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.

The Impact of Beehive Proximity, Human Activity and Agricultural Intensity on Diptera Diversity in a Mediterranean Mosaic of Agroecosystems, with a Focus on Pest Species.

Diptera, with their participation in pollination, significantly contribute to the maintenance of plant diversity, and they also have great potential for assessing habitat health and preserving it. A decline in their abundance and diversity has been recorded worldwide as a consequence of biotic, abiotic, and anthropic alterations. In addition to pollinators, these orders include agricultural and forestry pests, which are a threat to both cultivated and wild plants that are very important to the economy. Many pests have escaped from their native areas, and it is important to monitor their spread to implement sustainable means of control. Our study provides baseline information on Diptera and Vespidae diversity in the Mediterranean mosaic of agroecosystems, giving information on the importance of human influence on insect diversity. We carried out an insect inventory in Istria, Croatia, using a set of traps placed in the proximity of beehives. This study was also important in determining the presence of pests and newly introduced species. A total of 94 species from 24 families were recorded-7 important agricultural pests of Diptera and 17 new records for Croatia. The correlation between species diversity and environmental and anthropogenic factors leads to the conclusion that total insect species richness, pest species richness, and the first findings depend on human activities. The number of honeybee colonies negatively correlated with species richness, while anthropic influence positively affected total and pest species richness.

Polymer Chemical Identity as a Key Factor in Microplastic-Insecticide Antagonistic Effects during Embryogenesis of Sea Urchin Arbacia lixula.

As a proxy for pollutants that may be simultaneously present in urban wastewater streams, the effects of two microplastics-polystyrene (PS; 10, 80 and 230 µm diameter) and polymethylmethacrylate (PMMA; 10 and 50 µm diameter)-on fertilisation and embryogenesis in the sea urchin Arbacia lixula with co-exposure to the pyrethroid insecticide cypermethrin were investigated. Synergistic or additive effects were not seen for plastic microparticles (50 mg L-1) in combination with cypermethrin (10 and 1000 µg L-1) based on evaluation of skeletal abnormalities or arrested development and death of significant numbers of larvae during the embryotoxicity assay. This behaviour was also apparent for male gametes pretreated with PS and PMMA microplastics and cypermethrin, where a reduction in sperm fertilisation ability was not evidenced. However, a modest reduction in the quality of the offspring was noted, suggesting that there may be some transmissible damage to the zygotes. PMMA microparticles were more readily taken up than PS microparticles, which could suggest surface chemical identity as potentially modulating the affinity of larvae for specific plastics. In contrast, significantly reduced toxicity was noted for the combination of PMMA microparticles and cypermethrin (100 µg L-1), and may be related to less ready desorption of the pyrethroid than PS, as well as cypermethrin activating mechanisms that result in reduced feeding and hence decreased ingestion of microparticles.

Molecular Study of Selected Taxonomically Critical Taxa of the Genus Iris L. from the Broader Alpine-Dinaric Area.

Some wild, morphologically diverse taxa of the genus Iris in the broad Alpine-Dinaric area have never been explored molecularly, and/or have ambiguous systematic status. The main aims of our research were to perform a molecular study of critical Iris taxa from that area (especially a narrow endemic accepted species I. adriatica, for which we also analysed genome size) and to explore the contribution of eight microsatellites and highly variable chloroplast DNA (ndhJ, rpoC1) markers to the understanding of the Iris taxa taxonomy and phylogeny. Both the microsatellite-based UPGMA and plastid markers-based maximum likelihood analysis discriminated three main clusters in the set of 32 analysed samples, which correspond well to the lower taxonomic categories of the genus, and support separate status of ambiguous regional taxa (e.g., I. sibirica subsp. erirrhiza, I. x croatica and I. x rotschildii). The first molecular data on I. adriatica revealed its genome size (2C = 12.639 ± 0.202 pg) and indicated the existence of ecotypes. For future molecular characterisation of the genus we recommend the utilisation of microsatellite markers supplemented with a combination of plastid markers.

Western mosquitofish as a bioindicator of exposure to organochlorine compounds.

The evaluation of the alochthonous and cosmopolitan mosquitofish species Gambusia affinis suitability as a bioindicator species and the induction of its liver cytochrome P450-dependent mixed function oxygenase (MFO), measured as the 7-ethoxyresorfin-O-deethylase (EROD) activity, as well as changes in DNA integrity, measured by the Fast Micromethod, for the monitoring of organochlorine fresh water pesticide contamination, were the main aims of the study. The test mosquitofish were exposed under laboratory conditions to several doses (0.1, 10 and 100 microg l(-1)) of lindane in experimental basins for up to 7 days, and a subsequent field study was carried out at five natural ponds in the south-western Istrian peninsula, Croatia, where up to 10 fish were collected from each pond. Results obtained during the studies showed positive correlations between the measured biomarkers in G. affinis liver (EROD activity and DNA integrity status) and lindane (laboratory experiment) or persistent organochlorine pollutant amounts in natural pond sediments (field study). The clear dose-responses of EROD activity and DNA integrity deterioration in G. affinis were recorded after exposure to 0.1-10 microg/l lindane and 96 h exposure to lindane, respectively. The results indicate that the mosquitofish G. affinis, due to its biological-ecological characteristics and the biomarker dose-response, is suitable for the monitoring of fresh water organochlorine pesticide contamination in general and lindane in particular.

[The black widow spider--its appearance and control]. / Crna udovica--pojavnost i suzbijanje.

Between 1995 and 2002, a massive appearance of black widow (Latrodectus mactans tredecimguttatus, Rossi 1790) was recorded along the Croatia coast. This paper gives a historical review of latrodectism and observations from Istria and Dalmatia, paying particular attention to the spider's habitat. There are several reasons for the black widow to appear in a new habitat such as the introduction of leguminous plants, watermelons and melons where crops were grown earlier. Black widow can also be found near buildings and gardens. Human contacts with the spider are usually without consequences for humans, thanks to education and a relatively good visibility of the spider. The authors describe their experience in fighting black widow and emphasise the importance of education in preventing lactrodectism.