On the presence of Pseudo-nitzschia calliantha Lundholm, Moestrup et Hasle and Pseudo-nitzschia delicatissima (Cleve) Heiden in the Southern Adriatic Sea (Mediterranean Sea, Italy)

Abstract

This investigation included a 2-year monitoring program aimed at assessing the abundance and distribution of harmful marine phytoplankton along the Southern Adriatic coast of Italy. Monthly sampling was conducted from April 1995 to March 1997 at four sampling stations along four transects, to determine the temporal and spatial presence/abundance of the potentially toxic Pseudo-nitzschia species. The study focussed on the most abundant taxa Pseudo-nitzschia calliantha and Pseudo-nitzschia delicatissima, the identities of which were confirmed by TEM on cleaned net material. The distribution patterns of these potential Amnesic Shellfish Poisoning (ASP) toxin producers were statistically analysed by Generalised Linear Model ANalysis Of VAriance, Principal Component Analysis (GLM ANOVA, PCA) and Spearman’s correlation analysis in order to address relationships between environmental variable and population dynamics. Abundances displayed horizontal and vertical structure in the study area. Inter-annual variability was also observed for both species that appeared to respond differently to the environmental factors investigated. Distributions of P. calliantha showed a stronger seasonality and was more correlated with winter water conditions than P. delicatissima, which in turn exhibited a broader temporal distribution and appeared independent from major environmental constraints. This is the first report of the occurrence and dynamics of P. calliantha and P. delicatissima populations in Southern Adriatic coastal waters.

Received April 4, 2005; accepted in principle June 7, 2005; accepted for publication July 19, 2005; published online July 27, 2005

INTRODUCTION

Planktonic diatoms belonging to the genus Pseudo-nitzschia are widely distributed in all oceans of the world. Several species of this genus have been associated with the production of domoic acid (DA), a neurotoxic amino acid responsible for Amnesic Shellfish Poisoning (ASP) in humans (Bates et al., 1998), and for extensive sea-bird (Work et al., 1993) and mammal deaths (Scholin et al., 2000). This toxicity has aroused considerable interest in Pseudo-nitzschia spp. after the first ASP event had been detected in Canada in 1987 (Bates et al., 1989). DA-producing species seem to be cosmopolites (Hasle, 2002) and, in some cases, their intense proliferation has resulted in toxin accumulation through food chains impacting marine organisms, humans and ultimately ecosystems and the economy. Thus, field studies elucidating nutrient and physical requirements and also the biotic interactions of Pseudo-nitzschia spp., at both regional and global scales, are essential for the prediction and prevention of mass occurrence and toxicity. Recently, Italian strains of the species Pseudo-nitzschia galaxiae and Pseudo-nitzschia multistriata have proved toxic (Sarno and Dahlmann, 2000; Cerino et al., 2005), increasing the awareness of possible harmful outbreaks along Italian coasts. In addition, DA was detected several times in concentrated field samples along the Middle Tyrrhenian coasts proving that there is production in natural situations (Congestri et al., 2004). Although it is known that Pseudo-nitzschia spp. occur recurrently in Italian seas, difficulties encountered by operators during routine surveys and monitoring activities have resulted in the information on single species spatio-temporal distribution and autoecology to be scant and heterogeneous. In fact, identification at the species level of these phytoplankton relies, in many cases, on ultrastructural features evidenced only on cleaned frustules observed with an electron microscope (Hasle and Syvertsen, 1997).

Long-term observations on phytoplankton assemblages and their physico-chemical environment are not commonly available for the Southern Adriatic coastal waters of Italy. Along this 150 km stretch of coast the hydrological features are uneven, as it is an exchange area between the Ionian and the Adriatic Seas. The circulation is always cyclonic, and the ingression of Ionian waters through the Otranto Channel, responsible for the mixing events, is highly variable showing seasonal and annual fluctuations.

Within a wider study aimed at assessing the structure and diversity of potentially toxic phytoplankton along these coastal waters, the spatio-temporal distribution of DSP (Diarrhetic Shellfish Poisoning) toxin producers have been already analysed (Caroppo et al., 1999a, 2001a, 2001c). The aims of this study were to determine for the first time the distribution patterns of two potentially toxic Pseudo-nitzschia species, to identify Pseudo-nitzschia populations using electron microscopy, and to determine the response of the dominant taxa, Pseudo-nitzschia calliantha Lundholm, Moestrup et Hasle and Pseudo-nitzschia delicatissima (Cleve) Heiden, to changes of a range of environmental factors over a 2-year period. Thus, mechanisms conducive towards population development relative to environmental conditions of this Mediterranean oligotrophic area were addressed.

METHOD

Sampling procedures and environmental factors

Seawater samples were collected monthly from April 1995 to March 1997 at four sampling stations along four transects (Brindisi, S. Cataldo, Otranto and S. M. di Leuca) at a distance of 0.5, 3, 5 and 10 km from the coastline (Fig. 1). Samples were collected using a 5 L Niskin bottle at four discrete depths (0, 5, 10 and 20 or 50 m where possible according to bathymetry). In addition, vertically drawn net samples (20 μm mesh) were taken and stored in 4% buffered formaldehyde for taxonomic assessment of Pseudo-nitzschia species and confirmation of specific attribution.

Water temperature, salinity and dissolved oxygen were measured in situ with an Idronaut 501 multiprobe. Nutrient analyses were performed according to Strickland and Parsons (Strickland and Parsons, 1972); chlorophyll a (Chl a) was determined following Parsons et al. method (Parsons et al., 1984). Diatom abundances were performed following the Utermöhl (Utermöhl, 1958) method.

Measurements of abiotic and biotic factors were reported as transect, station and depth averaged values.

Pseudo-nitzschia densities and identification

Pseudo-nitzschia calliantha and P. delicatissima cell counts were performed on 50-mL sedimented subsamples, preserved with Lugol’s iodine solution following Utermöhl (Utermöhl, 1958) within 1 month of sampling.

For transmission electron microscopy (TEM) observation of morphometric and ultrastructural features of Pseudo-nitzschia frustules, samples were treated as follows: net material aliquots were acid cleaned (HNO₃ and H₂SO₄) and washed in distilled water, a drop of cleaned material was placed on Formvar-coated grids (300 mesh), air-dried and examined with a ZEISS CEM 902 microscope at 80 kV.

Statistical analyses

Statistical analyses were performed to evaluate any relationships among species abundance and occurrence, environmental variables and other community parameters. In order to establish whether time of the year, location (transect), distance relative to the coast (station) and depth had a significant effect on density distribution of P. calliantha and P. delicatissima (dependent variables), a Generalised Linear Model ANalysis Of VAriance (GLM ANOVA) was applied. To assess whether the interactions between these factors affected species density distributions, a two-way analysis was also performed. Principal Component Analysis (PCA) was conducted to analyse similarities and differences between the dynamics of the two species and the most relevant environmental factors responsible for any differences. The variables were normalized: the overall mean of each variable was subtracted from each value and the result was divided by the overall standard deviation of that variable. The first three principal components, PC1, PC2 and PC3, were selected. The Spearman’s correlation analysis was then performed to address the effect of the external conditions (biotic and abiotic data set, which showed binomial distributions) and the extracted PCs on species distributions. Statistics were carried out using NCSS 2000 Statistical Software.

RESULTS

Hydrobiological data

The physico-chemical factors were comparable among transects, except for nutrient concentrations (Fig. 2, Table I). Average concentrations of ammonia, nitrite, nitrate and phosphate were highest at S. Cataldo, while maximum silicate content was detected at S. M di Leuca (Table I). The annual trend of water temperature was sinusoidal, values ranged from 8.79°C (winter) to 26.91°C (summer) (Fig. 2a). Water warming began in May, reaching temperatures around 16°C and peaking in July–August, when a sharp thermocline was established. Water stratification came to an end in November, with thermal homogeneity throughout the column, then, in winter (January and February), a thermal inversion was observed. Salinity was lowest during winter (36.07–37.70). Dissolved oxygen showed an inverse sinusoidal trend to temperature, varying between 0.01 (August 1995 and 1996) and 0.80 mM (January 1997) (Fig. 2a).

The nutrient analysis of the seawater indicated a general oligotrophic status with regard to inorganic nitrogen (NH₄ + NO₂ + NO₃) -N and phosphorus (PO₄-P) concentrations. Overall, nutrient distributions and concentrations were widely influenced by the large scale circulation in the basin resulting in very high nutrient concentrations in winter. NH₄-N showed significant fluctuation throughout the year and reached its maximum level in August and November 1995 (Fig. 2b). NO₂-N and NO₃-N reached maxima in February and January 1996, respectively (Fig. 2b). PO₄-P levels were also constantly low, and at times undetectable, with average values below 0.10 μM (Fig. 2c). Silica concentrations (SiO₄-Si) also had a winter maximum, with average value of 5.02 μM (January 1996) (Fig. 2d).

Chl a values were consistently below 0.34 mg m⁻³ and average phytoplankton densities generally showed moderate values, below 1.0 × 10⁵ cells L^–1, with the exception of winter levels when cells were more abundant (Table I). Maximum values of Chl a and diatom abundance were measured during winter (Fig. 2e). Seasonal patterns of Chl a and diatom abundance were similar in the two sampling years and did not show significant differences among transects.

Pseudo-nitzschia identification

P. calliantha Lundholm, Moestrup et Hasle

Chains of linear, narrow cells of about 1.5–2.2 μm in width and 80–110 μm in length with pointed frustule ends (Fig. 3a) were attributed to the recently described P. calliantha (Lundholm et al., 2003) after TEM observations that revealed the presence of approximately two striae (36/10μm) per fibula (18–20/10 μm) and that of the central nodule and larger central interspace. Moreover, one row of large, almost square poroids with a central unperforated area surrounded by 5–9 finely perforated sectors was visible in each stria (Fig. 3c).

P. delicatissima (Cleve) Heiden

Chains of linear, narrow cells of about 0.6–1.8 μm in width (transapical axis) and 50–70 μm in length (apical axis) with a very small overlap of the truncate-rounded apices (Fig. 3b) were attributed to P. delicatissima after TEM observations due to the presence of approximately two striae per fibula (18–26 fibulae/10 μm) and that of the central nodule and larger interspace. Additionally, two rows of triangular to hexagonal poroids (8–9/1μm) with characteristically finely hymenate vela were visible in each stria (Fig. 3d).

Other Pseudo-nitzschia species

Fusiform cells wider than the other Pseudo-nitzschia spp. (5–6 μm) and 76–114 μm long, formerly reported as Pseudo-nitzschia pungens (Caroppo et al., 2001b), were here attributed to Pseudo-nitzschia fraudulenta (P. T. Cleve) Hasle, after TEM observations of few valves that showed the central larger interspace, striae and fibulae equal in numbers and two or three rows of poroids per stria. Poroid ultrastructure revealed the presence of a branched unperforated central part surrounded by hymenate sectors. Finally, narrow, lanceolate cells approximately 2 μm wide and 60 μm long, with a slight central inflation and very short overlap of frustule ends were distinguished with an inverted microscope. These forms, referred to as Pseudo-nitzschia cf. prolongatoides during cell counts, were a posteriori attributed to P. cf. galaxiae in light of the most recent literature (Lundholm and Moestrup, 2002; Cerino et al., 2005).

Pseudo-nitzschia density distribution

Pseudo-nitzschia calliantha and P. delicatissima contribution to total planktonic community was generally moderate, never exceeding, on average, 1.4 and 3.8%, respectively. When considered within the diatom assemblage P. calliantha abundance accounted for 3.1% and P. delicatissima for 7.0 %.

Spatial and temporal variations of species abundances were observed across the sampled stretch of coast in both vertical and horizontal dimensions. Temporal distributions showed that P. calliantha was mainly present in samples taken in April and during the winter months. This species revealed a stronger seasonality than P. delicatissima and its dynamics followed that of diatoms (Fig. 2e). Absolute densities were highest at North (S. Cataldo) and ranged between 0.4 and 131.4 × 10³ cells L^–1 (February, S. Cataldo, stn. 3, at surface). In April, average abundances were high at Brindisi, surface layer and 10 m depth in 1995 and 1996, respectively. Densities fell at all transects in summer and sharply increased in winter along the whole study area during both years. Peak average densities occurred in February, a maximum value was measured at S. Cataldo (94.9 × 10³ cells L^–1, at surface) in 1996. Seasonal patterns were similar in both years, although cell numbers were generally lower in winter 1997 (Fig. 4).

Pseudo-nitzschia delicatissima had a broader spatio-temporal distribution than P. calliantha. Abundance showed marked fluctuations and inter-annual variability, with higher values in the first year of study. Absolute densities ranged between 0.4 and 148.0 × 10³ cells L^–1 (April 1995, Brindisi, station 1, –10 m and –50 m), with the highest values at North (Brindisi). Average maximum densities were recorded in April 1995 at all transects, except S. Cataldo, after which they declined until late summer, September 1996, when average abundances of 36.6 × 10³ cells L^–1 were recorded at Brindisi (surface layer). In autumn, cell numbers remained below 24.8 × 10³ cells L^–1 (S. M. di Leuca, October 1995) until February when an increase was detected at S. Cataldo (19.0 × 10³ cells L^–1, –50 m) and Otranto (10.4 × 10³ cells L^–1, at surface). Seasonal patterns in the second year showed slightly higher values in October and January, but overall densities were consistently lower than those of the previous year (Fig. 5).

Populations were rather homogeneously dispersed throughout the water column, although some records deviated from this pattern. The peak abundance of P. calliantha occurred in February 1996 at the surface (S. Cataldo) and at –50 m (S. M. di Leuca) (Fig. 4). Pseudo-nitzschia delicatissima also appeared to aggregate at 10 m depth (Brindisi and Otranto), especially in April 1995, and at –50 m (S. M. di Leuca) in February 1996 (Fig. 5).

Statistical analysis

It was shown that time (month) and transect of sampling significantly affected the density variations of P. calliantha and P. delicatissima, whereas depth appeared to influence only P. delicatissima distribution (Table II). It was also shown that the interactions between these variables (time, transect, station and depth) had significant effects on distribution of both species, suggesting that the effect of a given variable was modulated by the influence of another.

The PCA analysis generated 10 principal components, the first three of which had eigenvalues >1 and explained 76% of the variance of the original data set. These PCs were obtained as weighted linear combinations of the original variables and were used for further analysis. PC1 accounted for 45.3% of total variance within the data set. It was primarily related to nitrite, Chl a, dissolved oxygen, salinity, temperature, diatom abundance and nitrate, as evidenced by the Table III. In particular, PC1 was related to the winter hydrodynamic features of the Southern Adriatic Sea, characterized by low temperature and salinity and high nutrient, especially nitrite, concentrations. The second principal component (PC2) expressed the 17.6% of variance, which was due to silicate, nitrate, salinity and diatom abundances. The third component (PC3) (13.0% of variance) was significantly related to phosphate and ammonia.

The dispersion in the score plot between PC1 versus PC2 (April 1995–March 1997 = 1–24) appeared to be related to the summer–winter transition of hydrological features (Fig. 6). There was a higher score dispersion during the winter season; indicated by the clear differentiation in the plot between winter (9, 10, 11, 12, 22, 23 and 24) and the other seasons. The scores during the winter months (November–February) were clearly distinguishable from the other dates, and during this period P. calliantha had the highest abundances.

The densities of P. calliantha were negatively correlated with temperature and positively correlated with dissolved oxygen (Table IV). Pseudo-nitzschia delicatissima density was significantly positively correlated with salinity. As for nutrients, P. calliantha density was positively correlated with nitrite and nitrate. In addition, it was indicated that both species were significantly related to the diatom assemblage and the entire phytoplankton community. Density of P. delicatissima was also correlated to Chl a content. Significant correlations were also found between both Pseudo-nitzschia species and PC1.

DISCUSSION

The morphology of the Pseudo-nitzschia species observed during the present research fitted their classical description and with the most recent revisions. However, uncertainty still exist about attributing the small, lanceolate cells characterized by a central inflation to P. galaxiae Lundholm et Moestrup. Unfortunately due to deterioration of the samples over time it was not possible to reassess the taxonomy of the cells in light of the recent description of this species (Lundholm and Moestrup, 2002). However, new data on P. galaxiae distribution in Italian waters (Cerino et al., 2005) would indicate that the last designation is highly likely to be correct. In addition, P. prolongatoides has been reported only for Antarctica so far (Hasle, 1965).

There is a paucity of data on single species distributions in Italian waters because of difficulties in species determination during routine monitoring programs, and this is the first known assessment of the distribution of this planktonic genus in the Southern Adriatic Sea. Conversely, it is well known that Pseudo-nitzschia, as a genus, is widespread in the Mediterranean basin (e.g. Socal et al., 1999; Orsini et al., 2002) and is a persistent component of the phytoplankton assemblages along Italian coasts, with cell densities up to 10⁷ cells L^–1 along the Middle Tyrrhenian Sea (Congestri et al., 2004). In coastal areas of the Northern Ionian Sea (Gulf of Taranto) Pseudo-nitzschia spp. are responsible for the winter peak, accounting for up to 68% phytoplankton community (Caroppo, unpublished data). In addition, it is known that Pseudo-nitzschia species significantly contribute to the subsurface chlorophyll maximum (SCM) in coastal areas of the Northern and Middle Adriatic sub-basins (Revelante and Gilmartin, 1995; Totti et al., 2000).

The four species distinguished during this study are potential DA producers making investigation on annual dynamics of the two most frequent and abundant taxa particularly interesting in terms of their bloom periodicity and intensity.

The investigated coastal system revealed a high hydrological variability which dictated P. calliantha and P. delicatissima distribution to a degree, dependent of the spatio/temporal scale used and the species endogenous features. The Southern Adriatic Sea is characterized by the mixing up of water masses with different thermo-haline features: the surface layer, which is cold, less saline and has a high nutrient level (Adriatic Surface Waters, ASW), the Ionian Surface Waters (ISW), along with the Levantine Intermediate Waters (LIW), which in turn exhibit high temperature and salinity. At transect-scale level, two groups of sampling locations could be distinguished based on inland inputs and anthropogenic impact, especially in summer. The northernmost transects of Brindisi and S. Cataldo are most impacted by pollution associated with industry, agriculture and tourism. Such factors affected the water nutrient content, these sites had the highest levels of nitrite and nitrate throughout the year and the highest levels of ammonia, during the tourist season (August). This was reflected by the phytoplankton composition which was dominated by microphytoplanktonic diatoms, which are typical of the nutrient-rich Adriatic waters (Vilicic et al., 1995). The Otranto transect, due to its geographical location, is particularly affected by LIW, mainly in winter, or by ASW in spring and summer. The prevailing phytoplankton are diatoms and phytoflagellates, and the community physiognomy largely differed from other transects. The southernmost site, S. M. di Leuca, is an oligotrophic ecosystem, where water quality is more strongly affected by general circulation than inland inputs. Phytoplankton is dominated throughout the year by nano-sized phytoflagellates, which are recognized as being characteristic of Adriatic oligotrophic systems (Vilicic et al., 1995).

In accordance with the environmental conditions, the spatial patterns of P. calliantha and P. delicatissima differed among the four transects, with the more northerly locations exhibiting higher abundances. This was also confirmed by the one-way ANOVA that highlighted a significant effect of the transect factor on species dynamics. Increasing abundance of Pseudo-nitzschia spp. in response to coastal eutrophication was also found to occur in the Gulf of Mexico (Parsons et al., 2002).

The study area here, not exceeding 200 m depth, is entirely considered a coastal system, as the whole Adriatic Sea which comprises the largest shelf area of the Mediterranean Sea (Ott, 1992). Thus, distance relative to the coast was not expected to exert a role in species distribution, as shown by the one-way ANOVA.

It was shown that depth of sampling influenced P. delicatissima distribution patterns. It is possible that the water column profile showed vertical segregation in the deeper layers due to the ingression of salty deep LIW, that could produce more favourable conditions for species proliferation. In fact, P. delicatissima was positively correlated with salinity by the Spearman’s correlation analysis. Moreover, Rines et al. (Rines et al., 2002) highlighted the possibility of Pseudo-nitzschia populations of a fjord system (Washington, USA) to be concentrated into thin horizontal layers, from centimetres to a few metres within the water column or near to the bottom due to the physical transport of water masses. It must be noted that the significance of factor interactions (time, transect, station and depth) revealed by two-way ANOVA would ultimately indicate that a complex of interaction terms regulated population growth in the study area, preventing direct and clear conclusion about single variable effect on species development. In any case, further analyses contributed to better depict population dynamics in this ecosystem.

Temporal patterns of P. calliantha occurrence and abundance showed similarity between transects except for the northernmost (Brindisi). A stronger seasonality was observed at the southern transects showing a winter maxima. Socal et al. (Socal et al., 1999) observed that diatom, and particularly Pseudo-nitzschia, species blooms occur in winter in the Southern Adriatic Sea and that this was related to the ingression of ASW from the Northern Adriatic. Pseudo-nitzschia calliantha was negatively correlated with water temperature while it was positively related to nutrient availability, especially nitrogen sources, hence the density maxima occurrence in the winter period.

The temporal patterns of P. delicatissima were similar among the different transects during the first year. A seasonal trend was recorded too, with minimum cell numbers in summer, once again in agreement with the study of Socal et al. (Socal et al., 1999). While this seasonal pattern was mostly maintained through the second year of study along the southernmost transects, a more complicated scenario was observed at North, where a higher inter-annual variability was evident. The existence of this inter-annual variation was also found in the Gulf of Naples, where long-term time series data of Pseudo-nitzschia species, including P. delicatissima, have been recorded (Zingone et al., 2002).

Statistical analyses indicated that P. calliantha development was more significantly restricted by environmental conditions than P. delicatissima. More specifically, PCA revealed that major environmental forces acting on the population development were low temperature, high salinity and nutrient levels that are typical of the winter period. During winter, when both species were present, the score plot clearly indicated the particular hydrological features (Fig. 6). During the months where the planktonic community was characterized by the presence of predominately P. delicatissima, there were no clear characteristic hydrological features detected in the plot. This further indicated that P. delicatissima populations exhibited lower environmental constraints than P. calliantha.

Although 2-year observations are not sufficient to trace accurate temporal trends and regularities of a species, the present field research indicated for the first time that P. calliantha and P. delicatissima had distinct patterns of occurrence and responded differently to environmental variability in the coastal system investigated. The P. delicatissima dynamics appeared to be less significantly correlated to the environmental features, supporting the hypothesis that a stronger endogenous regulation might act on controlling its distribution in space and time (Zingone et al., 2002).

It must be acknowledged that irradiance (Fehling et al., 2005) and pH (Hinga, 2002; Lundholm et al., 2004) could significantly affect the population dynamics, but these data were not taken. Pseudo-nitzschia calliantha appeared to be a more adaptable species, depending on prevailing environmental conditions, in agreement with the observations of Phlips et al. (Phlips et al., 2004) in a lagoon, Florida (USA). Our data are also in concordance with a study on the eurythermal and euryhaline characteristics of a Pseudo-nitzschia pseudodelicatissima clone isolated from Danish coastal waters (Lundholm et al., 1997). P. pseudodelicatissima is a complex of species from which P. calliantha has recently been distinguished (Lundholm et al., 2003). The competition effect on density by other phytoplankton could not be excluded, bearing in mind that the centric diatom Skeletonema costatum is responsible for most of the winter blooms in the study area (Caroppo et al., 1999b; Bernardi Aubry et al., 2004).

We gratefully thank Dr. Neil Ellwood for improving the English in this manuscript.

REFERENCES

Author notes

¹Institute for Coastal Marine Environment, Taranto Section, Italian National Research Council, via Rome, 3-74100 Taranto, Italy, ²Department of Biology, University of Rome “Tor Vergata”, via Della Ricerca Scientifica, 00133 Rome, Italy, ³Department of Chemical and Biosystem Sciences, Laboratory of Environmental Spectroscopy, University of Siena, via Aldo Moro, 2-53100 Siena, Italy and ⁴CSGI, via Della Lastruccia, 3-50010 Sesto Fiorentino, Florence, Italy