Metagenomics to paleogenomics: large-scale sequencing of mammoth DNA

We sequenced 28 million base pairs of DNA in a metagenomics approach, using a woolly mammoth (Mammuthus primigenius) sample from Siberia. As a result of exceptional sample preservation and the use of a recently developed emulsion polymerase chain reaction and pyrosequencing technique, 13 million base pairs (45.4%) of the sequencing reads were identified as mammoth DNA. Sequence identity between our data and African elephant (Loxodonta africana) was 98.55%, consistent with a paleontologically based divergence date of 5 to 6 million years. The sample includes a surprisingly small diversity of environmental DNAs. The high percentage of endogenous DNA recoverable from this single mammoth would allow for completion of its genome, unleashing the field of paleogenomics.     

Prevalence and severity of foot lesions in Danish Holstein heifers through first lactation

Several studies have shown that foot lesions and clinical lameness occur before first calving and develop further during the lactation period. Lameness may cause production losses, but the relationship between foot lesions, particularly in the claw horn, and lameness in heifers is unclear. The objectives of this study were to describe the development of and evaluate the relationship between lameness and foot lesions in Danish Holsteins before and after first calving. In a longitudinal study, 147 heifers were examined for lameness and foot lesions 2–5 times over an 18-month period. Lameness was assessed by means of a visual locomotion score and foot lesion severity was recorded.The prevalence of a locomotion score 3 was 25% before calving, and 90% at approximately 250 days in milk (DIM). Prevalence of moderate to severe sole haemorrhage (SH) was 27% before calving and 56% at 250 DIM, and that of moderate to severe white line lesion (WLL) 44% before calving with a peak of 70% at 200 DIM. There was one case of white line abscess but SH was seen throughout the entire study period. Digital dermatitis (DD) was prevalent prior to first calving (15%) and peaked at 39% at 0–100 DIM. Heel horn erosion (HHE) occurred in almost all cows (93–100%) and was strongly correlated with DD (r = 0.51). The correlation coefficient between SH and WLL was also high (0.42). The relatively high correlations between WLL and both DD and HHE were more surprising (0.38 and 0.35, respectively), those between SH and both DD and HHE were moderate (around 0.18). Interdigital dermatitis was significantly correlated with both HHE and DD, but completely unrelated to SH and WLL.The overall average locomotion score increased by about one-half of a score unit from 1 month prior to calving until 250 DIM, with a large difference between herds, although this was unsurprising as cows may alter their locomotion pattern with management factors (e.g. floor properties). DD and WLL were both associated with a locomotion score 3 but of the cows with severe WLL there was no clear association between a locomotion score 3 and DD. The highest locomotion scores occurred among cows with DD but without WLL.     

Adenylate cyclases of Trypanosoma brucei inhibit the innate immune response of the host

The parasite Trypanosoma brucei possesses a large family of transmembrane receptor-like adenylate cyclases. Activation of these enzymes requires the dimerization of the catalytic domain and typically occurs under stress. Using a dominant-negative strategy, we found that reducing adenylate cyclase activity by about 50% allowed trypanosome growth but reduced the parasite's ability to control the early innate immune defense of the host. Specifically, activation of trypanosome adenylate cyclase resulting from parasite phagocytosis by liver myeloid cells inhibited the synthesis of the trypanosome-controlling cytokine tumor necrosis factor-α through activation of protein kinase A in these cells. Thus, adenylate cyclase activity of lyzed trypanosomes favors early host colonization by live parasites. The role of adenylate cyclases at the host-parasite interface could explain the expansion and polymorphism of this gene family.     

Biodegradation of the herbicide mecoprop-p with soil depth and its relationship with class III tfdA genes

Mecoprop-p [(R)-2-(4-chloro-2-methylphenoxy) propanoic acid) is widely used in agriculture and poses an environmental concern because of its susceptibility to leach from soil to water. We investigated the effect of soil depth on mecoprop-p biodegradation and its relationship with the number and diversity of tfdA related genes, which are the most widely known genes involved in degradation of the phenoxyalkanoic acid group of herbicides by bacteria. Mecoprop-p half-life (DT₅₀) was approximately 12 days in soil sampled from <30 cm depth, and increased progressively with soil depth, reaching over 84 days at 70-80 cm. In sub-soil there was a lag period of between 23 and 34 days prior to a phase of rapid degradation. No lag phase occurred in top-soil samples prior to the onset of degradation. The maximum degradation rate was the same in top-soil and sub-soil samples. Although diverse tfdAα and tfdA genes were present prior to mecoprop-p degradation, real time PCR revealed that degradation was associated with proliferation of tfdA genes. The number of tfdA genes and the most probable number of mecoprop-p degrading organisms in soil prior to mecoprop-p addition were below the limit of quantification and detection respectively. Melting curves from the real time PCR analysis showed that prior to mecoprop-p degradation both class I and class III tfdA genes were present in top- and sub-soil samples. However at all soil depths only tfdA class III genes proliferated during degradation. Denaturing gradient gel electrophoresis confirmed that class III tfdA genes were associated with mecoprop-p degradation. Degradation was not associated with the induction of novel tfdA genes in top- or sub-soil samples, and there were no apparent differences in tfdA gene diversity with soil depth prior to or following degradation.     

Prey-predator dynamics in communities of culturable soil bacteria and protozoa: differential effects of mercury

We investigated whether the prey-predator dynamics of bacteria and protozoa were affected by inorganic mercury at concentrations of 0, 3.5 and 15 mg Hg(II) kg soil⁻¹. The amount of bioavailable Hg was estimated using a biosensor-assay based on the mer-lux gene fusion. The numbers of bacterial CFUs on the general medium 1/100 tryptic soy agar (TSA) were significantly decreased when the soil had been amended with Hg. In contrast, no effect was seen on the number of CFUs on the Pseudomonas-specific medium Gould's S1 agar. Protozoan numbers estimated by the most probable number (MPN) method with 1/100 TSB as growth medium were also negatively affected by Hg. The different fractions of protozoa were affected to different degrees suggesting that amoebae were less sensitive than slow-growing flagellates, which again were less sensitive than the fast-growing flagellates. In contrast, Hg did not induce any detectable changes in the diversity of flagellate morphotypes. In the treatment with 15 mg Hg kg⁻¹ a transiently increased number of bacteria was seen at day 6 probably concomitant with a decrease in the numbers of protozoa. This might indicate that Hg affected the prey-predator dynamics in communities of culturable bacteria and protozoa in soil. Furthermore, we showed that the number of Pseudomonas spp. was not affected by Hg whereas the number of bacteria growing on a general medium was.     

Using farmland prices to evaluate cost-efficiency of national versus regional reserve selection in Denmark

The current study focuses on the influence of geopolitical coordination of conservation strategies on cost and efficiency in terms of species representation when selecting network of protected areas. Conservation policies in the EU are implemented at many different administrative levels: from the European Union and national levels, to regional or/county levels within member countries. This arise the question what size of efficiency gains could be achieved if planning of conservation priorities could be coordinated between geopolitical units. Using data for the nationwide distribution of 763 species, representing all Danish species within eight taxa, we compared illustrative costs for the addition of new areas to the existing conservation network in order to ensure full coverage of all species. We found that the cost of independent regional planning is 20-fold higher than an inter-regional and nationally co-ordinated strategy. We also found that substituting land prices for a simple land-area measure in our analyses increased the expected conservation costs differential significantly, without increasing coverage of species representations. We suggest that in economic and biodiversity terms it can largely be a win-win situation to set a common goal, to develop priority-strategies, and to coordinate actions at higher rather than lower levels of administration.     

Ridging in autumn as an alternative to mouldboard ploughing in a humid-temperate region

In the original ridge tillage system as practiced in the US Corn Belt, ridges are formed during the growing season. Several studies have documented that this can reduce leaching of nutrients and improve fertilizer efficiency. This study was conducted to determine whether ridges formed in autumn can be used as an alternative to ploughing to reduce N leaching during autumn and winter, and thereby increase growth and N uptake of a subsequent unfertilized crop. A factorial field experiment with tillage and residues as factors was conducted during 1998–2000. Tillage treatments were autumn ridging and ploughing. Residue treatments were stubble, stubble + straw and stubble + liquid manure in order to create a gradient of C/N ratios. From the time of harvest until planting of a subsequent barley crop (Hordeum vulgare L.), inorganic N was determined 11 times in 1998–1999 and 10 times in 1999–2000 in the 0–10, 10–30, 30–60 and 60–90 cm soil layers. Growth and N uptake of barley was quantified nine times in both 1999 and 2000. Barley grain and straw yields were determined. Ridging resulted in lower levels of inorganic N in the 30–90 cm soil layer in November and a significantly higher level of inorganic N in the 0–30 cm soil layer in late April, indicating reduced leaching and increased N availability for the subsequent crop. Ridging significantly increased growth, yield and N uptake of barley whereas incorporation of straw generally reduced growth, yield and N uptake. It is concluded that ridging in autumn has a N conserving effect, and it is suggested that the potential of ridging in autumn as an alternative to ploughing is further investigated in detailed studies of solute movement, N immobilization/mineralization and crop performance.     

Tolerance of genetically characterized <Emphasis Type="Italic">Folsomia candida</Emphasis> strains to phenmedipham exposure

Background, Aims, and Scope  The springtail Folsomia candida is a commonly used model species in ecotoxicological soil testing. The species reproduces parthenogenetically and, thus, laboratories use different clonal lineages. In this study, we investigated if genetic divergence between F. candida strains impacts the reaction to chemical stress and may thus affect the outcome of toxicity tests. Methods  In two exposure assays (life-cycle reproduction test and avoidance behaviour test), three laboratory strains of F. candida from Portugal (PTG), Spain (SPN) and Denmark (DNK) were exposed to different concentrations of the reference chemical phenmedipham. Genetic divergence among strains was estimated based on mitochondrial COI sequence data. Results  No significant differences between tolerance towards phenmedipham exposure were observed in a reproduction test. In contrast, one strain (SPN) showed a decreased susceptibility to phenmedipham compared to the other strains (PTG and DNK) in the avoidance assay. Discussion  A phylogenetic tree based on mitochondrial COI sequences revealed clear genetic differentiation between both ‘reaction types’. Thus, we found a potential lineage dependent stress reaction in avoidance behaviour towards the pesticide. Conclusions  Our findings have implications for the comparability of test results among laboratories. Reproduction tests seem to be more robust towards interclonal genetic differentiation than avoidance tests. Recommendations and Perspectives  We recommend the use of molecular tools for simple and cost effective genetic characterization of F. candida strains used in chemical avoidance tests. Closer investigations concerning the relation between genetic relatedness and chemical response will provide a more detailed and comprehensive picture on the role of intraspecific genetic differentiation in stress tolerance. ESS-Submission Editor: Prof. Dr. Henner Hollert (henner.hollert@bio5.rwth-aachen.de)     

Bacterial Diversity and Aerobic Biodegradation Potential in a BTEX-Contaminated Aquifer

In order to gain insight into the community structure and the biodegradative potential of bacteria residing in a BTEX-contaminated field site, with benzene as main pollutant, an investigation strategy with a variety of cultivation-independent and -dependent approaches was undertaken. Based on 16S ribosomal DNA sequence analysis, sampling stations in the center of the pollution plume were found to be dominated by a bacterial consortium affiliated with various members of the class of Proteobacteria and Firmicutes, including different sporulating and non-sporulating sulfate-reducing bacteria and members of the genus Geobacter. Non-polluted samples retrieved from outside the plume revealed several phylotypes which were also observed in the centre of the plume. Bacterial sequences retrieved from the fringe of the plume were dominated by several genera of beta Proteobacteria subclass which were not associated with other phylotypes obtained in this study. All sampling stations were tested positive for catechol 2,3-dioxygenase genes, indicating the presence of microorganisms with the genetic potential degrading aromatic compounds via the meta-cleavage pathway. The groundwater system at the down-gradient edge of the plume was characterized, in contrast to all other sampling stations in the center and outside the plume, by high numbers of cultivable bacteria and cultivable aerobic benzene and toluene degraders. These findings are also in accordance with the investigations based on fluorescent in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes, suggesting the presence of an actively benzene degrading bacterial community of beta Proteobacteria at the fringe of the pollution plume.