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BACKGROUND: Mosquitoes pose a risk to human health worldwide, and correct species identification and detection of cryptic species are the most important keys for surveillance and control of mosquito vectors. In addition to traditional identification based on morphology, DNA barcoding has recently been widely used as a complementary tool for reliable identification of mosquito species. The main objective of this study was to create a reference DNA barcode library for the Croatian mosquito fauna, which should contribute to more accurate and faster identification of species, including cryptic species, and recognition of relevant vector species. METHODS: Sampling was carried out in three biogeographical regions of Croatia over six years (2017-2022). The mosquitoes were morphologically identified; molecular identification was based on the standard barcoding region of the mitochondrial COI gene and the nuclear ITS2 region, the latter to identify species within the Anopheles maculipennis complex. The BIN-RESL algorithm assigned the COI sequences to the corresponding BINs (Barcode Index Number clusters) in BOLD, i.e. to putative MOTUs (Molecular Operational Taxonomic Units). The bPTP and ASAP species delimitation methods were applied to the genus datasets in order to verify/confirm the assignment of specimens to specific MOTUs. RESULTS: A total of 405 mosquito specimens belonging to six genera and 30 morphospecies were collected and processed. Species delimitation methods assigned the samples to 31 (BIN-RESL), 30 (bPTP) and 28 (ASAP) MOTUs, with most delimited MOTUs matching the morphological identification. Some species of the genera Culex, Aedes and Anopheles were assigned to the same MOTUs, especially species that are difficult to distinguish morphologically and/or represent species complexes. In total, COI barcode sequences for 34 mosquito species and ITS2 sequences for three species of the genus Anopheles were added to the mosquito sequence database for Croatia, including one individual from the Intrudens Group, which represents a new record for the Croatian mosquito fauna. CONCLUSION: We present the results of the first comprehensive study combining morphological and molecular identification of most mosquito species present in Croatia, including several invasive and vector species. With the exception of some closely related species, this study confirmed that DNA barcoding based on COI provides a reliable basis for the identification of mosquito species in Croatia.
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Culicidae , Código de Barras del ADN Taxonómico , Culicidae/anatomía & histología , Culicidae/clasificación , Culicidae/genética , Mosquitos Vectores/anatomía & histología , Mosquitos Vectores/clasificación , Mosquitos Vectores/genética , Código de Barras del ADN Taxonómico/métodos , Ciclooxigenasa 1/genética , ADN Espaciador Ribosómico/genética , FilogeniaRESUMEN
The goal of the present study was to investigate differences in biomarker responses related to metal(loid)s in white stork (Ciconia ciconia) nestling's blood from continental Croatia. To achieve this, a battery of biomarkers that can be affected by environmental pollutants, including metal(loid)s, was assessed (esterase activity, fluorescence-based oxidative stress biomarkers, metallothionein levels, glutathione-dependent enzyme activity). The research was conducted during the white stork breeding season in diverse areas (a landfill, industrial and agricultural sites, and an unpolluted area). White storks' nestlings near the landfill exhibited reduced carboxylesterase (CES) activity, elevated glutathione (GSH) concentration, as well as high Pb content in the blood. Increased As and Hg concentrations in blood were attributable to environmental contamination in agricultural area and an assumed unpolluted area, respectively. Furthermore, agricultural practices appeared to affect CES activity, as well as elevate Se levels. In addition to the successful implementation of biomarkers, present research showed that agricultural areas and a landfill are areas with increased metal(loid) levels possibly causing adverse effects on the white storks. This first-time heavy metal and metalloid analyses in the white stork nestlings from Croatia point to the necessary monitoring and future assessments of pollution impact to prevent irreversible adverse effects.
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Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Contaminantes Ambientales , Metales Pesados , Animales , Monitoreo del Ambiente , Metales Pesados/toxicidad , Metales Pesados/análisis , Contaminantes Ambientales/análisis , Aves/fisiología , Glutatión , BiomarcadoresRESUMEN
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.
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Aedes/genética , Distribución Animal , Especies Introducidas , Animales , Europa OrientalRESUMEN
Improvement of morphological and molecular identification methods allows the detection of new species of mosquitoes. The mosquito fauna of Croatia currently includes 52 species, belonging to eight genera, including Anopheles (12 species), Aedes (24 species), Coquillettidia (one species), Culex (seven species), Culiseta (six species), Orthopodomyia (one species), and Uranotaenia (one species). This is an updated checklist, which includes five new species found in Croatian mosquito fauna. Two of these are invasive mosquito species, Aedes albopictus (Skuse, 1895) and Aedes japonicus (Theobald 1901), which are spreading across Europe and Croatia. The other three species, Culex laticinctus (Edwards 1913), Culex torrentium (Martini 1925), and Anopheles daciae (Linton, Nicolescu & Harbach 2004) are autochthonous species which haven't been recorded so far. Since there are several more invasive species spreading across Europe, we assume that this is not the final list.
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Especies Introducidas , Aedes , Animales , Anopheles , Croacia , CulicidaeRESUMEN
Invasive species can encounter environments different from their source populations, which may trigger rapid adaptive changes after introduction (niche shift hypothesis). To test this hypothesis, we investigated whether postintroduction evolution is correlated with contrasting environmental conditions between the European invasive and source ranges in the Asian tiger mosquito Aedes albopictus. The comparison of environmental niches occupied in European and source population ranges revealed more than 96% overlap between invasive and source niches, supporting niche conservatism. However, we found evidence for postintroduction genetic evolution by reanalyzing a published ddRADseq genomic dataset from 90 European invasive populations using genotype-environment association (GEA) methods and generalized dissimilarity modeling (GDM). Three loci, among which a putative heat-shock protein, exhibited significant allelic turnover along the gradient of winter precipitation that could be associated with ongoing range expansion. Wing morphometric traits weakly correlated with environmental gradients within Europe, but wing size differed between invasive and source populations located in different climatic areas. Niche similarities between source and invasive ranges might have facilitated the establishment of populations. Nonetheless, we found evidence for environmental-induced adaptive changes after introduction. The ability to rapidly evolve observed in invasive populations (genetic shift) together with a large proportion of unfilled potential suitable areas (80%) pave the way to further spread of Ae. albopictus in Europe.
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During the August and September 2012, seven human cases of the West Nile neuro-invasive disease were reported in Croatia. Medical entomology research on a potential vectors during the outbreak was supported by the Ministry of Health. A mosquito survey has been done in 64 sites in three eastern Croatian counties (Osijek-Baranja County, Vukovar-Srijem county and in Brod-Posavina county). Dry ice baited CDC traps were used for mosquito sampling in a period from the 10th to 25th September 2012. A total of 1785 mosquitoes were collected and 5 species were determined. The most numerous species were Aedes vexans with 1634 specimens, a Culex pipiens c., the potential vector of WNV, was present with 6.39%, in 114 specimens. That species was present in 43 out of 64 investigated sites. Vector control included both the control of mosquito larvae and the adults. Treatments have been done on 184 small breeding sites and on 2900 ha of an urban area.
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Culicidae , Brotes de Enfermedades , Insectos Vectores , Fiebre del Nilo Occidental/epidemiología , Animales , Croacia/epidemiología , Recolección de Datos , HumanosRESUMEN
During August and September 2012, seven cases of West Nile neuroinvasive disease were identified in three north-eastern counties of Croatia. Four cases were reported in Osijek-Baranja County, two in Brod-Posavina County and one in Vukovar-Srijem County. The median age of the patients was 62.7 years. All patients were hospitalized for 2-5 weeks. The patients from Slavonski Brod had more severe clinical presentation of disease with prolonged hospitalization. Medical entomological research was carried out in 64 localities, where 1785 mosquitoes were captured. Among the analyzed mosquitoes, 114 were determined to be Culex pipiens and subjected to molecular characterization for the presence of virus. No viral RNA was detected in mosquitoes. Subsequent public health measures taken include mosquito control in all settlements where disease was detected.
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Culicidae/virología , Brotes de Enfermedades/estadística & datos numéricos , Fiebre del Nilo Occidental/epidemiología , Anciano , Animales , Croacia/epidemiología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios SeroepidemiológicosRESUMEN
The West Nile Virus (WNV) is a zoonotic arbovirus that has recently been causing outbreaks in many countries in southern and Central Europe. In 2012, for the first time, it caused an outbreak in eastern Croatia with total of 7 human clinical cases. With an aim of assisting public health personnel in order to improve survey protocols and vector control, the high risk areas of the WNV transmission were estimated and mapped. The study area included cities of Osijek and Slavonski Brod and 8 municipalities in Vukovarsko-Srijemska County. Risk estimation was based on seroprevalence of WNV infections in horses as an indicator of the virus presence, as well as the presence of possible WNV mosquito vectors with corresponding vector competences. Four mosquito species considered as possible WNV vectors are included in this study: Aedes vexans, Culex modestus, Culex pipiens and Ochlerotatus caspius. Mosquitoes were sampled using dry-ice baited CDC trap, twice a month, between May and October. This study suggests that the two mosquito species present the main risk of WNV transmission in eastern Croatia: the Culex pipiens--because of good vector competence and the Aedes vexans--because of the very high abundances. As a result, these two species should be focus of future mosquito surveillance and a vector control management.