Factors affecting daily ranges of red deer<Emphasis Type="Italic">Cervus elaphus</Emphasis> in Białowieża Primeval Forest,Poland

Daily ranges of 19 (6 males, 13 females) adult red deerCervus elaphus Linnaeus, 1758 were studied using 24-h tracking sessions in Białowieża Primeval Forest (BPF), Poland, from 2001 to 2004. Overall, size of mean (± SE) daily ranges was larger for males (1.22 ± 0.10 km²) than females (1.00 ± 0.09 km²), although the difference was not significant. Similarly, mean daily ranges were 6–46% larger for males than females in each season, although there were no statistical differences in mean daily ranges among seasons for each sex. Abiotic factors, especially temperature, significantly affected daily ranges of females, but not males, suggesting sexual differences in response to weather variables. On a daily basis, males used 3% of their annual home range, whereas females used 12% of their annual home range, indicating females used their annual home ranges more intensely than males. Consecutive daily ranges overlapped little for each sex. Daily ranges of red deer in BPF were considerably larger than previously reported in Europe, suggesting factors unique to BPF also influenced size of daily ranges.     

Adaptation to cold and predation—shelter use by invasive raccoon dogs <Emphasis Type="Italic">Nyctereutes procyonoides</Emphasis> in Białowieża Primeval Forest (Poland)

Based on radio-tracking of raccoon dogs Nyctereutes procyonoides in Białowieża Primeval Forest (E Poland) in 1997–2002, we addressed the hypothesis that pattern of shelter use by this invasive carnivore is an adaptation to both climatic conditions and predation. Raccoon dogs used various types of shelters but were invariably concealed in dense vegetation or in the shelters created by trunks, branches, or roots of fallen trees (58% of days). Burrows (mainly badger setts) and hollow trees (mainly fallen limes) were selected on 27% and 14% of days, respectively. The pattern of shelter use varied throughout the year. In crucial periods (breeding period and winter), raccoon dogs used shelter offering better protection: burrows and hollow trees (use of which increased with decreasing ambient temperature) in winter, and hollow trees and dense vegetation during breeding and raising pups. The pattern of shelter use by raccoon dogs was also strongly modified by forest structure. Raccoon dogs living in the pristine old-growth stands (with large amounts of dead wood) utilized mainly natural shelters created by fallen trees (trunks, branches, and roots) and sedge tussocks (38% and 37%, respectively). Individuals inhabiting the managed part of the Forest used mainly burrows (43%) and thicket (23%). We recorded very strong monogamous relationships between pair mates, manifested by shared resting on 84% of days per year. We conclude that hiding behavior and plasticity in shelter use is a response to high mortality due to predation and to unfavorable climatic conditions. High adaptability to various environmental conditions may be one of the factors underlying successful range expansion of raccoon dogs in Europe.     

Seasonal and spatial pattern of shelter use by badgers<Emphasis Type="Italic">Meles meles</Emphasis> in Białowieża Primeval Forest (Poland)

In 1997–2001, we investigated the use of day-time shelters by radio-collared badgersMeles meles (Linnaeus, 1758) in the Białowieża Primeval Forest, eastern Poland. Each social group of badgers utilised, on average, 9 different shelters per territory (range: 4–20). The main setts, occupied for breeding and winter sleep, were also most frequently used for day-time rest throughout the year (73% of days). Badgers living in the pristine oldgrowth stands utilised larger number of shelters and spent more days in hollow trees (mainly limeTilia cordata), compared to badgers inhabiting younger secondary tree stands. Number of shelters used by individuals varied between seasons and depended on sex and age of animals. In summer, badgers used more shelters than in spring and autumn. In winter, they stayed in their main setts only. Adult males occupied more shelters and spent fewer days in the main sett than other badgers. In spring, females rearing young used only the main setts. The average underground space used by badgers within the main sett was 128 m². It was largest in summer and smallest in winter, and also varied between males and females. We proposed that, in a low-density population, badgers used several setts and other daily shelters to reduce energy expenditure when exploring their large territories and foraging. Furthermore, setts may play a role of marking sites. Analysis of the biogeographical pattern of sett use by European badgers showed that the number of setts used by social groups increased with increasing territory size, whereas the density of setts (n setts/km²) was negatively correlated with territory size. We proposed that different factors could shape the utilisation of setts by badgers in low- and high-density populations.     

Soil and vegetation nutrient response to bison carcasses in Białowieża Primeval Forest,Poland

Ungulate carcasses can have important effects on the surrounding soil and vegetation. The impact of six carcasses of European bison (Bison bonasus) was investigated for the first time in a natural temperate forest (Białowieża, Poland) by measuring soil and plant nutrient concentrations along a gradient extending from the centre of each carcass. Calcium concentration and pH were found to be higher at the centre of the carcass, decreasing towards the periphery. This effect lasted up to 7 years after the death of the animal. The concentration of most nutrients in the soil and plants varied irregularly, suggesting an effect of the carcass at its centre but the absence of a clear pattern of variation along the gradient. Concentrations of NO₃⁻ in the soil differed only at the 1-year old carcass, suggesting a fast turnover of nitrate in temperate forests. Our results show that the effects of large herbivore carcasses on soil and plant nutrient concentrations are not easily detectable in a temperate forest as in more homogeneous habitats, such as tundra and prairie. This may be due to the high activity of scavengers and nutrient recycling in the study area, but it may also be a consequence of a more complex and patchy interaction between nutrient availability and other limiting factors in temperate forests.     

Resting site selection by large herbivores – The case of European bison (Bison bonasus) in Białowieża Primeval Forest

Resting site selection by European bison (Bison bonasus, L., 1758), the largest terrestrial mammal of Europe, was studied in the free-ranging population in Bia?owie?a Primeval Forest (Poland) in 2009–2010. In total, 104 sites of 21 bison (both collared and uncollared) were analysed to determine the most important microhabitat characteristics selected by resting bison during summer and winter and to study the influence of supplementary feeding on resting behaviour of this herbivore. Resting sites were identified on the basis of GPS locations and activity records collected by GPS collars, as well as direct observations of bison, and were compared with control sites. Microhabitat selection by bison did not differ significantly between the sexes. During summer and winter, bison resting sites displayed a high tree density, low visibility and high complexity (structures providing cover). Summer resting sites were also characterised by a significantly lower abundance of blood-sucking insects and denser canopy than control sites. Winter resting sites showed a lower complexity and higher visibility than summer sites, and were less often located in mixed forest habitats. During winter, bison rested more frequently in forest below 50 years of age than in older forest. Resting sites of non-fed bison were more often located in young coniferous forests, were lower in visibility and situated closer to open areas than sites of bison using supplementary winter feeding, suggesting a trade-off between food and cover. The results indicate that European bison select their resting sites in areas of mosaic habitat structure providing cover from disturbances with access to profitable natural forage grounds.     

Howling activity of free-ranging wolves (<Emphasis Type="Italic">Canis lupus</Emphasis>) in the Białowieża Primeval Forest and the Western Beskidy Mountains (Poland)

We investigated spontaneous howling by radio-collared wolves Canis lupus inhabiting the Białowieża Primeval Forest (BPF), eastern Poland, and elicited howling behavior in wolves of the BPF and the Western Beskidy Mountains, southern Poland. Over half (58%) of all spontaneous howls recorded throughout a year occurred in the period from July to October, with a peak in August. The daily pattern of vocal activity by wolves was characterised by a peak between 1800 and 0000 hours, which coincided with the first (dusk) peak of wolf mobility. Wolves howled from the core areas of their territories and not from the peripheries. Howls served as communication between temporarily separated pack mates (43% of cases), after re-union (18%) and before setting out for a hunt (22%). Very few spontaneous howls (2%) were targeted at a neighbouring pack. Wolves responded to human-simulated howling in June–September, with a peak in August (reply rate: 39%). The duration of elicited howling increased significantly with group size: howls by single wolves or pairs lasted, on average, 34–40 s, whereas those of five to seven wolves (including pups) had an average duration of 67–95 s, with a maximum length of nearly 4 min. In the populations of Polish wolves studied here, spontaneous howling served primarily for intra-pack communication. Nonetheless, the high reply rate to howling simulation showed that – if necessary – wolves readily advertised their presence in a territory to strangers.     

Reproductive behaviour of wild-living wolves in Białowieża Primeval Forest (Poland)

Characteristics of the reproductive behaviour of wolves (Canis lupus) were studied by radio-tracking and snow-tracking of four packs in Białowieża Primeval Forest (BPF), Poland, in 1995–1999. Signs of mating occurred between 12 January and 22 March. Parturition occurred between 19 April and 12 May, and the denning period lasted for 49–64 days. During that time, wolves used 1–3 den sites, spending on average 27 days at each site. The dens were never reused in consecutive years, but year after year the breeding sites were located in the same parts of the pack’s territory. Ten days before parturition pregnant females reduced their normal mobility by half (from 23 to 13.5 km day⁻¹, on average). During the ten days post-partum, they spent 85% of their time with pups and travelled 3.9 km day⁻¹, only. On days 11–30 after parturition, females spent 74% of their time tending pups and increased their daily movement distance to a mean of 13.3 km. The females resumed full mobility 50–70 days after parturition, which coincides with termination of the weaning process. Anecdotal observations indicated that pups were tended by other pack members while the mother was absent. Compared with the years 1947–1950, in 1995–1999 the breeding season of wolves in BPF occurred two weeks earlier. A possible reason was the 1 to 1.5-degree increase in the mean annual temperature during the last 50 years.     

The process of a wolf pack splitting in Białowieża Primeval Forest,Poland

In 1998, the pack of 7 wolvesCanis lupus Linnaeus, 1758, radio-tracked in Białowieża Primeval Forest, East Poland, split into 2 packs (2 and 5 wolves), when an 8-year-old alpha female ceased breeding. The two sister-packs subdivided their original territory, but their ranges overlapped extensively (49%) for one year after the split, except for May-June, when both new packs reared pups. We propose that food related factors could have been the ultimate cause of splitting of a large pack. In European temperate forests, pack size of 5–6 wolves is optimal for the consumption of the red deerCervus elaphus.     

Spatial interactions between grey wolves and Eurasian lynx in Białowieża Primeval Forest, Poland

Various species of large predators are reported to influence each other through interference or exploitation competition that may affect demography and survival of the subordinate species. We analyzed spatial relationships between grey wolf (Canis lupus) and Eurasian lynx (Lynx lynx) in Białowieża Primeval Forest (BPF, eastern Poland) to determine how they partitioned the space. The wolves (n = 8) and lynx (n = 14) were radio-tracked in 1991–1999. Three wolves and seven lynx were radio-tracked simultaneously in 1994–1996. Territories of wolf packs and home ranges of lynx overlapped considerably (76% of wolf territories and 50% of lynx home ranges, on average). In three cases, their core areas were also overlapping. Wolf-lynx dyads with overlapping home ranges were simultaneously located at distances from 0 to 28 km from each other. We found neither avoidance nor attraction between wolves and lynx occupying the same areas. We concluded that in BPF, the two large predators coexist due to specialization on different preferred prey and heterogeneous habitat.     

Regeneration of transformed verbena (<Emphasis Type="Italic">Verbena </Emphasis>× <Emphasis Type="Italic">hybrida</Emphasis>) by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

Verbena (Verbena x hybrida), an important floricultural species, was successfully regenerated from stem segments on Murashige and Skoog's basal medium supplemented with thidiazuron and indole-3-acetic acid. A transformation system was developed using cvs. Temari Scarlet, Temari Sakura, Tapien Rose and TP-P2. Agrobacterium tumefaciens strain Agl0 harboring the sGFP gene was infected into stem segments. Transformation efficiency was improved by evaluating and manipulating the age of the plant material, the concentration of kanamycin in the medium during selection, and the length of the culture period in the dark. After 2-3 months of culture on the selection medium, GFP-positive shoots were obtained in all four of the cultivars tested. These shoots were successfully acclimated and set flowers within 2-3 months in a greenhouse. GFP was expressed in all of the organs including the floral parts. Stable genomic transformation was confirmed by Southern blot analysis. No morphological differences were observed between the transformed plants and their host plants.     

Brood cell size of <Emphasis Type="Italic">Apis</Emphasis> <Emphasis Type="Italic">mellifera</Emphasis> modifies the reproductive behavior of <Emphasis Type="Italic">Varroa</Emphasis> <Emphasis Type="Italic">destructor</Emphasis>

We undertook a field study to determine whether comb cell size affects the reproductive behavior of Varroa destructor under natural conditions. We examined the effect of brood cell width on the reproductive behavior of V. destructor in honey bee colonies, under natural conditions. Drone and worker brood combs were sampled from 11 colonies of Apis mellifera. A Pearson correlation test and a Tukey test were used to determine whether mite reproduction rate varied with brood cell width. Generalized additive model analysis showed that infestation rate increased positively and linearly with the width of worker and drone cells. The reproduction rate for viable mother mites was 0.96 viable female descendants per original invading female. No significant correlation was observed between brood cell width and number of offspring of V. destructor. Infertile mother mites were more frequent in narrower brood cells.     

Functional expression of the lipase gene Lip2 of <Emphasis Type="Italic">Pleurotus</Emphasis> <Emphasis Type="Italic">sapidus</Emphasis> in <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis>

The lipase Lip2 of the edible basidiomycete, Pleurotus sapidus, is an extracellular enzyme capable of hydrolysing xanthophyll esters with high efficiency. The gene encoding Lip2 was expressed in Escherichia coli TOP10 using the gene III signal sequence to accumulate proteins in the periplasmatic space. The heterologous expression under control of the araBAD promoter led to the high level production of recombinant protein, mainly as inclusion bodies, but partially in a soluble and active form. A fusion with a C-terminal His tag was used for purification and immunochemical detection of the target protein. This is the first example of a heterologous expression and periplasmatic accumulation of a catalytically active lipase from a basidiomycete fungus.     

Pistil-function breakdown in a new <Emphasis Type="Italic">S</Emphasis>-allele of European pear, <Emphasis Type="Italic">S</Emphasis><Subscript><Emphasis Type="Italic">21</Emphasis></Subscript>°, confers self-compatibility

European pear exhibits RNase-based gametophytic self-incompatibility controlled by the polymorphic S-locus. S-allele diversity of cultivars has been extensively investigated; however, no mutant alleles conferring self-compatibility have been reported. In this study, two European pear cultivars, ‘Abugo’ and ‘Ceremeño’, were classified as self-compatible after fruit/seed setting and pollen tube growth examination. S-genotyping through S-PCR and sequencing identified a new S-RNase allele in the two cultivars, with identical deduced amino acid sequence as S ₂₁ , but differing at the nucleotide level. Test-pollinations and analysis of descendants suggested that the new allele is a self-compatible pistil-mutated variant of S ₂₁ , so it was named S ₂₁ °. S-genotypes assigned to ‘Abugo’ and ‘Ceremeño’ were S ₁₀ S ₂₁ ° and S ₂₁ °S ₂₅ respectively, of which S ₂₅ is a new functional S-allele of European pear. Reciprocal crosses between cultivars bearing S ₂₁ and S ₂₁ ° indicated that both alleles exhibit the same pollen function; however, cultivars bearing S ₂₁ ° had impaired pistil-S function as they failed to reject either S ₂₁ or S ₂₁ ° pollen. RT-PCR analysis showed absence of S ₂₁ °-RNase gene expression in styles of ‘Abugo’ and ‘Ceremeño’, suggesting a possible origin for S ₂₁ ° pistil dysfunction. Two polymorphisms found within the S-RNase genomic region (a retrotransposon insertion within the intron of S ₂₁ ° and indels at the 3′UTR) might explain the different pattern of expression between S ₂₁ and S ₂₁ °. Evaluation of cultivars with unknown S-genotype identified another cultivar ‘Azucar Verde’ bearing S ₂₁ °, and pollen tube growth examination confirmed self-compatibility for this cultivar as well. This is the first report of a mutated S-allele conferring self-compatibility in European pear.     

Sarcoptic mange vulnerability in carnivores of the Białowieża Primeval Forest,Poland: underlying determinant factors

Sarcoptic mange caused by Sarcoptes scabiei, is a highly contagious worldwide mite infection responsible for epizootic skin disease in populations of wild and domestic mammals. It causes skin disorders that may lead to severe generalized skin disease (mange). We investigated the spread and dynamics of sarcoptic mange in one of the best preserved carnivore communities in the Bia?owie?a Primeval Forest over the last 20 years. Sarcoptic mange was found in 6 of 10 investigated species. The highest percentage of infected individuals was recorded in red foxes (19 %), European badgers (9 %), grey wolves (7 %), raccoon dogs (6 %), and stone martens (5 %). Other species were not infected (least weasel, American mink, European polecat, pine marten) or infected sporadically (Eurasian lynx––1 case). Rate of infection was correlated with the sum of ecological and behavioral features, which may increase the spread of infection. Outbreaks of sarcoptic mange occurred simultaneously in several species of carnivores, which indicates the occurrence of between species transmission. We found that the source of investigated individuals (trapped, culled and found dead) may influence the results of the analysis. Our study shows that in carnivore communities such as in Bia?owie?a Forest, numerous species may serve as a reservoir of the pathogen and favor its persistence in the environment. This study indicates that sociality and use of den sites are important factors affecting sarcoptic mange spread and prevalence in wild populations of carnivores.     

Rapid induction of <Emphasis Type="Italic">Agrobacterium</Emphasis> <Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transgenic roots directly from adventitious roots in <Emphasis Type="Italic">Panax</Emphasis> <Emphasis Type="Italic">ginseng</Emphasis>

Root segments from seedlings of Panax ginseng produced adventitious roots directly when cultured on 1/2 MS solid medium lacking NH₄NO₃ and containing 3.0 mg l⁻¹ IBA. Using this adventitious root formation, we developed rapid and efficient transgenic root formation directly from adventitious root segments in P. ginseng. Root segments were co-cultivated with Agrobacterium tumefaciens (GV3101) caring β-glucuronidase (GUS) gene. Putative transgenic adventitious roots were formed directly from root segments on medium with 400 mg l⁻¹ cefotaxime and 50 mg l⁻¹ kanamycin. Kanamycin resistant adventitious roots were selected and proliferated as individual lines by subculturing on medium with 300 mg l⁻¹ cefotaxime and 50 mg l⁻¹ kanamycin at two weeks subculture interval. Frequency of transient and stable expression of GUS gene was enhanced by acetosyringon (50 mg l⁻¹) treatment. Integration of transgene into the plants was confirmed by the X-gluc reaction, PCR and Southern analysis. Production of transgenic plants was achieved via somatic embryogenesis from the embryogenic callus derived from independent lines of adventitious roots. The protocol for rapid induction of transgenic adventitious roots directly from adventitious roots can be applied for a new Agrobacterium tumefaciens-mediated genetic transformation protocol in P. ginseng.     

<Emphasis Type="Italic">Carex</Emphasis> × <Emphasis Type="Italic">involuta</Emphasis> and <Emphasis Type="Italic">Carex juncella</Emphasis> in the flora of Slovakia

The paper brings information on an isolated occurrence and morphological characters of Carex × involuta and C. juncella populations in the Vel’ká Fatra Mts. Their presence has been known neither from the territory of Slovakia nor from the whole Western Carpathians till now.     

Gill cell toxicity of northern boreal scyphomedusae <Emphasis Type="Italic">Cyanea</Emphasis> <Emphasis Type="Italic">capillata</Emphasis> and <Emphasis Type="Italic">Aurelia</Emphasis> <Emphasis Type="Italic">aurita</Emphasis> measured by an in vitro cell assay

Scyphozoan medusae are very successful foragers which occasionally occur in high abundances in boreal waters and may impact many different groups in the marine ecosystem by means of a variety of toxins. A rainbow trout gill cell line, RTgill-W1, was tested for its suitability as quantitative indicator of the cytotoxicity of Cyanea capillata and Aurelia aurita; the major scyphozoan species in the North and Baltic seas. Cultures of rainbow trout gill cells were exposed to whole venoms extracted from fishing tentacles and oral arms at increasing protein concentrations. The venom caused detachment, clumping and lysis of cells, as well as a drop in vitality, in a dose-dependent manner. Morphological changes in the cells were evident within 1 h after venom addition. The damage to gill cells was quantified by measuring the metabolic activity of the cells by means of the fluorescence of resorufin derived from the nonfluorescent substrate, resazurin. In general, a decrease in the metabolic activity of the cells was detected at a venom (protein) concentration above 2.0 μg ml⁻¹ (corresponding to 0.2 μg 10⁴ cells⁻¹), and a total loss of activity was observed above 40.0 μg ml⁻¹ (corresponding to 4.0 μg 10⁴ cells⁻¹). C. capillata venoms had increased cytotoxic activity as compared to A. aurita venoms at the same concentration. Cnidocyst extracts from oral arms of A. aurita induced an 85% loss of gill cell viability at concentrations of 0.2 μg 10⁴ cells⁻¹, whereas crude venoms from fishing tentacles reduced cell viability by 18% at the same concentration. Gel electrophoresis of the venoms indicated that these consist of a large number of proteins in a fairly wide size range, from 6 to 200 kDa, including some that are the same size as those found in cubomedusae. It also appears that larger (i.e., older) medusae have more complex venoms and, in some cases, more potent venoms than smaller animals.     

Stereoselective Inhibition of Cholesterol Esterase by Enantiomers of <Emphasis Type="Italic">exo</Emphasis>- and <Emphasis Type="Italic">endo</Emphasis>-2-Norbornyl-<Emphasis Type="Italic">N</Emphasis>–<Emphasis Type="Italic">n</Emphasis>-butylcarbamates

Four stereoisomers of 2-norbornyl-N–n-butylcarbamates are characterized as the pseudo substrate inhibitors of cholesterol esterase. Cholesterol esterase shows enantioselective inhibition for enantiomers of exo- and endo-2-norbornyl-N–n-butylcarbamates. For the inhibitions by (R)-(+)- and (S)-(−)-exo-2-norbornyl-N–n-butylcarbamates, the R-enantiomer is 6.8 times more potent than the S-enantiomer. For the inhibitions by (R)-(+)- and (S)-(−)-endo-2-norbornyl-N–n-butyl-carbamates, the S-enantiomer is 4.6 times more potent than the R-enantiomer. The enzyme-inhibitor complex models have been proposed to explain these different enantioselectivities.     

Carbohydrate and Ethane Release with <Emphasis Type="Italic">Erwinia carotovora</Emphasis> Subspecies <Emphasis Type="Italic">betavasculorum</Emphasis><Emphasis Type="Italic">–</Emphasis>Induced Necrosis

Erwinia carotovora subspecies betavasculorum, also known as E. betavasculorum and Pectobacterium betavasculorum, is a soil bacterium that has the capacity to cause root rot necrosis of sugarbeets. The qualitatively different pathogenicity exhibited by the virulent E. carotovora strain and two avirulent strains, a Citrobacter sp. and an Enterobacter cloacae, was examined using digital analysis of photographic evidence of necrosis as well as for carbohydrate, ethane, and ethylene release compared with uninoculated potato tuber slices. Visual scoring of necrosis was superior to digital analysis of photographs. The release of carbohydrates and ethane from potato tuber slices inoculated with the soft rot necrosis-causing Erwinia was significantly greater than that of potato tuber slices that had not been inoculated or that had been inoculated with the nonpathogenic E. cloacae and Citrobacter sp. strains. Interestingly, ethylene production from potato slices left uninoculated or inoculated with the nonpathogenic Citrobacter strain was 5- to 10-fold higher than with potato slices inoculated with the pathogenic Erwinia strain. These findings suggest that (1) carbohydrate release might be a useful measure of the degree of pathogenesis, or relative virulence; and that (2) bacterial suppression of ethylene formation may be a critical step in root rot disease formation.