Bacterial pathogens deliver water- and solute-permeable channels to plant cells.

Many animal- and plant-pathogenic bacteria use a type III secretion system to deliver effector proteins into host cells1,2. Elucidation of how these effector proteins function in host cells is critical for understanding infectious diseases in animals and plants3-5. The widely conserved AvrE-family effectors, including DspE in Erwinia amylovora and AvrE in Pseudomonas syringae, have a central role in the pathogenesis of diverse phytopathogenic bacteria6. These conserved effectors are involved in the induction of 'water soaking' and host cell death that are conducive to bacterial multiplication in infected tissues. However, the exact biochemical functions of AvrE-family effectors have been recalcitrant to mechanistic understanding for three decades. Here we show that AvrE-family effectors fold into a ß-barrel structure that resembles bacterial porins. Expression of AvrE and DspE in Xenopus oocytes results in inward and outward currents, permeability to water and osmolarity-dependent oocyte swelling and bursting. Liposome reconstitution confirmed that the DspE channel alone is sufficient to allow the passage of small molecules such as fluorescein dye. Targeted screening of chemical blockers based on the predicted pore size (15-20 Å) of the DspE channel identified polyamidoamine dendrimers as inhibitors of the DspE/AvrE channels. Notably, polyamidoamines broadly inhibit AvrE and DspE virulence activities in Xenopus oocytes and during E. amylovora and P. syringae infections. Thus, we have unravelled the biochemical function of a centrally important family of bacterial effectors with broad conceptual and practical implications in the study of bacterial pathogenesis.

Identification of multiple odorant receptors essential for pyrethrum repellency in Drosophila melanogaster.

Pyrethrum extract from dry flowers of Tanacetum cinerariifolium (formally Chrysanthemum cinerariifolium) has been used globally as a popular insect repellent against arthropod pests for thousands of years. However, the mechanistic basis of pyrethrum repellency remains unknown. In this study, we found that pyrethrum spatially repels and activates olfactory responses in Drosophila melanogaster, a genetically tractable model insect, and the closely-related D. suzukii which is a serious invasive fruit crop pest. The discovery of spatial pyrethrum repellency and olfactory response to pyrethrum in D. melanogaster facilitated our identification of four odorant receptors, Or7a, Or42b, Or59b and Or98a that are responsive to pyrethrum. Further analysis showed that the first three Ors are activated by pyrethrins, the major insecticidal components in pyrethrum, whereas Or98a is activated by (E)-ß-farnesene (EBF), a sesquiterpene and a minor component in pyrethrum. Importantly, knockout of Or7a, Or59b or Or98a individually abolished fly avoidance to pyrethrum, while knockout of Or42b had no effect, demonstrating that simultaneous activation of Or7a, Or59b and Or98a is required for pyrethrum repellency in D. melanogaster. Our study provides insights into the molecular basis of repellency of one of the most ancient and globally used insect repellents. Identification of pyrethrum-responsive Ors opens the door to develop new synthetic insect repellent mixtures that are highly effective and broad-spectrum.

Transfluthrin enhances odorant receptor-mediated spatial repellency in Aedes aegypti.

Mosquito-borne diseases are an increasing global health challenge and vector-based interventions remain the most important methods for reducing the public health burden of most mosquito-borne diseases. Transfluthrin, a volatile pyrethroid insecticide, is used widely and effectively as an insecticide and as a repellent. In a recent study, we showed that at very low concentrations transfluthrin repels Aedes aegypti mosquitoes in a hand-in-cage assay without detectable stimulation of antennal olfactory responses. Furthermore, activation of sodium channels by transfluthrin enhances repellency by DEET, which has been reported to repel mosquitoes via odorant receptor (Or)-dependent and Or-independent mechanisms. However, whether activation of sodium channels by transfluthrin can serve as a general mechanism for synergizing the activities of other repellents remain unknown. In this study, we found that, in hand-in-cage assay, transfluthrin enhanced repellency by geranyl acetate and (E)-ß-farnesene, which activate AaOr31. Such enhancement was not observed in AaOr31-knockout mosquitoes and dampened in a pyrethroid-resistant strain carrying two sodium channel mutations, which reduce the action of transfluthrin on sodium channels. In addition, transfluthrin also enhanced repellency by (-)-borneol, (±)-citronellal, camphor, and eucalyptol, which activate Or-mediated repellency. Our study has uncovered the ability of transfluthrin to enhance the repellency to a variety of mosquito repellents, beyond DEET, and provided experimental support for the emerging paradigm of synergistic interactions between repellency mediated by sodium channel activation and Or activation. These findings have potential implications in the development of more effective mosquito repellent mixtures.

Bacterial pathogens deliver water/solute-permeable channels as a virulence strategy.

Many animal and plant pathogenic bacteria utilize a type III secretion system to deliver effector proteins into the host cell 1,2 . Elucidation of how these effector proteins function in the host cell is critical for understanding infectious diseases in animals and plants 3-5 . The widely conserved AvrE/DspE-family effectors play a central role in the pathogenesis of diverse phytopathogenic bacteria 6 . These conserved effectors are involved in the induction of "water-soaking" and host cell death that are conducive to bacterial multiplication in infected tissues. However, the exact biochemical functions of AvrE/DspE-family effectors have been recalcitrant to mechanistic understanding for three decades. Here we show that AvrE/DspE-family effectors fold into a ß-barrel structure that resembles bacterial porins. Expression of AvrE and DspE in Xenopus oocytes results in (i) inward and outward currents, (ii) permeability to water and (iii) osmolarity-dependent oocyte swelling and bursting. Liposome reconstitution confirmed that the DspE channel alone is sufficient to allow the passage of small molecules such as fluorescein dye. Targeted screening of chemical blockers based on the predicted pore size (15-20 Å) of the DspE channel identified polyamidoamine (PAMAM) dendrimers as inhibitors of the DspE/AvrE channels. Remarkably, PAMAMs broadly inhibit AvrE/DspE virulence activities in Xenopus oocytes and during Erwinia amylovora and Pseudomonas syringae infections. Thus, we have unraveled the enigmatic function of a centrally important family of bacterial effectors with significant conceptual and practical implications in the study of bacterial pathogenesis.

Novel Lactone-Based Insecticides and Drosophila suzukii Management: Synthesis, Potential Action Mechanisms and Selectivity for Non-Target Parasitoids.

Drosophila suzukii, an invasive insect pest, poses a significant threat to various fruit crops. The use of broad-spectrum insecticides to control this pest can reduce the effectiveness of biological control agents, such as the parasitoid Trichopria anastrephae. Here, we evaluated the toxicity of newly synthesized lactone derivatives on D. suzukii and their selectivity towards T. anastrephae. We used in silico approaches to identify potential targets from the most promising molecules in the D. suzukii nervous system and to understand potential differences in susceptibilities between D. suzukii and its parasitoid. Of the nine molecules tested, (rac)-8 and compound 4 demonstrated efficacy against the fly. Exposure to the estimated LC90 of (rac)-8 and compound 4 resulted in a mortality rate of less than 20% for T. anastrephae without impairing the parasitoid's functional parasitism. The in silico predictions suggest that (rac)-8 and compound 4 target gamma amino butyric acid (GABA) receptors and transient receptor potential (TRP) channels of D. suzukii. However, only the reduced interaction with TRP channels in T. anastrephae demonstrated a potential reason for the selectivity of these compounds on the parasitoid. Our findings suggest the potential for integrating (rac)-8 and compound 4 into D. suzukii management practices.

Bioallethrin activates specific olfactory sensory neurons and elicits spatial repellency in Aedes aegypti.

BACKGROUND: Use of pyrethroid insecticides is a pivotal strategy for mosquito control globally. Commonly known for their insecticidal activity by acting on voltage-gated sodium channels, pyrethroids, such as bioallethrin and transfluthrin, are used in mosquito coils, emanators and other vaporizers to repel mosquitoes and other biting arthropods. However, whether specific olfactory receptor neurons are activated by pyrethroids to trigger spatial repellency remains unknown. RESULTS: We used behavioral and electrophysiological approaches to elucidate the mechanism of bioallethrin repellency in Aedes aegypti, a major vector of dengue, yellow fever, Zika and chikungunya viruses. We found that bioallethrin elicits spatial (i.e. non-contact) repellency and activates a specific type of olfactory receptor neuron in mosquito antennae. Furthermore, bioallethrin repellency is significantly reduced in a mosquito mutant of Orco, an obligate olfactory co-receptor that is essential for the function of odorant receptors (Ors). These results indicate that activation of specific Or(s) by bioallethrin contributes to bioallethrin repellency. In addition, bioallethrin repellency was reduced in a pyrethroid-resistant strain that carries two mutations in the sodium channel gene that are responsible for knockdown resistance (kdr) to pyrethroids, indicating a role of activation of sodium channels in bioallethrin repellency. CONCLUSION: Results from this study show that bioallethrin repellency is likely to be the result of co-activation of Or(s) and sodium channels. These findings not only contribute to our understanding of the modes of action of volatile pyrethroids in spatial repellency, but also provide a framework for developing new repellents based on the dual-target mechanism revealed. © 2021 Society of Chemical Industry.

Implications of Sublethal Insecticide Exposure and the Development of Resistance on Mosquito Physiology, Behavior, and Pathogen Transmission.

For many decades, insecticides have been used to control mosquito populations in their larval and adult stages. Although changes in the population genetics, physiology, and behavior of mosquitoes exposed to lethal and sublethal doses of insecticides are expected, the relationships between these changes and their abilities to transmit pathogens remain unclear. Thus, we conducted a comprehensive review on the sublethal effects of insecticides and their contributions to insecticide resistance in mosquitoes, with the main focus on pyrethroids. We discuss the direct and acute effects of sublethal concentrations on individuals and populations, the changes in population genetics caused by the selection for resistance after insecticide exposure, and the major mechanisms underlying such resistance. Sublethal exposures negatively impact the individual's performance by affecting their physiology and behavior and leaving them at a disadvantage when compared to unexposed organisms. How these sublethal effects could change mosquito population sizes and diversity so that pathogen transmission risks can be affected is less clear. Furthermore, despite the beneficial and acute aspects of lethality, exposure to higher insecticide concentrations clearly impacts the population genetics by selecting resistant individuals, which may bring further and complex interactions for mosquitoes, vertebrate hosts, and pathogens. Finally, we raise several hypotheses concerning how the here revised impacts of insecticides on mosquitoes could interplay with vector-mediated pathogens' transmission.

Behavioral and physiological responses of Drosophila melanogaster and D. suzukii to volatiles from plant essential oils.

BACKGROUND: Insects rely on their sense of smell to locate food and hosts, find mates and select sites for laying eggs. Use of volatile compounds, such as essential oils (EOs), to repel insect pests and disrupt their olfaction-driven behaviors has great practical significance in integrated pest management. However, our knowledge on the olfaction-based mechanisms of EO repellency is quite limited. RESULTS: We evaluated the repellency of peppermint oil and nine plant EO components in Drosophila melanogaster, a model insect for olfaction study, and D. suzukii, a major fruit crop pest. All nine volatiles, menthone, (-)-menthol, menthyl acetate, (R)-(+)-limonene, nerol, (+)-fenchone, (-)-α-thujone, camphor, norcamphor and peppermint oil, elicited repellency in D. melanogaster in a dose-dependent manner. Most of the compounds, except camphor, also elicited repellency in D. suzukii. Menthone, (R)-(+)-limonene and (+)-fenchone were the most potent repellents against D. suzukii. Repellency was reduced or abolished in two D. melanogaster mutants of the odorant receptor co-receptor (Orco), indicating that the observed repellency is odorant receptor (Or)-mediated. Repellency by peppermint oil, menthone, (R)-(+)-limonene, (-)-α-thujone and norcamphor also involves Or-independent mechanism(s). Single sensillum recording from both species revealed that common and distinct Ors and olfactory receptor neurons were activated by these compounds. CONCLUSIONS: The tested plant EO components evoke repellency by activating multiple Ors in both Drosophila species. Our study provides a foundation for further elucidation of the mechanism of EOs repellency and species-specific olfactory adaptations. © 2021 Society of Chemical Industry.

Sodium channel activation underlies transfluthrin repellency in Aedes aegypti.

BACKGROUND: Volatile pyrethroid insecticides, such as transfluthrin, have received increasing attention for their potent repellent activities in recent years for controlling human disease vectors. It has been long understood that pyrethroids kill insects by promoting activation and inhibiting inactivation of voltage-gated sodium channels. However, the mechanism of pyrethroid repellency remains poorly understood and controversial. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show that transfluthrin repels Aedes aegypti in a hand-in-cage assay at nonlethal concentrations as low as 1 ppm. Contrary to a previous report, transfluthrin does not elicit any electroantennogram (EAG) responses, indicating that it does not activate olfactory receptor neurons (ORNs). The 1S-cis isomer of transfluthrin, which does not activate sodium channels, does not elicit repellency. Mutations in the sodium channel gene that reduce the potency of transfluthrin on sodium channels decrease transfluthrin repellency but do not affect repellency by DEET. Furthermore, transfluthrin enhances DEET repellency. CONCLUSIONS/SIGNIFICANCE: These results provide a surprising example that sodium channel activation alone is sufficient to potently repel mosquitoes. Our findings of sodium channel activation as the principal mechanism of transfluthrin repellency and potentiation of DEET repellency have broad implications in future development of a new generation of dual-target repellent formulations to more effectively repel a variety of human disease vectors.

A dual-target molecular mechanism of pyrethrum repellency against mosquitoes.

Pyrethrum extracts from flower heads of Chrysanthemum spp. have been used worldwide in insecticides and repellents. While the molecular mechanisms of its insecticidal action are known, the molecular basis of pyrethrum repellency remains a mystery. In this study, we find that the principal components of pyrethrum, pyrethrins, and a minor component, (E)-ß-farnesene (EBF), each activate a specific type of olfactory receptor neurons in Aedes aegypti mosquitoes. We identify Ae. aegypti odorant receptor 31 (AaOr31) as a cognate Or for EBF and find that Or31-mediated repellency is significantly synergized by pyrethrin-induced activation of voltage-gated sodium channels. Thus, pyrethrum exerts spatial repellency through a novel, dual-target mechanism. Elucidation of this two-target mechanism may have potential implications in the design and development of a new generation of synthetic repellents against major mosquito vectors of infectious diseases.

Population genomics of Drosophila suzukii reveal longitudinal population structure and signals of migrations in and out of the continental United States.

Drosophila suzukii, or spotted-wing drosophila, is now an established pest in many parts of the world, causing significant damage to numerous fruit crop industries. Native to East Asia, D. suzukii infestations started in the United States a decade ago, occupying a wide range of climates. To better understand invasion ecology of this pest, knowledge of past migration events, population structure, and genetic diversity is needed. In this study, we sequenced whole genomes of 237 individual flies collected across the continental United States, as well as several sites in Europe, Brazil, and Asia, to identify and analyze hundreds of thousands of genetic markers. We observed strong population structure between Western and Eastern US populations, but no evidence of any population structure between different latitudes within the continental United States, suggesting that there are no broad-scale adaptations occurring in response to differences in winter climates. We detect admixture from Hawaii to the Western United States and from the Eastern United States to Europe, in agreement with previously identified introduction routes inferred from microsatellite analysis. We also detect potential signals of admixture from the Western United States back to Asia, which could have important implications for shipping and quarantine policies for exported agriculture. We anticipate this large genomic dataset will spur future research into the genomic adaptations underlying D. suzukii pest activity and development of novel control methods for this agricultural pest.

Sex-dependent locomotion and physiological responses shape the insecticidal susceptibility of parasitoid wasps.

The adaptive fitness of insect species can be shaped by how males and females respond, both physiologically and behaviorally, to environmental challenges, such as pesticide exposure. In parasitoid wasps, most toxicological investigations focus only on female responses (e.g., survival and especially parasitism abilities), leaving the male contributions to adaptive fitness (survival, locomotion, mate search) poorly investigated. Here, we evaluated the toxicity of the spinosyn insecticide spinosad against the South American fruit fly, Anastrepha fraterculus, and we used the parasitoid wasp Diachasmimorpha longicaudata (Ashmead) to evaluate whether sex-linked locomotory and physiological responses would influence the susceptibility of these organisms to spinosad. Our results revealed that D. longicaudata males were significantly more susceptible (median lethal time (LT50) = 24 h) to spinosad than D. longicaudata females (LT50 = 120 h), which may reflect the differences in their locomotory and physiological (e.g., respiratory) responses to mitigate insecticide exposure. Compared to D. longicaudata females, male wasps were lighter (P < 0.001), walked for longer distances (P < 0.001) and periods (P < 0.001), and exhibited higher sensilla densities in their tarsi (P = 0.008), which may facilitate their intoxication with the insecticide. These findings indicate that male parasitoids should not be exempt from insecticide selectivity tests, as these organisms can be significantly more affected by such environmental challenges than their female conspecifics.

A Phylogeographic Approach to the Drosophila suzukii (Diptera: Drosophilidae) Invasion in Brazil.

Biological invasions have reached large parts of the globe, due to human actions across the planet. Drosophila suzukii (Matsumura, 1931) is a globally invasive species, always associated with enormous and costly damage to agricultural crops. Native to Southeast Asia, D. suzukii recently (i.e., 2013) invaded and is dispersing through South America. Here, we used a phylogeographic approach based on the cytochrome c oxidase subunit I gene fragment to explore the invasion dynamics of D. suzukii populations in Brazil. We identified five haplotypes and moderate genetic diversity in Brazilian populations, which are undergoing demographic and spatial expansion. The analyses of molecular variance indicated a high genetic structure among the populations, which is partially explained by their morphoclimatic origin and invasion history. Drosophila suzukii expanded from southern to southeastern Brazil, aided by human-mediated transport of fruits from region to region. The sharing of haplotypes among Brazilian and other invaded regions of the world suggests a single invasion event of D. suzukii in Brazil, originating from previously invaded areas (e.g., North America and Europe). The rapid geographic dispersal and wide variety of fruits attacked by of D. suzukii require immediate implementation of control strategies (legal and phytosanitary) to manage this pest in Brazil.

Host Potential and Adaptive Responses of Drosophila suzukii (Diptera: Drosophilidae) to Barbados Cherries.

Biological invasions are a global threat to agricultural crops worldwide. In the Neotropical region, the spotted-wing Drosophila [Drosophila suzukii (Matsumura)] has rapidly expanded its geographical range spreading throughout South America in recent years. Besides climatic factors, the remarkable success of its establishment and subsequent distribution in this region is closely dependent on the diversity and availability of host plants. We evaluated the host potential (e.g., as food and oviposition sources) of fruits of jabuticaba [Plinia cauliflora (Mart.) Kausel (Myrtales: Myrtaceae)], Barbados cherry (Malpighia emarginata DC) (Malpighiales: Malpighiaceae), bonnet pepper (Capsicum chinense Jacq.) (Solanales : Solanaceae), and coffee (Coffea arabica L.) (Gentianales : Rubiaceae) and their effects on the biological and physiological traits of D. suzukii. For the fruit types where fly emergence occurred, we assessed the biological and physiological performance of the flies and compared these parameters with those recorded for flies reared on strawberries (Fragaria × ananassa Duchesne) (Rosales : Rosaceae) and an artificial diet. Our results revealed that oviposition into fruits and completion of the life cycle occurred on Barbados cherries only. Furthermore, field surveys revealed a higher emergence rate of D. suzukii on undamaged ripe Barbados cherries than damaged ones. Moreover, flies developing on Barbados cherries and an artificial diet presented earlier emergence, shorter developmental time, lower number of adults per female, and a female-biased sex ratio compared to flies developing on strawberries. Overall, our findings demonstrated suitability of Barbados cherry as a host for D. suzukii, which renders management of D. suzukii in Neotropical region an even more challenging task.

Toxicity to and egg-laying avoidance of Drosophila suzukii (Diptera: Drosophilidae) caused by an old alternative inorganic insecticide preparation.

BACKGROUND: The application of synthetic insecticides remains the most used tool for the management of spotted-wing drosophila, Drosophila suzukii (Matsumura) (Diptera: Drosophilidae). However, management of this pest in the organic production of soft-skinned fruits is a complex task due to the restricted number of registered products. Here, we assess the toxicity of lime sulfur and evaluate whether lime sulfur-treated strawberry plants affected the oviposition and development of D. suzukii. RESULTS: Lime sulfur exhibited adequate toxicity to D. suzukii (LC50 = 26.6 mL L-1 ) without phytotoxicity to strawberry plants. When D. suzukii females were exposed to lime sulfur-treated plants in no-choice bioassays, oviposition was significantly (t-test, P < 0.05) reduced compared with that on untreated plants. In free-choice bioassays, D. suzukii females laid significantly (paired t-test, P < 0.05) more eggs on untreated plants. Furthermore, in the free-choice bioassays, immature development was slower for adults that originated from eggs laid on lime sulfur-treated plants than from those laid on untreated plants. CONCLUSIONS: Lime sulfur showed adequate control and, therefore, has potential for use as a management tool against D. suzukii infestations in organic production systems. This old, alternative insecticide preparation not only caused adult fly mortality, but also reduced the number of eggs laid on lime sulfur-treated plants. © 2017 Society of Chemical Industry.

Toxicities and effects of insecticidal toxic baits to control Drosophila suzukii and Zaprionus indianus (Diptera: Drosophilidae).

BACKGROUND: Drosophila suzukii is a primary insect pest that causes direct damage to fruits with a thin epidermis such as strawberries, cherries and blueberries. In strawberry fields, the co-occurrence of D. suzukii and Zaprionus indianus has increased production losses. This study evaluated the toxicities and effects of insecticidal baits to control adults and larvae of both D. suzukii and Z. indianus. RESULTS: Organophosphate (dimethoate and malathion), spinosyn (spinosad and spinetoram), pyrethroid (lambda-cyhalothrin) and diamide (cyantraniliprole) insecticides exhibited high toxicity to both adults and larvae of D. suzukii and Z. indianus (mortality >80%) in topical and dip bioassays. However, when the insecticides were mixed with a feeding attractant, a positive effect was observed only for adults of D. suzukii. Insecticides containing neonicotinoids (acetamiprid and thiamethoxam) and pyrolle (chlorfenapyr) caused intermediate mortality to adults of D. suzukii (40-60%) and low mortality for Z. indianus (mortality <23%); however, these compounds reduced the larval infestation of the two species by 55-86%. Botanical (azadirachtin) and sulphur insecticides exhibited low toxicity (mortality <40%) on adults and larvae of both species. CONCLUSION: Dimethoate, malathion, spinosad, spinetoram, lambda-cyhalothrin and cyantraniliprole are highly toxic to both larvae and adults of D. suzukii and Z. indianus. The use of toxic baits for adults of D. suzukii could be an alternative in management of this species. © 2016 Society of Chemical Industry.

Susceptibility of Bonagota salubricola (Lepidoptera: Tortricidae) to Insecticides in Brazilian Apple Orchards: Implications for Resistance Management.

The Bonagota salubricola (Meyrick) is a major pest in apple orchards in Brazil, and chemical control has been the primary tool for insect management. To support the development of an insect resistance management (IRM) program, baseline studies of the susceptibility of a reference (laboratory) B. salubricola population were conducted; seven wild B. salubricola populations were monitored for susceptibility to insecticide; and the toxicity of some new chemicals to third-instar larvae and adults was evaluated by a leaf dip and ingestion bioassay, respectively. Neonates from the susceptible (laboratory) population exposed to insecticide showed an LC50 ranging from 0.34 (spinetoram) to 30.19 (novaluron) µg of a.i. ml(-1) (88.8-fold variation), so the diagnostic concentrations for an IRM program in Brazil based on the LC99 were as follows: 19.0 µg of a.i./ml chlorantraniliprole, 510.0 novaluron, 72.0 phosmet, 4.1 spinetoram, 12.8 spinosad, and 110.0 tebufenozide. Based on the LC99, significant differences were not observed in the susceptibility of the field and laboratory populations to chlorantraniliprole, phosmet, spinetoram, spinosad, and tebufenozide insecticides, but there were significant differences in the survival rates of the two populations to novaluron insecticide (3.3%). All insecticides at the diagnostic concentrations showed high toxicity to third-instar larvae (mortality rates between 73 to 97%). Phosmet, spinetoram, and spinosad insecticides were toxic to B. salubricola adults (mortality >85%), while chlorantraniliprole, novaluron, and tebufenozide insecticides caused mortality below 5%. The evaluated insecticides showed high toxicity to different developmental stages of B. salubricola, so the diagnostic concentrations may be used in IRM programs in Brazil.