Nucleolar fragmentation in polytene trichogen cells of Lucilia cuprina and Chrysomya bezziana (Diptera: Calliphoridae)

Abstract

The location of genes coding for 18S and 28S ribosomal RNA in mitotic and polytene cells of Lucilia cuprina and Chrysomya bezziana was investigated using in situ hybridization of an 18 + 28S ribosomal gene probe and silver staining. In both species ribosomal genes were localized to secondary constriction regions in sex chromosome heterochromatin. In L. cuprina mitotic cells the probe hybridizes to a distal secondary constriction region in the short arms of the X and Y chromosomes. In C. bezziana mitotic chromosomes ribosomal genes were located in distal secondary constriction regions in the long arms of the X and Y chromosomes. In polytene trichogen cells of both species, hybridization results varied with the level of polyteny. Cells of low polyteny have a single hybridization site, but with greater polytenization, increasing numbers of extrachromosomal fragments strongly hybridize to the ribosomal gene probe. No hybridization occurs in structures representing the sex chromosomes or in the autosomes. These results indicate that fragmentation and dispersal of the nucleolus occurs during polytenization. Silver staining of both unsquashed and squashed polytene nuclei show identical behaviour of multiple, varied-sized nucleolar bodies, thus confirming the in situ hybridization results. Uridine incorporation studies in L. cuprina indicate that transcription occurs in extrachromosomal bodies similar to nucleolar fragments. Nucleolar fragmentation is more pronounced in L. cuprina males, particularly in those with the translocation T(Y;2)540. Chromosomally normal C. bezziana show nucleolar fragmentation levels similar to that in L. cuprina males. Ribosomal genes are disproportionately replicated in trichogen cells to a much greater extent than surrounding heterochromatin. Nucleolar fragmentation may be a gene amplification system, but it is not known to what degree, relative to diploid amounts, ribosomal genes replicate in trichogen cells.Key words: Lucilia cuprina, Chrysomya bezziana, polytene cells, ribosomal genes, nucleolar fragmentation, disproportionate replication, heterochromatin.