Widespread use of proton-pumping rhodopsin in Antarctic phytoplankton.

Photosynthetic carbon (C) fixation by phytoplankton in the Southern Ocean (SO) plays a critical role in regulating air-sea exchange of carbon dioxide and thus global climate. In the SO, photosynthesis (PS) is often constrained by low iron, low temperatures, and low but highly variable light intensities. Recently, proton-pumping rhodopsins (PPRs) were identified in marine phytoplankton, providing an alternate iron-free, light-driven source of cellular energy. These proteins pump protons across cellular membranes through light absorption by the chromophore retinal, and the resulting pH energy gradient can then be used for active membrane transport or for synthesis of adenosine triphosphate. Here, we show that PPR is pervasive in Antarctic phytoplankton, especially in iron-limited regions. In a model SO diatom, we found that it was localized to the vacuolar membrane, making the vacuole a putative alternative phototrophic organelle for light-driven production of cellular energy. Unlike photosynthetic C fixation, which decreases substantially at colder temperatures, the proton transport activity of PPR was unaffected by decreasing temperature. Cellular PPR levels in cultured SO diatoms increased with decreasing iron concentrations and energy production from PPR photochemistry could substantially augment that of PS, especially under high light intensities, where PS is often photoinhibited. PPR gene expression and high retinal concentrations in phytoplankton in SO waters support its widespread use in polar environments. PPRs are an important adaptation of SO phytoplankton to growth and survival in their cold, iron-limited, and variable light environment.

The requirement for external carbonic anhydrase in diatoms is influenced by the supply and demand for dissolved inorganic carbon.

Photosynthesis by marine diatoms contributes significantly to the global carbon cycle. Due to the low concentration of CO2 in seawater, many diatoms use extracellular carbonic anhydrase (eCA) to enhance the supply of CO2 to the cell surface. While much research has investigated how the requirement for eCA is influenced by changes in CO2 availability, little is known about how eCA contributes to CO2 supply following changes in the demand for carbon. We therefore examined how changes in photosynthetic rate influence the requirement for eCA in three centric diatoms. Modeling of cell surface carbonate chemistry indicated that diffusive CO2 supply to the cell surface was greatly reduced in large diatoms at higher photosynthetic rates. Laboratory experiments demonstrated a trend of an increasing requirement for eCA with increasing photosynthetic rate that was most pronounced in the larger species, supporting the findings of the cellular modeling. Microelectrode measurements of cell surface pH and O2 demonstrated that individual cells exhibited an increased contribution of eCA to photosynthesis at higher irradiances. Our data demonstrate that changes in carbon demand strongly influence the requirement for eCA in diatoms. Cell size and photosynthetic rate will therefore be key determinants of the mode of dissolved inorganic carbon uptake.

Pervasive iron limitation at subsurface chlorophyll maxima of the California Current.

Subsurface chlorophyll maximum layers (SCMLs) are nearly ubiquitous in stratified water columns and exist at horizontal scales ranging from the submesoscale to the extent of oligotrophic gyres. These layers of heightened chlorophyll and/or phytoplankton concentrations are generally thought to be a consequence of a balance between light energy from above and a limiting nutrient flux from below, typically nitrate (NO3). Here we present multiple lines of evidence demonstrating that iron (Fe) limits or with light colimits phytoplankton communities in SCMLs along a primary productivity gradient from coastal to oligotrophic offshore waters in the southern California Current ecosystem. SCML phytoplankton responded markedly to added Fe or Fe/light in experimental incubations and transcripts of diatom and picoeukaryote Fe stress genes were strikingly abundant in SCML metatranscriptomes. Using a biogeochemical proxy with data from a 40-y time series, we find that diatoms growing in California Current SCMLs are persistently Fe deficient during the spring and summer growing season. We also find that the spatial extent of Fe deficiency within California Current SCMLs has significantly increased over the last 25 y in line with a regional climate index. Finally, we show that diatom Fe deficiency may be common in the subsurface of major upwelling zones worldwide. Our results have important implications for our understanding of the biogeochemical consequences of marine SCML formation and maintenance.

Influence of Temperature and CO2 On Plasma-membrane Permeability to CO2 and HCO3- in the Marine Haptophytes Emiliania huxleyi and Calcidiscus leptoporus (Prymnesiophyceae).

Membrane permeabilities to CO2 and HCO3- constrain the function of CO2 concentrating mechanisms that algae use to supply inorganic carbon for photosynthesis. In diatoms and green algae, plasma membranes are moderately to highly permeable to CO2 but effectively impermeable to HCO3- . Here, CO2 and HCO3- membrane permeabilities were measured using an 18 O-exchange technique on two species of haptophyte algae, Emiliania huxleyi and Calcidiscus leptoporus, which showed that the plasma membranes of these species are also highly permeable to CO2 (0.006-0.02 cm · s-1 ) but minimally permeable to HCO3- . Increased temperature and CO2 generally increased CO2 membrane permeabilities in both species, possibly due to changes in lipid composition or CO2 channel proteins. Changes in CO2 membrane permeabilities showed no association with the density of calcium carbonate coccoliths surrounding the cell, which could potentially impede passage of compounds. Haptophyte plasma-membrane permeabilities to CO2 were somewhat lower than those of diatoms but generally higher than membrane permeabilities of green algae. One caveat of these measurements is that the model used to interpret 18 O-exchange data assumes that carbonic anhydrase, which catalyzes 18 O-exchange, is homogeneously distributed in the cell. The implications of this assumption were tested using a two-compartment model with an inhomogeneous distribution of carbonic anhydrase to simulate 18 O-exchange data and then inferring plasma-membrane CO2 permeabilities from the simulated data. This analysis showed that the inferred plasma-membrane CO2 permeabilities are minimal estimates but should be quite accurate under most conditions.

Plasma Membrane-Type Aquaporins from Marine Diatoms Function as CO2/NH3 Channels and Provide Photoprotection.

Aquaporins (AQPs) are ubiquitous water channels that facilitate the transport of many small molecules and may play multiple vital roles in aquatic environments. In particular, mechanisms to maintain transmembrane fluxes of important small molecules have yet to be studied in marine photoautotrophic organisms. Here, we report the occurrence of multiple AQPs with differential cellular localizations in marine diatoms, an important group of oceanic primary producers. The AQPs play a role in mediating the permeability of membranes to CO2 and NH3 In silico surveys revealed the presence of five AQP orthologs in the pennate diatom Phaeodactylum tricornutum and two in the centric diatom Thalassiosira pseudonana GFP fusions of putative AQPs displayed clear localization to the plasma membrane (PtAGP1 and PtAQP2), the chloroplast endoplasmic reticulum (CER; PtAGP1 and PtAQP3), and the tonoplast (PtAQP5) in P. tricornutum In T. pseudonana, GFP-AQP fusion proteins were found on the vacuole membrane (TpAQP1) and CER (TpAQP2). Transcript levels of both PtAQP1 and PtAQP2 were highly induced by ammonia, while only PtAQP2 was induced by high (1%[v/v]) CO2 Constitutive overexpression of GFP-tagged PtAQP1 and PtAQP2 significantly increased CO2 and NH3 permeability in P. tricornutum, strongly indicating that these AQPs function in regulating CO2/NH3 permeability in the plasma membrane and/or CER. Cells carrying GFP-tagged PtAQP1 and PtAQP2 had higher nonphotochemical quenching under high light relative to that of wild-type cells, suggesting that these AQPs are involved in photoprotection. These AQPs may facilitate the efflux of NH3, preventing the uncoupling effect of high intracellular ammonia concentrations.

The potential for co-evolution of CO2-concentrating mechanisms and Rubisco in diatoms.

Diatoms are a diverse group of unicellular algae that contribute significantly to global photosynthetic carbon fixation and export in the modern ocean, and are an important source of microfossils for paleoclimate reconstructions. Because of their importance in the environment, diatoms have been a focus of study on the physiology and ecophysiology of carbon fixation, in particular their CO2-concentrating mechanisms (CCMs) and Rubisco characteristics. While carbon fixation in diatoms is not as well understood as in certain model aquatic photoautotrophs, a greater number of species have been examined in diatoms. Recent work has highlighted a large diversity in the function, physiology, and kinetics of both the CCM and Rubisco between different diatom species. This diversity was unexpected since it has generally been assumed that CCMs and Rubiscos were similar within major algal lineages as the result of selective events deep in evolutionary history, and suggests a more recent co-evolution between the CCM and Rubisco within diatoms. This review explores our current understanding of the diatom CCM and highlights the diversity of both the CCM and Rubisco kinetics. We will suggest possible environmental, physiological, and evolutionary drivers for the co-evolution of the CCM and Rubisco in diatoms.

The diversity of CO2-concentrating mechanisms in marine diatoms as inferred from their genetic content.

Marine diatoms are one of the most ecologically significant primary producers in the ocean. Most diatoms use a CO2-concentrating mechanism (CCM) to overcome the scarcity of CO2 in the ocean and limitations of the carbon-fixing enzyme Rubisco. However, the CCMs in model diatoms differ substantially in their genetic make-up and structural organization. To assess the extent of CCM diversity in marine diatoms more generally, we analyzed genome and transcriptome data from 31 diatom strains to identify putative CCM genes, examine the overall CCM architecture, and study CCM development in the context of the evolutionary history of these diatoms. Key CCM genes [carbonic anhydrases (CAs) and solute carrier 4 (SLC4) bicarbonate transporters] identified in the diatoms were placed into groups of likely orthologs by sequence similarity (OrthoMCL) and phylogenetic methods. These analyses indicated that diatoms seem to share similar HCO3- transporters, but possess a variety of CAs that have either undergone extensive diversification within the diatom lineage or have been acquired through horizontal gene transfer. Hierarchical clustering of the diatom species based on their CCM gene content suggests that CCM development is largely congruent with evolution of diatom species, despite some notable differences in CCM genes even among closely related species.

Low temperature reduces the energetic requirement for the CO2 concentrating mechanism in diatoms.

The goal of this study is to investigate the CO2 concentrating mechanism (CCM) of the dominant phytoplankton species during the growing season at Palmer station in the Western Antarctic Peninsula. Key CCM parameters (cellular half-saturation constants for CO2 fixation, carbonic anhydrase activity, CO2 /HCO3 (-) uptake, δ(13) Corg ) in natural phytoplankton assemblages were determined. Those results, together with additional measurements on CO2 membrane permeability from Fragilariopsis cylindrus laboratory cultures, were used to develop a numerical model of the CCM of cold water diatoms. The field data demonstrate that the dominant species throughout the season possess an effective CCM, which achieves near saturation of CO2 for fixation. The model provides a means to examine the role of eCA activity and HCO3 (-) /CO2 uptake in the functioning of the CCM. According to the model, the increase in δ(13) Corg during the bloom results chiefly from decreasing ambient CO2 concentration (which reduces the gross diffusive flux across the membrane) rather than a shift in inorganic carbon uptake from CO2 to HCO3 (-) . The CCM of diatoms in the Western Antarctic Peninsula functions with a relatively small expenditure of energy, resulting chiefly from the low half-saturation constant for Rubisco at cold temperatures.

The minimal CO2-concentrating mechanism of Prochlorococcus spp. MED4 is effective and efficient.

As an oligotrophic specialist, Prochlorococcus spp. has streamlined its genome and metabolism including the CO2-concentrating mechanism (CCM), which serves to elevate the CO2 concentration around Rubisco. The genomes of Prochlorococcus spp. indicate that they have a simple CCM composed of one or two HCO3(-) pumps and a carboxysome, but its functionality has not been examined. Here, we show that the CCM of Prochlorococcus spp. is effective and efficient, transporting only two molecules of HCO3(-) per molecule of CO2 fixed. A mechanistic, numerical model with a structure based on the CCM components present in the genome is able to match data on photosynthesis, CO2 efflux, and the intracellular inorganic carbon pool. The model requires the carboxysome shell to be a major barrier to CO2 efflux and shows that excess Rubisco capacity is critical to attaining a high-affinity CCM without CO2 recovery mechanisms or high-affinity HCO3(-) transporters. No differences in CCM physiology or gene expression were observed when Prochlorococcus spp. was fully acclimated to high-CO2 (1,000 µL L(-1)) or low-CO2 (150 µL L(-1)) conditions. Prochlorococcus spp. CCM components in the Global Ocean Survey metagenomes were very similar to those in the genomes of cultivated strains, indicating that the CCM in environmental populations is similar to that of cultured representatives.

Internal carbonic anhydrase activity in the tissue of scleractinian corals is sufficient to support proposed roles in photosynthesis and calcification.

Reef-building corals import inorganic carbon (Ci) to build their calcium carbonate skeletons and to support photosynthesis by the symbiotic algae that reside in their tissue. The internal pathways that deliver Ci for both photosynthesis and calcification are known to involve the enzyme carbonic anhydrase (CA), which interconverts CO2 and HCO3 (-). We have developed a method for absolute quantification of internal CA (iCA) activity in coral tissue based on the rate of (18)O-removal from labeled Ci. The method was applied to three Caribbean corals (Orbicella faveolata, Porites astreoides and Siderastrea radians) and showed that these species have similar iCA activities per unit surface area, but that S. radians has ∼10-fold higher iCA activity per unit tissue volume. A model of coral Ci processing shows that the measured iCA activity is sufficient to support the proposed roles for iCA in Ci transport for photosynthesis and calcification. This is the case even when iCA activity is homogeneously distributed throughout the coral, but the model indicates that it would be advantageous to concentrate iCA in the spaces where calcification (the calcifying fluid) and photosynthesis (the oral endoderm) take place. We argue that because the rates of photosynthesis and calcification per unit surface area are similar among the corals studied here, the areal iCA activity used to deliver Ci for these reactions should also be similar. The elevated iCA activity per unit volume of S. radians compared with that of the other species is probably due to the thinner effective tissue thickness in this species.

Size scaling of extracellular carbonic anhydrase activity in centric marine diatoms.

Many microalgae have a surface-associated extracellular carbonic anhydrase (eCA) that converts HCO3 (-) to CO2 for uptake and subsequent photosynthetic fixation. We investigated eCA activity and assessed its importance for photosynthetic CO2 supply in six centric diatom species spanning nearly the full range of cell sizes for centric diatoms (equivalent spherical radius 3-67 µm). Since larger cells are more susceptible to diffusion limitation, we hypothesized that eCA activity would increase with cell size as would its importance for CO2 supply. eCA activity did increase with cell size, increasing with cell radius by a size-scaling exponent of 2.6 ± 0.3. The rapid increase in eCA activity with cell radius keeps the absolute CO2 concentration difference between bulk seawater and the cell surface very low (<~0.2 µM) allowing high rates of CO2 uptake even for large diatoms. Although inhibiting eCA did reduce photosynthesis in the diatoms, there was no overall relationship between the extent of inhibition of photosynthesis and cell size. The only indication that eCA may be more important for larger diatoms was that photosynthesis in the smallest diatoms (<4 µm radius) was only affected by eCA inhibition when CO2 concentrations were very low, while photosynthesis in some larger diatoms was affected even at typical seawater CO2 concentrations. eCA is ubiquitous in centric marine diatoms, in contrast to other taxa where its presence is irregularly distributed among different species, and plays an important role in supplying CO2 for photosynthesis across the size spectrum.

Quantification of extracellular carbonic anhydrase activity in two marine diatoms and investigation of its role.

Many microalgae induce an extracellular carbonic anhydrase (eCA), associated with the cell surface, at low carbon dioxide (CO2) concentrations. This enzyme is thought to aid inorganic carbon uptake by generating CO2 at the cell surface, but alternative roles have been proposed. We developed a new approach to quantify eCA activity in which a reaction-diffusion model is fit to data on (18)O removal from inorganic carbon. In contrast to previous methods, eCA activity is treated as a surface process, allowing the effects of eCA on cell boundary-layer chemistry to be assessed. Using this approach, we measured eCA activity in two marine diatoms (Thalassiosira pseudonana and Thalassiosira weissflogii), characterized the kinetics of this enzyme, and studied its regulation as a function of culture pH and CO2 concentration. In support of a role for eCA in CO2 supply, eCA activity specifically responded to low CO2 rather than to changes in pH or HCO3(-), and the rates of eCA activity are nearly optimal for maintaining cell surface CO2 concentrations near those in the bulk solution. Although the CO2 gradients abolished by eCA are small (less than 0.5 µm concentration difference between bulk and cell surface), CO2 uptake in these diatoms is a passive process driven by small concentration gradients. Analysis of the effects of short-term and long-term eCA inhibition on photosynthesis and growth indicates that eCA provides a small energetic benefit by reducing the surface-to-bulk CO2 gradient. Alternative roles for eCA in CO2 recovery as HCO3(-) and surface pH regulation were investigated, but eCA was found to have minimal effects on these processes.

A chloroplast pump model for the CO2 concentrating mechanism in the diatom Phaeodactylum tricornutum.

Prior analysis of inorganic carbon (Ci) fluxes in the diatom Phaeodactylum tricornutum has indicated that transport of Ci into the chloroplast from the cytoplasm is the major Ci flux in the cell and the primary driving force for the CO2 concentrating mechanism (CCM). This flux drives the accumulation of Ci in the chloroplast stroma and generates a CO2 deficit in the cytoplasm, inducing CO2 influx into the cell. Here, the "chloroplast pump" model of the CCM in P. tricornutum is formalized and its consistency with data on CO2 and HCO3 (-) uptake rates, carbonic anhydrase (CA) activity, intracellular Ci concentration, intracellular pH, and RubisCO characteristics is assessed. The chloroplast pump model can account for the major features of the data. Analysis of photosynthetic and Ci uptake rates as a function of external Ci concentration shows that the model has the most difficulty obtaining sufficiently low cytoplasmic CO2 concentrations to support observed CO2 uptake rates at low external Ci concentrations and achieving high rates of photosynthesis. There are multiple ways in which model parameters can be varied, within a plausible range, to match measured rates of photosynthesis and CO2 uptake. To increase CO2 uptake rates, CA activity can be increased, kinetic characteristics of the putative chloroplast pump can be enhanced to increase HCO3 (-) export, or the cytoplasmic pH can be raised. To increase the photosynthetic rate, the permeability of the pyrenoid to CO2 can be reduced or RubisCO content can be increased.

Localization of putative carbonic anhydrases in the marine diatom, Thalassiosira pseudonana.

Thirteen putative carbonic anhydrase (CA) genes have been identified in the marine multipolar centric diatom, Thalassiosira pseudonana, and two of these CAs have been localized previously. The first, an alpha CA (TpαCA1), was localized in the chloroplast stroma; the second, a zeta-type CA (TpζCA1), was localized to the periplasmic space. In the present study, cloning and localization of the remaining CAs were carried out. TpγCA2, TpγCA3, TpγCA4, TpγCA5, TpδCA1, TpδCA2, TpδCA3, and TpζCA1 were responsive to CO2 availability at the transcriptional level, being significantly reduced in cells grown at 0.4 % CO2, whereas TpαCA1, TpαCA2, TpαCA3, TpγCA1, and TpδCA4 transcript levels were constitutive with respect to CO2 concentration. Full-length cDNAs for TpγCA1, TpγCA2, TpγCA3, TpγCA4, TpδCA1, and TpδCA2 were isolated and fused with the enhanced-green fluorescent gene at their 3' termini. These GFP-fusion constructs were transformed into T. pseudonana, and the resulting GFP fusion products were localized using fluorescence microscopy. The δ-type TpδCA1 was localized on the periphery of the cell, strongly suggesting localization to the periplasmic space or the frustule. The δ-type TpδCA3 and the γ-type TpγCA2 were, respectively, localized in a periplastidal compartment and the cytosol. The δ-type TpδCA2, and the γ-types TpγCA1, 3, and 4 were localized in the mitochondria. The distribution of CAs in T. pseudonana contrasts notably with that of the raphid pennate diatom P. tricornutum, with likely consequences for CCM function including modes of CO2 acquisition, regions in which DIC is accumulated, and needs for minimizing CO2 leakage from the chloroplast.

Efficiency of the CO2-concentrating mechanism of diatoms.

Diatoms are responsible for a large fraction of CO(2) export to deep seawater, a process responsible for low modern-day CO(2) concentrations in surface seawater and the atmosphere. Like other photosynthetic organisms, diatoms have adapted to these low ambient concentrations by operating a CO(2) concentrating mechanism (CCM) to elevate the concentration of CO(2) at the site of fixation. We used mass spectrometric measurements of passive and active cellular carbon fluxes and model simulations of these fluxes to better understand the stoichiometric and energetic efficiency and the physiological architecture of the diatom CCM. The membranes of diatoms are highly permeable to CO(2), resulting in a large diffusive exchange of CO(2) between the cell and external milieu. An active transport of carbon from the cytoplasm into the chloroplast is the main driver of the diatom CCM. Only one-third of this carbon flux is fixed photosynthetically, and the rest is lost by CO(2) diffusion back to the cytoplasm. Both the passive influx of CO(2) from the external medium and the recycling of the CO(2) leaking out of the chloroplast are achieved by the activity of a carbonic anhydrase enzyme combined with the maintenance of a low concentration of HCO(3)(-) in the cytoplasm. To achieve the CO(2) concentration necessary to saturate carbon fixation, the CO(2) is most likely concentrated within the pyrenoid, an organelle within the chloroplast where the CO(2)-fixating enzyme is located.

13C NMR metabolomics: J-resolved STOCSY meets INADEQUATE.

Robust annotation of metabolites is a challenging task in metabolomics. Among available applications, 13C NMR experiment INADEQUATE determines direct 13C-13C connectivity unambiguously, offering indispensable information on molecular structure. Despite its great utility, it is not always practical to collect INADEQUATE data on every sample in a large metabolomics study because of its relatively long experiment time. Here, we propose an alternative approach that maintains the quality of information but saves experiment time. In this approach, individual samples in a study are first screened by 13C homonuclear J-resolved experiment (JRES). Next, JRES data are processed by statistical total correlation spectroscopy (STOCSY) to extract peaks that behave similarly among samples. Finally, INADEQUATE is collected on one internal pooled sample to select STOCSY peaks that originate from the same compound. We tested this concept using the 13C-labeled endometabolome of a model marine diatom strain incubated under various settings, intending to cover a range of metabolites produced under different external conditions. This scheme was able to extract known diatom metabolites proline, 2,3-dihydroxypropane-1-sulfonate (DHPS), ß-1,3-glucan, choline, and glutamate. This pipeline also detected unknown compounds with structural information, which is valuable in metabolomics where a priori knowledge of metabolites is not always available. The ability of this scheme was seen even in sugar regions, which are usually challenging in 1H NMR due to severe peak overlap. JRES and INADEQUATE were highly complementary; INADEQUATE provided directly-bonded 13C networks, whereas JRES linked INADEQUATE networks within the same compound but broken by nitrogen or sulfur atoms, highlighting the advantage of this integrated approach.

Iron transporters in marine prokaryotic genomes and metagenomes.

In the pelagic environment, iron is a scarce but essential micronutrient. The iron acquisition capabilities of selected marine bacteria have been investigated, but the recent proliferation of marine prokaryotic genomes and metagenomes offers a more comprehensive picture of microbial iron uptake pathways in the ocean. Searching these data sets, we were able to identify uptake mechanisms for Fe(3+), Fe(2+) and iron chelates (e.g. siderophore and haem iron complexes). Transport of iron chelates is accomplished by TonB-dependent transporters (TBDTs). After clustering the TBDTs from marine prokaryotic genomes, we identified TBDT clusters for the transport of hydroxamate and catecholate siderophore iron complexes and haem using gene neighbourhood analysis and co-clustering of TBDTs of known function. The genomes also contained two classes of siderophore biosynthesis genes: NRPS (non-ribosomal peptide synthase) genes and NIS (NRPS Independent Siderophore) genes. The most common iron transporters, in both the genomes and metagenomes, were Fe(3+) ABC transporters. Iron uptake-related TBDTs and siderophore biosynthesis genes were less common in pelagic marine metagenomes relative to the genomic data set, in part because Pelagibacter ubique and Prochlorococcus species, which almost entirely lacked these Fe uptake systems, dominate the metagenomes. Our results are largely consistent with current knowledge of iron speciation in the ocean, but suggest that in certain niches the ability to acquire siderophores and/or haem iron chelates is beneficial.

The BBSome restricts entry of tagged carbonic anhydrase 6 into the cis-flagellum of Chlamydomonas reinhardtii.

The two flagella of Chlamydomonas reinhardtii are of the same size and structure but display functional differences, which are critical for flagellar steering movements. However, biochemical differences between the two flagella have not been identified. Here, we show that fluorescence protein-tagged carbonic anhydrase 6 (CAH6-mNG) preferentially localizes to the trans-flagellum, which is organized by the older of the two flagella-bearing basal bodies. The uneven distribution of CAH6-mNG is established early during flagellar assembly and restored after photobleaching, suggesting that it is based on preferred entry or retention of CAH6-mNG in the trans-flagellum. Since CAH6-mNG moves mostly by diffusion, a role of intraflagellar transport (IFT) in establishing its asymmetric distribution is unlikely. Interestingly, CAH6-mNG is present in both flagella of the non-phototactic bardet-biedl syndrome 1 (bbs1) mutant revealing that the BBSome is involved in establishing CAH6-mNG flagellar asymmetry. Using dikaryon rescue experiments, we show that the de novo assembly of CAH6-mNG in flagella is considerably faster than the removal of ectopic CAH6-mNG from bbs flagella. Thus, different rates of flagellar entry of CAH6-mNG rather than its export from flagella is the likely basis for its asymmetric distribution. The data identify a novel role for the C. reinhardtii BBSome in preventing the entry of CAH6-mNG specifically into the cis-flagellum.

Molecular composition and biodegradation of loggerhead sponge Spheciospongia vesparium exhalent dissolved organic matter.

Sponges are critical components of marine reefs due to their high filtering capacity, wide abundance, and alteration of biogeochemical cycling. Here, we characterized dissolved organic matter (DOM) composition in the sponge holobiont exhalent seawater of a loggerhead sponge (Spheciospongia vesparium) and in ambient seawater in Florida Bay (USA), as well as the microbial responses to each DOM pool through dark incubations. The sponge holobiont removed 6% of the seawater dissolved organic carbon (DOC), utilizing compounds that were low in carbon and oxygen, yet high in nitrogen content relative to the ambient seawater. The microbial community accessed 7% of DOC from the ambient seawater during a 5-day incubation but only 1% of DOC from the sponge exhalent seawater, suggesting a decrease in lability possibly due to holobiont removal of nitrogen-rich compounds. If this holds true for other sponges, it may have important implications for DOM lability and cycling in coastal environments.

Automated classification of three-dimensional reconstructions of coral reefs using convolutional neural networks.

Coral reefs are biologically diverse and structurally complex ecosystems, which have been severally affected by human actions. Consequently, there is a need for rapid ecological assessment of coral reefs, but current approaches require time consuming manual analysis, either during a dive survey or on images collected during a survey. Reef structural complexity is essential for ecological function but is challenging to measure and often relegated to simple metrics such as rugosity. Recent advances in computer vision and machine learning offer the potential to alleviate some of these limitations. We developed an approach to automatically classify 3D reconstructions of reef sections and assessed the accuracy of this approach. 3D reconstructions of reef sections were generated using commercial Structure-from-Motion software with images extracted from video surveys. To generate a 3D classified map, locations on the 3D reconstruction were mapped back into the original images to extract multiple views of the location. Several approaches were tested to merge information from multiple views of a point into a single classification, all of which used convolutional neural networks to classify or extract features from the images, but differ in the strategy employed for merging information. Approaches to merging information entailed voting, probability averaging, and a learned neural-network layer. All approaches performed similarly achieving overall classification accuracies of ~96% and >90% accuracy on most classes. With this high classification accuracy, these approaches are suitable for many ecological applications.