Chemical induction in mangrove crab megalopae, Ucides cordatus (Ucididae): Do young recruits emit metamorphosis-triggering odours as do conspecific adults?

Estuarine, Coastal and Shelf Science 131 (2013) 264e270

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Chemical induction in mangrove crab megalopae, Ucides cordatus (Ucididae): Do young recruits emit metamorphosis-triggering odours as do conspecific adults? Darlan de Jesus de Brito Simith a, b, *, Fernando Araújo Abrunhosa a, Karen Diele c, d a

Laboratório de Carcinologia, Instituto de Estudos Costeiros (IECOS), Universidade Federal do Pará (UFPa), Campus Universitário de Bragança, Alameda Leandro Ribeiro s/n, Aldeia, 68600-000 Bragança, Pará, Brazil Laboratório de Ecologia de Manguezal (LAMA), Instituto de Estudos Costeiros (IECOS), Universidade Federal do Pará (UFPa), Campus Universitário de Bragança, Alameda Leandro Ribeiro s/n, Aldeia, 68600-000 Bragança, Pará, Brazil c Edinburgh Napier University, School of Life, Sport and Social Sciences, Edinburgh EH11 4BN, UK d Leibniz-Center for Tropical Marine Ecology (ZMT), Fahrenheitstrasse 6, 28359 Bremen, Germany b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 12 March 2013 Accepted 24 July 2013 Available online 3 August 2013

In many brachyuran species, including the mangrove crab Ucides cordatus, water-soluble chemicals (odours) emitted by adult residents trigger metamorphosis of megalopae, probably facilitating habitat selection and settlement near conspecific crab population. New field findings revealed that early benthic crab stages co-inhabit burrows of both juveniles and adults of U. cordatus which raised the question whether megalopae are also stimulated by sexually immature juveniles. Therefore, we tested in an experimental laboratory study the hypothesis that small benthic recruits and older juveniles also emit metamorphosis-stimulating odours as do conspecific adult crabs. U. cordatus megalopae were cultivated in eight conspecific odour-treatments containing seawater previously conditioned with crabs of different carapace widths (CW 0.15e5.0 cm) and in a control treatment with filtered seawater not conditioned with crabs. In all odour-treatments, including those with small immature crabs, the percentage of metamorphosed larvae was significantly higher (
74%) and the average development was shorter (15.8 e19.3 days) than in the control group, where only 30% moulted after 25.6  6.6 days of megalopal development. In addition, megalopae developed 2.7 days faster when exposed to odours from young and older juveniles compared to those larvae kept in contact with odours from conspecific adults. Our results clearly demonstrate that the emission of metamorphic odours in U. cordatus is independent of size/age or sexual maturity. The responsiveness of megalopae to chemicals emitted by resident crabs of varying ages should aid the natural recovery of U. cordatus populations in areas significantly affected by size-selective fishery where only large conspecific adults are harvested. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: juveniles larval settlement mangrove crab metamorphosis recruitment sexual maturity

1. Introduction Most marine and estuarine brachyuran decapod crustaceans develop in the planktonic environment before settling in their definitive benthic habitat (Anger, 2001, 2006). This ontogenetic change in lifestyle is often accomplished when their physiologically competent megalopal stage is stimulated and attracted by natural physical and/or chemical cues associated with the parental adult habitat (for an extensive review see Anger, 2001, 2006; Forward et al., 2001; Gebauer * Corresponding author. Laboratório de Carcinologia, Instituto de Estudos Costeiros (IECOS), Universidade Federal do Pará (UFPa), Campus Universitário de Bragança, Alameda Leandro Ribeiro s/n, Aldeia, 68600-000 Bragança, Pará, Brazil. E-mail address: [email protected] (D.deJ.deB. Simith). 0272-7714/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ecss.2013.07.015

et al., 2003). The crucial plankton-to-benthos transition is accompanied by an active larval ‘decision’ by which larvae selectively choose a favourable habitat for the early juvenile growth until sexual maturity (Anger, 2006). The ability of the larvae to selectively respond to specific environmental cues greatly increases the survival rates and consequently enhances the likelihood of successful recruitment in coastal marine ecosystems such as mangroves and estuaries (Anger, 2001; Forward et al., 2001; Gebauer et al., 2003). Several investigations have shown that diverse types of substrata characteristic of the conspecific benthic juvenile and adult habitat or even substratum-associated microorganisms (i.e. benthic microbial biofilms: e.g. O’Connor, 2007; Krimsky and Epifanio, 2008; Steinberg et al., 2008; Anderson and Epifanio, 2009) and water-soluble chemical substances (referred to as ‘odours’ in the

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literature) released by conspecific or interspecific adult crabs (e.g. Gebauer et al., 1998, 2002; Kopin et al., 2001; Steinberg et al., 2007; Simith and Diele, 2008b) can trigger settlement and metamorphosis in megalopae of many brachyuran crab species (for review see Anger, 2001, 2006; Forward et al., 2001; Gebauer et al., 2003). There is, however, a gap in knowledge for most crab species regarding the potential of juvenile stages in triggering larval metamorphosis. If stimulatory habitat cues such as those described above are absent, the larvae delay their metamorphosis and hence, the meroplanktonic larval period is lengthened which may carry highly significant costs for the post-metamorphic performance and fitness of early benthic juveniles (Gebauer et al., 1999, 2003; Pechenik, 2006). For megalopae of the mangrove crab Ucides cordatus (Linnaeus,   1763; Family Ucididae, Stev ci c, 2005; Ng et al., 2008) we recently showed in the laboratory that waterborne chemical substances (odours) emanating from conspecific adult crabs and mud-substratum from burrow openings (or combination thereof) accelerate the developmental time to metamorphosis (TTM) (Diele and Simith, 2007; Simith and Diele, 2008b). However, it is still unknown whether newly settled recruits and older juveniles also emit metamorphosisstimulating odours. U. cordatus is an ecologically important large burrow-dwelling semi-terrestrial crab (see Koch and Wolff, 2002; Schories et al., 2003; Nordhaus et al., 2006; Araújo et al., 2012) and also a key fishery resource for local populations in coastal Brazil (e.g. Nóbrega and Nishida, 2003; Glaser and Diele, 2004; Alves et al., 2005; Diele et al., 2005, 2010). Its larval development comprises five or six zoeal stages and one megalopal stage (Rodrigues and Hebling, 1989). After their release into estuarine waters, the larvae are dispersed offshore (Santarosa-Freire, 1998; Diele, 2000) where they encounter higher salinity conditions crucial for their zoeal development (Diele and Simith, 2006; Simith and Diele, 2008a). After three to five weeks, the megalopae return to the coast and re-invade the parental mangrove habitat (Diele, 2000). Our laboratory studies suggested that Ucides cordatus megalopae preferentially settle in mangrove areas near conspecific crabs, attracted and stimulated by their chemical odours (Diele and Simith, 2007; Simith and Diele, 2008b). Recent fieldwork indeed showed that small benthic recruits (carapace width, CW < 1.0 cm) co-inhabit burrows of conspecifics. The small recruits were found in burrows of both juvenile and adult crabs (Schmidt and Diele, 2009), suggesting that the emission of metamorphic inducers is independent of size, age or sexual maturity. In order to better understand the recruitment process of U. cordatus and respective intraspecific interactions between the megalopal stage and the benthic juvenile and adult population, we experimentally investigated in the laboratory whether odours of sexually immature juveniles and adolescent crabs also trigger metamorphosis in U. cordatus megalopae as do large conspecific adults through the emissions of their chemical inducers. The responsiveness of megalopae to odours originating from crabs of different size/ages could accelerate natural recovery of U. cordatus populations that suffer significantly reduced numbers of large conspecific adults, e.g. through size-selective fishery. 2. Materials and methods 2.1. Larval obtainment and rearing conditions Larvae of Ucides cordatus were obtained from five ovigerous females (average CW 4.4  0.2 cm) collected one day prior to hatching, at full moon in March 2010, in the mangroves of the Caravelas River estuary (Northeastern Brazil). Immediately after capture, the berried females were individually transported in aquaria filled with muddy substratum and estuarine water (salinity 20) to the Laboratory of the Mangrove Project at the CEPENE

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(Centro de Pesquisa e Gestão de Recursos Pesqueiros do Litoral Nordeste) in Caravelas city (State of Bahia). In the laboratory, the egg-bearing females were washed and gradually acclimatized to seawater with salinity 30 (increasing 5 units of salinity at each hour), which is the salinity which yields the highest survival rates of the zoea larvae in the cultivation (see Diele and Simith, 2006; Simith and Diele, 2008a). The females were kept in separated glass aquaria (filled with 10 L of gently aerated filtered seawater) at ambient temperature of 27.5  C, pH of 8.2, with natural light cycle and without food until larval release. The five females released their larvae synchronously. Upon hatching, actively swimming zoea-larvae of all females were mixed in a glass beaker (5000 mL) and groups of 50 larvae were then transferred to 250 mL plastic vials for mass-cultivation. The larvae were reared to megalopal stage at constant seawater temperature (27.0  0.4  C), salinity 30, pH (8.2  0.5), photoperiod regime (12 h light/12 h dark) and without aeration in the cultivation medium. Seawater for the larval cultivation was obtained 80 km off the Caravelas River estuary; it was filtered (Eheim and Diatom Filter, 1 mm), sterilized with sodium hypochlorite 2.5% (0.5 mL/L seawater) and stored in tanks (500 L) with constant aeration in the laboratory. The zoea-larvae were fed daily with planktonic microalgae Dunaliella salina. In an early study, it was shown that Ucides larvae exhibit high survival rate (81.7%) when they are exclusively fed with D. salina (see Abrunhosa et al., 2002). Larvae were also additionally fed with newly-hatched brine-shrimp Artemia sp. at a density of approximately 6 nauplii mL1. The cultivation water was changed every two days and new food was immediately added. After the larvae had developed to zoea VI stage, they were monitored daily for appearance of megalopae. Megalopae were reared under the same conditions as zoea-larvae but they were kept individually in 200 mL plastic vials to avoid cannibalism and competition for space and food (Forward et al., 2001). The megalopal stage was exclusively fed ad libitum with Artemia sp. (ca. 2 nauplii mL1).

2.2. Experimental procedures and design To assess whether small benthic recruits and older juveniles emit metamorphosis-triggering odours cues as do conspecific adults, 450 freshly-moulted Ucides cordatus megalopae were randomly distributed and cultivated in eight odour-treatments (T1eT8) containing seawater conditioned with crabs ranging in size classes between 0.15 and 5.0 cm CW, and in one control treatment (C) containing odourfree seawater (see Table 1). For determining the sexual maturation

Table 1 Overview of the different experimental treatments for cultivations of Ucides cordatus megalopae. (T1eT8) conspecific odour-treatments: cultivation in seawater previously conditioned with crabs of different carapace width (CW) classes including early juvenile IeIII crab stages (T1); (C) control treatment: cultivation of megalopae in pure filtered seawater not conditioned with crabs. Treatment

CW class (cm)

N crabs*

M:F

Weight (g)**

Total crabs***

T1 T2 T3 T4 T5 T6 T7 T8 C

0.15e0.25 1.0e1.5 1.6e2.0 2.1e2.5 2.6e3.0 3.1e3.5 3.6e4.0 4.1e5.0 e

3 82 39 20 12 8 5 2 e

e 39:43 21:18 15:5 3:9 3:5 2:3 1:1 e

0.0023  0.0001 116.2  3.8 116.9  3.5 116.9  2.9 117.8  3.1 118.2  2.8 118.7  1.6 117.4  2.6 e

150 1804 858 440 264 176 110 44 e

*Number and **average total wet weight (g) of crabs immersed in 200 mL (T1) and 20 L (T2eT8) for producing fresh odour-seawater at each water renewal; ***Total number of crabs used throughout the experiment for producing the odour-seawater and analysed for sexual maturation (except for specimens utilized in T1). M:F ¼ proportion of males and females used for each new seawater-conditioning.

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of the crabs that had been used for producing the odour-seawater, specimens with CW of 1.0 cm onwards (treatments T2eT8, see Table 1) were dissected and their gonads analysed under a stereoscopic microscope (Zeiss) using the methods established by MotaAlves (1975), Pinheiro and Fiscarelli (2001), Wunderlich et al. (2008). Three stages of sexual maturity were differentiated based on morphological aspects of the reproductive system following Pinheiro and Fiscarelli (2001): (1) immature (¼juvenile), (2) at stage of maturation (¼adolescent) and (3) sexually mature (¼adult). All conspecific odour-treatments and the control were carried out with 50 megalopae each. The newly moulted megalopae were 25e46 days old (the average length of the development period from hatching to megalopa was 33.3  4.1 days). For avoiding possible bias, different aged-megalopae were equally distributed among treatments. The specimens were assigned to the respective treatments at the day of moulting to the megalopal stage and cultivated individually in 200 mL plastic vials. They were monitored daily for mortality or moults to first juvenile crab stage. The culture medium was renewed every 2 days in all treatments. The experiment was conducted until the last megalopa had metamorphosed to juvenile or died in the respective treatments. For producing the odour-seawater, wild-captured young Ucides cordatus recruits and older juveniles were used except for the odour-treatment T1 (see Table 1). The latter involved very small first, second and third crab stage-juveniles (JIeJIII; 0.15e0.25 cm CW) which are difficult to find in sufficient numbers in the field due to their small size and cryptic behaviour (see Schmidt and Diele, 2009; Diele and Koch, 2010). Instead, they were cultivated in the laboratory from freshly hatched larvae. The small crabs (juvenile Ie III stages) could not be sexed and due to their young age (JI ¼ 15.2  5.8 days; JII ¼ 31.1  9.6 days; JIII ¼ 56.6  15.7 days) it was assumed that they were sexually immature. The respective juvenile crab stages (JIeJIII) were kept separately in glass aquaria containing 20 L of filtered seawater and 2 mm of mud on the bottom until they were used for producing the odour-seawater. Water and substratum were changed daily and Artemia sp. nauplii were provided as food item. For producing the odour-seawater for T1, the crabs were taken from the aquaria and carefully washed with fresh seawater. Three individuals (one per stage) were placed together in 200 mL seawater for 24 h without aeration. Thereafter, they were removed and the water was immediately used for the cultivation of the megalopae. The juvenile crabs were put back into the aquaria and in contrast to the other odour-treatments (T2eT8, see below), in T1 the same specimens were re-utilized for producing fresh odour-seawater for subsequent water changes. Cannibalism between different crab stages occurred in some plastic vials during the conditioning of seawater. Preyed or injured juveniles were replaced by healthy and intact specimens. Conspecific odours from crabs with carapace widths ranging from 1.0 to 5.0 cm CW (odour-treatments T2eT8, respectively; see Table 1) were obtained by previous immersion of field-captured males and females in 20 L of seawater for a 24 hour-period with constant aeration. On the following day the crabs were removed, the conditioned water was sieved (200-mm mesh size) and immediately used for individual cultivation of the megalopae. At each seawater change, freshly prepared crab-conditioned seawater from newly captured intact crabs was used. According to some authors, the magnitude of the stimulatory effect of chemical cues on metamorphosis of crab megalopae is dose-dependent and the concentration of emitted cues seems to be proportional to the weight of the organism (Fitzgerald et al., 1998; Kopin et al., 2001; O’Connor, 2005). Therefore, the total wet weight (g) of the crabs used for preparing the conditioned seawater was kept constant for the odour-treatments T2eT8 (see Table 1); however, for T1 it was different (see below). Depending on their density and size, the

crabs were kept in several aquaria (20 L) during the process of seawater-conditioning to avoid the release of stress-related chemicals under high-density conditions. The conditioned-water of the different aquaria was then mixed and used in the respective treatments except in T1. As this treatment comprised very small and thus light crabs (0.25 cm CW), it was not possible to reach the same total wet weight of crabs as for conditioning the seawater in the odour-treatments T2eT8. The small crabs of T1 were therefore placed in a small volume of water only (200 mL). Thus, the total weight of crabs used in T2eT8 was 505.2e516.1 times greater than in the odour-treatment T1. The latter was included to study whether the effectiveness of chemical cues on the megalopal stimulation of Ucides cordatus is dose-dependent and related to the weight of the odour-emitting organism. In addition, the odour-treatment T1 also allows to verify whether smallest benthic recruits (JIeJIII) are already able to emit metamorphosisstimulating odours as do conspecific adults. 2.3. Statistical analysis The effect of odours emitted by juvenile, adolescent and adult crabs on megalopal metamorphosis of Ucides cordatus was primarily analysed comparing the percentages of moulted megalopae and their average developmental time to metamorphosis (TTM). The day of moulting to megalopa was defined as day 0 for the determination of age and development TTM in the respective treatments. All statistical analysis followed Sokal and Rohlf (1995). The moulting rates (¼% of metamorphosed specimens) were analyzed by R  C contingency tables (rows  columns) followed by Chisquare tests. For the development TTM data, normality and homogeneity of variance were tested a priori through the KolmogoroveSmirnov’s test and the Levene median’s test, respectively. As some data did not show the prerequisites for parametrical statistics (e.g. non-normal distribution and variances heterogeneous), the KruskaleWallis’s H-test was chosen for the analyses. Multiple comparison analyses were carried out with Dunn’s test for data sets with different sample sizes, otherwise the ManneWhitney’s U-rank sum test was used to identify pairwise differences among treatments. The critical level (a) to reject the null hypothesis was fixed at 0.05. To avoid the probability of performing ‘Type I’ errors, the Bonferroni’s multiple-significance-test correction (a/n) was applied (see Hochberg, 1988; Bland and Altman, 1995; Abdi, 2007). Data were presented as average values  one standard deviation (SD). 3. Results Ucides cordatus megalopae metamorphosed in response to seawater conditioned with crabs of different carapace width (CW) classes (0.15e5.9 cm) (Fig. 1). The percentage of metamorphosed megalopae was significantly higher (p < 0.05) in the eight conspecific odour-treatments (T1eT8) than in the odour-free seawater control (C), where only 30% of the specimens underwent metamorphosis to first juvenile crab stage (Fig. 1). The moulting response in T1 was significantly lower (74%) than in all other odourtreatments, while T2 showed the highest percentage of moulted specimens (96%; p < 0.05). No significant difference was found among odour-treatments T3 to T8 (p > 0.05), all presenting moulting rates over 80% (Fig. 1). The average developmental time to metamorphosis (TTM) of Ucides cordatus megalopae varied significantly (KruskaleWallis Htest ¼ 36.2; n ¼ 355; df ¼ 8; p < 0.00001) among treatments (Fig. 2). In the conspecific odour-treatments T1 to T8, the average TTM was significantly shorter (n ¼ 52e63; df ¼ 2; p < 0.00001) than in the filtered seawater control, where the megalopal stage lasted for an average of 25.6  6.6 days (Fig. 2). Multiple comparisons showed that

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Fig. 1. Percentage of metamorphosed Ucides cordatus megalopae to first juvenile crab stage in response to seawater conditioned with crabs of different carapace width (CW) classes (odour-treatments T1eT8) and filtered seawater (control C). See Table 1 for abbreviations and explanations. Different letters above bars indicate statistically heterogeneous groups (p < 0.05).

megalopae developed 2.7 days significantly faster (n ¼ 79e91; df ¼ 2; p  0.04) when reared in the odour-treatments T1eT6 (average TTM of 16.4  4.8 days pooled data) in contrast to cultivations in the odourtreatments T7 and T8 (19.1  6.5 days; pooled data) (Fig. 2). In T1, where only three small juveniles (0.15e0.25 cm CW) were previously conditioned in 200 mL seawater, megalopae developed 3.1 and 3.5 days faster (n ¼ 79e80; df ¼ 2; p < 0.03) compared to those larvae reared in the odour-treatments T7 and T8, respectively, where a high density of larger conspecific crabs (3.6e5.0 cm CW) were incubated in 20 L seawater (Fig. 2). Gonad analysis revealed that both males and females with CW classes ranging between 1.0 and 2.5 cm (odour-treatments T2eT4) were immature (juvenile) (Fig. 3). All females in the 2.6e3.0 cm CW class (T5) were also immature, whereas 33.3% of the males in the respective size class were already at the stage of maturation (adolescent) (Fig. 3). In the size class with 3.1e3.5 cm CW (T6), the number of adolescent crabs increased in both sexes (Fig. 3). Fully sexually mature crabs were firstly found in the 3.6e4.0 cm CW class (T7), where 65.9% of the males and 50.0% of the females were adults. In the last size class (4.1e5.0 cm CW, odour-treatment T8), all crabs were sexually mature in both sexes (Fig. 3).

Fig. 2. Developmental time to metamorphosis (in days, average  standard deviation) of Ucides cordatus megalopae in response to seawater conditioned with crabs of different carapace width (CW) classes (odour-treatments T1eT8) and filtered seawater (control C). See Table 1 for abbreviations and explanations. Numbers inside bars refer to the number of metamorphosed specimens with initial n ¼ 50 megalopae/treatment. Different letters above bars indicate significant differences (p < 0.05) after pair-wise comparisons using Dunn’s or ManneWhitney’s U-test.

Fig. 3. Stages of sexual maturation of the crabs (males and females) of different carapace width classes (0.15e5.0 cm CW) used for producing the conspecific odourseawater for cultivations of Ucides cordatus megalopae in the treatments T1eT8. Early juvenile IeIII crab stages (0.15e0.25 cm CW) used in T1 were not analysed as they were logically considered sexually immature. See Table 1 for details.

4. Discussion During their meroplanktonic existence, competent megalopae of many marine and estuarine brachyuran crabs respond to physical and/or chemical stimuli indicating a suitable habitat for definitive settlement in the parental adult habitat (reviewed by Anger, 2001; Forward et al., 2001; Gebauer et al., 2003). In earlier laboratory studies, we demonstrated that metamorphosis of Ucides cordatus megalopae is triggered by water-soluble chemical substances (odours) released by conspecific adult males and females (see Diele and Simith, 2007; Simith and Diele, 2008b). We then suggested that competent megalopae settle inside burrows of conspecific adult crabs attracted by their stimulatory chemical cues. This assumption was corroborated by recent field findings of young recruits coinhabiting burrows of conspecific adults (Schmidt and Diele, 2009). Furthermore, young recruits were also found in burrows of immature conspecific crabs (e.g. 2.0e2.5 cm CW) (Schmidt and Diele, 2009). In the present study, we therefore hypothesized that conspecific odours induce larval metamorphosis irrespective of the status of sexual maturity of the odour-emitting crabs. In our experiment, significantly higher moulting rates and faster developmental time to metamorphosis (TTM) occurred when megalopae were reared in seawater conditioned with crabs of different size classes

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(1.0e5.0 cm CW, odour-treatments T2eT8) compared to the control group (odour-free seawater). Moreover, megalopae also responded (74% moulting rates and fast development TTM with 15.8  1.5 days) to seawater conditioned with laboratory-reared juveniles in the first, second and third crab stage (JIeJIII; 0.15e0.25 cm CW, odourtreatment T1), clearly demonstrating that recently moulted and small benthic recruits of U. cordatus are already able to release metamorphosis-stimulating cues. Furthermore, these findings also showed that waterborne chemical odours of immature juveniles and adolescent crabs also trigger metamorphosis in U. cordatus as do conspecific adult odours. This indicates that the nature and emission of conspecific odours is independent from sexual maturity in this species. Recently, Anderson et al. (2010) showed that chemical cues in exudates produced by juvenile crabs trigger metamorphosis in conspecific megalopae of the Asian shore crab Hemigrapsus sanguineus. To our best knowledge, this is the only other crab species investigated in this context. The outcomes of the present study also showed that developmental TTM was 2.7 days faster when megalopae were reared in exposure to odours emitted by immature crabs ranging from 0.15 to 3.5 cm CW (odour-treatments T1eT6) than when they were kept in contact with odours from larger conspecific adults ranging from 3.6 to 5.0 cm CW (T7 and T8). This fact showed that chemical odours from sexually immature crabs induce larval metamorphosis with different orders of magnitude than those from conspecific adults of Ucides cordatus. The observed pattern in responsiveness of megalopae to conspecific cues suggests that the presence of young juveniles has a strong influence on larval habitat choice. In contrast to our study, Anderson et al. (2010) did not observe any significant difference in the effectiveness, e.g. on TTM, of conspecific odours between juveniles and adults of Hemigrapsus sanguineus. In U. cordatus, however, conspecific attraction of megalopae by watersoluble chemical molecules produced by both juvenile and adult residents may significantly enhance the likelihood of finding an adequate habitat favouring post-metamorphic survival and growth of newly settled recruits within the mangrove ecosystem. The fact that a high percentage of megalopae had positively responded to conspecific odours emitted by immature crabs corroborates the field findings of Schmidt and Diele (2009). As in Ucides cordatus, crabs of several other brachyuran species host small conspecific juveniles in their burrows (e.g. Neohelice granulata: Spivak et al., 1994, 2001; Luppi et al., 2002; Neosarmatium meinerti: Emmerson, 2001; Cardisoma carnifex: Vannini et al., 2003). In the fiddler crab Uca maracoani, up to 7 small benthic recruits (e.g. 0.2e0.7 cm C.W.) were found inside burrows of young conspecifics (e.g. 1.1 cm CW) in the mid-intertidal mud banks in the northern Brazilian mangroves (Simith DJB, unpublished data). Conspecific burrows including those from young specimens may provide shelter and possibly increased food availability. On the other hand, gregarious settlement should be reduced or inhibited by the presence of newly moulted and pre-existent recruits to avoid intra- and intercohort competition for space and food (see Iribarne et al., 1994; Saint-Marie and Lafrance, 2002). However, for U. cordatus the density of juveniles within conspecific burrows is relatively low (e.g. 1e4 juveniles per host burrow; Schmidt and Diele, 2009) and food seems to be abundant in the muddy habitat. Diele and Koch (2010) observed that early U. cordatus recruits mostly remain below the sediment surface feeding upon the capitellid polychaete Notomastus lobatus. The density of the latter is high (>600 individuals m2) in undisturbed mangrove habitats (Beasley et al., 2010) and may be increased in areas bioturbated, and consequently oxygenated by U. cordatus. Additionally, the small recruits may also feed upon pieces of leaf litter remaining from sloppy feeding of the conspecific burrow owners (Diele, 2000; Diele and Koch, 2010).

Despite the advantages (shelter and food) of living within and around crab burrows, the risk of cannibalism between conspecific crabs of the same or different intraspecific cohorts (see Fernandez et al., 1993a; Moksnes et al., 1997; Luppi et al., 2001) could select against gregarious settlement of Ucides cordatus. However, despite the fact that newly moulted recruits and well established juveniles share the same benthic microhabitat (Schmidt and Diele, 2009) as result of successive events of reproduction followed by subsequent re-immigrations of the megalopae and low growth rate (Diele, 2000; Diele and Koch, 2010), it is unlikely that cannibalism occurs in U. cordatus in the mangroves. In the laboratory, Ventura et al. (2011) demonstrated that megalopae are not cannibalized by conspecific juveniles. Beside this, several other reasons support the hypothesis that cannibalism is rare in U. cordatus. Firstly, older juveniles and adults have large robust chelae, which decreases the likelihood of successful predation upon conspecific megalopae and tiny early juveniles (e.g. crab IeV stages). Secondly, the density of juveniles co-inhabiting conspecific burrows is low (see Schmidt and Diele, 2009). This should reduce the frequency of encounters between juveniles of the same or different intraspecific cohorts. Finally, megalopae (Diele, 2000; Ventura et al., 2008) and early juveniles of U. cordatus (Diele and Koch, 2010) have a cryptic life-style by digging and dwelling in small burrows in the muddy substratum. Such behaviour probably significantly reduces the risk of intra- and interspecific predation in the mangrove habitat. In Hemigrapsus sanguineus, predation of juveniles of the first crab stage by the killifish Fundulus majalis is significantly decreased in the presence of substratum (Kim and O’Connor, 2007). Following some authors, effects of inter- and intraspecific predation are density-dependent and determined by limitation of refuge (see Fernandez et al., 1993a; Iribarne et al., 1994; Eggleston and Armstrong, 1995; Dittel et al., 1995, 1996; Lovrich and Sainte-Marie, 1997; Moksnes et al., 1997,1998; Luppi et al., 2001, 2002; Saint-Marie and Lafrance, 2002). The survival rate of Ucides cordatus megalopae and early recruits has not yet been studied in the field and clearly is a major gap of knowledge for a better understanding of the population dynamics of the species. Also, the impact of interspecific predation by cooccurring species has been rarely investigated and should be addressed in future studies. For example, Ventura et al. (2010) demonstrated that fiddler crab species of different size classes (>0.5 cm CW) prey upon megalopae and early juveniles (JI) of U. cordatus under laboratory conditions. Intra- and interspecific predation have been considered as an important cause of early postsettlement mortality in benthic marine invertebrates including brachyuran crabs (Fernandez et al., 1993a,b; Dittel et al., 1995, 1996; Hunt and Scheibling, 1997; Luppi et al., 2001; Zmora et al., 2005). An elevated mortality, particularly for the newly settled recruits that have a higher frequency of moulting and become vulnerable during ecdysis could negatively affect successful recruitment (Gosselin and Qian, 1997; Hunt and Scheibling, 1997). In contrast to the numerous experimental studies on the effects of conspecific adult odours on brachyuran metamorphosis (reviewed by Anger, 2001; Forward et al., 2001; Gebauer et al., 2003), information on the effect of juvenile odours is still scarce (e.g. Anderson et al., 2010). Furthermore, the chemical identity and molecular size of water-born conspecific metamorphosis-inducing substances is unknown for most species, including Ucides cordatus. Molecular size was shown to be <10 kDa in Callinectes sapidus (Forward et al., 1996), <14 kDa in Hemigrapsus sanguineus (Steinberg et al., 2007) and <1 kDa in Panopeus herbstii (Andrews et al., 2001). In addition, the magnitude of metamorphic response in crab megalopae seems to be dose-dependent (e.g. Andrews et al., 2001; O’Connor, 2005; Anderson et al., 2010). Here, we demonstrated that 200 mL seawater in which only three small benthic juveniles (JIeIII) were incubated for 24 h (odour-treatment T1) was

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already enough to mediate a strong larval response. The same was also true when a high density of larger immature crabs was conditioned in 20 L for producing the odour-seawater (T2eT6). Our findings further suggest that the effectiveness of chemical odours is not dose-dependent above a certain minimum threshold concentration since the total wet weight of crabs used in T2eT8 was 505e 516 greater than that of crabs utilized in T1 and even so the average developmental TTM in T1 was 3.1 and 3.5 days shorter than in the odour-treatments T7 and T8, respectively. Therefore, it appears that only the presence of conspecific cues is important for larval stimulation. However, the minimum threshold concentration for the detection of the water-soluble cues by U. cordatus megalopae is unknown and should be determined in a future study. This should be important as the chemical substances emitted by resident crabs could be diluted during flood tides resulting in a decrease in cue concentration in the benthic environment. 5. Conclusions In summary, we demonstrated that Ucides cordatus megalopae respond not only to the water-soluble chemicals released by conspecific adult crabs, but also to those emanating from young recruits and older juveniles. This indicates that the emission of metamorphic chemical cues is independent of sexual maturity. Our results are coherent with recent field observations of Schmidt and Diele (2009) who firstly reported that young recruits co-inhabit juvenile (and adult) conspecific crab burrows. The responsiveness of crab megalopae to juvenile and adult odours probably results in increased recruitment rates and may thus favour the growth and maintenance of viable crab populations. The fact that resident juveniles induce gregarious megalopal settlement may have important consequences for the recovery of disturbed populations of the mangrove crab U. cordatus. Crab fishery, for example, targets large adult crabs only, thereby, reducing their numbers in intensively fished areas (Glaser and Diele, 2004; Diele et al., 2005, 2010). Hence, the presence of U. cordatus in different benthic life-history stages (from immature juveniles to sexually mature adult crabs) may accelerate larval settlement in disturbed mangrove areas. Acknowledgements We thank Angélica Otoni Pereira de Jesus, Lotta Kluger, Géssica, Loly, Zezinho, Edimilson, José, Gal, Josenildo, Marcão and Adelson Silva de Souza for their help during the practical work. Thanks to ‘Centro de Pesquisa e Gestão de Recursos Pesqueiros do Litoral Nordeste’ (CEPENE) e Advanced base of Caravelas city, ‘Projeto Manguezal’ (sponsored by FIBRIA) and Ulisses Scofield for the technical and logistical support. The present work was funded by the ‘Fundação de Amparo à Pesquisa do Estado do Pará’ (FAPESPA). The paper resulted from the scientific cooperation between the Center for Tropical Marine Ecology (ZMT), Bremen, Germany and the ‘Universidade Federal do Pará’ (UFPa), Belém, Brazil. This study is part of the PhD thesis of the first author. The co-author K. Diele received funding from the MASTS pooling initiative (The Marine Alliance for Science and Technology for Scotland) and its support is gratefully acknowledged. MASTS is funded by the Scottish Funding Council (grant reference HR09011) and contributing institutions. References  Abdi, H., 2007. The Bonferonni and Sidák corrections for multiple comparisons. In: Salkind, N.J. (Ed.), Encyclopedia of Measurement and Statistics. Sage, Thousand Oaks (CA), pp. 103e107. Abrunhosa, F.A., Silva-Neto, A.A., Melo, M.A., Carvalho, L.O., 2002. Importance of the food and feeding in the first larval stage of Ucides cordatus cordatus (Linnaeus,1763) (Decapoda: Ocypodidae). (Written in Portuguese). Revista Ciência Agronômica 33 (2), 5e12.

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