lack of noted negative impacts on human health. Ostreopsis cf. ovata has been reported along the Italian coasts since the late 1990s and, with a few exceptions, almost all Italian regions are seasonally affected by O. cf. ovata blooms [28]. Cases of respiratory problems and skin irritations in humans associated with massive blooms of O. cf. ovata in Croatian waters were reported for the first time in the northern Adriatic Sea in 2010 [31]. In 2015, complaints of similar signs and symptoms came from the beaches along the Kaštela Bay coasts, indicating the development of another O. cf. ovata bloom, this time in the central part of the Adriatic Sea.    Based on this event, the aim of this study is: (1) Taxonomical identification of the Ostreopsis species that cause massive blooms and affect human health; (2) determination of the toxin profile of these species; (3) reporting a new site where blooms occur to improve global mapping of the genus Ostreopsis; and (4) raising awareness about the necessity of introducing targeted monitoring of Ostreopsis species by reporting its occurrence.

Microscopy Analyses
Microscopic analyses of field samples showed an intensive bloom of Ostreopsis species (Figure 2A-C). Ostreopsis cf. ovata was identified on the basis of its specific cellular shape (like a pumpkin seed with an expanded oval dorsal side and a narrowed ventral part), morphological characteristics, and the ratio of dorsoventral (DV) and anteroposterior (AP) diameter ( Table 2). The ventral portion is characterized by a protrusion which is usually less pigmented due to mucus material. Based on this event, the aim of this study is: (1) Taxonomical identification of the Ostreopsis species that cause massive blooms and affect human health; (2) determination of the toxin profile of these species; (3) reporting a new site where blooms occur to improve global mapping of the genus Ostreopsis; and (4) raising awareness about the necessity of introducing targeted monitoring of Ostreopsis species by reporting its occurrence.

Microscopy Analyses
Microscopic analyses of field samples showed an intensive bloom of Ostreopsis species ( Figure  2A-C). Ostreopsis cf. ovata was identified on the basis of its specific cellular shape (like a pumpkin seed with an expanded oval dorsal side and a narrowed ventral part), morphological characteristics, and the ratio of dorsoventral (DV) and anteroposterior (AP) diameter ( Table 2). The ventral portion is characterized by a protrusion which is usually less pigmented due to mucus material.  Epifluorescence and SEM microscopy showed a plate pattern Po 3'7''5'''2'''' ( Figure 2D,E and Figure 3A   Epifluorescence and SEM microscopy showed a plate pattern Po 3'7"5"'2"" ( Figure 2D,E and Figure 3A

Molecular Analyses
The

Molecular Analyses
The molecular PCR amplifications were carried out on field samples to detect the presence of the

Molecular Analyses
The

Ostreopsis cf. ovata Abundance and Phytoplankton Community Composition
The abundance of O. cf. ovata in seawater in September ranged from 1.5 × 10 4 to 2.9 × 10 4 cells L −1 . Epiphytic cells recorded on macroalgae ranged from 2.25 × 10 6 to 2.67 × 10 6 cells g −1 of fresh weight of macroalgae and 11.4 × 10 6 to 14.4 × 10 6 cells g −1 of dry weight of macroalgae. The analysis of the benthic macroalgal assemblage showed the prevalence of the red macroalga Spyridia filamentosa (Wulfen) Harvey (1833) in the sampling area. The maximum abundance of O. cf. ovata was recorded in September during calm weather and with a surface seawater temperature of 23.4 • C. At the beginning of October, with the surface seawater temperature decreasing to 20.8 • C and SE winds of 2 Bf, the abundance of O. cf. ovata decreased by an order of magnitude, with abundances in seawater from 1.28 × 10 3 to 1.92 × 10 3 cells L −1 . Abundances of epiphytic cells of O. cf. ovata on macroalgae also decreased by an order of magnitude and ranged from 1.66 × 10 5 to 4.29 × 10 5 cells g −1 of fresh weight of macroalgae and 6.99 × 10 5 to 2.66 × 10 6 cells g −1 of dry weight of macroalgae.
In September, the phytoplankton community in the seawater was dominated by the diatoms Pseudo-nitzschia spp.; Chaetoceros sp.; Guinardia delicatula (Cleve) Hasle, 1997; G. striata (Stolterfoth) Hasle, 1996; Leptocilyndrus danicus Cleve, 1889; Navicula sp.; and Pleurosigma sp. (Table 3). The contribution of O. cf. ovata cells in the water column (2.9 × 10 4 cells L −1 ) in September represented up to 10% of the total phytoplankton community, decreasing to less than 1% in October with abundances up to 1.9 × 10 3 cells L −1 . October was also characterized by a strong prevalence of diatoms in the phytoplankton community. Ostreopsis cf. ovata was accompanied by the epiphytic dinoflagellate Coolia monotis. Taxonomical identification of C. monotis was based on size and morphological features obtained by SEM ( Figure 5). Tabulation was determined according to Balech [82]. Plate 7" is characterized by the ratio of width and length of approximately 1. Plate 1' is placed left of the center. While the abundances of C. monotis in seawater were lower than those of Ostreopsis cells, they were of the same order of magnitude throughout the sampling period. In September, abundances in seawater ranged from 3.20 × 10 2 to 1.12 × 10 3 cells L −1 . Epiphytic cells recorded on macroalgae ranged from unrecorded to 2.67 × 10 5 cells g −1 of fresh weight of macroalgae and unrecorded to 1.15 × 10 6 cells g −1 of dry weight of macroalgae. In October, abundances of C. monotis in seawater ranged from 4.80 × 10 2 to 1.6 × 10 3 cells L −1 . Epiphytic cells recorded on macroalgae ranged from 2.68 × 10 4 to 1.35 × 10 5 cells g −1 of fresh weight of macroalgae and 1.12 × 10 5 to 8.35 × 10 5 cells g −1 of dry weight of macroalgae. Ostreopsis cf. ovata was accompanied by the epiphytic dinoflagellate Coolia monotis. Taxonomical identification of C. monotis was based on size and morphological features obtained by SEM ( Figure  5). Tabulation was determined according to Balech [82]. Plate 7'' is characterized by the ratio of width and length of approximately 1. Plate 1' is placed left of the center. While the abundances of C. monotis in seawater were lower than those of Ostreopsis cells, they were of the same order of magnitude throughout the sampling period. In September, abundances in seawater ranged from 3.20 × 10 2 to 1.12 × 10 3 cells L −1 . Epiphytic cells recorded on macroalgae ranged from unrecorded to 2.67 x 10 5 cells g −1 of fresh weight of macroalgae and unrecorded to 1.15 × 10 6 cells g −1 of dry weight of macroalgae. In October, abundances of C. monotis in seawater ranged from 4.80 × 10 2 to 1.6 × 10 3 cells L −1 . Epiphytic cells recorded on macroalgae ranged from 2.68 × 10 4 to 1.35 × 10 5 cells g −1 of fresh weight of macroalgae and 1.12 × 10 5 to 8.35 × 10 5 cells g −1 of dry weight of macroalgae.

Concentration and Characterization of Ostreopsis Toxins
An indirect sandwich immunoenzymatic assay (ELISA) for palytoxin detection carried out on field microalgal samples showed 5.6 pg PLTX equivalents per Ostreopsis cell. A parallel investigation of the detailed toxin profile of O. cf. ovata was carried out by LC-HRMS and quantitative results were compared. Extracted ion chromatograms (XICs) for all the known PLTX congeners revealed the presence of OVTX-a-e and isobaric palytoxin (Figure 6), the identity of which was ascertained by: (i) Comparison of the retention times of individual compounds with those of ovatoxins contained in a reference sample available at the University of Naples Federico II; (ii) the diagnostic ion profile of ovatoxins and palytoxin analogs contained in full HRMS spectra (mass range m/z 800-1400) of each molecule, which represents a fingerprint for this class of molecules ( Figure 7); and (iii) elemental formula assigned to the monoisotopic ion peak of each ion (mass tolerance < 3 ppm) and isotopic pattern. The total toxin content measured by LC-HRMS was 6.3 pg cell −1 (Table 4) with OVTX-a being the major component, accounting for 57.1% of the total toxin content, followed by OVTX-b (20.6%), OVTX-d/e (17.5%), OVTX-c (3.2%), and isobaric PLTX (1.6%).

Concentration and Characterization of Ostreopsis Toxins
An indirect sandwich immunoenzymatic assay (ELISA) for palytoxin detection carried out on field microalgal samples showed 5.6 pg PLTX equivalents per Ostreopsis cell. A parallel investigation of the detailed toxin profile of O. cf. ovata was carried out by LC-HRMS and quantitative results were compared. Extracted ion chromatograms (XICs) for all the known PLTX congeners revealed the presence of OVTX-a-e and isobaric palytoxin (Figure 6), the identity of which was ascertained by: (i) Comparison of the retention times of individual compounds with those of ovatoxins contained in a reference sample available at the University of Naples Federico II; (ii) the diagnostic ion profile of ovatoxins and palytoxin analogs contained in full HRMS spectra (mass range m/z 800-1400) of each molecule, which represents a fingerprint for this class of molecules ( Figure 7); and (iii) elemental formula assigned to the monoisotopic ion peak of each ion (mass tolerance < 3 ppm) and isotopic pattern. The total toxin content measured by LC-HRMS was 6.3 pg cell −1 (Table 4) with OVTX-a being the major component, accounting for 57.1% of the total toxin content, followed by OVTX-b (20.6%), OVTX-d/e (17.5%), OVTX-c (3.2%), and isobaric PLTX (1.6%).   Table 4. Individual and total toxin concentration measured in O. cf. ovata cells by LC-HRMS (pg cell −1 ) and total toxin content measured by the indirect sandwich ELISA (pg PLTX eq cell −1 ).

Discussion
For the first time, the benthic dinoflagellates O. cf. ovata and C. monotis from Kaštela Bay were morphologically characterized. Both species were identified on the basis of morphological features, including thecal plate pattern, shape, and size. The thecal plate tabulation of O. cf. ovata cells described in this study (Po 3'7''5'''2'''') fit well with the original description by Fukuyo [83]. The designation of the thecal plates of O. cf. ovata have changed and been reinterpreted over time. Besada et al. [84] redetermined the first precingular plate determined by Fukuyo [83] as the first apical plate and completed the formula with sulcal and cingular plates (Po 4'6''6C8S5'''2''''). This new designation that considered the homology of the plates more than the relationship with the apical pore was supported by Fraga et al. [85] and Escalara et al. [86]. In this study, we adopted the original 12

Discussion
For the first time, the benthic dinoflagellates O. cf. ovata and C. monotis from Kaštela Bay were morphologically characterized. Both species were identified on the basis of morphological features, including thecal plate pattern, shape, and size. The thecal plate tabulation of O. cf. ovata cells described in this study (Po 3'7''5'''2'''') fit well with the original description by Fukuyo [83]. The designation of the thecal plates of O. cf. ovata have changed and been reinterpreted over time. Besada et al. [84] redetermined the first precingular plate determined by Fukuyo [83] as the first apical plate and completed the formula with sulcal and cingular plates (Po 4'6''6C8S5'''2''''). This new designation that considered the homology of the plates more than the relationship with the apical pore was supported by Fraga et al. [85] and Escalara et al. [86]. In this study, we adopted the original

Discussion
For the first time, the benthic dinoflagellates O. cf. ovata and C. monotis from Kaštela Bay were morphologically characterized. Both species were identified on the basis of morphological features, including thecal plate pattern, shape, and size. The thecal plate tabulation of O. cf. ovata cells described in this study (Po 3'7"5"'2"") fit well with the original description by Fukuyo [83]. The designation of the thecal plates of O. cf. ovata have changed and been reinterpreted over time. Besada et al. [84] redetermined the first precingular plate determined by Fukuyo [83] as the first apical plate and completed the formula with sulcal and cingular plates (Po 4'6"6C8S5"'2""). This new designation that considered the homology of the plates more than the relationship with the apical pore was supported by Fraga et al. [85] and Escalara et al. [86]. In this study, we adopted the original tabulation by Fukuyo [83], which is in accordance with Kofoidean plate nomenclature and accepted by most authors with slight modifications [6,[87][88][89]. The identification of O. cf. ovata was further confirmed by the DV/AP ratio, which is for O. siamensis either higher than 4 according to Penna et al. [6] or about 3 according to Aligizaki and Nikolaidis [43], as opposed to the congeneric species O. cf. ovata, which is characterized by a DV/AP ratio lower than 2. In Kaštela Bay, O. cf. ovata was accompanied by C. monotis as it has been observed in other Mediterranean areas [1,4,34,43,53,67,90,91] where O. cf. ovata appeared in association with other benthic dinoflagellates, such as C. monotis and Prorocentrum lima.
In addition, the identification of O. cf. ovata was confirmed by molecular PCR amplification using species-specific primers. In fact, due to the morphological plasticity and variability of Ostreopsis cells with consequent difficulty of species-specific identification, a PCR-based assay was applied to field samples in order to accurately identify the Ostreopsis species, which confirmed the microscopy analysis [69,92,93]. The molecular PCR assay is widely and successfully used because it is accurate, rapid, and reliable when applied to environmental samples [3,94]. It was found that only O. cf. ovata was present in the analyzed samples.
In order to determine the toxin profile of the Ostreopsis species found in Kaštela Bay, we used an indirect sandwich immunoenzymatic assay (ELISA) and LC-HRMS. While ELISA allowed us to measure the total toxin content (5.6 pg PLTXeq cell -1 ), LC-HRMS analyses provided the individual and total toxin contents. As a result, 6.3 pg of the sum of OVTXs and isobaric PLTX per Ostreopsis cell was measured, showing a prevalence of OVTX-a (3.6 pg cell −1 ). A comparison between the measurements made by the two approaches (LC-HRMS and ELISA) points to a toxin content of the same order of magnitude. However, due to a lack of replicates, no actual correlation can be extrapolated from the data. These toxin concentrations are similar to those recorded in Ostreopsis cells from the Ligurian Sea [24] but significantly lower than those recorded in the algal cells from the Conero Riviera (NW Adriatic), Catalan Sea, and NE Adriatic Sea [31,67,68] or those obtained from cultured Ostreopsis cells [95]. The absence of human poisoning associated with seafood consumption during O. cf. ovata blooms in the Mediterranean area could be tentatively related to the lower oral toxicity of ovatoxins (mainly OVTX-a) with respect to that of PLTX, as suggested by in vitro studies showing that OVTX-a cytotoxicity is about 100-fold lower than that of PLTX and also has lower hemolytic potency [29]. Nevertheless, the toxin content in O. cf. ovata cells recorded in Kaštela Bay could be related to the health problems recorded in humans exposed to marine aerosol and/or directly to seawater concomitantly with Ostreopsis bloom.
In the last two decades, Ostreopsis blooms have become common in the Mediterranean Sea, regularly occurring during the summer-autumn period (Table 1). According to the available literature, the highest abundances of Ostreopsis species in the Mediterranean Sea were recorded in the Ligurian Sea, along the Marche and Apulia coasts in the Adriatic Sea, the Balearic Sea, and the Catalan Sea. The highest abundances of Ostreopsis cells on macroalgae were reported in 2008 and 2009, while the highest abundances in seawater were reported in 2006, 2010, and 2016. It is interesting to note that all the reported maximal abundances of Ostreopsis species listed in Table 1, occurred during the negative phase of the North Atlantic Oscillation (NAO) index. The exception was in 2016, when blooms occurred during the positive phase of the NAO index, but this was preceded by a strong negative phase. A negative phase of the NAO index is characterized by a reduced pressure gradient, resulting in fewer and weaker winter storms that bring moist air into the Mediterranean. The analysis of precipitation data along the Croatian coast has shown a significant negative correlation with the NAO index [96].
In comparison with previously reported Ostreopsis occurrence in the Mediterranean Sea ( Table 1) the abundance of epiphytic cells of O. cf. ovata recorded in this study was one of the highest recorded abundances and was accompanied by citizen complaints. At the same time, in the summer of 2015, a massive occurrence of Ostreopsis species in the northern Adriatic near Rovinj, Croatia was recorded by a scientist from the Ruđer Bošković. Many complaints from citizens on a Facebook page that was opened regarding that event were received. Several years ago, there was a mass appearance of Ostreopsis species in the same area in the vicinity of Rovinj [31]. These findings point to the importance of introducing beach monitoring regarding the presence of Ostreopsis bloom along the Croatian coast, as is already done along the Italian coast [79].
Since Ostreopsis sp. bloom events are commonly associated with summer periods, some authors have proposed global warming as being the determining influence on Ostreopsis events [97,98]. The reported bloom of O. cf. ovata in Kaštela Bay in 2015 was associated with a trend of increasing sea surface temperatures in the bay. A linear trend analysis of sea surface temperature in the area of the eastern middle Adriatic shows the existence of an upward summer sea surface temperature trend (July-September) (Figure 8). In the last few decades (1979-2015), a positive trend has been observed in the entire Eastern Adriatic Sea [99], with several records of extreme sea surface temperatures in the warming season as a result of heat waves passing over Europe. Those heat waves hit Europe, North Africa, and the Middle East in the late spring and summer, where many new temperature records were measured. The heat continued in September, spreading across Eastern Europe. Modeling experiments suggest that anthropogenic forcing was a major factor in setting the conditions for the development of the 2015 heat wave [100]. According to the Croatian National Meteorological and Hydrological Service (DHMZ), the summer of 2015 in the middle Adriatic was generally dry, except for a rainy August, compared with the climatological average (http://meteo.hr/index_en.php). importance of introducing beach monitoring regarding the presence of Ostreopsis bloom along the Croatian coast, as is already done along the Italian coast [79]. Since Ostreopsis sp. bloom events are commonly associated with summer periods, some authors have proposed global warming as being the determining influence on Ostreopsis events [97,98]. The reported bloom of O. cf. ovata in Kaštela Bay in 2015 was associated with a trend of increasing sea surface temperatures in the bay. A linear trend analysis of sea surface temperature in the area of the eastern middle Adriatic shows the existence of an upward summer sea surface temperature trend (July-September) (Figure 8). In the last few decades (1979-2015), a positive trend has been observed in the entire Eastern Adriatic Sea [99], with several records of extreme sea surface temperatures in the warming season as a result of heat waves passing over Europe. Those heat waves hit Europe, North Africa, and the Middle East in the late spring and summer, where many new temperature records were measured. The heat continued in September, spreading across Eastern Europe. Modeling experiments suggest that anthropogenic forcing was a major factor in setting the conditions for the development of the 2015 heat wave [100]. According to the Croatian National Meteorological and Hydrological Service (DHMZ), the summer of 2015 in the middle Adriatic was generally dry, except for a rainy August, compared with the climatological average (http://meteo.hr/index_en.php). In contrast, some studies have shown that the growth of this species is not exclusively related to temperature [2]. These results are supported by the fact that in various parts of the Mediterranean, blooms of Ostreopsis appeared in different seasons contrary to the expectations regarding to the summer temperature of sea water [2,4]. Namely, according to the previous studies and the results of this research, the bloom in the Adriatic occurred in September, while in the Ligurian and Tyrrhenian Seas highest cell abundances are reported to occur earlier, in midsummer (July and August) [22,48,62] although summer temperatures are higher in the Adriatic Sea [2]. Based on these findings Mangalajo et al. [2] hypothesized that threshold temperature is required for Ostreopsis proliferation and maximal abundance is site specific related beside the temperature with others environmental factors as nutrients, substrate characteristic including macroalgal communities, biotic interactions as well as waves and currents. Hydrodynamics is an important factor involved in the ending of Ostreopsis bloom as demonstrated by earlier reports and results of this study. The observed intense bloom of O. cf. ovata in Kaštela Bay occurred during calm weather in September and decreased by an order of magnitude over a 12-day period, with lower temperatures and windy weather in October, confirming the importance of specific hydrodynamic conditions for the dynamics of Ostreopsis blooms previously reported by Accoroni and Totti [81]. The significant effect of hydrodynamics for Ostreopsis cells in seawater was confirmed by an investigation in the Ligurian Sea, while benthic stocks seem much more resistant to wave motion [101]. The benthic stock in this study also decreased by an order of magnitude but still remained high. In contrast, some studies have shown that the growth of this species is not exclusively related to temperature [2]. These results are supported by the fact that in various parts of the Mediterranean, blooms of Ostreopsis appeared in different seasons contrary to the expectations regarding to the summer temperature of sea water [2,4]. Namely, according to the previous studies and the results of this research, the bloom in the Adriatic occurred in September, while in the Ligurian and Tyrrhenian Seas highest cell abundances are reported to occur earlier, in midsummer (July and August) [22,48,62] although summer temperatures are higher in the Adriatic Sea [2]. Based on these findings Mangalajo et al. [2] hypothesized that threshold temperature is required for Ostreopsis proliferation and maximal abundance is site specific related beside the temperature with others environmental factors as nutrients, substrate characteristic including macroalgal communities, biotic interactions as well as waves and currents. Hydrodynamics is an important factor involved in the ending of Ostreopsis bloom as demonstrated by earlier reports and results of this study. The observed intense bloom of O. cf. ovata in Kaštela Bay occurred during calm weather in September and decreased by an order of magnitude over a 12-day period, with lower temperatures and windy weather in October, confirming the importance of specific hydrodynamic conditions for the dynamics of Ostreopsis blooms previously reported by Accoroni and Totti [81]. The significant effect of hydrodynamics for Ostreopsis cells in seawater was confirmed by an investigation in the Ligurian Sea, while benthic stocks seem much more resistant to wave motion [101]. The benthic stock in this study also decreased by an order of magnitude but still remained high.

Conclusions
Ostreopsis species are generally occurred in tropical waters, but its occurrence spread world-wide and its massive occurrence is well documented in the Mediterranean Sea (Table 1). Kaštela Bay is reported as a new site where O. cf. ovata blooms occurred causing the brown floating aggregate appeared in shallow parts. Since a massive occurrence of this species was recorded in the northern Adriatic Sea (near Rovinj) in the same year that this bloom occurred, as well as a few years ago, a monitoring program of toxic Ostreopsis species along the eastern Adriatic coast should be introduced to prevent health problems. OVTX-a was found to be the dominant toxin in the toxin profile, accounting for 57.1% of the total toxin content followed by OVTX-b (20.6%), OVTX-d/e (17.5%), OVTX-c (3.2%), and isobaric PLTX (1.6%), which was in good agreement with the toxin profile identified in the frame of a previous study on a number of different Mediterranean O. cf. ovata strains [102].

Sampling
Based on complaints of citizens about adverse effects associated with the bloom of Ostreopsis, sampling of phytoplankton and the macroalgal community was performed in September and October 2015 near the beach in Kaštela Bay ( Figure 9). Seawater samples were taken by a Niskin sampler to determine the abundance of Ostreopsis spp. cells. Substrate macroalgae at a 1-m depth were scraped from stones using a rectangular frame (20 × 20 cm) and shaken in 6 L of seawater. Seawater samples and substrate macroalgae were taken in triplicates with about a 3-m distance, making a total of 12 samples. Two subsamples of each final shake were fixed for the taxonomical identification of Ostreopsis, using both light and electron microscopes. The rest of the final shake was filtered by gravity on a 0.45-µm filter (Millipore membrane filters) to separate algal cells from the seawater. Pellets on the filter were frozen at −20 • C for subsequent toxin analyses and taxonomic identification by molecular analyses.

Conclusions
Ostreopsis species are generally occurred in tropical waters, but its occurrence spread worldwide and its massive occurrence is well documented in the Mediterranean Sea (Table 1). Kaštela Bay is reported as a new site where O. cf. ovata blooms occurred causing the brown floating aggregate appeared in shallow parts. Since a massive occurrence of this species was recorded in the northern Adriatic Sea (near Rovinj) in the same year that this bloom occurred, as well as a few years ago, a monitoring program of toxic Ostreopsis species along the eastern Adriatic coast should be introduced to prevent health problems. OVTX-a was found to be the dominant toxin in the toxin profile, accounting for 57.1% of the total toxin content followed by OVTX-b (20.6%), OVTX-d/e (17.5%), OVTX-c (3.2%), and isobaric PLTX (1.6%), which was in good agreement with the toxin profile identified in the frame of a previous study on a number of different Mediterranean O. cf. ovata strains [102].

Sampling
Based on complaints of citizens about adverse effects associated with the bloom of Ostreopsis, sampling of phytoplankton and the macroalgal community was performed in September and October 2015 near the beach in Kaštela Bay (Figure 9). Seawater samples were taken by a Niskin sampler to determine the abundance of Ostreopsis spp. cells. Substrate macroalgae at a 1-m depth were scraped from stones using a rectangular frame (20 × 20 cm) and shaken in 6 L of seawater. Seawater samples and substrate macroalgae were taken in triplicates with about a 3-m distance, making a total of 12 samples. Two subsamples of each final shake were fixed for the taxonomical identification of Ostreopsis, using both light and electron microscopes. The rest of the final shake was filtered by gravity on a 0.45-µm filter (Millipore membrane filters) to separate algal cells from the seawater. Pellets on the filter were frozen at −20 °C for subsequent toxin analyses and taxonomic identification by molecular analyses.

Microscopy Determinations
Phytoplankton community composition and abundance were analyzed according to the Utermöhl method [103]. Taxonomic identification of Ostreopsis species was performed using

Microscopy Determinations
Phytoplankton community composition and abundance were analyzed according to the Utermöhl method [103]. Taxonomic identification of Ostreopsis species was performed using epifluorescence microscopy after Calcofluor treatment and SEM. For epifluorescence microscopy, cells were fixed with 2% EM-grade glutaraldehyde dissolved in filtered seawater, stained with Calcofluor White M2R (Sigma-Aldrich, St. Louis, MO, USA) and SYBR Green (Lonza, Rockland, ME, USA)), and observed at 400× magnification using the epifluorescence microscope Zeiss AxioObserver Z1 (Carl Zeiss AG, Oberkochen, Germany) with Zeiss Filter Set 34 (excitation: 379-401 nm, emission: 435-485 nm, and beam splitter: 420 nm) and image acquisition by a Zeiss AxioCam MR M3 camera and epifluorescence microscope Leica DMI4000 B (Leica Microsystems; Wetzlar, Germany). For SEM observations, samples were preserved with 2% EM-grade glutaraldehyde, which was dissolved in filtered seawater. Subsequently, the samples were washed in 1:1 seawater/distilled water and then in distilled water. After that, samples were dehydrated in a gradual series of ethanol solutions and then critical-point dried with liquid carbon dioxide. Finally, samples were sputter-coated with gold and observed with LEICA STEREOSCAN 430i (Leica Microsystems; Wetzlar, Germany), FEI Quanta 200 (FEI, Thermo Fisher Scientific, Hillisbo, OR, USA), and MIRA 3 (Tescan, Brno, Czech Republic) scanning electron microscopes.

Molecular Analyses
Filter samples containing Ostreopsis cells were rinsed with sterile filtered seawater, the recovered volume (4 mL) was centrifuged at 4000× g for 10 min, and the supernatant was discharged. A second rinse with 1 mL of sterile filtered seawater was performed and the suspension was centrifuged at 1000× g for 10 min. Total genomic DNA was extracted from the obtained cell pellets using the DNeasy Plant Kit, and species-specific PCR assays for O. cf. ovata, O. cf. siamensis, and O. fattorussoi were carried out by amplifying 1 ng of genomic DNA according the protocols described by Battocchi et al. [3] and Vassalli et al. [104]. Expected amplicon size were 210, 223, and 104 base pair (bp) for O. cf. ovata, O. cf. siamensis, and O. fattorussoi, respectively. The PCR products were resolved on a 1.8% (w/v) agarose gel, 1× TAE buffer gel and were visualized by GelRed staining under UV light.

Extraction
A cell pellet was added to 3 mL of methanol/water (1:1, v/v) and extraction was performed by pulse sonication for 10 min in an ice bath. Centrifugation (6500 rpm for 1 min) was carried out to separate the supernatant from the residue. The extraction procedure was repeated twice on the pellet with 2 mL of methanol/water, combining the extracts to a final volume of 7 mL. The extract was analyzed by an indirect sandwich immunoenzymatic assay and liquid chromatography-high-resolution mass spectrometry.

Indirect Sandwich Immunoenzymatic Assay (ELISA)
The microalgal extract was analyzed by an indirect sandwich ELISA, as described by Boscolo et al. [105]. Briefly, ELISA multiwell strips were coated with the capture antibody by overnight incubation with 100 µL well −1 of mouse monoclonal anti-PLTX 73D3 antibody (20 µg mL −1 ) at 4 • C. Then, the wells were blocked with 200 µL of 2% skimmed milk (w/v) dissolved in PBS containing 0.1% Tween 20 (PBS-Tw) for 1 h at room temperature (RT) and incubated for 2 h at RT with 100 µL of PLTX solution (at different PLTX concentrations to obtain a calibration curve) or the microalgal extract solution in PBS-Tw (1:10). The wells were washed and incubated with the secondary antibody (100 µL well −1 of purified rabbit polyclonal anti-PLTX antibodies, 0.17 µg mL −1 ) for 2 h at RT. After washings, each well was incubated with the detection antibody (100 µL of horseradish peroxidase-conjugated goat anti-rabbit polyclonal antibodies, 1:2000) for 1 h at RT. After washings, the substrate and chromogen solution (3,3',5,5'-tetramethylbenzidine, 60 µL) was added to each well and the colorimetric reaction was stopped after 30 min by 30 µL of 1 M H 2 SO 4 . The absorbance of each well solution was measured at 450 nm (Spectra photometer; Tecan Italia; Milan, Italy). PLTX equivalents in the microalgal extract were determined by translating the absorbance into concentration by extrapolation from a PLTX calibration curve and are reported as mean of three independent experiments performed in triplicate.

Liquid Chromatography-High-Resolution Multiple Stage Mass Spectrometry (LC-HRMS n )
A hybrid linear ion trap LTQ Orbitrap XL TM Fourier transform MS (FTMS) with an ESI ION MAX TM source (Thermo-Fisher, San Josè, CA, USA) system coupled to a Dionex Ultimate 3000 quaternary system was used for analyzing the crude algal extract (injection volume = 5 µL). A Poroshell 120 EC-C18 (2.7 µm, 2.1 × 100 mm) (Agilent, USA) column kept at room temperature was used eluted with mobile phases (A = water and B = 95% acetonitrile/water), both added of 30 mM acetic acid. Flow was set at 0.2 mL min -1 . A good chromatographic separation among most PLTX congeners was obtained by using a slow gradient elution: 28-29% B over 5 min, 29-30% B over 10 min, 30-100% B in 1 min, and held for 5 min [23].
Positive ion HR full scan MS experiments were acquired in the range m/z 800-1400 at a resolving power of 60,000 (FWHM at m/z 400). Ionization source parameters were the followings: Spray voltage = 4.8 kV, capillary temperature = 290 • C, capillary voltage = 17 V, sheath gas = 32 and auxiliary gas = 4 (arbitrary units), and tube lens voltage = 145 V. HR collision-induced dissociation (CID) MS 2 experiments were acquired at a resolving power of 60,000 using a collision energy of 35%, isolation width of 4.0 Da, activation Q of 0.250, and activation time of 30 ms. The most intense peaks of the [M + H + Ca] 3+ ion cluster of isobaric palytoxin and individual ovatoxins were used as precursors. The monoisotopic peak of each ion cluster was used for calculating elemental composition (Xcalibur software v2.0.7 at a mass tolerance constraint of 5 ppm). The isotopic pattern of each ion cluster was considered in ion assignment.
Extracted ion chromatograms of the [M + H + Ca] 3+ ion of each known PLTX congener were used for quantitation. A not certified PLTX standard was used to prepare a calibration curve at five levels of concentration (100, 50, 25, 12.5, and 6.25 ng mL −1 ), which was used for OVTX and isobaric PLTX determination in the crude extract by assuming that their molar responses were similar to that of PLTX. Calibration curve equation was y = 31657x − 211166 and its linearity was expressed by R 2 = 0.9987.