First molecular detection of Borrelia burgdorferi sensu lat o in Ixodes ricinu s ticks collected from humans in the Sarajevo Canton (Bo snia and Herzegovina)

Polyploidy is a powerful mechanism driving genetic, physiological, and phenotypic changes among cytotypes of the same species across both large and small geographic scales. These changes can significantly shape population structure and increase the evolutionary and adaptation potential of cytotypes. Alyssum moellendorfianum, an edaphic steno-endemic species with a narrow distribution in the Balkan Peninsula, serves as an intriguing case study. We conducted a comprehensive analysis of genetic diversity and population structure across the species’ range, employing an array of genetic techniques (nuclear microsatellites, amplified fragment length polymorphisms, and plastid DNA sequences), flow cytometry (FCM), morphometry, and pollen analysis. The study reveals two genetic lineages: spatially distributed diploid and tetraploid cytotypes. Clear divergence between diploids and tetraploids was shown by AFLP, while plastid DNA sequences confirmed private haplotypes in each of the studied populations. Higher genetic diversity and allelic richness following the north-south pattern were documented in tetraploids compared to diploids, as indicated by nuclear microsatellites. Morphometric analysis via principal component analysis (PCA) and canonical discriminant analysis (CDA) did not reveal any divergence between diploid and tetraploid cytotypes. Nonetheless, a distinction in pollen size was clearly observed. The results suggest an autopolyploid origin of tetraploids from diploid ancestors. Despite the population fragmentation in a very small geographic range, these populations harbour high genetic diversity, which would allow them to remain stable if natural processes remain undisturbed.

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.

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.

Borrelia burgdorferi sensu lato complex comprises 20 species, from which B. afzelii, B. garinii, B. burgdorferi sensu stricto, B. bavariensis, and B. spielmanii are directly associated with Lyme borreliosis, while B. bissettiae, B. lusitaniae, and B. valaisiana were detected in individual cases. Their main vector in Europe is the hard tick species Ixodes ricinus. To date, two species, B. spielmanii and B. lusitaniae, have been molecularly detected in Bosnia and Herzegovina. To test for the presence of other Borrelia species, we performed nested PCR targeting intergenic region rrf (5S) - rrl (23S) on DNA isolates from 49 ticks collected from vegetation by flagging method and 43 removed from humans in The Center for Emergency Medical Assistance of the Sarajevo Canton and The Healthcare Centers of the Sarajevo Canton. Borrelia species were detected by one-directional Sanger sequencing of the amplified region using the same forward primer as in PCR. Out of six Borrelia species detected in the present study, this is the first record of B. afzelii, B. garinii, B. burgdorferi s.s. B. bavariensis, and B. valaisiana in Bosnia and Herzegovina.

Introduction: The COVID-19 pandemic and the restrictions from routine life habits had a tremendous impact on psychological and physical health of youth. It is known that stress, anxiety and depression can be associated with the development of gastrointestinal (GI) symptoms and known to exacerbate present GI symptoms. The pandemic has forced many changes in the behavior of student population such as the studying in an asocial environment. The aim of this study was to examine and quantify the influence of stress onto the quality of life and GI symptoms in the student population in Bosnia and Herzegovina (B&H), before and during the pandemic lockdown. Methods: A total of 279 students from B&H were assessed for their GI and emotional status in pre-COVID period and during the COVID period using validated instruments: GI symptom rating scale (GSRS), Visceral Sensitivity Index, and the Patient Health Questionnaire 15-item Somatic Symptom Severity Scale. Results: The results showed that moderate and severe GI symptoms were more frequently present among student population at the time of the pandemic than in period before pandemic. The most pronounced symptoms were bloating syndrome and abdominal pain syndrome according to the GSRS. Conclusions: We concluded that concern for one’s health and changed way of life are directly related to a worsening of the symptoms of GI disorders in the student population. Further research should go in the direction of early prevention of GI disorders that take root in early youth and later develop into chronic forms.