GENETIC CHARACTERISTICS OF BROOK TROUT Salvelinus fontinalis (MITCHILL, 1848) BROODSTOCKS FROM HATCHERIES IN BOSNIA AND HERZEGOVINA

Najčešće uzgajane riblje vrste u Bosni i Hercegovini su iz porodice Salmonidae, uključujući i potočnu zlatovčicu (Salvelinus fontinalis) koju se uzgaja za prehranu i za potrebe poribljavanja. Na uzgajalištima je primjećena povećana učestalost anatomskih deformacija kod mlađi potočne zlatovčice koje uzrokuju smanjenje kondicije kod riba, a time i značajne financijske gubitke. S obzirom da je dokazano da je pojava deformacija u korelaciji s niskom genetičkom raznolikošću i visokom stopom uzgoja u srodstvu, ova studija je imala za cilj procijeniti unutar i međupopulacijsku raznolikost Salvelinus fontinalis iz različitih ribogojilišta u Bosni i Hercegovini na osnovu varijacija mitohondrijskog i nuklearnog genoma. Primjenom mitohondrijskog kontrolnog područja i sedam nuklearnih mikrosatelitnih lokusa analizirano je ukupno 109 uzoraka potočne zlatovčice iz tri ribogojilišta locirana na rijeci Neretvi. Metoda PCR-RFLP i metoda sekvenciranja pokazale su samo jedan haplotip kontrolnog područja u svim istraživanim jedinkama. Uočen je mali broj genotipova za sve promatrane mikrosatelitne lokuse. Vrijednost genetičke raznolikosti i sadržaj informativnog polimorfizma pratili su porast u broju alela po lokusu. Općenito, vrijednost koeficijenta uzgoja u srodstvu bio je generalno veoma visok, dok su genetska raznolikost i uočena heterozigotnost imali niske stope. Rezultati provedene studije u suglasnosti su s rezultatima prethodnih istraživanja u kojima su razvojne deformacije bile praćene niskom genetičkom raznolikošću i inbreeding depresijom. Stoga se posebno preporuča redovno nadopunjavanje matičnog jata novim, nesrodnim jedinkama s obzirom da je to od presudnog značaja za održanje zadovoljavajuće razine genetičke raznolikosti i sprečavanje inbreeding depresije. Također, održavanje dobre prakse upravljanja u pogledu fluktuacije temperature vode, izloženosti zagađenju, prehrane itd. će dodatno prevenirati nastanak ovog štetnog stanja.The most commonly farmed fish species in Bosnia and Herzegovina’s aquaculture are from the family Salmonidae, including brook trout Salvelinus fontinalis which is reared both for consumption and stocking purposes. A number of farmers complained about the elevated frequency of anatomical deformities in the smolts and fingerlings of brook trout, decreasing their fitness rate and causing significant financial loss. Since it has been shown that occurrence of deformities is correlated with the low genetic diversity and high inbreeding, this study aimed to assess intra- and interpopulation diversity of Salvelinus fontinalis from different freshwater fish farms in Bosnia and Herzegovina by observing variation in mitochondrial and nuclear genome. Total of 109 samples of brook trout from three hatcheries located at the Neretva River were analyzed for the mitochondrial control region and seven nuclear microsatellite loci. Both PCR-RFLP and sequencing revealed only one haplotype of the control region in all investigated trout. Overall, a low number of genotypes was evident across all the observed loci. Values of genetic diversity and polymorphic information content followed the increase in the number of alleles per locus. In general, values of inbreeding coefficient were generally very high, while the genetic diversity and observed heterozygosity had low rates. The results of our study are congruent with the findings of previous studies in which developmental deformities were concomitant with the low genetic diversity and inbreeding depression. It is, therefore, strongly advised to regularly supplement the broodstock with new, unrelated individuals, as it is of vital importance for sustaining a satisfying level of genetic diversity and preventing inbreeding depression. Additionally, maintaining good management practices regarding the fluctuation of water temperature, exposure to pollution, nutrition, etc., will further contribute to the prevention of this detrimental condition

The first DNA barcode record for Rhyacophila bosnica Schmid, 1970 and pairing of adult and larval life stages

Rhyacophila Pictet 1834 is globally distributed and highly diverse genus of caddisflies (Trichoptera), characterized by numerous regionally endemic species. In the Balkan Peninsula, the highest number of Rhyacophila species (23) was recorded for Bosnia and Herzegovina. Rhyacophila bosnica Schmid, 1970 is found only in the Balkan Dinaric region, with a locus typicus in Vučja Luka, Bosnia and Herzegovina. Like with many species of Trichoptera, the morphology of its larva is still unknown. Therefore, DNA barcoding approach was used to link two developmental stages. In this paper, we report on the first DNA barcode record for this species

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

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

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

LONG-TERM TRENDS IN THE STRUCTURE AND DYNAMICS OF THE FISH COMMUNITIES IN BUŠKO BLATO RESERVOIR

Akumulacija Buško Blato, treća najveća hidroakumulacija u Europi, smještena je u jugoistočnom dijelu krškog Livanjskog polja. Ovaj vodeni ekosustav odlikuje se vrlo bogatom ihtiofaunom i prisutnošću četiri endemske vrste riba. Ihtiološka istraživanja navedene akumulacije bila su u fokusu brojnih istraživanja. Ihtiofauna Buškog Blata bila je izložena promjenama različitih ekoloških čimbenika, što se odrazilo na strukturu i sastav ribljih populacija. Glavni cilj ovog rada bio je procijeniti trenutno stanje i predvidjeti buduće trendove u strukturi i dinamici ihtiofaune na temelju prikupljenih terenskih podataka i sveobuhvatnih analiza iz literaturnih podataka. Rezultati istraživanja pokazali su prisutnost 11 vrsta riba iz četiri porodice, što je najveći broj vrsta riba ikada zabilježenih u ovom ekosustavu. Sander lucioperca, Lepomis gibbosus, Pseudorasbora parva, i Tinca tinca prvi su put zabilježeni u ovom ekosustavu, dok neke prethodno zabilježene vrste nisu pronađene. Rezultati analiza jasno ukazuju na prisutnost prirodne interspecifične konkurencije i značajnu razinu ugroženosti endemskih vrsta riba uzrokovanu ljudskim aktivnostima.Buško Blato Reservoir, the third largest hydro accumulation in Europe, is situated in the south-eastern part of the karstic Livanjsko Polje Valley. This aquatic ecosystem is distinguished by a very rich ichthyofauna and the presence of four endemic fish species. Numerous studies focus on ichthyological researches of Buško Blato Reservoir. Its ichthyofauna has been exposed to changes in ecological factors, which in turn reflected on the structure and composition of fish populations. The main objective of this paper was to assess the current state and predict future trends in the ichthyofauna structure and dynamics based on the field data and comprehensive analyses of literature data. The results of the research indicated the presence of 11 fish species from four families, which is the largest number of fish species ever recorded in this ecosystem. Sander lucioperca, Lepomis gibbosus, Pseudorasbora parva and Tinca tinca were recorded for the first time in this ecosystem, while some previously recorded species were not found. The results of the analyses clearly indicate the presence of natural interspecific competition and significant level of threats to the endemic fish species caused by human activities

GENETIC IDENTITY OF RASPBERRY ‘POLANA’ PLANTLINGS EXAMINED USING MICROSATELLITE MARKERS

Raspberry cultivars are clonally propagated and therefore all plants belonging to a single cultivar represent the same genotype. Cultivar integrity of raspberry plantlings placed on the market in Bosnia and Herzegovina (B&H) is based on examining of morphological traits, which is not a reliable tool for genetic identification. In this study plantlings declared as cultivar ‘Polana’ were genotyped using seven microsatellites, in order to gain preliminary insight into the genetic integrity of raspberry plantlings marketed in B&H. Plant tissue (leaves) from 10 raspberry plants were randomly sampled from a batch of plantlings sold by major fruit nursery in Bosnia and Herzegovina. Along with these samples, four reference cultivars with confirmed identity (‘Polka’, ‘Autumn Bliss’, ‘Heritage’ and ‘Polana’) were also included in the study. Seven primer pairs amplified 31 alleles, or on average 4.4 alleles per locus. UPGMA cluster analysis, based on the Jaccard similarity coefficient, revealed that among the ten samples declared as ‘Polana’ plantlings only five were genetically identical to any of the other samples. The cluster analyses also exposed that none of the ten samples declared as ‘Polana’ seedlings were in fact identical or even closely related to the ‘Polana’ reference cultivar or any of the other reference cultivars. These findings clearly show that the genetic identity of primocane raspberry plantlings , currently sold in Bosnia and Herzegovina, needs to be tested using objective and reliable methods rather than simple morphologic observation

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

Genetic structure and diversity of European chestnut (Castanea sativa Mill.) populations in western Balkans: On a crossroad between east and west

European chestnut (Castanea sativa Mill.) is highly valued in the western Balkans as a source of timber and fruit, but also as an important source of nectar and pollen for the production of honey. In this study, four chestnut populations, covering a major portion of the western Balkans, and a reference population from the northern Italy were examined using 21 microsatellite markers. The highest genetic diversity was detected within the populations geographically closest to the Italian Peninsula, which also displayed the highest level of admixture with the samples from Italy. The strongest genetic differentiation was noted among the southern and eastern chestnut populations from Bosnia and Herzegovina (B&H) (Gst=12.05%). This pronounced differentiation is probably caused by the genetic adaptations to notably different climatic conditions present in the south (Mediterranean climate) and east (Continental climate) of B&H. The clear genetic differentiation of the southern and eastern B&H chestnut populations from the Italian population, determined by pairwise Gst, FCA and Bayesian Structure analyses, indicates that these populations most likely originated from independent shelter zones (refugia), after the last glaciation period. Based on these results we propose a presence of an introgression zone in the northwestern Balkans, established through gene flow from the Italian and the Balkan Peninsula. The obtained insights into the structure of all analyzed populations will significantly contribute towards establishing a regional conservation and utilization strategy for European chestnut in western Balkans