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In wolves Canis lupus, scent marking plays an important role in territory defence. In Europe, studies on patterns of scent marking in wolves have mostly been conducted in mountains or primeval forests, but since these areas are characterised by low human activity, the impact of people on this behaviour has been neglected. We conducted a study that combined genetic methods with an analysis of the spatial distribution of wolf territory markings in lowland managed forests with high human activity. We found that scent markings are deposited by all members of wolf family groups. Wolves most intensively marked crossroads and their vicinity, especially on roads only accessible for four-wheel drive cars. Our study provides further evidence that crossroads of forest roads play a crucial role in wolf scent marking. The results of our study may be useful during inventories of wolf populations based on collecting indirect signs of their presence or non-invasive genetic sampling.

Herring (Clupea harengus) shows the unique behavior of reproductive biology in which spermatozoa remains in the surrounding media for extended periods. It is an excellent model for studying the malic enzyme (ME) and creatine kinase (CK) biochemical properties because of their high activity and variability of molecular isoforms. The specific activity of NAD-preferring ME in herring spermatozoa is the highest among other fish spermatozoa and is localized in its large mitochondrion. Two different CK isoforms, dimer and octamer, were detected in herring spermatozoa. It has already been shown that CK isoforms play an important role in energy homeostasis by catalyzing a reversible transfer of the phosphate of ATP to creatine to yield ADP and creatine phosphate (CP) (creatine/CP circuit). Two lactate dehydrogenase (LDH) isoenzymes were also shown in herring spermatozoa, LDH-B4 and LDH-A2B2. In this mini-review, the role of ME and energy transport system with easily diffusible creatine and CP in herring spermatozoa is discussed.

The gray wolf Canis lupus range in central Europe is dynamically expanding, reconnecting previously isolated populations. Thus, a recent paper has proposed to merge the current Baltic and Central European (CE) wolf management units, which are no longer isolated by distance. However, recent genetic findings indicate that these two populations are not genetically homogenous. Here we review the most recent data on wolf genetic structure in central Europe and show that even though the CE and Baltic wolves represent the same phylogeographic lineage, their demographic history has resulted in significant genetic structure between these two populations. While the groups are interconnected by moderate gene flow, it is not high enough to reduce the strong founder signal observed in the CE population, suggesting that population dynamics within the CE wolf range are largely independent from those of its source (Baltic) population. Consequently, a management unit combining the CE and Baltic wolves would not form a demographically coherent entity. Thus, we recommend that conservation management units maintain their separate status.

Monitoring of the wolf Canis lupus is a demanding task as it lives in low densities, utilizes vast home ranges and disperses over large areas. These factors make obtaining accurate data about population parameters over the whole distribution area of the species impossible. Thus detailed local studies on socio-spatial organization are essential to calibrate information obtained over a larger area. We applied GPS/GSM telemetry, non-invasive genetic sampling, year-round tracking, camera trapping and howling stimulations to determine the number of family groups, population density and home-range sizes of wolves in the Drawa Forest (DF, western Poland, 2500 km2), an area recently recolonized by the species. Home ranges of three collared male wolves ranged from 321.8 to 420.6 km2 (MCP 100%) and from 187.5 to 277.5 km2 (Kernel 95%), but core areas had a size of 30.5–84.7 km2 (MCP50%) and 35.0–88.8 km2 (Kernel 50%). Mean near neighbour distance between centres of 6 tracked pack homesites was 15.3 km. The number of wolves in DF increased from 14 individuals in 2013/2014 to 30 in 2016/2017. The annual rate of increase varied from 43% in 2014/2015 to 7% in the final year. Population density for the whole study area was relatively low (1.2 indiv./100 km2 in 2016/2017), but densities within territories of two packs studied with telemetry were 1.9 and 1.5 indiv./100 km2. Mean pack size varied between 3.5 and 5.6 individuals, with the largest pack comprising 8 wolves. Mean number of pups observed in summers (June–August) was 4.5. Differences in home range sizes between wolves in western and eastern Poland indicate that results of regional studies cannot be freely extrapolated despite close genetic relationships. Thus, decisions related to management of wolf habitats should be based on intensive local studies.

Local extinction and recolonization events can shape genetic structure of subdivided animal populations. The gray wolf (Canis lupus) was extirpated from most of Europe, but recently recolonized big part of its historical range. An exceptionally dynamic expansion of wolf population is observed in the western part of the Great European Plain. Nonetheless, genetic consequences of this process have not yet been fully understood. We aimed to assess genetic diversity of this recently established wolf population in Western Poland (WPL), determine its origin and provide novel data regarding the population genetic structure of the grey wolf in Central Europe. We utilized both spatially explicit and non-explicit Bayesian clustering approaches, as well as a model-independent, multivariate method DAPC, to infer genetic structure in large dataset (881 identified individuals) of wolf microsatellite genotypes. To put the patterns observed in studied population into a broader biogeographic context we also analyzed a mtDNA control region fragment widely used in previous studies. In comparison to a source population, we found slightly reduced allelic richness and heterozygosity in the newly recolonized areas west of the Vistula river. We discovered relatively strong west-east structuring in lowland wolves, probably reflecting founder-flush and allele surfing during range expansion, resulting in clear distinction of WPL, eastern lowland and Carpathian genetic groups. Interestingly, wolves from recently recolonized mountainous areas (Sudetes Mts, SW Poland) clustered together with lowland, but not Carpathian wolf populations. We also identified an area in Central Poland that seems to be a melting pot of western, lowland eastern and Carpathian wolves. We conclude that the process of dynamic recolonization of Central European lowlands lead to the formation of a new, genetically distinct wolf population. Together with the settlement and establishment of packs in mountains by lowland wolves and vice versa, it suggests that demographic dynamics and possibly anthropogenic barriers rather than ecological factors (e.g. natal habitat-biased dispersal patterns) shape the current wolf genetic structure in Central Europe.

Herring spermatozoa exhibit a high activity of NAD-preferring malic enzyme (NAD-ME). This enzyme is involved in the generation of NADH or NADPH in the decarboxylation of malate to form pyruvate and requires some divalent cations to express its activity. In order to confirm that NAD-ME isolated from herring sperm cells is localized in mitochondria, we performed immunofluorescent analysis and assayed spectrophotometrically the malic enzyme reaction. Production of polyclonal rabbit antibodies against NAD-ME from herring spermatozoa enabled identification of mitochondrial localization of this enzyme inside herring spermatozoa. The kinetic studies revealed that NAD-ME was competitively inhibited by ATP up to tenfold. Addition of fumarate reversed ATP-dependent inhibition of NAD-ME to 55 % of its maximum activity. The pH-dependent regulation of malic enzyme activity was also examined. Malic enzyme showed maximum activity at pH near 7.0 in all studied conditions. Finally, the role of malic enzyme activity regulation in mitochondria of herring sperm cells was discussed.

Creatine kinases (CKs) constitute a large family of isoenzymes that are involved in intracellular energy homeostasis. In cells with high and fluctuating energy requirements ATP level is maintained via phosphocreatine hydrolysis catalyzed by creatine kinase. In contrast to invertebrates and higher vertebrates, in poikilothermic vertebrates the adaptations for the regulation of energy metabolism by changes in the oligomeric state of CK isoforms are not well known. The present study aimed at identification of herring eye CK isoforms and focuses on factors affecting the CK-octamer stability. In addition to the CK octamer, three different dimeric isoforms of CK were detected by cellulose acetate native electrophoresis. Destabilization of octamer was studied in the presence of TSAC substrates and about 50% of octamers dissociated into dimers within 24h. Moreover, we found that the increase of temperature from 4 °C to 30 °C caused rapid inactivation of dimers in TSAC-treated samples but did not affect octameric structures. In a thermostability assay we demonstrated that octamers retain their activity even at 50 °C. Our results indicate that destabilization of the octameric structure can lead to loss of enzyme activity at higher temperatures (above 30 °C). Furthermore, our results based on N-terminal sequence analysis suggest that probably the mitochondrial s-type CK, rather than u-type, is predominantly expressed in herring eye. In conclusion the existence of four various CK isoforms in one organ may reflect complex regulation of energy metabolism in the phototransduction process in teleost fishes.

If protected areas are to remain relevant in our dynamic world they must be adapted to changes in species ranges. In the EU one of the most notable such changes is the recent recovery of large carnivores, which are protected by Natura 2000 at the national and population levels. However, the Natura 2000 network was designed prior to their recent recovery, which raises the question whether the network is sufficient to protect the contemporary ranges of large carnivores. To investigate this question we evaluated Natura 2000 coverage of the three wolf Canis lupus populations in Poland. Wolf tracking data showed that wolves have recolonised almost all suitable habitat in Poland (as determined by a recent habitat suitability model), so we calculated the overlap between the Natura 2000 network and all wolf habitat in Poland. On the basis of published Natura 2000 criteria, we used 20% as the minimum required coverage. At the national level, wolves are sufficiently protected (22% coverage), but at the population level, the Baltic and Carpathian populations are far better protected (28 and 47%, respectively) than the endangered Central European Lowland population (12%). As Natura 2000 insufficiently protects the most endangered wolf population in Poland, we recommend expansion of Natura 2000 to protect at least an additional 8% of wolf habitat in western Poland, and discuss which specific forests are most in need of additional coverage. Implementation of these actions will have positive conservation implications and help Poland to fulfil its Habitats Directive obligations. As it is likely that similar gaps in Natura 2000 are arising in other EU member states experiencing large carnivore recoveries, particularly in Central Europe, we make the case for a flexible approach to Natura 2000 and suggest that such coverage evaluations may be beneficial elsewhere.

Herring spermatozoa exhibit higher activity of malic enzyme (ME) than Atlantic salmon (Salmo salar), brown trout (Salmo trutta), carp (Cyprinus carpio) and African catfish (Clarias gariepinus) spermatozoa. Two molecular forms of ME are present in herring spermatozoa: an NAD-preferring malic enzyme with very high activity and an NADP-specific malic enzyme with much lower activity (ratio about 33:1). NAD-preferring ME was purified by chromatography on DEAE-Sepharose, Red Agarose and Sephadex G-200 to a specific activity of 36 μmol/min/mg protein and NADP-specific ME on DEAE-Sepharose and 2'5'-ADP Sepharose. The molecular mass for NAD-preferring and NADP-specific ME determined by SDS-PAGE was equal to 61 and 64 kDa, respectively. High activity of ME suggests adaptation of herring spermatozoa to metabolism at high oxygen tension for herring spawn.