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Different types of polyphagous true bug pests are present in Serbia and countries in the region, many of which stand out as significant pests of agricultural crops. Among the harmful true bugs, the green vegetable bug, Nezara viridula Linnaeus, 1758 and the brown marble bug Halyomorpha halys Stål, 1855 (Hemiptera: Pentatomidae) are the species that currently attract attention the most, primarily because of their invasive and polyphagous nautre and ability to migrate rapidly from one crop to another. In many regions, where harmful bugs cause large economic losses, control strategies are primarily based on the application of chemicals. In addition to chemical control measures, biological control measures are increasingly present, which are based on the application of natural enemies of pests. Species from the genus Trissolcus (Hymenoptera: Scelionidae) show the best results in the biological control of polyphagous true bug. The species that has been commercially used for many years to control harmful Hemiptera is Trissolcus basalis Wollaston, 1958, which is one of the most important natural enemies of the green vegetable bug in the World. The species Trissolcus japonicus Ashmead, 1904 and Trissolcus mitsukurii Ashmead, 1904 have been identified as the predominant parasitoids of the brown marble bug and show great potential in the biological control of this harmful species. Representatives of the genus Trissolcus are also registered on the territory of Serbia, which opens the possibility of including native species of the genus Trissolcus in the strategies of biological control of true bug pests on the territory of Serbia and the region.

Invasive mosquito species surveillance has been carried out in Europe since late 1990s, and the results revealed that their distribution range and population density have been increasing every year (ECDC, 2020). Majority of European countries are affected by at least one of invasive mosquito species, which are considered as important vector species of public health concern. Aedes invasive species are very aggressive daily biters and highly competitive with native mosquito species coexisting in the same breeding sites. Although pathogens transmitted by mosquitoes are much bigger problem in tropical areas, imported and autothonous cases of these diseases have been recorded every year in Europe. International and intercontinental transport of humans and goods increase the likely-hood of outbreaks caused by vector-borne pathogens. Cases of imported invasive mosquito species and cases of imported human infection (with Dengue and Chikungunya virus, imported and autohtonous) are increasing every year. Together with the presence of invasive mosquito species, outbreaks caused by vector-borne pathogens are significantly driven by human behaviour, ecosystem and climat changes. Two invasive mosquito species were present in Serbia so far: Aedes albopictus (Asian tiger mosquito) and Aedes japonicus (Japaneese bush mosquito). Populations of Ae. albopicus has been succesfully spreading in many urban and suburban areas in our country, while Ae. japonicus was identified in only two localities up today. According to the experience from Croatia, where this mosquito species is widely spread, similar scenario could be expected in Serbia as well.

A study on the occurrence of Dirofilaria immitis and its vectors was carried out in order to assess the prevalence of the disease in dogs in previously non-endemic areas of southern Italy. Blood samples (n = 385) and mosquitoes (n = 1540) were collected in two dog shelters and analysed by Knott’s test and duplex real-time PCR, respectively. Dirofilaria immitis was the most prevalent filarioid (44.2%), while Culex pipiens was the most prevalent mosquito species (68.8%). This high prevalence of D. immitis infection confirms this location as one of the most hyperendemic foci of dirofilariosis in Europe.

The aim of the study was to evaluate the bio-efficacy of two different acaricides against mobile stages of hard ticks Ixodes ricinus, Dermacentor marginatus and Haemaphysalis punctata in their natural habitats. The study was conducted during 2020 and 2021 at localities populated by I. ricinus as the predominant species, at which the presence of Borrelia afzeli, B. garini and B.lusitaniae was confirmed. During the first investigation year, a combination of two pyrethroids, permethrin and tetramethrin, with an insecticide synergist piperonyl butoxide (trade name: Perme Plus®) was tested. At the first evaluation, 24h after the treatment with Perme Plus®, the efficacy expressed as reduction rate of the population density was within the interval of satisfying performance (70% − 90%) at all localities, while the highest efficacy (97.80%) was recorded on the 14th post-treatment day. In the second investigation year, the formulation based on lambdacyhalothrin (trade name: Icon® 10CS) was used. At the first post-treatment evaluation day satisfying effects were also demonstrated. The highest recorded efficacy rate of lambda cyhalothrin (94.68%) was recorded on the 14th post-treatment day. Both tested acaricides manifested satisfying initial acaricidal effects against mobile stages of ticks and provided long-term effects. Comparison of the regression trend lines of population reduction revealed that satisfying effects of treatment with Perme Plus® lasted until the 17th post-treatment day, while the in the case of Icon® 10CS the residual effects were significantly prolonged (30 days).

Rodents belong to the group of occasionally economically significant to very significant pests. Some species, especially synanthropic species, cause material damage in various ways. Successful and sustainable management of rodent control strategies requires different procedures such as prevention and rodent control measures. The present study gives an overview of the most common rodent species and methods for estimating the rodent population to assess the risk of economic damage that may occur due to rodents feeding in field crops, gardens, orchards, and young forest plantations, as well as contamination of stored food. As a prerequisite for effective integrated control of mice and voles, it is necessary to implement an adequate long-term monitoring system of these species, as they are primary pests. The integrated approach improves the treatment efficacy and reduces the treatment costs but also is considered ecologically friendlier compared to conventional measures. An integrated approach should provide an effective strategy for rodent management and control in all types of rodent habitats, from agricultural and forestry production fields to residential and public areas. By combining different preventive measures, it is possible to prevent the presence of pests, which will therefore result in a reduction of pesticide use.

The results of the Serbian national integrated West Nile virus (WNV) surveillance program conducted in 2018 and funded by the Serbian Veterinary Directorate are presented. The WNV surveillance program encompassed the entire territory of Serbia and was conducted by the veterinary service in collaboration with entomologists and ornithologists. The objective of the program was early detection of WNV circulation in the environment and timely reporting to the public health service and local authorities to increase clinical and mosquito control preparedness. The program was based on the detection of WNV presence in wild birds (natural hosts) and mosquitoes (virus vectors) and on serological testing of sentinel horses (WNV-specific IgM antibodies). The season 2018 was confirmed to be the season of the most intensive WNV circulation with the highest number and severity of human cases in Serbia ever reported. The most intense WNV circulation was observed in the northern and central parts of Serbia including Vojvodina Province, the Belgrade City area, and surrounding districts, where most positive samples were detected among sentinel animals, mosquitoes and wild birds. The majority of human cases were preceded by the detection of WNV circulation during the surveillance. The WNV surveillance program in 2018 showed satisfactory results in the capacity to indicate the spatial distribution of the risk for humans and sensitivity to early detection of WNV circulation in the environment.

Aquatain® mosquito formulation (AMF) is a silicone‐based monomolecular film, which has recently been approved for use in the European Union. The physical mode of action based on lowering water surface tension prevents mosquito larvae/pupae respiration. Additionally, AMF disables gravid females from landing on the water surface and obstructs the natural oviposition process. Due to multistage effects on mosquitoes, AMF could be a product of choice for defined water body and container breeders such as Culex pipiens L. complex, principal vector of West Nile virus in Europe, and the invasive Aedes albopictus (Skuse) (both Diptera: Culicidae), vector of dengue and chikungunya viruses. The primary objectives of this study were to evaluate the efficacy of AMF, to determine the susceptibility of the immature forms of C. pipiens and A. albopictus, and the persistence/longevity of the product to suppress the eclosion of adults. AMF achieved high mortality rates of juvenile A. albopictus and C. pipiens under laboratory conditions. However, in the field C. pipiens larvae showed higher susceptibility to AMF than A. albopictus. Pupae of the two mosquito species were highly susceptible to the presence of AMF. When C. pipiens juveniles were exposed to AMF in the wild, effects lasted for 21 days in densely covered water bodies and 56 days in water recipients with less vegetation. In both breeding sites, natural habitat and artificial water recipient, the two mosquito species with high impact on public health in Europe could successfully be suppressed by application of AMF (1 ml m⁻²).

Vegetable production is one of the most intensive parts of agricultural production in Serbia. There are several different classifications of vegetable species, but the most common is based on the edible part of the plant. This classification emphasizes root vegetables as a special and very important category. During the entire vegetation period, root vegetables are exposed to numerous pests, which reduce the value and quality of the final product. In addition to insects, which represent the most numerous group of animals, vegetables are also damaged by mites, nematodes, snails, rodents and some species of birds. One of the ecologically acceptable methods in the protection of vegetables is trap cropping, whereby certain plant species, the so-called trap plants are used to attract pests, in order to reduce the damage of the main crop. In modern agricultural production, trap crops are used as a common tool in Integrated Pest Management. Rising interest of agricultural producers for trap cropping is primarily attributed to the public concern about the potential negative effects of pesticides to human and environmental health, as well as to the increasing problem of pest resistance to insecticides. Since root vegetables intended for the market are primarily in fresh condition, an important segment of production is the effective protection of vegetable crops, which implies production of healthy and safe food. In that sense, the consideration of trap crops application in protection of root vegetables is more than justified, because trap cropping represents an environmentally friendly method and an effective alternative to conventional protection of vegetables from economically important pests.

Summary The housefly Musca domestica is a cosmopolitan insect nuisance pest, also known as a carrier of numerous pathogens both to humans and animals. Animal farms, as a very important source of house flies, simultaneously allow for all stages of their development. Having vast quantities of constantly present manure, pig units represent perfect environment for house fly breeding. This fact, coupled with the known resistance to majority of available insecticides, creates difficulties in house fly control. The present study was performed to evaluate different types of monitoring methods for indoor use: (a) spot deposit records on the cardboard; (b) fly trappings by: glue coated cardboard; (c) sticky fly strips; (d) yellow sticky cards; (e) visual records of flies on the cardboard. This study provided a clear differentiation of efficacy/usefulness of the tested methods for various house fly densities. In animal breeding units or other areas with very abundant fly population, less sensitive methods (traps are less efficient) should be selected. For these circumstances, glued cardboard or yellow sticky cards should be chosen. The “more sensitive” methods, spot cards and sticky fly strips, should be used for lower abundance of the fly population. Except for this purpose, these highly sensitive methods should be selected also in the areas where the flies should not be tolerated at any density. Although the levels of the observed fly activity significantly differ from each other, in the majority of cases they depict the similar trend of the population dynamics and relative density. The only exception to the rule was the visual method, which could not reflect the changes in the population density in the current study.

International audience; The current study extends the faunistic survey ofTrichogrammaspecies parasitizing ECB to all agricultural growing regions in Serbia. Specimens ofTrichogrammawere reared from parasitized egg masses of ECB collected from field-grown corn and pepper crops. The number of egg masses parasitized varied by location. Using sequences of mitochondrial cytochrome oxidase I (COI) gene, we examined intra and interspecific variation inTrichogrammaparasitizing the ECB egg masses. Seventy specimens were successfully sequenced of which, 65 were identified asT. brassicae. The remaining five were identified asT. evanescens, and this second species was only collected from pepper.

Paper description: Internal transcribed spacer 2 (ITS2) sequences have proved to be useful in providing reliable identification of Trichogramma species. Identification of resident populations of Trichogramma wasps to species level has not been done in Serbia and has limited the potential for large scale use of these parasitoids. The current study used DNA sequences of the ITS2 gene to identify the most prevalent species in Vojvodina as Trischogramma brassicae . Accurate identification of native populations of Trichogramma wasps should help in the choice of the most suitable species for mass rearing and field release. Abstract: The European corn borer Ostrinia nubilalis (Hubner, 1796) (Lepidoptera: Pyralidae) is a major pest of corn ( Zea mays Linnaeus, 1753) in Serbia. Regular monitoring of O. nubilalis in the region of Kikinda (province of Vojvodina, Serbia) demonstrated a high percentage of parasitized eggs of this pest. Preliminary identification of the egg parasitoids based on morphological traits revealed that they belong to the genus Trichogramma Westwood, 1833 (Hymenoptera, Trichogrammatidae). The objective of this study was to identify the autochthonous Trichogramma species present in the region of Kikinda by using sequences of ribosomal DNA. Specimens of Trichogramma were reared from parasitized eggs of O. nubilalis collected at different localities in the region of Kikinda. The ITS2 gene was sequenced and subjected to BLAST searches of GenBank NCBI (National Center for Biotechnology Information). The majority of collected specimens were identified as Trichogramma brassicae Bezdenko, 1968, and a single specimen as T. evanescens Westwood, 1833. Intraspecific variation of T. brassicae was assessed by amplifying the mitochondrial cytochrome oxidase I (COI) gene . Wasps were also tested for the presence of the endosymbiotic bacteria Wolbachia Hertig, 1936 using Wolbachia -specific 16S and wsp primers as a preliminary test for evidence of thelytoky. Understanding of the diversity and biology of autochthonous Trichogramma species is a key step in the potential commercial implementation of these egg parasitoid wasps in the protection of corn fields in Serbia. https://doi.org/10.2298/ABS171103002I Received: November 13, 2017; Revised: January 12, 2018; Accepted: January 18, 2018; Published online: January 23, 2018 How to cite this article: Ivezic A, Rugman-Jones P, Stouthamer R, Ignjatovic-Cupina A. Molecular identification of Trichogramma egg parasitoids of Ostrinia nubilalis in northeastern Serbia. Arch Biol Sci. 2018;70(3):…

Motivated by the One Health paradigm, we found the expected changes in temperature and UV radiation (UVR) to be a common trigger for enhancing the risk that viruses, vectors, and diseases pose to human and animal health. We compared data from the mosquito field collections and medical studies with regional climate model projections to examine the impact of climate change on the spreading of one malaria vector, the circulation of West Nile virus (WNV), and the incidence of melanoma. We analysed data obtained from ten selected years of standardised mosquito vector sampling with 219 unique location-year combinations, and 10 years of melanoma incidence. Trends in the observed data were compared to the climatic variables obtained by the coupled regional Eta Belgrade University and Princeton Ocean Model for the period 1961-2015 using the A1B scenario, and the expected changes up to 2030 were presented. Spreading and relative abundance of Anopheles hyrcanus was positively correlated with the trend of the mean annual temperature. We anticipated a nearly twofold increase in the number of invaded sites up to 2030. The frequency of WNV detections in Culex pipiens was significantly correlated to overwintering temperature averages and seasonal relative humidity at the sampling sites. Regression model projects a twofold increase in the incidence of WNV positive Cx. pipiens for a rise of 0.5°C in overwintering TOctober-April temperatures. The projected increase of 56% in the number of days with Tmax ≥ 30°C (Hot Days-HD) and UVR doses (up to 1.2%) corresponds to an increasing trend in melanoma incidence. Simulations of the Pannonian countries climate anticipate warmer and drier conditions with possible dominance of temperature and number of HD over other ecological factors. These signal the importance of monitoring the changes to the preparedness of mitigating the risk of vector-borne diseases and melanoma.

In the past decade, leishmaniasis seems to be re-emerging in Balkan countries. There are serious implications that Kosovo is a visceral leishmaniasis endemic region with autochthonous transmission; nevertheless, surveillance of vectors, reservoirs or the disease is not yet established. Gaining knowledge about sandfly vector species is a prerequisite for the development of a monitoring and control plan in the future. After a long gap in research of over 70 years, sandfly studies in Kosovo were resumed in 2014. During this presence/absence study, nine sandfly species were detected: Phlebotomus papatasi, Ph. perfiliewi, Ph. tobbi, Ph. neglectus, Ph. simici, Ph. balcanicus, Ph. alexandri, Ph. mascittii and Sergentomyia minuta. Three species are new with regard to the fauna of Kosovo - Ph. alexandri, Ph. balcanicus and Ph. mascittii. Besides increased diversity, changes in the number of collected specimens and distribution range of species were recorded, with Ph. neglectus being the most dominant species with the widest distribution. Testing of randomly chosen females for Leishmania spp. DNA resulted the in detection of L. tropica in a specimen of Ph. neglectus. the presence of numerous vector species in the sandfly fauna of Kosovo pose a threat for the re-emergence of vector-borne diseases. Therefore, continuous surveillance is recommended with regular updates on vector distribution and abundance., EurNegVec COST ActionEuropean Cooperation in Science and Technology (COST) [TD1303]; VectorNet project - European Food Safety Authority (EFSA); European Centre for Disease Prevention and Control (ECDC) contract [OC/FSA/AHAW/2013/02-FWC1]; European Virus Archive goes Global project (EVAg) - European Union's Horizon 2020 research and innovation programme [653316]; Ministry of Education and Science of the Republic of Serbia [III43007, TR31084]; European Commission, Horizon 2020 Infrastructure Infravec2 projectEuropean Commission, The authors express their gratitude to Vid Srdic who designed the map for this manuscript. the work of Slavica Vaselek, Yusuf Ozbel, Bulen Alten and Dusan Petric was carried out under the frame of EurNegVec COST Action TD1303. the work was supported by VectorNet project funded by the European Food Safety Authority (EFSA) and the European Centre for Disease Prevention and Control (ECDC) contract OC/FSA/AHAW/2013/02-FWC1; European Virus Archive goes Global project (EVAg) funded by the European Union's Horizon 2020 research and innovation programme (grant agreement No 653316); and projects III43007 and TR31084 financed by the Ministry of Education and Science of the Republic of Serbia and European Commission, Horizon 2020 Infrastructure Infravec2 project (https://infravec2.eu).

The Asian bush mosquito, Aedes japonicus japonicus (Theobald, 1901), a potential vector of several pathogens, has recently established in North America and Central Europe. In 2013, it was found on the Slovenian-Croatian border, and during the following years, it emerged in more and more counties of northwestern Croatia. Surveillance of Ae. j. japonicus and other invasive mosquito species was subsequently extended both spatially and temporally in Croatia and neighbouring Bosnia and Herzegovina and Serbia. Mosquito collections were conducted in 2017 and 2018, based on adult trapping through dry ice-baited CDC traps and BG- Lure- baited BG-Sentinel traps, larval sampling through dippers and nets, and ovitrapping. Aedes j. japonicus specimens from collected samples were subjected to population genetic analysis by comparing microsatellite signatures and nad4 DNA sequences between sampled locations and with data previously obtained from more western European distribution areas. Aedes j. japonicus immature stages were found at 19 sites in Croatia, two sites in Bosnia and Herzegovina and one site in Serbia. In Croatia, four new counties were found colonised, two in the east and two in the south of the previously known distribution area. A spread of 250 km could thus be documented within five years. The findings in Bosnia and Herzegovina and Serbia represent the first records of Ae. j. japonicus in these countries. Genetic analysis suggests at least two introduction events into the surveyed area. Among the locations analysed, Orahovica can be considered a genetic border. The individuals collected west of this point were found to be similar to samples previously collected in the border regions of Southeast Germany/Austria and Austria/Slovenia, while the specimens from more eastern Croatian localities, together with those from Bosnia and Herzegovina and Serbia, were genetically different and could not be assigned to a probable origin. Thus, introduction from Central Europe, possibly by vehicular traffic, into the study area is likely, but other origins, transportation routes and modes of entry appear to contribute. Further dispersal of Ae. j. japonicus to other parts of southeastern Europe is anticipated.

In mainland Europe, the mosquito species Culex modestus Ficalbi (1890) is a bridge vector for West Nile virus (WNV) from its natural bird-mosquito cycle to mammals. The present study assessed the genetic diversity of Cx. modestus, as well as related Culex species, using the mitochondrial COI DNA barcoding region and compared this with the population structure across Europe. A haplotype network was mapped to determine genealogical relationships among specimens. The intraspecific genetic diversity within individual Culex species was below 2%, whereas theinterspecificgeneticdivergencevariedfrom2.99%to13.74%.Intotal,76haplotypes were identified among 198 sequences. A median-joining network determined from 198 COI sequences identified two major lineages that were separated by at least four mutation steps. A high level of intraspecific genetic diversity was not detected in Cx. modestus in samples submitted from different European populations, which indicates that morphologically identified specimens represent a single species and not a species complex. Therefore, it is deduced that different populations of Cx. modestus will show a similar potential to transmit WNV, lending support to concerns that the population present in southeast England represents a risk of transmission to humans. The authors thank the Department for Environment and Rural Affairs (DEFRA), Scottish Government and Welsh Government (grants SV3045 and SE4113) for funding the present study. EU Framework Horizon 2020 Innovation Grant, European Virus Archive (EVAg, grant no. 653316) is also acknowledged for providing funds for sequencing. Ignacio Ruiz-Arrondo thanks the European Regional Development Fund (ERDF) for funding. info:eu-repo/semantics/publishedVersion

Efforts to detect West Nile virus (WNV) in the Vojvodina province, northern Serbia, commenced with human and mosquito surveillance in 2005, followed by horse (2009) and wild bird (2012) surveillance. The knowledge obtained regarding WNV circulation, combined with the need for timely detection of virus activity and risk assessment resulted in the implementation of a national surveillance programme integrating mosquito, horse and bird surveillance in 2014. From 2013, the system showed highly satisfactory results in terms of area specificity (the capacity to indicate the spatial distribution of the risk for human cases of West Nile neuroinvasive disease - WNND) and sensitivity to detect virus circulation even at the enzootic level. A small number (n = 50) of Culex pipiens (pipiens and molestus biotypes, and their hybrids) females analysed per trap/night, combined with a high number of specimens in the sample, provided variable results in the early detection capacity at different administrative levels (NUTS2 versus NUTS3). The clustering of infected mosquitoes, horses, birds and human cases of WNND in 2014-2015 was highly significant, following the south-west to north-east direction in Vojvodina (NUTS2 administrative level). Human WNND cases grouped closest with infected mosquitoes in 2014, and with wild birds/mosquitoes in 2015. In 2014, sentinel horses showed better spatial correspondence with human WNND cases than sentinel chickens. Strong correlations were observed between the vector index values and the incidence of human WNND cases recorded at the NUTS2 and NUTS3 levels. From 2010, West Nile virus was detected in mosquitoes sampled at 43 different trap stations across Vojvodina. At 14 stations (32.56%), WNV was detected in two different (consecutive or alternate) years, at 2 stations in 3 different years, and in 1 station during 5 different years. Based on these results, integrated surveillance will be progressively improved to allow evidence-based adoption of preventive public health and mosquito control measures.

The aim of this study is to summarize the data on the occurrence, presence and prevalence of some zoonotic flaviviruses, which have been actively circulating in the territory of the Republic of Serbia during past decade. The genus Flavivirus, family Flaviviridae,encompasses vector-transmitted viruses with zoonotic potential. The genus is comprised of more than 70 viruses transmitted to humans by mosquitoes and ticks. Some of those are associated only with human diseases (such as yellow fever and dengue fever), whereas others can cause diseases in both humans and animals. Animals are major reservoirs and primary hosts for the latter group. The virus transmission to other animal species or humans occurs via diverse vectors – mostly mosquitoes and ticks. A range of these virus species is widely distributed worldwide, especially in tropical and sub-tropical climatic zones. Some representatives of these species have only recently been identified at the territory of Republic of Serbia. This paper gives an evidence on the presence and distribution of West-Nile virus, tick-borne encephalitis (TBE) virus and Usutu virus that were conducted within the study in Serbia. The research pointed out the presence of recently detected West-Nile virus and Usutu virus as well as the current circulation of tick-borne encephalitis virus, for which only limited serological evidence on the presence of virus-specific antibodies in humans was available so far. In the aspect of public health, the full understanding of the importance of identification and prevalence of different flaviviruses still require further comprehensive entomologicalacarological, seroepidemiological, clinical and virological research.

Background Blackflies have negative impact on public and animal health due to the haematophagous habit of females. In recent times, in some regions in Spain, blackfly outbreaks are becoming more and more frequent, threatening the public health. However, there is still a paucity of data concerning the Spanish blackfly fauna. Correct identification of species is of paramount importance in order to provide correct information on species distribution, biology and behaviour, so that control measures could be implemented appropriately. Methods Blackflies specimens (larvae, pupae, reared adults and biting females) were collected in the period 2015–2017 in and near rivers and streams from different regions in Spain. A modified Hotshot technique was used for the DNA extraction and the cox1 DNA barcoding region of the cytochrome c oxidase subunit 1 was sequenced from the specimens collected. Results In total, we collected 239 specimens representing 22 species. Of these, six species are new records for the Aragón region: P. tomosvaryi, S. bertrandi, S. galloprovinciale, S. lineatum, S. rubzovianum and S. xanthinum. Cox1 DNA barcode sequences for 21 species were recovered, including four species of the genus Prosimulium and 17 species of the genus Simulium [Boophthora (1 species), Eusimulium (1 species), Nevermannia (4 species), Simulium (s.s.) (6 species), Trichodagmia (1 species) and Wilhelmia (4 species)]. For the first time the complete DNA barcodes for five species (P. tomosvaryi, S. carthusiense, S. brevidens, S. monticola and S. sergenti) were registered. Most of the specimens belonging to the same recognized species were clustered together in the neighbour-joining tree, except for S. argyreatum, S. monticola and S. variegatum. The overall genetic distance in the dataset was 0.14%. The average of the intraspecific genetic divergence within the different taxa was 1.47% (0.05–3.96%). In contrast, the interspecific divergence varied between 2.50–22.0%. Conclusions In this study we assessed the use of the cox1 DNA barcoding region for the identification of species of blackflies in Spain. Our results showed that combining DNA barcoding with morphology enhanced our taxonomic rationale in identifying the blackflies in the country. Electronic supplementary material The online version of this article (10.1186/s13071-018-3046-7) contains supplementary material, which is available to authorized users.

uring a hot Mediterranean summer, an expedition brought parasitologists from Brazil, France, Greece, Italy, and Serbia to a wooded area near Xanthi, Thrace, northeastern Greece, near the Turkish border, on the track of the vector of the little-known nematode Onchocerca lupi. The scientifi c purposes of the expedition blended then with stories of humans, animals, and parasites in this rural area. The Beginnings: What’s that Worm in the Turkish Blue Eye? In the early months of 2010, Nermin Sakru, a microbiologist from the Medical School of Trakya University, Erdine, Turkey, contacted one of the authors (D.O.) seeking advice on how to identify a nematode extracted from the eye of an 18-year-old girl who had never traveled out of her native Trakya. Nematodes that might have caused such an infestation were many (1), including Thelazia callipaeda. This helminth, which infests carnivores and humans, has been studied for more than a decade at the University of Bari in southern Italy (2–5); this research was what led the Turkish colleague to establish original contact. The patient complained of pain and redness in the left eye and reported being bitten by a fl y on the left eyelid in the evening (around 5:00 PM), ≈30 days before onset of symptoms. Pain caused by a biting insect was suggestive of infestation other than by T. callipaeda, which is transmitted by Phortica variegata (Diptera, Drosophilidae), an insect that gently feeds on the ocular secretions of its hosts during the pleasantly warm Mediterranean summers (6). Some days later, the nematode was morphologically and molecularly identifi ed as a little spirurid, Onchocerca lupi, known to infest dog eyes, inducing an acute or chronic ocular disease characterized by conjunctivitis, photophobia, lacrimation, discharge, and exophthalmia. At that time, this helminth infestation had never been reported in dogs in Turkey, and information on the biologic features of the nematode was still meager, despite its wide distribution in Greece, Germany, Hungary, Portugal, and Switzerland and the increasing number of reported cases (7). O. lupi nematode infestation in humans (8) and its biologic and pathogenic affi nity with Onchocerca volvulus, the agent of river blindness, heightened the interest of D.O. and F.D.T. in the life cycle of this nematode. The idea of investigating the biologic features of O. lupi soon began to move across the convoluted pathways of their brains like larvae of Oestrus ovis (the nasal bot fl y) migrating toward the central nervous system, the main decisional center of all animals!

The European black fly Simulium (Simulium) colombaschense (Scopoli), once responsible for as many as 22,000 livestock deaths per year, is chromosomally mapped, permitting its evolutionary relationships and pest drivers to be inferred. The species is 12 fixed inversions removed from the standard sequence of the subgenus Simulium. Three of these fixed inversions, 38 autosomal polymorphisms, and a complex set of 12 X and 6 Y chromosomes in 29 zygotic combinations uniquely characterize S. colombaschense and reveal 5 cytoforms: 'A' in the Danube watershed, 'B' in Italy's Adige River, 'C' in the Aliakmonas River of Greece, 'D' in the Aoös drainage in Greece, and 'E' in the Belá River of Slovakia. 'C' and 'D' are reproductively isolated from one another, and 'B' is considered a cytotype of 'A,' the probable name bearer of colombaschense. The species status of 'E' cannot be determined without additional collections. Three derived polytene sequences, based on outgroup comparisons, place S. colombaschense in a clade of species composed of the S. jenningsi, S. malyschevi, and S. reptans species groups. Only cytoforms 'A' and 'B' are pests. Within the Simuliidae, pest status is reached through one of two principal pathways, both of which promote the production of large populations of blood-seeking flies: (1) colonization of the world's largest rivers (habitat specialization) or (2) colonization of multiple habitat types (habitat generalization). Evolutionary acquisition of the ability to colonize large rivers by an ancestor of the S. jenningsi-malyschevi-reptans clade set the scene for the pest status of S. colombaschense and other big-river members of the clade. In an ironic twist, the macrogenome of S. colombaschense reveals that the name associated with history's worst simuliid pest represents a complex of species, two or more of which are nonpests potentially vulnerable to loss of their limited habitat.

Research in the field of vector borne diseases and zoonozes became a topic of interest in Serbia, during the last decade. Climate changes in the country (and the region) are evident. Also, significantly is higher frequency of human and animal movement and travel, especially of dogs, within the European countries, but with overseas countries as well. The presence of vectors has already been confirmed in the country and all the surrounding countries. Current research in the domain of infectious diseases in dogs mostly includes diseases which drastically endanger health and population of dogs. Some of those infectious diseases, like dirofilariosis and leishmaniasis, which are found more or less often in dogs, cause clinical symptoms which are not obvious and therefore they represent a danger for public health with dogs acting as reservoirs of the infection. Vectors necessary for the transmission of dirofilariosis are mosquitoes and for leishmaniasis are sand flies. Vectors of dirofilariosis are mosquitoes. Female mosqui‐ toes which feed on mammals can transfer microfilaria from one infected organism to another non infected one. Dirofilaria immitis is a nematode, intravascular parasite that lives in bloodstream of host, usually pulmonary vessels. Prepatent period is at least 6-7 months in definitive hosts. Maturation of organisms in mosquitoes is temperature dependant and over 14oC is needed. Diagnostic methods for dirofilariosis are many, but several serological methods can be used: ELISA modified Knott test, immunoem‐ zyme fast test and then molecular method (PCR), etc. Leishmaniasis is a vector borne zoonotic disease caused by a pathogen of Leishmania species. For the transmission of the disease, sand flies are needed as vectors from © 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Lutzomyia spp. Female sand flies are bloodsucking organisms which can transfer the pathogen from one host to another during their feeding time. The presence of Phlebotominae (commonly known as “sand flies”) has been identified in Serbia. The most certain method for diagnostic is demonstration of the parasite from bone marrow, splenic or lymph node aspirates, but there are other less invasive methods, like IFAT (immunofluorescent test) and ELISA (enzyme-linked immunosorbent assay). Material for the research were samples from dogs and samples of vectors. In total, 292 samples of mosquitoes were collected and identified and 170 of blood samples from dogs were examined for dirofilariosis and leishmaniasis. Methods used in the study were modified Knott test and PCR for dirofilariosis and ELISA test for leismaniasis. For dirofialriosis a total prevalence of the disease in dogs was found to be 15,29%, (with different values from 3-22%) for different groups of dogs (hunting and military dogs, dogs from asylum and pet dogs). Total seroprevalence for all 170 blood samples was 10,59% for leishmaniasis. Overall, there is acually no difference in seroprevalnce for leishmaniosis, between different groups of dogs (hunting and military dogs, dogs from asylum and pet dogs). There is a reasonable doubt that leismaniasis appears as a disease in the Northern part of Serbia, in region of Vojvodina. The presence of vectors has been identified (Phlebotomus papatasi, Laroussius tobbi) as well as existing seropre‐ valence in dogs with and without clinical symptoms. All of this suggests that there is an existence of the reservoirs of infection.

Introduction. Flies – by feeding on decaying matter, human waste and food – have been implicated in the spread of numerous animal and human diseases. Excessive fly populations are generally associated with livestock units and domestic waste due to decaying organic matter. A large number of flies cause extreme disturbance in the behavior of the host, resulting in skin irritation, lesions, wounds, and secondary infections are likely to appear. Objective. The aim of this study was to evaluate the effects of combined applications of larvicide (cyromazine) and adulticides (acetamiprid in formulation with pheromone and thiamethoxam) on the suppression of fly populations. Materials and methods. The study was conducted on a pig farm. The piglet farms are one of the most favorable places for fly breeding. Three units were used for biocide applications and a fourth unit as the control where biocides were not applied. The monitoring of pre- and post-treatment of adult fly populations was carried out by glued cardboards. The cards were hung on metal rods above piglet’s cage. This monitoring method served as a parameter for the estimation of biological effectiveness. Results. The highest degree of fly control (88.4% mortality 8 days after treatment) was achieved when a combination of cyromazine and thiamethoxam was used. A biocide based on sex pheromone (Z)-9-tricosene + acetamiprid was the most effective on flies 3 days after biocide application, with a mortality rate of 69.1 %. Thiamethoxam achieved the highest reduction of flies 6 days after treatment, with 78.19% obtained mortality. Conclusion. Biological efficacy of the applied biocides in combination ciromazine + thiamethoxam and thiamethoxam alone was justified.

Background Among the arthropod-borne nematodes infesting dogs, Onchocerca lupi (Spirurida: Onchocercidae) is of increasing zoonotic concern, with new human cases of infection diagnosed in Turkey, Tunisia, Iran and the USA. Knowledge of the biology of this nematode is meagre. This study aimed at assessing the distribution and periodicity of O. lupi microfilariae from different body regions in naturally infested dogs. Methodology/Principal Findings Skin samples were collected from six dogs infested with O. lupi but without apparent clinical signs. Two skin samples were collected from 18 anatomical regions of dog 1 at necropsy. In addition, single skin biopsies were performed from the forehead, inter-scapular and lumbar regions of dogs 2–6, in the morning, afternoon, and at night. Two aliquots of the sediment of each sample were microscopically observed, microfilariae counted and morphologically and molecularly identified. Most of the 1,667 microfilariae retrieved from dog 1 were in the right ear (59.6%), nose (26.5%), left ear (6.7%), forehead (3.0%), and inter-scapular (2.9%) regions. In dogs 2–6, the overall mean number of microfilariae was larger on the head (n = 122.8), followed by the inter-scapular (n = 119.0) and lumbar (n = 12.8) regions. The overall mean number of microfilariae was larger in the afternoon (153.4), followed by night (75.4) and morning (25.8). Conclusions Onchocerca lupi microfilariae were more common in the head (i.e., ears and nose) than in the remaining part of the dog's body, indicating they tend to aggregate in specific body regions, which are the best sites to collect skin samples for diagnostic purposes. The periodicity pattern of microfilariae of O. lupi and their concentration in specific body regions is most likely a result of the co-evolution with their as-yet-unknown vector. The detection of skin microfilariae in asymptomatic animals, suggests the potential role of these animals as carriers and reservoirs of O. lupi., Author Summary Onchocerca lupi is a little known arthropod-borne helminth infesting dogs of increasing interest to the scientific community due to the recent demonstration of its zoonotic potential. Nonetheless, knowledge of the biology of this nematode is exiguous. In this study the distribution and periodicity of O. lupi microfilariae was investigated from different body regions in naturally infested dogs. Data indicate that O. lupi microfilariae were more common in the head (i.e., ears and nose) followed by the inter-scapular region than in the remaining part of the dog's body suggesting that these parasites aggregate in these anatomical sites. These regions might be the best sites to collect skin samples for diagnostic purposes. Finally, the periodicity pattern of microfilariae of O. lupi and their concentration in specific body regions is most likely a result of the co-evolution with their as-yet-unknown vector.

Traditional pulse crops such as pea, lentil, field bean, bitter vetch, chickpea and common vetch originate from Middle East, Mediterranean and Central Asia^1^. They were a part of human diets in hunter-gatherers communities^2^ and are one of the most ancient cultivated crops^3,4^. Europe has always been rich in languages^5^, with individual families still preserving common vocabularies related to agriculture^6,7^. The evidence on the early pulse history witnessed by the attested roots in diverse Eurasian proto-languages remains insufficiently clarified and its potential for supporting archaeobotanical findings is still non-assessed. Here we show that the paleolinguistic research may contribute to archaeobotany in understanding the role traditional Eurasian pulse crops had in the everyday life of ancient Europeans. It was found that the Proto-Indo-European language^8,9^ had the largest number of roots directly related to pulses, such as *arnk(')- (a leguminous plant), *bhabh- (field bean), *erəgw[h]- (a kernel of leguminous plant; pea), *ghArs- (a leguminous plant), *kek-, *k'ik'- (pea) and *lent- (lentil)^10,11,12^, numerous words subsequently related to pulses^13,14^ and borrowings from one branch to another^15^, confirming their essential place in the nutrition of Proto-Indo-Europeans^16,17,18^. It was also determined that pea was the most important among Proto-Uralic people^19,20,21^, while pea and lentil were the most significant in the agriculture of Proto-Altaic people^22,23,24^. Pea and bean were most common among Caucasians^25,26^, Basques^27,28^ and their hypothetical common forefathers^29^ and bean and lentil among the Afro-Asiatic ancestors of modern Maltese^30^. Our results demonstrate that pulses were common among the ancestors of present European nations and that paleolinguistics and its lexicological and etymological analysis may be useful in better understanding the earliest days of traditional Eurasian crops. We believe our results could be a basis for advanced multidisciplinary approach to the pulse crop domestication, involving plant scientists, archaeobotanists and linguists, and for reconstructing even earlier periods of pulse history.

The accelerating rate of outbreaks from mosquito borne diseases are urging the development of updated and effective tools for the surveillance of insect populations and their larval habitats. Harmonized field protocols help to build a comprehensive picture on species-specific vector ecology and generate important knowledge for implementing coordinated mosquito surveillance programs at regional scales and across continents. In this study, we test the efficiency and potential barriers of available harmonized protocols from earlier EU project VectorNet. As a kind of litmus test for such protocols, we specifically aim to capture the ecoregional variation of breeding site characteristics and population density of five mosquito vectors in Europe and Africa. As expected, the five species considered show different aquatic habitat preferences. Culex pipiens, Aedes albopictus in Europe and Ae. aegypti in Africa select breeding habitats within specific volume classes, while Anopheles gambiae and An. coluzzii may select breeding habitats based on seasonal availability. Population densities in aquatic habitats greatly varied across species and countries, but larval production sites of Ae. albopictus generate populations with higher ratio of pupae compared to the other species. This result underlines the fundamental ecological difference between the selected vector species disregarding of the ecoregion. Mean water temperatures had limited variation across species and higher among countries. Understanding the ecology of native and non-native mosquito vectors is key in evaluating transmission risks for diseases such as West Nile, chikungunya and dengue fevers, zika and malaria. The available harmonized field protocols are a valuable tool for achieving homogeneous mosquito surveillance in Europe and Africa.