Diatom induction of reproductive failure in copepods: The effect of PUAs versus non volatile oxylipins

Diatom induction of reproductive failure in copepods: The effect of PUAs versus non volatile oxylipins

Journal of Experimental Marine Biology and Ecology 401 (2011) 13–19 Contents lists available at ScienceDirect Journal of Experimental Marine Biology...

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Journal of Experimental Marine Biology and Ecology 401 (2011) 13–19

Contents lists available at ScienceDirect

Journal of Experimental Marine Biology and Ecology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j e m b e

Diatom induction of reproductive failure in copepods: The effect of PUAs versus non volatile oxylipins A. Barreiro a,⁎, Y. Carotenuto b, N. Lamari c, F. Esposito b, G. D'Ippolito c, A. Fontana c, G. Romano b, A. Ianora b, A. Miralto b, C. Guisande a a b c

Facultad de Ciencias, Universidad de Vigo, Lagoas-Marcosende, 36310 Vigo, Spain Stazione Zoologica Anton Dohrn, Villa Comunale, 80121 Napoli, Italy Instituto di Chimica Biomolecolare (ICB) del CNR, Via Campi Flegrei 34, 80078, Pozzuoli, Napoli, Italy

a r t i c l e

i n f o

Article history: Received 30 November 2010 Accepted 11 March 2011 Keywords: Chemical defence Copepods Diatoms Egg hatching Oxylipins PUAs

a b s t r a c t Several species and strains of diatoms were tested for their effects on egg production, hatching success, faecal pellet production, ingestion rate and food selection in the copepod Temora stylifera. Two of the diatom species tested were well known polyunsaturated aldehyde (PUA) producers: Skeletonema marinoi (SM) and Thalassiosira rotula (TR1), while the other species tested, Skeletonema pseudocostatum, was a non-PUA producing species. Another T. rotula strain (TR2) was also tested since previous studies had reported contradictory results, due either to absence of PUA production or production in small amounts. Our results showed strong inhibitory effects on reproductive parameters of copepods with all diatom species tested. Although we confirm that S. pseudocostaum did not produce PUAs, this diatom produced large quantities of other oxylipins such as (5Z,8Z,11Z,13E,15S,17Z)-15-hydroxy-5,8,11,13,17-eicosapentaenoic acid (15S-HEPE) and 13,14-13R-hydroxy-14S,15S-trans-epoxyeicosa-5Z,8Z,11Z,17Z-tetraenoic acid (13,14-HEpETE) and 15oxo-5Z,9E,11E,13E-pentadecatetraenoic acid (15-oxoacid), all of which have already been found in S. marinoi (Fontana et al., 2007a) and Pseudonitzschia delicatissimia (d'Ippolito et al., 2009). Another unidentified oxylipin was also present in high quantities in LC–MS profiles. Some of these oxygenated fatty acid derivatives were also found, together with PUAs, in TR1 and in TR2. Both PUA-producing and non-producing diatoms caused negative effects on hatching success with induction of apoptosis in newly hatched nauplii. This suggests that negative diatom effects can also depend on the production of non volatile oxylipins. Copepod feeding behaviour was found to be non-selective when each diatom species was offered together with the non toxic dinoflagellate Prorocentrum minimum suggesting that grazers are not aware of the toxicity of their food. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Several diatom species have been shown to induce reproductive failure in copepods under laboratory conditions by reducing egg production, hatching success, and naupliar survival to adulthood (reviewed by Ianora and Miralto (2010)). Inhibition of copepod egg hatching success has been shown to be diatom density-dependent (Chaudron et al., 1996): with decreasing diatom concentrations, deleterious effects on hatching diminish and these effects take longer to induce (Ban et al., 1997). Mixed diets dilute but do not delete the negative effects of

Abbreviations: PUA, polyunsaturated aldehyde; PUFA, polyunsaturated fatty acids; FAH, fatty acid hydroperoxide; LOX, lipoxygenase; TR1, Thalassiosira rotula strain CCMP 1647; TR2, Thalassiosira rotula strain CCMP 1018; SM, Skeletonema marinoi; SPC, Skeletonema pseudocostatum; PRO, Prorocentrum minimum; FSW, filtrated sea water. ⁎ Corresponding author at: Current address: Department of Ecology and Evolutionary Biology, Corson Hall, Cornell University, 14850 Ithaca, NY, USA. E-mail address: [email protected] (A. Barreiro). 0022-0981/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jembe.2011.03.007

diatoms on copepod reproduction (Kang and Poulet, 2000; Turner et al., 2001). Diatoms not only interfere with egg maturation but also induce strong developmental aberrations in those nauplii that do develop to hatching. Teratogenic (abnormal) nauplii show a variety of birth defects such as asymmetrical bodies and malformed or reduced number of swimming and feeding appendages (Ianora et al., 2004) and usually die soon after birth because they are unable to swim or feed properly. Pioneer studies (Miralto et al., 1999) led to the characterization of short-chained polyunsaturated aldehydes (hereafter PUAs) as the main molecules responsible for the toxic effects observed in copepods. These molecules are derived from lipoxygenase oxidation of polyunsaturated fatty acids (PUFAs), mainly C16 and C20, liberated from chloroplast galactolipids and membrane phospholipids after membrane damage or disruption (Pohnert et al., 2002; d'Ippolito et al. 2004; Reviewed by Pohner, (2005) and Fontana et al. (2007a)). Wichard et al. (2005) explored potential PUA production among 51 diatom isolates, reporting that 30% are PUA producers. But whether PUA production is widespread over different taxonomic groups of diatoms, from the genera to the strain level, has yet to be clarified. Furthermore, some differences may

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not be due to genetic differentiation, but to environmental conditions or particular physiological status of the cells (Ribalet et al., 2007). Recently, an entirely new group of other oxygenated derivatives of PUFAs (oxylipins) was identified in marine diatoms that were able (Thalassiosira rotula and Skeletonema marinoi) or unable (Chaetoceros socialis, Chaetoceros affinis, and Pseudonitzschia delicatissima) to produce PUAs (d'Ippolito et al., 2005; Fontana et al., 2007b; d'Ippolito et al. 2009). The presence of these compounds (here referred as nonvolatile oxylipins) demonstrated that different oxygenase pathways are active in marine diatoms and that the production of PUAs is complemented by species-specific synthesis of other products derived from the enzymatic oxidation of PUFAs. The process is mediated by massive synthesis of fatty acid hydroperoxides (FAHs) and highly reactive oxygen species (hROS). Both FAHs and non-volatile oxylipins induce reproductive failure in copepods even though the latter compounds seem to be less active (Fontana et al., 2007b). PUA synthesis is therefore not sufficient to assess the potential deleterious effects of diatoms on copepod reproduction since compounds other than PUAs can also be involved in the defence against potential predators (Fontana et al., 2007b; d'Ippolito et al. 2009). Here we continue to explore this possibility in the copepod Temora stylifera, a dominant copepod species in the Mediterranean Sea, by testing the potential effect of a known PUA (Skeletonema marinoi) and non-PUA (Skeletonema pseudocostatum) producing species on egg production, egg hatching success, female survival and faecal pellet production. S. pseudocostatum is considered a non-PUA producing species according to Wichard et al. (2005). But it was unknown whether the strain used in our experiment produced PUAs or not, since our strain was isolated from the Gulf of Naples in 2002. We also tested the potential effect on those reproductive parameters when T. stylifera was fed with two different strains of the diatom T. rotula: Strain CCMP 1647, known as a PUA producer, and Strain CCMP 1018, which did not produce PUAs in Pohnert et al. (2002), but showed minimal PUA production in Wichard et al. (2005). Finally, we studied the feeding behaviour of T. stylifera when offered with a mixture of each of these diatoms with the non-oxylipin producing dinoflagellate, Prorocentrum minimum, to better understand the response of copepods under more natural mixed diet conditions.

2. Methods

2.3. Reproduction experiments Healthy females and males of T. stylifera were sorted and transferred to crystallising dishes containing 100 ml of 50 μm filtered seawater (N = 10–15, Table 1) and placed at ≈20 °C. Each dish contained an individual couple to ensure re-mating and production of viable eggs (Ianora et al., 1989). Animals were maintained in this food suspension during 24 h. The next day, animals were transferred to new food suspensions with each of the target algae offered as the only food. The five microalgae tested were the diatoms SPC, SM, TR1, TR2 and the control dinoflagellate PRO, (Table 1). Food suspensions were renewed daily. Experiments were terminated after 12–15 days. PRO was used as a control diet since this alga was shown to induce high and constant egg hatching success in T. stylifera (Turner et al., 2001) and did not produce PUAs or other oxylipins (Fontana et al., 2007a). Cell concentrations of each alga were adjusted to keep the same carbon concentration between treatments (0.98–1.24 μg C ml−1, Table 1) in 100 ml of food suspension. This carbon concentration was food saturating and cell abundances were close to natural bloom conditions. Faecal pellet production, egg production and crumpled egg membranes due to cannibalism were counted daily under a binocular inverted microscope. Female survival was estimated as the % of accumulated survival of the initial population. Hatching was estimated daily by maintaining egg production containers for an additional 48 h at ≈20 °C. Eggs were then fixed with 25 ml of 95% ethanol. Hatching success was calculated as the percentage of nauplii with regards to egg production excluding eggs with crumpled membranes. Groups of treatments were performed on different dates during the period from 4 October to 14 December. 2.4. Fluorescence labelling and confocal microscopy Newly-hatched nauplius 1 and 2 embryos produced by females fed PRO, SKE or SPC were washed three times in FSW before fixation in 2–4% formaldehyde for 24 h. Fixed embryos were then rinsed several times in PBS and incubated for 24 h in 250 μl of 0.5 U ml−1 chitinase enzyme (EC3.2.1.14; Sigma-Aldrich) dissolved in 50 cmmol l−1 citrate buffer, pH 6, at 25 °C, to permeabilize the chitinous wall. After rinsing several times in PBS, embryos were incubated for 10 min in 0.1% Triton X-100 at room temperature, rinsed in PBS containing 1% BSA, and further incubated for 90 min in TUNEL (terminal-deoxynucleotidyl-transferase-mediated dUTP Nick End Labelling; Roche Diagnostics GmbH, Mannheim,

2.1. Phytoplankton cultures The diatom species S. pseudocostatum (Strain SZN B77, hereafter SPC), S. marinoi (Strain SZN B118, hereafter SM), T. rotula Strain CCMP 1647 (hereafter TR1), Strain CCMP 1018 (hereafter TR2) and the dinoflagellate P. minimum, (hereafter PRO) were cultured in 2 l jars with 1 l of 0.22 μm filtered and autoclaved seawater enriched with f/2 medium (Leftley et al., 1987) at 20 °C under 12 h:12 h light:dark cycle. Cultures were diluted daily by replacing ≈25% of the culture with fresh media. Cell carbon content of SM, TR1 and 2 and PRO was considered to be the same as in Carotenuto et al. (2002). Cell carbon content of SPC was not determined but was assumed to be the same as SM due to its similar size. This assumption is supported by similar values measured in SPC cells isolated from field samples collected in the Gulf of Naples (~20 pg C cell−1, D. Sarno, pers. comm.).

Table 1 Protocol details of the experiments. SPC (Skeletonema pseudocostatum), SM (Skeletonema marinoi), TR1 (Thalassiosira rotula Strain CCMP 1647), TR2 (Thalassiosira rotula Strain CCMP 1018), PRO (Prorocentrum minimum). Reproduction experiment

Cell abundance (cell ml−1)

Cell carbon content (pg C cell−1)

Total carbon content (μg C ml−1)

N° replicates

Days

SPC SM TR1 TR2 PRO

60 × 103 60 × 103 8 × 103 8 × 103 6 × 103

20.7a 20.7b 121.9b 121.9c 177.1b

1.24 1.24 0.98 0.98 1.06

15 15 15 15 10

15 14 15 15 12

Ingestion experiment

Mean cell abundance (cell ml−1) 1095 PRO 8700 SPC 874 PRO 800 TR1 1045 PRO 1212 TR2

3 2 3 2 4 2

4

PRO–SPC

2.2. Zooplankton collection PRO–TR1

Samples were collected weekly during 4 October to 30 November 2005 at a fixed station of the Gulf of Naples (South Tyrrhenian, Italy) by towing a 250 μm mesh net with a non-filtering cod obliquely from ≈50 m to the surface. Samples were poured in an insulated box and transported to the laboratory within 1 h of collection.

PRO–TR2 a b c

0.19 0.18 0.15 0.10 0.18 0.15

PRO SPC PRO TR1 PRO TR2

Assumed to be the same as SM (see Methods section). From Carotenuto et al., 2002. Assumed to be the same as TR1.

feeding controls feeding controls feeding controls

4 4

A. Barreiro et al. / Journal of Experimental Marine Biology and Ecology 401 (2011) 13–19 Table 2 ANCOVA analysis of egg production rate in Temora stylifera using time (LN transformed) as covariate, daily egg production rate as dependent variable and algal species as factor. Covariate was always significative.

TR1–TR2 TR1–SM TR1–SPC TR1–PRO TR2–SM TR2–SPC TR2–PRO SM–SPC SM–PRO SPC–PRO

df (factor, error, total)

F

p

1, 1, 1, 1, 1, 1, 1, 1, 1, 1,

1.14 69.46 34.55 78.93 106.86 56.29 139.46 0.04 4.37 5.34

0.286 b 0.001 b 0.001 b 0.001 b 0.001 b 0.001 b 0.001 0.841 0.035 0.022

289, 311, 240, 273, 291, 220, 253, 242, 275, 204,

292 314 243 275 294 223 256 245 278 207

Germany) solution, at 37 °C. Whole-mount copepod embryos were observed with a Zeiss Axio-Imager M1 microscope equipped with a 20´ objective and GFP filter to detect TUNEL green fluorescence. 2.5. Grazing experiments A mixture of PRO with each diatom was offered as food to T. stylifera in 25 ml bottles (0.10–0.18 μg C ml−1, Table 1). Food concentrations were much lower in the grazing experiments (below saturation levels) to better appreciate the removal of diatom cells by females. Three adult females were placed in each of three or four 25 ml bottles whereas two 25 ml bottles without copepods were used as a control for algal growth. Bottles were maintained on a rotating wheel (1 rpm to ensure continuous algal suspension) at 20 °C under 12 h:12 h light:dark cycle. Food suspensions were renewed daily and

A

140 120

15

experiments were terminated after 4 days. If any of the animals died, the sample was not considered for the calculation of ingestion rates. Two replicates of a sample of 1 ml for cell counting was taken from each bottle, fixed with 300 μl of Lugol iodide solution and placed into 2 ml Eppendorf tubes. Triplicates of 1 ml samples for cell counting were taken from initial cell suspensions, fixed with 300 μl of Lugol iodide solution and placed into 2 ml Eppendorf tubes. Samples were counted under a direct microscope in 1 ml Sedgewick–Rafter chambers. Ingestion values were calculated following Frost's (1972) equations. Selection of food was calculated with Manly's α index (Manly, 1974). 2.6. Chemical analysis 10 l of cultures of each diatom species were centrifuged 10 min at 1500 g at 4 °C. The pellet was resuspended in distilled water, sonicated for 1 min and, after 10 min, suspended in acetone (1 ml/g cell pellet). The hydro-acetone suspension was sonicated for 1 min and then centrifuged at 2000 g for 5 min at 5 °C. The resulting pellet was resuspended in H2O/acetone 1:1 (2 ml/g cell pellet), sonicated for 1 min and centrifuged at 2000 g for 5 min. The supernatants were combined prior to extraction with an equal volume of CH2Cl2. The organic layer was dried over dry Na2SO4, filtrated and evaporated at reduced pressure. A fraction (2/3 of the extract) of this material was submitted to PUAs analysis by GCMS in agreement with d'Ippolito et al. (2002a). After addition of 16-hydroxy-hexadecanoic acid as an internal standard, the remaining extract was methylated with ethereal diazomethane. The excess of organic solvent was removed at a reduced pressure and the residue dissolved in MeOH (1 μg/μl) was analysed by LCMS analysis on a Qtof-micro mass spectrometer (Waters SpA, Milan, Italy) equipped with an ESI source (positive mode) and coupled with a Waters Alliance HPLC system (reverse phase column with a gradient from MeOH/H2O 70/30 to MeOH/H2O 80/20 in 15 min, followed by isocratic elution MeOH/H2O 80/20 at a flow of 1 ml/min). Content of PUAs and non-volatile oxilipins was established by integration of area peaks against internal standards, on an average of four measurements.

100 3. Results

80

3.1. Egg production

60 40 20 0 1

Egg viability (% hatched eggs)

B

2

3

4

5

6

7

8

9

10 11 12 13 14 15

100 80

Several ANCOVA analyses (Table 2) showed significant differences in egg production between all pair wise comparisons except TR1–TR2 and SM–SPC. Strains TR1 and TR2 induced highest egg production rates, even if there was a decrease from the first half of the experiment (mean days 2–7: 61.9 ± 12 SD and 59.5 ± 11.7 SD eggs female−1 day−1 respectively) with respect to the second half (mean since day 8 to the end: 31.8 ± 9.8 SD and 35.4 ± 15.5 SD eggs female−1 day−1 respectively), representing a decrease of 48.7% and 40.4% respectively (Fig. 1A). PRO supported moderate–low but constant egg production rates over the whole experimental period (mean days 2–7: 13.6 ± 5.3 Table 3 ANCOVA analysis of egg hatching success in Temora stylifera using time (LN transformed) as covariate, hatching success as dependent variable and algal species as factor. Covariate was always significative.

60 40 20 0 1

2

3

4

5

6

7

8

9

10 11 12 13 14 15

Days Fig. 1. Egg production rates (A) and hatching success (B) of Temora stylifera during the reproduction experiments (mean ± SD). Symbols: open circle (PRO), dark square (TR1), grey square (TR2), dark diamond (SM), and grey diamond (SPC).

TR1–TR2 TR1–SM TR1–SPC TR1–PRO TR2–SM TR2–SPC TR2–PRO SM–SPC SM–PRO SPC–PRO

df (factor, error, total)

F

p

1, 1, 1, 1, 1, 1, 1, 1, 1, 1,

57.15 25.84 39.83 28.84 0.157 0.318 207.29 0.41 93.9 131.94

b 0.001 b 0.001 b 0.001 b 0.001 0.639 0.538 b 0.001 0.52 b 0.001 b 0.001

247, 184, 222, 215, 176, 214, 207, 151, 144, 182,

250 187 225 218 179 217 210 154 147 185

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Fig. 2. Apoptotic TUNEL-positive nauplii spawned by Temora stylifera females fed with the oxylipin producing diatoms Skeletonema pseudocostatum (A,B) and Skeletonema marinoi (C, D) in fluorescent and transmitted light.

SD eggs female−1 day−1, mean as of day 8 to the end: 14.4 ± 5.7 SD eggs female−1 day−1) (Fig. 1A). In contrast, SM and SPC resulted in higher initial egg production rates that decreased sharply with time (mean days 2–7: 21.7 ± 9.6 SD and 26.4 ± 17.5 SD eggs female−1 day−1, respectively mean as of day 8 to the end: 4.5 ± 2.2 SD and 1.5 ± 2.7 SD eggs female−1 day−1, respectively), representing a decrease of 94.1% and 79.2% respectively, and the lowest egg production rates recorded in this set of experiments (Fig. 1A). 3.2. Egg viability Optimal hatching rates were observed on day 1 with all diets (mean 87.3% ± 12.6 SD, Fig. 1B), but decreased thereafter with all tested diatoms. Only PRO supported optimal egg hatching rates throughout the experiment (mean 89.9%± 8.9 SD). Using an ANCOVA analysis, egg

viability with PRO was significantly different compared to all other treatments (Table 3). With TR1 and TR2 diets egg hatching success decreased slowly (mean day 2–day 7 50.7% ± 7.3 SD and 19.4% ± 8.7 SD respectively; mean as of day 8 to the end: 37.6% ± 14 SD and 15.7%± 5.3 SD, respectively). There were statistically significant differences in egg viability between TR1 and TR2. SPC and SM induced strong egg hatching inhibition after the first few days, with a drastic negative trend (mean day 2–day 7 20.5% ± 20.18 SD and 22% ± 29.4 SD respectively; mean as of day 8 to the end: 4.6% ± 9.2 SD and 0%± 0 SD, respectively) reaching values of about 0% by day 5 (Fig. 1B). There were no differences in egg viability between TR2, SM and SPC (Table 3). 3.3. Apoptotic nauplii 88% of the nauplii that hatched after T. stylifera was fed SPC for 48 h were TUNEL-positive, indicating apoptotic tissues and imminent death

20 18

80

16 14

60

µ g mg C-1

Survival (% female survival)

100

40

12 10 8 6

20

4 2 0

0 1

2

3

4

5

6

7

8

9 10 11 12 13 14 15

Days Fig. 3. Female survival of Temora stylifera during reproduction experiments. Symbols: open circle (PRO), dark square (TR1), grey square (TR2), dark diamond (SM), and grey diamond (SPC).

TR1

TR2

SM

SPC

Algae Fig. 4. Ingestion rates and Manly's α coefficient of food selection of Temora stylifera on each alga during the whole feeding period (mean 4 days ± SD). Symbols: black bars (PRO) and white bars (TR1, TR2 and SPC, respectively).

A. Barreiro et al. / Journal of Experimental Marine Biology and Ecology 401 (2011) 13–19

17

Fig. 5. LC/MS profiles of phycooxylipins (PHOXYs) in Skeletonema pseudocostatum (A) and Thalassiosira rotula strain CCMP 1018 (B).

(Fig. 2A,B). Similarly, females fed with the known apoptotic-inducer SM after 48 h spawned 100% TUNEL-positive nauplii (Fig. 2C,D). No TUNEL-positive areas were observed in nauplii spawned by females fed the non-oxylipin producing control diet PRO (data not shown, see Ianora and Miralto (2010)). 3.4. Female survival PRO supported 100% survival throughout the experimental period, and diatoms, in contrast, showed lower values at the end of the experiment: 50%, 6.6%, 40.7%, and 30% respectively in TR1, TR2, SM and SPC (Fig. 3). An ANCOVA analysis with time (LN transformed) as covariate, female survival as dependent variable and algal treatment

20

as factor was performed with the set of treatments without including PRO. No significant differences were found (F3, 51 = 0.08, p = 0.97). 3.5. Ingestion rates and food selection Ingestion rates with each alga were quite similar between PRO, TR1 and TR2 (Fig. 4). The presence of SPC seemed to produce some kind of interference on feeding, since ingestion rates were simultaneously reduced with both PRO and SPC in that treatment. Food selection between each algae showed feeding behaviours close to a non selective pattern (α = 0.5) for PRO–TR1 and PRO–TR2. A Wilcoxon test showed that there were no statistical differences between pairs of Manly's α values, so they were not different from 0.5 (PRO–TR1 n = 12, Z = 0.94, p = 0.34; PRO–TR2 n = 14, Z = 0.47, p = 0.64; PRO–SPC n = 9, Z = 1.84, p = 0.06). This indicates that our results do not show a selective feeding behaviour.

µ g mg C-1

18 16

3.6. Metabolite analysis

14

In agreement with previous studies (d'Ippolito et al., 2002a,b; Wichard et al., 2005), octatrienal, octadienal and heptadienal were the main PUAs in SM and TR2, whereas TR1 was the only species to produce a decatrienal. The other oxylipins in TR1 and SM were identical to those previously described (d'Ippolito et al., 2005; Fontana et al., 2007a) and are not reported here. Eeicosapenatnoate-dependent 15-LOX products, such as 15S-hydroxy-5Z,8Z,11Z,13E,17Z-eicosapentaenoic acid (15S-HEPE) and 13-hydroxy-14,15-epoxyeicosa5Z,8Z,11Z,17Z-tetraenoic acid (13,14-HEpETE) and the aldehydecontaining compound 15-oxo-5Z,9E,11E,13E-pentadecatetraenoic acid (15-oxoacid) were predominant in SPC, with very small amounts of C16-derivatives (Fig. 5A). On the other hand, in TR2 9-LOX metabolism of C16 fatty acids was predominant, with 9-hexadeca6Z,10E,12Z-trienoic acid (9-HHTrE), 9-hexadeca-6Z,10E,12Z,15-tetraenoic acid (9-HHTE) and the corresponding epoxy alcohols, 11hydroxy-9,10-epoxy-hexadeca-6Z,12Z-dienoic (11,9-HEpHDE) and 11-hydroxy-9,10-epoxy-hexadeca-6Z,12Z,15-trienoic acid (11,9-

12 10 8 6 4 2 0 TR1

TR2

SM

SPC

Algae Fig. 6. Quantitative analysis of non volatile oxilipins (white bars) and PUAs (black bars) in SPC (Skeletonema pseudocostatum), SM (Skeletonema marinoi), TR1 (Thalassiosira rotula Strain CCMP 1647), and TR2 (Thalassiosira rotula Strain CCMP 1018). No LOX products were detectable in Prorocentrum minimum. (n ≥ 3 ± SD).

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HEpHTE), as the major products (Fig. 5B). Neither PUAs nor other oxylipins were detectable in PRO (data not shown). 3.7. Dosage of LOX products Non volatile oxylipins and PUA content normalised to total carbon are shown in Fig. 6. Production of oxylipins differed among the diatom species tested (one-way analysis of variance ANOVA, F3,11 = 7.8, p b 0.01). This difference was due to the much lower amount of nonvolatile oxylipins produced by TR1 (4.2 ± 2.4 μg mg C−1) compared to SM (13.2 ± 4.3 μg mg C−1) and SPC (10.0 ± 2.3 μg mg C−1), since both Skeletonema species and TR2 were not significantly different from each other (Tukey's post-hoc test, p N 0.05). Interestingly, PUAs accounted for 39% of the total end-products in SM (8.5 ± 2.5 μg mg C−1), and 68% of the total end-products in TR1 (9.0 ± 3.5 μg mg C−1). However, the PUA content in both diatoms was not statistically different from non-volatile oxylipin content (Student's t-test: t4 = 1.64, p N 0.05 for SM, and Student's t-test: t8 = 2.1, p = 0.07 for TR1, respectively), though at the edge of the p value for TR1. Cultures of TR2 that have been reported as either able or unable to synthesise PUAs (Pohnert et al., 2002; Wichard et al., 2005) showed very low levels of PUA production (1.2 ± 0.5 μg mg C−1) but much higher levels of other oxylipins (7.1 ± 1.6 μg mg C−1) (Student's t-test t4 = 6.2, p b 0.01). No PUAs were detected in SPC. 4. Discussion Results of ingestion experiments showed no clear evidence for selective feeding behaviour with PRO, TR1 and TR2 which were ingested in similar amounts, as were PRO and SPC but at much lower concentrations. Manly's coefficients were not significantly different thus indicating no selective behaviour even though with PRO + SPC there was a p value which was at the edge of statistical significance. Turner et al. (2001) also reported similar ingestion rates of TR1 and PRO when offered together, although food selection coefficients were not reported. All these findings suggest that the chemicals produced by diatom species do not protect them from their grazers if the alternative prey is not of high food quality, as may be in the case of PRO. Of course, field conditions will differ from this scenario because selective grazers may be present, as well as alternative good quality food. To date, little research has been conducted in the field to study copepod feeding behaviour in the presence of PUA-producing diatoms. The only study performed, by Leising et al. (2005a,b) showed a selective behaviour of Calanus pacificus, rejecting a PUA producing species of the genus Thalassiossira. TR1 and TR2 induced higher egg production in T. stylifera compared to PRO, SM and SPC (Fig. 1, Table 2). High egg production rates with diets of TR have already been reported in the past for this copepod species (Turner et al., 2001; Pohnert et al., 2002; Ceballos and Ianora, 2003). On the other hand, both SM and SPC induced low fecundity similar to previous studies (Ban et al., 1997; Ceballos and Ianora, 2003). Intermediate egg production rates with PRO could be explained by the poor quality of PRO as food (see review by Heil et al. (2005)) or by reduced “catchability” of the swimming PRO cells rather than the sinking TR2 and TR1 cells. Female survival did not show differences among the diatoms tested compared to the control diet PRO. On the contrary, egg viability in Temora was affected by all diatom species tested (Fig. 1, Table 3). SM was already known to produce a strong reduction in egg hatching (Ceballos and Ianora, 2003; Ianora et al., 2004; reviewed by Ianora and Miralto (2010)). SPC and TR2 had significantly stronger negative effects on hatching viability than TR1 (Table 3) in Temora. In Pohnert et al. (2002), on the contrary, TR2 (with no PUA production) and SPC did not induce a significant decrease in egg viability in another copepod, Calanus helgolandicus. To date, such contrasting results have been explained as a result of a different sensitivity of different species or strains of

copepods to PUAs (Ianora et al., 2003) and/or to variations in PUA production (Ribalet et al., 2007) in response to environmental or physiological conditions. In agreement with previous reports (Wichard et al., 2005; Fontana et al., 2007b; Dutz et al., 2008; Poulet et al. 2008), PUAs by themselves cannot explain reduced hatching success with all diatom diets. On the other hand, the negative effect of diatoms on copepod reproduction becomes much clearer when other LOX products, namely oxygenated long chain fatty acid derivatives (hydroxyacids and epoxyalcohols), are taken into consideration (Fontana et al., 2007b). Of the two species (SM and SPC) with strongest negative effects on copepod egg production and egg hatching success, only one, SM, was a PUA producer. In the absence of PUAs, the strong activity of SPC on copepod egg viability can be explained by the large quantities of other oxylipins, such as (5Z,8Z,11Z,13E,15S,17Z)-15-hydroxy-5,8,11,13,17-eicosapentaenoic acid (15S-HEPE) and 13,14-13R-hydroxy-14S,15S-trans-epoxyeicosa5Z,8Z,11Z,17Z-tetraenoic acid (13,14-HEpETE) and 15-oxo5Z,9E,11E,13E-pentadecatetraenoic acid (15-oxoacid), deriving from the oxidation of C20 fatty acids. Interestingly these metabolites were recently reported in the pennate diatom P. delicatissima which does not produce PUAs but only these other oxylipins (d'Ippolito et al. 2009). 15S-HEPE has already been shown to induce reproductive failure in T. stylifera (Ianora et al., 2010) whereas similar aldehydic 9oxo-nonadienoic and 12-oxo-dodecatienoic acids in Phaeodactylum tricornutum have been suggested to block cell divisions in sea urchin embryos (Pohnert et al., 2002). Here we confirm that diatoms such as SPC (S. pseudocostatum) that produce these oxylipins can induce negative effects comparable to those of SM (S. marinoi) even though they do not produce PUAs. Such a response confirms previous results with SM and two species of Choetoceros that were not able to synthesise PUAs (Fontana et al., 2007b). Also in the case of the two T. rotula strains, TR2 was more toxic even if it produced significantly lower quantities of PUAs compared to TR1. TR2 produced mainly 9-hexadecatrienoic acid (9-HHTrE), 9hexadecatetraenoic acid (9-HHTE) and the corresponding epoxy alcohols which were present in lower quantities in TR1, possibly explaining the stronger activity of TR2 on hatching success. Another interesting finding that has emerged from this study regards with the chemical diversity of oxylipins, not only at the species but also at the strain level, as already noted by Wichard et al. (2005) for PUAs. The observed genetic differences between the ITS regions of TR1 (CCMP 1647) and TR2 (CCMP1018) support results by Pohnert et al. (2002) that these two strains have been raised from genetically distinct individuals within T. rotula (Kooistra pers. comm.) This finding is not surprising because the strains originate from cells collected ca. 25 years apart in different oceans (TR1, Gulf of Naples, Tyrrhenian Sea, Nov. 1993; TR2, La Jolla, California, 1968). What is interesting is that different individuals exhibit different levels of bioactive compounds and different cocktails of these, as a result of different enzymatic pathways committed to their production. In terms of oxylipin uptake rates, Buttino et al. (2008) calculated that T. stylifera females feeding on decadienal-loaded liposomes ingested about 9.7 ng of decadienal daily to reduce hatching success after 10 days. Considering ingestion rates reported by Turner et al. (2001) in experiments performed with the same copepod species under similar conditions as in our egg production experiments (with the species T. rotula, TR1), 13.2 μg mg C−1 of total oxylipins for TR1 (see our Fig. 5) × 300 ng C ind−1 h−1 ingested (see Fig. 3 in Turner et al. (2001)) × 24 h corresponds to 95 ng of total oxylipins ingested ind−1 day−1, which is one order of magnitude higher than the concentrations that were active in Buttino et al. (2008). We do not have data for the other diatom species (genus Skeletonema), but the expected values should be similarly high. The copepods' inability to detect oxylipins may render diatomgrazer relationships unsuitable for co-evolution processes (Ianora et al., 2006). Instead, evolution of diatom toxins could be driven by kin

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selection, as these compounds protect the entire population against grazers by reducing the number of grazer offspring. In this case, the benefit would be on inclusive fitness (of genetically related individuals or clonal members) relatively nearby rather than exclusive fitness of individual cells or chains of genetically identical cells. In conclusion, the negative impact of diatoms is not due to a single class of molecules (i.e. PUAs), as previously believed, but rather to a cocktail of bioactive compounds, most of which have been discovered only recently. The “rapid response” production process of these compounds probably evolved to deter grazers (Ianora et al., 2006). Acknowledgements We are grateful to Mario di Pinto, Massimo Perna and Antonio Maiello for their technical assistance. A.B. thanks the Stazione Zoologica Anton Dohrn in Naples for providing general facilities. A.B. was funded with a FPU grant from the Spanish government. Funds were also obtained from the EU Network of Excellence Marine Biodiversity and Ecosystem Functioning (contract number GOCE-CT-2003-505446). [SS] References Ban, S., Burns, Castel J., Chaudron, Y., Christou, E., Escribano, R., Fonda Umani, S., Gasparini, S., Guerrero Ruiz, F., Hoffmeyer, M., Ianora, A., Kang, H.K., Laabir, M., Lacoste, A., Miralto, A., Ning, X., Poulet, S., Rodríguez, V., Runge, J., Shi, J., Starr, M., Uye, S.I., Wang, J., 1997. The paradox of diatom–copepod interactions. Mar. Ecol. Prog. Ser. 157, 287–293. Buttino, I., De Rosa, G., Carotenuto, Y., Mazzella, M., Ianora, A., Esposito, F., Vitiello, V., Quaglia, F., La Rotonda, M.I., Miralto, A., 2008. Aldehyde-encapsulating liposomes impair marine grazer survivorship. J. Exp. Biol. 211, 1426–1433. Carotenuto, Y., Ianora, A., Buttino, I., Romano, G., Miralto, A., 2002. is postembyonic development in the copepod Temora stylifera negatively affected by diatom diets? J. Exp. Mar. Biol. Ecol. 276, 49–66. Ceballos, S., Ianora, A., 2003. Different diatoms induce contrasting effects on the reproductive success of the copepod Temora stylifera. J. Exp. Mar. Biol. Ecol. 294, 189–202. Chaudron, Y., Poulet, S.A., Laabir, M., Ianora, A., Miralto, A., 1996. Is hatching success of copepod eggs diatom density-dependent? Mar. Ecol. Prog. Ser. 144, 185–193. D'Ippolito, G., Romano, G., Iadicicco, O., Miralto, A., Ianora, A., Cimino, G., Fontana, A., 2002a. New birth-control aldehydes from the marine diatom Skeletonema costatum: characterization and biogenesis. Tetrahedron Lett. 43, 6133–6136. D'Ippolito, G., Iadicicco, O., Romano, G., Fontana, A., 2002b. Detection of short-chain aldehydes in marine organisms: the diatom Thalassiossira rotula. Tetrahedron Lett. 43, 6137–6140. D'Ippolito, G., Cutignano, A., Briante, R., Febbraio, F., Cimino, G., Fontana, A., 2005. New C16 fatty-acid-based oxylipin pathway in the marine diatom Thalassiosira rotula. Org. Biomol. Chem. 3, 4065–4070. Dutz, J., Koski, M., Jónasdóttir, S.H., 2008. Copepod reproduction is unaffected by diatom aldehydes or lipid composition. Limnol. Oceanogr. 53, 225–235. Fontana, A., D'Ippolito, G., Cutignano, A., Romano, G., Ianora, A., Miralto, A., Cimino, G., 2007a. Chemistry of oxylipins pathways in marine diatoms. Pure Appl. Chem. 79 (4), 481–490.

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Fontana, A., D'Ippolito, G., Cutignano, A., Romano, G., Lamari, N., Massa-Gallucci, A., Cimino, G., Miralto, A., Ianora, A., 2007b. LOX-induced lipid peroxidation mechanism responsible for the detrimental effect of marine diatoms on zooplankton grazers. Chembiochem 8 (15), 1810–1818. Frost, B.W., 1972. Effects of size and concentration of food particles on the feeding behaviour of the marine planktonic copepod Calanus pacificus. Limnol. Oceanog. 17, 805–815. Heil, C.A., Glibert, P.M., Fan, C., 2005. Prorocentrum minimum (Pavillard) Schiller. A review of a harmful algal bloom species of growing worldwide importance. Harmful. Algae. 4, 449–470. Ianora, A., Miralto, A., 2010. Toxigenic effects of diatoms on grazers, phytoplankton and other microbes: a review. Ecotoxicology 19, 493–511. Ianora, A., Scotto di Carlo, B., Mascellaro, P., 1989. Reproductive biology of the planktonic copepod Temora stylifera. Mar. Biol. 101, 187–194. Ianora, A., Poulet, S.A., Miralto, A., 2003. The effects of diatoms on copepod reproduction: a review. Phycologia 42 (4), 351–363. Ianora, A., Miralto, A., Poulet, S., Carotenuto, Y., Buttino, I., Romano, G., Casotti, R., Pohnert, G., Wichard, T., Colucci-D'Amato, L., Terrazzano, G., Smetacek, V., 2004. Aldehyde suppression of copepod recruitment in blooms of a ubiquitous planktonic diatom. Nature 429, 403–407. Ianora, A., Boersma, M., Casotti, R., Esposito, F., Harder, J., Hoffmann, F., Pavia, H., Potin, P., Poulet, S.A., Toth, G., 2006. New trends in marine chemical ecology. Estuaries Coasts 29 (4), 531–551. Ianora, A., Romano, G., Carotenuto, Y., Esposito, F., Roncalli, V., Buttino, I., Miralto, A., 2010. Impact of the diatom oxylipin 15S-HEPE on the reproductive success of the copepod Temora stylifera. Hydrobiologia 666 (1), 265–275. Kang, H.K., Poulet, S.A., 2000. Reproductive success in Calanus helgolandicus as a function of diet and egg cannibalism. Mar. Ecol. Prog. Ser. 201, 241–250. Leftley, J.W., Keller, D.K., Selvin, R.C., Claus, W., Guillard, R.R.L., 1987. Media for the culture of oceanic ultraphytoplankton. J. Phycol. 23, 633–638. Leising, A.W., Pierson, J.J., Halsband-Lenk, C., Horner, R., Postel, J., 2005a. Copepod grazing during spring blooms: does Calanus pacificus avoid harmful diatoms? Prog. Oceanog. 67, 384–405. Leising, A.W., Pierson, J.J., Halsband-Lenk, C., Horner, R., Postel, J., 2005b. Copepod grazing during spring blooms: can Pseudocalanus newmani induce trophic cascades? Prog. Oceanog. 67, 406–421. Manly, B.F.J., 1974. A model for certain types of selection experiments. Biometrics 30, 281–294. Miralto, A., Barone, G., Romano, G., Poulet, S.A., Ianora, A., Russo, G.L., Buttino, I., Mazzarella, G., Laabir, M., Cabrini, M., Giacobbe, M.G., 1999. The insidious effect of diatoms on copepod reproduction. Nature 402, 173–176. Pohnert, G., 2005. Diatom/Copepod interactions in plankton: the indirect chemical defence of unicellular algae. Chembiochem 6, 1–14. Pohnert, G., Lumineau, O., Cueff, A., Adolph, S., Cordevant, C., Lange, M., Poulet, S., 2002. Are volatile unsaturated aldehydes from diatoms the main line of chemical defence against copepods? Mar. Ecol. Prog. Ser. 245, 33–45. Ribalet, F., Wichard, T., Pohnert, G., Ianora, A., Miralto, A., Casotti, R., 2007. Age and nutrient limitation enhance polyunsaturated aldehyde production in marine diatoms. Phytochemistry 68 (15), 2059–2067. Turner, J.T., Ianora, A., Miralto, A., Laabir, M., Esposito, F., 2001. Decoupling of copepod grazing rates, fecundity and egg-hatching success on mixed and alternating diatom and dinoflagellate diets. Mar. Ecol. Prog. Ser. 220, 187–199. Wichard, T., Poulet, S., Halsband-Lenk, Albaina A., Harris, R., Liu, D., Pohnert, G., 2005. Survey of the chemical defence potential of diatoms: screening of fifty one species for α, β, γ, δ-unsaturated aldehydes. J. Chem. Ecol. 31, 949–958.