in response to benthic microbial biofilms

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Feb 7, 2017 - crab Uca maracoani is common (Diele and Simith, 2007). We hypothe- ..... associated biofilms (e.g. Uca vocator: Simith et al., 2010; U. pugnax:.
Journal of Experimental Marine Biology and Ecology 492 (2017) 132–140

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Metamorphosis of the edible mangrove crab Ucides cordatus (Ucididae) in response to benthic microbial biofilms 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 b 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 Applied Sciences, Edinburgh EH11 4BN, UK d St Abbs Marine Station, St Abbs TD14 5PW, UK

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Available online 7 February 2017 Keywords: Conspecific odours Decapod crustaceans Estuarine indicators Larval recruitment Megalopal settlement Microbial assemblages

a b s t r a c t Benthic microbial communities can play an important role in the induction of larval metamorphosis in marine invertebrates. The role of biofilms in recognizing the parental habitat is unknown for the ecologically and economically important mangrove crab Ucides cordatus, a species suffering from environmental pressures such as habitat degradation and disease. In the laboratory, we experimentally tested the influence of biofilms grown in offshore and estuarine waters on the moulting rates and developmental time to metamorphosis (TTM) of the last larval stage (megalopa) of U. cordatus. Here, we specifically studied whether: (i) megalopae of this larval-exporting species respond differently to marine (MB) and estuarine biofilms (EB), (ii) biofilms previously immersed (24 h) in adult crab-conditioned seawater (ACSW; i.e. conspecific chemical cues) exhibit a greater effectiveness (i.e. by inducing higher moulting rates and shorter TTM) on larval metamorphosis than non-immersed ones and (iii) biofilms pre-immersed (24 h) in ACSW decrease their effectiveness when incubated (24 h) in pure filtered seawater (FSW) before offering them to megalopae. U. cordatus megalopae metamorphosed to the first juvenile crab stage (JI) in response to both MB and EB, however the moulting rates were significantly higher and development TTM shorter in the presence of EB compared to MB. The metamorphosis-inducing effects were significantly enhanced when both types of biofilms were pre-immersed in ACSW, suggesting that conspecific cues can be absorbed and stored by microbial matrix. The higher inductiveness of EB compared to MB persisted after previous contact with ACSW. Furthermore, EB exhibited a significantly greater biomass production (measured as dry weight) than MB, and when immersed in ACSW, both groups of biofilms significantly increased in biomass (maintaining the hierarchy EB N MB), suggesting that water-soluble chemical substances emitted by the adult crabs may have been absorbed and metabolized by the biofilms. The metamorphic inductiveness and biomass production decreased when both groups of biofilms that were previously kept (24 h) in ACSW were thereafter immersed (24 h) in FSW. All megalopae successfully moulted to JI after accelerated TTM when reared in ACSW, regardless of presence or absence of biofilms, corroborating that conspecific stimuli are the most effective metamorphosis-stimulating cues tested so far in U. cordatus. The fact that EB improved the moulting rates and shortened the development TTM indicates that megalopae can recognize and respond to microbial assemblages typical for specific environments. This should be ecologically important during larval recruitment by facilitating settlement in appropriate habitats for the post-metamorphic development of early benthic recruits. In addition, the influence of EB could encourage colonization of new estuarine areas and aid natural recovery of U. cordatus in mangrove habitats where crab populations have suffered significant reduction due to deforestation, fishing pressure or diseases. © 2017 Elsevier B.V. All rights reserved.

1. Introduction ⁎ Corresponding author at: 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.J.B. Simith).

http://dx.doi.org/10.1016/j.jembe.2017.01.022 0022-0981/© 2017 Elsevier B.V. All rights reserved.

Larvae of many marine and estuarine benthic invertebrates respond to a broad range of naturally occurring physical, chemical and/or biological cues that indicate an appropriate habitat for definitive settlement near the conspecific population (Forward et al., 2001; Hadfield and

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Paul, 2001; McClintock and Baker, 2001; Hadfield, 2011). Such cues include diverse types of substratum characteristics of the parental adult habitat including chemical substances produced by conspecifics and benthic microbial communities (see review in Pawlik, 1992; Anger, 2001; Forward et al., 2001; Hadfield and Paul, 2001; Gebauer et al., 2003; Hadfield, 2011). Benthic biofilms are ubiquitous in marine and freshwater aquatic ecosystems (Decho, 1990, 2000). When natural and/or artificial substrata are immersed in water, they are rapidly colonized and coated with a thin microbial film, usually composed of bacteria, diatoms, protozoa and fungi living in a matrix of extracellular polymeric substances or ‘EPS’ (Decho, 1990, 2000; Pawlik, 1992; Costerton et al., 1995). Several studies have demonstrated that mono-species or multi-species biofilms can play an important ecological role by mediating larval settlement and metamorphosis of benthic marine invertebrate species (reviewed by Pawlik, 1992; Rodríguez et al., 1993; Wieczorek and Todd, 1997, 1998; Unabia and Hadfield, 1999; Hadfield and Paul, 2001; Steinberg et al., 2001). Conversely, biofilms or their chemical compounds have also been reported as inhibitors (i.e. antifouling) of larval attachment in some benthic invertebrates (see Holmström et al., 1992; Pawlik, 1992; Wieczorek and Todd, 1997, 1998; Dobretsov and Qian, 2004, 2006; Dobretsov et al., 2006). Biofilms can also substantially modify the physical and chemical properties of the substratum and serve as a source of food for the newly settled and well-established benthic recruits (Baird and Thistle, 1986; Decho, 1990, 2000; Hoskins et al., 2003). The biofilms themselves work as settlement cues for substratum selection of planktonic larvae. However, their inductiveness may result from two different types of chemical signalling: surface-bound cues (see Szewzyk et al., 1991; Huang and Hadfield, 2003; Khandeparker et al., 2003; Lau et al., 2003; Hadfield, 2011) and waterborne cues (i.e. bacterial metabolites) emanating from the biofilms (see Fitt et al., 1990; Rodriguez and Epifanio, 2000; Lau and Qian, 2001; Harder et al., 2002a; Lau et al., 2002; Bao et al., 2007a). In addition, large amounts of EPS produced by bacteria seem to be important for modulating larval settlement in some species (see Szewzyk et al., 1991; O'Connor and Richardson, 1998; Harder et al., 2002b; Lau et al., 2003). Compared to a large number of studies on the influence of biofilms on larval metamorphosis of marine invertebrates, e.g. bryozoans, polychaetes, mussels and ascidians (see Pawlik, 1992; Wieczorek and Todd, 1998; Hadfield and Paul, 2001; Steinberg et al., 2001; Hadfield, 2011; and references cited therein), few studies have been designed to investigate the inductive (or inhibitory) effects of benthic biofilms on larval metamorphosis of brachyuran decapod crustaceans. For example, some laboratory studies have suggested that settlement and metamorphosis of the last larval stage (i.e. megalopa) of decapod species can be triggered by substratum-associated biofilms (see Weber and Epifanio, 1996; O'Connor and Van, 2006; Krimsky and Epifanio, 2008; Steinberg et al., 2008; Anderson and Epifanio, 2009). Furthermore, it was proposed that the biochemical characteristics of the microbial communities (or bacterial products) associated with the benthic substratum may serve as settlement-inducing cues, rather than the physical properties or chemical cues produced by the substrata themselves (Weber and Epifanio, 1996; Rodriguez and Epifanio, 2000). In cases where there are multiple cues acting on larval metamorphosis, synergistic effects have been reported. For example, Steinberg et al. (2008) and Anderson and Epifanio (2009) have both used experiments to demonstrate that a combination of biofilms and textured artificial substrates or exudates from conspecific crabs can reduce the developmental time of megalopae of the Asian shore crab, Hemigrapsus sanguineus. For the mangrove crab Ucides cordatus (Linnaeus, 1763) (Family Ucididae, Števčić, 2005; Ng et al., 2008), we previously demonstrated that megalopal metamorphosis can be induced by chemical odours released by conspecific juveniles/adults and also by mud taken from near the openings of conspecific crab burrows in the mangrove forest habitat (Diele and Simith, 2007; Simith and Diele, 2008a; Simith et al., 2013a). It is not yet known, however, whether it is the substratum per

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se, or its association with surface-bound crab odours, that triggers larval metamorphosis. Chemical substances emanating from adult crabs may be sequestered and concentrated in biofilms on the mud surface as suggested by O'Connor and Van (2006). Recently, we demonstrated that U. cordatus megalopae settle on mud taken from a mid-intertidal channel mud-bank where conspecifics are not found, but where the fiddler crab Uca maracoani is common (Diele and Simith, 2007). We hypothesized that biofilms (e.g. diatom-dominated biofilms) were the source of stimulatory cues from the atypical habitat (i.e. channel mud bank void of conspecifics) rather than the interspecific cues, since the odours of U. maracoani isolated from substratum do not induce metamorphosis of Ucides megalopae (see Simith and Diele, 2008a). Thus, knowledge on the potential interactions between pre-established benthic microorganisms and settlement-competent megalopae is important for a better understanding of the pre-settlement determinants and recruitment patterns of this ecologically (Koch and Wolff, 2002; Schories et al., 2003; Nordhaus et al., 2006, 2009; Pülmanns et al., 2014, 2015) and economically important (Glaser and Diele, 2004; Diele et al., 2005, 2010) long-lived crab species (Diele and Koch, 2010). Ucides cordatus develops through five to six zoeal and a megalopal larval stage (Rodrigues and Hebling, 1989). After larval release in estuarine waters, their newly hatched zoeae are dispersed offshore by ebb-tide currents (Santarosa-Freire, 1998; Diele, 2000) for development in more stable and higher salinity conditions (Diele and Simith, 2006; Simith and Diele, 2008b). Megalopae migrate back upstream three to five weeks later (Diele, 2000) and settle in mangrove habitats induced by chemical ‘odours’ emitted by the conspecifics (Diele and Simith, 2007; Simith and Diele, 2008a; Simith et al., 2013a). During the larval recruitment period, specific benthic biofilms, particularly those growing in the estuarine environment, could have important ecological implications by encouraging colonization of virgin areas that are void of conspecifics and/or by aiding natural recovery of impacted crab populations in mangrove habitats exhibiting low crab density due to anthropogenic activities. Here, we experimentally investigated the influence of biofilms on the induction of metamorphosis in Ucides cordatus megalopae. We specifically studied whether megalopae respond differently to biofilms cultivated in offshore and estuarine waters, and assessed the magnitude of the effectiveness of these respective groups of biofilms in the laboratory. We hypothesized that more larvae metamorphose when exposed to microbial assemblages originating from the estuarine than those from the marine environment. The ability of larvae to differentiate between microbial groups of different composition and origin has been demonstrated in some marine invertebrate species (see Qian et al., 2003; Lau et al. 2005; Thiyagarajan et al. 2005; Dobretsov and Qian, 2006; Hung et al., 2007). Furthermore, the effectiveness on the induction of larval settlement may vary between biofilms of different species composition (see Holmstrøm et al., 1996; Lau et al., 2002, 2005; Huang and Hadfield, 2003) and between those developed under different environmental conditions (see Lau et al., 2005; Dobretsov and Qian, 2006; Dobretsov et al., 2006; Chiu et al., 2007; Hung et al., 2007). We further investigated whether megalopae cultivated in contact with marine and estuarine biofilms pre-treated for 24 h with conspecific crab odours (i.e. adultconditioned seawater) or with biofilms and adult-conditioned seawater together show a stronger metamorphic response (i.e. higher moulting rates and faster developmental time to metamorphosis) than when they are cultivated in filtered seawater with biofilms alone. Biofilms are composed of large concentrations of EPS that have a high absorptive capacity and hence, may absorb and store water-soluble chemicals from aquatic environments (Decho, 1990, 2000). Finally, we assessed if marine and estuarine biofilms pre-immersed in adult-conditioned seawater (24 h) decrease their effects on larval metamorphosis when placing them into pure filtered seawater for a further 24 h prior to being offered to U. cordatus megalopae in the cultivation. Here, we raise the hypothesis that during the 24-h period submerged in filtered seawater the biofilms could potentially release back into the aquatic

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medium or metabolize the chemical substances from crabs and, thus reducing the larval response of U. cordatus due to absence of conspecific cues in the biofilms. 2. Materials and methods 2.1. Seawater for larval cultivation in the laboratory Seawater (salinity 35 psu) was obtained 90 km offshore from the Caravelas River estuary (north-eastern Brazil) to minimise the likelihood of environmental chemical cues leaching from mangroves and estuaries (Simith and Diele, 2008a; Simith et al., 2010). The water was filtered (Eheim and Diatom Filter: 1 μm) and stored in constantly aerated polycarbonate tanks (500 L) in the laboratory. Sodium hypochlorite (2.5%) was added weekly (0.5 mL per litre seawater) for sterilization. The filtered seawater (hereafter referred to as FSW) was diluted with distilled tap water in order to obtain a salinity 30 psu for larval cultivation (after Diele and Simith, 2006; Simith and Diele, 2008b). Salinity was measured with a WTW-LF 197 conductivity meter and a hand refractometer (Atago). As the diluted seawater became slightly acidic (pH = 6.5), the pH value was then adjusted to 8.0 by using biological filters (i.e. biofilters) composed of calcareous substratum (e.g. crushed Mollusca shells) that provides sodium carbonate and sodium bicarbonate ions (buffering substances), thus increasing and stabilising pH. The same biofilters were also used to keep ammonium and nitrite levels low of the stored seawater. Detailed information about construction, functioning and types of biofilters can be found in New (2002), Timmons et al. (2002) and Valenti et al. (1998, 2009, 2010). The water remained in such filters for several weeks before larval cultivation. pH values were measured with a pH-meter probe (pH–710, Instrutherm). 2.2. Larval origin and rearing conditions Ucides cordatus zoea-larvae were obtained from five ovigerous females (5.4 ± 0.4 cm carapace width) captured two days before hatching, in April 2010, in the mangroves of the Caravelas River estuary, Bahia, north-eastern Brazil. The specimens were transported in individual aquaria filled with mud substratum and estuarine water (salinity 25 psu) to the laboratory at CEPENE (Centro de Pesquisa e Gestão de Recursos Pesqueiros do Litoral Nordeste), Caravelas City, State of Bahia. Ovigerous females were then carefully washed with saltwater and maintained individually in 10 L of gently aerated FSW at an ambient temperature of 27 °C, salinity 30 psu, pH 8.0 and under a natural light:dark cycle. All crabs were held without food until they released their larvae. All five ovigerous females released their larvae synchronously. Immediately after hatching, actively swimming zoea-I larvae were pooled in a glass beaker (5000 mL). Larvae were then collected using wide-bore pipettes and transferred into 500 mL plastic recipients for masscultivation at a density of 50 larvae per recipient until moulting to the

megalopal stage. Zoeae were reared at a constant water temperature of 27.9 ± 1.4 °C, salinity 30 psu (Diele and Simith, 2006; Simith and Diele, 2008b), pH 8.1 ± 0.2, photoperiod cycle of 12 h light and 12 h dark, and without aeration in the cultivation water. They were fed daily with planktonic microalgae (Dunaliella salina) and newlyhatched brine shrimp (Artemia sp. nauplii; INVE) ad libitum (ca. 10 nauplii·mL−1). When larvae had reached the zoea V stage, they were checked every day (at 2-h intervals) for moults to the megalopal stage. The cultivation medium was changed every 2 to 3 days. Megalopae were reared under the same conditions as zoea-larvae, but kept individually in 200 mL plastic vials to avoid cannibalism and competitive interactions (Forward et al., 2001). This larval stage was exclusively fed with Artemia sp. (ca. 2 nauplii·mL−1). 2.3. Development and growth of benthic microbial biofilms Benthic biofilms were grown in natural seawater (see above) and estuarine water under laboratory conditions and termed marine (MB) and estuarine (EB) biofilms, respectively. Estuarine water was collected during low tide from a large tidal channel (6 m water depth) in the Caravelas estuary. The two water types were filtered through a 100 μm nylon mesh to remove material in suspension (large particles, detritus and zooplankton) and stored separately in polycarbonate tanks (500 L). Sterile rubber tubes (1.5 cm diameter × 1.0 cm height) placed in each tank were used as an artificial substratum for development and growth of MB and EB. The water in each tank was kept under constant gentle aeration and the biofilms were allowed to grow for 30 days under controlled and constant temperature (27.3 ± 0.7 °C), salinity (30 psu), and pH (7.9 ± 0.5) conditions and a natural photoperiod regime (12 h/12 h: light/dark cycle). The water in each tank was replaced every 2 or 3 days. After 30 days, the biofilmcovered rubber tubes were removed from their respective tanks using sterile forceps and (1) transferred immediately to the cultivation vials containing Ucides cordatus megalopae, or (2) they were pre-treated with conspecific chemical cues from adult crabs (= adult-conditioned seawater) prior to being placed into the cultivation vials containing megalopae (see Table 1 and section 2.4 below). 2.4. General experimental procedures To investigate the metamorphic response of Ucides cordatus to marine and estuarine biofilms with or without exposure to conspecific adult odours, 330 recently-moulted megalopae (maximum of 1 h after moulting) were randomly distributed among the eight experimental treatments and three control groups and cultivated until reaching the first juvenile crab stage (see Table 1 for description of treatments). Treatments with megalopae reared in the presence of biofilms are termed as ‘biofilm-treatments’ (i.e. ‘biofilm-T1 to biofilm-T8’, see Table 1), and those without referred to as control treatments (C1–C3, see Table 1). Thirty 200 mL plastic vials were then established for each biofilm-treatment and control group, each one containing a single

Table 1 Description of experimental biofilm-treatments (T1–T8) and control groups (C1–C3) for cultivations of Ucides cordatus megalopae under laboratory conditions. ACSW: adult-conditioned seawater = water that was previously conditioned with conspecific male and female adults; FSW: filtered seawater not conditioned with crabs; MB: marine biofilm; EB: estuarine biofilm. Treatments

Description

T1: MB T2: EB T3: MB + ACSW/24 h T4: EB + ACSW/24 h T5: MB + ACSW/24 h + FSW/24 h T6: EB + ACSW/24 h + FSW/24 h T7: MB + ACSW T8: EB + ACSW C1: ACSW (Control 1) C2: FSW (Control 2) C3: FSW + rubber tube (Control 3)

Cultivation of megalopae in FSW containing MB. Cultivation of megalopae in FSW containing EB. Megalopae reared in FSW with MB immersed previously in ACSW for 24 h. Megalopae reared in FSW with EB immersed previously in ACSW for 24 h. Cultivation of megalopae in FSW with MB immersed previously in ACSW for 24 h and later immersed in FSW for a further 24 h. Cultivation of megalopae in FSW with EB immersed previously in ACSW for 24 h and later immersed in FSW for a further 24 h. Megalopae reared in ACSW with MB. Megalopae reared in ACSW with EB. Cultivation of megalopae in ACSW without biofilm. Cultivation of megalopae in FSW without biofilm. Cultivation of megalopae in FSW containing a sterile rubber tube not colonized by biofilm.

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megalopa reared in (= experimental replicate; see Forward et al., 2001). Megalopae varied in age from 31 to 37 days (33.3 ± 1.5 days) since hatching from egg. Specimens of different age were then interspersed among the respective treatments. The water in each vial was replaced every 2 days and the rubber tubes replaced by new 30-d old biofilm-covered tubes cultivated under the specific conditions. All rubber tubes removed from the vials were weighed to determine biofilm biomass (see section 2.5 below). During water changes of the C3 control (FSW + sterile rubber tubes), the rubber tubes were replaced by new sterile tubes to minimise the likelihood of biofilm formation during megalopal cultivation. Each individual megalopa reared in their respective plastic vial received only one tube colonized (biofilm-treatments T1 to T8) or not (control C3) by biofilm, except for the control groups C1 and C2 containing megalopae reared in complete absence of biofilm and tubes (see Table 1). The percentage of successfully metamorphosed megalopae was monitored and recorded daily. The experiment ended when the last megalopae had either moulted into juvenile crab stage or died. Adult-conditioned seawater (hereafter referred to as ACSW) containing chemical odours of Ucides cordatus was obtained by placing two male and two female adults (carapace width = 6.4 ± 0.7 and 5.8 ± 0.4 cm, respectively) in glass aquaria filled with 20 L of constantly aerated FSW for 24 h. Each crab was carefully washed with natural seawater (see section 2.1 above) and their carapace brushed to remove mud and likely natural biofilms covering their body prior to being placed in the aquarium. The crabs were then removed and the water was filtered through a 200 μm mesh filter prior to use in the treatments (Table 1). Fresh ACSW was prepared using newly captured crabs for each water change. For biofilm-treatments T3 and T4 (see Table 1), the rubber tubes colonized by 30-d old biofilms were placed in 20 L of gently aerated ACSW in a glass aquarium for 24 h. After incubation, the biofilm-covered tubes were collected, gently washed with FSW and immediately placed in the cultivation vials with megalopae. For the biofilm-treatments T5 and T6 (see Table 1), the same procedure was performed; after previous incubation (24 h) in ACSW, however, the biofilms were immersed in 20 L of gently aerated FSW for a further 24 h in a glass aquarium. The biofilms were then retrieved and offered to megalopae in the experiment.

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Kolmogorov-Smirnov's test and Levene's median test (applied after modifications proposed by Brown and Forsythe, 1974), respectively and when necessary, data were transformed [e.g. √ x, √ (x + 1)] to achieve these prerequisites for parametric statistics. One-way ANOVA tests were then used to compare TTM and biofilm dry weigh data from each treatment. Post-hoc Student-Newman-Keuls (SNK) pairwise comparison tests were used to identify significant differences among the respective treatments. Development TTM data obtained in the control groups C2 and C3 were not included in the analysis due to the low number of surviving specimens and, hence, small sample size (see Results). Differences were considered to be significant when p b 0.05.

3. Results 3.1. Larval metamorphosis in response to benthic microbial biofilms Ucides cordatus megalopae moulted to the first juvenile crab stage (JI) in response to benthic microbial communities developed in both marine and estuarine waters (Fig. 1). Significantly more megalopae metamorphosed to JI (χ2 = 6.0–105.46; p b 0.05) when cultivated in FSW in the presence of both marine (MB; biofilm-treatment T1) and estuarine (EB; biofilm-treatment T2) biofilms than in the treatments containing FSW only (control C2) or FSW and sterile rubber tubes (control C3) (Fig. 1). The metamorphic response was significantly lower (χ2 = 43.05; p b 0.0001) when megalopae were reared in exposure to MB (biofilm-T1, 33.3%) compared with EB (biofilm-T2, 80% of moulted specimens) (Fig. 1). The effect of MB and EB on metamorphosis of Ucides cordatus megalopae also differed significantly (χ2 = 26.92–125.27; p b 0.001) from the control treatments (C2 and C3) when the biofilms were immersed in ACSW containing conspecific adult odours (biofilm-treatments T3–T6; Fig. 1). The percentage of metamorphosed megalopae varied significantly (χ2 = 5.41; p = 0.02) between biofilm-T3, containing MB previously maintained in ACSW for 24 h and biofilm-T5, with MB also previously kept in ACSW for 24 h, but immersed in FSW for a further 24 h (Fig. 1). These treatments resulted in significantly higher moulting rates (70 and 53.3%, respectively; T3 vs. T1: χ2 = 25.94; p = 0.001; T5 vs. T1: χ2 = 7.36; p = 0.0067) compared to those

2.5. Measurement of the biofilm dry weight (biomass production) To determine if larval metamorphosis of Ucides cordatus was affected by the quantity of biofilm biomass, we measured the dry weight of the biofilm (MB and EB) throughout the period of megalopal culture. The rubber tubes removed from the cultivation vials during water changes were carefully rinsed with distilled tap water to remove saltwater, dried in an oven at 80 °C for 48 h, and then cooled in an acclimatized room in desiccators prior to weighing (Marte–AL200C, nearest 0.001 g). The rubber tubes were then washed and scrubbed to remove any biofilm, dried in an oven for a further 24 h, and then reweighed. Dry weight (mg·cm−2) was determined by subtracting the weight of the dried and cleaned rubber tube from the weight of the biofilmcovered tube. 2.6. Statistical analysis Percentage survival (= moulting rates) and average developmental time to metamorphosis (TTM in days) of the megalopal stage of Ucides cordatus were compared among treatments. The day of moulting to megalopa was termed as day 0 for age determination and obtainment of development TTM in the respective treatments. All statistical analysis followed standard techniques as described by Sokal and Rohlf (1995). The moulting rates were analyzed by R × C (rows × columns) contingency tables followed by Yates corrected Chi-square test (χ2). For data of development TTM and biofilm dry weight, assumptions of normality and homogeneity of variances were tested using

Fig. 1. Percentage of successfully metamorphosed Ucides cordatus megalopae to first juvenile crab stage in response to marine (MB) and estuarine (EB) benthic biofilms (alone or associated with conspecific crab odours), adult-conditioned seawater (ACSW) and filtered seawater (FSW). T1 = MB, T2 = EB, T3 = MB + ACSW/24 h, T4 = EB + ACSW/24 h, T5 = MB + ACSW/24 h + FSW/24 h, T6 = EB + ACSW/24 h + FSW/24 h, T7 = MB + ACSW, T8 = EB + ACSW, C1 = ACSW, C2 = FSW, C3, FSW + sterile rubber tube. See Table 1 for descriptions of the treatments. Different letters above bars indicate significant differences (Yates corrected Chi-Square test χ2; p b 0.05) among treatments.

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cultivated with MB alone (biofilm-T1 = 33.3%) (Fig. 1). The same pattern was also observed when comparing the biofilm-treatments containing EB (T2, T4 and T6), except that there was no significant difference (χ2 = 1.31; p = 0.253) between biofilm-T2 (EB alone not treated with ACSW, 80% of moulted specimens) and biofilm-T4 (EB previously immersed in ACSW for 24 h, 86.7% of metamorphosis) (Fig. 1). All megalopae successfully moulted to JI stage when reared in ACSW with the presence of MB and EB (biofilm-treatments T7 and T8) or without biofilms (control C1) (Fig. 1). In contrast, very few individuals underwent metamorphosis in the control groups containing FSW only (control C2) or FSW plus sterile rubber tubes (control C3) (Fig. 1). 3.2. Developmental time to metamorphosis (TTM) in response to benthic microbial biofilms The average developmental time to metamorphosis (TTM) of the megalopal stage of Ucides cordatus differed significantly (F8, 198 = 4.61; p b 0.001) among the respective treatments (C2 and C3 were not included in the analysis) (Fig. 2). The development TTM ranged from 10 to 33 days in the specimens that had successful moulted to the first juvenile crab stage (JI) in the respective biofilm-treatments (T1–T8) and control groups (C1–C3) (Fig. 2). TTM was shorter when megalopae were reared in exposure to MB (biofilm-T1) and EB (biofilm-T2) than when cultivated without biofilms in FSW (control C2) or in FSW plus sterile rubber tubes (control C3) (Fig. 2). Average TTM was significantly shorter in the presence of EB (biofilm-T2; 18.9 ± 2.7 days) than MB (biofilm-T1; 22.5 ± 2.0 days) (Fig. 2). With EB present, megalopae began to moult 8 days earlier than treatments with MB, and over 50% of the specimens had already undergone metamorphosis after 20 days of culture (Fig. 3A). The shortest TTM was recorded in the biofilm-treatments T4, T7 and T8 and in the control treatment C1, which all involved short-term or permanent exposure to ACSW (Fig. 2), with no significant difference between these treatments (pooled data, 17.4 ± 3.8 days) (Fig. 2). In biofilm-T3 and biofilm-T4, where MB and EB had previously been immersed in ACSW for 24 h, the first juveniles were recorded on day 12

of cultivation (Fig. 3B). In these biofilm-treatments, 57% (T3, MB) and 50% (T4, EB) of the specimens had already moulted to juvenile crab stage by day 22 and 17 of the megalopal cultivation, respectively (Fig. 3B). In the biofilm-treatments T5 and T6, where the previously ACSW-immersed MB and EB had been maintained in FSW for 24 h, megalopae began to metamorphose on day 14 of cultivation, and 53 and 50% of the specimens reached the first crab stage by day 26 and 21 of cultivation, respectively (Fig. 3C). In treatments where megalopae were permanently reared in ACSW with biofilms, the first specimens underwent metamorphosis 2 days later in biofilm-T7 containing MB than in biofilm-T8 with EB (Fig. 3D). In biofilm-T7, 80% of the specimens had moulted to juvenile by day 19, while in biofilm-T8, the 50% value was already reached by day 17 of cultivation (Fig. 3D). The last megalopae moulted on day 21 and 33 in biofilm-T7 and biofilm-T8, respectively (Fig. 3D). In the control group C1 that contained ACSW but no biofilm, the first and last megalopae metamorphosed on day 10 and 23, respectively, and over 50% of the specimens had moulted by day 19 of cultivation (Fig. 3E). In contrast, in treatments with FSW devoid of conspecific adult odours and biofilms (control group C2) and with FSW plus sterile rubber tubes (control C3), the first event of metamorphosis was only observed on day 26 and no megalopae metamorphosed after day 29 (control C3) and 30 (control C2) of cultivation (Fig. 3E). 3.3. Biomass production of the benthic microbial biofilms The average biomass (mg·cm− 2) production (expressed as dry weight) of the marine (MB) and estuarine (EB) biofilms differed significantly (F7, 230 = 37.65; p b 0.001) among treatments (biofilm-T1 to biofilm-T8) (Fig. 4). EB biomass (biofilm-T2) was significantly greater than MB biomass (biofilm-T1) (Fig. 4); the same pattern (EB N MB) was apparent among biofilm-treatments T3–T8, where biofilms were combined with conspecific chemical odours released by adult crabs (Fig. 4). Average dry weight was significantly higher when MB and EB were previously immersed in ACSW for 24 h (biofilm-T3, biofilm-T4) or kept in ACSW for 24 h, but immersed in FSW thereafter for 24 h (biofilm-T5, biofilm-T6), compared to the dry weight of biofilms not exposed to conspecific adult odours (see biofilm-T1 and biofilm-T2) (Fig. 4). However, the dry weight of EB did not differ significantly between biofilm-T4 and biofilm-T8 (Fig. 4). In contrast, the dry weight of EB from the biofilm-T8 was significantly higher when compared with that of EB from the biofilm-treatments T2 and T6 (Fig. 4). 4. Discussion

Fig. 2. Developmental time to metamorphosis (TTM; in days) of Ucides cordatus megalopae in response to marine (MB) and estuarine (EB) benthic biofilms (alone or associated with conspecific crab odours), adult-conditioned seawater (ACSW) and filtered seawater (FSW). T1 = MB, T2 = EB, T3 = MB + ACSW/24 h, T4 = EB + ACSW/24 h, T5 = MB + ACSW/24 h + FSW/24 h, T6 = EB + ACSW/24 h + FSW/24 h, T7 = MB + ACSW, T8 = EB + ACSW, C1 = ACSW, C2 = FSW, C3, FSW + sterile rubber tube. See Table 1 for descriptions of the treatments. Numbers inside bars refer to the number of successfully metamorphosed specimens from initially n = 30 megalopae/treatment. Different letters above bars indicate significant differences (p b 0.05) after statistical pairwise comparisons using the posthoc test of Student-Newman-Keuls (SNK). Absence of letters above bars in the control treatments C2 and C3 was due to the insufficient sample size (n = 2 and 5, respectively) and, therefore, these treatments were not included in the analyses. Data were expressed as average values ± standard deviation.

Biofilms are ubiquitous in marine aquatic ecosystems and play an important role as modulators of larval settlement and metamorphosis in many marine invertebrate taxa, including brachyuran decapod crustaceans (Pawlik, 1992; Wieczorek and Todd, 1998; Hadfield and Paul, 2001; McClintock and Baker, 2001; Steinberg et al., 2001, 2008; Anderson and Epifanio, 2009). In the present laboratory study, we demonstrated that megalopae of the mangrove crab Ucides cordatus undergo metamorphosis to first juvenile crab stage (JI) in response to benthic biofilms. The metamorphic response was mediated by biofilms that had developed on artificial substrata (rubber tubes) submerged in both marine and estuarine waters. However, a higher percentage of megalopae moulted and developed faster when reared with EB than when they were cultivated with MB. These differences persisted when both types of biofilms were immersed for 24 h in adult-conditioned seawater (ACSW) or immersed in both ACSW and filtered seawater (FSW) for 24 h (except for development TTM in which no significant difference was detected between the biofilm-treatments T5 and T6; see Fig. 2). Whether metamorphosis of U. cordatus megalopae reared in contact with MB and EB is triggered by the biofilms themselves (e.g. surfacebound cues, extracellular polymeric secretions - EPS) or by watersoluble products released by the microorganisms (e.g. bacterial metabolites) into seawater remains unresolved. Some studies have

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Fig. 3. Cumulative percentage of successfully metamorphosed Ucides cordatus megalopae in response to marine (MB) and estuarine (EB) benthic biofilms (alone or associated with conspecific crab odours), adult-conditioned seawater (ACSW) and filtered seawater (FSW). T1 = MB, T2 = EB, T3 = MB + ACSW/24 h, T4 = EB + ACSW/24 h, T5 = MB + ACSW/24 h + FSW/24 h, T6 = EB + ACSW/24 h + FSW/24 h, T7 = MB + ACSW, T8 = EB + ACSW, C1 = ACSW, C2 = FSW, C3, FSW + sterile rubber tube. See Table 1 for descriptions of the treatments. Dashed lines indicate 50% of metamorphosed larvae.

demonstrated that EPS (Szewzyk et al., 1991; O'Connor and Richardson, 1998; Lau et al., 2003) and microbial metabolites from biofilms induce larval settlement and metamorphosis in many marine invertebrates

Fig. 4. Biofilm biomass (mg·cm−2) production, expressed as dry weight, of the marine (MB) and estuarine (EB) benthic biofilms (alone or associated with conspecific crab odours) in the respective biofilm-treatments (T1–T8) containing adult-conditioned seawater (ACSW) and filtered seawater (FSW). T1 = MB, T2 = EB, T3 = MB + ACSW/24 h, T4 = EB + ACSW/24 h, T5 = MB + ACSW/24 h + FSW/24 h, T6 = EB + ACSW/24 h + FSW/24 h, T7 = MB + ACSW, T8 = EB + ACSW. See Table 1 for descriptions of the treatments. Data were based on the weighing of 30 biofilm-covered rubber tubes per treatment. Different letters above bars indicate significant differences (p b 0.05) after statistical pairwise comparisons using the post-hoc test of StudentNewman-Keuls (SNK). Data were expressed as average values ± standard deviation.

(see Fitt et al., 1990; Hadfield and Paul, 2001; Harder et al., 2002a; Hadfield, 2011). For the mud crab Panopeus herbstii, the induction of metamorphosis did not depend on physical interactions between megalopae and biofilm (Rodriguez and Epifanio, 2000), suggesting that waterborne cues derived from the biofilm were instead involved in the larval response. By contrast, in the polychaete Hydroides elegans, the contact with biofilms is necessary for larval settlement and metamorphosis (Hadfield et al., 2014). Our results clearly show that Ucides cordatus megalopae react differently to biofilms originating from two distinct environments, i.e. marine and estuarine. It appears that invertebrate larvae respond in a different way to biofilms derived from geographically distant habitats (see Keough and Raimondi, 1996; Wieczorek and Todd, 1998; Chiu et al., 2007). In the marine slipper limpet Crepidula onyx, for example, larval attachment is mediated by biofilm from a habitat where the species is commonly found, but not by biofilm from an atypical habitat devoid of conspecifics (Chiu et al., 2007). In brachyuran crabs, the effectiveness of settlement cues decreases with increasing distance from the adult habitat (see O'Connor and Judge, 2004; O'Connor and Van, 2006; Anderson and Epifanio, 2010) and differential responses to habitatspecific biofilms may occur. For example, for megalopae of the mud crab Panopeus herbstii, biofilms from the habitat of the conspecific adults can trigger metamorphosis whereas those from a non-adult habitat did not cause larval stimulation (see Rodriguez and Epifanio, 2000). In contrast to the former, megalopae of an Asian shore crab Hemigrapsus sanguineus react similarly to biofilms from different habitats (Anderson and Epifanio, 2009). Likewise, fiddler crab megalopae settle and metamorphose in response to substrata from different typical and atypical sources, possibly attracted and stimulated by substratum-

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associated biofilms (e.g. Uca vocator: Simith et al., 2010; U. pugnax: O'Connor and Van, 2006). Responses to cues from multiple habitats should facilitate settlement and colonization of new areas devoid of conspecifics (O'Connor and Van, 2006; Anderson and Epifanio, 2009). However, for U. cordatus, a species exhibiting several planktonic lifehistory stages (Rodrigues and Hebling, 1989), larval dispersal strategy (Santarosa-Freire, 1998; Diele, 2000), non-random settlement in mangrove forest habitats (Diele, 2000; Diele and Simith, 2007; Simith and Diele, 2008a; Schmidt and Diele, 2009) and very narrow habitat requirements (e.g. availability and type of preferred food: Nordhaus et al., 2006, 2009; Nordhaus and Wolff, 2007; degree of tidal flooding, tree-species composition in mangrove forest habitat: Piou et al., 2009; Wunderlich and Pinheiro, 2013), the larval ability to identify biofilm characteristic of the parental estuarine habitat may therefore be particularly relevant for successful recruitment and maintenance of the crab populations. Higher moulting rates and shorter TTM of U. cordatus megalopae in response to EB is therefore not necessarily surprising since the benthic habitats of this species are mangrove estuaries, suggesting an adaptive capacity to discriminate among biofilms of different origin. The observed differences in the magnitude of larval response towards the presence of EB and MB also suggest differences in the taxonomic composition of these benthic microbial communities. The community composition of biofilm has been shown to affect settlement of marine invertebrate larvae (see Huang and Hadfield, 2003; Webster et al., 2004; Lau et al., 2005; Whalan and Webster, 2014). In this experiment, metamorphosis of Ucides cordatus megalopae was associated with relatively well-developed (30-d old) biofilms. For several other invertebrate species, the magnitude of the larval response can be affected by the age of the biofilm, with older biofilms appearing more stimulating than younger biofilms (see Keough and Raimondi, 1995; Wieczorek and Todd, 1997; Rodriguez and Epifanio, 2000; Hamer et al., 2001; Bao et al., 2007b; Anderson and Epifanio, 2009). We also observed that the biomass (dry weight) of EB was greater than of MB (e.g. biofilm-T1 vs. biofilm-T2), despite being the same age, suggesting differences in biofilm growth rates, EPS productions and/or microbial community composition. The quantity of EPS can vary substantially among and within microbial groups under different environmental conditions (reviewed by Decho, 1990) and the larval metamorphosis of invertebrate species can be positively (e.g. Wieczorek et al., 1995; Hamer et al., 2001; Huang and Hadfield, 2003; Rahim et al., 2004; Bao et al., 2007a,b) or negatively correlated (e.g. Dahms et al., 2004; Anderson and Epifanio, 2009) with microbial density in multi-species biofilms. Here, the highest effects of EB on the induction of metamorphosis in U. cordatus megalopae may have been caused by one or several factors such as habitat-specific biofilms, higher biomass, concentration of EPS, and density of microorganisms. On the other hand, during larval recruitment and location of suitable habitats, only the presence of estuarine benthic indicators (i.e. microbial assemblages) should be sufficient for U. cordatus megalopae that have a limited time to settle and metamorphose before showing detrimental effects on early juvenile performance resulting from a prolonged larval life (see Simith et al., 2013b). The combination of biofilms with other stimulatory cues (e.g. textured substrata, adult exudates) can increase the effects on larval metamorphosis of decapod species (e.g. Steinberg et al., 2008; Anderson and Epifanio, 2009). In our laboratory study, when we immersed biofilms (MB and EB) in ACSW (24 h) with conspecific cues present, the metamorphic moult of Ucides cordatus megalopae (e.g. moulting rates and/ or TTM) increased significantly (see biofilm-T1 vs. biofilm-T3 in Figs. 1 and 2 and biofilm-T2 vs. biofilm-T4 in Fig. 2). We propose that conspecific cues attached to the biofilm surface or stored in the EPS matrix may be recognized by the megalopae after physical contact, thereby improving the attractiveness of and response to biofilms. In addition to the enhanced metamorphic response of the larvae, we observed an increase in biofilm biomass in the treatments where they were associated with ACSW (see biofilm-treatments T3–T8 in Fig. 4). Although our laboratory

experiment was not designed to test the hypothesis that chemical substances emitted by the crabs (e.g. urine, ammonium, chemical cues, pheromones) during the conditioning with seawater were absorbed by the biofilms, the 24-h period exposed to ACSW has unquestionably caused an increase in microbial biomass (see biofilm-treatments T3– T8) compared to biofilms not exposed to conspecific crab cues (see biofilm-T1 and biofilm-T2) (Fig. 4). Some authors have suggested that EPS produced by microorganisms may absorb, store and thereby increase the concentration of water-soluble chemical substances from the aquatic medium, including crab-derived molecules (see Decho, 1990; O'Connor and Van, 2006). Our results would support this hypothesis and, therefore, we cannot exclude the fact that the observed increase in biomass may have been caused by microbial metabolisation of the crab substances or by increased production of secondary microbial metabolites. Furthermore, the fact that EB was more stimulatory on U. cordatus megalopae and presented the highest biomass production when exposed to ACSW suggests that this microbial group is more absorptive and/or grows faster than MB. Moreover, the relatively weak response of U. cordatus megalopae and decrease in biomass of biofilms pre-treated (24 h) with ACSW and later immersed (24 h) in FSW (biofilm-T5 and biofilm-T6) suggests that crab derived molecules may be rapidly utilized in metabolic process or released back into the aquatic medium, thus reducing its concentration within the EPS and changing the larval response. An alternative explanation may be that biofilms experienced a ‘starvation regime’ when placed into FSW, resulting in a decrease in biomass. However, further studies involving biochemical analysis are needed to formally test the hypothesis that biofilms can sequester, release or metabolize crab substances. The stronger effect of ACSW on the moulting rates and development TTM corroborates earlier findings that chemical substances emitted by conspecific crabs are the most effective metamorphosis-stimulating cues tested so far in Ucides cordatus megalopae (Diele and Simith, 2007; Simith and Diele, 2008a; Simith et al., 2013a). In the present study, moulting success is increased (to 100%) and development TTM is accelerated when cultivating megalopae in ACSW with (biofilm-T7 and biofilm-T8) or without biofilms (control C1), while metamorphosis is significantly delayed and most megalopae die in FSW without biofilms (control C2) or with sterile rubber tubes (control C3). High mortality rates and the delay of metamorphosis in the absence of suitable habitat or conspecific adult cues have also been shown in previous studies (see Diele and Simith, 2007; Simith and Diele, 2008a; Simith et al., 2013a,b). Delayed metamorphosis can enhance the risk of predation in the planktonic environment (Morgan, 1995) and negatively affect post-larval performance and fitness of early benthic juveniles of decapod crabs (see Gebauer et al., 1999, 2003; Simith et al., 2013b), such that more broadly, the dynamics of U. cordatus populations within the mangrove ecosystem may be negatively affected. In summary, we experimentally demonstrated that Ucides cordatus megalopae metamorphose to first juvenile crab stage in response to biofilms originating from both offshore and estuarine waters. However, EB presented a stronger inductiveness and a higher biofilm biomass production than MB. The same hierarchy was also observed when biofilms were previously exposed to crab-associated cues. Our results further showed that U. cordatus megalopae respond differently to benthic biofilms from different habitats. The ability to identify habitatspecific biofilms is interpreted as an adaptation to facilitate the choice of an appropriate habitat for the early post-metamorphic development and growth of benthic juveniles in the mangroves. We also found that both metamorphosis-stimulating effect (except for moulting rates between biofilm-T2 and biofilm-T4) and microbial biomass were significantly enhanced when biofilms were previously immersed in ACSW. These findings suggest that biofilms can absorb, store, and/or metabolize crab-derived molecules, resulting in an increasing in biomass. Furthermore, the conspecific crab odours seem to be the most effective exogenous cues for the induction of larval metamorphosis in U. cordatus. The influence of multiple environmental cues on the

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attractiveness of megalopae should be particularly important for the mangrove crab U. cordatus, which ‘exports’ its larvae to offshore waters with subsequent re-immigrations to the estuarine system. Thus, a larval response to a wide range of environmental cues should increase the likelihood of finding a particular microhabitat (e.g. inner walls of crab burrows: Schmidt and Diele, 2009) for definitive settlement close to the conspecific population. In addition, the effectiveness of estuarine benthic biofilms on the induction of larval metamorphosis could stimulate colonization of virgin estuarine areas and aid natural recovery of U. cordatus populations previously impacted by habitat destruction, fishing pressure or diseases. Future studies need to investigate whether the inductiveness of biofilms for the metamorphosis in this important species is impaired (or masked) by environmental pollutants such as oil spills or sewage. 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 water collections, sampling of mangrove crabs, and larval and biofilm cultivation in the laboratory. Thanks to the ‘Centro de Pesquisa e Gestão de Recursos Pesqueiros do Litoral Nordeste’ (CEPENE) - Advanced base of Caravelas city, Projeto Manguezal (sponsored by FIBRIA) and Ulisses Scofield by the technical and logistical support. Thanks to Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio) by the authorization granted to the first author (SISBIO Authorization number: 22951–1) to perform activities with scientific purposes within the Extractive Reserve of the Cassurubá/Ba. This work was supported by the ‘Fundação Amazônia de Amparo a Estudos e Pesquisas do Pará’ (FAPESPA) through a doctorate scholarship granted to the first author. This study is part of the PhD thesis of the first author. Diele K. received funding from The Marine Alliance for Science and Technology for Scotland (MASTS) pooling initiative and its support is gratefully acknowledged. MASTS is funded by the Scottish Funding Council (grant reference HR09011) and contributing institutions. [SS] References Anderson, J.A., Epifanio, E., 2009. Induction of metamorphosis in the Asian shore crab Hemigrapsus sanguineus: characterization of the cue associated with biofilm from adult habitat. J. Exp. Mar. Biol. Ecol. 382, 34–39. Anderson, J.A., Epifanio, E., 2010. Response of the Asian shore crab Hemigrapsus sanguineus to metamorphic cues under natural field conditions. J. Exp. Mar. Biol. Ecol. 384, 87–90. Anger, K., 2001. The Biology of Decapod Crustacean Larvae, Crustacean Issues 14. A.A. Balkema, Lisse, The Netherlands. Baird, B.H., Thistle, D., 1986. Uptake of bacterial extracellular polymer by a depositfeeding holothurian (Isostichopus badionotus). Mar. Biol. 92, 183–187. Bao, W.Y., Yang, J.L., Satuito, C.G., Kitamura, H., 2007a. Larval metamorphosis of the mussel Mytilus galloprovincialis in response to Alteromonas sp. 1: evidence for two chemical cues? Mar. Biol. 152, 657–666. Bao, W.Y., Satuito, C.G., Yang, J.L., Kitamura, H., 2007b. Larval settlement and metamorphosis of the mussel Mytilus galloprovincialis in response to biofilms. Mar. Biol. 150, 565–574. Brown, M.B., Forsythe, A.B., 1974. Robust tests for the equality of variances. J. Am. Stat. Assoc. 69, 364–367. Chiu, J.M.Y., Thiyagarajan, V., Pechenik, J.A., Hung, O.S., Qian, P.Y., 2007. Influence of bacteria and diatoms in biofilms on metamorphosis of the marine slipper limpet Crepidula onyx. Mar. Biol. 151, 1417–1431. Costerton, J.W., Lewandowski, Z., Caldwell, D.E., Korber, D.R., Lappin-Scott, H.M., 1995. Microbial biofilms. Annu. Rev. Microbiol. 49, 711–745. Dahms, H.U., Dobretsov, S., Qian, P.Y., 2004. The effects of bacterial and diatom biofilms on the settlement of the bryozoan Bugula neritina. J. Exp. Mar. Biol. Ecol. 313, 191–209. Decho, A.W., 1990. Microbial exopolymer secretions in ocean environments: their role(s) in food webs and marine processes. Oceanogr. Mar. Biol. Annu. Rev. 28, 73–153. Decho, A.W., 2000. Microbial biofilms in intertidal systems: an overview. Cont. Shelf Res. 20, 1257–1273. Diele, K., 2000. Life history and population structure of the exploited mangrove crab Ucides cordatus cordatus (L.) (Decapoda: Brachyura) in the Caeté estuary, North Brazil. Center for Tropical Marine Ecology. ZMT — Contribution 09, Bremen (103 pp.). Diele, K., Koch, V., 2010. Growth and mortality of the exploited mangrove crab Ucides cordatus (Ucididae) in N-Brazil. J. Exp. Mar. Biol. Ecol. 395, 171–180.

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