Salivary polytene chromosome mapping of Anopheles (Cellia) subpictus Grassi (Culicidae: Diptera).

With the introduction of molecular taxonomy of mosquitoes, polytene chromosome maps have become indispensable as standard references for locating genes, puffs, and inversion breakpoints of unique DNA sequences. We present a line map and a photomap of the salivary polytene chromosomes of Anopheles (Cellia) subpictus Grassi, an important emerging vector of malaria in India. In addition, we discuss the nature of this species complex consisting of sibling species A, B, C, and D. The comparative study is in relevance to the X chromosome heterozygous inversion differences between 2 allopatric populations of the species and the recognition of 4 X-chromosome inversion genotypes viz: species A-X(+a+b), B-X(ab), C-X(a+b) and D-X(+ab).     

Polytene chromosomes in pupal and adult blackflies (Diptera: Simuliidae)

A number of pupal and adult tissues of eight Australian blackfly species representing three genera, Austrosimulium, Cnephia and Simulium, were examined for the presence of polytene chromosomes. Banded polytene chromosomes were found in malpighian tubules, hind gut, fat body, and ovary, but only those from the malpighian tubules of female adults and pupae were of good quality. A detailed comparison of polytene chromosomes from larval salivary glands and adult malpighian tubules was made in S. ornatipes and, to a limited extent, in S. melatum. The banding patterns of chromosomes from both tissues were found to be identical with minor differences in puffing patterns in S. ornatipes and chromocenter characteristics in S. melatum. A survey of the remaining six species shows five of them to have malpighian chromosomes suitable for detailed cytological analysis. Simultaneous studies of larval, pupal and adult polytene chromosome systems offer a novel approach to the analysis of population problems in blackflies. The ability to recognise sibling species in adults also has potential practical significance in efforts to control vectors of onchocerciasis.     

C banding in polytene chromosomes of Simulium ornatipes and S. melatum (Diptera: Simuliidae)

Polytene and mitotic chromosomes of Simulium ornatipes and S. melatum were subjected to C banding procedures. In both species polytene chromosomes consistently show C banding of centromere regions, telomeres, nucleolar organiser and, unexpectedly, numerous interstitial sites. The interstitial C banding sites correspond to morphologically single polytene bands. Their response is graded and independent of band size. Interstitial C bands in S. ornatipes are scattered throughout the complement, whereas in S. melatum they are clustered. Supernumerary heterochromatic segments in S. ornatipes also exhibit strong C banding and inverted segments can differ from standard in C banding pattern. — Mitotic chromosomes of both species show a single centric C band with indications of two weak interstitial bands in S. ornatipes, suggesting that many C band regions, detectable in polytene chromosomes, are not resolved by present techniques in mitotic chromosomes. — Contrary to current opinion that C banding is diagnostic for constitutive heterochromatin, the interstitial C band sites of polytene chromosomes are regarded as euchromatic. Conversely, the heterochromatic pericentric regions of S. ornatipes are not C banded. — It appears that polytene chromosomes offer a promising system for the elucidation of C banding mechanisms.     

Heterochromatic chromosomes and satellite DNAs of Drosophila nasutoides

Drosophila nasutoides is distinguished from other Drosophila species in that the metaphase karyotype shows a pair of very large V-shaped chromosomes. With Giemsa, a distinctive C-banding pattern is revealed along the arms of this large chromosome, indicating a largely heterochromatic nature. Furthermore, the banding patterns of the arms are symmetrical, indicating that it is an iso-chromosome. A comparison between the metaphase karyotype and polytene chromosomes suggests that the large V chromosome appears as the dot chromosome in polytene squash. One autosome has twice the arm length of typical Drosophila polytene chromosomes and arose either by centric fusion and a pericentric inversion, or by translocation connecting distal ends with a subsequent loss of one centromere. This chromosome appears to have a short arm which ectopically pairs with the proximal region of the long arm, representing a duplication of about ten bands. When the nuclear DNA is examined by neutral CsCl gradient, four satellites are observed. As much as sixty percent of the total DNA appears as satellites in the lysate of larval brains. No satellite was detectable in the lysate of salivary glands. These observations led us to suggest that the heterochromatic nature of the large V chromosome is due to the presence of all four satellites in this chromosome and that this large chromosome appears as the dot because of the under-reduplication of the satellites during polytenization.     

Interrelationships among three derivative forms of the immigrans-Hirtodrosophila radiation: evidence from heterochromatin and polytene chromosomes

The phylogenetic relationships among three derivative forms of the immigrans-Hirtodrosophila radiation viz. Chaetodrosophilella, Zaprionus and the immigrans species group are examined, by comparing the banding patterns of their polytene chromosomes and by analysing the nature of their heterochromatin. Based on the results of these studies it is concluded that D. quadrilineata has a very strong affinity with the members of the immigrans species group, while the genus Zaprionus represents a very distinct evolutionary lineage. This study further indicates that D. quadrilineata is a karyotypically primitive species having five pairs of rods and one pair of dots, while the karyotype of other members of the immigrans species group appears to have undergone modifications through fusions, fissions, inversions and the addition or deletion of heterochromatin to dots and other chromosomes.     

Cytogenetics and evolution of Simulium ornatipes skuse (Diptera:Simuliidae) I. Sibling speciation

Larval populations of the morphologically distinct species Simulium ornatipes and S. neornatipes and a subspecies of the former, S. ornatipes norfolkense, were analysed using polytene chromosome cytology. Fixed inversion differences, distinct differentiated sex chromosomes and unique arrays of chromosomal polymorphisms reveal two sibling species, S. ornatipes A and B, within S. ornatipes, and two, S. neornatipes 1 and 2, within S. neornatipes. Chromosomally S. ornatipes norfolkense is similar to S. ornatipes A. An unprecedented level of shared inversion polymorphism is shown by S. ornatipes A and B indicating a very close relationship between them. While it may be postulated that such a situation has arisen by independent origin of the shared inversions within each species, or by introgression of these rearrangements between the two species, it is concluded that the shared polymorphisms in fact originate from a common ancestor. Within the four chromosomally defined species, differentiated sex chromosome systems occur which may involve any of the three chromosome pairs. Simulium ornatipes A is particularly interesting because there are indications that it may be polymorphic for differentiated sex chromosomes on two different elements and that it may also share a sex chromosome marker with S. ornatipes B, a unique situation in the Simuliidae. The evolution of sex chromosome systems in the four species is compared with that of other Simulids and its intimate connection with speciation emphasised. Finally the data on fixed inversions, differentiated sex chromosomes and shared polymorphisms is used to construct a phylogeny of the four species outlining their patterns of speciation.     

Electron microscopy of surface spread polytene chromosomes of Drosophila and Stylonychia

The ultrastructure of polytene chromosomes of Drosophila and Stylonychia were compared in whole-mount spread preparations. In Drosophila the chromomeres appear as dense, unresolvable structures interconnected by 10-nm interband fibers. In contrast, chromomeres of Stylonychia polytene chromosomes are formed by aggregates of 30-nm loops laterally attached to 10-nm interband fibers. It is suggested that the polytene chromosomes in these two species are analogous rather than homologous structures.     

Detection and location of three simple sequence DNAs in polytene chromosomes from virilis group species of Drosophila

In vitro synthesized RNAs complementary to the three satellite DNAs of Drosophila virilis have been used in a series of in situ hybridization experiments with polytene chromosomes from virilis group species. Gall and Atherton (1974) demonstrated that each of the satellites of D. virilis is comprised of many repeats of a distinct, seven base pair long, simple sequence. With few exceptions, copies of each of these simple sequences are detected in the chromocenters of all virilis group species. This is true even in species which do not possess satellite DNAs at buoyant densities corresponding to those of the satellite DNAs of D. virilis. Small quantities of the three simple sequences are also detected in euchromatic arms of several different species. The same euchromatic location may contain detectable copies of one, two, or all three simple sequence DNAs. The amounts of simple sequences at each location in the euchromatin may vary between species, between different stocks of the same species, and even between individuals of the same stock. The simple sequences located in the euchromatin appear to undergo DNA replication during formation of polytene chromosomes unlike those in heterochromatin. The locations of the euchromatic sequences are not the results of single chromosomal inversion events involving heterochromatic and euchromatic breakpoints.     

INVERSION LENGTH AND BREAKPOINT DISTRIBUTION IN THE DROSOPHILA BUZZATII SPECIES COMPLEX: IS INVERSION LENGTH A SELECTED TRAIT?

Length and position of breakpoints are characteristics of inversions that can be precisely determined on the polytene chromosomes of Drosophila species, and they provide crucial information about the processes that govern the origin and evolution of inversions. Eighty-six paracentric inversions described in the Drosophila buzzatii species complex and 18 inversions induced by introgressive hybridization in D. buzzatii were analyzed. In contrast to previous studies, inversion length and breakpoint distribution have been considered simultaneously. We conclude that: (1) inversion length is a selected trait; rare inversions are predominantly small while evolutionarily successful inversions, polymorphic and fixed, are predominantly intermediate in length; a nearly continuous variation in length, from small to medium sized, is found between less and more successful inversions; (2) there exists a significant negative correlation between length and number of polymorphic inversions per species which explains 39% of the inversion length variance; (3) natural selection on inversion length seems the main factor determining the relative position of breakpoints along the chromosomes; (4) the distribution of breakpoints according to their band location is non-random, with chromosomal segments that accumulate up to eight breakpoints.     

Differences in the quinacrine staining of the chromosomes of a pair of sibling species: Drosophila melanogaster and Drosophila simulans

The patterns of intense fluorescence after staining with quinacrine dihydrochloride were studied in both the polytene chromosomes and mitotic chromosomes of a pair of sibling species, Drosophila melanogaster and D. simulans. Consistent differences between the two species were found in the pattern of fluorescence of both polytene and mitotic chromosomes. In addition, it was discovered that our stock of D. melanogaster (Oregon-R) is polymorphic at one autosomal position for the property of intense fluorescence after quinacrine staining. On the basis of these findings, the usefulness of quinacrine staining in the study of the cytogenetic structure of evolutionarily interesting populations is discussed.     

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.     

兴义维蚋多线染色体研究（英文）

首次在国内对兴义维蚋Simulium (Wilhelmia) xingyiense的多线染色体进行研究, 并提供其多线染色体标准图。选取兴义维蚋的成熟幼虫, 用改良苯酚品红染色法进行唾腺多线染色体制备, 并进行测量、 描述及分析。结果表明： 兴义维蚋多线染色体数目为3对（2n=6）。Ⅰ号染色体具中央着丝粒, Ⅱ和Ⅲ号染色体均为亚中央着丝粒染色体。核仁组织者区位于Ⅰ号染色体短臂近着丝粒端。巴尔比尼氏环和双泡位于Ⅱ号染色体短臂近中央位置。3对染色体的着丝粒区可形成明显的染色中心。兴义维蚋多线染色体具有多态性的倒位, 倒位频率为0.64。兴义维蚋多线染色体的着丝粒、 核仁组织区、 巴氏环、 双泡等主要特征性结构的位置及形态恒定一致,可作为该种的重要鉴别特征。其多态性的倒位可为该蚋种在细胞水平上进行蚋类分类鉴别和系统发育等研究提供基础资料。     

Polytene chromosome sof monogenic and amphogenic Chrysomya species (Calliphoridae,diptera): analysis of banding patterns and in situ hybridization with Drosophila sex determining gene sequences

Standard maps for the five banded polytene chromosomes found in trichogen cell nuclei of the monogenic blowfly Chrysomya rufifacies and the amphogenic Chrysomya pinguis are presented. The chromosomes are highly homologous in the two species; differences in banding patterns are predominantly caused by one pericentric and ten paracentric inversions. In chromosome 5 of the amphogenic Chrysomya phaonis, also analysed in this paper, an additional paracentric inversion was observed. The distribution of species specific inversions indicates that the monogenic C. rufifacies is phylogenetically older than the amphogenic species. The maternal sex realizer locus F'/f on polytene chromosome 5 of C. rufifacies is not associated with a structural heterozygosity. Chromosome pair 6 of C. rufifacies and the sex chromosome pair of C. pinguis are under-replicated in polytene nuclei; they consist of irregular chromatin granules, frequently associated with nucleolus material. Evolution of heteromorphic sex chromosomes in Chrysomya is probably correlated with heterochromatin accumulation. A search for sex determining genes in Chrysomya was initiated using sex determining sequences from Drosophila melanogaster for in situ hybridization. The polytene band 41A1 on chromosome 5 of monogenic and amphogenic Chrysomya species contains sequences homologous to the maternal sex determining gene daughterless (da). Homology to the zygotic gene Sex-lethal (Sxl) of Drosophila is detected in band 39A1 on chromosome 5 of C. rufifacies. The findings reported here are the first evidence for a possible homology between the da gene of Drosophila and the maternal sex realizer F of C. rufifacies. An hypothesis for the evolution of the maternal effect sex determination of C. rufifacies is proposed.Dedicated to Professor Dr. Fritz-Helmut Ullerich on the occasion of his 60th birthday     

Interspecific comparisons of polytene chromosomes in the genus Parasarcophaga (Sarcophagidae: Diptera)

A comparison of the polytene chromosomes of Parasarcophaga argyrostoma, P. ruficornis, P. misera and P. knabi, is presented. There is close resemblance in the banding patterns of the four species. Two independent paracentric inversions in chromosome arm IIR, a small paracentric inversion in chromosome arm IIIR and a pericentric inversion in chromosome V were found to be fixed as interspecific differences.     

Cytogenetics of New Zealand blackflies of the genus Austrosimulium (Diptera: Simuliidae) II. Heterozygote deficiency and non-random association of inversion heterozygotes

We present population cytogenetic data from an analysis of polytene chromosomes of individuals of the common New Zealand blackfly Austrosimulium australense (Schiner). These data show that, in an area of the central North Island of New Zealand (Tarawera), there are, firstly, a number of abrupt changes in inversion polymorphisms between this area and adjacent collection sites. Secondly, there is non-random association of heterozygous inversion pairs in samples from the Tarawera area. Thirdly, we present evidence of a deficiency of heterozygotes for a number of inversions found in this area. We conclude that this may provide evidence for the existence of cryptic species within our samples. We discuss this possibility in terms of the morphological and genetical changes which are likely to occur at speciation, and briefly discuss the mounting evidence for the ubiquity of cryptic species in Diptera.Publication No. 10 from the Evolutionary Genetics Laboratory, University of Auckland.     

Cytological differences and chromosomal rearrangements in four members of the Anopheles dirus complex (Diptera: Culicidae)

A reference photomap of the larval salivary gland, polytene chromosomes of the Anopheles dirus complex (species A) is presented. Samples of species A, B, C, and D from natural populations in Thailand were compared to this standard map using the larval progeny of wild-caught females. All species show differences in their chromosome banding patterns involving band size, number, and shape, particularly at the free ends of the X, 2R, and 2L. These differences provide useful diagnostic characters for separating members of the species complex. However, overall banding patterns are conservative in the group: species A, B, and C are virtually homosequential. Species D is highly polymorphic for a single paracentric inversion in each of the four autosomal arms and has a fixed inversion on the X chromosome. This same X chromosome inversion occurs at low frequency in species A.     

Cytogenetic analysis of the Ethiopian fruit fly <Emphasis Type="Italic">Dacus ciliatus</Emphasis> (Diptera: Tephritidae)

The Ethiopian fruit fly, Dacus ciliatus, is an important pest of cucurbits, which recently invaded the Middle East. The genetics and cytogenetics of D. ciliatus have been scarcely studied. Such information is, however, an essential basis for understanding the biology of insect pests, as well as for the design of modern control strategies. We report here the mitotic karyotype and detailed photographic maps of the salivary gland polytene chromosomes of this species. The mitotic metaphase complement consists of six pairs of chromosomes, including one pair of heteromorphic sex (XX/XY) chromosomes. The heterogametic sex is ascribed to the male. The analysis of the salivary gland polytene complement shows a total number of five long chromosomes (10 polytene arms), which correspond to the five autosomes of the mitotic nuclei, and a heterochromatic mass corresponding to the sex chromosomes. Banding patterns, as well as the most characteristic features and prominent landmarks of each polytene chromosome are presented and discussed. Chromosomal homologies between D. ciliatus and Bactrocera oleae are proposed by comparing chromosome banding patterns and by in situ hybridization of the hsp70 gene.     

Polyploidy and polyteny induced by a hymenopteran parasite in Dasyneura urticae (Diptera,Cecidomyiidae)

Summary Larvae of Dasyneura urticae parasitized by an unidentified species of the Platygasteridae were found to contain giant polyploid nuclei of a polytene type. Comparison of polytene chromosomes of the giant nuclei with those of salivary-gland nuclei of D. urticae has shown that the giant nuclei are derived from the host nuclei, polyploidy and polyteny of these nuclei being, therefore, induced by the parasite.     

Polytene chromosomes of Oxytricha: Biochemical and morphological changes during macronuclear development in a ciliated protozoan

After conjugation in the ciliated protozoan, Oxytricha, polytene chromosomes are formed during the development of a macronucleus from a micronucleus. Here we report a microscopic study of these chromosomes and an analysis of their DNA. The polytene chromosomes of Oxytricha bear a strong morphological resemblance to the polytene chromosomes of the Dipteran salivary gland. The nucleus of a developing macronuclear anlage contains 120±2 polytene chromosomes and each chromosome has an average of 81 bands; a total of about 10,000 bands per nucleus. At a later stage in development, the number of bands per chromosome is reduced by a factor of four, presumably due to fusion of adjacent bands. The polytene chromosomes then break up into their constituent bands, each of which is encased in a vesicle. There are about 2,700 vesicles per nucleus. — During the growth of polytene chromosomes, there is a change in the relative proportion of sequences in the DNA. The DNA from polytene nuclei has a buoyant density of 1.695 g/cc, significantly lighter than the density of the original micronuclear DNA (1.698 g/cc to 1.702 g/cc). We interpret this buoyant density change to be the result of differential replication of DNA sequences during polytene chromosome growth. A second change in DNA composition occurs after the polytene stage of development, shown by a shift in buoyant density to 1.701 g/cc in the DNA of the mature macronucleus. During this second process, the molecular weight of the DNA is reduced from greater than 50×10⁶ daltons to about 2×10⁶ daltons.This paper is No. VI in the series, DNA of Ciliated Protozoa.