Genetic diversity and identification of symbiotic community of Q Bemisia tabaci from Mediterranean countries

Introduction: We investigated a) the genetic diversity and population structure, b) the secondary symbiotic bacterial community and c) the frequencies of target site mutations conferring resistance to pyrethroids and organophospates in Q Bemisia tabaci collected from 5 Mediterranean countries. Material and Methods: Polymorphism at seven microsatellite loci and a 676-bp fragment of the cytochrome oxidase I (COI) gene were investigated for the genetic analysis of B. tabaci populations. Simple PCR based detection assays were used for the discrimination of B. tabaci subgroups and the detection of mutations associated with target site insecticide resistance (iAChE, kdr). Secondary symbionts were detected using specific primers. Wolbachia strains were characterized by Multilocus Sequence Typing (MLST) analysis. Results: Analyses of the 676-bp COI fragment distinguished seventeen haplotypes which delineated two groups (Q1 and Q2), within the Q B. tabaci. Both Q1 and Q2 were found in Spain and France, whereas only Q1 was observed in Greece, Morocco and Tunisia. The analyses of the microsatellite loci polymorphism revealed a high level of genetic differentiation even between some neighbouring samples belonging to the same sub-group within a country. In B. tabaci from Greece, Bayesian analysis revealed two main genetic groups within Q1, the first with populations from South Crete, and the second with populations from continental Greece and North Crete. Genetic differentiation was not correlated with host plant species or habitat. The secondary symbionts Wolbachia and Hamiltonella were present at high frequency while Arsenophonus, Cardinium and Rickettsia were absent. MLST analysis identified two Wolbachia strains which were found together in most of the Greek populations but never in the same host individual. The frequency of pyrethroid resistance mutations L925I and T929V in the para sodium channel gene and the organophosphate resistant mutation F331W in the ace1 gene was determined. The higher frequencies of the resistance mutations were found in France, Spain, and Greece, where in some samples the resistant alleles were fixed. In Morocco and Tunisia, the resistant alleles were less frequent and in some localities even undetected. Conclusion: Our findings suggest that the Mediterranean Q B. tabaci is more diverse and structured than reported so far and suggest that human activities play a major role in the genetic structure and as well as in the dynamics of resistance genes. The role of the symbionts, and in particular of Wolbachia W1 and W2 strains in the shaping of the population genetic structure, is currently under investigation. (Résumé d'auteur

Genetic variability in Tetranychus urticae (Acari : Tetranychidae) from Greece : insecticide resistance and isozymes

Etude de la résistance au méthyl-parathion, au méthidathion et au méthomyl chez une population de #Tetranychus urticae$ Koch originaire de Grèce. A la CL 50 le taux de résistance est hautement variable avec le méthidathion (5 à 63 fois) et avec le méthomyl (6 à 34 fois). Les courbes de mortalité avec le méthyl-parathion présentent un plateau net à une mortalité d'environ 20% et le taux de résistance est d'environ 50 fois à CL 50, dans tous les échantillons. Par utilisation de la focalisation isoélectrique sur des membranes d'acétate de cellulose, les études électrophorétiques de 5 loci codant les estérases (Est-1 et Est-2), l'isomérase gluco-phosphate (Gpi), l'enzyme malique (Me) et la phosphoglucomutase (Pgm), indiquent les différences génétiques importantes parmi les échantillons. (Résumé d'auteur

Reproductive incompatibility between genetically differentiated populations of Tetranychus urticae from different host plants

Η μελέτη της προσαρμογής των εχθρών των καλλιεργούμενων φυτών στα φυτά ξενιστές τους παρουσιάζει μεγάλο ενδιαφέρον, καθώς σχετίζεται με την απομόνωση και γενετική διαφοροποίηση των πληθυσμών, με αποτελέσματα που ποικίλλουν από τον περιορισμό της γονιδιακής ροής μέχρι και το σχηματισμό νέων ειδών. Με έναυσμα προηγούμενες μελέτες μας με ισοένζυμα, που κατέδειξαν γενετική διαφοροποίηση μεταξύ πληθυσμών Tetranychus urticae (Koch) από εσπεριδοειδή σε σχέση με άλλους ξενιστές, μελετήσαμε την συμβατότητα των διασταυρώσεων μεταξύ Τ. urticae που συλλέχθηκε σε λεμονιές (ELCI) και Τ. urticae που συλλέχτηκε από το φυτό Mercurialis annua (ELMA) στο ίδιο χωράφι. Η συμβατότητα των διασταυρώσεων όταν πρόκειται για απλοδιπλοειδές είδος, όπου τα αρσενικά προέρχονται από αγονιμοποίητα ωά, μελετάται λαμβάνοντας υπόψη τον αριθμό των απογόνων αλλά και την αναλογία φύλου μεταξύ τους. Η γονιμότητα των θηλυκών ELMA δεν επηρεάστηκε όταν διασταυρώθηκαν με αρσενικά ELCI, όμως η αναλογία θηλυκών : αρσενικών ήταν σημαντικά μειωμένη (16,6% θηλυκά, σε σύγκριση με 66% στην διασταύρωση αναφοράς ELMA x ELMA). Όταν τα θηλυκά ELCI διασταυρώθηκαν με αρσενικά ELMA, η γονιμότητα ήταν κατά 30% μειωμένη και το ποσοστό των θηλυκών απογόνων μειώθηκε από 67% σε 52%, σε σχέση με τη διασταύρωσης αναφοράς (ELCI x ELCI). Τα F1 θηλυκά που προέκυψαν από διασταυρώσεις θηλυκών και αρσενικών από διαφορετικούς πληθυσμούς γέννησαν λιγότερα ωά, που είχαν μεγαλύτερη θνησιμότητα, σε σχέση με τα F1 θηλυκά προερχόμενα από ενδοπληθυσμιακές διασταυρώσεις, μειώνοντας επιπλέον το δυναμικό αναπαραγωγής μεταξύ των πληθυσμών.The study of host plant adaptation in arthropods, and especially agricultural pests, presents great interest, as it relates to patterns of population isolation and differentiation, with effects ranging from gene flow restriction to speciation. Prompted by our earlier isoenzyme studies that revealed genetic differentiation of Tetranychus urticae (Koch) collected on citrus, compared to other host plants, we investigated crossing compatibility between T. urticae collected from citrus (lemon) trees (ELCI) and T. urticae collected from the weed Mercurialis annua (ELMA), in the same citrus grove. Crossing compatibility in haplodiploid species where unfertilised eggs develop into males, like T. urticae, is assessed based on both the number and the sex ratio of the offspring. When ELMA females were crossed with ELCI males, fecundity was not affected, however the sex ratio was significantly biased towards males (16.6 % females, compared to 66% in the control cross ELMAxELMA). In the reciprocal crosses (ELCIxELMA), fecundity was lower by 30% and the proportion of female offspring was reduced to 52% from 67 %, compared to the control. The fecundity of the F1 hybrid females was significantly reduced and the eggs they laid were less viable, compared to the non-hybrids, further reducing the reproductive potential of inter-strain crosses. Combined with previous data, these results suggest the existence of a citrus-associated T. urticae host race

Reproductive incompatibility between genetically differentiated populations of Tetranychus urticae from different host plants

Η μελέτη της προσαρμογής των εχθρών των καλλιεργούμενων φυτών στα φυτά ξενιστές τους παρουσιάζει μεγάλο ενδιαφέρον, καθώς σχετίζεται με την απομόνωση και γενετική διαφοροποίηση των πληθυσμών, με αποτελέσματα που ποικίλλουν από τον περιορισμό της γονιδιακής ροής μέχρι και το σχηματισμό νέων ειδών. Με έναυσμα προηγούμενες μελέτες μας με ισοένζυμα, που κατέδειξαν γενετική διαφοροποίηση μεταξύ πληθυσμών Tetranychus urticae (Koch) από εσπεριδοειδή σε σχέση με άλλους ξενιστές, μελετήσαμε την συμβατότητα των διασταυρώσεων μεταξύ Τ. urticae που συλλέχθηκε σε λεμονιές (ELCI) και Τ. urticae που συλλέχτηκε από το φυτό Mercurialis annua (ELMA) στο ίδιο χωράφι. Η συμβατότητα των διασταυρώσεων όταν πρόκειται για απλοδιπλοειδές είδος, όπου τα αρσενικά προέρχονται από αγονιμοποίητα ωά, μελετάται λαμβάνοντας υπόψη τον αριθμό των απογόνων αλλά και την αναλογία φύλου μεταξύ τους. Η γονιμότητα των θηλυκών ELMA δεν επηρεάστηκε όταν διασταυρώθηκαν με αρσενικά ELCI, όμως η αναλογία θηλυκών : αρσενικών ήταν σημαντικά μειωμένη (16,6% θηλυκά, σε σύγκριση με 66% στην διασταύρωση αναφοράς ELMA x ELMA). Όταν τα θηλυκά ELCI διασταυρώθηκαν με αρσενικά ELMA, η γονιμότητα ήταν κατά 30% μειωμένη και το ποσοστό των θηλυκών απογόνων μειώθηκε από 67% σε 52%, σε σχέση με τη διασταύρωσης αναφοράς (ELCI x ELCI). Τα F1 θηλυκά που προέκυψαν από διασταυρώσεις θηλυκών και αρσενικών από διαφορετικούς πληθυσμούς γέννησαν λιγότερα ωά, που είχαν μεγαλύτερη θνησιμότητα, σε σχέση με τα F1 θηλυκά προερχόμενα από ενδοπληθυσμιακές διασταυρώσεις, μειώνοντας επιπλέον το δυναμικό αναπαραγωγής μεταξύ των πληθυσμών.The study of host plant adaptation in arthropods, and especially agricultural pests, presents great interest, as it relates to patterns of population isolation and differentiation, with effects ranging from gene flow restriction to speciation. Prompted by our earlier isoenzyme studies that revealed genetic differentiation of Tetranychus urticae (Koch) collected on citrus, compared to other host plants, we investigated crossing compatibility between T. urticae collected from citrus (lemon) trees (ELCI) and T. urticae collected from the weed Mercurialis annua (ELMA), in the same citrus grove. Crossing compatibility in haplodiploid species where unfertilised eggs develop into males, like T. urticae, is assessed based on both the number and the sex ratio of the offspring. When ELMA females were crossed with ELCI males, fecundity was not affected, however the sex ratio was significantly biased towards males (16.6 % females, compared to 66% in the control cross ELMAxELMA). In the reciprocal crosses (ELCIxELMA), fecundity was lower by 30% and the proportion of female offspring was reduced to 52% from 67 %, compared to the control. The fecundity of the F1 hybrid females was significantly reduced and the eggs they laid were less viable, compared to the non-hybrids, further reducing the reproductive potential of inter-strain crosses. Combined with previous data, these results suggest the existence of a citrus-associated T. urticae host race

Population genetics of the Bemisia tabaci Med species from Mediterranean countries

Within the Bemisia tabaci species complex, the Middle East-Asia Minor I (MEAMI) and the Mediterranean (Med) species are the most invasive and widespread. These last years, Med has become the predominant, or even the only species, in the Mediterranean basin- its presumed area of origin- and has spread to many other parts of the world. Combining mitochondrial (COI gene) and nuclear (microsatellite loci) markers, an extensive survey of Med B. tabaci (i.e. more than 3500 females studied) collected from five Mediterranean countries was undertaken to infer the genetic diversity and the possible role of ecological and environmental factors in shaping the population genetic structure observed. Based on sequence polymorphism of a 676-bp fragment of the COI gene, eighteen haplotypes were obtained which delineated two mitochondrial groups (named Q1 and Q2 which also correspond to Western and Eastern Mediterranean countries, respectively). Simple PCR/RFLP-based molecular assays were used to examine the geographic distribution of these two mitochondrial types by monitoring approximately 1000 individuals: both Q1 and Q2 were found in Spain and France, in sympatry in the field or not respectively, whereas in Greece, Morocco and Tunisia only Q1 was observed. Based on nuclear markers, Bayesian analysis of seven microsatellite loci polymorphism revealed three genetic clusters (France, Spain, Morocco _ Greece _ Tunisia) and a high level of genetic differentiation even between neighbouring samples belonging to the same mitochondrial group within a country. In some cases, subsequent Bayesian analysis within each genetic cluster disclosed additional sub-clusters, as in Greece where two genetic groups were found: the first with populations from South Crete, and the second with populations from continental Greece and North Crete. In this country, genetic differentiation is rather explained by differences in landscape structure than host plant species or habitat type. In B. tabaci from Tunisia, however, host plant species seems to play a role in the Q1 population structure. In France and Spain, where both Q1 and Q2 were observed, comparison with an Israeli Q2 provided confirmation of hybridization and evidence for asymmetrical gene flow between the two mitochondrial types. Overall, our findings suggest that the Med B. tabaci species is more diverse and structured in its original geographic area than reported so far. Acknowledgements: We are grateful to C. Clouet, A. Tahiri, J.B. Kristoffersen for their valuable assistance. (Résumé d'auteur

Recent emergence and worldwide spread of the red tomato spider mite, [i]Tetranychus evansi[/i]: genetic variation and multiple cryptic invasions

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699Plant biosecurity is increasingly challenged by emerging crop pests. The spider mite Tetranychus evansi has recently emerged as a new threat to solanaceous crops in Africa and the Mediterranean basin, with invasions characterized by a high reproductive output and an ability to withstand a wide range of temperatures. Mitochondrial (868 bp of COI) and nuclear (1,137 bp of ITS) loci were analyzed in T. evansi samples spanning the current geographical distribution to study the earliest stages of the invasive process. The two sets of markers separate the samples into two main clades that are only present together in South America and Southern Europe. The highest COI diversity was found in South America, consistent with the hypothesis of a South American origin of T. evansi. Among the invaded areas, the Mediterranean region displayed a high level of genetic diversity similar to that present in South America, that is likely the result of multiple colonization events. The invasions of Africa and Asia by T. evansi are characterized by a low genetic variation associated with distinct introductions. Genetic data demonstrate two different patterns of invasions: (1) populations in the Mediterranean basin that are a result of multiple cryptic introductions and (2) emerging invasions of Africa and Asia, each likely the result of propagules from one or limited sources. The recent invasions of T. evansi illustrate not only the importance of human activities in the spread of agricultural pests, but also the limits of international quarantine procedures, particularly for cryptic invasion

Ryanodine receptor point mutations confer diamide insecticide resistance in tomato leafminer, Tuta absoluta (Lepidoptera: Gelechiidae).

This is the author accepted manuscript. The final version is available from Elsevier via the DOI in this record.Insect ryanodine receptors (RyR) are the molecular target-site for the recently introduced diamide insecticides. Diamides are particularly active on Lepidoptera pests, including tomato leafminer, Tuta absoluta (Lepidoptera: Gelechiidae). High levels of diamide resistance were recently described in some European populations of T. absoluta, however, the mechanisms of resistance remained unknown. In this study the molecular basis of diamide resistance was investigated in a diamide resistant strain from Italy (IT-GELA-SD4), and additional resistant field populations collected in Greece, Spain and Brazil. The genetics of resistance was investigated by reciprocally crossing strain IT-GELA-SD4 with a susceptible strain and revealed an autosomal incompletely recessive mode of inheritance. To investigate the possible role of target-site mutations as known from diamondback moth (Plutella xylostella), we sequenced respective domains of the RyR gene of T. absoluta. Genotyping of individuals of IT-GELA-SD4 and field-collected strains showing different levels of diamide resistance revealed the presence of G4903E and I4746M RyR target-site mutations. These amino acid substitutions correspond to those recently described for diamide resistant diamondback moth, i.e. G4946E and I4790M. We also detected two novel mutations, G4903V and I4746T, in some of the resistant T. absoluta strains. Radioligand binding studies with thoracic membrane preparations of the IT-GELA-SD4 strain provided functional evidence that these mutations alter the affinity of the RyR to diamides. In combination with previous work on P. xylostella our study highlights the importance of position G4903 (G4946 in P. xylostella) of the insect RyR in defining sensitivity to diamides. The discovery of diamide resistance mutations in T. absoluta populations of diverse geographic origin has serious implications for the efficacy of diamides under applied conditions. The implementation of appropriate resistance management strategies is strongly advised to delay the further spread of resistance.The work of Hellenic Agricultural Organisation - 'Demeter' was partially supported by an ARIMnet2 StomP grand to A.T and E.R. This work was also partially funded from a fellowship granted to H.A.A.S. (CNPq - PQ - Proc 308461/2013-4). The Universidad Politécnica de Cartagena group would like to thank for partial financial support the Ministerio de Economía y Competitividad of Spain and FEDER (AGL2011-25164). Lidia García-Vidal holds a grant from the MECD (FPU13/01528). The Tuta absoluta strain from Gela, Sicily was collected under the frame a resistance monitoring program established among the Hellenic Agricultural Organisation - 'Demeter' and DuPont De Nemurs (data published in 2015). Finally, the Hellenic Agricultural Organisation - 'Demeter' would like to thank Fytochem S.A., Neo Mirtos, Ierapetra for supplies of plant material

The druggability of the ATP binding site of glycogen phosphorylase kinase probed by coumarin analogues

Glycogen phosphorylase kinase (PhK) converts by phosphorylation, the inactive glycogen phosphorylase (GPb) into active GPa in the glycogenolytic pathway. It is a complex enzyme comprising of the catalytic (γ) and three regulatory subunits (α, β, δ) forming a hexadecamer with stoichiometry (αβγδ)4. Several studies have indicated PhK as a promising target for the development of antihyperglycemics as its inhibition blocks glycogenolysis in liver and a potential therapeutic target for cancer against pathological angiogenesis and tumor progression. The identification of compounds that inhibit the kinase through their direct binding to its catalytic site is an effective approach to identify bioactive molecules of therapeutic significance. Towards this, the structure of the N-terminal kinase domain (residues 1–298) of the catalytic γ subunit of PhK (PhKγtrnc) has been determined by X-ray crystallography while staurosporine and indirubin analogues have been characterized as potent inhibitors targeting the ATP binding site. In this study, a series of 38 synthetic analogues of naturally occurring coumarins were screened for inhibition of PhKγtrnc, in vitro, using a photometric assay. The IC50 values of the two most potent compounds were determined for PhKγtrnc and the pharmacologically relevant target, human liver isoform (PHKG2A). Their cellular efficacy and toxicity in HepG2 cells were further assessed ex vivo. Docking experiments and the structural comparison with previously described inhibitors reveal the binding mode of the coumarin scaffold at a no hinge region of the ATP site of PhK and the role of a conserved β3-Lys in binding. The experimental findings provide structural insights with implications to the kinase targeting and drug design

Spider mite web mediates anti-predator behaviour

Herbivores suffer significant mortality from predation and are therefore subject to natural selection on traits promoting predator avoidance and resistance. They can employ an array of strategies to reduce predation, for example through changes in behaviour, morphology and life history. So far, the anti-predator response studied most intensively in spider mites has been the avoidance of patches with high predation risk. Less attention has been given to the dense web produced by spider mites, which is a complex structure of silken threads that is thought to hinder predators. Here, we investigate the effects of the web produced by the red spider mite, Tetranychus evansi Baker & Pritchard, on its interactions with the predatory mite, Phytoseiulus longipes Evans. We tested whether female spider mites recognize predator cues and whether these can induce the spider mites to produce denser web. We found that the prey did not produce denser web in response to such cues, but laid more eggs suspended in the web, away from the leaf surface. These suspended eggs suffered less from predation by P. longipes than eggs that were laid on the leaf surface under the web. Thus, by altering their oviposition behaviour in response to predator cues, females of T. evansi protect their offspring