Using the rDNA Internal Transcribed Spacer 1 to Identify the Invasive Pest Rhagoletis cerasi (Diptera: Tephritidae) in North America

The cherry-infesting fruit fly Rhagoletis cerasi Loew is a significant commercial pest in Europe that has recently invaded North America. To date, it has been trapped only in Canada and northwestern counties of New York. It has the potential to spread further and threaten production and movement of cherry commodities. Timely diagnosis of the pest will facilitate surveys and quick response to new detections. Adult morphology of the pest is distinct from other flies in North America. However, when flies are significantly damaged on traps or the immature life stages are found in fruits, molecular methods of identification are important to confirm presence and host-use records. Other than DNA sequencing of genes from flies which takes over a day to complete, there are no timely methods of molecular identification for this pest. In this study, we report the first sequence record of the internal transcribed spacer 1 (ITS1) from R. cerasi and develop two diagnostic tests for the pest based on ITS1 differences among species in North America. The tests use loop-mediated isothermal amplification (LAMP) and multiplex, conventional polymerase chain reaction (mcPCR) technologies that target the same region of the R. cerasi ITS1 sequence. Both tests performed well when tested against collections of R. cerasi from North America and Europe, generating Diagnostic Sensitivity estimates of 98.4–99.5%. Likewise, the tests had relatively high estimates of Diagnostic Specificity (97.8–100%) when tested against Rhagoletis Loew species present in North America that also use cherry as a developmental host

Use of ITS‑1 to identify Bactrocera dorsalis and Bactrocera occipitalis (Diptera: Tephritidae): a case study using flies trapped in California from 2008 to 2018

Molecular methods are necessary to diagnose immature life stages of the agricultural pest fruit fly Bactrocera dorsalis (Hendel), and are useful to corroborate identifications based on adults because morphological variation within the species can overlap with congeners. DNA sequencing of the nuclear ribosomal internal transcribed spacer 1 (ITS‑1) has been adopted by the International Plant Protection Convention as an internationally accepted method to distinguish between the 2 pestiferous fruit fly species Bactrocera dorsalis and Bactrocera carambolae (Drew & Hancock). Reported ITS‑1 sequences also are distinct and diagnostically informative to distinguish several other Bactrocera species related to B. dorsalis. In this study, we applied DNA sequencing of ITS‑1 to a collection of 513 adult flies trapped in California, USA, in the yr 2008 to 2018. Internal transcribed spacer 1 sequences were successfully recovered from 504 (98%) of these flies. One fly had an ITS‑1 sequence that matched B. occipitalis (Bezzi) records. Re-examination of that fly using cytochrome c oxidase I, elongation factor 1‑alpha, and morphology supports it as the second record of B. occipitalis trapped in California. The other 503 flies had ITS‑1 sequences consistent with B. dorsalis. Six unique ITS‑1 sequences (or DNA types) were observed in the collection of 503 B. dorsalis. Three of the ITS‑1 sequences (types A, B, and C) were present in 84% of the 503 flies and match ITS‑1 records reported in prior publications on B. dorsalis. The other 3 sequences (types D, E, and F) observed in 4% of the 503 B. dorsalis have not been reported in publications. Ambiguous nucleotides were observed from 12% of the 503 B. dorsalis flies, precluding designation of a sequence type. Including the 3 new types from the current study, a total of 15 unique ITS‑1 sequences now are known for B. dorsalis. The study, therefore, documents additional intraspecific variation of ITS‑1 that aids in future applications for species identification. - Los métodos moleculares son necesarios para diagnosticar los estadios de vida inmaduras de la plaga agrícola mosca de la fruta Bactrocera dorsalis (Hendel) y son útiles para corroborar identificaciones basadas en adultos por la variación morfológica dentro de la especie puede superponerse con congéneres. La secuenciación del ADN del espaciador transcrito interno ribosómico nuclear 1 (ITS-1) ha sido adoptada por la Convención Internacional de Protección Fitosanitaria como un método aceptado internacionalmente para distinguir entre las dos especies de moscas de la fruta, Bactrocera dorsalis y Bactrocera carambolae (Drew & Hancock). Las secuencias de ITS-1 notificadas también son distintas y proporcionan información diagnóstica para distinguir varias otras especies de Bactrocera relacionadas con B. dorsalis. En este estudio, aplicamos la secuenciación de ADN de ITS-1 a una colección de 513 moscas adultas atrapadas en California, EE. UU. desde el 2008 hasta el 2018. Se recuperaron las secuencias espaciadoras transcritas internas1 con éxito de 504 (98%) de estas moscas. Una mosca tenía una secuencia ITS-1 que coincidía con los registros de B. occipitalis (Bezzi). El reexamen de esa mosca usando la citocromo c oxidasa I, el factor de elongación 1-alfa y la morfología lo respalda como el segundo registro de B. occipitalis atrapada en California. Las otras 503 moscas tenían secuencias de ITS-1 compatibles con B. dorsalis. Se observaron seis secuencias únicas de ITS-1 (o tipos de ADN) en la colección de 503 B. dorsalis. Tres de las secuencias de ITS-1 (tipos A, B, y C) estaban presentes en el 84% de las 503 moscas y coinciden con los registros de ITS-1 informados en publicaciones anteriores sobre B. dorsalis. Las otras 3 secuencias (tipos D, E, y F) observadas en el 4% de las 503 B. dorsalis no han sido reportadas en publicaciones. Se observaron nucleótidos ambiguos en el 12% de las 503 moscas B. dorsalis, lo que excluye la designación de un tipo de secuencia. Incluyendo los 3 nuevos tipos del estudio actual, ahora se conocen un total de 15 secuencias ITS-1 únicas para B. dorsalis. Por lo tanto, el estudio documenta una variación intraespecífica adicional de ITS-1 que ayuda en futuras aplicaciones para la identificación de especies

Episodic Radiations in the Fly Tree of Life

Flies are one of four superradiations of insects (along with beetles, wasps, and moths) that account for the majority of animal life on Earth. Diptera includes species known for their ubiquity (Musca domestica house fly), their role as pests (Anopheles gambiae malaria mosquito), and their value as model organisms across the biological sciences (Drosophila melanogaster). A resolved phylogeny for flies provides a framework for genomic, developmental, and evolutionary studies by facilitating comparisons across model organisms, yet recent research has suggested that fly relationships have been obscured by multiple episodes of rapid diversification. We provide a phylogenomic estimate of fly relationships based on molecules and morphology from 149 of 157 families, including 30 kb from 14 nuclear loci and complete mitochondrial genomes combined with 371 morphological characters. Multiple analyses show support for traditional groups (Brachycera, Cyclorrhapha, and Schizophora) and corroborate contentious findings, such as the anomalous Deuterophlebiidae as the sister group to all remaining Diptera. Our findings reveal that the closest relatives of the Drosophilidae are highly modified parasites (including the wingless Braulidae) of bees and other insects. Furthermore, we use micro-RNAs to resolve a node with implications for the evolution of embryonic development in Diptera. We demonstrate that flies experienced three episodes of rapid radiation—lower Diptera (220 Ma), lower Brachycera (180 Ma), and Schizophora (65 Ma)—and a number of life history transitions to hematophagy, phytophagy, and parasitism in the history of fly evolution over 260 million y

Chronic Infection Drives Expression of the Inhibitory Receptor CD200R, and Its Ligand CD200, by Mouse and Human CD4 T Cells

Certain parasites have evolved to evade the immune response and establish chronic infections that may persist for many years. T cell responses in these conditions become muted despite ongoing infection. Upregulation of surface receptors with inhibitory properties provides an immune cell-intrinsic mechanism that, under conditions of chronic infection, regulates immune responses and limits cellular activation and associated pathology. The negative regulator, CD200 receptor, and its ligand, CD200, have been shown to regulate macrophage activation and reduce pathology following infection. We show that CD4 T cells also increase expression of inhibitory CD200 receptors (CD200R) in response to chronic infection. CD200R was upregulated on murine effector T cells in response to infection with bacterial, Salmonella enterica, or helminth, Schistosoma mansoni, pathogens that respectively drive predominant Th1- or Th2-responses. In vitro chronic and prolonged stimuli were required for the sustained upregulation of CD200R, and its expression coincided with loss of multifunctional potential in T effector cells during infection. Importantly, we show an association between IL-4 production and CD200R expression on T effector cells from humans infected with Schistosoma haematobium that correlated effectively with egg burden and, thus infection intensity. Our results indicate a role of CD200R:CD200 in T cell responses to helminths which has diagnostic and prognostic relevance as a marker of infection for chronic schistosomiasis in mouse and man

The Effect of Repetitive Transcranial Magnetic Stimulation on Gamma Oscillatory Activity in Schizophrenia

Gamma (γ) oscillations (30-50 Hz) have been shown to be excessive in patients with schizophrenia (SCZ) during working memory (WM). WM is a cognitive process that involves the online maintenance and manipulation of information that is mediated largely by the dorsolateral prefrontal cortex (DLPFC). Repetitive transcranial magnetic stimulation (rTMS) represents a non-invasive method to stimulate the cortex that has been shown to enhance cognition and γ oscillatory activity during WM.We examined the effect of 20 Hz rTMS over the DLPFC on γ oscillatory activity elicited during the N-back task in 24 patients with SCZ compared to 22 healthy subjects. Prior to rTMS, patients with SCZ elicited excessive γ oscillatory activity compared to healthy subjects across WM load. Active rTMS resulted in the reduction of frontal γ oscillatory activity in patients with SCZ, while potentiating activity in healthy subjects in the 3-back, the most difficult condition. Further, these effects on γ oscillatory activity were found to be specific to the frontal brain region and were absent in the parieto-occipital brain region.We suggest that this opposing effect of rTMS on γ oscillatory activity in patients with SCZ versus healthy subjects may be related to homeostatic plasticity leading to differential effects of rTMS on γ oscillatory activity depending on baseline differences. These findings provide important insights into the neurophysiological mechanisms underlying WM deficits in SCZ and demonstrated that rTMS can modulate γ oscillatory activity that may be a possible avenue for cognitive potentiation in this disorder

The short-term effect of high versus moderate protein intake on recovery after strength training in resistance-trained individuals

Background: Dietary protein intakes up to 2.9 g.kg-1.d-1 and protein consumption before and after resistance training may enhance recovery, resulting in hypertrophy and strength gains. However, it remains unclear whether protein quantity or nutrient timing is central to positive adaptations. This study investigated the effect of total dietary protein content, whilst controlling for protein timing, on recovery in resistance trainees. Methods: Fourteen resistance-trained individuals underwent two 10-day isocaloric dietary regimes with a protein content of 1.8 g.kg-1.d-1 (PROMOD) or 2.9 g.kg-1.d-1 (PROHIGH) in a randomised, counterbalanced, crossover design. On days 8-10 (T1-T3), participants undertook resistance exercise under controlled conditions, performing 3 sets of squat, bench press and bent-over rows at 80% 1 repetition maximum until volitional exhaustion. Additionally, participants consumed a 0.4 g.kg-1 whey protein concentrate/isolate mix 30 minutes before and after exercise sessions to standardise protein timing specific to training. Recovery was assessed via daily repetition performance, muscle soreness, bioelectrical impedance phase angle, plasma creatine kinase (CK) and tumor necrosis factor-α (TNF-α). Results: No significant differences were reported between conditions for any of the performance repetition count variables (p>0.05). However, within PROMOD only, squat performance total repetition count was significantly lower at T3 (19.7 ± 6.8) compared to T1 (23.0 ± 7.5; p=0.006). Pre and post-exercise CK concentrations significantly increased across test days (p≤0.003), although no differences were reported between conditions. No differences for TNF-α or muscle soreness were reported between dietary conditions. Phase angle was significantly greater at T3 for PROHIGH (8.26 ± 0.82°) compared with PROMOD (8.08 ± 0.80°; p=0.012). Conclusions: When energy intake and peri-exercise protein intake was controlled for, a short term PROHIGH diet did not improve markers of muscle damage or soreness in comparison to a PROMOD approach following repeated days of intensive training. Whilst it is therefore likely that protein intakes (1.8g.kg-1.d-1) may be sufficient for resistance-trained individuals, it is noteworthy that both lower body exercise performance and bioelectrical phase angle were maintained with PROHIGH. Longer term interventions are warranted to determine whether PROMOD intakes are sufficient during prolonged training periods or when extensive exercise (e.g. training twice daily) is undertaken

Divergent responses of viral and bacterial communities in the gut microbiome to dietary disturbances in mice

To improve our understanding of the stability of mammalian intestinal communities, we characterized the responses of both bacterial and viral communities in murine fecal samples to dietary changes between high- and low-fat (LF) diets. Targeted DNA extraction methods for bacteria, virus-like particles and induced prophages were used to generate bacterial and viral metagenomes as well as 16S ribosomal RNA amplicons. Gut microbiome communities from two cohorts of C57BL/6 mice were characterized in a 6-week diet perturbation study in response to high fiber, LF and high-refined sugar, milkfat (MF) diets. The resulting metagenomes from induced bacterial prophages and extracellular viruses showed significant overlap, supporting a largely temperate viral lifestyle within these gut microbiomes. The resistance of baseline communities to dietary disturbances was evaluated, and we observed contrasting responses of baseline LF and MF bacterial and viral communities. In contrast to baseline LF viral communities and bacterial communities in both diet treatments, baseline MF viral communities were sensitive to dietary disturbances as reflected in their non-recovery during the washout period. The contrasting responses of bacterial and viral communities suggest that these communities can respond to perturbations independently of each other and highlight the potentially unique role of viruses in gut health

Towards a conceptual framework demonstrating the effectiveness of audiovisual patient descriptions (patient video cases): a review of the current literature

Background: Technological advances have enabled the widespread use of video cases via web-streaming and online download as an educational medium. The use of real subjects to demonstrate acute pathology should aid the education of health care professionals. However, the methodology by which this effect may be tested is not clear. Methods: We undertook a literature review of major databases, found relevant articles relevant to using patient video cases as educational interventions, extracted the methodologies used and assessed these methods for internal and construct validity. Results: A review of 2532 abstracts revealed 23 studies meeting the inclusion criteria and a final review of 18 of relevance. Medical students were the most commonly studied group (10 articles) with a spread of learner satisfaction, knowledge and behaviour tested. Only two of the studies fulfilled defined criteria on achieving internal and construct validity. The heterogeneity of articles meant it was not possible to perform any meta-analysis. Conclusions: Previous studies have not well classified which facet of training or educational outcome the study is aiming to explore and had poor internal and construct validity. Future research should aim to validate a particular outcome measure, preferably by reproducing previous work rather than adopting new methods. In particular cognitive processing enhancement, demonstrated in a number of the medical student studies, should be tested at a postgraduate level

Newly identified climatically and environmentally significant high-latitude dust sources

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