FISH analysis and cytogenetic characterization of male meiotic prophase I in Acricotopus lucidus (Diptera, Chironomidae).

In the chironomid Acricotopus lucidus, two cells with quite different chromosome complements arise from the last unequal spermatogonial mitosis, as a consequence of monopolar migration of the so-called germ line limited chromosomes (Ks). The cell receiving all the Ks, in addition to two sets of the regularly segregating somatic chromosomes (Ss), develops into the primary spermatocyte, while the cell getting only Ss differentiates into an aberrant spermatocyte. Only the primary spermatocyte enters meiosis. These nuclear events in the primary spermatocytes of A. lucidus during prophase I were analyzed by carmine-orcein staining, silver impregnation, live-cell RNA fluorescence labeling, and fluorescence in situ hybridization, using painting probes of the three Ss and the K centromeres. Early prophase I nuclei display large condensed chromatin blocks showing intense carmine staining, dark silver nitrate impregnation and bright DAPI fluorescence. The first clear signs of meiotic prophase progression are loops arising at early pachytene, which originate from the gradually decondensing chromatin blocks. The blocks presumably represent facultative heterochromatin. Chromosome painting demonstrates that the pachytene loops are composed of the two closely paired homologues. Conspicuous telomere attachments of differently painted non-homologous chromosomes were detected. The centromeres of the Ks group together, indicating a classical bouquet arrangement of the paired homologues in pachytene. The clustered centromeres may function as pairing centers to initiate synapsis of the homologues. Nucleolus expression data support the idea that the aberrant spermatocyte nourishes the primary spermatocyte via a connecting cytoplasmic canal.

Isolation of a new germ line-specific repetitive DNA family in Acricotopus by microdissection of polytenized germ line-limited chromosome sections from a permanent larval salivary gland preparation.

In Acricotopus lucidus (Diptera, Chironomidae) the germ line-limited chromosomes (Ks) have developed from the soma chromosomes (Ss) by endoreduplication, rearrangements and accumulation of germ line-specific repetitive sequences. For molecular analysis of specific small K sections, microdissection of metaphase Ks generally yields very limited amounts of DNA. In this study, K-specific DNA was microdissected from defined polytenized K sections of X-ray induced K-S-rearrangements of permanent salivary gland chromosome preparations and was then amplified by DOP-PCR. A new germ line-specific tandem repetitive DNA family was isolated by this way from a heterochromatic K segment, characterized and localized on the Ks by FISH. The repetitive elements are related to sequences of earlier described K-specific tandem repetitive DNA families in A. lucidus, but are located mainly in terminal heterochromatin bands of the two largest Ks and only to a limited degree in the paracentromeric K heterochromatin. This demonstrates that a collection of permanent preparations of K-S-rearrangements with polytenized heterochromatic and S-homologous K sections of A. lucidus can be used as a source for obtaining K sequences of defined K parts to investigate the molecular evolution of the Ks.

Immunocytological and FISH analysis of pole cell formation and soma elimination of germ line-limited chromosomes in the chironomid Acricotopus lucidus.

In the chironomid Acricotopus lucidus, germ line-soma differentiation becomes evident with the formation of the pole cells and the elimination of the germ line-limited chromosomes (Ks) from the future somatic nuclei of the embryo. Unlike in Drosophila, the early nuclear divisions do not proceed synchronously in A. lucidus. Usually, only one nucleus, the future pole nucleus, penetrates into the pole plasm, always at a telophase stage in the course of a regular mitosis. This happens by chance, depending on the orientation of the mitotic spindles of the early syncytial nuclei. Consequently, the time and the cell cycle at which a nucleus reaches the pole plasm, and pole cells arise, vary between embryos of the same oviposition. When entering the first germ line mitosis, while polar plasm and syncytial plasm are still not separated, some future somatic nuclei begin to eliminate their Ks. While the soma chromosomes (Ss) undergo normal anaphasic migration to the opposite poles, the K chromatids do not separate and remain in the equatorial plane, as demonstrated by fluorescence in situ hybridization using germ line-specific DNA probes. The elimination of the Ks does not occur at the same time in all future somatic nuclei. Nondisjunction of Ks was observed in the first mitosis of the pole nucleus, leading to primordial germ cells with different compositions of their K complements. The pattern and timing of elimination mitoses in the embryos indicate that each of the future somatic nuclei seems to regulate the elimination of the Ks autonomously.

Structural evolution of the germ line-limited chromosomes in Acricotopus.

The elimination of chromatin or whole chromosomes from the future somatic nuclei during germ line-soma differentiation in early embryogenesis is a genetic phenomenon found in a wide variety of animal species. Less is known about the origin, structure, and function of the germ line-limited chromosomes. In the chironomid Acricotopus lucidus fluorescence in situ hybridization (FISH) with labeled soma DNA to "Keimbahn" chromosomes (Ks) and soma chromosomes (Ss) of spermatogonial mitoses revealed that each of the nine different K types possesses large S-homologous sections, mostly in the distal parts of both chromosome arms. Painting probes of the three Ss and of each of their chromosome arms were generated by microdissection of polytene salivary gland chromosomes and subsequent amplification by the degenerate oligonucleotide-primed polymerase chain reaction. Multicolor FISH demonstrated that each of the Ks, with the exception of one K type, was painted by only one of the three S probes. Furthermore, in seven Ks, one chromosome arm was painted by the long-arm probe and the other by the short-arm probe of the S concerned. The hybridization pattern strongly suggests that each of these K types is derived from a specific S. One function of the S-homologous K sections is thought to be determination of the regular occurrence of crossover events, with the resulting chiasmata in these sections ensuring correct segregation of the K homologs during meiosis. Reverse chromosome painting on polytene S sets with a probe generated from metaphase Ks corroborates the above results and produces conclusive evidence for the hypothesis that during evolution the Ks have developed from the Ss by endopolyploidization and rearrangements followed by the accumulation of germ line-specific repetitive DNA sequences in the centromeric regions.

Isolation and chromosomal localization of a germ line-specific highly repetitive DNA family in Acricotopus lucidus (Diptera, Chironomidae).

In the chironomid Acricotopus lucidus, parts of the genome, the germ line-limited chromosomes, are eliminated from the future soma cells during early cleavage divisions. A highly repetitive, germ line-specific DNA sequence family was isolated, cloned and sequenced. The monomers of the tandemly repeated sequences range in size from 175 to 184 bp. Analysis of sequence variation allowed the further classification of the germ line-restricted repetitive DNA into two related subfamilies, A and B. Fluorescence in situ hybridization to gonial metaphases demonstrated that the sequence family is highly specific for the paracentromeric heterochromatin of the germ line-limited chromosomes. Restriction analysis of genomic soma DNA of A. lucidus revealed another tandem repetitive DNA sequence family with monomers of about 175 bp in length. These DNA elements are found only in the centromeric regions of all soma chromosomes and one exceptional germ line-limited chromosome by in situ hybridization to polytene soma chromosomes and gonial metaphase chromosomes. The sequences described here may be involved in recognition, distinction and behavior of soma and germ line-limited chromosomes during the complex chromosome cycle in A. lucidus and may be useful for the genetic and cytological analysis of the processes of elimination of the germ line-limited chromosomes in the soma and germ line.

Immunofluorescence study of spindle microtubule arrangements during differential gonial mitosis of Acricotopus lucidus (Diptera, Chironomidae).

The changes in microtubule arrangements correlated with the behavior of the germ line limited and soma chromosomes were studied during the last unequal gonial mitosis, the so-called differential mitosis, of the chironomid Acricotopus lucidus by indirect immunofluorescence using a monoclonal anti-beta-tubulin antibody, and by simultaneous staining with the DNA-specific fluorescence dye DAPI. An impressive difference in microtubule density between both half spindles was determined in metaphase and during the monopolar anaphasic migration of the germ line limited chromosomes. In the following normal separation of the soma chromosomes, a similar microtubule density in both half spindles occurred. In each of the half spindles, chromosome movement and spindle elongation occurred independently, and in one half spindle two anaphasic chromosome movements ran off one after another, the second without a simultaneous spindle elongation.

Effects of Nosema algerae infections on the gene activity of the salivary gland chromosomes of Acricotopus lucidus (Diptera, Chironomidae).

Larvae of Acricotopus lucidus (Diptera, Chironomidae) were successfully infected with Nosema algerae (Microsporidia, Nosematidae). Treatment of newly hatched larvae with 2-3 x 10(5) spores/ml produced a 59.7%-83.8% rate of microsporidia-infected animals within 6 weeks. One of the host tissues infected was the polytene salivary gland; 31.3%-35.3% of the larvae showed infections in the gland cells. This made it possible to investigate the reaction of the puffing pattern of the polytene host-cell chromosomes to the presence of an intracellular parasite. In slightly or moderately infected salivary gland cells, no change in the regular puffing patterns was observed. Only in heavily infected cells did an inactivation of cell-type-specific Balbiani rings and puffs occur, resulting in a change in the cell-type-specific genetic programme.