Isolation of sperm cells from mature pollen grains of Spinacia oleracea L.

Summary When mature pollen grains of Spinacia oleracea were squashed in a 25% sucrose solution and subsequently centrifuged on a percoll layer, sperm cells were isolated in high numbers. All steps were carried out at 4° C. Isolated sperm cells could be kept alive for several hours.     

The microtubule cytoskeleton and the rounding of isolated generative cells ofNicotiana tabacum

Summary Upon squashing of the pollen grain, the isolated generative cell ofNicotiana tabacum looses its spindle shape to become spherical; this phenomenon is independent of the sucrose concentration used. The time necessary for this change can vary from 1 min (0% sucrose) to 20 min (30% sucrose). The microtubular cytoskeleton was studied by means of immunofluorescence and electron microscopy. Just after isolation, 5 to 15 clearly visible bundles in microtubules organized in a basket-like structure are present. After 15 min in medium with 15% sucrose, the microtubular cytoskeleton disappears, and a diffusely spread tubulin can be observed. Neither the addition of 10–20 M taxol to the medium, nor the omission of Ca²⁺ to the medium has any effect on the changes in cell shape and loss of microtubular bundles after isolation.Abbreviations GC Generative cell - SC sperm cell - BK Brewbaker and Kwack - CLSM confocal laser scanning fluorescence micros copy     

A simple and low‐cost method to estimate spatial positions of shorebirds: the Telescope‐Mounted Angulator

ABSTRACT Estimating the spatial position of birds in open habitats like intertidal mudflats is important for many studies, for example, detailed density estimates or linking predation pressure to resource availability. To date, several methods have been used to estimate the positions of birds, including density counts in predetermined plots, range finders, photography, and tracking individuals tagged with GPS‐equipped transmitters, and each method has advantages and shortcomings. Counts in premarked plots are possible over relatively long distances, but small‐scale information is lost due to within‐plot averaging. Other methods accurately determine the position of individuals, but can only be used at relatively short distances or involve capturing birds. We describe a simple and low‐cost method to estimate the spatial position of individual birds in open habitats using a telescope‐mounted instrument that measures the scope's viewing angle. Using this Telescope‐Mounted Angulator (TMA), the distance to focal birds can be calculated by simple trigonometry, requiring only the viewing angle and mounting height of the telescope. Laboratory tests revealed that the TMA was most accurate when calibrated for individual observers. Field experiments performed on a 4‐m high observation platform showed that the TMA can estimate the position of shorebirds with an accuracy of 18 to 36 m up to a distance of 500 m. By also including the direction, determined with a compass, the spatial position of birds can be reliably estimated. The TMA can be a valuable tool for estimating the spatial position of animals in various flat landscapes, providing detailed measurements in a relatively short period of time.     

Disease‐limited distributions? Contrasts in the prevalence of avian malaria in shorebird species using marine and freshwater habitats

Migratory shorebirds show strong dichotomies in habitat choice during both the breeding and nonbreeding season. Whereas High Arctic breeding species are restricted to coastal marine and saline habitats during the nonbreeding season, more southerly breeding species tend to use freshwater habitats away from coasts. It has been proposed that this co-variation in habitat use is a consequence of a single axis of adaptation to pathogens and parasites, which are hypothesized to be relatively scarce in High Arctic, marine, and saline habitats and relatively common at lower latitudes and in freshwater habitats. Here we examine this contrast by comparing the prevalence of avian malaria infections in shorebirds occupying different habitats. We used a PCR-based assay on 1319 individuals from 31 shorebird species sampled in the Arctic, in temperate Europe and in inland and marine habitats in West Africa. Infections mainly occurred in tropical wetlands, with the shorebirds in freshwater inland habitats having significantly higher prevalence of malaria than birds in marine coastal habitats. Infections were not found in birds migrating through Europe even though conspecifics did show infections in tropical Africa. Adults should resist infection better than juveniles, but showed higher malaria prevalence, suggesting that infection probability increases with cumulative exposure. We argue that exposure to vectors is the main factor explaining the habitat-related differences in malaria prevalence.     

Constitutive immune function responds more slowly to handling stress than corticosterone in a shorebird

Ecological immunologists are interested in how immune function changes during different seasons and under different environmental conditions. However, an obstacle to answering such questions is discerning the effects of biological factors of interest and investigation artifacts such as handling stress. Here we examined handling stress and its effects on constitutive (noninduced) immune function via two protocols on captive red knots (Calidris canutus). We investigated how constitutive immunity responds to handling stress, how quickly these changes take place, and the practical implications for researchers interested in sampling baseline immune levels. We found that Staphylococcus aureus and Candida albicans killing increased with handling stress while total leukocyte and lymphocyte concentrations decreased. However, although corticosterone increased significantly and rapidly in response to handling stress, none of the 10 measures of constitutive immunity that we tested differed significantly from baseline within 20 or 30 min of capture. Thus, researchers interested in baseline immune function should sample animals as soon as possible after capture, but studies in species not easily sampled in less than 3 min (such as red knots) could still yield useful results.     

Characterization of polymorphic microsatellite DNA markers in the black-tailed godwit (Limosa limosa: Aves)

We isolated and tested 16 microsatellite loci in black-tailed godwits from the Netherlands (Limosa limosa limosa), and from Australasia (subspecies melanuroides). One locus was monomorphic, two loci had null-alleles and one was significantly heterozygote deficient. The remaining 12 polymorphic loci had on average 7.9 alleles (range 5-11) and the mean expected heterozygosity was 0.69. No significant linkage disequilibrium between the loci was observed and all loci were autosomal. Fourteen loci were successfully cross-amplified in bar-tailed godwit (Limosa lapponica).     

Reinterpretation of gizzard sizes of red knots world-wide emphasises overriding importance of prey quality at migratory stopover sites

The size of digestive organs can be rapidly and reversibly adjusted to ecological circumstances, but such phenotypic flexibility comes at a cost. Here, we test how the gizzard mass of a long-distance migrant, the red knot (Calidris canutus), is adjusted to (i) local climate, (ii) prey quality and (iii) migratory fuelling demands. For eight sites around the world (both wintering and stopover sites), we assembled data on gizzard masses of free-living red knots, the quality of their prey and the local climate. Using an energetic cost-benefit approach, we predicted the gizzard size required for fastest fuelling (net rate-maximization, i.e. expected during migration) and the gizzard size required to balance daily energy budgets (satisficing, expected in wintering birds) at each site. The measured gizzards matched the net rate-maximizing predictions at stopover sites and the satisficing predictions at wintering sites. To our surprise, owing to the fact that red knots selected stopover sites with prey of particularly high quality, gizzard sizes at stopovers and at wintering sites were nevertheless similar. To quantify the benefit of minimizing size changes in the gizzard, we constructed a model incorporating the size-dependent energy costs of maintaining and carrying a gizzard. The model showed that by selecting stopovers containing high-quality prey, metabolic rates are kept at a minimum, potentially reducing the spring migratory period by a full week. By inference, red knots appear to time their stopovers so that they hit local peaks in prey quality, which occur during the reproductive seasons of the intertidal benthic invertebrates.     

Synthesis, antimalarial activity and cytotoxicity of 10-aminoethylether derivatives of artemisinin

In this study, a series of 11 10-aminoethylether derivatives of artemisinin were synthesised and their antimalarial activity against both the chloroquine sensitive (D10) and resistant (Dd2) strains of Plasmodium falciparum was determined. The compounds were prepared by introducing aliphatic, alicyclic and aromatic amine groups with linkers of various chain lengths through an ethyl ether bridge at C-10 of artemisinin using conventional and microwave assisted syntheses, and their structures were confirmed by NMR and HRMS. All derivatives proved to be active against both strains of the parasite. The highest overall activity was displayed by the short chain aromatic derivative 8 (IC(50)=1.44nM), containing only one nitrogen atom, while long chain polyamine derivatives were found to have the lowest activity against both strains. An interesting correlation between the IC(50), pK(a) values and resistance index (RI) was found.