Polytene Chromosome Banding Patterns Research Articles

Polytene Chromosome Banding Pattern three Drosophila species from Bangladesh

Polytene Chromosome Banding Pattern three Drosophila species from Bangladesh

The Chironomus species studied by Letha Karunakaran in Singapore, with a review of the status of selected South-East Asian Chironomus

In the 1960s Letha Karunakaran studied the chironomid fauna of Singapore but faced a lack of sufficiently detailed descriptions to enable identification of her material with any certainty. She recognized seven species of Chironomus (s.s) but sent me fixed larval material of only four of these which she tentatively identified as C. apicatus Johannsen 1932, C. costatus Johannsen 1932, C. javanus Kieffer 1924, and C. stupidus Johannsen 1932. She sent fixed larvae to me for confirmation of her identifications, but died before I was able to determine accurate identities from morphology alone. With additional comparative material, along with polytene chromosome banding patterns and DNA barcode sequence from the mitochondrial COI gene, the species have been identified as a form of C. flaviplumus (auct, not Tokunaga)(here called C. flaviplumus Type B), C. circumdatus Kieffer 1916, probably C. striatipennis Kieffer 1910, and Kiefferulus barbatitarsis (Kieffer 1911), respectively. The identification of one species as a form of C. flaviplumus required an assessment of the present state of knowledge of this species where the name has been applied to at least five different species. Determination of a valid name for this species is not currently possible. The confusion of species identification is an indication that there are a number of closely related species which constitute a “C. flaviplumus group”.

A new species of black fly, formerly cytoform C of the Simulium angulistylum complex (Diptera: Simuliidae), from a high mountain in northeastern Thailand.

Cytoform C, one of three cytoforms in the Simulium angulistylum Takaoka & Davies complex from a high mountain in northeastern Thailand was morphologically and molecularly investigated. All known life stages (larva, pupa, adult male and female except egg) were morphologically similar to, but distinguishable from S. angulistylum s. str. and S. isanense Takaoka, Srisuka & Saeung in the adults by the relative length of the fore and hind basitarsi and relative length of the tooth to the claw. It is also morphologically distinct from other species of the S. epistum species-group. Here, it is formally described as a new species, S. prayooki. Molecular genetic data based on mitochondrial cytochrome c oxidase subunit I (COI) also supported the morphological similarity between the new species and the two related known species (S. angulistylum s. str. and S. isanense) as phylogenetic analysis retrieved them all from a single clade and with a low level of interspecific genetic divergence (1.74%). This might possibly have resulted from incomplete lineage sorting as they are likely to share a recent common ancestor. Despite limitation of molecular genetic differentiation, the new species was distinctly different from two other cytoforms of S. angulistylum complex based on polytene chromosome banding patterns and ecology of the immature stages. Thus, this study highlights the necessity of using an integrated approach for fully understanding black fly biodiversity.

Chromosomal and Molecular Diversity in the Simulium ornatum Group (Diptera: Simuliidae) in the Western Tian Shan Range of Central Asia.

Simple SummaryMembers of the Simulium ornatum group are among the most abundant and widely distributed black flies in the Palearctic region. We investigated the chromosomal, molecular, and morphological diversity of this group across the varied landscape of Kyrgyzstan. Morphology and chromosomal band patterns suggested the presence of one or two species, whereas mitochondrial DNA sequences indicated up to three species. We linked the name Simulium mesasiaticum with cytologically undifferentiated sex chromosomes, one of three mitochondrial DNA clusters, and higher elevations. We tentatively associated the name Simulium ferganicum with differentiated sex chromosomes, a second DNA cluster, and lower elevations. One Kyrgyz larva in a third DNA cluster could not be linked to a formal species name. The analyses also indicated that Simulium ornatum, the namesake of the entire group, does not occur in Kyrgyzstan despite previous records. The results demonstrate that using only one source of data does not provide a complete picture of the biodiversity and integrated taxonomy is often necessary for species resolution in the family Simuliidae.By any measure, such as abundance, species diversity or geographic range, the Simulium ornatum species group is one of the most successful Palearctic taxa of black flies. To explore potential diversity in this group in the Tian Shan range of Central Asia, we focused on Kyrgyzstan, in which three nominal morphospecies have been recorded. Among our samples, we morphologically identified S. mesasiaticum Rubtsov and a second possible species tentatively identified as S. ferganicum Rubtsov. By analyzing banding patterns of the larval polytene chromosomes, we discovered two fixed inversions, two sex-linked rearrangements, and 19 autosomal rearrangements, including supernumerary B chromosomes. The chromosomal data indicate minimal diversity of only one or two species across the surveyed area of nearly 50,000 km2. Mitochondrial DNA (CO1) sequences fell into three distinct clusters, possibly representing separate species. The chromosomal, molecular, and morphological data indicate that Kyrgyz populations are unique within the S. ornatum group, but the data sets are not entirely congruent. Thus, reconciling data sets and assigning existing names is tentative. Simulium mesasiaticum is linked with undifferentiated sex chromosomes, one of the three CO1 clades, and higher elevations, whereas S. ferganicum is tenuously associated with differentiated sex chromosomes, a separate CO1 clade, and lower elevations. These associations leave one Kyrgyz larva, which is in a third CO1 clade, unlinked to a formal species name. Our analyses also indicate that S. ornatum Meigen sensu stricto, contrary to previous reports, does not occur in Kyrgyzstan and should be deleted from the country’s faunal list.

A cytogenetic photomap from ovarian nurse cell polytene chromosome of Anopheles stephensi (Diptera: Culicidae): a comparative study

Polytene chromosome banding patterns have long been used to infer the degree of relatedness among taxa and as a standard reference for locating genes, puffs, and inversion breakpoints of unique gene arrangements. Chromosome comparisons between the species over the time have shown organizational changes in patterns which have resulted in adaptive advantages. An attempt has been made to prepare chromosome map from the ovarian nurse cell of Anopheles (Cellia) stephensi Liston, and to re-evaluate the physical map of this species. The photomaps prepared, were compared with other available maps of the past for the same species. The images of the polytene chromosomes have been straightened and divided by numbered divisions and lettered subdivisions. Tentative inversion breakpoints occurring naturally and that recorded from different insecticide resistant strains of An. stephensi is described in the present map. Some of the re-arrangements acquired have been highlighted in the paper in view to provide an overview of the importance of cytogenetic maps to Anopheles biology and the concept of chromosome evolution.

Taxonomic status of the black fly Prosimulium italicum Rivosecchi(Diptera: Simuliidae) based on genetic evidence.

The black fly Prosimulium italicum Rivosecchi, distributed in the Apennines and Sicily, was described as a subspecies of Prosimulium hirtipes (Fries), based on a few morphological details. It subsequently was considered conspecific with P. hirtipes and the name was synonymized. Analyses of polytene chromosome banding patterns and sequences of mitochondrial DNA (COI and COII) revealed deep genetic divergence between P. italicum from Italy and P. hirtipes from northern and central Europe and confirmed the species status of P. italicum. Populations of P. italicum either lack chromosomal inversion IS-9 or carry it as an X-chromosome polymorphism, whereas all analyzed populations of P. hirtipes (Slovakia, Sweden, England, and Scotland) are fixed for IS-9. The average K2P genetic distance was 3.7% between P. italicum and P. hirtipes from northern Europe (Sweden) and 4.3 % between P. italicum and P. hirtipes from central Europe (Slovakia). Cytogenetic analysis showed the presence of two cytoforms of P. hirtipes ('A' in Sweden and Slovakia and 'B' in England and Scotland) and two cytoforms of P. italicum ('A' in Sicily and 'B' in Campania and Basilicata), all of which differ in their sex chromosomes and autosomal polymorphisms, suggesting that P. hirtipes and P. italicum might each be a complex of cryptic species.

Chironomus strenzkei Fittkau, 1968 is a junior synonym of C. striatipennis Kieffer, 1910

Two species of Chironomus with essentially identical adult wing patterns, C. strenzkei and C. striatipennis, have been reported from Brazil. Specimens were collected from the same region in the Manaus area some 50 years apart. Morphological, cytological and DNA Barcode comparisons all confirm that these two species are inseparable on any of the characteristics studied. Moreover, for the mitochondrial COI region investigated, the sequences are completely identical, and polytene chromosome banding patterns are shared between C. strenzkei and C. striatipennis populations from Japan. I therefore argue that the former species must be a junior synonym of C. striatipennis. As a result of the synonymy, C. striatipennis now becomes a new record for California and North America, and hence has a Holarctic distribution. The DNA sequence comparisons suggest that the Brazilian population may have derived from China, rather than Korea as suggested previously, and that the Californian population may not have been introduced from South America, but could equally likely have come from Asia.

Tests of conspecificity for closely related black fly (Diptera: Simuliidae) species of the Simulium striatum group in Thailand.

Four black fly species of the Simulium striatum species group have been recorded in Thailand. These species are morphologically highly similar in all developmental stages except for the number and arrangement of the pupal gill filaments. In this study, we used multiple characters sources, including morphology, cytology, molecular biology and ecology, to test the hypothesis of conspecificity for S. quinquestriatum (Shiraki), S. nakhonense Takaoka & Suzuki and S. chiangmaiense Takaoka & Suzuki. A molecular study based on the cytochrome c oxidase subunit I (COI) was unable to separate these taxa. In contrast, the elongation complex protein 1 (ECP1) sequences clearly differentiate S. quinquestriatum from S. chiangmaiense and S. nakhonense. However, the latter two taxa could not be differentiated based on molecular DNA sequences. Simulium chiangmaiense and S. nakhonense are also similar in the larval habitat characteristics and have undifferentiated polytene chromosome banding patterns. Morphological examination of the number and arrangement of the pupal gill filaments found a number of intermediate forms. Therefore, S. chiangmaiense and S. nakhonense are apparently the same biological species that is polymorphic for the number and arrangement of gill filaments. Thus, we synonymized S. chiangmaiense with S. nakhonense.

<span>Sporozoite Infection Rate and Identification of the Infective and Refractory Species of <i>Anopheles gambiae</i> (Giles) Complex</span>

The ability of Anopheles gambiae complex mosquitoes to transmit Plasmodium infection is known to be variable within sibling species of the complex with strains that cannot transmit the parasite. High sporozoite infection rate recorded showed that A. gambiae mosquitoes are potent malaria vectors in southwestern Nigeria. The aim of this study was to identify the infective and refractory strains of A. gambiae mosquitoes and to determine the sporozoite infection rate in this area. The infective strains were A. gambiae (sensu stricto) and A. arabiensis, while the refractory strains were A. gambiae (sensu stricto). However, ovarian polytene chromosome banding patterns could not be used to distinguish between the infective and refractory strains of A. gambiae (sensu stricto). This study showed that the refractory strains of Anopheles gambiae complex are present, but in low frequencies, in southwestern Nigeria, and that the sibling species of Anopheles gambiae (A. gambiae s.s. and A. arabiensis) are potent malaria vectors.

Bionomic aspects of the Anopheles subpictus species complex in Sri Lanka.

Anopheles subpictus Grassi s.l. (Diptera: Culicidae) functions as a secondary malaria vector to Anopheles culicifacies Giles s.l. (Diptera: Culicidae) in Sri Lanka. The taxon A. subpictus is reported to exist as a species complex comprising four sibling species (A-D) that can be differentiated through polytene chromosome banding patterns and stage-specific morphometric traits in India. Based on the morphological characteristics described for the Indian Subpictus Complex, the presence of all four sibling species has been described in Sri Lanka. As sibling species show distinct bio-ecological characteristics that are important for devising appropriate vector control measures, a study was carried out in six districts in the dry zone of Sri Lanka. The results confirm the presence of all four sibling species, with species C predominating in inland areas and species B in coastal areas. Species C and D were indoor-resting and indoor-feeding, while species B was outdoor-resting with no significant preference for indoor- or outdoor-resting. Species B showed distinct morphological variation in the ornamentation of wings and palpi. Blood meal analysis revealed that species B, C, and D can feed on humans as well as cattle. The differential bio-ecological traits shown by the members of the Subpictus Complex are important for developing appropriate vector control measures in Sri Lanka.

Bionomic aspects of the Anopheles subpictus species complex in Sri Lanka

Anopheles subpictus Grassi s.l. (Diptera: Culicidae) functions as a secondary malaria vector to Anopheles culicifacies Giles s.l. (Diptera: Culicidae) in Sri Lanka. The taxon A. subpictus is reported to exist as a species complex comprising four sibling species (A–D) that can be differentiated through polytene chromosome banding patterns and stage-specific morphometric traits in India. Based on the morphological characteristics described for the Indian Subpictus Complex, the presence of all four sibling species has been described in Sri Lanka. As sibling species show distinct bio-ecological characteristics that are important for devising appropriate vector control measures, a study was carried out in six districts in the dry zone of Sri Lanka. The results confirm the presence of all four sibling species, with species C predominating in inland areas and species B in coastal areas. Species C and D were indoor-resting and indoor-feeding, while species B was outdoor-resting with no significant preference for indoor- or outdoor-resting. Species B showed distinct morphological variation in the ornamentation of wings and palpi. Blood meal analysis revealed that species B, C, and D can feed on humans as well as cattle. The differential bio-ecological traits shown by the members of the Subpictus Complex are important for developing appropriate vector control measures in Sri Lanka.

Fluorescent <em>in situ</em> Hybridization on Mitotic Chromosomes of Mosquitoes

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. One important application of this method is the development of high-quality physical maps useful for improving the genome assemblies for various organisms. The natural banding pattern of polytene and mitotic chromosomes provides guidance for the precise ordering and orientation of the genomic supercontigs. Among the three mosquito genera, namely Anopheles, Aedes, and Culex, a well-established chromosome-based mapping technique has been developed only for Anopheles, whose members possess readable polytene chromosomes 1. As a result of genome mapping efforts, 88% of the An. gambiae genome has been placed to precise chromosome positions 2,3 . Two other mosquito genera, Aedes and Culex, have poorly polytenized chromosomes because of significant overrepresentation of transposable elements in their genomes 4, 5, 6. Only 31 and 9% of the genomic supercontings have been assigned without order or orientation to chromosomes of Ae. aegypti 7 and Cx. quinquefasciatus 8, respectively. Mitotic chromosome preparation for these two species had previously been limited to brain ganglia and cell lines. However, chromosome slides prepared from the brain ganglia of mosquitoes usually contain low numbers of metaphase plates 9. Also, although a FISH technique has been developed for mitotic chromosomes from a cell line of Ae. aegypti 10, the accumulation of multiple chromosomal rearrangements in cell line chromosomes 11 makes them useless for genome mapping. Here we describe a simple, robust technique for obtaining high-quality mitotic chromosome preparations from imaginal discs (IDs) of 4th instar larvae which can be used for all three genera of mosquitoes. A standard FISH protocol 12 is optimized for using BAC clones of genomic DNA as a probe on mitotic chromosomes of Ae. aegypti and Cx. quinquefasciatus, and for utilizing an intergenic spacer (IGS) region of ribosomal DNA (rDNA) as a probe on An. gambiae chromosomes. In addition to physical mapping, the developed technique can be applied to population cytogenetics and chromosome taxonomy/systematics of mosquitoes and other insect groups.

Fluorescent <em>in situ</em> Hybridization on Mitotic Chromosomes of Mosquitoes

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. One important application of this method is the development of high-quality physical maps useful for improving the genome assemblies for various organisms. The natural banding pattern of polytene and mitotic chromosomes provides guidance for the precise ordering and orientation of the genomic supercontigs. Among the three mosquito genera, namely Anopheles, Aedes, and Culex, a well-established chromosome-based mapping technique has been developed only for Anopheles, whose members possess readable polytene chromosomes 1. As a result of genome mapping efforts, 88% of the An. gambiae genome has been placed to precise chromosome positions 2,3 . Two other mosquito genera, Aedes and Culex, have poorly polytenized chromosomes because of significant overrepresentation of transposable elements in their genomes 4, 5, 6. Only 31 and 9% of the genomic supercontings have been assigned without order or orientation to chromosomes of Ae. aegypti 7 and Cx. quinquefasciatus 8, respectively. Mitotic chromosome preparation for these two species had previously been limited to brain ganglia and cell lines. However, chromosome slides prepared from the brain ganglia of mosquitoes usually contain low numbers of metaphase plates 9. Also, although a FISH technique has been developed for mitotic chromosomes from a cell line of Ae. aegypti 10, the accumulation of multiple chromosomal rearrangements in cell line chromosomes 11 makes them useless for genome mapping. Here we describe a simple, robust technique for obtaining high-quality mitotic chromosome preparations from imaginal discs (IDs) of 4th instar larvae which can be used for all three genera of mosquitoes. A standard FISH protocol 12 is optimized for using BAC clones of genomic DNA as a probe on mitotic chromosomes of Ae. aegypti and Cx. quinquefasciatus, and for utilizing an intergenic spacer (IGS) region of ribosomal DNA (rDNA) as a probe on An. gambiae chromosomes. In addition to physical mapping, the developed technique can be applied to population cytogenetics and chromosome taxonomy/systematics of mosquitoes and other insect groups.

Colonization of Pacific Islands by Black Flies (Diptera: Simuliidae)

Abstract. Surveys of black flies on four Pacific islands revealed four species on Okinawa, two on Guam, and none on Rota or Saipan. Simulium aureohirtum Brunetti represents a new record for Guam and the only black fly that is not precinctive to Oceania. The banding patterns of the larval polytene chromosomes of this species are identical among populations from Guam, Okinawa, and Thailand, suggesting that the insular colonizations might be recent.

Partial mitochondrial DNA sequences suggest the existence of a cryptic species within the Leucosphyrus group of the genus Anopheles (Diptera: Culicidae), forest malaria vectors, in northern Vietnam

BackgroundDuring the last decade, Southeast Asian countries have been very successful in reducing the burden of malaria. However, malaria remains endemic in these countries, especially in remote and forested areas. The Leucosphyrus group of the genus Anopheles harbors the most important malaria vectors in forested areas of Southeast Asia. In Vietnam, previous molecular studies have resulted in the identification of only Anopheles dirus sensu stricto (previously known as An. dirus species A) among the Leucosphyrus group members. However, Vietnamese entomologists have recognized that mosquitoes belonging to the Leucosphyrus group in northern Vietnam exhibit morphological characteristics similar to those of Anopheles takasagoensis, which has been reported only from Taiwan. Here, we aimed to confirm the genetic and morphological identities of the members of the Leucosphyrus group in Vietnam.ResultsIn the molecular phylogenetic trees reconstructed using partial COI and ND6 mitochondrial gene sequences, samples collected from southern and central Vietnam clustered together with GenBank sequences of An. dirus that were obtained from Thailand. However, samples from northern Vietnam formed a distinct clade separated from both An. dirus and An. takasagoensis by other valid species.ConclusionsThe results suggest the existence of a cryptic species in northern Vietnam that is morphologically similar to, but phylogenetically distant from both An. dirus and An. takasagoensis. We have tentatively designated this possible cryptic species as Anopheles aff. takasagoensis for convenience, until a valid name is assigned. However, it is difficult to distinguish the species solely on the basis of morphological characteristics. Further studies on such as karyotypes and polytene chromosome banding patterns are necessary to confirm whether An. aff. takasagoensis is a valid species. Moreover, studies on (1) the geographic distribution, which is potentially spreading along the Vietnam, China, Laos, and Myanmar borders; (2) morphological and ecological characteristics; and (3) vectorial capacity of this newly identified cryptic species of An. dirus, which is one of the most important malaria vectors in the mainland of Southeast Asia, are necessary for planning efficient malaria vector control programs in this region.

An intra- and inter-island study of the polytene chromosomes of Simulium exasperans (Diptera: Simuliidae)

The polytene chromosome banding pattern of Simulium exasperans (Craig, 1987) is described for the first time. Three populations of S. exasperans from Moorea and Tahiti were examined cytologically. Interspecific comparisons revealed that S. exasperans is homosequential in chromosomal banding pattern to Simulium cataractarum (Craig, 1987), but contains three species-characteristic floating inversions: IL-1ex.2ex, IIL-1ex,2ex, and IIIL-1ex. No sex-linked inversions were identified; males and females had undifferentiated sex chromosomes. The IIL-1ex,2ex inversion was in Hardy-Weinberg disequilibrium in two of the three populations. From this preliminary data, two cytotypes may exist: cytotype A (Belvedre Cascade population) and cytotype B (1st Afareaitu Cascade and Jardin Public Vaipahi populations). Contrary to phylogenetic and biogeographic data, it is hypothesized that the Belvedre Cascade population (cytotype A) is ancestral to the populations containing cytotype B. Furthermore, a heterozygote advantage may exist in cytotype B.

The polytene dot chromosome of Drosophila: D. melanogaster and D. subobscura.

The chromosome arms are assumed to be homologous within the genus Drosophila. Homology at the level of the polytene chromosome banding pattern between non-sibling species is, however, almost impossible to establish as different processes such as inversion, transposition and unequal crossing over, have disturbed it. Even though the band sequences cannot be followed, we may ask whether there is a correlation in the total number of bands between species. The polytene dot chromosome is an excellent starting point for such an approach. Here we present the detailed cytology of polytene chromosome 4 of D. melanogasterand the polytene dot chromosome of D. subobscura using electron microscopy. The results show that the number of bands is about the same, around 30, in both species. We predict that by using thin sections and electron microscopy for the longer polytene chromosome arms, both species will turn out to have approximately equal band numbers.

Polytene chromosomes of Simulium craigi (Diptera: Simuliidae).

Simulium craigi Adler and Currie is a polymorphic species based on polytene chromosome banding patterns in the long arm of chromosome III (IIIL). Three cytotypes are described based on the predominant IIIL sequences. These correspond to three broad geographic areas: cytotype 'CC' from Pennsylvania; cytotype 'AF' from Ontario and Manitoba; and cytotype 'ACF/BCF' from New Hampshire. In the absence of sympatric populations, these cytotype differences are best explained by clinal variation within a single species. The relationship of S. craigi to other described members of the S. vernum group is discussed.

Polytene chromosome interband DNA is organized into nucleosomes.

The molecular basis that underlies the maintenance of polytene chromosome banding pattern remains unclear. To test the possibility that the decondensed state of interbands is provoked by the absence of nucleosomes, we have subjected chromatin from the previously defined 61C7/C8 interband to digestion with micrococcal nuclease. We have demonstrated that interband DNA forms nucleosomes both in salivary glands and in the bulk of larval tissues. This finding strongly suggests that the difference in compaction between DNA in polytene chromosome bands and interbands results from differences that appear at the higher levels of chromatin organization.

Chromosome differentiated populations of Anopheles cruzii: evidence for a third sibling species.

Anopheles cruzii is the most common species of mosquito in Southeast Brazil and a vector of human and monkey malaria. The banding pattern of the ovarian polytene chromosomes and the frequencies of paracentric inversions of individuals from two populations were studied. A new sequence of bands on the sex chromosome, defined as form C, was disclosed. In both populations where forms A (considered as standard) and C are sympatric no heterozygotes were detected. A sequence of events that could account for the observed changes in the banding sequences of the X chromosome forms was proposed. The frequencies of 22 paracentric inversions were used to assess panmixia and the results indicated the presence of two distinct genetic pools in each population. We consider these results as evidence of another sibling species in the taxon cruzii, characterized by a distinctive form of the X chromosome and provisionally designated Anopheles cruzii species C.