FROM INDO-PACIFIC TO ATLANTIC OCEAN

In: Oysters Editor: Judith P. Turner

ISBN: 978-1-62948-806-6 © 2014 Nova Science Publishers, Inc.

Chapter 3

FROM INDO-PACIFIC TO ATLANTIC OCEAN: PROBLEMS AND RISKS RELATED TO AN EXOTIC OYSTER INTRODUCTION CASE Mauro André Damasceno de Melo* 1 and Guilherme da Cruz Santos Neto2 1

Instituto Federal de Educação Ciência e Tecnologia do Pará, Campus Bragança, Brasil 2 Instituto Federal de Educação Ciência e Tecnologia do Pará, Campus Abaetetuba, Brasil

ABSTRACT The genus Crassostrea is comprised of a large number of oyster species distributed worldwide, the exact number of which remains uncertain due to high morphological plasticity in shell characteristics as a result of environmental influence. Transplantation of non-native species among new habitats involves a great deal of concern about potential environmental problems and it is extremely important to ensure that the economic benefits of these non-native species are balanced with the potential risks. The arrival of a non-native species in a new environment may cause unpredictable changes in coastal ecosystems. Studies have shown that oyster introductions often disturb habitat structure, causing changes in trophic dynamics and reducing the native populations through *

[email protected].

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M. A. Damasceno de Melo and G. da Cruz Santos Neto competition. Along the Brazilian coast two native (Crassostrea gasar and Crassostrea rhizophorae) and two exotic (Crassostrea gigas and Crassostrea sp.) cupped oyster species are observed in natural beds. The Asian origins of the species Crassostrea sp. is confirmed by genetic and its introduction in Brazilian coast occurred probably due to the ballast seawater. Frequent monitoring is necessary to identify oysters, especially near harbor areas where discharges of ballast water and hull encrustations are potential sources of exotic oysters. In this study we not only confirmed the introduction of the Asian specie Crassostrea sp., in terms of rRNA 16S and cytochrome c oxidase I (COI) genes, but also presented a review for the distribution data of this species along the Brazilian coast and tested a protocol for a fast identification without DNA sequencing. Phylogenetic results exhibited high levels of similarity between Brazilian and Chinese samples of Crassostrea sp. and the reaction using Internal Transcribed Spacer I (ITS1) fragment presented as a suitable procedure for fast identification of this species, providing a 621bp fragment for Crassostrea sp. and 718bp fragment for the species C. gasar, C. rhizophorae and C. gigas. It is already known that this exotic species is considered a problem for oyster culture in the northern coast of Brazil. Seeds of native (C. gasar) and non-native (Crassostrea sp.) species are collected simultaneously in artificial spat collectors near natural beds and grow on tables. However, the non-native species does not reach commercial length representing a considerable economic loss for the farmers. The same miss-identification of the seeds in terms of morphological traits observed in this region, may produces an equal waste of effort and time during the cultivation process, which may occur in other cultivation sites along the Brazilian coast. These results reinforce the need of appropriate regulations to control the release of ballast water in order to prevent environmental problems and exhibited an efficient genetic procedure to monitoring and management programs.

INTRODUCTION Transplants of non-native species among new habitats involves a great deal of concern about potential environmental problems and it is extremely important to ensure that the economic benefits of these non-native species are balanced with the potential risks (Guo et al. 2009). Exotic bivalves are widely used in aquaculture programs around the world mainly for better growth or disease resistance, among other characteristics that reduce costs in comparison to native species (Mckindsey et al. 2007). The last two decades were marked by research involving the relationship between aquaculture and the expansion of exotic species (Carlton 1992, Carlton 1999, Naylor et al. 2001, Streftaris et


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al. 2005). Some countries have implemented laws and eradication programs regulating the introduction of non-native species of oysters, for example Crassostrea ariakensis Fujita, 1931 in Chesapeake Bay, USA (http://www.dnr.state.md.us/dnrnews/infocus/oysters.asp) and Crassostrea gigas (Thunberg 1793) in Brazil (IBAMA Administrative Edict n. 145-n, of October 29, 1998) and Australia (Ayres, 1992; Nell and Perkins, 2005). The arrival of a non-native species in a new environment may cause unpredictable changes in coastal ecosystems (Crooks, 2002). Studies have shown that oyster introductions often disturb habitat structure, causing changes in trophic dynamics and reducing the native populations through competition (Ruesink et al., 2005; McKindsey et al., 2007; Krassoi et al., 2008). C. gigas has been introduced all over the world for oyster-farming and its accidental spread to the natural environment has been observed in Denmark, Norway, Sweden (Wrange et al., 2010), New Zealand (Dinamani 1971), the southwestern Atlantic (Orensanz et al., 2002) and South Africa (Robinson et al., 2005). In Australia, the Pacific oyster (Crassostrea gigas) competes with the native Sydney rock oyster (Saccostrea glomerata) and regulations were implemented to control its importation in some areas (Nell and Perkins, 2005). In the Wadden Sea, in Belgium and in France, C. gigas formed dense reefs, changing the original habitat and significantly altering the overall biodiversity and biomass (Kerckhof et al., 2007; Cognie et al., 2006). According to Robinson et al., (2005), the increase in the population size of the naturalized oyster C. gigas in Breede estuary (South Africa) probably caused a decrease in their food supply, since the condition index of this population was lower when compared with populations of the same species from two other estuaries. The intentional introduction of C. gigas in Australia reduced the market value of the native oyster Saccostrea glomerata due to the faster growth rate of C. gigas, which reaches a market size in almost fifteen months compared to three years for S. glomerata (Smith et al., 1986). The use of non-native species is usually supported by the oyster culture production worldwide reaching 4.5 million tons in 2010 with the genus Crassostrea representing the fourth largest fisheries resource in the Western Atlantic (FAO, 2010). C. gigas is the most commonly farmed species of oyster worldwide and its cosmopolitan range is the result of decades of anthropic human introductions. The records compiled by Ruesink et al., (2005) revealed that 66 out of 168 introductions were of C. gigas, mostly to temperate zones worldwide. Most of these introductions and translocations of the Pacific oyster were for aquaculture (Ruesink et al., 2005). In Brazil, C. gigas was first introduced at Rio de Janeiro (Cabo Frio) in 1974 (Littlepage and Poli, 1999).


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M. A. Damasceno de Melo and G. da Cruz Santos Neto

In 1975 the São Paulo State Fisheries Institute brought C. gigas seed from Japan to Cananéia, São Paulo (Akaboshi 1979, Akaboshi et al. 1983) and in 1981 the Bahia Biology Institute imported C. gigas seed from Great Britain to implement oyster culture in north-eastern Brazil (Ramos et al. 1986). Currently is the main commercial oyster farmed in the cooler waters of southern Brazil (Santos et al., 2011). Recently, Melo et al., (2010a) detected the occurrence of the Pacific oyster in natural habitats in Brazil's southern coast. According to the latter authors, seed selection processes for greater growth rates and survival over several generations, may also have selected for better adaptation to higher summer temperatures. The existence of a second non-native species of oyster was molecularly identified by Varela et al., (2007) at Canela Island (municipality of Bragança, Pará state, northern Brazil). In terms of 16S rRNA and cytochrome oxidase c sub-unit I (COI) fragments, the species Crassostrea sp. has been defined as a non-native species very similar to Indo-Pacific oysters (Varela et al., 2007; Melo et al., 2010b) and its arrival at the mangrove coast of northern Brazil remains unclear. Recently, this species was identified in the southern coast of Brazil (Galvão et al. 2012; Neto et al. 2012). Its restricted range at sampling sites along the Brazilian coast may indicate that it was recently introduced by international shipping traffic. Various examples of accidental introductions of exotic species through the release of ballast seawater and via external fouling and boring communities on ships have been documented (Ó Foighil et al., 1998; Farrapeira et al., 2011). Frequent monitoring is necessary to identify oysters, especially near harbor areas where discharges of ballast water and hull encrustations are potential sources of exotic oysters. However, because of strong phenotypic plasticity, the use of morphological traits has been considered an unreliable method of differentiation among oyster species (Gunter, 1950). The external characteristics of these bivalves are highly variable and influenced by the environment (Lam and Morton, 2003). Taxonomic uncertainties as a result of variable external characteristics have been recently clarified using molecular approaches (Lam and Morton, 2003; Wang et al., 2004; Varela et al., 2007; Reece et al., 2008; Melo et al., 2010b). The correct identification of the species is an essential step in oyster research and aquaculture (Liu et al., 2011). Molecular markers have become important tools for the identification of marine organisms. In particular, a number of genetic markers have been applied in order to answer questions about identification and taxonomy of the Crassostrea genus. DNA sequencing (Ó Foighil et al., 1998; Huvet et al., 2000; Boudry et al., 2003; Wang et al., 2008; Melo et al., 2010a) and



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restriction fragment length polymorphism (Patil et al., 2005; Pie et al., 2006; Wang and Guo, 2008), are two examples of such molecular procedures. However, these approaches are usually time-consuming, not always readily accessible and are expensive. Fast, effective and cheaper methods of identification are needed for monitoring the presence of exotic oysters in natural habitats along the Brazilian coast. The present study not only describes the development of a rapid and low cost molecular protocol, for the identification of the non-native oyster Crassostrea sp. in Brazil using only polymerase chain reactions (PCR), but also reports its distribution along the Brazilian coast and confirms, in terms of phylogenetic study, the Chinese origins of this species.

MATERIAL AND METHODS Fast Detection Protocol for Crassostrea sp. Oysters A total of 158 samples, collected along the Brazilian coast, were tested for ITS1 primers (Table 1). The adductor muscle of all oysters was preserved in 100% ethanol and stored at -20°C until genomic DNA was extracted using the phenol-chloroform protocol of Sambrook et al., (1989). All collected samples were previously identified by means of COI and/or rRNA16S sequencing according to Melo et al., (2010b) and Varela et al., (2007), respectively. Primers for the Internal Transcribed Spacer 1 (ITS1) region were originally designed and described by Pleyte et al., (1992) using the conserved 18S and 5.8S ribosomal DNA genes flanking the spacer region in salmonids: ITS1F 5'- AAA AAG CTT TTG TAC ACA CCG CCC GTC GC -3' and ITS1R 5'-AGC TTG CTG CGT TCT TCA TCG A -3'. This pair of primers was tested in different species of oysters (C. gasar, C. rhizophorae, C. gigas and Crassostrea sp.) found along the Brazilian coast (Figure 1). To measure interspecific variation in the ITS1 fragment between Crassostrea and Ostrea species, a set of 10 sequences (nine of Crassostrea and one of Ostrea) from different species were obtained from GenBank and compared according to the ITS1 region, including the 18S rRNA and 5.8S rRNA flanking regions amplified by the primers (Figure 2). PCR reaction for the ITS1 primers was performed in a 25µL reaction volume with 0.25 µL(5 U/µL) of Taq Polymerase (Invitrogen®, Carlsbad, CA, USA), 2.5 µL (100 ng) of template DNA, 0.75 µL (1.5 mM) of MgCl2, 4.0 µL (2 mM) of dNTPs, 2.5 µL of 1 x buffer solution and 0.25 µL (0.3 µM) of each


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primer. The reaction protocol for the ITS1 fragment consisted of an initial denaturing of 94°C for 2 min, 35 cycles of 94°C for 1 min, 60°C for 1 min, 72°C for 1 min, with an additional extension of 72°C for 5 min during the last cycle. A 3µL PCR product of each sample was subjected to electrophoresis running for 80 minutes (50V) in a 1.5% agarose gel dyed in ethidium bromide and subsequently visualized under UV-light.

Figure 1. Map of Brazil indicating the sampling locations for mangrove oysters.

Figure 2. Length variability of ITS1 fragment from 10 different species obtained from GenBank, including Crassostrea and Ostrea. 18S rRNA and 5.8S rRNA exhibited 187bp and 58bp respectively in both genus analyzed.



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Table 1. Species, number of oysters and origin of the samples used for testing the efficiency of the primer ITS1 as a marker Specie Crassostrea gasar Crassostrea sp. Crassostrea sp.

Number of Municipality (locality) samples 25 Augusto Corrêa (Nova Olinda) 25 Augusto Corrêa (Nova Olinda) 25 Bragança (Canela Island)

Crassostrea rhizophorae 50 Crassostrea gasar

25

Crassostrea gigas

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Coordinates

01°05’27.2”S, 46°28’28.6”W 01°05’27.2”S, 46°28’28.6”W 00°47’02”S, 46°43’32.9”W São Francisco do Conde 12°40'0.0"S, 38°38'60"W (Ilha das Fontes) Santos 23°52'18.55''S, 46°22'18.10''W Florianópolis oyster farm (matrices in captivity)

Phylogenetic Analysis COI mitochondrial gene was amplified according to procedures described by Melo et al., (2010b). Partial COI strands (538bp fragment) previously sequenced for species of Brazilian coast Crassostrea gasar, Crassostrea rhizophorae, Crassostrea gigas and Crassostrea sp. were submitted to the sequencing reaction (ABI Prism™ Dye Terminator Cycle Sequencing Ready Reaction, Applied Biosystems) and were carried out into an automatic sequencers (Applied Biosystems model 3500), according to the manufacturer’s protocols. Partial COI sequences of Crassostrea angulata (AF152567), Crassostrea nippona (AF300616), Crassostrea iredalei (AY038078), Crassostrea belcheri (AY038077), Crassostrea ariakensis (AF152569), Crassostrea virginica (AF152566), Crassostrea sp. (HQ661024), Saccostrea sp. 1 (HQ661030), Saccostrea sp. 2 (HQ661031), Saccostrea cucullata (AY038076) and Ostrea edulis (AF120651) were obtained from GenBank and aligned with the four Brazilian species using the programs BIOEDIT 7.0.5.3 sequence editor (Hall 1999) and Clustal X 1.82 (Thompson et al. 1997). The same analysis were performed for the rRNA16S mitochondrial gene (391bp fragment) in Atlantic (Crassostrea gasar, Crassostrea rhizophorae, Crassostrea gigas, Crassostrea sp. and Crassostrea virginica AF092285) and Indo-Pacific (Crassostrea ariakensis AY160757, Crassostrea hongkongensis AY160756, Crassostrea iredalei HQ660981, Crassostrea sp 1 China


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HQ660983, Crassostrea sp 2 China HQ660984, Crassostrea sp 3 China HQ660988, Ostrea sp HQ661001 and Saccostrea sp HQ660994) oyster species, according to procedures described by Varela et al. (2007). Haplotypes were identified by DnaSP 4.0 (Rozas et al., 2003). Phylogenetic analyses were performed with PhyML 3.0 program (Guindon & Gascuel, 2003) using the maximum likelihood (ML) methods. jMODELTEST 2.1.2 software (Posada, 2008) was used to obtain the most appropriate model for use in the ML analysis by AIC correction (Posada, 2008). Evaluation of statistical confidence was based on bootstrapping with 1000 pseudo-replicates for ML (Felsenstein, 1985). The criterion used to estimate robustness was to consider nodes with bootstrap values equal or superior to 90% as well supported.

Figure 3. ITS1 length polymorphism among native and non-native oyster species. Lane 1 and 8, Phi X 174 DNA/Hae III Digest; lane A, B and C, Crassostrea sp. lane D, Crassostrea gigas; lane E, C. gasar; lane F, Crassostrea rhizophorae.

RESULTS Amplification of the ITS1 and flanking regions, using primers ITS1F and ITS1R, were successful in all 158 samples tested producing a PCR fragment length equivalent to 718 bp for the species C. gasar, C. rhizophorae and C. gigas and a shorter fragment of 621 bp for Crassostrea sp. (from northern Brazil) (Figure 3). All 50 samples of Crassostrea sp. from Nova Olinda and Canela Island were amplified, and no evidence of length polymorphism was



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observed. Regarding the 718-bp fragment, all 108 collected samples including C. gasar, C. rhizophorae and C. gigas were analyzed and no evidence of length polymorphism was observed. Considering only the ITS1, the length variation of the Crassostrea and Ostrea sequences obtained from GenBank ranged from 455 bp to 526 bp. Fragments of 18S rRNA and 5.8S rRNA with 187 bp and 58 bp, respectively, were only available in GenBank for the species C. ariakensis, C. gigas, C. nippona and O. edulis and no evidence of length polymorphism was detected after alignment.

Figure 4. Tree of maximum likelihood (ML) method for genus Crassostrea based on rRNA 16S, Saccostrea and Ostrea were used as outgroup. Crassostrea sp. samples from Canela Island and China (Beihai) represent a single clade (bootstrap values: ML 100%). The map represents the cosmopolitan distribution of the genus, indicating the Brazilian North Coast (Nova Olinda and Canela Island samples) and the South China Sea (Beihai and Wenchang samples).


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Figure 5. Mitochondrial rRNA 16S Distance Matrix.

Figure 6. Mitochondrial COI Distance Matrix.

The rRNA 16S phylogenetic results exhibit an evident division of species in Atlantic and Indo-Pacific groups as previously suggested by Varela et al. (2007). The ML tree joined C. rhizophorae, C. gasar and C. virginica in the same clade (bootstrap values: ML 100%) and grouped Crassostrea sp. samples from Canela Island and Beihai in a single clade (Figure 4). Distances between



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Crassostrea sp. species inside this group ranged from 0.000 to 0.003. An increased genetic distance was observed when compared to Crassostrea sp. China 1 sample collected in Wenchang, Hainan, China ranging from 0.095 to 0.099. COI analysis suggested the same topology presenting high levels of genetic distance between Wenchang and Canela Island (0.0215). Both phylogenetic markers not only confirms the high similarity between Crassostrea sp. species from Nova Olinda/Canela Island and Beihai but also suggest that Brazilian North coast and Wenchang Crassostrea sp. species are different due to the high levels of genetic distance observed for rRNA 16S (Figure 5) and COI (Figure 6) fragments .

DISCUSSION Due to the phenotypic plasticity of Crassostrea genus, DNA sequencing is the most common approach used in species identification of oysters (Littlewood, 1994; Ó Foighil et al., 1995; Ó Foighil et al., 1998; Huvet et al., 2000; Lapègue et al., 2002; Boudry et al., 2003; Lam and Morton, 2003; Wang et al., 2004; Varela et al., 2007; Wang et al., 2008; Melo et al., 2010a; Melo et al., 2010b; Liu et al., 2011) and is certainly the most powerful. On the other hand, this technique is also the most expensive, time-consuming and not always the most appropriate for routine identification of large numbers of oysters. Another common approach for oyster identification is RFLP-PCR, a widely used technique for species identification when the DNA sequence has different targets recognized by restriction enzymes (Klinbunga et al., 2005; Pie et al., 2006; Melo et al., 2010a). However, this procedure usually uses more than one gene or enzyme which increases the cost of species identification. The identification of a conserved COI region at intraspecific level with high inter-specific variability made possible the design of a primer for C. gigas, which does not amplify COI in other oysters from the Brazilian coast. The development of a species-specific genetic marker for this non-native species is undoubtedly important for research in oyster culture and ecology as well as for environmental agencies that monitor exotic species in natural habitats. The recent identification of C. gigas in natural beds in southern Brazil (Melo et al., 2010a) is another example of the spread of this exotic oyster to natural habitats. Selection processes used to obtain higher growth and survival rates over several generations may have resulted in greater resistance of these oysters to higher temperatures (Melo et al., 2010a). The main concern over C. gigas recent range expansion is the potential for direct or indirect competition


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with native oysters and other benthic species. There is thus an urgent need for frequent monitoring of exotic oysters and their interactions with the native coastal biota in southern Brazil. Further south, in Argentina, C. gigas was introduced in 1982 to start oyster culture programs. However, between 1998 and 2000, an increase in the settlement of this species was observed in the wild, producing a more complex architecture of intertidal communities when compared to the same environment before the introduction, due to the development of oyster reefs (Orensanz et al., 2002). The non-native species Crassostrea sp. has been a problem for oyster farmers in northern coast of Brazil. The inappropriate identification of this species in terms of morphological traits provides a considerable economic loss during the cultivation procedure. Seeds of Crassostrea sp. and C. gasar are collected simultaneously from artificial spat collectors and placed to grow in tables positioned in the sub tidal zone (Figure 7). However, the exotic oyster Crassostrea sp. does not reach commercial length incoming, an economic loss of 70-80% during the dry season (Figure 8). The ITS1 protocol presented in this paper may be a suitable tool for the prior identification of potential sites for C. gasar spat collection in natural habitats and where localities with high concentrations of Crassostrea sp. may be avoided. In this study, C. gasar, C. rhizophorae and C. gigas did not show intraspecific variation in ITS1 length. Moreover, all individuals were indistinguishable according to their ITS1 size in the agarose gel, except for Crassostrea sp. and so this marker is suitable for the identification of the latter species along the Brazilian coast and probably in other parts of the world. The 621 bp length fragment of the exotic oyster Crassostrea sp. was specific for this species and clearly distinguishable from the other three species of Crassostrea from the Brazilian coast when analyzed with the 718 bp length fragment. The approach presented here should unambiguously allow identification of the exotic oyster Crassostrea sp. species, as its isolated fragment is smaller in length than those of the other species of Crassostrea. The phylogenetic results presented in this study confirm the Chinese origins of the exotic oyster Crassostrea sp. The same was detected by Galvão et al. (2012) comparing Crassostrea sp. samples from Cananéia, São Paulo and sequences of Crassostrea sp. from Beihai, China (Liu et al. 2011), reporting the establishment of this species in southeast coast of Brazil. Neto et al. (2012) observed an increased number of larvae and spat in the estuary of Guaratuba (South coast of Brazil) during the summer, with the prevalence being the Crassostrea sp. species. The same situation is observed in the North Coast when this species is frequently observed in artificial spat collectors,



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probably due to the high levels of salinity observed during this period. Ballast water may be the most reasonable cause for the arrival of this Indo-Pacific species at the northern and southern coast of Brazil (Melo et al., 2010b, Neto et al., 2012, Galvão et al. 2012). In the North coast cargo ship routes link Vila do Conde harbor (1°32'22.62''S, 48°45'12.17''W) in Pará state, Brazil with diverse parts of the world (Companhia Docas do Pará, 2012), the same situation observed in Santos harbor, São Paulo (southeast coast) (Galvão et al., 2012) and Paranaquá Harbor, Paraná (south coast) (Neto et al., 2012). Currently, shipping is considered the most common vector for introductions of benthic marine exotic species (Farrapeira et al., 2011).

Figure 7. Artificial spat collector and fixed tables in Nova Olinda oyster farm.

The use of molecular tools to identify the correct species of oyster during the cultivation process is extremely necessary to avoid economic loss associated to growth performance, since the same adverse problem observed in Nova Olinda oyster farm may occur in any oyster stock. However, to obtain a better improvement of oyster farms along the Brazilian coast, we suggest the


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implementation of more effective laws and eradication programs regulating the release of ballast waters, not only avoiding future introductions of exotic oysters but also preventing the introduction of non-native predators and parasites.

Figure 8. Samples from oyster farm in Nova Olinda (Brazilian North coast) after 8 months of cultivation process. In A, Crassostrea gasar and B, Crassostrea sp.

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