Proteomics Analysis of Bactroceraoleae with Emphasis in Sex Separation and Insecticide

Ανακοινώσεις: 10

6th International Symposium on Molecular Insect Science, 2-5 October 2011. NH Grand Krasnapolsky, Amsterdam, The Netherlands

“Proteomics analysis of Bactroceraoleae with emphasis in sex separation and insecticide resistance systems”.

E. Sagri1, G. Stravodimos1, M. Samiotaki2, G. Panagiotou2, K.D. Mathiopoulos1 and M. Kontou1

1 University of Thessaly- Department of Biochemistry and Biotechnology, Greece; 2Biomedical Sciences Research Center"Alexander Fleming", Greece

The olive fruit fly, Bactroceraoleae, is the most important enemy of the olive. Its economic importance makes this insect a target of alternative control methods, among which is the Sterile Insect Technique. Development of effective SIT protocols, however, presupposes an understanding of the species at the genetic, molecular and population level. Towards this purpose, we departed in the analysis of the proteome of the insect, in an effort to indentify pathways involved either in sex determination cascades or in insecticide resistance. Till now, all efforts to identify genetic loci involved in different functions in B. oleae were based on homology comparisons with other diptera. The proteomic analysis intends to investigate the differential expression of the proteome at selected conditions and identify mechanisms acting at the post-transcriptional or post-translational level. In a first attempt to study the differential expression of the proteome in the different sexes and in insects of different levels of insecticide resistance, head extracts were collected from flies and proteins were separated with 2D-PAGE. Spots corresponding to differentially expressed proteins from both male and female insects or between resistant and susceptible individuals wereexcisedfromthe gel, digestedwithtrypsinandfollowed by peptide fingerprinting with mass spectrometry.

34th Scientific Conference E.E.B.E., Trikala, 17- 19 May 2012

“Proteomics analysis of Bactroceraoleae with emphasis in sex separation and insecticide resistance systems”.

E. Sagri1, G. Stravodimos1, M. Samiotaki2, G. Panagiotou2, K.D. Mathiopoulos1 and M. Kontou1

1 University of Thessaly- Department of Biochemistry and Biotechnology, Greece; 2Biomedical Sciences Research Center"Alexander Fleming", Greece

XXIV International Congress of Entomology, Daegu, Korea , 19-25 August 2012

Spinosad resistance in the olive fly, Bactroceraoleae

Maria Eleni Gregoriou1, Efthimia Sagri2, Evdoxia G Kakani3, Klelia Salpea4, Vaggelis Harokopos5, Frank G Zalom6, JiannisRagoussis7, Kostas D Mathiopoulos8

1University of Thessaly, Greece, 2University of Thessaly, Greece, 3Harvard School of Public Health, U.S.A., 4Alexander Fleming - Biomedical Sciences Research Center, Greece, 5Alexander Fleming - Biomedical Sciences Research Center, Greece, 6University of California, Davis, U.S.A., 7Alexander Fleming - Biomedical Sciences Research Center, Greece, 8University of Thessaly, Greece

The olive fruit fly Bactroceraoleae is the most important and destructive pest of cultivated olive trees, in almostevery country where olive trees grow. For the last 40 years, the management of the pest has been based on the useof organophosphate insecticides. More recently, pyrethroids and the naturalytespinosad have been added in thearsenal against the olive fly. However, the extensive use of any insecticide inevitably leads to the development ofresistance. Low level spinosad resistance to the olive fly has been demonstrated in flies caught in several Californiacounties where the drug is the only insecticide used for the control of the fly. In Drosophila, the α6 subunit of thenicotine acetylcholine receptor (nAChR) has been implicated in spinosad resistance. On the contrary, the α6 subunitof the nAChR does not seem to be spinosad’s target in Muscadomestica. In order to investigate the mechanism ofspinosad resistance in the olive fly, we developed a ~30-fold spinosad resistant strain in the laboratory undercontinuous selection. By virtue of homology we isolated B. oleaeα6 nAChR both from the laboratory sensitive andresistant strains in an effort to identify possible sequence variants in this locus that could be responsible for theobserved resistance. At the same time, whole transcriptome analysis was performed in heads of the sensitive andresistant strains in order to study the differential expression of the two strains and identify the loci involved inspinosad resistance.

2nd International Symposium of Team. Kolymbari, 3-6 July 2012

“Sex-specific proteome differences in the olive fruit fly, Bactroceraoleae’’EfthymiaSagri1,GeorgeStravodimos1, Martina Samiotaki2, George Panagiotou2, Kostas D. Mathiopoulos1 and Maria Kontou1

1Department of Biochemistry and Biotechnology, University of Thessaly, Greece; 2Biomedical Sciences Research Center"Alexander Fleming", Greece

The olive fruit fly, Bactroceraoleae, is the most important enemy of the olive. Its economic importance makes this insect a target of alternative control methods, among which is the Sterile Insect Technique. Development of effective SIT protocols, however, presupposes an understanding of the species at the genetic, molecular and population level. Towards this purpose, we departed in the analysis of the proteome of the insect, in an effort to identify pathways involved in sex determination cascades. Till now, all efforts to identify genetic loci involved in different functions in B. oleae were based on homology comparisons with other diptera. The proteomic analysis intends to investigate the differential expression of the proteome at selected conditions and identify mechanisms acting at the post-transcriptional or post-translational level. In a first attempt to study the differential expression of the proteome in the different sexes, head extracts were collected from flies and proteins were separated with 2D-PAGE. Spots corresponding to differentially expressed proteins from both male and female insects or between resistant and susceptible individuals wereexcisedfromthe gel, digestedwithtrypsinandfollowed by peptide fingerprinting with mass spectrometry. Observed differences will be presented.

2nd International Symposium of Team. Kolymbari, 3-6 July 2012

“Spinosad resistance in Bactroceraoleae”

Maria-Eleni G Gregoriou1, EfthymiaSagri1,ValantisKouimanis1, Evdoxia G Kakani1, Nikos E Zygouridis1, Frank G Zalom2, Kostas D Mathiopoulos1

1Department of Biochemistry and Biotechnology, University of Thessaly, Larissa, Greece;2DepartmentofEntomology, UniversityofCalifornia-Davis, USA

The olive fruit fly Bactroceraoleae is the most important and destructive pest of cultivated olive trees, in almost every country where olive trees grow. For the last 40 years, the management of the pest has been based on the use of organophosphate insecticides. However, the increasing awareness of the society against environmental problems turned the industry to the development of environmentally friendly insecticides. A very promising naturalyte insecticide with particular targeted toxicity towards the olive fly is Spinosad. As it is known for all types of insecticides, the extensive use of Spinosad leads to the development of resistance. Low level of spinosad resistance has been demonstrated in flies caught in several California counties where the drug is the only insecticide used for the control of the fly.In Drosophila melanogaster, it was shown that the α6 subunit of the nicotinic acetylocholine receptor (nAChR) was involved in spinosad resistance. On the contrary, the α6 subunit of the receptor does not seem to be spinosad’s target in Muscadomestica. In order to determine the mechanism of spinosad resistance in the olive fly, we developed a ~30-fold spinosad resistant strain in the laboratory under continuous selection. Firstly, esterase biochemical assays did not demonstrate any differences in esterase activity between sensitive and resistant individuals. Secondly, by virtue of homology we isolated and compared the sequences of the Boα6 nAChR subunits from sensitive and resistance strains. Observed differences will be discussed. Finally, whole transcriptome analysis was performed in heads of the sensitive and resistant strains in order to study the differential expression of the two strains and identify possible loci involved in spinosad resistance.

35th Scientific Conference E.E.B.E., Nauplio, 23- 25 May 2013

‘‘Transcriptomics analysis of bactroceraoleae with emphasis in sex separationand insecticide resistance systems’’

E. Sagri1, K. Salpea2, M. Reczko2, V. Harokopos2, J. Ragoussis2 and K.D. Mathiopoulos1

1 University of Thessaly- Department of Biochemistry and Biotechnology, Larissa-Greece;2Biomedical Sciences Research Center"Alexander Fleming", Athens-Greece

Greece is the third olive producing country worldwide, both with regard to table olives and olive-oil. The most important enemy of the olive tree is the olive fruit fly, Bactroceraoleae, an insect which belongs to the Tephritidae family. Its economic importance makes the insect a target of alternative control methods such as the Sterile Insect Technique. However, an effective SIT necessitates an understanding of the species at the genetic, molecular and population level. Towards this purpose, we departed in the analysis of the transcriptome of the insect, in an effort to indentify pathways and mechanisms involved in insecticide resistance in the naturalytespinosad.Till now, all efforts to identify genetic loci involved in different functions in B. oleae were based on homology comparisons with other diptera. The whole transcriptomic analysis intends to investigate the differential expression of the transcriptome at selected conditions and identify mechanisms acting at the post-transcriptional level. In a first attempt to study the differential expression of the transcriptome of sensitive and resistant flies in spinosad, specific tissues with the expected gene expression were collected and followed by RNA seq analysis. Results were further bioinformatically analyzed and the genes that were overexpressed in resistant and sensitive flies were functionally studied by real time PCR in order to investigate their differentially expressed profile.

15thNational Entomological Conference, Kavala, 22-25 October 2013

‘‘Transcriptomics analysis of bactroceraoleae with emphasis in insecticide resistance systems’’

E. Sagri1, K. Salpea2, M. Reczko2, V. Harokopos2, J. Ragoussis2 and K.D.Mathiopoulos1

1 University of Thessaly- Department of Biochemistry and Biotechnology, Larissa-Greece;2Biomedical Sciences Research Center "Alexander Fleming", Athens-Greece

Greece is the third olive producing country worldwide, both with regard to table olives and olive-oil. The most important enemy of the olive tree is the olive fruit fly, Bactroceraoleae, an insect which belongs to the Tephritidae family. Its economic importance makes the insect a target of alternative control methods such as the Sterile Insect Technique. However, an effective SIT necessitates an understanding of the species at the genetic, molecular and population level. Towards this purpose, we departed in the analysis of the transcriptome of the insect, in an effort to indentify pathways and mechanisms involved in insecticide resistance in the naturalytespinosad. Till now, all efforts to identify genetic loci involved in different functions in B. oleaewere based on homology comparisons with other diptera. The whole transcriptomic analysis intends to investigate the differential expression of the transcriptome at selected conditions and identify mechanisms acting at the post-transcriptional level. In a first attempt to study the differential expression of the transcriptome of sensitive and resistant flies in spinosad, RNA from headsfromsensitive and resistant flies was extracted and followed by RNAseq analysis. Results were bioinformatically analyzed and the genes that were overexpressed in resistant and sensitive flies were functionally studied by qRT-PCR in order to investigate their differentially expressed profile. Interestingly, genes that are involved in production of metabolic energy were found to be up-regulated in spinosad resistant flies. This might indicate that insects under insecticide stress require an increased metabolism in order to compensate the costs of energy-consuming detoxification processes.

10th European Congress of Entomology, York, United Kingdom, 3-8 August 2014

“Olive fly transcriptomics analysis implicates energy metabolism genes in spinosad resistance”

E. Sagri1, M. Reczko2, M-E Gregoriou1, K.T. Tsoumani1, N Zygouridis1, K.D. Salpea2, F.G. Zalom3, J. Ragoussis2 and K.D.Mathiopoulos1

1 Department of Biochemistry and Biotechnology, University of Thessaly, Larissa, Greece; 2 Institute of Molecular Biology and Genetics, Biomedical Sciences Research Centre “Alexander Fleming”, Athens, Greece; 3Department of Entomology, University of California, USA

The olive fly, Bactroceraoleae, is the most devastating pest of the olive tree. Its control has been traditionally based on insecticides, mainly organophosphates and pyrethroides. In recent years, the naturalytespinosad is being employed against the olive fly. Inevitably, this has led to resistance development. Mutations in the α6 subunit of the nicotinic acetylcholine receptor (nAChR) have been implicated in spinosad resistance in several insect species (e.g., Drosophila melanogaster) but excluded in others (e.g., Muscadomestica). Yet, more mechanisms involving enhanced metabolism of detoxification enzymes have also been reported. In order to clarify the spinosad resistance mechanisms in the olive fly we both searched for mutations in the nAChR-α6 subunit, as well as looked for up-regulated genes in the entire transcriptome of spinosad resistant olive flies. The differences in thenAChR-α6 subunit reflected mainly silent nucleotide substitutions or conserved amino acid changes. Additionally, whole transcriptome analysis was performed in the two strains in order to reveal any underlying resistance mechanisms. Comparison of over 13,000 genes pointed at nine over-expressed and ~40 under-expressed loci. Further functional analyses confirmed that several immune system loci as well as elevated energy requirements of the resistance flies are required to lever the detoxification process.

65th National Conference of the Greek Society of Biochemistry and Molecular Biology, Thessaloniki, 28-30 November 2014

“Immunity and energy metabolism genes of the olive fly are implicated in spinosad resistance”.

E. Sagri1, M. Reczko2, M-E Gregoriou1, K.T. Tsoumani1, N Zygouridis1, K.D. Salpea2, F.G. Zalom3, J. Ragoussis2 and K.D. Mathiopoulos1

The olive fruit fly, Bactroceraoleae, is the most important enemy of the olive tree. Most control efforts are based on chemical insecticides, mainly organophosphates and pyrethroides. Recently, the use of the naturalytespinosad has been introduced against the olive fly. As expected, its use is gradually leading to resistance development, as has been documented in California. Mutations in the α6 subunit of the nicotinic acetylcholine receptor (nAChR) have been implicated in spinosad resistance in several insect species (e.g., Drosophila melanogaster) but excluded in others (e.g., Muscadomestica). However, more mechanisms involving enhanced metabolism of detoxification enzymes have also been reported. In order to clarify the spinosad resistance mechanisms in the olive fly we searched for mutations in the nAChR-α6 subunit, as well as looked for up-regulated genes in the entire transcriptome of spinosad resistant olive flies. The differences in the nAChR-α6 subunit reflected mainly silent nucleotide substitutions or conserved amino acid changes. Instead, the comparison of the entire transcriptomes of over of 13,000 genes of a sensitive and a resistant to spinosad strain resulted in nine over-expressed and ~40 under-expressed loci. Further functional analyses supported the notion that the immune system as well as energy related genes of the resistant flies are involved in the detoxification process.

65th National Conference of the Greek Society of Biochemistry and Molecular Biology, Thessaloniki, 28th - 30th November 2014

“Does the reproductive system “smell”?

M-E Gregoriou1, S. Tastsoglou1, K. Athanasiadis1, E. Sagri1, J. Ragoussis2 and K.D. Mathiopoulos1

1Department of Biochemistry and Biotechnology, University of Thessaly, Greece; 2Department of Human Genetics, McGill University, Montreal, QC, Canada

Insect odorant receptors (ORs) form a receptor system that consists of the actual receptor protein and an ion channel. After an odor molecule binds an Odorant Binding Protein (OBP), a receptor protein and the ion channel trigger the neural electrical response. OR expression in olfactory tissues is obvious and well-established. Interestingly, expression of ORs has also been documented in non-olfactory tissues. Specifically, OR expression has been documented in human and mouse germ cells, in mosquito sperm and in male-specific serum proteins of Ceratitiscapitata. We opted to explore the expression of various olfactory-related genes in the reproductive system of the olive fly, Bactroceraoleae, the major pest of the olive tree. Olfactory-related genes were present in the annotated list that resulted from the transcriptome analysis of male and female reproductive tissues. Nine of them presented various levels of overexpression in the male tissues, whereas the three others in the female. In order to get a deeper insight, the relative expression of five of these genes (obp83a, obp19a, os-d, obp8a, or10) was further analyzed in female accessory glands/spermathecae, male testes and male accessory glands, before and twelve hours after mating. The presence of these OBPs in the reproductive tissues demonstrates that OBPs are not restricted to olfaction and might have general carrier capabilities with a broad specificity for lipophilic compounds; thus, they could be involved in broader physiological functions.