Larva of Glyptotendipes (Glyptotendipes) glaucus (Meigen 1818) (Chironomidae, Diptera)-morphology by Scanning Electron Microscope (SEM), karyotype, and biology in laboratory conditions.

Larvae belonging to the family Chironomidae are difficult to identify. The aim of the present study was to describe the larval morphology of G. (G.) glaucus with the aid of a Scanning Electron Microscope (SEM), the karyotype and biology based on materials obtained from laboratory culture. Describing the morphology of larvae, special attention was paid to rarely or never described structures like the maxilla (lacinia and maxillary palp), the long plate situated below the ventromental plate, and plate X situated between lacinia and mentum. The use of SEM allowed also to obtain better images of labrum and ventromental plate. Morphological features of this species have been supplemented by karyotype and biology of larvae in laboratory conditions. Under controlled experimental conditions we found non-synchronous development of G. (G.) glaucus larvae hatched from one egg mass reflected in different lengths of larvae and emerged imagoes.

A new record of Chironomus (Chironomus) acidophilus Keyl (Diptera, Chironomidae) from the Uzon volcanic caldera (Kronotsky Reserve, Kamchatka Peninsula, Russia), its karyotype, ecology and biology.

Morphology, cytology, ecology and biology of Holarctic Chironomus (Chironomus) acidophilus Keyl, 1960 (Diptera, Chironomidae) was examined from material collected in the geothermal Vosmerka Lake (pH=2.0-2.5). An illustrated redescription of C. acidophilus is given on the basis of adult males reared from field-collected pupae, and of simultaneously collected larvae. Additional larvae belonging to the pseudothummi-complex were identified as C. acidophilus on the basis of their karyotype. The karyotype of C. acidophilus (2n=8) and detailed mapping of the 4 chromosome arms A, E, D and F are provided. The population of C. acidophilus from Kamchatka was found to be karyologically monomorphic. Information on distribution and ecology of C. acidophilus from Vosmerka Lake (total mineralization 1583.5 mg/l) is also given. Chironomus acidophilus is the only species of aquatic insects recorded in this lake. Lack of competition and a richness of food resources contribute to the high abundance (35161 ind./m2) and biomass (11.342 g/m2) of the larvae of C. acidophilus in Vosmerka Lake.

[Non-histone skeleton of mitotic chromosome in situ].

Studying giant nuclei of Chironomus plumosus in situ (Makarov, Chentsov, 2010), we concluded that polythene chromosome structure appears after 2 M NaCl and DNase treatment in presence of 2 mM CuCl2. Cu2+ -ions may stabilize bonds between specific non-histone components, arranged into non-histone matrix of polythene chromosome. Here, we investigated the non-histone matrix of pig embryo mitotic chromosomes in situ, using 2 mM CuCl2-stabilization method. In 2 mM CuCl2-stabilized cells the residual chromosome body (non-histone matrix) could be visualized in every stage of mitosis. Mitotic chromosome non-histone matrix had the same reaction on preliminary hypotonic treatment as normal chromosome: different decondensation of non-histone material was observed. Topoisomerase IIalpha and SMC 1 had uniform localization inside chromosomal body and did not form any axial structures.

[Nuclear protein matrix from giant nuclei of Chironomus plumosus determinates polythene chromosome organization].

Giant nuclei from salivary glands of Chironomus plumosus were treated in situ with detergent, 2 M NaCl and nucleases in order to reveal residual nuclear matrix proteins (NMP). It was shown, that preceding stabilization of non-histone proteins with 2 mM CuCl2 allowed to visualize the structure of polythene chromosomes at every stage of the extraction of histones and DNA. Stabilized NPM of polythene chromosomes maintains their morphology and banding patterns, which is observed by light and electron microscopy, whereas internal fibril net or residual nucleoli are not found. In stabilized NPM of polythene chromosomes, topoisomerase IIalpha and SMC1 retain their localization that is typical of untreated chromosomes. NPM of polythene chromosomes also includes sites of DNA replication, visualized with BrDU incubation, and some RNA-components. So, we can conclude that structure of NPM from giant nuclei is equal to NPM from normal interphase nuclei, and that morphological features of polythene chromosomes depend on the presence of NMP.

Exclusion of mRNPs and ribosomal particles from a thin zone beneath the nuclear envelope revealed upon inhibition of transport.

We have studied the nucleocytoplasmic transport of a specific messenger RNP (mRNP) particle, named Balbiani ring (BR) granule, and ribosomal RNP (rRNP) particles in the salivary glands of the dipteran Chironomus tentans. The passage of the RNPs through the nuclear pore complex (NPC) was inhibited with the nucleoporin-binding wheat germ agglutinin, and the effects were examined by electron microscopy. BR mRNPs bound to the nuclear basket increased in number, while BR mRNPs translocating through the central channel decreased, suggesting that the initiation of translocation proper had been inhibited. The rRNPs accumulated heavily in nucleoplasm, while no or very few rRNPs were recorded within nuclear baskets. Thus, the transport of rRNPs had been blocked prior to the entry into the baskets. Remarkably, the rRNPs had been excluded both from baskets and the space in between the baskets. We propose that normally basket fibrils move freely and repel RNPs from the exclusion zone unless the particles have affinity for and bind to nucleoporins within the baskets.

Centrosome hyperamplification with the formation of multiple asters and programmed chromosome inactivation in aberrant spermatocytes during male meiosis in Acricotopus.

In the germ line of the midge Acricotopus lucidus, an unequal chromosome segregation occurs in the last gonial mitosis prior to meiosis. This results in one daughter cell receiving only somatic chromosomes (Ss), whereas the other cell is given all the so-called germ line limited chromosomes (Ks) in addition to the Ss. The cytokinesis following this differential mitosis is incomplete and the daughter cells remain connected by a permanent cytoplasmic bridge. The cell with the Ss and Ks develops into a primary oocyte or spermatocyte, whereas the cell containing only Ss differentiates as a nurse cell in the female or as an aberrant spermatocyte in the male. When the primary spermatocyte enters meiosis, the Ss in the connected aberrant spermatocyte undergo chromosome condensation but the aberrant spermatocyte remains undivided, with the condensed metaphase status and inactivation of the Ss persisting during both meiotic divisions. These events indicate a programmed inactivation of all chromosomes in the aberrant spermatocyte at the beginning of meiosis. The alterations in the microtubule arrangements and of the distribution of mitochondria in the spermatocytes during meiosis have been followed via live-cell fluorescence labelling with the TubulinTracker and MitoTracker reagents and by transmission electron microscopy. The observations reveal a hyperamplification of the centrosomes and the formation of tetrapolar asters in the non-dividing aberrant spermatocytes containing the condensed Ss. The programmed inactivation of the Ss in the aberrant spermatocyte is suggested to have developed during evolution to inhibit the entry of the aberrant spermatocytes into meiosis, thereby preventing the formation of sperms containing only Ss but no Ks.

The association of Brahma with the Balbiani ring 1 gene of Chironomus tentans studied by immunoelectron microscopy and chromatin immunoprecipitation.

Many steps of gene expression take place during transcription, and important functional information can thus be obtained by determining the distribution of specific factors along a transcribed gene. The Balbiani ring (BR) genes of the dipteran Chironomus tentans constitute a unique system for mapping the association of specific factors along a eukaryotic gene using immuno-electron microscopy (immuno-EM). The chromatin immunoprecipitation (ChIP) technique has provided an alternative, more general method for studying the association of proteins with specific genomic sequences. The immuno-EM and the ChIP methods suffer from different limitations, and thus a combination of both is advantageous. We have established optimal conditions for ChIP on chromatin extracted from the salivary glands of C. tentans , and we have analyzed the association of the SWI/SNF chromatin remodelling factor Brahma (Brm) with the BR1 gene by combined immuno-EM and ChIP. We show that Brm is not restricted to the promoter of the BR1 gene but is also associated with sequences in the middle and distal portions of the gene, which suggests that Brm has additional roles apart from regulating transcription initiation.

[The geographic varyability of the polytene chromosome banding sequence of non-biting midje Chironomus pseudothummi str. (Diptera, Chironomidae)].

The karyotypes and chromosomal polymorphism of Chironomus pseudothummi were investigated in different parts of its range. It was established that chromosomal variability in the natural populations of this species was represented mainly by the inversion polymorphism of arm G. Only rare and unique inversions were found as heterozygous in arms C, D, and E. In total, the 14 banding sequences of polytene chromosomes form the banding sequences pool of Ch. pseudothummi. Geographic differences in distribution of chromosomal banding sequences throughout the range were established. The presence of banding sequences pstG1 and pstG2 is characteristic of European populations. Banding sequence pstG1 was completely vanished with simultaneous increase in frequency of pstG2 and appearance of new inversion banding sequence pstG3 in Siberian populations. The differences in the set of the rare and unique inversions in arms C, D, and E between west-European and west-Siberian populations were revealed.

[Gytogenetics of Chironomus riparius L. from the fishpond in Borok village (Diptera, Chironomidae)].

We have found three inherited inversions in Chironomus riparius populations from the Borok fishpond, namely: (A3d-B1a) in the arm A (C5a-C6a) in the arm D and (B3b-4d/e) in the arm F. Increase of heterochromatin in some bands of chromosome F (B3h, B3h + B3c--C1a) and puffs appearance in the arms C, D and E have been observed. We saw also changes in functional activity of nucleolar organizer (N) and Balbiani rings (BRe/BRb). It has been found that some of inversion breakpoints coincide with the Alu and Hinf satellite DNA localization sites.

A lunar clock changes shielding pigment transparency in larval ocelli of Clunio marinus.

Living in the tidal zones of the sea requires synchronization with the dominant environmental influences of tidal, solar, and lunar periodicity. Endogenous clocks anticipate those geoclimatic changes and control the respective rhythms of vital functions. But the underlying mechanisms are only partly understood. While the circadian clocks in animals are investigated employing neurobiological, molecular, and genetic approaches, clocks with a lunar periodicity have been studied with reference to development and behavior only. Sites of their pacemakers, zeitgeber receptors, and coupled endocrine components are unknown. Here, a lunar-rhythmic change of shielding pigment transparency in the larval ocelli of the intertidal midge Clunio marinus is demonstrated for the first time as a possible access to the neurobiology of lunar timing mechanisms. We studied third instar larvae (Vigo strain) throughout the lunar cycle by light- and electron-microscopy as well as by x-ray fluorescence analysis for the identification of the pigment. Moonlight detection is a prerequisite for photic synchronization of the lunar clock. The larval ocelli of Clunio putatively may function as moonlight receptors and are also controlled by the circalunar clock itself, hence being primary candidates for tracing input and output pathways of the lunar pacemaker. Additionally, the demonstration of a reversible optical change of shielding pigment transparency in Clunio is a novel finding, not reported so far in any other animal species, and reveals a mechanism to enhance photosensitivity under the condition of very dim light. It represents a remarkable change of a sense organ from an imaging device to a radiometer. Its restriction to the developmental stage susceptible to lunar timing elucidates a unique sensory strategy evolved at the level of sensory input. It also raises basic questions about the biochemistry of optically active pigments, like melanin, and their intracellular control.

[A phototube connecting digital cameras with light optic microscopes].

We present here a phototube model making possible connection of a digital camera with light optic microscopes in order to obtain images of microobjects and for their further computer treatment. The advantage of this model is simplicity of its manufacturing and small required expenses as well as an increase in information density for microobject studies. This phototube has been covered by a patent for a useful model N2 48228 registered in the Public Register of RF on September 27, 2005.

Sperm ultrastructure in Chironomoidea (Insecta, Diptera).

The fine structure of spermatozoa from several species of chironomids, of Culicoides sp. (Ceratopogonidae) and of Odagmia pontina (Simulidae) was studied. A synapomorphic feature, consisting of nine kidney-shaped structures forming the centriole adjunct, was found in the chironomid species. All members of Chironomoidea share a mono-layered acrosome and a flagellar axoneme, provided with accessory tubules with 15 protofilaments in their tubular wall. The axoneme has a 9+9+2 pattern, but in an unidentified species of chironomid, a 9+9+0 model was observed where the central complex and the spokes are missing. Sperm motility is, however, maintained in all the examined species. The spermatozoa of this taxon have the tendency to complete maturation during their progression along the deferent ducts. Thus, in the proximal region of these ducts, they often show remnants of the spermatid cytoplasm.

Genotoxic effect of copper on salivary gland polytene chromosomes of Chironomus riparius Meigen 1804 (Diptera, Chironomidae).

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.

In vitro lethal efficacy of leaf extract of Cannabis sativa Linn on the larvae of Chironomous samoensis Edward: an insect of public health concern.

Leaf extract of C. sativa causes paralysis leading to death in larvae of C. samoensis. The extract brought a drastic change in the morphology of sensilla trichoidea and the general body cuticle. The larvae exposed to the leaf extract also showed a significant reduction in the concentration of Mg and Fe, while Mn showed only slight average increase. Since the sensilla trichoidea has nerve connection, it is expected that the toxic principle of the leaf extract has affected the central nervous system. The significant reduction of the level of Fe indicates that the extract could cause the reduction in oxygen binding capacity of the haemolymph, thereby acting as a respiratory poison in addition to its known role as a neurotoxic substance.

DNA in the centromeric heterochromatin of polytene chromosomes is topologically open.

Heterochromatin differs from euchromatin by a set of specific features. We suggested earlier that specific features of heterochromatin result from differences in DNA topology of these two chromatin types and provided explanations for the majority of them (Gruzdev 2000). We proposed that, unlike topologically closed euchromatic DNA, the DNA of heterochromatin is topologically open, i.e. it likely contains single- or doublestrand breaks. In this work, we studied the topological state of DNA in a block of centromeric heterochromatin and in a euchromatic banded region of Chironomus melanotus polytene chromosomes by microfluorimetric methods using the fluorescent intercalating dye ethidium bromide (EB). It was demonstrated that the fraction of topologically closed DNA in heterochromatin blocks is five-fold smaller than in the banded region. The data obtained support the hypothesis proposed.

[The functional morphology of polytene chromosomes in the midge Chironomus pilicornis F. from the permafrost zone reservoirs]. / Funktsional'naia morfologiia politennykh khromosom khironomidy Chironomus pilicornis F. iz vodoemov kriolitozony.

The karyotype and chromosomal polymorphism of Chironomus pilicornis from the reservoirs of Yakutian permafrost zone are described. In the Yakutian populations, of 10 inversion sequences 11 genotypical combinations of these were registered. The level of inversion heterozygosity is 50-70%, but in several populations it makes only 11-29%. In comparison with the Scandinavian populations, significant functional modifications of chromosomal morphology were found. They are associated with the increase in centromeric heterochromatin amount, facultative chromocentre formation, and the appearance of numerous B-chromosomes and nucleoli, including the facultative ones. A possible adaptive significance of such functional modifications conditioned by climate peculiarities is discussed.

[Karyotype of chironomid Xenochironomus xenolabis (diptera, chironomidae)--obligatory miner of fresh water sponges]. / Kariotip khironomidy Xenochironomus xenolabis (diptera, chironomidae)--obligatnogo minera presnovodnykh gubok.

The karyotype of chironomid Xenochironomus xenolabis has been first described. Chromosomes of X. xenolabis have a peculiar spatial organization: subcentromere areas of all chromosomes join together in one common chromocenter. Seven chromosome arms (A = B = C > D > E > F > G) are conventionally recognized in the karyotype. Each arm is subdivided into sections, beginning with the centromere area. The nucleoli are located in arm B and probably D. No chromosome reorganization was found in X. xenolabis. The strong conservatism and ordered organization of the chromosome set in X. xenolabis seem to be determined by a specific life style of its larvae and, consequently, by a relatively constant habitation conditions.

Leucine transport in membrane vesicles from Chironomus riparius larvae displays a mélange of crown-group features.

Leucine uptake into membrane vesicles from larvae of the midge Chironomus riparius was studied. The membrane preparation was highly enriched in typical brush border membrane enzymes and depleted of other membrane contaminants. In the absence of cations, there was a stereospecific uptake of l-leucine, which exhibited saturation kinetics. Parameters were determined both at neutral (Km 33 +/- 5 microM and Vmax 22.6 +/- 6.8 pmol/7s/mg protein) and alkaline (Km 46 +/- 5 microM and Vmax 15.5 +/- 2.5 pmol/7s/mg protein) pH values. At alkaline pH, external sodium increased the affinity for leucine (Km 17 +/- 1 microM) and the maximal uptake rate (Vmax 74.0 +/- 12.5 pmol/7s/mg protein). Stimulation of leucine uptake by external alkaline pH agreed with lumen pH measurements in vivo. Competition experiments indicated that at alkaline pH, the transport system readily accepts most L-amino acids, including branched, unbranched, and alpha-methylated amino acids, histidine and lysine, but has a low affinity for phenylalanine, beta-amino acids, and N-methylated amino acids. At neutral pH, the transport has a decreased affinity for lysine, glycine, and alpha-methylleucine. Taken together, these data are consistent with the presence in midges of two distinct leucine transport systems, which combine characters of the lepidopteran amino acid transport system and of the sodium-dependent system from lower neopterans.