Introduction

Environmental factors strongly affect all phases of the life cycle of many marine invertebrates, and in particular of species inhabiting coastal and estuarine regions.

Temperature and salinity are primary abiotic variables affecting survival, activity and distribution of marine organisms (Kinne 1964), and environmental tolerance to salinity may not be the same for gametes as for adults (Fong 1998). Most euryhaline animals are not immune to the effects of salinity changes. Documented sublethal effects of altered salinity include reduced rates of growth and development (Qiu and Qian 1997 ; Lambert et al. 1994; Rosas et al. 1999; Specker et al. 1999) and reduced fecundity (Morritt and Stevenson 1993; Pechenik et al. 2007).

The effects of diet, sediment organic content, pollutants and prolonged larval life on growth and life history characteristics have been well documented for capitellid species (e.g. Pechenik and Cerulli 1991; Qian and Chia 1992a,b; Bridges 1996; Levin et al. 1996; Cohen and Pechenik 1999), but the effects of salinity on survival and somatic development of the Eunicidae have not been reported previously.

The iteroparous and long-lived polychaete M. sanguinea has been recorded from many localities around the world, often without an exhaustive description or deposition of voucher material. Recently, there has been some debate on whether M. sanguinea from different geographical regions may in fact encompass a series of cryptic species, morphologically difficult to distinguish (Hutchings and Karageorgopoulos 2003; Lewis and karageorgopoulos 2008).

In Portugal, the species is present along the Sado estuary, located in the south of the Setúbal peninsula, 25 km south-east of Lisbon (Fig. 1), particularly within former oyster production areas. In this estuary, M. sanguinea lives in deep borrows, 25–40 cm into the intertidal mudflats. It is a polychaete of considerable size, attaining a maximum length of about 40–50 cm and is a gonochoric species with an annual iteroparous reproductive strategy. According to Castro (1993), it has a contagious distribution, spreading in aggregates of different sizes. Concerning the dietary habits, the same author, through an analysis of the digestive contents, observed that this worm ingests preferentially benthic macroalgae (Ulva, Enteromorpha, Fucus vesiculosos) as well as fine sediment with benthic microalgae normally abundant in the intertidal mudflats of the estuaries.

Fig. 1
figure 1

Sado estuary, Central-Western Portugal

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Potential uses

The commercial interest in polychaetes mainly arises from their value as bait for recreational fishing and as food source for penaeid crustaceans and finfish in aquaculture (Dinis 1986; Olive 1994). This has lead to the development of small but economically viable aquaculture facilities providing a supply of different species (Gambi et al. 1994; Olive et al. 2000) for a variety of uses. Simultaneously, traditional bait digging exploitation continues to occur, presenting an ill-defined boundary between anglers collecting strictly for their own use and those collecting for direct profit. The activity therefore represents a form of parallel economy, directly conflicting with a sustainable and controlled use (Olive 1993).

M. sanguinea is commonly known in Portugal by the names of “ganso” and “minhocão”. It is one of the most appreciated bait species among those collected and commercialized in Portugal due to the high resistance it presents on the hook and to its reputed ability to attract valuable fish species such as the seabreams Sparus aurata and Diplodus sargus. Its harvest activity is one of the most important socio-economic resources for local fishermen. The price per litre of “minhocão” in 2007 ranged between 150 and 200 Euros, and in 2008 between 250 and 300 Euros, and it is estimated that between 2007 and 2009 the annual sales volume of the “minhocão” in the Sado estuary has fluctuated between 600 and 750 thousand Euros (Directorate General for Fisheries and Aquaculture -DGPA-, personal communication). More exact quantification is difficult because a substantial component is sold through a parallel economy in which sales are not declared.

The long-term exploitation of this species requires a good knowledge of biological and ecological characteristics of the natural populations that may allow optimized harvest and minimized collection impact. The main aim and the scientific challenge of the research on the biology of this bait polychaete species should be to develop rearing techniques for intensive production that may increase resource supply and potentially reduce harvest and its impact on natural populations. However, the same acquired knowledge can also serve to provide the tools for a sustainable utilization of the resource form the wild.

With this purpose in mind, a small-scale experiment was conducted in order to elucidate an important aspect of the role of salinity on the life cycle of M. sanguinea and more specifically the salinity level most suitable to its somatic growth. Further development of polychaete culture is considered of economic importance beyond that associated with the bait supply industry which acted as the initial catalyst for this research.

Materials and methods

In March 2006, 720 juveniles were produced in the laboratory from a population collected from the Sado estuary. At six to nine setigerous segments, their length was determined from the measurement of the width of the buccal segment (W.S.B.) (Fig. 2) according to Castro (1993). They were thence used for 2 months to study the development and growth rate in relation to salinity under two diet regimes.

Fig. 2
figure 2

Dorsal view of the anterior region of M. sanguinea, adapted from Castro (1993). W.S.B—Width of buccal segment

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The experimental set-up consisted in placing the animals in 36 containers (1 L) and maintaining them at six different salinities (15, 20, 25, 30, 35 and 40) in a laboratory (22 ± 0.5°C) with constant 12/12 light/dark photoperiod. All the containers were filled with a fine layer of sediment (1 cm) collected from the natural environment of the animals along the Marateca channel. The sediment was filtered through a 0.5-mm sieve in order to remove macroalgae and any associated macrofauna, and it was then placed in an oven at 90°C for 24 h and first divided into 6 groups according to the salinity range. For each salinity treatment, one half of the containers received an Ulva lattuca supplement. A population of 20 worms was placed randomly in each of the recipients, resulting in three replicates for each salinity and sediment type. The used water was renewed daily. Before adding to the recipients, new water was filtered through a biological filter and a cartridges series (<10, <5 and <1 μm) and maintained under constant oxygenation. During the first 48 h, the polychaetes were allowed to settle (construct and establish burrows). U. lattuca were provided daily, fresh and “ad libitum” after this period. For the biometric analysis, three recipients from each salinity were randomly selected weekly. While taking the measurements, worms were extracted and the recipients were placed back in the circuits in order to keep the flows constant. Length was always determined from the number of setigers formed and from the W.S.B. The linear measurements of W.S.B obtained were taken under a stereomicroscope equipped with a micrometric ocular at 2× magnification. The dimensions obtained were processed by the image-processing software system Qwin Lite 2.4 (Leica Mycrosystems Imaging Aquisition). Statistical analysis was performed using SPSS (statistical package for the social sciences) software (version 14; SPSS Inc, Chicago, Il). The relationships between salinities and subtracts and the effects of salinity and subtract on growth were analysed using a non-parametric two-way analysis of variance (ANOVA), followed by a multiple comparison of means (LSD), when the treatment means showed significant differences. A significance level of α = 0.05 was chosen.

Results

Although juvenile survival was generally high in all the salinities tested, the worms reared at 15, 20 and 40 displayed more elevated mortalities than the remaining ones in which the mortality remained more or less constant throughout the experiment. Under the experimental conditions, mortality was only relatively high under the lowest and highest salinities in the range (15, 40) in the first weeks of rearing and then tended to level off. No differences exclusively attributable to U. lattuca were observed (Fig. 3).

Fig. 3
figure 3

Survival, size patterns, and setigerous segment development of Marphysa sanguinea juveniles at each of a series of salinity and two sediment treatments conducted over 8 weeks. Numbers on top of each panel correspond to salinity values. Length was determined by the difference between the total length (TL) obtained weekly (TLW) and the initial total length (TL0): Length = TLW−TL0. Length was estimated from the width of the buccal segment (W.S.B.) from the equation TL = 3.9303WSB 1.3307 according to Castro (1993)

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At the start of the experiment, the animals measured 0.19 ± 0.2 cm.

Throughout the experiment, no significant differences in growth were obtained among replicates. However, significant differences (P < 0.01) were observed in both the development and the growth rates of M. sanguinea juveniles influenced by different salinities, whereas no effect was noted between the two different sediment types, but a significant interaction was found between them (Table 1). The comparison of means with the LSD test indicates that salinity effects can be individualized in three groups with different influences (P < 0.05): in the first one, constituted by the worms reared at 15, 20 and 40, growth was usually reduced (P < 0.05 and P < 0.01) when compared to 25, 30 and 35, respectively. In fact, after 4 weeks (see Fig. 3), juveniles maintained in salinity 15 practically did not grow, reaching a length of 0.4 ± 0.2 cm in both sediment set-ups. At 20 and 40, growth was still low (0.7 ± 0.4 cm; 0.9 ± 0.4 cm and 0.7 ± 0.2 cm; 0.4 ± 0.2 cm in length, respectively), which contrasts with the results obtained at 30 (1.4 ± 0.6 cm; 1.7 ± 0.4 cm) and 35 (1.3 ± 0.6 cm; 1.6 ± 0.6 cm). In fact, at 30 and 35, the worms grew more in 4 weeks than during the whole experiment at the other salinity treatments. On the other hand, the results obtained at 25 (1 ± 0.5 cm; 1.2 ± 0.5 cm) suggest that this salinity represents an intermediate level between the upper and lower salinities.

Table 1 Results of the two-way non-parametric ANOVA testing the effects of salinity and diet on the growth rate of M. sanguinea juveniles
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The results previously discussed seem supported when the number of setigers developed is considered. (see Fig. 3). However, here, well-defined extremes and a clear preference for U. lattuca exist mainly at salinities 25, 30 and 35 where growth rates are high. In fact, it is clear that at salinities 15 (18 ± 3; 20 ± 2 setigers) and 35 (33 ± 4; 40 ± 4 setigers), respectively, lie the worst and the best suitable conditions observed in the experiments when this developmental feature is considered. In the group reared at 20 and 40, the worms developed 22 ± 4; 24 ± 4 and 23 ± 3; 25 ± 3, respectively, and at 25 and 30 after 8 weeks, the worms reached 30 ± 3; 34 ± 4 and 34 ± 4; 36 ± 3, respectively. A significant linear correlation (P < 0.001) between total length and number of setigers formed was obtained. Morphological development appears consistent with growth rates. In worms exposed to 15, 20 and 40, the jaws are well developed, and 4 eyes, capillary setae, 4 antennae and simple gills appeared at the end of the experiment. However, worms exposed to salinities 25, 30, and 35 at 4 weeks showed well-developed jaws, 2 pairs of eyes and 5 antennae, and at the end of the experiment, a red pigmentation and well-developed gills were clearly visible from the 10th segment extending out to the 30th.

Discussion

Almost all polychaetes are known to be euryhaline, although salinity tolerance has not previously been well defined for M. sanguinea.

This study showed that juveniles of this species are very resistant to salinity fluctuations. In fact, the largest mortality occurred in the first week, which suggests it may be related with adaptation to laboratory conditions and not to the experimental treatments. On the other hand, juveniles exhibit a very territorial and aggressive behaviour with high rates of cannibalism if the environmental conditions are disadvantageous (pers. obs.), so that the mortality under experimental conditions may also derive from cannibalism and not directly from the conditions. It also appears that the tolerance of the juveniles of M. sanguinea to different salinities changes with age, so that differential survival along the experiment may derive to an extent from the changing tolerance. Therefore, mortality patterns remain largely unexplained and require further research.

Growth rates, on the other hand, were significantly (P < 0.05) influenced by salinity, and although there was not a clear preference for U. lattuca, growth rates under a diet supplemented by the alga were higher at all salinities, indicating a clear benefit accrued from the diet. Prevedelli (1994) in Marphysa juveniles and Pechenik et al. (2000) in Capitella juveniles obtained a significantly higher growth at salinity of 30. The influence of salinity on growth has also been demonstrated in other species. For example, at low salinity Nereis diversicolor has a greater growth rate than Nereis succinea (Neuhoff 1979). It is also known that under laboratory conditions, a few aquatic invertebrates can attain a maximum growth rates at salinity levels which are lower or higher than those of natural waters (Kinne 1971).

The significant correlation (P < 0.001; R 2 = 0.86) between total length and the number of setigerous segments obtained in this study is in agreement with previous findings reporting setigerous development to be a good parameter for estimating total length of juveniles. However, this parameter cannot always be used in older stages or adults due to the elevated autotomy that occurs in this species (Fauvel 1923; Castro 1993).

M. sanguinea, similarly to the majority of the known polychaetes, colonizes brackish waters. In the Sado estuary, colonies are found in biotypes of high and low natural salinities; more precisely, they are found in the Marateca (about 10% freshwater inflow—>36 at times) and Alcácer (80% freshwater inflow—<15 at times) channels (see Fig. 1). It has been shown that there are different degrees of adaptation to salinity variations. According to Abbiati (1996), N. diversicolor is the Mediterranean species that shows the highest genetic adaptation to brackish environments, but adaptation to salinity fluctuation has been found to be maximum in Nereis limnicola due to a strategic reproductive feature that protects larvae from the lowest salinities (Smith 1957, 1964). Krishnamoorthi (1951) demonstrated experimentally that the gelatinous masses observed in Marphysa gravelyi provide the eggs and embryos with an osmotic protection under conditions of salinity fluctuation. Garcês (2002) also observed in the laboratory in spawns that occurred in galleries inside oyster shells that the eggs of M. sanguinea were encapsulated in a gelatinous mass and protected by a jelly mucous forming “cocoon” where the larval development occurred. Aiyar (1931) observed in India a maximum reproductive activity in M. gravelyi after the monsoon season. Thus, conditions of low and variable salinity may serve to stimulate spawning in these species, possibly in anticipation of favourable trophic conditions, and the gelatinous masses may then provide the necessary environment in which embryonic development can proceed amidst a generally unfavourable environment.

We suggest that this species colonizes brackish waters and has a high capacity to tolerate variations in environmental parameters, in particular salinity, but that the most favourable developmental conditions lie within relatively limited parameters as shown.

Conclusion

The present experiment, which was conducted under laboratory conditions, utilizing specimens hatched in the laboratory from eggs layed by mature worms collected in Sado estuary demonstrates that in M. sanguinea juveniles, salinity can have an immediate and significant effect on growth. A salinity range outside 25–35 will significantly reduce growth rates. Salinities close to 15 and 40 seem to be the lower and upper physiological limit for this species. Thus, a salinity range between 30 and 35 is recommended for the juvenile culture of this polychaete.

The results obtained in this study have permitted a better understanding of larval development and population dispersion patterns of M. sanguinea juveniles in the Sado estuary and possibly elsewhere, but also bear significant implications for the aquaculture of the species, concerning farm site selection and salinity maintenance in order to maximize commercial productivity.