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.

Publisher Correction: Genetic tool development in marine protists: emerging model organisms for experimental cell biology.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Genetic tool development in marine protists: emerging model organisms for experimental cell biology.

Diverse microbial ecosystems underpin life in the sea. Among these microbes are many unicellular eukaryotes that span the diversity of the eukaryotic tree of life. However, genetic tractability has been limited to a few species, which do not represent eukaryotic diversity or environmentally relevant taxa. Here, we report on the development of genetic tools in a range of protists primarily from marine environments. We present evidence for foreign DNA delivery and expression in 13 species never before transformed and for advancement of tools for eight other species, as well as potential reasons for why transformation of yet another 17 species tested was not achieved. Our resource in genetic manipulation will provide insights into the ancestral eukaryotic lifeforms, general eukaryote cell biology, protein diversification and the evolution of cellular pathways.

Tissue-specific expression of p53 and ras genes in response to the environmental genotoxicant benzo(α)pyrene in marine mussels.

Marine mussels can develop hemeic and gonadal neoplasia in the natural environment. Associated with these diseases are the tumor suppressor (TS) p53 and the proto-oncogene ras coded proteins, both of which are highly conserved among molluscs and vertebrates. We report, for the first time, tissue-specific expression analysis of p53 and ras genes in Mytilus edulis by means of quantitative RT-PCR. A tissue-specific response was observed after 6 and 12 days exposure to a sublethal concentration of a model Polycyclic Aromatic Hydrocarbon (PAH), benzo(α)pyrene (B(α)P). This sublethal concentration (56 µg/L) was selected based on an integrated biomarker analysis carried out prior to gene expression analysis, which included a 'clearance rate' assay, histopathological analysis, and DNA strand break measurements. The results indicated that the selected concentration of B(α)P can lead to the induction of DNA strand breaks, tissue damage, and expression of tumor-regulating genes. Both p53 and ras are expressed in a tissue-specific manner, which collaborate with tissue-specific function in response to genotoxic stress. The integrated biological responses in Mytilus edulis strengthen the use of this organism to investigate the fundamental mechanism of development of malignancy in invertebrate which could be translated to other organisms including humans.

Detection and Replication of Moku Virus in Honey Bees and Social Wasps.

Transmission of honey bee viruses to other insects, and vice versa, has previously been reported and the true ecological importance of this phenomenon is still being realized. Members of the family Vespidae interact with honey bees via predation or through the robbing of brood or honey from colonies, and these activities could result in virus transfer. In this study we screened Vespa velutina and Vespa crabro collected from Europe and China and also honey bees and Vespula vulgaris from the UK for Moku virus (MV), an Iflavirus first discovered in the predatory social wasp Vespula pensylvanica in Hawaii. MV was found in 71% of Vespulavulgaris screened and was also detected in UK Vespa crabro. Only seven percent of Vespa velutina individuals screened were MV-positive and these were exclusively samples from Jersey. Of 69 honey bee colonies screened, 43% tested positive for MV. MV replication was confirmed in Apis mellifera and Vespidae species, being most frequently detected in Vespulavulgaris. MV sequences from the UK were most similar to MV from Vespulapensylvanica compared to MV from Vespa velutina in Belgium. The implications of the transfer of viruses between the Vespidae and honey bees are discussed.

Deformed wing virus implicated in overwintering honeybee colony losses.

The worldwide decline in honeybee colonies during the past 50 years has often been linked to the spread of the parasitic mite Varroa destructor and its interaction with certain honeybee viruses. Recently in the United States, dramatic honeybee losses (colony collapse disorder) have been reported; however, there remains no clear explanation for these colony losses, with parasitic mites, viruses, bacteria, and fungal diseases all being proposed as possible candidates. Common characteristics that most failing colonies share is a lack of overt disease symptoms and the disappearance of workers from what appears to be normally functioning colonies. In this study, we used quantitative PCR to monitor the presence of three honeybee viruses, deformed wing virus (DWV), acute bee paralysis virus (ABPV), and black queen cell virus (BQCV), during a 1-year period in 15 asymptomatic, varroa mite-positive honeybee colonies in Southern England, and 3 asymptomatic colonies confirmed to be varroa mite free. All colonies with varroa mites underwent control treatments to ensure that mite populations remained low throughout the study. Despite this, multiple virus infections were detected, yet a significant correlation was observed only between DWV viral load and overwintering colony losses. The long-held view has been that DWV is relatively harmless to the overall health status of honeybee colonies unless it is in association with severe varroa mite infestations. Our findings suggest that DWV can potentially act independently of varroa mites to bring about colony losses. Therefore, DWV may be a major factor in overwintering colony losses.

Distinct Oceanic Microbiomes From Viruses to Protists Located Near the Antarctic Circumpolar Current.

Microbes occupy diverse ecological niches and only through recent advances in next generation sequencing technologies have the true microbial diversity been revealed. Furthermore, lack of perceivable marine barriers to genetic dispersal (i.e., mountains or islands) has allowed the speculation that organisms that can be easily transported by currents and therefore proliferate everywhere. That said, ocean currents are now commonly being recognized as barriers for microbial dispersal. Here we analyzed samples collected from a total of six stations, four located in the Indian Ocean, and two in the Southern Ocean. Amplicon sequencing was used to characterize both prokaryotic and eukaryotic plankton communities, while shotgun sequencing was used for the combined environmental DNA (eDNA), microbial eDNA (meDNA), and viral fractions. We found that Cyanobacteria dominated the prokaryotic component in the South-West Indian Ocean, while γ-Proteobacteria dominated the South-East Indian Ocean. A combination of γ- and α-Proteobacteria dominated the Southern Ocean. Alveolates dominated almost exclusively the eukaryotic component, with variation in the ratio of Protoalveolata and Dinoflagellata depending on station. However, an increase in haptophyte relative abundance was observed in the Southern Ocean. Similarly, the viral fraction was dominated by members of the order Caudovirales across all stations; however, a higher presence of nucleocytoplasmic large DNA viruses (mainly chloroviruses and mimiviruses) was observed in the Southern Ocean. To our knowledge, this is the first that a statistical difference in the microbiome (from viruses to protists) between the subtropical Indian and Southern Oceans. We also show that not all phylotypes can be found everywhere, and that meDNA is not a suitable resource for monitoring aquatic microbial diversity.

Pentaplacodinium saltonense gen. et sp. nov. (Dinophyceae) and its relationship to the cyst-defined genus Operculodinium and yessotoxin-producing Protoceratium reticulatum.

Strains of a dinoflagellate from the Salton Sea, previously identified as Protoceratium reticulatum and yessotoxin producing, have been reexamined morphologically and genetically and Pentaplacodinium saltonense n. gen. et sp. is erected to accommodate this species. Pentaplacodinium saltonense differs from Protoceratium reticulatum (Claparède et Lachmann 1859) Bütschli 1885 in the number of precingular plates (five vs. six), cingular displacement (two widths vs. one), and distinct cyst morphology. Incubation experiments (excystment and encystment) show that the resting cyst of Pentaplacodinium saltonense is morphologically most similar to the cyst-defined species Operculodinium israelianum (Rossignol, 1962) Wall (1967) and O. psilatum Wall (1967). Collections of comparative material from around the globe (including Protoceratium reticulatum and the genus Ceratocorys) and single cell PCR were used to clarify molecular phylogenies. Variable regions in the LSU (three new sequences), SSU (12 new sequences) and intergenic ITS 1-2 (14 new sequences) were obtained. These show that Pentaplacodinium saltonense and Protoceratium reticulatum form two distinct clades. Pentaplacodinium saltonense forms a monophyletic clade with several unidentified strains from Malaysia. LSU and SSU rDNA sequences of three species of Ceratocorys (C. armata, C. gourreti, C. horrida) from the Mediterranean and several other unidentified strains from Malaysia form a well-supported sister clade. The unique phylogenetic position of an unidentified strain from Hawaii is also documented and requires further examination. In addition, based on the V9 SSU topology (bootstrap values >80%), specimens from Elands Bay (South Africa), originally described as Gonyaulax grindleyi by Reinecke (1967), cluster with Protoceratium reticulatum. The known range of Pentaplacodinium saltonense is tropical to subtropical, and its cyst is recorded as a fossil in upper Cenozoic sediments. Protoceratium reticulatum and Pentaplacodinium saltonense seem to inhabit different niches: motile stages of these dinoflagellates have not been found in the same plankton sample.

Schrödinger's Cheshire Cat: Are Haploid Emiliania huxleyi Cells Resistant to Viral Infection or Not?

Emiliania huxleyi is the main calcite producer on Earth and is routinely infected by a virus (EhV); a double stranded DNA (dsDNA) virus belonging to the family Phycodnaviridae. E. huxleyi exhibits a haplodiploid life cycle; the calcified diploid stage is non-motile and forms extensive blooms. The haploid phase is a non-calcified biflagellated cell bearing organic scales. Haploid cells are thought to resist infection, through a process deemed the "Cheshire Cat" escape strategy; however, a recent study detected the presence of viral lipids in the same haploid strain. Here we report on the application of an E. huxleyi CCMP1516 EhV-86 combined tiling array (TA) that further confirms an EhV infection in the RCC1217 haploid strain, which grew without any signs of cell lysis. Reverse transcription polymerase chain reaction (RT-PCR) and PCR verified the presence of viral RNA in the haploid cells, yet indicated an absence of viral DNA, respectively. These infected cells are an alternative stage of the virus life cycle deemed the haplococcolithovirocell. In this instance, the host is both resistant to and infected by EhV, i.e., the viral transcriptome is present in haploid cells whilst there is no evidence of viral lysis. This superimposed state is reminiscent of Schrödinger's cat; of being simultaneously both dead and alive.

ABC Assay: Method Development and Application to Quantify the Role of Three DWV Master Variants in Overwinter Colony Losses of European Honey Bees.

Deformed wing virus (DWV) is one of the most prevalent honey bee viral pathogens in the world. Typical of many RNA viruses, DWV is a quasi-species, which is comprised of a large number of different variants, currently consisting of three master variants: Type A, B, and C. Little is known about the impact of each variant or combinations of variants upon the biology of individual hosts. Therefore, we have developed a new set of master variant-specific DWV primers and a set of standards that allow for the quantification of each of the master variants. Competitive reverse transcriptase polymerase chain reaction (RT-PCR) experimental design confirms that each new DWV primer set is specific to the retrospective master variant. The sensitivity of the ABC assay is dependent on whether DNA or RNA is used as the template and whether other master variants are present in the sample. Comparison of the overall proportions of each master variant within a sample of known diversity, as confirmed by next-generation sequence (NGS) data, validates the efficiency of the ABC assay. The ABC assay was used on archived material from a Devon overwintering colony loss (OCL) 2006-2007 study; further implicating DWV type A and, for the first time, possibly C in the untimely collapse of honey bee colonies. Moreover, in this study DWV type B was not associated with OCL. The use of the ABC assay will allow researchers to quickly and cost effectively pre-screen for the presence of DWV master variants in honey bees.

Change in Emiliania huxleyi Virus Assemblage Diversity but Not in Host Genetic Composition during an Ocean Acidification Mesocosm Experiment.

Effects of elevated pCO2 on Emiliania huxleyi genetic diversity and the viruses that infect E. huxleyi (EhVs) have been investigated in large volume enclosures in a Norwegian fjord. Triplicate enclosures were bubbled with air enriched with CO2 to 760 ppmv whilst the other three enclosures were bubbled with air at ambient pCO2; phytoplankton growth was initiated by the addition of nitrate and phosphate. E. huxleyi was the dominant coccolithophore in all enclosures, but no difference in genetic diversity, based on DGGE analysis using primers specific to the calcium binding protein gene (gpa) were detected in any of the treatments. Chlorophyll concentrations and primary production were lower in the three elevated pCO2 treatments than in the ambient treatments. However, although coccolithophores numbers were reduced in two of the high-pCO2 treatments; in the third, there was no suppression of coccolithophores numbers, which were very similar to the three ambient treatments. In contrast, there was considerable variation in genetic diversity in the EhVs, as determined by analysis of the major capsid protein (mcp) gene. EhV diversity was much lower in the high-pCO2 treatment enclosure that did not show inhibition of E. huxleyi growth. Since virus infection is generally implicated as a major factor in terminating phytoplankton blooms, it is suggested that no study of the effect of ocean acidification in phytoplankton can be complete if it does not include an assessment of viruses.

Choice of molecular barcode will affect species prevalence but not bacterial community composition.

The rapid advancement of next generation sequencing protocols in recent years has led to the diversification in the methods used to study microbial communities; however, how comparable the data generated from these different methods are, remains unclear. In this study we compared the taxonomic composition and seasonal dynamics of the bacterial community determined by two distinct 16s amplicon sequencing protocols: sequencing of the V6 region of the 16s rRNA gene using 454 pyrosequencing vs the V4 region of the 16s rRNA gene using the Illumina Hiseq 2500 platform. Significant differences between relative abundances at all taxonomic levels were observed; however, their seasonal dynamics between phyla were largely consistent between methods. This study highlights that care must be taken when comparing datasets generated from different methods.

How many Coccolithovirus genotypes does it take to terminate an Emiliania huxleyi bloom?

Giant viruses are known to be significant mortality agents of phytoplankton, often being implicated in the terminations of large Emiliania huxleyi blooms. We have previously shown the high temporal variability of E. huxleyi-infecting coccolithoviruses (EhVs) within a Norwegian fjord mesocosm. In the current study we investigated EhV dynamics within a naturally-occurring E. huxleyi bloom in the Western English Channel. Using denaturing gradient gel electrophoresis and marker gene sequencing, we uncovered a spatially highly dynamic Coccolithovirus population that was associated with a genetically stable E. huxleyi population as revealed by the major capsid protein gene (mcp) and coccolith morphology motif (CMM), respectively. Coccolithoviruses within the bloom were found to be variable with depth and unique virus populations were detected at different stations sampled indicating a complex network of EhV-host infections. This ultimately will have significant implications to the internal structure and longevity of ecologically important E. huxleyi blooms.

Effect of Metals on the Lytic Cycle of the Coccolithovirus, EhV86.

In this study we show that metals, and in particular copper (Cu), can disrupt the lytic cycle in the Emiliania huxleyi - EhV86 host-virus system. E. huxleyi lysis rates were reduced at high total Cu concentrations (> approximately 500 nM) in the presence and absence of EDTA (ethylenediaminetetraacetic acid) in acute short term exposure experiments. Zinc (Zn), cadmium (Cd), and cobalt (Co) were not observed to affect the lysis rate of EhV86 in these experiments. The cellular glutathione (GSH) content increased in virus infected cells, but not as a result of metal exposure. In contrast, the cellular content of phytochelatins (PCs) increased only in response to metal exposure. The increase in glutathione content is consistent with increases in the production of reactive oxygen species (ROS) on viral lysis, while increases in PC content are likely linked to metal homeostasis and indicate that metal toxicity to the host was not affected by viral infection. We propose that Cu prevents lytic production of EhV86 by interfering with virus DNA (deoxyribonucleic acid) synthesis through a transcriptional block, which ultimately suppresses the formation of ROS.

Global honey bee viral landscape altered by a parasitic mite.

Emerging diseases are among the greatest threats to honey bees. Unfortunately, where and when an emerging disease will appear are almost impossible to predict. The arrival of the parasitic Varroa mite into the Hawaiian honey bee population allowed us to investigate changes in the prevalence, load, and strain diversity of honey bee viruses. The mite increased the prevalence of a single viral species, deformed wing virus (DWV), from ~10 to 100% within honey bee populations, which was accompanied by a millionfold increase in viral titer and a massive reduction in DWV diversity, leading to the predominance of a single DWV strain. Therefore, the global spread of Varroa has selected DWV variants that have emerged to allow it to become one of the most widely distributed and contagious insect viruses on the planet.