A DNA barcode reference library of Croatian mosquitoes (Diptera: Culicidae): implications for identification and delimitation of species, with notes on the distribution of potential vector species.

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.

Diversity of mosquito fauna (Diptera, Culicidae) in higher-altitude regions of Croatia.

Global climate change and the accompanying rise in temperature could affect the biology and ecology of a number of vectors, including mosquitoes. High altitude areas that were previously unsuitable for the spread of mosquito vector populations could become suitable. The aim of this research was to study the distribution of mosquito species in higher altitude regions of Croatia. Samples were collected in three areas: Slavonian Mountains, Gorski Kotar, and Middle Velebit. Specimens were morphologically determined and confirmed by DNA barcoding and other genetic markers and showed the presence of 16 species belonging to six genera. The most abundant species were the Culex pipiens complex with 50% of the collected specimens. Both pipiens (Linnaeus, 1758) and molestus (Forskal, 1775) biotypes and their hybrids were identified within the complex, followed by Culex torrentium (Martini, 1925) (20.2%), Culiseta longiareolata (Macquart, 1838) (8.5%), and the invasive species Aedes japonicus (Theobald, 1901) (7.8% of the total number of collected specimens). The remaining 12 species made up 14.7% of the collected specimens. Intraspecific COI p-distances were within the standard barcoding threshold for OTUs, while interspecific genetic distances were much higher, confirming the existence of barcoding gaps. Mosquito fauna of Croatian mountains showed a moderate variety and made 30.8% of the total number of recorded mosquito species in Croatia thus far.

Rapid spread and population genetics of Aedes japonicus japonicus (Diptera: Culicidae) in southeastern Europe (Croatia, Bosnia and Herzegovina, Serbia).

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.

Updated checklist of the mosquitoes (Diptera: Culicidae) of Croatia.

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.

Practical management plan for invasive mosquito species in Europe: I. Asian tiger mosquito (Aedes albopictus).

Aedes albopictus, also known as the "Asian Tiger Mosquito", is an invasive mosquito species to Europe causing high concern in public health due to its severe nuisance and its vectorial capacity for pathogens such as dengue, chikungunya, yellow fever and Zika. Consequently, the responsible authorities implement management activities to reduce its population density, possibly to below noxious and epidemiological thresholds. In urban areas, these aims are difficult to achieve because of the species' ability to develop in a wide range of artificial breeding sites, mainly private properties. This document (Management Plan) has been structured to serve as a comprehensive practical and technical guide for stakeholders in organizing the vector control activities in the best possible way. The current plan includes coordinated actions such as standardized control measures and quality control activities, monitoring protocols, activities for stakeholders and local communities, and an emergency vector control plan to reduce the risk of an epidemic.

An overview of mosquitoes and emerging arboviral infections in the Zagreb area, Croatia.

Mosquito control in the Zagreb area has been conducted for many years, whereas the fauna has only been investigated in the last 20 years. So far 30 mosquito species have been detected in the city area. Culex pipiens form molestus is the dominant mosquito species in indoor breeding sites. In forested areas and areas exposed to flooding, the active period is early spring and the dominant species are Ochlerotatus sticticus, Ochlerotatus cantans, Ochlerotatus geniculatus and Aedes vexans. The eudominant mosquito species found in the artificial breeding sites are Culex pipiens and the Asian tiger mosquito, Aedes albopictus. Invasive Ae. albopictus, present in the Zagreb area since 2004, has expanded to a larger area of the city during the last three years. The recent emergence of the human West Nile virus and Usutu virus neuroinvasive disease in Zagreb and its surroundings highlighted the role of mosquitoes as vectors of emerging arboviruses. The paper focuses on mosquito species and arboviral infections detected in humans and animals in the Zagreb area, Croatia.

Dengue virus infection in Croatia: seroprevalence and entomological study.

During 2011-2012, a total of 1,180 sera samples were collected from residents of seven Croatian counties located on the Adriatic Coast and four counties in northeastern Croatia and tested for the presence of dengue virus (DENV) IgG antibodies using ELISA. Reactive samples were further tested by IFA. Seven samples (0.59%) tested positive for DENV antibodies. Seroprevalence rates by county varied from 0-2.21%. The highest seropositivity rate (2.21%) was found in the Dubrovnik-Neretva County where autochthonous dengue cases were recorded in 2010. Additionally,3,699 mosquitoes were collected from 126 localities along the Adriatic coast in August-September, 2011. Aedes albopictus was the most prevalent species (81.37%). No evidence of DENV RNA was detected by RT-PCR among 1,748 female mosquitoes.

Recently discovered Aedes japonicus japonicus (Diptera: Culicidae) populations in The Netherlands and northern Germany resulted from a new introduction event and from a split from an existing population.

BACKGROUND: Originally native to East Asia, Aedes japonicus japonicus, a potential vector of several arboviruses, has become one of the most invasive mosquito species in the world. After having established in the USA, it is now spreading in Europe, with new populations emerging. In contrast to the USA, the introduction pathways and modes of dispersal in Europe are largely obscure. METHODS: To find out if two recently detected populations of Ae. j. japonicus in The Netherlands and northern Germany go back to new importations or to movements within Europe, the genetic makeup of mosquito specimens from all known European populations was compared. For this purpose, seven microsatellite loci from a representative number of mosquito specimens were genotyped and part of their mitochondrial nad4 gene sequenced. RESULTS: A novel nad4 haplotype found in the newly discovered Dutch population of Ae. j. japonicus suggests that this population is not closely related to the other European populations but has emanated from a further introduction event. With five nad4 haplotypes, the Dutch population also shows a very high genetic diversity indicating that either the founder population was very large or multiple introductions took place. By contrast, the recently detected North German population could be clearly assigned to one of the two previously determined European Ae. j. japonicus microsatellite genotypes and shows nad4 haplotypes that are known from West Germany. CONCLUSION: As the European populations of Ae. j. japonicus are geographically separated but genetically mixed, their establishment must be attributed to passive transportation. In addition to intercontinental shipment, it can be assumed that human activities are also responsible for medium- and short-distance overland spread. A better understanding of the processes underlying the introduction and spread of this invasive species will help to increase public awareness of the human-mediated displacement of mosquitoes and to find strategies to avoid it.

Seasonal and spatial oviposition activity of Aedes albopictus (Diptera: Culicidae) in Adriatic Croatia.

Seasonal and spatial ovipositional activity of Aedes albopictus (Skuse, 1894) was investigated in Split, South Croatia. During 2009 and 2010, 35 oviposition traps were used in this research. For studying oviposition behavior, mean egg density and Lloyd's mean crowding were used to define the dispersion of eggs into ovitraps and, together with Taylor's power law, to show aggregation degree. To show monthly distribution of egg density, Kriging interpolation was used. Oviposition activity started in April (week 13) and was completed at the beginning December (week 48). Mean egg density reaches the highest values from June to early September (week 25-35). Slope of regressions (mean crowding on mean density and log variance and log mean density) indicated a clumped distribution of eggs. Sampling sites were divided in four groups based on quartiles of median and maximum of mean density. There was no significant difference in measured abiotic factors (temperature, humidity, and rainfall) and measured mean egg density, total amount of eggs, and percentage of positive ovitraps between investigated years, except in mean egg density in some localities.

Mosquito survey during West Nile virus outbreak 2012 in northeast Croatia.

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.

Environmentally Dependent Density-Distance Relationship of Dispersing Culex tarsalis in a Southern California Desert Region.

The direct inclusion of environmental factors into the empirical model that describes a density-distance relationship (DDR) is demonstrated on dispersal data obtained in a capture-mark-release-recapture experiment (CMRR) with Culex tarsalis conducted around the community of Mecca, CA. Empirical parameters of standard (environmentally independent) DDR were expressed as linear functions of environmental variables: relative orientation (azimuthal deviation of north) of release point (relative to recapture point) and proportions of habitat types surrounding each recapture point. The yielded regression model (R(2)  =  0.5373, after optimization on the best subset of linear terms) suggests that spatial density of recaptured individuals after 12 days of a CMRR experiment significantly depended on 1) distance from release point, 2) orientation of recapture points in relation to release point (preferring dispersal toward the south, probably due to wind drift and position of periodically flooded habitats suitable for species egg clutches), and 3) habitat spectrum in surroundings of recapture points (increasing and decreasing population density in desert and urban environment, respectively).

West Nile virus outbreak in humans in Croatia, 2012.

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.

Equine seroprevalence rates as an additional indicator for a more accurate risk assessment of the West Nile virus transmission.

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.

Spatiotemporal monitoring of floodwater mosquito dispersal in Osijek, Croatia.

This paper demonstrates the possibility of using geostatistics to monitor the dispersal of mosquitoes for mosquito control programs at the municipal level. The case study objective was to quantify the dispersal of floodwater mosquitoes from the natural marshland Kopacki rit into the city of Osijek, Croatia, and to analyze the main factors controlling it. Fifty thousand adult Aedes vexans, Ochlerotatus sticticus, and Ochlerotatus caspius mosquitoes were marked with a powdered fluorescent pigment and released from the southern part of Kopacki rit on April 28, 2004. Forty CO2-baited Centers for Disease Control and Prevention traps were set in the area of the municipality of Osijek (171 km2) and were monitored for 10 days. A total of 582,471 mosquitoes were captured and examined in the laboratory. The mosquito counts from different sites were then interpolated using ordinary kriging and visualized dynamically to detect the dominant migrational directions. Mosquito dispersal and frequency were greatly influenced by wind speed (r = 0.82). The marked mosquitoes were found at 12 sites located from 1 km to 11.7 km away from the release point. The recapture rate was 0.044% (54% Oc. sticticus, 32% Ae. vexans, and 14% Oc. caspius). Based on the Lincoln index, the estimated total population size for floodwater mosquitoes in the study area ranged from 875 million to 2.0 billion mosquitoes. Limitations of the approach, recommendations for the improvement of the monitoring network, and spatial predictions are further discussed.

First record of Aedes albopictus in Croatia.

The first record of Aedes albopictus in Croatia was made in Zagreb, Croatia, on October 28, 2004. A total of 13 larvae were collected in a discarded ceramic toilet bowl in a forested area in the southwest suburbs. Because the winter temperatures in Zagreb are not favorable for survival, this finding of Ae. albopictus may be temporary. Its survival in northern Croatia remains to be studied.

Response of Tabanidae (Diptera) to different natural attractants.

The response of female tabanids to natural attractants was studied in the Monjoros Forest along the Nature Park Kopacki rit in eastern Croatia. Tabanids were caught in canopy traps baited with either aged cow, horse, sheep, or pig urine and also in unbaited traps. Tabanids were collected in a significantly higher numbers in traps baited with natural attractants compared to unbaited traps. The number of females of Tabanus bromius, Tabanus maculicornis, Tabanus tergestinus, and Hybomitra bimaculata collected from canopy traps baited with cow urine and traps baited with other natural attractants differed significantly. Females of Haematopota pluvialis were also collected more frequently in canopy traps baited with aged cow urine than in those with aged horse urine, but this difference was not significant. However, the number of females of Haematopota pluvialis collected from canopy traps baited with other natural attractants (sheep and pig urine) differed significantly when compared with aged cow urine baited traps. Canopy traps baited with aged cow urine collected significantly more Tabanus sudeticus than did traps baited with aged pig urine. Finally, the aged cow urine baited canopy traps collected 51 times more tabanids than unbaited traps, while aged horse, aged sheep, and aged pig urine baited traps collected 36, 30, and 22 times as many tabanids, respectively, than unbaited traps.

Seasonal dynamics of the Anopheles maculipennis complex in Osijek, Croatia.

Malaria was once endemic in the Osijek region of Croatia and although it has been eradicated, there are still several vector species of the Anopheles maculipennis complex in this area. During an eight-year investigation, using CDC traps with CO2 as an attractant, we collected a total of 3,508 mosquitoes. We determined the dynamics of members of this complex and found that they varied considerably with an average of two generations per season. Cross-correlations indicated that the Spearman's Index between the abundance rate and the water level was the highest 12 and 24 d before traps were set. This suggests that the long-term high water level created better conditions for continuous breeding of mosquitoes, which contributed to a significant increase in abundance rates of all species in the complex.