Publications

Aridgides, LJ, Doblin, MA, Berke, T, Dobbs, FC, Matson, DO & Drake, L 2004, 'Multiplex PCR allows simultaneous detection of pathogens in ships' ballast water', Marine Pollution Bulletin, vol. 48, no. 11-12, pp. 1096-1101.
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There is enormous potential for global transfer of microorganisms, including pathogens, in ships' ballast water. We contend that a major advancement in the study of ballast-water microorganisms in particular, and of aquatic pathogens in general, will be expedited sample analysis, such as provided by the elegant technology of DNA microarrays. In order to use DNA microarrays, however, one must establish the appropriate conditions to bind target sequences in samples to multiple probes on the microarrays. We conducted proof-of-concept experiments to optimize simultaneous detection of multiple microorganisms using polymerase chain reaction (PCR) and Southern hybridization. We chose three target organisms, all potentially found in ballast water: a calicivirus, the bacterium Vibrio cholerae, and the photosynthetic protist Aureococcus anophagefferens. Here, we show simultaneous detection of multiple pathogens is possible, a result supporting the promising future use of microarrays for simultaneous detection of pathogens in ballast water.

Baines, SB, Fisher, NK, Doblin, MA, Cutter, GA, Cutter, LS & Cole, B 2004, 'Light dependence of selenium uptake by phytoplankton and implications for predicting selenium incorporation into food webs', Limnology and Oceanography, vol. 49, no. 2, pp. 566-578.
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The potentially toxic element selenium is first concentrated from solution to a large but highly variable degree by algae and bacteria before being passed on to consumers. The large loads of abiotic and detrital suspended particles often present in rivers and estuaries may obscure spatial and temporal patterns in Se concentrations at the base of the food web. We used radiotracers to estimate uptake of both selenite (Se(IV)) and C by intact plankton communities at two sites in the Sacramento/San Joaquin River Delta. Our goals were to determine (1) whether C and Se(IV) uptake were coupled, (2) the role of bacteria in Se(IV) uptake, and (3) the Se: C uptake ratio of newly produced organic material. Se(IV) uptake, like C uptake, was strongly related to irradiance. The shapes of both relationships were very similar except that at least 42-56% of Se(IV) uptake occurred in the dark, whereas C uptake in the dark was negligible. Of this dark Se(IV) uptake, 34-67% occurred in the 0.2-1.0-mum size fraction, indicating significant uptake by bacteria. In addition to dark uptake, total Se(IV) uptake consisted of a light-driven component that was in fixed proportion to C uptake. Our estimates of daily areal Se(IV): C uptake ratios agreed very well with particulate Se: C measured at a site dominated by phytoplankton biomass. Estimates of bacterial Se: C were 2.4-13 times higher than for the phytoplankton, suggesting that bacteriovores may be exposed to higher dietary Se concentrations than herbivores.

Chen, M, Zeng, H, Larkum, AWD & Cai, ZL 2004, 'Raman properties of chlorophyll d, the major pigment of Acaryochloris marina: Studies using both Raman spectroscopy and density functional theory', Spectrochimica Acta - Part A: Molecular and Biomolecular Spectroscopy, vol. 60, no. 3, pp. 527-534.
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The Raman spectroscopy of purified chlorophyll (Chl) d extracted from Acaryochloris marina has been measured over the wide region of 250-3200 cm -1 at 77 K following excitation of its Soret band at 488 nm and analyzed with the aid of hybrid density-functional vibrational analyses. A Raman peak specific to Chl d, which arises from the formyl group 31 C = O stretching, was clearly observed at 1659 cm-1 with medium intensity. Peaks due to other C = O stretching vibrations of the 131 keto-, 133 ester- and 173 groups were also observed. Four very strong peaks were observed in the range of 1000-1600 cm-1, assigned to the CC stretching and mixtures of the CH3 bend and CN stretching. CCC and NCC bending contribute to medium intensity peaks at 986 and 915 cm-1. Out-of-plane CH bending at Chl d methine sites 10, 5 and 20 contribute to observed peaks at 885, 864 and 853 cm-1, respectively. A few modes involving the MgN stretching and MgNC bending motions were observed in the very low frequency range. Density functional theory (DFT) calculations have been used to make assignments on the observed Raman spectrum and the DFT results have been found to be in good agreement with the experimental results. © 2003 Elsevier B.V. All rights reserved.

Doblin, MA, Popels, LC, Coyne, K, Hutchins, DA, Cary, S & Dobbs, FC 2004, 'Transport of the harmful bloom alga Aureococcus anophagefferens by oceangoing ships and coastal boats', Applied and Environmental Microbiology, vol. 70, no. 11, pp. 6495-6500.
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It is well established that cyst-forming phytoplankton species are transported in ships' ballast tanks. However, there is increasing evidence that other phytoplankton species which do not encyst are also capable of surviving ballast transit. These species have alternative modes of nutrition (hetero- or mixotrophy) and/or are able to survive long-term darkness. In our studies of no-ballast-on-board vessels arriving in the Great Lakes, we tested for the presence of the harmful algal bloom species Aureococcus anophagefferens (brown tide) in residual (i.e., unpumpable) ballast water using methods based on the PCR. During 2001, the brown tide organism was detected in 7 of 18 ballast water tanks in commercial ships following transit from foreign ports. Furthermore, it was detected after 10 days of ballast tank confinement during a vessel transit in the Great Lakes, a significant result given the large disparity between the salinity tolerance for active growth of Aureococcus (>22 ppt) and the low salinity of the residual ballast water (similar to2 ppt). We also investigated the potential for smaller, recreational vessels to transport and distribute Aureococcus. During the summer of 2002, 11 trailered boats from the inland bays of Delaware and coastal bays of Maryland were sampled. Brown tide was detected in the bilge water in the bottoms of eight boats, as well as in one live-well sample. Commercial ships and small recreational boats are therefore implicated as potential vectors for long-distance transport and local-scale dispersal of Aureococcus.

Hill, R, Larkum, A, Frankart, C, Kuehl, M & Ralph, PJ 2004, 'Loss of functional Photosystem II reaction centres in zooxanthellae of corals exposed to bleaching conditions: using fluorescence rise kinetics', Photosynthesis Research, vol. 82, pp. 59-72.
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Hill, R, Schreiber, U, Gademann, R, Larkum, A, Kuehl, M & Ralph, PJ 2004, 'Spatial heterogeneity of photosynthesis and the effect of temperature-induced bleaching condition in three species of corals', Marine Biology, vol. 144, pp. 633-640.
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Huang, EP, Marquis, CP & Gray, PP 2004, 'Process development for a recombinant Chinese hamster ovary (CHO) cell line utilizing a metal induced and amplified metallothionein expression system.', Biotechnology and bioengineering, vol. 88, no. 4, pp. 437-450.
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The suspension Chinese Hamster Ovary cell line, 13-10-302, utilizing the metallothionein (MT) expression system producing recombinant human growth hormone (hGH) was studied in a serum-free and cadmium-free medium at different fermentation scales and modes of operation. Initial experiments were carried out to optimize the concentration of metal addition to induce the MT promoter. Subsequently, the cultivation of the 13-10-302 cell line was scaled up from spinner flasks into bioreactors, and the cultivation duration was extended with fed-batch and perfusion strategies utilizing 180 microM zinc to induce the promoter controlling expression of recombinant hGH. It was shown that a fed-batch process could increase the maximum cell numbers twofold, from 3.3 to 6.3 x 10(6) cell/mL, over those obtained in normal batch fermentations, and this coupled with extended fermentation times resulted in a fourfold increase in final hGH titer, from 135 +/- 15 to 670 +/- 70 mg/L at a specific productivity q(hGH) value of 12 pg cell(-1)d(-1). The addition of sodium butyrate increased the specific productivity of hGH in cells to a value of approximately 48 pg cell(-1)d(-1), resulting in a final hGH titer of over a gram per liter during fed-batch runs. A BioSep acoustic cell recycler was used to retain the cells in the bioreactor during perfusion operation. It was necessary to maintain the specific feeding rates (SFR) above a value of 0.2 vvd/(10(6) cell/mL) to maintain the viability and productivity of the 13-10-302 cells; under these conditions the viable cell number increased to over 10(7) cell/mL and resulted in a volumetric productivity of over 120 mg(hGH) L(-1)d(-1). Process development described in this work demonstrates cultivation at various scales and sustained high levels of productivity under cadmium free condition in a CHO cell line utilizing an inducible metallothionein expression system.

Jermiin, LS, Ho, SYW, Ababneh, F, Robinson, J & Larkum, AWD 2004, 'The biasing effect of compositional heterogeneity on phylogenetic estimates may be underestimated', Systematic Biology, vol. 53, no. 4, pp. 638-643.
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Macinnis-Ng, CM & Ralph, PJ 2004, 'In situ impact of multiple pulses of metal and herbicide on the seagrass| Zostera capricorni', Aquatic Toxicology, vol. 67, pp. 227-237.
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Macintyre, H, Lomas, M, Cornwell, J, Suggett, DJ, Gobler, C, Koch, E & Kana, T 2004, 'Mediation Of Benthic-pelagic Coupling By Microphytobenthos: An Energy- And Material-based Model For Initiation Of Blooms Of Aureococcus Anophagefferens', Harmful Algae, vol. 3, no. 4, pp. 403-437.
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We present a conceptual model for initiation of blooms of the estuarine brown-tide pelagophyte Aureococcus anophagefferens. The model is based on the observation that in addition to its well-documented stimulation by organic nutrients, Aureococcus is pie

Ostrowski, M, Fegatella, F, Wasinger, V, Guilhaus, M, Corthals, GL & Cavicchioli, R 2004, 'Cross-species identification of proteins from proteome profiles of the marine oligotrophic ultramicrobacterium, Sphingopyxis alaskensis', PROTEOMICS, vol. 4, no. 6, pp. 1779-1788.
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Runcie, J, Larkum, A & Ritchie, RJ 2004, 'Uptake kinetics and assimilation of phosphorus by Catenella nipae and Ulva lactuca can be used to indicate ambient phosphate availability', Journal of Applied Phycology, vol. 16, no. 3, pp. 181-194.
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Uptake, assimilation and compartmentation of phosphate were studied in the opportunist green macroalgaUlva lactucaand the estuarine red algal epiphyteCatenella nipae. The MichaelisMenten model was used to describe uptake rates of inorganic phosphate (Pi) at different concentrations. Maximum uptake rates (V max) of P-starved material exceededV maxof P-enriched material; this difference was greater forC. nipae. Uptake and allocation of phosphorus (P) to internal pools was measured using trichloroacetic acid (TCA) extracts and32P. Both species demonstrated similar assimilation paths: when P-enriched, most32P accumulated as free phosphate. When unenriched,32P was rapidly assimilated into the TCA-insoluble pool.C. nipaeconsistently assimilated more32P into this pool thanU. lactuca, indicatingC. nipaehas a greater P-storage capacity. In both species,32P release data showed two internal compartments with very different biological half-lives.

Ryan, KG, Ralph, PJ & McMinn, A 2004, 'Acclimation of Antarctic bottom-ice algal communities to lowered salinities during melting', Polar Biology, vol. 27, pp. 679-686.
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Seymour, JR, Mitchell, JG & Seuront, L 2004, 'Microscale heterogeneity in the activity of coastal bacterioplankton communities', Aquatic Microbial Ecology, vol. 35, no. 1, pp. 1-16.
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Microscale sampling techniques and flow cytometry were employed to measure the distribution patterns of 8 subpopulations of bacteria separated according to variations in cell fluorescence and light scatter properties. Subpopulations of bacteria defined on the basis of these parameters have recently been shown to represent cells exhibiting dissimilar activity levels, and we therefore assume that the subpopulations of bacteria identified here represent metabolically diverse groups. Microscale distribution patterns of these subpopulations were measured at a resolution of 4.5 and 12 mm, within 2 dissimilar coastal habitats, A mean 2-fold change in the abundance of the total bacterial community across sample sets was observed. However, levels of spatial heterogeneity were consistently higher for the cytometrically defined subpopulations than total counts. In most samples, the population of bacteria exhibiting the highest levels of green fluorescence, or DNA content, and hence assumed to represent the most active bacteria in the community, also showed the highest levels of microscale spatial variability, with a maximum change in abundance of 14.5-fold observed across a distance of 9 nun. Where Zipf rank-size analysis was conducted, the microscale distributions of subpopulations differed significantly (p < 0.05) in 79% of cases, implying that bacterial communities are made up of physiologically distinct compartments, perhaps influenced by different microscale features of the environment. We suggest that these results provide the first evidence for the existence of microscale heterogeneity in the metabolic activity of aquatic bacterial communities.

Siboni, N, Fine, M, Bresler, V & Loya, Y 2004, 'Coastal coal pollution increases Cd concentrations in the predatory gastropod Hexaplex trunculus and is detrimental to its health', Marine Pollution Bulletin, vol. 49, no. 1-2, pp. 111-118.
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Parameters of environmental health, including paracellular permeability of external epithelia, functional state of lysosomes and the level of metallothioneins (MTs), were examined using fluorescent markers and vital microfluorometry in different tissues of the marine gastropod, Hexaplex trunculus, from a coal-polluted and coal-free site. Vital microfluorometrical examinations exhibited enhanced paracellular permeability of external epithelia to the anionic marker, fluorescein (FLU), lower lysosomal accumulation of neutral red (NR) as well as higher levels of MTs, when compared with epithelia of gastropods from the coal-free site. Those differences were particularly marked in the foot epithelium, which is in direct contact with the substrate. In addition, cadmium was measured by ICP-AES in the hepatopancreas of gastropods sampled from the coal-polluted site and two coal-free sites. Significantly higher levels of Cd were found in gastropod hepatopancreas from the coal-polluted site. In addition, two months feeding experiments conducted in aquaria containing: (a) coal pieces covered by barnacles; (b) natural rocks covered by barnacles; and (c) natural rocks with barnacles+bare coal pieces, demonstrated significant increase of Cd concentration in the hepatopancreas of the gastropods exposed to coal. We suggest that coal in the marine environment has detrimental effects on marine gastropods, both directly through contact with the organisms and indirectly through the food web. © 2004 Elsevier Ltd. All rights reserved.

Siboni, N, Fine, M, Bresler, V & Loya, Y 2004, 'Coastal coal pollution increases Cd concentrations in the predatory gastropod Hexaplex trunculus and is detrimental to its health', MARINE POLLUTION BULLETIN, vol. 49, no. 1-2, pp. 111-118.
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Stewart, AR, Luoma, SN, Schlekat, CE, Doblin, MA & Hieb, KA 2004, 'Food web pathway determines how selenium affects aquatic ecosystems: A San Francisco Bay case study', Environmental Science & Technology, vol. 38, no. 17, pp. 4519-4526.
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Chemical contaminants disrupt ecosystems, but specific effects may be under-appreciated when poorly known processes such as uptake mechanisms, uptake via diet, food preferences, and food web dynamics are influential. Here we show that a combination of food web structure and the physiology of trace element accumulation explain why some species in San Francisco Bay are threatened by a relatively low level of selenium contamination and some are not. Bivalves and crustacean zooplankton form the base of two dominant food webs in estuaries, The dominant bivalve Potamocorbula amurensis has a 10-fold slower rate constant of loss for selenium than do common crustaceans such as copepods and the mysid Neomysis mercedis (rate constant of loss, k(e) = 0.025, 0.155, and 0.25 d(-1), respectively). The result is much higher selenium concentrations in the bivalve than in the crustaceans. Stable isotope analyses show that this difference is propagated up the respective food webs in San Francisco Bay. Several predators of bivalves have tissue concentrations of selenium that exceed thresholds thought to be associated with teratogenesis or reproductive failure (liver Se > 15 mug g(-1) dry weight). Deformities typical of selenium-induced teratogenesis were observed in one of these species. Concentrations of selenium in tissues of predators of zooplankton are less than the thresholds. Basic physiological and ecological processes can drive wide differences in exposure and effects among species, but such processes are rarely considered in traditional evaluations of contaminant impacts.

Suggett, DJ, MacIntyre, HL & Geider, RJ 2004, 'Evaluation of biophysical and optical determinations of light absorption by photosystem II in phytoplankton', Limnology And Oceanography-methods, vol. 2, pp. 316-332.
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Fast repetition rate (FRR) fluorescence can provide highly resolved estimates of light absorption by photosystem II ( PSII), a variable that is critical to bio-optical determinations of phytoplankton productivity. We compared estimates of chlorophyll a -

Salih, A, Larkum, A, Cronin, T, Wiedenmann, J, Szymczak, R & Cox, G 2004, 'Biological properties of coral GFP-type proteins provide clues for engineering novel optical probes and biosensors', Proceedings of SPIE - The International Society for Optical Engineering, pp. 61-72.
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In recent years, a variety of Green Fluorescent Protein (GFP)-like pigments have been discovered from corals and other marine organisms. They are widely used to expand the range of available GFP-type proteins in imaging applications, such as in vivo markers for gene expression and protein localization studies, FRET-based (Förster resonance energy transfer) multicolor imaging and biosensors. They have known diverse optical and biochemical properties but their in vivo spectral properties and biological function in marine organisms is only beginning to be understood We have investigated their spectral diversity, optical properties and cellular microstructure in corals of the Great Barrier Reef with the aim of elucidating their photo-biological function/s as well as to identify novel proteins suitable for GFP-based technologies. We found numerous spectral variants, with emissions covering almost the full range of the visible spectrum. Many of these GFP-like proteins, especially in corals from the more extreme habitats, such as sun-exposed shallows or in deep water, showed a range of light-related spectral characteristics: high photostability, spectral tuning for energy transfer and dynamic photo-induced transformation properties. Intra-cellularly they were organized into spectral donor-acceptor pairs or even arrays, tuned for FRET. Coral color proteins thus offer an exciting potential to expand the use of the available GFPs in bio-imaging applications and as a basis for improved protein engineering.

Seuront, L, Leterme, S, Seymour, JR & Mitchell, JG 2004, 'Sampling the sampling unit: A world in a bottle.', United States.

Seymour, JR & Mitchell, JG 2004, 'Microscale distributions of bacterioplankton and virus-like particles assessed using flow cytometry', United States.