A complex species complex: The controversial role of ecology and biogeography in the evolutionary history of Syllis gracilis Grube, 1840 (Annelida,Syllidae)

The cryptic diversity in the polychaete Syllis gracilis Grube, 1840, in the Mediterranean Sea was examined with an integrative morpho-molecular approach. Individuals of S. gracilis were collected at eleven Mediterranean localities to provide an insight into the role of brackish environments in inducing cryptic speciation. The examination of morphological features combined with a molecular genetic analysis based on a partial sequence of the 16S rRNA gene highlighted discrepancies between morphological and molecular diversity. Morphological data allowed to identify a morphotype with short appendages occurring in coralline algae communities and another one with long appendages observed in brackish-water environments and Sabellaria reefs. Multivariate analyses showed that sampling localities were the greatest source of morphological divergence, suggesting that phenotypic plasticity may play a role in local adaptations of S. gracilis populations. Molecular data showed the occurrence of four divergent lineages not corresponding to morphological clusters. Different species delimitation tests gave conflicting results, retrieving, however, at least four separated entities. Some lineages occurred in sympatry and were equally distributed in marine and brackish-water environments, excluding a biogeographic or ecological explanation of the observed pattern and suggesting instead ancient separation between lineages and secondary contact. The co-occurrence of different lineages hindered the identification of the lineage corresponding to S. gracilis sensu stricto. The discrepancy between morphological and molecular diversity suggests that different environmental and biogeographic features may interact in a complex and unpredictable way in shaping diversity patterns. An integrative approach is needed to provide a satisfactory insight on evolutionary processes in marine invertebrates.     

Effect of EGF-receptor tyrosine kinase inhibitor on Rab5 function during endocytosis

Tyrosine autophosphorylation within the cytoplasmic tail of EGF-receptor is a key event, which in turn recruits several factors including Shc, Grb2 and Rin1 that are essential activities for receptor-mediated endocytosis and signaling. In this study, we demonstrated that treatment with AG1478, an EGF-receptor kinase inhibitor, blocked the formation of Rab5-positive endosomes as well as the activation of Rab5 upon addition of EGF. We also found that EGF-receptor catalytically inactive mutant failed to activate Rab5 upon EGF stimulation. Additionally, endosomal co-localization of Rab5 and EGF-receptor was inhibited by AG1478. Interestingly, AG1478 inhibitor did not block the formation of enlarged Rab5-positive endosomes in cells expressing Rab5 GTP hydrolysis defective mutant (Rab5:Q79L). AG1478 inhibitor also blocked the in vitro endosome fusion in a concentration-dependent manner, and more importantly, Rab5:Q79L mutant rescued it. Furthermore, addition of Rin1, a Rab5 guanine nucleotide exchange factor, partially restored endosome fusion in the presence of AG1478 inhibitor. Consistent with these observations, we also observed that Rin1 was unable to localize to membranes upon EGF-stimulation in the presence of AG1478 inhibitor. These results constitute first evidence that the enzymatic activity of a tyrosine kinase receptor is required endosome fusion via the activation of Rab5.     

Defining the mode, energetics and specificity with which a macrocyclic hexaoxazole binds to human telomeric G-quadruplex DNA

Oxazole-containing macrocycles represent a promising class of anticancer agents that target G-quadruplex DNA. We report the results of spectroscopic studies aimed at defining the mode, energetics and specificity with which a hexaoxazole-containing macrocycle (HXDV) binds to the intramolecular quadruplex formed by the human telomeric DNA model oligonucleotide d(T2AG3)4 in the presence of potassium ions. HXDV binds solely to the quadruplex nucleic acid form, but not to the duplex or triplex form. HXDV binds d(T2AG3)4 with a stoichiometry of two drug molecules per quadruplex, with these binding reactions being coupled to the destacking of adenine residues from the terminal G-tetrads. HXDV binding to d(T2AG3)4 does not alter the length of the quadruplex. These collective observations are indicative of a nonintercalative 'terminal capping' mode of interaction in which one HXDV molecule binds to each end of the quadruplex. The binding of HXDV to d(T2AG3)4 is entropy driven, with this entropic driving force reflecting contributions from favorable drug-induced alterations in the configurational entropy of the host quadruplex as well as in net hydration. The 'terminal capping' mode of binding revealed by our studies may prove to be a general feature of the interactions between oxazole-containing macrocyclic ligands (including telomestatin) and intramolecular DNA quadruplexes.     

The role of lateral roots in bypass flow in rice (Oryza sativa L.)

Although an apoplastic pathway (the so‐called bypass flow) is implicated in the uptake of Na⁺ by rice growing in saline conditions, the point of entry of this flow into roots remains to be elucidated. We investigated the role of lateral roots in bypass flow using the tracer trisodium‐8‐hydroxy‐1,3,6‐pyrenetrisulphonic acid (PTS) and the rice cv. IR36. PTS was identified in the vascular tissue of lateral roots using both epifluorescence microscopy and confocal laser scanning microscopy. Cryo‐scanning electron microscopy and epifluorescence microscopy of sections stained with berberine‐aniline blue revealed that the exodermis is absent in the lateral roots. We conclude that PTS can move freely through the cortical layers of lateral roots, enter the stele and be transported to the shoot via the transpiration stream.     

Polynucleotide:adenosine glycosidase activity of saporin-L1: effect on various forms of mammalian DNA

Saporin-L1 from the leaves of Saponaria officinalis belongs to a group of plant polynucleotide:adenosine glycosidases, known as ribosome-inactivating proteins due to their property of depurinating the major rRNA. Previous experiments indicated that saporin-L1 and other ribosome-inactivating proteins depurinate also DNA [Barbieri et al. (1994) Nature 372, 324; and (1996) Biochem. J. 319, 507-513]. Here we describe the effects of highly purified nuclease-free saporin-L1 on mammalian nuclear and mitochondrial DNA. Saporin-L1 had less activity on mitochondrial DNA than on nuclear DNA. A low, although significant, depurination of both chromatin and whole nuclei was observed. Mitochondrial nucleic acids are heavily depurinated in intact mitochondria, although the contribute of mtDNA to the deadenylation events is not known. The kinetic constants for several substrates were determined.     

Bacillus subtilis as a host for mosquitocidal toxins production

Aedes albopictus transmits several arboviral infections. In the absence of vaccines, control of mosquito populations is the only strategy to prevent vector-borne diseases. As part of the search for novel, biological and environmentally friendly strategies for vector control, the isolation of new bacterial species with mosquitocidal activity represents a promising approach. However, new bacterial isolates may be difficult to grow and genetically manipulate. To overcome these limits, here we set up a system allowing the expression of mosquitocidal bacterial toxins in the well-known genetic background of Bacillus subtilis. As a proof of this concept, the ability of B. subtilis to express individual or combinations of toxins of Bacillus thuringiensis israelensis (Bti) was studied. Different expression systems in which toxin gene expression was driven by IPTG-inducible, auto-inducible or toxin gene-specific promoters were developed. The larvicidal activity of the resulting B. subtilis strains against second-instar Ae. albopictus larvae allowed studying the activity of individual toxins or the synergistic interaction among Cry and Cyt toxins. The expression systems here presented lay the foundation for a better improved system to be used in the future to characterize the larvicidal activity of toxin genes from new environmental isolates.     

Temporal and spatial dynamics of grapevine anthracnose and its relationship to pathogen survival

Anthracnose, caused by Elsinoë ampelina, is an economically important grapevine disease in south and southeast Brazil. Control is achieved by lime sulphur application during grapevine dormancy and foliar fungicide sprays until the berries are half-grown. This study assessed the temporal and spatial progress of grapevine anthracnose under field conditions in order to describe the disease dynamics and its relationship to pathogen survival. The experiment was carried out in a vineyard of table grape Vitis labrusca in Brazil, during the 2014 and 2015 growing seasons. The incidence of vines with diseased leaves, stems and berries and the disease severity on leaves were recorded from bud break to veraison. Monomolecular, logistic and Gompertz models were fitted by non-linear regression to the incidence and severity data over time to characterize the temporal progress. Ordinary runs, dispersion index, modified Taylor's power law and spatial hierarchy analyses were used to characterize the spatial pattern of diseased plants. The monomolecular model showed the best fit for the incidence progress, with disease progress rates ranging from 0.051 to 0.136 per day. In both seasons, the incidence of diseased plants reached 100% 1 month after bud break. However, the incidence of diseased leaves per plant was around 60% and leaf disease severity was lower than 5% for both years. Ordinary runs and dispersion index analyses revealed that diseased grapevines were distributed randomly on the majority of the assessment dates. Meanwhile, a slight aggregation of diseased vines was observed in the modified Taylor's power law analysis. Our results suggested that the progress of anthracnose incidence and severity over time was governed mainly by the income of the primary inoculum, which survived in the vineyard. Therefore, anthracnose control measures in Brazilian vineyards should be focused on the reduction in inoculum within the vineyard.     

Ancient Substructure in Early mtDNA Lineages of Southern Africa

Among the deepest-rooting clades in the human mitochondrial DNA (mtDNA) phylogeny are the haplogroups defined as L0d and L0k, which are found primarily in southern Africa. These lineages are typically present at high frequency in the so-called Khoisan populations of hunter-gatherers and herders who speak non-Bantu languages, and the early divergence of these lineages led to the hypothesis of ancient genetic substructure in Africa. Here we update the phylogeny of the basal haplogroups L0d and L0k with 500 full mtDNA genome sequences from 45 southern African Khoisan and Bantu-speaking populations. We find previously unreported subhaplogroups and greatly extend the amount of variation and time-depth of most of the known subhaplogroups. Our major finding is the definition of two ancient sublineages of L0k (L0k1b and L0k2) that are present almost exclusively in Bantu-speaking populations from Zambia; the presence of such relic haplogroups in Bantu speakers is most probably due to contact with ancestral pre-Bantu populations that harbored different lineages than those found in extant Khoisan. We suggest that although these populations went extinct after the immigration of the Bantu-speaking populations, some traces of their haplogroup composition survived through incorporation into the gene pool of the immigrants. Our findings thus provide evidence for deep genetic substructure in southern Africa prior to the Bantu expansion that is not represented in extant Khoisan populations.     

Phylogeographical pattern of Euplotes nobilii,a protist ciliate with a bipolar biogeographical distribution

Nuclear (18S and ITS) and mitochondrial (16S) ribosomal RNA gene sequences were determined from genetically distinct wild‐type strains of Antarctic (nine strains), Fuegian (four strains), Greenland (nine strains) and Svalbard (three strains) populations of the marine ciliate, Euplotes nobilii, and analysed for their nucleotide polymorphisms. A close genetic homogeneity was found within and between the Antarctic and Fuegian populations, while more significant levels of genetic differentiation were detected within and between the two Arctic populations, as well as between these populations and the Antarctic/Fuegian ones. The phylogeographical pattern that was derived from these data indicates that gene flow is not limited among Arctic populations; it equally connects the Arctic and Antarctic populations either directly, or through the Fuegian population. This indication reinforces previous evidence from laboratory assays of mating interactions between some of the strains analysed in this work that Southern and Northern polar populations of E. nobilii belong to a unique, panmictic population that substantially share the same gene pool.