Suppressing the expression of glutathione S-transferase gene GSTd10 increases the sensitivity of Zeugodacus cucurbitae against ß-cypermethrin.

Zeugodacus cucurbitae Coquillett (Diptera: Tephritidae) is an agriculturally and economically important pest worldwide that has developed resistance to ß-cypermethrin. Glutathione S-transferases (GSTs) have been reported to be involved in the detoxification of insecticides in insects. We have found that both ZcGSTd6 and ZcGSTd10 were up-regulated by ß-cypermethrin induction in our previous study, so we aimed to explore their potential relationship with ß-cypermethrin tolerance in this study. The heterologous expression of ZcGSTd6 and ZcGSTd10 in Escherichia coli showed significantly high activities against 1-chloro-2,4-dinitrobenzene (CDNB). The kinetic parameters of ZcGSTd6 and ZcGSTd10 were determined by Lineweaver-Burk. The Vmax and Km of ZcGSTd6 were 0.50 µmol/min·mg and 0.3 mM, respectively. The Vmax and Km of ZcGSTd10 were 1.82 µmol/min·mg and 0.53 mM. The 3D modelling and molecular docking results revealed that ß-cypermethrin exhibited a stronger bounding to the active site SER-9 of ZcGSTd10. The sensitivity to ß-cypermethrin was significantly increased by 18.73% and 27.21%, respectively, after the knockdown of ZcGSTd6 and ZcGSTd10 by using RNA interference. In addition, the inhibition of CDNB at 50% (IC50) and the inhibition constants (Ki) of ß-cypermethrin against ZcGSTd10 were determined as 0.41 and 0.33 mM, respectively. The Ki and IC50 of ß-cypermethrin against ZcSGTd6 were not analysed. These results suggested that ZcGSTd10 could be an essential regulator involved in the tolerance of Z. cucurbitae to ß-cypermethrin.

Oviposition behaviour and biochemical response of an insect pest, Zeugodacus cucurbitae (Coquillett) (Diptera: Tephritidae) to plant phenolic compound phloroglucinol.

Phenolic compounds are the secondary metabolites (SMs) present in plants carrying different bioactivities. In the present study, we explored the influence of a phenolic compound namely phloroglucinol on oviposition behaviour and different biochemical entities of an insect pest Zeugodacus cucurbitae (Coquillett) (Diptera: Tephritidae) using artificial diet. Phloroglucinol (IUPAC name: benzene-1,3,5-triol) affected the activity of antioxidant and detoxifying enzymes viz. superoxide dismutases (SOD), catalase (CAT), ascorbate peroxidases (APOX). dehydroascorbate reductase (DHAR), peroxidases (POX), phenol oxidase (PO), glutathione peroxidase (GPOX), glutathione S-transferase peroxidase (GSTpox), glutathione reductase (GR), glutathione S-transferase (GST) and esterases (EST) as well as the biological antioxidants viz. ascorbate content and glutathione. The lipid peroxide content (LP) and hydrogen peroxide content (H2O2) were significantly enhanced in the treated larvae indicating oxidative stress in the insect. Significant inhibition in oviposition was observed and effective repellency percentage increased with phloroglucinol treatment as compared to control. The oviposition deterrent activity and toxic effects of phloroglucinol on various biochemical parameters of Z. cucurbitae larvae revealed in the present study clearly confirms its suitability for use in pest management.

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.

Defensive Mechanisms in Cucurbits against Melon Fly (Bactrocera cucurbitae) Infestation through Excessive Production of Defensive Enzymes and Antioxidants.

Melon fly (Bactrocera cucurbitae) is the most common pest of cucurbits, and it directly causes damage to cucurbit fruits in the early developmental stage. The infection of fruit tissues induces oxidative damage through increased generation of cellular reactive oxygen species. The effects of melon fly infestation on the production of defensive enzymes and antioxidant capabilities in five cucurbit species, namely, bottle gourd, chayote, cucumber, snake gourd, and bitter gourd, were investigated in this study. The total phenolic and flavonoid content was considerably higher in melon fly infestation tissues compared to healthy and apparently healthy tissues. The chayote and bottle gourd tissues expressed almost 1.5- to 2-fold higher phenolic and flavonoid contents compared to the tissues of bitter gourd, snake gourd, and cucumber upon infestation. Defensive enzymes, such as peroxidase (POD), superoxide dismutase (SOD), polyphenol oxidase (PPO), and catalase (CAT), were high in healthy and infected tissues of chayote and bottle gourd compared to bitter gourd, snake gourd, and cucumber. The activity of POD (60-80%), SOD (30-35%), PPO (70-75%), and CAT (40-50%) were high in infected chayote and bottle gourd tissue, representing resistance against infestation, while bitter gourd, snake gourd, and cucumber exhibited comparatively lower activity suggesting susceptibility to melon fly infection. The antioxidant properties were also high in the resistant cucurbits compared to the susceptible cucurbits. The current research has enlightened the importance of redox-regulatory pathways involving ROS neutralization through infection-induced antioxidative enzymes in host cucurbit resistance. The melon fly infestation depicts the possible induction of pathways that upregulate the production of defensive enzymes and antioxidants as a defensive strategy against melon fly infestation in resistant cucurbits.

Toxics or Lures? Biological and Behavioral Effects of Plant Essential Oils on Tephritidae Fruit Flies.

The family Tephritidae (Diptera) includes species that are highly invasive and harmful to crops. Due to globalization, international trade, and human displacement, their spread is continuously increasing. Unfortunately, the control of tephritid flies is still closely linked to the use of synthetic insecticides, which are responsible for detrimental effects on the environment and human health. Recently, research is looking for alternative and more eco-friendly tools to be adopted in Integrated Pest Management (IPM) programs. In this regard, essential oils (EOs) and their main compounds represent a promising alternative to chemical insecticides. EOs are made up of phytoconstituents formed from the secondary metabolism of many plants and can act as attractants or toxics, depending on the dose. Because of this unique characteristic, EOs and their main constituents are promising tools that can be used both in Sterile Insect Technique (SIT) programs and in the "lure and kill" technique, exploiting the attractiveness of the product in the former case and its toxicity in the latter. In this article, current knowledge on the biological and behavioral effects of EOs and their main constituents on tephritid fruit flies is reviewed, mainly focusing on species belonging to the Anastrepha, Bactrocera, Ceratitis, and Zeugodacus genera. The mechanisms of action of EOs, their real-world applications, and challenges related to their use in IPM are critically discussed.

Odour Cues from Fruit Arils of Artocarpus heterophyllus Attract both Sexes of Oriental Fruit Flies.

The Oriental fruit fly, Bactrocera dorsalis (Hendel) is an economically devastating pest of fruit crops across the globe with stringent quarantine restrictions to limit its further spread. The current management programs increasingly depend on male annihilation but trapping female flies is equally important to reduce fruit damage. Considering the importance of kairomones in courtship and oviposition site selection behavior of B. dorsalis, the aim of this work was to isolate and identify potential cues from the volatiles of arils of jackfruit, Artocarpus heterophyllus. Using olfactometer assays and gas-chromatography linked to electroantennographic detection, attraction of both female and male B. dorsalis to specific jackfruit volatiles was demonstrated. Ethyl 3-methylbutanoate, ethyl hexanoate, pentyl butanote, 2-methylbutyl 3-methylbutanoate, 2-methylpropyl hexanoate, (Z)-3-hexenyl 3-methylbutanoate and dodecanal were found to attract female B. dorsalis specifically. Butyl acetate, 2 phenylethanol and pentyl 3-methylbutanoate elicited attraction in male B. dorsalis only. Synthetic blends of these compounds were found to attract female and male B. dorsalis in laboratory as well as field conditions. Using specific cues common to each set, a blend of methyl 3-methylbutanoate, butyl acetate, 3-methylbutyl acetate and hexyl acetate attracted both sexes of B dorsalis. This study demonstrates the use of kairomone-based lures for sex-specific as well as bisexual attraction for the first time.

Comparative proteomics provides insights into diapause program of Bactrocera minax (Diptera: Tephritidae).

The Chinese citrus fly, Bactrocera minax, is a notorious univoltine pest that causes damage to citrus. B. minax enters obligatory pupal diapause in each generation to resist harsh environmental conditions in winter. Despite the enormous efforts that have been made in the past decade, the understanding of pupal diapause of B. minax is currently still fragmentary. In this study, the 20-hydroxyecdysone solution and ethanol solvent was injected into newly-formed pupae to obtain non-diapause- (ND) and diapause-destined (D) pupae, respectively, and a comparative proteomics analysis between ND and D pupae was performed 1 and 15 d after injection. A total of 3,255 proteins were identified, of which 190 and 463 were found to be differentially abundant proteins (DAPs) in ND1 vs D1 and ND15 vs D15 comparisons, respectively. The reliability and accuracy of LFQ method was validated by qRT-PCR. Functional analyses of DAPs, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, and protein-protein interaction (PPI) network construction, were conducted. The results revealed that the diapause program of B. minax is closely associated with several physiological activities, such as phosphorylation, chitin biosynthesis, autophagy, signaling pathways, endocytosis, skeletal muscle formation, protein metabolism, and core metabolic pathways of carbohydrate, amino acid, and lipid conversion. The findings of this study provide insights into diapause program of B. minax and lay a basis for further investigation into its underlying molecular mechanisms.

A First Glimpse of the Mexican Fruit Fly Anastrepha ludens (Diptera: Tephritidae) Antenna Morphology and Proteome in Response to a Proteinaceous Attractant.

Anastrepha ludens is a key pest of mangoes and citrus from Texas to Costa Rica but the mechanisms of odorant perception in this species are poorly understood. Detection of volatiles in insects occurs mainly in the antenna, where molecules penetrate sensillum pores and link to soluble proteins in the hemolymph until reaching specific odor receptors that trigger signal transduction and lead to behavioral responses. Scrutinizing the molecular foundation of odorant perception in A. ludens is necessary to improve biorational management strategies against this pest. After exposing adults of three maturity stages to a proteinaceous attractant, we studied antennal morphology and comparative proteomic profiles using nano-LC-MS/MS with tandem mass tags combined with synchronous precursor selection (SPS)-MS3. Antennas from newly emerged flies exhibited dense agglomerations of olfactory sensory neurons. We discovered 4618 unique proteins in the antennas of A. ludens and identified some associated with odor signaling, including odorant-binding and calcium signaling related proteins, the odorant receptor co-receptor (Orco), and putative odorant-degrading enzymes. Antennas of sexually immature flies exhibited the most upregulation of odor perception proteins compared to mature flies exposed to the attractant. This is the first report where critical molecular players are linked to the odor perception mechanism of A. ludens.

Effects of fatty acids and vitamin E in larval diets on development and performance of Queensland fruit fly.

Tephritid fruit flies are commonly reared on artificial larval diets for laboratory studies and for sterile insect technique pest management programs. While significant effort has been invested in developing artificial larval diets, surprisingly little is known about the specific nutritional requirements of tephritid flies. Recently developed gel larval diets have provided new opportunities for nutritional studies in Queensland fruit fly, Bactrocera tryoni ('Q-fly'). Wheat germ oil (WGO) is the main source of fatty acids and vitamin E in this diet, and is key for production of high-quality adults. To identify the importance of nutritional components of WGO for Q-fly productivity and quality, linoleic, linolenic, oleic and palmitic fatty acids as well as α-tocopherol (vitamin E) were included in the diet individually and in combination. Diets that included all of the tested fatty acids or just unsaturated fatty acids performed as well as diets containing WGO in most quality control parameters except fecundity, and addition of vitamin E reduced the pupal productivity. Considering individual fatty acids, larval diets containing only linolenic acid produced adults with higher percentage of fliers than did larval diets containing only palmitic acid or oleic acid. Compared with diets containing WGO, nutritional requirements for egg production in Q-fly were not entirely met by either grouped fatty acids or individual polyunsaturated, monounsaturated or saturated fatty acids, however, diets containing linoleic acid alone produced more eggs than any other fatty acid. The present study is a significant advance in understanding of the role of fatty acids as a component of WGO in larval diet in meeting the needs of developing Q-fly for somatic performance, but highlight also that other, untested, components of WGO appear to be important for reproduction.

Microbiome Hijacking Towards an Integrative Pest Management Pipeline.

Pesticides are necessary to fight agricultural pests, yet they are often nonspecific, and their widespread use is a hazard to the environment and human health. The genomic era allows for new approaches to specifically target agricultural pests, based on analysis of their genome and their microbiome. We present such an approach, to combat Bactrocera oleae, a widespread pest whose impact is devastating on olive production. To date, there is no specific pesticide to control it. Herein, we propose a novel strategy to manage this pest via identifying novel pharmacological targets on the genome of its obligate endosymbiotic bacterium Candidatus Erwinia dacicola. Three genes were selected as pharmacological targets. The 3D models of the Helicase, Polymerase, and Protease-C gene products were designed and subsequently optimized by means of molecular dynamics simulations. Successively, a series of structure-based pharmacophore models were elucidated in an effort to pave the way for the efficient high-throughput virtual screening of libraries of low molecular weight compounds and thus the discovery of novel modulating agents. Our methodology provides the means to design, test, and identify highly specific pest control substances that minimize the impact of toxic chemicals on health, economy, and the environment.

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.

Integrated LC-MS and GC-MS-based untargeted metabolomics studies of the effect of azadirachtin on Bactrocera dorsalis larvae.

Azadirachtin exhibits excellent bioactivities against several hundred arthropods. However, current knowlege of its biochemical effect on B. dorsalis larvae is not deep enough. In this study, integrated LC-MS and GC-MS-based untargeted metabolomics were used to analyze the changes of endogenous metabolites and the biochemical effects of azadirachtin on B. dorsalis larvae. Azadirachtin has excellent bioactivities against B. dorsalis larvae in this study, leading to a longer developmental duration, lower survival rate, and low pupa weight. The effect of azadirachtin was investigated on a total of 22 and 13 differentially abundant metabolites in the LC-MS and GC-MS-based metabolomics results, are selected respectively. Pathway analysis indicated that 14 differentially enriched metabolic pathways, including seven influential pathways, are worthy of attention. Further integrated key metabolic pathway analysis showed that histidine metabolism, D-glutamine and D-glutamate metabolism, biotin metabolism, ascorbate and aldarate metabolism, pentose and glucuronate interconversions, and alanine, aspartate and glutamate metabolism in B. dorsalis larvae are significantly relevant pathways affected by azadirachtin. Although extrapolating the bioactivity results in this study to the practical project of B. dorsalis pest management in the field has limitations, it was found that azadirachtin has a significant effect on the primary metabolism of B. dorsalis larvae.

Assessment of Bactrocera dorsalis (Diptera: Tephritidae) Diets on Adult Fecundity and Larval Development: Insights Into Employing the Sterile Insect Technique.

Bactrocera dorsalis (Hendel) is a notorious insect pest that attacks diverse vegetables and fruits worldwide. The sterile insect technique has been developed as an environmentally friendly and effective control method that depends on the mass production of target flies. Because dietary yeast (protein) and sucrose (carbohydrate) are important in adult diets, yeast:sucrose (Y:S) mixtures are crucial for the mass-rearing of B. dorsalis. In this study, we found adult diets with different ratios of yeast to sucrose-influenced fecundity, and an extremely high or low Y:S ratios significantly decreased egg production of B. dorsalis. Additionally, the maximum oviposition efficiency was realized at dietary yeast to sucrose ratios of 1:1 and 1:3, suggesting their potential use to produce more eggs for the mass production of B. dorsalis. Here, new gel diets having different yeast concentrations (g/L water) were also assessed for rearing B. dorsalis larvae. Gel diets containing 20 g/L yeast led to a higher pupation, pupal weight and adult eclosion rate, and a shorter developmental time than other yeast concentrations. Moreover, the present gel diet also resulted in greater pupal production and adult emergence rates than previously used liquid and solid artificial diets, revealing that it is suitable for rearing B. dorsalis larvae. This research provides a useful reference on artificial diets mixtures for mass rearing B. dorsalis, which is critical for employing the sterile insect technique.

Ammoides verticillata Essential Oil as Biocontrol Agent of Selected Fungi and Pest of Olive Tree.

BACKGROUND: Ammoides verticillata Briq is an aromatic and medicinal herb that has been widely used in folk medicine for treatment of several types of pathologies such as respiratory problems, colds, fever, headache, gastric disorders and renal infections. OBJECTIVE: Essential oils have been subjected of numerous works for their antimicrobial and insecticidal properties which have been valued by hundreds of publications and patents. The main objective of this work was to evaluate the chemical composition, and the in vitro and in vivo antifungal, and insecticidal properties of essential oil of Ammoides verticillata. METHODS: In this work, the air-dried aerial parts of Ammoides verticillata were hydrodistilled in a Clevenger-type apparatus. The essential oil isolated was analyzed using gas chromatography (GC) and mass spectrometry (GC/MS). The in vitro antifungal activity of the essential oil was investigated against four plant fungi using radial growth technique. The effect of the essential oil on disease development of olive caused by Penicillium crustosum and Aspergillus niger in the in vivo conditions. The fumigation test of the essential oil was evaluated against L3 stages, pupas and adults of Bactrocera oleae known as the olive fly. RESULTS: The essential oil of A. verticillata was characterized mainly by carvacrol (44.3%), limonene (19.3%) and p-cymene (19.2%). The result of in vitro antifungal activity of essential oil showed an interesting antifungal inhibition against Alternaria alternata and Fusarium solani strains with percentage inhibition of 89%. Furthermore, oil of A. verticillata has demonstrated promising in vivo antifungal activity to control infections of olives caused by Aspergillus niger and Penicillium crustosum. In addition, the essential oil exhibited fumigation toxicity against Bactrocera oleae with mortality percentage of 100% at 2 µL/L air. CONCLUSION: The results showed that essential oil of A. verticillata has interesting biological activities, which suggests that oil have the potential to be used as biocide to protect olives of pathogenic fungi and pests.

Divergent molecular evolution in glutathione S-transferase conferring malathion resistance in the oriental fruit fly, Bactrocera dorsalis (Hendel).

Insect glutathione S-transferases (GSTs) are important in insecticide detoxification and Insect-specific GSTs, Epsilon and Delta, have largely expanded in insects. In this study, we functionally expressed and characterized an epsilon class GST gene (BdGSTe8), predominant in the adult Malpighian tubules of Bactrocera dorsalis. This gene may be associated with malathion resistance based on transcriptional studies of resistant and susceptible strains. RNA interference-mediated knockdown of this gene significantly recovered malathion susceptibility in the adults of a malathion-resistant strain, and overexpression of BdGSTe8 enhanced resistance in transgenic Drosophila. Analysis of BdGSTe8 polymorphism showed that several point mutations may be associated with metabolic resistance to malathion. A cytotoxicity assay in Escherichia coli indicated that both of the recombinant BdGSTe8 proteins may play a functional role in protecting cells from toxicity. The allele of BdGSTe8-B conferred higher levels of malathion detoxification capability. Liquid chromatography and ultra-performance liquid chromatography-tandem mass spectrometry analysis showed that the BdGSTe8-A allele did not metabolize malathion directly. However, the BdGSTe8-B allele was involved in the direct metabolism of malathion, which was caused by a mutation in V128A. Further analysis of the sequence suggests that BdGSTe8 evolved rapidly. It maybe play the role of a backup gene and could become a new gene in the future in order to retain the ability of detoxification of malathion, which was driven by positive selection. These results suggest that divergent molecular evolution in BdGSTe8 has played a role in metabolic resistance to malathion in B. dorsalis.

Wild bacterial probiotics fed to larvae of mass-reared Queensland fruit fly [Bactrocera tryoni (Froggatt)] do not impact long-term survival, mate selection, or locomotor activity.

Queensland fruit fly [Bactrocera tryoni (Froggatt), Diptera, Tephritidae] is the most devastating insect pest impacting Australian horticulture. The Sterile Insect Technique (SIT) is an important component of tephritid pest management programs. However, mass-rearing and irradiation (to render insects sterile) may reduce the fitness and performance of the insect, including the ability of sterile males to successfully compete for wild females. Manipulation of the gut microbiome, including the supplementation with bacterial probiotics shows promise for enhancing the quality of mass-reared sterile flies, however there are fewer published studies targeting the larval stage. In this study, we supplemented the larval stage of mass-reared B. tryoni with bacterial probiotics. We tested several individual bacteria that had been previously isolated and characterized from the gut of wild B. tryoni larvae including Asaia sp., Enterobacter sp., Lactobacillus sp., Leuconostoc sp. We also tested a consortium of all four of these bacterial isolates. The fitness parameters tested included adult survival in field cages, laboratory mate selection of bacteria supplemented males by bacteria nonsupplemented females, and laboratory locomotor activity of adult flies. None of the bacterial probiotic treatments in the current study was significantly different to the control for field survival, mate selection or locomotor activity of adult B. tryoni, which agree with some of the other studies regarding bacterial probiotics fed to the larval stage of tephritids. Future work is needed to determine if feeding the same, and/or other probiotics to adults, as opposed to larvae can positively impact survival, mating performance, mating competitiveness and locomotor activity of B. tryoni. The bacterial group(s) and function of bacterial species that increase fitness and competitiveness is also of interest to tephritid mass-rearing programs.

The comparative metabolic response of Bactrocera dorsalis larvae to azadirachtin, pyriproxyfen and tebufenozide.

Azadirachtin, as the most promising and effective botanical insecticide, exhibits significant growth inhibition activity against agricultural and forestry pests. However, its biochemical effects at the metabolic level compared with those of other insect growth regulators have not been studied. Therefore, in this study, a GC-MS based untargeted metabolomics approach was applied to compare azadirachtin with pyriproxyfen (a juvenile hormone analog) and tebufenozide (a molting hormone analog) in terms of their metabolic effects on Bactrocera dorsalis larvae. The bioactivity of azadirachtin against B. dorsalis larvae was significantly different than those of pyriproxyfen and tebufenozide. A total of 693 mass features were recognized, and 112 metabolites were identified in this study. The results showed that a total of 16, 13 and 10 differentially regulated metabolites corresponding to 12, 5 and 8 pathways occur in Aza versus CK, Pyr versus CK and Teb versus CK group, respectively. Further analysis showed that 6 differentially regulated metabolites corresponding to 5 key pathways could be the primary differential metabolic response of B. dorsalis larvae to the three insect growth regulators. The pathways were myo-inositol corresponding to ascorbate and aldarate metabolism as the specific response of B. dorsalis larvae to azadirachtin; xylitol, xylulose and 3-aminopropionitrile corresponding to pentose and glucuronate interconversions, and cyanoamino acid metabolism as the common responses to azadirachtin and pyriproxyfen; and 3-hydroxypropionic acid and beta-alanine corresponding to propanoate metabolism and beta-alanine metabolism as the specific responses to tebufenozide. The results showed that the metabolic response of B. dorsalis larvae to azadirachitin is closer to that of pyriproxyfen than tebufenozide. The differentially regulated metabolites and pathways responsible for this difference are discussed.

Gut bacterial diversity and physiological traits of Anastrepha fraterculus Brazilian-1 morphotype males are affected by antibiotic treatment.

BACKGROUND: The interaction between gut bacterial symbionts and Tephritidae became the focus of several studies that showed that bacteria contributed to the nutritional status and the reproductive potential of its fruit fly hosts. Anastrepha fraterculus is an economically important fruit pest in South America. This pest is currently controlled by insecticides, which prompt the development of environmentally friendly methods such as the sterile insect technique (SIT). For SIT to be effective, a deep understanding of the biology and sexual behavior of the target species is needed. Although many studies have contributed in this direction, little is known about the composition and role of A. fraterculus symbiotic bacteria. In this study we tested the hypothesis that gut bacteria contribute to nutritional status and reproductive success of A. fraterculus males. RESULTS: AB affected the bacterial community of the digestive tract of A. fraterculus, in particular bacteria belonging to the Enterobacteriaceae family, which was the dominant bacterial group in the control flies (i.e., non-treated with AB). AB negatively affected parameters directly related to the mating success of laboratory males and their nutritional status. AB also affected males' survival under starvation conditions. The effect of AB on the behaviour and nutritional status of the males depended on two additional factors: the origin of the males and the presence of a proteinaceous source in the diet. CONCLUSIONS: Our results suggest that A. fraterculus males gut contain symbiotic organisms that are able to exert a positive contribution on A. fraterculus males' fitness, although the physiological mechanisms still need further studies.

Systematic Modification of Zingerone Reveals Structural Requirements for Attraction of Jarvis's Fruit Fly.

Tephritid fruit flies are amongst the most significant horticultural pests globally and male chemical lures are important for monitoring and control. Zingerone has emerged as a unique male fruit fly lure that can attract dacine fruit flies that are weakly or non-responsive to methyl eugenol and cuelure. However, the key features of zingerone that mediate this attraction are unknown. As Jarvis's fruit fly, Bactrocera jarvisi (Tryon), is strongly attracted to zingerone, we evaluated the response of B. jarvisi to 37 zingerone analogues in a series of field trials to elucidate the functional groups involved in attraction. The most attractive analogues were alkoxy derivatives, with isopropoxy being the most attractive, followed by ethoxy and trifluoromethoxy analogues. All of the phenolic esters tested were also attractive with the response typically decreasing with increasing size of the ester. Results indicate that the carbonyl group, methoxy group, and phenol of zingerone are key sites for the attraction of B. jarvisi and identify some constraints on the range of structural modifications that can be made to zingerone without compromising attraction. These findings are important for future work in developing and optimising novel male chemical lures for fruit flies.

Exploration of anti-insect potential of trypsin inhibitor purified from seeds of Sapindus mukorossi against Bactrocera cucurbitae.

Peptidase inhibitors (PIs) are defense proteins of plants which are active against gut peptidases of different insects. Sapindus mukorossi was identified as a source of bioactive PIs which could confer resistance against Bactrocera cucurbitae, a most devastating pest of several economically important crops. In the present study, a trypsin inhibitor was purified from mature dry seeds of S. mukorossi and characterized for its biochemical properties as well as its potential for bio control of B. cucurbitae. The purified fractions from RP- HPLC through SDS-PAGE gave an apparent molecular weight of ~29 kDa. S. mukorossi trypsin inhibitor (SMTI) was found to be a non-competitive inhibitor which was active over a broad range of temperature (10-100 °C) and pH (6-11). SMTI when incorporated in artificial diet inhibited the growth and development of B. cucurbitae larvae. Gene expression analysis of trypsin and chymotrypsin genes via qRT-PCR indicated that their mRNA expression was down-regulated while that of other genes namely, Catalase, Elastase, Superoxide Dismutase, Glutathione -S-transferase and Alkaline Phosphatase was up regulated. SMTI also showed deleterious effects against different bacterial strains. The results of this study indicated that S. mukorossi trypsin inhibitor has potential to be used as a bio control agent that can reduce the harm caused by melon fruit fly and other devastating pests.