Bemisia tabaci is one of the most damaging agricultural pests world-wide. Although its control is based on insecticides, B. tabaci has developed resistance against almost all classes of insecticides, including neonicotinoids. We employed an RNA-seq approach to generate genome wide expression data and identify genes associated with neonicotinoid resistance in Mediterranean (MED) B. tabaci (Q1 biotype). Twelve libraries from insecticide resistant and susceptible whitefly populations were sequenced on an Illumina Next-generation sequencing platform, and genomic sequence information of approximately 73 Gbp was generated. A reference transcriptome was built by de novo assembly and functionally annotated. A total of 146 P450s, 18 GSTs and 23 CCEs enzymes (unigenes) potentially involved in the detoxification of xenobiotics were identified, along with 78 contigs encoding putative target proteins of six different insecticide classes. Ten unigenes encoding nicotinic Acetylcholine Receptors (nAChR), the target of neoinicotinoids, were identified and phylogenetically classified. No nAChR polymorphism potentially related with the resistant phenotypes, was observed among the studied strains. DE analysis revealed that among the 550 differentially (logFC > 1) over-transcribed unigenes, 52 detoxification enzymes were over expressed including unigenes with orthologues in P450s, GSTs, CCE and UDP-glucuronosyltransferases. Eight P450 unigenes belonging to clades CYP2, CYP3 and CYP4 were highly up-regulated (logFC > 2) including CYP6CM1, a gene already known to confer imidacloprid resistance in B. tabaci. Using quantitative qPCRs, a larger screening of field MED B. tabaci from Crete with known neonicotinoid phenotype was performed to associate expression levels of P450s with resistance levels. Expression levels of five P450s, including CYP6CM1, were found associated with neonicotinoid resistance. However, a significant correlation was found only in CYP303 and CYP6CX3, with imidacloprid and acetamiprid respectively. Our work has generated new toxicological data and genomic resources which will significantly enrich the available dataset and substantially facilitate the molecular studies in MED B. tabaci. No evidence of target site neonicotinoid resistance has been found. Eight P450 unigenes, including CYP6CM1, were found significantly over-expressed in resistant B. tabaci. This study suggests at least two novel P450s (CYP303 and CYP6CX3) as candidates for their functional characterization as detoxification mechanisms of neonicotinoid resistance in B. tabaci.

The brown marmorated stink bug, Halyomorpha halys (Hemiptera: Pentatomidae), is an invasive species in North America and Europe that damages many different host plants. Substantial work has been conducted on the genetic diversity and invasion pathways of H. halys in some of the countries where it has been found, based on mitochondrial sequences. The main objective of the present study was to further explore the genetic diversity of invasive populations of H. halys exploiting both mitochondrial and nuclear markers. We used two molecular markers: the mitochondrial Cytochrome Oxidase I (COI) gene, an ideal standardized molecular marker for distinguishing closely related species, and the ribosomal Internal Transcribed Spacer 1 (ITS1), because only a few sequences of H. halys exist to this point in global databases. We used specimens from eight populations from Greece, Italy, Canada, and the US. Among the 14 haplotypes retrieved based on the mtCOI gene, two of them (H162–H163) were detected for the first time. These two haplotypes were found in specimens from Canada, Italy, and the US. Concerning the ITS1 region, 24 haplotypes were identified, with 15 being unique for a sampled population. In Greece and the US, 14 and 12 haplotypes were found, respectively, with 7 and 6 of them being unique for Greece and the US, respectively. Our analysis of the nuclear genes of H. halys indicates high genetic diversity of the invading populations in North America and Europe.

To predict further invasions of pests it is important to understand what factors contribute to the genetic structure of their populations. Cosmopolitan pest species are ideal for studying how different agroecosystems affect population genetic structure within a species at different climatic extremes. We undertook the first population genetic study of the greenhouse whitefly (Trialeurodes vaporariorum), a cosmopolitan invasive herbivore, and examined the genetic structure of this species in Northern and Southern Europe. In Finland, cold temperatures limit whiteflies to greenhouses and prevent them from overwintering in nature, and in Greece, milder temperatures allow whiteflies to inhabit both fields and greenhouses year round, providing a greater potential for connectivity among populations. Using nine microsatellite markers, we genotyped 1274 T. vaporariorum females collected from 18 greenhouses in Finland and eight greenhouses as well as eight fields in Greece.

The plant-pathogenic bacteriumXylella fastidiosawhich causes significant diseases to various plant species worldwide, is exclusively transmitted by xylem sap-feeding insects. Given the fact thatX. fastidiosaposes a serious potential threat for olive cultivation in Greece, the main aim of this study was to investigate the genetic variation of Greek populations of three spittlebug species (Philaenus spumarius,P. signatusandNeophilaenus campestris), by examining the molecular markers Cytochrome Oxidase I, cytochrome b and Internal Transcribed Spacer. Moreover, the infection status of the secondary endosymbiontsWolbachia,Arsenophonus,Hamiltonella,CardiniumandRickettsia, among these populations, was determined. According to the results, theITS2region was the less polymorphic, while the analyzed fragments ofCOIandcytbgenes, displayed high genetic diversity. The phylogenetic analysis placed the Greek populations ofP. spumariusinto the previously obtained Southwest clade in Europe. The analysis of the bacterial diversity revealed a diverse infection status.Rickettsiawas the most predominant endosymbiont whileCardiniumwas totally absent from all examined populations.Philaenus spumariusharboredRickettsia,Arsenophonus,HamiltonellaandWolbachia,N. campestriscarriedRickettsia,HamiltonellaandWolbachiawhileP. signatuswas infected only byRickettsia. The results of this study will provide an important knowledge resource for understanding the population dynamics of vectors ofX. fastidiosawith a view to formulate effective management strategies towards the bacterium.

Citrus tristeza virus (CTV) is a vector-borne virus that poses a significant threat to citrus production worldwide, inducing a variety of symptoms. Therefore, a detailed knowledge of local aphids, identification of viruliferous species, and the development of new monitoring tools are necessary to improve CTV control strategies. Herein, a 2-year survey was conducted to assess the frequency of aphid species infesting several citrus pilot orchards. Plot findings based on morphological and molecular identification revealed Aphis spiraecola (ranged from 44–100%) as the most abundant aphid species, followed by A. gossypii (<50%). Toxoptera aurantii, Myzus persicae, and A. craccivora were present in low numbers, and A. citricidus was not detected. Due to the absence of CTV detection in aphids and citrus trees from the pilot orchards, a complementary survey was conducted in CTV-infected fields. Three aphid species were identified as CTV-positive by RT-PCR, suggesting that they may be viruliferous, with A. spiraecola as predominant, followed by A. gossypii and T. aurantii. Additionally, we developed a non-invasive procedure for identifying aphid species using wingbeat analysis. This method provides a faster alternative to traditional identification techniques by taxonomic keys based on morphological features or PCR, although its accuracy is lower (approximately 95% for the two species tested). Overall, this work provides a detailed study of aphid species composition in citrus orchards, identifies the predominant local putative CTV vector, and introduces a novel sensor for aphid monitoring, contributing to improved epidemic forecasting and sustainable disease management strategies.