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Genome of antarctic endemic Belgica antarctica Jacobs has been sequenced. However, no set of inversion diagnostic markers has ever been assigned for the species. Using the classical method of polytene chromosome squash preparation, we found three heterozygous inversions located on the second (two heterozygous inversions) and third chromosomes (one heterozygous inversion) in the Belgica antarctica population of a cape o f Wiencke Island, 5 00 m to SW from Port Lockroy. The chromosome set and chromosome variability did not differ from those described in the literature (Atchley and Davis 1979). Every salivary gland chromosome ha d its own markers by which it can be determined. However, we did not find a sex-linked inversion on chromosome III and heterozygous inversion on chromosome I, reported in earlier studies . For the first time , we observed a strong heterochromatin band in chromosome III at the telomere of one arm. Our data show not only the stability of the described inversions in the population but also the usefulness of the squash preparation technique in the studies of genetic variability of Belgica antarctica in present time .

This study was aimed to show whether subfossil Chironomidae (Diptera) may be useful tool for assessing toxicity of heavy metals in the aquatic environment. Investigations were carried out in subsidence ponds affected by the activity of metal mining: (1) the older ones formed before mining activity and (2) the younger one formed after the mine was closed. Waters of ponds were analyzed for physico-chemical parameters, whereas sediments were studied for metals (Cd, Pb, and Zn), pH, organic matter, nutrients (TOC, N-tot, and P-tot), and subfossil Chironomidae. High concentrations of Cd 6.7–612 μg g−1, Pb 0.1–10.2 mg g−1, and Zn 0.5–23.1 mg g−1 were found in the seven analyzed sediment cores. In total, 374 head capsules of Chironomidae larvae, belonging to four subfamilies Chironominae, Orthocladiinae, Tanypodinae, and Prodiamesinae, were determined. Both the diversity and density of Chironomidae change in particular sediment cores and layers. However, these changes were not related to metal concentrations, as shown in the statistical calculations (dendrogram of similarities, Mann-Whitney test, Spearman correlations). The only exception was the negative correlations between Cd, Pb, and Zn concentrations and the density of head capsules of Polypedilum sp. We found that organic matter and nutrient contents were important factors that control the spatial distribution of subfossil Chironomidae, mainly Orthocladiinae. Other factors which may influence subfossil Chironomidae distribution in sediment cores are discussed. To sum up, despite very high Cd, Pb, and Zn concentrations, the Chironomidae community was not clearly altered, therefore subfossil Chironomidae taxa appeared to be not useful for reconstructing dozen-years scale changes in the toxicity of the aquatic environment.

Simple Summary The chironomid midge Belgica antarctica Jacobs is endemic to the western Antarctic Peninsula and South Shetland Islands. We provide the first detailed photomicrographic images of the fourth-instar larval head capsule and posterior parapods. We assessed variation in the morphology of larvae from two different collection locations off the coast of the western Antarctic Peninsula and compared it with that available in the literature. A number of differences were identified relating to the size of the larvae, the number of teeth on the mandibles, the number of antennal segments and the length of the antennal blade. Malformations of the mandible and mentum are reported for the first time in this species. Abstract The external morphology of the fourth-instar larva of the Antarctic endemic chironomid midge Belgica antarctica is described. Larvae were collected from Jougla Point (Wiencke Island) and an un-named island close to Enterprise Island, off the coast of the western Antarctic Peninsula. Light microscopy was used to examine and document photographically the structures of the mouthparts (mandible, mentum, premandible, labrum), antennae, pecten epipharyngis, clypeus, frontal apotome and posterior parapods. Measurements of the mouthparts are presented. The data obtained are compared with that available in the literature. A number of differences were identified relating to the size of the larvae, the number of teeth on the mandibles, the number of antennal segments and the length of the antennal blade. Malformations of the mandible and mentum are reported for the first time in this species. Features of larvae of taxonomic value that can be used to determine the species in larval stages are presented. These are of utility in using the larvae to reveal relationships with other species. Larvae are also important in ecological and genotoxicological studies, which require accurate species level identification.

In Memoriam: Professor Iya Kiknadze (1930–2017)

The genome response, realized by structure chromosome rearrangements in the polytene chromosomes of two sibling species Prodiamesa bureshi Michailova and Prodiamesa olivacea Mg., was studied. The larvae of the species were collected in May and September, 2016, from Biala Przemsza River, a metal-mine-affected site in southern Poland, where Zn, Cd, and Pb concentrations in the sediment exceeded many times the reference data and those from unpolluted sites. The water had high contents of different major ions and nutrients. A high spectrum of somatic chromosome aberrations was detected in the salivary gland chromosomes of both species, which defined a high somatic index (from 1.2 to 7), indicating the sensitivity of both genomes to anthropogenic stress. The cells with somatic rearrangements of both species were significantly higher (P. bureshi: G = 25.636, P

We studied the effect of heavy metal contaminations on genome instability of Chironomus annularius sensu Strenzke from three sampling sites on the River Chechło valley in southern Poland and a control site in Saraya, Bulgaria. The analysis of the heavy metal concentrations in sediments at the three river sites indicated concentrations higher than those in the fossil reference data (Zn 83-160 times, Pb 64-250 times, and Cd 210-793). According to the geoaccumulation index (Igeo), the sediments at all Chechło stations were classified as extremely polluted (Igeo class 6) by Cd, Pb and Zn while they were much less polluted by Cu (classes 1-3). The concentration of Cd, Pb and Zn in the sediments studied exceeded 21-79, 11-44 and 18-34 times the toxic effect threshold (TET) respectively; therefore, the sediments potentially have detrimental effects on the fauna. Genome instability was determined by somatic structural chromosome alterations (mainly heterozygous inversions) in the salivary gland chromosomes of C. annularius. On the basis of the number and frequency of these aberrations the somatic (S) and cytogenetic (C) indices were calculated as these indices allow a better quantification of the impact of heavy metals on organisms living in sediments. In the three Chechło sampling sites somatic aberrations occurred at a frequency significantly higher than in the control site (P

This study assesses the effect of sediment contaminants as indicated by the measurement of trace metal levels in two large Bulgarian rivers (river Maritsa and a tributary and the river Chaya) on the genome of the abundant chironomid species Chironomus bernensis Klȍtzli. Six trace metals: Cd, Cr, Cu, Mn, Pb and Zn were analysed in the sediments plus the overlying water. The polytene chromosomes of C. bernensis were very sensitive to the pollutants on the basis of numerous somatic chromosome alterations. Two biomarkers based on the degree of somatic structural chromosome alterations—the somatic and cytogenetic indices were applied. Both indices are a sensitive and reproducible approach for assessing the genotoxic effect of contaminants in the sediments. The somatic index from the differentially contaminated sites varied from 1.93 to 2.61, indicating the sensitivity of the C. bernensis genome to anthropogenic stress. The cytogenetic index revealed a high level of pollution in the rivers with a score between 0.27 and 0.37 (

The constitutive heterochromatin of two homosequential sibling species, Chironomus riparius and Chironomus piger, was studied. The salivary gland chromosomes of both species were analyzed using three staining methods: orcein and C band staining combined with DAPI and CMA3 fluorochrome staining. Both species have the chromosome set 2n = 8, with the same banding pattern and chromosome arm combinations: AB, CD, EF, G, but they differed in number and distribution of heterochromatic bands, AT-rich sequences (DAPI+) and GC-rich sequences (CMA3+). In the polytene chromosomes of C. piger, C-bands were found in centromeres only. They contain two types of repetitive DNA sequences: DAPI+ (very weak) and CMA3+ sequences. However, the polytene chromosomes of C. riparius have many interstitial heterochromatic bands in addition to the centromeric heterochromatin. Some of these bands contain both AT-rich and GC-rich sequences, while others are either AT-rich (DAPI+) or GC-rich (CMA3+). Therefore, these closely related species differ both in the content and localization of constitutive heterochromatin. The species-specific organization of the constitutive heterochromatin can be used as an additional cytogenetic marker for species differentiation.

This paper reports on studies of the effect of heavy metals on the Chironomidae that inhabit the Matylda stream, which has been contaminated for about 100 years by discharge water from a zinc and lead ore mine. Stream sediment was strongly polluted by Cd, Pb, Cu and Zn. These metals did not affect the Chironomidae community but strongly changed the genome system of the Chironomidae species that inhabited the sediment. The salivary gland chromosomes of six species belonging to the two genera Chironomus and Prodiamesa are analyzed. In all species the somatic index is calculated on the basis of somatic chromosome alterations. Chironomus riparius has the highest numbers of somatic alterations and the highest somatic index — 9.67. The smallest chromosome G carries the key structures known as “Balbiani rings”, which play an important role in species development. This chromosome is very sensitive in the genome of the most studied species. The high sensitivity of the C. riparius genome is discussed in light of its DNA organization. The results show a high response of the salivary gland chromosomes to heavy metal pollution, and this makes them a valuable indicator in the assessment of water quality and detection of mutagenic agents in the aquatic environment.

The evolutionary speed hypothesis (ESH) proposes a causal mechanism for the latitudinal diversity gradient. The central idea of the ESH is that warmer temperatures lead to shorter generation times and increased mutation rates. On an absolute time scale, both should lead to an acceleration of selection and drift. Based on the ESH, we developed predictions regarding the distribution of intraspecific genetic diversity: populations of ectothermic species with more generations per year owing to warmer ambient temperatures should be more differentiated from each other, accumulate more mutations and show evidence for increased mutation rates compared with populations in colder regions. We used the multivoltine insect speciesChironomus ripariusto test these predictions with cytochrome oxidase I (COI) sequence data and found that populations from warmer regions are indeed significantly more differentiated and have significantly more derived haplotypes than populations from colder regions. We also found a significant correlation of the annual mean temperature with the population mutation parameterθthat serves as a proxy for the per generation mutation rate under certain assumptions. This pattern could be corroborated with two nuclear loci. Overall, our results support the ESH and indicate that the thermal regime experienced may be crucially driving the evolution of ectotherms and may thus ultimately govern their speciation rate.

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

Intra and interspecific variation was evaluated in two Bulgarian populations (Pancharevo and Koka- lijane) 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 in- stability 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 somatic 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 popu- lation, somatic aberrations were distributed at different points along the chromosomes while in the C. piger popula- tion, 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 individ- ual 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.

The constitutive heterochromatin of two homosequential sibling species, Chironomus riparius and Chironomus piger, was studied. The salivary gland chromosomes of both species were analyzed using three staining methods: orcein and C band staining combined with DAPI and CMA3 fluorochrome staining. Both species have the chromosome set 2n = 8, with the same banding pattern and chromosome arm combinations: AB, CD, EF, G, but they differed in number and distribution of heterochromatic bands, AT-rich sequences (DAPI+) and GC-rich sequences (CMA3+). In the polytene chromosomes of C. piger, C-bands were found in centromeres only. They contain two types of repetitive DNA sequences: DAPI+ (very weak) and CMA3+ sequences. However, the polytene chromosomes of C. riparius have many interstitial heterochromatic bands in addition to the centromeric heterochromatin. Some of these bands contain both AT-rich and GC-rich sequences, while others are either AT-rich (DAPI+) or GC-rich (CMA3+). Therefore, these closely related species differ both in the content and localization of constitutive heterochromatin. The species-specific organization of the constitutive heterochromatin can be used as an additional cytogenetic marker for species differentiation.

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 μg 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.

The effect of environment contaminants on genome instability and changes in enzyme activity (acetylcholinesterase (AChE), glutathione S-transferase activities (GST), etoxyresorufin O-deethylase (EROD) and metallothionein (MT)) of Chironomus riparius Mg. from Bulgarian and Turkish stations over two years (2009, 2010) as well as laboratory reared larvae were studied. Physicochemical analysis of the sediments from the field stations indicated the presence of heavy metal pollutants (Cr, Cu, Mn, Pb, and Cd) whose concentrations were higher than the reference data. Genome instability was determined by somatic structural and functional alterations of the polytene chromosomes. In the field sites of both countries somatic aberrations occurred at a significantly higher frequency (p < 0.001) compared with control and laboratory material. C. riparius in sediments with higher concentrations of trace metals (Derincay River, Turkey and Chaya River, Bulgaria), was found to possess a high spectrum of somatic chromosome rearrangements with a somatic index of 2.53 and 3.25 respectively. Changes in functional activity included decreased activity of the Balbiani rings (BRs) and nucleolar organizer (NOR). The observed chromosome alterations agree with the high degree of trace metal pollution and high activity of the studied enzymes. However, no correlation between single somatic chromosome rearrangements and concentrations of specific metal ions was defined. The data are discussed in the light of the wide variety of interactions of metals in nature. The results show that the genome response and biochemical markers are sensitive markers of toxicity and provide early warning indicators of contaminants in the environment. Bulgarian Ministry of Education and Sciences [02-259/08]; Bulgarian Academy of Sciences [108Y215]; TUBITAKTurkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [108Y215] This study was supported by a grant of the Bulgarian Ministry of Education and Sciences to Project DO, 02-259/08 as well as through an exchange Scientific Program between the Bulgarian Academy of Sciences and TUBITAK 108Y215. WOS:000310431900012

The genome response of model invertebrate and vertebrate species to stress agent in the environment was studied. The both group of species were collected from contaminated water stations along Marisa (Kemera) and Chaya (Asenovgrad) Rivers and terrestrial areas near Asenovgrad. The structural and functional alterations of the salivary gland chromosomes of Chironomus riparius as well as the aberrations of mitotic chromosomes of Apodemus flavicollis and Microtus arvalis were analyzed. In the polytene chromosomes of C. riparius a high spectrum of somatic aberrations were detected, which appeared in significantly higher frequency in comparison of the control (Kemera: G=37.99, df=1, P

Structural aberrations, their frequency and distribution as well as distribution of the tandem repetitive minisatellite DNA clusters of Alu and Hinf elements and two retroelements, the LINE NLRCth1 and the SINE CTRT1, were analyzed in the genome of the chironomid C. piger Strenzke larvae from a Bulgarian population. A consistent somatic variability in the structure of the polytene chromosomes was detected, showing that the C. piger genome is more actively rearranging than supposed before. Breakpoints were concentrated in proximal parts of chromosomes significantly more often than in distal parts. By FISH analysis we could detect only one locus containing Alu elements and 38 Hinf cluster loci which appear to be dispersed equally all over the chromosomes. The retrotransposons NLRCth1 and CTRT1 are present only in a few loci, but highly variant among different individuals. The mean number of NLRCth1 sites per individual was 18.4 +/- 2.09 and of CTRT1 was 54.8 +/- 8.42. A third of breakpoint locations were close to or coincide with a locus occupied by a retroelement (either NLRCth1 or CTRT1). Nineteen percent of breakpoints coincided with Hinf repetitive DNA elements. Some breakpoints were identical in the two sibling species C. piger and C. riparius Meigen (syn.: C. thummi thummi) and are considered as conserved hot spots of chromosome breakage.

The genotoxic action of copper (Cu) on the polytene chromosomes of Chironomus riparius was investigated by analysing structural and functional chromosome aberrations of fourth instars larvae hatched from eggs subject to acute (48 h) exposure with three environmentally relevant concentrations of aqueous Cu (0.005, 0.01, 0.05 mg/l). A dose dependent relationship was observed between Cu concentration and frequency of chromosomal aberrations. A significantly higher frequency of functional alterations, specifically decondensed centromeres and telomeres, and reduction in activity of Balbiani rings, was observed in treated material compared to control. A comparison of breakpoints resulting from treatment with chromium and lead from earlier studies with those Cu-induced identified a series of chromosomal weak points particularly vulnerable to trace metals. We also show that the appearance of structural and functional chromosome aberrations are more sensitive indicators of acute Cu toxicity in chironomid larvae than changes in external morphology.

Changes in the salivary gland chromosomes are described in sibling species Prodiamesa olivacea Mg. and P. bureshi Michailova larvae collected from trace metal polluted and unpolluted areas in Bulgaria and the United Kingdom. Although both species from polluted and unpolluted areas show a high frequency of ectopic conjugations, those between arm B (telomere) and arm F occur at a significantly higher frequency in P. bureshi from unpolluted sites. The reasons for chromosome conjugations and their changes under stress conditions are discussed. Regression of the puffin section 2 of chromosome arm A was significantly higher in P. bureshi from polluted sites, suggesting that trace metal pollution represses transcription at that site. Also, somatic heterozygous inversions and deficiencies occur in chromosomes of both species at the polluted areas. Some deformities ofthe head capsule were observed in both species at the metal-polluted sites. However, our data shows that the genome is a much more sensitive indicator of trace metal pollution than external morphology. The present authors suggest that measuring genotoxic damage at a cellular level provides a subtle and cost-effective indicator of trace metal pollution.

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.