Chantal Reusken 1*, Ankje De Vries 1, Wietse Den Hartog 2, Marieta Braks 1 and Ernst-Jan Scholte 1,2

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1 European Mosquito Bulletin 28 (2010), Journal of the European Mosquito Control Association ISSN ; w.w.w.e-m-b.org First published online 23 April 2010 A study of the circulation of West Nile virus in mosquitoes in a potential high-risk area for arbovirus circulation in the Netherlands, De Oostvaardersplassen. Chantal Reusken 1*, Ankje De Vries 1, Wietse Den Hartog 2, Marieta Braks 1 and Ernst-Jan Scholte 1,2 1. Laboratory for Zoonoses and Environmental Microbiology, Centre for Infectious Disease Control Netherlands, Bilthoven, the Netherlands. 2. National Centre of Vector Monitoring, Plant Protection Service, Wageningen, the Netherlands. *Corresponding author. Laboratory for Zoonoses and Environmental Microbiology. Centre of Infectious Disease Control Netherlands, RIVM. P.O. Box 1; 3720BA Bilthoven the Netherlands; chantal.reusken@rivm.nl Abstract The Dutch national nature reserve De Oostvaardersplassen is considered a potential high-risk area for West Nile virus (WNV) circulation in the Netherlands: over 6000 hectares of wetland accommodating a diverse and abundant mosquito population, and a wide variety of wildlife. This wildlife includes migratory birds arriving from WNV endemic areas in Africa and Central Europe. Here we have conducted a combined mosquito and virological survey as a first step to assess the risks for public and veterinary health of this nature reserve as ecosystem for enzootic WNV circulation. Thirteen different mosquito species, covering five different genera were collected. Remarkable is the presence of Anopheles algeriensis, a mediterranean species. Eleven of the 13 species are reported field or laboratory vectors for a variety of infectious pathogens, including WNV, Usutu virus, Tahyna virus, Sindbis virus, Batai virus, Lednice virus, Francisella tularensis and Dirofilaria immitis. Although seven potential WNV vector species were collected, viz. Anopheles maculipennis s.l., Culex modestus, Cx. pipiens s.l., Ochlerotatus cantans and Coquillettidia richiardii, no evidence for WNV circulation in mosquitoes was found in this study. Keywords: Anopheles algeriensis, West Nile virus, Culicidae, ecology Introduction West Nile virus (WNV) is the aetiologic agent of West Nile Fever (WNF), a vector-borne disease endemic in Africa, East Asia, North-, Central- and South America and Southern Europe (Dauphin et al., 2004). The virus was first isolated from a woman with febrile illness in the West Nile province of Uganda in 1937 (Smithburn et al., 1940) and is classified in the genus flavivirus of the family of the Flaviviridae (Burke & Monath, 2001). West Nile virus is maintained in an enzootic cycle with birds as amplifying reservoir hosts and mosquitoes as transmitting vectors. Humans and horses are sensitive dead-end hosts (Hurlbut et al., 1956). The majority of human WNV infections are asymptomatic. American studies estimated that approximately 20% of human infections will result in a mild disease characterized by fever, headache, myalgia and fatigue. Less than 1% of all infected persons develop severe neuroinvasive disease, with a case fatality of approximately 10% (Kramer et al., 2007). 69

2 The first recognised outbreak in Europe occurred in 1962 in France with clinical symptoms in humans and horses (Murgue et al., 2001). A major outbreak of human WNV infections took place in 1996 in Romania (Tsai et al., 1998). In subsequent years enhanced surveillance in Romania identified some WN fever cases each year between 1997 and 2000 but there were no major outbreaks (Ceianu et al., 2001). In 1997 some sporadic cases were reported in the Czech Republic (Hubálek et al., 2000). A second severe human outbreak of WNV took place in the Volgograd region in Russia in 1999 (Lvov et al., 2000, Platonov et al., 2001). The epidemiological picture of human WNV infections in Europe appears to be changing. In recent years sporadic cases of human WNV infections have been identified in Hungary, Italy, France, Portugal, Romania, Russia and Spain (Hubálek et al., 2000, Mailles et al., 2003, Connell et al., 2004, Del Giudice et al., 2004, Zeller & Schuffenecker 2004, Kaptoul et al., 2007, Krisztalovics et al., 2008, Popovici et al., 2008, Barzon et al., 2009, Rizzo et al., 2009). In 2008 for the first time outbreaks of human infections were reported simultaneously from three European countries, viz. Hungary, Italy and Romania (Krisztalovics et al., 2008, Popovici et al., 2008, Barzon et al., 2009). In addition to the known circulation of lineage 1 of WNV in Europe, the circulation of lineage 2 of WNV was reported from Hungary in 2005 and Austria in 2008 (Bakonyi et al., 2006, Weissenbock et al., 2009). Mosquitoes (Diptera: Culicidae) are the principal vectors for WNV transmission. The main route of introduction of WNV in the regions of Europe with a temperate climate is most likely by infected migratory birds from Africa (Hubálek & Halouzka, 1999, Rappole et al., 2000, Rappole & Hubálek, 2003, Koopmans et al., 2008). Moreover, WNV could be introduced in western Europe by birds arriving from central Europe (Koopmans et al., 2008). For an efficient local transmission, mosquito species are needed that are capable of sustaining and transmitting the virus among the indigenous bird population. Ornithophilic mosquito species will be the most relevant species for WNV establishment in an enzootic cycle in temperate Europe (Rappole et al., 2000, Higgs et al., 2004). For transmission to humans and horses, vectors are needed that have a more opportunistic feeding behaviour; feeding both on birds and mammals and serving as bridge vectors. In Europe WNV has been detected in seven different mosquito species, covering 4 genera, in France, Portugal, Moldavia, Romania, Slovakia, the Czech Republic, Ukraine and Belarus. Vectors reported positive in the field are Culex pipiens s.l., Cx. modestus, Cx. univittatus, Coquillettidia richiardii, Ochlerotatus cantans, Anopheles maculipennis s.s. and An. atroparvus (Hannoun et al., 1964, Filipe, 1972, Labuda et al., 1974, Hubálek & Halouzka, 1996, Fernandes et al., 1998, Savage et al., 1999, Esteves et al., 2005, Almeida et al., 2008, Hubálek, 2008). The ornithophilic members of the species complex Cx. pipiens s.l. are the most common vector species for WNV both in Europe and the USA. Because WNV in Europe is most likely introduced by bird migration and WNV enzootic circulation and outbreaks are often focused in or near wetlands where large numbers of birds are bitten by large numbers of mosquitoes (Koopmans et al., 2008), the Dutch national park De Oostvaardersplassen, is considered a high risk area for introduction and enzootic circulation of WNV in the Netherlands. De Oostvaardersplassen, located in the province of Flevoland (Figure 1), is one of the many areas of reclaimed land in the Netherlands. Nowadays, the Oostvaardersplassen is a 6000 hectares nature development reserve of international importance as wetland and habitat for migratory birds. Over 190 different bird species have been observed in the reserve since 1999, including over 45 bird species of which evidence for WNV infection exists from foreign field studies (Koopmans et al., 2008). De Oostvaardersplassen has diverse landscapes including marshes, lakes, ponds, ditches, trenches, plains, shrubs, and forests, which create a large diversity of potential mosquito breeding sites. This, combined with the presence of large numbers and variety of hosts (birds and wildlife consisting amongst others of approximately 500 Heck cattle, 1100 Konik horses and 2300 red deer), makes De Oostvaardersplassen an area where high mosquito species richness and mosquito abundances are expected. 70

3 Here we describe a combined mosquito and WNV survey in De Oostvaardersplassen as a first step towards an assessment of the risks of this nature reserve as ecosystem for enzootic WNV circulation in the Netherlands. Survey design Materials and methods Adult mosquitoes were collected in the Oostvaardersveld, an area of approximately 328 ha in the southeastern corner of De Oostvaardersplassen. The Oostvaardersveld consists of open grassland with sparse shrubs, shallow ponds, of which the smaller ones contain little or no aquatic vegetation, 5-10m wide canals with still standing water most of which are lined with reed (Phragmites communis), and deciduous forest. A group of approximately 100 Konik horses are present, spending most of their time in an open grassland area in the northeastern part of the Oostvaardersveld. In the middle of this open grassland lies a shallow, permanent pond without aquatic plants with high numbers of birds (mostly geese and various duck species). This area of open grassland is surrounded by deciduous forest (mostly willow). In some parts of this forest, especially near canals, flooding occurs, creating marshes. Due to the high groundwater level in the entire Oostvaardersplassen, puddles and pools remain in these marshes, even in relatively dry periods (Kooijman et al., 2005). Adult mosquitoes were collected using carbon dioxide baited traps of the type Liberty Plus (American Biophysics), with octenol as an additional lure. Where possible, the traps were placed in the lee of trees. All traps were placed on wind-protected locations, with ample space around the traps entry point. In total, nine traps ran continuously during the experimental period. One mosquito was collected when landing on one of the researchers. 71

4 Oostvaardersplassen 5 km Oostvaardersveld A D EB F C G I H 500m Figure 1. The study area, Oostvaardersveld, where nine traps were placed. 72

5 Trap Table 1. Presence or absence of several landscape parameters and estimated host availability of the immediate area surrounding the trap (< 25 m) deciduous forest a) open grassland b) Marshes c) Canals d) Ponds e) estimated presence of hosts (large mammals) A no yes no yes no regular always B yes no no no no occasionally always C yes no yes no no rarely always D no yes no no yes very often always E no yes no no yes very often always F yes no no no no occasionally always G yes no no no yes occasionally always H no yes no no yes rarely always I yes no yes no no occasionally always estimated presence of hosts (birds) a) Consisting mainly of willows (Salix alba), bramble (Rubus fruticosus), nettle (Urtica dioica), and at the margins elder (Sambucus nigra). b) Mainly grass (mostly Lolium and Poa spp.), nettle, thistles (Cirsium and Carduus spp.) and occasionally hawthorns (Crataegus laevigata). c) Consisting mainly of willow and at the margins dense nettle fields. d) m deep, with few or no aquatic plants. Often lined with reed (Phragmites communis). e) The ponds near traps D and E were relatively small, shallow and contained no aquatic plants. The pond near traps G and H measured approximately m., and contained some aquatic plant species, such as water lily (Nuphar lutea). No information is available on submerged aquatic plants in the Oostvaardersplassen. As WNV outbreaks in Europe in general occur during the summer (July-August-September) we conducted our survey in August. Mosquitoes were collected twice for two consecutive days; in week 33 (10-12 August 2009) and in week 35 (24-26 August 2009). For the entire duration of the experiment, the nine traps ran continuously. The traps were placed in the morning of the 10 th of August. The nets from the mosquito magnets were retrieved from all traps after 24 hrs (on the morning of the 11 th August), and replaced with empty nets, which were subsequently retrieved 24hrs later (12 th August). Retrieved nets with (still alive) mosquitoes were placed in a sealed bag, labelled, and placed in an equally labelled cardboard box for protection (one net per box). These labelled boxes were kept in the car for later mosquito species identification. In week 33, the same procedure was followed: traps were switched on, on the 24th of August, and retrieved subsequently 24 and 48 hrs later (25 th and 26 th August). Within a maximum of three hours after collection from the trap, the nets with mosquitoes were removed from the box and placed by -20 C for approximately 3-10 minutes to kill them. Subsequently, they were gently taken from the nets, separated from other insects, and identified. Mosquitoes were morphologically identified using a Culicidae key specifically designed for rapid field-identification of Dutch Culicidae adult female mosquitoes (Scholte, 2009a modified after Schaffner et al., 2001, Becker et al., 2003, Verdonschot, 2002). Males were not identified. Differentiation of members of the Anopheles maculipennis complex (An. messeae, An. maculipennis senso stricto, and An. melanoon), and between the biotypes of Cx. pipiens (pipiens and molestus forms), was not carried out. The species identification was confirmed by a specialist on European mosquito species. 73

6 Monitoring for presence of West Nile virus Upon identification, the collected mosquitoes were immediately flash frozen using dry-ice and stored at - 80 C until analysis. Mosquitoes were pooled by sampling site, sampling date and species. Mosquitoes were ground using liquid N 2 and pestles, and RNA was extracted using the RNeasy RNA Isolation Minikit (Qiagen, Inc., Valencia, CA) according to the manufacturer s instructions and including a Qiashredder step for homogenization and DNaseI treatment. RNA was isolated from pools of no more than five mosquitoes. As a quality control for the isolation step, each RNA isolate was checked by an actin reverse transcriptasepolymerase chain reaction (RT-PCR) (Koopmans et al., 2008). Although these primers were selected based on alignments of known actin sequences from Aedes spp. available in GenBank, we have successfully used them on Cx. pipiens s.l., Cx. modestus, Cx. subochrea, Cx. torrentium, Cx. territans, Culiseta annulata, Aedes cinereus, An. maculipennis s.l., An. plumbeus, An. claviger, Oc. punctor, Oc. cantans, and Coquillettidia richiardii that are all endemic in the Netherlands (data not shown). The presence of WNV was analysed using a multiplex real-time RT-PCR assay, allowing detection and discrimination of lineages 1 and 2 (Chao et al., 2007). An additional lineage 2 probe was designed and added to the reaction, viz. WNV-2: 5 -FAM-tgaaggaagttggaacaaagcctgg-BHQ1. To avoid false negative results arising from inhibitory factors known to be present in mosquito total RNA preparations, each pool was also analysed in the presence of a spike-mix consisting of viral RNA from both lineage 1 and 2 of WNV at an experimentally pre-determined detectable concentration. Only the results from pools that were positive for both lineages in the spiked RT-PCR were used in this study. Entomological survey Results In total 36 trap collections were carried out. Four times a trap had failed (traps B, D, and I for the collections on 11 th of August, and trap E for the collection on the 26 th of August), resulting in a total of 32 effective collections. In these 32 samples, a total of 410 mosquitoes were collected. Mosquito identification up to species level was carried out for 396 specimens (Table 2): two specimens were too damaged (of which one could be identified only as Culicidae, and one only up to genus level), and 12 were males. The identified species are An. algeriensis, An. claviger, An. maculipennis s.l., An. plumbeus, Cq. richiardii, Cx. modestus, Cx. pipiens, Cx. torrentium, Cs. annulata, Cs. morsitans, Cs. subochrea, Oc. cantans and Dahliana geniculata. Very interesting is the finding of An. algeriensis, a Mediterranean mosquito species that is rare in northern regions, here newly identified for the Netherlands. By far the most abundant species was Cs. annulata (207 specimens), followed by Cq. richiardii (96 specimens) and An. claviger (55 specimens). Together, these three species made up 90% of the collected mosquitoes. Of four species, only one specimen was collected: Cx. modestus, Cs. morsitans, Cs. subochrea, and Da. geniculata. Nine specimens were collected of Cx. pipiens. Anopheles claviger, An. maculipennis s.l., Cq. richiardii, and Cs. annulata were found at most locations. From the traps located at D and E most mosquitoes were collected, with 96 and 119 specimens respectively. The lowest number of mosquitoes was collected from the trap located at location I, where only two mosquitoes were collected. The number of different species ranged between 2 and 8 species per trap location. The highest number of species was collected from the traps at location B and E, with eight different species each. 74

7 Table 2. Overview of collected mosquito species per trap mosquito species\trap # A B C D E F G H I Total Anopheles algeriensis Anopheles claviger Anopheles maculipennis sl Anopheles plumbeus 4 4 Coquillettidia richiardii Culex modestus 1 1 Culex pipiens Culex torrentium Culiseta annulata Culiseta morsitans 1 1 Culiseta subochrea 1 1 Dahliana geniculata 1 1 Ochlerotatus cantans Total Ochlerotatus sp. 1 1 Male Culicidae heavily damaged 1 1 Total West Nile virus survey In total 314 mosquitoes, belonging to five different genera and 13 different mosquito species, were analysed for the presence of WNV lineages 1 and 2 (Table 3). None of the analysed specimens were found positive for WNV. Table 3. Overview of mosquitoes collected in the nature reserve De Oostvaardersplassen Mosquito species No. captured No. tested for WNV Anopheles algeriensis 6 3 Anopheles claviger Anopheles maculipennis sl 6 6 Anopheles plumbeus 4 2 Coquillettidia richiardii Culex modestus 1 0 Culex pipiens 9 9 Culex torrentium 2 2 Culiseta annulata Culiseta morsitans 1 0 Culiseta subochrea 1 1 Dahliana geniculata 1 1 Ochlerotatus cantans 7 5 Ochlerotatus spp. 1 1 Total

8 Discussion Mosquito species For the Netherlands the presence of 36 different mosquito species has been documented, including the recently discovered Ochlerotatus atropalpus (Scholte et al., 2009). In addition, Stegomyia albopicta is occasionally introduced into the Netherlands from South-East China with the import of Lucky Bamboo plants. However this species has not established in the Netherlands (Scholte et al., 2007, 2008). Thirteen different mosquito species, covering five different genera were collected during the survey. This represents 37% of the total number of species known to be present in the Netherlands, and confirms the presence of high mosquito species richness in the Oostvaardersplassen. Remarkable is the presence of An. algeriensis in the nature reserve. Anopheles algeriensis is a mediterranean mosquito species. The species is rare in southern Europe (only two populations are known from southern France (Poncon et al., 2007), and isolated populations have been described for northern Europe, viz. Britain (Rees & Rees, 1989; Snow et al., 1998), Estonia (Remm, 1957), Germany (Mohrig, 1969), western France (Ramsdale & Snow, 2000) and Ireland (Ashe et al., 1991). In terms of spatial species composition; the collected species per location match with the mosquito fauna that could be expected at these types of locations. However one exception was observed: Four adults of the forest species An. plumbeus were collected at location E, a relatively open area, approximately 200 metres from the nearest forest. This species was not collected at any of the forested locations (B, C, I, F, G). In terms of temporal species composition; only eight specimens, belonging to two species of the genus Ochlerotatus were collected, suggesting an under-representation of the genus. This can most likely be explained by the timing of the study in the season : many Ochlerotatus species have only one generation, and are abundant in (early) spring, after the overwintering eggs have hatched. Examples are Oc. annulipes, Oc. cantans, Oc. communis, Oc. excrucians, Oc. riparius, Oc. leucomelas, and Oc. rusticus (all endemic species). The absence of these species is therefore easily explained. Ochlerotatus species that, based on their known ecology, could be expected in this study but were not collected, include Oc. sticticus, Oc. nigrinus, Oc. flavescens, and Oc. caspius. A relatively low number (nine) of specimens were collected of Cx. pipiens, a species that is generally an abundant species in Culicidae surveys in different habitats in the Netherlands (Takken et al., 2007). As the type of trap used in entomological surveys is one of the factors determining the relative abundance and diversity of the mosquitoes collected, one should keep in mind that the mosquito magnet used in this study might not be the optimal trap for all species present in the nature reserve. The following notes give some information on the five most commonly encountered species in this survey: Anopheles claviger. This species was encountered at all locations except H. This species occurs mostly in permanent pools, shallow canals and ditches, with a preference for shaded sites. This species hibernates as a larva. Coquillettidia richiardii. An interesting feature of the larvae of this species, is that they obtain their oxygen not directly from the air (as other mosquito larvae), but from plant tissues by penetrating the roots of certain aquatic plants. Culiseta annulata. This species was by far the most abundant species in the Oosvaardersveld at the time of the study. This species is a relatively large mosquito and an aggressive biter, also to humans. Larvae are known to be present almost throughout the year, although the species is known to overwinter as adults. 76

9 Anopheles maculipennis s.l. Anopheles maculipennis consists of a complex, of which seven sibling species occur in Europe and three in the Netherlands: An. messeae, An. atroparvus, and An. maculipennis sensu stricto. Females are often present in barns and stables, but bite humans as well. Adults hibernate in cool rooms such as cellars. Culex pipiens complex. The Cx. pipiens complex consists of two biotypes: the pipiens and molestus forms. The first is ornithophilic (not attracted to humans), whereas the latter is mainly zoophilic but occasionally bites birds as well. In the case of West Nile virus circulation, biotype pipiens can play a role in maintaining the virus circulating among birds only, whereas biotype molestus may form the bridge species, transmitting the virus to horses and humans. Biotype pipiens breeds in a large variety of waterbodies, including shallow ponds and artificial containers. The species overwinters as adults. Disease vectors Ten species endemic to the Netherlands are reported as field vectors for WNV in foreign studies of which five have been collected in this study, viz. An. maculipennis s.l., Cx. modestus, Cx. pipiens s.l., Oc. cantans and Cq. richiardii (Table 4). WNV has been isolated from all five species in Europe (Hannoun et al., 1964, Filipe, 1972, Labuda et al., 1974, Katsarov et al., 1980, Fyodorova et al., 2006, Koopmans et al., 2008). Two species are reported laboratory vectors for WNV, viz. An. plumbeus and Da. geniculata (Table 4). Although seven potential WNV vector species were collected in this nature reserve, no evidence for WNV circulation in mosquitoes was found in this study. This could mean that although there is WNV circulation in the nature reserve, it cannot be detected with the current survey set-up. Enzootic WNV transmission may occur only at a low intensity in certain birds and mosquito species. The number of mosquitoes collected in this study is most likely not enough to detect a low endemic WNV circulation in the area. In total 314 mosquitoes were analysed for the presence of WNV of which only 9 specimens of the Cx. pipiens complex which, together with Cx. modestus, are considered as the vector species with the highest vectorial capacity for WNV. Furthermore there might be WNV circulation in the reserve but not in the area of study. De Oostvaardersveld may not completely reflect the total of the other areas of the nature reserve. Even though the Oostvaardersveld has several waterbodies, these are not comparable in size to the lakes in the other areas. The number of birds visiting the Oostvaardersveld, is relatively low compared to the other areas of the nature reserve. However these areas are not open to the public and researchers. On the other hand the failure to detect WNV in the collected mosquitoes could reflect the absence of WNV circulation in De Oostvaardersplassen. Although the virus might be introduced (repeatedly) in the reserve by migratory birds, the local basic reproduction rate, R0, might be too low for WNV establishment in a sylvatic cycle. Further research into factors of influence like the vector competence of the local mosquito population, the susceptibility of the indigenous bird population and ecological factors like temperature, precipitation and occurrence of floods is necessary. In total 11 of the 13 different mosquito species collected in the Oostvaardersplassen are reported field or laboratory vectors for infectious pathogens, including Tahyna virus (TAHV), Sindbis virus (SINV), Usutu virus (USUV), Batai virus (BATV), Lednice virus (LEDV), Francisella tularensis and Dirofilaria immitis (Table 4). Especially SINV, USUV, LEDV and BATV are likely other candidates for circulation in the nature reserve. Like WNV, SINV, USUV and LEDV are maintained in an avian-mosquito cycle. The vertebrate reservoirs are largely passeriform (WNV, USUV) and anseriform (LEDV) birds, with migratory members being responsible for wide geographic distribution (Hubálek, 2008, Weissenbock et al., 2009). Natural foci of SINV and LEDV infections occur mainly in wetland ecosystems like the Oostvaardersplassen (Hubálek 2008). Although BATV has principally a domestic animal mosquito 77

10 cycle it can persistently infect birds and the virus is thought to spread across Europe through migratory birds (Hubálek & Halouzka, 1996). Sindbis virus circulates in Eurasia, Africa and Oceania (Taylor et al., 1955, Hubálek, 2008). Outbreaks in Europe of Pogosta disease (human Sindbis virus infections) have thus far emerged in Fennoscandia every seven years since the 1st outbreak was noted in 1974 with hundreds to thousands of clinical cases (Hubálek, 2008). However in 2009 this seven-year cycle did not recur (Sane et al., 2010). Human disease is characterized by fever, rash and (chronic) arthritis (Hubálek, 2008). In Europe evidence for USUV circulation in birds exists for Austria, Hungary, Italy, Switzerland, England, Poland and the Czech Republic (Weissenböck et al., 2008). In 2009 the first European human cases of USUV-related neurological disease occurred in Italy (Pecorari et al., 2009; Cavrini et al., 2009). Lednice virus circulates in birds in the Czech Republic and Romania. No evidence for disease in mammals, including man, exists sofar (Hubálek, 2008). Bataivirus is present in Europe, Asia and Africa. In Europe evidence for circulation exists for Norway, Sweden, Finland, Slovakia, the Czech Republic, Croatia, Serbia, Bosnia, Montenegro, Italy, Hungary, Romania, Portugal, Germany and Belarus (Hubálek, 2008). Vertebrate hosts are pigs, horses and ruminants. Closely related viruses cause stillbirths and congenital abnormalities in ruminants in the USA (Chung et al., 1990). However, European veterinary data are lacking. Human BATV infections are associated with influenza-like illness in Europe and febrile illness in Asia and Africa (Juricová et al., 2009, Hubálek, 2008). Considering the wide variety of wildlife, richness in mosquito species and frequent avian visitors from arbovirus endemic areas in Europe and Africa, De Oostvaardersplassen is considered a high risk ecosytem for arbovirus circulation in the Netherlands. The establishment of a sylvatic cycle of WNV in the Oostvaardersplassen creates an opportunity for WNV introduction into the urban cycle through peridomestic birds and subsequent WNV transmission to humans through bridge vectors. The city of Almere with 190,000 inhabitants borders directly on the nature reserve while the city of Lelystad with 75,000 inhabitants is less then 3 km away (Figure 1). Further research into arbovirus circulation in the nature reserve is essential for assessing the risks for public health by emerging arboviruses endemic elsewhere in Europe or even Africa. Acknowledgements The authors are indebted to Francis Schaffner for checking and confirmation of the species identifications in this study. The authors thank Joke van der Giessen for critical reading of the manuscript and Staatsbosbeheer for the kind cooperation in the nature reserve. 78

11 Table 4. Overview diseases putatively transmitted by mosquito species collected in De Oostvaardersplassen. mosquito species reported field vector reported laboratory vector probable vector for Anopheles algeriensis malaria. An. claviger malaria Tahyna, Batai, myxomatosis (virus), canine filariasis, Setaria labiatopapillosa (nematode), anaplasmosis, Lyme, Tularaemia (bacterium). An. maculipennis s.l. - sensu stricto malaria (minor role) Batai, Tahyna, West Nile, Dirofilaria immitis, myxomatosis, Tularaemia. - atroparvus malaria, West Nile, myxomatosis Tahyna, Dirofilaria immitis, Tularaemia - messeae malaria (minor role) Batai, Tahyna, West Nile, canine filarias, myxomatosis, Tularaemia An. plumbeus malaria, West Nile, filariasis Coquillettidia richiardii Batai, West Nile, Tahyna Culex modestus West Nile, Tahyna, myxomatosis, Sindbis, Lednice, Dirofilaria immitis Tularaemia Cx. pipiens West Nile, Sindbis, Batai, Usutu, Tahyna, Dirofilaria immitis Cx. torrentium Sindbis Culiseta annulata Usutu, myxomatosis Tahyna Cs. morsitans Sindbis (minor role) Da. geniculata Tularaemia West Nile, yellow fever Ochlerotatus cantans Tahyna, West Nile, myxomatosis Taken from (Schaffner et al., 2001) and adapted based on (Poncon et al., 2007, Weissenböck et al., 2008) 79

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