Transposition of the hobo element in Drosophila melanogaster somatic cells

Somatic mutation and recombination test on wing cells of Drosophila melanogaster showed that the recombination frequency in the somatic tissues of strains studied correlated with the presence of a full-length copy of the hobo transposable element in the genome. Transposition of hobo in somatic tissue cells at a frequency 3.5 × 10^?2 per site per X chromosome was shown by fluorescence in situ hybridization with salivary gland polytene chromosomes of larvae of one of the D. melanogaster strains having a full-length hobo copy.     

Cognate chitinoproteins are detected during Drosophila melanogaster development and in cell cultures from different insect species

Chitinase-sensitive glycoproteins (chitinoproteins) were detected by [³H]d-glucosamine incorporation and polyclonal antibodies against D. melanogaster chitinoprotein (Kramerov et al., Insect Biochem., 16, 417–432, 1986) in established cell cultures from different insect species (D. hydei, D. virilis, mosquito Aedes aegypti and silkworm Bombyx mori). It was shown that reversible oxidation of disulfide bonds results in two interconvertible monomeric and dimeric forms of D. melanogaster chitinoprotein with apparent molecular masses of 100,000 and 200,000, respectively. Similar glycoproteins with molecular masses corresponding to monomeric (80,000) and dimeric (160,000) forms were detected in the D. hydei, D. virilis and A. aegypti cells. The cognate chitinoprotein with a molecular mass of 100,000 was revealed in cultured cells of B. mori. These chitinoproteins were localized by immunostaining within cytoplasmic granules in all of the cultured cells tested.Both forms of the chitinoprotein with molecular masses of 80,000 and 160,000 were continuously detected throughout the development of D. melanogaster, with the pupal stage being the most abundant.     

Evolution of the LINE-like I element in the Drosophila melanogaster species subgroup

LINE-like retrotransposons, the so-called I elements, control the system of I-R (inducer-reactive) hybrid dysgenesis in Drosophila melanogaster. I elements are present in many Drosophila species. It has been suggested that active, complete I elements, located at different sites on the chromosomes, invaded natural populations of D. melanogaster recently (1920–1970). But old strains lacking active I elements have only defective I elements located in the chromocenter. We have cloned I elements from D. melanogaster and the melanogaster subgroup. In D. melanogaster, the nucleotide sequences of chromocentral I elements differed from those on chromosome arms by as much as 7%. All the I elements of D. mauritiana and D. sechellia are more closely related to the chromosomal I elements of D. melanogaster than to the chromocentral I elements in any species. No sequence difference was observed in the surveyed region between two chromosomal I elements isolated from D. melanogaster and one from D. simulans. These findings strongly support the idea that the defective chromocentral I elements of D. melanogaster originated before the species diverged and the chromosomal I elements were eliminated. The chromosomal I elements reinvaded natural populations of D. melanogaster recently, and were possibly introduced from D. simulans by horizontal transmission.     

Cyclic GMP production and excretion by Drosophila cells: Modulation by 20-hydroxyecdysone

Intracellular and extracellular concentrations of cyclic GMP were measured under different conditions in cultures of two 20-hydroxyecdysone sensitive clones of Drosophila melanogaster. Exponentially growing cells maintained a concentration of 0.15 pmol/10⁶ cells inside the cells (about 10⁻⁶ M) and 5 pmol/10⁶ cells in the medium. When the medium was changed equilibrium was restored in the cells in about 15 hr. Addition of 20-hydroxyecdysone arrested growth and induced several enzymes and morphological modifications. It also increased (up to 10-fold) both intracellular and extracellular cyclic GMP levels depending on the amount of hormone. The ratio of the two levels remained constant.     

Karyotype polymorphism in a cell population of Drosophila melanogaster cultured in vitro

The karyotypic variation of a cell line (K _C ) established from primary cultures of embryonic cells of Drosophila melanogaster was studied. Aneuploidy and structural rearrangements were found consisting mainly in: 1) loss of one of the chromosomes of the IV pair, 2) presence of a heterochromatic centric fragment (Y ?), 3) enlargement of the heterochromatic portion of one X, 4) shortening of an arm of one element of the two large autosome pairs. Moreover, the presence of tetraploid cells (3–12%) was noticed. The polymorphism in this cell population can be explained as a result of different mechanisms, including hybridization between different cell types. Replication of DNA was studied by autoradiography with special reference to the timing of replication of heterochromatin.     

On the control of heat shock protein synthesis in Drosophila melanogaster and Ceratitis capitata

The regulation of heat shock protein synthesis has been studied in two dipteran species, Drosophila melanogaster and Ceratitis capitata. The synthesis of the heat shock proteins was studied in organ cultures and compared to the respective translatable mRNA levels determined by in vitro translation in cell-free system. The obtained results suggest regulation of these proteins at the level of translation.     

Studies on lectin-induced agglutination of Drosophila embryonic cell lines

The agglutination responses of three Drosophila cell lines to concanavalin A and wheat germ agglutinin have been examined. Although the cell lines were originally derived from late embryonic stages of the Ore-R strain of Drosophila melanogaster, they show quantitative differences in lectin-induced agglutination. Line 1 cells were least agglutinable with both lectins. All three cell lines reached maximum agglutination with concanavalin A concentrations at 25 μg/ml, but the agglutination response to wheat germ agglutinin was biphasic such that an initial rapid increase in agglutination with concentrations up to 25 μg/ml was followed by slower agglutination above this concentration. Cells of lines 1 and 2 from ten-day old cultures exhibited greater lectin-induced agglutination than cells from three-day old cultures. Age-dependent differences were not found for line 3 cells which gave maximum agglutination responses in both young and old cultures. Cell agglutination by concanavalin A was almost completely inhibited by pretreatment of the lectin with methyl-α-d-mannopyranoside, but preincubation of wheat germ agglutinin with N-acetyl-d-glucosamine caused only partial blockage. Lectin-induced agglutination was not reversible by treatment with the monosaccharide inhibitors. These observations have been discussed with reference to the origin of the three cell lines and their cell surface properties.     

Patterns of puffing activity in the salivary gland chromosomes of Drosophila

The autosomal salivary gland chromosome puffing patterns of Drosophila simulans are described and compared with the puffing patterns of the sibling species D. melanogaster. During the late third larval instar and the prepupal period the patterns of puffing activity of these two species are similar — approximately 50% of the puffs common to both species showing identical activities. The remaining puffs differ in their timing of activity, or in their mean sizes, or in both of these parameters. A number of puffs (14) found in D. simulans have not been regularly observed in the Oregon stock of D. melanogaster but are active in other D. melanogaster strains. One puff (46 A) of D. melanogaster was absent from D. simulans and forms a heterozygous puff in hybrids, when the homologous chromosomes are synapsed. When the homologues are asynapsed a puff at 46 A is restricted to the melanogaster homologue. The puff at 63E on chromosome arm 3L is considerably smaller in D. simulans than in D. melanogaster and this size difference is autonomous in hybrids. Other puffs not common to both species behave non-autonomously in the species hybrid, even when the homologous chromosomes are asynapsed.     

Effects of zinc on programmed cell death of Musca domestica and Drosophila melanogaster blood cells

Programmed cell death (PCD) and phagocytotic activity of immune cells play a pivotal role in insect development. We examined the influence of Zn²⁺, an important element to fundamental biological processes, on phagocytosis and apoptosis of hemocytes in two fly species: Musca domestica and Drosophila melanogaster. Hemocytes were isolated from the third instar larvae of both species and treated for 3 h with zinc chloride solutions, containing 0.35 mM or 1.7 mM of Zn²⁺, and untreated as control. Phagocytotic activity of hemocytes was examined by flow cytometry after adding latex fluorescent beads to the medium, while apoptosis was evaluated by application of annexinV-FITC and pan-caspase-FITC inhibitor. Mitochondrial viability was determined by measuring resazurin absorbancy in the cell medium. The obtained results showed that Zn²⁺ increases phagocytosis and affects PCD of both species hemocytes but each in a different way. Zinc decreases fraction of annexin-positive hemocytes in M. domestica but increases it in D. melanogaster. The pan-caspase analysis revealed low and high activity of caspases in hemocytes of M. domestica and D. melanogaster, respectively. Zn²⁺ also decreased the viability of hemocyte mitochondria but only in D. melanogaster. It suggests that flies use different pathways of PCD, or that Zn plays a different role in this process in M. domestica than in D. melanogaster.     

Genetic Analysis of Aspartate Aminotransferase Isozymes from Hybrids between DROSOPHILA MELANOGASTER and DROSOPHILA SIMULANS and Mutagen-Induced Isozyme Variants

The aspartate aminotransferases (designated GOT1 and GOT2) are two enzymes of Drosophila melanogaster for which naturally occurring electrophoretic variants were not found. There is an electrophoretic difference between D. melanogaster and D. simulans. Since the F ₁ hybrid offspring of these species are sterile, a genetic analysis of the ordinary type cannot be done on differences between the two species. A method was devised to make "partial hybrids" in which one chromosome arm is homozygous for melanogaster genes in an otherwise hybrid background. By using this method, Got1 was localized to 2R and Got2 to 2L. Once a gene can be assigned to a chromosome, it may be followed in crossing schemes and mutations from mutagen treatments may be looked for. At the locus of Got1 a mutation with low activity was recovered and designated Got1^lo. It was located at a genetic map position of 75 on 2R. A Got2 mutant with a greater migration to the anode was recovered and designated Got2 ^J. It was located at a genetic map position of 3.0, and in the salivary chromosome was between 22B1 and 22B4 inclusive.     

Inhibitory effects of oostatic hormone on ovarian maturation and ecdysteroid production in diptera

Extracts from post-vitellogenic ovaries of Musca domestica, Aedes atropalpus and Drosphila melanogaster were examined for the ability to inhibit the vitellogenic phase of growth in newly emerged A. atropalpus and D. melanogaster adult females. All extracts were inhibitory in A. atropalpus. D. melanogaster females were less sensitive than A. atropalpus females, only showing inhibition at a two-fold greater concentration of M. domestica extract. The titre of oostatic hormone in A. atropalpus ovaries correlated with the decline in ecdysteroid synthesis by these ovaries. A. atropalpus ovaries containing post-vitellogenic follicles were shown to inhibit ovarian ecdysteroid synthesis in vitro by vitellogenic ovaries of both A. atropalpus and D. melanogaster. D. melanogaster ovaries were not capable of eliciting such an inhibition in vitro, at least with the numbers of ovaries utilized in our experiments.     

Loss of reproductive parasitism following transfer of male-killing Wolbachia to Drosophila melanogaster and Drosophila simulans

Wolbachia manipulate insect host biology through a variety of means that result in increased production of infected females, enhancing its own transmission. A Wolbachia strain (wInn) naturally infecting Drosophila innubila induces male killing, while native strains of D. melanogaster and D. simulans usually induce cytoplasmic incompatibility (CI). In this study, we transferred wInn to D. melanogaster and D. simulans by embryonic microinjection, expecting conservation of the male-killing phenotype to the novel hosts, which are more suitable for genetic analysis. In contrast to our expectations, there was no effect on offspring sex ratio. Furthermore, no CI was observed in the transinfected flies. Overall, transinfected D. melanogaster lines displayed lower transmission rate and lower densities of Wolbachia than transinfected D. simulans lines, in which established infections were transmitted with near-perfect fidelity. In D. simulans, strain wInn had no effect on fecundity and egg-to-adult development. Surprisingly, one of the two transinfected lines tested showed increased longevity. We discuss our results in the context of host-symbiont co-evolution and the potential of symbionts to invade novel host species.     

Increasing the data size to accurately reconstruct the phylogenetic relationships between nine subgroups of the Drosophila melanogaster species group (Drosophilidae, Diptera)

Previous phylogenetic analyses of the melanogaster species group have led to conflicting hypotheses concerning their relationship; therefore the addition of new sequence data is necessary to discover the phylogeny of this species group. Here we present new data derived from 17 genes and representing 48 species to reconstruct the phylogeny of the melanogaster group. A variety of statistical tests, as well as maximum likelihood mapping analysis, were performed to estimate data quality, suggesting that all genes had a high degree of contribution to resolve the phylogeny.Individual locus was analyzed using maximum likelihood (ML), and the concatenated dataset (12,988 bp) were analyzed using partitioned maximum likelihood (ML) and Bayesian analyses. Separated analysis produced various phylogenetic relationships, however, phylogenetic topologies from ML and Bayesian analysis based on concatenated dataset, at the subgroup level, were completely identical to each other with high levels of support. Our results recovered three major clades: the ananassae subgroup, followed by the montium subgroup, the melanogaster subgroup and the oriental subgroups form the third monophyletic clade, in which melanogaster (takahashii, suzukii) forms one subclade and ficusphila [eugracilis (elegans, rhopaloa)] forms another. However, more data are necessary to determine the phylogenetic position of Drosophila lucipennis which proved difficult to place.     

Coevolution of Drosophila melanogaster mtDNA and Wolbachia Genotypes

Maternally inherited microorganisms can influence the mtDNA pattern of variation in hosts. This influence is driven by selection among symbionts and can cause the frequency of mitochondrial variants in the population to eventually increase or decrease. Wolbachia infection is common and widespread in Drosophila melanogaster populations. We compared genetic variability of D. melanogaster mitotypes with Wolbachia genotypes among isofemale lines associated with different geographic locations and time intervals to study coevolution of the mtDNA and Wolbachia. Phylogenetic analysis of D. melanogaster mtDNA revealed two clades diverged in Africa, each associated with one of the two Wolbachia genotype groups. No evidence of horizontal transmission of Wolbachia between maternal lineages has been found. All the mtDNA variants that occur in infected isofemale lines are found in uninfected isofemale lines and vice versa, which is indicative of a recent loss of infection from some maternal fly lineages and confirms a significant role of Wolbachia in the D. melanogaster mtDNA pattern of variation. Finally, we present a comparative analysis of biogeographic distribution of D. melanogaster mitotypes all over the world.     

Geographical Distribution and Diversity of Bacteria Associated with Natural Populations of Drosophila melanogaster

Drosophila melanogaster is one of the most widely used model systems in biology. However, little is known about its associated bacterial community. As a first step towards understanding these communities, we compared bacterial 16S rRNA gene sequence libraries recovered from 11 natural populations of adult D. melanogaster. Bacteria from these sequence libraries were grouped into 74 distinct taxa, spanning the phyla Proteobacteria, Bacteroidetes, and Firmicutes, which were unevenly spread across host populations. Summed across populations, the distribution of abundance of genera was closely fit by a power law. We observed differences among host population locations both in bacterial community richness and in composition. Despite this significant spatial variation, no relationship was observed between species richness and a variety of abiotic factors, such as temperature and latitude. Overall, bacterial communities associated with adult D. melanogaster hosts are diverse and differ across host populations.     

Effects of the Sex Ratio Organism on IN VITRO Differentiation of Drosophila Embryonic Cells

Effects of Sex Ratio Organism (SRO) on the development of embryonic cells of Drosophila melanogaster were studied in vitro. The single embryo culture technique and a histochemical staining method were employed to distinguish male embryo cultures from female ones. SRO-infected Zwⁿ females were crossed to wild-type males, and their embryos were separately dissociated and cultured in vitro. Cell growth and differentiation in each culture were examined by phase contrast microscopy and described both qualitatively and quantitatively. Neurons, imaginal disk cells and plasmatocyte-like cells barely differentiated in male (Zw ⁿ/Y) cultures, whereas muscle and fatbody cells were not so severely affected. These symptoms were generally specific to male cultures. Female (Zwⁿ/+) cultures were relatively normal. These observations are consistent with the results of mosaic analysis. The susceptibility of cells to SRO is discussed in relation to the dose of X chromosome in the cells.     

Molecular and cytogenic analysis of pericentromeric heterochromatin in ovarian nurse cells of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> subgroup species

Evolutionary rearrangements of pericentromeric heterochromatin among Drosophila melanogaster subgroup species have been investigated. A region-specific DNA library from Drosophila orena ovarian nurse cell chromocenter was obtained by the microdissection of polythene chromosomes. The probe has been localized on chromosomes of ovarian nurse cells of Drosophila melanogaster subgroup species using fluorescent hybridization in situ. Sequences homologous to the sequences of the DNA probe were detected in the chromocenter and pericentromeric regions of D. orena polythene chromosomes, in all pericentromeric regions of other species with several exceptions. There was no labeling on one of the arms of the D. simulans chromosome 2; however, these sequences were present on the telomere of D. erecta chromosome 3 and in regions adjacent to the brightly DAPI-stained heterochromatin blocks of D. yakuba, D. santomea and D. teissieri chromosomes 2 and 3. At the S₆ stage (secondary reticulate nucleus), labeled chromatin can be found mostly within a restricted territory in D. orena nucleus; no such chromatin can be detected throughout the rest of the nucleus. On the contrary, at this stage, in nuclei of other species, labeled DNA is spread diffusely.     

Nuclear envelope attachment sites of interphase chromosomes: Barrier elements but not insulators

A neDNA fragment that protects a transgene from position effect variegation when flanking it was tested for insulator properties. The fragment did not act as an insulator. A similarity of neDNA and various barrier elements was examined, and Drosophila melanogaster and Mus musculus chromosome DNA regions homologous to neDNA were analyzed. Additional conserved sites and DNA duplex destabilization sites were found in the neDNA sequence, and DNA conformational specifics were assumed for the chromosomal neighborhood of neDNA sites of the D. melanogaster and M. musculus genomes.     

Actin Heterogeneity in primary embryonic culture cells from Drosophila melanogaster.

We have investigated actin heterogeneity in differentiating primary embryonic cell cultures from Drosophila melanogaster. Proteins labeled with [³⁵S]methionine have been separated using O'Farrell two-dimensional gel electrophoresis. Cultures of heterogeneous cell types synthesize at least three major forms of actin, each differing slightly in isoelectric point. We have used a cell separation technique based on differential cell adhesion in the presence of ethylene glycol-bis(β-aminoethyl ether) N,N′-tetraacetate to prepare cultures either highly enriched for, or highly depleted of, myogenic cells. Postfusion myogenic cells synthesize predominantly the most acidic actin form (actin I), while nonmyogenic cells synthesize almost exclusively the two more basic forms (actins II and III). Synthesis of actins at earlier intervals in myogenic cell differentiation in vitro has also been examined. Immediately prior to the onset of myoblast fusion, the synthesis of actin I represents approximately 40% of total actin synthesis. As myogenic cell differentiation progresses this actin form is synthesized at an increasing rate, relative to actins II and III. Drosophila appears to be quite similar to vertebrates with regard to the number of actin species synthesized, as well as to cell and developmental specificity of actin synthesis.