The interaction between Elminius modestus Darwin cyprids and biofilms of Deleya marina NCMB1877

ELSEVIER J. Exp. Mar. Biol. Ecol. 176 (1994) 127-139 JOURNAL OF EXPERIMENTAL MARINE BIOLOGY AND ECOLOGY The interaction between Elminius modestus Darwin cyprids and biofilms of Deleya marina NCMB 1877 Andrew L. Neal*, Andrew B. Yule n4uritze Science Laboratories, University of Wales, Askew Street, Mewi Bridge, Gw:\wedd LL57 5E Y. Wales, UK (Received 12 January 1993; revision received 11 October 1993; accepted 3 November 1993) Abstract The interaction between Elminius modestus Darwin cypris larvae and biofilms of Dele)tu tnaritza NCMB1877 covering a range of surface types has been studied by the measurement of cypris temporary adhesion. A > 16 kDa water-soluble partial fraction of bacterial exudate significantly reduced temporary adhesion on all surfaces in a manner which was independent of the under- lying surface wettability. In contrast, the underlying surface wettability influenced the effects produced by both 4-day and l-month-old, whole biofilms. Cyprids adhered more strongly to 4-day-old films compared to control surfaces, but less strongly to l-month-old films. We suggest that bacterium/substratum adhesion plays a significant role in the adhesion and settlement of cypris larvae. The production of stimulatory or inhibitory factors within the biofilm is dependent upon biofilm age. The interaction between underlying substratum properties and biofilm age together with the local bacteria1 community structure, will help to determining the degree to which a submerged surface will become fouled. Key words: Biofilm; Cypris larva; Deleya marina; Eltninius modestus; Surface wettability; Tem- porary adhesion 1. Introduction Marine invertebrate larvae employ a wide range of biological and physical cues to indicate suitable settlement substrata (for reviews see Scheltema, 1974; Crisp, 1984; Pawlik, 1992). One possible cue, first proposed by Zobell & Allen (1935), is the na- ture of the biofilm covering a surface. Despite early evidence suggesting that bacteria * Corresponding author 0022-0981/94/$7.00 0 1994 Elsevier Science B.V. All rights reserved SSDI 0022-0981(93)E0161-Q 128 A.L. Ned. A.B. Yule 1 J. Exp. Mar. Bid. Ed 176 (1994) 127-139 form only a small proportion of the initial colonising assemblage (Ferguson-Wood, 1950; Daniel, 1955) it is now widely accepted that new surfaces in the sea can quickly develop, and sustain, diverse microbial communities at high population densities (Marshall, 1980). The literature, however, provides no simple model to describe the interaction between settling larvae and the biofilms adhering to submerged substrata. Although biofilms may not be essential precursors to settlement of all invertebrate species (Miller et al., 1948; Crisp & Ryland, 1960), there is considerable evidence to suggest that biofilms nearly always enhance settlement (Crisp, 1974). The individual species components of bacterial films differ in their capability to induce or enhance settlement of Balanus amphitrite (Maki et al., 1988). Single-species biofilms of strain DLSl induce settlement, whereas Vibrio spp. inhibit it. Pseudomonas atlantica on the other hand, has no appreciable effect on settlement. Such results indicate a potential role of biofilm community structure in determining the response of an exploring larva. Maki et al. (1988) considered the bacterial exopolymer to mediate settlement behaviour through chemosensory action, but the possibility exists for consideration of the three- dimensional structure, either of the exopolymer itself or the bacterial cells embedded within it as physical cues in determining behaviour. Many compounds associated with bacterial exopolymers affect the settlement of invertebrate larvae. For example, glycolipids have been implicated in the induction of settlement of planula larvae of the scyphozoan Aureliu aurita (Schmahl, 1982) whilst Janus brasiliensis larvae are induced to settle by D-glucose molecules present in the exopolymer of a Deleya marina biofilm (Kirchman et al., 1982) a response postulated to be mediated by lectins present on the surface of the larva (Mitchell & Kirchman, 1984; Maki & Mitchell, 1985). The involvement of peptides, associated with bacterial exopolymers in the settlement of B. amphitrite, has also been suggested in the settle- ment of B. amphitrite cyprids although no direct evidence has been put forward (Maki et al., 1990). The present study investigates the effect of biofilm age and underlying surface hy- drophobicity and attempts to elucidate the role of three-dimensional structure of a biofilm upon temporary adhesion of barnacle cypris larvae. The measurement of cypris temporary adhesion affords an indirect, but powerful appreciation of a surface’s fitness for barnacle settlement, whilst at the same time, avoiding the over-estimation of pref- erence inherent in settlement assays due to the gregarious nature of cypris larvae (Gotelli, 1990). Surfaces upon which higher levels of settlement are likely to occur elicit higher levels of temporary adhesion (Neal & Yule, 1992). Moreover, barnacle larvae are excellent animals with which to study the interactions between larvae and bacte- ria because of their ease of culture and the insight already gained into cypris behav- iour. 2. Materials and methods Cypris larvae were reared following methods described by Moyse (1960) and Yule (1984). Adult Elminius modestus Darwin were collected from Menai Bridge and Beau- maris piers in the Menai Strait, North Wales and transported to the laboratory where ripe egg masses were placed in UV-irradiated, cartridge filtered (0.2 pm) seawater (UVFS) to hatch, after which the nauplii were collected and placed in 2-l flasks of UVFS. Cultures were kept at room temperature (22-24 “C) under constant slow aera- tion and natural light conditions. The larvae were fed on Skefetonema costatum Greville (Cleve) which was grown semi-continuously in a manner similar to that described by Walne (1966). Larval development was followed with reference to Knight-Jones & Waugh (1949). On appearance in the cultures, cyprids were transferred to clean seawater. The day when metamorphosis occurred from naupliar stage VI to cyprid was designated as Day 0. All cyprids were kept in the dark at 6-8 “C where they remained inactive and did not readily settle. Before preparation for adhesion measurenlents, the cyprids were slowly brought to room temperature and prepared using techniques developed by Yule & Crisp (1983) as described by Neal & Yule (1992). The force of adhesion of 4-day- old cyprids to test surfaces was measured following methods described by Neal & Yule (1992). The area of the anntenular disc of E. modes&s was measured at 537.9 ,un? ()I = 30, SE = 17.6 pm2). Pure cultures of Deleya (= Pseudomonas) murina (NCMB1877, ATCC25374) were obtained from the National Collection of Industrial and Marine Bacteria (Torry Research Station, Aberdeen) and Dr. J. Maki (Harvard University, Mass., USA). Dmarina is a Gram-negative rod-shaped bacterium. Cultures were grown routinely in Zobell’s 2216E medium (Zobell, 1941) containing 0.1 g ferric citrate (BDH, Poole}, 1.0 g yeast extract (MCI, LabM, Bury), and 5.0 g peptone (MC4, LabM) in 1 1 of 75?; (v/v distilled deionised water) filtered, aged seawater, adjusted to pH 7.5 with NaOH. Fresh sub-cultures were taken every 14 days. For experimental purposes, batch cultures of 250 ml were grown in Erlenmeyer flasks at 30 “C for 24 h and then surfaces to be filmed were placed in sterile poly(styrene) Petri dishes and D. marina culture added. Petri dishes were incubated at 30 “C for either 1 month or 4 days, after which time the plates were stored at 6-8 “C before use within 24 h. Direct cell counts were not un- dertaken but surfaces were inspected with a binocular microscope ( x 20 mag.) revealing an amorphous filamentous matrix covering the surface. Partial water-soluble fractions of auotoclaved 2216E growth medium and D. murirza exudates were prepared using methods outlined by Christensen et al. (1985). Batch cultures were grown at 2.5 “C under constant aeration in 5 1 round bottom flasks. The growth medium contained 6 g of peptone and 4 g of D-glucose (BDH) in 2-l UVFS, corrected to pH 7.5. After 48 h incubation, cells were removed by centrifugation at 4800 g for 20 min at 4 “C. The supernatant containing suspended and dissolved bac- terial exudate was dialysed for 36 h at 8 “C in dialysis tubing with a molecular mass cut-off of 16 kDa. No chemical analysis of the supernatant was performed, hence the proportion of intracellular to extracellular components was not determined. The > 16 kDa fraction resultant from each preparation was freeze-dried and resuspended in 250 ml distilled deionised water. Three 25 11 aliquots of each fraction were then allowed to air dry onto experimental surfaces. Once dry, the surfaces were washed in seawater and then rinsed with distilled water to remove any material which was not strongly adsorbed. Surfaces were then redried before wettibility was measured. Surfaces on which biofilms were to develop were first cleaned with a ditute sodium 130 AL Ned, A.B. Yule! J. E.up. Mar. Bid. Ed. 176 119941 127-139 hypochlorite solution and then rinsed thoroughly in tap water. Glass surfaces were treated in a muffle furnace at 400 “C for 12 h for organic cleanliness. Twelve different surfaces or surface preparations were used covering a wide range of surface-free en- ergies. Untreated Borosilicate and soda glass surfaces were used along with Poly(sty- rene), Poly(methylmethacrylate), Poly(tetrafluoroethylene) and Nylon 6. In addition, six different treatments of soda glass were employed: sulphonated soda glass (soaked in concentrated H2S0, for 24 h), 1,1,2-Trichlorotrifluoroethane (CFE) (soaked 4-6 h) and four silane preparations: N-Trimethyoxysilylpropyl-N,N,~,-trin~ethylammonium chloride (QAP), trimethylchlorosilane (TMS), 3-Aminopropyltriethoxysilane (APS) and Diphenylchlorosilane (DPS). Each silane was used as a 17; aqueous or metha- nol solution in which the glass surfaces were soaked for 4-6 h. All glass surfaces were baked in an oven at 100 “C for 12 h before use. Surface-free energy was measured by exploiting the ability of a series of binary liquids of decreasing surface tension to spread to differing degrees over each surface (see Gerhart et al., I992) and expressed as a relative wettability based upon the simple harmonic mean (SHM) of the drop diameters. The wettabilities of all the surfaces used were measured before biofilm development or exudate application. For experiments involving water-soluble partial fractions of D. nzarirzn, control measurements were first made on the surfaces, the fraction allowed to adsorb to the same surface and the wettability then re-measured. Temporary adhe- sion measurements of cyprids were then made. Clearly, intact biofilms could not be treated in the same manner. Instead, a range of surfaces were prepared and their wettabilities measured prior to biofilm formation. Surfaces were chosen for biofilming on the basis of their wettability measurements relative to control surfaces. Reproduc- ing surface wettability measurements on the same surface proved so difficult, due presumably to the difference in cleaning procedures and time between cleaning and measuring, that comparisons had to be made on the basis of relative wettability rather than surface type. 3. Results Results from initial experiments conducted with a partial fraction of autoclaved 2216E medium are given in Table 1. A two-way analysis of variance between surface treatment and individual cyprids showed no significant differences between surfaces (p = 0.290), but there were significant differences between the mean adhesion of indi- vidual cyprids (p = 0.037). Interaction however, was not significant (P = 0.092). That there are significant differences between individual cypris larvae is not surprising if for no other reason than tenacity measurements are based on an average antennular disc area. The fact that there was no effect of the 2216E partial fraction on cypris tempo- rary adhesion confirms that standard glass surfaces are adequate controls for experi- ments using 2216E as the bacterial growth medium. The L). ~7u~i~ff partial fraction was used to simulate an adsorbed layer of bacterial exopolymers. Measurement of surface wettability before and after treatment with the partial fraction showed that the bacterial exudates have clear surface-active properties, Table 1 A.L. Neul. A.B. Yule i J. Exp. Mar. Biol. Ecol. 176 11994) 127-139 131 The effect of a water-soluble partial fraction of 2216E bacterial growth medium upon the temporary adhe- sion (1tN.m -‘) of E. modestus cypris larvae Surface II Mean ( & SE) Control 40 89.02 (3.75) 2216E 40 94.53 (4.22) Source df Sum of squares Mean square F P Surface I 60.9 x IO’ 60.9 x 10’ 1.14 0.290 Cyprid 7 86.0 x IO’ 12.3 x 10’ 2.30 0.037 Interaction 7 69.5 x 10’ 99.2 x 10’ 1.86 0.092 Error 64 34.2 x 10” 53.5 x 10’ Total 79 50.4 x 10’ reducing wettability by as much as 39% on the highest energy surface (see Fig. 1). Above a wettability of about 20, a reduction in wettability is apparent, wettability dropping to between 20 and 30. Below an initial wettability of 20, the reduction due to bacterial exudates is probably negligible within the limits of precision of this method. Surface wettability and forces of temporary adhesion are given in Table 2. A 2-factor analysis of variance (Table 2) demonstrates that both main effects, surface wettability and bacterial exudate, were significant (p 132 Table 2 A.L. Neal, A.B. Yule, .I ICYp. Mar. Biol. Ecol. 176 (1994) 127-139 The effect of a water-soluble partial fraction of D. mu&cc exudate upon wettabihty (SHM) and the force of temporary adhesion (kN,m- ‘) of E. modestus cypris larvae before and after treatment with exudate. Con- trol surface materials l-7 are the same as those for the exudate surfaces Surface Wettability II Mean ( k SE) Control Exudate 84 59 53 42 19 17 6 50 50 50 50 50 50 50 51 50 31 50 21 50 27 50 18 50 16 50 5 50 123.6 (4.4) 100.7 (7.0) 80.1 (4.8) 111.7(7.4) 97.6 (4.3) xx.9 (1.8) 110.5 (5.2) 68.7 (4.7) 65.1 (3.1) 78.4 (3.9) 73.0 (4.2) 70.7 (4.1) 59.1 (2.1) 64.8 (2.4) Source df Sum of squares Surface 6 34.3 x 10” Exudate 1 19.4 x 10”’ Interaction 6 42.8 x 10’ Error 686 70.2 x 10’” Total 699 97.4 x 1o1” Mean square 57.2 x 10’ 19.4 x 10”’ 71.3 x IO’ 10.2 x lox F I’ 5.59 to.001 190.03 < 0.00 1 6.97 A.L. Neal, A.B. Yule/J. Exp. Mar. Biol. Ecol. 176 (1994) 127-139 133 1.3 1 la* -__-____--____----_--LI t . Control 2 0.9 - ii m I- 0.8 - -_-----__---..“~___-_____ 2 . ‘G m 0.7 - . A z A Exudate Treated K . * 0.6 - . 0.5,,,, L , , , $1 0 10 20 30 40 50 60 70 a0 90 Wettability (SHM) Fig. 2. The relative (value/control mean) temporary adhesion of E. modestus cypris larvae to a range of different wettability surfaces, before and after the adsorption of a D. mar&a exudate. Surface wettabihty was measured before and after exudate adsorption. The 95?6 CI for the difference between the control mean and any other mean is also shown. 1.6 1 I .g 1.4 " .4d. Old Film : S 4 A d 1.2 - k I S .______--___-____-_c_,__ g 2 1.0 - conlrol nun it -_~~~----__,--l_-L--_-_ ‘G 9 ; 0.8 1 .lm. Old Film 0.6 _I, ; , 6 I I I1 0 10 20 30 40 50 60 70 Wettability (SHMI Fig. 3. The relative (value/control mean) temporary adhesion of E. modestus cypris larvae to biofilms of D. marina grown on a range of surface wettabilities. Wettability of each surface was measured prior to biofilming. The largest 95% CI for the difference from the control mean is also given. 134 A.L. Neal, A.B. Yule 1 .I. Exp. Mar. Biol. Ecol. 176 (1994) 127-139 a more uniform nature than were the control surfaces. Such uniformity could have arisen as a consequence of surface preparation (air drying) alone, resulting in surface uniformity with no differential adsorption of supernatant components. Fig. 2, however, shows that the resulting wettabilities after adsorption and washing were not uniform (ranging from 5 to 51). If we presume that the reduction in wettability results from different concentrations of exudate adsorbing to the treated surfaces according to ini- tial surface wettability, the apparent effect of the bacterial exudates does not appear to be concentration-dependent, or the threshold concentration is lower than those achieved in this instance. Table 3 shows the mean force of temporary adhesion of cyprids to control surfaces and to surfaces covered with 4-day-old and l-month-old biofilms, along with wettability estimates for the surfaces taken prior to adhesion measurements. The results of a 2-factor analysis of variance are also given, demonstrating a significant (pt0.001) effect of biofilm and a marginally significant main effect (p = 0.05) of surface wettability. Table 3 The effect of a D. mavinu biofilm, and biofilm age, upon the force of temporary adhesion (kN,m-‘) of E. modestus cypris larvae Initial surface wettability II Mean (+ SE) Control 59 53 42 17 13 50 50 50 50 50 4-day-old film 63 43 52 50 45 44 17 50 13 57 66.61 (3.11) 53.60(1.10) 59.23 (2.56) 67.31 (4.47) 48.17 (2.91) 35.67 (1.85) 53.48 (3.17) 53.48 (2.98) 40.15 (2.03) 36.62(1.12) l-month-old film 56 60 65.43 (2.92) 50 60 73.40 (4.63) 46 39 68.08 (3.48) 15 55 84.75 (5.29) 13 30 93.00 (4.82) Source df Sum of squares Mean square F I’ Surface 4 13.6 x 10” 68.0 x lo9 2.38 0.05 Biofilm age 2 51.6 x 10’ 12.9 x 10’ 125.50 A.L. Neal. A.B. Yule !.I. Exp. Mm. Bid. Ecol. 176 11994) ID-139 135 However, the presence, once again, of significant interaction (y < 0.001) required de- tailed analysis of the simple effect means. The lack of pattern in the control means with wettability again prompted an analy- sis by Bonferoni contrast comparing the simple mean adhesion to the two biofilms against a grand control mean. The results of such comparisons are illustrated in Fig. 3 and show generally, that the younger biofilms tended to increase temporary adhesion over the range of wettabilities studied, whilst the older biofilms reduced temporary adhesion in three out of five cases. The observed effects were greatest on the lower energy surfaces (relative wettability ~30) for both films. At higher relative wettabilities mean temporary adhesion to either film converges towards the mean control value. It is thus apparent that despite there being no direct effect of surface wettability upon the force of temporary adhesion (as seen on control surfaces) the wcttability of the surface to which the biofilm is adhered has an appreciable effect upon the expression of the film on the force of temporary adhesion of cyprids. 4. Discussion Deleya marinu biolilms inhibit settlement of B. amphitrite cyprids (Maki et al., 1988) and the older the film, the greater the inhibition [Maki et al., 1990). It was suggested that some inhibitory factor may accumulate within the biofilm as it aged, with settle- ment dependent upon the concentration of the factor. The effect of D. manna biofilm was, however, also mediated by the underlying surface wettability such that inhibition of settlement was only demonstrable upon low wettability substrata yet enhanced settlement was observed with D. marina films on high wettability substrata (Maki et al., 1990). Elminius larvae show a very similar response to D. marina films as interpreted through temporary adhesion measurements although there are subtle differences from those reported for B. amphitrite settlement. The age of the biofilm, rather than the wettability, determined whether adhesion was enhanced or reduced (potentially high/ low settlement, Neal & Yule, 1992) yet an increase in wettability of the underlying surface tended to result in adhesion much closer to that observed on unfilmed surfaces. Results for both B. amph~trite settlement and E. modestus temporary adhesion, although not contradicting the accumulation of an inhibitor factor in older films, are more suggestive of two separate active agents, one stimulatory, the other inhibitory, with the balance of production of the agents either closely associated with the development stage of the biofilm (present data) or exhibited under differing conditions of underlying surface-free energies (Maki et al., 1990). Growth-related production of different ex- opolymers by bacteria is very common, for example, Pseudomonas NCMB2021 pro- duces two strikingly different exopolysaccharides according to growth stage (Chris- tensen et al., 1985). Stationary phase cells of D. marina exhibit higher cell-surface hydrophobocity than mid-exponential phase cells (Little et al., 1986), further suggest- ing qualitative differences between growth phases. Differences in cell-surface hydro- phobicity alone, however, do not appear to affect the settlement of B. omphitrite larvae (Maki et at., 1992). The apparent inhibitory effect of the older films could result from 136 A.L. Neal, A.B. Yule /J. Exp. Mar. Biol. Ecol. 176 (1994) 127-139 production of an exopolymer similar to that produced by Pseudomonas S9 (Wrangstadh et al., 1986) during starvation which reduces the adhesion of the bacterium to the substratum. The lower forces of cypris temporary adhesion noted may result simply by detachment of the film itself from the surface, the cypris glue having a stronger hold on the film than the film has on the substratum. The lack of pattern evident for unfilmed surfaces clearly indicates that the influence of surface wettability is primarily on the biofilm, and only indirectly upon the cyprids. Two surfaces with relative wettabilities < 20 and one > 60 exhibit a negative effect of l-month-old biofilms upon cypris tenacity. The effect is largely mirrored by that pro- duced by 4-day-old biofilms, except that no surface with a wettability in excess of 60 was used. Surface hydrophobicity affects bacterial adhesion to surfaces as shown by Fletcher & Pringle (with freshwater bacteria, 1985) and Meyer et al. (in fresh, brack- ish and sea water, 1988), concluding that bacterial adhesion is hindered by highly polar, hydrophobic surfaces and is deterred by the large amount of energy needed to displace the adherent water (Fletcher & Pringle, 1985). Elminius tenacity shows a very similar trend on older biofilms of D. marina. The values of tenacity in this instance could equally well be reduced due to physical detachment of the slime layer from the surface as opposed to postulating an inhibitory factor incorporated within the biofilm which in- creased the cyprid’s willingness to detach. Barnacle cypris adhesion to the 4-day-old biofilms was much greater, but followed a similar trend with hydrophobicity, in as much as the difference to unfilmed surfaces was reduced at higher wettabilities. Surfaces with the lowest wettabilities gave the highest cypris tenacity. The biofilm on low wettability substrata might well be less affected by the underlying surface forces and hence present a different three-dimensional structure under these conditions, a structure which pre- sumably is conducive to barnacle temporary adhesion, or at least reduces the cyprid’s willingness to detach. Perhaps the larvae are more responsive to the three-dimensional structure of the whole film, rather than a specific orientation of a single molecule. Support for such a view comes from the observation that the indirect effect of surface hydrophobicity, described above for both films, could not be demonstrated using the high molecular weight bacterial exudates (see Fig. 2) and in this respect, the exudates showed some similarity to the control surfaces. At all but one hydrophobicity, adhesion to exudate- treated surfaces was significantly less than control surfaces, and therefore, quite dif- ferent from the enhanced adhesion shown on whole biofilms of similar age. These re- sults suggest the inclusion of the living cells, (whole biofilm) as crucial to the enhancement of temporary adhesion seen in the younger film with the concomitant inference of increased settlement potential. Cell activity and Maki et al.‘s (1990) ob- servations on altered states with surface energy, taken together, hint at three-dimensional structural effects. The bacterial exudate resulted in lower cypris adhesion irrespective of the hydrophobicity of the surfaces to which it was adsorbed, thus the wettability of the surface could not have affected the orientation of surface active exudate molecules in the manner described by Maki et al. (1990) for whole D. marina films, at least for those molecules of molecular mass > 16 kDa. Old D. marina biofilms produced lower cypris tenacity equivalent to that shown by the exudate treatment, but most markedly on substrata of low wettability. A.L. Neal. A.B. Yule/J. Exp. Mar. Biol. Ecol. 176 (1994) 127-139 137 The reduction in adhesion (either physical, or behaviourally mediated through the cyprid’s willingness to detach) is in some way related to the bacterial exopolymers, and it may prove that the older bacterial films had a far lower cell to exopolymer ratio than the younger films. Enhancement of adhesion (again, either physical, or behaviourally mediated) seems dependent on the presence of living cells, probably in large numbers. The effect of surface-free energy is only clearly manifest when cells are present, lead- ing to the hypothesis that the three-dimensional structure of the slime layer is largely determined by the presence of living cells, and that a whole biofilm is more likely to be affected by the underlying surface-free energy than are the higher molecular weight exudates alone. Temporary adhesion and settlement have been shown to be closely allied for cyprids of B. balunoides (Neal & Yule, 1992). Assuming a similar relationship exists with E. modestus, our results suggest that substrata with the characteristics of young D. ma- rina biofilms are more conducive to the settlement of E. modestus than equivalent unfilmed substrata. Conversely, substrata with the characteristics of older D. marina films would be less conducive to E. modestus settlement than equivalent unfilmed subtrata. That our results lead to similar, but subtly different, conclusions from those obtained from B. amphitrite cyprids (Maki et al., 1988, 1990) suggests a possible role for characteristic biofilms mediating zonation in barnacles. However, single-species biofilms are unrealistic models of natural biofilms and we should be cautious in ex- tending findings made on single-species films to multi-species films in the natural en- vironment. 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