Ticks and spirochetes of the genus Borrelia in urban areas of Central-Western Poland.

Due to the extensive use of green urban areas as recreation places, city residents are exposed to tick-borne pathogens. The objectives of our study were (i) to determine the occurrence of ticks in urban green areas, focussing on areas used by humans such as parks, schools and kindergartens, and urban forests, and (ii) to assess the prevalence of Borrelia infections in ticks in Zielona Góra, a medium-sized city in western Poland. A total of 161 ticks representing the two species Ixodes ricinus (34 males, 51 females, 30 nymphs) and Dermacentor reticulatus (20 males, 26 females) were collected from 29 of 72 (40.3%) study sites. In total, 26.1% of the ticks (85.7% of I. ricinus and 14.3% of D. reticulatus) yielded DNA of Borrelia. The difference in the infection rate between I. ricinus and D. reticulatus was significant. Among infected ticks, the most frequent spirochete species were B. lusitaniae (50.0%) and B. afzelii (26.2%), followed by B. spielmanii (9.5%), B. valaisiana (7.1%), B. burgdorferi sensu stricto, (4.8%) and B. miyamotoi (2.4%). No co-infections were found. We did not observe a correlation in the occurrence of Borrelia spirochetes in ticks found in individual study sites that differed in terms of habitat type and height of vegetation. Our findings demonstrate that the Borrelia transmission cycles are active within urban habitats, pointing the need for monitoring of tick-borne pathogens in public green areas. They could serve as guidelines for authorities for the proper management of urban green spaces in a way that may limit tick populations and the potential health risks posed by tick-borne pathogens.

Basidiomycota strains as whole-cell biocatalysts for the synthesis of high-value natural benzaldehydes.

Substituted benzaldehydes are the most commonly used natural-occurring flavours in the world. The consumer's preference for 'natural or organic' aromas has increased the request for flavours possessing the 'natural' status. The resulting shortage of aromatic aldehydes of extractive origin, such as vanillin, veratraldehyde and piperonal, can be offset by developing a new biotechnological synthesis method. Here, we report a study on the microbiological reduction of five natural benzoic acid derivatives, namely p-anisic, vanillic, veratric, piperonylic and eudesmic acids, to produce the corresponding fragrant aldehydes. We found that different Basidiomycota strains can efficiently perform this transformation, with good chemical selectivity and tolerance to the toxicity of substrates and products. Besides confirming the carboxylic acid reductase activity of the already studied fungi Pycnoporus cinnabarinus, we discovered that other species such as Pleurotus eryngii, Pleurotus sapidus and Laetiporus sulphureus as well as the non-ligninolytic fungi Lepista nuda are valuable microorganisms for the synthesis of anisaldehyde, vanillin, veratraldehyde, piperonal and 3,4,5-trimethoxybenzaldehyde from the corresponding acids. According to our findings, we propose a reliable process for the preparation of the above-mentioned aldehydes, in natural form. KEY POINTS: • Fragrant benzaldehydes were obtained by biotransformation. • Basidiomycota strains reduced substituted benzoic acid to the corresponding aldehydes. • Anisaldehyde, vanillin, veratraldehyde, piperonal and 3,4,5-trimethoxybenzaldehyde were prepared in natural form.

Microbial Metabolites of 3-n-butylphthalide as Monoamine Oxidase A Inhibitors.

Novel compounds with antidepressant activity via monoamine oxidase inhibition are being sought. Among these, derivatives of 3-n-butylphthalide, a neuroprotective lactone from Apiaceae plants, may be prominent candidates. This study aimed to obtain the oxidation products of 3-n-butylphthalide and screen them regarding their activity against the monoamine oxidase A (MAO-A) isoform. Such activity of these compounds has not been previously tested. To obtain the metabolites, we used fungi as biocatalysts because of their high oxidative capacity. Overall, 37 strains were used, among which Penicillium and Botrytis spp. were the most efficient, leading to the obtaining of three main products: 3-n-butyl-10-hydroxyphthalide, 3-n-butylphthalide-11-oic acid, and 3-n-butyl-11-hydroxyphthalide, with a total yield of 0.38-0.82 g per g of the substrate, depending on the biocatalyst used. The precursor-3-n-butylphthalide and abovementioned metabolites inhibited the MAO-A enzyme; the most active was the carboxylic acid derivative of the lactone with inhibitory constant (Ki) < 0.001 µmol/L. The in silico prediction of the drug-likeness of the metabolites matches the assumptions of Lipinski, Ghose, Veber, Egan, and Muegge. All the compounds are within the optimal range for the lipophilicity value, which is connected to adequate permeability and solubility.

Universal mtDNA fragment for Cervidae barcoding species identification using phylogeny and preliminary analysis of machine learning approach.

The aim of the study was to use total DNA obtained from bone material to identify species of free-living animals based on the analysis of mtDNA fragments by molecular methods using accurate bioinformatics tools Bayesian approach and the machine learning approach. In our research, we present a case study of successful species identification based on degraded samples of bone, with the use of short mtDNA fragments. For better barcoding, we used molecular and bioinformatics methods. We obtained a partial sequence of the mitochondrial cytochrome b (Cytb) gene for Capreolus capreolus, Dama dama, and Cervus elaphus, that can be used for species affiliation. The new sequences have been deposited in GenBank, enriching the existing Cervidae mtDNA base. We have also analysed the effect of barcodes on species identification from the perspective of the machine learning approach. Machine learning approaches of BLOG and WEKA were compared with distance-based (TaxonDNA) and tree-based (NJ tree) methods based on the discrimination accuracy of the single barcodes. The results indicated that BLOG and WEKAs SMO classifier and NJ tree performed better than TaxonDNA in discriminating Cervidae species, with BLOG and WEKAs SMO classifier performing the best.

Trametes hirsuta as an Attractive Biocatalyst for the Preparative Scale Biotransformation of Isosafrole into Piperonal.

Piperonal is a compound of key industrial importance due to its attractive olfactory and biological properties. It has been shown that among the fifty-six various fungal strains tested, the ability to cleave the toxic isosafrole into piperonal through alkene cleavage is mainly found in strains of the genus Trametes. Further studies involving strains isolated directly from different environments (decaying wood, fungal fruiting bodies, and healthy plant tissues) allowed the selection of two Trametes strains, T. hirsuta Th2_2 and T. hirsuta d28, as the most effective biocatalysts for the oxidation of isosafrole. The preparative scale of biotransformation with these strains provided 124 mg (conv. 82%, isolated yield 62%) and 101 mg (conv. 69%, isolated yield 50.5%) of piperonal, respectively. Due to the toxic impact of isosafrole on cells, preparative scale processes with Trametes strains have not yet been successfully performed and described in the literature.

Relationship between semen quality and level of heterozygosity on the model of endangered population of Western capercaillie.

Sperm quality is a very important population survival factor, which can be influenced by the level of male's genetic diversity and can influence reproduction effectiveness, what may be crucial in terms of decision about local, endangered population protection plans. Thus, we analysed if heterozygosity and age influence sperm quality in the engendered European capercaillie (Tetrao urogallus) population in Polish breeding stations. Obtained results showed no relationship between normal sperm morphology and heterozygosity, as well as age and sperm morphology, but it was shown that both high and low level of heterozygosity significantly influenced the percentage of abnormal sperm. Thus, heterozygosity level can be a useful tool for population reproduction potential prediction, however, further research on how those abnormalities influence fertilization process are needed.

Mitogenomes of Accipitriformes and Cathartiformes Were Subjected to Ancestral and Recent Duplications Followed by Gradual Degeneration.

The rearrangement of 37 genes with one control region, firstly identified in Gallus gallus mitogenome, is believed to be ancestral for all Aves. However, mitogenomic sequences obtained in recent years revealed that many avian mitogenomes contain duplicated regions that were omitted in previous genomic versions. Their evolution and mechanism of duplication are still poorly understood. The order of Accipitriformes is especially interesting in this context because its representatives contain a duplicated control region in various stages of degeneration. Therefore, we applied an appropriate PCR strategy to look for duplications within the mitogenomes of the early diverged species Sagittarius serpentarius and Cathartiformes, which is a sister order to Accipitriformes. The analyses revealed the same duplicated gene order in all examined taxa and the common ancestor of these groups. The duplicated regions were subjected to gradual degeneration and homogenization during concerted evolution. The latter process occurred recently in the species of Cathartiformes as well as in the early diverged lineages of Accipitriformes, that is, Sagittarius serpentarius and Pandion haliaetus. However, in other lineages, that is, Pernis ptilorhynchus, as well as representatives of Aegypiinae, Aquilinae, and five related subfamilies of Accipitriformes (Accipitrinae, Circinae, Buteoninae, Haliaeetinae, and Milvinae), the duplications were evolving independently for at least 14-47 Myr. Different portions of control regions in Cathartiformes showed conflicting phylogenetic signals indicating that some sections of these regions were homogenized at a frequency higher than the rate of speciation, whereas others have still evolved separately.

Characteristics of capercaillie (Tetrao urogallus) semen analysed with flow cytometry combined with fertility results.

In order to increase the reproductive indices of capercaillie kept in closed breeding facilities, it is necessary to constantly expand the methods of better understanding the characteristics of sperm and their fertilizing potency. The aim of the study was to analyse selected features of capercaillie sperm using flow cytometry and their connection with fertility results. The study included five males, three of which were kept in a family group with eight females and two were kept alone. For sperm viability, acrosome integrity, mitochondrial potential and DNA defragmentation were assessed. Paternity analyses were performed in order to confirm the paternity of the individual and to link the evaluated semen traits with reproductive success. Analyses carried out in the flow cytometer showed any significant differences between males in sperm characteristics. In the semen of male No. 101, the father of all chicks from the analysed family group, 91.3% of live sperm, 91.5% with intact acrosome, 83.6% with active mitochondria and 2.0% with DNA defragmentation were observed. The average fertility rate was 71.0%, and chick hatchability was 100%. Using flow cytometry in the analysis of capercaillie semen and its connection with the results of natural mating, we were able to obtain deeper knowledge about new sperm characteristics that were not examined before and which in the future may be helpful in selecting males for the reproductive flocks and developing assisted reproduction techniques.

Female-Male and Female-Female Social Interactions of Captive Kept Capercaillie (Tetrao Urogallus) and Its Consequences in Planning Breeding Programs.

Capercaillie behavior, both in the wild and in captivity, is poorly known due to this species' secretive way of life. Female-male and female-female social organization and interactions are especially poorly documented. The research was conducted in Capercaillie Breeding Center in Wisla Forestry District where a breeding flock is kept throughout the year. Thanks to video monitoring, we were able to observe mate choice, and then later, female-female interactions during laying and incubation period. Male individual variation in tooting latency and duration were recorded. Females' interest in males was related to males' tooting activity, but when males became too insistent and started to chase the females, the females avoided contact with them. There was a significant relationship between calendar date and when tooting starts, and between the tooting duration the female spent with a male. Two incidents of female-male aggression caused by competition for food were observed. Female intruder presence and competition for nesting place was observed in 66.67% nests. Most female-female interactions were limited to threat posturing, but fights and attempts to push out the intruder from the nest occurred as well. Such interactions may lead to nest abandonment and egg destruction, lowering the breeding success.

Cepaea spp. as a source of Brachylaima mesostoma (Digenea: Brachylaimidae) and Brachylecithum sp. (Digenea: Dicrocoeliidae) larvae in Poland.

Parasite diagnostics were carried out on 11 Polish populations of Cepaea spp. In three of them, coming from the roadside ditches of a village (Rytel, northern Poland), very high (up to 60%) prevalence of Brachylaima mesostoma was observed. This study provides the first molecular evidence of the presence of B. mesostoma inside Cepaea spp. in Europe. In a few snails from a population found in a private garden in a small town (Chelmza, northern Poland), larvae of Brachylecithum sp. were present. Cercariae and/or metacercariae of B. mesostoma were observed in both species of Cepaea: C. hortensis and C. nemoralis, whereas larvae of Brachylecithum sp. were found only in C. nemoralis. Both species of parasites inhabited snail hepatopancreas whose structure was significantly damaged by larvae. There was no significant connection between parasite invasion and snail host morphotype. The research did not allow the reasons for the high prevalence of B. mesostoma in Cepaea spp. to be explained, and also did not explicitly indicate how the parasite invaded Cepaea spp. individuals making them, at the same time a second intermediate host. However, it poses important questions about the life cycle of the parasite that may threaten extensively kept small-size farms of poultry.

Genetic diversity and relationship between cultivated, weedy and wild rye species as revealed by chloroplast and mitochondrial DNA non-coding regions analysis.

The genus Secale is small but very diverse. Despite the high economic importance, phylogenetic relationships of rye species have not been fully determined, and they are extremely important for the process of breeding of new cultivars that can be enriched with functional traits derived from wild rye species. The study analyzed the degree of relationship of 35 accessions of the genus Secale, representing 13 most often distinguished species and subspecies, originating from various seed collections in the world, based on the analysis of non-coding regions of the chloroplast (cpDNA) and mitochondrial genome (mtDNA), widely used in phylogenetic and population plant studies, because of a higher rate of evolution than the coding regions. There was no clear genetic structure between different species and subspecies, which may indicated the introgression between these taxa. The obtained data confirmed that S. vavilovii was very similar to S. cereale, which confirmed the assumption that they might share a common ancestor. The results also confirmed the divergence of S. sylvestre from other species and subspecies of rye. Areas that may be useful molecular markers in studies on closely related species of the genus Secale were also indicated.

Agents of swimmer's itch-dangerous minority in the Digenea invasion of Lymnaeidae in water bodies and the first report of Trichobilharzia regenti in Poland.

Trichobilharzia spp. have been identified as a causative agent of swimmers' itch, a skin disease provoked by contact with these digenean trematodes in water. These parasites have developed a number of strategies to invade vertebrates. Since we have little understanding of the behavior of these parasites inside the human body, the monitoring of their invasion in snail host populations is highly recommended. In our research, lymnaeid snails were collected from several Polish lakes for two vegetation seasons. The prevalence of bird schistosomes in snail host populations was significantly lower than that of other digenean species. We were the first to detect the presence of the snails emitted Trichobilharzia regenti (potentially the most dangerous nasal schistosome) in Poland. In addition, by sequencing partial rDNA genes, we confirmed the presence of the snails positive with Trichobilharzia szidati in Polish water bodies, showing that swimmer's itch is more frequent during summer months and that large snails are more often infected with bird schistosomes than small ones.

Salt mine microorganisms used for the biotransformation of chlorolactones.

The aim of the project was to find new catalysts capable of chlorolactone biotransformation. Three bicyclic chlorolactones with structures possessing one or two methyl groups in their cyclohexane ring were subjected to screening biotransformation using seven bacterial strains and one fungal strain from a salt mine. Three strains of bacteria (Micrococcus luteus Pb10, Micrococcus luteus WSP45, Gordonia alkanivorans Pd25) and one fungal strain (Aspergillus sydowii KGJ10) were able to catalyse hydrolytic dehalogenation of one substrate. The classification of the strains that were effective biocatalysts was confirmed by 16S rDNA analysis. The best result (76%) was obtained using Aspergillus sydowii KGJ10. All strains catalysed hydrolytic dehalogenation without changing the conformation. The equatorial position of the chlorine atom in the substrate turned out to be warrant of the positive result of the biotransformation process.

Ongoing Speciation and Gene Flow between Taxonomically Challenging Trochulus Species Complex (Gastropoda: Hygromiidae).

Geographical isolation, selection and genetic drift can cause the geographical diversification of populations and lead to speciation. Land snail species in the genus Trochulus show overlaps in geographical ranges as well as in morphology, but genetic data do not always support the species-level taxonomy based on morphological characters. Such a group offers an excellent opportunity to explore the processes involved. We have addressed the problem by determining the status of the restricted endemic T. graminicola within the larger context of Trochulus taxonomy. We used an integrated approach based on morphological features, ecological preferences and two molecular markers: mitochondrial COI sequences and microsatellites. Comparison of these results demonstrated: (i) conchological distinction of T. striolatus and T. sericeus; (ii) anatomical, ecological and genetic differentiation of T. graminicola and (iii) concordance between morphological characters and mtDNA markers in T. striolatus. Moreover, our data showed an intricate evolutionary history within the genus Trochulus, which can be best explained by: (i) recent or ongoing gene flow between taxa or (ii) their large ancestral polymorphism. Both of these hypotheses suggest that diversification within this group of snails has occurred relatively recently. The mismatches between species defined on morphology and on molecular genetics indicate the complexity of the processes involved in the diversification of this genus.

Complete mitochondrial genome of golden conure (Guaruba guarouba).

Arini tribe with 19 genera is the most diversified tribe of neotropical parrots. Six of them are classified as macaws and nine as conures. The presence of bare facial area distinguishes macaws from conures and other members of this tribe. However, such morphological division seems to be disputable as the smallest macaw (monotypic Diopsittaca genus) turned out to be more closely related to three monotypic conures genera (Guaruba, Leptosittaca, Thectocercus) than to other macaws. We sequenced the complete mitochondrial genome of Guaruba guarouba to enrich the resource of molecular markers for examination of phylogenetic relationships between macaws and conures.

Complete mitochondrial genome of the Eastern slow worm, Anguis colchica (Nordmann, 1840).

Here, we present complete mitochondrial genome of the Eastern Slow Worm, Anguis colchica (Nordmann, 1840). Mitogenome complete sequence is 17,097 bp long and consists of 13 protein-coding genes, 22 tRNA genes, two rRNA genes and one control region. Anguis colchica mitochondrial genome has the same gene order as other mitogenomes of Anguis spp. Their analyzed genome has base composition as: A (30.4%), T (24.6%), C (30.4%), G (14.6%), with an A + T bias (55%). Length of the all 22 tRNA genes varies from 65 to 73 bp with an average of 69 bp. Presented mitogenome will provide new data for phylogenetic analysis within the genus Anguis.

Complete mitochondrial genome of the Italian slow-worm Anguis veronensis Pollini, 1818, and its comparison with mitogenomes of other Anguis species.

In this paper, we present complete mitochondrial genome of the Italian legless lizard species Anguis veronensis Pollini, 1818. The complete mtDNA consisted of 13 protein-coding genes, 22 tRNAs, and two rRNA genes which in total formed a DNA strand of 17,322 bp. Anguis veronensis mitogenome had the same gene order as two other compared Anguis spp., i.e. A. cephallonica and A. fragilis. The base composition of A. veronensis mitochondrial genome was A - 30.8%, T - 24.9%, C - 29.9%, G - 14.4%, with an A + T bias (55.7%). The newly described genome provides valuable data for future comparative mitogenomic analysis within Anguis genus.

Complete mitochondrial genome of Blue-and-yellow Macaw (Ara ararauna): the species morphologically similar to Blue-throated Macaw (Ara glaucogularis).

Six genera of the Arini tribe form morphologically diverse group termed as Macaws. The presence of bare facial area distinguishes them from other members of the tribe. Genera and species of Macaw group differ in pattern of this bare skin as well as in body size. The genus Ara contains eight extant species. Two of them are classified as medium-sized Macaws and other six species are classified as large Macaws. Based on morphological similarities and differences, large Macaw species can be segregated into three pairs according to their plumage coloration. So far, representative mitochondrial genomes were sequenced only for Ara glaucogularis (blue and yellow coloration), Ara macao (predominantly red/scarlet) and Ara militaris (predominantly green) species. Ara ararauna is the second of two Ara species with predominant blue and yellow plumage. Hitherto, close phylogenetic relation of these two species was shown using only ND2 genes or incomplete combined COI/Cytb/ND2 sequences. Therefore, Ara ararauna mitogenome, which was sequenced in this study, will be indispensable to refine the phylogenetic relationships within Macaws group.

Complete mitochondrial genome of Mitred Conure (Psittacara mitratus): its comparison with mitogenome of Socorro Conure (Psittacara brevipes).

The Mitred Conure (Psittacara mitratus) is a species native to the forests and woodlands in the Andes in central Peru, south through west-central Bolivia, to northwestern Argentina. The genus Psittacara have recently been distinguished on the basis of molecular revision of the Aratinga species. In consequence, it became one of the nine genera, which form morphologically diverse group termed as Conures. Although, the number of species/subspecies belonging to Psittacara remains controversial, it is now believed that the genus is represented by 11 species. Taxonomic position of Mitred Conure was determined by molecular research and makes the species crucial for examination of evolutionary diversification of the genus. Therefore, we sequenced complete mitochondrial genome of P. mitratus mitogenome to gain a source of comprehensive molecular data. It will be indispensable to refine the phylogenetic relationships within "Psittacara group" as well as within the tribe Arini.

Complete mitochondrial genome of Red-throated Conure (Psittacara rubritorquis): its comparison with mitogenome of Socorro Conure (Psittacara brevipes).

According to some taxonomists the Red-throated Conure (Psittacara rubritorquis) is considered a subspecies of Green Conure (Psittacara holochlora). Some other classifications treat rubritorquis as a separate species based on relatively minor morphological differences between both species/subspecies. So far, taxonomic position of P. rubritorquis was determined by molecular researches using only ND2 gene sequence or incomplete combined mitochondrial ND2, COI and CYTB gene sequences. Obtained outcomes found that P. rubritorquis should be treated as a subspecies of P. holochlora. However, the lack of P. h. brewsterii and P. h. strenua samples as well as incompleteness of combined mitochondrial sequence do not exclude opposite scenario. Therefore, we sequenced P. rubritorquis mitogenome to gain a source of molecular data appropriate for future examination of evolutionary diversification of the P. holochlora group.