Habitat partitioning in endangered Cantabrian capercaillie Tetrao urogallus cantabricus

The endangered Cantabrian capercaillie (Tetrao urogallus cantabricus) lives at the southern edge of tetraonids’ distribution range, in entirely deciduous forests. Its conservation planning has been always lek-centred. There is very little information about the specific habitat requirements of hens and broods, even though reproductive success appears to be a limiting factor. We analysed summer surveys from 1997 to 2004, carried out to estimate the reproductive success of the population. We compared the habitat characteristics at different spatial scales of hens with broods, broodless hens, and cocks in summer, with the better known spring habitat in display areas. Summer habitat showed higher proportion of open areas and was associated with more rugged zones at moderate spatial scales (78 ha) than spring habitat at display areas. Cocks and hens showed summer habitat partitioning; hens were associated with higher proportions of open and shrubby habitats. Furthermore, broodless hens preferred areas with higher slope variability than the display and summer areas preferred by cocks. These differences may reflect the sexual dimorphism of the species in reproductive role, energetic demands and conspicuousness. At larger spatial scales a previously developed habitat suitability model performed well to predict good brood-rearing areas. Hens with broods were located in the best-preserved areas in the range, mainly characterized by higher proportion of forest cover at a large (50 km²) scale. We suggest that these characteristics indicate refuge habitats where Cantabrian capercaillie can still breed successfully.     

MAP kinase-like activity in transformed Catharanthus roseus hairy roots varies with culture conditions such as temperature and hypo-osmotic shock.

Mitogen activated protein (MAP) kinase-like activity was determined in extracts obtained from transformed Catharanthus roseus hairy roots by the ability to phosphorylate myelin basic protein (MBP). Both in solution and in gel kinase assays showed variation in activity, depending on root developmental stage. In gel kinase assays, using the extract soluble fraction, revealed a 56 kDa polypeptide with phosphorylation activity on MBP. In addition, another 75 kDa polypeptide was observed in the particulate fraction. Immunodetection with monoclonal antibodies against ERK-1, a mammalian MAP kinase, and with anti-phosphotyrosine antibodies cross-reacted with the 56 kDa polypeptide, named SMK56, from the soluble fraction, suggesting that this polypeptide could be related with members of the MAP kinase family. Antibodies against the dually phosphorylated threonine-tyrosine motif, characteristic of active forms of MAP kinases, also cross-reacted with this 56 kDa polypeptide. Changes in the levels of SMK56 were detected within the first 30 min of root exposure to low temperatures or hypo-osmotic shock, suggesting that this protein may be involved in the perception of environmental changes.     

Interactions between herbivory and resource availability on grazing tolerance of Leymus chinensis

Herbivory and resource interact to influence plant regrowth following grazing, but few detailed investigations on grazing tolerance at population levels are available. We conducted two pot experiments along a simulated grazing gradient (0%, 25%, 50% and 75% of shoot removal) at three water or nutrient levels to determine the interaction of resource and herbivory on Leymus chinensis, a perennial, dominant species in the eastern Eurasian steppes. Interactions between water availability and clipping intensity on the relative height growth rate (RHGR) and bud number were significant. Significant interactions between nutrient and clipping on RHGR, total biomass and specific leaf area (SLA) were also found. Total biomass and bud number, showing a unimodal curve along the clipping gradient in resource-rich environments, were highest at light clipping level, suggesting that this species has the plastic compensatory responses from under- to overcompensation. Interactions between herbivory and water or nutrient were opposite to each other. The “cooperative” interactions between water and herbivory magnified the difference in grazing tolerance of L. chinensis between high and low water treatments. The “antagonistic” interactions between nutrient and herbivory, on the other hand, were reflected in the lower tolerance to heavy clipping in the high nutrient than low nutrient treatments. Results partly support the limiting resource model (LRM). A modified and simplified graphic model of the LRM was proposed based on our results. The new LRM clearly demonstrated that “cooperative” interactions between varying water levels and clipping intensities aggravate the detrimental impacts of herbivores on plant growth and reproduction, whereas “antagonistic” interactions between nutrient and grazing alleviate the negative effects of herbivores. Biomass compensation and density compensation were identified as main mechanisms of herbivory tolerance in this clonal species.     

Ornamental characters,ion accumulation and water status in Arbutus unedo seedlings irrigated with saline water and subsequent relief and transplanting

Arbutus unedo seedlings were grown in a greenhouse and submitted to three irrigation treatments (salinity period) using solutions with an EC of 0.85 dS m^?1 (control treatment), 5.45 dS m^?1 (S1) and 9.45 dS m^?1 (S2). After 16 weeks, growth and ornamental characters, leaf water potentials, gas exchange and ion concentrations were determined. After the salinity period, plants were exposed to a relief period for 1 month, whereby half of the plants were transplanted to field conditions and the other half into 24 cm diameter plastic pots. Salinity induced a significant decrease in shoot biomass and leaf area but root/shoot ratio was increased. Plant height was significantly inhibited by salinity. The ornamental characters were affected in the treated plants, with symptoms of salt injury, such as burning of leaf margin. Leaf water potentials decreased with increasing salinity, more significantly at predawn than at midday. The relationship between net photosynthesis (P_n) and leaf conductance (g_l) was linear for all treatments and the same values of P_n are associated with lower values of g_l for the saline treatments than for control treatment. The concentration of Cl^? in leaves increased with increasing salinity and was higher than the corresponding concentration of Na⁺. Na⁺ and Cl^? contents were higher in the leaves than in the roots in both saline treatments. The K⁺ and Ca²⁺ levels were lower in the treated plants than in control plants and applied salinity reduced the K⁺/Na⁺ ratio in leaves, stems and roots, the decrease being much greater for leaves than for roots. The Ca²⁺/Na⁺ ratio fell with salinity in all parts of the plants. At the end of the relief period leaf water potentials were recovered mainly in field conditions. S2 treatment showed lower values of P_n and g_l than control and S1 treatments in pot conditions and in field conditions S1 showed the lowest values for P_n and g_l.     

Oscheius tipulae, a widespread hermaphroditic soil nematode, displays a higher genetic diversity and geographical structure than Caenorhabditis elegans

The nematode Oscheius tipulae belongs to the same family (Rhabditidae) as the model species Caenorhabditis elegans . Both species reproduce through self-fertilizing hermaphrodites and facultative males. Recent studies have shown that the self-fertile C. elegans and C. briggsae displayed a 20-fold lower genetic diversity than the male–female species C. remanei . Several explanations have been put forward to account for this difference, including their mode of reproduction and dynamic population structure. Here, we present the results of extensive worldwide sampling of O. tipulae , which we previously used as a laboratory organism for developmental genetics. We found that O. tipulae is much more widespread and common in soil throughout the world than Caenorhabditis species. We analysed 63 O. tipulae isolates from several continents using amplified fragment length polymorphism (AFLP). We found that O. tipulae harbours a 5-fold higher genetic diversity than C. elegans and C. briggsae . As in C. elegans , a high proportion of this diversity was found locally. Yet, we detected significant geographical differentiation, both at the worldwide scale with a latitudinal structure and between three localities in France. In summary, O. tipulae exhibited significantly higher levels of genetic diversity and large-scale geographical structure than C. elegans , despite their shared mode of reproduction. This species difference in genetic diversity may be explained by a number of other differences, such as population size, distribution, migration and dynamics. Due to its widespread occurrence and relatively high genetic diversity, O. tipulae may be a promising study species for evolutionary studies.     

Identification of human T cell receptor gammadelta-recognized epitopes/proteins via CDR3delta peptide-based immunobiochemical strategy

Human T lymphocytes, bearing T cell receptor (TCR) gammadelta, play an important role in anti-tumor/microbe immune responses. However, few tumor antigens recognized by TCRgammadelta have been defined so far. To investigate antigenic epitopes/proteins recognized by gammadeltaT cells, we have established a new immunobiochemical strategy that uses complementarity-determining region 3 of TCR delta chain (CDR3delta) peptide-mediated epitope/protein-binding assays. CDR3delta peptides synthesized using the CDR3 region in TCR Vdelta2 chain were validated for their binding specificity to target cells or tissues. These CDR3delta peptides were then employed as probes to pan putative epitopes in a 12-mer random peptide phage-displayed library and to identify putative protein ligands within tumor protein extracts by affinity chromatography and liquid chromatography/electrospray ionization-tandem mass spectrometry analysis. As a result, we have identified nine peptides and two proteins for TCRgammadelta, including human mutS homolog 2 (hMSH2) and heat shock protein (HSP) 60. All nine tested epitope peptides not only bind to gammadeltaT cells but also functionally activate gammadeltaT cells in vitro. Identification of HSP60 confirms the validity of this method as HSP60 is an identified ligand for TCRgammadelta. We show that hMSH2 is expressed on the surface of SKOV3 tumor cells, and cytotoxicity of Vdelta2 gammadeltaT cells to SKOV3 cells was blocked by anti-hMSH2 antibody, suggesting that hMSH2 may be a new ligand for TCRgammadelta. Taken together, our findings provide a novel immunobiochemical strategy to identify epitopes/proteins recognized by gammadeltaT cells.     

Evaluation of short estrus synchronization methods in dairy cows

In the present study, two new short estrus synchronization methods have been developed for lactating dairy cows. The study was completed in three consecutive phases. In experiment (Exp) 1, 32 cows, that were not detected in estrus since calving between the 50th and 84th post-partum days, were treated with PGF2alpha (PGF, d-cloprostenol, 0.150 mg), estradiol propionate (EP, 2mg) and GnRH (lecirelina, 50 microg) at 24h intervals, respectively, and timed artificial insemination (TAI) was performed 48 h after PGF. Different from Exp 1, EP and GnRH were given at 48 and 60 h, respectively after PGF in Exp 2 (n=20), instead of 24 and 48 h. Ovulations were investigated by ultrasound for 7 days starting from the day of PGF treatment, and ovulation rates were compared with the ones obtained in Exp 1. In Exp 3, cows were given the same treatments as Exp 2, but treatments started at certain estrus stages. Cows detected in estrus and with a confirmed ovulation (n=27) after the second PGF given 11 days apart were assigned to three treatment groups. Treatment was initiated at Day 3 (group metestrus, n=9), Day 12 (group diestrus, n=9) and Day 18 (group proestrus, n=9) after ovulation. All cows included in Exp 3 were TAI between 16 and 20 h after GnRH treatment. In Exp 2 and 3, blood samples were obtained once every 2 days, starting from Day 0 to the 10th day after GnRH injection, and once every 4 days between the 10th and the 22nd days after GnRH to examine post-treatment luteal development. During the study, animals exhibiting natural estrus were inseminated and served as controls (n=85). The rate of estrus was found to be significantly higher in cows with an active corpus luteum (CL) at the start of Exp 1 (72.7% vs. 30.0%, P<0.05) and the pregnancy rate tended to be higher than cows without an active CL (40.9% vs. 10.0%, P=0.08). Compared to those in Exp 1, cows in Exp 2 had higher rates of synchronized ovulation (94.1% vs. 59.1%, P=0.013). In Exp 3, estrus (P<0.001) and pregnancy rates (P=0.01) were found to be significantly higher in cows in the proestrus group than in those in the metestrus group. Comparable pregnancy rates were obtained from the first and second inseminations in Exp 1 and 3 with results from those inseminated at natural estrus (P>0.05). It was concluded from the study that the treatment in Exp 1 and 3 could result in comparable pregnancy rates after timed AI of lactating dairy cows at random stages of the estrus cycle relating to those inseminated at natural estrus, but the stage of the estrus cycle can have significant effects on pregnancy rates.     

Vector soup: high‐throughput identification of Neotropical phlebotomine sand flies using metabarcoding

Phlebotomine sand flies are haematophagous dipterans of primary medical importance. They represent the only proven vectors of leishmaniasis worldwide and are involved in the transmission of various other pathogens. Studying the ecology of sand flies is crucial to understand the epidemiology of leishmaniasis and further control this disease. A major limitation in this regard is that traditional morphological‐based methods for sand fly species identifications are time‐consuming and require taxonomic expertise. DNA metabarcoding holds great promise in overcoming this issue by allowing the identification of multiple species from a single bulk sample. Here, we assessed the reliability of a short insect metabarcode located in the mitochondrial 16S rRNA for the identification of Neotropical sand flies, and constructed a reference database for 40 species found in French Guiana. Then, we conducted a metabarcoding experiment on sand flies mixtures of known content and showed that the method allows an accurate identification of specimens in pools. Finally, we applied metabarcoding to field samples caught in a 1‐ha forest plot in French Guiana. Besides providing reliable molecular data for species‐level assignations of phlebotomine sand flies, our study proves the efficiency of metabarcoding based on the mitochondrial 16S rRNA for studying sand fly diversity from bulk samples. The application of this high‐throughput identification procedure to field samples can provide great opportunities for vector monitoring and eco‐epidemiological studies.