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Anticancer, antichemotactic and antimicrobial activities of marine sponges collected off the coast of Santa Catarina, southern Brazil
Noel R. Monks a,*, Clea Lerner b, Amelia T. Henriques c, c Fabiane M. Farias , Elfrides E.S. Schapoval c, Edna S. Suyenaga c, Adriana B. da Rocha a,d, Gilberto Schwartsmann a,d, Beatriz Mothes b
a
Centro Integrado do Cancer (CINCAN), Universidade Luterana do Brazil (ULBRA), Rua Miguel Tostes 101, Canoas 92420-280, RS, Brazil b Fundacao Zoobotanica, Museu de Ciencias Naturais, Rio Grande Sul, Rua Dr. Salvador Franca 1427, Porto Alegre 90690-000, RS, Brazil c Faculdade de Farmacia, Universidade Federal do Rio Grande do SUL (UFRGS), Avenida Ipiranga 2752, Porto Alegre 90610-000, RS, Brazil d South American Office for Anticancer Drug Development (SOAD), Hospital de Clnicas de Porto Alegre, Rua Ramiro Barcelos 2350, Porto Alegre 90035-003, RS, Brazil Received 10 July 2002; received in revised form 22 August 2002; accepted 30 August 2002
Abstract This study reports the in vitro screening of 10 marine sponges (Porifera) collected from the coastline of Santa Catarina, southern Brazil, in the search for novel pharmaceuticals. Organic and aqueous extracts were tested for anticancer, antibacterial, antifungal and antichemotactic activities. Eight of the ten species tested demonstrated activity in one or more of the bioassays. Organic extracts of Polymastia janeirensis Boury-Esnauls, 1973, Haliclona aff tubifera George and Wilson, 1919, Mycale arcuiris Lerner and Hajdu, 2002 and Raspailia (syringella) sp. each demonstrated cytotoxicity at 100 Ag/ml in an in vitro screening assay against the HT29 colorectal tumour cell line. Further analysis against three human tumour cell lines (HT29, U373 and NCI-H460) demonstrated IC50 concentrations ranging from 25 to 50 Ag/ml. Aqueous extracts of six species P. janeirensis, M. arcuiris, Raspailia (syringella) sp., Guitarra sp., Tedania ignis Duchassaing and Michelotti, 1864 and Pseudaxinella reticulata Ridley and Dendy, 1886 each significantly ( p V 0.05) retarded the migration of polymorphonuclear (PMN) leukocytes in a chemotactic assay. In antibacterial assays,
* Corresponding author. Current address: Dana-Farber Cancer Institute, Smith Building Rm 936A, 44 Binney Street, Boston, MA 02115, USA. Tel.: +1-617-632-4172; fax: +1-617-632-4680. E-mail address: Noel_Monks@dfci.harvard.edu (N.R. Monks). 0022-0981/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 0 9 8 1 ( 0 2 ) 0 0 3 8 0 - 5
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only H. aff tubifera (four of five bacterial strains) and Axinella corrugata George and Wilson, 1919 (one of five bacterial strains) demonstrated activity. None of the 10 species demonstrated measurable antifungal activity. These extracts are currently undergoing further analysis to identify the active constituents. D 2002 Elsevier Science B.V. All rights reserved.
Keywords: Polymastia janeirensis; Haliclona aff tubifera; Mycale arcuiris; Raspailia (syringella) sp.
1. Introduction Without doubt, natural products have been, and still are, the cornerstone of the health care armamentarium. Indeed, at the last estimate, 80% of the worlds population still rely on traditional medicines for their health care needs (Farnsworth et al., 1985). Considering prescription medicines alone, microbial and plant-derived drugs account for greater than 30% of the worldwide sales (Grabley and Thiericke, 1999). Some of the most notable include the analgesics aspirin (Filipendula ulmaria) and morphine and codeine (Papaver somniferum), the malaria prophylaxis, quinine (Cinchona pubescens) and the cardiotonic drugs, digoxin and digitoxin (Digitalis purpurea) (reviewed in Cox, 1994; da Rocha et al., 2001). The popularity of drug discovery programs based on nature are associated to a number of factors. Firstly, the diversity and complexity of the chemical structures go far beyond those, which can be synthesized in a laboratory. Secondly, the molecules isolated from nature are, more often than not, small ( < 1000 Da), with existing drug-like properties (Harvey, 1999). Added to this, as a result of evolutionary pressures, many organisms, both terrestrial and marine, have developed chemical defense mechanisms, secondary metabolites, which confer a selective advantage and often have distinct biological activities against enzymes and receptors which makes them ideal candidates for pharmacological investigation (Faulkner, 2000b). In the search for new pharmaceuticals, a number of approaches are used by natural product researchers in the selection of candidate species. These include (a) Ethnomedical information, which refers to species used in popular medicine to treat ailments; (b) Chemotaxonomy, which involves selection of species due to promising biological activity or the presence of a particular class of molecule(s) with the desired activity, in congeneric species and (c) Random collection (Farnsworth, 1994). Generally speaking, there is little or no ethnobotanical information regarding the use of marine species for medical ailments, therefore the screening of marine organisms generally fall into the latter category, although once an interesting species/compound is discovered, chemotaxonomy can be used to select related species. Covering around 70% of the planet surface, the oceans possess a huge potential for the new discovery often on novel molecules (Cragg et al., 1997). Compounds isolated from marine sources are often highly complex structures, which are frequently difficult to synthesize, which can often lead to problems in the supply of sufficient quantities of material for preclinical and clinical development. These compounds are usually a part of
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highly toxic defense mechanisms, which is a reflection of the highly competitive and solute environment in which the organism resides (Grabley and Thiericke, 1999). The most interesting phyla with respect to pharmacologically active marine compounds include bacteria, fungi, algae, sponges, soft corals and gorgonians, sea hares and nudibranchs, bryozoans and tunicates (Faulkner, 2000b). At present, there are a number of compounds from marine origin which are under investigation and/or are being developed as new pharmaceuticals (Faulkner, 2000a,b; da Rocha et al., 2001; Schwartsmann et al., 2001). Examples include: Cytarabine (Cryptothethya crypta), Halichondrin B (Halichondria okadai), Bryostatin 1 (Bugula neritina), Dolastatin 10 (Dolabella auricularia) and Ecteinascidin 743 (Ecteinascidia turbinata), which are all under evaluation as new anticancer therapies although none, as yet, are commercially available. A number of compounds have been identified with anti-inflammatory activity, including Manoalide (Luffariella variabilis), which is commerically available, Pseudopterosins (Pseudopterogorgia elisabethae), Topsentins (Topsentia genitrix, Spongosorites sp. and Hexadella sp.), Scytonemin (Cyanobacteria) and Debromohymenialdisine (DBH) (Phakellia flabellata, Hymeniacidon aldis and Stylotella aurantia). Aurantosides (Siliquariaspongia japonica and Homophymia conferta) and Spongistatin 1 (Hyrtios erecta) are commerically available antifungal agents which were also discovered from marine sponges. In this report, we describe the screening of marines sponge extracts collected from the coastline of Santa Catarina, southern Brazil, for antitumour, antichemotactic, antibacterial and antifungal activity. This study is part of a collaborative program between a number of Brazilian institutions (Fundacao Zoobotanica Rio Grande do Sul, Faculdade de Farmacia Universidade Federal do Rio Grande do Sul and the South American Office for Anticancer Drug Development (SOAD)) for the collection and screening of Brazilian marine sponges for a variety of biological activities, with the aim of identifying new sponges species and novel molecules with interesting and potentially useful therapeutic activities.
2. Materials and methods 2.1. Sponge sampling and identification Sponges samples were collected manually from exposed and semi-exposed habitats, at depths of between 0.5 and 14 m, from locations on the coastline of Santa Catarina (southern Brazil). Taxonomic designation was based on scanning electron microscope studies and on skeletal slides and dissociated spicule mounts. Specimens of all materials are deposited in the Museu de Ciencias Naturais Porifera (MCNPOR) collection of the Fundacao Zoobotanica do Rio Grande do Sul, Brazil. The species investigated in this study are detailed in Table 1. 2.2. Extract preparation Aqueous extracts were produced by the following procedure. Sponge materials were ground together with sand and water three times for 30 min. The resulting extract (collected after each 30 min) was subsequently filtered and freeze-dried. The remaining material was
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Table 1 Sponge species examined in this study Species Guitarra sp. P. citrina T. ignis P. reticulata P. janeirensis A. corrugata H. aff tubifera Guitarra sp. M. arcuiris Raspailia (Syringella) sp.
a
Family Guitarridae Halichondriidae Tedaniidae Axinellidae Polymastiidae Axinellidae Chalinidae Guitarridae Mycalidae Raspailiidae
Authors Muricy et al., 2001 Duchassaing and Michelotti, 1864 Ridley and Dendy, 1886 Boury-Esnauls, 1973 George and Wilson, 1919 George and Wilson, 1919 Lerner and Hajdu, 2002
Extracts testeda O+A O+A O+A O+A O+A O+A O+A O+A O+A O+A
sequentially extracted five times with a methanol/toluene mixture (3:1, v/v) by maceration over 5 days. The resulting extract solution was then filtered and concentrated in a Rotavapor. For the chemotatic, antibacterial and antifungal assays, both the aqueous and organic extracts were suspended in Hanks buffer and Tween 80 (9:1, v/v) at a concentration of 100 Ag/ml (w/v). The preparation of the extracts for the in vitro antitumour assays is described in Section 2.4. 2.3. Cell culture maintenance The HT29 human colon adenocarcinoma (ATCC No. HTB-38), NCI-H460 human large cell lung carcinoma (ATCC No. HTB-177) and U373 human glioblastoma astrocytoma (ECACC No. 89081403) cell lines were maintained as exponentially growing cultures in RPMI 1640 culture medium, supplemented with 10% foetal bovine serum, pH 7.4. All cell lines were cultured at 37 jC in an atmosphere of 5% CO2 in air (100% humidity). 2.4. Cytotoxic screening assay As part of our general anticancer screening programme, all extracts were initially tested at a concentration of 100 Ag/ml against HT29 cells to eliminate extracts which did not demonstrate cytotoxic activity. HT29 cells were seeded into 96-well cell culture plates and incubated overnight to allow adherence. The extracts were dissolved in 100% DMSO and added to the wells in triplicate at a final concentration of 100 Ag/ml (final DMSO concentration 0.25% (v/v) at which no growth inhibitory effects were observed). Both culture medium alone and culture medium plus vehicle (0.25% DMSO) controls were used. Following addition of the extracts, plates were incubated for 72 h, after which cellular growth was determined using the sulforhodamine B (SRB) colourimetric protein assay (Skehan et al., 1990). Extracts which produced an SRB absorbance lower than that of the time-zero value (approximately 10% of the control growth), generated by cellular fixation, using 50% TCA, immediately prior to the addition of the extracts, were considered to be cytotoxic and submitted for further investigation.
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2.5. Growth inhibition assay The IC50 (concentration at which cellular growth is inhibited by 50%) was determined against HT29, NCI-H460 and U373 cells (3.5, 1 and 2.5 103 cells/well, respectively) using the methods described above (Section 2.4). Cells were treated in triplicate with a log10 concentration range (0.1, 1, 10 and 100 Ag/ml) of each extract for 72 h. The IC50 values were estimated from a semi-log plot of extract concentration against SRB absorbance as a percentage of vehicle control growth (0.25% DMSO). 2.6. Antichemotactic assay Chemotaxis was measured by the method described previously by Zigmond and Hirsch (1973). Prior to the chemotactic assay, rat leukocytes were treated with 100 Ag/ml of sponge suspension at 37 jC for 1 h. Plasma collected from rats was incubated at 37 jC for 30 min with 65 Ag/ml of lipopolysaccharide (from Escherichia coli), following which the plasma was diluted in Hanks buffer 1:5 (v/v). Chemotaxic migration of leukocytes through an 8.0-Am nitrocellulose filter, towards the chemotactic stimulant (lipopolysaccharidetreated plasma), was measured after incubation for 1 h at 37 jC using the micrometer on the fine-focus knob of the microscope. The distance from the top of the filter to the farthest plane of focus still containing two cells, in five microscopic fields, allowed the evaluation of leukocyte migration. The assay was carried out in duplicate. 2.7. Antibacterial assay Antibacterial activity was determined against cultures of E. coli (ATCC 25922), Staphylococcus aureus (ATCC 6538P), Staphylococcus epidermidis (ATCC 12228), Bacillus subtilis (ATCC 6633) and Micrococcus luteus (ATCC 9341) using the agardiffusion assay method (Limberger et al., 2001). Ten plates were tested against each sponge sample at 2.5 mg/ml (five per aqueous extract and five per organic extract). Chloramphenicol (400 Ag/ml) was used as a positive control. Following incubation at 37 jC for 24 h, the diameters (mm) of the growth inhibition halos were determined using a pachymeter. 2.8. Antifungal assay Cultures of Candida albicans (ATCC 10231) and Saccharomyces cerevisiae (ATCC 1600) were treated with sponge extracts (2.5 mg/ml) as previously described in the antibacterial assay (see Section 2.7). Nistatin was used as a positive control at a concentration of 3 mg/ml. Following incubation at 25 jC for 18 h, the diameters (mm) of the growth inhibition halos were measured using a pachymeter.
3. Results and discussion Table 2 shows the results of the in vitro testing of sponge extracts against the HT29 colorectal tumour cell line. This is the first step in our anticancer drug development
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Table 2 In vitro screening of sponge extracts (100 Ag/ml) against the HT29 human colorectal tumour cell line Species Cell growtha (% of control growth) Organic Guitarra sp. P. citrina T. ignis P. reticulata P. janeirensis A. corrugata H. aff tubifera Guitarra sp. M. arcuiris Raspailia (Syringella) sp. 92b 68 F 19 44 F 9 91 F 8 0.4 F 0.3* na 1 F 1* 44 F 5 2 F 1* 1 F 1* Aqueous 89 F 13 97 F 3 90 F 10 94 F 4c 65 F 17 94 F 2 37 F 5 103 F 3 63 F 17 104 F 6
a Values were determined using the SRB protein assay after 72 h continuous exposure to the extracts. All values given are the mean F S.D. of z 3 separate experiments, unless otherwise stated. na = Not tested. b n = one experiment. c n = two experiments. * Extracts with cytotoxic activity at 100 Ag/ml (i.e. those extracts whose growth is less than the time-zero control = 10% of control growth).
programme and is designed to identify those extracts with cytotoxic activity. Four sponge species (organic extracts) were found to be cytotoxic at 100 Ag/ml, namely, Polymastia janeirensis, Haliclona aff tubifera, Mycale arcuiris and Raspailia (syringella) sp. These four active extracts were further tested against three human tumour cell lines, derived from different tumour types, over a concentration range (0.1 100 Ag/ml) to determine their potency (IC50 50% inhibition of cell growth), the results of these tests are shown in Table 3. The four extracts displayed similar levels of activity across the panel of three cell lines, the IC50 values ranged from 25 to 50 Ag/ml. These extracts are currently undergoing further investigation through collaboration with the Natural Products Branch of the American National Cancer Institute (NCI). Using a 60-cell line panel derived from a range of different tumour types, the NCI can help identify extracts with interesting activities by the pattern and degree of activity across their human tumour cell line panel. To date, a number of growth inhibitory, cytotoxic and other pharmacologically active molecules have been isolated from species of Porifera related to those tested in this study; these include alkaloids, macrolides and peptides and are summarized in Table 4.
Table 3 In vitro growth inhibitory activity of sponge extracts against human tumour cell lines Species Extract type IC50 (Ag/ml)a HT29 H. aff tubifera P. janeirensis M. arcuiris Raspailia (Syringella) sp. Organic Organic Organic Organic 28 F 4 42 F 7 31 F 1 27 F 6 U373 31 F 3 33 F 3 50 F 17 32 F 2 NCI-H460 30 F 1 28 F 5 27 F 1 25 F 0
a IC50 values were determined using the SRB protein assay after 72 h continuous exposure to the extracts. All values given are the mean F S.D. of z 3 separate experiments.
N.R. Monks et al. / J. Exp. Mar. Biol. Ecol. 281 (2002) 112 Table 4 Literature reports of cellular activity Species Axinella sp. Compounds Chemical class Pharmacological activity Reference Pettit et al., 1994a Pettit et al., 1994a,b Pettit et al., 1994b Supriyono et al., 1995 Luduena et al., 1995 Rudi et al., 1997, 1999 Charan et al., 1996; Clark et al., 1998 Blackburn and Faulkner, 2000; Blackburn et al., 1999 Randazzo et al., 2001 Erickson et al., 1997 Shen et al., 1996 Shen et al., 1996 Fusetani et al., 1989 Sevcik et al., 1994
A. carteri
Halichondrin B, Macrocyclic Growth inhibitory Homohalichondrin B lactones Axinastatins Cycloheptapeptides Growth inhibitory Hymenistatin Cyclo-octapeptide Growth inhibitory Hymenialdisines Alkaloids Cytotoxicity Halistatin 2 Macrocyclic Binds tubulin lactones Sodwanones Triterpenes Cytotoxicity Haliclonacyclamines Alkaloids Cytotoxicity Adociasulfates Meroterpenoid Inhibits Kinesin
H. viridis
Alkylpyridine Hexapeptides
Mycale sp.
Mycalamides
Pateamine
Macrolide
Peloruside A Thiomycalolides Onnamide Mycalisines A and B M. magellanica Mycalolides M. micracanthoxea Mycalazols Raspailia sp. Asmarines
Cytotoxicity Cytotoxicity Cytotoxicity Cytotoxicity Cytotoxicity Nerve potassium permeability In vivo antitumour Baslow and Turapaty, 1969 Cytotoxicity Rashid et al., 2000 Inhibitor of nitric Venkateswara Rao oxide synthetase et al., 1998 Cytotoxicity Kashman et al., 1999 Cytotoxicity, Shin et al., 1998 inhibitors of Na+/K+-ATPase. Cytotoxicity Ogawara et al., 1991; Perry et al., 1988, 1990; West et al., 2000b Cytotoxicity, Hood et al., 2001; Immunosupressant Remuinan and Pattenden, 2000 Cytotoxicity West et al., 2000a Cytotoxicity Matsunaga et al., 1998a Cytotoxicity Burres and Clement, 1989 Inhibitors of Kato et al., 1985 cell division Cytotoxicity Matsunaga et al., 1998b Cytotoxicity Ortega et al., 1997 Cytotoxicity Yosief et al., 2000, 1998
The inflammatory reaction consists of three fundamental processes: (1) hemodynamic changes, (2) alterations in vessel permeability and (3) accumulation of inflammatory cells. The directed migration of inflammatory cells along a chemical gradient is termed che-
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motaxis. The activation of this process appears to be an important mechanism by which the immune effector cells are located at sites of inflammation. Based on this, extracts from marine sponges were analyzed for their activity on polymorphonuclear (PMN) leukocytes chemotaxis. Six species, P. janeirensis, M. arcuiris, Raspailia (syringella) sp., Guitarra sp., Tedania ignis and Pseudaxinella reticulata all significantly ( p V 0.05) reduced the migration of the leukocytes through a nitrocellulose filter towards the chemotatic stimulant (Table 5). Interestingly, the antichemotactic activity was seen solely in the aqueous extracts, whereas extracts which demonstrated in vitro cytotoxicity were all from the organic phase. Two of the sponge species tested demonstrated antibacterial activity (Table 6). The aqueous extract from Axinella corrugata showed weak activity (7 11 mm halo) against M. luteus. The organic extract from H. aff tubifera demonstrated activity against each of the bacteria tested (weak, 7 11 mm), with the exception of B. subtilis, the strongest activity (moderate, 11 16 mm halo) was seen against E. coli. The aqueous extract from H. aff tubifera showed weak activity against M. luteus. A number of antimicrobial molecules have already been isolated from related sponge species, these are outlined in Table 7. None of the species tested demonstrated activity in antifungal tests against the species C. albicans and S. cerevisiae (NistatinC. albicans and S. cerevisiae, 7 11 and 11 16 mm halos, respectively).
Table 5 Antichemotatic activity of marine sponge extracts Sponge species Control Guitarra sp. P. citrina T. ignis P. reticulata P. janeirensis A. corrugata H. aff tubifera Guitarra sp. M. arcuiris Raspailia (syringella) sp. Extracta O A O A O A O A O A O A O A O A O A O A PMN migrationb(Am) 123.0 F 0.1 122.7 F 2.1 40.0 F 0.1* 121.0 F 1.1 121.2 F 2.0 122.3 F 1.5 18.9 F 0.0* 121.3 F 1.1 25.3 F 0.0* 122.3 F 1.5 19.0 F 0.02* C Cy C 122.17 F 2.19 Cy 119.0 F 3.0 122.67 F 1.3 13.0 F 0.02* Cy 35.33 F 1.03*
All values given are the mean F S.D. of 10 separate experiments. a AAqueous extract, OOrganic extract. b CCellular wall damage, CyCytotoxic. * Statistically significantp < 0.05 (Students t-test).
N.R. Monks et al. / J. Exp. Mar. Biol. Ecol. 281 (2002) 112 Table 6 Antibacterial activity of the Brazilian marine sponges Species Chloroamphenicol Guitarra sp. P. citrina T. ignis P. reticulata P. janeirensis A. corrugata H. aff tubifera Guitarra sp. M. arcuiris Raspailia (Syringella) sp. Extract O A O A O A O A O A O A O A O A O A O A E. coli *** ** S. aureus ** * S. epidermidis *** * B. subtilis **
M. luteus *** * * *
( ) No activity, (*) weak activity (7 11 mm halo), (**)moderate activity (11 16 mm halo), (***) high activity (>16 mm halo).
Over the past decade, interest in marine natural products has dramatically increased, and consequently, a number of novel molecules have, or are being, developed as pharmaceuticals, e.g. Cytarabine, Ecteinascidin 743, Manoalide and Spongistatin 1 to name only a few.
Table 7 Literature reports of antimicrobial activity Species Compounds Chemical class Pharmacological Reference activity Antibacterial Antimicrobial Antifungal Antibacterial, Antifungal. Antibacterial Antimicrobial Antifungal Inhibitors of reverse transcriptase Antiviral Urban et al., 1999 Wratten and Meinwald, 1981 Bhosale et al., 1999 Charan et al., 1996 Crews and Harrison, 2000 Fahy et al., 1988 Baker et al., 1988 Shin et al., 1998
Axinella sp. Axinellamines Alkaloids A. polycapella Hydroxyhydroquinone, 2,2V ,5,5V ,4,4V -Hexahydro-biphenyl Haliclona sp. Crude extract Haliclonacyclamines Alkaloids Haliclotriol Haliclonadiamine Papuamine Osirisynes Terpeneketides Alkaloid Alkaloid Oygenated polyacetylenes Mycalamides
H. osiris
Mycale sp.
Mycalamides
10
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Often present as defense mechanisms, these potent marine compounds provide pharmaceutical researchers with a novel platform for the development of new drugs to treat serious diseases such as cancer, bacterial infection and arthritis. Here, we have shown that a number of sponge species have activity in in vitro models systems, which are directly relevant to human disease. Studies of this nature highlight the potential of marine product screening programmes through which, without question, will identify new drugs from the vast, untapped, resources that are within our oceans.
4. Conclusions Sponges species collected from the State of Santa Catarina in southern Brazil have been shown to posses a number of biological activities. The most interesting species are H. aff tubifera, P. janeirensis, M. arcuiris and Raspailia (syringella) sp.antineoplastic; P. janeirensis, M. arcuiris, Raspailia (syringella) sp., Guitarra sp., T. ignis and P. reticulataantichemotactic and A. corrugata and H. aff tubiferaantibacterial. To the best of our knowledge, this is the first report demonstrating antineoplastic, antichemotactic and antibacterial activity of these species of porifera. These species are currently undergoing detailed investigations with the objective of isolating and identifying the molecular species responsible for the activities demonstrated in this report. Furthermore, the encouraging biological activities seen in this study show that the Brazilian coastline is a potential source of sponge species worthy of further investigation. In light of the work presented here, we have initiated further collection programs off the coastlines of Sao Paulo (southeast) and Pernambuco (northeast). Acknowledgements This work was supported by grants from FAPERGS, CNPq, CAPES and the SOAD foundation. The authors would also like to thank Dr. Miriam Anders Apel for her technical assistance with this work. [SS] References
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