Variations in ectoenzymatic hydrolytic activity in an oligotrophic environment (Southern Tyrrhenian Sea, W Mediterranean)

Available online at www.sciencedirect.com

Journal of Marine Systems 73 (2008) 123 – 137 www.elsevier.com/locate/jmarsys

Variations in ectoenzymatic hydrolytic activity in an oligotrophic environment (Southern Tyrrhenian Sea, W Mediterranean) Cristina Misic ⁎, Michela Castellano, Nicoletta Ruggieri, Anabella Covazzi Harriague Dipartimento per lo Studio del Territorio e delle sue Risorse, Università di Genova, C.so Europa 26, 16132 Genova, Italy Received 5 July 2007; received in revised form 5 October 2007; accepted 10 October 2007 Available online 24 October 2007

Abstract The variations in the expression of two hydrolytic ectoenzymes (leucine aminopeptidase – LA – and β glucosidase — BG) were studied in the southern Tyrrhenian Sea during spring 2004. This area is characterised by a complex morphology and hydrodynamism, which generate significant differences between different sectors, particularly in the 0–100 m layer. However, the area generally exhibits oligotrophic features such as low autotrophic pigment and organic matter concentrations and a higher bacterial biomass than the phytoplanktonic one. Despite this general bottom-up pressure, adaptations by the microbial consumers were indicated by the ectoenzymatic activities and by the relationships between the enzymes, their organic substrates and their producers (namely the bacteria). In particular, bacteria were able to exploit the inorganic N supply (nitrite + nitrate provided by irregular intrusions of intermediate waters) to escape the bottom-up limitation and produce enzymes such as BG devoted to the degradation of cellulose remnants and, therefore, also able to take advantage on this refractory organic matter. In the 200–800 m layer, where trophic limitation was strong due to the low values of potentially-labile organic matter (namely proteins), the peculiar hydrodynamism led to the formation of nepheloid layers rich in organic matter, which provided the bacteria with substrates and allowed the development of a significant correlation between LA activity and its own organic substrate. Furthermore, a reduction of the bottom-up pressure was also indicated by a higher mean bacteria cell size in the entire water column of the central and eastern sectors, and a significantly increased expression of BG related to the increase in the cell size. The ectoenzymatic activities, therefore, suggested that the southern Tyrrhenian Sea should be considered as a mosaic of subsystems, where the peculiar hydrological features stimulate bacterial adaptations and enhance the channelling of energy embedded in refractory materials into the food web. © 2007 Elsevier B.V. All rights reserved. Keywords: Leucine aminopeptidase; b glucosidase; Bacteria; Organic matter recycling; Oligotrophic sea; W Mediterranean; Southern Tyrrhenian Sea

1. Introduction Aquatic environments have been extensively studied for their trophic characteristics. Inorganic nutrient concentrations and their ratios have been used to define the ⁎ Corresponding author. Tel.: +0039 0103538068; fax: +0039 0103538140. E-mail address: [email protected] (C. Misic). 0924-7963/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jmarsys.2007.10.003

trophic status of aquatic ecosystems (Bethoux et al., 1998; Gomez et al., 2000; Moutin and Raimbault, 2002) as has chlorophyll-a, thanks also to remote sensing (Bosc et al., 2004), and both have increased our knowledge of production pathways. In oligotrophic areas such as the Mediterranean Sea the microbial food web is generally well developed and dominant throughout the year with the exception of spring (Schmidt et al., 2002). Several studies have reported significant differences in the activity and

124

C. Misic et al. / Journal of Marine Systems 73 (2008) 123–137

biomass of key-stone decomposers such as bacteria in variable trophic states. Although heterotrophic bacterial biomass decreases from eutrophic to oligotrophic waters, its contribution to total microbial biomass increases and even exceeds the biomass of phytoplankton in the euphotic zones of oligotrophic systems (Herndl, 1991; Robarts et al., 1996; Misic and Fabiano, 2006). Bacterial production also declines in oligotrophic systems (Sorokin and Mamaeva, 1991), but bacteria from oligotrophic environments have the ability to grow fast and exhibit high turnover times (Cho and Azam, 1988; Peduzzi and Herndl, 1992). Based on the regression analysis of bacterial biomass vs. production (see Billen et al., 1990), Dufour and Torréton (1996) suggested a moderate but increasing bottom-up control of bacteria from eutrophic to oligotrophic sites in the NE Atlantic. Rath et al. (1993) provided deeper insights into the bacterial degradation processes (namely the enzymatic hydrolytic activities), observing different enzyme expression trends in changing trophic conditions. While glycosidic enzymes tended to decrease from eutrophic to oligotrophic sites, the proteases showed no variations. A strong induction-suppression mechanism regulated by the catabolite concentration was observed by Münster (1991), who found a high expression of leucine aminopeptidase (LA) in low free amino-acid concentrations typical of oligotrophic situations. The specific LA activity, namely the LA per bacterial cell, shows a notable increase in oligotrophic environments, indicating that the bacteria are more closely adapted to high molecular weight polymer hydrolysis (Tamburini et al., 2002). The southern Tyrrhenian Sea is influenced by the exchanges of waters between the western and eastern Mediterranean through the Strait of Sicily, and the water movements, balances and anomalies have been reported (Millot, 1999; Astraldi et al., 2002; Gasparini et al., 2005), although little information is available on the eastern area of the southern Tyrrhenian (namely the Aeolian Islands and the Strait of Messina) (Hopkins, 1988; Tucci and Budillon, 1996). The southern Tyrrhenian Sea consists of a mosaic of subsystems, differently influenced by neighbouring sea and terrestrial inputs, and has been considered the most oligotrophic area of the western Mediterranean in terms of nutrient concentrations, primary productivity and vertical fluxes (Moutin and Raimbault, 2002; Speicher et al., 2006). Actually, very little information is provided on the biological processes involved in organic matter production and recycling (Povero et al., 1990; Innamorati et al., 1996; Tucci et al., 1997; Povero et al., 1998a, 1998b). The aims of the present paper are: 1) to identify the main physical and chemical characteristics of the southern Tyrrhenian Sea and define the patchiness of the environ-

ment in the upper and deeper layers of the water column, 2) to study the potential response of microbial heterotrophs (namely bacteria) to variable ecological features in terms of two hydrolytic enzymatic activities devoted to the degradation of organic matter to highlight adaptations to the strong oligotrophy and to the occurrence of variable ecological conditions in the area. 2. Materials and methods 2.1. Study area The sampling area (Fig. 1) covered several physical and morphological subsystems. The Aeolian Islands are the emerged end of the Patti Ridge, which starts in Sicily (in the area between the cities of Patti and Milazzo) and runs northwards, dividing the Cefalù Basin (western side) from the Gioia Basin (eastern side). The latter basin is heavily influenced by the input of Ionian waters, which follow a tidal regime, through the Strait of Messina. The currents of the entire water column come from the western side, flowing eastwards past Sicily and then turning north past Calabria. 2.2. Sampling Samples of seawater were collected during the Vulcano04 cruises of the SOLMAR (Sound, Oceanography and Living MArine Resources) Project (NATO, NURC La Spezia, Italy). The cruises were conducted during spring (1–11 May) 2004 using the R/V Universitatis and R/V Leonardo. Samples were collected from a total of 36 stations using Niskin bottles fitted onto rosette samplers with Sea Bird CTD probes (Fig. 1). The sampling depths were, generally, the following: surface, oxygen maximum, chlorophyll-a maximum, 200 m, salinity minimum (which identifies the core of the Levantine Intermediate Waters — LIW), and two or more depths in the 600 m — bottom layer. The main physical parameters (temperature and salinity) of all the stations were recorded and water samples were collected for the determination of nutrient (phosphate and nitrite + nitrate), photosynthetic pigment (chlorophyll-a and phaeopigments) and particulate protein concentrations. Additional analyses were carried out over a grid of 12 stations, centred on the Aeolian Islands (Fig. 1, circled stations). Turbidity and dissolved oxygen were detected with a multiparametric Sea Bird probe, and water samples were collected for the determination of dissolved proteins, particulate carbohydrates, bacterial parameters and ectoenzymatic hydrolytic activities (leucine aminopeptidase and β glucosidase). The

C. Misic et al. / Journal of Marine Systems 73 (2008) 123–137

125

Fig. 1. Study area and location of the stations. The longitudinal transect (stations #111 to #22) is indicated. The circled stations were investigated for a larger number of parameters (see Section 2.2 for details).

water samples were immediately treated as described below. 2.3. Inorganic nutrients The sample water was prefiltered through a 0.4 μm pore membrane (Albet, cellulose acetate) and stored at − 20 °C until analysis. Nutrient analysis was performed with a SYSTEA Nutrient Probe Analyser, following Hansen and Grasshoff (1983). The relative standard deviation between the replicates was, on average, about 13% for phosphate and 19% for nitrite + nitrate. 2.4. Particulate proteins and carbohydrates For the particulate organic matter determinations 1500 ml of seawater were filtered through Whatman

GFF glass-fibre filters and stored at − 20 °C until analysis. The samples were analysed following Hartree (1972) and Dubois et al. (1956) for proteins and carbohydrates respectively. Bovine serum albumin and δ(+)glucose were used as standards for proteins and carbohydrates respectively. The relative standard deviation between the replicates was about 17% for proteins and 12% for carbohydrates. 2.5. Autotrophic pigments (chlorophyll-a and phaeopigments) The water samples (500 to 1500 ml, depending on the depth) were taken in duplicate and filtered through precombusted Whatman GFF filters (nominal pore diameter 0.4 μm). The filters for autotrophic pigment analysis were immediately processed.

126

C. Misic et al. / Journal of Marine Systems 73 (2008) 123–137

The pigment was extracted in neutralised 90% v/v acetone, according to Holm-Hansen et al. (1965). A Varian Cary Eclipse spectrofluorometer was calibrated with a solution of chlorophyll-a from spinach. The relative standard deviation between the replicates was about 10% for the chlorophyll-a and 12% for the phaeopigments. The chlorophyll-a concentrations were converted into carbon equivalents by applying the coefficients 18.5 μgC μg chlorophyll-a− 1 to the deep chlorophyll maximum and 32.5 μgC μg chlorophyll-a− 1 to the other depths of the surface layer (Van Wambeke et al., 2002). 2.6. Dissolved protein-like compounds The samples were collected by filtering the seawater through a 0.4 μm pore-size membrane (Albet, cellulose acetate). Traditionally, marine organic matter has been divided into DOM and POM on the basis of its behaviour during filtration. Organic matter retained by filters with pores varying between 0.2 and 1.0 μm is considered POM, whereas the filterable material is considered DOM (Verdugo et al., 2004). Nevertheless, it must be kept in mind that the 0.4 μm cut-off we chose allowed a fraction of the colloids and small bacteria to pass through the filter and thus be incorporated in the DOM. Samples were processed immediately using synchronous fluorescence spectroscopy, an instrument technique that involves the simultaneous scanning of excitation and emission monochromators, keeping a constant difference between them. Generally, the lower the difference, the higher the resolution. We utilised a difference of 25 nm, as suggested by Ferrari and Mingazzini (1995). The 2D spectra, obtained with a Varian Carey Eclipse spectrofluorometer, were corrected by comparison with the spectrum of ultrapure water (Coble, 1996). We focused on one category of dissolved organic matter, defined by an excitation-emission wavelength of about 275–300 nm (protein-like signal). The filters were composed of cellulose acetate, which may retain proteins. Therefore our results on dissolved proteinlike compounds may be underestimations. However, we considered the differences between stations and areas rather than the absolute values (on the other hand no calibration was done with standards and we report these data as fluorescence units), and assumed that the error due to filter retention was constant. The relative standard deviation between the replicates was about 13%. 2.7. Total bacteria abundance and biomass Ten-millilitre aliquots of seawater were taken in duplicate, fixed immediately with 0.2 μm-filtered formalde-

hyde (2% final concentration) and stored at 4 °C in the dark until analysis, which was performed within 2 months. Measured volumes of fixed samples were filtered through 0.2 μm-pore black polycarbonate filters, stained with acridine orange and mounted on slides (Hobbie et al., 1977). The filters were examined with epifluorescence microscopy, using a Zeiss microscope, until a minimum of 10 fields or 100 bacteria were counted for each sample. This method may also reveal archeal cells, therefore when we report “bacterial abundance and biomass” we actually intended “prokaryote abundance and biomass”. Bacterial biovolume was determined by assigning bacteria to three different class sizes (small — volume less than 0.065 μm3, medium — volume ranging from 0.065 to 0.320 μm3, large — volume ranging from 0.320 to 0.572 μm3) (Palumbo et al., 1984), then converted to carbon content assuming 310 fgC/μm3 (Fry, 1990). The relative standard deviation between the replicates was, on average, about 11%. 2.8. Rates of potential hydrolytic ectoenzymatic activity Ectoenzymatic activities were immediately estimated with fluorescently tagged substrate analogs, as described by Hoppe (1983). Two types of enzyme were estimated: leucine aminopeptidase (LA), with L-leucine 7-amido-4methylcoumarin hydrochloride as a substrate analog, and β-glucosidase (BG), with 4-methylumbelliferyl β-Dglucoside as a substrate analog. It should be kept in mind that the incubation conditions were different from those of the natural in-situ ones, both physically (i.e. pressure) and chemically (medium of incubation, artificial substrates, see below for details). Therefore, the ectoenzymatic activities have to be considered as potential. The substrates were dissolved in 0.2 μm-filtered (Puradisc TM25AS), sonicated and autoclaved artificial seawater with the addition of methylcellosolve. The fluorescently tagged substrates were added at saturation concentrations (50 μM), as determined by shipboard substrate-saturation experiments. Aliquots from each water sample were split into sub-samples (from 50 to 100 ml each) to determine the ectoenzymatic activities in duplicate. In order to concentrate the samples to obtain measurable activities (this especially refers to BG, which in oligotrophic environments tends to be low — Rath et al., 1993) the water was filtered through 0.2 μm-pore polycarbonate filters (Huston and Deming, 2002). The filters were then added to the substrate solutions (5 ml final volume). In order to evaluate the contamination due to the sample handling during the analytical procedures and abiotic cleavage of the inorganic substrates, we processed

C. Misic et al. / Journal of Marine Systems 73 (2008) 123–137

127

Fig. 2. Cluster analysis of the surface layer (0–100 m) based on temperature, salinity, nutrient, autotrophic pigments and particulate proteins. The four main sectors are identified.

increase MUF fluorescence was not necessary. The relative standard deviation between the replicates of each sample was about 20%. The ectoenzymatic activity was also scaled to total bacterial abundance for later comparative analyses.

blank filters (not used for filtration) and sample filters containing formalin (Hoch and Bronk, 2007) with the samples. The filters were incubated in the dark at in-situ temperature for 4 h. After centrifugation (5′, 4000 rpm), the fluorescence of the supernatant was measured with a Varian Carey Eclipse spectrofluorometer, calibrated with 4-methylumbelliferone and 7-amino-4-methylcoumarin solutions. The comparisons of the readings of the formalin-killed samples and the blanks obtained with the clean filters showed that the latter were, on average, 2.0 and 1.9 times higher than the former for LA and BG, respectively. Despite the low activity characteristic of the open sea sites, the instrument readings for the samples were always significantly higher than the respective filter-containing blanks (t-test, p b 0.0001 both for LA and BG). Therefore, the addition of an alkaline buffer to

2.9. Statistics We tested differences between stations or groups of stations for the same parameter with Student's t-test. To test the relationships between the various parameters, a Spearman-rank correlation analysis was performed. We used the PRIMER 6β programme package to perform cluster analyses to reveal similarities between stations (Clarke and Warwick, 1994). Similarities were calculated using Euclidean distances. Normalisation was

Table 1 Mean values of the parameters and results of the SIMPER analysis (percentage of the difference expressed by each single parameter) of the surface layer (0–100 m) Sector

W C E N W vs. C C vs. E E vs. N C vs. N

Temperature °C

SD

15.17 14.58 14.68 14.95 30%

0.14 0.07 0.18 0.13

12% 19%

Salinity

37.70 37.80 37.88 37.67 14% 32% 15%

PO4

NO2 + NO3 −1

Proteins

SD

μmol l

SD

μg l

SD

μg l

SD

μg l− 1

SD

0.12 0.08 0.06 0.02

0.10 0.11 0.07 0.07

0.03 0.02 0.02 0.02

1.23 1.16 1.81 0.98

0.50 0.44 0.51 0.28

77.0 43.3 44.1 45.4 23%

3.5 9.7 12.4 9.2

0.23 0.14 0.13 0.07 14%

0.05 0.08 0.07 0.01

0.11 0.08 0.08 0.03

0.03 0.03 0.05 0.01

31%

W: western sector, C: central sector, E: eastern sector, N: northern sector.

−1

Phaeopigments

μmol l

29% 31%

−1

Chlorophyll-a

SD

31%

−1

128

C. Misic et al. / Journal of Marine Systems 73 (2008) 123–137

Fig. 3. Cluster analysis of the intermediate layer (200–800 m) based on temperature, salinity, nutrient and particulate proteins. The stations are mainly grouped according to depth. The average depth of each identified group is reported.

Fig. 4. Horizontal temperature (°C) and salinity trends of the surface layer (0–100 m). The four sectors are indicated by the station symbols: circles (western), triangles (central), crosses (eastern), squares (northern).

C. Misic et al. / Journal of Marine Systems 73 (2008) 123–137

performed for all the data. The complete group-linkage method was used to calculate dendrograms. Similarity percentage analysis (SIMPER) was used to analyse parameter dominance. A few stations were excluded from these analyses because not all the data were available. The clusters were based on the physical characteristics (temperature and salinity) and on nutrient, chlorophyll-a, phaeopigment and particulate protein concentrations. 3. Results The cluster analysis of the surface layer (namely the 0–100 m layer) indicated that the study area was not homogeneous (Fig. 2). The stations were grouped according to their geographical location, identifying four main sectors: western, central, eastern and northern. Table 1 reports the results of the SIMPER analysis. The differences between sectors were mainly due to the physical and chemical features of the water masses, and only marginally to chlorophyll-a and particulate protein concentrations. In order to summarise the information related to the 200–800 m layer, we performed another cluster analysis based on the same parameters utilised for the surface layer. However, the autotrophic pigments, that were under the detection limits in the mesopelagic layer, were not considered. Fig. 3 reports the cluster analysis results and indicates that the stations were mainly grouped

129

according to their bathymetry. The two groups with an average depth around 1000 m were different, mainly due to their phosphate and protein concentrations (SIMPER analysis, these two parameters explained 44% and 31% of the difference respectively). 3.1. Main physical and physico-chemical features Fig. 4 shows the thermoaline fronts dividing the western and central sectors (centred on stations #3 and #15) and the eastern and northern sectors (separating the area of stations #42 and #33 from that of stations #53 and #55). The central and eastern sectors were characterised by significantly lower temperatures and higher salinities (t-tests, p b 0.0001). Fig. 5 reports the vertical salinity profiles of the longitudinal transect. The LIW core, identified by salinity values higher than 38.7, was placed between 400 and 800 m and showed a slight “shallowing” at stations #17 and #19 (central-eastern sectors). The subsurface dissolved oxygen maximum (ca. 50– 60 m depth) had the lowest values in the central and eastern sectors (on average 5.53 ml l− 1) and the highest in the western sector (up to 5.76 at station #113). The lowest values in the deep layer (down to 4.04 ml l− 1) were found at ca. 1000 m in the western section and at ca. 800 m in the central and eastern sections. Station #17 of the central area was characterised by higher turbidity values (average of 0.27 tu for the first

Fig. 5. The vertical salinity and turbidity (tu) profiles of the longitudinal transect.

130 C. Misic et al. / Journal of Marine Systems 73 (2008) 123–137 Fig. 6. Horizontal trends of nutrient (nitrite + nitrate and phosphate, μmol l− 1), chlorophyll-a (μg l− 1) and particulate protein (μg l− 1) concentrations of the surface layer (0–100 m). The four sectors are indicated by the station symbols: circles (western), triangles (central), crosses (eastern), squares (northern).

C. Misic et al. / Journal of Marine Systems 73 (2008) 123–137

50 m) than the western (on average 0.11 tu) and eastern (0.12 tu) stations (Fig. 5). In the 100–800 m layer, instead, stations #17, #18 and #19 showed values lower than 0.04 tu, while the western stations in the same layer had turbidity values between 0.04 and 0.08 tu. 3.2. Nutrient concentrations The mean inorganic nutrient concentrations of the 0–100 m layer are shown in Fig. 6. The nitrite + nitrate and phosphate concentrations were not significantly correlated. The nitrite + nitrate showed higher concentrations in the area nearest the Strait of Messina (Table 1), significantly higher than those of the central, western and northern sectors (t-tests, p = 0.02, p = 0.001 and p = 0.04, respectively). In the central sector some stations had values lower than 1 μmol l− 1 (lowest value at station #17, 0.73 μmol l− 1). The nitrite + nitrate concentrations were significantly correlated to the salinity values (r = 0.38, n = 30, p b 0.05). The phosphate concentrations, instead, showed their maxima in the central sector, whose concentrations were significantly higher than those of the eastern and northern sectors (t-tests, p = 0.01 and p = 0.003, respectively). The distribution of nutrient concentrations with depth showed increases in the intermediate and deep layers of

131

the water column in all the sectors (Fig. 7). Higher concentrations of nitrite + nitrate were observed in the mesopelagic layer of the stations nearest Sicily (#8, #9 and #20) and a rise of waters characterised by high concentrations (Nthan 6 μmol l− 1) up to 600 and 400 m was observed at stations #15 and #18, respectively. The phosphate concentrations, instead, irregularly followed an increasing trend towards Sicily and the deep phosphate values were lower in the area of stations #2–#20 than near Calabria (#22) or in the extreme western sector (#111). 3.3. Organic matter The mean values of the autotrophic pigments (chlorophyll-a and phaeopigments) of the 0–100 m layer were highly correlated (r = 0.89, n = 32, p b 0.001) and showed similar trends. The high values of the chlorophyll-a: phaeopigment ratio (on average 2.2) were in accordance with the seasonal period of enhanced primary production. The western sector showed the highest chlorophyll-a values (Fig. 6 and Table 1), significantly higher than those of the other sectors (t-tests, p = 0.047, p = 0.007 and p b 0.001 for the central, eastern and northern respectively). On the contrary, the northern sector had the lowest values. The deep chlorophyll maximum was placed

Fig. 7. Vertical nutrient (nitrite + nitrate and phosphate, μmol l− 1) and particulate protein concentration profiles.

132

C. Misic et al. / Journal of Marine Systems 73 (2008) 123–137

Fig. 8. Horizontal trends of the ectoenzymatic activity (leucine aminopeptidase and β glucosidase, nmol l− 1 h− 1) and of the bacterial abundance (cells ⁎ 108 l− 1) of the surface layer (0–100 m) of the 12 stations around the Aeolian Islands.

C. Misic et al. / Journal of Marine Systems 73 (2008) 123–137

133

3.4. Bacterial parameters and ectoenzymatic activity

Fig. 9. Vertical profiles of the bacterial cell size (fgC cell− 1) for the western and central stations (filled circles) and eastern stations (empty circles).

between 60 and 90 m, deeper in the western sector and shallower in the eastern one. The mean values of the particulate proteins were not correlated to the chlorophyll-a ones, although the particulate protein concentrations were significantly higher in the western sector (Fig. 6 and Table 1) than in the others (t-tests, p b 0.001). A few stations next to Sicily (#8, #9 and #19) displayed mean values around 50 μg l− 1. In the surface layer the particulate proteins were correlated to particulate carbohydrates (r = 0.63, n = 12, p b 0.05), which showed values ranging from 22.4 to 45.2 μg l− 1, with mean values of 41 μg l− 1 for the western sector and ca. 30 μg l− 1 for the central and eastern ones. The protein: carbohydrate ratio was lower in the central sector (1.4) than in the western (2.0) and eastern (1.7) sectors and the ratio was correlated to the chlorophyll-a concentration (r = 0.68, n = 12, p b 0.05). The stations next to Sicily and Calabria, on the eastern side of the Patti Ridge, also showed higher particulate protein concentrations in the 200–800 m layer (Fig. 7). In this layer the background concentrations were about 10 μg l− 1 in both the central and eastern sectors, but the stations located in the Gulf of Gioia showed values of 18 μg l− 1, and other stations (#52, #55, #64 and #104) showed concentrations up to 19.5 μg l− 1. The stations next to the Aeolian Islands also showed the lowest protein:carbohydrate values in the 200–800 m layer, ranging from 0.5 to 1.1. Focusing on the 0–100 m layer of the 12 stations centred on the Aeolian Islands, the highest values of the dissolved proteins were found in the eastern sector (up to 9 fu), while the lowest values were found in the central sector (below 6 fu).

The LA ectoenzymatic activity (mean values, Fig. 8) showed no significant correlations with the dissolved and particulate proteins. The mean BG values showed a gradual increase from the western to the eastern sectors, similar to that of the bacterial abundance although they were not significantly correlated. Instead, the correlation between the BG and the dissolved oxygen was significant and inverse (r = − 0.81, n = 12, p b 0.01). The LA: BG ratio was highest in the western sector (ranging from 61 to 73), and decreased significantly (t-test, p = 0.04) in the central sector, where the values ranged between 24 (station #17) and 53 (#5). In the 0–100 m layer the bacterial abundance (Fig. 8) showed increasing values from the western (1.18 ⁎ 108 cells l− 1) to the central-eastern sectors (1.36–1.53 ⁎ 108 cells l− 1), although stations #8 and #30 showed values of 0.87 and 0.75 ⁎ 108 cells l− 1. The vertical profiles of the bacterial abundance showed low values below the 200 m depth (below 0.5 ⁎ 108 cells l− 1), with slight increases at stations #3, #5 and #7. The mean size of the bacteria had a different profile. The central-eastern stations had larger sizes (more than 55 fg C cell− 1), especially in the deep layer, and station #17 had the highest surface mean value (89 fg C cell− 1) (Fig. 9). The BG activity in the deep layer partially followed this trend. The correlation between all the observations relating to the BG activity and the mean size of the bacteria was significant (r = 0.30, n = 75, p b 0.01). The correlation between the LA and the bacterial size was also significant (r = 0.23, n = 77, p b 0.05), although less than the correlation between BG and bacterial size. The mean LA ectoenzymatic activity in this layer was significantly correlated to the dissolved and Table 2 Average specific activity (ectoenzymatic activity: total bacterial number, amol cell− 1 h− 1) of the water column at the 12 stations Sector

Station

LA/TBN

Avg (SD)

BG/TBN

Avg (SD)

Western

#3 #15 #5 #7 #17 #27 #29 #40 #18 #19 #30 #8

16.1 23.1 7.9 6.5 20.6 8.9 9.7 8.2 9.1 10.2 20.1 22.5

19.6 (4.9)

0.20 0.17 0.16 0.18 0.85 0.18 0.16 0.16 0.26 0.39 0.51 0.63

0.18 (0.02)

Central

Eastern

8.2 (1.2)

15.4 (6.8)

0.17 (0.01)

0.45 (0.16)

Station #17 values are limited to the surface layer, therefore the data were not used for the average sector value (avg). Standard deviations (SD) are reported in brackets.

134

C. Misic et al. / Journal of Marine Systems 73 (2008) 123–137

particulate proteins (r = 0.68 and r = 0.69 respectively, n = 12, p b 0.05). The ratio between the phytoplanktonic carbon (calculated using the chlorophyll-a values) and the bacterial carbon was quite variable in the study area, although it was always below 1 (except for station #30, 1.46). On average, the ratio value was higher for the western sector (0.80 ± 0.14) than for the other sectors (0.34 ± 0.21 for the central sector and 0.26 ± 0.10 for the eastern sector, excluding station #30). The LA specific activity (namely the ectoenzymatic activity per bacterial cell) showed the lowest values in the central sector (Table 2), while the BG specific activity showed significantly higher values (t-test, p = 0.039) in the eastern sector than in the other sectors (t-test, p = 0.044). 4. Discussion The Tyrrhenian Sea has been classified as one of the most oligotrophic areas of the western Mediterranean due to its low chlorophyll-a values, particularly in summer (Bosc et al., 2004). However, during our study, the spring pigment concentrations recorded in the southern Tyrrhenian Sea were similar to those recorded in other western Mediterranean areas, such as the Ligurian Sea (Misic and Fabiano, 2006). Despite the favourable seasonal period, the phytoplanktonic C was generally lower than the bacterial one (below 50% for 2/3 of the stations), in agreement with the features of oligotrophic areas (Robarts et al., 1996; Misic and Fabiano, 2006). Like the eastern Mediterranean (Moutin and Raimbault, 2002), the southern Tyrrhenian was P-limited during our study. Assuming that the upper layer nutrient input mainly came from the LIW, even though the vertical fluxes were limited to the winter season (Hopkins, 1988), the different distributions of the inorganic nutrient concentrations suggested that the consumption and recycling of the phosphate and nitrite + nitrate had different rates. Despite these general remarks, from a physical and morphological point of view, the southern Tyrrhenian Sea is complex and subjected to exchanges, often irregular, with the neighbouring oceanographic areas. Focusing on the enzymatic activities, Rath et al. (1993) observed that the LA specific activity was significantly higher and the BG specific activity was significantly lower at oligotrophic sites than at eutrophic sites. In our study the LA specific activity did not change significantly between the sectors, indicating a generalised oligotrophic condition, while the significant changes in the BG specific activity suggested high adaptation and

efficiency for the exploitation of the available trophic resources in the central and, mainly, eastern sectors. The significant and inverse correlation between the dissolved oxygen and BG indicated that the expression of this enzyme was linked to waters that had been segregated from the surface layer for a long time and did not host any notable primary productivity. The degradative processes had taken place and the labile components of the organic matter had been exploited, leaving recalcitrant organic matter such as cellulose remnants, which could be degraded only by specialised (but energy-expensive) enzymes. Therefore, although the bacteria in these waters were subjected to a number of trophic limitations, when one of these limitation was suppressed, the bacteria were able to respond very quickly and take advantage of the new environmental conditions. For instance, it is likely that the higher inorganic N availability (namely nitrite + nitrate) found in the eastern sector (where the LIW came from the Ionian Sea, passing over the Strait of Messina sill during high tide, Povero et al., 1998a) may fuel the bacteria with N, as observed in other very different but highly oligotrophic systems such as beaches, where an increase in inorganic N is strictly related to a notable decrease in LA:BG ratio values (Misic and Covazzi Harriague, 2007). In addition, intense water movement along the bottom in the eastern sector led to the formation of nepheloid layers in the intermediate layer at the stations to the east of the current flow. In fact, the cluster analysis for the 200–800 m layer indicated the general influence of depth in the grouping of the stations. A sign of the nepheloid layer was given by the increased particulate protein concentrations in the 200–800 m layer, which provided additional organic substrates for the heterotrophic microorganisms. In the 200–800 m layer the LA activity was correlated to the dissolved and particulate proteins, which might represent a further N input for bacterial metabolism (knowing that LA is directly associated with bacteria, Andersen-Elvehoy and Thingstad 1991). This suggested a notable adaptation of deep bacterial communities because the organic materials in the nepheloid layer are generally, refractory, as observed for the deep sediments (Gambi and Danovaro, 2006). One of the direct consequences of these processes was that bacteria showed a greater mean size in the eastern sector. The greater dimensions of the bacteria might have allowed them to host a wider spectra of different ectoenzymatic activity, that was not only devoted to protein exploitation (as generally found in oligotrophic environments, especially N-limited ones) but also to the consumption of other, less labile compounds such as cellulose. The BG activity, in fact,

C. Misic et al. / Journal of Marine Systems 73 (2008) 123–137

was more expressed in the eastern sector where the specific activity (namely BG:bacterial abundance) was also significantly higher. On the western side of the Patti Ridge and near the Aeolian Islands the morphology of the bottom facilitates the rise of intermediate waters (namely the LIW), driven by the general eastward circulation (Speicher et al., 2006 and citations therein). A slight rise in higher salinity waters towards the surface layer and the low dissolved oxygen values of the 0–100 m layer in the central sector (namely stations #17 and #19) indicated the occurrence of such a process, which was, however, irregular (Tucci and Budillon, 1996). The upward movements of the LIW along the Patti Ridge generated higher instability in the central area and led to a different mixing of the water masses. This process led to lower particulate organic matter concentrations in the central sector. The high turbidity of the surface layer at station #17 was in agreement with previous observations that indicated this area as a “concentration site” due to surface water movements (Tucci et al., 1997) and the low quantity and quality of the organic matter we found suggested that this particulate was mainly inorganic and refractory organic. The low trophic quality of the particulate organic matter and, moreover, the lower concentrations of the dissolved proteins were the result of the LIW component of the water column, which was more important here than in other areas, keeping in mind that the LIW has a generally low organic matter content (both dissolved and particulate) and the particulate fraction is probably already largely oxidised (Santinelli et al., 2002). This condition would stimulate the bacteria to produce higher LA activity, because of the greater gain given by LA in adverse trophic conditions (Münster, 1991; Christian and Karl, 1995). Nevertheless, we hypothesise that the inorganic N again supplied the bacteria with available N, thus allowing a relatively higher expression of BG able to exploit the available resources (namely the high carbohydrate proportion of the particulate matter). 5. Conclusions During spring the southern Tyrrhenian Sea maintained its oligotrophic features, with low nutrient and autotrophic pigment concentrations and a higher bacterial biomass than phytoplanktonic one. Nevertheless, the physical and morphological characteristics of this area generated significant differences between sectors. The ectoenzymatic activity was differently expressed, according to the physical and chemical features of the sectors. The oligotrophy led to a higher expression

135

of LA, but the occurrence of a particular hydrodynamism, which generated resuspension or the shallowing of the intermediate water masses, allowed the expression of glycosydic enzymes such as BG where the inorganic and organic N supply was higher (nitrite + nitrate and proteins, respectively) and the bacterial cells exhibited higher mean sizes. These processes helped the bacteria to overcome the limiting trophic conditions of the central area (located around the Aeolian Islands), where organic matter of lower quantity and quality (as reported by the protein:carbohydrate ratio) was faced with a decrease in the LA:BG ratio values, thus enhancing the expression of very specific and efficient enzymes (BG), although energy-expensive, in order to exploit the main part of the available organic matter. The ectoenzymatic activities, therefore, suggested that the southern Tyrrhenian Sea should be considered as a mosaic of subsystems, where the peculiar hydrological features stimulated bacterial adaptations and enhanced the channelling of energy embedded in refractory materials into the food web. Acknowledgements We wish to thank the Captains and crews of the R/V Leonardo and R/V Universitatis for their unstinting assistance during the cruises and the students of the Environmental Sciences course of the Università di Genova for their help in the sampling and analyses. This work was carried out within the framework of the Sound, Oceanography and Living Marine Resources (SOLMaR) Project of NATO-NURC (La Spezia, Italy). References Andersen-Elvehoy, I.L., Thingstad, T.F., 1991. Detection of limiting factors for bacterial activity using protease activity. Kiel Meeresforsch Sonderh 8, 392–398. Astraldi, M., Gasparini, G.P., Vetrano, A., Vignudelli, S., 2002. Hydrographic characteristics and interannual variability of water masses in the central Mediterranean: a sensitivity test for long-term changes in the Mediterranean Sea. Deep-Sea Res., Part I 49, 661–680. Bethoux, J.P., Morin, P., Chaumery, C., Connan, O., Gentili, B., RuizPino, D., 1998. Nutrients in the Mediterranean Sea, mass balance and statistical analysis of concentrations with respect to environmental change. Mar. Chem. 63, 155–169. Billen, G., Servais, P., Becquevort, S., 1990. Dynamics of bacterioplankton in oligotrophic and eutrophic aquatic environments: bottom-up or top-down control? Hydrobiol. 207, 37–42. Bosc, E., Bricaud, A., Antoine, D., 2004. Seasonal and interannual variability in algal biomass and primary production in the Mediterranean Sea, as derived from 4 years of SeaWiFS observations. Glob. Biogeochem. Cycles 18 (1). Cho, B.C., Azam, F., 1988. Major role of bacteria in biogeochemical fluxes in the ocean's interior. Nature 332, 441–443.

136

C. Misic et al. / Journal of Marine Systems 73 (2008) 123–137

Christian, J.R., Karl, D.M., 1995. Bacterial ectoenzymes in marine waters: activity ratios and temperature responses in three oceanographic provinces. Limnol. Oceanogr. 40, 1042–1049. Clarke, K.R., Warwick, R.M., 1994. Change in Marine Communities: An Approach to Statistical Analysis and Interpretation, 140. Natural Environment Research Council, UK. 144 pp. Coble, P.G., 1996. Characterization of marine and terrestrial DOM in seawater using excitation–emission matrix spectroscopy. Mar. Chem. 51, 325–346. Dubois, M., Gilles, K., Hamilton, J.K., Rebers, P.A., Smith, F., 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28, 350–356. Dufour, P., Torréton, J.-P., 1996. Bottom-up and top-down control of bacterioplankton from eutrophic to oligotrophic sites in the tropical northeastern Atlantic Ocean. Deep-Sea Res., Part 1 43, 1305–1320. Ferrari, G.M., Mingazzini, M., 1995. Synchronous fluorescence spectra of dissolved organic matter (DOM) of algal origin in marine coastal waters. Mar. Ecol., Prog. Ser. 125, 305–315. Fry, J.C., 1990. Direct methods and biomass estimation. Methods in Microbiology. Academic Press, New York, pp. 41–85. Gambi, C., Danovaro, R., 2006. A multiple-scale analysis of metazoan meiofaunal distribution in the deep Mediterranean Sea. Deep-Sea Res., Part I 53, 1117–1134. Gasparini, G.P., Ortona, A., Budillon, G., Astraldi, M., Sansone, E., 2005. The effect of the Eastern Mediterranean Transient on the hydrographic characteristics in the Strait of Sicily and in the Tyrrhenian Sea. Deep-Sea Res., Part I 52, 915–935. Gomez, F., Gonzalez, N., Echevarria, F., Garcia, C.M., 2000. Distribution and fluxes of dissolved nutrients in the Strait of Gibraltar and its relationships to microphytoplankton biomass. Est. Coast. Shelf Sci. 51, 439–449. Hansen, H.P., Grasshoff, K., 1983. Automated chemical analysis, In: Grassoff, K., Ehrhardt, M., Kremling, K. (Eds.), Methods of Seawater Analysis, 2nd ed. Verlag Chemie, Weinheim, pp. 347–379. Hartree, E.F., 1972. Determination of proteins: a modification of the Lowry method that gives a linear photometric response. Anal. Biochem. 48, 422–427. Herndl, G.J., 1991. Microbial biomass dynamics along a trophic gradient at the Atlantic Barrier Reef off Belize (Central America). P.S.Z.N. I: Mar. Ecol. 12, 41–51. Hobbie, J.E., Daley, R.J., Jasper, S., 1977. Use of nuclepore filters for counting bacteria by fluorescence microscopy. Appl. Environ. Microbiol. 33, 1225–1228. Hoch, M.P., Bronk, D.A., 2007. Bacterioplankton nutrient metabolism in the Eastern Tropical North Pacific. J. Exp. Mar. Biol. Ecol. 349, 390–404. Holm-Hansen, O., Lorenzen, C.J., Holmes, R.W., Strickland, J.D.H., 1965. Fluorimetric determination of chlorophyll. J. Cons. — Cons. Perm. Int. Explor. Mer 30, 3–15. Hopkins, T.S., 1988. Recent observation on the intermediate and deep water circulation in the southern Tyrrhenian Sea. In: Minas, H.-J., Nival, P. (Eds.), Océanographie pélagique méditerranéenne. Oceanol. Acta, pp. 41–50. N° sp. Hoppe, H.G., 1983. Significance of exoenzymatic activities in the ecology of brackish water measurement by means of methylumbrelliferyl-substrates. Mar. Ecol., Prog. Ser. 11, 299–308. Huston, A.L., Deming, J.W., 2002. Relationships between microbial extracellular enzymatic activity and suspended and sinking particulate organic matter: seasonal transformations in the North Water. Deep-Sea Res., Part II 49, 5211–5255. Innamorati, M., Massi, L., Nuccio, C., 1996. Indipendenza delle cenosi fitoplanctoniche dalle masse d’acqua circostanti le Isole Eolie.

In: Albertelli, A., De Maio, A., Picazzo, M. (Eds.), Proceedings XI Congress of the Associazione Italiana di Oceanologia e Limnologia. Genova, pp. 645–654. Millot, C., 1999. Circulation in the Western Mediterranean Sea. J. Mar. Syst. 20, 423–442. Misic, C., Fabiano, M., 2006. Ectoenzymatic activity and its relationship to chlorophyll-a and bacteria in the Gulf of Genoa (Ligurian Sea, NW Mediterranean). J. Mar. Syst. 60, 193–206. Misic, C., Covazzi Harriague, A., 2007. Enzymatic activity and organic substrates on a sandy beach of the Ligurian Sea (NW Mediterranean) subjected to anthropogenic pressure. Aquat. Microb. Ecol. 47, 239–251. Moutin, T., Raimbault, P., 2002. Primary production, carbon export and nutrients availability in western and eastern Mediterranean Sea in early summer 1996 (MINOS cruise). J. Mar. Syst. 33–34, 273–288. Münster, U., 1991. Extracellular enzyme activity in eutrophic and polyhumic lakes. In: Chrost, R.J., Overbeck, J. (Eds.), Microbial Enzymes in Aquatic Environments. Springer Verlag, New York, pp. 96–122. Palumbo, R., Ferguson, J.E., Rublee, P.A., 1984. Size of suspended bacterial cells and association of heterotrophic activity with size fraction of particles in estuarine and coastal waters. Appl. Environ. Microbiol. 48, 157–164. Peduzzi, P., Herndl, G.J., 1992. Zooplankton activity fueling the microbial loop: differential growth response of bacteria from oligotrophic and eutrophic waters. Limnol. Oceanogr. 37, 1087–1092. Povero, P., Hopkins, T.S., Fabiano, M., 1990. Oxygen and nutrient observations in the Southern Tyrrhenian Sea. Oceanol. Acta 13, 299–305. Povero, P., Accornero, A., Misic, C., Paschini, E., 1998a. Caratterizzazione del particellato organico sospeso nello stretto di Messina in relazione alle condizioni idrologiche. In: Picazzo, M. (Ed.), Proc. XII Congress of the Associazione Italiana di Oceanologia e Limnologia. Genova, pp. 65–75. Povero, P., Misic, C., Accornero, A., Fabiano, M., 1998b. Distribuzione e caratterizzazione biochimica della sostanza organica particellata nelle acque del Tirreno sud-orientale. In: Picazzo, M. (Ed.), Proc. XII Congress of the Associazione Italiana di Oceanologia e Limnologia. Genova, pp. 227–237. Rath, J., Schiller, C., Herndl, G.J., 1993. Ectoenzymatic activity and bacterial dynamics along a trophic gradient in the Caribbean Sea. Mar. Ecol., Prog. Ser. 102, 89–96. Robarts, R.D., Zohary, T., Waiser, M.J., Yacobi, Y.Z., 1996. Bacterial abundance, biomass and production in relation to phytoplankton biomass in the Levantine Basin of the Southeastern Mediterranean Sea. Mar. Ecol., Prog. Ser. 137, 273–281. Santinelli, C., Gasparini, G.P., Nannicini, L., Seritti, A., 2002. Vertical distribution of dissolved organic carbon (DOC) in the Western Mediterranean Sea in relation to the hydrological characteristics. Deep-Sea Res., Part I 49, 2203–2219. Schmidt, S., Andersen, V., Belviso, S., Marty, J.-C., 2002. Strong seasonality in particle dynamics of north-western Mediterranean surface waters as revealed by 234Th/238U. Deep-Sea Res., Part I 49 (8), 1507–1518. Sorokin, Y.I., Mamaeva, T.I., 1991. Role of planktonic bacteria in productivity and cycling of organic matter in the Eastern Pacific Ocean. Hydrobiol. 209, 39–50. Speicher, E.A., Moran, S.B., Burd, A.B., Delfanti, R., Kaberi, H., Kelly, R.P., Papucci, C., Smith, J.N., Stavrakakis, S., Torricelli, L., Zervakis, V., 2006. Particulate organic carbon export fluxes and size-fractionated POC/234Th ratios in the Ligurian, Tyrrhenian and Aegean Seas. Deep-Sea Res., Part I 53, 1810–1830.

C. Misic et al. / Journal of Marine Systems 73 (2008) 123–137 Tamburini, C., Garcin, J., Ragot, M., Bianchi, A., 2002. Biopolymer hydrolysis and bacterial production under ambient hydrostatic pressure through a 2000 m water column in the NW Mediterranean. Deep-Sea Res., Part II 49, 2109–2123. Tucci, S., Budillon, G., 1996. Caratteristiche fisiche, distribuzione e flussi del materiale particolato sospeso nel Tirreno Sud-orientale. Ann. Ist. Univ. Orient. Napoli 62, 75–90. Tucci, S., Ferrari, M., Capello, M., 1997. Caratteristiche e distribuzione del materiale particolato sospeso nel Tirreno Sud-orientale (1994 e 1995). In: Picazzo, M. (Ed.), Proc. XII Congress of the

137

Associazione Italiana di Oceanologia e Limnologia. Genova, pp. 275–286. Van Wambeke, F., Heussner, S., Diaz, F., Raimbault, P., Conan, P., 2002. Small-scale variability in the coupling/uncoupling of bacteria, phytoplankton and organic carbon fluxes along the continental margin of the Gulf of Lions, Northwestern Mediterranean Sea. J. Mar. Syst. 33–34, 411–429. Verdugo, P., Alldredge, A.L., Azam, F., Kirchman, D.L., Passow, U., Santschi, P.H., 2004. The oceanic gel phase: a bridge in the DOMPOM continuum. Mar. Chem. 92, 67–85.