Wild habitats adjoining farmland are potentially valuable sources of natural enemies, but also of pests. Here we tested the utility of birds as ‘sampling devices’, to identify the diversity of prey available to predators and particularly to screen for pests and natural enemies using natural ecosystems as refugia. Here we used PCR to amplify prey DNA from three sympatric songbirds foraging on small invertebrates in Phragmites reedbed ecosystems, namely the Reed Warbler (Acrocephalus scirpaceus), Sedge Warbler (Acrocephalus schoenobaenus) and Cetti's Warbler (Cettia cetti). A recently described general invertebrate primer pair was used for the first time to analyse diets. Amplicons were cloned and sequenced, then identified by reference to the Barcoding of Life Database and to our own sequences obtained from fresh invertebrates. Forty-five distinct prey DNA sequences were obtained from 11 faecal samples, of which 39 could be identified to species or genus. Targeting three warbler species ensured that species-specific differences in prey choice broadened the range of prey taken. Amongst the prey found in reedbeds were major pests (including the tomato moth Lacanobia oleracea) as well as many potentially valuable natural enemies including aphidophagous hoverflies and braconid wasps. Given the mobility of birds, this approach provides a practical way of sampling a whole habitat at once, providing growers with information on possible invasion by locally resident pests and the colonization potential of natural enemies from local natural habitats.

Triatomines are vectors that transmit the protozoan haemoflagellate Trypanosoma cruzi, the causative agent of Chagas disease. The aim of the current review is to provide a synthesis of the immune mechanisms of triatomines against bacteria, viruses, fungi and parasites to provide clues for areas of further research including biological control. Regarding bacteria, the triatomine immune response includes antimicrobial peptides (AMPs) such as defensins, lysozymes, attacins and cecropins, whose sites of synthesis are principally the fat body and haemocytes. These peptides are used against pathogenic bacteria (especially during ecdysis and feeding), and also attack symbiotic bacteria. In relation to viruses, Triatoma virus is the only one known to attack and kill triatomines. Although the immune response to this virus is unknown, we hypothesize that haemocytes, phenoloxidase (PO) and nitric oxide (NO) could be activated. Different fungal species have been described in a few triatomines and some immune components against these pathogens are PO and proPO. In relation to parasites, triatomines respond with AMPs, including PO, NO and lectin. In the case of T. cruzi this may be effective, but Trypanosoma rangeli seems to evade and suppress PO response. Although it is clear that three parasite-killing processes are used by triatomines – phagocytosis, nodule formation and encapsulation – the precise immune mechanisms of triatomines against invading agents, including trypanosomes, are as yet unknown. The signalling processes used in triatomine immune response are IMD, Toll and Jak-STAT. Based on the information compiled, we propose some lines of research that include strategic approaches of biological control.

The lack of biological knowledge of many invasive species remains as one of the greatest impediments to their management. Here I detail targeted research into the biology of the yellow crazy ant Anoplolepis gracilipes within northern Australia and detail how such knowledge can be used to improve the management outcomes for this species. I quantified nest location and density in three habitats, worker activity over 24 h, infestation expansion rate, seasonal variation of worker abundance and the timing of production of sexuals. Nests were predominantly (up to 68%) located at the bases of large trees, indicating that search efforts should focus around tree bases. Nest density was one nest per 22, 7.1 and 6.3 m2 in the three habitats, respectively. These data form the baselines for quantifying treatment efficacy and set sampling densities for post-treatment assessments. Most (60%) nests were underground, predominantly (89%) occurring in an open area rather than underneath a rock or log. Some seasonality was evident for nests within leaf litter, with most (83%) occurring during the ‘wet season’ (October–March). Of the underground nests, most were shallow, with 44% being less than 10 cm deep, and 67% being less than 20 cm deep. Such nest location and density information serves many management purposes, for improving detection, mapping and post-treatment assessments, and also provided strong evidence that carbohydrate supply was a major driver of A. gracilipes populations. Just over half of the nests (56%) contained queens. Of the 62 underground nests containing queens, most queens (80%) were located at the deepest chamber. When queens were present, most often (38%) only one queen was present, the most being 16. Queen number per nest was the lowest in July and August just prior to the emergence of virgin queens in September, with queen numbers then remaining steadily high until April. Nothing is known for any ant species about how the queen number per nest/colony affects treatment efficacy, but further research would no doubt yield important breakthroughs for treating ants. Activity occurred predominantly nocturnally, ceasing during mid-day. These activity data determined the critical threshold above which work must be conducted to be considered reliable, and also suggests that treatments are best applied in the afternoon. Total brood production peaked in February and was the lowest around August and September. These abundance data form the baselines for quantifying treatment efficacy, and may have implications for treatment efficacy. Males were found every month, predominantly between August and November. Queen pupae were found in September. The reproductive timing of sexuals determines the treatment schedule. Targeted, site-specific research such as that described here should be an integral part of any eradication program for invasive species to design knowledge-based treatment protocols and determine assessment benchmarks.

Classical biological control is a key method for managing populations of pests in long-lived crops such as plantation forestry. The execution of biological control programmes in general, as the evaluation of potential natural enemies remains, to a large extent, an empirical endeavour. Thus, characterizing specific cases to determine patterns that may lead to more accurate predictions of success is an important goal of the much applied ecological research. We review the history of introduction, ecology and behaviour of the parasitoid Ibalia leucospoides. The species is a natural enemy of Sirex noctilio, one of the most important pests of pine afforestation worldwide. We use an invasion ecology perspective given the analogy between the main stages involved in classical biological control and the biological invasion processes. We conclude that success in the establishment, a common reason of failure in biocontrol, is not a limiting factor of success by I. leucospoides. A mismatch between the spread capacity of the parasitoid and that of its host could nevertheless affect control at a regional scale. In addition, we suggest that given its known life history traits, this natural enemy may be a better regulator than suppressor of the host population. Moreover, spatial and temporal refuges of the host population that may favour the local persistence of the interaction probably reduce the degree to which S. noctilio population is suppressed by the parasitoid. We emphasize the fact that some of the biological attributes that promote establishment may negatively affect suppression levels achieved. Studies on established non-native pest–parasitoid interactions may contribute to defining selection criteria for classical biological control which may prove especially useful in integrated pest management IPM programmes of invasive forest insects.

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.

Spatial distribution studies in pest management identify the locations where pest attacks on crops are most severe, enabling us to understand and predict the movement of such pests. Studies on the spatial distribution of two mite species, however, are rather scarce. The mites Polyphagotarsonemus latus and Tetranychus bastosi are the major pests affecting physic nut plantations (Jatropha curcas). Therefore, the objective of this study was to measure the spatial distributions of P. latus and T. bastosi in the physic nut plantations. Mite densities were monitored over 2 years in two different plantations. Sample locations were georeferenced. The experimental data were analyzed using geostatistical analyses. The total mite density was found to be higher when only one species was present (T. bastosi). When both the mite species were found in the same plantation, their peak densities occurred at different times. These mites, however, exhibited uniform spatial distribution when found at extreme densities (low or high). However, the mites showed an aggregated distribution in intermediate densities. Mite spatial distribution models were isotropic. Mite colonization commenced at the periphery of the areas under study, whereas the high-density patches extended until they reached 30 m in diameter. This has not been reported for J. curcas plants before.

This is the first report of microsatellite markers (simple sequence repeats, SSR) for fall webworm, Hyphantria cunea (Drury) (Lepidoptera: Arctiidae), an important quarantine pest in some European and Asian countries. Here, we developed 48 microsatellite markers for H. cunea from SSR enrichment libraries. Sequences isolated from libraries were sorted into four categories and analyzed. Our results suggest that sequences classified as Grouped should not be used for microsatellite primer design. The genetic diversity of microsatellite loci was assessed in 72 individuals from three populations. The number of alleles per locus ranged from 2 to 5 with an average of 3. The observed and expected heterozygosities of loci ranged from 0 to 0.958 and 0 to 0.773, respectively. A total of 18 out of 153 locus/population combinations deviated significantly from Hardy–Weinberg equilibrium. Moreover, significant linkage disequilibrium was detected in one pair of loci (1275 pairs in total). In the neutral test, two loci were grouped into the candidate category for positive selection and the remainder into the neutral category. In addition, a complex mutation pattern was observed for these loci, and FST performed better than did RST for the estimation of population differentiation in different mutation patterns. The results of the present study can be used for population genetic studies of H. cunea.

Zizina emelina (de l'Orza) is listed on Japan's Red Data List as an endangered species because of loss of its principal food plant and habitat. We compared parts of the mitochondrial and nuclear genes of this species to investigate the level of genetic differentiation among the 14 extant populations. We also examined infection of the butterfly with the bacterium Wolbachia to clarify the bacterium's effects on the host population's genetic structure. Mitochondrial and nuclear DNA analyses revealed that haplotype composition differed significantly among most of the populations, and the fixation index FST was positively correlated with geographic distance. In addition, we found three strains of Wolbachia, one of which was a male killer; these strains were prevalent in several populations. There was linkage between some host mitochondrial haplotypes and the three Wolbachia strains, although no significant differences were found in a comparison of host mitochondrial genetic diversity with nuclear genetic diversity in Wolbachia-infected or -uninfected populations. These genetic analyses and Wolbachia infection findings show that Z. emelina has little migratory activity and that little gene flow occurs among the current populations.

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.

The brown planthopper Nilaparvata lugens is a serious phloem-feeding pest of rice in China. The current study focuses on a saccharopine dehydrogenase (SDH) that catalyzes the penultimate reaction in biosynthesis of the amino acid lysine (Lys), which plays a role in insect growth and carnitine production (as a substrate). The protein, provisionally designated as NlylsSDH [a SDH derived from yeast-like symbiont (YLS) in N. lugens], had a higher transcript level in abdomens, compared with heads, wings, legs and thoraces, which agrees with YLS distribution in N. lugens. Ingestion of Nlylssdh targeted double-stranded RNA (dsNlylssdh) for 5, 10 and 15 days decreased the mRNA abundance in the hoppers by 47, 70 and 31%, respectively, comparing with those ingesting normal or dsegfp diets. Nlylssdh knockdown slightly decreased the body weights, significantly delayed the development of females, and killed approximately 30% of the nymphs. Moreover, some surviving adults showed two apparent phenotypic defects: wing deformation and nymphal cuticles remained on tips of the legs and abdomens. The brachypterours/macropterours and sex ratios (female/male) of the adults on the dsRNA diet were lowered compared with the adults on diets without dsRNA. These results suggest that Nlylssdh encodes a functional SDH protein. The adverse effect of Nlylssdh knockdown on N. lugens implies the importance of Lys in hopper development. This study provides a proof of concept example that Nlylssdh could serve as a possible dsRNA-based pesticide for planthopper control.

The effects of two aphidophagous predators, the larvae of Chrysoperla carnea and adults of Adalia bipunctata, on the spread of cucumber mosaic virus (CMV) transmitted in a non-persistent manner by the cotton aphid Aphis gossypii were studied under semi-field conditions. Natural enemies and aphids were released inside insect-proof cages (1 m ×1 m ×1 m) with a central CMV-infected cucumber plant surrounded by 48 healthy cucumber seedlings, and the spatiotemporal dynamics of the virus and vector were evaluated in the short and long term (1 and 5 days) in the presence and absence of the natural enemy. The spatial analysis by distance indices methodology together with other indices measuring the dispersal around a single focus was used to assess the spatial pattern and the degree of association between the virus and its vector. Both natural enemies significantly reduced the number of aphids in the CMV-source plant after 5 days but not after 1 day. The CMV transmission rate was generally low, especially after 1 day, due to the limited movement of aphids from the central CMV-source plant, which increased slightly after 5 days. Infected plants were mainly located around the central virus-infected source plant, and the percentage of aphid occupation and CMV-infected plants did not differ significantly in absence and presence of natural enemies. The distribution patterns of A. gossypii and CMV were only coincident close to the central plant. The complexity of multitrophic interactions and the role of aphid predators in the spread of CMV are discussed.

Leafmining flies (Diptera: Agromyzidae) can be serious economic pests of horticultural crops. Some genera such as Liriomyza are particularly problematic with numerous species, some of which are highly polyphagous (wide host range), which can only be confidently identified morphologically from adult males. In our study, DNA barcoding was employed to establish new locality records of the vegetable leafminer fly, Liriomyza sativae, from the islands of Torres Strait (Queensland, Australia) and the central highlands of Papua New Guinea (PNG). These records represent significant range extensions of this highly invasive plant pest. Specimens of immature leafminers (from leaf mines) were collected over a 5-year period during routine plant health surveys in ethanol or on FTA® filter paper cards, both methods proved effective at preserving and transporting insect DNA under tropical conditions, with FTA cards possessing some additional logistical benefits. Specimens were identified through sequencing two sections of the cytochrome oxidase I gene and the utility of each was assessed for the identification of species and intra-specific genetic lineages. Our study indicates that multiple haplotypes of L. sativae occur in PNG, while a different haplotype is present in the Torres Strait, with genetic regionalization between these areas apart from a single possible instance – one haplotype ‘S.7’ appears to be common between these two regions – interestingly this has also been the most common haplotype detected in previous studies of invasive L. sativae populations. The DNA barcoding methods employed here not only identified multiple introductions of L. sativae, but also appear generally applicable to the identification of other agromyzid leafminers (Phytomyzinae and Agromyzinae) and should decrease the likelihood of potentially co-amplifying internal hymenopteran parasitoids. Currently, L. sativae is still not recorded from the Australian mainland; however, further sampling of leafminer flies from Northern Australia and surrounding areas is required, as surveillance for possible Liriomyza incursions, as well as to characterize endemic species with which Liriomyza species might be confused.

Because immune defenses are often costly employed, insect immunocompetence cannot be always maintained at its maximum level. Here, the oriental fruit fly, Bactrocera dorsalis (Hendel), was used as a study object to investigate how its immune defenses varied with the developmental stage change and mating access. Our data indicated that both phenoloxidase (PO) activity and antibacterial activity significantly increased from new larvae to pupae but decreased in adults after emergence. Furthermore, both the PO activity and antibacterial activity in the hemolymph of copulated male and female adults were dramatically higher than that of virgin male and female ones, respectively. It provided the evidence that copulation could increase the magnitude of immune defense in hemolymph of B. dorsalis. Together, these results suggest that B. dorsalis possess a flexible investment strategy in immunity to meet its specific needs based on the endo- and exogenous factors, such as their distinct food source and living environments.

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.

The presence of the yellow-necked drywood termite, Kalotermes flavicollis Fabr., has been reported along most of the Mediterranean coasts of Europe, Africa and Asia. While morphological and genetic data exist on European K. flavicollis populations from western and central Mediterranean regions, data on eastern Mediterranean Kalotermes are scarce, and no genetic data exist to date. In this study, we analyzed 17 Kalotermes sp. colonies from 11 localities in the Levant (Cyprus, Lebanon, and Israel), in order to characterize genetically (mitochondrial DNA: COII, 16S, and control region) these populations. We found that samples from the Levant are genetically different from K. flavicollis, with distance values falling in the range of interspecific distances. In the phylogeny of European Kalotermes populations, samples from the Levant form a clade of their own, sister to a clade including K. flavicollis and Kalotermes italicus. Inside the eastern Mediterranean clade, all the samples from Cyprus are included in a well-supported subclade, suggesting that the colonization of the island might have occurred in a single event. These findings show that the populations we examined do not belong to the species K. flavicollis, but to a new species peculiar to the Levant, that we describe as Kalotermes phoeniciae sp. nov. It is possible that previous reports of K. flavicollis in this region can be attributed to K. phoeniciae.

Glutathione S-transferases (GSTs, EC 2.5.1.18) are a family of super enzymes with multiple functions that play a major role in the detoxification of endogenous and xenobiotic compounds. In our previous study, we have predicted 23 putative cytosolic GSTs in the silkworm genome using bioinformatic methods. In this study, we cloned and studied the insect-specific epsilon-class GST gene GSTe4 from the silkworm, Bombyx mori. The recombinant BmGSTe4 (Bac-BmGSTe4) was overexpressed in SF-9 cell lines, and it was found to have effective GST activity. We also found that the expression of BmGSTe4 was especially down-regulated after the silkworms were fumigated with or ingested phoxim. Moreover, BmGSTe4 protected HEK293 cells against UV-induced cell apoptosis. These results demonstrated that BmGSTe4 has GST activity, is sensitive to phoxim, and plays a role in inhibition of UV-induced cell apoptosis.

The response of Grapholita molesta (Busck) males to three-component sex pheromone blends containing a 100% ratio of the major sex pheromone component, (Z)-8-dodecenyl acetate and a 10% ratio of (Z)-8-dodecenol, but with varying ratios of (E)-8-dodecenyl acetate (0.4, 5.4, 10.4, 30.4, and 100.1% E-blends) was tested with populations in eight stone and pome fruit orchards in Europe, Asia, and North and South America. Traps baited with the 5.4% E-blend caught significantly more males than traps with any other blend with all populations. Significantly more males were caught in traps baited with the 10.4% E-blend than in traps with the remaining blends, except with the 0.4% E-blend in Turkey. Significant differences in male moth catches occurred between the other blends with the 0.4>30.4% E-blend, and the 30.4>100.1% E-blend. Male moth catches with the 100.1% E-blend only differed from the hexane control in Chile. No apparent differences were noted to these blends in populations collected from pome or stone fruits. Flight tunnel assays to synthetic blends with a subset of populations were similar to the field results, but the breadth of the most attractive E-blends was wider. Flight tunnel assays also demonstrated a high level of male–female cross-attraction among field-collected populations. Female gland extracts from field-collected populations did not show any significant variation in their three-component blends. The only exceptions in these assays were that long-term laboratory populations were less responsive and attractive, and produced different blend ratios of the two minor components than recently collected field populations.

The soft scales (Hemiptera: Coccoidea: Coccidae) are a group of sap-sucking plant parasites, many of which are notorious agricultural pests. The quarantine and economic importance of soft scales necessitates rapid and reliable identification of these taxa. Nucleotide sequences of the mitochondrial cytochrome c oxidase subunit I (COI) gene (barcoding region) and 28S rDNA were generated from 340 individuals of 36 common soft scales in China. Distance-based [(best match, Automated Barcode Gap Discovery (ABGD)], tree-based (neighbor-joining, Bayesian inference), Klee diagrams, and general mixed Yule coalescent (GMYC) models were used to evaluate barcoding success rates in the data set. Best match showed that COI and 28S sequences could provide 100 and 95.52% correct identification, respectively. The average interspecific divergences were 19.81% for COI data and 20.38% for 28S data, and mean intraspecific divergences were 0.56 and 0.07%, respectively. For COI data, multiple methods (ABGD, Klee, and tree-based methods) resulted in general congruence with morphological identifications. However, GMYC analysis tended to provide more molecular operational taxonomic units (MOTUs). Twelve MOTUs derived from five morphospecies (Rhodococcus sariuoni, Pulvinaria vitis, Pulvinaria aurantii, Parasaissetia nigra, and Ceroplastes rubens) were observed using the GMYC approach. In addition, tree-based methods showed that 28S sequences could be used for species-level identification (except for Ceroplastes ceriferus – Ceroplastes pseudoceriferus), even with low genetic variation (<1%). This report demonstrates the robustness of DNA barcoding for species discrimination of soft scales with two molecular markers (COI and 28S) and provides a reliable barcode library and rapid diagnostic tool for common soft scales in China.

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.

Mortality estimates are central to understanding tsetse fly population dynamics, but are difficult to acquire from wild populations. They can be obtained from age distribution data but, with limited data, it is unclear whether the assumptions required to make the estimates are satisfied and, if not, how violations affect the estimates. We evaluate the assumptions required for existing mortality estimation techniques using long-term longitudinal ovarian dissection data from 144,106 female tsetse, Glossina pallidipes Austen, captured in Zimbabwe between 1988 and 1999. At the end of the hot-dry season each year, mean ovarian ages peaked, and maximum-likelihood mortality estimates declined to low levels, contrary to mark-recapture estimates, suggesting violations of the assumptions underlying the estimation technique. We demonstrate that age distributions are seldom stable for G. pallidipes at our study site, and hypothesize that this is a consequence of a disproportionate increase in the mortality of pupae and young adults at the hottest times of the year. Assumptions of age-independent mortality and capture probability are also violated, the latter bias varying with capture method and with pregnancy and nutritional status. As a consequence, mortality estimates obtained from ovarian dissection data are unreliable. To overcome these problems we suggest simulating female tsetse populations, using dynamical modelling techniques that make no assumptions about the stability of the age distribution.