Functional genomics of a symbiotic community : shared traits in the olive fruit fly gut microbiota

The olive fruit fly Bactrocera oleae is a major pest of olives worldwide and houses a specialized gut microbiota dominated by the obligate symbiont “Candidatus Erwinia dacicola”. Ca. E. dacicola is thought to supplement dietary nitrogen to the host, with only indirect evidence for this hypothesis so far. Here, we sought to investigate the contribution of the symbiosis to insect fitness and explore the ecology of the insect gut. For this purpose, we examined the composition of bacterial communities associated with Cretan olive fruit fly populations, and inspected several genomes and one transcriptome assembly. We identified, and reconstructed the genome of, a novel component of the gut microbiota, Tatumella sp. TA1, which is stably associated with Mediterranean olive fruit fly populations. We also reconstructed a number of pathways related to nitrogen assimilation and interactions with the host. The results show that, despite variation in taxa composition of the gut microbial community, core functions related to the symbiosis are maintained. Functional redundancy between different microbial taxa was observed for genes involved in urea hydrolysis. The latter is encoded in the obligate symbiont genome by a conserved urease operon, likely acquired by horizontal gene transfer, based on phylogenetic evidence. A potential underlying mechanism is the action of mobile elements, especially abundant in the Ca. E. dacicola genome. This finding, along with the identification, in the studied genomes, of extracellular surface structure components that may mediate interactions within the gut community, suggest that ongoing and past genetic exchanges between microbes may have shaped the symbiosis

Transcriptomics and disease vector control

Next-generation sequencing can be used to compare transcriptomes under different conditions. A study in BMC Genomics applies this approach to investigating the effects of exposure to a range of xenobiotics on changes in gene expression in the larvae of Aedes aegypti, the mosquito vector of dengue fever

PCR-based detection of Plasmodium in Anopheles mosquitoes: a comparison of a new high-throughput assay with existing methods.

Published onlineComparative StudyEvaluation StudiesJournal ArticleResearch Support, Non-U.S. Gov'tBACKGROUND: Detection of the four malaria-causing Plasmodium species (Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale and Plasmodium malariae) within their mosquito hosts is an essential component of vector control programmes. Several PCR protocols have been developed for this purpose. Many of these methods, while sensitive, require multiple PCR reactions to detect and discriminate all four Plasmodium species. In this study a new high-throughput assay was developed and compared with three previously described PCR techniques. METHODS: A new assay based on TaqMan SNP genotyping was developed to detect all four Plasmodium species and discriminate P. falciparum from P. vivax, P. ovale and P. malariae. The sensitivity and the specificity of the new assay was compared to three alternative PCR approaches and to microscopic dissection of salivary glands in a blind trial of 96 single insect samples that included artificially infected Anopheles stephensi mosquitoes. The performance of the assays was then compared using more than 450 field-collected specimens that had been stored on silica gel, in ethanol or in isopropanol. RESULTS: The TaqMan assay was found to be highly specific when using Plasmodium genomic DNA as template. Tests of analytical sensitivity and the results of the blind trial showed the TaqMan assay to be the most sensitive of the four methods followed by the 'gold standard' nested PCR approach and the results generated using these two methods were in good concordance. The sensitivity of the other two methods and their agreement with the nested PCR and TaqMan approaches varied considerably. In trials using field collected specimens two of the methods (including the nested protocol) showed a high degree of non-specific amplification when using DNA derived from mosquitoes stored in ethanol or isopropanol. The TaqMan method appeared unaffected when using the same samples. CONCLUSION: This study describes a new high-throughput TaqMan assay that very effectively detects the four Plasmodium species that cause malaria in humans and discriminates the most deadly species, P. falciparum, from the others. This method is at least as sensitive and specific as the gold standard nested PCR approach and because it has no requirement for post-PCR processing is cheaper, simpler and more rapid to run. In addition this method is not inhibited by the storage of mosquito specimens by drying or in ethanol or isopropanol.BBSRCInnovative Vector Control Consortiu

Cytochrome P450associated with insecticide resistance catalyzes cuticular hydrocarbon production in Anopheles gambiae.

The role of cuticle changes in insecticide resistance in the major malaria vector Anopheles gambiae was assessed. The rate of internalization of 14C deltamethrin was significantly slower in a resistant strain than in a susceptible strain. Topical application of an acetone insecticide formulation to circumvent lipid-based uptake barriers decreased the resistance ratio by ∼50%. Cuticle analysis by electron microscopy and characterization of lipid extracts indicated that resistant mosquitoes had a thicker epicuticular layer and a significant increase in cuticular hydrocarbon (CHC) content (∼29%). However, the CHC profile and relative distribution were similar in resistant and susceptible insects. The cellular localization and in vitro activity of two P450 enzymes, CYP4G16 and CYP4G17, whose genes are frequently overexpressed in resistant Anopheles mosquitoes, were analyzed. These enzymes are potential orthologs of the CYP4G1/2 enzymes that catalyze the final step of CHC biosynthesis in Drosophila and Musca domestica, respectively. Immunostaining indicated that both CYP4G16 and CYP4G17 are highly abundant in oenocytes, the insect cell type thought to secrete hydrocarbons. However, an intriguing difference was indicated; CYP4G17 occurs throughout the cell, as expected for a microsomal P450, but CYP4G16 localizes to the periphery of the cell and lies on the cytoplasmic side of the cell membrane, a unique position for a P450 enzyme. CYP4G16 and CYP4G17 were functionally expressed in insect cells. CYP4G16 produced hydrocarbons from a C18 aldehyde substrate and thus has bona fide decarbonylase activity similar to that of dmCYP4G1/2. The data support the hypothesis that the coevolution of multiple mechanisms, including cuticular barriers, has occurred in highly pyrethroid-resistant An. gambiae.Fil: Balabanidou, Vasileia. Foundation for Research and Technology-Hellas; Grecia. Universidad de Creta; GreciaFil: Kampouraki, Anastasia. Universidad de Creta; GreciaFil: Mac Lean, Marina. University of Nevada; Estados UnidosFil: Blomquist, Gary J.. University of Nevada; Estados UnidosFil: Tittiger, Claus. University of Nevada; Estados UnidosFil: Juarez, Marta Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata ; ArgentinaFil: Mijailovsky, Sergio Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata ; ArgentinaFil: Chalepakis, George. Universidad de Creta; GreciaFil: Anthousi, Amalia. Universidad de Creta; GreciaFil: Lynd, Amy. Liverpool School of Tropical Medicine; Reino UnidoFil: Antoine, Sanou. Liverpool School of Tropical Medicine; Reino UnidoFil: Hemingway, Janet. Liverpool School of Tropical Medicine; Reino UnidoFil: Ranson, Hilary. Liverpool School of Tropical Medicine; Reino UnidoFil: Lycett, Gareth J.. Liverpool School of Tropical Medicine; Reino UnidoFil: Vontas, John. Foundation for Research and Technology-Hellas; Grecia. Agricultural University of Athens; Greci

The Vector Population Monitoring Tool (VPMT): High-Throughput DNA-Based Diagnostics for the Monitoring of Mosquito Vector Populations

Regular monitoring of mosquito vector populations is an integral component of most vector control programmes. Contemporary data on mosquito species composition, infection status, and resistance to insecticides are a prerequisite for effective intervention. For this purpose we, with funding from the Innovative Vector Control Consortium (IVCC), have developed a suite of high-throughput assays based on a single “closed-tube” platform that collectively comprise the “Vector Population Monitoring Tool” (VPMT). The VPMT can be used to screen mosquito disease vector populations for a number of traits including Anopheles gambiae s.l. and Anopheles funestus species identification, detection of infection with Plasmodium parasites, and identification of insecticide resistance mechanisms. In this paper we focus on the Anopheles-specific assays that comprise the VPMT and include details of a new assay for resistance todieldrin Rdl detection. The application of these tools, general and specific guidelines on their use based on field testing in Africa, and plans for further development are discussed

Neonicotinoid Insecticides Alter Induced Defenses and Increase Susceptibility to Spider Mites in Distantly Related Crop Plants

Chemical suppression of arthropod herbivores is the most common approach to plant protection. Insecticides, however, can cause unintended, adverse consequences for non-target organisms. Previous studies focused on the effects of pesticides on target and non-target pests, predatory arthropods, and concomitant ecological disruptions. Little research, however, has focused on the direct effects of insecticides on plants. Here we demonstrate that applications of neonicotinoid insecticides, one of the most important insecticide classes worldwide, suppress expression of important plant defense genes, alter levels of phytohormones involved in plant defense, and decrease plant resistance to unsusceptible herbivores, spider mites Tetranychus urticae (Acari: Tetranychidae), in multiple, distantly related crop plants.Using cotton (Gossypium hirsutum), corn (Zea mays) and tomato (Solanum lycopersicum) plants, we show that transcription of phenylalanine ammonia lyase, coenzyme A ligase, trypsin protease inhibitor and chitinase are suppressed and concentrations of the phytohormone OPDA and salicylic acid were altered by neonicotinoid insecticides. Consequently, the population growth of spider mites increased from 30% to over 100% on neonicotinoid-treated plants in the greenhouse and by nearly 200% in the field experiment.Our findings are important because applications of neonicotinoid insecticides have been associated with outbreaks of spider mites in several unrelated plant species. More importantly, this is the first study to document insecticide-mediated disruption of plant defenses and link it to increased population growth of a non-target herbivore. This study adds to growing evidence that bioactive agrochemicals can have unanticipated ecological effects and suggests that the direct effects of insecticides on plant defenses should be considered when the ecological costs of insecticides are evaluated

Functional validation of target-site resistance mutations against sodium channel blocker insecticides (SCBIs) via molecular modeling and genome engineering in Drosophila.

Sodium channel blocker insecticides (SCBIs) like indoxacarb and metaflumizone offer an alternative insecticide resistance management (IRM) strategy against several pests that are resistant to other compounds. However, resistance to SCBIs has been reported in several pests, in most cases implicating metabolic resistance mechanisms, although in certain indoxacarb resistant populations of Plutella xylostella and Tuta absoluta, two mutations in the domain IV S6 segment of the voltage-gated sodium channel, F1845Y and V1848I have been identified, and have been postulated through in vitro electrophysiological studies to contribute to target-site resistance. In order to functionally validate in vivo each mutation in the absence of confounding resistance mechanisms, we have employed a CRISPR/Cas9 strategy to generate strains of Drosophila melanogaster bearing homozygous F1845Y or V1848I mutations in the para (voltage-gated sodium channel) gene. We performed toxicity bioassays of these strains compared to wild-type controls of the same genetic background. Our results indicate both mutations confer moderate resistance to indoxacarb (RR: 6-10.2), and V1848I to metaflumizone (RR: 8.4). However, F1845Y confers very strong resistance to metaflumizone (RR: >3400). Our molecular modeling studies suggest a steric hindrance mechanism may account for the resistance of both V1848I and F1845Y mutations, whereby introducing larger side chains may inhibit metaflumizone binding

Identification and geographical distribution of pyrethroid resistance mutations in the poultry red mite Dermanyssus gallinae

The poultry red mite (PRM) Dermanyssus gallinae is the most common ectoparasite on poultry and causes high economic losses in poultry farming worldwide. Pyrethroid acaricides have been widely used for its control and, consequently, pyrethroid resistance has arisen. In this study we aim to investigate the occurrence of resistance and study the geographical distribution of pyrethroid resistance mutations across PRM populations in Europe

Transcriptomic analysis of resistance and short-term induction response to pyrethroids, in Anopheles coluzzii legs.

BACKGROUND:Insecticide-treated bed nets and indoor residual spraying comprise the major control measures against Anopheles gambiae sl, the dominant vector in sub-Saharan Africa. The primary site of contact with insecticide is through the mosquitoes' legs, which represents the first barrier insecticides have to bypass to reach their neuronal targets. Proteomic changes and leg cuticle modifications have been associated with insecticide resistance that may reduce the rate of penetration of insecticides. Here, we performed a multiple transcriptomic analyses focusing on An. coluzzii legs. RESULTS:Firstly, leg-specific enrichment analysis identified 359 genes including the pyrethroid-binder SAP2 and 2 other chemosensory proteins, along with 4 ABCG transporters previously shown to be leg enriched. Enrichment of gene families included those involved in detecting chemical stimuli, including gustatory and ionotropic receptors and genes implicated in hydrocarbon-synthesis. Subsequently, we compared transcript expression in the legs of a highly resistant strain (VK7-HR) to both a strain with very similar genetic background which has reverted to susceptibility after several generations without insecticide pressure (VK7-LR) and a lab susceptible population (NG). Two hundred thirty-two differentially expressed genes (73 up-regulated and 159 down-regulated) were identified in the resistant strain when compared to the two susceptible counterparts, indicating an over-expression of phase I detoxification enzymes and cuticular proteins, with decrease in hormone-related metabolic processes in legs from the insecticide resistant population. Finally, we analysed the short-term effect of pyrethroid exposure on An. coluzzii legs, comparing legs of 1 h-deltamethrin-exposed An. coluzzii (VK7-IN) to those of unexposed mosquitoes (VK7-HR) and identified 348 up-regulated genes including those encoding for GPCRs, ABC transporters, odorant-binding proteins and members of the divergent salivary gland protein family. CONCLUSIONS:The data on An. coluzzii leg-specific transcriptome provides valuable insights into the first line of defense in pyrethroid resistant and short-term deltamethrin-exposed mosquitoes. Our results suggest that xenobiotic detoxification is likely occurring in legs, while the enrichment of sensory proteins, ABCG transporters and cuticular genes is also evident. Constitutive resistance is primarily associated with elevated levels of detoxification and cuticular genes, while short-term insecticide-induced tolerance is linked with overexpression of transporters, GPCRs and GPCR-related genes, sensory/binding and salivary gland proteins

Contemporary status of insecticide resistance in the major Aedes vectors of arboviruses infecting humans (PLoS Negl Trop Dis)

Publisher Copyright: © 2021 Moyes et al.After the publication of this article [1] the authors noticed citation errors in Table 2. The citations for item 5 listed under pyrethroids and items 2, 3, and 4 listed under temephos refer to the wrong references and these citations have been corrected in the updated Table 2 below. The citations for items 1, 3 and 4 listed under pyrethroids and item 1 listed under temephos are also incorrect and should cite references that have been omitted from the reference list. These citations have been corrected in the updated Table 2 below and the following corre-sponding references 79–82 should be added to the reference list: 79. Bariami V, Jones CM, Poupardin R, Vontas J, Ranson H. Gene amplification, ABC trans-porters and cytochrome P450s: unraveling the molecular basis of pyrethroid resistance in the dengue vector, Aedes aegypti. PLoS Negl Trop Dis. 2012;6: e1692. pmid:22720108 80. Saavedra-Rodriguez K, Suarez AF, Salas IF, Strode C, Ranson H, Hemingway J, et al. Transcription of detoxification genes after permethrin selection in the mosquito Aedes aegypti. Insect Mol Biol. 2012;21: 61–77. pmid:22032702 81. David J-P, Faucon F, Chandor-Proust A, Poupardin R, Riaz MA, Bonin A, et al. Comparative analysis of response to selection with three insecticides in the dengue mosquito Aedes aegypti using mRNA sequencing. BMC Genomics. 2014;15: 174. pmid:24593293 82. Strode C, de Melo-Santos M, Magalhaes T, Araujo A, Ayres C. Expression profile of genes during resistance reversal in a temephos selected strain of the dengue vector, Aedes aegypti. PloS One. 2012;7: e39439. pmid: 22870187.publishersversionpublishe