Marine Pollution Bulletin 77 (2013) 210–219
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Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul
Ecology of the ciguatera causing dinoflagellates from the Northern Great Barrier Reef: Changes in community distribution and coastal eutrophication Mark P. Skinner a, Richard J. Lewis b, Steve Morton c,⇑ a b c
University of Queensland, Entox (National Research Centre for Environmental Toxicology), 39 Kessels Road, Coopers Plains, Queensland 4108, Australia University of Queensland, Institute of Molecular Bioscience, St. Lucia, Queensland 4072, Australia NOAA, Marine Biotoxins Program, Charleston, SC, USA
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Keywords: Ciguatera Dinoflagellates Gambierdiscus Prorocentrum Ostreopsis Nutrients
a b s t r a c t Ciguatera fish poisoning (CFP) is known to be caused by the ciguatoxins from the dinoflagellate genus Gambierdiscus, however, there is the potential for other toxins such as okadaic acid and dinophysistoxins from the genus Prorocentrum, and palytoxin from the genus Ostreopsis, to contaminate seafood. These genera may also be indicators of ecosystem health and potentially impact on coral reef ecosystems and the role they may play in the succession of coral to macroalgae dominated reefs has not been researched. Sixteen GBR field sites spanning inshore, mid-lagoon and outer lagoon (offshore) regions were studied. Samples were collected from September 2006 to December 2007 and abundance of benthic dinoflagellates on different host macroalgae and concentration of nutrients present in the water column were determined. The maximum abundance of Prorocentrum, Ostreopsis and Gambierdiscus found was 112, 793 and 50 cells per gram wet weight of host macroalgae, respectively. The average level of Dissolved Inorganic Nitrogen (DIN) in the water column across all sites (0.03 mg/L) was found to be more than double the threshold critical value (0.013 mg/L) for healthy coral reefs. Compared to a previous study 1984, there is evidence of a major shift in the distribution and abundance of these dinoflagellates. Inshore reefs have either of Prorocentrum (as at Green Island) or Ostreopsis (as at Magnetic Island) dominating the macroalgal surface niche which was once dominated by Gambierdiscus, whilst at offshore regions Gambierdiscus is still dominant. This succession may be linked to the ongoing eutrophication of the GBR lagoon and have consequences for the sources of toxins for ongoing cases of ciguatera. Published by Elsevier Ltd.
1. Introduction The past few decades have seen a significant increase in coastal eutrophication globally, leading to widespread hypoxia and anoxia, habitat degradation, alteration of food web structures, loss of biodiversity, and the increased frequency, spatial extent and duration of harmful algal blooms, (Howarth, 2008). In this context, changing land-use practices in North Queensland have resulted in the clearing of extensive tracks of land for agricultural production in the last 150 years, resulting in dramatic increases in inputs of sediments, nutrients and pesticides into the Great Barrier Reef (GBR) lagoon, and the widespread decline in lagoon water quality (Smith et al., 2005). Furnas et al. (2005) further suggest that terrestrial runoff of sediment and nutrients to the GBR lagoon has increased 2–4fold over the last century. In view of these inputs, the GBR lagoon should be considered a partially enclosed sea with the associated ⇑ Corresponding author. Tel.: +1 8437628857. E-mail address: [email protected] (S. Morton). 0025-326X/$ - see front matter Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.marpolbul.2013.10.003
potential to accumulate nutrients discharged from ever-increasing anthropogenic activities (Bell, 1992). The evidence that nutrient enrichment, increased siltation and excess turbidity can lead to the local degradation of coral reefs is unequivocal (Fabricius et al., 2005). Elevated nutrient levels and higher suspended sediment loads have been cited as the cause of reductions in coral growth and a shift in the relative abundance and composition of coral and algae; particularly in coastal areas adjacent to catchments with intensive agricultural activities (Alongi and McKinnon, 2005). On some near shore reefs on the GBR, high nutrient availability, in conjunction with substrate availability (low coral cover) and insufficient grazing pressure, has also lead to altered benthic communities with high macroalgal cover (Schaffelke et al., 2005). Similarly, increased nutrient levels (and/or reduced herbivory) can also lead to more substrate for toxigenic dinoflagellates, exacerbating their impacts (Parsons and Preskitt, 2007). As noted by Parsons et al. (2010) nutrient enrichment and warming sea surface temperatures can stimulate Gambierdiscus growth and result in higher cell densities. By corollary, elevated sea surface
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temperatures (SSTs) associated with global warming may further exacerbate the growing extent and range of ciguatera distribution being driven by eutrophication and other anthropogenic influences. Ciguatera field studies have largely concentrated on dinoflagellates of the genus Gambierdiscus, well known to be the producer of ciguatoxin precursors, but have largely ignored the potential role of toxins from the genera Prorocentrum and Ostreopsis as causative agents in ciguatera fish poisoning (CFP). Little is known about the extent to which ciguatera related toxins (from benthic dinoflagellates) may be affecting trophic levels, or their potential to alter coral reef ecosystem function. This highlights the need to monitor changes in water quality and various indicators of ecosystem health to help determine if management plans on land are having a noticeable impact on the ecosystems and water quality of the GBR (Udy et al., 2005). Such monitoring might be augmented with assessments of the abundance of potentially toxic benthic HABs that have the potential to provide a useful bio-indicator of coral reef ecosystem health. Toxic benthic dinoflagellates, like their macroalgal hosts, are at the bottom of the food chain. Whether the succession to algal dominated reefs, either initiated due to natural disturbances (cyclonic weather conditions, predation, etc.) or from anthropogenic causes (global warming, eutrophication, sedimentation) or a combination of such influences, the loss of coral cover results in more macroalgal surface for epiphytic benthic dinoflagellates. For example, phase shifts that involve coral bleaching water temperatures provide macroalgal habitat for Gambierdiscus at temperatures highly favorable for their growth (Tester et al., 2010). In fact, coral reef succession to algal dominance might even be reinforced by the effects of benthic dinoflagellate toxins on herbivore fecundity or feeding. Such effects have been little studied but may contribute to increased incidence of ciguatera as recently shown for Pacific Island nations (Skinner et al., 2011). There has been only one previous published study of these three genera on the Northern Great Barrier Reef (Gillespie et al., 1985). The objective of this study is to determine the distribution and abundance of the ciguatera and other potential causative dinoflagellates across inshore island fringing reefs, middle GBR lagoon reefs and outer continental shelf reefs. We then correlated nutrient levels (ammonia, nitrites or nitrates) and benthic dinoflagellate abundance in relation to macroalgae sampled. Finally, a comparison to a previous study (Gillespie et al., 1985) of benthic dinoflagellates from similar field sites was undertaken.
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at a distance of 100 m from shore (or pontoon at reef sites), in a water depth of 1–2 m at all field sites. 2.2. Description of field sites The sites covered a three main reef types, inshore GBR coral reefs, fringing continental islands: Magnetic Island at over 5000 ha of land surface (more than half of which is national park) is inhabited with approx. 2000 people and is the only permanently inhabited site and is located close to Townsville, Snapper Island at 56 ha is 4 km east of the Daintree river, Double Island similar to Snapper and offshore from Palm Cove, Fitzroy Island at 339 ha (mostly national park), King reef that is only 200 m off the mainland coast at Kurrimine, Cape Richards on Hinchenbrook Island and, Dunk Island at 970 ha is also mostly national park; Mid-lagoon platform coral cays and reefs: Green Island at 15 ha land surface and with 710 ha of reef that is mostly sea grass, Lizard Island at 1000 ha is 240 km north of Cairns, Michelmas Cay is only 2 ha and 30 km NE of Cairns, Low Isles has 22 ha of reef found 15 km NE of Port Douglas, Upolu Cay is 28 km NE of Cairns, and Normanby Island of the Frankland group which is 10 km east of Russell river; and outer barrier coral reefs included: Norman Reef has 430 ha of reef located 70 km NE of Cairns, Moore Reef which is 2650 ha and 45 km SE from Cairns, Flynn Reef with 420 ha, found 50 km SE of Cairns and Thetford Reef at 200 ha, is 60 km SE of Cairns. Please note that the benthic HAB abundance is not presented in this paper for each site, as only those with multiple samples or representative abundances are shown. 2.3. Protocol of microalgal sampling
2. Methods
Many similar methods for epiphytic microalgal collection and enumeration have been used following the original sampling of Yasumoto et al. (1980). Divers collected a broad diversity of reef macroalgae harvested using plastic bags. Three or more of the most common species of macroalgae; in this study Sargussum flaricans, Padina australis, Halimedia opunita, Turbinaria ornata, Laurencia intricata, Dignea simplex, Dictyota bartayresi, Amphiroa foliacea, Hypnea saidana, Jania crassa and seagrass if present, were collected at a site. The macroalgae sample bags were vigorously shaken for 1 min to dislodge the epiphytic flora. The resulting dislodged epiphytes were sieved and the 38 lm wash back, transferred to a 50 ml vials and fixed with 10% Lugol’s solution. All fixed samples were stored at 12 °C in the dark until dinoflagellate abundance could be enumerated. The weight of each macrophyte sample was blotted dry and weighted for comparison.
2.1. Sampling regime
2.4. Abundance and identification of ciguatera dinoflagellates
Sixteen Great Barrier Reef field sites were sampled: Inshore, mid-lagoon and outer lagoon (offshore) from Lizard, Snapper and Low Islands (offshore of Port Douglas) in the north, Double and Green Islands, Upolu and Michelmas Cays, Fitzroy and Normanby Island (Frankland Islands), centrally located (near to Cairns) to King reef, Dunk and Magnetic (next to Townsville) Islands in the south (see Fig. 1; Table 1). Sampling occurred at ten sites being sampled three or more times, between September 2006 and December 2007. Green Island (two sites on either side of the island, included 9 monthly collections) and Magnetic Island (Nelly, Geoffrey and Arthur Bays sampled at six, monthly collections) where the most intensively sampled of all the field sites. At all other multiply sampled sites at least 3 samplings occurred, including 3 separate sites at Dunk Island and two separate sites at Low, Normanby Islands and Upolu Cay, nearly all of which where 200 m apart, except at Upolu Cay which was 1 km apart. Sampling generally took place
Ciguatera dinoflagellates were identified to genus level by morphology using the light microscope (a CE, XSZ-107BN Series). For abundance a 0.5 ml portion of the shaken wash back sample was pippetted onto a Sedgewick Rafter counting slide and diluted to 1 ml and viewed at 64, for counting and 160, for identification. Abundance was calculated by taking the number of each genus present and multiplying this by the amount of dilution and the size of the sample and dividing by the wet weight of the sample macroalgae to arrive at the number of cells per gram wet weight of macroalgae. Photographs of different species were taken with a digital camera (Nikon) focused down the tube of the microscope for further identification. Some samples were returned to the Centre for Microscopy and Microanalysis (CMM), University of Queensland for SEM analysis. Fixed samples were desalted using a 10% step gradient from seawater to freshwater on polycarbonate filter paper and dehydrated by using a step gradient of ethanol
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Fig. 1. Map of the Great Barrier Reef, Queensland, showing the field site locations.
(50–100%), fixed to stubs and sputter coated with 1.5 nm of platinum and viewed with a JOEL JCM5000 SEM. 2.5. Water column nutrient sample analysis Soluble Nutrients: Water column samples were collected with a 60 ml syringe, about 30 cm above the lagoon floor, whilst snorkeling slowly for 10 m, after rinsing three times with the water from the site to be collected, filtered (using a 0.45 lm membrane Millipore sterile disposable filter unit) into a sterile falcon tube and stored frozen until analysis at the Nutrient Laboratory at the Queensland Health and Scientific Services, Brisbane, Australia; a National Association of Testing Authorities (NATA) accredited laboratory. Analyses for FRP, NO2, NO3, and NHþ 4 were performed simultaneously using an automated LACHAT 8000QC flow injection system using methodology based on (see APHA, 2005): (a) ascorbic acid reduction of phosphomolybdate for FRP, (b) cadmium reduction of nitrate to nitrite by diazotizing the nitrite with sulfanilamide and coupling with N-(1-naphthyl)ethylenediamine dihydrochloride for NOx, (c) production of the indophenol blue color complex for NHþ 4. 2.6. Statistical analysis Statistical correlations of selected GBR field sites of benthic dinoflagellate abundance from macroalgae sampled on each occa-
sion and water column nutrients (DIN and separately for ammonia and nitrates), undertaken using SPSS 19 (SPSS Inc., Chicago 2010) for significant correlation wither using the Pearson correlation or the Spearman correlation (n = 3–6).
3. Results 3.1. Dinoflagellate abundance, Green Island, east and west, mid lagoon field sites At the field site on the eastern side of Green Island (Fig. 2), the most abundant dinoflagellate was Prorocentrum (24 samples) Ostreopsis (6 samples) and Gambierdiscus was most abundant on three occasions (all of which occurred on the one sampling date in June). Ostreopsis did not occur on any samples in May or June. On only 2 samples did all three genera occur at the same time. On 12 samples Prorocentrum and Ostreopsis occurred together. On only three samples Ostreopsis and Gambierdiscus occurred together and on eight Prorocentrum and Gambierdiscus occurred together. On fourteen samples Prorocentrum appeared by itself and on no samples did Ostreopsis or Gambierdiscus appear in a sample by themselves. Prorocentrum was the most dominant of the three dinoflagellates at the Green Island East field site. From Scanning electron microscopy, Gambierdiscus australis was the dominate species found (Fig. 3a).
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M.P. Skinner et al. / Marine Pollution Bulletin 77 (2013) 210–219 Table 1 Summary of the Great Barrier Reef field sites. GBR location
Number of sampling sessions and site location Solitary sampling
Latitude/longitude (°/0 )
Multiple sampling
Latitude/longitude
Inshore of lagoon
Snapper Is. Double Is. Hinchenbrook Is.
16 17 43/145 29 36 16 44 00/145 41 00 18 11 40/146 13 28
Magnetic Is. Fitzroy Is. King reef Dunk Is.
19 16 17 17
09 55 47 56
15/146 30/145 00/146 00/146
51 59 08 08
50 25 00 30
Middle of lagoon
Lizard Is. Michelmas Cay
14 41 00/145 27 00 16 36 16/145 58 30
Green Is. Low Isle Upolu Cay Normanby Is.
16 16 16 17
45 23 40 12
30/145 00/145 30/145 15/146
58 33 56 04
10 30 00 35
Outer lagoon
Flynn reef
16 53 20/146 11 30
Norman reef Thetford reef.
16 25 40/145 59 30 16 48 30/146 11 00
In the field site on the western side of Green Island (Fig. 4), 17 samples Prorocentrum was the most abundant dinoflagellate whilst Ostreopsis was the most abundant on 11 occasions and Gambierdiscus was most abundant on five occasions, three of which occurred on the one sampling date (June). Ostreopsis did not occur on any samples in December. Only 3 samples showed all three genera occurring at the same time, on Dignea (October and September 2006) and Laurencia (July 2007). On 15 samples Prorocentrum and Ostreopsis occurred together. On only eight samples Ostreopsis and Gambierdiscus occurred together and on seven Prorocentrum and Gambierdiscus occurred together. On eight samples Prorocentrum appeared by itself and on only two samples did Ostreopsis appear by itself, Gambierdiscus never appearing in a sample as the sole dinoflagellate. At Green Island all samples of macroalgae sampled host the three genera of dinoflagellates. In all 11 samples of seagrass no Gambierdiscus was to found, only Prorocentrum and Ostreopsis. Prorocentrum was the most dominant dinoflagellate on all macroalgae, appearing in more samples than Ostreopsis, except on Turbinaria, and yet Gambierdiscus was still present amongst all genera of macroalgae. The presence of Sinophysis (Fig. 3b), similar in form to the pelagic Dinophysis, is a newly described benthic species, whose toxicity has not been studied and was found in the samples on Green Island and some other GBR sites. Not previously reported on the GBR, it may be invasive, its toxicity and its role in the ecosystem are unknown. 3.2. Dinoflagellate abundance, Geoffrey, Arthur and Nelly bays, Magnetic island, inshore GBR lagoon field sites In the Geoffrey Bay field site on Magnetic Island (Fig. 4), Prorocentrum was the most abundant dinoflagellate on only one sample, Ostreopsis was the most abundant on 17 occasions (all but two) and Gambierdiscus was most abundant on one occasion. Gambierdiscus did not occur on any samples in October. On six samples all three genera occurred at the same time. On 14 samples Prorocentrum and Ostreopsis occurred together. On seven samples Ostreopsis and Gambierdiscus occurred together, whenever Gambierdiscus was to be found and on six samples Prorocentrum and Gambierdiscus occurred together. On one only sample did Prorocentrum appear by itself and on four samples did Ostreopsis appear by itself, Gambierdiscus never appeared in a sample by itself. Ostreopsis was the most dominant of the three dinoflagellates. In the Arthur and Nelly Bay field sites on Magnetic Island (Fig. 5), Prorocentrum was the most abundant dinoflagellate on only two samples, whilst Ostreopsis was the most abundant on 25 occasions (all but four) and Gambierdiscus was most abundant on no occasion. On eight samples all three genera occurred at the same time (Gambierdiscus occurred only 9 out of 29 samples). On 14 samples Prorocentrum and Ostreopsis occurred together. On
one only sample did Prorocentrum appear by itself and on twelve samples Ostreopsis appeared by itself. Ostreopsis was the most dominant of the three dinoflagellates. All types of macroalgae on Magnetic Island host the three genera of dinoflagellates. Ostreopsis was the most dominant dinoflagellate on all macroalgae, appearing in more samples than Prorocentrum, except for Turbinaria, and yet Gambierdiscus was still present amongst all genera of macroalgae sampled. Out of a total of 49 samples Ostreopsis was found 46 times, Prorocentrum 31 times and Gambierdiscus 13 times. 3.3. Dinoflagellate abundance, other field site examples (multiple sampling) Upolu Cay and reef show signs of a macroalgal phase shift (Fig. 6): Gambierdiscus dominate on some macroalgae (Dignea), occur on 11 out of 25 samples; Ostreopsis dominating 9 out of 25 samples and occur on 20; Prorocentrum dominate on only 3 samples and found on 16 out of 25 samples; whilst Sinophysis became more dominant in the December sampling. At Normanby island, in December there was a bloom of Ostreopsis on the west reef whilst on the north reef, Gambierdiscus dominated the samples with Prorocentrum occurring on both reefs at this time. At Norman reef (Fig. 7) of 13 samples, Sinophysis was found on 9 samples, Gambierdiscus was found on 7 (always found on Dictyota), Ostreopsis on 5 (always found on Turbinaria) and Prorocentrum on only 4 samples. Across all field sites (Fig. 8), of all 70 inshore samples, Prorocentrum, Ostreopsis and Gambierdiscus genera were found in 48%, 57% and 20% of all samples, respectively. In the 78 mid lagoon samples Prorocentrum, Ostreopsis and Gambierdiscus were found in 55%, 56% and 40% of all samples respectively. In the 23 offshore samples Prorocentrum, Ostreopsis and Gambierdiscus were found in 35%, 39% and 57% of samples respectively. Padina, Amphiroa and Sargassum were often found in the inshore and mid-lagoon sites but not often in the offshore sites (see Fig. 9). The green algae Halimeda although found across all three sites was not such a good host for dinoflagellates compared to Padina, Amphiroa and Sargassum. Of 30 Halimeda samples across all sites, only 5 had Gambierdiscus present and four of these were from offshore sites. Turbinaria not often found in the inshore sites was a good host to all dinoflagellate genera in the offshore sites. Dignea, a fine haired alga, generally found in the mid lagoon sites was a good host to all three dinoflagellate genera. 3.4. Nutrient results Four of the five highest DIN readings occurred in the wet season whilst the four lowest readings occurred in the dry season, on the two Green Island sites (Table 3). On three of the seven occasions
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Green Island (East) Benthic HAB abundance Prorocentrum
Osteropsis
Gambierdiscus
Sinophysis
Abundance cells/g wet wt.
1000
100
10
Padina Amphiroa Turbinaria Laurencia Dignia Seagrass Padina Amphiroa Turbinaria Laurencia Dignia Seagrass Padina Amphiroa Turbinaria Laurencia Dignia Seagrass Amphiroa Turbinaria Laurencia Seagrass Amphiroa Laurencia Halimeda Seagrass LYNGBYA Amphiroa Laurencia Dignia Seagrass LYNGBYA Amphiroa Laurencia Dignia Padina Amphiroa Turbinaria Laurencia Seagrass Padina Amphiroa Turbinaria Laurencia Dignia Seagrass
1
Sep-06
Oct-06
Dec-06
Feb-07
May-07
Jun-07
Jul-07
Oct-07
Dec-07
Month of Sampling & Substrate Fig. 2. Abundance of benthic dinoflagellates, cells/g wet weight, on differing substrates of macroalgae and seagrass from the eastern field site of Green Island at various months between September, 2006 and December 2007.
when samples were taken on either side of the island, there were some large differences between DIN readings, from twice to five times as strong, the sites only 400 m apart. Of the 17 samples from Green Island, 10 (59%) were over the critical Nitrogen point threshold (0.014 mg/L), for maintaining a healthy coral reef (Bell, 1992; Lapointe, 1997). The average for both sites on Green Islands from the 17 samples was 0.029 mg/L, more than double the critical threshold. Three of the four highest DIN water column readings occurred in the wet season and three of the four lowest Nitrogen readings occurred in the dry season, across the three sites on Magnetic Island. Nelly Bay had on the average higher readings than the other two bays, all three bays having an average over the threshold (0.013 mg/L) for critical Nitrogen levels for a healthy coral reef. Of the 14 samples, 8 (57%) were over the critical level. Of the 19 nutrient samples taken from the inshore GBR lagoon sites, samples were over the critical Nitrogen threshold for healthy coral levels, on only four occasions were the sites under the threshold, twice at Fitzroy Island, once at Dunk Island and once at King reef, all of which were in the dry season. The highest readings occurred in March towards the end of the wet season, at Snapper, Double and Dunk Islands, all three of which are influenced by river plumes, from the Daintree, Barron and Tully rivers respectively. On only one occasion was there a phosphate reading (0.012 mg/L) of any significance, at Snapper Island in March, all other samples remained below the level of detection (0.003 mg/L) except one, at King reef (0.007 mg/L).
Of the 26 nutrient samples taken from the middle GBR lagoon sites samples were over the critical Nitrogen threshold for healthy coral levels, and on eleven occasions the sites were under the threshold, this occurred at seven different sites with no site recording below the threshold on more than two occasions, out of the three. The lowest readings occurred towards the end of the dry season. Four of the five highest readings occurred in the wet season. All Phosphorus readings stayed below the level of detection (0.003 mg/L). Sample sites further offshore than the mid GBR lagoon sites also have raised, on average, Nitrogen water column levels higher than the critical threshold level. Of the 53 samples from all sites, 35 (66%) have raised Nitrogen levels above the critical threshold level and 18 (34%) sites below. Sampling was undertaken without the influence of any cyclones, and generally under calmer conditions which may have biased the readings to be lower than expected due to the lack of re suspension from more turbulent waters.
4. Discussion The two intensively studied field sites of Green Island and the bays of Magnetic Island showed a major difference in the benthic dinoflagellate assemblage. Green Island fringing reef macroalgae and seagrass were dominated by Prorocentrum and Magnetic Island macroalgae were dominated by Ostreopsis. Often both these genera are found together and whilst Ostreopsis is seldom found singularly
Fig. 3. (a) and (b) Scanning electron micrograph of Gambierdiscus austalis (a) and Sinophysis sp. (b).
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Green Island (west) Benthic HAB abundance Prorocetrum
Ostreopsis
Gambierdiscus
Sinophysis
10
1
Sargassum Laurencia Dignia Halimeda Seagrass Padina Amphiroa Sargassum Laurencia Dignia Halimeda Seagrass Amphiroa Sargassum Dignia Halimeda Seagrass Amphiroa Laurencia Dignia Halimeda Seagrass Padina Amphiroa Sargassum Laurencia Dignia Halimeda Seagrass Padina Amphiroa Sargassum Laurencia Dignia Halimeda Seagrass
Abundance cells/g wet wt.
100
Feb-07
May-07
Jun-07
Jul-07
Oct-07
Dec-07
Month of Sampling and Substrate Fig. 4. Abundance of benthic dinoflagellates, cells per gram wet weight, on differing substrates of macroalgae and seagrass from the western field site of Green Island on February, May, June, July, October and December 2007.
Magnetic Island Geoffrey Bay Benthic HAB Abundance Prorocentrum
Osteropsis
Gambierdiscus
Abundance cells/g wet wt.
100
10
Jan-07
Mar-07
Aug-07
Turbinaria
Sargassum
Padina
Amphiroa
Dictyota
Sargassum
Padina
May-07
Amphiroa
Turbinaria
Amphiroa
Sargassum
Padina
Turbinaria
Sargassum
Padina
Amphiroa
Sargassum Feb-07
Turbinaria
Padina
Turbinaria
Padina
Sargassum
1
Oct-07
Month of Sampling and Substrate Fig. 5. Abundance of benthic dinoflagellates, cells/g wet weight of macroalgae, on differing substrates of macroalgae and seagrass from Geoffrey Bay, Magnetic Island in January, February, March, May, August and October 2007.
in samples. Harmful algae that thrive in eutrophic habitats are mixotrophs that respond both directly to nutrient inputs, and indirectly through high abundance of bacterial and algal prey that are stimulated by the elevated nutrients (Burkholder et al., 2008). Toxins from Prorocentrum have the potential to decimate populations of grazing communities (Ajuzie, 2007) in addition to the possibility of an allelopathic role, as exudates from Prorocentrum are also known to inhibit the growth of co-occurring species (Sugg and Vandolah, 2002), which could also keep in check possible blooms of predators. Available external nutrient supplies can have a major influence on microalgal allelopathic activity (Graneli et al., 2008). No blooms of Prorocentrum where found in any samples, the highest abundance recorded were only 112 cells per gram of macroalgae (Green Island eastern site). It is hypothesized therefore that the three genera could be keeping populations of each other in check, by allelopathy and species competition. However there appears to be many benthic invertebrates that could be predating on the dinoflagellates, which have been found in samples. The
predation of such invertebrates, let alone normal fish herbivory, feeding directly on both the macroalgae and the biodetritus off the macroalgal surface, would make it more complex to find correlations between dinoflagellate abundance and the existence of nutrients in the field. In assessing the eutrophication HAB relationship, the collective grazing behavior of the herbivorous copepods, filter feeding benthos and benthic larvae must be considered (Smayda, 2008). The results from this study show that where nutrient levels are sufficient, Prorocentrum and Ostreopsis can be dominant over Gambierdiscus populations (Table 2), as found in inshore and mid lagoon reefs as opposed to offshore reefs. In 1985 a similar GBR study showed only 5% of samples contained Prorocentrum, now this genera is found in seven (35%, offshore) to nine (55% mid lagoon) times as many samples. Ostreopsis has doubled its presence in samples, whilst Gambierdiscus has halved its overall presence; however, it still remains dominant in outer reef sites. In the 22 year gap between comparative studies there has been a major shift in
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Benthic Dinoflagellates at Nelly & Arthur Bays, Magnetic Island Osteropsis
Gambierdiscus
100
10
Arthur Bay
Mar-07
May-07
Nelly Bay
Arthur Bay
Aug-07
Turbinaria
Sargassum
Padina
Amphiroa
Dictyota
Sargussum
Padina
Amphiroa
Dictyota
Amphiroa
Sargassum
Dictyota
Arthur Bay
Laurencia
Amphiroa
Sargussum
Dictyota
Nelly Bay
Laurencia
Sargassum
Padina
Arthur Bay
Amphiroa
Dictyota
Sargussum
Padina
Nelly Bay
Amphiroa
Amphiroa
Sargassum
Turbinaria
Amphiroa
1 Sargussum
Cells/g wet wt. macroalgae
Prorocentrum
1000
Nelly Bay
Oct-07
Sampling month, site and macroalgal substrate Fig. 6. Abundance of benthic dinoflagellates, cells per gram wet weight, on differing substrates of macroalgae from Nelly and Arthur Bays, Magnetic Island in March, May, August and October 2007.
Upolu Cay & Reef HAB Abundance Prorocentrum
Osteropsis
Gambierdiscus
Sinophysis
Abundance cells/g wet wt.
1000
100
10
Upolu Cay
Upolu Reef
Feb-07
Upolu Reef
Upolu Cay
Jul-07
Upolu Reef
Upolu Cay
Oct-07
Sargassum
Turbinaria
Amphiroa
Padina
Dictyota
Dignea
Padina
Sargassum
Turbinaria
Amphiroa
Sargassum
Dignea
Padina
Sargassum
Padina
Amphiroa
Upolu Cay
Turbinaria
Dignea
Sargassum
Padina
Halimeda
Turbinaria
Dignea
Sargassum
Dictyota
1
Upolu Reef Dec-07
Sampling month, site & macroalgal substrate Fig. 7. Upolu Cay and reef, mid GBR lagoon site, sampled on four occasions.
Osteropsis
Gambierdiscus
Turbinaria
Amphiroa
Norman Reef Benthic HAB Abundance Prorocentrum
Sinophysis
Jul-07
Oct-07
Dec-07
Sampling month and Macroalgal substrate Fig. 8. Normanby Island sampled on three occasions on the west and north side reefs.
Rubble
Halimeda
Dictyota
Turbinaria
Padina
Rubble
Halimeda
Dictyota
Padina
Rubble
Halimeda
Amphiroa
1
Dictyota
10
Turbinaria
Abundance cellls/g wet wt.
100
217
M.P. Skinner et al. / Marine Pollution Bulletin 77 (2013) 210–219
Halimeda
Sargussum
Amphiroa
Dictyota
Turbinaria
Dignea
Laurencia
20 18 16 14 12 10 8 6 4 2
Inshore
Midshore
Total
Gambierdiscus
Ostreopsis
Prorocentrum.
Total
Gambierdiscus
Ostreopsis
Prorocentrum.
Total
Gambierdiscus
Ostreopsis
0 Prorocentrum.
Number of times dinoflagellate found in a type of macroalgae sample
Dinoflagellate genera, total times found on macroalgal types in GBR sites Padina
Offfshore
GBR site and dinoflagellate type with totals for macroalgae sampled Fig. 9. The number of times the benthic dinoflagellate genera found on the different macroalgal substrate samples across all the GBR field sites either inshore (n = 78), mid lagoon (n = 70) or offshore (n = 28).
the GBR dinoflagellate assemblage, with Prorocentrum apparently becoming more dominant over time. Changes in nutrient stoichiometry are known to have drastic changes not only in cellular composition but in toxin production by these benthic dinoflagellates. Toxin production in both Prorocentrum and Gambierdiscus has been shown to be inversely related to nutrient limitation (Aikman, 1992; Sperr and Doucette, 1996; Varkitzi et al., 2010; Vanucci et al., 2010). Imbalances in N:P ratios also promote increase in exopolysacharides (Vanucci et al., 2010). Exopolysacharides are utilized by Prorocentrum, Gambierdiscus, and Ostropsis to form large aggregates within mucilage. This combination of increased toxin production plus increase excopolysacharides was hypothesized by Glibert et al. (2012) to decrease grazers’ ability to consume these benthic dinoflagellates. The potential link between nutrients and dinoflagellates is an important one as suggested by a study undertaken on Hawaiian coral reefs (Parsons and Preskitt, 2007). In this study correlations with Nitrogen and Ostreopsis only occurred at one site, Nelly Bay, Magnetic Island (see Table 2) and with Gambierdiscus at two sites, Nelly Bay and Fitzroy Island, both correlations with the abundance of ammonia. Toxic strains of Gambierdiscus spp. are known to grow faster on ammonium rather than other Nitrogen sources and they are able to take advantage of different Nitrogen sources on short time scales to support growth (Lartigue et al., 2009). All sites having large standing masses of macroalgae (personnel observations) consistent with eutrophication. There would appear to be some significance to eutrophication of the GBR lagoon and the presence of the epiphytic dinoflagellate genus Prorocentrum as there were eight correlations found between this genus abundance and either ammonia and/or nitrates found at five different field sites (Table 2). Four of those sites showed a direct correlation with the Prorocentrum abundance and the water column DIN. A similar study of a smaller lagoon in Nusa Dua, Bali, Indonesia also found a significant correlation between the dinoflagellate genera Prorocentrum with the DIN in the water column (Skinner et al., 2012), and a benthic Prorocentrum gillespii bloom in the sediment of Muri lagoon, Rarotonga was also correlated with nutrients (Skinner et al., 2009), where critical levels for nutrient thresholds were also exceeded at both sites. The genus Prorocentrum is known to be involved in ciguatera (Rongo and Van Woesik, 2010), so nutrient thresholds set for coral reefs should be employed, as these levels once exceeded, can assist to create HABs.
This may provide evidence for a succession in the dinoflagellate community as an ecosystem becomes more degraded (from coral to macroalgal phase shift), with increasing nutrients, leading to the prevalence of toxins from Prorocentrum and/or Ostreopsis into the ecosystem, rather than Gambierdiscus that is more likely present in the initial phase shift as it is presence now is more likely found at less impacted reefs on the outer GBR. The high density of benthic dinoflagellates from the genus Prorocentrum collected from sediments coincided with a sharp increase in cases of CFP in Rarotonga (Cook Islands) and may have explained increased poisonings associated with soft bottom benthic invertivores (e.g. mullet and goatfish) as well as filter feeders (i.e. Tridacna), as an example where an algal community shift and the assemblage of toxic dinoflagellates was altered (Rongo and van Woesik, 2010). These changes occurred in a small, highly eutrophic lagoon with high water retention rates. The result of such succession could then appear to be a shift away from Gambierdiscus as the cause of CFP to potentially an Ostreopsis/Prorocentrum dominated benthic dinoflagellate community at the exclusion of Gambierdiscus. P. lima and other Prorocentrum spp. are known to cause diarrhetic shellfish poisoning caused by okadaic acid, dinophysistoxins, prorocentralides as well as a fast acting toxin (Faust and Gulledge, 2002) although levels of these and related toxins likely to affect human health have not been detected in fish flesh or are rarely researched. Palytoxin is one of the more toxic members of a family of ciguatera related toxins as it appears to have the same marine food chain etiology (Kodama et al., 1989). The genus Ostreopsis are known to produce Ostreocin, a palytoxin (Ukena et al., 2001). There was one bloom of Ostreopsis at Normanby Island, that can be considered an inshore GBR field site, as its proximity to a river delta is quite close (4 km), where there was an increase in abundance within the four macroalgal samples collected. This bloom was found at a site sampled on three occasions, and on the last sampling period in December 2007 its persistence was not recorded. This bloom occurred on the western side of the island, protected from the regular SE trade winds, and was localized, as at the same sampling of the northern site, 400 m away, showed no increases in normal abundances (less than 100 cells/g macroalgae). At a temperate reef in New Zealand, Ostreopsis blooms were most prevalent at sites protected from prevailing swells (Shears and Ross, 2009). It would be no surprise then that more blooms would be found to
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Table 2 Percentage of benthic dinoflagellate genera were observed among the different sites compared to previous study by Gillespie et al. (1985). Year
GBR locality
Prorocentrum (%)
Ostreopsis (%)
Gambierdiscus (%)
Total average nitrogen (mg/l) (n)
2007
Inshore (n = 70) Mid lagoon (n = 78) Offshore (n = 23)
48 55 35
57 56 39
20 40 57
0.030 (33) 0.016 (43) 0.015 (8)
2007
All sites Ave (n = 171)
46
51
39
0.020 (84)
1985
All sites Ave (n = 280)
5
26
79
na
Table 3 Dissolved Inorganic Nitrogen (NHþ 4 plus NOx), mg/L of water column, 1st column at: GBR inshore sites (Fitzroy, Snapper, Hinchenbrook, Double, Dunk Islands and King Reef), sampled between September 2006 and November 2007 from two to up to four occasions each site; 2nd column at GBR Middle lagoon sites (Upolu Cay, Upolu reef, Michelmas Cay, Michelmas reef, Low Isle East, Low Isle West, Normanby Is. North, Normanby Is. West), sampled between February 2007 and December 2007 on three occasions at each site; 3rd column at Offshore sites (Thetford, Moore, and Norman). Sample month
Inshore site
DIN
Sept06 Nov06 Jan07 Feb
Fitz Snap Hinc
0.005 0.02 0.015
Mar
Snap Doub Dunk Snap Fitz Dunk Doub Dunk
0.139 0.082 0.064 0.018 0.026 0.038 0.02 0.015
Jul
KingR
0.01
Sept Oct
KingR Dunk Fitz
0.039 0.004 0.022
Nov
Doub Fitz KingR Hinc
0.013 0.01 0.016 0.019
Apr
Jun
Dec
AVERAGE
Inshore
0.030
occur in a year when there were long breaks between the times when the trade winds are blowing, which was not the case in this year of sampling. It has been suggested that the combination of El Nino events and cyclones, within the positive PDO (Pacific Decadal Oscillation) phase, may facilitate CFP in Rarotonga, Cook Islands (Rongo and van Woesik, 2010). It would be of interest, if a record of CFP was kept in North Queensland, to see whether an increase of CFP will occur after 2011s violent cyclone season. Cyclonic rainfalls bringing more nutrients to the lagoon combined with higher than normal water temperatures and calm periods, rather than consistent trade winds may initiate and sustain more blooms of toxic dinoflagellates. It is of interest that this one Ostreopsis bloom at Normanby Island occurred in December when water temperatures were towards their warmest; high water temperatures, relative to summer water temperatures on the GBR, increased the growth
Middle site
DIN
UpoC UpoR MicC MicR
0.025 0.031 0.01 0.036
LowE LowW NorN NorW UpoR UpoC
0.016 0.018 0.057 0.01 0.014 0.009
LowE LowW NorN NorW UpoC UpoR MicC MicR MicC MicR
0.013 0.009 0.014 0.022 0.008 0.016 0.02 0.018 0.004 0.003
LowE LowW NorN NorW UpoC UpoR Mid Lagoon
0.004 0.01 0.015 0.007 0.048 0.006 0.017
Offshore site
DIN
Moor Thet
0.012 0.014
Norm
0.014
Thet NormR
0.020 0.018
Thet Moor
0.006 0.012
NormR
0.027
Offshore
0.015
rate and biomass of O. ovata (Graneli et al., 2011). Spikes of Nitrogen in the water column were recorded earlier in the year that had the 4th highest average Nitrogen concentration over all sites. The other three highest (Snapper, Double and Dunk islands) are also in close proximity to river mouths and no doubt being impacted by river plumes as a source of the water column DIN, and not just the recycling and re suspension within the GBR lagoon. 5. Conclusion This study has found a shift in the assemblage of potentially toxic benthic dinoflagellates in the GBR lagoon, with one genus, Prorocentrum, becoming more dominant with increasing DIN in the water column at inshore reef sites. Levels of DIN at inshore reef sites were more than double the threshold level for coral reef ecosystem health and if persistent could potentially result in
M.P. Skinner et al. / Marine Pollution Bulletin 77 (2013) 210–219
ecosystem change (Furnas et al., 2005). There appears to be at least two stages in the succession/phase shift of coral to macroalgal reef in terms of benthic dinoflagellate abundance. Secondly, any effect of ciguatoxins on the fecundity of fishes would contribute to a decline in fish biomass and decreased herbivory (other than over fishing and a consequence of the decline of coral abundance), with either Prorocentrum (e.g. Green Island) and/or Ostreopsis (e.g. Magnetic Is. and Normanby Is.) dominating the benthic community. We believe that inshore reefs of the GBR have reached the second stage. Thus the implementation of management plans to halt or reverse a decline in water quality, through improved upstream land use practices and waste water treatment is vital to ensure the long term health of inshore reefs of the GBR (Fabricius et al., 2005). We predict that coral reefs are more likely to become macroalgal and benthic HAB dominated reefs if stressors such as eutrophication are not removed. Acknowledgements The authors are gratitude to the late Dan Wruck for his support in nutrient analysis at QHSS and for the field work support from Doug, Jeff, Samantha, Simon and Mica and the support from staff of the many tourist vessels which made the collection of samples possible. This research was supported by a grant to MPS from the Edward Koch Foundation. This research was carried out under Great Barrier Reef Marine Park Authority; Marine Parks permit G07/22433.1 and was endorsed by the IOC/UNESCO, GEOHAB program. References Aikman, K.E., 1992. The physiology, biochemistry, and toxicity of the marine dinoflagellae, Prorocentrum hoffmannianum Faust (Phyrophyta) and its role in seafood-borne human disease. Ph.D. Dissertation. Southern Illinois University, Carbondale, p. 124 Ajuzie, C.C., 2007. Palatability and fatality of the dinoflagellate Prorocentrum lima to Artemia salina. Journal Applied Phycology 19, 513–519. Alongi, D.M., McKinnon, A.D., 2005. The cycling and fate of terrestrially-derived sediments and nutrients in the coastal zone of the Great Barrier Reef shelf. Marine Pollution Bulletin 51, 239–252. APHA, 2005. Standard methods for the examination of water and waste water Edited by A.D. Eaton, L.S. Clesceri, E.W. Rice and A.E. Greenberg, 21st ed. APHAAWWA-WPCF. Bell, P.R.F., 1992. Eutrophication and coral reefs – some examples in the Great Barrier Reef lagoon. Water Resources 26 (5), 553–568. Burkholder, J.M., Glibert, P.M., Skelton, H.M., 2008. Mixotrophy, a major mode of nutrition for harmful algal species in eutrophic waters. Harmful Algae 8, 77–93. Fabricius, K., De’arth, G., Mc Cook, L., Turak, E., McWilliams, D., 2005. Changes in algal, coral and fish assemblages along water quality gradients on the inshore Great Barrier Reef. Marine Pollution Bulletin 51, 384–398. Faust, M.A., Gulledge, R.A., 2002. Identifying harmful marine dinoflagellates. Smithsonian institution. Contributions from the US National Herbarium 42, 1–144. Furnas, M., Mitchell, A., Skuza, M., Brodie, J., 2005. In the other 90%: Phytoplankton responses to enhanced nutrient availability in the Great Barrier Reef. Marine Pollution Bulletin 51, 253–265. Gillespie, N.C., Holmes, M.J., Burke, J.B., Doley, J., 1985. Distribution and periodicity of Gambierdiscus toxicus in Queensland, Australia. In: Anderson, White, Baden (Eds.), Toxic Dinoflagellates, Elsevier Science Publishing Co. Inc. Glibert, P.M., Burkholder, J.M., Kana, T.M., 2012. Recent insights about relationships between nutrient availability, forms, and stoichiometry, and the distribution, ecophysiology, and food web effects of pelagic and benthic Prorocentrum species. Harmful Algae 14, 231–259.
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Graneli, E., Weberg, M., Salomon, P.S., 2008. Harmful algal blooms of allelopathic microalgal species: the role of eutrophication. Harmful Algae 8, 94–102. Graneli, E., Vidyarathna, Funari.E., Cumaranatunga, P.R.T., Scenati, R., 2011. Can Increases in temperature stimulate blooms of toxic benthic dinoflagellate Ostreopsis ovata? Harmful Algae 10, 165–172. Howarth, R.W., 2008. Coastal nitrogen pollution: a review of sources and trends globally and regionally. Harmful Algae 8, 14–20. Kodama, A.M., Hokama, Y., Yasumoto, T., Fukui, M., Manea, S.J., Sutherland, N., 1989. Clinical and laboratory findings implicating Palytoxin as cause of ciguatera poisoning due to Decapterus macrosoma (mackerel). Toxicon 27 (9), 1051–1053. Lapointe, B.E., 1997. Nutrient thresholds for bottom-up control of macroalgal blooms on coral reefs in Jamaica and southwest Florida. Limnology and Oceanography 42, 1119–1131. Lartigue, L., Jester, E.L.E., Dickey, R.W., Villareal, T.A., 2009. Nitrogen source effects on the growth and toxicity of two strains of the ciguatera causing dinoflagellate Gambierdiscus toxicus. Harmful Algae 8 (5), 781–791. Parsons, M.L., Preskitt, L.B., 2007. A survey of epiphytic dinoflagellates from the coastal waters of the island of Hawaii. Harmful Algae 6 (5), 658–669. Parsons, M.L., Settlemier, C.J., Bienfang, P.K., 2010. A simple model capable of simulating the population dynamics of Gambierdiscus, the benthic dinoflagellate responsible for ciguatera fish poisoning. Harmful Algae 10, 71–80. Rongo, T., van Woesik, R., 2010. Ciguatera poisoning in Rarotonga, southern Cook Islands. Harmful Algae 10 (4), 345–355. Schaffelke, B., Mellors, J., Duke, N.C., 2005. Water quality in the Great Barrier Reef region: responses of mangrove, seagrass and macroalgal communities. Marine Pollution Bulletin 51, 279. Shears, N.T., Ross, P.M., 2009. Blooms of the genus Ostreopsis; an increasing and ecologically important phenomenon on temperate reefs in New Zealand. Harmful Algae 8, 916–925. Skinner, M.P., Smith L.L., Johnstone, R., Turua T., Shaw, G., Lewis, R.J., Morton, S.L., 2009. Description of a new epipsammonic Prorocentrum species Prorocentrum gillespii sp. nov. (Dinophyceae), and ciguatera from Muri Lagoon, Rarotonga, Cook Islands. In: Proceedings of the Pacific Science Inter Congress, Tahiti, 2009. Skinner, M.P., Brewer, T.D., Johnstone, R., Fleming, L.E., Lewis, R.J., 2011. Ciguatera Fish Poisoning in the Pacific Islands (1998 to 2008). PLoS Negl Trop Dis 5 (12), e1416. http://dx.doi.org/10.1371/journal.pntd.0001416. Skinner, M.P., Lewis, R.J., Morton, S., 2012. The abundance of potentially toxic epiphytic dinoflagellates and nutrients from Bali and Gili Trawangan, Indonesia. Marine Research in Indonesia 36 (2). Smayda, T.J., 2008. Complexity in the eutrophication-harmful algal bloom relationship, with comment on the importance of grazing. Harmful Algae 8, 140–151. Smith, L.D., Devlin, M., Haynes, D., Gilmour, J.P., 2005. A demographic approach to monitoring the health of coral reefs. Marne Pollution Bulletin 51, 399–407. Sperr, A.E., Doucette, G.J., 1996. Variation in growth rate and ciguatera toxin production among geographically distinct isolates of Gambierdiscus toxicus. Sugg, L.M., VanDolah, F.M., 2002. No evidence for an allelopathic role of Okadaic Acid among ciguatera associated dinoflagellates. Journal of Phycology 35 (1), 93–103. Tester, P.A., Feldman, R.L., Nau, A.W., Kibler, S.R., Litaker, W.R., 2010. Ciguatera fish poisoning and sea surface temperatures in the Caribbean Sea and the West Indies. Toxicon 56, 698–710. Udy, J., Gall, M., Longstaff, B., Moore, K., Roelfsema, C., Spooner, D.R., Albert, S., 2005. Water quality monitoring: a combined approach to investigate gradients of change in the Great Barrier Reef, Australia. Marine Pollution Bulletin 51, 224– 238. Ukena, T., Satake, M., Usami, M., Oshima, Y., Naoki, H., Fujita, T., Kan, Y., Yasumoto, T., 2001. Structural Elucidation of Ostreocin D, a palytoxin analog isolated from the dinoflagellate Ostreopsis siamensis. Bioscience, Biotechnology, and Biochemistry 65 (11), 2585–2588. Vanucci, S., Guerrini, F., Milandri, A., Pistocchi, R., 2010. Effects of different levels of N- and P-deficiency on cell yield, okadaic acid, DTX-1, protein and carbohydrate dynamics in the benthic dinoflagellate, Prorocentrum lima. Harmful Algae 9, 590–599. Varkitzi, J., Pagou, K., Graneli, E., Harzianestis, J., Pyrgaki, C., Pavilidou, R., Montesanto, B., Ecanomou-Amilli, A., 2010. Unbalanced N:P ratios and nutrient stress controlling growth and toxin production of the harmful dinoflaellate Prorocentrum lima (Ehrenberg) Dodge. Harmful Algae 9, 304–311. Yasumoto, T., Inoue, A., Ochi, T., Fuyimoto, K., Oshima, Y., Fukuyo, Y., Adachi, R., Bagnis, R., 1980. Environmental studies on a toxic dinoflagellate responsible for ciguatera. Bulletin Japan Society Science Fisheries 46, 1397–1404.
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Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul
Ecology of the ciguatera causing dinoflagellates from the Northern Great Barrier Reef: Changes in community distribution and coastal eutrophication Mark P. Skinner a, Richard J. Lewis b, Steve Morton c,⇑ a b c
University of Queensland, Entox (National Research Centre for Environmental Toxicology), 39 Kessels Road, Coopers Plains, Queensland 4108, Australia University of Queensland, Institute of Molecular Bioscience, St. Lucia, Queensland 4072, Australia NOAA, Marine Biotoxins Program, Charleston, SC, USA
a r t i c l e
i n f o
Keywords: Ciguatera Dinoflagellates Gambierdiscus Prorocentrum Ostreopsis Nutrients
a b s t r a c t Ciguatera fish poisoning (CFP) is known to be caused by the ciguatoxins from the dinoflagellate genus Gambierdiscus, however, there is the potential for other toxins such as okadaic acid and dinophysistoxins from the genus Prorocentrum, and palytoxin from the genus Ostreopsis, to contaminate seafood. These genera may also be indicators of ecosystem health and potentially impact on coral reef ecosystems and the role they may play in the succession of coral to macroalgae dominated reefs has not been researched. Sixteen GBR field sites spanning inshore, mid-lagoon and outer lagoon (offshore) regions were studied. Samples were collected from September 2006 to December 2007 and abundance of benthic dinoflagellates on different host macroalgae and concentration of nutrients present in the water column were determined. The maximum abundance of Prorocentrum, Ostreopsis and Gambierdiscus found was 112, 793 and 50 cells per gram wet weight of host macroalgae, respectively. The average level of Dissolved Inorganic Nitrogen (DIN) in the water column across all sites (0.03 mg/L) was found to be more than double the threshold critical value (0.013 mg/L) for healthy coral reefs. Compared to a previous study 1984, there is evidence of a major shift in the distribution and abundance of these dinoflagellates. Inshore reefs have either of Prorocentrum (as at Green Island) or Ostreopsis (as at Magnetic Island) dominating the macroalgal surface niche which was once dominated by Gambierdiscus, whilst at offshore regions Gambierdiscus is still dominant. This succession may be linked to the ongoing eutrophication of the GBR lagoon and have consequences for the sources of toxins for ongoing cases of ciguatera. Published by Elsevier Ltd.
1. Introduction The past few decades have seen a significant increase in coastal eutrophication globally, leading to widespread hypoxia and anoxia, habitat degradation, alteration of food web structures, loss of biodiversity, and the increased frequency, spatial extent and duration of harmful algal blooms, (Howarth, 2008). In this context, changing land-use practices in North Queensland have resulted in the clearing of extensive tracks of land for agricultural production in the last 150 years, resulting in dramatic increases in inputs of sediments, nutrients and pesticides into the Great Barrier Reef (GBR) lagoon, and the widespread decline in lagoon water quality (Smith et al., 2005). Furnas et al. (2005) further suggest that terrestrial runoff of sediment and nutrients to the GBR lagoon has increased 2–4fold over the last century. In view of these inputs, the GBR lagoon should be considered a partially enclosed sea with the associated ⇑ Corresponding author. Tel.: +1 8437628857. E-mail address: [email protected] (S. Morton). 0025-326X/$ - see front matter Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.marpolbul.2013.10.003
potential to accumulate nutrients discharged from ever-increasing anthropogenic activities (Bell, 1992). The evidence that nutrient enrichment, increased siltation and excess turbidity can lead to the local degradation of coral reefs is unequivocal (Fabricius et al., 2005). Elevated nutrient levels and higher suspended sediment loads have been cited as the cause of reductions in coral growth and a shift in the relative abundance and composition of coral and algae; particularly in coastal areas adjacent to catchments with intensive agricultural activities (Alongi and McKinnon, 2005). On some near shore reefs on the GBR, high nutrient availability, in conjunction with substrate availability (low coral cover) and insufficient grazing pressure, has also lead to altered benthic communities with high macroalgal cover (Schaffelke et al., 2005). Similarly, increased nutrient levels (and/or reduced herbivory) can also lead to more substrate for toxigenic dinoflagellates, exacerbating their impacts (Parsons and Preskitt, 2007). As noted by Parsons et al. (2010) nutrient enrichment and warming sea surface temperatures can stimulate Gambierdiscus growth and result in higher cell densities. By corollary, elevated sea surface
M.P. Skinner et al. / Marine Pollution Bulletin 77 (2013) 210–219
temperatures (SSTs) associated with global warming may further exacerbate the growing extent and range of ciguatera distribution being driven by eutrophication and other anthropogenic influences. Ciguatera field studies have largely concentrated on dinoflagellates of the genus Gambierdiscus, well known to be the producer of ciguatoxin precursors, but have largely ignored the potential role of toxins from the genera Prorocentrum and Ostreopsis as causative agents in ciguatera fish poisoning (CFP). Little is known about the extent to which ciguatera related toxins (from benthic dinoflagellates) may be affecting trophic levels, or their potential to alter coral reef ecosystem function. This highlights the need to monitor changes in water quality and various indicators of ecosystem health to help determine if management plans on land are having a noticeable impact on the ecosystems and water quality of the GBR (Udy et al., 2005). Such monitoring might be augmented with assessments of the abundance of potentially toxic benthic HABs that have the potential to provide a useful bio-indicator of coral reef ecosystem health. Toxic benthic dinoflagellates, like their macroalgal hosts, are at the bottom of the food chain. Whether the succession to algal dominated reefs, either initiated due to natural disturbances (cyclonic weather conditions, predation, etc.) or from anthropogenic causes (global warming, eutrophication, sedimentation) or a combination of such influences, the loss of coral cover results in more macroalgal surface for epiphytic benthic dinoflagellates. For example, phase shifts that involve coral bleaching water temperatures provide macroalgal habitat for Gambierdiscus at temperatures highly favorable for their growth (Tester et al., 2010). In fact, coral reef succession to algal dominance might even be reinforced by the effects of benthic dinoflagellate toxins on herbivore fecundity or feeding. Such effects have been little studied but may contribute to increased incidence of ciguatera as recently shown for Pacific Island nations (Skinner et al., 2011). There has been only one previous published study of these three genera on the Northern Great Barrier Reef (Gillespie et al., 1985). The objective of this study is to determine the distribution and abundance of the ciguatera and other potential causative dinoflagellates across inshore island fringing reefs, middle GBR lagoon reefs and outer continental shelf reefs. We then correlated nutrient levels (ammonia, nitrites or nitrates) and benthic dinoflagellate abundance in relation to macroalgae sampled. Finally, a comparison to a previous study (Gillespie et al., 1985) of benthic dinoflagellates from similar field sites was undertaken.
211
at a distance of 100 m from shore (or pontoon at reef sites), in a water depth of 1–2 m at all field sites. 2.2. Description of field sites The sites covered a three main reef types, inshore GBR coral reefs, fringing continental islands: Magnetic Island at over 5000 ha of land surface (more than half of which is national park) is inhabited with approx. 2000 people and is the only permanently inhabited site and is located close to Townsville, Snapper Island at 56 ha is 4 km east of the Daintree river, Double Island similar to Snapper and offshore from Palm Cove, Fitzroy Island at 339 ha (mostly national park), King reef that is only 200 m off the mainland coast at Kurrimine, Cape Richards on Hinchenbrook Island and, Dunk Island at 970 ha is also mostly national park; Mid-lagoon platform coral cays and reefs: Green Island at 15 ha land surface and with 710 ha of reef that is mostly sea grass, Lizard Island at 1000 ha is 240 km north of Cairns, Michelmas Cay is only 2 ha and 30 km NE of Cairns, Low Isles has 22 ha of reef found 15 km NE of Port Douglas, Upolu Cay is 28 km NE of Cairns, and Normanby Island of the Frankland group which is 10 km east of Russell river; and outer barrier coral reefs included: Norman Reef has 430 ha of reef located 70 km NE of Cairns, Moore Reef which is 2650 ha and 45 km SE from Cairns, Flynn Reef with 420 ha, found 50 km SE of Cairns and Thetford Reef at 200 ha, is 60 km SE of Cairns. Please note that the benthic HAB abundance is not presented in this paper for each site, as only those with multiple samples or representative abundances are shown. 2.3. Protocol of microalgal sampling
2. Methods
Many similar methods for epiphytic microalgal collection and enumeration have been used following the original sampling of Yasumoto et al. (1980). Divers collected a broad diversity of reef macroalgae harvested using plastic bags. Three or more of the most common species of macroalgae; in this study Sargussum flaricans, Padina australis, Halimedia opunita, Turbinaria ornata, Laurencia intricata, Dignea simplex, Dictyota bartayresi, Amphiroa foliacea, Hypnea saidana, Jania crassa and seagrass if present, were collected at a site. The macroalgae sample bags were vigorously shaken for 1 min to dislodge the epiphytic flora. The resulting dislodged epiphytes were sieved and the 38 lm wash back, transferred to a 50 ml vials and fixed with 10% Lugol’s solution. All fixed samples were stored at 12 °C in the dark until dinoflagellate abundance could be enumerated. The weight of each macrophyte sample was blotted dry and weighted for comparison.
2.1. Sampling regime
2.4. Abundance and identification of ciguatera dinoflagellates
Sixteen Great Barrier Reef field sites were sampled: Inshore, mid-lagoon and outer lagoon (offshore) from Lizard, Snapper and Low Islands (offshore of Port Douglas) in the north, Double and Green Islands, Upolu and Michelmas Cays, Fitzroy and Normanby Island (Frankland Islands), centrally located (near to Cairns) to King reef, Dunk and Magnetic (next to Townsville) Islands in the south (see Fig. 1; Table 1). Sampling occurred at ten sites being sampled three or more times, between September 2006 and December 2007. Green Island (two sites on either side of the island, included 9 monthly collections) and Magnetic Island (Nelly, Geoffrey and Arthur Bays sampled at six, monthly collections) where the most intensively sampled of all the field sites. At all other multiply sampled sites at least 3 samplings occurred, including 3 separate sites at Dunk Island and two separate sites at Low, Normanby Islands and Upolu Cay, nearly all of which where 200 m apart, except at Upolu Cay which was 1 km apart. Sampling generally took place
Ciguatera dinoflagellates were identified to genus level by morphology using the light microscope (a CE, XSZ-107BN Series). For abundance a 0.5 ml portion of the shaken wash back sample was pippetted onto a Sedgewick Rafter counting slide and diluted to 1 ml and viewed at 64, for counting and 160, for identification. Abundance was calculated by taking the number of each genus present and multiplying this by the amount of dilution and the size of the sample and dividing by the wet weight of the sample macroalgae to arrive at the number of cells per gram wet weight of macroalgae. Photographs of different species were taken with a digital camera (Nikon) focused down the tube of the microscope for further identification. Some samples were returned to the Centre for Microscopy and Microanalysis (CMM), University of Queensland for SEM analysis. Fixed samples were desalted using a 10% step gradient from seawater to freshwater on polycarbonate filter paper and dehydrated by using a step gradient of ethanol
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Fig. 1. Map of the Great Barrier Reef, Queensland, showing the field site locations.
(50–100%), fixed to stubs and sputter coated with 1.5 nm of platinum and viewed with a JOEL JCM5000 SEM. 2.5. Water column nutrient sample analysis Soluble Nutrients: Water column samples were collected with a 60 ml syringe, about 30 cm above the lagoon floor, whilst snorkeling slowly for 10 m, after rinsing three times with the water from the site to be collected, filtered (using a 0.45 lm membrane Millipore sterile disposable filter unit) into a sterile falcon tube and stored frozen until analysis at the Nutrient Laboratory at the Queensland Health and Scientific Services, Brisbane, Australia; a National Association of Testing Authorities (NATA) accredited laboratory. Analyses for FRP, NO2, NO3, and NHþ 4 were performed simultaneously using an automated LACHAT 8000QC flow injection system using methodology based on (see APHA, 2005): (a) ascorbic acid reduction of phosphomolybdate for FRP, (b) cadmium reduction of nitrate to nitrite by diazotizing the nitrite with sulfanilamide and coupling with N-(1-naphthyl)ethylenediamine dihydrochloride for NOx, (c) production of the indophenol blue color complex for NHþ 4. 2.6. Statistical analysis Statistical correlations of selected GBR field sites of benthic dinoflagellate abundance from macroalgae sampled on each occa-
sion and water column nutrients (DIN and separately for ammonia and nitrates), undertaken using SPSS 19 (SPSS Inc., Chicago 2010) for significant correlation wither using the Pearson correlation or the Spearman correlation (n = 3–6).
3. Results 3.1. Dinoflagellate abundance, Green Island, east and west, mid lagoon field sites At the field site on the eastern side of Green Island (Fig. 2), the most abundant dinoflagellate was Prorocentrum (24 samples) Ostreopsis (6 samples) and Gambierdiscus was most abundant on three occasions (all of which occurred on the one sampling date in June). Ostreopsis did not occur on any samples in May or June. On only 2 samples did all three genera occur at the same time. On 12 samples Prorocentrum and Ostreopsis occurred together. On only three samples Ostreopsis and Gambierdiscus occurred together and on eight Prorocentrum and Gambierdiscus occurred together. On fourteen samples Prorocentrum appeared by itself and on no samples did Ostreopsis or Gambierdiscus appear in a sample by themselves. Prorocentrum was the most dominant of the three dinoflagellates at the Green Island East field site. From Scanning electron microscopy, Gambierdiscus australis was the dominate species found (Fig. 3a).
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M.P. Skinner et al. / Marine Pollution Bulletin 77 (2013) 210–219 Table 1 Summary of the Great Barrier Reef field sites. GBR location
Number of sampling sessions and site location Solitary sampling
Latitude/longitude (°/0 )
Multiple sampling
Latitude/longitude
Inshore of lagoon
Snapper Is. Double Is. Hinchenbrook Is.
16 17 43/145 29 36 16 44 00/145 41 00 18 11 40/146 13 28
Magnetic Is. Fitzroy Is. King reef Dunk Is.
19 16 17 17
09 55 47 56
15/146 30/145 00/146 00/146
51 59 08 08
50 25 00 30
Middle of lagoon
Lizard Is. Michelmas Cay
14 41 00/145 27 00 16 36 16/145 58 30
Green Is. Low Isle Upolu Cay Normanby Is.
16 16 16 17
45 23 40 12
30/145 00/145 30/145 15/146
58 33 56 04
10 30 00 35
Outer lagoon
Flynn reef
16 53 20/146 11 30
Norman reef Thetford reef.
16 25 40/145 59 30 16 48 30/146 11 00
In the field site on the western side of Green Island (Fig. 4), 17 samples Prorocentrum was the most abundant dinoflagellate whilst Ostreopsis was the most abundant on 11 occasions and Gambierdiscus was most abundant on five occasions, three of which occurred on the one sampling date (June). Ostreopsis did not occur on any samples in December. Only 3 samples showed all three genera occurring at the same time, on Dignea (October and September 2006) and Laurencia (July 2007). On 15 samples Prorocentrum and Ostreopsis occurred together. On only eight samples Ostreopsis and Gambierdiscus occurred together and on seven Prorocentrum and Gambierdiscus occurred together. On eight samples Prorocentrum appeared by itself and on only two samples did Ostreopsis appear by itself, Gambierdiscus never appearing in a sample as the sole dinoflagellate. At Green Island all samples of macroalgae sampled host the three genera of dinoflagellates. In all 11 samples of seagrass no Gambierdiscus was to found, only Prorocentrum and Ostreopsis. Prorocentrum was the most dominant dinoflagellate on all macroalgae, appearing in more samples than Ostreopsis, except on Turbinaria, and yet Gambierdiscus was still present amongst all genera of macroalgae. The presence of Sinophysis (Fig. 3b), similar in form to the pelagic Dinophysis, is a newly described benthic species, whose toxicity has not been studied and was found in the samples on Green Island and some other GBR sites. Not previously reported on the GBR, it may be invasive, its toxicity and its role in the ecosystem are unknown. 3.2. Dinoflagellate abundance, Geoffrey, Arthur and Nelly bays, Magnetic island, inshore GBR lagoon field sites In the Geoffrey Bay field site on Magnetic Island (Fig. 4), Prorocentrum was the most abundant dinoflagellate on only one sample, Ostreopsis was the most abundant on 17 occasions (all but two) and Gambierdiscus was most abundant on one occasion. Gambierdiscus did not occur on any samples in October. On six samples all three genera occurred at the same time. On 14 samples Prorocentrum and Ostreopsis occurred together. On seven samples Ostreopsis and Gambierdiscus occurred together, whenever Gambierdiscus was to be found and on six samples Prorocentrum and Gambierdiscus occurred together. On one only sample did Prorocentrum appear by itself and on four samples did Ostreopsis appear by itself, Gambierdiscus never appeared in a sample by itself. Ostreopsis was the most dominant of the three dinoflagellates. In the Arthur and Nelly Bay field sites on Magnetic Island (Fig. 5), Prorocentrum was the most abundant dinoflagellate on only two samples, whilst Ostreopsis was the most abundant on 25 occasions (all but four) and Gambierdiscus was most abundant on no occasion. On eight samples all three genera occurred at the same time (Gambierdiscus occurred only 9 out of 29 samples). On 14 samples Prorocentrum and Ostreopsis occurred together. On
one only sample did Prorocentrum appear by itself and on twelve samples Ostreopsis appeared by itself. Ostreopsis was the most dominant of the three dinoflagellates. All types of macroalgae on Magnetic Island host the three genera of dinoflagellates. Ostreopsis was the most dominant dinoflagellate on all macroalgae, appearing in more samples than Prorocentrum, except for Turbinaria, and yet Gambierdiscus was still present amongst all genera of macroalgae sampled. Out of a total of 49 samples Ostreopsis was found 46 times, Prorocentrum 31 times and Gambierdiscus 13 times. 3.3. Dinoflagellate abundance, other field site examples (multiple sampling) Upolu Cay and reef show signs of a macroalgal phase shift (Fig. 6): Gambierdiscus dominate on some macroalgae (Dignea), occur on 11 out of 25 samples; Ostreopsis dominating 9 out of 25 samples and occur on 20; Prorocentrum dominate on only 3 samples and found on 16 out of 25 samples; whilst Sinophysis became more dominant in the December sampling. At Normanby island, in December there was a bloom of Ostreopsis on the west reef whilst on the north reef, Gambierdiscus dominated the samples with Prorocentrum occurring on both reefs at this time. At Norman reef (Fig. 7) of 13 samples, Sinophysis was found on 9 samples, Gambierdiscus was found on 7 (always found on Dictyota), Ostreopsis on 5 (always found on Turbinaria) and Prorocentrum on only 4 samples. Across all field sites (Fig. 8), of all 70 inshore samples, Prorocentrum, Ostreopsis and Gambierdiscus genera were found in 48%, 57% and 20% of all samples, respectively. In the 78 mid lagoon samples Prorocentrum, Ostreopsis and Gambierdiscus were found in 55%, 56% and 40% of all samples respectively. In the 23 offshore samples Prorocentrum, Ostreopsis and Gambierdiscus were found in 35%, 39% and 57% of samples respectively. Padina, Amphiroa and Sargassum were often found in the inshore and mid-lagoon sites but not often in the offshore sites (see Fig. 9). The green algae Halimeda although found across all three sites was not such a good host for dinoflagellates compared to Padina, Amphiroa and Sargassum. Of 30 Halimeda samples across all sites, only 5 had Gambierdiscus present and four of these were from offshore sites. Turbinaria not often found in the inshore sites was a good host to all dinoflagellate genera in the offshore sites. Dignea, a fine haired alga, generally found in the mid lagoon sites was a good host to all three dinoflagellate genera. 3.4. Nutrient results Four of the five highest DIN readings occurred in the wet season whilst the four lowest readings occurred in the dry season, on the two Green Island sites (Table 3). On three of the seven occasions
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Green Island (East) Benthic HAB abundance Prorocentrum
Osteropsis
Gambierdiscus
Sinophysis
Abundance cells/g wet wt.
1000
100
10
Padina Amphiroa Turbinaria Laurencia Dignia Seagrass Padina Amphiroa Turbinaria Laurencia Dignia Seagrass Padina Amphiroa Turbinaria Laurencia Dignia Seagrass Amphiroa Turbinaria Laurencia Seagrass Amphiroa Laurencia Halimeda Seagrass LYNGBYA Amphiroa Laurencia Dignia Seagrass LYNGBYA Amphiroa Laurencia Dignia Padina Amphiroa Turbinaria Laurencia Seagrass Padina Amphiroa Turbinaria Laurencia Dignia Seagrass
1
Sep-06
Oct-06
Dec-06
Feb-07
May-07
Jun-07
Jul-07
Oct-07
Dec-07
Month of Sampling & Substrate Fig. 2. Abundance of benthic dinoflagellates, cells/g wet weight, on differing substrates of macroalgae and seagrass from the eastern field site of Green Island at various months between September, 2006 and December 2007.
when samples were taken on either side of the island, there were some large differences between DIN readings, from twice to five times as strong, the sites only 400 m apart. Of the 17 samples from Green Island, 10 (59%) were over the critical Nitrogen point threshold (0.014 mg/L), for maintaining a healthy coral reef (Bell, 1992; Lapointe, 1997). The average for both sites on Green Islands from the 17 samples was 0.029 mg/L, more than double the critical threshold. Three of the four highest DIN water column readings occurred in the wet season and three of the four lowest Nitrogen readings occurred in the dry season, across the three sites on Magnetic Island. Nelly Bay had on the average higher readings than the other two bays, all three bays having an average over the threshold (0.013 mg/L) for critical Nitrogen levels for a healthy coral reef. Of the 14 samples, 8 (57%) were over the critical level. Of the 19 nutrient samples taken from the inshore GBR lagoon sites, samples were over the critical Nitrogen threshold for healthy coral levels, on only four occasions were the sites under the threshold, twice at Fitzroy Island, once at Dunk Island and once at King reef, all of which were in the dry season. The highest readings occurred in March towards the end of the wet season, at Snapper, Double and Dunk Islands, all three of which are influenced by river plumes, from the Daintree, Barron and Tully rivers respectively. On only one occasion was there a phosphate reading (0.012 mg/L) of any significance, at Snapper Island in March, all other samples remained below the level of detection (0.003 mg/L) except one, at King reef (0.007 mg/L).
Of the 26 nutrient samples taken from the middle GBR lagoon sites samples were over the critical Nitrogen threshold for healthy coral levels, and on eleven occasions the sites were under the threshold, this occurred at seven different sites with no site recording below the threshold on more than two occasions, out of the three. The lowest readings occurred towards the end of the dry season. Four of the five highest readings occurred in the wet season. All Phosphorus readings stayed below the level of detection (0.003 mg/L). Sample sites further offshore than the mid GBR lagoon sites also have raised, on average, Nitrogen water column levels higher than the critical threshold level. Of the 53 samples from all sites, 35 (66%) have raised Nitrogen levels above the critical threshold level and 18 (34%) sites below. Sampling was undertaken without the influence of any cyclones, and generally under calmer conditions which may have biased the readings to be lower than expected due to the lack of re suspension from more turbulent waters.
4. Discussion The two intensively studied field sites of Green Island and the bays of Magnetic Island showed a major difference in the benthic dinoflagellate assemblage. Green Island fringing reef macroalgae and seagrass were dominated by Prorocentrum and Magnetic Island macroalgae were dominated by Ostreopsis. Often both these genera are found together and whilst Ostreopsis is seldom found singularly
Fig. 3. (a) and (b) Scanning electron micrograph of Gambierdiscus austalis (a) and Sinophysis sp. (b).
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Green Island (west) Benthic HAB abundance Prorocetrum
Ostreopsis
Gambierdiscus
Sinophysis
10
1
Sargassum Laurencia Dignia Halimeda Seagrass Padina Amphiroa Sargassum Laurencia Dignia Halimeda Seagrass Amphiroa Sargassum Dignia Halimeda Seagrass Amphiroa Laurencia Dignia Halimeda Seagrass Padina Amphiroa Sargassum Laurencia Dignia Halimeda Seagrass Padina Amphiroa Sargassum Laurencia Dignia Halimeda Seagrass
Abundance cells/g wet wt.
100
Feb-07
May-07
Jun-07
Jul-07
Oct-07
Dec-07
Month of Sampling and Substrate Fig. 4. Abundance of benthic dinoflagellates, cells per gram wet weight, on differing substrates of macroalgae and seagrass from the western field site of Green Island on February, May, June, July, October and December 2007.
Magnetic Island Geoffrey Bay Benthic HAB Abundance Prorocentrum
Osteropsis
Gambierdiscus
Abundance cells/g wet wt.
100
10
Jan-07
Mar-07
Aug-07
Turbinaria
Sargassum
Padina
Amphiroa
Dictyota
Sargassum
Padina
May-07
Amphiroa
Turbinaria
Amphiroa
Sargassum
Padina
Turbinaria
Sargassum
Padina
Amphiroa
Sargassum Feb-07
Turbinaria
Padina
Turbinaria
Padina
Sargassum
1
Oct-07
Month of Sampling and Substrate Fig. 5. Abundance of benthic dinoflagellates, cells/g wet weight of macroalgae, on differing substrates of macroalgae and seagrass from Geoffrey Bay, Magnetic Island in January, February, March, May, August and October 2007.
in samples. Harmful algae that thrive in eutrophic habitats are mixotrophs that respond both directly to nutrient inputs, and indirectly through high abundance of bacterial and algal prey that are stimulated by the elevated nutrients (Burkholder et al., 2008). Toxins from Prorocentrum have the potential to decimate populations of grazing communities (Ajuzie, 2007) in addition to the possibility of an allelopathic role, as exudates from Prorocentrum are also known to inhibit the growth of co-occurring species (Sugg and Vandolah, 2002), which could also keep in check possible blooms of predators. Available external nutrient supplies can have a major influence on microalgal allelopathic activity (Graneli et al., 2008). No blooms of Prorocentrum where found in any samples, the highest abundance recorded were only 112 cells per gram of macroalgae (Green Island eastern site). It is hypothesized therefore that the three genera could be keeping populations of each other in check, by allelopathy and species competition. However there appears to be many benthic invertebrates that could be predating on the dinoflagellates, which have been found in samples. The
predation of such invertebrates, let alone normal fish herbivory, feeding directly on both the macroalgae and the biodetritus off the macroalgal surface, would make it more complex to find correlations between dinoflagellate abundance and the existence of nutrients in the field. In assessing the eutrophication HAB relationship, the collective grazing behavior of the herbivorous copepods, filter feeding benthos and benthic larvae must be considered (Smayda, 2008). The results from this study show that where nutrient levels are sufficient, Prorocentrum and Ostreopsis can be dominant over Gambierdiscus populations (Table 2), as found in inshore and mid lagoon reefs as opposed to offshore reefs. In 1985 a similar GBR study showed only 5% of samples contained Prorocentrum, now this genera is found in seven (35%, offshore) to nine (55% mid lagoon) times as many samples. Ostreopsis has doubled its presence in samples, whilst Gambierdiscus has halved its overall presence; however, it still remains dominant in outer reef sites. In the 22 year gap between comparative studies there has been a major shift in
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Benthic Dinoflagellates at Nelly & Arthur Bays, Magnetic Island Osteropsis
Gambierdiscus
100
10
Arthur Bay
Mar-07
May-07
Nelly Bay
Arthur Bay
Aug-07
Turbinaria
Sargassum
Padina
Amphiroa
Dictyota
Sargussum
Padina
Amphiroa
Dictyota
Amphiroa
Sargassum
Dictyota
Arthur Bay
Laurencia
Amphiroa
Sargussum
Dictyota
Nelly Bay
Laurencia
Sargassum
Padina
Arthur Bay
Amphiroa
Dictyota
Sargussum
Padina
Nelly Bay
Amphiroa
Amphiroa
Sargassum
Turbinaria
Amphiroa
1 Sargussum
Cells/g wet wt. macroalgae
Prorocentrum
1000
Nelly Bay
Oct-07
Sampling month, site and macroalgal substrate Fig. 6. Abundance of benthic dinoflagellates, cells per gram wet weight, on differing substrates of macroalgae from Nelly and Arthur Bays, Magnetic Island in March, May, August and October 2007.
Upolu Cay & Reef HAB Abundance Prorocentrum
Osteropsis
Gambierdiscus
Sinophysis
Abundance cells/g wet wt.
1000
100
10
Upolu Cay
Upolu Reef
Feb-07
Upolu Reef
Upolu Cay
Jul-07
Upolu Reef
Upolu Cay
Oct-07
Sargassum
Turbinaria
Amphiroa
Padina
Dictyota
Dignea
Padina
Sargassum
Turbinaria
Amphiroa
Sargassum
Dignea
Padina
Sargassum
Padina
Amphiroa
Upolu Cay
Turbinaria
Dignea
Sargassum
Padina
Halimeda
Turbinaria
Dignea
Sargassum
Dictyota
1
Upolu Reef Dec-07
Sampling month, site & macroalgal substrate Fig. 7. Upolu Cay and reef, mid GBR lagoon site, sampled on four occasions.
Osteropsis
Gambierdiscus
Turbinaria
Amphiroa
Norman Reef Benthic HAB Abundance Prorocentrum
Sinophysis
Jul-07
Oct-07
Dec-07
Sampling month and Macroalgal substrate Fig. 8. Normanby Island sampled on three occasions on the west and north side reefs.
Rubble
Halimeda
Dictyota
Turbinaria
Padina
Rubble
Halimeda
Dictyota
Padina
Rubble
Halimeda
Amphiroa
1
Dictyota
10
Turbinaria
Abundance cellls/g wet wt.
100
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M.P. Skinner et al. / Marine Pollution Bulletin 77 (2013) 210–219
Halimeda
Sargussum
Amphiroa
Dictyota
Turbinaria
Dignea
Laurencia
20 18 16 14 12 10 8 6 4 2
Inshore
Midshore
Total
Gambierdiscus
Ostreopsis
Prorocentrum.
Total
Gambierdiscus
Ostreopsis
Prorocentrum.
Total
Gambierdiscus
Ostreopsis
0 Prorocentrum.
Number of times dinoflagellate found in a type of macroalgae sample
Dinoflagellate genera, total times found on macroalgal types in GBR sites Padina
Offfshore
GBR site and dinoflagellate type with totals for macroalgae sampled Fig. 9. The number of times the benthic dinoflagellate genera found on the different macroalgal substrate samples across all the GBR field sites either inshore (n = 78), mid lagoon (n = 70) or offshore (n = 28).
the GBR dinoflagellate assemblage, with Prorocentrum apparently becoming more dominant over time. Changes in nutrient stoichiometry are known to have drastic changes not only in cellular composition but in toxin production by these benthic dinoflagellates. Toxin production in both Prorocentrum and Gambierdiscus has been shown to be inversely related to nutrient limitation (Aikman, 1992; Sperr and Doucette, 1996; Varkitzi et al., 2010; Vanucci et al., 2010). Imbalances in N:P ratios also promote increase in exopolysacharides (Vanucci et al., 2010). Exopolysacharides are utilized by Prorocentrum, Gambierdiscus, and Ostropsis to form large aggregates within mucilage. This combination of increased toxin production plus increase excopolysacharides was hypothesized by Glibert et al. (2012) to decrease grazers’ ability to consume these benthic dinoflagellates. The potential link between nutrients and dinoflagellates is an important one as suggested by a study undertaken on Hawaiian coral reefs (Parsons and Preskitt, 2007). In this study correlations with Nitrogen and Ostreopsis only occurred at one site, Nelly Bay, Magnetic Island (see Table 2) and with Gambierdiscus at two sites, Nelly Bay and Fitzroy Island, both correlations with the abundance of ammonia. Toxic strains of Gambierdiscus spp. are known to grow faster on ammonium rather than other Nitrogen sources and they are able to take advantage of different Nitrogen sources on short time scales to support growth (Lartigue et al., 2009). All sites having large standing masses of macroalgae (personnel observations) consistent with eutrophication. There would appear to be some significance to eutrophication of the GBR lagoon and the presence of the epiphytic dinoflagellate genus Prorocentrum as there were eight correlations found between this genus abundance and either ammonia and/or nitrates found at five different field sites (Table 2). Four of those sites showed a direct correlation with the Prorocentrum abundance and the water column DIN. A similar study of a smaller lagoon in Nusa Dua, Bali, Indonesia also found a significant correlation between the dinoflagellate genera Prorocentrum with the DIN in the water column (Skinner et al., 2012), and a benthic Prorocentrum gillespii bloom in the sediment of Muri lagoon, Rarotonga was also correlated with nutrients (Skinner et al., 2009), where critical levels for nutrient thresholds were also exceeded at both sites. The genus Prorocentrum is known to be involved in ciguatera (Rongo and Van Woesik, 2010), so nutrient thresholds set for coral reefs should be employed, as these levels once exceeded, can assist to create HABs.
This may provide evidence for a succession in the dinoflagellate community as an ecosystem becomes more degraded (from coral to macroalgal phase shift), with increasing nutrients, leading to the prevalence of toxins from Prorocentrum and/or Ostreopsis into the ecosystem, rather than Gambierdiscus that is more likely present in the initial phase shift as it is presence now is more likely found at less impacted reefs on the outer GBR. The high density of benthic dinoflagellates from the genus Prorocentrum collected from sediments coincided with a sharp increase in cases of CFP in Rarotonga (Cook Islands) and may have explained increased poisonings associated with soft bottom benthic invertivores (e.g. mullet and goatfish) as well as filter feeders (i.e. Tridacna), as an example where an algal community shift and the assemblage of toxic dinoflagellates was altered (Rongo and van Woesik, 2010). These changes occurred in a small, highly eutrophic lagoon with high water retention rates. The result of such succession could then appear to be a shift away from Gambierdiscus as the cause of CFP to potentially an Ostreopsis/Prorocentrum dominated benthic dinoflagellate community at the exclusion of Gambierdiscus. P. lima and other Prorocentrum spp. are known to cause diarrhetic shellfish poisoning caused by okadaic acid, dinophysistoxins, prorocentralides as well as a fast acting toxin (Faust and Gulledge, 2002) although levels of these and related toxins likely to affect human health have not been detected in fish flesh or are rarely researched. Palytoxin is one of the more toxic members of a family of ciguatera related toxins as it appears to have the same marine food chain etiology (Kodama et al., 1989). The genus Ostreopsis are known to produce Ostreocin, a palytoxin (Ukena et al., 2001). There was one bloom of Ostreopsis at Normanby Island, that can be considered an inshore GBR field site, as its proximity to a river delta is quite close (4 km), where there was an increase in abundance within the four macroalgal samples collected. This bloom was found at a site sampled on three occasions, and on the last sampling period in December 2007 its persistence was not recorded. This bloom occurred on the western side of the island, protected from the regular SE trade winds, and was localized, as at the same sampling of the northern site, 400 m away, showed no increases in normal abundances (less than 100 cells/g macroalgae). At a temperate reef in New Zealand, Ostreopsis blooms were most prevalent at sites protected from prevailing swells (Shears and Ross, 2009). It would be no surprise then that more blooms would be found to
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Table 2 Percentage of benthic dinoflagellate genera were observed among the different sites compared to previous study by Gillespie et al. (1985). Year
GBR locality
Prorocentrum (%)
Ostreopsis (%)
Gambierdiscus (%)
Total average nitrogen (mg/l) (n)
2007
Inshore (n = 70) Mid lagoon (n = 78) Offshore (n = 23)
48 55 35
57 56 39
20 40 57
0.030 (33) 0.016 (43) 0.015 (8)
2007
All sites Ave (n = 171)
46
51
39
0.020 (84)
1985
All sites Ave (n = 280)
5
26
79
na
Table 3 Dissolved Inorganic Nitrogen (NHþ 4 plus NOx), mg/L of water column, 1st column at: GBR inshore sites (Fitzroy, Snapper, Hinchenbrook, Double, Dunk Islands and King Reef), sampled between September 2006 and November 2007 from two to up to four occasions each site; 2nd column at GBR Middle lagoon sites (Upolu Cay, Upolu reef, Michelmas Cay, Michelmas reef, Low Isle East, Low Isle West, Normanby Is. North, Normanby Is. West), sampled between February 2007 and December 2007 on three occasions at each site; 3rd column at Offshore sites (Thetford, Moore, and Norman). Sample month
Inshore site
DIN
Sept06 Nov06 Jan07 Feb
Fitz Snap Hinc
0.005 0.02 0.015
Mar
Snap Doub Dunk Snap Fitz Dunk Doub Dunk
0.139 0.082 0.064 0.018 0.026 0.038 0.02 0.015
Jul
KingR
0.01
Sept Oct
KingR Dunk Fitz
0.039 0.004 0.022
Nov
Doub Fitz KingR Hinc
0.013 0.01 0.016 0.019
Apr
Jun
Dec
AVERAGE
Inshore
0.030
occur in a year when there were long breaks between the times when the trade winds are blowing, which was not the case in this year of sampling. It has been suggested that the combination of El Nino events and cyclones, within the positive PDO (Pacific Decadal Oscillation) phase, may facilitate CFP in Rarotonga, Cook Islands (Rongo and van Woesik, 2010). It would be of interest, if a record of CFP was kept in North Queensland, to see whether an increase of CFP will occur after 2011s violent cyclone season. Cyclonic rainfalls bringing more nutrients to the lagoon combined with higher than normal water temperatures and calm periods, rather than consistent trade winds may initiate and sustain more blooms of toxic dinoflagellates. It is of interest that this one Ostreopsis bloom at Normanby Island occurred in December when water temperatures were towards their warmest; high water temperatures, relative to summer water temperatures on the GBR, increased the growth
Middle site
DIN
UpoC UpoR MicC MicR
0.025 0.031 0.01 0.036
LowE LowW NorN NorW UpoR UpoC
0.016 0.018 0.057 0.01 0.014 0.009
LowE LowW NorN NorW UpoC UpoR MicC MicR MicC MicR
0.013 0.009 0.014 0.022 0.008 0.016 0.02 0.018 0.004 0.003
LowE LowW NorN NorW UpoC UpoR Mid Lagoon
0.004 0.01 0.015 0.007 0.048 0.006 0.017
Offshore site
DIN
Moor Thet
0.012 0.014
Norm
0.014
Thet NormR
0.020 0.018
Thet Moor
0.006 0.012
NormR
0.027
Offshore
0.015
rate and biomass of O. ovata (Graneli et al., 2011). Spikes of Nitrogen in the water column were recorded earlier in the year that had the 4th highest average Nitrogen concentration over all sites. The other three highest (Snapper, Double and Dunk islands) are also in close proximity to river mouths and no doubt being impacted by river plumes as a source of the water column DIN, and not just the recycling and re suspension within the GBR lagoon. 5. Conclusion This study has found a shift in the assemblage of potentially toxic benthic dinoflagellates in the GBR lagoon, with one genus, Prorocentrum, becoming more dominant with increasing DIN in the water column at inshore reef sites. Levels of DIN at inshore reef sites were more than double the threshold level for coral reef ecosystem health and if persistent could potentially result in
M.P. Skinner et al. / Marine Pollution Bulletin 77 (2013) 210–219
ecosystem change (Furnas et al., 2005). There appears to be at least two stages in the succession/phase shift of coral to macroalgal reef in terms of benthic dinoflagellate abundance. Secondly, any effect of ciguatoxins on the fecundity of fishes would contribute to a decline in fish biomass and decreased herbivory (other than over fishing and a consequence of the decline of coral abundance), with either Prorocentrum (e.g. Green Island) and/or Ostreopsis (e.g. Magnetic Is. and Normanby Is.) dominating the benthic community. We believe that inshore reefs of the GBR have reached the second stage. Thus the implementation of management plans to halt or reverse a decline in water quality, through improved upstream land use practices and waste water treatment is vital to ensure the long term health of inshore reefs of the GBR (Fabricius et al., 2005). We predict that coral reefs are more likely to become macroalgal and benthic HAB dominated reefs if stressors such as eutrophication are not removed. Acknowledgements The authors are gratitude to the late Dan Wruck for his support in nutrient analysis at QHSS and for the field work support from Doug, Jeff, Samantha, Simon and Mica and the support from staff of the many tourist vessels which made the collection of samples possible. This research was supported by a grant to MPS from the Edward Koch Foundation. This research was carried out under Great Barrier Reef Marine Park Authority; Marine Parks permit G07/22433.1 and was endorsed by the IOC/UNESCO, GEOHAB program. References Aikman, K.E., 1992. The physiology, biochemistry, and toxicity of the marine dinoflagellae, Prorocentrum hoffmannianum Faust (Phyrophyta) and its role in seafood-borne human disease. Ph.D. Dissertation. Southern Illinois University, Carbondale, p. 124 Ajuzie, C.C., 2007. Palatability and fatality of the dinoflagellate Prorocentrum lima to Artemia salina. Journal Applied Phycology 19, 513–519. Alongi, D.M., McKinnon, A.D., 2005. The cycling and fate of terrestrially-derived sediments and nutrients in the coastal zone of the Great Barrier Reef shelf. Marine Pollution Bulletin 51, 239–252. APHA, 2005. Standard methods for the examination of water and waste water Edited by A.D. Eaton, L.S. Clesceri, E.W. Rice and A.E. Greenberg, 21st ed. APHAAWWA-WPCF. Bell, P.R.F., 1992. Eutrophication and coral reefs – some examples in the Great Barrier Reef lagoon. Water Resources 26 (5), 553–568. Burkholder, J.M., Glibert, P.M., Skelton, H.M., 2008. Mixotrophy, a major mode of nutrition for harmful algal species in eutrophic waters. Harmful Algae 8, 77–93. Fabricius, K., De’arth, G., Mc Cook, L., Turak, E., McWilliams, D., 2005. Changes in algal, coral and fish assemblages along water quality gradients on the inshore Great Barrier Reef. Marine Pollution Bulletin 51, 384–398. Faust, M.A., Gulledge, R.A., 2002. Identifying harmful marine dinoflagellates. Smithsonian institution. Contributions from the US National Herbarium 42, 1–144. Furnas, M., Mitchell, A., Skuza, M., Brodie, J., 2005. In the other 90%: Phytoplankton responses to enhanced nutrient availability in the Great Barrier Reef. Marine Pollution Bulletin 51, 253–265. Gillespie, N.C., Holmes, M.J., Burke, J.B., Doley, J., 1985. Distribution and periodicity of Gambierdiscus toxicus in Queensland, Australia. In: Anderson, White, Baden (Eds.), Toxic Dinoflagellates, Elsevier Science Publishing Co. Inc. Glibert, P.M., Burkholder, J.M., Kana, T.M., 2012. Recent insights about relationships between nutrient availability, forms, and stoichiometry, and the distribution, ecophysiology, and food web effects of pelagic and benthic Prorocentrum species. Harmful Algae 14, 231–259.
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