Résultat de la recherche
Isolation of Serratia marcescens involved in chitin degradation in the bulb mite Rhizoglyphus robini
There is an increasing awareness of the importance of the microbiome of arthropods to understand their host’s biology. In the bulb mite, Rhizoglyphus robini, associated bacteria have been found to be involved in its chitinolytic abilities. The bulb mite, a plant pest feeding on below- ground parts of mostly Liliaceae crops, prefers fungus- infested plants. Moreover its fitness is higher when feeding on a fungal food source than when feeding on non-infected plants. In this study we isolated a chitinolytic bacterium from mite homogenate and identified it molecularly as Serratia marcescens (Bizio 1823), which is a model organism for chitin degradation. Precise identification of the bacterium can be important for the development of biological control programs of the mite as well as for further studies investigating Serratia marcescens and its chitinolytic machinery.
Symbiontes et arthropodes: quelles implications pour la lutte biologique?
Arthropod symbioses: a neglected parameter in pest- and disease-control programmes
1. Arthropods are important players in biological control as pests, control agents and transmitters of invertebrate diseases. Arthropods are frequently infected with one or several micro-organisms, serving as micro-ecosystems in which multiple interactions can take place. These micro-organisms include disease agents and symbiotic micro-organisms. The latter are usually vertically transmitted and can have a broad spectrum of effects on their hosts, ranging from reproductive manipulations to protection against natural enemies. These interactions may directly or indirectly alter the biology of many arthropods in agriculturally, medically and ecologically relevant ecosystems.
2. Symbiotic micro-organism-induced reproductive manipulations such as cytoplasmic incompatibility and parthenogenesis induction can substantially affect the rearing of biological control agents. Many insects, and recently also mites and nematodes, have been found to be infected, displaying a wide range of effects. We discuss examples of arthropod-micro-organism interactions and effects, which could have consequences for the practical application of arthropods in biological control.
3. Symbiotic micro-organisms can also be involved in host protection against natural enemies such as parasitoids, pathogenic bacteria, fungi and viruses.
4. Symbiotic bacteria can influence the vectorial capacity of disease-vectoring arthropods and may be very helpful in decreasing the transmission of disease agents.
5. Synthesis and applications. The effect of micro-organisms on the outcome of biological control programmes is usually not considered in risk assessments and failure analyses. This review emphasizes the importance of endosymbiotic micro-organisms in comprehensive biological control programmes and provides recommendations on how to recognize, avoid or benefit from these influential tenants.
Predation of native coccinellids by the invasive alien Harmonia axyridis (Coleoptera: Coccinellidae): detection in Britain by PCR-based gut analysis
. 1. The harlequin ladybird, Harmonia axyridis, is an invasive alien species that arrived in Britain in 2003 and has spread across most of the country. 2. This spread has been accompanied by a decline in some native coccinellid species, in particular, the two spot ladybird Adalia bipunctata and the ten spot ladybird Adalia decempunctata. One reason for this decline could be intraguild predation (IGP) of the Adalia species by H. axyridis. 3. A DNA-based approach was used to look for evidence of coccinellid IGP in the wild. Adalia bipunctata- and A. decempunctata-specific Polymerase chain reaction primers were developed to analyse the gut contents of field collected H. axyridis larvae for the presence of Adalia DNA. Harmonia axyridis larvae (156) were collected from lime trees at four sites in eastern England over 3 years. 4. Adalia bipunctata DNA was detected in 7.7% and A. decempunctata DNA in 4.5% of H. axyridis larvae. DNA from both Adalia species was detected in two larvae. 5. This is the first demonstration of specific IGP interactions between H. axyridis and other coccinellid species in wild populations in Britain.
Mites and endosymbionts: towards improved biological control
Le microbiome vivant en association avec certains arthropodes est très divers et peut significativement influencer la biologie de nombreux agents de lutte biologique ou de ravageurs de culture. Certains endosymbiontes bactériens peuvent même être utilisés contre des populations de insectes ravageurs de ou de vecteurs de maladies dans un programme de lutte biologique.
Les espèces de la sous-classe des Acari (acariens et tiques) sont très diverses en forme, habitats et comportements. Il y a des acariens ravageurs, des parasites d’animaux domestiques ou de l’homme et même des acariens utiles, prédateurs pouvant s’attaquer à d’autres ravageurs de culture.
Le but du deuxième chapitre est de faire la synthèse de la littérature concernant les symbiontes d’acariens et en particulier sur l’incidence et la prévalence d’endosymbiontes maternellement transmis comme Arsenophonus spp., Cardinium spp., Flavobacteria spp., Rickettsia spp., Spiroplasma spp. et Wolbachia spp. chez les acariens (Chapitre 2). Bien que de nombreux articles aient été publié sur ce sujet, la plupart des études portent sur la présence d’endosymbiontes chez les tétranynques alors que de nombreuses espèces d’acariens ou des études sur les effets de ces bactéries sur leurs hôtes ont reçu moins d’attention. Ces données ont ensuite été complétées par notre propre étude ou des acariens phytophages, prédateurs et parasites ont été analysés pour la présence d’endosymbiontes (Chapitre 3). L’étape suivante de cette thèse a été de décrire les différents mécanismes par lesquelles les endosymbiontes peuvent influencer le succès de programme de contrôle biologique de ravageurs de culture ou de vecteur de maladies. Nous nous sommes également penchés sur l’apport de ces connaissances afin de mieux appréhender ou comprendre la biologie de ces arthropodes. Dans le chapitre un, nous faisons des recommandations pratiques sur les façons de prendre en compte les endosymbiontes dans le cadre de la lutte biologique (Chapitre 1).
L’acarien phytophage Rhizoglyphus robini s’attaque principalement aux racines de plantes cultivées de la famille des Liliacées. Bien que la plupart des animaux ne possèdent pas les enzymes nécessaires à la digestion d’hydrates de carbones d’origine fongique, cette espèce note une préférence alimentaire pour les plantes infectées par des champignons sur lesquelles elle est principalement trouvée. Par une approche multidisciplinaire, nous avons testé l’hypothèse que le bactériome était nécessaire à l’acarien pour digerer la chitine. Une analyse génétique préliminaire réalisée avec des sondes moléculaires générales a révélé une la présence d’une riche communauté de bactéries vivant avec cet acarien. Une analyse plus détaillée de cette communauté bactérienne par une approche métagénomique (454) a révélé la présence de plusieurs genres de bactéries connue pour être capables de dégrader la chitine. Des expériences en laboratoire ont confirmé la préférence des acariens pour une source de nourriture contenant des champignons et un taux de reproduction plus élevé sur ce type de régime alimentaire. Nous avons donc pu faire le lien entre la présence des bactéries et leur activité chitinolytique en démontrant l’activité de digestion d’homogénats d’acariens ainsi que de bactéries isolées de ces derniers (4). Nous avons enfin identifié la bactérie isolée comme Serratia marcescens (chapitre 5), une bactérie possédant une machinerie efficace pour dégrader la chitine des champignons.
Les détails de la biologie de l’acarien Rhizoglyphus robini ne peuvent donc être comprise qu’en tenant en compte de la biologie des bactéries qui lui sont associées. Cette étude complète nous a amené à reconsidérer le statut de peste de l’acarien. En effet R. robini était considéré comme une peste primaire, se nourrissant directement sur les racines des plantes alors que maintenant il est considéré comme une peste secondaire s’attaquant préférentiellement aux plantes préalablement infectées par un champignon. Le fait que l’acarien dépende de la présence de bactéries pour pouvoir digérer les champignons ouvre la voie pour de nouvelles stratégies de lutte. Nous sommes persuadés qu’une meilleure connaissance des communautés bactériennes vivant en association avec la plupart des acariens phytophages et des acariens bénéficiaires amèneront des informations cruciales pour le développement de nouvelles stratégies de lutte ou pour mieux comprendre les problèmes dans des programmes de lutte biologique existants. Dans le chapitre un, nous expliquons en détail pourquoi nous recommandons de se pencher sur les interactions entre les arthropodes utilisés en lutte biologique et leur microbiome (Chapitre 1)., Gliederfüsser (Arthropoden) beherbergen sehr diverse Bakteriengesellschaften in ihrem Innern. Sie können durch ihre Wirte grosse Auswirkungen auf die biologische Schädlingsbekämpfung haben. Vor allem dann nämlich, wenn sie ihren Wirt auf eine Weise beeinflussen, dass dieser entweder in seiner Funktion als Nützling beeinträchtigt wird oder aber, wenn sie einem Schädling zur erhöhten Resistenz gegen seine natürlichen Feinde verhelfen. Manchmal werden bakterielle Endosymbionten (ES) sogar gezielt zur Bekämpfung von Schädlingen eingesetzt.
Die Arthropoden-Untereinheit Acari (Milben und Zecken) ist sehr vielfältig in Form, Verhalten und Lebensraum. Es gibt Milben, die als Schädlinge auf Pflanzen leben, solche, die parasitisch auf Haustieren oder sogar Menschen leben und dann gibt es Milben-Nützlinge, die zur Eindämmung von Spinnmilben (Raubmilben) oder von Pflanzenschädlingen (Eriophyidae) eingesetzt werden. Milben-Symbionten wurden bisher wenig untersucht, trotz der Tatsache, dass alle Erkenntnisse aus diesem Forschungszweig für die biologischen Schädlingsbekämpfung von grossem Nutzen sein können.
Das erste Ziel der vorliegenden Arbeit bestand darin, alle vorhandenen Studien (und ihre Resultate) zum Thema zu sammeln und eine gute Übersicht über ihre Resultate zu erstellen. Wir haben alle Literatur zur Häufigkeit und Verbreitung mütterlich-vererbter Endosymbionten (Arsenophonus spp., Cardinium spp., Flavobacteria spp., Rickettsia spp., Spiroplasma spp. und Wolbachia spp.) in Milben, sowie alles, was über die Auswirkungen der ES auf ihre Wirte bekannt ist zusammengetragen (Kapitel 2 dieser Arbeit) und sie durch unsere eigenen Resultate (Kapitel 3) ergänzt. Zusammenfassend kann gesagt werden, dass es mehr Studien zu Milben-ES gibt als erwartet, dass die Forschung sich allerdings mehrheitlich darauf beschränkt, die An- bzw. Abwesenheit dieser ES in einem bestimmten Wirt festzustellen. Weitaus am besten untersucht sind die Spinnmilben und die ihnen zugehörigen Bakterien, während andere Milbenfamilien bisher wenig wissenschaftliche Aufmerksamkeit erhalten haben.
In einem nächsten Schritt haben wir an Beispielen aufgezeigt auf welche unterschiedlichen Weisen biologische Schädlingsbekämpfungsprogramme von ES beeinflusst werden können (Kapitel 1). Das geht von unausgewogenen Geschlechterverhältnissen in den Zuchten bis zur Inkompatibilität von eingeführten Nützlingen mit den ansässigen Populationen. Auch in der Kontrolle der Vektoren von Krankheitserregern können ES-Wirkungen auf die Wirte Folgen haben. Schlussendlich empfehlen wir die Präsenz von ES in Nützlingen und auch in den Schädlingen zu untersuchen, vor allem wenn Probleme wie Resistenzen oder Zuchtschwierigkeiten auftreten (Kapitel 1).
Unsere Theorie wird praktisch untermauert mit der Untersuchung der Wurzelmilbe (Rhizoglyphus robini). Die Wurzelmilbe ist ein Pflanzenschädling, der vor allem an den unterirdischen Knollen von Liliengewächsen frisst. Es wurde beobachtet, dass R. robini meistens auf Pflanzen gefunden wird, die zusätzlich auch von einem Pilz befallen sind. Nicht vielen Tieren jedoch sind Kohlenstoffe aus Pilzen als Nahrung zugänglich, da sie nicht die richtigen Enzyme besitzen für den Verdau von Chitin. Wir haben uns nun gefragt, ob die Bakterien im Innern der Milbe bei der Verdauung eine Rolle spielen könnten. Eine Voruntersuchung (Denaturing gradient gel electrophoresis (DGGE) mit breitamplifizierenden Bakterienprimern) hatte gezeigt, dass R. robini eine grosse Bakterienvielfalt beherbergt. In einer mehr detaillierten Untersuchung durch 454 Sequenzierung der nächsten Generation konnten wir dann mehrere Bakteriengattungen finden, in denen Arten mit chitinolytischer Aktivität vorkommen. Mithilfe von Bio-Assays konnten wir die Vorliebe für Pilz- Nahrung und hohe Fortpflanzungsrate der Milben bestätigen. Den aktiven Chitinverdau des Milbenhomogenates wiesen wir nach, indem wir jeweils 1μl davon auf eine Chitin-beschichtete Platte gaben. Schon nach 12 Stunden bildeten sich klare Zonen, sogenannte „haloes“ (Heiligenscheine) um den Tropfen (Kapitel 4). Wir konnten Bakterien aus den klaren Zonen isolieren, kultivieren und molekular (durch Sequenzierung eines grossen 16S-Fragmentes) der Bakterienart Serratia marcescens zuweisen (Kapitel 5).
Man kann die Biologie von Rhizoglyphus robini nur verstehen, wenn man die dazugehörigen Bakterien in die Betrachtung miteinbezieht. Der Pflanzenschädlingsstatus dieser Milbe muss nun überarbeitet werden, da sie wahrscheinlich vor allem den Pilz frisst oder zumindest für eine Pflanze eine viel grössere Gefahr darstellt, wenn diese schon von einem Pilz befallen ist. Die am Beispiel der Wurzelmilbe gewonnen Erkenntnisse sind wichtig für die weitere Forschung in der biologischen Schädlingsbekämpfung, denn sie eröffnen viele neue Möglichkeiten. Die Bakterien von Milben in der biologischen Schädlingsbekämpfung sollten unter, The microbiome associated with arthropods is very diverse and may significantly influence the biology of biological biocontrol agents and arthropod pests. Some endosymbiont bacteria may even be used to achieve pest or disease vector biological control.
The subclass Acari (mites and ticks) is very diverse in form, habitat and behaviour. Mites can be plant pests, parasites of domestic animals and humans or predators of major crop-pests.
The aim of this second chapter was to collect and present an overview of the present literature and knowledge on the prevalence and distribution of maternally-inherited ES (Arsenophonus spp., Cardinium spp., Flavobacteria spp., Rickettsia spp., Spiroplasma spp. and Wolbachia spp.) in mite species (chapter 2). We completed this knowledge by adding the results of our own screening (chapter 3), including mite species of three different lifestyles (herbivorous, predatory and parasitic). We found that mites and their ES have been studied, but the focus has clearly been on presence and distribution of insect ES in spider mites, whereas many other mite families as well as the further characterization of effects of ES on their hosts have received less attention. As a next step we describe the different ways microorganisms associated with arthropods can have an influence on biological pest- and disease vector control, through the many ways they can be involved in their hosts biology and we then provide practical recommendations on the handling of ES in biological control agents and pests (chapter 1).
The bulb mite Rhizoglyphus robini is below-ground pest mostly on Liliacean crop plants, which has been observed mainly on plants already infested with a fungus. As not many animals carry the enzymatic machinery to digest fungal carbohydrates, we tested the hypothesis that associated bacteria are involved in the chitin digestion. Preliminary DGGE assessment with general bacterial primers, revealed a high diversity. Investigating the bacterial community associated with this mite in more detail by 454 metagenomic analysis, we found several genera which contain chitin-degrading species. Bioassays confirmed the mite’s preference for - and high fitness on - a fungal food source. Finally we could make the link between the fungal food source of the mite and the bacterial chitinolytic ability by demonstrating the digestion of chitin by mite homogenate and isolating bacteria from the chitin-free zones (chapter 4). We molecularly identified these isolated clones as Serratia marcescens (chapter 5), a bacterium well known for its chitinolytic machinery.
The biology of Rhizoglyphus robin i can be understood only if the associated bacteria are also considered. The mites’ status has to be reassessed now, as it was previously described as a primary pest, feeding on the bulbs and tubers of plants. If the mite attacks plants preferentially after previous plant- infection with a fungus, this could be exploited in biological control. The fact that the mite might need the bacteria to digest the fungus also opens up new possibilities for control of the mite. We believe that the bacterial community in most pest and beneficial mites will deliver valuable information, which can be used in the development of new control strategies or to explain and potentially solve problems in existing control programs (chapter 1). In chapter two we explain in detail why we recommend including an investigation of the associated microbiome in standard assessments of pests and control agents.
Detecting arthropod intraguild predation in the field
The process of biological control carries a distinct risk that an alien biological control agent (BCA) will become established as an invasive alien species with an associated threat to the local ecosystem biodiversity. It is imperative that a wide-ranging environmental risk assessment (ERA) is performed before the release of any BCA. This should include considering various potential but difficult to observe ecological interactions between the BCA and members of the native community, including disruption of intraguild relationships. Detection of intraguild predation (IGP) events involving predatory arthropods in the field can be done by analyzing their gut contents. Polymerase chain reaction (PCR) is a sensitive and specific tool to identify target prey DNA within a predator’s gut. This paper reviews the efficiency of a DNA based approach for detecting IGP in the field, compared with detection by the use of monoclonal antibodies or gas chromatography. Prey specificity, detection times after prey consumption, capacity for quantification, multiple prey targeting and the time and costs involved in developing and using the different methods are considered.
The role of the bacterial community in the nutritional ecology of the bulb mite Rhizoglyphus robini (Acari: Astigmata: Acaridae)
The biology of many arthropods can only be understood when their associated microbiome is considered. The nutritional requirements of the bulb mite Rhizoglyphus robini Claparede (Acari: Astigmata: Acaridae) in the laboratory have been shown to be very easily satisfied, and in the field the mites prefer fungus-infected over uninfected plants. To test whether symbiotic bacteria facilitate the survival of R. robini on a temporarily nutritionally unbalanced diet, we investigated the composition of its microbiome. Using 454 pyrosequencing of 16S rRNA gene fragments, 3 genera were found to dominate the bacterial community: Myroides (41.4%), Serratia (11.4%), and Alcaligenes (4.5%); the latter 2 are known to include chitinase-producing species. Laboratory experiments demonstrated that mite fecundity is significantly higher (2 times) on fungus than on controls (sterilized potato dextrose agar and filter paper). Also, when mite homogenate was applied to a chitin layer, the halo produced through degradation was clearly visible, while the saline control did not produce a halo. We thus concluded that R. robini utilizes fungal chitin, at least to a certain extent, as a food source with the help of its associated bacteria. This information supports the general concept of multigenome organisms and the involvement of bacteria in the mite's nutritional ecology.-Zindel, R., Ofek, M., Minz, D., Palevsky, E., Zchori-Fein, E., Aebi, A. The role of the bacterial community in the nutritional ecology of the bulb mite Rhizoglyphus robini (Acari: Astigmata: Acaridae). FASEB J. 27, 1488-1497 (2013). www.fasebj.org
Arthropod symbioses: a neglected parameter in pest- and disease-control programmes
1. Arthropods are important players in biological control as pests, control agents and transmitters of invertebrate diseases. Arthropods are frequently infected with one or several micro-organisms, serving as micro-ecosystems in which multiple interactions can take place. These micro-organisms include disease agents and symbiotic micro-organisms. The latter are usually vertically transmitted and can have a broad spectrum of effects on their hosts, ranging from reproductive manipulations to protection against natural enemies. These interactions may directly or indirectly alter the biology of many arthropods in agriculturally, medically and ecologically relevant ecosystems. 2. Symbiotic micro-organism-induced reproductive manipulations such as cytoplasmic incompatibility and parthenogenesis induction can substantially affect the rearing of biological control agents. Many insects, and recently also mites and nematodes, have been found to be infected, displaying a wide range of effects. We discuss examples of arthropod-micro-organism interactions and effects, which could have consequences for the practical application of arthropods in biological control. 3. Symbiotic micro-organisms can also be involved in host protection against natural enemies such as parasitoids, pathogenic bacteria, fungi and viruses. 4. Symbiotic bacteria can influence the vectorial capacity of disease-vectoring arthropods and may be very helpful in decreasing the transmission of disease agents. 5. Synthesis and applications. The effect of micro-organisms on the outcome of biological control programmes is usually not considered in risk assessments and failure analyses. This review emphasizes the importance of endosymbiotic micro-organisms in comprehensive biological control programmes and provides recommendations on how to recognize, avoid or benefit from these influential tenants.
Predation of native coccinellids by the invasive alien Harmonia axyridis (Coleoptera: Coccinellidae): detection in Britain by PCR-based gut analysis
1. The harlequin ladybird, Harmonia axyridis, is an invasive alien species that arrived in Britain in 2003 and has spread across most of the country.
2. This spread has been accompanied by a decline in some native coccinellid species, in particular, the two spot ladybird Adalia bipunctata and the ten spot ladybird Adalia decempunctata. One reason for this decline could be intraguild predation (IGP) of the Adalia species by H. axyridis.
3. A DNA-based approach was used to look for evidence of coccinellid IGP in the wild. Adalia bipunctata- and A. decempunctata-specific Polymerase chain reaction primers were developed to analyse the gut contents of field collected H. axyridis larvae for the presence of Adalia DNA. Harmonia axyridis larvae (156) were collected from lime trees at four sites in eastern England over 3 years.
4.  Adalia bipunctata DNA was detected in 7.7% and A. decempunctata DNA in 4.5% of H. axyridis larvae. DNA from both Adalia species was detected in two larvae.
5. This is the first demonstration of specific IGP interactions between H. axyridis and other coccinellid species in wild populations in Britain.