Prodiamesa olivacea Meigen and Prodiamesa bureshi Michailova (Diptera, Chironomidae, Prodiamesinae) as a candidate for assessing the genotoxicity of trace metals in fluvial sediments.

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 < 0.001 May, G = 32.722, P < 0.001 September; P. olivacea: G = 47.863, P < 0.001 May, G = 38.742, P < 0.001 September) than the control. Both species from polluted and unpolluted sites showed a high frequency of ectopic conjugations, as between arms B, CD (centromere regions), and E (NOR). Some deformities of mentum and mandibles of P. bureshi (20%) and P. olivacea (35%) were detected. We postulate that the appearances of somatic chromosome aberrations are more sensitive indicators of genotoxicity in the studied species than changes in external morphology. The sensitivity of the P. olivacea and P. bureshi genomes shows that these species are good candidates for detecting the presence of genotoxic compounds in aquatic basins and evaluating their genotoxic effects.

Support for the evolutionary speed hypothesis from intraspecific population genetic data in the non-biting midge Chironomus riparius.

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 species Chironomus riparius to 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 parameter θ that 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.

Characterization of Heterochromatin in the Salivary Gland Chromosomes of Chironomus riparius Meigen and Chironomuspiger Strenzke (Diptera, Chironomidae) by Differential and Fluorochrome Staining.

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.

External Morphology of Larvae of Belgica antarctica Jacobs, 1900 (Diptera, Chironomidae) Obtained from Two Locations in Maritime Antarctica.

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.

Somatic breakpoints, distribution of repetitive DNA and non-LTR retrotransposon insertion sites in the chromosomes of Chironomus piger Strenzke (Diptera, Chironomidae).

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.

Cytogenetic alterations in Prodiamesinae species (Diptera, Chironomidae) from different polluted regions.

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.