Feulgen staining in malpighian tubules of meliponid bees: a methodological contribution

Whole-mounted Malpighian tubules of larvae from two meliponid bee species fixed in acetic acid-ethanol showed apositive cytoplasmic staining with Schiff reagent when submitted to the Feulgen reaction in which acid hydrolysiswas done in 4 M HCl at room temperature. The ability of various treatments applied before the Feulgen acid hydrolysisto abolish this cytoplasmic staining was examined. The aldehyde groups of phospholipids present in the cytoplasm ofthe Malpighian tubules were blocked or removed by sequential treatment with 5% sodium borohydride and acetonechloroform(1:1, v/v) for 15 min each prior to HCl hydrolysis. This treatment is recommended in order to abolish thecytoplasmic (plasmal) reaction and to allow the reliable quantification of DNA by the Feulgen reaction and thediscrimination of nuclear phenotypes in the Malpighian tubules of meliponid bees.

Cell cycle and DNA content of mitotic cells in brain ganglia of Drosophila larvae.

The programmes of replication of hetero- and euchromatin regions, mitotic cell cycle and the DNA content in metaphases in brain ganglia from late third instar larvae of Drosophila melanogaster (wild type and a tumour bearing mutant, 1(2)gl, strain) and of Drosophila nasuta were examined by autoradiography of [3H]thymidine labelled (continuous or pulse) cells and by cytophotometry, respectively. Brain ganglia labelled continuously with [3H]thymidine for 24 h in vitro showed a significantly high proportion of cells with incorporation of radioactivity restricted to heterochromatin only. Pulse labelling of brain ganglia from larvae of Drosophila melanogaster and Drosophila nasuta followed by chase for different time intervals showed that (i) the frequency of labelled metaphases was more than 50% within 15 to 30 min of chase and remained higher than 50% in nearly all the chase samples till 24 h, (ii) euchromatin labelled metaphases appeared with a low frequency within 1 to 4 h chase period but the heterochromatin labelled metaphases continued to be more common in the later chase samples also, (iii) single chromatid labelled second cycle metaphases were seen within 1 to 4 h after the pulse, but their frequency did not increase in the later samples. Cytophotometry of feulgen-DNA and Hoechst 33258 stained metaphases in late third instar larval brain ganglia revealed a greater variation in the DNA content of individual metaphases, although the means were close to the expected 4 C content. It appears that in relation to the known asymmetric cell divisions of neuroblast and other neural cells, the mitotically active cells in brain ganglia comprise a heterogenous population with widely varying lengths of the different phases of cell cycle; some of them may not cycle regularly and may possibly have a discontinuous S-phase.