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coral tissue, while vertical migrations within the band were documented by ... tissue is lysed and degraded, leaving the calcium carbonate coral skeleton ...
Microb Ecol (1996) 32:323-335

MICROBIAL ECOLOGY © YorkInc. 1996 Springer-Verlag New

Horizontal and Vertical Migration Patterns of Phormidium corallyticum and Beggiatoa spp. Associated with Black-Band Disease of Corals L.L. Richardson Department of Biological Sciences and Drinking Water Research Center, Florida International University, Miami, Florida 33199, USA Received: 17 October 1995; Accepted: 15 January 1995

Abstract. An in situ field study of the motility patterns exhibited by Phormidium corallyticum and Beggiatoa spp. in black-band disease of corals was conducted over a 5-day period. Measurements were made at a spatial resolution of 50 ~m to document the horizontal migration of black-band across living coral tissue, while vertical migrations within the band were documented by observation and macrophotography of the black-band surface. It was determined that horizontal migration occurred both day and night, with the fastest movements by the front of the band during the day and the back of the band at night. Beggiatoa would rise to the band surface at night, and would often remain above the cyanobacterial population during extended periods of illumination the following day. The migration patterns are discussed in terms of motility cues and microbial physiology.

Introduction Motile benthic microorganisms have been investigated in many aquatic environments, including sediments and microbial mats associated with freshwater, estuarine, marine, hypersaline, and hot-spring outflow ecosystems [8]. Such studies have generally focused on three aspects: discernment of motility patterns, often on a diel basis [15, 18]; determination of the environmental cue to which the microorganisms are responding and that control the motility patterns [5, 27, 31]; and the relationship between motility and microbial physiology, which may include the specific sensing of environmental cues and the physiological benefit to the microorganism from motility [7, 18, 31]. Two of the most well-studied groups of motile microorganisms are the cyanobacteria [6], and members of the sulfide-oxidizing genus Beggiatoa [24]. Migrating populations of cyanobacteria and Beggiatoa spp. commonly occur together in benthic environments that are sulfide-rich and illuminated, and there have been many ecological studies documenting motility patterns of these microbes in both microbial mats and sediments [5, 7, 15, 19, 23, 25-27, 31, 37].

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Fig. 1. Black-band disease on a colony of Diploria strigosa (D. strigosa #2 of Table 1, photographed at the end of the study). The white areas are exposed coral skeleton where the coral tissue has been destroyed during the progression of the disease. The two small circles off of the largest band are areas where new black band infections have coalesced with the older infection. Pairs of nails, inserted at t = 0 (100 h previously) at the edge of the band, were used to track migration of the band at specific sites. Nail diameter = 9 ram.

A unique pattern of migrating cyanobacteria and Beggiatoa is present in a microbial consortium known as black-band disease of corals. Black-band disease, first reported in 1973 [1], is a phenomenon found on coral reefs of the Caribbean [12, 35], the Florida Keys [21], the Indo-Pacific [2] and the Red Sea [3]. The disease consists of a microbial community that forms an obvious band, 1 millimeter to several centimeters wide, between bare coral skeleton and living coral tissue, as shown in Fig. 1. The thickness of the band is normally about 1 ram. The main characteristic of black-band disease is its destructive migration across coral colonies, which can occur at rates >1 cm day -l. As the band migrates, live coral tissue is lysed and degraded, leaving the calcium carbonate coral skeleton exposed. Black-band disease can cause the death of entire coral colonies due to the fast rate of tissue destruction as the band migrates vs. the much slower growth rate of the scleractinian corals that are most susceptible. Such corals grown on the order of 1 cm in height and 2 cm in diameter per year [17] and cannot outgrow the migrating band. As a result, black-band disease is considered to be a contributing factor to observed global coral reef degradation [29, 39]. Black-band disease consists of a well characterized microbial population dominated in terms of biomass by the gliding, phycoerythrin-rich, filamentous cyanobacterium Phormidium corallyticum [34] (originally described as Oscillatoria submembranaceae [1]), numerous heterotrophic bacteria [16], marine fungi [30], and both sulfide-oxidizing and sulfate-reducing bacteria [11]. The horizontal migration of this microbial consortium across living animal tissue is apparently unique. Yet, there have been no detailed studies of black-band microbial migration patterns with the exception of documentation of the distance moved by the entire consortium per day [1, 35]. Only one study was conducted that examined night movement [3], in which it was concluded that there was no migration during darkness. While it has been reported that Beggiatoa is commonly present in the black-band community [ 11, 28] such reports are limited to microscopic examination of samples collected from black-band; there are no reported studies of motility patterns of Beggiatoa associated with the intact black-band community. Also unknown is the environmental cue that causes the consortium to migrate across coral, although it has been reported that filaments of P. corallyticum will migrate toward samples

Motility Patterns of Phormidium and Beggiatoa

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Table 1. Description of corals studied, and numbers of measurements recorded for specific study sites on each band. Measurements consisted of distance moved from a stationary nail embedded in (dead) coral skeleton Size (m) Coral species

Montastraea cavernosa Colpophyllia natans (#1) Colpophyllia natans (#2) Diploria strigosa (#1) Diploria strigosa (#2) Diploria strigosa (#3)

Colony height

Colony diameter

No. of sites on banda

No. of sampling periodsb

1.0 1.5 3.0 2.0 0.8 1.0

0.6 1.7 4.0 1.3 0.5 0.5

5 3 3 4 5 6

11 5c 11 11 11 11

aAt each site, both the front and the back of the band were measured, so the actual number of sites measured (52) was 2 x the number of sites (26) on the bands bTotal number of measurements (with 5 or 11 sample sets) was 536 COnly five sample sets were taken of this coral due to extreme difficulty in sampling due to the position of black band in relation to other corals in the area

of coral tissue [35], a response observed microscopically to occur on microscope slide preparations of coral tissue and P. corallyticum filaments placed together under coverslips. In contrast, much is known about environmental motility cues that control motility of both cyanobacteria [6] and Beggiatoa [24] in other aquatic systems. Such cues include variations in light, oxygen, sulfide, and both organic and inorganic chemicals. A study was conducted to determine the motility patterns of Phormidium corallyticum and Beggiatoa spp. within in situ black-band in terms of previously reported tactic responses. The research reported here is the first documentation and analysis of a detailed study of the horizontal and vertical motility patterns performed by Phormidium and Beggiatoa populations in association with in situ black-band disease. Materials and Methods

Research Site Field research was carried out at Algae Reef, approximately 7 km off shore from Key Largo, Florida in the Florida Keys National Marine Sanctuary. Algae Reef is a patch reef, with water depth ranging from 1 to 7 m. During the study (12-16 August 1991) water temperature was 30°C. All data gathering was conducted underwater using SCUBA. Six coral colonies with active black-band disease were studied, representing three genera of scleractinian, or stony, reef-building corals (Montastraea cavernosa, Colpophyllia natans, and Diploria strigosa). Colonies ranged in size from less than 1 m to 4 m in height, and were within 15 m of each other. Table 1 summarizes the corals in this study.

Motility Measurements Twenty-six specific sites on active black-bands on the 6 coral colonies were studied over a time period of 100 h from 12 August to 16 August 1991. A complete sampling (52 individual measurements of

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the relative positions of the fronts and backs of the bands) required approximately 1 h, and was always performed following the same sequence of sites. The sites were sampled 11 times for a total of 536 individual measurements, with the sampling period occurring at different times throughout the day. To sample horizontal migration of black-band, measurements of the distance moved at each specific site were made at both the front (leading edge) of the band and at the back of the band next to exposed coral skeleton. Measurements were made at a spatial resolution of 50 Ixm, using Manostat plastic vernier calipers. Specific sites on the band were marked with 9 mm diameter stainless steel nails. To ensure that the same site was measured at each time point, two nails were placed in a line perpendicular to the band. During a measurement, the calipers were lined up against both nails with the reference edge of the calipers placed immediately behind the nail farthest from the band. Nails were inserted only in dead areas of coral that had been recently killed by black-band (see Fig. 1). Patterns present in the horizontal migration of black-band were analyzed by statistics performed on the measured distances, whereas documentation of vertical migrations was limited to noting when Beggiatoa replaced Phormidium as the surface microorganism. No effort was made to section the < I-ram-thick band to determine vertical positioning of the populations within the band itself.

Chemotaxis Gradients of several test substances (10 mM solutions of glucose, fructose, NH4CI,Na-acetate, Na2PO4; 1 mM sulfide; and coral tissue and mucous) were set up in agar and inoculated with fresh black-band samples. Gradients were made using sterile solutions of test substance, which were either placed (5 Ixl drop) on the side of agar plates made with the marine cyanobacterial mineral medium ASNIII, or at the base of test tubes (1 ml solution) and overlain with 5 ml of 0.5 strength agar (again made with ASNIII). Small clumps of freshly collected black-band were placed either in the center of the agar plates or on the surface of the gradient tubes, and observed over time for migration responses.

Light Light measurements were made using a Biospherical Instruments, Inc. (San Diego, California) diveroperated averaging quantum meter (Model QSI- 140), that measured photosynthetically active radiation (PAR) between 400 and 700 nm. The instrument was calibrated at Biospherical Instruments, Inc.

Results

Horizontal Migration Patterns A n example of the horizontal migration of b l a c k - b a n d disease at one of the 26 sites studied is shown in Fig. 2. This series of photographs of b l a c k - b a n d was taken in situ on Diploria strigosa (study coral D. strigosa #1 in Table 1) during 58 h of the 100-h study. The d o m i n a n c e of Phormidium corallyticum is evident b y the dark color (due to high concentrations of phycoerythrin) of the band. Total horizontal distance m o v e d by b l a c k - b a n d was 13.3 m m _+ 5.4 m m (range 2.3-23.0) for the fronts and 12.9 m m + 5.9 m m (range 2.15-28.4) for the backs during 100 h (n = 22). W h i l e overall m o v e m e n t b e t w e e n sites was highly variable, both on the same coral colony (data not shown) and b e t w e e n colonies, the differences in average distance m o v e d were not statistically significant (one-way ANOVA, P > 0.05). There was highly significant forward m o v e m e n t (toward living coral) during the day at the fronts of the bands (P < 0.0001, Table 2), and significant forward

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Fig. 2. The migration of black-band across coral (D. strigosa #1). These photographs were taken between August 13 (09:52) and August 15 (19:35), which represents less then 58 h of the 100-h study. Beggiatoa can be seen (white spots) on the band surface during early to late morning time periods. The last photograph (H) was taken 30 rain after dark, and shows swollen coral tissue (polyps are extended for nocturnal feeding). The bright white areas between the black band and the coral tissue are coral mesenterial filaments, which contain nematocysts and are a coral defense mechanism against other coral colonies competing for the same substrate. They do not, however, fend off black-band. Beggiatoa had not yet migrated to the band surface. (Time of photographs: A-B, 09:52, 14:58 (August 13); C-E; 10:33, 12:30, 16:00 (August 14); F - H : 09:10, 11:30, 19:35 (August 15). Nail diameter = 9 mm.

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Table 2. Horizontal movement of black band during the day (August 12) vs. night (August 1213). N = 25

Band site

Mean (ram)

%"

Standard deviation

Minimum

Maximum

P (t)b

Front, day Front, night Back, day Back, night

2.76 1.04 -0.5 2.22

73 27 0 100

2.56 2.87 1.57 3.84

- 1.95c -6.00 -4.45 -2.10

9.15 6.35 1.30 5.65

0.05 >0.05