Mar Biol (2007) 150:565–574 DOI 10.1007/s00227-006-0383-4
R E SEARCH ART I CLE
Larval settlement and metamorphosis of the mussel Mytilus galloprovincialis in response to bioWlms Wei-Yang Bao · Cyril Glenn Satuito · Jin-Long Yang · Hitoshi Kitamura
Received: 31 January 2006 / Accepted: 7 June 2006 / Published online: 4 July 2006 © Springer-Verlag 2006
Abstract BioWlms were allowed to develop on glass slips immersed 1.0–1.5 m below the sea surface in Tachibana Bay, Nagasaki, Japan, for diVerent periods of time from November 2003 to January 2005. The eVects of age, immersion month, dry weight, bacterial and diatom densities of these bioWlms on the settlement and metamorphosis of pediveliger larvae of the mussel Mytilus galloprovincialis were investigated in the laboratory. Furthermore, bioWlms were subjected to various treatments to investigate the nature of the settlement and metamorphosis cue in the bioWlm. Pediveliger larvae of the mussel settled and metamorphosed in response to bioWlms. Settlement and metamorphosis to the post-larval stage signiWcantly increased with the bioWlm age. In addition, the bioWlm activity varied depending on the immersion month (season), e.g., for bioWlms with the same age, those immersed between June and August had higher activities than those immersed between November and March. The activity of the bioWlm also positively correlated with the dry weight, bacterial and diatom densities. These three quantitative parameters of the bioWlm were signiWcantly aVected by the Wlm age but were not
Communicated by S. Nishida, Tokyo W. Y. Bao (&) · C. G. Satuito · J. L. Yang Graduate School of Science and Technology, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan e-mail:
[email protected] H. Kitamura Faculty of Fisheries, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan
aVected by the immersion month, suggesting that other parameters (e.g., community structures, extracellular products) also aVected the inductive activity of the bioWlm. The Wxative agents (formalin and glutaraldehyde), heat, ethanol, ultraviolet irradiation and antibiotics treatments of the bioWlm resulted in signiWcant reduction or loss of its inductive activity. The survival of bacterial cells in the treated Wlms where activities were either reduced or lost also decreased signiWcantly. No settlement and metamorphosis were obtained when larvae were exposed to the conditioned water of the bioWlm. Thus, larvae of M. galloprovincialis settled and metamorphosed in response to a cue produced by living bacteria in the bioWlm. The cue may be a bacterial extracellular product which was susceptible to the above treatments.
Introduction Settlement and metamorphosis of larvae of many marine invertebrate species are known to be mediated by bioWlms (see reviews by Pawlik 1992; Wieczorek and Todd 1998; HadWeld and Paul 2001). A large volume of references is available on this subject, many of these documenting the inductive eVect of monospecies bioWlms. Studies have demonstrated that monospecies bacterial or diatom bioWlms induced settlement and metamorphosis of larvae in coelenterates (Leitz and Wagner 1993), bryozoans (Maki et al. 1989; Dahms et al. 2004), polychaetes (Kirchman et al. 1982; Unabia and HadWeld 1999; Harder et al. 2002), mussels (Satuito et al. 1995, 1997), oysters (Weiner et al. 1989), barnacles (Maki et al. 1990; O’Connor and Richardson 1998) and ascidians (Szewzyk et al. 1991). On the other
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hand, researchers are also investigating the eVects of natural multi-species bioWlms on larval settlement and metamorphosis, either through Weld observations (e.g., Keough and Raimondi 1995, 1996; Wieczorek et al. 1996; Olivier et al. 2000) or in the laboratory using cultured organisms (e.g., Brancato and Woollacott 1982; HadWeld et al. 1994; Wieczorek et al. 1995; Wieczorek and Todd 1997; Tsurumi and Fusetani 1998; Rahim et al. 2004). In natural situations, the formation of bioWlms on surfaces can be viewed as a net result of a number of processes involving a complex series of physical, chemical and biological phenomena (Characklis and Cooksey 1983). Changes in the successional composition, physiological condition and growth phase of the bioWlm community can also alter their inXuence on larval settlement (Wieczorek et al. 1995; Wieczorek and Todd 1997). Keough and Raimondi (1995) demonstrated in their Weld study that in almost all cases, larval recruitment on Wlmed panels was positively correlated with the Wlm age, although some taxa showed little or no relationship to the age of bioWlm in their settlement density. For two sea urchin species, settlement and metamorphosis of Pseudocentrotus depressus larvae positively correlated with the age of the bioWlm, whereas Anthocidaris crassispina exhibited a bellshaped response curve to bioWlm age (Rahim et al. 2004). For the barnacle Balanus amphitrite, the change in the bioWlm volume aVected cyprid settlement, with the settlement increasing until a certain volume was reached, but decreasing thereafter (Tsurumi and Fusetani 1998). Wieczorek et al. (1996) observed that seasonal changes in bioWlms also aVected the settlement response of larvae in some species; that is, the eVect of the bioWlm reversed from inhibitory to facilitatory and vice versa. The mussel Mytilus galloprovincialis is a dominant species in the intertidal zones in most parts of Japan (Sakaguchi 1987). It is considered an important fouling species and causes serious problems in electric power stations, where seawater is used in their cooling water systems (Sakaguchi and Kazihara 1988). The mussel has a planktonic larval stage and its larvae will settle on suitable substrates once they become competent. Bayne (1964) postulated for larval settlement of Mytilus edulis that it consists of a primary and secondary phase, where competent larvae will settle and metamorphose on Wlamentous algae during primary settlement, thereafter juveniles (0.9 » 1.5 mm) undergo a migratory phase to settle on adult beds during secondary settlement. McGrath et al. (1988), however, showed that M. edulis larvae will settle directly from the plankton on adult beds, without an initial growth
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phase on Wlamentous algae. Cáceres-Martínez et al. (1993) also reported that the settlement pattern of M. galloprovincialis observed on an exposed rocky shore in Ría de Vigo, NW Spain indicated direct settlement of larvae from the plankton onto adult beds. In both the Weld and laboratory, M. edulis larvae have been observed to settle preferentially on Wlamentous substrates, especially on Wlamentous algae (e.g., Eyster and Pechenik 1987; Dobretsov 1999). Larvae of M. galloprovincialis settled directly on various substrates showing preference for natural Wlamentous substrates, but also settled on rugous hard surfaces (CáceresMartínez et al. 1994). Dobretsov (1999) showed that M. edulis larvae were attracted to the Wlamentous green alga Cladophora rupestris and its bioWlm, while Satuito et al. (1997) found settlement and metamorphosis of M. galloprovincialis to be strongly dependent on the presence of bacterial bioWlms. From these observations, it is apparent that although Mytilus exhibits varying modes of settlement, the mechanism of larval settlement in this genus is not fully understood. In the present study, the authors investigated the settlement and metamorphosis response of M. galloprovincialis larvae to bioWlms of diVerent ages obtained during diVerent months between November 2003 and January 2005. In addition, the eVects of some quantitative parameters (dry weight and densities of bacteria and diatoms) of the bioWlm on its inductive activity were investigated. The eVects of age and immersion month (season) on these three quantitative parameters were also assessed, in order to deWne the eVects of age and immersion month on the inductive activity of the bioWlm. Furthermore, in order to clarify the nature of the settlement and metamorphosis cue of the bioWlm, the activity of the potential cue was evaluated after treatments with Wxative agents (formalin, glutaraldehyde), ultraviolet irradiation, heat, ethanol and antibiotics. Simultaneously, the eVects of these treatments on the cell survival of constituent organisms of the bioWlm (bacteria, diatoms) were investigated. The inductive activity of the conditioned water of the bioWlm was also investigated.
Materials and methods Spawning and larval culture Spawning and larval culture of M. galloprovincialis were conducted following the method described by Satuito et al. (2005). Adults of M. galloprovincialis used for spawning were collected from populations
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growing on wharfs in Makishima-Machi (129°59⬘E; 32°45⬘N) and Taira-Machi (129°51⬘E; 32°43⬘N), Nagasaki, or were purchased from a culture farm in Matoya, Isobe-Machi, Mie (136°52⬘E; 34°22⬘N), Japan. They were packed in crushed ice overnight and placed into a 30 l polycarbonate tank Wlled with GF/C (Whatman glass Wber Wlter; pore size: 1.2 m) Wltered seawater (FSW) adjusted to 24°C. The Wnal temperature of the FSW was approximately 21°C. Mussels that started spawning were immediately separated from the group and were let to spawn individually in 2 l glass beakers Wlled with FSW. Eggs derived from a single female were fertilized by sperms derived from a single male. Fertilized eggs were washed with FSW and left undisturbed for 2 days inside an incubator adjusted to 17 § 1°C. After 2 days, swimming straight-hinged veligers were collected, gently washed with FSW and cultured as described below. Larvae were cultured in 2 l glass beakers at an initial stocking density of 5 larvae/ ml and fed a diet of Chaetoceros gracilis (5 £ 104 cells/ ml per day). The culture temperature was maintained at 17 § 1°C in an incubator and the culture water was changed every 2 days throughout the culture period. Larvae reached the pediveliger stage after 16–19 days from the start of culture and were used in assays after these reached an average shell height (SH) and shell length (SL) of >288 and >309 m, respectively (Satuito et al. 2005). The supply of pediveligers used in assays was ensured almost all year round by storing straighthinged veligers that were obtained during the spawning season in a refrigerator for a maximum period of 3 months and culturing these refrigerated larvae in an incubator to the pediveliger stage when needed. Refrigeration was found to have no adverse eVect on the survival, growth, settlement behavior and metamorphosis of the larvae (Satuito et al. 2005). Conditions for storing larvae in a refrigerator are described elsewhere (Satuito et al. 2005). Conditions for culturing refrigerated larvae are the same as described above. Larval settlement and metamorphosis assays Twenty pediveliger larvae were released into each glass Petri dish (ø60 mm £ 15 mm height) containing 20 ml FSW and the test slip (e.g., bioWlm slip, treated bioWlm slip). Details of the preparation of bioWlm and treated bioWlm slips are described in the next section. In the assay with the conditioned water of the bioWlm, Petri dishes were each Wlled with 20 ml conditioned water instead of the FSW. In this case, no test slip was placed in the Petri dish. Preparation of the conditioned
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water of the bioWlm is also described below. Subsequent metamorphosis was determined after 48 h from the commencement of the experiment by counting the number of individuals with post-larval shell growth under a microscope and was expressed as the percentage of post-larvae from the total number of individuals in each Petri dish. Petri dish containing a clean glass slip (half portion of a microscopic glass slide) and 20 larvae was set as the control in each assay. All assays were conducted at 17 § 1°C in a dark environment. Assays to test the eVects of age, immersion month, dry weight, and bacterial and diatom densities of bioWlms were conducted between November 2003 and January 2005. Each experimental condition was conducted in four to six replicate Petri dishes. In assays with treated (formalin, glutaraldehyde, UV, heat, ethanol and antibiotics) bioWlms and with the conditioned water, more than 12 replicate Petri dishes using larvae from three diVerent culture batches were prepared. Preparation and treatment of bioWlms BioWlm slips were prepared by immersing clean glass slips (half portions of microscopic glass slides; 38 £ 26 mm) in seawater in Tachibana Bay from a pontoon bridge adjacent to the Fisheries Center of Nagasaki City, Makishima-Machi (129°59⬘E; 32°45⬘N), Nagasaki. The glass slips were placed on PVC holders and immersed at a depth of 1.0–1.5 m below the sea surface for 1–30 days, between November 2003 and January 2005. These were brought back to the laboratory on the day of the assay and were thoroughly washed with FSW prior to use. Throughout the investigation period, the attachment of macrofoulers (e.g., barnacles, hydrozoans) on bioWlm slips was also observed, but attached individuals were few. These bioWlm slips were used as they were, with the attached individuals left undisturbed. However, when bivalves were present in the bioWlms, only these were removed prior to use in assays, the other macrofoulers left untouched. In June and August of 2004, glass slips became densely fouled by ascidians when immersed for more than 14 days. During this period, bioWlms older than 14 days were not prepared. Daily seawater temperature data were kindly provided by the Fisheries Center of Nagasaki City. The monthly seawater temperature was determined by taking the average of the daily seawater temperature during the immersion period. BioWlm slips that were 21–30 days old were obtained between September 2004 and January 2005 and subjected to the following treatments. BioWlms were soaked in 5% formalin solution for 24 h to
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prepare formalin treated (FA) Wlms. BioWlms were soaked in 2.5% glutaraldehyde solution for 24 h to prepare glutaraldehyde treated (GA) Wlms. Ultraviolet treated (UV) bioWlms were prepared by exposing bioWlms to ultraviolet (15 W/m2) irradiation for 1 h. Ethanol treated (5E, 10E, 50E, 100E) bioWlms were prepared by soaking bioWlms in 5, 10, 50 or 100% ethanol solutions for 30 min. Heated (30T, 40T, 50T, 100T) bioWlms were prepared by heating bioWlms at 30, 40, 50 or 100°C for 30 min. Antibiotics treated (0.2A, 2A, 20A) bioWlms were prepared by soaking bioWlms in solutions of 0.2, 2 or 20 mg/ml antibiotic mixtures (AM) for 2 h. At each concentration level of AM, the proportion of penicillin G potassium and streptomycin sulfate was the same; e.g., 0.2 mg/ml of AM contained 0.1 mg/ml each of penicillin G potassium and streptomycin sulfate. For FA and GA bioWlms, treated Wlms were rinsed in groups of the same treatment condition, each group rinsed nine times using a total amount of 9 l FSW prior to assays. For ethanol and AM treated bioWlms, each group was rinsed six times using 2 l of 0.22 m Wltered (Millipore) FSW prior to use. For the heated and UV treated bioWlms, each group was rinsed three times using 1 l of 0.22 m FSW. Measurement of the bioWlm dry weight The bioWlm on each glass slip was scraped oV by using a clean glass slide and separately suspended in FSW. Each suspension was collected on a pre-weighed GF/C Wlter by Wltration. Each Wlter paper holding the bioWlm was washed with 50 ml of 0.22 m Wltered distilled water, dried for 48 h in an oven at 80°C and cooled to room temperature in a desiccator before weighing. The dry weight of the bioWlm was determined after subtracting the weight of the Wlter. For young bioWlms, dry weight was determined by collecting the bioWlm suspension scraped from several bioWlm slips of the same age on a pre-weighed GF/C Wlter and dividing the dry weight by the number of Wlm slips scraped. Enumeration of densities of bacteria and diatoms BioWlms were Wxed in 5% formalin solution for a maximum duration of 3 weeks. These were washed with 0.22 m FSW and stained by acridine orange (AO, 0.1%) for 5 min. Bacterial densities of stained samples were counted directly at £1,000 magniWcation under an Olympus BH-2 epiXuorescence microscope. Densities of diatoms were counted directly at £200 magniWcation under a light microscope immediately after bioWlms were brought back to the laboratory. The den-
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sities of bacteria and diatoms in each sample were enumerated from ten random Welds of view. Determination of the cell survival of bacteria and diatoms The survival of bacterial cells was determined following the method of Kogure et al. (1979). Treated and untreated bioWlms were placed in separate A60 mm Petri dishes and were incubated in the dark at 20°C for 6 h, with each of these Petri dishes containing 15 ml solution of Bacto yeast extract [0.025% (W/V)] and nalidixic acid [0.002% (W/V)] dissolved in 0.22 m FSW. After incubation, bacteria were stained using AO and counted as described above. Living bacteria grew considerably in size and were recognized as elongated cells. By contrast, cells that did not change in size were counted as dead. The cell survival of diatoms was determined following the staining method reported by GaV and Okong’o-Ogola (1971) using Evan’s blue. BioWlms were stained with 15 ml of 0.25% Evan’s blue solution (w/v) dissolved in FSW and incubated for 30 min. After thorough rinsing of the stained Wlms, cells that were not stained blue by the dye were counted as living. Bacto yeast extract was purchased from Becton, Dickinson and Company (Sparks, MD, USA). Nalidixic acid and AO were purchased from Sigma Co. (St Louis, MO, USA). Evan’s blue was purchased from Wako Pure Chemical Industries (Osaka, Japan). Preparation of the conditioned water of the bioWlm Petri dishes, each containing 20 ml FSW and a bioWlm slip, were placed in an incubator at 17 § 1°C for 48 h. After 48 h, the FSW conditioned in the Petri dish with the bioWlm slip was collected and Wltered through a 0.22 m syringe Wlter to obtain 100% concentration of the conditioned water of the bioWlm. Concentrated conditioned water (e.g., 200, 300% concentrations) was obtained by adjusting the volume of FSW in each Petri dish with the bioWlm slip; 200% concentration was prepared by adding only 10 ml FSW in the dish with the bioWlm; and 300% concentration, by adding 6.7 ml in the dish with the bioWlm. Data analysis Activities of the bioWlm and conditioned water were evaluated by the percentages of post-larvae that settled and metamorphosed in the Petri dishes. The eVects of age and immersion month on the bioWlm activity were assessed using a two-way ANOVA. DiVerences in activities of bioWlms between immersion months were
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further analyzed by an LS means student’s t test. Correlations between the bioWlm activity and dry weight, bacterial and diatom densities of bioWlms were analyzed using a Pearson correlation test. The eVects of age and immersion month on the dry weight, bacterial and diatom densities of the bioWlm were assessed by a two-way ANOVA. The eVects of treatments on the Wlm activity and cell survival of bacteria and diatoms were assessed by a one-way ANOVA followed by Tukey’s multiple comparison test. All data were arcsine-transformed prior to analysis with ANOVA and student’s t test. DiVerences were considered signiWcant at P < 0.05.
Results Throughout all experiments, the percentage of postlarvae on clean glass pieces (control) was always 0%.
100 Nov`03 Dec Jan`04 Feb Mar May Jun Jul Aug Sept Nov Dec Jan`05
Post-larvae (%)
80 60 40 20 0 0
10
20 Age (days)
30
Fig. 1 Mytilus galloprovincialis. Percentages of post-larvae that settled and metamorphosed in response to bioWlms of diVerent ages formed during the period between Nov (November) 2003 and Jan (January) 2005. Jan, Feb, Mar, Jun, Jul, Aug, Sept, Nov and Dec indicate January, February, March, June, July, August, September, November and December, respectively. `03, `04 and `05 indicate 2003, 2004 and 2005, respectively. Each value is the mean of four to six replicates
Age and immersion month of bioWlm versus activity Percentages of post-larval metamorphosis on bioWlms with diVerent ages obtained from November 2003 to January 2005 are shown in Fig. 1. In general, the percentage of post-larvae increased with the age of the bioWlm (Fig. 1). During the months of June and August 2004, >80% post-larvae were obtained on the bioWlms immersed for 7–10 days. During May and July of 2004, >80% post-larval metamorphosis was achieved on 17and 18-day-old bioWlms. In other months, however, either >80% post-larval metamorphosis was achieved only in bioWlms with the ages of >20 days or >80% post-larvae was not achieved at all (e.g., Dec of 2003, Feb, Mar and Nov of 2004, and Jan of 2005, Fig. 1). Data were grouped into three age groups (1 » 10, 11 » 20 and 21 » 30 days) and the eVects of age and immersion month were assessed by the two-way ANOVA as shown in Table 1. The analysis revealed that the activity of bioWlm, expressed in percentage of post-larval metamorphosis, was signiWcantly aVected by both age and immersion month (Table 1). Furthermore, the analysis of monthly data by student’s t test showed three signiWcantly diVerent groups, i.e., A (Jun and Aug), B (Sept, Jul and May) and C (Nov, Dec, Mar, Feb and Jan) (Table 2). Average monthly temperatures during the months of experimentation are also shown in Table 2. Except for September, monthly temperatures were highest in group A, followed by group B and lowest in group C (Table 2). Hence, although the activity of the bioWlm on larval settlement and metamorphosis was signiWcantly aVected by the bioWlm age, this age-eVect was also modulated by the water temperature of the month.
Table 1 The result of a two-way ANOVA to assess the eVects of age and immersion month on the activity, expressed in percentage of post-larvae, of the bioWlm Factor
Level
df
F
P
Age
1 » 10; 11 » 20; 21 » 30 days Nov, Dec (`03,`04); Jan (`04, `05); Feb, Mar, May, Jun, Jul, Aug, Sept (`04)
2
67.921
