Polytene Research Articles

Black flies (Diptera: Simuliidae) in the Galapagos Islands: Native or adventive?

Invasive species are a threat to ecosystems worldwide, but determining if a species is adventive or native is not always straightforward. The black flies that inhabit the Galapagos Islands, long known as Simulium ochraceum, are blood-feeding pests of humans and livestock. They first came to the attention of residents in 1989, suggesting a recent arrival. Earlier colonization, however, has been suggested, based largely on polymorphic genetic loci. To address questions of origin, provenance, and length of residency, we conducted a macrogenomic analysis of the polytene chromosomes of the S. ochraceum complex from seven sites in the Galapagos Islands and 30 sites in mainland Ecuador, Central America, and the Caribbean. Among 500 analyzed larvae, we discovered 88 chromosomal rearrangements representing 13 cytoforms, at least seven of which are probably full species. All evidence points to a single, cohesive cytoform with full species status in the Galapagos, conspecific with mainland populations, and widely distributed in the Neotropical Region. It has an identical, nearly monomorphic banding sequence with 10 novel fixed inversions and a subtle but unique Y-linked chromosomal rearrangement across all populations sampled in the Galapagos, the mainland, and the Caribbean. We recalled the name Simulium antillarum from synonymy with S. ochraceum and applied it to the Galapagos black flies, and we established that wolcotti is a junior synonym of antillarum. The time(s) and mode(s) of arrival of S. antillarum in the Galapagos remain uncertain, although the wide geographic distribution, including islands in the Caribbean, suggests that the species is an adept colonizer. Regardless of how long it has been in the archipelago, S. antillarum might have assumed a functional role in the streams of San Cristobal, but otherwise has had a detrimental effect on humans and livestock and potentially on the unique birds and mammals of the Galapagos Islands.

Drosophila Protein Z4 Possesses ZAD Dimerization Domain.

The transcription factor Z4 (putzig) is one of the key proteins that determine the chromatin structure in Drosophila. Z4 is found at the boundaries of bands on polytene chromosomes, and the bands are currently thought to correlate with chromatin domains. Z4 is a component of a protein complex that additionally includes Chromator and BEAF-32, and a conserved domain is necessary to occur at the N end of Z4 to ensure its interaction with the two proteins. In this study, a zinc finger-associated domain (ZAD) domain was identified in Z4. The capability of dimerization was confirmed for the domain by biochemical methods. A dimer model of the domain was obtained using AlphaFold2, and the model structure was confirmed using small-angle X-ray scattering (SAXS). The dimer structure shows a fold typical of ZAD domains.

Markers in the polytene chromosomes of freshwater Chironomus Meigen (Diptera, Chironomidae) species

Based on the morphology of the polytene chromosomes, 21 species of the genus Chironomus Meigen from Bulgaria have been identified. Original pictures of the polytene chromosomes are presented for each species. “Basic” sequences were determined for each arm of the polytene chromosomes which are used to determine the corresponding arms of the chromosomes, and the cytocomplex to which the species belongs. The species are distributed in five cytocomplexes: thummi, pseudothummi, parathummi, lacunarius and modified thummi cytocomplexes. Marker sequences have been revealed on chromosome G that are proposed as a species identifier. Cytogenetic characters by which homosequential species of the genus Chironomus can be distinguished are discussed. This study emphasizes the importance of polytene chromosomes for the taxonomy of the species of Chironomus.

The southernmost member of the Simulium venustum species group (Diptera: Simuliidae) in the Old World: a new species from the Rif Mountains of Morocco

The polytene chromosomes, larva, pupa, and male are described for the black fly Simulium chaouikaidi n. sp., a member of the Simulium venustum group from Morocco. Chromosomally, this new species is one fixed inversion removed from the standard banding sequence for the subgenus Simulium and has microscopically undifferentiated sex chromosomes and minimal autosomal polymorphism. It is homosequential with two other species in the S. venustum species group: S. bergi Rubtsov and S. paramorsitans Rubtsov. Structurally, each known life stage of the new species is distinct from that of all other simuliids in North Africa. This new species is the southernmost member of the S. venustum group in the Old World and is apparently endemic to the Rif Mountains. Its restricted distribution includes Bou Hachem Natural Park where its habitat is afforded some protection.

Two Nested Inversions in the X Chromosome Differentiate the Dominant Malaria Vectors in Europe, Anopheles atroparvus and Anopheles messeae.

The Maculipennis subgroup of malaria mosquitoes includes both dominant malaria vectors and non-vectors in Eurasia. Understanding the genetic factors, particularly chromosomal inversions, that differentiate Anopheles species can provide valuable insights for vector control strategies. Although autosomal inversions between the species in this subgroup have been characterized based on the chromosomal banding patterns, the number and positions of rearrangements in the X chromosome remain unclear due to the divergent banding patterns. Here, we identified two large X chromosomal inversions, approximately 13 Mb and 10 Mb in size, using fluorescence in situ hybridization. The inversion breakpoint regions were mapped by hybridizing 53 gene markers with polytene chromosomes of An. messeae. The DNA probes were designed based on gene sequences from the annotated An. atroparvus genome. The two nested inversions resulted in five syntenic blocks. Only two small syntenic blocks, which encompass 181 annotated genes in the An. atroparvus genome, changed their position and orientation in the X chromosome. The analysis of the An. atroparvus genome revealed an enrichment of gene ontology terms associated with immune system and mating behavior in the rearranged syntenic blocks. Additionally, the enrichment of DNA transposons was found in sequences homologous to three of the four breakpoint regions. This study demonstrates the successful application of the physical genome mapping approach to identify rearrangements that differentiate species in insects with polytene chromosomes.

Study of the association of <i>ouib</i> and <i>nom</i> with heterochromatin in <i>Drosophila melanogaster</i>

In Drosophila, a large group of actively transcribed genes is located in pericentromeric heterochromatin. It is assumed that heterochromatic proteins attract transcription factors to gene promoters. Two proteins, ouib and nom, were previously shown to bind to the promoters of the heterochromatic genes nvd and spok. Interestingly, ouib and nom are paralogs of the M1BP protein, which binds to the promoters of euchromatic genes. We have shown that, like M1BP, the quib and nom proteins bind to CP190, which is involved in the recruitment of transcription complexes to promoters. Unlike heterochromatic proteins, ouib and nom do not interact with the major heterochromatic protein HP1a and bind to euchromatic promoters on polytene chromosomes from the larval salivary glands. The results suggest a new mechanism for the recruitment of transcription factors into the heterochromatic compartment of the nucleus.

<i>Otu</i> and <i>Rif<sup>1</sup></i> double mutant enables analysis of satellite DNA in polytene chromosomes of ovarian germ cells in <i>Drosophila melanogaster</i>

Polytene chromosomes in Drosophila serve as a classical model for cytogenetic studies. However, heterochromatic regions of chromosomes are typically under-replicated, hindering their analysis. Mutations in the Rif1 gene lead to additional replication of heterochromatic sequences, including satellite DNA, in salivary gland cells. Here, we investigated the impact of the Rif1 mutation on heterochromatin in polytene chromosomes formed in ovarian germ cells due to the otu gene mutation. By the analysis of otu11; Rif11 double mutants, we found that, in the presence of the Rif1 mutation, ovarian cells undergo additional polytenization of pericentromeric regions. This includes the formation of large chromatin blocks composed of satellite DNA. Thus, the effects of the Rif1 mutation were similar in salivary gland and germ cells. The otu11; Rif11 system opens new possibilities for studying factors associated with heterochromatin during oogenesis.

Developmental and Housekeeping Genes: Two Types of Genetic Organization in the Drosophila Genome.

We developed a procedure for locating genes on Drosophila melanogaster polytene chromosomes and described three types of chromosome structures (gray bands, black bands, and interbands), which differed markedly in morphological and genetic properties. This was reached through the use of our original methods of molecular and genetic analysis, electron microscopy, and bioinformatics data processing. Analysis of the genome-wide distribution of these properties led us to a bioinformatics model of the Drosophila genome organization, in which the genome was divided into two groups of genes. One was constituted by 65, in which the genome was divided into two groups, 62 genes that are expressed in most cell types during life cycle and perform basic cellular functions (the so-called "housekeeping genes"). The other one was made up of 3162 genes that are expressed only at particular stages of development ("developmental genes"). These two groups of genes are so different that we may state that the genome has two types of genetic organization. Different are the timings of their expression, chromatin packaging levels, the composition of activating and deactivating proteins, the sizes of these genes, the lengths of their introns, the organization of the promoter regions of the genes, the locations of origin recognition complexes (ORCs), and DNA replication timings.

North African Endemism: A New Species of Black Fly (Diptera: Simuliidae) from the Djurdjura Mountains of Algeria.

Discoveries of endemic species highlight areas of biogeographic and conservation interest. Endemic species, however, are often morphologically disguised as more common and widespread species. The larval polytene chromosomes revealed a new species of black fly, Prosimulium fungiforme, from the Djurdjura Mountains of northern Algeria, and its female, male, pupa, and larva are described. The species is chromosomally unique; none of its 11 chromosomal rearrangements are shared with other species. Although the new species structurally resembles Prosimulium rufipes (Meigen) with which it previously has been confused, it can be distinguished from all other known species of Prosimulium in the Western Palearctic based on at least one character in each described life stage. Symbiotic organisms included two species of microsporidia, at least one of which is probably undescribed, one unknown protozoan pathogen novel in simuliids, and the trichomycete fungus Harpella melusinae Léger and Duboscq. Associated simuliid species included at least one new species of the genus Helodon. The new species of Prosimulium is tentatively considered endemic to the mountains of northern Algeria but might be expected in the mountains of eastern Morocco and northern Tunisia and perhaps in Sicily. If its endemic status holds, it would be the only nominal species of black fly unique to Algeria.

Pseudolycoriella hygida (Sauaia and Alves)-An Overview of a Model Organism in Genetics, with New Aspects in Morphology and Systematics.

Pseudolycoriella hygida (Sauaia & Alves, 1968) is a sciarid that has been continuously cultured in the laboratory for nearly 60 years. Studies on this species have contributed to the understanding of DNA puffs, which are characteristic of Sciaridae, and to the knowledge of more general aspects of insect biology, including cell death, nucleolar organization, and the role of the hormone ecdysone during molting. The genome of Psl. hygida has now been sequenced, and it is the third publicly available sciarid genome. The aim of this work is to expand the current knowledge on Psl. hygida. The morphology of the adults is revisited. The morphology of larvae and pupae is described, together with the behavior of immature stages under laboratory conditions. Cytogenetic maps of the salivary gland polytene chromosomes are presented, together with a comparative analysis of the mitotic chromosomes of six different sciarid species. Pseudolycoriella hygida was originally described as a species of Bradysia and recently moved to Pseudolycoriella. We examine here the systematic position of Psl. hygida in the latter genus. Our results extend the characterization of an unconventional model organism and constitute an important resource for those working on the cytogenetics, ecology, taxonomy, and phylogenetic systematics of sciarids.

Цитогенетическая характеристика видов рода Chironomus группы ‘‘plumosus’’ (Diptera, Chironomidae) природно-территориальных комплексов Уральского региона

The polytene chromosomes of the cells of the salivary glands of the larvae of Ch. plumosus L. 1758, Ch. entis Shobanov 1989, Ch. borokensis Kerkis et al. 1988 and Ch. curabilis Beljanina et al. 1990 from 20 reservoirs of six natural-territorial complexes of the Ural region were studied. On the basis of a cytotaxonomic study, it was established that natural complexes differ from each other in the species composition of the “plumosus” group, the ratio (in %) between species and, certain parameters of cytogenetic variability of Ch. plumosus, Ch. entis and Ch. borokensis. Comparative analysis revealed differences between groups of natural complexes. A significantly lower frequency of occurrence of sequence of chromosome disks pluA1.2 was found within the Ural plain-mountainous country (Western foothill and Middle Ural low-mountain regions) compared with natural complexes on the eastern slopes of the Southern Urals and the West Siberian Plain at the junction with the Ural mountain range. Populations of Ch. plumosus of the Ural plain-mountain country have lower cytogenetic distances between themselves compared to populations of Ch. plumosus on the eastern slopes of the Southern Urals and the West Siberian Plain at the junction with the Ural Mountain Range. With respect to Ch. entis, these territories differ from each other in the absence of sequence of chromosome disks entА1.2 within the Ural plain-mountain country. According to cytogenetic distances, the populations of Ch. entis on the eastern slopes of the Southern Urals and the West Siberian Plain country at the junction with the Ural Mountain Range differ more from the population of Ch. entis of the Ural plain-mountain country than among themselves. In the north-south direction, it is necessary to indicate a significantly lower frequency of occurrence of sequence of chromosome disks borB1.2 within the Ural plain-mountain country (Eastern Podgorny region) relative to a natural complex with a more southern location within the West Siberian Plain country (Tura Plain region). For Ch. borokensis the interpopulation cytogenetic distances were calculated for the first time (0.0128±0.0017). Differences in this indicator between the natural complexes of the Ural region have not been established.

Aeroplane wing, a new recessive autosomal phenotypic marker in the malaria vector, Anopheles stephensi Liston

A novel and distinct mutant with a phenotype, aeroplane wing (ae) is reported for the first time in the urban malaria vector Anopheles stephensi. The main aim of this study was to establish the mode of inheritance of the ae gene performing genetic crossings between the mutants and wild types. These mutants show extended open wings that are visible to naked eyes in both the sexes. Mutants were first noticed in a nutritionally stressed isofemale colony. Strategic genetic crosses revealed that the ae gene is a recessive, autosomal, and monogenic trait having full penetrance with uniform expression in its adult stage. Egg morphometric analysis confirmed that these mutants were intermediate variant. No significant differences were observed in the wing venation and size of ae mutants compared to their control parental lines. Further cytogenetic analysis on the ovarian polytene chromosome of ae mutant showed an inversion (3Li) on the 3L arm like its parental line. This ae mutant would be a prominent marker and could be useful to study the functions of related specific genes within its genome.

Otu and Rif1 Double Mutant Enables Analysis of Satellite DNA in Polytene Chromosomes of Ovarian Germ Cells in Drosophila melanogaster.

Polytene chromosomes in Drosophila serve as a classical model for cytogenetic studies. However, heterochromatic regions of chromosomes are typically under-replicated, hindering their analysis. Mutations in the Rif1 gene lead to additional replication of heterochromatic sequences, including satellite DNA, in salivary gland cells. Here, we investigated the impact of the Rif1 mutation on heterochromatin in polytene chromosomes formed in ovarian germ cells due to the otu gene mutation. By the analysis of otu11; Rif11 double mutants, we found that, in the presence of the Rif1 mutation, ovarian cells undergo additional polytenization of pericentromeric regions. This includes the formation of large chromatin blocks composed of satellite DNA. Thus, the effects of the Rif1 mutation are similar in salivary gland and germ cells. The otu11; Rif11 system opens new possibilities for studying factors associated with heterochromatin during oogenesis.

Study of the Association of Ouib and Nom with Heterochromatin in Drosophila melanogaster.

In Drosophila, a large group of actively transcribed genes is located in pericentromeric heterochromatin. It is assumed that heterochromatic proteins recruit transcription factors to gene promoters. Two proteins, Ouib and Nom, were previously shown to bind to the promoters of the heterochromatic genes nvd and spok. Interestingly, Ouib and Nom are paralogs of the M1BP protein, which binds to the promoters of euchromatic genes. We have shown that, like M1BP, the Quib and Nom proteins bind to CP190, which is involved in the recruitment of transcription complexes to promoters. Unlike heterochromatic proteins, Ouib and Nom do not interact with the major heterochromatic protein HP1a and bind to euchromatic promoters on polytene chromosomes from the larval salivary glands. The results suggest a new mechanism for the recruitment of transcription factors into the heterochromatic compartment of the nucleus.

A hybrid RNA FISH immunofluorescence protocol on Drosophila polytene chromosomes

ObjectivesInvestigating protein-DNA interactions is imperative to understanding fundamental concepts such as cell growth, differentiation, and cell development in many systems. Sequencing techniques such as ChIP-seq can yield genome-wide DNA binding profiles of transcription factors; however this assay can be expensive, time-consuming, may not be informative for repetitive regions of the genome, and depend heavily upon antibody suitability. Combining DNA fluorescence in situ hybridization (FISH) with immunofluorescence (IF) is a quicker and inexpensive approach which has historically been used to investigate protein-DNA interactions in individual nuclei. However, these assays are sometimes incompatible due to the required denaturation step in DNA FISH that can alter protein epitopes, hindering primary antibody binding. Additionally, combining DNA FISH with IF may be challenging for less experienced trainees. Our goal was to develop an alternative technique to investigate protein-DNA interactions by combining RNA FISH with IF.ResultsWe developed a hybrid RNA FISH-IF protocol for use on Drosophila melanogaster polytene chromosome spreads in order to visualize colocalization of proteins and DNA loci. We demonstrate that this assay is sensitive enough to determine if our protein of interest, Multi sex combs (Mxc), localizes to single-copy target transgenes carrying histone genes. Overall, this study provides an alternative, accessible method for investigating protein-DNA interactions at the single gene level in Drosophila melanogaster polytene chromosomes.

ZFH-2 is required for Drosophila ovarian follicle development and is expressed at the band/interband boundaries of polytene chromosomes

The transcription factor ZFH-2 has well-documented roles in Drosophila neurogenesis and other developmental processes. Here we provide the first evidence that ZFH-2 has a role in oogenesis. We demonstrate that ZFH-2 is expressed in the wild-type ovary and that a loss of zfh-2 function produces a mutant ovary phenotype where egg chambers are reduced in number and fused. We also show that a loss of zfh-2 function can suppress a daughterless loss-of-function ovary phenotype suggesting a possible genetic relationship between these two genes in the ovary. We also show that ZFH-2 is located at the boundary between bands and interbands on polytene chromosomes and that at a subset of these sites ZFH-2 colocalizes with the insulator/promoter cofactor CP190.

DIFFERENT PROTEIN GROUPS INVOLVED IN TRANSCRIPTION REGULATION IN GENES OF DEVELOPMENT AND HOUSEKEEPING IN <i>DROSOPHILA</i>

Antibodies to histone modifications and an insulator protein involved in the processes of transcription initiation and elongation are mapped in Drosophila polytene chromosomes. The CHRIZ protein (chromatin insulator) and H3K36me3 histone modification (RNA elongation) are detected only in the localization of housekeeping genes (interbands and gray bands of polytene chromosomes) and never in the regions of development genes (black bands and large puffs arising from them). Antibodies to H3S10P histone modification, which is associated with the initial elongation of the RNA strand during transcription, are found exclusively in small puffs, but not in housekeeping gene localization sites or large ecdysone-induced puffs, where housekeeping genes are localized. Antibodies to H4R3me2 histone modification (a co-repressor of the ecdysone receptor) are detected only in large α-induced puffs.

Seasonal variations and other changes in the geographical distributions of different cytospecies of the Simulium damnosum complex (Diptera: Simuliidae) in Togo and Benin

Simulium damnosum s.l., the most important vector of onchocerciasis in Africa, is a complex of sibling species described on the basis of differences in their larval polytene chromosomes. These (cyto) species differ in their geographical distributions, ecologies and epidemiological roles. In Togo and Benin, distributional changes have been recorded as a consequence of vector control and environmental changes (e.g. creation of dams, deforestation), with potential epidemiological consequences. We review the distribution of cytospecies in Togo and Benin and report changes observed from 1975 to 2018. The elimination of the Djodji form of S. sanctipauli in south-western Togo in 1988 seems to have had no long-term effects on the distribution of the other cytospecies, despite an initial surge by S. yahense. Although we report a general tendency for long-term stability in most cytospecies’ distributions, we also assess how the cytospecies’ geographical distributions have fluctuated and how they vary with the seasons. In addition to seasonal expansions of geographical ranges by all species except S. yahense, there are seasonal variations in the relative abundances of cytospecies within a year. In the lower Mono river, the Beffa form of S. soubrense predominates in the dry season but is replaced as the dominant taxon in the rainy season by S. damnosum s.str. Deforestation was previously implicated in an increase of savanna cytospecies in southern Togo (1975–1997), but our data had little power to support (or refute) suggestions of a continuing increase, partly because of a lack of recent sampling. In contrast, the construction of dams and other environmental changes including climate change seem to be leading to decreases in the populations of S. damnosum s.l. in Togo and Benin. If so, combined with the disappearance of the Djodji form of S. sanctipauli, a potent vector, plus historic vector control actions and community directed treatments with ivermectin, onchocerciasis transmission in Togo and Benin is much reduced compared with the situation in 1975.

A comparative analysis of the chromosomes of three FARQ species complex members, Ceratitis rosa, C. quilicii, and C. fasciventris F2 (Diptera: Tephritidae).

The Ceratitis FARQ species complex consists of four highly destructive agricultural pests of Africa, namely C. fasciventris, C. anonae, C. rosa, and C. quilicii. The members of the complex are considered very closely related and the species limits among them are rather obscure. Their economic significance and the need for developing biological methods for their control makes species identification within the complex an important issue, which has become clear that can only be addressed by multidisciplinary approaches. Chromosomes, both mitotic and polytene, can provide a useful tool for species characterization and phylogenetic inference among closely related dipteran species. In the current study, we present the mitotic karyotype and the polytene chromosomes of C. rosa and C. quilicii together with in situ hybridization data. We performed a comparative cytogenetic analysis among the above two species and C. fasciventris, the only other cytogenetically studied member of the FARQ complex, by comparing the mitotic complement and the banding pattern of the polytene chromosomes of each species to the others, as well as by studying the polytene chromosomes of hybrids between them. Our analysis revealed no detectable chromosomal rearrangements discriminating the three FARQ members studied, confirming their close phylogenetic relationships.

Development of N.K. Koltsov’s Idea about Genetic Organization of Interbands in Drosophila melanogaster Polytene Chromosomes

Development of N.K. Koltsov’s Idea about Genetic Organization of Interbands in Drosophila melanogaster Polytene Chromosomes