Hybrid lethal systems in theDrosophila melanogaster species complex

Lethal phases of the hybrids betweenDrosophila melanogaster and its sibling species,D. simulans are classified into three types: (1) embryonic lethality in hybrids carryingD. simulans cytoplasm andD. melanogaster X chromosome, (2) larval lethality in hybrids not carryingD. simulans X, and (3) temperature-sensitive pupal lethality in hybrids carryingD. simulans X. The same lethal phases are also observed when either of the two other sibling species,D. mauritiana orD. sechellia, is employed for hybridization withD. melanogaster. Here, we describe genetic analyses of each hybrid lethality, and demonstrate that these three types of lethality are independent phenomena. We then propose two models to interpret the mechanisms of each hybrid lethality. The first model is a modification of the conventional X/autosome imbalance hypothesis assuming a lethal gene and a suppressor gene are involved in the larval lethality, while the second model is for embryonic lethality assuming an interaction between a maternal-effect lethal gene and a suppressor gene.     

The evolutionary genetics of thehobo transposable element in theDrosophila melanogaster complex

Hobo elements are a family of transposable elements found inDrosophila melanogaster and its three sibling species:D. simulans, D. mauritiana andD. sechellia. Studies inD. melanogaster have shown thathobo may be mobilized, and that the genetic effects of such mobilizations included the general features of hybrid dysgenesis: mutations, chromosomal rearrangements and gonadal dysgenis in F1 individuals. At the evolutionary level somehobo-hybridizing sequences have also been found in the other members of themelanogaster subgroup and in many members of the relatedmontium subgroup. Surveys of older collected strains ofD. melanogaster suggest that completehobo elements were absent prior to 50 years ago and that they have recently been introduced into this species by horizontal transfer. In this paper we review our findings and those of others, in order to precisely describe the geographical distribution and the evolutionary history ofhobo in theD. melanogaster complex. Studies of the DNA sequences reveal a different level of divergence between the groupD. melanogaster, D. simulans andD. mauritiana and the fourth speciesD. sechellia. The hypothesis of multiple transfers in the recent past into theD. melanogaster complex from a common outside source is discussed.     

The pattern of proliferation of the neuroblasts in the wild-type embryo of Drosophila melanogaster

Summary The pattern of neuroblast divisions was studied in thoracic and abdominal neuromeres of wild-type Drosophila melanogaster embryos stained with a monoclonal antibody directed against a chromatin-associated antigen. Since fixed material was used, our conclusions are based upon the statistical evaluation of a large number of accurately staged embryos, covering the stages between the formation of the cephalic furrow up to shortened germ band. Our observations point to a rather stereotypic pattern of proliferation, consisting of several parasynchronous cycles of division. The data suggest that all SI neuroblasts divide at least eight times, all SII neuroblasts six or seven times and all SIII neuroblasts at least five times. This conclusion is based on the mapping of mitotic neuroblasts and is supported by the progressive reduction of the neuroblast volume and by the results of cell countings performed on embryos of increasing age. No conclusive evidence was obtained concerning the fate of the neuroblasts after their last mitosis, i.e. it cannot be decided whether the neuroblasts degenerate or become incorporated as inconspicuous cells in the larval ventral cord. The duration of the cycles of division of the neuroblasts was found to be 40–50 min each, while in the case of ganglion mother cells about 100 min are required to complete one cell cycle.     

The isolation of functional pole cells from theDrosophila melanogaster maternal effect mutantmat(3)1

Summary A procedure for pole cell isolation has been developed that takes advantage of theDrosophila melanogaster maternal effect mutantmat(3) 1. Embryos derived from homozygousmat(3)1 mothers form exclusively pole cells. By outcrossing we could substantially increase the expressivity of the original mutant stock. We further introduced theTM8 balancer chromosome, which carries the dominant temperature sensitive mutationDTS-4. This allows the accumulation of large homozygousmat(3) 1 fly populations by eliminating the heterozygous flies at the restrictive temperature.Early embryos were mechanically fragmented and the cells were isolated by means of metrizamide step gradients. The isolated cells were demonstrated to exhibit the various ultrastructural and histochemical characteristics of pole cells. The isolated cells were transplanted into genetically marked host embryos. The germ line mosaics that were obtained indicate that the isolated cells represent functional pole cells.Proteins synthesized by the isolated pole cells during short term in vitro labelling with³⁵S-methionine were compared to the proteins synthesized by blastoderm cells fromOregon-R embryos. At least one protein could be demonstrated in the pole cell samples that is not synthesized byOregon-R blastoderm cells.The method allows a fast and gentle isolation of highly enriched pole cell populations which are a prerequisite for the biochemical analysis of germ cell determination and differentiation.     

Development time and pupation behavior in theDrosophila melanogaster subgroup (Diptera: Drosophilidae)

This study is an in-depth analysis of intersexual, intraspecific, and interspecific variability in larvopupal developmental time, pupation site preference, and larval and pupal survival of a number of isofemale lines of the speciesDrosophila mauritiana, D. melanogaster, D. sechellia, D. simulans, D. teissieri, andD. yakuba. There was no significant sex differences in pupation height, but females eclosed significantly earlier than males in all species. In addition, the suggestion of a strong negative correlation between larval developmental time and pupation height could not be confirmed in this study. The hypothesis that differences in pupation height provide a basis for niche partitioning between closely related species with overlapping distributions was tested by three planned orthogonal contrast analyses of variance. First, the two speciesD. teissieri andD. yakuba, with largely overlapping distribution, were significantly different in pupation height. Second, the two allopatric, nonoverlapping island speciesD. mauritiana andD. sechellia did not significantly differ in pupation height. However, the absence of a significant difference in the final contrast between the two cosmopolitan speciesD. melanogaster andD. simulans, which are often found together, makes us cautious to accept the hypothesis.     

Genetic analysis of pattern-formation in the embryo ofDrosophila melanogaster

Summary The mutationbicaudal (Bull, 1966) causes embryos to develop a longitudinal mirror image duplication of the posteriormost abdominal segments, while head and thorax are missing. These embryos occur with varying frequencies among eggs laid by mutant females, irrespective of the paternal genotype. Recombination and deletion mapping indicate thatbicaudal (bic) is a recessive, hypomorphic, maternal-effect mutation mapping at a single locus on the second chromosome ofDrosophila melanogaster close tovg (67.0±0.1). The frequency of bicaudal embryos depends on the age of the mother, her genetic constitution and the temperature at which she is raised. Best producers are very young females hemizygous forbic (bic/Df(2)vg ^B ) at 28° C. Under these conditions 80% to 90% of the eggs which differentiate can show the bicaudal embryo phenotype. Upon ageing of the mother the frequency of bicaudal embryos declines rapidly, and most of the eggs develop the normal body pattern. Temperature shift experiments suggest a temperature-sensitive period at the onset of vitellogenesis.The mutation causes several types of abnormalities in the segment pattern of theDrosophila embryo, which are interpreted as various degrees of expression of the mutant character. The most frequent abnormal phenotype is the symmetrical bicaudal embryo with one to five abdominal segments duplicated. Less frequent are asymmetrical types, in which the smaller number of segments is always in the anterior reversed part. Other phenotypes are embryos with missing or rudimentary heads, and embryos with irregular gaps in the segment pattern. In bicaudal embryos, the pole cells, formed at the posterior pole of the egg prior to blastoderm formation, are not duplicated at the anterior. The significance of thebicaudal phenotypes for embryonic pattern-formation inDrosophila is discussed.     

Courtship inDrosophila sechellia: Its structure,functional aspects,and relationship to those of other members of theDrosophila melanogaster species subgroup

The courtship behavior of Drosophila sechelliais described. Male wing displays are mainly vibration and scissoring, with low levels of rowing. As courtship proceeds the proportion of courtship spent in male wing vibration and licking increases, whereas female movement decreases. The male courtship song of sechelliacontains pulse song but no sine song. This species also shows a distinctive copulation song associated with mounting and copulation. The main cuticular hydrocarbon in females is 7,11-heptacosadiene. The number of copulations increased when flies were placed in the presence of food. Visual and acoustic stimuli appear to be important for mating. A multidimensional comparison was used to compare members of the melanogaster species subgroup, based upon courtship behavior, song characteristics, and cuticular hydrocarbons. A multidimensional comparison of courtship sequences in sechellia, melanogaster, simulans,and mauritianashowed differences in variability between the two island species as compared to the two cosmopolitan species. The courtship song of D. orenais described: it shows both sine and pulse song; there is also a copulation song in this species.     

Spontaneous interchanges in females of Drosophila melanogaster

Summary Herein is described an attempts to establish chromosome pairing-interchange relationships in Drosophila melanogaster female. For this purpose, the formation of half-translocations was studied in XXY and XX females bearing compounds of the second pair of autosomes. With respect to XXY females, it was expected that the free Y chromosome would pair with these compounds and that half-translocations involving 2L would arise. In as much as compound chromosomes in XX females had no partner for pairing, the formation of half-translocations involving 2L was not expected.Half-translocations were registered in the F₁ from crosses of XX and XXY females to b j pr cn/T(Y;2)C males. The cross was designed to permit the detection of very rarely occurring non-homologue interchanges.Offspring number was 335 in XX females and 550 in XXY females. The majority of offspring consisted of individuals arisen from the spontaneous restitution of compounds and the formation of 2n egg cells. Based on phenotype, the offspring of XX females contained 4 individuals with half-translocations involving 2L; there were 48 such flies among the offspring of XXY females. As confirmed by progeny analysis, 38 half-translocations occurred in XXY females and none in XX females. Of the 31 spontaneous interchanges in XXY females 28 were recorded between the Y and the left compound, one between the Y and the right compound, and one between the X and the left compound. Non-homologue interchanges were of oogonial origin judging by the fact that individuals with half-translocations arose in clusters. Unlike Y — left compound interchanges, the interchanges between autosomal compounds seem to be of meiotic origin.     

Distribution of two GABA receptor-like subunits in theDrosophila CNS

Previously we have described the distribution of theRdl GABA receptor subunit in theDrosophila CNS. Knowing thatRdl can coassemble with LCCH3 (aDrosophila GABA receptor-like subunit showing sequence similarity to vertebrate subunit GABA_A receptors) in baculovirus infected insect cells, we compared the localization of these two receptor subunits in order to identify any potential overlap in their spatial or temporal distribution. The two subunits show very different patterns of localization. Early in development LCCH3 is found in the majority of developing neuroblasts and later is localized to the cell bodies of the embryonic nerve cord and brain, and the neuronal cell bodies surrounding the adult brain. In contrast,Rdl receptor subunits appear confined to the neuropil in all developmental stages. These results have two important implications. Firstly, they suggest that although these two subunits can coassemble in heterologous expression systems, they may not be found in the same tissues in the nervous system. Secondly, production of LCCH3 before neuronal differentiation leads us to speculate on the role of that LCCH3 containing receptors in the developing nervous system.     

The DNA content of polytene nuclei in midgut and Malpighian tubule cells of adultDrosophila melanogaster

Summary The amounts of DNA in midgut and Malpighian tubule cells of adult maleDrosophila melanogaster have been determined by Feulgen-DNA cytophotometry. The DNA values fall into discrete classes reflecting different levels of polyteny. The maximum level is 64C in the midgut, 256C in Malpighian tubules, and the modal values are 32C and 128C respectively. The data provide no evidence for extensive underreplication of heterochromatin. It is suggested that the reduced amount of satellite DNA found in the tissues of young adult flies may be a consequence of the fact that cycles of DNA replication started in the pre-adult stages are not completed until some hours after eclosion.     

Genetic Variants Affecting Phenoloxidase Activity in Drosophila melanogaster

In Drosophila melanogaster, two new variants affecting the activity of phenoloxidase were found in natural populations at Gomel in Belorussia and at Krasnodar in Russia. Prophenoloxidases, A ₁ and A ₃ , in these variants had the same mobilities on native electrophoresis as the wild type. However, enzymatic activities in their activated states were much lower than in the wild type, whereas the existence of prophenoloxidase proteins was demonstrated. Egg-to-adult and relative viabilities in the variants did not decrease at temperatures between 18 and 29°C. Genetic analyses indicated that the genes showing the phenotype of variants are new alleles of Mox and Dox-3 on the second chromosome.     

An approach to study the evolution of theDrosophila 5S ribosomal genes using P-element transformation

Summary The P-element-mediated gene transfer system was used to introduceDrosophila teissieri 5S genes into theDrosophila melanogaster genome. Eight transformedD. melanogaster strains that carryD. teissieri 5S mini-clusters consisting of 9–21 adjacent 5S units were characterized. No genetic exchanges betweenD. melanogaster andD. teissieri 5S clusters were detected over a 2-year survey of the eight strains. The occurrence of small rearrangements within theD. melanogaster 5S cluster was demonstrated in one of the transformed strains.     

Differential characterization of two leucine aminopeptidases in Drosophila melanogaster

Two leucine aminopeptidases from Drosophila melanogaster larvae have been partially purified. The LAP A and D enzymes have similar biochemical characteristics including molecular weights of 280,000 daltons, Michaelis-Menten constants of 0.05 mM, associations with metal cofactors, and specificities toward natural and chromogenic substrates. They differ in their pH optima and spatial distributions. If the closely linked genes that code for these enzymes have resulted from a tandem gene duplication event, it is suggested that there has been subsequent evolutionary divergence. This would provide Drosophila larvae with two related, but functionally distinct enzymes.Virginia K. Walker was supported by an NRC Predoctoral Scholarship and a Killam Merit Scholarship.     

The eggshell ofDrosophlla melanogaster

Summary The micropylar apparatus (MA) inDrosophila melanogaster (Diptera: Drosophilidae) is a 25 m long protrusion on the anterior pole of the eggshell, and it contains the 0.8 m wide micropylar canal through which the spermatozoon penetrates the eggshell. The canal terminates in the paracrystalline structure thus forming the pocket. The MA is secreted during oogenesis by the micropylar follicle cells (border cells and peripheral cells). Morphogenesis of the micropylar canal starts before stage 11A, when two of the border cells form two thin extensions containing microfilaments and penetrate the paracrystalline structure. Microtubules found at the base of the extensions participate in the formation of two projections. Adjacent follicle cells secrete the chorionic part of the MA, whilst the two projections elongate and twist during stages 12 and 13. Microtubules run parallel to the long axis of the projections and probably are related to cellular elongation during the formation of the projections. The paracrystalline structure is composed of lamellae having a periodicity of about 50 nm, which is maintained possibly due to the lateral fibrils connecting the lamellae. These lamellae consist of small fragments secreted during stages 10–11 by the border cells. The paracrystalline structure and the spongy vitelline membrane are condensed after egg maturation.     

The occurrence of the transposable elementpogo inDrosophila melanogaster

We examined the genomic occurrence of the transposable elementpogo in over 120 strains ofDrosophila melanogaster, from around the world and from different eras. All had multiple copies of a 2.1 kilobase (kb)pogo element, and multiple copies of several size classes between 1.0 and 1.8 kb. There were differences between strains in intensities or presences of deletion-derivative size classes, suggesting current or recent mobility in the species. We were unable to find anypogo-hybridization in eight other species in the genus, in three subgenera, or in the relatedScaptomyza pallida. Thepogo element may be a ‘middle-aged’ element in the genome ofD. melanogaster, having entered the species since its divergence from its sibling species, but long before theP andhobo elements.     

The size of poly(A)-containing RNAs in Drosophila melanogaster embryos

The size range of poly(A)-containing RNA from Drosophila melanogaster embryos has been estimated by hybridization with ³H-labeled poly(U) and subsequent fractionation on sucrose gradients. The median size of nuclear poly(A)-containing RNA is about 30 S (6000 nucleotides), and the median size of cytoplasmic poly(A)-containing RNA is about 17 S (1800 nucleotides). The relationship of these sizes to messenger RNA needed to code for protein and to the length of DNA contained in a chromomere is discussed.Research grant support was provided by NIH (6M35558; HD-00266) and NSF (GB-30600).     

Rudimentary mutants ofDrosophila melanogaster

Summary The X-linkedrudimentary (r) mutants ofDrosophila melanogaster are pyrimidine auxotrophs and require exogenous pyrimidines (Nørby, 1970; Falk, 1976). We have established a set ofrudimentary cell lines that are derived from embryos, homozygous for eitherr ¹ orr ³⁶. The enzymatic activities of the pyrimidine synthesizing enzymes were measured in the mutant lines. We have further investigated the nutritional requirements of the mutant cells in vitro by using a pyrimidine free culture medium.Ther ¹ cell lines were found to express 3–7%dihydroorotase (DHOase) activity as compared to a wildtype cell line. Reducedaspartate transcarbamylase (ATCase) activity was measured in somer ¹ cell lines whereas wildtypecarbamylphosphate synthetase (CPSase) activity is expressed in allr ¹ cell lines. Ther ³⁶ cell line expresses wildtype activity ofDHOase andCPSase. ATCase activity was found to be reduced to 10% of the wildtype activity.The mutant cell lines do not proliferate in pyrimidine free minimal medium and cell proliferation is obtained by the addition of crude RNA. Proliferation of ther ¹ cells is restored by the supplementation of the minimal medium withdihydroorotate whereas proliferation of ther ³⁶ cells is restored by supplementation with eitherdihydroorotate orcarbamylaspartate.The results demonstrate that therudimentary phenotypesr ¹ andr ³⁶ are expressed at the cellular level and that the two mutant cell types behave as cellular pyrimidine auxotrophs in vitro.