Microbes Environ. Vol. 20, No. 1, 69–80, 2005 http://wwwsoc.nii.ac.jp/jsme2/ Characteristics of the Mucus Layer on the Surface of the Bluegill (Lepomis macrochirus) and the Bacterial Flora in the Mucus TAKEAKI HASHIZUME1, CHIKAKO TAKAI1, MANAMI NAITO1 and HISAO MORISAKI1* 1 Department of Bioscience and Biotechnology, Faculty of Science and Engineering, Ritsumeikan University, 1–1–1 Nojihigashi, Kusatsu, Shiga 525–8577, Japan (Received October 9, 2004—Accepted December 24, 2004) The layer of mucus on the surface of bluegills (Lepomis macrochirus) captured in Lake Biwa was character- ized as 1) large enough to host microbes (ca. 76 m thick), 2) a physically different environment from the sur- rounding lake water in viscosity and buffering capacity, and 3) chemically rich in organic substances, which may be utilized as nutrients. Based on DAPI staining and on the number of colonies formed respectively, it was found that ca. 103 times and 3 to 7 times the number of microbial cells were present in the mucus layer, as compared with the lake water. The bacterial flora of the mucus was greatly different from that of the lake water, according to a phylogenetic analysis. About 60% of the isolates from the mucus were Gram-positive. These Gram-positive isolates could be divided into two major groups. Each group consisted of strains sampled in one season, i.e., the strains sampled in July were closely related to the genus Staphylococcus, while the strains sampled in November were close to the genus Mycobacterium. In contrast, most isolates from the lake water were Gram-negative (72%); with all the strains closely related to - and -Proteobacteria sampled in July. With the exception of one strain, the Gram-positive isolates from the lake water (6 strains) were all sampled in November. Almost all of the isolates from the mucus could metabolize glucose, whereas only half of the isolates from the lake water could do the same. Key words: mucus layer, bacterial flora, bluegill (Lepomis macrochirus), Lake Biwa, 16S rDNA sequence Microbes inhabiting fish were reported as early as 110 The purpose of the present study was 1) to characterize years ago (the first was a Vibrio isolated from a diseased the mucus layer of fish, as a habitat for microbes, and 2) to fish5)). Since then, many kinds of microbes causing diseases reveal the characteristics of the bacterial flora in the layer in in fish have been investigated3,7,14,15,23). These, as well as relation to the properties of the mucus, in comparison with non-pathogenic microbes, seem to inhabit fish and interact the relationship between the microbes in the adjacent water with the surrounding environment. The surface of the fish is and the properties of that water. The bluegill Lepomis mac- the outermost boundary of that organism, and interacts di- rochirus was selected, because 1) bluegills, which have rectly with the outside environment. Fish are covered by a been causing a serious ecological problem by expelling in- layer of mucus, which has different characteristics from the digenous species, now account for 80–90% of the fish popu- environment outside, water; it is a place where various sub- lation in Lake Biwa19), which means that we could readily stances or possibly microbes are exchanged with the water obtain experimental animals for our study; and 2) the spread outside. However, few attempts have been made to charac- of this species over various areas of Japan makes it possible terize the microbes in mucus in connection with the proper- to investigate the environmental factors affecting the micro- ties of the mucus. bial flora on fish in further studies. In the present paper, we report the physicochemical fea- * Corresponding author; E-mail: [email protected], Tel: tures and seasonal changes of the mucus layer of the blue- 81–77–561–2767, Fax: 81–77–561–2659 gill. We also report that the bacterial flora in the mucus dif- 70 HASHIZUME et al. fer in character from those in the surrounding water, and NaCl (pH7.2). The DNB was a 100-fold dilution of the NB undergo seasonal changes. medium. NB plates and DNB plates were prepared for each sample in triplicate and incubated aerobically at 20LC for 30 Materials and Methods days, and the number of colonies appearing every day was counted. The colonies were distinguished each day by pen Sampling markings of different colors and shapes made on the back of The fish (bluegills, or Lepomis macrochirus) used in this the Petri dishes. After the incubation period, strains were study were extracted from their environment without being randomly isolated from the colonies. touched by bare hands and without disturbance of the ground at the site (34L58'30'' N, 135L54'30'' E), where the DNA extraction river Seta flows out from Lake Biwa (the largest lake in Ja- Each isolate was cultured in 10 mL of NB medium, with pan). The fish were placed in a box containing lake water shaking at 100 rpm and at 27LC, for 1 to 2 days. The cell and brought back to the laboratory within several hours. A suspension at the exponential growth-phase was centrifuged 300-mL sample of water was also obtained. The fish were (4LC, 10 min, 12000Pg). Then, the cell pellet was re-sus- kept in a state of syncope by cooling to 4LC, and then cov- pended in distilled water. The cell suspension was frozen ered with a perforated polyethylene rubber sheet (for large with liquid nitrogen and thawed three times. After thawing, fish, the perforated area was rectangular, 5 cmP3 cm, and 10 L of proteinase K (10 mg/mL) and 50 L of BL for small fish, 5 cmP2 cm). Surface mucus from each fish buffer12) (containing 40 mM of Tris aminomethane, 1 mM was gathered through the perforated area, as shown in Fig. of EDTA·2Na, 1% of Tween 20 and 0.5% of nonidetP-40) 1, with a sterilized rubber scraper. were added to 50 L of the suspension. After incubating at 60LC for 30 min, the supernatant (4LC, 10 min, 12000Pg) Total number of particles was used for amplification by PCR. The total number of particles showing bright blue fluo- rescence after DAPI (4',6-diamidino-2-phenylindole; 1 g/ PCR amplification and sequencing of the 16S rRNA gene mL final concentration) staining of each sample was count- The PCR mixture consisted of 0.75 units of Takara Ex- ed under an Olympus model BX50 BX-FLA epifluores- Taq, 1x Taq polymerase buffer, 200 M of dNTPs, 50 pmol cence microscope. of each primer and the extracted DNA (50 ng to 100 ng), all in a 20- L reaction mixture. The PCR primers used for am- Isolation of microbes plifying the 16S rDNA of the isolated bacteria were 25F17) The mucus samples used for the isolation of microbes (5'-AGTTTGATCCTGGCTC-3') as the forward primer and were collected on July 5 and November 7, 2002. The mucus 1510R20) (5'-GGCTACCTTGTTACGA-3') as the reverse was diluted 103-fold with sterilized water. NB or DNB agar primer, corresponding to positions 10–26 and 1495–1510, (1.5 wt%) medium (ca. 20 mL) was poured onto the diluted respectively, in the 16S rRNA gene sequence of Escheri- mucus samples (1 mL) and mixed. The NB medium con- chia coli. tained (per liter) 10 g of polypepton from Nihon Seiyaku, 10 The thermal cycling program used was as follows: 5 min g of bonito extract from Wako Pure Chemicals and 5 g of initial denaturation at 95LC, then 30 cycles at 95LC for 1 min, 52LC for 2 min, and 72LC for 2 min, and a final exten- sion for 10 min at 72LC. The PCR products were analyzed by electrophoresis on 1% Takara Agarose LO3 gels in 1PTAE. A 200-bp Bexel DNA Marker was used as the mo- lecular weight standard, and stained with ethidium bromide (1 g/mL with 1PTAE buffer) for visualization. The PCR products were purified with a Viogene PCR-M Clean-Up System in accordance with the manufacturer’s instructions. The sequences were determined with the primer 907R24) (5'-CCGTCAATTCCTTTGAGTTT-3') and an ABI PRISM AVANT 3100 genetic analyzer (PE Biosystems, Foster Fig. 1. The surface area (a rectangle, 3 cm×5 cm) from which the City, USA). The BigDye Terminator Cycle Sequencing mucus was obtained. Ready Reaction Kit, ver. 3.1 (PE Biosystems, Foster City, Bacteria in the Mucus Layer of Bluegills 71 USA), was used in accordance with the manufacturer’s di- fish on October 18. Each sample of mucus collected was rections. centrifuged (4LC, 14000Pg, 30 min) to remove black sub- stances. The mucus was diluted 10, 50 and 100 fold with Phylogenetic analysis distilled water. The viscosity of each diluted mucus solution Approximately 500 bp were used for the phylogenetic was determined three times with a Sibata No. 2 Ostwald vis- analysis. Unaligned sequences were submitted to the Ad- cometer at 25LC, and the results were averaged. The viscos- vanced BLAST search program at the website of the Na- ity of each mucus solution relative to that of distilled water tional Center of Biotechnology Information (NCBI), in or- was plotted against the logarithmic value of the dilution rate der to find closely related sequences. The sequences were of the mucus (e.g., log 50 for a 50-fold dilution). Then the aligned with the CLUSTAL X program (version 1.83)32). viscosity of non-diluted mucus was deduced from the linear Nucleotide positions containing ambiguous alignments and relation (confirmed beforehand in another experiment) be- gaps were omitted from the subsequent phylogenetic analy- tween the relative viscosity and the dilution rate.