Automated image-based analysis unveils acute effects due to sub-lethal pesticide doses exposure.

Pesticides are still abused in modern agriculture. The effects of their exposure to even sub-lethal doses can be detrimental to ecosystem stability and human health. This work aims to validate the use of machine learning techniques for recognizing motor abnormalities and to assess any effect post-exposure to a minimal dosage of these substances on a model organism, gaining insights into potential risks for human health. The test subject was the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), exposed to food contaminated with the LC30 of Carlina acaulis essential oil. A deep learning approach enabled the pose estimation within an arena. Statistical analysis highlighted the most significant features between treated and untreated groups. Based on this analysis, two learning-based algorithms, Random Forest (RF) and XGBoost were employed. The results were compared through different metrics. RF algorithm generated a model capable of distinguishing treated subjects with an area under the receiver operating characteristic curve of 0.75 and an accuracy of 0.71. Through an image-based analysis, this study revealed acute effects due to minimal pesticide doses. So, even small amounts of these biocides drifted far from distribution areas may negatively affect the environment and humans.

Chemical Composition of Essential Oils from Leaves and Fruits of Juniperus foetidissima and Their Attractancy and Toxicity to Two Economically Important Tephritid Fruit Fly Species, Ceratitis capitata and Anastrepha suspensa.

The present study analyzed the chemical composition of Juniperus foetidissima Willd. essential oils (EOs) and evaluated their attractancy and toxicity to two agriculturally important tephritid fruit flies. The composition of hydrodistilled EOs obtained from leaves (JFLEO) and fruits (JFFEO) of J. foetidissima was analyzed by GC-FID and GC-MS. The main compounds were α-pinene (45%) and cedrol (18%) in the JFLEO and α-pinene (42%), α-thujone (12%), and ß-thujone (25%) in the JFFEO. In behavioral bioassays of the male Mediterranean fruit fly, Ceratitis capitata (Wiedemann), both JFLEO and JFFEO showed strong attraction comparable to that observed with two positive controls, Melaleuca alternifolia and Tetradenia riparia EOs. In topical bioassays of the female Caribbean fruit fly, Anastrepha suspensa (Loew), the toxicity of JFFEO was two-fold higher than that of JFLEO, with the LD50 values being 10.46 and 22.07 µg/µL, respectively. This could be due to differences in chemical components between JFLEO and JFFEO. The JFFEO was dominated by 48% monoterpene hydrocarbons (MH) and 46% oxygenated monoterpenes (OM), while JFLEO consisted of 57% MH, 18% OM, and 20% oxygenated sesquiterpenes (OS). This is the first study to evaluate the attractancy and toxicity of J. foetidissima EOs to tephritid fruit flies. Our results indicate that JFFEO has the potential for application to the management of pest tephritid species, and further investigation is warranted.

Insecticidal and Attractant Activities of Magnolia citrata Leaf Essential Oil against Two Major Pests from Diptera: Aedes aegypti (Culicidae) and Ceratitis capitata (Tephritidae).

In this study, Magnolia citrata Noot and Chalermglin (Magnoliaceae) essential oil (MCEO) was evaluated for insecticidal activity against the yellow fever mosquito Aedes aegypti and attractant activity for the Mediterranean fruit fly Ceratitis capitata. The leaves of Magnolia citrata (Gioi chanh) were collected from northwestern Vietnam, and the water-distilled MCEO was analyzed by gas-chromatography and mass spectrometry (GC-MS). The major constituents of MCEO were identified as linalool 19%, geranial 16%, citronellal 14%, neral 14%, and sabinene 12%. MCEO showed 100% mortality at 1 µg/µL against 1st instar larvae of Ae. aegypti (Orlando strain, ORL), and the oil exhibited 54% (ORL) and 68% (Puerto Rico strain) mortality at 5 µg/mosquito against Ae. aegypti adult females. Initial screens showed that MCEO had weak insecticidal activity compared to the positive control permethrin. In bioassays with sterile male C. capitata, MCEO exhibited moderately strong attraction, comparable to that observed with a positive control, Tetradenia riparia essential oil (TREO). Herein, the insecticidal and attractant activities of MCEO are reported for the first time.

Phylogenomics Reveals that Asaia Symbionts from Insects Underwent Convergent Genome Reduction, Preserving an Insecticide-Degrading Gene.

The mosquito microbiota is composed of several lineages of microorganisms whose ecological roles and evolutionary histories have yet to be investigated in depth. Among these microorganisms, Asaia bacteria play a prominent role, given their abundance in the gut, reproductive organs, and salivary glands of different mosquito species, while their presence has also been reported in several other insects. Notably, Asaia has great potential as a tool for the control of mosquito-borne diseases. Here, we present a wide phylogenomic analysis of Asaia strains isolated from different species of mosquito vectors and from different populations of the Mediterranean fruit fly (medfly), Ceratitis capitata, an insect pest of worldwide economic importance. We show that phylogenetically distant lineages of Asaia experienced independent genome reductions, despite following a common pattern, characterized by the early loss of genes involved in genome stability. This result highlights the role of specific metabolic pathways in the symbiotic relationship between Asaia and the insect host. Finally, we discovered that all but one of the Asaia strains included in the study possess the pyrethroid hydrolase gene. Phylogenetic analysis revealed that this gene is ancestral in Asaia, strongly suggesting that it played a role in the establishment of the symbiotic association between these bacteria and the mosquito hosts. We propose that this gene from the symbiont contributed to initial pyrethroid resistance in insects harboring Asaia, also considering the widespread production of pyrethrins by several plants.IMPORTANCE We have studied genome reduction within several strains of the insect symbiont Asaia isolated from different species/strains of mosquito and medfly. Phylogenetically distant strains of Asaia, despite following a common pattern involving the loss of genes related to genome stability, have undergone independent genome reductions, highlighting the peculiar role of specific metabolic pathways in the symbiotic relationship between Asaia and its host. We also show that the pyrethroid hydrolase gene is present in all the Asaia strains isolated except for the South American malaria vector Anopheles darlingi, for which resistance to pyrethroids has never been reported, suggesting a possible involvement of Asaia in determining resistance to insecticides.

TLC-Based Bioassay to Isolate Kairomones from Tea Tree Essential Oil That Attract Male Mediterranean Fruit Flies, Ceratitis capitata (Wiedemann).

The Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) poses a major threat to fruit and vegetable production in the United States and throughout the world. New attractants and detection methods could improve control strategies for this invasive pest. In this study, we developed a method that combined thin-layer chromatography (TLC) of tea tree essential oil (TTO) (Melaleuca alternifolia) with short-range bioassays to isolate attractive kairomones for male C. capitata. After development, the TLC chromatogram indicated that TTO separated into five major spots, designated as zones 1 to 5. When the TLC plate was exposed to flies, zones 1 and 3 were strongly attractive to male C. capitata. To confirm activity, the developed TLC plate was cut into five zones which were then tested in short-range bioassays. Again, flies were observed to aggregate around zones 1 and 3, which corresponded with Rf values of 0.93 and 0.59. In addition, zones 1 to 5 were separated using preparative-TLC, and olfactory responses to volatile emissions from the five fractions were quantified by electroantennography (EAG). Highest amplitude EAG responses were recorded with fractions 1 and 3, further supporting the bioactivity of these samples. In conclusion, a TLC-based bioassay system can provide an effective, rapid screening protocol for initial isolation of insect kairomones from complex mixtures such as essential oils or plant extracts. Further analysis of TTO fractions 1 and 3 is needed to identify the specific constituents attractive to male C. capitata.

Exposure to essential oils and ethanol vapors affect fecundity and survival of two frugivorous fruit fly (Diptera: Tephritidae) pest species.

Plant-derived compounds can be an environmentally friendly alternative to synthetic pesticide use for pest management. Essential oils (EOs) in several plant families have been found to be toxic to various pest species of insects through topical application, ingestion, and as fumigants. Previous studies revealed that, among various environmentally friendly insecticides, the EOs of Baccharis dracunculifolia and Pinus elliottii and an ethanol extract of Solanum granulosoleprosum plus Ricinus communis, were toxic to Ceratitis capitata and Anastrepha fraterculus (Diptera: Tephritidae) when applied topically to pupae or when ingested by adults. Here, we aimed to examine the potentially toxic effects of these plant-derived compounds when these two pestiferous fruit fly species were exposed to their vapors. We also examined their fumigant effect on female fecundity and fertility and compared it with water and ethanol controls. Exposure of C. capitata and A. fraterculus sexually mature adults to volatiles and vapors of both B. dracunculifolia and P. elliottii EOs resulted in lower longevity (half-life), survivorship, and female fecundity than the water vapor control. Toxicity of C. capitata was greater for P. elliottii than for B. dracunculifolia while the reverse was true for A. fraterculus. Exposure to vapors of S. granulosoleprosum + R. communis (S + R) had no effect on longevity but reduced survivorship of adults of both species. Interestingly, exposure to vapors of S + R, 50% (v/v) and pure ethanol resulted in greater fecundity of females of both frugivorous fly species than the water control. By contrast, fertility (% egg hatch) was in all cases high (>85%) and not different than the water control. Exposure to ethanol vapors appears to have similar effects on frugivorous tephritids as those reported on saprophagous and frugivorous species of Drosophila, a novel finding that may have important practical implications.

Effects of lipopolysaccharide and juvenile hormone III treatments on cell growth and gene expression in the Ceratitis capitata (Diptera: Tephritidae) CCE/CC128 cell line.

The Mediterranean fruit fly Ceratitis capitata is one of the most important insect pest species in the world. It has a high colonization capacity and population variety, giving it considerable genetic diversity. Strategies for its control have included the sterile insect technique and insect growth regulators. Many studies have analyzed the medfly transcriptome, and along with the medfly genome sequence, the sequences of multiple genes related to sex determination, mating, development, pheromone detection, immunity, or stress have been identified. In this study, the medfly CCE/CC128 cell line was used to assess cell growth variation and changes in the expression of genes covering different functions, after lipopolysaccharide (LPS) and juvenile hormone III (JHIII) treatments. No significant effects on cell growth and gene expression were observed in the cells treated with LPS. In the cells treated with JHIII, the results showed significant effects on cell growth, and an overexpression was found of the Shade gene, one of the Halloween gene members of the cytochrome p450 family, which is involved in development and the synthesis of 20-hydroxyecdysone. This study shows preliminary results on the insect cell line in combination with whole-genome sequencing, which can facilitate studies regarding growth, toxicity, immunity, and transcriptome regulations as a response to different compounds and environmental alterations.

Laboratory Evaluation of Natural and Synthetic Aromatic Compounds as Potential Attractants for Male Mediterranean fruit Fly, Ceratitis capitata.

Ceratitis capitata, the Mediterranean fruit fly, is one of the most serious agricultural pests worldwide responsible for significant reduction in fruit and vegetable yields. Eradication is expensive and often not feasible. Current control methods include the application of conventional insecticides, leading to pesticide resistance and unwanted environmental effects. The aim of this study was to identify potential new attractants for incorporation into more environmentally sound management programs for C. capitata. In initial binary choice bioassays against control, a series of naturally occurring plant and fungal aromatic compounds and their related analogs were screened, identifying phenyllactic acid (7), estragole (24), o-eugenol (21), and 2-allylphenol (23) as promising attractants for male C. capitata. Subsequent binary choice tests evaluated five semisynthetic derivatives prepared from 2-allylphenol, but none of these were as attractive as 2-allylphenol. In binary choice bioassays with the four most attractive compounds, males were more attracted to o-eugenol (21) than to estragole (24), 2-allylphenol (23), or phenyllactic acid (7). In addition, electroantennography (EAG) was used to quantify antennal olfactory responses to the individual compounds (1-29), and the strongest EAG responses were elicited by 1-allyl-4-(trifluoromethyl)benzene (11), estragole (24), 4-allyltoluene (14), trans-anethole (9), o-eugenol (21), and 2-allylphenol (23). The compounds evaluated in the current investigation provide insight into chemical structure-function relationships and help direct future efforts in the development of improved attractants for the detection and control of invasive C. capitata.

Functional characterization of CYP6A51, a cytochrome P450 associated with pyrethroid resistance in the Mediterranean fruit fly Ceratitis capitata.

Overexpression of the cytochrome P450 monooxygenase CYP6A51 has been previously associated with pyrethroid resistance in the Mediterranean fruit fly (medfly) Ceratitis capitata, an important pest species worldwide; however, this association has not been functionally validated. We expressed CYP6A51 gene in Escherichia coli and produced a functional enzyme with preference for the chemiluminescent substrate Luciferin-ME EGE. In vitro metabolism assays revealed that CYP6A51 is capable of metabolizing two insecticides that share the same mode of action, λ-cyhalothrin and deltamethrin, whereas no metabolism or substrate depletion was observed in the presence of spinosad or malathion. We further expressed CYP6A51 in vivo via a GAL4/UAS system in Drosophila melanogaster flies, driving expression with detoxification tissue-specific drivers. Toxicity bioassays indicated that CYP6A51 confers knock-down resistance to both λ-cyhalothrin and deltamethrin. Detection of CYP6A51 - associated pyrethroid resistance in field populations may be important for efficient Insecticide Resistance Management (IRM) strategies.

Quantification of dose-mortality responses in adult Diptera: Validation using Ceratitis capitata and Drosophila suzukii responses to spinosad.

Quantitative laboratory bioassay methods are required to evaluate the toxicity of novel insecticidal compounds for pest control and to determine the presence of resistance traits. We used a radioactive tracer based on 32P-ATP to estimate the volume of a droplet ingested by two dipteran pests: Ceratitis capitata (Tephritidae) and Drosophila suzukii (Drosophilidae). Using blue food dye it was possible to distinguish between individuals that ingested the solution from those that did not. The average volume ingested by C. capitata adults was 1.968 µl. Females ingested a ~20% greater volume of solution than males. Adults of D. suzukii ingested an average of 0.879 µl and females ingested ~30% greater volume than males. The droplet feeding method was validated using the naturally-derived insecticide spinosad as the active ingredient (a.i.). For C. capitata, the concentration-mortality response did not differ between the sexes or among three different batches of insects. Lethal dose values were calculated based on mean ingested volumes. For C. capitata LD50 values were 1.462 and 1.502 ng a.i./insect for males and females, respectively, equivalent to 0.274 and 0.271 ng a.i./mg for males and females, respectively, when sex-specific variation in body weight was considered. Using the same process for D. suzukii, the LD50 value was estimated at 2.927 ng a.i./insect, or 1.994 ng a.i./mg based on a mean body weight of 1.67 mg for both sexes together. We conclude that this technique could be readily employed for determination of the resistance status and dose-mortality responses of insecticidal compounds in many species of pestiferous Diptera.

The parasporal crystals of Bacillus pumilus strain 15.1: a potential virulence factor?

Bacillus pumilus strain 15.1 was previously found to cause larval mortality in the Med-fly Ceratitis capitata and was shown to produce crystals in association with the spore. As parasporal crystals are well-known as invertebrate-active toxins in entomopathogenic bacteria such as Bacillus thuringiensis (Cry and Cyt toxins) and Lysinibacillus sphaericus (Bin and Cry toxins), the B. pumilus crystals were characterized. The crystals were composed of a 45 kDa protein that was identified as an oxalate decarboxylase by peptide mass fingerprinting, N-terminal sequencing and by comparison with the genome sequence of strain 15.1. Synthesis of crystals by a plasmid-cured derivative of strain 15.1 (produced using a novel curing strategy), demonstrated that the oxalate decarboxylase was encoded chromosomally. Crystals spontaneously solubilized when kept at low temperatures, and the protein produced was resistant to trypsin treatment. The insoluble crystals produced by B. pumilus 15.1 did not show significant toxicity when bioassayed against C. capitata larvae, but once the OxdD protein was solubilized, an increase of toxicity was observed. We also demonstrate that the OxdD present in the crystals has oxalate decarboxylate activity as the formation of formate was detected, which suggests a possible mechanism for B. pumilus 15.1 activity. To our knowledge, the characterization of the B. pumilus crystals as oxalate decarboxylase is the first report of the natural production of parasporal inclusions of an enzyme.

Toxic and hormetic-like effects of three components of citrus essential oils on adult Mediterranean fruit flies (Ceratitis capitata).

Plant essential oils (EOs) and a wide range of their individual components are involved in a variety of biological interactions with insect pests including stimulatory, deterrent, toxic and even hormetic effects. Both the beneficial and toxic properties of citrus EOs on the Mediterranean fruit fly (medfly) have been experimentally evidenced over the last years. However, no information is available regarding the toxic or beneficial effects of the major components of citrus EOs via contact with the adults of the Mediterranean fruit fly. In the present study, we explored the toxicity of limonene, linalool and α-pinene (3 of the main compounds of citrus EOs) against adult medflies and identified the effects of sub-lethal doses of limonene on fitness traits in a relaxed [full diet (yeast and sugar)] and in a stressful (sugar only) feeding environment. Our results demonstrate that all three compounds inferred high toxicity to adult medflies regardless of the diet, with males being more sensitive than females. Sub-lethal doses of limonene (LD20) enhanced the lifespan of adult medflies when they were deprived of protein. Fecundity was positively affected when females were exposed to limonene sub-lethal doses. Therefore, limonene, a major constituent of citrus EOs, induces high mortality at increased doses and positive effects on life history traits of medfly adults through contact at low sub-lethal doses. A hormetic-like effect of limonene to adult medflies and its possible underlying mechanisms are discussed.

To Catch a Fly: Landing and Capture of Ceratitis capitata in a Jackson Trap with and without an Insecticide.

Attractant-based traps are a cornerstone of detection, delimitation and eradication programs for pests such as tephritid fruit flies. The ideal trap and lure combination has high attraction (it brings insects to the trap from a distance) and high capture efficiency (it has a high probability of capturing the insect once it arrives at the trap). We examined the effect of an insecticide (DDVP) in combination with a pheromone lure (trimedlure) on capture of Ceratitis capitata using 1) digital images of surfaces of a Jackson trap analyzed via computer vision, and 2) counts of the number of flies caught in the trap and in the area under the trap. Our results indicate no significant difference in trap capture without or with insecticide (means ± SD = 324 ±135 and 356 ±108, respectively). However, significantly more dead flies were found around the trap with insecticide (92 ±53 with insecticide compared with 35 ±22 without), suggesting a possible decrease in trap efficiency due to mortality before insects enter the trap. Indeed, the average number of flies detected on all surfaces of the traps with insecticide was lower than that for lure-only (4.15±0.39 vs 8.30±1.18), and both were higher than control (no lure: 0.76 ±0.08). We found that the majority of fly sightings, 71% of the total, occurred on the inside panels of the lure-only traps, suggesting that increased efficiency of the Jackson trap may be obtained by adding a contact insecticide to those surfaces.

Ammonium Acetate Enhances the Attractiveness of a Variety of Protein-Based Baits to Female Ceratitis capitata (Diptera: Tephritidae).

Ammonia and its derivatives are used by female fruit flies (Diptera: Tephritidae) as volatile cues to locate protein-rich food needed to produce their eggs. This need for external protein sources has led to the development of behaviorally based control strategies such as food-based lures and insecticidal baits targeting pestiferous fruit fly species. In field cage studies conducted in Hawaii, we examined the behavioral response of laboratory-reared male and female Mediterranean fruit fly, Ceratitis capitata (Wiedemann), to seven commercially available protein baits and to beer waste, a relatively inexpensive and readily available substance. Each material was tested alone or in combination with either ammonium acetate or ammonium carbonate. For the majority of baits evaluated, the presence of ammonium acetate, but not ammonium carbonate, elicited a significantly greater level of response of female C. capitata compared with the protein baits alone. The addition of ammonium acetate to selected baits increased bait attractiveness to a level comparable with that elicited by the most widely used spinosad-based protein bait, GF-120. Our findings indicate that the addition of ammonium acetate to commercially available proteinaceous baits and to beer waste can greatly improve their attractiveness to C. capitata, potentially increasing the bait's effectiveness for fruit fly monitoring and suppression.

Exploitation of the Medfly Gut Microbiota for the Enhancement of Sterile Insect Technique: Use of Enterobacter sp. in Larval Diet-Based Probiotic Applications.

The Mediterranean fruit fly (medfly), Ceratitis capitata, is a pest of worldwide substantial economic importance, as well as a Tephritidae model for sterile insect technique (SIT) applications. The latter is partially due to the development and utilization of genetic sexing strains (GSS) for this species, such as the Vienna 8 strain, which is currently used in mass rearing facilities worldwide. Improving the performance of such a strain both in mass rearing facilities and in the field could significantly enhance the efficacy of SIT and reduce operational costs. Recent studies have suggested that the manipulation of gut symbionts can have a significant positive effect on the overall fitness of insect strains. We used culture-based approaches to isolate and characterize gut-associated bacterial species of the Vienna 8 strain under mass rearing conditions. We also exploited one of the isolated bacterial species, Enterobacter sp., as dietary supplement (probiotic) to the larval diet, and we assessed its effects on fitness parameters under the standard operating procedures used in SIT operational programs. Probiotic application of Enterobacter sp. resulted in improvement of both pupal and adult productivity, as well as reduced rearing duration, particularly for males, without affecting pupal weight, sex ratio, male mating competitiveness, flight ability and longevity under starvation.

Field Estimates of Attraction of Ceratitis capitata to Trimedlure and Bactrocera dorsalis (Diptera: Tephritidae) to Methyl Eugenol in Varying Environments.

Measuring and modeling the attractiveness of semiochemical-baited traps is of significant importance to detection, delimitation, and control of invasive pests. Here, we describe the results of field mark-release-recapture experiments with Ceratitis capitata (Wiedemann) and Bactrocera dorsalis (Hendel) to estimate the relationship between distance from a trap baited with trimedlure and methyl eugenol, respectively, and probability of capture for a receptive male insect. Experiments were conducted using a grid of traps with a central release point at two sites on Hawaii Island, a Macadamia orchard on the East side of the island and a lava field on the West side. We found that for B. dorsalis and methyl eugenol there is a 65% probability of capture at ∼36 m from a single trap, regardless of habitat. For C. capitata, we found a 65% probability of capture at a distance of ∼14 m from a single trap in the orchard and 7 m in the lava field. We also present results on the spatial and temporal pattern of recaptures. The attraction data are analyzed via a hyperbolic secant-based capture probability model.

Toxic effect of citrus peel constituents on Anastrepha fraterculus Wiedemann and Ceratitis capitata Wiedemann immature stages.

The toxicity of essential oils from the citrus peel has been proposed as the major resistance mechanism offered by citrus to fruit fly infestation. We evaluated the insecticidal activity of the ether extracts from the lemon (Citrus limon [L.] Burm.) and grapefruit (C. paradisi Macfadyen) peel as well as from limonene and citral against Anastrepha fraterculus (Wiedemann) and Ceratitis capitata (Wiedemann) immature stages. We also evaluated the toxicity of the extracts at two ripening stages. Extracts proved toxic to A. fraterculus egg and larvae. The lemon and grapefruit extracts showed the same toxicity in both fruit fly species. For A. fraterculus eggs, citral was more toxic than limonene; for larvae, they showed equal toxicity. Anastrepha fraterculus eggs were more sensitive than C. capitata eggs. In conclusion, we provide evidence of chemical resistance mechanisms that could account for the nonhost condition of lemon for A. fraterculus.

Evaluation of candidate systems for mass trapping against Ceratitis spp. on La Réunion island.

BACKGROUND: Two Ceratitis spp. coexist on the island of La Réunion, Ceratitis rosa (Karsch) and C. capitata (Wiedemann). The effectiveness of candidate systems based on attractants and traps to control both species was evaluated through comparative studies of trap types, attractants, insecticides and commercial complete systems. RESULTS: The Ceratitis spp. most captured in all trials was C. rosa. Captures of C. rosa and C. capitata were not significantly different when Maxitrap® or Tephri-trap® were used. Captures with the Easy-trap® were lower for both species. The BioLure® Med Fly lure showed higher catches of C. rosa than Ferag® CC D TM. The proportion of dead C. rosa did not differ significantly when deltamethrin or dichlorvos was used. There were no significant differences in the capture of C. rosa and C. capitata between commercial systems composed of the lure Ferag® CC D TM, the Maxitrap® and dichlorvos and the BioLure® Unipak, the Tephri-trap® and dichlorvos. The Cera Trap® system performed less effectively. CONCLUSION: The most effective traps for the capture of C. rosa and C. capitata were Maxitrap® and Tephri-trap®; the most effective attractants were BioLure® Med Fly for C. rosa and BioLure® Med Fly, BioLure® Unipak and Ferag® CC D TM for C. capitata. However, for both species the most effective commercial combinations of trap and attractant were Maxitrap® with Ferag® CC D TM and Tephri-trap® with BioLure® Unipak, both using dichlorvos. The insecticide deltamethrin showed good efficacy when used inside traps.

Detection of the Acetylcholinesterase Insecticide Resistance Mutation (G328A) in Natural Populations of Ceratitis capitata.

Wild Mediterranean fruit fly specimens collected from various regions worldwide were screened for the glycine to alanine (Gly->Ala) point mutation (G328A) in the acetylcholinesterase enzyme, presumably causing resistance to organophosphates. We found that the single nucleotide polymorphism (SNP) responsible for this amino acid change is located at the beginning of exon 6 of the Ccace2 gene. The identification of the exact location of the SNP permitted PCR primer design around this site and direct sequencing of the corresponding genomic region. We detected the resistance allele in natural Mediterranean fruit fly populations from Brazil and Spain, but not from other sites in four continents. The known treatment history of sites suggests that the resistance buildup is linked to organophosphate application in the field. The PCR-based detection provides a screening method useful for monitoring Mediterranean fruit fly insecticide resistance in local populations and improving pest management strategies accordingly.

Effects of pesticides used on citrus grown in Spain on the mortality of Ceratitis capitata (Diptera: Tephritidae) Vienna-8 strain sterile males.

Vienna-8 sterile males are currently released in Spain to reduce wild populations of the medfly. Because pesticides are required to maintain populations of some citrus key pests below economic thresholds, there is a need to evaluate the effects of pesticides commonly used in citrus on Vienna-8 males. Males were exposed to differently aged residues of eight pesticides. Abamectin, etofenprox, etoxazole, petroleum spray oil, pymetrozine, and pyriproxyfen resulted harmless to Vienna-8 males. However, fresh residues of chlorpyrifos and spinosad caused high mortalities and had residual effects until 21 and 28 d after treatment, respectively. Following the same method, the lethal effects of chlorpyrifos and spinosad on Ceratitis capitata (Wiedemann) wild-type (wt) males were determined. Surprisingly, these pesticides resulted more toxic for wt than for Vienna-8 males. To determine whether these results could be attributed to intrinsic characteristics of the pesticides or to behavioral differences among Vienna-8 and wt males, a topical application trial was conducted. Vienna-8 males were twice as susceptible to chlorpyrifos as wt males, whereas their susceptibility to spinosad was slightly lower. These results in intrinsic toxicity did not directly explain the differences observed in the extended-laboratory tests with these pesticides. We hypothesize that the lower flight activity ofVienna-8 males relative to wt conspecifics can explain the lower risk observed for Vienna-8 males in the residual tests. Our results should be taken into account when planning area-wide Sterile Insect Technique programs against C. capitata especially in those areas where treatments with chlorpyrifos are approved.