Paraskeva Michailova

CYTOGENETIC CHARACTERISTICS OF A POPULATION OF CHIRONOMUS RIPARIUS MEIGEN 1804 (DIPTERA, CHIRONOMIDAE) FROM A POLLUTED PO RIVER STATION

A population of Chironomus riparius from a Po river station near Moncalieri (a trace-metal polluted station) was studied. In this population was established a great variability of band structure of polytene chromosomes as well as paracentric heterozygous inversions, deletions, deficiencies, partial breaks, diploid chromosome fragments, and changes in functional activity and appearance of heterochromatin. In arms A through F, some bands had an increased size compared to the standard chromosomic map. Some bands appeared in a heterozygous or normal homozygous state or were amplified. In all arms, many condensed stable bands appeared in the decondensed state when compared to the standard map. Asynaptic zones in arms E and G as well as heterozygous Balbiani rings and NORs were established. Very often the 4th chromosome was almost completely heteropycnotic and looded like a ‘pompon’ chromosome. For the first time in this species, a high frequency of ectopic pairings of different arms was observed. Telomeric regions involved in ectopic pairings had a granular appearance, as did some centromeres. The hypothesis is advanced that such a high frequency of structural rearrangements could be correlated with genomic distribution of specific mobile elements.

Functional and structural rearrangements of salivary gland polytene chromosomes of Chironomus riparius Mg. (Diptera,Chironomidae) in response to freshly neutralized aluminium

Although recent work has shown that environmentally relevant concentrations of freshly neutralized aluminium (AI) are bioavailable and toxic to freshwater invertebrates, the genotoxicity of Al has not been examined. Here we show that freshly neutralized Al affects structure and function of the salivary gland polytene chromosomes of the ubiquitous chironomid larva Chironomus riparius over three generations. Exposure to 500 microg l-1 added Al for 24-25 days resulted in a significantly higher frequency of numerous somatic aberrations, while no structural aberrations were found in F1 controls and few in the second and third generation. Aberrations also included deletions of sections of chromosome G of C. riparius larvae as well as deletions of one or more Balbiani rings. Changes in functional activity included decreased activity of the Balbiani rings (BR), and an increase in the number of decondensed centromeres. The activity of the nucleolar organizer (NOR) significantly decreased in F1 chironomids exposed to Al, while in the F2 and F3 generations the NOR showed normal (high) activity. First generation chironomids were generally more susceptible to Al although no clear evidence of tolerance was apparent over three generations. The possible use of alterations in chironomid polytene chromosomes as biomarkers of trace metal pollution is discussed.

Structural-functional rearrangements in chromosome G in Chironomus riparius (Diptera, Chironomidae) collected from a heavy metal-polluted area near Turin, Italy

A natural population of Chironomus riparius from the heavy metal-polluted Piedmont station (Italy) was analysed. Chromosome G, on which the Balbiani rings BRa, BRb and BRc are located, was very sensitive to the pollution. The BR system was considered to be an interesting model for studying the response of the genome to the heavy metal pollution. Together, with a standard activity of BRs (BRc>BRb), we observed a clear reversed level of activity of BRb and BRc: BRc was repressed while BRb became activated. Also, there were a number of cells in which BRb and BRc were collapsed. A change in the activity of the nucleolar organizing region (N) was found, from a very high activity till completely collapsed. For the first time it was established that there was an activation at the telomere region of chromosome G. Additional active sites in sections Dc and E2de were observed. The conjugation of both homologues was often disturbed. A high frequency of ectopic pairings was also observed. In addition, structural rearrangements as inversions and deletions were found. Some deletions led to the formation of pompon-like chromosome G. Chromosome G of C. riparius is suggested to be used to test for the presence of genotoxic concentrations of polluting agents in an aquatic ecosystem.

Nonrandom Chromosomal Distribution of Spontaneous Breakpoints and Satellite DNA Clusters in two Geographically Distant Populations of Chironomus Riparius (Diptera: Chironomidae)

Two geographically distant populations of Chironomus riparius (syn. C. thummi) from two environmentally polluted sites (Santena, Italy and Varna, Bulgaria) show numerous somatic and inherited chromosomal aberrations (inversions, deletions and deficiencies). Fifty-five percent of the observed breakpoints occurred in at least two larvae from both populations. Breakpoints occurring twice or more were considered as ‘common’ structural chromosomal breakpoints. We tested whether such common breakpoints in larvae of the two polluted populations had a random chromosomal distribution or occurred preferentially in specific heterochromatic regions. Distribution of common breakpoints was not random, and proximal regions of first and third chromosome had significantly more common breakpoints than distal ones. By FISH we identified and mapped 56 chromosomal sections containing clusters of two tandem-repetitive satellite DNA families called Hinf and Alu elements. Like the common breakpoints, these repetitive DNA clusters appeared to be significantly more abundant in regions of constitutive heterochromatin such as the pericentromeric regions, while in distal sections of chromosomal arms they were rare or absent. Twenty-four out of 45 common breakpoints (i.e., 53.3%) occurred in cytogenetic sections where Alu and Hinf satellite DNA probes hybridized. The frequency of co-localization between common breakpoints and repetitive DNA hybridization signals was significantly higher than expected by chance. We hypothesize that spontaneous or induced breaks occur more frequently in sections containing blocks of repetitive DNA.

Rearrangements in the salivary gland chromosomes of Chironomus riparius Mg. (Diptera, Chironomidae) following exposure to lead

Abstract The effect of lead (Pb) nitrate on the structural and functional organization of the polytene chromosomes of Chironomus riparius larvae was examined. No specimens with standard polytene chromosomes were found in the material treated with different concentrations of Pb ions. The polytene chromosomes of all individuals exposed to Pb showed various somatic chromosome rearrangements (heterozygous inversions, deletions, duplications and deficiencies), which were not detected in the studied controls. Deletions in chromosome G occurred in a high frequency resulting in the formation of so called “pompons”. The activity of Balbiani rings (BRs) and nucleolar organizer (NOR) significantly decreased with increasing the concentrations of Pb ions. The number of BRc/BRb showing little or no activity (-/-) increased following exposure of the F2 generation to low and high concentrations of Pb ions. Sections of the polytenechromosomes where somatic aberrations were concentrated are considered as “weak” sites. It was shown that in these sites were distributed repetitive DNA elements (Alu and Hinf) that might be activated by stress agents and generated many chromosome rearrangements. It is evident that lead nitrate has a marked genotoxic effect on the salivary polytene chromosomes of Chironomus riparius.

Chironomids (Diptera) and their salivary gland chromosomes as indicators of trace-metal genotoxicity

Abstract The long-term impact of pollution in freshwater aquatic environments can be assessed realistically only if the mutagenic effects of pollutants on organisms inhabiting aquatic biotopes are evaluated. To do this, the larvae of Chironomids (Diptera) can serve as suitable indicators of potential environmental genotoxicity. These larvae exhibit aberrations in their polytene chromosomes, which, when analyzed, indicate an early response to toxicity more efficiently than morphological analysis, up to now the usual technique. Chironomids have excellent salivary gland chromosomes with well-defined bands and permanent somatic pairing, where there are two important regions of active transcription—Balbiani rings (BRs) and Nucleolar organizers (NOR). We have described the responses of different Chironomid species in two situations: in the field and in the laboratory, where we performed Cr, Al, Pb and Cu trace-metal-exposure experiments with Chironomus riparius. In both situations, the genome response consisted in a statistically significant increase in somatic chromosome aberrations and decrease in BR and NOR activity to levels lower than those of larvae under standard conditions. The main chromosome aberrations consisted in inversions, amplifications, deletions and deficiencies. Deletions in the chromosome G in C. riparius transformed it into a pompon-like structure. Chromosome aberrations were not randomly distributed: they occurred mainly in sites with repetitive DNA elements that have a highly interspersed location in the C. riparius genome. Hence C. riparius is the species most likely to produce aberrations and can be used as a model for cost-effective monitoring of the early genomic response to trace metals and other stress agents.

Chromosome variability in Chironomus acidophilus Keyl, 1960 from the Afon Goch, UK - a river subject to long-term trace metal pollution.

The structure and function of the salivary gland polytene chromosomes of the larvae of the chironomid Chironomus acidophilus in a UK river subject to long-term acid (pH < 3.0) mine drainage (AMD) was examined. Two types of chromosome rearrangements (inherited and somatic) were identified. The inherited aberrations in chromosome arms B, D, F as well as all somatic rearrangements (heterozygous inversions, deficiencies and deletions) were observed for the first time in this species. Asynapsis affected all chromosomes and a new region of activity in chromosome G, designated ‘Balbiani ring (BR2)’, was detected. Variations in the activity of the Balbiani rings and NOR were evaluated. Changes in the degree of ‘puffing’ in the telomere region of chromosome G are considered as a compensatory mechanism in response to the environmental conditions in the Afon Goch. As somatic chromosome rearrangements can be caused by stress agents we suggest that the somatic alterations in C. acidophilus indicate the existence of pollution-induced stress.

Genome instability of Chironomus riparius Mg. and Chironomus piger Strenzke (Diptera, Chironomidae)

Abstract Intra and interspecific variation was evaluated in two Bulgarian populations (Pancharevo and Kokalijane) of the two sibling and homosequential species Chironomus riparius Mg. and Chironomus piger Strenzke, by analyzing structural and functional alterations in salivary gland polytene chromosomes. In both species genome instability was demonstrated, which was expressed by structural and functional somatic chromosomal alterations. In the C. riparius population from Pancharevo, living in sediments containing high concentrations of Cu, Pb and Zn, salivary gland cells containing romatic rearrangements appeared at a significantly higher frequency (51.92%) than in the Kokalijane C. piger population, living in heavy metal-unpolluted sediments (21.3%). In the C. riparius population, somatic aberrations were distributed at different points along the chromosomes while in the C. piger population, somatic rearrangements were concentrated in the pericentromeric regions of chromosomes CD, EF and in proximal part of arms D and F. At the cytological level using FISH analysis, both species can be identified also by the different location of tandem-repetitive minisatellite DNA clusters of Alu and Hinf families, and insertion sites of the LINE retrotransposon NLRCTh1. While location of the former is fixed, NLRCth1 appeared to have both fixed and inter-individually variable positions in both species. On average, 19.0 ± 9.5 insertions of NLRCth1 per individual were observed in C. riparius and 5.57 ± 2.09 in C. piger. In both species locations of minisatellite DNA clusters, NLRCth1 retrotransposons and aberration breakpoints concentrated in proximal regions of chromosomes, and the majority of the breakpoints were located in sections containing blocks of repetitive DNA clusters.

A new species of Chironomus Meigen, 1803 (Diptera, Chironomidae) from the southeast of Brazil

The article describes the larva, pupa, adults and karyotype of Chironomus reissi, sp. n., which live on leaf detritus beds of small temporary waters in the southeast of Brazil. The species belongs to the pseudothummi cytocomplex with the chromosome arm combination AE, BF, CD, G.

Comparative Cytogenetic Variation of the Salivary Gland Polytene Chromosomes in Chironomus riparius Mg., 1804 (Diptera, Chironomidae) from Two Polluted Biotopes of Bulgaria and Russia

The structural–functional variation ofChironomus riparius salivary gland polytene chromosomes was studied in two geographically isolated Palearctic regions, Bulgaria (village Pancharevo) and Russia (St. Petersburg). The two biotopes, where larvae were collected, were polluted with various heavy metals from anthropogenic sources. Hereditary paracentric heterozygous inversions were characteristic of the Russian population, whereas somatic paracentric or pericentric heterozygous inversions were more common in the Bulgarian one. All inversions occurred at low frequencies. Other aberrations found in the two populations included somatic deletions resulting in a pompon structure of chromosome IVG, heterozygous translocation between chromosomes IVG and IIIF, enlargement of individual disks, and the appearance of a heterozygous block close to the centromere of chromosome IVG. In addition, changes in functional activity of the nucleolus organizer and Balbiani rings (BRc/BRb) were observed. Several aberration breakpoints proved to coincide with satellites of the Alu and Hinf families.