Sex determination and dosage compensation in Drosophila melanogaster: production of male clones in XX females

Sex determination and dosage compensation in Drosophila are implemented by the ratio of X-chromosomes to sets of autosomes (X:A ratio). Our aim was to change this X:A ratio during development, and to assess the response of the affected cells in sexually dimorphic structures. For this purpose, clones of XO constitution were produced in female embryos and larvae of two genotypes in which almost the entire euchromatic arm of one X-chromosome was translocated to the third chromosome. Genotype I was heterozygous for the X-linked recessive mutations Sxl^fLS, genotype II was homozygous for Sxl⁺. The Sxl⁺ gene (sex-lethal) is involved in mediating sex determination and dosage compensation. In genotype I (Sxl^fLS), male clones could be generated up to 48 h in genitalia and analia, up to 72 h in the sex comb region and up to 96 h in 5th and 6th tergites. In genotype II (Sxl⁺), male clones only appeared in the tergites, and only up to 24 h. The difference in these results is ascribed to the presence of Sxl^fLS in genotype I: when homozygous, this mutation causes XX clones to differentiate male structures; most of the male clones produced in genotype I must therefore be XX. In contrast, male clones produced in genotype II must be XO. Since these were only found when generated in embryos we conclude that the X:A ratio expresses itself autonomously in clones by setting the state of activity of the Sxl gene around blastoderm stage. Once this is achieved, the X:A signal is no longer needed, and the state of activity of the Sxl⁺ gene determines sex and dosage compensation.     

The gene fl(2)d is required for various Sxl-controlled processes in Drosophila females

Summary In Drosophila melanogaster, the gene Sex-lethal (Sxl) controls the processes of sex determination, dosage compensation, oogenesis and sexual behaviour. The control of Sxl is by alternative splicing of its primary RNA. We have identified a gene, female-lethal-2-d (fl(2)d), which is needed for the female-specific splicing of Sxl RNA and which also has a vital function independent of Sxl. Here we analyse other aspects of the gene fl(2)d. Specifically, we have analysed the effect of the temperature-sensitive mutation fl(2)d ¹ on the viability of adult flies homozygous for this mutation. We have found that the viability of the mutant females is reduced, while that of the mutant males is not affected. In addition, the capacity of the mutant females to be inseminated is considerably reduced, whilst all the mutant males are able to inseminate females. These effects on females are suppressed by Sxl ^M1. However, the fat body cells of fl(2)d ¹ homozygous females are able to synthesize yolk proteins at the restrictive temperature. We have also carried out, in males, a clonal analysis of fl(2)d ², a mutation lethal in both sexes. We have found that the clones are fully viable. We conclude that the gene fl(2)d seems to be necessary during the adult life of females for the processes that require Sxl ⁺ activity. Moreover, the Sxl-independent vital function of fl(2)d seems to be required in both sexes only during larval development. Offprint requests to: L. Sánchez     

Requirement offlex (femalelethal onX) in the development of the female germ line ofDrosophila melanogaster

Drosophila melanogaster females homozygous forflex, an X-linked recessive mutation, do not survive. Hemizygous males are unaffected. Homozygous embryos appear to lack SXL, the product of theSex-lethal (Sxl) gene, apparently as a result of disruption ofSxl splicing. It is known that bothSxl and its somatic splicing regulators [snf andfl(2)d] also function in the development of the female germ line. For this reason, we investigated the role offlex in the germ line by generatingflex/flex clones inflex/+ females. Females carrying such clones in their germ lines do not lay eggs whereas females carryingflex+ eggs lay viable eggs. Additionally, DAPI staining of ovarioles showed that diploid germ cells that are homozygous mutant forflex do not complete oogenesis. These results indicate that theflex+ gene product may be required for the development of the female germ line.     

Biological Parameters for Four Coccinellid Species in Central Saudi Arabia

Developmental and reproductive rates ofAdonia variegataGoeze,Coccinella novemnotataHerbst,C. septempunctataL., andC. undecimpunctataL. were studied in wheat and barley in Central Saudi Arabia. Beetles were reared in the laboratory at 25 ± 2°C onBrevicoryne brassicae(L.) andRhopalosiphum padiL.A. variegataandC. undecimpunctatahad significantly higher egg hatching (X = 81.8 and 74.5%), adult longevity (X = 71.8 and 70.0 days), and survival (X = 61.8 and 61.5%), respectively.Coccinella undecimpunctatahad the highest fecundity (X = 370.5 eggs/female) and longest oviposition period (X = 29.8 days).C. septempunctataexhibited the lowest values for these biological parameters. No differences were evident in the total developmental time or egg and pupal duration, although species differences occurred in the larval duration. Larvae ofA. variegatadeveloped faster (X = 11.8 days) than Larvae ofC. septempunctata(X = 12.0 days),C. undecimpunctata(X = 12.2 days), orC. novemnotata(X = 13.1 days). The intrinsic rate of increase (r_m) and predicted population density were higher forC. undecimpunctata(X = 0.093 and 98553) after 60 days than forC. novemnotata(X = 0.088 and 59405),C. septempunctata(X = 0.086 and 35033), orA. variegata(X = 0.082 and 37991), respectively.     

Suppression of distinct ovo phenotypes in the Drosophila female germline by maleless− and Sex-lethalM

Mutations in ovo result in several different phenotypes, which we show are due to the regulation of distinct developmental pathways. Two X (female) germ cells require ovo⁺ activity for viability, but 1X (male) germ cells do not. In our study, we observed suppression of the ovo germline-lethality phenotype in loss-of-function maleless (mle) females indicating that ovo⁺ and mle⁺ have opposing effects in female germ cells; or that they are hierarchically related. Gain-of-function Sex-lethal (Sxl) alleles and male specific lethal-2 alleles did not suppress the ovo germline death phenotype. Many of the surviving germ cells in females mutant for both ovo and mle showed ovarian tumors. In contrast to the germline viability phenotype, we did observe suppression of the tumor phenotype in females heterozygous for gain-of-function alleles of Sxl. Further, females mutant for some hypomorphic ovo alleles were rendered fertile by Sxl gain-of-function alleles. Thus, ovo⁺ is required for at least two distinct functions, one involving mle⁺, and one mediated by Sxl⁺ gene products. The existence of ovo⁺ functions independent of mle⁺ and Sxl⁺ is likely. Dev. Genet. 23:335–346, 1998. © 1998 Wiley-Liss, Inc.     

Intraspecific host discrimination and larval competition inMicroplitis croceipes,Microplitis demolitor,Cotesia kazak (Hym.: Braconidae) andHyposoter didymator (Hym.: Ichneumonidae), parasitoids ofHeliothis virescens (Lep.: Noctuidae)

Intraspecific host discrimination and larval competition were studied forMicroplitis croceipes (Cresson),Microplitis demolitor Wilkinson,Cotesia kazak (Telenga), andHyposoter didymator (Thunberg), solitary endoparasitoids of the tobacco budworm,Heliothis virescens (F.). In ovipositional choice tests between unparasitized and parasitized hosts, the mean number of ovipositions for unparasitized hosts was significantly higher than the mean number of ovipositions for hosts parasitized once by a conspecific female forC. kazak andH. didymator, demonstrating that females of these 2 species discriminate against hosts recently (within a few seconds) parasitized by a conspecific female. No significant difference in oviposition occurred between these 2 kinds of hosts forM. croceipes andM. demolitor. Mean percent parasitization by a 2^nd conspecific female was determined at 24, 48, and 72-h delays in time between the 1^rst and 2^nd female attack, and with no delay. Except for the 0 h time delay forC. kazak andH. didymator, percent parasitization by a 2^nd conspecific female generally decreased as the delay in time between the 1^rst and 2^nd female attack increased. When the 2^nd parasitization immediately followed the 1^rst, one parasitoid larva always eliminated the other by physical combat. With a 24 or 48 h delay between the 1^rst and 2^nd parasitization, the younger larva was the victor over the older larva forM. croceipes, M. demolitor andC. kazak in at least 50% of the cases. Elimination of older larvae by younger larvae was by physical attack. However, forH. didymator, the older instar was the victor, and elimination of younger larvae by older larvae was probably through physiological processes. Further, older larvae ofH. didymator apparently killed the eggs of the 2^nd female by physiological processes.      

Bristle patterns,clones and cell competition along the anterior margin ofNotch wings ofDrosophila hydei

Summary The bristle pattern along the anterior margin ofNotch (N1-22.3) wings ofDrosophila hydei and the occurrence ofyellow (y 1–38.8) marked clones induced by X-ray irradiation during various larval stages are described. UnirradiatedN/N ⁺ wings show gaps (notches) in the longitudinal bristle rows along the 1st longitudinal vein, with tufts of bristles particularly near gaps. X-ray irradiation increases the number and total length of the gaps. The patterning of bristles along the margin depends on theN ⁽⁺⁾ genotype of the induced clones. RecombinantN ⁺/N ⁺ clones from irradiated wings show excessive growth with an autonomous wildtype bristle pattern. Characteristically, these clones do not respect the dorso-ventral compartment boundary along the wing margin, do not follow an exponential (2ⁿ) growth pattern, tend to fill the gaps with bristles and theiryellow medial row bristles are less often interspersed withy ⁺ bristles than described forN ⁺/N ⁺ wings. HomozygousN appears to be a cell lethal condition inD. hydei as it is inD. melanogaster. When y clones were kept phenotypicallyNotch (viz.,N/N/N ⁺) as the background cells, we found a lower number ofy bristles, a lower percentage of mosaic wings but also a reltive deficiency ofy ⁺ interspersions. The latter is discussed in relation to a possible clonal originof the notches.     

A male-specific lethal mutation in Drosophila melanogaster that transforms sex.

Sex-lethal, male-specific allele #1 (Sxl^M#1, 1–19.2) is a dominant, X-linked mutation that is lethal to males. It has no effect in females other than to rescue them from the otherwise lethal maternal effect of the autosomal mutation, daughterless. From a study of the effects of Sxl^M#1 on the development of sexually mosaic flies (gynandromorphs), it was observed that this lethal mutation can cause genetically male (haplo-X) tissue to differentiate as if it were female. With respect to its effect on sexual differentiation, the mutation transformer (tra, 3–45) is epistatic to Sxl^M#1, though the lethal effects of Sxl^M#1 are not modified by tra. In addition to its effect on sexual differentiation, Sxl^M#1 reduces the size of haplo-X imaginal disc and histoblast derivatives in general in a cell autonomous fashion. The viability of gynandromorphs with Sxl^M#1 tissue is very low, and the surviving mosaics have relatively little haplo-X tissue, suggesting that there is no single localized “lethal focus” for this mutation. The relationship between Sxl and daughterless is discussed, as well as the possible involvement of the Sxl locus in X-chromosome dosage compensation.     

A Device for Infecting Adult Tsetse Flies,Glossinaspp., with an Entomopathogenic Fungus in the Field

Various chamber designs for infecting natural populations ofGlossina pallidipes, G. longipennis,andG. fuscipes fuscipeswith the entomopathogenic fungusMetarhizium anisopliaewere tested in the field. All three species of tsetse flies entered the chambers and became infected with the fungus. Mortality attributed to infection byM. anisopliaeranged from 0 to 76% forG. pallidipes/G. longipennisand from 0 to 80% forG. fuscipes.One design proved to be more efficient than the others in permitting the passage of flies and contaminating them with fungal conidia. Dry conidia ofM. anisopliaein the infection chamber retained their infectivity for more than 21 days in the field.     

Synergistic interaction between particular X-chromosome deletions andSex-lethal causes female lethality inDrosophila melanogaster

We studied the effect on female viability oftrans-heterozygous combinations of X-chromosome deficiencies andSxl ^f1, a null allele ofSex-lethal. Twentyfive deficiencies, which together covered 80% of the X chromosome, were tested. Seven of thesetrans-hcterozygous combinations caused significant levels of female lethality. Two of the seven interacting deficiencies include the previously known sex determination genessans fills andsisterless-a. Four of the remaining uncover X-chromosomal regions that were not hitherto known to contain sex determination genes. These newly identified regions are defined by deficienciesDf(1)RA2 (7D10; 8A4-5),DJ(1)KA14 (7F1-2; 8C6),Df(1)C52 (8E; 9C-D) andDf(1)NI9 (17A1; 18A2). These four deficiencies were characterized further to determine whether it was the maternal or zygotic dosage that was primarily responsible for the observed lethality of female embryos,daughterless andextra macrochaetae, two known regulators ofSxl, influence the interaction of these deficiencies withSxl.     

The suppressor of Hairy-wing binding region is required for gypsy mutagenesis

Summary We undertook a deletional analysis of the gypsy retrotransposon in order to determine which sequences of the element are required for its mutagenic effect. We show that a phenotype indistinguishable from that ofy ² flies can be generated by transformingy ^– flies with a construct containing theyellow gene and a gypsy element located at the same insertion site inyellow as found iny ² flies. When flies are transformed with similar constructs in which increasing amounts of the 5 transcribed untranslated region of gypsy have been removed, either a partialy ² revertant or a completely revertant phenotype is obtained. These results yield direct proof that the region of gypsy to which thesu(Hw) protein binds is required for the generation of mutant phenotypes by this retrotransposon.     

Sex determination in<Emphasis Type="Italic"> Drosophila melanogaster</Emphasis> and<Emphasis Type="Italic"> Musca domestica</Emphasis> converges at the level of the terminal regulator<Emphasis Type="Italic"> doublesex</Emphasis>

Sex-determining cascades are supposed to have evolved in a retrograde manner from bottom to top. Wilkins 1995 hypothesis finds support from our comparative studies in Drosophila melanogaster and Musca domestica, two dipteran species that separated some 120 million years ago. The sex-determining cascades in these flies differ at the level of the primary sex-determining signal and their targets, Sxl in Drosophila and F in Musca. Here we present evidence that they converge at the level of the terminal regulator, doublesex (dsx), which conveys the selected sexual fate to the differentiation genes. The dsx homologue in Musca, Md-dsx, encodes male-specific (MdDSX^M) and female-specific (MdDSX^F) protein variants which correspond in structure to those in Drosophila. Sex-specific regulation of Md-dsx is controlled by the switch gene F via a splicing mechanism that is similar but in some relevant aspects different from that in Drosophila. MdDSX^F expression can activate the vitellogenin genes in Drosophila and Musca males, and MdDSX^M expression in Drosophila females can cause male-like pigmentation of posterior tergites, suggesting that these Musca dsx variants are conserved not only in structure but also in function. Furthermore, downregulation of Md-dsx activity in Musca by injecting dsRNA into embryos leads to intersexual differentiation of the gonads. These results strongly support a role of Md-dsx as the final regulatory gene in the sex-determining hierarchy of the housefly.Edited by D. Tautz     

Rearing phonotactic parasitoid flies [Diptera: Tachinidae,ormiini, ormia spp.]

Females of ormiine tachinids fly to their hosts' calling songs and deposit larvae on the host or nearby. Two species,Ormia ochracea (Bigot) andO. depleta (Wiedemann), were reared for at least 8 generations, making them the first ormiines to be laboratory-propagated. Both were reared on natural hosts:Gryllus spp. field crickets (principallyG. rubens) forO. ochracea, andScapteriscus spp. mole crickets forO. depleta. Commercially rearedAcheta domesticus tested as hosts were less satisfactory. Hosts were parasitized manually or by confinement with flies or planidia (infective larvae). Transparent, cylindrical, sleeved cages were designed to accommodate parasitized hosts and pupae and adults ofO. ochracea. Cages were joined to allowO. ochracea to cycle through its stages with minimum handling and care. Parasitized hosts and pupae ofO. depleta were held in containers of damp sand; adults were held in cages developed forO. ochracea. Adults of both species were maintained on applesauce, sugar cubes, powdered milk, and water. The life cycle ofO. ochracea was about 31 days and ofO. depleta about 36 days, with the principal difference being the time required for planidia to complete development. InO. ochracea the adults emerged synchronously but inO. depleta males preceded females. In both species sex ratio was generally I: 1 and females lived slightly longer than males.O. depleta from our laboratory colony have been released for biological control of mole crickets.      

Autoregulatory Functioning of a Drosophila Gene Product That Establishes and Maintains the Sexually Determined State

Sxl appears to head a regulatory gene hierarchy that controls Drosophila sexual dimorphism in response to the X chromosome/autosome balance. Only XXAA cells normally have Sxl⁺ activity. It maintains both the female morphogenetic sequence and a level of X-linked dosage-compensated gene expression compatible with diplo-X cell survival. In the absence of this activity, male sexual development and dosage-compensated gene hyperactivation ensure. Loss-of-function Sxl mutations generally have female-specific lethal effects caused by upsets in dosage compensation. New female-viable Sxl mutant alleles and combinations which lack Sxl's female sex determination function, yet still provide sufficient dosage compensation function for diplo-X survival, are described here. Consequently, such mutants cause genotypic females to develop as phenotypic males. Some of these sex-transforming Sxl mutants do not require the maternally produced da⁺ activity that is normally essential for the functioning of zygotic Sxl alleles. In this paper, products of these unusual alleles are shown to act in trans to induce the expression of zygotic Sxl⁺ alleles that would otherwise be unable to function due to a lack of maternal da⁺ activity. This result indicates a third function for Sxl⁺ product: a positive autoregulatory role. Controls for the autoregulation experiments demonstrated the sex-trans-forming epigenetic effect of the da mutation for the first time in diploids. In these experiments the female-specific zygotic lethal effects that normally would have accompanied loss of maternal da⁺ activity were suppressed by mutations known to block dosage-compensation gene hyperactivation—the autosomal, male-specific lethals. Three types of abnormal sexual phenotypes were produced in the experiments described here, each with important implications for the mechanism of sex determination: (1) a true intersex phenotype produced by one particular Sxl allele shows that Sxl⁺ must be involved in the cellular response to the X/A balance rather than in its establishment; (2) a maternally induced, female-sterile phenotype indicates that either the process of autoregulation or the mutants used to demonstrate it are tissue specific and (3) a mosaic intersexual phenotype whose character implies that the Sxl⁺ activity level is set early in development, both by the da ⁺-mediated X/A balance signal and by autoregulation, and is maintained subsequently in a cell autonomous fashion, independent of the initiating X/A balance signal. Thus, this study supports the view that sex determination is truly determinative in the standard developmental sense, and that Sxl is the carrier of the sexually determined state.     

The culture ofEntomophthora muscae (C) Fres. in carrot flies (Psila rosae F.) and the effect of temperature on the pathology of the fungus

A method for maintaining anin vivo culture ofEntomophthora muscae (C) Fres. on its original host, adult carrot flies (Psila rosae F.), is described. The lethal time for adult carrot flies was greatly influenced by temperature, both for infected and for uninfected flies. In the range 8.2°C–20.2°C the LT₅₀ for infected flies was about 5.4 times shorter than the estimated average life-span for uninfected flies. The discharge of primary spores was also strongly dependent on temperature. The total number of primary spores discharged per fly at 100% RH and in darkness ranged between 1.2×10⁴ and 9.6×10⁴ with a mean of 5.1×10⁴.      

Selection and micropropagation of nodulating and non-nodulating clones ofAlnus crispa (Ait.) Pursh

Summary 600,000 seedlings ofAlnus crispa were inoculated with a 111 mixture of theFrankia strains ACN1 ^AG , AGN1 _exo ^AG and MGP10i. After 3 successive inoculations and screenings, one individual, AC-4, was selected as non-nodulating (Nod^–) with Frankiae. This selected individual AC-4 (Nod^–) and two other clones ofA. crispa, AC-2 and AC-5, known for their ability to nodulate (Nod⁺) and two other clones ofA. crispa, AC-2 and AC-5, known for their ability to nodulate (Nod⁺) withFrankia werein vitro propagated. The different clones ofA. crispa in culture required different kinds and concentrations of sugar during the in vitro multiplication and rooting stages. Nodulation tests using 7Frankia strains indicated that the clone AC-4 (Nod^–) was non-nodulating with 6 of the 7Frankia strains tested. One strain,Frankia ANNI, isolated from one unique nodule produced on the mother-plant AC-4, induced 38% of the AC-4 plantlets to nodulate but with a number of nodules 10 to 20 times less than the clones AC-2 (Nod⁺) and AC-5 (Nod⁺). Morphological observations of the roots of AC-4 (Nod^–) indicated that this clone had few and abnormally short root hairs.     

The sex-lethal gene homologue in Chrysomya rufifacies is highly conserved in sequence and exon-intron organization

A great variety of sex determination mechanisms exists in insect species. In Drosophila melanogaster sex is determined by the ratio between X chromosomes and autosomes, while in the blowfly Chrysomya rufifacies it is maternally determined. A cascade of genes which are involved in sex determination has been identified in D. melanogaster with the Sex-lethal gene (Sxl) as the key gene. We screened genomic libraries of C. rufifacies with a probe of the Sxl gene from D. melanogaster and isolated a genomic region that included most of the homologous gene. DNA- and protein-sequence comparison showed a high percent identity between the Chrysomya and the Drosophila gene. Up to 90% identity of the amino acid sequences was found in the region that contained the RNA-binding domains. The degree of identity is much lower outside of this functionally important region (18% identity). cDNA analysis showed a highly conserved exon-intron structure between the two species, although sex-specific splicing as used in D. melanogaster for the regulation of Sxl activity, could not be detected in C. rufifacies.