webmailSCHEDA AUTORI: Serena Felline

45° Congresso della Società Italiana di Biologia Marina
Venezia, 19-23 maggio 2014
webmailSCHEDA
AUTORI: Serena Felline, Marianna Carbone, Roberto Caricato, Adele Cutignano, Biagio
d’Aniello, Giuseppe d’Errico, Alessandra Ferramosca, Maria Elisa Giuliani, Stefania Gorbi,
Laura Grauso, Maria Giulia Lionetto, Laura Magliozzi, Ernesto Mollo, Lucia Pittura, Gianluca
Polese, Francesco Regoli, Vincenzo Zara, Antonio Terlizzi
TITOLO: Caulerpa racemosa and Diplodus sargus: a model for an integrated study of
invasive pest metabolites impact on Mediterranean coastal marine systems
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COMUNICAZIONE
Tema 1: Potenzialità del plancton nell’economia del mare
Tema 2: Specie aliene: casi di studio
Tema 3: Livelli di diversità del benthos marino in relazione alle pressioni antropiche
Tema 4: Zone rifugio e possibile resilienza di alcuni stock demersali mediterranei
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Autore referente
Nome e Cognome Antonio Terlizzi
e-mail: [email protected]
Autore che paga la quota di iscrizione al Congresso
Nome e Cognome: Serena Felline
e-mail: [email protected]
Autore che presenta il lavoro al Congresso
Nome e Cognome: Serena Felline
e-mail: [email protected]
46_Congr SIBM form testi v. 11_feb_2010
45° Congresso della Società Italiana di Biologia Marina
Venezia, 19-23 maggio 2014
S. FELLINE1, M. CARBONE2, R. CARICATO1, A. CUTIGNANO2, B. D’ANIELLO3, G. D’ERRICO4,
A. FERRAMOSCA1, M.E. GIULIANI4, S. GORBI4, L. GRAUSO2, M.G. LIONETTO1, L.
MAGLIOZZI3, E. MOLLO2, L. PITTURA4, G. POLESE3, F. REGOLI4, V. ZARA1, A. TERLIZZI1*
1
Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali (Di.S.Te.B.A.), Università del Salento,
Prov.le Lecce - Monteroni – 73100 Lecce, Italia.
2
ICB, CNR, Pozzuoli, Naples, Italy
3
Dip. di Biologia, Università degli Studi di Napoli Federico II, Naples, Italy
4
Di.S.V.A., Universita` Politecnica delle Marche, Ancona, Italy
*
[email protected]
CAULERPA RACEMOSA AND DIPLODUS SARGUS: A MODEL FOR AN INTEGRATED
STUDY OF INVASIVE PEST METABOLITES IMPACT ON MEDITERRANEAN COASTAL
MARINE SYSTEMS
CAULERPA RACEMOSA E DIPLODUS SARGUS: UN MODELLO PER LO STUDIO
INTEGRATO DELL'IMPATTO DI METABOLITI SECONDARI DA SPECIE INVASIVE SU SISTEMI
MARINO-COSTIERI DEL MEDITERRANEO
Abstract – In the Mediterranean Sea, the biological invasion by the green algae Caulerpa racemosa is
maybe the most striking marine sea-bottom landscape change of the last decades. In this paper, we have
synthesized the results of 5 years of studies about the C. racemosa effects on fish of ecological and
economic importance, such as the white sea bream Diplodus sargus. We found that secondary metabolite of
C. racemosa has the potential to enter in trophic chains and accumulate in fish tissues. Ecotoxicological
analyses made on tissues of the D. sargus specimens showed alterations of some of main antioxidant
systems, such as catalase, glutathione peroxidases and total glutathione, and a significant induction of
P450 biotrasformation system. We found also that the C. racemosa based-diet can alter muscle fatty acids
composition in the white sea bream by reducing the percentage of polyunsaturated fatty acids (PUFA) of
the n-3 and n-6 series, such as eicosapentaenoic (EPA, C20:5), docosahexaenoic (DHA, C22:6) and
arachidonic acids (AA, C20:4). On the whole, this work sheds light on an unexplored and critical aspect of
biological invasions with implication on the health of both humans and the environment.
Key-words: Introduces species, diet deficiencies, fatty acids, biomarkers, fish physiology.
Introduction - Biological invasions are deemed as one of the most severe threats to
terrestrial and marine biodiversity and can result in huge economic impacts on natural
resources. In the Mediterranean Sea, the biological invasion by the green alga
Caulerpa racemosa is maybe the most striking marine sea-bottom landscape change of
the last decades, due to its high potential of dispersal and to deep modifications
produced in the invaded benthic communities (Klein and Verlaque, 2008). Although
many studies have assessed the effects of C. racemosa spread on the benthic
community structure, less clear are the complex indirect effects of invasive pests on
functional properties of marine ecosystems.
In the last 5 years, we carried out several sampling surveys along the coast of Apulia
(South Italy) in order to explore the effects of C. racemosa on functional properties of
marine subtidal systems such as those represented by trophic relationships and cascade
effects. Because of its ecological importance in modulating trophic cascade effects and
thus the diversity of benthic assemblages, the white sea bream Diplodus sargus has
been chosen as a organism model. Through this paradigmatic example, the resulted
46_Congr SIBM form testi v. 11_feb_2010
45° Congresso della Società Italiana di Biologia Marina
Venezia, 19-23 maggio 2014
presented here aimed at providing further insights to the subtle mechanisms by which
biological invasions could affect marine biodiversity.
Materials and methods - Results are based on analyses performed on D. sargus
individuals sampled in the late summer of 2008, October 2009 and June and October
2012, along the coast between Brindisi and Lecce (BR) and the Marine Protected Areas
of Porto Cesareo (PC) and Torre Guaceto (TG). Once speared, individuals were
immediately stored in a polystyrene box containing dry ice until transportation to the
laboratory, where they were measured and weighed. Overall, a total of 155 specimens
were collected, with a mean weight of 350 g (SE = ±16.9) and a mean length of 23.4
cm (SE = ±1.7). Liver, spleen, gills, brain and muscle were excised, frozen in liquid
nitrogen and maintained at -80°C till processed for chemical and biochemical analyses.
Stomach contents were sorted under magnification and identified to the lowest possible
taxonomic level, depending on the type and digestion level of each prey item. The
percentage of occurrence (O%) and the modified index of relative importance (MI%)
were calculated for all items as described in Terlizzi et al. (2011).
For each individual, liver, white and red muscle, skin and brain were separately
extracted and organic extracts were analyzed in reverse phase high-performance liquid
chromatography mass spectrometry (RP-HPLC-MS) to quantify algal metabolite. The
enzymatic activities of catalase (CAT), glutathione peroxidase (GPx), glutathione
reductase (GR), glutathione S-transferase (GST), total glutathione (TGSH), total
oxyradical scavenging capacity (TOSC), Acyl CoA oxidase (AOX),
acetylcholinesterase (ACH), micronuclei frequencies (MN), Na+/K+ - ATPase (Na+/K+)
and 7-ethoxyresorufin O-deethylase (EROD), were measured. Details of chemical and
biochemical analyses are provided in Felline et al. (2012).
Muscle lipids were extracted by the Bligh and Dyer procedure and the fatty acid
composition of lipid fractions of muscle samples determined by gas chromatography of
the corresponding methyl esters (Felline et al., in press).
Results - Fish revealed a high frequency of occurrence of C. racemosa in the stomach
contents at invaded areas (72.7 and 85.7%) while the alga was not detected in
organisms from control, non-invaded area. We demonstrated that D. sargus
accumulates one of the metabolites synthesized by C. racemosa at concentrations
ranging from zero to more than one hundred of µg/g, depending on the analyzed tissue,
with the highest concentrations found in brain, liver and red muscle. The presence of C.
racemosa in the diet of D. sargus, as indicated by the significant accumulation of
caulerpin in tissues, caused the onset of various biological effects, including activation
of some enzymatic pathways (catalase, glutathione peroxidases, glutathione Stransferases, total glutathione and the total oxyradical scavenging capacity, 7-ethoxy
resorufin O-deethylase), the inhibition of others (acetylcholinesterase and acylCoA
oxidase), an increase of hepatosomatic index and the decrease of gonadosomatic index
(Fig. 1A) (Felline et al., 2012; Gorbi et al., in press).
The C. racemosa based-diet has moreover, the potential to induce changes in the
nutritional properties of D. sargus. Fish feeding on C. racemosa showed indeed, an
altered muscle fatty acids composition, with a lower percentage of polyunsaturated
fatty acids (PUFA) of the n-3 and n-6 series, such as eicosapentaenoic (EPA, C20:5),
46_Congr SIBM form testi v. 11_feb_2010
45° Congresso della Società Italiana di Biologia Marina
Venezia, 19-23 maggio 2014
docosahexaenoic (DHA, C22:6) and arachidonic acids (AA, C20:4) than fish not
feeding on this seaweed (Fig. 1B) (Felline et al., in press).
Fig.1 - Canonical analysis of principal coordinates representing differences in cellular and molecular
responses (A) and fatty acid composition (B) across the different levels of caulerpin
bioaccumulation. Green circles represent increasing caulerpin concentration in liver, expressed as
µg/g on dry weight. A clear-cut separation was between fish with medium or high content of
caulerpin, clustered together on the left-hand side of the graph, from those with low or absent
levels. The vectors in the plots represented the contribution of each variable to differences between
these two groups. The longer is the line, the greater is the contribution to the observed differences.
Overall, biomarkers highly related to the first and second axes (A) indicate the occurrence of an
altered health status in fish with medium and high caulerpin accumulation compared with controls.
In the second plot (B) a worse fatty acid profile for fish accumulating caulerpin is represented by
the long chain polyunsaturated fatty acids (C20:4, C20:5, C22:6), which most characterizes fish
without caulerpin (From: Felline et al., 2012 and Felline et al., in press, modified).
Analisi canonica delle coordinate principali raffigurante le differenze nelle risposte cellulari e
molecolari (A) e nella composizione in acidi grassi (B) tra diversi livelli di accumulo di caulerpina.
I cerchi verdi sovraimposti su ogni campione rappresentano concentrazioni crescenti di caulerpina
nel fegato, espresse in µg/g su peso secco. In generale, si nota una netta separazione tra i pesci con
medio o elevato contenuto di caulerpina nel fegato, e quelli con bassi o nulli livelli di accumulo. Il
contributo di ogni variabile alle differenze tra i due gruppi è rappresentato dai vettori sui plot. Più
lungo è il vettore, maggiore è il contributo alle differenze osservate. In generale, i biomarker
indicano la comparsa di uno stato alterato nei pesci con medio o elevato accumulo di caulerpina
rispetto ai controlli. Nel secondo plot (B) è possibile osservare come i pesci con caulerpina
risultano avere un peggiore profilo acidico come indicato dagli acidi grassi polinsaturi a lunga
catena (C20:4, C20:5, C22:6) che caratterizzano soprattutto i pesci senza caulerpina (da: Felline
et al., 2012 e Felline et al., in stampa, modificato)
Conclusions – Invasive alien species can have multiple effects ranging from changes
in individual behavior to altered nutrient and energy fluxes in ecosystems. Through an
integrated approach which conjugated organic chemistry, cellular biology,
neurobiology, ecotoxicology and ecology, these studies supports the view that the
entering of alien species in subtidal systems can alter trophic webs and can represent an
important, indirect mechanism that might affect marine biodiversity with, also, critical
implications of nutritional interest for human health.
The observed alterations in cellular and molecular responses in fact, might lead to a
detrimental health status and altered behaviors, potentially preventing the reproductive
success of fish populations. Particularly, a higher activity of the cytochrome P450
biotrasformation pathway was found in fish exposed to algal metabolite. Considering
that cytochrome P450 has a key role in metabolism of environmental xenobiotics (i.e.
polycyclic aromatic and halogenated hydrocarbons) direct effects of algal metabolism
46_Congr SIBM form testi v. 11_feb_2010
45° Congresso della Società Italiana di Biologia Marina
Venezia, 19-23 maggio 2014
on the efficiency of this pathway might indirectly influence the sensitivity of local fish
populations toward chemical pollutants.
The lower levels of AChE in fish accumulating caulerpin suggested an involvement in
nervous systems impairment that could affect swimming, researching of food or
predator avoidance.
Gonadosomatic index resulted affected by a C. racemosa-based diet, suggesting
possible detrimental effect on gross gonadal morphology.
The activation of antioxidant systems confirmed the ability of Caulerpales to increase
production of radical oxygen species in fish (e.g. Box et al., 2009), which may lead to
oxidative damages.
Peroxisomes have a key role in lipid metabolism and beta-oxidation of fatty acids,
being modulated by a variety of natural molecules, synthetic products and
anthropogenic pollutants (Gorbi et al., 2012). Therefore, the alteration in the AOX
activity found in this study in fish accumulating caulerpin could mechanistically
explain variations in the content of fatty acids observed in fish exposed to C. racemosa.
The alteration in fatty composition of flesh fish not only could represent a risk for the
health of fish unable to biosynthesize these essential fatty acids, but also lowered the
nutritional value of white seabream flesh because of the key role of long-chain n-3
PUFA in the prevention of cardiovascular diseases, diabetes, hepatic steatosis, cancer
and neurological disorders (Nicholson et al., 2013).
References
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physiological responses of Spondyliosoma cantharus (Linnaeus, 1758) to the Caulerpa racemosa var.
cylindracea invasion. J. Exp. Mar. Biol. Ecol., 380: 11-19.
FELLINE S., CARICATO R., CUTIGNANO A., GORBI S., LIONETTO M.G., MOLLO E.,
REGOLI F., TERLIZZI A. (2012) Subtle Effects of Biological Invasions: Cellular and Physiological
Responses of Fish Eating the Exotic Pest Caulerpa racemosa. PLoS One, 7: e38763.
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46_Congr SIBM form testi v. 11_feb_2010

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