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.     

Low genetic diversity and population connectivity fuel vulnerability to climate change for the Tertiary relict pine Pinus bungeana

Endemic species are important components of regional biodiversity and hold the key to understanding local adaptation and evolutionary processes that shape species distributions. This study investigated the biogeographic history of a relict conifer Pinus bungeana Zucc. ex Endl. confined to central China. We examined genetic diversity in P. bungeana using genotyping-by-sequencing and chloroplast and mitochondrial DNA markers. We performed spatial and temporal inference of recent genetic and demographic changes, and dissected the impacts of geography and environmental gradients on population differentiation. We then projected P. bungeana's risk of decline under future climates. We found extremely low nucleotide diversity (average π 0.0014), and strong population structure (global F_ST 0.234) even at regional scales, reflecting long-term isolation in small populations. The species experienced severe bottlenecks in the early Pliocene and continued to decline in the Pleistocene in the western distribution, whereas the east expanded recently. Local adaptation played a small (8%) but significant role in population diversity. Low genetic diversity in fragmented populations makes the species highly vulnerable to climate change, particularly in marginal and relict populations. We suggest that conservation efforts should focus on enhancing gene pool and population growth through assisted migration within each genetic cluster to reduce the risk of further genetic drift and extinction.     

Systematics of Mukdenia and Oresitrophe (Saxifragaceae): Insights from genome skimming data

Oresitrophe and Mukdenia (Saxifragaceae) are epilithic sister genera used in traditional Chinese medicine. The taxonomy of Mukdenia, especially of M. acanthifolia, has been controversial. To address this, we produced plastid and mitochondrial data using genome skimming for Mukdenia acanthifolia and Mukdenia rossii, including three individuals of each species. We assembled complete plastomes, mitochondrial CDS and nuclear ribosomal ETS/ITS sequences using these data. Comparative analysis shows that the plastomes of Mukdenia and Oresitrophe are relatively conservative in terms of genome size, structure, gene content, RNA editing sites and codon usage. Five plastid regions that represent hotspots of change (trnH-psbA, psbC-trnS, trnM-atpE, petA-psbJ and ccsA-ndhD) are identified within Mukdenia, and six regions (trnH-psbA, petN-psbM, trnM-atpE, rps16-trnQ, ycf1 and ndhF) contain a higher number of species-specific parsimony-informative sites that may serve as potential DNA barcodes for species identification. To infer phylogenetic relationships between Mukdenia and Oresitrophe, we combined our data with published data based on three different datasets. The monophyly of each species (Oresitrophe rupifraga, M. acanthifolia and M. rossii) and the inferred topology ((M. rossii, M. acanthifolia), O. rupifraga) are well supported in trees reconstructed using the complete plastome sequences, but M. acanthifolia and M. rossii did not form a separate clade in the trees based on ETS + ITS data, while the mitochondrial CDS trees are not well-resolved. We found low recovery of genes in the Angiosperms353 target enrichment panel from our unenriched genome skimming data. Hybridization or incomplete lineage sorting may be the cause of discordance between trees reconstructed from organellar and nuclear data. Considering its morphological distinctiveness and our molecular phylogenetic results, we strongly recommend that M. acanthifolia be treated as a distinct species.     

First chemical synthesis of a scorpion alpha-toxin affecting sodium channels: the Aah I toxin of Androctonus australis hector.

Aah I is a 63-residue alpha-toxin isolated from the venom of the Buthidae scorpion Androctonus australis hector, which is considered to be the most dangerous species. We report here the first chemical synthesis of Aah I by the solid-phase method, using a Fmoc strategy. The synthetic toxin I (sAah I) was renatured in DMSO-Tris buffer, purified and subjected to thorough analysis and comparison with the natural toxin. The sAah I showed physico-chemical (CD spectrum, molecular mass, HPLC elution), biochemical (amino-acid composition, sequence), immunochemical and pharmacological properties similar to those of the natural toxin. The synthetic toxin was recognized by a conformation-dependent monoclonal anti-Aah I antibody, with an IC50 value close to that for the natural toxin. Following intracerebroventricular injection, the synthetic and the natural toxins were similarly lethal to mice. In voltage-clamp experiments, Na(v) 1.2 sodium channel inactivation was inhibited by the application of sAah I or of the natural toxin in a similar way. This work describes a simple protocol for the chemical synthesis of a scorpion alpha-toxin, making it possible to produce structural analogues in time.     

Linking life history traits in successive phases of a complex life cycle: effects of larval biomass on early juvenile development in an estuarine crab, Chasmagnathus granulata

In marine benthic invertebrates with complex life cycles, recruitment success, juvenile survival, and growth may be affected by variation in both maternal factors and environmental conditions prevailing during preceding embryonic or larval development. In an estuarine crab, Chasmagnathus granulata, previous investigations have shown that initial larval biomass is positively correlated with the biomass of recently extruded eggs, and it depends also on the salinity experienced during embryogenesis. Biomass at hatching has consequences for the subsequent larval development which, in this species, comprises two alternative developmental pathways with four or five zoeal instars (short or long pathway) and a megalopa. Larvae hatching with a lower than average biomass tend to develop through the long pathway and metamorphose to megalopae with higher biomass. In the present study, we show experimentally that the long pathway produces also significantly larger juveniles (crab size measured as carapace width, biomass as dry mass, carbon and nitrogen contents). Compared with juveniles originating from the short pathway, those from the long pathway showed in successive instars longer moulting cycles and larger carapace width, but lower size increments at ecdysis. In consequence, differences in size or biomass of long pathway vs short pathway crabs tended to disappear in later instars (after stage V). Furthermore, we tested in juveniles the tolerance of starvation at three salinities (5‰, 15‰, 32‰). Tolerance of starvation was significantly higher in juveniles originating from the long pathway, indicating higher energy reserves. While salinity played only a minor role for survival, it exerted significant effects on the time of moulting to the second juvenile instar, regardless of the preceding developmental pathway. The biomass of first juveniles obtained from the short pathway showed a significant positive correlation with the biomass of the freshly hatched zoea I, but not in those from the long pathway. In conclusion, the fitness of juvenile C. granulata is linked with previous developmental processes and environmental conditions during the embryonic and larval phase. Hence, a better understanding and prediction of the recruitment success of marine benthic invertebrates with a complex life cycle may require more comprehensive life‐history investigations.     

Characterization of xanthans from selected Xanthomonas strains cultivated under constant dissolved oxygen

Seventeen wild-type Xanthomonas isolates were screened in terms of broth viscosity in shake-flasks. As culture conditions affect polymer characteristics, a fair comparison among isolates required their cultivation in a fermenter under controlled dissolved oxygen tension. Three isolates and a reference strain were studied. The mean molecular weights and molecular weight distributions of their xanthans were determined. Products showed different pyruvate (0.2–7%), acetate (5–10%) and proteinaceous nitrogen (1–3%) contents. The selected isolates exhibit properties which could improve xanthan gum production and some could be used to produce polymers with specific characteristics.     

Establishment of the Dorso-ventral Axis in Xenopus Embryos Is Presaged by Early Asymmetries in β-Catenin That Are Modulated by the Wnt Signaling Pathway

Eggs of Xenopus laevis undergo a postfertilization cortical rotation that specifies the position of the dorso-ventral axis and activates a transplantable dorsal-determining activity in dorsal blastomeres by the 32-cell stage. There have heretofore been no reported dorso-ventral asymmetries in endogenous signaling proteins that may be involved in this dorsal-determining activity during early cleavage stages. We focused on β-catenin as a candidate for an asymmetrically localized dorsal-determining factor since it is both necessary and sufficient for dorsal axis formation. We report that β-catenin displays greater cytoplasmic accumulation on the future dorsal side of the Xenopus embryo by the two-cell stage. This asymmetry persists and increases through early cleavage stages, with β-catenin accumulating in dorsal but not ventral nuclei by the 16- to 32cell stages. We then investigated which potential signaling factors and pathways are capable of modulating the steady-state levels of endogenous β-catenin. Steadystate levels and nuclear accumulation of β-catenin increased in response to ectopic Xenopus Wnt-8 (Xwnt-8) and to the inhibition of glycogen synthase kinase-3, whereas neither Xwnt-5A, BVg1, nor noggin increased β-catenin levels before the mid-blastula stage. As greater levels and nuclear accumulation of β-catenin on the future dorsal side of the embryo correlate with the induction of specific dorsal genes, our data suggest that early asymmetries in β-catenin presage and may specify dorso-ventral differences in gene expression and cell fate. Our data further support the hypothesis that these dorso-ventral differences in β-catenin arise in response to the postfertilization activation of a signaling pathway that involves Xenopus glycogen synthase kinase-3.     

Application of immunoprecipitation techniques to the diagnosis of cutaneous and extracutaneous forms of sporotrichosis

Sporotrichosis is a mycosis which in our country has been described as only producing cutaneous and subcutaneous infections; in other countries it has been reported as causing pulmonary, bone, joint and meningeal infections.Possibly the systemic form also occurs in Venezuela since, along with the causative agent, all other factors considered as predisposing to systemic infection exist here.Through immunodiffusion (ID) and immunoelectrophoresis (IEP) and using a filamentous form metabolic antigen from Sporothrix schenckii, we were able to demonstrate precipitating antibodies against this fungus both in patients with cutaneous sporotrichosis, diagnosed by culture of the infecting agent, and in patients with pulmonary lesions. The IEP test showed the presence of an anodic arc which we have called the S arc, which could be one of the specific antigens of S. schenckii.

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Resumen En Venezuela, el diagnóstico de la esporotricosis solo se plantea ante lesiones cutáneas y subcutáneas, sin embargo, no deja de llamar la atención que siendo esta micosis la más frecuente en nuestro medio y existiendo concomitantemente en la población todos los factores predisponentes para que el S. schenckii invada órganos internos, estas localizaciones no han sido descritas.La incorporación del antígeno del S. schenckii para detectar anticuerpos circulantes en la ID en nuestros estudios serológicos de rutina, nos permitió observar la presencia de bandas de precipitación contra este antígeno en sueros de pacientes con lesiones pulmonares, igualmente fueron detectados anticuerpos en pacientes con esporotricosis cutáneas diagnosticadas por cultivo.El estudio de la estructura antigénica de este hongo, puso de manifiesto la presencia de un arco de localización anódica, que podría ser producido por uno de los antígenos específicos de este hongo; este arco que denominamos S fue visualizado en todos los casos positivos de esporotricosis cutáneas, al ser estudiados sus sueros en la IEF.Los estudios realizados en sueros de pacientes con lesiones pulmonares pusieron de manifiesto anticuerpos precipitantes en la ID e IEF, con las caractéristicas de evidenciar más de una línea de precipitación en la ID y un arco S más largo en la IEF, pudiendo ser esto originado por mayor producción de anticuerpos circulantes. Se plantea la necesidad de obtener el cultivo del S. schenckii de las posibles lesiones extracutáneas de esta micosis.