USE OF ARTIFICIAL COLLECTORS TO OBTAIN OYSTER SEEDS IN BABITONGA BAY, SANTA CATARINA, BRAZIL

The aim of this study was to evaluate oyster seed settlement pattern in artificial collectors affixed along Babitonga Bay (26°28’S – 48°50’W), Santa Catarina state, Brazil. Artificial collectors were installed in the upper tidal range (supralittoral), in the line of intermediate variation of the tide (mesolittoral), and below the limit region of the low tide (infralittoral). A total of 7,579 seeds were obtained over a year, presenting clear variations of quantities and species according to the seasons, depths and collection sites. Among the total number of seeds collected, 284 were submitted to molecular analyses for species identification, among which 5.63% belonged to Crassostrea gasar, 16.9% to C. rhizophorae, 0.35% to C. gigas, 24.29% of Ostrea sp. and 52.81% to C. talonata. Species identification through morphological analysis is difficult because of the inherent polymorphism of shells. Results demonstrate that artificial collectors present a higher performance for the settlement of oyster species inappropriate for farm cultivation (C. talonata and Ostrea sp.). Moreover, C. talonata is an exotic species from Indo-Pacific seas recently confirmed along Brazilian coasts, and it already possesses a high recruitment rate in the estuarine region. This raises environmental and commercial concerns about the maintenance of the natural oyster population in Babitonga Bay.


INTRODUCTION
The collection of native oyster seeds in the natural environment for cultivation purposes has already been used in some brazilian regions such as the estuarine complex-lagoa de Iguape-Cananéia-Paranaguá (Pereira et al., 2003). Recent studies conducted in the northern region of Brazil have determined the period of the year for seed collection, defined types of collectors as well as environmental conditions that maximize the recruitment of seeds (Garduncho et al., 2012;Funo et al., 2019). However, more than one species of native oyster is expected to occur sympatrically among our littoral zone (Carranza et al., 2009). Each one possesses different growth rates, but similar continuum of morphological variation. This complicates the organization and cultivation of seeds and adults (Absher, 1989;Nascimento, 1991;Pereira et al., 2003;Galvão et al., 2009;Melo et al., 2010b;Lazoski et al., 2011).
One native oyster species known as Crassostrea gasar (Dautzenberg, 1891) (= C. brasiliana/C. paraibanensis; see Lazoski et al., 2011) has well-known commercial potential as it is a preferable species for ostreiculture in tropical areas (Legat et al., 2017) where the cultivation of C. gigas (Thunberg, 1793) is impractical . Although hatchery protocol for production of seeds is available, their attainment through artificial collectors can be a viable practice as in Senegal ecosystems (Diadhiou and Ndour, 2017), or in Brazil, Guaratuba Bay/Paraná state (Christo et al., 2016) and Curuça/Pará state (Funo et al., 2015(Funo et al., , 2019, where only C. gasar is expected to settle in the plate structures. Evaluation of potential oyster larvae settlement per species is, then, a requirement to test the feasibility of obtaining seeds in artificial collectors in the environment. In particular, it is critical that the correct molecular identification of species in the seed phase be made as a fundamental tool for the development of mangrove oyster farming activities. For decades, studies have suggested the existence of at least two species of oysters of the genus Crassostrea in Brazilian estuaries (Absher, 1989): C. rhizophorae (Guilding, 1828) and C. gasar, a fact later confirmed by genetic marker analysis (Ignacio et al., 2000;Lapègue et al., 2002;Lazoski, 2004;Pie et al., 2006).
However, more recently, the number of invasive oyster species in Brazilian natural stocks has increased substantially. Varela et al. (2007) reported to Ilha Canela in northern Brazil (Bragança, State of Pará) an exotic species of the genus Crassostrea with a strong phylogenetic relationship to Indo-Pacific seas (Wu et al. 2013). Such species was also noted in other areas of the Brazilian coastline (Galvão et al., 2013;Melo et al., 2013), the western coast of Peru (Li et al., 2017), and now in Argentina and southern Brazil, having recently been identified as C. talonata Li and Qi (1994) (Cavaleiro et al., 2019). Melo et al. (2010b) detected the first banks of C. gigas in the natural environment of southern Brazil. Galvão et al. (2017) confirmed the invasion of a lineage of Saccostrea Dollfuss and Dautzenberg (1920) in the Itaguaré River, São Paulo. Therefore, based on the variation in the number of species, the correct identification of oysters in regions with potential for cultivation has fundamental importance for the development of aquaculture, especially in tropical estuarine.
Molecular markers have been used successfully in the delimitation of native oyster stocks. Thus, it has been demonstrated that C. rhizophorae is present from the southern Caribbean to Uruguay, usually found in mangrove roots and rocks in upper mesolithic regions, while C. gasar is generally found in the infralittoral regions from northern South America to the southern region of the state of Santa Catarina, Brazil (Ignacio et al., 2000;Lapègue et al., 2002;Lazoski, 2004;Pie et al., 2006;Varela et al., 2007;Melo et al., 2010a;Lazoski et al., 2011;Galvão et al., 2013) and on the African continent from Mauritania to Angola (Carpenter and De Angelis, 2016).
In southern Brazil, one of the sites with potential for cultivation of native oysters from the genus Crassostrea is Babitonga Bay (26 ° 28'S -48 ° 50'W) where artisanal farmers observed that these animals settle themselves on several natural and artificial substrates. However, the difficulty lies in separating species with growth potential, a problem which stems from the morphological similarities, mainly in the seed phase.
Thus, the present work aimed to identify the recruitment pattern of seeds of oysters in Babitonga Bay as a way to gauge the possibility of obtaining seeds at low cost and also infer the best condition and places where the attainment of seeds of commercial oyster species is maximal.

MATERIAL AND METHODS
The experiment was carried out in Babitonga Bay, which is located on the northern coast of the state of Santa Catarina (26°28'S -48°50'W) ( Figure 1). The bay possesses 160 km 2 with extensive mangrove areas and significant natural oyster stocks (IBAMA, 1998).
At Iperoba, the collectors were installed in a region characterized by sand-muddy shoals under the influence of a small river. At Linguado, the collectors were installed in the margins of a channel 15 m in depth (actual max. depth of 2 m owing to siltation) characterized by intense variation in salinity (IBAMA, 1998). Vila da Glória is located in the central portion of Babitonga Bay and receives fresh water from the rivers relative to the other sampling points, as well as a strong influence of seawater from the effect of tides.
The collectors were made of PVC plates (polyvinyl chloride) with a rough texture (3 mm thick), comprising a set of 5 plates of 10 × 25 cm fixed inside a PVC pipe frame. At each collection point, three (03) sets of plates were distributed from the bottom to the surface of the water column, tied along two bamboo sticks previously buried in the sediment.
The collectors were installed on the surface, immersed only in the high tides, and denominated Surface (supralittoral), in the range of the tide and denominated Middle (mesolittoral) and below the low-water line and denominated Bottom (infralittoral). At each sampling point, structures were installed in triplicate, totaling 45 plates per point or 15 plates in each range of tidal variation. The collection period lasted 12 months. Temperature and salinity were recorded weekly at 30 cm below the surface. Averages and standard deviations were calculated monthly to characterize the summer, autumn, winter and spring seasons. The collection structures were placed and removed at each season of the year and then packed in separate plastic bags, identified and frozen. Once thawed, each plate was analyzed, and the oyster seeds were detached and counted.
The numbers of seeds obtained among the sites (Iperoba, Linguado and Vila da Glória), seasons (summer, autumn, winter and spring), deep level (surface, middle and bottom) at each sampling point and among the points were compared using non-parametric t-test using the PROC MULTITEST (Westfall et al., 1999) with SAS.
The Principal Component Analysis (PCA) was performed using the set of variables and linear combinations that were ordered and placed in descending order by their variances. Pearson Correlation was adopted to obtain the maximum possible information contained in the original variables. For PCA analysis, the XLSTAT 2014 statistical package was used. From the total number of seeds collected, 353 individuals were photographed, and tissue samples were collected for molecular analyses. The mitochondrial 16S sequence was used in the PCR/RFLP process for identification of species.

RESULTS
During the study period, water temperature showed a similar and characteristic seasonal variation in the three sites evaluated with means and standard annual deviations of 23.6 ± 3.07 ºC in Iperoba, 24.1 ± 3.73 ºC in Vila da Glória, and 24.5 ± 2.68 ºC in Linguado ( Figure 2). For salinity, a greater variation was observed, according to the place and time of the year with averages of 27.34 ± 3.0 in Iperoba, 26.8 ± 2.94 in Vila da Glória and 27 ± 6.2 in Linguado ( Figure 2). The maximum and minimum values of salinity occurred in Linguado and ranged from 2 to 34 gL -1 .
A total of 7,579 oyster seeds attached in the artificial collectors throughout the experimental year (Table 1) with height varying from 0.5 to 3.0 cm.  A total of 284 seeds were analyzed genetically, and 52.81% of them were identified as Crassostrea talonata, 16.9% as C. rhizophorae, 5.63% as C. gasar, 24.29% as Ostrea sp. and 0.35% as C. gigas (Table 2). The diversity of external aspects of shells of different species can be seen in Figure 3. Analyzing the results of oysters settled by season, it could be seen that differences in the number of specimens occurred when compared to other seasons. Similarity in recruitment was noticed during autumn and spring ( Table 2). The number of oysters attached to the collectors was higher for C. rhizophorae and Ostrea sp.
In the analyses of total seed quantities attached to artificial collectors per climatic season, significant differences were observed between spring and winter and between autumn and winter (Table 3). No recruitment of C. gasar occurred during winter.
Total seed quantities obtained in artificial plates placed at the surface, middle and bottom strata in all sites showed values with significant differences only between surface and bottom. However, when the total amount of seeds per site was analyzed separately, significant differences were detected only between the surface and bottom strata at the Linguado and Vila da Glória sites (Table 4).
PCA analysis demonstrated a high correlation between the distribution of C. talonata (CT) with bottom stratum (B) in the summer season (SU) at Iperoba (IP). Such grouping of variables was responsible by the formation of component 1, sustaining 78.94% of the total variability of the distribution. The influence of the component was also detected, albeit with less intensity, in the middle (M) and surface (S) strata, though not presenting a significant difference between both strata. These descriptors had a higher correlation with Vila da Glória (VG), which, together with winter (W), were the main factors responsible for the variability of component 2 with 12.67%. For C. gasar (BR), a higher occurrence was noted at the bottom (B) stratum ( Figure 4).
In winter (W) and autumn (A), no significant differences were seen in species fixation. The low fixation rates of C. rhizophorae

DISCUSSION
Differences in the rate of seed attainment in Crassostrea and Ostrea were quite evident among collector sites. Such differences are related to the oceanographic characteristic of each location, following the environmental gradient of the estuary. The physicochemical data at Babitonga Bay presented salinity variation at each site, which is a limiting parameter for the distribution of oyster larvae (Gosling, 2003;Nalesso et al., 2008).
The results showed a higher intensity of larvae recruitment at Vila da Glória, a collection point in the median section of the bay with greater density of individuals during the hotter times of the year. We found the polypropylene plate collectors to be suitable for the recruitment of oysters in the region.
Although Iperoba is closer to the ocean, it has a cove conformation and is strongly influenced by the Monte de Trigo River. Vila da Glória is located in the central body of the bay, and it is characterized by a strong current with a large water flow from both mountain and sea. Linguado exhibits the greatest variation of salinity, as recorded in the present study.
When evaluating the mechanism of dispersal and retention of larvae in estuarine environments, studies suggest the existence of an interaction among local conditions, water circulation and larval behavior (Galtsoff, 1964;Andrews, 1983;Mann, 1988;Dekshenieks et al., 1996). Environmental factors that determine the horizontal transport of larvae are mainly temperature, salinity, wave action, and the velocity of currents and tides. (RH) and Ostrea sp. (OS) in autumn was conditioned and limited by the distribution of C. talonata. However, a greater trend of occupation could be observe during winter (W) in Vila da Glória (VG) (Figure 4).

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In their previous studies with oyster larvae in Paranaguá Bay/Paraná state, Brazil, Boehs and Absher (1997) suggest that settling individuals maintain active locomotion in order to persist in the interior of the estuary. On the other hand, studies involving Crassostrea virginica larvae imply that transportation of larvae in estuaries can either occur actively or passively owing to currents and tides, which explains the heterogeneity in larvae distribution in this environment (Mann, 1988;Newell et al., 2000).
In the present study, continuous seed recovery through the seasons is correlated with the reproductive pattern observed for some species of oyster collected. During winter, the number of specimens collected differed from other seasons of the year, showing a different recruitment pattern throughout the year. The gamete production of some species, such as C. gasar and C. gigas, is strongly affected by temperature pattern when they occur in places where the temperature is more pronounced according to season ; therefore, these species are mostly recruited in warmer seasons. Otherwise, C. rhizophorae and Ostrea sp. were the most recruited species during the winter. C. rhizophorae concentrated the recruitment of larvae during winter, while Ostrea sp. presented high larvae recruitment during both summer and winter. It is also important to note that settlements during winter involved specimens of little interest in aquaculture.
More than fifty percent of this recruitment is from C. talonata, a species described for the Indo-Pacific regions. In the last decade, this species expanded its distribution to the coastal zone of Peru (Li et al., 2017), Brazil and Argentina, forming large populations in estuaries, thus impacting natural stocks of native species (Cavaleiro et al., 2019). In fact, Canela Island/Pará state, where the species was first reported in Brazil (Varela et al., 2007), the recruitment of oysters through collectors was completely compromised since C. talonata was the only ostreid found in those structures (Garduncho et al., 2012). Castilho-Westphal et al. (2013) also account for a species denominated Crassostrea sp. alongside native mangrove oyster species. This species is, most likely, C. talonata since it is now the exotic species expected to occur in estuaries of Brazil, and its confirmation to Brazil's littoral zone came much later than the study in question. Under the worst set of circumstances, this occurrence signals that C. talonata is probably present in most impacted Brazillian estuaries.
The potential compromise of the natural recruitment of oysters of Babitonga Bay for cultivation as a result of competition from C. talonata with native species is expected (Ruesink et al., 2005) since the species is considered rare in its natural distribution in China (Wang et al., 2008), while, in Babitonga Bay, it has the highest abundance among other ostreids. Even the fact that C. rhizophorae and C. gasar present a continuous spawning and recruitment of larvae throughout the year in Guaratuba Bay (Christo and Absher, 2006) and Paranaguá Bay/Paraná state (Absher, 1989), nearby regions with conditions similar to those of Babitonga Bay, cannot overcome the larvae settlement rate of C. talonata.
The preponderance of settlement of C. talonata in Babitonga Bay, especially in the warmer seasons, is a new concerning fact as the seeds of Crassostrean oysters possess the same morphological conformation (Figure 3), hindering the possibility of visual identification of the species. Although the spawning cycle in this species is yet to be evaluated, it is possible to determine the potential continuous partial spawning of individuals in the warmer seasons with a high recruitment rate of seeds.
When it comes to evaluating the native mangrove oyster species C. gasar and C. rhizophorae, several studies suggest that the species possess a continuous reproduction during the whole year in the tropical littoral zone of Brazil with spawning concentrated in warmer seasons (Wakamatsu, 1973;Nascimento and Lunetta, 1978;Lenz and Boehs, 2011).
In the present study, a higher settlement of C. gasar seeds in Babitonga Bay was also observed in the warmer seasons. C. gasar is mainly associated with the tropical Atlantic (Carpenter and De Angelis, 2016), inhabiting regions significantly influenced by freshwater inflow possessing large variations in salinity (Castilho-Westphal, 2013), and Santa Catarina state (area of the present study) is the southern boundary of its distribution.
A limiting colder temperature of the water acts as a barrier for the species distribution, and different from tropical zones, it reduces the maturation of gonadal tissues completely as adult individuals build glycogen reserves, especially in the winter Ramos et al., 2014), reducing the density of C. gasar larvae in this season.
Such pattern is also corroborated by Christo and Absher (2006) and Castilho-Westphal et al. (2013) for the same species in Guaratuba Bay. Both estuaries are located in the subtropical area of Brazil and possess well-defined seasons; thus, the reproduction process of this species under such circumstances presents a direct correlation with an enhanced spawning process in bivalves as temperature rises in the summer season, according to Quayle (1980), Moura et al. (2008), Pereira et al. (2003), and Gomes et al. (2014), implying a higher density of seeds in this period.
However, in tropical areas, where the temperature is rather constant, salinity is the major factor that triggers spawning in C. gasar, as well as maturation (Paixão et al., 2013).
C. rhizophorae demonstrated a higher seed settlement rate occurring in winter than in the warmer seasons. This conformation points out the wider physiological plasticity of such species as C. rhizophorae which can maintain its reproduction process in several partial spawnings throughout the whole year in tropical Atlantic seas without any retractions (Lennz and Boehs, 2011), with peaks suggested to occur between March (autumn) and October (spring) (Menzel, 2018) or July (winter) and December (summer) (Velez, 1977). Since the species is distributed in southern regions up to Uruguay (Carranza et al., 2009), gonadal tissue maturation and spawning process are maintained, even in colder seasons.
The detection of one specimen of C. gigas through genetic analysis in the artificial collector may be a reflection of an unexpected settlement resulting from a few surviving larvae that came from spawned cultivated adults from farms located inside Babitonga Bay. Natural stocks of C. gigas are reported for Santa Catarina state where sea conditions probably do not permit the establishment of a viable population. Although recruitment has been observed in some places in Florianopolis Bay (Melo et al., 2010b), the environmental factors in estuaries, in particular low salinity and high turbidity, can interfere with the development and maturation of the species, preventing its reproduction or adaptation of larvae (Akaboshi, 1979;Christo and Absher, 2006).
The settlement of oysters in our artificial collectors showed a tendency for affixation in deep level quadrants (inferior quadrants). Frequently, this pattern is related to the preference of larvae for shady substratum for occupation (Siqueira, 2008), suggesting that aritificial dark collectors should be test for C. gasar.
In field and laboratory studies, Baker (1997) has shown that gravity is a factor for the primary settlement of Crassostrea larvae. According to the author, the larvae preferentially settle on the lower surfaces of shells. This strategy is more advantageous for nesting of larvae in coastal and estuarine regions with high sediment input, as the lower surfaces of the substrates are probably free of sediment (Buitrago and Alvarado, 2005).
The distribution of the larvae in the water column during their cycle contributes to settlement configuration. Larvae of ostreids are abundant near the surface during the early stages of larval development, but, often, they are more abundant near the bottom at the end of the larval cycle when they seek a substrate for settlement (Dinamani, 1973;Andrews, 1983;Absher, 1989;Boehs and Absher, 1997;Baker, 2003;Finelli and Wethey, 2003;Christo and Absher, 2006). Diadhiou and Ndour (2017), otherwise, suggest that settlement of larvae was more efficient when plates were positioned in a sagittal way as water currents carry larvae laterally at the plates, expanding the contact surface of oysters to the substratum.
In the recruitment process of larvae, it should be considered that the interaction between genetic and environmental factors may also contribute to the definition of the substrate and the level of seed affixation (Connel, 1985;Mann et al., 1991;Baker, 2003;Finelli and Wethey, 2003).
Thus, within this context, several factors need to be considered in the selection of areas for oyster larvae collection, among them materials, salinity, temperature, tidal level, flow, wave action, turbidity, epibionts, and predators. Therefore, the monitoring of environmental variables is fundamental in the search for the best locations for each stage of the crop, since they will directly influence the biological cycle of the species (Ramos and Castro, 2004).
Further studies are needed to assess which factors are decisive for obtaining oyster seeds in Babitonga Bay, within a set of factors such as larval flow, ecological issues, predation, and oceanographic aspects for each species. Up to the present study, no information had been available regarding the efficiency of artificial collectors from oyster seeds originating in Babitonga Bay. Artisan producers in the region observe that there are several materials with which seeds can be obtained. In the present study, however, the difficulty in the separation of the species with real potential for cultivation became clear, making this method of capture of seeds impracticable for commercial needs. Galvão et al. (2009) note at least more than one species in an artificial collection plate placed in the Cananéia region of São Paulo, making it difficult to perform a precise evaluation of growth performance of oyster species. Nalesso et al. (2008) and Christo et al. (2016) also reported the same difficulties in correctly identifying oyster seeds attached to artificial collectors in the littoral zone of Espirito Santo state and Guaratuba Bay/Paraná state, respectively. Additionally, the sympatric occurrence of C. gasar, C. rhizophorae and, now, most recently, C. talonata in estuaries configures a common assembly along the Brazilian littoral, increasing, still more, the difficulty of attaining suitable oysters for commercial purposes at low cost.
Getting economically important oyster seeds in artificial collectors is still possible in some localities, especially where the variation of salinity is higher, with values reaching the limits of the freshwater condition in rainy seasons. These areas, most of them located in northeastern and northern municipalities of Brazil, are denominated "sementeiras", or oyster sowing, in which oyster farmers place artificial collectors with a higher chance of getting only the target species for cultivation. Indeed, Funo et al. (2015) showed through genetic analysis that such practice is possible as 100% of oysters obtained in one of those areas situated in Curuça/Pará state belonged to C. gasar.
The results of this research certainly contribute to the planning of oyster crops in the Babitonga Bay region. Thus, despite the ability to get oyster seeds with the tested system, it should still be considered that most of the specimens belong to C. talonata, in addition to Ostrea sp. and C. rhizophorae, i.e., animals that reach small size and are not suitable for commercial farming in the molds currently used in southern Brazil. Therefore, profitable oyster cultivation may rely on seeds provided by hatcheries, discounting the fact that hatchery techniques are not well developed for getting C. gasar seeds (FAO, 2014).

CONCLUSIONS
Genetic analyses indicated the presence of Crassostrea gasar, C. rhizophorae, C. gigas, C. talonata, and Ostrea sp., in the collectors arranged along Babitonga Bay.
It was not possible to identify, by morphological analysis, the species of the genus Crassostrea in the seed phase, mainly owing to the enormous plasticity of shells.
Seeds of the genus Crassostrea were obtained mainly in the summer season, followed by spring and autumn. In winter, the highest amounts of seeds were observed for C. rhizophorae and Ostrea oysters.
The results of this work showed the current impossibility of obtaining seed quantities of oyster species with zootechnical potential to supply the commercial farms by artificial collectors in the study region since C. talonata, a highly invasive species in the Brazilian estuaries with no commercial value, now corresponds to 52% of the total settlement rate in the these collectors.