Glabralysins, potential New β-pore-forming toxin family members from the schistosomiasis vector snail biomphalaria glabrata

Biomphalaria glabrata is a freshwater Planorbidae snail. In its environment, this mollusk faces numerous microorganisms or pathogens, and has developed sophisticated innate immune mechanisms to survive. The mechanisms of recognition are quite well understood in Biomphalaria glabrata, but immune effectors have been seldom described. In this study, we analyzed a new family of potential immune effectors and characterized five new genes that were named Glabralysins. The five Glabralysin genes showed different genomic structures and the high degree of amino acid identity between the Glabralysins, and the presence of the conserved ETX/MTX2 domain, support the hypothesis that they are pore-forming toxins. In addition, tertiary structure prediction confirms that they are structurally related to a subset of Cry toxins from Bacillus thuringiensis, including Cry23, Cry45, and Cry51. Finally, we investigated their gene expression profiles in snail tissues and demonstrated a mosaic transcription. We highlight the specificity in Glabralysin expression following immune stimulation with bacteria, yeast or trematode parasites. Interestingly, one Glabralysin was found to be expressed in immune-specialized hemocytes, and two others were induced following parasite exposure

Candidate genes revealed by a genome scan for mosquito resistance to a bacterial insecticide: sequence and gene expression variations

<p>Abstract</p> <p>Background</p> <p>Genome scans are becoming an increasingly popular approach to study the genetic basis of adaptation and speciation, but on their own, they are often helpless at identifying the specific gene(s) or mutation(s) targeted by selection. This shortcoming is hopefully bound to disappear in the near future, thanks to the wealth of new genomic resources that are currently being developed for many species. In this article, we provide a foretaste of this exciting new era by conducting a genome scan in the mosquito <it>Aedes aegypti </it>with the aim to look for candidate genes involved in resistance to <it>Bacillus thuringiensis </it>subsp. <it>israelensis </it>(<it>Bti</it>) insecticidal toxins.</p> <p>Results</p> <p>The genome of a <it>Bti</it>-resistant and a <it>Bti</it>-susceptible strains was surveyed using about 500 MITE-based molecular markers, and the loci showing the highest inter-strain genetic differentiation were sequenced and mapped on the <it>Aedes aegypti </it>genome sequence. Several good candidate genes for <it>Bti</it>-resistance were identified in the vicinity of these highly differentiated markers. Two of them, coding for a cadherin and a leucine aminopeptidase, were further examined at the sequence and gene expression levels. In the resistant strain, the cadherin gene displayed patterns of nucleotide polymorphisms consistent with the action of positive selection (e.g. an excess of high compared to intermediate frequency mutations), as well as a significant under-expression compared to the susceptible strain.</p> <p>Conclusion</p> <p>Both sequence and gene expression analyses agree to suggest a role for positive selection in the evolution of this cadherin gene in the resistant strain. However, it is unlikely that resistance to <it>Bti </it>is conferred by this gene alone, and further investigation will be needed to characterize other genes significantly associated with <it>Bti </it>resistance in <it>Ae. aegypti</it>. Beyond these results, this article illustrates how genome scans can build on the body of new genomic information (here, full genome sequence and MITE characterization) to finally hold their promises and help pinpoint candidate genes for adaptation and speciation.</p

A MITE-based genotyping method to reveal hundreds of DNA polymorphisms in an animal genome after a few generations of artificial selection

<p>Abstract</p> <p>Background</p> <p>For most organisms, developing hundreds of genetic markers spanning the whole genome still requires excessive if not unrealistic efforts. In this context, there is an obvious need for methodologies allowing the low-cost, fast and high-throughput genotyping of virtually any species, such as the Diversity Arrays Technology (DArT). One of the crucial steps of the DArT technique is the genome complexity reduction, which allows obtaining a genomic representation characteristic of the studied DNA sample and necessary for subsequent genotyping. In this article, using the mosquito <it>Aedes aegypti </it>as a study model, we describe a new genome complexity reduction method taking advantage of the abundance of miniature inverted repeat transposable elements (MITEs) in the genome of this species.</p> <p>Results</p> <p><it>Ae. aegypti </it>genomic representations were produced following a two-step procedure: (1) restriction digestion of the genomic DNA and simultaneous ligation of a specific adaptor to compatible ends, and (2) amplification of restriction fragments containing a particular MITE element called <it>Pony </it>using two primers, one annealing to the adaptor sequence and one annealing to a conserved sequence motif of the <it>Pony </it>element. Using this protocol, we constructed a library comprising more than 6,000 DArT clones, of which at least 5.70% were highly reliable polymorphic markers for two closely related mosquito strains separated by only a few generations of artificial selection. Within this dataset, linkage disequilibrium was low, and marker redundancy was evaluated at 2.86% only. Most of the detected genetic variability was observed between the two studied mosquito strains, but individuals of the same strain could still be clearly distinguished.</p> <p>Conclusion</p> <p>The new complexity reduction method was particularly efficient to reveal genetic polymorphisms in <it>Ae. egypti</it>. Overall, our results testify of the flexibility of the DArT genotyping technique and open new prospects as regards its application to a wider range of species, including animals which have been refractory to it so far. DArT has also a role to play in the current burst of whole-genome scans carried out in various organisms, which track signatures of selection in order to unravel the basis of genetic adaptation.</p

Interaction between Insects, Toxins, and Bacteria: Have We Been Wrong So Far?

Toxins are a major virulence factor produced by many pathogenic bacteria. In vertebrates, the response of hosts to the bacteria is inseparable from the response to the toxins, allowing a comprehensive understanding of this tripartite host-pathogen-toxin interaction. However, in invertebrates, this interaction has been investigated by two complementary but historically distinct fields of research: toxinology and immunology. In this article, I highlight how such dichotomy between these two fields led to a biased, or even erroneous view of the ecology and evolution of the interaction between insects, toxins, and bacteria. I focus on the reason behind such a dichotomy, on how to bridge the fields together, and on confounding effects that could bias the outcome of the experiments. Finally, I raise four questions at the border of the two fields on the cross-effects between toxins, bacteria, and spores that have been largely underexplored to promote a more comprehensive view of this interaction

Devenir du bioinsecticide Bti dans l'environnement et impact sur le développement de résistances chez le moustique

Bti is a bioinsecticide used worldwide for mosquito control. Bti toxicity is due to a toxins-containing crystal, produced by the bacteria Bacillus thuringiensis var. israelensis. Bti is known to have a low persistence in the environment and no firm resistance has been found yet in the field, therefore representing a “safe” alternative to chemical insecticides. Nevertheless, leaf litters collected several months after a treatment exhibited high toxicity against mosquito larvae, due to persistent Bti. The selection of a laboratory mosquito strain of Aedes aegypti with this litter (LiTOX strain) led to a moderate 3.5-fold resistance to Bti but to higher resistance (until 60-fold) to Bti toxins tested separately. This thesis is structured in three main axes. 1. A test has been developed and patented to follow the fate of Bti in the environment. We showed that Bti has recycled in the collected toxic leaf litters and that the different Bti toxins persist differently. 2. The Bti-resistance of the LiTOX strain has been studied by global and functional approaches, leading to a better understanding of the multigenic mechanisms implicated in the resistance phenotype. 3. Cryptic Bti-resistance has been investigated performing bioassays with separate Bti Cry toxins, showing that some populations exhibit increased Cry tolerance. This thesis gives the knowledge and new tools necessary to better understand how Bti can persist and recycle in the environment and how mosquitoes can develop resistance.Le Bti est un bioinsecticide mondialement utilisé pour contrôler les populations de moustiques. La toxicité du Bti est due à un cristal constitué de plusieurs toxines, produit par la bactérie éponyme Bacillus thuringiensis var. israelensis. Qualifié de peu persistant dans l'environnement et sans résistance connue en populations naturelles, le Bti représente une alternative « propre » aux insecticides chimiques. Cependant, une litière végétale prélevée plusieurs mois après un traitement insecticide a montré une forte toxicité contre les larves de moustiques, due à la présence de Bti. La sélection d'une souche de moustique de laboratoire Aedes aegypti avec cette litière (souche LiTOX) a permis d'obtenir une résistance modérée au Bti (3,5 fois) mais plus élevée aux toxines testées séparément (jusqu'à 60 fois). La présente thèse se décompose en trois axes. 1. Un test permettant le suivi du Bti dans l'environnement a été développé et breveté. Il a permis de mettre en évidence un recyclage du Bti dans les litières toxiques et une persistance différentielle des toxines. 2. La résistance au Bti de la souche LiTOX a été étudiée par des approches globales et fonctionnelles, permettant de mieux caractériser les mécanismes de résistance multigéniques impliqués. 3. La résistance a été recherchée en populations naturelles par des bioessais aux toxines du Bti testées séparément, outil plus sensible que les bioessais « classiques » au Bti, et a permis de mettre en évidence une tolérance accrue de certaines populations. Cette thèse apporte les connaissances et les outils nécessaires pour mieux caractériser les paramètres favorisant la prolifération du Bti en milieu naturel et les mécanismes de résistance potentiellement sélectionnés chez les moustiques

Fate of the bioinsecticide Bti in the environment and impact on the development of resistance in mosquito species

Le Bti est un bioinsecticide mondialement utilisé pour contrôler les populations de moustiques. La toxicité du Bti est due à un cristal constitué de plusieurs toxines, produit par la bactérie éponyme Bacillus thuringiensis var. israelensis. Qualifié de peu persistant dans l'environnement et sans résistance connue en populations naturelles, le Bti représente une alternative « propre » aux insecticides chimiques. Cependant, une litière végétale prélevée plusieurs mois après un traitement insecticide a montré une forte toxicité contre les larves de moustiques, due à la présence de Bti. La sélection d'une souche de moustique de laboratoire Aedes aegypti avec cette litière (souche LiTOX) a permis d'obtenir une résistance modérée au Bti (3,5 fois) mais plus élevée aux toxines testées séparément (jusqu'à 60 fois). La présente thèse se décompose en trois axes. 1. Un test permettant le suivi du Bti dans l'environnement a été développé et breveté. Il a permis de mettre en évidence un recyclage du Bti dans les litières toxiques et une persistance différentielle des toxines. 2. La résistance au Bti de la souche LiTOX a été étudiée par des approches globales et fonctionnelles, permettant de mieux caractériser les mécanismes de résistance multigéniques impliqués. 3. La résistance a été recherchée en populations naturelles par des bioessais aux toxines du Bti testées séparément, outil plus sensible que les bioessais « classiques » au Bti, et a permis de mettre en évidence une tolérance accrue de certaines populations. Cette thèse apporte les connaissances et les outils nécessaires pour mieux caractériser les paramètres favorisant la prolifération du Bti en milieu naturel et les mécanismes de résistance potentiellement sélectionnés chez les moustiques.Bti is a bioinsecticide used worldwide for mosquito control. Bti toxicity is due to a toxins-containing crystal, produced by the bacteria Bacillus thuringiensis var. israelensis. Bti is known to have a low persistence in the environment and no firm resistance has been found yet in the field, therefore representing a “safe” alternative to chemical insecticides. Nevertheless, leaf litters collected several months after a treatment exhibited high toxicity against mosquito larvae, due to persistent Bti. The selection of a laboratory mosquito strain of Aedes aegypti with this litter (LiTOX strain) led to a moderate 3.5-fold resistance to Bti but to higher resistance (until 60-fold) to Bti toxins tested separately. This thesis is structured in three main axes. 1. A test has been developed and patented to follow the fate of Bti in the environment. We showed that Bti has recycled in the collected toxic leaf litters and that the different Bti toxins persist differently. 2. The Bti-resistance of the LiTOX strain has been studied by global and functional approaches, leading to a better understanding of the multigenic mechanisms implicated in the resistance phenotype. 3. Cryptic Bti-resistance has been investigated performing bioassays with separate Bti Cry toxins, showing that some populations exhibit increased Cry tolerance. This thesis gives the knowledge and new tools necessary to better understand how Bti can persist and recycle in the environment and how mosquitoes can develop resistance

Devenir du bioinsecticide Bti dans l\u27environnement et impact sur le développement de résistances chez le moustique

Lors de cette étude est présentée une thèse sur le devenir du bioinsecticide Bti dans l\u27environnement et son impact sur le développement des résistances chez le moustique. On partira d\u27une interrogation principale : le Bti est-il vraiment cet insecticide parfait, respectueux de l\u27environnement