Rainfall and sea level drove the expansion of seasonally flooded habitats and associated bird populations across Amazonia.

Spatial arrangement of distinct Amazonian environments through time and its effect on specialized biota remain poorly known, fueling long-lasting debates about drivers of biotic diversification. We address the late Quaternary sediment deposition that assembled the world's largest seasonally flooded ecosystems. Genome sequencing was used to reconstruct the demographic history of bird species specialized in either early successional vegetation or mature floodplain forests. Sediment deposition that built seasonally flooded habitats accelerated throughout the Holocene (last 11,700 years) under sea level highstand and intensification of the South American Monsoon, at the same time as global increases in atmospheric methane concentration. Bird populations adapted to seasonally flooded habitats expanded due to enlargement of Amazonian river floodplains and archipelagos. Our findings suggest that the diversification of the biota specialized in seasonally flooded habitats is coupled to sedimentary budget changes of large rivers, which rely on combined effects of sea level and rainfall variations.

Astronomical tuning of the Aptian stage and its implications for age recalibrations and paleoclimatic events.

The Aptian was characterized by dramatic tectonic, oceanographic, climatic and biotic changes and its record is punctuated by Oceanic Anoxic Events (OAEs). The timing and duration of these events are still contentious, particularly the age of the Barremian-Aptian boundary. This study presents a cyclostratigraphic evaluation of a high-resolution multiproxy dataset (δ13C, δ18O, MS and ARM) from the Poggio le Guaine core. The identification of Milankovitch-band imprints allowed us to construct a 405-kyr astronomically-tuned age model that provides new constraints for the Aptian climato-chronostratigraphic framework. Based on the astronomical tuning, we propose: (i) a timespan of ~7.2 Myr for the Aptian; (ii) a timespan of ~420 kyr for the magnetic polarity Chron M0r and an age of ~120.2 Ma for the Barremian-Aptian boundary; and (iii) new age constraints on the onset and duration of Aptian OAEs and the 'cold snap'. The new framework significantly impacts the Early Cretaceous geological timescale.

Evidence-based radiography: A new methodology or the systematisation of an old practice?

INTRODUCTION: Evidence based radiography (EBR) is the logical development of evidence based practice applied to radiography. The aim of this study was to investigate the opinion of a cohort of Portuguese radiographers in Southern Portugal working in public hospitals regarding evidence based practice (EBP), namely about the levels of knowledge about EBR, how they access information and how they use it within daily practice. METHODS: A self-administered questionnaire was applied to a sample of 40 radiographers in the Portuguese region of Algarve. This questionnaire was validated for Portuguese speakers using the translation-retranslation method. RESULTS: The final response rate was 69% (40/58). Results suggest that most radiographers trained EBR during their undergraduate training. Although, no statistically significant correlations were found in the practice of EBR against participant gender, age, training, career level, reading papers and workplace. The most frequent reason to read papers is the "interest" to do so, and national professional journals are read more often. It was found that radiographers that read scientific papers more frequently know more about research (p = 0.005), understand the importance of research for the professional activity (p = 0.023), and know more on how to conduct research papers (p = 0.034). CONCLUSION: EBR within radiography is not yet well established, and radiographers' have varying viewpoints. Radiographers that read scientific papers more frequently understand better the philosophy behind this concept but it is very important to deepen the knowledge on this area. IMPLICATIONS FOR PRACTICE: When practicing radiography based on the best available scientific knowledge, professionals are ensuring the best for patients and for profession. To achieve this, and before taking any action, it is important to evaluate the current situation, and this research presents a way to do so.

Addressing the New Global Threat of Xylella fastidiosa.

Xylella fastidiosa is one of the most important threats to plant health worldwide. This bacterial pathogen has a long history, causing disease in the Americas on a range of agricultural crops and trees, with severe economic repercussions particularly on grapevine and citrus. In Europe, X. fastidiosa was detected for the first time in 2013 in association with a severe disease affecting olive trees in southern Italy. Subsequent mandatory surveys throughout Europe led to discoveries in France and Spain in various host species and environments. Detection of additional introductions of X. fastidiosa continue to be reported from Europe, for example from northern Italy in late 2018. These events are leading to a sea change in research, monitoring and management efforts as exemplified by the articles in this Focus Issue . X. fastidiosa is part of complex pathosystems together with hosts and vectors. Although certain X. fastidiosa subspecies and environments have been well studied, particularly those that pertain to established disease in North and South America, this represents only a fraction of the existing genetic, epidemiological, and ecological diversity. This Focus Issue highlights some of the key challenges that must be overcome to address this new global threat, recent advances in understanding the pathosystem, and steps toward improved disease control. It brings together the broad research themes needed to address the global threat of X. fastidiosa, encompassing topics from host susceptibility and resistance, genome sequencing, detection methods, transmission by vectors, epidemiological drivers, chemical and biological control, to public databases and social sciences. Open communication and collaboration among scientists, stakeholders, and the general public from different parts of the world will pave the path to novel ideas to understand and combat this pathogen.

Phylogeny of the Trichogramma endosymbiont Wolbachia, an alpha-proteobacteria (Rickettsiae) / Filogenia do endosimbionte Wolbachia em Trichogramma, an alpha-proteobacteria (Rickettsiae)

Abstract Wolbachia (Hertig) endosymbionts are extensively studied in a wide range of organisms and are known to be transmitted through the egg cytoplasm to the offsping. Wolbachia may cause several types of reproductive modifications in arthropods. In Trichogramma species, parthenogenesis-inducing Wolbachia bacteria allow females wasps to produce daughters from unfertilized eggs and these bacteria are present in at least 9% of all Trichogramma species. Phylogenetic studies have led to the subdivision of the Wolbachia clade in five supergroups (A, B, C, D and E) and Wolbachia from Trichogramma belong to supergroup B. Here, using the wsp gene, four groups of Wolbachia that infect Trichogramma species were distinguished and the addition of a new group "Ato" was suggested due to the addition of Wolbachia from Trichogramma atopovirilia (Oatman and Platner). Specific primers were designed and tested for the "Ato" group. Seventy-five percent of all evaluated Wolbachia strains from Trichogramma fell within "Sib" group.

Resumo Endosimbiontes do gênero Wolbachia (Hertig) são extensivamente estudados em uma ampla gama de organismos e são conhecidos por serem transmitidos via citoplasma do ovo hospedeiro para seu descendente. Wolbachia pode causar vários tipos de alterações reprodutivas nos artrópodes. Nas espécies de Trichogramma, a reprodução partenogenética induzida por Wolbachia, possibilita as fêmeas dos parasitoides a produção de fêmeas a partir de ovos não fertilizados e estas bactérias estão presentes em pelo menos 9% de todas as espécies de Trichogramma. Estudos filogenéticos têm levado a subdivisão do clado Wolbachia em cinco supergrupos (A, B, C, D and E). Wolbachia em Trichogramma pertence ao supergrupo B. Com o gene wsp foi possível se distinguir quatro grupos de Wolbachia que infectam Trichogramma e adicionar um novo grupo (Ato) devido a inclusão de Wolbachia detectada em Trichogramma atopovirilia (Oatman and Platner, 1983). Primers específicos foram construídos e testados para o grupo "Ato". Setenta e cinco por cento de todas as linhagens de Wolbachia que infectam Trichogramma se enquadraram dentro do grupo "Sib".

Phylogeny of the Trichogramma endosymbiont Wolbachia, an alpha-proteobacteria (Rickettsiae).

Wolbachia (Hertig) endosymbionts are extensively studied in a wide range of organisms and are known to be transmitted through the egg cytoplasm to the offsping. Wolbachia may cause several types of reproductive modifications in arthropods. In Trichogramma species, parthenogenesis-inducing Wolbachia bacteria allow females wasps to produce daughters from unfertilized eggs and these bacteria are present in at least 9% of all Trichogramma species. Phylogenetic studies have led to the subdivision of the Wolbachia clade in five supergroups (A, B, C, D and E) and Wolbachia from Trichogramma belong to supergroup B. Here, using the wsp gene, four groups of Wolbachia that infect Trichogramma species were distinguished and the addition of a new group "Ato" was suggested due to the addition of Wolbachia from Trichogramma atopovirilia (Oatman and Platner). Specific primers were designed and tested for the "Ato" group. Seventy-five percent of all evaluated Wolbachia strains from Trichogramma fell within "Sib" group.

Distinct Roles of Th17 and Th1 Cells in Inflammatory Responses Associated with the Presentation of Paucibacillary Leprosy and Leprosy Reactions.

It is well established that helper T cell responses influence resistance or susceptibility to Mycobacterium leprae infection, but the role of more recently described helper T cell subsets in determining severity is less clear. To investigate the involvement of Th17 cells in the pathogenesis of leprosy, we determined the immune profile with variant presentations of leprosy. Firstly, IL-17A, IFN-γ and IL-10 were evaluated in conjunction with CD4+ T cell staining by confocal microscopy of lesion biopsies from tuberculoid (TT) and lepromatous leprosy (LL) patients. Secondly, inflammatory cytokines were measured by multiplex assay of serum samples from Multibacillary (MB, n = 28) and Paucibacillary (PB, n = 23) patients and household contacts (HHC, n = 23). Patients with leprosy were also evaluated for leprosy reaction occurrence: LR+ (n = 8) and LR- (n = 20). Finally, peripheral blood mononuclear cells were analysed by flow cytometry used to determine the phenotype of cytokine-producing cells. Lesions from TT patients were found to have more CD4+ IL-17A+ cells than those from LL patients. Higher concentrations of IL-17A and IL-1ß were observed in serum from PB than MB patients. The highest serum IFN-γ concentrations were, however, detected in sera from MB patients that developed leprosy reactions (MB LR+ ). Together, these results indicate that Th1 cells were associated with both the PB presentation and also with leprosy reactions. In contrast, Th17 cells were associated with an effective inflammatory response that is present in the PB forms but were not predictive of leprosy reactions in MB patients.

Transmission of Xylella fastidiosa to Grapevine by the Meadow Spittlebug.

There is little information available on Xylella fastidiosa transmission by spittlebugs (Hemiptera, Cercopoidea). This group of insect vectors may be of epidemiological relevance in certain diseases, so it is important to better understand the basic parameters of X. fastidiosa transmission by spittlebugs. We used grapevines as a host plant and the aphrophorid Philaenus spumarius as a vector to estimate the effect of plant access time on X. fastidiosa transmission to plants; in addition, bacterial population estimates in the heads of vectors were determined and correlated with plant infection status. Results show that transmission efficiency of X. fastidiosa by P. spumarius increased with plant access time, similarly to insect vectors in another family (Hemiptera, Cicadellidae). Furthermore, a positive correlation between pathogen populations in P. spumarius and transmission to plants was observed. Bacterial populations in insects were one to two orders of magnitude lower than those observed in leafhopper vectors, and population size peaked within 3 days of plant access period. These results suggest that P. spumarius has either a limited number of sites in the foregut that may be colonized, or that fluid dynamics in the mouthparts of these insects is different from that in leafhoppers. Altogether our results indicate that X. fastidiosa transmission by spittlebugs is similar to that by leafhoppers. In addition, the relationship between cell numbers in vectors and plant infection may have under-appreciated consequences to pathogen spread.

Ferrisia gilli (Hemiptera: Pseudococcidae) Transmits Grapevine Leafroll-Associated Viruses.

Several mealybug species are vectors of grapevine leafroll-associated viruses (GLRaV), which cause the economically important grapevine leafroll disease in grape-producing regions worldwide. The mealybug Ferrisia gilli Gullan is a new pest of grapevines in El Dorado County, located in the Sierra Foothill wine-growing region of California. GLRaV species 1, 2, 3, and 4LV have been detected in vineyards with symptomatic vines in the Sierra Foothills. We conducted controlled virus acquisition and transmission experiments using source vine accessions infected with different combinations of GLRaV. We determined that F. gilli acquired GLRaV 1, 2, 3, and 4LV, and transmitted GLRaV-3 and GLRaV-4LV to uninfected recipient vines. Like numerous other mealybug species, in addition to causing direct damage to vines, F. gilli poses a threat to the grape industry as a vector of economically damaging viruses.

ITS-2 sequences-based identification of Trichogramma species in South America.

ITS2 (Internal transcribed spacer 2) sequences have been used in systematic studies and proved to be useful in providing a reliable identification of Trichogramma species. DNAr sequences ranged in size from 379 to 632 bp. In eleven T. pretiosum lines Wolbachia-induced parthenogenesis was found for the first time. These thelytokous lines were collected in Peru (9), Colombia (1) and USA (1). A dichotomous key for species identification was built based on the size of the ITS2 PCR product and restriction analysis using three endonucleases (EcoRI, MseI and MaeI). This molecular technique was successfully used to distinguish among seventeen native/introduced Trichogramma species collected in South America.

ITS-2 sequences-based identification of Trichogramma species in South America / Identificação molecular de espécies de Trichogramma na América do Sul via sequenciamento da região ITS-2

Abstract ITS2 (Internal transcribed spacer 2) sequences have been used in systematic studies and proved to be useful in providing a reliable identification of Trichogramma species. DNAr sequences ranged in size from 379 to 632 bp. In eleven T. pretiosum lines Wolbachia-induced parthenogenesis was found for the first time. These thelytokous lines were collected in Peru (9), Colombia (1) and USA (1). A dichotomous key for species identification was built based on the size of the ITS2 PCR product and restriction analysis using three endonucleases (EcoRI, MseI and MaeI). This molecular technique was successfully used to distinguish among seventeen native/introduced Trichogramma species collected in South America.

Resumo Sequências do Espaço Transcrito Interno 2 (ITS2) têm sido utilizadas em estudos taxonômicos e sua utilidade constatada pela confiabilidade que o método confere à identificação das espécies de Trichogramma. Esta técnica molecular foi bem sucedida em distinguir dezessete espécies nativas e introduzidas de Trichogramma, coletadas na América do Sul. As sequências do DNAr variaram de 379 a 632 pb. Em 11 linhagens de T. pretiosum estudadas, o endosinbionte Wolbachia foi detectado pela primeira vez. Estas linhagens telítocas foram encontradas no Peru (9), Colômbia (1) e Estados Unidos (1). Uma chave dicotômica para identificação de espécies foi construída baseada no tamanho do produto da PCR do ITS2 e em análises de restrição utilizando-se três endonucleases (EcoRI, MseI and MaeI).

Interactions Within Susceptible Hosts Drive Establishment of Genetically Distinct Variants of an Insect-Borne Pathogen.

Coinfections are common, leading to pathogen interactions during transmission and establishment in a host. However, few studies have tested the relative strengths of pathogen interactions in vectors and hosts that determine the outcome of infection. We tested interactions between two genetically distinct variants of the mealybug-transmitted Grapevine leafroll-associated virus 3. The transmission efficiency of each variant in single variant inoculations by two vector species was determined. The effects of vector species, a coinfected source, and simultaneous inoculation from multiple hosts to one host on variant establishment were examined. Within-vector interactions could have a role in transmission from hosts containing mixed infections, but not when vectors were moved from separate singly infected source plants to a single recipient plant. The invasive Planococcus ficus (Signoret) was a more efficient vector than Pseudococcus viburni (Signoret). Transmission efficiency of the two variants did not differ in single variant inoculations. Overall infections were the same whether from singly or coinfected source plants. In mixed inoculations, establishment of one variant was reduced. Mixed inoculations from two singly infected source plants resulted in fewer mixed infections than expected by chance. Therefore, the observed outcome was determined subsequent to host inoculation rather than in the vector. The outcome may be due to resource competition between pathogens. Alternatively apparent competition may be responsible; the pathogens' differential ability to overcome host defenses and colonize the host may determine the final outcome of new infections. Detailed knowledge of interactions between pathogens during transmission and establishment could improve understanding and management of disease spread.

Expression of glyceraldehyde 3-phosphate dehydrogenase is enhanced in Leishmania spp naturally resistant to nitric oxide.

Leishmania spp are the causative agents of a spectrum of diseases termed leishmaniasis that affect mammals, including humans and dogs. Although reactive nitrogen species are employed in the control of parasitism by the immune system, it is known that Leishmania can withstand this oxidative stress. As the mechanism by which these species are resistant to nitric oxide (NO) is poorly understood, the main objective of this study was to evaluate the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in Leishmania amazonensis and Leishmania chagasi promastigotes showing natural resistance to NO. GAPDH transcript levels were quantified by real-time polymerase chain reaction amplification, and GAPDH activity (assessed by levels of NADH oxidation) was measured by spectrophotometry. The level of nitration in total protein was assessed by immunoblotting. The results demonstrated an increase in GAPDH expression in resistant isolates of both species compared to susceptible isolates. The increase in GAPDH expression led to an increase in the activity of GAPDH in L. amazonensis human isolates resistant to NO. The pattern of protein nitration did not differ between sensitive and resistant isolates. Our results suggest that changes in expression of GAPDH may be responsible, at least in part, to natural resistance to NO found in human and canine Leishmania spp.

Molecular identification of Trichogramma species from regions in Brazil using the sequencing of the ITS2 region of ribosomal DNA.

The objective of this work was the identification and differentiation of Trichogramma exiguum Pinto and Platner species, T. pretiosum Riley, and T. galloi Zucchi using sequences of the ITS2 region of ribosomal DNA. After extracting DNA from the studied species, a PCR reaction was performed, where the amplified samples were subjected to sequencing. The sequences obtained were submitted to a similarity search in GenBank (NCBI - National Center for Biotechnology Information) using the BLAST program, aiming to determine the similarity of these sequences with the species already deposited in the referenced database, and then multiple sequences were aligned using version 2.0 of the ClustalX program. According to the results of the multiple alignments of all sequences obtained, it was possible to observe the differences between the T. pretiosum, T. galloi and T. exiguum species. It was concluded that using the sequences of the ITS2 region of the ribosomal DNA was efficient in the differentiation of the studied Trichogramma species, which suggests a strong inter-specific variation among species.

The exopolysaccharide of Xylella fastidiosa is essential for biofilm formation, plant virulence, and vector transmission.

Exopolysaccharides (EPS) synthesized by plant-pathogenic bacteria are generally essential for virulence. The role of EPS produced by the vector-transmitted bacterium Xylella fastidiosa was investigated by knocking out two genes implicated in the EPS biosynthesis, gumD and gumH. Mutant strains were affected in growth characteristics in vitro, including adhesion to surfaces and biofilm formation. In addition, different assays were used to demonstrate that the mutant strains produced significantly less EPS compared with the wild type. Furthermore, gas chromatography-mass spectrometry showed that both mutant strains did not produce oligosaccharides. Biologically, the mutants were deficient in movement within plants, resulting in an avirulent phenotype. Additionally, mutant strains were affected in transmission by insects: they were very poorly transmitted by and retained within vectors. The gene expression profile indicated upregulation of genes implicated in cell-to-cell signaling and adhesins while downregulation in genes was required for within-plant movement in EPS-deficient strains. These results suggest an essential role for EPS in X. fastidiosa interactions with both plants and insects.

Leishmania chagasi naturally resistant to nitric oxide isolated from humans and dogs with visceral leishmaniasis in Brazil.

Nitric oxide (NO) plays an important role as a leishmanicidal agent in murine macrophages. NO resistant Escherichia coli and Mycobacterium tuberculosis have been associated with poor outcomes of their resulting diseases. NO resistant Leishmania braziliensis has also been identified and exacerbates the clinical course of human leishmaniasis. We report, for the first time, natural resistance of Leishmania chagasi promastigotes to NO. These parasites were isolated from humans and dogs with visceral leishmaniasis. We also demonstrate that this resistance profile was associated with a greater survival capacity and a greater parasite burden in murine macrophages, independent of activation and after activation by IFN-γ and LPS.

Outbreak of Neisseria meningitidis C in workers at a large food-processing plant in Brazil: challenges of controlling disease spread to the larger community.

SUMMARYAn outbreak of meningococcal disease (MD) with severe morbidity and mortality was investigated in midwestern Brazil in order to identify control measures. A MD case was defined as isolation of Neisseria meningitidis, or detection of polysaccharide antigen in a sterile site, or presence of clinical purpura fulminans, or an epidemiological link with a laboratory-confirmed case-patient, between June and August 2008. In 8 out of 16 MD cases studied, serogroup C ST103 complex was identified. Five (31%) cases had neurological findings and five (31%) died. The attack rate was 12 cases/100 000 town residents and 60 cases/100 000 employees in a large local food-processing plant. We conducted a matched case-control study of eight primary laboratory-confirmed cases (1:4). Factors associated with illness in single variable analysis were work at the processing plant [matched odds ratio (mOR) 22, 95% confidence interval (CI) 2·3-207·7, P<0·01], and residing <1 year in Rio Verde (mOR 7, 95% CI 1·11-43·9, P<0·02). Mass vaccination (>10 000 plant employees) stopped propagation in the plant, but not in the larger community.

Does Wolbachia infection affect Trichogramma atopovirilia behaviour?

Unisexual Trichogramma forms have attracted much attention due to their potential advantages as biocontrol agents. Fitness studies have been performed and understanding the cost that Wolbachia may inflict on their hosts will help in deciding if Wolbachia infected (unisexual) forms are indeed better than sexual forms when used in biological control programmes. The influence of Wolbachia on the foraging behaviour (including walking activity and speed) of T. atopovirilia is reported in this paper. Temperature strongly affected T. atopovirilia female walking activity, but Wolbachia infected and uninfected females differed in none of the behavioural components that were measured such as walking activity and walking speed. Walking activity was highest at 25 masculineC and differed significantly from that at 20 and 15 masculineC. Trichogramma wasps were highly affected at 15 masculineC. Behaviour analysis with females showed that female wasps spend most of the time on drilling + ovipositing on host eggs followed by host drumming and walking while drumming. The parasitism rate and number of offspring did not differ significantly between infected and cured Trichogramma females. Biological control implications of these findings are discussed.

Comparative susceptibility of two banana cultivars to Banana bunchy top virus under laboratory and field environments.

Field and laboratory experiments were carried out on the island of Oahu, HI, to compare the susceptibility of the two most commonly grown banana (Musa sp.) cultivars in the state ('Dwarf Brazilian' or Santa Catarina [locally known as dwarf apple] and 'Williams') to the aphid-borne Banana bunchy top virus (genus Babuvirus, family Nanoviridae, BBTV). Several morphological and physiological features of the two cultivars were monitored to determine whether the banana aphid, Pentalonia nigronervosa Coquerel (Hemiptera: Aphididae), transmits BBTV to both cultivars at a similar rate; and whether after successful inoculation, does each cultivar respond similarly to viral infection. Results from the laboratory experiment showed that a similar percentage of both cultivars were infected with BBTV by aphid vectors (> 90% for both cultivars). However, field results showed a significantly lower percentage of dwarf apple (39%) infected with BBTV compared with Williams (79%). We also found that all physiological and morphological features measured (i.e., plant height, leaf area, canopy, chlorophyll level, and moisture content) for both cultivars were impacted similarly by BBTV. The incubation period, or the time between plant infection and initial appearance of disease symptoms, was similar for both cultivars. Results also showed that BBTV transmission efficiency was lower in the field than in the laboratory, despite that more aphids per plant were used for field than laboratory inoculation tests. The results highlight the potential use of less susceptible cultivars to help manage BBTV and the importance of screening banana varieties in the field to determine their response to vectors and associated diseases.

Transmission of grapevine leafroll-associated virus 3 by the vine mealybug (Planococcus ficus).

Grapevine leafroll disease is caused by grapevine leafroll-associated viruses (GLRaVs). Within this virus complex, GLRaV-3 is the predominant species in the world. Several GLRaVs have been shown to be transmitted from vine to vine by mealybugs although a detailed characterization of transmission biology is lacking. The introduction of the vine mealybug (Planococcus ficus) in California and other regions of the world may result in increasing disease incidence of established GLRaVs. We studied the characteristics of GLRaV-3 transmission by the vine mealybug. Our results indicate that the vine mealybug transmits GLRaV-3 in a semipersistent manner. First instars were more efficient vectors than adult mealybugs. GLRaV-3 transmission lacked a latent period in the vector. Virus transmission occurred with a 1-h acquisition access period (AAP) and peaked with a 24-h AAP. Mealybugs inoculated GLRaV-3 with a 1-h inoculation access period (IAP), and transmission efficiency increased with longer plant access period up to 24 h, after which transmission rate remained constant. After an AAP of 24 h, mealybugs lost GLRaV-3 and infectivity 4 days after virus acquisition. In addition, GLRaV-3 was not transovarially transmitted from infected females to their progeny as detected by reverse transcription polymerase chain reaction. In summary, we systematically analyzed transmission parameters of GLRaV-3 by the vine mealybug and showed that transmission of this virus occurs in a semipersistent manner. This research fills in important gaps in knowledge of leafroll virus transmission, which is critical for development of leafroll disease management practices.