Identifying hybridizing taxa within the <Emphasis Type="Italic">Daphnia longispina</Emphasis> species complex: a comparison of genetic methods and phenotypic approaches

Daphnia galeata Sars, D. longispina O. F. Müller and D. cucullata Sars (Crustacea: Cladocera) are closely related species which often produce interspecific hybrids in natural populations. Several marker systems are available for taxon determination in this hybridizing complex, but their performance and reliability has not been systematically assessed. We compared results from identifications by three molecular methods. More than 1,200 individuals from 10 localities in the Czech Republic were identified as parental species or hybrids by allozyme electrophoresis and the analysis of the restriction fragment length polymorphism of the internal transcribed spacer (ITS-RFLP); over 440 of them were additionally analyzed and identified by 12 microsatellite loci. Identification by microsatellite markers corresponded well with allozyme analyses. However, consistent discrepancies between ITS-RFLP and other markers were observed in two out of 10 studied localities. Although some marker discrepancies may have been caused by occasional recent introgression, consistent deviations between ITS-RFLP and other markers suggest a long-term maintenance of introgressed alleles. These results warn against its use as a sole identification method in field studies. Additionally, we quantitatively evaluated the discriminatory power of geometric morphometric (elliptic Fourier) analysis of body shapes based on photos of over 1,300 individuals pre-classified by allozyme markers. Furthermore, a randomly selected subset of 240 individuals was independently determined from photos by several experts. Despite a tendency for morphological divergence among parental Daphnia species, some taxa (especially D. galeata, D. longispina, and their hybrids) substantially overlapped in their body shapes. This was reflected in different determination success for particular species and hybrids in discriminant analysis based on shape data as well as from photographs.     

Advanced fertility diagnosis in stallion semen using transmission electron microscopy

Routine semen analysis of stallions is based on light microscopy (LM). However, there are still a number of animals that are subfertile or even infertile not being identified with conventional semen analysis. The objective of this study was to investigate the suitability of transmission electron microscopy (TEM) for advanced fertility diagnosis in stallion. We examined ejaculates of 46 stallions with known fertility. Animals were divided into three different groups: group 1, fertile stallions (pregnant mares> or =70%, n=29); group 2, subfertile stallions (pregnant mares 10-69%, n=14); group 3, infertile stallions (pregnant mares<10%, n=3). Ejaculates were collected in spring 2002. Conventional semen analysis (volume, sperm concentration, motility, live:dead ratio and percentage of morphologically normal sperm) was immediately performed after semen collection. Ultrastructural analysis included the evaluation of 200 acrosomes, heads, midpieces and cross-sections of tails as well as 100 longitudinal sections of tails from every ejaculate. Using LM, we found a significant increase of morphological deviations from 24.5% (x ) in group 1 to 34.5% in group 2 and 73.5% in group 3. Using TEM, we found a significant increase of detached acrosomes from 6.1% in group 1 to 7.6% in group 2 and 21.4% in group 3. Deviations in tubule pattern were also increased (but not significant) from 2.7% in fertile and 2.8% in subfertile to 11.4% in infertile stallions as well as multiple tails from 1.9% in fertile to 2.0% in subfertile and 8.9% in infertile. Our data indicate that TEM is suitable for advanced fertility diagnostic in stallions, giving a connection between fertility and morphology. It suggests that the most likely reason for sub- and infertility in stallion in case of increased LM pathomorphology of semen are acrosomal alterations, especially detached acrosomes.     

Occurrence of tetraploidy in Nicotiana attenuata plants after Agrobacterium-mediated transformation is genotype specific but independent of polysomaty of explant tissue

Genotypes of Nicotiana attenuata collected from Utah and Arizona were transformed with 17 different vectors (14 unpublished vectors based on 3 new backbone vectors) using an Agrobacterium-mediated procedure to functionally analyze genes important for plant–insect interactions. None of the 51 T1–T3 transgenic Utah lines analyzed by the flow cytometry were tetraploid, as opposed to 18 of 33 transgenic Arizona lines (55%). Analysis of T0 regenerants transformed with the same vector carrying an inverted repeat (IR) N. attenuata pro-systemin construct confirmed the genotype dependency of tetraploidization: none of the 23 transgenic Utah lines were tetraploid but 31 (72%) of 43 transgenic Arizonas were tetraploid. We tested the hypothesis that the differences in polysomaty of the explant tissues accounted for genotype dependency of tetraploid formation by measuring polysomaty levels in different seedling tissues. Hypocotyls, cotyledons, and roots of Utah and Arizona genotypes contained similar percentages of 4C nuclei (61 and 60; 7 and 5; and 58 and 61%, respectively). Since we used hypocotyls as explant sources and the nonoccurrence of tetraploid Utah transformants does not correspond to the high percentage of 4C nuclei in Utah hypocotyls, we can rule out a direct relationship between tetraploid formation and polysomaty level. We hypothesize that the difference between the Utah and Arizona genotypes results from the failure of polyploid Utah callus to regenerate into fully competent plants. We propose that future work on post-transformation polyploidy concentrate on the processes that occur during callus formation and plant regeneration from callus.Electronic Supplementary Material Supplementary material is available for this article at     

Lens‐anomalies and other ophthalmic findings in a group of closely‐related angola lions (Panthera leo bleyenberghi)

After the diagnosis of bilateral, immature, nuclear, and posterior cortical cataracts in one Angola lioness, and because of the possible implications of the cataracts for a breeding program, complete ophthalmic examinations on a group of related adult Angola lions and their offspring were carried out. Five adult lions, ranging in age from 1.5–5.5 years, and five lion cubs were studied clinically. The examination included slit‐lamp biomicroscopy, indirect ophthalmoscopy, and photography. The eyes of three of the offspring were submitted for histopathologic examination and examined by light microscopy. The most significant findings were cataracts of various stages, which were observed in four adult lions and one male cub. Mild lenticular abnormalities were noted in the histopathologic examination of the lion cubs' eyes. Additional ophthalmic findings, of lesser clinical consequence, were also noted. This breeding program would benefit from further investigation by animal nutritionists and geneticists, and the animals in this group should undergo periodic ophthalmologic examinations. Zoo Biol 0:1–7, 2006. © 2006 Wiley‐Liss, Inc.     

F-actin-based Ca signaling-a critical comparison with the current concept of Ca signaling

A short comparative survey on the current idea of Ca signaling and the alternative concept of F-actin-based Ca signaling is given. The two hypotheses differ in one central aspect, the mechanism of Ca storage. The current theory rests on the assumption of Ca-accumulating endoplasmic/sarcoplasmic reticulum-derived vesicles equipped with an ATP-dependent Ca pump and IP3- or ryanodine-sensitive channel-receptors for Ca-release. The alternative hypothesis proceeds from the idea of Ca storage at the high-affinity binding sites of actin filaments. Cellular sites of F-actin-based Ca storage are microvilli and the submembrane cytoskeleton. Several specific features of Ca signaling such as store-channel coupling, quantal Ca release, spiking and oscillations, biphasic and "phasic" uptake kinetics, and Ca-induced Ca release (CICR), which are not adequately described by the current concept, are inherent properties of the F-actin system and its dynamic state of treadmilling.     

Exchange of a single amino acid switches the substrate properties of RhoA and RhoD toward glucosylating and transglutaminating toxins

Rho GTPases are the preferred targets of various bacterial cytotoxins, including Clostridium difficile toxins A and B, Clostridium sordellii lethal toxin, the cytotoxic necrotizing factors (CNF1) from Escherichia coli, and the dermonecrotizing toxin (DNT) from Bordetella species. The toxins inactivate or activate specific sets of Rho GTPases by mono-O-glucosylation and deamidation/transglutamination, respectively. Here we studied the structural basis of the recognition of RhoA, which is modified by toxin B, CNF1, and DNT, in comparison with RhoD, which is solely a substrate for lethal toxin. We found that a single amino acid residue in RhoA and RhoD defines the substrate specificity for toxin B and lethal toxin. Change of serine 73 to phenylalanine in RhoA turned RhoA into a substrate for lethal toxin. Accordingly, change of the equivalently positioned phenylalanine 85 in RhoD with serine allowed glucosylation by toxin B. Comparable results were achieved with the Rho-activating and transglutaminating enzymes CNF1 and DNT. Here, amino acid glutamate 64 of RhoA and the equivalent aspartate 76 of RhoD define substrate specificity for CNF1 and DNT, respectively. These data indicate that single amino acid residues located in the switch II region of Rho proteins determine enzyme specificity for diverse bacterial toxins.