Analysis of the olive fruit fly Bactrocera oleae transcriptome and phylogenetic classification of the major detoxification gene families

he olive fruit fly Bactrocera oleae has a unique ability to cope with olive flesh, and is the most destructive pest of olives worldwide. Its control has been largely based on the use of chemical insecticides, however, the selection of insecticide resistance against several insecticides has evolved. The study of detoxification mechanisms, which allow the olive fruit fly to defend against insecticides, and/or phytotoxins possibly present in the mesocarp, has been hampered by the lack of genomic information in this species. In the NCBI database less than 1,000 nucleotide sequences have been deposited, with less than 10 detoxification gene homologues in total. We used 454 pyrosequencing to produce, for the first time, a large transcriptome dataset for B. oleae. A total of 482,790 reads were assembled into 14,204 contigs. More than 60% of those contigs (8,630) were larger than 500 base pairs, and almost half of them matched with genes of the order of the Diptera. Analysis of the Gene Ontology (GO) distribution of unique contigs, suggests that, compared to other insects, the assembly is broadly representative for the B. oleae transcriptome. Furthermore, the transcriptome was found to contain 55 P450, 43 GST-, 15 CCE- and 18 ABC transporter-genes. Several of those detoxification genes, may putatively be involved in the ability of the olive fruit fly to deal with xenobiotics, such as plant phytotoxins and insecticides. In summary, our study has generated new data and genomic resources, which will substantially facilitate molecular studies in B. oleae, including elucidation of detoxification mechanisms of xenobiotic, as well as other important aspects of olive fruit fly biology

Substrate specificity and promiscuity of horizontally transferred UDP-glycosyltransferases in the generalist herbivore Tetranychus urticae

Uridine diphosphate (UDP)-glycosyltransferases (UGTs) catalyze the addition of UDP-sugars to small hydrophobic molecules, turning them into more water-soluble metabolites. While their role in detoxification is well documented for vertebrates, arthropod UGTs have only recently been linked to the detoxification and sequestration of plant toxins and insecticides. The two-spotted spider mite Tetranychus urticae is a generalist herbivore notorious for rapidly developing resistance to insecticides and acaricides. We identified a set of eight UGT genes that were overexpressed in mites upon long-term acclimation or adaptation to a new host plant and/or in mite strains highly resistant to acaricides. Functional expression revealed that they were all catalytically active and that the majority preferred UDP-glucose as activated donor for glycosylation of model substrates. A high-throughput substrate screening of both plant secondary metabolites and pesticides revealed patterns of both substrate specificity and promiscuity. We further selected nine enzyme-substrate combinations for more comprehensive analysis and determined steady-state kinetic parameters. Among others, plant metabolites such as capsaicin and several flavonoids were shown to be glycosylated. The acaricide abamectin was also glycosylated by two UGTs and one of these was also overexpressed in an abamectin resistant strain. Our study corroborates the potential role of T. urticae UGTs in detoxification of both synthetic and natural xenobiotic compounds and paves the way for rapid substrate screening of arthropod UGTs

Transcriptomic responses of the olive fruit fly Bactrocera oleae and its symbiont Candidatus Erwinia dacicola to olive feeding

The olive fruit fly, Bactrocera oleae, is the most destructive pest of olive orchards worldwide. The monophagous larva has the unique capability of feeding on olive mesocarp, coping with high levels of phenolic compounds and utilizing non-hydrolyzed proteins present, particularly in the unripe, green olives. On the molecular level, the interaction between B. oleae and olives has not been investigated as yet. Nevertheless, it has been associated with the gut obligate symbiotic bacterium Candidatus Erwinia dacicola. Here, we used a B. oleae microarray to analyze the gene expression of larvae during their development in artificial diet, unripe (green) and ripe (black) olives. The expression profiles of Ca. E. dacicola were analyzed in parallel, using the Illumina platform. Several genes were found overexpressed in the olive fly larvae when feeding in green olives. Among these, a number of genes encoding detoxification and digestive enzymes, indicating a potential association with the ability of B. oleae to cope with green olives. In addition, a number of biological processes seem to be activated in Ca. E. dacicola during the development of larvae in olives, with the most notable being the activation of amino-acid metabolism

Multiple recombination events between two cytochrome P450 loci contribute to global pyrethroid resistance in Helicoverpa armigera

The cotton bollworm, Helicoverpa armigera (Hübner) is one of the most serious insect pest species to evolve resistance against many insecticides from different chemical classes. This species has evolved resistance to the pyrethroid insecticides across its native range and is becoming a truly global pest after establishing in South America and having been recently recorded in North America. A chimeric cytochrome P450 gene, CYP337B3, has been identified as a resistance mechanism for resistance to fenvalerate and cypermethrin. Here we show that this resistance mechanism is common around the world with at least eight different alleles. It is present in South America and has probably introgressed into its closely related native sibling species, Helicoverpa zea. The different alleles of CYP337B3 are likely to have arisen independently in different geographic locations from selection on existing diversity. The alleles found in Brazil are those most commonly found in Asia, suggesting a potential origin for the incursion of H. armigera into the Americas.Additional co-authors: Mary E A Whitehouse, Pierre Jean Silvie, Sharon Downes, Lori Nemec, David G. Hecke

Multiple recombination events between two cytochrome P450 loci contribute to global pyrethroid resistance in Helicoverpa armigera

The cotton bollworm, Helicoverpa armigera (Hübner) is one of the most serious insect pest species to evolve resistance against many insecticides from different chemical classes. This species has evolved resistance to the pyrethroid insecticides across its native range and is becoming a truly global pest after establishing in South America and having been recently recorded in North America. A chimeric cytochrome P450 gene, CYP337B3, has been identified as a resistance mechanism for resistance to fenvalerate and cypermethrin. Here we show that this resistance mechanism is common around the world with at least eight different alleles. It is present in South America and has probably introgressed into its closely related native sibling species, Helicoverpa zea. The different alleles of CYP337B3 are likely to have arisen independently in different geographic locations from selection on existing diversity. The alleles found in Brazil are those most commonly found in Asia, suggesting a potential origin for the incursion of H. armigera into the Americas.Financial support in Australia (to TKW) was provided by the Commonwealth Science and Industry Research Organisation, Land and Water, Health and Biosecurity, Agriculture. In Germany financial support (to DGH) was provided by the Max-Planck-Gesellschaft. NJ was supported by a grant from the Deutsche Forschungsgemeinschaf (DFG, Germany; JO 855/1-1)

Multiple recombination events between two cytochrome P450 loci contribute to global pyrethroid resistance in Helicoverpa armigera

The cotton bollworm, Helicoverpa armigera (Hübner) is one of the most serious insect pest species to evolve resistance against many insecticides from different chemical classes. This species has evolved resistance to the pyrethroid insecticides across its native range and is becoming a truly global pest after establishing in South America and having been recently recorded in North America. A chimeric cytochrome P450 gene, CYP337B3, has been identified as a resistance mechanism for resistance to fenvalerate and cypermethrin. Here we show that this resistance mechanism is common around the world with at least eight different alleles. It is present in South America and has probably introgressed into its closely related native sibling species, Helicoverpa zea. The different alleles of CYP337B3 are likely to have arisen independently in different geographic locations from selection on existing diversity. The alleles found in Brazil are those most commonly found in Asia, suggesting a potential origin for the incursion of H. armigera into the Americas

Multiple recombination events between two cytochrome P450 loci contribute to global pyrethroid resistance in Helicoverpa armigera

The cotton bollworm, Helicoverpa armigera (Hubner) is one of the most serious insect pest species to evolve resistance against many insecticides from different chemical classes. This species has evolved resistance to the pyrethroid insecticides across its native range and is becoming a truly global pest after establishing in South America and having been recently recorded in North America. A chimeric cytochrome P450 gene, CYP337B3, has been identified as a resistance mechanism for resistance to fenvalerate and cypermethrin. Here we show that this resistance mechanism is common around the world with at least eight different alleles. It is present in South America and has probably introgressed into its closely related native sibling species, Helicoverpa zea. The different alleles of CYP337B3 are likely to have arisen independently in different geographic locations from selection on existing diversity. The alleles found in Brazil are those most commonly found in Asia, suggesting a potential origin for the incursion of H. armigera into the Americas

Use of Mutagenesis, Genetic Mapping and Next Generation Transcriptomics to Investigate Insecticide Resistance Mechanisms

Insecticide resistance is a worldwide problem with major impact on agriculture and human health. Understanding the underlying molecular mechanisms is crucial for the management of the phenomenon; however, this information often comes late with respect to the implementation of efficient counter-measures, particularly in the case of metabolism-based resistance mechanisms. We employed a genome-wide insertional mutagenesis screen to Drosophila melanogaster, using a Minos-based construct, and retrieved a line (MiT[w−]3R2) resistant to the neonicotinoid insecticide Imidacloprid. Biochemical and bioassay data indicated that resistance was due to increased P450 detoxification. Deep sequencing transcriptomic analysis revealed substantial over- and under-representation of 357 transcripts in the resistant line, including statistically significant changes in mixed function oxidases, peptidases and cuticular proteins. Three P450 genes (Cyp4p2, Cyp6a2 and Cyp6g1) located on the 2R chromosome, are highly up-regulated in mutant flies compared to susceptible Drosophila. One of them (Cyp6g1) has been already described as a major factor for Imidacloprid resistance, which validated the approach. Elevated expression of the Cyp4p2 was not previously documented in Drosophila lines resistant to neonicotinoids. In silico analysis using the Drosophila reference genome failed to detect transcription binding factors or microRNAs associated with the over-expressed Cyp genes. The resistant line did not contain a Minos insertion in its chromosomes, suggesting a hit-and-run event, i.e. an insertion of the transposable element, followed by an excision which caused the mutation. Genetic mapping placed the resistance locus to the right arm of the second chromosome, within a ∼1 Mb region, where the highly up-regulated Cyp6g1 gene is located. The nature of the unknown mutation that causes resistance is discussed on the basis of these results

Μελέτη προσαρμογής των αρθροπόδων στις τοξίνες των φυτών (plant allelochemicals) και τα εντομοκτόνα, με έμφαση στον ρόλο των μηχανισμών αποτοξικοποίησης

Arthropod pests seriously threaten food security and human health, as theyattack agricultural crops and transmit various diseases. Their control has beenlargely based on chemical insecticides. However the intense use of insecticides hasled in the development of resistance, mainly achieved by target site resistancemutations and detoxification, enzymes, such as the Cytochrome P450s, theGlutathione S-transferases GSTs and the Carboxylesterases (CCEs). The samedetoxification enzymes also participate in the adaptation of arthropod pests to theirhosts, as they also metabolize – inactivate phytotoxins. In this thesis, I usedtransgenic approaches, bioassays, transcriptomics and biochemical/ functionaltechniques to investigate detoxification mechanisms of major arthropod pests, suchas the Aedes aegypti, the Bactrocera oleae and the Tetranychus urticae, againstxenobiotics.First, the A. aegypti cytochrome P450 CYP9J28 was successfully expressed inDrosophila melanogaster and shown to confer significant levels of resistance in vivo,providing solid evidence for its role in pyrethroid resistance and showing that ectopicexpression in D. melanogaster may be a robust approach for validation of candidateresistance genes.Second, a large transcriptomic dataset of B. oleae was generated and morethan 130 putative detoxification genes were identified and phylogeneticallyclassified. The transcriptome was used for the construction of a microarray tool,which was used, in its pilot application, to study detoxification and adaptationmechanisms of the olive fly against insecticides and olive flesh/phytotoxins,respectively. The pyrethroid resistance study indicated the association of twocytochrome P450 genes with the phenotype. Several detoxification and digestivegenes were found over-expressed upon development in olives (green versus blackversus artificial diet), providing a useful starting point for further investigation.In the last chapter, my study dealt and focused on T. urticae GSTs that hadbeen associated with insecticide resistance by microarray studies. Four GSTs werefunctionally expressed and characterized. TuGSTd14 was found to interact withabamectin, supporting earlier work that GSTs are may play a role in abamectinresistance. Strong evidence were provided that TuGSTd05 catalyzes the conjugationof glutathione (GSH) to cyflumetofen in vitro and the possible site of attack as well askey amino acids possibly implicated in the interaction were identified. This studyrepresents the first functional convincing report for the implication of an acari GST inresistance.Τα αρθρόποδα παράσιτα αποτελούν απειλή για την ασφάλεια των τροφίμωνκαι τη δημόσια υγεία καθώς καταστρέφουν τις καλλιέργειες και μεταφέρουνεντομομεταδιδόμενες ασθένειες. Ο έλεγχός τους βασίζεται κυρίως στη χρήσηεντομοκτόνων. Ωστόσο, η συχνή τους εφαρμογή έχει οδηγήσει στην ανάπτυξηανθεκτικότητας, η οποία επιτυγχάνεται κυρίως μέσω μεταλλαγών στις πρωτεΐνες-στόχους των εντομοκτόνων και μέσω της δράσης των ενζύμων αποτοξικοποίησης,όπως είναι οι κυτοχρωμικές P450 μονοοξυγενάσες (P450s), οι μεταφοράσες τηςγλουταθειώνης (GSTs) και οι καρβοξυλεστεράσες (CCEs). Τα ίδια ένζυμααποτοξικοποίησης συμμετέχουν και στην προσαρμογή των αρθροπόδων στα φυτά-ξενιστές, καθώς έχουν την ικανότητα να μεταβολίζουν- απενεργοποιούν τιςφυτοτοξίνες. Στην παρούσα διατριβή χρησιμοποιήθηκαν προσεγγίσεις μεδιαγονιδιακούς οργανισμούς, βιοδοκιμές, μεθόδοι ανάλυσης μεταγραφώματοςκαθώς και βιοχημικές/ λειτουργικές μελέτες για τη διερεύνηση των μηχανισμώναποτοξικοποίησης ξενοβιοτικών ουσιών στο κουνούπι Aedes aegypti, το δάκο τηςελιάς Bactrocera oleae και το φυτοφάγο άκαρι Tetranychus urticae.Αρχικά, το γονίδιο CYP9J28 του A. aegypti που κωδικοποιεί για κυτοχρωμικήοξειδάση P450 εκφράστηκε επιτυχώς στη Drosophila melanogaster και δείχθηκε ότιπροσδίδει σημαντικά επίπεδα ανθεκτικότητας in vivo, παρέχοντας ισχυρέςαποδείξεις για το ρόλο του στη ανθεκτικότητα στα πυρεθροειδή και δείχνοντας ότιη έκφραση γονιδίων στη D. melanogaster είναι μια ισχυρή προσέγγιση για τηνεπιβεβαίωση του ρόλου υποψηφίων γονιδίων στην ανθεκτικότητα.Στο δεύτερο κεφάλαιο της διατριβής, δημιουργήθηκε ένα μεγάλο σύνολοδεδομένων μεταγραφώματος για το B. oleae και ταυτοποιήθηκαν καικατηγοριοποιήθηκαν φυλογενετικά πάνω από 130 πιθανά γονίδιααποτοξικοποίησης. Τα δεδομένα του μεταγραφώματος χρησιμοποιήθηκαν για τηνκατασκευή μιας πλατφόρμας μικροσυστοιχιών, η οποία εν συνεχείαχρησιμοποιήθηκε πιλοτικά για την μελέτη των μηχανισμών αποτοξικοποίησης καιπροσαρμογής του δάκου έναντι των εντομοκτόνων και του ελαιοκάρπου/φυτοτοξινών, αντίστοιχα. Η ανάλυση της ανθεκτικότητας σε πυρεθροειδή υπέδειξετη συσχέτιση δύο κυτοχρωμικών οξειδασών P450 με το φαινότυπο. Επίσης,μελετήθηκαν οι μοριακοί μηχανισμοί προσαρμογής στον ελαιόκαρπο και ηαξιοποίηση του μεσοκαρπίου. Αρκετά γονίδια αποτοξικοποίησης και γονίδια τηςπέψης βρέθηκαν να υπερ-εκφράζονται κατά τη διάρκεια της ανάπτυξης στονελαιόκαρπο, παρέχοντας ένα χρήσιμο σημείο εκκίνησης για περαιτέρω διερεύνηση.Στο τελευταίο κεφάλαιο της διατριβής εκφράστηκαν λειτουργικά καιχαρακτηρίστηκαν τέσσερις μεταφοράσες της γλουταθειώνης (GSTs) του T. urticae,οι οποίες είχαν προηγουμένως συσχετιστεί με την ανθεκτικότητα σε εντομοκτόνααπό μελέτες μικροσυστοιχιών. Η TuGSTd14 βρέθηκε να αλληλεπιδρά με τοabamectin, ενισχύοντας προηγούμενες μελέτες που υποστηρίζουν ότι οι GSTsενδεχομένως εμπλέκονται στην ανθεκτικότητα στο abamectin. Ισχυρές αποδείξειςδόθηκαν για την in vitro κατάλυση της σύζευξης της γλουταθειώνης (GSH) στοcyflumetofen από την TuGSTd05 και αναγνωρίστηκε το πιθανό σημείο τηςπροσβολής καθώς και σημαντικά αμινοξέα που πιθανόν να συμμετέχουν στηναλληλεπίδραση. Η συγκεκριμένη μελέτη αποτελεί την πρώτη πειστική αναφορά γιατη συμμετοχή μιας GST από ακάρεα στην ανθεκτικότητα

The role of glutathione S-transferases (GSTs) in insecticide resistance in crop pests and disease vectors

Insecticide resistance seriously threatens efficient arthropod pest management. Arthropod glutathione S-transferases (GSTs) confer resistance via direct metabolism or sequestration of chemicals, but also indirectly by providing protection against oxidative stress induced by insecticide exposure. To date, GST activity has been associated with resistance to all main classes of insecticides. However, recent advances in genome and transcriptome sequencing, together with modern genetic, functional and biochemical techniques, facilitate the unraveling of specific GST-mediated resistance mechanisms. Recently, the role of a number of GSTs (BdGSTe2, BdGSTe4, AfGSTe2) has been validated by (reverse) genetic methods in vivo, while a number of GSTs (BmGSTu2, TuGSTd05, AfGSTe2) have now been shown to metabolize insecticides in vitro