The effect of earthworms (Lumbricidae) on nitrogenase activity in soil

Summary Nitrogenase activity associated with earthworms, their faeces and activity in soil was measured by the acetylene reduction technique. A clear increase in nitrogenase activity was found in field-deposited casts of Aporrectodea caliginosa in comparison with surrounding soil, although potential nitrogenase activity was significantly higher in soil than in casts. Nitrogenase activity associated directly with earthworms (Lumbricus rubellus ) was detected, indicating the presence of active N₂-fixing bacteria on the body surface and/or in the gut. Laboratory experiments showed that nitrogenase activity in the casts of L. rubellus was higher than in unmodified soil, and that nitrogenase activity in soil was significantly increased by the burrowing and feeding activity of these worms. This paper discusses the possible causes of these earthworm effects on soil nitrogenase activity and some methodological problems of determining the nitrogenase activity.     

Synthesis of Novel Thin-Film Materials by Pulsed Laser Deposition

Pulsed laser deposition (PLD) is a conceptually and experimentally simple yet highly versatile tool for thin-film and multilayer research. Its advantages for the film growth of oxides and other chemically complex materials include stoichiometric transfer, growth from an energetic beam, reactive deposition, and inherent simplicity for the growth of multilayered structures. With the use of PLD, artificially layered materials and metastable phases have been created and their properties varied by control of the layer thicknesses. In situ monitoring techniques have provided information about the role of energetic species in the formation of ultrahard phases and in the doping of semiconductors. Cluster-assembled nanocrystalline and composite films offer opportunities to control and produce new combinations of properties with PLD.     

Macroinvertebrate Community and Chemistry of the Most Atmospherically Acidified Streams in the Czech Republic

We performed a comparative study of strongly acidified mountain streams covering 10 sites in the Czech Republic in the season 1999/2000. The aim of the study was to determine how acidification influenced macroinvertebrate community structure within a relatively narrow pH range. We focused on strongly acidified, non-humic running waters with low ionic content (pH < 4.6, total organic carbon < 10 mg l^?1, specific conductivity < 100 μS cm^?1) and minimum human influence in the catchment. The actual pH values ranged from 3.98 to 4.65, and concentrations of reactive aluminium ranged from 0.2 to 2.0 mg l^?1. Characteristic macroinvertebrates were the stoneflies Leuctra nigra, Nemurella pictetii, and Protonemura spp.; the chironomids Corynoneura spp.; and the caddisfly Plectrocnemia conspersa – the former two stoneflies formed together 46% of total organisms. The sites were divided into three groups with use of divisive classification. Individual groups reflected similarity in water chemistry, catchment characteristics, and geographical proximity. The largest biotic difference detected by PCA was in the abundance of stoneflies, mainly Diura bicaudata and Leuctra major. The strongest correlation with this gradient was shown by pH (and associated heavy metals), followed by the distance from source. The results show that even in such narrow pH range, the number of taxa is determined by the low pH value and related high concentrations of aluminium and heavy metals.     

Leukocytic responses and intestinal mucin dynamics of broilers protected with Enterococcus faecium EF55 and challenged with Salmonella Enteritidis

The protective effect of Enterococcus faecium EF55 in chickens challenged with Salmonella enterica serovar Enteritidis phage type 4 (SE PT4) was assessed. The antibacterial effect on the bacterial microflora in the small intestine in relation to white blood cell count, phenotyping of peripheral blood and intestinal lymphocytes, functional activity of lymphocytes and phagocytes and mucin quantitation were investigated. Day-old chicks (85) were randomly divided into four groups. The probiotic group (EF) and Salmonella + probiotic group (EFSE) received E. faecium EF55 (10⁹ CFU – 3 g/group/day) for 21 days. The Salmonella group (SE) and EFSE group were infected with Salmonella Enteritidis (10⁸ CFU in 0.2 ml PBS) in a single dose per os on day four of the experiment. The control group chicks (C) were fed a commercial diet without added bacteria. Supplementation of EF55 in the diet of the chickens in the EFSE group, challenged with S. Enteritidis, caused the density of the intestinal mucin layer to increase significantly in non-specific regions (duodenum and jejunum), but decrease significantly in target regions (caeca) for S. Enteritidis. Probiotic treatment also appeared to result in a significantly higher number of lymphocytes in peripheral blood and a tendency to increase CD3, CD4, CD8, and IgM positive cells 3 days post-infection with S. Enteritidis. The results demonstrated an antibacterial effect and suggested that EF55 had a moderating effect on intestinal mucin production and leukocytic response in the early phase of S. Enteritidis infection.     

High throughput phenotyping for aphid resistance in large plant collections

ABSTRACT: BACKGROUND: Phloem-feeding insects are among the most devastating pests worldwide. They not only cause damage by feeding from the phloem, thereby depleting the plant from photo-assimilates, but also by vectoring viruses. Until now, the main way to prevent such problems is the frequent use of insecticides. Applying resistant varieties would be a more environmental friendly and sustainable solution. For this, resistant sources need to be identified first. Up to now there were no methods suitable for high throughput phenotyping of plant germplasm to identify sources of resistance towards phloem-feeding insects. RESULTS: In this paper we present a high throughput screening system to identify plants with an increased resistance against aphids. Its versatility is demonstrated using an Arabidopsis thaliana activation tag mutant line collection. This system consists of the green peach aphid Myzus persicae (Sulzer) and the circulative virus Turnip yellows virus (TuYV). In an initial screening, with one plant representing one mutant line, 13 virus-free mutant lines were identified by ELISA. Using seeds produced from these lines, the putative candidates were re-evaluated and characterized, resulting in nine lines with increased resistance towards the aphid. CONCLUSIONS: This M. persicae-TuYV screening system is an efficient, reliable and quick procedure to identify among thousands of mutated lines those resistant to aphids. In our study, nine mutant lines with increased resistance against the aphid were selected among 5160 mutant lines in just 5 months by one person. The system can be extended to other phloem-feeding insects and circulative viruses to identify insect resistant sources from several collections, including for example genebanks and artificially prepared mutant collections.     

The pre-emptive effect of epidural ketamine on wound sensitivity in horses tested by using von Frey filaments

Objective To evaluate the pre‐emptive analgesic effect of pre‐incisional epidural ketamine. Study Design A blinded, randomized experimental study. Animals Sixteen mixed breed mares, 7.6 ± 2.8 years old, weighing 352 ± 32 kg. Methods In a pilot study, an incision was made on one lateral thigh using a lidocaine block and no further analgesics, and it was verified that the nociceptive threshold was lower on the incised side than nonincised side (p ≤ 0.05), and that von Frey filaments evoked a pain response. The 16 animals were divided into group A (ketamine, n = 9) and B (saline, n = 7). An epidural catheter was inserted 24 hours before the trials. The thigh was shaved bilaterally, and the right side was blocked (incised side) using lidocaine. Twenty‐five minutes later, ketamine (A) or saline (B) was administered epidurally. Five minutes later, a 10‐cm skin incision was made on the right side, and then sutured. Nociceptive threshold was determined with von Frey filaments at 1, 3, and 5 cm around the incision at 15‐minute intervals for 2 hours, then at 4, 6, and 8 hours. Behavioral alterations, heart and respiratory rates were recorded. Nociceptive thresholds from these points were averaged to obtain mean values at each time, converted to a logarithmic scale, and submitted to a nonparametric analysis (Mann–Whitney and one‐way repeated measures anova test, p ≤ 0.05). Results After 8 hours, the global range score revealed reduced hyperalgesia (p < 0.01) around the incision in 92% (4.65–4.27) of evaluated intervals in group A (ketamine). There were no significant changes in behavior, heart and respiratory rates. Conclusions It was concluded that pre‐emptive epidural ketamine reduced post‐incisional pain in the horse, and that von Frey filaments were able to quantify cutaneous sensitivity after tissue damage. Clinical relevance Epidural ketamine injection can reduce post‐incisional sensitivity in the horse.     

Chloroplast DNA polymorphism of an exotic P. mugo Turra population introduced to seaside spit of Kursiu Nerija in Lithuania

The aim of this study was to elucidate the introduction history of P. mugo in the unique landscape of the Lithuanian seaside spit of Kursiu Nerija by assessing its genetic structure and the genetic diversity. The individuals were sampled in 12 populations within an area of 3 km × 50 km along the Lithuanian part of Kursiu Nerija. P. mugo was introduced over 200 years ago to prevent sand erosion by establishing a forest cover. Chloroplast DNA polymorphism of 220 individuals of P. mugo together with 18 P. sylvestris and 11 putative P. sylvestris × P. mugo hybrids was assessed by the aid of five microsatellite markers. The standard intra-population diversity indexes were calculated. The intra-specific variation between distinct morphotypes as well as the population differentiation within the most spread P. mugo ssp. rotundata morphotype was assessed based on the haplotype frequencies by hierarchical AMOVA, G_ST/R_ST test, UPGMA clustering and PCA methods. The genetic diversity of P. mugo in Kursiu Nerija was high (H_e = 0.95; 83 different haplotypes). All except one of the P. mugo populations sampled contained a notable share of private haplotypes. AMOVA revealed high intra-specific diversity but low differentiation between the P. mugo populations. Most of the haplotypic variance was within populations. The UPGMA clustering produced groups more corresponding to the sub-species morphotypes than the geography of the populations. There was no geographical pattern of reduction in genetic diversity towards the younger plantations. A strong candidate for a species-specific DNA marker was found. After several events of introduction, the genetic diversity of P. mugo in Kursiu Nerija is very high and is structured based on the sub-species morphotypes rather than geography. The high frequency of shared and notable frequency of private haplotypes in most of the populations indicate that the major part of the P. mugo material originates from a number of geographically and genetically related sources, which more likely are introductions from abroad that the local collections. The high frequency of private haplotypes in the northernmost populations leaves a possibility for minor introductions from other genetically distinct sources. The absence of private haplotypes in one of the sampled populations indicates the use of local seed collections. The large number of shared haplotypes provides a strong evidence for a geneflow among the P. mugo taxa.     

Postpartum Variations of Plasma IGF and IGFBPs,Oocyte Production and Quality in Dairy Cows: Relationships With Parity and Subsequent Fertility

The aim of this study was to determine whether postpartum variations of plasma IGF‐1 and IGFBP concentrations, oocyte production and quality were related to parity and subsequent conception rate in Holstein dairy cows. Holstein dairy cows [10 primiparous (PP) and 22 multiparous (MP)] were allotted in six batches and sampled once weekly between calving and oestrous synchronization treatment started at 71.2 ± 2.0 days postpartum. During the 3 weeks before treatment, ovum pick‐up (OPU) was performed twice weekly. Oocytes were scored on a 4‐point scale, and oocytes from OPU1, 3 and 5 were fertilized in vitro. Seventeen cows became pregnant after first and second AI and were considered as fertile (F), while the others were considered to be subfertile (SF). Logistic regression was carried out to investigate the relationships between repeated measurements and fertility including parity and batch effects in the models. Likelihood of fertility significantly increased when plasma urea and IGFBP‐3 concentrations decreased and was higher in PP compared with MP cows. There was a trend for fertility to increase when plasma IGF‐1 concentrations increased (p = 0.07). In vitro cleavage and development rates were similar between SF and F cows (46.4% and 28.3% in SF vs 55.0% and 22.1% in F). Parity had an effect on plasma IGF‐1 concentrations (PP: 61.65 ± 2.67 vs MP: 41.63 ± 5.81 ng/ml, p < 0.001), mean number of follicles aspirated per session (PP: 5.7 ± 1.3 vs MP: 9.5 ± 0.8, p < 0.05) and fertility (PP: 8/10 = 80% vs MP: 9/22 = 41%, p < 0.05) but not on the number of oocytes recovered per session nor their quality. In conclusion, postpartum plasma urea and IGFBP‐3 concentrations, but not oocyte production and quality before breeding, were related to subsequent conception rate in our experimental design. Parity had a significant effect on energy status, follicular growth and fertility and needs to be considered when investigating relationships between nutrition and reproduction.     

Morphological characteristics of blood cells in monitor lizards: is erythrocyte size linked to actual body size?

Blood cell morphology and count are not uniform across species. Recently, between‐species comparisons revealed that the size of red blood cells is associated with body size in some lizard taxa, and this finding was interpreted in the context of the metabolic theory. In the present study, we examined the numbers and the size of blood cells in 2 species of monitor lizards, the mangrove‐dwelling monitor (Varanus indicus) and the savannah monitor (V. exanthematicus), and we compared these traits in individuals of different body size. The results revealed that during the course of ontogeny, the size of red blood cells increases with body mass. Because the mass‐specific metabolic rate decreases with body size and the cell volume‐to‐surface ratio decreases with the cell size, changes in the erythrocyte size might be the result of oxygen transport adjustment.     

Genotyping of Bacillus anthracis Isolated from Croatia and Bosnia and Herzegovina

Anthrax is a serious disease caused by Bacillus anthracis. Humans can become infected by handling products from infected animals, by breathing spores and rarely by eating undercooked meat from infected animals. The genome of B. anthracis is highly monomorphic and thus shows very low DNA sequence variation. We analysed the molecular characteristics of 12 B. anthracis isolates from outbreaks in Croatia and Bosnia and Herzegovina, which have occurred during the past 10 years along with two vaccine strains. Genotyping system based on variable‐number tandem repeat analysis at six loci revealed that six isolates belong to genotype from the A1.a cluster whilst six isolates relate to the B2 cluster, compared to 89 previously described genotypes. The distribution of two evolutionarily distant clusters suggests an introduction of B. anthracis to this area in at least two separate events.