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PUFAs and PUAs production in three benthic diatoms from the northern Adriatic Sea
Phytochemistry 142 (2017) 85e91
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PUFAs and PUAs production in three benthic diatoms from the northern Adriatic Sea Laura Pezzolesi a, *, Salvatore Pichierri b, Chiara Samorì c, Cecilia Totti b, Rossella Pistocchi a di Bologna, via Sant'Alberto 163, 48123, Ravenna, Italy Dipartimento di Scienze Biologiche, Geologiche e Ambientali - Universita Politecnica delle Marche, via Brecce Bianche, 60131, Ancona, Italy Dipartimento di Scienze della Vita e dell'Ambiente - Universita c di Bologna, via Selmi 2, 40126, Bologna, Italy Dipartimento di Chimica “Giacomo Ciamician” - Universita a
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 March 2017 Received in revised form 27 June 2017 Accepted 30 June 2017
The production of polyunsaturated aldehydes (PUAs) has been reported by many planktonic diatoms, where they have been implicated in deleterious effects on copepod reproduction and growth of closeby microbes or suggested as infochemicals in shaping plankton interactions. This study investigates the production of PUAs by diatoms commonly occurring in the microphytobenthic communities in temperate regions: Tabularia affinis, Proschkinia complanatoides and Navicula sp. Results highlight the production of PUAs by the three benthic diatoms during stationary and decline phases, with intracellular concentrations from 1.8 to 154.4 fmol cell1, which are within the range observed for planktonic species. The existence of a large family of PUAs, including some with four unsaturations, such as decatetraenal, undecatetraenal and tridecatetraenal, was observed. Since particulate and dissolved PUAs were positively correlated, together with cell lysis, equivalent concentrations may be released during late growth stages, which may affect benthic invertebrates grazing on them and other microalgae. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Proschkinia complanatoides Tabularia affinis Navicula sp. Proschkiniaceae Ulnariaceae Naviculaceae Benthic diatoms GC-MS Fatty acids Polyunsaturated aldehydes
1. Introduction Aquatic organisms have been shown to produce several chemical compounds responsible for the regulation of numerous biological processes (Gross, 2003; Ianora and Miralto, 2010; Ianora et al., 2012). These molecules include various specialised metabolites that are not directly involved in the control of basic life mechanisms (Demain and Fang, 2000), i.e. low-molecular weight organic compounds such as organic acids, phenolic substances, alkaloids, nitrogen-containing compounds, and polyunsaturated aldehydes (PUAs) (Ianora et al., 2011). Recently, several studies have been paying increasing attention to PUAs due to their deleterious effect on fecundity and/or hatching success of copepods (Ianora ^ et al., 2014; Lavrentyev et al., 2015; Miralto et al., 2003, 2012; Ka et al., 1999; Wichard et al., 2007), described as the “paradox of diatom-copepod interactions” by Ban et al. (1997). In addition,
* Corresponding author. E-mail address: [email protected] (L. Pezzolesi). http://dx.doi.org/10.1016/j.phytochem.2017.06.018 0031-9422/© 2017 Elsevier Ltd. All rights reserved.
other important ecological effects have been reported for PUAs, such as competitive inhibition of algal growth (Ribalet et al., 2007a), antibacterial activity (Ribalet et al., 2008), bloom termination (Ribalet et al., 2014; Vidoudez et al., 2011) and intrapopulation signaling (Casotti et al., 2005; Vardi et al., 2006). These volatile aldehydes are oxygenated derivatives of fatty acids (oxylipins) widely produced by plants (Delory et al., 2016), macrophytes (Alsufyani et al., 2014), cyanobacteria (Watson, 2003) and microalgae (d'Ippolito et al., 2009; Ianora et al., 2012; Wichard et al., 2005a). Diatoms are known to contribute about 40% of the marine primary production, constituting half of entire organic material produced in the planet (Rousseaux and Gregg, 2014). Interestingly, under certain conditions, diatoms produce the highest amounts of PUAs among phytoplankton groups (Leflaive and Ten-Hage, 2009). Wichard et al. (2005a) reported a PUA-release by 27 of the 71 algal isolates, with concentrations ranging from 0.01 to 9.8 fmol per cell. In addition to being strain- and speciesspecific (Wichard et al., 2005a), PUAs production and composition can also depend on environmental factors, such as growth stage and nutrient limitation (Pohnert, 2002; Ribalet et al., 2007b).
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PUAs are produced from an oxidative degradation of free polyunsaturated fatty acids (PUFAs), synthesized in chloroplasts and plasmatic membranes by lipase-mediated hydrolysis of glycolipids and phospholipids that occurs after cell disruption (Pohnert, 2000). While describing the origin of PUAs produced by Skeletonema costatum with labeled precursors, d'Ippolito et al. (2004) showed that the metabolism of glycolipids leads to the production of hexadecatrienoic acid (C16:3), hexadecatetraenoic acid (C16:4) and eicosapentaenoic acid (C20:5) which are the direct precursors of octadienal (C8:2), octatrienal (C8:3) and heptadienal (C7:2), respectively, while phospholipids are involved only in the production of heptadienal. The same pool of PUFAs can be a precursor of different PUAs, such as C20:5 acid for decatrienal (C10:3), as observed in Thalassiosira rotula (Barofsky and Pohnert, 2007). Despite the clear correlation between PUFA and PUA composition, the variety of aldehydes that a species is capable of producing depends on its pool of lipoxygenases (LOXs) (Cutignano et al., 2011). These molecules are iron nonheme enzymes that can catalyse the addition of molecular oxygen to the carbon chain of PUFAs, giving fatty acid hydroperoxides (FAHs) as intermediates (d'Ippolito et al., 2009). LOXs have a species-dependent specificity and are responsible for further breaking down FAHs into several derivatives, i.e. PUAs, mediated by hydroperoxide-lyase (HPLs) (d'Ippolito et al., 2009). Vidoudez and Pohnert (2008) observed concentrations of dissolved PUAs during the stationary-early declining phase of diatom cultures and indicated that PUAs can be released by cells independently from grazing, thus affecting surrounding organisms and potentially acting as infochemicals. However, although the pathways and the effect of these oxylipins have been widely investigated for various photosynthetic organisms (Ianora and Miralto, 2010; Ribalet et al., 2007a), almost all of the diatoms investigated belong to the planktonic domain and only a few studies have evidenced PUAs production by benthic diatoms (Jüttner et al., 2010; Scholz and Liebezeit, 2012). The microphytobenthos assemblages can be highly productive (Pinckney and Zingmark, 1993) and benthic primary producers represent an important source of organic carbon in some littoral areas (Allen et al., 2016). The real capacity of benthic organisms to produce PUAs, as well as the effect that these compounds may have on the same benthic ecosystems remain largely unknown. In the present study, we investigated the production of PUFAs and PUAs in three of the most common benthic diatoms (Proschkinia complanatoides (Hustedt ex R.Simonsen) D.G.Mann, Proschkiniaceae; Tabularia affinis (Kützing) Snoeijs, Ulnariaceae and Navicula sp., Naviculaceae) of the Conero Riviera (northern Adriatic Sea, Italy) (Accoroni et al., 2016), providing the production of some compounds that have not yet been reported.
Fig. 1. Cell abundances of Proschkinia complanatoides, Tabularia affinis and Navicula sp. along the growth curve. Data are means of independent replicates (n ¼ 2) and the error bars represent standard deviations.
relevant from day 14 in each species. The highest values were observed in Tabularia affinis (1.17 ± 0.30 ng cell1) and Navicula sp. (0.92 ± 0.05 ng cell1) during the decline phase (day 44). Similarly, the highest fatty acid concentration in Proschkinia complanatoides was reached at day 44 but the total amount never exceeded 0.12 ± 0.01 ng cell1, about 10-times lower than the other species. Generally, the contribution of unsaturated fatty acids resulted higher than that of saturated chains along the growth curve for all the tested species (Fig. 2). Total fatty acids ranged between 1.4 (day 14) and 6.0 (day 37) mg mL1 in P. complanatoides, 2.1 (day 14) and 32.9 (day 35) mg mL1 in T. affinis and between 3.8 (day 14) and 26.5 (day 35) mg mL1 in Navicula sp., while the maximum concentration of unsaturated compounds in the three diatoms was 4.1, 20.2 and 17.9 mg mL1, respectively (Table 1). From a qualitatively point of view, C16 and C20 fatty acids were the prominent compounds in all the species (Fig. 3). In P. complanatoides culture, hexadecatrienoic acid (C16:3) resulted the main PUFA at the beginning of the growth (0.014 ± 0.003, day 14), while hexadecenoic acid (C16:1), and eicosapentaenoic acid (EPA, C20:5) were the most abundant compounds at day 44, reporting maximum values of 0.017 ± 0.002 and 0.019 ± 0.002 ng cell1, respectively (Fig. 3a). C18 unsaturated fatty acids (oleic acid C18:1, linoleic acid C18:2 and linolenic acid C18:3) were also observed along the growth curve, but they were less abundant than C16 and C20 species. A comparable PUFAs composition was observed in
2. Results 2.1. Algal growth The growth curves of Tabularia affinis, Proschkinia complanatoides and Navicula sp. obtained in this study are illustrated in Fig. 1. The curves were characterized by an exponential growth phase until day 9 for T. affinis and Navicula sp. and until day 7 for P. complanatoides. A stationary phase until day 35 for Navicula sp. and T. affinis, until day 37 for P. complanatoides and a subsequent decline phase (from day 35 for T. affinis and Navicula sp. and from day 37 for P. complanatoides) were observed. The highest cell abundances reached were 61 ± 5.6 103, 63 ± 0.8 103 and 57 ± 7.9 103 cells mL1 for P. complanatoides, Navicula sp. and T. affinis, respectively. 2.2. Fatty acids analysis The production of saturated and unsaturated fatty acids became
Fig. 2. Saturated and unsaturated (mono- plus poly-) fatty acids production in Proschkinia complanatoides, Tabularia affinis and Navicula sp. at different days (14, 21, 28, 35, 37, 44). Data are means of independent replicates (n ¼ 2).
L. Pezzolesi et al. / Phytochemistry 142 (2017) 85e91 Table 1 Total and mono- plus polyunsaturated (UFAs) fatty acid concentrations in Proschkinia complanatoides, Tabularia affinis and Navicula sp. at different days (14, 21, 28, 35, 37, 44). UFAs (mg mL1) Proschkinia complanatoides day 14 0.9 day 21 2.2 day 28 3.4 day 35 4.0 day 37 4.1 day 44 2.9 Tabularia affinis day 14 1.3 day 21 3.9 day 28 9.8 day 35 20.2 day 37 13.7 day 44 19.5 Navicula sp. day 14 2.2 day 21 5.8 day 28 6.6 day 35 17.9 day 37 6.9 day 44 14.2
Total fatty acids (mg mL1) 1.4 3.2 5.1 5.8 6.0 4.7 2.1 6.3 16.5 32.9 20.7 31.2 3.8 10.9 12.0 26.5 9.6 19.8
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were revealed only in T. affinis (Fig. 3b and c). Regarding the saturated fatty acids, hexadecanoic acid (palmitic acid, C16:0) was the main compound produced by P. complanatoides and Navicula sp. while tetradecanoic acid (myristic acid, C14:0) the one by T. affinis (data not shown).
2.3. Aldehydes analysis The results of aldehydes quantification, that includes saturated and unsaturated compounds, are shown in Fig. 4. Some aldehydes were not identified as their fragmentation pattern in GC-MS resulted very complex and not comparable with the commercial standards or with compounds present in the NIST libraries. No aldehydes were detected until day 28 in each diatom culture, then a marked increase of these compounds was observed in two of the tested cultures, except for Proschkinia complanatoides. The maximum amount was achieved at day 28 for P. complanatoides (0.61 ± 0.43 pg cell1), 44 for Tabularia affinis (0.67 ± 0.15 pg cell1) and 44 for Navicula sp. (20.44 ± 4.03 pg cell1) (Fig. 4). Moreover, the percentage of unsaturated compounds on the total aldehydes was higher in Navicula sp. (20.4e95.5%) than in P. complanatoides (64.2e67.5%) and T. affinis (61.7e74.1%) cultures. Total aldehydes, in fact, ranged between 0.016 (day 44) and 0.036 (day 28) mg mL1 in P. complanatoides, 0.018 (day 28 and 44) and 0.024 (day 37) mg mL1 in T. affinis and between 0.078 (day 28) and 0.441 (day 44) mg mL1 in Navicula sp., while unsaturated aldehydes reported maximum concentrations of 0.023, 0.015 and 0.421 mg mL1 at day 28, 37 and 44 in the three diatoms, respectively (Table 2). From a qualitative point of view, medium-chained PUAs were the most abundant aldehydes produced by Navicula sp. (Fig. 5c). Although hexadienal (C6:2), octadienal (C8:2), decatetraenal (C10:4), undecatetraenal (C11:4), undecapentenal (C11:5) and tridecatetraenal (C13:4) were also detected, octenal (C8:1) was the most abundant compound (93.8%) corresponding to 18.40 ± 3.34 pg cell1 at day 44. On the contrary, PUAs composition of the other two species was more variable, but results were similar between the two diatoms: hexadienal (C6:2) contributed up to 50.56% and 39.04%, for T. affinis and for P. complanatoides, respectively (Fig. 5a and b). Decatetraenal (C10:4) reached a maximum value of 0.09 ± 0.01 pg cell1 for T. affinis and 0.13 ± 0.04 pg cell1
Fig. 3. Mono- and polyunsaturated fatty acids production in (a) Proschkinia complanatoides, (b) Tabularia affinis and (c) Navicula sp. at different days (14, 21, 28, 35, 37, 44). Data are means of independent replicates (n ¼ 2) and the error bars represent standard deviations. Hexadecenoic acid (C16:1), hexadecadienoic acid (C16:2), hexadecatrienoic acid (C16:3), octadecenoic acid (C18:1), octadecadienoic acid (C18:2), octadecatrienoic acid (C18:3), eicosatetraenoic acid (C20:4), eicosapentaenoic acid (C20:5) and docosahexaenoic acid (C22:6).
Navicula sp. and T. affinis; C16:1 acid was the main compound in both cultures, with the highest concentrations reached at day 44 (0.503 ± 0.178 ng cell1 for T. affinis and 0.564 ± 0.035 ng cell1 for Navicula sp.). Trace amounts of C18 fatty acids (C18:1 and C18:3)
Fig. 4. Saturated and unsaturated (mono- plus poly-) aldehydes production in Proschkinia complanatoides (left Y axis), Tabularia affinis (left Y axis) and Navicula sp. (right Y axis) at different days (28, 35, 37, 44). Data are means of independent replicates (n ¼ 2).
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Table 2 Total and mono- plus polyunsaturated (UAs) aldehyde concentrations in Proschkinia complanatoides, Tabularia affinis and Navicula sp. at different days (28, 35, 37, 44). UAs (mg mL1)
UAs (pmol mL1)
Proschkinia complanatoides day 28 0.023 188.3 day 35 0.012 107.9 day 37 0.019 153.9 day 44 0.011 96.7 Tabularia affinis day 28 0.014 126.0 day 35 0.013 120.2 day 37 0.015 131.4 day 44 0.011 101.6 Navicula sp. day 28 0.016 135.4 day 35 0.225 1771.9 day 37 0.383 3011.7 day 44 0.421 3331.6
Total aldehydes (mg mL1)
Total aldehydes (pmol mL1)
0.036 0.018 0.029 0.016
273.6 150.1 223.4 134.7
0.018 0.021 0.024 0.018
161.3 187.9 205.7 154.2
0.078 0.243 0.397 0.441
705.7 1925.5 3107.3 3441.0
for P. complanatoides at day 44 and 28, respectively. 3. Discussion The intracellular unsaturated aldehydes concentration ranged from 1.8 to 154.4 fmol cell1, varying either with the algal stage and among the diatom species. Such values are within the range observed for other planktonic species (Wichard et al., 2005a,b), such as Skeletonema marinoi (d'Ippolito et al., 2004; Ribalet et al., 2007b, 2009; Vidoudez and Pohnert, 2008) and Melosira
Fig. 5. Mono- and polyunsaturated aldehydes production in (a) Proschkinia complanatoides, (b) Tabularia affinis and (c) Navicula sp at different days (28, 35, 37, 44). Data are means of independent replicates (n ¼ 2) and the error bars represent standard deviations. Hexadienal (C6:2), octenal (C8:1), octadienal (C8:2), octatrienal (C8:3), nonatetraenal (C9:4), decatetraenal (C10:4), undecatetraenal (C11:4), undecapentenal (C11:5) and tridecatetraenal (C13:4). Right Y axis in Navicula sp. refers only to C8:1.
nummuloides (Taylor et al., 2007). A survey performed by Wichard et al. (2005a) on five Navicula species did not reveal the production of any PUAs; however, the authors observed that PUAs production within different isolates of some species ranged widely and depended on the growth phase of the culture, as later confirmed by other studies (Ribalet et al., 2007b; Vidoudez and Pohnert, 2008). More recently, the variability in PUAs production has been confirmed also by Scholz and Liebezeit (2012), which report aldehydes in cultures of benthic intertidal diatoms of the southern North Sea, including Navicula phyllepta. Moreover, most of the studies focused only on the production of some common PUAs, such as heptadienal, octadienal, octatrienal, decadienal and decatrienal (i.e., d'Ippolito et al., 2004; Jüttner et al., 2010; Ribalet et al., 2007b, 2009; Taylor at al., 2007; Vidoudez and Pohnert, 2008; Wichard et al., 2005a,b). However, d'Ippolito et al. (2002) suggested the existence of a large family of PUAs involved in the chemical interactions within planktonic communities. A similarly wide range of production was also found in the benthic community in our study. We thus confirm for the first time the high potential in the production of these metabolites in three benthic diatoms: a total of 9 compounds were identified, and additional PUAs remain unknown. Although heptadienal represents one of the most common PUAs released by planktonic diatoms (Wichard et al., 2005a), based on the time-frame where C7 aldehydes are typically detected by GCMS and due to the lack of peak signals in the present study, we exclude the potential production of this aldehyde by Proschkinia complanatoides, Tabularia affinis and Navicula sp. Other studies have focused on the effects of the PUAs octadienal and decadienal, which are commonly reported in other algal species (e.g. Skeletonema costatum, d'Ippolito et al., 2004; Thalassiosira rotula, Wichard et al., 2005a,b). In this study, these PUAs were not observed either, except for low levels of octadienal in Navicula sp. Our results highlight that a variety of PUAs can be produced and released by diatoms, including some with four unsaturations, such as decatetraenal, undecatetraenal and tridecatetraenal. In a previous study, a positive correlation between the length of PUAs chain and their bioactivity was observed (Adolph et al., 2003; Hansen et al., 2004; Pichierri et al., 2016; Ribalet et al., 2007a), thus these compounds (i.e., C10:4, C11:4 and C13:4) could have considerable effects on the benthic community, in particular at the concentrations produced by Navicula sp; however further investigations to better understand their toxic effects on benthic organisms are needed. A difference between intracellular and released PUA amounts was observed during a PUA surface mapping performed in the Adriatic Sea (Vidoudez et al., 2011), where the concentrations of dissolved PUAs never reached those released from ruptured cells. More recently, a field study performed in the northern Adriatic Sea during different years reported total particulate and dissolved PUA concentrations from 0 to 5.37 fmol cell1 and from 0 to 2.53 nM, respectively (Ribalet et al., 2014). These authors observed a positive correlation between particulate and dissolved PUAs, and cell lysis, suggesting that PUAs are released in the surrounding seawater upon cell rupture. Intracellular levels of PUAs found in the present study suggest a significant release by the three benthic diatoms, particularly by Navicula sp., with a major release during the late growth stage which may have an effect on grazers or, for instance, on meiobenthic organisms. The fatty acids analysis performed on the three species revealed a composition mainly constituted by myristic and palmitic acid (C14:0 and C16:0) among saturated acids, and C16-chains and eicosapentaenoic acid (C20:5, EPA) among unsaturated ones; low amounts of stearic acid (C18:0) and other C18 fatty acids were detected. These results appear consistent with fatty acid profiles reported for diatoms and other microalgae (Jüttner, 2001; Medina
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et al., 1998; Wichard et al., 2007). Dunstan et al. (1994) described EPA as the main PUFA in 14 diatom species, and high amounts of palmitoleic acid (C16:1) were measured in 28 microalgae by other authors (Viso and Marty, 1993). On the contrary, although hexadecatetraenoic acid (16:4) was among the main PUFAs identified in diatoms (Jüttner, 2001; Wichard et al., 2007), no traces of this compound were observed in each of the tested diatoms. Fatty acids concentrations increased with culture age, particularly for EPA, which has been previously found to increase during the stationary growth phase (Stonik and Stonik, 2015). Despite the presence of PUFAs in each growth phase (except for the lag one) and their decisive function in the PUAs metabolic pathway as precursors, the aldehydes production clearly occurred only during the stationary and decay growth phases in each of the three tested diatoms. In addition, no clear correlation between PUAs and PUFAs content has been observed, as different patterns were observed during the growth phases, confirming that the fatty acid decrease cannot be entirely attributed to the production of PUAs (Wichard et al., 2007). This result could be due to an inactivation state of those enzymes essential for catalyzing biochemical transformations, such as phospholipases that make PUFAs available for lipoxygenase/hydroperoxide lyases oxidation. In fact, enzyme activity, together with available lipid resources, seem to control the absolute and relative amount of PUAs, as previously suggested by other authors (Ribalet et al., 2007b). This may explain why in Navicula sp. and T. affinis we observed on one hand similar PUFA amounts, and on the other hand, differences in PUFAs composition and in PUAs concentration and composition. As observed for other diatom species, the diffusion of woundactivated compounds, such as PUAs, may indicate abundance of possible food, thus attracting some invertebrates in search for food, or serve as a repellent for other invertebrates, such as crustaceans, providing a chemical defense towards the grazing (Jüttner, 2005; Jüttner et al., 2010; Romano et al., 2010). This may in turn lead to variable effects on individual benthic invertebrate species and to the formation of complex behavioural reactions among benthic diatoms and their associated invertebrates, especially if considering the variety of PUAs compounds found in the present study and their variable toxicity, which has been reported either to vary among the different PUAs (i.e. Pichierri et al., 2016; Ribalet et al., 2007a) and to be target-specific (Gallina et al., 2014). In addition, PUAs produced by benthic diatoms could also affect the population dynamics of cooccurring microalgae and/or regulate the natural bloom successions, including toxic bloom-forming species (i.e. Ostreopsis spp., which are usually blooming in this area). In conclusion, these results underline the importance of designing studies to understand the interactions which occur between chemical signals and the various organisms, especially in benthic biofilms which are particular micro-environments where species are exposed to more concentrated chemicals exuded by neighbouring cells (Allen et al., 2016). Field studies are needed to better clarify the role of these specialised metabolites in the structure of the microphytobenthic communities. 4. Experimental 4.1. Reagents All reagents were purchased from Sigma-Aldrich (Milan, Italy) and used without further purification. 4.2. Cultivation and sampling The diatom strains, Proschkinia complanatoides (Hustedt ex R.Simonsen) D.G.Mann family Proschkiniaceae (strain PCAPS0313),
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Tabularia affinis (Kützing) Snoeijs family Ulnariaceae (strain TAAPS0313) and Navicula sp. family Naviculaceae (strain NAPS0313), were obtained from microphytobenthos samples collected before summer period in 2013 in the Passetto station (Conero Riviera, Italy, northern Adriatic Sea, 43 370 0900 N, 13 3105400 E), which is a semi-enclosed and shallow (mean depth 2 m) area, sheltered by a natural reef and characterized by a mostly rocky benthic surface. Single cells were isolated by the capillary pipette method (Hoshaw and Rosowski, 1973) using an inverted microscope; monoclonal cultures were set up using multi-well plates and were grown using f/2 medium (Guillard, 1978) with a salinity of 30. Cultures were maintained at 20 ± 1 C under a 16:8 L:D photoperiod and irradiance of 100e110 mmol m2 s1. Two replicate flasks (final culture volume of 2000 mL) for each diatom were established and maintained in a growth chamber. The experiment lasted 44 days and an aliquot of 200 mL was collected from each flask at the sampling days 3, 7, 9, 14, 21, 28, 35, 37 and 44. The algal aliquot was centrifuged at 3000 x g for 10 min to collect the algal pellet for PUFAs and PUAs extraction and analysis. 4.3. Fatty acids analysis The determination and quantification of PUFAs were carried out following the method of Samorì et al. (2012). Gas chromatographyemass spectrometry (GC-MS) analyses were performed using a 6850 Agilent HP gas chromatograph connected to a 5975 Agilent HP quadrupole mass spectrometer. The injection port temperature was 280 C. Analytes were separated by a HP-5 fused-silica capillary column (stationary phase poly[5% diphenyl/95% dimethyl]siloxane, 30 m, 0.25 mm i.d., 0.25 mm film thickness), with helium as the carrier gas (at constant pressure, 33 cm s1 linear velocity at 200 C). Mass spectra were recorded under electron ionization (70 eV) at a frequency of 1 scan s1 within the 12-600 m/z range. The temperature of the column was increased from 50 C up to 180 C at 50 C min1, then from 180 C up to 300 C at 5 C min1. Methyl nonadecanoate was utilized as internal standard for quantification of free and bounded fatty acids converted into fatty acid methyl esters (FAMEs). The relative response factor used for the quantitation was obtained by injecting solutions of known amounts of methyl nonadecanoate and commercial FAMEs mixture. Each analysis was repeated in duplicate. 4.4. Aldehydes analysis The quantification of PUA concentration was carried out following the method of Wichard et al. (2005b). After removing the supernatant, 1 mL of derivatisation reagent (O-(2,3,4,5,6pentafluorobenzyl) hydroxylamine hydrochloride, PFBHA HCl, 25 mM in Tris/HCl 100 mM, pH 7) and 25 mL of internal standard (0.968 mM benzaldehyde in hexane) were added to the algal pellet. The sample was cooled at 4 C, then ultrasonicated for 1 min and finally incubated at room temperature for 30 min. 0.5 mL of methanol and 1 mL of hexane were added to the sample that was vortexed for 1 min and acidified with 20 mL of H2SO4. After that, the hexane upper layer was removed, dehydrated over Na2SO4, evaporated under N2 and resuspended in 50 mL hexane. PUA concentration was determined by GC-MS (same equipment described in 4.3 Fatty acids analysis); the molecular identification was done by comparison of retention times and mass spectra with those of commercial standards when possible: propionaldehyde, 2,4heptadienal, octanal, 2-octenal, 2,4-octadienal, 6-nonenal, 2,6nonadienal, 4-decenal, 2,4-decadienal, undecanal, 8-undecenal and 2,4-undecadienal. If PUAs standards were not available, the identification was performed by comparison with NIST libraries.
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Each analysis was repeated in duplicate. Note The authors declare no competing financial interest. Acknowledgements This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. We would like to thank the anonymous reviewers for their careful reading of our manuscript and the insightful comments, which led us to considerably improve the work. We wish to thank Dr. Tiziana Romagnoli for providing the SEM images and Dr. Veronica Lo for English revision. References Accoroni, S., Romagnoli, T., Pichierri, S., Totti, C., 2016. Effects of the bloom of harmful benthic dinoflagellate Ostreopsis cf. ovata on the microphytobenthos community in the northern Adriatic Sea. Harmful Algae 55, 179e190. Adolph, S., Poulet, S.A., Pohnert, G., 2003. Synthesis and biological activity of a, b, g, d-unsaturated aldehydes from diatoms. Tetrahedron 59, 3003e3008. Allen, J.L., Ten-Hage, L., Leflaive, J., 2016. Allelopathic interactions involving benthic phototrophic microorganisms. Environ. Microbiol. Rep. 8, 752e762. Alsufyani, T., Engelen, A.H., Diekmann, O.E., Kuegler, S., Wichard, T., 2014. Prevalence and mechanism of polyunsaturated aldehydes production in the green tide forming macroalgal genus Ulva (Ulvales, Chlorophyta). Chem. Phys. Lipids 183, 100e109. Ban, S., Burns, C., Castel, C., Christou, E., Escribano, R., Fonda Umani, F., Gasparini, S., Guerrero Ruiz, F., 1997. The paradox of diatom-copepod interactions. Mar. Ecol. Prog. Ser. 157, 287e293. Barofsky, A., Pohnert, G., 2007. Biosynthesis of polyunsaturated short chain aldehydes in the diatom Thalassiosira rotula. Org. Lett. 9, 1017e1020. Casotti, R., Mazza, S., Brunet, C., Vantrepotte, V., Ianora, A., Miralto, A., 2005. Growth inhibition and toxicity of the diatom aldehyde 2-trans, 4-trans-decadienal on Thalassiosira weissflogii (Bacillariophyceae). J. Phycol. 41, 7e20. Cutignano, A., Lamari, N., d'ippolito, G., Manzo, E., Cimino, G., Fontana, A., 2011. Lipoxygenase products in marine diatoms: a concise analytical method to explore the functional potential of oxylipins. J. Phycol. 47, 233e243. Delory, B., Delaplace, P., Jardin, P., Fauconnier, M., 2016. Barley (Hordeum distichon L.) roots synthesise volatile aldehydes with a strong age-dependent pattern and release (E)-non-2-enal and (E,Z)-nona-2,6-dienal after mechanical injury. Plant Physiol. Biochem. 104, 134e145. Demain, A., Fang, A., 2000. The natural functions of secondary metabolites. Adv. Biochem. Eng. Biot. 69, 1e39. d'Ippolito, G., Iadicicco, O., Romano, G., Fontana, A., 2002. Detection of short-chain aldehydes in marine organisms: the diatom Thalassiosira rotula. Tetrahedron Lett. 43, 6137e6140. d'Ippolito, G., Tuccia, S., Cutignano, A., Romano, G., Cimino, G., Miralto, A., Fontana, A., 2004. The role of complex lipids in the synthesis of bioactive aldehydes of the marine diatom Skeletonema costatum. Biochim. Biophys. Acta 1686, 100e107. d'Ippolito, G., Lamari, N., Montresor, M., Romano, G., Cutignano, C., Gerecht, A., Cimino, G., Fontana, A., 2009. 15S-Lipoxygenase metabolism in the marine diatom Pseudo-nitzschia delicatissima. New Phytol. 183, 1064e1071. Dunstan, G., Volkman, J., Barret, S., Leroi, J., Jeffrey, S., 1994. Essential polyunsaturated fatty acids from 14 species of diatom (Bacillariophyceae). Phytochemistry 35, 155e161. Gallina, A., Brunet, C., Palumbo, A., Casotti, R., 2014. The effect of polyunsaturated aldehydes on Skeletonema marinoi (Bacillariophyceae): the involvement of reactive oxygen species and nitric oxide,. Mar. Drugs 12, 4165e4187. Gross, E.M., 2003. Allelopathy of aquatic autotrophs. Plant Sci. 22, 313e339. Guillard, R.R.L., 1978. Counting slides. In: Sournia, A. (Ed.), Phytoplankton Manual. Monographs on Oceanographic Methodology, vol. 6. UNESCO, Paris, pp. 182e189. re, A., 2004. The a, b, g, d-unsaturated aldehyde 2Hansen, E., Even, Y., Genevie trans-4-trans-decadienal disturbs DNA replication and mitotic events in early sea urchin embryos. Toxicol. Sci. 81, 190e197. Hoshaw, R.W., Rosowski, J.R., 1973. Methods for microscopic algae. In: Stein, J.R. (Ed.), Handbook of Phycological Methods. Cambridge University Press, New York, pp. 53e67. Ianora, A., Poulet, S.A., Miralto, A., 2003. The effect of diatoms on copepod reproduction: a review. Phycologia 42, 351e363. Ianora, A., Miralto, A., 2010. Toxigenic effects of diatoms on grazers, phytoplankton and other microbes: a review. Ecotoxicology 19, 493e511. € J., € m-Ost, Ianora, A., Bentley, M.G., Caldwell, G.S., Casotti, R., Cembella, A.D., Engstro €, A., Halsband, C., Sonnenschein, E., Legrand, C., Llewellyn, C.A., Paldavi ciene Pilkaityte, P., Pohnert, G., Razinkovas, A., Romano, G., Tillmann, U., Vaiciute, D.,
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Ser. 400, 63e73. ^, S., Carotenuto, Y., Romano, G., Hwang, J., Buttino, I., Ianora, A., 2014. Impact of Ka the diatom-derived polyunsaturated aldehyde 2-trans,4-trans decadienal on the feeding, survivorship and reproductive success of the calanoid copepod Temora stylifera. Mar. Environ. Res. 93, 31e37. , G., Pierson, J., Stoecker, K., 2015. The effect of dissolved Lavrentyev, P., Franze polyunsaturated aldehydes on microzooplankton growth rates in the chesapeake bay and atlantic coastal waters. Mar. Drugs 13, 2834e2856. Leflaive, J., Ten-Hage, L., 2009. Chemical interactions in diatoms: role of polyunsaturated aldehydes and precursors. New Phytol. 184, 794e805. Medina, A., Grima, E., Gimenez, A., Gonzales, M., 1998. Downstream processing of algal polyunsaturated fatty acids. Biotechnol. Adv. 16, 517e580. Miralto, A., Barone, G., Romano, G., Poulet, S., Ianora, A., Russo, G., Buttino, I., Mazzarella, G., Laabir, M., Cabrinik, M., Giacobbe, M., 1999. The insidious effect of diatoms on copepod reproduction. Nature 402, 173e176. Pichierri, S., Pezzolesi, L., Vanucci, S., Totti, C., Pistocchi, R., 2016. Inhibitory effect of polyunsaturated aldehydes (PUAs) on the growth of the toxic benthic dinoflagellate Ostreopsis cf. ovata. Aquat. Toxicol. 179, 125e133. Pinckney, J.L., Zingmark, R.G., 1993. Modeling the annual production of intertidal benthic microalgae in estuarine ecosystems. J. Phycol. 29, 396e407. Pohnert, G., 2000. Wound-actived chemical defence in unicellular planktonic algae. Angew. Chem. Int. Ed. 39, 4352e4354. Pohnert, G., 2002. Phospholipase A2 activity triggers the wound-activated chemical defense in the diatom Thalassiosira rotula. Plant Physiol. 129, 103e111. Ribalet, F., Berges, J.A., Ianora, A., Casotti, R., 2007a. Growth inhibition of cultured marine phytoplankton by toxic algal-derived polyunsaturated aldehydes. Aquat. Toxicol. 85, 219e227. Ribalet, F., Wichard, T., Pohnert, G., Ianora, A., Miralto, A., Casotti, R., 2007b. Age and nutrient limitation enhance polyunsaturated aldehyde production in marine diatoms. Phytochemistry 68, 2059e2067. Ribalet, F., Intertaglia, L., Lebaron, P., Casotti, R., 2008. Differential effect of three polyunsaturated aldehydes on marine bacterial isolates. Aquat. Toxicol. 86, 249e255. Ribalet, F., Vidoudez, C., Cassin, D., Pohnert, G., Ianora, A., Miralto, A., Casotti, R., 2009. High plasticity in the production of diatom-derived polyunsaturated aldehydes under nutrient limitation: physiological and ecological implications. Protist 160, 444e451. Ribalet, F., Bastianini, M., Vidoudez, C., Acri, F., Berges, J., Ianora, A., Miralto, A., Pohnert, G., Romano, G., Wichard, T., Casotti, R., 2014. Phytoplankton cell lysis associated with polyunsaturated aldehyde release in the northern Adriatic Sea. PLoS ONE 9, e85947. Romano, G., Miralto, A., Ianora, A., 2010. 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Short-term impacts of polyunsaturated aldehyde-producing diatoms on the harpacticoid copepod, Tisbe holothutiae. J. Exp. Mar. Biol. Ecol. 341, 60e69. Vardi, A., Formiggini, F., Casotti, R., De Martino, A., Ribalet, F., Miralto, A., Bowler, C., 2006. A stress surveillance system based on calcium and nitric oxide in marine diatoms. PLoS Biol. 4 (3), e60. Vidoudez, C., Pohnert, G., 2008. Growth phase-specific release of polyunsaturated aldehydes by the diatom Skeletonema marinoi. J. Plankton Res. 30, 1305e1313. Vidoudez, C., Casotti, R., Bastianini, M., Pohnert, G., 2011. Quantification of dissolved and particulate polyunsaturated aldehydes in the Adriatic Sea. Mar. Drugs 9, 500e513. Viso, A., Marty, J., 1993. Fatty acids from 28 marine microalgae. Phytochemistry 34, 1521e1533. Watson, S., 2003. Cyanobacterial and eukaryotic algal odour compounds: signals or by-products? A review of their biological activity. Phycologia 42, 332e350. Wichard, T., Poulet, S.A., Halsband-Lenk, C., Albaina, A., Harris, R., Liu, D., Pohnert, G., 2005a. Survey of the chemical defence potential of diatoms: screening of fifty-one species for a, b, g, d-unsaturated aldehydes. J. Chem. Ecol. 31, 949e958.
L. Pezzolesi et al. / Phytochemistry 142 (2017) 85e91 Wichard, T., Poulet, S.A., Pohnert, G., 2005b. Determination and quantification of a, b, g, d-unsaturated aldehydes as pentafluorobenzyl-oxime derivates in diatom cultures and natural phytoplankton populations: application in marine field studies. J. Chromatogr. B 814, 155e161.
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PUFAs and PUAs production in three benthic diatoms from the northern Adriatic Sea Laura Pezzolesi a, *, Salvatore Pichierri b, Chiara Samorì c, Cecilia Totti b, Rossella Pistocchi a di Bologna, via Sant'Alberto 163, 48123, Ravenna, Italy Dipartimento di Scienze Biologiche, Geologiche e Ambientali - Universita Politecnica delle Marche, via Brecce Bianche, 60131, Ancona, Italy Dipartimento di Scienze della Vita e dell'Ambiente - Universita c di Bologna, via Selmi 2, 40126, Bologna, Italy Dipartimento di Chimica “Giacomo Ciamician” - Universita a
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 March 2017 Received in revised form 27 June 2017 Accepted 30 June 2017
The production of polyunsaturated aldehydes (PUAs) has been reported by many planktonic diatoms, where they have been implicated in deleterious effects on copepod reproduction and growth of closeby microbes or suggested as infochemicals in shaping plankton interactions. This study investigates the production of PUAs by diatoms commonly occurring in the microphytobenthic communities in temperate regions: Tabularia affinis, Proschkinia complanatoides and Navicula sp. Results highlight the production of PUAs by the three benthic diatoms during stationary and decline phases, with intracellular concentrations from 1.8 to 154.4 fmol cell1, which are within the range observed for planktonic species. The existence of a large family of PUAs, including some with four unsaturations, such as decatetraenal, undecatetraenal and tridecatetraenal, was observed. Since particulate and dissolved PUAs were positively correlated, together with cell lysis, equivalent concentrations may be released during late growth stages, which may affect benthic invertebrates grazing on them and other microalgae. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Proschkinia complanatoides Tabularia affinis Navicula sp. Proschkiniaceae Ulnariaceae Naviculaceae Benthic diatoms GC-MS Fatty acids Polyunsaturated aldehydes
1. Introduction Aquatic organisms have been shown to produce several chemical compounds responsible for the regulation of numerous biological processes (Gross, 2003; Ianora and Miralto, 2010; Ianora et al., 2012). These molecules include various specialised metabolites that are not directly involved in the control of basic life mechanisms (Demain and Fang, 2000), i.e. low-molecular weight organic compounds such as organic acids, phenolic substances, alkaloids, nitrogen-containing compounds, and polyunsaturated aldehydes (PUAs) (Ianora et al., 2011). Recently, several studies have been paying increasing attention to PUAs due to their deleterious effect on fecundity and/or hatching success of copepods (Ianora ^ et al., 2014; Lavrentyev et al., 2015; Miralto et al., 2003, 2012; Ka et al., 1999; Wichard et al., 2007), described as the “paradox of diatom-copepod interactions” by Ban et al. (1997). In addition,
* Corresponding author. E-mail address: [email protected] (L. Pezzolesi). http://dx.doi.org/10.1016/j.phytochem.2017.06.018 0031-9422/© 2017 Elsevier Ltd. All rights reserved.
other important ecological effects have been reported for PUAs, such as competitive inhibition of algal growth (Ribalet et al., 2007a), antibacterial activity (Ribalet et al., 2008), bloom termination (Ribalet et al., 2014; Vidoudez et al., 2011) and intrapopulation signaling (Casotti et al., 2005; Vardi et al., 2006). These volatile aldehydes are oxygenated derivatives of fatty acids (oxylipins) widely produced by plants (Delory et al., 2016), macrophytes (Alsufyani et al., 2014), cyanobacteria (Watson, 2003) and microalgae (d'Ippolito et al., 2009; Ianora et al., 2012; Wichard et al., 2005a). Diatoms are known to contribute about 40% of the marine primary production, constituting half of entire organic material produced in the planet (Rousseaux and Gregg, 2014). Interestingly, under certain conditions, diatoms produce the highest amounts of PUAs among phytoplankton groups (Leflaive and Ten-Hage, 2009). Wichard et al. (2005a) reported a PUA-release by 27 of the 71 algal isolates, with concentrations ranging from 0.01 to 9.8 fmol per cell. In addition to being strain- and speciesspecific (Wichard et al., 2005a), PUAs production and composition can also depend on environmental factors, such as growth stage and nutrient limitation (Pohnert, 2002; Ribalet et al., 2007b).
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PUAs are produced from an oxidative degradation of free polyunsaturated fatty acids (PUFAs), synthesized in chloroplasts and plasmatic membranes by lipase-mediated hydrolysis of glycolipids and phospholipids that occurs after cell disruption (Pohnert, 2000). While describing the origin of PUAs produced by Skeletonema costatum with labeled precursors, d'Ippolito et al. (2004) showed that the metabolism of glycolipids leads to the production of hexadecatrienoic acid (C16:3), hexadecatetraenoic acid (C16:4) and eicosapentaenoic acid (C20:5) which are the direct precursors of octadienal (C8:2), octatrienal (C8:3) and heptadienal (C7:2), respectively, while phospholipids are involved only in the production of heptadienal. The same pool of PUFAs can be a precursor of different PUAs, such as C20:5 acid for decatrienal (C10:3), as observed in Thalassiosira rotula (Barofsky and Pohnert, 2007). Despite the clear correlation between PUFA and PUA composition, the variety of aldehydes that a species is capable of producing depends on its pool of lipoxygenases (LOXs) (Cutignano et al., 2011). These molecules are iron nonheme enzymes that can catalyse the addition of molecular oxygen to the carbon chain of PUFAs, giving fatty acid hydroperoxides (FAHs) as intermediates (d'Ippolito et al., 2009). LOXs have a species-dependent specificity and are responsible for further breaking down FAHs into several derivatives, i.e. PUAs, mediated by hydroperoxide-lyase (HPLs) (d'Ippolito et al., 2009). Vidoudez and Pohnert (2008) observed concentrations of dissolved PUAs during the stationary-early declining phase of diatom cultures and indicated that PUAs can be released by cells independently from grazing, thus affecting surrounding organisms and potentially acting as infochemicals. However, although the pathways and the effect of these oxylipins have been widely investigated for various photosynthetic organisms (Ianora and Miralto, 2010; Ribalet et al., 2007a), almost all of the diatoms investigated belong to the planktonic domain and only a few studies have evidenced PUAs production by benthic diatoms (Jüttner et al., 2010; Scholz and Liebezeit, 2012). The microphytobenthos assemblages can be highly productive (Pinckney and Zingmark, 1993) and benthic primary producers represent an important source of organic carbon in some littoral areas (Allen et al., 2016). The real capacity of benthic organisms to produce PUAs, as well as the effect that these compounds may have on the same benthic ecosystems remain largely unknown. In the present study, we investigated the production of PUFAs and PUAs in three of the most common benthic diatoms (Proschkinia complanatoides (Hustedt ex R.Simonsen) D.G.Mann, Proschkiniaceae; Tabularia affinis (Kützing) Snoeijs, Ulnariaceae and Navicula sp., Naviculaceae) of the Conero Riviera (northern Adriatic Sea, Italy) (Accoroni et al., 2016), providing the production of some compounds that have not yet been reported.
Fig. 1. Cell abundances of Proschkinia complanatoides, Tabularia affinis and Navicula sp. along the growth curve. Data are means of independent replicates (n ¼ 2) and the error bars represent standard deviations.
relevant from day 14 in each species. The highest values were observed in Tabularia affinis (1.17 ± 0.30 ng cell1) and Navicula sp. (0.92 ± 0.05 ng cell1) during the decline phase (day 44). Similarly, the highest fatty acid concentration in Proschkinia complanatoides was reached at day 44 but the total amount never exceeded 0.12 ± 0.01 ng cell1, about 10-times lower than the other species. Generally, the contribution of unsaturated fatty acids resulted higher than that of saturated chains along the growth curve for all the tested species (Fig. 2). Total fatty acids ranged between 1.4 (day 14) and 6.0 (day 37) mg mL1 in P. complanatoides, 2.1 (day 14) and 32.9 (day 35) mg mL1 in T. affinis and between 3.8 (day 14) and 26.5 (day 35) mg mL1 in Navicula sp., while the maximum concentration of unsaturated compounds in the three diatoms was 4.1, 20.2 and 17.9 mg mL1, respectively (Table 1). From a qualitatively point of view, C16 and C20 fatty acids were the prominent compounds in all the species (Fig. 3). In P. complanatoides culture, hexadecatrienoic acid (C16:3) resulted the main PUFA at the beginning of the growth (0.014 ± 0.003, day 14), while hexadecenoic acid (C16:1), and eicosapentaenoic acid (EPA, C20:5) were the most abundant compounds at day 44, reporting maximum values of 0.017 ± 0.002 and 0.019 ± 0.002 ng cell1, respectively (Fig. 3a). C18 unsaturated fatty acids (oleic acid C18:1, linoleic acid C18:2 and linolenic acid C18:3) were also observed along the growth curve, but they were less abundant than C16 and C20 species. A comparable PUFAs composition was observed in
2. Results 2.1. Algal growth The growth curves of Tabularia affinis, Proschkinia complanatoides and Navicula sp. obtained in this study are illustrated in Fig. 1. The curves were characterized by an exponential growth phase until day 9 for T. affinis and Navicula sp. and until day 7 for P. complanatoides. A stationary phase until day 35 for Navicula sp. and T. affinis, until day 37 for P. complanatoides and a subsequent decline phase (from day 35 for T. affinis and Navicula sp. and from day 37 for P. complanatoides) were observed. The highest cell abundances reached were 61 ± 5.6 103, 63 ± 0.8 103 and 57 ± 7.9 103 cells mL1 for P. complanatoides, Navicula sp. and T. affinis, respectively. 2.2. Fatty acids analysis The production of saturated and unsaturated fatty acids became
Fig. 2. Saturated and unsaturated (mono- plus poly-) fatty acids production in Proschkinia complanatoides, Tabularia affinis and Navicula sp. at different days (14, 21, 28, 35, 37, 44). Data are means of independent replicates (n ¼ 2).
L. Pezzolesi et al. / Phytochemistry 142 (2017) 85e91 Table 1 Total and mono- plus polyunsaturated (UFAs) fatty acid concentrations in Proschkinia complanatoides, Tabularia affinis and Navicula sp. at different days (14, 21, 28, 35, 37, 44). UFAs (mg mL1) Proschkinia complanatoides day 14 0.9 day 21 2.2 day 28 3.4 day 35 4.0 day 37 4.1 day 44 2.9 Tabularia affinis day 14 1.3 day 21 3.9 day 28 9.8 day 35 20.2 day 37 13.7 day 44 19.5 Navicula sp. day 14 2.2 day 21 5.8 day 28 6.6 day 35 17.9 day 37 6.9 day 44 14.2
Total fatty acids (mg mL1) 1.4 3.2 5.1 5.8 6.0 4.7 2.1 6.3 16.5 32.9 20.7 31.2 3.8 10.9 12.0 26.5 9.6 19.8
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were revealed only in T. affinis (Fig. 3b and c). Regarding the saturated fatty acids, hexadecanoic acid (palmitic acid, C16:0) was the main compound produced by P. complanatoides and Navicula sp. while tetradecanoic acid (myristic acid, C14:0) the one by T. affinis (data not shown).
2.3. Aldehydes analysis The results of aldehydes quantification, that includes saturated and unsaturated compounds, are shown in Fig. 4. Some aldehydes were not identified as their fragmentation pattern in GC-MS resulted very complex and not comparable with the commercial standards or with compounds present in the NIST libraries. No aldehydes were detected until day 28 in each diatom culture, then a marked increase of these compounds was observed in two of the tested cultures, except for Proschkinia complanatoides. The maximum amount was achieved at day 28 for P. complanatoides (0.61 ± 0.43 pg cell1), 44 for Tabularia affinis (0.67 ± 0.15 pg cell1) and 44 for Navicula sp. (20.44 ± 4.03 pg cell1) (Fig. 4). Moreover, the percentage of unsaturated compounds on the total aldehydes was higher in Navicula sp. (20.4e95.5%) than in P. complanatoides (64.2e67.5%) and T. affinis (61.7e74.1%) cultures. Total aldehydes, in fact, ranged between 0.016 (day 44) and 0.036 (day 28) mg mL1 in P. complanatoides, 0.018 (day 28 and 44) and 0.024 (day 37) mg mL1 in T. affinis and between 0.078 (day 28) and 0.441 (day 44) mg mL1 in Navicula sp., while unsaturated aldehydes reported maximum concentrations of 0.023, 0.015 and 0.421 mg mL1 at day 28, 37 and 44 in the three diatoms, respectively (Table 2). From a qualitative point of view, medium-chained PUAs were the most abundant aldehydes produced by Navicula sp. (Fig. 5c). Although hexadienal (C6:2), octadienal (C8:2), decatetraenal (C10:4), undecatetraenal (C11:4), undecapentenal (C11:5) and tridecatetraenal (C13:4) were also detected, octenal (C8:1) was the most abundant compound (93.8%) corresponding to 18.40 ± 3.34 pg cell1 at day 44. On the contrary, PUAs composition of the other two species was more variable, but results were similar between the two diatoms: hexadienal (C6:2) contributed up to 50.56% and 39.04%, for T. affinis and for P. complanatoides, respectively (Fig. 5a and b). Decatetraenal (C10:4) reached a maximum value of 0.09 ± 0.01 pg cell1 for T. affinis and 0.13 ± 0.04 pg cell1
Fig. 3. Mono- and polyunsaturated fatty acids production in (a) Proschkinia complanatoides, (b) Tabularia affinis and (c) Navicula sp. at different days (14, 21, 28, 35, 37, 44). Data are means of independent replicates (n ¼ 2) and the error bars represent standard deviations. Hexadecenoic acid (C16:1), hexadecadienoic acid (C16:2), hexadecatrienoic acid (C16:3), octadecenoic acid (C18:1), octadecadienoic acid (C18:2), octadecatrienoic acid (C18:3), eicosatetraenoic acid (C20:4), eicosapentaenoic acid (C20:5) and docosahexaenoic acid (C22:6).
Navicula sp. and T. affinis; C16:1 acid was the main compound in both cultures, with the highest concentrations reached at day 44 (0.503 ± 0.178 ng cell1 for T. affinis and 0.564 ± 0.035 ng cell1 for Navicula sp.). Trace amounts of C18 fatty acids (C18:1 and C18:3)
Fig. 4. Saturated and unsaturated (mono- plus poly-) aldehydes production in Proschkinia complanatoides (left Y axis), Tabularia affinis (left Y axis) and Navicula sp. (right Y axis) at different days (28, 35, 37, 44). Data are means of independent replicates (n ¼ 2).
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Table 2 Total and mono- plus polyunsaturated (UAs) aldehyde concentrations in Proschkinia complanatoides, Tabularia affinis and Navicula sp. at different days (28, 35, 37, 44). UAs (mg mL1)
UAs (pmol mL1)
Proschkinia complanatoides day 28 0.023 188.3 day 35 0.012 107.9 day 37 0.019 153.9 day 44 0.011 96.7 Tabularia affinis day 28 0.014 126.0 day 35 0.013 120.2 day 37 0.015 131.4 day 44 0.011 101.6 Navicula sp. day 28 0.016 135.4 day 35 0.225 1771.9 day 37 0.383 3011.7 day 44 0.421 3331.6
Total aldehydes (mg mL1)
Total aldehydes (pmol mL1)
0.036 0.018 0.029 0.016
273.6 150.1 223.4 134.7
0.018 0.021 0.024 0.018
161.3 187.9 205.7 154.2
0.078 0.243 0.397 0.441
705.7 1925.5 3107.3 3441.0
for P. complanatoides at day 44 and 28, respectively. 3. Discussion The intracellular unsaturated aldehydes concentration ranged from 1.8 to 154.4 fmol cell1, varying either with the algal stage and among the diatom species. Such values are within the range observed for other planktonic species (Wichard et al., 2005a,b), such as Skeletonema marinoi (d'Ippolito et al., 2004; Ribalet et al., 2007b, 2009; Vidoudez and Pohnert, 2008) and Melosira
Fig. 5. Mono- and polyunsaturated aldehydes production in (a) Proschkinia complanatoides, (b) Tabularia affinis and (c) Navicula sp at different days (28, 35, 37, 44). Data are means of independent replicates (n ¼ 2) and the error bars represent standard deviations. Hexadienal (C6:2), octenal (C8:1), octadienal (C8:2), octatrienal (C8:3), nonatetraenal (C9:4), decatetraenal (C10:4), undecatetraenal (C11:4), undecapentenal (C11:5) and tridecatetraenal (C13:4). Right Y axis in Navicula sp. refers only to C8:1.
nummuloides (Taylor et al., 2007). A survey performed by Wichard et al. (2005a) on five Navicula species did not reveal the production of any PUAs; however, the authors observed that PUAs production within different isolates of some species ranged widely and depended on the growth phase of the culture, as later confirmed by other studies (Ribalet et al., 2007b; Vidoudez and Pohnert, 2008). More recently, the variability in PUAs production has been confirmed also by Scholz and Liebezeit (2012), which report aldehydes in cultures of benthic intertidal diatoms of the southern North Sea, including Navicula phyllepta. Moreover, most of the studies focused only on the production of some common PUAs, such as heptadienal, octadienal, octatrienal, decadienal and decatrienal (i.e., d'Ippolito et al., 2004; Jüttner et al., 2010; Ribalet et al., 2007b, 2009; Taylor at al., 2007; Vidoudez and Pohnert, 2008; Wichard et al., 2005a,b). However, d'Ippolito et al. (2002) suggested the existence of a large family of PUAs involved in the chemical interactions within planktonic communities. A similarly wide range of production was also found in the benthic community in our study. We thus confirm for the first time the high potential in the production of these metabolites in three benthic diatoms: a total of 9 compounds were identified, and additional PUAs remain unknown. Although heptadienal represents one of the most common PUAs released by planktonic diatoms (Wichard et al., 2005a), based on the time-frame where C7 aldehydes are typically detected by GCMS and due to the lack of peak signals in the present study, we exclude the potential production of this aldehyde by Proschkinia complanatoides, Tabularia affinis and Navicula sp. Other studies have focused on the effects of the PUAs octadienal and decadienal, which are commonly reported in other algal species (e.g. Skeletonema costatum, d'Ippolito et al., 2004; Thalassiosira rotula, Wichard et al., 2005a,b). In this study, these PUAs were not observed either, except for low levels of octadienal in Navicula sp. Our results highlight that a variety of PUAs can be produced and released by diatoms, including some with four unsaturations, such as decatetraenal, undecatetraenal and tridecatetraenal. In a previous study, a positive correlation between the length of PUAs chain and their bioactivity was observed (Adolph et al., 2003; Hansen et al., 2004; Pichierri et al., 2016; Ribalet et al., 2007a), thus these compounds (i.e., C10:4, C11:4 and C13:4) could have considerable effects on the benthic community, in particular at the concentrations produced by Navicula sp; however further investigations to better understand their toxic effects on benthic organisms are needed. A difference between intracellular and released PUA amounts was observed during a PUA surface mapping performed in the Adriatic Sea (Vidoudez et al., 2011), where the concentrations of dissolved PUAs never reached those released from ruptured cells. More recently, a field study performed in the northern Adriatic Sea during different years reported total particulate and dissolved PUA concentrations from 0 to 5.37 fmol cell1 and from 0 to 2.53 nM, respectively (Ribalet et al., 2014). These authors observed a positive correlation between particulate and dissolved PUAs, and cell lysis, suggesting that PUAs are released in the surrounding seawater upon cell rupture. Intracellular levels of PUAs found in the present study suggest a significant release by the three benthic diatoms, particularly by Navicula sp., with a major release during the late growth stage which may have an effect on grazers or, for instance, on meiobenthic organisms. The fatty acids analysis performed on the three species revealed a composition mainly constituted by myristic and palmitic acid (C14:0 and C16:0) among saturated acids, and C16-chains and eicosapentaenoic acid (C20:5, EPA) among unsaturated ones; low amounts of stearic acid (C18:0) and other C18 fatty acids were detected. These results appear consistent with fatty acid profiles reported for diatoms and other microalgae (Jüttner, 2001; Medina
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et al., 1998; Wichard et al., 2007). Dunstan et al. (1994) described EPA as the main PUFA in 14 diatom species, and high amounts of palmitoleic acid (C16:1) were measured in 28 microalgae by other authors (Viso and Marty, 1993). On the contrary, although hexadecatetraenoic acid (16:4) was among the main PUFAs identified in diatoms (Jüttner, 2001; Wichard et al., 2007), no traces of this compound were observed in each of the tested diatoms. Fatty acids concentrations increased with culture age, particularly for EPA, which has been previously found to increase during the stationary growth phase (Stonik and Stonik, 2015). Despite the presence of PUFAs in each growth phase (except for the lag one) and their decisive function in the PUAs metabolic pathway as precursors, the aldehydes production clearly occurred only during the stationary and decay growth phases in each of the three tested diatoms. In addition, no clear correlation between PUAs and PUFAs content has been observed, as different patterns were observed during the growth phases, confirming that the fatty acid decrease cannot be entirely attributed to the production of PUAs (Wichard et al., 2007). This result could be due to an inactivation state of those enzymes essential for catalyzing biochemical transformations, such as phospholipases that make PUFAs available for lipoxygenase/hydroperoxide lyases oxidation. In fact, enzyme activity, together with available lipid resources, seem to control the absolute and relative amount of PUAs, as previously suggested by other authors (Ribalet et al., 2007b). This may explain why in Navicula sp. and T. affinis we observed on one hand similar PUFA amounts, and on the other hand, differences in PUFAs composition and in PUAs concentration and composition. As observed for other diatom species, the diffusion of woundactivated compounds, such as PUAs, may indicate abundance of possible food, thus attracting some invertebrates in search for food, or serve as a repellent for other invertebrates, such as crustaceans, providing a chemical defense towards the grazing (Jüttner, 2005; Jüttner et al., 2010; Romano et al., 2010). This may in turn lead to variable effects on individual benthic invertebrate species and to the formation of complex behavioural reactions among benthic diatoms and their associated invertebrates, especially if considering the variety of PUAs compounds found in the present study and their variable toxicity, which has been reported either to vary among the different PUAs (i.e. Pichierri et al., 2016; Ribalet et al., 2007a) and to be target-specific (Gallina et al., 2014). In addition, PUAs produced by benthic diatoms could also affect the population dynamics of cooccurring microalgae and/or regulate the natural bloom successions, including toxic bloom-forming species (i.e. Ostreopsis spp., which are usually blooming in this area). In conclusion, these results underline the importance of designing studies to understand the interactions which occur between chemical signals and the various organisms, especially in benthic biofilms which are particular micro-environments where species are exposed to more concentrated chemicals exuded by neighbouring cells (Allen et al., 2016). Field studies are needed to better clarify the role of these specialised metabolites in the structure of the microphytobenthic communities. 4. Experimental 4.1. Reagents All reagents were purchased from Sigma-Aldrich (Milan, Italy) and used without further purification. 4.2. Cultivation and sampling The diatom strains, Proschkinia complanatoides (Hustedt ex R.Simonsen) D.G.Mann family Proschkiniaceae (strain PCAPS0313),
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Tabularia affinis (Kützing) Snoeijs family Ulnariaceae (strain TAAPS0313) and Navicula sp. family Naviculaceae (strain NAPS0313), were obtained from microphytobenthos samples collected before summer period in 2013 in the Passetto station (Conero Riviera, Italy, northern Adriatic Sea, 43 370 0900 N, 13 3105400 E), which is a semi-enclosed and shallow (mean depth 2 m) area, sheltered by a natural reef and characterized by a mostly rocky benthic surface. Single cells were isolated by the capillary pipette method (Hoshaw and Rosowski, 1973) using an inverted microscope; monoclonal cultures were set up using multi-well plates and were grown using f/2 medium (Guillard, 1978) with a salinity of 30. Cultures were maintained at 20 ± 1 C under a 16:8 L:D photoperiod and irradiance of 100e110 mmol m2 s1. Two replicate flasks (final culture volume of 2000 mL) for each diatom were established and maintained in a growth chamber. The experiment lasted 44 days and an aliquot of 200 mL was collected from each flask at the sampling days 3, 7, 9, 14, 21, 28, 35, 37 and 44. The algal aliquot was centrifuged at 3000 x g for 10 min to collect the algal pellet for PUFAs and PUAs extraction and analysis. 4.3. Fatty acids analysis The determination and quantification of PUFAs were carried out following the method of Samorì et al. (2012). Gas chromatographyemass spectrometry (GC-MS) analyses were performed using a 6850 Agilent HP gas chromatograph connected to a 5975 Agilent HP quadrupole mass spectrometer. The injection port temperature was 280 C. Analytes were separated by a HP-5 fused-silica capillary column (stationary phase poly[5% diphenyl/95% dimethyl]siloxane, 30 m, 0.25 mm i.d., 0.25 mm film thickness), with helium as the carrier gas (at constant pressure, 33 cm s1 linear velocity at 200 C). Mass spectra were recorded under electron ionization (70 eV) at a frequency of 1 scan s1 within the 12-600 m/z range. The temperature of the column was increased from 50 C up to 180 C at 50 C min1, then from 180 C up to 300 C at 5 C min1. Methyl nonadecanoate was utilized as internal standard for quantification of free and bounded fatty acids converted into fatty acid methyl esters (FAMEs). The relative response factor used for the quantitation was obtained by injecting solutions of known amounts of methyl nonadecanoate and commercial FAMEs mixture. Each analysis was repeated in duplicate. 4.4. Aldehydes analysis The quantification of PUA concentration was carried out following the method of Wichard et al. (2005b). After removing the supernatant, 1 mL of derivatisation reagent (O-(2,3,4,5,6pentafluorobenzyl) hydroxylamine hydrochloride, PFBHA HCl, 25 mM in Tris/HCl 100 mM, pH 7) and 25 mL of internal standard (0.968 mM benzaldehyde in hexane) were added to the algal pellet. The sample was cooled at 4 C, then ultrasonicated for 1 min and finally incubated at room temperature for 30 min. 0.5 mL of methanol and 1 mL of hexane were added to the sample that was vortexed for 1 min and acidified with 20 mL of H2SO4. After that, the hexane upper layer was removed, dehydrated over Na2SO4, evaporated under N2 and resuspended in 50 mL hexane. PUA concentration was determined by GC-MS (same equipment described in 4.3 Fatty acids analysis); the molecular identification was done by comparison of retention times and mass spectra with those of commercial standards when possible: propionaldehyde, 2,4heptadienal, octanal, 2-octenal, 2,4-octadienal, 6-nonenal, 2,6nonadienal, 4-decenal, 2,4-decadienal, undecanal, 8-undecenal and 2,4-undecadienal. If PUAs standards were not available, the identification was performed by comparison with NIST libraries.
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