Grantee Research Project Results
2000 Progress 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 Period Covered by this Report: December 15, 1999 through December 14, 2000
Project Amount: $325,000
RFA: Harmful Algal Blooms (1997) RFA Text | Recipients Lists
Research Category: Water Quality , Water , Aquatic Ecosystems
Objective:
Our goals for Year 3 focused on Objectives 1-5 of the grant proposal and included: (1) continuing to monitor permanent transects for cyanobacterial abundance and diversity,( 2) making collections of cyanobacterial blooms, (3) identifying the cyanobacteria, (4) examining the natural products chemistry of these extracts for known and novel metabolites, (5) examining the capability of these extracts to deter feeding by generalist herbivores (fishes, urchins) and specialists such as sea hares, (6) examining the effects of Lyngbya majuscula on the settlement of coral larvae, (7) manipulating light and nutrients and determining the effects on cyanobacterial growth and secondary metabolite production, and (8) conducting a field experiment that manipulated herbivore abundance and nutrient availability and assessing the effects on cyanobacterial and macroalgal abundance.Progress Summary:
Objective 1:
To document the temporal and spatial patterns of cyanobacterial bloom formation in reef and seagrass habitats around the island of Guam.
We have continued to monitor 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, and Sails). We have continued to monitor these sites every 4 weeks. Over 25 strains of cyanobacteria have been recorded from our nine sites, and several occur at more than one location. Some patterns of seasonality are beginning to show up, but there is still variability from year to year.
We also have developed molecular techniques to obtain 16s rDNA sequences from these strains and characterize their genetic variation. Dr. Robert Thacker, a postdoctoral researcher on this project, has been instrumental in this work. We compared morphological and chemical characteristics with partial 16S ribosomal DNA sequences for several strains of L. majuscula, Lyngbya spp., and Symploca spp. found on Guam.
Morphology. Cell length and cell width of cyanobacteria were measured at 500x and 1250x magnification.
2-D TLC. Fresh collections of cyanobacteria were extracted in 1:1 CH2Cl2:MeOH (v:v). Solvents were removed by rotary evaporation. Two-dimensional TLC analysis was used to examine the chemical variation of the different collections. Each crude extract was dissolved in 1:1 CH2Cl2:MeOH and applied to form a single spot in a corner of an aluminum-backed Silica gel TLC sheet (10 cm x 10 cm). The TLC sheets were developed in 9:1 CH2Cl2:MeOH and removed after the solvent front traveled 8 cm. The TLC sheets were dried, then placed into another solvent chamber (50 percent EtOAc in hexanes) so that the developed extract was placed horizontally above the second solvent. The TLC sheets were removed after the solvent front traveled 8 cm. Pigmented compounds were marked by brackets directly on the TLC sheets. The sheets were placed in a UV viewing cabinet (254 nm) and locations of UV-fluorescing compounds were recorded on the sheets with circles. The sheets were coated with H2SO4 in EtOH (1:19) and heated with an air gun. Acid-charring compounds were marked on the sheets with arrows.
16S rDNA Sequencing. Genomic DNA was isolated from fresh collections of cyanobacteria using the G-NOME DNA isolation kit (Bio 101). 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.
Phylogenetic Analyses. All analyses were conducted using PAUP* (Swofford, 1999). Morphological measurements were entered as ordered characters, while 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. Numbers above branches indicate the number of bootstrap replicates that contained that clade.
Each strain of cyanobacteria has a distinct cellular morphology. Each strain has a unique chemotype, but common characters are shared among four strains of L. majuscula. Three closely related strains of L. majuscula showed 0.2 to 1.0 percent pairwise sequence divergence. Morphological characters distinguished Symploca from Lyngbya, 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 Lyngbya bouillonii that occurs at several sites around Guam has yielded a variety of known and new lipopeptides. A collection of Lyngbya 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 have tested the feeding deterrence of 17 of the extracted strains of cyanobacteria with parrotfish (Scarus schlegeli), urchins (Echinometra mathaei), and sea hares (Stylocheilus longicauda). 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 have 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. longicauda has been considered a specialist on Lyngbya 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. This work is now completed and a manuscript is in preparation.
Fish-Exclusion Experiment. In the past 2 years, we have found 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 this year's rabbitfish recruitment period (April-June). We placed fish-exclusion cages over 0.5 m x 0.5 m 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.
Another experiment was conducted at a depth of 10 m at two fore reef sites on
Guam, Piti Bombholes and Asan, which are located on the central eastern side of
Guam about 2 km apart. In this study, 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 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 data are now being analyzed, but preliminary results suggest
that both herbivory and fertilization had an important effect on algal and
cyanobacterial biomass on the tiles.
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 Lyngbya majuscula, a prolific cyanobacterium known to produce many toxic secondary metabolites, on the larvae of two species of coral, Acropora 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 and suggests the possibility of Lyngbya compounds acting as negative settlement cues.
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.
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. Two outdoor microcosm experiments were used to test the effects of iron, nitrate, and phosphate on three species of algae (Halimeda incrassata, Padina tenuis and Dictyota 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- (~ 6 µM), phosphate- (~ 1 µM), iron- (~ 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. A manuscript has been submitted to the journal Marine Ecology Progress Series summarizing this research.
References:
Nubel U, Garcia-Pichel F, Muyzer G. PCR primers to amplify 16S rRNA genes from cyanobacteria. Applied Environmental Microbiology 1997;63:3327-3332.
Swofford DL. PAUP*: Phylogenetic Analysis Using Parsimony (*and other methods), Version 4.0b3a. Sinauer, Sunderland, MA, 1999.
Future Activities:
We have made progress on many aspects of this research this past year and are now entering the final year of the project. We have addressed all of our original objectives, although there is still much that could be done for all aspects of this work. We will focus on writing and submitting all of the work that has been finished to date. The monitoring work will continue for at least the next 6 months; the data will then be analyzed and presentations and publications will be prepared.Journal Articles on this Report : 2 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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Thacker RW, Paul VJ. Are benthic cyanobacteria indicators of nutrient enrichment? Relationships between cyanobacterial abundance and environmental factors on the reef flats of Guam. Bulletin of Marine Science 2001;69(2):497-508. |
R826220 (2000) |
Exit |
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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 |
Supplemental Keywords:
harmful marine cyanobacteria, cyanotoxins, secondary metabolites, coral reefs, coastal management, harmful algal blooms, tropical HABs, ecological effects, natural toxins, marine, monitoring, Pacific Islands, Guam, herbivory, chemical defenses, ECOHAB., 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.