Mitochondrial DNA content of mature spermatozoa and oocytes in the genetic model Drosophila

Although crucial to the success of fertilization and embryogenesis, little is known about the mitochondrial DNA (mtDNA) content of mature spermatozoa and oocytes across taxa and across different fertilization systems. Oocytes are assumed to hold a large population of mtDNAs that populate emerging cells during early embryogenesis, whereas spermatozoa harbor only a limited pool of mtDNAs that is believed to sustain functionality but fails to contribute paternal mtDNA to the zygote. Recent work suggests that mature sperm of the genetic model Drosophila melanogaster lack mtDNA, questioning the significance of zygotic mechanisms for the selective elimination of paternal mtDNA and their necessity for fertilization success. This finding further contradicts previous observations of the inheritance of paternal mtDNA in drosophilids. Using quantitative polymerase chain reaction, we estimate the mtDNA content of several laboratory strains of D. melanogaster and D. simulans to shed light on this discrepancy and to describe the mitochondrial/mtDNA load of gametes within this system. These measurements led to an average estimate of 22.91±4.61 mtDNA molecules/copies per spermatozoon across both species and to 1.07E+07±2.71E+06 molecules/copies per oocyte for D. simulans. As a consequence, the ratio of paternal and maternal mtDNA in the zygote was estimated at 1:4.65E+05.     

Extensive diversity among Drosophila species with respect to nucleotide sequences within the adenine + thymine-rich region of mitochondrial DNA molecules.

Mitochondrial DNA (mtDNA) molecules from species of the genus Drosophila contain a region exceptionally rich in adenine + thymine (A+T). Using agarose gel electrophoresis and electron microscopy, we determined that in the mtDNA molecules of D. melanogaster, D. simulans, D. mauritiana, D. yakuba, D. takahashii, and D. virilis, the A+T-rich regions, which are 5.1, 4.8, 4.6, 1.1, 2.2, and 1.0 kilobase pairs in size, respectively, are at homologous locations relative to various common EcoRI and HindIII cleavage sites. Under conditions highly permissive for base pairing (35% formamide), heteroduplexes were constructed between EcoRI fragments and whole circular molecules of mtDNAs of the above mentioned six species in a variety of combinations. Complete pairing of molecules outside the A+T-rich region was found in all heteroduplexes examined. However, in contrast, A+T-rich regions of the different species failed to pair in all but those combinations of mtDNAs involving the three most closely related species. In heteroduplexes between D. melanogaster and D. simulans, and between D. melanogaster and D. mauritiana mtDNAs, up to 35% of the A+T-rich regions appeared double-stranded. These data indicate that much more extensive divergence of sequences has occurred in A+T-rich regions than in other regions of Drosophila mtDNA molecules.     

Drosophila mitochondrial DNA: Conserved sequences in the A+T-rich region and supporting evidence for a secondary structure model of the small ribosomal RNA

Summary The sequence of a segment of theDrosophila virilis mitochondrial DNA (mtDNA) molecule that contains the A+T-rich region, the small rRNA gene, the tRNA^f-met, tRNA^gln, and tRNA^ile genes, and portions of the ND2 and tRNA^val genes is presented and compared with the corresponding segment of theD. yakuba mtDNA molecule. The A+T-rich regions ofD. virilis andD. yakuba contain two correspondingly located sequences of 49 and 276/274 nucleotides that appear to have been conserved during evolution. In each species the replication origin of the mtDNA molecule is calculated to lie within a region that overlaps the larger conserved sequence, and within this overlap is found a potential hairpin structure. Substitutions between the larger conserved sequences of the A+T-rich regions, the small mt-rRNA genes, and the ND2 genes are biased in favor of transversions, 71–97% of which are AT changes. There is a 13.8 times higher frequency of nucleotide differences between the 5 halves than between the 3 halves of theD. virilis andD. yakuba small mt-rRNA genes. Considerations of the effects of observed substitutions and deletion/insertions on possible nucleotide pairing within the small mt-rRNA genes ofD. virilis andD. yakuba strongly support the secondary structure model for theDrosophila small mt-rRNA that we previously proposed.     

Discrepancy in divergence of the mitochondrial and nuclear genomes ofDrosophila teissieri andDrosophila yakuba

Summary Restriction sites were compared in the mitochondrial DNA (mtDNA) molecules from representatives of two closely related species of fruit flies: nine strains ofDrosophila teissieri and eight strains ofDrosophila yakuba. Nucleotide diversities amongD. teissieri strains and amongD. yakuba strains were 0.07% and 0.03%, respectively, and the nucleotide distance between the species was 0.22%. Also determined was the nucleotide sequence of a 2305-nucleotide pari (ntp) segment of the mtDNA molecule ofD. teissieri that contains the noncoding adenine+thymine (A+T)-rich region (1091 ntp) as well as the genes for the mitochondrial small-subunit rRNA, tRNA^f-met, tRNA^gln, and tRNA^ile, and portions of the ND2 and tRNA^val genes. This sequence differs from the corresponding segment of theD. yakuba mtDNA by base substitutions at 0.1% and 0.8% of the positions in the coding and noncoding regions, respectively. The higher divergence due to base substitutions in the A+T-rich region is accompanied by a greater number of insertions/deletions than in the coding regions. From alignment of theD. teissieri A+T-rich sequence with those ofD. yakuba andDrosophila virilis, it appears that the 40% of this sequence that lies adjacent to the tRNA^ile gene has been highly conserved. Divergence between the entireD. teissieri andD. yakuba mtDNA molecules, estimated from the sequences, was 0.3%; this value is close to the value (0.22%) obtained from the restriction analysis, but 10 times lower than the value estimated from published DNA hybridization results. From consideration of the relationships of mitochondrial nucleotide distance and allozyme genetic distance found among seven species of theDrosophila melanogaster subgroup, the mitochondrial nucleotide distance observed forD. teissieri andD. yakuba is anomalously low in relation to the nuclear genetic distance.     

Genetics of esterases in Drosophila. III. Influence of different chromosomes on esterase pattern in Drosophila

The present report presents the results of starch and polyacrylamide gel electrophoretic studies of the influence of the X chromosome on the expression of esterase-6 in D. melanogaster × D. simulans hybrids heterozygous for locus Est-6 as well as studies of the influence of autosomes on esterase expression in Drosophila of the virilis group. A differential expression of esterase-6 has been detected in D. melanogaster × D. simulans hybrid males. A differential decrease in the activity of esterase-6 (both F and S allozymes) derived from D. melanogaster has been noted. In hybrid females, the activity of parental esterases is the same. It is suggested that the X chromosome regulates the expression of esterase-6 in D. melanogaster. Analysis of individuals obtained in different schemes of crosses between different species of Drosophila of the virilis group by use of stocks marked with mutations in various chromosomes indicates that other autosomes (in particular, autosomes 4 and 5) also influence the phenotypic expression of esterases (which are controlled by genes located on the second chromosome).     

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.     

Contrasting patterns of genetic structure and evolutionary history as revealed by rnitochondrial DNA and nuclear gene-enzyme variation betweenDrosophila melanogaster andDrosophila simulans

Drosophila melanogaster and its sibling speciesD. simulans have a cosmopolitan distribution. Studies on nuclear gene-enzyme variation from natural populations of these species reveal that the two have almost equal overall heterozygosity, yetD. simulans populations are significantly less differentiated. However, it is not clear whether this difference in population structure represents a difference in the genetic strategy with which they respond to the same adaptive challenges, or is the result of difference in species history. To help answer this question, we have undertaken an intensive survey of restriction fragment length polymorphisms of mitochondrial DNA (mtDNA) fromD. simulans; the results are compared with those fromD. melanogaster. We surveyed 69 isofemale lines ofD. simulans from four continents and seven lines from the Seychelles Islands. Ten restriction enzymes detected 104 restriction sites in the continental mtDNAs, of which only threeHinf1 sites were variable and account for fourHlnf1 (restriction variants) haplotypes. These four variants were all found in geographically distant locations. By contrast, twenty-three haplotypes were observed inD. melanogaster, many of which were observed in only one population. It would seem, therefore, that these two species have had different histories. Specifically, cosmopolitan populations ofD: simulans are probably products of a comparatively recent expansion from a source population in Africa. These results do not negate differences in their genetic strategy of adaptation, but they do show the importance of historical contingency in the present-day pattern of geographic variation.     

Identification of cytoplasmically transferred mitochondrial DNA in female germlines of Drosophila and its propagation in the progeny

Summary The composition of mitochondrial DNA (mtDNA) was analyzed in single female flies that developed from fertilized Drosophila melanogaster eggs, into which germ plasm of D. simulans had been introduced. HpaII cleavage patterns showed that all 12 individual female flies examined had developed from eggs in which 37%–71% of the total mtDNA was D. simulans mtDNA (Ds mtDNA) and the rest was D. melanogaster mtDNA (Dm mtDNA). The stability of this heteroplasmic state in these isofemale lines was monitored for seven generations at both individual and population levels. Results showed that the heteroplasmy of Dm and Ds mtDNAs was stably transmitted for at least three generations at the population level, but showed stochastic segregation at the individual level. After 4–6 generations, all individuals lost Ds mtDNA. The mechanisms of preferential loss of Ds mtDNA and of transmission of heteroplasmic mtDNA to descendants are discussed.     

Aurora,a non-mobile retrotransposon in Drosophila melanogaster heterochromatin

A novel retrotransposon, aurora, containing 324 by long terminal repeats (LTRs) was detected in Drosophila melanogaster as a 5 kb insertion in the heterochromatic Stellate gene. This insertion causes a 5 bp duplication of the integration site. Southern analysis and in situ hybridization data show that all detectable copies of aurora are immobilized in the D. melanogaster heterochromatin. However, mobile copies of aurora were revealed in the cuchromatin of D. simulans. The element was also found in various species of the melanogaster subgroup and in the D. virilis genome.     

Evolutionary Changes in Gene Expression,Coding Sequence and Copy-Number at the Cyp6g1 Locus Contribute to Resistance to Multiple Insecticides in Drosophila

Widespread use of insecticides has led to insecticide resistance in many populations of insects. In some populations, resistance has evolved to multiple pesticides. In Drosophila melanogaster, resistance to multiple classes of insecticide is due to the overexpression of a single cytochrome P450 gene, Cyp6g1. Overexpression of Cyp6g1 appears to have evolved in parallel in Drosophila simulans, a sibling species of D. melanogaster, where it is also associated with insecticide resistance. However, it is not known whether the ability of the CYP6G1 enzyme to provide resistance to multiple insecticides evolved recently in D. melanogaster or if this function is present in all Drosophila species. Here we show that duplication of the Cyp6g1 gene occurred at least four times during the evolution of different Drosophila species, and the ability of CYP6G1 to confer resistance to multiple insecticides exists in D. melanogaster and D. simulans but not in Drosophila willistoni or Drosophila virilis. In D. virilis, which has multiple copies of Cyp6g1, one copy confers resistance to DDT and another to nitenpyram, suggesting that the divergence of protein sequence between copies subsequent to the duplication affected the activity of the enzyme. All orthologs tested conferred resistance to one or more insecticides, suggesting that CYP6G1 had the capacity to provide resistance to anthropogenic chemicals before they existed. Finally, we show that expression of Cyp6g1 in the Malpighian tubules, which contributes to DDT resistance in D. melanogaster, is specific to the D. melanogaster–D. simulans lineage. Our results suggest that a combination of gene duplication, regulatory changes and protein coding changes has taken place at the Cyp6g1 locus during evolution and this locus may play a role in providing resistance to different environmental toxins in different Drosophila species.     

Characterization of the length polymorphism in the A+T-rich region of the Drosophila obscura group species

Summary In the twelve Drosophila obscura group species studied, belonging to the affinis, obscura, and pseudoobscura subgroups, the mitochondrial DNA length ranges from 15.8 to 17.2 kb. This length polymorphism is mainly due to insertions/deletions in the variable region of the A+T-rich region. In addition, one species (D. tristis) possess a tandem duplication of a 470-bp fragment that contains the replication origin.The same duplication has occurred at least twice in the Drosophila evolutionary history due to the fact that the repetition is analogous to repetitions found in the four species of the D. melanogaster complex.By comparing the nucleotide sequence of the conserved region in D. ambigua, D. obscura, D. yakuba, D. teissieri, and D. virilis, we show the presence of a secondary structure, likely implied in the replication origin, which could favor the generation of this kind of duplications.Finally, we propose that the high A and T content in the variable region of the A + T-rich region favors the formation of less-stable secondary structures, which could explain the generation of minor insertion/deletions found in this region.Offprint requests to: A. Latorre     

Intraspecific diversity of nucleotide sequences within the adenine + thymine-rich region of mitochondrial DNA molecules of Drosophila mauritiana, Drosophila melanogaster and Drosophila simulans.

Mitochondrial DNA (mtDNA) molecules from Drosophila mauritiana, D. melanogaster, and D. simulans contain a single adenine + thymine (A+T)-rich region, which is similarly located in all molecules, but varies in size among these species. Using agarose gel electrophoresis and electron microscopy, a difference in occurrence of one EcoRI site, and a difference in size (approximately 0.7 kb) of the A+T-rich regions was found between mtDNA molecules of flies of two female lines of D. mauritiana. In heteroduplexes constructed between these two kinds of mtDNA molecules, two or three regions of strand separation, each comprising single strands of unequal length, were apparent near the center of the A+T-rich region. Using the structural differences between D. mauritiana mtDNA molecules it was demonstrated the mtDNA of this species is maternally inherited. Differences in length of A+T-rich regions were also found between mtDNA molecules of two geographically separated strains of D. melanogaster, and between mtDNA molecules of two geographically separated strains of D. simulans. However, in both cases, in heteroduplexes constructed between mtDNA molecules of different strains of one species, the A+T-rich regions appeared completely paired.     

Origin and direction of replication in mitochondrial DNA molecules from the genus Drosophila.

Mitochondrial DNA (mtDNA) obtained from ovaries of Drosophila simulans, D. mauritiana, D. takahashii, D. yakuba and D. virilis was examined by electron microscopy. From a consideration of the structural properties of replicative intermediates, it was concluded that in mtDNA molecules of each species, synthesis on one strand can be up to 97% complete before synthesis on the complementary strand is initiated. MtDNA molecules of each species contain a single A+T-rich region which shows species-specific size variation from 1.0 kb (D. virilis) to 4.8 kb (D. simulans), and maps at the same position in all molecules relative to three common EcoRI sites. The structural properties of complex forms, interpreted as having originated from replicative intermediates, and produced by either partial denaturation or EcoRI digestion, are consistent with the hypothesis that replication is initiated within the A+T-rich region and proceeds unidirectionally around the molecule towards the nearest common EcoRI site. The replication origin is located near the center of the A+T-rich region in D. simulans and D. mauritiana, but lies closer to that end of the A+T-rich region which is distal to the nearest common EcoRI site in D. takahashii, D. yakuba and D. virilis.     

Physiological tolerance of Drosophila simulans to dark environment: A comparison with D. melanogaster

According to ecological and behavioural studies, Drosophila simulans is considered to be less tolerant of darkness than its sibling species D. melanogaster which is well adapted both behaviourally and physiologically to darkness. The relationships between physiological and behavioural adaptations have been analysed by studying the developmental and the reproductive capacities of D. simulans submitted to various light regimes (LL, LD 12:12, DD). This species has a lower reproductive capacity than D. melanogaster but failed to react to light treatment. In particular, D. simulans showed no effect of darkness on either fertilization or ovarian function. The lack of differences between D. melanogaster and D. simulans as regards their physiological capacities in relation to light regime suggests that the selective pressures of light may act at different levels of regulation.     

MITOCHONDRIAL–NUCLEAR EPISTASIS AFFECTS FITNESS WITHIN SPECIES BUT DOES NOT CONTRIBUTE TO FIXED INCOMPATIBILITIES BETWEEN SPECIES OF DROSOPHILA

Efficient mitochondrial function requires physical interactions between the proteins encoded by the mitochondrial and nuclear genomes. Coevolution between these genomes may result in the accumulation of incompatibilities between divergent lineages. We test whether mitochondrial–nuclear incompatibilities have accumulated within the Drosophila melanogaster species subgroup by combining divergent mitochondrial and nuclear lineages and quantifying the effects on relative fitness. Precise placement of nine mtDNAs from D. melanogaster, D. simulans, and D. mauritiana into two D. melanogaster nuclear genetic backgrounds reveals significant mitochondrial–nuclear epistasis affecting fitness in females. Combining the mitochondrial genomes with three different D. melanogaster X chromosomes reveals significant epistasis for male fitness between X‐linked and mitochondrial variation. However, we find no evidence that the more than 500 fixed differences between the mitochondrial genomes of D. melanogaster and the D. simulans species complex are incompatible with the D. melanogaster nuclear genome. Rather, the interactions of largest effect occur between mitochondrial and nuclear polymorphisms that segregate within species of the D. melanogaster species subgroup. We propose that a low mitochondrial substitution rate, resulting from a low mutation rate and/or efficient purifying selection, precludes the accumulation of mitochondrial–nuclear incompatibilities among these Drosophila species.     

Comparison of dot chromosome sequences from D. melanogaster and D. virilis reveals an enrichment of DNA transposon sequences in heterochromatic domains

Background

Chromosome four of Drosophila melanogaster, known as the dot chromosome, is largely heterochromatic, as shown by immunofluorescent staining with antibodies to heterochromatin protein 1 (HP1) and histone H3K9me. In contrast, the absence of HP1 and H3K9me from the dot chromosome in D. virilis suggests that this region is euchromatic. D. virilis diverged from D. melanogaster 40 to 60 million years ago.

Results

Here we describe finished sequencing and analysis of 11 fosmids hybridizing to the dot chromosome of D. virilis (372,650 base-pairs) and seven fosmids from major euchromatic chromosome arms (273,110 base-pairs). Most genes from the dot chromosome of D. melanogaster remain on the dot chromosome in D. virilis, but many inversions have occurred. The dot chromosomes of both species are similar to the major chromosome arms in gene density and coding density, but the dot chromosome genes of both species have larger introns. The D. virilis dot chromosome fosmids have a high repeat density (22.8%), similar to homologous regions of D. melanogaster (26.5%). There are, however, major differences in the representation of repetitive elements. Remnants of DNA transposons make up only 6.3% of the D. virilis dot chromosome fosmids, but 18.4% of the homologous regions from D. melanogaster; DINE-1 and 1360 elements are particularly enriched in D. melanogaster. Euchromatic domains on the major chromosomes in both species have very few DNA transposons (less than 0.4 %).

Conclusion

Combining these results with recent findings about RNAi, we suggest that specific repetitive elements, as well as density, play a role in determining higher-order chromatin packaging.     

Functional conservation of a glucose-repressible amylase gene promoter from Drosophila virilis in Drosophila melanogaster

Summary Previous studies have demonstrated that the expression of the -amylase gene is repressed by dietary glucose in Drosophila melanogaster. Here, we show that the -amylase gene of a distantly related species, D. virilis, is also subject to glucose repression. Moreover, the cloned amylase gene of D. virilis is shown to be glucose repressible when it is transiently expressed in D. melanogaster larvae. This cross-species, functional conservation is mediated by a 330-bp promoter region of the D. virilis amylase gene. These results indicate that the promoter elements required for glucose repression are conserved between distantly related Drosophila species. A sequence comparison between the amylase genes of D. virilis and D. melanogaster shows that the promoter sequences diverge to a much greater degree than the coding sequences. The amylase promoters of the two species do, however, share small clusters of sequence similarity, suggesting that these conserved cis-acting elements are sufficient to control the glucose-regulated expression of the amylase gene in the genus Drosophila.Offprint requests to: D.A. Hickey     

Divergent strategies in low temperature environment for the sibling species Drosophila melanogaster and D. simulans: overwintering in extension border areas of France and comparison with African populations

The two sibling species D. /melanogaster and D. simulans adopt different overwintering strategies in northern border areas situated in France. If the winter is mild, both species reappear in early spring to refound the population. If the winter is cold, with several weeks of temperatures below 0 °C, D. melanogaster leave their shelters in April whereas D. simulans, which do not use shelters, reappear in late June, probably after returning from further south. Here, we tried to identify life-history characteristics responsible for this difference. For this, we studied developmental duration, viability, fecundity, fertility and longevity, and compared the abilities of French and African populations to survive when food supplies were inadequate, at different temperatures (14, 11, 7 °C). These temperatures are lower than those commonly used in the laboratory but closer to real conditions encountered in the wild. When the temperature was mild (14 or 11 °C) and the food supply was adequate, D. simulans performed better than D. melanogaster: it had a higher fecundity, a longer life expectancy and the males remained fertile, allowing outdoor reproduction late in winter. However, D. simulans was less resistant in more extreme conditions. At 7 °C D. simulans survived shorter on normal medium and its ability to survive when food supplies were inadequate was insufficient to allow outdoor overwintering. In contrast, D. melanogaster could not reproduce during winter: its fecundity was low and males were sterile at 11 °C. Nevertheless, if only protein-deficient resources were available, temperate D. melanogaster could survive for longer than D. simulans at all the temperatures tested. This greater resistance to underfeeding allows the species to survive until spring, in shelters for several months. A comparison of French and African population performances showed differences in the evolution of the two species during the colonization of more northern areas. African D. simulans, which are efficient at mild temperatures, underwent few modifications. In contrast, the viability of D. melanogaster improved at low developmental temperatures. This species also displayed higher fecundity, longer survival and higher underfeeding resistance at low temperatures. The relationship between the long retention genotype and underfeeding resistance or survival ability observed in French D. melanogaster populations may not exist in African populations.