Drosophila Bifasciata Research Articles

Chromosome-Level Assembly of Drosophila bifasciata Reveals Important Karyotypic Transition of the X Chromosome

The Drosophila obscura species group is one of the most studied clades of Drosophila and harbors multiple distinct karyotypes. Here we present a de novo genome assembly and annotation of D. bifasciata, a species which represents an important subgroup for which no high-quality chromosome-level genome assembly currently exists. We combined long-read sequencing (Nanopore) and Hi-C scaffolding to achieve a highly contiguous genome assembly approximately 193 Mb in size, with repetitive elements constituting 30.1% of the total length. Drosophila bifasciata harbors four large metacentric chromosomes and the small dot, and our assembly contains each chromosome in a single scaffold, including the highly repetitive pericentromeres, which were largely composed of Jockey and Gypsy transposable elements. We annotated a total of 12,821 protein-coding genes and comparisons of synteny with D. athabasca orthologs show that the large metacentric pericentromeric regions of multiple chromosomes are conserved between these species. Importantly, Muller A (X chromosome) was found to be metacentric in D. bifasciata and the pericentromeric region appears homologous to the pericentromeric region of the fused Muller A-AD (XL and XR) of pseudoobscura/affinis subgroup species. Our finding suggests a metacentric ancestral X fused to a telocentric Muller D and created the large neo-X (Muller A-AD) chromosome ∼15 MYA. We also confirm the fusion of Muller C and D in D. bifasciata and show that it likely involved a centromere-centromere fusion.

Males do not prolong copulation in response to competitor males in the polyandrous fly Drosophila bifasciata

Males in many taxa exhibit behavioural plasticity in response to the perceived threat of sperm competition. Drosophila males prolong mating in response to the presence of competitor males before copulation. The benefits of this behaviour to males are evident in Drosophila melanogaster. However, the adaptive nature of the trait is challenged by the observation that it is present in four other Drosophila species, two of which are largely monandrous, raising the possibility that this plasticity is not evolutionarily labile. In the present study, behavioural plasticity and the mating system in Drosophila bifasciata Pominini (Diptera, Drosophilidae) are analyzed. By contrast to other Drosophila species, male D. bifasciata do not exhibit plasticity in copulation duration when competitor males are present before mating. Thus, plasticity in mating duration is not fixed in the genus Drosophila. The mating system of D. bifasciata is also examined. The species is polyandrous but, uncommonly for the genus Drosophila, males transfer a mating plug composed of sperm to females, which forms very shortly after copulation and fills the female uterus. The absence of plasticity observed in D. bifasciata may arise from the sperm plug.

Encapsulation ability: Are all Drosophila species equally armed? An investigation in the obscura group

Unable to form cellular capsules around large foreign bodies, the species Drosophila subobscura Collin in Gordon, 1936 was previously shown devoid of lamellocytes, the capsule-forming hemocytes in Drosophila melanogaster Meigen, 1830. This unusual case of deficiency in encapsulation ability was remarkable enough to motivate further investigations in phylogenetically related species of the obscura group. Like D. subobscura, the species Drosophila azteca Sturtevant and Dobzhansky, 1936, Drosophila bifasciata Pomini, 1940, Drosophila guanche Monclus, 1976, Drosophila miranda Dobzhansky, 1935, Drosophila persimilis Dobzhansky and Epling, 1944, and Drosophila pseudoobcura Frovola and Astaurov, 1929 were found to be unable to encapsulate large foreign bodies and also to lack lamellocytes. Surprisingly, Drosophila affinis Sturtevant, 1916, Drosophila tolteca Patterson and Mainland, 1944, and Drosophila obscura Fallen, 1823 were capable of mounting cellular capsules, although their encapsulation abilities remained weak. These three species were free of lamellocytes but possessed small pools of never before described “atypical hemocytes” present in the hemolymph when capsules were formed.

Genic variation in Scandinavian populations of Drosophila bifasciata

Genetic variation at 21 enzyme loci in natural populations of Drosophila bifasciata has been studied by starch gel electrophoresis. The species has a trans-palearctic distribution. Data on the allelic variation are given for 23 natural populations in an area comprising Finland, Sweden and Norway. Within this area D. bifasciata is a rare but widespread species. In general, different populations are equally polymorphic. The amount of genetic variation differs widely from locuto locus. Most polymorphic loci have relatively uniform allele frequencies, but extensive fluctuations have also been observed. These differences are interpreted to be adaptive. The limited information obtained from three Japanese strains of D. bifasciata indicates that populations at the extreme ends of the distribution area of a widespread species may be genetically different to a very high degree.

Host resistance does not explain variation in incidence of male-killing bacteria in Drosophila bifasciata

BackgroundSelfish genetic elements that distort the sex ratio are found widely. Notwithstanding the number of records of sex ratio distorters, their incidence is poorly understood. Two factors can prevent a sex ratio distorter from invading: inability of the sex ratio distorter to function (failure of mechanism or transmission), and lack of drive if they do function (inappropriate ecology for invasion). There has been no test to date on factors causing variation in the incidence of sex ratio distorting cytoplasmic bacteria. We therefore examined whether absence of the male-killing Wolbachia infection in D. bifasciata in Hokkaido island of Japan, in contrast to the presence of infection on the proximal island of Honshu, was associated with failure of the infection to function properly on the Hokkaido genetic background.ResultsThe male-killer both transmitted and functioned well following introgression to each of 24 independent isofemale inbred lines carrying Hokkaido genetic backgrounds. This was maintained even under stringent conditions of temperature. We therefore reject the hypothesis that absence of infection is due to its inability to kill males and transmit on the Hokkaido genetic background. Further trap data indicates that D. bifasciata may occur at different densities in Hokkaido and Honshu populations, giving some credence to the idea that ecological differentiation could be important.ConclusionsThe absence of the infection from the Hokkaido population is not caused by failure of the male-killer to function on the Hokkaido genetic background.

What causes inefficient transmission of male-killing Wolbachia in Drosophila?

Selfish genetic elements that distort the sex ratio are common in arthropods. Theory predicts they will invade and spread to fixation if they are vertically transmitted with perfect fidelity, potentially leading to host extinction. For inherited microorganisms that distort the sex ratio, inefficient vertical transmission or incomplete sex ratio distorting ability is required for host persistence. However, the relative roles of genetic and environmental factors in permitting the survival of male hosts and preventing parasite transmission are poorly understood. We examined the causes of transmission infidelity and male survival for a male-killing Wolbachia strain in Drosophila bifasciata. Under standard laboratory conditions (18 degrees C), in its standard genetic background, males are produced very rarely, and no case of reversion has been observed in 20 generations of laboratory rearing. To investigate the role of host genetic factors, Wolbachia was crossed into 27 different inbred lines of D. bifasciata, but in no case was reversion observed at preferred environmental temperatures. The role of elevated temperature in inducing inefficient transmission was examined. Whilst vertical transmission was perfect over three generations of maintenance at 23.5 degrees, transmission infidelity was observed at 25 degrees. We conclude that there is no evidence for the presence of either fixed or polymorphic host genes that repress transmission at standard environmental temperatures. However, severe temperature treatment does make vertical transmission imperfect. We suggest that the case of Wolbachia in D. bifasciata is one that is naturally balanced, the population being maintained polymorphic without the evolution of host resistance genes.

Transcription and splicing patterns of M- and O-type P elements in drosophila bifasciata, D. helvetica, and scaptomyza pallida

RT-PCR was applied to analyze the splicing patterns of P-element-derived mRNAs in Drosophila bifasciata, D. helvetica, and Scaptomyza pallida. D. melanogaster was used as a control. The experiments revealed that P elements are transcribed in all species investigated. However, there are differences in the splicing patterns of IVS3, which has to be removed in order to produce transposase mRNA instead of repressor mRNA. These differences are observed among species as well as between the P element subfamilies, the M and the O type, which coexist in the genomes of D. bifasciata and S. pallida. In D. helvetica M-type transposase mRNA was found in the germline and repressor mRNA in the soma, as has been previously described for the canonical (M-type-related) P element of D. melanogaster. In contrast, in S. pallida only repressor mRNA of M-type elements was detected in all tissues. In D. bifasciata, M-type IVS3, although activated both in the soma and the germline, is never completely excised. Instead, two alternative double-spliced variants occur in which two small introns are removed within the IVS3 region. One of these variants codes for a protein 12 aa longer than the regular transposase. Taking these findings together, transposase production and transpositional activity of M-type elements seem to be limited to D. helvetica and D. melanogaster, whereas M-type elements have become immobile in D. bifasciata and S. pallida. Unlike the M type, the splicing of O-type transcripts in D. bifasciata and S. pallida follows the classical rules of tissue-specific P element regulation: transposase mRNA is produced exclusively in the germline whereas repressor mRNA is formed in somatic cells. Thus O-type elements are thought to be still transpositionally active in both species. This finding is in accordance with the postulated recent transfer of O-type elements between the gene pools of D. bifasciata and S. pallida. In addition, we were able to show that the IVS3 double-spliced variants of both P element types are produced regularily in all species of the genus Drosophila investigated so far, but not in S. pallida.

Repeated horizontal transfer of P transposons between Scaptomyza pallida and Drosophila bifasciata.

Two distinct P element subfamilies, designated M-type and O-type, reside in the genome of D. bifasciata. PCR-screening of 65 Drosophila species revealed that only D. bifasciata and its closest relative D. imaii possess O-type elements. Outside the genus, O-type elements were detected in Scaptomyza pallida. Restriction analyses show that the general structure of the O-type elements from S. pallida and D. bifasciata is the same. Sequence divergence turned out to be extremely low (0.43%). These results suggest that the O-type subfamily of D. bifasciata has been received by horizontal transfer from an external source, most probably from the genus Scaptomyza, as has been previously suspected for the M-type family. Since the sequence divergence between M-type elements from S. pallida and D. bifasciata is eighteen-fold higher than that between O-type elements, two independent intergeneric transfer events have to be postulated. In order to re-examine the taxonomic status of S. pallida, a partial sequence (489 bp) of the Adh gene was analysed. The data clearly prove that S. pallida has to be placed far outside the D. obscura group.

Different evolutionary behaviour of P element subfamilies: M-type and O-type elements in Drosophila bifasciata and D. imaii

Distribution and variation of two P-element subfamilies designated M-type and O-type elements were investigated in Drosophila bifasciata (Db) and its relatives. PCR screening revealed that full-sized and internally deleted elements of both types occur in three geographic Db strains and in the related species, D. imaii (D1). Molecular analyses indicate differences in the evolutionary behaviour of the two P-element types. Internally deleted M-type elements fall into two size classes present in all three Db strains. In contrast, internally deleted O-type elements vary between the strains in number and length. With respect to genomic location, M-type elements seem to be restricted to conserved euchromatic sites, whereas the positions of O-type elements appear to be geographically variable. In one strain of Db (Italy), O-type elements seem to accumulate in the heterochromatin. Sequencing of a 397-bp segment shows intra- and interspecific divergence of M-type elements. In a 452-bp segment of the O-type elements, no substitutions were found, neither within nor between species. This finding suggests recent introgression of O-type elements via hybridization between Db and Dl. Sequence identity and variation in chromosomal locations among different copies imply that O-type elements are transpositionally active. For M-type elements, genomic mobility cannot be proved. In a survey of several other taxa, no O-type-related sequences were detected so far. Therefore, the origin of the O-type subfamily remains unknown, whereas the source of M-type elements can be traced back to the genus Scaptomyza.

Identification of a complete P-element in the genome of Drosophila bifasciata.

A full-size P-element (IbifM3) was isolated from a genomic library of Drosophila bifasciata. The sequence has a length of 2935 bp and is flanked by 8 bp duplications of the target site. The termini are formed by 31 bp inverted repeats. The four exons have intact reading frames and possess the coding capacity for a protein of 753 amino acids and a molecular weight of 86.4 kd. The sections of the D. melanogaster transposase presumed to be functionally important (three leucine zippers and a helix turn helix motif) are conserved in the D. bifasciata P-element. Copy number and genomic distribution resemble the situation in true P-strains of D. melanogaster. Both findings support the idea that IbifM3 represents an active transposon. The sequence comparison between the P-elements of D. bifasciata, D. melanogaster and Scaptomyza pallida reveals relationships not in accordance with the phylogeny of the species. This result suggests a further case of horizontal transmission involving mobile elements in the genus Drosophila.

P-related sequences inDrosophila bifasciata: A molecular clue to the understanding of P-element evolution in the genusDrosophila

Two P-elements (bif1 and bif2) were isolated from a genomic library of Drosophila bifasciata. Both elements are internally deleted and have lost the coding capacity for a functional transposase. One of the elements (bif2) contains an insert consisting of a repetitive sequence. The terminal inverted repeats and the segments necessary for passive mobility are well conserved. Element bif2 has retained rudiments of the coding sequence of exon 0 and exon 3, but the reading frame is destroyed by insertions and deletions. The comparison of the D. bifasciata P-elements with P-elements of Drosophila melanogaster and Drosophila nebulosa reveals that the two latter sequences are more similar to each other than either of them is to the D. bifasciata elements. This finding contradicts the phylogenetic relationship of the species and can be taken as an indirect but unequivocal evidence for recent horizontal gene transfer from a relative of D. nebulosa to the gene pool of D. melanogaster. The P-elements of D. bifasciata are phylogenetically ancient and have evolved independently for about 50 million years. A higher substitution rate at the third codon position as well as a predominance of conservative replacements at the amino acid level indicates that the P-elements of D. bifasciata have been under selective constraint over a long period and that immunobilization has occurred only recently.

CHROMOSOMAL VARIATION IN NATURAL POPULATIONS OF <i>DROSOPHILA BIFASCIATA</i>

Chromosomal inversions in natural populations of Drosophila bifasciata were analyzed in distribution and frequency. A total of 13 inversions were observed in eleven populations, with each population showing high polymorphism. In view of results of comparison of geographical distributions and frequencies for each inversion, D. bifasciata forms “flexible” type chromosomal polymorphism.Judging from differences of the genetic constitutions between populations, it is suggested that the marginal sourthwestern range of this species is Kujyusan, Kyushu, while the central range is the north-east territories of Japan.

The sex ratio condition in Drosophila bifasciata; Its experimental transmission to several species of Drosophila

The following evidence has been accumulated in this study of the sex ratio ( SR) condition in Drosophila bifasciata: A distinct cytopathology is present in dying males. An infectious material is isolable from the ovaries of SR females. This material, injected into normal healthy hosts, significantly alters the sex ratios of the injected flies and their progenies. Therefore, in view of these transfer-experiment results and the distinctive cytopathology previously described, it is postulated that the SR condition in D. bifasciata is due to an infectious agent exhibiting many viral characteristics: submicroscopic size, intracellular site, nucleopathic effects when multiplying, contagious spread by cell lysis, and affinity for neural and epidermal tissues. The SR agent resembles the sigma virus of Drosophila melanogaster with a stabilized strain, SR, and non-stabilized strains, “ia” and “ib.” Most of the normal flies tested appear to be refractory; the transferred condition is difficult to establish and maintain.

INTERSPECIFIC TRANSFER OF THE "SEX-RATIO" AGENT OF DROSOPHILA WILLISTONI IN DROSOPHILA BIFASCIATA AND DROSOPHILA MELANOGASTER.

The maternally transmitted "sex-ratio" condition in several species of Drosophila appears to be due to infection by a microorganism of the genus Treponema. Drosophila bifasciata is an exception, since no microorganism has been found in the "sex-ratio" strains of this species. Normal D. bifasciata can be infected by injection of the hemolymph of a "sex-ratio" strain of D. willistoni containing treponemas. The progenies of the infected D. bifasciata, up to and including the F(4) and F(5) generations, have numerous treponemas in their hemolymph. Their progenies are, however, not unisexual, although both females and males are infected. The hemolymph of these D. bifasciata injected in D. melanogaster females causes typical "sex-ratio" symptoms in the progenies of the latter.

Salivary Gland Chromosomes of <i>Drosophila bifasciata</i>

1. In Drosophila bifasciata, a wild species distributed widely in “transpalaearctic” regions, the correspondence between the karyotype and nine elements in the salivary gland chromosomes is discussed.2. A composite map of the standard gene arrangement of the salivary chromosomes is drawn.3. Descriptions of eleven different inversions detected in the salivary cells of geographic strains are given.4. Qualitative variations of the gene arrangements from different geographical regions are preliminary investigated.We wish to express our thanks to Drs. Jean and Katsuma Dan for reading the manuscript.