Among the invasive mosquitoes registered all over the world, Aedes species are particularly frequent and important. As several of them are potential vectors of disease, they present significant health concerns for 21st century Europe. Five species have established in mainland Europe, with two (Aedes albopictus and Aedes japonicus) becoming widespread and two (Ae. albopictus and Aedes aegypti) implicated in disease transmission to humans in Europe. The routes of importation and spread are often enigmatic, the ability to adapt to local environments and climates are rapid, and the biting nuisance and vector potential are both an ecomonic and public health concern. Europeans are used to cases of dengue and chikungunya in travellers returning from the tropics, but the threat to health and tourism in mainland Europe is substantive. Coupled to that are the emerging issues in the European overseas territorities and this paper is the first to consider the impacts in the remoter outposts of Europe. If entomologists and public health authorities are to address the spread of these mosquitoes and mitigate their health risks they must first be prepared to share information to better understand their biology and ecology, and share data on their distribution and control successes. This paper focusses in greater detail on the entomological and ecological aspects of these mosquitoes to assist with the risk assessment process, bringing together a large amount of information gathered through the ECDC VBORNET project.

The distribution of phlebotomine sand flies is widely reported to be changing in Europe. This can be attributed to either the discovery of sand flies in areas where they were previously overlooked (generally following an outbreak of leishmaniasis or other sand fly-related disease) or to true expansion of their range as a result of climatic or environmental changes. Routine surveillance for phlebotomines in Europe is localized, and often one of the challenges for entomologists working in non-leishmaniasis endemic countries is the lack of knowledge on how to conduct, plan and execute sampling for phlebotomines, or how to adapt on-going sampling strategies for other haematophagous diptera. This review brings together published and unpublished expert knowledge on sampling strategies for European phlebotomines of public health concern in order to provide practical advice on: how to conduct surveys; the collection and interpretation of field data; suitable techniques for the preservation of specimens obtained by different sampling methods; molecular techniques used for species identification; and the pathogens associated with sand flies and their detection methods.

Aedes aegypti (L.) (Diptera: Culicidae), the main vector of dengue and urban yellow fever in the world, is highly adapted to the human environment. Artificial containers are the most common larval habitat for the species, but it may develop in tree holes and other phytotelmata. This study assessed whether tree holes in San Ramón de la Nueva Orán, a city located in subtropical montane moist forest where dengue outbreaks occur, are relevant as larval habitat for Ae. aegypti and if the species may be found in natural areas far from human habitations. Water holding tree holes were sampled during 3 years once a month along the rainy season using a siphon bottle, in urban and suburban sites within the city and in adjacent forested areas. Larvae and pupae were collected and the presence and volume of water in each tree hole were recorded. Finding Ae. aegypti in forested areas was an isolated event; however, the species was frequently collected from tree holes throughout the city and along the sampling period. Moreover, larvae were collected in considerably high numbers, stressing the importance of taking into account these natural cavities as potential reinfestation foci within dengue control framework.

Studies on insect natural enemies and their effects on host populations are of immense practical value in pest management. Predation and parasitism on a citrus pest, the leafminer Phyllocnistis citrella Stainton, were evaluated by sampling over 3 years in four locations within a world leading lemon producing area in Northwest Argentina. Both mortality factors showed seasonal trends consistent across locations, with predation exerting earlier and more sustained pressure than parasitism, which showed wider seasonal variations. The dominant parasitoids, native Cirrospilus neotropicus and introduced Ageniaspis citricola, showed different seasonal trends: C. neotropicus was dominant in spring whereas A. citricola superseded it in autumn and winter. Although parasitism rates were relatively low, the native C. neotropicus revealed favourable features as potential control agent, by showing density-dependence, parasitism rates comparable with those of the specific A. citricola during part of the cycle, and earlier synchronization with the host. The study provides highly relevant information for a sustainable management of this worldwide pest, for which biological control is considered the best long-term option.

Parthenogenesis is the main mode of reproduction of aphids. Their populations are therefore composed of clones whose frequency distribution varies in space and time. Previous population genetic studies on aphids have highlighted the existence of highly abundant clones (‘super-clones’), distributed over large geographic areas and persisting over time. Whether the abundance of ‘super-clones’ results from their ecological success or from stochastic forces, such as drift and migration, is an open question. Here, we looked for the existence of clines in clonal frequency along a climatic gradient in the cereal aphid Rhopalosiphum padi (Linnaeus, 1758) and examined the possible influence of geographical distance and environmental variables in the buildup and maintenance of such clonal clines. We investigated the spatial distribution of the commonest genotypes of R. padi by sampling populations along an east–west transect in maize fields in the northern half of France in both spring and late summer. Individual aphids were genotyped at several polymorphic loci, allowing the assessment of frequency distributions of multilocus genotypes (MLGs) across the cropping season. We found several MLGs showing longitudinal clines in their frequency distribution in both spring and summer. In particular, two dominant asexual genotypes of R. padi showed inverted geographical clines, which could suggest divergent adaptations to environmental conditions. We concluded that while the distribution of some ‘super-clones’ of R. padi seems most likely driven by the action of migration and genetic drift, selection could be also involved in the establishment of longitudinal clines of others.

In the present work, we carried out a comparative molecular study of Stenoponia tripectinata tripectinata isolated from Mus musculus from the Canary Islands, Spain. The Internal Transcribed Spacers 1 and 2 (ITS1, ITS2) and 18S ribosomal RNA partial gene and cytochrome c-oxidase 1 (cox1) mitochondrial DNA partial gene sequences of this subspecies were determined to clarify the taxonomic status of this subspecies and to assess inter-population variation and inter-specific sequence differences. In addition, we have carried out a comparative phylogenetic study with other species of fleas using Bayesian, Maximum Parsimony, Maximum Likelihood and Neighbor-Joining analysis. A geographical signal was detected between the cox1 partial gene sequences of S. t. tripectinata isolated from M. musculus from different islands and those isolated from Apodemus sylvaticus from the Iberian Peninsula. Our results assess the monophyletic origin of Stenoponiinae and a different genetic lineage from Ctenophthalmidae. Thus, the elevation of subfamily Stenoponiinae to family level (Stenoponiidae) is suggested.

Aphids are important agricultural pests worldwide. Their control is largely based on chemical insecticides. One species that shows important invasive abilities and host-plant-related differences is Therioaphis trifolii (Monell) (Hemiptera: Aphididae). T. trifolii maculata, also known as spotted alfalfa aphid (SAA), can be very injurious to alfalfa crops in certain regions, such as in Saudi Arabia for effective control it is essential to diagnose and monitor the resistance mechanisms in the SAA populations. In the present study, we analysed acetylcholinesterase (ace) target site insensitivity mechanisms. A 650 bp length DNA containing the putative acetylcholinesterase (ace1) precursor was obtained and compared with other Hemipteran species. The sequences of many individual aphids collected from alfalfa crops in Saudi Arabia were analysed for the presence of resistance mutations: no resistance mutations were found at the resistance mutation loci 302; however, the presence of a serine–phenylalanine substitution (S431F) was identified in one individual. The S431F substitution, has been shown to confer significant levels of both organophosphate and carbamate resistance in other aphid species, and is now found for the first time in T. trifolii. We subsequently developed a simple polymerase chain reaction–restriction fragment length polymorphism assays for the S431F mutation, using a TaqI restriction site destroyed by the S431F mutation. The novel diagnostic assay may support the implementation of Insecticide Resistance Management strategies, for the control of SAA in alfalfa crops in the Kingdom of Saudi Arabia, and other countries worldwide.

Metabarcoding, the coupling of DNA-based species identification and high-throughput sequencing, offers enormous promise for arthropod biodiversity studies but factors such as cost, speed and ease-of-use of bioinformatic pipelines, crucial for making the leapt from demonstration studies to a real-world application, have not yet been adequately addressed. Here, four published and one newly designed primer sets were tested across a diverse set of 80 arthropod species, representing 11 orders, to establish optimal protocols for Illumina-based metabarcoding of tropical Malaise trap samples. Two primer sets which showed the highest amplification success with individual specimen polymerase chain reaction (PCR, 98%) were used for bulk PCR and Illumina MiSeq sequencing. The sequencing outputs were subjected to both manual and simple metagenomics quality control and filtering pipelines. We obtained acceptable detection rates after bulk PCR and high-throughput sequencing (80–90% of input species) but analyses were complicated by putative heteroplasmic sequences and contamination. The manual pipeline produced similar or better outputs to the simple metagenomics pipeline (1.4 compared with 0.5 expected:unexpected Operational Taxonomic Units). Our study suggests that metabarcoding is slowly becoming as cheap, fast and easy as conventional DNA barcoding, and that Malaise trap metabarcoding may soon fulfill its potential, providing a thermometer for biodiversity.

Granivory can play a pivotal role in influencing regeneration, colonization as well as abundance and distribution of plants. Due to their high abundance, nutrient content and longevity, seeds are an important food source for many animals. Among insects, carabid beetles consume substantial numbers of seeds and are thought to be responsible for a significant amount of seed loss. However, the processes that govern which seeds are eaten and are therefore prevented from entering the seedbank are poorly understood. Here, we assess if DNA-based diet analysis allows tracking the consumption of seeds by carabids. Adult individuals of Harpalus rufipes were fed with seeds of Taraxacum officinale and Lolium perenne allowing them to digest for up to 3 days. Regurgitates were tested for the DNA of ingested seeds at eight different time points post-feeding using general and species-specific plant primers. The detection of seed DNA decreased with digestion time for both seed species, albeit in a species-specific manner. Significant differences in overall DNA detection rates were found with the general plant primers but not with the species-specific primers. This can have implications for the interpretation of trophic data derived from next-generation sequencing, which is based on the application of general primers. Our findings demonstrate that seed predation by carabids can be tracked, molecularly, on a species-specific level, providing a new way to unravel the mechanisms underlying in-field diet choice in granivores.

Obolodiplosis robiniae is native to North America and is an important introduced insect pest that forms leaf margin roll galls on species of genus Robinia (Fabaceae) in China. It was first detected in China in 2004, but subsequently spread and provoked local outbreaks. An analysis of a 676-bp sequence of the mitochondrial DNA cytochrome oxidase subunit I was conducted in 560 individuals from 28 populations, in order to (1) assess population genetic structuring and (2) explore possible explanations for the rapid spread and invasion success of O. robiniae. Yet, only four haplotypes were identified and the nucleotide diversity was low (π = 0.00005) and among the 560 specimens studied, only ten showed haplotypic variation involving no more than three substitutions. The result showed a low degree of genetic diversity among populations of the successful invasive gall midge, which suggested that the pest experienced a severe genetic bottleneck and a loss of genetic diversity after its introduction. The successful establishment and spread of O. robiniae in China is attributed to the wide distribution of its host plant, thus allowing ample opportunities for gene flow in the pest species, and to the advantageous life history characteristics of O. robiniae.

Anastepha ludens (Diptera: Tephritidae) is a highly polyphagous fruit fly that is able to develop in a wide range of hosts. Understanding the limits of this pest's host range could provide valuable information for pest management and plant breeding for pest resistance. Previous studies have shown that guavas (Psidium guajava (Myrtaceae) L.), are not attacked under natural conditions by A. ludens. To understand this phenomenon, guavas were exposed to natural infestation by A. ludens and to other fruit fly species that infest guavas in nature (Anastrepha striata Schiner, Anastepha fraterculus (Wiedemann), Anastepha obliqua (Macquart)). Once the susceptible phenological stage of guavas was determined, fruit infestation levels were compared between A. ludens and A. striata. Choice and non-choice tests were performed under field-cage conditions. Under field conditions, guavas were susceptible to A. striata and A. fraterculus attack all the way from when fruit was undeveloped to when fruit began to ripen. No infestation by A. ludens was recorded under natural conditions. Similar results were obtained when forced exposures were performed, indicating that unripe guavas were preferred by A. striata over ripe fruit, and that infestation rates were higher at early fruit maturity stages. Under forced oviposition conditions, A. ludens larvae were unable to develop in unripe guavas but did so in fully ripe fruit. However, A. ludens fitness parameters were dramatically affected, exhibiting reduced survival and reduced pupal weight compared to conspecifics that developed in a natural host, grapefruit. We confirm that P. guajava should not be treated as a natural host of this pestiferous species, and suggest that both behavioral aspects and the fact that larvae are unable to adequately develop in this fruit, indeed represent clear limits to A. ludens's broad host range.

Planthoppers are important worldwide crop pests as well as vectors of numerous diseases. Different species transmit Mal de Río Cuarto virus, which causes the most economically important corn disease in central Argentina. Epidemiological studies rely on the accurate identification of the species present in the field. Presently, morphological identification of planthoppers requires taxonomic expertise and there are no taxonomic keys for females and nymphs. Nevertheless, no molecular protocols are available for accurate species identification of most frequent delphacid species from central Argentina. In this context, the aim of this study was to evaluate the utility of the cytochrome oxidase I gene (COI) as a DNA barcode and its digestion with restriction enzymes (Restriction Fragment Length Polymorphism, RFLP) for the identification of the most common species of planthoppers in central Argentina. We amplified and sequenced a 843 bp fragment of the COI gene of taxonomically identified specimens and evaluated its use as a DNA barcode. Restriction enzymes were also selected for digesting the COI fragment via RFLP. The high interspecific variability (20.79%; ± 2.32%) and low intraspecific divergence (0.12%; ± 0.17%) observed in the studied species, demonstrate the effectiveness of the COI gene for species identification of major vector delphacids affecting corn crops in Argentina. Moreover, the digestion of this COI gene fragment with Bfa I and Apo I enzymes allows a fast and cost-effective species identification method when numerous specimens need to be processed. Both molecular techniques developed here, allow the accurate identification of planthopper species at regional scale. These new tools would assist traditional identification of these insects, especially for aiding non-experts in morphological taxonomy.

Understanding the biological parameters of some triatomine subspecies of Meccus phyllosomus (Burmeister) is a crucial first step in estimating the epidemiological importance of this group. Biological parameters related to egg eclosion, egg-to-adult development time, number of blood meals to moult, percentage of females at the end of the cycle, number of laid eggs, and the accumulative mortality for each instar of three M. phyllosomus subspecies [Meccus phyllosomus pallidipennis (Stål), Meccus phyllosomus longipennis (Usinger), and Meccus phyllosomus picturatus (Usinger)] as well as their laboratory hybrids were evaluated and compared. No significant differences (P > 0.05) were recorded among the experimental hybrids (M. p. longipennis × M. p. pallidipennis, M. p. longipennis × M. p. picturatus, M. p. pallidipennis × M. p. picturatus) and reciprocal cohorts. In five of the six studied parameters (egg eclosion, egg-to-adult development time, number of blood meals to moult, number of laid eggs and accumulative mortality), with the exception of the non-significant percentage of females obtained among all the studied cohorts, at least one of the parental cohorts in each set of crosses exhibited better fitness results than by those of their hybrid descendants. The lack of hybrid fitness in our study indicates the maintenance of reproductive isolation of parental genotypes. Moreover, the results lead us to propose that an incipient speciation process by distance is currently developing among the three studied subspecies, increasing the differences between them that modify the transmission efficiency of Trypanosoma cruzi to human beings in Mexico.

Quercetin is ubiquitous in terrestrial plants. The cotton bollworm Helicoverpa armigera as a highly polyphagous insect has caused severe crop losses. Until now, interactions between this pest and quercetin are poorly understood at the biochemical and molecular levels. In this study, we investigated the in vivo effects of quercetin on performance of cotton bollworm and on cytochrome P450 (P450) expression. Deleterious effects of quercetin on the performance of the cotton bollworm, including growth, survival, pupation and adult emergence were observed after oral administration of 3 and 10 mg g−1 quercetin to larvae since the third instar, whereas no significant toxic effect was found at 0.1 mg g−1 quercetin treatment. Piperonyl butoxide treatment enhanced the toxicity of quercetin. In vitro metabolism studies showed that quercetin was rapidly transformed by gut enzymes of fifth instar larvae of the cotton bollworm. qRT–PCR results revealed that the effect of quercetin on P450 expression was tissue- and dose-specific. Quercetin regulated P450 expression in a mild manner, and it could serve as P450 inducer (CYP337B1, CYP6B6) or repressor (CYP337B1, CYP6B7, CYP6B27, CYP9A14, CYP6AE11, and CYP4M7). These findings are important for advancing our understanding of the biochemical and molecular response of insects to plant toxins and have implications for a smart pest control.