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.

Abstract.In the last few decades, Aedes albopictus (Skuse) (Diptera: Culicidae) (= Stegomyia albopicta), the so-called "Asian tiger mosquito", has spread from its native range in southeast Asia to Africa, the Middle East, Europe, the Americas, and Pacific islands. The spread of this species poses a risk to human health as it is considered to be one of the main vectors of dengue and other arboviruses. Aedes albopictus was reported in Croatia in 2004, thereafter it was discovered at several coastal localities in 2005 and to date it has spread to most coastal areas and islands in Croatia. Here we investigate the genetic variability of A. albopictus based on 39 individuals collected during the summer of 2009 along the East-Adriatic coast and islands of Croatia and Montenegro and using two mitochondrial molecular markers: cytochrome oxidase I (COI) and NADH dehydrogenase 5 (ND5). We identified a single ND5 haplotype, corresponding to the previously reported and worldwide-distributed haplotype H3. The COI marker was more variable and we identified four COI haplotypes. In order to identify the geographic origin of the populations that colonized Croatia, we performed phylogenetic analyses of ND5 and COI haplotypes in Croatian populations and other A. albopictus populations retrieved from the GenBank. The phylogenetic tree based on ND5 haplotypes revealed two well supported clades where the unique Croatian ND5 haplotype clustered with the majority of haplotypes originating from South-Asia, America, Europe, and Africa. Another smaller cluster consisted of only Brazilian haplotypes. The phylogenetic tree and haplotype network that resulted from the COI analysis also indicates that the three Croatian COI haplotypes cluster with European and American haplotypes. However the fourth Croatian COI haplotype was the only European haplotype that occurred in a separate clade (group) with Indian, South-Asian, and Brazilian haplotypes. This data suggests there have been several independent introduction events in 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.

Background Colonization of large part of Europe by the Asian tiger mosquito Aedes albopictus is causing autochthonous transmission of chikungunya and dengue exotic arboviruses. While pyrethroids are recommended only to reduce/limit transmission, they are widely implemented to reduce biting nuisance and to control agricultural pests, increasing the risk of insurgence of resistance mechanisms. Worryingly, pyrethroid resistance (with mortality < 70%) was recently reported in Ae. albopictus populations from Italy and Spain and associated with the V1016G point mutation in the voltage-sensitive sodium channel gene conferring knockdown resistance (kdr). Genotyping pyrethroid resistance-associated kdr mutations in field mosquito samples represents a powerful approach to detect early signs of resistance without the need for carrying out phenotypic bioassays which require availability of live mosquitoes, dedicated facilities and appropriate expertise. Methods Here we report results on the PCR-genotyping of the V1016G mutation in 2530 Ae. albopictus specimens from 69 sampling sites in 19 European countries. Results The mutation was identified in 12 sites from nine countries (with allele frequencies ranging from 1 to 8%), mostly distributed in two geographical clusters. The western cluster includes Mediterranean coastal sites from Italy, France and Malta as well as single sites from both Spain and Switzerland. The eastern cluster includes sites on both sides of the Black Sea in Bulgaria, Turkey and Georgia as well as one site from Romania. These results are consistent with genomic data showing high connectivity and close genetic relationship among West European populations and a major barrier to gene flow between West European and Balkan populations. Conclusions The results of this first effort to map kdr mutations in Ae. albopictus on a continental scale show a widespread presence of the V1016G allele in Europe, although at lower frequencies than those previously reported from Italy. This represents a wake-up call for mosquito surveillance programs in Europe to include PCR-genotyping of pyrethroid resistance alleles, as well as phenotypic resistance assessments, in their routine activities. Graphical Abstract

Distribution of fecal microorganisms in water, periphyton, and sediment was studied along the Žrnovnica river (Croatia) over a 1.5-year period. It was found that periphyton was inhabited by the highest number of investigated bacteria, while lower numbers of them were found in sediment and the lowest in surface water of the river. The concentrations of fecal microorganisms in periphyton and partly in sediment were found to be significantly higher in the middle of the river course, near the town of Žrnovnica, while according to the analysis of surface water the highest degree of pollution was reached on its estuary. The results were explained with respect to bacterial-algal associations. Considering the fact that most of the river microorganisms are associated with periphyton and sediment particles and only a small number of them is in the free-living form, microbiological analysis of both periphyton and sediment together with water samples has been suggested when fecal pollution of a river is concerned.