EXTRACELLULAR MATRIX PROTEO‐GLYCANS INVOLVED IN DIATOM ADHESION AND MOTILITY

Although diatom extracellular matricies are usually thought of exclusively in terms of the beautiful, architecturally complex silicious frustule, polymers exuded through the frustule are critical mediators of interactions with the external environment. In several species, complex proteoglycans appear to be the primary components involved in adhesion and motility. When viewed with high‐resolution cryo‐scanning electron microscopy methods, the ubiquity and pervasiveness of these polymers was revealed in both freshwater and marine taxa. Monoclonal antibody mapping of carbohydrate epitopes characterized by NMR, methylation and monosaccharide analysis and correlated with structural observations by EM revealed an organizational pattern far more complex than previously proposed. Modeling assembly of extracellular “stalks” in the marine biofouling diatom Achnanthes longipes involves intracellular sequestering of multiple components, deposition at the protoplasmic membrane/diatotepum interface, transport through the multilayered diatotepum and holes in the silica, extrusion from the frustule, and assembly into a very complex multi‐laminate biocomposite structure. The mechanism of extracellular polymer participation in motility is complex in a different way, as some current models of raphe associated motility involve cytoskeletal interactions and molecular motors.     

Extracellular matrix assembly in diatoms (Bacillariophyceae). iv. ultrastructure of Achnanthes longipes and Cymbella cistula as revealed by high-pressure freezing/freeze substituton and cryo-field emission scanning electron microscopy

Extracellular matrix (ECM) polymers secreted by the diatoms Achnanthes longipes Ag. and Cymbella cistula (Ehr.) Kirchn. completely encase the cell and are responsible for adhesion and other interactions with the external environment. To preserve details of the highly hydrophilic ECM in the native state and to preserve, with a high degree of fidelity, the intracellular structures involved in synthesis of extracellular polymers, we applied a suite of cryotechniques. The methods included high‐resolution visualization of surfaces using cryo‐field emission SEM (cryo‐FESEM) and preservation for TEM observation of thin sections by high‐pressure freezing (HPF) and freeze substitution (FS). The extracellular structures of diatoms plunge‐frozen in liquid ethane, etched at low temperature, and observed on a cryostage in the FESEM showed overall dimensions and shapes closely comparable to those observed with light microscopy. Cryo‐FESEM demonstrated the pervasive nature of the extracellular polymers and their importance in cell–substratum and cell–cell associations and revealed details of cell attachment processes not visible using other SEM techniques or light microscopy. The layer of ECM coating the frustule and entirely encapsulating cells of A. longipes and C. cistula was shown to have a significant role in initial cell adhesion and subsequent interaction with the environment. Trails of raphe‐associated ECM, generated during cell motility, were shown at high resolution and consist of anastomoses of coiled and linear strands. Cryo‐FESEM revealed a sheet‐like mucilage covering stalks. HPF/FS of A. longipes resulted in excellent preservation of intra‐ and extracellular structures comparable to previous reports for animals and higher plants and revealed several organelles not described previously. Three distinct vesicle types were identified, including a class closely associated with Golgi bodies and postulated to participate in formation of the extracellular adhesive structures. HPF/FS showed a number of continuous diatotepic layers positioned between the plasma membrane and the silicon frustule and revealed that extracellular adhesive extrusion through frustule pores during stalk production was closely related to the diatotepum. The stalks of A. longipes consist of highly organized, multilayered, fine fibrillar materials with an electron‐opaque layer organized as a sheath at the stalk periphery.     

Extracellular Matrix Assembly in Diatoms (Bacillariophyceae) (II. 2,6-Dichlorobenzonitrile Inhibition of Motility and Stalk Production in the Marine Diatom Achnanthes longipes)

The cellulose synthesis inhibitor 2,6-dichlorobenzonitrile (DCB) and the DCB analogs 2-chloro-6-fluorobenzonitrile, 3-amino-2,6-dichlorobenzonitrile, and 5-dimethylamino-naphthalene-1-sulfonyl-(3-cyano-2, 4-dichloro)aniline (DCBF) inhibited extracellular adhesive production in the marine diatom Achnanthes longipes, resulting in a loss of motility and a lack of permanent adhesion. The effect was fully reversible upon removal of the inhibitor, and cell growth was not affected at concentrations of inhibitors adequate to effectively interrupt the adhesion sequence. Video microscopy revealed that the adhesion sequence was mediated by the export and assembly of polymers, and consisted of initial attachment followed by cell motility and eventual production of permanent adhesive structures in the form of stalks that elevated the diatom above the substratum. A. longipes adhesive polymers are primarily composed of noncellulosic polysaccharides (B.A. Wustman, M.R. Gretz, and K.D. Hoagland [1997] Plant Physiol 113: 1059-1069). These results, together with the discovery of DCB inhibition of extracellular matrix assembly in noncellulosic red algal unicells (S.M. Arad, O. Dubinsky, and B. Simon [1994] Phycologia 33: 158-162), indicate that DCB inhibits synthesis of noncellulosic extracellular polysaccharides. A fluorescent probe, DCBF, was synthesized and shown to inhibit adhesive polymer production in the same manner as DCB. DCBF specifically labeled an 18-kD polypeptide isolated from a membrane fraction. Inhibition of adhesion by DCB and its analogs provides evidence of a direct relationship between polysaccharide synthesis and motility and permanent adhesion.     

NANOSTRUCTURE OF THE DIATOM FRUSTULE AS REVEALED BY ATOMIC FORCE AND SCANNING ELECTRON MICROSCOPY

The cell wall (frustule) of the freshwater diatom Pinnularia viridis (Nitzsch) Ehrenberg is composed of an assembly of highly silicified components and associated organic layers. We used atomic force microscopy (AFM) to investigate the nanostructure and relationship between the outermost surface organics and the siliceous frustule components of live diatoms under natural hydrated conditions. Contact mode AFM imaging revealed that the walls were coated in a thick mucilaginous material that was interrupted only in the vicinity of the raphe fissure. Analysis of this mucilage by force mode AFM demonstrated it to be a nonadhesive, soft, and compressible material. Application of greater force to the sample during repeated scanning enabled the mucilage to be swept from the hard underlying siliceous components and piled into columns on either side of the scan area by the scanning action of the tip. The mucilage columns remained intact for several hours without dissolving or settling back onto the cleaned valve surface, thereby revealing a cohesiveness that suggested a degree of cross-linking. The hard silicified surfaces of the diatom frustule appeared to be relatively smooth when living cells were imaged by AFM or when field-emission SEM was used to image chemically cleaned walls. AFM analysis of P. viridis frustules cleaved in cross-section revealed the nanostructure of the valve silica to be composed of a conglomerate of packed silica spheres that were 44.8 ± 0.7 nm in diameter. The silica spheres that comprised the girdle band biosilica were 40.3 ± 0.8 nm in diameter. Analysis of another heavily silicified diatom, Hantzschia amphioxys (Ehrenberg) Grunow, showed that the valve biosilica was composed of packed silica spheres that were 37.1 ± 1.4 nm and that silica particles from the girdle bands were 38.1 ± 0.5 nm. These results showed little variation in the size range of the silica particles within a particular frustule component (valve or girdle band), but there may be differences in particle size between these components within a diatom frustule and significant differences are found between species.     

Utilizing QCM-D to characterize the adhesive mucilage secreted by two marine diatom species in-situ and in real-time

The quartz crystal microbalance with dissipation monitoring (QCM-D) was used to monitor the deposition of adhesive extracellular polymeric substances (EPS) employed by the marine biofouling diatoms Craspedostauros australis Cox and Amphora coffeaeformis Cleve during initial adhesion and subsequent motility. Upon injection into the QCM chamber, initial negative frequency (f) shifts and positive dissipation (D) shifts were measured that correlated to cells impacting and adhering to the QCM sensor surface. Following this "initial adhesion" response, f continued to decrease while D increased logarithmically. Rather than the result of any cell morphological alterations at the substrate surface, the shifts were correlated to the time-dependent deposition of EPS upon the substrate surface as a result of cellular motility, or gliding. Experiments utilizing comparable cell concentrations of the diatom species C. australis and A. coffeaeformis revealed significant differences between the parameter responses recorded, where A. coffeaeformis produced Deltaf and DeltaD values of -32 Hz and 6.6, and C. australis produced values of -82 Hz and 42, respectively, after 20 h post-inoculation. The viscoelastic properties of the adhered EPS adlayer from both species were examined via a Deltaf/DeltaD plot, providing reproducible signature "ratio" values for each species that likely correlate to differences in EPS interactions with the substrate that may be associated directly to differences in the fouling potential of the two species. There is a distinct lack of knowledge regarding the chemical nature of the adhesive polymers engaged, and few quantitative techniques are applicable to the study of diatom EPS. We propose that QCM-D may be a useful tool in identifying differences in the EPS employed by diatoms of different fouling potential.     

Use of fluorophore-conjugated lectins to study cell-cell interactions in model marine biofilms

Biofilms dominated by pennate diatoms are important in fields as diverse as ship biofouling and marine littoral sediment stabilization. The architecture of a biofilm depends on the fact that much of its mass consists of extracellular polymers. Although most illuminated biofilms in nature are dominated by phototrophs, they also contain heterotrophic bacteria. Given the close spatial association of the two types of organisms, cell-cell interaction is likely. Fluorophore-conjugated lectins were used to demonstrate the sites of the various extracellular polymers in three species of diatoms. Based on their lectin staining properties, the polymers in different species appeared to be similar, but their involvement in the process of attachment to a surface differed. In a coculture Pseudoalteromonas sp. strain 4 or its sterilized spent medium reduced the ability of Amphora coffeaeformis and Navicula sp. strains 1 and D to adhere, inhibited motility, and caused agglutination and eventually diatom cell lysis. Diatoms could be protected from the negative effects of the bacterial spent medium if D-galactose or mannan was included in the incubation medium. The active principle of the spent medium is probably a lectin/agglutinin that is able to bind to the extracellular polymers of the diatoms that are involved in adhesion and motility. Awareness of interactions of this type is important in the study of natural biofilms.     

Micro‐photoluminescence of single living diatom cells

Diatoms are single‐celled microalgae that possess a nanostructured, porous biosilica shell called a frustule. This study characterized the micro‐photoluminescence (μ‐PL) emission of single living cells of the photosynthetic marine diatom Thalassiosira pseudonana in response to UV laser irradiation at 325 nm using a confocal Raman microscope. The photoluminescence (PL) spectrum had two primary peaks, one centered at 500–510 nm, which was attributed to the frustule biosilica, and a second peak at 680 nm, which was attributed to auto‐fluorescence of photosynthetic pigments. The portion of the μ‐PL emission spectrum associated with biosilica frustule in the single living diatom cell was similar to that from single biosilica frustules isolated from these diatom cells. The PL emission by the biosilica frustule in the living cell emerged only after cells were cultivated to silicon depletion. The discovery of the discovery of PL emission by the frustule biosilica within a single living diatom itself, not just its isolated frustule, opens up future possibilities for living biosensor applications, where the interaction of diatom cells with other molecules can be probed by μ‐PL spectroscopy. Copyright © 2016 John Wiley & Sons, Ltd.     

OBSERVATIONS ON THE ULTRASTRUCTURE OF THE MALI GAMETES OF BIDDULPHIA LEVIS EHR.1

The marine centric diatom Biddulphia levis produced uniflagellate fusiform male gametes completely within the parent cell frustule. These gametes lacked both a central pair of microtubules in the flagellar axoneme and chloroplasts but did contain a cone of microtubules which passed posteriorly from the base of the kinetosome along the nuclear envelope. The gametes were released through a specialized pore in the girdle band leaving behind a cytoplasmic mass which contained chloroplasts and other cytoplasmic components. Tubules which resembled the flimmer hairs on the gamete flagellum occurred in cisternae of the cytoplasmic reticulum in the residual cytoplasm and in the nuclear envelope of the gametes. Gametogenesis in B. levis is compared with similar processes in other centric diatoms.     

Ultrastructure of Anaulus creticus Drebes & Schulz with special reference to its reduced ocelli

The ultrastructure of the vegetative cell of the centric diatom Anaulus creticus Drebes & Schulz is described. Exhibiting the organic wall component (diatotepum) separated from the siliceous parts of the cell wall this species belongs to the diatom group with so-called split-walls [17]. A most interesting feature of the Anaulus cell is the horn region and its internal structure. As there is no silicified sieve-plate at the horn top but just a big hole sealed only by a thin diatotepum with a hexagonal meshwork, this wall structure is termed a "reduced ocellus". The inner part of the horn region is largely occupied by a conspicuous protoplasmic plug of proteinaceous nature. The possible function of this peculiar structure is discussed as well as new ideas about the role of the labiate process are provided.     

On the role of cell surface associated,mucin-like glycoproteins in the pennate diatom Craspedostauros australis (Bacillariophyceae)

Diatoms are single-celled microalgae with silica-based cell walls (frustules) that are abundantly present in aquatic habitats, and form the basis of the food chain in many ecosystems. Many benthic diatoms have the remarkable ability to glide on all natural or man-made underwater surfaces using a carbohydrate- and protein-based adhesive to generate traction. Previously, three glycoproteins, termed FACs (F rustule A ssociated C omponents), have been identified from the common fouling diatom Craspedostauros australis and were implicated in surface adhesion through inhibition studies with a glycan-specific antibody. The polypeptide sequences of FACs remained unknown, and it was unresolved whether the FAC glycoproteins are indeed involved in adhesion, or whether this is achieved by different components sharing the same glycan epitope with FACs. Here we have determined the polypeptide sequences of FACs using peptide mapping by LC–MS/MS. Unexpectedly, FACs share the same polypeptide backbone (termed CaFAP1), which has a domain structure of alternating Cys-rich and Pro-Thr/Ser-rich regions reminiscent of the gel-forming mucins. By developing a genetic transformation system for C. australis, we were able to directly investigate the function of CaFAP1-based glycoproteins in vivo. GFP-tagging of CaFAP1 revealed that it constitutes a coat around all parts of the frustule and is not an integral component of the adhesive. CaFAP1-GFP producing transformants exhibited the same properties as wild type cells regarding surface adhesion and motility speed. Our results demonstrate that FAC glycoproteins are not involved in adhesion and motility, but might rather act as a lubricant to prevent fouling of the diatom surface.     

Diatom motility and low frequency electromagnetic fields--a new technique in the search for independent replication of results

The hypothesis that exposure to a certain combination of static and alternating electromagnetic fields (EMFs) results in an increase in motility of the marine diatom Amphora coffeaeformis was tested. Diatom motility in three strains of A. coffeaeformis was positively correlated with extracellular calcium ion (Ca2+) concentration. The test apparatus consisted of two pairs of Helmholtz coils supported around the stage of a microscope linked to a video recorder and monitor. This system allowed real-time in vivo recordings of diatom speed under EMF and control exposures. The EMFs were calculated at calcium resonance values, previously found to cause enhanced motility. Computerised image analysis was used to calculate the distance moved by individual diatoms in 2-min periods before, during and after EMF or sham-EMF (control) exposure. The addition of EMF caused no significant increase in diatom motility. The results are discussed in relation to the use of diatom motility to measure EMF exposure effects.     

Substratum adhesion and gliding in a diatom are mediated by extracellular proteoglycans

Diatoms are unicellular microalgae encased in a siliceous cell wall, or frustule. Pennate diatoms, which possess bilateral symmetry, attach to the substratum at a slit in the frustule called the raphe. These diatoms not only adhere, but glide across surfaces whilst maintaining their attachment, secreting a sticky mucilage that forms a trail behind the gliding cells. We have raised monoclonal antibodies to the major cell surface proteoglycans of the marine raphid diatom Stauroneis decipiens Hustedt. The antibody StF.H4 binds to the cell surface, in the raphe and to adhesive trails and inhibits the ability of living diatoms to adhere to the substratum and to glide. Moreover, StF.H4 binds to a periodate-insensitive epitope on four frustule-associated proteoglycans (relative molecular masses 87, 112, and >200 kDa). Another monoclonal antibody, StF.D5, binds to a carbohydrate epitope on the same set of proteoglycans, although the antibody binds only to the outer surface of the frustule and does not inhibit cell motility and adhesion. Received: 2 December 1996 / Accepted: 6 March 1997     

Diatom Cells Grown and Baked on a Functionalized Mica Surface

We demonstrate the cultivation of diatom cells on a functionalized mica surface and the preparation of frustules on a mica surface by baking. Diatom cells were successfully grown on a mica surface treated with 3-aminopropyltriethoxysilane. After baking at 400?C for 2 h, frustule structures without the organic components of the diatom cells were successfully observed by scanning electron microscopy and atomic force microscopy. Furthermore, the frustules deformed and became slender when a sample was baked at 800?C for 2 h. Our method is effective for the direct characterization of frustule structures and physical properties without changing the configuration of the diatom cells grown on the mica surface.     

Algicidal activity and gliding motility of Saprospira sp. SS98-5

A marine bacterium, Saprospira sp. SS98-5, which was isolated from Kagoshima Bay, Japan, was able to kill and lyse the cells of the diatom Chaetoceros ceratosporum. The multicellular filamentous cells of this bacterium captured the diatom cells, formed cell aggregates, and lysed them in an enriched sea water (ESS) liquid medium. Strain SS98-5 also formed plaques on double layer agar plates incorporating diatom cells. The diatom cell walls were partially degraded at the contact sites with the bacteria, the bacteria invaded from there into the diatom cells, and then the diatom cells were completely lysed. The strain possessed gliding motility and grew as spreading colonies on ESS agar plates containing lower concentrations of polypeptone (below 0.1%) while forming nonspreading colonies on ESS agar plates containing 0.5% polypeptone. Electron micrographs of ultrathin sections demonstrated that microtubule-like structures were observable only in gliding motile cells. Both the gliding motility and the microtubule-like structures were diminished by the addition of podophyllotoxin, an inhibitor of microtubule assembly, suggesting that the microtubule-like structures observed in these bacterial cells are related to their gliding motility.     

Pleuralins are involved in theca differentiation in the diatom Cylindrotheca fusiformis

Diatom cells are encased within a silica-based cell wall (frustule) that serves as armour-like protection for the enclosed protoplast. Maintaining the integrity of the frustule requires a precise coupling between the biogenesis of new frustule components and the cell cycle. Thus far, the molecular mechanisms by which this coupling is achieved are unknown. This study demonstrates that pleuralins (formerly HEPs), a previously characterized family of diatom cell wall proteins, are involved in cell cycle-dependent frustule development. The frustule is made up of two, overlapping half-shells termed the epitheca and hypotheca. Both thecae are morphologically identical, yet immunolocalisation with anti-pleuralin antibodies demonstrates that their protein composition is clearly different. During interphase, pleuralins are associated only with the epitheca, where they are confined to the inner surface of the terminal elements (pleural bands) in the region of overlap with the hypotheca. At cell division, pleuralins also become associated with the newly formed pleural bands of the hypotheca. Remarkably, this process is concomitant with the functional conversion of the parental hypotheca into the epitheca of one of the progeny cells. These results indicate that developmentally controlled association of pleuralins with the frustule is involved in hypotheca-epitheca differentiation, which is a crucial process to ensure proper frustule development.     

Shaping the phycosphere: Analysis of the EPS in diatom-bacterial co-cultures

The phycosphere is a unique niche that fosters complex interactions between microalgae and associated bacteria. The formation of this extracellular environment, and the associated bacterial biodiversity, is heavily influenced by the secretion of extracellular polymers, primarily driven by phototrophic organisms. The exopolysaccharides (EPS) represent the largest fraction of the microalgae-derived exudates, which can be specifically used by heterotrophic bacteria as substrates for metabolic processes. Furthermore, it has been proposed that bacteria and their extracellular factors play a role in both the release and composition of the EPS. In this study, two model microorganisms, the diatom Phaeodactylum tricornutum CCAP 1055/15 and the bacterium Pseudoalteromonas haloplanktis TAC125, were co-cultured in a dual system to assess how their interactions modify the phycosphere chemical composition by analyzing the EPS monosaccharide profile released in the culture media by the two partners. We demonstrate that microalgal–bacterial interactions in this simplified model significantly influenced the architecture of their extracellular environment. We observed that the composition of the exo-environment, as described by the EPS monosaccharide profiles, varied under different culture conditions and times of incubation. This study reports an initial characterization of the molecular modifications occurring in the extracellular environment surrounding two relevant representatives of marine systems.     

Pleuralins are Involved in Theca Differentiation in the Diatom Cylindrotheca fusiformis

Diatom cells are encased within a silica-based cell wall (frustule) that serves as armour-like protection for the enclosed protoplast. Maintaining the integrity of the frustule requires a precise coupling between the biogenesis of new frustule components and the cell cycle. Thus far, the molecular mechanisms by which this coupling is achieved are unknown. This study demonstrates that pleuralins (formerly HEPs), a previously characterized family of diatom cell wall proteins, are involved in cell cycle-dependent frustule development. The frustule is made up of two, overlapping half-shells termed the epitheca and hypotheca. Both thecae are morphologically identical, yet immunolocalisation with anti-pleuralin antibodies demonstrates that their protein composition is clearly different. During interphase, pleuralins are associated only with the epitheca, where they are confined to the inner surface of the terminal elements (pleural bands) in the region of overlap with the hypotheca. At cell division, pleuralins also become associated with the newly formed pleural bands of the hypotheca. Remarkably, this process is concomitant with the functional conversion of the parental hypotheca into the epitheca of one of the progeny cells. These results indicate that developmentally controlled association of pleuralins with the frustule is involved in hypotheca-epitheca differentiation, which is a crucial process to ensure proper frustule development.     

A STUDY OF Si DEPOSITION SYNCHRONY IN RHIZOSOLENIA (BACILLARIOPHYCEAE) MATS USING A NOVEL 32Si AUTORADIOGRAPHIC METHOD

A ³²Si autoradiographic technique using a liquid photographic emulsion was developed for the study of diatom silica deposition in culture or in natural water samples. The method was used in the Central North Pacific to study silica deposition by diatoms of the genus Rhizosolenia. The species examined form centimeter-sized aggregates commonly referred to as mats. The Rhizosolenia mats examined were composed of a matrix of R. fallax Sundström chains, embedded with chains of larger cells, either R. debyana H. Peragallo or R. acuminata H. Peragallo. The autoradiographs revealed distinct rings of labeled intercalary bands and/or labeled valves. A greater proportion of the frustule of the larger species was labeled during the incubations with ³²Si, implying higher rates of silicification by R. debyana and R. accuminata compared to R. fallax. A quantitative consideration of these differences in species-specific Si production combined with abundance and surface area estimates for each species indicates that cells of the larger species carry out the majority of silica production in Rhizosolenia mats. The large cell size (pervalvar axis 240 to 3000 μm) and elongate frustule morphology of Rhizosolenia cells enabled us to localize the deposition of silica along the pervalvar axis. Positions of labeled bands along this axis indicate progress through the Si deposition cycle, and the results suggest that cell division is phased, with either a bimodal or unimodal age distribution of cells within the cell cycle for all species in a mat. Species-specific doubling times from 25 to 60 h were implied by the mean fractions of frustule that were labeled. ³²Si autoradiography revealed unique species-specific differences in diel patterns of cell division and silica deposition and has potential for studies of Si deposition by other diatom species and assemblages.     

OBSERVATIONS OF A TUBE-DWELLING DIATOM: NAVICULA HAMULIFERA (BACILLARIOPHYCEAE)1

Colonies of the tube-dwelling diatom Navicula hamulifera Grunow living on mangrove prop roots in Indian River, Florida and at La Parguera, Puerto Rico, were studied using light and electron microscopy. Observations of the tube morphology and cell structure of this diatom from fresh samples and cultures are described, as well as the ultrastructural morphology of its frustule. The formation of tubes by this diatom is reported for the first time. Comparisons are made with the closest species; Navicula delognei V.H. and Navicula pseudoco moides Hendey.     

Grazing-induced changes in cell wall silicification in a marine diatom

In aquatic environments, diatoms (Bacillariophyceae) constitute a central group of microalgae which contribute to about 40% of the oceanic primary production. Diatoms have an absolute requirement for silicon to build-up their silicified cell wall in the form of two shells (the frustule). To date, changes in diatom cell wall silicification have been only studied in response to changes in the growth environment, with consistent increase in diatom silica content when specific growth rates decrease under nutrient or light limitations. Here, we report the first evidence for grazing-induced changes in cell wall silicification in a marine diatom. Cells grown in preconditioned media that had contained both diatoms and herbivores are significantly more silicified than diatoms grown in media that have contained diatoms alone or starved herbivores. These observations suggest that grazing-induced increase in cell wall silicification can be viewed as an adaptive reaction in habitats with variable grazing pressure, and demonstrate that silicification in diatoms is not only a constitutive mechanical protection for the cell, but also a phenotypically plastic trait modulated by grazing. In turn, our results corroborate the idea that plant-herbivore interactions, beyond grazing sensu stricto, contribute to drive ecosystem structure and biogeochemical cycles in the ocean.