Monitoring population and environmental parameters of invasive mosquito species in Europe.

To enable a better understanding of the overwhelming alterations in the invasive mosquito species (IMS), methodical insight into the population and environmental factors that govern the IMS and pathogen adaptations are essential. There are numerous ways of estimating mosquito populations, and usually these describe developmental and life-history parameters. The key population parameters that should be considered during the surveillance of invasive mosquito species are: (1) population size and dynamics during the season, (2) longevity, (3) biting behaviour, and (4) dispersal capacity. Knowledge of these parameters coupled with vector competence may help to determine the vectorial capacity of IMS and basic disease reproduction number (R0) to support mosquito borne disease (MBD) risk assessment. Similarly, environmental factors include availability and type of larval breeding containers, climate change, environmental change, human population density, increased human travel and goods transport, changes in living, agricultural and farming habits (e.g. land use), and reduction of resources in the life cycle of mosquitoes by interventions (e.g. source reduction of aquatic habitats). Human population distributions, urbanisation, and human population movement are the key behavioural factors in most IMS-transmitted diseases. Anthropogenic issues are related to the global spread of MBD such as the introduction, reintroduction, circulation of IMS and increased exposure to humans from infected mosquito bites. This review addresses the population and environmental factors underlying the growing changes in IMS populations in Europe and confers the parameters selected by criteria of their applicability. In addition, overview of the commonly used and newly developed tools for their monitoring is provided.

Two cases of Plasmodium falciparum malaria in the Netherlands without recent travel to a malaria-endemic country.

Recently, two patients of African origin were given a diagnosis of Plasmodium falciparum malaria without recent travel to a malaria-endemic country. This observation highlights the importance for clinicians to consider tropical malaria in patients with fever. Possible transmission routes of P. falciparum to these patients will be discussed. From a public health perspective, international collaboration is crucial when potential cases of European autochthonous P. falciparum malaria in Europe re considered.

Development of guidelines for the surveillance of invasive mosquitoes in Europe.

BACKGROUND: The recent notifications of autochthonous cases of dengue and chikungunya in Europe prove that the region is vulnerable to these diseases in areas where known mosquito vectors (Aedes albopictus and Aedes aegypti) are present. Strengthening surveillance of these species as well as other invasive container-breeding aedine mosquito species such as Aedes atropalpus, Aedes japonicus, Aedes koreicus and Aedes triseriatus is therefore required. In order to support and harmonize surveillance activities in Europe, the European Centre for Disease Prevention and Control (ECDC) launched the production of 'Guidelines for the surveillance of invasive mosquitoes in Europe'. This article describes these guidelines in the context of the key issues surrounding invasive mosquitoes surveillance in Europe. METHODS: Based on an open call for tender, ECDC granted a pan-European expert team to write the guidelines draft. It content is founded on published and grey literature, contractor's expert knowledge, as well as appropriate field missions. Entomologists, public health experts and end users from 17 EU/EEA and neighbouring countries contributed to a reviewing and validation process. The final version of the guidelines was edited by ECDC (Additional file 1). RESULTS: The guidelines describe all procedures to be applied for the surveillance of invasive mosquito species. The first part addresses strategic issues and options to be taken by the stakeholders for the decision-making process, according to the aim and scope of surveillance, its organisation and management. As the strategy to be developed needs to be adapted to the local situation, three likely scenarios are proposed. The second part addresses all operational issues and suggests options for the activities to be implemented, i.e. key procedures for field surveillance of invasive mosquito species, methods of identification of these mosquitoes, key and optional procedures for field collection of population parameters, pathogen screening, and environmental parameters. In addition, methods for data management and analysis are recommended, as well as strategies for data dissemination and mapping. Finally, the third part provides information and support for cost estimates of the planned programmes and for the evaluation of the applied surveillance process. CONCLUSION: The 'Guidelines for the surveillance of invasive mosquitoes in Europe' aim at supporting the implementation of tailored surveillance of invasive mosquito species of public health importance. They are intended to provide support to professionals involved in mosquito surveillance or control, decision/policy makers, stakeholders in public health and non-experts in mosquito surveillance. Surveillance also aims to support control of mosquito-borne diseases, including integrated vector control, and the guidelines are therefore part of a tool set for managing mosquito-borne disease risk in Europe.

Prevalence of Neoehrlichia mikurensis in ticks and rodents from North-west Europe.

BACKGROUND: Neoehrlichia mikurensis s an emerging and vector-borne zoonosis: The first human disease cases were reported in 2010. Limited information is available about the prevalence and distribution of Neoehrlichia mikurensis in Europe, its natural life cycle and reservoir hosts. An Ehrlichia-like schotti variant has been described in questing Ixodes ricinus ticks, which could be identical to Neoehrlichia mikurensis. METHODS: Three genetic markers, 16S rDNA, gltA and GroEL, of Ehrlichia schotti-positive tick lysates were amplified, sequenced and compared to sequences from Neoehrlichia mikurensis. Based on these DNA sequences, a multiplex real-time PCR was developed to specifically detect Neoehrlichia mikurensis in combination with Anaplasma phagocytophilum in tick lysates. Various tick species from different life-stages, particularly Ixodes ricinus nymphs, were collected from the vegetation or wildlife. Tick lysates and DNA derived from organs of wild rodents were tested by PCR-based methods for the presence of Neoehrlichia mikurensis. Prevalence of Neoehrlichia mikurensis was calculated together with confidence intervals using Fisher's exact test. RESULTS: The three genetic markers of Ehrlichia schotti-positive field isolates were similar or identical to Neoehrlichia mikurensis. Neoehrlichia mikurensis was found to be ubiquitously spread in the Netherlands and Belgium, but was not detected in the 401 tick samples from the UK. Neoehrlichia mikurensis was found in nymphs and adult Ixodes ricinus ticks, but neither in their larvae, nor in any other tick species tested. Neoehrlichia mikurensis was detected in diverse organs of some rodent species. Engorging ticks from red deer, European mouflon, wild boar and sheep were found positive for Neoehrlichia mikurensis. CONCLUSIONS: Ehrlichia schotti is similar, if not identical, to Neoehrlichia mikurensis. Neoehrlichia mikurensis is present in questing Ixodes ricinus ticks throughout the Netherlands and Belgium. We propose that Ixodes ricinus can transstadially, but not transovarially, transmit this microorganism, and that different rodent species may act as reservoir hosts. These data further imply that wildlife and humans are frequently exposed to Neoehrlichia mikurensis-infected ticks through tick bites. Future studies should aim to investigate to what extent Neoehrlichia mikurensis poses a risk to public health.

Aedes aegypti mosquitoes imported into the Netherlands, 2010.

During summer 2010, Aedes aegypti mosquitoes were discovered in the Netherlands. Using genetic markers, we tracked the origin of these mosquitoes to a tire shipment from Miami, Florida, USA. Surveillance of tire exports from the United States should be included as part of a comprehensive surveillance system.

Parasites of vectors--Ixodiphagus hookeri and its Wolbachia symbionts in ticks in The Netherlands.

BACKGROUND: Ixodiphagus hookeri is a parasitic wasp of ixodid ticks around the world. It has been studied as a potential bio-control agent for several tick species. We suspected that the presence of Wolbachia infected I. hookeri eggs in ticks is responsible for incidental detection of Wolbachia DNA in tick samples. METHODS: The 28S rRNA and 16S rRNA genes of a specimen of I. hookeri was amplified and sequenced. PCR on part of the 28S rRNA gene was used to detect parasitic wasp DNA in 349 questing Ixodes ricinus ticks from various sampling sites. Furthermore, the wsp gene of Wolbachia was sequenced from the I. hookeri specimen and a subset of ticks was tested using this marker. RESULTS: Several sequences from tick specimens were identical to the Wolbachia sequence of the I. hookeri specimen. Ixodiphagus hookeri was detected in 9.5% of all tested ticks, varying between 4% and 26% depending on geographic location. Ten out of eleven sampling sites throughout the Netherlands were positive for I. hookeri. Eighty-seven percent of I. hookeri-positive but only 1.6% of I. hookeri-negative ticks were Wolbachia positive. Detection of I. hookeri DNA was strongly associated with the detection of Wolbachia in ticks. CONCLUSION: This is the first reported case of I. hookeri in the Netherlands. Furthermore I. hookeri harbours Wolbachia species and is broadly distributed in the Netherlands. While detection of Wolbachia DNA in ticks might often be due to parasitism with this wasp, other sources of Wolbachia DNA in ticks might exist as well.

Towards an integrated approach in surveillance of vector-borne diseases in Europe.

Vector borne disease (VBD) emergence is a complex and dynamic process. Interactions between multiple disciplines and responsible health and environmental authorities are often needed for an effective early warning, surveillance and control of vectors and the diseases they transmit. To fully appreciate this complexity, integrated knowledge about the human and the vector population is desirable. In the current paper, important parameters and terms of both public health and medical entomology are defined in order to establish a common language that facilitates collaboration between the two disciplines. Special focus is put on the different VBD contexts with respect to the current presence or absence of the disease, the pathogen and the vector in a given location. Depending on the context, whether a VBD is endemic or not, surveillance activities are required to assess disease burden or threat, respectively. Following a decision for action, surveillance activities continue to assess trends.

Studies on Aedes albopictus larval mass-rearing optimization.

To set up a sterile male technique program to control Aedes albopictus (Skuse) in areas in northern Italy, a pilot mass-rearing facility is under development. For this purpose, experiments were carried out to find the optimal larval density for the optimization of the rearing parameters, i.e., to obtain the fastest larval development, the highest larval and pupal survival rate, and large-sized pupae. Several different larval densities, from 40 to 2874 larvae per liter, were tested. For densities from 40 to 600 larvae per liter significant size differences were found among pupae obtained under different larval densities. The larvae raised at the lowest density tended to be smaller and to develop most slowly, i.e., longer pupation time. Also, increasing water volume and depth seemed to negatively affect the pupation success. Compared with the other larval densities tested, the larvae reared at a density of 2874 larvae per liter developed slightly faster and showed higher survival rates, indicating this density as appropriate for the development of a mass rearing, at least using the current larval diet.

Entomopathogenic fungus as a biological control for an important vector of livestock disease: the Culicoides biting midge.

BACKGROUND: The recent outbreak of bluetongue virus in northern Europe has led to an urgent need to identify control measures for the Culicoides (Diptera: Ceratopogonidae) biting midges that transmit it. Following successful use of the entomopathogenic fungus Metarhizium anisopliae against larval stages of biting midge Culicoides nubeculosus Meigen, we investigated the efficacy of this strain and other fungi (Beauveria bassiana, Isaria fumosorosea and Lecanicillium longisporum) as biocontrol agents against adult C. nubeculosus in laboratory and greenhouse studies. METHODOLOGY/FINDINGS: Exposure of midges to 'dry' conidia of all fungal isolates caused significant reductions in survival compared to untreated controls. Metarhizium anisopliae strain V275 was the most virulent, causing a significantly decrease in midge survival compared to all other fungal strains tested. The LT(50) value for strain V275 was 1.42 days compared to 2.21-3.22 days for the other isolates. The virulence of this strain was then further evaluated by exposing C. nubeculosus to varying doses (10(8)-10(11) conidia m(-2)) using different substrates (horse manure, damp peat, leaf litter) as a resting site. All exposed adults were found to be infected with the strain V275 four days after exposure. A further study exposed C. nubeculosus adults to 'dry' conidia and 'wet' conidia (conidia suspended in 0.03% aq. Tween 80) of strain V275 applied to damp peat and leaf litter in cages within a greenhouse. 'Dry' conidia were more effective than 'wet' conidia, causing 100% mortality after 5 days. CONCLUSION/SIGNIFICANCE: This is the first study to demonstrate that entomopathogenic fungi are potential biocontrol agents against adult Culicoides, through the application of 'dry' conidia on surfaces (e.g., manure, leaf litter, livestock) where the midges tend to rest. Subsequent conidial transmission between males and females may cause an increased level of fungi-induced mortality in midges thus reducing the incidence of disease.

Introduction, scenarios for establishment and seasonal activity of Aedes albopictus in The Netherlands.

The Asian tiger mosquito Aedes albopictus was detected for the first time in the Netherlands in the summer of 2005. Aedes albopictus is a competent vector of several human viral diseases, and therefore the recent appearance of the vector is a concern to local public health authorities. In 2006 and 2007, the mosquito was found repeatedly and regularly at Lucky bamboo import companies. To assess whether imported Ae. albopictus could establish to produce subsequent generations in the following years or whether the winter conditions in the Netherlands would prove too cold to allow overwintering of diapausing eggs, predictions were made using a Geographic Information Systems (GIS) model based on January average temperature and the annual precipitation recorded in 2006. Seasonal activity of overwintering Ae. albopictus was estimated for temperate strains based on the weekly average temperature and weekly photoperiod using spring egg hatching thresholds of 10.5 degrees C and 11.25 hours, and egg diapause and adult survival thresholds of 9.5 degrees C and 13.5 hours. The analyses indicate that the climate conditions in the Netherlands over the past 10 years were favorable to allow overwintering of diapausing eggs of temperate strains of Ae. albopictus, particularly in the western coastal region. This region was also the area where adult Ae. albopictus were intercepted inside and surrounding plant glasshouses. The estimated number of weeks elapsing between first egg hatching in spring and the production of diapausing eggs in autumn ranged between 17 and 22 weeks in 2006.

The phenology and population dynamics of Culicoides spp. in different ecosystems in The Netherlands.

The Netherlands has enjoyed a relatively free state of vector-borne diseases of economic importance for more than one century. Emerging infectious diseases may change this situation, threatening the health of humans, domestic livestock and wildlife. In order to be prepared for the potential outbreak of vector-borne diseases, a study was undertaken to investigate the distribution and seasonal dynamics of candidate vectors of infectious diseases with emphasis on bluetongue vectors (Culicoides spp.). The study focused primarily on the relationship between characteristic ecosystems suitable for bluetongue vectors and climate, as well as on the phenology and population dynamics of these vectors. Twelve locations were selected, distributed over four distinct habitats: a wetland area, three riverine systems, four peat land areas and four livestock farms. Culicoides populations were sampled continuously using CO(2)-baited counterflow traps from July 2005 until August 2006, with an interruption from November 2005 to March 2006. All vectors were identified to species level. Meteorological and environmental data were collected at each location. Culicoides species were found in all four different habitat types studied. Wetland areas and peat bogs were rich in Culicoides spp. The taxonomic groups Culicoides obsoletus (Meigen) and Culicoides pulicaris (Linnaeus) were strongly associated with farms. Eighty-eight percent of all Culicoides consisted of the taxon C. obsoletus/Culicoides scoticus. On the livestock farms, 3% of Culicoides existed of the alleged bluetongue vector Culicoides dewulfi Goetghebuer. Culicoides impunctatus Goetghebuer was strongly associated with wetland and peat bog. Many Culicoides species were found until late in the phenological season and their activity was strongly associated with climate throughout the year. High annual variations in population dynamics were observed within the same study areas, which were probably caused by annual variations in environmental conditions. The study demonstrates that candidate vectors of bluetongue virus are present in natural and livestock-farm habitats in the Netherlands, distributed widely across the country. Under favourable climatic conditions, following virus introduction, bluetongue can spread among livestock (cattle, sheep and goats), depending on the nature of the viral serotype. The question now arises whether the virus can survive the winter conditions in north-western Europe and whether measures can be taken that effectively halt further spread of the disease.

African water storage pots for the delivery of the entomopathogenic fungus Metarhizium anisopliae to the malaria vectors Anopheles gambiae s.s. and Anopheles funestus.

We studied the use of African water storage pots for point source application of Metarhizium anisopliae against the malaria vectors Anopheles gambiae s.s. and An. funestus. Clay pots were shown to be attractive resting sites for male and female An. gambiae s.s. and were not repellent after impregnation with fungus. M. anisopliae was highly infective and virulent after spray application inside pots. At a dosage of 4 x 10(10) conidia/m(2), an average of 95 +/- 1.2% of An. gambiae s.s. obtained a fungal infection. A lower dosage of 1 x 10(10) conidia/m(2) infected an average of 91.5 +/- 0.6% of An. gambiae s.s. and 91.8 +/- 1.2% of An. funestus mosquitoes. Fungal infection significantly reduced mosquito longevity, as shown by differences between survival curves and LT(50) values. These pots are suitable for application of entomopathogenic fungi against malaria vectors and their potential for sustainable field implementation is discussed.

Infection of adult Aedes aegypti and Ae. albopictus mosquitoes with the entomopathogenic fungus Metarhizium anisopliae.

This study describes a laboratory investigation on the use of the insect-pathogenic fungus Metarhizium anisopliae against adult Aedes aegypti and Ae. albopictus mosquitoes. At a dosage of 1.6 x 10(10)conidia/m(2), applied on material that served as a mosquito resting site, an average of 87.1+/-2.65% of Ae. aegypti and 89.3+/-2.2% of Ae. albopictus became infected with the fungus. The life span of fungus-contaminated mosquitoes of both species was significantly reduced compared to uninfected mosquitoes. LT(50)-values of fungus-contaminated mosquitoes ranged between 3.1+/-0.2 days (male Ae. aegypti) and 4.1+/-0.3 days (female Ae. aegypti). LT(50)-values of uncontaminated mosquitoes ranged from 17.7+/-0.4 days (female Ae. albopictus) to 19.7+/-0.6 days (male Ae. albopictus). These results indicate that both mosquito species are highly susceptible to infection with this entomopathogen. Requirements for developing and incorporating this biological control method into current strategies to control major diseases vectored by these species, such as dengue fever, are discussed.

Infection of the malaria mosquito Anopheles gambiae with the entomopathogenic fungus Metarhizium anisopliae reduces blood feeding and fecundity.

The entomopathogenic fungus Metarhizium anisopliae is being considered as a biocontrol agent against adult African malaria vectors. In addition to causing significant mortality, this pathogen is known to cause reductions in feeding and fecundity in a range of insects. In the present study we investigated whether infection with M. anisopliae affected blood feeding and fecundity of adult female malaria vectors Anopheles gambiae Giles sensu stricto. Mosquitoes were contaminated with either a low or a moderately high dose of oil-formulated conidia of M. anisopliae, and offered a single human blood meal 48, 72, or 96 h later to assess feeding propensity and individual blood meal size. In a second experiment, individual fungus-infected females were offered a blood meal every third day (to a total of 8 gonotrophic cycles), and allowed to oviposit after each cycle in order to quantify feeding propensity and fecundity. Infected females took smaller blood meals and displayed reduced feeding propensity. It was found that mosquitoes, inoculated with a moderately high dose of fungal conidia, exhibited reduced appetite related to increasing fungal growth. Of the fungus-infected females, the proportion of mosquitoes taking the second blood meal was reduced with 51%. This was further reduced to 35.3% by the 4th blood meal. During 8 feeding opportunities, the average number of blood meals taken by uninfected females was 4.39, against 3.40 (low dose), and 2.07 (high dose) blood meals for the fungus-infected females. Moreover, infected females produced fewer eggs per gonotrophic cycle and had a lower life-time fecundity. Epidemiological models show that both blood feeding and fecundity are among the most important factors affecting the likelihood of a mosquito transmitting malaria, which suggests that this fungus may have potential as biocontrol agent for vector-borne disease control.

An entomopathogenic fungus for control of adult African malaria mosquitoes.

Biological control of malaria mosquitoes in Africa has rarely been used in vector control programs. Recent developments in this field show that certain fungi are virulent to adult Anopheles mosquitoes. Practical delivery of an entomopathogenic fungus that infected and killed adult Anopheles gambiae, Africa's main malaria vector, was achieved in rural African village houses. An entomological inoculation rate model suggests that implementation of this vector control method, even at the observed moderate coverage during a field study in Tanzania, would significantly reduce malaria transmission intensity.

Autodissemination of the entomopathogenic fungus Metarhizium anisopliae amongst adults of the malaria vector Anopheles gambiae s.s.

BACKGROUND: The entomopathogenic fungus Metarhizium anisopliae is being considered as a biocontrol agent for adult African malaria vectors. In the laboratory, work was carried out to assess whether horizontal transmission of the pathogen can take place during copulation, as this would enhance the impact of the fungus on target populations when compared with insecticides. METHODS: Virgin female Anopheles gambiae sensu stricto were exposed to conidia whilst resting on fungus-impregnated paper. These females were then placed together for one hour with uncontaminated males in proportions of either 1:1 or 1:10 shortly before the onset of mating activity. RESULTS: Males that had acquired fungal infection after mating indicate that passive transfer of the pathogen from infected females does occur, with mean male infection rates between 10.7 +/- 3.2% and 33.3 +/- 3.8%. The infections caused by horizontal transmission did not result in overall differences in survival between males from test and control groups, but in one of the three experiments the infected males had significantly shorter life spans than uninfected males (P < 0.05). CONCLUSION: This study shows that autodissemination of fungal inoculum between An. gambiae s.s. mosquitoes during mating activity is possible under laboratory conditions. Field studies are required next, to assess the extent to which this phenomenon may augment the primary contamination pathway (i.e. direct contact with fungus-impregnated targets) of vector populations in the field.

Entomopathogenic fungi for mosquito control: a review.

Fungal diseases in insects are common and widespread and can decimate their populations in spectacular epizootics. Virtually all insect orders are susceptible to fungal diseases, including Dipterans. Fungal pathogens such as Lagenidium, Coelomomyces and Culicinomyces are known to affect mosquito populations, and have been studied extensively. There are, however, many other fungi that infect and kill mosquitoes at the larval and/or adult stage. The discovery, in 1977, of the selective mosquito-pathogenic bacterium Bacillus thuringiensis Berliner israelensis (Bti) curtailed widespread interest in the search for other suitable biological control agents. In recent years interest in mosquito-killing fungi is reviving, mainly due to continuous and increasing levels of insecticide resistance and increasing global risk of mosquito-borne diseases. This review presents an update of published data on mosquito-pathogenic fungi and mosquito-pathogen interactions, covering 13 different fungal genera. Notwithstanding the potential of many fungi as mosquito control agents, only a handful have been commercialized and are marketed for use in abatement programs. We argue that entomopathogenic fungi, both new and existing ones with renewed/improved efficacies may contribute to an expansion of the limited arsenal of effective mosquito control tools, and that they may contribute in a significant and sustainable manner to the control of vector-borne diseases such as malaria, dengue and filariasis.

Infection of malaria (Anopheles gambiae s.s.) and filariasis (Culex quinquefasciatus) vectors with the entomopathogenic fungus Metarhizium anisopliae.

BACKGROUND: Current intra-domiciliary vector control depends on the application of residual insecticides and/or repellents. Although biological control agents have been developed against aquatic mosquito stages, none are available for adults. Following successful use of an entomopathogenic fungus against tsetse flies (Diptera: Glossinidae) we investigated the potency of this fungus as a biological control agent for adult malaria and filariasis vector mosquitoes. METHODS: In the laboratory, both sexes of Anopheles gambiae sensu stricto and Culex quinquefasciatus were passively contaminated with dry conidia of Metarhizium anisopliae. Pathogenicity of this fungus for An. gambiae was further tested for varying exposure times and different doses of oil-formulated conidia. RESULTS: Comparison of Gompertz survival curves and LT50 values for treated and untreated specimens showed that, for both species, infected mosquitoes died significantly earlier (p < 0.0001) than uninfected control groups. No differences in LT50 values were found for different exposure times (24, 48 hrs or continuous exposure) of An. gambiae to dry conidia. Exposure to oil-formulated conidia (doses ranging from 1.6 x 10(7) to 1.6 x 10(10) conidia/m2) gave LT50 values of 9.69 +/- 1.24 (lowest dose) to 5.89 +/- 0.35 days (highest dose), with infection percentages ranging from 4.4-83.7%. CONCLUSION: Our study marks the first to use an entomopathogenic fungus against adult Afrotropical disease vectors. Given its high pathogenicity for both adult Anopheles and Culex mosquitoes we recommend development of novel targeted indoor application methods for the control of endophagic host-seeking females.

Field experiments on the distributions of eggs of different phosphoglucomutase (PGM) genotypes in the yellow dung fly Scathophaga stercoraria (L.).

Female yellow dung flies can, in the laboratory, influence the probability that stored sperm from different males are used to fertilize eggs. This matches offspring phosphoglucomutase genotypes to the environmental conditions in which the larvae will grow, increasing larval growth success. We conducted field experiments in which dung topology or shading conditions were controlled. The proportions of the five common phosphoglucomutase alleles in eggs laid in north-facing slopes or in shaded conditions was related to their electrophoretic mobility. We suggest that females lay eggs of different genotypes, by appropriately choosing their fathers, in different places.