New fluorescence reactions in DNA cytochemistry. 2. Microscopic and spectroscopic studies on fluorescent aluminum complexes

Metal-dye complexes are widely applied in light microscopic techniques for chromatin staining (e.g., hematoxylin and carmine), but fluorescent complexes between phosphate-binding cations and suitable ligands have been little used. Preformed and postformed Al complexes with different anionic dyes induced strong and selective fluorescence reactions in nuclei from chicken blood smears, frozen sections, paraffin-embedded sections and Epon-embedded sections of mouse and rat tissues, mitotic chromosomes, meiotic chromosomes and kinetoplasts of Trypanosoma cruzi epimastigotes. The DNA-dependent fluorescence of these structures showed a very low fading rate. The emission colors were related to the ligand. The most suitable compounds for forming fluorescent Al chelates were 8-hydroxyquinoline, morin, nuclear fast red and purpurin. Staining with diluted carmine solutions and InCl3 mordanting, followed by 8-hydroxyquinoline, also induced chromatin fluorescence. After treating isolated mouse chromosomes with the preformed complex Al-nuclear fast red, x-ray microanalysis indicated a P:Al:dye binding ratio of about 40:15:1. The selectivity, stability and easy formation of these fluorescent Al complexes are obvious advantages for their use as new cytochemical probes in cytologic studies.     

Chromatin Fluorescence after Carmine Staining

After staining with dilute solutions (0.1 mg/ml in distilled water) of commercial carmine, a strong reddish orange fluorescence was observed in nuclei from cell smears and frozen and paraffin tissue sections. Optimal exciting light ws 436 nm (violet-blue) or 450-490 nm (blue). Compact chromatin from interphase nuclei, mitotic and meiotic chromosomes and the kinetoplast of Trypanosoma cruzi showed the highest fluorescence, while the basophilic cytoplasm appeared weakly fluorescent. No emission was observed in cartilage matrix, mast cell granules or goblet cell mucin. This selective method could be valuable in microscopic and cytochemical studies on chromatin because the carmine fluorescence is stable and preparations can be dehydrated and mounted permanently without changes in the fluorescence pattern.     

Effects of the non-native amphibian species Discoglossus pictus on the recipient amphibian community: niche overlap, competition and community organization

The painted frog, Discoglossus pictus, was introduced into the Iberian Peninsula 100 years ago and its distribution has steadily increased since then. We studied the effects of this non-native amphibian species on the native ones in the northeastern area of the peninsula. We compared amphibian assemblages in regions with and without D. pictus to estimate niche overlap between species. Additionally, we carried out a laboratory evaluation of the effects of competition between the non-native and the two native species with which it overlaps most commonly: Bufo calamita and Pelodytes punctatus. The presence of D. pictus larvae reduced the survival, body mass and activity of B. calamita, and increased time to metamorphosis. Furthermore, D. pictus showed the highest consumption rate while P. punctatus showed the lowest. One possible consequence of these competitive interactions is an alteration of species co-occurrence patterns in the recipient community on a regional scale. In the non-invaded area, the checkerboard score (C-score) of co-occurrence indicated that the community was structured, whereas the standardized C-score in the invaded area indicated that the community did not differ significantly from having a random structure. These results suggest that competition between native and non-native species can cause recipient communities to become less structured.