Cytogenetic and isozyme profile of Sabethes cyaneus: a mosquito of the neotropical canopy

Sabethes cyaneus, a newly colonized culicine mosquito from the Panamanian forest canopy, has a distribution range from Honduras to Argentina. Cytogenetic studies, the first on any Sabethes species, revealed a karyotype of three pairs of homomorphic chromosomes (2n = 6). Well-developed polytene chromosomes were discovered in the larval salivary glands and a photomap standard was constructed. Clear banding patterns and consistent landmarks distinguished each of the six arms. Substantial asynapsis occurred in the three polytene chromosomes, although the banding pattern of the homologous regions appeared homosequential. A nucleolar organizer was localized in region 5A of the shortest chromosome by the recurrent association of this region with the nucleolus. The large band in region 5A was found heterozygous for width and a deletion. Additional, less conspicuous, polymorphisms for band width and staining intensity were distributed throughout the genome. Biochemical studies of 31 enzyme loci revealed 10 loci to be polymorphic, with an average heterozygosity of 13 percent. Differential expression in developmental stages occurred for 11 loci involving six enzymes.     

Karyotype structure in species of Propsilocerus genus (Diptera, Chironomidae)

Karyotype structure and polytene chromosome banding patterns were studied in two Orthocladiinae siblings--Propsilocerus akamusi (China) and Propsilocerus jacuticus (Russia). Both species have haploid number of chromosome typical for Orthocladiinae (n = 3). An unusual structure of centromeric regions was observed in all three chromosomes of karyotypes in both species. Photomaps of polytene chromosomes are presented. A comparison of karyotypes of P. akamusi and Propsilocerus jacuticus revealed a high level of homology in their banding sequences, however, the presence of fixed paracentric inversions in chromosomal arms IR, IIR, IIIR of Propsilocerus jacuticus has shown a clear-cut phylogenetic divergence. No chromosomal polymorphism was found in both species.     

Cytogenetics of Zaprionus indianus Gupta (Diptera: Drosophilidae): Nucleolar organizer regions,mitotic and polytene chromosomes and inversion polymorphism

Zaprionus indianus, a member of the family Drosophilidae, is one of the commonest and most widespread species in India. It exploits a variety of fermenting fruits in nature. The nucleolar organizer regions (NORs), mitotic and polytene chromosomes were studied. A standard map of the polytene chromosomes has been constructed in order to locate break-points precisely for naturally occurring chromosomal rearrangements. Analysis of population samples of this species from four different geographical areas has revealed the presence of a single paracentric inversion in the second chromosome. Our quantitative data on the above inversion have confirmed the excess of inversion heterozygotes in nature.     

Linear and spatial organization of polytene chromosomes of the African malaria mosquito Anopheles funestus

Anopheles funestus Giles is one of the major malaria vectors in Africa, but little is known about its genetics. Lack of a cytogenetic map characterized by regions has hindered the progress of genetic research with this important species. This study developed a cytogenetic map of An. funestus using ovarian nurse cell polytene chromosomes. We demonstrate an important application with the cytogenetic map for characterizing various chromosomal inversions for specimens collected from coastal Kenya. The linear and spatial organization of An. funestus polytene chromosomes was compared with the best-studied malaria mosquito, An. gambiae Giles. Comparisons of chromosome morphology between the two species have revealed that the most extensive chromosomal rearrangement occurs in pericentromeric heterochromatin of autosomes. Differences in pericentromeric heterochromatin types correlate with nuclear organization differences between An. funestus and An. gambiae. Attachments of chromosomes to the nuclear envelope strongly depend on the presence of diffusive beta-heterochromatin. Thus, An. funestus and An. gambiae exhibit species-specific characteristics in chromosome-linear and -spatial organizations.     

Homology of Balbiani Ring DNA in two closely related Chironomus species

Cytogenetic analysis indicates that Balbiani Ring 2 (BR 2) in the two sibling species Chironomus tentans and Chironomus pallidivittatus arises from identifically banded segments in the salivary gland polytene chromosomes, although chromosomal rearrangements have occurred. In situ hybridization of BR 2 RNA to the polytene chromosomes of each individual species, as well as their F1 hybrids, reveals that the repetitious BR 2 DNA in the two species has, within the limits of the technique, retained identity of nucleotide sequences and degree of repetition. The DNA of the naturally expressed BR 1 and BR 3 in both species and that ot the galactose induced BR 6 in C. pallidivittatus did not hybridize with BR 2 RNA, indicating that these BR's are different from BR 2 with regard to sequence content.     

Characterization of extrachromosomal DNA in the flesh fly Sarcophaga bullata

The polytene pupal foot pad cells of the flesh fly Sarcophaga bullata contain numerous extrachromosomal DNA containing granules. We have determined both the origin and the nature of the DNA sequences present in these granules. Studies done with quinacrine staining of seven day old pupal foot-pad polytene nuclei showed that the granules fluoresced very brightly while the chromosomal bands to which the granules were attached did not. The only other highly fluroescent regions of the polytene karyotype were the centromeric heterochromatin of chromosomes C and E and several bands associated with the nucleolus of Chromosome A. When polytene nuclei were hybridized in situ with cRNA made from highly repetitive DNA, many of the granules positively labeled. Most of the label on these slides was concentrated on the centromeric heterochromatin of chromosomes C and E. Quinacrine staining of the foot-pad cells at very early stages of pupal development showed that when granules were present, they were always closely associated with the same two centromeric regions, those of chromosomes C and E. Since the highly repetitive DNA located in these centromeric regions is underreplicated, we conclude that the granules result from an extrusion process which takes place early during the polytenization of these cells. The chromosomal integrity of the centromeric heterochromatin of chromosomes C and E is apparently disrupted and repetitive sequences are dissociated from the chromosomes as DNA granules which then secondarily become associated with chromosomal bands throughout the nucleus.     

Maintenance of chromosome arm integrity between two Anopheles mosquito subgenera

Low-resolution chromosomal homology between Anopheles gambiae and A. albimanus was determined by polytene chromosome in situ cross hybridization of 17 recombinant DNA and PCR products hybridizing to 23 loci. Hybridization results reflect that the chromosomes have rearranged in the form of autosomal whole-arm translocations and numerous paracentric inversions and not by large detectable pericentric inversions or partial arm translocations. An. gambiae and An. albimanus chromosomes hence differ from each other by possessing alternative autosomal arm associations and rearranged internal structure of each arm, but the integrity of the whole arms has remained conserved. In addition, a photomap of the larval salivary gland polytene chromosomes of An. albimanus that we used to identify sites of hybridization in this species is presented that delineates further banding details than maps published in the past.     

Species-specific localization of DNA from pericentromeric heterochromatin on polytene chromosomes in the salivary gland cells and 3D-nuclear organization nurse cells in Drosophila virilis and Drosophila kanekoi (Diptera: Drosophilidae)

Microdissection of the chromocenter of D. virilis salivary gland polytene chromosomes has been carried out and the region-specific DNA library (DvirIII) has been obtained. FISH was used for DvirIII hybridization with salivary gland polytene chromosomes and ovarian nurse cells of D. virilis and D. kanekoi. Localization of DvirIII in the pericentromeric regions of chromosomes and in the telomeric region of chromosome 5 was observed in both species. Moreover, species specificity in the localization of DNA sequences of DvirIII in some chromosomal regions was detected. In order to study the three-dimensional organization of pericentromeric heterochromatin region of polytene chromosomes of ovarian nurse cells of D. virilis and D. kanekoi, 3S FISH DvirIII was performed with nurse cells of these species. As a result, species specificity in the distribution of DvirIII signals in the nuclear space was revealed. Namely, the signal was detected in the local chromocenter at one pole of the nucleus in D. virilis, while the signal from the telomeric region of chromosome 5 was detected on another pole. At the same time, DvirIII signals in D. kanekoi are localized in two separate areas in the nucleus: the first belongs to the pericentromeric region of chromosome 2 and another to pericentromeric regions of the remaining chromosomes.     

Cytogenetics of New Zealand blackflies of the genus Austrosimulium (Diptera: Simuliidae) 1. The cytogenetics of Austrosimulium australense

Abstract

This study undertakes a cytogenetic analysis of the New Zealand blackfly species Austrosimulium australense (Schiner). The principles of such an approach are outlined, and previous taxonomic studies of the genus Austrosimulium, in particular the taxonomic position of A. australense, are discussed. Populations from North Island localities covering a wide area were sampled and analysed for polymorphisms in the polytene chromosomes, taken from salivary glands of larvae. In all, 1018 larvae from 49 sites were analysed. A polytene chromosome map of A. australense is presented, with details of chromosomal inversions found in salivary gland cells. Three geographical zones are designated, according to the incidence of certain chromosomal polymorphisms.     

Cytotaxonomic characteristics of the genus Glyptotendipes Kieffer (Chironomidae, Diptera) from fish and retention ponds (Silesia, southern Poland)

The cytotaxonomic characteristics of species of the genus Glyptotendipes (Chironomidae): G. glaucus Meigen, G. paripes Edwards, and G. barbipes (Staeger) are described. The studied material was collected from a fish pond at Go?ysz and a sewage retention pond at Chybie in Silesia. All the studied species have the chromosome set 2n = 8, but with many specific structural and functional changes. The cytogenetic data of G. glaucus showed that the studied specimens of this species have been produced by the introgressive hybridization of two sibling species: G. glaucus x G. pallens, and subsequent crossing-over in the hybrid chromosome CD. Owing to this process, the band pattern of chromosome arm D coincided with those of G. pallens. New aberrations (homo- and heterozygous deletions in arm G as well as heterozygous inversions in chromosome arm B) were detected in G. glaucus. Specific band sequences were discovered in chromosomes of G. paripes. The band patterns established in chromosomes AB and G of this species were identical with those of Siberian populations. The banding patterns of the polytene chromosomes of G. barbipes do not differ from the standard. However, high frequency of pericentric inversion of chromosome AB was established. Many new puffs were found in the polytene chromosomes of all the studied species. Their nucleolar organizer was very sensitive to environmental changes. In G. glaucus it appeared in three different states: very active, slightly active, and heterozygous state. The organic pollution existing in the sewage retention pond may contribute to possible mutations and chromosomal damage in Glypotendipes species. Structural and functional rearrangements of the polytene chromosomes of these species mobilized their genomes and provide for survival under polluted conditions.     

Chromosomal and cross-mating evidence for two species within Anopheles (A.) coustani (Diptera: Culicidae)

Abstract Examination of the polytene chromosomes of the fourth instar larvae provides evidence for the existence of two sibling species within the taxon Anopheles (Anopheles) coustani Laveran. Crosses between the two chromosomal types produces sterile male offspring. Since no classical morphological differences can be demonstrated in the adults, the probable existence of such characters in the larvae and pupae is discussed.     

On the origins of tandemly repeated genes: Does histone gene copy number inDrosophila reflect chromosomal location?

Widely regarded beliefs aboutDrosophila histone gene copy numbers and developmental requirements have been generalized from fairly limited data since studies on histone gene arrangements and copy numbers have been largely confined to a single species,D. melanogaster. Histone gene copy numbers and chromosomal locations were examined in three species:D. melanogaster, D. hydei andD. hawaiiensis. Quantitative whole genome blot analysis of DNA from diploid tissues revealed a tenfold variability in histone gene copy numbers for these three species. In situ hybridization to polytene chromosomes showed that the histone DNA (hDNA) chromosomal location is different in all three species. These observations lead us to propose a relationship between histone gene reiteration and chromosomal position.     

Cytogenetic studies of Chironomus circumdatus from India (Diptera: Chironomidae)

Chironomus circumdatus is one of the most common and wide-spread species in India. Cytogenetic studies pertaining to the mitotic and polytene chromosomes, nucleolar organizer regions, C-banding and naturally occurring chromosomal polymorphism have been carried out for the first time in this Indian species. Altogether seven inversions comprising six paracentric and one pericentric are detected in the Indian populations. The distribution of inversions in relation to different environmental conditions is discussed.     

Immunological identification of a chromocenter-associated protein in polytene chromosomes of Drosophila

An antiserum directed against a non-histone chromosomal protein was found to stain preferentially the heterochromatic chromocenter of the polytene chromosomes of the salivary glands of several Drosophila species. The highly condensed α-heterochromatin stained most prominently. The chromocenter-associated antigen probably consists of a single polypeptide of molecular weight of 38000. This protein may play a role in the specific chromatin condensation of this chromosomal region.     

Malpighian tubule polytene chromosomes of Culex quinquefasciatus (Diptera,Culicinae)

Dipteran polytene chromosomes provide an excellent model for understanding in species complexes, as well as for structural and functional cytogenetics. The status of species in the Culex pipiens complex is controversial and the use of polytene chromosomes for cytogenetic analysis in the subfamily Culicinae has been difficult because of methodological problems. In this study, Malpighian tubule polytene chromosomes were obtained from young (0 to 12 h, 20 C) and old (20 to 42 h, 28 C) laboratory-bred C. pipiens quinquefasciatus pupae. The chromosome maps for this species were constructed and compared with published data for C. pipiens pipiens and C. p. quinquefasciatus. Although the banding patterns were conserved between subspecies, analysis of the structural variations in the bands and interbands revealed differences apparently related to the physiological stage and ecogeographical strain. The organization of the centromeric regions in larval and pupal chromosomes showed greater similarity to each other than did those of pupal and adult chromosomes. The use of pupal polytene chromosomes for in situ hybridization with vector competence probes is discussed.     

The use of surface spread polytene chromosomes for high resolution fluorescence microscopic analyses

The technique of surface spreading of polytene chromosomes is applied to fluorescence microscopy. With bisbenzimide Hoechst 33258 stained surface spread polytene chromosomes from the dipteran species Chironomus thummi piger, depiction of the band-interband structure is close to that of electron micrographs of the same enlargement.     

Mitotic and polytene chromosomes analysis of the oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae)

The Oriental fruit fly, Batrocera dorsalis s.s. (Hendel) is one of the most destructive agricultural pests, belonging to a large group of difficult to distinguish morphologically species, referred as the B. dorsalis complex. We report here a cytogenetic analysis of two laboratory strains of the species and provide a photographic polytene chromosome map from larval salivary glands. The mitotic complement consists of six chromosome pairs including a heteromorphic sex (XX/XY) chromosome pair. Analysis of the polytene complement has shown a total of five polytene chromosomes (10 polytene arms) that correspond to the five autosomes. The most important landmarks of each polytene chromosome and characteristic asynapsis at a specific chromosomal region are presented and discussed. Chromosomal homology between B. dorsalis and Ceratitis capitata has been determined by comparing chromosome banding patterns. The detection of chromosome inversions in both B. dorsalis strains is shown and discussed. Our results show that the polytene maps presented here are suitable for cytogenetic analysis of this species and can be used for comparative studies among species of the Tephritidae family. They also provide a diagnostic tool that could accelerate species identification within the B. dorsalis complex and could shed light on the ongoing speciation in this complex. Polytene chromosome maps can facilitate the development of biological control methods and support the genome mapping project of the species that is currently in progress.