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Acetylcholinesterase (AChE), encoded by the ace gene, is a key enzyme of cholinergic neurotransmission. Insensitive acetylcholinesterase (AChE) has been shown to be responsible for resistance to OPs and CBs in a number of arthropod species, including the most important pest of olives trees, the olive fruit fly Bactrocera oleae. In this paper, the o...
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Citations
... This amino acid change was associated with a 35-40% reduction in AChE catalytic efficiency. The second one is a point mutation located in the exon IV that changes the amino acid valine for isoleucine next to the catalytic center (I214V) and reduces the insecticide's ability to disable this site [12,13]. The substitution G488S, together with the substitution I214V (I199V substitution in Drosophila, which confers low levels of OP resistance) produced up to a 16-fold decrease in insecticide sensitivity and, then, an important enhancement of insecticide resistance [12]. ...
... Finally, a deletion of three glutamines in the C-terminal of the protein (∆Q3) was described. This modification increases the affinity for the phosphatidil inositol present at the cell membranes and prolongs the presence of this enzyme in the synaptic gap [13][14][15]. Therefore, resistance of the olive fruit fly to OPs is related to three polymorphisms placed at three of the ten exons of ace gene, I214V at exon IV, G488S at exon VII and ∆Q3 at exon X [13]. ...
... This modification increases the affinity for the phosphatidil inositol present at the cell membranes and prolongs the presence of this enzyme in the synaptic gap [13][14][15]. Therefore, resistance of the olive fruit fly to OPs is related to three polymorphisms placed at three of the ten exons of ace gene, I214V at exon IV, G488S at exon VII and ∆Q3 at exon X [13]. ...
The olive fruit fly (
olf
)
Bactrocera oleae
is the most damaging olive pest. The intensive use of organophosphates (OPs) to control it, led to an increase in resistance in field populations. This study assesses the presence and distribution of three mutations at the
ace
gene related to target site insensitivity to OPs in Spain. Samples from other Mediterranean countries were included as external references. Resistance-conferring alleles (from exons IV and VII of the
ace
gene) reached almost an 80% frequency in
olf
Spanish populations. In total, 62% of them were homozygous (
RR/RR
), this being more common in eastern mainland Spain. High frequencies of
RR/RR
individuals were also found in North Mediterranean samples. Conversely, in Tunisia, only sensitive alleles were detected. Finally, the exon X mutation ∆Q3 had an extremely low frequency in all samples. The high frequency of genotype
RR/RR
in Spain indicates high fitness in an agroecosystem treated with pesticides, in contrast to ∆Q3. At exon IV all flies carried the same haplotype for the allele conferring resistance. The sequence analysis at this exon suggests a unique origin and fast expansion of the resistant allele. These results provide evidence that OPs appropriate use is needed and prompt the search for alternative methods for
olf
pest control.
... The availability of the B. oleae polytene chromosome maps enabled the in situ localization of a considerable number of molecular markers (Table 1). More specifically, 18 gene sequences (Zambetaki et al., 1999(Zambetaki et al., , 2000Kakani et al., 2012;Drosopoulou et al., 2014), two anonymous genomic clones (Zambetaki et al., 1999), 13 microsatellites , and 35 ESTs (Tsoumani et al., 2011) have provided molecular markers for all B. oleae chromosomes (nine out of 10 chromosome arms). Based on the chromosome distribution pattern of the above sequences, the correspondence of the polytene arms and the significant synteny conservation between B. oleae and C. capitata, B. tryoni (Froggatt), and D. melanogaster have been revealed (Zambetaki et al., 1999(Zambetaki et al., , 2000Tsoumani et al., 2011;Drosopoulou et al., 2014) (Tables 2 and 3). ...
Cytogenetics of Tephritidae has contributed for more than 30 years to the efficient control of pest members of this family and the species delimitation among them. The sterile insect technique (SIT) is a species-specific and environmentally friendly method for the population control of insect pest species, always as a component of integrated pest management approaches. The construction of polytene chromosome maps facilitated the development, characterization, and improvement of genetic sexing strains, which have boosted the effectiveness of SIT and are considered as models for developing genetic sexing methodologies in other target species. In parallel, characterization of mitotic karyotypes and the availability of polytene chromosomes in Diptera have been and are being used as supplementary evidence in taxonomic studies. Lately, under the common understanding that speciation can be achieved via various pathways and under the umbrella of integrative taxonomy, cytogenetic studies have contributed to multidisciplinary taxonomic approaches. Such studies can shed light on the borders between closely related species and/or incipient speciation, and this is crucial both for the implementation of SIT and the revision/improvement of quarantine policies. In this review, we summarize the cytogenetic status of Tephritidae and discuss the contribution of cytogenetics to the development of genetic sexing strains in this family.
... Preselected library fractions of an adult olive fly library in λ DASH II [41,42] was screened with a probe containing an amplified Achilles-fragment. This PCR fragment (Achill400), targeting part of the gag gene of Achilles (S1 Table), was 338 bp in length and further labeled with biotin-11-dUTP using a random primer DNA labeling kit (Fermentas, Burlington, Canada). ...
Sex chromosomes have many unusual features relative to autosomes. The in depth exploration of their structure will improve our understanding of their origin and divergence (degeneration) as well as the evolution of genetic sex determination pathways which, most often are attributed to them. In Tephritids, the structure of Y chromosome, where the male-determining factor M is localized, is largely unexplored and limited data concerning its sequence content and evolution are available. In order to get insight into the structure and organization of the Y chromosome of the major olive insect pest, the olive fly Bactrocera oleae, we characterized sequences from a Pulse Field Gel Electrophoresis (PFGE)-isolated Y chromosome. Here, we report the discovery of the first olive fly LTR retrotransposon with increased presence on the Y chromosome. The element belongs to the BEL-Pao superfamily, however, its sequence comparison with the other members of the superfamily suggests that it constitutes a new family that we termed Achilles. Its ~7.5 kb sequence consists of the 5'LTR, the 5'non-coding sequence and the open reading frame (ORF), which encodes the polyprotein Gag-Pol. In situ hybridization to the B. oleae polytene chromosomes showed that Achilles is distributed in discrete bands dispersed on all five autosomes, in all centromeric regions and in the granular heterochromatic network corresponding to the mitotic sex chromosomes. The between sexes comparison revealed a variation in Achilles copy number, with male flies possessing 5-10 copies more than female (CI range: 18-38 and 12-33 copies respectively per genome). The examination of its transcriptional activity demonstrated the presence of at least one intact active copy in the genome, showing a differential level of expression between sexes as well as during embryonic development. The higher expression was detected in male germline tissues (testes). Moreover, the presence of Achilles-like elements in different species of the Tephritidae family suggests an ancient origin of this element.
... The polytene complement consists of five polytene chromosomes, corresponding to the submetacentric autosomes of the mitotic nuclei, and a heterochromatic mass corresponding to the sex chromosomes (Zambetaki et al. 1995;Mavragani-Tsipidou 2002, Drosopoulou et al. 2012. The availability of well-characterized cytological maps enabled the exact localization of nine gene (Zambetaki et al. 1999(Zambetaki et al. , 2000Kakani et al. 2013), 13 microsatellite (Augustinos et al. 2008), and 35 EST markers (Tsoumani et al. 2011) by in situ hybridization on the polytene chromosomes of B. oleae. ...
... The above data doubles the number of the mapped gene probes on B. oleae polytene complement and further supports the proposed chromosomal homologies among B. oleae, Ceratitis capitata, and Drosophila melanogaster, which have been based on banding pattern similarities and molecular marker localization data Scott et al. 1993;Zhao et al. 1998;Zambetaki et al. 1999;Gariou-Papalexiou et al. 2002;Mavragani-Tsipidou 2002, Tsoumani et al. 2011. Distribution of gene markers on the salivary gland polytene chromosome arms of the above mentioned species are given in Table 2 and Fig. 2 (data derived from Banks et al. 1995;Zwiebel et al. 1995;Papadimitriou et al. 1998;Zambetaki et al. 1999Zambetaki et al. , 2000Gariou-Papalexiou et al. 2002;Theodoraki and Mintzas 2006;Kakani et al. 2013;St. Pierre et al. 2014;present report). ...
... Solid lines connect the relative positions of the markers localized in B. oleae in the present study. Dashed lines connect the relative positions of the markers localized in B. oleae previously (Zambetaki et al. 1999(Zambetaki et al. , 2000Kakani et al. 2013). C in grey circles indicates the centromeres. ...
Four homologous and five heterologous gene-specific sequences have been mapped by in situ hybridization on the salivary gland polytene chromosomes of the olive fruit fly, Bactrocera oleae. The nine genes were dispersed on four of the five autosomal chromosomes, thus enriching the available set of chromosome landmarks for this major agricultural pest. Present data further supports the proposed chromosome homologies among B. oleae, Ceratitis capitata, and Drosophila melanogaster and the idea of the conservation of chromosomal element identity throughout dipteran evolution.
... Data on haplotype frequencies and distributions at the Ace locus was also obtained. Unfortunately, the chromosomal distance between the two substitutions screened for (at least 13 kb, and likely >16 kb; Kakani et al., 2013) and the lack of genomic sequence information made it unpractical to ascertain the gametic phase for all individuals. Nevertheless, it could be inferred for homozygous individuals in at least one of the two sites, representing 52% of the Iberian flies tested (68/130). ...
The olive fly, Bactrocera oleae (Rossi, 1790) (Diptera: Tephritidae), is the most important pest of olive trees globally, causing
losses that, in the absence of control measures, can exceed 90% of the crop. In the Mediterranean basin, where the overwhelming
majority of production is concentrated (~ 98%), organophosphate insecticides (OPs) have been the main tool for B. oleae control for
the last four decades, leading to the development of resistance to these compounds. Mutations of the Ace gene, which codes for acetylcholinesterase,
the target enzyme of OPs and other insecticides, have been identified as the underlying cause, with studies reporting
mid to very high frequencies of resistance alleles in several countries. Interestingly, no resistance alleles were detected in Portugal, at
the Western end of the Mediterranean basin. As the original study was done almost a decade ago and did not include many samples, we
decided to re-evaluate the situation, by analysing a larger number of individuals from multiple locations in Western and Southern Iberia
(Portugal and Spain). In our present study, resistance-associated Ace alleles were found to have become widespread in both regions,
but with highly varying frequencies. Together with the observed haplotype distributions, this finding is consistent with previous suggestions
of a recent, selection-driven spread and has implications for the importance of Ace mutations in organophosphate resistance in
the field as well as the importance of gene flow between Mediterranean populations of B. oleae.
... Screening of an adult olive fly library in l DASH II [21] was performed on preselected library fractions, as described in [22]. The probe used for the screening was a ,400 bp PCR product of a retrotransposon fragment (Tsoumani et al. unpublished data) after labeling with 11-dUTP-biotin by random priming (DecaLabel TM DNA Labeling Kit, Fermentas, Burlington, Canada) at a hybridization temperature of 65uC. ...
Satellite repetitive sequences that accumulate in the heterochromatin consist a large fraction of a genome and due to their properties are suggested to be implicated in centromere function. Current knowledge of heterochromatic regions of Bactrocera oleae genome, the major pest of the olive tree, is practically nonexistent. In our effort to explore the repetitive DNA portion of B. oleae genome, a novel satellite sequence designated BoR300 was isolated and cloned. The present study describes the genomic organization, abundance and chromosomal distribution of BoR300 which is organized in tandem, forming arrays of 298 bp-long monomers. Sequence analysis showed an AT content of 60.4%, a CENP-B like-motif and a high curvature value based on predictive models. Comparative analysis among randomly selected monomers demonstrated a high degree of sequence homogeneity (88% - 97%) of BoR300 repeats, which are present at approximately 3,000 copies per haploid genome accounting for about 0.28% of the total genomic DNA, based on two independent qPCR approaches. In addition, expression of the repeat was also confirmed through RT-PCR, by which BoR300 transcripts were detected in both sexes. Fluorescence in situ hybridization (FISH) of BoR300 on mitotic metaphases and polytene chromosomes revealed signals to the centromeres of two out of the six chromosomes which indicated a chromosome-specific centromeric localization. Moreover, BoR300 is not conserved in the closely related Bactrocera species tested and it is also absent in other dipterans, but it's rather restricted to the B. oleae genome. This feature of species-specificity attributed to BoR300 satellite makes it a good candidate as an identification probe of the insect among its relatives at early development stages.
... is located at the synapses of cholinergic neurons in the central and peripheral nervous systems in all animals. It is essential for catalyzing the hydrolysis of the neurotransmitter acetylcholine (ACh) and terminating neurotransmission [1,2]. Based on the strategy of inhibiting AChE, both organophosphate and carbamate insecticides have been developed to control various insect species such as the diamondback moth (DBM), Plutella xylostella (Lepidoptera: Plutellidae) [3,4]. ...
Background:
Over the last 60 years, synthetic chemical pesticides have served as a main tactic in the field of crop protection, but their availability is now declining as a result of the development of insect resistance. Therefore, alternative pest management agents are needed. However, the demonstration of RNAi gene silencing in insects and its successful usage in disrupting the expression of vital genes opened a door to the development of a variety of novel, environmentally sound approaches for insect pest management.
Methodology/principal findings:
Six small interfering RNAs (siRNAs) were chemically synthesized and modified according to the cDNA sequence of P. xylostella acetylcholine esterase genes AChE1 and AChE2. All of them were formulated and used in insecticide activity screening against P. xylostella. Bioassay data suggested that Si-ace1_003 and Si-ace2_001 at a concentration of 3 µg cm(-2) displayed the best insecticidal activity with 73.7% and 89.0%, mortality, respectively. Additional bioassays were used to obtain the acute lethal concentrations of LC50 and LC90 for Si-ace2_001, which were 53.66 µg/ml and 759.71 µg/ml, respectively. Quantitative Real-time PCR was used to confirm silencing and detected that the transcript levels of P. xylostella AChE2 (PxAChE2) were reduced by 5.7-fold compared to the control group. Consequently, AChE activity was also reduced by 1.7-fold. Finally, effects of the siRNAs on treated plants of Brassica oleracea and Brassica alboglabra were investigated with different siRNA doses. Our results showed that Si-ace2_001 had no negative effects on plant morphology, color and growth of vein under our experimental conditions.
Conclusions:
The most important finding of this study is the discovery that chemically synthesized and modified siRNA corresponding to P. xylostella AChE genes cause significant mortality of the insect both under laboratory and field conditions, which provides a novel strategy to control P. xylostella and to develop bio-pesticides based on the RNA interference technology.
... We have purified several batches of the AChE2 at various ratios of intact and cleaved forms and found that the same amount of the cleaved AChE2 hydrolyzed ATC at a rate ~20% higher than the intact one did (data not shown). In D. melanogaster, Bactrocera oleae and Musca domestica, the orthologous AChEs were partially cleaved at a similar position (Kakani et al., 2012;Harel et al., 2000;Mutero and Fournier, 1992;Haas et al., 1988;Gnagey et al., 1987;Steele and Smallman, 1976). Interestingly, although the insert sequence is hypervariable, the cleavage site sequence (RHGR*GLN) is nearly identical to RHGRGAN in the fly AChEs. ...
Acetylcholinesterases (AChEs) catalyze the hydrolysis of acetylcholine, a neurotransmitter for cholinergic neurotransmission in animals. Most insects studied so far possess two AChE genes: ace-1 paralogous and ace-2 orthologous to Drosophila melanogaster ace. We characterized the catalytic domain of Anopheles gambiae AChE1 in a previous study (Jiang et al., 2009) and report here biochemical properties of A. gambiae AChE2 expressed in Sf9 cells. An unknown protease in the expression system cleaved the recombinant AChE2 next to Arg(110), yielding two non-covalently associated polypeptides. A mixture of the intact and cleaved AChE2 had a specific activity of 72.3 U/mg, much lower than that of A. gambiae AChE1 (523 U/mg). The order of V(max)/K(M) values for the model substrates was acetylthiocholine > propionylthiocholine ≈ acetyl-(β-methyl)thiocholine > butyrylthiocholine. The IC(50)'s for eserine, carbaryl, BW284C51, paraoxon and malaoxon were 1.32, 13.6, 26.8, 192 and 294 nM, respectively. A. gambiae AChE2 bound eserine and carbaryl stronger than paraoxon and malaoxon, whereas eserine and malaoxon modified the active site Ser(232) faster than carbaryl or paraoxon did. Consequently, the k(i)'s were 1.173, 0.245, 0.029 and 0.018 μM(-1)min(-1) for eserine, carbaryl, paraoxon and malaoxon, respectively. Quantitative polymerase chain reactions showed a similar pattern of ace-1 and ace-2 expression. Their mRNAs were abundant in early embryos, greatly decreased in late embryos, larvae, pupae, and pharate adult, and became abundant again in adults. Both transcripts were higher in head and abdomen than thorax of adults and higher in male than female mosquitos. Transcript levels of ace-1 were 1.9- to 361.8-fold higher than those of ace-2, depending on developmental stages and body parts. Cross-reacting polyclonal antibodies detected AChEs in adult brains, thoracic ganglia, and genital/rectal area. Activity assays, immunoblotting, and tandem mass spectrometric analysis indicated that A. gambiae AChE1 is responsible for most of acetylthiocholine hydrolysis in the head extracts. Taken together, these data indicate that A. gambiae AChE2 may play a less significant role than AChE1 does in the mosquito nervous system.
