Quantifying the damage of red deer (Cervus elaphus) grazing on grassland production in southeastern Slovenia

The influence of red deer (Cervus elaphus) grazing on grassland production for forage conservation at the forest border during the vegetation period was studied on three locations (Mala gora, Cvišlerji, and Mačkovec) in the Kočevje region (SE Slovenia). The experiment lasted from the 25th of March until the 8th of October 2002. Portable cages were used to exclude red deer from grazing the herbage. At four sampling dates in the season, herbage air dry matter (DM) yield was measured at three different observations (cage-protected plot, cage-protected plot only two to three weeks before sampling date, otherwise freely grazed (removed), and unprotected plot). The results from the experiment showed that red deer grazed on grassland through the entire season and that the regeneration capability of sward was the highest in summer, middle in spring and smallest in autumn. On unprotected plots, an average reduction of 50% of herbage DM yield was found with the most damaged sites also up to 80% reduction.     

Presence of a unique male-specific extension of C-terminus to the cytochrome c oxidase subunit II protein coded by the male-transmitted mitochondrial genome of Venustaconcha ellipsiformis (Bivalvia: Unionoidea)

Analyses of unionoidean bivalve male-transmitted (M) mtDNA genomes revealed an approximately 555 bp 3' coding extension to cox2. An antibody was generated against this predicted C-terminus extension to determine if the unique cox2 protein is expressed. Western blot and immunohistochemistry analyses demonstrated that the protein was predominantly expressed in testes. Weak expression was detected in other male tissues but the protein was not detected in female tissues. This is the first report documenting the expression of a cox2 protein with a long C-terminus in animals. Its universal presence in unionoidean bivalve testes suggests a functional significance for the protein.     

Extra‐mitochondrial localization and likely reproductive function of a female‐transmitted cytochrome c oxidase subunit II protein

Our previous study documented a reproductive function for the male‐transmitted mitochondrial DNA (mtDNA)‐encoded cytochrome c oxidase subunit II (MCOX2) protein in a unionoid bivalve. Here, immunoblotting, immunohistochemistry and immunoelectron microscopy analyses demonstrate that the female‐transmitted protein (FCOX2) is: (i) expressed in both male and female gonads; (ii) maximally expressed in ovaries just prior to the time of the annual fertilization event; (iii) displayed in the cytoplasm and more strongly in the plasma membrane (microvilli), vitelline matrix and vitelline envelope of mature ovarian eggs; and (iv) strongly localized to the vitelline matrix of some eggs just prior to fertilization. These findings represent evidence for the extra‐mitochondrial localization of an mtDNA‐encoded gene product and are consistent with multifunctionality for FCOX2 in eggs.     

Investigation on the optimal term of one-fold insecticide application for decreasing onion thrips ( Thrips tabaci Lindeman, Thysanoptera, Thripidae) damage on early white cabbage

An impact of the term of one-fold insecticide application to reduce the damage of the onion thrips in early white cabbage was established. During the growing season plants were treated with insecticide (abamectin) in three different terms (treatment 1: 9 June, treatment 2: 16 June, treatment 3: 23 June, and treatment 4: untreated). On the exterior leaves of cabbage heads, treated with the insecticide, statistically significantly lower mean index of damage was determined as compared to the untreated plants. No significant differences were found between three different terms of application. The majority of the economically important damages in all of the treatments was found between the 3rd and 6th exterior leaf in the head. The highest mean weight of heads (1517.3 g) and mean net weight of heads I = mean weight of heads - weight of damaged and removed leavesl (1166.3 g) were established in plants which were treated the last. These parameters were the lowest in untreated plants (1083.3 g / 805.6 g). The yield loss due to damaged leaves removal amounted from 22.9% (treatment 2) to 25.6% (treatment 4). Based on the results obtained in this research we concluded that yield loss due to onion thrips attack in plants with one-fold insecticide application is not substantially lower as in untreated plants (though statistically significant differences between them were established), because insecticide cannot reach the interior leaves in the head. Substantial differences in the total and net weight of heads between the treatments and especially between the treated and untreated plants are explained by the fact that feeding of numerous thrips populations in the heads and on the exterior cabbage leaves negatively affects plant physiology and yield. The highest average yield in plants which were treated the last indicates a possibility that insecticide also inhibits plant growth and development to a certain extent.     

Determination of raloxifene and its glucuronides in human urine by liquid chromatography-tandem mass spectrometry assay

A selective, sensitive, accurate and precise liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for determination of raloxifene and its three glucuronides: raloxifene-6-β-glucuronide (M1), raloxifene-4'-β-glucuronide (M2), raloxifene-6,4'-diglucuronide (M3) in urine samples is presented in this paper. To our knowledge the developed analytical method is the first fully validated method capable of simultaneous determination of raloxifene and its glucuronides in real urine samples. Moreover, for the first time a method for determination of raloxifene diglucuronide in relevant biological samples was introduced. Metabolites were obtained by a bioconversion process of raloxifene to its glucuronides using the microorganism Streptomyces sp. and were used as standards for validation. Urine samples were introduced to a simple solid phase extraction prior to the analysis by LC-MS/MS. The method was linear in a wide range with high determination coefficient (r(2) > 0.997). The limits of quantification achieved were 1.01, 1.95, 2.83 and 4.69 nM for raloxifene, M1, M2 and M3, respectively. The recoveries were higher than 92.5%, the accuracy was within 100 ± 8.8% and the precision was better than 12% for all compounds. The developed method was successfully applied to the real urine samples and showed to be appropriate for use in further research of still not completely discovered raloxifene pharmacokinetics. Furthermore, the presented method could also serve for a potential application in anti-doping analysis.     

Efficacy of Three Natural Chemicals to Reduce the Damage of Erysiphe cichoracearum on Chicory in Two Meteorologically Different Growing Seasons

In two meteorologically quite different years, 2002 and 2003, efficacy of three natural substances with a fungicidal activity (sulphur, soya lecithin, salicylic acid) for control of Erysiphe cichoracearum on chicory was tested. In a block designed field experiment, there were statistically significant differences in the mean index of infection of exterior leaves, in the mean total weight and mean net weight of the plants (= total weight − weight of removed damaged leaves). There was also a significant difference in firmness of the heads of nine chicory varieties that was established in both years. In 2002, when no extreme weather conditions occurred, the fungus spread to a substantially larger extent. Yet no statistically significant differences were found in the efficacy of environmentally friendly fungicidal substances for control of the fungus. In the hotter and drier year of 2003, which was less favourable for spread of the pathogen, sulphur‐containing fungicide was the most effective. In 2002, chicory yield was lower than in 2003. Generally, yield loss due to removal of damaged leaves was from 30.7 to 45.0% in the first year, and from 11.3 to 24.8% in the second. The highest mean total yield and net yield of chicory were in plants, which were treated three times with sulphur‐containing fungicide. In 2002, the most firm heads were in plants treated with sulphur. In 2003, no significant differences among substances were detected. The most infected with the powdery mildew caused by E. cichoracearum, were the less productive varieties of chicory. A statistically significant relationship was not found between the firmness of the heads and the degree of infection. Among different parameters in this experiment, the only relevant and high statistically significant positive correlation (r² = 0.71) was determined between the total weight of the plants and yield loss of chicory due to infection with the fungus in question.     

Novel protein genes in animal mtDNA: a new sex determination system in freshwater mussels (Bivalvia: Unionoida)?

Mitochondrial (mt) function depends critically on optimal interactions between components encoded by mt and nuclear DNAs. mitochondrial DNA (mtDNA) inheritance (SMI) is thought to have evolved in animal species to maintain mito-nuclear complementarity by preventing the spread of selfish mt elements thus typically rendering mtDNA heteroplasmy evolutionarily ephemeral. Here, we show that mtDNA intraorganismal heteroplasmy can have deterministic underpinnings and persist for hundreds of millions of years. We demonstrate that the only exception to SMI in the animal kingdom, that is, the doubly uniparental mtDNA inheritance system in bivalves, with its three-way interactions among egg mt-, sperm mt- and nucleus-encoded gene products, is tightly associated with the maintenance of separate male and female sexes (dioecy) in freshwater mussels. Specifically, this mother-through-daughter and father-through-son mtDNA inheritance system, containing highly differentiated mt genomes, is found in all dioecious freshwater mussel species. Conversely, all hermaphroditic species lack the paternally transmitted mtDNA (=possess SMI) and have heterogeneous macromutations in the recently discovered, novel protein-coding gene (F-orf) in their maternally transmitted mt genomes. Using immunoelectron microscopy, we have localized the F-open reading frame (ORF) protein, likely involved in specifying separate sexes, in mitochondria and in the nucleus. Our results support the hypothesis that proteins coded by the highly divergent maternally and paternally transmitted mt genomes could be directly involved in sex determination in freshwater mussels. Concomitantly, our study demonstrates novel features for animal mt genomes: the existence of additional, lineage-specific, mtDNA-encoded proteins with functional significance and the involvement of mtDNA-encoded proteins in extra-mt functions. Our results open new avenues for the identification, characterization, and functional analyses of ORFs in the intergenic regions, previously defined as "noncoding," found in a large proportion of animal mt genomes.     

In vitro bioactivation of bazedoxifene and 2-(4-hydroxyphenyl)-3-methyl-1H-indol-5-ol in human liver microsomes

Bazedoxifene is a selective estrogen receptor modulator (SERM) that has been developed for use in post-menopausal osteoporosis. However, it contains a potentially toxic 5-hydroxy-3-methylindole moiety. Previous studies on the 5-hydroxyindole and the 3-alkylindole-containing drugs indometacine, zafirlukast and MK-0524 structural analogs have shown that they are bioactivated by cytochrome P450s through a dehydrogenation process to form quinoneimine or 3-methyleneindolenine electrophilic species. In the present study, bazedoxifene was synthesized and then evaluated, together with raloxifene and 2-(4-hydroxyphenyl)-3-methyl-1H-indol-5-ol (13), a 3-methyl-5-hydroxyindole-based structural fragment of bazedoxifene, for its ability to form reactive electrophilic species when incubated with human liver microsomes (HLMs) or recombinant CYP isozymes. We showed that bazedoxifene was bioactivated only in trace amounts with recombinant CYP isozymes. In contrast, the N-dealkylated fragment of bazedoxifene (2-(4-hydroxyphenyl)-3-methyl-1H-indol-5-ol) was bioactivated in considerable amounts to an electrophilic intermediate, which was trapped with glutathione and identified by LC-MS/MS. This suggests that bazedoxifene would require initial N-dealkylation, which could subsequently lead to the formation of the reactive intermediate. However, such an N-dealkylated metabolite of bazedoxifene was not detected after the incubation of bazedoxifene in HLM or recombinant CYP isozymes.     

Population dynamics of banded thrips (Aeolothrips intermedius Bagnall, Thysanoptera, Aeolothripidae) and its potential prey Thysanoptera species on white clover

In 2002, the occurrence of banded thrips (Aeolothrips intermedius Bagnall) and some other Thysanoptera species on white clover (Trifolium repens L.) was monitored at two locations in the continental part of Slovenia. White clover presents in many countries important intercrop in integrated vegetable production. Light blue sticky boards were placed on grasslands (one parcel on each location) with high percentage of white clover. Sticky boards were changed in about 10-days intervals from the end of April till the beginning of October. Number of caught individuals on the boards was counted. They were classified in three different groups: 1. Aeolothrips intermedius, 2. representatives of Haplothrips, Odontothrips and Frankliniella genera, 3. representatives of Thrips genus. We stated that, compared with the other Thysanoptera species in the open, predatory thrips occurs in lower number. Predatory species Aeolothrips intermedius was the most numerous during the flowering of white clover. It was established that other Thysanoptera species (the most of them are facultative phytophagous species) were more numerous also in the periods of less favourable weather conditions and during the non-flowering growing stages of white clover. Based on the results of present research we concluded that A. intermedius has a potential to control onion thrips (Thrips tabaci Lindeman), especially in July and August, when in the open both species occur in high numbers.     

Effect of Cannabis sativa L. root,leaf and inflorescence ethanol extracts on the chemotrophic response of entomopathogenic nematodes

Plant and Soil - Soils represent the natural habitat of entomopathogenic nematodes (EPNs). When moving in soil, EPNs are oriented to follow a chemical signal (chemotaxis). Cannabis sativa L. is...