Larval development and post-settlement metamorphosis of the barnacle Balanus albicostatus Pilsbry and the serpulid polychaete Pomatoleios kraussii Baird: Impact of a commonly used antifouling biocide,Irgarol 1051

Abstract

The impact of a commonly-used antifouling algicide, Irgarol 1051, on the larval development and post-settlement metamorphosis of the barnacle, Balanus albicostatus Pilsbry (Crustacea: Cirripedia), and the larval metamorphosis of a serpulid polycheate, Pomatoleios kraussii Baird, was evaluated. In the case of B. albicostatus, larval mortality increased with an increase in the concentration of Irgarol 1051, and there was a shift in the larval stage targeted from advanced instars to early instars. Nauplii that survived to the cyprid instar stage when reared in the presence of Irgarol 1051 showed prolonged instar and total naupliar duration when compared to the controls. The post-settlement metamorphosis of cyprids significantly varied with Irgarol concentration and also with biofilm age. One and 2-d-old untreated biofilms showed higher metamorphosis when compared to 5-d-old biofilms. However, when the biofilms that promoted cyprid metamorphosis were treated with Irgarol 1051 at low concentrations, metamorphosis rates decreased. Cyprids were prevented from metamorphosing completely by biofilms treated at the highest concentration of Irgarol 1051. Inhibition of metamorphosis was also observed in the case of competent polychaete larvae when exposed to Irgarol 1051 compared to those exposed to metamorphosis inducers such as 3-iso-butyl-1-methylxanthine (IBMX) and natural biofilms. Identification of the pathway(s) that caused the promotory biofilms to become toxic when exposed to Irgarol 1051 is discussed.     

Impact of Irgarol 1051 on the larval development and metamorphosis of Balanus amphitrite Darwin, the diatom Amphora coffeaformis and natural biofilm

The effect of Irgarol 1051 on the biofilm-forming diatom, Amphora coffeaformis, and on natural biofilm (NBF) was assessed. A reduction in the number of A. coffeaformis cells within a biofilm was observed after treatment with Irgarol 1051, confirming its role as an inhibitor of photosynthetic activity. The impact of this compound on the development of nauplii of Balanus amphitrite was evaluated through its impact on Chaetoceros calcitrans, which was provided as food for the larvae. A reduction in the number of cells of C. calcitrans was observed when treated with Irgarol 1051. When larvae of B. amphitrite were reared using C. calcitrans in the presence of Irgarol 1051, their mortality increased with an increase in the concentration of Irgarol 1051 (13% at 1 microg l(-1) to 40% at 1000 microg l(-1)) compared with the control (6%). Nauplii reared in the presence of Irgarol 1051 developed more slowly (6-7 days) compared with control larvae (4-5 days). Cyprid bioassay results indicated an increase in percentage metamorphosis (76%) when NBFs were treated with the highest concentration of Irgarol 1051, compared with untreated biofilm (28%). The enhanced rate of metamorphosis appeared to be related to an increase in bacterial numbers in the biofilm, which may have been due to lysis of diatoms caused by Irgarol 1051. A. coffeaformis biofilms grown in the presence of antibiotics showed a significant reduction in cell numbers, which on further treatment with Irgarol 1051 showed an increase in cell numbers. Thus, it can be hypothesised that A. coffeaformis cells that were subjected to stress twice may have expressed resistant genes. Furthermore, if plasmids are present in the biofilms, they may enhance transfer to the surviving cells making them more resistant to hostile conditions.     

Larval development and post-settlement metamorphosis of the barnacle Balanus albicostatus Pilsbry and the serpulid polychaete Pomatoleios kraussii Baird: Impact of a commonly used antifouling biocide, Irgarol 1051

AbstractThe impact of a commonly-used antifouling algicide, Irgarol 1051, on the larval development and post-settlement metamorphosis of the barnacle, Balanus albicostatus Pilsbry (Crustacea: Cirripedia), and the larval metamorphosis of a serpulid polycheate, Pomatoleios kraussii Baird, was evaluated. In the case of B. albicostatus, larval mortality increased with an increase in the concentration of Irgarol 1051, and there was a shift in the larval stage targeted from advanced instars to early instars. Nauplii that survived to the cyprid instar stage when reared in the presence of Irgarol 1051 showed prolonged instar and total naupliar duration when compared to the controls. The post-settlement metamorphosis of cyprids significantly varied with Irgarol concentration and also with biofilm age. One and 2-d-old untreated biofilms showed higher metamorphosis when compared to 5-d-old biofilms. However, when the biofilms that promoted cyprid metamorphosis were treated with Irgarol 1051 at low concentrations, metamorphosis rates decreased. Cyprids were prevented from metamorphosing completely by biofilms treated at the highest concentration of Irgarol 1051. Inhibition of metamorphosis was also observed in the case of competent polychaete larvae when exposed to Irgarol 1051 compared to those exposed to metamorphosis inducers such as 3-iso-butyl-1-methylxanthine (IBMX) and natural biofilms. Identification of the pathway(s) that caused the promotory biofilms to become toxic when exposed to Irgarol 1051 is discussed.     

Larval settlement and metamorphosis of the mussel Mytilus coruscus in response to natural biofilms

Settlement and metamorphosis of pediveliger larvae of Mytilus coruscus in response to natural biofilms was investigated in the laboratory. Pediveliger larvae settled and metamorphosed in response to biofilms and post-larval settlement and metamorphosis increased with biofilm age. The activity of the biofilm was positively correlated with biofilm age, dry weight, bacterial density and diatom density, but had no apparent relationship with chlorophyll a concentration. The change in bacterial community composition corresponding to biofilm age may explain differences in the age-dependent inducing activities of biofilms, which in turn may play an important role in larval settlement in this species.     

The interplay of substrate nature and biofilm formation in regulating Balanus amphitrite Darwin, 1854 larval settlement

The settlement of marine larvae is influenced by a wide range of physical and biological factors. It is still poorly known how the nature of substrate and the biofilm can interact in regulating settlement patterns of invertebrate larvae. Here we use laboratory experiments focused on settlement behaviour of the barnacle Balanus amphitrite. The aim of this work is to understand whether: (i) the nature of substratum can affect biofilm formation and its structure, (ii) the nature of substratum can affect B. amphitrite larval settlement, (iii) the age of the biofilms and the nature of substrate can interact in influencing larval settlement.Four kinds of substrata (marble, quartz, glass, and cembonit) were biofilmed under laboratory conditions for 5, 10 and 20 days at the temperature of 28 °C. Settlement response was investigated with 5-day-old cyprids. Biofilms were quantitatively and qualitatively analysed by scanning electron microscopy. The settlement of B. amphitrite larvae significantly differed among substrata; also, the patterns of development of biofilm assemblages changed with substrate. In addition, the larval attractiveness of different substrates tends to disappear with biofilm age.     

The effect of butenolide on behavioral and morphological changes in two marine fouling species,the barnacle Balanus amphitrite and the bryozoan Bugula neritina

Butenolide [5-octylfuran-2(5H)-one] is a very promising antifouling compound. Here, the effects of butenolide on larval behavior and histology are compared in two major fouling organisms, viz. cypris larvae of Balanus amphitrite and swimming larvae of Bugula neritina. Butenolide diminished the positive phototactic behavior of B. amphitrite (EC₅₀ = 0.82 μg ml^?1) and B. neritina (EC₅₀ = 3 μg ml^?1). Its effect on the attachment of cyprids of B. amphitrite was influenced by temperature, and butenolide increased attachment of larvae of B. neritina to the bottom of the experimental wells. At concentrations of 4 μg ml^?1 and 10 μg ml^?1, butenolide decreased attachment of B. amphitrite and B. neritina, respectively, but the effects were reversible within a certain treatment time. Morphologically, butenolide inhibited the swelling of secretory granules and altered the rough endoplasmic reticulum (RER) in the cement gland of B. amphitrite cyprids. In B. neritina swimming larvae, butenolide reduced the number of secretory granules in the pyriform-glandular complex.     

Induction of metamorphosis of pediveliger larvae of the mussel Mytilus galloprovincialis Lamarck, 1819 using neuroactive compounds,KCl, NH4Cl and organic solvents

Pediveliger larvae of Mytilus galloprovincialis were subjected to a series of bioassays to investigate the induction of metamorphosis using neuroactive compounds, K⁺, NH₄ ⁺ and organic solvents. Growth and survival of post-larvae obtained using ethanol and methanol were also observed. Epinephrine, phenylephrine, clonidine and metanephrine induced larval metamorphosis at 10^?6 to 10^?4 M in both 24-h and continuous exposure assays. In 24-h exposure assays, α-methyldopa at 5×10^?5 M and methoxyphenamine at 5×10^?5?10^?4 M induced 55?94% metamorphosis. Similarly, excess K⁺ at 3×10^?2 M induced 39% metamorphosis and NH₄ ⁺ at 1?5×10^?2 M induced 63–78% metamorphosis. The EC50s of seven organic solvents ranged from 0.04 to 0.82 M. Post-larvae that metamorphosed using ethanol and methanol survived as juveniles and grew at the same rate as those from microbial biofilm. Thus, the above compounds can be useful inducers of metamorphosis for antifouling studies using larvae and juveniles of M. galloprovincialis.     

Metamorphosis of a Scleractinian Coral in Response to Microbial Biofilms

Microorganisms have been reported to induce settlement and metamorphosis in a wide range of marine invertebrate species. However, the primary cue reported for metamorphosis of coral larvae is calcareous coralline algae (CCA). Herein we report the community structure of developing coral reef biofilms and the potential role they play in triggering the metamorphosis of a scleractinian coral. Two-week-old biofilms induced metamorphosis in less than 10% of larvae, whereas metamorphosis increased significantly on older biofilms, with a maximum of 41% occurring on 8-week-old microbial films. There was a significant influence of depth in 4- and 8-week biofilms, with greater levels of metamorphosis occurring in response to shallow-water communities. Importantly, larvae were found to settle and metamorphose in response to microbial biofilms lacking CCA from both shallow and deep treatments, indicating that microorganisms not associated with CCA may play a significant role in coral metamorphosis. A polyphasic approach consisting of scanning electron microscopy, fluorescence in situ hybridization (FISH), and denaturing gradient gel electrophoresis (DGGE) revealed that coral reef biofilms were comprised of complex bacterial and microalgal communities which were distinct at each depth and time. Principal-component analysis of FISH data showed that the Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Cytophaga-Flavobacterium of Bacteroidetes had the largest influence on overall community composition. A low abundance of Archaea was detected in almost all biofilms, providing the first report of Archaea associated with coral reef biofilms. No differences in the relative densities of each subdivision of Proteobacteria were observed between slides that induced larval metamorphosis and those that did not. Comparative cluster analysis of bacterial DGGE patterns also revealed that there were clear age and depth distinctions in biofilm community structure; however, no difference was detected in banding profiles between biofilms which induced larval metamorphosis and those where no metamorphosis occurred. This investigation demonstrates that complex microbial communities can induce coral metamorphosis in the absence of CCA.     

Reversible anti-settlement activity against Amphibalanus (=Balanus) amphitrite,Bugula neritina,and Hydroides elegans by a nontoxic pharmaceutical compound,mizolastine

Mizolastine, an antihistamine pharmaceutical, was found to significantly inhibit larval settlement of the barnacle Amphibalanus (=Balanus) amphitrite, the bryozoan Bugula neritina, and the polychaete Hydroides elegans with EC₅₀ values of 4.2, 11.2, and 4.1 µg ml^?1, respectively. No toxicity against the larvae of these three species was observed at the concentration range tested during incubations with mizolastine. To determine whether the anti-settlement activity of mizolastine is reversible, recovery bioassays using these three species were conducted. More than 70% of the larvae that had been exposed for 4 h to mizolastine at concentrations four-fold greater than their respective EC₅₀ values completed normal metamorphosis. The results of the recovery bioassay provide evidence that the anti-settlement effect of mizolastine is reversible in addition to being nontoxic. The anti-settlement activities of several intermediates of the synthesis process of mizolastine were also examined. One of the intermediates, 2-chloro-1-(4-fluorobenzyl)-1H-benzo[d]imidazole, inhibited larval settlement and metamorphosis with low toxicity. These results may improve the understanding of the key functional group responsible for the anti-settlement activity of mizolastine.     

Characterisation of the bacteria associated with barnacle,Balanus amphitrite,shell and their role in gregarious settlement of cypris larvae

The acorn barnacle Balanus amphitrite (syn. Amphibalanus amphitrite) is a model organism to investigate pelago-benthic transitions in marine invertebrates. A driver for larval settlement in this organism is the need to attach close to conspecifics, to allow reproduction to take place. Adult barnacles are covered by microbial biofilms and the contribution of these biofilms to conspecific recognition is not fully understood. Little information is available on microbial communities associated with B. amphitrite. We compared biofilm communities from the barnacle shell surface with those from the surrounding rocks using the culture-independent methods of quantitative PCR and denaturing gradient gel electrophoresis. Quantification of the relative abundances of higher bacterial taxa showed that barnacles hosted a greater proportion of α-Proteobacteria compared to rock-associated biofilms (p < 0.01). Differences in relative abundances of other taxa were not observed but DGGE profiling suggested that differences were present at lower taxonomic levels. The capacity of these communities to influence larval settlement was assessed by growing multispecies biofilms on artificial medium, obtained by extracting nutrients from adult barnacles. Biofilms composed of shell-associated bacteria were capable of promoting conspecific settlement by 67% compared to control surfaces (p < 0.05), while rock-associated communities showed contrasting effects. A taxonomic comparison of settlement-stimulating and -inhibiting bacteria was performed by DGGE and band sequencing. All partial 16S rRNA genes sequenced were similar to members of the Vibrio and Pseudoalteromonas genera, suggesting that larvae can detect and respond to variations in the composition of microbial biofilms at low taxonomic levels. Our results indicate that barnacle larvae may be able to detect parentally-associated biofilms and use this information to settle close to members of its own species.     

Biofilm form and function: carbon availability affects biofilm architecture, metabolic activity and planktonic cell yield

Aims: To investigate carbon transformation by biofilms and changes in biofilm architecture, metabolic activity and planktonic cell yield in response to fluctuating carbon availability. Methods and Results: Pseudomonas sp. biofilms were cultured under alternating carbon‐replete and carbon‐limited conditions. A shift to medium without added carbon led to a 90% decrease in biofilm respiration rate and a 40% reduction in planktonic cell yield within 1 h. Attached cell division and progeny release were shown to contribute to planktonic cell numbers during carbon limitation. Development of a significantly enlarged biofilm surface area during carbon limitation facilitated a rapid increase in whole‐biofilm metabolic activity, cell yield and biomass upon the re‐introduction of carbon after 8 days of limitation. The cumulative number of planktonic cells (>10¹⁰ CFU) released from the biofilm during the cultivation period contained only 1·0% of the total carbon available to the biofilm, with 6·5% of the carbon retained in the biofilm and 54% mineralized to CO₂. Conclusions: Biofilm‐derived planktonic cell yield is a proliferation mechanism. The rapid response of biofilms to environmental perturbations facilitates the optimal utilization of resources to promote both proliferation and survival. Biofilms function as efficient catalysts for environmental carbon transformation and mineralization. Significance and Impact of the study: A greater understanding of the relationship between biofilm form and function can inform strategies intended to control and/or promote biofilm formation.     

Comparative assessment of single and joint effects of diuron and Irgarol 1051 on Arctic and temperate microalgae using chlorophyll a fluorescence imaging

Ship groundings and ice-breakers can cause pollution of the polar environment with antifouling biocides such as diuron and Irgarol 1051. The present study used pulse amplitude modulated fluorometry to compare single and joint toxicities of diuron and Irgarol 1051 on two freshwater taxa of microalgae (Chlorella and Chlamydomonas) originating from Arctic and temperate regions. 30 min acute toxicity tests using chlorophyll a (Chl a) fluorescence revealed that Arctic strains of microalgae were more sensitive to herbicides than their temperate counterparts. Diuron and Irgarol 1051 had equal toxicities in the Arctic species, while Irgarol 1051 was more toxic (EC₅₀ = 5.55–14.70 μg L⁻¹) than diuron (EC₅₀ = 12.90–>40 μg L⁻¹) in the temperate species. Toxicity assessment of various mixtures of diuron and Irgarol 1051 revealed antagonistic, additive, and synergistic effects. Our data suggest that herbicides can adversely affect photosynthesis in Arctic microalgae at relatively low levels, and their impact can increase under complex mixture conditions.     

Larval settlement and metamorphosis of the mussel Mytilus coruscus in response to natural biofilms

Settlement and metamorphosis of pediveliger larvae of Mytilus coruscus in response to natural biofilms was investigated in the laboratory. Pediveliger larvae settled and metamorphosed in response to biofilms and post-larval settlement and metamorphosis increased with biofilm age. The activity of the biofilm was positively correlated with biofilm age, dry weight, bacterial density and diatom density, but had no apparent relationship with chlorophyll a concentration. The change in bacterial community composition corresponding to biofilm age may explain differences in the age-dependent inducing activities of biofilms, which in turn may play an important role in larval settlement in this species.     

Combined treatment of Pseudomonas aeruginosa biofilms with bacteriophages and chlorine

Bacterial biofilms are a growing concern in a broad range of areas. In this study, a mixture of RNA bacteriophages isolated from municipal wastewater was used to control and remove biofilms. At the concentrations of 400 and 4 × 10⁷ PFU/mL, the phages inhibited Pseudomonas aeruginosa biofilm formation by 45 ± 15% and 73 ± 8%, respectively. At the concentrations of 6,000 and 6 × 10⁷ PFU/mL, the phages removed 45 ± 9% and 75 ± 5% of pre‐existing P. aeruginosa biofilms, respectively. Chlorine reduced biofilm growth by 86 ± 3% at the concentration of 210 mg/L, but it did not remove pre‐existing biofilms. However, a combination of phages (3 × 10⁷ PFU/mL) and chlorine at this concentration reduced biofilm growth by 94 ± 2% and removed 88 ± 6% of existing biofilms. In a continuous flow system with continued biofilm growth, a combination of phages (a one‐time treatment at the concentration of 1.9 × 10⁸ PFU/mL for 1 h first) with chlorine removed 97 ± 1% of biofilms after Day 5 while phage and chlorine treatment alone removed 89 ± 1% and 40 ± 5%, respectively. For existing biofilms, a combined use of a lower phage concentration (3.8 × 10⁵ PFU/mL) and chlorination with a shorter time duration (12 h) followed by continuous water flushing removed 96 ± 1% of biofilms in less than 2 days. Laser scanning confocal microscopy supplemented with electron microscopy indicated that the combination treatment resulted in biofilms with lowest cell density and viability. These results suggest that the combination treatment of phages and chlorine is a promising method to control and remove bacterial biofilms from various surfaces. Biotechnol. Bioeng. 2013; 110: 286–295. © 2012 Wiley Periodicals, Inc.     

Kinetics of biofilm formation and desiccation survival of Listeria monocytogenes in single and dual species biofilms with Pseudomonas fluorescens,Serratia proteamaculans or Shewanella baltica on food-grade stainless steel surfaces

This study investigated the dynamics of static biofilm formation (100% RH, 15 °C, 48–72 h) and desiccation survival (43% RH, 15 °C, 21 days) of Listeria monocytogenes, in dual species biofilms with the common spoilage bacteria, Pseudomonas fluorescens, Serratia proteamaculans and Shewanella baltica, on the surface of food grade stainless steel. The Gram-negative bacteria reduced the maximum biofilm population of L. monocytogenes in dual species biofilms and increased its inactivation during desiccation. However, due to the higher desiccation resistance of Listeria relative to P. fluorescens and S. baltica, the pathogen survived in greater final numbers. In contrast, S. proteamaculans outcompeted the pathogen during the biofilm formation and exhibited similar desiccation survival, causing the N_{21 days} of Serratia to be ca 3 Log₁₀(CFU cm^?2) greater than that of Listeria in the dual species biofilm. Microscopy revealed biofilm morphologies with variable amounts of exopolymeric substance and the presence of separate microcolonies. Under these simulated food plant conditions, the fate of L. monocytogenes during formation of mixed biofilms and desiccation depended on the implicit characteristics of the co-cultured bacterium.     

Proteomic analysis during larval development and metamorphosis of the spionid polychaete Pseudopolydora vexillosa

Background  

While the larval-juvenile transition (metamorphosis) in the spionid polychaete Pseudopolydora vexillosa involves gradual morphological changes and does not require substantial development of juvenile organs, the opposite occurs in the barnacle Balanus amphitrite. We hypothesized that the proteome changes during metamorphosis in the spionids are less drastic than that in the barnacles. To test this, proteomes of pre-competent larvae, competent larvae (ready to metamorphose), and juveniles of P. vexillosa were compared using 2-dimensional gel electrophoresis (2-DE), and they were then compared to those of the barnacle.     

Analysis of the mechanical stability and surface detachment of mature Streptococcus mutans biofilms by applying a range of external shear forces

Well-established biofilms formed by Streptococcus mutans via exopolysaccharide matrix synthesis are firmly attached to tooth surfaces. Enhanced understanding of the physical properties of mature biofilms may lead to improved approaches to detaching or disassembling these highly organized and adhesive structures. Here, the mechanical stability of S. mutans biofilms was investigated by determining their ability to withstand measured applications of shear stress using a custom-built device. The data show that the initial biofilm bulk (~ 50% biomass) was removed after exposure to 0.184 and 0.449 N m^?2 for 67 and 115 h old biofilms. However, removal of the remaining biofilm close to the surface was significantly reduced (vs initial bulk removal) even when shear forces were increased 10-fold. Treatment of biofilms with exopolysaccharide-digesting dextranase substantially compromised their mechanical stability and rigidity, resulting in bulk removal at a shear stress as low as 0.027 N m^?2 and > a two-fold reduction in the storage modulus (G′). The data reveal how incremental increases in shear stress cause distinctive patterns of biofilm detachment, while demonstrating that the exopolysaccharide matrix modulates the resistance of biofilms to mechanical clearance.     

Larval experience of stable fly,Stomoxys calcitrans Linneaus, 1758 (Diptera: Muscidae) does not influence oviposition preference in gravid females

1. Hopkins' host selection principle (HHSP) proposes that, in holometabolous insects, gravid females prefer to oviposit on their developmental substrate. This hypothesis is widely explored in phytophagous insects, but few studies have considered blood‐feeding insects. In this study, the HHSP was tested using the haematophagous stable fly, Stomoxys calcitrans (Diptera: Muscidae). Using no‐choice and two‐choice tests, the study first sought to demonstrate whether S. calcitrans larvae recognise their rearing substrate. 2. It was found that when a rearing substrate is offered to S. calcitrans larvae singly (no‐choice) or associated with a non‐rearing substrate (two‐choice), they were able to recognise the substrate in which they developed. This ability disappeared when larvae were transferred to another substrate for 5 h. 3. Next, using oviposition bioassays, it was investigated whether information gathered by pre‐imaginal stages during their development can persist into the adult stages after metamorphosis. It was hypothesised that gravid females emerging from clean and uncleaned pupae reared on sheep and camel dung would still prefer to lay on their rearing substrate. The results revealed that gravid females did not prefer to oviposit on their developmental substrate. In addition, failure to recognise and prefer their developmental substrate was found in gravid females that had emerged from puparia that were cleaned as well as those contaminated with sheep dung. 4. It is concluded that S. calcitrans adult preference is not influenced by their offspring's experience. Overall, the HHSP was not supported by our study, suggesting that this principle may not apply to generalist insects characterised by high plasticity in oviposition substrate acceptance.     

Sessile Legionella pneumophila is able to grow on surfaces and generate structured monospecies biofilms

Currently, models for studying Legionella pneumophila biofilm formation rely on multi-species biofilms with low reproducibility or on growth in rich medium, where planktonic growth is unavoidable. The present study describes a new medium adapted to the growth of L. pneumophila monospecies biofilms in vitro. A microplate model was used to test several media. After incubation for 6 days in a specific biofilm broth not supporting planktonic growth, biofilms consisted of 5.36 ± 0.40 log (cfu cm^?2) or 5.34 ± 0.33 log (gu cm^?2). The adhered population remained stable for up to 3 weeks after initial inoculation. In situ confocal microscope observations revealed a typical biofilm structure, comprising cell clusters ranging up to ～300 μm in height. This model is adapted to growing monospecies L. pneumophila biofilms that are structurally different from biofilms formed in a rich medium. High reproducibility and the absence of other microbial species make this model useful for studying genes involved in biofilm formation.     

Structural changes in S. epidermidis biofilms after transmission between stainless steel surfaces

Transmission is a main route for bacterial contamination, involving bacterial detachment from a donor and adhesion to receiver surfaces. This work aimed to compare transmission of an extracellular polymeric substance (EPS) producing and a non-EPS producing Staphylococcus epidermidis strain from biofilms on stainless steel. After transmission, donor surfaces remained fully covered with biofilm, indicating transmission through cohesive failure in the biofilm. Counter to the numbers of biofilm bacteria, the donor and receiver biofilm thicknesses did not add up to the pre-transmission donor biofilm thickness, suggesting more compact biofilms after transmission, especially for non-EPS producing staphylococci. Accordingly, staphylococcal density per unit biofilm volume had increased from 0.20 to 0.52 μm^–3 for transmission of the non-EPS producing strain under high contact pressure. The EPS producing strain had similar densities before and after transmission (0.17 μm^–3). This suggests three phases in biofilm transmission: (1) compression, (2) separation and (3) relaxation of biofilm structure to its pre-transmission density in EPS-rich biofilms.