The Mixoplankton Database (MDB): Diversity of photo-phago-trophic plankton in form, function, and distribution across the global ocean.

Protist plankton are major members of open-water marine food webs. Traditionally divided between phototrophic phytoplankton and phagotrophic zooplankton, recent research shows many actually combine phototrophy and phagotrophy in the one cell; these protists are the "mixoplankton." Under the mixoplankton paradigm, "phytoplankton" are incapable of phagotrophy (diatoms being exemplars), while "zooplankton" are incapable of phototrophy. This revision restructures marine food webs, from regional to global levels. Here, we present the first comprehensive database of marine mixoplankton, bringing together extant knowledge of the identity, allometry, physiology, and trophic interactivity of these organisms. This mixoplankton database (MDB) will aid researchers that confront difficulties in characterizing life traits of protist plankton, and it will benefit modelers needing to better appreciate ecology of these organisms with their complex functional and allometric predator-prey interactions. The MDB also identifies knowledge gaps, including the need to better understand, for different mixoplankton functional types, sources of nutrition (use of nitrate, prey types, and nutritional states), and to obtain vital rates (e.g. growth, photosynthesis, ingestion, factors affecting photo' vs. phago' -trophy). It is now possible to revisit and re-classify protistan "phytoplankton" and "zooplankton" in extant databases of plankton life forms so as to clarify their roles in marine ecosystems.

A grazing-driven positive nutrient feedback loop and active sexual reproduction underpin widespread Noctiluca green tides.

The mixoplankton green Noctiluca scintillans (gNoctiluca) is known to form extensive green tides in tropical coastal ecosystems prone to eutrophication. In the Arabian Sea, their recent appearance and annual recurrence have upended an ecosystem that was once exclusively dominated by diatoms. Despite evidence of strong links to eutrophication, hypoxia and warming, the mechanisms underlying outbreaks of this mixoplanktonic dinoflagellate remain uncertain. Here we have used eco-physiological measurements and transcriptomic profiling to ascribe gNoctiluca's explosive growth during bloom formation to the form of sexual reproduction that produces numerous gametes. Rapid growth of gNoctiluca coincided with active ammonium and phosphate release from gNoctiluca cells, which exhibited high transcriptional activity of phagocytosis and metabolism generating ammonium. This grazing-driven nutrient flow ostensibly promotes the growth of phytoplankton as prey and offers positive support successively for bloom formation and maintenance. We also provide the first evidence that the host gNoctiluca cell could be manipulating growth of its endosymbiont population in order to exploit their photosynthetic products and meet critical energy needs. These findings illuminate gNoctiluca's little known nutritional and reproductive strategies that facilitate its ability to form intense and expansive gNoctiluca blooms to the detriment of regional water, food and the socio-economic security in several tropical countries.

Differential expression of PEPCK isoforms is correlated to Aedes aegypti oogenesis and embryogenesis.

The mosquito Aedes aegypti undertakes a shift in carbohydrate metabolism during embryogenesis, including an increase in the activity of phosphoenolpyruvate carboxykinase (PEPCK), a key gluconeogenic enzyme, at critical steps of embryo development. All eukaryotes studied to date present two PEPCK isoforms, namely PEPCK-M (mitochondrial) and PEPCK-C (cytosolic). In A. aegypti, however, these proteins are so far uncharacterized. In the present work we describe two A. aegypti PEPCK isoforms by sequence alignment, protein modeling, and transcription analysis in different tissues, as well as PEPCK enzymatic activity assays in mitochondrial and cytoplasmic compartments during oogenesis and embryogenesis. First, we characterized the protein sequences compared to other organisms, and identified conserved sites and key amino acids. We also performed structure modeling for AePEPCK(M) and AePEPCK(C), identifying highly conserved structural sites, as well as a signal peptide in AePEPCK(M) localized in a very hydrophobic region. Moreover, after blood meal and during mosquito oogenesis and embryogenesis, both PEPCKs isoforms showed different transcriptional profiles, suggesting that mRNA for the cytosolic form is transmitted maternally, whereas the mitochondrial form is synthesized by the zygote. Collectively, these results improve our understanding of mosquito physiology and may yield putative targets for developing new methods for A. aegypti control.

Inhibition of Aedes aegypti DNA topoisomerase II by etoposide: Impact on survival and morphology of larvae and pupae.

DNA topoisomerase II enzymes maintain DNA stability during vital processes, such as genome replication, transcription and chromosomal segregation during mitosis and meiosis. In the present work, we analyzed functional aspects of the DNA topoisomerase II (AeTopII) enzyme of the mosquito Aedes aegypti. Here, we show that AeTopII mRNA is expressed at all stages of mosquito development. By in situ hybridization, we found that the AeTopII mRNA is concentrated along the ovarian follicular cells as well as in the region of the follicles. The observed expression profiles likely reflect increased topoisomerase II cellular requirements due to the intense ovarian growth and egg production following blood feeding in Ae. aegypti females. The drug etoposide, a classic inhibitor of topoisomerase II, was used for in vivo testing with 2nd stage larvae, in order to investigate the functional importance of this enzyme in Ae. aegypti survival and development. Inhibition of topoisomerase II activity with etoposide concentrations ranging from 10 to 200 µM did not leads to the immediate death of larvae. However, after 10 days of observation, etoposide treatments resulted in 30-40% decrease in survival, in a dose dependent manner, with persisting larvae and pupae presenting incomplete development, as well as morphological abnormalities. Also, approximately 50% of the treated larvae did not reach the pupal stage. Thus, we conclude that AeTopII is a vital enzyme in the development of Ae. aegypti and its sensitivity to inhibitors should be explored for potential chemical agents to be used in vector control.

A physiologic overview of the organ-specific transcriptome of the cattle tick Rhipicephalus microplus.

To further obtain insights into the Rhipicephalus microplus transcriptome, we used RNA-seq to carry out a study of expression in (i) embryos; (ii) ovaries from partially and fully engorged females; (iii) salivary glands from partially engorged females; (iv) fat body from partially and fully engorged females; and (v) digestive cells from partially, and (vi) fully engorged females. We obtained > 500 million Illumina reads which were assembled de novo, producing > 190,000 contigs, identifying 18,857 coding sequences (CDS). Reads from each library were mapped back into the assembled transcriptome giving a view of gene expression in different tissues. Transcriptomic expression and pathway analysis showed that several genes related in blood digestion and host-parasite interaction were overexpressed in digestive cells compared with other tissues. Furthermore, essential genes for the cell development and embryogenesis were overexpressed in ovaries. Taken altogether, these data offer novel insights into the physiology of production and role of saliva, blood digestion, energy metabolism, and development with submission of 10,932 novel tissue/cell specific CDS to the NCBI database for this important tick species.

Modulation of phosphatidylinositol 3-kinase activity during in vitro oocyte maturation increases the production of bovine blastocysts.

This study aimed to evaluate the effect of regulating phosphatidylinositol 3-kinase (PI3K) activity on the kinetics of oocyte nuclear maturation and the blastocyst rate. To evaluate oocyte viability, nuclear maturation rate and in vitro embryo production, cumulus-oocyte complexes (COCs) were maintained for 0, 10 min, 6 h or 22 h in TCM 199 medium supplemented with 20 nM wortmannin, an inhibitor of PI3K. After each period, COCs were transferred to the same medium without wortmannin and kept under the same conditions until completion of 22 h of in vitro maturation (IVM). To evaluate the effect of time on progression of nuclear maturation, COCs cultivated with 20 nM wortmannin was maintained for 22, 28 or 34 h of IVM. To determine the effect of wortmannin on the activity of maturation-promoting factor (MPF), COCs were kept under IVM conditions in the presence of the inhibitor for 0, 1, 3, 6, or 8 h. Exposure of COCs to wortmannin decreased (P < 0.05) the percentage of oocytes that reached metaphase II (MII) up to 22 h, MPF activity and reduced PI3K activity by 30%. However, after 28 and 34 h, 70% of oocytes reached the MII stage in the presence of inhibitor Moreover, COCs matured in the presence of wortmannin showed an increase (P < 0.05) in the blastocyst rate. These findings suggested that the regulation of the PI3K activity during IVM of bovine COCs interfered with the meiotic progression due to control of MPF activity, positively affecting the blastocyst rate.

Ecosystem state change in the Arabian Sea fuelled by the recent loss of snow over the Himalayan-Tibetan Plateau region.

The recent trend of global warming has exerted a disproportionately strong influence on the Eurasian land surface, causing a steady decline in snow cover extent over the Himalayan-Tibetan Plateau region. Here we show that this loss of snow is undermining winter convective mixing and causing stratification of the upper layer of the Arabian Sea at a much faster rate than predicted by global climate models. Over the past four decades, the Arabian Sea has also experienced a profound loss of inorganic nitrate. In all probability, this is due to increased denitrification caused by the expansion of the permanent oxygen minimum zone and consequent changes in nutrient stoichiometries. These exceptional changes appear to be creating a niche particularly favorable to the mixotroph, Noctiluca scintillans which has recently replaced diatoms as the dominant winter, bloom forming organism. Although Noctiluca blooms are non-toxic, they can cause fish mortality by exacerbating oxygen deficiency and ammonification of seawater. As a consequence, their continued range expansion represents a significant and growing threat for regional fisheries and the welfare of coastal populations dependent on the Arabian Sea for sustenance.

Robust learning algorithms for capturing oceanic dynamics and transport of Noctiluca blooms using linear dynamical models.

The blooms of Noctiluca in the Gulf of Oman and the Arabian Sea have been intensifying in recent years, posing now a threat to regional fisheries and the long-term health of an ecosystem supporting a coastal population of nearly 120 million people. We present the results of a local-scale data analysis to investigate the onset and patterns of the Noctiluca blooms, which form annually during the winter monsoon in the Gulf of Oman and in the Arabian Sea. Our approach combines methods in physical and biological oceanography with machine learning techniques. In particular, we present a robust algorithm, the variable-length Linear Dynamic Systems (vLDS) model, that extracts the causal factors and latent dynamics at the local-scale along each individual drifter trajectory, and demonstrate its effectiveness by using it to generate predictive plots for all variables and test macroscopic scientific hypotheses. The vLDS model is a new algorithm specifically designed to analyze the irregular dataset from surface velocity drifters, in which the multivariate time series trajectories are having variable or unequal lengths. The test results provide local-scale statistical evidence to support and check the macroscopic physical and biological Oceanography hypotheses on the Noctiluca blooms; it also helps identify complementary local trajectory-scale dynamics that might not be visible or discoverable at the macroscopic scale. The vLDS model also exhibits a generalization capability (as a machine learning methodology) to investigate important causal factors and hidden dynamics associated with ocean biogeochemical processes and phenomena at the population-level and local trajectory-scale.

The effects of Roundup® in embryo development and energy metabolism of the zebrafish (Danio rerio).

Roundup® is currently the most widely used and sold agricultural pesticide in the world. The objective of this work was to investigate the effects of Roundup® on energy metabolism during zebrafish (Danio rerio) embryogenesis. The embryo toxicity test was performed for 96 h post-fertilisation and the sublethal concentration of Roundup® was defined as 58.3 mg/L, which resulted in failure to inflate the swim bladder. Biochemical assays were performed with viable embryos following glyphosate exposure, and no significant effects on protein, glucose, glycogen, triglyceride levels or the enzymatic activities of alanine aminotransferase and aspartate aminotransferase were observed. However, the activity of hexokinase was significantly altered following exposure to 11.7 mg/L Roundup®. Through molecular docking we have shown for the first time that the interactions of glucokinase and hexokinases 1 and 2 with glyphosate showed significant interactions in the active sites, corroborating the biochemical results of hexokinase activity in zebrafish exposed to the chemical. From the results of molecular docking interactions carried out on the Zfishglucok, ZfishHK1 and ZfishHK2 models with the glyphosate linker, it can be concluded that there are significant interactions between glyphosate and active sites of glucokinase and hexokinase 1 and 2 proteins. The present work suggests that Roundup® can induce problems in fish embryogenesis relating to the incapacity of swim bladder to inflate. This represents the first study demonstrating the interaction of glyphosate with hexokinase and its isoforms.

Inhibition of energy metabolism by 3-bromopyruvate in the hard tick Rhipicephalus microplus.

The cattle tick R. microplus is the biggest obstacle to livestock rearing in tropical countries. It is responsible for billions of dollars in losses every year, affecting meat and milk production, beef and dairy cattle, and the leather industry. The lack of knowledge and strategies to combat the tick only increases the losses, it leads to successive and uncontrolled applications of acaricides, favouring the selection of strains resistant to commercially available chemical treatments. In this paper, we tested 3­bromopyruvate (3­BrPA), an alkylating agent with a high affinity for cysteine residues, on the R. microplus metabolism. We found that 3-BrPA was able to induce cell death in an assay using BME26 strain cell cultures derived from embryos, it was also able to reduce cellular respiration in developing embryos. 3-BrPA is a nonspecific inhibitor, affecting enzymes of different metabolic pathways in R. microplus. In our experiments, we demonstrated that 3-BrPA was able to affect the glycolytic enzyme hexokinase, reducing its activity by approximately 50%; and it strongly inhibited triose phosphate isomerase, which is an enzyme involved in both glycolysis and gluconeogenesis. Also, the mitochondrial respiratory chain was affected, NADH cytochrome c reductase (complex I-III) and succinate cytochrome c reductase (complex II-III) were strongly inhibited by 3-BrPA. Glutamate dehydrogenase was also affected by 3-BrPA, showing a gradual inhibition of activity in all the 3-BrPA concentrations tested. Altogether, these results show that 3-BrPA is a harmful compound to the tick organism.

Novel and Selective Rhipicephalus microplus Triosephosphate Isomerase Inhibitors with Acaricidal Activity.

The cattle tick Rhipicephalus microplus is one of the most important ectoparasites causing significant economic losses for the cattle industry. The major tool of control is reducing the number of ticks, applying acaricides in cattle. However, overuse has led to selection of resistant populations of R. microplus to most of these products, some even to more than one active principle. Thus, exploration for new molecules with acaricidal activity in R. microplus has become necessary. Triosephosphate isomerase (TIM) is an essential enzyme in R. microplus metabolism and could be an interesting target for the development of new methods for tick control. In this work, we screened 227 compounds, from our in-house chemo-library, against TIM from R. microplus. Four compounds (50, 98, 14, and 161) selectively inhibited this enzyme with IC50 values between 25 and 50 µM. They were also able to diminish cellular viability of BME26 embryonic cells by more than 50% at 50 µM. A molecular docking study showed that the compounds bind in different regions of the protein; compound 14 interacts with the dimer interface. Furthermore, compound 14 affected the survival of partially engorged females, fed artificially, using the capillary technique. This molecule is simple, easy to produce, and important biological data-including toxicological information-are available for it. Our results imply a promising role for compound 14 as a prototype for development of a new acaricidal involving selective TIM inhibition.

Patterns of Transcript Abundance of Eukaryotic Biogeochemically-Relevant Genes in the Amazon River Plume.

The Amazon River has the largest discharge of all rivers on Earth, and its complex plume system fuels a wide array of biogeochemical processes, across a large area of the western tropical North Atlantic. The plume thus stimulates microbial processes affecting carbon sequestration and nutrient cycles at a global scale. Chromosomal gene expression patterns of the 2.0 to 156 µm size-fraction eukaryotic microbial community were investigated in the Amazon River Plume, generating a robust dataset (more than 100 million mRNA sequences) that depicts the metabolic capabilities and interactions among the eukaryotic microbes. Combining classical oceanographic field measurements with metatranscriptomics yielded characterization of the hydrographic conditions simultaneous with a quantification of transcriptional activity and identity of the community. We highlight the patterns of eukaryotic gene expression for 31 biogeochemically significant gene targets hypothesized to be valuable within forecasting models. An advantage to this targeted approach is that the database of reference sequences used to identify the target genes was selectively constructed and highly curated optimizing taxonomic coverage, throughput, and the accuracy of annotations. A coastal diatom bloom highly expressed nitrate transporters and carbonic anhydrase presumably to support high growth rates and enhance uptake of low levels of dissolved nitrate and CO2. Diatom-diazotroph association (DDA: diatoms with nitrogen fixing symbionts) blooms were common when surface salinity was mesohaline and dissolved nitrate concentrations were below detection, and hence did not show evidence of nitrate utilization, suggesting they relied on ammonium transporters to aquire recently fixed nitrogen. These DDA blooms in the outer plume had rapid turnover of the photosystem D1 protein presumably caused by photodegradation under increased light penetration in clearer waters, and increased expression of silicon transporters as silicon became limiting. Expression of these genes, including carbonic anhydrase and transporters for nitrate and phosphate, were found to reflect the physiological status and biogeochemistry of river plume environments. These relatively stable patterns of eukaryotic transcript abundance occurred over modest spatiotemporal scales, with similarity observed in sample duplicates collected up to 2.45 km in space and 120 minutes in time. These results confirm the use of metatranscriptomics as a valuable tool to understand and predict microbial community function.

Biological response of Costa Rica Dome phytoplankton to Light, Silicic acid and Trace metals.

The Costa Rica Dome (CRD) is a unique open-ocean upwelling system, with picophytoplankton dominance of phytoplankton biomass and suppressed diatoms, yet paradoxically high export of biogenic silica. As a part of Flux and Zinc Experiments cruise in summer (June-July 2010), we conducted shipboard incubation experiments in the CRD to examine the potential roles of Si, Zn, Fe and light as regulating factors of phytoplankton biomass and community structure. Estimates of photosynthetic quantum yields revealed an extremely stressed phytoplankton population that responded positively to additions of silicic acid, iron and zinc and higher light conditions. Size-fractioned Chl a yielded the surprising result that picophytoplankton, as well as larger phytoplankton, responded most to treatments with added silicic acid incubated at high incident light (HL + Si). The combination of Si and HL also led to increases in cell sizes of picoplankton, notably in Synechococcus. Such a response, coupled with the recent discovery of significant intracellular accumulation of Si in some picophytoplankton, suggests that small phytoplankton could play a potentially important role in Si cycling in the CRD, which may help to explain its peculiar export characteristics.

Vaccination with cyclin-dependent kinase tick antigen confers protection against Ixodes infestation.

Among arthropods, ticks lead as vectors of animal diseases and rank second to mosquitoes in transmitting human pathogens. Cyclin-dependent kinases (CDK) participate in cell cycle control in eukaryotes. CDKs are serine/threonine protein kinases and these catalytic subunits are activated or inactivated at specific stages of the cell cycle. To determine the potential of using CDKs as anti-tick vaccine antigens, hamsters were immunized with recombinant Ixodes persulcatus CDK10, followed by a homologous tick challenge. Though it was not exactly unexpected, IpCDK10 vaccination significantly impaired tick blood feeding and fecundity, which manifested as low engorgement weights, poor oviposition, and a reduction in 80% of hatching rates. These findings may underpin the development of more efficacious anti-tick vaccines based on the targeting of cell cycle control proteins.

Non-classical gluconeogenesis-dependent glucose metabolism in Rhipicephalus microplus embryonic cell line BME26.

In this work we evaluated several genes involved in gluconeogenesis, glycolysis and glycogen metabolism, the major pathways for carbohydrate catabolism and anabolism, in the BME26 Rhipicephalus microplus embryonic cell line. Genetic and catalytic control of the genes and enzymes associated with these pathways are modulated by alterations in energy resource availability (primarily glucose). BME26 cells in media were investigated using three different glucose concentrations, and changes in the transcription levels of target genes in response to carbohydrate utilization were assessed. The results indicate that several genes, such as glycogen synthase (GS), glycogen synthase kinase 3 (GSK3), phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6 phosphatase (GP) displayed mutual regulation in response to glucose treatment. Surprisingly, the transcription of gluconeogenic enzymes was found to increase alongside that of glycolytic enzymes, especially pyruvate kinase, with high glucose treatment. In addition, RNAi data from this study revealed that the transcription of gluconeogenic genes in BME26 cells is controlled by GSK-3. Collectively, these results improve our understanding of how glucose metabolism is regulated at the genetic level in tick cells.

Massive outbreaks of Noctiluca scintillans blooms in the Arabian Sea due to spread of hypoxia.

In the last decade, the northern Arabian Sea has witnessed a radical shift in the composition of winter phytoplankton blooms, which previously comprised mainly of diatoms, the unicellular, siliceous photosynthetic organisms favoured by nutrient-enriched waters from convective mixing. These trophically important diatom blooms have been replaced by widespread blooms of a large, green dinoflagellate, Noctiluca scintillans, which combines carbon fixation from its chlorophyll-containing endosymbiont with ingestion of prey. Here, we report that these massive outbreaks of N. scintillans during winter are being facilitated by an unprecedented influx of oxygen deficient waters into the euphotic zone and by the extraordinary ability of its endosymbiont Pedinomonas noctilucae to fix carbon more efficiently than other phytoplankton under hypoxic conditions. We contend that N. scintillans blooms could disrupt the traditional diatom-sustained food chain to the detriment of regional fisheries and long-term health of an ecosystem supporting a coastal population of nearly 120 million people.

Identification and structural-functional analysis of cyclin-dependent kinases of the cattle tick Rhipicephalus (Boophilus) microplus.

Cyclin-dependent kinases (CDKs) are a family of serine/threonine kinases essential for cell cycle progression. Herein, we describe the participation of CDKs in the physiology of Rhipicephalus microplus, the southern cattle tick and an important disease vector. Firstly, amino acid sequences homologous with CDKs of other organisms were identified from a R. microplus transcriptome database in silico. The analysis of the deduced amino acid sequences of CDK1 and CDK10 from R. microplus showed that both have caspase-3/7 cleavage motifs despite their differences in motif position and length of encoded proteins. CDK1 has two motifs (DKRGD and SAKDA) located opposite to the ATP binding site while CDK10 has only one motif (SLLDN) for caspase 3-7 near the ATP binding site. Roscovitine (Rosco), a purine derivative that inhibits CDK/cyclin complexes by binding to the catalytic domain of the CDK molecule at the ATP binding site, which prevents the transfer of ATP's γphosphoryl group to the substrate. To determine the effect of Rosco on tick CDKs, BME26 cells derived from R. microplus embryo cells were utilized in vitro inhibition assays. Cell viability decreased in the Rosco-treated groups after 24 hours of incubation in a concentration-dependent manner and this was observed up to 48 hours following incubation. To our knowledge, this is the first report on characterization of a cell cycle protein in arachnids, and the sensitivity of BME26 tick cell line to Rosco treatment suggests that CDKs are potential targets for novel drug design to control tick infestation.

Glycogen and glucose metabolism are essential for early embryonic development of the red flour beetle Tribolium castaneum.

Control of energy metabolism is an essential process for life. In insects, egg formation (oogenesis) and embryogenesis is dependent on stored molecules deposited by the mother or transcribed later by the zygote. In oviparous insects the egg becomes an isolated system after egg laying with all energy conversion taking place during embryogenesis. Previous studies in a few vector species showed a strong correlation of key morphogenetic events and changes in glucose metabolism. Here, we investigate glycogen and glucose metabolism in the red flour beetle Tribolium castaneum, an insect amenable to functional genomic studies. To examine the role of the key enzymes on glycogen and glucose regulation we cloned and analyzed the function of glycogen synthase kinase 3 (GSK-3) and hexokinase (HexA) genes during T. castaneum embryogenesis. Expression analysis via in situ hybridization shows that both genes are expressed only in the embryonic tissue, suggesting that embryonic and extra-embryonic cells display different metabolic activities. dsRNA adult female injection (parental RNAi) of both genes lead a reduction in egg laying and to embryonic lethality. Morphological analysis via DAPI stainings indicates that early development is impaired in Tc-GSK-3 and Tc-HexA1 RNAi embryos. Importantly, glycogen levels are upregulated after Tc-GSK-3 RNAi and glucose levels are upregulated after Tc-HexA1 RNAi, indicating that both genes control metabolism during embryogenesis and oogenesis, respectively. Altogether our results show that T. castaneum embryogenesis depends on the proper control of glucose and glycogen.

Warming of the Eurasian landmass is making the Arabian Sea more productive.

The recent trend of declining winter and spring snow cover over Eurasia is causing a land-ocean thermal gradient that is particularly favorable to stronger southwest (summer) monsoon winds. Since 1997, sea surface winds have been strengthening over the western Arabian Sea. This escalation in the intensity of summer monsoon winds, accompanied by enhanced upwelling and an increase of more than 350% in average summertime phytoplankton biomass along the coast and over 300% offshore, raises the possibility that the current warming trend of the Eurasian landmass is making the Arabian Sea more productive.