Screening of mosquitoes for filarioid helminths in urban areas in south western Poland-common patterns in European Setaria tundra xenomonitoring studies.

In recent years, numerous studies screening mosquitoes for filarioid helminths (xenomonitoring) have been performed in Europe. The entomological monitoring of filarial nematode infections in mosquitoes by molecular xenomonitoring might serve as the measure of the rate at which humans and animals expose mosquitoes to microfilariae and the rate at which animals and humans are exposed to the bites of the infected mosquitoes. We hypothesized that combining the data obtained from molecular xenomonitoring and phenological studies of mosquitoes in the urban environment would provide insights into the transmission risk of filarial diseases. In our search for Dirofilaria spp.-infected mosquitoes, we have found Setaria tundra-infected ones instead, as in many other European studies. We have observed that cross-reactivity in PCR assays for Dirofilaria repens, Dirofilaria immitis, and S. tundra COI gene detection was the rule rather than the exception. S. tundra infections were mainly found in Aedes mosquitoes. The differences in the diurnal rhythm of Aedes and Culex mosquitoes did not seem a likely explanation for the lack of S. tundra infections in Culex mosquitoes. The similarity of S. tundra COI gene sequences found in Aedes vexans and Aedes caspius mosquitoes and in roe deer in many European studies, supported by data on Ae. vexans biology, suggested host preference as the most likely cause of the mosquito genus-biased infections. High diversity of the COI gene sequences isolated in the city of Wroclaw in south western Poland and the presence of identical or almost identical sequences in mosquitoes and roe deer across Europe suggests that S. tundra has been established in most of Europe for a very long time.

Assessment of productivity of Culex spp. larvae (Diptera: Culicidae) in urban storm water catch basin system in Wroclaw (SW Poland).

In urban environments, catch basins serve as major developmental and resting sites for anthropophilic and zoophilic mosquitoes. However, the use of this habitat is inconsistent, with abundance of larvae varying significantly across catch basins at a fine spatial scale. During seasonal summer investigations on mosquito species composition, their spatial and temporal distribution and the environmental characteristic of the breeding sites in the underground storm drain systems of the Wroclaw urban area (SW Poland) were assessed from May to September in 2012-2013. The study was conducted in order to develop a rational strategy to control mosquito populations and prevent the potential human exposure to mosquito-transmitted pathogens. Mosquito larvae and pupae were collected and identified weekly from 100 regularly inspected street catch basins located in the town center. All existing and potential breeding habitats in the study area were recorded using a GPS receiver (Magellan MobileMapper CX) and transferred to the computer database. Collected data on the geographical location of inspected breeding places, water quality parameters in inspected catch basins, daily temperature, and precipitation were imposed on orthophotomap in ArcGIS (ESRI, USA). Water quality parameters including pH, electrical conductivity, and water temperature were measured by standard methods. Chemical water analysis of cations (Na(+), NH4 (+), K(+), Mg(2+), Ca(2+)) and anions (Cl(-), NO2 (-), NO3 (-), SO4 (2-)) were carried out using Waters Alliance high-performance liquid chromatograph (HPLC) 2695 with 432 Conductivity Detector and 2998 Photodiode Array Detector, an IC-Pak Anion HR column (glauconate/borate eluent) and IC-Pak Cation M/D column (EDTA/HNO3 eluent). Over two seasonal studies and 3739 samplings in total, 3669 mosquito larvae and 274 pupae/1 dip (from 0 to 110 individuals/dip) were collected by dipper. Culex pipiens s.l. (L.) and Cx. torrentium (Martini) prevailed at all catch basins of the study area as the predominant species. In all examined catch basins, autogenous individuals dominated by far. Breeding activity was first detected in early May. Peak abundance of Culex spp. population in many catch basins was observed in June 2012 and August 2013 when average daily temperatures were increasing and rainfall had declined. Dry periods between rainfalls varied during 2 years of the study period and were noted on June 2012 as well as on July and August 2013. Organically enriched catch basins with significant higher concentrations of Na(+) and NO3 (-) were found to be more productive breeding habitats. Differences in the Culex immature stage density based on the variables of habitat type, temperature, and precipitation support the need for ongoing surveillance in communities to guide public health officials in planning for and prioritizing mosquito control efforts.

Culex pipiens s.l. and Culex torrentium (Culicidae) in Wroclaw area (Poland): occurrence and breeding site preferences of mosquito vectors.

Both ornithophilic mosquito species, Culex pipiens s.l. (L.) and Culex torrentium (Martini, 1925), occur sympatric in temperate Europe. They are presumed to be primary vectors of West Nile and Sindbis viruses. Differentiation of these morphologically similar Culex species is essential for evaluation of different vector roles, for mosquito surveillance and integrated control strategies. Cx. torrentium has been neglected or erroneously determined as Cx. pipiens s.l. in some previous studies, because only males of both species can be diagnosed reliably by morphology. Thus, knowledge about species abundance, geographical distribution, breeding site preferences and the zoonotic risk assessment is incomplete also in Poland. In Wroclaw area (Silesian Lowland), besides typical urban breeding sites, huge sewage irrigation fields provide suitable breeding conditions for Culex species. They are also inhabited by 180 resident and migratory bird species serving as potential virus reservoirs. In this study, morphology of larvae and males as well as species diagnostic enzyme markers, namely adenylate kinase (AK) and 2-hydroxybutyrate dehydrogenase (HBDH), were used to discriminate Cx. pipiens s.l. and Cx. torrentium. In a total of 650 Culex larvae from 24 natural and artificial breeding sites, Cx. pipiens s.l. had a proportion of 94.0% and Cx. torrentium only 6.0%. It could be shown that both species are well adapted to various breeding site types like ditches, catch basins, flower pots and buckets with diverse water quality. Cx. torrentium preferred more artificial water containers in urban surrounding (12% species proportion), whereas in semi-natural breeding sites, Cx. torrentium was rare (3%). In 12 of 24 breeding sites, larvae of both species have been found associated.

Methods of mosquito plague control in Swinoujscie area based on the analysis of species distribution.

OBJECTIVE: Studies conducted in 2013 in Swinoujscie area aimed at determining mosquito species distribution and analyzing sites, productivity and dynamics of larva and adult breeding as well as developing guidelines for effective mosquito control in this area. MATERIALAND METHODS: Entomological studies were conducted from March to September 2013. Distribution of mosquito larvae and adults was investigated. Mosquito larvae were observed mainly in April in transient snow-melt pools. RESULTS: Ochlerotatus cantans, Oc. communis, Oc. annulipes, Oc. punctor were the most predominant mosquito species. Since July, larvae of the following genera: Culex spp., Culiseta spp., Anopheles spp. were collected in standing water bodies. A total of 17 species adult mosquito were identified. Of them, Oc. communis, Aedes vexans, Culex pipiens/torrentium, Oc. Cantans, Oc. caspius predominated over other species. CONCLUSIONS: Mosquito plague in Swinoujscie area can be controlled if constant actions would be undertaken from April (use of microbial biocides for snow-melt mosquito larva control) to August (use of microbial biocides for polygeneration larva species control and use of chemical biocides for adult mosquito control). Furthermore, there is a need for actions that would make environment less friendly to mosquitoes and would help to identify breeding sites of aggressive mosquito species - Aedes vexans. There is also a necessity to educate the inhabitants of Swinoujscie area on the danger resulting from uncontrolled use of insecticides in protected areas and those which are unique from a faunistic perspective.

First record of Anopheles (Anopheles) hyrcanus (Pallas 1771) (Diptera: Culicidae) in Poland.

The spatial distribution of mosquito species in the course of globalization and climate warming is highly dynamic. Different studies have demonstrated the spread and establishment of thermophilic mosquito species, potentially increasing the prevalence of 'nuisance' mosquitoes and the local transmission of pathogens. Here we report the first recorded sampling of Anopheles hyrcanus in Wroclaw, southwest Poland. This is the most northern detection of this species to date in Europe. Future spread and population development of this potential vector of malaria parasites, viruses or zoonotic helminths, such as Dirofilaria spp., must be monitored carefully. Potential factors underlying the spread of this species are discussed.

Field efficacy of granular formulations of Bacillus thuringiensis Israelensis--strain AM65-52 against floodwater mosquitoes in Poland and Germany.

This paper presents results of studies evaluating the efficacy of various granular formulations of VectoBac (Bacillus thuringiensis israelensis de Barjac [Bti] H-14 strain AM65-52) against immature mosquitoes in 2 distinct habitats in Poland and Germany. Meadows intermittently flooded with sewage water in the city of Wroclaw, Poland, provide ideal habitats for immature stages of Aedes caspius, Ae. vexans, and Culex pipiens pipiens. Helicopter applications of VectoBac G and VectoBac TP sand granules (VectoBac TP-SG) at rates of 5 and 10 kg/ha to the meadows resulted in between 70.9% and 97.4% larval mortality. In Germany, some swampy woodlands in the upper Rhine Valley provide ideal habitats for snowmelt mosquitoes, Aedes cantans, Ae. punctor, Ae. rusticus, and Ae. communis. Ground applications of VectoBac G, VectoBac TP-SG, and VectoBac WG IcyPearls (VectoBac WG-IP) were made to these habitats when the water temperature was approximately 7 degrees C. Larval mortality at 7-day post-application ranged from 90.9% (VectoBac TP-SG: 10 kg/ha) to 98.0% (VectoBac WG-IP: 15 kg/ha). In a separate microcosm trial within the same habitat, all 3 granule formulations controlled larvae of Ae. cantans for 3 wk.

[Ecological safety of mosquitocidal biocides based on Bacillus thuringiensis israelensis]. / Bezpieczenstwo ekologiczne komarobójczych biocydów Bacillus thuringiensis israelensis.

Bacillus thuringiensis israelensis (Bti) has been developed into many products for the biological control of dipteran larvae, including mosquitoes (Culicidae), black flies (Simuliidae), and midges (Chironomidae) in various parts of the World. Bti appears to pose significantly less of a risk than other chemical pesticides used for mosquito control and eradication programs. Bioproducts based on Bti are highly selective with short environmental persistence, and thus they have very little potential to cause damage to populations of non-target organisms. So far, no example of an unexpected pathogenic organism being developed in the field as well as no examples of resistance to Bti both laboratory and field populations of mosquitoes have been documented. There are some indications that large declines in insect biomass can occur after long-term use of Bti in freshwater wetlands. However, no evidence for permanent damage to ecosystem function has been found. Organisms that utilized insects for food, adapted to the declines and either switched to other food sources or migrate (birds) outside of the treated zones to acquire insects. Even though over 40 tons of Bti have been applied in West Africa alone, no indications of human health or non-target effects have been reported.

[Ecological aspects of mosquito biocontrol with implementation of GPS/GIS]. / Ekologiczne aspekty biokontroli komarów z wykorzystaniem technik GPS/GIS.

Mosquitoes bionomics and vector role as well as integrated control methods with GPS/GIS techniques were presented. Special attention was put on GIS which enables analysis of biological and environmental data generated by GPS (Global Positioning System). Combined with data from surveillance and management activities, those techniques provide a powerful tool for the precise analysis of mosquito development, breeding sites, and effective biocontrol effects on maps.

Evaluation of three microbial formulations against Culex pipiens pipiens larvae in irrigation fields in Wroclaw, Poland.

Two evaluations of microbial mosquito larvicides were conducted in irrigation fields located along the Odra River near the city of Wroclaw, Poland. In the first evaluation, 3 dosages each of Bacillus thuringiensis israelensis (Bti)--VectoBac water dispersible granule (WDG) and VectoBac aqueous suspension formulation (12AS)--and Bacillus sphaericus (Bs)--VectoLex WDG--were tested against larvae of Culex pipiens pipiens. There was little difference between formulations and rates of application between day 2 and 7 following the treatment. VectoBac WDG at 300 g/ha resulted in 100% mortality of Cx. p. pipiens. Between 0.5 and 1 liter of VectoBac 12AS caused sufficient mortality for up to 7 days. The effective shortterm application rate of VectoLex WDG was found to be 400 g/ha. The above doses can be recommended for up to 7-day control in this habitat. In the second evaluation, using VectoBac WDG at 800 g/ha, the residual efficacies of 2 ground application methods, a knapsack sprayer configured for large droplet application and a motorized backpack blower configured for small droplet application, were compared. The results indicated no significant difference between the 2 application methods, but a significant long-term effect of VectoBac WDG was observed at the rate of 800 g/ha.

Current procedures of the integrated urban vector-mosquito control as an example in Cotonou (Benin, West Africa) and Wroclaw area (Poland).

Current strategy of Integrated Vector Management (IVM) comprises the general approach of environmentally friendly control measures. With regard to mosquitoes it includes first of all application of microbial insecticides based on Bacillus thuringiensis israelensis (Bti) and B. sphaericus (Bs) delta-endotoxins as well as the reduction of breeding habitats and natural enemy augmentation. It can be achieved thorough implementation of the interdisciplinary program, i. e., understanding of mosquito vector ecology, the appropriate vector-diseases (e. g., malariometric) measurements and training of local personnel responsible for mosquito abatement activities, as well as community involvement. Biocontrol methods as an alternative to chemical insecticides result from the sustainability development concept, growing awareness of environmental pollution and the development of insecticide-resistant strains of vector-mosquito populations in many parts of the world. Although sustainable trends are usually considered in terms of the monetary and training resources within countries, environmental concerns are actually more limiting factors for the duration of an otherwise successful vector control effort. In order to meet these new needs, increasing efforts have been made in search of and application of natural enemies, such as parasites, bacterial pathogens and predators which may control populations of insect vectors. The biological control agent based on the bacterial toxins Bti and Bs has been used in the Wroclaw's University and Municipal Mosquito Control Programs since 1998. In West-Africa biocontrol appears to be an effective and safe tool to combat malaria in addition to bed-nets, residual indoor spraying and appropriate diagnosis and treatment of malaria parasites which are the major tools in the WHO Roll Back Malaria Program. IVM studies carried out 2005-2008 in Cotonou (Benin) as well those in Wroclaw Irrigated Fields during the last years include the following major steps: 1. Mapping of all breeding sites in the project area and recording data in a geographical information system (GIS/relational database). All districts, streets and houses are numbered for quick reference during the operation; 2. Studying mosquito vector bionomics, migration and vectorial capacity in the project area, before, during and after the routine Bti treatments; 3. Assessment of the optimum for effective larvicide insecticide dosages at major breeding sites against the different target mosquito species; 4. Implementation of the microbial control agents in the integrated routine program. Adaptation of the application equipment to the local situation, training of the field staff, and routine treatments; 5. Conducting surveillance of vector-disease (e. g., malariometric) parameters in the control and experimental area before, during, and after the application of biocontrol agents.

[Mapping of parasitological environmental data: the tick Ixodes ricinus--a case of study]. / Kartowanie srodowiskowych badan parazytologicznych na przykladzie kleszczy pospolitych Ixodes ricinus.

While the mapping of health data is not new for epidemiologists the incorporation of differentiated environmental factors, e.g., temperature, rainfall, humidity, elevation, vegetation type, host abundance and distribution, zoonotic reservoirs of infection can create a new opportunities for parasitologists. Suitable tools for spatial modeling of health problems and pathogen occurrence in space and time are provided by geographic information system (GIS). It is computer-based system which integrates, storages, edits, analyses, shares and displays information. This software system is based on connection between information--data and their location. GIS applications allow users to create interactive queries, analyze spatial information, edit data and maps. GIS is very useful to define the habitats of parasites, especially for the ticks which are strong depended on environmental conditions. Mapping not only enables to create maps based on field monitoring but also to create forecasting maps for prevention and control strategies on small and large scale. Up to now ticks and tick-borne diseases (TBD) having strong relationship with the ecosystem are highly amenable to predictive mapping. The aim of study is the characterization of procedural steps with regard to entering field environmental data to GIS database and their visualization on digital maps. The field date of tick monitoring conducted in April 2008 in the Wroclaw area (the Osobowicki Forest) made possible to create digital database. ArcView as one of three separate software products of ArcGIS (a scalable framework for implementing GIS) was used to create an interactive maps. Visualization of the data which are stored in tables of attributes made possible to show legibly the distribution of I. ricinus on the analysed area. Mapping of I. ricinus occurrence on digital maps enable to indicate areas of the highest risk of biting and potential tick-borne diseases.

[Changes in range of mosquito-borne diseases affected by global climatic fluctuations]. / Zmiany zasiegu chorób transmitowanych przez komary pod wplywem globalnego ocieplenia klimatu.

Climate models suggest the strong possibility of range increase of the diseases transmitted by parasitic arthropods, mostly mosquitoes. In predicting processes of malaria and Dengue diseases dispersion the estimation of risk is based mostly on reproduction rate of vector species. These models allow to calculate the critical threshold of host density which is necessary to maintain parasites and pathogens transmission. Such studies based on integrated mathematical modelling indicate widespread increase of risk due to expansion of the areas suitable for mosquito-borne diseases transmission. This predicted increase is the most pronounced at the borders of the endemic areas and at higher altitudes within malaria and Dengue areas. The simulated change in mosquito-borne diseases risk must be interpreted on the basis of local environmental conditions as well as the effects of socio-economic developments and control disease programs. Apart from mathematical models the sequencing of proteins and DNA of vectors and their pathogens as well as satellite technology (GIS) are taken into consideration. It is supposed that potential impact of global climate change on malaria and Dengue risk can be reduced by constant warning system based on biological monitoring of mosquito vector species and their pathogens. Efficient care system connected with full diagnosis, treatment and prophylaxis of transmission diseases are also required.

[Environmental conditions of evening activity of synanthropic mosquito species (Diptera: Culicinae) in the Wroclaw area]. / Srodowiskowe uwarunkowania wieczornej aktywnsci synantropijnych gatunków komarów (Diptera: Culicinae) na terenie Wroclawia.

MATERIALS AND METHODS: Temporal variations in the evening activity pattern of adult mosquito females of locally common mosquito were studied at Wroclaw area using CO2 charged olfactory traps. RESULTS: There has been 4856 synanthropic individuals which belonged to Culex pipiens (L.) i Culiseta annulata (Schrank.) species distinguished from the general population of 5997 individuals caught in the three sampling sites. The number of active mosquito females increased from 4 pm to 9 pm creating one distinct peak during the evening activity maximum. Statistical analysis of the results showed the important correlation between mosquito activity of locally common populations and air relative humidity supported by variation of light intensity.

[Environmental monitoring and control strategy of urban mosquito Culicidae (Diptera: Culicidae) populations in Wroclaw]. / Monitoring srodowiskowy i zwalczanie miejskich populacji komarów Culicinae (Diptera: Culicidae) we Wroclawiu.

Among dipterans of medical and veterinary importance, the mosquitoes (Culicidae) play the most important role both as vectors and nuisance insects. Mass occurrence of floodwater mosquito species in the flooded area in Wroclaw district (Lower Silesia, Poland) in 1997 has enhanced the already existed problem of mosquito control. A successful model of control strategy for the city based on the currently recommended integrated methodologies with special emphasis on preventive treatment of aquatic larvae with microbial insecticides has been conducted. Bacillus thuringiensis israelensis-based formulations replaced chemicals to control Culex pipiens and Culiseta annulata--two dominant species out of eight noticed during field study in Wroclaw area in the years 1998-2000. Use of Bti larvicide or other biocontrol agent, conserving biodiversity, requires collecting entomological data, mapping and treating all breeding sources as well as the designing appropriate strategies every year.

[Transgenic bioinsecticides inimical to parasites, but imical to environment]. / Nieprzyjazne pasozytom a przyjazne srodowisku transgeniczne bioinsektycydy.

Identification of Bacillus thuringiensis (Bt) parasporal crystalline inclusions composed of Cry proteins (=delta-endotoxins) resulted in introduction of microbial pesticides for biological control of some parasites. Delta-endotoxins are encoded by cry genes and are active against pest and nuisance insects (mostly mosquitoes and black flies--vectors of still important infectious diseases). The recent significant progress in DNA recombination technique may overcome limitations (a short residual persistence and a narrow spectrum of activity) associated with application of Bt conventional products. An introduction of cry genes from mosquitocidal subspecies B. th. israelensis (Bti) to the aquatic microorganisms inhabiting the same water bodies as mosquito and fly larvae (Diptera), has considerably improved the toxin delivery system to target insects. However, in the first experiments, in which Bti genes were cloned in cyanobacteria (Agmenellum quadruplicatum, Synechocystis PCC6803), a low gene expression was observed. Thus, it was necessary to integrate cry genes with strong promoters or to increase the number of vector-introduced copies. To overcome the obstacles of low gene expression and regulatory restriction for recombinant organisms, Bti spore/crystal formulations were encapsulated in the aquatic protozoan, Tetrahymena pyriformis. Large numbers of crystals (180 to 240/cell) were accumulated in its food vacuoles. This system resulted also in an increase in toxin persistence from 24 to 71 h. Cloning Bti genes in B. sphaericus (which also produces mosquitocidal proteins) was another way of an increasing Bt crystal residual activity. In this case, the crystals were additionally protected by B. sphaericus exosporium. These transgenic bacteria produced large amounts of delta-endotoxins that remained under water surface longer than the wild B. sphaericus strains. Moreover, they had a broader spectrum of insecticidal activity, because B. sphaericus is toxic mostly to Culex and Anopheles, and Bti--mostly to Culex, Aedes and some Simmulidae. Gram-negative bacteria (Asticcacaulis excentricus, Caulobacter crescentus and Ancylobacter aquaticus) turned out also to be effective delta-endotoxin producers. They grow on simple media and do not contain proteases which could degrade Cry proteins. In some cases, 100% mosquito larvae mortality was observed as a result of an exposure to transgenic microorganisms containing Bti genes. However, transgenic techniques are still not very popular in the world, despite their efficacy in biological control of insects. The transgenic organism construction is expensive and time-consuming. Genetic engineering is still raising a lot of anxieties and doubts concerning inappropriate use of modified organisms. On the other hand, this technology could solve many problems associated with vectors of important diseases, which are still unapproachable to contemporary medicine.

Environmental factors associated with the distribution of floodwater mosquito eggs in irrigated fields in Wroclaw, Poland.

A survey of distribution patterns of floodwater mosquito eggs related to environmental conditions such as moisture and plant associations was conducted by using soil samples from irrigated fields in Wroclaw, Poland. Mosquito egg distribution was determined by repeatedly flooding the soil samples with aerated water at a temperature of 25° C. Under laboratory conditions, hatching in installments of Aedes caspius (Pallas) and Aedes vexans (Meigen) were commonly observed. The results show that ∼75% of the larvae of Ae. caspius and Ae. vexans hatched after the first flooding under summer-like conditions, whereas, following the second and third flooding, the numbers of hatched larvae were significantly lower. In our study, within one intermediate flooded field, a total of 66 plant species was identified and classified into six communities. All vegetation types were associated by varied egg densities and showed differences both in richness and Shannon-Wiener diversity index as well as in ecological indices for moisture, soil reaction, and nutrient level. Small changes in elevation along the slope within the study area showed a large difference in the distribution of mosquito eggs. The highest average egg density was observed in zones with high occurrence of Phalaris arundinacea, usually prevalent in intermediate flooded and fertile areas. Knowledge of the indicators for the distribution of floodwater mosquito eggs in temporary breeding sites may be essential for organizing a successful, integrated mosquito control program with special regard to microbial control agents.