Biofilm Journal Pontificia Universidad Católica de Valparaíso ISSN: 1360-3655 Vol. 4, Num. 1, 1999

V Thiyagarajan¹, P Sriyutha Murthy¹, Y V Nancharaiah², V P Venugopalan^2*, K V K Nair³ and T Subramoniam¹

1Department of Zoology, University of Madras, Chennai 600 025, INDIA
²Macrofoulants Studies Group, Water and Steam Chemistry Laboratory, BARC Facilities, Kalpakkam 603 102, INDIA
³National Institute of Ocean Technology, Chennai - 600036, INDIA
^*corresponding author - E.mail: vpv@igcar.ernet.in

Date received: October 23rd 1999
Date accepted: March 23rd 2000
Date published: March 28th 2000

Microbial films, which develop on submerged artificial surfaces, elicit variable responses in settling invertebrate larvae. There is no information on the response of the larvae of the tropical fouling barnacle, Balanus reticulatus to biofilms. Therefore, the influence of biofilms and their components (such as bacteria, diatoms and bacterial exopolymer) on settlement of cyprid larvae of B. reticulatus has been studied. Biofilms significantly reduced larval settlement when compared to clean polystyrene surfaces (control). No significant correlation was found between percentage settlement and biofilm age. Inhibition or induction of settlement was observed, depending on the cell density and growth phase of the bacterial films. Diatom films, regardless of cell density, were inhibitory to cypris settlement. Bacterial exopolymers did not influence settlement at low concentrations (0.001 to 0.0001 %), but inhibited settlement at higher concentrations (0.1 to 0.01%). Our study indicates that presence of natural biofilms and their components such as bacteria, diatoms and bacterial exopolymers on polystyrene render an otherwise attractive surface unsuitable for settlement by larvae of B. reticulatus.

Keywords:biofilm; bacteria; diatoms; exopolymer; Balanus reticulatus; cyprid settlement

Artificial surfaces immersed in seawater are soon coated by organic molecules, leading to the formation of a conditioning film (Loeb and Neihof, 1975). Subsequently, bacteria, diatoms and other organisms colonize the surface, ultimately resulting in the formation of a complex biofilm (Keough and Raimondi, 1995). Biofilms in coastal marine habitats are generally dominated by bacteria, fungi, microalgae and exopolymeric substances of microbial origin (Holmstrom and Kjelleberg, 1994). Invertebrate larvae which settle on immersed surfaces generally encounter and interact with such biofilms (Keough and Raimondi, 1995).

Biofilms have been reported to influence the settlement and metamorphosis of a wide range of marine invertebrate larvae (see review of Holmstrom and Kjelleberg, 1994; Wieczorek and Todd, 1998). The effects of biofilms on invertebrate larval settlement depend on their composition (Roberts et al., 1991), age (Maki et al., 1988, 1990), film volume (Tsurumi and Fusetani, 1998) and underlying substratum (Maki et al., 1988). Anderson and Underwood (1994) suggested that the pattern of the fouling community in a habitat is influenced by the nature of the biofilm.

Knowledge about the cues provided by biofilms to settling larvae would be useful in understanding spatial variations in larval settlement and developing control strategies for biofouling (Richmond and Seed, 1991). Moreover, any broad interpretation of settlement dynamics needs knowledge of the responses to biofilms of all or most fouling species (Keough and Raimondi, 1995). Earlier studies have examined barnacle larval settlement (henceforth "settlement") response to biofilms in the field (Roberts et al., 1991; Keough and Raimondi, 1995; Wieczorek et al., 1996) and, in the laboratory, to individual films of bacteria and their exopolymers (Maki et al., 1988; 1990; 1994; Holmstrom et al., 1992; Mary et al., 1993; O'Connor and Richardson, 1996). Earlier studies indicate a lack of any uniformity of species response to biofilms. Biofilms were found to elicit a facilitatory response in B. amphitrite cyprids in the laboratory until the film volume grew to 0.1-1 mm³m^-2, and the effect decreased thereafter (Tsurumi and Fusetani, 1998). Biofilms developed in the aquarium inhibit barnacle settlement in the laboratory (Maki et al., 1988), while they enhance the settlement in field conditions (Meenakumari and Nair, 1994). Settlement of B. variegatus and Elminius modestus cyprids was found to be greater on unfilmed (or less filmed) surfaces, when compared to filmed surfaces, in field conditions (Keough and Raimondi, 1995). B. improvisus larvae preferred to settle on bacterial films which were coated on hydrophilic surfaces and the same bacterial film inhibited larval settlement when it was coated on hydrophobic surfaces (O'Connor and Richardson, 1996).

The objective of this study was to assess the influence of natural biofilm and its components (such as bacteria, diatoms and bacterial exopolymers) on barnacle larval settlement. In this study, cyprids of Balanus reticulatus were chosen as test material for the following reasons, i) Balanus reticulatus is a dominant fouling species in the east and west coasts of India (Thiyagarajan et al., 1997a), and ii) the larval settlement behaviour of this species is not known yet (Thiyagarajan et al., 1997b).

Adult barnacles were collected (using a chisel) from the jetty piers of Madras Atomic Power Station. First (sometimes second) - stage nauplii were obtained by immersing the adult barnacles in clean seawater, after a few hours of desiccation. Nauplii were reared up to the cypris stage, using the procedure of Thiyagarajan et al. (1996). Chaetoceros wighamii, a commonly available diatom, was used as larval food. Cyprids were harvested from the culture container using 240 mm plankton net. The larvae were used immediately or stored at 6^oC in the dark for later use (Rittschof et al., 1992).

The assay procedure outlined by Rittschof et al. (1992) was followed with some modifications. Sterile polystyrene petri dishes (35mm diameter) (Tarson, India) were used as the substratum for settlement. Five ml of aged (in the dark for 10 days) and membrane filtered (Millipore, 0.22 mm) seawater (30 ppt) in an assay petri dish was inoculated with 25-50 cyprids. After incubation for 24 hours in complete darkness at room temperature (28± 2^oC), dishes were emptied and rinsed with filtered seawater.

Biofilms were developed in polystyrene petri dishes by suspending sterile dishes in the coastal waters (at Kalpakkam, East Coast of India) at 3 m depth for 1 to 5 days. The dishes were placed inside a metal cage and covered with 60 µm plankton net, to avoid larval settlement (Keough and Raimondi, 1995). The number of bacteria attached on the dish was counted using epifluorescence microscopy after staining the dish with acridine orange (Daley and Hobbie, 1975). The bacterial numbers are represented as a mean of ten random field counts.

Three different strains of bacteria were isolated from biofilms developed in coastal waters, following the procedure of Mary et al. (1993). Films of the bacteria on polystyrene petri dishes were prepared by following the method of Maki et al. (1988). Individual films were developed using cells harvested from logarithmic (3-6 h old culture) and stationary growth phases (24 h old culture). Petri dishes were filled with 5 ml of bacterial suspension and incubated for 2 hours. Films with different bacterial density were developed by incubating serially diluted bacterial suspensions.

Two pure cultures of marine fouling diatoms, Nitzschia sp. and Amphora sp. were maintained in f/2 medium (Guillard and Ryther, 1962). Late logarithmic phase cultures (5 to 7 day old culture) were taken from the flask wall using a bottlebrush. Diatom films were developed on sterile polystyrene dishes by incubating a diatom suspension under light. Films of different diatom densities were obtained by incubating the suspension for 1, 3, 6, 12 and 24 hours. The number of attached diatoms was counted using light microscopy. The diatom numbers are represented as a mean of ten random field counts.

A film of bacterial exopolymer was prepared according to the procedure of Maki et al. (1990). Five ml of 0.1 to 0.0001 % solution (dissolved in double distilled water) of exopolysaccharide isolated from Pseudomonas sp. (supplied by T. Subba Rao) was placed in a polystyrene dish for 2 hours. After incubation, the dishes were rinsed with filtered seawater.

The differences between treatments were tested using one-way ANOVA (Sokal and Rohlf, 1987). Data were tested for homogeneity using Bartlett's test. Differences between individual treatments were tested by the SNK test. Student's t-test was used to test the difference between treated and control. The differences were considered significant at a level of P<0.05.

Natural biofilms developed in the coastal waters comprised 1.6 to 3.1x10⁶ bacteria/cm² in 1 to 5 days. In addition to bacteria, diatoms and detritus were also observed under the microscope. Vibrio spp. were dominant in the biofilms. The bacterial density was relatively high in the 2-day old film (3.1x10⁶ bacteria/cm²) and low in the 4-day old biofilm (1.6x10⁶ bacteria/cm²). Cypris settlement was significantly reduced by biofilms, irrespective of the film age, when compared to unfilmed control dishes (Fig.1). There was no significant relationship between percentage settlement and either biofilm age or bacterial density in the biofilm (P>0.05).

Logarithmic phase cells of strain I reduced larval settlement at cell densities ranging from 4.4 x10⁵ to 7.1x10⁶ cells/cm², when compared to the control (Fig. 2). Films of strain I derived from stationary phase cells showed both inhibition and induction of larval settlement depending on its density in the film (Fig. 3). Settlement was significantly different between the films derived from logarithmic and stationery phase cells of strain II at all densities tested (Figs 4 and 5). Settlement was not strongly induced by logarithmic phase cells of stain III at 1.5x10⁵ cells/cm², whereas at a density of 7.1x10⁵ to 2x10⁶ cell/cm² the stationary phase cells inhibited settlement (Figs. 6 and 7). Even though, the films of stain-III developed from logarithmic phase cells at the densities ranging from 2.1-3.1x10⁶ cell/cm² led to reduced settlement values, the values were not significantly different from the control (P>0.05).

Inhibition/induction of larval settlement by bacterial films, in general, depends on the growth phase of the cells. All the bacterial films, irrespective of the strain and growth phase, inhibited settlement at low cell density and had no effect at higher densities except in a few instances (Figs. 2-7).

Diatom films, irrespective of cell density, inhibited settlement (Figs. 8 and 9). In contrast to bacterial films, the inhibition of cypris settlement was positively correlated with diatom density (r = 0.96 - Nitzschia sp; r = 0.86 - Amphora sp).

Data showed that exopolymeric films adsorbed on polystyrene dishes at the concentration of 0.001% to 0.0001% (w/v), did not have any effect on settlement, when compared to the control, whereas films developed at 0.1 to 0.01% (w/v) strongly inhibited settlement (Fig. 10).

Physical factors such as vibration, light, substratum type, colour of the surface, surface energy of the substratum, salinity, water current, chemical cues originating from biofilms and cypris age have significant influence on barnacle larval settlement (Branscomb and Rittschof, 1984; O'Connor and Richardson, 1994; Holm et al., 1997; Rittschof et al, 1998; Wieczorek and Todd, 1998). Barnacle larvae receive both negative and positive cues (Holmstrom and Kjelleberg, 1994) from the environment. Laboratory static experiments demonstrated that the presence of natural biofilms, or films composed of single species of marine bacteria, could stimulate or inhibit the settlement of B. amphitrite (Maki et al., 1990) and B. reticulatus (present study) larvae.

Polystyrene dishes immersed in the coastal waters were soon coated by organic detritus, bacteria and diatoms. This is the generally reported pattern of microfouling in coastal waters (Roberts et al., 1991; Tsurumi and Fusetani, 1998). In the biofilms, the bacterial density reached 10⁶ cells /cm² within a day and remained almost constant for several days (Maki et al., 1988; Wieczorek et al., 1995). Our studies showed that biofilms which developed on polystyrene dishes constitute a surface which is not preferable for barnacle settlement. The presence of biofilms consisting of bacteria, diatoms and organic polymers render the surfaces more wettable (Mihm and Banta, 1981). Since B. reticulatus larvae do not prefer a wettable surface (Thiyagarajan, unpublished), the presence of biofilms on a hydrophobic (polystyrene) surface might have reduced the cypris settlement when compared to clean, untreated hydrophobic surfaces, due to a change in surface wettability. O'Connor and Richardson (1996) reported that the settlement of B. improvisus cyprids decreased in the presence of bacterial cells when filmed on a hydrophobic (polystyrene) surface whereas settlement was facilitated when bacteria were coated on hydrophilic (glass) surface. Types of surface (surface energy of a substratum) and cues (positive/negative) associated with biofilms, determine the strength of adhesion of barnacle larvae (tenacity) (Neal and Yule, 1994). That is, in the absence of a suitable (hydrophobic surface in the case of B. reticulatus) substratum and a strong positive cue for settlement and metamorphosis, cyprids undergo voluntary detachment which leads to reduced settlement (Neal and Yule, 1994), as in this case of B. reticulatus (present study).

In the present study, Vibrio-type bacteria were dominant in the biofilm. Among the bacterial types, vibrios have been reported to be the most potent inhibitors of cypris settlement (Mary et al., 1993). The inhibition of settlement by biofilms, therfore, may not only be due to change in surface energy and production of negative cues but also be related to the species composition of the biofilm .

A large number of studies on the effect of biofilms on barnacle larval settlement have used single-species cultures of microorganisms (Maki et al., 1988; 1990; 1994; Mary et al., 1993; O'Connor and Richardson, 1996), which is inappropriate if inferences on larval responses to biofilms under natural condition are to be drawn. Nonetheless, it is clear that only by utilizing a single species film in the laboratory can one identify the specific components of a biofilm that may be an important cue to larval settlement in the field (Wieczorek and Todd, 1998). Therefore, we studied the effect of single species bacterial/diatom films on B. reticulatus larval settlement. Our data show that settlement of B. reticulatus cyprids on bacteria adsorbed onto a polystyrene surface can vary with the growth phase of the cells (inhibition, no difference and induction). This observation is similar to that reported by O'Connor and Richardson (1996) for B. amphitrite and B. improvisus larvae. Since exopolymers are involved in bacterial adhesion (Maki et al., 1990), the production of qualitatively different exopolymers may be attributed to the effect of growth phase on settlement.

Results of the present study revealed that bacterial films at higher densities (<10⁶ cells/cm²) were not inhibitory to settling cyprids. These results are in accordance to those reported by Maki et al. (1990) for B. amphitrite larvae. However, the nature of bacterial density and barnacle larval interaction is not clear.

Diatom cells secrete glue (usually acidic polysaccharides) for firm attachment to surfaces (Webster et al., 1985). Therefore, settling cyprids are not only exposed to bacterial exopolymers but also to biomolecules of diatom origin. Results of this study show that barnacle larvae are strongly inhibited by fouling diatoms, probably due to their surface bound molecules. However, this point has to be studied in detail.

Finally, this study demonstrates that biofilms and their components (bacteria and diatoms), which cover surfaces immersed in coastal waters, may act as a negative cue or make a surface unsuitable for B. reticulatus cyprid settlement in the laboratory. Even though large number of studies have emphasized the interaction between barnacle larvae and biofilms, still it is not known how bacteria and diatoms interact with settling cyprid larvae and influence their attachment.

This work was supported by a grant from BRNS, Department of Atomic Energy (No.4/13/93-G) and Council of Scientific and Industrial Research (CSIR), New Delhi. Thanks are due to Prof. V. N. Raja Rao, CAS in Botany, University of Madras, who supplied the inoculum of diatoms, to Dr. S. V. Narasimhan, Head, Water and Steam Chemistry Laboratory and to Shri K. Hariharan, Station Director, Madras Atomic Power Station for laboratory facilities and support.

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