Molecular analysis of the fungal microbiome associated with the olive fruit fly Bactrocera oleae

A molecular approach was used to investigate the fungal microbiome associated with Bactrocera oleae a major key pest of Olea europea, using the ITS2 region of the ribosomal DNA (rDNA) as barcode gene. Amplicons were cloned and a representative number of sequenced fragments were used as barcode genes for the identification of fungi. The analysis of the detected sequence types (STs) enabled the identification of a total of 34 phylotypes which were associated with 10 fungal species, 3 species complexes and 8 genera. Three phylotypes remained unresolved within the order Saccharomycetales and the phylum Ascomycota because of the lack of closely related sequences in GenBank. Cladosporium was the most abundantly detected genus, followed by Alternaria and Aureobasidium, well-known components of olive sooty moulds. Interestingly, Colletotrichum sp. and other fungal plant pathogens were also detected, leading to potential new insights into heir epidemiology.     

Polymorphic microsatellite markers in the olive fly,Bactrocera oleae

Bactrocera oleae is the single most important insect pest of the olive fruit, causing extensive of the losses in the olive production annually. Nonetheless, there has never been an analysis of the homogeneity of B. oleae populations, the extent of gene flow, the genetic differentiation and/or reproductive isolation of marginal populations or the nature of the invasive populations in newly established olive tree cultivations. Here we describe the development of 10 novel polymorphic microsatellite markers that can be used in the analysis of natural B. oleae populations as well as to create a basis for its genome analysis.     

Targeted somatic mutagenesis through CRISPR/Cas9 ribonucleoprotein complexes in the olive fruit fly,Bactrocera oleae

The olive fruit fly, Bactrocera oleae (Diptera: Tephritidae), is the most destructive insect pest of olive cultivation, causing significant economic and production losses. Here, we present the establishment of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 methodology for gene disruption in this species. We performed targeted mutagenesis of the autosomal gene white (Bo-we), by injecting into early embryos in vitro preassembled and solubilized Cas9 ribonucleoprotein complexes loaded with two gene-specific single-guide RNAs. Gene disruption of Bo-we led to somatic mosaicism of the adult eye color. Large eye patches or even an entire eye lost the iridescent reddish color, indicating the successful biallelic mutagenesis in somatic cells. Cas9 induced either indels in each of the two simultaneously targeted Bo-we sites or a large deletion of the intervening region. This study demonstrates the first efficient implementation of the CRISPR/Cas9 technology in the olive fly, providing new opportunities towards the development of novel genetic tools for its control.     

Genetic and cytogenetic analysis of the olive fruit fly Bactrocera oleae (Diptera: Tephritidae)

The genetic and cytogenetic characteristics of one of the major agricultural pests, the olive fruit fly Bactrocera oleae, are presented here. The mitotic metaphase complement of this insect consists of six pairs of chromosomes including one pair of heteromorphic sex chromosomes, with the male being the heterogametic sex. The analysis of the polytene complements of three larval tissues, the fat body, the salivary glands and the Malpighian tubules of this pest has shown (a) a total number of five long chromosomes (10 polytene arms) that correspond to the five autosomes of the mitotic nuclei and a heterochromatic mass corresponding to the sex chromosomes, (b) the constancy of the banding pattern of the three somatic tissues, (c) the absence of a typical chromocenter as an accumulation of heterochromatin, (d) the existence of reverse tandem duplications, and (e) the presence of toroid tips of the chromosome arms. The in situhybridization of genes or DNA sequences to the salivary gland polytene chromosomes of B. oleaeprovided molecular markers for all five autosomes and permitted the establishment of chromosomal homologies among B. olea, B. tryoniand Ceratitis capitata. The heat shock response of B. oleae, as revealed by heat-inducible puffing and protein pattern, shows a higher thermotolerance than Drosophila melanogaster.     

Effect of acetone feeding on alcohol dehydrogenase activity in the olive fruit fly,Bactrocera oleae

The purpose of this study is to demonstrate a clear connection between the presence of acetone in larval diet and alcohol dehydrogenase (ADH) activity in laboratory raised populations of Bactrocera oleae. ADH activity of B. oleae is depressed in acetone-impregnated diets. At the same time the change of activity is accompanied by a change in the relative proportions of the multiple forms of ADH. The bulk of activity in the most cathodally migrating form is lost, and all the activity becomes localized in the less cathodally migrating forms of the enzyme. Moreover, ADH activity, expressed in vivo, appears to drop after exposure to acetone, as shown by the fact that larvae become less sensitive to pentenol poisoning. Our results show clear selective differences imposed by acetone on three homozygous genotypes involving the ADH alleles F, S and I in B. oleae. The directions of these differences were found to vary with the fitness component under test. Acetone treatment seems to affect developmental time and larva's viability as well as allele frequencies of ADH under artificial rearing. The effect of acetone on the maintenance of ADH polymorphism in artificially reared populations of B. oleae is further discussed.     

Relation of fruit color,elongation, hardness,and volume to the infestation of olive cultivars by the olive fruit fly,Bactrocera oleae

The susceptibility of olive cultivars to the olive fruit fly, Bactrocera oleae (Rossi) (Diptera: Tephritidae), has seldom been studied. This article examines factors associated with olive fruit fly infestation of 16 commonly planted Sicilian olive cultivars. Total infestation data were simultaneously correlated with categorical and quantitative factors using ordinal logistic regression. When all factors were included in the analysis, year, sampling date, cultivar, and fruit color were highly significant, but the quantitative factors fruit volume, fruit elongation, and fruit hardness were not. When the analysis was repeated excluding cultivar, all quantitative factors were significant, and elongation and volume were highly significant. Spherical, large, and hard fruit seemed to be preferred by B. oleae over fruit that are elongate, small, and soft. Therefore, fruit color, elongation, volume, and hardness provide useful information regarding the susceptibility of cultivars. In both organic and conventional olive cultivation, information about olive cultivar susceptibility to olive fruit fly will help orchard managers to produce quality oil and table olives while reducing treatments for olive fruit fly control.     

Spatio‐temporal analysis of the relationship between landscape structure and the olive fruit fly Bactrocera oleae (Diptera: Tephritidae)

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Genetic population structure in olive fly Bactrocera oleae (Gmelin): gene flow and patterns of geographic differentiation

Four wild Spanish populations of Bactrocera oleae (Gmelin) (Dipt., Tephritidae) were studied by gel electrophoresis, in order to find out their population genetic structure and the relative importance of gene flow in that structure. A great amount of genetic variability was observed. In terms of the pattern of the genetic variability in our populations, the most noticeable characteristics were the similarity of the configuration of allelic frequencies from locality to locality, the existence of exceptions and quantitative differences within that similarity, and the heterogeneity among the variable loci. Gene flow estimated from Wright's method gave a value of Nm = 8.9. However, the F _ST statistic in three of the loci ranged from 0.0451 to 0.0620 indicating a substantial degree of differentiation among populations (P < 0.001). The present observations seem to support the hypothesis that although gene flow could be high ( Nm = 8.9), 'natural' selection, probably due to agricultural practices, may be the major factor responsible for the pattern of the genetic variability observed in B. oleae .     

The olfactory circuit of the fruit fly Drosophila melanogaster

The olfactory circuit of the fruit fly Drosophila melanogaster has emerged in recent years as an excellent paradigm for studying the principles and mechanisms of information processing in neuronal circuits. We discuss here the organizational principles of the olfactory circuit that make it an attractive model for experimental manipulations, the lessons that have been learned, and future challenges.     

Mitochondrial haplotypes reveal olive fly (Bactrocera oleae) population substructure in the Mediterranean

The olive fly (Bactrocera oleae) is the most important olive tree (Olea europaea) pest. In the Mediterranean basin, where 98?% of its main hosts are concentrated, it causes major agricultural losses, due to its negative effect on production and quality of both olive and olive oil. Previous phylogeographic analyses have established that Mediterranean olive fly populations are distinct from other Old World populations, but did not agree on the specific population substructure within this region. In order to achieve a higher resolution of the diversity of olive fly populations, particularly in Central and Western Mediterranean (home to 70?% of the world production), we comparatively analyzed a set of samples from Portugal in the context of published mitochondrial sequences across the species' worldwide range. Strong evidence of population substructure was found in the Central and Western Mediterranean area, with two clearly separate phylogenetic branches. Together with previously published data, our results strongly support the existence of at least three distinct Mediterranean populations of the olive fly, raise the possibility of additional regional substructure and suggest specific avenues for future research. This knowledge can be instrumental in the development of better management and control strategies for a major pest of Mediterranean agriculture.     

Transinfection of the olive fruit fly Bactrocera oleae with Wolbachia: towards a symbiont‐based population control strategy

The olive fruit fly Bactrocera oleae is responsible for worldwide economic damage. In this report, we describe the first B. oleae lines transinfected with the Wolbachia strain wCer2, an endosymbiont of the cherry fruit fly Rhagoletis cerasi. Immunostaining followed by confocal microscopy, detects high numbers of Wolbachia in embryos as well as in ovarioles and sperm from individuals of both transinfected lines. wCer2 was uniformly distributed in B. oleae egg chambers and the cortex of preblastoderm embryos. Wolbachia is known to manipulate host reproduction with several strategies, one of which is cytoplasmic incompatibility (CI), resulting in embryonic mortality in incompatible crosses. Wolbachia was found to induce complete CI in the novel host, suggesting that symbiont‐based approaches can be used as novel environmentally friendly tools for the control of natural olive fruit fly populations.     

Cloning and structural characterization of the 6-phosphogluconate dehydrogenase locus of the medfly Ceratitis capitata and the olive fruit fly Bactrocera oleae

The pentose phosphate cycle is considered as a major source of NADPH and pentose needed for nucleic acid biosynthesis. 6-Phosphogluconate dehydrogenase (6PGD), an enzyme participating in this cycle, catalyzes the oxidative decarboxylation of 6PGD to ribulose 5-phosphate with the subsequent release of CO(2) and the reduction of NADP. We have determined the genomic sequences of 6PGD of two species of Tephritidae, the medfly Ceratitis capitata and olive fruit fly Bactrocera oleae, and constructed a three-dimensional model of 6PGD of C. capitata based on the homologous known sheep structure. In a comparative study of 6PGD sequences from seven species, all the conserved and variable sites of the enzyme were analyzed and the regions of functional importance were localized, an attempt promoted also by the direct involvement of the enzyme in various human diseases. The enzymes between the two species of Tephritidae have a very high homology and further examination of the variable positions with respect to the highly conserved binding site residues enabled their grouping in three distinct categories, with possible association to dimer formation, functional specificity, and antigenicity. Moreover, placement of sequence differences on the 3-D model suggests probable sites accommodating variations appearing at the allozymic variants of both species.     

A kaolin‐based particle film for suppression of the olive fruit fly Bactrocera oleae Gmelin (Dip., Tephritidae) in olive groves

The efficacy of a kaolin‐based particle film formulation M‐99‐099 to control olive fruit fly, Bactrocera oleae Gmelin, field infestations was investigated in north‐western Syria. The results showed that fruit infestation levels were significantly reduced on kaolin‐treated trees compared with untreated trees. Kaolin particle film successfully suppressed B. oleae populations and provided season‐long insect control (>14 weeks) whereas the insecticide dimethoate failed to protect olives for as long a period after the last spray. Consistent with previous findings, the M‐99‐099 kaolin particle film proved to be a promising alternative method to synthetic insecticides and could be used to control B. oleae in olive groves.     

Geographical distribution and evolutionary history of organophosphate-resistant Ace alleles in the olive fly (Bactrocera oleae)

Acetylcholinesterase (Ace) is the molecular target of organophosphate (OP) insecticides, and two mutations that confer different levels of OP insensitivity have previously been identified in the olive fly, Bactrocera oleae. Numerous sensitive and two insensitive alleles (including one convergent acquisition) are described from the entire worldwide distribution of the fly. Most of the variation is harbored in the native range of the species and in the Middle East and consists of numerous low-frequency sensitive alleles. The insensitive alleles likely came to high frequency more recently in the Mediterranean region or in the Middle East, reaching frequencies as high as 100% in some populations, and determined a corresponding decline in overall genetic variation. We hypothesize that the major force that shaped the current distribution of resistant and non-resistant acetylcholinesterase alleles is natural selection, likely responsible for the high frequency of insensitive alleles in areas where organophosphates have been used extensively. We also discuss a role for historical contingency, that can explain why sensitive alleles are absent altogether in the species ancestral range and present in areas of recent expansion, such as California, despite the limited use of OPs.     

Effect of the presence of olive fruit on ovarian maturation in the olive fruit fly, Dacus oleae, under laboratory conditions

Ovarian maturation was prevented in 7-, 22-, 23- and 28-day-old females of Dacus oleae (Gmelin) (Diptera: Tephritidae), developed in the pre-imaginal stages at LD 12:12, 19±1°C and kept as adults at LD 16:8, 26±1°C without access to olive fruits. In females of the above ages having continuous access to olive fruits and held under the same photoperiod and temperature conditions, 54, 87 and 100%, respectively, had mature oocytes. When the females had access to domes of paraffin wax, the percentages of females with mature oocytes were intermediate between those with and those without access to olive fruits. Under the above photoperiod and temperature conditions unfavorable for maturation, 50% of 4-week-old females had mature oocytes if exposed for one week to olive fruits during their first week and 91% if exposed during their 4th week of adult life. The respective percentages with wax domes in the cages were again intermediate between those with olives and those without olives or wax domes. The presence of olive fruits had also a strong positive effect on the ovarian maturation of females which developed from egg through the adult stage at LD 16:8, 26±1°C, a condition favoring ovarian maturation. Access to wax domes had again an intermediate effect on maturation. It is concluded therefore that the lack of ovarian maturation of D. oleae females which is observed under a short photophase during the pre-imaginal stages if followed by a long photophase and an increase of temperature during the adult stage, continues at least till the end of the 4th week of adult life in the absence of olive fruits but is averted when such fruits are offered to the adult flies.

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Résumé La maturation ovarienne des femelles adultes agées de 7, 22–23 et 28 jours de D. oleae ayant accompli leur développement embryonnaire, larvaire et nymphal en LD 12:12, 19±1°C et maintenues en LD 16:8, 26±1°C, sans accès à des olives, est inhibée. Dans les mêmes conditions, mais avec accès permanent à des olives, 54% des femelles ont des ovocytes mûrs à 7 jours, 87% à 22–23 jours et 100% à 28 jours. Avec des femelles ayant accès à des domes de paraffine, les pourcentages d'individus ayant des ovocytes mûrs sont intermédiaires entre ceux des deux situations précédentes. Dans les conditions cidessus défavorables à la maturation ovarienne, 50% des femelles de 28 jours ont des ovocytes mûrs si elles sont en présence d'olives durant leur première semaine de vie imaginale et 91% si elles sont en présence la 4éme semaine. Les pourcentages respectifs concernant les femelles pourvues de dômes de paraffine sont encore intermédiaires entre ceux des femelles avec olives et ceux de femelles privées de tout substrat. La présence d'olives a aussi un effet fortement positif sur la maturation ovarienne de femelles ayant accompli leur développement pre-imaginal et maintenues aussi à l'etat adulte en conditions LD 16:8, 26±1°C, favorisant la maturation ovarienne. L'accès à des dômes de paraffine a, là encore, un effet intermédiaire. On conclut par conséquent que la non maturation ovarienne des femelles de D. oleae qui est causée par une photopériode à jour court pendant la vie pre-imaginale accompagné par une photopériode à jour long et une augmentation de la température pendant la vie imaginale persiste au moins jusqu'à la fin de la quatrième semaine de la vie imaginale en absence d'olives, mais ne se produit pas si on met les femelles adultes en présence d'olives.

     

Population structure and colonization history of the olive fly, Bactrocera oleae (Diptera, Tephritidae)

The olive fly, Bactrocera oleae, is the major pest of olives in most commercial olive-growing regions worldwide. The species is abundant in the Mediterranean basin and has been introduced recently into California and Mexico, creating problems for quarantine protection and international trade. Here, we use nuclear microsatellite markers and mitochondrial sequences to examine the history of olive fly range expansion and colonization. Sampled populations span the current distribution of the olive fly worldwide, including South and Central Africa, Pakistan, Mediterranean Europe and Middle East, California, and Mexico. The Pakistani populations appear to be genetically well differentiated from the remaining populations, though rooting the origins of the species is problematic. Genetic similarity and assignment tests cluster the remaining populations into two genetic groups--Africa and a group including the Mediterranean basin and the American region. That Africa, and not the Mediterranean, is the origin of flies infesting cultivated olive is supported by the significantly greater genetic diversity at microsatellite loci in Africa relative to the Mediterranean area. The results also indicate that the recent invasion of olive flies in the American region most likely originated from the Mediterranean area.     

Cation-distribution in developing follicles of the fruit fly Drosophila melanogaster

Abstract. Cations were precipitated with potassium antimonate in ovarian follicles of Drosophila and the distribution of the formed precipitates was studied. The precipitates were analyzed with a laser microprobe mass analyzer (LAMMA) and found to contain a high concentration of calcium; potassium and sodium were also detected. On counting the antimon precipitates in stage 10B follicles with the electron microscope, few precipitates per unit area were found in anterior nurse cells, but more in posterior nurse cells; the highest precipitate density occurred consistently in the oocyte. When follicles of different stages were compared, the precipitate density was found to increase in the ooplasm and in the posterior nurse cells during vitellogenesis, whereas it remained nearly constant in the anterior nurse cells. Thus, the ratio of precipitates between the posterior and anterior end of the follicle increases during vitellogenesis. It begins to decrease at the time when the nurse cells collapse. These results suggest that the electrical polarity observed in polytrophic ovarioles may be based on differences in the cation distribution along the antero-posterior axis of the follicle.     

Effect of pyridalyl on mortality,fecundity and physiological performance of olive fruit fly,Bactrocera oleae Rossi (Diptera: Tephritidae)

Toxicity and physiological effects of pyridalyl were studied on the adults of Bacterocera oleae via oral exposure and biochemical experiments. First, adults were fed on a protein hydrolysate diet which has been amended by five concentrations of pyridalyl. Then, another cohort of flies was fed on LC₅₀ concentration to find potential effects on fecundity, detoxifying enzymes and intermediary metabolism. Results of bioassay showed a concentration of 0.517 μg/ml as the LC₅₀ value with confidence limit of 0.305–0.791 μg/ml at 95%. Fecundity of the females fed on pyridalyl-treated diet showed no significant differences with controls following 7 days of exposure while a significant higher mortality was recorded in laid eggs by the treated females. Activities of esterase and glutathione S-transferase significantly increased in the treated adults compared to control when α-naphthyl acetate and CDNB (1-chloro-2,4-dinitrobenzene) were used as substrates. Activities of alanine- and aspartate aminotransferases significantly increased in the pyridalyl-treated adults compared to control while no significant difference was observed in activity of ɤ-glutamyl transferase. Although lactate dehydrogenase showed no statistically different activity but both acid- and alkaline phosphatases had statistically higher activity in the control adults compared to treatment. Finally, low density lipoprotein and total protein were the only non-enzymatic components which showed statistical difference between control and treated flies. Our results indicated that pyridalyl had toxicity on B. oleae and intervened in the physiological performance of flies.     

Interaction between sexes and between different circadian phenotypes affects lifespan in the fruit fly Drosophila melanogaster

Circadian clocks regulate the daily temporal structure of physiological and behavioural functions. In the fruit fly Drosophila melanogaster Meigen, disruption of daily rhythms is suggested to reduce the fly's lifespan. In the present study, because pairs of mixed‐sex flies are known to show an activity pattern different from that of individual flies, this hypothesis is tested by measuring the lifespan of flies housed same‐sexually or mixed‐sexually under an LD 12 : 12 h photocycle at a constant temperature of 25 °C. The effect of housing wild‐type (Canton‐S) flies with period (per) circadian clock mutant flies is also examined because the mutant flies have different daily activity patterns. When males and females of wild‐type flies are housed together, their lifespan is substantially lengthened (males) or shortened (females) compared with same‐sex housed flies. The shortening of the lifespan in females is significantly enhanced when mated with per mutant males. The shortening effects are significantly reduced when the mixed‐sex interaction is limited for the first 5 days after emergence. A slight elongation in lifespan, rather than a reduction, occurs when wild‐type females are housed same‐sexually with per⁰ or per^L mutant flies. In male flies, the elongation of lifespan occurs not only when wild‐type males are housed with wild‐type, per⁰ or per^L females, but also when housed with per⁰ or per^S mutant males. Mixed‐sex couples always show altered daily locomotor rhythms with an enhanced night‐time activity, whereas same‐sex couples show daily behavioural profiles slightly altered but essentially similar to a sum of the respective two flies. No significant correlation is found between the lifespan and reproductive capacity. These results suggest that the alteration of daily activity rhythms and sexual interaction may have significant impact on the fly's lifespan.