A chromosome-level genome assembly enables the identification of the follicule stimulating hormone receptor as the master sex-determining gene in the flatfish Solea senegalensis

Sex determination (SD) shows huge variation among fish and a high evolutionary rate, as illustrated by the Pleuronectiformes (flatfishes). This order is characterized by its adaptation to demersal life, compact genomes and diversity of SD mechanisms. Here, we assembled the Solea senegalensis genome, a flatfish of great commercial value, into 82 contigs (614 Mb) combining long- and short-read sequencing, which were next scaffolded using a highly dense genetic map (28,838 markers, 21 linkage groups), representing 98.9% of the assembly. Further, we established the correspondence between the assembly and the 21 chromosomes by using BAC-FISH. Whole genome resequencing of six males and six females enabled the identification of 41 single nucleotide polymorphism variants in the follicle stimulating hormone receptor (fshr) consistent with an XX/XY SD system. The observed sex association was validated in a broader independent sample, providing a novel molecular sexing tool. The fshr gene displayed differential expression between male and female gonads from 86 days post-fertilization, when the gonad is still an undifferentiated primordium, concomitant with the activation of amh and cyp19a1a, testis and ovary marker genes, respectively, in males and females. The Y-linked fshr allele, which included 24 nonsynonymous variants and showed a highly divergent 3D protein structure, was overexpressed in males compared to the X-linked allele at all stages of gonadal differentiation. We hypothesize a mechanism hampering the action of the follicle stimulating hormone driving the undifferentiated gonad toward testis.     

Morphology and ultrastructure of the pharynx in Solenofilomorphidae (Acoela)

The homology of pharynges within the mostly pharynx-less Acoela has been a matter of discussion for decades. Here, we analyze the pharynges of three members of the Solenofilomorphidae, Myopea sp. and two species of the genus Solenofilomorpha, by means of light and transmission electron microscopy. Special focus is placed on the ultrastructure of the pharyngeal musculature, epidermis surrounding the mouth, pharyngeal epithelium, and junction with the digestive parenchyma. The main goal of this study was to evaluate the usefulness of certain characters for broader comparisons within the Acoela. Among the three species, characters relating to position of the mouth, presence and elaboration of sphincter muscles, presence of pharyngeal glands, and ultrastructure of epitheliosomes proved to be variously species- and genus-specific. The arrangement of pharyngeal muscles and their connection with body wall musculature, ultrastructure of receptor cells, and morphology of a nonciliated glandular region in the posterior pharynx, in contrast, appear to be characteristic of the family Solenofilomorphidae and thus of predominant interest for comparisons with other acoel families.     

The Type II NADPH Dehydrogenase Facilitates Cyclic Electron Flow,Energy-Dependent Quenching,and Chlororespiratory Metabolism during Acclimation of Chlamydomonas reinhardtii to Nitrogen Deprivation

When photosynthetic organisms are deprived of nitrogen (N), the capacity to grow and assimilate carbon becomes limited, causing a decrease in the productive use of absorbed light energy and likely a rise in the cellular reduction state. Although there is a scarcity of N in many terrestrial and aquatic environments, a mechanistic understanding of how photosynthesis adjusts to low-N conditions and the enzymes/activities integral to these adjustments have not been described. In this work, we use biochemical and biophysical analyses of photoautotrophically grown wild-type and mutant strains of Chlamydomonas reinhardtii to determine the integration of electron transport pathways critical for maintaining active photosynthetic complexes even after exposure of cells to N deprivation for 3 d. Key to acclimation is the type II NADPH dehydrogenase, NDA2, which drives cyclic electron flow (CEF), chlororespiration, and the generation of an H⁺ gradient across the thylakoid membranes. N deprivation elicited a doubling of the rate of NDA2-dependent CEF, with little contribution from PGR5/PGRL1-dependent CEF. The H⁺ gradient generated by CEF is essential to sustain nonphotochemical quenching, while an increase in the level of reduced plastoquinone would promote a state transition; both are necessary to down-regulate photosystem II activity. Moreover, stimulation of NDA2-dependent chlororespiration affords additional relief from the elevated reduction state associated with N deprivation through plastid terminal oxidase-dependent water synthesis. Overall, rerouting electrons through the NDA2 catalytic hub in response to photoautotrophic N deprivation sustains cell viability while promoting the dissipation of excess excitation energy through quenching and chlororespiratory processes.Oxygenic photosynthesis involves the conversion of light energy into chemical bond energy by plants, green algae, and cyanobacteria and the use of that energy to fix CO₂. The photosynthetic electron transport system, located in thylakoid membranes, involves several major protein complexes: PSII (water-plastoquinone oxidoreductase), cytochrome b₆f (cyt b6f; plastoquinone-plastocyanin oxidoreductase), PSI (plastocyanin-ferredoxin oxidoreductase), and the ATP synthase (CF_oCF₁). Light energy absorbed by the photosynthetic apparatus is used to establish both linear electron flow (LEF) and cyclic electron flow (CEF), which drive the production of ATP and NADPH, the chemical products of the light reactions needed for CO₂ fixation in the Calvin-Benson-Bassham (CBB) cycle.With the absorption of light energy by pigment-protein complexes associated with PSII, energy is funneled into unique chlorophyll (Chl) molecules located in the PSII reaction center (RC), where it can elicit a charge separation that generates a large enough oxidizing potential to extract electrons from water. In LEF, electrons from PSII RCs are transferred sequentially along a set of electron carriers, initially reducing the plastoquinone (PQ) pool, then the cyt b₆f complex, and subsequently the lumenal electron carrier plastocyanin (PC). Light energy absorbed by PSI excites a special pair of Chl molecules (P700), causing a charge separation that generates the most negative redox potential in nature (Nelson and Yocum, 2006). The energized electron, which is replaced by electrons from PC, is sequentially transferred to ferredoxin and ferredoxin NADP⁺ reductase, generating reductant in the form of NADPH.Electron transport from water to NADPH in LEF is accompanied by the transport of H⁺ into the thylakoid lumen. For each water molecule oxidized, two H⁺ are released in the thylakoid lumen. In addition, H⁺ are moved into the lumen by the transfer of electrons through cyt b₆f (Q cycle). H⁺ accumulation in the thylakoid lumen dramatically alters the lumenal pH, and the transmembrane H⁺ gradient (ΔpH) together with the transmembrane ion gradient constitute the proton motive force (pmf), which drives ATP formation by ATP synthase (Mitchell, 1961, 1966, 2011). This pmf also promotes other cellular processes, including the dissipation of excess absorbed excitation energy as heat in a photoprotective process (see below; Li et al., 2009; Erickson et al., 2015). The NADPH and ATP molecules generated by LEF and CEF fuel the synthesis of reduced carbon backbones (in the CBB cycle) used in the production of many cellular metabolites and fixed carbon storage polymers.A basic role for CEF is to increase the ATP-NADPH ratio, which can satisfy the energy requirements of the cell and augment the synthesis of ATP by LEF, which is required to sustain CO₂ fixation by the CBB cycle (Allen, 2003; Kramer et al., 2004; Iwai et al., 2010; Alric, 2014). There are two distinct CEF pathways identified in plants and algae. In both pathways, electrons flow from the PQ pool through cyt b₆f to reduce the oxidized form of P700 (P700⁺). In one CEF pathway, electrons are transferred back to the PQ pool prior to the formation of NADPH. This route involves the proteins PGR5 and PGRL1 (DalCorso et al., 2008; Tolleter et al., 2011; Hertle et al., 2013) and is termed PGR5/L1-dependent CEF. A second route for CEF includes an NADPH dehydrogenase that oxidizes NADPH (product of LEF) to NADP⁺, simultaneously reducing the PQ (Allen, 2003; Kramer et al., 2004; Rumeau et al., 2007). The reduced PQ pool is then oxidized by cyt b₆f, causing H⁺ translocation into the thylakoid lumen, followed by the transfer of electrons to P700⁺ via PC. In the green alga Chlamydomonas reinhardtii, this second route for CEF involves a type II NADPH dehydrogenase (NDA2; Jans et al., 2008; Desplats et al., 2009).Oxygenic photosynthetic organisms have inhabited the planet for approximately 3 billion years and have developed numerous strategies to acclimate to environmental fluctuations. These acclimation processes confer flexibility to the photosynthetic machinery, allowing it to adjust to changes in conditions that impact the metabolic/energetic state of the organism and, most importantly, the formation of reactive oxygen species that may damage the photosynthetic apparatus and other cellular components (Li et al., 2009). Several ways in which the photosynthetic apparatus adjusts to environmental fluctuations have been established. A well-studied acclimation process, nonphotochemical quenching (NPQ), reduces the excitation pressure on PSII when oxidized downstream electron acceptors are not available (Eberhard et al., 2008; Li et al., 2009; Erickson et al., 2015). Several processes constitute NPQ, as follows. (1) qT, which involves the physical movement of light-harvesting complexes (LHCs) from one photosystem to another (this is also designated state transitions; Rochaix, 2014). (2) qE, which involves thermal dissipation of the excitation energy. This energy-dependent process requires an elevated ΔpH and involves an LHC-like protein, LHCSR3 (in C. reinhardtii) or PSBS (in plants), as well as the accumulation of specific xanthophylls (mainly lutein in C. reinhardtii and zeaxanthin in plants; Niyogi et al., 1997b; Li et al., 2000, 2004; Peers et al., 2009). (3) qZ, which is energy independent and involves the accumulation of zeaxanthin (Dall’Osto et al., 2005; Nilkens et al., 2010). (4) qI, which promotes quenching following physical damage to PSII core subunits (Aro et al., 1993). Additional mechanisms that can impact LEF and CEF are the synthesis and degradation of pigment molecules, changes in levels of RC and antenna complexes, and the control of electron distribution between LEF and CEF as the energetic demands of the cell change (Allen, 2003; Kramer et al., 2004). In addition, electrons can be consumed by mitochondrial and chlororespiratory activities (Bennoun, 1982; Peltier and Cournac, 2002; Johnson et al., 2014; Bailleul et al., 2015). The latter mainly involves the plastid terminal oxidase PTOX2, which catalyzes the oxidation of the PQ pool and the reduction of oxygen and H⁺ to form water molecules (Houille-Vernes et al., 2011; Nawrocki et al., 2015).Photosynthetic processes also must be modulated as organisms experience changes in the levels of available nutrients (Grossman and Takahashi, 2001). The macronutrient nitrogen (N), which represents 3% to 5% of the dry weight of photosynthetic organisms, is required to synthesize many biological molecules (e.g. amino acids, nucleic acids, and various metabolites) and also participates in posttranslational modifications of proteins (e.g. S-nitrosylation; Romero-Puertas et al., 2013). Importantly, N is highly abundant in chloroplasts in the form of DNA, ribosomes, Chl, and polypeptides (e.g. Rubisco and LHCs; Evans, 1989; Raven, 2013). Furthermore, there is a strong integration between N and carbon assimilation. During N limitation under photoautotrophic conditions, the inability of the organism to synthesize amino acids and other N-containing molecules necessary for cell growth and division can feed back to inhibit both carbon fixation by the CBB cycle and electron transport processes and also can negatively impact the expression of genes encoding key CBB cycle enzymes (Terashima and Evans, 1988; Huppe and Turpin, 1994; Nunes-Nesi et al., 2010).C. reinhardtii is a well-established model organism in which to study photosynthesis and acclimation processes, including acclimation to nutrient limitation (Wykoff et al., 1998; Grossman and Takahashi, 2001; Moseley et al., 2006; Grossman et al., 2009; Terauchi et al., 2010; Aksoy et al., 2013). This unicellular alga grows rapidly as a photoheterotroph (on fixed carbon in the light) or as a heterotroph (on fixed carbon in the dark), has completely sequenced nuclear, chloroplast, and mitochondrial genomes, can be used for classical genetic analyses, and is haploid, which makes some aspects of molecular manipulation (e.g. the generation of knockout mutants) easier (Merchant et al., 2007; Blaby et al., 2014). In the past few years, there have been many studies on the ways in which C. reinhardtii responds to N deprivation (Bulté and Wollman, 1992; Blaby et al., 2013; Goodenough et al., 2014; Schmollinger et al., 2014; Wei et al., 2015; Juergens et al., 2015). Cells deprived of N under photoheterotrophic conditions (i.e. acetate as an external carbon source) minimize the use of N (referred to as N sparing) and induce mechanisms associated with scavenging N from both external and internal pools, all of which eventually lead to proteome modifications and an elevated carbon-N ratio (Schmollinger et al., 2014). Acclimation under photoheterotrophic conditions also causes dramatic modifications of cellular metabolism and energetics: photosynthesis is down-regulated at multiple levels, with a portion of its N content recycled (mainly Chl and polypeptides of the photosynthetic apparatus), while there is enhanced accumulation of mitochondrial complexes leading to increased respiratory activity (Schmollinger et al., 2014; Juergens et al., 2015). Additionally, while fixed carbon cannot be used for growth in the absence of N, it may be stored as starch and triacylglycerol (Work et al., 2010; Siaut et al., 2011; Davey et al., 2014; Goodenough et al., 2014).In contrast to the acclimation of photoheterotrophically grown C. reinhardtii to N deprivation, little is known about how the photosynthetic machinery in this alga adjusts in response to N deprivation under photoautotrophic conditions, when the cells absolutely require photosynthetic energy generation for maintenance. Specifically, we sought to understand how photosynthesis adjusts to metabolic restrictions that slow down the CBB cycle, which in turn could cause the accumulation of photoreductant, particularly NADPH, as the demand for electrons declines (Peltier and Schmidt, 1991; Rumeau et al., 2007). Based on analyses of mutants and the use of spectroscopic and fluorescence measurements, we established a critical role for NDA2 in the acclimation of C. reinhardtii to N deprivation under photoautotrophic conditions, including (1) an augmented capacity for alternative routes of electron utilization (which decrease the NADPH-NADP⁺ ratio) based on increased NDA2-dependent CEF and chlororespiration, and (2) elevated qE, which relies on the H⁺ gradient generated by NDA2-dependent CEF.     

Early Indicators of Fatal Leptospirosis during the 2010 Epidemic in Puerto Rico

Background

Leptospirosis is a potentially fatal bacterial zoonosis that is endemic throughout the tropics and may be misdiagnosed as dengue. Delayed hospital admission of leptospirosis patients is associated with increased mortality.

Methodology/Principal Findings

During a concurrent dengue/leptospirosis epidemic in Puerto Rico in 2010, suspected dengue patients that tested dengue-negative were tested for leptospirosis. Fatal and non-fatal hospitalized leptospirosis patients were matched 1:1–3 by age. Records from all medical visits were evaluated for factors associated with fatal outcome. Among 175 leptospirosis patients identified (4.7 per 100,000 residents), 26 (15%) were fatal. Most patients were older males and had illness onset during the rainy season. Fatal case patients first sought medical care earlier than non-fatal control patients (2.5 vs. 5 days post-illness onset [DPO], p < 0.01), but less frequently first sought care at a hospital (52.4% vs. 92.2%, p < 0.01). Although fatal cases were more often diagnosed with leptospirosis at first medical visit (43.9% vs. 9.6%, p = 0.01), they were admitted to the hospital no earlier than non-fatal controls (4.5 vs. 6 DPO, p = 0.31). Cases less often developed fever (p = 0.03), but more often developed jaundice, edema, leg pain, hemoptysis, and had a seizure (p ≤ 0.03). Multivariable analysis of laboratory values from first medical visit associated with fatal outcome included increased white blood cell (WBC) count with increased creatinine (p = 0.001), and decreased bicarbonate with either increased WBC count, increased creatinine, or decreased platelet count (p < 0.001).

Conclusions/Significance

Patients with fatal leptospirosis sought care earlier, but were not admitted for care any earlier than non-fatal patients. Combinations of routine laboratory values predictive of fatal outcome should be considered in admission decision-making for patients with suspected leptospirosis.     

Plankton populations of Lake Leake and Tooms Lake—Oligotrophic Tasmanian Lakes

The characteristic feature of Lake Leake and Tooms Lake is their great similarity in physical and chemical limnology. This similarity extends to the plankton populations in general terms, both lakes being dominated by Cyclotella stelligera Cleve et Grun. and having few other species. Floristic differences are minimal. Despite this floristic similarity distinct differences exist in plankton periodicity, principally in that of Cyclotella stelligera. The extreme oligotrophy of the lakes, already suspected from their low nutrient levels, is confirmed by the extremely meagre standing crops.     

Altered sexual maturation and gamete production in wild roach (Rutilus rutilus) living in rivers that receive treated sewage effluents.

Disruption in gonadal development of wild roach living in U.K. rivers receiving large volumes of treated sewage effluent is manifest in a variety of ways, ranging from malformation of the germ cells and/or reproductive ducts to altered gamete production. Intersex fish were also found to have an altered endocrine status and an elevated concentration of plasma vitellogenin. Gonadal growth was inhibited only in severely intersex fish, whereas progression of spermatogenesis was delayed in a large proportion of all intersex and exposed male fish. In contrast to the effects observed in the intersex and exposed male fish, the maturation of ovaries in female fish inhabiting effluent-contaminated rivers appeared to be less obviously affected, although a higher incidence of oocyte atresia was found in the effluent-exposed fish compared with the reference fish. A positive correlation was found between the proportion of female tissue in the gonads of intersex fish and their plasma vitellogenin concentration, suggesting that vitellogenin can be an indicator for the level of gonadal disruption in intersex roach. The estradiol-17beta concentration in intersex fish was intermediate between the concentration found in males and females, and the plasma testosterone was between 2- and 3-fold higher in intersex fish compared with male fish. These data suggest a link between altered endocrine status in intersex and female fish and gonadal disruption. Spermiation was also affected in roach living in effluent-impacted rivers: a lower proportion of fish were found releasing sperm, and in those intersex fish that were spermiating, a reduced milt volume and a reduced sperm density were found. All intersex fish had malformations of the reproductive duct(s), and in severely affected fish, the ducts were occluded, thus preventing release of gametes. In view of the widespread occurrence of intersexuality in wild fish populations in rivers throughout the United Kingdom, assessment of the reproductive capabilities of these intersex roach is clearly needed to understand the impact of this phenomenon on roach fertility.     

Isotope-coded affinity tag (ICAT) approach to redox proteomics: identification and quantitation of oxidant-sensitive cysteine thiols in complex protein mixtures

An approach is described for the simultaneous identification and quantitation of oxidant-sensitive cysteine thiols in a complex protein mixture using a thiol-specific, acid-cleavable isotope-coded affinity tag (ICAT) reagent (Applied Biosystems, USA). The approach is based on the fact that only free cysteine thiols are susceptible to labeling by the iodoacetamide-based ICAT, and that mass spectrometry can be used to quantitate the relative labeling of free thiols. Applying this approach, we have identified cysteine thiols of proteins in a rabbit heart membrane fraction that are sensitive to a high concentration of hydrogen peroxide. Previously known and some novel proteins with oxidant-sensitive cysteines were identified. Of the many protein thiols labeled by the ICAT, only relatively few were oxidized more than 50% despite the high concentration of oxidant used, indicating that oxidant-sensitive thiols are relatively rare, and denoting their specificity and potential functional relevance.     

Adenosine A2A receptor activation reduces infarct size in the isolated, perfused mouse heart by inhibiting resident cardiac mast cell degranulation

Mast cells are found in the heart and contribute to reperfusion injury following myocardial ischemia. Since the activation of A2A adenosine receptors (A2AARs) inhibits reperfusion injury, we hypothesized that ATL146e (a selective A2AAR agonist) might protect hearts in part by reducing cardiac mast cell degranulation. Hearts were isolated from five groups of congenic mice: A2AAR+/+ mice, A2AAR(-/-) mice, mast cell-deficient (Kit(W-sh/W-sh)) mice, and chimeric mice prepared by transplanting bone marrow from A2AAR(-/-) or A2AAR+/+ mice to radiation-ablated A2AAR+/+ mice. Six weeks after bone marrow transplantation, cardiac mast cells were repopulated with >90% donor cells. In isolated, perfused hearts subjected to ischemia-reperfusion injury, ATL146e or CGS-21680 (100 nmol/l) decreased infarct size (IS; percent area at risk) from 38 +/- 2% to 24 +/- 2% and 22 +/- 2% in ATL146e- and CGS-21680-treated hearts, respectively (P < 0.05) and significantly reduced mast cell degranulation, measured as tryptase release into reperfusion buffer. These changes were absent in A2AAR(-/-) hearts and in hearts from chimeric mice with A2AAR(-/-) bone marrow. Vehicle-treated Kit(W-sh/W-sh) mice had lower IS (11 +/- 3%) than WT mice, and ATL146e had no significant protective effect (16 +/- 3%). These data suggest that in ex vivo, buffer-perfused hearts, mast cell degranulation contributes to ischemia-reperfusion injury. In addition, our data suggest that A2AAR activation is cardioprotective in the isolated heart, at least in part by attenuating resident mast cell degranulation.