Grantee Research Project Results
Final Report: Chemical Ecology of Cyanobacterial Blooms on the Tropical Reefs of Guam
EPA Grant Number: R826220Title: Chemical Ecology of Cyanobacterial Blooms on the Tropical Reefs of Guam
Investigators: Paul, Valerie J.
Institution: University of Guam
EPA Project Officer: Packard, Benjamin H
Project Period: December 15, 1997 through December 14, 2000 (Extended to December 14, 2001)
Project Amount: $325,000
RFA: Harmful Algal Blooms (1997) RFA Text | Recipients Lists
Research Category: Water Quality , Water , Aquatic Ecosystems
Objective:
This project examined the chemical ecology of cyanobacterial blooms on Guam. We studied the temporal and spatial patterns of bloom formation and persistence, the natural products produced by bloom cyanobacteria, the effects of these compounds on potential consumers, and the environmental factors regulating the production of toxic and deterrent compounds by the harmful cyanobacterial blooms (HCBs). These research questions coincide with the ECOHAB program objective, which is to "understand the fundamental processes underlying the population dynamics of HABs and the impacts they precipitate".The research objectives of the project were to: (1) document the temporal and spatial patterns of cyanobacterial bloom formation in reef and seagrass habitats around the island of Guam; (2) characterize and quantify the natural products chemistry of these cyanobacterial blooms; (3) determine the effects of the natural products (secondary metabolites) isolated from cyanobacterial blooms on potential consumers, including herbivorous reef fishes and specialist mesograzers such as sea hares; (4) examine the types and amounts of compounds released by cyanobacteria and their effects on consumers, competitors, and other microorganisms; and (5) manipulate light and nutrients (nitrogen, phosphorus, and iron) in field and laboratory experiments to determine their effects on secondary metabolite production by cyanobacteria under natural conditions. The progress made in addressing each of these objectives is described below.
Summary/Accomplishments (Outputs/Outcomes):
Objective 1: To document the temporal and spatial patterns of cyanobacterial bloom formation in reef and seagrass habitats around the island of Guam.We established permanent transects at nine locations around Guam: Tanguisson, Pago Bay, Togcha, Cocos Lagoon, Fingers Reef, Piti Bombholes, and three transects in Tumon Bay (Ypao, Pia Marine, and Sails). Our initial plan for biweekly surveys was changed to once every 3-4 weeks, because most sites did not change quickly.
The transects were monitored at these locations for more than 3 years. We are continuing to monitor sites at Pago Bay, Togcha, Cocos Lagoon, Piti Bombholes, Ypao, Pia, and Sails in Tumon Bay every 4 weeks. We will continue to monitor these sites until July 2002, which will give us 4 years of data. More than 25 different species or strains of cyanobacteria have been recorded from our nine sites, and several occur at more than one location. The abundance of cyanobacteria at our field sites has ranged from 0 to 70 percent cover. Some field sites are dominated by one or a few abundant cyanobacteria, while others have low numbers of cyanobacteria, but high diversity. Our data show no clear associations between cyanobacterial, abundance and nutrient concentrations in the water column (Thacker and Paul, 2001).
We developed molecular techniques to obtain 16S ribosomal DNA sequences from these strains and characterize their genetic variation. We compared morphological and chemical characteristics with partial 16S ribosomal DNA sequences for several strains of Lyngbya (L.) majuscula, L. spp., and Symploca spp. found on Guam (Thacker and Paul, in review). Genomic DNA was isolated from fresh collections of cyanobacteria using the G-NOME DNA isolation kit (Bio 101). Polymerase Chain Reaction (PCR) amplification with cyanobacteria-specific primers followed the protocol of Nubel, et al.(1997). PCR products were cleaned with preparatory columns, then directly sequenced at a commercial facility. All phylogenetic analyses were conducted using Phylogenetic Analysis Using Parsimony (PAUP*) (Swofford, 1999). Morphological measurements were entered as ordered characters, and chemical characters were entered as unordered characters. Trees represent the consensus tree of 100 bootstrap replicates with a heuristic search for the most parsimonious tree.
Each chemotype of cyanobacteria has a distinct cellular morphology, but common characters are shared among four strains of L. majuscula. Three closely related chemotypes of L. majuscula showed 0.2 to 1.0 percent pairwise sequence divergence. Morphological characters distinguished Symploca from L., while chemical characters established phylogenetic relationships among closely related L. majuscula. 16S sequences reconstructed relationships among distantly related taxa, but did not resolve relationships among closely related L. majuscula.
Objective 2: To characterize and quantify the natural products chemistry of these cyanobacterial blooms.
Twenty-five of the most common strains in our study sites have been extracted, yielding from 0.5 to 10 percent crude organic extract per dry mass. Many of these have been extracted in bulk, providing enough extract for chemical studies and bioassays with herbivores. Compounds have been isolated including dolastatin 11, symplostatins 1 and 2, malyngamides A and B, and majusculamides A and B. A red strain of L. bouillonii that occurs at several sites around Guam has yielded a variety of known and new lipopeptides including a toxic substance named apratoxin A. A collection of L. majuscula from Piti Bomb Holes, Guam, yielded two new compounds, pitipeptolides A and B.
Objective 3: To determine the effects of the natural products (secondary metabolites) isolated from cyanobacterial blooms on potential consumers, including herbivorous reef fishes and specialist mesograzers such as sea hares.
We tested the feeding deterrence of 17 of the extracted strains of
cyanobacteria with the parrotfish Scares schlegeli, the sea urchin
Echinometra mathaei, and the sea hare Stylocheilus (S.) striatus.
We have used two types of assays: (1) comparing consumption of freeze-dried,
powdered cyanobacteria with consumption of freeze-dried, powdered
Enteromorpha clathrata (a palatable green alga); and (2) comparing
consumption of crude organic extracts of cyanobacteria coated onto powdered
Enteromorpha with consumption of uncoated Enteromorpha. All of the
powdered cyanobacteria that we have tested significantly reduced feeding by all
three consumers. Of the assays with crude extracts, 11 deterred feeding by
parrotfish, while 6 had no significant effect. Urchins were deterred by 8
extracts, while 6 had no significant effect. Sea hares were deterred by only 3
extracts, while 2 had no significant effect and 12 stimulated feeding. S.
striatus has been considered a specialist on L. majuscula, but it
clearly can consume other cyanobacteria, including Tolypothrix,
Oscillatoria, and Hormothamnion. Sea hare consumption of
cyanobacteria was not significantly correlated with crude extract yields or
percent nitrogen content of the cyanobacteria.
Fish-Exclusion Experiments. We have observed a correlation between cyanobacterial bloom formation and seasonal fish recruitment. To determine if there is a direct effect of herbivorous fishes on the formation of cyanobacterial blooms, we conducted a field experiment during the rabbitfish recruitment period of April-June 1998. We placed fish-exclusion cages over 0.5 times 0.5 meter quadrats on a reef flat for 3 months. We also monitored control cages that allowed fishes inside and control quadrats without cages. Higher palatability macroalgae, including Acanthophora spicifera and Caulerpa racemosa, showed dramatic increases in abundance in the exclusion cages. A bloom of the cyanobacterium Hormothamnion enteromorphoides occurred in all three treatments, with higher abundances in the no-cage quadrats than in either caged treatment. Thus, alterations in light or water flow due to the cages could have decreased Hormothamnion growth. Herbivory did not restrict the growth of Hormothamnion.
In 1999, we examined the impacts of both herbivory (again using uncaged, partially caged, and fully caged quadrats) and nutrient enrichment (using a commercial fertilizer, Osmocote Extended Time Release Fertilizer, containing nitrogen and phosphorus) on cyanobacterial and macroalgal community structure (Thacker, et al., 2001). We conducted the experiment on a reef flat that was initially dominated by the cyanobacteria Oscillatoria spp. and Tolypothrix sp. In the absence of cages, surface cover of the cyanobacterium Tolypothrix sp. declined, while cover of the cyanobacteria Oscillatoria spp. increased over the 4-month duration of the study. Lower cyanobacterial cover and biomass in the partial and full cages were correlated with higher macroalgal cover and biomass. The macroalga Dictyota (D.) bartayresii dominated the partial cages, while the macroalgae Padina tenuis and Tolypiocladia glomerulata recruited into the full cages. Palatability assays demonstrated that herbivore-exclusion shifted macroalgal species composition from unpalatable to palatable species. Nutrient enrichment increased cover of D. bartayresii in the uncaged and fully caged plots, but had no other significant effects. These results stress the critical role of herbivory in determining coral reef community structure and suggest that herbivores may buffer the effects of nutrient enrichment when the dominant algae are palatable. When the dominant algae are unpalatable, herbivores may be unable to compensate for increased levels of productivity due to nutrient enrichment.
Another experiment was conducted at a depth of 10 meters at two fore reef sites on Guam, Piti Bombholes and Asan, which are located on the central eastern side of Guam about 2 kilometers apart. The purpose of the study was to examine the combined interactions of reduced herbivory and increased nutrients on the colonization of crustose coralline algae (CCA) and fleshy algae, including cyanobacteria. Caging was again used to manipulate herbivory, and fertilization also was conducted simultaneously in a factorial design. Individual terra cotta tiles were used as replicates to study algal biomass and community structure in response to the various treatments. Herbivory was manipulated with three levels of herbivore exclusion: fully caged tiles, partially caged tiles, and uncaged tiles. Two levels of nutrient enrichment were used, with and without bags of slow-release fertilizer (Osmocote, N-P-K, 14-14-14). Ten replicates of each of the six treatments were placed at both the Piti and Asan sites. The treatments were left in place for 6 weeks during late June to early August 2000. The responses of the following variables were measured: percent cover of CCA, fleshy algal biomass, coral recruitment, and sediment load. Fleshy algal biomass and sediment were greatest on tiles where herbivores were excluded, whereas CCA were greatest on tiles exposed to herbivores. Nutrients did not have a significant effect on fleshy algae; however, CCA decreased in response to increased nutrients at one site. Coral recruitment was low, and preferential settlement to tiles with more CCA was only marginally significant at P equals 0.06. This study demonstrated that herbivory, in particular, can have a marked response on the initial composition of the benthic community, even over a relatively short time span. Moreover, the inverse relationship between CCA, and both fleshy algae and sediment, has implications for the recruitment of organisms, such as corals, with reliance on CCA. This work was conducted by graduate student Stephanie Belliveau for her Master's thesis research (Belliveau, 2001) and is now in press (Belliveau and Paul, 2002).
Objective 4: To examine the types and amounts of compounds released by cyanobacteria and their effects on consumers, competitors, and other microorganisms.
The settlement of coral larvae to suitable substrate is a critical component of the life histories of scleractinian corals. Research has shown that crustose coralline algae and biological films are sources of positive settlement cues for coral larvae, but little research has been conducted on possible negative cues. We examined the effects of L. majuscula, a prolific cyanobacterium known to produce many toxic secondary metabolites, on the larvae of two species of coral, Acropora (A.) surculosa (broadcast spawner) and Pocillopora damicornis (brooder). Larvae were reared from fertilization (A. surculosa) or collected from adult colonies (P. damicornis) and placed in specially designed larval settlement chambers for 1 week at 10 m depth. One hundred larvae per chamber were allowed to settle to biologically conditioned terra cotta settlement tiles in the presence of either L. majuscula or a plastic aquarium plant (control). The presence of L. majuscula had a negative effect on larval survival and settlement of both species of coral. Also, P. damicornis larvae showed preferential settlement to the empty area of the tile when L. majuscula was present, but settled all over the tile in chambers with the control plastic plants. These results suggest that L. majuscula could potentially inhibit the successful recruitment of coral larvae to the reef.
Objective 5: To manipulate light and nutrients (nitrogen, phosphorus, and iron) in field and laboratory experiments to determine their effects on secondary metabolite production by cyanobacteria under natural conditions.
Two separate experiments have been conducted to begin to address this objective. Cyanobacteria and blue-green algae are primary producers that can respond to physical environmental factors such as light and nutrients in ways similar to plants. In the first study, which was conducted by Ronald Pangilinan for his Master's thesis research (Pangilinan, 2000), light and nutrient resources available to the benthic marine cyanobacterium L. majuscula, which is known to produce differing amounts and types of secondary metabolites, were manipulated in laboratory experiments. Light significantly affected the growth and the concentration of organic extract and the major secondary metabolite pitipeptolide A. Conversely, enhanced nitrogen and phosphorus did not influence growth or secondary metabolite production. These results suggest that light may be the major factor influencing secondary metabolite formation for L. majuscula.
The observed high abundance of algae and cyanobacteria on Guam's coral reefs raises concern regarding a possible shift from coral- to algal-dominated communities. Possible increased nutrient supply to macroalgae and cyanobacteria via the watershed due to anthropogenic disturbance could be a partial cause. In a second study, two outdoor microcosm experiments were used to test the effects of iron, nitrate, and phosphate on three species of algae (Halimeda incrassata, Padina (P.) tenuis, and Dictyota (D.) bartayresiana) and three species of cyanobacteria (Tolypothrix sp., Schizothrix sp., and Lyngbya majuscula) from Cocos Lagoon, Guam. The six species were cultured together sewn to an artificial substrate for 9 days, with either nitrate- (approximately 6 µM), phosphate- (approximately 1 µM), iron- (approximately 0.5 µM) enriched or control (ambient nutrients) conditions. Overall, gram-specific growth was greatest for L. majuscula, which grew at nine times the rate of the other species. Algae did not show statistically significant nutrient limitation, although results with D. bartayresiana and P. tenuis suggested iron and nitrate limited growth in the first and second experiment, respectively. Two species of cyanobacteria showed phosphate limitation. The growth of L. majuscula was enhanced with phosphate enrichment, whereas the release of hormogonia by Tolypothrix sp., not the growth of the colonies themselves, also may have been enhanced. Patterns of Tolypothrix sp. hormogonia release also suggested possible direct competition between macroalgae and cyanobacteria; the hormogonia aggregated upon some species but not others. The results of this study suggest that L. majuscula may have more efficient growth and/or nutrient uptake mechanisms compared to the other species and that it is capable of increased growth in response to phosphate in the water column (Kuffner and Paul, 2001).
Journal Articles on this Report : 5 Displayed | Download in RIS Format
Other project views: | All 30 publications | 6 publications in selected types | All 6 journal articles |
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Type | Citation | ||
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Belliveau SA, Paul VJ. Effects of herbivory and nutrients on the early colonization of crustose coralline and fleshy algae. Marine Ecology Progress Series 2002, Volume: 232, Page: 105-114. |
R826220 (Final) |
not available |
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Kuffner IB, Paul VJ. Effects of nitrate, phosphate and iron on the growth of macroalgae and benthic cyanobacteria from Cocos Lagoon, Guam. Marine Ecology-Progress Series 2001;222:63-72. |
R826220 (Final) |
not available |
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Nubel U, Garcia-Pichel F, Muyzer G. PCR primers to amplify 16S rRNA genes from cyanobacteria. Applied and Environmental Microbiology 1997;63(8):3327-3332. |
R826220 (Final) |
not available |
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Thacker RW, Ginsburg DW, Paul VJ. Effects of herbivore exclusion and nutrient enrichment on coral reef macroalgae and cyanobacteria. Coral Reefs 2001;19(4):318-329. |
R826220 (1999) R826220 (2000) R826220 (Final) |
not available |
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Thacker RW, Paul VJ. Morphological, chemical, and genetic diversity of tropical marine cyanobacteria, Lyngbya spp. and Symploca spp. Applied and Environmental Microbiology 2004;70(6):3305-3312. |
R826220 (Final) |
not available |
Supplemental Keywords:
cyanobacteria, marine, ecological effects, chemical ecology, monitoring, western Pacific., RFA, Geographic Area, Water, Ecosystem Protection/Environmental Exposure & Risk, Ecosystem/Assessment/Indicators, Ecosystem Protection, Ecological Effects - Environmental Exposure & Risk, algal blooms, Ecological Indicators, ecological exposure, chemical ecology, ecological effects, spectroscopic methods, bloom dynamics, marine biotoxins, coral reefs, harmful algal blooms, cyanobacterial blooms, tropical reefs, bioassay, benthic algae, natural toxins, cyanobacteriaProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.