David Yellowlees

ENCORE: The effect of nutrient enrichment on coral reefs. Synthesis of results and conclusions

Coral reef degradation resulting from nutrient enrichment of coastal waters is of increasing global concern. Although effects of nutrients on coral reef organisms have been demonstrated in the laboratory, there is little direct evidence of nutrient effects on coral reef biota in situ. The ENCORE experiment investigated responses of coral reef organisms and processes to controlled additions of dissolved inorganic nitrogen (N) and/or phosphorus (P) on an offshore reef (One Tree Island) at the southern end of the Great Barrier Reef, Australia. A multi-disciplinary team assessed a variety of factors focusing on nutrient dynamics and biotic responses. A controlled and replicated experiment was conducted over two years using twelve small patch reefs ponded at low tide by a coral rim. Treatments included three control reefs (no nutrient addition) and three + N reefs (NH4Cl added), three + P reefs (KH2PO4 added), and three + N + P reefs. Nutrients were added as pulses at each low tide (ca twice per day) by remotely operated units. There were two phases of nutrient additions. During the initial, low-loading phase of the experiment nutrient pulses (mean dose = 11.5 microM NH4+; 2.3 microM PO4(-3)) rapidly declined, reaching near-background levels (mean = 0.9 microM NH4+; 0.5 microM PO4(-3)) within 2-3 h. A variety of biotic processes, assessed over a year during this initial nutrient loading phase, were not significantly affected, with the exception of coral reproduction, which was affected in all nutrient treatments. In Acropora longicyathus and A. aspera, fewer successfully developed embryos were formed, and in A. longicyathus fertilization rates and lipid levels decreased. In the second, high-loading, phase of ENCORE an increased nutrient dosage (mean dose = 36.2 microM NH4+; 5.1 microM PO4(-3)) declining to means of 11.3 microM NH4+ and 2.4 microM PO4(-3) at the end of low tide) was used for a further year, and a variety of significant biotic responses occurred. Encrusting algae incorporated virtually none of the added nutrients. Organisms containing endosymbiotic zooxanthellae (corals and giant clams) assimilated dissolved nutrients rapidly and were responsive to added nutrients. Coral mortality, not detected during the initial low-loading phase, became evident with increased nutrient dosage, particularly in Pocillopora damicornis. Nitrogen additions stunted coral growth, and phosphorus additions had a variable effect. Coral calcification rate and linear extension increased in the presence of added phosphorus but skeletal density was reduced, making corals more susceptible to breakage. Settlement of all coral larvae was reduced in nitrogen treatments, yet settlement of larvae from brooded species was enhanced in phosphorus treatments. Recruitment of stomatopods, benthic crustaceans living in coral rubble, was reduced in nitrogen and nitrogen plus phosphorus treatments. Grazing rates and reproductive effort of various fish species were not affected by the nutrient treatments. Microbial nitrogen transformations in sediments were responsive to nutrient loading with nitrogen fixation significantly increased in phosphorus treatments and denitrification increased in all treatments to which nitrogen had been added. Rates of bioerosion and grazing showed no significant effects of added nutrients. ENCORE has shown that reef organisms and processes investigated in situ were impacted by elevated nutrients. Impacts were dependent on dose level, whether nitrogen and/or phosphorus were elevated and were often species-specific. The impacts were generally sub-lethal and subtle and the treated reefs at the end of the experiment were visually similar to control reefs. Rapid nutrient uptake indicates that nutrient concentrations alone are not adequate to assess nutrient condition of reefs. Sensitive and quantifiable biological indicators need to be developed for coral reef ecosystems. The potential bioindicators identified in ENCORE should be tested in future research on coral reef/nutrient interactions. Synergistic and cumulative effects of elevated nutrients and other environmental parameters, comparative studies of intact vs. disturbed reefs, offshore vs. inshore reefs, or the ability of a nutrient-stressed reef to respond to natural disturbances require elucidation. An expanded understanding of coral reef responses to anthropogenic impacts is necessary, particularly regarding the subtle, sub-lethal effects detected in the ENCORE studies.

Metabolic interactions between algal symbionts and invertebrate hosts

Some invertebrates have enlisted autotrophic unicellular algae to provide a competitive metabolic advantage in nutritionally demanding habitats. These symbioses exist primarily but not exclusively in shallow tropical oceanic waters where clear water and low nutrient levels provide maximal advantage to the association. Mostly, the endosymbiotic algae are localized in host cells surrounded by a host-derived membrane (symbiosome). This anatomy has required adaptation of the host biochemistry to allow transport of the normally excreted inorganic nutrients (CO2, NH3 and PO43-) to the alga. In return, the symbiont supplies photosynthetic products to the host to meet its energy demands. Most attention has focused on the metabolism of CO2 and nitrogen sources. Carbon-concentrating mechanisms are a feature of all algae, but the products exported to the host following photosynthetic CO2 fixation vary. Identification of the stimulus for release of algal photosynthate in hospite remains elusive. Nitrogen assimilation within the symbiosis is an essential element in the hosts control over the alga. Recent studies have concentrated on cnidarians because of the impact of global climate change resulting in coral bleaching. The loss of the algal symbiont and its metabolic contribution to the host has the potential to result in the transition from a coral-dominated to an algal-dominated ecosystem.

Analysis of an EST library from the dinoflagellate (Symbiodinium sp.) symbiont of reef-building corals

Dinoflagellates (Symbiodinium sp. Freud.) are an obligatory endosymbiont of the reef‐building corals. Recent changes to the environment surrounding coral reefs (e.g., global warming) have demonstrated that this endosymbiotic relationship between corals and Symbiodinium is particularly sensitive to environmental changes. Therefore, understanding gene expression patterns of Symbiodinium is critical to understanding why coral reefs are susceptible to global climate change. This study identified 1456 unique expression sequence tags (ESTs) generated for Symbiodinium (clade C3) from the staghorn coral Acropora aspera following exposure to a variety of stresses. Of these, only 10% matched previously reported dinoflagellate ESTs, suggesting that the conditions used in the construction of the library resulted in a novel transcriptome. The function of 561 (44%) of these ESTs could be identified. The majority of these genes coded for proteins involved in posttranslational modification, protein turnover, and chaperones (12.3%); energy production and conversion (12%); or an unknown function (18.6%). The most common transcript found was a homologue to a bacterial protein of unknown function. This algal protein is targeted to the chloroplast and is present in those phototrophs that acquired plastids from the red algal lineage. An additional 48 prokaryote‐like proteins were also identified, including the first glycerol‐phosphate:phosphate antiporter from dinoflagellates. A protein with similarity to the fungi–archael–bacterial heme catalase peroxidases was also found. A variety of stress genes, in particular heat‐shock proteins and proteins involved in ubiquitin cascades, were also identified. This study is the first transcriptome from the unicellular component of a eukaryote–eukaryote symbiosis.

Moisture can be the dominant environmental parameter governing cadaver decomposition in soil

Forensic taphonomy involves the use of decomposition to estimate postmortem interval (PMI) or locate clandestine graves. Yet, cadaver decomposition remains poorly understood, particularly following burial in soil. Presently, we do not know how most edaphic and environmental parameters, including soil moisture, influence the breakdown of cadavers following burial and alter the processes that are used to estimate PMI and locate clandestine graves. To address this, we buried juvenile rat (Rattus rattus) cadavers (approximately 18 g wet weight) in three contrasting soils from tropical savanna ecosystems located in Pallarenda (sand), Wambiana (medium clay), or Yabulu (loamy sand), Queensland, Australia. These soils were sieved (2mm), weighed (500 g dry weight), calibrated to a matric potential of -0.01 megapascals (MPa), -0.05 MPa, or -0.3 MPa (wettest to driest) and incubated at 22 degrees C. Measurements of cadaver decomposition included cadaver mass loss, carbon dioxide-carbon (CO(2)-C) evolution, microbial biomass carbon (MBC), protease activity, phosphodiesterase activity, ninhydrin-reactive nitrogen (NRN) and soil pH. Cadaver burial resulted in a significant increase in CO(2)-C evolution, MBC, enzyme activities, NRN and soil pH. Cadaver decomposition in loamy sand and sandy soil was greater at lower matric potentials (wetter soil). However, optimal matric potential for cadaver decomposition in medium clay was exceeded, which resulted in a slower rate of cadaver decomposition in the wettest soil. Slower cadaver decomposition was also observed at high matric potential (-0.3 MPa). Furthermore, wet sandy soil was associated with greater cadaver decomposition than wet fine-textured soil. We conclude that gravesoil moisture content can modify the relationship between temperature and cadaver decomposition and that soil microorganisms can play a significant role in cadaver breakdown. We also conclude that soil NRN is a more reliable indicator of gravesoil than soil pH.

Inorganic Nitrogen Uptake by Symbiotic Marine Cnidarians: A Critical Review

The physiological data support host involvement in net ammonium uptake by intact symbioses. The evidence for nitrate assimilation by intact symbioses is equivocal. The depletion-diffusion model can account for net ammonium uptake by intact symbioses, but is inadequate to account for phosphate or nitrate uptake by symbioses. There is no evidence for nitrogen limitation as the means by which the host regulates algal growth in symbiosis; phosphorus limitation appears to be more likely.

Differential Responses of the Coral Host and Their Algal Symbiont to Thermal Stress

The success of any symbiosis under stress conditions is dependent upon the responses of both partners to that stress. The coral symbiosis is particularly susceptible to small increases of temperature above the long term summer maxima, which leads to the phenomenon known as coral bleaching, where the intracellular dinoflagellate symbionts are expelled. Here we for the first time used quantitative PCR to simultaneously examine the gene expression response of orthologs of the coral Acropora aspera and their dinoflagellate symbiont Symbiodinium. During an experimental bleaching event significant up-regulation of genes involved in stress response (HSP90 and HSP70) and carbon metabolism (glyceraldehyde-3-phosphate dehydrogenase, α-ketoglutarate dehydrogenase, glycogen synthase and glycogen phosphorylase) from the coral host were observed. In contrast in the symbiont, HSP90 expression decreased, while HSP70 levels were increased on only one day, and only the α-ketoglutarate dehydrogenase expression levels were found to increase. In addition the changes seen in expression patterns of the coral host were much larger, up to 10.5 fold, compared to the symbiont response, which in all cases was less than 2-fold. This targeted study of the expression of key metabolic and stress genes demonstrates that the response of the coral and their symbiont vary significantly, also a response in the host transcriptome was observed prior to what has previously been thought to be the temperatures at which thermal stress events occur.

Dinoflagellate symbioses: strategies and adaptations for the acquisition and fixation of inorganic carbon

Dinoflagellates exist in symbiosis with a number of marine invertebrates including giant clams, which are the largest of these symbiotic organisms. The dinoflagellates (Symbiodinium sp.) live intercellularly within tubules in the mantle of the host clam. The transport of inorganic carbon (Ci) from seawater to Symbiodinium (=zooxanthellae) is an essential function of hosts that derive the majority of their respiratory energy from the photosynthate exported by the zooxanthellae. Immunolocalisation studies show that the host has adapted its physiology to acquire, rather than remove CO2, from the haemolymph and clam tissues. Two carbonic anhydrase (CA) isoforms (32 and 70 kDa) play an essential part in this process. These have been localised to the mantle and gill tissues where they catalyse the interconversion of HCO3- to CO2, which then diffuses into the host tissues. The zooxanthellae exhibit a number of strategies to maximise Ci acquisition and utilisation. This is necessary as they express a form II Rubisco that has poor discrimination between CO2 and O2. Evidence is presented for a carbon concentrating mechanism (CCM) to overcome this disadvantage. The CCM incorporates the presence of a light-activated CA activity, a capacity to take up both HCO3-and CO2, an ability to accumulate an elevated concentration of Ci within the algal cell, and localisation of Rubisco to the pyrenoid. These algae also express both external and intracellular CAs, with the intracellular isoforms being localised to the thylakoid lumen and pyrenoid. These results have been incorporated into a model that explains the transport of Ci from seawater through the clam to the zooxanthellae.

Using Ninhydrin to Detect Gravesoil

Abstract:  Some death scene investigations commence without knowledge of the location of the body and/or decomposition site. In these cases, it is necessary to locate the remains or the site where the body decomposed prior to movement. We hypothesized that the burial of a mammalian cadaver will result in the release of ninhydrin reactive nitrogen (NRN) into associated soil and that this reaction might have potential as a tool for the identification of clandestine graves. Juvenile rat (Rattus rattus) cadavers were buried in three contrasting soil types in Australian tropical savanna ecosystems and allowed to decompose over a period of 28 days. Soils were sequentially harvested and analyzed for NRN. Cadaver burial resulted in an approximate doubling (mean = 1.7 ± 0.1) in the concentration of soil NRN. This reaction has great potential to be used as a presumptive test for gravesoil and this use might be greatly enhanced following more detailed research.

Role of carbonic anhydrase in the supply of inorganic carbon to the giant clam—zooxanthellate symbiosis

The mechanism whereby inorganic carbon (Ci) is acquired by the symbiotic association between the giant clam (Tridacna derasa) and zooxanthellae (Symbiodinium sp.) has been investigated. Ci in the haemolymph of the clam is in equilibrium with the surrounding sea water. The photosynthesis rate exhibited by the intact clam varies as a function of the Ci concentration in the clam haemolymph. The gill tissue contains high carbonic anhydrase activity which may be important in adjusting the Ci equilibrium between haemolymph and sea water. Zooxanthellae (Symbiodinium sp.) isolated from the clam mantle prefer CO2 to HCO3-as a source of inorganic carbon. The zooxanthellae have low levels of carbonic anhydrase on the external surface of the cell; however, mantle extracts display high carbonic anhydrase activity. Carbonic anhydrase is absent from the mantle of aposymbiotic clams (T. gigas), indicating that this enzyme may be essential to the symbiosis. The enzyme is probably associated with the zooxanthellae tubes in the mantle. The results indicate that carbonic anhydrase plays an important role in the supply of carbon dioxide within the clam symbiosis.

PRELIMINARY INVESTIGATIONS INTO THE STRUCTURE AND ACTIVITY OF RIBULOSE BISPHOSPHATE CARBOXYLASE FROM TWO PHOTOSYNTHETIC DINOFLAGELLATES1

Ribulose bisphosphate carboxylase / oxygenase (Rubisco) from the dinoflagellates Symbiodinium sp. Freudenthal and Amphidinium carterae Hulburt rapidly loses activity following cell lysis. Evidence presented indicates that this is not due to proteolysis. Using the tight binding inhibitor [14C] carboxyarabinitol bisphosphate as a marker, the Rubisco large subunit (LSu) from Symbiodinium sp. was purified. The subunit molecular weight was 56 kDa, while non‐denaturing polyacrylamide gel electrophoresis indicated that the purified protein had a molecular weight significantly less than that expected of the intact hexadecameric protein. No trace of the small subunit was apparent. The initial loss of carboxylase activity following cell lysis may be due to instability of the quaternary structure of the enzyme. Antibodies prepared to the purified LSU cross‐reacted with LSus from other dinoflagellates but not with the LSus of higher plants, diatoms, and other chromophytic algae. This suggests that the LSu of at least some dinoflagellates is antigenically different from that of other eukaryotes.