Hybrid Lethal Systems in the Drosophila Melanogaster Species Complex. II. the Zygotic Hybrid Rescue (Zhr) Gene of D. Melanogaster

Hybrid females from Drosophila simulans females X Drosophila melanogaster males die as embryos while hybrid males from the reciprocal cross die as larvae. We have recovered a mutation in melanogaster that rescues the former hybrid females. It was located on the X chromosome at a position close to the centromere, and it was a zygotically acting gene, in contrast with mhr (maternal hybrid rescue) in simulans that rescues the same hybrids maternally. We named it Zhr (Zygotic hybrid rescue). The gene also rescues hybrid females from embryonic lethals in crosses of Drosophila mauritiana females X D. melanogaster males and of Drosophila sechellia females X D. melanogaster males. Independence of the hybrid embryonic lethality and the hybrid larval lethality suggested in a companion study was confirmed by employing two rescue genes, Zhr and Hmr (Hybrid male rescue), in doubly lethal hybrids. A model is proposed to explain the genetic mechanisms of hybrid lethalities as well as the evolutionary pathways.     

tudor, a gene required for assembly of the germ plasm in Drosophila melanogaster

Developmental analysis of a newly isolated maternal effect grandchildless mutant, tudor (tud), in Drosophila melanogaster indicates that tud+ activity is required during oogenesis for the determination and/or formation of primordial germ cells (pole cells) and for normal embryonic abdominal segmentation. Regardless of their genotype, progeny of females homozygous for strong alleles (tud1 and tud3) never form pole cells, apparently lack polar granules in the germ plasm, and approximately 40% of them die during late embryogenesis exhibiting severe abdominal segmentation pattern defects. Females carrying weak allele, tud4, produce progeny with some functional pole cells and form polar granules approximately one-third the size of those observed in wild-type oocytes and embryos. No segmentation abnormalities are observed in the inviable embryos derived from tud4/tud4 females.     

Interspecific transplantation of polar plasm between Drosophila embryos

Posterior polar plasm of the Drosophila egg has been shown to function autonomously in germ cell determination after transplantation to either the anterior or mid-ventral region of the early embryo. By means of similar transplantations, we have tested the ability of polar plasm of Drosophila immigrans to induce the formation of pole cells in a Drosophila melanogaster embryo. After the transplantation of polar plasm, "hybrid" pole cells were found in which both pole cell-specific organelles, the polar granules and nuclear body, were structurally similar to those characteristic of the transplanted cytoplasm. In order to determine whether these hybrid cells can function as germ cell precursors, these cells were transplanted to the posterior tip of genetically marked embryos. Approximately 5% of the flies obtained from embryos receiving potential pole cells produce offspring derived from the induced pole cells. This result demonstrates that polar plasm can function in interspecific species combinations and indicates that the molecular mechanisms of germ cell determination are conservative in evolution. Finally, in order to test whether there is any evidence for cytoplasmic inheritance of polar granules, embryos derived from hybrid pole cells were examined for their polar granule morphology. The fine structure of the granules conformed to that of the nucleus. Thus, no evidence was found for the cytoplasmic inheritance of these particular organelles.     

A novel system of fertility rescue in Drosophila hybrids reveals a link between hybrid lethality and female sterility

Hybrid daughters of crosses between Drosophila melanogaster females and males from the D. simulans species clade are fully viable at low temperature but have agametic ovaries and are thus sterile. We report here that mutations in the D. melanogaster gene Hybrid male rescue (Hmr), along with unidentified polymorphic factors, rescue this agametic phenotype in both D. melanogaster/D. simulans and D. melanogaster/D. mauritiana F(1) female hybrids. These hybrids produced small numbers of progeny in backcrosses, their low fecundity being caused by incomplete rescue of oogenesis as well as by zygotic lethality. F(1) hybrid males from these crosses remained fully sterile. Hmr(+) is the first Drosophila gene shown to cause hybrid female sterility. These results also suggest that, while there is some common genetic basis to hybrid lethality and female sterility in D. melanogaster, hybrid females are more sensitive to fertility defects than to lethality.     

以ND4L和ND4基因为标记探讨黑腹果蝇种组的系统发育关系

多年来的形态学、染色体组学以及DNA序列几个方面的研究均没有很好地阐明黑腹果蝇种组内的系统发育关系。本实验测定了33个样品的ND4和31个样品的ND4L基因序列,以D．obscuroides为外群,用最大简约法和Bayesian法分别构建进化树。结果表明两种方法构建的拓扑结构一致,而且大部分支系的支持率较高。整个黑腹果蝇种组分成三大谱系：1）montium种亚组;2）ananssae种亚组;3）Oriental种亚组（melanogaster、ficsphila、eugracilis、elegans、suzukii、takahashii）。montium是最早分化的种亚组。在第三谱系中,melanogaster分化得最早;然后依次是ficsphila,eugracilis,elegans;suzukii与takahashii为姐妹种亚组,最后分化。     

The Drosophila melanogaster hybrid male rescue gene causes inviability in male and female species hybrids

The Drosophila melanogaster mutation Hmr rescues inviable hybrid sons from the cross of D. melanogaster females to males of its sibling species D. mauritiana, D. simulans, and D. sechellia. We have extended previous observations that hybrid daughters from this cross are poorly viable at high temperatures and have shown that this female lethality is suppressed by Hmr and the rescue mutations In(1)AB and D. simulans Lhr. Deficiencies defined here as Hmr(-) also suppressed lethality, demonstrating that reducing Hmr(+) activity can rescue otherwise inviable hybrids. An Hmr(+) duplication had the opposite effect of reducing the viability of female and sibling X-male hybrid progeny. Similar dose-dependent viability effects of Hmr were observed in the reciprocal cross of D. simulans females to D. melanogaster males. Finally, Lhr and Hmr(+) were shown to have mutually antagonistic effects on hybrid viability. These data suggest a model where the interaction of sibling species Lhr(+) and D. melanogaster Hmr(+) causes lethality in both sexes of species hybrids and in both directions of crossing. Our results further suggest that a twofold difference in Hmr(+) dosage accounts in part for the differential viability of male and female hybrid progeny, but also that additional, unidentified genes must be invoked to account for the invariant lethality of hybrid sons of D. melanogaster mothers. Implications of our findings for understanding Haldane's rule-the observation that hybrid breakdown is often specific to the heterogametic sex-are also discussed.     

Analysis of the Drosophila maternal effect mutant MAT(3)1 by pole cell transplantation experiments

Summary Pole cell transplantations were used to construct germ line mosaics of the Drosophila melanogaster maternal effect mutant mat(3)1. The mutant is of particular interest since the development of embryos derived from homozygous mat(3)1 females is arrested at the pole cell stage. Such embryos form exclusively pole cells and no blastoderm cells. By means of germ line mosaics we could demonstrate the primary target tissue of mutant gene expression. For normal development the mat(3)1 ⁺gene has to be expressed in the germ line. Pole cells formed in defective embryos derived from homozygous mutant mothers were transplanted into normal recipient embryos to test their developmental potential. Heterozygous mat(3)1 pole cells were found to form fertile gametes in both sexes whereas homozygous mat(3)1 pole cells form fertile gametes only in males. The lack of progeny derived from homozygous mat(3)1 donor pole cells in recipient females further demonstrates the germ line autonomy of the mat(3)1 mutation. Pole cells from defective embryos that are transplanted into normal hosts colonize the gonads with the same frequency as donor pole cells derived from normal embryos. This indicates that mat(3)1 derived pole cells are normal with respect to their function as germ cells and that the mat(3)1 mutant might therefore offer a convenient source for the mass isolation of functional pole cells.     

线粒体ND4-ND4L基因在黑腹果蝇种组中的进化特征

本实验对黑腹果蝇种组(melanogaster species group)中8个种亚组33个样品两个线粒体基因ND4和ND4L进行了测序,并分析了ND4基因的序列差异和碱基替换特点,发现近缘物种中存在很明显的转换倾向,而在远缘物种中由于重复替换导致转换数处于饱和状态,我们的实验数据证实了线粒体基因较核基因有较快的进化速度。最后根据D．melanogaster与D．yakuba的遗传距离推算了8个种亚组的分化时间,ananassae种亚组最先分化,然后依次是montium,melanogaster,ficsphila,eugracilis,elegans,suzukii和takahashii最后分化。     

A Genetic Basis for the Inviability of Hybrids between Sibling Species of Drosophila

A mutation of Drosophila melanogaster whose only known effect is the rescue of otherwise lethal interspecific hybrids has been characterized. This mutation, Hmr, maps to 1-31.84 (9D1-9E4). Hmr may be the consequence of a P element insertion. It rescues hybrid males from the cross of D. melanogaster females to males of its three sibling species, D. simulans, D. mauritiana and D. sechellia. This rescue is recessive, since hybrid males that carry both Hmr and a duplication expected to be Hmr+ are not rescued. Hmr also rescues the otherwise inviable female hybrids from the cross of compound-X D. melanogaster females to males of its sibling species. This rescue is also recessive, since a compound-X heterozygous for Hmr does not rescue. Another mutation, discovered on the In(1)AB chromosome of D. melanogaster, is also found to rescue normally inviable species hybrids: unlike Hmr, however, In(1)AB rescues hybrid females from the cross of In(1)AB/Y males to sibling females, as well as hybrid males from the cross of In(1)AB females to sibling males. These data are interpreted on the basis of a model for the genetic basis of hybrid inviability of complementary genes.     

Misregulation of sex-lethal and disruption of male-specific lethal complex localization in Drosophila species hybrids

A major model system for the study of evolutionary divergence between closely related species has been the unisexual lethality resulting from reciprocal crosses of Drosophila melanogaster and D. simulans. Sex-lethal (Sxl), a critical gene for sex determination, is misregulated in these hybrids. In hybrid males from D. melanogaster mothers, there is an abnormal expression of Sxl and a failure of localization of the male-specific lethal (MSL) complex to the X chromosome, which causes changes in gene expression. Introduction of a Sxl mutation into this hybrid genotype will allow expression of the MSL complex but there is no sequestration to the X chromosome. Lethal hybrid rescue (Lhr), which allows hybrid males from this cross to survive, corrects the SXL and MSL defects. The reciprocal cross of D. simulans mothers by D. melanogaster males exhibits underexpression of Sxl in embryos.     

Basal relationships in the Drosophila melanogaster species group

The Drosophila melanogaster species group is a popular model for evolutionary studies due to its morphological and ecological diversity and its inclusion of the model species D. melanogaster. However, phylogenetic relationships among major lineages within this species group remain controversial. In this report, the phylogeny of 10 species representing each of the well-supported monophyletic clades in the melanogaster group was studied using the sequences of 14 loci that together comprise 9493 nucleotide positions. Combined Bayesian analysis using gene-specific substitution models produced a 100% credible set of two trees. In the strict consensus of these trees, the ananassae subgroup branches first in the melanogaster species group, followed by the montium subgroup. The remaining lineages form a monophyletic clade in which D. ficusphila and D. elegans branch first, followed by D. biarmipes, D. eugracilis, and the melanogaster subgroup. This strongly supported phylogeny resolves most basal relationships in the melanogaster species group, and provides a framework that can be extended in the future to encompass more species.     

Current status of the Drosophila melanogaster species-group (Diptera)

Abstract. The Drosphila melanogaster species-group, established by Sturtevant (1942) for fourteen species, is now known to contain 115 described species here divided into twelve named subgroups (including one newly proposed), as well as further undescribed species. Three of the species, melanogaster, simulans and ananassae , are cosmopolitan; two others, kikkawai and malerkotliana , are widespread in the southern hemisphere, the latter apparently a recent introduction to South America. The greatest numbers of species otherwise occur in the Oriental region with smaller numbers in the Ethiopian, eastern Palaearctic and Australian regions and in several islands of the South Pacific. D.rajasekari and D.raychaudhurii are synonymized with D.biamipes;also D.andamanensis Parshad & Singh is synonymized with D.andamanensis Gupta & Raychaudhuri.     

我国三地区黑腹果蝇中Wolbachia的系统发育关系及其对宿主生殖的影响

【目的】Wolbachia 是广泛存在于节肢动物和丝状线虫体内的一类共生菌, 能够以多种方式对宿主产生影响。精卵细胞质不亲和（CI）是其引起的最普遍的表型, 即感染Wolbachia的雄性宿主与未感染或感染不同品系的雌性宿主交配后, 不能产生后代或后代极少, 而感染同品系Wolbachia的雌雄宿主交配后则能正常产生后代。我们前期研究发现, 湖北武汉、 云南六库和天津3个地区黑腹果蝇Drosophila melanogaster被Wolbachia感染。本研究旨在明确这3个地区黑腹果蝇中Wolbachia的系统发育关系及其对宿主生殖的影响。【方法】利用Clustal X软件对Wolbachia的wsp基因序列进行比对, 利用MEGA软件构建系统发育树。采用多位点序列分型（MLST）的方法对Wolbachia进行分型。通过区内交配和区之间杂交的方式研究不同地区黑腹果蝇体内Wolbachia 的关系及其对果蝇生殖的影响。【结果】湖北武汉、 云南六库和天津3个地区黑腹果蝇中感染的Wolbachia都是属于A大组的Mel亚群。这3个地区果蝇感染的Wolbachia的序列类型（ST）不同, Wolbachia之间存在一定的差异。湖北武汉和天津果蝇中的Wolbachia能引起强烈的CI表型, 而云南六库果蝇中的Wolbachia引起的CI强度相对较弱。武汉果蝇中Wolbachia不能完全挽救天津果蝇中Wolbachia引起的CI表型, 而天津果蝇中Wolbachia也不能完全挽救武汉果蝇中Wolbachia引起的CI表型。【结论】武汉和天津地区黑腹果蝇中的Wolbachia可能距离较远。Wolbachia的长期共生可能对黑腹果蝇的进化产生了一定的影响, 湖北武汉与云南六库的黑腹果蝇中感染的Wolbachia属于不同的序列类型, 这2个地区的黑腹果蝇已发生了一定的分歧, 产生了一定的生殖隔离。     

Delocalization of polar plasm components caused by grandchildless mutations, gs(1)N26 and gs(1)N441, in Drosophila melanogaster

Two maternal-effect grandchildless (gs) mutations of Drosophila melanogaster, gs(1)N26 and gs(1)N441, cause delay in nuclear arrival at the polar plasm. In mutant embryos, polar plasm loses its ability to induce pole cells during retarded nuclear migration to the posterior pole of embryos. In the present study, it was shown that in N26 and N441 embryos, mitochondrial large rRNA (mtlrRNA), an essential factor for pole cell formation, is delocalized during the delay in nuclear arrival. This suggests that the loss of mtlrRNA causes failure of the mutants to form pole cells. Furthermore, it was shown that all of the other polar plasm components examined, namely Vasa protein, Germ cell-less protein, nanos mRNA and Polar granule component RNA start to be delocalized during the delay in nuclear arrival. This suggests that polar plasm integrity is not maintained in mutant embryos. It was finally shown that Vas is also delocalized in embryos that are inhibited to form pole cells by reducing the amount of mtlrRNA. This indicates that the segregation of polar plasm into pole cells is required to maintain polar plasm integrity. The mechanism regulating polar plasm integrity in embryos is discussed.     

Nup96-dependent hybrid lethality occurs in a subset of species from the simulans clade of Drosophila

The cross of Drosophila melanogaster females to D. simulans males typically produces lethal F(1) hybrid males. F(1) male lethality is suppressed when the D. simulans Lhr(1) hybrid rescue strain is used. Viability of these F(1) males carrying Lhr(1) is in turn substantially reduced when the hybrids are heterozygous for some mutant alleles of the D. melanogaster Nup96 gene. I show here that similar patterns of Nup96-dependent lethality occur when other hybrid rescue mutations are used to create F(1) males, demonstrating that Nup96 does not reduce hybrid viability by suppressing the Lhr(1) rescue effect. The penetrance of this Nup96-dependent lethality does not correlate with the penetrance of the F(1) hybrid rescue, arguing that these two phenomena reflect genetically independent processes. D. simulans, together with two additional sister species, forms a clade that speciated after the divergence of their common ancestor from D. melanogaster. I report here that Nup96(-) reduces F(1) viability in D. melanogaster hybrids with one of these sister species, D. sechellia, but not with the other, D. mauritiana. These results suggest that Nup96-dependent lethality evolved after the speciation of D. melanogaster from the common ancestor of the simulans clade and is caused by an interaction among Nup96, unknown gene(s) on the D. melanogaster X chromosome, and unknown autosomal gene(s), at least some of which have diverged in D. simulans and D. sechellia but not in D. mauritiana. The genetic properties of Nup96 are also discussed relative to other hybrid lethal genes.     

Mutations in a newly identified Drosophila melanogaster gene, mago nashi, disrupt germ cell formation and result in the formation of mirror-image symmetrical double abdomen embryos.

The mago nashi (mago) locus is a newly identified strict maternal effect, grandchildless-like, gene in Drosophila melanogaster. In homozygous mutant mago females reared at 17 degrees C, mago+ function is reduced, the inviable embryos lack abdominal segments and 84-98% of the embryos die. In contrast, at 25 degrees C, some mago alleles produce a novel gene product capable of inducing the formation of symmetrical double abdomen embryos. Reciprocal temperature-shift experiments indicate that the temperature-sensitive period is during oogenetic stages 7-14. Furthermore, embryos collected from mago1 homozygous females contain no apparent functional posterior determinants in the posterior pole. In viable F1 progeny from mago mutant females, regardless of genotype and temperature, polar granules are reduced or absent and germ cells fail to form (the grandchildless-like phenotype). Thus, we propose that the mago+ product is a component of the posterior determinative system, required during oogenesis, both for germ cell determination and delineation of the longitudinal axis of the embryo.     

Incompatibility between X chromosome factor and pericentric heterochromatic region causes lethality in hybrids between Drosophila melanogaster and its sibling species

The Dobzhansky-Muller model posits that postzygotic reproductive isolation results from the evolution of incompatible epistatic interactions between species: alleles that function in the genetic background of one species can cause sterility or lethality in the genetic background of another species. Progress in identifying and characterizing factors involved in postzygotic isolation in Drosophila has remained slow, mainly because Drosophila melanogaster, with all of its genetic tools, forms dead or sterile hybrids when crossed to its sister species, D. simulans, D. sechellia, and D. mauritiana. To circumvent this problem, we used chromosome deletions and duplications from D. melanogaster to map two hybrid incompatibility loci in F(1) hybrids with its sister species. We mapped a recessive factor to the pericentromeric heterochromatin of the X chromosome in D. simulans and D. mauritiana, which we call heterochromatin hybrid lethal (hhl), which causes lethality in F(1) hybrid females with D. melanogaster. As F(1) hybrid males hemizygous for a D. mauritiana (or D. simulans) X chromosome are viable, the lethality of deficiency hybrid females implies that a dominant incompatible partner locus exists on the D. melanogaster X. Using small segments of the D. melanogaster X chromosome duplicated onto the Y chromosome, we mapped a dominant factor that causes hybrid lethality to a small 24-gene region of the D. melanogaster X. We provide evidence suggesting that it interacts with hhl(mau). The location of hhl is consistent with the emerging theme that hybrid incompatibilities in Drosophila involve heterochromatic regions and factors that interact with the heterochromatin.     

Wolbachia transfer from Drosophila melanogaster into D. simulans: Host effect and cytoplasmic incompatibility relationships.

Wolbachia are maternally transmitted endocellular bacteria causing a reproductive incompatibility called cytoplasmic incompatibility (CI) in several arthropod species, including Drosophila. CI results in embryonic mortality in incompatible crosses. The only bacterial strain known to infect Drosophila melanogaster (wDm) was transferred from a D. melanogaster isofemale line into uninfected D. simulans isofemale lines by embryo microinjections. Males from the resulting transinfected lines induce >98% embryonic mortality when crossed with uninfected D. simulans females. In contrast, males from the donor D. melanogaster line induce only 18-32% CI on average when crossed with uninfected D. melanogaster females. Transinfected D. simulans lines do not differ from the D. melanogaster donor line in the Wolbachia load found in the embryo or in the total bacterial load of young males. However, >80% of cysts are infected by Wolbachia in the testes of young transinfected males, whereas only 8% of cysts are infected in young males from the D. melanogaster donor isofemale line. This difference might be caused by physiological differences between hosts, but it might also involve tissue-specific control of Wolbachia density by D. melanogaster. The wDm-transinfected D. simulans lines are unidirectionally incompatible with strains infected by the non-CI expressor Wolbachia strains wKi, wMau, or wAu, and they are bidirectionally incompatible with strains infected by the CI-expressor Wolbachia strains wHa or wNo. However, wDm-infected males do not induce CI toward females infected by the CI-expressor strain wRi, which is found in D. simulans continental populations, while wRi-infected males induce partial CI toward wDm-infected females. This peculiar asymmetrical pattern could reflect an ongoing divergence between the CI mechanisms of wRi and wDm. It would also confirm other results indicating that the factor responsible for CI induction in males is distinct from the factor responsible for CI rescue in females.     

Pole cells of Drosophila paulistorum: embryologic differentiation with symbionts.

The pole cells of young D. paulistorum embryos are destined to form the germinal cells of both male and female imagoes. In addition, specialized portions of the midgut may be derived from pole cell progenitors. In this initial study of their embryogenesis by means of electron microscopy, various stages of pole cell development are shown in both non-hybrid (potentially fertile) and intersemispecific hybrid (potentially sterile as males) materials. Originally, approximately 5 or 6 cells emerge to form the early polar cap and subsequently divide asynchronously until the 35-50 cells of the late polar cap are derived. Unlike other Drosophila species, however, mycoplasma-like symbionts, apparently an hereditary infection, have been traced to locations within the cytoplasm of these pole cells. They are depicted as arriving there after transmission via the egg cytoplasm, implicating this as their probable route of entry into the future germinal tissues of adult flies. It is postulated that these microorganisms function as an infectious reproductive isolating mechanism fostering hybrid male sterility between D. paulistorum semispecies.