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SYMBIONTS TODAY, PROBIOTICS TOMORROW: MICROBE-BASED STRATEGY FOR IMPROVING HONEYBEE HEALTH
Full text linkIl più importante tra gli insetti impollinatori è l’ape, Apis mellifera, il quale svolge un ruolo fondamentale in molti raccolti orto-frutticoli ma anche contribuisce al mantenimento della biodiversità delle colture selvatiche. Nelle ultime decadi è stata registrata una grossa moria di alveari in tutto il pianeta. Le cause di questo fenomeno non sono ancora state del tutto chiarite e sembra esserci alla base una interazione di fattori biotici e abiotici, come il largo uso di agrofarmaci (es. neonicotinoidi), patogeni, parassiti e i cambiamenti climatici che stressano la salute dell’animale.
Negli ultimi anni le ricerche hanno dimostrato che i batteri simbionti sembrano essere determinanti nel proteggere la salute animale ma purtroppo il loro ruolo non è ancora stato studiato in modo approfondito. I simbionti sono dei microrganismi che stabiliscono delle forti interazioni con il loro animale “ospite”, e tra questi anche gli insetti (compresa l’ape). Tali sono coinvolti in molti aspetti della fisiologia dell’ospite, come la nutrizione, la riproduzione, l’omeostasi delle difese immunitarie, svolgendo un ruolo fondamentale nell’evoluzione dell’ospite stesso. La manipolazione e l’utilizzo del microbiota batterico può essere utile per lo sviluppo di strategie per la gestione di problemi legati agli insetti. Infatti, questo approccio, generalmente definito come ‘Microbial Resource Management’ (MRM), è stato descritto anche come ‘Symbiont Resource Management’ (SMR), quando applicato ai simbionti. L’importanza dei commensali microbici dell’ape per il mantenimento e il miglioramento della salute dell’ape è il tema principale di questa tesi di dottorato. In particolare, questo studio ha come obiettivo, inizialmente, di approfondire la diversità microbica associata all’apparato digerente delle api dell’area Mediterranea, la sua interazione con l’ospite e con un modello patogeno e, infine, sviluppare una strategia di biocontrollo basata sull’uso dei simbionti microbici così da migliorare la salute dell’ospite e non permettere lo sviluppo della malattia.
Usando il patogeno Paenibacillus larvae, agente eziologico della Peste Americana (AFB), è stata determinata la capacità di diversi batteri simbionti dell’ape in modo da verificare se possano co-esistere differenti attività sinergiche tra le varie classi batteriche nel preservare la salute dell’ape. L’AFB è una delle malattie più virulenti della larva dell’ape. E’ stata identificata in numerose aree, provocando enormi perdite economiche, ma purtroppo sono ancora poche informazioni sulla diversità del fattore eziologico. Settantacinque isolati di P. larvae ottenuti da larve sintomatiche e collezionate in differenti siti della Tunisia sono stati analizzati tramite test biochimici e sequenziamento del gene ribosomiale 16s. Tramite la metodologia della BOX-PCR, sono stati definiti dei profili specifici di P. larvae (comparandoli a quelli di altre specie di Paenibacillus) e che possono essere utili per la loro identificazione. Alcune bande, specifiche di P.larvae, possono rappresentare dei nuovi modelli molecolari per la identificazione delle specie. Il pattern ottenuto dalla BOX-PCR ha identificato una certa diversità intraspecifica. Ciononostante, le analisi di virulenza effettuate in-vivo di tre genotipi di P.larvae selezionati non hanno differito fortemente tra di loro, suggerendo che la patogenicità non è l’unico effetto connesso alla diversità genotipica e fenotipica.
Il microbiota associato all’intestino di api di diversa età sia sane che affette dal patogeno è stato studiato tramite l’analisi del gene 16s ribosomiale tramite la tecnica della elettroforesi con gradiente denaturante (DGGE). I taxa più rappresentativi identificati sono α-, β-, γ- Proteobatteri e Firmicutes sia nelle api adulte che nelle larve. Inoltre, è stato osservato un crescente sbilanciamento del microbioma all’avanzare della malattia. Uno studio più approfondito della diversità microbica delle larve del quinto stadio di crescita è stata fatta con l’analisi del gene ribosomiale 16s tramite la metodologia del pirosequenziamento. I dati hanno confermanto i risultati ottenuti dalle DGGE: le larve sintomatiche creano un unico cluster, separatamente dalle larve sane, mostrando anche una dominanza di sequenze dell’ordine dei Firmicutes, dalla quale fa parte P.larvae. Contrariamente, nelle larve sane α-, γ- Proteobatteri e Firmicutes sono gli ordini dominanti.
I metodi cultura-dipendenti hanno permesso di isolare differenti gruppi batterici, tra i quali batteri acetici (AAB), batteri lattici (LAB) e batteri sporigeni (SFB). Quindi, allo scopo di valutare se questi isolati potessero inibire la crescita del patogeno, è stato effettuato un test di inibizione in vitro contro due ceppi di P.larvae; in particolare P.larvae 20it e P.larvae BMG93. Gli esperimenti hanno dimostrato che numerosi ceppi tra gli AAB, LAB e SFB sono stati capaci di inibire fortemente lo sviluppo del patogeno.
Successivamente alcuni di questi ceppi sono stati saggiati in-vivo: la capacità dei simbionti selezionati nel proteggere le giovani larve (prima fase larvale) dall’infezione patogena è stata determinata confrontando la mortalità degli insetti con il patogeno a cui è stato somministrato il simbionte attraverso la dieta. E’ stata dimostrata la capacità dei due SFB di fermare l’invasione di P.larvae, abbassando la mortalità fino alla mortalità basale (cioè la mortalità delle larve alimentate con la sola dieta sterile). L’effetto di protezione sembra essere addirittura maggiore quando i due SFB erano somministrati alle larve contemporaneamente.
Alla base della protezione mediata dai batteri simbionti esistono diversi meccanismi, come sostiene Hamdi et al. (2011), tra i quali: 1) inibizione diretta del patogeno con il rilascio di molecole antimicrobiche; 2) induzione del sistema immunitario; 3) esclusione competitiva.
In modo da sviluppare una strategia di biocontrollo efficace e praticabile, questa ricerca ha focalizzato l’attenzione sullo studio di tali meccanismi svolti dai simbionti.
In primis, l’abilità dei simbionti di inibire la crescita del patogeno è stata dimostrata con un test di inibizione in vitro usando come surnatante competente al patogeno P.larvae, intestini omogeneizzati ottenuti da larve alimentate con i probiotici. I risultati hanno confermato che esiste un effetto diretto dei surnatanti intestinali nei confronti del patogeno.
Quindi, per valutare la capacità di aumentare l’espressione del sistema immunitario dell’ape, larve alimentate con diete arricchite con i probiotici sono state analizzate per l’espressione del sistema immunitario innato, tramite l’uso della tecnica della RT-Real Time-PCR quantitativa. Il trascritto mRNA di alcuni dei peptidi antimicrobici (AMPs) espressi dal sistema immunitario dell’ape; imenoptaecina, abaecina e defensina, hanno mostrato un aumento della trascrizione quando le larve erano alimentate in presenza del mix BT e BL, confermando un’attività probiotica sinergica dei due simbionti. Al contrario, il trascritto del gene lisozima ha mostrato di essere sotto espresso in tutti i trattamenti, rispetto alle larve alimentate con la sola dieta sterile. Infine, è stata valutata la capacità dei due probiotici di spiazzare il patogeno tramite competizione esclusiva. E’ stato difatti dimostrato che i due probiotici sono capaci di colonizzare l’intestino dell’ape. Questo è stato dimostrato tramite l’uso di tecniche molecolari come la BOX-PCR, che ha evidenziato come BT e BL erano presenti dopo 6 giorni dalla somministrazione. La colonizzazione dei probiotici è stata quindi confermata tramite la tecnica della ibridazione fluorescente in situ (FISH). BT e BL sembrano colonizzare l’intestino della larva, rallentando lo sviluppo della malattia quando co-somministrati in presenza del patogeno PL.
Inoltre, ulteriori esperimenti sono stati effettuati per dimostrare l’efficacia dei trattamenti probiotici nello prevenire l’attacco patogeno anche “in campo”. E’ stata quindi definita una modalità di somministrazione del prodotto probiotico direttamente sugli alveari, ed è stato effettuato anche un test di mortalità affiancato a ciascuna applicazione in campo. I risultati hanno confermato che i trattamenti con il mix BT/BL diminuivano fortemente la mortalità larvale, indicando che questo rappresenta un metodo efficace per prevenire lo sviluppo della malattia.
Inoltre, tramite l’uso della RT-Real time PCR quantitativa, sono stati analizzati i livelli dei trascritti di AMPs (abaecina ed imenoptaecina) di larve trattate in campo confrontate con quelle non trattate con il prodotto probiotico e sottoposti al patogeno. Una risposta immunitaria preventiva è evidente, misurando gli aumenti dei trascritti dei geni abaecina ed imenoptaecina quando le larve erano in presenza del prodotto probiotico. Quando le larve trattate erano invece in presenza del ceppo patogeno, una diminuzione dei livelli di trascritto di abaecina e di imenoptaecina rispetto alle larve non trattate avviene dopo il quinto trattamento, nonostante un forte abbassamento della mortalità larvale mostrato dagli esperimenti di mortalità. Questa diminuzione dei trascritti dei due geni erano invece non più osservati dopo il settimo trattamento con i ceppi probiotici, coerentemente con quanto mostrato dall’esperimento di mortalità. Questo indica che l’influenza dei due ceppi probiotici nella espressione degli AMP, quando la larva era trattata con i due ceppi per lungo tempo, sembra prevalere sull’influenza guidata dal patogeno e che inoltre i due probiotici favoriscono l’omeostasi immunitaria quando in presenza del patogeno.
Concludendo, la ricerca enfatizza l’importanza dei batteri simbionti intestinali dell’ape con un effetto probiotico nella prevenzione di una patologia e soprattutto suggerisce che i probiotici possono migliorare lo stato di salute dell’ospite implementando la protezione contro i diversi tipi di stress a cui l’ospite è sottoposto.Among pollinators the honeybee Apis mellifera is the most important one playing an essential role in many crops, fruit and wild plants and contributing in the maintenance of biodiversity. In the last decades, a large-scale loss of honeybee colonies is occurring worldwide. The causes of this decline are yet not completely clear and are believed to stem from the interaction of several biotic and abiotic stress factors, such as insecticides, pathogens and parasites and the ongoing climate changes. In the recent years bacterial gut symbionts have been revealed a very important but yet understudied factor in protecting animal health. Symbionts are microorganisms establishing close interactions with their animal host, including insects and honeybees. They are involved in many aspects of the host physiology, including nutrition, reproduction, immune homeostasis and defense and have played major role in the host evolution. The manipulation and exploitation of the insect microbiota could be effective for the development of strategies for the management of insect-related problems. Indeed, this approach, generally defined as ‘Microbial Resource Management’ (MRM), was described as ‘Symbiont Resource Management’ (SMR) when applied to insect symbionts. The importance of the honeybee microbial commensals for the maintenance and improvement of honeybee health is the main topic of this PhD thesis. In particular, this study aims to dissect, first, the microbial diversity associated to Mediterranean honeybee gut, its interaction with the host and a model honeybee pathogen and, finally, to develop a pathogen biocontrol strategy, based on the use of honeybee symbionts, in order to improve the host health and to counter face the pathogen infection.
Using as pathogenic model Paenibacillus larvae, the causative agent of the American Foulbrood Disease (AFB), the ability of different intestinal honeybee symbionts has been assessed in order to verify if synergistic activities of different classes of bacteria can occur in preserving host health.
AFB is one of the most virulent disease of honeybee larvae. It was detected in many beekeeping areas, where it causes important economic losses, but little is known about the diversity of the causing agent. Seventy-five isolates of P. larvae, identified by biochemical tests and 16S rRNA gene sequencing, were obtained from fifteen contaminated broods showing typical AFB symptoms and collected in different locations in Tunisia. Using BOX-PCR, distinct profiles of P. larvae with respect to related Paenibacillus species were detected and may be useful for its identification. Some P. larvae-specific bands represented novel potential molecular markers for the identification of the species. BOX-PCR fingerprints indicated a relatively high intraspecific diversity. Nonetheless, the in vivo evaluation of virulence of three selected P. larvae genotypes did not differ significantly one another, suggesting that pathogenicity is not the only effect related to the genotypic and phenotypic diversity.
The microbiota associated to the gut of healthy and P. larvae-infected honeybees of different stage was characterized by 16S rRNA gene based Denaturing Gradient Gel Electrophoresis (DGGE). Proteobacteria of the α-, β- and γ- subgroups and Firmicutes were identified as the major bacterial taxa associated to A. mellifera larvae and adults. Moreover, an increasing intestinal unbalance in the larval microbiome (dysbiosis) associated with the development of the disease was observed. An in-depth analysis of the microbial diversity from 5th instar larvae collected from healthy and AFB infected hives was performed by 16S rRNA gene barcoding pyrosequencing. Data analysis confirmed DGGE results: symptomatic larvae clustered clearly together, separately from healthy ones, and showed dominance of sequences of the order Bacillales, to which P. larvae belongs. Conversely, in healthy larvae members of Firmicutes, Alpha and Gammaproteobacteria were detected.
Culture-based methods allowed the isolation of bacteria belonging to different taxa, including Acetic Acid (AAB), Lactic Acid (LAB), and Spore Forming (SFB) Bacteria. In order to evaluate if the isolates may hinder the growth of P. larvae, an inhibition test was performed in vitro against two strains of P. larvae, namely P. larvae 20it and P. larvae BMG93. The experiments demonstrated that several strains, among which one AAB, one LAB and two SFB, were capable of strongly inhibiting the growth of two pathogen strains.
An in-vivo rearing assay was performed. The capacity of the selected symbionts to protect young honeybee larvae from P. larvae infection was assessed by challenging the animals with the pathogen after administering to the larvae, reared in 96-well plates, the symbionts through the diet. It was demonstrated the capability of the two SFB strains (BT and BL) to counter face the pathogen, lowering the larvae mortality to the background mortality measured under normal diet. The protection action resulted stronger when the two bacteria were administered together to the larvae.
Different mechanisms mediated by the microbial symbionts are involved in the honeybee protection (Hamdi et al., 2011), among which 1) direct inhibition of pathogen by the release of antimicrobial compounds; 2) stimulation of the immune system; and 3) competitive exclusion. In order to develop a suitable and feasible biocontrol strategy this research focused on the evaluation of the symbionts-mediated mechanisms.
First, the symbiotic ability to inhibit the growth of the pathogen was analysed measuring directly the pathogen inhibition "in vivo" by the symbionts. Smashed guts obtained from larvae fed with the probiotics were evaluated for their inhibition capability against the two strains of P. larvae, confirming that a direct inhibition activity is produced in the larval gut.
In order to understand whether the candidate probiotic bacteria enhance the honeybee larval immune system, honeybee brood response to bacterial-enriched diets was detected by assessing the expression of innate-immune system-related genes using quantitative Real Time RT-PCR. The main antimicrobial peptides (AMPs) hymenoptaecin, abaecin, and defensin, showed an increase of transcription when larvae were fed with the mixture of BT and BL, confirming a synergistic activity between the two probiotics. Conversely, the lysozyme transcripts were down regulated in all the treatments, in comparison to the larvae fed with the artificial sterile diet. Ultimately, it has been evaluated the capability of the probiotics to outcompete with the pathogen by competitive exclusion. The two probiotics were able to successfully recolonize the larval gut and by the use of molecular techniques, such as BOX PCR, it was demonstrated that BT and BL were present after six days from the initial administration. The colonization of the probiotics in the larval gut was confirmed by Fluorescence In Situ Hybridization (FISH). BT and BL colonized the honeybee midgut, hindering the development of the disease in co-administration experiments.
Finally, experiments to evaluate the efficacy of the probiotic treatment in counteracting the pathogen were performed in real field conditions. An approach to administer the probiotics to honeybee larvae directly on the hives was developed, and a mortality test was coupled for the 7 week-applications of the probiotics. The results confirmed that the treatment with the BT/BL mix significantly decreased the larval mortality, indicating the approach as an effective method to prevent the disease development.
The levels of AMP transcripts (abaecin and hymenoptaecin) of larvae treated or not in field condition with the two probiotic strains and then challenged with the pathogen, were measured by Real Time RT-PCR. A disease prevention response, measured as significant increases of abaecin and hymenoptaecin transcript levels, occurred when the larvae were treated with the probiotic bacteria. When the larvae treated with the probiotic strains were exposed to the pathogen, a decrease in the levels of the abaecin and hymenoptaecin transcripts respect to the non treated larvae occurred at the fifth week of treatments with the probiotic strains, despite such a treatment significantly decreased the larval mortality induced by the pathogen. Such decreases of the two transcript levels induced by the pathogen were abolished at the seventh week of treatment with the two probiotic strains, in coherence with the maintained decreased mortality. This indicates that the influence of the two probiotic strains on the AMP expression, when the larvae were continuously treated with the two strains overtime, prevailed on that driven by the pathogen and that the two probiotics support the immune response homeostasis even in presence of the pathogen challenge.
In summary, the research emphasized the importance of probiotic gut symbionts in the prevention of a honeybee disease and the overall results suggest that probiotics may in general improve host health possibly by helping in protecting it from different kinds of stresses
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
Author-wise bibliometric analysis based on entropy.
No full textAuthor-wise bibliometric analysis based on entropy.</p
Author Under Sail The Imagination of Jack London, 1893-1902
No full textIn Author Under Sail, Jay Williams offers the first complete literary biography of Jack London as a professional writer engaged in the labor of writing. It examines the authorial imagination in London's work, the use of imagination in both his fiction and nonfiction, and the ways he defined imagination in the creative process in his business dealings with his publishers, editors, and agents. In this first volume of a two-volume biography, Williams traverses the years 1893 to 1902, from London's "Story of a Typhoon" to The People of the Abyss. The Jack London who emerges in the pages of Author Under Sail is a writer whose partnership with publishers, most notably his productive alliance with George Brett of Macmillan, was one of the most formative in American literary history. London pioneered many author models during the heyday of realism and naturalism, blurring the boundaries of these popular genres by focusing on absorption and theatricality and the representation of the seen and unseen. London created an impassioned, sincere, and extremely personal realism unlike that of other American writers of the time. Author Under Sail is a literary tour de force that reveals the full range of London as writer, creative citizen, and entrepreneur at the same time it sheds light on the maverick side of machine-age literature.Intro -- Title Page -- Copyright Page -- Dedication -- Contents -- Acknowledgments -- Introduction -- 1. Spirit Truth -- 2. From Absorption to Theatricality and Back Again -- 3. "I Will Build a New Present" -- 4. Sons as Authors -- 5. Fathers as Publishers -- 6. The Daughter as Author -- 7. Lovers as Authors -- 8. At Sea with the Family -- 9. Yellow News, Yellow Stories -- 10. The Return Home -- Notes -- Bibliography -- Index -- About Jay WilliamsIn Author Under Sail, Jay Williams offers the first complete literary biography of Jack London as a professional writer engaged in the labor of writing. It examines the authorial imagination in London's work, the use of imagination in both his fiction and nonfiction, and the ways he defined imagination in the creative process in his business dealings with his publishers, editors, and agents. In this first volume of a two-volume biography, Williams traverses the years 1893 to 1902, from London's "Story of a Typhoon" to The People of the Abyss. The Jack London who emerges in the pages of Author Under Sail is a writer whose partnership with publishers, most notably his productive alliance with George Brett of Macmillan, was one of the most formative in American literary history. London pioneered many author models during the heyday of realism and naturalism, blurring the boundaries of these popular genres by focusing on absorption and theatricality and the representation of the seen and unseen. London created an impassioned, sincere, and extremely personal realism unlike that of other American writers of the time. Author Under Sail is a literary tour de force that reveals the full range of London as writer, creative citizen, and entrepreneur at the same time it sheds light on the maverick side of machine-age literature.Description based on publisher supplied metadata and other sources.Electronic reproduction. Ann Arbor, Michigan : ProQuest Ebook Central, YYYY. Available via World Wide Web. Access may be limited to ProQuest Ebook Central affiliated libraries
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