The efficacy of three entomopathogenic nematodes (EPN) of family Steinernematidae was tested in different laboratory conditions in this study. The virulence of Steinernema arenarium, S. carpocapsae and S. feltiae was evaluated to fourth-instar larvae of Galleria mellonella and Tenebrio molitor. Standard temperature (25°C) and humidity (12,5 %) was used in bioassay No I. Trial No II focused on impact of temperature 10, 15 and 25°C on effectivity of S. feltiae and S. carpocapsae. Bioassay No III examined the influence of lower humidity and in consequence rehydration on virulence nematodes.

Entomopathogenic Steinernema nematodes (Nematoda: Steinernematidae) have a mutualistic relationship with Xenorhabdus bacteria (Gamma-Proteobacteria Enterobacteriaceae). The two partners form an insecticidal alliance that is successful in killing a wide range of insects. A few studies have shown that Steinernema IJs have an enhanced virulence and reproductive fitness when they associate with their cognate symbionts. However, there are unanswered questions regarding the physiological interactions that govern and perpetuate the interactions between different nematode developmental stages and their bacterial partners. In this study, we evaluated gonad development and maturation time of first-generation adults of S. carpocapsae and S. feltiae adults when reared under four bacterial scenarios: a) cognate symbiotic, b) non-cognate symbiotic bacterial strain, c) non-cognate symbiotic bacterial species and d) non-symbiotic bacteria (Serratia proteamaculans). For comparative purposes, we also considered adult nematodes reared in vivo in Galleria mellonella larvae to assess nematode development under natural conditions. Furthermore, in this study we also measured production of nematode pheromones (ascarosides), which play a key role in mating and reproduction. For this purpose, we considered in vitro rearing methods (with cognate and non-cognate Xenorhabdus symbionts) to qualitatively and quantitatively characterize ascarosides produced by first-generation adults. Our data showed that for both Steinernema spp. tested, time to adult maturation and gonad development was tightly dependent on the bacterial conditions under which juveniles were reared. However, contrasting results were observed when assessing total body length and gonad size. S. feltiae males and females size (body length and width) and respective gonad length were smaller when reared with a non-cognate symbiotic species. Additionally, non-symbiotic bacteria did not sustain S. feltiae maturation to adult stages. Contrarily, S. carpocapsae juveniles developed to adults when reared with any of the bacterial conditions tested, including with non-symbiotic Serratia proteamaculans. Additionally, S. carpocapsae adults, unlike S. feltiae, did not exhibit enhanced body and gonad size when reared with their cognate symbiont. In fact, S. carpocapsae males and females had larger gonad lengths when reared with a non-cognate symbiotic strain, XnAna (X. nematophila associated with S. anatoliense). S. carpocapsae males and females had significantly underdeveloped gonads when reared with non-symbiotic bacteria. In both Steinernema spp., sex ratio was not impacted by the bacterial condition. However, sex ratio (female:male) S. carpocapsae, decreased from 2:1 to 1:1 when reared with non-symbiotic bacteria. The body and gonad sizes of Steinernema spp. reared in vitro with their cognate symbiont were significantly smaller than those grown in vivo. Ascaroside production in either Steinernema spp. was not significantly impacted by the rearing conditions. In S. carpocapsae, a significant increase in glucoside-1 was observed when the nematodes were reared with cognate or non-cognate bacteria. No detectable quantities of asc-C11 were produced by S. feltiae nematodes when reared with a non-cognate symbiotic bacterial species. We conclude that bacterial symbionts influenced maturation and development of first-generation adults’ in both Steinernema spp. tested in this study. However, response to the bacterial symbionts was species specific. Additionally, this study showed that Xenorhabdus as a food source plays an important role in the type and amount of ascarosides produced by Steinernema spp.

Nematodes in the genus Steinernema (Nematoda: Steinernematidae) and their associated bacteria Xenorhabdus spp. (Gamma-Proteobacteria) are an emergent model of terrestrial animal-microbe symbiosis. Although interest in this association initially arose out of their potential as biocontrol agents against insect pests (Tanada and Kaya, 1993), this mutualistic partnership is currently viewed more broadly under the umbrella of basic sciences to inform ecology, evolution, biochemistry, molecular, among other disciplines (Burnell and Stock, 2000; Forst and Clarke, 2002).Despite advances in the discovery and field application of this nematode-bacterium partnership, and the growing popularity of this model system, relatively little has been published to uncover the evolutionary facets of their association. This study adds to the body of knowledge regarding nematode-bacteria symbiosis by 1) producing novel, multi-gene phylogenies for Steinernema and Xenorhabdus; 2) proposing a possible scenario for historical association in the form of a cophylogenetic hypothesis; 3) describing a newly discovered Steinernema species from France.

The biological control of subterranean termites (Isoptera: Rhinotermitidae; Termitidae) using entomopathogenic nematodes (Nematoda: Steinernematidae; Heterorhabditidae) (EPN) was investigated. The desert subterranean termite Heterotermes aureus Snyder was found to be very susceptible to Steinernema riobrave Cabanillas, Poinar and Raulston. In laboratory bioassays S. riobrave (355, TP, 3-8b and 7-12 strains), S. carpocapsae Weiser (Mexican 33 strain), S. feltiae Filipjev (UK76 strain), and Heterorhabditis bacteriophora Poinar (HP88 strain) were all capable of infecting and killing H. aureus, Reticulitermes flavipes Kollar, R. virginicus Banks, Coptotermes formosanus Shiraki and Gnathamitermes perplexus Banks. In sand assays, S. riobrave caused > 90% H. aureus mortality in 3 days and 100% mortality by day 5 at 22 °C. TP strain of S. riobrave caused 75% R. flavipes mortality and 90.91% C. formosanus mortality in 7 days. EPNs utilizing termites as hosts produced smaller sized offspring, with the exception of S. feltiae. Stunted females of S. feltiae were frequently found in termite cadavers, but no progeny. Small IJs of S. carpocapsae, S. riobrave and H. bacteriophora infect, reproduce and form normal size IJs after subsequent infection in Galleria mellonella L. The progeny of small IJs were as effective as the normal size IJs, with regard to subsequent induced mortality, under the conditions tested. In laboratory two-container choice experiments, H. aureus were repelled by EPN treated areas for up to 10 days at 10,000 IJs per device. The repellency threshold was found to vary among nematodes species. We hypothesis that it is the physical movement of the nematodes that repels the termites. Temperature is a key factor affecting nematode pathogenicity. Temperature tolerance of the nematodes varied between species. After a gradual heat adaptation process, S. riobrave and H. bacteriophora caused significantly higher H. aureus mortality at 32 °C compared with original laboratory cultured strains. Further work may result in the contribution of commercially available strains with enhanced heat tolerance. Preliminary field studies confirmed EPN protection of a structure, however, termites began to reinfest 4 weeks after the application. Additional tests are necessary to provide more evidence before we can conclude nematodes as useful in the field.

Made available in DSpace on 2017-07-10T17:37:38Z (GMT). No. of bitstreams: 1 Lilian_Bortoluzzi.pdf: 601021 bytes, checksum: 32fb8cf95dc69539e950c2ecdd8f63ad (MD5) Previous issue date: 2009-06-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The black weevil (Cosmopolites sordidus) is the main pest of banana crops in several regions of the world, causing significant losses in productivity of the culture. The larvae build galleries in the rhizome or pseudostem base, affecting the development of the plant and the fruits, beside favoring the installation of pathogenic microrganisms. The use of nematodes to control of the pest can be an alternative, mainly because the insect's habits make them a potential target of entomopathogenic nematodes. So, this study had the objectives to evaluate isolates of entomopathogenic nematodes in laboratory conditions to their use in controlling the borer. Sixteen isolates Sterinernematidae and Heterorhabditidae were tested, applied on pseudostem (100JIs/cm2). The evaluation was performed 7 days after application. After, the most virulent isolates were compared with each other for production of nematodes in cadavers of wax moth (Galleria mellonella) and also about his compatibility with the insecticide carbofuran. All isolates were pathogenic to the adults of C. sordidus, except the isolate SC (Steinernema carpocapsae). The most virulent isolates were CB24 and CB40 (both of the Heterorhabditidae), which caused respectively 33,3% and 36,7% of mortality. The two isolates showed high production of JIs. The insecticide was compatible with isolated CB40 and incompatible with CB24, nevertheless, CB24 showed better performance in combination with carbofuran / A broca-da-bananeira (Cosmopolites sordidus) é a principal praga dos cultivos de banana em várias regiões do mundo, acarretando perdas significativas na produtividade da cultura. As larvas constroem galerias no rizoma ou base do pseudocaule, afetando o desenvolvimento da planta e dos frutos, além de favorecer a instalação de microrganismos patogênicos. O uso de nematóides para o controle da praga pode ser uma alternativa, principalmente devido aos hábitos do inseto que os tornam um potencial alvo dos nematóides entomopatogênicos. Assim, este trabalho teve como objetivos avaliar isolados de nematóides entomopatogênicos em condições de laboratório visando sua utilização no controle da broca. Foram testados 16 isolados pertencentes às famílias Sterinernematidae e Heterorhabditidae, aplicados sobre pseudocaule (100JIs/cm2). A avaliação foi realizada 7 dias após a aplicação. Os isolados mais eficientes foram comparados entre si quanto à produção de nematóides em cadáveres da traça dos favos (Galleria mellonella) e também quanto a sua compatibilidade com o inseticida carbofurano. Todos os experimentos foram realizados em delineamento inteiramente casualizado. Verificou-se que exceto o isolado SC (Steinernema carpocapsae), todos os demais foram patogênicos aos adultos de C. sordidus. Os isolados mais virulentos foram o CB24 e CB40, os quais provocaram respectivamente 33,3% e 36,7% de mortalidade, ambos da família Heterorhabditidae. Os dois isolados apresentaram alta produção de JIs, não diferindo estatisticamente entre si. O inseticida foi compatível com o isolado CB40 e incompatível com CB24, mesmo assim, CB24 apresentou melhor desempenho em associação com carbofurano

Les entérobactéries du genre Xenorhabdus sont pathogènes de larves d'insectes et symbiotiques de nématodes du genre Steinernema. En lutte biologique, les couples Steinernema-Xenorhabdus sont utilisés contre un large spectre d'insectes ravageurs de culture. Les deux partenaires du couple modèle Steinernema carpocapsae-Xenorhabdus nematophila peuvent être expérimentalement dissociés tout en restant pathogènes pour les insectes. En revanche, certaines souches de Xenorhabdus sont non-virulentes lorsqu'elles sont injectées directement dans une larve d'insecte. L'objectif de cette thèse est de caractériser deux souches non-virulentes de Xenorhabdus, X. poinarii G6 (Xp G6) et X. bovienii CS03 (Xb CS03). Les souches appartenant à l'espèce non-virulente X. poinarii possèdent des génomes de petite taille. Nous avons mis en évidence un phénomène de réduction génomique due à la délétion de larges régions génomiques chez la souche Xp G6. Cette évolution pourrait avoir eu lieu suite à un transfert des fonctions bactériennes de virulence à son nématode hôte et/ou à sa spécialisation envers certains coléoptères. Au sein de l'espèce X. bovienii, Xb CS03 est non-virulente par injection dans les lépidoptères Spodoptera littoralis et Galleria mellonella. Par rapport à d'autres couples némato-bactériens Steinernema sp.-X. bovienii, le couple formé par Xb CS03 et son nématode symbiotique S. weiseri 583 présente également une virulence atténuée sur ces lépidoptères. Le génome de Xb CS03 est de très grande taille et contient un grand nombre de gènes dégradés (pseudogènes). Une comparaison génomique entre Xb CS03 et une souche virulente appartenant à la même espèce, X. bovienii SS-2004 (Xb SS-2004), montre que Xb CS03 est plus riche que Xb SS-2004 en gènes codant des chaînes d'assemblage enzymatiques NRPS/PKS (non-ribosomal peptide synthase/polyketide synthethase) produisant des métabolites antimicrobiens potentiels. A l'inverse, Xb SS-2004 contient davantage de gènes codant des facteurs de virulence de type hémolysine, adhésine ou systèmes de sécrétion. Ceci suggère deux scénarios évolutifs différents, privilégiant une forte virulence pour Xb SS-2004 et l'élimination des compétiteurs au sein du cadavre de l'insecte pour Xb CS03. Enfin, une recherche de facteurs de virulence potentiels a été effectuée par une approche de génomique comparative entre les souches non-virulentes Xp G6 et Xb CS03, d'une part et trois souches de Xenorhabdus virulentes, d'autre part. L'analyse fonctionnelle de gènes candidats a été entamée. En conclusion, la caractérisation de nouveaux modèles bactériens dans le genre Xenorhabdus ouvre le champ à l'identification de nouvelles stratégies de virulence et de nouveaux facteurs de virulence chez les bactéries entomopathogènes. / Xenorhabdus are enterobacteria pathogenic of insect larvae and symbiotic of nematodes from the Steinernema genus. The Steinernema-Xenorhabdus associations are used against a wide range of insect pests. The two partners of the model Steinernema carpocapsae-Xenorhabdus nematophila association can be experimentally dissociated. Each partner is pathogenic for insect larvae. Contrarily, some other Xenorhabdus strains are non-virulent when injected directly into insect larvae. In this thesis, we characterized two non-virulent Xenorhabdus strains, X. poinarii G6 (Xp G6) and X. bovienii CS03 (Xb CS03). Strains from the X. poinarii species had small-sized genomes. We showed that the Xp G6 strain had undergone a genome reduction due to the deletion of large genomic regions. Transfer of virulence functions from the bacteria to the nematode and/or the specialization of the association towards coleopteran insects are likely the cause of this evolution. Within the X. bovienii species, Xb CS03 was non-virulent strain when injected into the Spodoptera littoralis and Galleria mellonella lepidopteran insects. When compared to other Steinernema-X. bovienii pairs, the association between Xb CS03 and its symbiotic nematode S. weiseri 583 had also a lower virulence on those insects. Xb CS03 had a large-sized genome and harbored numerous degraded genes (pseudogenes). Genome comparison between Xb CS03 and a virulent strain from the same species, X. bovienii SS-2004 (Xb SS-2004), showed that Xb CS03 contained more loci encoding NRPS/PKS enzymes (non-ribosomal peptide synthase/polyketide synthethase), producing potential antimicrobial metabolites, than Xb SS-2004. On the other hand, Xb SS-2004 contained more genes encoding virulence factors such as hemolysins, adhesins or secretion systems. This suggests that the two strains followed different evolutionary scenarios, favoring strong virulence in Xb SS-2204 and elimination of competitors for Xb CS03.Finally, we searched for potential virulence factors by comparing the genomes of the non-virulent strains Xp G6 and Xb CS03 with three virulent strains. Functional analyses of the candidates are in progress. In conclusion, characterizing new bacterial models in the Xenorhabdus genus paves the way for the identification of new virulence strategies and new virulence genes in entomopathogenic bacteria.

Surveys for entomopathogenic bacteria and their associated nematode hosts were conducted locally (University of Bath campus) and across southern England. Sampling involved trialing a novel Android app. (Epicollect) to manage sample collection data. Galleria larvae were used to bait UK soil samples. Insects which became infected were placed on White traps to collect any emerging nematodes, from which bacteria were isolated. Bacteria were also isolated from the haemolymph of any infected larvae. Bacterial isolates were classified on the basis of 16s rDNA and recA gene sequences. Serratia proteamaculans-like strains dominated the samples, and Multilocus sequence analysis (MLSA) was developed for the characterization of these Serratia isolates. We determined the sequences of (350-450-bp) fragments from five housekeeping genes of 84 isolates of Serratia proteamaculans. MLSA was shown to be effective for distinguishing closely related strains found in the insects’ haemolymph and from different nematodes. goeBURST was used to visualize the relationships between the STs, and the data showed a high level of discrimination, resolving 69 STs from the 84 isolates. In addition, the data derived from this study were represented in a phylogenetic network using the Splits Tree-network methods, to show the rate of recombination within and between the genes. From a total of 256 infected Galleria 23.04% were nematode positive. The nematodes were identified based on 18S rDNA 19 isolates were close relatives of the species Pristionchus entomophaga and Diplogasteriodes magnus (Diplogastridae). A further 16 isolates were more closely related to Steinernema glaseri (Steinernematidae). All three nematode types were isolated from diverse habitats and soil types, but were isolated more frequently in cold seasonal conditions. The bacterial sequence data suggest that the nematode- associated strains of bacteria belong to specific clades, distinct from the free living infective strains, which hints at ecological diversity within the S. proteamaculans population. Two of the Serratia proteamaculans-like strains had been chromosomally labeled with GFP to confirm the specifics of their association with the nematode hosts. The associated S. proteamaculans-like isolates isolated from Bath and Chepstow soils were examined further for their pathogenicity to Galleria mellonella and Manduca sexta larvae. Serratia Bath isolates, isolated from Pristionchus were more virulent toward both insect hosts than the Serratia from the Chepstow isolates associated with Steinernema nematodes. This suggests that host specificity may play important role in the virulence of the strain.

University, Diss., 2005--Kiel.

It is widely assumed that current phylogenetic methods are fairly accurate at recovering the evolutionary relationships among different species, but evaluating the relative success of this enterprise is a difficult task. This study addresses some fundamental questions associated with generating phylogenetic trees. The complete genomes of five species of Steinernema were sequenced and assembled. Genes were predicted in AUGUSTUS and orthologous genes were found from those data using OrthoMCL. I aligned 3890 genes in MAFFT and eliminated poorly aligned positions with GBlocks. I created individual trees for each gene as well as a supermatrix tree in PAUP*, using a closely related taxon from another genus, Panagrellus redivivus. In the resulting gene trees, I found only a small subset of all the possible topologies. I discovered that the supermatrix tree has the same topology as the topology with the most gene trees in the gene-topology distribution. There are only a small number of histories for all of the genes and many of the genes have the same lineage. I bootstrapped the gene-topology distribution and found that the best-supported topology was sampled 22.1% of the time. I show that many genes must be sampled in order to converge on the topology with the most support from the gene trees in this dataset.

Les Nématodes Entomopathogènes (NEP) de la famille des Steinernematidae et Heterorhabditidae sont des parasites pour les insectes. Ils hébergent dans leur intestin un symbiote bactérien (genres Xenorhabdus et Photorhabdus) essentiel au succès parasitaire. Les NEP sont présents dans les sols des cinq continents de la planète. Leur diversité génétique et leurs propriétés biologiques constituent une intéressante ressource biologique. Grâce à leur entomotoxicité, les NEP sont de bons outils de lutte biologique en agriculture et en culture ornementale un peu partout dans le monde. La diversité et la biogéographie des NEP dans les cinq continents de la Terre (à l'exception de l'Antarctique) ont été étudiées partout dans le monde mais le Liban est parmi les rares pays du moyen orient où aucune prospection de ces nématodes n'a été réalisée alors que des NEP en Turquie, Syrie, Jordanie, Palestine et Egypte ont déjà été trouvés et caractérisés. L'objet de la thèse est de procéder à une étude biogéographique dans le but de connaître la diversité des NEP au Liban. L'enjeu scientifique est donc de combler un « vide » dans la connaissance de la répartition et de la biodiversité mondiale des NEP. Pour cela, un échantillonnage à l'échelle des étages de végétation est mené au Liban. Des échantillons de sol sont ainsi prélevés, mis en contact avec des larves de Galleria mellonella pour isoler les nématodes entomopathogènes et leurs symbiotes. Les nématodes et leurs symbiotes sont par la suite identifiés morphologiquement et moléculairement. Par la suite, une approche à l'échelle de l'habitat fait l'objet de cette thèse également pour étudier les interactions biotiques et abiotiques influençant la présence des nématodes entomopathogènes dans le sol. Les enjeux technologiques, exposés au second volet de la thèse, sont liés aux propriétés biologiques des nématodes et de leurs symbiotes afin de valoriser leur entomotoxicité en lutte biologique. Dans ce cadre, la sensibilité des Cephalcia tannourinensis, ravageur des cédraies au Liban, par rapport aux nématodes entomopathogènes est exploitée in vitro ; différentes espèces de nématodes entomopathogènes sont testées pour suivre leur cycle à l'intérieur des Cephalcia. / Entomopathogenic nematodes (EPNs) are parasites of soil-dwelling insects that occur in natural and agricultural soils around the world. Thanks to their entomotoxicity, EPNs are good tools for biological control in agriculture almost everywhere in the world. They are ubiquitous, having been isolated from every inhabited continent (except Antartica) from a wide range of ecologically diverse soil habitats including cultivated fields, forests, grasslands, deserts, and even ocean beaches. Biogeographic assessments of EPNs in the Eastern Mediterranean basin have been conducted in several countries such as Turkey, Syria, Jordan, Israel, Palestine and Egypt. Lebanon is among the few countries of the Middle East for which no survey of EPNs has been done. The scientific stake is thus to fill a gap in our knowledge of EPNs distribution in the Mediterranean basin. Survey of EPNs was conducted in this framework to cover the different vegetation levels defined in Lebanon. Soil samples were removed placed in contact with Galleria mellonella to isolate entomopathogenic nematode and their symbiotic bacteria. EPNs and their bacteria were then identified morphologically and molecularly. On the other hand, despite the different national surveys conducted on EPNs distribution around the world, habitat preferences remain inadequately known for entomopathogenic nematodes. Therefore, a comprehensive understanding of their distribution and the various biotic and abiotic factors influencing their presence is also a second object of our work. Beside a technological approach related to the biological properties of the nematodes and their symbiotics: valorisation of the entomotoxicity in biological control will be part of the third shutter of the thesis. In this framework, the sensibility of cedar pests, Cephalcia tannourinensis against entomopathogenic nematodes is exploited in vitro; different EPNs species were tested to study their life cycle inside Cephalcia larvae.