Determination of an efficient and reliable method for PCR detection of borrelial DNA from engorged ticks

Since ticks have been recognized as one of the most important vectors of pathogens causing serious diseases in humans, a number of studies have focused on identifying the pathogen composition as well as transmission and infection mechanisms. Although a plethora of detection methods is available today, PCR-based approach is regarded as the most sensitive and rapid. However, common challenges in molecular analyses conducted on ticks are weak amplification results because of present inhibitors, either from a mammalian bloodmeal or a male DNA in female reproductive organs. Present study aimed to evaluate which body part of an engorged tick is the most preferable as a starting material in DNA extraction for molecular detection of Borrelia burgdorferi sensu lato, causative agent of Lyme borreliosis. We analyzed 58 Ixodes ricinus ticks removed from patients in The Center for Emergency Medical Assistance of the Sarajevo Canton, Bosnia and Herzegovina. Our findings suggest using the anterior half of semi-engorged and fully-engorged ticks for DNA extraction with the purpose of Borrelia detection

Status of the BOLD reference library of DNA barcodes of caddisflies (Insecta: Trichoptera) from the Western Balkans

Background and purpose: Available data in research literature suggest that the Western Balkan countries hold a rich diversity of caddisflies. Assessment and biomonitoring of such rich diversity could be facilitated through DNA-based high-throughput approaches like DNA metabarcoding that depend on the availability of comprehensive reference libraries. Materials and methods: We assessed the status of the COI barcode sequence data for a total of 112 caddisflies species in the investigated region by determining the gaps in representative sequences in the Barcode of Life Data System (BOLD) and examining the accuracy of available records using the Barcode, Audit and Grade System (BAGS). Results: Results revealed a considerable underrepresentation of surveyed geographic region in BOLD records for the target insect group. Moreover, the large majority of the species records were rated “discordant” (72.80% grade E), and only 15.20% were classified as “consolidated concordance or basal concordance” (3.20% grade A and 12.00% B). Approximately 3.20% of the records pertaining to species occurring in multiple BINs (Barcode Index Number) and 8.80% were poorly represented (i.e., less than three specimens, grade D). A fraction of the species graded discordant were deemed concordant after detailed inspection of individual data, decreasing by 14.07%. Conclusions: The assessment of the current state of BOLD entries indicated that DNA barcoding is still not widely applied in Albania, Bosnia and Herzegovina, Montenegro, North Macedonia, Serbia, and Slovenia, emphasizing that Croatia has the most barcoded caddisflies species. The finding that available BOLD Trichopteran records for investigated countries were mainly graded as “discordant” indicates the need for better quality control of reference libraries

MOLECULAR STRUCTURE AND HYBRIDIZATION PATTERNS OF Abramis brama × Rutilus rutilus HYBRIDS FROM MODRAC RESERVOIR, BOSNIA AND HERZEGOVINA

Interspecijska hibridizacija u porodici Cyprinidae zabilježena je širom svijeta, a Abramis brama (deverika) i Rutilus rutilus (bodorka) su jedan od često prijavljenih hibridizirajućih parova. Jedini opis takvog događaja u Bosni i Hercegovini potiče iz akumulacije Modrac. Koristeći morfološke i molekularne markere, istražili smo prisutnost hibrida, odredili smjer hibridizacije i procijenili strukturu hibridnih skupina u ovom ekosustavu. Naša su otkrića potvrdila nesmetanu prirodnu hibridizaciju bodorke i deverike u akumulaciji Modrac. Primjenom NewHybrids softvera, preko 50% hibridnih primjeraka klasificirano je kao F2 hibridi, dok su ostali kategorizirani kao čisti roditeljski oblik, što je prvi takav nalaz u Europi. Analiza mitohondrijskog citokroma b pokazala je da 90% hibridnih jedinki ima majčinsko podrijetlo od deverike. Hibridna skupina izrazila je veće srednje vrijednosti promatrane heterozigotnosti i raznolikosti gena u odnosu na obje roditeljske vrste. Otkriveni su znakovi introgresivne hibridizacije između roditeljskih vrsta. Čini se da hibridna zona akumulacije Modrac slijedi intermedijarni ili "ravni" hibridni model zasnovan na uravnoteženoj raspodjeli roditeljskih i hibridnih genotipova. Potrebna su daljnja istraživanja kako bi se rasvijetlili čimbenici koji omogućuju preživljavanje i uspjeh u parenju jedinki poslije F1 generacije.Interspecific hybridization in the Cyprinidae family has been recorded worldwide, with Abramis brama (bream) and Rutilus rutilus (roach) as one of the often-reported hybridizing pairs. The only account of such an event in Bosnia and Herzegovina has been in Modrac Reservoir. Using morphological and molecular markers, the presence of hybrids was surveyed, the hybridization direction was determined and the hybrid group structure in this ecosystem was evaluated. Our findings confirmed unhindered natural hybridization between roach and bream in Modrac Reservoir. Over 50% of the hybrid specimens were classified as F2 hybrids by the NewHybrids software, while the rest were categorized as pure parental form, making it the first such finding in Europe. The analysis of mitochondrial cytochrome b showed that 90% of hybrid individuals were of bream maternal origin. The hybrid group expressed higher mean values of observed heterozygosity and gene diversity than both parental species. Signs of introgressive hybridization between parental species were detected. The hybrid zone of Modrac Reservoir appears to follow the intermediate or “flat” hybrid model based on the balanced distribution of parental and hybrid genotypes. Further investigation is needed to elucidate the factors that enable the survival and mating success of post-F1 individuals

An assessment of regulation, education practices and socio-economic perceptions of non-native aquatic species in the Balkans

Alongside climate change, the introduction of non-native species (NNS) is widely recognized as one of the main threats to aquatic biodiversity and human wellbeing. Non-native species and biodiversity are generally low priority issues on the political agendas of many countries, particularly in European countries outside the European Union (EU). The objectives and tasks of this study were to address the policy regulation, education level, education practices, and socioeconomic perceptions of NNS in the Balkans. A questionnaire-based survey was conducted in Albania, Bosnia and Herzegovina, Montenegro, North Macedonia and Turkey (Balkan EU candidate and potential candidate members), in Croatia and Greece (Balkan EU Member States) and Italy (non-Balkan EU Member State). The EU Alien Regulation (1143/2014) concerning NNS is implemented in EU Member States and Montenegro, whereas Albania, Bosnia and Herzegovina and Turkey have not reported specific policy regulations for NNS. Permanent monitoring programmes specifically designed for NNS have not yet been established in the EU Member States. Most countries tackle the issue of NNS through educational activities as part of specific projects. Education level is indicative of the implementation of NNS policy regulation, and efforts are needed for the proper development of relative study programmes. Public awareness and educational preparedness concerning NNS in the Balkans were identified as poor. Strong programmes for management and education should be developed to increase public awareness to prevent further biodiversity losses in the Balkan region

Unheeded biodiversity: perspectives of DNA barcoding of Trichoptera in Bosnia and Herzegovina

Bosnia and Herzegovina has valuable natural resources with a high percentage of endemic and autochthonous species (Kučinić et al. 2008, Đug and Drešković 2012). The freshwater fauna of Trichoptera in this area is under-investigated, with a lack of morphological description of different life stages and DNA barcode data. Public data show 58,993 barcode entries for Trichoptera in the Barcode of Life Data Systems (BOLD) submitted from 92 countries, and none from Bosnia and Herzegovina (B&H) (BOLD 2021). Previous research in Bosnia and Herzegovina has provided the first DNA barcode for the endemic species Rhyacophila bosnica, stored in GeneBank, under accession number MK211322 by a domestic institution (Kalamujić Stroil et al. 2018). A few DNA barcodes of adult individuals of Trichoptera from Bosnia and Herzegovina were found in BOLD. However, these specimens were collected on B&H territory, but analyzed, processed, and stored by foreign institutions. To change the current state of DNA barcoding of Trichoptera in Bosnia and Herzegovina, we aimed to employ this approach in investigating caddisflies in selected habitats in the Sarajevo Canton.Our fieldwork was done in all five protected areas (spring of the Bosna River, Bijambare, Trebević, Skakavac, and Bentbaša) in which larvae samples were collected according to the AQEM sampling methodology. The standard animal DNA barcode was successfully obtained using degenerated primers LCO1490- JJ and HCO2198-JJ (Astrin and Stüben 2008).Out of 684 collected individuals (313 Trebević, 130 spring of the Bosna River, 117 Bijambare, 71 Bentbaša, 53 Skakavac), a subset of specimens were sequenced. We uncovered 14 different taxa, 11 genera and six families (Limnephilidae, Glossosomatidae, Rhyacophilidae, Goeridae, Hydropsychidae, Polycentropodidae). The preliminary data of Trichoptera composition in the Sarajevo Canton indicated species richness. Based on our sequential data, a new subspecies was discovered in two investigated areas (Valladolid et al. 2020), proving that Trichoptera species diversity in our country is far from entirely uncovered. The benefit and power of the DNA barcoding approach are that it can pinpoint the areas of vast and unknown species diversity more economically, both financially and temporarily, than the morphological approach. Therefore, we believe that it is critical to support the development of DNA barcoding for the bioassessment of freshwater ecosystems in Bosnia and Herzegovina.Several problems prevented us from exploiting sequential data to the fullest. Despite a general notion among scientists that European Trichoptera species are well covered in the BOLD database, most of the sequences we obtained were absent from the database. Secondly, we recognized that morphological data about the larval developmental stage of B&H Trichoptera species are largely missing. The unified, updated, and complete data on this order of insects is urgently needed. However, insufficient financial support by governmental institutions and lack of systematic approach to barcoding the wildlife of Bosnia and Herzegovina hampers this process.Further attempts to collaborate with the stakeholders can be crucial with profound and substantial implications for biomonitoring of aquatic macroinvertebrates in general. New approaches, such as novel DNA barcoding-based methodology can fill an important gap in our knowledge of Balkan caddisflies haplotypes, lineages, and their diversification and distribution patterns

Is cytochrome c oxidase subunit I (COI) the right DNA barcoding marker for the Chaetopteryx villosa group?

Chaetopteryx villosa (Fabricius, 1798) is a caddisfly species distributed throughout Europe, except in the Balkan and Apennine Peninsula. However, phylogenetically close species belonging to the C. villosa group are widespread throughout entire Europe. Species of this group (C. villosa, C. gessneri, C. fusca, C. sahlbergi, C. atlantica, C. bosniaca, C. vulture, and C. trinacriae) have distinct distributions with some overlaps. Adult forms of these species are morphologically similar, whereas larval morphology is only known for some species. There are also indications of species hybridization (e.g., C. villosa x fusca). Presumably, the molecular approach for the species determination of this group would be highly beneficial. In the BOLD database, there are 154 specimens with COI-5P barcodes of C. villosa species. Out of the remaining species, C. sahlbergi has 27 specimens with a barcode, C. fusca 20, C. gessneri 5, C. bosniaca 5, and C. atlantica 1, whereas sequences from the species C. vulture and C. trinacriae are missing. Therefore, we tested the power of discrimination of the COI-5P marker in the C. villosa group, as the most common barcoding markers for species identification in animals.Only sequences from public records originating from experienced research groups or taxonomists and containing a specimen photograph were taken as input. A total of 75 sequences from the BOLD database were obtained. Out of these sequences, 11 belonged to C. fusca, 5 to C. gessneri, 52 to C. villosa, 5 to C. bosniaca, and 2 to C. sahlbergi. For the generation of overview trees, COI-5P barcodes of Rhyacophila fasciata and Rh. nubila were used as outgroups. All sequences were trimmed at 5’ and 3’ ends, resulting in a final alignment length of 516 base pairs. Multiple sequence alignments and editing were done in the MEGA-X software. Analysis of nucleotide polymorphism was done in DNASP6 software. MEGA-X was used to calculate the pairwise distance and overall mean p-distance, and to construct the overview trees.Analysis of DNA polymorphism revealed 14 haplotypes of C. villosa, 3 haplotypes of C. fusca, 2 haplotypes of C. gessneri, and one for species C. bosniaca and C. sahlbergi. There were no significant interspecific and intraspecific differences among haplotypes based on pairwise distances. The p-distance between one of the haplotypes of C. fusca and C. villosa was 0.000, whereas the p-distance among haplotypes of C. villosa varied from 0.001 to about 0.055. The mean overall p-distance among haplotypes of all species equaled 0.03. No species-specific clusters were observed when phylogenetic trees were constructed except for C. gessneri, regardless of the method used (i.e., NJ, UPGMA, ML, ME, or MP).To minimize the possibility of species misidentification, we used only records submitted by NTNU-Norwegian University of Science and Technology (Norway), SNSB-Zoologische Staatssammlung Muenchen (Germany), Zoologisches Forschungsmuseum Alexander Koenig (Germany), University of Oulu, Zoological Museum (Finland), prof Hans Malicky and prof Mladen Kučinić. No records identified as hybrids were included in the analyses. With the exception of C. gessneri, COI-5P marker failed to separate the species of the C. villosa group. However, it is highly unlikely that poor species determination was the basis for such a result. To enable the comprehensive and unbiased evaluation of the relationships within this group, data coverage in BOLD database for most of the studied species should be enhanced, encompassing different geographical distribution of samples. Further studies are needed to detect the array of molecular markers suitable for the species delineation in a complex group such as C. villosa