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Effects of small abalone, Haliotis diversicolor, pedal mucus on bacterial growth, attachment, biofilm formation and community structure
Aquaculture 293 (2009) 35–41
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Aquaculture j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e
Effects of small abalone, Haliotis diversicolor, pedal mucus on bacterial growth, attachment, biofilm formation and community structure Feng Guo, Zhao-bin Huang, Miao-qin Huang, Jing Zhao, Cai-huan Ke ⁎ State Key Laboratory of Marine Environmental Science, Xiamen University, Fujian Province 361005, PR China College of Oceanography and Environmental Science, Xiamen University, Xiamen 361005, PR China
a r t i c l e
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Article history: Received 23 November 2008 Received in revised form 15 March 2009 Accepted 16 March 2009 Keywords: Pedal mucus Haliotis diversicolor Bacteria Attachment Biofilm
a b s t r a c t Pedal mucus is important for locomotion, protection and signal transmission in the Gastropoda. It also provides nutrients and a habitat for microbes. In this study, we examined the effects of pedal mucus and the mucus trail of the small abalone, Haliotis diversicolor, on bacterial growth, attachment, biofilm formation and community structure. The results showed that pedal mucus enhanced the growth of bacteria, and that more opportunistic pathogens attached to a substrate covered with pedal mucus (for Vibrio alginolyticus, the cell density was up to 5 times higher than on a clean substrate). During microplate assay, when pedal mucus was added as the sole nutrient source, V. alginolyticus formed a biofilm, but few cells grew on the bottom of the wells if no mucus was added. Under flow-cell culture conditions, differently structured biofilms were formed in the clean and pedal mucus conditioned glass slides. The mucus trail induced a heterogeneous biofilm with cell clusters. Moreover, pedal mucus could support much more CFUs than the clean substrates. Using denatured gradient gel electrophoresis comparison of the bacterial community on a clean substrate and in the mucus trail, we found several specific species in the latter. At least one species, Klebsiella pneumoniae, is potentially harmful to the small abalone. In summary, the pedal mucus of Haliotis diversicolor enriched bacterial occurrence, including some pathogens, which deserves to be considered in high density culture conditions. © 2009 Elsevier B.V. All rights reserved.
1. Introduction In gastropods, pedal mucus is important for their locomotion, protection and signal transmission (Davies and Hawkins, 1998; Hutchinson et al., 2007). In some intertidal species, the energetic budget for producing pedal mucus accounts for over 70% of their total energy expenditure (Horn, 1986). Besides water, the other major components of mucus are protein, carbohydrate and inorganics (Davies et al., 1990), which can be useful nutrients for microbes. Pedal mucus conditioned seawater promotes bacterial growth and extracellular enzymatic activity in the water column (Peduzzi and Herndl, 1991). On the other hand, in some gastropod species, certain glycoproteins in the mucus show antimicrobial activity (Kubota et al., 1985). To our knowledge, there is no report of the relationship between microorganisms and the pedal mucus of gastropods living in shallow subtidal zones. This may be attributed to their relatively small mucus secretion as compared with intertidal species, which need to secrete much more mucus in order to avoid dehydration in air. Haliotis spp. live in the shallow sea and many of them are important economic species all over the world. In south China, small abalone, Haliotis diversicolor, is one of the key mariculture species. ⁎ Corresponding author. State Key Laboratory of Marine Environmental Science, Xiamen University, Fujian Province 361005, PR China. Tel.: +86 592 2187420. E-mail address: [email protected] (C. Ke). 0044-8486/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2009.03.033
Unfortunately, this species can suffer many serious diseases during growing out, which can lead to high mortality. Several reports reveal that some diseases of small abalone are caused by infection with Vibrio spp. (Liu et al., 2001; Huang et al., 2001; Cai et al., 2007). However, the details of infection are not clear. During the farming of small abalone, the density of the abalones (about 80–150 individuals per m2) is much higher than in natural conditions. They are all semi-sealed in confined cages, and so the pedal mucus and mucus trails of H. diversicolor, which accumulate on the surface of the cages day by day, may continuously provide nutrients and/or a habitat for microbes including pathogens. In this study, we examined the effects of the pedal mucus and mucus trail of H. diversicolor on bacterial growth, attachment, biofilm formation and the community structure in vitro. The results should be meaningful for amendment of farming methods during mariculture of the small abalone, since our results also suggested that pedal mucus could serve as a substrate for growth of pathogens and a possible route of infection. 2. Material and methods 2.1. Strains and medium Four bacterial strains were used in this study. Escherichia coli DH5α is a commercially available strain. Staphylococcus epidermidis, Vibrio alginolyticus and Pseudomonas sp. were isolated from dying, disease
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Haliotis diversicolor and seawater of abalone culture pond respectively. The sample site of the three isolates is an abalone culture factory in Zhangzhou, Fujian province, China. All the cultures were shaken at 180 rpm overnight at 30 °C. E. coli (gram-negative) and S. epidermidis (gram-positive) were cultured with LB medium. V. alginolyticus and Pseudomonas sp. grew in marine 2216E medium. Before the experiments, the bacteria were collected by centrifugation (6000 ×g, 10 min) and washed twice with autoclaved artificial seawater (AASW, Lyma & Flemming). Finally, the cell suspensions were adjusted to an OD600 = 0.2 (If not mentioned, the suspensions with such density were used in all concerned experiments). For the CFU counting assay, 2216E marine agar and TCBS medium were used. CFU counting was done by serial dilution and spread plating. 2.2. Effect of mucus on bacterial growth In this study, abalones of 4–5 cm shell length were used. After cleaning the abalone pedal surface using sterile gauze, 12 individuals were put into two polystyrene Petri dishes (150 mm diameter) and allowed to attach. The mucus was collected by pipettes with 1–10 μL tips from the side faces of the feet. In this method, the abalones were kept in air, since it was shown that exposure to air increases pedal mucus production in Haliotis midae (Vosloo and Vosloo, 2006). Approximately 1 mL of mucus was collected within 1 h, and this was mixed with an equal volume of AASW because the original mucus was too ropy to perform subsequent operation. The 50% mucus solution was centrifuged and the suspension was filtered (0.22 μm Syringe Driven Filter Unit, Millipore, USA) to be a sterile fluid. A portion of the fluid was diluted to 10% and 2% mucus content using AASW, and 100 μL of these dilutions were added to a 96-well plate. Each treatment of the 50%, 10% and 2% mucus involved four replicates, and the AASW was used as the control. One microlitre of an E. coli DH5α or S. epidermidis suspension was added to the 96well plate and it was cultured at 30 °C. The OD600 values of the wells were recorded at 4, 8, 16, 24, 36 and 48 h using a microplate reader (680XR, Bio-Rad, USA). 2.3. Effect of mucus on bacterial attachment In order to quantify the numbers of attached cells and to eliminate the original bacteria in or on the mucus, we carried out the experiment in the following way. After cleaning the pedal surface of the abalones, 24 × 24 × 1 mm quartz slides were affixed to the surface for a few seconds. Next, the quartz slides with mucus and some without mucus were stained using calcofluor white (10 μg mL− 1 in AASW, a dye which specifically binds to β-linked polysaccharides, Maeda and Ishida, 1967) for 30 min and then washed 3 times with AASW. Meanwhile 1 mL suspensions of Vibrio alginolyticus and Pseudomonas sp. were stained for 10 min with the SYTO9 probe for live cells, (using 1 μL to 1 mL AASW, 1/3 of standard dosage; Baclight dead/live bacterial viability kit, Invitrogen, Molecular Probes, USA). The prestained cells were then washed twice and resuspended in AASW to a final concentration of 2 × 107 cell mL− 1. Next, the test slides with mucus were placed in the wells of 6-well plates which contained 3 mL suspensions of prestained V. alginolyticus or Pseudomonas sp. in each well, and slides without mucus were placed as the control. The wells containing only AASW and mucus-covered slides were used as the negative control (without bacteria, to confirm there was no miscounting of the original bacteria in or on the mucus). All treatments had three replications. The plates were kept at room temperature (25 °C) for 1 h. Finally, the slides were washed with AASW and examined under epifluorescence microscopy (DM4500, Leica Corp., Germany). The excitation wave lengths were 488 nm for SYTO 9, and 400 nm for calcofluor white, respectively. Only SYTO 9 stained cells (green) were counted as
attached bacteria on 9 random fields (348 × 260 μm). Images were captured under the two excitation wave lengths, and two images of the same field under each excitation wave length were composed using Leica FW4000 software. 2.4. Effect of pedal mucus on biofilm formation Two culture systems were used to form biofilms. For flow-cell assay, multi-specific biofilms were examined. This system is homemade based on Sternberg et al. (1998). Quartz slides were affixed to the pedal surface for a few seconds and clean slides were used as the control. After being immersed in natural seawater for 1 h, the clean and mucus-covered slides were placed in the same flow-cell for continuous culture. The flow rate was 1 mL min− 1. The culture medium was AASW without adding any carbon source. After 48 h, the slides were taken out and stained using live/dead dye and examined under the microscope as above. For microplate assay, the pedal mucus was collected and 100 μL 50% sterile mucus was added to each well of the 96-well plate, with AASW used as the control. One microlitre of Vibrio alginolyticus suspension was inoculated into each well. After 36 h, the optical density at 600 nm of the wells was measured to indicate the biomass of planktonic cells. Then the suspensions were discarded. The wells were gently washed 3 times by AASW, stained with 0.1% crystal violet for 5 min, and then washed with ddH2O until the water became colorless. Finally, 200 μL 95% ethanol was added to the wells to dissolve the dye binding with the biofilm, and then the OD600 was measured to calculate the biomass of the biofilms (Haase et al., 2006). The mucus solution without inoculation was used as the negative control. 2.5. Counting CFU in pedal mucus trails In this experiment, we used glass slides (80 × 25 × 1 mm) as substrates to load pedal mucus as above. The slides with or without mucus were put into a tank containing 20 L natural nearshore seawater from abalone culture pond (salinity 30). The tank was kept at room temperature (25 °C). Then the slides were sampled in triplicate after 0, 6, 12, 24 and 48 h. After gently washing with AASW, the microorganisms were scraped off from the slides and collected in 50 mL centrifuge tubes using cell scrapers. Finally, the suspensions (20 mL) were vortexed and spread on 2216E agar and TCBS medium. The plates were cultivated at 30 °C for 24–48 h, and then the CFU were counted and transformed to viable cell number per cm− 2. 2.6. Comparison between the bacterial community structure of mucus trails and clean substrates using denatured gradient gel electrophoresis (DGGE) Glass slides (2.5 × 8 cm) with or without mucus conditioning were used as substrates for bacterial attachment, and they were then placed in a tank containing 100 L natural sea water (NSW). The slides were sampled after days 2 and 5. The biofilms on the slides were collected using cell scrapers. The biofilms from triplicate slides were mixed to form one sample for DNA extraction. At the same time a 500 mL NSW sample was filtered through a 0.22 μm filter to obtain the bacteria in the water column. The following operations were then performed: 1) DNA extraction: the samples were handled following exactly the reported method (Narváez-Zapata et al., 2005), for the sample of filters, they were scraped into centrifuge tubes with blades and extracted. 2) PCR amplification: Bacterial 16s rRNA of the samples was amplified using the primers GC-341f and 534r as previously reported (Gillan et al., 1998). 3) DGGE: PCR products were run on an 8.0% acrylamide gel with a 40–60% linear gradient of urea and formamide and electrophoresed at 100 V and 60 °C for 10 h in 1 × TAE buffer. Then the gel was stained with silver dye (Ausubel et al., 1999). The bands of
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interest in the samples of the mucus trail were excised, and DNA was eluted with an equal volume of TE buffer (10 mM Tris–HCl, 1 mM EDTA, pH = 8.0) at 4 °C overnight. The resulting solution (2 μL) was used as template DNA for a subsequent PCR amplification. Finally, the reamplified PCR products were cloned to PMD 18T vector (Takara, China) and sequenced. 2.7. Data analysis All data analyses were carried out using SPSS 11.0 software. A t-test was used to determine the difference between the mucus or mucus trail treatments and the control. Significance for all analyses and comparisons was set at p b 0.05. For the results of DGGE, sequences were aligned to the closest relatives in the NCBI database using the BLAST algorithm (Altschul et al., 1997). 3. Results 3.1. Effect of pedal mucus on bacterial growth The effect of pedal mucus on bacterial growth is shown in Fig. 1. The cell density in the control showed little increase in either E. coli or S. epidermidis, but it was enhanced in the wells containing seawater amended with mucus, especially in the 10% and 50% mucus treatments. Moreover, their growth yield increased obviously with increase in the concentration of mucus. For E. coli, the cell density was maximum in 50% mucus after 36 h cultivation, which is significantly higher than seawater and 2% mucus (p b 0.001). For S. epidermidis cultured in 50% mucus, the cell density did not decrease even after 48 h.
Fig. 2. Bacterial attachment to mucus trails. A: Pseudomonas sp.; B: Vibrio alginolyticus. The bacteria were allowed to attach for 1 h to mucus conditioned and clean slides. In these study, the bacteria in the suspension were prestained with STYO9, which should not cause the cell death. Finally, only the stained cells (with green fluorescence) were counted as attached cell.
3.2. Effect of pedal mucus on bacterial attachment As show in Fig. 2, the attachment of both species of bacteria increased significantly if the substrate was covered with pedal mucus (for Pseudomonas sp., p b 0.05; for Vibrio alginolyticus, p b 0.001). For the latter strain, its ability to attach to clean quartz slides was very poor, but when the slides were covered by a layer of mucus, the attached cell density could increase approximately 5 times. The images of mucus stained with calcofluor white showed that the pedal mucus was not uniform at the microscopic level. Newly secreted abalone pedal mucus had fractional filaments about 10 μm wide. When counting the attached bacterial density, we found that their distribution was not uniform on the mucus-covered slides either (Fig. 3). Interestingly, bacteria were more prone to attach to the filaments than to the blank zones. The increase of bacterial attachment on mucus-covered slides was contributed by the cells obviously aggregating on these filaments. 3.3. Effect of pedal mucus on biofilm formation
Fig. 1. Bacterial growth yields in different pedal mucus concentrations. A: E. coli; B: S. epidermidis. The bacteria were culture in seawater amended with 0%, 2%, 10% and 50% pedal mucus of Haliotis diversicolor.
Under the same flow-cell culture condition, different phenotypes of biofilms were formed on the quartz slides with or without mucus (Fig. 4). Loosely built and thin biofilms were formed on clean quartz slides, while compact and heterogenous structures with cell clusters appeared on mucus-covered slides. The cell clusters were about 20– 60 μm in diameter. Very few dead cells were found either on mucuscovered or clean slides. In microplate assay, we found that V. alginolyticus could form biofilm in wells containing 50% mucus, but this failed when mucus was absent (Fig. 5). The difference was very significant (p b 0.001). This result was in accordance with the attachment assay. Meanwhile, mucus also greatly stimulated planktonic cell growth (p b 0.01).
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same to that on the control treatment at 48 h (Fig. 6B, C; p N 0.05). However, by considering the CFU on 2216E plates, which indicated the most cultivable bacteria, mucus supported 5 times more CFU than did clean substrates even at 48 h (Fig. 6A). 3.5. Analysis of the bacterial community in mucus trails by DGGE The bacterial community in mucus trails showed a different profile to that on a clean substrate (Fig. 7). At day 2, there were about 6 specific bands in the mucus trail (band 1 also existed in the clean substrate, but at much lower abundance). But at day 5, the difference became less obvious. Bands 1, 4, and 5 were also found in the water column, which meant that these species of bacteria could be attracted from seawater and grow in mucus trails but not (or weakly) on clean slides. Band 4, Klebsiella pneumoniae (100% similarity, see Table 1) is a pathogenic bacterium, and it existed on mucus-covered slides but not the clean ones. Bands 2, 3 and 6 are more likely to be derived from the pedal surface of the small abalone, because they were not detected in the water column. 4. Discussion
Fig. 3. Direct observation of attachment of V. alginolyticus to mucus trails by fluorescent stain. A: Mucus trail stained by calcofluor white. This dye was a specific stain for the βlink polysaccharides. It showed that the pedal mucus is fractionally filamentous at microcosmic level; B: Attached bacteria prestained with SYTO 9; C: Composite of A and B. This photo shows that the increase of bacterial attachment occurred on the filaments that could be stained by calcofluor white.
3.4. CFU counting in mucus trail Whether on 2216E or TCBS plates, the numbers of CFU on mucuscovered slides were always greater than those on the clean ones during the entire experimental period (Fig. 6). For both 2216E and TCBS plates, CFUs in mucus trails were at a maximum at 12 h, which were generally 5–8 times higher than those on clean substrates, then they decreased with culture time. We speculated that this was due to the mucus were fast consumed by the bacteria on it. For Vibrio spp. counting on TCBS plates, either the CFU of the yellow colonies (sucrose-fermenting species) or the green ones (species which cannot ferment sucrose) on the mucus-covering glass slides was almost the
Pedal mucus plays a key role in gastropod movement (Denny, 1980; Donovan and Carefoot, 1997). To be a better lubricant, mucus must have improved viscosity. Generally, it contains some glycoproteins (Hunt, 1970). Undoubtedly, such organic matter could be a carbon and nitrogen source for microorganisms, if it does not contain sufficient antimicrobial matter. In our study, the pedal mucus of small abalones could be the sole nutrient source for E. coli and S. epidermidis and promote bacterial growth. Furthermore, the CFU count on mucus trails of the small abalone was about 5-8 times more than that on clean substrate. The pedal mucus of intertidal limpets contains about 2% protein and 1% carbohydrate by wet weight (Connor, 1986). According to our previous study, although the protein and carbohydrate concentrations of pedal mucus in H. diversicolor are about one order of magnitude less than that of limpets (about 0.14% for protein and 0.08% carbohydrate, unpublished data), it is still much higher than NSW. Recently, researchers found that the mouse gastrointestinal mucus act as a nutrient source to pathogenic E. coli O157:H7 (Fabich et al., 2008). Among the aquatic animal, the surface mucus of salmon promotes the growth of a pathogen (Flavobacterium columnare) and its protease activity (Staroscik and Nelson, 2008). It seems that some specific components in the salmon mucus induced this result, because even in a rich medium, the growth rate of the bacterium is significantly higher when mucus is added. On the other hand, Salmon intestinal mucus could also support the growth of its pathogen, Vibrio anguillarum (Garcia et al., 1997). In addition, researchers have shown that adding coral mucus to the medium can result in the culture of more bacterial species (Koren and Rosenberg, 2006). Therefore, it could be a common phenomenon whereby the mucus might provide a highly active micro-environment for microorganisms. In abalones, it is well known that the pedal mucus of adults induces larval settlement and is utilized by postlarvae as transitional food (Seki and Kan-no, 1981; Slattery, 1992; Takami et al., 1997). In the present study, more cells of V. alginolyticus attached on the pedal mucus of H. diversicolor than on the clean slides. It should be noted that V. alginolyticus is a prevalent pathogen in many marine organisms. Several researchers showed it cause disease or death in H. diversicolor supertexta (Liu et al., 2001; Wang et al., 2005). Increased attachment only occurred in the filamentous locations, while no increase was seen in the blank areas. In our opinion, mucus, as a polymer layer, changed some characteristics of the substrate. Polymers on bacterial cell surfaces facilitate their attachment by overcoming the energy barrier between cell surface and substrate (Morisaki et al., 1999). The results suggested that this principle may also work when the substrate was coated with the pedal mucus
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Fig. 4. Biofilm formation on the mucus trails in flow-cell culture. After 48 h culture, biofilm formed on glass coverslips and was visualized by fluorescence microscopy after being dyed with the dead/live stain kit. The upper two images show the biofilm structure forming on clean slides (A: 100×; B: 400×). The bottom images show the biofilm forming on the mucus trail (C: 100×; D: 400×). The arrows illustrate that the bacterial cell clusters were much larger and denser on the mucus trail than on the clean glass surface.
polymers of the abalone. The mucus of vertebrates facilitates bacterial attachment is well known (Ofek et al., 2003). Bacteria inhabit the mucus covering animal tissues because of its viscosity and binding activities. Some bacteria are able to degrade mucus using specific glycosidases and then utilize them as nutrients (Hoskins et al., 1985). To our knowledge, facilitation of bacterial invasion by pedal mucus may due to its viscous character. Swimming motility in V. parahaemolyticus, which is driven by the rotation of a polar flagellum, is inhibited in highly viscous media, and so lateral flagella gene expression is induced (Kawagishi et al., 1996). The latter process is very important to bacterial invasion and particularly to one type of surface motility termed “swarming” (McCarter and Wright, 1993), which causes direct contact between pathogens and hosts. The pedal mucus of gastropods is innately viscous, and it could be an important physical factor explaining why Vibrio spp., or other bacteria, switch their life style from free living to colonization of a host or its mucus trails. This mechanism could also explain the results of our microplate assay. The pedal mucus of limpets can trap particles from the water column and enhance the growth of benthic diatoms as their food supply (Connor and Quinn, 1984; Davies et al., 1992). Calliostoma zizyphinum even obtains an additional food supply by wiping its mucus-coated shell twice a day (Holmes et al., 2001). Undoubtedly, in the natural environment, this is a classic and positive effect in community ecology in order to recycle the highly energetic mucus. Our results also showed that when trapping organic particles from the water column, some bacteria (including pathogens) could also settle on mucus-covered substrates, either actively or passively. This may not be an important phenomenon in the natural environment because of the low density of animals and the high water exchange, but in
mariculture conditions, a high density of abalone and a low water exchange rate would result in pathogenic microorganisms entering and growing quickly. Thus, regular cleaning of the surfaces of the cages containing abalone should be considered, as should an increase in the water exchange rate and a lowering of the culture density, in order to decrease the chance of pathogenic infection on the small abalone. The pedal mucus of marine gastropods persists on substrates for some time, even more than a month (Davies and Williams, 1992), during which time the bacteria could form biofilm on it by utilizing the
Fig. 5. Effect of pedal mucus on biofilm formation in V. alginolyticus. V. alginolyticus was inoculated to the wells contain 200 μL 50% mucus or AASW. After 36 h, the OD600 of planktonic cell and biofilm (stained by 0.1% crystal and washed by 200 μL 95% ethanol) were measured. Four replications were done in this experiment.
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Fig. 7. DGGE profiling of bacterial communities in the water column, on clean slides and on the mucus trail after immersion in seawater for 2 and 5 d. M: marker DL2000; a: 2d water sample; b: biofilm sample on clean slides at 2d; c: biofilm sample on mucus trail at 2d; d: 5d water sample; e: biofilm sample on clean slides at 5d; f: biofilm sample on mucus trail at 5d. The marked numbers show the specific bands in mucus trail samples. They were cut, amplified, cloned and sequenced.
Fig. 6. Change in CFU on mucus trail and clean glass slides. A: 2216E plates; B: Yellow colonies on TCBS plates; C: Green colonies on TCBS plates. The glass slides with mucus trail or not were put into a tank containing 20 L natural seawater from abalone culture pond. They were triple sampled with time lapse. The bacteria on the slides were scrapped off and counting CFU by serial dilution and spread on the plates.
nutrients in it. In our study, after 2 days of culture, a heterogeneous biofilm formed on the mucus-covered surface but a homogenous biofilm formed on the clean slides. This phenomenon may have resulted from differences in nutrient supply or in the initial colonizer. Biofilms, especially those pathogenic bacterial biofilms with highly heterogeneous structure, are notorious for their ability to resist drugs and cause chronic infections (Costerton et al., 1999). It is very hard to eradicate them using routine chemicals or antibiotics. In Chinese abalone farming, antibiotics are used after disease symptoms appear, but often with little effect. This could be a result of antibiotic over-use during culture. However, we also speculate that this is related to the living mode of the pathogens. Antibiotics, for example penicillin, could repress the pathogenic bacteria in the water column significantly, but its effect on bacteria in the biofilm (forming on the abalone cage surface or pedal surface) is difficult to evaluate. These bacteria in the biofilm are, of course, closest to the abalones, and such dense and
compact accumulation of bacteria could more easily break into the host's tissues and cause disease. As the DGGE profile shows, at least one potential pathogen, Klebsiella pneumoniae, was found in the mucus trail of the small abalone, but was not detected on the clean substrate. This bacterium is a common pathogen in Southeast Asian aquaculture facilities (Huys et al., 2007). A recent study shows that another close species, Klebsiella oxytoca, is virulent to small abalone postlarvae (Cai and Wang, 2008). On the other hand, pedal mucus may enrich some bacteria that could utilize mucus and grow more easily than the ones that cannot consume it. Observations of Haliotis spp. showed that their pedal surface is slightly tinted and relatively clean in most species. But the small abalone, H. diversicolor, in south China is an exception. Its foot was dark brown and often fouled. The fouling condition was different even between the two small abalone populations from distinct geographic
Table 1 Phylogenetic identification of selected bands from the DGGE bacterial profiles of the biofilm. Band⁎
Closest relative and database accession number⁎⁎
Sequence similarity
Alignment
1 2 3 4 5 6
Uncultured bacterium (AY160846) Tamlana agarivorans (EU221275) Uncultured gamma proteobacterium (AY792268) Klebsiella pneumoniae (EU419756) Uncultured Pseudoalteromonas sp.(EU167413) Uncultured bacterium (EF379757)
89% 94% 100% 100% 99% 93%
172/192 180/190 192/192 194/194 165/166 161/172
⁎For the band number see Fig. 7. ⁎⁎Sequences were aligned to the closest relative using BLAST. If the same similarity sequences are listed, the names of identified species are presented.
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Fig. 8. Different foot color and fouling condition between H. diversicolor from south China (upper) and from Japan (bottom).
areas, south China and Japan (Fig. 8). The essential reason for this phenomenon is unclear and needs further study. Interestingly, the fouling of the abalone foot seems to be related to their mortality, because the mortality rate of the south China population (N70%) is much more than that of the Japanese population (b30%) after 8 months in the same culture cage. We speculate that the different composition of the mucus in the two populations of small abalone may induce such a result, and so, comparison of the composition of the pedal mucus in the two populations will be the next step in our work. Acknowledgements This research was supported by grants from the National Natural Science Foundation of China (Project No. 40676081) and the Hi-Tech Research and Development (863) Program of China (Project No. 2006AA10A407). Professors John Hodgkiss is thanked for his assistance with English. References Altschul, S.F., Madden, T.L., Schaeffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402. Ausubel, F.M., Kingston, R.E., Seidman, J.G., Struhl, K., Brent, R., Moore, D.D., Smith, J.A., 1999. Short protocols in molecular biology, 4th ed. John, Wiley & Sons, Inc. Cai, J.P., Wang, Z.X., 2008. Characterization and identification of virulent Klebsiella oxytoca isolated from abalone (Haliotis diversicolor supertexta) postlarvae with mass mortality in Fujian, China. J. Invertebr. Pathol. 97, 70–75. Cai, J., Li, J., Thompson, K.D., Li, C., Han, H., 2007. Isolation and characterization of pathogenic Vibrio parahaemolyticus from diseased post-larvae of abalone Haliotis diversicolor supertexta. J. Basic Microbiol. 47 (1), 84–86. Connor, V.M., 1986. The use of mucous trails by intertidal limpets to enhance food resources. Biol. Bull. 171, 548–564. Connor, V.M., Quinn, J.F., 1984. Stimulation of food species growth by limpet mucus. Science 225 (4664), 843–844. Costerton, J.W., Stewart, P.S., Greenberg, E.P., 1999. Bacterial biofilms: a common cause of persistent infections. Science 284 (5418), 1318–1322. Davies, M.S., Hawkins, J.S., 1998. Mucus in marine molluscs. Adv. Mar. Biol. 34, 1–71. Davies, M.S., Williams, G.A., 1992. Pedal mucus of a tropical limpet, Cellana grata (Gould): energetics, production and fate. J. Exp. Mar. Biol. Ecol. 186 (1), 77–87. Davies, M.S., Jones, H.D., Hawkins, S.J., 1990. Seasonal variation in the composition of pedal mucus from Patella vulgata (L.). J. Exp. Mar. Biol. Ecol. 144, 101–112. Davies, M.S., Hawkins, S.J., Jones, H.D., 1992. Pedal mucus and its influence on the microbial food supply of two intertidal gastropods, Patella vulgata L. and Littorina littorea (L.). J. Exp. Mar. Biol. Ecol. 161, 57–77. Denny, M., 1980. Locomotion: the cost of gastropod crawling. Science 208 (4449), 1288–1290. Donovan, D., Carefoot, T., 1997. Locomotion in the abalone Haliotis kamtschatkana: pedal morphology and cost of transport. J. Exp. Biol. 200, 1145–1153.
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Identification of genes encoding components of the swarmer cell flagellar motor and propeller and a sigma factor controlling differentiation of Vibrio parahaemolyticus. J. Bacteriol. 175 (11), 3361–3371. Morisaki, H., Nagai, S., Ohshima, Ikemoto, E., Kogure, K., 1999. The effect of motility and cell-surface polymers on bacterial attachment. Microbiology 145, 2797–2802. Narváez-Zapata, J.A., Rodríguez-Avila, N., Ortega-Morales, B.O., 2005. Method for recovery of intact DNA for community analysis of marine intertidal microbial biofilms. Mol. Biotechnol. 30 (1), 51–56. Ofek, I., Hasty, D.L., Doyle, R.L., 2003. Bacterial adhesion to animal cells and tissues. ASM press. Peduzzi, P., Herndl, G.J., 1991. Mucus trails in the rocky intertidal: a highly active microenvironment. Mar. Ecol., Prog. Ser. 75, 267–274. Seki, T., Kan-no, H., 1981. Induced settlement of the Japanese abalone, Haliotis discus hannui, veliger by the mucous trails of the juvenile and adult abalones. Bull. Tohoku Reg. Fish. 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Aquaculture j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e
Effects of small abalone, Haliotis diversicolor, pedal mucus on bacterial growth, attachment, biofilm formation and community structure Feng Guo, Zhao-bin Huang, Miao-qin Huang, Jing Zhao, Cai-huan Ke ⁎ State Key Laboratory of Marine Environmental Science, Xiamen University, Fujian Province 361005, PR China College of Oceanography and Environmental Science, Xiamen University, Xiamen 361005, PR China
a r t i c l e
i n f o
Article history: Received 23 November 2008 Received in revised form 15 March 2009 Accepted 16 March 2009 Keywords: Pedal mucus Haliotis diversicolor Bacteria Attachment Biofilm
a b s t r a c t Pedal mucus is important for locomotion, protection and signal transmission in the Gastropoda. It also provides nutrients and a habitat for microbes. In this study, we examined the effects of pedal mucus and the mucus trail of the small abalone, Haliotis diversicolor, on bacterial growth, attachment, biofilm formation and community structure. The results showed that pedal mucus enhanced the growth of bacteria, and that more opportunistic pathogens attached to a substrate covered with pedal mucus (for Vibrio alginolyticus, the cell density was up to 5 times higher than on a clean substrate). During microplate assay, when pedal mucus was added as the sole nutrient source, V. alginolyticus formed a biofilm, but few cells grew on the bottom of the wells if no mucus was added. Under flow-cell culture conditions, differently structured biofilms were formed in the clean and pedal mucus conditioned glass slides. The mucus trail induced a heterogeneous biofilm with cell clusters. Moreover, pedal mucus could support much more CFUs than the clean substrates. Using denatured gradient gel electrophoresis comparison of the bacterial community on a clean substrate and in the mucus trail, we found several specific species in the latter. At least one species, Klebsiella pneumoniae, is potentially harmful to the small abalone. In summary, the pedal mucus of Haliotis diversicolor enriched bacterial occurrence, including some pathogens, which deserves to be considered in high density culture conditions. © 2009 Elsevier B.V. All rights reserved.
1. Introduction In gastropods, pedal mucus is important for their locomotion, protection and signal transmission (Davies and Hawkins, 1998; Hutchinson et al., 2007). In some intertidal species, the energetic budget for producing pedal mucus accounts for over 70% of their total energy expenditure (Horn, 1986). Besides water, the other major components of mucus are protein, carbohydrate and inorganics (Davies et al., 1990), which can be useful nutrients for microbes. Pedal mucus conditioned seawater promotes bacterial growth and extracellular enzymatic activity in the water column (Peduzzi and Herndl, 1991). On the other hand, in some gastropod species, certain glycoproteins in the mucus show antimicrobial activity (Kubota et al., 1985). To our knowledge, there is no report of the relationship between microorganisms and the pedal mucus of gastropods living in shallow subtidal zones. This may be attributed to their relatively small mucus secretion as compared with intertidal species, which need to secrete much more mucus in order to avoid dehydration in air. Haliotis spp. live in the shallow sea and many of them are important economic species all over the world. In south China, small abalone, Haliotis diversicolor, is one of the key mariculture species. ⁎ Corresponding author. State Key Laboratory of Marine Environmental Science, Xiamen University, Fujian Province 361005, PR China. Tel.: +86 592 2187420. E-mail address: [email protected] (C. Ke). 0044-8486/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2009.03.033
Unfortunately, this species can suffer many serious diseases during growing out, which can lead to high mortality. Several reports reveal that some diseases of small abalone are caused by infection with Vibrio spp. (Liu et al., 2001; Huang et al., 2001; Cai et al., 2007). However, the details of infection are not clear. During the farming of small abalone, the density of the abalones (about 80–150 individuals per m2) is much higher than in natural conditions. They are all semi-sealed in confined cages, and so the pedal mucus and mucus trails of H. diversicolor, which accumulate on the surface of the cages day by day, may continuously provide nutrients and/or a habitat for microbes including pathogens. In this study, we examined the effects of the pedal mucus and mucus trail of H. diversicolor on bacterial growth, attachment, biofilm formation and the community structure in vitro. The results should be meaningful for amendment of farming methods during mariculture of the small abalone, since our results also suggested that pedal mucus could serve as a substrate for growth of pathogens and a possible route of infection. 2. Material and methods 2.1. Strains and medium Four bacterial strains were used in this study. Escherichia coli DH5α is a commercially available strain. Staphylococcus epidermidis, Vibrio alginolyticus and Pseudomonas sp. were isolated from dying, disease
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Haliotis diversicolor and seawater of abalone culture pond respectively. The sample site of the three isolates is an abalone culture factory in Zhangzhou, Fujian province, China. All the cultures were shaken at 180 rpm overnight at 30 °C. E. coli (gram-negative) and S. epidermidis (gram-positive) were cultured with LB medium. V. alginolyticus and Pseudomonas sp. grew in marine 2216E medium. Before the experiments, the bacteria were collected by centrifugation (6000 ×g, 10 min) and washed twice with autoclaved artificial seawater (AASW, Lyma & Flemming). Finally, the cell suspensions were adjusted to an OD600 = 0.2 (If not mentioned, the suspensions with such density were used in all concerned experiments). For the CFU counting assay, 2216E marine agar and TCBS medium were used. CFU counting was done by serial dilution and spread plating. 2.2. Effect of mucus on bacterial growth In this study, abalones of 4–5 cm shell length were used. After cleaning the abalone pedal surface using sterile gauze, 12 individuals were put into two polystyrene Petri dishes (150 mm diameter) and allowed to attach. The mucus was collected by pipettes with 1–10 μL tips from the side faces of the feet. In this method, the abalones were kept in air, since it was shown that exposure to air increases pedal mucus production in Haliotis midae (Vosloo and Vosloo, 2006). Approximately 1 mL of mucus was collected within 1 h, and this was mixed with an equal volume of AASW because the original mucus was too ropy to perform subsequent operation. The 50% mucus solution was centrifuged and the suspension was filtered (0.22 μm Syringe Driven Filter Unit, Millipore, USA) to be a sterile fluid. A portion of the fluid was diluted to 10% and 2% mucus content using AASW, and 100 μL of these dilutions were added to a 96-well plate. Each treatment of the 50%, 10% and 2% mucus involved four replicates, and the AASW was used as the control. One microlitre of an E. coli DH5α or S. epidermidis suspension was added to the 96well plate and it was cultured at 30 °C. The OD600 values of the wells were recorded at 4, 8, 16, 24, 36 and 48 h using a microplate reader (680XR, Bio-Rad, USA). 2.3. Effect of mucus on bacterial attachment In order to quantify the numbers of attached cells and to eliminate the original bacteria in or on the mucus, we carried out the experiment in the following way. After cleaning the pedal surface of the abalones, 24 × 24 × 1 mm quartz slides were affixed to the surface for a few seconds. Next, the quartz slides with mucus and some without mucus were stained using calcofluor white (10 μg mL− 1 in AASW, a dye which specifically binds to β-linked polysaccharides, Maeda and Ishida, 1967) for 30 min and then washed 3 times with AASW. Meanwhile 1 mL suspensions of Vibrio alginolyticus and Pseudomonas sp. were stained for 10 min with the SYTO9 probe for live cells, (using 1 μL to 1 mL AASW, 1/3 of standard dosage; Baclight dead/live bacterial viability kit, Invitrogen, Molecular Probes, USA). The prestained cells were then washed twice and resuspended in AASW to a final concentration of 2 × 107 cell mL− 1. Next, the test slides with mucus were placed in the wells of 6-well plates which contained 3 mL suspensions of prestained V. alginolyticus or Pseudomonas sp. in each well, and slides without mucus were placed as the control. The wells containing only AASW and mucus-covered slides were used as the negative control (without bacteria, to confirm there was no miscounting of the original bacteria in or on the mucus). All treatments had three replications. The plates were kept at room temperature (25 °C) for 1 h. Finally, the slides were washed with AASW and examined under epifluorescence microscopy (DM4500, Leica Corp., Germany). The excitation wave lengths were 488 nm for SYTO 9, and 400 nm for calcofluor white, respectively. Only SYTO 9 stained cells (green) were counted as
attached bacteria on 9 random fields (348 × 260 μm). Images were captured under the two excitation wave lengths, and two images of the same field under each excitation wave length were composed using Leica FW4000 software. 2.4. Effect of pedal mucus on biofilm formation Two culture systems were used to form biofilms. For flow-cell assay, multi-specific biofilms were examined. This system is homemade based on Sternberg et al. (1998). Quartz slides were affixed to the pedal surface for a few seconds and clean slides were used as the control. After being immersed in natural seawater for 1 h, the clean and mucus-covered slides were placed in the same flow-cell for continuous culture. The flow rate was 1 mL min− 1. The culture medium was AASW without adding any carbon source. After 48 h, the slides were taken out and stained using live/dead dye and examined under the microscope as above. For microplate assay, the pedal mucus was collected and 100 μL 50% sterile mucus was added to each well of the 96-well plate, with AASW used as the control. One microlitre of Vibrio alginolyticus suspension was inoculated into each well. After 36 h, the optical density at 600 nm of the wells was measured to indicate the biomass of planktonic cells. Then the suspensions were discarded. The wells were gently washed 3 times by AASW, stained with 0.1% crystal violet for 5 min, and then washed with ddH2O until the water became colorless. Finally, 200 μL 95% ethanol was added to the wells to dissolve the dye binding with the biofilm, and then the OD600 was measured to calculate the biomass of the biofilms (Haase et al., 2006). The mucus solution without inoculation was used as the negative control. 2.5. Counting CFU in pedal mucus trails In this experiment, we used glass slides (80 × 25 × 1 mm) as substrates to load pedal mucus as above. The slides with or without mucus were put into a tank containing 20 L natural nearshore seawater from abalone culture pond (salinity 30). The tank was kept at room temperature (25 °C). Then the slides were sampled in triplicate after 0, 6, 12, 24 and 48 h. After gently washing with AASW, the microorganisms were scraped off from the slides and collected in 50 mL centrifuge tubes using cell scrapers. Finally, the suspensions (20 mL) were vortexed and spread on 2216E agar and TCBS medium. The plates were cultivated at 30 °C for 24–48 h, and then the CFU were counted and transformed to viable cell number per cm− 2. 2.6. Comparison between the bacterial community structure of mucus trails and clean substrates using denatured gradient gel electrophoresis (DGGE) Glass slides (2.5 × 8 cm) with or without mucus conditioning were used as substrates for bacterial attachment, and they were then placed in a tank containing 100 L natural sea water (NSW). The slides were sampled after days 2 and 5. The biofilms on the slides were collected using cell scrapers. The biofilms from triplicate slides were mixed to form one sample for DNA extraction. At the same time a 500 mL NSW sample was filtered through a 0.22 μm filter to obtain the bacteria in the water column. The following operations were then performed: 1) DNA extraction: the samples were handled following exactly the reported method (Narváez-Zapata et al., 2005), for the sample of filters, they were scraped into centrifuge tubes with blades and extracted. 2) PCR amplification: Bacterial 16s rRNA of the samples was amplified using the primers GC-341f and 534r as previously reported (Gillan et al., 1998). 3) DGGE: PCR products were run on an 8.0% acrylamide gel with a 40–60% linear gradient of urea and formamide and electrophoresed at 100 V and 60 °C for 10 h in 1 × TAE buffer. Then the gel was stained with silver dye (Ausubel et al., 1999). The bands of
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interest in the samples of the mucus trail were excised, and DNA was eluted with an equal volume of TE buffer (10 mM Tris–HCl, 1 mM EDTA, pH = 8.0) at 4 °C overnight. The resulting solution (2 μL) was used as template DNA for a subsequent PCR amplification. Finally, the reamplified PCR products were cloned to PMD 18T vector (Takara, China) and sequenced. 2.7. Data analysis All data analyses were carried out using SPSS 11.0 software. A t-test was used to determine the difference between the mucus or mucus trail treatments and the control. Significance for all analyses and comparisons was set at p b 0.05. For the results of DGGE, sequences were aligned to the closest relatives in the NCBI database using the BLAST algorithm (Altschul et al., 1997). 3. Results 3.1. Effect of pedal mucus on bacterial growth The effect of pedal mucus on bacterial growth is shown in Fig. 1. The cell density in the control showed little increase in either E. coli or S. epidermidis, but it was enhanced in the wells containing seawater amended with mucus, especially in the 10% and 50% mucus treatments. Moreover, their growth yield increased obviously with increase in the concentration of mucus. For E. coli, the cell density was maximum in 50% mucus after 36 h cultivation, which is significantly higher than seawater and 2% mucus (p b 0.001). For S. epidermidis cultured in 50% mucus, the cell density did not decrease even after 48 h.
Fig. 2. Bacterial attachment to mucus trails. A: Pseudomonas sp.; B: Vibrio alginolyticus. The bacteria were allowed to attach for 1 h to mucus conditioned and clean slides. In these study, the bacteria in the suspension were prestained with STYO9, which should not cause the cell death. Finally, only the stained cells (with green fluorescence) were counted as attached cell.
3.2. Effect of pedal mucus on bacterial attachment As show in Fig. 2, the attachment of both species of bacteria increased significantly if the substrate was covered with pedal mucus (for Pseudomonas sp., p b 0.05; for Vibrio alginolyticus, p b 0.001). For the latter strain, its ability to attach to clean quartz slides was very poor, but when the slides were covered by a layer of mucus, the attached cell density could increase approximately 5 times. The images of mucus stained with calcofluor white showed that the pedal mucus was not uniform at the microscopic level. Newly secreted abalone pedal mucus had fractional filaments about 10 μm wide. When counting the attached bacterial density, we found that their distribution was not uniform on the mucus-covered slides either (Fig. 3). Interestingly, bacteria were more prone to attach to the filaments than to the blank zones. The increase of bacterial attachment on mucus-covered slides was contributed by the cells obviously aggregating on these filaments. 3.3. Effect of pedal mucus on biofilm formation
Fig. 1. Bacterial growth yields in different pedal mucus concentrations. A: E. coli; B: S. epidermidis. The bacteria were culture in seawater amended with 0%, 2%, 10% and 50% pedal mucus of Haliotis diversicolor.
Under the same flow-cell culture condition, different phenotypes of biofilms were formed on the quartz slides with or without mucus (Fig. 4). Loosely built and thin biofilms were formed on clean quartz slides, while compact and heterogenous structures with cell clusters appeared on mucus-covered slides. The cell clusters were about 20– 60 μm in diameter. Very few dead cells were found either on mucuscovered or clean slides. In microplate assay, we found that V. alginolyticus could form biofilm in wells containing 50% mucus, but this failed when mucus was absent (Fig. 5). The difference was very significant (p b 0.001). This result was in accordance with the attachment assay. Meanwhile, mucus also greatly stimulated planktonic cell growth (p b 0.01).
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same to that on the control treatment at 48 h (Fig. 6B, C; p N 0.05). However, by considering the CFU on 2216E plates, which indicated the most cultivable bacteria, mucus supported 5 times more CFU than did clean substrates even at 48 h (Fig. 6A). 3.5. Analysis of the bacterial community in mucus trails by DGGE The bacterial community in mucus trails showed a different profile to that on a clean substrate (Fig. 7). At day 2, there were about 6 specific bands in the mucus trail (band 1 also existed in the clean substrate, but at much lower abundance). But at day 5, the difference became less obvious. Bands 1, 4, and 5 were also found in the water column, which meant that these species of bacteria could be attracted from seawater and grow in mucus trails but not (or weakly) on clean slides. Band 4, Klebsiella pneumoniae (100% similarity, see Table 1) is a pathogenic bacterium, and it existed on mucus-covered slides but not the clean ones. Bands 2, 3 and 6 are more likely to be derived from the pedal surface of the small abalone, because they were not detected in the water column. 4. Discussion
Fig. 3. Direct observation of attachment of V. alginolyticus to mucus trails by fluorescent stain. A: Mucus trail stained by calcofluor white. This dye was a specific stain for the βlink polysaccharides. It showed that the pedal mucus is fractionally filamentous at microcosmic level; B: Attached bacteria prestained with SYTO 9; C: Composite of A and B. This photo shows that the increase of bacterial attachment occurred on the filaments that could be stained by calcofluor white.
3.4. CFU counting in mucus trail Whether on 2216E or TCBS plates, the numbers of CFU on mucuscovered slides were always greater than those on the clean ones during the entire experimental period (Fig. 6). For both 2216E and TCBS plates, CFUs in mucus trails were at a maximum at 12 h, which were generally 5–8 times higher than those on clean substrates, then they decreased with culture time. We speculated that this was due to the mucus were fast consumed by the bacteria on it. For Vibrio spp. counting on TCBS plates, either the CFU of the yellow colonies (sucrose-fermenting species) or the green ones (species which cannot ferment sucrose) on the mucus-covering glass slides was almost the
Pedal mucus plays a key role in gastropod movement (Denny, 1980; Donovan and Carefoot, 1997). To be a better lubricant, mucus must have improved viscosity. Generally, it contains some glycoproteins (Hunt, 1970). Undoubtedly, such organic matter could be a carbon and nitrogen source for microorganisms, if it does not contain sufficient antimicrobial matter. In our study, the pedal mucus of small abalones could be the sole nutrient source for E. coli and S. epidermidis and promote bacterial growth. Furthermore, the CFU count on mucus trails of the small abalone was about 5-8 times more than that on clean substrate. The pedal mucus of intertidal limpets contains about 2% protein and 1% carbohydrate by wet weight (Connor, 1986). According to our previous study, although the protein and carbohydrate concentrations of pedal mucus in H. diversicolor are about one order of magnitude less than that of limpets (about 0.14% for protein and 0.08% carbohydrate, unpublished data), it is still much higher than NSW. Recently, researchers found that the mouse gastrointestinal mucus act as a nutrient source to pathogenic E. coli O157:H7 (Fabich et al., 2008). Among the aquatic animal, the surface mucus of salmon promotes the growth of a pathogen (Flavobacterium columnare) and its protease activity (Staroscik and Nelson, 2008). It seems that some specific components in the salmon mucus induced this result, because even in a rich medium, the growth rate of the bacterium is significantly higher when mucus is added. On the other hand, Salmon intestinal mucus could also support the growth of its pathogen, Vibrio anguillarum (Garcia et al., 1997). In addition, researchers have shown that adding coral mucus to the medium can result in the culture of more bacterial species (Koren and Rosenberg, 2006). Therefore, it could be a common phenomenon whereby the mucus might provide a highly active micro-environment for microorganisms. In abalones, it is well known that the pedal mucus of adults induces larval settlement and is utilized by postlarvae as transitional food (Seki and Kan-no, 1981; Slattery, 1992; Takami et al., 1997). In the present study, more cells of V. alginolyticus attached on the pedal mucus of H. diversicolor than on the clean slides. It should be noted that V. alginolyticus is a prevalent pathogen in many marine organisms. Several researchers showed it cause disease or death in H. diversicolor supertexta (Liu et al., 2001; Wang et al., 2005). Increased attachment only occurred in the filamentous locations, while no increase was seen in the blank areas. In our opinion, mucus, as a polymer layer, changed some characteristics of the substrate. Polymers on bacterial cell surfaces facilitate their attachment by overcoming the energy barrier between cell surface and substrate (Morisaki et al., 1999). The results suggested that this principle may also work when the substrate was coated with the pedal mucus
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Fig. 4. Biofilm formation on the mucus trails in flow-cell culture. After 48 h culture, biofilm formed on glass coverslips and was visualized by fluorescence microscopy after being dyed with the dead/live stain kit. The upper two images show the biofilm structure forming on clean slides (A: 100×; B: 400×). The bottom images show the biofilm forming on the mucus trail (C: 100×; D: 400×). The arrows illustrate that the bacterial cell clusters were much larger and denser on the mucus trail than on the clean glass surface.
polymers of the abalone. The mucus of vertebrates facilitates bacterial attachment is well known (Ofek et al., 2003). Bacteria inhabit the mucus covering animal tissues because of its viscosity and binding activities. Some bacteria are able to degrade mucus using specific glycosidases and then utilize them as nutrients (Hoskins et al., 1985). To our knowledge, facilitation of bacterial invasion by pedal mucus may due to its viscous character. Swimming motility in V. parahaemolyticus, which is driven by the rotation of a polar flagellum, is inhibited in highly viscous media, and so lateral flagella gene expression is induced (Kawagishi et al., 1996). The latter process is very important to bacterial invasion and particularly to one type of surface motility termed “swarming” (McCarter and Wright, 1993), which causes direct contact between pathogens and hosts. The pedal mucus of gastropods is innately viscous, and it could be an important physical factor explaining why Vibrio spp., or other bacteria, switch their life style from free living to colonization of a host or its mucus trails. This mechanism could also explain the results of our microplate assay. The pedal mucus of limpets can trap particles from the water column and enhance the growth of benthic diatoms as their food supply (Connor and Quinn, 1984; Davies et al., 1992). Calliostoma zizyphinum even obtains an additional food supply by wiping its mucus-coated shell twice a day (Holmes et al., 2001). Undoubtedly, in the natural environment, this is a classic and positive effect in community ecology in order to recycle the highly energetic mucus. Our results also showed that when trapping organic particles from the water column, some bacteria (including pathogens) could also settle on mucus-covered substrates, either actively or passively. This may not be an important phenomenon in the natural environment because of the low density of animals and the high water exchange, but in
mariculture conditions, a high density of abalone and a low water exchange rate would result in pathogenic microorganisms entering and growing quickly. Thus, regular cleaning of the surfaces of the cages containing abalone should be considered, as should an increase in the water exchange rate and a lowering of the culture density, in order to decrease the chance of pathogenic infection on the small abalone. The pedal mucus of marine gastropods persists on substrates for some time, even more than a month (Davies and Williams, 1992), during which time the bacteria could form biofilm on it by utilizing the
Fig. 5. Effect of pedal mucus on biofilm formation in V. alginolyticus. V. alginolyticus was inoculated to the wells contain 200 μL 50% mucus or AASW. After 36 h, the OD600 of planktonic cell and biofilm (stained by 0.1% crystal and washed by 200 μL 95% ethanol) were measured. Four replications were done in this experiment.
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Fig. 7. DGGE profiling of bacterial communities in the water column, on clean slides and on the mucus trail after immersion in seawater for 2 and 5 d. M: marker DL2000; a: 2d water sample; b: biofilm sample on clean slides at 2d; c: biofilm sample on mucus trail at 2d; d: 5d water sample; e: biofilm sample on clean slides at 5d; f: biofilm sample on mucus trail at 5d. The marked numbers show the specific bands in mucus trail samples. They were cut, amplified, cloned and sequenced.
Fig. 6. Change in CFU on mucus trail and clean glass slides. A: 2216E plates; B: Yellow colonies on TCBS plates; C: Green colonies on TCBS plates. The glass slides with mucus trail or not were put into a tank containing 20 L natural seawater from abalone culture pond. They were triple sampled with time lapse. The bacteria on the slides were scrapped off and counting CFU by serial dilution and spread on the plates.
nutrients in it. In our study, after 2 days of culture, a heterogeneous biofilm formed on the mucus-covered surface but a homogenous biofilm formed on the clean slides. This phenomenon may have resulted from differences in nutrient supply or in the initial colonizer. Biofilms, especially those pathogenic bacterial biofilms with highly heterogeneous structure, are notorious for their ability to resist drugs and cause chronic infections (Costerton et al., 1999). It is very hard to eradicate them using routine chemicals or antibiotics. In Chinese abalone farming, antibiotics are used after disease symptoms appear, but often with little effect. This could be a result of antibiotic over-use during culture. However, we also speculate that this is related to the living mode of the pathogens. Antibiotics, for example penicillin, could repress the pathogenic bacteria in the water column significantly, but its effect on bacteria in the biofilm (forming on the abalone cage surface or pedal surface) is difficult to evaluate. These bacteria in the biofilm are, of course, closest to the abalones, and such dense and
compact accumulation of bacteria could more easily break into the host's tissues and cause disease. As the DGGE profile shows, at least one potential pathogen, Klebsiella pneumoniae, was found in the mucus trail of the small abalone, but was not detected on the clean substrate. This bacterium is a common pathogen in Southeast Asian aquaculture facilities (Huys et al., 2007). A recent study shows that another close species, Klebsiella oxytoca, is virulent to small abalone postlarvae (Cai and Wang, 2008). On the other hand, pedal mucus may enrich some bacteria that could utilize mucus and grow more easily than the ones that cannot consume it. Observations of Haliotis spp. showed that their pedal surface is slightly tinted and relatively clean in most species. But the small abalone, H. diversicolor, in south China is an exception. Its foot was dark brown and often fouled. The fouling condition was different even between the two small abalone populations from distinct geographic
Table 1 Phylogenetic identification of selected bands from the DGGE bacterial profiles of the biofilm. Band⁎
Closest relative and database accession number⁎⁎
Sequence similarity
Alignment
1 2 3 4 5 6
Uncultured bacterium (AY160846) Tamlana agarivorans (EU221275) Uncultured gamma proteobacterium (AY792268) Klebsiella pneumoniae (EU419756) Uncultured Pseudoalteromonas sp.(EU167413) Uncultured bacterium (EF379757)
89% 94% 100% 100% 99% 93%
172/192 180/190 192/192 194/194 165/166 161/172
⁎For the band number see Fig. 7. ⁎⁎Sequences were aligned to the closest relative using BLAST. If the same similarity sequences are listed, the names of identified species are presented.
F. Guo et al. / Aquaculture 293 (2009) 35–41
Fig. 8. Different foot color and fouling condition between H. diversicolor from south China (upper) and from Japan (bottom).
areas, south China and Japan (Fig. 8). The essential reason for this phenomenon is unclear and needs further study. Interestingly, the fouling of the abalone foot seems to be related to their mortality, because the mortality rate of the south China population (N70%) is much more than that of the Japanese population (b30%) after 8 months in the same culture cage. We speculate that the different composition of the mucus in the two populations of small abalone may induce such a result, and so, comparison of the composition of the pedal mucus in the two populations will be the next step in our work. Acknowledgements This research was supported by grants from the National Natural Science Foundation of China (Project No. 40676081) and the Hi-Tech Research and Development (863) Program of China (Project No. 2006AA10A407). Professors John Hodgkiss is thanked for his assistance with English. References Altschul, S.F., Madden, T.L., Schaeffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402. Ausubel, F.M., Kingston, R.E., Seidman, J.G., Struhl, K., Brent, R., Moore, D.D., Smith, J.A., 1999. Short protocols in molecular biology, 4th ed. John, Wiley & Sons, Inc. Cai, J.P., Wang, Z.X., 2008. Characterization and identification of virulent Klebsiella oxytoca isolated from abalone (Haliotis diversicolor supertexta) postlarvae with mass mortality in Fujian, China. J. Invertebr. Pathol. 97, 70–75. Cai, J., Li, J., Thompson, K.D., Li, C., Han, H., 2007. Isolation and characterization of pathogenic Vibrio parahaemolyticus from diseased post-larvae of abalone Haliotis diversicolor supertexta. J. Basic Microbiol. 47 (1), 84–86. Connor, V.M., 1986. The use of mucous trails by intertidal limpets to enhance food resources. Biol. Bull. 171, 548–564. Connor, V.M., Quinn, J.F., 1984. Stimulation of food species growth by limpet mucus. Science 225 (4664), 843–844. Costerton, J.W., Stewart, P.S., Greenberg, E.P., 1999. Bacterial biofilms: a common cause of persistent infections. Science 284 (5418), 1318–1322. Davies, M.S., Hawkins, J.S., 1998. Mucus in marine molluscs. Adv. Mar. Biol. 34, 1–71. Davies, M.S., Williams, G.A., 1992. Pedal mucus of a tropical limpet, Cellana grata (Gould): energetics, production and fate. J. Exp. Mar. Biol. Ecol. 186 (1), 77–87. Davies, M.S., Jones, H.D., Hawkins, S.J., 1990. Seasonal variation in the composition of pedal mucus from Patella vulgata (L.). J. Exp. Mar. Biol. Ecol. 144, 101–112. Davies, M.S., Hawkins, S.J., Jones, H.D., 1992. Pedal mucus and its influence on the microbial food supply of two intertidal gastropods, Patella vulgata L. and Littorina littorea (L.). J. Exp. Mar. Biol. Ecol. 161, 57–77. Denny, M., 1980. Locomotion: the cost of gastropod crawling. Science 208 (4449), 1288–1290. Donovan, D., Carefoot, T., 1997. Locomotion in the abalone Haliotis kamtschatkana: pedal morphology and cost of transport. J. Exp. Biol. 200, 1145–1153.
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